New Foundation of Biology Neuroimmune Biology, Volume 1
Neuroimmune Biology Series Editors
I. Berczi, A. Szentivanyi
Advisory Board
B.G. Arnason, Chicago, IL E. Artzt, Buenos Aires, Argentina P.J. Barnes, London, UK T. Bartfai, La Jolla, CA L. Bertok, Budapest, Hungary H.O. Besedovsky, Marburg, Germany J. Bienenstock, Hamilton, Canada C.M. Blatteis, Memphis, TN J. Buckingham, London, UK Ch. Chawnshang, Rochester, NY R. Dantzer, Bordeaux, France M. Dardenne, Paris, France N. Fabris, Ancona, Italy R.C. Gaillard, Lausanne, Switzerland Ch. George, Bethesda, MD R. Good, Tampa, FL R.M. Gorczynski, Toronto, Canada C. Heijnen, Utrecht, The Netherlands T. Hori, Fukuoka, Japan H. Imura, Kyoto, Japan
M.D. Kendall, Cambridge, UK E.A. Korneva, St. Petersburg, Russia K. Kovacs, Toronto, Canada G. Kunkel, Berlin, Germany L.A. Laitinen, Helsinki, Finland B. Marchetti, Catania, Italy L. Matera, Turin, Italy H. Ovadia, Jerusalem, Israel C.P. Phelps, Tampa, FL L.D. Prockop, Tampa, FL R. Rapaport, New York, NY S. Reichlin, Tucson, AZ R. Schmidt, Hannover, Germany A. Shmakov, Novosibirsk, Russia K. Skwarlo-Sonta, Warsaw, Poland E.M. Sternberg, Bethesda, MD D.W. Talmage, Denver, CO S. Walker, Columbia, MO A.G. Zapata, Madrid, Spain
New Foundation of Biology Neuroimmune Biology, Volume 1
Volume Editors Istvan Berczi Reginald M. Gorczynski
University of Manitoba, Winnipeg, Canada and University of Toronto, Toronto, Canada
2001 ELSEVIER AMSTERDAM
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Acknowledgements This volume contains the presentations by invited speakers at the first Canadian Symposium on Neroimmune Biology, held in Winnipeg, June 9-11, 2000. Scientific Committee: Berczi I (Chair), Anisman H, Baines MB, Befus AD, Bienenstock J, Chow AD, Gorczynski RM, Moldofsky H, Nance D, Pittman Q, Pomerantz DK, Rivest S. Organizing Committee: Berczi I, Chow DA, Nance D, Baral E, Dawood M, Kisil FT, Kroeger E, Nagy E, Paraskevas F, Sabbadini ER, Warrington RJ. The symposium was followed by a workshop (June 12) on which the Canadian Network for Neuroimmune Biology (CANIB) was initiated. Website: http://cyboard.com/canib/ This conference has been supported by the Canadian Institutes of Health Research through the CIHR Opportunity Program. The University of Manitoba, the Faculty of Medicine and the Faculty of Graduate studies, University of Manitoba provided additional funding. We are grateful to Mrs. Carol Funk, who provided excellent service as secretary to the conference and also assisted us with the preparation of this volume. Ms. Valentina Tautkus, served as secretary and Technical Editor for this volume with much skill, diligence and devotion.
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Editorial to Volume I: Why Neuroimmune Biology? The importance of proper mindset for the maintenance of health and for general well-being has been known since prehistoric times [1]. Jesus Christ actually practised, perhaps unknowingly, healing of the sick and miserable, simply by giving them hope for recovery. References to faith healing are also present in the Koran and in other religious texts. Similar practices exist in primitive societies, where the "medicine man" provides spiritual and physical support to the sick. Darwin described the theory of evolution of the species over a hundred years ago [2], which is now regarded as scientifically proven [3], yet religion is still going strong, satisfying the "spiritual need" of enormous masses of people, especially in poor societies. Although there are suggestions for emotions similar to religion in animals, it is reasonably safe to suggest that the true religious mindset is only present in homo sapiens. Why this seemingly obligatory dependence on religion? Why spiritual satisfaction seems to be a compulsive need for so many people? The most fundamental difference between higher animals and man lies in intellectual capacity. Only man has to survive and prosper with the knowledge of certain death. Even today for most people on our planet life poses enormous difficulties that may include starvation, homelessness, devastating diseases, and no hope for improvements in the future. One may suggest that religion was, and still is, essential for providing hope for all those people who need help to maintain a balanced mindset that enables them to cope with the harsh realities of life. It appears that an optimistic mindset for these people is only possible through believing in God and Heaven, where there is eternal life and happiness without any suffering. Throughout history severe crisis situations, such as war, created terrible epidemics of infectious disease. Although not proven, the epidemics of deranged mindset may have contributed significantly to the spread of disease under these conditions. It is now emerging that emotional crisis may lead to severe depression, which is associated with disturbed neuroendocrine and immune functions. If these conditions persist, disease may follow [4-7]. Therefore, there is scientific evidence to indicate that the "spiritual need" of many people may actually stem from the enormous regulatory power of the human neuroimmune regulatory system over bodily functions. It needs to be set properly, in spite of unfavourable circumstances, so that maintenance of health and survival is maximally supported. That a strong belief in recovery from a serious illness has survival value stands the rigor of scientific scrutiny. Modern clinical trials of new drugs are conducted with control groups of patients that receive ineffective substances (placebo). Repeatedly it has been observed on the basis of objective parameters, that a significant percentage of placebo-treated patients show clinical improvements [8]. This may be interpreted as proof for the healing power of the proper mindset. Pathologists observed first that emotional factors and hormonal alterations have a major influence on the size of the thymus [9]. In 1936 Hans Selye discovered that noxious agents, when injected into rats, activate the ACTH-adrenal axis, which leads to the shrinkage of the thymus and of lymphoid organs [11, 12]. He produced evidence that glucocorticoids released by the adrenal gland caused the thymus atrophy. A similar "stress response" could be observed in rats by the emotional upset of being restrained from movement. Selye established that the hypothalamus-pituitary-adrenal-thymus axis was always activated
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under stressful conditions. Therefore, it was postulated by him that this axis was involved in the adaptation of animals to survive life-threatening challenges by various 'nocuous' agents [10-12]. It is only now that we are beginning to understand that indeed he was right. What he saw was the acute phase response (APR), which may indeed be regarded as an emergency defence reaction. Selye's legacy has been extended by our studies on the role of the pituitary gland in the regulation of the immune system. Growth hormone and prolactin was shown to maintain immunocompetence, whereas the ACTH-adrenal axis was found to exert an immunosuppressive effect. Sex steroid hormones have been designated as immunomodulators. These conclusions were made in reference to the adaptive immune response [13, 14]. It is now emerging that during febrile illness there is immunoconversion from the adaptive mode of immune reactivity to boosting natural immune mechanisms. The activation of the hypothalamus-pituitary-adrenal axis and the sympathetic outflow actually help the production of acute phase proteins by the liver and of natural antibodies by CD5+ B lymphocytes, which in turn command the immune system during acute illness [15, 16]. These developments fully support Selye's conclusion that the bodies defence mechanisms are mobilized after stress. It was discovered in 1949 by Szentivanyi and colleagues that the hypothalamus regulates the anaphylactic response in guinea pigs. Subsequent observations revealed that in laboratory animals, where the hypothalamus was imbalanced by lesions or by electrical stimulation, anaphylactic reactivity and antibody formation were altered significantly [17-21]. These experiments revealed that the nervous system has a dominant regulatory power over immune reactivity. In 1964 Korneva and Khai made similar observations [22]. The potential of sensory nerves to induce inflammation has been discovered by Jancso and co-workers [23]. This discovery ties in nicely with the above findings, indicating that the nervous system is capable of both causing and inhibiting inflammatory reactions. A compelling body of experimental evidence is available today, indicating the regulatory role of nerves in the inflammatory process. There is little doubt that inflammatory diseases have a significant input from the nervous system. The task is now to understand the mechanisms involved and to use the insights gained to the benefit of patients. The work of Pavlov called attention to the role of the mind in alimentary physiology by demonstrating that in dogs the expectation of receiving food leads to salivation (conditioned reflex). Later, the phenomenon of conditioning has been extended to numerous other bodily functions. In 1926 Metalnikov and Chorine showed that the Pavlovian rules of conditioning also apply to the immune system [24]. In modern times Ader and co-workers [25], MacQuin et al., [26] and Gorczynski and colleagues [27] provided rigorous scientific proof, indicating that the expectation of an immunological insult has a significant modulatory effect on subsequent immune responses. Therefore, immune responses may be conditioned in the classical Pavlovian sense. Moreover, it is now emerging that saliva itself has major immunoregulatory substances. In laboratory rodents the submandibular gland is a major site of production of these substances, which participate in the regulation of both mucosal and systemic immune reactions [28, 29]. In Persia, in Egypt and in the Roman Empire a healing power was attributed to fever. This view, which was supported by empirical observations, persisted till modern times. During the early nineteen hundreds an active search has been done by scientists for pyrogenic substances that could be used for fever therapy of diseases [30]. Such a substance was isolated by Boivin and colleagues from gram-negative bacteria [31], which is now known as bacterial lipopolysaccharide (LPS), or endotoxin. Now it is clear that LPS, a harmless substance by itself, is instantaneously recognized by the immune system. LPS induces cell activation, proliferation,
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cytokine production and the activation of immune-effector mechanisms. It also affects directly the central nervous system. If given systemically, LPS induces APR and boosts host defence. Clearly, there is evidence to support the idea that LPS has many beneficial effects, and that it can be used to good advantage in many life-threatening situations [15]. Similar homologous epitopes (homotopes) that are capable of instantaneous activation of the innate immune system exist in other microorganisms and in self-components [ 15, 16]. In 1975 Wannemacker and co-workers isolated the leukocyte endogenous mediator (LEM) of fever [32]. This was the first immune-derived molecule that mediated feedback signals towards the central nervous system. Later LEM was found to be identical with interleukin-1. It is now clear that IL-1 also serves as a feedback signal for pituitary hormone release [33-38]. Subsequently other cytokines, especially IL2, IL6, TNF-~t and interferon gamma were shown to regulate the secretion of pituitary hormones during systemic immune/inflammatory reactions [39]. It is also clear by now that the nerves have immunoregulatory function and provide feedback signals from lymphoid organs and from sites of immune/inflammatory reactions towards the central nervous system [40-43]. The Science of Immunology has evolved from observations that higher animals and man will acquire specific immunity after previous exposure to an infectious agent or toxin. Naturally microbiologists were most interested in this phenomenon as their major concern has been to fight infectious diseases. In order to take advantage of the body's phenomenal capability to develop specific resistance against pathogenic microbes after exposure, Jenner developed vaccination. This was a major advance in preventing infectious diseases and even today, still is a very important part of preventive medicine. Therefore, the traditional thinking in Immunology has revolved around the specific stimulus (antigen) that is capable of inducing immunity and it's interaction with the cells (lymphocytes) that are able to produce antibodies [44, 45]. It took some time to realize that cells, not antibodies, mediated some forms of immunity. With the advent of Cellular Immunology it has been discovered that lymphocytes are capable of producing antibodies in culture systems [46]. This fortified the view that the immune system was a largely autonomous system that went about the business of fighting 'foreign' intruders, while sparing 'self' from immune attack [45]. Seemingly there was no need for other control mechanisms, nor did it occur to the scientists pre-occupied with the prevention of infectious disease, that higher regulation of the immune system is in order, or actually it is required for normal function. Clearly, this system was mysteriously intelligent, capable of deciding with remarkable precision what to do. No other tissues/organs/systems were capable of self-non-self discrimination with such a remarkable precision and to display memory when stimulated by the same antigen/pathogen for the second time. However, the case for Neuroimmune interaction, which was first advanced by pathologists a century ago, grew stronger and stronger and by the mid-seventies half a dozen, or so, laboratories were preoccupied with studies in this area. The term 'bi-directional communication' between the Nervous and Immune Systems has been coined by Blalock and accepted enthusiastically by many people in the field. At the same time, it became obvious that both the immune system (which was watching self integrity) and the nerveous system, which innervated all tissues and organs, including the immune system, were in fact communicating with the entire organism. Indeed, it seems clear by now, there is much more to this interaction than 'bi-directional'. It is emerging, that we are dealing with a truly multi-directional, all-inclusive systemic regulatory network formed by the nervous-, endocrine- and immune systems, which controls all bodily functions of higher animals and man. This system is involved in conception and in the entire process of reproduction, in the growth and development of the fetus and of the newborn, in aging, in the process of daily life rhythms, in the sleep-wake cycle, in seasonal adjustments
and in most, if not all, pathological conditions, where defense, healing and regeneration are all influenced [47-49]. Clearly, the entire biology of higher organisms is based on this highly evolved and incredibly sophisticated regulatory system that is able to sense outside stimuli, including danger signs as well as to monitor and patrol the body for intruders, abnormalities and aberrations and correct, protect, heal and regenerate the organism as it may be required for the optimal maintenance of health and recovery from disease. Historic observations, the healing power of God and Jesus Christ, as well as every day events indicating the association of emotional difficulties and ill health maintain a very strong popular belief in the importance of mind-body interaction. In contrast, scientists pride themselves to only accept phenomena as true when sufficient scientific data are available in support of their validity. So far the scientific community at large does not fully appreciate the fundamental importance of the neuroimmune regulatory network. However, the time has arrived, when the role of this fundamental regulatory system may be submitted to scientific scrutiny. The human genome has been mapped and the experimental tools and sophistication, as well as the capacity of handling the vast amount of information that needs to be evaluated, are all available to undertake this task. There is little doubt, that fitting together the puzzle will soon become the next, and perhaps the last, frontier of Vertebrate Biology. Clearly, what is also required is to organize and interpret the scientific data as we go along. This is especially important because the relevant information is published in diverse specialty journals. The Science of Neuroimmune Biology deals with this systemic regulatory network, coordinating, organizing and interpreting the rapidly accumulating knowledge. The ultimate goal is to understand the function of higher organisms, including man, in their entire complexity. The objective of the book series, Neuroimmune Biology, is to provide regular assessments and interpretation of accumulated experimental evidence. It is hoped that this publication will enable the scientific community to keep abreast with essential advancements of our knowledge in a quest for understanding the Biology of higher organisms. We are pleased to present to the interested readers the introductory volume of this publication series and our plans for the forthcoming issues. We feel that it is high time to turn our attention to the organization and interpretation of the knowledge that has been accumulated in Biology. A new scientific field called Genomics has emerged recently, as attention is focused on the interaction of individual genes in the genome. In contrast with Genomics that still deals largely with events at the molecular and cellular level, our interest focuses on Integrative Physiology and Pathophysiology, never forgetting the milieu in which the cells (and their genes) of the body have to exert their functions. The term Neuroimmune Biology expresses this overall objective.
Istvan Berczi and Andor Szentivanyi
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Preface Observations indicating the dominant role of the central nervous system in the maintenance of health have been made since prehistoric times and the proverb "healthy body -- healthy mind" exists in many languages. Hans Selye was the first to study experimentally this mind-body interaction at McGuill University in Montreal. While attempting to isolate a hormone from the placenta, he injected his fractions into rats and observed the enlargement of the adrenal glands, the shrinkage of the thymus and of lymphoid organs. However, despite repeated attempts to purify the putative hormone, the activity was invariably lost. Finally, in 1936, he came to the conclusion that he was dealing with a non-specific reaction whose nature remained unknown (Nature, 138; 32, 1936). Supporting this conclusion, he even found that when he injected formalin into rats a similarly dramatic reaction occurred that included gastrointestinal haemorrhages. All 'noxious' agents and even emotional upset (restraining rats on the laboratory bench) could elicit this syndrome. He called this phenomenon "stress", and the eliciting agents/situations "stressors". Selye argued that stress elicited a defence reaction, which he named "the general adaptation syndrome". After an initial enthusiastic response from scientists to these ideas, contradictions and confusion prevailed and regrettably Selye's achievements went unrecognised during the 50 years he dedicated to the understanding of this phenomenon. It has now been recognized that he was the first to "discover" the existence of a hypothalamicpituitary-adrenal-thymus axis, and he consistently maintained till the end of his life that this axis played an important role in the adaptation of higher animals and man to various physical, chemical, biological and emotional challenges. Only over the past 2-3 decades has Selye's work been appreciated and interpreted. Although he knew little about the immune system, he discovered its conversion from the adaptive mode of reactivity to the development of the so-called acute phase response, which can be understood as an amplification of natural immune defence. The neuroendocrine response he observed is fundamental to this conversion. He was fully correct in concluding that this reaction is a general and adaptive defence reaction. The first Canadian Conference on Neuroimmnune Biology, and this volume which reports the papers presented at that conference, are dedicated to Selye's memory and to his life-time achievements. Andor Szentivanyi was the guest of honour and gave a conference-opening lecture entitled: "Studies on the hypothalamic regulation of histamine synthesis". In this discussion he reported the demonstration, by contemporary scientific methodology, of the mechanism(s) for a fundamentally important discovery he and his colleagues made more than 50 years ago. In 1949 his group discovered that the central nervous system seemed to have broad regulatory power over immune reactions (Acta. Med. Hungarica 3(2): 163, 1952). As a young medial student, Szentivanyi observed catecholamine-resistance in an asthmatic patient, who died in spite of aggressive treatment with adrenaline. This incident inspired him to do animal experiments and to dedicate his entire research career to the clarification of the role of the central nervous system in immune and inflammatory reactions. His subsequent experiments, published in a wide range of Internationally acclaimed journals, demonstrated the important role of the beta-adrenergic receptor in the regulation of immune and inflammatory conditions. It was humbling to hear from this distinguished guest that finally, after a long (over 50 years) career in science, he was able,
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for the first time, to present his experimental results to an audience that was genuinely interested in the subject. The conference and delegates were privileged to honour Dr. Szentivanyi for his fundamental discoveries and remarkable achievements in Nueroimmune Biology, and to welcome him as an Editor for the Proceedings. He has been instrumental to the formulation of the idea of this series as well as to its realization. Only now, some 65 years after Selye's discovery of the stress reaction and 50 years after Szentivanyi's unequivocal demonstration of immunoregulation by the CNS, are we beginning to understand so-called "mind-body" interactions at a cellular and molecular level. There is now a growing consensus amongst the general scientific community that the nervous-, endocrine-, and immune systems form a systemic regulatory network. This network is fundamental to the maintenance of the entire life cycle of higher animals and man in health and disease. It seems clear this regulatory network coordinates and maintains all physiological functions, including reproduction, and further commands host defence mechanisms in life-threatening circumstances and in disease. The term, Neuroimmune Biology, has been adopted to define this new scientific discipline. The realization that the immune system is part of the systemic regulatory network that regulates the function of higher organisms provides important new foundations to Biology. The objective of this book, and the book series it has spawned, termed Neuroimmune Biology, is to present a coordinated and integrated view of the growing body of knowledge which is rapidly accumulating in this area. Our ultimate goal is to achieve a more thorough understanding of higher organisms in their entire complexity.
Istvan Berczi Reginald Gorczynski .,
.:
! Hans Selye
Andor Szentivanyi
XV
List of Corresponding Authors
Jack P. Antel
Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal QC, Canada Malcom G. Baines
Department of Microbiology & Immunology, McGill University, Room 44, 3775, University Street, Montreal QC, Canada H3A-2B4 A. Dean Befus
Pulmonary Research Group, Department of Medicine, The University of Alberta, Room 574 Heritage Medical Research Centre, Edmonton, AB, Canada T6G-2S2 lstvan Berczi
Department of Immunology, Faculty of Medicine, The University of Manitoba, 795 McDermot Avenue, Winnipeg, MB, Canada R3E-OW3 John Bienenstock
Faculty of Health Sciences, McMaster University, 1200 Main Street West, Room 2El, Hamilton, ON, Canada L8N-3Z5 Peter Bretscher
The University of Saskatchewan, Department of Microbiology and Immunology College of Medicine, A231 Health Sciences Building, 107 Wiggins Road, Saskatoon, SK, Canada S7N-5E5 Donna A. Chow
Department of Immunology, Faculty of Medicine, The University of Manitoba, 795 McDermot Avenue, Winnipeg, MB, Canada R3E-OW3 Joe S. Davison
Department of Physiology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive N.W., Calgary, AB, Canada T2N-4N1 Judah A. Denburg
Director, Division of Clinical Immunology & Allergy, Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, ON, Canada Gordon Ford
University of Calgary, 4A186 Holy Cross Ambulatory Care Centre, Rocky View, General Hospital 7007-14th Street, Calgary, AB, Canada T2V-1P9
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Reginald M. Gorczynski Department of Surgery & Immunology, University of Toronto, The Toronto Hospital, CCRW 2-855, 200 Elizabeth Street, Toronto, ON, Canada M5G-2C5 Kent HayGlass Department for Immunology, Faculty of Medicine, The University of Manitoba, 730 William Avenue, Winnipeg, MB, Canada R3E-OW3 Teresa Krukoff Department of Cell Biology, Faculty of Medicine & Dentistry, The University of Alberta, Edmonton, AB, Canada T6G-2H7 Alexander Kusnecov Department of Psychology, Biopsychology and Behavioral Neuroscience Program Rutgers University, 152 Frelinghuysen Road, Piscataway, NJ, USA 08855 Julio Licinio UCLA Department of Psychiatry and Biobehavioral Sciences, 3357A Gonda (Goldschmied) Center, Los Angeles, CA, USA 90095-1761 Giamal N. Luheshi Department of Neuroscience, Douglas Hospital Research Centre, 6875 Boulv. LaSalle, Verdun, QC Canada H4H-1R3 Brian MacNeil Department of Pathology, P220 Pathology Building, Faculty of Medicine, The University of Manitoba, Winnipeg, MB, Canada R3E-OW3 Zul Merali Psychology & Molecular Medicine, 11 Marie Curie Room, 214 Vanier Building, Ottawa, ON, Canada K1N-6N5 Harvey Moldofsky The University of Toronto, Centre for Sleep & Chronobiology, Toronto Western Hospital University Health Network, 399 Bathurst Street, Room MP14-308, Toronto, ON, Canada M5T-2S8 Eva Nagy Department of Immunology, Faculty of Medicine, The University of Manitoba, 795 McDermot Avenue, Winnipeg, MB, Canada R3E-OW3 Dwight Nance Department of Pathology, University of Manitoba, P220 Pathology Bldg., Winnipeg, MB, Canada R3E-OW3 Trevor Owens Neuroimmunology Unit, Montreal Neurological Institute, 3801 University Street, Montreal, QC, Canada H3A-2B4
xvii
Quentin Pittman Neuroscience Research Group and Department of Medical Physiology, The University of Calgary, 3330 Hospital Drive N.W., Calgary, AB, Canada T2N-4N1 David K. Pomerantz Department of Physiology, University of Western Ontario, London, ON Canada, N6A-3K7 Robert J. Rapaport Mount Sinai Diabetes Center, 1200 Fifth Avenue (101 st Street), First Floor, New York, NY, USA 10029 Serge Rivest Molecular Endocrinol Lab., CHUL Res Ctr., Laval University, 2705 Boul. Laurier, Quebec, QC, Canada G1V-4G2 Edris R. Sabbadini Department of Immunology, Faculty of Medicine, The University of Manitoba, 795 McDermot Avenue, Winnipeg, MB, Canada R3E-OW3 Vijendra Singh Department of Biology, Utah State University, 5305 Old Main Hill Logan, UT USA 84322-5305 Andrzej Stanisz HSC-3N5C, McMaster University, 1200 Main Street West, Hamilton, ON, Canada L8N-3Z5 Lucia Stefaneanu Division of Pathology, St. Michael's Hospital, 30 Bond Street, Toronto, ON Canada M5B-1 W8 Esther M. Sternberg Section of Neuroendocrine Immunology and Behavior, National Institutes of Health, Bldg 10, Room 2D46, Bethesda, MD, USA 20892-1284 Andor Szentivanyi Department of Internal Medicine, University of South Florida, Box 9, 12901 Bruce B. Downs Blvd., Tampa, FL, USA 33612-4799 Richard J. Warrington Departments of Internal Medicine & Immunology, Faculty of Medicine, The University of Manitoba, Winnipeg, MB, Canada R3E-OW3
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xix
Contents
Acknowledgements ..........................................................
v
Editorial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vii
Andor Szentivanyi and Istvan Berczi Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xiii
Istvan Berczi and Reginald M. Gorczynsld
List of C o r r e s p o n d i n g A u t h o r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
xv
Introduction
Neuroimmune Biology -- An introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
Istvan Berczi Studies on the Hypothalamic Regulation of Histamine Synthesis . . . . . . . . . . . . . . . . . .
45
Andor Szentivanyi, Istvan Berczi, Denyse Pitak, Allen Goldman
II. N e u r o i m m u n e R e g u l a t o r y M e c h a n i s m s
Introduction: II. Neuroimmune Regulatory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
57
Reginald M. Gorczynsld Dynamics of Immune Responses: Historical Perspectives in our Understanding of Immune Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
61
Kent T. HayGlass Cell-to-cell Interaction and Signaling within the Immune System: Towards Integrating Mechanism and Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
71
Peter A. Bretscher, Nahed Ismail, Nathan Peters, Jude Uzonna Regulation of the Immune Response within the Central Nervous System . . . . . . . . . . . .
87
Jack Antel Regulatory Circuits of the Pituitary Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lucia Stefaneanu
99
XX
Neuroendocrine Stress and Inflammatory Disease: From Animal Model to Human Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
115
Esther M. Sternberg, Mojdeh Moghaddam Immunoregulation by the Sympathetic Nervous System . . . . . . . . . . . . . . . . . . . . . . . . .
121
Dwight M. Nance, Brian J. MacNeil Behavioral and Central Neurochemical Consequences of Cytokine Challenge: Relationship to Stressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
141
Zul Merali, Hymie Anisman, Shawn Harley Proinflammatory Signal Transduction Pathways in the CNS During Systemic Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
163
Serge Rivest, Sylvain Nadeau, Steve Lacroix, Nathalie Laflamme Nitric Oxide in Neuroimmune Feedback Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . .
175
Teresa L. Krukoff, Wen@ W. Yang
III. Neuroimmune Mechanisms in Physiology Introduction: III. Neuroimmune Mechanisms in Physiology . . . . . . . . . . . . . . . . . . . . .
207
Reginald M. Gorczynski A Model of Neuroimmune Communication: Mast Cells and Nerves . . . . . . . . . . . . . . .
195
John Bienenstock Immunomodulation by the Submandibular Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
203
A. Dean Befus, Paul Forsythe, Rene E. Dgry, Ronald Mathison, Joseph S. Davison Glandular Kallikrein in Immunoregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
225
Edris Sabbadini, Eva Nagy, Alexander Viir6s, Gertrude V6r6sova, Fred T. Kisil, Istvan Berczi Understanding Classical Conditioning of Immune Responses . . . . . . . . . . . . . . . . . . . .
237
Reginald M. Gorczynski Sleep, Health and Immunocompetence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
255
Harvey Moldofsky, Wah-Ping Luk, Jodi Dickstein Interactions Between the Immune System and the Testis . . . . . . . . . . . . . . . . . . . . . . . .
269
David K. Pomerantz Leptin and Cytokines: Actions and Interactions in Fever and Appetite Control . . . . . .
Giamal N. Luheshi
283
xxi
IV. Neuroimmune Host Defence Introduction: IV. Neuroimmune Host Defence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
295
Reginald M. Gorczynski Fever and Antipyresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
297
Quentin J. Pittman, Abdeslam Mouihate, Marie-Stephanie Clerget The Salivary Gland Peptides: Their Role in Anaphylaxis and Lipopolysaccharide (LPS)-Induced Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . .
307
Joe S. Davison, Dean Befus, Ronald Mathison Olfactory Stimuli and Allo-Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
313
Malcolm G. Baines Natural Immune Regulation of Activated Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
331
Donna A. Chow, Ricky Kraut, Xiaowei Wang
V. Neuroimmune Pathology Introduction: V. Neuroimmune Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
347
Istvan Berczi Stress, Health and the Immune Response: Reciprocal Interactions Between the Nervous and Immune Systems . . . . . . . . . . . . . . .
351
Alexander W. Kusnecov, Alba Ross#George, Scott Siegel Cytokines in the Brain: From Localization and Function to Clinical Implications . . . . . . . . . . . . . . . . . . . . . . . .
365
Julio Licinio, Ma-Li Wong Neurogenic Inflammation: Role of Substance P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
373
Andrew M. Stanisz Lupus as a Model of Neuroimmune Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
379
Judah A. Denburg, Boris SaMc, Henry Szechtman, Susan D. Denburg The Pathogenesis of Encephalitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Trevor Owens, Elise H. Tran, Mina Hassan-Zahraee, Alicia Babcock, Michelle L. Krakowski, Sylvie Fournier, Michael B. Jensen, Bente Finsen
387
xxii
VI. Clinical Neuroimmune Biology Introduction: VI. Clinical Neuroimmune Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
401
Istvan Berczi Growth Hormone Therapy and Immune Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
403
Robert Rapaport, Robert Moghaddas The Role of Prolactin in Systemic Lupus Erythematosus . . . . . . . . . . . . . . . . . . . . . . . .
409
Richard Warrington, Tim McCarthy, Eva Nagy, Kingsley Lee, Istvan Berczi Combination Immunotherapy of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
417
Eva Nagy, Istvan Berczi, Edward Baral, John Kellen The Influence of Reproductive Hormones on Asthma . . . . . . . . . . . . . . . . . . . . . . . . . .
433
Gordon T. Ford, Candice L. Bjornson, lan Mitchell, M. Sarah Rose Neuro-Immunopathgenesis in Autism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
447
Vijendra K. Singh Skin inflammation and Immunity After Spinal Cord Injury . . . . . . . . . . . . . . . . . . . . . .
459
Brian J. MacNeil, Dwight M. Nance
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
475
INTRODUCTION
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New Foundation of Biology Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
N e u r o i m m u n e B i o l o g y -- A n I n t r o d u c t i o n
ISTVAN BERCZI
Department of Immunology, Faculty of Medicine, The University of Manitoba, Bannatyne Campus, 32-795 McDermot Avenue, Winnipeg, Manitoba R3E OW3, Canada
ABSTRACT That a healthy mind is fundamental to general well being has been recognized since prehistoric times and proverbs analogous to "Healthy body - healthy mind" exist in many languages. A century ago pathologists noted first that the size of the thymus was profoundly influenced by emotional events and by neuroendocrine aberrations. Hans Selye discovered first (1936) that the hypothalamus-pituitary-adrenal axis, which is activated by diverse 'nocuous' stimuli, leads to the rapid involution of the thymus. He coined this phenomenon as the 'stress' response. Selye established that stress results in the development of the general adaptation syndrome which is characterized by elevated resistance to diverse insults. Andor Szentivanyi and colleagues discovered (1949) that hypothalamic lesions prevent anaphylactic death in guinea pigs. This is the first experimental evidence for the sweeping regulatory power of the nervous system over violent, life threatening immune reactions. That the nervous system also controls the inflammatory response was first demonstrated by Milos Jancso and co-workers (1964). These fundamental discoveries were not followed by a burst of research activity. Progress has been slow because of the lack of basic knowledge and because of the immense technical difficulties encountered. In the seventies a handful of laboratories started to re-examine various aspects of neuroimmune-interaction. It was established that pituitary hormones have the capacity to stimulate, inhibit and modulate immune responses. Placental and pituitary hormones are also involved in the development of the immune system and maintenance of immunocompetence. It was also described that lyphoid organs are innervated and that neurotransmitters and neuropeptides are important immunomodulators. It became gradually apparent that immune derived cytokines and nerve impulses serve as feedback signals towards the neuroendocrine system. Compelling evidence was produced, indicating that immune reactions may be conditioned in the classical pavlovian sense and that emotions affect immune function of various organs and tissues, and in reproduction. It is also becoming obvious that Selye's general adaptation syndrome really corresponds to the acute phase response. This is a multi-faceted and highly co-ordinated systemic defence reaction, which involves the conversion of the immune system from a specific, adaptive mode of reactivity to a rapidly amplifiable polyspecific reaction mediated by natural immune mechanisms. Immunological (poly)specificity is assured by profoundly elevated levels of natural antibodies and liver derived proteins.
Much has been learned about the regulation of cell activation, growth and function from immunological studies. Burnet's clonal selectional theory designates antigen as the sole activator. Bretcher and Cohn recognised first that at least 2 signals are required. This was followed by numerous studies on cell-to-cell interaction within the immune system and led to our current understanding of the importance of cell adhesion molecules and cytokines in cell activation and proliferation. This, coupled with the available information about the mechanisms of action of hormones and neurotransmitters, of signal transduction and nuclear regulatory pathways paves the way to understanding how higher organisms function in their entire complexity. It is now apparent that the Nervous- Endocrine- and Immune-systems form a systemic regulatory network, which is capable of regulating all aspects of bodily functiuons in health and disease. Thus, Neuroimmune Biology provides new foundations to Biology.
1.
INTRODUCTION
Observations indicating that the central nervous system has a fundamental role in the maintenance of health has been made since prehistoric times and is referred to in proverbs of many languages. The healing power of mind and faith provides one of the important foundations of religion and is described in many religious texts. These phenomena are also observed in modern medicine and is known as the placebo effect. It has been demonstrated repeatedly by exact scientific methodology that patients treated with placebo in controlled medical trials do in fact show significant improvement clinically in the absence of effective treatment. In ancient Persia, Egypt and in the Roman Empire fever has been regarded as a reaction with healing power. This view was maintained until modern times and during the early nineteen hundreds pyrogenic substances have been developed for the purposes of fever therapy [1-3]. About a century ago pathologists observed that acromegaly was frequently associated with thymic hyperplasia. Hammar [4] described that the thymus frequently showed involution under the influence of environmental or emotional factors. In contrast, thymic hyperplasia was associated with castration, Graves' Disease, Addison's Disease and acromegaly. Smith described in 1930 that in hyposectomyzed (Hypox) rats the thymus regressed in weight to less than half of that of controls. In partially Hypox rats there was no involution [5]. In 1936 Hans Selye documented that the pituitary-adrenal-thymus axis was activated by various nocuous stimuli, which led to the involution of the thymus and of the lymphoid organs [6, 7]. Moreover, Selye has established that the bursa of Fabricius in chickens was extremely sensitive to steroid hormones [8]. Within ten years Selye has proposed the theory of general adaptation syndrome (GAS) [9] on the basis of his experiments. He pointed out that this is a general reaction that leads to resistance of the organism to various insults. Selye's scheme of GAS is shown in Figure 1, updated with current information. In 1949 Selye discovered that the inflammatory response is regulated by corticosteroids [10]. In his article entitled "Stress and Disease" he proposed that deficient host defense due to abnormalities of neuroendocrine factors may lead to disease [11 ]. Selye recognized the importance of mast cells in pathology and performed numerous studies in this respect. He summarized the knowledge about mast cell in a book [ 12], which is a lasting contribution on the subject.
