Superantigens: Molecular Biology, Immunology, and Relevance to Human Disease

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SUPERANTIGENS

National Jewish Center for Immunology and Respiratory Medicine. and University of Colorado Health Sciences Center, Denver, Colorado

Tufts University School of Medicine, Boston, Massachusetts

University of Minnesota Medical School, Minneapolis, Minnesota

MARCELDEKKER, INC.

NEWYORK BASELHONGKONG

Library of Congress Cataloging-in-Publication Data

Superantigens : Molecular Biology, Immunology, and Relevance to Human Disease / [edited by] DonaldY. M. Leung, Brigitte T. Huber, PatrickM. Schlievert. cm. p. Includes bibliographical references and index. ISBN 0-8247-9813-9 (hardcover : alk. paper) 1.Superantigens. I. Leung,Donald Y. M. 11. Huber, Brigitte T. 111. Schlievert, Patrick M. [DNLM: Superantigens-physiology. 2. Receptors, Antigen, B -Cell-immunology. 3. Receptors, Antigen, T-Cell-immunology. QW573S959519971 QR186.6.S94S88 1997 6 16.07'92-421 DNLMDLC for Libraryof Congress 96-40407 CIP The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the address below. This book is printed on acid-free paper. Copyright

1997 by Marcel Dekker, Inc. All Rights Reserved.

Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronicor mechanical, including photocopying, microfilming, and recording, or by anyinformation storage and retrieval system, without permission in writing from the publisher. Marcel Dekker, Inc. 270 Madison Avenue, New York,

New York 10016

Current printing (last digit): l 0 9 8 7 6 5 4 3 2 1

PRINTED IN THE UNITED STATES OF AMERICA

Preface

Over the past seven to eight years, a new group of immunogens has been defined that are of microbial origin, i.e., bacterial or viral gene products. These agents have been termed superantigens, because they have a profound impact on the immune system of the host. Similar to mitogens, they lead to massive lymphocyte and macrophage activation. However, in contrast to mitogens, superantigens are specifically recognizedby receptors expressedon T and lymphocytes, and use different strategies for T-cell activation than conventional peptide antigens. A detailed understanding of their structure and mechanisms of action is important for an understanding of how infectious agents cause disease, and for the development of new approaches for treating diseases triggered by superantigens. A substantial amount of literature has accumulated in this field, defining the functional, biochemical, and molecular properties these proteins. Our book is intended to provide the reader with an up-to-date overview of this work, including new insights into the impact of microbial superantigens on human diseases. This text is intended to serve as a valuable resource for basicand clinical immunologists, physicians, cell biologists, biochemists, microbiologists, and graduate students interested in the role of superantigens in biology and human disease.

...

Preface

We would like to thank each of the authors for their outstanding contributions. We are grateful to Maureen Sandoval for her secretarial assistance, and Michele Sinoway and Janet Sachs for their editorial assistance.

Donald Y M. Leung Brigitte T. Huber Patrick M.Schlievert

Contents

iii

Preface Contributors

ix

1. HistoricalPerspective of SuperantigensandTheir Biological Activities Donald Y. M . Leung, Brigitte T. Huber,

1

and Patrick M. Schlievert 2. Immunobiology of

Susan R. Ross

“ T V

Superantigens

15

3. StructuralFeatures of MMTVSuperantigens Gary Winslow, John W. Kappler, and Philippa Marrack 4. Interaction of SuperantigenswithMHCClass

11 Molecules pascal M. Lavoie, Rafick-Pierre Skkaly, Jacques Thibodeau, and Francois Denis

5. SuperantigeninRabiesVirusand

Monique Lafon

Its Involvement in Paralysis

61

85

vi

Contents

6. SuperantigenAssociatedwithEpstein-BarrVirus:Potential

in

Role

103

Natalie Sutkowski and Brigitte T. Huber 7. Toxic Shock Syndrome Toxin-l: Molecular Structure and Basis

for

Recognition

127

Cathleen A. Earhart, David T. Mitchell, Debra L. Murray, Patrick M. Schlievert, and Douglas H. Ohlendorf 8. Comparison of Structures of Toxic Shock SyndromeToxin-l Unbound and Bound to a Class I1 Major Histocompatibility Molecule

149

David T. Mitchell, Patrick M. Schlievert, Douglas H. Ohlendor$ Jongsun Kim, Don C. Wiley, Robert G. Urban, and Jack L. Strominger 9. StaphylococcalEnterotoxins B and C: Structural Requirements for Superantigenic and Entertoxigenic Activities

167

Gregory A. Bohach D, and E Structure of T-cellSuperantigenicity L. Anders Svensson, Elinor M. Schad, Michael Sundstrom, Per Antonsson, Terje Kalland, and M i k e 1 Dohlsten

10. StaphylococcalEnterotoxins

andFunction,IncludingMechanism

11. The Exfoliative Toxins of Staphylococcus aureus John J. Iandolo and Stephen Keith Chapes

199

23 1

12. MolecularGenetics, Structure, andImmunobiology

of Streptococcal Pyrogenic Exotoxins A and C

257

Michael H. Kim and Patrick M. Schlievert 13. StreptococcalSuperantigen,Mitogenic Factor, andPyrogenic

Exotoxin B Expressed by Streptococcus pyogenes: Structure and

28 1

James M. Musser 14. Streptococcal M Protein: Role in Post-Streptococcal

Autoimmunity

311

Malak Kotb

15. TheSuperantigen Mycoplasma arthritidis Mitogen (MAM): mmunobiology andProperties Physical Kevin L. Knudtson, Allen D. Sawitzke, and Barry C. Cole

339

vii

Contents

16. Characterization of a Superantigen Produced by

Yersinia pseudotuberculosis Jun Abe and Tae Takda

369

17. B-Cell Superantigens and Their Biological Implications

405

Arnold I. Levinson and Lisa M . Kozlowski 435

18. Staphylococcal Toxic Shock Syndrome

Robert L. Deresiewicz 48 1

19. Streptococcal Toxic Shock Syndrome

Dennis L. Stevens 20. Viral Superantigens in Humans: A Potential Role in HIV and CMV Infection

503

David N. Posnett 21. Superantigens in Autoimmunity: Their Role as Etiologic and Therapeutic Agents

Joel Schiffenbauer, Howard Johnson, and Jeanne

525

Soos

22. Superantigens in Inflammatory Skin Diseases: A Role in Disease Maintenance and Induction David A. Norris and Donald Y. M. Leung

55 1

23. Superantigens in Human Disease: Future Directions in Therapy and Elucidation of Disease Pathogenesis Donald Y. M. Leung and Patrick M . Schlievert

58 1

Index

603

This Page Intentionally Left Blank

Contributors

Jun Abe Department of Child Ecology, The National Children’s Hospital Medical Research Center, Tokyo, Japan Per Antonsson Lund Research Center, Pharmacia & Upjohn, Lund, Sweden Gregory A. Bohach Department of Microbiology, Molecular Biology, and Biochemistry, University Idaho, Moscow, Idaho Stephen Keith C h a p Division of Biology, Kansas State University, Manhattan, Kansas

Barry C. Cole Division of Rheumatology, Department Internal Medicine, University Utah School of Medicine, Salt Lake City, Utah Frangois Denis Department of Immunology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada Robert L. Deresiewicz Brigham and Women’s Hospital, and Department Medicine, Harvard Medical School, Boston, Massachusetts Mikael Dohlsten

Lund Research Center, Pharmacia & Upjohn, Lund, Sweden ix

Contributors

Cathleen A. Earhart Department of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota Brigitte T. Huber Departmeht of Pathology, Tufts UniversitySchoolof Medicine, Boston, Massachusetts

John J. Iandolo Department of Diagnostic Medicine/Pathobiology, College Veterinary Medicine, Kansas State University, Manhattan, Kansas Howard Johnson Department of Microbiology and Cell Sciences, University of Florida College of Medicine, Gainesville, Florida Terje Kalland Pharmacia & Upjohn,Lund,Sweden John W. Kappler Department of Immunology,HowardHughesMedical Institute, National Jewish Center for Immunology and Respiratory Medicine, Denver,Colorado Michael H. Kim Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota Jongsun Kim Howard Hughes Medical Institute and Laboratory of Molecular Medicine, Children’s Hospital, Boston, and Hmard University, Cambridge, Massachusetts KevinL.Knudtson Division of Rheumatology,Department Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah Malak Kotb Departments of Surgery and Microbiology/Immunology, The University Tennessee, Memphis, and Veterans Administration Medical Center, Memphis, Tennessee LisaM.Kozlowski Pennsylvania School

DivisionofAllergyandImmunology, Medicine, Philadelphia, Pennsylvania

University

Monique Lafon Department of Virology, Neurovirology Unit, Pasteur Institute, Paris, France Pascal M. Lavoie Clinical Research Institute of Montreal, and Division of Experimental Medicine, School of Medicine, McGill University, Montreal, QuCbec,Canada

Contributors

Donald Y. M.Leung

DivisionofPediatricAllergy-Immunology,National Jewish Center for Immunology and Respiratory Medicine, and University of Colorado Health Sciences Center, Denver, Colorado

Arnold I. Levinson DivisionofAllergyandImmunology,Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Philippa Marrack Department of Medicine, Howard Hughes Medical Institute, NationalJewish Center for Immunologyand Denver,Colorado

Respiratory Medicine,

David T. Mitchell Department of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota

DebraL.Murray

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota

JamesM.Musser

Department of Pathology, Baylor College of Medicine,

Houston, Texas

David A. Norris Department of Dermatology, University of Colorado School of Medicine, and Department of Veterans Affairs Hospital, Denver, Colorado

Douglas H.Ohlendorf Department of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota

David N. Posnett Department of Medicine, Cornel1 University Medical College, New York, New York

Susan R. Ross Department of Microbiology, University Philadelphia, Pennsylvania

of Pennsylvania,

Allen D. Sawitzke Division of Rheumatology, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah ElinorM.%had

Department of Molecular Biophysics, Center try and Chemical Engineering, Lund University, Lund, Sweden

JoelSchiffenbauer DivisionofRheumatology,Departmentof University of Florida College of Medicine, Gainesville, Florida

for Chemis-

Medicine,

xii

Contributors

Patrick M. Schlievert Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota Rafick-Pierre Skaly Clinical Research Institute of Montreal, and Division of Experimental Medicine, Schoolof Medicine, McGill University, Montreal, Quebec,Canada Jeanne Soos Department of Microbiology andCellStudies,University Florida College of Medicine, Gainesville, Florida

of

Dennis L. Stevens Veterans Affairs Medical Center, Boise, Idaho, and Department of Medicine, University of Washington School of Medicine, Seattle, Washington Jack L. Strominger Department of Molecular and Cellular Biology, Harvard University, Cambridge,Massachusetts Michael Sundstrom PreclinicalResearchandDevelopment,Department Structural Biochemistry, Pharmacia & Upjohn, Stockholm, Sweden

of

NatalieSutkowski Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts

L. Anders Svensson Department of Molecular Biophysics, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden TaeTakeda Department of Infectious Diseases Research, The National Children’s Hospital Medical Research Center, Tokyo, Japan Jacques Thibodeau Analyfic Immunochemistry Unit, Pasteur Institute, Paris, France Robert G. Urban Department of Molecular and Cellular Biology, University, Cambridge,Massachusetts

Harvard

Don C. Wiey Howard Hughes Medical Institute and Laboratory of Molecular Medicine, Children’s Hospital, Boston, and Harvard University, Cambridge, Massachusetts Winslow .Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York

Historical Perspective of Superantigens and Their Biological Activities Donald Y. M. Leung National Jewish Center for Immunology and Respiratory Medicine, and University of ColoradoHealthSciencesCenter,Denver, Colorado Brigitte T. Huber University School

of Medicine, Boston, Massachusetts

Patrick M. Schlievert University of Minnesota Medical School, Minneapolis, Minnesota

HISTORY OF SUPERANTICENS

The term "superantigen" was first used by Kappler and Marrack and their co-workers in 1989 to describe the massive T-cell-receptor (TCR) Vp-restricted primary response to bacterial toxins (1). This was followed quickly by the insight that a class of murine endogenous antigens, known since the early 1970s as minor lymphocyte stimulating (Mls) antigens (21, had the same functional properties Mls antigens were originally described by Festenstein as unmapped genetic loci, encoding antigens that were inherited by Mendelian fashion and generated vigorous mixed leukocyte reactions (MLR) between major histocompatibility complex (MHCkidentical strains of

2

Leung et al.

mice (2). In these reactions, the responding cells were T lymphocytes, and the stimulatory cells were B lymphocytes. Since these antigenic loci mapped outside the MHC, they were named "minor" lymphocyte stimulating antigens. The designation of "minor" was somewhat of a misnomer, however, as it only reflected the fact that Mls antigens were described after the MHC antigens. Indeed, these Mls determinants are known to stimulate a high level T-cell proliferation. The function of Mls antigens did not become apparent until 1988, when their intimate relationship to the TCR was reported simultaneouslyby Kappler, Marrack, and their colleagues and MacDonald et al. (4), both groups demonstrating deletion of T-cell subsets from mice expressing Mls antigens. Importantly, these T-cell subsets were defined by the Vp domain of the TCR. Thus, the Mls antigens were clearly recognized bythe TCR, and the major determinant Mls reactivity appeared to be the expression of a particular BV (Vp)* gene segment. This latter property distinguished Mls antigens from nominal peptide antigens, in that the recognition of a specific peptide/MHC complex is generally dependent on all of the variable elements of the TCR, i.e., Vu, Ja,Vp, Dp, and JP regions. Furthermore, the Mls antigens were recognized as self-antigens, since Mls-reactive T cells are deleted from the repertoire during thymic maturation. Almost in parallel with the Mls story, staphylococcal enterotoxins, toxic shock syndrome toxin-l (TSST-l), and streptococcal scarlet fever toxins, known for years to be the cause of food poisoning and toxic shock syndrome in humans, were found to have immunological properties with striking similarities to Mls antigens In this regard, they were also found to stimulate a high frequency of T cells expressing a particular set of BV gene segments. a result, Mls antigens, staphylococcal enterotoxins, TSST-1, and streptococcal scarlet fever toxins were all designated as "superantigens."At the time, however, it was enigmatic that the gene productsderived from pathogenic bacteria causing disease in humans could have similar properties to endogenous Mls antigens of mice. *New nomenclature for T-cell-receptor(TCR) gene segments: The WHO-IUISnomenclature subcommittee onTCR designation has recommended the following procedures and criteria for TCR designations (Immunogenetics 1995; 42:451-453): B, G, and D to describe the genes (formerly designatedQ, p, y, 6) and V, D,J and C to describe the gene segments. We have adopted this terminology throughout book; e.g., TCRA is used to describe a TCR a gene, TCRAVlSl or VAL1 is used to describe the Val, subgroup gene, BV6S7 describe Vp6.7, etc. References at the protein level will use greek symbols for the variable elementsof the TCR regions, e.g., Vp.

Historical Perspectiveof Superantigens

II.

HALLMARKS OF SUPERANTICENS

Superantigens differ in four major ways from conventional peptide antigens: 1) They elicit a strong primary response, which is never seen with normal peptide antigens. This feature has led to the term "superantigen" for this new class of immunogens (l).'2) The Vp chain of the TCR is sufficient recognition of a superantigen, while a conventional peptide antigen requires a very specific interaction with the third hypervariable region of the TCR (1).This latter region of the TCR consists of the joining elements of both VJa and VDJP; thus, T cells responding to conventional peptide antigen have an extremely low precursor frequency. On the other hand, the murine genome contains only about functional BV genes, allowing of unprimed T cells to mount a response to any superantigen. In the case of human peripheral blood T cells, between 5 and 30% of T cells can react to the various superantigens, whereas most conventional peptide antigens stimulate less than 0.1% circulating T cells. While all superantigens discovered so far require MHC class I1 proteins for presentation to T cells, the T-cell response is not class I1 restricted violating the golden rule of MHC restriction seen for all other peptide antigen-specific T-cell responses. 4) Superantigens associate with MHC class I1 in unprocessed form (9-11). In contrast, conventional antigens are broken down into peptides before being loaded into the MHC peptide binding groove. The combinationof these four features unequivocally defines a superantigen. Superantigen-induced activation of T cells is dependent on expression of costimulatory molecules (see Chapter similar to that of conventional peptide antigens. Lackof accessory signals can lead to anergy induction in vivo and in vitro, because full T-cell stimulation cannot proceed. This latter point is much more obvious for superantigens than conventional antigens, because the former stimulate a large subset of lymphocytes that can easily be identified by serology. Thus, superantigens have served as valuable tools for immunologies to follow the fate of T cells exposed to antigen in vivo and in vitro. MICROBIAL ORIGIN OF SUPERANTICENS A.

Virally EncodedSuperantigens

1.

At the beginning of the 1990s the exciting discovery was made that murine Mls superantigens are encoded by murine mammary tumor

et al.

virus (MMTV) proviral DNA that had been integrated into the germline (12-14). This demonstrated a link between endogenous Mls antigens and exogenous bacterial toxins; i.e., these molecules were all derived from infectious agents. Furthermore, it was shown that infectious MMTV encodes such a superantigen and makes use of this molecule for facilitating transmission in its host, as will be discussed in Chapters 2-4 The microbial origin of superantigens in general implies that these proteins provide an advantage for the respective pathogen by engagingthe immune system.of the host (17,18). 2. SuperantigensAssociated with OtherViruses

While only the MMTV-encoded viral superantigen has been defined in detail, there are reports of association of superantigen activity with other viruses. Thus, the rabies virus nucleocapsid protein has been shown to be a superantigen, and models of its functional relevance have been proposed, as discussed in Chapter 5. Furthermore, superantigen activity has been attributed to two human tumor viruses, CMV (see Chapter 20) and EBV (see Chapter Although the functional significanceof the superantigen activity for theseherpes viruses has not yet been fully elucidated, it is likely that these molecules play a role in the viral life cycle, by facilitating infection and/or viral spread. Thus, we strongly believe that we have only reached the tip of the iceberg in recognition of virally encoded superantigens, and that the next few years will bring an explosion of new data in this field. The importanceof these findings is that they may provide completely new insights into the pathogenesis of these infectious agents, as well as into the mechanism of tumor transformation associated with these viruses. B.

Pyrogenic Toxin Superantigens(PTSAgs)

PTSAgs form a subfamily within the larger superantigen family and include TSST-l, staphylococcal enterotoxins, and streptococcal scarlet fever toxins (also known as erythrogenic toxins and streptococcal pyrogenic exotoxins, SPES). The term "pyrogenic toxin" is used for three reasons for these toxins: theyareamongthe most potent pyrogens known, historically the streptococcal toxins were referred to and this term unified as pyrogenic exotoxins in the early the toxins into the same family in the early 1980s (20). These bacterial toxins differ from other superantigens in that the PTSAgs are highly lethal, for example 3-5 pg of SPE type A is capable of caus-

Perspective Historical

of

Superantigens

5

ing streptococcal toxic shock syndrome in humans as observed in laboratory accidents. Despite having variable amounts of primary sequence similarity, PTSAgs also share a typical three-dimensional structure that is likely to be different from that of other superantigens. The streptococcal scarletfever toxins have been known since the early to cause scarlet feverand a fulminant illness that has the same features as streptococcal toxic shock syndrome, the latter illness first described by Cone et al. in and Stevens et al. in This illness is discussed in detail in Chapter 19 of this book. years it was thought the only property of these scarlet fever toxins was rash production. It is now clear the toxins have many other biological effects on the host, as discussed in Chapters and and rash production, although characteristic, may be one of the effects least harmful to the host. Barber in is generally credited with first providing evidence for enterotoxin production by Staphylococcus aureus. Today, seven major serotypes have been identified (A-H, excluding F). These toxins were originally recognized for their ability to induce vomiting and diarrhea, and thus their association with food poisoning. However, in addition to that unique activity, the enterotoxins share many other biological properties with PTSAgs. These are discussed in detail in Chapters and 10 in which we have broken the large enterotoxin group into two subgroups based on sequence similarities. as Finally, TSST-l was first identified by Schlievert et al. staphylococcal pyrogenic exotoxin C and by Bergdoll and colleagues as enterotoxin F in as the principal cause of staphylococcal toxic shock syndrome (Chapter Schlievert proposed the name "pyrogenic exotoxin C" since the toxin shared many properties with streptococcal pyrogenic exotoxin type C and toxic shock syndrome resembles severe scarlet fever. Bergdoll proposed the name "enterotoxin F" since the toxin resembled enterotoxins and toxic shock syndrome-like illness had anecdotally been reported associated with enterotoxins. In both investigators agreed upon the name "toxic shock syndrome toxin-l'' for this toxin TSST-l is the prototypical PTSAg, as discussed in detail in Chapters 7 and in that it contains all the shared PTSAg biological activities but lacks unique activities such as the ability to induce emesis as characterizes the enterotoxins. A summary of the biological properties of the PTSAgs is shown in Table 1.

Leung et al.

Table 1 Biological Properties of Pyrogenic Toxin Superantigens Toxin subfamily Staphylococcal Streptococcal scarlet TSST-l enterotoxins fever toxins

Property Superantigenicity Capacity to enhance: Lethal endotoxin shock Cardiotoxicity Skin reactivity resulting in a scarlet fever rash Suppression of immunoglobulin production Emesis Renal tubular and immune cell in the presence of endotoxin Ability to bind endothelial cells Ability to bind endotoxin

,

+

+

+

-

+

-

+

+ ?

?

+

? ?

+ +

The role of PTSAgs in the life cycle of bacteria has been the subject of much discussion and remains incomplete. The structural similarity among PTSAgs suggests these molecules have a common origin. Two questions arise in this regard: 1) there a selective advantage offered to the microbes by production of PTSAgs? 2) did the PTSAgs arise? 1.

SelectiveAdvantage

Production

PTSAgs

It is important to remember that S. aureus and group A streptococci are pathogens animals only, including humans, and indeed, group A streptococci are pathogens only of humans. Furthermore, human S. aureus strains are found only in humans, and strains isolated from other animals are restricted to thoseother hosts. These bacteriaare not found in plant or soil environments in nature. Presumably these ganisms have adopted those traits (such as PTSAgs) that permit survival in the presence of a host immune system and tissues for a sufficient period of time to ensure transmission to the next host. Note that the majority of the toxic biological properties of PTSAgs result from effects on the immune system. are variable traits in that some S. aureus and group A streptococci make the toxins and contain the genes for their production while other strains lack these traits and their genes. The toxins


of Superantigens

7

are not required forgrowth of the organisms on laboratory media, but production of PTSAgs in laboratory media does not put on the microbes at a growth disadvantage compared to nonproducers. The PTSAgs are contained on mobile genetic elements, forexample streptococcal pyrogenic exotoxins A and C and staphylococcal enterotoxin A are encoded by bacteriophage genes (28,291. Thus,the PTSAgs may be transferred horizontally from strain to strain. As discussed in Chapter 12, it has indeed been proposed that streptococcal pyrogenic exotoxin A arose from bacteriophage acquisition of a staphylococcal gene. Because of the epidemic of staphylococcal toxic shocksyndrome in the early 1980s, investigations were done to evaluate whether or not there was an emergence of a TSST-l-producing S. aureus strain that resulted in the illness (30,31). Several studies showed that the major S. aureus associated with toxic shock syndrome belonged to phage group I, 29/52 complex. This organism, with the capability making TSST-l, appears to have emerged since 1972, replacing nontoxin-producing organisms vaginally prior to that time. Thus, there must have been a selective advantage to this TSST-l-producing organism in humans. Incidentally, the emergence of the TSST-l-producing organism preceded by 4 years introduction to the marketplace of high-absorbency tampons, most associated with menstrual toxic shock syndrome. A similar emergence of SPEA-producing group A streptococci, notably of M type 1, appears to have occurred worldwide in the 1980s (32-34), in association with the reemergence of streptococcal toxic shock syndrome (Chapter 19) and other invasive diseases. Thus, from the above examples it is likely that the ability to "knock down" the host for a sufficient period of time to ensure transmission to the next host provides a selective advantage PTSAgproducing organisms. Toxin-mediated alteration of the immune system with consequent cytokine release appears to be that mechanism, either by preventing elimination from the host by up-regulating host cell receptors for the organisms. example, staphylococci and streptococci are eliminated from the host by development of opsonic antibodies combined with complement activation and PMN phagocytosis. Major effectsof PTSAgs in the host involve interference both with development of antibody responses (35) and with normal recruitment of PMNs (36,37) as a consequence of cytokine.release. It would be These properties are discussed in Chapters 7 and of particular importance to the microbe whether not the host dies as a result of exposure to PTSAg as often happens, provided the host remains alive for sufficient time for effective transmission. While we

Leung

al.

think of toxic shock syndrome as highly severe, the illnesses only become severe after several days of host exposure to the toxins. Thus, the typical case of staphylococcal toxic shocksyndrome occurs on day of menstruation, although flu-like symptoms areusually present earlier. But, the causative, S aureus grow to high numbers vaginally in general in the absence of a pyogenic response on day 1-2 of menstruation, presumably to ensure transmission. In a similar way streptococcal toxic shocksyndrome with necrotizing fasciitis most often occurs days after exposure of adults to the causative organism. However, in the preceding days the organism is rapidly spreading typically along a limb such that by the time full-blown streptococcal toxic shock syndrome illness occurs, there is massive necrotizing fasciitis. It is also clear that the majority of individuals who are exposed to PTSAg-producing staphylococci or streptococci do not develop toxic shock syndrome. Thus, toxic shock syndrome may be incidental to transmission of the PTSAg-producing organisms. In the case of atopic dermatitis (see Chapter it is known that large numbers of toxin-producing S. uureus attach to and chronically colonize the inflamed skin of these patients. The capacity of these organisms to secrete toxinsthat trigger or maintain skin inflammation in these patients may therefore provide a survival advantage for uureus 2.

Where

ComeFrom?

It is fairly easy to construct a family tree of relatedness among PTSAgs. The relationships among these toxins are discussedin Chapters 10, 12, and 13. However, a more basic question arises-where did these toxins come from in the first place? This is still the subject of considerable debate, but Mitchell et al. (39) have offered a possible answer. These and other investigators have noted that the PTSAgs exist as two domains, A and B, with domain A consisting of a grasp structure and domainB consisting of an OB fold structure (see Chapters 10, 12, and It can be argued that these two folds had different ancestral origin and came together in PTSAgs through gene recombination. For example, it i s highly unusual among bacterial toxins that molecules have sites for recognition of two receptors, as is the case with PTSAg, namely class I1 MHC molecules on antigenpresenting cells and the T-cell receptor. Toxins such as diphtheria toxin and others have a single host cell receptor recognition site. In addition, both the OB and grasp folds are seen in a variety of other microbial products. The OB fold is present in staphylococcal nuclease


of Superantigens

9

(40), the host cell receptor-binding domains of cholera and Escherichia coli heat-labile enterotoxins (411, and verotoxin (Shiga-like toxin)

(42). Furthermore, the p grasp fold is found in immunoglobulin-binding molecules such as protein G (43). C.

Other BacterialSuperantigens

Superantigens made by bacteria and which do not belong to the PTSAg family include staphylococcal exfoliative toxins, Mycoplasma arthritidis mitogen, Yersinia enterucolitica and pseudotuberculosis superantigens, and streptococcal M protein. Melish et al. (44) reported on characterization of exfoliative toxin A in 1972 and established an animal model for staphylococcal scalded skin syndrome (SSSS) (see Chapter 11). Many other investigators also reported on the characterization this toxin and identified serotype B exfoliative toxin. These two toxins are clearly recognized as causing SSSS, but the mechanism disease causation remains controversial. M. arthritidis mitogen (MAM) was first identified in the early 1980s by Cole and co-workers (45). The protein has now been cloned, sequenced, and compared to other superantigens (Chapter 15). As indicated in Chapter 15, the genus Mycoplasma is thought to have arisen as a result of degenerative evolution of Gram-positive bacteria. That M.arthritidis has retained the gene for MAM despite "downsizing" of its genome suggests MAM is an important survival property for the organism. Y. enterocolitica superantigen (YES) and a superantigen from Y pseudotuberculosis (Y. pseudotuberculosis mitogen, YPM) are thus far the only major superantigens produced by Gram-negative bacteria. YES was first demonstrated by Stuart and Woodward (46) in 1992, and the Y. pseudotuberculosis superantigen by Abe et al. (47) and others (48) in 1993. These superantigens are discussed in detail in Chapter 16. Finally, M proteins have been extensively studied since the turn of this century. These proteins are recognized as major streptococcal factors that are antiphagocytic, but against which protective host antibody responses are directed. These molecules have long been studied for the immunological cross-reactivitywith mammalian heart tissue, and thus are likely contributors to development of rheumatic fever. Kotb and colleagues (49) proposed M proteins were superantigens and suggested this may further enhance their role in disease causation. Whether not M protein has superantigenic activity remains controversial (50) and will be discussed further in Chapter 14.

Leung et al.

10

W.

CONCLUDING REMARKS RELEVANCE TO HUMAN DISEASE

The discovery of superantigens and characterization of their biological activities has provided new insights into potential mechanisms by which infectious agents may cause disease. In the last section of this book (Chapters 18-23), we will review the role of superantigens in human disease. The explosive nature of staphylococcal and streptococcal toxins make them likely causes for toxic shock and toxic shocklike syndromes. Potentially more exciting, however, is the possibility that their actions may differ in. various clinical settings, thus contributing to autoimmune and inflammatory responses. Furthermore, virally encoded superantigens may play a major role in the development of malignancies, especiallyin immunosuppressed patients (see Chapter 6 ) . Understanding the conditions that alter the host response to superantigens is a goal many laboratories. However, identification of microbial molecules involved in the pathogenesis of diseases, many of which have no definitive cure, is an important area of future investigation and will undoubtedly lead to new approaches for treatment of illnesses currently plaguing us. REFERENCES 1. White J, Herman A, Pullen AM, Kubo R, Kappler JW, Marrack P. The

2. 3. 4.

5. 6. 7.

VP-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell 1989; 56~27-35. Festenstein H. Immunogenetic and biological aspects of in vitro lymphocyte allotransformation (MLR) inthemouse.TransplantRev1973; 15:62-88. Kappler JW, Staerz JD, White Marrack PC. Self-tolerance eliminates T cells specific for Mls-modified productsof the major histocompatibility complex. Nature 1988; 332:35-40. MacDonald HR, Schneider Lees RK, et al. T-cell receptorV beta use predicts reactivity and tolerance to Mlsa-encoded antigens. Nature 1988; 332:40-45. Marrack P, Kappler J. The staphylococcal enterotoxins and their relatives.Science1990;248:705-711. Janeway CA Jr, Yagi J, Conrad PJ, Katz ME, Jones B, Vroegop S, Buxser S. T cell responses toMls and to bacterial proteins that mimic its behavior. Immunol Rev 1989; 107:61-88. Lynch DH, Gress RE, Needleman BW, Rosenberg SA, Hodes RJ. T cell responses to Mls determinants are restricted by cross-reactive MHC determinants. J Immunol 1985; 134:2071-2078. Janeway CJ, Katz ME. The immunobiology of the T cell response to Mlslocus-disparate stimulator cells.I.Unidirectionality, new strain combi-


9.

12. 13.

14. 15. 16. 17.

19. 20. 21. 22. 23.

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nations, and the role of la antigens. Journal of Immunology 1985; 134:2057-2063. Dellabona P, Peccoud J, Kappler J, Marrack P, Benoist C, Mathis D. Superantigens interact with MHC class I1 molecules outside of the antigen groove. Cell 1990;62:1115-1121. Fleischer B, Wrezenmeir H. T cellstimulation by staphylococcal enterotoxins: clonally variable response and requirement for major histocompatibility complex class I1 molecules on accessory or target cells. J Exp Med 1988; 167:1697-1707. Fleischer B. Stimulation of human T cells by microbial “superantigens.” [Review]. Immunol Res 1991; 10:349-355. Frankel WN, Rudy C, Coffin JM, Huber BT. Linkage of Mls genes to endogenous mammary tumour viruses of inbred mice. Nature 1991; 349:526-528. Dyson PJ, Knight AM, Fairchild S, Simpson Tomonari K. Genes encoding ligands for deletion of V beta l1 T cells cosegregatewith mammary tumour virus genomes. Nature 1991; 349:531-532. Woodland DL, Happ MP,Gollob KJ, Palmer E. An endogenous retrovirusmediatingdeletion of alphabeta T cells? Nature 1991a; 349:529-530. Marrack P, Kushnir E, Kappler A maternally inherited superantigen encoded by a mammary tumour virus. Nature 1991; 349:524-526. Golovkina TV, Chervonsky A, Dudley JP, Ross SR. Transgenic mouse mammary tumor virus superantigen expression prevents viral infection. Cell 1992; 69:637-645. Beutner U, Kraus E, Kitamura D, Rajewsky K, Huber BT. B cells are essential for murine mammary tumor virus transmission, but not for presentation of endogenous superantigens. J Exp Med 1994; 179:14571466. Held W, Waanders GA, Shakhow AN, Scarpellino L, Acha-Orbea H, MacDonald HR. Superantigen-induced immune stimulation amplifies mouse mammary tumor virus infection and allows virus transmission. Cell 1993; 74:529-540. Watson DW. Host-parasite factors in group A streptococcal infections: pyrogenic and other effects of immunologic distinct exotoxins related to scarlet fever toxins. J Exp Med 1960; 111:255-284. Schlievert PM. Alteration of immune function by staphylococcal pyrogenic exotoxin type. C: possible role in toxic-shock syndrome. J Infect Dis 1983; 147:391-398. Dick GF, Dick GH. A skin test for susceptibility to scarlet fever. JAMA 1924;82:256-266. Cone, LA, Woodard DR, Schlievert PM, Tomory GS. Clinical and bacteriologic observations of a toxic shock-like syndromedue to Streptococcus pyogenes. N Engl J Med 1987; 317:146-149. Stevens DL, Tanner MH, Winship J, Swarts R, Ries KM, Schlievert PM, Kaplan EL. Reappearance of scarlet fever toxin among streptococci in

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31. 32. 33. 34.

35. 36.

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the Rocky Mountain West: association with severe streptococcal soft tissue infection, sepsis and the toxic shock-like sylldrome. N Engl J Med 1989; 321:l-7. Holmberg SD, Blake PA. Staphylococcal food poisoning in the United States. JAMA 1984; 251:487-489. Schlievert PM, K.N. Shands KN, Dan BB, Schmid GP, Nishimura RD. Identification and characterization an exotoxin from Staphylococcus aureus associated with toxic-shock syndrome. J Infect Dis 1981; “EMJ516. Bergdoll MS, Crass BA, Reiser RF, Robbins RN, Davis JP. new staphylococcal enterotoxin, enterotoxin F, associated with toxic-shock-syndrome Staphylococcus aureus isolates. Lancet 1981;1:1017-1021. Bergdoll MS, Schlievert PM. Toxic-shock syndrome toxin. Lancet 1984; 2:691. Johnson LP, Tomai MA, Schlievert PM. Molecular analysis of bacteriophage involvement in group A streptococcal pyrogenic exotoxin A production. J Bacteriol 1986; 166:623-627. Betley MJ, Mekalanos JJ. Staphylococcal enterotoxin A is encoded by phage. Science 1985; 229:185-187. Altemeier WA, Lewis S, Schlievert PM, Bergdoll MS, Bjornson HJ, Staneck JL. Studies on the correlation of phage typing and toxin production of toxic-shock syndrome-associated Staphylococcus aureus. Ann Intern Med. 1982; 96:978-982. Todd J, Fishaut M, Kapral F, Welch Toxic-shock syndrome associated with phage-group-l staphylococci. Lancet 1978;2:1116-1118. Cleary PP, Kaplan EL, Handley JP, Wlazlo A, Kim MH, Hauser AR, Schlievert PM.Clonalbasis for resurgence of serious Streptococcus pyogenes disease in the 1980s. Lancet 1992; 339:518-521. Schlievert PM, Assimacopoulos AP, Cleary PP. Severe invasive group A streptococcal disease: clinical description and mechanisms of pathogenesis. J Lab Clin Med 1996; 127:13-22. Musser JM, Kapur V, Szeto J, Pan X, Swanson DS, Martin’DR. Genetic diversity and relationships among Sfreptococcus pyogenes strains expressing serotype M1 protein: recent intercontinental spread of a subclone causing episodes invasive disease. Infect Immun 1995; 63:994-1003. Davis JP, Chesney PJ, Wand PJ, Laventure M, Investigation and Laboratory Team. Toxic- shock syndrome: epidemiological features, recurrence, risk factors, and prevention. N Engl J Med 1980; 1429-1435. Fast DJ, Schlievert PM, NelsonRD. Nonpurulent response to toxic shock syndrome toxin-l producing Staphylococcus aureus: relationship to toxinstimulated production of tumor necrosis factor. J Immunoll988; 140:949953. Fast DJ, Schlievert PM, Nelson RD. Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production. Infect Immun 1989; 57:291-294.


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Reiser RF,Yang E, Norris AD, 38. Leung DYM, HarbeckH,BinaP, Hanifin JM, Sampson HA. Presence of IgE antibodies to staphylococcal enterotoxins on the skinof patients with atopic dermatitis: evidence for a new group of allergens. J Clin Invest. 1993; 92: 1374-80. 39. Mitchell DT, Schlievert PM, Ohlendorf DH. Structural evidence for the evolution of pyrogenic toxin superantigens (submitted). 40. Loll PJ, Lattman EE. The crystal structure of the ternary complex cf staphylococcal nuclease, Ca+2,and the inhibitor pdTp, refined at 1.65 A. Proteins 1989;5:183-201. 41. Merritt EA, Sarfaty S, van den Akken F, L'Hoir C, Martial JA, Hol WG. Crystal structure of cholera toxin P-pentamer bound to receptor GM1 pentasaccharide. Prot Sci 1994; 166-175. 42. Stein PE, Boodhoo A, Tyre11 GJ, Brenton JL, Read RJ. Crystal structure of the cell-binding j3 oligomer of verotoxin-l from E. coli. Nature 1992; 355:748-750. Achari A, Whitlow M, 43. Gronenborn AM, Filpula DR,EssigNA, Wingfield PT, Clore GM. A novel, high stable fold of the immunoglobulin binding domain of streptococcal protein G. Science 1991; 253:657661. 44. Melish ME, Glasgow LA, Turner MD. The staphylococcal scalded skin syndrome: isolation and partial characterization of the exfoliative toxin. J Infect Dis 1972; 125:129-140. 45. Cole BC, Daynes RA, Ward JR. Stimulation of mouse lymphocytes by amitogen derived fromMycoplasma arthritidis. I. Transformation is associated with an H-2-linked gene that maps to the I-ED-C subregion. Immun01 PM, Woodward JG. Yersinia enterocolitica produces superantigenicactivity. J Immunol Abe J, Takeda T, Watanabe Y, Nakao H, Kobayashi N, Leung DYM, Kohsaka Evidence for superantigen production by Yersinia pseudotuberculosis. J Immunol Miyoshi-Akiyama T, Imanishi K, Uchiyama T. Purification and partial characterization of a product from Yersinia pseudotuberculosis with the ability to activate human cells. Infect Immun TomaiM,KotbM,MajumdarG,BeachyEH.Superantigenicityof streptococcal Mprotein. J Exp Med Fleisher B, Schmidt KH, Gerlach D, Kohler Separation of mitogenic activity from streptococcal M protein.Infect Immun


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of

INTRODUCTION

Mouse mammary tumor virus (MMTV) has served as a model for the study of breast cancer since its discovery in the late 1920s as an infectious agent that could be transmitted to newborn mice through milk (1,2) (Fig. 1). Genetic studies also showed that there was an inherited form of breast cancer in mice and that foster-nursing hightumor-incidence pups on low-tumor-incidence mothers did not always free the offspring of mammary tumors The agent associated with the milk-borne transmission of mammary tumors was found to be a retrovirus (4). When the replication pathway of retroviruses was elucidated and it was shown that their genomic RNA was reversetranscribed to a DNA copy that integrated into chromosomes, the basis of the inherited form of MMTV-induced mammary cancer was understood (5). It became apparent for a number species, including mice, that there were germline copies of retroviruses inherited from one generation to the next (6). Virtually all inbred mice were shown to contain endogenous copiesof MMTV and the chromosomal location and copy number of these proviruses differed from strain to 15

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"

" Mutated provirus Functional provirus

-v

ON

Figure 1 Genetic and exogenous transmission of MMTV.

strain (4). Moreover, it was recognized that in some mouse strains expression of a functional endogenous provirus resulted in the inherited form of the disease (7) (Fig. 1 ) . More recently, endogenous and exogenous MMTVs have generated interest because one of the virus-encoded proteins possesses superantigen (Sag) activity. The expression of Sags expressed from endogenous MMTVs has profound effects on the immune repertoire of mice, as discussed elsewhere in this volume. In this chapter, the role that this protein plays in the MMTV infectious cycle and the retention of germline, MMTV-encoded Sags as an antiviral defense mechanism will be described. Brief Description of MMTV 1. Viral

andProteins

MMTV has a typical retrovirus genome, in which the coding regions for the viral capsid (Gag), polymerase (Pol), and envelope (Env) proteins are flanked by two long terminal repeats (LTRs) (Fig. 2). Two major RNAs are transcribed, both of which initiate in the 5' LTR, a full-length genomic transcript of 8.5 kilobases (kb) and a spliced RNA of 3.8.kb (5).The genomic transcript, which is packaged in the virion, encodes the group-specific antigen (Gag) capsid and

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Figure 2 Map of MMTV and transcripts. The large arrow denotes the major transcription initiation site, the small arrows minor initiation sites. The black boxes show the relative position the glucocorticoid response elements.

reverse transcriptase proteins (Pol). The virion envelope proteins (Env) are translated from the spliced mRNA. Two Envpolypeptides are produced by protease cleavageof a polyprotein precursor. The cell surface (SU) and transmembrane (TM) polypeptides associateto the two domains of the viral protein responsible MMTV binding to its cellular receptor ( 8 ) . A third transcript that may encode the Sag protein has been described (9,lO). This 1.7-kb RNAinitiates in the 5' LTR, using the same splice donor site as the env mRNA and a splice acceptor site just upstream of the LTR (Fig. 1). Although the major transcription start site is found in the LTR, there have been several reports of additional initiation sites within "TV. Phorbol ester-inducible transcripts that initiate in the env gene have been found in some T-cell lymphomas (11,12) (Fig. 1). More recently, Reuss and Coffin have proposed that the sag RNA could initiate within env, because they observed transcription initiation in a cultured early B-cell line transfected with a construct lacking the 5' LTR (13). Finally, an additional promoter located upstream of the major transcription initiation site in the LTR has been described (14) (Fig. 2). Whether any of these novel promoters playa role in the biological activity of Sag or viral infectivity awaits experiments in which mutations are introduced into the novel promoter sequences within the context the virus. For most retroviruses, including MMTV, the LTRs contain the major transcription start site and the regulatory regions necessary for determining the level of RNA produced by cellscontaining integrated proviruses. One of the earliest observations about MMTV was that

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pregnancy or glucocorticoid and progesterone hormones dramatically increased viral transcription and, as a consequence, virus production (15). These sequences, termed the glucocorticoid response elements (GRE), are responsible for the up-regulation of virus production that occurs during lactation, when glucocorticoid and progesterone levels are elevated (16) (Fig. 2). As expected for a virus transmitted through milk, there are sequences within the LTR that confer mammary gland-specific expression (17-19). Viral transcripts can also be found in lymphoid tissues ( 20) ; the sequences controlling MMTV expression in B and T cells have not yet been defined. 2. The MMTV LTR Encodes a Protein

When sequencing of the virus began in the late 1980s, it was discovered that the MMTV LTR contained an open reading frame (ORF) (21). This ORF peptide was the first retroviral protein shown to be encoded in an LTR and for many years its function was unknown. It was suggested that the protein could play a role in the oncogenic process (21). Several studies indicated the protein was involved in the regulation of viral transcription. For example, van Klaveren and Bentvelzen showed that cotransfection of a plasmid containing the Sag coding region and one with the MMTV LTR driving expression of the CAT reporter gene resulted in elevated transcription of the reporter gene (22). In contrast, Giinzburg and colleagues showed in similar types of studies that transfection of a plasmid with part of the gag as well as the sag gene down-regulated transcription from the MMTV promoter (23). With the discovery that the ORF protein possessed Sag activity, the significance of its role in transcription regulation has become unclear. The major role of the ORF or Sag protein is most likely to facilitate MMTV infection in pups nursing on viremic mothers, as described below. 3. Mechanism of Tumorigenesis

MMTV is a nonacute transforming retrovirus; that is, it does not encode its own oncogene. Instead, MMTV causes mammary tumors by integrating next to cellular oncogenes and activating their transcription (24,25). MMTV works through a unique set of oncogenes that were not previously identified prior to their association with MMTV-induced tumors. These int (for integration site) oncogenes are for the most part not normally expressed in mammary or any other adult tissue. Instead, several int genes have been shown to be important in prenatal development (24,26,27).


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Both virgin and multiparous females, but not males, develop MMTV-induced mammary gland tumors (1). However, pregnancy reduces the latency and increases the incidence of mammary tumor induction (1).The effect of pregnancy is twofold. First, as described in the preceding section, the induction of MMTV transcription increases virus production, leading to higher infection levels. Second, the mammary gland epithelial cells undergo cell division in response to lactogenic hormones, which may allow for more efficient infection. Because MMTV integrates relatively randomly in the genome, as do all retroviruses, the activation of cellular oncogenes is a stochastic event. Thus, the more MMTV particles produced, the more cells become infected, increasing the likelihood that oncogene activation and hence mammary tumors will be induced. Prior to the availability of molecular biological techniques used to detect MMTV RNA or DNA, an effective measurement of virus load in a mouse was the incidence and latency of tumor formation (1). The offspring of an animal that shed high-titer virus in its milk would show a high tumor incidence, with a latency of less than 6-7 months. In contrast, the offspring of a mouse that produced little virus would not all develop tumors and the latency of this tumor formation was increased. As will be seen below, a delay in tumor incidence is probably significant for the reproductive life span of the mouse. 4.

Endogenous and Exogenous MMTVs

In addition to acquiring MMTV through milk, virtually all inbred strains of mice contain endogenous copies of MMTV as integrated proviruses. As with all endogenous retroviruses, these represent infection of germ cells or early embryos with exogenous viruses that were then maintained in the germline. Approximately 50 different M t v loci located at different chromosomal sites have been identified. Many of the M t v loci are transcribed; however, they often show a different tissue-specific pattern and timing of expression. For example, M t u 7 and -9 are only expressed at high levels in lymphoid tissue, while Mtu17 RNA is found at significant levels only in mammary gland (28,29). The differences in expression are somewhat surprising, because the LTRs of the different endogenous MMTVs are remarkably homologous to each other outside the superantigen hypervariable coding region (30 ; described in Chapter 3 ) . Indeed, the promotors and hormone regulatory elements among the different MMTVs are almost identical (30). Small sequence differences in transcription factor-binding sites important for the regulation of MMTV

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transcription and integration site effects probably account for this variable expression. Most endogenous MMTV proviruses do not produce infectious particles. Some Mtv loci are known to have mutations or deletions in protein-coding regions (31) (Fig. l), while others are not expressed in the mammary gland, as described in the preceding paragraph. The GR mouse strain, on the other hand, has an infectious endogenous provirus, M t v 2 , that can be transmitted to other strains of mice as an exogenous, milk-borne virus (32) (Fig. 1). As a result, female GR mice develop mammary tumors after their first pregnancy and do not survive past 1 year of age (33). In summary, inbred mouse strains contain several germline copies of"TV, which in most strains are not expressed, are not transcribed in the mammary gland, contain coding region mutations that preclude production of infectious virus. These endogenous viruses affect the immune repertoire, but otherwise are not deleterious for their hosts. In contrast, it may be advantageous for mice to retain defective endogenous MMTVs in their genome to prevent infection by exogenous milk-borne virus, as described below. II. THE ROLE OF THE IMMUNE SYSTEM IN MMTV TRANSMISSION A.

Historical Perspective

1. GeneticStudies

After the discovery that there was an infectious agent that could be transmitted through milk to nursing pups, it was recognized that not all strains of mice were equally able to be infected with MMTV. Notably, the C57BL mouse strain and its derivatives were shown to have very low mammary tumor incidence when foster-nursed on C3H/He mice known to transmit virus either to their own pups or to other mouse strains, such as BALB/c (34). Genetic studies mapped one major resistance gene to the major histocompatibility complex(MHC) locus in C57BL mice (34). It is now known that the Sag encoded by C3H/He"TV+ mice is only efficiently presented by the MHC class I1I-E chain, as discussed elsewhere in this volume. Since C57BL mice have a genomic deletion of the I-E gene, it is thought that these early genetic studies mapped the resistance to the gene for this molecule. Indeed, it has recently been shown that C57BL/6 mice transgenic for I-E are easily infected by milk-borne virus (35).


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In addition to the MHC class I1 genes, C57BL mice may contain an additional virus resistance gene. Classic backcrossingstudies demonstrated that were was an additional resistance locus in C57 mice that could be genetically segregatedfrom the MHC locus (36). These genetic studies have recently been confirmed using B1O.BR mice, which have the MHC H-2k alleleon a C57BL/10 background; B1O.BR mice are less infected by MMTV(C3H) in comparison with C3H/HeN (H-2k) mice when nursed on the same C3H/HeN "TV+ mothers (Golovkina et al., in preparation). Backcrosses between C3H/HeN and B1O.BR mice indicate that there is a single, recessive resistance gene in the C57BL/10 background that controls resistanceto infection. It is not.yet known, however, what this gene is what step of the virus life cycle it affects. It has been reported, however, that a gene in the C57BL/6 background decreasesthe effectiveness of endogenous Sag presentation (37). At least two other loci were identified that affect the ability of mice to be infected with MMTV and that appear to involve the immune system. The resistancegene in the I strain mice did not map to the MHC to the other C57BL locus, since I x C57BL F1 mice are highly susceptible to mammary tumors (38). It was thought that the resistance of this strain to MMTV is due to the high titer its antibody response to virus infection (38). An additional gene thought to contribute to resistance to MMTV infection is the lipopolysaccharide sensitivity allele (Ips). A substrain of C3H mice maintained at the Jackson Laboratory, C3H/HeJ, had a spontaneous mutation of this allele in the 1960s; C3H/HeJ mice are 60 times less sensitive to killing by Ips than are other substrains of C3H mice, such as C3H/HeN. This resistance allele (Ips') also results in decreased B-cell proliferation and other immune system effects in response to this endotoxin (39). Interestingly, C3H/HeJ mice show a much lower level of infection by MMTV(C3H) than do C3H/HeN mice and they develop fewer mammary tumors with a much longer latency (40). This indicates that there is overlap between the Ips response and MMTV infection pathways. One possible mechanism by which this occurs is at the level of transcription. Lps can up-regulate transcription from the endogenous Mtv9 locus in B cells (41,42). Stimulation of T cells by Sag B cells by T-cell cytokines may also increase viral RNAsynthesis through the same pathways as Ips. example, if virus transcription in C3H/HeJ mice is lower than in C3H/HeN mice, decreased virion production would result. As a result, C3H/HeJ would be less infected than C3H/

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HeN mice and develop fewer mammary tumors. It is not known, however, whether all MMTVs, including the exogenous viruses such as MMTV(C3H), are transcriptionally regulated by Ips nor what DNA sequences within Mtu9 confer Ips responsiveness. OtherStudiesImplicatingtheImmuneSystem In addition to the genetic studies, a number of investigators reported that MMTV antigens could be detected in cells of the immune system, such as B and T cells (43). Similarly, transplantation experiments in which thymic splenic lymphocytes from infected mice were injected into na'ive animals showed that virus transfer could occur (44,45). However, these studies did not show that cells of. the immune system were required for MMTV infection. The first direct evidence that the immune system was involved in the MMTV infection pathway was obtained when Squartini and colleagues showed in 1970 that neonatal thymectomyof newborn mice that nursed on viremic mothershad a much lower incidenceof mammary tumors than sham-operated mice (46). Similarly, nude mice also were also shown to be relatively resistant to MMTV-induced tumors (44). Transplantation experiments into nude mice indicated that T cells were the most efficient at transmitting virus (44); however, other investigators have found that injection of MMTV-infected B cells into na'ive mice also results in the infection of other lymphoid and mammary gland cells (47)B.

The Finding that the ORF Gene Encoded a Superantigen

A major breakthrough in the understanding of the transmission of MMTV came with the discovery of the ORF protein function. In the early 1990s, mapping studies by several groups showed that the endogenous MMTVs cosegregated genetically with the endogenous superantigens called the MZs loci (described in Chapter 1). Similarly, it was shown that there was maternal but not paternal transmission of a Vpl4-deleting element by C3H/HeJ mice infected with MMTV(C3H) (48,49). Shortly thereafter, Choi et al. showed that the MMTV (C3H) ORF encoded Sag activity, since cells transfected with constructs containing this viral gene alone could stimulate cognate Vp14+ and Vp15+ T cells (50). Moreover, Acha-Orbea and colleagues demonstrated that transgenic mice containing the gene from GR virus showed specific deletion of their VplCbearing T cells (51).

These discoverieswere a major breakthrough in both the immunology and virology fields. The role of the MIS loci in shaping im-

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mune repertoire had long been a subject of investigation for immunologists and these results explained their genetic segregation. For virologists, the identification of a viral protein that affected cells of the immune system helped explain how lymphoid cells were involved in virus transmission. The remainderof this chapter will describe how the Sag protein works to facilitate MMTV infection and how the endogenous loci confer protection against exogenous virus. C.

How the MMTV Sag Protein Functions in the infection Pathway

The Role

T Cells

The Mls loci were originally identified becauseof their ability to stimulate the cellular division of a large percentage T cells in mixed lymphocyte cultures (52). In addition to stimulating cell division, viral and bacterial Sags cause T cells to secrete cytokines that cause B-cell expansion. Thus, a major effect of Sag presentation is the creation of dividing T and B cells. It is well known that most retroviruses, with the exception of human immunodeficiency virus (HIV), can only infect activelydividing cells (53-55). Recent evidence indicates that retroviral replication complexes are not able to crossthe nuclear membrane; asa result, the double-stranded DNA copy of genomic RNA that will integrate into the chromosomes can only enter the nucleus during the period of nuclear membrane breakdown (54,551. Thus, it was hypothesized that a major role for the MMTV Sag protein during the acquisition of milk-borne virus by uninfected pups is to cause T-cell stimulation (Fig. 3). This would result in the creation of actively dividing populations of T, B, and other bystander cells that could be easily infected by virus. In the absence of Sag-responsive T cells, viral infection would be inefficient, because there would be few, if any, actively dividing, infection-competent cells. The hypothesis that Sag-cognate T cells were required for virus infection was tested using mice transgenic for the MMTV(C3H) (56). The presence of endogenous MMTVs or loci in the genome causes the deletion of all V@-bearingT cells that recognize the Sags encoded by these loci during the shaping of the immune repertoire. Transgenic mice that expressed the MMTV (C3H) Sag protein under the control of the MMTV LTR deleted cognate VplGbearing T cells. When these mice were nursed on C3H/HeN mothers that produced milk-borne MMTV (C3H), they were resistant to infection relative to their nontransgenic littermates that retained cognate T cells. More-

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+ -+--”

PREGNANCY

GLUCOCORTICOIDS PROGESTERONE

Figure 3 Model of acquisition

MMTV by susceptible

over, trangenic mice that had incomplete deletion of their Vp14+ T cells due to low transgene expression levels were still susceptible to virus infection (56), while those that had very efficient deletion were highly protected against infection (28). These results were later confirmed for a different exogenous MMTV, called SW virus (57). Unlike MMTV(C3H), this virus interacts with Vp6+ T cells, because of sequence differences in its Sag hypervariable region. The Sag protein encoded by Mtu7, which is highly homologous to that of SW virus, also causes the deletion of Vp6+ T cells. Held and colleagues showed that inbred mouse strains that contained Mtu7 were resistant to SW but not MMTV(C3H) virus (57). In addition, transgenic mice that express only the Vp8.2specific chain of the T-cell receptor on their T cells could not be infected with SW virus because they lacked cognate T cells (57). It also became clear why C57BL mice were resistant to milkborne MMTV(C3H). Because of the lack of the class I1 I-E molecule, there is no Sag presentation in C57BL mice, and as a result, little or no stimulation of cognate T cells can occur. Hence, MMTV infection is inefficient. In contrast, C57BL/6 mice transgenic for I-E were efficiently infected, since there was Sag presentation to T cells (35).


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T cells also become infected with MMTV (58-60). Studies have shown that MMTV infection occurs after the transfer of T cells from infected animals to na'ive mice (44). It is also known that T-lymphoma cell lines are capable of producing virus particles (61) and it has recently been shown that primary T cells also shed virus L. Dzuris et al., submitted). Moreover, variant "TV's that have deletions in their LTRs are known to cause T-cell lymphomas (62), presumably through integration next to cellular oncogenes. These deletions may remove negative transcription-regulatory regions and cause increased lymphoid-cell expression of the virus and the adjacent oncogene (63,64). The deletions also encompassthe Sag hypervariable coding region; it remains to be determined whether the lack of a functional Sag protein plays a role in lymphomagenesis. It is also not known if MMTV infection of T cells is required for virus transmission to the mammary gland. Because MMTV infection occurs during the shaping of the immune repertoire in neonatal life and because infection is persistent, there is a gradual deletion of Sag-cognate T cells (49). Mice infected with MMTV(C3H), for example, have only about 10% the normal VplGbearing T cells by about 10 weeks of age and the remaining cognate T cells are anergic (49). It is possible that this loss of functional cognate T cells could result in an immune system that is tolerant to "TV-infected cells later in life and prevent recognition, for example, of mammary tumors. There is some precedent for this effect ofMMTV in viral immunity. It has been proposed that the deletion ofVp6+ T cells associated with the M t u 7 locus results in no immune responses to polyoma virus; inbred strains of mice that lack the M t u 7 locus are resistant to the tumorigenic effects of this virus (65). It remains to be shown whether a similar loss of immune response occurs during MMTV infection. Therefore, at a minimum, the role of the MMTV-encoded Sag protein is to stimulate cognate T cells and this stimulation is a requisite step in the infection pathway. Whether T cells are important for other steps i s yet to be determined. B-cell infection is also critical, as described in the next section. 2. TheRole

Cells

Sags are presented by a number of different class 11+antigen-presenting cells (APCs) to cognate T cells, including B cells, macrophages, and dendritic cells. B cells present in the Peyer's patches of the gut have been shown to be the first cells that are infected when newborn mice acquire MMTV through suckling on viremicmothers (66). Simi-

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lar infection of cells is seen in the draining lymph node when MMTV virions are injected in the footpad of adult mice (47). It was proposed that cells were the first to be infected during "TV infection and these then served as APCs for Sag-cognate T cells (66). This hypothesis was confirmed by Beutner et al. (67). They showed that mice that lacked cells because of targeted mutagenesis of the immunoglobulin heavy-chain gene (Igp) were unable to be infected with MMTV and that no deletion of cognateT cells by milkborne virus occurred in these animals. More recently, it has been shown that mice with a targeted deletion of the CD40 ligand (CD40L) gene also are unable to be infected with MMTV (68). Presumably, in the absence of T-cell help in the CD4OL-deficient mice, there is no induction of B7-2 on cells and Sag cannot be presented. As a consequence, no stimulation of T cells can occur. Interestingly, there is deletion of cognate T cells by endogenous Sags in both the Igp and CD4OL mice, indicating that there are other APCs such as dendritic cells, epithelial cells, macrophages, and so forth that function during the shaping of the immune repertoire. That these other APCs only present endogenous Sags also indicates that they do not become infected during the course of an exogenous MMTV infection, perhaps because they lack the receptor for the virus do not actively undergo cell division during this process. Therefore, infection of cells in the Peyer's patches of the gut is the first step in the MMTV infection pathway (Fig. 3). After stimulation of cells, both CD4+ and CD8+ T cells become infected (47), and presumably, cytokine production occurs. Thisin turn leads to the proliferation of cells (69). example, it has been shown that B cells of the IgG class are amplified in the draining lymph node of mice injected with SW virus and that these cells are "TV-infected (69). This amplification is not dependent on retroviral infection of additional cells, since it occurs in the presence of 3'azido-3'-deoxythymidine (AZT), an inhibitor of retrovirus replication (69). However, as is the case with T cells, it is still not known whether B-cell infection is required for transmission of the virus to the mammary gland. There is no evidence for a Sag-independent pathway of MMTV transmission and only MMTVs with functional sag genes can be transmitted through milk (70). The acquisitionof a sag gene in the MMTV genome probably allows this virus to take maximum advantage of its host's biology. The main target tissue for MMTV is the mammary gland. The epithelial cells of the mammary gland undergo proliferation at about 3-4 weeks of age (during puberty) and in response to


27

the hormonal stimulus provided by pregnancy. Because of the requirement for cell division, MMTV most likely only efficiently infects cells during these two periods (Fig. 3). Thus, MMTV can reside and amplify in the cells of the immune system during the neonatal period until efficient infection of the mammary gland can occur. The Role

EndogenousViruses

The mouse genome contains a number of retroviruses that are passed from generation to generation and some of these have been shown to confer resistance to exogenous viruses, primarily through expression of endogenous envelope proteins resulting in receptor interference. In contrast to some of the other endogenous viruses, it is thought that expression of the MMTV envelope protein does not prevent reinfection of a cell. MMTV-induced mammary tumors often have multiple independent proviral integrations characteristic of a cell that has been reinfected (71,72). Thus, if endogenous MMTVs conferred resistance to exogenous virus infection, this protection would likely involve a different mechanism. MMTV is unique among the murine and perhaps other retroviruses in encoding a Sag whose activity it uses as part of its infection pathway. Because this protein causes profound deletion of cognate T cells when expressed from the Mtzl loci, any mouse that is infected with an exogenous virus encoding a Sag with the same specificityas its endogenous loci cannot be infectedwith this virus. a result, these mice do not acquire exogenousMMTV in the mammary gland nor do they develop mammary tumors (56,57). Since the discovery that MMTV encodes a Sag, a number of new endogenous and exogenous viruses with different VP specificities have been described. These viruses have been found in previously existing stocks of mice (73-75). Interestingly, most of the infectious viruses encode Sags that interact with different VP-bearing T cells than those encoded by the endogenous loci (Table 1). One exception is virus, which is only weakly tumorigenic (76). This predicts that there is a natural selection for tumorigenic or highly infectious MMTVs that produce Sag proteins capable of stimulating cognate T cells and against viruses that do not have this ability. . This prediction has beenborne out in the laboratory. In the case of the MMTV (C3H) Sag transgenic mice, MMTV (C3H) virus was totally lost froma pedigree after three generations (77). Similarly, SW virus was eliminated from Mtv-7-containing mice after two generations (57).

Ross

28

Table 1 Infectious Viruses EncodeSag Proteinswith Novel Vp Specificities Endogenous virus with Exogenous MMTV MMTV (C3H), TES 14 GR ( M f ~ 2 ) ~ SW BALB/cV, TES 2, MMTV ((241, Mfv51b MMTV (FM)

Vpspecificity specificity same 14, 15a

None

6 2

M t ~ 7 -43 ,

8.2

None

None None

(C3H) has been shown to interact with Vp15+ hybridomas (52). bMtv-2 and MW51 produce infectious virus and are therefore both endogenous and

*"TV

exogenous.

This elimination of infectious virus is beneficial to mice. Inheritance of an endogenous sag with the same Vp specificity as an exogenous virus should increase the reproductive lifespan of the mouse. MMTV(C3H) very efficiently causes tumors in both BALB/c and C3H/HeN mice, with about 95% incidence and an average latency of 6-7 months in breeding females (1). In MMTV(C3H) Sag transgenic mice, the virus load is reduced several orders of magnitude and the average latency of tumor formation more than doubles As a result, breeding females develop mammary tumors after their peak reproductive period. Although this increase in latency has little effect on litter number and size in the laboratory setting, it is probably significant in the wild, where the average life span is less than 1 year (78). Moreover, tumor-bearing animals are less able to nurse their young and are more susceptible to opportunistic infection. Interestingly, there is historical evidence that the acquisition a novel Mfv in the germline of the MA/My mouse also caused the loss an exogenous, tumor-causing MMTV. The MA mouse was originally identified as a high-tumor-incidence mouse strain; however, in the 1940s a substrain was developed that no longer gotmammary tumors (79). This substrain, MA/My, has a novel endogenous MMTV, Mtu43, that interacts with Vp6-bearing T cells. A novel integration into the germline of this substrain probably caused this substrain to be resistant to a mammary-tumor-causing exogenous MMTV similar to SW virus In this case, however, there must have been a mutation in one of the coding regions of the virus, because Mfu43 is still expressed in mammary gland and yet does not cause tumors.


29

Balanced against the selective pressure to retain endogenous copies ofMMTV to protect against exogenous infection is the pressure to retain only those copies of the virus that are not infectious. Inbred strains of mice that retain an infectious endogenous provirus, such as GR, very rapidly succumb to mammary tumors. This would imply that the M t u 2 and other loci that encode functional MMTVs represent recent integrations into the mouse genome and that with time, they would accumulate mutations that would inactivate their tumorigenic capability. Inactivation can result from mutations that prevent functional virus capsid protein synthesis or the modulation of the tissue-specific expression of the virus. Interestingly, all of the known defective Mtv proviruses have mutations in their protein coding regions or in the transcriptional regulatory regions but not in their genes, further confirming that it is advantageous for mice to retain these genes. CONCLUDING REMARKS

MMTV represents a unique virus, in that its sole route of natural infection is through milk. Since the mammary gland cell is the ultimate target for this virus, it is not surprising that MMTV acquired the ability to replicate and amplify in cells of the lymphoid system of newborn, nursing pups. At least one other virus, rabies, has also been shown to encode such a protein. Whether MMTV is the only virus that uses the activity of a virally encoded Sag to facilitate its infection of lymphoid cells is not currently known. A number questions remain to be resolved for MMTV. It has not yet been shown whether lymphoid cells themselves are required to carry MMTV virions to the developing mammary gland and whether cell-cell contact is required for this transmission. Moreover, the cellular receptor for MMTV has not yet been identified. Whether the virus uses the same cell surface molecule to infect all cell types awaits its identification. It is also possible that the MMTV Sag protein plays additional roles in the virus life cycle. Future studies will determine whether the MMTV Sag protein is involved in immune recognition ofMMTVinduced mammary tumors or in the transformation process itself. REFERENCES

1. Nandi S, McGrath CM. Mammary neoplasia in mice. Adv Cancer Res 1973; 17:353-414.

30

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2. Bittner JJ. Some possible effects of nursing on the mammary gland tumor incidence in mice. Science 1936; 84:162. 3. Bentvelzen P, Daams JH. Hereditary infections with mammary tumor viruses in mice. Natl Cancer Inst 1969; 43:1025-1035. 4. Duesberg PH, Cardiff RD. Structural relationships between the RNA of mammary tumor virus andthose of other RNA tumor viruses. Virology 1968;49:92-101. 5. Coffin JM. Retroviridae and their replication. In: Fields BN, Knipe DM, eds. Virology. New York: Raven Press, 1990:1437-1500. 6. Weiss R, TeichN, Varmus H,Coffin J. RNA Tumor Viruses. Cold Spring Harbor, NY: CSHL Press, 1984. 7. Varmus HE, Bishop ]M, Nowinski RC, Sarker NH. Mammary tumour virus specific nucleotidesequences in mouse DNA. Nature 1972; 238:189-191. Buijs F, Kroezen V, Bluemink N, Hilgers 8. Hilkens J, van der B u s t Identification of a cellular receptor for mouse mammary tumor virus and mapping of its gene to chromosome 16. J Virol 1983; 45:140-147. 9. van Ooyen AJJ,MichalidesRJAM, Nusse R. Structural analysis of a 1.7kb mouse mammary tumor virus-specific RNA. J Viroll983; 46:362270. 10. Wheeler DA, Bute1 JS, Medina D, Cardiff RD, Hager GL. Transcription of mouse mammary tumor virus: identification of a candidate mRNA for the long terminal repeat gene product. J Virol 1983; 46:42-52. 11. Elliott J, Pohajdak B, Talbot D, etal.Phorboldiester-inducible, cyclosporine-suppressibletranscription from a novel promoter within the mouse mammary tumor virus env gene. J Viroll988; 62:1373-1380. 12. Miller CL, Garner R, Paetkau V. An activation-dependent, T-lymphocyte-specific transcriptional activator in the mouse mammary tumor virus env gene. Mol Cell Biol 1992; 12:3262-3272. 13. Reuss FU, CoffinJM. Stimulation of mouse mammary tumor virus superantigen expression by an intragenic enhancer. Proc Natl Acad Sci USA 1995; 92:9293-9297. 14. Gunzburg WH, Heinemann F, Wintersperger S, et al. Endogenous superantigen expression controlled by a novel promoter in the MMTV long terminal repeat. Nature 1993; 364:154-158. 15. Bittner JJ. Genetic concepts in mammary cancer in mice. Ann NY Acad Sci 1958; 71:943-975. 16. Yamamoto K. Steroid receptor regulated transcription of specific genes and gene networks, Annu Rev Genet 1985; 19:209-252. 17. Choi YC, Henrard DH, Lee I, RossSR.Themouse mammary tumor virus long terminal repeat directs expression in epithelial and lymphoid cells of different tumors in transgenic mice. J Virol 1987; 61:3013-3019. 18. Ross SR, Hsu C-L, Choi Y, et al. Negative regulation in correct tissue-


19.

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specific expression of mouse mammary tumor virus in transgenic mice. Mol Cell Biol 1990; 10:5822-5829. Mok E, Golovkina TV,RossSR.A mousemammarytumorvirus (MMTV) mammary gland enhancer confers tissue-specific, but not lactation-dependent expression in transgenic mice. J Virol 1992; 66:75297532. Henrard D, Ross SR. Endogenous mouse mammary tumor virus is expressed in several organs in addition to the lactating mammary gland. J Virol 1988; 62:3046-3049. Donehower LA, Huang AL, Hager GL. Regulatory and coding potential of the mouse mammary tumor virus long terminal redundancy. J Virol 1981;37:226-238. van Klaveren P, Bentvelzen P. Transactivating potential of the 3’ open reading frame of murine mammary tumor virus. J Virol 1988; 62:44104413. Salmons B, Erfle V, Brem G, Gtinzburg WH. naf, a trans-regulating negative-acting factor within the mouse mammary tumor virus open reading frame region. J Viroll990; 64:6355-6359. Nusse R. The int genes in mammary tumorigenesis and in normal development. Trends Genet 1988;4:291-295. Peters G. Inappropriate expression of growth factor genes in tumors induced by mouse mammary tumor virus. Semin Viroll991; 2:319-328. Rijsewijk F, Schuermann M, Wagenaar et al. The Drosophila homolog of the mouse mammary oncogene inf-l is identical to the segment polarity gene wingless. Cell 1987; 50:649-657. Thomas KR, Capecchi MR. Targeted disruption of the murine ink1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 1990; 346:847-850. Ross SR, Golovkina TV. The role of endogenous Mtvs in resistance to MMTV-induced mammary tumors. In: Tomonari. K, ed. Viral Superantigens. Boca Raton, FL: CRC Press (in press). Golovkina TV, Prakash 0, Ross SR. Endogenous mouse mammary tumor virus Mtvl7 is involved in Mtv2-induced tumorigenesis in GR mice. Virology 1996;218:14-22. Brandt-Carlson C, Bute1 JS, Wheeler D. Phylogenetic and structural analysis of MMTV LTR ORF sequences of exogenous and endogenous origins. Virology 1993; 185:171-185. Kozak C. et al. A standardized nomenclature for endogenous mouse mammary tumor viruses. J Virol 1987; 61:1651-1654. Michalides R, van Deemter L, Nusse R, van Nie R. Identification of the Mtv2 gene responsible for early appearance mammary tumors in the GR mouse by nucleic acid hybridization. Proc Natl Acad Sci USA 1978; 2368-2372.

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33. Muhlbock 0. Note on a new inbred mouse strain GR/A. Eur J Cancer 1965;1:123-124. 34. Muhlbock Dux A. MTV-variants and histocompatibility.In: Mouriquand ed., Fundamental Research on Mammary Tumour. Paris: INSERM, 1972:ll-20. 35. Pucillo C, Cepeda R, Hodes RJ. Expression of a MHC Class I1 transgene determines superantigenicity and susceptibility to mouse mammary tumor virus infection. J Exp Med 1993; 178:1441-1445. 36. Dux A. Genetic aspects in the genesis of mammary cancer. In: Emmelot P, Bentvelzen P, eds. RNA Viruses and Host Genome in Oncogenesis. Amsterdam: North-Holland Publishers, 1972:301-308. 37. Pullen AM, Marrack P, Kappler J W .Evidence that MIS-2 antigens which delete Vp3+ T cells are controlled by multiple genes. J Immunol 1989; 142:3033-3037. 38. Bentvelzen P. Interaction between hose and viral genomes in mouse mammary tumors. Annu Rev Genet 1982; 16:273-295. 39. Coutinho A. Identification of the spleen B-cell defect in C3H/HeJ mice. Scand J Immunol 1976; 5:129-140. 40. Outzen HC, Corrow D, Shultz LD. Attenuation exogenous murine mammary tumor virusvirulence in the C3H/HeJ mouse substrain bearing the Lps mutation. Natl Cancer Inst 1985; 75:917-923. 41. King LB, Corley RB. Lipopolysaccharide and dexamethasone induce mouse mammary tumor proviral gene expression and differentiation in B lymphocytes through distinct regulatory pathways. Mol Cell Biol 1990; 10:4211-4220. 42. Carr J, Traina-George VL, Cohen Mouse mammary tumor virus gene expression regulated in trans by Lps locus. Virology 1985; 147:210-213. 43. Moore DH, Long CA, Vaidya AB, et al. Mammary tumor viruses. Adv Cancer Res 1979; 29:347-418. 44. Tsubura A, Inaba M, Imai S, et al. Intervention of T-cells in transportation of mouse mammary tumor virus (milk fador) to mammary gland cells in vivo. Cancer Res 1988; 48:6555-6559. 45. Held W, Shaknow AN, Izui S, et al. Superantigen-reactive C D 4 T cells are required to stimulate B cells after infection with mouse mammary tumor virus. J Exp Med 1993; 177:359-366. 46. Squartini F, Olivi M, Bolis GB. Mouse strain and breeding stimulation as factors influencing the effect of thymectomy on mammary tumorigenesis. Cancer Res 1970; 30:2069-2072. 47. Waanders GA, Shakhov AN, Held W, et al. Peripheral T cell activation and deletion induced by transfer lymphocyte subsets expressing endogenous or exogenous mouse mammary tumor virus.J Exp Med 1993; 177:1359-1366. 48. Marrack P, Kushnir E, Kappler A maternally inherited superantigen encoded by mammary tumor virus. Nature 1991; 349:524-526.

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Superantigens

Ignatowicz L, Kappler J, Marrack P. The effects of chronicinfection with a superantigen-producing virus. J Exp Med Choi Y, Kappler JW, Marrack P. A super antigen encoded in the open reading frame of the 3’ long terminal repeat of the mouse mammary tumor virus. Nature Acha-Orbea H, Shakhov AN, Scarpellino L, et al. Clonal deletion of Vpl4-bearing T cells in mice trangenic for mammary tumor virus. Nature Festenstein H. Immunogeneticand biological aspects ofin vitro lymphocyte allotransformation (MLR) in the mouse. Transplant Rev Fritsch EF, Temin HM. Inhibition of viral DNA synthesis in stationary chicken embryo fibroblasts infectedwith avian retroviruses. J Virol Roe T, Reynolds TC, Yu G, Brown PO. Integration of murine leukemia virus DNA depends on mitosis. EMBO J Lewis PF, Emerman M. Passagethrough mitosis is required for oncoretroviruses but not for human immunodeficiency virus. J Virol Golovkina TV, Chervonsky A, Dudley JP, Ross SR. Transgenic mouse mammary tumor virus superantigen expression prevents viral infection. Cell Held W, Waanders G, Shakhov AN, et al. Superantigen-induced immune stimulation amplifies mouse mammary tumor virus infection and allows virus transmission. CeIl Dudley J, Risser R. Amplification and novel locations of endogenous mouse mammary tumor virus genomes in mouse T-cell lymphomas. J Virol Ball J, Arthur L, Dekaban G. The involvement of type-B retrovirus in the induction of thymic lymphomas. Virology Michalides R, Wagenaar E, Hilkins J, et al. Acquisition of proviral DNA of mouse mammary tumor virus in thymic leukemia cells fromGR mice. J Virol Meyers Gottlieb PD, Dudley JP. Lymphomas with acquired mouse mammary tumor virus proviruses resemble distinct prethymic and intrathymic phenotypes defined in vivo. J Immunoll989; Yanagawa S-I, Kakimi K, Tanaka H, et al. Mouse mammary tumor virus with rearranged long terminal repeats causes murine lymphomas. J Virol Theunissen HJM, Paardekooper M, Maduro LJ, et al. Phorbol ester-inducible T-cell-specific expression of variant moust mammary tumor virus long terminal repeats. J Virol Hsu C-LL, Fabritius C, Dudley J. Mouse mammary tumor virus proviruses in T-cell lymphomas lack a negative regulatory element inthe long terminal repeat. J Virol

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65. Lukacher AE, Ma Y, Carroll JP, et al. Susceptibility to tumors induced by polyoma virus is conferred by an endogenous mouse mammary tumor virus superantigen. J Exp Med 1995; 181:1683-1692. 66. Karapetian Shakhov Kraehenbuhl J-P, Acha-Orbea H. Retroviral infection of neonatal Peyer's patch lymphocytes: the mouse mammary tumor virus model. J Exp Med 1994; 180:1511-1561. 67. Beutner U, Draus E, Kitamura D, et al. B cells are essential for murine mammary tumor virus transmission, but not for presentation of endogenous superantigens. J Exp Med 1994; 179:1457-1466. 68. Chervonsky AV, Xu J, Barlow AK, et al. Direct physical interaction involving CD40 ligand on T cells and CD40 on B cells is required to propagate MMTV. Immunity 1995; 3:139-146. 69. Held W, Waanders GA, Acha-Orbea H, MacDonald HR. Reverse transcriptase-dependent and -independent phases of infection with mouse mammary tumor virus: implications for superantigen function. J Exp Med 1994; 180:2347-2351. 70. Golovkina TV, Dudley JP, Jaffe A, Ross SR. Mouse mammary tumor viruses with functional superantigen genes are selected during in vivo infection. Proc Natl Acad Sci USA 1995; 92:4828-4832. 71. Nusse R, van Ooyen A, Rijsewijk et al. Retroviral insertional mutagenesis in murine mammary cancer. Proc SOCLond (Biol) 1985; 226:3-13. 72. Nusse R., Varmus H.E. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell 1982;31:99-109. 73. Yoshimoto T, Nagase H, Nakano H, et al. A VP8.2-specific superantigen from exogenous mouse mammary tumor virus carried by FM mice. Eur J Immunol 1994; 24:1612-1619. 74. Ando Y, Wajjwalku W, Niimi N, et al. Concomitant infection with exogenousmousemammarytumorvirusencodingI-E-dependent superantigen in I-E-negative mouse strain. J Immunol 1995; 154:62196226. 75. Shakhov AN, Wang H, Acha-Orbea H, et al. A new infectious mammary tumor virus in the milk mice implanted with C4 hyperplastic alveolar nodules. Eur J Immunol 1993; 23:2765-2769. 76. Held W, Shakhov AN, Waanders G, et al. An exogenous mouse mammary tumor virus with properties of Mls-la (Mtv7). J Exp Med 1992; 175:1623-1633. 77. Golovkina TV, Prescott JA, Mouse mammary tumor virus-induced tumorigenesis in sag transgenic mice; a laboratory model of natural selection. J Virol 1993; 67:7690-7694. 78. Sage RD.Wild mice. In: Foster HL, Small JD, JG, eds. The Mouse. Vol I. History, Genetics and Wild Mice. New York: Academic Press, 1981:40-90.

lmmunobiology of

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35

79. Murray WS. MA/My strain of the Marsh albino mouse. Natl Cancer Inst 1963; 30:605-610. Rudy CK, Kraus E, Palmer E, Huber BT. Mlsl-like superantigen in the MA/MyJ mouse is encoded by a new mammary tumor provirus that is distinct from Mtv7. Exp Med 1992; 175:1613-1621.


Structural Features of Superantigens Gary Winslow

John W. Kappler and Philippa Marrack

INTRODUCTION

Although the Mls antigens have been studied for over 20 years, since their first descriptionby Festenstein in 1973 (l),OUT knowledge of the biochemistry of these important proteins has lagged far behind. The identification of the MIS antigens as components of the mouse mammary tumor virus (MMTV) occurred only recently as a result of genetic and immunological analyses. Prior to the availability of the amino acid sequence of the MMTV superantigens, no investigators had been successful in obtaining monoclonal antibodies that recognized these proteins, nor had any of the MMTV superantigens been analyzed using conventional biochemical methods. Thus, the biochemical nature of the MMTV superantigens has been an enigma. 37

Winslow

38

et ai.

This was due in no small part to the fact that the superantigens are expressed at the cell surface at very low levels, and to difficulties encountered in attempts to produce functional proteins in recombinant form. Concomitant with the development of monoclonal antibodies and the access to increasingly sophisticated molecular tools, physical characterizationof the MMTV superantigens has begun. This review will summarize our current understanding of the structural features and biochemistry of the MMTV superantigens. II.

NOMENCLATURE

Prior to the knowledge of the biological function of the protein encoded by the open reading frame in the 3’ long terminal repeat (LTR) of the MMTV, the coding region had been referred to asthe orf (open reading frame). Subsequent to the discovery that the open reading frame encoded a superantigen, several investigatorshave retained this nomenclature and refer to the orf genes and the ORF proteins. Others have renamed the genes as sag (superantigen) and the gene products the SAg proteins. This latter nomenclature will be used in this chapter. The MMTV superantigens form a family of proteins, so individual SAg proteins will be indicated with a suffix to indicate the MMTV of origin, as in SAgl (from the provirus Mtvl). Superantigens of exogenous origin will be referred to using a suffix indicating the mouse strain of origin, as in SAg(C3H). The letter “v” is often used to precede the abbreviation to distinguish the viral superantigens from the bacterial superantigens, as in vSAgl. STRUCTURAL FEATURES OF VSAGS A.

Protein-Encoding Potential of the sag Gene

Shortly after the discovery that MMTVs encoded the Mls determinants, it was determined that the open reading frame in the LTR of the virus encoded the SAg protein Inspection of the coding potential of the sag gene revealed a number of important clues to the structure of the vSAgs, some of which will be described here. The amino acid sequences of 29 vSAgs of endogenous and exogenous origin are shown in Fig. 1. If a typical sag open reading frame is translated in its entirety, it would encode for a protein of about 320 amino acids with a predicted molecular weightof about kilodaltons (kDa). However, five methionine codons are found in the amino terminal amino acids of most vSAgs, so it is possible that shorter

Features Structural

of

Superantigens

39

versions of the vSAg proteins are produced in vivo. Evidence to date has indicated that the first ATG is indeed utilized in vivo, but the possibility that the MMTV might utilize internal initiation codons to generatetruncated vSAgs cannot be discounted. An engineered amino-terminal truncation of the sag(C3H) gene that required translation to initiate at the methionine at position 38 was found to produce a functional vSAg, but further amino-terminal truncations did not yield any functional vSAgs (6). Immunoprecipitation from cells with anti-vSAg antibodies directed against an epitope containing the first methionine in the open reading frame precipitatedvSAg proteins (7), indicating that the initial methionine was utilized in some, if not all, vSAgs. On the basis of a large body of experimental evidence, it was predicted that the vSAgs would be class I1 MHC-associated cell surface proteins, although some earlystudies had suggested a role for the gene products in transcriptional regulation. Hydrophobicityanalysis of vSAg proteins failed to identify an amino-terminal signal sequence but revealed a 23-amino-acid hydrophobic region (positions 45-67) that was predicted to be a transmembrane domain (Fig. 1). These observations, combinedwith the prediction that the bulk the protein, including the carboxy-terminal T-cell-recognition region, was likely to be extracellular, suggestedthat the vSAg proteins were type I1 integral membrane proteins. The topological orientation of type I1 proteins with respect to the cell membrane is such that their amino termini are intracellular and their carboxyl termini are extracellular. This topology is shared by other proteins such as the class II-associated invariant chain, but is unlike the majority of membrane proteins, which share the opposite type I orientation. Amino acid sequence analysis also revealed the presence five candidate N-linked glycosylation sites in most vSAg proteins. The consensus glycosylation motif eukaryotic proteins is NXS/T (in single-letter amino acid code, where X is any amino acid). N-linked glycosylation motifs canbe found at positions 79, 94, 132, and 147 (Fig. 1).It is unlikely that closely apposed asparagine residues are both glycosylated, however, so N-linked glycosylation probably occurs at as many as four positions in the protein. Six to seven cysteine residuesare found in most vSAgs, suggesting the possibility for intramolecular disulfide bonding. However, one to two cysteines are found in the intracellular portion (positions 115, 264), and three in the putative transmembrane domain. Thus, the extracellular portion of most vSAgs contains only two cysteine resi-

Winslow et al.

dues that might form disulfide bonds. Three vSAgs (SAgl, DDO, IITES14) do not contain a cysteine at position 115 and thus have only a single sulfhydryl group in the extracellular portion of the protein, indicating that disulfide bonding cannot be critical for function. In addition, experimental evidence to be summarized below has suggested that intramolecular disulfide bonding does not occur. Amino acid sequence analysis has also revealed the presence of several basic amino acid motifs in the vSAgs (Fig. 1). These motifs are recognition sequences for processing endoproteases, also known as protein convertases (PCs), a family of mammalian enzymes that recognize dibasic or tetrabasic amino acids (reviewed in The presence of the recognition sites raised the possibility that the vSAgs undergo proteolytic processing, a characteristics of a number of viral glycoproteins, including MMTV envelope proteins (10). Experimental evidence suggeststhat the vSAgs are indeed processed by PCs, and that the processing may be important for function (see below). The structural features that have emerged from analyses of the vSAg sequence data indicate a protein not atypical from other viral

Figure 1 Amino acid sequence comparison of vSAgs. The vSAg designation indicates the provirusorigin or strain of origin of exogenous viruses (in parentheses). Vp specificities are indicated at the endof the sequences and include modifications as described in Ref. 49. vSAg7 was arbitrarily used for sequence comparisons of the different vSAgs. Dashes refer to sequence identities and dots are used to represent unavailable sequence or to fill gaps in the alignment. Cysteine residues are in bold; N-linked glycosylation motifs are underlined. The transmembrane region is boxed and shaded; protein convertase recognition sites are cross-hatched; sequence differences between some vSAg families are boxed; critical amino acid residues in vSAg44 are encircled. This is an update of Refs. 50 and 51 with the addition of the following sequences (reference followed by Genbank Accession number): SAg50 (52; 573770); SAg(JYG) (53; accession number not available); SAg44 (46; 219515);SAg(MA1)(54; 222552); SAg(DD0) (55; n/a); SAg(I1-TES2)(56; D45409); SAg(FM) (57; D26359); SAg(RCS) (58; L11933); SAg(SHN) (59; X78950);SAg(11-TES14)(60;D38639);SAg23(61;S67365); SAg(M12)(61; S67367); SAg30 (49; 1049303). (Obtained from the output of the program Pileup from the Program Manual for the Wisconsin Package, Version8, September 1994, Genetics Computer Group, 575 Science Drive, Madison, W1 53711. Amino acid numbering may differ slightly from other reports due to alignment gaps.)

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Structural Featuresof

SAg7 SAg43 SAg50 SAS(=) SAg(JYG) SAS1 SAg3 SAg6 SAgl3 SAg44 SA9 (MA1)

Superantigens

43

270 290 250 NGYKVLYRSL PFRERLARAR PpWCVLTQEE KDDMKQQVtn, YIYLGTGMNF WGKIFDYTEE " " " " " " " " " " " " " " "

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Winslow et al.

44

glycoproteins encoded by MMTV and other retroviruses (11,12). Many of theses viral glycoproteins are highly glycosylated and are produced as large precursorproteins.that undergo proteolytic processing. IV. IDENTIFICATION A.EarlyPhysical

OF THE VSAC. PROTEINS

Studies of vSAgs

The amino acid sequenceof the protein encoded by a MMTV 3' open reading frame was available for over years before the function of the gene was determined (13,14) and before the first biochemical characterization of a vSAg. Biochemical characterization of the components of the MMTVs had been performed (15,16; reviewed in 17), but these reports did not describe any novel proteins that might now be identified as vSAgs. This had led to the idea that vSAgs are not present in virions, but the possibility that vSAgs are components of virions but present at levels that are below the limits of biochemical detection cannot be discounted. MMTV antigens were known to be expressed in mouse leukemia cells and in vivo products of the gene were first detected in phorbol ester-treated EL4 cells using an antipeptide antiserum (18). Genomic analyses of the EL-4 cells had determined that the long terminal repeat was highly amplifiedand contained a deletion, and thus was unlikely to encode a functional superantigen. Metabolic radiolabeling and immunoprecipitation of the truncated vSAgs identified glycoproteins of 37 and 34 kDa, along with several other, less prominent polypeptides. It was not determined whether these proteins were found on the cell surface It is not clear why the vSAg proteins expressed by the leukemia cells have invariably been found to be truncated at the carboxy-terminus (19), a modification that almost is certain to destroy superantigen function. Prior to the discovery that the genes encoded superantigens, in vitro translation products were produced to demonstrate the protein-coding potential of the MMTV open reading frame (20). The studies were later extended by several groupswho performed in vitro translation of genes in the presence and absence of microsomal membranes (6, 21, 22). In the absence of microsomes the vSAg proteins were synthesized as nonglycosylated proteins as large as 38 kDa. The 38-kDa translation products resulted from translation of the engene, which was predicted from the primary sequence to yield tire a protein of approximately this size. In the presence of microsomal

Features Structural

of MMTV Superantigens

45

membranes, which permit glycosylation,a 45-kDa N-glycosylated and membrane-associated vSAg was detected. Membrane association required the presence of the amino-terminal hydrophobic region, indicating that this region did indeed function as a transmembrane domain. Protease digestion of the microsomal membrane-associated vSAgs demonstrated that the carboxy-terminus was extravesicular, confirming the hypothesis that the vSAgs were type I1 membrane proteins. These studies provided the first data on the physical properties of the vSAgs and demonstrated that they were indeed type I1 membrane glycoproteins. B.

Generation of Antibodies

One of the technical limitations that hindered the physical characterization of the vSAgs was the lack useful antibodies. Although a number of immunologists had attempted to generate antisera and monoclonal antibodies against vSAgs, before the discovery that they were encoded in the MMTV genome, none succeeded. This was probably due to the very low abundance of the vSAgs on the cells used in the attempts to generate antibodies. As soon as the amino acid sequences of the vSAgs were known, three groups were able to produce antipeptide monoclonal antibodies against carboxy-terminalpeptides. These antibodies recognized the native vSAgs, as determined by the ability of the monoclonal antibodies to inhibit vSAg-specific T-cell proliferation (23-25). The antibodies also detectedvSAgs on the cell surface of vSAg-expressing baculovirus-infected insect cells and B-cell lymphomas (see also 26). One group succeeded in detecting vSAg7 expression on lipopolysaccharide-stimulatedB cells using flow cytometry, but not on unstimulated B cells, nor on T cells, or in the thymus (25). Thus, the antibodies proved to be of limited use for detecting vSAgs in normal mouse cells, again indicating that the vSAgs were present at very low levels on the cell surface. C.

Biochemical Characterization of vSAg Proteins In Vivo

1.

MetabolicRadiolabeling

The availability of anti-vSAg antibodies made a biochemical analysis of vSAg proteins possible. The problem low expression of the vSAgs was partially overcome by the use sensitive radiolabeling experiments and by the use of cell lines that were engineered to overexpress vSAg proteins. Using35S-radiolabeled amino acids, two groups detected vSAgs after a brief metabolic radiolabeling followed

46

Winslow et al.

by immunoprecipitation with monoclonal or polyclonal anti-carboxyterminal vSAg antibodies (25,271. The vSAgs migrated in polyacrylamide gels at a molecular weight of 45-46 kDa, which was consistent with the earlier results from the in vitro translation experiments. The observed mobilityof the radiolabeled vSAgs was in excess of that predicted for the core protein (37 kDa) and suggested that posttranslational modification had occurred. This modification was shown to be the addition of high-mannose oligosaccharides (25,27), characteristic of endoplasmic reticulum (ER) resident proteins. The metabolically radiolabeled vSAgshad a short half-life [less than 2 hr (27); G. Winslow, unpublished observations]. The radiolabeledvSAgs were of low abundance, and it was not possible to use pulse-chase analyses to monitor the progress of the vSAgs after egress from the ER and transit through the secretory pathway. Most of the cellular vSAg detected after radiolabeling remained in the high-mannose form and was presumably degraded in the ER (7), like HIV gp160 (28). 2.

Structure

CellSurface

The cell surface form of vSAg7 was biochemically detected after cell surface iodination (7,25). These studies utilized two monoclonal antibodies, one specific for the conserved amino-terminus of the vSAgs and the other specific for the carboxy-terminus of vSAg7. Both antibodies immunoprecipitated a radiolabeled protein of18.5 kDa from the cell surface. Western analyses of vSAg7 precipitated from wholecell detergent lysates determined that the 18.5-kDa proteins contained the carboxy-terminus ofvSAg7 (7). The carboxy-terminal fragment was determined to be a proteolytic cleavage product of vSAg7, and not due to internal translation initiation in the open reading frame, because the 18.5-kDa protein was precipitated with both the amino- and carboxy-terminal-specific antibodies. The mobility of the carboxy-terminal fragment predicted an approximate site of proteolytic cleavage in the region of residues 150-200. Examination of the amino acid sequence of vSAg7 revealed that most (12of 15) of the iodinatable tyrosines were found in the predicted carboxy-terminal portion of the protein (after amino acid 200). The amino-terminal portion of the protein therefore in all likelihood was weakly radiolabeled and was difficult to detectin the surface radiolabeling experiments. The most plausible explanation of the surface radiolabelling data was that on the cell surface some of the amino- and carboxyterminal proteolytic cleavage products were bound noncovalently, but only the radiolabeled carboxy-terminus was detected by immunoprecipitation.

Structural Features of MMTV Superantigens

47

Although the presence of two cysteines in the putativeextracellular portion of most vSAgs offered the possibility for intramolecular disulfide bonding, the electrophoretic migrationof the radiolabeled vSAg7 proteins precipitated by either antibody was not significantly altered by the presence of reducing agents, indicatingthat the aminoand carboxy-termini were not disulfide-bonded to each other or to other proteins. Three functional vSAgs contain substitutions for the cysteine found in most vSAgs at position 115 (Fig. l), indicating that disulfide bonding in the extracellular domain is not obligatory in vSAgs. Thus, vSAg7 appeared to be similar toa number of other viral glycoproteins that undergo proteolytic processing, such as HIV gp160 (11).In several cases, it has similarly been shown that the proteolytic processing products remained noncovalently associated on the cell surface (10,29,30). The data summarized above indicated that at least some of the vSAg7 carboxy-terminal proteolytic cleavage product was present on the cell surface noncovalently associated with the amino-terminus. The data did not rule out the possibility that the carboxy-terminus might also be found free of the amino-terminus. To test this hypothesis, immunoprecipitation of the 18.5-kDa carboxy-terminal vSAg7 processing product was performed using lysates that had been first completely cleared of the amino-terminus of vSAg7 (Fig. 2). The carboxy-terminus of vSAg7 was present in the lysate after complete clearing using the amino-terminal-specific antibody. The data suggested that the carboxy-terminus can be found on the cell surface independent of the amino-terminus, perhaps bound to class I1 MHC proteins, or to other surface proteins. Most important, the data suggest that the carboxy-terminus of vSAg7 may function independently of the amino-terminus to stimulate T cells. Thus the 18.5-kDa form of vSAg7 may be sufficient onits own to act as a superantigen. Some features of vSAgs may .berequired for functions other than stimulating T cells, and once the vSAg delivered to the cell surface these features may be dispensable. The structure of the vSAgs may therefore reflect not only the generation of superantigen activity, but requirements peculiar to production of a superantigen in a eukaryotic cell. Although the vSAg7 detected on the cell surface was found to be proteolytically processed, it was possible that low or undetectable levels of full-length vSAg7 were expressed at the cell surface of the B-cell lymphoma. Some radiolabeling experiments revealed a diffuse streak of high-molecular-weight proteins in polyacrylamide gels that appeared to be specifically precipitated bythe amino-terminal-specific vSAg antibodies (25). This material may represent unprocessedvSAg,

Winslow et al. ...

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Figure 2 Some of the vSAg7 carboxy-terminal processing product is found free the amino-terminus on the cell surface. CH12/S7 cells expressing vSAg7 were surface-radiolabeled and lysed as described previously (7). Lysates were passed over Sepharose beads that had been bound with an irrelevant antibody (control) or an anti-amino-terminal vSAg antibody (VS1). The cleared lysates were then immunoprecipitated with VS1 or an anticarboxy-terminal vSAg7 antibody (VS71 in the absence (-) or presence (+) specific competitor peptide, as controls. The arrow indicates the carboxyterminus of vSAg7, precipitated with either VS1 or VS7 (lanes and Passage the lysate over a antibody column effectively cleared all vSAg7 that had been bound to theamino-terminus (compare lanes l and 5), but did not clear all of the carboxy-terminal fragment. The data indicate that the carboxy-terminus vSAg7 can be found on the cell surface independent the amino-terminus.

or amino-terminal cleavage products. It is possible, therefore, that some unprocessed vSAg7 may be present at low levels on the surface of the B cells and may be presented to T cells. The identity of the functional form of vSAg7 remains unresolved, although data will be summarized below regarding the possibility that proteolytic processing is required for presentation. Cell The metabolic radiolabelingstudies described above indicatedthat the nascent vSAg proteins were first detected as 45-kDa glycoproteins containing high-mannose-type oligosaccharides, characteristic of ERresident proteins. Western analyses of vSAg7 partially purified from

Structural Features of

Superantigens

49

whole-cell lysates also indicatedthat the majority of vSAg7 was modified by the addition of high-mannose oligosaccharides, asdetermined by the sensitivity of 'the 45-kDa form to endoglycosidase H (7). vSAg7 was also detected at lower levels in cell lysates as a full-length endoglycosidase H-resistant glycoprotein of approximately 82 kDa 97). This high-molecular-weight form was proposed to contain cornplex-type oligosaccharides, characteristic of a protein that had transited the secretory pathway, an indication that this form of vSAg7 had exited the ER. However, because most cellular vSAg7was found in a form characteristic of proteins found in the ER, it appeared that the bulk ofvSAg7 was retained and degraded there. This notion is supported by the metabolic radiolabeling studies, which indicated a short half-life for the nascent vSAg (27; G. Winslow, unpublished data). It is presently unclear what mechanism may operate to limit egress of vSAg7 from the ER. One possibility is that most of the ERresident vSAg is improperly folded and is subsequently degraded by ER-resident proteases. Alternatively, the vSAgs may compete with the class 11-associated invariant chain or other proteins, such as calnexin (31,321, for binding to class I1 proteins, and export from the ER may be limited by the available class I1 protein-binding sites. Surface expression of vSAg7 was not detected immunochemically in mouse strains that did not express an appropriate class I1 protein, which suggested that class I1 was required for vSAg surface expression in vivo (25). In another study, increased levels of class I1 led to increased vSAg activity, in the absence of any changes in vSAg mRNA levels (33). In contrast, class 11-negative fibroblast or B-cell lines continued to express vSAgs on their cell surface, albeit at slightly reduced levels (7; G. Window, unpublished data), indicating that class I1 expression was not obligatory, at least in cells that overexpress a vSAg. Finally, mice that lack class 11-associated invariant chain show reduced levels of vSAg activity (341, suggesting that the invariant chain may play a role in vSAg trafficking. In addition to the full-length, 82-kDa form of vSAg7, additional endoglycosidase H-resistant vSAg7 ranging 45 to 82 kDa was detected by Western analysis (7). In contrast to the 82-kDa form, these other forms of vSAg7 were not detected by monoclonalantibodies directed against the carboxy-terminus, suggesting that they were the amino-terminal proteolytic cleavage products that were not detected or were weakly detected in the surface radiolabeling experiments. After digestion with N-glycanase (also known as Endo F, PNGaseF), which removed all N-linked oligosaccharides, the hetero-

Winslow et al.

50

geneous amino-terminal cleavage products migrated at approximately 27 kDa. Thus, Western analyses revealed the vSAg7 amino-terminal proteolytic cleavage product that had been predicted from the radiolabeling experiments. The size of the 27-kDa amino-terminal proteolytic cleavage product was greater than that predicted for the core molecular weight of the predicted amino-terminal proteolytic cleavage product (approximately 19 kDa) and suggested that additional modifications other than N-linked glycosylation occurred. Treatment with O-glycanase (which removes 0-linked oligosaccharides) did not alter the migration of the 27-kDa amino terminal product (G. Winslow, unpublished data). The results of the carbohydrate analyses indicated that the amino-terminal cleavage products were only observed among vSAg7 containing complex-type oligosaccharides. This suggested that proteolytic processing occurred after exit of vSAg7 from the ER, possibly in the Golgi, where candidate vSAg-processing endoproteases are known to be located (35). The biochemical analysis of vSAg7 indicated that a considerable amount of carbohydrate was present on vSAg7. Six asparagine-linked consensus glycosylation sites are found in most vSAgs (Fig. 1, positions 80, 81, 90, 94, 132, 147). However, only four of the possible six sites appear to be used, probably because two pairs of sites are adjacent or very close to one another (80, 81; 90, 94). The nascent vSAgs carry a total of about 8 kDa of oligosaccharide, and mutagenesis studies have indicated that this is due to glycosylation at four positions, each contributing about 2 kDa (C. McMahon and A. Pullen, personal communication). Mutagenesis studies have indicated that at least one N-linked site is required for vSAgl presentation to T cells, possibly because glycosylation is important for intracellular trafficking of vSAgl (C. McMahon and A. Pullen, personal communication; 36). D.

Binding to Class I I MHC

It has been known for some time that vSAgs, like other superantigens, require class I1 MHC proteins for presentation to T cells. The role of MHC proteins for vSAg presentation is the subject of another chapter. Little is known, however, regarding how the vSAgs actually contact the class I1 proteins. Physical evidence for the direct binding of vSAg7 with class I1 IA proteins was demonstrated by the capture of surface radiolabeled vSAg-class I1 protein complexes using anticlass I1 antibodies. Because the carboxy-terminus of vSAg7 has been detected on the cell surface in the absence of the amino-terminus (Fig.

Structural Features of MMTV Superantigens

51

2), the carboxy-terminus may by itself bind to class I1 proteins. However, one group has reported that class I1 binding sites exist in the amino-terminal portion of the protein, based on the binding of relatively short peptides from the amino-terminus of vSAgl (residues 76119; 37). Full-length (unprocessed) vSAg7 purified from whole-cell lysates was shown to bind to class I1 proteins (7), so it is possible that binding is mediated by both amino-terminal and carboxy-terminal portions of the protein. The role that vSAg7 proteolytic processing plays in its binding to class I1 proteins is presently unclear, but available evidence suggests that processing may facilitate class I1 binding, because the carboxy-terminus was overrepresented, relative to other forms of vSAg7, among those forms that were found bound to class I1 proteins ( 7 ) . E.

Proteolytic Processing

1.

Recognition Sites for Processing Endoproteases

The observation that cell surface vSAg7 was proteolytically processed suggested the presence of endoprotease recognition motifs in the coding region. Indeed, examination of the amino acid sequence revealed the existence of several highly conserved multibasic motifs that are putative recognition sites for a family of endoproteases, the subtilisin-like serine proteases, also known as proprotein convertases (PCs; 8,s).Among the mammalian members of this family are the protein convertases furin (also known as PACE), PC2, PCl/PC3, PACE4, and PC5/6 (9). The PCs typically cleave sequences containing the motif RXK/RR and are known to be responsible for the processing of cellular and viral precursor proteins in both the constitutive and regulated secretory pathways. The distribution of several of the PCs is restricted to neuroendocrine and germ cells. The expression of furin and PACE4, however, is widespread among cell types, although only furin is expressed ubiquitously (38). Furin, and possibly PACE4, are therefore candidate vSAg-processing endoproteases. Furin recognition motifs can be found at two to three positions in most vSAgs. The first motif is found just after the putative transmembrane region (RARR; residues 68-71; Fig. 1) and has been shown to be furin substrate in vitro (39). This furin motif is conserved in all vSAgs except vSAg(SW), vSAg(RCS), and vSAg(C3H-K), which contain the sequences RACR, RAHR, and SARR, respectively (Fig. 1).The RACR and RAHR motifs conform to a more degenerate furin recognition motif (RXXR; 40) and therefore SAg(SW) and SAg(RCS)

52

Winslow et al.

may nonetheless be processed at this site, The SARR sequence in vSAg(C3H-K) clearly violates the known requirement of furin for an arginine at position -4 (numbering in reverse from the cleavage site, which occurs afterthe carboxy-terminal arginineinthe motif). v S A ~ ( C ~ H - Kwhich ), was isolated from a kidney adenocarcinoma (411, appears to be incapable of reacting with T cells (42). One explanation for the apparent lack of activity could be that processing at this position is required for vSAg function. The available evidence suggests that vSAg7 is indeed processed in Vivo at position 71. Western analysisof vSAg7 isolated from wholecell lysates demonstrated the presence of an amino-terminal polypeptide with an electrophoretic mobilityof about kDa that most likely resulted from proteolytic processing at position71 (7). Because the site is immediately distal to the transmembrane region, proteolytic processing would likely generate a soluble vSAg. Cell transfer of has been demonstrated to occur in vivo and might occur as a result of processing at this position. A second PC recognition site occurs at position 169-172 in most vSAgs (RKRR in vSAg7; Fig. l).This site has also been shown for vSAg7 to be a substrate for furin in vitro (39). The observed 18.5-kDa surface form of vSAg7 has been predicted to result from processing at this position. Mutagenesis of this recognition sequence (RKRR > GEEF) eliminated production of a carboxy-terminal vSAg7 polypeptide in vivo, indicating that this motif is indeed a site of proteolytic processing. However, the furin recognition site at this position has not been not retained in four functional vSAg of exovirus origin (RKRR has been replaced by RKRH; Fig. 1).It is presently unknown whether these vSAgs are processed at this position or if this in any way affects vSAg function. A third PC recognition motif is found in 16 of 28 vSAgs in a highly basic region at position 192-195(RGKR; Fig. 1). The lack of complete conservation in this region suggests either that processing important for function. does not occur at this position or that it is substitutions in this region are to other charged residues, in most cases other basic residues, Which ma)’ indicate a requirement simply for a high charge density in this region. Proteolytic processing not observed at this position after treatment of a recombinant form of vSAg7 with furin in vitro(39), perhaps indicating that these sites are not utilized by furin in any VSAgS. However, Western analysis has revealed the existence of an additional carboxy-terminal vSAg7 polpeptide that, based on its electrophoretic migration, may have resulted if processing occurred at this position (7)-

Structural Featuresof

Superantigens

2. TheRole and Requirement

53

Proteolytic Processing

Although it seems clear that vSAgs are proteolytically processed, it remains uncertain whether proteolytic processingis required for normal vSAg function in vivo. Data in support of this notion have come from one group who analyzed a single vSAg7 mutant protein in which the motif at position 168-171 was disrupted (39). The mutant protein was not processed at this position and was not expressed at the cell surface, suggesting that proteolytic processing was obligatory for vSAg activity. It was possible, however, that the mutation interfered with some other function of the vSAg, such as class I1 association or intracellular trafficking, and it was not determined whether an unprocessed vSAg could activate T cells had the protein been expressed on the surface. In another study, substitution of the RKRR motif (position 168171) in vSAgl with the nonconsensus sequences RKRK or RERE was accompanied by no loss or only a partial loss of T-cell stimulatory activity, although proteolytic cleavage of vSAgl at an undetermined position was still observed (C. McMahon and A. Pullen, unpublished data). Neither mutated recognition site was likely to be a substrate for a PC, so the data suggested that processing at this site was not required for function, or that a non-PC endoprotease was responsible. Because processing of the mutated vSAgl was observed, the data suggested that processing may have occurred at the PC recognition site at position 192-195 (RGKR in vSAgl). If proteolytic processing is required for vSAg function, it is possible that processing at either of the two PC recognition sites may be sufficient foractivity. In support of this notion, note that the five vSAgs that have lost a consensus PC recognition site at position at position 168-171have all retained a PC recognition site at position 192-195 (vSAgs44, 11-TES2,FM,BR6,IITES14; Fig. 1).The likelihood of this occurring by chance is approximately 1 in 25. Thus, all known vSAgs have retained at least one PC recognition motif in this portion of the protein. The existing data do not rule out the possibility, however, that unprocessed vSAgs can be presented to T cells. It is possible, for example, that proteolytic processing is required not for presentation, but for intercellular transfer from one antigen presenting cell to another. Transfer of vSAgs has been documentedin vivo, but the mechanism whereby this occurs has not been determined. vSAg processing might liberate a soluble and functional vSAg that retains activity. The active moiety might consist of the entire extracellular domain the carboxy-terminal processing product.

Winslow et al.

54

V.

TCR SPECIFICITY

After the determination of the amino acid sequence of a number of different genes, it became clear that the vSAgs could be grouped into subfamilies based on their T-cell-receptor variable region (Vp) specificities and sequence similarities at their immediate carboxy-termini (Fig. The carboxy-terminus sequence conservation occurs among vSAgs that share T-cell-receptor specificities and suggested that the carboxy-terminus (distal to residue 280) was the site of interaction of the vSAgs with the T-cell receptor. This was confirmed using chimeric vSAgs where it was shown that the carboxy-terminal 108 amino acids of vSAg7 were sufficient for recognition Vp8.l when placed in the context of vSAgl, which normally recognizes VP3-bearing T cells (45). Six to seven vSAg subfamilies can be defined on the basis of their T-cell-receptor V@specificities, although the amino acid sequence differences between the subfamilies are sometimes subtle. For example, comparison of the Vp3- and Vp2-specific vSAg families identified only five amino acid residues that are unique to each vSAg family: an isoleucine-to-valine change at position282 (I282V), W291L, A302V, I306L, and H309S (Fig. 1). Three of the differences represent very conservative amino acid substitutions. In addition, vSAg44, which by sequence comparison is clearly a member the family of vSAgs that recognize Vp3, is unique in its ability to also recogni,ze Vp6, 8.1, and (46). This relaxed specificity can beattributed to differences at only two positions (312 and 313; Fig. l). Similarly, two amino acid residues define differences between Vp7- and Vp5/ll-TCR specificities: R318S and K321G. Recent data suggest some overlap in the specificities among the two subfamilies, so the differences may notbe significant, and the two families should perhaps be grouped as one. Mutagenesis studies coupled with a three-dimensional vSAg structure will allow a precise identification of the residues critical for the interaction of the vSAgs with the TCR and will determine how the problem of T-cell-receptor recognition is solved differently by the various vSAgs. VI. OTHER

Comparison of vSAg sequences with the protein database has failed, with one exception, to reveal any significant similarities with any known proteins, including the bacterial SAgs. The vSAgs do show

Features Structural

of MMTV Superantigens

55

significant similarity to the IE-G protein from herpesvirus saimiri (up to 59% amino acid similarity in one region of the protein; 47). Few of the predicted structural features of the vSAgs, such as the proteolytic processing sites and transmembrane region, appear to be conserved between MMTV and the herpesvirus. However, the herpesvirus protein, like the vSAgs, has four N-linked glycosylationsites in the amino-terminal portion of the protein and has retained the two cystine residues found at homologous positions in the vSAgs. It is not known, however, if the herpesvirus protein also acts as a superantigen. VII.

CONCLUDING REMARKS

Because the difficulties encounteredin analyzing the vSAg proteins isolated from cells, future studies will certainly utilize a recombinant source of these proteins. Recombinant vSAgs wilI facilitate direct binding studies of the vSAgs, in processed and unprocessed forms, to MHC and TCR proteins, and will ultimately provide a determination of their three-dimensional structure. To date, most attempts at producing a functional vSAg in bacterial or insect cells have met with difficulty due to poor solubilityand lack of activity Choi, personal communication; G. Winslow, unpublished data). One group has reported direct binding of a bacterially produced vSAg to class I1 proteins, but was unable to demonstrate functionality (48). Production of functional vSAgs in the yeast Pichia pustoris appears to hold some promise (N. Gascgoine, personal communication; G. Winslow, unpublished data). Our understanding of how the MMTV SAgs function at the molecular level, and how they are similar and different to the bacterial SAgs, will be important for our ability to mediate disease induced by pathogens, both bacterial and viral, that use superantigens to subvert the immune response. REFERENCES 1. Festenstein H. Immunogenetic and biological aspects in vitro lymphocyte allotransformation (MLR) in the mouse. Transplant Rev1973; 15:62-88. 2. Acha-Orbea H, Shakhov AN, Scarpellino L, et al. Clonal deletion of 14-bearing T cells in mice transgenic for mammary tumour virus. Nature 350:207-211. Choi Y, Kappler JW, Marrack P. A superantigen encoded in the open

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reading frame of the 3' long terminal repeat of mouse mammary tumour virus. Nature 1991;350:203-207. 4. Beutner U, Frankel WN, Code MS, Coffin JM, Huber BT. Mls-l is encoded by the long terminal repeat open reading frame of the mouse mammary tumor provirus Mtu7. Prcc Natl Acad Sci USA 1992; 89:54325436. 5. Pullen AM, Choi Y, Kushnir E, Kappler J, Marrack P. The open reading frames in the 3' long terminal repeat of several mouse mammary tumor virus integrants encode VP3-specific superantigens. J Exp Med 1992;175:41-47. 6. Choi H, Marrack P, Kappler J. Structural analysis of a mouse mammary tumor virus superantigen. J Exp Med 1992; 175:847-852. 7. Winslow GM, Marrack P, Kappler JW. Processing and major histocompatibility complex binding the MTV7 superantigen. Immunity 1994; 1:23-34. 8. Barr PJ. Mammaliansubtilisins: the long-sought dibasic processing endoproteases. Cell 1991; 66:l-3. 9. Steiner DF, Smeekens SP, Ohagi S, Chan SJ. The new enzymology of precursor endoproteases. Biol Chem 1992; 267:23435-23438. 10. Dickson C, Atterwill M. Structure and processing of the mouse mammary tumor virus glycoprotein precursor Pr73env,J . Virol 1980; 35:349361. 11. VeroneseFD,DeVico AL, Copeland TD, Oroszlan S, Gallo R, Sarngadharan MG. Characterization of gp41 as the transmembraneprotein coded by the HTLV-III/LAV envelope gene. Science 1985; 229:14021405. 12. Stieneke-Gr6ber A, VeyM, Angliker H, et al. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisinlike endoprotease. EMBO 1992; 11:2407-2414. 13. Donehower LA, Huang AL, Hager GL. Regulatory and coding potential of the mouse mammary tumor virus long terminal redundancy. J Virol 1981;37:226-238. 14. Majors JE, Varmus HE. Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression the mouse mammary tumor virus env gene. J Virol 1983; 47:495-504. 15. Yagj MJ, Compans R. Structural components of mouse mammary tumor virus. I. Polypeptides of the virion. Virology 1977; 76:751-766. 16. Cardiff D, Puentes MJ, Young LJ, etal. Serological and biochemical characterization of the mouse mammary tumorvirus with localization of p10. Virology 1978; 85:157-167. 17. Dickson C, Peters G. Proteins encoded by mouse mammary tumor virus. Curr Topics Micro Immunol 1983; 106:l-34. 18. Racevskis J. Expression of the protein product the mouse mammary tumor virus long terminal repeat gene in phorbol ester-treated mouse T-cell-leukemia cells. J Viroll986; 58:441-449.

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19. Michalides R, Wagenaar E, Weijers P. Rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in T-cell leukemias of mouse strain GR result in a novel enhancer-like structure. Mol Cell Biol 1985; 5:823-830. 20. Dickson C, Smith R, Peters G. In vitro synthesis of polypeptides encoded by thealong terminal repeat region of mouse mammary tumor virus DNA. Nature 1981;291:511-513. 21. Korman AJ, Bourgarel P, Meo T, Rieckhof GE. The mouse mammary tumour virus long terminal repeat encodes a type I1 transmembrane glycoprotein. EMBO J 1992;11:1901-1905. 22. Knight AM, Harrison GB, Pease RJ, Robinson PJ, Dyson PJ. Biochemical analysis of the mouse mammary tumor virus long terminal repeat product:evidence for the molecular structure of an endogenous superantigen. Eur J Immunoll992; 222379-882. 23. Acha-Orbea H, Scarpellino L, Shakhov AN, Held W, MacDonald M . Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein. J Exp Med 1992; 176:1769-1772. 24. Mohan N, Mottershead D, Subramanyam M, Beutner U, Huber BT. Production and characterization of an Mls-l-specific monoclonal antibody. J Exp Med 1993; 177:351-358. 25. Winslow GM, Scherer MT, Kappler J W , Marrack P. Detection and biochemical characterization of the mousemammarytumorvirus 7 superantigen (Mls-l"). Cell 1992;71:719-730. Detection and characterization of a glycopro26. Brandt-Carlson C, Butel tein encoded by the mouse mammary tumor virus long terminal repeat gene. Virol 1991;65:6051-6060. 27. Krummenacher C, Diggelmann H. The mouse mammary tumor virus long terminal repeat encodes a 47kDa glycoprotein with a short half-life in mammalian cells. Mol Immunol 1993;30:1151-1157. 28. Willey RL, Bonifacino JS, Potts BJ, Martin MA, Klausner RD. Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelopeglycoprotein gp160.Proc Natl AcadSciUSA1988; 85:9580-9584. 29. Kowalski M, Potz Basiripour L, et al. Functional regions of the envelope glycoprotein of human immunodeficiency virus type I. Science 1987;237:1351-1355. 30. Thomas DJ, Wall JS, Hainfeld JF, et al. gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of125kilodalton subunits stabilized through interactions between their gp41 domains. J Virol 1991; 65:3797-3803. 31. Degen E, Williams DB. Participation of a novel 88-kD protein in the biogenesis of murine class I histocompatibilitymolecules. J Cell Bioll991; 112:1099-1115. 32. Rajagopalan S, Xu Y, Brenner MB. Retention of unassembled compo-

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36. 37. 38. 39. 40. 41.

42. 43. 44.

45.