Clinical shock Loss of body weight+N Gastrointestinal ulcers Temporary rise in plasma potassium level Temporary fall in plasma
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Figure 1. Functional interrelations during the general adaptation syndrome. This figure is modified from Selye [9] by updating it with recent information. Solid arrows and the two broken arrows on the top with bold text is Selye's original figure. Recently identified pathways are indicated with dotted arrows and with text in italics. The text below is also from Selye. "Schematized drawing indicating that non-specific damage causes clinical shock, loss of body weight and nitrogen, gastro-intestinal ulcers, temporary rise in plasma potassium with fall in plasma C1, through unknown pathways (nervous stimulus?, deficiency?, toxic metabolites?) but manifestly not through the stimulation of the hypophyseoadrenal mechanism. This is proven by the fact that the above manifestations are not prevented either by hypophysectomy or by adrenalectomy; they even tend to be more severe in the absence of either or both of these glands. Non-specific damage, again through unknown pathways, also acts upon the hypophysis and causes it to increase corticotropic hormone production at the expense of a decreased gonadotropic, lactogenic and growth hormones. The resulting corticotropic hormone excess causes enlargement of the adrenal cortex with signs of increased corticoid hormone production. These corticoids in turn cause changes in the carbohydrate (sugar active corticoids) and electrolyte metabolism (salt-active corticoids) as well as atrophy of the thymus and the other lymphatic organs. It is probable that the cardiovascular, renal, blood pressure and arthritic changes are secondary to the disturbances in electrolyte metabolism since their production and prevention are largely dependent upon the salt intake. The changes in y-globulin, on the other hand, appear to be secondary to the effect of corticoids upon the thymicolymphatic apparatus. We do not know as yet, whether the hypertension is secondary to the nephrosclerosis or whether it is a direct result of the disturbance in electrolyte metabolism caused by the corticoids. Similarly, it is not quite clear, as yet, whether corticoids destroy the circulating lymphocytes directly, or whether they influence the lymphocyte count merely by diminishing lymphocyte formation in the lymphatic organs. Probably both these mechanisms are operative".
Selye made all his contributions without knowing the function of the thymus, lymph nodes or the bursa of Fabricius. The function of these organs was understood in the sixties and early seventies, decades after he published his seminal papers on stress. With the advent of the science of Immunology it became clear that stress has a profound immunosuppressive effect and increases the susceptibility to infectious disease. These findings seemed to contradict Selye's conclusion that the response to stress was an adaptive defense reaction which increased the resistance of the body to various noxious agents. Andor Szentivanyi and his colleagues were the first to document that the nervous system has a dominant regulatory power over immune reactions. As a medical student Szentivanyi observed that adrenaline treatment was ineffective to alleviate an asthmatic attack in a patient. This clinical observation inspired him to do experiments in guinea pigs using anaphylactic shock as a model system. Hypothalamic lesions inhibited the development of anaphylactic shock in immunized animals [13]. Tuberal lesions (TBL) of the hypothalamus were effective in preimmunized guinea pigs and in later experiments also in rabbits to inhibit anaphylactic reactions elicited by the intravenous application of the antigen. Antibody production was also inhibited if the lesions were induced prior to immunization. The reaction of antibodies with the specific antigen was not affected by such lesions, nor was the release of tissue material mediating anaphylaxis. TBL temporarily increased the resistance of the animals to histamine and inhibited the anaphylactic reaction even when the animals were provided with passively transferred antibody, which elicited lethal shock in normal animals. The Schultz-Dale test, which was performed with small pieces of intestine in vitro, was also inhibited when the animals were subjected to TBL. The Arthus reaction, turpentine induced inflammation and the Sanarelli-Schwartzmann phenomenon were unaffected by hypothalamic lesions. Lesions inflicted in other areas of the hypothalamus or the central nervous system were ineffective in modulating immune phenomena. Electrical stimulation of the mamillary region of the hypothalamus had an inhibitory effect on the anaphylactic response and increased the resistance of animals to histamine [ 14-16]. Szentivanyi devoted his entire career to the study of allergy and asthma. Animal experiments pointed to the importance of the beta-adrenergic receptor in these reactions [17]. In 1968 Szentivanyi had synthesized the knowledge and all his findings in a review article, entitled, "The beta-adrenergic theory of the atopic abnormality in bronchial asthma" [ 18]. He concluded that bronchial asthma, whether it is due to "extrinsic" or "intrinsic" causes, is ultimately elicited by the same mediators, such as histamine, serotonin, catecholamines, slow reactive substances plus cytokines. These are released during asthmatic reactions and should be considered as additional group of mediators in many tissues and in most species. Glucocorticoids are natural inhibitors of inflammation. He proposed that the atopic abnormality in asthma is due to the abnormal function of the [3-adrenergic system, irrespective of what triggered the reaction: "The beta adrenergic theory regards asthma not as an 'immunological disease' but as a unique pattern of bronchial hypersensitivity to a broad spectrum of immunological, psychic, infectious, chemical and physical stimuli. This gives to the antigen-antibody interaction the same role as that of a broad category of non-specific stimuli which function only to trigger the same defective homeostatic mechanism in the various specialized cells of bronchial tissue". Szentivanyi remained faithful to the idea of beta-adrenergic malfunction in atopy and asthma. This is the common thread that connects the numerous papers reviews, book chapters and books he published. He studied c~- and [3-adrenergic receptors; adenylcyclase, cyclic-AMP and signal transduction; isolated, characterized and pharmacologically modulated phosphodiesterase; observed the systemic effect of immunization and of endotoxin on the adrenergic and cholinergic systems, on metabolism and on immune inflammatory mediators; performed clinical studies on asthma and related conditions. His major observations were:
1. Beta-adrenergic sub-sensitivity did exist in patients with atopic dermatitis who never received adrenergic medication. This indicates that therapeutic desensitization cannot account for the dysfunction of the beta-adrenergic system [19]. 2. The beta- adrenergic reactivity of lung tissue of lymphocytes and bronchocytes from patients with atopic asthma was found to be abnormal and various patterns of drug vs. disease-induced sub-sensitivity could be recognized [20-25]. 3. Bronchial hyper-reactivity to cholinergic agents in asthma was not mediated through cholinergic mechanisms but it was caused by the adrenergic abnormality, which was due to the so called "denervation super-sensitivity" [26-29]. Lymphocytes of asthmatic patients showed a significant decrease in adrenaline binding to beta-adrenergic receptors, which was independent of therapy [21, 22, 25]. Szentivanyi also studied the effects of inflammation on [3-adrenergic receptors [30-33]. .
In 1964 Korneva and Khai [34] described that hypothalamic lesions in commonly used laboratory rodents (e.g. rabbits, guienea pigs, rats) inhibited the production of complement fixing antibodies. In 1960 Miklos Jancso and co-workers reported that capsaicin is a sensory irritant and that repeated local or systemic administration to rats, mice and guinea pigs causes desensitization, which involves interference with pain receptors. Systemic pretreatment of animals with capsaicin or repeated local applications prevented the inflammatory response, indicating the involvement of the nervous system. This was later confirmed by experiments performed on denervated tissues. These observations indicated the existence of a distinct form of inflammation, which depends on sensory nerve innervation. The stimulation of C-fibers was necessary to induce this inflammatory response. The neurogenic inflammatory response was also demonstrated in man [35, 36]. It was known for some time that hormones, including those secreted by the pituitary gland, affect immune reactions [37]. However, only after the publication of systematic studies performed on hypophysectomized rats and in animals treated with bromocriptine [38-42], was the role of pituitary hormones seriously considered in immunoregulation by the scientific community. In 1975 Wannemacker and co-workers isolated the leukocyte endogenous mediator (LEM) of fever [43], which was the first immune-derived molecule identified, that mediated feedback signals towards the central nervous system. Later LEM was found to be identical with interleukin-1. That IL-1 also serves as a signal for pituitary hormone release was shown by a number of investigators in the early 1980's [44-49]. Subsequently other cytokines, especially IL2, IL6, TNF-~t and interferon gamma were shown to regulate the secretion of pituitary hormones during systemic immune/inflammatory reactions [50]. It is also clear by now that the nerves have immunoregulatory function and provide feedback signals from lymphoid organs and from sites of immune/inflammatory reactions towards the central nervous system (CNS) [51-54]. In 1926 Metalnikov and Chorine proposed first the behavioral modification of the immune response [55]. In 1933 Smith and Salinger [56] observed that asthmatic attacks were provoked in some patients with visual stimuli in the absence of the allergen. That immune reactions can be conditioned in the Pavlovian sense was demonstrated by Ader, MacQueen et al and by Gorczynski et al [57-59]. It was also observed that various cells in the immune system produce classical hormones and neurotransmitters. Smith and Blalock, Montgomery et al and DiMathia et al. [60-62] pioneered these observations.
2.
NEUROIMMUNE INTERACTIONS
2.1.
Cell-to-cell interaction
Traditionally the cells of all tissues and organs have been divided into stromal cells, which were thought to provide for the structure of organs and the frame for the functioning cells, which were called parenchymal cells. It is now evident that stromal cells interact actively with parenchymal elements and this interaction leads to functional regulation of the tissue/organ. Moreover, invariably the stroma of all tissues and organs contain immune derived elements such as lymphocytes, macrophages or more specialized cells that include the glia cells in the nervous system, Kupffer cells in the liver, the Langerhans cells in the skin, etc. These cells contribute to function both in health and disease. Blood vessels and endothelial cells lining the blood vessels are also active participants in lymphocyte recirculation and in local immune/inflammatory reactions. These cells interact both with the circulatory elements of the immune system and locally with elements of the tissue/organ. Cell-to-cell regulation in tissues is mediated by adhesion molecules that have complementary binding sites. These molecules are capable of delivering activation or inhibitory signals in a tissue and cell-specific manner [63-76]. Adhesion molecules and other cell membrane receptors have the capacity to co-aggregate within the semi-fluid cell membrane (capping) and allow the interaction of immunoreceptor thyrosin based activation motifs (ITAM) and -inhibitory motifs (ITIM). These motifs promote phosphorylation and dephosphoylation of signal transuding molecules, respectively. The cell may be activated or inhibited depending on the outcome of receptor interactions after capping. The relevance of these regulatory motifs to cell function is especially well established for the antigen receptors of NK cells and of T lymphocytes and for the function of Fc receptors. However, the phenomena of "receptor crosstalk" has been observed in many other systems [77-85]. These developments indicate that numerous receptors are involved in cell signaling, and that these receptors interact by multiple mechanisms that may lead to activation, inhibition or even inactivation (apoptosis) [ 130]. Numerous receptors in immunology and several hormone receptors need to be cross-linked by the ligand in order to deliver an activation signal to a cell. This mechanism provides an important regulatory function in that cross linking may take place only at an optimal concentration of the ligand, whereas low or high concentrations would not be able to signal the cells. When more than one receptor isotype is available, the homo- and hetero-diamers formed by the specific ligand may have different regulatory functions. In addition, cross-linking may be one of the important mechanisms that promotes capping of the receptors prior to activation [77, 87]. The immune system consists of mobile cells that are able to home readily to specific target tissues and also to sites of infection, injury, regeneration and healing. Stromal lymphoid cells play physiological roles and are very important for host defense, regeneration and repair. Adhesion molecules mediate immunocyte homing and lymphocyte recirculation. Blood vessels also provide important barrier function in some tissues and organs that are known as immunologically privileged sites. The blood-brain barrier is very important from the point of view of neuroimmune interaction and is being extensively studied at the present time [71, 86, 88-93].
2.2.
Innervation
The central nervous system has the capacity to deliver neurotransmitters and neuropeptides to all tissues and cells in the body. For a long time the immune system was considered as an exception to this rule. However, it is now clear that the thymus and the spleen and other lymphoid organs are innervated. Interestingly, the spleen contains only sympathetic efferent nerve fibers [94, 95]. Tissue mast cells are also innervated and the formation of synapses with nerve fibers and lymphocytes can be readily demonstrated in tissue culture. Neurogenic inflammation is the direct result of the discharge of inflammatory mediators from mast cells after stimulation by mediators (primarily substance P) released from sensory nerve terminals. Neural mediators, such as growth factors, neurotransmitters, and neuropeptides, (e.g. substance-P, somatostatin) play major roles in the regulation of immune/inflammatory responses. Nerve fibers are capable of rapid and specific local delivery of mediators that are suitable of mounting an instantaneous reaction by initiating inflammation. In other situations nerves may exert an anti-inflammatory effect. The local modulation of immune reactions is equally possible by neurotransmitters and neuropeptides [94-96]. During the acute phase response there is a massive release of catecholamines into the circulation, which is known as "sympathetic outflow". Catecholamines are important regulators in the acute phase response, which is an emergency defense reaction. Sensory nerves provide feedback signals towards the CNS from sites of injury, inflammation, and infection. The vagus nerve carries feedback signals to the CNS from visceral organs [94-97]. 2.3.
Humoral communication
Historically the humoral mediators of cell-communication have been classified as hormones that act at distant targets, neurotransmitters and neuropeptides, and locally produced hormone-like mediators, now called cytokines. One may also include here immunoglobulins, which originate from B-lymphocytes within the immune system. Immunoglobulins have evolved from adhesion molecules. In addition, virtually every cell membrane bound molecule is present in the serum, which includes MHC molecules and receptor-like-binding proteins. By now it is clear that "classical" hormones, neurotransmitters and neuropeptides are widely synthesized at various ectopic sites, including the immune system. Moreover, cytokines, which have been originally discovered within the immune system are now known to be synthesized in other tissues and organs, including the neuroendocrine system. Therefore, the historical definition of hormones, neurotransmitters and neuropeptides no longer applies. Rather, systemic and locally produced mediators complement each other, so that optimal function is assured both under physiological and pathophysiological conditions. In addition to the blood stream, lymphatic drainage of tissues, including the CNS, is important for humoral communication. The immune system receives signals from all tissues via the lymphatic system [86, 93, 98, 99].
3.
NEUROIMMUNE REGULATORY PATHWAYS
3.1.
The TRH-PRL, GH, IGF-I, TSH-thyroid axis
Thyrotropin releasing hormone (TRH) stimulates prolactin (PRL), thyroid stimulating hormone (TSH) and under some pathophysiological conditions, growth hormone (GH) release [ 100, 101 ]. Moreover, GH, PRL and TSH producing pituitary cells share the nuclear regulatory factor,
10
Pit-1 [102]. This suggests that these hormones represent an interdependent regulatory unit. Indeed in rats immunized with sheep red blood cells the increase of TRH mRNA was found in the hypothalamus at 4-24 hours after immunization. Pituitary TRH receptor mRNA and plasma PRL levels were also increased at the same time, while TSH and GH did not change. The hypothalamus-pituitary-adrenal (HPA) axis was activated 5-7 days after immunization. Antisense oligonucleotides complementary to TRH mRNA, given i.c.v, inhibited PRL secretion and decresed the titer of antibodies produced [ 103].
3.1.1. Thyrotropin releasing hormone (TRH) TRH affects directly lymphocyte proliferation and the development of T lymphocytes in the gastrointestinal tract [104, 105]. In man, serum interleukin-2 (IL-2) levels rose significantly during the standard TRH test [106]. The treatment of patients in critical illness repeatedly with TRH increased serum TSH, PRL, GH, T4 and T3 levels, and may correct the euthyroid sick syndrome [ 101 ]. 3.1.2. Growth and Lactogenic Hormones (GLH) Growth hormone, PRL and placental lactogen (PL) are referred to collectively as GLH. All three hormones show molecular heterogeneity and the variant forms of GH and PRL differ in their biological activity. GLH hormones are produced by a variety of cells in the body, including lymphocytes [107-117]. Our recent observations indicate that PRL production in lymphoid tissues is pituitary dependent (Figure 2). GLH and cytokines (e.g. G-CSF, GM-CSF, EPO, IL-2, -3, -4, -5, -6, -7,-9, -11, -13) share receptor structure [ 118-121 ]. Receptors for PRL and GH show heterogeneity and require cross-linking for signal delivery. At high hormone concentrations, cross-linking will not take place, but rather each receptor molecule will be bound to a separate hormone molecule, which leads to the self-inhibition of signal delivery. Homo- and heterodiamerization may take place after receptor-ligand interaction and some of the heterodiamers lead to inhibition, rather than stimulation. More than one signaling pathways play a role in GH and PRL action [87, 118-124]. Both GH and PRL induce the production of insulin-like growth factor-I (IGF-I) in cells of the immune system. IGF-I receptors belong to the transmembrane thyrosine kinase receptor family and are ubiquitously displayed on immunocytes [ 125]. The fetal pituitary gland does not play a role in the development of the immune system. There is evidence to suggest that maternal and placental lactogenic hormones fulfil this role [126-128]. After parturition, the function of the bone marrow, the thymus and the maintenance of immunocompetence all become pituitary dependent. The bone marrow deficiency of hypophysectomized rats can be normalized by treatment with purified PRL, GH or PL [ 129-131 ]. IGF-I plays a role in the mediation of GH action on bone marrow [132, 33]. Colony stimulating factor-1 (GM-CSF) and interleukin-3 are capable of stimulating IGF-I production in bone marrow cells and thus might function similarly to GLH in this organ [134]. GH, PRL, PL and IGF-I all stimulate thymus growth [116, 127, 135-139]. This stimulatory effect is directly related to the maintenance of immunocompetence [ 136]. GH, PRL and PL all promote the antibody response [128, 141]. Human pituitary dwarf individuals have normal immune function, which can be explained by the presence of normal serum PRL levels [140]. The dopaminergic drug, bromocriptine, suppressed humoral immunity which could be reversed by treatment with either GH or PRL. ACTH induced immunosuppression was also reversed by these hormones [142]. PRL enhanced the antibody response in mice in a biphasic manner [ 141 ]. Cell mediated immune reactions, including contact sensitivity reactions, graft rejection, graft versus host reaction, and killer cell activity were
11
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7.
NEUROPEPTIDE VARIATIONS
As indicated earlier, Tilders and his associates demonstrated that IL-1 [3 administration provoked time-dependent variations of CRH and AVP coexpression within the external zone of the median eminence. Thus, it was of interest to establish whether TNF-ot would likewise promote such an outcome. Moreover, given that stressors markedly affected amygdala functioning, in our experiments we also assessed the changes of CRH that occurred within this region. In these
155
experiments TNF- (4.0 ~tg) was administered to mice, and then at 1, 7, 14 or 28 days afterward half the mice received saline and half received reexposure to the cytokine (1.0 ~tg). Thus, we could determine whether the reexposure session was essential for particular peptide variations. As seen in Figure 7, within the external zone of the median eminence, immunoreactivity for both CRH and AVP was increased at 7 and 14 days following the initial TNF-ct treatment. Interestingly, these neuropeptide alterations were not further augmented upon reexposure to TNF-~, suggesting that the passage of time rather than sensitized cellular responses were responsible for these variations. Interestingly, while initial TNF-~ treatment itself did not markedly affect c-fos-immunoreactive (c-fos-ir) measured after 7 and 14 day intervals, reexposure to TNF-ct at these times provoked a sensitization of the immediate early gene. Moreover, double labeling revealed that the c-fos-ir cells also were immunoreactive for AVP within the neuroendocrine regulatory PVN and supraoptic nucleus, suggesting that the cytokine may have been having protracted actions on magnocellular AVP circuits [58]. Unlike the effects observed in the hypothalamus, within the central amygdala, increased staining of CRH immunoreactive fibers was only evident if mice were reexposed to TNF-c~ after the 1 and 7 day intervals following the initial cytokine treatment. In effect, the passage of time alone was a necessary, but not sufficient condition, to elicit the sensitization. Yet, it appeared that amygdaloid c-fos immunoreactivity was increased upon reexposure to TNF-~ 7 and 14 days following initial challenge with the cytokine. Thus, it is likely that neural responses within the central amygdala involving non-CRH processes are also subject to more protracted time-dependent sensitization.
Figure 7. Immunoreactivity for AVP and CRH (top and bottom panels, respectively) within the median eminence of mice receiving saline (A and D) or those treated with 4.0 ~tg of rhTNF- and sacrificed 7 (B or E) or 14 (C and F) days later. Pretreatment with a single dose of TNF-ct maximally increased stores of the neuropeptides in the external terminals after the 7-day interval (B and E). In contrast to the increased AVP within the external terminals, immunoreactivity for the peptide was reduced within the internal zone of the median eminence 14 days following the single cytokine exposure (C).
The neuroplasticity of the central CRH and AVP systems to both IL-1 and TNF-ct raises the possibility that these peptides may be responsible for the long term actions on hormonal and behavioral processes associated with cytokine or stressor treatments. Moreover, there is evidence to suggest that a cross-sensitization between immune (e.g., cytokine or infection) and non-immune (e.g., stressor) stimuli may exist with respect to behavioral and neuroendocrine
156
activity [57, 60]. It might be noted at this juncture that the effects of chronic stressors on CRH/AVP co-expression were comparable to those associated with the passage of time following an acute stressor. This should not be misconstrued to suggest, however, that the two have comparable outcomes with respect to other systems, nor that chronic unpredictable and intermittent events have effects like those associated with predictable stressors. Furthermore, while cytokine administration may influence neuroendocrine responses to later acute stressors, the effects elicited upon exposure to chronic stressors remain to be established. Likewise, data are unavailable concerning the impact of chronic stressors on the response to later cytokine or endotoxin challenge. The protracted CRH and AVP variations associated with acute and chronic challenges may be important for a wide array of pathological states, including depression, anxiety, neurodegenerative diseases and drug abuse, wherein neuropeptide involvement is suspected and some degree of neural memory may underlie the disorder [61 ]. Indeed, the possibility ought to be considered that individuals suffering from viral or bacterial infections may be at increased risk for subsequent stressor-related pathology. In this respect, it may be important to consider the frequency and timing of the stressors relative to the period of infection, as well as the number of infections previously encountered. In parallel with such neuroendocrine changes, repeated release of acute phase reactants (which might possibly include 13-amyloid) after multiple infections, may ultimately contribute to the progression of Alzheimer's disease [21].
8.
BEHAVIORAL EFFECTS OF CYTOKINE TREATMENTS
It will be recalled that proinflammatory cytokines, such as IL-I[5 and TNF-c~, induce a behavioral profile which reflects sickness. These behaviors have been thought to minimize energy expenditure, conserve body temperature, and generally allow the organism to maximize its ability to contend with viral and bacterial insults [10, 11]. In effect, such behaviors may reflect the manifestation of a highly organized motivational state critical to the survival of the organism [10]. In addition, it seems that cytokine treatments may engender anhedonic-like effects, wherein ordinarily reinforcing stimuli are not perceived as rewarding as they might otherwise be. Thus, when animals exhibit a decline in the consumption of palatable snacks, it may represent the anorexic effects associated with illness, but it may also be indicative of a general anhedonia. Unfortunately, it is exceedingly difficult to disentangle the anorexic and anhedonic effects of cytokine treatments. However, it has been shown that endotoxin administration may provoke a disruption of responding for rewarding brain stimulation (a behavior independent of the anorexic actions of cytokines), and this effect could not simply be attributed to illness [41 ]. Although psychogenic stressors and cytokines influence CNS functioning through different neural circuitry, the cytokines might be part of a regulatory loop that, by virtue of their effects on the CNS, contribute to the symptoms of behavioral pathologies, including mood and anxiety-related disorders [40, 44]. Indeed, depressive illness is associated with elevated levels of IL-I[5, IL-2, IL-6, TNF-c~, and soluble cytokine receptors [44]. Furthermore, the effects of endotoxin on consumption of palatable food substances could be attenuated by chronic antidepressant administration [62], possibly indicating that the treatment acts to temper the anhedonia, a fundamental characteristic of depressive illness. It might be noted at this juncture that humans u~ ~lergoing immunotherapy (e.g., with IL-2 or with interferon-~t) display numerous adverse neuropsychological, neurologic and psychiatric disturbances, including depression, which may be sufficiently severe to necessitate discontinuation of treatment [63, 64, 65].
157
Interestingly, it has been reported that the selective serotonin reuptake inhibitor, paroxetine, attenuated the depressive-like effects associated with interferon-or treatment of malignant melanoma [65]. Although TNF-ot has been used in cancer treatment, its extreme toxicity and shock inducing properties limit the systemic application of this cytokine. There have also been suggestions that elevated circulating TNF-ot may be associated with psychiatric illness [44] and such effect could come about by virtue of the cytokine's effects on central monoamine turnover [66]. Indeed, antidepressant medication has been reported to alter central levels of TNF-c~ [66].
9.
SUMMARY
Both IL-I[5 and TNF-c~ engender a profile of stressor-like effects. In addition to eliciting sickness-like behaviors, these cytokines may also promote depressive and anxiogenic-like responses, and elicit marked neuroendocrine and central monoamine changes. The latter effects were not restricted to the hypothalamus, but also occurred at mesolimbic sites. As in the case of stressors, cytokine challenge may engender a sensitization effect, wherein the response to subsequent cytokine and stressor challenges are augmented [57]. Despite the similarity between stressor and cytokine actions, analysis of the behavioral and neurochemical alterations associated with cytokines have been largely limited to the effects of acute treatments. Yet, viral and bacterial insults are sustained over protracted periods, and it is likely that the effects of traumatic events may engender at least some central cytokines alterations that are fairly persistent [57]. To assess adequately the impact of cytokine challenge on psychological processes it is essential to evaluate the impact of protracted treatments. In the case of processive stressors, chronic insults may promote a compensatory increase of amine synthesis, permitting the organism to deal with environmental challenges e.g., [5]. Yet, if the stressor is sufficiently protracted the wear and tear induced by attempts to adapt (allostatic load) may have adverse behavioral repercussions [6]. Although it is unclear what behavioral and central neurochemical alterations are introduced by sustained cytokine administration, analyses regarding immunebrain interactions and the ramifications for psychopathology need to consider the impact of continuous and sustained insults.
ACKNOWLEDGEMENTS Supported by the Medical Research Council of Canada. H.A. is a Senior Research Fellow of the Ontario Mental Health Foundation.
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New Foundation of Biology Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
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Proinflammatory Signal Transduction Pathways in the CNS During Systemic Immune Response
SERGE RIVEST, SYLVAIN NADEAU, STEVE LACROIX and NATHALIE LAFLAMME
Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, 2705, boul. Laurier, Qudbec, Canada, G1V 4G2
ABSTRACT Circulating lipopolysaccharide (LPS) causes a rapid transcriptional activation of its transmembrane receptor mCD14 within the circumventricular organs (CVOs), brain regions that contain a rich vascular plexus with specialized arrangements of the blood vessels. Parenchymal cells located in the anatomical boundaries of the CVOs exhibit a delayed response, which is followed by a positive signal for CD 14 transcript in microglia across the brain parenchyma. The constitutive expression of the toll-like receptor 4 (TLR4) in the CVOs is likely to be a key element allowing the proinflammatory signal transduction pathways (MyD88/IRAK/NIK/NF-KB) to take place rapidly in these organs in response to circulating LPS. These results strongly suggest that the endotoxin first reaches organs devoid of the blood brain barrier (BBB) to induce the transcription of its own receptor and thereafter increases CD14 biosynthesis within parenchymal structures surrounding the CVOs and then the entire brain of severely challenged animals. Brain CD 14 expression may be a key step in the transcription of proinflammatory cytokines primarily within accessible structures from the blood and subsequently through scattered parenchymal cells during severe sepsis. However, CD14 synthesis in parenchymal cells of the brain is also dependent on the production of proinflammatory cytokines. Of interest is the data that systemic injection of the bacterial endotoxin induces a strong expression of CD14 mRNA in a pattern that is closely related to the induction of tumor necrosis factor alpha (TNF-ct) transcript with a rapid and delayed response. Although there is a large body of evidence that CD 14 (and now TLR4) is necessary for the role of LPS on the induction of cytokine transcription from different myeloid cells, the possibility remains that the cytokine itself acts as an autocrine and paracrine factor to up regulate the LPS receptor. The binding of TNF to its type I receptor (p55) leads to the activation and translocation of p50/65 NF-~:B into the nucleus, which seems a key player in activating CD 14 transcription in the CNS. Central injection of recombinant rat TNF-ct causes a robust expression of the genes encoding IKBct, TNF-~t and CD14 in microglial cells of the brain parenchyma. The time-related induction of these transcripts suggested a potential role of NF-KB in mediating TNF-induced transcriptional activation of the LPS receptor. Systemic injection with the endotoxin LPS provoked a similar microglial activation that was
164
prevented in inhibiting the biological activity of the proinflammatory cytokine in the CNS. Together these data provide the evidence that centrally-produced TNF-c~ plays an essential autocrine/paracrine role in triggering parenchymal microglial cells during severe endotoxemia. These events may be determinant for orchestrating the neuroinflammatory responses that take place in a well coordinated manner to activate the resident phagocytic population of cells in the brain. The physiological outcomes of this innate immune response of the CNS are likely to include a rapid elimination of LPS particles via an increased opsonic activity of the transmembrane CD 14 receptor to prevent potential detrimental consequences on neuronal elements during blood sepsis.
1.
INTRODUCTION
The clinical manifestations of endotoxemia are characterized by hyperventilation, hypercoagulation, pain, fever, cachexia, tachycardia, hypotension, somnolence, change in oxygen consump, tion and multiple organ failure [1 ]. Most of these symptoms can be mimicked by the stimulation of host monocytes/macrophages in the presence of the endotoxin lipopolysaccharide (LPS) [2] or prevented with anti-CD14 antibody [3, 4, 5]. The endotoxin is released by the outer membrane of the Gram-negative bacteria during sepsis and is detected by cells of myeloid origin, which bear the LPS receptor CD 14 at their membrane surfaces [6]. CD 14 is considered as the key player in the induction of the septic shock provoked by Gram-negative bacteria. Two forms of the CD14 receptor can be found; the first one is present at the surface of myeloid cells (mCD14) and acts as a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein. The other form is soluble in the serum (sCD14) and lacks the GPI properties, although it can bind LPS to activate cells devoid of mCD14, such as endothelial and epithelial cells, astrocytes and vascular smooth muscle cells [7]. The spontaneous binding of the endotoxin to its transmembrane and soluble receptor occurs slowly, but with high affinity [8]. However, the binding rate is accelerated by the presence of LPS-binding protein (LBP), a serum protein that binds the endotoxin [9, 10]. Although LBP is not essential for the LPS signaling, the LPS/LPB complex is particularly powerful to activate cells of myeloid origin including monocytes, macrophages, neutrophils, and microglia. One of the well known consequences of such activation is the production of proinflammatory molecules, namely interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF) and different prostaglandins (PGs). It is not well known how cell activation is triggered after binding between the LBP-LPS complex and the GPI-anchored mCD14, although there is now evidence that activation of tyrosine kinase leads to signal transduction and cytokine gene transcription through nuclear factor kappa B (NF-KB). The recent characterization of human homologues of Toll, especially the Toll-like receptor 4, may be the missing link for the transduction events leading to NF-KB activity in response to the LPS/mCD14 interaction (see below). NF-~zB is normally present in the cytoplasm forming an inactive complex with an inhibitor known as I~;Bc~ (see Figure 1). Following extracellular stimulation by growth factors, mitogens and cytokines that activate mitogen-activated protein (MAP) kinases, I~:Bc~ is phosphorylated by NF-KB-inducible kinase (NIK)/IKBc~ kinases (IKK), ubiquitinated and degraded by cytoplasmic proteasomes [11, 12]. Free active NF-KB (the commonest complex is the p50/p65 heterodimer) is then translocated into the nucleus where it is able to regulate transcription of various genes by binding to an ~;B consensus sequence. Following its degradation, I~:Bc~ is rapidly re-synthesized to act as an endogenous inhibitory signal for NF-~zB, and monitoring its d e n o v o expression is
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Figure 1. The proinflammatory signal transduction pathways evolving the nuclear factor kappa B (NF-•B). p50 and p65 are the two DNA-binding subunits of the NF-KB dimer that is a potent transcription factor for numerous proinflammatory genes. See text for details and abbreviations.
a powerful tool to investigate the activity of the transcription factor within the CNS (for reviews, see [ 13, 14]).
2.
DO THESE EVENTS TAKE PLACE IN THE BRAIN?
We have recently reported that circulating LPS causes a rapid expression of CD14 mRNA within the circumventricular organs (CVOs), brain regions that contain a rich vascular plexus with specialized arrangements of the blood vessels [15]. The tight junctions normally present between the endothelial cells are shifted in part to the ventricular surface and partly to the boundary between the CVOs and adjacent structures, explaining the diffusion of large molecules into the perivascular region [16]. Parenchymal cells located in the anatomical boundaries of the CVOs exhibited a delayed response, followed by a positive signal for CD14 transcripts in microglia throughout the brain parenchyma [15]. These results strongly suggest that endotoxin first reaches organs devoid of the blood brain barrier (BBB) to induce the transcription of its own receptor and thereafter increased CD 14 biosynthesis occurs within parenchymal structures surrounding the CVOs and subsequently the entire brain of severely challenged animals. Brain CD14 expression is likely to be a key step in the transcription of proinflammatory cytokines [17, 18, 19] first within accessible structures from the blood and thereafter through scattered parenchymal cells during severe sepsis.
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Like systemic phagocytes [20], CD14 synthesis in parenchymal cells of the brain may be dependent on the production of proinflammatory cytokines. Of interest is the data that systemic injection of the bacterial endotoxin induced strong expression of CD14 mRNA [15] in a pattern that was closely related to the induction of TNF-c~ transcript [17] with a rapid and delayed response. Although there is a large body of evidence that CD14 is necessary for an effect of LPS on the induction of cytokine transcription from different myeloid cells, the possibility remains that the cytokine itself acts as an autocrine and paracrine factor to upregulate the LPS receptor. The binding of TNF to its type I receptor (p55) leads to the activation and translocation of p50/65 NF-KB into the nucleus [21], an event that has been reported to modulate CD14 expression. Indeed, TNF-c~ is able to induce a transient increase in plasma CD14 levels with a peak at 6-8 hour and this elevation in levels of CD14 antigen was shown to be accompanied by increased levels of CD14 mRNA in lung, liver and kidney [22]. Pretreatment of mice with an anti-TNF-c~ antibody significantly prevented LPS-induced mCD14 transcription [22, 23]. We have recently investigated the role of microglial-derived TNF in the regulation of CD14 in the brain during endotoxemia [24]. Central injection of recombinant rat TNF-c~ caused a robust expression of the genes encoding IKB~t, TNF-c~ and CD14 in microglial cells of the brain parenchyma. The time-related induction of these transcripts suggested a potential role of NF-KB in mediating TNF-induced transcriptional activation of the LPS receptor. Systemic injection with the endotoxin LPS provoked a similar microglial activation that was prevented by inhibiting the biological activity of the proinflammatory cytokine in the CNS. Together these data provide the evidence that centrally-produced TNF-c~ plays an essential autocrine/paracrine role in triggering parenchymal microglial cells during severe endotoxemia. These events may be determinant for orchestrating the neuroinflammatory responses that take place in a well coordinated manner to activate the resident phagocytic population of cells in the brain. The physiological outcomes of this innate immune response of the CNS are likely to include a rapid elimination of LPS particles via an increased opsonic activity of the transmembrane CD14 receptor to prevent potential detrimental consequences to neuronal elements during blood sepsis.
3.
THE AUTOCRINE/PARACRINE ACTION OF TNF-c~ ACROSS THE CNS
As mentioned, systemic LPS injection induced mCD14 expression first within structures devoid of BBB and thereafter throughout the brain parenchyma of animals that received high doses of LPS [ 15]. Microscopic analysis of emulsion-dipped slides revealed that CD 14-positive cells spread over the anatomical boundaries of the CVOs in a migratory-like pattern during the course of endotoxemia. The direct action of LPS on myeloid-derived cells expressing mCD14 and present in structures that are accessible from the systemic circulation may allow a rapid production of proinflammatory cytokines within these organs. The rapid induction of TNF-~ mRNA in the CVOs by i.p. LPS clearly indicates that such events take place in specific populations of cells in the brain. Like CD14, small scattered TNF-expressing cells can be found across the brain parenchyma in response to LPS, although this depends on the dose of the endotoxin and the route of administration [17]. Indeed, the CVOs displayed low but positive signal as soon as 1 hour after the LPS challenge and increased to reach maximal levels at 6 hour, but positive cells gradually became apparent in the boundary of these organs and spread over the entire brain from time 6 to 24 hour during severe endotoxemia. However, this phenomenon is only provoked by a high dose of bacterial LPS (2.5 mg/kg i.p.); a lower
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dose (250/,tg/kg i.p.) caused a more restricted transcriptional activation of the gene encoding the proinflammatory cytokine within the CVOs and the adjacent areas [17]. The same dose injected i.v. is capable of inducing CD14 and TNF across the brain parenchyma, whereas these transcripts are localized to the CVOs and choroid plexus (chp) in response to 5 jug LPS i.v. (N. Laflamme and S. Rivest, unpublished data). This clearly indicates that the endotoxin first reaches available targets devoid of BBB, which in return, depending on the severity of the challenge, primes adjacent cells within the parenchymal brain to stimulate TNF-ot transcription. The bacterial endotoxins are among the most powerful agents known to stimulate circulating monocytes and tissue macrophages, which leads to the synthesis and release of a variety of proinflammatory cytokines [7]. The most important target of macrophage-derived secretory products is the macrophage itself [1]. The early production of TNF may be an essential step in this autocrine and paracrine loops, as this cytokine is able to induce its own production by an autocrine stimulation that is followed by the synthesis of other proinflammatory cytokines, such as IL-113 and IL-6 [7]. TNF has also been found to be a major factor inducing shock, and passive immunization against the cytokine can attenuate the appearance of IL-I[3 and IL-6 [25]. Surprisingly however, cytokine production and circulating IL-6 and IL-I[3 induced by i.p. LPS injection is intact in TNF-deficient mice [26], which suggests that TNF production is certainly not the sole primary event that leads to production of subsequent cytokines in response to endotoxin and other models of inflammation. In the brain, the cytokine seems to act as a key ligand to activate parenchymal microglia in a paracrine manner during endotoxemia. It is suggested here that circulating LPS targets its transmembrane receptor in CVOs/chp resident macrophages and microglia, which may stimulate the NF-KB signaling events and trigger TNF transcription (see Figure 2). The cytokine may in return bind to its cognate p55 receptor and lead to the formation of the TNF-Rl-associated death domain (TRADD)/TRAF2 complex, which activates the NF-KB signaling events in adjacent microglia. TNF-~ is actually one of the most potent effectors of NF-KB activity through the 55 kd TNF type I receptor [11, 12]. Such events are likely to contribute to the transcriptional activation of both CD14 and TNF genes in the brain of endotoxin-treated animals. Central production of TNF-c~ is a key mechanism controlling CD 14 expression in the brain parenchyma, but not in the CVOs and chp. The anti-TNF did not significantly change the relative CD14 mRNA levels in these organs, but prevented quite specifically the parenchymal expression of the LPS receptor during endotoxemia. The constitutive expression of CD14 in regions that can be reached by the bloodstream may allow a rapid production of TNF that in return acts as the endogenous ligand to activate adjacent myeloid cells. An important question however is what mechanisms control the spreading of the message from regions close to the CVOs to deep parenchymal elements. As mentioned, the spreading of CD14 and TNF-c~-expressing cells depends on the severity of the endotoxin challenge [15, 17]. It is therefore possible that the paracrine influence of the cytokine remains localized in response to low circulating levels of LPS, but takes place across the cerebral tissue during severe endotoxemia. Such effects of TNF in activating CD14 expression have previously been reported in different systemic organs and like the CNS, pretreatment of mice with anti-TNF-~ antibody significantly prevented LPS-induced mCD14 transcription in the lung, liver and kidney [22, 23]. IL-113 has also been reported to stimulate CD 14 expression in different organs and anti-IL-113 antibody attenuated the induction of the LPS receptor in response to the endotoxin [22, 23]. IL-I[3 and TNF-c~ are known to have numerous overlapping activities and inhibiting one cytokine may frequently be associated with redundant mechanisms because of the presence of
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Figure 2. The lipopolysaccharide (LPS)/LPS-binding protein (LBP) complex has the ability to trigger accessible organs through its membrane CDI4 (mCD14) receptor. The constitutive expression of mCD14 together with Toll-like receptor 4 (TLR4) may allow the signaling events to take place in the circumventricular organs (CVOs) and activate transcription of proinflammatory cytokines. The subsequent release of tumor necrosis factor alpha (TNF-ct) acts as an autocrine and paracrine factor for the synthesis of the LPS receptor CD14 in the brain microglial cells during blood endotoxemia. See text for details.
the other cytokine [27]. Although IL-l[3 may have the ability to stimulate CD14 in the brain microglia, its involvement depends most likely on the prior production of TNF as the anti-TNF completely inhibited LPS-induced CD14 transcription. On the other hand, IL-1[3 is the key inflammatory signal in the brain to stimulate the production of growth factors by astrocytes during brain trauma, while TNF is not essential for such response. Indeed, we have recently observed a strong and rapid production of numerous proinflammatory molecules in cells lining the lesion site that was followed by a robust increase in ciliary neurotrophic factor (CNTF) biosynthesis [28]. The release of CNTF was completely inhibited in IL-1 [3-deficient mice while TNF-ct was still produced by microglial cells lining the corticectomy [28]. Circulating IL-I[3 has also been recently shown to be a key mediator of the NF-KB activity and COX-2 transcription in cells of the BBB during a systemic model of inflammation [29]. These data, together with the present study, support the concept that despite the recognized overlapping activities of both NF-KB-signaling cytokines in the systemic immune system, IL-I[3 and TNF-ct seem to have a distinct role in orchestrating the inflammatory events that take place in the brain. The possibility that circulating TNF may influence CNS CD14 expression was also investigated, as the cytokine is rapidly detected into the bloodstream during endotoxemia [30]. Intravenous injection of recombinant rat TNF-ct caused up regulation mCD14 transcript
169
quite selectively in the CVOs and not within parenchymal cells adjacent to these organs. Therefore, circulating TNF may not contribute to the robust transcriptional activation of the LPS receptor in parenchymal microglia of endotoxin-challenged rats. The positive autoregulatory loop is therefore not always associated with a paracrine influence of the cytokine to trigger CD14 in surrounding cerebral tissues. This unexpected result provides the evidence that microglial-derived TNF-c~ only is responsible for activating the biosynthesis of the LPS receptor in deep parenchymal cells of the brain in response to high circulating levels of the bacterial endotoxin.
4.
TOLL-LIKE RECEPTORS IN THE CNS
Host organisms detect the presence of infection by recognizing specific elements produced by micro-organisms, such as Gram-negative bacteria, Gram-positive bacteria and mannans of fungi [31]. These elements are called the pathogen-associated molecular patterns (PAMPs) that are recognized by specific cells of the immune system as innate mechanisms to mount a rapid response to bacterial infection. The endotoxin LPS is a major component of the outer membranes of Gram-negative bacteria, which is the best characterized example of innate recognition associated with a robust inflammatory response by phagocytic cells [32]. As mentioned, secretion of cytokines by circulating monocytes/neutrophils and tissue macrophages by LPS requires a series of mechanisms in cascade, the first step being the binding of the endotoxin with the serum proteins LPS-binding protein (LBP) or septins. The newly formed complex may then activate different populations of cells in binding to its CD 14 receptor [5]. Until recently, the exact mechanisms involved in the activation of the proinflammatory signal transduction pathways after binding between the LBP-LPS complex and the GPI-anchored mCD14 was unknown. Indeed, studies in CD14-deficient mice suggested the existence of a coreceptor to mediate LPS-induced NF-•B activity and cytokine gene transcription [2, 33]. The recent characterization of human homologues of Toll may be the missing link for the transduction events leading to NF-~:B activity and cytokine production in response to bacterial cell wall components. A large family of Toll-like receptors (TLRs) has already been characterized, which share similar extracellular and cytoplasmic domains [31]. The extracellular domains include 18-31 leucine-rich repeats (LRRs), whereas the cytoplasmic domains are similar to the cytoplasmic portion of the IL-1 receptor and is named the Toll/IL-l-receptor homologous region [31, 34]. Distinct TLRs have now been proposed as the key molecules to recognize quite selectively one of the major PAMPs produced by either Gram-negative or Gram-positive bacteria. The data that mutation of the mouse Lps locus abolishes the LPS response and that Lps encodes the TLR4 provided the first evidence that this particular receptor may play a key role in the innate immune response to Gram-negative bacteria (for review, see [35]). Further support for this concept comes from the TLR4-deficient mice that are unresponsive to LPS, whereas TLR2-deficient mice exhibit a normal inflammatory response to the endotoxin [36]. These results demonstrate that while TLR2 makes no contribution to LPS signaling, TLR4 is critical to recognize the PAMP produced by Gram-negative bacterial cell wall components. It is not yet known how LBP, CD14 and TLR4 interact together to function as the LPS signal transducer leading to activation of NF-KB and MAP kinases. It is possible that CD 14 acts as the principal LPS binding protein on the surface of monocytic cells and the newly formed complex reaches adjacent TLR4 receptors, which transduce the LPS signal via the general adaptor protein MyD88 [35, 37]. These events may also take place in the brain because
170
mRNAs encoding mCD14 and TLR4 are present in structures that can be reached by the bloodstream, namely the CVOs, chp and leptomeninges [38]. In contrast to the profound transcriptional activation of the LPS receptor CD 14 and the indicator of NF-KB activity I~:Bc~, the endotoxin and circulating IL-l[3 caused a significant decrease of TLR4 transcript in most of the constitutively-expressing parenchymal and non-parenchymal regions of the brain [38]. The basal expression of CD14 and TLR4 in the CVOs is likely to be a key mechanism in the proinflammatory signal transduction events that originate from these structures during innate immune response. Indeed, cell wall components of the Gram-negative bacteria may be selectively recognized by the TLR4/CD14-bearing cells of the CVOs, which allows the LPS signaling and then the rapid transcription of proinflammatory cytokines; the subsequent microglial activation in the brain parenchyma is, however, dependent on TNF-c~ (Figure 2). Therefore, TLR4 may be essential in this innate immune reaction that originates from the CVOs in response to cell wall components of Gram-negative bacteria. Although a strong increase in CD14 transcription is generally detected after systemic LPS injection, the endotoxin failed to stimulate the gene encoding TLR4 [38]. CD14-expressing cells were clearly devoid of TLR4 transcript in microglia across the brain parenchyma during moderate and severe endotoxemia. It is possible that TLR4 is the recognizing molecule for Gram-negative bacterial components only in response to systemic infection, whereas CD14 has a more complex role in the proinflammatory signal transduction events in the brain parenchyma. Nomura and colleagues have recently reported that TLR4 mRNA expression in mouse peritoneal macrophages significantly decreased within a few hours of LPS treatment and returned to the original level at 24 hours [39]. A rapid decrease of surface TLR4 expression was seen as early as 1 hour and remained suppressed over 24 hours in cells pre-exposed with LPS. These authors suggested that the down-regulation of the surface TLR4 expression may be responsible for the decrease in inflammatory cytokine production in tolerant macrophages, which may explain one of the mechanisms for LPS tolerance [39]. These data obtained from systemic macrophages are in complete agreement with our study that shows convincing down regulation of TLR4 gene in response to a single LPS bolus [38]. The phenotype of TLR4 cells in the CVOs was not determined due to the rather low levels of TLR4 transcript, making interpretation of the agglomeration of silver grains within immunoreactive cells arbitrary. Because LPS has the ability to increase CD14 mRNA in these organs, it was possible to perform the dual-labeling for the LPS receptor and numerous resident macrophages/microglia were positive for the transcript [ 15]. Although both transcripts may not be expressed within the same cells, we speculate here that TLR4 is located at the surface of the phagocytic population of cells of the CVOs, chp as well as the leptomeninges. TLR4 transcript levels are quite low in the cerebral tissue under basal conditions [38]. The signal was nevertheless specific, as we did perform numerous controls to ensure that what was being seen may not be related to an artifact of the in situ hybridization procedure. Actually, we had to adjust and maximize the hybridization conditions to detect this transcript in situ by generating the riboprobe just after the pre-hybridization step on freshly mounted brain sections. This very low level in the brain fits however quite well with the fact that the copy number of TLR4 is extremely low in systemic phagocytes compared to the more abundant membrane protein CD14 [35]. It is nevertheless remarkable that so few TLR4 receptors (perhaps 1000 or fewer per cell), residing on macrophages alone, have such an important influence in the LPS signaling and the coordination of the biological responses to Gram-negative infections [35]. It is expected that CVO TLR4 acts as a sensor for engaging the cerebral innate immune response in case of invasion during such systemic bacterial infection that may have detrimental consequences for the neuronal material.
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5.
CONCLUDING REMARK
Our data strongly suggest that endotoxin first reaches organs devoid of the BBB to induce the transcription of its own receptor and thereafter increased CD 14 biosynthesis within parenchymal structures surrounding the CVOs and then the entire brain of severely challenged animals. TLR4 is likely to play a key role in LPS signaling and the innate immune response that is triggered in a very well organized manner from specific structures of the brain during endotoxemia. The constitutive expression of CD 14 in the CVOs and its up regulation in the brain parenchyma suggest a potential role in protecting the neural elements against LPS particles. The resident macrophages and microglia in the CVOs are strategically well positioned to respond rapidly to circulating endotoxin or bacteria, while parenchymal microglia are the phagocytic population of cells in the brain in case of invasion. There is alteration of the BBB during severe endotoxemia [40], which may allow diffusion of molecules that normally have no access to the parenchymal elements and be detrimental for neurons. Activation of the microglial cells across the CNS may rapidly eliminate this foreign material, although a sustained activity of these cells is not suitable as it may have opposite effects and be associated with neurodegenerative disorders [41 ]. A better understanding of this innate immune response in the cerebral tissue may lead us to the fundamental mechanisms underlying how the brain is capable of mounting an inflammatory response that either protects or contributes to damage neurons.
ACKNOWLEDGMENTS Our work on this subject is currently supported by Canadian Institutes of Health Research (CIHR) formely the the Medical Research Council (MRC) of Canada. Serge Rivest is a Canadian MRC Scientist. Sylvain Nadeau is supported by a Ph.D. studentship from the Canadian MRC, whereas Steve Lacroix was supported by a Ph.D. studentship from the Natural Sciences and Engineering Research Council of Canada. Dr. Lacroix is currently an MRC postdoc fellow at UCSD in La Jolla, California.
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New Foundationof Biology Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
175
Nitric Oxide in Neuroimmune Feedback Signaling
TERESA L. KRUKOFF and WENDY W. YANG
Department of Cell Biology and Division of Neuroscience, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7
ABSTRACT The gaseous neurotransmitter, nitric oxide (NO), has been implicated in regulation of the hypothalamo-pituitary-adrenal (HPA) axis. NO donors attenuate lipopolysaccharide (LPS)-induced release of corticotropin releasing factor (CRF) in vitro and NO synthase (NOS) inhibitors potentiate and prolong activation of the HPA axis by LPS in vivo. Changes in activities of the NO synthase isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), have been reported in response to immune challenge. High doses of LPS administered either intravenously or intraperitoneally lead to increased activity of iNOS in perivascular microglia and in endothelial cells of microvascular origin. The NO produced by iNOS may in turn stimulate release of pro-inflammatory cytokines from cells of the brain. At much lower doses of endotoxin, however, where septic shock is not induced nor is the blood-brain barrier disrupted, nNOS and/or eNOS may play more important roles in NO production and signaling. Our work has shown that, in rats receiving 100 pg/kg intravenous LPS, blockade of NO production in the brain leads to elimination of the drop in body temperature and increased neuronal activation (including NO-producing neurons) in the paraventricular nucleus of the hypothalamus (PVN). The location of activated neurons in functionally distinct subdivisions of the PVN suggests that NO is involved in inhibition of the HPA axis, of sympathetic output, and of vasopressinand/or oxytocin-producing neurons in response to LPS. We also found that inhibition of NO production leads to increased gene expression of the cytokine, interleukin-1 ct (IL-1 ct), in non-neuronal cells of the PVN 4 hours after LPS injection and a return to baseline levels at 8 hours. While IL-1 t~ affects secretion of corticotropin releasing factor (CRF) from the PVN, the mechanism is controversial since neither IL-1 ct receptors nor IL-1 t~ binding have been reported in the PVN. NO's inhibition of IL-1 ct gene expression may, therefore, be mediated through an intermediate molecule (e.g. prostaglandins, cytokines, NO). Using inhibitors of the NOS isoforms, we provide evidence that eNOS is the isoform responsible for the effects described above. While it is most likely that eNOS found in the vasculature of the brain is responsible for these effects, other possible sources include neurons or glial cells because eNOS has been shown to be present in hippocampal neurons and in astrocytes activated by cytokines. In conclusion, NO is a strong messenger candidate as a mediator of signaling between the immune system and the brain, both in septic and non-septic conditions. Our data suggest that NO from eNOS inhibits neuronal
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activation and IL-1 ot gene expression in the PVN and affects temperature regulation in response to relatively mild levels of immune stress.
1.
INTRODUCTION
The gas, nitric oxide (NO), is recognized as a versatile signalling molecule which has a wide array of effects throughout the body depending on its site and level of production, and the molecules with which it interacts. In the presence of oxygen and nicotinamide adenine dinucleotide phosphate, NO is produced from L-arginine by NO synthase (NOS). NO diffuses in aqueous and lipid environments and, because of spread in three dimensions, can potentially influence activity in many nearby cellular elements. Three isoforms of NOS have been identified. Neuronal NOS (nNOS/NOS I) and endothelial NOS (eNOS/NOS III) are constitutively expressed, calcium-dependent, and commonly regarded as being produced in neurons and endothelial cells, respectively. Activity of inducible NOS (iNOS/NOS II) is calcium-independent and almost undetectable under basal conditions; appropriate stimuli induce this enzyme at the transcriptional level, nNOS, eNOS, and iNOS are encoded by genes on chromosomes 7, 12, and 17, respectively [74]. nNOS and eNOS produce NO in the nanomolar range in response to intermittent increases in calcium concentration; iNOS can produce NO in the micromolar range for extended periods of time [14, 74]. This review will address two primary issues. First, the effects of immune challenges on activity of each NOS isoform within the brain will be discussed. Second, the current state of knowledge about the role of brain NO in regulating activity of the hypothalamo-pituitary-adrenal axis in response to immune challenges will be presented.
2.
IMMUNE CHALLENGE AFFECTS CENTRAL NO
2.1.
nNOS
Neurons producing NO are found in many autonomic centers in the brain, including in neurons of the parvocellular PVN (pPVN) which have the capacity to directly affect activity of the HPA axis through their projections to the median eminence. Enkephalin and corticotropinreleasing factor (CRF) are co-localized with nNOS in neurons of the pPVN [reviewed in 40]. In addition, NO neurons in the PVN possess the NMDA R~ subunit [4], supporting a role for glutamate as a regulator of NO neurons in this hypothalamic nucleus. Intravenous (i.v.) injections of lipopolysaccharide (LPS) at doses which do not cause endotoxic shock (100 pg/kg) have been shown to stimulate nNOS gene expression and NO content in the PVN [44; 79]; intracerebroventricular (i.c.v.) injections of IL-I[3 stimulate nNOS expression within the PVN [43]. Relatively large numbers of NO neurons in the pPVN are also activated by LPS (100 pg/kg, i.v.) as assessed by expression of the immediate early gene, c-fos [86]. In addition to its neuroimmune effects, this dose of LPS leads to changes in body temperature and arterial pressure [85] and to activation of neurons in the pPVN [41] including NO neurons [55]. Therefore, the potential exists for LPS to affect the central neuronal NO system through several routes, directly through neuroimmune pathways and indirectly through other autonomic pathways. As will be discussed below, one of the important roles that NO plays in the pPVN is to affect activity of the HPA axis.
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High doses of LPS which cause endotoxic shock have been shown to stimulate or have no effect on nNOS gene expression in the hypothalamus. Using homogenates of hypothalamus, no differences in nNOS mRNA were found after 20 mg/kg intraperitoneal (i.p.) LPS [30; 66]. More selective assessment of nNOS mRNA with in situ hybridization, however, showed that i.p. LPS (25 mg/kg) stimulated nNOS gene expression in the PVN with a peak in expression at 5 hours after LPS injection [24]. These neurons were found primarily in the magnocellular PVN suggesting an interaction with vasopressin-and/or oxytocin-producing neurons. In addition, smaller numbers of neurons with increased levels of nNOS were found in the parvocellular PVN and about one-third of these neurons also expressed the CRF gene [24]. 2.2.
eNOS
Like nNOS, eNOS is a constitutive enzyme whose activity depends on the presence of Ca 2+. The primary source of eNOS in the brain is the vasculature where it is produced by endothelial cells. It has been claimed that neurons and glia do not normally produce eNOS [68; 72]. On the other hand, eNOS has been demonstrated in hippocampal [18; 32; 76], cortical [76] and thalamic neurons [76], and in astrocytes [2; 82; 10]. Viral infection has been shown to stimulate eNOS gene expression in glia both in vivo and in vitro [2], suggesting that cells other than endothelial cells may act as sources of eNOS in the brain during periods of immune challenge. Because of the association of astrocytes with blood vessels, astrocytic production of eNOS may provide a means through which blood-borne signaling molecules affect NO production which, in turn, may affect activity of nearby neurons. Until our recent study, surprisingly little attention had been paid to the effects of immune stimuli on eNOS activity. Studies focussed on identifying the source of NO which affects activity of the HPA axis often ignored eNOS [24; 30, 66]. Only in one study using i.v. injections of NOS inhibitors was it shown that a constitutive form of NOS, likely eNOS, suppresses the ACTH response to systemic injections of IL-I[3 [78]. Because of the systemic injections of inhibitors and IL-1 [3, however, this study did not differentiate whether the effects were central in nature or whether the effects were mediated at the level of the anterior pituitary gland. Our study using i.c.v, injections of NOS inhibitors has provided data which implicate eNOS in the brain as the source of NO which inhibits neuronal activation (as assessed by expression of Fos, the protein product of the immediate early gene, c-fos) and IL-I[3 gene expression in the PVN in response to i.v. LPS [85]. In addition, NO from eNOS is involved in mediating the drop in body temperature that occurs approximately 80 minutes after i.v. injection 100 ktg/kg of LPS [85]. A recent study has shown that brain astrocytes may be one of the sources of this eNOS as i.p. LPS (2.5 mg/kg) stimulated eNOS, but not iNOS, activity in these cells [29]. It is interesting that in vivo inhibition of NO production led to the conclusion that NO inhibits LPS-induced IL-I[3 gene expression in the PVN [85] while an in vitro study showed that NO stimulated IL-I[3 release from hypothalamic explant cultures under basal and KCl-stimulated conditions [49]. This discrepancy requires further investigation but, as pointed out in Section 3, may be related to inherent differences between studies using whole animals and those using hypothalamic explants. 2.3.
iNOS
iNOS is transcriptionally regulated and, after expression of the gene, iNOS is active for 4 to 24 hours to generate levels of NO which are 100 to 1000-fold greater than those produced by nNOS or eNOS [83]. Various cellular elements within the brain have the ability to produce iNOS.
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LPS or cytokine-induced iNOS production occurs in perivascular cells [83], microglia [53, 11], neonatal glial cell cultures [36], adult astrocyte cultures [22, 70], and endothelial cells of microvascular origin [8]. High levels of NO have been implicated in neurotoxicity in vitro [16, 27]. I.c.v injections of LPS lead to iNOS-associated neuronal death in the vicinity of the injection site and to spatial memory deficits [87]. In primary cultures of hippocampal neurons, IL-I[3, TNF-c~, and interferon attenuated astrocytic high-affinity glutamate uptake through an NO-dependent process [88]. The resulting excess of glutamate at the synaptic cleft is, in turn, responsible for causing widespread neurotoxicity [ 12]. Furthermore, production of free radicals and subsequent damage and disruption of metabolic processes also contribute to the toxicity of high levels of NO [7]. Finally, TNF-ot and LPS-induced apoptosis in cultured PC12 cells was found to be dependent on NO from iNOS [26]. In addition to the direct damage to neurons and glia by NO from iNOS, it has been hypothesized that recurrent bouts of systemic infection may play a role in the pathogenesis of neuronal disease associated with aging and may impair the brain's ability to respond appropriately to stressors and infection [52]. Increases in the permeability of the blood-brain barrier (BBB) occur during systemic infection [6] and L-NAME administration attenuated LPS-induced changes in the BBB [9, 69]. That NO from iNOS is specifically important in this response was shown when topical LPS (200 ng/ml) applied onto pial arterioles caused opening of the BBB which could be blocked by aminoguanidine, a specific inhibitor of iNOS [51]. High doses of LPS associated with endotoxic shock stimulate iNOS production and activity within the brain. After 20 mg/kg of i.p. LPS, perivascular cells increased their iNOS gene expression 6 hours after injection [83]. High-dose i.p. LPS (20-25 mg/kg) induced gene expression of iNOS in homogenates of the hypothalamus [30, 66] and in tissue sections [24]. iNOS mRNA was first detected at 2 hours, was significantly elevated at 3 hours, and returned to basal levels at 12 hours. Gene expression of iNOS in the brain after i.p. LPS was attenuated by i.p. administration of dexamethasone, suggesting that glucocorticoids are directly or indirectly involved in the iNOS response [75]. LPS (10 mg/kg, i.v.) has also been shown to lead to increased iNOS expression in the brainstem nucleus of the tractus solitarius beginning at 5 hours after LPS administration [46]. iNOS expression after LPS treatment is dependent on time of exposure. LPS at 0.25 mg/kg i.p. stimulated a significant increase in iNOS mRNA at 8 hours but not at 3 hours [66]. iNOS was expressed in cells of the circumventricular organs, preoptic area, hippocampus, arcuate nucleus, and nucleus of the tractus solitarius [37]. Angiotensin II, a neuropeptide known best as a neurotransmitter and renal hormone, attenuates LPS and IL-1 [3-induced increases in iNOS activity in cultures of adult rat astrocytes [38]. As angiotensin II is regarded as an important regulator of central cardiovascular responses and of body fluid balance it may, therefore, participate in the integration of autonomic and neuroimmune responses to immune challenges. 2.4.
Molecular mechanisms of neuroimmune signaling by NO
NO activates guanylyl cyclase to catalyze production of the second messenger, cGMP, in target cells [20, 21, 47]. cGMP can then affect ion channel function or phosphodiesterase activity, or activate cGMP-dependent protein kinases to affect other cellular events [1] including gene transcription [56]. NO has been shown to inhibit constitutive NOS activity in cerebellar extracts, possibly through indirect inhibition of NMDA receptors or through interactions with ferric heme which is required for NOS activity [reviewed in 40].
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NO affects activity of the DNA binding factor, NF-~B, which is essential for activation of several inflammatory mediators including TNF-c~, IL-I[3, IL-2, IL-6, IL-8, and interferon-j3 [5]. NO's effects may depend on the cell type in question. In glial cultures, NO from nNOS inhibited NF-~cB activity [77]. In cultured microglia, NO donors prevented LPS and TNF-c~-inducible NO synthesis from iNOS [13]. These effects were thought to be mediated through inhibition of NK-KB activity [13] since the iNOS promoter has a NK-KB binding site [84]. On the other hand, exposure of primary neuronal cultures of rat striatum to NO donors led to increased nuclear expression of the NK-KB subunits, p50 and p65, within 30 minutes [71]. As these subunits dimerize to form the NK-~zB which binds to DNA, these results were interpreted to indicate that NO has the potential to stimulate NK-KB activity [71], although a direct relationship to immune-stimulated changes in NK-KB activity in neurons remains to be demonstrated. Basal levels of NO have been measured in several types of tissue including endothelial cells and invertebrate ganglia [48]. Because addition of L-NAME resulted in a decrease in this basal level, it was assumed that the NO produced is physiologically relevant [48; 74]. It has been speculated that "tonal" NO from constitutive NOS tonically inhibits NF-~:B under basal conditions and that NO has, therefore, the ability to inhibit proinflammatory responses [74]. These investigators further hypothesized that, when the stimulus is sufficiently strong, a threshold of activation is surpassed, iNOS induction is not prevented, and relatively large amounts of NO are produced [74] to stimulate the effects described for iNOS above.
NITRIC OXIDE AFFECTS THE RESPONSE OF THE HPA AXIS TO IMMUNE CHALLENGE 3.1.
Effects of lipopolysaccharide and interleukin- 1[3
In the 1990's, investigators began to turn their attention to the role of NO in regulating activity of the HPA axis. In vitro studies have yielded contradictory results and have demonstrated both inhibitory and stimulatory roles for NO. In hypothalamic explants, it was shown that NO donors attenuated endotoxin-induced release of CRF but not basal CRF secretion [15]. On the other hand, inhibition of NO with Na-nitro-L-arginine or hemoglobin (a NO scavenger) in explant cultures attenuated the stimulatory effect of IL-1 on CRF secretion [64]. Finally, further confusion has been added to the area by a study in which inhibition of NO production reversed the inhibitory effects of LPS on release of CRF in hypothalamic explants [39]. In favor of an inhibitory role for NO on activity of the HPA axis, in vivo experiments showed that systemic L-NAME, a non-specific inhibitor of NOS, potentiated and prolonged the ACTH response to systemic LPS, IL-I[3, IL-6, or tumor necrosis factor-c~ [63; 62; 78; 34]. The proposed inhibitory role for NO on LPS-stimulated activity of the HPA axis proposed by the Rivier group was later contradicted by the same group when they showed that systemic L-NAME attenuated the ACTH response to i.v. LPS [79]. The reasons for this difference in results are not apparent at this time. Systemic L-NAME was shown to have no effect on the ACTH response to peripherally injected CRF or i.c.v. IL-I[3 which is thought to stimulate the HPA axis through CRF neurons in the PVN [61]. CRF mRNA levels were also unaffected [63], arguing against a direct action of NO on CRF neurons in the PVN. It has been suggested that NO's effects may occur at the level of the median eminence where CRF terminals are found or at the anterior pituitary [34]. In contradiction of the latter possibility, however, ACTH release stimulated by IL-I[3, CRF, vasopressin, or phorbol myristate acetate from anterior pituitary cell cultures was not affected
180
by application of the NOS inhibitor, N omega-Nitro-L-arginine (Nitro-arg) [25]. A further possible explanation [24] is that NO activates both guanylyl cyclase and cyclo-oxygenase [23] and that these enzymes have opposing effects on CRF secretion [24]. Some of the confusion with NOS inhibitor studies may arise from their systemic application, so that NO inhibition likely occurs, not only in the brain, but in peripheral sites including the pituitary gland. For example, i.v. or i.p. administration of L-NAME is well-known for its hypertensive effect due to systemic NO depletion. Activity in the PVN and the HPA axis may then be affected by changes in cardiovascular activity which have not been well controlled. Indeed, our results using i.c.v, inhibitors of NOS showed that, while arterial pressure responses were not affected, NO inhibited LPS-induced neuronal activation (Fos expression) in those subdivisions of the PVN which regulate activity of the HPA axis and inhibited IL-I[3 gene expression in the PVN [85]. Because IL-1[3 is stimulatory to CRF production and release [3, 65, 54, 31 ], our results support an inhibitory role for NO on the HPA axis. Finally, because NOS inhibition led to increased numbers of magnocellular neurons which were activated in response to LPS, we speculate that NO from eNOS inhibits LPS-induced secretion of vasopressin and/or oxytocin [85]. NO's effects on activity of the HPA axis may depend on the state of activation of the system. In the non-stimulated state, for example, direct injection of the NO donor, 3-morpholinosydnonimine (SIN-l), stimulated ACTH release and the response was blunted by injections of CRF or vasopressin antibodies [45]. In addition, increased gene expression of NGFI-B, CRF, and vasopressin were also noted. The authors hypothesized that, during unstimulated states, exogenous application of NO stimulates the HPA axis [45]. It is, however, probably too early to determine if this hypothesis is correct. First, the authors discounted the possibility that SIN-l-induced early increases in arterial pressure may have been responsible for some of the changes in gene transcription. On the contrary, increased pressure has been shown to be associated with changes in CRF gene expression in the PVN [42]. Second, the amount of NO released by SIN-1 is not known [45] and may have reached levels which are not characteristic of those released during responses to non-septic levels of immune challenge. The type of stressor may also affect NO's effects on activity of the HPA axis. Although outside the scope of this review, NO has been reported to have inhibitory [81] and stimulatory [35; 78] effects on activation of the HPA axis to non-immune physical stressors. 3.2.
Effects of other cytokines
NO has been implicated as a mediator of the IL-2-stimulated release of vasopressin and CRF from hypothalamic and amygdalar slices [57, 58]. Application of the NO donor, sodium nitroprusside, stimulated vasopressin and CRF release from these areas and application of the NOS inhibitor, NC-methyl-L-arginine (L-NMA), blocked the stimulatory effect of IL-2 on their release [57]. Similarly, the substrate for NOS, L-arginine, has been shown to enhance interleukin 2-induced CRF release in hypothalamus explants [33]. In explant cultures of median eminence, IL-10 stimulated NO production and CRF release through an NO-dependent process that was demonstrated by blocking NO production using L-NAME [73]. 3.3.
Vagal transmission
Information transfer about inflammation and cytokine levels in the peritoneum is mediated by the vagus nerve [80]. Little information is available about NO's role in transmission of
181
these signals, although intraperitoneal IL-I[3 stimulation of NO production in the PVN was significantly attenuated by subdiaphragmatic vagotomy [28]. 3.4.
NO and LPS-induced changes in body temperature
The effects of LPS on body temperature very much depend on the dose and route of administration. While low doses of i.v. LPS (5 or 10 pg/kg) stimulated increases in body temperature 2 to 6 hours after injection [19, 67], higher doses (100 or 125 pg/kg i.v.) produced a drop in temperature 80 to 100 minutes after injection [19; 85]. Intraperitoneal LPS at 100 jug/kg caused increased body temperature with a peak at about 5 hours [50]; this effect could be blocked with vagotomy [80], suggesting that the fever response is related to peritoneal inflammation. Most of the studies focussed on the role of NO in LPS-induced changes in body temperature have utilized systemic injections of NOS inhibitors; pyretic, antipyretic, or no effects have been described [59, 67, 60]. As systemic L-NAME may have its primary effects on LPS-induced changes in body temperature through its actions on brown fat thermogenesis [ 17], these earlier results may not be relevant to the role of brain NO in thermal regulation. On the other hand, we have used i.c.v. NOS inhibitors to show that central NO mediates the drop in body temperature which occurs 80 to 100 minutes after i.v. LPS (100 pg/kg) [85].
eNOS
.
PVN nNOS
iNOS
Anterior Pituitary
Adrenal glands Figure 1. Nitric oxide (NO) is produced from neuronal NO synthase (nNOS), endothelial NOS (eNOS), or inducible NOS (iNOS) within several cellular elements found in the paraventricular nucleus of the hypothalamus (PVN) including neurons (N), astrocytes (A), endothelial cells of blood vessels (B), and microglia (M). Most available data support the hypothesis that NO from nNOS and eNOS inhibits the hypothalamo-pituitary-adrenal axis as a feedback mechanism to limit chronic activation of the immune system.