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nents of integral membrane proteins by calnexin. Science 1994; 263:387390. Lund FE, Randall TD, Woodland DL, Corley RB. MHC class I1 limits the functional expression of endogenous superantigens in B cells. J Immunol 1993;150:78-86. Viville S, Neefjes J, Lotteau V, et al. Mice lacking the MfIC class IIassociated invariant chain. Cell 1993; 72:635-648. Molloy SS, Thomas L, VanSlyke JK, Stenberg PE, Thomas G. Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. J Biol Chem 1994; 13:18-33. Hammond C, Braakman I, Helenius A. Role of N-linked oligosaccharide recognition, glucose trimming and calnexin in glycoprotein folding and quality control. Proc Natl Acad Sci USA 1994; 91:913-917. Torres BA, Griggs ND, Johnson HM. Bacterial and retroviral superantigens share a common binding region on' class I1 MHC antigens. Nature 1993;364:152-154. Seidah NG, Chr6tien M, Day R. The family of subtilisin/kexin like proprotein and pro-hormone convertases: divergent or shared functions. Biochimie 1994; 76:197-209. Park CG, Jung M-Y, Choi Y, Winslow GM. Proteolytic processing is required for viral superantigen activity. J Exp Med 1994; 181:1899-1904. Matthews DJ, Goodman LJ, Gorman CM, Wells JA. A survey of furin substrate specificity using substrate phase display. Prot Sci 1994; 3:11971205. Wellinger RJ, Garcia M, Vessaz A, Diggelmann H. Exogenous mouse mammary tumor virus proviral DNA isolated from a kidney adenocarcinoma cell line contains alterations in the U3 region of the long terminal repeat. J Virol 1986;6O:l-11. Shakhov AN, Wang H, Acha-Orbea H, Pauley RJ, Wei-Zen W. A new infectious mammary tumor virus in themilk of mice implanted with C4 hyperplastic alveolar nodules. Eur J Immunol 1993; 23:2765-2769. Pullen AM, Marrack P, Kappler JW. The T-cell repertoire is heavily influenced by tolerance to polymorphic self-antigens. Nature 1988; 335:796-801. Speiser DE, Schneider R, Hengartner H, MacDonald HR, Zinkernagel RM. Clonal deletion of self-reactive cells in irradiation bone marrow chimeras and neonatally tolerant mice: evidence for intercellular transfer of Mlsa. ] Exp Med 1989; 170:595-600. Yazdanbakhsh K, Park CG, Winslow GM, Choi Y. Direct evidence for the role of carboxyl terminus of mousemammarytumorvirus superantigen in determining TCR Vp specificity. J ExpMed 1993; 178:737-741. Rosenwasser OA, Fairchild S, Tomonari K. New superantigen specific-

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47. 48. 49.

50. 51. 52. 53. 54. 55. 56.

57. 58,

59.

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ity created by two amino acid replacements. Immunogenetics 1993; 38:367-369. Nicholas H, Smith EP, Coles L, Honess R. Gene expression in cells infected with gammaherpesvirus saimiri: properties of transcripts from two immediate-early genes. Virology 1990; 179:189-200. Mottershead DG, Hsu P-N, Urgan RG, Strominger JL, Huber BT. Direct binding of the Mtv7 superantigen, (Mls-l) to soluble MHC class I1 molecules. Immunity 1995;2:149-154. Scherer MT, Ignatowicz L, Pullen A, Kappler J, Marrack P. The use of mammary tumor virus (Mtvl-negative and single-Mtv mice to evaluate the effects of endogenous viral superantigens on theT cell repertoire. J Exp Med 1995; 182:1493-1504. Acha-Orbea H, MacDonald HR. Superantigens of mouse mammary tumor virus. Annu Rev Immunol 1995; 13:459-486. Brandt-Carlson C,Bute1JS, Wheeler D. Phylogenetic and structural analyses of MMTV LTR ORF sequences of exogenous and endogenous origins. Virology 1993;193:171-185. Niimi N, Wajjwalku W, Ando Y, Tomida S, Ueda M, Yoshikai Y. A new gene encodingthe ligand for deletion of T cells bearing Tcrb-V6 and V8.1 (Mtv-50). Immunogenetics 1994; 40:312. Xu L, Haga S, Imai S, Sarkar NH. Cloning in a plasmid of an MMTV from a wild Chinese mouse: sequencingof the viral LTR. Virus Res 1994; 33:167-178. Jouvin-Marche E, Cazenave P-E, Voegtle D, Marche PN. Vp17 T-cell deletion by endogenous mammary tumor virus in wild-type-derived mouse strain. Proc Natl Acad Sci USA 1992; 89:3232-3235. Jouvin-Marche E, Marche PN, Six A, Liebe-Gris C, Voegtle D, Cazenave P-A. Identification of an endogenous mammary tumor virus involved in the clonal deletion ofVj32 T cells. Eur J Immunol 1993; 23:2758-2764. Ando Y, Wajjwalku W, Niimi N, Hiromatsu K, Morishima T, Yoshikai Y. Concomitant infection with exogenous mouse mammary tumor virus encoding I-E-dependent superantigen in I-E-negative mouse strain. J Immunol 1995;154:6129-6226. Yoshimoto T, NagaseH, Nakano H, Matsuzawa A, Nariuchi H. A VP8.2-specific superantigen from exogenous mouse mammary tumor virus carried by FM mice. Eur J Immunol 1994; ,24:1612-1619. Tsiagbe VK, Yoshimoto T, Asakawa J, Cho SY, Meruelo D, Thorbecke GJ. Linkage of superantigen-like stimulation of syngeneic T cells in a mouse model of follicular center B cell lymphoma to transcription of endogenous mammary tumor virus. EMBO J 1993; 12:2313-2320. New Luther S, Shakhov AN, Xenarios I, Haga S, Imai S, Acha-Orbea infectious mammary tumor virus superantigen with VP-specificity identical to staphylococcal enterotoxin B (SEB). Eur J Immunoll994; 24:17571764.

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60. Wajjwalku W, Ando Y, Nimi N, Yoshikai Y. A novel exogenous mammary tumor virus encoding MHC class 11 H2E-independent superantigen specific for Tcr-Vpl4. Immunogenetics 1995; 41:156-158. 61. Ignatowicz L, Kappler JW, Marrack P, Scherer MT. Identification of two 'Vp7 specific viral superantigens. J Immunol 1994; 152:65-71.

Interaction of Superantigens with MHC Class II Molecules Pascal M. Lavoie and Rafick-Pierre

of

of

Jacques Thibodeau

Franqois Denis

of

INTRODUCTION

Over the course of evolution, pathogenic organisms have developed creative mechanisms of circumventing hosts’ immune systems. Bacteria, for example, secretemultiple types of toxins, including a group proteins called superantigens, capable of triggering an exaggerated activation and proliferation T cells. They do so by bridging the variable (V) region the beta chain of the T-cell receptor (TCR) and the MHC class I1 molecule, thereby mimicking antigen recognition and inducing signaling cascades through both receptors (1). The benefit to the pathogen this T-cell stimulation is not always appar61

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ent at first sight, but in the case of viruses such as the mouse mammary tumor virus (MMTV) results have suggested that they promote a proliferative response of the target cell allowing efficient replication of viral DNA (2-4). However, the reason why bacteria have developed superantigens is still unclear, though they may somehow downregulate an eventual immune response against the parasite. SAGs produced by Staphylococcus aureus and streptococcal strains secrete exotoxins that have a high affinity for human and murine MHC class I1 molecules with characteristic superantigen properties These bacterial species cause a variety of medical conditions ranging from food poisoning and scalded-skin syndrome to pneumonia severe hemorrhagic gangrene and may result in full-blown toxic shock. The characteristic pattern of superantigen response by the host implies massive T-cell activation and malignant secretion of pyrogenic cytokines (IL-l, TNf-a). This eventually leads to septic shock and in most cases to anergy and deletion of SAG-responsive T lymphocytes bearing the particular Vp element(6). Recent efforts of various groups have allowed the characterization of the mechanisms of action of these powerful toxins. Bacterial SAGs produced by S. aureus are the most frequently studied and include a family structurally related proteins (SEA, SEB, SEC,,, SED, SEE, SEH) termed "enterotoxins" because of their emetic properties, but also toxic shock syndrome toxin-l (TSST-l), the sole member of its category (7,8). The recent crystallization of staphylococcal enterotoxin B (SEB) or TSST-l complexed with the human MHC class I1 molecule HLA-DR1, together with mutagenesis of their binding sites, has shed further light on the way SAGs make contact with their MHC class I1 receptor (9-11). SAGs seem to interact with conserved elements both on the alpha (a)chain and beta (0) chain outside the conventional antigen-binding groove (12). In spite of the fact that they all share a comparable mode of interaction with MHC class I1 molecules, most of the bacterial SAGs studied to date demonstrate unique functional characteristics. They are rarely all secreted simultaneously by the same strain of S. aureus and tend to show characteristic patterns of tissue distribution depending on the pathological context in which they are expressed. Little is known about the determinants involved in the production of the various SAGs by different strains of S. aureus but reports tend to indicate that their synthesis is highly regulated according to distinct conditions inherent to the host (13,14). The recent observationthat'the affinity of staphylococcal enterotoxins for their MHC class I1 receptor depends on the cell type on which they are expressed raised the

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possibility that the structure of cell surface MHC class I1 molecules might be influenced by the expression of cellular factors but, also, that different bacterial SAGs may demonstrate tropism for a specific target tissue ((11,151; P. M. Lavoie and R. P. Sekaly, manuscript in preparation). In this chapter, we discuss this possibility in the light of the structural interactions between bacterial superantigens and MHC class I1 molecules. The last section of this chapter will describe current knowledge concerning the interaction between viral superantigen from "TV's and MHC class I1 molecules. Very little is known about the latter interaction and analogies will be made with the interaction of bacterial superantigens with MHC class I1 molecules. II. INTERACTION OF BACTERIALSUPERANTICENS WITH MHC CLASS II MOLECULES

At the beginning of the efforts to define the exact binding site bacterial SAGs, results from Dellabonaet al. suggested that they bind outside the conventional antigen groove (12). This led to extensive site-directed mutagenesis of MHC class I1 proteins to identify critical residues involved in the stabilization of the interaction with SAGs Although the presentation of superantigens is generally described as not being restricted to particular MHC class I1 proteins, alleles differ in their ability to bind and present them Thus, comparisons of the SAG-binding affinities of different alleles of HLADR implicated certain conserved residues on the outer framework of MHC class I1 molecules. Whereas in general, human HLA molecules of the DR isotype bind bacterial SAGs with more affinity, HLA-DQ and murine H-2 class I1 molecules still present them with reasonable efficiency (21). Staphylococcal enterotoxin C, seems to be an exception to this rule and previous studies suggested that this SAG had better affinity for HLA-DQ (22). However, the functional relevance of this observation needs to be investigated further. The interaction of SAGs with MHC class I1 molecules will be briefly described in the following sections. The reader can refer to other chapters for a detailed description of the structure the toxins. The structure of HLADRl can be found in Ref. 23. A.

Staphylococcal Enterotoxin A (SEA)Crosslinks M H C Class II Molecules

Early characterization of the binding site of staphylococcal enterotoxin A (SEA) on MHC class I1 molecules, using synthetic peptides, map-

Lavoie et al.

ped its contact site on the C-terminal end of the alpha helix, on the p1 domain of DRl (24). These results suggestedthat although the beta chain is sufficient for binding of the toxin, both the alpha and beta chains are required for full T-cell activation (25). More precisely, the histidine at position 81 in the chain (PSlHis), conserved among all DR alleles but DRw53, was later defined as the critical contact site of the toxin (Fig. 1) (16,17). Analysis of the crystal structure ofSEA suggests that this residue is involvedin a high-affinity Zn-coordinated interaction with residues lSer, 187His, 225His, and 227Asp in the Cterminal domain of SEA (26). In contrast to SEB and TSST-l, the DR1binding interface of SEA is in the C-terminal domain of the molecule (26-29). Protein sequence alignments between staphylococcal enterotoxins demonstrated that SEA and SEB share significant homologyin the corresponding region involved in the binding ofSEB to DR1. Moreover, an excess ofSEA displaces efficiently the binding ofSEB to HLA-DR1 although the converse i s not true (11,301. These observations prompted several workers to predict the existence of a second binding site for SEA in a SEB-like configuration, involving residue lysine 39 of the alpha chain of DRl (a39Lys) (9,31). This hypothesis was later verified by independent investigators and confirmed that SEA has two binding.sites for DRl (32,331, supporting the previous observation recorded by J. K. Russell et al. Furthermore, these results suggested that a molecule ofSEA bound to the p 81His binding site (Kd = M) cooperates to favor the binding of another molecule of SEA on a low-affinity site (Kd = lo” M) on the a1 domain ofDR1 (residue a 39Lys) (32,33). Thelatter would result in the actual highaffinity interactionthat has been described by several workers (16,34). SEA is therefore a functionally trivalent molecule and has the ability to bind two MHC class I1 molecules plus the TCR. The importance of the second DR1 binding site in the pathogenesis of SEA is not yet clear. Although the beta chain is sufficient for high-affinity binding, experiments using SEA mutated in the Znbinding site suggest that binding at the a1 binding site is necessary for T-cells expressing specific Vps. (I. Cloutier and R. P. Sekaly, submitted). A direct implication of the cooperativity model proposed by K. Hudson and J. D. Fraser suggests that a stoichiometry of two SEA for one DRl molecule may be favoredover the formation of two DRl crosslinked by one molecule ofSEA. The recent observation that SEA,:DRl trimers exist in solution (35), together with results showing that SEA mediates signaling through the antigen-presenting cells

Interaction with MHC Class I t Molecules

65

(low affinity), ??(low affinity), SEC2

Figure 1 Top view of the MHC class I1 molecule HLA-DR1 showing the

binding sites for SEA (low affinity), SEB, and TSST-l involving residue a39Lys, al8Gln, and al3Tyr (in black) on the alpha chain. High-affinity binding ofSEA involving residues P81His on the beta chain is also represented (see text). SEB and both bind to HLA-DRl through the alpha chain only. The suggested binding sites for SEE and SEC, are also indicated according to references cited in the text and Roseanna and Lechler (Eur J Immunol 1995; 25:3437-3444). The peptide in the antigen groove is represented as a helical arrow. [Generated using Molscript(Kraulis PJ, Appl Crystallogr 1991; 24:946-950), according to the crystal coordinates published by Stern et al. (Nature 1994;368:215-2211.1

(APC) by direct crosslinking ofDR1 molecules suggests that a mixture of oligomers of SEA/DRl with distinct stoichiometries exists at the cell surface and may contribute to complex activation events of immune effector cells. Crosslinking of MHC class I1 molecules by SEA induces the APC to up-regulate costimulatory molecule expression and cytokine release with a subsequent enhancement of T-cell

Lavoie et al.

stimulator activity (36; W. Mourad, personal communication). This may partly explain the high potency of SEA compared to other SAGs. The presence on SEAof two cooperative DR1-binding sites of greatly different affinities taken individually results in the stepwise building a multimeric complex between SEA and DRl molecules and the TCR. Hence, the interaction on the DR-beta chain may serve the role a checkpoint or intermediate step in this T-cell-activation process by SEA. Considering that SEA is functionally efficient in activating T cells when bound to the alpha chain, this mechanism ensures that crosslinking of MHC class I1 molecules effectively precedes T-cell activation. At the same time, however, the model proposed by Hudson et al. implies that crosslinking of MHC class I1 molecules may not be a favored event in terms of stoichiometry. To assure adequate occupancy of the low-affinity a-chain binding site, a saturating amount of SEA would be required and would result instead in a SEA,:DR, stoichiometry with only minimal, although functionally efficient, crosslinking (37). The kinetics of T-cell activation by SEA and possibly other SAGs constitutes a highly regulated processinvolving distinct entities of SEAn:DRln complexes in a concentrationdependent and MHC class 11-expressing, cell-type-dependent fashion. This particular mode of interaction involving crosslinking and multimerization of SEA/DRl complexes does not seem to be shared by other SAGs except SEE which shows a high homology with' SEA and SED (see Chapter 10). Previous observations suggested that SEB and TSST-l exist as dimerized entities in solution and consequently may crosslink MHC class I1 molecules (40). However, this hypothesis has not been confirmed. The reason why SEA evolved toward this original mode of interaction is not clear but it is possible that S. aweus may recruit SEA in particular pathological situations where the use ofSEB or TSST-1 is not appropriate. The understanding of the topology of interaction of this SEA/DRl complex with the T-cell receptor may help to resolve this issue. B.

Overlapping Binding Sites for SEB and TSST-1 O n the Alpha Chain of HLA-DR1

Early observations that SEB and TSST-1 did not compete with each other for their binding to cell surface HLA-DRl and HLA-DQ molecules on DAP fibroblasts led Scholl and co-workers to conclude that these two SASGs did not share a common binding site (41). Later, the binding site for SEB was characterized by site-directed mutagenesis

Interaction with MHC Class

Molecules

67

and crystallography (9,ll). Staphylococcal enterotoxin B binds to the external surface of the a1 domain ofDR1. The wide interface of interaction is composed of the alpha helix and the first and third loops of the beta pleated sheet on the a1 domain, which delimits a shallow hydrophobic pocket where residue 44Phe ofSEB binds (for a complete description, see Ref. 9). Residue a39Lys, which is at the tip of the third loop of the a1 domain ofDR1 (Fig. 1) appears to be critical, forming a salt bridge with residues 67Glu, 89Tyr, and 115Tyr in SEB. Residues al3Tyr and a18Gln ofDR1 also make tight hydrogen bonds with the toxin, stabilizing its N-terminal domain on the MHC class I1 molecule. This structure of a bacterial superantigen complexed with MHC class I1 molecules confirmed the previous hypothesis put forward by Dellabona et al. (12). Subsequently, the binding site for TSST-1 was determined and, surprisingly, overlaps the actual site defined for SEB (10). Hence, amino acid a39Lys in the a1 domain is also critical forthe binding TSST-1, and mutation of this residue abrogates the binding of both SEB and TSST-1 (11). However, TSST-1 does not bind the MHC class I1 molecule exactly the same way and the toxin is located more on top of the groove, making hydrogen bond interactions with the peptide backbone of the antigen (Fig. 2). These results suggested that the affinity ofTSST-1 for the class I1 molecules might be influenced by the nature of the peptides in the groove (10,ll). The absence of competition between SEB and TSST-l on DR1-transfected cells, while both toxins can be efficiently displaced bySEA as well as anti-DR1 monoclonal antibody (MAb) L-243, was later confirmed by Thibodeau et al. (11). These results provided the first evidence that bacterial SAGS bind to structurally distinct subsets of HLA-DRl molecules and now raise the question whether these subsets can be functionally distinguished. Ill. THE STRUCTURE O F CELLSURFACE MHC CLASS II MOLECULES INFLUENCES THE BINDING OF SAGS

As mentioned above, the data obtained by Thibodeau et al. supported the concept that different populations of HLA-DRl molecules existon the cell surface and that they are mutualIy exclusive as to their binding to SEB and TSST-1. Another group has recently observed a similar pattern of heterogeneity concerning human MHC antigens of the DQ isotype using MAb (42). The idea that cells may have the ability to express MHC class I1 molecules derived from the same mRNA

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but having a slightly different posttranslationally modified structure reveals another level of polymorphism that goes beyond the diversity generated by haplotypic or allelic variations. Whether this phenomenon relates to real functional differences among different subsets

with Interaction

MHC Class

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DR1 molecules simply reflects sensitivity of SAGS tide-induced epitopes remains to be determined. A.

69

MAb for pep-

Affinity of SACS for D R 1 is Influenced by the Nature of the Cell Type

Subsequent to the identification of different subpopulations of HLADRl antigens,at the cell surface, the affinity of bacterial superantigens for cell surface MHC class I1 molecules was compared on different HLA-DR-expressing cell types. Interestingly, the EBV-B lymphoidderived Raji cell line, which expressed HLA-DR constituvely (DR3, DRwlO) (431, does not bind staphylococcal enterotoxin B (SEB) with significant affinity (11).LG-2 cells, the cell line used to purify papaincleaved soluble HLA-DRl molecules, and peripheral blood lymphocytes isolated from a DR1-homozygous individual bind SEB in the same range-of affinity (Kd > M) (44). On the other hand, these cell lines express high levels of cell surface HLA-DR molecules and bind SEA and TSST-1 with good affinity (11,44). The binding ofSAGS was also studied on human HeLa cells. Interestingly, DR1-transfected HeLa cells, which are not classified as professional APCs but do have the capacity to express endogenous HLA class I1 molecules in appropriate conditions, have a very good affinity for SEB but bind poorly TSST-1 and SEA (lL44). These results illustrate very well the structural heterogeneity of HLA-DR1 antigens. Furthermore, they suggest that a cell type can express one population while another one expresses the other. In the experiments described above, LG-2, Raji cells, peripheral blood lymphocytes, and other B-lymphoid-derived cell types tested can be grouped together on the basis of their SAG-binding characteristics. HeLa cells display a completely different phenotype and constitute the opposing pole. One possible interpretation is that professional APCs display the TSST-l-binding phenotype and nonprofessional APCs show SEB-binding characteristics. MHC class 11-expressing cell types isolated from DR1-homozygous individuals need to be tested to confirm this hypothesis. The idea that nonprofessional antigen-presenting cells producing low levels of invariant chain may express MHC class I1 antigens with a distinctive structure has been proposed previously (45). Our current results suggest that SAGS bind specifically to these distinct subsets of MHC class I1 molecules (11). In the light of the results showing that cell binding of SAGS to surface MHC class I1 molecules is influenced by the cell type, internal cellular factors mediating this phenomenon need to be better characterized.

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B.

Cellular Factors Influence the Binding of Bacterial Superantigens to HLA-DR1

1. ThePeptideInfluencesthe

Binding

BacterialSuperantigens

Cellular factors that may possibly influence the binding of bacterial superantigens at the cell surface were investigated. First, peptide may influence the conformation of MHC class I1 molecules and thus their affinity for SAGs for several reasons. The first two pieces of evidence came when we demonstrated a lower affinity of the TSST-l toxin for a DR1 mutant (residue allGlu to a lysine) implicated in peptide specificity ofDR molecules (11). Computer modeling of this mutant showed that the structure of the hydrophobic pocket involved in the binding of EST-l is well preservedbut the configuration of the 6th pocket of the peptide groove, according to the model of Stern et al. (46), is modified (P. M. Lavoie, unpublished observations). Other evidence came from the crystal structure of the TSST-1:DRl complex (10). This result shows that TSST-1 contacts the peptide in its C-terminal end (P7-Pl3) and suggests that bulky side chains in this region may prevent the binding of TSST-l. Finally Scholl et al. showed that allelic polymorphism of DR antigens influences the binding of SAGs: DR4 DwlO efficiently bound SEB but not TSST-1 and the reverse situation was observed for DR7 (19). Since these two alleles share the same alpha chain, this result can be explained by the different conformation of these class I1 molecules imposed by the binding of peptides to polymorphic residues of the beta chain. A precise molecular description of the way the peptide may affect the binding site of TSST1 is not available at the moment, the difficulty being that peptide loading on cell surface MHC class I1 molecules barely attains 5-10% in the best conditions (47; unpublished observations), thus rendering comparisons of SAG binding on DRl molecules substituted with different peptides difficult. However, small differences in TSST-1 binding to DRl can be well estimatedin functional assays usingTSST1responsive T-cell hybridomas. This was recently attempted by Von Bonin et al. (48). This result suggests that some peptides may affect the presentation TSST-1 but not SEB in accordance with their respective mode of binding to DR1. Current information predicts that SEA will also be affected by the nature of the peptide in the groove. The exact description of how SEA contacts HLA-DR1 awaits description of the crystal structure. However, the observation that SEA masks the accessibility of antigenspecific T cells to MHC class 11-bound peptides suggests that

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may also bind by extending over the peptide groove (49). Residue PSlHis, which is the critical contact site between SEA and DRI, is also involved in stabilization of the antigen backbone in the peptide groove (46). Long side chains in position P4 (309Val), according to the HA/DRl crystal, peptides extendingat their N-terminal end are likely to interfere with the binding of the toxin. To confirm this hypothesis, a recent study involving the use of peptide loaded in covalently linked to soluble murine H-2 class I1 molecules showed that some peptides were permissive, while other were not, to the binding ofSEA (50). In this study, binding ofSEB was also tested and, again, did not seem to be influenced by the nature of the peptide. According to the crystal structure, SEB binds HLA-DR1 well away from the antigen-binding groove (9) and is less likely tobe influenced by peptide-induced conformational changes. The recent crystal structure ofCLIP peptide complexed with HLA-DR3 shows a long side chain extending over SEA-binding residue P81His (51). This suggests that DR molecules loaded with CLIP peptide may not be able to interact with the toxin. At the moment, it is not clear if the influence of the peptide on the binding of SAGS reflects a direct adaptive mechanism merely a structural incapacity of SAGS to bind to all MHC classI1 molecules. It is interesting to consider that SEA and TSST-1 may bind to MHC class I1 loaded with immunodominant antigens a specific setof selfpeptides. This mechanism may target the activation a particular population of APCs that have encountered antigens produced by S. uureus itself. However, these theories remain at an embryonic stage as an eventual immunodominant peptide has not yet been identified. Resolution refinement of both the TSST-1 and SEB cocrystals may help in the detection of subtle changes in their binding sites induced by a particular set of peptides. 2.

Influence of the Invariant Chain

The invariant chain (Ii) is a 31-44-kDa protein that assembles with MHC class I1 molecules in the endoplasmic reticulum and targets the complex to endosomal compartments It is widely accepted that Ii occupies and masks the peptide groove before MHC classI1 vesicles merge with endocytic vesicles. This critical step forms the basis for the discrimination process of MHC class I1 molecules, which bind peptides of an exogenous origin, and MHC class I molecules, which do not associate with Ii and bind endogenous peptides early in the ER. This role of invariant chain was recently emphasized by Busch

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et al., who demonstrated that in the absence of Ii, MHC class I1 molecules agglutinate large polypeptides in the ER and are subsequently transported to the cell surface (53). Because of the antigen groove of MHC class I1 molecules is open at both ends proteins present in the ER supposedly get trapped in MHC class I1 molecules in the process of folding. In their system, Busch et al. used HeLa cells, which do not express endogenous invariant chain. For these reasons, the idea arose that invariant chain may be a factor influencing the nature of the peptides bound in the antigen groove and, thus, the binding of SAGs at the cell surface. The presence of these large polypeptides at the cell surface is thought to mask the binding site for SEA and possibly TSST-l, which binds more on top of the peptide cleft. Transfection of Ii in HeLa cells resultedin the formation of SDSstable, oligopeptide-loaded MHC classI1 molecules depicting increased affinity,for SEA (I. Cloutier, P. M. Lavoie, and R. P. Sbkaly, submitted). However, the binding of TSST-1 could not be restored by transfection of Ii, suggesting that this toxin is affected by additional factors. Other

A number of years ago, the very first biochemical characterizationof MHC class I1 molecules suggested that the glycosylation moieties on DR antigens were heterogenousin structure and size (54). In general, human and murine MHC class I1 molecules bear two glycosylation sites (a78Arg and nll8Arg) on the alpha chain and one on the beta chain (pl9Arg; according to DR-amino acid numbering) (55). Moreover, asparagine-linked glycosylationsites are well conserved among MHC class I1 molecules from many vertebrate species. The importance of these highly hydrophilic compounds for protein folding is well established (56). As for the nature of the peptide in the groove, glycosylation patterns may influence the affinity of the toxins for their binding sites for reasons that include folding stability, targeting, direct steric interference. It has been reported that the nature of the sugar structure influences the recognition of MHC class I1 antigens by the T-cell receptor (57,581, The observation that these glycosylation structures on DR antigens are different depending on the cell type on which they are expressed (59) raised the possibility that, as for the peptide in the groove, carbohydrate structures may influence the binding of at the cell surface. Although studies have suggested that carbohydrate structures contribute minimally in the folding process of MHC class I1 molecules (60), results from our group have

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73

suggested that mutation of the glycosylation sites on HLA-DRl affects the binding of SAGs partially, providing only a partial explanation for the opposed phenotypes we observe between LG-2 and HeLa cells (44). This issue awaits further investigation. As discussed above, SEA and TSST-1 can be influenced by peptide-induced modulation the conformation of MHC class I1 molecules. But interestingly, the peptide has not been shown so far to influence the binding ofSEB (48,50). This correlates with the nature the binding site ofSEB, which extends away from the antigen groove. In the cocrystal ofSEB complexed with DR1, the electron density in the groove corresponded to a mixture of peptides and it was not possible to identify any particular peptide conformational motif (9). Unlike LG-2 cells, HeLa cells do not express invariant chain the nonclassical MHC class I1 molecules HLA-DM. However,transfection of these molecules does not seemto restore the binding of SEB (44). Reports have shown that a number of molecules are associated with MHC class I1 molecules at the cell surface of B lymphocytes possibly responsible forinteraction with the T-cell receptor signaling through the MHC class I1 molecule (61,62). An interesting possibility is that the binding of SEB may be stericallyhindered by these DR-associated molecules present on Raji and LG-2 but not on HeLa cells. Considering the DR1-binding location ofSEB closer to the cell surface, expression of these DR-associated molecules is likely to interfere with the binding of this toxin.

W.

TROPISMOF BACTERIAL SUPERANTICENSFORDIFFERENT TISSUES

The crystal structure of SEB, TSST-l, SEA, and most recently SEC, and SED showed that all SAGs produced by S. aureus share a high degree of structural similarity. The enterotoxins can be separated into two groups based on protein sequence homology. The first group contains SEA and SEE, which share 92.2% similarity, while SEB and the SEC,-, subfamily form the other group. SED is a particular member in which it is homologous toSEA and SEE in amino acid sequence but shows a Vp specificity profile more similar to SEB and SEC (7). Despite their considerable similarity in structure, their mode interaction with the MHC class I1 molecules appears to be completely different. The location of their MHC class 11-binding sites shows surprising diversity both in position in the common structural motifs and in chemical composition. Surprisingly, SEA, SEE but also SEC, and

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SED have in common a Zn-coordination motif, but in a different place for each toxin. While sequence homology suggests that they were all developed from closely related genes, their great variety in mode of action suggests that they may have distinctive features implicated in a complex pathological process. Reasons why an organism such as S. aureus maintained such a diversified arsenal of MHC class 11-binding proteins include the requirement for a larger spectrum of action upon the T-cell repertoire of infected hosts. But also,the affinity of SAGS for different cell types suggest that distinct toxins may be recruited differentially to adapt well to different tissue-dependent structures of MHC class I1 molecules. Interestingly, among strains of staphylococcus aureus isolated from infected individuals, most of the ones infecting the blood compartment, eventually leading to toxic shock, expressed TSST-1. Interestingly, the one restricted to food poisoning in the gut expressed SEB extensively (14). We postulate that this difference in distribution may not be solely due to the presence of emetic properties in enterotoxins that are not present in TSST1..Of note, studies indicate that the expression of these two toxins (SEB, TSST-l) by strains of S. aureus seems to be mutually exclusive as is their binding to HLA-DRl subpopulations. Considering that SEB has a very poor affinity for peripheral blood lymphocytes or B lymphocytes, a bacterium that proliferates into the blood stream would gain a selective advantage in expressing TSST-1 rather than SEB. It is interesting to correlate these observations with the affinity of the different SAGS for cell surface HLA-DRl molecules. Hence,the term "tropism" can be applied to describe this selectivity of bacterial SAGS toward different cell types. V.

OTHER RECEPTORS FOR BACTERIAL SUPERANTICENS

Observations that class 11-negative colon-carcinoma cells efficiently presented SEB to cytotoxic T lymphocytes prompted workers to suggest the existence of a receptor different than MHC class I1 molecules for SAGS including SEB (81). Another experiment showed that macrophages isolated from 11-deficient CD2 micecan bind SEA, SEB, and the exfoliate toxins A and B (ETA, ETB). This binding induced signaling events in macrophages without requirement for the presence MHC class I1 molecules In these experiments a set of proteins immunoprecipitated with SEA implicated non-MHC class11 molecules as novel receptors for SAGS. One of these molecules was later identified as the murineH-2Dk MHC classI protein (83). Whether SAGS bind to these additional receptors using the same molecular determi-

lymorphic

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75

nants as MHC class I1 is not known. However, these results demand a reevaluation of the importance of MHC class I1 molecules in the pathogenesis of staphylococcal enterotoxins. VI.

INTERACTION OF MMTVSUPERANTICENSWITHMHC MOLECULES

CLASS II

Compared to bacterial superantigens, little is known about the tertiary structure ofMMTV superantigens (v-SAGS)due to difficulties in obtaining sufficient amounts of protein from cells expressing them naturally. Some data about the interactions between v-SAGS and class I1 molecules are beginning to be presented in the literature but the information is still sketchy.MMTV is a type B retrovirus that encodes in its 3'-LTR for a protein of about 320 amino acids having superantigenic properties (v-SAG) (31). The MMTVv-SAGS are type I1 transmembrane glycoproteins of a molecular weight of KDa in the glycosylated form (Fig. 3) Only four of the five potential glycosylation sites are used, presumably because of the close proximity the second and third sites The primary amino acid sequences between different MMTV isolates are highly conserved except for a stretch of about amino acids located at the C-termi0

40

80

l

1

I

sites

320 120 280 160240

I

200

I

I

I

I

glycosylation region

RKRR KR 45 kDa 36 kDa 18.5 kDa 16 kDa

Figure 3 Diagram of the superantigen protein encoded by mouse mammary

tumor viruses (MMTVs). The numbers at the top represent amino acid numbering. The C-terminal polymorphic region is represented. TM = transmembrane. The putative sequence of the processing sites is shown together with the molecular weight of cleaved fragments generated.

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Lavoie et al.

nus which imparts VP specificity (66,67). Another salient feature in the structure of MMTV v-SAGS is the presence of conserved potential dibasic endoprotease processing sites. While bacterialsuperantigens do not require processing for their mitogenic activity, it has been demonstrated that v-SAGS are processed at some of their dibasic endoprotease sites(68). Cleavage of the protein would occur in the Golgi by furin-like proteases, and the unglycosylated 18.5-kDa C-terminus has been found to be associated to MHC class I1 molecules at the cell surface (68). It was shown that coexpression of furin and V-SAG7in insect cells resulted in cleavage at the second conserved processing (generating the 18.5-kDa fragment), and it was suggested that this processing is necessary for biological activity since mutation of this site abrogated cleavage, cell surface expression, and T-cell reactivity (69). However, it is difficult to evaluate what effect these drastic amino acid substitutions (RKRR>EEEE) could have had on the overall conformation of the protein class I1 association. v-SAGS can also be processed at another dibasic processing site, which is not a "consensus" furin site, generating a 16kDa polypeptide (70). While this fragment could not be detected associated to IEk classI1 molecules at the cell surface (681, it was able to bind to IEk + IAk-loaded affinity columns (70). Thus, residues located between the 18.5-kDa and the 16-kDa fragments might play a role in hierarchical class I1 binding. The entire extracellular portion of V-SAG7produced in Escherichia coli (thus unglycosylated) was shown to be able to bind labeled soluble class I1 molecules (711, indicating that processing is not essential for classI1 binding. When sets of short overlapping peptides covering the entire V-SAG7were tested for their ability to inhibit binding of a bacterial superantigen to class I1 molecules, a peptide (76-119) located in the membrane proximal region was able to compete for binding (72). It thus appears that several regions on v-SAGS are involved in class I1 binding. In early studies of Mls, a class I1 hierarchy in the efficiency for T-cell stimulation was established: I-E > I-Ak > I-Ad > I-Ab >>> I-AY The I-As allele apparently cannot interact with v-SAGS, since no VPrestricted deletions are seen in DBA/l mice (73). Cells from these mice can still transfer the deletion potential to cells bearing permissive class I1 alleles (suggesting again that v-SAGS can be processed). Several results suggest that both chains of MHC class I1 molecules contribute to presentation of v-SAGS to T cells. Transgenic mice expressing EaAP mixed pairs can partially restore T-cell deletion (74), and since the Ab allele is a poor presenter of v-SAGS, the contribution the a chain is apparent. On the other hand, HLA-DQP single

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MHC Class

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77

transgenic mice in an H-2E-negative background did delete v-SAGresponsive T cells (75,76), suggesting a role for the chain. Interestingly, no clonal deletions were observed in DQa/DQP double transgenic mice, indicating that DQais not permissive for Mls presentation (77). From the transgenic data, it became apparent that human class I1 molecules could interact with v-SAGS and this observation was later extended to other alleles and isotypes. When murine fibroblasts were cotransfected with HLA-DR1 class I1 molecules and v-SAGS, these cells acquired the ability to stimulate T cells in a Vpspecific manner (78). Interestingly, transfectants generated with other alleles like DR2A, DR2B, and DRw53 were not able to stimulate T cells (78). Since all DR alleles have a common a chain, these results point out the role of the chain in v-SAG binding. Again, DQaDQP double transfectants could not present v-SAGS in this system. Since no significant sequence homology is apparent between v-SAGS and bacterial SAGs, it is not surprising that class I1 mutations that abrogate toxin binding have no effect on v-SAG stimulation of T cells (79). Sequence alignments between different murine and human class I1 alleles should allow identification of key residues involved in interaction with v-SAGS. Altogether, these results point out that interactions between vSAGs and class I1 molecules are complex, with several regions of vSAGs involved in binding and both chains of class I1 molecules contributing to the interaction. Other cofactors could also beinvolved in this interaction as exemplified by the recent demonstration that the env gene product ofMMTV can enhance T-cell response by a still unknown mechanism By analogy to bacterial superantigens, it is also possible that peptides found in the groove of class I1 molecules could affect v-SAG presentation, and this might explain some of the differences observed when using various alleles of HLA-DR. Invariant chain is .another type I1 glycoprotein that is known to interact with class I1 molecules in a hierarchical manner and it is tempting to speculate that v-SAGS could interact directly through the groove of class I1 molecules via a CLIP-like sequence. Extensive mutagenesis will have to be performed on both v-SAGS and class I1 molecules to precisely define their mode of interaction. REFERENCES 1. Kappler

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49. Dowd JE, Jennkins RN, Karp DR. Inhibition of antigen-specific T cell activation by staphylococcal enterotoxins. J Immunoll995; 154:1024-1031. 50. Kozono H, Parker D, White J, et al. Multiple binding sites for bacterial superantigens on soluble class I1 major histocompatibility complex molecules. Immunity 1995;3:187-196. 51. Ghosh P, Amaya M, Mellins E, et al. The structure of an intermediate in class I1MHC maturation: CLIP bound to HLA-DR3. Nature 1995; 378:457-462. 52. Cresswell P. Assembly, transport, and function of MHC class I1 molecules. Annu Rev Immunol 1994; 12:259-293. 53. Busch R, Cloutier I, Sekaly R-P, et al. Invariant chain protects class I1 histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum. EMBO J 1996; 15:418-428. 54. Jones PP. Analysisof H-2 and Ia molecules by two-dimensional gel electrophoresis. J Exp Med 1977; 146:1261-1279. 55. Parham P, Alpert BN, Orr HT, et al. Carbohydrate moiety of HLA antigens: antigenic properties and amino acid sequences around the site of glycosylation. J Biol Chem 1977; 252:7555-7567. 56. Helenius A. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol Biol Cell 1994; 5:253-265. 57. Boog CJ, Neefjes JJ, Ploegh HL, et al. Specific immune response restored by alteratoin in carbohydrate chains of surface molecules on antigenpresenting cells. Eur J Immunol 1989; 19:537-542. 58. Ishikawa S, Kowal C, Cole B, et al. Replacement of N-glycosylation sites on the MHC class I1 E a chain: effect on thymic selection and peripheral T cell activation. J Immunol 1995; 54:5023-5029. 59. Shackelford DA, Strominger JL. Analysis of the oligosaccharides on the HLA-DR and D C 1 B cell antigens. J Immunol 1983; 130:274-282. 60. Altman JD, Reay PA, Davis MM. Formation of functional peptide complexes of class I1 major histocompatibility complex proteins from subunits produced in Escherichia coli. Proc Natl SciUSA1993;90:1033010334. 61. Schick MR, Levy S. The TAPA-l molecule is associated on the surface of B cells with HLA-DR molecules. J Immunol 1993; 151:4090-4097. 62. Angelisova P, Hilgert I, Horejsi V. Association of four antigens of the tetraspans family (CD37, CD53, TAPA-l, and R2/C33) with MHC class I1 glycoproteins. Immunogenetics 1994; 39:249-256. 63. De Boer ML, Chow AW. Toxic shock syndrome toxin l-producing Staphylococcus aureus isolates contain the staphylococcal enterotoxin B genetic element but do not express staphylococcal enterotoxin B. J Infect Dis 1994;170:818-827. 64. Choi Y, Kappler JW, Marrack P. A superantigen endoded in the open reading frame of the 3' long terminal repeat of mouse mammary tumour virus. Nature 1991;350:203-207. 65. Korman AJ, Bourgarel P, Meo T, et al. The mouse mammary tumour

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virus long terminal repeat encodes a type I1 transmembrane glycoprotein. EMBO J 1992;11:1901-1905. Yazdanbakhsh K, Park CG, Winslow G, et al. Direct evidence for the role of COOH terminus mouse mammary tumor virus superantigen in determining T cell receptor VP specificity. J Exp Med 1993; 178:737741. Brandt-Carlson C,Bute1JS, Wheeler D. Phylogenetic and structural analyses of MMTV LTR ORF sequences of exogenous and endogenous origins. Virology 1993;193:171-185. Winslow GM, Scherer MT, Kappler J W , et al. Detection and biochemical characterization of the mouse mammary tumor virus 7 superantigen (Mls-la). Cell 1992;71:719. Park CG, Jung M-Y, Choi Y, et al. Proteolytic processing is required for viral superantigen activity. Exp Med 1995; 181:1899-1904. Winslow GM, Marrack P, Kappler JW. Processing and major histocompatibility complex binding the MTV7 superantigen. Immunity 1994; k23-33. Mottershead DG, Hsu P-N, Urgan RG, et al. Direct binding the Mtv7 superantigen (Mls-l) to soluble MHC class I1 molecules. Immunity 1995; 2:149-154. Torres BA, Griggs ND, Johnson HM. Bacterial and retroviral superantigens share a common binding region on class I1 MHC antigens. Nature 1993;364:152-154. MacDonald HR, Glasebrook AL, Schneider R, et al. T-cell reactivity and tolerance to Mlsa-encoded antigens. Immunol Rev 1989; 107:89-108. Anderson GD, David CS. In vivo expression and function of hybrid Ia dimers (EaAP) in recombinant and transgenic mice. J Exp Med 1989; 170:1003-1008. Zhou P, Anderson GD, Savarirayan S, et al. Thymic deletion of VP11+, Vp5+ T cells in H-2E negative, HLA-DQP+ single transgenic mice. J Immunol 1991; 146:854-859. Zhou P, Smart MK, Cheng S, et al. HLA-DQP chain can present mouse endogenous provirus MTV-9 product and clonally delete TCR VP5+ and VP11+ T cells in transgenic mice. Immunogenetics 1992; 35:219-223. Nishimura Y, Iwanaga T, Inamitsu T, et al. Expression of the human MHC, HLA-DQw6 genes alters the immuneresponse in C57BL/6 mice. J Immunol 1990; 145:353-360. Subramanyam M, McIellan B, Labrecque N, et al. Presentation the Mls-l superantigen by human HLA class I1 molecules to murine T cells. Immunol 1993; 151:2538-2545. Thibodeau J, Labrecque N, Denis F, et al. Binding sites for bacterial and endogenous retroviral superantigens can be dissociated on major histocompatibility complex class I1 molecules. J Exp Med 1994; 179:1029-1034. Golovkina TV, Chervonsky A, Prescott JA, et al. The mouse mammary tumour virus envelope gene product is required for superantigen presentation to T cells. T EXD Med 1994: .179:439-449.

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Molecules

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81. Dohlsten M, Hedlund G, Segren S, et al. Human major histocompatibility complex class 11-negative colon carcinoma cells present staphylococcal superantigens to cytotoxic T lymphocytes: evidence for a novel enterotoxin receptor. Eur J Immunol 1991; 21:1229-1233. 82. Beharka AA, Armstrong JW, Iandolo et al. Binding and activation of major histocompatibility complex class 11-deficient macrophages by staphylococcal exotoxins. Infect Immun 1994; 62:3907-3915. Chapes SK. Staphylococal enterotoxins bind H83. Beharka AA, Iandolo 2Db molecules on macrophages. Proc Natl Acad Sci USA 1995; 92:62946298.


5 Superantigen in Rabies Virus. and Its Involvement in Paralysis Monique Lafon

INTRODUCTION

Many viruses have developed invasive strategies based on the subversion of the immune system (1).Viruses such as influenza, herpes simplex, Sendai, human lymphotropic, or lactate dehydrogenase elevating virus act as potent B-cell mitogens. These infections are characterized by a strong polyclonal activation of Ig with irrelevant specificities that do not participate in the clearance of the virus and misguide the specific immune response (2-7). These viruses activate B lymphocytes by the means different mechanisms. Influenza virus hemagglutinin and lactate deshydrogenate virus proteins display proliferation of B cells by binding to MHC class I1 Ia molecules, respectively, and do not require the presence of T cells In contrast, for other viruses that are T-cell-dependent B-cell mitogens, Bcell proliferation can be mediated by IL-6, a strong B-cell activator cytokine (10,ll). Superantigens (SAg) that are also strong T-cell-dependent B-cell mitogens may be regarded asanother powerful mechanism leading to the stimulation of polyclonal Ig. Therefore, it can be suspected that pathogens encoding forSAg take advantage of the SAg property to circumvent the host immune response too 85

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Like other viruses that multiply in the nervous system, rabies virus is protected from the host reaction, partly by the limited capacity of the nervous system to mount an efficient immune response and partly by the nervous system isolation (14). However, the impermeability of the blood-nerve-and-brain barrier to activated lymphocytes and antibodies is incomplete (15,16). In particular, antibodies directed against rabies or Sindbis virus were found to mediate clearance of virus from the infected neurons (17,181. It seems therefore unlikely that the neurotropism by itself accounts for the ability of rabies virus to escape immune surveillance. It is possible that rabies virus has adopted an additional invasive strategy by decreasing the host immune reaction. We hypothesize that the rabies SAg, the nucleocapsid, helps the virus to quickly move through the nervous system because it shuts off the rabies-specific antibody response. Additionally, because SAg-induced T- and B-cell hyperactivation may contribute to the appearance of an autoimmune reaction (19-21), we envisaged that rabies SAg plays a role in the development of flaccid paralysis often associated with the rabies infection (22-24). In this chapter, after summarizing the properties of rabies SAg (25,26), in particular its ability to activate B cells in the presence of T cells (271, we address two issues: 1) whether, as predicted, mice bearing the rabies SAg target Vp6 T cells are susceptible to rabies infection and immunopathology, whereas mice lacking these Vp6 T cells are difficult to infect and resistant to immunopathology, and 2) whether neutralization of the SAg renders mice resistant to rabies. Our results are consistent with the idea that NC plays a role in making the host susceptible to rabiesvirus infection and that target T cells of the rabies SAg are crucial factors in the pathogenesis and the immunopathology of rabies. The resultsstrongly support the hypothesis that coding for a SAg could be a strategem used by microorganisms to favor their invasion of the host. II. STRUCTURE AND LIFECYCLE O F THE RABIES VIRUS

The rabies virus is an enveloped, bullet-shaped virus the Rhabdoviridae family, genus Lyssavirus, which possesses a negative-stranded RNA genome 11,932 nucleotides long. The genome consists of a leader RNA and five genes that code in the to the 5' end, the N, nonstructural NS, matrix M, and glyco G and L proteins (28). The rabies virus particle is composed of a viral membrane surrounding a helical nucleocapsid (NC). NC results from the association of three internal proteins, the N, NS, and L proteins, together with the viral

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RNA. The length ofNC varies according to its conformation; NC is 10-18 nm long and 2-6.5 nm wide when tightly coiled and is 3.8-4.6 pm long when uncoiled (29). The major protein in NC is the N protein (30), a phosphorylated (31), not glycosylated (321, protein 450 amino acids in length. N protein, tightly associated with the rabies virion RNA genome, protects the RNA from ribonucleases (33) and ensures a suitable configuration for transcription. G and M, together with host lipids, constitute the virus envelope. Only G is exposed on the surface of the virion and forms spikes protruding from the virus envelope. This protein is the only virus protein that induces and reacts with neutralizing antibodies. Antigenic structure of the N protein was studied by using monoclonal antibodies and synthetic peptides. Several antigenic sites and both T and B epitopes were identified along the N-protein structure and sequence (34-37). Infection of the host by rabies virus occurs by bites or scratches, by contact of preexisting wounds with infected saliva, or after inhalation of aerosols containing rabies virus. The virus then enters into the peripheral nerve endings of sensory nerves, into stretch proprioceptor or motor-end plates, or into the neuroepithelial cells of olfactory neuroepithelium. In some cases nerve penetration can be preceded by a local multiplication in the striated muscle cells at the site of inoculation (38,39). Following the attachment of G to membrane receptor, the nature of which has not yet been clearly identified (4045), the rabies virus enters cells by endocytosis. Itis assumed that the viral genome travels in the form of NC within the axoplasma before it reaches the perikaryon or the proximal parts of dendrites. In these ribosome-rich areas of the neuron, the genome is transcribed by the NS-L complex into mRNAs for the five viral proteins and subsequently into a full-length positive mRNA. The positive strands of RNA serve as templates for the production of infectious negativestrand genomic RNAs, Newlysynthesized G associate with the rough endoplasmic reticulum. Concomitantly, N, NS, and L proteins associate with progeny negative-strand genomic RNAs to formNC aggregates, the Negri bodies, that accumulate in perikaryons and proximal dendrites. Association ofNC with the G-containing membranes to form new virus particules may take place in late infection stages. It is postulated that neuron-to-neuron spread of rabies virus occurs in the form ofNC complexes that spread from the synapse into the postsynaptic neuron (46). Complete viruses are produced only in the late stages of infection when the virus is excreted in saliva before being transmitted to a new host.

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RABIES NC

SAG

We found evidence that NC of the rabies virus is an exogenous SAg in humans and also in mice (25,26). This was established after we showed: 1) NC binds to HLA class I1 molecules; 2) NC induces specific Vp T-lymphocyte proliferation (Vp8 in humans; Vp6 and 7 in mice), and NC causes clonal deletion of some entire Vp subsets, a function that is regarded as a prerequisite for a SAg. It was demonstrated that the major protein of NC, the N protein, resumes the SAg activity. We addressed in humans the question of whether NC can induce cognate T-B interactions in vitro by using human tonsil B and T lymphocytes (27). The capacity to stimulate antibody production is well documented for the Mycoplasma-derived SAg, MAM, which was found to stimulate IgM production both in vivo and in vitro (47,481. We analyzed the culture requirements and the nature of the antibodies produced upon NC activation. By comparing the properties of rabies SAg with those of two Staphylococcus-derived SAg: SEE, which, like NC, targets Vp8 T cells, and TSST-l, which targets Vp2 T cells, we found that NC triggers human lymphocytes to produce IgG to a similar extent as and TSST-l. Despite its weak T-lymphocyte mitogenic activity, NC requires the presence of Vp-specific T cells, with a preferential T/B ratio of l/ 5, to aid B cells to produce IgG. The fact that IgG production depends on the presence of T cells argues in favor of the hypothesis that NC does not activate B cells directly. Instead, NC could promote cognate T-B-cell interactions, mediated by the SAg bridge, resulting in polyclonal IgG secretion. Similar amounts and frequencies IgG production were obtained independently of the stimulus used, whether it was NC or the Staphylococcus-derived SAg, and TSST-l. This suggests that cognate T-B-cell interactions induced by rabies SAg are similar to those induced by bacterial SAg. The risk that the rabies SAg, by inducing polyclonal activation, could stimulate autoimmune reactions was studied in humans (49). Peripheral lymphocytes from asymptomatic patients infected with HIV-l were stimulated ex vivo with NC and autoantibodies were measured. NC was found to induce polyclonal activation of B cells including the secretion of autoantibodies (antibodies directed against single-strand DNA, actin, myosin). This secretion requires the presence of rabies SAg T-cell target (VPST cells). After we showed that rabies virus encodes a SAg, the next step was to investigate the biological significanceit may have in rabies infection and immunopathology.

Superantigen in Rabies Virus RABIES PATHOGENESIS, ROLE OF RABIESSAC A. Rabies Pathogenesis

Death of the host in rabies infection seems to result from the alteration of neuronal functions rather than from neuronal destruction. Sleep alteration, a decrease in opiate and serotonin binding, and a drop in proenkephalin concentrations are observed in the course of rabies infection (50-53). Encephalitic rabies is characterized by the low increase of inducible nitric oxide synthetase, a marker of inflammation, indicating that rabies infection, in contrast to other viral neurological infections, provokes only a weak inflammation of the nervous tissue (54,55). There is evidence that a rapid and successful invasion of the host nervous system requires a low immune surveillance (23,56). This early host reaction is supposed to prevent infection of the nervous system or to block the first steps of nerve infection. In studies on natural mouse resistance to rabies, it was observed that susceptibility to rabies infection correlates with the low capacity to mount an efficient neutralizing immune response under the control of CD4+,T lymphocytes (57,581. Virus could be detected earlier and at higher concentrations in brains of immunosuppressed mice compared to those of immunocompetent mice (56,59,60). On another hand, the attenuated pathogenicity exhibited by mutants of laboratory rabies virus strains correlates with their ability to trigger a strong specific neutralizing antibody response from the host: viruses that do not successfully infect the host induce a strong immune response, whereas fully pathogenic rabies virus strains trigger a weak immune response (61-63). The production of neutralizing antibodies therefore seems beneficial for fighting a rabies infection. However, in some instances, the capacity to produce antibodies is deleterious and hastens the course of disease. This happens during the so called earlydeath phenomenon where animals with low levels of antibodies or with antibodies of low affinity died earlier than nonimmunized controls when challenged with a lethal dose of virus (64). This suggests that the nature of antibodies is an important issue in disease and that antibodies of low affinity or of irrelevant specificity favor infection (65,66). It is striking to note that dogs injected with a rabies N-protein vaccinia recombinant were sicker and had a shorter incubation period when compared to control dogs (67), suggesting that N protein can be implicated in mechanisms hastening rabies death or morbidity.

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B.

Role of

in Rabies Infection

We postulated that NC helps the virus to infect the host in the presence of Vp target T cells by weakening the immune response and stimulating antibodies irrelevant specificity. The immunosuppression controlled by the rabies SAg is expected in mice expressing the Vp6 T cells but not in mice lacking these cells. As a consequence, mice expressing Vp6 T cells should be susceptible to rabies infection and develop low levels of rabies-specific antibodies, whereas mice lacking Vp6 T cells should be resistant to rabies infection and develop an efficient protective antibody response. In addition, mice susceptible to rabies infection should develop a strong polyclonal antibody response. First, we addressed the question whether Vp may interfere with rabies resistance by comparing rabies mortality in congenic or closely related mice expressing or lacking the rabies SAg Vp T-cell targets. We compared the susceptibility of CBA/J mice and CBA/Ca mice to rabies infection. CBA are IE+, H2k mice that lack Vp5, 11, and 12 T cells because of the integration of Mtv-8, 9, and 14 in their genome; in addition, CBA/J mice lack Vp6, 7, 8.1, and 9 T cells as a result of the integration Mtv 6, 7, and 17. It was found that indeed the Vp6depleted CBA/J mice were resistant to rabies infection, whereasCBA/ Ca, the VP6-positive mice, were highly susceptible. To demonstrate the role of rabies SAg target VPT cells in rabies pathology, Vp6 and Vp8.1-3 T cells were transfused into BALB/D2 mice, a congener of BALB/c(H~~, IE +), that also lack Vp6 and 7 T cells (68). All the Vp6reconstituted mice died of rabies, whereas none of the Vp8-reconstituted or the nonreconstituted BALB/D2 mice had died by day 18 (26). These results clearly demonstrate that rabies susceptibility is dependent on the presence ofVp6 T cells. Then, we checked whether Vp6positive mice are rabies-specific-antibodieslow responders in comparison with VP6-negative mice, by measuringthetiters of rabies-specific antibodies in the course of rabies infectionin the CBA/ J and CBA/Ca mice. As shown in Fig. lA, Vp6-positive mice are low responders for rabies-specific antibodies, in contrast to VP6-negative mice, which are high responders. As expected, VP6-positive mice develop a strong polyclonal response in the course of rabies infection significantly different from that of VP6-negative mice (Fig. 1B). Finally, to test the role of rabies SAg in rabies susceptibility, we compared the rabies virus infection in VP6-positive mice injected either with an antibody directed against the rabies SAg or with an irrelevant antibody specific for the N protein of the lyssavirus, Mokola


91

virus (Fig. 2). Groups of mice injected with antibodies or injected with saline buffer as controls on day -1, 0, and were challenged on day 0 with infectious rabies virus. Ten days after infection, 70% of mice treated with monoclonal antibodies directed against the N protein remained healthy, whereas all of the nontreated mice or mice that had received an irrelevant monoclonal antibody for resistance to rabies presented rabies symptoms. This suggests that neutralization of the SAg renders mice resistant to rabies infection. Conversely, the addition of SAg to viral inoculum hastens death by 2 days. This was observed only when minute amounts ofNC (0.05 per mouse) were added, whereas higher concentrations of NC (>5 per mouse) increased survival (data not shown). Altogether these observations emphasized that 1) in IE+ mice, rabies susceptibility is dependent on the presence of Vp6 T cells, and conversely, that natural resistance of rabies requiresthe absence of the rabies SAg target T cells, 2) development of polyclonal antibody response is detrimental to the protective specificantibody response and is linked to the presence of Vp6 T cells, and neutralization of NC renders mice resistant to rabies infection. Theseresults are consistent with the hypothesis that, in the presence of Vp6 T cells, SAg can block the rabies-specific antibody response and therefore may help the virus to colonize the host. V.

RABIES IMMUNOPATHOLOGY, ROLE OF RABIES SAG

A.

Paralytic Rabies

In addition to encephalitic rabies symptoms, the rabies infection can induce symptoms of flaccid paralysis. Paralyzed limbs show evidence of neuronal inflammation and injury. Enlargement of the dorsal root ganglia of the inoculated limb and a motor neuron degeneration the sciatic nerve are observed (24,56). The reason why rabies develops as an encephalitic or paralytic disease seems, at least in mice, to depend on the rabies strain. Paralytic rabies is observed with viruses of low pathogenicity such as virus strains of bat origin or attenuated laboratory rabies virus strains (56,59,60). Virus replication is required for the development of paralysis since neuritis is not observed when inactivated virus is injected (24). However, the neuropathology is not linked to the viral replication itself, since there is no correlation with the axonal accumulation of NC and the severity of the neuronal destruction (24). T cells play a key role in the immunopathological paralytic process. This was concluded from experiments showing that

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rabies-specific lg2a antibodies (UA/ml)

*

Vp6- positive mice

*VpS- negative mice

0

1

2

3

4

5

6

7

8

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13

days after infection

Figure 1 (a) Comparison of rabies-specific IgG2a antibody titers in Vp6positive mice and VP6-negative mice. Antibody titers expressed in AU/ml were measured by ELISA using purified rabies virus antigen as solid phase in serum of CBA/J, the VP6-negative mice or CBA/Ca, the VP6-positive mice (*l. Titers of VP6-positive mice were significantly lower than those of VPbnegative mice. Each point represents the mean titers of at least six mice. (b) Comparison of polyclonal Ig production in the course of rabies infection of VP6-positive and VP6-negative mice. Total antibodies were measured in serum of CBA/J, the VP6-negative mice or CBA/Ca, the VP6-positive mice (*l, by capture ELISA. Ability ofCBA/Ca, the VP6-positive mice, to produce Ig during rabies infection was significantly higher than that of CBA/ J, the VP6-negative mice.

paralysis is not observed either in immunosuppressed mice, in nude mice, or after depletion of CD4+ T and CD8+ T cells before infection, butis seen after T-cell reconstitution before infection (23,24,57,69). However, pathology cannot be transferred into nayve mice solely by the injection of T cells from paralyzed mice (24). Several mechanisms can be proposedto explain rabies-associated immunopathology. The most evident hypothesis is that rabies neu-

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total IgG (UA/ml)

300 -

*

250

Vp6- positive mice

* Vp6- negative mice

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50 -

d-l

d5

d9

dl1

days postinfection

Figure 1 Continued

ritis results from the attack of infected neurons by cytotoxic T cells. This hypothesis is supported by the fact that depletion of CD8+ T cells, and CD4+ T cells as well, prevents the onset of immunopathology (24) and by the evidence that neurons can express MHC class I molecules inthe presence ofIFN-y, orwhenthey become bioelectrically silent (70). The second possibility is that neuritis results from neurotoxic phenomena mediated by cytokines such as IL-1, IL6, or TNF-a or noxious agents induced at the site of inflammation (71). The third mechanism .could be the involvement of an autoimmune reaction. In the viral mimicry model of autoimmunity, it is supposed that active virus infections present viral motifs similar to self-Ag motifs that trigger autoreactive T cells (72). When the nerve is the target of the autoimmune reaction, T cells that have been activated in the periphery pass through the blood-nerve barrier and attack some nervous Ag.

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number of animals

clinical symptoms healthy mild paralysis severe paralysis dead

irrelevant anti-SAg antibody treatment

Figure 2 Effect of SAg neutralization on rabies susceptibility. CBA/Ca, the VP6-positive mice, were injected with antibodies directed against the rabies SAg or with irrelevant antibody specific for the N protein of the lyssavirus, Mokola virus on day -1, 0, and and challenged on day with a lethal dose infectious rabies virus. Results are presented as cumulative score of clinical symptoms (healthy, mild severe paralysis, and death). Seventy percent of mice treatment with monoclonal antibody directed against the N protein (right column) survived, indicating that neutralization of the SAg renders mice resistant to rabies infection.

Whatever the mechanism involved in rabies immunopathology, we think that the rabies SAg, with its capacity to activate entire Tcell families bearing particular Vp TCR, regardless of their antigen specificity and to activate B cells to produce antibodies can aggravate the rabies-associated immunopathology. In particular, in the hypothesis of the autoimmune origin, the paralysis associated with the rabies infection may be regarded as the result of the NC property to stimulate the production of antibodies directed against self-motifs of the nerves. B.

Role of RabiesSAg in Rabies-AssociatedParalysis

We postulated that rabies SAg is able to aggravate rabies immunopathology directed against self-nerve motif.In this hypothesis, the property of SAg to aggravate immunopathology can be expected in mice


95

% of paralyzed +

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+

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-

60 40 -

20 -

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CP

l T

l

l

L

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7

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10

I

I

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12

days postinfection Figure 3 Comparative rabies virus immunopathology in VP6-positive and

-

negative mouse strains. Percentage of CBA/Ca and CBA/J mice showing symptoms hind-limb paralysis after intramuscular injection of 1 X lo7 infectious particles of Pasteur rabies virus strain in both hindlegs. Results are presented for groups of 12 mice.

expressing VPT cells targeted by rabies SAg but not in mice lacking these cells. As a consequence, mice expressing VP6 T cells should suffer from severe rabies-associated paralysis, whereas mice lacking the Vp6 T cells should remain healthy. In addition, reconstitution of VP6-negative mice with VP6T cells should restore immunopathology. These questions were addressed by comparing the percentages of paralytic mice in congenic strains of mice expressing or lacking Vp6 T cells. In our initial experiments, paralysis following rabies infection was compared in Vp6-positive CBA/Ca and in the closely-related VP6negative CBA/J mice. During the rabies infection, the VP6-depleted CBA/J mice did not show any paralysis symptoms, while CBA/Ca, the Vp6-positive mice, were severely paralyzed (Fig. In a second set experiments, rabies paralysis was compared in VP6-positive BALB/c and in congenic Vp6-negativeBALB/D2 mice.

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Significantly fewer BALB/D2 were paralyzed following rabies infection, in comparison with BALB/c mice, the Vp6 T-cell-positive mice. To demonstrate the role of rabies SAg target VPT cells in rabies pathology, Vp6 and Vp8.1-2-3 T cells were transfused separately into BALB/D2 mice, which normally lack these lymphocyte subsets. All the VP6-reconstituted mice becameparalyzed in comparison with the nonreconstituted BALB/D2 mice (26). These results clearly demonstrate that rabies immunopathology is dependent on the presence of Vp6 T cells, the rabies SAg-specific T-cell targets. VI.

CONCLUSION

Our results clearly establish that rabies NC triggers a preferential stimulation of Vp T cells both in humans after in vitro activation (Vp8) and in mice in vitro after local injection (Vp6, Vp7). Moreover, in newborn mice, repeated injections ofNC lead to the peripheral elimination of the particular Vp (Vp6 and' Vp7) preferentially stimulated by NC. Since NC binds to surface class I1 molecules and does not require processing, the results obtained in the mouse 'model strengthen our conclusion that rabies NC is a SAg. Our experiments showed that host susceptibility to rabies infection is linked to the presence ofVp6 T cells. This strongly supports the hypothesis that mice that integrated MMTV in their genome, MMTV-7 in particular, such as BALB/D2 or CBA/J, are resistant to rabies infection because rabies SAg cannot exert its deleterious effect on theimmune response. Similarly, susceptibility to Toxoplasma gondii, which codes for a VP5-specific SAg (731, was found to be linked to the presence Vp5 SAg target T cells: survival of mice following T. gondii infection was higher in mice lacking the SAg target T cells than in mice bearing these T cells (74). These findings emphasize that SAg could be important in modulating the resistance and susceptibility to pathogens. In addition, SAg properties may be more complex than expected. In particular, immunostimulant properties have been described for the of rabies SAg, which was found to boost associated antigenic responses, whether directed against the rabies virus or against an antigenically unrelated antigen such as influenza virus (75,761. On one hand, rabies SAg can immunosuppress the associated immune response; this happens when it is added in minute amounts or injected with the viral inoculum. On the other hand, when injected in large amounts concomitantly with an antigen, rabies virus SAg has an adjuvant property. Elucidation of the mechanisms governing these two opposite behaviors deserves further attention.


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ACKNOWLEDGMENTS

Mireille Lafage is acknowledged forher excellent technical assistance. This work was supported by internal grants from the Institut Pasteur, Paris, and by Specific Grant 930607 from the Institut National de la Sante et de la Recherche Medicale (INSERM). REFERENCES 1. Marrack P, Kappler

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13. Palmei E. Infectious origin of superantigens. Curr Biol 1991; k74-76. 14. Joly E, Mucke L, Oldstone MBA. Viral persistence in neurons explained by lack of major histocompatibility class I expression. Science 1991; 253:1283-1285. 15. Hickey WF, Hsu BI, Kimura H. T lymphocyte entry into the central nervous system. J Neurosci Res 1991; 28:254-260. 16. Tuomanen EI, Prasad SM, George Hoepelman AIM, Ibsen P, Heron I, Starzyk RM. Reversible opening of the blood-brain barrier by antibacterial antibodies. Proc Natl Acad Sci USA 1993; 90:7824-7828. 17. Levine B, Hardwick JM, Trapp BD, Crawford TO, Bollinger RC, Griffin DE. Antibody-mediated clearance of alphavirus infection from neurons. Science 1991;254:856-860. 18. Dietzschold B. Antibody mediated clearance of viruses from the mammalian central nervous system. Trends Microboll993; 1:63-66. Gaur A,PiercyC, Gautam A, Gijbels K, Fathman CG, 19. Brocke Steinman L. Induction of relapsing paralysis in experimental autoimmune encephalomyelitisby bacterial superantigen. Nature 1993; 365542644. 20. Cole BC, Atkins CL. The Mycoplasma arthritidis T-cell mitogen, MAM: a model superantigen. Immunol Today 1991; 12:271-276. 21. Wucherpfennig KW, Weiner HL, Hafler DA. T cell recognition of myelin basic protein. 22. Hemachuda Phanuphak P, Swriwanthana B. Immunologic study of human encephalitic and paralytic rabies: a preliminary study of 16 patients. Am J Med 1988; 84:673-677. 23. Iwasaki Gerhard W, Clarke HF. Role of host immune response in the development either encephalitic or paralytic disease after experimental rabies infection in mice. Infect Immun 1977; 18:220-225. 24. Weiland F, Cox Meyer S, Dahme Reddehase MJ. Rabies virus neuritic paralysis: immunopathogenesis of non fatal paralytic rabies. J Virol 1992; 66:5096-5099. 25. Lafon M, Lafage M, Martinez-Arends A, Ramirez R, Vuillier F, Charron D, Lotteau V, Scott-Algara D. Evidence in humans of a viral superantigen. Nature 1992; 358:507-509. 26. Lafon M, Scott-Algara D, Marche PN, Cazenave P-A, Jouvin-Marche E. Neonatal deletion and selective expansion of mouse T cells by exposure to rabies virus nucleocapsid superantigen. J Exp Med 1994; 180:1207-1215. 27. Martinez-Arends A, Astoul E, Lafage M, Lafon M. Activation of human tonsil lymphocytes by rabies virus nucleocapsid superantigen. Clin Immunol Immunopathol 1995; 77:177-184. 28. Tordo N, Poch 0, Ermine A, Keith G, Rougeon F. Walking along the rabies genome: is the large G-L intergenic region remnant gene? Proc Natl Acad Sci USA 1986; '83:3914-3918. 29. Sokol F, Schlumberger HD, Wiktor TJ, Koprowski H, Hummeler K. Biochemical and biophysical studies on the nucleocapsid and on the RNA of rabies virus. Virology 1969; 38:651-665.


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30. Sokol F, Koprowski H. Structure-function relationships and mode of replication of animal rhabdoviruses. Proc Natl AcadSciUSA1975; 72:933-936. 31. Dietzschold B, LafonM,Wang H-H, Otvos L, Celis E, Wunner H, Koprowski H. Localization and immunological characterization of antigenic domains of the rabies N and NS protein. Virus Res 1987; 8:103125. 32. Dietzschold B. Oligosaccharides of the glycosylation of rabies virus. J Virol 1977; 23:286-293. 33. Wunner WH. The chemical composition and molecular structure of rabies viruses. In: Baer GM, ed. The Natural History of Rabies, 2nd ed. New York: Academic Press, 1994:31-67. 34. Celis E, Miller RW, Wiktor TJ, Dietzschold B, Koprowski H. Isolation and chartacterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interfon-gamma. J Immunol 1986;136:692-697. 35. Ertl HCJ, Dietzschold B, Gore M, Otvos LJR, Larson JK, Wunner W, Koprowski H. Induction of rabies virus-specific T-helper cells by synthetic peptides that carry dominant T-helper cell epitopes of the viral ribonucleoprotein. J Virol 1989; 63:2885-2892. 36. Goto H, Minamoto N, Ito H, Luo TR, Sugiyama M, Kinjo T, Kawai A. Expression of the nucleoprotein of rabies virus in Escherichia coli and mapping of antigenic sites. Arch Virol 1985; 140:1061-1074. 37. Lafon M, Wiktor TJ. Antigenic structure of the rabies virus nucleocapsid. J Gen Virol 1985; 66:2125-2133. 38. Charlton KM, Casey GA. Experimental rabies in skunks: immunofluorescent, light and electron microscopic studies. Lab Invest 1979; 41:3644. 39. Murphy FA, Bauer SP. Early street rabies virus infection in striated muscle and later progression to the central nervous system. Intervirology 1974;3:256-268. 40. Conti C, Superti F, Tsiang H. Membrane carbohydrates requirement for rabies virus binding to CER cells. Intervirology 1986; 26:164-168. 41. Hanham CA, Zhao F, Tignor GH. Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor. J Vir01 1993;67:530-542. 42. Lentz TL, Burrage TG, Smith AL, Crick J, Tignor GH. Is the acetylcholine receptor a rabies virus receptor? Science 1982; 215:182-184. 43. Reagan KJ, Wunner WH. Rabies virus interaction with various cell lines is independent of the acetylcholine receptor. Arch Viol 1985; 84:277-282. 44. Superti F, Seganti L, Tsiang H, Orsi N. Role of phospholipids in rhabdovirus attachment to CER cells. Arch Virol 1984; 81:47-56. 45. Wunner WH, Reagan KJ, Koprowski H. Characterization of saturable binding sites for rabies virus. J Virol 1984; 50:691-697. 46. Gosztonyi G, Dietzschold B, Kao M, Rupprecht CE, Koprowski H.Ra-

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bies and borna disease: a comparativepathogenetic study of two neurovirulent agents. Lab Invest 1993; 68:285-295. Crow MK, Zagon G, Zhongqiang CH, Ravina B, Tumang JR, Cole BC, Friedman SM. Human B cell differentiation induced by microbial superantigens: unselected peripheral blood lymphocytes secrete polyclonal immunoglobulin in response to Mycoplasma arthritidis mitogen. Autoimmunology 1992; 14:23-32. Cole BC, Elsayed Araneo BA, Shelby J, Kamerath C, Wei S, McCall S, Atkin CL. Immunomodulation in vivo by the Mycoplasma arthritidis superantigen, MAM. Clin Infect Dis 1993;17:S163-169. Scott-Algara D, Lafon M, Vuillier F, Pialous G, Dauguet C, Dighiero G. Viral superantigen-induced hyporesponsivenessof T cells and polyclonal B cell activation in HIV-l infection. Eur J Immunol 1994; 24:2595-2601. Ceccaldi P-E, Fillion "P, Ermine A, Tsiang H, Fillion G. Rabies virus selectively alters 5-HT1 receptor subtypes in rat brain. Eur J Pharm 1993; 245:129-138. FuZF, Weihe E, Zheng YM, Schlfer MK-H, Sheng H, Corisdeo S, Rauscher FJ, Koprowski H, Dietzschold B. Differential effects of rabies and borna disease viruses on immediate-early and late-response gene expression in brain tissues. J Virol 1993; 67:6674-6681. Gourmelon P, Briet D, Clarencon D, Court T, Tsiang H. Sleep alterations in experimental street rabiesvirus infection in the absence of major EEG abnormalities. Brain Res 1991; 554:159-165. Koschel K, Munzel P. Inhibition of opiate receptor-mediated signal transmission by rabies virus persistently infected mouse neuroblastoma rat glioma hybrid cells. Proc Natl Acad Sci USA 1984; 81:950954. Akaike T, Weihe E, Dchaefer M, Fu ZF, Zheng YM, Vogel W, Schmidt H, Koprowski H, Dietzschold B. Effect of neurotropic virus infection on neuronal and inducible nitric oxide synthetase activity in rat brain. J Neurovirol 1995; M18-125. Koprowski H, Zheng Y M, Heber-Katz E, Fraser N, Rorke L, Fu ZF, Hanlon C, Dietzschold B. In vivo expression of inducible nitric oxide synthase in experimentally induced neurologic disease. Proc Natl Acad SciUSA 1993; 90:3024-3027. Smith J, McClelland CL, Reid FL, Baer GM. Dual role of the immune response in street rabies virus infection of mice. Infect Immun 1982; 35:213-221. Perry L, Lodmell DL. Role of CD4+ and CD8+ T cells in murine resistance to street rabies virus. J Virol 1991; 65:3429-3433. Templeton JW, Holmberg C, Garber T, Sharp M. Genetic control of serum neutralizing-antibody response to rabies vaccination and survival after rabies challenge infection in mice. J Virol 1986; 59:98-102. Lodmell DL. Genetic control of resistance to street rabies virus in mice. J Exp Med 1983; 157:451-460.


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60. Lodmell DL, Chesebro J. Murine resistance to street rabies virus: genetic analysis by testing second-backcross progeny and verification of allelic resistance genes in SJL/J and CBAIJ mice. Virol 1984; 50:359-362. 61. Flamand A, Coulon P, Pepin M, Blancou J, Rollin P, Portnoi D. Immunogenic and protective power of avirulent mutants of rabies virus selected with neutralizing monoclonal antibodies. In: Chanok RM, Lerner RA, eds. Modern Approaches to Vaccines: Molecular and Chemical Basis of Virus Virulence and Immunogenicity. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1984:345-356. 62. Yang C, Jackson AC. Basis of neurovirulence of avirulent rabies virus variant AvOl with stereotaxic brain inoculation in mice. J Gen Viroll992; 73:895-900. 63. Marcowitz R, Leal EC, De Souza Matos DC, Tsiang H. Interferon production and immune response induction in apathogenic rabies virus infected mice. Acta Virol 1994; 38:193-197. 64. Blancou J, Andral B, Andral L. A model for the studyof the early death phenomenon after vaccination and challenge with rabies virus. J Gen Virol 1980; 50:433-435. 65. Andral B, Blancou J. Study of themechanisms of the early death occuring after vaccination in mice inoculated with street rabies virus. Ann Virol Inst Pasteur 1981;132E:503-517. 66. Prabakhar BS, Nathanson N. Acute rabies death mediated by antibody. Nature 1981;290:590-591. 67. Fekadu M, Sumner JW, ShaddockJH,Sanderlin DW, Baer GM. Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein. J Virol 1992; 66:2601-2604. 68. Berumen L, Halle-Pannenkoo 0, Festentein H. Strong histocompatibility and cell-mediated cytotoxic effects of a single Mls difference demonstrated using congenic mouse strain. Eur J Immunol 1983; 13:292-300. 69. Sugamata M, Miyazawa M, Mori S, Spangrude GJ, Ewalt LC, Lodmell DL. Paralysis of street rabies virus-infected mice is dependent on T lymphocytes. J Virol 1992; 66:1252-1254. 70. Neumann H, Cavalib A, Jenne DE, Wekerle H. Induction of MHC class I genes in neurons. Science 1995; 269:549-552. 71. Rothwell NJ, Hopkins SJ. Cytokines and the nervous system11: actions and mechanisms of action. Trends Neurosci 1995; 18:130-136. 72. Wucherpfennig KW, Strominger J. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 1995; 80:695-705. 73. Denkers EY, Caspar P, Sher Toxoplasma gondii possesses a superantigen activity that selectively expands murine T cell receptor Vp5 bearing CD8+ lymphocytes. J Exp Med 1994; 180:985-994. 74. McLeod R, Mark DG, Brown C, Skamene E. Secretory IgA antibody to SAG-l, H-2 class I restricted CD8+ T lymphocytes and the INT-l locus

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in protection against Toxoplasma gondii. In: Smith J, ed. Toxoplasmosis. Series H, Cell Biology, Vol78. New York: Springer-Verlag, 1993:131-151. 75. Dietzschold B, Wang H, Rupprecht CE, Celis Tollis M, Ertl H, HeberKatz Koprowski H. Induction of protective immunity against rabies by immunization with rabies virus nucleoprotein. Proc Natl Acad Sci USA 1987;84:9165-9169. 76. Astoul Lafage M, Lafon M. Rabies superantigen as a Vp6 T dependent adjuvant. Exp Med 1996; 183:1628-1631.

Superantigen Associated with Epstein-Barr Virus

Natalie Sutkowski and Brigitte T. Huber

INTRODUCTION

It is estimated that more than 90% of adults are latently infected with Epstein-Barr virus (EBV). This herpesvirus is the causative agent of infectious mononucleosis (IM), a self-limiting lymphoproliferative disease characterized by extensive T-cell activation. EBV is also an oncogenic virus. Early studies associated EBV with African Burkitt’s lymphoma and with nasopharyngeal carcinoma in Asian patients (1; reviewed in More recently, EBV has been associated with the development of lymphoproliferativedisorders in AIDS patients (4,5), as well as in bone marrow and organ transplant recipients Although it was suspected for some time that an EBV-associated superantigen might be responsible for the massive T-cell proliferation seen during acute infection, only recently has there been solid evidence to indicate that this is indeed the case. In this chapter we will discuss how the superantigen manipulates the immune response to EBV. We will concentrate on two points: 1) The role of the superantigen in the 103

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establishment of persistent infection; and 2) the role of the superantigen in the various EBV-associated diseases. II. BACKGROUND

Epstein-Barr virus (EBV) is a highly successful virus, which has evolved to inhabit and manipulate the immune system to its own advantage. EBV has a strong tropism-for B lymphocytes and has the capacity to activate them to proliferate continuously (reviewed in 8,9). During acute infection, EBV causes polyclonal B-cell activation, and this is accompanied by a rapid activation of nonimmune T cells. Initially, both CD4+ and CD8+ T cells are activated (10); however, later the response comprises mainly CD8+ lymphoblastoid cells (11). The massive immune response causes the symptoms of IM. The symptoms associated with the acute infection regress slowly over a period of months. Virus shedding decreases, and there is a falloff in the number of infected B cells, coinciding with the onset of virusspecific cytotoxic T cells (CTL) (12). As occurs with all of the herpesvirus family members, EBV enters a state of latency that persist for the lifetime the host. The EBV genome can be roughly divided into twosets of genes: the lytic cycle genes, necessary for viral replication, and the latent cycle genes, responsible for B-cell transformation and persistent infection. The 172-kb viral genomehas been entirely sequenced (13,141, and is composed mainly of genes encoding lytic infection proteins, although the function of many of the genes remains unclear. During latency, the viral genome exists as a circular episome in the nucleus of the B cell (Fig. 1); however, during the lytic cycle, EBV is replicated as a linear genome (Fig. 2). The linear genome is bounded by direct terminal repeats and i s divided into five unique regions, termed U1-U5, which are interspersed between four internal repeats, termed IRI-IR4. EBV has a complex replication cycle. Thevirus enters B cells by receptor-mediated polar endocytosis, dockingon the CR2 complement receptors, CD21, via the major EBV outer-envelope glycoprotein, gp350/220 The virus is internalized in smooth membrane vesicles, and once inside the cell, the envelope fuses with the vesicle, releasing the nucleocapsid into the cytoplasm. Capsid'dissolution follows, and the linear genome is transported to the nucleus, where circularization occurs. reviews, see Refs. 9,171. EBV is unique in its ability to transform B cells. In vitro infection with EBV results in the generation of lymphoblastoid cell lines (LCL), which proliferate indefinitely, independent of cytokine

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antigen stimulation. generally express only latent-cycle genes, including, but not limited to, six nuclear proteins, termed EBNAs, two integral membrane proteins, the LMPs, and two small RNAs, termed EBERs (18). It has been determined by deletion mapping that of these genes, EBNA3B, LMP2, and the EBERs are not essential for growth transformation The LCL can be stimulated by a variety of means to enter the lytic cycle. As the name suggests, lytic infecTR

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Sutkowski and Huber

tion results in lysis of the B cell and the production and release of infectious virus. Although many of the latency geneswere defined by expression in LCL, the site of viral latency in vivo is not a proliferating, growthtransformed B cell, but instead a resting B cell, which does not express the majority of the latent-cycle genes (20,21). During acute infection, or IM, there is evidence for both latent-cycle and lytic-cycle gene expression. The development of EBV-specific CTL limits the growth of the activated B cells expressing the latent-cycle genes (22,231. It is believed that a subpopulation of the B cells downregulates latent-cycle gene expression, thereby evadingthe CTL response (21). These cells become a reservoir for persistent infection. Speculation that a superantigen might manipulate the immune response to EBV arose as a result of several observations. The first hint was garnered from the fact that like the prototypic murine mammary tumor virus (MMTV) superantigen, Mls-l, EBV infects antigenpresenting B cells. Mls-l is expressed in B cells in association with MHC class I1 molecules. Superantigens are dependent on MHC class I1 presentation to exert their effects on T cells. A somewhat more obvious indication of superantigen activity is the clinical observation that during IM an immediate activation of nonspecific T cells, both CD& and CD8+, takes place, which is followed by an expansion CD8+ cells (11,12). Furthermore, the activated T cells have an atypical phenotype, lacking common markers of activation, such as CD28 and the IL-2R, CD25, but expressing high levels of Fas. The EBVactivated T cells are both anergic and apoptotic (24-26). These are precisely the characteristics that would be predicted as a result of superantigen stimulation. An additional line of evidence suggesting the presence of a superantigen was based on recent studies indicatingthatanotherherpesvirus family member, cytomegalovirus (CMV), exhibits superantigen activity. CMV infects antigen-presenting monocytes and preferentially stimulates Vp12+ T cells (27-29). Since superantigens are evolutionarily conservedin certain pathogens, the likelihood was increased that an EBV superantigen might confer a selective advantage for the virus. EXPERIMENTAL OBSERVATIONS A.

In VitroModel System

Evidence that EBV contains a superantigen consists mainly of results obtained from an in vitro model system Prior studies by a num-

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ber of investigators attempting to detect a T-cell receptor (TCR) VPrestricted response in the blood of IM patients yielded inconsistent results. Presumably these endeavors failed because by the time the IM patients developed noticeable clinical symptoms, the disease had already progressed to the lymphoproliferative stage, at which point bystander T-cell activation, resulting from cytokine production and a generally activated immune status, obscured the TCR VP-restricted response. An in vitro model was, therefore, adopted to reproduce the primary steps that occur during IM. To replicate in vitro the early T-cell response to infected B cells, a classic T-cell proliferation assay was established using EBV-transformed B LCL as antigen-presenting cells (APC) and autologous T cells as'responders. A strong T-cell proliferative response could be detected, with a maximal response at 72 hr, only after the LCL were stimulated with phorbol ester to undergo lytic cycle viral replication (Fig. T cells from the peripheral blood of healthy adult volunteers and from human umbilical cord blood proliferated as vigorously as T cells stimulated with the mitogen phytohemagglutinin (PHA). It was subsequently demonstrated that this T-cell activation was dependent upon MHC class I1 presentation and was not MHC-restricted. Preincubation of the LCL with antibodies specific for a nonpolymorphic region on all HLA-DR molecules completely blockedautologous T-cell proliferation (Fig. 4). T-cell proliferation was also demonstrated in response to autologous and allogeneic LCL transformed by a number of different viral isolates of EBV. All viral strains tested induced a strong T-cell stimulation, indicating that the viral superantigen is expressed in common laboratory strains, such as B95-8 and Raji, as well as in wild-type virus derived from the peripheral blood of IM patients. In addition, LCL transformed with recombinant EBV viruses containing up to kb genome deletions also induced T-cell proliferation, indicating that the superantigen was not in the regions deleted in the constructs (Fig. 5) (T. Palkama et al., unpublished observations). The deleted regions comprise most of the viral unique regions designatedU3,U4, and US. Moreover, since the Raji cell line has a small deletion in the EBNA 3C gene, there is an early block in lytic cycle gene expression and consequently in virus production; thus, it is likely that the .superantigen is either a latent cycle gene, or an immediate early or early gene. Arguing against it being a latent cycle gene is the fact that in all cases, T-cell activation could only be demonstrated after treatment

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of the LCL with phorbol ester. EBV is generally maintained in a latent form in LCL, and phorbol ester inducesthe lytic cycle (31). However, it also up-regulates viral transcription. It is, therefore, possible that the gene is expressed only at a very low level prior to induction.


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It is also possible that an EBV gene transactivates expression of a cellular gene that is not normally expressed, or expressed only at a very low level prior to infection. From the experiments performed, this possibility cannot be excluded. That EBV is responsible for the T-cell stimulation is certain, since the response is not seen against PMA-treated EBV-negative Burkitt's lymphoma lines; however, after infection of the same cell lines with EBV, a rapid T-cell proliferative response ensues (30). 1. TCR Vpl3-RestrictedResponse

Preferential CD69 Expression A defining characteristic of a superantigen-induced immune response is the activation of specific subsets T cells expressing a particular TCR Vp gene. The human IR2 TR

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genome contains 47 TCR Vp family members; thus approximately 1 in 47 T cells would be expected to respond to a given superantigen. Hence, although superantigens selectively activate T cells, they actually generate a massive T-cell proliferative response, which is further amplified by cytokine produced from the activated cells. The activation of bystander T cells can make it difficult to identify the superantigen activated T cells, since it is likely that at the time maximal T-cell response, the majority of participating T cells are nonspecifically activated and are not VP-restricted. Therefore, to identify the T cells that are directly activated by the EBV-associated superantigen, T-cell activation was assayed at an early time point, before the activated T cells could produce cytokines. To accomplish this,a novel method for detecting a VP-restricted response was developed that measures the appearance of the early activation antigen CD69 on individual Vp subsets 4 hr after stimulation (30). Cytokines are generally not produced until 6 hr after T-cell activation. Also, the absence of proliferation at 4 hr assured that a polyclonal response was detected and eliminated skewing artifacts introduced by maintaining the cells in vitro for long periods. Using the in vitro system already described, autologous T cells were stimulated for 4 hr with EBV LCL induced to undergo lytic cycle replication. CD69 was found to be preferentially expressed on T cells expressing TCRVb13 genes (Fig. 6). As a control, T cells stimulated with the bacterial superantigen StuphyZococcus enterotoxin B (SEB) did not stimulate Vp13 T cells but, as previously documented, stimulated Vp12 T cells. Stimulation of VpZ3 T-cell Hybridomas The preferential expression of CD69 on Vp13 T cells was the first evidence that an EBV-associated superantigen existed. The human Vp13 family containsnine members, including two identical copies of Vp13.2, and 1 pseudogene (Vp13.8) comprising a large portion of the overall T-cell repertoire consisting of 47 Vp genes. The Vp13 MAb used for staining in the CD69 experiments, H131,was originally developed against thehuman Vp13.1 gene expressed in a murine T-cell hybridoma lacking endogenous murine Vp genes (32). To confirm that the EBV-transformed LCL preferentially activate Vp13 T cells, this murine T-cell hybridoma was used as well as twoother T hybridomas expressing Vp13.2 and Vp13.6 (32,33). These cell lines express a chimeric TCR consisting of a human Vp chain in combination with a murine a and CD3 chains. As predicted, the Vp13.1 T-cell hybridoma produced IL-2 in response to stimulation with lytically induced LCL. The Vp13.2 and Vp13.6 T-cell hybridomas also produced IL-2 in response to LCL, although IL-2 production was greatly decreased for the Vp13.6 hybridoma, suggesting

111


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that it does not bind the EBV superantigen as strongly as Vp13.1 or Jurkat cells, which express human Vp8.2, were not stimulated by the EBV LCL, although they produced IL-2 in response to the bacterial superantigen SEE, as previously documented, while the Vp13 hybrids did not. Addition of an antimurine CD28 MAb significantly enhanced IL-2 production by all of the Vp13 T-cell hybri-

13.2 (Fig.

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VP13 genes produce IL-2 in response to B95-8 and IM-l LCL, as assessed by proliferation of the IL-2-dependent cell line HT-2. Vj38.2+ Jurkat cells were not stimulated by EBV LCL, but did respond to SEE, while the V013 T-cell hybrids did not.

domas, supplying the costimulatory signal that is absent between human APC and murine T cells. Taken together, the early activation of a large proportion of nonimmune T cells in the in vitro model system, combined with the fact that the stimulation is HLA-DR dependent, but not restricted, and the specific activation of Vp13 T cells convincingly demonstrates the expression a superantigen in lytically infected EBV-transformedLCL. While the studies described above adequately demonstrate that EBV can activate Vp13 T cells, the possibility exists that it can also stimulate other Vps, which were not tested due to the limitation of antiVp MAb. The ultimate confirmation still remains, however, that cloning the superantigen gene itself. IV. MODEL OF BIOLOGICALSIGNIFICANCE A.

Establishment of PersistentInfection

What are the possible advantages for EBV expressing a superantigen in infected B cells? It is predicted that the superantigen is required for the establishment and/or the maintenance persistent infection.

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Since the EBV latent genes have the capacity to directly induce B-cell proliferation in vitro without T-cell help, superantigen stimulation would appear to be superfluous. However, the site of viral persistence in vivo is a resting B cell that does not express these growth-promot-

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ing latent genes (20). Therefore, it is likely that the role of superantigen-activated T cells is to provide signals necessary for the survival of these persistently infected B cells in vivo, possibly through CD40/CD40L interaction. The T-cell signaling could periodically induce expression of the growth-promoting viral latency genes, result-


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ing in replication of the viral genome during cellular division. This interaction would be a means of replenishing the pool of latently infected B cells, which does not decrease over time, but instead remains remarkably stable for years. Moreover, T-cell regulatory signals might also induce differentiation of the infected B cells, driving some of them to terminally differentiate, reactive the virus, produce more superantigen, and thereby complete the cycle. This model, proposing that superantigen-driven activation of T cells is essential for viral persistence, is depicted in Fig. 8. V.

ROLE OF THE EBV SUPERANTIGEN I N DISEASE

A.

Infectious Mononucleosis

The EBV-specific cytotoxic T-cell response counterbalances the superantigen-driven T-cell response. In EBV-positive adults, CTL specific for the growth-promoting latent cycle genes are known to regulate the expansion of infected B cells. To escape becoming a CTL target, a subset of B cells down-regulates viral transcription, and assumes a resting phenotype, i.e., CD19+, CD23-, and CD80-. These B cells are thought to express EBNA1, the only latency gene that has never been described as a target of CTL (2). Thus, in healthy adults, persistent infection with EBV can be seen as a balance between the superantigen-driven, T-cell-dependent B-cell growth, and the elimination of infected B cells by CTL. The clinical symptoms of IM would arise as a result of the disruption of this balance. During IM there is a rapid activation of both CD4+ and CD8+ T cells early after infection, presumably as a consequence of superantigen stimulation, either directly through specific activation of Vp13+ T cells, or through bystander activation resulting from lymphokine production. A characteristically massive expansion of atypical, nonspecifically activated CD8-t T cells ensues. These cells were shown to be apoptotic and to exhibit nonspecific cytotoxic activity (25,26). The nonspecific activation of apoptotic cells might be a mechanism for EBV to evade a specific immune response. The inappropriate activation would delay the development of EBV-specific CTL, promoting establishment of latent infection. B.

Asymptomatic EBV Infection

Paradoxically, although the vast majority of adults are latently infected with EBV, most people do not develop IM. Infection with EBV during childhood instead usually leads to seroconversion with limited or no clinical symptoms; however, if infection is delayed until adolescence or adulthood, approximately 50% of peopIe develop IM. This

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Infection

Figure 8 The role of the superantigen in persistent infection is to provide T-cell help to latently infected B cells, providing the signals necessary to initiate growth and differentiation. This results in periodic viral replication. EBVspecific CTL work in an antagonistic manner, killing actively dividing, latently infected B cells, and thus selecting for a population of cells that downregulates EBV gene expression. This population becomes the site of viral persistence. During immunosuppression, the memory CTL response is compromised, while the superantigen-driven T cells are unaffected. As a consequence, unregulated B-cell growth can result in tumor formation.

apparent incongruity would be explained by the following scenario. In adolescents exposed for the first time to this pathogen,, the mature T-cell pool is poised to mount a rapid superantigen response, while the development of virus-specific CTL requires priming and is thus only effective after an initial lag period. CTL priming might be further delayed by the superantigen-induced activation of nonspecific T cells. The rapid activation of superantigen-driven T cells and the delayed CTL response would result in the symptoms of IM as described above. Children, on the other hand, would predictably mount a suboptimal T-cell response to the EBV superantigen, as inferred from

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murine studies, indicating an impaired immune response to viral superantigens in immature animals (34). Thus, in children the slower and weaker superantigen response, necessary for establishing persistent infection, would be balanced by the induction ofCTL, which control the spread of infected cells. As both responses develop simultaneously, clinical manifestations are prevented. So does IM result from an atypical immune response? The answer to this is unclear. For the host, although the symptoms of IN are self-limiting, clearly IM is not beneficial; however, from the virus’s perspective, persistent infection is established, and the host survives the immune system attack. It is therefore likely that the induction of IM in a mature immune environment actually favors the establishment of persistent infection. C.

1.

Oncogenesis

Requirement

T

in

Formation

A role for T cells influencing EBV infection was suggested from earlier studies demonstrating that the spontaneous outgrowth of LCL in vitro from EBV-seropositive PBMC is more efficient in the presence of T cells (35). This requirement for T cells was obscured by the seemingly converse finding that EBV-specific CTL, which are also present in culture, must be depleted because they will eliminate the newly generated LCL (36,37). Recently, an in vivo correlate to these experiments was demonstrated in SCID mice transplanted with EBVseropositive human PBMC (38-40). These mice spontaneously developed EBV-associated B-cell lymphomas only when T cells were cotransferred. Transplantation of highly purified B cells never yielded lymphomas, even in the presence of cytokines known to be secreted by activated T cells suggesting that a direct T cell-B cell interaction is requisite. Development of EBV-specific CTL limited the spread of the lymphomas in some of the mice, resembling the block to LCL development seen in the in vitro outgrowth assays. It has recently been demonstrated that the tumors arising in SCID mice are a mixture of terminally differentiated B cells, which replicate the virus, and proliferating B cells, which are latently infected (39,41). It is likely that the nondividing differentiated cells provide an essential growth component for the tumor, namely superantigen activation of T cells. These T cells would push the latently infected B cells to continuously proliferate, while simultaneously allowing some fraction to terminally differentiate, leading to growth arrest and production of virus, as well as more superantigen. Thus, tumor growth may represent a type of equilibrium between prolif-

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eration and differentiation. Long-term growth selection, however, would lead to the outgrowth of cells that have mutated away from the requirement for the superantigen, resulting in tumors no longer containing differentiated cells. This is precisely the characteristic of tumors that arise in SCID mice after long periods (41). We propose, therefore, that the SCID/hu mouse model supports an essential role of the viral superantigen in EBV-induced oncogenesis.

Immunosuppression EBV reactivation inimmunosuppressed AIDS patients and transplant recipients sometimes leads to lymphomas and other lymphoproliferative disorders. It is well known that virus-specific CTL control the level of EBV-infected B cells, ordinarily preventing the development of EBV-associated tumors During times of immunosuppression,the memory CTL compartmentis compromised to a greater extent than the naNe T-cell compartment, which can participate in a superantigen response. Therefore, the balance that exists in a healthy individual between viral-specific memory CTL and superantigen-reactive T cells is shifted in favor of the k t ter in an immunosuppressed patient. The superantigen-driven T cells would perpetuate division of B cells expressing the latent genes. The unregulated B-cell growth would result in the subsequent accumulation of mutations in cellular genes such as myc and bcl-2, allowing for the development of lymphomas. Consequently, it is not so surprising that Burkitt’s lymphomas arise in HIV-l-infected patients at a stage when a significant number of CD4+ T cells are still present (42). The deficiency in virus-specific CTL, combined with the T-cell help provided by superantigen expression,.would favor this form of EBVassociated oncogenesis. An analogous murine disease model has been described in detail for the development of reticular cell sarcoma in SJL mice (43,441. This B-cell lymphoma expresses elevated levels of an endogenous MMTV superantigen, causing T-cell stimulation, which, in turn, drives the proliferation of the newly transformed B cells, promoting tumor formation. This phenomenon was termed ’’reverse immunesurveillance,” because the T cells encourage tumor growth. The identification of EBV superantigen activity could be important from a clinical statldpoint, as it provides a basis for the formulation of specific immunotherapies for the treatment and prevention of lymphoproliferative disease. Treatments designed to prevent activation of Vp13 T cells would in theory block tumor formation during immunosuppression. It is possible that other T-cell subsets may be stimulated by the EBV-associated superantigen, but due to the lack

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of specific MAbs, these subsets are as yet unidentified. For an immunotherapy to be successful, all superantigen-reactive TCR Vp subsets would need be inhibited. Alternatively, once the viral gene is identified, therapies could be designed to block expression of the superantigen or prevent its association with HLA-DR. Rule in HZV Recently, it has been suggested that the presence of a superantigen in another herpesvirus, cytomegalovirus (CMV), causes enhanced HIV-l replication in Vp12 T cells, which function as a reservoir for HIV-lin infected patients (28). It is plausible that EBV might play a similar role as CMV in AIDS, particularly since EBV infection is more prevalent than CMV, and it has been reported that perturbations in the VD13 compartment occur in patients with HIV infection In addition, a recent report describes increased HIV1 replication as a consequence of EBV lymphoma development, in SCID mice transplanted with EBV-seropositive PBMC from HIV-l-infected patients (46). Although it .is likely that HIV-1 derives an advantage from the immune activation resulting from the herpesviruses, there is potentially a more important clinical consequence of EBV reactivation in HIV infection. We would predict that some of the anergy and apoptosis of T cells seen in patients infected with HIV might actually be due to activation-induced cell death, resulting as an indirect consequence of EBV-associated superantigen stimulation. It is our opinion that the atypical, apoptotic CD8+ cells, which are massively expanded during IM (25,26), are also augmented in HIV infection In both diseases, elimination of CD4+ T cells is seen, possibly due in part to the nonspecific cytotoxic activity of these activated CD8+ T cells. It is conceivable that these cells arise during HIV infection as a result of EBV reactivation by HIV-1 Tat leading to superantigen expression. In support of our postulate, a correlation was recently .found between T-cell apoptosis in HIV-l-infected children and an increased burden of EBV (56). Role in Autoimmunity

Superantigens have long been linked to the concept of autoimmunity. Since superantigens simultaneously activate many T-cell clones, the possibility is increased after superantigen stimulation for activating autoreactive clones. Furthermore, the activated immune environment with increased cytokine production could be capable of sustaining autoreactive T cells once they are activated. Likewise,it has long been postulated that viruses can induce autoimmunity. As EBV is a ubiq-

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uitous virus, there have been numerous remarks in the literature implicating EBV in the induction or aggravation of almost every known autoimmune disease. Of particular note are reports describing elevated levels of EBV in patients with Sjtigren’s syndrome (5762). Furthermore, there are a number of clinical case reports describing a significant increasein Vp13 T cells in the lesions of patients with this autoimmune disease (63-65). Taken together, these reports can be interpreted as indicative of the possible action of an EBV-associated superantigen in Sjtigren’s syndrome. Moreover, interfering with activation Vp13 T cells might help to ameliorate some the symptoms of the disease. VI. CONCLUDING REMARKS

The identification superantigen activity associated with EBV imposes a new way of thinking about this oncogenic herpesvirus. We have proposed a model suggesting an essential role for the superantigen response in the establishment of persistent infection. It our opinion that the superantigen-driven T-cell response perpetuates viral latency, and in a healthy individual, this response is counterbalanced by EBV-specific CTL, whichlimit the growth of infected B cells. During periods of immunosuppression, this balance is upset. The memory CTL response is compromised to a greater extent than the superantigen-driven T-cell response, leading to unregulated proliferation of infected B cells and eventual tumor formation. This novelway of viewing the virus provokes us to reevaluate existingtreatments for EBV-associated diseases, in particular lymphoproliferative disorders, and possibly autoimmunity. We suggest that interfering with the superantigen-induced T-cell response might have therapeutic benefit in EBV-associated oncogenesis. ACKNOWLEDGMENTS

The research work described in this review was supported by grants from the NIH, R 0 1 AL14910,5T32AR07570-04, and from Human Frontiers, RG-544/95M. REFERENCES 1. Nonoyama M, Pagano JS. Homology between Epstein-Barr virus DNA

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18. Middleton T, Gahn TA, Martin JM, Sugden B. Immortalizing genes of Epstein-Barr virus. Adv Virus Res 1991; 40:19-55. 19. Kieff E, Izumi K,Kaye K, Longnecker R, Mannick J, Miller C, Robertson E, Swaminathan S, Tomkinson B, Tong X, Yalamanchili R. Specifically mutated Epstein-Barr virus recombinants: defining the minimal genome for primary B lymphocyte transformation. In: Minson A, Neil J, McCare, M eds. Viruses and Cancer (Symposia for the Society for General Microbiology). Cambridge: Cambridge University Press, 1994: 123-147. 20. Miyashita EM,Yang B, LamKM, Crawford DH, Thorley-Lawson DA. A novel formof Epstein-Barr virus latency in normal B cells in vivo. Cell 1995;80:593-601. 21. Miyashita E, Thorley-Lawson DA. A new form of Epstein-Barr virus latency in vivo. Curr Topics Microbiol Immunol 1995; 194:135-44. 22. Khanna R, Burrows SR, Kurilla MG, Jacob CA, Misko Sculley TB, Kieff E, Moss DJ. Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development. J Exp Med 1992; 176:169-176. 23. Khanna R, Burrows SR,Moss DJ. Immune regulation in Epstein-Barr virus associated diseases. Microbiol Rev 1995; 59:387-405. 24. Bishop CJ, Moss DJ, Ryan JM, Burrows SR. T lymphocytes in infectious mononucleosis. I1 Response in vitro to interleukin-2 and establishment of T cell lines. Clin Exp Immunol 1985; 60:70-77. 25. Moss DJ, Burrows SR, Baxter JD, Lavin MF. T cell-T cell killing is induced by specific epitopes: evidence for an apoptotic mechanism. J Exp Med 1991;173:681-686. 26. Uehara T, Miyawaki T, Ohta K, Tamaru Y, YokoiT, Nakamura S, Taniguchi N. Apoptotic cell death of primed CD45RO+ T lymphocytes in Epstein Barr virus-induced infectious mononucleosis. Blood 1993; 80:452-458. 27. Laurence J, Hodtsev AS, Posnett DN. Superantigen implicated in dependence of HN-1replication in T cells on TCR Vp expression. Nature 1992; 358:255-259. 28. Dobrescu D, Ursea B, Pope M, Asch AS, Posnett DN. Enhanced HIV-l replication in Vp12 T cells due to human cytomegalovirus in monocytes: evidence for a putative herpesvirus superantigen. Cell 1995; 82:753-763. 29. Dobrescu D, Kabak S, Mehta K, SuhCH, Asch A,Cameron PU, Hodtsev AS, Posnett DN. Human immunodeficiency virus 1 reservoir in CD4+ T cells is restricted to certain Vp subsets. Proc Natl Acad Sci USA 1995; 92:5563-556~7. 30. Sutkowski N, Palkama T, Ciurli C, Sekaly R-P, Thorley-Lawson DA, Huber BT. An Epstein Barr virus-associated superantigen. J Exp Med (in press) 1996. 31. zur Hausen H, ONeill FJ, Freese UK. Persisting oncogenic herpesvirus induced by the tumour.promoter TPA. Nature 1978; 272:373-375.

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45. Rebai N, Pantaleo G, Demarest JF, Ciurli C, Soudeyns H, Adelsberger JW, Vaccarezza M, Walker RE, Sekaly RP, Fauci AS. Analysis of the Tcell receptor beta-chain variable-region (VD) repertoire in monozygotic twins discordant for human immunodeficiency virus: evidence for perturbations of specific VP segments in CD4+ T cells of the virus-positive twins. Proc Natl Acad Sci USA 1994; 91:1529-1533. 46. Van Kuyk R, Mosier DE. Lack of pseudotype formation between human immunodeficiency virus type 1 and Epstein-Barr virus in productively coinfected B lymphoblastoid cell lines. Virology 1995; 209:643-648. 47. Prince HE, Jensen ER. Three-color cytofluorometric analysis of CD8 cell subsets in HIV-l infection. J AIDS 1991; 4:1227-1232. 48. Levacher M, Hulstaert F, Tallet S, Ullery S, Pocidalo JJ, Bach BA. The significance of activation markers on CD8 lymphocytes in human immunodeficiency syndrome: staging and prognostic value. Clin Exp Immunol 1992;90:376-382. 49. Ho HN, Hultin LE, Mitsuyasu RT, Matud JL, Hausner MA, Bockstoce D, Chou CC, O’Rourke S, Taylor JM, Giorgi JV. Circulating HIV-specific CD8+ cytotoxic T cells express CD38 and HLA-DR antigens. J Immunol 1993;150:3070-3079. 50. Janossy G, Borthwick N, Lomnitzer R, Medina E, Squire SB, Phillips AN, Lipman M, Johnson MA, Lee C, Bofill M. Lymphocyte activation in HIV-1 infection. I. Predominantproliferative defects among CD45RO+ cells of the CD4 and CD8 lineages. AIDS 1993; 7:613-624. 51. Saukkonen JJ, Kornfeld H, Berman JS. Expansion of a CD8+CD28- cell population in theblood and lung of HIV-positive patients. J AIDS 1993; 6:1194-204. 52. Giorgi JV, Ho HN, Hirji K, Chou CC, Hultin LE, ORourke S, Park L, Margolick JB, Ferbas J, Phair JP. CD8+ lymphocyte activation at human immunodeficiency virus type 1 seroconversion: development of HLADR+ CD38- CD8+ cells is associated with subsequent stable CD4+ cell levels. The Multicenter AIDS Cohort Study Group. J Infect Dis 1994; 170:775-781. 53. Lewis DE, Tang DS, Adu OA, Schober W, Rodgers JR. Anergy and apoptosis in CD8+ T cells from HIV-infected persons. Immunoll994; 153:412-420. 54. Vingerhoets JH, Vanham GL, Kestens LL, Penne GG, Colebunders RL, Vandenbruaene MJ, Goeman J, Gigase PL, De BM, Ceuppens JL.. Increased cytolytic T lymphocyte activity and decreased B7 responsiveness are associated with CD28 down-regulation on CD8+ T cells from HIVinfected subjects. Clin Exp Immunol 1995; 100:425-433. 55. Astrin SM, Laurence J. Human immunodeficiency virus activates c-myc and Epstein-Barr virus in human B lymphocytes. Ann NY Acad Sci 1992; 651:422-432. 56. Lauener RP, Huttner S, Buisson M, Hossle JP, Albisetti M, Seigneurin JM, Seger RA, Nadal D. T cell death by apoptosis in vertically human immunodeficiency virus-infected children coincides with expansion of

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Toxic

Syndrome Toxin-l

Cathleen A. Earhart, David T. Mitchell, Debra L. Murray, Patrick M. Schlievert, and Douglas Ohlendorf

of

INTRODUCTION

Toxic shock syndrome toxin-l (TSST-l), the major cause of staphylococcal toxic shock syndrome (TSS), is a member of a large family of toxins knownas pyrogenic toxin superantigens (PTSAgs) (1,2). PTSAgs are small, nonglycosylated proteins, with molecular weights ranging from 22,000 to secreted by Staphylococcus aureus, group A streptococci, and certain non-group A streptococci. Wellcharacterized members of this family include streptococcal pyrogenic exotoxins (SPEA, SPEB,SPEC, and SPEF) and streptococcal superantigen (SSA) from Streptococcus pyogenes, TSST-1 from aureus, and the staphylococcal enterotoxins (SEA, SEB, SEC,, SED, SEE, SEG and SEH). PTSAgs are produced with an amino-terminal signal peptide sequence that is removed during toxin secretion. They are generally highly resistant to proteases and stable over a range of pH 2.5-11 and to temperatures of 60°C or higher For example, TSST-1 stored for years at room temperature shows no loss of biological activity and 127

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still produces large-diffraction-quality crystalsafter being subjectedto repeated freezing, thawing, lyophilization, and incubation at 100°C. The genes for most of the staphylococcal and group A streptococcal PTSAgs have been cloned and sequenced. The levelof sequence homology between PTSAgs varies widely, and they may be divided into groups based on primary sequence similarities (Fig. 1). The group showing the highest level of sequence homology, between and 81%, consists of SEA, SED, and SEE. A second group, composed of SEB, the SECs, SPEA and SSA, have homologies between and 66%. In addition, SPEC is homologous to SPEA but has no statistically significant homologywith the other members of this group. On the other hand, SEH is homologous with SEB, 27% homologous with the SECs, and homologous with SEA, SED, and SEE, while showing no homology to SPEA TSST-1, SPEB, and SPEF show no primary sequence similarity to any other toxin or each other. The three-dimensional structures of several PTSAgs have been solved. These include SEA SEB (71, SEC2, SEC3 (lo), and TSST-1 (11,12). All of the PTSAgs have similar structures despite a low level of sequence homology. However, there are significant differences between the structure of TSST-l and the enterotoxins, with TSST-1 having the prototypical structure. II. GENETICS AND BIOPHYSICAL PROPERTIES OF TSST-1

TSST-l was the first toxin shown to be associated with TSS and today is considered to be the cause of all or nearly all menstrual TSS and of nonmenstrual cases The toxin was identified and purified by Schlievert etal. and Bergdoll and colleagues The toxin has a molecular weightof approximately 22,000 and an isoelectric point of 7.2 with two interconvertible forms identified (16). A.

Genetics of TSST-1

Despite the difference in isoelectric point between the two forms TSST-1, both forms arise from a single gene. Coagulase-negative staphylococci do not make TSST-l and lack the gene for the toxin. occurs in two major forms, fstH, where H refers to human isolate of S. aureus and the gene encodes TSST-l, and where 0 refers to ovine isolate of S. aureus and the gene encodes TSST-0 (17,lS). occurs as a variable trait within a large heterologous DNA insert in the chromosome. Production of TSST-1 as well as many other secreted S. aureus virulence factors is under the control of at least three glo-

4 El

e

El

8 4 El

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bal regulatory systems, designated as the accessory gene regulator (ugr) (191, staphylococcal accessory regulator (sur) (20), and extracellular protein regulator (xpr) (21). ugr is a locus of at least five genes that positively regulate extracellular virulence factors but negatively regulate cell-associated virulence factors (22). TSST-1 is tightly regulated by ugr in that the toxin is made in large amounts in ugr' strains but only minimally made in ugr- mutants. SEB and and exfoliative toxins are less tightly regulated by ugr with SEA production independent of ugr in some strains. Interestingly, SPEA when cloned into S. uureus is partially under ugr control. SPEA shares nearly 50% sequence similarity with SEB and SEC and has been hypothesized to have been transferred to group A streptococci by bacteriophagefrom S. uureus. The positive regulatory activityof ugr appears to depend on a two-component prokaryotic regulatory system, requiring the genes ugrA as a transcription activator and ugrB as the signaling component. ugr also yields a 514-nucleotide transcript designated RNAIII, which functions both to initiate transcription of virulence factors and to encode delta hemolysin. xpr and sur also regulate virulence factor production positively at the level of mRNA. Furthermore, both of these loci regulate the levels of RNAIII. B.

Protein Chemistry

TSST-l and TSST-0 are both translated as proteins consisting of 234 amino acids in which the first 40 residues comprise signal peptides (17,181. Like many other S. uureus exotoxins, TSST-1 and TSST-0 are made primarily during the postexponential phase of growth. These two proteins differ from each other by seven amino acids at positions 19, 55, 57, 69, 132, and 140. The consequence of these differences is that TSST-l is biologically active whereas TSST-0 lacks significant biological activity. There are no cysteines in either protein, and both contain a high percentage of hydrophobic amino acids. TSST-l is easily obtained from S. uureus culture fluids by several methods (1). We use ethanol precipitation of toxin from cultures of high toxin producers, such as MN8 and FRI1169, followed by resolubilization in water and preparative thin-layer isoelectric focusing in pH gradients of3-10 and 6-8 (16). TSST-1 thus purified is homogeneous when tested by SDS-PAGE and protein sequencing. The toxin is made in complex media containing animal protein, at pHs of 6.58, at temperatures 37-40°C, and under aerobic but not anaerobic conditions (23). Glucose functions a catabolite to repress TSST-l production.

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TSST-0 is produced comparably, but it is our experience that strains that make TSST-0 also produce SEC. Both proteins have PIS between pH 8.0 and 9.0 and thus only partial separation is achieved by isoelectric focusing. Final purification of TSST-0 is achieved by reverse-phase HPLC. C. Structure Analysis

We have crystallized TSST-l in four crystal foTms (see Table 1) that diffract to resolutions ranging from 1.9 to 4.2 A (24). A fifth crystal form requiring Zn2+has been grown by W. Cook,-.University of Alabama (personal communication), that diffracts to a resolution of 2.8 A. In addition, several mutants of TSST-1, including a tetramutant (T691, Y80W,E132K,I140T) (see Table l), have been crystallized and their structures are currently under refinement. Overall these mutations and the various native forms have shown no large-scale structural differences. Accordingly, we can limit our attention to the form, which is the most highly ordered native structure. The structure of the form was first reported by our group in 1993 (11) and by Acharya et al. in 1994 (12). Working independently, both groups reported structures refined to 2.5A resolution with R factors of 0.226 (F > 20) and 0.213 > 30), respectively. We have subsequently refined the form to an R factor of0.154 ( F > lo) to 2.05 A resolution. The crystal form has three TSST-l molecules in the asymmetric unit (unique portion of the crystal unit cell). All 582 residues of these three TSST-1 molecules are present in the refined model, as are 405 solvent molecules. The three TSST-1 molecules have essentially identical structuresowith a root mean square agreement between molecules of0.3-0.4A using Ca’s and 11.1A using all atoms. Since this similarity is near the level of experimental error, no distinction between molecules will be made in the subsequent discussion. Ribbon drawings of the structural elements ofTSST-1 are presented in Figure 1. TSST-1 is a kidney-shaped molecule, divided into two dom$ns, with the approximate overall dimensions of 55 A X 40 A 35 A. Domain A, the larger of the two domains, is composed of residues 1-15 and 89-194. The main features of domain A (Table 2) are the amino-terminal a helix A composed of residues 4-15 and positioned over the carboxyl end of a helix a 17-residue a helix. a Helix rests against a five-stranded p sheet with mixed topology. p strands 10, 11, and 13 are adjacent and antiparallel, as are p strands 7 and 8, while p strands 7 and 10 are adjacent and parallel. The p

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Table 2 TheSecondary Structure Elements of TSST-l Secondary Residues structures Helix A p strand 1 strand p strand p strand p strand strand

p strand p strand strand Helix B p strand 10 strand 11 Helix C strand p strand

sheet and a helix a motif known as the grasp (25). This motif is present in a number of proteins including ferredoxin, ubiquitin, and the immunoglobulin-binding domain of streptococcal protein G All these proteins interact with other proteins as a key part of their function suggesting that the p-grasp motif may play a role in protein-protein interactions. Domain (residues 18-89) consists a five-stranded mixed p barrel. With the exception strands 3 and 5, which are parallel, all of the strands are antiparallel to their neighbors. This structural motif, known as an fold (27), is present in all known PTSAg structures, and several other proteins including staphylococcal nuclease (28), verotoxin-l in the anticodon-binding domain aspartyl-tRNA synthetase in the major cold-shock protein from Bacillus subtilis in the active domain tissue inhibitor metalloproteinase? (31), and in the B subunits of heat-labile enterotoxin (32). All of these proteins bind oligosaccharide or oligonucleotides and it has been suggested that the OB fold plays a role in this binding (27).

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Key features of the TSST-l structure are a small groove on the front surface and a large groove onthe back surface of domain A. The small groove is bounded by a helix A and its subsequent loop, domain B, and a front flap composed of a helix C with its preceding loop (see Fig. 2A). While it may appear from Fig. 2 that helix B is accessible to solvent in the front groove, it is actually buried by the side chains of neighboring residues. The large groovei s bounded by helix A with its loop and a rear flap composed of p strand 9 with its preceding loop running down the middle of the back surface of the TSST-l molecule (see Fig. 1B). The rear flap is approximately parallel to the axis of helix B and covers most of the bottom of helix B to the center of the helical axis. The residues on the backside of helix B that are at least 20% accessible to solvent in this groove are E132, H135, Q139, and 1140. The residues in the interface between domain A and B are noteworthy in being as hydrophilic as the rest of the molecular surface. Most intermolecular interfaces are either neutral or hydrophobic. In

T cell

presenting cell Figure 2 Schematic representation

PTSAg (SAg) stimulation T-cell superantigenicity as a result toxin interaction with the a chain class I1 MHC molecules on antigen-presenting cells and the variable part the p chain the a:p T-cell receptor.