182
4.
CONCLUSIONS
While it is clear that NO participates in communication between the immune system and the brain, the precise role(s) for NO in neuroimmune signaling appear(s) to depend on the relative amounts of NO released in the brain. Low to moderate levels of immune challenge stimulate NO production from nNOS and eNOS whereas more intense immune challenges stimulate production of larger amounts NO from iNOS. All three types of NO release occur within the PVN (Figure 1). Most of the data published to date support the hypothesis that NO from nNOS and/or eNOS inhibits activity of the HPA axis. NO from iNOS, on the other hand, has toxic or inflammatory effects within the brain which may or may not lead to tissue damage. Challenges to homeostasis by immune stressors lead to activation of the HPA axis as part of the "host defence response", and recovery of an animal from disease is dependent on the acute neural and hormonal activation of the immune system through this pathway. On the other hand, chronic stimulation of these potent systems is deleterious to the long-term survival of the animal, making a negative feedback mechanism necessary. The current knowledge about the central NO system in the brain leads to the hypothesis that the inhibitory effects of NO from nNOS and eNOS on the HPA axis are related to the feedback loop which returns the animal to homeostatic balance through central inhibition of the immune system.
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Ill.
NEUROIMMUNE MECHANISMS IN PHYSIOLOGY
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Introduction
REGINALD M. GORCZYNSKI
Department of Surgery & Immunology, University of Toronto, The Toronto Hospital, CCRW 2-855, 200 Elizabeth Street, Toronto, ON, Canada M5G-2C5
In this following section, the reader will find a series of papers in which authors have examined different physiological systems in order to uncover the mechanisms(s) whereby CNS control of function may be mediated. We have already encountered some preliminary thoughts in this area with the previous chapters on inflammation and CNS: immune system regulation (see for instance, Nance et al., (chapter II-7) and Merali et al., Rivest et al., (chapters II-8 and II-9 respectively). The section begins with a timely review from B ienenstock and colleagues of their elegant work, over nearly two decades, which has focused on the communication between mucosal mast cells and the nervous system. There follows the first in a number of discussion to be found in this volume of an issue many readers will find one of the most provocative and exciting in this field at the present time, namely the key role of molecules produced by the submandibular gland (SMG) in neuroimmunology. Much of this work stems from the laboratory of Befus in Edmonton. In this chapter, the first to introduce the concepts, Forsythe et al., characterize the structure, including the peptides produced by the SMG, and regulation of this gland by hormones (e.g. by androgens and the thyroid gland especially), documenting the integration of the gland within the context of the body's endocrine system. There is also evidence for autonomic system control of the SMG, which leads immediately to consideration of its role in immunoregulation, given the knowledge that surgical sympathectomy has long been favoured as a way to study CNS: immune system interactions. The authors discuss the role for the SMG in inflammation, and regulation of mast cell function, and proceed to characterize some of the peptides, and the active moieties thereof, which are implicated in these functions (see also Davison et al., (Chapter II-3)). They conclude by stressing that investigations of this gland "have the potential to provide valuable insights into the mechanisms underlying psychological and neuroendocrine control of the immune system". This chapter is followed, appropriately, by a discussion by Sabbadini et al., on the evidence for the importance of another salivary gland peptide, kallikrein, in CNS-mediated immunoregulation. This molecule, a member of the serine protease family of enzymes, was active in modifying lymphocyte proliferation in vitro, and suppressing a variety of immune responses in vivo, including allograft rejection and contact hypersensitivity reactions-in all cases, activity was dependent upon enzyme activity being retained. These data suggest that there is a crucial substrate, in vivo, which when acted upon by kallikrein, releases immunoregulatory peptides. Even more provocative is the data from this group concerning the potential role for kallikrein in oral tolerance induction. Arguing that its high levels in saliva might have a physiological
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role, the authors tested the effect of removal of the salivary gland on oral tolerance induction to collagen in rats. No tolerance developed, but oral replacement of kallikrein fully restored tolerance induction. The target molecules for these effects remain unknown. The two following chapters represent somewhat of a recursion to more phenomenological studies of CNS: immune interactions, though not wholely so. Gorczynski attempts to provide some logic, and even molecular understanding, of studies which have documented so-called "classical conditioning" of immune responses. There are a number of such experiments in the literature, all of which might lead the reader to believe that in principle, any immune response system might be "brought under" the control of the CNS (by biobehavioural techniques). If this is the case, Gorczynski asks, what might be happening within the CNS? Taking production of inflammatory cytokines (TNFet and IL-1) in response to LPS (or its perceived presence, in conditioned mice) as a measuring system, and using monoclonal antibodies and quantitative PCR technology as a measuring instrument, Gorczynski argues that both peripherally, and in the CNS, the same two peptides, somatostatin and substance P, play important regulatory roles. This can be seen as supporting of those studies, which have documented redundancy in the mediators used within the CNS and immune system (see the previous section of this book). Nevertheless their relative importance in the conditioned mice is quite different from that seen in mice where immunity is induced by conventional immunological tools. This discussion of TNFc~ and IL-1 induction in the CNS is timely given the thrust of the next chapter, by Moldofsky and colleagues, concerning an investigation of the role of sleep, and perturbations of it, in general health and immunocompetence. It has become increasingly evident that disruption of sleep has profound consequences to the function of the immune system as a whole, in addition to its effects on other physiological systems. As an example, sleep deprived rats die from systemic sepsis. Furthermore, data from Krueger and co-workers over the last decade have clearly established a role for TNFc~ and IL-1 production, within the CNS itself, in regulation of sleep induction. The potential communication between such cytokines and the CNS/immune system, and the additional role of the HPA axis and neuroendocrine circuitry, in sleep physiology, offers an important avenue of approach for those interested in manipulating those disruptions in sleep which have been implicated in human pathology and disease. Few areas of contemporary research are likely to provoke as much interest to the lay and scientific community as those in which the subject matter involves the genital organs, or obesity. These topics are introduced by our last two chapters in this section. Pomerantz lays to rest the notion that all physiological regulation of the testis can be considered in terms of the HPA axis, and instead forces us to consider the importance of the nervous system, the immune system and even a number of intragonadal factors. As an example, a number of groups have reported on peptides contained in Sertoli-cell-conditioned-medium with immunoregulatory properties; the expression of FasL by Sertoli cells in rats has been implicated as the critical factor explaining (in this species) the immunoprivilige accorded to this site for tumor growth. And finally, Pomerantz's group has also documented an important role for iNOS induction and NO production by Sertoli cells (like immune macrophages) in response to inflammatory stimuli, which can in turn decrease androgen production by neighbouring Leydig cells. Luheshi concludes this section with a discussion of the interactions between leptin and cytokines in both fever and control of obesity. Leptin was believed to be produced predominantly by adipocytes in relation to body mass, to gain access to the brain via a saturable transport mechanism, and to act on hypothalamic receptors to suppress appetite. IL-1 (in the periphery) is implicated in leptin production, while secondary mediators of leptin action in the CNS include members of the pro-opiomelanocortin family, CRF and neuropeptide-Y. Most interestingly, however, Luheshi suggests that leptin can induce IL-1 in the hypothalamus, and this in turn leads
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him to speculate that actions of leptin on food intake might be directly related to production and action of IL-1 within the CNS. In addition, doses of leptin which induce appetite suppression are pyrogenic (a known effect of IL-1 within the CNS), suggestive of widespread ramifications for leptin as a mediator of neuroimmune regulation in human disease.
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Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
A Model of Neuroimmune Communication Mast Cells and Nerves
JOHN BIENENSTOCK
Departments of Medicine, Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario
ABSTRACT Communication between mast cells and nerves is perhaps the best current example of neuroimmune interaction. This occurs in a bidirectional fashion and has been demonstrated in a variety of animal and human tissues in vitro, in vivo and ex vivo. Examples of this interaction and its importance have been produced in experimental models as well as in human diseases, and the unit of mast cell-nerve is thought to play an important homeostatic role in several tissues such as skin, lung, intestine and urinary bladder. The unit is involved in complex interactions which include psychological stress in which corticotropin releasing factor (CRF) plays a role. Nerve growth factor (NGF), a member of the neurotrophin family, which is synthesized by many cell types, both structural and inflammatory, also plays an important role in maintaining the physiological state, and in addition is involved in as yet unknown fashion in inflammation and disease. NGF has an enormously pleiotropic activity and can be both pro and antiinflammatory. Since NGF is synthesized by mast cells and nerves, and because of its neuronal and non-neuronal effects, it plays a significant role in the maintenance of health, and an as yet unknown role in disease.
1.
INTRODUCTION
The purpose of this review is to outline some of the interactions between the nervous and immune systems as seen through the eyes of myself and my many colleagues who have helped me in my research over the years. This short review is far from exhaustive, but nevertheless serves to highlight some of the experimental evidence which supports Blalock's general thesis of bidirectional communication between the nervous and immune systems [ 1].
2.
NERVE-MAST CELL COMMUNICATION
While there had been, in the literature, some previous references to occasional connections between mast cells and nerves, we carried out the first detailed morphometric study in this area [2].
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We showed that mast cells and nerves were commonly in association in the small intestinal villi of rats. These nerves contained substance P (SP) and calcitonin gene related peptide (CGRP). The analysis showed that the association was not by chance alone and therefore was purposeful. In many subsequent studies, others have shown this association to hold true in tissues from different animal species and various body sites which include small and large bowel, mesentery, portal tracts of the liver, skin, lung, blood vessels, urinary bladder, etc. [3, 4]. That this anatomical association had some functional attributes was shown by Baird and Cuthbert in guinea pigs sensitized to lactoglobulin [5]. In an in vitro system, antigen was shown to have a pronounced physiological affect on the epithelium. These observations have been extended enormously since that time mainly through the use of the Ussing chamber. Thus, in mouse, rat, guinea pig, human and primate tissues a number of investigators have shown that antigen is capable of causing short circuit current changes whether placed on the luminal or serosal side of the tissue, using pharmacological approaches [6-9]. In this model, antigen causes chloride and water secretion by the epithelium as a result of interaction between mast cells and nerves. Various mediators derived from mast cells have been shown to be involved in whole or in part, and these include histamine and serotonin. The process can be inhibited by atropine and substance P antagonists. Thus, an axon reflex is thought to be initiated in both intestine and lung [10] which involves antigen, sensitized mast cells, local nerves and target tissue. Mast cells are an obligatory component of this reaction, as shown by Perdue et al., using the mast cell deficient w/w v strain [7]. Conclusive evidence followed from studies of the repopulation of these animals with mast cells derived from bone marrow culture, which restored the response to antigen in Ussing chambers to its former level. Cooke and co-workers [6] showed in guinea pig intestine, again using the Ussing chamber, that in exactly the same time course as the development of a short circuit current response to antigen, acetylcholine was released, indicating that antigen-dependent neurotransmitter release had occurred. We have performed co-culture using sympathetic superior cervical ganglion neurons which change their phenotype in the culture conditions used, to those of cholinergic neurones. Mast cells (both peritoneal and rat basophil leukemic cells, an exemplar of mucosal mast cells) have been shown to purposefully associate with such nerves in culture, with both trophic and tropic effects [11-14]. There are electrical consequences to mast cell association with nerves, once contact has been made. The contacts have been maintained for 72 hours in vitro and associated mast cells matured, increased their granulation and released more mediator on challenge. In the associated nerve, the appearance of dense core vesicles as well as their accumulation, again suggested a meaningful and significant association. The contacts between the neuron and mast cell were intimate (less than 20 nm). However no specialized synaptic structural changes occurred either in the associated neurone or mast cell. We have used this model to further study communication between mast cells and nerves and have co-cultured RBL-2H3 with superior cervical ganglion neurites which, in the culture conditions employed, synthesize and release substance P (SP). Cells were loaded with the calcium fluorphore fluo-3 and examined by confocal laser scanning microscopy [15]. Scorpion venom and bradykinin, in a dose response manner showed calcium to increase in associated neurones invariably before it increased in the associated RBL cell. Neither scorpion venom or bradykinin had any effect on RBL alone. The most important communication seemed to occur via SP since a neutralizing antibody to it as well as an NK-1 receptor antagonist (CP90-994-1) but not an NK2 antagonist (SR48968), interrupted this direct neurite mast cell communication. An antibody to the IgE receptor caused an appropriate lag response with an increase in calcium in the associated neurite. Previous work by Cooke had shown the presence of NK-1 receptors on RBL [16].
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While substance P has generally been shown to have some physiological effect on mast cells, its role has been questioned since the concentrations required to degranulate the mast cell are relatively high, i.e., 10 -5 M. We have shown in patch clamp studies of mast cells and RBL cells that stimulation with very low concentrations of substance P (picomolar) have been able to promote whole cell current oscillations and even degranulation after a very significant and prolonged lag period [17]. The lag period was proportionate to the concentrations used, i.e., >25 minutes in the case of repeated 5 pM concentrations of substance P. Similar results were obtained by initial priming of mast cells with 5 pM substance P followed by subthreshold amounts of anti-IgE. These experiments revealed that the effects described were not just in vitro artifacts with neuropeptides, but had some biological and physiological meaning.
3.
NERVE GROWTH FACTOR
Nerve growth factor is involved in neuroimmune interactions in a variety of ways and I will review some of these briefly. N G F is found in secretions, especially those in the male submandibular gland but probably in all secretions [18]. It is essential for the growth, survival and differentiation of sympathetic sensory afferent and some cortical neurons. The predominant message for its up regulation is IL-1. It is made by many different cell types which include structural cells in the nervous system, a variety of immune cells which include mast cells [19], lymphocytes (especially TH2) [20], and eosinophils [21]. N G F has been shown to be synthesized by fibroblasts, keratinocytes [22] as well as constitutively by various epithelial cells.The effects of nerve growth factor are pleiotropic (Table I) and they range from synergy with G M - C S F and IL-5 in terms of promotion of human stem cell colony growth [23, 24], to the priming of basophils and mast cells for subsequent degranulation by mast cell secretogogues [25, 26]. In these latter experiments N G F was shown to be a significant molecule involved in priming of cells for subsequent agonist activity in the formation and release of leukotrienes and histamine. In addition, N G F has an effect on B-cells [27], promotes synthesis by human B-cells of IgG4 [28], promotes wound healing [29], repair of human corneal ulceration [30], and prevents mast cell, eosinophil and neutrophil apoptosis [31-33]. Table I
9 9 9 9 9 ~ 9 9
Non-neuronaleffects of nerve growth factor.
Promoteswound healing, [58], prevents carrageenin-induced inflammation [61] Heals corneal ulcers [75] Promotesgrowth, differentiation, proliferation and survival of B cells [27, 51]. EnhancesIgG4 production [28]. Prevents apoptosis in mast cells, eosinophils and neutrophils [31-33]. Strongmastopoietic effect in rodents, partly through mast cell degranulation [34, 52]. Primes basophils and mast cells for agonist action (e.g., C5a) [25, 26]. Enhances human haematopoietic colony growth of basophils and eosinophils; synergistic with GM-CSF and IL-5 [23, 24].
N G F has been shown to be one of the most potent mastopoietic substances known. Injections of small amounts of NGF, especially in the neonatal period, promote significant increases in
198
mast cells in peripheral tissues [34]. The effects are in part due to mast cell degranulation, since treatment of animals with disodium cromoglycate which stabilizes connective tissue mast cells, prevented this increase, while leaving unaffected the increase in mucosal mast cells. Interestingly, NGF causes upregulation of substance P message and synthesis in autonomic ganglia [35] and causes airway hyper-reactivity in a variety of experimental models. These include transgenic mice which over-express NGF in the lung [36]. In human nasal secretions NGF has been shown to be released upon specific antigen challenge in perennial rhinitis patients [37] and surprisingly, antibody to NGF to abrogate at least one model of experimental asthma and the brochial hyperreactivity which occurs in it [38]. We have shown that NGF causes an increase in IL-6 production by rat peritoneal mast cells at the same time as it inhibits TNFc~ synthesis and release [39]. This effect appears to be mediated via PGE2 through autocoid release since indomethacin treated peritoneal mast cells failed to respond in the fashion described. The response was restored by the addition of PGE2 to indomethacin treated mast cells. These results offer a possible explanation for why NGF may have protective (anti-inflammatory) activities (Table II) since the inhibition of TNF~t synthesis and secretion would be expected to have decidedly anti-inflammatory effects through inhibition of its known pro-inflammatory biological role such as the promotion of neutrophil influx into inflammatory sites. It is possible that this may also be responsible for the reduced inflammatory response of animals treated with NGF in the experimental model of autoimmune encephalomyelitis [40]. Table II
Protective effects of nerve growth factor.
9 Promotes wound healing [29]. 9 Prevents carrageenan inflammation [53]. 9 Repairs human corneal ulceration [30]. 9 Inhibits intestinal inflammation in hapten induced colitis [50]. 9 Inhibits autoimmune encephalomyelitis [40].
One of the many examples of neuroimmune interactions which appears to involve the mast cell-nerve physiological unit is that of stress. It has been well established now that stress effects cause dura mater intracerebral mast cell degranulation through the release and direct action of corticotrophin releasing factor (CRF) [41 ]. This molecule is essential for the signal to the pituitary gland to synthesize and release ACTH, and subsequently influence the up regulation and discharge of cortisone from the adrenal cortex. CRF causes mast cell degranulation in vivo and in vitro [41-43]. Both acute and chronic stress causes mast cell activation and degranulation in intestinal tissue and the effect on mucus discharge by goblet cells, as well as the changes in motility and increase in chloride ion secretion by the epithelium appear all to be mediated through this interaction [43-46]. The intestinal effects of stress can be interrupted by pharmacological blockade of a variety of autonomic and ganglionic nerves through the use of bretylium, atropine and hexamethonium [47]. The whole process can also be interrupted by NK1 antagonists which block the effect of substance P, and also by blockade of neurotensin [43, 45]. Finally, the intracerebral or peripheral injection of CRF can mimic most of the effects of stress on the intestine [43]. These complex interactions affect epithelial function, barrier integrity and smooth muscle motility and involve mast cells and nerves. We wished to test
199
whether NGF was involved in any of these activities and chose to look at epithelial function using the Ussing chamber as a read-out. In brief, the injection of anti-NGF before and at the time of acute stress caused a significant change (increase) of short circuit current in the colon of Wistar Kyoto animals. This preliminary observation suggests a role for NGF in stabilizing or protecting the epithelium and may likely fit the pattern of protection and anti-inflammatory effects which were alluded to earlier. Most recently, we have examined intestinal epithelial cells for their constitutive production of NGF. We have shown that human intestinal epithelial T84 cells have a low constitutive NGF expression, but message for NGF is markedly up regulated if the cells are incubated for 1 hour with physiologic concentrations of human recombinant IL-10. Furthermore, the message is translated and T84 cells synthesize NGF protein. This action of IL-10 is reciprocated since 10 or 100 ng of NGF markedly and selectively up regulated IL-10 synthesis, and again, IL-10 protein was increased in the cells subsequently. Thus, there appears to be a reciprocal autocoid up regulation of IL-10 by NGF, and in turn of NGF by IL-10 in this intestinal epithelial cell line. These actions appear to be very selective since no effects by these molecules on other cytokines have been seen with respect to TGF[3, IL-6, IL-8 or themselves in the test systems employed. Interestingly, Brodie et al., [48] have shown that IL-10 similarly causes astrocytes to increase their synthesis of NGF. These experiments promote further support for the protective effects of NGF in addition to giving an additional emphasis to the possible role of IL-10 in providing protection to the intestine. It has been known for some time that transgenic IL-10 knockout mice develop spontaneously a form of colitis that is caused by conventional intestinal flora. Colonization of IL-10 KO mice with lactococci which synthesize IL-10 prevents the colitis [49]. Furthermore, in a model of hapten induced colitis, treatment of mice with anti-NGF strikingly increased the inflammation induced by challenge with the hapten, confirming the likely protective effect of NGF in this model [50]. The whole way in which the brain and the nervous system interact, regulate and in turn are regulated by the cells of the immune and inflammatory systems is being slowly defined. Perhaps the best example is one given in terms of stress where even injection of CRF into the brain of experimental rats can mimic the peripheral effects of stress in animals. It can reasonably be expected that we will begin to have the same sense of molecular events and an understanding of them in many, if not all, physiologic and disease conditions and in this manner, may manage to offer new alternative therapeutic possibilities where they are needed.
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Bischoff SC, Dahinden CA. c-kit Ligand: A Unique Potentiator of Mediator Release by Human Lung Mast Cells. J Exp Med 1992; 175: 237-244. B ischoff SC, Dahinden CA. Effect of nerve growth factor on the release of inflammatory mediators by mature human basophils. Blood 1992; 79: 2662-2669. Torcia M, Bracci-Laudiero L, Lucibello M, Nencioni L, Labardi D, Rubartelli A, Cozzolino F, Aloe L, Garaci E. Nerve growth factor is an autocrine survival factor for memory B lymphocytes. Cell 1996; 85: 345-356. Kimata H, Yoshida A, Ishioka C, Kusunoki T, Hosoi S, Mikawa H. Nerve growth factor specifically induces human IgG4 production. Eur J Immunol 1991; 21: 137-141. Matsuda H, Koyama H, Sato H, Sawada J, Itakura A, Tanaka A, Matsumoto M, Konno K, Ushio H, Matsuda K. Role of nerve growth factor in cutaneous wound healing: accelerating effects in normal and healing-impaired diabetic mice. J Exp Med 1998; 187: 297-306. Lambiase A, Rama P, Bonini S, Caprioglio G, Aloe L. Topical treatment with nerve growth factor for corneal neurotrophic ulcers [see comments]. N Engl J Med 1998; 338: 1174-1180. Kawamoto K, Okada T, Kannan Y, Ushio H, Matsumoto M, Matsuda H. Nerve growth factor prevents apoptosis of rat peritoneal mast cells through the trk proto-oncogene receptor. Blood 1995; 86: 4638-4644. Kannan Y, Usami K, Okada M, Shimizu S, Matsuda H. Nerve growth factor suppresses apoptosis of murine neutrophils. Biochem Biophys Res Commun 1992; 186: 1050-1056. Hamada A, Watanabe N, Ohtomo H, Matsuda H. Nerve growth factor enhances survival and cytotoxic activity of human eosinophils. Br J Haematol 1996; 93: 299-302. Marshall JS, Stead RH, McSharry C, Nielsen L, Bienenstock J. The role of mast cell degranulation products in mast cell hyperplasia. I. Mechanism of action of nerve growth factor. J Immunol 1990; 144:1886-1892. Hart RP, Shadiack AM, Jonakait GM. Substance P gene expression is regulated by interleukin- 1 in cultured sympathetic ganglia. J Neurosci Res 1991; 29:282-291. Hoyle GW, Graham RM, Finkelstein JB, Nguyen KP, Gozal D, Friedman M. Hyperinnervation of the airways in transgenic mice overexpressing nerve growth factor. Am J Respir Cell Mol Biol 1998; 18: 149-157. Sanico AM, Stanisz AM, Gleeson TD, Bora S, Proud D, Bienenstock J, Koliatsos VE, Togias A. Nerve growth factor expression and release in allergic inflammatory disease of the upper airways. Am J Respir Crit Care Med 2000; 161: 1631-1635. Braun A, Appel E, Baruch R, Herz U, Botchkarev V, Paus R, Brodie C, Renz H. Role of nerve growth factor in a mouse model of allergic airway inflammation and asthma. Eur J Immunol 1998; 28:3240-3251. Marshall JS, Gomi K, Blennerhassett MG, Bienenstock J. Nerve growth factor modifies the expression of inflammatory cytokines by mast cells via a prostanoid-dependent mechanism. J Immunol 1999; 162: 4271-4276. Villoslada P, Hauser SL, Bartke I, Unger J, Heald N, Rosenberg D, Cheung SW, Mobley WC, Fisher S, Genain CP. Human nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system [see comments]. J Exp Med 2000; 191: 1799-1806. Theoharides TC, Spanos C, Pang X, Alferes L, Ligris K, Letourneau R, Rozniecki JJ, Webster E, Chrousos GP. Stress-induced intracranial mast cell degranulation: a corticotropin-releasing hormone-mediated effect. Endocrinology 1995; 136: 5745-5750.
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Theoharides TC, Singh LK, Boucher W, Pang W, Letourneau R, Webster E, Chrousos G. Corticotropin-releasing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects. Endocrinology 1998; 139: 403-413. Castagliuolo I, LaMont JT, Qiu B, Fleming SM, Bhaskar KR, Nikulasson ST, Kornetsky C, Pothoulakis C. Acute stress causes mucin release from rat colon: role of corticotropin releasing factor and mast cells. Am J Physiol 1996; 271: G884-G892. Castagliuolo I, Wershil B K, Karalis K, Pasha A, Nikulasson ST, Pothoulakis C. Colonic mucin release in response to immobilization stress is mast cell dependent. Am J Physiol 1998; 274:G1094-G1100. Castagliuolo I, Leeman SE, Bartolak-Suki E, Nikulasson S, Qiu B, Carraway RE, Pothoulakis C. A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats. Proc Natl Acad Sci USA 1996; 93: 12611-12615. Santos J, Benjamin M, Yang PC, Prior T, Perdue MH. Chronic stress impairs rat growth and jejunal epithelial barrier function: role of mast cells. Am J Gastrointest. Liver Physiol 2000; 278: G847-G854. Santos J, Saunders PR, Hanssen NP, Yang PC, Yates D, Groot JA, Perdue MH. Corticotropin-releasing hormone mimics stress-induced colonic epithelial pathophysiology in the rat. Am J Physiol 1999; 277:G391-G399. Brodie C. Differential effects of Thl and Th2 derived cytokines on NGF synthesis by mouse astrocytes. FEBS Lett 1996; 394:117-120. Madsen KL, Doyle JS, Jewell LD, Tavernini MM, Fedorak RN. Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice [see comments]. Gastroenterology 1999; 116:1107-1114. Reinshagen M, Rohm H, Steinkamp M, Lieb K, Geerling I, Von Herbay A, Flamig G, Eysselein VE, Adler G. Protective role of neurotrophins in experimental inflammation of the rat gut. Gastroenterology 2000; 119: 368-376. Otten U, Ehrhard P, Peck R. Nerve growth factor induces growth and differentiation of human B lymphocytes. Proc Natl Acad Sci USA 1989; 86: 10059-10063. Aloe L, Levi-Montalcini R. Mast cells increase in tissues of neonatal rats injected with the nerve growth factor. Brain Res 1977; 133: 358-366. Banks BE, Vernon CA, Warner JA. Nerve growth factor has anti-inflammatory activity in the rat hindpaw oedema test. Neurosci Lett 1984; 47: 41-45.
New Foundationof Biology
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Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
Immunomodulation by the Submandibular Gland
PAUL FORSYTHE 1, RENE E. DIARY1, RONALD MATHISON 2, JOSEPH S. DAVISON 2 and A. DEAN BEFUS 1
1pulmonary Research Group, Department of Medicine, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada, T6G 2S2 2Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada, T2N-4N1
ABSTRACT We have established that decentralization (cutting sympathetic nerve trunk) of the superior cervical ganglia bilaterally reduces the magnitude of allergic inflammation in the airways of rats. The magnitude of anaphylactic and endotoxic hypotension, and of gastrointestinal inflammation was also reduced. This anti-inflammatory activity was dependent upon intact submandibular glands. Reconstitution of sialadenectomized (removal of submandibular glands) rats with soluble extracts of the submandibular glands identified two polypeptides with antiinflammatory activities. The sequences of these polypeptides were found within a prohormone, submandibular gland rat 1 (SMR1). The C-terminal peptide TDIFEGG, has been studied extensively. Using sequential amino acid substitutions and systematic removal of C terminal or N terminal amino acids, we established that the tripeptide FEG is biologically active. Modification of FEG to the D-isomeric feG, enhances its activity in some assay systems. We postulated that feG would inhibit airways inflammation, and tested this using a model of allergic asthma, namely the Brown Norway rat sensitized to ovalbumin (OA). Sensitized rats were challenged 14 to 21 days later with aerosolized OA. This challenge markedly increased numbers of inflammatory cells recovered from the airways after 24 hour (29 x 106, n = 23) compared to saline controls (1 x 106, n = 4). The infiltrating cells included macrophages (10 x 106), neutrophils (9 x 106) and eosinophils (9 x 106). Intravenous (0.25 mg/kg) or oral feG (1 mg/kg) given 30 min prior to OA significantly inhibited influx of inflammatory cells by 50 to 70%. feG reduced inflammatory cell infiltration when given 30 min before to 3 to 6 hour post allergen exposure. Oral feG reduced the numbers of macrophages, neutrophils and eosinophils. One of the mechanisms underlying the effects of feG may be its ability to inhibit PAF-induced expression of CD1 l b on purified human neutrophils. It is possible that feG may be useful in the treatment of allergic asthma, given either as an oral prophylactic, or as a post exposure therapeutic.
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The cervical sympathetic nerve trunk-submandibular gland axis of neuroendocrine regulation of inflammation may be dysfunctional in inflammatory diseases and provide opportunities for new therapeutic intervention. This axis may be sensitive to modulation by central and peripheral neural mechanisms that influence its function/dysfunction. Supported by MRC, Salpep Biotechnology Inc and Heart & Stroke Fdn of Canada.
1.
INTRODUCTION
To most, the salivary glands are simply exocrine organs concerned with aiding digestion and maintaining oral health. This view is held despite the fact that endocrine secretion from salivary glands was first reported almost 50 years ago. Since then it has emerged that salivary gland factors aid in the maintenance and integrity of the esophageal and gastrointestinal mucosa, promote hepatic regeneration and mammary gland tumorigenesis, are essential for the maintenance of the reproductive system and have regulatory effects on the immune system. Similarily, while regulation of salivary gland function by the autonomic nervous system has been well known since Pavlov's classical experiments, the significance of the cervical sympathetic trunk-submandibular gland (CST-SMG) axis as an effector of neuroendocrine-immune regulation has only recently come to light [1]. What follows is a description of the immunoregulatory activities of the submandibular gland (SMG), the endocrine factors responsible for these effects and mechanisms that control the production and release of such factors. The potential implications of the CST-SMG axis in neuroendocrine immunology and psychoneuroimmunology are also discussed.
2.
STRUCTURE OF THE SUBMANDIBULAR GLAND
Structurally the SMG comprises four major epithelial compartments: acinar cells, intercalated ducts, granular convoluted tubule (GCT) cells and striated excretory ducts. The acini are connected to each other by intercalated ducts that lead to the GCT, which in turn join into the striated secretory ducts [2] (Figure 1). The acinar cells secrete the important digestive enzyme amylase, and produce saliva. The intercalated duct cells include stem cells that produce acinar and GCT cells during development. The cells of the striated excretory ducts regulate water content and ionic composition of saliva. GCT cells represent a major source of bioactive polypeptides produced in the SMG [3]. The potential importance of GCT cells in homeostatic mechanisms is reflected in the number of hormonal systems that exert control over their development and content of biologically active polypeptides.
3.
BIOLOGICALLY ACTIVE POLYPEPTIDES
A large number of biologically active polypeptides have been identified in the SMG many of which have been localized to the GCT [3, 4]. These factors, released as both exocrine and endocrine agents, can be classified into three groups: a) growth factors such as nerve growth factor (NGF), epidermal growth factor (EGF) and transforming growth factor-J3 (TGF-[3), b) processing enzymes such as kallikrein-like proteinases and renin and c) regulatory peptides including glucagon, insulin, erythropoietin, somatostatin, angiotensin II,
205
Acinar cells Intercalated ducts
i
Granulated Convoluted tubule
i
1
L
o 0
0 0
0
0
0
~
o o _o
0
0
o
0 010 10101010101 101010101011
1010101010101q 01010101010101
Striated excretory duct
Figure 1. A schematic representation of the relationship between the four major epithelial compartments of the submandibular gland.
vasoactive intestinal peptide and neuropeptide Y. In the following sections a number of wellcharacterized SMG derived peptides with biological functions relevant to immunoregulation will be described. 3.1.
Nerve growth factor
Nerve growth factor was first described in the mouse SMG and identified as a neurotrophic agent. Subsequent investigations have revealed properties of NGF that suggest it may play a role in immunoregulation [5, 6]. NGF increases the number and size of mast cells in tissues of neonatal mice [7], and stimulates histamine release from mast cells, both in vivo and in vitro [8-10]. NGF also increases phagocytosis and chemotaxis of neutrophils, promotes the development of hemopoietic colonies and stimulates lymphocyte growth in vitro [11-15]. Other effects include the up-regulation of IgM and IgG 4 production by human B cells [ 16, 17]. NGF may also aid in neuronal control of the immune system as it stimulates the growth of sympathetic ganglia that innervate immune organs [5]. While these effects suggest a proinflammatory role for NGF the mediator has been shown to suppress in vivo inflammatory reactions in several models [ 14]. The reason for this apparent paradox is, as yet, unknown. 3.2.
Transforming growth factor [5
Transforming growth factor [5 (TGF-[3) is a multifunctional regulator of cell growth and differentiation in a wide variety of normal and neoplastic systems [18]. A homodimeric
206
polypeptide originally purified from human platelets, TGF-[5 shares structural and functional homology with epidermal growth factor (EGF). These factors share a receptor [19]. Binding of TGF-[5 or EGF to the 170 kDa plasma membrane receptor on the target cell stimulates intrinsic tyrosine kinase activity. The subsequent phosphorylation cascade leads to increased proliferation and differentiation of skin tissues, corneal epithelium, lung and tracheal epithelium [20-22]. TGF-[5 exerts a range of effects on inflammatory and immune responses, acting as a chemoattractant for monocytes, neutrophils and lymphocytes and activating monocytes to secrete cytokines and growth factors [23-25]. TGF-[5 is a stimulatory factor in the early stages of inflammation but later supports its resolution and contributes to healing. CD8+ T cells are stimulated, while activated CD4+ T cells are suppressed [26]. B cell proliferation and the production of IgG and IgM are also suppressed [27]. 3.3.
Epidermal growth factor
One of the most widely studied aspects of SMG function is the exocrine release of epidermal growth factor (EGF) and its regenerative and reparative properties on oral, oesophageal and gastric mucosa. The importance of EGF in tissue repair is emphasized by its diverse effects that include gastric epithelial cell division, DNA synthesis, collagen synthesis, matrix deposition, neovascularization and stimulation of protein and hyaluronic acid synthesis in epithelial cells [28]. Through these multiple activities SMG-derived EGF promotes the healing of gastric ulcers and tongue lesions and maintains the integrity of oesophageal mucosa [29, 30]. EGF can also be released into the blood stream and its actions extend beyond the oral and gastrointestinal mucosa to other organs. EGF enhances liver regeneration after partial hepatectomy and is important in maintaining uterine growth and fertility [31, 32]. Immunoregulatory functions of EGF include stimulation of T cell proliferation, up-regulation of interferon-~, production and reduction of T suppressor cell activity [33-35]. EGF also stimulates macrophage chemotaxis and phagocytosis [36, 37]. These actions suggest that EGF stimulates the immune system and is proinflammatory. However, as with NGF, immunosuppressive activity of EGF has been reported [38]. 3.4.