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TSST-1 this interface has several buried charged residues. Buried charged residues are usually paired with opposing charged residues forming salt bridges sinceburying a charged residue without a counterion can be very destabilizing (33). There are two examples of this in the domain A/domain B interface. Asp 18 is paired with Arg 68 and Asp 63 is paired with Lys 121 and Lys91. However, two other residues are >95% buried with no formal counterion. One is Lys 67 where only the very tip of the side chain sticks out, forming a hydrogen bond with a water molecule. The other residue is Glu 177, which is buried near the amino-terminus of helix B and uses the helix dipole to satisfy the requirement for a counterion. Owing to the hydrophilic nature of the domain A/domain B interface, the separation of the two domains may occur as a breathing mode of TSST-1 in solution. Evidence to support this idea comes from Edwin and Kass (34), who found that digestion with papain produces a 12-kDa fragment corresponding to residues 88-194. Since residues86-89 are completely buried within the interface, some form of breathing must occur to allow papain access to the site. A comparison of TSST-1 with other PTSAgs whose structures are known can provide insight into the functionally significant structural features of the superantigens. Despitelimited sequence homology, the overall fold of TSST-1 is very similar to that of SEA, SEB, SEC2, and SEC3. However, close analysis of the structures reveals four significant differences between the TSST-1 structure and the structures of the enterotoxins: The amino terminus ofTSST-l is shorter than those found in the other PTSAgs. example, the amino terminus of SEA,SEB,SEC2, and SEC3 all have about 20 amino acids preceding the amino-terminal a helix. These residues form a short additional a helix and loop that extends down the outside of the five-strand sheet of domain A. In SEA, these residues participate in the formation of a zinc-binding site important for binding to class I1 MHC molecules (6). 2.TSST-1 has no cysteine residues and has a regular duplex at the top of domain B. SEA, SEB, SEC2, and SEC3 all have a motif containing a disulfide bridge referred to as the "disulfide loop" in this region. The disulfide loop has been proposed to play a role in TCR binding (as will be discussed later) and in the emetic activity PTSAgs. The absence of the disulfide loop in TSST-l has been suggested to explain the lack of emetic activity in TSST-1 (35). Mutational stud-

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ies of SEC1 by Hovde et al. have shown that the disulfide bond itself is not a requirement for emesis. Mutations substituting serine for cysteine were emeticbut substitutions of alanine were not. The investigators concluded that the conformation of residues within adjacent to the loop was important for emetic activity and that this conformation was maintained by the substitution of cysteine with residues that hydrogen-bond. Analysis of the known enterotoxin structures reveals that the conformation of the disulfide loop is different in each. These differences, however, may be the result of crystal packing forces. The disulfide loop could adopt a consensus conformation in solution. Another important difference between TSST-1 and the other PTSAgs whose structures are known is the size of the flaps on the front and back of the toxin that cover the central a helix, a helix B in TSST-1. The thin layer of side chains covering the front of a helix in TSST-1 results in a pronounced indentation in the surface of the toxin. In SEB and the other enterotoxins, the carbon backbone of the front flap covers the entire a helix extending nearly to helix A. The enterotoxins allhave a much larger flap extending to the top of the molecule and producing a significant bulge onthe front surface. The front flap is purported to play a role in the binding to the TCR. On the back of TSST-l, a flap covers the quadrant of helix B below the helical axis and to the rear of . the molecule.Theenterotoxins have a much smaller rear flap exposing a larger portion of the back of the helix B (helix 4 in terms). 4. The connector between p strands and 4 in TSST-l forms an extended loop at the bottom of domain B. This loop is replaced in SEA,SEB,SEC2, and SEC3 with a 10-residue a helix. The three prolines present in the TSST-l sequence may prevent the formation of an a helix by this segment. The absence of this a helix in TSST-l allows p strands 1 and 2 to wrap smoothly around the p barrel. In known enterotoxin . structures, the amino-terminal end of p strand 2 curls away from the barrel to avoid steric clashes with the helix. BIOLOGICAL PROPERTIES O F TSST-1

TSST-1 shares numerous biological properties with other PTSAgs, including pyrogenicity, ability to enhance lethal endotoxin shock,


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superantigenicity, and ability to cause capillary leak through direct interaction with endothelial cells (1). In addition, TSST-1 has been shown to synergize with endotoxin to kill renal tubular cells Each of these properties will be discussed below. Pyrogenicity

TSST-1 and all other PTSAgs induce fever in experimental animals that is characterized by a fairly linear rise in body temperature with a peak hr after injection TSST-1 may be administered intravenously, intramuscularly, subcutaneously, intradermally, and intracisternally (into the cerebrospinal fluid) without affecting the shape of the fever curve. The minimum pyrogenic dose of TSST-1 by all routes except intracisternal is 0.15 &kg; intracisternal injections require 1000-fold less toxin consistent with the fever response control center being the hypothalamus. Interestingly, staphylococcal enterotoxins and SPEA are much less able to cause fever when given intradermally, possibly asa consequence these toxins being more charged molecules and thus lacking mobility through the skin. The mechanism of fever production byTSST-1 and other PTSAgs probably occurs as a result of toxins' release of interleukin-l and tumor necrosis factor-a (TNF-a) from macrophages, whether peripheral within the central nervous system, and through direct effects on the hypothalamus Subsequently, the hypothalamus is stimulated resulting in elevated levels of prostaglandin E,, alteration of the ratio of norepinephrine and serotonin, and stimulation of a-adrenergenic nerve receptors As expected, agents such as aspirin and indomethacin, which block protaglandins, interfere with fever production. It is interesting that the shape PTSAg fever curves differ from that induced by endotoxin, another well-known pyrogen. As indicated above, PTSAgs cause fever characterized by a steady rise with a peak 4 hr after injection. In contrast, endotoxin causes fever characterized by peaks at both 1and hr postinjection. The mechanism behind this difference is unclear, though the pathways of fever production are probably similar. TSST-0 is nonpyrogenic when injected into rabbits (18). The protein has not been evaluatedin sheep for this activity, but the toxin is nearly always associated with S. aureus strains that cause mastitis in sheep.

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B.

Enhancement of Susceptibility to Endotoxin Shock

The ability of TSST-l and other PTSAgs to enhance susceptibility to the lethal effects of endotoxin'appears to result from the PTSAgs' ability to block liver clearance function (38,39). Thus, in rabbits the LD,of endotoxin alone is approximately 500 pg/kg. When animals are preteated with 50 pg/kg TSST-l, the LD,of endotoxin drops to 0.01 pg/kg, a 50,000-fold enhancement. The LD,ofTSST-1 alone in rabbits, given a bolus intravenous injection, is greater than 1mg/kg. The enhancement phenomenon is best described mathematically by the following: The log PTSAg pretreatment graphed versus the log LD,of endotoxin gives a straight line with a slope of -1. Thus far, this has been shown experimentally for both TSST-1 and SPEA, but since all PTSAgs enhance endotoxin shock,it is likely the mathematical relationship will hold for all members of the family. The mechanism underlying the enhancement phenomenon is currently under investigation. It is clearthat TSST-1 and other PTSAgs must be given to animals1-2 hr prior to administration of endotoxin, unless high doses of PTSAg are injected (38,39,44). Also, the ability of the liver to clear colloidal carbonor endotoxin from the circulation is significantly reduced by such PTSAg pretreatment. Finally, streptococcal pyrogenic exotoxinshave been shown to interfere with liver RNA synthesis in animals treated with the exotoxins, in isolated liver hepatocytes (containing both Kupffer cells and parenchymal cells), and in isolated liver nuclei (45). All RNA types appeared to be affected. Whether this inhibition of RNA synthesis is a direct or indirect effect of the exotoxins is unclear. Many other agents also simplify the lethal effects of endotoxin, including CCl,, lead, cycloheximide, hepatitis virus, and a amanitin (46). Of these agents, a amanitin requires pretreatment of animals 12 hr prior to administration of endotoxin similar to PTSAg. a Amanitin has been shown to bind and inactivate mRNA polymerase as its major mechanism of action. Our attempts to show binding of streptococcal pyrogenic exotoxins to polymerase have been unsuccessful. Although the role of endotoxin enhancement in human TSS is not clear, the liver autopsy findings together with acute TSS sera containing endotoxinare consistent with a role (47,48). Autopsy studies of staphylococcal TSS patients reveal three fairly distinctive findings: 1) triaditis and fatty replacement of the liver seen also in patients with endotoxin shock, 2) sloughing of mucous membranes, and erythrophagocytosis indicative of significant macrophage activation.


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The reduced clearance function of the liver as seen in rabbits treated with TSST-1 or other PTSAg would mean that endotoxin derived from the gut or other sources could “spill over” into the circulation, bind lipopolysaccharide-bindingprotein, and cause release of TNF-a from macrophages and capillary leak. Currently, the most widely accepted model for the study of TSS is to administer approximately TSST-1 in subcutaneously implanted miniosmotic pumps in rabbits (49). This provides rabbits with continual exposure to toxin and, as opposed to bolus injections, renders the toxin highly lethal. In this model, it is our experience that rabbits succumb to TSST-1 in two major groups, one group succumbing on day 1-2 and another on day 4-5 postimplantation. We hypothesize that the animals that die on day 1-2 succumb as a result of endotoxin enhancement, whereas those that die on days 4-5 die as a result of superantigenicity. One other animal model has recently become popular for the study of staphylococcal TSS, i.e., mice, where it is easier to study immune effects of TSST-1 and other PTSAgs. However, mice are highly resistant to development ofTSS despite being highly susceptible to superantigenicity (1,141. To overcome this resistance of mice, investigators administer D-galactosamine first to the animals to interfere with liver clearance function prior to administration of PTSAg (50). These studies also point to the significant role of interference with normal liver clearance mechanisms as central to induction ofTSS symptoms. TSST-1 also enhances the lethality of endotoxin in vitro for renal tubular cells (37).This effect depends on TSST-1 interaction with cell receptors, endocytosis, and generation of toxic oxygen radicals. Whether or not this effect is specific for TSST-l or shared by all PTSAgs is unclear. C.

Superantigenicity

Like other PTSAgs, TSST-l induces a dramatic T-cell proliferation, which is now referred to as superantigenicity. The mitogenic activity ofTSST-l was first shown by Schlievert et al. in 1981 (14). Several other investigators confirmed this finding and showed the T-cell proliferative activity results in massive release of cytokines, including those from T cells and macrophages (51-53). In the late Marrack and Kappler (54) showed that TSST-1 induced T-cell proliferation by a novel mechanism, which they referred to as superantigenicity. TSST-1 and other PTSAgs induced T-cell proliferation depen-

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dent on interaction of toxin with invariant regions on class I1 major histocompatibility complex (MHC) molecules and the toxin:MHC complex interaction with T-cell receptor (TCR) to cause cytokine release. A schematic representation ofPTSAg interaction with antigen-presenting cells and T cells that results in superantigenicity is shown in Fig. 2. T-cell stimulation is dependent primarily on the composition of the variable part of the chain of the TCR and without regard to the antigenic peptide specificity of the T cell. In this way 5-50%of all T cells can be stimulated by a PTSAg as opposed to stimulated by a peptide presented through typical antigen presentation. Interestingly, each PTSAg has its own unique profile of T cells stimulated. TSST-1 stimulates human VP 2+T cells. In patients with acute TSS, VP 2+ T cells may account for 60-70% of all circulating T cells (55). Recently, the three-dimensional structure of TSST-l complexed to HLA-DR1 has been solved (56). In agreement with other lines of evidence, including mutational analysis, which will be discussed later, TSST-1 residues within the P-barrel structure of domain B bind primarily to the a chain with few interactions with the chain of HLADR1. The specificinteractions of TSST-l with HLA-DR1 are discussed in detail elsewhere in this volume. The massive release of cytokines by both T cells and macrophages induced by TSST-1 and other PTSAgs contributes significantly to capillary leak, rash, and failure to make neutralizing antibody responses in TSS patients. D. Effects on EndothelialCells

TSST-1 has been shown to bind specifically to both human (57) and porcine (58) endothelial cells. Furthermore, the toxin is cytotoxic to porcine endothelial cells at high toxin concentrations but causes the cells to contract without lethality at low toxin concentrations. There are 104-l@ receptors/cell on endothelial cells. The toxic effect of TSST1 on these cells depends on receptor-mediated endocytosis and generation of oxygen radicals. It is possible this direct effect TSST-l contributes to the capillary leak seen in TSS. IV. MUTATIONAL ANALYSIS

A large number of mutations of TSST-1 have been made and provide detailed information on the functional role of various residues of the toxin (59-65). Analysis of these mutations coupled with knowledge


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of the structure can delineate functionally important regions of TSST1. The majority of these mutations affect T-cell superantigenicity and can be divided into two classes. In one class are those mutations that display altered binding to class I1 MHC molecules. In the second class are those mutations that show wild-type binding to class I1 MHC molecules and thus are inferred to alter binding t o the TCR.For TSST-1, these mutations cluster into separate regions (see Fig. 1). Mutations affecting binding to the class I1 MHC molecule are grouped on the p barrel of domain B. This binding site has been confirmed by the solution of the TSST-kclass I1 MHC complex (56). The mutations located along the back of a helix B near the carboxyl end at the top of domain A in a helix A and its subsequent loop affect binding to the TCR. Mutations have also been described along the back of a helix B that alter either superantigenicity lethality without significantly affecting the other property. Mutations ofSEB are also clustered into two distinct regions. As is the case with TSST-1, mutations affecting binding to MHC class I1 molecules are predominantly in the amino-terminal p-barrel domain (62). These data are consistent with the structural analysis of the SEB:class I1 MHC complex (66). For SEA, there are data (67,68) supporting binding class I1 MHC molecules at the same site. However, there is evidence for a second Zn2+-dependentMHC binding site on the left of domain A involving the p sheet in the p-grasp motif (6). ForSEA,SEB, and SEC, studies (54,69-74) suggest that the TCRbinding site is found on the top of the molecule in a shallow cavity formed by helix 2 (helix A in TSST-l), the loop connecting p strands 2 and p strand 4 and the loop connecting p strand 4 with p strand 5 (the disulfide loop), and helix 5 (helix D in TSST-l). There is no structure in TSST-1 that is homologous to the TCR-binding cavity of the enterotoxins. a Helix C (equivalent to helix 5 in SEB) is shorter and does not extend to the top of the molecule. Thus for SEA, SEB, and SEC the putative TCR-binding site is on the front side of the amino-terminal a helix while for TSST-l it is on the back. V.

CONCLUSION

Despite poor amino acid homology, TSST-1 has the same structural components as found in the enterotoxins. However, as suggested by the low homology, there are significant structural differences between TSST-1 and the enterotoxins focused on regions suggested a critical for biological function. TSST-1 and other PTSAgs have been implicated in a variety of allergic and autoimmune diseases in addition to their

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role in TSS and scarlet fever. For example, studies have suggested TSST-l and SPEC contribute to cases of Kawasaki syndrome SPES have been implicated in guttate psoriasis (77,781. Furthermore, any staphylococcal PTSAg may induce atopic dermatitis (79). Finally, in experimental models TSST-l reactivates arthritis (80) and enterotoxins may induce or reactivate allergic encephalomyelitis It i s clear that only through the analysisof engineered molecules in light of their three-dimensional structures can the functional consequences of these differences be discovered. ACKNOWLEDGMENTS

This work was supported in part by USPHS Grants A122159 and 2T32-HD07381. Melodie Bahan is gratefully acknowledged for preparation of the manuscript. The authors thank the Minnesota Supercomputer Center for providing computational resources. REFERENCES

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39. Schlievert PM. Enhancement of host susceptibility to lethal endotoxin shock by staphylococcal pyrogenic exotoxintype C. Infect Immun 1982; 36:123-128. 40. Schlievert PM, Watson DW. Group A streptococcal pyrogenic exotoxins: pyrogenicity, alteration of blood brain barrier, separation of sites for pyrogenicity and enhancementof lethal endotoxin shock. Infect Immun 1978;21:753-763. 41. Fast DJ, Schlievert PM, Nelson RD. Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production. Infect Immun 1989; 57:291-294. 42. Schlievert PM, Bettin KM, Watson DW. Effect of antipyretics on group A streptococcal pyrogenic exotoxin fever production and ability to enhance lethal endotoxin shock. Proc SOC Exp Biol Med 1977; 157:472-475. 43. Schlievert PM, Watson DW. Biogenic amine involvement in pyrogenicity and enhancement of lethal endotoxin shock by group A streptococcal pyrogenic exotoxin. Proc SOC ExpBiol Med 1979; 162:269-274. 44. Schlievert PM. Alteration of immune function by staphylococcal pyrogenic exotoxin type C: possible role in toxic-shock syndrome. Infect Dis 1983;147:391-398. 45. Schlievert PM, Bettin KM, Watson DW. Inhibition of ribonucleic acid synthesis by group A streptococcal pyrogenic exotoxins. Infect Immun 1979;27:542-548. 46. Fiume L, Stirpe F. Deceased RNA content in mouse liver nuclei after intoxication with a-amanitin. Biochim Biophys Acta 1966; 123:643-645. 47. Larkin SM, Williams DN, Osterholm MT, Tofte RW, Posalaky Z. Toxic shock syndrome: clinical, laboratory, and pathological findings in nine fatal cases. Ann Intern Med 1982; 96:858-864. 48. Stone RL, Schlievert PM. Evidence for the involvement of endotoxin in toxic-shock syndrome. J Infect Dis 1987; 155:682-689. 49. Parsonnet J, Gillis ZA, Richter AG, Pier GB. A rabbit model of toxic shock syndrome that uses a constant, subcutaneous infusion of toxic shock syndrome toxin 1. Infect Immun 1987; 55:1070-1076. 50. Miethke T, Wahl C, Heeg K, Echtenacher B, Krammer PH, Wagner H. T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. J Exp Med1992;175:91-98. 51. Parsonnet J,Gillis ZA. Production of tumor necrosis factor by human monocytes in response to toxic shock syndrome toxin-l. J Infect Dis 1988; 158:1026-1033. 52. Poindexter NJ, Schlievert PM. Toxic-shock syndrome toxin-1 induced proliferation of lymphocytes: comparison of the mitogenic response of human, murine, and rabbit lymphocytes. J Infect Dis 1985; 151:65-72. 53. Fast DJ, Schlievert PM, Nelson RD. Nonpurulent response to toxic shock syndrome toxin-l producing Staphylococcus uureus: relationship to toxin-

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stimulated production of tumor necrosis factor.J Immunoll988; 140:949953. Marrack P, Kappler JW. The staphylococcalenterotoxins and their relatives. Science 1990;248:705-711. Choi Y, Lafferty JA, Clements JR, Todd JK, Gelfand EW, Kappler J, Marrack P, Kotzin BL. Selective expansion of T cells expressing in toxic shock syndrome. J Exp Med 1990; 172:981-984. Kim J,Urban RG, Strominger JL, Wiley DC. Crystallographic structure of toxic shock syndrome toxin-l complexed with a human class 11 major histocompatibility molecule, HLA-DRl Science 1994; 266:1870-1874. Kushnaryov VM, MacDonald HS, Reise RF, Bergdoll MS. Reaction of toxic shock syndrome toxin1with endothelium of human umbilical cord vein. Rev Infect Dis 1989;11:282-288. Lee PK, Vercellotti GM, Deringer JR, Schlievert PM. Effects of staphylococcal toxic shock syndrome toxin 1 on aortic endothelial cells. J Infect Dis 1991;164:711-719. Murray DL, Prasad GS, Earhart CA, Leonard BAB, Kreiswirth BN, NovickRP,OhlendorfDH, Schlievert PM. Immunobiological and biochemical properties of mutants of toxic shock syndrome toxin-l. J Immunol 1994;152:87-95. Murray DL, Earhart CA, Mitchell DT, OhlendorfDH,NovickRP, Schlievert PM. Localization of biologically important regions on toxic shock syndrome toxin-l. Infect Immun 1996;64:371-374. Deresiewicz RL, Wood J, Chan M, Finberg RW, Kasper DL. Mutations affecting the activity of toxic shock syndrome toxin-l. Biochemistry1994; 33:12844-12851. Hurley JM, Shimonkevitz R, Hanagan A, Enney K, Boen E, Kotzin BL, Matsumura M. Identification of class I1 MHC and T cell receptor binding sites in the superantigen toxic shock syndrome toxin-l. J Exp Med 1995;181:2229-2235. Blanco L, Choi EM, Connolly K, Thompson MR, Bonventre PF. Mutants of staphylococcal toxic shock syndrome toxin 1: mitogenicity and recognition by a neutralizing monoclonal antibody. Infect Immun 1990; 58:3020-3028. Bonventre PF, Heeg H, Cullen C, Lian C. Toxicity of recombinant toxic shock syndrome toxin 1 and mutant toxins produced by Staphylococcus aureus in a rabbit infection model of toxic shock syndrome. Infect Immun 1993;61:793-799. Bonventre PF, Heeg H, Edwards CD 111, Cullen CM. A mutation at histidine residue 135 of toxic shock syndrome toxin yields an immunogenic protein with minimal toxicity. Infect Immun 1995; 63:509-515. Jardetsky TS, Brown JH, GorgaJC, Stern LJ, Urban RG, Chi Y-I, Stauffacher CV, Strominger JL, Wiley DC. Three-dimensional structure of a human class I1 histocompatibility molecule complexed with superantigen. Nature 1994;368:711-718.


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67. Harris TO, Grossman D Kappler JW, Marrack P, Rich RR, Betley, MJ. Lack of complete correlation between emetic and T-cell stimulatory activities of staphylococcal enterotoxins. Infect Immun 1993; 61:2059-2068. Abrahamsen L, Dohlsten M, Segren S, Bjork P, Jonsson E, Kallard Characterization of two distinct MHC class I1 binding sites in the superantigen staphylococcal enterotoxin A. EMBO J 1995; 14:2978-2986. 69. Kappler JW, Herman A, Clements J, Marrack P. Mutations defining functional regions of tho superantigen staphylococcal enterotoxin B. J Exp Med 1992; 175:387-396. 70. Grossman D, Van M, Mollick JA, Highlander SK, Rich RR. Mutation of the disulfide loop in staphylococcal eneterotoxin A. J Immunol 1991; 147:3274-3281. 71. Irwin, MJ, Hudson KR, Fraser, JD Gascoigne, NRJ. Enterotoxin residues determining T-cell receptor Vp binding specificity. Nature 1992; 359:841843. 72. Hudson KR, Robinson H, Fraser JD. Two adjacent residues in staphylococcal enterotoxins A and E determine cell receptor Vp specificity. J Exp Med 1993; 177:175-184. 73. Mollick JA, McMasters RL, Grossman D, Rich RR. Localization of a site on bacterial superantigensthatdetermins T-cell receptor P-chain specifity. J Exp Med 1993; 177:283-293. 74. Hoffmann ML, Jablonski LM, Crum KK, Hackett SP, Chi Y-I, Stauffacher CV, Stevens DL, Bohach GA. Predictions of T-cell receptor and major histocompatibility complex-binding sites on staphylococcal enterotoxin Cl. Infect Immun 1994; 62:3396-3407. 75. Abe J, Kotzin BL, Jujo K, Melish ME, Glode MP, Kohsaka T, Leung DY. Selective expansion of T cells expressing T-cell receptor variable regions Vp2 and Vp8 in Kawasaki disease. Proc Natl AcadSciUSA1992; 89:4066-4070. 76. Leung DYM, Meissner HC, Fulton DR Murray DL, Schlievert PM. Toxic shock syndrome toxin-secreting Staphylococcus aureus in Kawasaki syndrome. Lancet 1993; 342:1385-8. 77. Baker BS, Bokth S, Powles A, Garioch JJ,Lewis H, Valsimarsson H, Fry L. Group A streptococcal antigen-specific T lymphocytes in guttate psoriatic lesions. Br J Dermatol 1993; 128(5):493-499. 78. Leung DYM, Traver JB, Giorno R, Norris DA, Skinner R, Aelion J, Kazemi LV, Kim MH, Trumble AE, Kotb M, Schlievert PM. Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis. J Clin Invest 1995; 96:2106-2112. 79. Hofer MF, Lester MR, Schlievert PM, Leung DYM. Upregulation of IgE synthesis by staphylococcal toxic shock syndrome toxin-l in peripheral blood mononuclear cells from patients with atopic dermatitis. Clin Exp Allergy 1995;25:1218-1227. Schwab JH, Brown RR, Anderle SK, Schlievert PM. Superantigen can reactivate bacterial cell wall-induced arthritis. J Immunol1993; 150:41514159.

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81. Brocke S, Gaur A, Piercy C, Gautam A, Gijbels K, Fathman G, Steinman L. Induction of relapsing paralysis in experimental autoimmune encephalomyelitis by bacterial superantigen. Nature 1993; 365:642-644. 82. Schiffenbauer J, Johnson H, Butfiloski E, Wegrzyn L, Soos JM. Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis. Proc Natl Acad Sci USA 1993; 90:8543-8546.

Comparison of Structures of Toxic Shock Syndrome Toxin-l Unbound and Bound to a Class II Major Histocompatibility Molecule David

Mitchell, Patrick M. Schlievert, and Douglas

Ohlendorf

of Jongsun Kim and Don C. Wiley

of Robert G. Urban and Jack L. Strominger

INTRODUCTION

Bacterial pyrogenic toxin superantigens are a family of exogenous toxins from Staphylococcus aureus and Streptococcus pyogenes that combine with major histocompatibility (MHC) class I1 molecules and Tcell receptors to stimulate the release of cytokine molecules tumor necrosis factors-a and -p, interleukin-2, and interferon?. This overstimulation can contribute significantly to fever and shock resulting from capillary leak and to death. Toxicshock syndrome toxin-l 149

Mitchell et al.

(TSST-1) is produced by certain S. aureus strains and can be isolated from patients suffering from toxic shock syndrome (TSS) (1,2), AIDS patients exhibiting a recalcitrant, erythematous, dequamating skin disorder known as RED syndrome (31, and many children suffering from a toxic shock-like condition without hypertension known as Kawasaki syndrome (4). Similarly, staphylococcal enterotoxins, notably serotypes B (SEB) and C (SEC), are associated with approximately 25% of TSS patients (5). Streptococcal bacteria have long been associated with a number of illnesses including rheumatic and scarlet fevers. Streptococcal pyrogenic exotoxins (SPES),the cause of scarlet fever, have recently been associated with streptococcal TSS (6). Early reviews of microbial superantigens and their properties can be found in Refs. 7-13. Clearly, the medical significance of this whole family of toxins and their ability to act as superantigens merits further attention. Superantigens are molecules with four defining characteristics. First, these toxins bind MHC class I1 molecules as intact molecules (14-17). Second, once bound to an MHC class I1 molecule, superantigens promote the release of lymphokines by activating entire families ofCD4+ T cells as well as CD8+ T cells that display particular Vp chains in their receptors This T-cell activation is largely independent of the peptide antigen concurrently bound to the class I1 MHC molecule; thus the term "superantigen." For example, in acute cases of TSST-l-induced TSS, Vp2+ T cells can account for up to 60% of all host circulating T cells (18). Third, superantigens are powerful stimulators mitosis, acting at femtomolar concentrations (19-21). Finally, superantigens act as immunosuppressive agents on B cells, reducing the ability to develop protective immunity against these toxins(22,231. TSST-l is synthesized as a 234-amino-acid precursor (24); the first 40 residues are cleaved to produce the mature, 22-kDa form of the toxin. The mature toxin is a very stable molecule that can withstand radiation, extremes in temperature and pH, and is generally protease resistant. In addition to its superantigenic activity, TSST-1 potentiates the sensitivity of the host to lipopolysaccharides (25), resulting in lethal TSS. TSST-1 is also able to bind aortic endothelial cells (26). Experiments with mutants of TSST-1 (27,281 and the ovine S. aureus variant TSST-0 have demonstrated Glu 132, His 135, and Gln 136 as critical residues in producing host lethality. TSST-l has been crystallized in multiple crystal forms (291, and the three-dimensional structures of the native and mutant forms of

Comparison ofand Unbound

Bound TSST-1

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the toxin have been determined at resolutions between 2.9 and 1.9 A (30,31; manuscripts in preparation). Similarly, three-dimensional structures of superantigens SEB (32), SEC3 (331, SEA (341, and SEC2 (35-36) from S. aureus have been determined. In all cases these molecules have a two-domain, kidney-shaped fold with a large amount of regular secondary structural elements. In addition, crystal structures of binary complexes of TSST-l and SEB each with the class I1 MHC molecule HLA-DR1 have been determined (16,171. The combination of TSST-1 X-ray models and mutational analysis provides a framework for understanding some of the biology of this family of toxins. Experiments suggest that although the class I1 MHC binding sites of TSST-1 and SEB (as well as SEA) overlap, the toxins do not compete with each other because they bind different subsets of DRl molecules (37-42). Differences in the electrostatic character of homologous residues inthe toxins, DRl molecules, or both would be the likely cause of such specificity. Multiple class I1 MHC binding sites on the toxins have been predicted and confirmed by mutational analysis (43) and molecular modelling studies (44) in the case of SEA. Here the structures of TSST-1 unbound and bound to the human class I1 major histocompatibility molecule are compared to assess the effect of complex formation on the structure of TSST-1. II.

MODELBUILDINGANDREFINEMENT

The structure ofTSST-1 in space group C222, was originally determined at 2.5 A resolution using molecular isomorphous replacement (MIR) techniques and density averaging of the three molecules in the asymmetric unit (30,31). The three molecules ofTSST-1 associate to form a pseudo-P6,22 cell. The structures of all three TSST-l molecules in the C222, cell have been refined to an overall R factor of0.156 (F > lo, 5770 protein atoms, 405 solvent molecules) at 2.0 A resolution (manuscript in preparation). The structurp of the three molecules ofTSST-1 have RMS differences of 1-1.1 A for all atoms. The structure of the complex of TSST-l to HLA-DRl was determined to 3.5 A resolution from a crystal of a 1:l complex (17) using molecular replacement methodswith HLA-DRl coordinates refined at 2.75 resolution and TSST-1 coordinates at 2.5 A resolution as probes. The positionsof the HLA-DR1 and TSST-l molecules could be placed with confidence based both on rotation and translation functions for each molecule and on separate six-dimensional searches of

a

.

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152

a-carbon models of each molecule in a single isomorphous replacement, solvent-flattened A electron-density map. However, kecause significant intensity data could not be measured beyond A, crystallographic refinement was difficult, yielding an R factor of 22% A data; R free = The unit cell of the TSST-1:HLADRl complex contains 4 molecules ofTSST-1 near the screw axis. High-resolution details such asthe presence of hydrogen bonds salt bridges could not be assessed with complete certainty. Refinement of the 2.5 A resolution structure ofTSST-1 to 2.0 A resolution suggested a revised interpretation of a five-residue loop (TSST-l residues 142-146). This loop is distant from the HLADR1 contact region. The A resolution electron-density map of the TSST-1:HLA-DRl complex is consistent with this change, although TSST-l Leu has no side-chain electron density. High-resolution refinement also suggested the repositioning of a number of carbonyl groups (residues 15, 26, and 48) and the side chain of Asp 27. These changes allow additional hydrogen bonds in the complex (TSST-l Asp 27 to HLA-DR1 Gln 18a and Lys as well as TSST-1 Tyr to TSST-1' Tyr The A resolution limit to the TSST1:HLA-DRl data, however, precludes checking these changes independently. Crystallographic refinement was carried out using X-PLOR and model building with (46), Insight (Biosym Corp, La Jolla, CA), and TOM/FRODO (47). Superposition of the two TSST-1 molecules was done in (46). Figures l and were made using the program MOLSCRIPT (48) and solvent accessibility calculations were carried out by the method of Lee and Richards (49). STRUCTURALDIFFERENCESBETWEENTSST-1UNBOUND AND BOUND TO HLA-DR1

Superposition of the unbound TSST-l model and TSST-1 bound to HLA-DR1 is shown in Fig. 1. TSST-1 is presented in the standard orientation and so defines descriptors such as front and top. TSST-l is composed of two basic folding domains. Domain A consists of residues and 90-194 with a central a helix (residues 125-141) resting against a wall of five p strands. Across the top of this central a helix lies a three-turn amino-terminal a helix (residues 4-14). Domain B contains residues 18-89, which form a five-strand p barrel The residues of TSST-1 interacting with FILA-DR1 cover nearly all of the front face of domain B (17). The three major TSST-1:HLA-DR1

Comparison of Unbound and Bound TSST-1

153

Mitchell et al.

154

contact regions in domain B are the loop between p strands 1and 2 and an extended segment after p strand 3, the hydrophobic surface at the front of this domain including p strand 3, and the loop between strands 4 and 5. Figure 2 shows the mean RMS difference for all atoms in each residue between unbound and MHC-bound TSST-l. Overall the RMS difference between alpha carbons is 1.49 A while that between all nonhydrogen atoms is 1.83 A. Neglecting NH,-terminal and COOHterminal residues, wbich are involved in lattice contacts, the RMS differences are 0.87 A and 1.38 respectively. Elements of regular secondary structure and residues forming the TSST-1:HLA-DRl interface are indicated in Fig. 2. TSST-1:HLA-DRl complex formation involves the intercalation of turns from each molecule (see Fig. 3). The first of the three TSST1:HLA-DRl contact regions involves TSST-lpstrands 1and (residues 27-32) and the turn between p strand 3 and an extended segment (residues 49-52). The solvent-accessible surface buried in this contact region is 380 Both turns in this contact region retain their overall conformation (type I1 and I,respectively) despite being forced

A*.

o ! . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . { 0 10 20 SO 40 50 6 0 70 80 SO 100110120 130140150 l60170180 1 9 0 2 0 0

Residue Numbers

Figure 2 RMS difference in CQ position between unbound and bound TSST1. Thick bars labeled with letters indicate Q helices, thin bars labeled with numbers indicate p strands

Comparison Bound ofand Unbound

TSST-1

155

45 a

Pro Figure Intercalating turns in TSST-1:HLA-DR1 complex. TSST-l is on the right and shown using black (white) atoms for the bound (unbound) toxin. Ca trace for the bound (unbound) TSST-1 is shown in dashed (solid) lines HLA-DR1 is on the left and shown in gray.

about 2 A further apart in the complex. The differenc? in the first turn is centered at Leu 30 whose alpha carbon moves1.5 A and whose side chain x1 angle changes by 138" to avoid a steric clash with the main chain HLA-DR1 Met 37a and Ala 38a. An alignment of the amino acid sequences of TSST-l, the staphylococcal enterotoxins, and the streptococcal pyrogenic exotoxins based onthe structures of TSST1, SEA,SEB, and SEC3 (30-36) reveals a conservation of this Leu residue and suggests it may serve as an important contact for superantigen:MHC class I1 binding. The change at the second turn results in a 1.7 A movement of the carbonyl oxygen of TSST-l Pro 50 allowing it to accept a hydrogen bond from N1; of HLA-DR1 Lys 39a (see Fig. 3). Additional evidence for the importance of the loop between p strands 1 and 2 of TSST-l in complex formation comes from a mut-

Mitchell et al.

156

ational study (50) where a TSST-lG31S/S32P double mutant was unable to bind an HLA-DRl molecule stimulate human peripheral blood lymphocytes. The second TSST-1:HLA-DRl contact region centers on Ile 46 in the middle p strand 3. The solvent-accessible surface of TSST-l buried in this interface is 210 A2.This region is largely hydrophobic and undergoes no significant rearrangements during complex formation. It is interesting to note that while TSST-1 and SEB both have a significant hydrophobic patch on domain B, the positions of these patches are only partially overlapping. This difference may contribute to the observed differences in class I1 MHC binding. The TSST-1 mutation I45V (50) has reduced HLA-DR1 affinity, which most likely reflects a charge in the packing of the p barrel since this residue is buried. The largest conformational changeof TSST-1 at the TSST-1:HLADRl interface occursat the third contact region or strand 4-P strand 5 loop (TSST-l residues 75-80) at the top of domain B (Fig. 4). In unbound TSST-l, the electron density for Ser 76 is weak; however, the conformation for the neighboring residues infers its placement. In forming the complex, the end of this loop moves about 5.2 A to avoid a collision between TSST-l Glu 77 and the backbone of residue P13 of the bound peptide antigen. HLA-DR1 Tyr 60p moves 1 closer to TSST-1 and disrupts one hydrogen bond in the HLA-DR1 a helix. In this shift the loop bends uniformly without disrupting the hydrogen bonds of the unbound TSST-1. In this new orientatiop hydrogen bonds may form between P13 N and Thr 75 Oyl (3.71 A), between P13 0 and Glu 77 N (2.95 and between HLA-DR1 Tyr 60P OH and Gln 63 Ne2 (2.60 A) and TSST-1 His 74 0 (3.23 A). The interactions found between TSST-1 and the HLA-DRl chain and bound peptide bury 295 A2 of solvent-accessible surfacearea of TSST-1 and differentiates this model from the SEB:HLA-DR1 complex structure where no similar contacts are observed (51).

A

A),

IV.

LAlTlCE INTERACTIONS

The X-ray models of unbound and bound TSST-1 used in this comparison have multiple copies TSST-1 in their respective unit cells. Thus some of the differences between unbound TSST-l and TSST-1 found in the complex with the class I1 MHC can be attributed to changes in lattice packing interactions. These interactions can be broken down into TSST-l:TSST-l* interactions in either the unbound


TSST-1

157

TSST-

P3

Domain B

HLA-DR

Figure 4 Difference in the turn at residues 75-80 at the top domain Peptide antigen (coil) andportion HLA-DR1P chain are shown in gray. Ca trace bound (unbound) TSST-I is shown using dashed (solid) lines.

or bound lattice, and TSST-l:HLA-DRl* interactions where the asterisk denotes symmetry-related molecules in the crystal. AnHLADR1:HLA-DRY interaction seen in other HLA-DRl crystals is also briefly discussed. Unbound Form

Several interactions are 'made with neighboring TSST-1 molecules in forming the unit cell. The most important is the interaction around a twofold axis involving nearly the entire top TSST-1, as shown in Fig. 1. This surface i s used in forming interfaces with other TSST-1 molecules in all known crystals forms ofTSST-l as well as TSST-1 tetramutant (Fig. 5). Mutational analysis indicates that this surface is critical to.the superantigenic and lethal properties of TSST1 Reduced superantigenic activity has been reported when Tyr

Mitchell et al.

Figure 5 Top view ofTSST-1 showing conserved intermolecular interface. The two domains ofTSST-1 are labeled. Residues in dark gray with white letters are inboth the unbound TSST-l and TSST-1:HLA-DR interfaces. Residues in white with black letters (black with white letters) are only in the unbound (bound) interface. Smaller residues in white with dark letters are in the interfaces in one of the other native or mutant crystal forms.


TSST-1

159

115, His 135, His 141 is changed into alanines (52,531. The observed difference in Ca position of TSST-1 Asn 194 (C-terminal residue) may be due to the presence of TSST-l* Tyr 115. A second region of difference involves TSST-1 residues 158-161, which make a type I turn between p strands 10 and 11at the bottom right corner of domain A. In unbound TSST-l this regions interacts with a neighboring molecule just under the p hairpin that contact region 1 in the TSST-1:HLA-DRl complex. In the complex, the same 158-161 turn is interacting with the top of domain B of aoneighboring molecule. This interaction moves the turn by nearly 1A placing the side chain of Asp 160 close to three positively charged residues-Arg 68', Lys 71', and His 82'. B.

TSST-l:TSST-l* Bound Form

Within crystals of the TSST-1:HLA-DRl complex a very significant interaction is that between TSST-1 molecules in neighboring complexes (at in Fig. 6). comparison, the area the TSST-1:TSSTl*contact surface is 500 A2 where the area of the TSST-1:HLA-DR1 surface is 885 A*. This second contact is almost certainly not physiological, but it does involve a TSST-1 surface that has been proposed to interact with a T-cell receptor (TCR; 31,32). The putative TSST-l TCR interaction site faces up away from the HLA-DRl molecule in the TSST-1:HLA-DR1 complex (Fig. 3 in Ref. 18) in a position accessible to an approaching T cell. Of the six TSST-1 residues implicated in TCR recognition by mutagenesis (Tyr 115, Glu 132, His 135, Ile 140, His 141, and Tyr 144) (28,29,49,50), two (Tyr 115 and Tyr 144) are in the TSST-l:TSST-l* contact and three (His 135, Ile 140, and His 141) are in the immediate vicinity. In Fig. 6, the potential TCR-binding residues are marked using #'S on the symmetry-related TSST-l* molecule for convenience. C. TSST-1 Bound:HLA-DR1 *

The largest differences betweenthe unbound and bound BST-l structures are in the NH,-terminal region (residues 1-17) where a close contact is seen between TSST-1 and HLA-DR1* the equivalent TSST-l* and HLA-DR1, see Fig. 7). In unbound TSST-1 an 11-residue a helix beginswith Asp 4. In bound TSST-1 this a helix begins almost one full turn later at Lys 7 because the unwound extended NH, terminus is stabilized by several electrostatic interactions with a neighboring HLA-DRP molecule (Ser 1 N with Glu 134a* and Glu 46a*,

HLA-DRl

-

Mitchell et al.

HLA-DR1

Figure 6 Stereo view of HLA-DRl (thin lines) interacting with two TSST-l (thick lines) molecules in the crystal lattice. TSST-l and *TSST-l are related by a 4, screw axis and a second HLA-DR1 molecule is located 'at the label *HLA-DR1, but not shown for simplicity. All Ca positions are marked by small opencircles. The residues at the HLA-DRl:*TSST-l interface (HLA-DR1 Asp 35a, Lys 38a, Glu Val Trp Glu Gly and Arg 50a; *TSST-l Ser Thr 2, Asn 3, Asp 4, Lys 7, Ser 146, Ser 147, Asp 148, and Asn are represented by larger closed circles. The interface is small compared to thqmajor contact regions with the buried solvent accessible surface area of A2 The residues at the TSST-FTSST-1 interface (TSST-l Pro Gln 165, Glu 173, Thr 50, Tyr 51, Asn 159, Asp Gly 161, Ser 162, Thr 176, Lys 178, Lys 187, and Asn *TSST-l Asp Lys 7, Leu Asp Ser 15, Gly 16, Asp Lys 71, Ser 72, Tyr 80, His 82, Tyr 115, Gln and Tyr are represented by larger op$n circles. The buried solvent accessible surface area of this interface is 500 A2 *TSST-l residues Tyr 115, Glu 132, His 135, Ile His and Tyr which are important for mitogenic activity TSST-l, are labeled # on the second *TSST-l molecule for convenience.

and Asp 4 with Lys This interaction surface is fairly small at 330 Az. The unraveling of the NH,-tenninal a helix cannot occur in crystals of unbound TSST-l because the steric constraints with neighboring molecules. HLA-DRl:HLA-DRl*

A 650 Az surface of HLA-DR1 forms the dimeric contact with a neighboring HLA-DRY. This interaction is equivalent to that previously

Comparison of Unbound and Bound TSST-1

TSST-

*

Figure 7 NH,-terminal region in the TSST-1:HLA-DR1 complex. TSST-1 molecules are shown with black Ca trace. White TSST-l* atoms are those in the symmetry-related molecule. HLA-DR1 is shown with gray atoms and trace. Asterisks indicate symmetry-related molecules.

observed in other crystals HLA-DRl (17,51,54). Although the crystal lattice of TSST-1:HLA-DR1 has no other contacts in common with the three earlier HLA-DR1 crystals, all four crystals were grown at pH’s below 6.0. Whether this DR1:DRl contact is a crystallization artifact, has a role in T-cell signaling (51,55), or indicates an HLADR1 surface involved in binding other proteins (calnexin, invariant

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chain, HLA-DM, CD4) during assembly, peptide loading, or T-cell signaling is unknown (56-59). V.

CONCLUSION

Binding of HLA-DR1 to TSST-l requires the intercalation of TSST-l turns with those of the HLA-DRl molecule, but produces no largescale rearrangements in either molecule. Structural differences between the two TSST-l molecules due to HLA-DRl binding are limited to lateral shifts and rotations of turns and loops. Other structural differences canbe attributed to differences in the interactions responsible for crystal formation. Interestingly, the putative TCR binding site matches nearly exactly a portion of TSST-l involved in contacting neighboring molecules in the TSST-1:HLA-DR1 complex as well as in several crystal forms unbound TSST-l. ACKNOWLEDGMENTS

This work has been partially supported by grants from the National Institutes of Health (AI-22159 to P.M.S. and HD-17461 to D.C.W.), Kimberly-Clark Corp., Neenah WI, Personal Products Co., New Brunswick NJ, and Tambrands, Palmer MA. We are grateful to the National Institute of Health (NIH) for training grant support (HDfor D.T.M. J.K. acknowledges support from the NIH. In addition, the authors would like to thank Dr. G. S. Prasad and Dr. R. Radhakrishnan for discussions and for their efforts in the refinement of unbound TSST-1 structures to be reported elsewhere. D.C.W. is an investigator of the Howard Hughes Medical Institute. Coordinates unbound TSST-1(1TSS) have been deposited with the Protein Data Bank at Brookhaven National Laboratory. Coordinates of the TSST1:HLA-DR1 complex will be deposited in the Protein Data Bank and are available before their release by e-mail ([email protected]). REFERENCES

Schlievert PM, Shands KN, Dan BB, Schmid GP, Nishimura RD. Identification and characterization of an exotoxin from Staphylococcus aureus associated with toxic shock syndrome. J Infect Dis 1981; 2. Bergdoll MS, Crass BA, Reiser RF, Robbins RN. A new staphylococcal enterotoxin, enterotoxin associated with toxic-shocksyndrome Sfaphylococcus aureus isolates. Lancet 1981; Cone LA, Woodard DR, Byrd RG, Schulz K, Kopp Schlievert PM.


4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18. 19.

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A recalcitrant, erythematous, desquamating disorder associated with toxin-producing staphylococci in patients with AIDS. J Infect Dis 1992; 165:638-643. Leung DYM, MeissnerHC, Fulton DR, Murray DL, Kotzin BL, Schlievert PM.Toxicshock syndrome toxin-secreting Staphylococcus uureus in Kawasaki syndrome. Lancet 1993; 342:1385-1388. Schlievert PM. Role of toxic shock syndrome toxin 1 in toxic shock syndrome: overview. Rev Infect Dis 1989; ll:S107-S109. Stevens DL, Tanner MH, Winship J, Swarts R, Ries KM, Schlievert PM, Kaplan E. Severe group A streptococcal infections associated with a toxic shock-like syndrome and scarlet fever toxin A. N Engl J Med 1989; 321:l7. Marrack P, Kappler JW. The T cell receptors. Chem Immunol 1990; 49:69-81. Herman A, Kappler JW, Marrack P, Pullen AM. Superantigens: mechanism of T-cell stimulation and role in immune responses. Annu Rev Immunol 1991; 9:745-772. Fleischer B, Hartwig U. T-lymphocytestimulation by microbial superantigens. Chem Immunoll992; 55:36-64. Kotzin BL, Leung DY, Kappler J, Marrack P. Superantigens and their potential role in human disease. Adv Immunol 1993; 54:99-166. Bohach GA, Fast DJ, Nelson RD, Schlievert PM. Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses. Crit Rev Microbiol 1990; 17:251-272. Johnson HM, Russell JK, Pontzer CH. Superantigens in human disease. Sci Am 1992; 266:92-101. Schlievert PM. Roleof superantigens in human disease. J Infect Dis 1993; 167:997-1002. Dellabona P, Peccoud J, Kappler J, Marrack P, Benoist C, Mathis D. Superantigens interact with MHC class I1 molecules outside of the antigen groove. Cell 1990;62:1115-1121. Karp DR, Teletski CL, Scholl P, Geha R, Long EO. The a 1 domain of the HLA-DR molecule is essential for high-affinity bindingof the toxic shock syndrome toxin-l. Nature 1990; 346:474-476. Jardetsky TS, Brown JH, GorgaJC, Stern LJ, Urban RG, Chi Y-I, Stauffacher CV, Strominger JL, Wiley DC. Three-dimensional structure of a human class I1 histocompatibility molecule complexed with superantigen. Nature 1994; 368:711-718. Kim J, Urban RG, Strominger JL, Wiley DC. Crystallographic structure of toxic shock syndrome toxin-l complexed with a human class 11 major histocompatibility molecule, HLA-DRI. Science 1994; 266:1870-1874. White J, Herman A, Pullen AM, Kubo R, Kappler JW, Marrack P. The VP-specific superantigen staphylococcal enterotoxin B: stimulation mature T cells and clonal deletion in neonatal mice. Cell 1989; 56:27-35. Langford MP, Stanton GJ, Johnson HM. Biological effects of staphvlo-

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coccal enterotoxin A on human peripheral lymphocytes. Infect Immun 1978; 22:62-68. Carlsson R, Sjggren HO. Kinetics of IL-2 and interferon7 production, expression of IL-2 receptors, and cell proliferation in human mononuclear cells exposed to staphylococcal enterotoxin A. Cell Immunol 1985;96:175-183. Cole BC, Atkin CL. The Mycoplasma arthritidis T-cell mitogen, MAM: a model superantigen. Immunol Today 1991; 12:271-276. Schlievert PM. Alteration of immune function by staphylococcal pyrogenic exotoxin type C: possible role in toxic-shock syndrome. J Infect Dis 1983;147:391-398. Poindexter NJ, Schlievert PM. Suppression of immunoglobulin-secreting cells from human peripheral blood by toxic shock syndrome toxin-l. J Infect Dis 1986; 153:772-779. Blomster-Hautamaa DA, Kreiswirth BN, Kornblum Novick RP, Schlievert PM. The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-l. J Biol Chem 1986; 261:15783-15786. Keane WF, Gekker G, Schlievert PM, Peterson PK. Toxic-shock syndrome-l sensitizes renal tubular cells to lipopolyssaccharide induced necrosis. Am J Pathol 1986; 122969-1761. Lee PK, Vercellotti GM, Deringer JR, Schlievert PM. Effects of staphylococcal toxic shock syndrome toxin 1on aortic endothelial cells. J Infect Dis 1991;164:711-719. Murray DL, Prasad GS, Earhart CA, Leonard BAB, Kreiswirth BN, Novick RP, Ohlendorf DH, Schlievert PM. Immunological and biochemical properties of mutants of toxic shock syndrome toxin-l. J Immunol 1994; 152:87-95. Murray DL, Earhart CA, Mitchell DT, Ohlendorf DH, Novick RP, Schlievert PM. Localization of biologically important regions on toxic shock syndrome toxin-l. Infect Immun 1996; 64:371-374. Earhart CA, Prasad S, Murray DL, Novick DP, Schlievert PM, Ohlendorf DH. Growth and analysis of crystal forms of toxic shock syndrome toxin 1. Prot Struct Funct Genet 1993; 17:329-334. Prasad GS, Earhart CA, Murray DL, NovickRP, Schlievert PM, Ohlendord DH. Structure toxic shock syndrome toxin-l. Biochemistry 1993;32:13761-13766. Acharya KR, Passalacqua EF, Jones EY, Harlos K, Stuart DI, Brehm RD, Tranter HS. Structural basis of superantigen action inferred from crystal structure of toxic-shock syndrome toxin-l. Nature 1994; 367:94-98. Swaminathan S, Furey Pletcher Sax M. Crystal structure of staphylococcal enterotoxin B, a superantigen. Nature 1992; 359:801-806. Chi Y-I, Bohach GA, Stauffacher CV. The crystal structure of staphylococcal enterotoxin at 2.3 A resolution. 1994 (submitted). Schad EM, Zaitseva Zaitsev VN, Dohlsten M, Kalland T, Schlievert


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PM, Ohlendorf DH, Svensson LA. Crystal structure of the superantigen staphylococcal enterotoxin type A. EMBO J 1995; 14:3292-3301. Swaminathan S, Furey W, Pletcher J, Sax M. Residues defining Vp specificity in staphylococcalenterotoxins. Nature Struc Bioll995; 2:680-686. Papageorgiou AC, Acharya KR, Shapiro R, Passalacqua EF, Brehn RD, Tranter HS. Crystal structure of the superantigen enterotoxin C2 from Staphylococcus aureus reveals a zinc-binding site. Structure 1995; 3:769779. Panina-Bordignon P, Fu X-T, Lanzavecchia R, Karr W. Identification of HLA-DRa chain residues critical for binding the toxic shock syndrome toxin superantigen. J Exp Med 1992; 176:1779-1784. Thibodeau J, Labrecque N, Denis F, Huber B, Sekaly R-P. Binding sites for bacterial and endogenous retoviral superantigens can be dissociated on major histocompatibility complex class I1 molecules. J Exp Med 1994; 179:1029-1034. Thibodeau J, Cloutier I, Lavoie PM, Labrecque N, Mourad W, Jardetzky T, Sekaly R-P. Subsets HLA-DR1 moleculesdefined by SEB and TSST1 binding. Science 1994; 266:1874-1878. Scholl PR, Diez A, Geha RF. Staphylococcal enterotoxin B and toxic shock syndrome toxin-l bind to distinct site on HLA-DR and HLA-DQ molecules. J Immunol 1989; 143:2583-2588. Chintagumpala MM, Mollick JA, Rich RR. Staphylococcal toxins bind to different sites on HLA-DR. J Immunol 1991;147:3876-3881. Braunstein NS, Weber DA, Wang X-C, Long EO, Karp D. Sequences in both class I1 major histocompatibility complex a and p chains contribute to the binding of the superantigen toxic shock syndrome toxin 1. J Exp Med 1992;175:1301-1305. Fraser JD, Lowe S, Irwin MJ, Gascoigne NRJ, Hudson KR. Structural model of staphylococcal enterotoxin A interaction with MHC class I1 antigens. In: Current Communication in Cell and Molecular Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1993:7. Ulrich RG, Bavari S, Olson MA. Staphylococcal enterotoxins A and B share a common structural motif for binding class I1 major histocompatibility complex molecules. Nature Struct Biol 1995; 2:554-560. Brtinger AT, Kuriyan J, Karplus M. Crystallographic R factor refinement by molecular dynamics. Science 1987; 235:458-460. Jones TA, Zou JY, Cowan SW, Kjeldgaard M. Improved methods for finding protein models in electron density maps and the location of error in these models. Acta Cryst 1991;A47:llO-119. Jones TA. A graphics model building and refinement system for macromolecules. J Appl Cryst 1978;11:268-272. Kraulis PJ, MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Cryst 1991; 24:946-950. Lee B, Richard FM. The interpretation of protein structures: estimation of static accessibility. J Mol Biol 1971; 55:379-400.

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Staphylococcal Enterotoxins B and C

Gregory

of

INTRODUCTION

The staphylococcal enterotoxins (SEs) are a subgroup of related protein exotoxins inthe pyrogenic toxin (PT) family produced by phyZococcus aureus and Streptococcus pyogenes (1).Like other members of the PT family, the SEs are superantigens and elaborate a set of biological activities linked to their ability to stimulate cells of the immune system (2). These activities contribute to their ability to induce toxic shock syndrome, immunosuppression, and probably other diseases However, as is evident from the fact that they are designated as enterotoxins, the SEs are distinguishable from other members of the PT family by their ability to induce gastroenteritis when ingested. Hence, they are the causative agents in staphylococcal food poisoning (SFP),a very common form of food-associated gastroenteritis in the United States and worldwide (4). Barber is generally credited with being the first to provide convincing evidence for enterotoxin production by S. aureus in 1914 (5). However, the identity of the toxin caused a great deal of confusion 167

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for several decades because antisera prepared by immunizing animals with toxin produced by one strain often did not react with toxins from other staphylococcal isolates. Itwas eventually determined that staphylococci can produce more than one antigenic form of SE (6). Therefore, the present-day SE classification schemeis based primarily on immunological differences among the SEs The seven currently recognized major antigenic forms ofSEs (A,B,C,D,E,G, and H) have been designated according to standard nomenclature in which SEs are assigned a letter designation in order of their discovery. While in general there has been little reported molecular heterogeneity among proteins within a single antigenic type ofSE, that is not the case for SEC. Shortly after the initial report of SEC, Bergdoll's group reported that the toxins produced by S. aureus strains FRI137 and FRI361 both reacted with the SECspecific antisera despite the fact that they have significantly different isoelectric points (9,101. When further analysis revealed that the toxins from these two strains were only partially identical in immunological reactivity, they were referred to as SEC1 and SEC2, respectively. It is interesting to note that SEE antisera cross-react strongly with SEA, even in relatively insensitive assays such as agar immunodiffusion assays. Unlike the SEC subtypes, these cross-reactive SEs have been assigned separate letter designations. It is now know that the molecular heterogeneity among toxins reacting with SEC antisera is significantly more complexthan initially reported (11). In this chapter, SEC is generally used in reference to a group of toxins including SEC subtypes 1-3 and several speciesspecific SEC molecular variants. When it is relevant, specific SEC molecules will be designated with additional detail according to their subtype, molecular variant, or strain designation. The purpose of this review is to discuss the structure-function relationships in SEB and SEC. It is customary and advantageous to discuss these two general types of SEs together. The factthat all known SECSshare at least 65% amino acid sequence identity with SEB and greater than 93% identity with each other is reflected in antigenic, chemical, and biological similarities that are not uniformly shared with other SEs (Table 1). The relatedness of SEB and SEC to other SEs is significantly lower ( 8 ) . In fact, SEB and SEC are more closely related to several streptococcal PTs than to the five other presently known forms of SEs. For a detailed review of the SEB and SEC relatedness to other members of the PT family, one should refer to separate chapters in this volume in addition to Refs. 8 and 12.

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II. SEB AND SEC SEQUENCERELATEDNESS

Like other PTs, SEB and the SEC molecular variants are single polypeptides that have not been shown to be arranged functionally into a classical A-B subunit organization. The primary sequences have been determined for SEB and SECl by direct amino acid sequencing and through inference from nucleotide sequence analysis the of cloned genes (13-16). The other SEC variants have been sequenced only at the nucleotide level (11,171. SEB and all forms ofSEC are expressed as precursor proteins containing 266 residues, which include a 27residue signal peptide. During export, the signal peptide is cleaved to produce a mature cell-free toxin with 239 residues (Fig. 1). Although an alternative cleavage site has been proposed for SEC from S. aureus 1230, the N-terminus of the toxin purified from this strain was found by other investigators to be glutamic acid, identical to other known SEC molecular variants (18,19). The toxins in this group are neutral basic proteins with PIS ranging from 7 to 8.6, but they display a high degree of microheterogeneitywhen assessed by isoelectric focusing (Table 1). For example, SEC2 focuses into at least eight bands ranging in p1 from 5.50 to 7.35 (20). Although this heterogeneity has been attributed to enzymatic deamidation, it could also be explained by spontaneous deamidation since the putative deamidase enzyme has not been identified. From a functional standpoint the classification of SEC based on antigenicity, without considering the sequences and biological properties, has several shortcomings. For example, the literature cites at least three different toxins classified as SEC3 based on their immunological reactivity (11,18). Although they may be indistinguishable immunologically, the toxins currently classified as SEC3 have significantly different sequences. Since the genotype is widely spread among human, animal, and food isolates of S. aureus (111, it is likely that the observed heterogeneity SEC reflects the ability of S. aureus to adapt to host cell receptors. This ability is presumably of crucial importance to S. aureus considering its broad host range. The best evidence for this hypothesis has been an analysis ofSEC variants produced by staphylococci implicated in bovine and ovine mastitis (SEC-bovine and SEC-ovine, respectively). SEC-ovine and SEC-bovine, which share 236 of 239 residues, are most closely related to each other and to SECl. Despite differing by only three residues, the two mastitis variants differ significantly in regard their ability to stimulate T cells from humans, cattle, and sheep (11).

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SECl SECl SEC3-FRI913 SEC3-FR1909 SEC-bovine SEC-ovine SEC-canine SEB

GO GO GO 60 60 60 GO

SECl SEC2 SEC3-FRI913 SEC3-FR1909 SEC-bovine SEC-ovine SEC-canine SEE

117 117 117 117 117 117 117 120

SECl SEC2 SEC3-FRI913 SEC3-FRI909 SEC-bovine SEC-ovine SEC-canine SEB

177 171 177 177 177 177 177 178

SECl SEC2 SEC3-FRI913 SEC3-FRI909 SEC-bovine SEC-ovine SEC-canine SEB

239 239 239 239 239 239 239 239

60

Figure 1 Alignment of primary sequences of SEB and the known SEC variants from human, animal, and food isolates (11, 13, 14, and 17). Dashes

(-1 designate the location of residues conserved with SEC1. Dots (.l Indicate gaps introduced to obtain maximal alignment. Amino acid residue numbers are shown on the right. MAPPING OF RELEVANT ANTIGENIC EPITOPES IN RELATION TO SEB AND SEC STRUCTURE

In addition to its importance in classification of the toxins, SE antigenicity has been of interest for its potential relevance to other applications. For example, reactivity with antibodies has been used to study the stability SEB, and other SEs, since loss of antigenicity roughly correlates with inactivation by heat or protease (10). Also, knowledge of the location of antigenic epitopes in relation to regions involved in toxicity could have practical application toward

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the development of vaccines and other therapeutic reagents. One potential strategy that has been discussed is to produce recombinant toxoids that express a protein retaining significant levels of antigenicity but which are devoid of toxicity. As discussed above, the SEC molecular variants are very similar even indistinguishable based on their antigenic properties. SEB is less closely related toSEC. Using agar gel diffusion assaysand other relatively insensitive methods, SEB and SEC usually appear to be antigenically distinct, although minor cross-reactivity can occasionally be demonstrated between these two toxins (21). On the contrary, more sensitive assays such as immunoblotting and coprecipitation clearly show that SEB and SEC possess some common epitopes. This is not surprising, considering the extensive primary sequence similarity shared by SEB and SEC, since it has also been possibleto demonstrate a small, albeit reproducible, cross-reactivity among SEB or SEC and far less similar toxins such as SEA and SEE (22,23). Over the years a great deal of attention has been focused on the mapping of shared and unique antigenic determinants of the SEs, especially SEB and SEC1. Based on their initial experiments to test the binding ofSEB and SECl protease-generated fragments with polyclonal antisera, Spero and Morlock (24) concluded that binding to reciprocal antibodies by these two toxins was determined predominantly through N-terminal residues whereas binding by the two toxins to their own homologous antibodies appeared to be occurring mostly in central and C-terminal portions of the toxins. In contrast, two separate studies using monoclonal antibodies and protease-generated toxin fragments foundthat binding to heterologous monoclonal antibodies was more likely to occur by toxin fragments from the Cterminus (25,261. In reality, considering these discrepancies in the literature and what is currently known in regard to the three-dimensional structures ofSEB and SEC (discussed below) it is likely that cross-reactive and unique epitopes on SEB and SEC are both distributed throughout the proteins. This issue of SEB and SEC antigenic relatedness is further complicated by the fact that SECl subtype-specific monoclonal antibodies recognize SEB, but not SEC2 or SEC3 (25-27). Sequencing data suggest that the epitope shared by SEB and SECl, and therefore responsible for these observations, was acquired by genetic recombination of a portion of the genes encoding at least residues 14-26 In this stretch of residues, SECl is nearly identical to SEB but differs from SEC2 (and SEC3) by five residues (Fig. 2). Thus, recombination between the genes for SEB and SEC2 (or SEC3) could explainthe generation of SECl. The specific residues that determine the epitope are

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o P 15 16 17 18 19 20 21 22 23 24 75 26 27 SEB SEC1 SEC2 SEC3(FRI909) SEC3(FRI913) SECl (V26Y)

S

K

F

T

G

L

M

- - - - - - - E - - - T - E - - - T - E - - - T -"""""

E

-

G G G

N

M

K

V

L

Y

-

- - - - - - - - - Y - - - Y -

Figure 2 Sequence alignment comparing part of the region proposed to have been involved in genetic recombination between SEB and SEC. Dashes (-1 designate residues identical to SEB. Residues 20,22, and 26 determine the subtype-specific epitope differentiating SECl from SEC2 and SEC3. This stretch of residues is located on an a-helix delineating a shallow cavity involved in binding to the TCR. SECl (V26Y) is an SECl mutant used to implicate this stretch of residues in determining Vf3 specificity (see text for details).

located at positions 20,22, and 26 (27). Interestingly, the SEC subtype-specific epitope that SEB shares with SECl only is located in an important functional regionof the molecules involved in superantigen binding to the T-cell receptor (see below). As a result, SECl mutants containing single site replacements with the analogous residues of SEC2 have altered antigenicity in addition to T-cell-receptor (TCR) stimulation patterns characteristic of SEC2 (27). These combined results provide further evidence that heterogeneity among the SEC group of toxins is the result of the adaptability of the organism in its attempt to interact with the immune system of its many potential hosts. Obviously, the degree of cross-protection induced in vivo among the SEC subtypes and molecular variants is very high. In addition, neutralizing antibodies that can cross-protect against SEB and SEC have been demonstrated using in vivo models for both TSS and SFP. For instance, immunization of rabbits with either SEB or SECl protects the animals against enhanced lethal endotoxin shock induced by the heterologous toxin (28). Similarly, in the monkey-feeding assay, at least partial protection against challenge with SEB or SEC can be demonstrated in monkeys immunized against the heterologous toxin (29). Information regarding the location of cross-reactive epitopes that induce protective immunity in vivo is limiting. However, it has been suggested that neutralization of the superantigenic activity of the toxins by antibodies in vitro could be used as an indicator that protection may be achieved in vivo. Although this has not been confirmed, it has prompted attempts to map and characterize epitopes

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in vitro using antibodies generated by immunizing with synthetic peptides encompassing highly conserved stretches ofSE primary sequence. One such region that induces neutralizing antibodies has been identified independently by two groups of investigators. Peptides corresponding to residues ofSEB, or an overlapping region of SEC1 (residues induced antibodies that neutralized the Tcell proliferative activity of the toxins The highly conserved SE sequence K-K-X-V-T-X-Q-E-L-D (see SEC residues in Fig. l),encompassed by both peptides, may represent part of an epitope that could be useful for immunization against several SEs. It is unlikely that all of the major SEB and SEC antigenic epitopes induce neutralizing antibodies. Using a panel of nonoverlapping monoclonal antibodies, it has been possible to differentiate epitopes that correspond to functional molecular regions (i.e.,T-cell and antigen-presenting cell binding) from other parts of the toxins that appear to have apparent role in superantigenicity IV. SEB AND SEC THREE-DIMENSIONAL STRUCTURES A.

General Attributes of the SEB and SEC Crystal Structures

The first published crystal structure for a superantigen was that of SEB, reported by Swaminathan et al. in This achievenlent was a significant advance in our understanding of the structure and function relationships for SEB and for superantigens in general. The structure of related SEC3, subsequently described in confirmed the prediction that folding ofSEB and SEC are very similar. The SEB and SEC proteins are ellipsoidal, tightly compact, and fold into two unequal-sized domains of mixed structure (Fig.

Figure (A) Ribbon diagram showing the crystal structure of SEC3 with its major structural features. j3 and a structures are numbered sequentially. The diagram is oriented so that the large p-grasp domain with the N- and Ctermini is on the left. The small domain on the right contains the disulfide bond shown as a ball-and-stick linkage and has topology equivalent to an OB domain (see Fig. 4 ) . The interdomain a3 cavity and a5 groove are also labeled. (Adapted from Refs. 30, 34, and 47.) (B) Schematic diagram of the SEC3 molecule rotated compared to (A) showing the back the molecule, which contains a zinc site at the base of the a5 groove. The zinc is coordinated by three residues including two histidines at positions 118 and within H-E-X-X-H motif typical of metalloproteases such as thermolysin. (Adapted from Refs 47, 49, and 50.)


(a)

B and C

a5 Groove

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The similar structures of SEB and SEC3 are not unexpected considering the high degree of sequence identity shared by the two toxins. In fact, spectral techniques and computer-assisted structural analyses have long predicted certain features common to all the SEs that also extend to other members of the PT family such as TSST-1 (35-39). Although there is some discrepancybetween the crystal and solution structures of SEs (40), in general the predicted PT structural similarities have been confirmed by the recent reporting of the SEA, SEC2, and TSST-l three-dimensional crystal structures (41-44). Despite the fact that the SEs have a shared folding motif, each toxin seems to have its own unique structural features. The most noticeable differences among the SEs are the length and composition of their disulfide loops and the presence and location of a bound zinc atom. SEB and SEC differ from each other and from SEA in regard to both these properties. B.

The Small Domain

The smaller domain ofSEB and SEC (domain 1) contains SEC3 residues 35-120 near to, but not including, the N-terminus. The fold of this domain can be described as a Greek-key P-barrel, capped at one end by an a-helix. It therefore has the same size and topology as the oligonucleotide/oligosaccharide binding (OB) fold (Fig. 4 and Ref. 45). The internal portion of the P-barrel is rich in hydrophobic residues and the potential oligomer binding surface is covered with mainly hydrophilic residues. It is interesting that several bacterial exotoxins have the same general folding motif as the small domain, despite lacking significant homology with SEs at the primary sequence level. Examples of such bacterial toxins with activity in the gastrointestinal tract include the heat-labile toxin (LT) and Shiga-like toxin (SLT), two AB5 toxins produced by coli. The OB fold is a property of the LT and SLT B subunits, which are responsible for interacting with oligosaccharide portions of receptors on susceptible host cells. Despite its apparent uniform presence among the SEs, there is no indication that their small domain performs an analogous function in staphylococcal pathogenesis. Currently, the only known SE receptors are major histocompatibility complex (MHC) class I1 molecules and the TCR. The available evidence suggests that interaction with these two glycosylated receptors does not involve binding to their carbohydrate moieties (see below). However, since other potentially significant

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B Figure 4 Ribbon diagrams to compare the SEC3 small domain (A) with that of the OB folding motif described by Murzin (45) on (B). This motif is generally found in proteins that bind to nucleic acids or carbohydrate portions of cellular receptors, including enterotoxins from several gram-negative pathogens. A similar function for this domain in SEs has not been identified.

receptors, such as those involved in the emetic response, have not been identified, a role for the SEOB binding motif cannot be ruled out at this point. At the opposite end of domain 1 from the helix cap is the disulfide loop, which is presumed to be present in all SEs. Crystallographic analyses of both SEB and SEC indicate that the loop residues are quite flexible. In SEB and SEC, the second of the two cysteine residues (based on primary sequence) appears to be an integral part of the adjacent strand (p5 in Fig. 3A). The other cysteine residue is located within the flexible disulfide loop and connects the loop to the rest of the molecule through the disulfide linkage. The extent of loop flexibility and protease susceptibility seems to be related to the loop length, which is a variable feature among the SEs. For example, the SEB and SEC loops are both highly susceptibleto proteolysis. The loops of these two toxins (containing 19 and 16 residues, respectively) are approximately twice the length of those in other SEs, which usually contain only nine residues and are resistant to proteolysis. There is some indication that even the three-residue difference in length between SEB and SEC3 loops has a significant effect on flexibility

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since the longer loop ofSEB was not modeled in the published crystal structure. C.The

large Domain andlnterdomain Features

The larger domain2 ofSEB and SEC3 contains the N-terminus (SEC3 residues 1-33) in addition to the remainder of the structure including the C-terminus (SEC3 residues 123-239). It is a P-grasp motif composed of a five-strand antiparallel P-sheet wall over which a group of a-helices is laid. The N-terminal 20 residues of SEC3 form a loosely attached structure, which drapes over the edge of the domain. It is interesting that, despite being far removed in relation to primary sequence, the N- and C-termini are brought into close proximity as a result the toxin-folding properties. Residues immediately downstream from the N-terminal 20 residues ofSEB and SEC3 form an important a-helix (SEC3 a3-helix), which contains residues that determine the SEC subtype-specific epitope discussed above. Furthermore, this helix also helps to delineate part of the interface between the large and small domains of the toxins. Thus, the a3 helix of SEC3 and the analogous helix in SEB (a2) are among several structures that line a shallow cavity at the top of the SE molecules. The significance of this cavity in superantigen activity is discussed below. Therest of the interdomain interface can be described as two large (approximately15A wide) grooves running the along the length the SEC3 and SEB, each containing one the interdomain helices. The groove defined by the a5 helix in SEC3 (a4 in SEB) contains most of the highly conserved residues shared throughout the PT family (30) and also is the location of the crossreactive protective epitope discussed above. Thepotential roles of this part of the molecules in superantigen and enterotoxic biological ac. tivities are still uncertain and are discussed below. D. Cation Binding

Consistent with a potential importance for the role of the a5 groove in SEC biological activity was the discovery of a zinc atom and a common zinc-binding motif at the base of the groove in SEC3 (Fig. 3B). The presence of zinc in the SEC3 structure was somewhat unexpected. Whereas Fraser et al. (46) implicated zinc-mediated ligand binding by SEA and SEE as a requirement for T-cell stimulation, their results were inconclusive for SEB and SEC. Since the same zinc-bind-


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ing site has also been demonstrated for both SEC2 and SEC3 (43,471, it appears to be a uniform feature of several SEC subtypes that is not apparently present in other SEs. While severalSEs are known to bind zinc, it is now recognized that the mechanism and location of cation binding by SEC is unique compared to that of SEA, SEE, and possibly SED (41,43,47,48). The crystal structure did not indicate the presence of zinc in SEB, which lacks the SEC zinc-binding motif discussed below. The residues in SEC2 and SEC3 involved in zinc atom coordination are aspartate 83, histidine 118, and histidine 122. The zinc sits in the center of these residues approximately 0.75w above the three liganding atoms; the fourth tetrahedral ligand site of the zinc is not occupied in the SEC3 structure. It is interesting that some of the features of the environment of the zinc atom in SEC are very similar to those found in a group of zinc metalloenzymes,of which thermolysin is its prototype. One shared characteristic is the invariant zinc binding motif H-E-X-X-H (Table 2 ) . This motif contains two histidine coordination sites and encompasses residues 118-122 of SECS. The other property shared with proteases is that zinc is coordinated by only three residues. However, in molecules containing zinc-dependent protease activity, the fourth site is typically occupied by water. SEC3 differs from the proteases in this regard since a water molecule is absent from the zinc atom coordination shell in the SEC3 structure at a resolution of 1.9w (47). The presence of the of zinc site in SEC and its characteristics raise a number of questions regarding its potential significance in the activity of the toxins. Several other protease bacterial exotoxins (Table 2) possess thermolysin-like zinc binding motifs. While in these other cases there is ample evidence that the toxins act as zinc metalloproteases and that metalloprotease activity is required for pathogenesis of the disease, protease activity has not been demonstrated for SEC3. If zinc-mediated catalysis is necessary for SEC activity, it is unlikely to be important for all other SEs that lack the interdomain motif. Furthermore, it is now clear that SEs have evolved at least two mechanisms for zinc coordination. UnlikeSEC, other SEs such as SEA and SEE have an alternate zinc-binding region on the external region of the molecule on domain 2 (46,48) that does not involve a H-E-XX-H motif. There is convincing evidence in these cases that zinc is necessary for binding to MHC class I1 and probably serves as a bridge between the toxin and its receptor.

Bohach

Staphylococcal EnterotoxinsB and C V.

STRUCTURE-FUNCTION RELATIONSHIPS IN TCR-BINDING ACTIVITY

A.

Vs Usage by SEB and SEC

The ability to interact with a defined TCR repertoire, determined by the variable region of the chain (Vp) on the receptors, is a unique characteristic of each superantigen (2). A considerable amount of effort has been devoted toward evaluating the Vp repertoire used by SEB and the various molecular forms of SEC. Although these studies have produced similar results, some discrepancies are noted in the literature. While these could bedue in part to the different techniques used to evaluate the Vp repertoire of T-cell cultures, in some cases the discrepancies result from small amounts of contaminating superantigens. Since staphylococcal and streptococcal strains could potentially produce more than one superantigen, the most reliable comparative results are obtained using recombinant toxins. Recombinant SEB and all three SEC subtypes consistently stimulate cells expressing Vps 12, 13.2, 14, 15, 17, and 20 (Fig. 5). Furthermore SEC1, similar to SEB, stimulates high levels of VP3-bearing cells but not those expressing Vp13.1. SEB and SECl differ from the other SEC subtypes in regard to these two Vp chains since SEC2 and SEC3 cause a high level stimulation of cells expressingVp13.1, but are drastically reduced in Vp3 stimulation compared to SECl (51). B.

SEB and SEC Molecular Regions Involved in TCR Binding

SECl and SEC2 differ by only seven residues but have widely differing affinities in Vp3 and vp13.1 interactions. A cluster divergent residues-near the N-terminus SEC1, which are thought to have been acquired from the gene by recombination (see above), may be responsible for this difference. SECl residues occupying positions 16,20,22, and 26 are identical to SEB, but differ from the analogous residues in SEC2 (and SEC3). Since SEB and SECl are identical in regard to stimulation of T cells expressing Vp3 and Vp13.1, one or more of these residues are presumably critical for this response. A single residue controllingthe inverse ability of SEC subtypes to stimulate these two Vps has been shown to be located at position 26 by site-directed mutagenesis (51). An SECl mutant, in which the valine at position 26 is changed to the corresponding SEC2 residue (tyrosine), induces a profile of T cells identical to that of SEC2 (Fig. 5). Residue 26 is located at the base of the shallow interdomain a 2 or a 3 cavities in the SEB and SEC3 crystal structures, respectively.

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RSEB .SEC1

T

OSECl SEC2 .SEW FR1913

1

2

3

4

5.1

5.2

6

7

8

9

10

11 15 12 1413.2 13.1

16

17

18

I9

Primer Figure 5 Vp expansion profiles for in vitro stimulation of human T lymphocytes induced in vitro by recombinant SEB and SEC subtypes. Vp expression was quantified by a polymerase chain reaction (PCR) technique using primers specific for each major human Vp (51). The PCR value represents the average relative increase in Vp expression induced by each toxin or mutant compared to the basal Vp level (determined upon stimulation with CD3 antisera). Note the inverse stimulation ofVp3 and Vp13.1 by SECl compared to the other SEC subtypes. Also shown are the profiles produced upon stimulation by an SECl single-site substitution mutant in which the residue in position of SECl (valine) was changed to the corresponding residue of SEC2 (or SEC3) indicating the importance of this residue in Vp specificity. (Adapted from Ref. 51.)

This putative TCR-binding cavity is a common feature of all PTs; a similar cavity has been demonstrated in the SEA and TSST-1 crystal structures The critical role of this cavity in TCR recognition by SEC was first suspected when it was shown that SEC-l N-terminal deletions had little effect on T-cell stimulation, provided residues very near or within the a3-helix defining the floor of the TCR binding cavity are not deleted. Since deletion of residues in the SEC a3 helix produced nonmitogenicmutants that were not affected in ability to bind to MHC classI1 (30), the defect was likelyin their TCR-binding ability.

20


183

Studies with SEB have also implicated its analogous a2 cavity in TCR binding. For instance, random mutagenesis produced several mutants that influenced T-cell proliferation, predominantly through VP recognition (52). In particular, the conserved asparagine residue at position 23 was strongly implicated. This residue was later shown to be located in the SEB a2 helix of the TCR binding cavity when the crystal structure was solved (33). The second location implicated was further downstream in the SEB primary sequence at positions 60 and 61. Alteration of these residues produced mutants that had depressed T-cell stimulatory ability. This effect seemed to result from defective TCR recognition, since their ability to bind HLA-DR was unaffected. Hayball et al. (53) extended these observations and showed that it was possible to alter SEB VP specificity by substituting residues 60 and 61. Although these residues are not close to asparagine 23 in the primary sequence, they are brought close together by folding of the SE Ca backbone. This causes residues 60 and 61 to be positioned directly across from the SEB a2 helix (and SEC3 a3 helix) at the top the TCR-binding.cavity. These results,and the results described above for SEC, strongly suggest that several structural components of the TCR-binding cavity are involved in recognition of the VP receptor. C.

Molecular Interactions Between SEB or SEC and the TCR

Despite earlier predictions to the contrary, there is now ample evidence that SEB, SEC, and other superantigens can bind directly to the TCR in vitro and in vivo. The interactions ofSEB and SEC with the TCR have been extensively analyzed in vitro. Seth et al. (54) studied the interaction ofSEB with a soluble TCR a/P heterodimer containing a VP 3.1 P-chain using native polyacrylamide gel electrophoresis and plasmon resonance affinity analysis. They were able to estimate the equilibrium dissociation constant for the SEB:TCR complex (Kd = 0.82 PM). Malchiodi et al. (55) extended these results by comparing the binding ofSEB and SEC subtypes to murine VP 8.2 TCR a/P heterodimer and also to purified TCR P-chain derived by papain digestion. These investigators found that SECI, SEC2, and SEC3 bound to the purified P-chain with Kds ranging from 0.9 to 2.5 PMSeveral regions of the TCR VP element have been reported to have an influence on the interaction with SEB, SEC, and other PT and viral superantigens. These include the complementary-determining region 1 (CDRl), CDR2, and the hypervariable loop (HV4). However, not all three loop regions were implicated in any one study (56-

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Based on the crystal structure of a murine P-chain containing the Vp element, these loops are in close proximity to each other and distal to the transmembrane region One likely possibility, based on the proximity and orientations of these three loop regions, is that all three contribute to TCR binding by the SEs (51). There is currently no indication that carbohydrate residues on the P-chain contribute to the recognition of either SEB or SEC. In fact, an unglycosylated murine VP mutant bound to several SEC subtypes with affinity equal to or greater than that of the native VP chain (55). VI.

MHC

II INTERACTIONS WITH SEB AND SEC

A.

Binding Affinity and Competition Studies

The specific binding of SEs to MHC class I1 molecules on the surface of antigen-presenting cells was reported independently by at least three separate laboratories in Although binding to MHC class I1 is a shared property, it becoming clear that there is a significant degree of heterogeneity in the mechanisms of binding from one superantigen to another. This heterogeneity is observed in regard to: 1. Affinity ofSE binding to MHC class I1

Repertoire of compatible MHC class I1 molecules 3. Molecular regions of MHC class I1 involved in binding to SEs SE molecular regions involving in binding to MHC class I1 5. Requirement formetal atoms The ability of various human and murine MHC class I1 molecules to present SEB, SEC, and various other SEs with different efficiencies has been well documented. In general, HLA-DR1 is the most effective human HLA molecule for SE presentation, although in some SE::MHC::TCR combinations, equal or enhanced T-cell stimulation is produced by other haplotypes The binding affinities of SEB and SECl for HLA-DR are lower than that of SEA but substantially higher than that ofSEE or SED The reported Kd values obtained at for SEB and SECl are lo” M and M, respectively. Suggesting a functional significance for these differences, Chitagumpala et al. (73) noted a correlation between the levels of Tcell proliferation induced by SEA, SEB, and SECl and their binding affinities for MHC class 11, which showed the hierarchy of effectiveness to be SEA > SEB > SEC1. The effect of temperature on SE binding is noteworthy. Whereas SEB and TSST-l binding is affected only minimally by increasingthe temperature the assay conditions from


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4°C to SEA and SECl binding is increased by at least twofold at the higher temperature. This and several other early observations led to the speculation that the mechanisms of binding to HLA-DR by SEA and SECl differ from those ofSEB and TSST-1 Results of competition assays also suggestthat PT evolution has led to several potential types ofSE-MHC class I1 interactions. The similarities between any two toxins in their interactions with MHC partially parallel the degree of molecular relatedness shared by the toxins. SEB and TSST-1, which are not related at the primary sequence level, only partially compete with each other for HLA-DR1. Their binding sites on the receptor overlap but are not identical (see below). In contrast, SEA is an efficient competitor of both toxins. Since SEA-binding sites, a mixture of TSST-1 and SEB does not compete it has been proposed that SEA has multiple binding sites, one of which overlaps with SEB and TSST-1, and one that is unique. While SECl is able to compete with SEB, the entire SEC group of toxins has not been subjected to the same degree of competitive studies as SEA and SEB. Considering that SEB and SECl compete for binding to HLA-DR, plus their extensive sequence and structural homologies, these two toxins likely have at least one MHC-binding site in common to their molecular structures. However, there is increasing evidence that additional unique mechanisms exist forinteraction of both toxins with the receptor. The SEB::HLA-DR1 Crystal Complex

'

The most direct information on the interaction of SEs with MHC class I1 has been provided by Jardetzky et al. who reported the threedimensional crystal structure of the SEB::HLA-DR1 complex (Fig. In their structure, SEB interacts entirely with the a1 domain of HLADRI. The specific site binding on the receptor is a concave surface on the side of the molecule close to, but outside of, the peptide-binding groove. The interface between SEB and the receptor is comprised of hydrophobic and polar regions and involves SEB residues at positions and (see Fig. l) and residues in the HLA-DRl a-chain. The major contacts are provided by SEB residues within four stretches of primary sequence on the outside and toward the top of the small domain of the toxin. This orients the larger P-grasp domain away from the HLA-DR1 a1 chain. The orientation of the SEB in this complex is similar to that TSST-1 in the TSST-1::HLA-DRl complex

Bohach

HLA-DR1

SEB Figure 6 Crystal complex of bound to human HLA-DRI. Relevant portions of the toxin and receptor molecules are indicated. See text for details. (Adapted from Ref. 74.)

with one major exception. Specifically, TSST-l extends further over the top of the a1 domain into the peptide-binding site. Unlike TSST1, does not interact with the'peptide (75). There was no indication that contacts carbohydrate moieties on glycosylated residues


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of the receptor, or that cations facilitate the interaction. There is ample biologic!l data' to validate the SEB::HLA-DR1 crystal complex described aboire. For example, site-directed mutagenesis studies have implicated residues on the outside face of the small domain near and within the disulfide loop as being important for MHC binding. Mutations in SEB affecting residues generally cause a 100- to 1000-fold reduction in SEB binding to MHC class I1 Similarly, severalstudies involving the use of synthetic peptides have predicted a biological significance for this portion of the SEB molecule in interactions with MHC class I1 (76-78). C. Evidence

for a Second Binding Site on SE6 for M H C Class II

Although only one MHC class I1 binding site on SEB has been conclusively demonstrated by crystallographic methods, evidence for the existence of a second binding site continues to accumulate. Several independent investigations have repeatedly implicated residues far removed from the binding interface defined in the crystal complex discussed above. Residuesoutside the TCR binding cavity nearthe Nterminus at positions 14 and 17 of SEB caused nearly a 100-fold reduction in HLA-DR binding Similarly, peptides derived from the N-terminus, and containing residues or compete with SEB for binding to HLA-DR and possibly other MHC class I1 molecules Other peptides derived from sequences within the large domain ofSEB also inhibit binding ofSEB. Although the most consistently implicated residuesare far removed from eachother in the SEB primary sequence, they are close together on the outside edge and back of the large P-grasp domain (according to the view of the SE structure shown in Fig. 3A). Interestingly, there is some overlap between this putative alternative SEB-binding site and that implicated for SEA binding to the receptor (48). However, whereas SEA binding apparently requires zinc, this cation is absent from the SEB crystal structure (33,411. D. Potential Molecular Interactions Between SEC and M H C Class II

Compared to SEB, less is known about the interaction ofSEC with MHC class 11. Considering their extensive sequence identity and data from competition assays discussed above, it is reasonable to predict overlap between the mechanisms by which SEB and SEC bind to the receptor. Althoughits presence has not been confirmed,a binding site

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188

on the outside of the small domain nearly identical to that identified in the SEB::HLA-DRl crystal complex has been suggested for SEC (74). In contrast, one possible differencebetween SEC and SEB is the location of a second potential MHC class I1 binding site. Although it has been predicted that residues in the N-terminus ofSEB and adjacent structures are involved in binding to HLA-DR1, the analogous region ofSEC does not appear to have the same function. For example, N-terminal deletion mutagenesis of SEC affects TCR binding if residues in the a 3 cavity are deleted, but has no significant affect binding to MHC class I1 (30). Furthermore, unlike SEB, N-terminal peptides have not been shown to inhibit SEC1-induced T-cell proliferation (30). On the contrary, biologically active synthetic peptides derived from the SEC1 a 5 groove (residues 74-86 and 148-171) have been proposed to affect T-cell proliferation bybinding to MHC class I1 (30; Fig. 3A). Furthermore, the zinc atom in the a 5 groove base provides, with the empty liganding site, a potential mechanism for MHC class I1 to bind in this region of the toxin (Fig. 3B). This possibility is currently being actively investigated. Although it is still unclear whether SEC binding to MHC class I1 requires zinc, the importance of this cation in binding to SEA and SEE has been confirmed (see above and Ref. 46). VII. A.

SEB AND SEC REGIONS INVOLVED IN THE EMETIC RESPONSE General Considerations in Dissociation of Emetic and Superantigenic Properties

Although patients with SFP present a variety of nonspecific symptoms, the ability ofSEs to induce an emetic response when orally ingested is the hallmark symptom associated with this intoxication. Thus, emesis is the usual indicator for assessing biological relevance to SFP in structure-function studies. However, several complicating factors affect investigation of structural aspects required for emesis. First, the cells and receptors in the gut that interact with SEs following their ingestion have not been clearly defined. Despite the early demonstration that nerve transmission is required, binding of SEs to nerves has not been shown Recent studies have suggested roles for mast cells, inflammatory mediators, and neuropeptide generation in the emetic response (80,81). Also, the expense of the monkey-feeding assay, the only reliable animal model for SE-induced emesis, pre-


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cludes its extensive use. Another shortcoming of in vivo feeding assays in emesis structure-function studies is that, when interpreting results, one must consider that alteration of the protein might reduce its stability in the harsh conditions of the gastrointestinal tract. Thus, the lack of an emetic response by a mutant chemically modified toxin may not necessarily be attributed to their inability to bind appropriate cellular receptors. Another question to consider is whether emesis in SFP is due to the superantigen activity of SEs. Following the demonstration that SEs and other PTs were superantigens and recognition of the importance of superantigen activity in certain diseases of humans and animals, some investigators proposed that gastroenteritis in SFP results from their function as superantigens. In addition, patients with TSS may have gastrointestinal symptoms including vomiting and diarrhea. Despite the circumstantial evidence tothe contrary, most early experimental data, acquired from testing protease-generated SE fragments, suggested that the emetic and T-cell proliferative activities of the SEs .are determined by separate molecular regions (82-84). Alber et al. (85) used an anti-idiotype antibody to provide further evidence that the superantigenic and emetic activities SEB are separable. They found that an antibody directed against the binding site of an SEB-specific monoclonalantibody was nonemetic, but was mitogenic for human and monkey T lymphocytes. Similarly, a carboxymethylated SEB preparation was nonemetic, despite being unaltered compared to native SEB in ability to stimulate T-cell proliferation. Using a mutagenesis approach, Harris et al. (86) reached the same conclusion for SEB. They showed that an SEB mutant altered in its MHC binding site (serine substitution for phenylalanine 44) was emetic but almost completely devoid of mitogenic activity. The same conclusion was obtained in a study of SECl site-directed mutants. Hovde et al. (34) constructed a series of single and double substitutions for the cysteine residues 93 and 110 in SECl. They were unable to demonstrate a correlation between the emetic ability of any class of mutants with T-cell proliferation. B.

SEB and SEC Molecular Regions Implicated in the EmeticResponse

Initial work toward definingthe molecular requirements necessary for SE-induced emesis focused on testingthe biological activities of chemically modified toxins and fragments generated from various SEs,

Bohach

including SECl SEB. Spero and Morlock concluded in 1978 that the N-terminus of SECl was not required for emesis (84). Proteolytic removal of the 59 N-terminal residues produced a polypeptide containing 180 central, disulfide loop, and C-terminal residues, which retained the ability to induce emesis. The disulfide bond appears to contribute indirectly in the emetic response of bothSEB and SECl (34,861. SECl mutants, unable to form the linkage, have been constructed by substituting alanine serine for cysteine at positions 93 or 110. Interestingly, retention of emetic activity by the mutants was dependent upon whether serine alanine substituted for cysteine. Mutants with alanine substitutions were nonemetic, whereas those substituted with serine retained emetic activity when administered to monkeys. These results confirmedthat the disulfide bond is not required for emesis, since serine mutants, unable to form the bond, retained activity. However, the loss of activity in the analogous set of mutants with alanine substitutions suggests that the residues occupying positions 93 and 110 played a critical role in preserving a certain SE structure necessary in emesis. Presumably, the ability of serine to form hydrogen bonds can substitute for the disulfide linkage, thus retaining the proper orientation of critical residues. Based on the requirement for structural stability provided by serine substitutions, a region close to the disulfide linkage is probably needed for emesis. It is unlikely that conformation of the loop itself is responsible for emesis since this region is highly flexible and not conserved in either length composition among the SEs. Furthermore, proteolysis of the SEB and SECl loops does not affect emetic activity (82,841. Instead, it is more likely that the stretch of conserved residues (Fig. 71, immediately downstream from the second cysteine ofSEB and SECl (residues 111-122 in SECl), is involved. These residues form the P5-strand and an adjacent loop in the SEC3 structure; their orientation could be affected by the disulfide linkage. This region is also conserved among nonemeticPT toxins. Thus, one hypothesis is that only toxins with 1) critical residues in this region plus 2) proper orientation of these residues by the disulfide bond are able to induce emesis when orally ingested. Interestingly, histidine residues 118 and 122, which make up the zinc-binding motif of SEC3, are located within this conserved stretch of residues downstream from the disulfide bond. The role of zinc in SEC-induced emesis has not been investigated. However, it is not likely to be a uniform requirement

(A) Figure 7 The disulfide loop and adjacent regions ofSEB and SEC. (A) Schematic diagram of the disulfide loop region of the SEC3 molecule showing details of the conformation of the cysteine-cysteine bond and loop in relation to the downstream conserved residues in the strand (Adapted from Ref. 34). The bound zinc atom in SEC is coordinated by two histidines at positions 118 and 122 within and adjacent to the strand. (B) Sequence alignment of the cysteine loop and downstreamsequences for SEB and the major SEC subtypes. Selected residues are numbered for orientation. Residues in the region downstream from the cysteine loop that are most conserved among all PTs are indicated (*). Arrows designate the SEC histidine residues that are part of the zinc-binding motif. Note that the absence of zinc in the SEB structure is consistent with a substitution of one of the histidine residues to glutamine (position 125).

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Cystine loop residues Downstream conserved residues 4 4 SEC1 93 CYFSSKDNVGKVTGG---KTC 111 M Y G G I T K H E G N H 122 111 M Y G G I T K H E G N H 122 SEC2 93 CYFSSKDNVGKVTGG---KTC SEC3 93 CYFSSKDNVGKVTGG---KTC 111 M Y G G I T K H E G N H 122 SEB 93 CYFSKKTNDINSHQTDKRKTC 114 M Y G G V T E H N G N Q 125

* * * *

*

*

(B)

Figure 7 Continued

in the emetic activity of all SEs since only has the complete binding motif in this position of their structure (Fig. 7B). ACKNOWLEDGMENTS

The author’s efforts in preparation of this manuscript were supported by grants from the Public Health Service(A128401and RR00166), U.S. Department of Agriculture(94-023991, the United Dairymen of Idaho, and the Idaho Agriculture Experiment Station. James Deringer and Jana Joyce assisted in the preparation several figures. Cynthia Stauffacher is acknowledged for critically reviewing portions of the manuscript and for providing original artwork. REFERENCES 1. BohachGA,FastDJ,NelsonRD,

Schlievert PM.Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses. CRC Crit Rev Microbiol 2. Marrack P, Kappler The staphylococcal enterotoxins and their relatives. Science Schlievert PM. Role of superantigens in human disease. J Infect Dis Holmberg SD, Blake PA. Staphylococcal food poisoning in the United States. JAMA BergdollMS.Staphylococcal intoxications. In: Riemann H, Bryan FL, eds. Food-Borne Infections and Intoxications. New York: Academic Press, Begdoll MS, Surgalla MJ, Dack GM. Staphylococcal enterotoxin: identification of a specific neutralizing antibody with enterotoxin neutralizing property. J Immunol Casman EP,BergdollMS, Robinson J. Designation of staphylococcal enterotoxins. J Bacteriol Jablonski LM,BohachGA. Staphylococcus uureus. In: Beuchat L, Doyle M, Montville T, eds. Fundamentals of Food Microbiology. Washington, DC:ASM Press. (in press).


and C

9. Avena RM, Bergdoll MS. Purification and some physicochemical properties of enterotoxin C Staphylococcus aureus strain 361. Biochemistry 1967; 6:1474-1480. 10. Borja CR, Bergdoll MS. Purification and characterization of enterotoxin Cproducedby Staphylococcus aureus strain 137. Biochemistry 1967; 6:1467-1473. 11. Marr JC, Lyon JD, Roberson JR, Lupher M, Davis WC, Bohach GA. Characterization of novel type C staphylococcalenterotoxins: biological and evolutionary implications. Infect Immun 1993; 61:4254-4262. 12. Betley MJ, Borst DW, Regassa LB. Staphylococcal enterotoxins, toxic shock syndrome toxinand streptococcal pyrogenic exotoxins: a comparative study of their molecular biology. Chem Immunol 1992; 55:l-35. 13. Jones CL, Khan SA. Nucleotide sequence of the enterotoxin B gene from Staphylococcus aureus. J Bacteriol 1986; 166:29-33. 14. Bohach GA, Schlievert PM. Nucleotide sequence of the staphylococcal enterotoxin C gene and relatedness to other pyrogenic toxins. Mol Gen Genet 1987;209:15-20. 15. Huang IY, Bergdoll MS. The primary sequence of staphylococcal enterotoxin B: the cyanogen bromide peptides of reduced and aminoethylated enterotoxin B, and the complete amino acid sequence. J Biol Chem 1970; 245:3518-3525. 16. Schmidt JJ, Spero J. The complete amino acid sequence of staphylococcal enterotoxin Cl. J Biol Chem 1983; 258:6300-6306. 17. Hovde CJ, Hackett SP, Bohach GA. Nucleotide sequence of the staphylococcal enterotoxin C3 gene: sequence comparison of all three type C staphylococcal enterotoxins. Mol Gen Genet 1990; 220:329-333. Couch JL, Betley MJ. Nucleotide sequence of the type C3 staphylococcal enterotoxin gene suggests that intergenic recombination causes antigenic variation. J Bacteriol 1989; 171:4507-4510. 19. Bohach GA, Dinges MM, Mitchell DT, Ohlendorf DH, Schlievert PM. Staphylococcal exotoxins In: Archer GL, Crossley KB. Staphylococci and Human Diseases Churchill Livingstone, New York (in press). 20. Dickie N, Yang Y, Robern H, Stavric S. On theheterogeneity of staphylococcal enterotoxin C2. Can J Microbiol 1972; 18:801-804. 21. Hynes WL, Weeks CR, Iandolo JJ, Ferretti JJ. Immunologic cross-reactivity of type A streptococcal exotoxin (erythrogenic toxin) and staphylococcal enterotoxins B and Cl. Infect Immun 1987; 55:837-838. 22. Bergdoll MS, Borja CR, Robbins R, Weiss KF. Identification of enterotoxin E. Infect Immun 1971;4:593-595. 23. Gruber J, Wright GG. Ammonium sulfate coprecipitation antibody determination with purified staphylococcal enterotoxins. J Bacteriol 1969; 99:18-24. 24. Spero L, Morlock BA. Cross-reactivity between tryptic polypeptides of staphylococcal enterotoxins B and Cl.J Immunol 1979; 122:1285-1289. 25. Thompson NE, Ketterhagen MJ, Bergdoll MS. Monoclonal antibodies to

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staphylococcalenterotoxins B and C: cross-reactivity and localization of epitopes on tryptic fragments. Infect Immun 1984; 45:281-285. 26.Bohach GA, Handley JP, Schlievert PM. Biological and immunological properties of the carboxyl terminus of staphylococcal enterotoxin Cl. Infect Immun 1989; 57:23-28. 27. Turner TN, Smith CL,BohachGA. Residues 20,22, and 26 determine subtype specificities of staphylococcal enterotoxins C3 and C2. Infect Immun 1992; 60:694-697. 28. Bohach GA, Hovde CJ, Handley JP, Schlievert PM. Cross-neutralization of staphylococcaland streptococcalpyrogenic toxins by monoclonal and polyclonal antibodies. Infect Immun 1988; 56:400-404. 29. Bergdoll MS, Borja CR, Avena RM. Identification of a new enterotoxin as enterotoxin C. J Bacteriol 1965; 90:1481-1485. 30. Hoffmann ML, Jablonski LM, Crum KK, Hackett SP, Chi Y-I, Stauffacher CV, Stevens DL, Bohach GA. Predictions of T cell receptor and major histocompatibility complex binding sites on staphylococcal enterotoxin Cl. Infect Immun 1994; 62:3396-3407. 31. Jett MR,Neil1 R, Welch C, Boyle T, Bernton Hoover D, Lowell G, Hunt RE, Chatterjee S, Gemski P. Identification of staphylococcal enterotoxin B sequences important for induction of lymphocyte proliferation by using synthetic peptide fragments of the toxin. Infect Immun 1994; 62:3408-3415. 32. Hamad ARA, Herman A, Marrack P, Kappler J W .Monoclonal antibodies defining functional sites on the toxin superantigen staphylococcal enterotoxin B. J Exp Med 1994; 180:615-621. 33. Swaminathan Furey W, Pletcher Sax M. Crystal structure of staphylococcal enterotoxin B, a superantigen. Nature (Lond) 1992; 359:801-806. 34. Hovde CJ, Marr JC, Hoffmann ML, Hackett SP, Chi Y-I, Crum KK, Stevens DL, Stauffacher CV, Bohach GA. Investigation of the role the disulfide bond in activity and structure of staphylococcal enterotoxin Cl. Mol Microbiol 1994; 139397-909. 35. Singh BR, Evenson ML, Bergdol, MS. Structural analysis of staphylococcal enterotoxins B and C1 using circular dichroism and fluorescence spectroscopy. Biochemistry 1988;2793735-8741. 36. Middlebrook JI, Spero L, Argos P. The secondary structures of staphylococcal enterotoxins A, B, and C. Biochim Biophys Acta 1980; 621:233240. 37. Singh BR, Kokan-Moore W, Bergdoll MS. Molecular topography toxic shock syndrome toxin as revealed by spectroscopic studies. Biochemistry 1988; 27:8730-8735. 38. Munoz PA, Warren JR, Noelken ME. p structure of aqueous staphylococcal enterotoxin B by spectropolarimetry and sequenced based conformational predictions. Biochemistry 1976; 15:4666-4671. 39. Singh, BR, Evenson, ML,Bergdoll, M S Structural analysis of staphylococcal enterotoxins B and C l using circular dichroism and fluorescence spectroscopy. Biochemistry 1988;2793735-8741.


Crystal and solution structures of 40. Singh, BR, Fu, F-N, Ledoux, DN. superantigenic staphylococcal enterotoxins compared. Nature Struct Biol 1994;1:358-360. 41. Schad EM, Zaitseva I, Zaitsev VN, Dohlsten M, Kalland T, Schlievert PM, Ohlendorf DH, Svensson LA. Crystal structure of the superantigen staphylococcal enterotoxin type A. EMBO J 1995; 14:3292-3301. 42. Prasad GS, Earhart CA, Murray DL, NovickRP, Schlievert PM, Ohlendorf DH. Structure of toxic shock syndrome toxin 1. Biochemistry 1993;32:13761-13766. 43. Papageorgiou AC, Acharya KR, Shapiro R, Passalacqua EF, Brehm RD, Tranter HS. Crystal structure of the superantigen enterotoxin C2 from Stuphylococcus uureus reveals a zinc-binding site. Structure 1995; 3:769779. 44. Swaminathan Furey W, Pletcher J, Sax M. Residues determining Vp specificity in staphylococcal enterotoxins. Nature Struct Bioll995; 2:680686. 45. Murzin AG. OB(oligonucleotide/oligosaccharidebinding)-fold: common structural and functional solution for non-homologous sequences.EMBO J 1993;12:861-867. 46. Fraser JD, Urban RG, Strominger JL, Robinson H. Zinc regulates the function of two superantigens. Proc Natl Acad Sci 1992; 89:55075511. 47. Bohach, GA, Jablonski, LM, Deobald CF, Chi, YI, and Stauffacher. CV. Functional domains of staphylococcal enterotoxins. In: Ecklund M, Richard JL, Mise K, eds. Molecular Approaches to Food Safety; Issues Involving Toxic Microorganisms. Fort Collins, CO: Alaken Inc, 1995:339356. 48. Fraser JD, Lowe S, Irwin MJ, Gascoigne NRJ, Hudson KR. Structural model of staphylococcal enterotoxin A interaction with MHC class I1 antigens. In: Hubert BT, Palmer E, eds. Superantigens: A Pathogen’s View of the Immune System. Plainview, NY: Cold Spring HarborLaboratory Press, 1993:7-30. 49. Klimpel, KR, Arora N, Leppla, SH. Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity. Mol Microbiol 1994; 13:1093-1100. Stauffacher CV, Ohlendorf DH,ChiY-I,VathGM, 50. Bohach, Schlievert PM. The staphylococcal and streptococcal pyrogenic toxin family. In: Singh BR, Tu AT, eds. Natural Toxins 11. New York: Plenum Press, 1995:131-154. 51. Deringer JR, Ely RJ, Stauffacher CV, Bohach GA. Subtype-specific interactions of the type Cstaphylococcal enterotoxin with the T cell receptor. Mol Microbiol; in press. 52. Kappler JW, Herman A, Clements Marrack P. Mutations defining functional regions of the superantigen staphylococcal enterotoxin B. J Exp Med 1992; 175:387-396.

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OHehir RE, Verhoef A, Lamb JR, Lake RA. 53. Hayball JD, Robinson Identification of two binding sites in staphylococcal enterotoxin B that confer specificity for TCR VP gene products. Int Immunol 1994; 6:199211. 54. Seth A, Stem LJ, Ottenhoff THM, Engel I, Owen MJ, Lamb JR, Klausner RD, Wiley DC. Binary and ternary complexes between T-cell receptor, class I1 MHC, and superantigen in vitro. Nature 1994; 369324-327. 55. Malchiodi, EL, Einstein E, Fields BA, Ohlendorf DH, Schlievert PM, Karjalainen K, Mariuzza RA. Superantigen binding to a T cell receptor $ chain of known three-dimensional structure. J Exp Med 1995; 182:18331845. 56. Choi Y, Herman A, DiGusto D, Wade T, Marrack P, Kappler J. Residues the variable region of the T-cell-receptor P-chain that interact with S. uureus toxin superantigens. Nature 1990; 346:471-473. 57. Gahm SJ, Fowlkes BJ, Jameson SC, Gascoigne NRJ, Cotterman MM, Kanagawa 0, Schwartz RH, Matis LA. Profound alteration in an Tcell antigen receptor repertoire due to polymorphism in the first complementarity-determining region of the p chain. Proc Natl Acad Sci USA 1991;88:10267-10271. 58. BellioM, Lone YC, de la Calle-Martin 0, Malissen B, Abastado JP, Kourilsky P. The VP complementarity determining region 1 of a major histocompatibility complex (MHC) class I-restricted T cell receptor is involved in the recognition of peptide/MHC I and superantigen/MHC I1 complex. J Exp Med 1994; 179:1087-1097. 59. White J, Pullen A, Choi K, Marrack P, Kapper JW. Antigen recognition properties of mutant V$3 T cell receptors are consistent with an immunoglobulin-like structure for the receptor. J Exp Med 1993; 177:119-125. 60. Patten PA, Rock EP, Sonoda T, de St. Groth BF, Jorgensen JL, Davis MM. Transfer putative complementarity-determining region loops of T cell receptor V domains confers toxin reactivity but not peptide/MHC specificity. J Immunol 1993; 150:2281-2294. 60a. Kang J, Chambers CA, Pawling J, Scott C, Hozumi N. Conserved amino acid residues in the complementarity-determining region 1 of the TCR P-chain are involved in the recognition of conventional Ag and Mls-l superantigen. J Immunol 1994; 152:5305-5317. 61. Herrman, T.,T. Hochgrebe, N.E. Torres-Nagel, B. T. Huber, and T. Hllnig. Controlof the rat T cell response to retroviral and bacterial superantigens by class I1MHC products and Tcrb-V8.2 alleles. J Immunol 1994;152:4300-4309. 62. Gold DP, Surh CD, Sellins KS, Schroder K, Sprent J, Wilson DB. Rat T cell responses to superantigens. 11. Allelic differences in VP8.2 and Vp 8.5 chains determine responsiveness to staphylococcal enterotoxin B and mouse mammary tumor virus-encoded products. J Exp Med 1994; 179:63-69. 63. Pullen AM,Bill J, Kubo RT, Marrack P, Kappler JW. Analysis of the


64.

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75. 76.

77. 78.

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interaction site for the self superantigen Mls-la on T cell receptor VP. J Exp Med 1991;173:1183-1192. Cazenave, PA, Marche PN, Jouvin-Marche E,Voegtlc!D, Bonhomme F, Bandeira A, Coutinho A. VP gene polymorphism in wild-derived mouse strains: two amino acid substitutions in the VP17 region greatly alter T cell receptor specificity. Cell 1990; 63:717-728. Pullen AM, Wade T, Marrack P, Kappler JW. Identification of the region of T cell receptor p chain that interacts with the self-superantigen Mls-la. Cell 1990;61:1365-1374. Irwin MJ, Hudson KR, Ames KT, Fraser JD, Gascoigne NRJ. T-cell receptor P-chain binding to enterotoxin superantigens. Immunol Rev 1993; 131:61-78. Bentley GA, Boulet G, Karjalainen K, Mariuzza RA. Crystal structure of the chain of a T cell antigen receptor. Science 1995; 267:1984-1987. Mollick JA, Cook RG,Rich RR. Class I1 MHC molecules are specific receptors for staphylococcal enterotoxin A. Science 1989; 244:817-821. Fraser JD. High affinity binding of staphylococcal enterotoxin A and B to HLA-DR. Nature 1989; 339:221-223. Fleischer B. Schrezenmeier H, Conradt P. T lymphocyte activation by staphylococcal enterotoxins: role of class I1 molecules and T cell structures. Cell Immunol 1989; 120:92-98. Herman A, Croteau G, S6kaly R-P, Kappler J, Marrack P. HLA-DR alleles differ in their ability to present staphylococcal enterotoxions to T cells. J Exp Med 1990; 172:709-717. Mollick JA, Chintagumpala M, Cook RG, Rich RR. Staphylococcal exotoxin activation of T cells. J Immunol 1991; 146:463-468. Chintagumpala MM, Mollick JA, Rich RR. Staphylococcal toxins bind to different sites on HLA-DR. J Immunol 1991;147:3876-3881. Jardetzky TS, Brown JH, GorgaJC, Stern LJ, Urban RG, Chi Y-I, Stauffacher CV, Strominger JL,Wiley DC. 1994. Three-dimensional structure of a human class I1 histocompatibility molecule complexed with superantigen. Nature (Lond) 1994;368:711-718. Kim J, Urban RG, Strominger JL, Wiley DC. Toxic shock syndrome toxin-l complexed with a class I1 major histocompatibility molecule HLA-DRl. Science. 1994;266:1870-1874. Jett M,Neil1 R, Welch C,Boyle T, Bernton Hoover D, Lowell G, Hunt RE, Chaterjee S, Gemski P. Identification of staphylococcal enterotoxion B sequences important for the induction of lymphocyte proliferation by using synthetic peptide fragments of the toxin. Infect Immun 1994;62:3408-3415. JM, Johnson HM. Multiple binding sites on the superantigen, staphylococcal enterotoxin B, imparts versatility in binding to MHC class I1 molecules. Biochem Biophys Res Commun 1994; 201:596-602. Komisar JL, Small-Harris S, Tseng J. Localization of binding sites of staDhvlococca1 enterotoxin B (SEB), . . . a suuerantigen, for HLA-DR1 by

Bohach

79. 80. 81.

82. 83. 84. 85.

86.

inhibition with synthetic peptides of SEB. Infect Immun 1994; 62:47754780. Beery JT, Taylor Schlunz LR, Freed RC, Bergdoll MS. Effects of staphylococcal enterotoxin A,pn the rat gastrointestinal tract. Infect Immun 1984; 44:234-240. Scheuber PH, Golecki JR, Kickhofen B, W e e l D, Beck G, Hammer DK. Cysteinyl leukotrienes as mediators of staphylococcal enterotoxin B in the monkey. Eur J Clin Invest 1987; 17:455-459. Jett M, Brinkley W,Neil1 R, Gemski P, Hunt Staphyl~~occus uureus enterotoxin B challenge monkeys: correlation of plasma levels of arachidonic acid cascade products with occurrence of illness. Infect Immun 1994; 58:3494-3499. Spero L, Metzger JF, Warren ]R, Griffin, BA. Biological activity and complementation the two peptides of staphylococcal enterotoxin B formed by limited tryptic hydrolysis. J Biol Chem 1975; 250:5026-5032. Noskova VP, Ezepchuk Noskov AN. Topology of the functions in molecule of staphylococcal enterotoxin type A. Int J Biochem 1984; 16:ZOl-206. Spero L, Morlock BA. Biological activities of the peptides of staphylococcal enterotoxin C generated by limitedtryptic hydrolysis. J Biol Chem 1978; 2538787-8791. Alber G, Hammer D, Fleischer B. Relationship between enterotoxic- and T lymphocyte-stimulating activity of staphylococcal enterotoxin B. J Immunol 1990; 144:4501-4506. Harris TO, Grossman D, Kappler JW, Marrack P, Rich RR, Betley, MJ. Lack of complete correlation between emetic andT-cell-stimulatory activities of staphylococcal enterotoxins. Infect Immun 1993; 61:3175-3183.

Staphylococcal Enterotoxins A, D, and E Structure and Function, Including Mechanism of T-cell Superantigenicity

L. Anders Svensson and Elinor

Schad

Michael Sundstrom &

Per Antonsson, Terje Kalland, and Mikael Dohlsten &

INTRODUCTION

Staphylococcal enterotoxins (SEs) are a family of structurally related exotoxin molecules produced by certain Gram-positive Staphylococcus aureus bacterial strains. SEs are a major cause of food poisoning and are involved in bacterial Gram-positive shockin humans. SEs bind to major histocompatibility complex (MHC) class I1 molecules on antigen-presenting cells (APCs) and subsequently activate a large fraction, 5-20%, T lymphocytes (1).This property has led to their classification of superantigens (SAg). The T cells are activated by SAg to proliferate and produce cytokines such as interleukin-2 (IL-21, inter199

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ferony (IFNy), and tumor necrosis factor-a and p (TNF-a and p) (2,3). Depending on origin, superantigens can be divided in two groups, viral and bacterial. reviews see Refs. 4-8. SEs can be divided into distinct serological types, SEA, SEB, SECl-,, SED, SEE, and SEH. Toxic shock syndrome toxin-l (TSST-l), another superantigen secreted by S. uureus, is also structurally related to the super family (Table 1).Comparison of sequence homologies allows division the SEs into two sub families. SEB and SEC,, have marked homology, forming one subfamily while SEA, SED, SEE, and SEH. forma second subfamily (7).The second subfamily is characterized by the presence of a Zn2+binding 'motif and coordination of Zn2+ in the SE molecule seems tobe required for efficient binding to MHC class I1 molecules (9,lO).Recently, the structures several bacterial superantigens have been determined by X-ray crystallography including SEA (111, SEB (121, SEC, (13,14), and TSST-1 (15,16). In addition, the crystal structures ofSEB complexed with the human MHC class I1 molecule HLA-DRl (17)and TSST-l complexed with HLA-DR1 (18) have been determined recently. The binding of superantigens to MHC class I1 molecules differs from normal antigen presentationwhere peptide fragments are bound into the MHC peptide-binding groove and presented to the T-cell receptor (TCR) (19). Superantigens bind, instead, as unprocessed proteins outside the antigen-binding grooveof the MHC class I1 molecules (17,18,20). SEA is the biologically most potent of the SEs and binds stronger to HLA-DR than SEB does (Table1).The crystal structure ofSEB and HLA-DR1 in complex showed that SEB binds to the side of the a-chain of the MHC class I1 molecule (17). In contrast, a number observations suggest that SEA contain two separate binding sites, one these binds in a Zn2+-dependentmanner to the chain of the MHC class I1 molecules, while the other SEB-like site interacts with the MHC class I1 a-chain (10,11,21-28). The immune system expresses three extremely polymorphic recognition receptors, the TCRs, the MHC/peptide complexes, and antibodies (Ab). The interplay between the mammalian immune system and bacteria has led to the evolution of the family of SAg immune recognition ligands, which have targeted two of the polymorphic immune recognition receptors.A certain degree of polymorphism has also developed within the SAg family. The SAg-TCR interaction is most likely the main driving force for this variability since each bacterial SAg displays a characteristic pattern ofTCR VP chain interaction. Recently, structural information has provided evidence that considerable variability exists in the mode of interaction with MHC class 11. Thus certain SAg (such as SEB) bind mono-

Toxin

S

I 233

10

23

3

8.6

23

31

§

7. 7.

228 230

53 81

SE TSST-1

5.7 7.2

218 194

37 24

7.3

S

SEC2 2 S

n.d. = So~~~e: et al. (

~~h~~~ et al. (47) nd r e ~ e r e ~ there ~es

1.1, 5.3, 6.3, 6

2

i

(3), 7, 8.1, 8.2, 8.3, W, 17 (3), 8.2, 10, 17

n.d.

ley et al. (7) (size and se for SEH are taken from

3, 8.2, 8.3, 11, 17 11, 15, 17

5, 12 5.1, 6.3, 6.4, 6.9, 8.1, 18

n.d. 2

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202

valently to one of the HLA-DR chains; others such as TSST-1, Seem to interact with the bound antigenic peptide as well as HLA-DR chains. Further, the Zn2+-dependentSEA/D/E subfamily is suggested to bind bivalently to HLA-DR. Among the Zn2+-dependentSAg we will discuss two bivalent binding patterns, one exemplified by a tentative trimeric DR,-SEA-DR, complex and another pattern by a DR,SED-SED-DR, tetrameric complex. Certain such as SEA and SEE show very high homology (82%). The variability between these two molecules has accumulated especiallyon exposed surface residues. We analyzed the human antibody response to these homologous toxins to record possible minor differences in antigenicity. Interestingly, a very low cross-reactivity between polyclonal human anti-SEA antibodies and SEE was noted, suggesting that escape from antibody recognition motifs in SEA has served as a driving force for the evolution of SEE-related variability. In this chapter we will discuss the structural features within the SEA/D/E subfamily and pinpoint the role of sequence divergences in changing the specificity of these proteins. Apparently, all three immune recognition receptors, TCR, MHC class I1 and Ab, have driven the evolution of extensive polymorphism both within this subfamily and in an interfamily (SEA/B) SAg perspective. THESTRUCTURE OF SEA

The crystallographic structure ofSEA has been determined to high resolution from two different crystallization conditions. Crystal form 1, of space group P2,, was grown at pH 6.4 in a solution containing 250 mM MES buffer, 20% polyethyleneglycol (PEG) and 60 PM CdS04. In crystal form 2, of space group P3,21, of SEA was cocrystalked with Zn2+in the pH range 5.9-6.5, the higher pH giving the best crystals. Further, 0.1 mM ZnS04 was added to the protein solution. In general, the two SEA structures (SEA crystal form 1 and 2) $re very similar with an root-mean-squares deviation (RMSD) of 0.66 A comparing 220 C,-atom pairs. A.

The Protein Fold

The two SEA structures reveal the same type of fold as that found in SEB, and TSST-l, characterized by two tightly packed domains that form a relatively flat molecule (Fig. 1). Domain 1, containing residues 31-116, is a closed P-barrel capped byan a-helix. In addition, domain 1contains a disulfide bridge between residues96 and 106. The

Staphylococcal EnterotoxinsA, D, and E

203

Figure 1 A Schematic representation of staphylococcal enterotoxin A produced by the MOLSCRIPT (87) program. The molecule shown is a model combined of the two crystal forms. The secondary structure elements, the disulfide bridge, and the Zn2+ion together with the coordinating residues are indicated. The likely TCR-binding region along with the low- and high-affinity MHC class I1 binding regions are indicated as TCR, M1,and M2, respectively.

nine amino acid residues intervening between the two cysteines form a highly exposed and mobile loop structure. The fold of domain 1has been observed in other proteins secreted by bacterial pathogenicproteins, which all bind oligosaccharides such as the staphylococcal nuclease the B-subunits of heat-labile enterotoxin and verotoxin-l The oligosaccharide-binding regionof these proteins corresponds well with a homologous MHC class I1 binding region described for SEB. Domain 2 ofSEA contains a P-grasp motif, an a-helix packed against a mixed P-sheet that connects the peripheral strands The P-grasp fold in SEA is formed by P strands and a-helix Moreover, the P-grasp fold is modified by an insertion between P-strand

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10 and 11. This insertion folds back toward domain 1 and forms an irregular a-helix (a-51, which is involved in the packing of the two domains. The P-grasp motif has been found (35) in immunoglobulinbinding domains from protein G. Streptomyces griseus (36,371, and protein L, Peptostreptococcus mugnus (38). Other proteins are Ubiquitin, c-Rafl, Ras-binding domain, and 2Fe-2S ferredoxin (35). In contrast, the immunoglobulin-binding domain B in protein A from S. uureus does not show the P-grasp fold (39). The fold domain 2 in SEA is completed with the 30 proximal tail residues. In the distal end of this N-terminal tail the a-helix 2 is close to the interface with domain 1, while the proximal part of the tail is embracing the P-sheet in the P-grasp fold. There is a one-turn a-helix (a-N) in the crystal form 1 structure, and in the same crystal form, the N-terminal amino group is involved in direct coordination of a bound zinc ion.

B.

Interactions Between SEs and MHC Class I I Molecules

The SEs bind to common structural motifs on MHC class I1 molecules (28,40,41). However, different isotypesof human MHC class 11, HLADP, HLA-DQ, and HLA-DR show preferences for individual SEs. Thus SEA, SEB, SED, SEE, and TSST-1 bind predominantly to HLADR while SEC, binds to HLA-DQ and SEC, to HLA-DR and HLADQ (42,431. In addition, certain HLA-DR alleles show heterogeneity in binding to particular SEs. This is particularly pronounced DRw53, which does not bind to SEA (24,26,44) but displays an intact affinity for SEB SEC. Most likely this discrepancy reflects an HLA-DR @-chain His/Tyrsubstitution, which disables the function of a critical zinc bridge betweenSEA and HLA-DR. Presently, two crystal structures SEs in complex with MHC class I1 molecules have been published. The published SEB/HLA-DR1 (17) and TSST-1/HLADR1 (18) structures seem to differ markedly in the binding to the HLA-DR a$-heterodimer/peptide complex. The superantigens do not compete for the same subsets of HLA-DR1 molecules (451, possibly explained by the interaction between the MHC-bound peptide and the TSST-1 molecule (45,461. The complex ofSEB and HLA-DR demonstrated that SEB have one binding site for MHC class I1 molecules, while several observations suggest that SEA contain two distinct binding sites. Binding studies have shown that SEA, SEE, and SED compete with SEB and TSST-l in binding to HLA-DR1 (45) while SEB and TSST-l do not inhibit SEA, SEE, SED binding. This indicates the existence one


and E

205

common binding region on HLA-DR for these SEs as well as additional binding region for the SEA subfamily (25). Synthetic peptides from the P-chain of mouse MHC class I1 isotype A (I-Ap), comprising residues 65-85, inhibited SEA binding to a MHC class 11+B-cell lymphoma, suggesting a role for the MHC class I1 P-chain in binding to SEA. Further, peptides of the MHC class I1 a-chain bound to SEA but did not block SEA binding to MHC class 11+ cells. It was concluded that the P-chain is both necessary and sufficient for SEA binding whereas the a-chain is a minor binding site that is not required for SEA binding (22,47). Herman et al. (24) andKarp and Long (26) used HLA-DR mutants to determine a role of residue His81 in the HLA-DR P1 domain in binding to SEA and SEE. Similarly, Jorgensen et al. (48) showed by site-directed mutagenesis of murine I-Ek MHC class I1 molecules the importance of the residuesHis 8lP and Glu 690 in binding and presentation of SEA. C. The High-Affinity MHC Class I I Binding Site

A recombinant C-terminal fragment of SEA containing residues 107233 was shown to bind to HLA-DR but failed to activate T cells (21). In addition, SEA and SEE binding was shown to be abolished in the presence of EDTA (9). Site-directed alanine substitution mutagenesis has confirmed that Zn2+is directly bound to SEA and that the major coordinating residues are His 187, His 225, and Asp 227 situated in domain 2. Substituting these residues abrogated Zn2+binding and reduced the binding to MHC class I1 molecules between 10- (His 187) and 10,000-fold (His 225, Asp 227) (10,271. The crystal structure determination ofSEA confirmed the proposed Zn2+ligands. Zinc is bound on the surface of the p-grasp motif in both crystal forms of SEA; however, some differences in the coordination of the zinc ion is observed. 1. ZincCoordination in

CrystalForm 1

The octahedral metal coordination seen in crystal form1SEA was first described with Cd2+ replacingthe naturally bound Zn2+ ion. Anidentical coordination figure was, however, seen in crystals grown with Zn2+ions bound to the protein (Schad et al., in preparation). Maximum resolution of the data from the Cd2+-and the Zn2+- containing crystals to form 1 is 1.9 and 2.2 A, respectively. In crystal form 1the Zn2+ ion ligated by the atoms Serl N (the a-amino group). Ser-l O y, His -187N,, His 225 N,, Asp-227 O,,, and, in the sixth position, a weakly bound oxygen atom of a water mol-

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ecule (Fig. 2a). The coordination by the residue 1 amino group was unexpected, but earlier experiments using large synthetic peptides SEA have implicated a role of the N-terminal in binding to MHC class I1 molecules (49-51). The Zn2+site is located toward the N-terminal part of @-strand12 on the surface of the P-grasp motif (Fig. 1). With small deviations when Ser-l is involved, the coordination figure octahedral. The deviations are caused by restraints from intramolecular bonds in the serine residue. Octahedral coordinationis unusual when zinc ions are bound by biological macromolecules. For structural zinc sites tetrahedral coordination seems to be the most common motif (52,531. In contrast, zinc ions bound to proteins and involved in catalysis do increase their coordination number momentarily during the catalytic process.

&

(a)

Figure 2 a ) The domain 2 zinc sit of crystal form 1 SEA. The residues His187, His-225, Asp-227, ser-l, and a loosely bound water molecule (W) form an octahedral coordination. b) The same site in crystal form 2 SEA. The residues His-187, His-225, Asp-227 and, from another molecule in the crystal lattice, His-61 form a tetrahedral coordination site.


A,

D, and

207

Figure 2 Continued

over, octahedrally coordinated zinc ions have frequently been observed in small molecules (54). In crystal structures the mobility of each atom indicates the strength in the interactions (lower temperature factors favor a tighter interaction). The crystallographically determined temperature factors of the side-chain residues that coordinate the zinc ion correlate with theresultsobtained from site-directed mutagenesis experiments (10,271. The least mobile, strongest bound, atoms are Asp-227 and His-225 N,. The most mobile, less tightly bound, atoms are the water oxygen, His-l87 NSl, Ser-l N, and Ser The tendency is the same in the two independent molecules in the SEA crystals.

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208

Site-directed mutagenesis showed, in addition to residues His187, His-225, and Asp-227, that Asn-128 also has a significant influence on the binding to MHC class I1 molecules (10). From the crystal structure ofSEA it is inferred that Asn-128 N,, hydrogen binds Asp-227 The bond is relatively strong, as shown by the bond length of 2.9 A. Collectively this suggests that the hydrogen bond of Asn-128 plays a role in directing the Asp-227 carboxyl group for an optimal ligation of the Zn2+ion. Alternatively, the Asn-128 residue may serve as a contact residue for MHC class I1 molecules. Mutation of the Asn to Ala would possibly result in loss of important interface hydrogen bonds. 2. ZincCoordination in

CrystalForm

The two independent SEA structures (crystal form 1 and 2) are very similar; however, one major difference is that the N-terminal nine residues in crystal form 2 lack electron density and subsequently are not involved in the coordination of the bound metal ion. In molecule 1 of the asymmetric unit, the amino acid residues His-187, Asp-225, and His-227, located on the surface of the P-sheet in domain 2 of the protein, coordinates the Zn2+ion. His-61 from a symmetry-related molecule is the fourth ligand and thus a normal tetrahedral coordination of the Zn2+ion is seen (Fig. 2b). This intermolecular coordinating Zn2+unit shows metal-ligand distances and coordination angles well within the range normally seen for tetrahedrally coordinated zinc ions. The symmetry molecule interaction in crystal form 2 seems to stabilize the loop region 59-63, which was not visible in the SEA crystal form 1 structure. In the asymmetric unit of SEA molecule 2 the three strong ligands His-187, Asp-225, and His-227 coordinate the metal ion and the fourth ligand apparently is a water molecule. Again, no involvement from the N-terminus is seen. Thus, in crystal form 2 both molecules in the asymmetric unit have normal tetrahedral coordination of the Zn2+. D. Zinc-Mediated SEA-MHC Class II Interactions

Histidine side chains are excellent binders of zinc ions. A probable explanation for the importance the His-8lP residue in HLA-DR1 and the metal coordination site in SEA for the SEA-DRl binding affinities is a Zn2+-modulatedinterface. In this interface the HLA-DR His-81P residue is most likely involved in direct coordination of the Zn2+ion bound to the SEA molecule (10,11,27). This connection is


A, D, and

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crucial for the complex to form, and substitutions of the involved residues lower the binding affinities drastically. Hudson et al. (27) have shown that extensive washing of the SEA-Znz+/HLA-DR complex with high concentrations of EDTA, which was sufficient to strip Zn2+from uncomplexed SEA, failed to remove Zn2+from the complex. This strongly implies that the Zn2+ion is sandwiched between the two molecules in the complex. A high-affinity binding site, formed by domain 2 ofSEA and the P-chain of HLA-DR, locks the Znz+ ionin the complex. A similar example of an intermolecular modulation of a Zn2+-bindingaffinity is found in the crystal structure of the growth hormone-prolactin receptor complex (55). E. The N-terminal of SEA

The N-terminus behaves differently in the two crystal forms of SEA. In crystal form 1the first nine residues are visible in the electron density maps while in crystal form 2 they are invisible. Why are differences seen, and do they influence the characteristics in the interaction with MHC class I1 molecules? The N-a atom of the N-terminal residue needs to be uncharged to be able to participate in coordination of a Zn2+ion as in crystal form 1. Crystal form 1 ofSEA only grows at high pH (6.4), while crystals of form 2 can grow also at low pH (down to 5.9). Therefore, a difference in pH duringcrystallization can, to some extent, explain the difference between the two structures. Crystal packing interaction is another factor that may influence the N-terminal conformation. In crystal form 2 the environment around the two zinc ions differs. At the zinc site of molecule 1 no symmetry-related molecules, which could stabilize the N-terminal, are found. In molecule 2, of the same crystal form, the zinc ion is coordinated by His-61 from a symmetryrelated molecule. The coordination by His-61 mayhave driven the Nterminal away from .the zinc ion. In contrastto crystal form 2, the Nterminal in crystal form 1 does interact with other molecules. There are both a hydrogen bond interaction and several hydrophobic interactions between residues in the N-terminal and symmetry-related molecules. The crystal form 1 structure of SEA demonstrate a role for the N-terminal in Znz+ coordination. However,the N-terminal has a high mobility, which suggests that it may easily be removed during docking ofSEA to the MHC class I1 molecule. Thus the N-terminal may have a dynamic role during SEA-MHC interaction. Indeed, it has been reported that deletions of the first three residues in SEA reduce

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the MHC class I1 binding affinity threefold (56). Synthetic peptides of residues 1-45 in SEA have also been shown to bind to HLA-DR (50). Recently, Kozono et al. (57) demonstrated a peptide dependency for SEA binding to MHC class 11. It is tempting to suggest that the docking of SEA to the HLC-DR P-chain forces the N-terminal tail to move away from the zinc-binding site and possibly participate in interaction with MHC-bound peptides. F.

Hydrophobic SurfaceResidues

on SEA

Hydrophobic amino acid residues build up a stable core structure in proteins. This is also seen in SEA where the inner parts of particularly the P-grasp motif of domain 2 show high stability (low-temperature factors) in the crystal structure. However, hydrophobic residues, like phenylalanines, usually avoid exposure in the solvent regions as this is thermodynamically unfavorable. Nevertheless, highly hydrophobic residues are found on the surface ofSEA. It is tempting to suggest that these hydrophobic regions are important in the interactions SEA forms with other molecules. Interactions between charged and polar residues will also be very important in the final complex interface, but the hydrophobic interactions will further stabilize the complexes. Examples of hydrophobic interface interactions are seen in the crystal packing of the SEA molecules. Someof the most important packing interactions in the crystal lattice are formed by hydrophobic surface residues. There are two highly pronounced hydrophobic regions on the surface of the SEA molecule. The most pronounced region is built of residues Val 174 and Phe 175. If one describes SEA as a flat box, these two residues project straight into the solvent from the corner this box. The residues are positioned on the edge of the P-grasp motif in the loop connecting the C-terminal part of a-helix 4 and P-strand 9. In protein G and L, also displaying the P-grasp motif, this region has been shown to be involved in binding to immunoglobulin domains (58-60). These findings make it likely that residues 174 and 175 are involved in interface interaction with target molecule like MHC class I1 or TCR. A second highly pronouncedhydrophobic surface regionon SEA involves amino acid residues Phe-47, Leu-48, Leu-53, and Ala-97. These residues are located in the domain 1 P-barrel, in the loop between P-strands l and 2, within P-strand 2, and in the disulfide loop. Phe-47 is structurally equivalent to Phe-44 in SEB, which in a crystallographic structure determination of the SEB/HLA-DR1 complexby


A,

D, and E

21 1

Jardetzky et al. (17) has been shown to directly interact with the HLA-DR1 molecule. C. The low-Affinity

Class II BindingSite

There is accumulated evidence for a second SEB-like MHC class I1 binding site in SEA (10,27,61,62). Site-directed mutagenesis experiments where residue Phe 47 in SEA was replaced with either Ala or Ser showed six- tosevenfold decrease in MHC class I1 binding (10,271. Further, alanine substitution of His 50 showed a threefold decrease in MHC class I1 binding (10). Proliferation of T cells was drastically reduced by Phe-47-Gly, Leu-48-Gly mutations (61,62) or Phe-47-Ala and Phe-47-Ser mutations (10,27). The residues Phe-47, Leu-48, and His-50 are found on the P-barrel in domain 1, opposite the Zn2+modulated MHC class I1 binding site of SEA (Fig. l).Phe-47, Leu-48, and His-50 of SEA are equivalent to the residues Phe-44, Leu-45, and Phe-47 on SEB, which are all important in the interface between SEB and HLA-DR1 (17). SEA-SEB competition studies along with site-directed mutagenesis on SEA and crystallographic studies on SEB strongly suggest that the second HLA-DR1 site of SEA equivalent to the SEB/HLA-DRl site. Mutation at Phe 47 in SEA has a far more drastic effect on T-cell proliferation compared to MHC class I1 binding. This suggests an important role of the domain 1 site in SEA in position SEA for optimal TCR interaction. In contrast, mutations in the Zn2+-dependentdomain 2 similarly affect T-cell proliferation and MHC binding. Binding studies of SEA mutated in the binding sites in domain 1 and 2 indicate that the affinity for MHC class I1 display K d values of to and M, respectively (10). This shows that the binding site located in domain 1has a weaker affinity in SEA than in SEB (Kd = to M). The observed differences in affinity canpossibly be explained from a structural investigation of an SEA/HLA-DRl complex, but such studies have not yet been reported. Modeling of the SEA domain 1 site in complex with HLA-DR1, however, can easily be made using the SEA and the SEB/HLA-DRl (17) crystal coordinates. The latter coordinates were kindly provided by D. C. Wiley and co-workers. In a docking experiment where SEA was superimposed on SEB in the HLA-DRl complex, significant structural similarities are seen, e.g., at Phe-47, (Phe-44; SEB residues in parentheses), Leu-48 (Leu-45), Tyr-92 (Tyr-89), Tyr-108 (Tyr-115), and His50 (Phe-47). However, differences that can explain the lower affinity ofSEA than SEB are also seen (11).The Asp-70 (Glu-67) residue

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shows such a difference. The shorter side chain of the Asp residue, and thereby the longer distance (DRl)Lys-39Nq-(SEA)Asp-7006 compared to (DRl)Lys-39Nq-(SEB)Glu-670&, will decrease the strength of the important salt bridge compared with the SEB/HLA-DR1 complex. However, a structural rearrangement of the SEA/HLA-DRl, compared to the SEB/HLA-DR interface, may decrease the negative dependence. Possibly, an extra water molecule can bridge the two residues and replace the salt bridge interaction with weaker hydrogen bonds. Another structural difference that can decrease the binding affinity in an SEA/HLA-DR1 complex, compared tothat for SEB, is the smaller van der Waals contact area obtained for the Ala-97 (Tyr94). H. The SEA M H C Class Il. Binding Sites Cooperate

SEA shows stronger binding affinity to MHC class I1 as well as higher potency in T-cell proliferation assays as compared to SEB. This is compatible with the bivalent binding properties of SEA to MHC class I1 molecules (10,27). It is possible to model a feasible complex where one SEA molecule is simultaneously binding two MHC class I1 molecules (SEA-DR1,). One docking site is identical to the SEB site, and, on the opposite side of the SEA molecule, there is another docking site where the HLA-DR1 His 8lp residue is coordinating the Zn2+ion of the SEA molecule. The HLA-DR1 molecule is then rotated around the Zn2+ion to avoid steric contacts (Fig. (11,27,63). Hudson et al. (27) have suggested that a complex consisting of two SEA and one HLA-DR molecule (SEA,.DRl), with a direct contact between the two SEA molecules during certain conditions would better explain the determined cooperativity. It will be interesting to see if the SEAiDRl complex is biologically relevant. Nevertheless, the existence of a SEA2-DRl complexprobably does not rule out the existence of the predicted SEA-DRI, complex. Mehindate et al. (64) has, for example, shown that crosslinking of two MHC class I1 molecules by one single SEA molecule is a requirement for cytokine gene expression in a monocytic cell line. This suggests an important role for bivalent SAg molecules in activation of MHC class 11+ monocytes. Zinc Site II in SEA

The crystal structure ofSEC, (13) reveals a zinc ion bound to the protein at a position completely different from the high-affinity site in SEA. The residues of SEC, involved in zinc coordination are Asp83,His-118, and His-122; in addition, Asp-9 from a neighboring

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Model of a likely SEA (HLA-DRl), complex. (Left) The SEB-like, low-affinity, HLA-DRl a-chain interaction where SEA has replaced SEB in the SEB/HLA-DR1 complex structure (Right) A possible high-affinity HLADR1 p-chain interaction where His-81P of HLA-DRl is coordinating the Zn2+ ion in SEA.

Figure

molecule in the crystal lattice occupy the last coordination site in the tetrahedral coordination of the zinc ion. This site is situated between the two domains; Asp-83 is found in P-strand 4, in domain 1, while His-l18 and His-l22 are found in the loop between P-strand 5 and 6 that connects the two domains. No biological function has so far been attributed to this site. A possible function of the bound Zn2+ion is to stabilize the two-domain structure ofSEC,. A crystal of SEA, form 1, was soaked at very high concentrations ofZnC1, m M ) and X-ray crystallographic data were collected to 2.9 A resolution. The results indicate a bound zinc ion in SEA at the same position as in SEC2. Asp-86 and His-l14 of SEA, equivalent to the SEC2 Asp-83and His-l18 residues, are Zn2+-coordinatingresidues in SEA. On the other hand, Arg-118 in SEA, equivalent to His-l22 in SEC2, is not close enough to participate in coordination of the zinc ion. Instead, its side chain lies like a lid over the zinc site. Residue 118 is replaced by residue Glu-39 in the coordination of the zinc in SEA and a well-ordered water molecule finalizes the tetrahedral co-

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ordination figure. A second zinc-binding site ofSEA has not been reported earlier as high concentration of zinc is needed for binding. It is uncertain if local zinc concentrationsin vivo can rise high enough to facilitate binding to the second zinc site. SE INTERACTIONSWITHTHE TCR MOLECULES

The highly polymorphic T-cell-receptor moleculesare important binding targets for the SEs. The TCRs are normally involved in recognition of peptides of processed antigens presented by the MHC molecule and bound to its peptide-binding groove (1,6,65). Peptide antigens bound to MHC class I1 are recognized by the hypervariable V, D, and segments in the TCR a- and P-chain. SEs, though, predominantly recognize the VP segments of the TCRs (66-70). There are about 20 different mouse and 60 different human VP elements and each SE shows preference for distinct VP chains. This results in interaction with one or a few of the TCR VP chains and specific fingerprints of each SE on the TCR repertoire (Table l).Collectively, a large percentage of T cells are stimulated by the particular superantigen. Highly homologous superantigens such as SEA, SED, and SEE show also a difference in Vp specificity, raising the question of which specific variable residues in these related SEs are responsible for interacting with the different VP segments. Crystallographic-determined structures of the a- and P-chains of the TCR have been reported (71,72); however, at the time of writing, crystallographic structure determinations of complexes between SEs and soluble parts of the TCR (73) have not been reported but are eagerly awaited (74). So far, regions of the SEs that are involved in TCR interactions have been reported to be situated in the interdomain groove, on the top of the molecule as seen in Fig. 1. This is true both for SEB (12,17,75,76) and for SEA. Kappler et al. (75) have shown that mutated SEB, where residue Asn-23 was replaced by other amino acid types, failed to stimulate a panel of T-cell hybridomas including four known SEB-reactive murine VP elements. The result indicated that Asn-23 in SEB is an important residue in VP interaction. This residue is conserved among all the SEs (equivalent to residue 25 in SEA). It is therefore possible that this Asn residue is important in VP interactions also for the other SEs. Moreover, it has been shown that residues Gly-200, Ser-206, and Asn-207 in SEA, equivalent to Asp-197, Pro-203, and Asp-204 in SEE, are important for activation of T cells (77,781. Hudson et al. (78) have


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shown that an exchange of residues 203 and 204 in SEE to the homologous residues in SEA converts the profile of responding TCR Vp families in human T lymphocytes from an SEE- to .an SEA-specific profile. Similarly, a reciprocal changeof residues 206 and 207 in SEA to the homologous SEE residues converts the TCR Vp profile to an SEE-like pattern. The importance of these residues is further indicated by the fact they are present in a region that deviates structurally between SEA and SEB. The loop region between P-strand and the irregular a-5 helix, residues 197-204 in SEA, folds closer to the a-5 helix than it does in SEB. The maximum distance between the two main chains is about 9 A. Residues in the 60-64 region also seemto be important for TCR interactions. A naturally occurring mutant of SEA, Asp-60-Asnr fails to stimulate the proliferation of murine splenic lymphocytes (79). Further, it has been shown that residues Asn-60 and Tyr-61 ofSEB, homologous to residues Trp-63 and Tyr-64 in SEA, affect the interactions with TCR Vp 7 and 8.1 (75). It has been shown that alanine substitution the disulfide bridge residues in SEA, Cys-96 or Cys-106, decreases mitogenicity by 100-fold without affecting MHC class I1 binding (80). As this type of substitution can substantially change the 3-D structure in a relatively large part of the molecule, one needs to be careful in the interpretation of what specific residues in this region really are important for mitogenicity.

W.

THE CRYSTALSTRUCTURE OF SED

SED is the shortest member of the staphylococcal enterotoxin protein family, predicted to consist of228 residues. Compared to SEA (233 residues) and SEE (230 residues), SED is truncated in the N-terminus of the protein. Apart from this difference, the rest of the sequences align without gaps. SED i s 51% identical at the amino acid level to SEA and 54% identical to SEE. SEA and SEE are 81% identical to each other. Since SED was expected to be dependent upon Zn2+for optimal biological activity, the protein was incubated with ZnSO, (100 PM) prior to crystallization. It was observed that SED is inclined to form crystals in the presence of Zn2+and various forms of polyethylene glycol at pH 6.8-7.5 but is virtually impossible to crystallize in the absence of Zn2+. Thus, the zinc ions seem to stabilize the molecule and promote ordered crystal growth. The SED crystal structure was

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solved using molecular replacement technique and refined to 2.25 A resolution (SundstrZJm et al., Embo in press). The asymmetric unit of the crystal contains two independent molecules. As revealed from the crystal structure, SED is very similar to SEA and other superantigens. The commonly observed two-domain architecture of the molecule is seen with a P-barrel in domain l (residues 15-110) and a P-grasp motif in domain 2 (residues 1-15 and 120228). The overall similarity forthe SED monomer to SEA crystal form 1 and 2 is very high with an overall RMSD approximately 0.87 A comparing 201 Ca atom pairs. Despite this high overall similarity, SED shows a unique property to crystallize as a Zn2+-dependent dimer. Two Zn2+-bindingsites are observed per monomer. The first Zn2+site resembles the metal-binding site seen in SEA and is located on the domain 2 P-sheet. In SED, in contrast to SEA where the site is exposed on the surface, this site is buried in the jnterface of an apparent homodimer (Fig. 4a). Approximately 1200 A2 between the two domain-2 P-sheets in the SED dimer interface are not accessible to solvent, compared to the free components. Three Zn2+ligands are in the same position as the corresponding high-affinity ligands in SEA. Asp-182, His-220, and Asp-222 (in SEA equivalent to residues His-187, His-225, and Asp-227, respectively) are the ligands from one molecule of the dimer, and His-218 (Asn-223 in SEA) from the second molecule is the fourth ligand in a normal tetrahedral coordination of the Zn2+ion. Other important residues in this interface are Asn-123,Trp-125,Asn-130,Lys-178,Glu-181,Lys-188, and Tyr-224, whose side chains create an extensive network polar and nonpolar interactionsaround the Zn2+ ion-bindingsite and thus stabilize this motif (Fig. 4b). The arrangement of the interactions in the dimer interface createsa twofold noncrystallographicsymmetry between the two molecules in the dimer (Fig. 4a). The metal-ligand distances and coordination angles are within the range seen for tetrahedrally coordinated zinc ions in proteins. A second Zn*+-bindingsite, similar to that reported for SEC, (13) and described for SEA above, is also seen but here the metal ion apparently has a lower occupancy compared to the site discussed above. The secondsite resembles a normal metalbinding site rather than a specific Zn2+-binding site. However, it should be pointed out that calorimetric titration of Zn2+at pH 6.57.0 verified the existence of two binding sites per molecule (data not shown) in contrast to similar experiments with SEA, where only one Zn2+-bindingsite was observed within the zinc concentration interval used.

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The hydrophobic motif around Phe-42, as seen in the SEB/HLADRl structure (17) (Phe-44) and in SEA mutagenesis studies (81) (Phe-47) and shown to be a binding site for MHC class 11, and is SED involved in crystal packing. Therefore, nofurther conclusions will be drawn from the local conformation of the main and side chains in this area. However, the loop is similar to the conformation observed in SEA. Other regions in SED are unordered and have weak absent electron density, i.e., the first six residuesin the N-terminus, loop 5459, the disulfide loop 92-98, loop 167-173, and loop 184-188. Thus, SED appears to have a higher degree of flexibility in the loop regions compared to SEA. Some of these regionsin SEA have been implicated as areas involved in TCR recognition, and thus might only be ordered upon binding of the receptor molecule. Is the observed SED dimer an artifact crystallization does dimerizing represent a unique property for this SAg? Preliminary experiments using laser light scattering indicated that SED dimerized aggregated in solution at concentrations of Zn2+ranging from 100 to 500 pM at pH 7.4. This was shown by a dramatic increase in particle size solution compared to EDTA-treated SED material (data not shown). To verify this observation, gel filtration chromatographywas absence of Zn2+.Approxiperformed in the presence (100 FM) mately 50% of the SED protein eluted as an apparent dimer in the presence of Zn2+. In contrast,an EDTA-treated sample eluted only as monomer. In a third approach, covalent crosslinkerswere used at pH 7.4 in the presence and absence of Zn2+. From these experiments it is clear that SED forms dimers at a concentration of 5 pM lower) Zn2+in solution. In preliminary experiments, mutants ofSED, Asp182-Ala, Asp-227-Ala, and His-218-Ala all abrogate dimer formation as judged from crosslinking experiments, whereas a mutant in the SEB-like MHC classI1 binding site Phe-42-Ala does not. In all these experiments SEA served as a negative control sinceSEA did not show a tendency to form dimers. The failure of SEA to form dimers most likely relates to the fact that the residue corresponding to His-218 in SEA is an asparagine ( Asn-223): However, even though asparagine could be a potential Zn2+ligand, histidine is determined to be stronger. Similarly, His-61 from a symmetry-related molecule may act as a fourth ligand in a normal tetrahedral coordination of the metal ion. Thus, the ability of SED to form a Zn2+-dependentdimer in solution as well as in crystals apparently resides in and around the Zn2+-binding site of the protein.

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(a)

Figure 4 MOLSCRIPT (87) representations of the crystal structure of staphylococcal enterotoxin D. a) The two independent molecules forming a zincmediated dimer; b) a closeup view showing the residues involved in highaffinity zinc coordination. Interactions of SED-MHC Class II

It is not clear whether SED appears mainly as a dimer in vivo. However, if the dimer also exists in plasma, could it then be important for the mode action this superantigen? Different ways of binding could then be considered. 1) The SED molecule is a monomer in solution and binds two separate MHC class I1 molecules in a HLA-


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Figure 4 Continued

DR,/SED/HLA-DR, chain trimeric complex, similarly to what is expected for SEA. 2 ) SED is a dimer and forms a tetrameric complex to the a chains on two separate MHC class I1 molecules utilizing the SEB-like binding motif around Phe-42. The SED dimer is dissociated upon binding to MHC classI1 and binds as a bivalent monomer as described in mode 1). Although at present highly speculative, SED dimers may modulate the interactions with MHC class I1 and TCRs to elicit unique biological responses. This may include extensive crosslinkingof MHC class I1 molecules on the surface of monocytes and potent induction of proinflammatory cytokines (64). Another interesting aspect is that

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more than one serotype of enterotoxin can be produced simultaneously by some strains of staph ylococcus. The combination SEA/SED is frequently seen and is commonly observed to cause food poisoning (82). Would a SEA/SED heterodimer possessing unique hybrid enterotoxin properties be possible to form in the presence of Zn2+? Albeit interesting, the functional relevance of the dimerizing capability can at the present stage only be hypothetical and further work is needed to clarify the importance of it. V.

I

A THREE-DIMENSIONAL MODEL OF SEE

The crystal structure of SEE has so far not been determined. The sequence identity with SEA is considerably larger than with the other SEs (Table 1). We therefore created a model structure of SEE on the basis of the SEA crystal structures (crystal from 1 and 2). Using the 0 program (83), the three first residues where deleted from the SEA structure and 19%of the side chains were replaced. Side-chain conformations were picked from rotamers pointing in similar orientations as in the SEA structure. In almost all cases side chains could be easily replaced without causing any collisions. The most profound change from the SEA structure was made at residue 139 (142), which is part of a so-called pbulge (84) between p-strands 7a and 7b in domain 2. (Residue numbering of the SEE model will hereafter be written as: SEE residue number and in parenthesis the equivalent SEA residue.) The side chain of residue 142 of SEA, a Thr residue, points toward the center of the protein, making it difficult to replace it with the larger Lys residue found in SEE. This problem makes it probable that this pbulge region, 137-139 (140-142), has a somewhat different structure in SEE than in SEA. The variable residues between SEE and SEA accumulate on the surface (Fig. 5a and b). However, the side chains of some of the variable hydrophobic residues are pointing into the hydrophobic core of SEE. The variable residues are more widely distributed over domain 2 than over domain 1. In domain 2 the variable residues seem to form paths on the molecule. In domain 1 a cluster of variable amino acids is formed by residues from the loop between P-strand 1 and 2, P-strand 2 and helix 3, namely Asp-41 (His-44), Glu-46 (Gln49), Asn-47 (His-50), Leu-68 (Phe-71), and Asn-75 (Asp-78). These residues appear in a region that in SEB binds to HLA-DR1 (17). Glu46 is equivalent to one of the residues directly involved in HLA-DR1 binding in SEB. The equivalent SEB residue is Tyr-46 for which a


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MHC class I1 interaction is made through a main-chain hydrogen bond connection to HLA-DR1. This main-chain connectionwould not be affected by a Tyr-to-Glu mutation. The same is true for SEA, which has a Gln residue at this position. The two most prominent changes between SEA and SEE in this region are the replacement of two His residues with a negatively charge and a polar side chain, Asp-41 (His-44) and Asn-47 (His-50). Of these two residues Asp-41 is far from the HLA-DR1 site in the SEB-MHC class I1 complex while Asn-47 is equivalent to Phe-47 in SEB, which is involved in van der Waals contacts with an Ala residue in the HLA-DRI. This region ofSEA has been shown to be involved in binding to HLA-DRI, although the affinity of the domain 1 site in SEA for HLA-DRl seems to be severalfold weaker than the corresponding region in SEB (11,17,63,85). Since SEA binds to HLADRl molecules, despite the SEB-Phe-47 to SEA-His-50 change, it is likely that the van der Waals contacts between the SEB-Phe-47 residue and HLA-DRl are of less importance in the complex formation. If so, it is also likely the HLA-DRl molecule can tolerate as Asn residue present in SEE and form an SEE/HLA-DR1 complex of the same type as seen in the SEB/HLA-DR1 structure. At the domain 2, Zn2+-mediated,MHC class I1 binding site, the major variance is the reported difference in the length of the SEA and SEE N-termini. Three residues less, nearby the important Zn2+ion, can significantly influence the interactions between SEE and MHC class 11. Other variations around the Zn2+ion are relatively distant and small. The Iargest variance, not counting the N-terminal difference, is the change at SEE residue 187 from Thr in SEA to Glu, however, the residue is about 15 A away from the Zn2+ion. The small structural variances observed make it likely that the binding interactions between SEE and HLA-DR are similar to those between SEA and HLA-DR. This agrees well with other observations (25,261 and the biological difference observed between SEA and SEE does not seem to be particularly dependent on MHC class I1 interactions. Instead the difference in TCR interactions is probably of greater importance. It has been shown that mutations at residues Pro-203 (Ser-206), Asp-204 (Asn-2071, and Asp-l97 (Gly-200) affect Vp-specific activation of T cells (77,78). Theseresidues are found at the top of domain 2, in proximity to domain 1 (Fig. 5a and b.) Other variable surface residues in this part of the model are Arg-17 (Gly-20), Asn-18 (Thr21), Ser-21 (Gly-24), Arg-24 (Lys-27), Gly-57 (Asp-60), and Pro-59 (Ser-62) (Fig. 5b). These residues are additional candidates for Vpspecific TCR interactions.

(b) Figure 5 Space-filling model ofSEE based on the crystal structure ofSEA (combination of both crystal forms). Side chains of residues variable between SEA and SEE are indicated in gray. One-letter amino acid codes along with SEE sequence numbers are included. a) An orientation similar to Fig. 1. b) The molecule rotated relative to a ) around a vertical axis.


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To summarize, there are several nonconservative amino acid replacements between SEE and SEA that can be critical in forming distinct structural and biological features of these SEs. One observation is that SEA in general is richer in Gly residues than SEE, which may contribute to mobility critical regions in SEA. The most other striking differences are the following substitutions: Arg-17 (Gly-20), Asn-18 (Thr-21),Ile-34(Lys-371,Asp-41(His-441,Asn-47 (His-50), Lys-139 (Thr-142), Ser-171 (Val-174), Asp-l97 (Gly-2001, and Pro-203 (Ser-206). However, analyses of nonconserving substitutions in SEA and SEE will only address differences in specificity between the proteins. Substitutions conserved residues will have importance to determine residues central in forming an appropriate backbone for interactions of SEs with various TCR chains. We have analyzed the titer of antibodies toward different staphylococcal superantigens in a panel of individual human serum samples from different parts of the world. Surprisingly, despite the 81%identity ofSEA and SEE, the antibody titer toward these superantigens showed a greater difference. The serum antibody titer was about 5-10 times higher for SEA compared to SEE. To further investigate this we analyzed the occurrence of cross-reactive epitopes in SEA and SEE with affinity-purified human anti-SEA antibodies. The SEE titer of these antibodies was about 5 times lower compared to reactivity against SEA, indicating a low degree of shared antibody epitopes between SEA and SEE albeit the high degree of identity. The differences betweenSEA and SEE in reactivity with human serum antibodies and the low degree of shared epitopes may reflect a natural occurrence superantigen polymorphism to evade .recognition by the host immune system upon successive staphylococcal infections. ACKNOWLEDGMENTS

We thank Prof. D. C. Wiley and co-workers for supplying the coordinates of the SEB-MHC class I1 complex and Dr. K. R. Acharya and co-workers for supplying the coordinates ofSEC,. Parts of the work presented were supported by the Swedish National Research Council (NFR). REFERENCES 1. Webb SR, Gascoigne RJ. T-cell activation by superantigens. Curr Opin

Immunol 2. Uchiyama T, Kamagata Y, Yan XJ, et al. Study the biological activities toxic shock syndrome -toxin-l.11. Induction the proliferative

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11 The Exfoliative Toxins of Staphylococcus aureus JohnJ. landolo and Stephen Keith Chapes

INTRODUCTION

Among the extracellular proteins produced bythe Gram-positive bacterium Staphylococcus aureus are the exfoliating toxins exfoliationA and B (ETA and ETB). The toxins were named because of the sloughing of the epidermis that each is known to produce. The clinical symptoms produced by the toxins were first described in infants in 1878 by Ritter von Rittershain as a bullous exfoliative dermatitis (1) of unknown origin. Ritter’s disease syndrome was eventually associated with S. aureus, as are a number of other epidermal infections. These syndromes, which include Ritter’s disease, are toxic epidermal necrolysis (TEN),staphylococcal scarlatiniform rash, and bullous impetigo and were all referred to as one disease complex, staphylococcal scalded skin syndrome (SSSS). Each these diseases exhibits a similar pattern of clinical symptoms (2).

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II. THE DISEASE

Most often a disease of newborns, SSSS may result in a denuding of one-half more of the epidermal surface of newborns and infants The extensive peelingof this surface layer and the looseness of the skin when gently stroked (Fig. l), has diagnostic significanceand is referred to as a Nikolsky sign (3,4). Large, flaccid bullae appear, which fill with clear fluid. As these bullae develop, they raise areas of the skin that separate, leaving the appearance of a red, scalded epidermis. SSSS (i.e., exfoliation) maycontinue for days and after 7-10 days there is usually complete recovery. Theinfection tends not to be systemic and the toxins appear to be specific to the epidermis. Curiously, even intraperitoneal inoculation of neonatal mice results in exfoliation, suggesting that the toxins are transported specifically to sites in the epidermis.

Figure 1 Newborn (c24 hr) C2D mice (MHCII-/-) injected subcutaneously with 2 lo* S. aureus (strain KSI709, ETB positive) with or without simultaneous injection of 70 or 140pg anti-H2Db monoclonal antibody (ATCC HB36; see Ref. 93). Animals were injected with bacteria + anti-H2Db antibody and scored Nikolsky signs (folding over epidermis uponstroking) 5 hr later. Neonates injected with an isogenic ETB negative strain do not induce Nikolsky responses (not shown).

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At’the tissue level, SSSS is characterized by its effects on the stratum granulosum (5). This layer resides below the stratum corneum and the stratum lucidium (5). Intraepidermal splitting occurs in the stratum granulosum, which results in the disruption of normal cell-to-cell forces of adhesion. Lillibridge et al. ( 6 ) noticed that small vesicles or ”bubbles” present between cellsof the stratum granulosum were affected by exfoliative toxin (ET). Within 20 min of ET injection into neonatal mouse skin, when viewed by electron microscopy the vesicles appeared to widen or disappear. Within 25 min, a positive Nikolsky sign appeared, followed by splitting of the desmosomes (thick cell membrane structures that enable granular cells to attach to each other). Presumably, cell-cementing forces are compromised, which leads to splitting of the desmosomes. Thereis no cytolysis and it is unknown whether desmosomal splitting is a primary or secondary event induced by exfoliative toxin. Cleft formation within the stratum granulosum was observed after 150 min and the desmosomes along the cleft were split. However, only cell separation was apparent and there seemed to be no cellular damage, with only the stratum granulosum layer affected (6). Because of the change in the intercellular “bubbles,” Lillibridge and co-workers proposed that an enzyme proenzyme activated by Et was released from intercellular vesicles. This putative enzyme could then cause splitting of the desmosomes. Studies by Elias et al. (5) andMcLay et al. (7), however, refute Lillibridge’s contention. They suggest that desmosomal splitting is a secondary event rather than a primary one. Elias et al. (5) used ultrastructural studies to show that desmosomes did not always separate in neonatal mice dosed with ET, but they found that the vesicles between granular cells widened. Using mouse neonates administered purified ET, McLay et al. (7) histologically identified large gaps between cells along a horizontal cleavage plane following a long lag period after toxin administration. The only region where contact remained between cell layers was at desmosomal junctions. Thesedata indicated that the primary action of ET is on intercellular forces of adhesion; only subsequently were desmosomes affected. It remains to be proven which group is correct. Nevertheless, ET seems to affect normal forces that hold cells together in the stratum granulosum. Progress continued when Koblenzer (8) demonstrated in 1967 that TEN and Ritter’s disease were histologically identical. The absence of polymorphonuclear leukocytesand stainable staphylococci in the skin lesions assoclated with these diseases prompted Elias et al. (9) and Lyell (10) to propose that a diffusible productof S. auras may be responsible the disease symptoms.

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It has been suggested that neonates are more susceptible to the action of the exfoliative toxins than adults because an infant’s immune system is incapable of producing antibodies to combat the infection Others speculate that there may be differences in adult and neonatal epidermis, and that adults may be able to clear the toxin from their systems more effectively than infants Recently, Cribier et al. reviewed in adults. Two major risk factors, kidney failure and immunosuppression, have been identified as critical for contractof disease. Although a total of only cases have been reported in adults, and the sample size is somewhat limited, the mortality rate is substantially higher than in infant cases. This may be due to the increased risk of septicemia, as blood cultures from adults are often positive for S. aureus. THE

An intensive search for the causative agent of scalded skin syndrome began in the when Lye11 reported Ritter’s diseaseand TEN were symptomologically identical and were potentially of the same etiology (11). However, it was not until that Parker (12) identified phage group I1 staphylococci as the etiological agent associated with blistering skin lesions and in Holzel and Jacobs and Tyson et al. (14) isolated a phage group I1 staphylococcal strain (phage type 71) from a patient with TEN. The majority of S. aureus isolates phage group I1 that are associated with SSSS contrasts sharply with the observation that the majority of pathogenic staphylococci belongto phage group 111. However, staphylococci of both phage groups I and 111 have been implicated in skin lesion formation, but to a lesser degree In a study in Parker et al. reported that of phage group I1 staphylococci isolated from patients with SSSS were of phage type Several other researchers have noted the abundance of this phage type in SSSS infections. Rasmussen (15) conducted studies with SSSS patients and found only one staphylococcal isolate belonging to phage group I. In agreement, Arbuthnott and Billcliffe reported that only of 11strains of SSSS producing S. aureus were non-phage group I1 isolates. A more comprehensive study by de Azavedo and Arbuthnott (17) further illustrated the prevalence phage group I1 strains in the production of skin diseases. They isolated strains of S. aureus from patients with exfoliative skin lesions. These included patients with those having impetigo and the rest

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(39%)with superficial skin lesions. Of these, 72% were of phage type 11, approximately 15% showed a mixed group I/III pattern, 8% belonged to phage group 111, and 3.5% were of phage type I. The epidemiology of the infectious strains appears to differ in Japan, however, as Kondo and co-workers (18) found that greater than 40% of their toxigenic isolates (19 or 43) were non-phage group 11. In another Japanese study, Sarai et al. (19) found that of patients with Ritter-type TEN harbored non-phage group I1 organisms. In their study, de Azavedo and Arbuthnott (17) also characterized the antigenic serotype of exfoliative toxin produced by 114 staphylococcal strains. They found that 85.5% of the phage group I1 strains were toxigenic and 44% produced only ETA, 17% produced only ETB, and 39% produced both serotypes. Of the non-phage group I1 strains, 31%were toxigenic. Sixty percent of the toxigenic isolates within this group produced only ETA, 10% produced only ETB, and 30% produced both serotypes. When correlated to phage group, it appears that there is no real preference for production of exfoliative toxin. The ETB phenotype is less commonthan ETA and this may be a reflection of the lesser stability of the plasmid genotype ofETB. ET-producing strains may be isolated from a variety of skin lesions. Moreover, without specificity as to the serotype, all three phage groups types are capable of ET production. While approximately 70% of S. aureus strains isolated from skin lesions were ET producers, as shown above, Piemont et al. (20) showed that only 6% of coagulase-positive S. aureus strains isolated from a variety of clinical sources produced ET. W.

ASSAY OF EXFOLIATIVE TOXIN

In 1970, Melish and Glasgow (2) isolated phage group I1 staphylococci from 17 patients between 5 days and years of age who exhibited symptoms of SSSS. The organisms from these patients were injected subcutaneously of intraperitoneally into neonatal mice, which resulted in the clinical manifestations of the disease seen in human patients. The Melish and Glasgow neonatal mouse assay of exfoliative toxin activity has become the standard animal model system (4) for detection of toxin (Fig. 1).Furthermore, the mouse is an appropriate model because epidermal tissue of newborn mice is organized into the same layers as human skin. Recently, additional assay systems have been proposed. Impetigo-like lesions have been produced in human skin explants in cul-

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ture (21) that were inoculated with exfoliative toxin-producing staphjilococci. Histologically,intraepidermal blisters at the granular layer with acantholytic cells could be seen after 10 hr of incubation, which suggested that adult impetigo could develop under the proper conditions. Gentilhomme et al.(22) also demonstrated that cultured human epidermal cells would serve as an experimental model of SSSS. Both direct microscopic and histological examination at various time intervals showed detachment of the epidermal sheets after treatment with various doses of exfoliative toxin A. Total exfoliation occurred at 24 hr at a concentration of500 &ml. Finally, piglets have shown susceptibility to exfoliation by a toxin(s) produced by Staphylococcus hyicus (37). These toxins are thought to be responsible for exudative dermatitis, which shares many of the symptoms of SSSS in humans. In fact, Sat0 et al. (23) have isolated and partially characterizeda new type of exfoliative toxin (which they named SET) from strains of S. aureus isolated from a horse. The purified toxin elicited general exfoliation in mice and in chicks. Consistent with other observations, intradermal splitting was observed at the granular layer of the epidermis. We have also consistently isolated ETA-producing strains S. aureus producing neonatal mouse exfoliating substances,which are not cross-reactive with ET antibody, from greyhound dogs exhibiting symptoms of Alabama rot, a skin infection with multiorgan involvement. It is not clear what the relationship of these ET-producing strains is to the manifestations of this disease, because the causative agents appear to be Gram-negative. It is rather puzzling, but not unprecedented, to find a reservoir of important human toxin in greyhound dogs since toxic shock syndrome toxin-l is known to occur in ovine strains of staphylococci. Reports vary widely as to the prevalence of ET-producing aureus strains (24) and the differences reported are most likely due to differences in the sensitivity the assay system used. These assays include the in vivo mouse system (4,251, double immunodiffusion (25,26), isoelectric focusing (16,25), radial immunodiffusion (16,25),SDS-PAGE analysis radioimmunoassay (251, and enzyme-linkedimmunosorbent assay (ELISA) (25). The latter two methods are the most sensitive, but are also more time consuming and therefore are not performed routinely when large numbers of isolates are being screened. However, regardless the assay, most estimates of S. aureus isolates from blistering skin lesions producing ET range from 50 to

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Today it is known with surety that the exfoliative toxins of S. Aureus are responsible for producing the scalded skin syndrome in susceptible individuals (4). V.

OF ACTION

The precise mode of action of the exfoliative toxins is unknown. However, mounting evidence suggests that the toxins may be involved in an enzymatic cleavage the epidermis. Recently, two reports described amino acid sequence similarities between ETA, ETB, and staphylococcal V8 protease (32,33) and also between ETA and lipase (34). In spite of these structural similarities, no lipase- or protease-like activity has ever been demonstrated for the exfoliative toxins. Phenylmethylsulfonyl fluoride and both potent protease inhibitors, have been shown to bind to a peptide containing the Ser-195 residue of both ETA and ETB, but did not reduce exfoliation produced byETA. Replacement of the Ser-195 residue with Cys brought about a 3000-fold decrease in the biological activity of ETA (34), but these workers were not able to demonstrate protease activity in either wild-type ormutant toxin forms, even with a variety of substrates. Several lines of evidence support the contention that the exfoliative toxins play a role in the induction of proteolytic enzymes that are responsible for the tissue destruction observed. Evidence in support of this hypothesis was, in part, presented by researchers in Japan (35). They showed that staphylococcal exfoliative toxin induced caseinolytic activity. When exfoliative toxin A was incubated in the presence of murine epidermis and casein, caseinolytic activity was observed. Furthermore, addition of alpha,-macroglobulin, a proteinase inhibitor, resulted in levels of casein activity similar to background. These findings suggest that ET is activating a proteinase(s1 or inactivating proteinase inhibitors and are consistent with the observations of Lillibridge et al. (6). However, it remains to be proven whether the vesicles in the intercellular spaces of granular cells described by them actually contain proteolytic enzymes. Current and future experimentation are expected to elucidate which portion(s) of the exfoliative toxins is/are responsible for the biological activity. In a search for susceptible cells, the work of Baker et al. (36) suggests that ET receptors are rare on several cell types examined. They failed to detect binding of Et to erythrocytes, leukocytes, trypsin-dispersed keratinocytes, heat-separated epidermis, or whole new-

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born mouse skin. Studies by Elias et al. (5) showed that ET did not remove or interfere with human leukocyte antigens on the surface of lymphocytes. Keratinocyteswere unaffected by Et when stained with ruthenium red (a dye specific for surface mucopolysaccharides), indicating that ET was not interacting detectably with surface acid mucopolysaccharides. In contrast, the S. exfoliative toxin described by Tanabe et al. and by Andresen et al. (38) was shown to bind to the GMGlike glycolipid extracted from the skin of l-dayold chickens, but not to glycolipid of adult birds or suckling mice The binding appears to be a potential receptor for the toxins, as incubation of exfoliative toxinswith purified glycolipid eliminated the toxicity of the toxins. Thus, a primary step in receptor binding may be necessary for further steps in exfoliation to be activated. Pemphigus is a disease similar to SSSS that is also characterized by bullous skin lesions. Antibodies from patients with pemphigus attach to particular areas within the intercellular space of squamous epithelia (3,5). ET also affects such sites, suggesting a connection exists between pemphigus antigen and a putative exfoliative toxin receptor site. However, Elias et al. showed that ET did not interfere in the binding of pemphigus antibody to its normal sites. Moreover, ET did not alter pemphigus antigen. These data suggest that ET is not binding to cell surfaces directly. Recent studies by Smith and Bailer (39) using SDS-PAGE and Western blot analysis show that ETB binds to keratohyalin granule extracts. Furthermore, ETB bound to purified profilaggrin, the major component of keratohyalin granules. A band corresponding to the molecular,weight of filaggrin was also visible in the Western blots of solubilized granules. Fillaggrin results from profillaggrin breakdown and may occur as a result of the activation of proteolytic cleavage by ET bound to the precursor. Sincekeratohyalin granules in the epidermis are linked to a keratin intermediate filament network that extends to surfaces cells and particularly to desmosomes,the relationship between keratohyalin granules and ET binding is in need of further examination. Such studies should provide much needed insights into the mechanism of action of these toxins. VI.

MOLECULAR BIOLOGY

Early investigations of the genetic carriage of the toxins focused on bacteriophages as the reservoir of the genes (40). However, Iysogeny was never convincingly shown to be responsible for the TOX+ pheno-

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type. Difficulties arose in interpreting phage carriage, curing, and transductional data largely as a result of the failure to recognize that two serotypes ( A and B) of exfoliative toxin existed in separate genetic loci. Rogolsky (3) was the first to note that the toxin-positive phenotype could be lost at high frequency by plasmid-curing agents. These studies led Rogolsky's group and Rosenblum and Tyrone (41) to the identification of a plasmid that appeared to carry a gene for exfoliative toxin. The plasmidprototype for exfoliative toxinthat they identified was pRW001, a 42-kbp plasmid that encodes the genes for exfoliative toxin B, cadmium, and other heavy metal resistances, and a bacteriocin and its immunity gene. Warren (43) used hybridization and restriction enzyme mapping to show that this plasmid is similar to other ET plasmids in phage group I1 strains. Among the plasmids present in the seven strains investigated, 19 22 Hind11 DNA fragments were identical. In addition, these plasmids alsoshare sequence similarities with the phage group I11 penicillinase plasmid pI258. One might speculate that formation ofpRWOOl probably occurred by a recombination event between a putative mobile element (bacteriophages transposon) and a plasmid similar to pI258. A similar event most likely occurred in enterotoxin B-positive strains wherein the gene resides on a unique DNA fragment of at least 27 kb (45). Although evidence to support this contention is not strong, there is ample precedent for such an event. example, the staphylococcal plasmid 0llde was formed bya recombination event between the bacteriophage 011 and the plasmid p1258 (44). Early studies to convincingly demonstrate the carriage of the ETB gene on plasmids by transduction transformation ofpRWOOl other related plasmids were'hampered because there was not a clearly associated resistance marker on these plasmids that could be used for selection.To further confuse the issue, heavy metal resistance was also shown to be associatedwith a small plasmid carriedin many ETB+ strains (3,40). Moreover, with respect to transduction, the plasmids were larger than the genome size of the transducing phages and hence could not be packaged efficiently. The final proof of the location of the gene was provided by the cloning and sequencing the gene for ETB (46-48). At present, there are no reports of instances of the ETB gene being localized to the chromosome, but again there is ample precedent for such occurrences. Jackson and Iandolo (46) mapped the etb gene and the cadmium resistance determinant on the plasmid pRW001. They also clonedthe toxin gene in an Escherichia coli shuttle vector, but curiously in the

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Gram-negative background, etb was not expressed from its ownpromoter. However, positioning the gene downstream from a strong Gram-negative promoter allowed readthrough transcription. Thisgene remains the sole exoprotein gene from aureus that is not expressed in E. coli. The reason for this remains unresolved but it reinforces the contention of Hudson and Stewart (53), who reported that at least two types of promoters existed in S. aureus, one of which is not expressible in E. coli. At about the time the plasmid locus ETB was being firmly established, Keyhani et al. (49) and Rosenblum and Tyrone (41) complicated these findings by showing that exfoliative toxin could also be produced by strains of aureus that did not harbor plasmids. The toxin type produced by these strains was different from that produced by strains cured of the TOX+ phenotype reported by Rogolsky (3). The Tox+phenotype of Rogolsky’s strains showed resistance to curing and provided a strong suggestion that a second variety exfoliative toxin was produced. Chemical, immunological, and partial genetic characterization of the toxin eventually led to the naming and assignment of the gene for exfoliative toxin A (eta) to the chromosome and the gene for exfoliative toxin B (etb) to a plasmid typified by pRW001. At present, only a rudimentary genetic and physical map of the phage group I1 genome exists (P. A. Pattee, personal communication), and as a result, the eta gene has never been mapped on the chromosome the phage group I1 strains. In contrast to other chromosomal toxin genes, such as staphylokinase staphylococcal enterotoxin A (51), staphylococcalenterotoxin B and toxic shock syndrome toxin-l (TSST-1) (52), the gene for exfoliative toxin A has not been shown to be directly associated with a bacteriophage or other mobile genetic element. However, considering the similarities of the two genes and their disparate loci, it is tempting to speculate that association with mobile genetic elements was evolutionarily responsible for their genetic configuration. The efa gene was first cloned and sequenced by Lee et al. (48) from chromosomal digests of the phage group I1 strain UT0002 and by O’Toole and Foster (54,55) from strain TC16 and later by Sakurai et al. (56). All three research groups obtained data that were similar. In addition, Foster’s group showed that fusion of the luxAB and the xylE reporter systems to the eta promoter was dependent on expression agr and could be influenced by high osmoticpressure as well. They also showed that an agr-independent regulatory control of the expression of eta was imparted by alteration in DNA topology.


241

The actual transcription start sites for either toxin gene have not been mapped but the promoter proximal region of both genes contains a large inverted repeat sequence that is highly conserved with 30 33 shared nucleotides. This inverted repeat region is also conserved, although less stringently, among other extracellular proteins such as enterotoxin B, enterotoxin D, and lipase. We have speculated on the significance of these repeats, but a potential regulatory role for this region has yet to be determined (55a). The DNA-derived protein sequences for both toxins have been deduced and have been partially confirmed by automated Edman degradation (48). ETA is comprised of280 amino acids and contains a 38-amino-acid signal sequence. Cleavage results in a mature form consisting of 242 amino acids with a molecular weight of 26,950 daltons. The precursor ofETB consists of 277 amino acids with a signal peptide of 31 residues. The mature form consists 246 amino acids and has a molecular weight of24,318 daltons. A comparison of the two proteins is presented in Fig. 2. Although there i s a high degree of similarity between the two molecules (i.e., >40%amino acid identities), three prominent regions are worth noting. The first is in the Eta Etb Eta Etb Eta Etb Eta Etb Eta Etb Etb Eta Etb Eta Etb

230 221

243 247

Figure 2 Comparison the sequences of the mature forms (i.e., not including the signal peptides) of ETA and ETB. Sequence identities are shown in boxes.

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N-terminal portion of the molecule at positions 42-66 [20 or 25 amino acids match (80%)], the second near the middle of the molecule at residues 100-128 [l7 of29 amino acids match (58%)], and the third near the C-terminus at positions 193-213 [l7 of21 residues match (81%)]. This high degree of similarity is somewhat unexpected because of the lack of shared antigenic determinants This might suggest that the shared sequences of the proteins are buried within the structure and not available as epitopes. These regions of similarity may be important sites controlling biological activity, and if buried, they may have difficulty in contacting the surface of susceptible cell types. However, even if buried, they would not necessarily prevent the shared regions .from participating in exfoliation becausethey may be exposed by conformational changeswhen the toxins encounter cell surface binding ligands. Further speculation concerning the shape of the toxins can be generated by comparisons of the hydropathicity of the proteins (Fig. The plots are virtually superimpossable, indicating that many of the sequences represent stable domains that contain only conservative amino acid differences. In fact, the hydrophobic prominences seen correspond to the regions of sequence identity. Considering that exfoliatins A and B have identical biological activity involving sites rich in lipids, such structural parity seems reasonable and perhaps necessary for toxicity. These speculations have been confirmed by the site-directed alteration the molecules described earlier. The Ser-195 residue of both toxins lies within the C-terminal region of similarity and its presence has a profound influence on activity. Further analysis awaits a detailed solution of the crystal structures. Preliminary crystallization of ETA (57) and ETB (58) has been reported, but in neither case was the level of resolution sufficient to allow conclusions about the shape of the molecules. VI1.

IMMUNOLOGICALAND SUPERANTIGENIC ACTIVITY

The classification of the exfolitive toxins as superantigens is controversial. Some have ascribed the superantigenic activity to contaminating enterotoxins (60,61). Moreover, the description of lipid-binding activity (62) and the age-dependent binding ETA and ETB to GM4 glycolipid in neonates susceptible to exfoliation (37) also suggestthat the exfoliative toxins are different from other staphylococcal superantigens. Nevertheless, there is compelling evidence thatthese exoproteins exert powerful biological activity on cells of the immune system. The mitogenic activity of ETA was observed as early as 1980

243


200

l00

-4

-4

Residue Number

200

200

3 2 2 .f l

2 1

2 0

U

I”-1

-1 -2

200

Residue Number Figure 3 Hydrophobicity plots of the precursors of ETA and ETB. Hydro-

phobicities were averaged over an 11-residue

window.

by Morlock et al. (63). These investigatorsdemonstrated that BALB/c T cells as well as B cells proliferated when incubated with ETA. Indeed, both ETA and ETB stimulate spleen cell proliferationin a dosedependent fashion (Fig. 4) with ETA having slightly higher biological activity than ETB. However, some of the differences in activity may occur because ETB is less stable than ETA (48,64). The biological activity of these preparations cannot be due to contaminating enterotoxins becausethey were isolated from S. Aureus strains UT0003 (ETA) and UT0007 (ETB), which do not carry genes for enterotoxins Iandolo, unpublished data). Furthermore, additional confirmation is provided by the fact that our exfoliative toxin

landolo and Chapes

244 30

48 Hr. Proliferative Response

U

-c

2ot l

10 1 0.1 0.01 Toxin Concentration (vglml)

Figure 4 Response murine spleen cells to activation by exfoliative toxin A and Cells were stimulatedwith various concentrations ETA and ETB and the proliferative response was assayed 48 hr later.

preparations neither react with antisera specific for SEA or SEB nor contain significant amounts of endotoxin or lipoteichoic acid (80%, ++++.Onlymacrophagesonthe C57B6/6J background are included in this summary. TNF, tumor necrosis factor; IL-6, interleukin6; NO, nitric oxide.

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Table 2 Effects of Exfoliative Toxin Injection on Splenic Hypertrophy Experiment l

2

Body Mouse Toxina strain C3HeB/FeJ C3HeB/FeJ BALB/c BALB/c BALB/c BALB/c

Spleen weight as % weightb PBS 0.511 ETA PBS ETA PBS ETA

0.408 +.c 0.55 + 0.496 5 0.52 0.565 5 0.492 5 0.042 0.582 .c 0.088'

pg of ETA or ETB injected i.p. in 200 p1 of pyrogen-free PBS 7 days before determination of spleen weights. bNumbers represent SD of 6 mice. Indicates significantly more thanPBS control: + < 0.01; 'pe0.08. Two experiments presented representativeof 5 independent experiments.

linking of MHCII molecules by toxins (83-87). obvious Zn2+binding sites are apparent in the eta or etb gene sequences (48). ETA and ETB may bind to MHCII similarly to SEB, which only binds MHCII at one site However, it is interesting that SEA as well as SEB, ETA, and ETB are able to induce immediate signal transduction in macrophages indicatingthatbinding to epitopesotherthanthe MHCII p chain can result in a productive functional response. Because superantigens act as powerful T-cell and macrophage stimulants, many have suggested that the immune system contributes to pathogenesis (79,89-91). Although this may readily occur in humans, it is less obvious how bacterial superantigens cause murine pathogenesis since physiological concentrations of exotoxin have minimal acute effects on adult mice. Some investigators have found that weight loss (up to 10% of original body weight) occurs in response to enterotoxin (89,92). Marrack et al. (92) determined that the activation of T cells by superantigens was responsible for the wasting, and Grossman et al. (89) presented data to suggest that macrophages exacerbate the response. Intraperitoneal injection 100 of ETA and ETB induced similar wasting responses in BALB/c and C3HeB/FeJ mice (Fig. 5). Peak wasting occurred 1-2 days after injection and was accompanied by significant splenic hypertrophy (Table l), indicative of immune system activation. Therefore, exfoliative toxins manifest similar in vivo activity to enterotoxin superantigens. The characterization of molecules as superantigens by virtue of specific T-cell-receptorVp stimulation and the lack of antigen process-

247

Exfoliative Toxins of S. aufeus

1VIlINI %

l H D I 3 M AaO8 1 V I l I N I %

Chapes 24%

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ing by MHCII molecules should not blind us to the fact that there are other factors that regulate superantigen responsiveness. Our studies with the exfoliative and other exotoxins have allowed us to identify two of these other variables. On macrophages, there appears to be at least one additional receptor epitope for exfoliative toxins that is not found within the MHCII molecule. ETA and ETB bind to macrophages from MHCII-deficient C2D knockout mice(81) in a manner that is concentration-dependent, saturable, and competitive (81). The toxins were able to induce immediate .signal transduction and substantial IL-6 secretion through the non-MHCII receptors. Furthermore, MHCI appears to be a functional superantigen receptor that regulates in vivo pathogenesis, as studies with SEA and SEB have identified a low-affinity receptor epitope in the a-2 domain of Db, an MHCI molecule (93,94). We have not yet determined whether this same receptor binds ETA ETB. However, all neonatal C2D (MHCII-/-) mice injected with ETB S. K. Chapes and Iandolo, unpublished results) exhibited a Nikolsky sign (4). Furthermore, monoclonal antibody specific for the 01-2 domain H-2Db blocks appearance of ETB-induced scalded skin syndrome. Therefore, MHCII receptors are unnecessary for manifestation of the disease and point to MHCI as a potentially important receptor in onset of the disease. These data confirm the findings of Baker et al. (361, who found monocytes in little binding of exfoliative toxin to lymphocytes exfoliative toxin-inducedskin lesions even when toxin concentrations approached levels (nM) that should bind MHCII. This should not be too surprising. Skin pathologies may not be dependent on MHCII receptor epitopes since epithelia and fibroblast-like cellsfound in the skin do not express MHCII molecules. However, MHCI moleculesare expressed on virtually all cells in the body. Indeed, even in those early studies, epidermal cells bound exfoliative toxin three times better than lymphocytes and monocytes (94). This does not mean that MHCII-positive Langerhans cells found in the skin will not respond to exfoliative toxin. Pickard et al. (95) found that ETA will activate Langerhans cells in a concentration-dependent way. This indicates that scalded skin syndrome may result from the activation several different cell types and includes processes that do not reflect superantigenic activity; e.g. proteaseactivity (35) binding to ganglioside receptors An additional variable that may affect superantigen responsiveness is the Ips gene. The Ips gene is located on the murine 4th chromosome. The gene has not yet been cloned, but its location has been mapped close to the rnupl, ps, and ifa genes (96). The Ips gene not

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only regulates macrophage responsiveness to LPS, but it appears to regulate responsiveness to a number of biological response modifiers, including the Gram-positive bacterium Propionibacterium acnes (97) and calcium ionophore (98). We found that macrophage stimulation by staphylococcal exoproteins, including ETA and ETB, is also controlled by Ips gene expression (65,99). Micethat are Ipsd are also significantly more resistant to superantigen-induced shock than I p s ” mice (99). Therefore, this is clearly an important regulatory gene in the responsiveness to superantigens. The recent association of the Ips gene with the sphingomyelinase signal transductionpathway (100) suggests that long-term activation of PKC is important to superantigen-mediated effects (101,102). This is consistent with other data indicating roles for PKC and tyrosine kinases in the activation of immune cells by superantigens 104). Clearly, we have a long way to go before we understand all the variables that contribute to superantigen responsiveness. However, our work with the exfoliative toxins has helped us toward that goal. REFERENCES 1. Ritter von Rittershain G. Die exfoliative dermatitis jungerer sauglinge.

Zent Z Kinderheilkd Melish ME, Glasgow LA. The staphylococcal scalded skin syndrome: the expanded clinical syndrome. J Pediatr Rogolsky M. Nonenteric toxins of Staphylococcus uureus. Microbiol Rev Melish M, Glasgow LA. The staphylococcal scaldedskin syndrome: development of an experimental model. N Engl J Med 1970; Elias PM, Fritsch P, Dah MV, Wolff K. Staphylococcal toxic epidermal necrolysis: pathogenesis and studies on the subcellular site of action of exfoliation. J Invest Dermatol Lillibridge CB, Melish ME, Glasgow LA. Site of action of exfoliative toxin in the staphylococcal scalded skin syndrome. Pediatrics McLay ALC, Arbuthnott JP, Lyell A. Action of staphylococcal epidermolytic toxin on mouse skin: an electron microscopic study. J Invest Dermatol. Koblenzer PK. Acute epidermal necrolysis (Ritter von RittershainLyell). Arch Dermatol Elias PM, Fritsch P, Epstein EH. Staphylococcal scalded skin syndrome. Arch Dermatol Lyell A. Toxic epidermal necrolysis (the scalded skin syndrome): a reappraisal. Br J Dermatol

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89.

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killer cell activity in the absence of major histocompatibility complex class I1 molecules. Infect Immun 193; 61:4013-4016. Machida K, Sakurai S, Kono I, Ikawa S. Genetic control of immune response to staphylococcal exfoliative toxin A in mice. Infect Immun 1987; 55:2768-2773. Kondo I, Sakurai S. Studies on toxicity and immunogenicity of staphylococcal exfoliatins A and B. In: Eaker D, Wadstrom T, eds. Natural Toxins. Elmsford, NY: Pergamon Press, 1980:379-387. Callahan J, Herman A, Kappler J, Marrack P. Stimulation of B1O.BR T cells with superantigenic staphylococcal toxins. J Immunol 1990; 144:2473-2479. Herman A, Kappler J, Marrack P, Pullen A. Superantigens: mechanism of T-cell stimulation and role in immune responses. Annu Rev Immunol 1991; 9:745-772. Choi Y, Kotzin B, Herron L, Callahan J, Marrack P, Kappler J. Interaction of staphylococcus aureus toxin "superantigens" with human T cells. Proc Natl Acad Sci USA 1989; 86:8941-8945. Beharka A, Armstrong J, Iandolo J, Chapes S. Binding and activation of major histocompatibility complex class 11-deficient macrophages by staphylococcal exotoxins. Infect Immun 1994; 62:3907-3915. Vroegop S, Buxser S. Cell surface molecules involved in early events in T-cell mitogenic stimulation by staphylococcal enterotoxins. Infect Immun 1989; 57:"-1824. Fraser J, Urban R, Strominger J, Robinson H. Zinc regulates the function of two superantigens. Proc Natl Acad Sci USA 1992; 89:5507-5511. Hudson K, Tiedemann R, Urban R, Lowe S, Strominger J, Fraser J. Staphylococcal enterotoxin A has two cooperative binding sites on major histocompatibility complex class 11. J Exp Med 1995; 182:711-720. Abrahmsen L, Dohlsten M, Segren S, Bjork P, Jonsson E, Kalland T. Characterization of two distinct MHC class I1 binding sites in the superantigen staphylococcal enterotoxin A. EMBO J 1995; 14:2978-2986. Schad E, Zaitseva I, Zaitsev V, Dohlsten M, Kalland T, Schlievert P, Ohlendorf D, Svensson L. Crystal structure of the superantigen staphylococcal enterotoxin type A. EMBO J 1995; 14:3292-3301. Kozono H, Parker D, White J, Marrack P,White J. Multiple binding sites for bacterial superantigens on soluble class I1 MHC molecules. Immunity 1995; 3:187-196. Jardetzky T, Brown J, Gorga J, Stern L, Urban R, Chi Y-I, Stauffacher C, Strominger J, Wiley D. Three-dimensional structure of a human class I1 histocompatibility molecule completed with superantigen. Nature 1994; 368:711-718. Grossman D, Lamphear J, Mollick J, Betley M, Rich R. Dual roles for class I1 major histocompatibility complex molecules in staphylococcal enterotoxin-induced cytokine production and in vivo toxicity. Infect Immun 1992; 60:5190-5196.


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90. Taub D, Blank K. Superantigens and microbial pathogenesis. Ann Intern Med 1993; 119239-90. 91. Jackett S, Stevens D. Superantigens associated with staphylococcal and streptococcal toxic shock syndrome are potent inducers of tumor necrosis factor-beta synthesis. J Infect Dis 1993; 168232-235. 92. Marrack P, Blackman M, Kushnir E, Kappler J. The toxicity of staphylococcal enterotoxin B in mice is mediated by T cells. J Exp Med 1990; 171:455-464. 93. Beharka A, Iandolo J, Chapes S. Staphylococcal enterotoxins bind H2Db molecules on macrophages. Proc Natl Acad Sci USA 1995; 9262946298. 94. Stiles B, Bavari S, Krakauer T, Ulrich R. Toxicity of staphylococcal enterotoxins potentiated by lipopolysaccharide: major histocompatibility complex class I1 molecules dependency and cytokine release. Infect Immun 1993; 61:5333-5338. 95. Pickard S, Shankar G, Burnham K. Langerhans cell depletion by staphylococcal superantigens. Immunology 1994; 83:568-572. 96. Vogel S, Wax J, Perera I?, Padlan C, Potter M, Mock B. Construction of a BALB/c congenic mouse, C.C3H-LPSd, that expresses the LPSd allele: analysis of chromosome 4 markers surrounding the LPSLPStgene. Infect Immun 1994; 62:4454-4459. 97. Jackson R, Lill P, Gangemi J. Role of pulmonary macrophages in resistance to experimental metastasis. J Leukoc Biol 1986; 40:575-587. 98. Shinomiya H, Nakano M. Calcium ionophore A23187 does not stimulate lipopolysaccharide nonresponsive C3H/HeJ peritoneal macrophages to produce interleukin 1. J Immunol 1987; 139:2730-2736. 99. Chapes SK, Beharka AA. Liposaccharide is required for the lethal effects of enterotoxin B after D-galactosamine sensitization. J Endotoxin Res 1995; 2263-271. 100. Barber S, Perera P-Y, Voge S. Defective ceramide response in C3H/HeJ ( LPSd) macrophages. J Immunol 1995; 155:2303-2305. 101. Borchardt R, Lee W, Kalen A, Buckley R, Peters C, Schiff S, Bell R. Growth-dependent regualtion of cellular ceramides in human T-cells. Biochim Biophys Acta 1994; 1212:327-336. 102. Joseph C, Wright S, Bornmann W, Randolph J, Kumar E, Bittman R, Liu J, Kolesnick R. Bacterial lipopolysaccharide has structural similarity to cerminde and stimulates ceramide-activated protein kinase in myeloid cells. J Biol Chem 1994; 269:17606-17610. 103. Scholl P, Trede N, Chatila T, Geha R. Role of protein tyrosine phosphorylation in monokine induction by the staphylococcal superantigen toxic shock syndrome toxin-1. J Immunol 1992; 148:2237-2241. 104. Chapes SK, Kopydlowski K, Fleming S, Iandolo J. Glucocorticoid effects in immune cell activation by staphylococcal exotoxins and lipopolysaccharide. Trans Kans Acad Sci 1992; 95:23-28.


12 Molecular Genetics, Structure, and Immunobiology of Streptococcal Pyrogenic Exotoxins A and C Michael H. Kim and Patrick M. Schlievert

University of Minnesota Medical School, Minneapolis, Minnesota

1.

INTRODUCTION

In 1924 Dick and Dick reported that filtrates of scarlet fever-associated streptococcal strains induced a rash when injected intradermally into healthy subjects (1). That was the beginning of a long road that has seen many milestones concerning the biological properties of what the Dicks called erythrogenic toxins. Over the next 45 years, many attempts were made to isolate the toxins that were part of these filtrates. The efforts of several investigators led to the identification of three toxins (2-5). It was shown that these purified scarlet fever toxins not only produced a rash, but, along with many other properties, had the capacity to induce a high fever when injected into animals (5). This distinctive fever-producing ability led Watson (5) to label these proteins streptococcal pyrogenic exotoxins (SPEs) A, B, and C. The SPEs were also shown to elicit a number of pathological effects in animals including structural changes in heart and liver tissue (3,6). 257

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Importantly, it was also noted that when SPE injections were followed by the administration of endotoxin, a potent synergistic effect occurred, drastically reducingthe quantity of both SPE and endotoxin needed for lethality A model of pathogenicity was proposed in which the SPEs made by an invading streptococcal strain cause damage to the host and amplify the effects of circulating endotoxin, leading to the deadly effects seen in severe scarlet fever cases. In a new property of the SPEs was discovered. The toxins were shown to have the ability to induce mitogenic effects on lymphocytes Approximately years later it was noted that these toxins had specificity for the variable portion of the beta chain of the T-cell receptor (11). This effect, now referred to as superantigenicity, did not reach the wider scientific community until only recently, although the Tlymphocyte proliferative effects of the toxins have been studied consistently through the and Although group A streptococci have a number of important virulence factors, including surface proteins and other exotoxins, the have particular relevance to severe disease in humans andconstitute the major exotoxins of these organisms The relatives of the SPEs found in Staphylococcus uureus have been associated with the severe and potentially fatal toxic shock syndrome (TSS) The SPEs have been linked to a streptococcal TSS (STSS) as well as milder scarlet fever illness, sometimes referred to as scarlatina The prevalence of these toxins in strains isolated from STSS cases is significantly higher than in strains isolated from uncomplicatedpharyngitis The SPEs belong to a family of true exotoxins, referred to as pyrogenic toxin superantigens (PTSAgs), that include SPE serotypes A-C, mitogenic factor (SPE F), streptococcal superantigen (SSA), the staphylococcal enterotoxins serotypes A-H but not F, and toxic shock syndrome toxin-l( Recently, non-group A streptococcal pyrogenic toxins have been described and may also be part of this group All of these toxins share many biological activities, including pyrogenicity, the ability to enhancethe effects endotoxin shock, and the ability to cause T-cell proliferation as superantigens This chapter will focus on the genetic control, structure, and function of two important and related SPEs, types A and C. II. STREPTOCOCCAL PYROGENIC EXOTOXIN A

The actual discoverydate of SPE A is ambiguous since itappears than many investigators may have worked with the toxin under different

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guises. The toxin we know today as SPE A was first definitively established by Stock (2). SPE A, however, is believed to be equivalent to the major toxic factor in what was previously described as erythrogenic toxin (30). SPE A as well as the other toxins in this family, whether staphylococcal streptococcal in origin, can easily bepurified after culture of organisms in a dialyzable beef heart medium (13,15,31). Toxin is purified from the medium by precipitation with ethanol, resolubilization in acetate-buffered saline atpH 4, followed by dialysis against water and then preparative thin-layer isoelectric focusing in successive pH gradients of 3-10 and 4-6 (for SPEA or with another narrow gradient for other toxins depending on their isoelectric point) (25). SPE A has a molecular weight of approximately 25,000 when tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and occurs as two forms based on isoelectric point (PI), with PIS of 4.5 and 5.5. The molecular basis for the two forms is unclear, but both forms can be convertedinto a single form bytreatment with 2-mercaptoethanol (32). Production of SPE A has been epidemiologicallylinked to The gene for SPE A is found in 50-90% of strains isolated from STSS compared to only about 15% of isolates from uncomplicated pharyngitis (21-24). The two major organisms associated with STSS are streptococci of M types 1 and Nearly all of these organisms have the gene for SPE A, designated speA, though significant differences exist in amounts of SPE A that is produced by M1 compared to M3 strains. Furthermore, recent studies suggest that there has been an emergence of invasive M1 isolates since the late 1980s when STSS was first described (33-35). The emergent invasive M1 strains differ from prior noninvasive M1 isolates in that the invasive strains carry the bacteriophage encoding the speA. SPE A also appears to be the most prevalent toxin in the severe scarlet fever cases described in the early part of this century (36,37). Significant experimental evidence supports the role of the SPEs, most notably SPE A, in the development of scarlet fever and STSS. example, historical strains of group A streptococci from patients with severe scarlet fever make SPE A This was the erythrogenic toxin described by the Dicks. We have shown that erythrogenic toxin make by A. Stock, a contemporary of the Dicks, is primarily SPE A. Furthermore, Lederle horse antibody against erythrogenic toxin, made for treatment of severe scarlet fever, is highly specific for SPE A. Recently, two researchers injected themselves with preparations of SPE A and induced clinical STSS.

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Experiments, performed using isogenic strains of streptococci, with and without the SPE A bacteriophage administered in subcutaneously implanted Wiffle balls in rabbits, showed that the SPE Aproducing strain caused STSS whereas the SPE A-negative strain did not (30). Interestingly, although the SPE A-positive organism caused STSS, none of the infected animals developed necrotizing fasciitis, often seen in association with STSS. Purified SPE A was also capable of inducing STSS when administered to rabbits in subcutaneous miniosmotic pumps (38). Approximately 200 pg of toxin given in the miniosmotic pumps represents the LD,, with an LD, of pg. Again, necrotizing fasciitis is not seen in this model. Finally, prior immunization of rabbits against SPE A provided protection from both STSS and necrotizing fasciitis when rabbits were challenged subcutaneously with viable M1 or M3 invasive group A streptococci (30). The majority of immune animals showed only purulent abscesses after challenge but no symptoms ofSTSS, whereas nonimmune animals lack pyrogenic responses and developed STSS with necrotizing fasciitis. The data from this last experiment suggested that SPE A-induced release of tumor necrosis factor (TNF)-a (39) may interfere with the normal PMN chemotactic response to infection. Antibody neutralization SPE A may prevent this TNF-a release, thus allowing for a normal PMN response.

Genetics of SPE It was first published in 1964 that a bacteriophage, designated T12, was responsible for the transmission of the ability to make SPE A from a toxin-positive strain (T25,T12) to a toxin-free strain (T25,) (40). The role of the bacteriophage was conclusively establishedboth with the isolation and characterization of the large 36-kb T12 bacteriophage (Fig. la) (41) and when the SPE A structural gene was localized onto the bacteriophage (42). The gene for SPE A, now designated speAl, was first cloned by Johnson and Schlievert (42) from the bacteriophage T12 and was later sequenced by Weeks and Ferretti (43). The structural gene consists of 756 base pairs (bps) encoding 252 amino acids, of which the first 30 are removed during toxin secretion, and thus, comprises a signal peptide (Fig. 2). Two putative promoter regions have been located as well as a putative ribosomal binding site. Approximately 850 bp 3' the structural gene is an inverted repeat that appears to be required for stable transcription. It may be part of a transcriptional termination region.

Molecular Genetics of SPES

261 Eco RI

oac

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Figure 1 Physical map of (A) the bacteriophages T12 (encodes speA) and (B) CS112 (encodes spec). Bacteriophages for these toxins are circularly permuted and terminally redundant and thus map as circular phages of kb for T12 and 40.8 kb for CS112. The position and direction transcription of the SPE genes are indicated. The location bacteriophage integration into the streptococcal chromosome is designated att.

Kim and Schlievert 1: ATGTTTGACA GCTTATCATC GATAAGCTTA CTTTTCGAATCAGGTCTATC

CTTGAAACAG GTGCAACATA GATTAGGGCA

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Figure 2 Nucleotide and inferred aminoacid sequences of speA and SPE A, respectively. Amino acidsare indicated by their single-letter designations. The bar indicates the cleavage site for removal of the signal peptidefrom mature SPE A.

S

Molecular Genetics of

263

Recently, Nelson et al. (44) have demonstrated the existence of three additional allelic forms of speA. The M type 1 SPE A gene has a nucleotide substitution at base pair 328of the structural gene, resulting in a serine instead of a glycine at amino acid 110. This gene is referred to as speA2. The M3 SPE A gene (speA3) has a nucleotide substitution at position 316, resulting in an isoleucine in place of a valine at amino acid Finally, speA4 encodes a toxin thathasseveralamino acid changes compared to the standard SPE A. Transcription of speA is probably under some form of genetic control. Expression of speA varies in a clonal manner, generally with respect to M type. For example, M3 strains are high producers SPE A while recently isolated M1 strains are low producers (at least fold different). The amount of toxin made by strains appears to be proportional to the amount of speA messenger RNA made. The variable expression among M types may be due to an element external to the bacteriophage. The SPE A gene is adjacent to the chromosomal insertion site of the bacteriophage (4X,and it is possible that a cisacting element near this site could be controlling SPE A expression. Alternatively, a global regulatory network may be controlling SPE A production. Precedence for this existswith virulence factors expressed the surface of Streptococcus pyogenes. Many of these factors are controlled by a global regulatory system known as mga (45). In addition, when speA is cloned into aureus, the gene is partially under control of the global regulator of virulence factor production referred to as the accessory gene regulatory (agr) system. Finally, like S. aureus exotoxins under agr control, SPE A is produced primarily in the postexponential phase of growth. B.

Structure of SPE A

Since the large PTSAg family shares many biological properties, it would be expected that they would have structural similarities. The primary nucleotide and amino acid sequences have been determined for nearly all of the toxins, and the amount of sequence similarity is surprisingly variable and often low (Fig. 3). Two subgroups within the family have been established, including an interesting one that links SPE A with the staphylococcal enterotoxins B and C. It is possible that SPE A arose as a result of gene transfer from S. clureus to group A streptococci. The other subgroup consists of staphylococcal enterotoxins A, D, and

Kim and Schlievert

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SPE A

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Figure Primary amino acid sequence similarities of the pyrogenic toxin superantigens. Numbers represent percent similarity; NA = not available. Bold numbers indicate significant homology.

The three-dimensional structure of several of the staphylococcal PTSAgs has been determined, including enterotoxins A, B, C,,C,, and toxic shock syndrome toxin-l (46-49). These proteins appear to have similar secondary and tertiary folds (see other chapters). Based on the sequence homologies of SPE A with enterotoxin C,, a structural model of SPE A (Fig. 4) has been created based on the coordinates for enterotoxinC,. This preliminary structure also has the characteristic features of the other PTSAgs. These features occur in two major domains of the toxin, designated A and B. Starting at the Nterminus and following along the peptide backbone, the similarities to the other toxins will be summarized. The first common feature is a short alpha helix in domain A (a31 leading into domain B, which consists of a large barrel of antiparallel beta strands. The structure then returns to domainA, where there i s a characteristic long diagonal central alpha helix(a5)followed by a wall of beta strands. Above the beta barrel of SPE A, there is a short region that contains three cysteine residues in close proximity, two of which may form a cysteine loop. Similar structures have been determined for the enterotoxins but not as yet for SPE C. The structures of the PTSAgs give some clues as to how they interact with T cells and antigen-presenting cells (APCs) and to where other biological activities arise. The PTSAgs form a ternary complex between themselves and the T-cell receptor (TCR) and the major histocompatability complex (MHC) class I1 molecules on APCs (11). This triad facilitatesa strong T-cell proliferative effect independent of

Molecular Geneticsof SPES

Domain A

265

Domain B

Figure 4 Ribbon diagram the modeled structure SPE structural coordinates staphylococcal enterotoxin type C.

based on the

the antigenic specificity of the TCR. The binding site on the TCR for the PTSAgs is the Vp region on the exterior of the typical antigen peptide recognition site (50). Each PTSAg stimulates a collection of T cells based on the Vp profile. Thus, each toxin can cause proliferation of 5-50% of all T cells as opposed to the conventional antigenic response of about 0.0001% of cells. Mutagenesis studies of PTSAgs along with cocrystallization and structural determination data have localized regions of toxin interaction with MHC class I1 molecules and with the TCR (see other chapters). PTSAgs appear to vary in their structural features required for binding to MHC class I1 molecules, and each has a preferential subset of MHC class I1 molecules it binds to. The TCR and MHC class I1 binding sites on SPE A are at present the subject of investigation. Site-directed mutagenesis of specific amino acids in the SPE A sequence has revealed some association$ It is clear that a deep groove that runs along the back side

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of the molecule, with the diagonal a helix at its base, is important for T-cell proliferation and probably encompasses the TCR interaction site. Also, the presumed cysteine loop that exists on the top of the beta barrel appears to be important. Abolishment of the residues that form this loop result in a nonmitogenic protein A recent study has suggested that the interaction ofSPE A with MHC class I1 molecules depends on amino acids in the beta barrel STREPTOCOCCAL PYROGENIC EXOTOXIN C

Less is known about SPE C than about the other PTSAgs. This is in part due to the poor immunogenicity, and thus the difficulty in the detection, ofSPE C. Only a small number of rabbits have been successfully immunized against this toxin, even when immunized for more than a year (unpublished observation). Also, SPE C is both produced in smaller quantities and is less stable than the other toxins. Under our standard culture and purification conditions described above, yields of toxin are approximately one-tenth of those for SPE A. SPE was first identified in group A streptococcal strains associated with scarlet fever in which animals were not protected by immunization with SPEs A and B SPE C is serologically distinct from SPEs A and B, but the toxin can be coproduced with either or both of these other toxins. Like SPE A, the structural gene for The toxin is most SPE C, spec, is encoded by bacteriophage easily purified from M type strains of group A streptococci, which typically produce SPE but not A and The protocols usedto purify SPE C are the same as those for SPE A, notably ethanol precipitation from stationary-phase culture fluids followed by isoelectric focusing. The p1ofSPE C is approximately 7.0, and its molecular weight is approximately Some studies suggest that SPE C is epidemiologically associated with development of severe invasive streptococcal diseases such as (55). In addition, all M18 streptococci associated with rheumatic fever, including recent outbreaks, make the toxin. Many other rheumatogenic strains also make SPE C However, whether or not this toxin has a causative effect in rheumatic fever remains to be determined. Finally, group .A streptococcal strains associated with guttate psoriasis consistently make SPE either alone or together with SPEs A and B

267

Molecular Geneticsof SPES

Genetics of

SPE C

As indicated above, the gene for SPE C is also carried within bacteriophage DNA (53,54). The SPE bacteriophage isolated fromstrain CS112 is of similar size to that ofT12 phage encoding speA (Fig. lb). As with SPE A, the structural gene for SPE C lies near the attachment site insertion of the bacteriophage into the bacterial chromosome (54).

The gene for SPE C has been cloned and characterized ( 5 7 ) . The gene consists 708 nucleotides that encode for 36 amino acids, the first 27 which are removed as the signal peptide (Fig. 5 ) (58,591. Polymorphism in the nucleotide sequence of the structural gene for l: CMCCTTGAC TATTTAAATG GMCTGCCAC TCCTAAAAAC TAMATATM ATACATTTAT AAAATTTCTAMTAAACAGA

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Figure 5 Nucleotide and inferred amino acid sequence of spec and SPE C, respectively. Amino acidsare indicated by their single-letter designations. the bar indicates the cleavage site for removal of the signal peptide from mature C.

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SPE C has been observed, although all base substitutions sequenced so far have resulted in silent mutations in the mature protein (59). As with SPE A, SPE C exhibits variability in the amount of toxin produced from strain to strain, generally dependent on M type. With this consideration, it is likely that SPE C is regulated in much the same way that SPE A is. Also, like SPE A, SPE C is made in the postexponential growth phase. B.

Structure of SPE C

The nucleotide and amino acid sequences of SPE C are relatively unrelated to those of other PTSAgs (Fig. Again, this was at first unexpected due to the similarities in biological activities. Itis expected that SPE C will have the same general secondary and tertiary structure as the other known PTSAg structures.

W.

IMMUNOBIOLOCY OF THE SPES

Many activities of the PTSAgs are shared, but each toxin group also has unique features. All of the PTSAgs are pyrogenic, superantigenic, and enhance host susceptibility to the lethal effects of endotoxin. The staphylococcal enterotoxins, however, have a unique emetic property and thus cause staphylococcal food poisoning. The have the unique property of predisposing the'host to significant cardiotoxicity. Also, SPE A (the only PTSAg evaluated so far) has been shown to bind to lipopolysaccharide. Many of the effects of the SPEs are the result of their interaction with cells of the immune system. Other activities may be due to direct effects on host tissues. The biologicalproperties of the SPEs are discussed below. A.

Pyrogenicity

There are conflicting views concerning the mechanism of fever production by SPEs, which are among the most potent pyrogens known. Injection of &kg of purified SPEs A and C into rabbits results in a peak fever as high as "C at 3-4 hr postinjection The minimum pyrogenic dose of these toxins in rabbits is 0.15 pg/kg. Traditionally, models for the fever response to exogenous pyrogens rely on the stimulation of the endogenous pyrogens IL-1 and TNF-a in monocytes. Evidence suggests that the SPEs also have a more direct effect on the fever response control center of the hypothalamus. The SPEs have the ability to break down and cross the

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blood-brain barrier (60,61). Other data implicating a direct effect look at the temporal features the SPE fever. Classically, endotoxin-induced fever is mediated by endogenous pyrogens and peaks at 1and hr postinjection. SPE-induced fever peaks atonly 4 hr (61). In SPEimmune animals, injection of SPE intravenously does not result in fever, but if toxin is administered intracisternally into the cerebrospinal fluid, fever results (61). The dose of toxin required to produce fever intracisternally is at last 1000 times lower than when the toxin is given intravenously. This central nervous system activity may take the form of direct prostaglandin stimulation in the hypothalamus and/or local production of cytokines. B.

Enhancement of EndotoxinShock

A key feature in the pathogenesis ofSTSS may be the ability of the SPES to act synergistically with endotoxin from Gram-negative bacteria. The presence of SPE reduces the LD,of endotoxin by as much as 100,000-fold (7). Interestingly, in a rabbit model, the timing of the injections is important. Exotoxin must be given at least 1-2 hr before endotoxin to achieve synergism. If endotoxin is given prior to at the same time as SPE, lethality is greatlydiminished (7,62). This may clarify part of the pathogenesis of this enhancement effect. The most obvious explanation is that the pretreatment with SPE results in decreased clearance of endotoxin. This is supported by the observation that the SPES cause damage to the liver (5,621 and more specifically block the reticuloendothelial system (RES) (63), which is responsible for endotoxin clearance (64). When these rabbits are challenged with SPEs, RES function, as measured by clearance of colloidal carbon clearance from the bloodstream, decreases within hours of toxin injection and continues over 2 days, slowly returning to normal (62,63). This is in contrast to the effects of endotoxin alone on the RES. Injection of endotoxin causesan initial decrease in activity, but at 1and 2 days, the activity increases to above-normal levels (63). Interestingly, a-amanitin, a potent inhibitor of mRNA polymerase, also has the same ability to enhance lethal endotoxin shock. Lile the SPEs, aamanitin must be given to animals 1-2 hr prior to endotoxin treatment. Studies have shown that the SPES also inhibit RNA synthesis in liver cells, but it is unknown whether this is a primary or secondary effect in liver toxicity. The final pathway of shock by the enhancement phenomenon is almost certainly mediated by endotoxin interaction with lipopolysaccharide (LPS)-binding protein, subsequent release of TNF-a from

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macrophages, and ensuing effects on the vasculature to induce capillary leak.

C. Cardiotoxicity Experimental animals that survive the ability of the SPES to enhance susceptibility to endotoxin show .extensive myocardial necrosiswithin 3-4 days (5). In addition, the SPES enhance the cardiotoxic effects of streptolysin 0 Because of these effects and the association of the SPES, notably SPE C, with rheumatic fever strains of group A streptococci, these toxins may be important in the early events leading to this autoimmune responses against other streptococcal products. Furthermore, the superantigenic effects of the SPES may amplify autoimmune processes. Finally, SPES are not made by strong hyaluronidaseproducing streptococci. The same strains do not appear to cause rheumatic fever (65). D. T-Cell Proliferation

First identified more than 20 years ago, this amazing property of the superantigens has been the main focus of the majority of recent research on the PTSAgs (9,661. Originally viewed as a nonspecific proliferation of T cells, it is now known that each toxin stimulates a subset of T cells with recognition of the Vj3 region of the T-cell receptor. SPE A preferentially stimulates T cells bearing Vp 8.2 in mice (67) and 2,4, 12, 14, and 15 in humans (68). SPE stimulates human Vps 2,5.1, 10, and 15 (68). As alluded to previously, PTSAgs create a large imbalancein the cytokine profile of the infected host. Principal cytokines released include IL-1 and TNF-a from macrophages and IL-2, interferony, and TNF-j3 from T cells. Animals injected with SPES in miniosmotic pumps succumb in three major groups. One set of animals die within 1-2 days, a large group on days 4-5, and a final set on days 7-10. It is possible that the first group of animals succumb as a result of the enhanced susceptibility to endotoxin whereas the second group die as a result of the cytokine release from superantigenicity. The mechanism underlying the late death is unclear. A model for the mechanism of hypotension and shock leading to death, induced by SPES and other PTSAgs, is shown in Fig. 6. E.

ScarletFever

Rash

Originally considered the result of primary toxicity, it now appears that the characteristic rash seenin scarlet fever and STSS is due to an

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Figure 6 Proposed mechanism of development of hypotension and shock by pyrogenic toxin superantigens (PTSAgs).

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amplified hypersensitivity reaction resulting from superantigenicity. Rash is not seen in those individuals in experimental .animals that have not had prior exposure to group A streptococci. Thus, skin reactivity is lower in the very young and in military recruits from the Southern United States where there is a lower incidence of scarlet fever. Thislack of skin reactivity upon initial exposure to SPEs is why Watson proposed the name “streptococcal pyrogenic exotoxins’’ the toxin family. Sincethe defining feature of erythrogenic toxin was the appearance of rash, he believed the SPEs were a separate family. The rash appears to be the result of cytokine release from a secondary antigen exposure that is then amplified by SPE superantigenicity. For example, animals injected intradermally with SPEs do not show scarlet fever rashes. However, if the animals are presensitized with either SPEs or other antigens likely to induce delayed hypersensitivity, rash occurs with a subsequent intradermal challenge plus the sensitizing antigen. Finally, if antibodies against with SPEs are present in the animal, the scarlet fever rash does not occur, though the animals will continue to show delayed hypersensitivity skin reactivity to the sensitizing antigen. F.

B-Cell lmmunosuppresion

Hanna and Watson first showed that SPEs suppress IgM antibody production but deregulate IgG responses When the spleens from SPE-treated animals were examined, the authors noted that, although the overall number of cells in the spleen was increased, the number of IgM antibody-producing cells was a sixth of control spleens. PTSAg activation of CD4+ T cells to release interferon7 appears to be the mechanism that results in antibody suppression The mechanism of deregulation of IgG synthesis is unclear. G.

Binding of SPE A to Endotoxin

When patients succumb to there is large-scale depletion of their lymphocytes. Depletion is not in a VP-specific manner, as might be expected after superantigenicity, but rather there is essentially a disappearance the immune system. The mechanism underlying this phenomenon has been partially investigated and may depend on SPE interaction with endotoxin late in disease to kill rather than stimulate lymphocytes (72). It has been shown that SPE A binds to endotoxin through ketodeoxyoctonate residues in the common core region. The complex formed has the ability to bind to lymphocytes and cause cell death.

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This effect does not appear to involve a SPE A endotoxin interaction with the TCR, since T cells with TCRs that do not bind SPE A are also killed. The molecular mechanism of cell death is unclear. V.

NON-GROUP A STREPTOCOCCALSUPERANTIGENS

Streptococcus agalactiae (group streptococci) are common pathogens of neonates but occasionally can causesevere infections in older children (73) and in adults (74). In some of these cases of severe disease, the symptomatology is similar to that ofSTSS, including fever, hypotension, rash, desquamation, and multiorgan involvement. An exotoxin was isolated from one strain of group B isolated from a patient with STSS. This toxin displayed the classic biological activities of the PTSAgs. This 12,000-molecular-weight protein induced high fever when injected into rabbits, enhanced the lethal effects of endotoxin, and was mitogenic for rabbit splenocytes (27). The toxin was different from the other toxins in that it was about half the size of the others (and thus may represent a cleavage product), its enhancement endotoxin took longer (4 days instead of 21, and animals developed hind-limb paralysis. T-cell proliferative effects were comparable to those of the other exotoxins. Anecdotal evidence suggeststhat there are also PTSAgs in group C, F, and G streptococci. Recently, three cases of necrotizing fasciitis and myositis, one of these patients having STSS, have been reported in association with group G streptococci (28,751. A 26,000-molecularweight protein with a p1of approximately 6 was obtained from one strain that was mitogenic for rabbit splenocytes, induced a high fever when injected into rabbits, and enhanced the lethal effects of endotoxin. This toxin showed no cross-reactivity with SPEs A, ,or C when tested by reactivity with polyclonal antibodies in Western immunoblot (28). Furthermore, chromosomal DNA from the group G streptococcal strain failed to hybridize with or C specific probes under stringent conditions. VI.

CONCLUSION

The bacterial PTSAgs are tremendously active. toxins with a wide range of immunological and direct toxic properties. Although many insights into the pathogenesis of these toxins have been gained, we are still a long way from fully understanding how these toxins cause disease. For example, the amount of SPE required to induce death in rabbits appears to be 100-200 pg. Mice and other animals appear to

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be even more resistant to SPES. In contrast, 2 may be lethal in humans. The reasons for these differences are unclear, but must be clarified if we are to fully understand the toxins’ pathogenic mechanisms. The possibilities for the understanding the pathogenesis of STSS will be valuable in the prevention, detection, and treatment of this severe disease. Insight into how these toxins may be involved into autoimmunity must also be researched. Finally,the immunological properties of the superantigens can be exploited for their therapeutic potential. VII.

ACKNOWLEDGMENT

This work was supported by USPHS Grant HL36611 from the National Heart, Lung, and Blood Institute. REFERENCES 1. Dick GF, Dick GH. A skin test for susceptibility to scarlet fever. JAMA

1924;82:256-266. 2. Stock AH, Verney E. Properties of scarlet fever toxin of the NY5 strain.

J Immunol 1952; 69:373-378. 3. Schwab JH, Watson DW, Cromartie WJ; Further studies of group A streptococcal factors with lethal and cardiotoxic properties. J Infect Dis 1955;96:14-18. 4. Stock AH, Lynn RJ. Preparation and properties partially purified erythrogenic toxin B of group A streptococci. J Immunol 1961;86:561566. 5. Watson DW. Host-parasite factors in group A streptococcal infections: pyrogenic and other effects on immunologic distinct exotoxins related to scarlet fever toxins. J Exp Med 1959; 111:255-283. 6. Schwab JH, Watson DW, Cromartie WJ. Production of generalized Schwartzman reaction with group Astreptococcal factors. ProcSOCExp Biol Med 1953; 82:754-761. 7. Kim YB, Watson DW. purified group A streptococcal pyrogenic exotoxin: physiochemical and biological properties including the enhancement of susceptibility to endotoxin lethal shock. J Exp Med 1970; 131:611628. Taranta A, Cuppari G, Quagliata F. Dissociation of hemolytic and lymphocyte-transforming activities of streptolysin S preparations. J Exp Med 1969;129:605-622. Peavy DL, Adler W, Smith RT. The mitogenic effectsof endotoxin and staphylococcal enterotoxin B on mouse spleen cells and human peripheral lymphocytes. J Immunol 1970; 105:1453-1458. 10. Plate J M , Amos B. Lymphocyte stimulation by a glycopeptide isolated

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from Streptococcuspyogenes C203S. I. Isolation and partial purification. Cell Immunol 1971; 1:476-487. 11. Marrack P, Kappler J. The staphylococcal enterotoxins and their relatives. Science 1990;248:705-711. 12. Hanna EE, Watson DW. Enhanced immune response after immunosuppression by streptococcal pyrogenic exotoxin. InfectImmun 1973; 7:1009and specific 13. Barsumian EL, Schlievert PM, Watson DW. Nonspecific immunological mitogenicity by group A streptococcal pyrogenic exotoxins. Infect Immun 1978;22:681-688. 14. Schlievert PM. Role of superantigens in human disease. J Infect Dis 1993; 167:997-1002. Schlievert PM, Shands KN, Dan BB, Schmid GP, Nishimura RD. Identification and characterization of an exotoxin from Staphylococcus uureus associated with toxic shock syndrome. J Infect Dis 1981;143:509-516. 16. Bergdoll MS, Crass BA, Reiser RF, Robbins RN, Davis JP. A new staphylococcal enterotoxin, enterotoxin associated with toxic shock drome. Lancet 1981;i:1017-1021. 17. Schlievert PM. Biological properties of toxic shock syndrome exotoxin. Surv Synth Pathol Res 1984; 3:54-62. 18. Schlievert PM, Bettin KM, Watson DW. Production of pyrogenic exotoxin by groups of streptococci: association with group A. J Infect Dis 1979;140:676-681. 19. Cone LA, Woodard Dr, Schlievert PM, Tomory GS. Clinical and bacteriologic observations of a toxic shock-like syndrome due to Streptococcus pyogenes. N Engl J Med 1987; 317:146-149. 20. Stevens DL, Tanner MH, Winship J, Swarts R, Ries KM, Schlievert PM, Kaplan E. Severe group A streptococcalinfections associated with a toxic shock-like syndrome and scarlet fever toxin A. N EnglJ Med 1989; 321:l7. 21. Musser JM, Hauser AR, Kim MH, Schlievert PM, Nelson K, Selander RK. Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression. Proc Natl Acad Sci USA 1991; 88:2668-2672. 22. Hauser AR, Stevens DL, Kaplan EL, Schlievert PM. Molecular analysis of pyrogenic exotoxins from Streptococcus pyogenes isolates associated with toxic shock-like syndrome. J Clin Microbiol 1991; 299562-1567. 23. Yu CE, Ferretti JJ. Frequency of the erythrogenic toxin B and genes (speB and spec) among clinical isolates of group A streptococci. Infect Immun 1991;59:211-215. 24. Norgren M, Norrby A, Holm Genetic diversity in TlMl group A streptococci in relation to clinical outcome of infection. J Infect Dis 1992; 166:1014-1020. 25. Bohach GA, Fast DJ, Nelson RD, Schlievert PM. Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses. Crit Rev Microbiol 1990; 17:251-272.

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26. Schlievert PM. The role of superantigens in human disease. Curr Opin Infect Dis 1995;8:170-174. 27. Schlievert PM,Gocke JE, Deringer JR. Group B streptococcal toxic shock-like syndrome: report of a case and purification of an associated pyrogenic toxin. Clin Infect Dis 1993; 17:26-31. 28. Wagner JG, Schlievert PM, Assimacopoulos AP, Stoehr JA, Carson PJ, Komadina K. Acute group G streptococcal myositis with streptococcal toxic shock syndrome: case report, microbiology, and review of the literature. J Infect Dis (in press). 29. Schlievert PM,BohachGA, Ohlendorf DH, Stauffacher CV, Leung DYM, Prasad GS, Earhart CA, Jablonski LM, Hoffmann ML, Chi YI. Molecular structure of Staphylococcus and Streptococcus superantigens. J Clin Immunol 1995; 15:4-10. 30. Schlievert PM, Assimacopoulos AP, Cleary PP. Severe invasive group A streptococcal disease: clinical description and mechanisms of pathogenesis. J Lab Clin Med 1996; 127:13-22. 31. Schlievert PM, Bettin KM, Watson DW. Purification and characterization of group A streptococcal pyrogenic exotoxin type C. Infect Immun 1977;16:673-679. 32. Nauciel C, Blass J, Mangalo R, Raynaud M. Evidence for 2 molecular forms of streptococcal erythrogenic toxin: conversion to a single form by 2-mercaptoethanol. Eur J Biochem 1969; 11:160-164. 33. Gaworzewska E, Colman G. Changes in the pattern of infection caused by Streptococcus pyogenes. Epidemiol Infect 1988; 1988:257-269. 34. Cleary PP, Kaplan EL, Handley JP, Wlazlo A, Kim MH, Hauser AR, Schlievert PM. Clonal basisforresurgence of serious Streptococcus pyogenes disease in the 1980s. Lancet 1992; 339:518-521. 35. Musser Jm, Kapur V, Szeto J, Pan Swanson Genetic diversity and relationships among Streptococcus pyqenes strains expressing serotype M1 protein: recent intercontinental spread a subclone causing episodes of invasive disease. Infect Immun 1995; 63:994-1003. 36. Birkhaug KE. Studies in scarlet fever. 11. Studies on the use of convalescent scarlet fever serum and Dochez’ scarlatinal antistreptococcal serum for treatment of scarlet fever. Bull Johns Hopkins Hosp 1925; 36:134-171. 37. Hooker SB, Follensby EM. Studies of scarlet fever. 11. Different toxins produced by hemolytic streptococciof scarlatinal origin. J Immunoll934; 18:177-193. 38. Lee PK, Schlievert PM. Quantification and toxicity of group A streptococcal pyrogenic exotoxins inan animal model of toxic shock syndromelike illness. J Clin Microbiol 1989; 27:1890-1892. 39: Fast DJ, Schlievert PM, Nelson RD. Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducersof tumor necrosis factor production. Infect Immun 1989; 57:292-294. 40. Zabriskie JB. The role of temperate bacteriophage in the production of erythrogenic toxin by group A streptococci. J Exp Med 1964; 119:761-779.

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41. Johnson LP, Tomai MA, Schlievert PM. Bacteriophage involvement in group A streptococcal pyrogenic exotoxin Aproduction. Bacterioll986; 166:623-627. 42. Johnson LP, Schlievert PM. Group A streptococcal phage Tl2 carries the structural gene for pyrogenic exotoxin type A. Mol Gen Genet 1984; 194:52-56. 43. Weeks CR, Ferretti JJ. Nucleotide sequence of the type A Streptococcus pyogenes bacteriophage T12. Infect Immun 1986; 52:144-159. 44. Nelson K, Schlievert PM, Wander RK, Musser JM. Characterization and clonal distribution of four alleles of the speA gene encoding exotoxin A (scarlet fever toxin) in Streptococcus pyogenes. J Exp Med 1991; 174:12711274. 45. Chen C, Bormann N, Cleary PP. VirR and Mry are homologous trans acting regulators of M protein and C5a peptidase expression in group A streptococci. Mol Gen Genet 1993; 241:685-693. 46. Swaminathan S, Furey W, Pletcher J, Sax M. Crystal structure of staphylococcal enterotoxin B, a superantigen. Nature 1992; 359:801-805. Schlievert PM, 47. Prasad GS, Earhart CA, Murray DL, NovickRP, Ohlendorf DH. Structure of toxic shock syndrome toxin 1. Biochemistry 1993;32:13761-13766. 48. Hovde CJ, Marr JC, Hoffmann ML, Hackett SP, Chi YI, Crum KK, Stevens DL, Stauffacher CV, Bohach GA. Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin Cl. Cl Mol Microbiol 1994; 132397-909. 49. Schad EM, Zaitseva Zaitsev VN, Dohlsten M, Kalland T, Schlievert PM, Ohlendorf DH, Svensson LA. Crystal structure of the superantigen staphylococcal exotoxin type A. EMBO J 1995; 14:3292-3301. 50. Kappler J, Kotzin B, Herron L, Gelfand EW, Bigler RD, Boylston A, Carrel S, Posnett DN, Choi Y, Marrack P. VD-specific stimulation of human T cells by staphylococcal toxins. Science 1989;2449311-813. 51. Kline JB, Collins CM. Analysis of the superantigenic activity of mutant and allelic forms of streptococcal pyrogenic exotoxin A. Infect Immun 1996;642361-869. 52. Rogiani M, Stoehr JA, Leonard BAB, Schlievert PM. Analysis of toxicity of mutants of streptococcal pyrogenic exotoxin A. Science (submitted). 53. Johnson LP, Schlievert PM, Watson DW. Transfer of group A streptococcal pyrogenic exotoxin productiontonontoxigenicstrainsby lysogenic conversion. Infect Immun 1980; 28:254-257. 54. Goshorn SC, Schlievert PM. Bacteriophage association of streptococcal pyrogenic exotoxin type C. Bacteriol 1989; 171:3069-3073. 55. Leggiadro RJ, Bugnitz MC, Peck BA, Luedtke GS, Kim MH, Kaplan EL, Schlievert PM. Group A streptococcal bacteremia in a mid-south children’s hospital. South Med J 1993; 86:615-618. 56. Leung DYM, Travers JB, Giorno R, Norris DA, Skinner R, Aelion J, Kazemi LV, Kim MH, Trumble AE, Kotb M, Schlievert PM. Evidence

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60. 61.

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for a streptococcal superantigen-driven process in acute guttate psoriasis. J Clin Invest 1995; 96:2106-2112. Goshorn SC, Bohach GA, Schlievert PM. Cloning and characterization of the gene, spec, for pyrogenic exotoxin type C from Streptococcus pyogenes. Mol Gen Genet 1988; 212:66-70. Goshorn SC, Schlievert PM. Nucleotide sequence of streptococcal pyrogenic exotoxin type C. Infect Immun 1988; 56:2518-2520. Kapur V, Nelson K, Schlievert PM, Selander RK, Musser JM. Molecular population genetic evidence of horizontal spread of two alleles of the pyrogenic exotoxin C gene ( s p e c ) among pathogenic clones of Stveptococcus pyogenes. Infect Immun 1992; 60:3513-3517. Okada K, Ayala GF, Sung JH. Ultrastructure of penicillin-induced epileptogenic lesion of the cerebral cortex in cats. J Neuropathol Exp Neurol 1971; 30:337-353. Schlievert PM, Watson DW. Group A streptococcal pyrogenic exotoxin: pyrogenicity, alteration of blood-brain barrier, and separation of sites for pyrogenicity and enhancement of lethal endotoxin shock. Infect Immun 1978; 21:753-763. Schlievert PM, Bettin KM, Watson DW. Inhibition of ribonucleic acid synthesis by group A streptococcal pyrogenic exotoxin. Infect Immun 1980; 27:542-548. Hanna EE, Watson DW. Host-parasite relationship among group A streptococci. 111. Depression of reticuloendothelial function by streptococcal pyrogenic exotoxins. J Bacteriol 1965; 89:154-158. Braude AI. Absorption, distribution, and elimination of endotoxins and their derivatives. In: Landy M, Braun W, eds. Bacterial Endotoxins. New Brunswick, NJ: Institute of Microbiology, 1964:98-109. Kuttner AG, Krumwiede E. Observations on the effect of streptococcal upper respiratory infections on rheumatic children: a three year study. J Clin Invest 1941; 20:273-287. Nauciel C. Mitogenic activity of purified streptococcal erythrogenic toxin on lymphocytes. Ann Immunol 1973; 124C:383-390. Imanishi K, Igarashi H, Uchiyama T. Activation of murine T cells by streptococcal pyrogenic toxin type A: requirement for MHC class I1 molecules on accessory cells and identification of Vp elements in the T cell receptor of toxin reactive cells. J Immunol 1990; 145:3170-3176. Tomai MA, Schlievert PM, Kotb M. Distinct T-cell receptor Vp gene usage by human T lymphocytes stimulated with streptococcal pyrogenic exotoxins and pep M5 protein. Infect Immun 1992; 60:701-705. Hanna EE, Watson DW. Host-parasite relationships among group A streptococci. IV. Suppression of antibody response by streptococcal pyrogenic exotoxin. J Bacteriol 1968; 95:14-21. Cunningham CM, Watson DW. Suppression of antibody response by group A streptococcal pyrogenic exotoxin and characterization of the cells involved. Infect Immun 1978; 19:470-476.

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71. Rogers TJ. Immunosuppressive activity of the bacterial superantigens. In: Thibodeau J, Sekaly R-P, eds. Bacterial Superantigens: Structure, Function and Therapeutic Potential. Austin, TX: R. G. Landes Co.,181-194 72. Leonard BA, Schlievert PM. Immune cell lethality induced by streptococcal pyrogenic exotoxinA and endotoxin. Infect Immun 1992; 60:37473755. Schlievert PM, Leggiadro RJ. Inva73. Hussain SM, Luedtke GS, Baker sive group B streptococcal disease in children beyond early infancy. Pediatr Infect Dis J 1995; 14:278-281. 74. Farley MM, Harvey RC, Stull T, Smith JD, Schuchat A, Wenger JD, Stephens A population-based assessment of invasive disease due to NEngl J Med 1993; group B Streptococcus innonpregnantadults. 328:1807-1811. 75. Gaunt N, Rogers K, Seal D, Denham M, Lewis J. Narcotising fasciitis due to group C and G haemolytic streptococcus after chiropody. Lancet 1984;1:516.


Streptococcal Superantigen, Mitogenic Factor, and Pyrogenic Exotoxin B Expressed Streptococcus pyogenes

James M. Musser of

BACKGROUND

Streptococcus pyogenes is a Gram-positive human bacterial pathogen that causes pharyngitis, tonsillitis, skin infections (impetigo, erysipelis, and other forms of pyoderma), acute rheumatic fever (ARF), scarlet fever (SF), poststreptococcal glomerulonephritis (PSGN), a streptococcal toxic shock'syndrome (STSS), and necrotizing fasciitis. These infections are some of the most economically and medically important conditions that affect humans.' For example, globally, ARF is the most common cause of pediatric heart disease. It is estimated that in India more than six million school-aged children suffer from rheumatic heart disease (1).In the United States, "sore 'throat" is the third most common reason for physician office visits and pyogenes 281

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is recovered from about 30% of children with this complaint ( 2 ) . It has been estimated that there are 25-35 million cases of streptococcal pharyngitis per year in the United States, and these infections cause 1-2 billion dollars per year in direct health care costs ( 3 , 4 ) . Although the continued great morbidity and mortality caused by S. in developing nations, the significant health care financial burden attributable to group A streptococci in the United States, and increasing levelsof antibiotic resistance (51, have highlighted the need for a fuller understanding the molecular pathogenesis of streptococcal infection, it has been the relatively recent intercontinental increase in streptococcal disease frequency and severity (6,7) that has resulted in renewed interest in S. pyogenes virulence factors and hostparasite interactions. II. OVERVIEW OF S. PYOGENES PATHOGENESIS

VIRULENCE

FACTORS

Despite decades of research, we are far from a comprehensive understanding of the molecular pathogenesis of S. pyogenes virulence. Putative colonization factors have been studied extensively by several investigators, and the results have shown that there are multiple extracellular adherence mechanisms (8,9). The following molecules have been implicated in adhesion: M protein (3,lO-14), M proteinlike molecules that bind immunoglobulins (15), lipoteichoic acid (161, and fibronectin- and vitronectin-binding proteins (17-26). Following attachment to host tissue, streptococci may either remain at the site of colonization initiate a more invasive infection involving tissue destruction and/or intravascular dissemination. Currently, it is unclear exactly how S. pyogenes gains access tothe blood stream, because many individuals with sepsis lack significant local disease. Both M protein and a hyaluronic acid capsule are antiphagocytic and therefore presumably assistin intravascular survival (27-30). In the blood, the organism may inactivate the complement cascade as a consequence of selective binding of M protein to complement control protein factor H (31-33). The pathogen also expresses a highly specific endopeptidase that selectively inactivates complement cascadeprotein C5a (34,35). Streptokinase may assist spread by virtue of its ability to enhance fibrinolysis (36-38). A gene (mga) has been identified and characterized that is involved in the positive regulation of expression of M protein and several other putative virulence factors (39-44). The mga gene encodes a trans-acting factor that has similarity to the re-

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ceptor proteins of two-component regulatory systems (42). Environmental regulation of streptococcal gene expression is also likely to be an important factor in host-parasite interactions (45-49). Other extracellular products have been studied that may also play a role in streptococcal pathogenesis (50-52). In addition to the molecules noted above, several S. pyogenes exoproteins have been reported to be superantigens, including pyrogenic exotoxin A (SPE A), pyrogenic exotoxin B (SPE B), pyrogenic exotoxin C (SPE C), M protein (53-551, streptococcal superantigen (SSA) (56-59), and mitogenic factor (MF) (60-64). This chapter will summarize current knowledge about the genetics, structure, and function ofSPE B, SSA, and MF (Table 1).The other molecules are discussed in other chapters.

STREPTOCOCCALSUPERANTICEN (SSA) A.

PurificationandCharacterization of SSA

SSA was identified, purified, and characterized from a serotype M3 strain as a result of a search for novel streptococcal superantigens (56,57). Because the chromatography matrix red dye A (RDA) had been successfully used to purify several staphylococcal enterotoxins (65,66), the RDA matrix was employed in an attempt to recover previously uncharacterized superantigens. A concentrate of supernatant from 16 L of stationary-phase growth of an M3 strain was chromatographed on an RDA column, and fractions were tested for the presence of a class 11-dependent T-cell mitogen. Subsequent gel filtration, ion-exchange chromatography, and amino-terminal protein sequencing of a 28-kDa protein yielded a 29-amino-acid NH, terminus that was about 60% identical to the NH,terminus of staphylococcal enterotoxin B (SEB), Cl (SECl), and C3 (SEC3). The 28-kDa molecule was designated streptococcal superantigen (SSA). Further purification of the 29-kDa protein was conducted by affinity chromatography with antiserum raised against a peptide composed of the first 19 amino acids ofSSA. In addition, SSA stimulated T-cell proliferation in a MHC class 11-dependent fashion. It was also demonstrated that SSA activated T cells in the context of HLA-DR, DQ, and H-2 I-E, but not HLA-DP or H-2 I-A, a usage pattern similar to SEB. Moreover, using TCR Vp-specific primers and PCR, purified SSA was shown to expand human T cells bearing Vpl-3, -5.2, and 15 (Table 2) (57).

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B.