Proteases and convertases
The polypeptides produced by the SMG are synthesized as inactive precursors that have the potential to become active following proteolytic processing. The submandibular glands contain an array of enzymes capable of hydrolysing peptide bonds including members of the prohormone convertase and kallikrein families [3, 39]. These proteases act to modulate the biological activities of growth factors and regulatory peptides. In addition to being vital for the production of biologically active polypeptides it is also apparent that at least one family of proteases found in the SMG may be directly involved in the modulation of immune responses. These enzymes, the kallikreins, will be considered here in more detail. 3.5.
Kallikreins
The SMG of the rat contains large amounts of kallikrein, a serine protease with the ability to release the vasodilatory peptide kallidin (Lys-bradykinin) from the plasma protein kininogen. Kallikrein is secreted into the saliva and blood. In the mouse, 14 genes have been confirmed to have the potential to encode functional kallikrein proteins. At least three of these are EGF binding proteins and can cleave EGF to its active form [40].
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The rat SMG expresses at least 6 kallikrein genes [41]. Glandular or tissue kallikreins differ in molecular weight and enzymatic activity from plasma kallikreins. Mouse glandular kallikrein strongly enhances spontaneous and mitogen induced proliferation of lymphocytes [42]. This function is independent of EGF as non-EGF binding forms of kallikrein also exhibit this activity. Serine proteinase inhibitors can block the response suggesting it is related to enzymatic activity of kallikreins. This is in accordance with reports that trypsin, chymotrypsin, thrombin and other proteinases mitogenically stimulate many kinds of cells including lymphocytes. In vitro the addition of kallikrein and other serine proteases to B cells stimulated with LPS and IL-4 enhances the production of IgE, IgG 1 and IgG 3 [43]. Rat glandular kallikrein has also been reported to suppress the DTH response to picryl chloride in mice [44].
4.
HORMONAL CONTROL OF THE SMG
It has been clearly demonstrated that androgens, thyroid and adrenocortical hormones are necessary for the normal development of the SMG and for the production of biologically active polypeptides [45-47]. Perhaps the most striking aspect of hormonal control of the SMG is the sexual dimorphism of the organ. This is reflected in the androgen-induced increase in the number of GCT cells and content of biologically active peptides [4]. Assays have revealed much greater concentrations of many polypeptides (e.g. EGF, NGF, kallikrein and renin) in the glands of male compared to female rodents [48-50]. The levels of these factors are androgen dependent. The NGF content of the SMG is reduced by castration and increased by administration of testosterone to castrated male or female animals. NGF levels also increase during pregnancy and lactation [51]. Similar observations have been made regarding EGF levels in the SMG. The gland of the female contains about 1/10 the EGF detected in the male. Administration of testosterone to both male and female animals leads to complete reversal of the drastic reduction in EGF levels observed following removal of the pituitary gland (hypophysectomy) [52]. Androgen receptors have been detected in mouse and rat SMG. Specific androgen binding is higher in homogenates of female SMG compared to males in both rats and mice [53, 54] despite the fact that glands of male mice contain three times as much total androgen receptor capacity as those of females. The reason for this apparent conflict was determined by Kyakamoto et al. They showed that in males 74% of receptor capacity was in nuclei (occupied) while in females 94% was in the cytosol (unoccupied). Castration results in a female distribution of the receptor that can be reversed by testosterone administration [53]. There is a significant decrease in plasma lutenizing hormone (LH) levels following sialectomy but this procedure does not significantly modify hypophyseal LH content [55]. The decrease in plasma LH causes changes in Leydig cells and reduces testosterone production. This observation indicates that while testosterone plays a crucial role in maintenance of the SMG there is also a feedback relationship between the SMG and the testis via hypophyseal LH secretion. Several studies have established that in addition to androgens, thyroid hormones are important in the development and maintenance of the GCT cells [4]. Aloe and Levi-Montalcini reported that thyroxine caused a precocious differentiation of cells [56]. Thyroid hormones have also been demonstrated to increase the levels of NGF and EGF in the SMG of the adult female mouse [57-61]. The SMG of Tfm/Y mice lacks specific androgen binding capacity [59] and subsequently these mice are deficient in NGF and EGF [62]. Administration of thyroxine but not testosterone greatly increases the level of both growth factors in the SMG of Tfm/Y mice. Thyroxine also restores EGF levels to normal in hypothyroid animals.
208
Hypophysectomy induces marked atrophy of the SMG and reduces both in vitro lymphocytestimulating activity and in vivo immunosuppressive activity. The important role of thyroid hormones and androgens in maintaining SMG function is further emphasized by the fact that thyroid stimulating hormone (TSH) and LH restore the ability of the SMG to modulate lymphocytes in hypophysectomized rats. Prolactin was also required for full restoration [63]. Feedback between the SMG and the hypothalamus-pituitary-adrenal (HPA) axis has been suggested, based on observations that NGF and EGF are able to stimulate release of ACTH and glucocorticoids [64, 65] while, in contrast, TGF-13 depresses acetylcholine induced CRH release from the hypothalamus [66]. These demonstrations of SMG dependence on pituitary function and the feedback relationships with other glands indicate that the SMG is a fully integrated component of the body's endocrine system (Figure 2). Stimulation -- i
- Inhibition
~hypo~t~alamu.q~ Cot,..
Exogenous LH
Immunomodulation
Exogenous TSH
Figure 2. The SMG as a fully integrated component of the endocrine system (see text for details).
5.
AUTONOMIC CONTROL OF THE SMG
In addition to endocrine control, the autonomic nervous system also participates in homeostatic responses to inflammation through the regulation of cardiovascular function and stimulation of release of certain stress hormones such as glucagon and renin. Given the role of the SMG in controlling inflammation it is not surprising that it is also under the control of the autonomic nervous system (ANS). It has been known for some time that the size of SMG is influenced by the ANS and that in adult rats prolonged electrical stimulation of the sympathetic branch of the ANS via the superior cervical ganglion (SCG) causes enlargement of SMG by increasing
209
both cell size and number [67]. Similarily the [3-adrenoreceptor agonist isoprenaline (IPR) induces hypertrophic and hyperplastic enlargements of rodent SMG and induces the expression of a number of genes. The SMG receives both sympathetic and parasympathetic innervation. Both branches regulate the volume and composition of saliva. The parasympathetic system stimulates the glandular acinus leading to the secretion of large volumes of saliva with low concentrations of biologically active polypeptides. [3-adrenergic stimulation increases the synthesis and release of these polypeptides, c~-adrenergic agents stimulate the secretion of growth factors and homeostatic proteases by the GCT cells and lead to the appearance of large amounts of kallikrein NGF, EGF and renin in the saliva, whereas [3-adrenergic agents exert a significantly lesser effect [68, 69]. The parasympathetic and sympathetic nerves differentially regulate the compartments into which kallikrein is secreted. Local parasympathetic nerve stimulation selectively releases kallikrein into the saliva (exocrine secretion) while activation of the sympathetic nerves increases kallikrein levels in the saliva and the blood through a process mediated by adrenergic receptors [70]. The kallikreins released from the SMG exert both local and distant effects in that they dilate veins of the glands as well as decrease systemic blood pressure in response to heat stress. The release of EGF is stimulated by both ct and [3 adrenergic mechanisms, although the ct-adrenergic response is more intense and prolonged. The sympathetic nerves also regulate gene expression for NGF and EGF [1, 2]. The increase in the release of kallikrein and EGF from SMG upon activation of the sympathetic nerves, suggests that autonomic regulation of inflammatory responses includes stimulation of the release of glandular factors involved in cardiovascular control and tissue repair. Cystatin S is a cysteine proteinase inhibitor that regulates proteolysis by endogenous and/or exogenous cysteine proteases such as the cathepsins. Rat cystatin S gene expression is tissue specific and occurs temporally during normal development [71]. The steady state level reaches a maximum level at 28 days of age and is not observed in the adult animal reaching barely detectable levels at 32 days of age. However, the cystatin S gene can be induced in the adult SMG by IPR [71, 72]. Data suggests that expression of the rat cystatin S gene is also controlled by tissue specific factors. This is reflected in a much greater IPR induced increase of cystatin S mRNA in the SMG compared to the parotid gland. Induction of cystatin mRNA is also more pronounced in the SMG of female compared to male rats. This difference is evident in 15 day old animals and is therefore thought to be linked to gender genotype rather than to circulating levels of steroid hormones [71 ]. Autonomic regulation of protease inhibitor levels offers another potential control mechanism for the production of biologically active polypeptides in the SMG. Changes in the ratios of polypeptides, proteases and protease inhibitors following hormonal or neuronal stimulation of the SMG have yet to be assessed. However, such investigation may provide important insights into, what is undoubtedly, a complex regulatory system (Figure 3).
6.
THE CERVICAL SYMPATHETIC TRUNK-SUBMANDIBULAR GLAND AXIS
Axons project down the cervical sympathetic trunk to the inferior and superior cervical ganglia (SCG). The postganglionic neurons leaving the inferior cervical ganglia predominately innervate the lungs and heart while axons leaving the SCG provide sympathetic innervation to the upper thorax, neck and skull as well as to facial structures [73]. The number of endocrine organs found in these areas, including the pineal, thyroid, parathyroid and salivary glands is an indication of the relevance of the SCG to the neuroendocrine system.
210
Physiological- Pathological- Psychological Stimuli
Central Processing
Sympathetic _ / ~L
SMG ProteasesCm=~Protease/ ~-~ inhibi7 Regulatory/ Bloodstreamrelease
Immunomodulation Figure 3. Mechanisms controlling bioactive peptide release from the SMG (see text for details).
Surgical sympathectomy has been used as an approach to study neural regulation of the immune system. These surgical denervations involve either SCG ganglionectomy (SCGx) or severing the connection between the inferior and superior cervical ganglia, so called decentralization. Unilateral removal of the SCG enhances contact hypersensitivity and delayed type reactions in the denervated submandibular lymph nodes [74]. These altered responses of the immune system indicate a direct modulation of inflammatory events by the sympathetic nervous system. 6.1.
A role for the CST-SMG axis in anaphlaxtic and endotoxic reactions
Rats sensitized by infection with the nematode Nippostrongylus brasiliensis have been used to investigate the modulatory role of the sympathetic nervous system in pulmonary inflammation. Within 8 hour of intravenous challenge with sensitizing allergen these rats develop a pronounced influx of macrophages and neutrophils into the lumen of the airway [75]. The cellular responses
211
can be used as a read out to determine the effects of surgical SCGx and decentralization. We have shown these surgical interventions have dramatic anti-inflammatory effects on lifethreatening anaphylaxis and subsequent pulmonary inflammation when compared to sham operated animals [75]. Macrophage and neutrophil influx into the lumen of the airways is markedly reduced. Peripheral blood neutrophils from treated animals exhibit a decreased phagocytotic ability and respiratory burst [76]. Chemotaxis to N-formyl-methionyl-leucylphenyalanine is also depressed, while TNFc~ production by alveolar macrophages is similarly compromised [77]. In marked contrast to the anti-inflammatory effects of SCGx and decentralization, the hypotensive effects of endotoxin are increased following these interventions in an animal model of endotoxic shock [78]. While the cells responsible for the modified responses to endotoxin in decentralized or SCGx animals have not been identified, neutrophils, platelets and monocytes/macrophages could all be involved. Neutrophils are known to play a significant role in determining the severity of hypersensitivity reactions, endotoxemia and postoperative hypoxia in the heart [79-81]. In addition a correlation exists between the severity of the response to endotoxin and neutrophil activity to nitroblue tetrazolium [82]. Platelets may attenuate the cytolytic activity of neutrophils through H202 scavenging by a glutathione cycle dependent process [83] and the monocyte/macrophage has a tremendous capacity to release mediators implicated in the immunophysiological effects of endotoxemia e.g. HzO2 and TNFc~ [84, 85]. At the time we first made these observations they highlighted the involvement of the SCG in modulation of responses to endotoxic and anaphylactic shock. However, given that the field of innervation of the SCG is limited to the upper thoracic and head regions it was deemed unlikely that the nerves affected by SCGx were innervating the organs primarily responsible for the immunophysiological reactions. These results suggested the involvement of an intermediary gland or organ. Subsequent experiments identified this intermediary as the SMG. Prior removal of the SMG prevented the depression of pulmonary inflammation and down regulation of macrophage and neutrophil function seen following surgical denervation [ 1]. These observations suggest that the SMG is a direct source of factors, which down regulate inflammation or control the release of anti-inflammatory factors from elsewhere in the body. Under normal circumstances the SCG exerts an inhibitory influence preventing the release of these factors from the gland while removal of the inhibitory tone through sympathetic decentralization or SCGx allows for an increased anti-inflammatory action as observed in the animal model of pulmonary inflammation. The enhanced hypotensive effects of endotoxin observed following sympathetic decentralization and SCGx were also observed following sialadectomy suggesting that the CST-SMG axis normally protects against potential hypotensive effects of endotoxin [78]. Taken together these results suggested a model in which postganglionic fibres arising from the SCG innervate cells within the SMG that synthesise both anti-hypotensive and anti-inflammatory factors. Under normal circumstances sympathetic signaling differentially regulates the release of these factors, stimulating the anti-hypotensive factor while inhibiting the release of the anti-inflammatory agents (Figure 4). 6.2.
CST-SMG control of mast cell function
Mast cell function is also modulated by the CST-SMG axis. However, the regulatory mechanism appears distinct from that involved in controlling neutrophil and macrophage responses [86]. TNF-ct-dependent cytotoxic activity of peritoneal mast cells (PMC) is reduced in rats following
212
Superior Cervical
A)
Ganglia decentralization
SCG stimulates release of factors leading to inhibition of endotoxic response
Q G
B) SCG exerts inhibitory tone on release of anti-inflammatory factors
G Figure 4. Immunomodulatory actions of the cervical sympathetic trunk-submandibular gland axis. A) Superior cervical ganglion decentralisation increases the hypotensive responses to endotoxin. This suggests that under normal circumstances the cervical sympathetic nerves stimulates the submandibular gland to release factors that down-regulate the response of immune cells to endotoxin. B) Superior cervical ganglion decentralisation down-regulatesinflammatory responses in sensitized rats. This suggests that intact sympathetic nerves suppress the release of anti-inflammatory agents from the submandibular gland.
decentralization but not SCGx. This suggests that the neural regulation of mast cell function probably occurs at the level of the SCG unlike neutrophils and macrophages where neural structures within the thoracic spinal cord are responsible for depressing function. Removal of the SMG also inhibits TNF-ot production by PMC indicating that salivary glands constitutively release a factor that upregulates mast cell function. However, since a combination of sialadenectomy and decentralization has no effect on PMC cytotoxicity, glands or organs other than the SMG probably participate in the regulation of mast cell activity. Taken as a whole these observations indicate that there are multiple mechanisms by which the CST-SMG can regulate immunological functions. 6.3.
A novel class of regulatory peptides
In an attempt to determine the SMG derived factors responsible for the modulation of endotoxic and anaphylaxtic reactions, extracts of SMG subjected to molecular weight cut-off filtration and high-performance liquid chromatography (HPLC) purification were tested for their ability to reduce the severity of endotoxin induced hypotension. Our initial expectation was that the agents involved would be well characterized regulatory factors such as NGF and EGF. However, utilizing these methods two novel peptides, which could attenuate the severity of endotoxin-induced hypotension, were isolated from purified extracts, sequenced and
213
synthesized. These peptides were: a pentapeptide, submandibular gland peptide S (SGP-S), with the sequence SGEGV and a heptapeptide, with the sequence TDIFEGG, named SGP-T [44, 87]. When given intravenously SGP-T can attenuate hypotension during cardiovascular anaphylaxsis, inhibit the disruption of intestinal myoelectric activity of the intestine and development of diarrhea during intestinal anaphylaxis, and downregulate neutrophil chemotaxis [88, 89]. These activities were also evident in the C-terminal fragment of the peptide, FEG. The D-isomeric form of FEG, denoted feG, was an effective inhibitor in both models of anaphylaxis when administered orally. SGP-S has not yet been assessed in a biological assay other than the endotoxic shock model. 6.4.
The VCS gene family
SGP-T was identified as a carboxy-terminal fragment (residues 138-144) of the submandibular gland rat 1 (SMR1) protein, while SGP-S is found closer to the amino terminal of the same polypeptide [44] (Figure 5). The sequence of the SMR1 protein was deduced from the cDNA sequence of the SMR1-VA1 gene, which encodes the prohormone-like protein in rat SMG [90]. The SMR1 polypeptide is found almost exclusively in the salivary glands and prostate and is one of several peptides generated by the variable coding sequence (VCS) multigene family that has been localized to chromosome 14 bands p21-p22 [91] [92]. The gene family has at least 10 members. Three of these, VCSA1 (SMR1 gene), VCSA2 and VCSA3 belong to the VCSA subclass and are found exclusively in the rat. These genes encode SMR1 and SMRl-related polypeptides that contain potential recognition sites for proteolytic enzymes. They can be considered potential preprohormones [93]. The structure of the VCSA1 gene is similar to the structure of several genes encoding prohormones such as genes for preprothyrotrophin-releasing hormone, preproenkephalins or preproopiomelanocortin [94]. It is not known whether this structure reflects the existence of a common ancestor, or convergent evolution. Seven genes belong to the VCSB subclass. The B subclass is found in several species and encodes a family of proline rich proteins found in rats, mice and humans [95]. A major characteristic of the VCS gene family is the presence of a hypervariable region inside the coding sequence [91]. In intraspecies pairwise comparisons a higher level of sequence divergence is observed in the hypervariable region than in the adjacent exonic or intronic sequences. Furthermore most of the mutations at the nucleotide level lead to amino acid substitutions. As a consequence the VCS family encodes proteins that are diverse in amino acid content, structure and probably function. Like many SMG derived polypeptides, the accumulation of mature SMR1 peptides appears to be dependent on the integrity of the hypothalamic-pituitarygonad axis. In 4 week old hypophysectomized or gonadectomized male rats the levels of mature peptide are greatly reduced, being close to two orders of magnitude less than in the SMG of sham operated animals [90].
6.5.
Processing of SMR1
The SMR1 prohormone contains an amino-terminal putative secretory signal sequence and a tetrapeptide (QHNP), located between dibasic amino acids, that constitutes the most common signal for prohormone processing. The proteolytic processing of SMR1 has been partially characterized by Rougeot et al. [96]. Cleavage at pairs of arginine residues close to the amino-terminus of the SMR1 polypeptide generates three structurally related peptides. The undecapeptide (23VRGPRRQHNPR33) is
214
SMR1 Prohormone and Peptide Fragments 146
1
SIGNALI~IPEPTI~RR DERF~ NH2
/
',,
19-SGEGV-23 (SGP-S)
23-VRGPRRQHNPR-33 28-RQHNPR-33 29-QHNPR-33
~
COOH
138-TDIFEGG-144 (SGP-T) 141-FEG-143
Figure 5. Schematic representation of the SMR1 preprotein and the peptides derived from it through proteolytic cleavage. Dibasic cleavage sites are indicated by small arrows and single-letter amino acid symbols.
generated by selective endoproteolysis at the Arg33-Arg34 bond and at the signal sequence. The hexapeptide (28RQHNPR33) and the pentapeptide (29QHNPR33) are generated by selective cleavages at both the Arg27-Arg28 and Arg33-Arg34 bonds (Figure 5). The biosynthesis of these peptides is subject to distinct regulatory pathways depending on the organ, sex and age of the rat. The peptides are differentially distributed within the SMG and in resting or epinephrine-elicited salivary secretions, suggesting distinct proteolytic pathways are involved in their maturation. In the male rat SMG the hexapeptide and the undecapeptide are found at increasing levels at 6-10 weeks of postnatal life [96]. This corresponds to the differentiation of acinar cells where the SMR-1 protein has been shown to localize. The 6 week old rat contains mostly the undecapeptide form, while the hexapeptide predominates in 10 week old animals. In 14 week old female SMG the undecapeptide is the major form. Protease activities are very low in the glands of newborn rats and mice and a rapid increase in enzyme activity coincides with the onset of puberty. Therefore the changes in ratio of processed peptides may be explained by the sex hormone dependant accumulation of SMR-1, processing enzymes or both, with fully active processing enzymes only being present in the male rat from 10 weeks onward. Although generated in the gland of both the male and female rats under basal conditions, the undecapeptide is only released into the saliva of the male. The hexapeptide is produced in large amounts in the gland of adult male rats and released into the saliva under both resting and epinephrine stimulated conditions. The pentapeptide appears only in the male saliva and is present mostly under stimulated conditions. Administration of epinephrine also induces the release of the hexapeptide into the blood stream. Therefore, it appears that the SMG can act as both an exocrine and endocrine organ for the SMR-1 derived peptides (Figure 6) [96]. Although no biological function has been ascribed to these peptides Rougeot et.al, were able to identify specific binding sites of the pentapeptide at physiological concentrations using radiolabelled peptide coupled to quantitative image analysis of whole rat body sections [97]. The peptide was detected in the renal outer medulla, bone and dental tissue glandular gastric mucosa and pancreatic lobules. Pentapeptide binding was localized to selective portions of the
215
Unstimulated
Stimulated
Epinephrine Saliva VRGPRRQHNPR RQHNPR
/
Saliva VRGPRRQHNPR
SMG
QHNPR
)
PRRQH
RQHNPR
-
/
RQHNPR
Figure 6. Differential processing and release of SMR 1-derived peptides in the SMG of male rats (ref. 96).
male rat nephron and in bone exclusively accumulated within the trabecular bone, remodeling unit. Based on these studies it has been suggested that the SMR-1 derived pentapeptide is primarily involved in the modulation of mineral balance between at least four systems: kidney, bone, tooth and circulation [97]. The enzymatic cleavage processes that generate SGP-T have not been identified. However, non-arginine-dependent serine proteases are abundant in salivary glands and could generate this peptide [98]. It has generally been believed that the immunomodulatory effects of the SMG are mediated predominantly by growth factors released into saliva and blood. However, more recent studies, described here, indicate that small peptides of salivary gland origin are also capable of modulating inflammatory reactions. The possibility that other pro-hormones in the salivary gland may yield small peptides capable of immunomodulation must be considered. Indeed, salivary glands contain chromogranin A and B, members of a family of highly acidic proteins, the chromogranins [99]. Chromogranins are prohormones and biological functions have been ascribed to many chromogranin-derived peptides. There are reports of antibacterial and antifungal activities and immunoregulatory functions including induction of monocyte chemotaxis and modulation of mast cell activity [100-103]. In rats, chromogranin A has been localized to the GCT and chromogranin-like immunoreactivity is detected in the saliva following stimulation with acetylcholine and noradrenaline [104]. Further investigations may reveal that these peptides also have a place in the immunomodulatory arsenal of the SMG.
216
7.
STRESS AND THE SUBMANDIBULAR GLAND
The widely held belief that the mind exerts significant effects on the health of an individual has stimulated research in an area termed psychoneuroimmunology. A major focus of psychoneuroimmunology has been a study of the effects of stress on the immune system. It is regarded as common knowledge that stressful experiences suppress the ability of the immune system to respond to antigenic challenge and thus increase susceptibility to infectious and neoplastic disease [105, 106]. Data from human and animal studies have confirmed the immunosuppressive effect of stress. However, there is evidence that stress may also enhance immune function, suggesting that the relationship between stress and the immune system is complex [107, 108]. These differences in the immune response to stress may be related to the stress inducing agent or the duration of stress (acute vs. chronic). During fighting or when under attack from a predator, immunological challenge in the form of a wound is likely. It would be advantageous for an organism if the capacity to respond to immunological challenge were enhanced in these acutely stressful situations. Increasing immune surveillance and preparing an organism for potential immune challenges arising from the action of stress inducing agents makes evolutionary sense. Indeed, antigen-specific cell mediated immunity can be significantly enhanced through activation of the physiological stress response [109]. The SMG may be one effector of the immunomodulatory actions of stress. Fighting is known to increase plasma NGF in mice, while other types of stress such as cold-water swim, escapable and inescapable footshock or physical restraint, do not affect plasma levels of this growth factor. Plasma NGF levels are related to the number of fighting episodes [110-112]. The NGF released from the SMG of adult male mice following intraspecific fighting is biologically active and causes degranulation of peritoneal mast cells. Administration of NGF antibodies or sialadenectomy prior to fighting blocks the mast cell degranulation [113]. Bilateral removal of the SMG leads initially to a significant increase in aggressive behaviour during social encounters. These behavioral changes decreased significantly during subsequent social encounters while defensive behaviors and elements of arrested flight increased progressively. These results suggested that sialadenectomy, perhaps by removing salivary NGF, interferes with the ability of mice to cope in stressful situations [113]. The exposure of rats to saturated ether vapour for 2 minutes is used as a model of acute stress. In adult male rats secretion of mature SMR1 peptides is rapidly stimulated in response to exposure to ether fumes (2 min), while no detectable increase in release is observed following long term exposure to stressful ambient temperatures (120 min at 4~ or 37~ [95]. The response to acute stress stimuli in conscious male rats results in an SMR-1 increase similar to that observed under pharmacologically induced adrenergic stimulation, suggesting that the surge in circulating SMR-1 is mediated by an endogenous adrenergic secretory response to acute stress. It would be of interest to examine changes in SMR-1 levels induced by behaviorally relevant stress such as fighting among males. It has been shown that adrenalectomy fails to block NGF release in fighting mice and injections of ACTH or extracts of adrenal gland or hypophysis do not result in NGF release into the bloodstream [112]. This suggests that the HPA axis is not involved in NGF release by the SMG. However, there are likely to be several mechanisms that control differential expression of biologically active peptides in response to various stimuli and the role of the HPA axis in modulating SMR-1 release should be examined.
217
8.
CONCLUSIONS
Some SMG factors show marked species related differences. For example, the serine proteinase, renin, was found in murine SMG but not in the rat [114]. Tonin, a member of the kallikrein family is present in large amounts in the rat SMG but has not been found in other species. High levels of NGF are detectable in both murine and rat SMG, whereas the human gland contains much smaller amounts [115]. Other factors such as kallikrein and EGF have been found in several species including humans [3, 4]. Immunoregulatory effects of the SMG have been observed in many species. However, the role of the human SMG in this regard is not clear. The qualitative and quantitative differences in protein content, coupled with the fact that many of the biologically active peptides can be synthesised elsewhere in the body, prompts the question; how necessary is the SMG for immune homeostasis? The possible redundancy of the gland's immunomodulatory actions is unclear, even in rodents. However, there are a number of observations that encourage further investigation of immuoregulation by the SMG in man. Sj6gren's syndrome (SS) is an inflammatory disease of the salivary and lacrimal glands. The disease results in failure of the glands leading to xerostomia and keratoconjunctivitis sicca [116, 117]. SS may occur as an isolated disease, but, it is frequently associated with systemic immune disorders, such as systemic lupus erythematous, rheumatoid arthritis, and sarcoidosis [118, 119]. These associations suggest that immunoregulation by salivary glands plays an important role in modulating systemic immunity. Strenuous exercise is followed by lymphopenia, neutrophilia, depressed NK cell function and lymphocyte proliferative responses to mitogens, and impaired natural immunity [120-122]. These exercise-induced immune changes may provide the physiological basis of altered resistance to infections. The mechanisms underlying exercise-induced immune changes are thought to be multifactorial and include neuroendocrinological and metabolic mechanisms. The contribution of the SMG to the immunological changes observed following exercise is unknown. However, it is interesting to note that salivary composition changes following strenuous exercise. Salivary IgA levels are decreased, while amylase, EGF and total protein have all been demonstrated to increase [123-125]. Further investigation of salivary and plasma levels of other SMG derived regulatory peptides may provide evidence for involvement of the gland in the immunological changes which follow exercise induced stress. As stated previously the gene encoding SMR1 (VCSA1) is found only in rats, while human salivary glands express genes from the VCSB subfamily. However it is possible that these, or as yet undiscovered, genes encode a human homologue of the SMR1. The observation that human neutrophils respond to, and thus may express receptors for, the tri-peptide FEG lends support to this hypothesis [44]. The use of degenerative primers for SMR1 mRNA to screen for humans homologues and the development of sensitive assay systems for FEG or SGP-T are two approaches which may lead to the discovery of novel anti-inflammatory peptides in man. From the information presented above it is clear the SMG constitutes a fully integrated component of the body's neuroimmunoendocrine network. The gland is under control of the CNS and regulated by the hypothalamus and sympathetic nervous system. The extent of the role played by the SMG in controlling systemic immune function remains to be seen. However, it is clear that investigations of what Barka describes as "that somewhat neglected "appendix" of the digestive system" [3] have the potential to provide valuable insights into the mechanisms underlying psychological and neuroendocrine control of the immune system.
218
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Rosinski-Chupin I, F. Rougeon. The gene encoding SMR1, a precursor-like polypeptide of the male rat submaxillary gland, has the same organization as the preprothyrotropinreleasing hormone gene. DNA Cell Biol 1990; 9: 553-559. Rougeot C, Rosinski-Chupin I, Rougeon F. Novel genes and hormones in salivary glands: from the gene for the submandibular rat 1 protein (SMR1) precursor to receptor sites for SMR1 mature peptides. Biomedical Reviews 1998; 9: 17-32. Rougeot C, Rosinski-Chupin I, Njamkepo E, Rougeon F. Selective processing of submandibular rat 1 protein at dibasic cleavage sites. Salivary and bloodstream secretion products. Eur J Biochem. 1994; 219: 765-773. Rougeot C, Vienet R, Cardona A, Le Doledec L, Grognet JM, Rougeon F. Targets for SMRl-pentapeptide suggest a link between the circulating peptide and mineral transport. m. J Physiol 1997; 273: R1309-20. Berg T., Wassdal I, Sletten K. Immunohistochemical localization of rat submandibular gland esterase B (homologous to the RSKG-7 kallikrein gene) in relation to other serine proteases of the kallikrein family. J Histochem Cytochem 1992; 40: 83-92. Letic-Gavrilovic A, Shibaike S, Niina M, Naruse S, Abe K. Localization of chromogranin A and B, beta-endorphin and enkephalins in the submandibular glands of mice. Shika. Kiso. Igakkai. Zasshi. 1989; 31: 453-462. Lugardon K, Raffner R, Goumon Y, Corti A, Delmas A, Bulet P, Aunis D, MetzBoutigue. Antibacterial and antifungal activities of vasostatin-1, the N-terminal fragment of chromogranin A. J Biol Chem 2000; Apr. 14; 275(15): 10745-53. 275: 10745-10753. Kong C, Gill BM, Rahimpour R, Xu L, Feldman RD, Xiao Q, McDonald TJ, Taupenot L, Mahata SK, Singh B, O'Connor DT, Kelvin DJ. Secretoneurin and chemoattractant receptor interactions. J Neuroimmunol 1998; 88: 91-98. Reinisch N, Kirchmair R, Kahler CM, Hogue-Angeletti R, Fischer-Colbrie R, Winkler H, Wiedermann CJ. Attraction of human monocytes by the neuropeptide secretoneurin. FEBS Lett 1993, 334: 41-44. Forsythe P, Curry WJ, Johnston CF, Harriott P, MacMahon J, Ennis M. 1The modulatory effects of WE-14 on histamine release from rat peritoneal mast cells. Inflamm. Res 1997; 46 Suppl 1: S13-4: S13-4. Kanno T, Asada N, Yanase H, Iwanaga T, Ozaki T, Nishikawa Y, Iguchi K, Mochizuki T, Hoshino M, Yanaihara N. Salivary secretion of highly concentrated chromogranin a in response to noradrenaline and acetylcholine in isolated and perfused rat submandibular glands. Exp Physiol 1999; 84: 1073-1083. Borysenko JZ. Behavioral-physiological factors in the development and management of cancer. Gen. Hosp. Psychiatry 1982; 4: 69-74. Kort WJ. The effect of chronic stress on the immune response. Adv Neuroimmunol 1994; 4:1-11. Dhabhar FS, Miller AH, McEwen BS, Spencer RL. Effects of stress on immune cell distribution. Dynamics and hormonal mechanisms. J Immunol 1995; 154:5511-5527. Dhabhar FS, Miller AH, Stein M, McEwen BS, Spencer RL. Diurnal and acute stressinduced changes in distribution of peripheral blood leukocyte subpopulations. Brain Behav Immun 1994; 8: 66-79. Dhabhar FS. Stress-induced enhancement of cell-mediated immunity. Ann N. Y. Acad. Sci 1998; 840: 359-72: 359-372. Alleva E, Aloe L, Bigi S. An updated role for nerve growth factor in neurobehavioural regulation of adult vertebrates. Rev Neurosci 1993; 4: 41-62. Maestripieri D, De Simone R, Aloe L, Alleva E. Social status and nerve growth factor
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serum levels after agonistic encounters in mice. Physiol Behav 1990; 47:161-164. 112. Aloe L., Alleva E, Bohm A, Levi-Montalcini R. Aggressive behavior induces release of nerve growth factor from mouse salivary gland into the bloodstream. Proc Natl Acad Sci USA 1986; 83: 6184-6187. 113. De Simone R, Alleva E, Tirassa P, Aloe L. Nerve growth factor released into the bloodstream following intraspecific fighting induces mast cell degranulation in adult male mice. Brain Behav Immun 1990; 4: 74-81. 114. Morris B J, de Zwart RT, Young JA. Renin in mouse but not in rat submandibular glands. Experientia 1980; 36: 1333-1334. 115. Levi-Montalcini R, Dal Toso R, della Valle F, Skaper SD, Leon A. Update of the NGF saga. J Neurol Sci 1995; 130:119-127. 116. St. Clair EW. New developments in Sjogren's syndrome, urr Opin Rheumatol 1993; 5:604-612. 117. Rhodus NL. Sjogren's syndrome. Quintessence Int 1999; 30: 689-699. 118. Lois M, Roman J, Holland W, Agudelo C. Coexisting Sjogren's syndrome and sarcoidosis in the lung. Semin Arthritis Rheum 1998; 28:31-40. 119. McDonagh JE, Isenberg DA. Development of additional autoimmune diseases in a population of patients with systemic lupus erythematosus. Ann Rheum Dis 2000. Mar; 59(3): 230-2. 2000; 59: 230-232. 120. Pedersen BK, Bruunsgaard H, Jensen M, Toft AD, Hansen H, Ostrowski K. Exercise and the immune system-influence of nutrition and ageing. J Sci Med Sport 1999; 2: 234-252. 121. Pedersen BK, Bruunsgaard H, Jensen M, Krzywkowski K, Ostrowski K. Exercise and immune function: effect of ageing and nutrition. Proc Nutr Soc 1999; 58: 733-742. 122. Nieman DC. Nutrition, exercise, and immune system function. Clin Sports Med 1999; 18: 537-548. 123. Nexo E, Hansen MR, Konradsen L. Human salivary epidermal growth factor, haptocorrin and amylase before and after prolonged exercise. Scand. J Clin Lab Invest 1988; 48: 269-273. 124. Mackinnon LT, Ginn E, Seymour GJ. Decreased salivary immunoglobulin A secretion rate after intense interval exercise in elite kayakers. Eur J Appl Physiol 1993; 67: 180-184.
125. McDowell SL, Hughes RA, Hughes RJ, Housh TJ, Johnson GO. The effect of exercise training on salivary immunoglobulin A and cortisol responses to maximal exercise. Int J Sports Med 13: 577-580.
New Foundationof Biology
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Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
Glandular Kallikrein in Immunoregulation
EDRIS SABBADINI 1, EVA NAGY 1, ALEXANDER FRED T. KISIL 1 and ISTVAN BERCZI 1
V(~RI~S 2, GERTRUDE VOROSOVA 3,
1Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada 2Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada 3Department of Anatomy, University of Manitoba, Winnipeg, Manitoba, Canada
ABSTRACT Glandular Kallikrein (GK) is an enzyme of the serine protease family capable of generating biologically active peptides by partially degrading various substrates. It is found in several tissues with particularly high concentrations in salivary glands, pancreas, kidney and the prostate gland. The physiological functions of this enzyme appear to vary according to the tissue in which it acts and the substrate(s) available in such tissues. Our interest in GK arose when we demonstrated that an immunoregulatory factor isolated from the salivary submandibular (SM) gland of rats, was rat GK (rGK). When added to cultures of lymphoid tissues, rGK induced a significant increase in lymphocyte proliferation manifested either in unstimulated cell cultures or in cultures of cells stimulated with suboptimal amounts of T cell mitogens. When injected subcutaneously, rGK induced a marked, albeit short lived, immunosuppressive effect in various experimental models including contact hyper-sensitivity to picryl chloride, allograft rejection, the production of antibody plaque-forming cells in the spleen of animals immunized with sheep red blood cells, and collagen-induced arthritis. All of the above effects were species-nonspecific inasmuch as murine GK and pig GK induced identical effects to rGK either in mice or in rats. Moreover, for these effects to take place, the enzymatic activity of GK had to be preserved. In the presence of inhibitors of proteolytic activity, both the in vivo and the in vitro immunoregulatory effects were abolished. This indicated that GK acts on a protein substrate to generate immunoregulatory peptides. If this is the case, the apparent conflict between in vitro stimulation and the suppression induced by subcutaneously injected GK can be explained as due to the accumulation of lymphocyte stimulating peptides in the in vitro cultures, while in vivo injection probably results in the rapid dispersal and degradation of such peptides leaving the organism depleted of substrate and unable to produce amounts of active peptides sufficient for a normal immune response. The effects described up to this point refer to in vitro phenomena or to the subcutaneous administration of GK. The fact that GK is found in high concentration in salivary glands and is secreted in saliva suggests that a significant physiological function of GK may occur following external salivary secretion. For this reason, we also tested the effect of orally administered GK in rats. Oral GK was shown to be required for the induction of tolerance to orally administered antigens. Rats subjected to the surgical removal of the SM gland (SMX) did
226
not develop tolerance to oral collagen given in doses that induced significant tolerance in normal controls. The oral administration of GK in the SMX animals fully restored the capacity to develop oral tolerance. This suggests that salivary GK is responsible for homeostasis in the mucosa associated lymphoid tissue which is responsible for the development of tolerance to oral antigens. The observation that GK may be used to enhance the induction of oral tolerance holds promises for the therapuetic application of oral GK in autoimmune diseases.
1.
INTRODUCTION
Kallikreins are a family of serine proteases capable of cleaving various substrates and generating biologically active peptides. In spite of the identity of names, tissue or glandular kallikreins should be distinguished from plasma kallikrein. They differ from plasma kallikrein in their genes of origin, molecular weight, amino acid sequences, substrates, peptide products and most probably physiological functions. There are at least 20 genes for tissue kallikrein in rodents [1], while in humans only 4 genes have been so far described. Of these, only one gene in each species codes for true glandular kallikrein. Of the rat genes at least 6-7 appear to be expressed in the submandibular (SM) gland [2]. These include true glandular kallikrein, tonin, c~ and ~, NGF, and the EGF-binding protein (EGF-BP), type A, B, and C. Here the term glandular kallikrein (GK) will apply to true GK only, while the general term kallikrein(s) will be used for any unspecified members of the tissue kallikrein family. True GK has been designated in various species as kallikrein-1 (K1) [3]. The best known substrates for GK action are hepatic-derived kininogens [3] which occur in two forms, low molecular weight kininogen (50 kDa) and high molecular weight kininogen (120 kDa). From the action of plasma kallikrein on high molecular weight kininogen a nonapeptide, bradikinin, is generated, while in most species GK gives rise to a decapeptide, kallidin (lys-bradikinin) from either low or high molecular weight kininogen. Kallidin is biologically active in itself but may also be further processed into bradikinin. An exception may be the GK of the rat SMG which was reported to produce bradikinin [4]. While the action of GK on kininogen is particularly well studied, the full range of GK substrates has not yet been investigated. Since GK is, and most of the times remains, localized in certain tissues, physiological substrates are likely to vary from tissue to tissue. Of particular interest here is the possibility that GK may activate or in some way regulate other immunologically active factors of the SM gland, including NGF, EGF/TGF~ and TGF[3. Thus, salivary gland GK may exert its immunological effects either via the production of classical kinins or via other immunologically active factors. Moreover, salivary GK is actively secreted in saliva [5] and would be expected to reach various points in the gastro-intestinal tract and act on various substrates there. Several in vitro effects of kallikreins on cells of the immune system have been reported. Thus, several authors have described mitogenic and co-mitogenic effects of kallikrein and other serine proteases. Such mitogenic effects were observed with thymocytes [6], T cells and B cells [7]. Although bradikinin may also have mitogenic effects [8], the involvement of this kinin in kallikrein-induced mitogenesis is not well investigated. Moreover, several proteases, including kallikrein, were shown to be involved in immunoglobulin isotype control. Thus Ishizaka described a kallikrein-like factor called glycosylation-enhancing factor, which induced CD4+ T cells to produce an IgE-potentiating factor and to favour the production of IgE by memory B cells [9]. Serine proteases from Schistosoma mansoni schistosomula were reported to enhance IgE production [10]. Moreover, the addition of kallikrein and other serine proteases in various
227
concentrations to cultures of B cells stimulated with LPS and IL 4 enhanced the production of IgE, IgG 1, or IgG3, depending on the enzyme concentration used [ 11 ]. Our interest in GK arose from studies on immunosuppressive factors in the SM gland of rats. The addition of crude extracts from rat SM glands to murine spleen and lymph node cultures stimulated with concanavalin A (Con A) induced either suppression (at high concentrations) or further stimulation (at lower concentrations) of proliferative activity [12]. This suggested that these extracts contained factors with suppressive effects as well as factors with the ability to enhance lymphocyte proliferation. Gel filtration of the crude extracts revealed that the in vitro suppressive activity was due to factors with molecular weight higher than 50 kilo Daltons (kDa), while stimulation was due to factors with molecular weight lower than 50 kDa (Figure 1). We tested the in vivo activity of both the higher and lower molecular weight fractions in the skin allograft, direct plaque forming cell response and in the delayed-type hypersensitivity (DTH) models [13]. As shown in Table I, and contrary to what one might have expected in view of their in vitro suppressive activity, the high molecular weight fractions did not have any significant effect in these models. On the other hand, the lower molecular weight fractions produced significant suppression in all three models. .I 6 0
-
120 =
,,7 o v
"(
80
ci.
40
!
!
o z's 8. 3 z- 8 0.93 0:3, o.,o Fresh hssue
(/J..g/ml)
Figure 1. Effects of pooled Sephacryl fractions of submandibular ( 0 , II) and parotid (O, F-I) glands on the Con A stimulated lymph node cell proliferation: ( 0 , 9 MW > 50 kDa; (m, D) MW < 50 kDa. Thesolid horizontal line is the value of mean c.p.m, for the controls and the shaded area represents the 95% confidence limits. From Ref. 12.
Fractionation of the lower molecular weight pool of fractions through successive steps of hydrophobic interaction, anion exchange chromatography and finally gel filtration, led to the isolation a single protein (Figure 2) which retained the properties of in vitro stimulation of lymphocyte proliferation and in vivo immunosuppression (results not shown). The amino acid sequence of the isolated protein was determined [14] using an automated Edman degradation procedure [15]. Figure 3 shows the partial N-terminal amino acid sequence of the 40 kDa protein and of the members of the kallikrein family represented in the rat SMG. The x's (unidentified amino acids) in our sequence are probably cysteines which are destroyed in the Edman degradation process. If this is taken into account, the first 25 amino acids of our protein
228
has identical sequence
with that of true GK and differ from those of other members
of the
kallikrein family. In view of the fact that no rat genes with the same amino acid sequence are k n o w n , it w a s c o n c l u d e d t h a t t h e i s o l a t e d p r o t e i n w a s t r u e r a t G K ( r G K ) . Table I
Effects of high molecular weight (HMW) and low molecular weight (LMW) pools of gel filtration fractions in three in vivo immunological assays. Modified from Ref. 13.
Exp Model
Treatment a
Groups
Results ___SD b
Skin transplantation
10 daily doses
PBS
12.2 + 0.37
CBA/2J to C57B1/6J
Days 0 to 9
HMW
13.0 • 0.44
NS
LMW
14.7 _+0.76
p < 0.05
Direct PFC
5 daily doses
PBS
237.0 • 19.7
Days 1 to 3
HMW
193.0 + 12.0
NS
DTH (A/J mice)
2 daily doses
Days 4 and 6
HMW
LMW
119.6 • 10.0
PBS
19.0 • 0.70
18.2 + 0.49
NS
LMW
9.0 • 1.00
Significancec
p < 0.05
p < 0.01
a. The animals received the subcutaneous injection of 200 ~tl of PBS or PBS containing either the HMW or the LMW fractions. The doses corresponded to one-half of a SM gland (0.965 mg or 0.53 mg, respectively) and to one gland in the other models (1.93 mg or 1.06 mg, respectively). b. Skin transplantation results are expressed as mean survival time; PFC response is expressed as the number of IgM anti-SRBC PFC per one million splenocytes; DTH results are expressed as increases of ear thickness in 1/10 mm units 24 hours after challenge. c. Significance was determined by two-sample t-test, using the PBS-treated group as the control; NS, not significant.
. . . .
97.4 kD 66.2 kD
I~
42.7 kD
~.
31kD
~,
21.5 kD
I~
i i 84184184 51 i.
14.4 kD
1
2
Figure 2. SDS-PAGE of the purified protein from rat SM gland (lane 2). Molecular weight standards are shown in lane 1. From Ref. 14.
229
Protein
40
1
i0
15
20
25
WGGYNxEMNSQPWQVAVYYFGEYLx
kDa
Gland.
5
Kallikrein
VVGGYNCEMNSQPWQVAVYYFGEYLC
GYNy~ ~ NSQPWQVA~
KLP-S3
K sQpwQvA
Tonin Antigen 7
:I VGG'i K rd~ Kt~SQPWQV'~
T-kininogenase
I VGG~ K ZE K NSQPWQV~
Proteinase
I
B
Proteinase A Figure 3. Partial N-terminal amino acid of the 40 kDa protein isolated from rat SM gland compared with those of members of kallikrein family expressed in the rat SM gland. The boxed areas represent regions of identity with the 40 kDa protein. Blank spaces are used to align homologous sequences in different proteins, x = not identified. From Ref. 14.
The esterase activity of the isolated rGK was approximately the same as that of a commercially obtained porcine GK (pGK) when measured in the 2-N-benzoyl-arginine ethyl estez (BAEE) assay [16]. Different concentrations of aprotinin induced different degrees of inhibition. Figure 4 demonstrates the effects of rGK and pGK in the presence or in the absence of aprotinin on the proliferative activity of Con A stimulated murine lymph node cells. The same concentrations of pGK induced similar co-mitogenic effects. The Con A concentration used in these experiments was such as to induce only suboptimal mitogenic effects, suitable for the demonstration of the co-mitogenic activity of rGK. The addition of different concentrations of aprotinin to the co-stimulated cultures induced dose dependent suppression. It should be noted that the highest concentration of aprotinin used in this experiment (1.5 ~tg/ml or 6 ~tg/culture) was capable of inducing some 90% inhibition in the B AEE assay. On the other hand, the lowest concentration of aprotinin (1.5 ~tg/culture) induced approximately 40% inhibition of the enzymatic activity and partial inhibition of the co-mitogenic activity. The results of an in vivo experiment performed along the same lines are presented in Figure 5. A DTH reaction was induced in mice sensitized with picryl chloride and challenged with the same agent six days later. The injection of rGK 24 hour before challenge resulted in nearly complete (~ 87%) suppression of the response. Similar suppression was obtained with pGK (-72%). The dose of rGK used in this experiment (57 ~tg/animal) was based on our previous experience. The higher of the two aprotinin doses (190 ~tg/animal) was selected so as to provide, after dilution in the blood stream, a concentration similar to that used in the in vitro experiments. The suppressive effects of rGK and pGK were almost completely removed by the injection of this higher dose of aprotinin given immediately before GK injection (inhibition nearly -8%). On the other hand, the lower dose (95 ~tg) of aprotinin induced only partial suppression of the rGK effects (inhibition still -60%). These two experiments clearly demonstrate that
230
the enzymatic activity of GK must be preserved in order to retain its in vivo and i n vitro immunological effects.
cpm xl,000 10 15 I
20
I
!
PBS Ill
rGK
II1|
II
I
-t-
I
§
Apr. (6 lag) rGK + Apr. (6 I~g) rGK + Apr. (3 I~g)
-q-
rGK + Apr. (1.5 I~g)
II
III
I
I
I
pGK
I
I
I
-t_ff
pGK + Apr. (6 Ixg)
Figure 4. Increased proliferative activity of Con A stimulated A/J lymph node cells induced by rGK and pGK and reversal of this effect with aprotinin. The rGK and pGK doses were 1.78 g/culture (final concentration 0.22 ~tM/1). Aprotinin (Apr.) doses were: 6 gg, 3 gg, or 1.5 gg per culture 4.6 M/i, 2.3 M/l, and 1.15 M/l, respectively). Results are expressed as counts per minute (C.P.M.) in triplicate cultures (+S.D.). From Ref. 14.
A Ear thickness ~rn rn) 0.5 1.0 I
I
+
PBS
+
rGK Apr. (190 I~g) rGK + Apr. (190 I~g)
II
el
rGK + Apr. (95 I~g) pGK pGK + Apr. (190 Jag)
II
, I
, , II
I
"t-
II
-I-
Figure 5. Effects of rGK and pGK on the DTH response of A/J mice immunized with picryl chloride and reversal of such effects with aprotinin. Aprotinin (Apr.) was injected subcutaneously as a full dose (190 pg per animal) or as a half dose (95 ~tg per animal). Fifteen minutes later the animals received a further subcutaneous injection of rGK or pGK in 0.2 ml of PBS or PBS only. Results are expressed in terms of the increase of the thickness of the challenged ear over the pre-challenge values. Unimmunized controls (not shown) did not show any increases of the ear thickness. From Ref. 14.
231
Table II demonstrates the effects of varying the time of GK injection with respect to the time of immunization or of challenge in the DTH model in mice. If given before immunization, GK had a suppressive effect lasting for at least fourteen days. This suggested that the animals did not develop any immunity when presented with the antigen. On the other hand, if GK was given after the development of immunity, it induced a short lived suppression of the skin reaction with a full recovery of reactivity a week after GK administration. This demonstrated that GK did not affect the state of immunity of an animal and suppressed the skin reaction with a mechanism that may be either immunological or anti-inflammatory. It should be noted that the half life of GK is such that 24 hours after injection, i.e. at the time of antigen administration for either immnunization or challenge, none of the injected GK is present in the animal. Thus, GK mediated inhibition probably occurs indirectly, following production of mediators dependent on GK enzymatic action. Table II
Effects of the intradermal injection o f r G K given before or after immunization in the DTH model.
Treatment a
None
Test on day: b
7
14
18.4 + 0.76
17.6 + 0.92
rGK, Day-1
6.0 + 1.2"
7.6 + 0,98*
rGK, Day+6
4.4 + 0.63*
16.8 + 1.12
rGK, Day+13
19.0 + 1.24
5.6 + 0.78*
a. A/J mice were immunized by the application of 0.1 ml of a 5% solution of picryl chloride in ethanol to the skin of the abdomen; the day of immunization is referred to as day 0; the animals a single dose of 60 ~tg of rGK in 200 ~tl of PBS on the days indicated. b. Animals were tested with the application of a 1% solution of picryl chloride in olive oil on one ear. Results are expressed in terms of the increase if the challenged ear over the pre-challenge values using units equivalent to 1/10 ram. * p < 0.05.
Figure 6 shows the results of an experiment in the collagen arthritis (CA) model in rats. A single injection of GK given at the time when the arthritic reaction begins to flare induced an almost complete suppression which lasted 4-5 days, followed by the return to central levels of arthritis. In contrast, repeated injections maintained suppression for the entire duration of the experiment. Thus, this experiment confirmed that the effects of GK in immune animals are short lived and do not reduce the state of immunity of the animals. The effects described up to this point refer to in vitro phenomena or to the subcutaneous administration of GK. The fact that GK is found in high concentration in salivary glands and is secreted in saliva suggests that a significant physiological function of GK may occur following external salivary secretion. For this reason, we tested the effects of orally administered GK in rats. Experiments of this nature were carried out in rats using the collagen arthritis model. This involves the injection of Type II collagen in an oil based adjuvant which induces an arthritic reaction beginning two weeks after immunization, which lasts for the next 3-4 weeks.
232
10 x --
8 6
9 w,,,,,i
4
i
"
12
18
9
24
u
30
9
36
Days Figure 6. Effects of a single (V, day 14) or multiple ( I , days 14, 18, and 24) injections of a semi-purified preparation of rGK in the CA model. Controls (O) received PBS only.
The variables we tested included the effects of a pre-treatment with oral collagen to induce tolerance to the subsequent immunization; the effects of the surgical removal of the submandibular gland; and the effect of oral administration of GK, given before tolerization and/or before immunization to normal and sialoadenectomized rats. Figure 6 demonstrates the results of one of such experiments. The oral pre-treatment with Type II collagen significantly reduced the arthritic response in normal rats but had no such effect in sialoadenectomized animals. In contrast, the oral administration of GK significantly reduced the arthritic reaction. Figure 7 shows the results of another experiment which confirmed that sialoadenectomy interferes with tolerance induction. Moreover, this experiment demonstrated that the oral administration of GK to sialoadenectomized rats restores the ability of the mucosal immune system of these animals to develop tolerance upon oral collagen administration. These results point to similarities and differences in the action of GK depending on whether it is used in vitro or in vivo, and whether it is injected or given orally. The most prominent in vitro effect was to induce stimulation of lymphocyte proliferation, while the most prominent in vivo effect was an immunosuppressive one. One hypothesis to explain this apparent discrepancy in vivo suggests that immune deviation occurs following administration of GK in vivo, involving a reduction in the responses we assessed, but a stimulation of other responses which we did not resume. One example of such an immune deviation would be an altered balance of TH1 to TH2 responses with a decrease of TH1 activity. The responses we found to be suppressed by GK treatment were cell- mediated ones (DTH, allograft rejection and CA) or T-dependent IgM production (direct PFC response). These responses would be suppressed by any mechanisms that reduce TH1 activity or favor the switch from IgM to any other immunoglobulin class. This explanation is consistent with the observation that oral GK favors the induction of oral tolerance, a reaction thought to involve a deviation from cell-mediated responses to IgA production and induced by increased activity of TGF- (and IL-4) producing
233
4.5
A
IE IE nr"
uJ I--
4.0
I..U
< a
3.5
o lo
2'0 DAYS AFTER IMMUNIZATION
Figure 7. Effects of SMX and semi-purified oral rGK on the arthritic response and on the induction of oral tolerance in Sprague Dawley rats. (O) immunized controls; ( 9 oral collagen followed by immunization; (A) SMX followed by immunization; (m) SMX, oral collagen, followed by immunization; (@) oral rGK, followed by immunization.
TH3_ type cells. Alternately suppressor cells may be activated by GK. Yet another possible explanation for the apparent discrepancy of in vitro versus in vivo effects would suggest that in vitro stimulation of lymphocyte proliferation was due to the formation of stimulatory peptides, formed by the action of GK on some substrate(s) contained in the foetal calf serum present in our cultures. Under in vivo conditions, the proteolytic action of GK may result in decreased concentration of the same substrate(s) and in a deficient production of stimulatory peptides for a few days, until the substrates are reconstituted in full. Differences in GK action were also observed when the route of GK administration changed from subcutaneous to oral. In both situations, some suppression of immune reactions was observed if the treatment was simultaneous with or shortly preceded, antigen administration. On the other hand, the oral administration of GK appeared to enhance the induction of tolerance if given together with the oral antigen. The ensuing suppression of the arthritic reaction exceeded in duration and in magnitude the "immunosuppressive" effect of GK alone, administered either by injection or by mouth. The same experiments also demonstrated that oral tolerance could not be induced by antigen alone in sialoadenectomized animals. Oral tolerance is a state of antigen specific hyporesponsiveness which follows oral delivery of an antigen. It represents a protective reaction by the gut associated mucosal tissue (GALT) to prevent unnecessary and potentially harmful reactions to food antigens. It involves more than one mechanism. High antigen doses induce clonal deletion, while lower doses induce an active form of suppression or immune deviation, mediated by TGF- and IL-4 producing T cells,
234
6.5
6.0
E E v nLU i'LU
5.5
< a
.,,..=
n 100 kDa in rat testicular extracts that inhibited in vitro lymphocyte proliferation and which they named "protectin". Selawry et al., [102] have reported that Sertoli cell-conditioned medium (SCM) contains a factor that inhibits T-cell proliferation. Conversely, tubule conditioned medium and testicular intersitial fluid contain an "interleukin-l-like" protein which augments lymphoproliferation [103, 104] and testicular interstitial fluid contains material of >30 kDa that stimulates lymphoproliferation [32]. Hedger et al., showed in vitro stimulation of lymphocyte proliferation by inhibin and reduction by activin [105]. A coherent picture does not arise from these studies; the testis may contain a mixture of immuno-stimulatory and inhibitory factors. The normal immuno-suppressed state may represent the net effect of these factors on the immune system of the testis and may be influenced by the functional state of the gonad. Fas (CD95, Apo-1) is a membrane protein which, upon contact with crosslinking antibodies or the natural Fas ligand (FasL) elicits rapid apoptosis in the FAS expressing cell. This has led to the notion of "death factors" and the "death gene" as components of this pathway for programmed cell death [106]. It is believed that the Fas/FasL system plays an important role in regulating clonal expansion and contraction in the immune system. Several groups have suggested that Fas mediates both activation induced T-cell death (viewed by some as a mechanism to eliminate autoreactive T-cells) as well as the cytolytic effects of CD8 + T-cells [107, 108, 109, 110, 111]. In papers submitted within weeks of one another, two groups suggested that Fas/FasL might be expressed in non-lymphoid tissue and be involved in immune privilege. Griffith et al., [112] noted that Fas+, but not Fas-, cells were killed by apoptosis when placed in the anterior chamber of the eye. Bellgrau's group [113] demonstrated that expression of FasL by Sertoli cells was necessary to prevent their rejection as allografts. Both groups contend that immune privilege can be explained in part by Fas/FasL systems mediating death of immune cells in these privileged sites. It is already known that NO can induce apoptosis in several cell types [82, 83, 84]. It was recently discovered that NO can up regulate Fas and apoptosis in vascular smooth muscle. Given that the Sertoli cell may express both constitutive and inducible NOS as well as FasL, we embarked on a series of experiments that examined the potential for NO involvement in Sertoli-Leydig cell interactions. We hypothesized that activation of Sertoli cells with cytokines and bacterial lipopolysaccharide would result in increased expression of induceable nitric oxide synthase (iNOS) and production of nitric oxide (NO); this in turn would act directly on adjacent Leydig cells to alter synthesis of androgens. Sertoli cells from immature mice were activated in vitro with a mixture of 500 U/mL, Interferon% 1 ~g/mL, E.
275
coli lipopolysccharide, and 500 U/mL Tumor-Necrosis Factor-~t (I/L/T). The Sertoli cells responded with increased expression of iNOS mRNA and increased production of NO. I'--!
Control
BB LH 6.0
m
(9
o
4.0
e~ o .,m
E
~
z.0
01
0.0 None
Quiescent
Activated
Quiescent
Activated
L-NAME Figure 1. The effect of activation of Sertoli cells on androgen production by mouse Leydig cells in co-culture. Sertoli cells were untreated or activated for 24 hour with a mixture of I/L/T which was then removed at the same time Leydig cells were added. Activated Sertoli cells caused a significant (P < 0.001) inhibition of LH-stimulated androgen production. This inhibition was completely reversed by inhibition of NO production with L - N A M E .
As shown in Figure 1, Sertoli cells that were induced to NO production had an inhibitory effect on the production of testosterone by Leydig cells that were co-cultured with these activated Sertoli cells. These results have led us to suggest that exposure of Sertoli cells to agents that are associated with inflammation and immune activation result in induction of iNOS expression, and an attendant NO release. This NO production may account for a significant component of the decreased androgen production noted in neighboring Leydig cells. Of particular interest is the similarity of response of a somatic cell-the Sertoli cell, and an immune cell-the macrophage to activation. These observations provide an additional entry in the lengthening catalogue of examples of interactions and similarity of physiologic regulation between immune cells and cells of the reproductive and endocrine systems.
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New Foundation of Biology Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
283
Leptin and Cytolines: Actions and Interactions in Fever and Appetite Control
GIAMAL N. LUHESHI
The Douglas Hospital Research Center, Department of Psychiatry, McGill University, Quebec, Canada H4H-1R3
ABSTRACT Leptin, the 16 kDa ob gene product is an important regulator of energy balance via direct action on the brain. Since its discovery in 1994, there has been an explosion in this area of research aimed mainly at leptin's role in the regulation of appetite and by inference its use as a potential treatment for obesity. This hormone is synthesised mainly by adipocytes in relation to body mass and is released into the circulation from which it gains access to the brain via a saturable transport mechanism. In the brain leptin acts on its hypothalamic receptors to suppress appetite and increase energy expenditure thus collectively resulting in weight loss. On activating its receptors, leptin has been proposed to act via the induction of a number of secondary mediators including members of the pro-opiomelanocortin (POMC) family, corticotrophin releasing factor (CRF) and neuropeptide-Y (NPY). More recently interaction between leptin and the proinflammatory cytokine interleukin (IL)-I has been proposed, suggesting that this cytokine is involved in leptin production in the periphery. Our own recent findings in rodent brain have suggested the converse by demonstrating that leptin can induce IL-I[3 production in the hypothalamus. These findings have led us to propose that actions of leptin on food intake are mediated by the production and action of IL-I[5 in the brain. We have also demonstrated, that leptin, at the same dose that induces appetite suppression, is pyrogenic and that this effect is also mediated by IL-I[5. These results suggest that leptin may be an important mediator of neuroimmune interactions which activates CNS responses to disease, and reveal novel mechanisms of leptin action in the brain that depend on the synthesis and action of IL-115.
1.
OBESITY AND LEPTIN
Obesity affects at least 20% of the adult population in the Western world and is fast becoming the leading cause of illness world wide. A major factor that can lead to the development of obesity is dysregulation of energy balance, which is dependent on the level, and control of food intake (appetite) and energy expenditure (thermogenesis). Energy balance is controlled by the brain, through actions and interactions of a variety of peptides and neurotransmitters acting mainly in the hypothalamus to regulate appetite, satiety and thermogenesis [45]. Leptin appears
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to be particularly important in the regulation of this process and since its discovery by Zhang et al. in 1994 [46], has received a great deal of attention relating to its role as a principle regulator of appetite and energy expenditure [16, 46]. Leptin, (the product of the ob gene), is produced mainly by adipocytes and acts as a hormonal link between peripheral fat mass and the appetite regulating centres in the brain. Leptin does this by conveying information to the brain on the amount of energy stored as fat, reflecting the nutritional state of the individual [7] thus helping to maintain weight stability by modulating food intake and energy expenditure. It is not surprising therefore that dysfunction in the leptin system could lead to the development of obesity as dramatically demonstrated in mice with recessive mutations in either genes encoding leptin (ob/ob) or its receptor (db/db), and rats possessing a dysfunctional leptin receptor (fa/fa). These animals exhibit an abnormal increase in body weight, several fold greater than normal controls and approximately five fold increase in body fat content [16]. Injection of leptin in ob/ob mice and in normal rodents results in a reduction in food intake and body weight [7]; inhibiting the actions of endogenous leptin with antibodies [6] or antagonists in the shape of inactive leptin mutant forms [41 ] results in an increase in body weight. The weight loss induced by leptin is specific to the depletion of adipose tissue [21], which appears to be mediated by apoptotic mechanisms [35]. This is qualitatively distinct from the weight loss resulting from restriction of food intake, which includes loss of both fat and lean body mass [21 ]. These results suggest that leptin would perhaps present a way of treating obesity in humans. However, the vast majority (90-95%) of obese humans exhibit normal plasma leptin levels [29] relative to their body weight [15]. It is therefore likely that the development of obesity could be associated with failure of leptin transport into the brain (reduced in some obese individuals, [8]), or to insensitivity to leptin at the level of the receptor or post-receptor pathways, which probably present the best targets for the development of therapeutic strategies.
2.
SITES AND MECHANISMS OF ACTION OF LEPTIN
The primary site of leptin action on appetite and energy metabolism is the hypothalamus, particularly in the arcuate nucleus [20, 40], though actions in the brain stem have been reported [11]. The hypothalamus is rich in leptin receptor expression and lesions of hypothalamic areas involved in appetite control [e.g. ventromedial hypothalamus (VMH) and the arcuate (ARC) nuclei] results in obesity (see [12]). Circulating leptin gains entry to the brain by crossing the blood brain barrier (BBB) via a saturable transport mechanism [2]. This form of transport is mediated via the short (non-signalling) form of the leptin receptor that is expressed in blood vessels associated with the choroid plexus, meninges, hypothalamus and cerebellum [4]. This transport mechanism acts by endocytosis of the leptin molecule and is a specific and temperature-dependent system [ 19]. In the brain the specific mechanisms of leptin action have not been elucidated fully. A recent study [ 17] demonstrated that leptin influences the state of the brain reward circuitry resulting in the reduction of appetite, probably via the induction of mediators implicated in the control of feeding. The most prominent of these mediators include NPY and CRF [43] but others have also been implicated and include insulin, POMC and its cleavage product c~-melanocyte-stimulating hormone (et-MSH), urocortin, orexins/hypocretins, melanin-concentrating hormone (MCH), noradrenergic and serotonergic pathways, cocaine and amphetamine-regulated transcript (CART), agouti and agouti-related peptide, glucagon-like peptide-1 (GLP), and galanin (see [16] for review). Leptin may of course stimulate food intake and energy expenditure directly, or may act via as yet undetermined mediators such as the product of the TUB gene [24].
285
Alternatively, other molecules known to be involved in regulating energy balance such as cytokines may mediate actions of leptin (see Figure 1, for overview of leptin expression and actions). NPY .................................................. ~:>
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3.
CYTOKINES AND NEUROIMMUNE INTERACTIONS
Cytokines are a heterogeneous family of endogenous proteins that are produced in response to a variety of physiological and pathophysiological stimuli. These molecules which include the interleukins, interferons, colony stimulating factors growth factors and tumor necrosis factors induce a variety of effects and are generally considered to influence cell growth, differentiation, survival and in some cases cell death [38]. Cytokines are produced by virtually all cell types in the body but are associated particularly with the motile cells of the peripheral immune system. More diverse actions have now been reported and cytokines are now known to be released by and to affect a variety of organs including the brain. Association with the brain has become an area of intense study and interest, and the neurobiology of cytokines is now a major research area, in particular their role as neuroimmune modulators of host defence responses in disease. Via direct action on the brain cytokines can elicit fever, sickness behavior, reduced food intake, increased energy expenditure and cachexia (see [22, 26, 37] for comprehensive reviews of this area). Perhaps the best known and most intensely studied member of the cytokine family is interleukin (IL)-I. This cytokine exists in two forms (c~ & [5) both of which act on the same receptor (IL-1RI) and induce identical biological responses. This molecule was originally described as a heat labile protein that induced fever when administered to experimental animals or humans [ 1], and consequently was named "endogenous pyrogen". Other members of the IL-1 family include the naturally occuring receptor antagonist (IL-lra), which acts by limiting the action of IL-1 via competitively preventing its actions on the IL-1RI. In the brain IL-1 is expressed primarily by microglia but is also found in astrocytes [10] and it is activated in these cells after local (brain) or systemic injury or infection. Although it acts on a number of different brain sites, the primary area of I L - l ' s action in mediating the
286
host defense response to disease is the hypothalamus. Application of exogenous IL-1 directly to the hypothalamus or into the cerebral ventricles proximal to it results in the induction of fever and other sickness like behaviours including appetite suppression [36]. Studies on the role of endogenous IL-1 either by investigating its induction in the brain following a systemic stimulus or by inhibiting its action using IL-lra have confirmed that this cytokine is a primary mediator of sickness responses via direct action on the hypothalamus [37]. The nature of the afferent signal responsible for the induction of brain IL-1 is however still controversial. A role for IL-1 in this respect is somewhat controversial owing to a lack of significant biologically active amounts in the circulation of sick e.g. febrile humans or experimental animals. Unlike IL-1, however another cytokine, IL-6, increases dramatically in the circulation of febrile subjects and this increase correlates favorably with the development of fever. Our recent studies in rats have demonstrated the importance of this cytokine in fever by using a neutralizing antiserum raised against rat IL-6 which abolished the febrile response to systemic LPS (Figure 2 [9]).
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Other studies favour a neuronal link between the periphery and the brain namely the vagus nerve. A number of recent reports have demonstrated a role for the vagus nerve in transmitting cytokine signals to the brain to elicit an array of host defence responses. These responses, which included fever, were shown to be significantly inhibited in vagotomized experimental animals injected peripherally with inflammatory stimuli including IL-1 (see [30, 42] for reviews of this area). This evidence would suggest that a number of mechanisms are involved in relaying cytokine induced signals from the periphery to the brain during infection injury or inflammation. The relative importance of a humoral versus a neuronal signal remains to be determined. Recent evidence has linked circulating leptin with cytokines, a relationship that could be an important one in neuroimmune inetraction.
287
5.