Musser Phylogenetic Distribution of SSA Production

The phylogenetic distribution ofSSA production was then assessed with an immunoblot assay (57). Assay of culture supernatants from strains of Lancefield group B, C, and G streptococci failedto identify evidence for SSA production. In contrast, screening of culture supernatants from 58 group A streptococci recoveredfrom patients with a variety of infections (pharyngitis, severe invasive disease, and toxic shock syndrome) found that production of SSA was restricted to organisms previously assigned to three distinct clonal lineages termed electrophoretic type 2 (ET2), ET 14, and ET 24, corresponding to some strains expressing serotype M3, M4, and M12 protein, respectively. The associationof SSA with the ET 2/M3 lineage was particularly intriguing because these isolates accounted for a significant percentage of contemporary invasive disease episodes occurring in the United States and Canada. C. Cloning of the SSA Gene ( s a ) and Restricted Phylogenetic Distribution Among S. pyogenes lineages

more precisely define the phylogenetic distribution of SSA and to characterize its structural relationship to other superantigens, the SSA gene was cloned and sequenced from an ET2/M3 strain The gene encodes a predicted 260-amino-acid protein with a molecular weight of29,771 daltons. The first 26 amino acids form a leader sequence that is cleaved to result in the mature 234 amino acid molecule with a predicted molecular weight of 26,892 daltons. Alignment of the amino acids sequences ofSSA and several staphylococcal and streptococcal superantigens showed that SSA is 60.2% and 59.2% identical to SEB and SEC3, respectively, but only 49% identical to SPE A. The sequence alignment also found that, like several other superantigens, has centrally located cysteine residues that may form a functionally important disulfide loop (67,681. Construction of a phylogenetic tree based on protein sequence identity revealed that SSA is clearly more related to the bacteriophage-encoded staphylococcal enterotoxins SEB, SEC1, and SEC3 than to other streptococcal superantigens such as SPE A and SPE C, a result suggesting the possibility of intergeneric transfer. To assess the occurrence of among group A streptococci, 138 isolates representing 65 distinct M protein serotypes and 15 nontypeable strains were screened by PCR with primers specific for the 5' and termini of and by Southern analysis (58). The gene was present in (12.5%) of the 80 clonal lineages tested, including

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all 52 isolates of ET 2/M3 analyzed. The other nine ssa-positive lineages included organisms expressing serotype M4, M15, M23, M33, M41,M43,M56, and provisional types PT5757 and PT4854 (Fig. 1). Importantly, based on multilocus enzyme electrophoretic analysis, strains with ssa on average were not more related to each other than they are to strains of other M types that lack ssa. This result suggested that ssa may have been acquired independently by several different streptococcal lineages, perhaps by bacteriophage-mediated transduction. Two additional noteworthy observations were made (58). First, all ssa-positive clones expressedSSA, a result suggesting that the protein is constitutively expressed. Second, interestingly, evidence for temporal variation in frequency of occurrence of ssa with members of the ET 2/M3 lineage was identified. All 38 contemporary (19691990) invasive ET2/M3 disease isolates, 11 strains cultured from patients with scarlet fever in the 1940s, and a puerperal sepsis isolate from 1937 had ssa. In contrast, two organisms causing scarletfever in the 1910s and 1920s lacked ssa. D. Allelic Variation of ssa in Natural Populations

Inasmuch as phylogenetic analyses found that ssa occurs in several well-differentiated clones, it was of interest to determine whether distinct clonal lineages carried the same ssa coding sequence harbored a group of allelic variants. To explore this question, an automated DNA sequencing strategy was used to characterizessa from 23 pyogenes strains representing 10 clones identified by multilocus enzyme electrophoresis (59). Three alleles of ssa were discovered, named ssa-l, ssa-2, and ssa-3. and ssa-3 differed from one another by a synonymous substitution in codon 94 (TT&->TTT; Phe), and both alleles encoded a protein designated SSA-1. These two alleles were present in nine phylogenetically diverse clones, including some that had not shared a recent common ancestor. ssa-2 occurred in an M43 strain assigned to a single streptococcal lineage. ssa-2 (encoding SSA-2) is identical to ssa-l except for the occurrence of a nonsynonymous mutation in codon 28 that changes the second amino acid of the mature protein from serine to arginine (AGA, Arg -> AGT, Ser) (Fig. 1). This amino acid substitution altered the predicted isoelectric point and was associated with a change in apparent molecular mass during SDS-PAGE. The comparative sequence data, coupled with t l e distribution patterns of ssa alleles among well-differentiated clonal lineages of pyogenes, added further data supporting the hy-

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pothesis that can be horizontally transferred in natural populations. It will clearly be of interest to determine whether SSA-1 and SSA-2 differ functionally, and to more fully investigate the role of horizontal transfer processes in distributing ssa alleles in natural populations. ssa Can BeAssociated with Streptococcal Insertion Sequence

IS1239

While allelic variation in ssa was studied, it was noted that several strains had aberrantly large PCR products a result demonstrating that nucleotide sequence polymorphism existed at the ssa locus (69). The ssa-positive organisms expressing M4,M23,M33,M41,M43, and PT4854 all had an amplicon that was 30-40 bp larger than expected, and the M15 organism yielded an amplicon that was more than 1kb larger than expected. Based on the location of the oligonucleotide primers used for amplification, and the observation that all of the isolates synthesize an approximately 28-kDa SSA protein (581, it was hypothesized that the size variation in the PCR products was due to sequence polymorphism located at the 5' noncoding end of the fragment. Subsequent molecular genetic studies confirmed that this was the case. The small size increase observed in six of the isolates was due to a 26-bp insert, immediately followed by an 8-bp apparent target sequence, which together constitute a 34-bp insertion. Sequence analysis of the amplification product from the M15 strain revealed a 1110-bp fragment with several noteworthy features (69). The complementary strand of the insert has a 981-bp open reading frame that potentially encodes a 326-amino-acid polypeptide with substantial homology to Escherichia coli IS30 transposase. Results of Southern blot analysis showed that at least 12 copies of the sequence were present in the M15 strain. The element was designated IS2239 and is thefirst simple insertion sequence described in group A Streptococcus. MITOGENIC FACTOR (MO, ALSO KNOWN AS STREPTOCOCCAL PYROGENIC EXOTOXIN F A.

Purification and Characterization of the Protein

Several groups have investigated the properties of a protein variably termed mitogenic factor streptococcal pyrogenic exotoxin (6062,64). The molecule was initially named mitogenic factor (601, but then later named streptococcal pyrogenic exotoxin based largely on

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the ability to induce cytokine production in vitro Because pyrogenicity tests of mitogenic factor have not been performed, the name mitogenic factor (MF) will be used in this chapter. Yutsudo et al. initially identified MF in the course of purifying known streptococcal pyrogenic exotoxins. The protein was recovered from supernatants of a stationary phase culture and purified to apparent homogeneity by ion-exchangechromatography, preparative isoelectric focusing, and reversed-phase high-performance liquid chromatography. A molecular weight of daltons was determined, and the protein was mitogenic in nanogram quantities in an assay employing rabbit peripheral blood lymphocytes. Aminoterminal sequencing showed that the protein was distinct from previously described streptococcal pyrogenic exotoxins. B.

Molecular Characterization of the MF Gene

Based on the amino-terminal sequence data, the structural gene encoding MF was cloned and characterized by standard molecular genetic strategies employing degenerate oligonucleotides and PCR An open reading frame of nucleotides that would encode a MF precursor protein with amino acids was identified. No significant homology was identified at either the gene protein level between MF and known streptococcal pyrogenic exotoxins. Surprisingly, no cysteine residues would occur in the mature protein. Hence, the protein would lack the well-conserved disulfide loop that has been shown to be functionally important in several superantigens Recombinant MF made in an coli expression system had mitogenic activity in a rabbit peripheral blood lymphocyte assay, and rabbit antiserum raised against secreted MF made by S. pyogenes reacted with the recombinant material. Characterization of the cloned MF gene permitted additional investigations to be conducted examining the distribution of the gene in natural populations of streptococci A total of clinical isolates of S. recovered from patients in Japan were studied, and the MF gene (mf) was identified in organisms byPCR and Southern hybridization. Analysis of isolates classified as group B, C, D, or G showed that the gene is confined to S. pyogenes isolates. The occurrence of mf among virtually all S. organisms studied led to the hypothesis that the gene is chromosomally encoded,rather than located on a bacteriophage However, the spectrum of M protein serotypes of these organisms was not provided; hence, it is unclear if the organisms studied represent the entire genetic span of the species S. pyogenes. Norrby-Teglund et al. have also reported that all

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S. pyogenes studied have the gene encoding MF. Forty-two strains expressing different M or serotypes, and recovered from patients with different disease manifestations between and were found to harbor the MF gene. C.

Functional Studies Conducted with MF

Norrby-Teglund et al. analyzed the in vitro proliferative response and cytokine production induced by MF in human peripheral blood mononuclear cells obtained from healthy blood donors. Purified MF was shown to induce a striking interferon7 and TNF-P response by the PBMCs after hr of cell stimulation. Marked production of IL-la, IL-1P, IL-lra, and IL-8 was also recorded, but in contrast, relatively little production of IL-2 and TNF-a was detected. The cytokineand proliferation-inducing capacity of MF was approximately equal to the level observed for SPE A and SPE B, a result that was interpreted as evidence that MF is also a superantigen. Interestingly, considerableinterindividual variation in cytokine inductionwas documented, and this difference was not due to occurrence of MFneutralizing antibody. To investigate the potential superantigenic properties of purified MF, Norrby-Teglund et al. studied its interaction with antigenpresenting cells and T cells in vitro. Antigen-presenting cells were required for induction of T-cell proliferation by MF. In addition, presentation to T cells was found to be HLA class I1 dependent, but not MHC restricted. Based on use of a semi-quantitative PCR assay, it was discovered that MF preferentially activated T cells bearing Vp 15, and Relatively little work has been done to investigate the potential role MF in human infections. However, it has been reported (70) that sera from patients with S. pyogenes bacteremia had significantly lower neutralizing ability against MF than did sera from 25 uncomplicated tonsillitis cases. Although this result could be interpreted to mean that MF plays an active role in some patients with invasive streptococcal disease, the lack of in vivo expansion or depletion of T cells bearing MF-specificVp markers does not strongly support this hypothesis (71). V.

STREPTOCOCCAL PYROGENIC EXOTOXIN

A.

Introduction

B (SPE B)

Several groups have published data consistent with the idea that a molecule termed streptococcal pyogenic exotoxinB (SPE B) is a super-

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antigen (72-88). As discussed below, SPE is initially made as a 40,000-kDa protein and under appropriate conditions becomes a potent cysteine protease (81). Conversion to the fully active protease occurs in reducing conditions and involves truncation of the 40-kDa zymogen to a 28-kDa enzyme. Studies conducted with a very limited number of strains have shown that the protease form is maximally present in the medium at a pH less than 6.5 (73,81,89). The literature pertaining to SPE B is somewhat confusing, in large part due to the many names given the protein over the years. Hence, considerable background is provided for the reader in an attempt to make the literature more readily understandable. The reader should be alerted to the fact that it is frequently difficult, if not impossible, to determine if the 4O-kDa, 28-kDa, any of several truncated intermediately sized forms, or a mixture of these proteins were used in many of the reported studies. B.

Biochemicaland EnzymologicalStudies

The initial isolation and characterization of streptococcal cysteine protease was accomplished by Elliott (90), who was investigating the observation that some strains of group A Streptococcus could not be typed with antisera directed against M protein, an antiphagocytic surface protein. Elliott was testing the hypothesis that these serologically nontypable isolates produced a substance that destroyed M protein when the organisms were grown under appropriate conditions, such as 37°C for 18 hr. It was first shown that culture filtrates obtained from two test strains destroyed the serological reactivity of the M substance of type 1. This capacity was abolished by heating to 70°C for 30 min. Subsequently, the enzyme was purified, and it was discovered that reducing agents such as thioglycollic acid, glutathione, and cysteine hydrochloride activated the enzyme, whereas iodoacetic acid inhibited the activity. Based on these general properties, it was noted that the streptococcal enzyme resembled papain and some of the cathepsins. Testingof several other potential substrates revealed that the enzyme degraded casein, gelatin, human milk, and fibrin, and the synthetic substrate benzoyl-l-arginineamide. Study of78 group A streptococcal strains found a perfect correlation between expression of protease and failure to be M typable. The capacity to produce enzyme was not limited to organisms of a particular M serological type. Soon after Elliott’s initial study, Elliott and Dole (91) demonstrated that the enzyme was made as an inactive precursor that could

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be converted to an active form by treatment with.trypsin. Moreover, it was shown that under appropriate conditions, the proteinase precursor underwent autocatalytic conversion to .form active enzyme. Importantly, in the strain employed, the precursor was elaborated mainly during the period of maximal bacterial growth, and it remained as precursor until later growth stages, at which point the reducing conditions essential for autocatalysis were achieved. Immunological and biochemical evidence confirmedthat an inactive precursor was converted to an active enzyme and showed that precursor and protease have distinct antigenic specificities The observation that the enzyme was made as an inactive precursor led to several studies examining the physical and enzymatic characteristics of the two molecules. Shedlovsky and Elliott separated the precursor and protease by electrophoresisand found that the precursor was converted to protease in the presence of sodium thioglycollate. Importantly, careful immunological studies suggested that the precursor was chemically modified during conversion to the active form. Liu et al. then used column chromatography, equilibrium ultracentrifugation, and amino acid analysis to show that the zymogen underwent reduction in apparent molecular weight from to during autocatalytic zymogen-to-enzyme transabout formation. Interestingly, it was also demonstrated that whereas the zymogen form was electrophoretically homogeneous, the enzyme derived by proteolytic action in culture medium was not. It was suggested, based on chemical studies, that proteolysis of the zymogen form occurred at both the amino- and carboxy-terminal ends. The amino acid analysis discoveredthat only one half-cysteine existedper molecule of zymogen or active protease, and the authors concluded that the streptococcal protease is a sulfhydryl enzyme. Additional study of the zymogen-to-enzyme transformation initiated by trypsin treatment found that the process involves formation of an intermediate protein that contains only residues less than the zymogen. Complete zymogen-to-enzymetransition occurred with a loss of a fragment of about amino acid residues. This peptide reacted with antiserum raised against zymogen,but not with antiserum made against active enzyme. C. Protein Sequence and Biochemical Evidence for Catalytic Site Residues

Considerable effort was devoted in the and to analysis of the primary amino acid sequence of the zymogen and enzyme, to

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enzymatic characterization of the specificity of cleavage of the protease, and to active site analysis. In an early study of the active site of the enzyme, Liu et al. (96) labeled the single cysteine molecule with [14C]-iodoacetic acid and determined the sequence of27 amino acid residues around the labeled residue. Based on analysis of tryptic and chymotryptic peptides, a primary sequence of the protease form was reported 20 years ago (97-99). Subsequently, a complete amino acid sequenceof the zymogen was reported that contained 337 residues The zymogen-to-enzyme conversion byeither trypsin or active streptococcal protease was found to involve removal of84 amino acid residues from the aminoterminus the molecule (100). The detailed biochemical specificity of the protease was first studied by Mycek et al. who found that the synthetic substrates benoyl-L-argininamide, benzoyl-L-lysinamide, benzoyl-L-histidinamide, carbobenzoxy-L-isoglutamine,and carbobenzoxy-L-isoasparagine were cleaved by the streptococcal enzyme. These investigators also demonstrated that presence of a glycyl residue at the proteolytic site of the enzyme markedly decreasedthe hydrolysis of the sensitive peptide bond. Examination of the ability of the protease to hydrolyze additional substrates resulted in the hypothesis that an unprotonated imidazole ring and the protonated form of the single sulfhydryl group were essential for enzymatic activity (102). This hypothesis was strengthened by the findings of Liu (103), who demonstrated that a histidine residue occurred at the active site of the enzyme. The presence of a tryptophan residue at the active site was suggested by the labeling results of Robinson (104). Strong evidence that the single sulfhydryl group in the protease is essential for enzymatic activity was provided by data demonstrating that the specific activity of the activated enzyme was directly proportional to its SH content (105). Interestingly, although virtually all experimental work with the protein has focused on its proteolytic activity, two groups have reported that the protease cleaves esters1000 times fasterthan amides (106,107). D. MolecularGenetic Studies

The structural gene for SPE B was cloned and characterized by the laboratory of Schlievert (82,108). The speB gene (1194 bp) encodes a 398-amino-acid protein. A presumed signal peptide of26 amino acids is cleaved to result in a mature extracellular protein (zymogen) with 372 residues. Subsequently, Kapur et al. (109) used an automated DNA sequencing strategy to study speB allelic variation in 67 strains expressing 39 M protein serotypes and five provisional M

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serological types and representing phylogenetically distinct clones identified by multilocus enzyme electrophoresis. It was discovered that the speB gene is well conserved and allelic variation is duesolely to accumulation of point mutations (Fig. 2). A total of distinct speB alleles was identified, and based on predicted amino acid sequences, of the alleles would encode one three mature protease variants that differ from one another at only one or two amino acids clustered in a 10-amino-acid region (residues that contains a linear B-cell epitope (110). A total of of 64 strains examined, including strains representing all alleles sequenced, expressed a product that reacted with polyclonal rabbit antisera specific for purified cysteine protease. The data demonstrated that the zymogen and protease are very well conserved, and virtually all strains expressed the speB gene under the conditions assayed. The percentage of strains expressing the speB gene was significantly higher than values reported by other investigators (111-115), a result probably due

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Mature Protease

Figure 2 Schematic representation of polymorphism at the speB locus and in the translated protein. The locations of polymorphic sites in 160 bp of upstream DNA and 1197 bp of the speB gene identified among 39 alleles in 67 isolates of S. pyogenes are shown. There is one deletion of a single adenine residue and 44 nucleotide substitutions, of which 12 result in amino acid replacements. The single-letter amino acid abbrevations immediately above the codon numbers refer to residues found in the variant protein arbitrarily designated as SPE B1. A, Ala; D, Asp; G, Gly; Ile; K, Lys; L, Leu; N, Asn; R, Arg; S, Ser; T, Thr; V, Val. (From Ref. 109.)

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to the use of a more sensitive assay and/or different strain growth conditions. In the initial publication describing the nucleotide sequence of speB, the investigators were unable to demonstrate proteolytic activity associated with the recombinant material (108). It was hypothesized that the lack of proteolytic activity was due to differences in amino acid sequence between the published protease sequence and inferred amino acid sequence encoded bythe cloned gene. However, characterization (109) of the speB gene from the S. pyogenes strain used to produce the protease that was sequenced showed that the published amino acid sequence (100) is incorrect. Thisresult was recently confirmed by Ohara-Nemoto et al. E. Virulence Role of the spell Gene Product: Abundant Evidence

In contrast to SSA and MF, abundant strong evidence has accumulated that the speB gene product is involved in the molecular pathogenesis of some streptococcal infections (116-124). Early clear insight into a toxic effect attributable to streptococcal protease was obtained by Kellner and Robertson (116), who showed that a single injection of crystalline material administered intravenously to rabbits, mice, and guinea pigs produced focal myocardial necrosis. Rabbits generally tolerated the injection without obvious reaction, and death ocovernight. Interestingly, little no inflamcurred within hours matory reaction was associated with early lesions, an observation suggesting a direct cytotoxic effect. The lesions were very similar to those induced by intravenous injection of papain, the canonical cysteine protease; Destructionof enzymatic activity by heating the streptococcal protease resulted in failure to induce these necrotic lesions. Inasmuch as several experimentalstudies suggest a role in pathogenesis of some streptococcal diseases, it is important to substantiate that the enzyme is actually expressedin the course of infections. Todd (125) showed that patients with streptococcal infections seroconvert to streptococcal protease, which clearly documented that the molecule is expressed in vivo in the course of host-parasite interactions. tease production by strains recovered from a variety of infections, including scarlet fever, puerperal fever, tonsillitis, and rheumatic fever, was documented. The majority of normal individuals lacked significant levels of antiprotease antibody, and importantly, no significant difference was found between the antiprotease titers of sera from patients with acute rheumatic fever and sera from other infections. This observation suggested that antibody directed against the streptococcal protease is not causally involved in ARF. Moreover,

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administration of horse antiprotease antibody to patients with ARF had no detrimental beneficial effect. Interestingly, Todd concluded that the streptococcal protease he studied was identical to human muscle- and tissue-degrading enzymes studied previously by other investigators (126,127). Studies by Rotta (128) documenting that humans with glomerulonephritishave antiprotease antibodies also demonstrate that this enzyme is expressed in vivo. Knoll's group demonstrated that SPE B was expressed during the course of experimental group A infection of rabbits using a tissue cage model (129,130). Moreover, Bjorck et al. (117) reported that cysteine protease was required for mouse virulence and bacterial growth. More recently, the author's group has documented that patients with invasive disease seroconvert to SPE B (130a) Several other observations are consistent with the hypothesis that the cysteine protease participates in one or more phases of hostparasite interactions (85,109,119-122,124). For example, Holm et al. (119) reported that patients with fatal streptococcal diseaseshave lower acute antibody levels to SPE B than .individuals with less serious infections. This finding implies that serum antibody against SPE B confers at least partial protection against invasive streptococcal disease in humans. Knoll et al. (85) found a positive correlation between expression of SPE B and occurrence of fever >38"C in children with scarlet fever. Virtually all organisms have the speB gene (113,114,131137) and express cysteine protease The speB gene is only nominally variable in the species, which suggeststhat significant functional constraints are acting on the protein product (109). The author's group discovered that streptococcal cysteine protease cleaves human IL-lp precursor to generate biologically active mature IL-lp, a major cytokine mediating inflammation and shock (120). The cysteine protease cleaves purified human fibronectin, degradesvitronectin in vitro, and causes striking cytopathic effect to human umbilical vein endothelial cells (HUVECs) grown in culture (109). Mouse passiveimmunization experiments with rabbit antibody raised against purified denatured cysteine protease significantly lengthened the time required for 50% mortality when challenged with live streptococci (121). Purified cysteine protease activates a latent human matrix metalloprotease, a process that could contribute to bacterial invasion, tissue destruction, inflammation, shock, and inhibition of wound healing (122). Recent experiments indicate that the cysteine protease releases biologically active fragments of the streptococcal cell surface moleculesM protein and C5a peptidase (123). Investigations with a rat lung model of inflammation have shown that streptococcal cysteine protease acts synergistically with either streptococcal cell wall antigen streptolysin

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0 to augment lung injury (124). Hence, taken together, data generated over five decadesare strongly consistent with the notion that the protein encoded by speB is involved in several types of streptococcal diseases, and that antibody directed against the molecule is partially protective. F.

Evidence Favoring Superantigen Function

The initial suggestion that SPE B had superantigen function was based on several observations. Cunningham et al. (74) demonstrated that material purified from cell-free streptococcal culture filtrates, and thought to be toxin B (SPE B), induced a pyrogenic response when injected intravenously into American Dutch rabbits. The material also enhanced susceptibility to endotoxin-mediated shock in rabbits and caused skin erythema in New Zealand rabbits when injected intradermally. Toxin B was considerably less active than streptococcal pyrogenic exotoxin A in these assays. Soon thereafter, Cunningham and Watson (77) reported that SPE B suppressed the in vitro ability of mouse spleen cells to make antibody against sheep red blood cells. SPE B was also far less active than either SPE A or SPE C in suppression ability. Additional work discovered that SPE B induced proliferation of lymph node cells recovered from guinea pigs and was a nonspecific mitogen forhuman cord blood lymphocytes, splenic lymphocytes from American Dutch belted rabbits, and splenic lymphocytes from BALB/cWAT mice (76,831. In addition, SPE B induced proliferation of human peripheral blood lymphocytes (76) and T lymphocytes (79). SPE B also induces interferon activity in vitro (80). Following the cloning of the speB gene, recombinant SPE B was purified from E. coli and shown to stimulate proliferation of rabbit spleen cells (82). Virtually equal levels of incorporation of [3Hlthymidine into cellular DNA were recorded for the S. pyogenes and E. coli derived material in the rabbit splenocyte stimulation assay used. Abe et al. (84) were the first investigators to carefully study the mechanism by which SPE B activates human T lymphocytes. Using SPE B purified to apparent homogeneity and two-color direct immunofluorescence and cytofluorographic analysis, the molecule was shown to stimulate expansion of T cells bearing Vp 8 from six separate healthy Quantitative PCR analysis confirmed expansion of Vp 8 cells and also found that Vp 2-bearing T cells were selectively expanded. Leonard et al. (86) also reported that purified SPE B had mitogenic activity for murine T cells, and that the proliferative activity was dependent on class I1 MHC molecules expressed on antigen-

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presenting cells. Imanishi et al. (87) examined the relative abilities of distinct isotypes of human MHC class I1 molecules to bind SPE B. Using a series of L cells transfected with genes encoding DR4, DR2, Dqwl, DP(Cp63), or Dpw4, evidence was presented that SPE B bound well to DQ molecules, less well to DR molecules, and very weakly to DP molecules. Purified SPE B was mitogenic for human T cells and also induced IL-2 production by peripheral blood mononuclear cells. Ohara-Nemoto et al. (88) have also presented evidence supporting the idea that SPE B (both zymogen and mature, proteolytically active form) is a human T-cell mitogen. Purified SPE B stimulated proliferation of peripheral blood mononuclear cells; however, no stimulatory activity was recorded when PBMCs were depleted of T cells. These data are consistent with the idea that SPE B is a T-cellspecific mitogen. Interestingly, antibody directed against the proteolytically active form of SPE B (28.5 kDa) did not totally inhibit Tcell mitogenicity, a result suggesting the possibility of presence of a contaminating proliferation-stimulating-activity. G.

EvidenceAgainstSuperantigen

Function

In striking contrast to the evidence cited above,two laboratories have reported that the human T-cell proliferation activity attributed to SPE B is due to contamination ofSPE B preparations with other streptococcal superantigens (138,139). Braun et al. (138) examined the ability of several streptococcal erythrogenic toxins to stimulate proliferation of human T cells. SPE B purified to apparent homogeneity, as assessed by SDS-PAGE, isoelectric focusing, and HPLC, caused expansion of Vp 8-bearing T cells. However, antiserum directed against SPE C specifically inhibited the Vp 8-stimulating activityof SPE B (but not Vp 2-proliferation induction). The authors concluded that trace amounts of a potent novel VP 8-stimulating activity (termed "SPEX") had contaminated the SPE B preparations. Although it was stated that further purification by reversed-phase HPLC yielded a preparation devoid of Vp 8 mitogenic activity, the actual data were not presented. No explanation was presented for the inability to neutralize Vp 2 proliferation. VI.

AREAS FOR FUTURE INVESTIGATION

For neither MF nor SSA has there been presented in vivo evidence of pyrogenicity, alteration in reticulocyte function, or enhancement of lethal shock by endotoxin.Future studies will need to be conducted

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to determine if MF or SSA mediates any all these processes. These investigations, including studies conducted with defined mutants are especially important because of the considerable controversy surrounding mitogenic and pyrogenic responsesattributed to these and other streptococcal molecules (for example, see Refs. 53, In addition, it will be important to determine the role, if any, of allelic variation in altering structure-function activities of SSA variants. Studies should also be undertaken to examine the possibility ofMF variation at the gene and protein level. Because SSA is variably present in natural populations of S. pyogenes, the structural gene should be carefully studied to determine if it is bacteriophage encoded horizontally transferred. Although molecular genetic approaches will be utilized to address the potential detrimental affect of MF, SSA, and SPE B on hosts, the lack of a universally accepted and relevant animal model system for studies of group A streptococcal pathogenesis limits the conclusions that can be drawn from data obtained from these experimental systems. Thus far, most research on superantigens has been conducted in the relatively limited context of potential role in streptococcal toxic shock syndrome. Inasmuch as host-parasite interactions, investigations there are many stages should be conducted to elucidate the role of these and other superantigens in processes such as colonization, bacterial survival despite apparently adequate antimicrobial agent treatment (1411, and potential in vivo modulation of local and systemic humoral responses in humans. Clearly, much work needs to be done to fully explain the role of these .products in human streptococcal diseases. ACKNOWLEDGMENTS

Research on S. pyogene in the author’s laboratory is supported by Public Health Services Grant AI-33119 and by an award from the Texas Advanced Technology Program. The author is an Established Investigator of the American Heart Association. REFERENCES

1. Aganval BL. Rheumatic heart disease unabated in developing countries. Lancet 1981;1:910-911. 2. Pichichero ME, Disney FA, Green ]L, Francis AB, Marsocci SM, Lynd AM, Wood GC. Comparative reliability of clinical, culture, and antigen detection methods for the diagnosis of group A beta-hemolytic streptococcal tonsillopharyngitis. Pediatr Ann 1992; 21:798-805.

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3. Fischetti VA. Streptococcal M protein: molecular design and biological

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Streptococcal M Protein

INTRODUCTION

It is well established that rheumatic fever (RF) and rheumatic heart disease (RHD) are nonsuppurative sequelae of group A streptococcal infections (1-5). However, the sequence of events leading from the infection to the autoimmune manifestations remains an enigma. Despite the evidence that these bacteria are capable of eliciting humoral and cellular immune responses in the mammalian host, the pathogenesis of these autoimmune disorders has not been fully elucidated. Of the many factors produced by group A streptococci that can possibly contribute to pathogenesis, the surface M protein is considered one of the major virulence factors for these organisms because it protects the bacteria from phagocytic cells (6-8) and has been shown to harbor epitopes that mimic ones found in human tissues, including heart, kidney, and brain (L9-14). In addition, recent studies revealed that certain M-protein serotypes belong to the family of microbial superantigens that trigger potent immunological reactions 31

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that can potentially lead to autoimmunity (15,161. In this chapter, the immunopathogenesis ofRF and RHD as well as the immunological characteristics and possible role of the M protein in these diseases will be reviewed. II. IMMUNOPATHOCENESIS OF RHEUMATIC FEVER AND RHEUMATICHEARTDISEASE

RF and RHD were historically considered diseasesof molecular mimicry, mediated by cross-reactive autoantibodies. This notion was based on the findings that the streptococcal bacteria express several proteins, including the M proteins, that share sequences with a number of host proteins (1,9-11,13,14,17-23) and are therefore capable of eliciting these autoantibodies. Heart-reactive antibodies that cross-react with streptococcal proteins have been detected in sera of patients with RHD (5,24). Several groups were able to determine that this reactivity was directed to proteins in the sarcolemmal structure of myocardial tissues and to the outer membrane proteins of group A streptococci (1,5,21,25,26). Additionalstudies revealed that certain Mprotein serotypes share sequences with myosin, vimentin, tropomyosin, phosphorylase, and laminin (9-13,27,28). Cross-reactivity was also found between valvular tissues and streptococcal carbohydrates (29,301 and between myocardial tissues and other streptococcal proteins (31). However, despite ample data on shared B-cell epitopes between the Sfreptococcus and the host, recent studies suggest that cross-reactive antibodies are not the primary mediators of RHD (32). The pathogenic significance of heart cross-reactive humoral immune responses is obscured by the fact that the level of these autoantibodies often shows little correlation with clinical and/or histopathological manifestations and many patients who contract streptococcalpharyngitis develop antibodies against cardiac tissue and yet most have no evidence of rheumatic fever or cardiac injury (18,25,33,34). Similarly, experimental animals injected with rheumatogenic streptococci purified streptococcal components generate high titers of heart crossreactive autoantibodies, but do not develop the heart damage characteristic of RHD in the human. This situation may be similar to other experimental models of autoimmune myocarditis where adoptive transfer of disease can be accomplishedwith T cells, and not autoantibodies, from affected animals (35-37). The involvement of cell-mediated immune responses to streptococcal components in the pathogenesis RF and RHD is supported by clinical and experimental data. Direct histopathological examina-


313

tion of heart tissues from RHD patients provided strong evidence that T cells have an active role in the pathogenesis of rheumatic carditis. Marboe et al. (38) analyzed an endomyocardial biopsy from a RHD patients and observed a heterogeneous infiltrate composed of T cells, macrophages, cells, and mast cells. The predominant cells were T lymphocytes with twice as many CD4 cells as CD8 T cells. Raizada et al. (39) also examined valvular tissues removed from the patients 10-20 years following the initial attack and noted that the majority of T cells in the pathogenic lesion belonged to the CD4 subset. These observations were confirmed by Kemeny et al. (40), who found that the cellular infiltrate in affected cardiac tissues consisted primarily of T cells and macrophages and noted an increase in HLA-DR expression on the majority of the infiltrating cells as well as on the vascular endothelium. The link between cellular responsesto streptococcal components and cardiac tissues.was demonstrated by the findings of Yang et al. (41), who observed that membrane antigens from group A, but not group C, streptococci can induce guinea pig T cells to differentiate into cytotoxic T lymphocytes (CTL) capable of destroying allogeneic cardiac heart cells. The target specificity of this CTL population was demonstrated by the lack of cytotoxicity toward other tissues such as skeletal muscle, skin fibroblasts, and liver cells. Heart cross-reactive antibodies failed to either mediate or augment the killing of cardiac cells. A clinical relevance of this phenomenon was provided by the study of Hutto and Ayoub (42), who documented the presence of circulating heart-reactive CTL in blood of patients with active rheumatic carditis. Interestingly,this cytotoxic activity was blocked by the patients’ plasma, suggesting that certain heart cross-reactive autoantibodies may have a protective rather than a pathogenic role in this disease. More recent studies by Yoshinaga et al. (43) showed that T-cell lines established from valvular tissues of RHD patients responded to cell wall and cell membrane antigens of rheumatogenic streptococci. Although, several streptococcal components can potentially stimulate T cells and induce the differentiation ofCTL, in vitro studies have shown that certain M-protein serotypes can elicit the differentiation of cytotoxic T cells capableof killing myocardial cells(44,45). Crossrecognition of M protein and heart epitopes was also shown by Pruksakorn et al. (46), who generated T-cell lines to different M-protein peptides and demonstrated that several of these clones also recognize heart proteins. These findings suggested that immune responses to M proteins may evoke cellular autoreactivity. However, more direct

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evidence forthe involvement of the M protein in RHD came fromthe recent studies of Guilherme et al. (471, who generated T-cell clones from valvular tissues ofRHD patients and demonstrated that these clones were responsive to specific epitopes of type 5 M proteins. Therefore, mounting evidence points to the M protein as a major player in the pathogenesis ofRF and RHD. To gain a clear understanding of this role, the immunological responses to this protein need to be elucidated. STRUCTURALFEATURES OF M PROTEINS

The M proteins represent a family of closely related serotypes that emanate as a-helical coiled-coil fibrous molecules from the surface of the streptococcus (48). Molecular studies revealed that the various serotypes of M protein are encoded by allelic genes that exhibit several remarkable features (49-53). M proteins have a pepsin cleavage site that conveniently cleaves the molecule into the amino- and carboxy-terminal halves (Fig. 1). The carboxyl-terminal half of the molecule is conserved, whereas the amino-terminal half is highly variable (54-57) and harbors the type-specific epitopes of the protein. M proteins contain a seven-residue motif with respect to placement of hydrophobic and hydrophilic residues, and this motif is repeated throughout most of the molecule (12,49). This seven-residue periodicity confers an a-helical coiled-coil structure upon the M-protein molecule (48) (Fig. l).In addition, most M proteins studied thus far contain varying lengths of internal sequence repeats; the most striking occur in type 24 M protein with up to five identical 35-aminoacid residuerepeats (49-51,58).Based on amino acid sequence homologies within the amino-terminal half of the molecule, the M protein superfamily may be grouped into subfamilies. For example, there is a high degree of homology between M5 and M6 proteins but both bear very little homology to M24 protein. However, little is known about the structure/function relation of particular regions of the various M proteins, particularly as they impact on the nature of cell-mediated immune responses elicited by these molecules. IV. THECONCEPT OF RHEUMATOGENICAND NONRHEUMATOCENICSEROTYPES

There are over 80 different serotypes of M protein, but not all are associated with these sequelae. Serotypes that show an association with rheumatic fever are casually called rheumatogenic, and those

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frequently isolated from glomerulonephritis outbreaks are considered nephritogenic (3,59,60). Among the rheumatogenic serotypes are types M1, M3, M5, M6, M12, M18, M19, and M24. These observations have led to speculation that the ability of M protein to elicit autoimmune responses may reside in the amino-terminal half of the molecule harboring the type-specific epitopes. However, despite the lack of primary sequence homology in this region between certain rheumatogenic serotypes such as M5 and M24, strains expressing these different serotypes seem equally capable of triggering RF in the susceptible host., Although this clinical classification of serotypes is by no way absolute, as shown by the existence of overlap between certain rheumatogenic and nephritogenic serotypes, common featuresare shared among rheumatogenic strains of Streptococcus The majority of rheumatogenic isolates lack the serum opacity factor (SORI, and recently, Bessen et al. (61) identified an epitope on a surface M-associated protein (MAP) that appears to correlate very well with rheumatogenicity. This epitope maps to the relatively conserved C repeat region of the molecule. Based on the presence absence of this epitope, the bacteria can be classified as class I and class 11, respectively. The rheumatogenic isolates all seem to express the MAP Iepitope that is found on SOR- strains. Interestingly, RF patients exhibit a high titer of serum immunoglobulins directed toward the MAP I epitope. Whether this M-protein epitope is directly involved in disease pathogenesis represents a marker for genetically related serotypes remains to be elucidated. V.

CELL-MEDIATEDIMMUNERESPONSESTO

M PROTEIN

Earlier studies by of Lawrence (62) demonstrated passive transfer of delayed cutaneous hypersensitivity to nonreactive subjects by injecting viable leukocytes from individuals presensitized to crude M type 1 protein. Beachey and his colleagues generated highly purified pepsin-extracted M proteins (pep M) anddemonstrated that these preparations elicit cell-mediated immune responses in humans (58,64). Studies by Dale et al. (64) demonstrated that human T cells respond briskly to purified, pep M preparations. The response was not merely due to prior exposure to streptococcal infections, inasmuch as cord blood lymphocytes were also responsive (64). The potency of the response to M protein was uncharacteristic of an antigen, suggesting that it may be a polyclonal T-cell mitogen. However, our studies revealed that the biochemical signals triggered in T cells by PHA and M protein were different (65-67) suggesting that the M protein was

Streptococcal

Protein

31 7

neither a regular antigen nor a mitogen. As will be detailed below, these observations led us to the finding that the M protein belongs to the family of microbial superantigens. In addition to their potent mitogenic effect, certain M-protein serotypes (e.g., pep M5, pep M6, and pep M19) are capable of eliciting the differentiation ofCTL (44). Consistent with superantigenactivated CTL, pep M-activated CTL consisted of both CD4 and CD8 cells and killed their targets in an MHC nonrestricted fashion (44). Interestingly, unlike the response to streptococcal pyrogenic exotoxins, none of the lymphocytes from nonimmunized laboratory animals, including mice and rabbits, responded to in vitro stimulation with pep M5, pep M6, or pep M24, suggesting the existence of species-specific responses to these proteins. VI.

MECHANISM BY WHICHMPROTEININDUCESCELLULAR RESPONSES

As mentioned above, the M-protein response was not characteristic of either antigen or polyclonal mitogen. To further investigate differences between these proteins and mitogens, we studied the response of pep M5 in some detail. Although both PHA and pep M5 required the presence of accessory cells to stimulate T cells, PHA could be presented by class 11-positive antigen-presenting cells (APC) as well as class 11-negative APC, whereas pep M5 required class I1 expression on the APC (15,16,68,69). However, unlike conventional antigen, the presentation of pep M5 was not restricted by MHC elements and could be presented by autologous as well as allogeneic APC including mouse fibroblasts that were transfected with various HLA-DR, DQ or -DP alleles (68). Anti-MHC class I1 monoclonal antibodies blocked the presentation of pep M5 by the HLA-transfected cells,but had no effect on the response T cells to PHA (68). Flow cytometric analysis revealed that pep M5 binds to HLA class 11-expressing fibroblasts, and this binding was also blocked by anti-MHC classI1 monoclonal antibodies. Synthetic peptides up to 41 amino acids in length copying various regions of the M protein failed to induce T-cell proliferation indicating that pep M5 did not activate human T cells via classic T-cellepitopes described for conventional antigens. Collectively these data suggested that pep M5 may be a superantigen requiring larger domainsof the protein to associate with class I1 MHC molecules and subsequent recognition by the T-cell receptor (TCR). Recently, studies from Kalil's group in Brazil have mapped the class II-binding regions ofM5 protein (personal communication).

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Superantigens interact with relatively invariant elements within the variable region of the chain (Vp) of the TCR heterodimer (70,71). Each superantigen has a characteristic affinity fora set of Vp elements and can potentially stimulate all T cells expressing those elements, which may be asmany as 5-40% of resting T cells, depending on the number of Vp elements recognizing a particular superantigen and the frequency of expression of these Vp elements in the T-cell repertoire of the responding individual (70,71). The entire set ofVps expanded in response to a superantigen (Vp spectrogram) serves as the fingerprint for this protein. To determine if pep M5 interacts with T cells in a VP-specific manner, we analyzed the relative Vp expression in T cells stimulated with either pep M5 or the polyclonal T-cell mitogen anti-CD3. These studies revealed that pep M5 preferentially stimulates T cells expressing Vp2, Vp4, and Vp8 elements. Furthermore, the hypervariable sequences of the expanded Vps exhibited extensive diversity in the CDR3 region with no apparent conservation of JP or Cp elements (Table 1). Accordingly, we concluded that pep M5 is a superantigen for human T cells. Analysis of other rheumatogenic serotypes, including pep M6,18,19, and 24, revealed that they were also superantigens for human T cells, but that each serotype stimulated a unique set of Vp specificity (16). Interestingly, all rheumatogenic serotypes tested stimulated VPGbearing T cells (Table 2), whereas, the nonrheumatogenic serotype, pep M2, stimulated only Vp2-bearing cells (16). We have recently mapped a region that seems to beinvolved in the interaction of pep M5 with TCR Vp elements (72) (and unpublished data). This region, which is located within the B repeats of the M5 molecule, harbors the motif KSKQXXGAXKQEL that is found in several superantigens and is homologous to a shared sequence also found in invariant chain of MHC class I1 antigens from human (residues 192-211) and mouse (residues 145-1641 (56) (Fig. 2). VII.

IS THE SUPERANTIGENIC ACTIVITY MEDIATED BY PEP M PROTEIN OR A CONTAMINANT?

In 1992, Fleischer et al. (73) suggested that the mitogenic activity of M proteins types 1 and 5 is due to contamination with a streptococcal pyrogenic exotoxin (SPE), namely, SPE C. In their study, M protein lost its mitogenic activity for T cells after a single purification step by affinity chromatography on immobilized albumin or fibrinogen (73). They conducted Vp analysis and determined that both pep M5 and SPE C stimulate Vp8-bearing T cells, and based on these

Streptococcal

Protein

319

Table 1 Diversity in Junctional Region Sequences Suggests a SAgDriven Response CSA CSA CSA SCA CSA CSA CSA

csv csv csv csv csv CAS CAS CAS CAS CAS CAS CSA CSA CSA

KDFSGIQET MRYEKLF RLVNTDT RPGQGVPET SGGVLGGET SILAGGTDT SLSDT AARGSPLH ESDGNTI IDRGSAGGTE QGVYE VPSGRYE SFRDRVYGYH SGTYNE SPTGAPY NSPLH TFSTGGEL TMGRDTEA TPTYSNQPQH GLGQGPLAL RPSGRRGTIE RVFLLSGGKET

QYFG FG QYFG QYFG QYFG QYFG QYFG FG YFG QYFG QYFG QFFG FG QFFG FG FFG FFG FG TFG QFFG QYFG

cDNA from pepM5-stimulated T cells was amplifiedby PCR using 5' forward primers specific for Vp2, Vp4, and Vp8 and a reverse Cp primer. The amplified product that spans the TCR VDJ region and covers part of the region was eluted and sequenced as described (16). Shown are the deduced aa sequences.

results, they concluded that pep M5 is contaminated with SPE That SPE is mediating the mitogenic activity in the pep M5 preparation used in this original study was later retracted when it was found that one of the strains lacked the gene for SPE and that neither highly purified native (74) nor recombinant SPE did stimulate Vp8 T cells (75). Consequently, these investigators concluded that yet another toxin, SPE X, must have a Vp8 specificity and is responsible for the mitogenic activity of pep M5 (75). However, a superantigen has to be characterized bythe entire Vp spectrogram and not just by one Vp element, because a particular Vp may be recognized by many superantigens, and there is considerable overlap between the Vp specificity of different superantigens.

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Table 2 Human TCRVp Specificity of Streptococcal pep M Proteins and Pyrogenic Exotoxins Streptococcal superantigen

Human TCR Vb specificity

Pep M 5 (15, 74) Pep M Pep M Pep M 19 Pep M 24 Pep M 2 SPE A (74, 94) SPE B (74, 94) SPE C (74) SPE F SSA

Vj312,Vp14,Vj315 VP8 VplS

Vp15,

Val5

8:

SM5-IO SEA & SED (147-155). (143-151)

T v T[--/!!I

:[

M

"

SEB (152-160)

V T A

--

T A

--

Q E L

SPEA (141-149)

ZJv

T

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h1 (192-211)

SEC1 & SEC3 (1522-160)

11- F V F I /-

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vp10

VP8

@I]; K S E L Q

K S E L Q R

-

T N

-

LI

N L K Q E - LI SM5-IO hl(192-211)

L S N L R Q

-

sm(15-29)

I SEE(12-26)

SEB (3943) K S - - Q

TSST-I(58-60)

Figure 2 Sequence motif shared by M5 and other superantigens. A sequence motif found in the B repeat region of M5 protein is shownaligned two different ways with sequences found in other superantigens (70) and human invariant chain (hI) (94). The motif is located in the region believed to be involved in the interaction of the molecule with TCR Vp elements (unpublished data).

Streptococcal

Protein

321

The claim that the superantigenicity of pep M5 is due to a contaminant (73) is very difficult to conceive given the large body of evidence pointing to the contrary. This evidence dates back to 1981 when Dale et al. (64) showed that antibodies to defined fragments of the pep M molecule could be used to precipitate out the mitogenic activity of this protein, and that inhibitors of the mitogenic activity of SPE did not affect the proliferative response elicited by the purified pep M used in their study. In addition, they showed that pep M proteins were mitogenic only to human T cells, and that none of the lymphocytes from nonimmunized laboratory animals, including mice and rabbits, respond to pep M5, pep M6, or pep M24 (64, and our unpublished data). We have observed that mouse T cells stimulated with 10, 30, and pg/ml pep M5 failed to proliferate, and that rabbit lymphocytes were unable to respond to10 pep M5 without prior immunization (76). By contrast, rabbit lymphocytes respond briskly to all known SPE preparations (77). This intriguing species specificity of pep M proteins was latter confirmed by others (781, who also found that mouse T cells do not respond to M protein unless they have been previously immunized with it. Despite the information from the earlier studies, we felt the issue of contamination needed to be revisited, and accordingly, we conducted a number of studies to investigate if the native, HPLCpurified pep M5 protein is contaminatedwith minute amounts of SPE that could not be detected on silver-stained gels. First, we demonstrated that the pattern of Vp specificity of pep M5 is quite distinct from the known SPES (15,16,79-81,93). The Vp specificity of the pep M proteins and the SPES studied to date is summarized in Table 2. If pep M5 was contaminated with SPE A, we should have seen expansion of Vp 12 and 14 in addition to Vp2, 4, and 8, which are specific for pep M5. If the pep M5 was contaminated with SPE C, we should have seen evidence for expansion of Vpl, 5.1, and 10, and if the pep M5 activity was due to SPE F, we should have seen the expansion of Vp 15 and 20 in addition to Vp2, 4, and 8. Finally, if all pep M preparations were contaminated with the same toxin, then we would expect the various pep M serotypes to have the same Vp specificity profile, and as can be seen in Table 2, this is not the case. Instead, the pattern for each pep M protein studied was unique and did not reflect contamination with any of the known toxins (Table 2 ) . Second, we used synthetic peptides copying primary sequences of pep M5 protein to localize immunologicallyfunctional areas of the molecule and determine whether the mitogenic activity seen is mediated by the M protein (72). Because superantigens require both the

322

Kotb

class 11- and TCR-binding domains to stimulate T cells, fragmentation of the protein into peptides results in loss of mitogenic activity. However, synthetic peptides can be used to block the mitogenic activity of the whole protein. Dose-dependent inhibition by pep M5 synthetic peptides can be taken as evidence for mitogenicity of this protein provided appropriate controls are included to rule out nonspecific suppression by peptides. Ten overlapping synthetic peptides, spanning the entire amino acid sequenceof pep M5, were used to delineate immunologically important regions (Figs. l and 3). Peptides representing residues 157-197 (SM5-10) of the mature pep M5 (based on strain B788 sequence) protein inhibited the response to whole pep M5 in a dose-dependent manner, while having little or no effect on the response to SPE A, SPE B, SPE C, anti-CD3 antibodies, or SEB (72) (Fig. 4). Furthermore, this peptide preferentially blocked the expansion of T cells expressing pep M5-specific Vp elements (72). These data are consistent with the conclusion that the mitogenic activity of the purified protein preparation is mediated by the M protein and not the SPEs. In addition to the above investigations, we conducted biochemi’ cal studies in which antibodies to the SM5-10 (residue 157-197) synthetic peptide were found to augment signal transduction in T cells stimulated with pep M5. We had shown that purified T cells can be stimulated with pep M5 in the absence of APC if costimulatory signals are provided exogenously (67,82-85). Proliferation and changes in intracellular Ca2+ levelswere monitored in T cells stimulated with pep M5, plus rIL-l, rIL-6, and the phorbol ester PMA. It is well known that crosslinked, but not soluble, anti-CD3 antibodies can stimulate T cells in the absence of APC, suggesting that antibodies to a specific ligand bound to the TCR may crosslink the ligand and result in a stronger activation signal. We found that when pep M5 was crosslinked by a specific antibody to whole native pep M5 or to the 41-amino-acid SM5-10 peptide, both the Ca2+ signaland the proliferative response were enhanced. The anti-pep M5 antibodies had not direct effect on T cells, and addition of antibodies directed to irrelevant proteins failed to augment the pep M5 signal (67,831. These results are inconsistent with the response being mediated by a contaminant unless this contaminant harbors the same epitope to which the antibody is directed, and this is unlikely because immunoblot analyses showed that this antibody does not react with other streptococcal proteins. Despite the strong evidence that the M-protein preparations used in our studies are free SPEs, we had to address the issue directly

323


0

12.5

25

50

100

SM5 -10 (mol)

Figure 3 Inhibition of pep M5-induced T-cell proliferation by SM5 peptides. The SM5 peptides listed in Fig. 1were tested for their ability to block T-cell activation by pep M5. Purified T cells (2 lo5) were preincubated for 1 hr at 37°C with the indicated doses of the peptides in the presence of fixed APC (5 lo4) and 50 p1 activated APC-derived SUP and then stimulated with 0.5 &m1 pep M5. T-cell proliferation was determined after 72 hr by measuring [3H]-thymidine uptake. Data are expressedas the meancpm of triplicate cultures ? SEM. (Data derived from data presented in Ref. 72.)

by testing the activity of recombinant pep M protein. We used to amplify 750 bp covering the entire region encoding pep M5 from genomic DNA of S. pyogenes type 5 Manfred0 strain (51). The purified PCR product was cloned and sequenced to ensure identity to the previously published pep M5 sequence (51). The clonedpep M5 DNA was then ligated into a pGEM-3Z and introduced into Escherichia coli AAEC 191 that was cotransfected with pGP1-2. Heat-inducible expression of pep M5 was verified by Western blot analysis, and the

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324

SM5-10(aa 157-197)

5

Pep M5

SPEA

SPEB

SPEC

Figure 4 Specific inhibition of pep M5-induced T-cell proliferation by the pep M5 synthetic peptide SM5-10. Purified T cells (2 lo5) were preincubated for 1 hr at 37°C50nmolSM5-10 (residues 157-197) in the presence fixed APC (5 lo4) and 50 pVm1 activated APC-derived supernatant and

then stimulated with the indicated concentrationsof anti-CD3 MAb or with pep M5, SPE SPE B, or SPEC. T-cell proliferation was determined after 72 hr by measuring [3H]-thymidine uptake. p 0.01 was considered statistically significant compared to cultures without SM5-10 peptide. (Data derived from data presented in Ref. 72.) protein was purified by HPLC using DEAE-anion exchange chromatography (79). Purification of rpep M5 was confirmed by Western blot analysis as detailed in Figure 5. Partially purified rpep M5 showed reduced mitogenic activity; however, after HPLC purification the activity was markedly enhanced, suggesting that an inhibitor from E. coli may have interfered with the mitogenic response (Fig. 6). The mitogenic activity or rpep M5 was comparable to that the native protein (Fig. 6). However, the most direct proof for the superantigenic nature of pep M5 came from the finding that the TCR Vp specificity of the native and recombinant proteins were identical (Fig.7 ) . Both the native and the recombinant pep M5 stimulated Vp2, Vp4, and Vp8. It should bementioned that Robinson and Kehoe (78) also reported that recombinant whole M5 protein was mitogenic only to human, and not to mouse, T cells; however, they failed to see expansion of Vp2 T cells in response to their preparation. This may be related to changes in the protein conformation when the carboxy-terminal half of the molecule is added. has been demonstrated for other superantigens, slight changes in superantigen can at times abolish and/or create new Vp specificities


325

for the protein. It is also important to realize that the activity of rM5 or ‘pep M5 expressed in E. coli seems to vary from batch to batch, and the basis for this variation is not known. For this reason we are planning to express the ‘pep M5 in genetically engineered S. pyogenes strains where the genes for M-protein expression are either blocked or deleted and examine the properties of the protein when expressed in its natural host. Therefore, given the evidence presented above, the concept that the mitogenic activity of pep M protein may be mediated by a contaminant is very difficult to accept.We have provided ample positive data and performed many studies to detect this contaminant and the evidence always supported the notion that the activity is mediated by the pep M protein. We do not have an explanation for the results of Fleischer et al. but believe that differences in purification procedures may have accounted for this discrepancy. We have experienced difficultieswith the purification of functional native and recombinant streptococcal proteins, including M proteins and a number of pyrogenic exotoxins, and had to optimize conditions for the purification of biologically active molecules. Expression in native host may alleviate most of these problems and resolve discrepancies. Meanwhile, we continue to pursue the activity of rheumatogenic and nonrheumatogenic strains in an effort to decipher the role of certain Mprotein serotypes in the pathogenesis ofRF and RHD. VIII.

INDUCTION OF AUTOREACTIVE T CELLS BY PEP

As mentioned above, human T cells stimulated in vitro with whole pep M5 (44,451, and T-cell lines generated to different M-protein peptides have been found to recognize heart proteins (46). Furthermore, as reported by Guilherme et al. (471, T-cell clones derived from heart biopsies obtained from RHD patients recognize M5 proteinderived synthetic peptides, whereas none of the T-cell clones derived from heart biopsies from patients with chronic Chagas’ cardiomyopathy and heart transplant patients with allograft rejection responded to these peptides. Most heart-derived T-cell clones generated by these investigators simultaneously recognized multiple protein fractions from both myocardium and aortic valve tissues. In addition, 95-150 kDa and 43-65 kDa aortic valve fractions were frequently recognized by M-protein cross-reactive T-cell clones, which led the authors to suggest that cross-reactivity mayinvolve proteins such as myosinand vimentin.

326 .

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Silver Stained SDS-PAGE

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49.5 kDa -+

32.5 kDa

+

18.5 kDa

-. ( II t

t

27.5 kDa -+

PepMS

-+ . : ; . , L .

a

h

h c

d e f

d e

Lane a = Mwt Std; lane b = extract of AAEC 191 plus vector without insert; lane c = extract of AAEC 191 plus vector with pep M5 insert; lane d = % Amm.SO4 supernatant; lane = e % Amm.SO4 pellet; lanef = native pepM5.

-+

PCR

0

+ +

pCR I1 Vector &OR I digest

pGEM-3Z

Sequenced

t

BamH I + Xba I

Spe I,t!zen BamH I

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AAECl91-

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rPep MS

l T

Streptococcal

Protein

327

Examination of the specificity of T-cell clones derived from heart biopsies of RHD patients revealed that the highest response was elicited by the 43-65 kDa valvular proteins as well by type M5 protein amino acid residues 163-177. Interestingly, we have reported that this region of the M5 protein is important for its ability to stimulate T cells (72) and may contain residues involved in the interaction with TCR VD elements (unpublished data). Furthermore, this region of the M5 molecule contains a myosin cross-reactiveepitope (87). Additional studies are required to investigate the molecular basis for the crossrecognition by human T cells of valvular autoantigens and specific regions of M proteins. M protein-induced T-cell proliferation and cytokine release may also furnish conditions that allow responsesto a dominant streptococcal epitopes to evoke autoimmune responses to cross-reactive cryptic self-epitope. The binding superantigens to the TCR and/or to class I1 molecules can triggerintracellular biochemical signals, which

,

Figure 5 Expression and purification of recombinant pep M5. the sequence ofPCR primers used to amplify pep M5 DNA was based on that described by Podbielski et al. (95). Two primers were synthesized: MJE-1 (5' forward primer: "GGGGGGGGATCCATAAGGAGCATAAAAATGGCTY)and MJE-2 (3' reverse primer: 5'GGGTGCGCATTATAATTCTTGTTTAAGGGCACC3'). Together these primers amplified 750 bp covering the entire Manfred0 pep M5 sequence (51). The purified PCR product was ligated to pCRI1 cloning vector and subcloned in INVaF' competent cells and the positive clones were sequenced by the dideoxy termination method using the Sequenase kit (UBI) according to the instructions of the manufacturer. The cloned pep M5 DNA was then ligated into a pGEM-3Z (Promega) and introduced into E. coli AAEC 191, which is derived from MG1655 recA strain (86). The AAEC 191 bacteria were cotransfected with pGEM-3Z with insert and pGP1-2 (vector pGP1-2, a derivative ofPACYC177, provided expression ofT7 RNA polymerase and consists of gene 1of phage l7 under thecontrol of the inducible promoter and the gene for the heat-sensitive N repressor; CI857). Expression of pep M5 at 42°C was verified by Western blot analysis. A band of the expected size (27 kDa) was detected in extracts of cells with insert, but not in extracts of cells transfected with both plasmids without insert. The cell extract was subjected to ammonium sulfate fractionation and the dialyzed fractions were further purified by HPLC using DEAE-anion exchange chromatography as described for native pepsin-extracted pep M5 from streptococci (79). Purification of 'pep M5 was confirmed by Western blot analysis using polyclonal rabbit anti-pep M5 antisera that was generated to a synthetic peptide copying residues 157-197 of the mature B788 pep M5 sequence (shown in Fig. 1).

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328

”

Medium

Pep M5

rPep M5

Figure 6 Mitogenic activity of native and recombinant pep MS. Peripheral blood lymphocytes (lo5) were stimulated for 3 days with 1 pg/ml of each of the HPLC-purified native or rpep M-protein preparations. Proliferation was measured by determining r3H]-thymidine uptake. The data are presented as mean cpm of triplicate culturesS E M . (Data derived from data presented in Ref. 16.)

+

Peripheral Blood T Cells SuperAg or anti-CD3 448h IL2(10U/ml), 24h

RNA

1

cDNA

-

Flow cytometry

VB analysisby

PCR

F i h e 7 PCR analysis ofTCR Vp specificity of native and recombinant pep M5. Cells from the same individual were stimulated separately with pep M proteins. T cells (106/ml) were stimulated for 3 days with either 2 pg/ml antiCD3 MAb or 1 &m1of each of the indicated pep M proteins. RNA was extracted from the cells; cDNA was prepared and analyzed using PCR. The autoradiograms were scanned to obtain a quantitative measurement for amplified Vps. The PCR value = [area Vp,/area CaJppM5 t [area VpJarea CaIantiCDJ. (Data derived from Ref. 16.)


329

in turn program a number of events leading to cell activation, differentiation, proliferation, and the release of inflammatory cytokines (reviewed in Refs. 70,71,88,89). A number of streptococcal superantigens, including pep M5, have been shown to cause the release of TH1 cytokines, namely interferon7 (IFNy) and tumor necrosis factor-p (TNF-P) (90), which can induce up-regulation chaperones, heat shock proteins, and MHC molecules resulting in abnormal processing and presentation of sequestered self-antigens (reviewed in Ref. 91). Furthermore, the induction of costimulatory molecules and cell adhesion proteins during an infection may also lower the threshold for the presentation of self-peptides and cause the activation of autoreactive T cells. M proteins may also actas adjuvants enhancing the response of autoreactive T cells to cross-reactive T-cell epitopes expressed on heart proteins. For example, we tested the ability of cardiac myosin, which is suspected to be a target autoantigen in RHD and shares several structural features with M proteins (10,11,57), and found that it was not capable of eliciting the proliferation of resting human T cells. They were able to recognize and proliferate in response to cardia1 myosin. Thus, it appears that priming with pep M5 enriched T cells that can recognize cardiac myosin (92) (Fig. The recognition of myosin by primed T cells was not seen when T cells were prestimulated with a polyclonal mitogen or a staphylococcal superantigen. Therefore, the ability of M protein, which harbors human cross-reactive B- and T-cell epitopes, to act as a superantigen may allow it to amplify the immune response and direct the response toward recognition of autoimmune epitopes expressed on heart proteins including, but not necessarily confined to, cardiac myosin. Finally, recent studies in our laboratory revealed that exposure to a mixture of rheumatogenic streptococcirenders T cells responsive to a number myocardial antigen. One such antigen was recognized only by T cells from RHD and RF patients, but not from patients with pharyngotonsillitis or healthy controls (96). The nature of this target protein and its relationship to streptococcalproteins, including M protein, is currently under investigation (96). IX.

CONCLUDING REMARKS

The role of M protein in the pathogenesis ofRF and RHD is supported by strong clinical and experimental evidence. However, the mechanism by which these proteins can trigger autoimmunity in the

330

Kotb

0Medium Myosin

Anti-CD3-primed PepM5+3d Anti-CD3 +3d

Pep MS -primed

+restfor 2 d +Myosin 3d -+Proliferation + rest for 2 d +Myosin3d +Proliferation

Figure 8 Priming of T cells with pep M5 renders them responsive to cardiac myosin. Purified cardiac myosin failedto stimulate resting human T cells to proliferate; however, when T cells were first primed with pep M5, they were then able to recognize myosin epitopes and proliferate in response to this protein. Priming with anti-CD3 did not have the same effect. cells (2 x 106/ml) were stimulated with either 1 pg/ml anti-CD3 MAb or 0.5 pg/ml of pep M5 protein. After 3 days in culture, the cells were washed and placed in fresh RPMI-complete medium for 48 hr to rest. Recovered cells (0.6 lo5) were then incubated in 96-well plates with either medium alone or with 0.2 pg purified cardiac myosin in a final volume 200 pl. Proliferation was measured 3 days later by determining t3H]-thymidine uptake. The data are presented as mean cpm of triplicate cultures f SEM.

susceptible host has not been fully elucidated. A number of M proteins have been shown to harbor human tissue cross-reactive B- and T-cell epitopes. In fact, several studies have shown that T cells derived from heart tissues of RHD patients respond to specific regions of M5 protein. However, despite extensive studies aiming to gain a better understanding of the differences between these serotypes and deduce the structure-function relationship of M proteins, the role of these specific serotypes in disease development is still unknown. Furthermore, the recent discovery that these proteins are superantigens suggests additional mechanisms by which these proteins may contrib-

Streptococcal

Protein

331

Ute to the development of poststreptococcal autoimmunity. It is not likely that these proteins are not the sole mediators of the pathogenesis of RHD; instead they probably synergize with other streptococcal components to elicit autoreactivity and heart damage. Immune responses elicited by these proteins involving the stimulation of T cells based on their VP specificity and the release of inflammatory cytokines can create conditionsthat are conducive of the development of autoimmunity in the susceptible host. In addition, host factors that are likely to regulate immunity responses to M proteins and other streptococcal superantigens may contribute to the risk of autoimmune sequelae of group A streptococcal infections. Although a number of autoimmune diseases are believed to be triggered or exacerbated by microbial infections, in most cases the inciting organism is not known (reviewed in Ref. 91). RF and RHD offer an excellent model to study the role of host/parasite interactions in the pathogenesis of postinfection autoimmunity. Deciphering the nature of immune responses elicited by these bacteria and understanding how host genetic factors regulate these responses will undoubtedly help us unravel underlying mechanisms in other autoimmune diseases that are also believed to have an infectious etiology. REFERENCES 1. Beachey EH, Majumdar G, Tomai M, Kotb M. Molecular aspects of autoimmune responses to streptococcal M proteins. In: Gallin, Fauci A, eds. Advances in Host Defense Mechanisms. 1990533-96. 2. Bisno AL. Group A streptococcal infections and acute rheumatic fever. N Engl J Med 1991; 325:783-793. 3. Stollerman, GH. Rheumatogenic group A streptococci and the return of rheumatic fever. Adv Intern Med 1990; 35:l-25. 4. Zabriskie JB. Rheumatic fever: a model for the pathological consequences of microbial-host mimicry. Clin Exp Rheumatol 1986; 4:65-73. 5. Kaplan MH. Rheumatic fever, rheumatic heart disease, and the streptococcal connection: the role of streptococcal antigens cross reactive with heart tissue. Rev Infect Dis 1979; 1:988-996. 6. Horstmann RD, Sievertsen HJ,Knobloch J, Fischetti, VA. Antiphagocytic activity of streptococcal M protein: selective binding of complement control protein factor H. Proc Natl Acad Sci USA 1988; 85:1657-61. 7. Whitnack E, Beachey EH. Antiopsonic activity of fibrinogen bound to M protein on the surface of group A streptococci. J Clin Invest 1982; 69:1042-. Whitnack E, Beachey Inhibition of complement-mediated

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opsonization and phagocytosis of streptococcus pyogenes by D fragments and fibrin bound to cell-surface M protein. J Exp Med 1985; 162:198397. 9. Dale JB, Beachey EH. Multiple, heart-cross-reactive epitopes streptococcal M proteins. J Exp Med 1985; 161:113-122. 10. Dale JB, Beachey EH. Epitopes of streptococcal M proteins shared with cardiac myosin. J Exp Med 1985; 162:583-591. 11. Kisher K, Cunningham MW. Myosin: a link between streptococci and heart. Science 1985;227:413-415. 12. Manjula BN, Fischetti VA. Tropomyosin-like seven residue periodicity in three immunologically distinct streptococcal M proteins and its implications for the antiphagocytic property of the molecule. J Exp Med 1980;151:695-708. 13. Kraus W, Dale TB, Beachey EH. Identification of an epitope of type 1 streptococcal M protein that is shared with a 43-kDa protein of human myocardium and renal glomeruli. J Immunol 1990; 145:4089-93. 14. Barnett LA, Fujinami RS. Molecular mimicry: a mechanism for autoimmune injury. FASEB J 1992; 6940-844. 15. Tomai MA, Aelion JA, Dockter ME, Majumdar G, Spinella DG, Kotb M. T cell receptor V gene usage by human T cells stimulated with the superantigen streptococcal M protein. J Exp Med 1991; 174:285-8. 16. Watanabe-Ohnishi R, Aelion J, Le Gros HL, Tomai MA, Sokurenko EV, Newton D, Takahara J, Irino S, Rashed S, Kotb M. Characterization of unique human TCR V beta specificities for a family of streptococcal superantigens represented by rheumatogenic serotypes of M protein. J Immunol 1994; 152:2066-73. 17. Kaplan MH, Suchy ML. Immunological relation of streptococcal and tissue antigens. 11. Cross reactions of antisera to mammalian heart tissue and the cell wall constituent of certain strains of group A streptococci. J Exp Med 1964; 119:643-650. 18. Zabriskie JB, Freimer EH. An immunological relationship between the group A Streptococcus and mammalian muscle. J Exp Med 1966; 124:661678. 19. Van De Rijn I, Zabriskie JB, McCarthy M. Group A streptococcal antigens cross reactive with myocardium: purification of heart reactive antibodies and isolation and characterization of the streptococcal antigen. J Exp Med 1977; 146:579-599. 20. Beachey EH, Bronze M, Dale JB, Kraus W, Poirier T, Sargent S. Protective and autoimmune epitopes of streptococcal M proteins. Vaccine 1988; 6:192-196. 21. Dell A, Antone SM, Gauntt CJ, Crossley CA, Clark WA, Cunningham MW. Autoimmune determinants of rheumatic carditis: localization of epitopes in human cardiac myosin. Eur Heart J 1991; 12:158-162. 22. Manjula BN, Trus BL, Fischetti VA. presence of two distinct regions in coiled-coil structure of the streptococcalpep M5 protein: relationship to

Streptococcal

23. 24. 25. 26. 27. 28. 29. 30. 31.

32.

33.

34. 35. 36. 37. 38.

Protein

mammalian coiled-coil proteins and implications to its biological properties. Proc Natl Acad Sci USA 1985; 82:1064-1068. Vashishtha A, Fischetti VA. Surface-exposed conserved region of the streptococcal M protein induces antibodies cross-reactive with denatured forms of myosin. J Immunol 1993; 150:4693-701. Shulman ST. Complications of streptococcal pharyngitis. Pediatr Infect Dis-J 1994; 13(Suppl l):S78-79.. Zabriskie JB, Hsu KC, Seegal BC. Heart-reactive antibody associated with rheumatic fever: characterization and diagnostic significance. Clin Exp Immunoll970; 7:147-159. Dale JB, Beachey EH. Protective antigen determinant of streptococcalM protein shared with sarcolemmal membrane protein of human heart. J Exp Med 1982;156:1165-1176. Dale JB, Courtney HS, Kotb M, Schifferli D. Phosphorylase-cross-reactive antibodies evoked by streptococcal M protein. Infect Immun 1990; 58:774-778. Fenderson PG, Fischetti VA, Cunningham MW. Tropomyosin shares immunologic epitopes with groupA streptococcal Mproteins. J Immunol 1989;142:2475-2481. Goldstein I, Halpren B, Robert L. Immunological relationship between streptococcus A polysaccharide and the structuralglycoprotein of heart valve. Nature 213:44 1967; 213:44. Taranta A. Of man’s heart valves and strep’s cell walls. Ann Intern Med 1967;66:1287-1288. Barnett LA, Cunningham MW. Evidence for actinlike proteins in an M protein-negative strain of Streptococcus pyogenes. Infect Immun 1992; 60:3932-3936. Ayoub EM, Shulman ST. Pattern of-antibody response to the streptococcal group A carbohydrate in rheumatic patients with or without carditis. In: Read SE, Zabriskie JB, eds. Streptococcal Disease and the Immune Response. New York: Academic Press, 1980:649-679. Ayoub EM, Kaplan E. Host-parasite interaction in the pathogenesis of rheumatic fever. J Rheumatol 1991; 3O(Suppl):6-13. Bahr GM, Yousof AM, Majeed H, Chedid L, Behbehani K. Antibodies to a streptococcal cell wall adjuvant structure persist in patient with chronic rheumatic heart disease. J Mol Cell Cardiol 1989; 21:61-66. Neu N, Ploier B, Ofner C. Cardiac myosin-induced myocarditis: heart autoantibodies are not involved in the induction of the disease. J Immunol 1990;145:4094-100. Neu N, Ploier B. Experimentally-induced autoimmune myocarditis: production of heart myosin-specific autoantibodies within the inflammatory infiltrate. Autoimmunity 1991; 8:317-22. Smith SC, Allen PM. Myosin-induced acute myocarditis is a T cell-mediated disease. J Immunol 1991;147:2141-2147. Marboe CC, Knowles Weiss MB, Fenoglio JJ. Monoclonal antibody

334

39.

40. 41. 42.

43. 44. 45. 46.

47.

48. 49. 50.

51.

Kotb

identification of mononuclear cells in endomyocardial biopsy specimens from a patient with rheumatic carditis. Hum Pathol 1985; 16:332-338. Raizada V, Williams RCJ, Chopra P, Gopinath N, Prakash K, Sharma KB, Cherian KM, Panday S, Arora R, Nigam M, Zabriskie JB, Husby G. Tissue distribution of lymphocytes in rheumatic heart valves as defined by monoclonal anti-T cell antibodies. Am J Med 1983; 74:90-96. Kemeny E, Grieve T, Marcus R, Sareli P, Zabriskie JB. Identification of mononuclear cells and T cell subsetsinrheumaticvalvulitis. Clin Immunol Immunopathol 1989; 52:225-237. Yang LC, Soprey PR, Wittner MK, Fox EN. Streptococcal induced cell mediated immune destruction of cardiac myofibers in vitro. J Exp Med 1977;146:344-359. Hutto JH, Ayoub EM. Cytotoxicity of lymphocytes from patients with rheumatic carditis to cardiac cells. In: Read SE, Zabriskie JB, eds. Streptococcal Disease and the Immune Response. New York: Academic Press, 1980:733-738. Yoshinaga M, Figueroa F, Wahid MR, Marcus RH, Suh E, Zabriskie JB. Antigenic specificity lymphocytes isolated from valvular specimens rheumatic fever patients. J Autoimmun 1995; 8:601-613. Dale JB, Beachey EH. Human cytotoxic T lymphocytes evoked by group A streptococcal M proteins. J Exp Med 1987; 166:1825-1835. Kotb M, Courtney HS, Dale JB, Beachey EH. Cellular and biochemical responses of human T lymphocytes stimulated with streptococcal M proteins. J Immunol 1989; 142:966-970. Pruksakorn S, Currie B, Brandt E, Phornphutkul C, Hunsakunachai S, Manmontri A, Robinson JH, Kehoe MA, Galbraith A, Good MF. Identification of T cell autoepitopes that cross-react with the C-terminal segment of the M protein of group A streptococci. Int Immunol 1994; 6:1235-1244. Guilherme L, Cunha-Net0 E, Coelho V, Snitcowsky R, Pomerantzeff PMA, Assis RV, Pedra F, Neumann Goldberg A, Patarroyo ME, Pileggi F, Kalil J. Human heart infiltrating T-cell clones from rheumatic heart disease patients recognize both streptococcaland cardiac proteins. Circulation 1995; 92:415-420. Phillips GNJ, Flicker PF, Cohen C, Manjula BN, Fischetti VA. Streptococcal M proteins: alpha-helical coiled coilstructure and arrangement on the cell surface. Proc Natl Acad Sci USA 1981; 78:4689-4693. Hollingshead SK, Fischetti VA, Scott JR. Complete nucleotide sequence of type 6 M protein of the group A Streptococcus: repetitive structure and membrane anchor. J Biol Chem 1986; 261:1677-1686. Mouw A, Beachey E, Burdett V. Molecular evolution of streptococcal M protein: cloning and nucleotide sequence of the type 24 M-protein gene and relation to other genes of Streptococcus pyogenes SL3261. J Bacteriol 1988;170:676-684. Miller L, Gray L, Beachey E, Kehoe M. Antigenic variation among group

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A streptococcal M proteins: nucleotide sequence of the serotype 5 M protein gene and its relationship with genes encoding types 6 and 24 M proteins. J Biol Chem 1988; 263:5668-5673. Fischetti V. Streptococcal M protein extracted by nonionic detergent. III. Correlation between immunological cross-reactions and structural similarities with implications for antiphagocytosis.J Exp Med 1978; 147:17711774. Scott J, Pulliam W, Hollingshead S, Fischetti V. Relationship of M protein genes in group A streptococci. Proc Natl AcadSciUSA1986; 82:1822-1826. Beachey EH, Seyer JM, Kang AH. Studies of the primary structure of streptococcal M protein-antigens. In: Read SE, Zabriskie JB, eds. Streptococcal Disease and the Immune Response. New York: Academic Press, 1980:149-160. Beachey EH, Seyer JM. Primary structure and immunochemistry of group A streptococcal M proteins. In: Robbins JB, Hill C, Sadoff JC, eds. Seminars in Infectious Disease, Vol IV: Bacterial Vaccines. New York Stuttgart: Georg Thieme Verlag, 1982:401-410. Fischetti VA, Jones KF, Holingshead SK, Scott JR. Structure, function, and genetics of streptococcal M protein. Rev Infect Dis 1988; 10:356-359. Fischetti VA. Streptococcal M protein. Sci Am 1991; 264:58-65. Beachey EH, Seyer JM, Kang AH. Primary structure of protective antigens of type 24 streptococcal M protein. J Biol Chem 1980; 255:62846289. Bisno AL. The concept of rheumatogenic and non-rheumatogenic group A streptococci. In: Read SE, Zabriskie JB, eds. Streptococcal Disease and the Immune Response. New York: Academic Press, 1980:789-803. Stollerman GH. Rheumatogenic streptococci and autoimmunity. Clin Immunol Immunopathol 1991;61:131-142. Bessen D, Veasy G, Hill HR, Augustine NH, Fischetti VA. Serological evidence for class I Group A streptococcal infection among rheumatic fever patients. J Infect Dis 1995;172:1608-11. Lawrence HS. The cellular transfer in humans of delayed cutaneous reactivity to hemolytic streptococci. J Immunol 1952; 68:159-. Cunningham MW, McCormack JM, Talaber LR, Harley JB, Ayoub EM, Muneer RS, Chun LT, Reddy DV. Human monoclonal antibodies reactive with antigens of the group A Streptococcus and human heart. J Immunol 1988;141:2760-2766. Dale JB, Simpson WA, Ofek I, Beachey EH. Blastogenic responses of human lymphocytes to structurally defined polypeptide fragments of streptococcal M protein. J Immunol 1981;126:1499-1505. Kotb M, Dale JB, Beachey EH. Stimulation of Sadenosylmethionine synthetaseinhumanlymphocytesby streptococcal M protein. J Immunol 1987; 139:202-206.

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66. Kotb M, Beachey EH. Serine and tyrosine phosphorylation of28- and 35-kDa proteins of human T lymphocytes stimulated by streptococcal M protein. Biochem Biophys Res Commun 1989; 158:803-810. Differential signal re67. Majumdar G, Beachey EH, Tomai M, Kotb M. quirements in T-cell activation by mitogen and superantigen. Cell Signal 1990;2:521-530. 68. Tomai M, Kotb M, Majumdar G,BeacheyEH. Superantigenicity of streptococcal M protein. J Exp Med 1990; 172:359-362. 69. Tomai MA, Beachey EH, Majumdar G, Kotb M. Metabolicallyactive antigen presenting cells are required for human T cell proliferation in response to the superantigen streptococcal M protein. FEMS Microbiol Immunol 1992;4:155-164. GM, Choi SchererM, Pullen A, White J, 70. MarrackP,Winslow Kappler JW. The bacterial and mouse mammary tumor virus superantigens; two different families of proteins with the same functions. Immunol Rev1993;131:79-92. 71. Herman A, Kappler JW, Marrack P, Pullen AM. Superantigens: mechanism of T-cell stimulation and role in immune responses. Annu Rev Immunol 1991; 9:745-772. 72. Wang B, Schlievert PM, Gaber AO, Kotb M. Localization of an immunologically functional region of the streptococcal superantigen pepsinextracted fragment of type 5 M protein. J Immunoll993; 151:1419-1429. 73. Fleischer B, Schmidt KH, Gerlach D, Kohler W. Separation ofT-cellstimulating activity from streptococcal M protein. Infect Immun 1992; 60:1767-1770. 74. Tomai M, Schlievert PM, Kotb M. Distinct T Cell receptor Vp Gene usage by human lymphocytes stimulated with the streptococcalpyrogenic exotoxins and M protein. Infect Immun 1992; 60:701-705. 75. Braun MA, Gerlach D, Hartwig UF, Ozegowski JH, Romagne F, Carrel S, Kohler W, Fleischer B. Stimulation of human T cells by streptococcal “superantigen”erythrogenictoxins(scarletfevertoxins). J Immunol 1993; 150:2457-2466. 76. Newton SM, Kotb M, Poirier TP, Stocker BA, Beachey EH. Expression and immunogenicity of a streptococcal M protein epitope inserted in Salmonella flagellin. Infect Immun 1991; 592158. 77. Leonard BA,LeePK, Jenkins MK, Schlievert PM. Cell and receptor requirements for streptococcal pyrogenic exotoxin T-cell mitogenicity. Infect Immun 1991;59:1210. 78. Robinson JH, Kehoe MA. Group A streptococcal proteins: virulence factors and protective antigens. Immunol Today 1992; 13:362-367. 79. Kotb M, Watanbe-Ohnishi R, Wang B, Tomai MA, Le Gros L, Schlievert P, El Demellawy M, Geller A. Analysis the TCR Vp specificities of bacterial superantigens using PCR. Immunomethods 1993; 2:33-40. Norrby-Teglund A, Newton D, Kotb M, Holm SE, Norgren M. Superantigenic properties of the group A streptococcal exotoxin SpeF (MF). Infect Immun 1994; 62:5227-5233.


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81. Reda KB, Kapur V, Mollick JA, Lamphear JG, Musser JM, Rich RR. Molecular characterization and phylogenetic distribution of the streptococcal superantigen gene (ssa) from Streptococcus pyogenes. Infect Immun 1994;62:1867-1874. 82. Kotb M, Majumdar G, Tomai M, Beachey EH. Accessory cell-independent stimulation of human T cells by streptococcal M protein superantigen. J Immunol 1990; 145:1332-1336. 83. Majumdar G, Ohnishi H, Tomai MA, Geller AM, Wang B, Dockter ME, Kotb M. Role of antigen-presenting cells in activation of human T cells by the streptococcal M protein superantigen: requirement for secreted and membrane-associated costimulatory factors. Infect Immun 1993; 61:785-790. 84. Ohnishi H, Tanaka T, Takahara J, Kotb M. CD28 delivers costimulatory signals for superantigen-induced activation of antigen-presenting celldepleted human T lymphocytes. J Immunoll993; 150:3207-3214. 85. Ohnishi H, Ledbetter JA, Kanner SB, Linsley PS, Tanaka T, Geller Am, Kotb M. CD28 crosslinking augments TCR-mediated signals and costimulates superantigen responses. J Immunol 1995; 154:3180-3193. 86. Blomfield IC, McClain MS, Eisenstein BI. Type 1 fimbriae mutants of Escherichia coli K12: characterization of recognized afimbriate strains and construction of new fim deletion mutants. Mol Microbiol 1991; 5:14391445. 87. Cunningham MW, McCormack JM, Fenderson PG, Ho MK, Beachey EH, Dale JB. Human and murine antibodies cross-reactive with streptococcal M protein and myosin recognize the sequence GLN-LYS-SERLYS-GLN in M protein. J Immunol 1989; 143:2677-2683. 88. Kotzin BL, Leung DY, Kappler J, Marrack P. Superantigens and their potential role in human disease. Adv Immunol 1993; 54:99-166. 89. Kotb M. Bacterial exotoxins as superantigens. Clin Microbiol Rev 1995; 8:411-426. 90. Kotb M, Ohnishi H, Majumdar G, Hackett S, Bryant A, Higgins G, Stevens D. Temporal relationship of cytokine release byperipheral blood mononuclear cells stimulated by the streptococcal superantigen pep M5. Infect Immun 1993;61:1194-201. 91. Kotb M. Infection and autoimmunity: a story of the host, the pathogen, and the copathogen. Clin Immunol Immunopathol 1995; 74:lO-22. 92. Kotb M. Post-streptococcal autoimmune sequelae: a link between infection and autoimmunity. In: Dalgleish AG, Albertini A, Paoletti R, eds. The Impact of Biotechnology on Autoimmunity. Dordrecht, The Netherlands: Kluwer Academic Publisher, 1994; 37-50. 93. Abe J, Forrester J, Takako N, Lafferty JA, Kotzin BL, Leung DYM. Selective stimulation of human T cells with streptococcal erythrogenic toxins A and B. J Immunol 1991; 146:3747-3750. 94. Cleasson L, Larhammar D, Rask L, Peterson PA. cDNA clone for the human invariant r chain of class I1 histocompatibility antigens and its implications for the protein structure. Proc Natl AcadSciUSA 1983; 80:7395-7399.

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95. Podbielski A, Melzer B, Lutticken R. Application of the polymerase chain reaction to study the M protein(-like) gene family in beta-homolytic streptococci. Med Microbiol Immunol (Berl) 1991; 180:213-227. 96. El-Demellawy M, El Ridi, R, Guirguis N, Kotb, M. Preferential recognition of human myocardial antigens by lymphocytes from rheumatic fever and rheumatic heart disease patients (submitted).

TheSuperantigen Mycoplasma arthritidis Mitogen (MM)

Kevin L. Knudtson, Allen

.

Sawitzke, and Barry C. Cole

University of Utah School of Medicine, Salt Lake City, Utah

INTRODUCTION

Mycoplasma arthritidis, which induces a spontaneous polyarthritis in rodents, produces a soluble immunomodulatory protein that demonstrates classical superantigenic activity. M.arthritidis mitogen (MAM) is a typical superantigen (SAg) in that it is presented to murine or human T cells by direct binding to major histocompatibility (MHC) molecules present on accessory cell surfaces and is recognized by specific Vp chain segments of the T-cell receptor for antigen (TCR) without MHC restriction. MAM was first characterized as a SAg in when it was shown that the T-cell mitogen contained in M. arthritidis culture supernatants specifically activated VP&bearing T cells (1). Thus, MAM was among the first SAgs described. M. arthritidis is a member of the class Mollicutes, a group of eubacteria characterized by the lack of a cell wall, possessing the minimum genomic capacityrequired to sustain an autonomous exist339

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ence, and a low G + C content (2). Due to their limited genetic potential, mycoplasmas possess onlylimited biosynthetic pathways and are dependent upon their host for synthesis of macromolecules (3). Mycoplasmas are believed to be a product of degenerative evolution from Gram-positive bacteria with low content of G + C base pairs (3,4). It is curious, therefore, that M. arthritidis has retained a gene encoding a protein with immunomodulatory properties despite the pressure to downsize its genome. The members within the genus Mycoplasma are further characterized by an alternative codon usage similar to that of mitochondria in which the UGA codon is read as a tryptophan instead of as a universal stop codon (5). This chapter will address the unique properties of M. arthritidis and MAM as they relate to other bacterial SAgs and the immunobiology of the MAM protein.

II.

PROPERTIES AND SEQUENCEANALYSIS OF M A M

A.

Production of SAg

arthritidis and Other Mycoplasma spp.

MAM was originally described as a soluble protein contained in M. arthritidis culture supernatants that promoted proliferation of murine T cells in a genetically restricted manner (6,7). Subsequent studies designed to optimize the production ofMAM for purification purposes revealed that MAM levels were elevated in cultures that had Thus, it appears just reached senescenceor were grown at 30°C that MAM is produced by M. arthritidis in response to an environmental stress and perhaps is involved in acquisition of necessary macromolecules through its ability to adhere to nucleic acids or proteins (C. L. Atkin and K. L. Knudtson, unpublished observations) MAM is produced by all strains of M. arthritidis tested irrespective of their virulence potential. Ultrafiltrates of culture supernatants from 31 virulent and avirulent strains of M. arthritidis all demonstrated T-cell mitogen activity (11).Furthermore, PCR analysis using primers to the 5‘- and 3’-ends of the MAM gene (rnam) showed that the appropriate ca. 650-base-pair fragment was amplified in all 22 strains that were examined (12). The presence of mam or mam-like genes in other Mycoplasma species was examined by DNA-DNA hybridization using mam as a probe (12). Genomic DNA from human mycoplasmas including: M. buccale, M. fermentans, M. hominis, M. orale, M . penetrans, M. pneumoniae, and M. salivarium failed to hybridize with the mam probe.

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of MAM

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Thus, MAM a MAM-like protein may be unique to M. arthritidis. It should be noted, however,that ultrafiltrates of culture supernatants from M. fermentans, M. orale, M. penetrans, and M. salivarium have demonstrated weak ability to induce proliferation of both human and murine T cells (B. C. Cole, M. Manohar, and K. L. Knudtson, unpublished observations). Therefore, the existence other SAgs in other species of mycoplasmas should not be discounted. B.

Physical Properties and Sequence of MAM

Partially purified preparations ofMAM from M. arthritidis culture supernatants were initially used to characterizeMAM as a basic, heatand acid-labile protein with a molecular mass of15-30 kilodaltons (kDa) anda p1 2 9.0 (8,lO). The high isoelectric point and hydrophobic nature of the protein made initial attempts at purification to homogeneity difficult because MAM could bind nucleic acidsand other proteins in the culture as well as to surfaces (11). Pure MAM was eventually obtained by using series of cationic and hydrophobic interaction columns with an approximate yield of20-50 MAM per liter of culture (11).Analysis of this purified protein by SDS-PAGE showed a single band with a molecular mass of approximately 27 . kDa. Moreover, mitogenic activity of this pure protein could be detected in the picogram range and it possessed the biological and physical properties previously described using the less pure preparations ofMAM (11). The sequence of the first 53 amino acids of the purified MAM was obtained by Edman degradation (11) and was used to design primers to generate a PCR product probe for screening an M. arthritidis genomic library (12). Themum gene was subsequently identified, subcloned, and sequenced. The nucleic acid sequence was translated in the reading frame giving the amino acid sequence previously determined by Edman degradation (Fig. 1). The deduced sequence of MAM predicts a protein of213 amino acids with a calculated molecular mass of25,193 Daltons (121, which is similar to the molecular mass of27 kDa observed by SDS-PAGE analysis (11). Interestingly, the molecular mass ofMAM is within the range of values reported for the staphylococcal and streptococcal SAgs (13). However, the calculated p1 of 10.1 makes the MAM the most basic of the known bacterial SAgs. To confirm that the cloned gene truly encodes MAM, the UGA codons at amino acid positions 132, 177, and 178 were converted to UGG tryptophan codons andtheresultingrecombinantprotein

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Figure 1 Amino acid sequence and structural predictions of MAM. The amino acid sequence shown at the topcorresponds to the matureMAM peptide reported by Cole et al. (12). Hydropathicity values are illustrated in the center and were determined by the method Hopp and Woods The bottom plot represents the predicted secondary structure of MAM as determined by using the algorithm Chou and Fasman Regions predicted to form a-helices, p-sheets, and turns are denoted by helix, sheet, and turn, respectively.

(rMAM) was expressed in Escherichia coli (K. L. Knudtson et al., submitted for publication). Recombinant MAM possesses the same physical properties and is biologically similar to native MAM in every parameter examined including: recognition by antibody, ability to induce lymphocyte proliferation, Vp and MHC usage, induction of immune suppression, and SAg bridge formation (K. L. Knudtson et al., submitted for publication) (12). Examination of the amino acid sequenceof MAM (Fig. 1 ) shows that MAM, like toxin shock syndrome toxin-l (TSST-1) (14), does not

Physical Properties lmmunobiology and

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contain cysteine residues. Thus, the disulfide bridge present in most other SAgs cannot be formed. While it has been shown that the formation the disulfide bridge may not be necessary for SAg activity (15), the inability to form the disulfide bridge by MAM may account for someof the apparent instability associatedwith this protein.

C.

Functional Domains in

To identify regions of MAM required for SAg activity, a panel of 11 overlapping peptides spanning the entire MAM protein was first synthesized (12). These peptides were then tested for their ability to block MAM-induced lymphocyte proliferation in a competition assay. Two peptides, MAM,,-,, and MAM,,,,, appeared to compete with MAM in a dose-dependent manner. Interestingly, these two peptides span two of the five predicted hydrophobic domains ofMAM (Fig. 1). The surface hydrophobic regions, of other bacterial SAgs in which the crystal structure has been solved, have also been shown to be possible interaction sites with MHCclass I1 and T-cell receptors (16-20). The results of sequence comparisons between .MAM and other known SAgs and proteins (12) revealed that the MAMI,-,, region could be aligned with regions of several bacterial SAgs including streptococcal pep M5 toxin (residues 101-112) (21), staphylococcal enterotoxin (SE) B (SEB, residues 38-82) (22), and C1 (SECI, residues 127-139) (23), as well as with a region of the endogenous SAg mouse mammary tumor virus-7 (MMTV-7, residues 80-99) (24). Interestingly, a region of the human immunodeficiency virus-l .(HIV-l, residues 550-570) gp160 envelope glycoprotein also showed a sequence similarity to MAM,,, (12). TheSEB region that shares sequence similarity with MAM is the region that interacts with MHC class I1 molecules (22). This region contains a hydrophobic area that includes important residues at SEB,, 45 and 47 and is followed by a hydrophilic or polar region containing SEB, also important for MHC binding (17). As is apparent from Fig. 1, MAM,-, has a similar profile in which a hydrophobic domain is followed by one of hydrophilicity, which also lies in a predicted P-strand. Analyses of this region by using site-directed mutagenesis have supported the findings obtained using the synthetic peptides and have narrowed the region thought to be associated with MAM activity. Amino acid base changes within MAM,, appear to reduce or eliminate the ability to induce lymphoproliferation (K. L. Knudtson et al, manuscript in preparation). Further studies are underway to determine the role this region plays in

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MAM’s ability to form the SAg ternary complex. However, because MAM,,, shares sequence similarity with regions of other SAgs that are thought to interact with MHC molecules, is it not unlikely that this region of MAM may be involved in MHC class I1 binding. The region of similarity between MAM,,-,, and the conserved retroviral sequence of the murine MMTV-?’, is of interest since a retroviral peptide containing a region of sequence similarity bound to MHC molecules and competitively blocked the binding of SEA to the same MHC molecules (25). While regions similar to the retroviral sequence were also present in the SEC SAgs and in streptococcal pep M5 toxin, there is no data to suggest that these regions play a role in the activity of these latter SAgs. It is noteworthy that HIV-l gp160 may exhibit SAg-like properties (26) and may expand some of the Tcells bearing thoseVp chain segments that are used by MAM (26,27). However, recent studies suggest that SAg present in cytomegaloviis responsible forthe enhanced replicationof HIV in human Vp12bearing cells (28). Regions of similarity between pep M5 and other bacterial SAgs have also been suggested (21). Therefore, despite the lack of extensive sequence homology betweenthe SAg proteins, there appear to be short regions of similar amino acid residues that may have a common function in generating the SAg ternary complex. This possibility remains to be established. Few sequence similarities were observed between MAM,,-,, and other SAgs. However, a search for motifs revealed that MAM83-89consisted of the seven-residue lectin-legume motif-p consensus sequence that is present in all legume lectins including concanavalin A (Con A) (29). This motif is not present in the other SAgs, suggesting that MAM may have the ability to activate lymphocytes in a novel manner. Interestingly, MAM,,-,, also inhibited proliferation induced by Con A and this inhibition was not due to toxicity, as subsequent addition of Con A restored the proliferative responses to the levels obtained with Con A alone (12). Conversely, a peptide representing Con A,+, which contains this motif, inhibited Con A-, phytohemagglutinin-, and MAM-induced lymphocyte proliferation. Thus, the Con A peptide and MAM,,-,, appear to be functionally cross-reactive. Recent experiments in our laboratory in which the only legume lectin motif in MAM was altered by site-directed mutagenesis have shown that the region encompassed by the motif is, in fact, necessary for MAM activity (K. L. Knudtson et al., manuscript in preparation).


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The role of the lectin-legume motif-8 in MAM is unclear. In plant lectins, the motif achieves and maintains a bioactive conformation through the binding of metals enabling the amino acid residues just downstream of the motif to be in a position to bind saccharides (29,301. Thus, the motif could be playing a similar role in MAM. Another possibility is that the motif is a site of metal binding to achieve a stable interaction between MHC class I1 or TCR molecules and MAM. Fraser et al. (31) have shown that SEA and SEE have a binding site for Zn2+ which facilitates SAg binding to MHC class I1 molecules (32). Driessen et al. (33) have suggested that MAM may interact with MHC class I1 in a similar manner because MAM-, SEA-, and SEE-, but not TSST-l-induced activation was affected by the addition of supplemental zinc. Finally, because the motif lies within a hydrophobic domain (Fig. l), this motif could be involved in a hydrophobic interaction with MHC class I1 and TCR. Current efforts in our laboratory are aimed at distinguishing these possibilities and closely examining the role of the two identified functional domains in the various MAM-associated activities. D. Relationship of M A M to Other SAgs

As noted previously, mycoplasmas are thought to have evolved from Gram-positive bacteria possessing a low mol% G + C content (4). Since the genomes of Staphylococcus spp. and Streptococcus spp. also have a low mol% G + C content, the possibility therefore exists that the SAg from M.arthritidis is related to the SAgs of staphylococci and streptococci. Considering a common origin for these genes, we conducted a phylogenetic analysis comparing the amino acid sequences of MAM and the pyrogenic toxin gene family (34). In addition, the Yersinia pseudotuberculosis mitogen (YPM) and representatives of the endogenous SAgs were included in the analysis. MAM was not closely related to other SAgs and alone occupied one of three main branches on an unrooted phylogram (Fig. 2). The closest similarity was between MAM and streptococcal pep M5 (21) at 24% sequence similarity. Interestingly, the analysis suggests that the SAg from Y. pseudotuberculosis (a Gram-negative bacterium) is more closely related to the SAgs from Gram-positive bacteria than MAM despite the hypothesis that mycoplasmas have evolved from Gram-positive bacteria. Therefore, even though MAM is similar to the pyrogenic family of toxins with respect to molecular massand superantigenic activity, the primary amino acid sequence of MAM is clearly phylogenetically unrelated to the other SAgs.

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TSST-1

SEC1 SEC2

SPEB

_c SPEC - YPM

Figure 2 Relationship of MAM to other SAgs. The SAg amino acid sequences were obtained from current PIR and Swiss-protein data bases, except the sequence of YPM, which was obtained from recent reports (101,102). All sequences were aligned using the Clustal V algorithm using the default settings and subsequently examined using PAUP 3.0. Trees showing relatedness were derived using a heuristic search and the single best fit is displayed here as .an unrooted phylogram. MAM, Mycoplasma arthritidis mitogen; TSST-l, toxin shock syndrome toxin-l; staphylococcal enterotoxins A, B, Cl, C2, and D; SPE, streptococcal pyrogenic toxins A, B, and C; MMTV, murine mammary tumor virus; YPM, Yersinia pseudotuberculosis mitogen; pep M5, peptide digest of streptococcal M5 protein. IMMUNOBIOLOCY OF THE MYCOPLASMAARTHRITIDIS SAG

A.

InteractionwithMurine M H C Molecules

The importance of class I1 recognition by SAg to T-lymphocyte activation has been well established Recently, many details of this interaction have become available resulting in a broadened understanding of the critical features. In the text that follows, the interactions of MAM and MHC molecules, especially with murine class 11, but also with human class I1 and with chimeric molecules, will be highlighted.

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The mitogen present in M. arthrifidis was first observed to be distinct from other known mitogens when lymphocytes from H-2Ebearing mouse strains were shown to undergo proliferation and develop cytolytic abilities in response to culture supernatants, whereas lymphocytes from H-2E negative mouse strains did not (6). The use of congenic and recombinant mouse strains suggested that H-2Ea chain is the determining factor, since lymphocytes bearing H-2Ea(A.TFR5) with H-2A,, but not H-2Aa and H-2E, (A.TFR41, could selectively adsorb mitogenic activity from mycoplasma supernatants (7). The use lymphocytes from transgenic mice or fibroblasts transfected with murine H-2 molecules and empioying highly purified preparations of MAM confirmed that expression of H-2Ea is preferred for presentation ofMAM to T cells (37). As is true for other SAgs, MAM binds directly to MHC-bearing accessory cellswithout the need for processing Both fixed accessory cells (AC) and AC membranes can present MAM to T hybridomas. In addition, glass beads or liposomes coated with class I1 molecules present MAM to T cell hybridomas only when is present (38). The observation that liposomes containing isolated H-2E can present MAM to T cells indicates that costimulatory moleculesare not required for activation of murine T cells. It remains to be shown whether they might enhance the response or effect specific SAg-mediated responses such as the SAg-bridge or SAg-dependent cytotoxicity. As for other SAgs, presentation ofMAM to T cells is not classically MHC-restricted. Early work indicated that only H-2E molecules could present MAM. Recently, the use of very potent preparations have indicated that T-cell activation can also occur by select H-2A molecules, Thus, splenocytes from H-2b mouse strains show weak or moderate responses to high levelsof MAM, while splenocytes from H-2k mice can respond very well to even low doses MAM. A comparison of lymphocytes from inbred, congenic, and recombinant mouse strains on a C57BL/10 background revealed that mice lacking H-2E but which expressed H-2Ab, H-2Ad, and H-2Ap exhibit weak or moderate responses to MAM, whereas those expressing H-2k,H-2q, H-2', and H2s are totally refractory to MAM. Thus, allelic polymorphisms at H2A influence lymphocyte responses toMAM (B. C. Cole, submitted). B.

InteractionwithHuman

MHC Molecules

Human cells can be activated by MAM (39) and antiserum to HLADR inhibits the response (40). Because of the known preference of

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MAM for the E, chain on murine MHC molecules, it was suggested that the homologous DR, chain on human MHC molecules would be similarly recognized (40). The use ofAC expressing human, mouse, or chimeric MHC class I1 molecules in transgenic systems has confirmed that DR, can present MAM (41-43). Therefore, the ability of nonresponder cells from H-2Af haplotype mice that have been transchain homolog) and hufected with the human DRA*0101 chain man DRB*0301 chain (EP chain homolog) to respond to MAM was examined. The DRA*0101 chain in combination with the murine H2Af3chain was recognized by MAM whereas the DRB*0301 chain in combination withthemurineH-2Aiwasnot. Moreover, the DRA*0101 and DRB*O301 double transfectant also responded toMAM, which further supports the preference of H-2Ea or an E,-like chain by MAM (41). Chang and co-workers have combined a chain genes that coded for the outer domain of E, with the inner domain of DR, and vice versa (42). These constructs were expressed in human 9.22.3 cells and their effect on MAM-induced proliferation noted. The 9.22.3 cellline normally expresses the DQ2 and DP4.1 molecules (44) and can present MAM in the absence of the transgene, albeit at low efficiency. Cells expressing intracellular DRBY0301 combined with the a chain transgene products to form HLA-DR3 surface antigen that can be detected by the use of monoclonal antibodies. Using this system, the outer domain was found to present better than that ofDR,. An excess of times as much MAM was required by DR,-containing cells as by E,+cells to get the same degree of activation, suggesting that the binding affinity toward MHC is particularly important to MAM-induced proliferation (42). Whether the same is true for SAgmediated T-cell deletion, B-cell activation, or other SAg-mediated events is not known. The fact that some murine H-2A molecules present MAM and others do not provides an opportunity to determine which amino acids of the MHC class I1 molecules are required for effective MAM presentation. Comparison of the responding H-2d and H-2b haplotypes to the poorly responsive k, f, q, and S haplotypes suggests that amino acids 65 and 66 of the a chain might be particularly important. Those haplotypes with a valine (like H-2Ea) or alanine at position 65 are responsive, whereas those with a tryptophan are not (B. C. Cole et al., unpublished). Studies by Chu and co-workers (43) used a panel of point mutations within the H-2Ea chain that contained H-2A amino acid substitutions and examined their effects on peptide and SAg presentation as measured by induction of Sur-


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prisingly, the change of amino acid at positions 65 and 66 to those associated with H-2A resulted in a slight increase in MAM-induced IL-2 production. Testing of MAM-induced proliferation was not performed, however. HLA-DQ class I1 molecules, the human homologs of H-2A, have also been tested for their ability to be recognized by MAM (B. C. Cole et al., submitted). Specifically, transgenic mice expressing HLA-DQ, and or -DQ, chains were constructed on the nonresponding BIO.M (H-2E-, H-2Af) background. Lymphocytes from DQw6 (DQAY0102DQB1*0601) double transfectants were very reactive to MAM, whereas those from DQw8 (DQA1*0301-DQB1*0302) micewere not. Because the two DQ molecules differ in both their a and p chains, it is impossible to determine which chain selects for MAM reactivity. As stated earlier, for H-2E and HLA-DR, it is the a chain product that reacts with MAM. Further studies should clarify whether it is also true for HLA-DQ. It is possible that some p chains can present MAM or influence peptide selection. Alternatively,the p chain may alterthe a-chain conformation in a manner that facilitates recognition by MAM. Most importantly, selective MAM presentation by specific class I1 types would be in keeping with autoimmune disease pathogenesis as such diseases are typically associated with only a limited subset of MHC class I1 types. Increasingly, it has been shown that a single mechanism of immune activation is not used by all SAgs (45). Further, it has also become apparent that the peptide present in the groove may influence SAg activity (46) and there is even evidence that the requirement for AC molecules can be bypassed (47). As peptide selectivity is based largely on the contributions of the p chain, the possibility of MHC P-chain influences in MAM presentation would have important ramifications for the known MHC associations with autoimmune disease. C.

T-cell ReceptorUsage by

The association of the VP8 chains the TCR with reactivity of lymphocytes to MAM was first shown by demonstrating cosegregation of these traits by analyzing the progeny of a test cross consistingof RIIIS mice (H-2E+, VpS-, poor responders to MAM) mated with (RIIIS B1O.RIII) F1 mice (Vp8+, H-2E+, high responders to MAM). Subsequently, we found that antibodies to the Vp8 TCR family inhibit MAM-induced proliferation. In addition, BALB/c splenocytes expanded with MAM express VP8.l and Vp8.2 and to a lesser extent

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VP8.3 and Vp6. However, the lymphocytes from V@-negative mice (C57BR) strongly express when stimulated with MAM (1).Later

studies used multiprobe RNase protection assays to confirm these findings and also showed that MAM uses the murine Vp5.1 TCR. In the rat, all of the above Vp chains are used by MAM except for Vp8.3 (48). These results suggest that there is a hierarchical usage of different murine Vp chains by MAM. Genomic compositionat the Vp chain segment of the TCR influences the pattern of Vp usage by MAM. Thus, by reacting V p and Vpb riboprobes with RNA extracts from the splenocytes of Vpa (C57BR) and VfF RIIIS haplotype mice, which lack various combinations of Vp5.1, Vp6, and Vp8 family of TCRs, it was shown that MAM could also engage Vpl, Vp3.1, and Vp16 (49). However, allelic polymorphisms may also play a role in MAM recognition since there are structural differences in Vpl, Vp3.1, and Vp6, between the VPb and V p haplotypes (50). It was also observed that different class I1 isotopes may engage in different Vp TCRs. Thus MAM-induced activation of Vp6- and VP8.1-bearing T cells is more dependent on expression of H-2E than is activation of cells bearing the Vp5.l chain or the Vp8.2 TCR. Human T cells are less responsive to MAM than are murine cells (7,51). H-2E has been shown to present MAM more effectively than HLA-DR, murine T cells (421, and the difference in the responses of murine and human cells to MAM may also suggest that factors other than presentation by MHC molecules are responsible. There is evidence that the requirement for AC molecules can be bypassed in the presence of phorbol myristate acetate. Also, the Jurkat T-cell line undergoes a rise in cytosolic Ca2+when MAM is the only stimulator present (52). Since human cells respond better to the staphylococcal SAgs than do murine cells, it has been proposed that the degree of responsiveness of lymphocytes to SAgs reflects an evolutionary adaptation of microbial flora to their natural hosts (51). As for murine T cells, MAM is recognized by human T cells via specific Vp chain segments of the TCR. Clonal expansion of peripheral blood lymphocytes by MAM followed by Vp TCR analysis by use of monoclonal antibodies (531, multiprobe RNase protection assay (411, or PCR (27,54) has shown that MAM can use human Vp3.1, 5, 7, 10, 11.1, 12.1, 13.1, 14, 17.1, 20.0f considerable interest is that these human Vp chain segments show that the most homology with the murine Vp chains that are used by MAM. However, there are a number of differences between human and murine Vp usage by MAM.


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Thus, MAM preferentially uses the murine Vp8 family, which is most closely related to human Vp3, and Vpll-14. In contrast, the most commonly encountered MAM-expanded human T-cell clones bear the Vp17 receptor, which exhibits most homology to the murine Vp6 chain (55). A comparison of MAM-reactive human and murine Vp chain sequences indicated that most share a motif in the CDR 4 region at residues 68-75 (55). However, the existence of MAM-reactive Vp clones that lack this motif suggests that there may be other MAMreactive Vp TCR sites. An alternative explanation is that other regions of the TCR may influence MAM reactivity. In support this notion, it has been shown that differences in the JP segment influence reactivity to MAM (56). D. B-Cell Activation by the SAg Bridge

Superantigen-mediated linkage of T cells via their TCRs to B cells by their MHC molecules not only activates the T cell, but also can lead to B-cell activation. Irradiated, MAM-reactive human T-cell lines exhibit the ability to induce resting, purified, peripheral blood or tonsillar B cells to secrete Ig and to increase expression of the CD23 antigen when cocultured in the presence MAM. This T-cell helper activity is restricted to the CDb T-cell subset (57,581. In the murine system, MAM mediates both Ig secretion and B-cell proliferation in the presence of MAM-reactive T cells. It was further shown that this cognate interaction also promoted secretion of specific antibody to sheep RBCs when the latter was present with MAM. It was hypothesized that SAg-mediated B-cell activation might also result in secretion of autoantibodies especially to polyvalent self-antigens (59). The ability to induce rheumatoid factor(RF) by SAg has been shown with (60) and earlier observations indicated that M. arthritidis culture supernatants induce human lymphocytes to secrete RF (61). As we discuss later, this pathway may contribute to autoimmune disease. The interaction MAM with unseparated human peripheral blood lymphocytes may differ in a significant manner as compared with the reaction seen with the staphylococcal SAgs. Crow and coworkers have shown that MAM and pokeweed mitogen induce high levels of polyclonal IgM and IgG secretion by peripheral blood mononuclear cells whereas the staphylococcal toxins fail to do so (62). Conversely, the staphylococcal SAgs promoteda higher level of T-cell proliferation than does MAM. In a related observation, low doses of SEB were found to promote B-cell activation whereas high doses pro-

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mote a T-cell proliferative responsethat leads to cytolysis of activated cells (63). Thus, the lower proliferation of human T cells exposed to MAM may facilitate a shift from a cell-mediated response to one of humoral immunity. This type shift is classically observed in autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus that are characterized by hypergammaglobulinemia. As proposed by Friedman et al., SAgs may be responsible for this feature of autoimmune disease (58,64). In vivo Effects of The ability to examine the in vivo properties of MAM has previously been limited by the. lack availability of homogeneous material. However, the results obtained usingthe partially purified materialare now being confirmed by using pure preparations of MAM. MAM injected iv results in a mild transient toxic syndrome after 1-2 days characterized by ruffledfur, lethargy, and ocular inflammation.There is a marked splenomegaly and lymphadenopathy even at low doses, which can last for several weeks. Subcutaneous injection of MAM into mice fails to evoke any clinical symptoms, while intra-articular injection into rats results in a marked arthritis that is more severe in D A than BN rats, perhaps correlating with the lesser activity of lymphocytes to MAM from the latter strain. Early joint lesions are characterized by partial shedding of the synovial membrane and a mild infiltration of polymorphonuclear cells and lymphoid cell foci. Subsequently, hyperplasia and hypertrophy of the subsynovium are seen along with infiltration of lymphocytes, macrophages, and fibroblasts. The entire process resolves by 14 days. Fibroplasia may be mediated by IL-l, which has been shown to be induced by MAM (65). Of note, intraperitoneal injection of MAM results in marked peritoneal adhesions in those strains of mice whose lymphocytes are strongly reactive to MAM. The mycoplasma SAg, like the other bacterial SAgs, profoundly affect immune functions following in vivo administration. First, MAM modulates VP TCR expression in vivo since when given iv to VPb haplotype mice, it selective expands VP8.l- and VP8.2-bearing T cells in lymph nodes whereas in VfF mice, which lack the VP8 TCR family, there is a selective expansionof T cells bearing Vp6 and VP16 (49). The relevance of this to disease will be described later. However, despite this expansion, lymphocytes collected from mice 1-2 days after iv injection of MAM are markedly inhibited in their ability to proliferate in vitro to MAM and to a lesser extent to PHA or

Physical Properties lrnrnunobiology and

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Con A. Whereas responsiveness to lectin mitogens is regained by 10 days, responsiveness to MAM is still depressed by (66). MAM does not inhibit subsequent responsiveness of lymphocytes to SEA. Conversely, lymphocytes collected from mice injected with SEA fail to respond to SEA, but respond normally to MAM (67). This anergic state appears not to be due to deletion of MAM-reactive cells, but is mediated by a CD4+ population of T cells that transfers suppression to lymphocytes from control mice (66). This is in marked contrast to the "suppressor" cells induced by ConA, which are CD8'. The inhibitory properties of MAM-induced cellsare also Vp specific, since these cells are unable to suppress proliferative responses to SEA but partially suppress responses to SEB, which shares some VP TCR usage with MAM (B. C. Cole and E. A. Ahmed, unpublished observations). Earlier work established that MAM could induce T cells to become cytolytic for transfected mouse fibroblastsin vitro (6). It was recently shown that MAM up-regulated Apo-l expression on malignant human B lymphocytes and that in the presence of CD4+, MAMreactive T cells resulted in apoptotic death of the B cells (68). Thus, one explanation for the induction of VP-specific CD4+ T-suppressor cells is that they induce apoptosis in the presence of MAM or SEB SAgs. The ability of MAM to inhibit T-cell responses to mitogens appears to be associated with suppression of other T-cell functions in vivo (67). It was shown that two to three iv injections of MAM prolong the subsequent survival rate of skin transplants between mice differing at both MHC and non-MHC loci. A single injection of MAM was also shown to inhibit contact hypersensitivity to dinitrofluorobenzene when administered 2 days before sensitization. In contrast to the inhibitory effect on some T-cell functions, MAM appears to increase B-cell functions. Splenocytes collected from mice injected iv with MAM exhibit an increased ability to secrete IgG and IgM when cultured in vitro. MAM enhances Ab formation to specific antigens if given after sensitization or suppresses Ab formation if given prior to sensitization. MAM enhances the plaque-forming (pfc) responses to sheep RBCs when given 2 days after the antigen. However, MAM had little effect or slightly suppressed the numbers of pfcs when given prior to the sheep red blood cells. MAM also increasedthe ELISA antibody response to ovalbumin when given after the antigen challenge. A key to understanding these changes might lie in MAM-induced alterations in cytokine profiles. Thus, splenocytes collected from mice previously injectedwith MAM show a marked decrease in

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their ability to produce IL-2 when challenged in vitro with MAM. The IL-2 responses to Con A are less inhibited. In contrast, the ability of cells from MAM-injected mice to produce IL-4 and IL-6 in response to in vitro stimulation with MAM was markedly increased. IL4 responses to ConA were slightly increased, but no change was seen in the IL-6 response to Con A. These observations suggest that MAM induces a shift from a THl-, to a TH2-associatedcytokine profile (67). This process favors the differentiation and proliferation of B cells and might act together with the cognate B/T-cell interaction to up-regulate antibody responses and autoimmunity. IV. A.

ROLE OF MAM IN DISEASE Mycoplasma arthritidis-Induced Disease

Large doses of viable M arthritidis cells are very toxic to mice and can lead to a severe toxic shocksyndrome associated with fecal impaction, paralysis of the hindlimbs, and death. This severe toxic reaction occurs predominantly in mice that express H-2E and which are high responders to MAM. Thus, C3H(H-2k)and B10.D2(H-2d) mice were susceptible whereas C3H.SW(H-2b), and C57BL/10(H-2b)(3H.BI (H-2b), mice were less affected (69). The severity of arthritis in the surviving mice was not significantly different. One explanation for the lack of difference is that surviving H-2E+ mice may have developed antibodies to MAM and hence were more resistant to the biological effects of MAM. This important issue needs to be resolved. All mouse and rat strains injected subcutaneouslywith viable M. arthritidis developseveresubdermal abscesses. However, mouse strains whose lymphocytes are strongly reactive to MAM develop an ulcerative dermal coagulation necrosis (70). A comparison of the response using inbred and congenic mouse strains differing at the MHC indicated that mice expressing H-2E were more susceptible to necrosis. When more virulent strains of M. arthritidis are used, some necrosis occurs in H-2E- mouse strains. The results are consistent with observations that MAM can induce tumor necrosis factor (71,721 and that lymphocytes from some mice that lack H-2E can also undergo a proliferative response. B.

MAM and Experimental Autoimmune Disease

Two major mechanisms have been proposed by which SAgs might play a role in autoimmune disease. First, by activating specific VD TCR-bearing autoreactive T cells, a SAg might increase the numbers

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of these cells above the threshold necessary for induction of overt clinical disease (73). Second, the cognate interaction between T and B cells mediated by some SAgs can result in polyclonal B-cell activation, which was hypothesized to lead to the generation of autoantibodies especially to polyvalent self-antigens (58). Collagen-induced arthritis (CIA) has been used as a model of autoimmune arthritis to test the hypothesis that SAgs might contribute to autoimmune disease. In this model, mice immunized with heterologous or homologous type I1 collagen, the predominant type of collagen found in articular cartilage, develop severe chronic arthritis approximately 4-5 weeks later (74). In the B1O.RIII mouse, T cells bearing the Vp6, Vp7, and Vp8 chains of the TCR have been shown to drive the inflammatory response since their removal by use of specific antibody, somatic or genomic deletions,or congenic mice that lack these Vp TCR receptors results in amelioration or resistance to disease (75-77). Since MAM uses these same VP-bearing T cells and has been shown to clonally expand Vp6 and Vp8 TCRs in vivo, experiments were undertaken to examine the effect ofMAM on CIA induced by porcine collagen in BlO.RII1 and susceptible F1 and test cross B1O.RIII hybrid mice. It was shown that convalescent mice immunized with collagen 130-200 days previously, and whose arthritis had largely resolved, rapidly develop a severe flare in arthritis when challenged iv, ip, or in the base of the tail with a single injection of MAM (78). MAM is not arthritogenic by these routes in normal mice. Of considerable interest was that some mice that had never developed arthritis in response to the collagen did develop severe disease when subsequently injected with MAM. The increase in disease is also associatedwith an increase in ELISA antibodies to mouse type I1 collagen. MAM also enhances the severity of disease in mice suboptimally immunized with collagen. SEB, which also uses the Vp7 and VD8 TCRs, also promotes disease activity, but SEA, which utilizes a different set of Vp TCRs, fails to do so. These observations strongly suggest that clonal expansion of, or interaction with, collagen-reactive T cells plays a key role in triggering or exacerbating CIA activity. Increased antibody production may also play a role and this would be consistent with a T-cell-dependent polyclonal B-cell activation by the bridge pathway, mediated in part by the production of the B-cell differentiation cytokines IL-4 and IL-6. However, nonspecific cytokine production appears insufficient to trigger CIA since mice injected with SEA, which activates CIA-irrelevant Vp chain-bearing T cells, fail to develop disease.