INTERACTIONS BETWEEN LEPTIN AND CYTOKINES
It has been suggested that leptin is a cytokine [44]. The primary leptin receptor resembles gpl30, the common signal transducing subunit of the IL-6 receptor [3, 33] and leptin shares some actions with pro-inflammatory cytokines, such as suppression of appetite, and stimulation of thermogenesis [16]. Leptin production by adipose tissue (and its release into circulation) in rats is stimulated by administration of bacterial LPS, which is a potent stimulus for cytokine production in vivo [31, 32]. Leptin release is also induced by proinflammatory cytokines such as IL-1[3 and tumor necrosis factor (TNF)-~t [39], which themselves inhibit food intake and stimulate metabolic rate [37]. IL-1 has also been shown to mediate leptin induction during inflammation and LPS fails to increase leptin levels in mice lacking the gene expressing IL-I[3 [14]. Conversely, exogenous leptin has been demonstrated to up-regulate LPS-induced phagocytosis and proinflammatory cytokine expression in ex vivo mouse macrophages [27]. Additionally, leptin-deficient (ob/ob) mice and the obese (fa/fa) Zucker rats exhibit attenuated levels of serum TNF-c~ and IL-6 in response to LPS administration [27]. Our own recent studies [28] revealed that peripheral or central injection of leptin not only suppresses appetite but also induces marked fever in rats at doses similar to, or lower than, those which inhibit food intake [21, 34]. We also found that obese Zucker rats which posses a dysfunctional leptin receptor fail to develop fever in response to injection of recombinant leptin. These previously unreported effects of leptin appeared to be IL-1 mediated in the brain since co-administration of leptin systemically or into the brain with intracerebroventricular IL-lra abrogated the response on fever and food intake (Figure 3). In addition, experiments on genetically modified IL-1RI receptor knock-out mice showed that these animals do not respond to leptin's effect on food intake. These observations, along with the fact that leptin induces hypothalamic IL-I[3 production, further verified the involvement of IL-1 in leptin's action on appetite control and fever [28]. We subsequently demonstrated that the effects of leptin on body temperature, like those of IL-1, are inhibited by administration of a cyclo-oxygenase inhibitor [28]. Since cyclo-oxygenase inhibitors do not modify effects of leptin on food intake [28], different pathways appear to mediate the effects of leptin on body temperature and food intake. Pyrogenic cytokines also depend on PG's for induction of fever [13], but not suppression of food intake. Both of these responses (fever and appetite suppression) also form part of generalized sickness behavior responses to disease in which brain IL-I[3 is involved [23]. Interestingly, there are no reports associating leptin with defined sickness behaviors other than fever, which would suggest a divergence in the pathways controlling food intake and fever and other behaviors such as depressed social interaction, which is also induced by brain IL-I[3 [23]. Our recent observations would support this hypothesis, since we demonstrated that the effect of leptin on appetite involves a direct action of CRF [18] a neuropeptide shown not to be involved in sickness behavior [5]. In addition, we have now demonstrated that IL-6, which mediates the pyrogenic action of IL-1, does not itself induce sickness behavior [25]. This would suggest that IL-6 could play an important part in the mediation of leptin's action on appetite and fever either directly or through IL-1 (see Figure 4 for schematic representation). In fact IL-6 has previously been shown to be involved in body weight control [15, 35] however this evidence is largely circumstantial and no direct evidence exists to link brain IL-6 with appetite regulation.
6.
SUMMARY
The original suggestion that leptin could be a possible treatment for obesity through its appetite suppressing actions has led to a great deal of interest from scientist across many fields.
288
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Many aspects of the mechanisms of action of leptin were investigated resulting in the discovery that apart from regulating energy balance leptin is also involved in other biological processes making it pleiotropic in nature. Leptin has now been implicated in for example reproduction and development and in the pathogenesis and progress of a number of diseases, most notable diabetes. It is interesting to note that some of these processes are also influenced by the immune system, which has now been described to be a major target for leptin. This association and particularly the interaction with cytokines will no doubt receive more attention in the future.
289
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4.
5. 6.
7.
8.
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Cartmell T, Poole S, Turnbull AV, Rothwell NJ, Luheshi GN. Circulating interleukin (IL)-6 mediates the febrile response to localized inflammation in rats. J Physiol 2000; 526: 653-661. Davies CA, Loddick SA, Toulmond S, Stroemer RP, Hunt J, Rothwell NJ. The progression and topographic distribution of interleukin-lbeta expression after permanent middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 1999; 19: 87-98. Elmquist JK, Ahima RS, Maratos-Flier E, Flier JS, Saper CB. Leptin activates neurons in ventrobasal hypothalamus and brainstem. Endocrinology 1997; 138: 839-842. Elmquist JK, Elias CF, Saper CB. From lesions to leptin: hypothalamic control of food intake and body weight. Neuron 1999; 22: 221-232. Elmquist JK, Scammell TE, Saper CB. Mechanisms of CNS response to systemic immune challenge: the febrile response. Trends Neurosci 1997; 20: 565-570. Faggioni R, Fantuzzi G, Fuller J, Dinarello CA, Feingold KR, Grunfeld C. IL-1 beta mediates leptin induction during inflammation. Am J Physiol 1998; 274: R204-R208. Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB, Flier JS. Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nat Med 1995; 1: 1311-1314. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998; 395: 763-770. Fulton S., B. Woodside and P. Shizgal. Modulation of brain reward circuitry by leptin [published erratum appears in Science 2000 Mar 17; 287 (5460): 1931]. Science 2000; 287:125-128. Gardner J. D., N. J. Rothwell and G. N. Luheshi. Leptin affects food intake via CRFreceptor-mediated pathways. Nat Neurosci 1998; 1: 103. Golden PL, Maccagnan TJ, Pardridge WM. Human blood-brain barrier leptin receptor. Binding and endocytosis in isolated human brain microvessels. J Clin Invest 1997; 99: 14-18. Hakansson ML, Hulting AL, Meister B. Expression of leptin receptor mRNA in the hypothalamic arcuate nucleus: relationship with NPY neurones. Neuroreport 1996, 7: 3087-3092. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995; 269: 543-546. Hopkins SJ, Rothwell NJ. Cytokines and the nervous system. I: Expression and recognition. Trends Neurosci 1995; 18: 83-88. Kent S, Bluthe RM, Kelley KW, Dantzer R. Sickness behavior as a new target for drug development. Trends Pharmacol Sci 1992; 13: 24-28. Kleyn PW, Fan W, Kovats SG, Lee JJ, Pulido JC, Wu Y, Berkemeier LR, Misumi DJ, Holmgren L, Charlat O, Woolf EA, Tayber O, Brody T, Shu P, Hawkins F, Kennedy B, Baldini L, Ebeling C, Alperin GD, Deeds J, Lakey ND, Culpepper J, Chen H, GlucksmannKuis MA, Moore KJ. Identification and characterization of the mouse obesity gene tubby: a member of a novel gene family. Cell 1996; 85: 281-290. Lenczowski MJ, Bluthe RM, Roth J, Rees GS, Rushforth DA, Van Dam AM, Tilders FJ, Dantzer R, Rothwell NJ, Luheshi GN. Central administration of rat IL-6 induces HPA activation and fever but not sickness behavior in rats. Am J Physiol 1999; 276: R652-R658. Liles WC, Van Voorhis WC. Review: nomenclature and biologic significance of cytokines involved in inflammation and the host immune response. J Infect Dis 1995; 172: 1573-1580. Loffreda S, Yang SQ, Lin HZ, Karp CL, Brengman ML, Wang DJ, Klein AS, Bulkley
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GB, Bao C, Noble PW, Lane MD, Diehl AM. Leptin regulates proinflammatory immune responses. FASEB J 1998; 12: 57-65. 28. Luheshi GN, Gardner JD, Rushforth DA, Loudon AS, Rothwell NJ. Leptin actions on food intake and body temperature are mediated by IL-1. 1999; Proc Natl Acad Sci USA 96: 7047-7052. 29. " Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1995; 1: 1155-1161. 30. Maier SF, Goehler LE, Fleshner M, Watkins LR. The role of the vagus nerve in cytokineto-brain communication. Ann NY Acad Sci 1998; 840: 289-300. 31. Miller AJ, Hopkins SJ, Luheshi GN. Sites of action of IL-1 in the development of fever and cytokine responses to tissue inflammation in the rat. Br J Pharmacol 1997; 120: 1274-1279. 32. Miller AJ, Luheshi GN, Rothwell NJ, Hopkins SJ. Local cytokine induction by LPS in the rat air pouch and its relationship to the febrile response. Am J Physiol 1997; 272:R857-R861. 33. Nakashima K, Narazaki M, Taga T. Overlapping and distinct signals through leptin receptor (OB-R) and a closely related cytokine signal transducer, gpl30. FEBS Lett 1997; 401: 49-52. 34. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995; 269: 540-543. 35. Qian H, Azain MJ, Compton MM, Hartzell DL, Hausman GJ, Baile CA. Brain administration of leptin causes deletion of adipocytes by apoptosis. 1998; Endocrinology 139: 791-794. 36. Rothwell NJ. Functions and mechanisms of interleukin 1 in the brain. Trends Pharmacol Sci 1991; 12: 430-436. 37. Rothwell NJ, Hopkins SJ. Cytokines and the nervous system II: Actions and mechanisms of action. Trends Neurosci 1995; 18:130-136. 38. Sachs L, Lotem J. The network of hematopoietic cytokines. Proc Soc Exp B iol Med 1994; 206: 170-175. 39. Sarraf P, Frederich RC, Turner EM, Ma G, Jaskowiak NT, Rivet DJ, Flier JS, Lowell BB, Fraker DL, Alexander HR. Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia. J Exp Med 1997; 185: 171-175. 40. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG. Identification of targets of leptin action in rat hypothalamus. J Clin Invest 1996; 98:1101-1106. 41. Verploegen SA, Plaetinck G, Devos R, van der Heyden J, Guisez Y. A human leptin mutant induces weight gain in normal mice. FEBS Lett 1997; 405: 237-240. 42. Watkins LR, Maier SF, Goehler LE. Cytokine-to-brain communication: a review & analysis of alternative mechanisms. Life Sci 1995; 57: 1011-1026. 43. Wettstein JG, Earley B, Junien JL. Central nervous system pharmacology of neuropeptide Y. Pharmacol Ther 1995; 65: 397-414. 44. White DW, Tartaglia LA. Leptin and OB-R: body weight regulation by a cytokine receptor. Cytokine Growth Factor Rev 1996; 7: 303-309. 45. Wilding J, Widdowson P, Williams G. Neurobiology. Br Med Bull 1997; 53: 286-306. 46. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372: 425-432.
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IV.
NEUROIMMUNE HOST DEFENCE
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Introduction
REGINALD M. GORCZYNSKI
Department of Surgery & Immunology, University of Toronto, The Toronto Hospital, CCRW 2-855, 200 Elizabeth Street, Toronto, ON, Canada M5G-2C5
In the preceding chapters of this volume we have been introduced to the role of cytokines, and neurohormones, as mediators of CNS: immune system interactions. We have seen evidence that these mediators can influence behaviour and vice-versa. Particular attention has already focused on inflammatory cytokines (TNFc~, IL-1 etc), given the evidence already extant which supports their role in phenomena as widely separate as sleep induction (Moldofsky et al., Chapter III-6), appetite control (Luheshi, Chapter III-8), and even autoimmune disease (Sternberg and Moghaddam, Chapter II-6). In the section which follows four authors present reviews which develop this theme of CNS: immune system interactions further, and show how such interactions play an important part in the regulation of breaches in host defence, the sine qua non of immunity. In an interesting and thought-provoking discussion, Pittman et al., review recent work from the author's laboratory which implicates the neuropeptides alpha melanocyte stimulating hormone (etMSH) and arginine vasopressin (AVP) in particular, in reduction of pyresis. Sex-related differences in fever regulation are suggested by the authors to be related in turn to a decreased utilization of AVP in females. An intriguing possibility is that increased AVP production near term may also underlie the suppression of fever in response to peripheral pyrogens (such as IL-1 and LPS) in pregnant animals. Nevertheless, as the authors indicate if, as current dogma suggests, fever has a survival value, what is the rationale for production of these endogenous anti-pyretics? The paper that follows from the Befus laboratory (Davison et al.,) should not be read in isolation, but in the context of the earlier discussion by Forsythe et al., (Chapter III-3). This current review provides more detail on the intriguing role of salivary gland peptides in anaphylaxis and LPS-induced inflammation, and the functional activity of a tripeptide analogue (FEG) of the critical submandibular gland (SMG) peptide. It seems these molecules inhibit expression of important regulatory integrins (such as CD14), thus blocking recruitment and activation of neutrophils and eosinophils, responsible for tissue damage during inflammation. Since secretion from the SMG is itself under sympathetic control, these studies in turn begin to describe, at the molecular level, the pathway whereby innervation by the sympathetic chain might directly regulate inflammation in vivo. As mentioned earlier in this book, few discussions so enliven an audience as those on sex or appetite. One might add, on reflection, cancer (and probably heart disease). Two of the former are covered again in the last two chapters of this section. Baines provides a lucid and thoughtful review of a large literature on olfactory control of allorecognition, particularly as it pertains to
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mate selection in animals. Perhaps the most well-known reproductive responses to pheromonal stimuli are the so-called Whitten effect and the Bruce effect. The former describes the onset of oestrus in unmated female mice by the presence of the odour of an alien male, while the latter documents the pre-implantation block of pregnancy in mated females by the odour of an alien male. Both effects are apparently a reflection of release of neuroendocrine mediators, and both are believed to result from a "down-stream" regulation of the (female) host cell-mediated uterine response to the potential fetal graft. From the male perspective, few could find fault with this as a method of improving survival of one's own gene pool! Chow and her colleagues conclude this section with an insightful consideration of the role of neuroendocrinological regulation of "natural immunity" in host resistance to tumors, and perhaps even to inflammation and autoimmune disease. Activation of the acute-phase response invariably increases the titre of polyclonal natural antibody (Nab), and activity of activated macrophages, both of which are likely important in immune surveillance against tumors, especially NK-resistance tumors. Interestingly, major epitopes recognized by Nabs seem to be those believed to be primarily associated with T cell activation (CD25 and asialo-forms of CD45RA). This raises intriguing possibilities concerning the mechanism(s) by which intravenous Ig might prove efficacious in the treatment of a myriad of autoimmune disorders, and Chow provides evidence that indeed natural human serum used for iv infusion does recognize CD45RA. The authors conclude that the epitope recognized on CD45RA "may be a highly conserved homologous epitope or homotope of the neuroimmune system implicated in health and disease".
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Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
Fever and Antipyresis
QUENTIN J PITTMAN, ABDESLAM MOUIHATE and MARIE-STEPHANIE CLERGET
Neuroscience Research Group and Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta, Canada T2N-4N1
ABSTRACT One of the most common manifestations of disease is a regulated elevation in body temperature known as fever. A variety of experiments now indicate that fever is an integral part of the host defense response, which acts in synergy with other participants to combat disease. In the light of its importance, the sequence of events leading to development of the fever has been intensively studied. It is thought that lipopolysaccharide (LPS) released by bacteria, or other antigenic substances are phagocytosed by cells of the immune system, which in turn synthesize and secrete a number of cytokine molecules, including Interleukin-lbeta, Interleukin-6, and tumour necrosis factor-oz. These cytokines then activate the brain in various ways, including activation of sensory afferent nerves such as the vagus, direct access to the brain via circumventricular organs where the blood brain barrier is leaky and direct activation of endothelial cells in the microvasculature of the brain. Subsequent elaboration of prostaglandins of the E series from endothelial cells and glia appears to be an obligatory step for most experimental fevers; in keeping with this idea is the ability of prostaglandin synthesis inhibitors such as aspirin to reduce fever. Prostaglandins act largely in the anterior, ventral hypothalamus to cause activation of heat production and conservation mechanisms through sympathetic, hormonal and behavioral outputs. There appears also to be within the brain secondary synthesis of some cytokines, but the involvement of these molecules in the neural response is not well understood. The reduction of febrile body temperature appears also to be an active process. At least two transmitter molecules have been implicated as neurotransmitters within the brain to lower fever. These molecules include arginine vasopressin (AVP) and alpha-melanocyte stimulating hormone (alpha MSH); interference with the synthesis, release or action of these molecules results in elevated fevers. In addition, peripherally released corticosteroids suppress fever. Such molecules have been called endogenous antipyretics or cryogens. There are times in an animal's life when the ability to develop a fever is compromised; such times include the early neonatal period, the peri-parturient period in the mother, and some hypertensive states. The fever appears to be suppressed due to a central (neural) mechanism, a part of which may involve endogenous antipyretics. In the light of the survival value of fever, one questions why there are periods in an animal's life in which fever suppressed.
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1.
INTRODUCTION
While fever has been studied for well over a hundred years, the recognition that there are endogenous systems that limit or reduce fever (antipyresis) came only in the last 30 years. While the focus of this brief review is to provide an understanding of antipyresis, it is important to know the basics of the cascade of events underlying the fever response. Several recent reviews cover this field in depth [1-5], but the following provides a relatively brief overview.
2.
FEVER
When microorganisms (gram negative infections are best understood) invade our bodies, they expose our immune system to large lipopolysaccharide molecules (LPS) often called exogenous pyrogens or endotoxins. LPS binds to a soluble, circulating LPS binding protein and this complex binds to the CD 14 surface receptor found on certain monocytes and macrophages. These in turn synthesize and release a variety of endogenous proteins; those thought to be most important in fever are interleukin-l[3 (IL-I[3), IL-6 and tumour necrosis factor (TNF). During fever, a number of humoral changes take place, collectively called the acute phase response. However, the regulated rise in body temperature characteristic of fever involves the CNS and the mechanism by which peripherally generated cytokines or other peptides activate the brain has been intensively investigated. Evidence exists for several possible avenues, depending upon the route and dose of administration of cytokines (reviewed in [3, 6]). These include direct transport across the blood brain barrier, entry at circumventricular organs, local stimulation of perivascular and meningeal cells, and activation of peripheral nerves. Whatever the route of administration, it appears that most cytokines activate an inducible cyclooxygenase (COX 2), most likely in glia, to cause intracerebral synthesis of prostaglandins, largely of the E series (PGE; reviewed in [3, 7]). Peripheral immune stimuli activate many autonomic and endocrine nuclei, as revealed by Fos expression [8-10], but it is difficult to distinguish which pathways are involved in the fever response and which are involved in the many other autonomic responses (cardiovascular, gastrointestinal, etc) associated with immune activation, especially at the high doses often employed in these studies. Prostaglandins are known to act in several sites to activate central sympathetic pathways (reviewed in [7]), but the most sensitive of these for the purposes of fever generation appears to be a small nucleus in the ventral medial preoptic area (VMPOA) [3, 11]. Among other projections of this nucleus, that to the paraventricular nucleus (PVN) and nearby perifornical area appear to be particularly important sites for activation of heat conservation and thermogenesis to cause fever. In addition to the prostaglandin link, intense (i.e. high dose) peripheral immune activation causes synthesis within the brain of a variety of cytokines and certain transcription factors [1]. While application of IL-1 or TNF directly to the brain by icv injection will cause a fever, and receptors of such cytokines are present in the brain, the involvement of this brain cytokine system in the responses to peripheral immune stimuli is not well understood. Nonetheless, for some models of fever, particularly those with long latencies, injection of IL-1 receptor antagonist (IL-lra) into the brain will inhibit fever due to peripheral inflammation [ 12, 13].
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3.
ANTIPYRESIS
Fevers subside, either naturally by inactivation of causative organisms, or due to active pharmacological intervention (i.e. aspirin inhibits PGE synthesis). There is now good evidence that defervescence and fever suppression is a controlled process involving release and action of the neuropeptides, arginine vasopressin (AVP) and alpha melanocyte stimulating hormone (MSH). While the evidence for MSH is not as extensive as that for AVP, its icv injection reduces fever and an antagonist elevates fever [14, 15]. Salient facts supporting a role for AVP as an antipyretic arise from several laboratories [16, 17], including our own and are summarized as follows: 1. AVP, introduced into the brain (ventral septal area (VSA) or amygdala) reduces febrile, but not normal body temperature via an action at V 1 type receptors. 2. Activation of endogenous AVP pathways causes antipyresis. 3. Interference with AVP release or action results in elevated, prolonged fevers. 4. During defervescence, increased quantities of AVP are released into the brain. While these observations provide support of AVP's role, for the remainder of this review we will discuss possible avenues for future research and unresolved questions, drawing upon additional observations that implicate AVP in the reduction of fever.
4.
WHAT IS THE STIMULUS FOR THE RELEASE OF AVP?
As work with AVP antagonists has implicated endogenous release of AVP in the control of fever height and duration, we know that it is released during fever. In keeping with this, studies using immunohistochemistry for the immediate early onset protein Fos indicate that cell bodies in the bed nucleus of the terminalis (BST) are activated during fever induced by LPS [18]. Unfortunately, we do not know if these Fos-immunoreactive neurons are AVP in nature, as immunohistochemistry of AVP in this nucleus usually requires pretreatment with colchicine to allow visualisation of the peptide and this would interfere with fever. We considered the possibility that it was the rise in body temperature that was responsible for its release. To test this we took advantage of the fact that one can manipulate body temperature in urethane anaesthetised rats. We found that AVP was released during PGE induced fever even when the body temperature was initially at relatively low levels, i.e. 35~ Administration of PGE at high body temperatures was not associated with release if temperature was not allowed to rise. Similarly, elevating temperature alone, without the pyretic stimulus, was not associated with AVP release as measured by push-pull perfusion. Thus we concluded that both the pyretic stimulus and the rise in body temperature were the triggers [19]. It should be emphasized that these studies with PGE as the pyretic stimulus may not be representative of what occurs after administration of a peripheral pyrogen. Indeed, there is some evidence that AVP may be involved in hypothermic responses to cytokines when body temperature is never elevated [20]. Within the BST itself, there appear to be neurons responsive to cytokines that activate AVP neurons [21]. We were able to carry out push-pull studies in the terminal areas of BST vasopressinergic cells and demonstrate that micro infusion of IL-I[3 caused release of AVP in the VSA [22]. Thus, one could envision a scenario where intracerebral synthesis of IL-I[3, possibly followed by PGE synthesis, activates vasopressinergic neurons in the BST, which then initiate antipyresis.
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5.
HOW DOES AVP INITIATE ANTIPYRESIS?
We do not know the exact locus of AVP action within the brain, nor the mechanism by which it acts to reduce fever. There are two ill-defined loci that have been identified-the VSA [23] and the medial amygdala [24]. They are ill-defined because, in working with conscious animals, the precision of the micro injection techniques previously utilized did not facilitate identification of a specific locus; thus hence the description of this region as a "VS Area". Although there may be more than one site and mechanism of action, a likely possibility is that AVP may interfere with the ability of PGE to activate the cell group in the VMPOA identified by Saper et al as a "hot spot" of PGE sensitivity. In recent unpublished experiments we have observed that local application of AVP in nanoliter quantities interferes with the pyretic action of locally applied PGE, making this a likely possibility. The febrile response is but one of a host of reactions to LPS which include elaboration of hepatic derived proteins (host defence response) [25]; induction of a type of social withdrawal termed "sickness behaviour" [26]; changes in food intake [27]; and alterations in hormone secretion [28, 29]. Most of these responses can occur independently. Just as it attenuates fever, AVP also attenuates sickness behaviour via an action in the VSA [30], but it is not known if it has an action upon other components of the response to LPS. One such action could occur within the brain where AVP may act on neural pathways important in controlling the hypothalamic-pituitary axis. In support of such a possibility is the observation that brain vasopressin is involved in stress-induced suppression of immune function in the rat [31]. It is also important to note that the source of the AVP involved in brain antipyresis is a group of AVP immunoreactive cell bodies located in the bed nucleus of the stria terminalis (BST) and is not the AVP found in the magnocellular PVN and supraoptic nuclei (SON). The latter cell bodies project to the pituitary where they release AVP into the circulation to regulate renal and cardiovascular function. It is interesting that, in addition to the release of AVP within the brain that we have described [19], it is also released during fever from the pituitary into the circulation [29, 32]. It would be important to know if this circulating AVP interacts with the neuroimmune response resulting from LPS exposure. It is possible that AVP's well known actions on cardiovascular and renal systems are important to counteract the cardiovascular collapse that can occur after high doses of LPS. Several other actions have also been proposed relating to an action of AVP to "restore homeostasis" [33]. Current dogma is that plasma AVP does not affect fever [34]. However, as fever is associated with high circulating levels of AVP, receptors are most likely saturated and it would appear unlikely that exogenously applied AVP would have much of an effect. However, experiments using peripheral AVP antagonists may reveal such an action. One could predict a possible action on ACTH, as AVP is a releasing factor in conjunction with CRH. Another possibility is a direct action on the adrenal cortex to enhance corticosterone secretion. V I receptors have been localized to the adrenal [35] and AVP increases corticosterone secretion of the isolated perfused adrenal gland [36].
6.
GENDER DIFFERENCES
It has now been reported in many laboratories, including our own, that fever size and duration can change dramatically between males and females [37] and also in association with the reproductive cycle [38]. While the mechanism for these changes is not known, there is circumstantial evidence that varying antipyretic action may underlie these changes n particular,
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we have evidence that females have higher fevers in response to central PGE than do males, and this appears to be associated with a reduced utilization of AVP as an antipyretic. We found that an AVP antagonist did not elevate PGE fever in females unlike the effect in males, and also did not display the elevation in VSA AVP release associated with fever. This is possibly because the AVP innervation arising from the BST is sexually dimorphic and much reduced in females [39]. As other substances exist in the brain with potential antipyretic action, it is possible that females may use a different strategy to lower body temperature. For example, an antagonist of MSH has recently become available and was shown to prolong fever in male rats [14]. It's function merits testing in females. Of course, it is also possible that females are simply more responsive to PGE because of differences in PGE receptor or numbers. There are at least 4 such receptors identified by binding studies and they are designated EP1-4 [40]. cDNAs encoding these receptors have been cloned and a number of isozymes are known to exist [41, 42]. There is evidence that gene expression for at least some of these receptors is enhanced by estrogen [43]. The EP3 subtype appears to be the predominant form expressed in rodent brain [44] and is found in high concentration in the medial POA [45]. Other data indicate a role for the EP1 receptor in fever [46]. Studies need to be carried out to ask if PGE receptors are more numerous or show different affinities in females. As pointed out above, PGE 1 induced fevers are higher in females than in males. Now it is necessary to look earlier in the fever cascade to ask if IL-I[3 and LPS fevers are similarly different in males and females. While this might seem obvious, there are reasons to suspect that it may not be so. Responses to peripherally injected pyrogens are known to be modulated by circulating steroids [47]; females have higher circulating glucocorticoids and enhanced steroid secretion in response to some kinds of stress [48].
7.
ENDOGENOUS ANTIPYRESIS
Central AVP may also be important in causing a condition we have called "endogenous antipyresis". This is a state in which the normal febrile response to a pyrogen is reduced. It can be seen in certain neonates; in some types of hypertension; in acute hypotension; and in parturient animals (reviewed in [49]). However, we still do not know what is responsible for the antipyresis seen at parturition. Suppression of fever due to peripheral pyrogens such as LPS, IL-I[3 and to centrally administered PGE in pregnant animals including rats has been reported by several labs, including our own [50-54]. The phenomenon is most evident when pregnancy is close to term. While the fact that PGE fever is suppressed at term [54] suggests a central mechanism, the fact that the suppression is actually most profound after injection of LPS [53] raises the possibility of more than one locus. Two possibilities which are not necessarily mutually exclusive may account for the antipyresis of parturition: 1. It may be due to a general reduction in central sympathetic drive, including that to sympathetic organs involved in thermogenesis and heat conservation. In favour of this possibility is the well known reduction in peripheral vascular responsiveness and baroreflex during pregnancy [55-57]. In addition, we have been able to show that cardiovascular responses to centrally injected PGE are also reduced at term [58]. 2. There may b e a specific endogenous antipyretic activity which manifests itself at term. In favour of this is our previous observation that there appears to be more AVP in the VSA in pregnant and post-parturient rats compared to non-pregnant females [59]. Several studies have tested this possibility. We were unable to demonstrate enhanced vasopressinergic
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'tone' at the time of parturition [58], but another laboratory reported that an AVP antagonist was effective in elevating fever at term, but not at other times [60]. However, this same laboratory was unable to find evidence for AVP involvement in the suppression of interleukin fever, which is also thought to involve a PGE step [61]. We have recently looked at the expression of the AVP receptor in the brain around parturition and have also found no differences (Clerget, unpublished observations).
8.
PERSPECTIVES
It is now appreciated that fever is an important part of the host defense response, but that it must be regulated at an optimum level to fight infection without deleterious effects upon the organism. It therefore makes good physiological sense that there are endogenous mechanisms to limit its height and duration. There are many questions which have been raised in the preceding discussion which will require investigation to resolve the mechanisms by which AVP acts as an endogenous antipyretic. With respect to the suppression of fever at certain times, one must ask why this takes place when fever is thought to have survival value.
9.
ACKNOWLEDGEMENTS
This work supported by MRC/CIHR.
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activates fever-producing autonomic pathways. J Neurosci 1996; 16: 6246-6254. Luheshi G, Hopkins SJ, Lefeuvre RA, Dascombe MJ, Ghiara P, Rothwell NJ. Importance of Brain IL-1 Type II Receptors in Fever and Thermogenesis in the Rat. American Journal of Physiology 1993; 265: E585-E591. 13. Luheshi G, Miller AJ, Brouwer S, Dascombe MJ, Rothwell NJ, Hopkins SJ. Interleukin-1 receptor antagonist inhibits endotoxin fever and systemic interleukin-6 induction in the rat. Am J Physiol Endocrinol Metab 1996; 270:E91-E95. 14. Huang QH, Entwistle ML, Alvaro JD, Duman RS, Hruby VJ, Tatro JB. Antipyretic role of endogenous melanocortins mediated by central melanocortin receptors during endotoxin-induced fever. J Neurosci 1997; 17:3343-3351. 15. Rajora N, Boccoli G, Burns D, Sharma S, Catania AP, Lipton JM. alpha-MSH modulates local and circulating tumor necrosis factor-alpha in experimental brain inflammation. J Neurosci 1997; 17:2181-2186. 16. Zeisberger E. Role of Vasopressin in Fever Regulation and Suppression. TIPS 1985; 6(11): 428-430. 17. Cridland RA, Kasting NW. A Critical Role for Central Vasopressin in Regulation of Fever During Bacterial Infection. Am J Physiol 1992; 263: R1235-R1240. 18. Hare AS, Clarke G, Tolchard S. Bacterial lipopolysaccharide-induced changes in FOS protein expression in the rat brain: correlation with thermoregulatory changes and plasma corticosterone. J Neuroendocrinol 1995; 7:791-799. 19. Landgraf R, Malkinson T, Veale WL, Lederis K, Pittman QJ. Vasopressin and oxytocin in the rat brain in response to prostaglandin fever. Amer J Physiol 1990; 259: R1056-R1062. 20. Derijk RH, Berkenbosch F. Hypothermia to Endotoxin Involves the Cytokine Tumor Necrosis Factor and the Neuropeptide Vasopressin in Rats. Am J Physiol 1994; 266: R9-R14. 21. Wilkinson MF, Mathison WB, and Pittman QJ. Interleukin-I[5 has excitatory effects on neurons of the bed nucleus of the stria terminalis. Brain Res 1993; 625: 342-346. 22. Wilkinson MF, Horn TFW, Kasting NW, Pittman QJ. Central interleukin-l[3 stimulation of vasopressin release into the rat brain: Activation of an antipyretic pathway. J Physiol (Lond.) 1994; 481: 641-646. 23. Naylor AM, Ruwe WD, Veale WL. Thermoregulatory actions of centrally- administered vasopressin in the rat. Neuropharmacology 1986; 25: 787-794. 24. Federico P, Veale WL, Pittman QJ. Vasopressin-induced antipyresis in the medial amygdaloid nucleus of conscious rats. Amer J Physiol: Regulatory, Integrative and Comparative Physiology 1992; 262: R901-R908. 25. Long NC. Evolution of infectious disease: How evolutionary forces shape physiological responses to pathogens. News Physiol Sci 1996; 11: 83-90. 26. Hart BL. Biological Basis of the Behavior of Sick Animals. Neurosci Biobehav Rev 1988; 12: 123-137. 27. Kent S, Bret-Dibat JL, Kelley KW, Dantzer R. Mechanisms of sickness-induced decreases in food-motivated behavior. Neurosci Biobehav Rev 1996; 20: 171-175. 28. Rivier C, Rivest S. Mechanisms mediating the effects of cytokines on neuroendocrine functions in the rat. Ciba Found Symp 1993; 172: 204-220. 29. Landgraf R, Neumann I, Holsboer F, Pittman QJ. Interleukin-l[5 stimulates both central and peripheral release of vasopressin and oxytocin in the rat. Eur J Neurosci 1995; 7: 592-598. 30. Dantzer R, Bluthe RM, Kelley KW. Androgen-dependent vasopressinergic neurotransmission attenuates interleukin-l-induced sickness behavior. Brain Res 1991; 557: 115-120. 12.
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Shibasaki T, Hotta M, Sugihara H, Wakabayashi I. Brain vasopressin is involved in stress-induced suppression of immune function in the rat. Brain Res 1998; 808: 84-92. 32. Kasting NW, Carr DB, Martin JB, B lume H, Bergland R. Changes in Cerebrospinal Fluid and Plasma Vasopressin in the Febrile Sheep. Can J Physiol Pharmacol 1983; 61: 427-431. 33. Martin, SM, Malkinson TJ, Veale WL, Pittman QJ. Prostaglandin Fever in Rats is Altered by Kainic Acid Lesions of the Ventral Septal Area. Brain Res 1988; 455: 196-200. 34. Cooper KE, Kasting NW, Lederis K, Veale WL. Evidence Supporting a Role for Endogenous Vasopressin in Natural Suppression of Fever in the Sheep. J Physiol 1979; 295: 33-45. 35. Balla T, Enyedi P, Spat A, Antoni FA. Pressor-type vasopressin receptors in the adrenal cortex: properties of binding, effects on phosphoinositide metabolism and aldosterone secretion. Endocrinology 1985; 117: 421-423. 36. Hinson JP, Vinson GP, Porter ID, Whitehouse BJ. Oxytocin and arginine vasopressin stimulate steroid secretion by the isolated perfused rat adrenal gland. Neuropeptides 1987; 10: 1-7. 37. Chen X, Landgraf R, Pittman QJ. Differential ventral septal vasopressin release is associated with sexual dimorphism in PGE 2 fever. Am J Physiol Regul Integr Comp Physiol 1997; 272: R 1664-R 1669. 38. Mouihate A, Chen X, Pittman QJ. Interleukin-lbeta fever in rats: gender difference and estrous cycle influence. Am J Physiol 1998; 275: R1450-R1454. 39. DeVries GJ, Buijs RM, van Leeuwen FW, Caffe AR, Swaab DF. The Vasopressinergic Innervation of the Brain in Normal and Castrated Rats. Journal of Comparative Neurology 1985; 233: 236-254. 40. Negishi M, Sugimoto Y, Ichikawa A. Prostanoid receptors and their biological actions. Prog Lipid Res 1993; 32:417-434. 41. Kawamura T, Yamauchi T, Koyama M, Maruyama T, Akira T, Nakamura N. Expression of prostaglandin EP2 receptor mRNA in the rat spinal cord. Life Sci 1997; 61: 2111-2116. 42. Manba T, Sugimoto Y, Negishi M, Irie, A, Ushikubi F, Kakizuka A, Ito S, Ichikawa A, Narumiya S. Alternative splicing of prostaglandin E receptor subtype EP 3 determines Gprotein specificity. Nature 1993; 365: 166-170. 43. Rage F, Lee BJ, Ma YJ, Ojeda SR. Estradiol enhances prostaglandin E 2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE 2 by activating a glia-to-neuron signaling pathway. J Neurosci 1997; 17: 9145-9156. 44. Sugimoto Y, Shigemoto R, Namba T, Negishi M, Mizuno N, Narumiya S, Ichikawa A. Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 in the mouse nervous system. Neuroscience 1994; 62: 919-928. 45. Ericsson A, Arias C, Sawchenko PE. Evidence for an intramedullary prostaglandindependent mechanism in the activation of stress-related neuroendocrine circuitry by intravenous interleukin- 1. J Neurosci 1997; 17:7166-7179. 46. Oka T, Hori T. EPl-receptor mediation of prostaglandin E2-induced hyperthermia in rats. Am J Physiol 1994; 267: R289-94. 47. Coelho MM, Luheshi G, Hopkins SJ, Pel~i IR, Rothwell NJ. Multiple mechanisms mediate antipyretic action of glucocorticoids. Am J Physiol Regul Integr Comp Physiol 1995; 269: R527-R535. 48. Kant GJ, Lenox RH, Bunnell BN, Mougey EH, Pennington LL, Meyerhoff JL. Comparison
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of stress response in male and female rats: pituitary cyclic AMP and plasma prolactin, growth hormone and corticosterone. Psychoneuroendocrinology 1983; 8:421-428. Pittman QJ, Wilkinson MF. Central arginine vasopressin and endogenous antipyresis. Can. J Physiol Pharmacol 1992; 70: 786-790. Cooper KE, B lahser S, Malkinson TJ, Merker G, Roth J, Zeisberger E. Changes in body temperature and vasopressin content of brain neurons, in pregnant and non-pregnant guinea pigs, during fevers produced by PolyI: Poly C. Pflugers Arch 1995; 412: 292-296. Simrose RL, Fewell JE. Body temperature response to IL-1 beta in pregnant rats. Am J Physiol 1995; 269: R 1179-82. Stobie-Hayes KM, Fewell JE. Influence of pregnancy on the febrile response to intracerebroventricular administration of PGE 1 in rats. J Appl Physiol 1996; 81: 1312- 1315. Martin SM, Malkinson TJ, Veale WL, Pittman QJ. Fever in pregnant, parturient, and lactating rats. Am J Physiol Regul Integr Comp Physiol 1995; 268: R919-R923. Martin SM, Malkinson TJ, Veale WL, Pittman QJ. Prostaglandin fever in rats throughout the estrous cycle, late pregnancy and parturition. J Neuroendocrinol 1996; 8: 145-151. Brooks VL, Quesnell RR, Cumbee SR, Bishop VS. Pregnancy attenuates activity of the baroreceptor reflex. Clin Exp Pharmacol Physiol 1995; 22: 152-156. Deng YM, Kaufman S. Effect of pregnancy on activation of central pathways following atrial distension. Am J Physiol Regul Integr Comp Physiol 1995; 269: R552-R556. Heesch CM, Rogers RC. Effects of pregnancy and progesterone metabolites on regulation of sympathetic outflow. Clin Exp Pharmacol Physiol 1995; 22:136-142. Pittman QJ, Chen X, Mouihate A, Hirasawa M, Martin S. Arginine vasopressin, fever and temperature regulation. Prog Brain Res 1998; 119: 383-92: 383-392. Landgraf R, Neumann I, Pittman QJ. Septal and Hippocampal Release of Vasopressin and Oxytocin During Late Pregnancy and Parturition in the Rat. Neuroendocrinology 1991; 54: 378-383. Eliason HL, Fewell JE. AVP mediates the attenuated febrile response to administration of PGE1 in rats near term of pregnancy. Am J Physiol 1998; 275: R691- R696. Eliason HL, Fewell JE. Arginine vasopressin does not mediate the attenuated febrile response to intravenous IL-lbeta in pregnant rats. Am J Physiol 1999; 276: R450-R454.