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C. MAM-like SAgs in Rheumatoid Arthritis

Autoimmunity is characterized by cellular and humoral immune defects, but the sine qua non is autoantibody formation. Diseases associated with autoantibodies abound and are typified by disproportionate involvement of females, MHC class I1 associations, and a chronic remittive natural history. That infections could trigger disease in such susceptible individuals is a recurrent theme. More recently, specific infection-related products including lipopolysaccharide and SAgs have been proposed to be involvedin induction of autoimmune disease (55). Rheumatoid arthritis (RA) is an example of a human autoimmune disease in which SAgs, particularly those with properties similar to MAM, may well be involved (55,58,79,80). As such, we will use the disease-associated properties that have been attributed to MAM as an example to present the accumulating evidence of SAg involvement in RA (Table 1). RA is predominantly a disease of reproductive-age women characterized by inflammatory joint swelling, bony erosion, disability, and deformity Rheumatoid factor (RF), an autoantibody directed against the Fc portion of immunoglobulin, is observed in about 70% ofRA cases. The production ofRF is generally associated with a worse prognosis and especially with extra-articular manifestations including: cutaneous vasculitis, interstitial fibrosis, and Felty’s syndrome (83). Many patients have a generalized hypergammaglobulinemia and most have at least one copyof an MHC class I1 molecule containing the shared epitope QKRAA (84). As the disease develops, symmetricalinvolvement of the smaller joints of the hands and feet is characteristic. These joints exhibit swelling, stiffness, and pain and later develop marked hyperplasia of the synovial cells. Inflammatory cells, including macrophages, endothelial cells, T and B lymphocytes, secrete cytokines resulting in erosion of bone and cartilage. Surface antigen expressionof HLA-DR and VCAM is increased as is the production of the cytokines IL-1, IL-6, TGF-P, and TNF-a (85). As noted previously, the proinflammatory cytokines can be induced in vitro by MAM as well as by other SAgs (86,87). A feature ofRA is the formation of RF. As outlined in the section dealing with the SAg-bridge, the SAgs have been shown capable of activating B cells to secrete antibody. In some cases, they can act as adjuvant to promote production of antigen-specific antibodies (59) and also to induce RF In fact, He and co-workers showed that SED skews the antibody repertoire of normal human B cells toward

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RF formation (60). No difference between HLA-DR4 positive and negative donors was observed and only about 8% of normal B cells could be induced to secrete Ig with SED. Interestingly, B cells from RA patients were less responsive to SED stimulation than normal cells. Studies of the mechanism and requirements for SAg-induced Ig synthesis are in progress. Thus far, it is apparent that not all B cells respond, that dose and timing of SAg may have an effect, and that the phospholipase C and K pathways are involved (89). MAM is known to be more effectivethan staphylococcal enterotoxinsin inducing human Ig synthesis, and its ability to induce RF is under investigation. Abnormalities of T-cell function are characteristic ofRA and many therapies have been directed against them. Examples include cyclosporin A, thoracic duct drainage, and monoclonal antibodies directed against CD4 or other surface antigens. Unlike traditional antigen-stimulated T cells, SAg stimulation results in activation of specific VP-chain-bearing clones.As a consequence, the occurrence of oligoclonality in the T-cell VP-chain repertoire of diseased joint tissues would constitute evidence for SAg inducement. Thus far, there have been many conflicting reports on the VpTCR repertoire in human RA perhaps reflecting the different sources of T cells used (i.e., peripheral blood vs. activated T cells from synovial tissue). To further complicate matters, the initiating process for RA need not be the same as that which maintains it or results in characteristic disease flares. Thus, the studies showing polyclonality and those showing oligoclonality might represent different stages of the disease process. However, despite these variables, a consensus is now emerging that oligoclonal expression of T cells bearing certain VP-chain segments of the TCR in the joint tissues is occurring. The most frequently encountered T cells in RA synovial tissues are those expressing Vp3 and Vp14, or Vp17, which share the most homology with murine Vp7 and the Vp8 family, or Vp6TCRs, respectively (55,79,90,91). It should be noted that MAM and SEB both use the same human Vp-chain segments. As part of the synovial hypertrophy that occurs in RA, an increase in type B synoviocytes is observed. These cells have been shownto be capable of SAg presentationand of SAg-induced cytokine production. Mourad and co-workers demonstrated that SEA activates IL-6 and IL-8 synthesis from type B synovial cells collected from RA patients (71). Consistent with AC functions, they express functionally relevant class 11,VCAM-1,CD28, and ICAM-1 (92). MAM and other SAgs are very effectively presented to human T cells

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by dendritic cells, which are present in increased numbers in rheumatoid synovium Another immune cell thought to be involved in human RA is the NK cell (94). RA patients typically have more circulating NK cells seen morphologically as large granular lymphocytes than control people and some RA subsets such as Felty’s syndrome and large granular lymphocytic leukemia are associated with even more substantial increases in large granular cells MAM has been shown to activate NK cytolysis in a direct fashion as well as to stimulate cytokines that indirectly enhance NK function If SAgs are to be involved in autoimmunity in humans, evidence of human exposure to SAg should be present in the form of SAg-specific antibodies. As indicated in Table 1, antibodies to MAM are present in humans especially those with RA. MAM-reactive antibodies of IgG, IgM, and IgA isotype are present in samples from normal and affected humans, but they are significantly higher in RA than in SLE or control patients. The difference is not related to RF titers or to the total Ig concentration present (A. D. Sawitzke et al., unpublished observations). Studies to define why or how humans develop antibodies to a protein produced by a mycoplasma that infects rodents are in progress. Origuchi and co-workers have also shown increased IgM against SEB in RA patients (97). We also found antibodies to SEB, but not SEA, to be elevated in RA as compared to controls. This is of interest in view of the similar Vp usage by SEB and MAM noted above. Anti-SE antibodies have also been found to be present in significant quantities in pooled human IgG, showing that humans are exposed to SE on a recurrent basis Further, these antibodies are able to inhibit in vitro activation of T cells by SE SAgs. V.

MAM appears to be phylogenetically unrelated to other SAgs on the basis of primary amino acid sequence similarity. Thus, MAM may represent a distinct class of SAg. Despite the apparent lack of sequence similarity, however, we suggest that short regions of sequence homology may predict functional domains that are common to several SAgs. The domains so identified require confirmation of crossreactive formation. Present work is aimed at solving the X-ray crystal structure of MAM, which, together with our mutation analyses, will enable us to determine whether the ternary structure of and putative functional domains in MAM and other SAgs are truly similar.

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MAM and other SAgs have been shown to effect many of the cells implicated in the pathogenesis of human RA. They are capable of inducing antibody and even autoantibody formation and can stimulate a cytokine profile consistent with that observed in RA joints. In addition, they have been shown capable of triggering autoimmune arthritis inanimal models and of causing disease flares in those models in a SAg-specific fashion. Considering that humans are repeatedly exposed to multiple SAgs, one cannot help but think they may play a role in triggering disease in genetically susceptible individuals. Further, we suggest parallels between human autoimmune disease and MAM effects on human immune cells, which together support the possibility of MAM-like SAgs in human RA pathophysiology. Of particular note is the different responses human lymphocytes to MAM as compared with the other SAgs. It has been well established that human T cells exhibit a lower ability to proliferate in response to MAM. On the other hand, MAM appears to have a greater ability to induce B-cell-associated responses. Thus,these characteristics place MAM in a unique position for studying how SAgs may contribute to human diseases associatedwith immunostimulation and hypergammaglobulinemia. Moreover,MAM, a mutant derivative thereof, may eventually be useful in treating these diseases. ACKNOWLEDGMENT

The authors’ work was supported by Grants A112103 and AR02255 from the National Institutes of Health and by a grant from the Nora Eccles Treadwell Foundation. REFERENCES 1. Cole BC, Kartchner DR, Wells DJ. Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. VII. Responsiveness is associated with expression of a product(s1 of the V08 gene family present onthe T cell receptor a@ forantigen. J Immunol 1989; 142:4131-4137. Razin S. Mycoplasma taxonomy and ecology. In: Maniloff J, McElhaney RN, Finch LR, Baseman JB, eds. Mycoplasmas: Molecular Biology and Pathogenesis. Washington, DC: American Society for Microbiology, 1992:3-22. 3. Samuelsson T, Boren T. Evolution of macromolecule synthesis. In: Maniloff J, McElhaney RN, Finch LR, Baseman JB, eds. Mycoplasmas:


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16 Characterization of a Superantigen Produced by Yersinia pseudotuberedosis Jun Abe and Tae Takeda

INTRODUCTION

Yersiniu species are Gram-negative coccobacilli consisting of three pathogenic species, Y. pestis, Y. pseudotuberculosis, X enterocoliticu, and five nonpathogenic species, X kristensenii, Y. frederiksenii, X intemzediu, Y. rohdei, and Y. uldovue. The former three species are primary pathogens of wild and domestic animals and birds. In the human, Y. pestis causes plague, or black death, while Y. pseudotuberculosis and Y. enterocoliticu produce milder forms of disease varying from diarrhea and abdominal pain to more systemic symptoms such as fever, scarlatiniform skin rash, conjunctivitis, erythema nodosum, and lymphadenopathy (1-3). Complications of reactive arthritis, acute uveitis, coronary aneurysms, and acute renal failure are not infrequently reported after the latter two Yersiniu infections (4-8). The mechanisms by which these organisms mediate these complicated symptoms are poorly understood. However, the preferential avidity for lymphoid tissues seen in these species and the characteristic histopathological finding of lymphoid hyperplasia mainly seen in mesenteric 369

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lymph nodes (9-10) suggest that the stimulation of a large proportion of T lymphocytes may be involved in the pathogenesis of this infection. Although the three pathogenic Yersiniu species invade the human body through different routes and causes diseases of different organs, they share many virulence factorsthat enable them to survive under the bactericidal environment of human serum and phagocytes (11-13). Most of the genes of these factors are encoded on a 70- to 75kb virulence plasmid commonly present amongthe three species (1115). In addition to these virulence factors, Y. enterocolificu and Y. pseudotuberculosis have been reported to produce superantigens (1618). So far, the identification of the superantigen gene is completed only on the chromosome of X pseudotuberculosis (YPM, Y. pseudotuberculosis-derived mitogen) (19,201. This chapter will focus on pseudotuberculosis infection and discuss the pathogenic role of YPM as well as other virulence factors in the disease process. II. VIRULENCEFACTORS A.

.

Y. PSEUDOTUBERCULOSIS INFECTION

Mode of Infection

Y. pestis enters directly into human blood vessels through flea bites. The bacteria colonizethe regional lymph node, where they resist the bactericidalactivity of macrophages and proliferate.The inflammatory response to this process causes the marked swelling of lymph nodes known as bubo. The infection progresses rapidly and systemic dissemination often leads to death. On the other hand, infection by Y. pseudotuberculosis and X enterocoliticu follows a slower course and affects more localized organs. In most cases the organisms are transmitted from infected animals and birds to humans by ingestion of contaminated food water. After entrance to the gastrointestinal tract, they adhere to the ileal mucosa and pass through M cells to the submucosal tissue of the intestine (9,211. In Peyer’s patches and mesenteric lymph nodes, the organisms can proliferate resisting phagocytic cells, similar to X pestis (22,23). The infectionis usually selflimiting and dissemination to blood stream (septicemia) is less frequent than in Y. pestis. However, the gastrointestinal symptoms are sometimes severe enough to mimic acute appendicitis. In such cases, ulcerative ileitis and mesenteric lymphadenitis are the major histopathological findings (24-26). While most of the Y. pseudotuberculosis strains isolated from humans can cause disease, isolates of Y. enterocolitica consist of both

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pathogenic and nonpathogenic strains. The pathogenic strains are characterized by the presence of a 75-kb Lcr (low calcium response) plasmid (27-29); The similar sizes of plasmids are also shared by most strains of the Y. pestis and Y. pseudotuberculosis and it has been shown that the strain deleted of this Lcr plasmid inevitably loses its virulence to mice (11,14,30). The Lcr plasmid encodes a number of genes for the excretory proteins, which act in favor of the organism to enhance adherence,toresistphagocytosis andcomplementmediated lysis (Table Y. pestis, but not Y. pseudotuberculosis or Y. enterocoliticu, has two additional virulence plasmids, 110-kb Tox (exotoxin) plasmid and 10-kb Pst (pesticin) plasmid, which may contribute to the mortality and greater invasiveness of the infection by this organism (31,32). Furthermore, there are other chromosomally located genes that may produce virulence factors amongeither pathogenic or nonpathogenic Yersiniu strains (Table 1). B.

Factors That Mediate Adherence

Four kinds of adhesion moleculesthat have been identified in Yersiniu species. Invasin, Ail, and PsaA are produced by the chromosomal genes and YadA (previously called YopA Yopl) originates fromLcr plasmid (33,34). The gene for Inv was first cloned by Isberg and Falkow from Y. pseudotuberculosis chromosome (35). Later, the homologous genes were found not only in Y. enterocoliticu and Y. pestis but enteropathogenic Escherichia coli as well (36,37). Ailgene is found only in enterocoliticu and PsaA gene is found in Y. pestis and Y. pseudotuberculosis but not in Y. enterocoliticu (38-40). Although Y. pestis has an inv and a yudA gene, both genes are nonfunctional because of the mutations (41). Thus, Y. pseudotuberculosis shares Invasin and YadA with Y. enterocoliticu, and PsaA with pestis. Invasin and YadA bind to integrins of the target cells (42,431. Because phagocytic cells are rich in integrins and absorptive apical mucosal cells usually lack p1 integrins, Y. pseudotuberculosis is thought to bind mainly to mucosal M cells followed by internalization and transportation through M cells to submucosal lymphoid tissues and Peyer’s patches (13,44,45). Within the lymphoid tissues, adherence of the organism to host cells also plays an important role because a close contact of the organism to macrophage is necessary to induce its antiphagocytic activity (46). In this regard, it is of interest that in an inv mutant strain of Y. pseudotuberculosis, although a lethal dose (LD,) was not altered in mice after oral challenge, the time to death was almost twice as long for the mutant (41).

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However, a contradictory result was also reported by the same authors. Using an inv and yudA double-mutated Y. pseudotuberculosis strain in the same protocol, they found that LD, was reduced by 1000-fold and 100-fold after intraperitoneal and oral challenge, respectively Moreover, the reduction of virulence was observed in a Y. pestis strain after reconstitution of yudA gene It seems that there is much to be learned about how invasin and YadA work in vivo, independently coordinately, on the other immune cells as well as macrophages and intestinal mucosal cells. PsaA is a pilus adhesin found in Y. pseudotuberculosis and Y. pestis. Because this molecule is produced only under acidic conditions (pH < it is speculated that this protein may work in the phagosome after the organism i s ingested by phagocytes C.

Plasmid-DerivedVirulence Factors, Yops

Most of the secretory virulence factorsof Yersiniu species are encoded on a to Lcr plasmid. They are called Yops (Yersiniu outer membrane proteins) and the expression of these proteins is coordinately regulated by environmental factors, temperature, and Ca2+ concentration Of these Yops, YopE and YopH are the most well-characterized virulence factors. YopE mediates cytotoxicity against cultured epithelial cells and macrophages When YopE is translocated into target cells, the epithelial cells round up and detach from the culture flask and die. This effect is accompanied by the destruction of actin monofilaments of the target cells YopH is a tyrosine phosphatase and also has an antiphagocytic effect It was shown that YopH, after introduced into macrophage, dephosphorylated a few specific macrophage proteins that had been activated in vivo Thus, the antiphagocytic activity of YopH may be related to interference the host cell signal transduction Similarly, YpkA has serinehhreonine protein kinase activity. Although this protein is indispensable in mice infection models, it has not been determined whether YpkA is translocated into target cells, whether it has the similar antiphagocytic activity as YopH There are several other Yops that confer virulencein Y. pseudotuberculosis. YopM is homologous to platelet glycoprotein GPIb and prevents platelet aggregation by competitive binding to a-thrombin It may contribute to virulence by enhancing the spread of the organism through blood vessels preventing the release of proinflammatory chemokines from the aggregated platelets. YadA works

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not only as adhesion but also as a serum resistance factor. It binds to factor H of the complement system and enhances C3b degradation by factor I (60). The functions ofYopK and LcrV are not known. A YopK mutant strain of Y. pseudotuberculosis colonized and persisted in Peyer's patches after oral challenge but failed to spread systemically (61). LcrV has been shown to be required for a regulated expression ofYops (62). It also provokes protective antibodies in the host (63). D.

Factors that Regulate Production and Secretion of Yops

In Y. pseudotuberculosis as well as in other pathogenic Yersinia species, the production and secretion ofYops are regulated by a number of proteins. The genes for these proteins are encoded on the same Lcr plasmid as Yops and coordinately regulated by two extracellular factors, temperature and calcium concentration, from whichthe term Icr (low calcium response) operons derived (64,65). The key regulatory proteins are listed in Table 2. A simplified scheme for the positive andthe negative regulatory signal pathways for yops and ysc (Yersinia secretion) genes is illustrated in Fig. 1. The positive pathway is regulated by LcrF (previously called VirF) and temperature (66,67). At 37OC in the absence of Ca2+, LcrF is maximally expressed. LcrF binds to yop promoters and activates yops and yscs transcription. The negative pathway is regulated by LcrH and Ca2+ (62,68). LcrHworks as a repressor, and at millimolar concentrations of calcium, it suppresses most yops and Icr expression. When Ca2+is depleted, this repression is released and LcrF-driven production of Yops as well as other ysc proteins follows. Cell surface protein YopN is thought to be involved in this Ca2+-dependentregulatory system because yopN mutants show derepressed yop gene ex-

Table 2 Regulatory Proteins for Production and Secretion of Yops Designation LcrF LcrH YopN (LcrE) for and

Ysc proteins and LcrD Binds YerA

Putative function expression Activates yop and ysc genes Represses expression of yop genes Calcium sensor, derepresses yop expression Forms a type 111 secretion system stabilize YopE

pestis

pdt.

en t.

+

+

+ +

+ + +

+

+

+

+

+

+

+ +


375

Ca

Figure Schematic diagram the regulation Yop expression and secretion. Positive (enhancer) signal is indicated by thick arrows and negative (repressor) signal is shown in thin lines.

pression independent of Ca2+concentration (69). Recently, Rosqvist et al. proposed that YopN might be also involved in the recognition mechanism of bacterial attachment to the target cell (70). After the organism adheres to macrophage in vivo, YopN might send a signal that depresses the lcrH gene expression, thus promoting Yops production even in the presence of Ca2+. The other .Zcr operon genes such as lcrD and encode a protein complex for secretion machinery Yops (71-73). In addition, YerA works as a YopE-specific chaperone for binding and stabilizing YopE in the bacterial cytoplasm (74,751. This protein secretion system of Yersinia species has been classified as type 111secretion system and has several specific features. Yops secretion does not require the cleavage of a typical signal sequenceor the recognition of a carboxylterminal domain (76). Similar secretion systems have been described among the other enteropathogenic bacteria suchas Shigella flaneri and Salmonella typhirnuriurn, and the homologies between the component proteins have recently been identified (77,781. Although the antiphagocytic function of Yops is characteristic of Yersinia species, these

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shared secretion mechanisms suggest that the other pathogenic bacteria might also include some virulence factors to host cells viathese pathways. E.

Superantigens

In the previous sections, we have reviewed the virulence factors that confer resistance againstthe host's innate immune system. Because Y. pseudotuberculosis infection is widely seen in a variety of animals and birds, it may not besurprising if the organism has adopted the means to communicate with the host's specific immune system, T cells andB cells. In fact, one of the candidate factors, in Y , enterocolitica and Y. pseudotuberculosis appears to be a superantigen (16-18). The superantigenic activity in Y , enterocolitica was detected in culture supernatants as well as membrane and cytoplasmic fractions of the organism It stimulates BALB/c T cells bearing Vp3, 6, 11, and mouse T-cell hybridomas selected from a panel of hybridomas, which express VP3, 7,8.1,9, and 11. Through partial purification procedures, it is suggested that the molecule is heat-stable and membrane-associated. Although it seems that the characteristics of T-cell stimulation, the TCR VP-specific expansion and requirement for MHC class 11-positive APC, are consistent with the properties of superantigens, further purification the substance has not been reported. Thus, it may be too early to conclude that Y. enterocolitica produces superantigens. On the other hand, characterization of the Y. pseudotuberculosisderived superantigen, YPM, has recently been done (19,20,79,80). It is a secretory protein with a 20-amino-acid signal sequence. The mature protein has mw of 14,529 and stimulates human T cells bearing TCR-VP 3, 9, and 13.1-2, the pattern which does not resemble those of prototypic superantigens produced by Staphylococcus aureus or Streptococcus pyogenes. The ypm gene is supposed to be on the bacterial chromosome, and no homologous gene, so far, has been found using EMBL-GDB data base. Thus, YPM seems to be a distinct superantigen. We will discuss other details about YPM in the remainder of this chapter. CLONING AND BIOCHEMICAL CHARACTERIZATION OF YPM A.

Purification and Biochemical Characterization of YPM

The superantigenic activity of Y pseudotuberculosis was first reported by two independent researchers either in the culture supernatants or


377

in the ultrasonicated bacterial cell lysates (17,18). Both groups used clinical strains isolated from patients in Japan andserotyped 4b. Although the source of the superantigenic molecules and the technique for the purification were different, they stimulated human PBMC at ng/ml order of the concentration and had the same TCR-VP preference of stimulation, i.e., Vp 3, 9, 13.1, and 13.2. Later, nucleotide sequence analysis revealed that these molecules originate from the identical gene, ypm (19,20). In this section, we briefly describe a method to purify YPM from the culture supernatants of the organism (79). The other method for purifying cellular YPM is described in Refs. 18 and 80. A clinical strain of Y. pseudotuberculosis belonging to the serotype 0:4b was cultured at 25°C for 5 days and culture supernatants were collected, followed by precipitation with 80% ammonium sulfate. The resulting pellets were resuspended, dialyzed, and then subjected to gel filtration on a Sephacryl S-200 Superfine column. The mitogenic fractions to human PBMC were concentrated and further purified by high-performance ion-exchange chromatography on a TSKgel DEAE5PW column. The mitogenic fraction (Fig. 2A) was then subjected to reversed-phase high-performance liquid chromatography (RP-HPLC) on a Develosil300C4-HG-5 column to give a final purified YPM (Fig. 2B, C). Proliferative response of human PBMC to purified YPM was detectable even at a concentration of 1 pg/ml (Fig. 3). This value is comparable to that of the known bacterial superantigens. To calculate the molecular weight of the YPM, electrospray ionization mass spectrometry (ESI-MS) was carried out using a JEOL JSM-HX/HX llOA four-sector tandem mass spectrometer (Tokyo, Japan) equipped with an ESI ion source (Analytica of Branford, Branford, CT). In the ESI mass spectrum of the purified YPM, four intense signals were observed (Fig. 4), from which the molecular weight of the YPM was calculated to be 14.524.4 f 0.3. Rabbit antiserum against YPM was prepared by repeated immunizations (3 times at 4 week-intervals) with 20 mg of the purified YPM at a time. Antiserum obtained 10 days after the last booster was tested for neutralizing activity against the purified YPM pg/ml). Rabbit antiserum could completely neutralize the YPM-induced proliferation, supporting that the purified YPM is highly homogeneous (Fig. 5). The N-terminal23-aminoacid residues were then determined by an automated Edman degradation of the purified YPM to be Thr-Asp-Tyr-Asp-Asn-Thr-Leu-AsnSer-Ile-Pro-Ser-Leu-Arg-Ile-Pro-Asn-Ile-Ala-Thr-Tyr-~r-Gly-.While the amino acid sequences of various superantigens produced by

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*

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h

I

%

m U)

2

.-+> m

U J

I

I

L

Retention l i m e (min)

Figure 2 Purification of YPM. (A) HPIECof thecrude YPM obtained by Sephacryl superfine gel filtration. (B) RP-HPLC of the mitogenic peak fraction indicated by an asterisk in (A). (C) Rechromatography of (B). (From Ref. 79.)


379

t

Figure 3 Dose-response curve of human PBMC proliferation stimulated by the purified W " . Values are the mean of three determinants f SE. (From Ref.

I

I Mobs.= .-

+l

.-> -m *.'

1800 1600 10001400 1200

2000

2200

m/z

Figure 4 ESI mass spectrum of the purified YPM. From each observed m/z value of four multiply charged ion signals ([M+"H]"+),molecular weight of purified YPM was calculated to be 14524.4 f 0.3. (From Ref. 79.)

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+

Normal Rabbll Serum

YPM

+

AnUBlank

Figure 5 Inhibition of the YPM-induced proliferation by antiserum against purified YPM. Values are the mean of three determinants ? SE. The number in a column denotes the percentageof inhibition, which was calculated as the ratio of the difference between the observed and blank cpm values to that between maximum (obtained in the incubation with only YPM) and blank cpm values. (From Ref. 79.)

Gram-positive cocci have been known (81-931, the N-terminal sequence of the purified YPM was not homologous to any sequence of previously characterized superantigens or any proteins. From these results, it was concluded that YPM is a novel superantigen whose molecular structure is different and the size 50-66% of Gram-positive coccal superantigens. B.

Cloning and Sequencing of ypm Gene

Based on the information about the N-terminal23 aminoacids of the purified YPM, cloning and sequencing of the ypm gene was reported in 1995 (19,20). The DNA sequenceof ypm together with the deduced amino acid sequence is shown in Fig. 6. The ypm has an open reading frame of456 nucleotides encoding a protein of 151 amino acids. A Met codon at position 1-3 in Fig. 6 is considered as the translation start point because a TGA stop codon is located six bases-upstream of this site in the same coding frame. Also a possible Shine-Dalgarno-~' sequence (AGGT) was found seven bases upstream from this Met codon (94). The protein deduced from this open reading frame has a 20-amino-acid stretch rich in hydrophobic residues at its N-terminal portion, after which follow a pair of amino acids, Ala-Thr. Because these amino acids have been considered to be a preferential site

GATGAACTGQTCCTGTTTTATCTGTTGGCTGCGCTTCAACTTTTGCTGACTTACC -142 TACGCTAATGCAACTGAGCCATTATTTCCACMCCAATCCCCCGAGGATGAGTTT -87

TATAAAATTTGAMTTAATCACAAAAllllhACMTAAAGATAGTGTAAATAATACli -32

-35

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-1 1

AATGAoAaTGATTATATTTAT~TGAGTTATG

SD TCA S

TTA CTA ACA L L T F

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TTT CTT ACA TTC T L F

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AAT AAA CTT TT0 18 N K L L -15

GC0 TTG GC0 60 TCT GGA GTA S G V A L A -1 -1

ACT GAT TAT + T D Y

GAT AAT ACA D N T L

CTA AAT TCA CCC ATC TCT CTTCGG N S I P S L R

102 14

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ATA CCC W T ATC GCA ACA I P N I A T GGA G E

GTA V

GGA GGG G G

E

TAT ACT GGT ACT Y T G T

TOT ATT ATA AAT AAA GAG GGC C I I G N K E G TTA L

TAT GCT GTA Y A V L

GCA GAT ATG ACG TTA ATT A D M T L I L GGA TGG G W

ATC GGA AAA I Q G K

144 28

K

ACG T

AGA 186 R 42

TTA CAT TCT H S T N

ACC V

AAT 228 GTA N 56

TTA CTA CGC AAT GGC GTA AAT GGA 270 L R N V G G N 70 ATT GAC AAA CCT CTT I D K P L

312 84

K

TAT GAG GAT TAT TAT ACTTCA GGG CTT AGT TGG ATT TGG Y E D Y Y T S G L S W I W

354 98

K

ATT AAA AAC I K N

AA0

GGA GAG ATA AGA M C GAT G E I K R N D

AAT N S

X T GAA ACA S E T

GAT GCT ACT D A T

GTACAT GAT GAC V H D D

ACG AAA TGT T K C

CCT GTG TGA P V *

AAT

TCT AAT TAT S N Y S

GAA GAT AGT GAC GTA

K

E

D

S

D

V

TCA 396 TTA L 112 TTG 438 L 12 6 481 131

AGTTGTATCCCTCCTTTATTTATTTGAGTAGCACTTTATATTTTGAAGGGAGCCT 542 ATTGTCTGAGCCCCCCCGATCTGATTAGGTTCCATCACAGCATATAGCGGTGTTC 597

CGGTTGCTGGCAACTGACGATATTCAGTTCTTTCATTATCTTACTGGCTCGCCAA 652

Figure 6 Nucleotide sequence of the p m and deduced amino acid sequence of YPM. The deduced amino acids are given below the triplets using,singleletter codes. An asterisk indicates the stop codon. Numbering of nucleotide is in reference to the ATG start codon. The most likely cleavage site for prokaryote signal peptidase is denoted with a vertical arrow and the aminoterminal Thr in the mature protein is referred to the first amino acid. The predetermined sequence of N-terminal 23 amino acids is underlined. The possible promoter (-10 and -35) and Shine-Dalgarno (SD)sequences are indicated by thick underlines. (From Ref. 17., copyright 1995, The American Association of Immunologists.)

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for cleavage by the prokaryote signal peptidase(951, we assumed that the protein is synthesized as a precursor and is subsequently cleaved at this site to produce a 131-amino-acid mature secretory protein. In support of this view, the 23-amino-acid sequence that followed this site completely matched the N-terminus of purified YPM described in the previous section. The calculated molecular weight of the precursor proteinwas 16,679 and that of thematureformwas 14,529,which almost completely matched the molecular weight of purified YPM (14,524) estimated by electrospray ionization mass spectrometry (79). The p1 value of the mature YPM was calculated to be 4.95. Homology search of the nucleotide sequence the ypm gene using the EMBL-GDB data base by themethod of Lipman and Pearson (96) did not reveal any significantly homologous gene. Furthermore, no homologous protein was found by the survey of the amino acid sequence of the YPM using the NBRF-PDB (PIR) data bank. Alignment of the amino acid sequence of mature YPM with that of other known superantigens using the same program did not reveal any particular homologous sequences except fora four-aminoacid stretch between YPM and toxic shock syndrome toxin-l (TSST1). Based on these results, together with the previous finding that the mitogenic activity of YPM was not neutralized by antibodies against the known staphylococcal superantigens, SEA, SEB, SEC2, SED, SEE, and TSST1, we concluded that YPM is a novel superantigen this species. In addition, later experiments suggested that the gene is not located on the virulence plasmid. C. Expression of Recombinant YPM and Characterization of Its Superantigenic Properties

To facilitate the studies of the superantigenic properties

the protein, we constructed the plasmid vector that expresses the mature form of YPM as a fusion protein with MBP (maltose binding protein) at its C-terminus (97). The expressed proteins were treated with a specific proteinase, factor Xa, to be separated from MBP and further purified by gel filtration and RP-HPLC. The original vector (pMALc2, New England Biolabs, Bevery, MA) expresses a fusion protein of MBP and the a-fragment of P-galactosidase, which served as a negative control. The purified recombinant YPM (rYPM) was tested for its mitogenic activity on human PBMC (Fig. 7). The rYPM had strongly mitogenic activity at concentrations as low as 1 pg/ml, while the MBP-a-


-

2

-

1

0

1

2

383

3

4

5

6

Log o Figure 7 Mitogenic activity of the rYPM. Data are expressed as the mean cpm of the triplicate cultures. (From Ref: 17.)

fragment that was obtained in parallel with rYPM had no activity even at higher concentrations (max. ng/ml). The capacity of murine fibroblasts, transfected with various human MHC class 11 molecules, to stimulate proliferation of purified T cells was tested (98100). The results shown in Table indicate that rYPM was able to induce T-cell proliferation in the presence of fibroblasts transfected with four different HLA class I1 molecules, HLA-DPw9, -DQw6, DRI,

Table 3 MHC Class 11-Dependent Presentation ofrYPM by Mouse Fibroblast Transfectantsa [aH]-TdR incorporation (cpm)

rYPM

Medium APC None L cells L-DPw9 L-DQw6 L-DR1 L-DR4Dw15

690 f 15 2,086 ? 126 825 f 219 878 f 224 908 f 414 1931 115

rYPM

3,175 f 444 3,015 231 11,674 1,612 16,768 f 756 11,684 536 32,225 f 2,010

+ Anti-DR 1063 f 45 973 f 44 ND ND 5974 ? 160 259 f 190

.Purified T cells (2 lo5) were cocultured mitomycin C-treated L cells or L cells transfected the class I1 MHC molecule ( 2 X lo4), or medium alone in the presence or absence of rYPM and anti-DR antibodies. All data are expressed as mean 13H]TdR uptake ? SEM (cpm). Source: From Ref. 17, copyright 1995, The American Association Immunologists.

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and -DR4Dw15 (101). Moreover, addition of anti-HLA-DR antibody to the cultures abolished the stimulatory activity of HLA-DR-transfectants. These results confirmthat YPM has MHC class 11-dependent but not restricted mitogenic activity like other bacterial superantigens. In addition, the paraformaldehyde-fixed APC, both HLA-transfected murine fibroblasts and EBV-transformed human B cells, were unable to support the mitogenic activity ofrYPM on human T cells (Table 4), suggesting that antigen processing was not required in the stimulation process by rYPM (102,103). Superantigens are distinguished from conventional antigens by their capacity to interact with T cells predominantly through the variable region of the p chain (Vp) ofTCR, and the other variable elements (Vu, Ja, Dp, and JP) ofTCR contribute very little to the interaction betweensuperantigens and T cells(98,104,105). This property of superantigens was confirmed in rYPM by analyzing the TCR-Vp repertoire of T cells after stimulation with rYPM (105,106). The nonclonotypicnature of the responding T cells after rYPM stimulation was also confirmed by analyzing the sequences of random cDNA clones that encode PCR-amplified TCR p chain junctional fragments of responding T cells (107,108). Figure 8 illustrates the percentages of T cells bearing Vp2, 8.2, and 13.6 within the CD4+and CD8+subsets after stimulation with either rYPM or MBP-a-fragment fusion protein as the negative control. In contrast to MBP-a-fragment fusion protein, rYPM induced a

Table 4 Paraformaldehyde-Fixation IndependentPresentation of rYPM by APCa [aH]-TdR incorporation (cpm) APC

Medium

rYPM

None L-DR4Dw15 (unfixed) L-DR4Dw15 (fixed) EBV-transformed B cells (unfixed) EBV-transformed B cells (fixed)

425 110 ND ND ND ND

1,175 f 181 31,426 f 8,467 10,514 f 1,160 49,381 f 6,709 11,719 f 1,673

OPurified T cells (2 X 105) were cocultured with either medium alone or mitomycinCtreated (unfixed) paraformaldehyde-treated (fixed) L cells transfected with the class I1 MHC molecule or EBV-transformed B cells (2 X 104) in the presence or absence ofrYPM (100 ng/ml). All data are expressed as mean t3H]TdR uptake f SEM (cpm). Source: From Ref. 17, copyright 1995, The American Association of Immunologists.


385

% 124 10

8 6 4

2

' 8.2

V62

I

Vp3'Vg8.1/'Vf313.6 8.2

'

CD 4 Figure 8 TCR-Vj3 expression ofPBMC after stimulation with rYPM. PBMC were stimulated with either rYPM or MBP-a-fragment (control) and the percentage of T cells bearing each VP was studied within CD4+ and CD8+ subsets using immunofluorescence andflow cytometry. All data are expressed as mean % SEM. pvalue was calculated by two-tailed paired t-test. (From Ref. 17.)

significant expansion of Vp3- and Vpl3.6-bearing T cells within both CD4+and CD8+subsets and also caused a relative reduction of Vp2and VP8-bearing T cells. Analysis by semiquantitative PCR with 26 different VP-specific primers confirmed the results obtained by the immunofluorescence analysis. A marked increase in the percentages of Vp3, Vp9, Vp13.1, and Vp13.2 cDNA was observed after stimulation with rYPM as compared with stimulation with MBP-a-fragment fusion protein (Fig. 9). Because the results obtained by semiquantitative PCR suggested that Vp3-bearing T cells were the most dominant repertoire stimulated by rYPM, we analyzed the sequence of the junctional region of the VP3-containing messages prepared from the rYPM-stimulated PBMC.Of the 21 independent cDNA clones bearing PCR-amplified Vp3 junctional fragment, none had an identical sequence and considerable diversity of JP usage was observed (Table 5). From these results, we concluded that rYPM stimulates T cells mainly through the relevant Vps, disregarding their CDR3 regions.

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Figure 9 Autoradiograms ofTCR transcripts amplified by PCR. Intensity of radioactivity each Vp band was quantitated with the radio image analyzer and normalized with the coamplified Ca band. Percentage of each Vp among all Vp families studied was indicated under each lane. (From Ref. 17, copyright 1995, The American Association of Immunologists.)

Thus, rYPM satisfied the generally accepted requirements of superantigenic properties, i.e., powerful mitogenic activity that i s dependent on but not restricted by the MHC class I1 molecules, no. need processing, Vp specificity, and a lack of clonality in responding T cells. Although SEB,SEC& and SEC3 share some of the VP specificity with rYPM the amino acid sequence of the mature YPM showed no significant homology to these superantigens. At present, data are insufficient to determine which domains in YPM interact with TCR and MHC class I1 molecules. Studies for further structural analysis using site-directed mutagenesis are being carried out in our laboratory. EPIDEMIOLOGY OF YPM GENE

We examined the distribution of the ypm gene among several strains of Y. pseudotuberculosis isolated from diverse geographical areas. A total of 204 strains of Y. pseudotuberculosis, which included 142 from Japan, 33 from Far Eastern Russia, 11 from Belgium, 10 from Germany, six from Italy, and two from the Netherlands, were investigated by colony hybridization using a Pagenerated DNA probe. Of the 76 clinical strains investigated, 59 (78%) hybridized with the


387

Table 5 Sequence Analysis of Junctional Regions ofTCR p Chains Containing Vp3 Element in T cells Stimulated with rYPM NDbN -LCASS -LCASS “LCAS -LCASS “LCASS -LCASS -LCASS “LCASS “LCAS “LCASS -LCAS -LCASS -LCASS -LCASS -LCAS -LCASS -LCAS -LCAS -LCASS -LCASS “LCAS Source: From Ref. 17,

LQ LTG ADSV HYAT PTV PSVG KTRDRY DRRTGV ASGSFE LGGGGY IGGRRWD LTG WGQGK ARLAA RN FAG AAGDG RSI LYGAGK PGTGN IRPGT

Jb NTEAFTEAFTEAFNYGYTNYGYTYGYTGNTINEKLEKLSNQPQSNQPQSNQPQQPQSYNEQYNEQNTGELNTGELTDTQETQYYEQYYEQY-

copyright 1995, The American Association of Immunolo-

gists.

probe. The culture supernatant fluids of all the ypm-positive strains were mitogenic against human PBMC, while none of the ypm-negative strains gave mitogenic activity. The complete correlation between the presence of the ypm gene and the mitogenic activity clearly indicates that all the strains harboring ypm gene produce the YPM. During screening of the strains, a geographical difference in the prevalence of the ypm gene among strains of Y. pseudotuberculosis was seen. The ypm gene was detected in all the clinical strains from Far Eastern Russia and in 95% the clinical’strains from Japan, while only 17%of the European clinical strains harbored the gene. A difference in the distribution of serotypes of Y. pseudotuberculosis between Japan and Europe has been reported (109). Most of the strains from Europe belong to the serotype O:la, whereas no strain belonging to this se-

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rotype has been isolated in Japan. By contrast, Y. pseudotuberculosis belonging to nine serotypes (O:lb, 2a, 2b, 2c, 3, 4a, 4b, 5a, and 5b) have been isolated from patients in Japan, of which serotypes 0:4b and 5b are dominant. The two dominant serotypes have never been detected in Europe From Fig. 10A, it is clear that the gene is not associated with serotype O:la, which comprises the majority of the strains from Europe. Therefore, it appears that the geographical pattern in the prevalence of the ypm gene is directly related to spatial distribution of serotypes. However, all the European strains belonging to serotypes O:lb, 2a, and 2b, were negative, while all the strains of the above serotypes fromJapan and Far Eastern Russia were positive. This implies that the geographical association of the gene is not always dependent on the geographical differences in the serotypes. The low prevalence of the ypm gene in Europe appears to be related to the geographical disparity in both serotype and the ypm gene itself. Next, 128 strains of Y. psatdotuberculosis isolated from animal and environmental sources were investigated. The geographical and serotype-related distribution of the ypm gene among the clinical strains was also reflected among the animal and environmental strains (Fig. 10B). The prevalenceof p m gene in strains belonging to the serotypes O:lc, 6, 7, 8, 9, 10, 11, 12, and 13, which are not associated with disease, was as low as 26%. This further reiterates the role ofYPM as a virulence factor of Y. pseudotuberculosis infection. The main clinical manifestationsof Y. pseudotuberculosis infection seen in Europe are fever and gastroenteric symptoms (110). By contrast, the clinical features in Japan (111,112), Far Eastern Russia and Korea (3,110) are usually more diverse and severe. The major differences in clinical symptoms between Far East and Europe are rash and desquamation, which are not seen in European patients but are common in the Far East. Moreover, approximately 10% of the infected children fulfill diagnostic criteria of Kawasaki syndrome including the coronary vasculitis (7). Acute renal failure, usually tubulointerstitial nephritis, is also seen in these patients (114). It appears that the difference in clinical manifestations of Y. pseudotuberculosis infection between FarEast and Europe is related to the heterogeneity in the distribution of the ypm gene. A total of 225 strains of Y. enterocolitica, mainly from Japan, were negative for the ypm gene, suggesting that YPM i s relevant to the clinical characteristic of Y. pseudotuberculosis infection. The viru-


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Figure 10 Prevalence the ypm gene among clinical(A) and environmental (B) strains Y. pseudotuberculosis in relation to serotype and area isolation. J, R, and E denote Japan, Far Eastern Russia, and Europe, respectively. Number strains in each area is shown by two columns. The left and right columns represent, respectively.

lence of Yersiniu, such as invasiveness, antiphagocytosis, and antiserum killing, is equally characterized for Y. enterocolitica and Y. pseudotuberculosis. However, these mechanisms cannot explain the severity of the Y. pseudotuberculosis infection in the Far East.

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V.

IMPLICATION OF YPM IN DISEASES ASSOCIATED WITH PSEuDOTUBERCULOSlS INFECTION

A.Systemic

Manifestations in the Acute Phase of Illness

The infection of Y. pseudotuberculosis is accompanied by multiple systemic symptoms as well as gastrointestinal tract symptoms. According to the study of pediatric patients in Japan (115), fever and polymorphous skin rash were observed in more than 80% of the affected patients and the incidence of cervical lymphadenopathy and injection of mucosal tissue such as conjunctiva and oropharynx was 10% and 40%, respectively. The actual mechanism by which Y. pseudotubercuIosis mediate these symptoms is currently unknown. Many factors that confer virulence in this species have been identified (Table 1). However, most of them are involved in the organism’s invasiveness and defense against the host’s epithelial barrier and phagocytosis. For example, YopE has been shown to mediate cytotoxicity on the host’s cells but its activity was strictly dependent on the bacterial attachment to the outer surface of the target cells. The secreted YopE within the bacterial culture supernatants had no effect on target cells(46). Therefore, it is not likely that these Yop proteins directly mediate cytotoxicity beyond the intestinal lymphoid tissues and Peyer’s patches where Y. pseudotuberculosis shows special avidity. Based on these observations, it is conceivable that YPM might take part in the manifestation of systemic illnesses by activating a large proportion of T cells and inducing an excessive amount of inflammatory cytokines, as has been suggested in toxic shock syndrome (TSS) and streptococcal TSS (116-119 In staphylococcal toxic shock syndrome, the experimental mice model has been established using either the constant subcutaneous infusion ofTSST-1 (120) or presensitization of mice with D-galactosamine (121). Miethke et al. (118,122) showed, using thepresensitization model, that TNF-a and TNF-P secreted from the TSSTI-stimulated T cells played an essential role in inducing shock. Cyclosporin A was able to prevent the onset of shock. The importance the T cells and T-cell-derived cytokines in TSS was also proved by the adoptive transfer experiment in which scid mice reconstituted with syngeneic T cells became susceptible to TSST-l-mediated shock (122). As described in Chapter 18, in human patients, the involvement of TSST-1 in disease pathogenesis has been suggested by the findings that the TSST-l-responsive VP2-bearing T cells were expanded in the patient’s peripheral circulation (123).

\


39 1

On the other hand, there has been little information about the involvement ofYPM in Y. pseudotubercubsis infections. In our preliminary experiments, anti-YPM IgG antibodies developed in more than half of acute-phase patients with Y. pseudotuberculosis infection. The antibody titer was higher in the patients with systemic symptoms than in the patients with fewer systemic symptoms (unpublished data). Although the higher anti-YPM titer does not necessarily mean a higher production ofYPM in the host, it implies, at least, that the host's immune cells reacted to YPM more vigorously in the patients with systemic illnesses. The alteration ofTCR-VP usage in vivo has also been observed in a limited number of patients studied in our laboratory. In five of 11patients studied, the percentage of VP3-bearing T cells, the major YPM-responsive VP family of T cells, was increased (manuscript in preparation). Moreover, in one patient who underwent a laparotomy becauseof severe gastrointestinalsymptoms, a hypertrophic mesenteric lymph node contained about times as high percentage of VP3-bearing T cells as PBMC (124). These data strongly argue the pathogenic role ofYPM in the infection of Y. pseudotuberculosis. In the context of the pathogenesis superantigens, it has been stressed that the activation of T cells by superantigens favors the overproduction of proinflammatory cytokines such as TNF-a, TNFp, IL-1, IFNy, and IL-6, which contribute to endothelial cell and gan damage (118,119). However, there have been reports that focused on the protective role of T cells in yersiniosis (125-127). In the experimental mice model of Y. enterocolitica infection, Autenrieth et a1 (125) reported that T cells could mediate protection in resistant C57BL/6 mice but not in susceptible BALB/c mice. Neutralization ofIFN-y abrogated resistanceto the bacteria in C57BL/6 mice. Also, it has been shown that some of the secreted Yop proteins could provoke the host's CTL against the Yop-derived peptide in an MHC class I-restricted manner (128). Currently, it is not clear whether the superantigens work'in concert with, or against, the host's T-cell-mediated protective immunity. In this regard, it is of interest to study how and to what degree YPM influences the cytokine production pattern and to understand the destiny of T cells stimulated with YPM in the patients, i.e., to undergo expansion driven by (auto)antigens or to undergo programmed cell death or anergy.

B.

late Sequelae in Y. pseudotuberculosis Infection

A variety of diseases have been reported as later sequelae of Y. pseudotuberculosis infection. These include reactive arthritis, Reiter's syn-

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drome, ankylosing spondylitis, acute uveitis, liver abscess, chronic pancreatitis, Crohn's disease, acute renal failure due to acute interstitial nephritis, and Kawasaki syndrome (KS) (4-8,111,114,129-134). It may be appropriate to include KS here because it is an acute febrile illness of unknown etiology that primarily affects children younger than 5 years of age (135-136). It has clinical features resembling toxic shock syndrome or scarlet fever and acute Y. pseudotuberculosis infection (111). The characteristic complication of coronary aneurysms in KS usually develop in the second week after onset of the illness and the process seems to be the same in Y. pseudotuberculosis infection (7). It has long been debated whether Y. pseudotuberculosis could be counted as one of the etiological agents of KS (137,138), since most KS patients lacked elevation of Yersinia-specific hemagglutinin antibodies. However, Konishi et al. (139) reported that 55 of 208 KS patients had positive culture and/or elevated hemagglutinin titer against Y. pseudotuberculosis, and 21 of the seropositive KS patients (38%) had dilatation or aneurysms of the coronary arteries. It seems, at least in the epide'mic area, that infection by this organism should be considered one of the possible etiological agents. Recently, Leung et al. (140) suggested that TSST-1-producing S. aureus and SPEC-producing streptococci might be causally involved in the pathogenesis of KS. It is, therefore, of particular interest whether YPM, acting as a superantigen, may have some pathogenic role in the formation of coronary aneurysms as well as multiple systemic illnesses seen in this disease (141). Ankylosing spondylitis, Reiter's syndrome, and reactive arthritis share several clinical features that suggest autoimmune pathogenesis. They include a chronic aseptic arthritis that develops several weeks after the gastrointestinal or urogenital infection (4,5). Y. enterocolitica and Y. pseudotu berculosis, as well as Salmonella, Shigella, Campylobacter, and Chlamydia species, have been identified as causative bacteria (142). Patients with these diseases have strong association with HLA-B27 (129,130). Extensive studies on the cross-reactivity of the monoclonal antibodies against HLA-B27 revealed the presence of shared epitopes between the B27 molecule and bacterial components (143-145). However, evidence that suggests a cytotoxic effect of such cross-reactive antibodies has not been obtained yet (146-148). In Yevsiizia-induced arthritis, it has been demonstrated that bacterial antigen could be carried over to the local joint and retained within the synovial fluid mononuclear cells and synovial membrane cells in the affected patients (149,150). These findings provoked much


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attention to the function of the local inflammatory T cells and antigen-presenting cells within the affected joint (151-153). far, it has been shown that synovial fluid T cells in the reactive arthritis patients consist of a variety of subsets. i.e, CD4+ or CD8+ aP-TCR+ T cells, and $3-TCR+ T cells, with multiple target epitopes (154-158). It is of interest that there has been marked predominanceof Thl-like (inflammatory) T cells compared with Th2-like (antigen-producing) T cells within the CD4 subset (155). It was also demonstrated that synovial fluid-derived CD8 T cells in Yersinia arthritis patients could mediate cytotoxicity on autologous HLA-B27-positive EB blast cells in the presence or absence of the bacteria (158). At present, it is not clear whether these T cells cause organ damage in response to bacterial antigen versus autoantigens. Inan experimental arthritis model of rats after infection with Y. enterocolitica, it has been shown that Lewis rats were especially susceptible to arthritis (159). This strain of rats harbored 10-fold more bacteria that persisted a longer period in the spleen and liver after inoculation of Y. enterocolitia than the arthritisresistant strains, suggesting the importance of the amount and the route of the bacterial antigen load in triggering arthritis. Thus, it is conceivable that YPM may have a special role in the pathogenesis of Y. pseudotuberculosis-induced arthritis by enhancing the systemic inflammatory response of macrophages and T cells as well as stimulating local resident T cells that may mediate direct tissue destruction. VI.

CONCLUSIONS

In the past, many virulence factors have been identified as Yersinia species. Most of them are the plasmid-encoded excretory proteins, which act against the host’s immune system by enhancing adherence and resisting phagocytosis and complement-mediatedlysis. Recently found superantigen produced by Y. pseudotuberculosis, YPM, is unique in that the primary target of the protein is the host’s specificimmune system, T cells. In the infection by Y. pseudotuberculosis, YPM may be involved not only in the pathogenesis of acute systemic illnesses but also in the autoimmune sequelae such as KS, reactive arthritis, Crohn’s disease, and acute interstitial nephritis. One of the. important points is the close similarity in the acute-phase symptoms between infection with this organism and with aureus and pyogenes, both of which are well-known producers of the superantigens. The other point is the finding in the epidemiological studies that the heterogeneity in the distribution ofYPM producing strains may account for

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the difference in the severity of clinical manifestations between patients from Europe and the Far East. However, it seems that there is much to be learned about the manner in which YPM is involved in the pathogenic immune cell responses. Whether YPM can influence the Thl-Th2 homeostasis maintained in the local tissues by inducing inflammatory cytokines, orwhether YPM can enhance local cytotoxic T-cell activity,or whether YPM can enhance the development of autoreactive T cells remains to be answered. Future studies directed to these questions will help to explain the possible autoimmune mechanism of the Yersinia-related diseases. In addition, more studies on the molecular structure and function ofYPM, along with the studies of the other prototypic superantigens, will provide a basis to understand how superantigens engage MHC class I1 molecules and the particular Vp repertoire of T cells. This information will be important in directing new investigations into the treatment of infections caused by Y. pseudotuberculosis. ACKNOWLEDGMENT

We wish to thank colleagues in the Departments of Immunology and Infectious Diseases Research, Drs. Yasuhiko Ito, Ken-ichi Yoshino, Takao Kohsaka, and G. Balakrish Nair, with whom the original studies on YPM were done. Preparation of this chapter was supported in part by a grant from the Japan Health Sciences Foundation and the Japan Intractable Disease Research Foundation. REFERENCES 1. Knapp W. Mesenteric adenitis due to Pasteurella pseudotuberculosis

in young people. N Engl J Med Winblad Nillehn B, Sternby NH. Yersinia enterocolifica (Pasteurella X) in human enteric infections. Br Med J Krober MS, Bass JW, Barcia PJ. Scarlatiniform rash and pleural effusion in a patient with Yersinia pseudotuberculosis infection. J Pediatr Leino R, Makela AL, Tiilikainen A, et al. Yersinia athritis in children. Scand J Rheumatol 1980; Tertti R, Granfors K, Lehtonen OP, et al. An outbreak of Yersinia pseudotuberculosis infection. J Infect Dis Saari KM, Maki M, Paivonsalo T, et al. Acute anterior uveitis and conjunctivitis following Yersinia infection in children. Int Ophthalmol


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7. Baba K, Takeda N, Tanaka M. Cases of Yersiniu pseudotuberculosis infection havingdiagnosticcriteria of Kawasaki disease.Contrib Microbiol Immunol 1991; 12:292-296. Davenport A, O'Connor B, Finn R. Acute renal failure following Yersiniu pseudotuberculosis septicaemia. Postgrad Med J 1987; 63915-6. 9. Une T. Studies on the pathogenicity of Yersinia enterocoliticu. 111. Comparative studies between Y. enterocoloticu and Y. pseudotuberculosis. Microbiol Immunol 1977; 21:505-516. 10. El MN, Mair NS. The histopathology of enteric infection with Yersiniu pseudotuberculosis. Am J Clin Pathol 1979; 71:631-639. 11. Cornelis G, Laroche Y, Ballingand G, et al. Yersinia enterocoliticu, a primary model for bacterial invasiveness. Rev Infec Dis 1987; 9:64-87. 12. Brubaker RR. Factors promoting acute and chronic diseases caused by yersiniae. Clin Microbiol Rev 1991; 4:309-324. 13. Salyers AA, Whitt DD. Yersiniu infection. In: Salyers AA, Whitt DD, eds. Bacterial pathogenesis, a molecular approach, Washington, DC: ASM Press, 1994:213-228. outer mem14. Bolin, I, Norlander L, Wolf WH. Temperature-inducible brane protein of Yersiniu pseudotuberculosis and Yersiniu enterocolitica is associated with the virulence plasmid. Infect Immun 1982; 37:506-512. et al. Characterization of common 15. Portnoy DA, Wolf WH, Bolin I, virulence plasmids in Yersiniu species and their role in the expression of outer membrane proteins. Infect Immun 1984;43:108-114. PM, Woodward JG.Yersiniu enterocoliticu produces superantigenic 16. activity. J Immunol 1992; 148:225-233. 17. Abe J, Takeda Watanabe Y, et al. Evidence for superantigen production by Yersiniu pseudotuberculosis. J Immunol 1993;151:4183-4188. 18. Uchiyama T, Miyoshi AT, Kat0 H, et al. Superantigenic properties of a novel mitogenic substance produced by Yersiniu pseudotuberculosis isolated from patients manifesting acute and systemic symptoms. J Immunol 1993;151:4407-4413. 19. Ito Y, Abe Yoshino K, et al. Sequence analysis of the gene for a novel superantigen produced by Yersiniu pseudotuberculosisand expression of the recombinant protein. J Immunol 1995; 154:5896-5906. 20. MiyoshiAT,Abe A, Kat0 H, et al. DNA sequencing of the gene encoding a bacterial superantigen, Yersiniu pseudotuberculosis-derived mitogen (YPM), and characterizationof the gene product, cloned YPM. J Immunol 1995; 154:5228-5234. 21. Simonet M, Richard S, Berche P. Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudotuberculosis harboring the pYV plasmid. Infect Immun 1990; 58:841-845. 22. Tertti R, Eerola E, Lehtonen OP, et al. Virulence-plasmid is associated with the inhibition of opsonization in Yersinia enterocoliticu and Yersiniu pseudotuberculosis. Clin Exp Immunol 1987; 68:266-274. 23, Rosqvist R, Bolin Wolf WH. Inhibition of phagocytosis in Yersiniu

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pseudotuberculosis: a virulence plasmid-encoded ability involving the YopZb protein. Infect Immun 1988; 562139-2143. Weber J, Finlayson NB, Mark JB. Mesenteric lymphadenitis andterminal ileitis due to Yersinia pseudotuberculosis. N Engl J Med 1970; 283:172174. Saari TN, Triplett DA. Yersinia pseudotuberculosis mesenteric adenitis. J Pediatr 1974; 85:656-659. Paterson IC, Grant IW., Crompton GK. Terminal ileitis due to Yersinia pseudotuberculosis. Br Med J 1976; 2:6041. Zink DL, Feeley JC, Wells JG, et al. Plasmid-mediated tissue invasiveness in Yersinia enterocolitica. Nature 1980; 283:224-226. Gemski P, Lazere JR, Casey T. Plasmid associated with pathogenicity and calcium dependency of Yersinia enterocolitica. Infect Immun 1980; 27:682-685. Portnoy, D. A., Moseley, L., Falkow, S. Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis. Infect Immun 1981; 31:775-782. Gemski P, Lazere JR, Casey T, et al. Presence of a virulence-associated plasmid in Yersinia pseudotuberculosis.Infect Immun 1980; 28:1044-1047. Ferver DM, Brubaker RR. Plasmids in Yersiniapestis. Infect Immun 1981;31:839-841. Sodeinde OA, Sample AK, Brubaker RR, et al. Plasminogen activator/ coagulase gene of Yersinia pestis is responsible for degradation of plasmid-encoded outer membrane proteins. Infect Immun 1988; 56:27492752. Bolin I, Wolf W. Molecular cloningof the temperature-inducibleouter membrane protein 1of Yersinia pseudotuberculosis. Infect Immun 1984; 43:72-78. Emody L, Heesemann Wolf WH, et al. Binding to collagen by Yersiniaenterocolitica and Yersinia pseudotuberculosis: evidencefor yopA-mediated and chromosomally encoded mechanisms. J Bacteriol 1989;171:6674-6679. Isberg RR, Falkow S. A singlegenetic locus encodedby Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12. Nature 1985; 317:262-264. Pepe JC, Miller VL. The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin. J Bacteriol 1990; 172:3780-3789. Jerse AE, Yu J, Tall BD, et al. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci USA 1990; 87:78397843. Miller VL. Yersinia invasion genes and their products. ASM News 1992; 58:26-33.


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39. Miller VL, Farmer I11 JJ, Hill WE, et al. The ail locus is found uniquely in Yersiniu enterocoliticu serotypes commonly associated with disease. Infect Immun 1989;57:121-131. 40. Lindler LE, Tall BD. Yersiniu pestis pH 6 antigen forms fimbriae and is induced by intracellular association with macrophages. Mol Microbiol 1993;8:311-324. 41. Rosqvist R, Skurnik M,Wolf WH.Increased virulence of Yersiniu independent mutations. Nature 1988; 334:522pseudotuberculosis by 524. 42. Isberg RR, Leong JM. Multiple beta 1chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells.Cell1990;60:861-871. 43. Tertti R, Skurnik M, Vartio T, et al. Adhesion protein YadA of Yersiniu species mediates binding of bacteria to fibronectin. Infect Immun 1992; 60:3021-3024. Sulrnonellu typhirnuriurn initiates murine 44. Jones BD, Ghori N, Falkow infection by penetrating and destroying the specialized epithelial M cells of the Peyer’s patches. J Exp Med 1994; 180:15-23. 45. Clark MA, Jepson MA, Simmons NL, et al. Preferential interaction of Sulrnonellu typhirnuriurn with mouse Peyer’s patch M cells. Res Microbiol 1994; 145:543-552. 46. Rosqvist R, Forsberg A, Rimpilainen M, et al. The cytotoxic protein YpoEof Yersiniu obstructs the primary host defence. Mol Microbiol 1990;4:657-667. 47. Higuchi K, Smith JL. Studies on the nutrition and physiology of Pusteurellu pestis. VI. A differential plating medium for the estimation of the mutation rate to avirulence. J Bacteriol 1961; 81:605-608. 48. Straley SC, Brubaker RR. Cytoplasmic and membrane proteins of yersiniae cultivated under conditions simulating mammalian intracellular environment. Proc Natl Acad Sci USA 1981; 78:1224-1228. 49. Bolin I, Norlander L, Wolf WH. Temperature-inducible outer membrane protein of Yersiniu pseudotuberculosis and Yersiniu enterocoliticu is associated with the virulence plasmid. Infect Immun 1982; 37:506-512. 50. Bolin I, Portnoy DA, Wolf WH. Expression of the temperature-inducible outer membrane proteins of yersiniae. Infect Immun 1985; 48:234240. 51. Rosqvist R, Bolin I, Wolf WH. Inhibition of phagocytosis in Yersiniu pseudotuberculosis: a virulence plasmid-encoded ability involving the Yop2b protein. Infect Immun 1988; 56:2139-2143. 52. Rosqvist R, Forsberg A, Wolf WH. Intracellular targeting of the Yersiniu YopE cytotoxin in mammalian cells induces actin microfilament disruption. Infect Immun 1991;59:4562-4569. 53. Guan K, Dixon JE. Protein tyrosine phosphatase activity of an essential virulence determinant in Yersiniu. Science 1990; 249:553-556. 54. Bliska JB, Clemens JC, Dixon JE, et al. The Yersinia tyrosine phos-

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phatase: specificity of a bacterial virulence determinant for phosphoproteins in the J774A.1 macrophage. J Exp Med 1992;176:1625-1630, 55. Bliska JB, Guan KL, Dixon JE, et al. Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci USA 1991;88:1187-1191. 56. Bliska JB, BlackDS. Inhibition of the Fc receptor-mediated oxidative burst in macrophages by the Yersinia pseudotuberculosis tyrosine phosphatase. Infect Immun 1995; 63:681-685. 57. Fallman M, Anderson K, Hakansson S, et al. Yersiniupseudotuberculosis inhibits Fc receptor-mediated phagocytosis in J774 cells. Infect Immun 1995;63:3117-3124. 58. Galyov EE, Hakansson S, Forsberg A, et al. A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature 1993;361:730-732. 59. Leung KY, Straley SC. The yopM gene of Yersiniu pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha. J Bacteriol 1989; 171:4623-4632. 60. China B, Sory MP, N’Guyen BT, et al. Role of the YadA protein in prevention of opsonization of Yersinia enterocolitica by C3b molecules. Infect Immun 1991;61:3129-3136. 61. Holmstrom A, Rosqvist R, W, et al. Virulence plasmid-encoded YopK is essential for Yersiniu pseudotuberculosis to cause systemic infection in mice. Infect Immun 1995; 632269-2276. 62. Bergman T, Hakansson S, Forsberg A, et al. Analysis of the V antigen 1crGVH-yopBD operon of Yersinia pseudotuberculosis: evidence for a regulatory role of LcrH and LcrV. J Bacteriol 1991; 173:1607-1616. 63. Motin VL, Nakajima R, Smirnov GB, et al. Passive immunityto yersiniae mediated by antirecombinant V antigen and protein A-V antigen fusion peptide. Infect Immun 1994; 62:4192-4201. 64. Straley SC, Plano GV, Skrzypek E, et al. Regulation by Ca2+ in the Yersiniu low-Ca2+ response.Mol Microbiol 1993; 8:1005-10. 65. Cornelis GR. Yersinia, finely tuned pathogens. In: Hormaeche CE, Penn CW, Smyth CJ, eds. Molecular Biology of Bacterial Infections: Current Status and Future Perspectives. Cambridge, UK: Cambridge University Press, 1992:231-265. 66. Cornelis GR, Sluiters C, Lambert-de-Rouvroit C, et al. Homology between virF, the transcriptional activator of the Yersinia virulence region, and AraC, the Escherichiacoli arabinoseoperon regulator. J Bacteriol 1989;171:254-262. 67. Lambert-de-Rouvroit. C, Sluiters C, Cornelis GR. Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocoliticu. Mol Microbiol 1992; 6:395-409. 68. Price SB, Straley SC. LcrH, a gene necessary for virulence of Yersiniu pestis and the normal response of Y. pestis to ATP and calcium. Infect Immun 1989;57:1491-1498.


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69. Forsberg A, Viitanen AM, Skurnik M, et al. The surface-locatedYopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis. Mol Microbiol 1991;5:977-986. 70. Rosqvist R, Magnusson KE, Wolf WH. Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J 1994; 13:964-972. 71. Michiels T, Vanooteghem Jc, Lambert-de-Rouvroit C, et al. Analysis of vi 0.1, paired t-test) More recently, we have established that this mod-SPA-induced complement consumption is associated with the appearance of sizable amounts of C3a in treated sera (72). Thus, we established that although SPA activates complement, mod-SPA (SPA abrogated of its IgG Fc-binding activity) retains its ability to activate the complement cascade.

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These results strongly suggested that the Fab-binding site on SPA is critical for complement consumption. directly determine if complement activation was 1) dependent on an interaction between the Fab-binding site on SPA and VH3+ Igs and proceeded via the classical complementpathway, we assessed the ability of human polyclonal IgM and a panel of monoclonal IgM proteins to bind following their interaction with SPA in an ELISA. We observed that polyclonal IgM and only VH3+, SPAreactive IgM proteins bound Clq following their interactionwith SPA (Fig. Therefore, we concluded that binding of the first component of complement and, presumably, the activation of the complement cascade are by-products of the superantigenic properties of SPA. To formally determine if the binding of SPA to VH3+ IgM proteins can lead to complement activation,we utilized serum (reconsti-

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Absorbance @ 450nm Figure 7 Binding of SPA-reactive IgM/SPA to Clq as detected by peroxidase conjugated anti-IgMFc. The binding of IgM/SPA reaction mixtures to Clq was analyzed in an ELISA. Polyclonal IgM, or monoclonal VH3 IgM proteins Pom, 14-3, Vin, 2-3, Riv, and Dau, preincubated with SPA (1 pg/ml), were added to Clq-fixed ELISA plates. Background absorbance wassubtracted from the raw data. The background absorbance was determined in Clq-coated wells in which IgM proteins and SPA were sequentially incubated and the ELISA then developed by standard protocol. All of the VH3 monoclonal proteins bound SPA in a separate ELISA except for Dau. The represent means of duplicate determinations from a representative experiment; similar results were obtained in three separate experiments.

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tutedwith selected monoclonal IgM proteins)from a severely hypogammaglobulinemic patient (72). Addition of mod-SPA to hypogammaglobulinemic serum reconstituted with an SPA-nonreactive, VH3+ IgM did not alter the CH, activity of the serum. By contrast, addition of mod-SPA to hypogammaglobulinemicserum reconstituted with an SPA-reactive, Clq-binding, VH3+ IgM did support complement consumption. In addition, incubation of mod-SPA alone with the hypogammaglobulinemic serum did not cause any complement consumption. The latter observation eliminated the possibility that direct activation of the alternative complement pathway contributed to the findings observed with the mod-SPA-treated, normal serum. Taken together with the Clq-binding data, the above results provided formal proof that the interaction of VH3+ Igs with the Fabbinding site on SPA leads to binding of Clq and activation of the classical complement cascade. Complement activation by protein Fv has also recently been described (74). However, our results provided direct evidence for the first time that the interaction of a model B-cell superantigen, mod-SPA, with its reactive (VH3+) Igs leads to activation of the classical complement cascade. Complement activation by a B-cell superantigen is also being examinedin the HIV gp120 system. Townsley-Fuchs and co-workers have suggested (75) that gp120 and VH3+ Igs may form immune complexes and activate complement, which may sequester HIV to phagocytic cells. They found that serum IgM could inhibit binding of gp120 to T cells, but only in the presence of complement. They suggested that the interaction between gp120, VH3+ Igs, and complement may cause phagocytic cells to more readily take up HIV. The interaction of a B-cell superantigen with the Fabs reactive Igs in vivo could lead to the formation of complement-activating immune complexes, which may have profound clinical significance. For example, patients with SPA+, S. aureus-induced endocarditis (76) and patients treated with autologous plasma perfused through a SPA-immunoabsorbent column develop immune complex-mediated glomerulonephritis and vasculitis, respectively. These manifestations may be the result of an interaction between SPA and VH3+ Igs with the formation of immune complexes and resultant complement activation. Moreover, some cases of glomerulonephritis, arthritis, and thrombocytopenia in HIV infected patients may be caused by deposition of complexes containing HIV gp120 and Igs containing VH3 heavy chains. Unlike a conventional antigen, a B-cell superantigen can react with a large fraction of serum Igs. This interaction can lead to activation of the classical complement cascade and

B-Cell Superantigens

427

thus has the potential to elicit prominent tissue inflammation in a

VII.

CONCLUSIONS

Evidence has been reviewed for a new class of antigens that stimulate B cells in an unconventional manner. These agents, largely microbial cell wall constituents, stimulate a high frequency of B cells. Activation is restricted to B cells that express a particular type of VH or immunoglobulin. Based on the example of SPA, it is likely that these unconventional antigens bind outside the conventional antigenbinding region to sites located in the V-domain of heavy or light chains. These properties are reminiscent of those of T-cell superantigens. Thus, these unconventional antigens have been dubbed Bcell superantigens. Limited functional data now available suggestthat B-cell superantigens may impact on the host immune system to shape the B-cell repertoire and perhaps contribute to the pathogenesis of allergic, inflammatory, and autoimmune diseases. ACKNOWLEDGMENTS

This work was supportedinpartby N.I.H. TrainingGrant 2T32CA09140, a Merit Review grant from the Veterans Administration, and a grant from the University of Pennsylvania Research Foundation. REFERENCES

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18 Staphylococcal Toxic Shock Syndrome Deresiewicz

INTRODUCTION

The toxic shock syndrome (TSS) is an acute, life-threatening intoxication characterized by high fever, hypotension, rash, multiorgan dysfunction, and cutaneous desquamation during the early convalescent period. While named and first detailed in 1978 (l),similar cases of so-called staphylococcal scarlet fever were described in the medical literature at least as far back as the The diseasegained great notoriety in after the occurrence of a large tampon-associated outbreak in menstrual young women (5-8). Despite the dominant impression left by that event, about half of cases ofTSS today occur in settings other than menstruation, are not linked to the use of catamenial or medical products, and are distributed among individuals of both sexes and all ages. is a clinically defined syndrome. It is caused by any of several related bacterial exotoxins, all of which share a similar mechanism of action. In classical TSS, the etiological bacterial agent is Sfaphylococcus aureus, and the toxin most frequently implicated is the toxic shock syndrome toxin-l (TSST-1). In the toxic shock-like syn435

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drome (TSLS streptococcal TSS), Streptococcus pyogenes is the etiological agent. TSLS is described elsewhere in this volume. This chapter will review the epidemiology and pathogenesis of staphylococcal TSS, its clinical presentation and differential diagnosis, and strategies its treatment and prevention. II. CASE DEFINITION

Although great strides have been made in our understanding .of the etiology and pathogenesis of TSS, its diagnosis continues to rest exclusively on clinicalgrounds. The accepted casedefinition was develand is detailed in Table 1 oped by investigators in the early A case is classified as "confirmed if it meets all six diagnostic criteria and "probable" if it meets five of the six. Because cutaneous desquamation does not occur until 1-2 weeks after onset of the disease, the status of "confirmed case" is essentially never achieved acutely. In otherwise-compatible fatal cases, the requirement desquamation is waived. Isolation of a toxigenic staphylococcusis not required by the case definition, nor i s demonstration of a serological response by the host. Staphylococcal bacteria is not an exclusionary criterion. A less stringent case definition has been formulated suspected recurrences in patients who have had at least one welldocumented episode of menstrual TSS 111.

A. 1.

ETIOLOGY,PATHOGENESIS,ANDPATHOPHYSIOLOGY Overview

S. aureus

TSS is caused by any of several related staphylococcal exoproteins. Although some reports have suggested that either coagulase-positive coagulase-negative staphylococci could cause the syndrome (lO,ll), the weight evidence now suggests that only coagulase-positive organisms produce the toxins that can cause TSS (12,131. The TSS toxins are a subset of the larger group of staphylococcal and streptococcal superantigens, which includes allthe staphylococcal enterotoxins, exfoliative toxins, and TSST-1, and the streptococcal pyrogenic exotoxins. These proteins are examples of nonenzymatic bacterial toxins: superantigenic peptides that effect immune dysregulation and presumably cause disease that basis. TSST-l [formerly termed both staphylococcalenterotoxin F and staphylococcal pyrogenic exotoxinC (14,1511 is the agent most frequently implicated in TSS and is respon-

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TablC 1 Staphylococcal Toxic Shock Syndrome: Case Definition I. Fever: temperature 2 102°F 11. Rash: diffuse macular erythroderma ("sunburn" rash) 111. Hypotension: systolic blood pressure I90 mmHg (adults) or c 5th percentile for age (children under 16 years of age), or orthostatic hypotension (orthostatic drop in diastolic blood pressure 2 15 mm orthostatic dizziness or orthostatic syncope) IV. Involvement of at least three the following organ systems: A. Gastrointestinal: vomiting or diarrhea at onset of illness B. Muscular: severe myalgias, or serum creatine phosphokinase level (CPK) at least twice the upper limit or normal C. Mucousmembranes: vaginal, oropharyngeal,or conjunctival hyperemia D. Renal: blood urea nitrogen (BUN)or creatinine at least twice the upper limit normal, or pyuria (25 leukocytes per high-power field), in the absence of a urinary tract infection E. Hepatic: totalserumbilirubinortransaminase level (alanine aminotransferase or aspartate aminotransferase) at least twice the upper limit of normal F. Hematologic: thrombocytopenia (platelets 100,000 per 111) G. Central nervous system: disorientation alteration or in consciousness but no focal neurological signs at a time when fever and hypotension are absent V. Desquamation: 1-2 weeks after the onset of illness (typically palms and soles) VI. Evidence against an alternative diagnosis: If obtained, negative cultures of blood, throat, or cerebrospinal fluid;a absence of a rise in antibody titers to the agents of Rocky Mountain spotted fever, leptospirosis, or rubeola aBlood culture may be positive for S. UUEUS. Source: Ref. 8.

sible for about 90% of menstrual cases and 60% of nonmenstrual cases. Staphylococcal enterotoxin B (SEB) causes mostof the remaining cases. Rare cases have been attributed to enterotoxin C (SEC) and perhaps enterotoxin A (SEA) A recent report described the cloningfrom a TSST-l-negative nonmenstrual-TSS strain of a novel staphylococcal exotoxin that is structurally and functionally homologous to the known enterotoxins 2.

in thePathogenesis

TSS

Broadly stated, the pathogenesis ofTSS proceeds as follows: 1) colonization infection of the host by a toxigenic strain, 2) toxin pro-

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duction, 3) toxin absorption, and 4) intoxication. Each of these steps is considered in greater detail below. Note that overt staphylococcal infection is neither required for the genesis ofTSS nor usually present. The often benign appearance of the primary site toxin production in TSS presents a great diagnostic challenge to clinicians. B.

Staphylococcal Colonization

1.

Frequency

StaphylococcalColonization

Human beings constitute the major reservoir of S. aureus in nature. At any given time, between and 40%of adults are colonized (21,22). The anterior nasopharynx is the principal site of carriage. Others include the axillae, vagina, perineum, and occasionally, the gastrointestinal tract. Colonization may be intermittent or persistent and is likely influenced both by microbialand by host factors, as well as by the nature of the competing nonstaphylococcal microflora(23). Carriage is most common among personswith frequent staphylococcal exposure and those with habitual or chronic disruption of cutaneous epithelial integrity. Thus, health care workers, dialysis patients, diabetics, intravenous drug users, and persons with chronic dermatological conditions are most frequently colonized (24-28). Among normal postmenarcheal American women, the rate vaginal colonizationby S. aureus is between about and 15% (29-32). It may be higher in developing countries (33). Both the rate of vaginal colonization and the vaginal staphylococcal burden in colonized subjects are greatest during the menses. A recent study of 509 healthy young women found that other than menstruation coincident with sample collection, the likelihood of vaginal staphylococcal colonization was influenced only by thewbject’s race and city of residence (32). Tampon use did not exert an independent effect on the rate of staphylococcal carriage in that study, nor are tampons the source of vaginal staphylococci (34). The staphylococcal cell-wall teichoic acid appears to mediate adherence to the nasal epithelium (35). Whether it also mediates vaginal adherence is unknown. 2.

Distribution Frequency

ToxinGenes

Strains

S. aureus

Most strains of aureus lack the structural genes for TSST-1 or any of the enterotoxins and are unable to cause Production of any of those proteins is therefore a variable genetic trait (36). Like cholera toxin, the TSST-1 gene (tstH) is encoded by a transposon-like mobile genetic element (37,38). The tstH element can occupy at least two positions on the staphylococcal chromosome and is presumably

k


Syndrome

439

transferrable between strains. The SEB gene (entB) is also borne on a mobile element, the exact nature of which (transposon-like or phage-like) remains unclear. About 10-20% of all human staphylococcal isolates, including vaginal isolates, carry the tstH gene, while 714%carry entB (39-43). Thus, considering both the frequency of vaginal staphylococcal carriage and the frequency distribution of tstH among clinical strains, about l-4% of postmenarcheal women are at risk for menstrual TSS at any given time. That the incidence ofTSS is far lower than those figures suggest implies that factors other than mere colonization by a toxigenic strain bear powerfully on the risk. 2.

Single Clone

S. aureus Causes the Majority

Cases

TSS

Multilocus enzyme electrophoresis has demonstrated that tstH is widely distributed in staphylococcal lineages collectivelyrepresenting the total breadth of genotypic diversity within the species aureus (44). Nevertheless, a single clonal electrophoretotype accounts forthe majority of TSST-l-associated menstrual cases and about half of TSSTl-associated nonmenstrual cases. In Musser’s study of 315 aureus strains collected in five countries on three continents, 88% of menstrual TSS strains and 53%of nonmenstrual TSS strains were electrophoretically indistinguishable (44). Chromosomal restriction-fragment-length-polymorphism (RFLP) analysis and ribotyping support that view (38,45). In Musser’s study, the same clone was also overrepresented among vaginal isolates from asymptomatic women (28% of isolates). Two hypotheses have been offered to explain these phenomena (44): that the clone in question is uniquely adapted to colonize the urogenital epithelium,as suggested byitsoverrepresentation in asymptomatic women, or 2) that the clone is uniquely adapted to disease, perhaps because of particularly exuberant toxin production in response to certain clinically relevant stimuli. Of course, both mechanisms may be operative. C.

Factors Influencing Toxin Production

1.

ThePerspective

theBacterium

Bacterial virulence factors serve to enhance the survival of the bacterium in the hostile and changing environment of the mammalian host (46). Their production typically is tightly and coordinately regulated by genetic apparati that sense and respond to environmental cues. Such coordinate regulation enablesthe organism rapidly to tailor its repertoire of proteins to suit its changing needs, as it passes from microenvironment to microenvironment, or as its microenvironment

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evolves around it. TSST-1 and SEB are typical virulence factorsin this regard (47). Both are produced during the late-log phase of growth in batch culture, at a time when nutrients become scarce and cell density saturates. Both are coordinately regulated in parallel with other staphylococcal exoproteins (e.g., a-, p-, and &hemolysins, serine protease, nuclease) and reciprocally to the principal staphylococcal cell-wall-associated proteins (protein A and coagulase) (47). Several genetic regulatory loci modulate toxin production, as do certain discrete environmental conditions-conditions that presumably operate through those genetic loci. Tampons, nasal packing devices, other foreign materials, the use of which increases the risk of TSS, likely do so by orchestrating environmental changes that stimulate toxin production. GeneticRegulation

ToxinProduction

To date, at least three separate genetic loci are known to regulate exoprotein production in S. uureus: ugr (accessory generegulator) (48), xpr (extracellular protein regulator) (49), and sur (staphylococcal accessory regulator) (50). All three affect gene expression primarily at the level of transcription, although the ugr effector (a small regulatory RNA molecule called RNAIII) also modulates the translation of certain proteins While there are clear differencesbetween the phenotypic effects of mutations at these loci, all three activate the expression of secreted proteins and diminish the expression of cellwall-associated proteins during the late log phase of bacterial growth. Recent data suggest that ugr may function primarily as a “quorum sensor,” an apparatus that informs the bacterium of the density of staphylococci in its environment (53). RNAIII is encoded by one of two divergent transcriptional units within ugr (47,51,54). The second transcriptional unit encodes four open reading frames (ugrA-D), the roles of which are to control the activation of RNAIII transcription (55,56). ugrC and are homologous to the sensory transducer and response regulator, respectively, of the family of two-component bacterial regulatory systems (57,58). The ugr locus reports on cell density by synthesizing and monitoring the concentration in the medium of an endogenous ectohormonal oligopeptide, which peptide is a ligand for the agrC gene product ( AgrC) (53’59). This peptide’s synthesis is directed by two of the agr genes and ugrD), and its concentration is monitored by the other two (ugrC and ugrA). When the peptide’s concentration achieves a certain threshold level, it activates the ugr response system, presumably by causing AgrC to signal AgrA, the response regulator. Cellular RNAIII concentration


Syndrome

441

increases rapidly, and the cells undergo their phenotypic shift in protein expression. In a wild-type (ugr+) strain, cell-wall-associated proteins are made during exponential growth in batch culture, and secreted proteins are suppressed. At about mid-log phase, RNAIII production becomes detectable Shortly thereafter, exoprotein production commences, and coagulase and protein A production cease. In ugU strains, the late-log phase phenotypic transition does not occur, and exoprotein production remains scant. While the synthesis of many exoproteins is coordinately regulated by ugr, not all are equally impacted by it. example, TSST-l production is augmented up to fold in an ugr' compared to an strain. SEB production is augmented only three- to fivefold Such regulatory differences might in part influence the propensities of TSST-1- and SEB-bearing strains to cause TSS in certain clinical settings. xpr and sur influence expression of RNAIII. Accordingly, at least part of their effects on protein synthesis is due to their effects on ugr sur clearly operates byother mechanisms, as well, and xpr might also. The details of the cross-talk between these and other putative regulatory loci remain to be elucidated, as do the particulars of the signals to which they respond. It is likely that exoprotein regulation in S. uureus results from a complex interplay between environmental factors and gene products, only one component of which is quorum sensing. Environmental A number of environmental factors influence the amount of TSST-1 produced by toxigenic strains, although the pathways by which they do so remain unclear. S. uureus is a hardy and adaptable organism, facultatively anaerobic, and capable of growth at a wide range of temperatures and pHs. Optimal toxin production occurs over a much more limited range of conditions. In batch culture, aerobiosis, carbon dioxide content supplemented to about 5%, high protein concentration, low glucose concentration, a starting pH in the neutral range that is allowed to drift with cell growth, and temperatures of rather than all enhance toxin synthesis According to some studies but not others, a limited but nonzero concentration of magnesium ion also enhances toxin production, especially when judged on a per-cell basis It is worth noting that associations such as those noted above, while well established, are not necessarily directly causal. example, the enhancement of toxin synthesis that occurs under aerobic

442

Deresiewicz

as compared to anaerobic conditions might actually be a quorum effect rather than a specific oxygen effect: Schlievertand Blomster studied the impact of aeration on TSST-1 production by seven TSS-associated strains of S. aureus. Under aerobic conditions the strains’ final average cell density was about 2.5 times greater than under anaerobic conditions, and they made on average 32-fold more toxin (65). Perhaps it is the greater bacterial density achieved under aerobic growth rather than aerobiosis per se that activates the cells’ regulatory machinery. Analysis of purulent fluid from the presumed focus of toxin production in several nonmenstrual TSS cases has suggested that environmental conditions in vivo approximate those found to enhance toxin synthesis in vitro (66). Similarly, the chemical composition of vaginal fluid during the menses more closelymatches the conditions that are conducive to toxin production in vitro than does the composition .of vaginal fluid at midcycle (65,72). 4.

.

Role

the Tampon or Other Foreign Device

in the Etiology

TSS

As noted earlier, tampons neither deliver staphylococci to the vagina nor enhance their growth there, nor do they increase the likelihood vaginal staphylococcalcarriage(31,34,73).Rather, the risk ofTSS associated with their use likely accrues from changes they effect to the vaginal microenvironment and the stimulus to TSST-l production resulting therefrom. The same is probably true of the risk imposed by certain surgical dressings, such as the Teflon splints and white .petrolatum or adaptic gauze favored by otorhinolaryngologists. For example, the use of tampons or other intravaginal devices introduces air into the normally anaerobic vagina and thereby ensures the presence of oxygen. Vaginal carbon dioxide tensionis only briefly affected by tampon insertion (74). It has been suggested that the air content of a given style or brand of tampon might be the variable that best correlates with the risk ofTSS attendant to its use (75). Rely brand of tampons, the product most strongly associated with TSS during the 1980 epidemic, and one that provoked exuberant toxin production in vitro, was composed of polyester foam and crosslinked carboxycellulose. Millset al. presented evidence that the polyester foam component bound magnesium, and that at least part of its stimulatory effect on toxin synthesis was attributable to that property (68). Tampons composed of materials that enhance TSST-1 production in vitro or shown epidemiologically to be associated with a higher-than-average risk for TSS, have been withdrawn from the


Syndrome

market. Products currently sold are quite safe menstrual disease for most users is low

443

and the risk

D. Toxin Uptake 1.

Evidence that ToxinUptake

NecessaryforIntoxication to Occur

There is no direct evidence that systemic toxin uptake is required for TSS to occur, but such uptake canbe demonstrated. Nanogram amounts TSST-l have been detected in breast milk, blood, and urine humans with TSS and in blood and urine in various animal models ofTSS Following i.v. injection in rabbits, TSST-1 has a plasma half-life of about 1.5 hr E.

Protective Role of Antitoxin Antibodies

While there is little direct evidence speaking to the issue of whether antibodies to TSST-l protect against TSST-1 mediated TSS, much indirect evidence suggests just that. This evidence includes the discrepancy between the low prevalence of TSST-l antibodies among TSS cases compared to matched controls, the protection of rabbits against experimental TSS by passive immunization with antibody to TSST1, and the neutralization in vitro certain biological properties TSST-1 by anti-TSST-l. 1. Human Data

Bergdoll's group first noted the frequent absence of antibody to TSST1 in the sera of TSS patients acutely and the frequent presence of such antibody in the sera of matched controls. For example, in one report they found low antibody levels (defined as anti-TSST-l titers of < 1:lOO) in of 92 TSS patients but in only 21of 111controls vs. 18.9%, p < Several other studies have since confirmed their findings Jacobson et al. sought to define risk factors for the development of TSST-l-associated TSS following nongenital staphylococcal infection. Of evaluable patients infected with toxigenic staphylococci, had anti-TSST-l titers of >1:100 and none of those developed a TSS-like illness. Of the eight patients whose titers were < 1:100, two had definite TSS and a third had probable TSS Finally, Arnow et al. described the case of a hospital nurse who endured three separate episodes ofTSS associated with intermittent nasal and vaginal colonization by a toxigenic strain of S. aureus. Her initial episode fulfilled the complete CDC criteria for TSS, and her

Deresiewicz

444

titer of anti-TSST-l was at that time. Eachof her subsequent episodes ofTSS was progressively milder. After the third, her antitoxin titer was >1:1000. While she continued to be intermittently colonized by the toxigenic strain, she had no further episodes of disease (87). 2. AnimalStudiesand In

Data

Polyclonal and monoclonal antisera to TSST-l are capable of protecting against morbidity and mortality in leporine models of TSS. Melish et al. protected animals against all manifestations of the illness in a toxin-depot model ofTSS by administering polyclonal anti-TSST-l antibody concurrently with toxin implantation. In a second group of four animals, they delayed administration of the antitoxin until hr after toxin implantation. While all the rabbits developed signs of TSS, none died. In contrast, six of six untreated animals died (88). Bonventre et al. developed a panel of murine monoclonal antibodies (MAbs) directed against primary sequence determinants of TSST-l Several blocked the ability of TSST-1 to induce IL-1 production by human peripheral blood mononuclear cells, and at least two neutralized TSST-l mitogenicity for murine splenocytes. The most potent of the MAbs offered significant protection against TSST-Finduced leporine TSS, both in the subcutaneous infusion model and in the implanted infection chamber model Hirose-Kumagai et al. demonstrated that polyclonal human antiTSST-l can neutralize the IL-l-inducing properties ofTSST-1 while Takei et al. showed that pooled human immunoglobulin blocks TSST-l- and SEB-induced proliferation of human peripheral blood lymphocytes in vitro Intoxication

Despite tremendous advances over the past 15 years in our understanding of the structure and biology of TSST-l, the issues of how exactly it causes the diverse manifestationsof TSS and whether it does so by itself requires cofactor(s) remain unsettled. Several hypotheses have been advanced: that TSST-1 is directly toxic to certain mammalian cells or tissues, that TSST-l is injurious by virtue of its enhancement of host susceptibility to Gram-negative endotoxin (lipopolysaccharide; LPS), that TSST-l is harmful because it induces a dysregulated endogenous inflammatory cascadethat ultimately causes TSS. These hypotheses are not mutually exclusive.

Staphylococcal Toxic Shock Syndrome 1.

445

DirectToxicity

No enzymatic toxic property has ever been ascribed to TSST-l, nor has direct cytotoxicityever convincingly beendemonstrated. The evidence that has been developed is as follows: In a study by Dmmm et al., TSST-1 caused cellular architectural distortion and subsequent detachment of chick embryo cells in tissue culture at a toxin concentration of 0.2 pg/ml. The effect was potentiated by bacterial LPS and was dependent on the means by which the cells were prepared. No such effect was seen on a variety of other cell types tested, including cells from both primary cultures and established lines (94). Kushnaryov et al. showed that TSST-1 binds specifically and saturably to normal human conjunctival epithelial cells in tissue culture, is internalized by them in coated pits, and decreases their net growth rate by 64% at a toxin concentration of pg/ml (95,961. Lee et al. showed that TSST-1 also binds to cultured porcine aortic endothelial cells and kills them in a time- and dose-dependent manner. They demonstrated cytotoxicity beginning at a toxin concentration of 1 pg/ml and were able to inhibit it with rabbit anti-TSST-l, but not with preimmune rabbit sera (97). Given the rather high concentrations ofTSST-1 necessary to demonstrate toxicity in all the above studies (at least two orders of magnitude higher than that which has been demonstrated to circulate during human disease), the clinical relevance of the above observations is uncertain. Deresiewicz et al. concluded that TSST-1 probably does not possess a discrete enzymatic property cytotoxic for eukaryotic cells: They placed tstH under the control of an inducible promoter in Saccharocerevisiae. Under similar circumstances, the known bacterial enzymatic cytotoxins Shiga-like toxin and diphtheria toxin are both highly lethal to the eukaryotic host. Although full-lengthstable TSST1 was demonstrated within the yeast cells, and although it retained mitogenicity for human T cells, it had no apparent effect on the yeast cells’ growth kinetics on their morphology (98). 2.

Endotoxin Enhancement

Shortly after Schlievert’s codiscovery ofTSST-1, he recognized the toxin’s ability markedly to enhance the susceptibility of rabbits to lethal endotoxic shock (15,99). Rabbitspretreated with a bolus injection of TSST-l are killed by doses of endotoxin approximately four orders of magnitude below those required to kill na’ive animals. Schlievert postulated that endotoxin enhancement, a property that

Deresiewicz

TSST-l shares with the streptococcal pyrogenic exotoxins (loo), might be the key event leading to TSS (101). Several studies have documented synergy between TSST-1 and LPS in the induction of the inflammatory cytokines tumor necrosis factor (TNF) and interleukin 1 (IL-l) (102-104) in the suppression of antibody responsiveness to erythrocyte antigens, and in the delaying of reticuloendothelial clearance of colloidal carbon (105). Stone and Schlievert examined the lethality of TSST-l for two breeds of rabbits: New Zealand White females (certified free of the Gram-negative organism Pasteurella rnultocida) and standard American Dutch belted females. Bolus infusion of TSST-1 was more lethal to the latter than the former and was preventable by prior and concurrent treatment with polymyxin an inactivator of endotoxin (106). While recognizing the validity of the above observations, others have argued that endotoxin is not required for the induction of a TSS-like illness in animals, and probably is not in humans, either. In Melish’s two leporine subcutaneous depot models of TSS, administration of neither polymyxin B nor anti-J5 (an antibody directed at the endotoxin core region) prevents death, while infusion of anti-TSST1does (80). In Parsonnet’s subcutaneous infusion model of TSS, polymyxin is similarly ineffective in preventing morbidity or mortality (107). All three of these models faithfully reproduce a TSS-like illness in the rabbits and share with human TSS the slow and sustained delivery of toxin to the subject. It is possible that the differences between these results and those of Stone and Schlievert relate to the method of toxin delivery. Because bolus injection of toxin does not reproduce the probable kinetics of toxin uptake in human TSS, i.v. infusion of the toxin may imperfectly model the disease (102). Finally, several authors have questioned the endotoxin-enhancement hypothesis on clinical grounds: TSS and Gram-negative sepsis are distinct clinical syndromes. Several of the cardinal manifestations TSS either do not occur or occur to a much lesser extent in septic shock than in toxic shock-for example, the erythroderma, prominent gastrointestinal symptoms, hypocalcemia, hypomagnesemia, accelerated renal failure, rhabdomyolysis, and late desquamation (80,108). DysregulatedCytokineRelease Although many of the immunomodulatory properties of TSST-1 and the other staphylococcal superantigens are well established, the link between these properties and disease is still a matter of debate. As noted at the outset, the TSS toxins are superantigens-Vprestricted T-cell mitogens. They bind strongly and without prior pro-

k


Syndrome

447

cessing to MHC class I1 molecules on mononuclear cells, and in that context, bind to those T cells whose receptors bear particular Vp sequences While the class I1 molecule is required for the integrity of this pathway, the identity of the particular class I1 allele that is expressed on the monocyte is relatively unimportant compared to the Vp haplotype of the T cell. One consequence of the interaction is proliferation of the T cells so stimulated- cells that are orders of magnitude more abundant than those stimulated by any conventional antigen, and that can constitute up to a few percent of the entire Tcell repertoire of a given individual (112). Superantigen-driven Vpspecific T-cell expansion can bedemonstrated both for normal human peripheral blood cells stimulated in vitro and in the blood ofTSS patients (111,113). There is evidence forother pathways of superantigen-induced Tcell activation that are class 11-independent (114,115). The consequences of activation of these pathways differ somewhat from that of the class 11-dependent pathway outlined above. Whether they play a role in TSS remains to be determined. The toxicity of for mice has been shown to be mediated by T cells. Administration of the toxin to normal animals results in significantly more weight loss and immune dysfunction than does administration to nude mice (which lack T cells), or to mice deficient in SEB-responsive T cells (116). Similarly, SEB is lethal to D-galactosamine (D-gabsensitized normal mice, but not to D-gal-sensitized SCID mice (lacking T and B cells). Lethality is restored by T-cell repopulation of the SCID mice The critical role of T cells in superantigen-mediated shock has also been demonstrated by site-directed mutagenesis of TSST-l. Bonventre constructed a point mutant of TSST-1 bearing the substitution alanine for histidine at residue 135 (TSST-1 H135A) (118). Residue 135 is in the region of the toxin that is believed to interact with the Tcell receptor Like wild-type TSST-I, mutant H135A retains resistance to trypsinization and is recognized by anti-TSST-l MAbs, indicating that its three-dimensional structure is not grossly perturbed by the point mutation (120). It is markedly attenuated in mitogenicity for T cells, does not induce production of T-cell-derived cytokines, and is toxic neither to rabbits in the subcutaneous infection chamber model ofTSS nor to D-gal-sensitized mice (118,121,122). Stimulation of T cells and monocytes by TSST-1 [or by products of TSST-l-negative nonmenstrual TSS strains (102,123,124)l induces production of an array of cytokines, including IL-l and TNF-a by monocytes, and IL-2,IFN-y, and TNF-a and by lymphocytes

Deresiewicz

448

(102,125-131). These products undoubtedly play a role in the pathogenesis of TSS, as they are believed to do in the sepsis cascade initiated by endotoxin. TNF-a occupies a central place in the Gram-negative sepsis cascade. It stimulates the release of IL-l, IL-6,IL-8, platelet-activating factor, leukotrienes, thromboxane A2, and prostaglandins, promotes leukocyte and endothelial cell adhesion, activatesthe complement and coagulation pathways, alters lipid and glucose metabolism, causes fever by directly affecting the hypothalamus, and is directly toxic to endothelial cells (132). It causeshypotension, acidosis, coagulopathy, and end-organ damagewhen infused into animals. Anti-TNF antibodies block the sequelae ofLPS administration (133,134). TNF-P, which is made primarily by lymphocytes, shares many of the properties of TNF-a Moreover, anti-TNF-a/P antibody protects D-gal-sensitized mice againstlethal challenge with SEB (117). TSST-l is a potent inducer of TNF-P synthesis; LPS is not. Moreover, the kinetics of TNF-P induction by TSST-l is quite different from that of TNF-a induction by LPS. It is attractive, therefore, and consonant with the observation that superantigen-mediated shock is dependent on T cells, to speculate that the clinical differences between toxic shock and septic shock in part derive from differences in the timing and type of TNF produced in the two syndromes (131). The cytokines produced in response to TSST-l interact in complex ways and produce a host of effects. Whiledysregulated cytokine release is probably the best available explanation for the toxicity of the staphylococcal superantigens, how exactly it leads to the diverse manifestations of TSS remains a matter of speculation.

W. A.

EPIDEMIOLOGY Overall Incidence of TSS

TSS is a reportable disease in the United States. The Centers for Disease Control and Prevention (CDC) monitors disease activity through passive reporting by state departments of health. According to CDC has dropped steadily and considerably figures, the incidence of since the peak of disease activity in 1980 (Fig. 1). Withdrawal of certain catamenial products highly associated with the disease, reformulation of products currently on the market, and more prudent patterns of tampon use were each partly responsible for that decrease. Nevertheless, it is likely that the disease is substantially underreported. example, Gaventa et al. studied the incidence ofTSS in

I

Staphylococcal Toxic Shock Syndrome

449

RelyTMWithdrawn

Menstrual Nonmenstrual

b

a

.-

a

a

a a

Year Figure Menstrual and nonmenstrualcases of TSS reported to the CDC, by year, 1979-1994. Indicated are the dates withdrawal of Rely-brand tampons and of tampons composed of polyacrylate rayon. Between the years 1980 and 1985, tampon absorbency was lowered considerably. (Data kindly provided by Dr. David A. Ashford, Division Emerging Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA).

five states and in Angeles County in 1986 using active biweekly surveillance of participating hospitals (136). They identified 179 definite or probable cases of TSS, compared with only 206 reported the national system by all states with passive surveillance during that same time period. In addition to the underreporting caused by weakness inherent in passive surveillance, cases (especially nonmenstrual cases) are undoubtedly also underreported due to misdiagnosis (136138). The overall annual incidence ofTSS was 0.53/100,000 in Gavenfa’s study and varied significantly by region: the peak rate was in Oklahoma (1.23 cases per while the lowest was in New Jersey (0.22 cases per Twenty-nine definite probable cases were reported by the Massachusetts State Department of Health between 1990 and 1994, of which at least 19 were menstrual cases (139). During that same period, CDC recorded 443 definite or probable cases, of which 253 were menstrual (140). Based on 1990 census data (U.S. population 248.7

450

Deresiewicz

million, Massachusetts population 6.02 million) (141), these passively reported figures imply current annual incidences of about 0.10 per 100,000 in Massachusetts and about 0.04 per nationally. Estimates of the incidence in several specific clinical settings and gathered over different time periods are presented in Table 2.

B.

.

Influence of Age and Race

TSS is primarily a disease of the young. The peak incidence of menstrual TSS is in the 15-19 year age group (142), reflectingthe fact that younger people are less likely than older people to have protective levels of antibody to TSST-l. While regional differences exist, the overall prevalence of protective antibody levels in adults is greater than 90%, as documented by several studies. For example, Vergeront et al. studied sera from both males and females obtained over two decades and found a fairly linear acquisition of protective antibody through adolescence (about 50% prevalence of protective levels at age 10 rising to greater than 80% by age 20) (143). Other work suggests that immunity may be acquired by most people by age 10 (32,144147). The prevalence of antibody to is similar to that of antiTSST-l. It, too, is acquired by most people by the end of adolescence (1481. The seemingly curious factthat antibody to TSST-1 is commonly acquired in the absence of identifiable disease is not without precedent. For example, asymptomatic cutaneous infection with Corynebacterium diphtheriae is often immunizing (149). In the case of acquisition of antibodies to TSST-1, immunization due to subclinical or mild TSS, to asymptomatic colonization by a toxigenic strain of S. aureus, or to immunization with cross-reactive proteins have all been postulated as mechanisms (22,150). A fraction of the' population never develops antibodies to TSST-1. The reasons for this are unclear, but may reflect a lack of exposure of those people to the toxin, or perhaps a lacunar defect in the ability of their immune systems to respond to it. Several studies suggest that the incidence ofTSS is higher in Caucasians than in members of other racial groups (5-7,151). A number of explanations for this have been proposed, including the possibility of differences in tampon usage patterns between racial groups, differences in intensity of exposure to toxigenic organisms or in levels of antitoxin antibodies as a function of socioeconomic status, differences in susceptibility to the effects of toxin, or perhaps even differences in the ease of recognition of the disease due to differences

Staphylococcal Toxic Shock Syndrome

45

Deresiewicz

452

in the ease of appreciation of the rash as a function of a patient's skin color C.

Mortality Rate

The case fatality rate for TSS has dropped over time, probably because of earlier recognition and better treatment of menstrual cases. Ten percent of all cases with onset before were fatal compared with 5% with onset in and with onset in Nonmenstrual cases have a higher fatality rate than menstrual ones. Of cases reported to the CDC between and the minimum case fatality rate for definite and probable menstrual cases was whereas the minimum case fatality rate for nonmenstrual cases was Similarly, the fatality rate for male cases with known outcome through was compared to a fatality rate of for female cases with known outcome over the same time period D. Clinical Settings in Which TSS Occurs

Menstruation remains the most common setting in which TSS occurs, presently accounting for over half of reported cases (Table Of menstrual cases, the vast majority are in tampon users. example, tampon users accounted for of the reported

Table

Distribution of Conditions Associated with ToxicShock Svndrome, bv Gender of Patient Number of cases (% of total)

Case type

Female

Male

Total

~

Menstruala Barrier contraceptiveb PostpartumC Nonsurgical wound Surgical wound Other, unknown Total

-

10 10 11

-

-

30 -

"Onset of illness during or within 24 hr of cessation of menstrual bleeding. bContraceptive sponge, diaphragm, or cervical cap in place at onset of illness. Consetwithin 30 days after termination of pregnancy (i.e., postpartum or following miscarriage abortion). Source: Ref. 136. (Copyright 1989, The University of Chicago.)

StaphylococcalToxic Shock Syndrome

453

menstrual TSS cases collected through June for which catamenial product usage information was known. The Tri-State ToxicShock Syndrome Study was the first to show that any tampon use increased the risk of TSS compared to no tampon use (O.R. C.I. Of tampons then on the market, the greatest risk by far was associated with the use of Rely brand tampons (O.R. vs. no tampon use; O.R. vs. use of all other brands). Other than the use of Rely brand, the only tampon-related variable shown in that study independently to increase the risk for TSS was tampon fluid capacity. Other studies suggest that continuous use of tampons, as opposed to the alternating use of tampons and pads, is an indeThe total number of tampons used per day pendent risk and the maximum length of wear of any given tampon have not been found to be independent risk factors for TSS, although one study suggested an increased risk associatedwith maximum length of wear in excess of hr ( p = Based on these and other studies, the tampons associated with a high risk TSS were withdrawn from the market, federally mandated absorbency limits were imposed on tampon manufacturers, and standard warnings were formulated for tampon packages advising users to avoid prolonged continuous use of tampons and to select a product with the minimum absorbency needed to control flow. is also associated with a number of conditions of the female genitourinary tract other than menstruation, including the use of barrier contraceptives (diaphragm contraceptive sponge), the puerperium (following either vaginal cesarean deliveries), septic abortion, and nonobstetric gynecological surgery Nonmenstrual TSS not associated with the urogenital tract occurs with approximately equal incidence in males and females. The single greatest risk seems to be antecedent nasal surgery and likely reflects the frequency of staphylococcal colonizationof the nasopharynx, the presence of operative tissue trauma, and the local conditions favorable to toxin production (ample oxygen, surgical packing) TSS has been described in the setting of skin lesions of many types, including chemical thermal burns, insect bites, varicella infections, mastitis, and following surgery Various staphylococcal upper lower respiratory infections have also been associated with TSS, including staphylococcal pneumonia (e.g., postinfluenzal), laryngotracheitis, sinusitis, pharyngitis, or odontogenic infection Cases have also been associated with musculoskeletal infections: example, with osteomyelitis, septic arthritis, muscle abscess. The earliest reports in the literature described cases of this type

454

Deresiewicz

Finally, cases have been associated with staphylococcal bacteremia or endocarditis, but only rarely (178-180), despite the fact that bacteremic strains of S. uureus are frequently TSST-l positive in vitro (86). The rarity with which TSS follows primary staphylococcal bacteremia or endocarditis may therefore reflect the absence in the blood stream of the signals or environmental conditions favorable to toxin production. V.

CLINICAL DESCRIPTION

A.

Overview

The case definition of TSS is strictly formulated and does not admit the possibility of mild cases. Nevertheless, mild cases are widely believed to occur, as evidenced by the histories antecedent perimenstrual flu-like illnesses reported by some women with full-blown TSS. Patients meeting the full criteria are by definition acutely and severely ill, and the impression of a severely ill patient immediately confronts the clinician. Like many toxin-mediated diseases, TSS follows a predictable, almost stereotypical course, as evidenced by the temporal unfolding of its signs and symptoms (Fig. 2). Menstrual disease most often begins between the second and fifth days of the menstrual period, the peak incidence occurring onthe fourth day ( 7 ) . Postoperative disease can begin within hours of the surgical procedure, or weeks later (169,178). Menstrual and nonmenstrual cases are clinically indistinguishable, except for the epidemiological settings in which they occur. The challenge to the clinician is first and foremost to recognize the disease-a challenge mandating vigilance in considering the diagnosis whenever confronted by a patient with a febrile exanthem and hypotension.

B. Prodromeand Acute Phase A minority of TSS patients report mild prodromal symptoms of malaise, low-grade fever, myalgia, or vomiting (181). The acute illness

typically begins precipitously., with high fever, nausea, vomiting, abdominal pain, severe muscular pain and tenderness, and headache, followed shortly by profuse, watery diarrhea. The temperature may reach 105"F, and rigors may be present. Sore throat, tender oral mucosa, and conjunctival and vaginal irritation are often also present (7,153,181-187).

Orthostasis or frank hypotension and the characteristic macular erythroderma develop over the next 48 hr (Fig. 3A) (183). The eryth-

455

Staphylococcal Toxic Shock Syndrome Desquamation

Erylhroderma

.

L E r y I h e m a t o u s Mucous

and

Skin and M u c:ous Membrane Changes Petechiae

Fever

Maculopapular Rash

I

I .

I

Myalgias Abdominal Pain Weakness Hypotension and Oliguria Confusion Headache and Vomiting

"

Diarrhea

1

l

2

3

4

5

6

7

8

9

I

1

1

0

1

I J h 1

? Hospitalization

Day of Illness

Figure 2 Temporal evolution of themajor clinical manifestations Of toxic shock syndrome. (From Ref. 181. Copyright 1981, American Medical Association. 1

roderma is usually generalized,but can be confined to a more discrete area, such as the trunk, axillae, or groin. It can be mild and fleeting or intense, is smooth, and typically blanches dramatically with pressure. Despite that, it often either overlooked or attributed simply to the flush of fever (7). Palatal or cutaneous petechiae are occasionally present, and subepidermal bullae have been described (188-191). By the time of admission the patient typically appears toxic, with hypotension, tachycardia, and oliguria. The patient may have arthralgias, arthritis, and/or meningismus (192-194). The mental status is often abnormal (e.g., confusion, agitation, or lethargy), even when hypotension is absent. Frank encephalopathy with diffuse EEG abnormalities has been described, and mild cerebrospinal fluid pleocytosis is common (181-185,195). Conjunctival suffusionwith or without hemorrhage (Fig. 3B), pharyngeal injection, and peripheral edema are present in the majority of cases. So-called strawberry tongue occurs in 25-50% of cases. In menstrual disease, the vaginal mucosa may

Figure Dermatologic and mucous membrane manifestations of toxic shock syndrome. (A): Diffuse erythroderma in a 7-year-old child with osteomyelitis of the fibula and nonmenstrual TSS. The rubor was particularly pronounced on the chest wall of this patient, and is accurately reproduced in this blackand-white photograph.Circumoral palor is evident. (Kindly provided byDr. P. Chesney, University of Tennessee Health Sciences Center, Memphis, TN). (B): Bulbar conjunctival injection with hemorrhage in a 24-year-old woman with nonmenstrual TSS. (From Ref. 188.) (C): Desquamation of the hand during early convalescence, same patient as in A. (Kindly provided by Dr. Chesney.)

k


457

F

Figure

Continued

be erythematous and inflamed, and a purulent discharge may be present. In nonmenstrual cases, intense erythema may be present adjacent to the site of toxin production, or the site may appear entirely benign. Initial examination of the heart, lungs, and circulatory system usually reveals only tachycardiaand peripheral cyanosis. Intravascular volume depletion is frequent. The abdomen is unremarkable except for diffuse tenderness of the muscles of the abdominal wall. Lymphadenopathy and hepatosplenomegaly are rare (187 A broad array of laboratory parameters is typically abnormal, as shown in Table 4. In particular, renal dysfunction, hypoalbuminemia, disproportionate hypocalcemia, hypophosphatemia, creatine phosphokinase elevation, thrombocytopenia, and pyuria are common (153,187). C.

Early Convalescent Phase

Many of the early signs and symptoms of TSS resolve within the first few days of illness: the fever, erythroderma, and gastrointestinal disturbance are typically short-lived, and chemical abnormalities re-

rness

ation

Deresiewicz

Table 4 CommonClinical

Manifestations and Laboratory Abnormalities in Toxic Shock Syndrome" Estimated frequency of occurrence (%) Symptoms" Myalgias Vomiting Diarrhea Headache Dizziness Sore throat SignsC Abdominal Pharyngitis/strawberry tongue Peripheral edema Conjunctivitis Central nervous dysfunction system Vaginal Laboratory abnormalities Hematology Leukocytosis Increased prothrombin time Anemia (within first 24 hr) Thrombocytopenia Increased partial thromboplastin 43 time Serum chemistry Hypoalbuminemia Hypocalcemia Increased SGOT Increased serum creatinine Increased urea blood nitrogen Increased total bilirubin Increased creatinine phosphokinase Hypophosphatemia Urinalysis Pyuria Hematuria

92 90 86 72 70 65 81 65 60 47 70 70 66

81 80 73 69 68 66 66 60 46

'These data were compiled from six reviews of clinical manifestations of TSS. reported symptoms: chills, cough, dyspnea, arthralgias, abdominal pain, vaginal discharge. CAll patients fulfilled criteria of fever, hypotension, rash, and desquamation. Other reported signs: joint effusion, meningismus, muscle tenderness. Source: Ref. 153. Reproduced with permission of the McGraw-Hill Companies.

k


459

solve promptly in many cases. While hypotension and oliguria can be prolonged, they typically last only a few days. In severe disease, the challenge becomes fluid and electrolyte management in the face of renal and myocardial dysfunction, massive edema, and the adult respiratory distress syndrome. Rhabdomyolysis can be severe,and myoglobinuria may compound the renal dysfunction. If the patient survives the acute stages of the illness, cardiopulmonary recovery is typically complete, and persistent renal or hepatic disease is rare (9,182-184,186,196). A short-lived maculopapular rash occurring in the second week of the illness has been described in some patients (181,197). About a week after the onset of illness, desquamation begins with a superficial flaking and peeling of the skin of the torso and extremities. This is followed by the characteristic full-thickness desquamation of the palms, soles, and digits, whereby the fifth criterion of the case definition is fulfilled (Fig. 3C). D. late Convalescent Phase

Common late sequelae include peripheral gangrene, reversible nail and hair loss (postfebrile telogen effluvium), sustained muscular weakness, and neuropsychiatric dysfunction (e.g., difficulty concentrating, memory loss, and emotional changes) (198-201).

E.

Fatal Cases

Literature on the clinical and pathological details of fatal is relatively sparse. Only two autopsy series and a handful of individual reports are published (202-207). Death typically occurs within the first few days of illness, most often from refractory shock or respiratory failure. Two patients died from tonsillar or uncal herniation due to acute cerebral edema (203,207). Heart failure, disseminated intravascular coagulation, or other bleeding disorders account for the late mortality. Postmortem findings have included periportal hepatic inflammation, .fatty liver and/or centrilobular necrosis, pulmonary congestion and alveolar edema, acute renal tubular necrosis, reticuloendothelial hemophagocytosis, and vaginal mucosal ulceration and inflammation (203-206).

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F.

Mild Cases

While not accounted for in the surveillance definition formulated by the CDC, and while not statistically linked to toxigenic staphylococci as full-blown TSS has been, mild cases ofTSS likely occur. Several groups have reported such cases in young female tampon users, the typical setting for menstrual disease (182,208-212). Those cases have lacked one or more of the diagnostic criteria, yet had certain features particularly. suggestive of for example, erythroderma, pronounced gastrointestinal symptoms, and/or convalescent desquamation. Some patients had full-blown TSS during a previous menstrual period. Others did during a subsequent menstrual period. Still others never did. It has been postulatedthat less exuberant toxin production by some strains partial immunity to the toxin in some hosts might account for a portion of these cases (213). Some patients might have forestalled development of the complete clinical picture by promptly discontinuing tampon use at the first signs of illness (209). While it is impossible rigorously to ascribe such cases to the clinician should consider the possibility in young women reporting a perimenstrual flu-like illness with erythroderma desquamation, particularly if the illness is substantial or recurrent. In those cases, anti-TSST-l .antibody titer should be determined, and isolation of toxigenic vaginal staphylococci attempted.Nonimmune subjects should be advised to avoid the use of tampons or barrier contraceptives. VI.

DIFFERENTIAL DIAGNOSIS

The differential diagnosis of full-blown TSS is that of the febrile exanthem with hypotension. In certain menstrual cases, particularly tampon-associated cases accompaniedby purulent vaginal discharge, the diagnosis may be readilyapparent. As noted above, the challenge is to recognize the less obvious cases, including mild cases, cases in which the exanthem is fleeting, and cases of nonmenstrual disease. Other diagnoses to consider include streptococcal staphylococcal scaled skin syndrome (caused by exfoliatin, a related staphylococcal exoprotein), Kawasaki syndrome (believed by some to be a variant of TSS), Rocky Mountain spotted fever, leptospirosis, meningococcemia, Gram-negative sepsis, exanthematous viral syndromes (e.g., rubeola, adenoviral infection, certain enteroviral infections, dengue), and severe allergic drug reactions. Careful questioning with attention to potential exposures and other epidemiological clues (e.g.,

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ill contacts, travel history, vocation and avocation, vaccination status, medication usage, and surgical and gynecological history) is critical to the accurate assessment of these patients and may focus the differential diagnosis considerably. So, too, may laboratory exclusion of other illnesses. Certain signs and symptoms are suggestive of TSS: severe myalgia, vomiting, profuse watery diarrhea, and profound renal dysfunction. Conjunctival suffusion is common, but is also seen in leptospirosis, Kawasaki syndrome, erythema multiforme, and certain viral syndromes (188). TSS and TSLS can be clinically indistinguishable. Necrotizing cellulitis, fasciitis, or myositis and exudative pharyngitis are more common in streptococcal than in staphylococcal disease, but none of those establishes a streptococcal etiology with certainty. For the seriously ill patient with an uncertain diagnosis, it is appropriate to employ broad-spectrum antibiotics and adjunctive measures to cover the patient against the variety of etiological possibilities until the diagnosis is clarified. While the isolation of S. aureus is neither required for, nor conclusive evidence of, TSS, it should be attempted (153). Its achievement is considered supportive of the diagnosis. If a strain is isolated, particularly from a menstrual case, it should be tested for the ability to produce TSST-1. Acute and convalescent sera should be tested for antibody to TSST-1. The isolation of a TSST-l-producing staphylococcal strain in the appropriate clinical setting from a patient who lacks protective antibody to TSST-1 is strong evidence for the veracity of the diagnosis (153). Seroconversion occasionally accompaniesTSS, but its utility is for prognostic rather than for diagnostic purposes. VII.

TREATMENT AND RECURRENCE Treatment

Treatment involves several key components: 1. Decontamination the site of toxin production. The primary site of toxin production should be carefully sought. If a site is identified, it should be drained and debrided, foreign material should be removed, and the wound should be copiously irrigated. These measures physically remove the causative agent, and interrupt the process of toxin accretion. ForTSS occurring in the postoperative period, the surgical wound should be explored and irrigated, its typically normal appearance not withstanding.

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2 Antistuphylococcul antibiotics. Antibiotic administration in TSS makes intuitive sense. The standafd of care for TSS historically has been the use of a p-lactamase-resistant semisynthetic penicillin (e.g., nafcillin or oxacillin), which in high dose is active against both staphylococci and streptococci. While some strains of methicillin-resistant uureus carry the TSST-1 genetic element, the predominant clone causing menstrual TSS is methicillin-sensitive (6,38,214). It should be recognized, though, that in TSS the immediate goal is not to eradicate the causative organism, but to shut off toxin production. The best agent to achieve the former task is not necessarily best for the latter. There is a growing body of evidence that in a disease such as TSS, where organisms may not be rapidly dividing and where illness is predicated on protein synthesis by the bacterium, the efficacy of a protein synthesis inhibitor (e.g., clindamycin) may be a superior to that of a cell-wall-active agent (e.g., the penicillins). This dependency of efficacy on the "physiological state of the organism" is the so-called Eagle effect, first described in the (215). It was again documented by Stevens et al., who showed the superior efficacy of clindamycin in experimental streptococcal myositis (216). Two groups have shown that concentrations of clindamycin below the MIC suppress TSST-l production in vitro (217,218), and anecdotal reports have described rapid improvement in human TSS following clindamycin therapy (174). Additionally, new and provocative data indicate that subinhibitory concentrations of p-lactam antibiotics actually increase TSST-1 production by uureus. For example; Parsonnet and colleagues have shown that nafcillin at a concentration of half the MIC can boost toxin production 10-fold over control conditions, and significant enhancement can be demonstrated even at nafcillin levels considerably below that (219). A similar phenomenon has also been described with staphylococcal a-toxin production and nafcillin (220). The effect is not seen with other classes of antibiotics, including the cell-wall-active agent vancomycin, suggesting a specificity beyond merely a cell-wall effect. The coadministration a protein synthesis inhibitor with the p-lactam blocks the effect (221). Thus, there may be a hidden deleterious effect of penicillins on the outcome ofTSS (and, indeed, on other staphylococcal disease, too). While, on balance, that deleterious effect of the 'penicillins may be outweighed by their beneficial bactericidal effect,it may nonetheless be present and clinically significant. Research in this area is ongoing. In the meantime, therapy with clindamycin, either alone, or


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in combination with a p-lactam agent, should be strongly considered for TSS (221). The optimal duration is undefined, but a 10-14-day course is reasonable. Aggressiverepletion of intravascular volume. This is a critical intervention. Although there is evidence myocardial dysfunction in some cases ofTSS (222,223), hypotension is primarily due to intravascular volume depletion caused by massive capillary leakage. Maintaining cardiac filling pressures is crucial to forestall the development of end-organ damage. Fluid should be administered aggressively, and the clinician should not be deterred by weight gain by the development of peripheral edema, both of which can be massive. An adult patient may require as much as 10 L of fluid in the first day to maintain intravascular volume (224). Central hemodynamic monitoring is appropriate if hypotension not immediately responsive to fluids. Animal data suggest that capillary leak syndrome i s the key component contributing to death in TSS, and fluid resuscitation the key measure to prevent it (225). 4. General supportive care. The severity of the case dictates the extent to which general supportive care is required. Intensive care monitoring is frequently necessary. Pressors and inotropic agents should be used for sustained hypotension not responsive to fluids (226). Electrolyte abnormalities should be corrected, and ventilatory support given as needed. Calcium should be repleted in accordance with the level of serum albumin; magnesium repletion may also be necessary. Dialysis has been usedin cases of protracted renal dysfunction. 5. !mmunoglobulin. An additional treatment option for refractory cases, for those associated with an undrainable focus of infection, is the administration of intravenous immunoglobulin. All commercial immunoglobulin preparations contain high levels of antibody to TSST-l. A single i.v. infusion of 400 mg/kg will generate a protective titer in a nonimmune patient. While evidence supporting this therapy is strictly anecdotal, some have reported dramatic improvements in certain clinical parameters followingits use, in particular the normalization of blood pressure (227). B.