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Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
Salivary Gland Peptides: Their Role in Anaphylaxis and Lipopolysaccharide (LPS)-Induced Inflammation
J.S. DAVISON, D. BEFUS 1 and R. MATHISON
Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N-4N1 1Asthma Research Institute, University of Alberta, Edmonton, Alberta
ABSTRACT About a decade ago, we published the first of a series of studies, which eventually led us to develop the concept of a sympathetic-superior cervical ganglion-submandibular gland axis, which appeared to regulate the release of immunosuppressive substances from the submandibular glands of rats. These putative mediators could suppress anaphylaxis and LPS-induced shock. We eventually discovered that the agents released from the salivary glands capable of producing the suppression of these inflammatory responses were novel, small molecular weight peptides and our current work has focused on the actions of one of these (submandibular gland peptide-T: SGP-T) and its analogs on animal models of anaphylaxis and LPS-induced inflammation. SGP-T, as well as the C terminal tri-peptide FEG, are both potent inhibitors of intestinal and cardiovascular anaphylaxis in egg albumen-sensitized Hooded-Lister or Sprague Dawley rats. They also inhibit endotoxin-induced hypotension in Sprague Dawley rats. These results are a striking demonstration of the ability of these salivary gland peptides to inhibit early phase immune responses. We have shown that the D-isomer of FEG (feG) prevents the infiltration of leukocytes following injection of LPS into the peritoneum. Similarly, in another presentation, at this meeting, we show that feG can also block late phase responses in anaphylaxis by preventing infiltration of pulmonary tissue by leukocytes. In other models, we have been able to show that these peptides inhibit carrageenan-induced neutrophilia within the skin and inhibit leukocyte rolling and adhesion in mesenteric venules. Current work is focusing on the molecular mechanisms which lead to recruitment and activation of leukocytes into intestinal tissue following anaphylaxis and LPS activation. We will present data showing inhibition of expression of identified cell markers involved in chemotaxis and activation in both these models, which reveals some interesting differences between the two, suggesting that these peptides may act on different receptor subtypes. In summary, our collective work to date implies that there is an important sympathetic pathway, involving the superior cervical ganglion, that regulates the release of novel peptides from the submandibular glands which play an important part in early and late phase immune responses in anaphylaxis and LPS-induced inflammation. In future work we hope, not only
308
to reveal the molecular mode of action of these peptides, but also to return to the point where we began and use our increasing molecular knowledge to study the neuroregulation of their production and release.
1.
INTRODUCTION
The salivary glands subserve a number of physiological functions [1]. Besides their wellrecognized role in carbohydrate digestion through the secretion of salivary amylase, they play an important role in the growth and development of the digestive tract in the young, and in the maintenance and repair of mucosal integrity in the adult through the secretion of trophic factors such as epidermal growth factor, nerve growth factor, and transforming growth factor [3. It has been long recognized that saliva plays an important role in wound healing involving not only the growth factors, but also suppression of local infection through the antibacterial properties of lyzozymes. It has become increasingly evident however, in the past decade, that the salivary glands also play a role in the regulation of the immune system at both a local and systemic level, due to the release of immunosuppressive agents that modulate the activity of leukocytes, in particular granulocytes, and perhaps other cells of the immune system and that the sympathetic nervous system regulates the release of these salivary gland immunoregulatory agents. Much of the work upon which this concept of a sympathetic nervous system-salivary gland-immune system axis is based was carried out in our laboratories [2]. The present paper will review some of the key findings which led to this concept and will present some new preliminary data that provide an explanation at the molecular level of the way in which immunosuppressive agents released from the salivary glands might suppress inflammatory responses.
THE ROLE OF THE SUPERIOR CERVICAL GANGLION AND THE SALIVARY GLANDS IN THE REGULATION OF ANAPHYLACTIC AND ENDOTOXIN-INDUCED SHOCK About a decade ago we published the first in a series of studies which eventually led us to develop the concept of a sympathetic-superior cervical ganglion-submandibular gland axis, which appeared to regulate the release of immunosuppressive substances from the submandibular glands of rats. These putative mediators could suppress the late phase pulmonary inflammation following anaphylaxis as well as LPS-induced shock [3, 4]. We showed that cutting the superior cervical sympathetic nerve, thereby decentralizing the superior cervical ganglion, modified the late phase pulmonary inflammation induced in rats previously parasitized with Nippostronglylus brasiliensis (Nb). The effect of the denervation procedure was eliminated by extirpation of the submandibular glands, suggesting that the cervical sympathetic nervous system was, in some way, regulating the release of immunomodulatory substances from the submandibular glands, which then modified the ability of leukocytes to infiltrate the pulmonary epithelium [3]. Subsequently we showed that endotoxin (LPS)-induced shock was exacerbated by cutting the superior cervical sympathetic trunks or by bilateral extirpation of the submandibular glands [4]. These observations again supported the view that the sympathetic nervous system regulated the immune system by modulating the release of immunoreactive agents from the salivary glands. Specifically we postulated that these agents were immuno-suppressive substances, whose secretion could either be inhibited or excited by the sympathetic nervous system, depending on the physiological, pathophysiological or experimental conditions pertaining at the time.
309
.
ISOLATION AND IDENTIFICATION OF NOVEL SUBMANDIBULAR GLAND PEPTIDES
On the basis of these earlier studies, we postulated that rat submandibular glands contained factors that would reduce the severity of endotoxin or anaphylaxis-induced hypertension. Therefore we carried out a series of studies using classical peptide isolation procedures in which extracts of rat submandibular glands were subjected to molecular weight cut off filtration followed by preparative, reverse phase high performance liquid chromatography (HPLC) and finally analytical HPLC purification. At each step in the process isolated fractions were tested for their ability to reduce the severity of endotoxin-induced hypertension. As a result, a novel heptapeptide was isolated from these extracts, which was subsequently sequenced and synthesized and shown to attenuate the severity of endotoxin-induced hypotension [5]. This peptide had the sequence TDIFEGG and will subsequently be referred to as submandibular gland peptide T (SGP-T). In this and subsequent studies, we confirmed that SGP-T would reduce the severity of cardiovascular and intestinal anaphylaxis in Nb and ovalbumen sensitized rats, as well as the severity of hypotension and fever induced by lipopolysaccharide [6-10]. Subsequent structure-activity relationship studies revealed that the inhibition of intestinal anaphylaxis required only the tripeptide FEG localized at the carboxyl terminal of the parent heptapeptide [11]. Our current studies, therefore, have focused on the parent molecule SGP-T and its tripeptide analogue FEG and the D-isomeric form-the tripeptide leG. During the period we were sequencing the active peptide, a search of the gen bank database revealed that TDIFEGG is a COOH-terminal fragment of the submandibular gland rat 1 (SMR1) protein at positions 138-144 of this 146 amino acid protein. The structure of this prohormone was deduced from the cDNA sequence of the SMR1-VA1 gene in the submandibular glands of Wistar rats (12). The SMR1-VA1 protein in Sprague-Dawley or Fischer rats differs by only one amino acid from that found in Wistar rats (13). This does not affect the TDIFEGG sequence, which is present in all rat strains studied thus far. The SMR1-VA1 gene is one of the variable coding sequence (VCS) multigene family of genes of which 3 belong to the VCSA subgroup and are found only in the rat. The gene coding for the anti-shock peptide SGP-T is one of these and our work provided the first description of a biological activity for a peptide product of a VCSA gene.
4.
MECHANISM OF ACTION
Since isolating SGP-T we have tested it in various models of shock and inflammation several of which have been alluded to above and another is reported in an article in these proceedings (Befus et al.). As a result we know that SGP-T and certain analogues such as FEG and feG can attenuate both immediate and later phases of shock or inflammation. Our studies of the mechanism of action of these novel antishock/antiinflammatory peptides has focused on the recruitment of inflammatory cells during the early to middle periods of inflammation. These cells are the source for the development of the late phase reaction. We have shown that SGP-T and analogues inhibit leukocyte rolling (14) an important first step in the recruitment and subsequent activation of leukocytes. This process of recruitment is initiated by chemotactic agents and inhibition of recruitment was one of the first properties we identified for our putative anti-shock hormone prior to its isolation (15). Recruitment and activation is regulated by a large family of integrin-associated proteins and some of our recent work has focused on the actions of feG on integrin expression. We have found that
310
intraperiteonal injection of LPS into rats increases the expression of CD18 on mesenteric tissue macrophages by 4-fold and that a single treatment with feG (100 ~tg/kg) abrogated the increase in this cell activation marker [16]. In addition, we found that feG reduces the expression of CDllb and CD16b (Fc~,RIIIb) on isolated human neutrophils provoked by platelet activating factor (PAF) [17]. Since the peptide did not have noticeable effects on CD43, CD62L or CD162, we are actively exploring the role of feG in regulating the expression and activity of the [3-integrins and IgG-receptors. These results are consistent with our original hypothesis and provide us with the beginning of an explanation for the action of these peptides. By blocking the expression of important regulatory integrins, including the LPS receptor CD14, these salivary gland peptides are able to inhibit recruitment and activation of leukocytes such as neutrophils and eosinophils which are responsible for tissue damage in late phase inflammation. In summary, our collective work to date implies that there is an important sympathetic pathway through the superior cervical ganglion, that regulates the release of novel peptides from the submandibular glands which play an important role in immediate and later phase immune responses during anaphylaxis and LPS-induced inflammation. In future work we hope to determine, not only the molecular mode of action of these peptides, but also to return to the point where we began and use our increasing knowledge of molecular mechanisms to develop the tools to study the neuroregulation of the production and release of these immunoregulatory hormones.
REFERENCES 1. 2. 3.
4. 5.
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Barka T. Biologically active polypeptides in submandibular glands. J Histochem Cytochem 1980; 28: 836-859. Mathison R, Befus D, Davison JS. Neuroendocrine regulation of inflammation and tissue repair by submandibular gland factors. Immunology Today 1994; 15: 527-532. Ramaswamy K, Mathison R, Carter L, Kirk D, Green F, Davison JS, Befus D. Marked antiinflammatory effects of decentralization of the superior cervical ganglia. J Exp Med 1990; 172: 1819-1830. Mathison R, Befus D, Davison JS. Removal of the submandibular glands increases the acute hypotensive response to endotoxin. Circ Shock 1993; 39: 52-58. Mathison RD, Befus AD, Davison JS. A novel submandibular gland peptide protects against endotoxic and anaphylactic shock. Am J Physiol 1997; 273 (Regulatory Integrative Comp Physiol 42): R1017-R1023. Mathison RD, Befus AD, Davison JS. Attenuation of cardiovascular anaphylaxis by submandibular gland peptide-T (SGP-T). Proc West. Pharmacol Soc 1997; 40: 5-7. Mathison RD, Tan D, Oliver M, Befus AD, Davison JS, Scott B. A novel peptide from submandibular glands inhibits intestinal anaphylaxis. Dig Dis Sci 1997; 442: 2378-2383. Mathison RD, Davison JS, Moore G. Submandibular Gland Peptide-T (SGP-T). Modulation of endotoxic and anaphylactic shock. Drug Discovery Research 1997; 42: 164-171. Mathison RD, Malkinson T, Cooper KE, Davison JS. Submandibular glands: Novel structures participating in thermoregulatory responses. Can J Physiol Pharmacol 1997; 75: 407-413. Mathison R, Kubera M, Davison JS. Submandibular Gland Peptide-T (SGP-T) modulates ventricular function in response to intravenous endotoxin. Pol J Pharmacol 1999; 51: 331-339. Mathison RD, Lo P, Davison JS, Scott B, Moore G. Attenuation of intestinal and
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12.
13.
14. 15.
16. 17.
cardiovascular anaphylaxis by the salivary gland tripeptide FEG and its D-isomeric analogue feG. Peptides 1998; 19: 1037-1042. Rosinski-Chupin I, Tronik D, Rougeon F. High level of accumulation of a mRNA coding for a precursor-like protein in the sub-maxillary gland of male rats. Proc Natl Acad Sci USA 1988; 85: 8553-8557. Rosinki-Chupin I, Rougeon F. One amino acid change in rate SMRI polypeptide induces a lkDa difference in its apparent molecular mass determined by electrophonetic analysis. FEBS Lett 1990; 267: 147-149. Mathison R, Sank C, Davison JS. Inhibition of leukocyte rolling by submandibular gland peptide-T (SGP-T). Proc West. Pharmacol Soc 1999; 42: 39-40. Carter L, Ferrari JK, Davison JS, Befus D. Inhibition of neutrophil chemotaxis and activation following decentralization of the superior cervical ganglia. J Leukoc Biol 1992; 51: 597-602. Mathison RD, Lo P. Attenuation of intestinal endotoxemia in rats by the salivary gland tripeptide FEG and its d-isomeric analog feG. INABIS Symposium. Mathison RD, Teoh D, Woodman R, Lo P, Davison JS, Befus D. Regulation of neutrophil function by SMR1 C-terminal peptides. Shock 2000; 13 (Suppl): 52.
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Edited by I. Berczi and R.M. Gorczynski 9 2001 Elsevier Science B.V. All rights reserved
Olfactory Stimuli and Allo-recognition
MALCOLM G. B AINES,
Dept. Microbiology and Immunology, McGill University, 3775 University St. Montreal, QC., Canada H3A-2B4
ABSTRACT A number of species have demonstrated the ability to recognise related individuals by scent alone in a manner which may relate to their major histocompatibility (MHC) genotype. Further, the scent of the members of the opposite sex can activate aggressive responses, affect mating preference, induce oestrus, induce implantation delay or loss, augment early embryo survival and even alter the quality of the recipients immune defences. Two well known reproductive responses to pheromonal stimuli include the Whitten and Bruce effects. The "Whitten effect" is initiated by the presence of odours of an alien male, resulting in the onset of oestrus in un-mated female mice. The "Bruce effect" defines a complete pre-implantation block of pregnancy in mated females exposed to odours of an alien male. Conversely, if an alien male is introduced to a pregnant mouse on the day after implantation the incidence of spontaneous early embryo losses may be reduced. Therefore, neuroendocrine mediators induced by pheromonal messages derived from the resident male can alter the maternal cell-mediated immune response in the uterus to the fetal graft, dramatically affecting the outcome of pregnancy.
1.
INTRODUCTION
Reproduction in mammals has long intrigued researchers in both basic and clinical immunology because the long-term acceptance of the semi-allogeneic fetal graft throughout gestation appears to violate the basic principles of transplantation biology. While it is obvious that mate selection is governed by the senses of the participants, recent data suggests that the sense of smell is arguably the most important of the senses (Table I). The senses of vision and hearing, which operate from a distance, provide only a general assessment of the attractions of a mate. However, the sense of smell can define the general health, the readiness of the subject to mate and the genetic suitability of that individual as a mate. The senses of taste and touch, which require much closer contact, may serve to confirm this evaluation. Most importantly, the sense of smell can define kinship as defined by the expression of antigens of the major histocompatibility complex (MHC). Part of the sense of smell resides in the vomeronasal organ (VNO). The VNO sensory tissue is located in a tubular organ in the base of the anterior part of the nasal septum where it is supplied with bipolar sensory neurones, which are connected to the accessory olfactory bulb [1]. This organ appears to be important but not essential for
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detection of MHC related odours. Endocrine hormones augment the activity of the VNO in gonadectomized animals [2] and test odours trigger neuronal activity [3]. Removal of the vomeronasal organ reduced but did not completely eliminate odour discrimination. However, the anterior bilateral transection of the lateral olfactory tract eliminated the identification of mice by odour, suggesting the involvement of the accessory olfactory system in the transmission of pheromonal stimuli [4, 5]. Table I
Involvement of the senses in reproduction.
Senses
Sensory organs
Reproduction
Vision
Eyes
General physical health
Sound
Ears
Acoustic attractants
Touch
Skin
Tactile stimulation
Taste
Tongue
Oral stimulation
Smell
Nose
Gender, health, kinship, oestrus, receptiveness
2.
OLFACTORY RESPONSES AND REPRODUCTION
Apanius et al and Penn have published recent reviews on odour discrimination, mating preference and MHC selection [6, 7]. As a general introduction to the sources of data on this subject, it is necessary to know that only some experiments involve the observation of wild or inbred mice in controlled 'semi-wild' natural settings. Most researchers have focused on the training of inbred mice and rats to recognise MHC specific odours in various olfactory testing devices in which the odours of 'donor' mice are alternately blown to, or sensed by, the recipient whose 'correct' responses are noted. Early experiments used a passive testing box where untrained subjects could move towards preferred odours and mate with preferred partners (Figure 1) [8, 9]. Subjects can now be placed in automated training and testing chambers which are supplied with test odours and responses are compiled by computer (Figure 2) [10, 11]. Whether these two basic types of experiments entirely accurately reflect the natural mating preferences of mice in the wild is not certain although the data obtained confirms responses observed in 'semi-wild' experiments and helps explain the unusual distribution of MHC alleles in wild populations. Further, there can be many confounding elements in experimental studies, which have to be controlled or accounted for in the final analysis. Such factors include the genetic selection against allo-MHC preference that is inevitably associated with the breeding of MHC inbred mouse strains which might alter responses of the subjects. Using 'outbred' mice avoids this problem. The olfactory 'noise' from non-MHC genes for gender and other genetic factors combined with exogenous factors such as diet in 'semi-wild' mice can influence responses [12]. The effects of gene dosage on donor odour concentration and the inherent strength or hierarchical dominance of odours must also be taken into account. However, it is clear that prior to mating, all animals tested thus far can detect the foreign scents of the presumptive mate and this can influence willingness to mate, the implantation of the blastocyst and the successful development of the implanted embryo.
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PASSIVE APPARATUS FOR TESTING ODOR PREFERENCE OF MICE.
30 cm
@ @ @ A and C contain the test and control odors. B is where the test subject starts the test.
Figure 1. A passive apparatus for testing odor preference of mice. This device is primarily used for assessing odor preferences of untrained mice without a reward to encourage correct choices. The test subject always starts at position "B" and has the choice of attractants "A" or "C" and the preference for "A" over "C" is noted. The attractant may be bedding scent marked by another mouse or it may be a mouse tethered so that it can not stray from the "home" cubicle. Adapted from Egid and Brown Anim. Behav 38 (548), 1989.
3.
ODOURS: G E N E R A L AND MHC SPECIFIC
A m o n g most animals, natural odours are used for kin recognition, scent marking of territory, aggression/dominance, defence/flight and reproduction. The basis for odour recognition or preference could be instinctive, learned or by serendipity (Table II). Such scents are found in the volatile fraction of sweat, saliva, urine and faeces. Odours can be defined by both exogenous and endogenous factors and are most certainly expressed simultaneously as a complex multifactorial signal. Exogenous factors affecting the odour of an individual can originate from diet, microbial flora, general body secretions and interactions between these factors. The diet of mice, rats and humans, can add m a n y volatile factors to urine (e.g. garlic, asparagus, meat diet versus vegetarian diet) [13]. In fact, animals can learn to detect the dietary differences between individuals quicker than they can learn strain specific differences and therefore, diet cues may occasionally mask M H C associated kinship scents [12, 14]. The familiar c o m m e n s a l microbial flora colonising the individual may also be detectable and could vary inversely with infection by pathogenic microbes. The health of the mate is important as infected mates are not accepted
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0
L.
0
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Computerized Olfactometric Apparatus for Odor Identification. 1
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Figure 7. Computerized olfactometric apparatus for odor identification. This device is primarily used for assessing odor preferences of mice or rats that have been trained with a reward to e n c o m g e correct choices. The volatile scents are injected into the air stream and when the subject identifies the correct odor. a reward of water is supplied. The rdt move5 forward in the chamber, breaking a light beam (Inset 1 and 2) and auto~naticallyrecording its response for the computer. Adapted from Beauchamp et al
317
even when they express an allogeneic MHC [15, 16]. Healthy males will avoid infected females and healthy females will avoid infected males [17]. When both are infected there is less discrimination against infection and kinship indicating that olfactory imprinting can be learned from sensing odours related to self [18]. Further, the scent of an infected male appears to 'suppress' the sensitivity or receptivity of the female to allogeneic male scents and even heat stimuli [15]. It has also been suggested that the action of commensal microbes may be required for the processing of both exogenous and endogenous macromolecules into the volatile fragments that are detected by the vomeronasal organ. However, as germ-free mice can identify MHC associated scents, the microbial component may complement but is not critical for recognition [19]. Endogenous factors detected by the sense of smell are genetically controlled factors specific for the individual, some of which may be related to products of the sex chromosomes and endocrine factors related to gender and maturity [20]. Species related factors are also detectable in the excretions of animals (e.g. rat versus mouse). Males are more interested in unrelated females or their bedding (non-kin) [21], which contains both common and specific factors [22]. Common female factors are those that indicate that the female is receptive or has commenced oestrus. While males strongly respond to the indicators of oestrus, it is also clear that the difference between female oestrus versus dioestrus scents is learned by juvenile male mice [23]. Whereas many reproduction related scents appear to relate to the MHC specificity of the individual, specific scents from specific genes such as the lethal t-complex gene can been detected, as +/+ females prefer +/+ males to +/t males [9]. However, the fact that both male and female can distinguish relatives from strangers and choose the latter, is perhaps one of the most interesting facets of mate selection. The role of MHC associated volatile compounds in defining the odourtype of the individual, still lacks many details. MHC associated odours and proteins have been shown to be present in urine and other secretions of individual mice but are not dominant over all other factors, such as diet and oestrus related odours [22]. Further, mouse serum and purified mouse MHC proteins or synthetic MHC peptides are not specific attractants and MHC proteins can be separated from the more volatile active attractants in natural excretions [24, 25]. The suggestion that MHC associated serum proteins may bind or carry volatile carboxylic acid odourants that could be liberated by cellular and/or microbial action, was supported by the observation that pronase digestion of MHC or serum proteins produced volatile attractants [11, 26, 27]. The source of MHC associated odours has been shown to be the bone marrow. Radiation chimeras were produced by repopulating the hemopoietic system of mice of one strain with the marrow of a second. The MHC chimerism was detectable in the urine of these mice by odour response tests [28]. Using the "Y"-maze habituation dis-habituation training process, mouse or rat males could learn to identify MHC specific odours from congenic donors with 80-90% accuracy (Figure 3) [22]. MHC detection can be trained for a difference at a single MHC class-I or MHC-II allele [29, 30]. Even the lack of an MHC allele in an MHC deletion mutant could be detected by trained rats [31]. In fact, the specificity of the MHC detection can be trained for a few amino acid differences in a single mutation in a class-I MHC allele H2 b bm or b m l [32]. In recent experiments, rats trained to detect MHC related odours in human urine, could even identify paternal odours derived from the fetal tissues in the uterus of pregnant donors [10]. The major remaining questions concern the nature of the normal endogenous process of 'volatilisation' of the MHC macromolecules, the chemical structure of the scents and whether microbial mediators are required or simply complement the process.
318
Y-MAZE APPARATUS FOR TESTING ODOR PREFERENCES Air
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Figure 3. The "Y"-maze apparatus is also primarily used for assessing odor preferences of mice or rats that have been trained with a reward to encourage correct choices. The volatile scents are injected into the air stream of one arm of the "Y" and when the subject identifies the correct odor by moving towards it, a reward of water is supplied. Each test is a choice of two odors. Adapted from Yamazaki et al., P.N.A.S 96 (1522), 1999.
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Table II
Basis and effects of odour recognition in reproduction.
Odour Response
Effect of allogeneic odour recognition.
Instinct
Avoidance of genetically related mice (littermates)?
Learned
Avoidance of MHC pheromones previously sensed?
Curiosity
Select an allogeneic mate who smells different or more interesting?
4.
MHC BASED MATE PREFERENCE SELECTION
It is now apparent that volatile derivatives or components associated with MHC are contained in the body secretions and excretions of all species tested thus far. Males prefer the odours of females with an MHC which is allogeneic versus syngeneic in tests using congenic females [33, 34]. However, some studies of actual mating in the passive chamber device appear to contradict this point as males occasionally mate without MHC preference [8]. One explanation is that in these tests, interest of a recipient in a donor scent is a one-way response that may be totally under the control of the recipient while mating is a two-way event that requires some physical co-operation by both parties. Consequently, in mating tests using tethered females in the passive test chamber, males seemed to show no preference for MHC, whereas females continued to show allo-MHC preference when the males were tethered [8]. Therefore, even though female preference for allogeneic MHC versus syngeneic MHC in congenic males appears stronger than male preference, this may be an artefact of the assay format [35]. Males show no preference for MHC-identical congenic females or MHC-identical females with different non-MHC background genes implying that non-MHC associated factors are either non-existent or are much weaker in this type of experiment [8]. Finally, the question as to the genetic v e r s u s environmental basis for self or kinship recognition was answered by cross-fostering experiments. Cross-fostering allogeneic mouse pups to MHC different dams changes the specificity of MHC based mate selection, supporting a mechanism based on familial imprinting [33, 34, 36, 37]. All the female mice derived from cross-fostered familial settings avoided mating with males expressing MHC alleles identical to the original a n d the cross-fostered members of the litter. The benefits of mating with healthy, allogeneic males provides a significant advantage both for the individual female and for the population by creating offspring with heterozygous resistance to disease and preserving existing and new MHC polymorphism in the gene pool (Table III). In conclusion, most mates instinctively prefer partners with an MHC different from their parents and their littermates. However, opportunity to mate may over-rule choice in situations where the female can not resist, or the signal indicating the oestrus state or receptiveness is stronger than the allo-recognition signal.
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5.
PREGNANCY: PHEROMONE-INDUCED EVENTS FOLLOWING MATING
5.1.
Induction of oestrus: the "Whitten effect"
Female receptivity is also altered in response to a number of pheromonal stimuli related to the Y chromosome, health, ovulation, endocrinology and other factors. While the male can naturally detect oestrus-related scents from female mice, the oestrous cycle of female mice can also be altered by the presence of male mice. The Whitten effect defines the induction and synchrony of oestrus by exposure of grouped female mice to corralled males. The volatile factor is present in male urine and is effective even when direct contact between male and female is prevented [38]. The response of female mice is also directly related to the allogeneicity of the males as previously described. Therefore, male scent induces females to ovulate and female scent of 'heat' attracts the males. 5.2.
Pre-implantation pregnancy block: the "Bruce Effect"
Once mating has taken place, the MHC alloantigens of the male can still have significant effects on pregnancy outcome. The most prominent pre-implantation example is known as the Bruce effect which describes a virtually total pregnancy block or the prevention of implantation by exposure to alien males so that the female mouse can return to oestrus (Figure 4) [39]. The corpora lutea degenerate and all fertilised ova fail to implant and are lost. The apparent value of this response would be to allow the female to quickly block the progression of the current pregnancy, so that she can mate with the new and more successful dominant male. Pregnancy block optimally requires from 48 to 72 hours of contact with the new male (Figure 5) and can be induced at any time up to the time of implantation (Figure 6) [40, 41]. The induction of the Bruce effect requires direct contact with the urine, soiled bedding or other male derived factors (Figure 7) [42]. The effect is greater for MHC different alien mice, whether they are male or female though responses to male factors appear stronger [43, 44, 45]. A castrated male does not induce these effects implying that testosterone may be required. However, castrated males injected with testosterone induce pregnancy block in mated mice, confirming this requirement. Direct contact with the 'resident' male is required for this effect, suggesting a poorly volatile factor [42]. If a cage within a cage format physically separates the male and female from each other, the volatile scents are less effective in proportion to the degree of separation (Figure 8). Urinary odours or factors associated with differences in the X and Y-chromosomes also induce pregnancy block. However, there is no convincing evidence of gender selective embryo loss that could affect the sex ratio of the offspring [20, 46, 47]. Pregnancy block occurs in entirely untrained mice, indicating that the fine discrimination of MHC associated odours is a natural event. Early studies by Bruce and Dominic clearly demonstrated that the alien male elicited the implantation blocking effect via androgendependent pheromones which appeared in male urine and acted via a maternal neuroendocrine pathway involving decreased prolactin and progesterone secretion (Figure 9) [44, 48, 49, 50]. The augmentation of prolactin or stimulation of endogenous prolactin secretion by reserpine, reduced pregnancy block (Figure 9) [50]. Similarly, supplementing progesterone levels also reduced pregnancy block in some experiments but not in others depending on the time of injection (Figure 9) [44, 51, 52, 53].
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MALE INDUCED PREGNANCY BLOCK THE "BRUCE" EFFECT: RETURN TO ESTRUS. Day of return to estrus
DAY 2 DAY 3 DAY4 DAY 5
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PERCE NT BLOCK 24 Hours with CBAJG on day 0 Figure 4. Male induced pregnancy block, the "Bruce" effect: Return to oestrus. Outbred Parkes strain albino female mice were mated to albino-P males and then introduced to a CBA/G mouse for 24 hours on the first day after the mating plug was detected. This normally causes about half the females to return to oestrus, mostly within two days. Adapted from Bruce, J Reprod Fert 2 (138-142) 1961.
MALE INDUCED IMPLANTATION BLOCK THE "BRUCE" EFFECT" INDUCTION TIME. HOURS OF STRESS Stud AIb-P male New male 12 Hrs New male 24 Hrs New male 48 Hrs New male 72 Hrs
. 0
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. 40
. 60
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PERCENT BLOCK Figure 5. Male induced implantation block, the "Bruce" effect: Induction time. Outbred Parkes strain albino female mice were mated to albino-P males and then introduced to a new CBA/G mouse for variable lengths of time starting on the first day after the mating plug was detected. This greatest incidence of pregnancy block was achieved by two or more days of exposure. Adapted from Parkes and Bruce, J Reprod Fert 4 (303-308) 1963.
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MALE INDUCED PREGNANCY BLOCK THE " B R U C E " EFFECT" S E N S I T I V E PERIOD. 90 80700 60~50 z40 uJ 30uJ 20 100
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DAYALIEN MALEINTRODUCED Figure 6. Male induced pregnancy block, the "Bruce" effect: The sensitive period extends from the first day after mating to the point of implantation. Once the blastocyst has implanted in the uterine wall, pregnancy block does not occur. Bruce, J Reprod Fert 2 ( 138-142) 1961.
MALE INDUCED IMPLANTATION BLOCK T H E " B R U C E " EFFECT: ( C O N T A C T F A C T O R ) STRESS FACTOR Stud AIb-P male New AIb-P male New CB.A~ male Castrated CBMG Testosterone S/C New female/none 0
20
40
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PE RCENT PREGNANCY BLOCK Figure 7. Male induced implantation block, the "Bruce" effect: Contact with the factor. Outbred Parkes strain albino female mice were mated to albino-P males and then introduced to a new albino-P or a CBA/G male starting on the first day after the mating plug was detected and continued until the female returned to oestrus or was obviously pregnant. If the CBA/G male had been castrated, the incidence of pregnancy block was reduced but could be partly restored by injections of testosterone to the castrated male. Adapted from Bruce, J Reprod Fert 1 (96-103) 1960.
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MALE INDUCED IMPLANTATION B L O C K THE "BRUCE" EFFECT: (ISOLATED FACTOR). FACTOR Stud AIb-P male
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20
40
60
80
100
PE RCENT BLOCK
Figure 8. Male induced implantation block, the "Bruce" effect: Isolated from the factor. If either the male or the female is placed in an internal cage, which prevents direct contact, pregnancy block is reduced. If both are kept apart in separate mini-cages, pregnancy block is further reduced indicating that the smell of the volatile component is less effective than direct contact with the scent marked bedding. Adapted from Bruce J Reprod Fert 1 (96-103) 1960.
MALE INDUCED IMPLANTATION BLOCK THE "BRUCE" EFFECT: ENDOCRINOLOGY.
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20
40
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80
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PERCENT P R E G N A N C Y B L O C K Figure 9. Male induced implantation block, the "Bruce" effect: Endocrinology. Outbred Parkes strain albino female mice were mated to albino-P males and then introduced to a new CBA/G male or CBA/G urine starting on the first day after the mating plug was detected. Two other groups of mated albino-P mice were injected with prolactin or reserpine, which increased prolactin secretion. A final group was injected with progesterone. Dominic, J Reprod Fert 11(415-421), 1966.
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6.
POST-IMPLANTATION ENHANCING EFFECTS ON PREGNANCY
The presence of MHC related odours from allogeneic males also affects post-implantation embryo survival. While most strains of inbred mice show a very low incidence of postimplantation spontaneous early embryo loss (