Recurrent Disease

Because vaginal staphylococcal carriage can besustained intermittent, and because TSS is frequently not immunizing, recurrent cases of menstrual TSS were common in the early days this illness. As expected, the greatest risk was in women who did not receive an

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antistaphylococcal antibiotic after the first episode, and who continued to use tampons. Women who did receive an antibiotic and who stopped using tampons had a much lower riskof recurrence, although it was still measurable (9). Accordingly, patients should be treated with an appropriate antibiotic, as described above. Testing seroconversion to TSST-l should be offered topatients who had menstrual disease. Those who develop high titers of antibody can probably resume tampon use without risk, regardless of whether they still harbor toxigenic vaginal staphylococci. While the length of persistence of such antibody is not known, it is probably long-lived. Those who do not seroconvert, who do not have access to testing for antibody to TSST-l, should avoid further use of tampons barrier contraceptives. Recurrent nonmenstrual disease is generally not an issue. ACKNOWLEDGMENTS

I gratefully acknowledge Dr. P. Joan Chesney for kindly sharing photographs of clinical material, and Dr. Jeffrey Parsonnet for generously sharing unpublished data, and for many helpful discussions during the preparation of this manuscript. REFERENCES

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157. h m i s L, Feder HM. Toxicshock syndrome associated with diaphragm (letter). use. N Eng J Med 1980; 305:1585 158. Jaffe R. Toxic-shock syndrome associated with diaphragm use. N Eng J Med 1981; 305:1585-1586 (letter). 159. Centers for Disease Control. Toxic-shock syndrome and the vaginal contraceptive sponge. MMWR 1984; 33:43-44. 160. Reingold AL. Toxic shock syndrome and the contraceptive sponge. JAMA 1986; 255:242-243(editorial). 161. Whitfield JW, Valenti WM, Magnussen CR. Toxic shock syndrome in the puerperium. JAMA 1981; 246:1806-1807. 162. Barcero L, Bowe E. Postpartum toxic shock syndrome. Am J Obstet Gynecol 1982; 143:478. 163. Jacobson KA, Kasworm EM. Toxic shock syndrome after nasal surgery: case reports and analysis of risk factors. Arch Otolaryngol Head Neck Surg 1986; 112:329-332. 164. Korcok M. Untoward effect of a face peel: toxic shock syndrome. (news). JAMA 1982; 248:23 165. Farmer BA, Bradley JS, Smiley PW. Toxic shock syndrome in a scald burn victim. J Trauma 1985; 25:1004-1006. 166. Klug R, Immerman R, Giron JA. Bee bite and the toxic shock syn(letter). drome. Ann Intern Med 1982; 96:382 167. Jacobson JA, Burke JP, Benowitz BA, Clark PV. Varicella zoster and staphylococcal toxic shock syndrome in a young man. JAMA 1983; 249:922-923. 168. Niessen GJ, Bartels CC, Degener JE, Stibbe J. Mastitis and toxic shock syndrome (tampon disease). Neth J Surg 1985; 37:lOl-104. 169. Bartlett P, Reingold AL, Graham DR, et al. Toxic shock syndrome associated with surgical wound infections. JAMA 1982; 247:1448-1450. 170. Graham DR, OBrien M, Hayes JM, Raub MG. Postoperative toxic shock syndrome. Clin Infect Dis 1995; 205395-899. 171. Marchant B, Brown J. Toxic shock syndrome and staphylococcal pneumonia. Lancet 1987; 2:578 (letter). 172. Centers for Disease Control. Toxic shock syndrome associated with influenza-Minnesota. MMWR 1986; 35:143-144. 173. Solomon R, Truman T, Murray DL. Toxic shock syndrome as a complication of bacterial tracheitis. Pediatr Infect Dis 1985; 4:298-299. 174. Dann EJ, Weinberger M, Gillis S, Parsonnet J, Shapiro M, Moses AE. Bacterial laryngotracheitis associated with toxic shock syndrome in an adult. Clin Infect Dis 1994; 18:437-439. 175. Griffith JA, Perkin RM. Toxic shock syndrome and sinusitis-a hidden site infection. West J Med 1988; 148:580-581. 176. Hirsch B, Stair T, Horowitz BZ, Brooks C. Toxic shock syndrome from staphylococcal pharyngitis. Ear Nose Throat J 1984; 63:494-497.

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177. Egbert GW, Simmons AK, Graham LL. Toxic shock syndrome: odontogenic origin. Oral Surg Oral Med Oral Pathol 1987;63:167-171. 178. Reingold AL, Hargrett NT,Dan BB, ShandsKN, Strickland BY, Broome CV. Nonmenstrual toxic shock syndrome: a reviewof 130 cases. Ann Intern Med 1982; 96(part 2):871-874. 179. Whitby M, Fraser S, Gemmell CG, Wright PA. Toxic shock syndrome and endocarditis. Br Med J [Clin Res] 1983;286:1613.

180. Pokriefka R, Rabah M, Saravolatz L, Ognjan A. Toxic shock syndrome in an injection drug user with Staphylococcus aureus endocarditis. Infect Med 1994;11:34-36,48-49. 181. Chesney PJ, David JP, Purdy WK, Wand PJ, Chesney RW. Clinical manifestations of toxic shock syndrome. JAMA 1981; 246:741-748. 182. McKennaUG,Meadows I11 JA,BrewerNS,WilsonWR, Perrault J. Toxic shock syndrome, a newly recognized disease entity: report of l1 cases. Mayo Clin Proc 1980; 55:663-672. 183. Tofte RW, Williams DN. Toxic shock syndrome: clinical and laboratory features in 15 patients. Ann Intern Med 1981;94:149-156. 184. Fisher RF, Goodpasture HC, Peterie JD, Voth DW. Toxic shock syndrome in menstruating women. Ann Intern Med 1981;94:156-163. 185. Fisher Jr CJ, Horowitz BZ, Nolan SM. The clinical spectrum of toxic shock syndrome. West J Med 1981;135:175-182. Goldring MacFarlane AM, BarlettKH.Toxic 186. Chow AW,WongCK, .shock syndrome: clinical and laboratory findings in 30 patients. Can Med Assoc J 1984; 130:425-430. 187. Chesney PJ, Bergdoll MS. Clinical Spectrum and Therapy: Toxic Shock Syndrome. Boca Raton FL: CRC Press, 1991:33-49. Bach MC. Dermatologic signs in toxic shock syndrome-clues to diagnosis. J Am Acad Dermatol 1983; 8:343-347. 189. Hurwitz RM, Rivera HP, Gooch MH, Slama TG, Handt A, Weiss J. Toxic shock syndrome or toxic epidermal necrolysis? Case reports showing clinical similarity and histologic separation. J Am Acad Dermatol 1982; 7~246-254. 190. Elbaum DJ,WoodC, 191. 192. 193. 194. 195.

Abuabara F, Morhenn VB. Bullae in a patient with toxic shock syndrome. J Am Acad Dermatol 1984; 10:267-272. Hurwitz RM, Ackerman AB. Cutaneous pathology of the toxic shock syndrome. Am J Dermatopathol 1985; 7:563-578. Soldin I1 JV, Stillman MT, Engberg K.Toxic shock syndrome associated with symmetrical polyarthritis. Minn Med 1981; 64:267-269. Gertner E, Inman RD. Aseptic arthritis in a man with toxic shock syndrome. Arthritis Rheum 1986;29:910-912. Lund L, Nielsen D, Andersen ES. Meningismus as main symptom in toxic shock syndrome. Acta Obstet Gynecol Scand 1988; 67:395. Barrett JA, Graham DR. Toxic shock syndrome presenting as encephalopathy. J Infect 1986; 12:276-278 (letter).

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196. Chesney RW, Chesney PJ, Davis JP, Segar WE. Renal manifestations of the staphylococcal toxicshock syndrome. J Med 1981; 71:583-588. 197. Deetz TR, Reves R, Septimus E. Secondary rash in toxic-shock syndrome. N Engl J Med 1981; 304:174 (letter). 198. Chesney PJ, Crass BA, Polyak MB, et al. Toxic shock syndrome: management and long-term sequelae. AnnInterm Med 1982; 96(Part 2):847-851. 199. Rosene KA, Copass MK, Kastne LS, Nolan CM, Eschenbach DA. Persistent neuropsychological sequelae of toxic shock syndrome. Ann Intern MEd 1982;-96(Part 2):865-870. 200. Chesney PJ, Bergdoll MS, Davis JP, Vergeront JM. The disease spectrum, epidemiology, and etiology of toxic-shock syndrome. Annu Rev Microbiol 1984; 38:315-338. 201. Davis JP, Vergeront JM, Amsterdam LE, Hayward J, StoLz-La Verriere SJ. Long-term effects of toxic shock syndrome in women: sequelae, subsequent pregnancy, menstrual history, and long-term trends in catamenial product use. Rev Infect Dis 1989; ll(Supp1 l):S50-S51. 202. Abdul-Karim FW, Lederman MM, Carter JR, Hewlett EL, Newman AJ, Greene BM. Toxic shock syndrome: clinicopathologic findings in a fatal case. Hum Pathol 1981; 12:16-22. 203. Blair JD, Livingston DG, Vongsnichakul Tampon-related toxic-shock syndrome: histopathologic and clinical findings in a fatal case. Am J Clin Pathol 1982; 78:372-376. 204. Larkin SM, Williams DN, Osterholm MT, Tofte RW, PosalakyZ. Toxic shock 'syndrome: clinical, laboratory, and pathologic findings in nine fatal cases. Ann Intern Med 1982; 96(Part 2):858-864. 205. Paris AL, Herwaldt LA, Blum D, Schmid GP, Shands KN, Broome CV. Pathologic findings in twelve fatal cases of toxic shock syndrome. Ann Intern Med 1982; 96(Part 2):852-857. 206. Wick MR, Bahn RC, McKenna UG. Toxic shock syndrome: a fatal case with autopsy findings. Mayo Clin Proc 1982; 57:583-589. 207. Smith DB, Gulinson Fatal cerebral edema complicating toxic shock syndrome. Neurosurgery 1988; 22:598-599. 208. Siklos P, Carmichael D, Rubenstein D. Toxic-shock syndrome. N Eng J Med 1981;304:1039 (letter). 209. Chesney PJ, Slama SL, Hawkins RL, et al. Outpatient diagnosis and management of toxic-shock syndrome. N Engl J Med 1981; 304:1426 (letter). 210. Tofte RW, Williams DN. Toxic shock syndrome: evidence of a broad clinical spectrum. JAMA 1981; 246:2163-2167. 211. Bass JW, Harden LB, Peixotto JH. Probable toxic shocksyndrome without shock and multisystem involvement. Pediatrics 1982; 70:279-281. 212. Kasper DL. Case records of the Massachusetts General Hospital: Case 4-1986 (CPC). N Eng J Med 1986; 314:302-309.

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213. Reingold AL. Toxic shock syndrome: an update.Am J Obstet Gynecol 1991;165:1236-1239. 214. Kreiswirth BN, Novick RP, Schlievert PM, Bergdoll M. Genetic studies on staphylococcal strains from patients with toxic shock syndrome. Ann Intern Med 1982; 96(Part 2):974-977. 215. Eagle H. Experimental approach to the problem of treatment failure with penicillin. I. Group A streptococcal infection in mice. Am J Med 1952;13:389-399. 216. Stevens DL, Gibbons AE, Bergstrom R, Winn The eagle effect revisited: efficacy of clindamycin, erythromycin, and penicillin in the treatment of streptococcal myositis. J Infect Dis 1988; 158:23-28. 217. Schlievert PM, Kelly JA. Clindamycin-induced suppression of toxicshock syndrome-associated exotoxin production. J Infect Dis 1984; 149:471. 218. Dickgiesser N, Wallach U. Toxic shock syndrome toxin-l (TSST-l): influence of its production by subinhibitory antibiotic concentrations. Infection 1987;15:351-353. 219. Parsonnet J, Modern PA, Giacobbe K. Effect of subinhibitory concentrations of antibiotics on production of toxic shock syndrome toxin-l (TSST-l) (abstract). Program and Abstracts of the 32nd Annual Meeting of the Infectious Disease Society of America. Washington, DC: Infectious Disease Society of America, 1994. Abstract 29. 220. Kernodle DS, McGrawPA, BargNL, Menzies BE, Voladri RKR, Harshman S. Growth of Staphylococcus aureus with nafcillin in vitro induces a-toxin production and increases the lethal activity of sterile broth filtrates in a murine model. J Infect Dis 1995; 172:410-419. 221. Andrews MM, Giacobbe KD, Parsonnet J. Induction of toxic shock syndrome toxin-l (TSST-l) and beta-lactamase by subinhibitory concentrations beta-lactam antibiotics. Biomedicine ‘96, Washington, DC, May 3-6, 1996. Abstract N-IN-0011. 222. Olson RD, Stevens DL, Melish ME. Direct efforts of purified staphylococcal toxic shock syndrome toxin 1on myocardial function of isolated rabbit atria. Rev Infect Dis 1989; ll(Supp1 l):S313-S315. 223. Crews JR, Harrison JK, Corey GR, Steenbergen C, Bashore TM. Stunned myocardium in the toxic shock syndrome. Ann Intern Med 1992;117:912-913. 224. Todd JK. Therapy of toxic shock syndrome. Drugs 1990;392456-861. 225. Lee PK, Deringer JR, Kreiswirth BN, Novick RP, Schlievert PM. Fluid replacement protection of rabbits challenged subcutaneously with toxic shock syndrome toxins. Infect Immun 1991; 592379484. Albertson TE. Cardiorespiratory failure in 226. Fisher Jr CJ, Horowitz toxic shock syndrome: effect of dobutamine. Crit Care Med1985; 13:160-165.

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Streptococcal Toxic Shock Syndrome Dennis L. Stevens of

THE EVOLUTION

STREPTOCOCCUSPYOCENES

Clinical descriptions of illnesses compatible with a variety of group A streptococcal (GAS) infections were recorded by Hippocrates (l), providing the earliest evidence that GAS and the human host have evolved together for at least thousands of years. The major consequence of this coevolution is that GAS has become a highly specialized pathogen. Evidence this statement may be derived from the observations that: 1) GAS has a restricted host range and is, in fact, a uniquely human pathogen; though family pets are occasionally colonized, no other animal species i s regularly and characteristically infected with GAS; and 2) GAS has developed an impressive armamentarium of molecules that interact with the human immune system in unique ways. The multiple consequences of the interaction of. these diverse molecules (virulence factors) with the humanhumoral and cellular immunological systems determine the array of clinical illnesses caused by GAS, such as pharyngitis, scarlet fever, erysipelas, cellulitis, lymphangitis, rheumatic fever, and poststreptococcal glomerulonephritis. The pathogenesisof many of these streptococcal infec481

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tions and their complications has been extensively investigated for over years, and though impressive progress has been made in the description of virulence factors, microbial genetics, typing of strains, pathological description, and epidemiology, definitive understanding of the pathogenesis of any GAS infection or its sequelae has not materialized. Investigations are complicated by the observations that multiple M types of GAS may be associated with a specific streptococcal clinical illnessesand yet a given M type may be associatedwith a variety of streptococcal diseases.

II. THE DECLINE O F SCARLET FEVER AND RHEUMATIC FEVER

'

The epidemiology of GAS is no less complicated than its virulence factors. Epidemics of scarlet fever have been well recorded since the 16th century andwere commonlyassociated with high mortality. In contrast, the prevalence and severity scarlet fever has greatly diminished throughout most of the 20th century and this decline began well before the development of penicillin. This phenomenon has been very dramatic in the Western world, though scarlet fever continues to be a significant, but poorly defined problem in underdeveloped countries. Similarly, rheumatic fever has become dramatically less common and less severe since WorldWar 11, in part owingtothe availability of penicillin in developed countries. Like scarlet fever, rheumatic fever continues to be a major problem in developing countries and as such is a leading cause death among patients less than 40. Thus, there is ample data to support the notion that either the virulence of GAS has waned or the resistance the human host has increased during the present century. As a consequence of the declining severity of GAS infections in the United States, the reporting of scarlet fever and rheumatic fever cases to public health agencies ceased ne&ly 20 years ago. THE EMERGENCE OF STREP TSS

Remarkably, in the latter half of the 1980s the Western world began to experience fulminant GAS infections associated with shock, organ .failure, necrotizing fasciitis, bacteremia, and death (reviewed in Ref. 2). Thus, the streptococcal toxic shock syndrome (Strep TSS) joined the ranks of newly emerging infectious diseases. Cases tend to be sporadic with a prevalence no more than 10-20 cases per 100,000 population (3-5). Secondary cases are rare but have been reported among family members where intimate contact with a patient has

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occurred (6,7), in nursing homes settings and among medical personnel caring for patients with invasive GAS infections (6,7,10). Since the first descriptions of Strep TSS (11-141, over additional reports have been published from the United States, Europe, Canada, Australia, New Zealand,' and the Orient (reviewed in Ref. 15). The case definition, clinical characteristics, pathogenesis, and treatment will be discussed in subsequent sections.

W.

CASE DEFINITION OF STREPTSS

It is quite clear that a wide variety of primary GAS infections may result in Strep TSS. In addition, not all the reported features of Strep occur in every patient, and finally, some patients with GAS infections may develop shock death, but only late in the course of illness. The spirit of the Working Group on Severe Group A Streptococcal Infections was to provide a case definition of Strep TSS requiring that shock and organ failure occur early in the course of infection (16) (Table 1). In addition, this definition acknowledges that the sites of infection may be diverse (Table 2) and not limited to necrotizing fasciitis, though the latter has been commonly associated with Strep V.

CLINICAL FEATURES OF STREP TSS

Symptoms

Strep TSS may begin insidiously, and approximately 20% of patients experience an influenza-like syndrome characterized by fever, chills, and myalgias (Table (14,171. Other patients may experience, nausea, vomiting, and diarrhea associated with fever prior to development of Strep TSS (author's unpublished observations). Pain, the most common initial symptom of Strep TSS, is abrupt and severe (14,17). Pain commonly precedestenderness any physical evidence of localized findings (Table Pain is most commonly localized to an extremity, though headache pain mimicking sinusitis, peritonitis, pelvic inflammatory disease, acute myocardial infarction, pericarditis has also been described (14,171.

B.

Physical Findings

Fever, the most common presenting sign, may range from99 to 106'F, except in patients with advanced disease who may develop profound hypothermia secondary to shock (14,17). Confusion is present in 55%

Stevens

Table 1 A Case Definition of Streptococcal Toxic Shock Syndrome Streptococcal toxic shock syndrome (Strep is defined as any group A streptococcal infection associated with the early onset of shock and organ failure. Definitions describing criteria for shock, organ failure, definite cases, and probable cases are included below.Because of the high profile of necrotizing fasciitisin the literature, in the press, and among the governments of the world, some health authorities have been charged with monitoring the incidence of necrotizing fasciitis as well. Recognizingthat some patients with Strep but not all, have necrotizing fasciitis, we have also included a case definition for necrotizing fasciitis. I. Isolation of group A Streptococcus A. From a sterile site B. From a nonsterile body site 11. Clinical signs of severity A. Hypotension and B. Clinical and laboratory abnormalities (requires two or more of the following): 1. Renal impairment 2. Coagulopathy Liver abnormalities 4. Acute respiratory distress syndrome 5. Extensive tissue necrosis, i.e., necrotizing fasciitis 6. Erythematous rash Definite case = 1A + 2(A+B). Probable case = 1B + 2(A+B). The following is a case definition for group A streptococcal necrotizing fasciitis. Definite necrotizing fasciitis. A. Necrosis of soft tissues with involvement of the fascia. Plus B. Serious systemic disease including one or more of the following: 1. Death 2. Shock (systolic blood pressure < 90 mmHg) Disseminated intravascular coagulopathy 4. Failure of organ systems a. Respiratory failure b. Liver failure c. Renal failure C. Isolation of group A streptococci from a normally sterile body site

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485

Table 1 Continued ~~~

11. Suspected necrotizing fasciitis:

A + B and serological confirmation of A streptococcal infection by a 4-fold titer rise against: A. Streptolysin 0 B. DNAase B A + and histological confirmation: Gram-positive cocci in a necrotic soft-tissue infection

Source: From Ref. 16.

of patients, and in some, coma or combativeness is manifest (14,171. Evidence of soft-tissue infection, such as localized swelling, tenderness, pain, and erythema, is often present at the time of admission and is apparent early where a cutaneous portal of entry is defined. The appearance of violaceous bullae suggestsdeeper soft tissue infection such as necrotizing fasciitis or myositis. In one series (17), 70% of patients required surgical debridement, fasciotomy, or amputation (17). The remainder of these patients displayed a variety of clinical presentations such as endophthalmitis, myositis, perihepatitis, peritonitis, myocarditis, puerperal sepsis, septic arthritis, pharyngitis, and overwhelming sepsis (Table 2) Hypotension and tachycardia out of proportion to the fever are present in most patients; however, in patientswith normal blood pressure (systolic pressure > 110 mmHg) at the time initial evaluation, hypotension developed rap-

Table 2 GAS Infections Associated with Strep 1. Necrotizing fasciitis

2. 3. 4. 5. 6. 7. 8. 9. 10.

Myonecrosis Pneumonia Septicjoint Peritonitis Sinusitis Epiglottitis Meningitis Cellulitis Pharyngitis (rare)

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T a b l e 3 Symptoms of Strep TSS

Pain Chills Myalgias

Pharyngitis (rare) Nausea Vomiting Diarrhea

idly (within 4 hr) (14,171. Massive local swelling occurs at the site of necrotizing fasciitis and myositis, and generalized interstitial edema develops in virtually all patients due to diffuse capillary leakiness, though this may not be clinically apparent until after intravenous fluid resuscitation. C.

laboratory TestResults

Though the initial white blood cell count may be normal or slightly elevated, the differential count demonstratesa strikingly high percentage of immature neutrophils including band forms, metamyelocytes, and myelocytes; the mean percentage exceeds 50% (14,17). Evidence of renal involvement is apparent in 80% of patients (mean serum creatinine > 2.5 times normal) (14,17). Hypoalbuminemia and profound hypocalcemia are prominent features at the time of admission and throughout the hospital course. Theserum creatinine kinase level may be useful in detecting the presence of deeper soft-tissue infections, and when the level is elevated or rising, there is a good correlation with the presence of deeper infections such as necrotizing fasciitis or myonecrosis (14,171. D. Clinical Course

Hypotension was apparent at the time of admission or within 4-8 hr in virtually all patients (Table 4). In 10% of patients systolic blood pressure normalized 4-8 hr after administration of antibiotics, albumin, and electrolyte solutions containing salts or dopamine. In other patients shock and renal dysfunction progressed persisted in all patients for 48-72 hr in spite of treatment, and several patients required dialysis for 10-20 days (14,17). Among survivors, serum creatinine values returned to normal within 4-6 weeks. Renal dysfunc-

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487

Table 4 Complications of Group A Streptococcal Soft-Tissue Infection Complication Percentage

Patients

Shock ARDS Renal impairment

95 55 80

Irreversible Reversible Bacteremia

10 70 60 30

Mortalitv Source: From Ref. 14.

tion preceded shock in many cases and was apparent early in the course of shock in all others. Acute respiratory distress syndrome (ARDS) occurred in 55% of patients in one series and generally developed after the onset of hypotension (14,171, which may in part be related to a diffuse capillary leak. Theseverity of ARDS was such that supplemental oxygen, intubation, and mechanical ventilation were necessary in 90% of patients who developed this syndrome (14,17). Sixty percent of patients with Strep TSS may be bacteremic and overall mortality may range from (14,171 to 60-70%(4,5). Morbidity has also been high, and approximately 50% of patients'require major surgical procedures, which include fasciotomy, surgical dbbridement, exploratory laparotomy, intraocular aspiration, amputation, and hysterectomy (14,171. Surgical exploration has demonstrated myositis/myonecrosis in over one-half of the patients with necrotizing fasciitis. VI.

FACTORSAFFECTINGTHEDEVELOPMENTANDPROGRESSION OF STREP TSS

Despite the fact that the strains of GAS associated with Strep TSS are found widely in communities, cases have remained sporadic and epidemics have not materialized, suggesting that simple contact with a virulent strain is not sufficient to cause Strep TSS. Thus, other predisposing factors may be necessary for Strep to occur (Table 5). Certain viral infections such as chicken pox can disrupt anatomical barriers in both mucosa and skin. Similarly, influenza virus infection alters respiratory epithelium sufficiently to provide a portal of entry. Viral infections may also have effects on the immune system, which

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Table 5 Predisposing Factors for the Development of Severe Group A Streptococcal Infection A. Antecedent virus infection 1. Varicella 2. Influenza B. Disruption ofanatomicalbarriers 1. Skin a. Chicken pox b. Lacerations c. Bums d. Bites (insects, dog, cat) e. Surgical wounds 2. Mucous membrane a. Virus infection b. Childbirth C. Nonpenetrating trauma 1. Muscletear 2. Hematoma D. Nonsteroidal anti-inflammatory agents

at this point have not been adequately studied. Disruption of anatomical barriers from any cause, such as lacerations, burns, slivers, surgical procedures, decubitus ulcers, intravenous drug abuse, bites (by insects, dogs, cats, etc.), provide cutaneous portals of entry. Childbirth, another risk factor, compromisesthe integrity of the uterine mucosa, allowing entry of The sourceof bacteria in this setting is usually GAS colonizing the vagina though GAS may also be introduced into the birth canal from the hospital environment or personnel In other patients with documented GAS bacteremia, a portal of entry cannot be ascertained and in some series, 50% of patients have cryptogenic bacteremia Since the these cases have frequently been associated with deep-seated infection such as necrotizing fasciitisand myositis In these instances, a clinical diagnosis may be difficult to establish until late in the course. In fact, a diagnosis of invasive GAS infection may not be entertained until shock and organ failure develop. In these cases, very specific predisposing factors are likely required. For example, these deep softtissue infections invariably occur at the exact site of blunt trauma, muscle strain, hematoma, joint effusion.

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These observations raise two important questions. First, how does nonpenetrating local trauma predispose to infection developing at that exact site? Second, in the absence of a penetrating injury, how does GAS arrive at the site of infection? The best hypothesis is that GAS translocates to the site of injury likely although a transient bacteremia from a mucous membrane i.e., pharyngeal source. The site of injury favors growth of the bacteria at the expense of clearance by professional phagocytes. Increasinglysevere pain develops within the tissue and precedes any of the other cardia1 manifestations of inflammation. At this point, many patients subject themselves to a second risk factor: the patient begins to take analgesics such as nonsteroidal anti-inflammatory agents. These agents adversely affect neutrophil chemotaxis, phagocytosis, and bacterial killing while promoting cytokine synthesis by macrophages (17). Though symptoms improve transiently, and fever (if any) is suppressed, the GAS infection progresses. A diagnosis may be difficultat this time because the infection is deep in muscle fascia and there may not be evidence of cellulitis, lymphangitis, erysipelas. VII. CLINICALCLUESANDDIFFERENTIALDIAGNOSIS

Patients with Strep initially present with shock a deep-seated infection such as necrotizing fasciitis. Thus, this section will discuss differential diagnosis of each of these entities. The abrupt onset of shock in a previously healthy individual has a limited number of causes. In addition to Strep TSS, staphylococcal TSS, Gram-negative sepsis, typhoid fever, Rocky Mountain spotted fever, (RMSF), meningococcemia, and overwhelming Streptococcus pneumoniue sepsis may cause fulminant shock in noncompromised patients. Heat stroke has been confused with some cases Strep TSS largely because.of the presence of elevated temperature, dehydration with evidence of renal impairment, confusion, and hypotension. The differential diagnosis of deep soft-tissue infection with without shock includes the following clinical entities: acute hemorrhage in the form of a retroperitoneal, intra-abdominal, deep softtissue bleed, deep vein thrombophlebitis, compartment syndrome, necrotizing fasciitis, and/or myonecrosis caused by mixed aerobic/ anaerobic bacteria Clostridium-species. Oncethe site of infection has been. localized by either clinical clues radiographic techniques, surgical intervention is not only useful for diagnostic purposes, but of major therapeutic importance. A decision to surgically explore a

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patient is easy when there is cutaneous evidence of an infectious process and the patient is either not responding to medical management is clinically deteriorating. It is more difficult to suspect necrotizing fasciitis in a patient when only increasingpain and fever are present and there is no cutaneous evidence of infection. Since about 50%of patients with Strep TSS have deep-seated soft-tissue infections, and only about 50% have obvious portals of entry, the aforementioned presentation is quite common. The physician’s job gets even more difficult when patients are taking nonsteroidal anti-inflammatory agents, aspirin, acetaminophen since theseagents reduce pain and suppress fever. Some of these agents may predispose patients to a more severe type of infection by their abilities to suppress phagocytic killing of bacteria and to alter the host’s response (21). Still, necrotizing fasciitis due to S. pyogenes progresses very rapidly and a delay in diagnosis for any reason is associated with a worse prognosis. In addition, the greater the delay, the greater the need for more extensive surgery. In some cases, the rapid onset of shock and organ failure may preclude surgical intervention. VIII.

PATHOGENIC MECHANISMS

Pyrogenic exotoxins induce fever in humans and animals and also participate in shock by lowering the threshold to exogenous endotoxin (17). Streptococcal pyrogenic exotoxins A (SPEA) and B (SPEB) induce human mononuclear cellsto synthesize not only tumor necrosis factor-a (TNF-a) (22) but also interleukin-lp (IL-lp) (23) and interleukin-6 (IL-6) (23), suggesting that TNF could mediate the fever, shock, and tissue injury observedin patients with Strep TSS (14). Interestingly, fever induced by injection of purified SPEA into rabbits is totally abolished by prior administration of a neutralizing monoclonal antibody against rabbit TNF (author’s unpublished observations). Pyrogenic exotoxin C has been associated with mild cases of scarlet fever in the United States (author‘s observations) and in England (24). The roles of two newly described pyrogenic exotoxins, streptococcal superantigen (SSA) (25) and mitogenic factor (MF) (26,271, in Strep TSS haves not been elucidated. M protein contributes to invasiveness through its ability to impede phagocytosis of streptococci by‘human polymorphonuclear leukocytes (PMNL) (28). Conversely, type-specific antibody against the M protein enhances phagocytosis (28). Following infection with a particular M type, specific antibody confers resistanceto challenge to

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viable GASof that M type (28). While M types 1 and 3 have been the most common strains isolated from cases of Strep TSS, many other M types, including M-12, M-28, M-6, M-18, and some nontypable strains, have also been isolated from such cases. M types 1 and 3 are also commonly isolated from asymptomatic carriers, patients with pharyngitis, and patients with mild scarlet fever (29,30). Using multilocus electrophoresis, Musser et al. (31) have shown that strains from patients with Strep TSS fit unique genetic patterns that correspond to M-l M3 strains. Cleary et al. (32) studied M-l strains from invasive cases and noninvasive cases and were able to demonstrate distinct DNA fingerprint patterns that they termed invasinpositive invasin-negative. The invasin-positive strains contained additional genetic elements, at least one of which coded for SPEA. Strains of GAS may acquire genetic information coding for SPEA or SPEC via certain bacteriophage, and following lysogenic conversion, toxin is synthesized synchronously with the growth cycle of the Strep tococcus (33-35). Still, the mere presence of a potential virulence factor by such strains may be less important than the quantity that is produced. Quantitation of putative virulence factors from strains of GAS isolated from patients with Strep TSS has not been definitive, but Cleary et al. (36) have proposed that GAS possess a virulence regulon that controls the production of a gene cluster of virulence factors such as M protein and C5a peptidase. Could the streptococcal toxic shock syndrome be related to the ability of streptococcal pyrogenic exotoxins M proteins type 1 3 to act as “superantigens” (37)? Some data suggest that SPEA and a number of staphylococcal toxins (TSST-1 and staphylococcal enterotoxins A, B, and C) can stimulate T-cell responses through their ability to bind to both the class I1 MHC complex of antigen presenting cells and the Vp region of the T-cell receptor (37). The net effect would be to induce T-cell stimulation with production of cytokines capable of mediating shock and tissue injury. Recently, Hackett and Stevens demonstrated that SPEA induced both TNF-a and TNF-P from mixed cultures of monocytes and lymphocytes (38), supporting an important role for lymphocyte-derived cytokines in shock associated with strains producing SPEA. Kotb et al. (39) have shown that a digest of M protein type 5 can also stimulate T-cell responses by this superantigen mechanism. Thus, in vitro results suggest that superantigens could play a role in the pathogenesis of Strep TSS. Proof would require demonstration of massive expansion of T-cell subsets bearing Vp repertoires specific for the putative superantigen(s1. Re-

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cently, quantitation of such T-cell subsets in patients with acute Strep demonstrated deletion rather than expansion, suggesting that perhaps the life span of the expanded subset was shortened by the process of apoptosis (40). In addition, the subsets deleted were not specific for SPEA, SPEB, SPEC or MF, suggesting that perhaps an as yet undefined superantigen may exist (40). Cytokine production by less exotic mechanisms may also contribute to the genesis of shock and organ failure. Peptidoglycan, lipoteichoic acid (41), and killed GAS organisms (42,43) are capable of stimulatingTNF-a production by mononuclear cells invitro (17,43,44). Exotoxins such as streptolysin 0 (SLO) are also potent inducers of TNF-a and IL-lp. SPEB, a proteinase precursor, has the ability to cleave pre-IL-lp to release preformed IL-lP (45). Finally, SLO and SPEA together have additive effects in the induction of ILl p by human mononuclear cells (38). Whatever the mechanisms, induction of cytokines in vivo is likely the cause of shock, and many GAS virulence factors such as SPEA, SPEB and SPEC, as well as cell wall components, are potent inducers of TNF and IL-1. Thus, the specific GAS virulence factors that stimulate cytokine production in patients may be complex, particularly among the 60% of Strep TSS patients who are bacteremic. Here the systemic immune system would be exposed to a barrage of GAS virulence factors all of which are capable of cytokine induction through a variety of cellular mechanisms. The interactionbetween these microbial virulence factorsand an immune or nonimmune host determines the epidemiology, clinical syndrome, and outcome. Since horizontal transmission ofGAS in general is well documented, then the only explanation for the absence of a high attack rate of invasive infection is the presence of significant herd immunity against one or more of the virulence factors responsible Strep This model explainswhy epidemics have not materialized and why a particular strain of GAS can cause different clinical manifestations in the same 'community (46). TREATMENT A.

Antibiotic Therapy-Importance of the Mechanism of Actio'n

S. pyogenes remains exquisitely susceptibleto p-lactam antibiotics, and numerous studies have demonstrated the clinical efficacy of penicillin preparations for treating erysipelas, impetigo, cellulitis, and pharyngitis and for prevention of acute rheumatic fever(47). Nonetheless,

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clinical failures of penicillin treatment of streptococcal infection do occur. In addition, penicillin fails to eradicate bacteria from the pharynx in 5-20% of patients with symptomatic GAS pharyngitis (48-50). Moreover, more aggressive GAS infections (such as necrotizing fasciitis, empyema, burn wound sepsis, subcutaneous gangrene, and myositis) respond less well to penicillin and continue to be associated with high mortality and extensive morbidity (14,17,20,51-54). Studies of experimental infection suggest that penicillin fails when large numbers of organisms are present as a result of either injection of large numbers of bacteria or delayed treatment (55,561. For example, in a mouse model myositis due to S. pyogenes, penicillin was ineffective when treatment was delayed 22 hr after initiation of infection (56). Survival of erythromycin-treated mice was greater than that of both penicillin-treated mice and untreated controls, but only if treatment was begun within 2 hr. Mice receiving clindamycin, however, had survival rates of loo%, and 70% when treatment was delayed 2, 6, and 16.5 hr, respectively (56,571. Eagle suggested that penicillin failed in this type of infection because of the ”physiologic state of the organism’’ (55). Recent studies suggest that this is due to an in vivo inoculum effect (58,591. Thus, early in the stages of severe infectionor later in the course of initially mild infections, organisms are growing rapidly and are present in rather small numbers. With delays in treatment higher concentrations of GAS accumulate and growth begins to slow. That high concentration S. pyogenes accumulate in deep-seated infection is supported by data from Eagle (55). These observations fit with the widely held knowledge that penicillin is most effective against rapidly growing bacteria. Why should penicillin lose its efficacy when large numbers of GAS are present or when they are making the transition from logarithmic growth to stationary? Since penicillin mediates its antibacterial action against GAS by interacting with the bacterial penicillin binding proteins we hypothesized that expression of PBPs may decline during stationary phase. Indeed, binding of radiolabeled penicillin by all PBPs was decreased in stationary cells (58). Thus, the loss of certain during stationary-phase growth in vitro may be responsible for the inoculum effect observedin vivo and may account for the failure of penicillin in both experimental and human cases of severe streptococcal infection. Though these studies suggest one explanation for penicillin failure, the greater efficacy of clindamycin in severe GAS infections is due to many factors. First, its efficacy is not affected by inoculum size or stage of growth (58,60). Second, clindamycin isa potent suppressor

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of bacterial toxin synthesis (61,62). Third, clindamycin facilitates phagocytosis of pyogenes by inhibiting M-protein synthesis (62). Fourth, clindamycin suppresses synthesis of penicillin-binding proteins, which, in addition to being targets for penicillin, are also enzymes involved in cell wall synthesis and degradation (60). Fifth, clindamycin has a longer postantibiotic effect than p-lactams such as penicillin. Finally, we have recently shown that clindamycin causes suppression of LPS-induced monocyte synthesis of TNF-a (63). Thus, clindamycin’s efficacy may also be related to its ability to modulate the host’s immune response.

B. Other Treatment Measures Though antibiotic selection is critically important, other measures such as prompt and aggressive exploration and debridement of suspected deep-seated pyogenes infection is mandatory. Initially, the patient may have only fever and excruciating pain. Later systemic toxicity develops and definite evidence of necrotizing fasciitis and myositis appears. Unfortunately, surgical debridement may be too late at this point. Prompt surgical exploration through a small incision with visualization of muscle and fascia and timely Gram stain of surgically obtained material may provide an early and definitive etiological diagnosis (64). It is critically important that our surgical colleagues be involved early in such cases, since later in the course surgical intervention may be impossible due to toxicity or because infection has extended to vital areas that are impossible to debride (i.e., the head and neck, thorax, or abdomen). Anecdotal reports suggest that hyperbaric oxygen has been used in a handful of patients, though no controlled studies are underway nor is it clear if this treatment is useful. Because of intractable hypotension and diffuse capillary leak, massive amounts of intravenous fluids (10-20 L/day) are often necessary. Pressors such as dopamine are used frequently, though no controlled trials have been performed in Strep TSS. In patients with intractable hypotension, potent vasoconstrictors such as epinephrine have been used, but symmetrical gangrene of digits seems to result frequently (author’s unpublished observations), often with loss multiple limbs. In these cases it is difficult to determine if symmetrical gangrene was due to pressors or infection or a combination of these factors. Neutralization of circulating toxins would be a desirable therapeutic modality, yet specific antibody preparations are not commer-

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cially available in the United States or Europe. Swedish investigators have demonstrated that some commercialintravenous gamma globulin (IVIG) preparations do, in fact, contain neutralizing antibodies against several GAS factors such as the pyrogenic exotoxins (SPEA, SPEB, SPEC, and MF) as well as and DNase B (5,65). Based on these data, clinicians have used gamma globulinpreparations to treat patients with Strep TSS. Recently, three case reports have been published that describe patients who dramatically improved following administration of IVIG (66-68). In another publication, four patients received either intramuscular or intravascular gamma globulin preparations and seven others received fresh frozen plasmaor whole blood, with or without plasma exchange (69). Of these 11patients, survived. I n a larger series published in abstract form, 15 patients were given IVIG and mortality was reduced from 66% to None of these studies were randomized and the latter one used historical controls for comparison. Thus, although administration ofIVIG appears promising in the treatment of Strep TSS, definitive proof awaits controlled clinical trials. Toxin neutralization may not be the only immune mechanism responsible for the efficacy gamma globulin preparations in the treatment of Strep TSS. For example, gamma globulin“nonspecifically’’ inhibits TNF-a production by either LPS-stimulated monocytes (71) or splenocytes from animals with adjuvant-induced arthritis (72). Thus, IVIG might be efficacious by specific neutralization of GAS toxins, by nonspecific inhibition of monocyte/T-cell ac-. tivation, or by inhibition other putative GAS virulence factors (Table 6). Recently a monoclonal antibody against TNF-a showed promising efficacy in a baboon model of Strep TSS In summary, if a wild ”flesh-eating strain” has recently emerged, a major epidemic with a high attack rate would be expected. Clearly epidemics of streptococcal infections, including impetigo, pharyngitis, scarlet fever, and rheumatic fever, have occurred in the past. In the last decade subsequent to early reports of Strep we conclude

Table 6 Factors Favoring a Poor Outcome 1; Delay in diagnosis

2. Ineffectiveness of cell-wall-active antimicrobials 3. Early onset of shock and organ failure 4. Lackof available antitoxins 5. Lackof effective anticytokine modalities

Stevens

that the incidence has remained relatively low. Large outbreaks have not occurred because 1 ) the vast majority of the population probably has immunity to one or more streptococcal virulence factors (17,74); 2) predisposing conditions (varicella, use of NSAIDs, etc.) are required in a given patient (21); and there may be only a small percentage of the population having an inherent predisposition to severe streptococcal infection by virtue of constitutional factors such as HLA class I1 antigen type (75,761, B-cell alloantigens (771, or specific Vp regions on lymphocytes.This hypothesis is further supported by the observation 'that secondary cases of Strep TSS, though reported (6), have been rare. REFERENCES 1. Hippocrates. Prognostic XXII. In: Capps E, Page TE, Rouse WHD, e&. Hippocrates, with an English translation by W. H. S. Jones. Cambridge, MA: Harvard University Press, 1923:47-49. 2. Stevens D. Streptococcal toxic shock syndrome: spectrum of disease, pathogenesis and new concepts in treatment. Emerg Infect Dis 1995; 1:69-78. 3. Schwartz B, Facklam RR, Brieman RF. Changing epidemiology of group A streptococcal infection in the USA. Lancet 1990;336:1167-1171. 4. Demers B, Simor AE, Vellend H, Schlievert PM, Byrne S, Jamieson F, et al. Severe invasive group A streptococcal infections in Ontario, Canada: 1987-1991.. Clin Infect Dis 1993; 16:792-800. 5. Holm SE, Norrby A, Bergholm AM, Norgren M. Aspects of pathogenesis of serious group A streptococcal infections in Sweden, 1988-1989. J Infect Dis 1992; 166:31-37. 6. Schwartz B, Elliot JA, Butler JC, Simon PA, Jameson BL, Welch GE, et al. Clusters of invasive group A streptococcal infections in family, hospital, and nursing home settings. Clin Infect Dis 1992; 15(2):277-284. 7. Dipersio JR, Define LA, Gardner W, Stevens DL, Kaplan EL, File TM. Use of pulsed field gel electrophoresis to investigate the clonal spread of severe group A streptococcal disease. ASM Abstracts 1995. Auerbach SB, Schwartz B, Williams D, Fiorilli MG, Adimora AA, Breiman RF, et al. Outbreak of invasive group A streptococcal infections . in a nursing home: lessons on prevention andcontrol. Arch Intern Med 1992;152(5):1017-1022. 9. Hohenboken JJ, Anderson F, Kaplan EL. Invasive group A streptococcal (GAS) serotype M-l outbreak in a long-term care facility (LTCF) with mortality. ICAAC 1994. Abstract #Jl89. 10. Valenzuela TD, Hooton TM, Kaplan EL, Schlievert PM. Transmission of "toxic strep" syndrome from aninfected child to a firefighter during CPR. Ann Emerg Med 1991; 20:1:123-125.

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Bloomster TG. Hypocalcemia and "toxic" syndrome associated with streptococcal fasciitis. South Med J 1983; 76 (7):916-918. Cone LA, Woodard DR, Schlievert PM, Tomory GS. Clinical and bacteriologic observations of a toxic shock-like syndrome due to Streptococcus pyogenes. N Engl J Med 1987; 317:146-149. Bartter T, Dascal A, Carroll K, Curley FJ."Toxic strep syndrome": manifestation of group A streptococcal infection. Arch Intern Med 1988; 148:1421-1424. Stevens DL, Tanner MH, Winship J, Swarts R, Reis KM, Schlievert PM, et al. Reappearance of scarlet fever toxin A among streptococci in the Rocky Mountain West: severe Group A streptococcal infections associated with a toxic shock-like syndrome. N Engl J Med 1989; 321(1):1-7. Stevens DL. Introduction to soft tissue infections. In: Stevens DL, ed. Mandell's Infectious Disease Teaching Atlas. Philadelphia: Churchill Livingstone, 1994. Working Group on Severe Streptococcal Infections. Defining the group A streptococcal toxic shock syndrome: rationale and consensus definition. JAMA 1993; 269(3):390-391. Stevens DL. Invasive group A streptococcus infections. Clin Infect Dis 1992;14:2-13. Stamm WE, Feeley JC, Facklam RR. Wound infections due to group A Streptococcus traced to a vaginal carrier. J Infect Dis 1978; 138:287-292: Bibler MR, Rouan GW. Cryptogenic group A streptococcal bacteremia: experience at an urban general hospital and review of the literature.Rev Infect Dis 1986; 8:941-951. Adams EM, Gudmundsson S, YocumDE, Haselby RC, Craig WA, Sundstrom WR. Streptococcal myositis.Arch Intern Med 1985; 145:10201023. Stevens DL. Could nonsteroidal anti-inflammatory drugs (NSAIDs) enhance the progression of bacterial infections to toxic shock syndrome? Clin Infect Dis 1995; 21:977-980. Fast DJ, Schlievert PM, Nelson RD. Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducersof tumor necrosis factor production. Infect Immun 1989; 57:291-294. Hackett SP, Schlievert PM, Stevens DL. Cytokine production by human mononuclear cells in response to streptococcal exotoxins. Clin Res 1991; 39:189A (abstract). Hallas G. The production of pyrogenic exotoxins by group A streptococci. J Hyg (Camb) 1985;95:47-57. Mollick JA, Miller GG, Musser JM, Cook RG, Grossman D, Rich RR. A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminalhomology to staphylococcal enterotoxins B and C. J Clin Invest 1993; 92:710-719. Iwasaki M, Igarashi H, Hinuma Y, Yutsudo T. Cloning, characterization

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and overexpression of a Streptococcus pyogenes gene encoding a new type of mitogenic factors. FEBS Lett 1993; 331:187-192. Norrby-Teglund A, Newton D, Kotb M, Holm SE, Norgren M. Superantigenic properties of the group A streptococcal exotoxin SpeF (MF). Infect Immun 1994; 625227-5233. Lancefield RC. Current knowledge of type specific M antigens of group A streptococci. J Immunol 1962; 89:307-313. Johnson DR, Stevens DL, Kaplan EL. Epidemiologic analysis of group A, streptococcal serotypes associated with severe systemic infections, rheumaticfever,oruncomplicatedpharyngitis. J Infect Dis 1992; 166:374-382. Kohler W, Gerlach D, Knoll H. Streptococcal outbreaks and erythrogenic toxin type A. Zentralbl Bakt Hyg 1987;266:104-115. Musser JM, Hauser AR, Kim MH, Schlievert PM, Nelson K, Selander RK. Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression. Proc Natl Acad Sci USA 1991; 88:2668-2672. Cleary PP, Kaplan EL, Handley JP, Wlazlo A, Kim MH, Hauser AR, et al. Clonal basis for resurgence of serious streptococcus pyogenes disease in the 1980s. Lancet 1992; 339:518-521. Nida SK, Ferretti JJ. Phage influence on the synthesis of extracellular toxins in group A streptococci. Infect Immun 1982; 36(2):745-750. Hauser AR, Goshorn SC, Kaplan E, Stevens DL, Schlievert PM. Molecular analysis of the streptococcal pyrogenic exotoxins. Third International ASM conference on Streptococcal Genetics 1990; Minneapolis, MN. Abstract. Johnson LP, Tomai MA, Schlievert PM. Bacteriophage involvement in group A streptococcal pyrogenic exotoxinA production. J Bacterioll986; 166:623-627. Cleary R, Chen C, Lapenta D, Bormann N, Heath D, Haanes E. A virulence regulon in Streptococcus pyogenes. Third International ASM conference on Streptococcal Genetics 1990; Minneapolis, MN. Abstract #19. Mollick JA, Rich RR. Characterization a superantigen from a pathogenic strain of Streptococcus pyogenes. Clin Res 1991; 39(2):213A. Hackett SP, Stevens DL. Streptococcaltoxic shock syndrome: synthesis of tumor necrosis factor and interleukin-l by monocytes stimulated with pyrogenic exotoxin A and streptolysin 0. J Infect Dis 1992; 1659379-885. Kotb M, Tomai M, Majumdar G, Walker Beachey EH. Cellular and biochemical responses of human T lymphocytes stimulated with streptococcal M protein. XI Lancefield International Symposium on Streptococcal Diseases, 1990; Sienna, Italy. Abstract. Watanabe-Ohnishi R, Low DE, McGeer A, Stevens DL, Schlievert PM, Newton D, et al. Selective depletion VP-bearing T cells in patients with severe invasive groupA streptococcal infections and streptococcal toxic shock syndrome. J Infect Dis 1995; 171:74-84.

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41. Stevens DL, Bryant AE, Hackett SP. Gram-positive shock. Curr Opin Infect Dis 1992; 5(3):355-363. 42. Hackett S, Ferretti J, Stevens D. Cytokine induction by viable group A streptococci: suppression by streptolysin 0. American Society of Microbiology, 1994. Abstract B-249. 43. Muller-Alouf H,Alouf JE, Gerlach D, Ozegowski JH, Fitting C, Cavaillon JM.Comparative study of cytokine release by human peripheral blood mononuclear cells stimulated with Streptococcuspyogenes superantigenicerythrogenictoxins,heat-killedstreptococci and lipopolysaccharide. Infect Immun 1994; 62:4915-4921. 44. Hackett SP, Stevens DL. Superantigens associated with staphylococcal and streptococcal toxic shock syndrome are potent inducers of tumor necrosis factor beta synthesis. J Infect Dis 1993; 168(1):232-235. Musser JM. Cleavage of 45. Kappur V, Majesky MW,LiLL,BlackRA, Interleukin 1B (IL-1B) precursor to produce active IL-1B by a conserved extracellular cysteine protease fromStreptococcus pyogenes. Proc Natl Acad SciUSA 1993; 90:7676-7680. 46. Stevens DL. Invasive group A streptococcal infections:the past, present and future. Pediatr Infect Dis J 1994; 13:561-566. 47. Wannamaker LW, Rammelkamp CH, Jr., Denny W ,Brink WR, Houser HB, Hahn EO, et al. Prophylaxis of acute rheumatic fever by treatment of the preceding streptococcal infection with various amounts of depot penicillin. Am J Med 1951; 10:673-695. 48. Kim KS, Kaplan EL. Association of penicillin tolerance with failure to eradicate group A streptococci from patients with pharyngitis.J Pediatr 1985; 107:681-684. 49. Gatanaduy AS, Kaplan EL, Huwe BB, McKay C, Wannamaker LW. Failure of penicillin to eradicate group Astreptococci during an outbreak of pharyngitis. Lancet 1980; 2:498-502. 50. Brook I. Role of beta-lactamase-producing bacteria in the failure of penicillin to eradicate group A streptococci. Pediatr Infect Dis 1985; 4:491495. 51. Martin PR, Hoiby EA. Streptococcal serogroup A epidemic in Norway 1987-1988. Scand J Infect Dis 1990; 22:421-429. 52. Kohler W. Streptococcal toxic shock syndrome. Zentralbl Bakt 1990; 272:257-264. 53. Hribalova V. Streptococcus pyogenes and the toxic shock syndrome. Ann Intern Med 1988; 108:772. 54. Gaworzewska ET, Coleman G. Correspondence: Group A streptococcal infectionsanda toxic shock-like syndrome.N Engl J Med 1989; 321( 22) 9546. 55. Eagle H. Experimental approach to the problem of treatment failure with penicillin. I. Group A streptococcal infection in mice. Am J M 4 1952; 13:389-399. 56. Stevens DL, Gibbons AE, Bergstrom R, Winn V. The Eagle effect revis-

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ited: efficacy of clindamycin, erythromycin, and penicillin in the treatment of streptococcal myositis. J Infect Dis 1988; 158:23-28. 57. Stevens DL, Bryant AE, Yan S. Invasive group A streptococcal infection: new concepts .in antibiotic treatment. Int J Antimicrob Agents 1994; 4:297-301. 58. Stevens DL, Yan S, Bryant AE. Penicillin- binding protein expression at different growth stages determines penicillin efficacy in vitro and in vivo: an explanation for the inoculum effect. J Infect Dis 1993; 167:14011405. 59. Yan S, Mendelman PM, Stevens DL. The in vitro antibacterial activity of ceftriaxone against Streptococcus pyogenes is unrelated to penicillinbinding protein 4. FEMS Microbiol Lett 1993; 110:313-318. 60.Yan S, Bohach GA, Stevens DL. Persistant acylation of high-molecular weight penicillin binding proteinsby penicillin induces the post antibiotic effect in Streptococcus pyogenes. J Infect Dis 1994; 170:609-614. 61. Stevens DL, Maier KA, Mitten JE. Effect of antibiotics on toxin productionand viability of Clostridium perfringens. AntimicrobAgents Chemother 1987; 31:213-218. 62. Gemmell CG, Peterson PK, Schmeling D, Kim Y, Mathews Wannamaker L, et al. Potentiation of opsonization and phagocytosis of Streptococcus pyogenes following growth in thepresence of clindamycin.. J Clin Invest 1981; 67:1249-1256. 63. Stevens DL, Bryant AE, Hackett SP. Antibiotic effects on bacterial viability,toxinproduction,andhostresponse.Clin Infect Dis 1995; 20:S154-S157. 64. Bisno AI, Stevens DL. Streptococcal infections in skin and soft tissues: N Engl J Med 1995. 65. Norby-Teglund A, Kaul R, LowDE,McGeerA, Kotb M. Intravenous immunoglobulin and superantigen-neutralizing activity in streptococcal toxic shock syndrome patients. 35th Annual ICAAC, 1995. Abstract. 66. Barry W, Hudgins L, Donta S, Pesanti E. Intravenous immunoglobulin therapy for toxic shock syndrome. JAMA 1992; 267:3315-3316. 67.Yong JM. Letter. Lancet 1994;343:1427. 68. Nadal D, Lauener RP,Braegger Cl', Kaufhold A, Simma B, Lutticken R, et al. T cell activation and cytokine release in streptococcal toxic shock-like syndrome. J Pediatr 1993; 122:727-729. 69. Stegmayr B, Bjorck S, Holm S, Nisell Rydvall A, Settergren B. Septic shock induced by group A streptococcal infection: clinical and therapeutic aspects. Scand J Infect Dis 1992; 24:589-597. 70. Kaul R, McGeerA, Norby-Teglund A, Kotb M, LowD. Intravenous immunoglobulin therapy instreptococcal toxic shock syndrome: results of a matched case-controlled study. 35th Annual ICAAC, 1995. Abstract. 71. Shimozato T, Iwata M, Tamura N. Suppression of tumor necrosis factor alpha production by a human immunoglobulin preparation for intravenous use. Infect Immun 1990; 58:1384-1390.

ehock

Toxic Streptococcal

. 501

72. Achiron A, Margalit R, Hershkoviz R, Markovits D, Reshef T, Melamed E, et al. Intravenous immunoglobulintreatment of experimental T cellmediated autoimmune disease: upregulation of T cell proliferation and downregulation of tumor necrosis factor a secretion. J Clin Invest 1994; 93:600-605. 73. Stevens DL, Bryant AE, Hackett SP, Chang A, Peer G, Kosanke S, et al. Group A streptococcal bacteremia: the role of tumor necrosis factor in shock and organ failure. J Infect Dis 1995. 74. Stegmayr B, Bjorck S, Holm S, Nisell Rydvall A, Settergren B. Septic shock induced bygroup A streptococcal infections: clinicaland therapeutic aspects. Scand J Infect Dis 1992; 24:589-597. 75. Greenberg LJ, Gray ED, Yunis E. Association of HL-A5 and immune responsiveness in vitro to streptococcal antigens. J Exp Med 1975; 141:934-943. 76. Weinstein L, Barza M. Gas gangrene. N Engl J Med 1972; 289:1129. 77. Zabriskie JB, Lavenchy D, Williams RCJ, et al. Rheumatic-fever associated B-cell alloantigens as identified by monoclonal antibodies. Arthritis Rherm 1985;28:1047-1051.


!

Viral Superantigens in Humans HIV

David N. Posnett

Cornell University Medical College, New York, New York

INTRODUCTION

From studies on mouse mammary tumor virus (Mtv) superantigens (SAG), it has become clear that viral SAG play a crucial role in the life cycle of infectious Mtv, which are vertically transmitted milkborne viruses. The gene of these viruses in the 3’ORF, directs activation of a targeted T-cell BV (Vp) subset, and T/B collaboration results in B-cell proliferation and 105-fold amplification of the viral load in B lymphocytes during the first week of infection (1,2). Without the gene effect, e.g., in a Mtv transgenic mouse that has deleted the target BV subset, viral infection is inefficient Thus, in this well-studied case the SAG benefits the virus. Both viral and bacterial SAGS cause T-lymphocyte proliferation usually among CD4 and CD8 cells. This may include not only the directly targeted BV subsets but also bystander cells such as lymphocytes that benefit from cognate T/B interactions and T-derived cytokines (1,2,4-6). Both transient acute exposure and chronic exposure to SAGS then cause variable degreesof depletion of the target BV T-cell subset or anergy. 503

504

When considering human acute viral infections for possible involvement of one usually considers thoseacute viral infections associated with absolute .lymphocytosisand an infectious mononucleosis-like syndrome. Relative lymphocytosis is common with most viral infections, but prominent absolute lymphocytosis is relatively scarce. Viral infections that cause prominent lymphocytosis may also be characterized by atypical lymphocytes seen on a peripheral blood smear. Infectious agents known to cause prominent clinical lymphocytosis include those listed in Table 1, and several reports have examined the evidence for associated in some cases. The available evidence for in human viral infections will be discussed in this chapter. The rabies virus nucleoprotein is discussed in Chapter 5 , and evidence for an EBV is presented in Chapter 6. The single report of activity associated with the parasite Toxoplasma gondii will not be further discussed as this is likely not a viral .It should be noted that acute infection with Toxoplasma is characterized by absolute.lymphocytosis. HTLV-l is a candidate for the production of a as it is known to induce TCR-mediated proliferation resting T cells without requiring infection of the T cells (8-10). T-cell stimulation can be blocked with anti-gp46 (env).These findings may in part explain the cofactor effect of HTLV-1 in HIV-1 infection (9). Surprisingly, there are no data on the mechanism this T-cell mitogenic effect of HTLV1 and no BV repertoire analysis. single report suggests that the Bel protein of the human foamy virus (spuma virus) acts as a (11). Bel isencoded in the

Table 1 Viral Infections with Possible EBV (HHV4) Cytomegalovirus (HHV5) Roseola infantum (HHV6) Herpes varicella/zoster (HHV3) Mumps Hepatitis A HTLV I infection HIV Rabies virus Spuma virus

Involved

Lymphocytosis

SAG reported

++ ++ ++ + + + +

+ +

+/? ?

6

Ref. Chapter 54

ND ND ND 83

ND

+ + +

41,43,48-50 Chapter 84-86, 5 11

ND: not done, but listed here becauseof associated lymphocytosis."-" means that a TCR reper-

toire analysis did not uncover skewed BV gene usage.

Viral Superantigens in Humans

505

3'ORF of this retrovirus at the equivalent location to the gene of MTv. The recombinant purified gene product, expressed in Escherichia coli, activated normal human Pbl over 5 days to proliferate and the BV18 subset was specifically expanded by RT-PCR in 6/9 unrelated donors. This preliminary report needs to be extended. Natural infection with human foamy virus initially induces a strong immune response and then causes a chronic persistent asymptomatic infection (12,13). It could lead to activation HIV-1 transcription (14). Simian foamy virus causes immunosuppression in experimentally infected monkeys and human foamy virus causes a transient immhnosuppression in inoculated rabbits and mice (15): II. ROLE OF SUPERANTICENS IN HIV INFECTION

TCR Repertoire

The topic of an HIV-associated SAG has been extensively covered in the literature. There is little consensus for a single HIV gene product acting as a SAG (16). The original observation of deleted BV 14-20 subsets in symptomatic HIV-l-infected patients (17) was basedon RTPCR methodology and has not been confirmed. When T-cell yields are low, as in the case of lymphopenic patients, the smaller BV subsets (such as BV14-20) may yield insufficient RNA forPCR amplification, providing a possible explanation for these results. A number of follow-up papers claimed specific changes in various BV subsets without reaching a consensus (18-211, and others found no specific BV subsets.either selectively expanded deleted in HIV-l+ patients (2225). Samples from both symptomatic AIDS patients and asymptomatic seropositive patients were studied. In the latter it is less likely that repertoire changes are due to opportunistic infections, but ubiquitous herpes viruses (and other clinically silent pathogens) could have effects on the TCR repertoire. Human BV repertoire studies that fail to show specific BV changes are difficult to interpret. Careful studies in mice have shown that numerous variables influence the extent of the BV expansion/ deletion that follows in vivo administration of a bacterial superantigen (26). These variables may includethe dose of SAG, the route of exposure (IV IP), single dose versus repeated exposures, the type of antigen-presenting cell (27), analysis of BV subsets in the CD4 versus CD8 T cells, analysis of T cells from. blood, spleen, peripheral blood, mesenteric LNs, analysis of "high-affinity" target BV sub-

Posnett

506

sets versus "low-affinity" target subsets, and the presence of endogenous SAGS that might have overlapping specificity for target BV subsets. The list of variables that have confounded an interpretation of the human repertoire studies in HIV+ patients is well discussed by Westby et al. (16) and may explain the lack of consensus in the literature. The list includes the technique used (PCR vs. cytofluorometry), the stage of disease (AIDS, asymptomatic stage, or acute infection), the tissue source (Pbl vs. lymph nodes), HLA-matched versks nonmatched populations, in vitro studies involving cell culture versus ex vivo analysis, and Ags/SAGs encoded by HIV itself versus derived from other microbial pathogens frequently associated with HIV infection. A comparison of BV subsets in blood and LNs of HIV patients has demonstrated striking differences (28,29), and even within the same organ-the spleen-the TCR repertoire of separate lymphoid aggregates differs dramatically (30). In contrast, the repertoire is quite uniform inmormal lymphnodes and blood from the same individual (D. N. Posnett, unpublished data). HLA differences in outbred human populations may result in different SAG responses, as HLA-DR alleles can differ in their efficiency for SAG presentation (31,32). This problem has beenaddressed in a few studies withtwin pairs discordant for HIV infection (33,341, but uniform BV perturbations could not be identified in these studies either. Another problem relates to the extensive heterogeneity of HIV sequences and isolates. Even within a single patient the numbers of pseudospecies present may be large, and i f each species were to encode a SAG with a different BV specificity, the BV-specific effect of a single viral species would be obscured. B.

CD8 Clones

In mice clonal CD8 T-cell responses can represent large percentages of the total CD8 population. For instance, the response to the HLA CW3 peptide 170-179 presented by H-2Kd is primari1.y composed of BVlO CD8 cells in DBA/2 mice (35). When the Ag is administered IP in the form of lo7 P815 cells transfected with HLA-CW3, the BVlO response 10 days after immunization comprises 85% ofCD8 cells in the peritoneum, 65% in the Pbl, 35% in the spleen, 25% in the mesenteric lymph nodes, and is barely detectable in axillary/inguinal


507

In humans CD8 cells also appear to readily undergo clonal expansion in response to Ag exposure, such that many normal individuals contain clones in their Pbl that are detectable with BV-specific antibodies (36). Some interesting repertoire studies have focused on patients with acute HIV infection. Pantaleoet al. describe expansionsof clonal CD8 cells in 4/6 patients (37). These do not involve a common BV gene and appear to represent CD8 clones reactivewith viral antigens. A study of eight patients with acute infectious mononucleosis-like syndrome followed shortly by seroconversion for HIV-1 (38,39) revealed no selective BV deletions in CD4 or CD8 subsets: 3/8 patients showed expansions of CD8 clones as in thePantaleo study. Such CD 8 BV expansions are mono- oligoclonal and may persist in vivo for 31 months (40). These shifts in the repertoire are unrelated to the presence of superantigens. However, it is not difficult to appreciate how they may confound analyses designedto detect the effect of a superantigen in vivo, especially since similar clonal expansions have now been described in some CD4+ subsets. C.

BV8 Anergy

Gougeon and co-workers have described anergic BV8 T cells in 56% of patients from a French HIV-seropositive cohort (41). The anergy was detected as a lack of response to the streptococcal SAG erythrogenic toxin A (ETA). A nice internal control was the BV12 response to ETA, which remained intact in all the patients. Both CD4 and CD8 BV8 cells were anergic, and the response to other BV8 specific ligands (SEE, BV8 MAb) was also lacking. BV8 anergy was not observed in any of their normal controls. The data are consistent with the effects of a SAG, but it is not yet clear whether HIV or some other opportunistic microbe is the cause. For example, Mycobacterium tuberculosis has been reported to produce a BV8-specific SAG (42). Nef

Torres and Johnson have shown that a synthetic HIV-1 nef peptide, 123-160, blocks binding ofSEE and SEA to Raji B cells and binds directly to HLA-DR1. Two different sources of purified Nef protein stimulated IL-2 production and proliferation of T cells, with a stimulation index of 5-10 using 1 pg/ml of Nef (43). The nef gene is known to enhance HIV-1 replication in resting human T cells (44,45) and this

Posnett

might thus be due to T-cell activation with more efficient viral replication. However, Nef does not appear to have BV specificity in activating T cells (431, which is the hallmark of known SAGS. The nef gene lies in the 3’ORF at the equivalent genomic site as the gene of MTv. Moreover, nef transgenic mice develop an immunodeficiency similar to what is seen in patients (46,471. However these transgenic micehave not been reported to have deletions of BV subsets, which would be expected if nef encoded a SAG, based on the results of mice transgenic for Mtv (3).

Perhaps the most promising studies in favor of an HIV-encoded SAG come from Akolkar et al. (48-50). These authors used gp160 and gp120 purified from an infected T-cell line (6D6). The gp160 preparation was only weekly mitogenic for Pbl, less than 1000 cpm after 7 days of stimulation (49), but consistently resulted in a skewed repertoire with overrepresentation of BV3,12,14,15, and sometimes 17 and 20. The authors surmise that targeted T-cell subsets are transformed to lymphoblasts without much T-cell proliferation. Lymphoblasts contain higher levels ofTCR BV RNA than resting cells, thus explaining the skewed RT-PCR results. By cytofluorometry the BV skewing was evident only if the analysis gates were set on lymphoblasts (49). The T-cell response satisfied several criteria forSAG a response. Thus, shifts in expression of BV subsets were observed in both CD4 and CD8 cells. The BV-specific responses appeared to be polyclonal as assessed by analyzing CDRIII size distribution. Paraformaldehyde-fixed APCs could be substituted for nonfixed APCs, indicatingthat Ag processing was not required (49). The BV-specific effectswere inhibited with anti-HLA DR but not with MHC class I antibody. Inhibition was also obtained with a MAb to gp120. Curiously, gp120 isolated from the same 6D6 cells used for the gp160 preparation did not have the same effect as gp 160. Rather, recombinant gp120 and 6D6-derived gp120 resulted primarily in expansion ofBV2 and BV3 with some variation in the response among four normal Pbl tested (50). The gp160 tested in these experiments was actually a mutated form ofgp160 with a premature termination codon that results in a secreted soluble protein that can be isolated from cell supernatants. It is therefore not yet clearthat natural gp160 possesses the same SAGlike effect. Nor is it clear that the SAG effect was not due to minute amounts of an unknown protein within the gp160 preparation. Therefore, the results need to be confirmed with other sources of gp160 and

.


509

with transfectants expressing Further questions are whether gp160 binds promiscuously to MHC class I1 alleles (like SAGS) and what percentage of the target BV subsets are actually activated. The latter point is important as it allows distinction between a promiscuous peptide with a BV-specific T-cell response (51) and a "true" SAG response, which involves a much larger fractionof the target BV subset. HIV VIRAL LOADANDCMVINFECTION HIV Targets BV Subsets for Efficient Replication

Our group took a different approach (52-54). We reasoned that an HIV-associated SAG would serve to establish a viral reservoir in host CD4 T cells just as MTv SAG aids in establishing the MTv reservoir in vertically infected mice and that this might be a common theme in the biology of viral SAGs. Activated, but not resting, CD4 T lymphocytes support HIV replication, and this difference is due in part to the presence of nuclear factors such as NFKBand SP1 that bind to the HIV-1 LTR resulting in transcription of viral genes. Thus CD4+ T-lymphocyte BV subsets activated by an HIV-l-associated SAG would be expected to replicate virus to a higher degree than control subsets. We found HIV-1 replication was up to 100-fold higher in Tcell lines expressing BV12TCRs as opposed to other TCRs (BV6.7, BV8,BV17). The increased virus production was dependent on the presence of MHC class 11-positive antigen-presenting cells( APCs) but was not MHC-restricted. High-level viral replication was thought to be due to activation of the BV12-expressing cells within the cultures, because other T-cell lines could be activated to yield similar high-level HIV-1 replication if treated with mitogens exogenous SAGs. NonT cells from HIV+ patients induced proliferation of BV12-expressing cell lines but not control cell lines, suggesting that the non-T cells obtained ex vivo may be expressing a BV12-specific SAG. We also found that normal Pbl, infected in vivo with HIV-1, preferentially replicate HIV-1 in BV12 cells. After 5-7 days of culture with IL-2 but without exogenous mitogens, HIV-1 replication was first detectable (by a sensitive radioactive PCR assay) in positively selected BV12-expressing cells as compared with 11 other other BV subsets (see Fig. 1) (53). Vpl2-selective HIV-l replication was indistinguishable from the effects known SAG such as Mtv-7 (specific for hBV12) or Mtv-9 (hBV6.7) or MAM (hBV17). In each case the SAG-directed preferential viral replication in the targeted BV subset

HLA-UU

-

>000r0

=! ..... .

.

Figure 1 HIV-1 load in 12 different BV subsets. T cells mixed with autologous non-T cells were infected in vitro with HIV-1 (TIIIB), 3000 TCID,, and then cultured for 7 days with IL-2 added on day 4 but no mitogens. The subsets were positively selected with specific monoclonal antibodies and magnetic beads, DNA prepared, and PCR performed with primers for HLADQ (the internal control)and for viral gag sequences. The percentages of each subset were also determined by FACS at the beginning of the culture. (From Ref. 53.)

and the magnitude and time course of the effect resembledthe "spontaneous" HIV replication in BV12 cells (54). Corrected for total cell numbers in each subset, viral replication was increased by approximately 100-fold in the SAG-targeted BV subset. This assay eventually allowed us to determine the source of the SAG-like activity, which derived from CMV-infected monocytes (see below) (54). This is therefore an example of a SAG derived from a different microbe, which influences HIV-1 viral replication. Bacterial SAGS have also been shown to up-regulate HIV and SIV replication (54-56) and have been discussed as possible causes for disease progression in vivo. CMV infection is one of the most frequent opportunistic infections in AIDS and thus likely contributes to an increased HIV-l viral load.


B.

BV12 CD4+ Cells as a Reservoir for HIV-1

Surprisingly, we found that BV12 CD4 T cells were not preferentially depleted from the blood ofHIV sero+ and AIDS patients (22), suggesting that the BV12 T cells serve as a reservoir of HIV-infected CD4 T cells. When this hypothesis was tested, HIV-1 DNA and infectious virus were found specifically concentrated in the BV12 subset, representing 1-2%of all CD4 T cells (22,57,58). For instance, in 16/23 HIV-infected patients there was a 2-360-fold higher viral loadin fresh, uncultured BV12 cells than in control BV6.7 cells (53). In several patients HIV-1 DNA was selectively present in BV12 cells at repeated time points. example, in patient M3 the viral load was strongly skewed to the Vp12 subset (Fig. 2). Several other BV subsets lacked detectable HIV-1 gag. This was a consistent feature in three samples taken over a 1.3-year period. It was not due solely to variable cell numbers in the positively selected BV subsets as the PCR contained an internal control for total cellular DNA in the selected subset and the results could be normalized to express HIV gag viral load per cell number. As in normal individuals, the BV12 subset was one of the smaller subsets in patient M3 (1-2% of CD3+CD4+T cells). The BV12 subset was not deleted in M3 any of our HIV+ patients (22). HIV1 in patients’ BV12 cells was competent in an infectivity assay (53). Therefore, this subset appears to act as a viral reservoir in patients like M3. One striking feature of this reservoir is its stability over time. In contrast to recent reports of massive viral production and T-cell turnover our findings suggesta small quiescent poolof T cells in the peripheral blood bearing viral cDNA. These cells appear to be protected from deletion and there is no evidence of dissemination of virus from one BV subset to another in patients such as M3. C.

CMV Drives H I V Replication in BV12 T Cells

The source of the putative BV12-specific SAG was originally a mystery: HIV-l itself, a ubiquitous virus, a host cell gene were all equally likely alternatives (61). It was appreciated all along that non T-cells were essential to observe BV12-specific HIV replication to activate BV12 cell lines to proliferate (52). A clue was provided when we examined what cell type among the non-T cells was essential. Only CD14+ monocytes were capable of supporting selective HIV-1 replication in Vp12 cells (54). CD19+ cells and CD19-14-dendritic cells were insufficient although both express MHC class I1 and can

512

Posnett

Figure 2 HIV-l load in BV subsets isolated from the PBL of a patient (M3). PBL were obtained at three differenttime points and the indicated BV subsets isolated with MAbs and magnetic beads. The percentage of cells in each BV subset (by FACS) is indicated for the December 1992 sample. The results show a strongly skewed distribution of the viral load to the BV12 subset 'without change over a l-year period. Positively selected CD4 cells represent about 10-fold more T cells than in the individualBV subsets. 1/10 of the CD4 cells represent a total number of T cells similar to the BV subsets. (From Refs. and 54.)

efficiently present exogenous SAG (62). Since monocytes from normal adults and their bone marrow progenitors are often persistently infected with human CMV (63-65) and can express immediate early genes of CMV after in vitro culture we considered the possibility that CMV was the source of the SAG.


,

513

Non-T cells were able to promote selective HIV-1 replication in BV12 cells only when obtained from CMV-seropositive donors (54). Non-T cells from human cord blood and from CMV-negative adult donors were unable to stimulate HIV-1 replication in BV12 cells, even when the non-T cells had received prior treatment with the mitogen NaI04. CMV-negative cord blood non-T cells were perfectly capable of presenting an exogenous SAG (MAM), resulting in HIV-1 replication in the targeted BV17 subset. The monocyte-like cell line, U937, is partially permissive for CMV infection (68). U937 cells transcribe CMV immediate early genes (IEI and IE2) within 24 hr after infection (54). U937 cells were infected with CMV (MO1 1) in a 3-day culture followed by treatment with mitomycin C to inhibit further cellular proliferation. The CMVinfected U937 cells were then added, at a 1:lO ratio, to freshly isolated normal T cells, infected in vitro with HIV-l. CMV-infected U937 cells efficiently stimulated selective HIV-1 replication in BV12 cells, but uninfected U937 cells NaIO4-treated U937 cells did not (54). CMV laboratory strain AD169 and clinical isolates 1165 and 2830 were equally capable of stimulating HIV-l replication in BV12 T cells from CMV negative donors. Thus several CMV isolates can stimulate HIV1replication in BV12 cells. This was also not dependent on the HIV1 isolates used (TIIIB, JR-FL, and Ba-L). In a transwell experiment it was shown that selective HIV-l replication in BV12 T cells requires cell contact with CMV-infected U937 cells and that CMV was not transferred from the infected U937 cells to the HIV-infected T cells. This i s consistent with the mode of action of a SAG, which requires presentation by an APC, but inconsistent with the effect of a cytokine, for example. Moreover, recombinant TNF-a, a cytokine known to potently up-regulate HIV-l replication, induced HIV-1 replication in all three BV subsets examined rather than in a BV12-selective pattern. The CMV effect could no longer be observed when the CMV viral stock was inactivated by exposure.to UV light prior to infection ofU937 cells. Thus live viruswas required. However, CMV DNA replication was not required as foscarnet and acyclovir did not inhibit the SAG-like effect. Finally, cycloheximide, added 12 hr prior to harvesting of CMV-infected U937 cells, partially inhibited the SAG-like effect in a dose-dependent manner. Proteinsynthesis in CMV-infected U937 cells was .therefore required. SAGS added to T-cell cultures produce an expansion of the targeted BV subsets (69,70). Normal T cells were cultured with either mitomycin-treated U937 CMV-infected and mitomycin-treated U937 cells in the absence of HIV-l for 6 days and then stained. There

.

Posnett

*

were 1.5%versus 8.1% BV12/CD3 cells, respectively, as assayed with two different BV12-specific MAb. There was no increase in the percentages of three other BV subsets. The relative expansionof the Vp12 subset involved both CD4 and CD8 cells. This result argues in favor of 3 SAG response, as opposed to a peptide/MHC response, which would be either class I class I1 restricted and therefore involve either CD8 CD4 cells, but not both. Serum antibodies to CMV were examined in 23 HIV+ patients (54). There was a highly significant correlation between CMV antibodies and high BV12/BV6.7 viral load ratios. In contrast, there was no correlation with antibodies to other viruses. Thus CMV infection is associated with HIV-l DNA selectively enriched in BV12 cells in vivo. To determine whether CMV immediate early gene products transactivate a host cell gene responsible for the BV12 SAG effect, IE1 and IE2 were transfected into U937 cells. Unlike infected U937 cells, which promoted HIV-1 replication specificallyin BV12 cells, IE2-transfected cells upregulated HIV-1 replication in all three BV subsets tested and IE-l transfected cells did not augment HIV-1 replication in any subset (54). These results and the failure to detect the SAG effect with mitogen-treated U937 cells, with U937 cells infected with other viruses (HSVI, influenza virus), suggest that a CMV-encoded gene is responsible for the SAG effect rather than a transcriptionally activated host cell gene. The BV12 subset does not appear to be preferentially deleted in vivo in CMV-seropositive individuals. However, in humans natural exposure to a SAG, like TSST-l in toxic shock syndrome, often does not lead to detectable deletion of the reactive (BV2) subset (71). A possible explanation is that presentation of a SAG by monocytes rather than B cells (the host cell for Mtv SAGS) results in a different outcome. Vella et al. have recently shown that SEA does not induce deletion of the targeted mBV3 subset in mice when presented by LPSstimulated B cells (27). This was.due in part to TNF-a production by these B cells.StaphylococcalSAGS are known to stimulate TNF-aexpression in APCs by signals induced through crosslinking of MHC class I1 (72,73). Moreover, CMV infection of monocytes induces TNFa expression (74). It is therefore possible that SAG production by CMV-infected monocytes does not readily lead to BV-specific deletion. Finally, in the setting of in vitro cultures, particularly with NaI04 activation of monocytes, expression of the SAG is likely activated. Moreover, with HIV infection, CMV genes are known to be


515

transcriptionally activated (75,76), which may result in SAG production and stimulate selective HIV-1 replication in the BV12 subset. D. Significance A CMV-driven SAG specific for BV12 cells would represent the first example of a herpes virus SAG, with wide-reaching implications for

host-pathogen relationshipsin humans, as several herpes viruses commonly infect the large majority of humans worldwide. A SAG might, for instance, stimulate the initial lymphoproliferation in the infectious mononucleosis syndrome of primary CMV or EBV (77) infection, and in the lymphocytosis observed in primary HIV infection associated with CMV coinfection (78). The proposed role of the CMV-driven SAG is similar to the role of SAGS produced by exogenous MTV, which are involved in amplification of the viral load in the earliest stages of infection after milkborne vertical transmission of virus from mothers to pups and where the target BV subset is required for the exogenous mouse mammary tumor virus to efficiently infectthe neonatal host (1,3). However, the MTV-encoded SAG benefits the original virus, rather than assisting a heterologous virus. Therefore, a role for activated BV12 cells in CMV infection itself is likely. One idea is that production of infectious CMV from latently infected monocytes requiresT-cell help. Previous attempts to reactivate latent CMV from blood monocytes in vitro to obtain infectious virus have been remarkably unsuccessful. However, in a culture system primary monocytes, which relies on contactswith mitogen-stimulated T cells, productive CMV infection was observed (66). A SAG could provide the same conditions. By bridging BV12 cells and monocytes (via TCR and MHC class 11) both cell types would be potently activated and local concentrations of lymphokines elevated. This also occurs in all alloresponse in which a large number of T cells bind the allogeneic target cells (via TCR/MHC). This is in effect what happens in allo-mismatched transplantation, a common clinical scenario for CMV reactivation. During natural CMV infection activated T cells could also play a role in facilitating CMV replication in monocytes or their bone marrow progenitors and in establishing a cellular reservoir. In a cohort of 23 HIV+ patients we found the HIV D N A load skewed to the BV12 subset in 70% of patients. There was an excellent correlation between the presence of serum CMV antibodies and skewed HIV-l load in the BV12 subset (53). Others have conducted epidemiological studies suggesting that CMV infection is correlated

516

Posnett

with progression to AIDS and shortened survival (79-81), although this is controversial (37,821. In our small cohort there was no correlation between clinical outcome and the presence of CMV antibodies skewed viral load to the BV12 subset. Could CMV play a role in establishing a reservoir of HIV-l during primary HIV infection? As most adults are already CMV-infected (especially if assayed by PCR), we hypothesize that reactivation of latent CMV may occur in the setting of acute HIV infection. Alternatively, some instances of HIV transmission may be associated with simultaneous transmission ofCMV (and/or other herpes viruses). A recent report describes patients with apparent cotransmission of HIV-l and CMV (78). These patients characteristically have marked lymphocytosis and are symptomatic. An important question is whether the minimal dose of HIV-1 required for transmission in the setting of CMV coinfection differs from the minimal dose required in the absence of CMV. The answer may depend on transmission studies in animal models. Certainly, the efficiency of in vitro infection of Pbl from normal donors differs dramatically from one person to another when Pbl are not prestimulated with mitogens (D. N. Posnett, unpublished data). One possibility is that endogenous cofactors, such as herpes viruses that produce superantigens, could be variably expressed in different donors and thus explain the variation in efficiency of HIV-1 infection. IV.

SUMMARY

Defined viral SAG with a clear biological rolehave been described in the murine Mtv system. It is not yet clear whether viral SAG are much more ubiquitous. Do they represent a commonly used mechanism in different species for viruses to assure survival in host cells? Among human viral pathogens there are several candidates for the production of SAG, as listed in Table 1. The evidence for each case is presented in this chapter (and in Chapters 5 and 6). Much of the evidence is still in need of molecular confirmation. However,the likelihood that herpes viruses encode genes makes this topic a high priority for future research. Future questions should address the role of SAGS in the pathogenesis of a wide variety of diseases due tothese viruses. This includes the infectious mononucleosis syndrome and its complications, neonatal CMV infection, CMV chorioretinitis and other CMV infections in immunodeficient patients and in AIDS, pulmonary CMV infection post bone marrow transplantations, oncogenic properties of herpes viruses including B-cell lymphomas, Kaposi’s sarcoma


517

in AIDS (HHV8), and nasopharyngeal carcinoma. Finally, the cofactor effect of herpes virus infection HIV-l infection requires further study. ACKNOWLEDGMENT

This work was supported by RO-1 A133322 from the National Institutes of Health. REFERENCES 1. Held W, Waanders GA, Shakhov AN, Scarpellino L, Acha-Orbea H,

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'

15. Santillana-Hayat M,Rozain F, Bittoun P, et al. Transient immunosuppressive effect induced in rabbits and mice by the human spumaretrovirus prototype HFV (human foamy virus). Res Virol 1993; 144:389-396. 16. Westby M, Manca F, Dlagleish AG. The role of host immune responses in determining the outcome of HIV infection. Immunol Today 1996; 17:120-126. 17. Imberti L, Sottini A, Bettinardi A, Puoti M, Primi D. Selective depletion in HIV infection of T cells that bear specific T cell receptor Vb sequences. Science 1991; 254:860-862. 18. Hodara VL, Jeddi-Tehrani M, Grunewald et al. HIV infection leads to differential expression of T-cell receptor beta genes in CD& and CD% T cells. AIDS 1993; 7:633-638. 19. Dalgleish AG, Wilson S, Gompels M, et al. T-cell receptor variable gene products and early HIV-1 infection [published erratum appears inLancet 1992; 339(8798):942] [see comments]. Lancet 1992;3399324-828. 20. Posnett DN, Tam J. Multiple antigenic peptide method for producing antipeptide site-specific antibodies. Meth Enzymol 1989; 178:739-745. 21. McCoy JP, Jr., Overton WR, Blumstein L,Baxter JD, Gekowski KM, Donaldson "I. Alterations of T-cell receptor variable region expression in human immunodeficiency virus disease. Cytometry 1995; 22:l-9. 22. Posnett DN, Kabak S, Hodtsev AS, Goldberg EA,AschA.TCR-Vb subsets are not preferentially deleted in AIDS. AIDS 1993; 7:625-631. 23. Boyer V, Smith LR, Ferre F, et al. T cell receptor Vb repertoire in HIVinfected individuals:lack of evidence for selective Vb deletion. Clin Exp Immunol 1993; 92:437-441. 24. Bahadoran P,Rieaux-Lecat F, LeDeist F, Blanche S, Fischer A, de Villartay JP. Lackof selective / deletion in peripheral CD4+ T cells of human immunodeficiency virus-infected infants. Eur J Immunol 1993; 23:2041-2044. 25. Boldt-Houle DM, Rinaldo CR, Jr., Ehrlich GD. Random depletion of T cells that bear specificT cell receptor V beta sequences in AIDS patients. J Leukoc Biol 1993; 54:486-491. 26. McCormack JE, Callahan JE, Kappler Marrack PC. Profound deletion of mature T cells in vivo by chronic exposureto exogenous superantigen. J Immunol 1993; 150:3785-3792.


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envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire. J Exp Med 1994; 179:1261-1271. 41. Dadaglio G, Garcia S, Montagnier L, Gougeon M-L. Selective anergy of Vb8+ T cells in human immunodeficiency virus-infected individuals. J Exp Med 1994; 179:413-424. 42. Ohmen JD, Barnes PF, Grisso CL, Bloom BR, Modlin RL. Evidence for a superantigen in human tuberculosis. Immunity 1994; 1:35-43. 43. Torres BA, Johnson HM. Identification of an HIV-1 Nef peptide that binds to HLA class I1 antigens. Biochem Biophys Res Commun 1994; 200:1059-1065. 44. Miller MD,WarmerdamMT,SatonI,GreeneWC,FeinbergMB.The human immunodeficiency virus-l nef gene product: a positive factor for viral infection and replication in primary lyphocytes and macrophages. J Exp Med 1994; 179:lOl-114. 45. Spina DA, Kwoh TJ, Chowers MY, Guatelli JC, Richman DD. The importance of nef in theinduction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. Exp Med 1994; 179:115-124. 46. Skowronski J, Parks D, Mariani R. Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO J 1993; 12:703-713. 47. Lindemann D, Wilhelm R, Renard P, Althage A, Zinkernagel R, Mous J. Severe immunodeficiency associatedwith a human immunodeficiency virus 1NEF/J-long terminal repeat transgene. J Exp Med 1994; 179:797807. 48. Akolkar PN, Chirmule N, Gulwani-Akolkar B, et al. V beta-specific activation of cells by the HIV glycoprotein gp 160. Scand J Immunol 1995;41:487-498. 49. Akolkar PN, Gulwani-Akolkar B, Chirmule N, et al. The HV glycoprotein gp160 has superantigen-like properties. Clin Immunol Immunopathol 1995; 76:255-265. 50. Akolkar PN, Gulwani-Akolkar B, Silver J. Differential patterns of T-cell receptor BV-specific activation of T cells by gp120 from different HIV strains. Scand Immunol 1995; 42:598-606. 51. Boitel B, Ermonval M, Panina-Bordignon P, Mariuzza RA, Lanzavecchia A, Acuto 0. Preferential Vb usage and lack of junctional sequence conservation among human cell receptors specific for a tetanus toxinderived peptide: evidence for a dominant role of a germline-encoded V region in antigen/major histocompatibility complex recognition. J Exp Med 1992; 765-777. 52. Laurence Hodtsev AS, Posnett DN. Superantigen implicated in dependence of HIV-1replication in T cells on TCR Vb expression. Nature 1992; 358:255-259. 53. Dobrescu D, Kabak S, Mehta K, et al. HIV-l reservoir in CD4+ T cells is restricted to certain Vb subsets. Proc Natl Acad Sci USA 1995; 92:55635567.


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54. Dobrescu D, Ursea B, Pope M, Asch AS, Posnett DN. Enhanced HIV-1 replication in Vb12 T cells due to human cytomegalovirus in monocytes: evidence for a putative herpesvirus superantigen. Cell 1995; 82:753-763. 55. Hashimoto K, Shigeta S, Baba M. Superantigen toxic shock syndrome toxin-l (TSST-l) enhances the replication of HIV-1 in peripheral blood mononuclear cells through selective activation of CD4+ T lymphocytes. J AIDS Hum Retrovirol 1995; 10:393-399 56. Firpo PP, Axberg I, Scheibel M, Clark EA. Macaque CD4+ T-cell subsets: influence activation on infection by simian immunodeficiency viruses (SIV). AIDS Res Hum Retroviruses 1992; 8:357-366. , 57. Bigler RD, Fisher D, Wang CY, Rinnooy-Kan E, Kunkel HG. Idiotype molecules on the cells of a human T cell leukemia. J Exp Med 1983; 158:lOOO. 58. Posnett DN, Wang CY, Friedman S. Inherited polymorphism the human T cell antigen receptor detected by a monoclonal antibody. Proc Natl Acad Sci USA 1986; 83:7888-7892. Chen W, Leonard JM, Markowitz 59. H o DD, Neumann AU, Perelson M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection [see comments]. Nature 1995;373:123-126. 60. Wei X, Ghosh SK, Taylor ME, et al. Viral dynamics in human immunodeficiency virus type 1infection comments]. Nature 1995; 373:117122. 61. Posnett DN, Kabak S, Asch A, Hodtsev AS. HIV-l replication in T cells dependent on TCR V-beta expression. In: Huber B, Palmer E, eds. Superantigens: A Pathogen’s View of the Immune System.Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1993; 163-177. 62. Bhardwaj N, Friedman SM, Cole BC, Nisanian AJ. Dendritic cells are potent antigen-presenting cells for microbial superantigqns. J Exp Med 1992;175:267-274. 63. Taylor-Wiedeman J, Sissons JGP, Borysiewicz LK, Sinclair JH. Monocytes are a major site persistence of human cytomegalovirus in peripheral blood mononuclear cells. J Gen Virol 1991; 72:2059-2064. 64. Kondo K, Kaneshima H, Mocarski ES. Human cytomegalovirus latent infection of granulocyte-macrophage progenitors. Proc Natl Acad Sci USA 1994;91:11879-11883. 65. Maciejewski JP,Bruening EE, Donahue RE, Mocarski ES, Young NS, St Jeor SC. Infection of hematopoietic progenitor cells by human cytomegalovirus. Blood 1992;80:170-178. 66. Ibanez CE, Schrier R, Ghazal P, Wiley C, Nelson JA. Human cytomegalovirus productively infects primary differentiated macrophages. J Virol 1991;65:6581-6588. Al-Daccak R, Chatila T, Geha Staphylococcal 67. Mourad W, superantigens as inducers of signal transduction in MHC class 11-posi; tive cells. Semin Immunol 1993; 5:47-55. 68. Numazaki K, Nagata N, Sato T, Chiba S. Replication human cytome-

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Superantigens in Autoimmunity Their Role

Etiologic and Therapeutic Agents

Schiffenbauer, Howard Johnson, and JeanneSoos

INTRODUCTION

Autoimmune disorders are the result of complex interactions'between genetic and environmental factors. In support this statement, studies examining the incidence of disorders such as systemic lupus erythematosus and rheumatoid arthritis in identical twins have shown a 30-50% concordance while in nonidentical twins or siblings the concordance is considerably lower, around (1). Although some genetic factors such as the MHC class I and I1 genes have been demonstrated to contribute to a number of autoimmune disorders the study of genetic contributions to these processes is just beginning to gain momentum with the development of genetic markers that span the entire genome. In the next several years, new information pertaining to the various genetic factors contributing to autoimmune disorders should become available. For some time, environmental agents have been implicated in the pathogenesis of autoimmunity. Clearly, agents such as drugs can have a profound influence on the development of autoimmune dis525

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orders. For example, specific drugs such as hydralazine or procainamide are known to cause a lupus-like illness in certain individuals and not others However, much attention over the years has been focused on infectious agents in the initiation and ongoing development of autoimmune disorders. For example, hepatitis C has been shown to cause an immune-complex disorder (4), and in some patients hepatitis is responsible for the development of polyarteritis nodosa, a systemic vasculitis ( 5 , 6 ) . Hepatitis B surface antigen has been found in immune complexes in the blood vessels of these patients. In addition, a group of disorders known as the reactive arthritides occurs following either a genitourinary or gastrointestinal bacterial infection (7). Therefore, infectious agents can lead to the onset of autoimmunity in specifically predisposed individuals. Although infectious agents have been proposed to be important in the pathogenesis of autoimmune disorders, the exact mechanisms by which these agents lead to the breakdown of immune tolerance and development of autoreactive T and cells is not known. Several theories have been put forth to explain the possible contribution of infectious agents to autoimmunity ( 8 ) . First, an individual may not possess the appropriate class I1 molecules to allow immune recognition of antigens produced by the infectious agent, and thus a chronic infection can lead to progressive destruction of the affected organ. , Although possible, this seems unlikely since the immune system is usually responsible for tissue destruction following infection. Alternatively, if the immune system can recognize the infectious agent as foreign but cannot clear the infection, then a chronic infection may lead to tissue damage. This situation would not truly be an autoimmune disorder since the host can mount an appropriate immune response against the invading organism. However, if the infectious agent cannot be identified by the investigator, the end result would appear to be an autoimmune disorder. In the above example, we have suggested that the infection is ongoing. However, a subset of this mechanism is that the infection can initiate the immune response but does not have to be present for the ongoing tissue destruction. This is known as the molecular mimicry theory. In this scenario, the infectious agent contains a protein that resembles a self-antigen and thus initiates an appropriate immune response that ultimately leads to the recognition of a self-antigen and a true autoimmune disorder. Some evidence exists for this in animal studies. For example, a transgenic mouse has been producedthat expresses the lymphocytic choriomeningitis viral glycoprdtein in the pancreas. If the mouse is then

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infected with the virus containing the same glycoprotein, pancreatic destruction ensues with the resulting development of diabetes (9). II.

SUPERANTICENSANDAUTOIMMUNITY

More recently, much attention has focused on the contribution of the products of a number of bacteria and viruses called the microbial superantigens (l0,ll). Considerable evidence supports the ability of these products to profoundly influence the immune system by interacting with and stimulating T cells to become activated and release inflammatory cytokines. Indeed, because of these profound effects on the immune system several mechanisms have been suggested by which these superantigens may influence the development of autoimmune disorders (Table 1).First, superantigens may activate normally quiescent autoreactive T cells. Once activated, these T cells may continue to proliferate due to .the presence of autoantigen along with antigen-presenting cells, resulting in a chronic autoimmune disorder. Alternatively, the autoimmune response may be down-regulated leading to a period of clinical remission. Repeatedexposures to the same related superantigens may then lead to further relapses of the autoimmune disorder. This hypothesis supposes the presence of autoreactive T cells before the exposure to a superantigen. In a related mechanism, the infecting agent contains both a superantigen and a peptide that acts as a molecular mimic and induces cross-reactive T cells. In this scenario neither the superantigen alone nor the peptide alone can induce a sufficient autoreactive T-cell response to produce

Table

Mechanisms

Superantigen-Mediated Autoimmunity

1. Superantigen-induced activation

autoreactive T cells. Superantigens can induce autoimmunity de novo or reactivate disease in an individual with a preexisting autoimmune disorder. Tissue damage may be secondary to the release toxic cytokines by the T cell. 2. Superantigen-induced activation autoreactive B cells. This may occur either due to direct activation the B cell by the superantigen or indirectly through the activation autoreactive T cells and subsequent release cytokines that activate the B cell. Superantigen activation o f antigen-presenting cells such as B cells or macrophages that leads to altered processing andpresentation autoantigens (cryptic epitopes) to T cells, or the release toxic cytokines, etc., by the activated macrophage.

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a clinical autoimmune disorder. However, the combination can lead to an amplification of the autoimmune response and produce clinical autoimmunity. Therefore, in the above scenarios, superantigens can either initiate an autoimmune disorder in an individual with preexisting autoreactive T cells (or in whom the autoreactive T cells are induced by the same infecting agent) or reactivate the disease in an individual otherwise in clinical remission. Indeed,we have shown (see below) in an animal model of multiple sclerosis (MS) that superantigens can reactivate autoimmune experimental allergic encephalomyelitis (EAE) when administered to an animal that has recovered from an episode of clinical disease (12). The final common effector pathways are likely secondary to the release of cytokines from CD4 or CD8+ T cells such as TNF-a or IFN-y in, for example, EAE or the release cytokines that activate cells to secrete antibodies; alternatively, activated CD8 cytolytic T cells may contribute to the pathogenesis of tissue damage. One potential confounding problem with superantigen-activated autoreactive T cells leading to autoimmunity is that superantigens appear to induce anergy or deletion in the very T cells they activate. However, we have shown that repeated administration of superantigen leads to repeated episodes of clinical EAE in mice, suggesting that superantigens will not induce anergy or deletion in previously activated autoreactive T cells (12). In addition, it has recently been demonstrated that lipopolysaccharide canblock the superantigen-mediated processes of anergy 'or deletion Therefore, in summary, it seems likely that in the appropriately predisposed individual infectious agents can induce autoreactive T cells, for example, through a mechanism of molecular mimicry. autoimmune disease may develop as either insufficient numbers of autoreactive T cells are stimulated or mechanisms to suppress the proliferation these cells develops. Through the action of superantigens, further expansion and activation of these autoreactiveT cells occurs without the development of T-cell anergy or deletion, ultimately leading the development of an autoimmune disorder. This could occur either during the initial infection or during a subsequent infection after the initial expansion of autoreactive T cells has occurred:In an individual with a preexisting autoimmune disorder who is in a clinical remission, superantigens would be capable of inducing an exacerbation of the disorder. In a similar fashion superantigens could lead to a worsening of the process in an individual who already has active disease. A second mechanism by which superantigens may induce an autoimmune disorder is by the activation of cells. This would most

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likely be through the activation of T cells leading to cognate T-B-cell interactions with the subsequent release of appropriate cytokines. It has been suggested that a graft-versus-host (GVH)-likeT-B-cell interaction may occur mediated by a superantigen "bridge" (8). In certain combinations of mouse strains, GVH can be induced by the transfer of splenocytes with the subsequent development of an autoimmune Furthermore, it appears disorder resembling systemic lupus that the binding of superantigens to class I1 molecules can induce activation signals in monocytes and B cells with the subsequent activation of B cells and the induction of autoantibodies. Tissue damage may occur as a result of immune complex formation, complement deposition, and neutrophil activation, and the activation of macrophages with the resultant release of cytokines and toxic molecules. As a consequence of macrophage activation through any number of mechanisms including activation by superantigen, enhanced antigen processing and presentation may lead to the generation of peptides derived from cryptic epitopes. This may also contribute to the development of autoimmunity. The above has focused on potential contributions of superantigens to development of the autoimmune process. As will be seen, the effects of superantigens on animal models of autoimmunity are evident. However, determining the role of superantigens in the pathogenesis of human autoimmune diseases has been more difficult. As described later, investigators have used TCR analyses to determine whether superantigens may be implicated in a disease process. For example, in Kawasaki disease it has been demonstrated that Vp2+ T cells are overrepresented This has suggested that a superantigen capable of activating Vp2+ T cells contributes to the development of Kawasaki disease. While the identification of overrepresented T-cell subsets suggests that superantigens are involved, the lack of such findings, or the demonstration of T cells with limited T-cell-receptor (TCR) junctional diversity, should not be taken as evidence of an antigen-specific processwithout involvement of superantigens. Based on the above discussion, one can envision a scenario whereby a superantigen activates both autoreactive and nonautoreactive T cells, but only the autoreactive T cells home to the appropriate organ containing antigens recognized by those T cells. Therefore, an examination of the diseased organ would demonstrate the presence of antigen-specific T cells bearing TCR with limited junctionaldiversity even though the process was initiated by a superantigen. Activated nonautoreactive T cells would ultimately be eliminated because lack of continued antigen stimulation. Alternatively, in an established

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ongoing autoimmune disease, superantigens can activate antigen-reactive T cells that are already present at the site of disease. Again, if one sequences the TCR, limited junctional diversity may be present even though a superantigen induced a flare of the disease process. This may occur because antigen-specific T cells were present in the affected organ at the time of superantigen activation. In summary, the finding expansion of specific Vp's suggests the role of superantigens, but the finding of limited junctional diversity in TCRs may not exclude such a contribution. The previous discussion can serve as a basis for understanding the potential role of superantigens in the pathogenesis of autoimmune disorders. The following discussion will focus on information demonstrating the role of superantigens in animal models of autoimmunity, and will present available information as it relates to the potential role of superantigens in thedevelopment of thehuman counterparts of these animal models. As will be seen, evidence forthe role of superantigens in various animal models of autoimmunity is clear. However, the role of superantigens in the pathogenesis of human disorders is for the most part circumstantial. RHEUMATOIDARTHRITISANDANIMALMODELS

Rheumatoid arthritis (RA) is an chronic autoimmune disorder characterized by inflammation of joints and often joint damage. Mononuclear cellsprimarily composed of CD4+ T lymphocytes and plasma cells initially accumulate around blood vessels. Although the pathogenesis of this disorder remains unknown, it appears that both genetic and environmental factors contribute to this process. In terms of genetics, there is much data to support the idea that specific MHC class I1 genes with shared epitopes are critical in disease predisposition or in the severity of disease Although a number of viruses, including the Epstein-Barr virus, have been implicated in the development of RA, no definite evidence of an environmental agent contributing to the pathogenesis of disease has been found. To examine the potential contribution of superantigens to the development of RA, two groups have studied the TCR Vp usage in patients with this disorder. Using a quantitative PCR method to analyze the TCR Vp chain repertoire in the periphery and synovial fluid of patients with RA, Paliard et a1 reported all RA patients examined had a lower percentage of Vpl4-positive T cells in the periphery than in synovial fluid. Indeed, in most cases, Vp14 could not be


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detected in the periphery. An examination of the junctional sequences of the Vp 14 gene segments revealed that one or a few clones dominated this VD14 population in the synovial fluid. Based on these results, the authors proposed a scheme whereby a patient encounter with a superantigen specific for Vp14 could initiate RA (Fig. 1). A subset of these Val4 cells cross-reactswith a joint specific antigen, which causes these cells to migrate to the joints and set up a local inflammatory response. This inflammation is capable of recruiting additional cells with multiple Vp’s leading to the chronic condition we recognize as RA. cells activated by the superantigen but not capable of recognizing a specific antigen would eventually become deleted from the periphery. In a second paper, Howell et al. (22) also examined Vp usage in cells from RA patients. In this work the authors examined interleukin-2 receptor-positive cells in the synovium (in the Paliard study, anti-CD3-activated cells were analyzed). Three gene families, Vp3, Vp14, and Vp17, were found in a majority of the synovial

smallsubset of activated Vbetal4+ T cells cross-react with a joint antigen

+

cross-reactive T cells migrate to the joints and produce local inflammation additional T cells

destructive joint inflammation

Figure 1 Proposed mechanism for the development Ref. 11by permission of authors.)

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of RA. (Adapted from

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samples analyzed. Given that in many instances the Vp repertoires were dominated by a single rearrangement, and given the sequence similarity between these Vp's, the authors postulated the potential role for a superantigen in the pathogenesis of RA. They went on to suggest that superantigen activation of T cells could occurin the periphery or in the joint if the superantigen was trophic forthe synovium and could be an exogenous or endogenous superantigen. Both reports examined RA patients that were either DR4 or DR1 positive. In summary, an analysis ofTCR usage in RA suggests that superantigens may contribute to disease. He et al. (23) examined the ability of superantigens to stimulate cells to produce rheumatoid factor. They demonstrated that SED could drive cells to secrete antibodies that preferentially bind the Fc portion of immunoglobulin, and that this skewing was dependent on the presence of CD4+ T-helper cells. In contrast, SEC or anti-CD3 stimulation of T cells did not elicit the preferential production rheumatoid factor (RF). Thus, superantigens may support the ongoing production ofRF in patients with RA. Of interest are several in vitro studies that demonstrate the ability of superantigens to activate synoviocytes.For example, Mehindate et al. (24) demonstrated that SEA could induce the expression of various chemokines by human fibroblast-like synoviocytes. Ligation of class I1 with Mycoplasma arthritidis mitogen (MAM) as well as anti-class I1 antibody could also trigger an increase in the mRNA levels of RANTES, MCP-1, and IL-8. Mourad et al. (25) demonstrated that SEA was capable of activating human synoviocytes treated with IFNy isolated from patients with RA to produce IL-6 and IL-8. Finally, Origuchi et al. (26) showed that activated synovial cells can act as potent antigen-presenting cells to induce T-cell proliferation and activation in the presence ofSEB. In each of the last two studies it was necessary to activate the synoviocytes to induce class I1 expression in order to observe an effect by the superantigen. Several studies in animal models of arthritis support the concept that superantigens may contribute to the pathogenesis of disease. Cole and Griffiths (27) examined the effects of superantigen "AM on the course of collagen-induced arthritis (CIA). Mice convalescing from CIA were given MAM systemically and evaluated for a flare of arthritis. In contrast to mice receiving phosphate-buffered saline (PBS), mice receiving MAM developed an exacerbation of their arthritis. In addition, mice receiving a suboptimal immunization of collagen developed clinical arthritis when challenged with MAM. MAM could


533

also enhance the severity of arthritis in mice given low doses of cob lagen. In a related study, Schwab et al. (28) examined the effects of toxic shock syndrome toxin-l (TSST-1) on bacterial cell-wall-induced arthritis. In this model, a peptidoglycan-polysaccharide isolated from the cell wall of group A streptococci is injected into the rat ankle joint to induce a monoarticular arthritis that recedes over several weeks. TSST-l was able to induce a recurrence of arthritis characterized by prolonged inflammation, pannus formation, and marginal erosions. Another superantigen, streptococcal pyrogenic exotoxin, was only able to induce a weak arthritis. Finally, Vp8 transgenic mice carrying the Ipr gene were used to examine the effects of intra-articular injection of superantigen. SEB injected intra-articularly was capable of inducing histological evidence of synovial cell hyperplasia, villus formation, and erosion and destruction of cartilage and bone (29). SEA, which does not activate Vp8 T cells, induced significantly less arthritis than SEB, which does stimulate Vp8 T cells. Thus, based on direct evidence from studies in animal models of autoimmune disorders, it appears that superantigens are capable of significantly altering the course of disease. However, the evidence that superantigens are important in the pathogenesis of RA in humans is circumstantial. Evidence fora direct effect of superantigen on the course of RA is lacking. There are no studies evaluating the exposure of RA patients to superantigen-producing bacteria, or to a potentially superantigen-producing virus. Without this evidence, the hypothesis that superantigens contribute to the pathogenesis ofRA remains unsupported, although intriguing. IV.

MULTIPLE SCLEROSIS A N D EAE

There is no evidence from human studies that superantigens play a role in the initiation of MS. Indeed, MS is felt to be an antigen-specific autoimmune process directed against a central nervous system antigen such as myelin, proteolipid protein, MOG, or others. Oligoclonal T cells have been demonstrated in the CSF of patients with MS, and autoreactive T cells that recognize CNS antigens have been obtained from patients with MS There is, however, considerabledata from animal models of MS to support a contribution superantigens to the course disease. Our group examined the effects of administration of SEB to PL/J mice that had recovered from an episode of EAE (12) (Fig. 2). SEB, which activates Vp8+ T cells, was able to induce a clinical relapse of EAE

534


Induction of Immunization with MEP in Pertussis toxin

Reactivation of

Recovery from

by superantigen in recovered mice

2ndImmunization with MEP in Pertussis toxin

Inject or Superantigen

Superantigen-induced Inject or Superantigen

No Recurrence of

Reactivation of

Recovery from

in mice thatdid not developan initial episode of disease Reactivation of

Recovery from

Reactivation of EAE by superantigens. PL/J mice immunized with myeline basic proteindevelop EAE. Following recovery, EAE maybe reinduced in these mice with superantigens SEB or SEA. In addition, PL/J mice immunized with MBP but which never develop clinical signs of EAE may be induced to develop an initial episode of EAE with SEB or SEA. (Reprinted from Ref. by permission of authors.)

Figure 2

in these mice. These results were confirmed by Brocke et al. (35). Matsumoto and Fujiwara (36) demonstrated that SED could induce relapses of EAE in rats. Interestingly, we also found that SEA, a superantigen that does not activate Vp8+ T cells, was also capable inducing relapses of EAE in these same mice (12). We postulated that Vp8+ and -T cells were present in the CNS of mice recovering from EAE, and that SEB

Superantigensin Autoimmunity

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or SEA could activateT cells present in these lesions leading to clinical relapses. Vp8+ T cells were not necessary at this stage because any T cells present in the CNS were capable of releasing the appropriate cytokines upon stimulation with superantigen to produce neurotoxicity. We also demonstrated that SEB could induce clinical disease in mice immunized with MBP but which never developed clinical signs of EAE. Thus, superantigens were capable of initiating disease symptoms in immunized but asymptomatic animals harboringautoreactive T cells. More recently, we demonstrated that the course of EAE could be accelerated by pretreating mice with SEB before immunization with MBP and administration ofSEA (Fig. In another study, Burns et al. showed that superantigens could activate human lymphocytes reactive with myelin and proteolipid protein. Finally, Racke et al. (39) demonstrated that MBPreactive T cells activated in vitro with SEB could transfer EAE to nai've mice. We concluded from thesestudies that in individuals with a previous history of an autoimmune illness, superantigens may induce acute flares of the disease. Alternatively, in individuals with autoreactive T cells but who have not yet developed the initial onset of clinical disease, superantigens may be capable of initiating the onset of a new clinical illness. A clinical or subclinical infection with the appropriate superantigen-producing infectious agent would be sufficient to initiate the clinical autoimmune process. V.

KAWASAKI DISEASE

Kawasaki disease (KD) is an acute febrile illness characterized by persistent fevers lasting 1-2 weeks, ranging from 38 to In addition, it is characterized by conjunctival injection, redness of the palms or soles, redness of the lips and oral mucosa, edema of the hands, cervical adenopathy, and a polymorphous skin rash. Desquamation of the skin also occurs. A pancarditis may develop acutely, but potentially the most devastating complication is the development of coronary aneurysms associated with stenotic lesions that may lead to sudden death. KD occurs most commonly among Japanese children and may occur as epidemics. Although the cause is unknown, the high incidence in children and the epidemiology of the disease strongly suggest an infectious etiology. InJapan and the United States, KD is the most common cause of acquired heart disease in children (40). Studies have demonstrated the presence of activated T cells as well


536

Superantigen pretreatment

B. Immunization with rat MBP

W

&

C. Induction of

Figure Modulation ofEAE by superantigens. PL/J mice are pretreated with SEB or SEA and then immunized with MBP. Those animals pretreated with SEB are protected from EAE, while those animals pretreated with SEA develop EAE. After resolution of clinical EAE, mice are treated with SEA or SEB. Those mice receiving a pretreatment with SEB and SEB again do not develop EAE. However, if those mice initially treated with SEB then receive SEA for their second treatment, they exhibit accelerated onset of EAE. If those mice pretreated with SEA develop EAE after MBP and then receive SEA for their second treatment, they do not develop EAE. However, if those mice initially receiving SEA develop EAE and then receive SEB, they redevelop EAE. Plus (+) sign or minus (-1 sign indicates development ofEAE or not. (Reprinted from Medical Hypothesis, in press, by permission of authors.)

as monocytes/macrophages. In addition, KD is associated with elevated serum levels a number inflammatory cytokines including IL-l, TNF-a,IFNy, and IL-6 (40,41). Based on the above information, studies were initiated to determine whether KD could be due to activation of the immune system


537

by a superantigen. Abe et al. (18) examined Vp expression in patients with KD using quantitative PCR on T cells cultured in vitro from patients with acute convalescent KD. These analyses revealed a significantly elevated levelof circulating Vp2+ and, to a lesser extent, Vp8.1+ T cells comparedto control populations in patients with acute KD. During the convalescent phase a reduction in the levels these Vp's was observed. In a follow-up study, Abe et al. (42) examined Vp usage in KD and compared quantitative PCR with an anti-VP2 monoclonal antibody and confirmed that VP2-bearing T cells were expanded in patients with acute KD. Furthermore, the percentages of Vp2 + T cells determined by RT-PCR and flow cytometry demonstrated a linear correlation. A decrease in Vp2 after the acute phase developed. V/38.1+ T cells were again also increased in acute KD. Sequencing Vp2 and Vp8.l TCR revealed extensive junctional region diversity, suggesting activationof these cells bysuperantigen and not a specific disease-associated antigen. A more recent study (43) also demonstrated an increase in Vp2 + T cells in KD patients compared to normal controls. By comparison, patients with toxic shock syndrome manifest an illness characterized by fever, rashwith desquamation, and hypotension, and may also develop diarrhea, mucous membrane hyperemia, CNS renal hepatic dysfunction, and thrombocytopenia. Choi et al. (44) showed a selective expansion of Vp2+ T cells during the acute phase. Taken together, the above results are consistent with the concept that KD is the result of superantigen activation of the immune system in a fashion similar to toxic shock syndrome, with the resultant clinical manifestations likely secondary to activation of T cells and macrophages and the release cytokines. To further examine the hypothesis that KD is caused by a superantigen, Leung et al. (45) investigated whether patients with KD harbored bacteria (e.g., StaphylococcusorStreptococcus) capable of producing toxins that activate Vp2+ T cells. S. aureus-producing TSST was isolated from 11/16 KD patients, and streptococcal pyrogenic exotoxin (SPE) B and C were found in two others. Therefore, bacteriaproducing toxins were isolated from 13/16 KD patients but only 1/15 of ,controls. More importantly, these toxins are capable of stimulating Vp2+ T cells. Finally, Leung et al. (46) recently reported on their studies of a patient who died of acute KD. They demonstrated a selective expansion of Vp2+ T cells in the myocardium and coronary arteries this individual. Both CD4 and CDS+ T cells showed extensive junctional diversity.

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A number of papers have demonstrated the efficacy of intravenous immunoglobulin (IVIG) for the treatment of toxic shock syndrome and KD (47). In this respect, Takei et al. (48) were able to demonstrate the ability of commercial preparations of IVIG to inhibit activation of T cells by staphylococcal superantigen in vitro. Taken together, the above data point to the possibility that superantigen-producingbacteria such as Staphylococcus and Streptococare capable of inducing KD. Nevertheless, two other groups were not able to document the expansion of any Vp family during the acute phase of KD (49,50), arguing against the hypothesis that superantigens mediate KD. However, there are several differences between the studies. These include populationsstudied (Abe examined Asians vs. Caucasians in the Pietra study), methods for collecting samples, timing of blood collection [Curtis et al. (43) found elevated Vp 2+ T cells only in week 2 of illness], activation of lymphocytes in vitro before analysis (Abe), and use ofRT-PCR rather than flow cytometry, and these could potentially explain the differences in results. In addition, the link between superantigen and the development of coronary aneurysms in some individuals is not immediately evident. In spite of these concerns, the similarity of the clinical manifestations between TSS and KD and the finding of bacteria capable of elaborating superantigens that activate Vp2+ T cells make the concept that superantigens are involved in the pathogenesis of KD very appealing. An animal model of this disorder has been developed that involves injecting Lactobacillus casei cell wall fragments intraperitoneally into mice. Mice develop an inflammatory coronaryarteritis (51). However, the relationship of this model to superantigen-induced coronary aneurysm formation is not clear. VI.

OTHER DISORDERS

A.

Insulin-DependentDiabetesMellitus

Insulin-dependent diabetes (IDDM) is characterized by the autoimmune T-cell-mediated destruction of the beta islets of the pancreas. A strong genetic predisposition exists, but in addition environmental factors are felt to play a role (52). Based on information both from the NOD animal model of IDDM and from human studies, specific antigens such as glutamic acid decarboxylase, insulin, or islet cell antigen are felt to play a role in the pathogenesis of the disease (53). No specific VD's were associated with the disease. However, Conrad et al. (54) examined the islet infiltrating T cells from two IDDM


539

patients who died at the onset of disease. They determined that there was a selective expansion of Vp7+ T cells exhibiting extensive junctional diversity. These Vp's were associated with unselected V a chains. Furthermore, flow cytometric analysis of PBLs from nondiabetic patients after exposureto patients' cell membranes demonstrated specific overexpression of Va7+ T cells. These data suggested that a pancreatic islet cell membrane-bound superantigen was responsible for their findings. Further identification of this putative islet-cell superantigen has not been described. A study in the NOD mouse demonstrated a diverse T-cell repertoire in islet infiltrates from 7-week-old mice (55). However, an examination TCR Vp types in the peri-islet infiltrates from 4-weekold mice revealed restricted Vp- and JP-gene usage. Vp3 T cells with limited junctional diversity were detected. Whether this indicates that T cells early in the course of the disease are specific for a limited set of autoantigens or possibly an intrinsic viral superantigen has not yet been established. Ellermanand Like (56) were able to passively transfer diabetes using staphylococcal enterotoxin-activated spleen cells from RT6.1 T-cell-depleted (presumably the anti-RT6.1 treatment removes regulatory T cells) diabetes-resistant rats. Finally, we (M. Atkinson and Schiffenbauer, unpublished) attempted to accelerate diabetes in the NOD mouse by administering SEB to NOD females at either 4 10 weeks of age, at a time when insulitis has begun. However, we were unable to induce either a more severe disease or a more rapid onset of the disease (see also the section on the use of superantigens as therapeutic agents). In summary, limited human and mouse data suggest that superantigens may play a role in the development of IDDM.

B. Wegener's Granulomatosis Wegener's granulomatosis is a systemic disease characterized by a necrotizing granulomatous vasculitis of the respiratory tract and kidneys. The pathogenesis is not known, although because of involvement of the respiratory tract, an infectious etiology has long been considered. Indeed, several reports have suggested that the antibiotic sulfamethoxazole-trimethoprim may be beneficial in milder disease especially if the disease process is restricted to the respiratory tract. Stegeman et al. (57) prospectively examined whether chronic nasal carriage of Stuphylococcus uureus was a risk factor for relapseof Wegener's. They determined that in a group of 57 patients with Wegener's followed for 1-3.5 years, chronic nasal carriageof S. aureus was indeed

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an independent risk factor for a relapse of the disease. However, they did not examine Vp usage in patients with a relapse, nor did they examine whether the S. aureus elaborated any specific enterotoxin. Further examination of these issues would beof great interest in linking superantigens to the course of Wegener’s.

C. Psoriasis Psoriasis is a skin disease characterized by increased proliferation of epidermal cells associated with an inflammatory component composed of neutrophils, T cells, macrophages, and keratinocytes expressing class I1 molecules. The mechanisms leading to these findings are not known, although it has been suggested that genetic and environmental agents contribute to the disorder. In this regard, there are several interesting findings that suggest infections may beimportant. First, upward 50% of patients with psoriasis carry S. aureus on their skin (58). Second, patients with a form of the disease called guttate psoriasis often have flares of psoriasis following streptococcal infections, and these flares are associated with a rise in serum antistreptococcal titers (59-61). These patients may improve with antibiotic therapy (62). Third, it has been reported that intradermal injections streptococcal material could induce psoriatic lesions (63). These data suggested that psoriatic lesions could be triggered byinfections, and one of the mechanisms might be superantigen activation of T cells in the psoriatic lesion. To evaluate this possibility, an examination of Vp usage in the psoriatic lesionswas performed. In one study, Leung et al. (64) demonstrated the Vp expansion in the psoriatic skin lesions of two patients corresponded to the superantigen cultured from the skin of the patients. For example, one patient who cultured S. aureus-producing SEB demonstrated an increased expression of Vp5.1 and Vp12. SEB is known to stimulate Vp 12 but not Vp 5. In a second paper by Lewis et al. (651, Vp usage was examined in patients with guttate or chronic plaque psoriasis. Using flowcytrometry, they found an overrepresentation of Vp2 and Vp5.1+ T cells in the lesions of these patients. Various streptococcal superantigens can stimulate Vp2 or Vp5 T cells, and the data are consistent with a streptococcal infection exacerbating the psoriasis. However, although several patients had group A or C p-hemolytic streptococci isolated from their throats, no attempt was made in this latter study to examine the production of superantigen by these organisms. Finally, one group examined the Vp repertoire of blood and skin in patients with chronic plaque-stage psoriasis using semiquantitative PCR (66). The authors

Superantigensin Autoimmunity

541

found a rather restricted TCR repertoire, which they interpreted to indicate that superantigens do not play a role in this form of psoriasis. Taken together, the data suggest that superantigens may activate T cells in the skin of patients with psoriasis leading tothe persistence of psoriatic lesions.On the other hand, superantigens may lead to the induction of psoriasis by activating T cells that cross-react with skin antigens (see Chapter 22). There is no information concerning the course of psoriatic arthritis and superantigens, although it certainly seems possible that factors that lead to flares of psoriatic skin lesions may influence the course of psoriatic arthritis affecting the peripheral joints. D. Other Disorders

Besides streptococcal-induced psoriatic flares, it has been known for some time that rheumatic heart disease and glomerulonephritis follow infections with group A streptococci. One hypothesis to explain this illness has been that the M protein of strep can induce a crossreaction with host antigens present in the heart or kidney. One additional factor now known is that streptococcal M protein has the properties of a superantigen Although no data for M protein activation of cross-reactive T cells exist, it is possible that the M protein activates cells with specificity for self-antigens. Finally, there are no data that demonstrate the contribution of superantigens to the pathogenesis of systemic lupus erythematosus. specific Q's were deleted equivalently by Indeed, in one study superantigen in control and lupus-prone mice. A further understanding of B-cell superantigens may lead to new insightsinto the relationship of these molecules to autoimmunity VII.

SUPERANTIGENS AS THERAPEUTICAGENTS IN THETREATMENT OF AUTOIMMUNE DISORDERS

There are no human studies to support the efficacy of superantigens in the treatment of autoimmune disorders. Indeed, based on the above discussion, one would predict that superantigens could potentially lead to the exacerbation of an autoimmune process. However, a number of studies in animals have demonstrated the ability of superantigens to prevent the initiation of an autoimmune disorder either induced by antigen or genetically programmed, such as animal models of diabetes or lupus.

542


Based on the ability of superantigens to induce anergy or deletion of specific T cells .bearing the appropriate TCR, several groups used superantigens to treat autoimmune disorders in mice. For example, Kim et al. (72) treated MRL-lpr/lpr mice, which develop a lupus-like illness, with SEB starting at 6 weeks of age, before the onset of clinical disease. They were able to demonstrate that SEB reduced Vp8+ T cells, In addition, levels of anti-DNA antibodies, circulating immune complexes, proteinuria, lymph node hyperplasia, and vasculitis were also reduced. Other superantigens were not used. Our group showed that SEB administered prior to immunization with MBP could protect PL/J mice from the development ofEAE (73). Again, it appeared that the mechanism of protection was the induction of anergy or the deletion of Vp8+ T cells. Kalman et al. (74) also found a reduction in the incidence of EAE in PL/Jmice pretreated with SEB. Similar results were obtained by treating Lewis rats with SEE (75). An interesting study by Kawamura et al. (76) demonstrated the ability of superantigens to prevent autoimmune type I diabetes in NOD mice. In this study, SEA or SEC was administered to NOD mice at 4 or at 4 and weeks of age. At 32 weeks of age, there was a significant reduction in the incidence of diabetes with either treatment. Interestingly, splenocytes from superantigen-treated mice were able to suppress the transfer of diabetes by splenocytes from acutely diabetic animals, and suppressor activity was diminished by the depletion of CD4+ T cells. These results suggest that superantigens can induce a state of immune suppression at least in some models of autoimmunity. Based on our studies involving superantigens and EAE, we wished to examine the effects ofSEB on the development of diabetes in the NOD mouse (Table We initially examined the effect of administering SEB to NOD mice at either 4 weeks of age or weeks of age. We anticipated that SEB administered at weeks of age, when insulitis is well established, might actually accelerate the disease process. Surprisingly, SEB administered at 10 weeks of age actually delayed the onset of overt diabetes, although by 20 weeks of age all groups of mice, treated or untreated, had developed the same incidence of diabetes (M. Atkinson and J. Schiffenbauer, unpublished). Therefore, unlike EAE where SEB was able to exacerbate disease in animals with autoreactive T cells, SEB not only did not exacerbate diabetes, but seemed to delay its onset. The mechanism for this delay was not further investigated.

oimmunity Superantigens in

543

Table 2 Onset of Diabetes (bs > 400) in Female NOD Mice Administered Superantigen at Various Intervals ~

12 weeks

14 weeks

~~~~

~

16 weeks

~

18 weeks

~~~~

20 weeks

Number in parentheses is the percentage of mice with diabetes. '(1) Saline at 4 and 10 weeks; (2) at 4 weeks; (3) SEB at 10 weeks.

Together with the results from Kawamura, thesestudies suggest that the effects of superantigens on autoimmunity in different systems cannot be predicted. Furthermore, the mechanisms by which superantigens may influence the course of autoimmune disorders have not been fully explored. It seems possible, therefore, that superantigens may yet have a place in the treatment of autoimmunity. VIII.

SUMMARYANDCONCLUSIONS

We have presented evidence that superantigens may-have the ability to initiate or exacerbate autoimmune disorders. Unfortunately, in most instances of human diseases, the evidence is circumstantial based on the ability of superantigens to leave their imprint on the immune system by activating, anergizing, or deleting specific T-cell subsets. With the exception of KD where one group has demonstrated not only the expansion of specific Vp subsets, but also cultured bacteria capable of producing superantigens from individuals with this disorder, there is no firm evidence to support the role of superantigens in the development of autoimmune disorders in humans. However, evidence from animal models demonstrating the ability of superantigens to alter the course of the autoimmune process is compelling. Based on this, it certainly seems reasonable to conclude that superantigens will play a role in the course of autoimmune disorders in humans. We believe it is most likely that these bacterial products may lead to the exacerbation of a preexisting autoimmune process in humans and will be one of the key environmental agents influencing the course of autoimmunity. If this is indeed proven to be the case, therapies aimed at eliminating these bacterial agents or neutralizing the products of these agents will have a significant impact on the course and treatment of autoimmune disorders in humans.


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28. Schwab JH, Brown RR, Anderle Schlievert PM. Superantigen can reactivate bacterial cell wall-induced arthritis. J Immunol 1993; 150:41514159. 29. Mountz JD, Zhou T, Long RE, Bluethmann H, Koopman WJ, Edwards CK. T cell influence on superantigen-induced arthritis in MRL-lpr/lpr mice. Arthritis Rheum 1994;37:113-124. 30. Giegerich G, Pette M, Meinl E, Epplen JT, Wekerle H, Hinkkhanen A. Diversity of T cell receptor a and chain genes expressed by human T cell specific for similar myelin basic protein peptide/major histocompatibility complexes. Eur J Immunol 1992; 22:753-758. 31. Martin R, Howell MD, Jaraquemada D, Flerlage M, Richert J, Brostoff S, Long EO, McFarlin DE, McFarland HF. A myelin basic protein peptide is recognized by cytotoxic T cells in the context four HLA-DR 173:19-24. types associated with multiple sclerosis. J Exp Med 32. Wucherpfennig KW, Newcombe Li H, Keddy C, Cuzner ML, Halfer DA. T cell receptor Vu-VP repertoire and cytokine gene expression in active multiple sclerosis lesions. J Exp Med 1992; 175:993-1002. 33. Oksenberg JR, Panzara MA,Begovich AB, MitchellD, Erlich HA, Murray RS, Shimonkevitz R, Sherritt M, Rothbard J, Bernard CCA, Steinman L. Selection T-cell receptor VP-DP-JP gene rearrangements with specificity for a myelin basic protein peptide in brain lesion sof multiple sclerosis. Nature 1993; 362:68-70. 34.HaflerDA,DubyAD,Lee BenjaminD,Seidman JG, WeinerHL. Oligoclonal T lymphocytes in the cerebrospinal fluid of patients with multiple sclerosis. J Exp Med 1988; 167:1313-1322. 35.Brocke Gaur A,PiercyC, GautmA, Gijbels K, Fathman CG, Steinman L. Induction of relapsing paralysis in experimental autoimmune encephalomyelitis by bacterial superantigens.Nature 1993; 365:642-644. 36. Matsumoto Y, Fujiwara M. Immunomodulation of experimental autoimmune encephalomyelitis by staphylococcal enterotoxin D. Cell Immunol 1993;149:268-278. 37. JM,Hobeika AC, Butfiloski EJ, Schiffenbauer J, Johnson HM. Accelerated induction of experimental allergic encephalomyelitis in PL/ J mice by a non-VP8-specific superantigen. Proc Natl Acad Sci USA 1995; 92:6082-6086. 38. Burns J, Littlefield K, Gill J, Trotter JL. Bacterial toxin superantigens activate human T lymphocytes reactive with myelin autoantigens. Ann Neurol 1992;32:352-357. 39.RackeMK, Quigley L, Cannella B, Raine CS, McFarlin DE, Scott DE. Superantigen modulation of experimental allergic encephaloymelitis: activation or anergy determines outcome. J Immunol 1994;152:20512059. 40. Leung DYM. Immunologic aspects of Kawasaki syndrome. J Rheum 1990;24:15-18.


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41. Leung DYM,Siege1 L, Grady S, Krensky A, Meade R, Reinherz EL, Geha RS. Immunoregulatory abnormalities in mucocutaneous lymph node syndrome. Clin Immune Immunopatholl982; 23:lOO-112. 42. Abe J, Kotzin BL, Meissner C, Melish ME, Takahashi M, Fulton D, Romagne F, Malissen B, Leung DYM, Characterization of T cell repertoire changes in acute Kawasaski disease. J Exp Med 1993; 177:791-796. 43. Curtis N, Zheng R, Lamb JR, Levin M. Evidence for a superantigen mediated process in Kawasaki disease. Arch Dis Child 1995; 72:308-311. 44. Choi Y, Lafferty JA, Clements JR, Todd JK, Gelfand EW, Kappler J, Marrack P, Kotzin BL. Selective expansion of T cells expressing Vp2 in toxic shock syndrome. J Exp Med 1990; 172:981-984. Fulton DR, Murray DL, Kotzin BL, 45. Leung DYM, MeissnerHC, Schliever PM.Toxicshock syndrome toxin-secreting staphylococcus aureus in Kawasaki syndrome. Lancet 1993; 342:1385-1388. 46. Leung DYM, Giorno RC, Kazemi LV, Flynn PA, Busse JB. Evidence for superantigen involvement in cardiovascular injury due to Kawasaki syndrome. J Immunol 1995; 155:5018-5021. 47. Leung DYM, Burns JC, Newburger JW, Geha RS. Reversal of lymphocyteactivation in vivo in the Kawasaki syndrome by intravenous gammaglobulin. J Clin Invest 1987; 468-472. 48. Takei S, Arora Y, Walker SM. Intravenous immunoglobulin contains specific antibodies inhibitory to activation of T cells by staphylococcal toxin superantigens. J Clin Invest 1993; 91:602-607. 49. Pietra BA, Inocencio JD, Giannini EH, Hirsch R. TCR Vp Family repertoire and T cell activation markers in Kawasaki disease. J Immunol 1994;153:1881-1888. 50. Sakaguchi M, Kat0 H, Nishiyori A, Sagawa K, Itoh K. Characterization of CD4+ T helper cells in patients with Kawasaki disease (KD): preferential production of tumour necrosis factor-alpha (TNF-a) by Vp2- or Vp8- CD& helper cells. Clin Exp Immunol 1995; 99:276-282. 51. Lehman TJA, Warren R, Gietl D, Mahnovski V, Prescott M. Variable expression of Lactobacillus casei cell wall-induced coronary arteritis: an animal model Kawasaki’s disease in selected inbred mouse strains. Clin Immunol Immunopathol 1988;48:108-118. 52. Leiter EH. The genetics of diabetes susceptibility in mice. FASEB J 1989; 32231-2241. 53. Atkinson MA, Maclaren NK. The pathogenesis of insulin-dependent diabetes mellitus. N Engl J Med 194; 331:1428-1435. 54. Conrad B, Weldmann E, Trucco G, Rudert WA, Behboo R, Ricordi C, Rodriguez-Rilo H, Finegold D, Trucco M. Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetiology. Nature 1994;371:351-355. 55. Galley KA, Danska JS. Peri-islet infiltrates of young non-obese diabetic mice display restricted TCR P-Chain diversity. J ImmunollFI5; 154:29692982.

Schiffenbauer et al. 56. Ellerman KE, Like AA. Staphylococcal enterotoxin-activated spleen cells passively transfer diabetes in BB/Wor rat. Diabetes 1992; 41:527-532. 57. StegemanCA, Tervaert JW, Sluiter WJ,Manson WL, de Jong PE, Kallenberg CGM. Association of chronic nasal carriage of staphylococcus aureus and higher relapse rates in Wegener granulomatosis. Ann Intern Med 1994; 120:12-17. 58. Marples RR, Heaton CL, Kligman AM. Staphylococcus aureus in psoriasis. Arch Dermatol 1973; 107:568-570. 59. Whyte HJ, Banghman RD. Acute guttate psoriasis and streptococcal infection. Arch Dermatol 1964; 89:350-356. 60. b u n g DYM, Harbeck R, Bina P, Reiser RF, Yang E, Norris DA, Hanifin JM, Sampson HA. Presence of IgE antibodies to staphylococcal exotoxins on the skin of patients with atopic dermatitis. J Clin Invest 1993; 92:1374-80. 61. Henderson CA, Highet AS. Acute psoriasis associated with Lancefield group C and group G cutaneous streptococcal infections. Br J Dermatol 1988;118:559-562. 62. Rosenberg EW, Noah PW, Zanolli MD, Skinner RB, Bond MJ, Crutcher N. Use of rifampin with penicillin and erythromycin in the treatment of psoriasis. J Am Acad Dermatol 1986; 14:761-764. 63. Rosenberg EW, Noah PW. The Koebner phenomenon and the microbial basis of psoriasis. J Am Acad Dermatol 1988; 18:151-158. 64. Leung DYM, WalshP, Giorno R, Norris DA.A potentialrolefor superantigens in the pathogenesis of psoriasis. J Invest Dermatoll993; 100:225-228. 65. Lewis I", Baker BS, Bokth S, Powles AV, Garioch JJ, Valdimarsson H, Fry L. Restricted T-cell receptor Vp gene usage in the skin of patients with guttate and chronic plaque psoriasis. Br J Dermatol 1993; 129:514520. 66. Boehncke WH, Dressel D, Manfras B, Zollner TM, Wettstein A, Bohm BO, Sterry W. T-cell-receptor repertoire in chronic plaque-stage psoriasis is restricted and lacks enrichment of superantigen-associated Vp regions. J Invest Dermatol 1995; 104:725-728. Majumdar G, Beachey EH. Superantigenicity of 67. Tomai M, Kotb M, streptococcal M protein. J Exp Med 1990; 172:359-362. 68. Tomai MA, Aelion JA, Dockter ME, Majumdar G, Spinella DG, Kotb M. T cell receptor V gene usage by human T cells stimulated with the superantigen streptococcal M protein. J Exp Med 1991; 174:285-288. 69. Scott DE, Kisch WJ, Steinberg AD. Studies of T cell deletion and T cell anergy following in vivo administration ofSEB to normal and lupusprone mice. J Immunol 1993; 150:664-671. 70. Zouali M. B-cell superantigens: implications for selection of the human antibody repertoire. Immunol Today 1995; 16:399-405. 71. Pascual V, Capra JD. B-cell superantigens? Curr Biol1991;1:315-317. 72. Kim C, Siminovitch KA, Ochi A. Reduction of lupus nephritis in MRL/


73.

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75. 76.

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lpr mice by a bacterial superantigen treatment. J Exp Med 1991; 174:14311437. Soos MJ, Schiffenbauer Johnson HM. Treatment of PL/J mice with the superantigen, staphylococcal enterotoxin B, prevents development of experimental allergic encephalomyelitis. J Neuroimmunol 1993; 43:3944. Kalman B, Lublin FD, Lattime Joseph Knobler RL. Effects of staphylococcal enterotoxin B on T cell receptor Vp utilization and clinical manifestations of experimental allergic encephalomyelitis. J Neuroimmunol 1993;45233-88. Rott Wekerle H, Fleischer B. Protection from experimental allergic encephalomyelitis by application of a bacterial superantigen.Int Immunol 1992; 4:347-353. Kawamura T, Nagata M, Utsugi T, Yoon JW. Prevention of autoimmune type I diabetes by CD4+ suppressor T cells in superantigen-treated nonobese diabetic mice. J Immunol 1993; 151:4362-4370.


Superantigens in Inflammatory SkinDiseases

David

Norris

of Donald Y. M. Leung

INTRODUCTION

The skin is a frequent site of infection, and the lesions of a number chronic skin diseases are commonly secondarily infected due to breakdown in the normal barrier function of the stratum corneum and to accumulation of serum-filled scale and crust. In the case psoriasis and eczema, clinicians have long postulated that bacterial infections might indeed trigger these diseases. Exanthems and reactive erythemas are also common cutaneous manifestations,of systemic bacterial and viral infection. The discovery of the functions of bacterial superantigens and toxins has provided an important step in un551

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derstanding how localized systemic bacterial infections could induce erythemas and trigger inflammatory skin diseases. The mechanisms by whichsuperantigens can activate largeT-cell populations are reviewed in detail elsewhere in this book. Superantigens have three major effects in vivo on T cells bearing the relevant Vp elements. First, exposure of developing thymocyles to superantigen causes intrathymic deletion of thymocytes bearing the appropriate Vp specificities, with resultant skewing of the T-cell repertoire (1,2). Second, exposure of peripheral T cells to superantigen leads to profound activation and lymphocytosis of T cells expressing the appropriate Vp specificity. This can lead to Vp skewing of the stimulated population, to deletion and anergy of the stimulated clones with time. Finally, exposure of T cells to superantigen produces profound systemic and local effects through the release of cytokines by T cells and antigen-presenting cells (APCs). The potential to expand or delete large populations of lymphocytes implicates superantigens as key mediators in a variety of diseases associated with immunodeficiency, autoimmunity, and/or inflammation (reviewed in Ref. 2). Superantigens also have profound effects on APC, on local endothelial cells, and on tissue targets such as epithethial cells. In this chapter we will review the local and systemic effect superantigens in the skin, as well as our current understanding of how superantigens trigger skin disease. II. IMMUNOBIOLOCY

SUPERANTICENS IN THESKIN

The cutaneous effects of bacterial toxins fallinto three categories. The first is the superantigenic effect, in which lymphocytes of particular Vp specificities are stimulated by superantigen to prdiferate, express cutaneous homing receptor, and infiltrate the skin. Alternatively, skin-infiltrating lymphocytes of particular Vp specificities may respond within the skin to superantigens presented by macrophages, cutaneous Langerhans cells, and MHC class 11+ keratinocytes. The second category is the capacity of bacterial toxins and superantigens to induce cytokine release, either systemically locally. Systemic cytokine release would augment leukocyte migration into the skin through induction of adhesion molecules on vascularendothelium and by the induction adhesion molecules, activation molecules, and homing receptors on leukocytes. Cutaneous toxin exposure would cause the same effect through local cytokine release, predominantly from epidermal keratinocytes. The third type of toxin-mediated in-

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tracutaneous activity is nonimmune and is exemplified by the superficial epidermal exfoliatin produced by the staphylococcal exfoliative toxin, which induces premature separation of the superficial epidermis. A.

SuperantigenicEffects of Bacterial Toxins in the Skin

The superantigenic effect of bacterial toxins is the key to their role in T-lymphocyte activation. Superantigens possess the unique capacity to stimulate a large fraction of T cells by circumventing the normal mechanisms of T-cell activation that determine clonal activation of lymphocytes whose TCR is engaged by the appropriate peptide sequence on the stimulating antigen. Nominal peptide antigens are usually processed and presented in the groove of the MHC molecule on the surface of the APC. Superantigens activate lymphocytes by directly bridging the molecular complexes of MHC class I1 on the surface of APC, and the Vp chain of the TCR on T cells, binding outside the normal antigen-binding groove of the TCR. In this way, they can activate many clones of T cells bearing the particular Vp specificity activated bythe superantigen in question. Toxins produced by Staphylococcus aureus and Streptococcus pyogenes are the most wellcharacterized bacterial superantigens and the specificities of the T lymphocytes activated by these toxins are summarized in Table 1.

Table 1 Human TCR Vp Specificities Activated by Particular Superantigens Exotoxin Staphylococcus aureus SEAa SEB SEC SED SEE TSST-lb Exfoliatin Strepytococcuspyogenes SPEAc SPEB SPEC "Staphylococcal enterotoxin bToxic shock syndrome toxin-l. Streptococcal pyrogenic exotoxin

TCR VS specificity

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Since S. aureus is a common skin pathogen and since toxins produced by S. aureus may be absorbed epicutaneously, there is great interest in the local effects of staphylococcal superantigens in the epidermis. Several laboratories have addressed the ability of Langerhans cells and/or keratinocytes to function as accessory cells in the T-cell response to staphylococcal superantigens. Epidermal Langerhans cells and IFNy-treated epidermal cells both acted as accessory cells in presenting staphylococcal enterotoxin B (SEB) to murine splenic T cells (4). Similarly, MHC class 11+ cultured human keratinocytes treated with IFNy acquire the ability to activate T cells in the presence of superantigen This interaction can be blocked by antibodies to MHC(HLA-DR, HLA-DQ), to intercellular adhesion molecule-l (ICAM-l), to lymphocyte function associated antigen-l (LFA-l), indicating that cell adhesion and accessibility of class I1 molecules are necessary for this activation. Class 11+ keratinocytes are not able to present conventional peptide antigens to na'ive T cells, presumably because keratinocyte cannot process antigen for inclusion in the antigen-binding groove of the class I1 molecule, and because they lack accessory molecules to deliver the second signal in T-cell activation Expression of ICAM-l on keratinocytes can be induced by the synergistic effects of TNF and IFNy and this is necessary to facilitate lymphocyte binding to keratinocytes, which are constitutively ICAM-1 negative. Class I1 expression on keratinocytes is induced by IFNy, but not by TNF-a or IL-l Recently, Elmets has demonstrated that a class I positive transformed keratinocytes cellline can present SEB superantigen to lymphocytes (11). An erythroid cell line negative for class Icould be induced to act as APC for SEB when transfected to express cell surface class I. This raises the possibility that keratinocytes induced by local release of TNF-a to express cell surface ICAM-1 might act as superantigen-presenting cells to epidermal lymphocyte, without the need for lymphocyte-derived IFNy. Conversely, established APC populations within the skin may be directly affected by superantigens. Pickard et al. (12) reported depletion epidermal Langerhans cells following treatment by SPEA ET but not TSST-1, producing the same type of changes in APC seen with UV radiation of the skin. Recently it has been shown that superantigenic stimulation lymphocytes may promote skin homing by up-regulation of homing receptors that favor localization subsets activated leukocytes to the skin. The heterogeneous expressionof lymphocyte homing receptors (HR) by the memory/effector T-cell [CD45RA(low)/RO(high)] population in the human is thought to define subsets with tissue-

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selective recirculatory potential. Picker et al. (13) have shown that skin T cells, compared to peripheral blood T cells, were highly enriched for the cutaneous lymphocyte-associated antigen (CLA) subset. In contrast, lung-homingT lymphocytes expresseddifferent homing receptors. Leung et al. have shown that in vitro stimulation of peripheral blood mononuclear cellswith staphylococcal enterotoxin B, toxic shock syndrome toxin-l, and streptococcal pyrogenic exotoxins A and C induces a significant increasein the numbers CLA+ T-cell blasts ( p < O.Ol), but not blasts bearing the mucosa-associated adhesion molecule aE P7-integrin, compared with T cells stimulated with phytohemaglutinin (PHA) anti-CD3 (14). Bacterial toxinswere also found to specifically induce interleukin (IL)-12 production. More importantly, induction of toxin-induced CLA expression was blocked by anti-IL-12, and the addition ofIL-12 to PHA-stimulated T cells induced CLA, but not aE B7-integrin, expression. These data suggest that bacterial toxins induce the expansion of skin-homing CLA+ T cells in an IL-12-dependent manner (14). This characteristic of superantigens would greatly favor the development of skin rashes in toxin syndromes mediated by superantigens. B.

Cytokine Release Triggered by Superantigens in the Skin

Cytokine release is a major factor in superantigen-mediated systemic toxicity. Staphylococcal superantigens have also been shown to release IL-1 and TNF-a from monocytes (15). Local cytokine release in the skin by superantigens has been proposed as a factor contributing to superantigen triggering of skin disease Murine keratinocytes release TNF-a but not IL-a when stimulated with SEB (4). The type of APC used to present superantigen to T cells also determines the cytokine profile following activation. Goodman et al. (17) compared the profile of cytokines produced by T cells stimulated by SEB in the presence dermal dendritic cells versus MHC class11+ keratinocytes. T cells stimulated in the presence of dermal dendricytes produced a TH1 pattern of IL2 and IFNy while T cells stimulated in the presence of MHC class 11+ keratinocytes produced onlyIL-4. It has been speculated that this difference is related to defective IL-12 production by keratinocytes during this activation. Although it is indeed intriguing that keratinocyte presentation of superantigen might favor a different T-cell pattern of cytokine release, these results have not been verified by other groups in any diseases where superantigen influence TH2 response is favored. In atopic dermatitis, other mechanisms fa-

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voring the TH2 phenotype have been described, including enhanced dermal macrophage production ofIL-10 C.

BacterialExfoliatin and the Skin

In the staphylococcal scalded skin syndrome, separation (acantholysis) occurs within the epidermis just below the stratum granulosum (19). The granular cell layer is the point at which differentiated keratinocytes undergo apoptosis and form the stratum corneum, which is the major barrier to transepidermal water loss and penetration of environmental irritants. The acantholysis in these patients is caused by the staphylococcal exfoliative toxin, present as two serotypes exfoliatin a (eta) andexfoliatin b (etb). The staphylococcal exfoliatins can act as classical superantigens, but their action in inducing epidermal exfoliation is unique and unlikely to occur asa result of its superantigenic properties. The mechanism by which the staphylococcal exfoliative toxininduces acantholysis is still a mystery. The toxin binds to skin when exfoliatin-producing strains of S. aureus are injected intraperitoneally into mice, but purified toxin does not bind to erythrocytes, leukocytes, trypsin, dispersed keratinocytes, or whole skin (20). Application of exfoliatin to cultures of keratinocytes or fibroblasts to lymphocyte or sperm cell suspensions or to skin sections does not alter the cell surface expression of proteins of lectins. Tracer studies and ultrastructural analysis do not demonstrate changes in cellular permeability or plasma membrane damage (21). Recent studies have indicated that keratohyalin granules might be a binding site for the staphylococcal exfoliative toxin,and that it can enter cells permeabilized by protease treatment (22). However, the mechanism induction acantholysis remains unclear. TOXINSYNDROMESANDTHESKIN

The skin is a common site of manifestations of many toxin-induced syndromes in humans. Indeed, the erythema and desquamation observed in many of the toxin syndromes can be quite similar. The fact that it may be difficult to separate the overlapping clinical characteristics of scarlet fever, toxic shock syndrome, Kawasakisyndrome, and staphylococcal scaldedskin syndrome has been noted (23). Since the superantigenic toxins that cause the cutaneous lesions in each of these syndromes have similar and overlapping effects, it is not surprising that each causes similar findings on the skin: redness, desquamation,


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and tenderness. It is useful to consider some of the characteristics of the major infectious toxin syndromes as they affect the skin. A.

Scarlet Fever

Many of the symptoms and signs of scarlet fever (scarlatina) are caused by erythrogenic toxin(s) produced by S. pyogenes infection. Scarlet fever can occur in patients with streptococcal pharyngitis, or in patients with generalized streptococcal infections. The scarlatiniform eruption of the skin appears as finely punctate erythema of the chest (”sunburn with goosepimples”) and generalizes over several days. In the creases, transverse red streaks (Pastia’s lines) appear due to capillary leakage. The mucous membranes are commonly bright red, and the tongue is initially coated; later, red swollen papillae of the tongue appear (strawberry tongue). Fever may last 7-10 days without treatment. The rash generalizes and is commonly followed by desquamation, with lamellar scaleson the palms and soles. Multiorgan involvement can be seen in severe scarlet fever. The presence of the genes encoding SPEA, SPEB, SPEC, and streptolysin 0 (SLO) in strains of pyogenes isolated from various clinical infection syndromes has strongly implicated SPEA in the induction of scarlet fever (24). Eight-one percent of S. pyogenes strains from patients with scarlet fever showed SPEA gene after amplification by PCR, compared to 42.9% from severe infections and only 18.4%from pharyngitis. No significant differences were seen in SPEC distribution, while SPEB and SLO genotypes were more common in pharyngitis (54.1%)than in scarlet fever (18.8%).Other investigators have found scarlet fever more strongly associatedwith particular betahemolytic streptococcal serotypes (25). B.

Toxic Shock Syndrome

In the classic toxic shock syndrome (TSS), patients present with fever, chills, vomiting, diarrhea, hypotension, and a diffuse red rash. A rash may be one of the first signs of disease. A diffuse erythema is most common, but scarlatiniform, pustular, and even vesicular characteristics may be seen. Edema of the hands and feet and bulbar conjunctival erythema are common (26), as are erythema and swelling of the mucous membranes. Desquamation, including shedding of the palms and soles, is usually seen weeks after initiation of toxic shock. The syndrome was characterized in menstruating women using tampons that were contaminated with high counts of strains of S. aureus producing a distinctive toxin (TSST-1) (26,27).

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Although TSST-l-producing strains of S. aureus have been associated historically with toxic shock syndrome, strains of beta-hemolytic Streptococcus have also been associated with a "toxic shock-like syndrome" difficult to differentiate from class TSS (28). As discussed previously, systemic absorption of large amounts of superantigens toxins can induce generalized cytokine (especiallyIL1, TNF, and IFNy) release, polyclonal activation of large cohorts of lymphocytes of appropriate Vp specificity, generation of large numbers skin-homing lymphocytes, and direct effects on many cell types, inducing cascades local cytokine release. In addition, the shock induced by TSST-1 appears to be potentiated by endotoxin. Lee et al. (29) showed that the capillary leakageand hypotension induced in rabbits by TSST-l or by SPEA injection was blocked by polymyxin treatment (to neutralize endogenous endotoxin) but not by cyclosporin A (to block toxin-induced T-cell proliferation). Staphylococcal exfoliative toxinand concanavalin A used as mitogen controls did not produce the lethal "toxic shock" in rabbits induced by TSST-1 or SPEA. It appears that selected toxins from both staphylococci and streptococci can induce toxic shock syndromes in which capillary leakage is potentiated by endogenous endotoxin.

C. Kawasaki Syndrome Kawasaki syndrome or the mucocutaneous lymph node syndrome (MCLNS) is characterized by swollen tongue similar to a strawberry tongue, dry red lips and conjunctival erythema, a diffuse red rash, swollen lymph nodes, arthritis and arthralgia, and cardiac arrhythmia (30). It is the main cause of acquired heart disease in children. The red rash in MCLNS has been described as urticarial, scarlatiniform, or morbilliform, and is followed by desquamation. There is now strong evidence to support the role of superantigens in Kawasaki syndrome (31). Superantigenic activation of T lymphocytes was suggested by the preferential expansion of Vp2 and Vp8 in Kawasaki syndrome (32). Evidence for involvement of toxinproducing strains of both Sfreptococcus and SfaphyZococcus has been presented, although identification of pharyngeal streptococcal infection has been variable in patients with Kawasaki syndrome (33). In some recent studies, TSST-l-secreting S. aureus has been the predominant organism isolated from the pharynx or rectal swabs of patients with Kawasaki syndrome (34,351.

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(SSSS)

Class SSSS is an acute toxic syndrome in children characterized by widespread erythematous, tender skin that separates in sheets spontaneously or with applied friction (19). The syndrome also occurs in adults (36) and is caused by one of two exfoliative toxins produced by S. aureus. A localized form of the syndrome, bullous impetigo, is seen in both children and adults Koch’s postulates have been fulfilled in linking SSSS to the exfoliative toxin: 1) S. aureus is isolated in every case, 2) S. aureus injected into experimental animals produces the disease, a specific extracellular toxin (ET) can reproduce the syndrome, and 4) antibody to toxin can protect experimental animals against the syndrome (38). However, purified ET does not cause erythema in either humans or experimental animals, and the full-blown SSSS may require other staphylococcal toxins for someof the systemic effectsobserved. Little is known about absorption, metabolism, or neutralization of the ET, and it is not clearly understood why children are more susceptible to development of SSSS. It is also likely that multiple staphylococcal toxins may synergize in patients to produce more or less erythema, fever, or even hypotension. E.

Differentiation

Toxin Syndromes

Clinical distinction of scarlet fever, toxic shock syndrome, Kawasaki syndrome, and early SSSS can be quite difficult clinically, due to similarity to the red rash and mucous membrane lesions seen in these syndromes, and the similarity of the systemic symptoms and signs in these patients (23,39). With better understanding of the role of bacterial toxins ininducing these syndromes,the reasons for the similarities among these syndromes may be better understood. How. ever, we do not yet understand the mechanistic differences between MCLNS, TSS, and scarlet fever, especially regarding susceptibility to multiorgan involvement. These toxin-mediated syndromes must also be differentiated from ”toxic erythemas” seen with many febrile illnesses, exanthems associated with acute viral illnesses, and reactive erythemas such as erythema chronicum migrans, erythema annulare centrifugum, and erythema multiforme, all of which are likely to be linked to infectious diseases. In someof these cases, such as severe erythema multiforme, the extent of disease can be as severe as the above toxin-mediated syndromes ( 40).

Norris and Leung

S K I N DISEASESTRIGGERED BY SUPERANTIGENS Psoriasis

Psoriasis is a complex heterogeneous disease with diverse clinical presentations and with polygenic inheritance. The pathogenesis of psoriasis involves four features: 1. Lymphocyte activation 2. Vascular activation Keratinocyte activation 4. Neutrophil activation

Lymphocyte activation is now believed to be the central feature of the pathogenesis of psoriasis, supported by the following evidence. Histological examination of early skin lesions of psoriasis shows that infiltration of lymphocytes and' macrophages into the skin precedes the characteristic epidermal proliferation that is characteristic of psoriasis (41). T-cell clones isolated from psoriasis plaques releasegrowth factors that stimulate keratinocyte proliferation (42); IFNy is one of the cytokines that trigger the regenerative phenotype in psoriasis (42). The lymphocyte cytokine pattern in lesional psoriatic skin is THl in type, characterized by IL-1, F N y , and TNF-a Although psoriasis appears to be polygenic, there is evidence that lymphocyte responses may be geneticallydetermined in psoriasis. The strong association with particular HLA genotypes in psoriasis suggests that MHC-influenced lymphocyte activation is involved in psoriasis (44). The strongest evidence linking lymphocyte activation to psoriasis has derived from the clinical observation that immunosuppressive drugs that inhibit T-cell activation and cytokine release, such as anti-CD3, corticosteroids, and cyclosporin A, are effective treatments for psoriasis (45,46). The most convincing demonstration of this point has come from the work of Krueger and Gottlieb, who demonstrated that an IL-2-fusion toxin that eliminated activated IL-2 receptor-positive lymphocytes causedstrong clinical resolutionof psoriasis. At the same time, the dermal and intradermal lymphocyte populationswere eliminated and the regenerative epidermal phenotype was reversed (46). It is now believed that elimination of lymphocyte populations by induction of apoptosis is a crucial mechanism of action of ultraviolet light therapy and PUVA treatment (47). The release of cytokines by activated lymphocytes and monocytes and secondary cytokine release from fibroblasts and keratinocytes are believed to be central in maintenance of endothelial cell


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activation and the regenerative epidermal keratinocytephenotype that are characteristic of psoriasis. Localized collectionsof HLA-DR+ keratinocytes suggest IFNy production by TH1 helper lymphocytes (48). IFNy production within psoriatic epidermis has been demonstrated by Nickoloff (49). There still debate concerning which activated lymphocyte populations are central to induction and maintenance the psoriatic phenotype. Because of the many clinical presentations of psoriasis (Table investigating the mechanisms of psoriasis has been difficult. The informative research addressingthis question has focused on two clinical types of psoriasis: acute guttate psoriasis as a model the initiation of the psoriatic lesion, and chronic type I plaque psoriasis as a model maintaining the psoriatic phenotype. We will discuss both models, although only acute guttate psoriasis has been shown quite convincingly to be a model of superantigen-driven induction of psoriasis. 1. Acute Guttate Psoriasis as a Model

Superantigenic Lymphocyte

Activation The mechanisms and triggers that activate T lymphocytes and maintain the activated state lymphocytes, keratinocytes, and macrophages in psoriasis skin lesions are incompletely understood. This pro-

Table 2 Heterogeneity in Psoriasis Vulgaris Clinical variant ~

Characteristics

~~~

Psoriasis vulgaris Type 1 Type I1 Guttate psoriasis Pustular psoriasis Generalized Palms and soles Acute (von Zumbusch) Erythroderma Psoriatic arthritis

Small or large plaques, knees, elbows, trunk, scalp Early onset in childhood, persistent, extensive Late adult onset, more limited Acute onset, small teardrop lesions, may become p. vulgaris Intraepidermal sterile pustules Extensive pustules on an erythematous base Limited to palms and soles Acute onset, hundreds pustules with erythema, fever, leukocytosis Generalized erythema and scale, often after steroid withdrawal Classical proximal digits Symmetrical, RA-like Axial

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cess is surely multifactorial, but there are a number of reports that indicate that one likely trigger, especially in acute guttate psoriasis, is bacterial infection (50-52). It has been demonstrated that 50% of patients with psoriasis harbor S. aureus on their skin (51,53). In acute guttate psoriasis, guttate psoriasis lesions are preceded by pharyngitis infected with S. pyogenes and are accompanied by rises in serum antistreptococcal titers (52,54). Patients with this form of psoriasis frequently show improvement of their skin disease when treated with antistreptococcal antibiotics. The first investigative support of these clinical observations was provided by Cole and Wuepper, who reported that alcohol extracts of cultures of S. pyogenes (strain NY-5) induced increased keratinocyte proliferation after injection into rabbit skin (55). These culture filtrates were strongly mitogenic for cultured human lymphoid cells and probably contained large amounts of the streptococcal enterotoxins SPEA,SPEB, and SPEC. It was subsequently demonstrated that intradermal injections of small amounts of streptococcal extracts into normal skin of psoriatic patients induced lesions with histological characteristics of psoriasis (56). Furthermore, lymphocytes isolated from both guttate and chronic plaque psoriasishave enhanced T-lymphocyte proliferative responsesto group A streptococcal antigens (57). These observations have led to the hypothesis that bacterial superantigenic toxins could induce activation of epidermal keratinocytes, infiltrating lymphocytes, and monocytes in some patients with psoriasis (16). As illustrated in Fig. 1, local action of superantigens could expand T lymphocytes bearing the specific Vp to which each superantigen binds. The APC presenting thesesuperantigens to T cells in the skin could be MHC class 11-bearing Langerhans cells in the epidermis, dermal dendricytes (58,59), or epidermal macrophages entering the epidermis following UVR (60,61) (Fig. 2). In the induction of psoriatic lesions, the APCs would likely be "professional" APCs in the dermis or the epidermis, presenting either circulating superantigen or epicutaneous superantigen delivered through broken skin. In persistent psoriasis, activated MHC class 11-bearing keratinocytes might also be important partners in superantigenic activation of lymphocytes. In the complex pathology of psoriasis, it is unlikely that any one APC acts alone. Lymphocyte activated systemically bysuperantigen or superantigen-released cytokines would be recruited to skin sites following CLA up-regulation (13,621. These LFA-l positive lymphocytes could bind to keratinocytes on which ICAM-1 has also been induced by IFNy or TNF-a or -B (9,63).

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VB 14

Local Production

of Cytokines

Julation

VB 2

VB 2

VB 2

Promoting the Psoriatic Phenotype

Figure 1 Vp expansion in psoriasis skin.

Local superantigens could induce further lymphocyte activation and cytokine release. . To prove this concept for acute induction guttate psoriasis, it was necessary to determine whether expansion of particular Vp populations of lymphocytes was seen in the peripheral blood skin of patients, to identify pathogenic bacteria and their superantigens from the patients, to determine whether the Vp pattern in tissue was what would be expanded by the superantigen isolated in the patient, and to induce new lesions with superantigen challenge. These steps have been successfully demonstrated in acute guttate psoriasis, with evidence provided by a number of different investigative groups. In 1993, Lewis et al. contrasted the percentages of nine TCR Vp specificities in the peripheral blood and skin lesions of patients with guttate psoriasis and chronic plaque psoriasis, usinga panel monoclonal antibodies (64). Both types of psoriasis showed a selective expansion of Vp2+ T lymphocytes in the skin lesions compared tothe peripheral blood. Less impressive expansions of Vp 5.1 and 12 were also seen in some patients. The expansion of Vp2 is consistent with activation by the superantigen streptococcal pyrogenic exotoxin C (SPEC) secreted during streptococcal pharyngitis In addition, cloned lymphocytes isolated from such psoriatic lesionsresponded to

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stimulation in vitro by streptococcal proteins (64). Another study of acute guttate psoriasis patients has convincingly linked pharyngeal streptococcal toxins with the lymphocyte activation seen in guttate psoriasis lesions, by demonstrating that streptococci isolated from the pharynx of patients with guttate psoriasis produced the superantigenic toxin SPEC, and that all patients studied had local expansion of Vp2 positive CD4 and CD8 lymphocytes in skin lesions, consistent with SPEC activation (65). In addition, Vp transcripts were cloned from the guttate skin lesions, the sequences of their TCR were analyzed, and it was demonstrated that there was polyclonal expansion of TCR Vp2 consistent with superantigenic activation by the SPEC toxin identified in the patients. Taken together with the previous finding that superantigens activate the expression of CLA in lymphocytes, these findings support the hypothesis that systemic lymphocyte activation by circulating SPEC in patients with streptococcal pharyngitis induces cutaneous localization of lymphocytes, further superantigenic activation, and expansion of SPEC-activated VP2+ lymphocytes, which initiate the lymphocyte-driven inflammation characteristic of psoriasis. Travers et al. (65a) have further demonstrated that application of the superantigens SEB, SPEC, on tape-abraded skin psoriatic patients induces an inflammatory lesion with a mononuclear infiltrate and epidermal thickening. Similar skin testing in normal subjects on normal skin of patients with atopic dermatitis does not produce the same type of inflammatory lesion. Interestingly,the lymphocytes in the dermis and epidermis in this positive patch test to superantigens do not show preferential Vp skewing corresponding to the superantigen applied. In addition, early up-regulation of TNF-a message is seen in the epidermis of these patients. These results are most consistent with the hypothesis that epicutaneous superantigens may activate local cytokine release preferentiallyin psoriasis, and that this is seen in patients with both guttate and plaque psoriasis. 2.

PlaquePsoriasis

In patients with long-standing chronic plaque psoriasis, local exacerbation of disease is commonly seen in the .intergluteal cleft, in the inframammary region, in the groin, and in the scalp. All of these areas have been shown to be reservoirs of bacterial fungi that might trigger local plaque expansion. The concept that the Vp pattern of leukocytes in psoriasis plaque might correspond to the same Vp expanded by microorganisms cultured from the plaques was demon-

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strated in two patients by Leung et al. (16). Unfortunately, in plaque psoriasis there is not yet the same type of hard evidence as there is in guttate psoriasis that bacterial superantigens selectively expand particular lymphocyte populations bearing the appropriate Vp specificities. The most convincing evidence that activated lymphocytes control and maintain the plaques of established type Ipsoriasis is the series of studies from Rockefeller University that systematically studied the effects of antilymphocyte treatments on the lymphocytic infiltrates in psoriasis and on the regenerative epidermal hyperproliferative phenotype of psoriasis (46,47,66). As discussed previously, these studies showed that major antipsoriatic treatments caused reductions in CD3+ lymphocyte and reversed the proliferative epidermal phenotype, decreasing hyperproliferative keratin expression, decreasing PCNA expression, normalizing a3pl integrin expression, normalizing vascular laminin expression, and normalizing filaggrin expression. The effect of antilymphocyte treatments was impressive on total CD8+cells, on dermal CD4+TH lymphocytes, and on the intraepidermal CD8+ lymphocytes. New evidence suggests that the CD8+ epidermal lymphocytes in psoriasis may be a central antigen-driven population necessary to maintain the established psoriasis lesion. Chang and colleagues (67) have analyzed the T-cell-receptor beta-chain variable gene segment (Vp) use of epidermal T cells in shave biopsies of psoriatic lesions. Theyshowed increased expression of Vp 3 and/or Vp 13.1 messages in the CD8+, but not CD4+, T cells in the lesions of a majority patients studied. Sequence analysis of region 3 (CDR3) these two Vp genes from the skin demonstrated monoclonality or marked oligoclonality. A second biopsy from the same or different lesions, performed 3.58 months later in four patients, again revealed increased Vp 3 and/ or Vp 13.1 expression and clonality. Moreover, in three of the four patients, the same Vp CDR3 rearrangement was found in both biopsies, although there was no Vp CDR3 homology between patients. In two patients in whom Vp 3 and/or Vp 13.1 was not increased, an increase in Vp 17 gene use and clonality was found. The clonality of Vp sequence data indicates these cells are recruited and expanded in situ. The persistence of Vp 3 and/or Vp 13.1-bearing CD8+ T cells in lesions that did not undergo resolution suggests their role as effector cells rather than as regulatory cells. The consistent clonal expansion of CD8+ lymphocytes of limited VB specificity strongly suggests a classic antigen, possibly driven by


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an antigen from an infectious agent that cross-reacts with a normal epidermal protein. This scenario would also be consistent with the HLA-A and -C associations described in psoriasis (68), which would activate CD8 lymphocytes. However, there is also evidence that other lymphocyte populations are necessary in psoriasis. Treatment of plaque psoriasis patients with anti-CD4 monoclonal antibodies leads to strong clinical responses. Antilymphocyte drugs depleteCD4 as well as CD8 cells (46). A limited Vp repertoire and lymphocytes that respond to bacterial superantigens have been described in plaque psoriasis as well as in guttate psoriasis (57,641. Evidence for lymphocyte activation and cytokine effects in guttate and plaque psoriasis is summarized in Table The relative importance of local and/or systemic cytokine release, polyclonal lymphocyte activation, and of clonal lymphocyte activation must be determined in these two forms of psoriasis. Additional data are needed before one can determine whether guttate psoriasis and plaque psoriasis are mechanismatically distinct or part of a continuum. Also, the sequence of steps in development of new lesions and in spreading of established lesions must be studied, focusing on the variables of cytokine effectsand lymphocyte activation, as well as on the endothelial cell and keratinocyte activation, and on the neutrophil mobilization that characterizes psoriasis lesions. OtherBacterialToxins and Psoriasis Ezepchuk et al. (69) have further examined the S. aureus isolates from the skin of patients with atopic dermatitis and psoriasis, finding that the bacterial isolates from thesepatients exhibited either characteristic superantigenic toxins or thermolabile toxins believed to be staphylococcal a-toxin. All of these staphylococcalstrains also secretedstaphylococcal protein A (SPA). There were significant differences in the action of these toxins on human keratinocytes and keratinocyte cell lines. The superantigenic toxins TSST-l, SEA, SEB, and ExT-A as well as SpA did not induce significant cytotoxic damage on the keratinocyte cell line HaCaT, while the staphylococcal a-toxin produced profound cytotoxicity. Keratinocyte cytotoxicity induced by staphylococcal a-toxin was time and dose dependent and demonstrated the morphological and functional characteristics of necrosis, not apoptosis. Addition of a-toxin to HaCaT cells induced TNF-a release into the media within 30 min, well before cell death was observed. Addition of superantigens also produced release of TNF-a, with maximal release at 6 or 12 hr. However, the greatest releaseof TNF-a from

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keratinocytes was produced by staphylococcal protein A. Thus SPA, a-toxin, and superantigenic toxins found in S. aureus isolates from patients with psoriasis and atopic dermatitis can produce direct proinflammatory effects on keratinocytes through the release TNF-a. We propose that these effects may be relevant to the induction and persistence of lesions in these two diseases. It is believed that TNF release is a principal trigger in the induction of psoriasis (43). In psoriasis, trauma, scratching, surgery, other stimuli commonly induce new psoriasis lesions, a characteristic termed the isomorphic, Koebner, phenomenon. It has been proposed that TNF-a release is a principal trigger of koebnerization psoriasis. The findings by Travers et al. (65a) raise the possibility that superantigens may locally trigger psoriasis by release of TNF-a from epidermal keratinocytes. The findings by Ezepchuk et al. (69) extend these observations by demonstrating that multiple staphylococcal toxins can induce TNF-a release in the epidermis and that such toxins are commonly found in S. aureus isolates from the skin of patients with psoriasis and atopic dermatitis. Atopic Dermatitis

Atopic dermatitis is a chronic pruritic inflammatory skin disease seen in association with asthma and hayfever in patients with evidence of type I immediate hypersensitivity reactions. It is characterized by local infiltration of monocytes and lymphocytes and a complex immunobiology involving mast cell degranulation and a combination of immediate and delayed hypersensitivity (reviewed in Ref. 70). Recent experimental evidence supports the hypothesis that activation and expansion of T-helper type 2 (Th2) lymphocytes and allergen sensitization are involved in the pathogenesis of atopic dermatitis. The majority of patients with atopic dermatitis have a personal or family history of either asthma or allergic rhinitis. Serum IgE levels are elevated in approximately 80% of patients with atopic dermatitis and these levels correlate with the skin disease activity. In clinical studies, the majority of subjects evaluated produced IgE to various food and inhalant allergens, and double-blind, placebo-controlled food challenges have triggered cutaneous reactions in some patients with atopic dermatitis. Mite-specific Th2 lymphocytes have been cloned from the peripheral blood and skin of atopic patients, corresponding to the mite-specific IgE identified in the same patients (71,72). Lymphocytes in the skin lesions atopic patients demonstrate cytokine RNA patterns most consistent with a Th2 phenotype, i.e., elevated

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IL-4 and IL-5 but not IFNy (73). However, in a more recent study of patch test reactions to house dust mite in atopic individuals, a biphasic cytokine mRNA pattern was observed, with IL-2 and IL-4 mRNA elevation seen at hr and IFNy mRNA elevation seen at 48 hr (74). The IFNy stimulation was attributed to increased IL-12 release seen in 48 hr. However, patients with severe atopic dermatitis do respond to therapeuticadministration recombinant IFN-y, which would be expected to down-regulate the Th2 lymphocyte population (75). There still remains skepticism regrading the relative importance of food and inhalant-induced eczema in atopic dermatitis, and it is argued that only a subset of atopic dermatitis patients clearly show this pattern of reactivity (76). However, there are numerous reports that S. aureus can exacerbate this disease. Leyden et al. first demonstrated that S. aureus could be isolated fromthe affected skin of more than 90% of the patients with atopic dermatitis (77). Up to lo7 colonyforming units of S. aureus have been identified by quantitative methods from the skin of atopic dermatitis patients (78). In clinical trials, patients treated with antibiotics plus corticosteroids fared better than patients treated with corticosteroids alone (79). To better study the mechanisms by which S. aureus might exacerbate atopic dermatitis, Leung and colleagues (53) isolated S. aureus from the skin of atopic dermatitis patients and characterized the toxins produced by these bacterial strains. More than half of the atopic dermatitis patients had S. aureus that secreted identifiable toxins, primarily the superantigenic toxins SEA, SEB, and TSST-1. Similar findings were also reported by McFadden et al. One can propose a number of credible ways in which these superantigens might induce or promote atopic dermatitis lesions. First, they might directly stimulate Langerhans cells, macrophages, dermal dendrocytes, or keratinocytes to secrete TNF-aor IL-la, promoting chemokine release from the epidermis and promoting leukocyteemigration into the skin (81). Second, they might stimulated lymphocyte proliferation via binding to TCR Vp, inducing release of additional cytokines that might mediate tissue inflammation and induce epidermal changes conducive to prolonged atopic dermatitis. Indeed, mouse Th2cells expanded in vitro by superantigen induce cutaneous inflammation when injected into the skin of mice (41). Results by Ezepchuk et al. indicate that there are also other staphylococcal proteins, such as atoxin and staphylococcal protein A, identified in S. aureus isolates from atopic dermatitis patients that might directly induce TNF-a release from keratinocytes (69).

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Since specific antigen-driven activation of T cells and IgE-producing B cells has been proposed as an important component of the pathogenesis of atopic dermatitis, and since superantigens are globular proteins of24-30 kDa, there has also been investigation of the role of staphylococcal proteins as allergens in atopic dermatitis. Leung et al. investigated the immune responses to TSST-1 SEB and SEA identified in S. aureus skin isolates from atopic dermatitis patients Sera from 57%of atopic dermatitis patients contained IgE specific for one more of these superantigenic toxins. In addition, basophils from these patients with IgE antitoxin showed significant degranulation when incubated in vitro with the specific toxin for whichthe IgE was specific. Basophils from normal controls from atopic dermatitis patients with IgE antitoxin did not show degranulation when exposed to toxins. These findings raise the intriguing possibility (16,531 that epicutaneous superantigenic toxins might induce specific IgE in atopic dermatitis patients, including mast celldegranulation in vivo when the toxins penetrate the disrupted epidermal barrier (82) and inducing IgE-triggered histamine release, which promotes the scratch- itch cycle prominent in atopic patients. V.

S K I N DISEASES IN WHICH SUPERANTIGENS MIGHT BE TRIGGERS

A number of common forms of eczema dermatitis have been found to favorably respond to antibiotic therapy, and in which local infections might be triggers of disease activity. 1: SeborrheicDermatitis

Seborrheic dermatitis is a common inflammatory disease of the scalp, face, central beard, chest, and groin. Culture of the scalp in severe seborrheic dermatitis produces very high yield the fungus pityrosporum ovale and of S. uureus, even higher than culture from the same regions in normal controls (83). The same organisms (plus Candida albicans) are seen in culture of infantile seborrheic dermatitis of the groin (84,85). 2. Nummular Eczema

Nummular eczema is a characteristic form of eczema consisting of symmetrical annular ("coin-like" nummular) lesions on the extremities. This form of eczema responds poorly to therapy, but often shows enhanced responses to oral antibiotics and has been proposed as a form of eczema triggered by local infection (86).

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InfectiousEczematoidDermatitis ”Infective dermatitis’’ or “infectious eczematoiddermatitis” is a form of eczema triggered by infection, which clearswith antibiotic therapy. It is commonly seen in the perianal area, in the periauricular area, and around leg ulcers on the lower legs. It isthe least well understood form of eczema. It is differentiated from “infected eczema’’ in which established eczema is worsened by secondary infection. In these patients, the responsible organism is usually S. aureus, often with multiple antibiotic resistances In either case, it appears that infections such as S. aureus can trigger or exacerbate eczema. In none of these syndromes has careful study of superantigen activation of eczema been performed. 4. AIDS-Related

Diseases

Patients with the acquired immunodeficiencysyndrome (AIDS) have a number of skin diseases that have been proposed to be triggered by infection of superantigens: eosinophilic folliculitis, AIDS-associated seborrhea, AIDS-associated psoriasis, AIDSpruritus. The mechanisms by which these clinical syndromes might be activated by viral bacterial or fungal superantigens are of great interest to investigators in dermatology VI.

CONCLUSIONS

Superantigens produce cutaneous effects by three major mechanisms. Presentation of superantigens by macrophages, dendritic cells, or keratinocytes induces promiscuous T-lymphocyte expansion and cytokine release. Superantigens also induce massive cytokine release from macrophages and keratinocytes and other parenchymal cells, producing a profound inflammatory cascade. Finally, some superantigens produce selective nonimmune effects such as exfoliation by the staphylococcal exfoliative toxins. These mechanisms induce systemic and cutaneous effects that present as toxin-mediated syndromes in scarlet fever, Kawasaki syndrome, SSSS, and TSS. In smaller amounts the same toxins that induce these syndromes appear to be important triggers of skin disease such as psoriasis and atopic dermatitis. It is believed that the effects of bacterial toxins will bedemonstrated in many other skin diseases, and it isalso speculated that similar effects for fungal toxins or viral superantigens may be observed (16).


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45. Gupta AK, Ellis CN, Nickoloff BJ, Goldfarb MT, Ho VC, Rocher LL, Griffiths CE, Cooper KD, Voorhees JJ. Oral cyclosporine in the treatment of inflammatory and noninflammatory dermatoses: a clinical and immunopathologic analysis. Arch Dermatol 1990; 126:339-350. 46. Gottlieb SL, Gilleaudeau P, Johnson R, Estes L, Woodworth TG, Gottlieb AB, Krueger JG. Response of psoriasis to a lymphocyteselective toxin (DAB38912-2) suggests a primary immune, but not a keratinocyte, pathogenesis. Nature Med 1995; 1:442-447. 47. Vallat VP, Gilleaudeau P, Battat L, Wolfe J, Nabeya R, Heftler N, Hodak E, Gottlieb AB, Krueger JG. PUVA bath therapy strongly suppresses immunological and epidermal activation in psoriasis: a possible cellular basis for remittive therapy. J Exp Med 1994; 180:283-296. 48. Kaplan G, Witmer MD, Nath I, Steinman RM, Laal S, Prasad HK, Sarno EN, Elvers U, Cohn ZA. Influence of delayed immune reactions on human epidermal keratinocytes. Proc Natl AcadSciUSA1986; 83:3469-3473. 49. Barker JN, Karabin GD, Stoof TJ, Sarma VJ, Dixit VM, Nickoloff BJ. Detection of interferon-gamma mRNA in psoriatic epidermis by polymerase chain reaction. J Dermatol Sci1991;2:106-111. 50. Henderson CA, Highet AS. Acute psoriasis associated with Lancefield group C and group G cutaneous streptococcal infections. Br J Dermatol 1988;118:559-561. 51. Marples RR, Heaton CL, Kligman AM. Staphylococcus aureus in psoriasis. Arch Dermatol 1973; 107:568-570. 52. Whyte JH, Baughman RD. Acute guttate psoriasis and streptococcal infection. Arch Dermatol 1964; 89:350-356. 53. Leung DY, Harbeck R, Bina P, Reiser RF, Yang E, Norris DA, Hanifin JM, Sampson HA. Presence of IgE antibodies to staphylococcal exotoxins on the skinof patients with atopic dermatitis: evidence for a new group of allergens. J Clin Invest 1993; 92:1374-1380. 54. Hintner H, Neises GR, Lawley TJ. Immunologic properties of enzymatically degraded human keratin intermediate filaments. J Invest Dermatol 1985;84:108-113. 55. Cole GW, Wuepper KD. Isolation and partial characterization of a keratinocyte proliferative factor produced by streptococcus pyogenes (strain NY-5). J Invest Dermatol 1978; 71:219-223. 56. Rosenberg EW, Noah PW. The Koebner phenomenon and the microbial basis of psoriasis. JAMA 1988; 18:151-158. 57. Baker BS, Bokth S, Powles A, Garioch JJ, Lewish H, Valdimarsson H, Fry L. Group A streptococcal antigen-specific T lymphocytes in guttate psoriatic lesions. Br J Dermatol 1993; 128:493-499. 58. Cerio R, Griffiths CE, Cooper KD, Nickoloff BJ, Headington JT. Characterization of factor XIIa positive dermal dendritic cells in normal and inflamed skin. Br J Dermatol 1989; 121:421-431. 59. Duraiswamy N, Tse Y, Hammerberg C, Kang S, Cooper KD. Distinction of class I1 MHC+ Langerhans cell-likeinterstitial dendritic antigen-


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presenting cells in murine dermis from dermal macrophages. J Invest Dermatol 1994;103:678-683. Cooper KD, Neises GR, Katz SI. Antigenpresenting OKM5+ melanophages appear in humanepidermis after UV radiation. J Invest Dermatol 1986; 86:363-370. Baadsgaard 0, Cooper KR, Lisby S, Wulf HC, Wantzin GL. Dose response and time course for induction ofT6-DR+ human epidermal antigen-presenting cells by in vivo ultraviolet A, B, and C irradiation. J Am Acad Dermatol 1987;17:792-800. Leung DY, Gately M, Trumble A, Ferguson Darnel1 B, Schlievert PM, Picker LJ. Bacterial superantigens induceT cell expression of the skinselective homing receptor, the cutaneous lymphocyte-associated antigen, via stimulation of Interleukin 12 production. J Exp Med 1995; 181:747-753. Norris DA. Cytokine modulation of adhesion molecules in the regulation of immunologic cytotoxicityof epidermal targets.J Invest Dermatol 1990;95:111S-l2OS. Lewis HM, Baker BS, Bokth S, Powles AV, Garioch JJ, Valdimarsson H, Fry L. Restricted T-cell receptor V beta gene usage in the skin of patients with guttate andchronic plaque psoriasis. Br J Dermatol 1993; 129:514-520. Leung DY, Travers JB, Giorno R, Norris DA, Skinner R, Aelion J, Kazemi LV, Kim MH, Trumble AE, Kotb M. Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis. J Clin Invest 1995;96:2106-2112. Travers JB, Norris DA, Leung DY. The cutaneous environment in psoriasis induces enhanced inflammatory reactions to epicutaneous application of bacterial superantigens. J Invest Dermatoll995; 104:648A. Gottlieb AB, Grossman RM, Khandke L, Carter DM, Sehgal PB, Fu SM, Granelli Piperno A, Rivas M, Barazani L, Krueger JG. Studies the effect of cyclosporine in psoriasis in vivo: combined effects on activated lymphocytes and epidermal regenerative maturation. J Invest Dermatol 1992;98:302-309. Chang JC, Smith LR, Froning KJ, Schwabe BJ, Laxer JA, Caralli LI, Kurland HH, Karasek MA, Wilkinson DI, Carlo DJ. CD8+ T cells in psoriatic lesions preferentially use T-cell receptor V beta 3 and/or V beta 13.1 genes. Proc Natl Acad Sci USA 1994; 91:9282-9286. Valdimarsson H, Baker BS, Jonsdottir I, Powles A, Fry L. Psoriasis: a T-cell-mediated autoimmune disease induced by steptococcal superantigens? Immunol Today 1995; 16:145-149. Ezepchuk YV, Leung DYM, Middleton MH, Bina P, Reiser R, Norris DA. Staphylococcal toxins and protein A differentially induce cytotoxicity and release of tumor necrosis factor-alphafrom human keratinocytes. 1996 J. Invest. Dermatol. (in press). Leung DY. Atopic dermatitis: the skin as a window into thepathogen-

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esis of chronic allergic diseases. J Allergy Clin Immunoll995; 96:30218;. 71. van der Heijden FL, WierengaEA,BosJD, Kapsenberg ML. High frequency of IL-4-producing CD4+ allergen-specific T lymphocytes in atopic dermatitis lesional skin. J Invest Dermatol 1991; 97:389-394. 72. Wierenga EA, Snoek M, de Groot C, Chretien I,Bos JD, Jansen HM, Kapsenberg ML. Evidence for compartmentalization of functional subsets of CD2+ T lymphocytes in atopicpatients. J Immunol 1990; 144:4651-4656. 73. Hamid Q, Boguniewicz M, Leung DY. Differential in situ cytokine gene expression in acute versus chronic atopic dermatitis. J Clin Invest 1994;94:870-876. 74. Grewe M, Walther S, Gyufko K, Czech W, Schopf E, Krutmann J. Analysis of the cytokine pattern expressed in situ in inhalant allergen patch test reactions atopic dermatitis patients. J Invest Dermatoll995; 105:407-410. 75. Boguniewicz M, Jaffe HS, Izu A, Sullivan MJ, York D, Geha RS, Leung DY. Recombinant gamma interferon in treatment of patients with atopic dermatitis and elevated IgE levels. Am J Med 1990; 88:365-370. 76. Halbert A, Morelli JG, Weston WL. Atopic dermatitis: is it an allergic disease? J Am Acad Dermatol 1995; 33:1008-1018. 77. Leyden JJ, Marples RR, Kligman AM. Stuphylococcus uureus in the lesions of atopic dermatitis. Br J Dermatol 1974; 90:525-530. 78. Hauser c, Wuethrich B, Matter L, Wilhelm JA, Schopfer K. Immune response to Staphylococcus uureus in atopic dermatitis. Dermatologica 1985;170:114-120. 79. Lever R, Hadley K, Downey D, MacKie Staphylococcal colonization in atopic dermatitis and the effect of topical mupirocin therapy. Br J Dermatol 1988;119:189-198. 80. McFadden JP, Noble WC, Camp RD. Superantigenic exotoxin-secreting potential of staphylococci isolated from atopic eczematous skin. Br J Dermatol 1993;128:631-632. 81. Schroder JM. Cytokine networks in the skin. J Invest Dermatol 1995; 105:20S-24S. 82. Werner Y, Lindberg M. Transepidermal water loss in dry and clinically normal skin in patients with atopic dermatitis. Acta Dermatol Venereol (Stockh) 1985;65:102-105. 83. McGinley KJ, Leyden JJ, Marples RR, Kligman AM. Quantitative microbiology of the scalp in non-dandruff, dandruff, and seborrheic dermatitis. J Invest Dermatol 1975; 64:401-405. 84. Leyden JJ, Kligman AM. The role of microorganisms in diaper dermatitis. Arch Dermatol 1978;114:56-59. 85.BrobergA, Faergemann J. Infantileseborrhoeicdermatitis and Pityrosporurn ovule. Br J Dermatol 1989; 120:359-362.

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86. Leyden JJ, Kligman AM. The case for steroid-antibiotic combinations. Br J Dermatol 1977; 96:179-187. 87. Leyden JJ. Mupirocin: a new topical antibiotic. Am Acad Dermatol 1990;22:879-883. Duvic M. Human immunodeficiency virus and the skin: selected controversies. J Invest Dermatol 1995; 105:117S-l21S. 89. Boehncke WH, Dressel D, Manfras B, Zollner TM, Wettstein A, Bohm BO, Steny W. T-cell-receptor repertoire in chronic plaque-stage psoriasis is restricted and lacks enrichment superantigen-associated V beta regions. J Invest Dermatol 1995; 104:725-728. 90. Schmitt-Egenolf M, Boehncke WH, Christophers Stander M, Sterry W. Type I and type I1 psoriasis show a similar usage of T-cell receptor variable regions. J Invest Dermatol 1991;97:1053-1056.


Superantigens in Human Disease future Directions in Therapy and Elucidation of Disease Pathogenesis

Y.

INTROdUCTlON

Despite the enormous strides that have been made in the field of medicine during the past century, the fundamental mechanisms by which infectious agents cause disease remain poorly understood. The studies reviewed in the first 17 chapters indicate that superantigens likely represent one important strategy by which bacteria and viruses alter T-cell responses and activate accessory cells. As a result, these molecules have provided a useful set of tools to probe and provide new insights into fundamental immunological processes. Recent attention, however, has increasingly shifted to the potential role of these molecules inthe pathogenesis of human diseases associatedwith 581

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immunodeficiency, autoimmunity, and/or inflammation. This is an area of critical importance because it may lead to new strategies in the prevention or treatment of many illnesses that are presently managed only by supportive care. Chapters 18-22 have provided an excellent overview of our current state of knowledge on the role of superantigens in a wide spectrum of human diseases. The current chapter will attempt to examine some of the issues thatrequireattentioninthefuture. In particular, we will discuss the challenges we face in establishing the role of superantigens in diseases beyond toxic shocksyndrome (TSS), e.g., autoimmune conditions, factors that may account for variability in host responses to superantigens, the development more effective treatments for superantigen-mediated diseases, and the strategies used to exploit the unique properties of superantigens for potential therapeutic benefit. Since the greatest evidence, to date, supporting a role for superantigens in human disease relates to staphylococcal and streptococcal toxins, we will restrict our review to these bacterial superantigens. II. SUPERANTICENS IN DISEASEPATHOGENESIS:BEYOND A.

TSS

Biological Responses to Superantigens

Several immune mechanisms have been proposed to explain how bacterial superantigens can participate in the pathogenesis of human diseases (reviewed in Ref. 1): First, superantigens bind directly without antigen processing to constitutively expressed HLA-DR molecules on professional antigen-presenting cells (APC) such as macrophages or dendritic cells, and to cytokine-induced HLA-DR molecules on nonprofessional APC such as keratinocytes. This can have profound physiological consequences due to the local or massive systemic release of cytokines and mediators of inflammation by these HLA-DR+ cells, or via the subsequent activation of T cells Second, superantigens may influence inflammatory or autoimmune responses via the activation of B cells. Recent studies suggest that superantigens stimulate autoantibody or IgE production by bridging the MHC class I1 molecule on B cells with the T-cell receptor (TCR) on T cells (4-6). This form of B-cell activation is likely to be non-antigen-specific, depending primarily on the release of cytokines in the vicinity of B cells previously stimulated by autoantigen or allergen. Importantly, stimulation as opposed to inhibition of immunoglobulin synthesis may depend on the concentration bacterial

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superantigen present in the local milieu. In this regard, picogram or femtogram concentrations of superantigen induce polyclonal B-cell activation (7). In contrast, nanogram or greater concentrationsof staphylococcal superantigen inhibit B-cell activation by inducing B-cell apoptosis. Third, the stimulation of T cells by superantigens results in the activation and expansion of lymphocytes expressing specificTCR V@ regions. Such T cells may include autoreactive T cellsthat migrate to the target tissue containing the autoantigen recognized by that T cell and mediate damage via cytotoxic mechanisms or the secretion of proinflammatory cytokines. While all bacterial superantigens can cause marked stimulation of T cells, they frequently cause the expansion of different portions of the T-cell repertoire (see Table 1 ) . Therefore, superantigens may differ in their capacity to expand autoreactive T cells with varying specificities. Under certain conditions, T-cell expansion by superantigens may be followedin time by anergy and/ or deletion of the stimulated T cells (8). The particular response of T cells to superantigens depends on a variety of factors, including their stage of development and activation, TCR avidity for the specific toxin as well as costimulatory signals provided by the superantigen-presenting cell. Recentstudies also indicate that, under certain circumstances, superantigen-mediated activation of T cells can occur in the absence of MHC class 11+ APC. In this regard, CD28 crosslinking synergizeswith signals deliveredby superantigens to induce the proliferation of T cells even in the ab-

Table 1 Human TCR Vp Specificities of Bacterial Superantigens” Exotoxin Staphylococcal enterotoxin (SE) A SEB SEC1 SED SEE Toxic shock syndrome toxin (TSST)-l

Exfoliative toxin Streptococcal pyrogenic exotoxin (SPEIA SPEB SPEC Mycoplasma arthritidis mitogen aReviewed in Ref. 1.

TCR Vp specificity

1.1,5,6,7.3-7.4,9.1 3,12,14,15,17,20 3,6.4,6.9,12,15 5,12 5.1,6,8,12 2 2

8,12,14,15 2,8 1,2,5.1,10 3.1,8, 10-14, 17.1,20

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sence ofAPC Furthermore, SEA- and SEB-induced T-cell activation can occur when class I1 MHC-negative colon carcinoma cell lines are used as APC In the latter study, the superantigen-binding molecule was not identified. However, Haffner et al. (12) observed a similar T-cell response when class I1 MHC-negative human epidermal cell lines were used as APC. In this study, they provided evidence that MHC class I molecules on these malignant squamous cell carcinomas served as ligands for SEB to stimulate T-cell proliferation. Interestingly, normal keratinocytes are poor APC for SEB. This discrepancy between the ability of normal keratinocytes versus squamous cell carcinoma cell lines to actas APC for SEB may relate to the expression of costimulatory signals, e.g.,ICAM-1 or B7, on tumor cell lines but not on normal keratinocytes. This allowsthe former but not the latter cell type to activate T cells. To further examine the requirement of class I1 MHC molecules in the presentation of staphylococcal toxins to T cells, Avery et al. studied the activation of T cells by superantigens in MHC class I- and class 11-deficient mice. They found that although T-cell activation by SEA and SEB was MHC class 11-dependent, SEC and SEE could also stimulate strong MHC-independent T-cell responses. T-cell specificity of these responses resembledconventional T-cell responses to superantigen associated with class 11, insofar as T cells bearing the same V s elements were selectively expanded by and SEE. However, the response seemedto differ from the MHC class 11-dependent response, because the initial expansion phase was not followed by clonal deletion, as is observed following SEB stimulation in mice. Interestingly, twofold to 10-fold more SEC or SEE was required for productive T-cell interaction with class 11-deficient cells. This raised the possibility that decreased avidity ofTCR binding to toxin presented by non-class I1 molecules does not allow the signaling that required for apoptosis. Taken together, these findings indicate that staphylococcal superantigens use MHC-dependentand MHC-independent T-cell activation pathways that lead to distinct types of immune responses. This functional heterogeneity may contribute to the induction of the wide range of in vivo clinical effects seenin superantigenmediated diseases. B.

Role of Superantigens in Human Disease

While staphylococcal and streptococcal toxins arepotentsuperantigens, it should be noted that bacterial toxins can also have biological activities distinct from their superantigen properties. For ex-

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ample, the capacity of exotoxins such as exfoliative toxin to induce scalded skin syndrome relates to their ability to cause intercellular separation of granular layer keratinocytes (14). Other examples where toxin effects are nonsuperantigenic include the production of vomiting and diarrhea, i.e., food poisoning, by staphylococcal enterotoxins in humans and primates. In this regard, the enterotoxic effectsof SEB can be separated from its T-cell-stimulatory properties by carboxymethylation of the SEB (15). Thus, to demonstrate that a particular disease is mediated by superantigen-mediatedimmune responses, we propose that the criteria listed in Table 2 should be fulfilled. The potential role superantigens in immune-mediated human disease is an area of intense investigation. Due to the complexities of studying cause-and-effect relationships in the clinical setting, however, information on the role of superantigens in mediating human disease remains limited. Menstrual TSS provides the best example of a disease most likely to be caused by superantigen(s) as it fulfills essentially all the criteria in Table 2. In this regard, TSS is associated with marked macrophage and T-cell activation, selective expansionof circulating Vp2+ T cells, and a focus of infection with toxic shock syndrome toxin (TSST)-l-secreting Staphylococcus aureus (reviewed in Chapter 18). The demonstration of lethal shock in rabbits that were injected with TSST-1 provided strong support for a role of this toxin in the pathogenesis ofTSS Following the removal of high-absorbency tampons from the market, reported cases of menstrual TSS has decreased dramatically. However, there remain a significant number of nonmenstrual cases of TSS associated with a variety of S, aureus infections including skin infections, postsurgical or postpartum infection, focal tissue infection, and pneumonia. TSST-1 accounts for approximately half of nonmen-

Table 2 Approach to Demonstrating a Role for Superantigens in a Particular Disease The disease is associated with T-cell and macrophage activation Correlation between exacerbation of illness with expansion deletoin of VP-specific T cells Isolation of a microorganism that produces a superantigen capable of inducing the relevant Vp-specific T-cell expansion 4. In vivo exposure to the superantigen in an experimental animal or human model should induce the disease 5. Elimination of the offending superantigen effectively treats the disease

1.

2.

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Leung and Schlievert

strual cases of TSS, and the enterotoxins SEB and, to a lesser extent, SEC account for the remainder. Thus, several staphylococcal toxins are capable of inducing TSS (see Chapter 18). Similarly, several streptococcal superantigens, including SPEA,SPEC, mitogenic factor, and streptococcal superantigen, have been implicated in the pathogenesis of streptococcal TSS (see Chapter 19). Although it is generally accepted that TSS is caused by bacterial superantigens, there is much less consensus for the role of superantigens in the pathogenesis of other diseases. However, it is important to note that when Kappler and Marrack first described the superantigenic properties of staphylococcal toxins, in 1989, it was already well established that staphylococcal TSST-1 was the primary cause of TSS. Thus the link between the superantigenic properties of TSST-1 and the immunological features of was logical and could be established relatively rapidly. Nevertheless, there have been recent reports demonstrating that superantigen-secreting staphylococci and/or streptococci can be isolated from patients with guttate psoriasis, atopic dermatitis, and Kawasaki syndrome (KS) at the time of disease exacerbation (reviewed in Chapters 21 and 22). The data implicating a role for superantigens are particularly convincing in guttate psoriasis, which is frequently associated with streptococcal throat infection and responds to treatment with penicillin. To test the hypothesis that T cells in the skin lesions these patients has been stimulated by superantigens, an analysis of the TCR repertoire in their skins lesions and peripheral blood has been analyzed by two different groups investigators (17,18). Both groups found marked expansion of Vp2+ T cells (up to 70% of the T cells) in the skin lesion but not the peripheral blood these patients. Furthermore, Leung et al. found that this expansion occurred in both the CD4+ and the CD8+ T-cell subsets, and sequence analysis of the BV2 genes of T cells from skin biopsies of guttate psoriasis showed extensive junctional region diversity compatible with a superantigen rather than a nominal antigen-driven T-cell response (18). Interestingly, all streptococcal throat isolates from patients with guttate psoriasis secreted streptococcal pyrogenic exotoxin C, a superantigen known to stimulate marked Vp2+ T-cell expansion. Taken in concert with earlier reports that intradermal infection streptococcal antigens can induce psoriatic lesions (19), these data strongly support the concept that acute guttate psoriasis is associated with superantigenic stimulation of T cells triggered by streptococcal superantigens and largely fulfills the criteria in Table

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Numerous reports have also shown that S. uureus can exacerbate atopic dermatitis (AD), and that the clinical severity of AD improves with antibiotic therapy. Leung et al. (20) found that S. uureus from more than half of AD patients secreted superantigens SEA, SEB, and TSST-l. Interestingly, these patients also produced IgE antibodies against staphylococcal toxins, and these same toxins could induce histamine release from mast cellsand basophils in an IgE-dependent manner, suggesting a novel mechanism by which toxins couldinduce skin inflammation. Recently, it has been demonstrated that patch testing with staphylococcal toxins induces local eczematoid skin reactions (21). Although these data support the concept that staphylococcal toxins are involved in the pathogenesis of AD, they do not demonstrate that they are acting via a superantigenic mechanism, as one study TCR Vp skewing has not yet been demonstrated. Indeed, in TCR Vp expansion was not observed following injection of mouse skin with superantigens, suggesting that acute local skin inflammation could occur as the result of toxins acting on keratinocytes and HLA-DR+ dendritic cells to release TNF-a and other inflammatory cytokines into the skin (22). Further studies are therefore needed to determine the mechanisms by which staphylococcal toxins induce eczema. Perhaps the most fascinating area of investigation relates to the potential role of superantigens in the pathogenesis of autoimmune diseases. In experimental animals, superantigens have been found to induce experimental autoimmune encephalomyelitis (EAE) and progressive joint destruction in rodents recovering from experimental arthritis (reviewed in Chapters 15 and 21). In humans, Paliard et al. (23) demonstrated increased numbers of Vp14+ T cells in the affected joints of patients with rheumatoid arthritis with an apparent depletion of such cells in the circulation. An analysis of the VJ314 cells in the joint revealed they were clonally expanded, suggesting a local response to conventional antigen. Based on these data;these investigators hypothesized that exposure to a Vpl4-specific superantigen had resulted in initial activation but then deletion of most of the peripheral Vp714+ T-cell population. However, in predisposed individuals, a small number of the activated Vp14 cells could then infiltrate into the joints and recognize a conventional self-antigen, with a consequent oligoclonal immune response. TOdate, a putative superantigen or infectious agent involved in the triggering of rheumatoid arthritis has not been identified, although M Y C O ~ ~urthritidis U S ~ Umay be a potential candidate in a subset

588


of patients with arthritis (see Chapter 15). This is likely because the infectious trigger is a distant event that has triggered a long-term autoimmune process sustained by T-cell recognition of local tissue antigens. KS is an acute, multiorgan vasculitis with many of the same clinical features as TSS. The epidemiological features of this disease strongly suggested an infectious etiology, leading to the speculation that this illness has a superantigenic basis. In this regard, several studies of peripheral blood mononuclear cells from patients with KS have demonstrated significantly elevated levels of Vp2+, and to a lesser extent Vp8.1+, T cells as compared to cells from age-matched normal donors and patients with other febrile illnesses (24-26). In the convalescence phase of KS, the proportion of VP2+ and Vp8.1+ T cells returned to normal levels. These results suggested that the immune stimulation of KS is triggered by a bacterial toxin with superantigenic activity. Superantigens stimulate T cells primarily through the Vp domain of the TCR and therefore induce T-cell expansion independent of the TCR P-chain junctional region (1). To determine whether Vp2+ T cells in acute KS were expanded in a superantigen-like, i.e., polyclonal rather than oligoclonal, manner, sequences of random cDNA clones containing BV2 gene segments were analyzed (25). In response to conventional peptide antigens, patients with a two- to three-fold increase in BV2 gene expression would be expected to have similar junctional sequences in up to 60% of their abnormally expanded T cells. None of the BV2 sequences, however, were identical to each other. These data indicate that in the expansion of Vp2+ T cells in acute KS, the BV region plays the dominant role in T-cell recognition. Together with the high frequency of responding Vp2+ T cells in the circulation, which was disproportionate to that expected for a response to a conventional antigen, the results were most consistent with a superantigen trigger in acute KS. To further address whether bacterial toxins might cause the immune activation associated with KS, Leung et al. (27) obtained cultures from the skin (groin, axilla) and mucous membranes (rectum, throat) of 16 acute KS patients and 15 control patients. All group A P-hemolytic streptococci and all coagulase-positive S. aweus isolates were screened for toxin production in a blinded fashion. Organisms secreting superantigens were found in 13 of 16 KS patients, but in only one of 15 control patients ( p c .0001): 11 of 13 toxin positive cultures from patients with KS were TSST-elaborating S. aureus and two of 13 were streptococci producing pyogenic exotoxins B and C.


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More recently we have extended these analyses tothree patients with coronary artery complications: TSST-l-secretingS. aureus was isolated from two of the three children. An exfoliative toxin-secretingS. aureus was isolated from the remaining patient. Importantly, each of these toxins possesses Vp2 stimulatory activity (see Table 2). These studies suggest the immune activation associated with the acute phase of KS may be caused by S. aureus or, to a lesser extent by streptococci that colonize mucous membranes and elaborate toxins with superantigenic activity. Furthermore, the observation that different bacterial toxins associated with KS are potent activators of Vp2+ T cells suggests an important role for this subset of T cells in the pathogenesis of this disease. Indeed, at a tissue level, there are two recent studies demonstrating the local infiltration of Vp2+ T cells in acute First, h u n g et al. (28) found selective expansion of Vp2+ T cells in the myocardium and coronary artery aneurysm of a patient who died weeks after the onset of KS. Interestingly, this selective expansion of Vp2+ T cells was not seen in the kidney or spleen. This was somewhat surprising since splenic cells generally reflect the peripheral blood T-cell repertoire. However, we and others have demonstrated that within 1 month into the convalescent phase of KS the percentage of Vp2+ T cells in the peripheral blood is normal (24-26). Thus, the increased numbers of Vp2+ T cells in the heart and coronary artery of this patient may reflect the selective retention of such cells in these tissues due to sustained autoantigenic stimulation. Second, Yamashiro and his colleagues (29) have recently reported a selective expansion of Vp2+ T cells in the small intestinal mucosa of patients with acute KS but not in control subjects. Interestingly, they did not observe Vp2+ T-cell expansion in the peripheral blood Yamashiro, personal communication), suggesting that local Vp2+ Tcell expansion can occur in the gastrointestinal (GI) tract of patients with acute without affecting the T-cell repertoire in the peripheral blood. Based on these observations, they suggested that the GI tract is the primary site entry for a superantigen-secreting organism causing acute This is consistent with our finding that essentially all the superantigen-producing bacteria isolated from acute patients are derived from either the pharnyx or the rectum (27). Although studies from three independent groups of investigators have demonstrated expansion of Vp2+ T cells in acute KS (24-26,28-. 29), there remains considerable controversy over the role of superantigens in because some investigators have not found expansion of Vp2+ T cells in the peripheral blood of these patients (30). How-

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ever, in serial analyses of individual patients Curtis et al. (26) were able to find elevated Vp2+ T cells in the peripheral blood of acute KS patients only during the second week of their illness. Indeed, even in staphylococcal TSS only five of eight patients were found to have increased Vp2+ T cells in their circulation despite the use of a sensitive PCR assay Furthermore, in studies examining cardiac or gastrointestinal tissue from acute KS patients, increased numbers of infiltrating Vp2+ T cells were seen despite normal numbers of Vp2+ T cells in the circulation (28,291. These observations are consistent with recent data demonstrating that superantigens stimulate the expression of homing receptors on memory T cells and therefore can induce the migration of T cells into tissue, e.g., the skin (32). Thus, there may only be a very narrow time interval in which to detect increased numbers of circulating Vp2+ T cells before such cellshave migrated into inflamed tissues. The other area of controversy is whether a single superantigen causes the Vp2+ T-cell expansion in acute KS. As noted above, in our experience the predominant superantigen involved is TSST-l. However, we have found exfoliative toxin and SPEC in a minority of cases, indicating that several toxins (see Table 1) can potentially induce the Vp2+ T-cell expansion in this disease. The observation that multiple superantigens can result in the same clinical phenotype in KS has its parallel in other diseases such as staphylococcal TSS, which can be caused by TSST-1, SEB, or Of note, certain investigators have used the failure to find seroconversion to TSST-1 as evidence against a role for this toxin in KS However, this approach is flawed because seroconversion rarely occurs in staphylococcal TSS. Furthermore, all their KS patients were treated with high-dose intravenous immunoglobulin, which is known to inhibit antibody synthesis. It is our view that ultimate acceptance of the "superantigen hypothesis" in KS will require a rapid, simple diagnostic test to detect superantigen-producing organisms during the acute phase of this illness as well as the demonstration that superantigens can induce coronary artery disease in experimental animals. These studies are currently in progress. Recent studies in a variety of clinical settings indicatethat superantigens are likely to beimportant molecules involved in mechanisms by which infectious agents trigger immune-mediated diseases. With the recent identification of superantigens produced by Mycoplasma Yersiniu, CMV, EBV, and rabies viruses discussed earlierin this book, it is likely that superantigens will be implicated in a number other


591

diseases. The ultimate demonstration that superantigens play a key role in the pathogenesis of a particular disease will likely require careful sampling of involved tissues as well as peripheral blood to find alterations in T-cell repertoire. Identification of the putative organism will require early culturing of body fluids from suchpatients. The dramatic clinical onset ofTSS and the expansion of circulating Vp2+ T cells due to toxemia facilitated the demonstration of superantigens in the pathogenesis of TSS. In other diseases, by contrast, the demonstration of Vp-specific T-cell expansions is less accessible tissues or the isolation of superantigen-secretingorganisms may be far more difficult. Nevertheless such studies are worth pursuing as they may lead to fundamental new approaches in the diagnosis and treatment of a large group of common diseases. HOST FACTORSDETERMININGRESPONSE

TO SUPERANTIGENS

The observation that patients with KS or AD can be colonized or infected with TSST-secreting S. aureus raises the important question of why they do not develop TSS. It is likely that several factors play an important role (Table First, immunological factors play a key role in determining the course of illness. Absence of circulating antiTSST antibody levels is a major risk factor for the development of shock following infectionwith TSST-l-producing S. aureus .In the case of older AD patients who are colonized with TSST-l-producing S. aureus, we have found them to have high levels of serum IgG antiTSST-1 (D. M. Leung, unpublished observations). Under such cir-

Table 1.

2. 3.

4. 5.

Factors Affecting Responses to Superantigens

Level of toxin exposure Focus of infection vs. colonization Toxin production level Environmental influences due to location of infection, e.g., pH, oxygen, glucose levels, etc. Age of host Immunological factors Low antitoxin levels T-cell repertoire Costimulatory signals Nature of toxin Concomitantvirulencefactorsbeingsecreted

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cumstances, the patient is likely protected from developing shock. However, local colonization or infection with S. aureus will not prevent superantigen-induced tissue inflammation as normal donors or patients with AD develop inflammatory skin lesions when patchtested with staphylococcal superantigens (21). Other factors that likely influence the course of cellular responses to superantigens undoubtedly include the nature of the costimulatory and accessory molecules on the superantigen-presenting cell as discussed earlier in this chapter. Second, age is an important factor in susceptibility to shock. In rabbits, the induction of shock with TSST occurs primarily in older, sexually mature rabbits. However, sexually immature rabbits under the age of 2 months are much more resistant to lethal shock induced by TSST-l (35). An important clinical parallel exists in humans exposed to TSST-l-secreting S. aureus: Children get KS whereas adults get TSS.Both diseases have overlapping clinical features, with hypotension in adults being the major distinguishing feature. The lack of shock in children, e.g., with KS, may allow the evolution of a cellular immune response resulting in vasculitis. In contrast, TSS is short-lived and high doses of TSST are immunosuppressive. These divergent immune responses may contribute to some of the differences in the clinical manifestations betweenTSS and KS, e.g., erythroderma in TSS versus inflammatory skin response and vasculitis in KS. Third, the induction of shock is concentration-dependent. Thus, in experimental models of TSS it has been well established that relatively low doses of TSST-l can induce fever, but not shock. Of interest, in a recent study we have found that low doses, but not high doses, ofTSST-1 induce vasculitis in rabbits (F. W. Quimby and D. M. Leung, unpublished observations). Both treatment groups, however, had significant febrile responses. Furthermore, we have recently found that low concentrationsof TSST-1 induce polyclonal Bcell activation, whereas high concentrations of TSST-1 inhibit immunoglobulin responses (7). The immunological basis for this finding appears to relate to increased interferon-gammaproduction, followed by the induction FAS expression and apoptosis of B cells when mononuclear cells are incubated with nanogram or higher concentrations of TSST-l. In contrast, low TSST-l concentrations enhance cognate T-B-cell interactions resulting in polyclonal B-cell activation. These observations may account for the observation of polyclonal Bcell activation in patients with acute KS where low TSST-l exposure is likely to occur. Inhibition of B-cell responses, however, is found in TSS where high TSST-1 production occurs.


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593

Finally, the location of the infection likely plays a critical role in toxin production by S. aureus. Previous studies have demonstrated that protein, neutral pH, oxygen, and low environmental glucose are required for high toxin production (36). These conditions are more likely to occur in menstrual TSS than KS. IV. TREATMENT OF SUPERANTIGEN-MEDIATEDDISEASE ANEMERGINGROLEFOR lVlG

The above-described studies suggest several possible approaches for the treatment of superantigen-mediated diseases that go beyond simple supportive therapy. First, particularly in the case of staphylococcal TSS antibiotic therapy and elimination of the focus of infection has been found to be quite effective (37). Second, since low antibody titers are a risk factor for TSS, the development of mutant vaccines that lack superantigenic activity but induce antibodies that neutralize the in vivo effects of bacterial toxins would be of interest. However, considering the diversity of staphylococcal and streptococcal superantigens that may trigger TSS, and the observation that other as yet unidentified virulence factors likely synergize with these superantigens to exacerbate clinical symptoms, efforts aimed at developing specific vaccines may be a problem. A third consideration is the use of intravenous immune globulin (IVIG) for treatment of superantigen-mediated diseases. Recent in vitro studies have demonstrated that pooled immunoglobulin used for IVIG therapy contains antibodies against multiple staphylococcal and streptococcal superantigens (38,39). Importantly, these antibodies neutralize and immunostimulatory activities of bacterial superantigens. This raises the possibility that IVIG may be an effective therapy for treatment of superantigen-mediated diseases such as TSS and Indeed, studies in experimental animal models of TSS have demonstrated that effectiveness of neutralizing antibodies in the prevention of lethal shock and cytokine generation due to superantigens such as TSST-1 (40). In support of a role for IVIG therapy in the treatment ofTSS, there have been several case reports of severely sick patients with either staphylococcal or streptococcal TSS in whom dramatic clinical improvement occurred shortly after treatment with IVIG (41,42). Furthermore, clinical improvement after IVIG therapy was associated with significant reduction of serum levels of tumor necrosis factoralpha and IL-6. More recently, a case-controlled study was carried out on 51 patients with streptococcal TSS, which has up to 70% mortal-

'

Schlievert 594

and

Leung

ity. In this study, 21 patients received IVIG and 30 patients received standard care (43). Patients receiving IVIG had a significantly improved 30-day survival (67% in the IVIG group vs. 30% receiving standard care, p = 0.004). Furthermore, IVIG administration promptly enhanced the ability of patient plasma to neutralize bacterial mitogenicity and reduce the production ofIL-6 and tumor necrosis factoralpha by circulating mononuclear cells. In the case of KS, well-controlled studies in Japan and the United States have demonstrated that treatment with IVIG plus aspirin given within the first 10 days of onset of fever significantly reduces the prevalence of coronary artery disease (44,45). In addition, high-dose IVIG therapy at a dose of 2 g/kg results in a significantly more rapid normalization of laboratory measures of acute-phase reactants such as a,-antitrypsin or C-reactive protein as well as faster resolution of clinical symptoms such as fever. Interestingly, a lower peak IgG level is associated with a worse clinical outcome, suggesting a therapeutic threshold of IgG was necessary to achieve benefit (45). In this regard, the duration of fever and the degree of inflammation were inversely relatedto the peak IgG level. Even more importantly, the peak serum IgG level following IVIG administration was lower in those childrenwho developed coronary artery disease as compared to children who did not develop cardiovascular complications. Thesedata suggest a specific concentration of antibody is necessary before inflammation is arrested. The mechanism forIVIG efficacy in the treatment of KS is poorly understood. However, successful treatment of acute KS with WIG is associated with significant reduction of immune activation (46). Aspirin and IVIG therapy is associated with a reduction in the number of activated helperT cells and B cells, which characterizethe acute stage of Several theorieshave been proposed as to the mechanism by which IVIG reverses the immune activation of KS (see Table 4). In general, it appears that IVIG works by reducing the immune activation with an associated reduction in cytokine secretion, reducing

Table 4 Mechanisms of Action ofIVIG in Superantigen-mediated Diseases ~~~~~~~

~

~

Antibodies that neutralize the causative toxin(s) 2. Transcriptional modulation cytokineproduction Inhibition cytokine-induced vascular endothelial activation 4. Anti-idiotypic antibody modulation autoimmune T cells and antibodies

1.


595

activation of vascular endothelial cells and thereby reducing vasculitis. It is likely that IVIG modifies the inflammatory response through more than one mechanism. For example, several reports suggest that IVIG production acts directly on cytokine gene expression via transcriptional and posttranscriptional modulation (47,48). This suggests that IVIG may down-regulate the immune response by a nonspecific immunomodulatory mechanism. In addition, IVIG may act by neutralizing the causative toxin that results in the massive immune stimulation that characterizes V.

POTENTIAL THERAPEUTIC APPLICATIONS OF SUPERANTIGENS

Exploitation of the unique T-cell capabilities of superantigens for therapeutic application is an emerging area interest. In this regard, the injection of superantigens, e.g., SEB, into mice has been demonstrated to result in the selective deletion inactivation of T cells expressing specific TCR VD regions. This selective inactivation of T cells has suggested a new approach for prevention of autoimmune disease. Indeed, Schiffenbauer and his colleagues have found intriguing results using experimental autoimmune encephalomyelitis (EAE) as a mouse model of human multiple sclerosis (see Chapter 21). In this model, mice are immunized with myelin basic protein (MBP) to induce a Vp8.2+ T-cell-dependent responseleading to demyelination. However, injection ofSEB prior to MBP immunization prevented the development of autoimmune encephalomyelitis. Thesedata suggested that superantigens could potentially be used for the prevention of human autoimmune diseases by selectively eliminating specific T-cell subpopulations. Other experiments, however, also carried out by Schiffenbauer and his colleagues have demonstrated that SEB or SEA could induce a relapse of EAE in mice that had recovered from a clinical episode of EAE. Thus, previously activated autoimmune T cells appear to be resistant to the induction of anergy or deletion by superantigens. Furthermore, .although pretreatment with SEB was able to block the induction of EAE with MBP, it did not prevent SEA from including EAE in these mice (50). These data document the complex and varied responses that autoimmune animals can have to superantigens and suggest that the effects of superantigens in humans are also likely to be quite complex. There are also a number of other concerns with the use of superantigens as therapeutic agents. First, systemicadministration of superantigens may stimulate T cells and macrophages, leading to the dan-

596


gerous release of high levels of cytokines, e.g., TNF, which can induce shock. Second, superantigens could stimulate clones of autoreactive T cells, which might result in autoimmune disease. Nevertheless, the heterogeneity of clinical responses tosuperantigens provides some glimmer of optimism that lethality resulting from shock or autoimmune responses may not always be linked to the superantigenic effects of these potent molecules. With these considerationsin mind, two encouraging approaches have been taken to engineer superantigens for potential treatment of human disease. First, a large number of mutations have been made in the TSST-1 or SEB molecules to identify functionally important regions of superantigens. The majority of mutations have been clustered in two distinct regions, with the class I1 MHC binding site located in the hydrophobic region of the amino-terminal domain, and the TCR binding site primarily in the major central groove of the carboxyl-terminal domain (51,521. In vivo studies ofTSST mutants have also identified some interesting functional regions. In particular, the ability of the toxin to cause TSS in rabbit models depends on the glutamic acid residue at amino acid position 132, whereas the ability ofTSST to cause T-cell mitogenicity depends not only on amino acid 132, but also on other amino acids within a central diagonal alpha helix and possibly amino acids within residues 16-80 (53). Using these approaches, superantigen mutants may possibly be engineered that generate beneficial effects, e.g. potential vaccines that induce neutralizing antitoxin responses, without the lethal effects associated with wild-type bacterial superantigens. A second interesting approach in this area has utilized antibody targeting of superantigens for the treatment of experimental tumors. These studies are based on the observation that T cells activated by superantigens exhibit potent cytotoxic activity against tumor cells (54,55). Since systemic administration of superantigens can induce massive activation of T cells, Dohlsten and his colleagues have conjugated superantigens to tumor-specific monoclonal antibodies, e.g., C242, which recognizes human colon carcinoma cells, thereby developing reagents that can target relatively small quantities of superantigens directly to the tumor site in the body (11). These fusion proteins had a reduced MHC class I1 binding and were capable of targeting T cells to MHC class 11-negative tumor cells. Using tumor-bearing SCID mice as a model, these investigators demonstrated that injection of C242Fab-SEA fusion proteins was accompanied by tumor infiltration of T cells and significantly reduced

e


597

the tumor load in these mice (56). While studies are still required in human cancer patients to address the efficacy and unforeseen difficulties, this is a novel and promising approach that could be used for the treatment of a number of cancers that are currently poorly responsive to therapy. VI.

SUMMARYANDCONCLUSIONS

The discovery superantigens and characterization of their biological activities has provided new insights into potential mechanisms by which infectious agents cause disease. In the last section of this book, we have reviewed the role of superantigens in human disease and their potential therapeutic applications. The explosive nature staphylococcal and streptococcal toxins has facilitatedtheir identification as the etiological agents involved in Potentially more exciting, however, is the possibility that these same toxins have different actions in other immunological clinical settings, thus contributing to autoimmune and inflammatory responses. Indeed, similar to other immunological triggers, the same toxins appear to cause a variety of different diseases. Furthermore, the same disease, e.g., can be triggered by different superantigens. In the future, identification of the bacterial viral superantigens that trigger particular diseasesand the immunological mechanisms by which this occurs will provide more effective and objective methods to diagnose and treat superantigen-mediated diseases. We envision that treatment of these diseases will change and they will become more amenable to early treatment with antimicrobial agents, hightitered antibodies directed against superantigens including those present in intravenous immune globulin, or that certain superantigenmediated diseases may be prevented altogether by the development of targeted vaccines. REFERENCES 1. Kotzin BL, Leung DYM, Kappler Marrack P. Superantigens and human disease. Adv Immunol 1993; 54:99-166.

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liferationin the presence of Langerhans cells or class 11-bearing keratinocytes and stimulate keratinocytes to produce T-cell-activating cytokines. J Invest Dermatol 1994; 102:31-38. 4. Hofer MF, Lester MR, Schlievert PM, Leung DYM. Effect of bacterial toxins on IgE synthesis. Clin Exp Allergy 1995; 25:1218-1227. Crow MK, Chu Z, Ravina B, et al. Human B cell differentiation induced by microbial superantigens: unselected peripheral blood lymphocytes secrete polyclonal immunoglobulin in response to Mycoplasma arthritidis mitogen. Autoimmunity 1992; 14:23-32. He Goronzy J, Wevand C. Selective induction of rheumatoid factors by superantigens and*humanhelper T cells. J Clin Invest 1992; 89:673-

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17. Lewis HM, Baker BS, Bokth S, et al. Restricted T-cell receptor V$ gene usage in the skin of patients with guttate and chronic plaque psoriasis. Br J Dermatol 1993; 129:514-520. 18. Leung DYM, Travers JB, Giorno R, et al. Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis. J Clin Invest 1995; 96:2106-2112. 19. ColeGW, Wuepper KD. Isolation and partial characterization of a keratinocyteproliferative factor producedby Streptococcus pyogenes (strain NY-5). J Invest Dermatol 1978; 71:219-223. 20. Leung DYM, Harbeck H, BinaP, et al. Presence ofIgE antibodies to staphylococcal enterotoxins on the skin of patients with atopic dermatitis: evidence for a new group of allergens. J Clin Invest 1993; 92:13741380. 21. Strange P, Skov L, Lisby S, Nielsen PL, Baadsgaard 0. Staphylococcal enterotoxin B applied on intact normal and intact atopic skin induces dermatitis. Arch Dermatol 1996; 13227-33. 22. Saloga J, Leung DYM, Reardon C, Giorno RC,BornW, Gelfand EW. The cutaneous inflammatory response to bacterial superantigen is T-cell dependent. J Invest Dermatol 1996; 106:982-988. West SG, Lafferty JA, et al. Evidence for the effects of a 23. Paliard superantigen in rheumatoid arthritis. Science 1991; 253:325-329. 24. Abe J, Kotzin BL, Jujo K, et al. Selective expansion T cells expressing T cell receptor variable regions Vp2 and V$8 in Kawasaki disease. Proc Natl Acad Sci USA 1992; 89,4066-4070. 25. Abe J, Kotzin BL, Meissner C, et al. Characterization of T cell repertoire changes in acute Kawasaki disease. J Exp Med 1993; 177:791-796. 26. Curtis N, Zheng R, Lamb JR, Levin M. Evidence for a superantigen mediated process in Kawasaki disease. Arch Dis Child 1995; 72:308-311. 27. Leung DYM, MeissnerHC, Fulton DR, Murray DL, Kotzin BL, Schlievert PM. Toxic shock syndrome toxin-secreting Staphylococcus aureus in Kawasaki syndrome. Lancet 1993; 342:1385-1388. 281 Leung DYM, Giorno Kazemi LV, Flynn PA, Bussel JB. Evidence for superantigen involvement in cardiovascular injury due to Kawasaki syndrome. J Immunol 1995;155:5018-5021. 29. Yamashiro Y, Nagata S, Oguchi S, Shimizu T. Selective increase of V$2+ T cells in the small intestinal mucosa in Kawasaki disease. Pediatr Res 1996;39:264-266. 30. Pietra BA, De Inocencio J, Giannini EH, Hirsch R. V$ family repertoire and T cell activation markers in Kawasaki disease. J Immunol 1994;153:1881-1888. 31. Choi Y, Lafferty JA, Clements JR, et al. Selective expansion of T cells expressing V$ 2 in toxic shock syndrome. J Exp Med 1990; 172:981-984. 32. Leung DYM, Gately M, Trumble A, Ferguson-Darnel1 B, Schlievert PM, Picker LJ. Bacterial superantigens induce T cell expression of the skinselective homing receptor, the cutaneous lymphocyte-associated antigen (CLA). J Exp Med 1995; 181:747-753.

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33. Terai M, Miwa K, Williams T, Kabat W, Fukuyama M, Okajima Y, Igarashi H, Shulman ST. The absenceof evidence of staphylococcal toxin involvement in the pathogenesis of Kawasaki disease. J Infect Dis 1995; 172:558-561. 34. Vergeront JM, Stolz SJ, Crass BA, Nelson DB, Davis JP, Bergdoll MS. Prevalence of serum antibody to staphylococcal enterotoxin F among Wisconsin residents: implications for toxic shock syndrome. J Infect Dis 1983;148:692-698. 35. Quimby FW, Nguyen HT. Animal studies of toxic shock syndrome. Crit Rev Microbiol 1985; 12:l-44. 36. Schlievert PM, Blomster DA. Production of staphylococcal pyrogenic exotoxin type C: influence of physical and chemical factors. J Infect Dis 1983;147:236-242. 37. Todd JK. Therapy of toxic shock syndrome. Drugs 1990; 39:856-861. 38. Takei Arora YK, Walker SM. Intravenous immunoglobulin contains specific antibodies inhibitory to activation of T cells by staphylococcal toxin superantigens. J Clin Invest 1993; 91:602-607. 39. Norrby-Teglund A, Kaul R, Low DE, et al. Plasma from patients with severe invasive group A streptococcal infections treated with normal polyspecific IgG inhibits streptococcal superantigen-induced T cell proliferation and cytokine production. J Immunol 1996; 156:3057-3064. 40. Best GK, Scott DF, Kling JM, Thompson MR, Adinolfi LE, Bonventre PF. Protection of rabbits in an infection model of toxic shock syndrome (TSS) by a TSS toxin-l specific monoclonalantibody. Infect Immun 1988; 56:998-999. 41. Barry W, Hudgins L, Donta ST, Pesanti EL. Intravenous immunoglobulin therapy for toxic shock syndrome. JAMA 1992; 267:3315-3316. 42. Nadal D, Lauener RP, Braegger DP, et al. T cell activation and cytokine release in streptococcal toxic shock-like syndrome. J Pediatr 1993; 122:727-729. 43. Kaul R, McGeer A, Norrby-Teglund A, Kotb MM. The Canadian Streptococcal Study Group, Low DE. Intravenous immunoglobulin (IVIG) therapy in streptococcal toxic shock syndrome (STSS): results of a matched case-control study, 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC 1995. 44. Furusho K, Kamiya T, Nakano et al. High dose intravenous gammaglobulin for Kawasaki disease. Lancet 1984; 2:1055-1058. 45. Newburger JW, Takahashi M, Beiser AS, et al. A single intravenous infusion of gamma globulin as compared with four infusions in the treatment of acute Kawasaki syndrome. N Engl J Med 1991; 324:1633-1639. 46. Leung DYM, Burns J, Newburger J, Geha Reversal of immunoregulatory abnormalities in Kawasaki syndrome by intravenous gammaglobulin. J Clin Invest 1987; 79:468-472. 47. Anderson UG, BjSrk L, Skansbn-Saphir U, Anderson JP. Down-regulation of cytokine production and receptor expression by pooled human IgG. Immunology 1993;79:211-216.


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Petrosian A, Galera OA, Wang S, Jordan SC. 48. Toyoda M, Zhang Modulation of immunoglobulinproduction and cytokine mRNA expresion in peripheral blood mononuclear cells byintravenous immunoglobulin. J Clin Immunol 1994; 14:178-188. 49. Schiffenbauer J, Johnson HM, Butfiloski EJ, Wegrzyn L, Soos JM. Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis. Proc Natl Acad Sci USA 1993; 90:8543-8546. 50. JM, Hobeika AC, Butfiloski EJ, Schiffenbauer J, Johnson HM. Accelerated induction of experimental allergic encephalomyelitis in PL/ J mice by a non-VP8-specific superantigen. Proc Natl Acad Sci USA 1991; 269:32063-32069. 51. Hurley JM, Shimonkevitz R, Hanagan A, Enney K, Boen E, Malmstrom S, Kotzin BL, Matsumura M. Identification of class I1 major histocompatibility complex and T cell receptor binding sites in the superantigen toxic shock syndrome toxin 1. J Exp Med 1995; 181:2229-2235. 52. Kappler JW, Herman A, Clements J, Marrack P. Mutations defining functional regions of the superantigen staphylococcal enterotoxin B. J Exp Med 1992; 175:387-396. 53. Murray DL, Prasad GS, Earhart CA, Leonard BAB. Immunobiologic and biochemical properties of mutants toxic shock syndrome toxin-l. J Immunol 1994; 15297-95. 54. Hedlund G, Dohlsten M, Petersson C, Kalland T. Superantigen-based tumor therapy: in vivo activation of cytotoxic T cells. Cancer Immunol Immunother 193; 36:89-93. 55. Shu Krinock RA, Matsumura T, et al. Stimulation tumor-draining lymph node cells with superantigenic staphylococcal toxins lead to the generation of tumor specific effector cells. J Immunol 1994; 152:12771288. 56. Litton MJ, Dohlsten M,P.A. L, et al. Antibody-targeted superantigen therapy induces tumor-infiltrating lymphocytes, excessive cytokineproduction, and apoptosis in human colon carcinoma. EurJ Immunoll996; 26:l-9.


Alpha toxin, Atopic dermatitis, (see also Autoimmunity, Kawasaki disease, rheumatoid arthritis, multiple sclerosis, diabetes, Wegener’s granulomatosis) B-Cell superantigens, (see also protein A, protein L, protein Fv, HIV g p role of autoimmunity, Bacterial superantigens interaction with MHC class II, other receptors, SEA, SEB, Tropism, TSST-l,

Colon carcinoma treatment, Cysteine protease (see Streptococcal pyrogenic exotoxins

B) Cytomegalovirus,

Diabetes mellitus,

Enterotoxins (see Staphylococcal enterotoxins) . Epstein-Barr virus autoimmunity, biologicalsignificance, oncogenesis, structure, superantigen associated, TCR Vp specificity, 603

Index

Exfoliative toxins, assays of, genetics of, mechanisms of action of, organisms of, scalded skin syndrome, sequences of, superantigenic activity, Experimental allergic encephalomyelitis, (see also autoimmunity) Human Immunodeficiency Virus,

HIV gp

(see also

B-cell superantigens) Intravenous immunoglobulin, treatment Kawasaki disease, treatment of staphylococcal TSS, treatment

streptococcal TSS,

Kawasaki disease,

M protein, introduction, rheumatic fever, serotypes, structure, T-cellresponses, autoreactive induction, mechanism, superantigenicity,

Mitogenic factor (see streptococcal pyrogenic exotoxins) Mouse Mammary Tumor Viruses (MMTV) endogenous, exogenous, immunobiology, interaction with MHC class 11, mechanism of tumorigenesis, nomenclature,

ow,

structural features, TCR Vfi specificity, transmission, virus and proteins, Multiple Sclerosis, Myocoplasma arthritidis mitogen, arthritis, biochemistry, disease association, functional domains, immunobiology, B-cell activation in, class I1 MHC binding, in vivo effects, T-cell receptor, introduction, production, rheumatoid arthritis, Nummular eczema, Protein A, complement activation, keratinocyte activation, mast cell/basophil interactions,

Index

Protein Fv, 416-417 Protein L, 414-416 Psoriasis, 540-541 586 guttate psoriasis, 561-565, plaque psoriasis, 565-567 Rabies virus immunopathology, 91 life cycle, 86 nucleocapsid cncodes superantigen, 88 pathogenesis, 89 structure, 86 Rheumatic fever, 541 Rheumatoid arthritis, 530-533 Scarlet fever, 557 Seborrheic dermatitis, 571

Staphylococcus aurew (see exfoliative toxins, toxic shock syndrome toxin-l , staphylococcal enterotoxins) Staphylococcal enterotoxins, 167 types A, D,and E, 199-230 class II MHC binding, 204209,211-214,218-220 general properties, 199-202 hydrophobic surface, 210-21 1 N terminus, 209-210 structure, 202-204,215-218, 220-223 T-cell receptor binding, 214215 zinc binding, 205-209, 212218, 221 types B and C, 167-198 class II MHC binding, 184188 emesis, 188-192 epitopemapping, 171-174

[Staphylococcal enterotoxins] general properties, 167- 169 sequence relatedness, 170-171 structure, 174-178 T-cell binding, receptor 181184 zinc binding, 178-180 Staphylococcal scalded skin syndrome, 556,559 Staphylococcal toxic shock drome, 435-464,557,585 (see also TSST-1) case definition, 436-437 clinical features, 454-460 epidemiology, 448-454 pathogenesis, 437-448 toxin association, 436-437 treatment, 461-464 Streptococcal M protein role in autoimmunity, 311-338 Streptococcal pyrogenic exotoxins, 257 B-cell immunosuppression, 272 cardiotoxicity, 270 endotoxin binding, 272-273 endotoxin enhancement, 269-270 mechanism of action, 271 mitogenic factor (SPE F), 28929 1 functional studies, 291 general properties, 284, 289290 genetics, 290-291 purification, 289-290 T-cell receptor binding, 285 non-group A toxins, 273 organisms, 281-283 pyrogenicity, 268-269 scarlet fever, 257-259, 270-272 streptococcal superantigen, 283289

606

Index

[Streptococcal pyrogenic exotoxins] alleles, general properties, genetics, phylogeny, T-cell receptor binding, superantigenicity, type A, bacteriophage association, general properties, genetics, sequence, structure, type B, biochemistry, cysteine protease, enzymology, general properties, genetics, superantigenicity, virulence, type C, bacteriophage association, general properties, genetics, sequence, structure,

Streptococcus pyogenes (see M protein, streptococcal pyrogenic exotoxins, streptococcal superantigens) Streptococcal superantigen (see also streptococcal pyrogenic exotoxins) alleles, general properties, genetics, phylogeny, T-cell receptor binding,

Streptococcal toxic shock syndrome, case definition, clinical features, pathogenesis, treatment. Superantigens . biological activities, co-stimulatory signals, factors affecting host response,

(see also staphylococcal and streptococcal TSS) hallmarks, immunobiology, microbial origin, pyrogenic toxin, relevance to human disease, selective advantage, as therapeutic agents, T-cell receptor Vp specificities, virally encoded superantigens, Toxic shock syndrome toxin-l, binding to class I1 MHC, biological properties, endothelial cell effects on, endotoxin enhancement, pyrogenicity, superantigenicity, general properties, genetics, mutational analysis, protein chemistry, structure,

Index

[Toxic shock syndrome toxin-l] enterotoxin comparison, 135136 latticeinteractions,156-162 model building and refinement, 151-152 secondaryelements,133 Treatment, 593-595 (see intravenousimmunoglobulin, superantigens) superantigens as therapeutic agents, 595-597 use of antibiotics in toxic shock syndromes, 462-463, 492494, 593

607

Viral superantigens, 503-517 (see also H I V , CMV, "TV,

EBV)

Wegener's granulomatosis, 539-540

Yersinia pseudotuberculosis,369394 disease association, 390-393 superantigen, 376-386 virulencefactors, 370-376

Superantigens: Molecular Biology, Immunology, and Relevance to Human Disease

Superantigens and Superallergens (Chemical Immunology)

Fungal disease: biology, immunology, and diagnosis

Parasitic Nematodes: Molecular Biology, Biochemistry and Immunology

Ubiquitin and Disease (Molecular Biology Intelligence Unit)

Hedgehog-Gli Signaling in Human Disease (Molecular Biology Intelligence Unit)

Human Molecular Biology Laboratory Manual

Human Molecular Biology Laboratory Manual

Human Molecular Biology Laboratory Manual

Molecular Biology in Plant Pathogenesis and Disease Management Disease Management

Molecular Biology in Plant Pathogeneses and Disease Management Disease Development

Molecular biology in plant pathogenesis and disease management. Disease development

Introduction to Human Disease

Human retrovirus protocols: virology and molecular biology

Introduction to Molecular Biology

Immunology of Behcet's Disease

Superantigens and Superallergens

Molecular Carcinogenesis and the Molecular Biology of Human Cancer

Transplantation Immunology: Methods and Protocols (Methods in Molecular Biology)

Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology and Physiology

Immunogenomics and Human Disease

Worms and Human Disease

Viruses and Human Disease

Human Demography and Disease

Viruses and human disease

Molecular Pathology: The Molecular Basis of Human Disease

Alternative Splicing and Disease (Progress in Molecular and Subcellular Biology)

Genetics and Molecular Biology

Molecular biology and genomics

Molecular Biology and Biotechnology

Molecular Biology and Biotechnology

Superantigens: Molecular Biology, Immunology, and Relevance to Human Disease

Superantigens and Superallergens (Chemical Immunology)

Fungal disease: biology, immunology, and diagnosis

Parasitic Nematodes: Molecular Biology, Biochemistry and Immunology

Ubiquitin and Disease (Molecular Biology Intelligence Unit)

Hedgehog-Gli Signaling in Human Disease (Molecular Biology Intelligence Unit)

Human Molecular Biology Laboratory Manual

Human Molecular Biology Laboratory Manual

Human Molecular Biology Laboratory Manual

Molecular Biology in Plant Pathogenesis and Disease Management Disease Management

Molecular Biology in Plant Pathogeneses and Disease Management Disease Development

Molecular biology in plant pathogenesis and disease management. Disease development

Introduction to Human Disease

Human retrovirus protocols: virology and molecular biology

Introduction to Molecular Biology

Immunology of Behcet's Disease

Superantigens and Superallergens

Molecular Carcinogenesis and the Molecular Biology of Human Cancer

Transplantation Immunology: Methods and Protocols (Methods in Molecular Biology)

Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology and Physiology

Immunogenomics and Human Disease

Worms and Human Disease

Viruses and Human Disease

Human Demography and Disease

Viruses and human disease

Molecular Pathology: The Molecular Basis of Human Disease

Alternative Splicing and Disease (Progress in Molecular and Subcellular Biology)

Genetics and Molecular Biology

Molecular biology and genomics

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