Advances in Immunology Volume 77

ADVANCES IN

Immunology VOLUME 77

This Page Intentionally Left Blank

ADVANCES IN

Immunology EDITED BY FRANK J. DIXON The Scripps Research Institute La Jolla, California ASSOCIATE EDITORS

Frederick Alt K. Frank Austen Tadamitsu Kishimoto Fritz Melchers Jonathan W. Uhr

VOLUME 77

San Diego San Francisco New York Boston London Sydney Tokyo

∞ This book is printed on acid-free paper. 

Copyright C 2001 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2001 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2776/01 $35.00 Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given.

Academic Press A Harcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com

Academic Press Harcourt Place, 32 Jamestown Road, London NW1 7BY, UK http://www.academicpress.com International Standard Book Number: 0-12-022477-1 PRINTED IN THE UNITED STATES OF AMERICA 01 02 03 04 05 06 EB 9 8 7 6 5 4 3

2

1

CONTENTS

ix

CONTRIBUTORS The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction

S. CELESTE POSEY MORLEY AND BARBARA E. BIERER

I. II. III. IV. V. VI.

Introduction Overview of T Cell Signaling Lipid Rafts Actin Cytoskeleton Actin Dynamics in Signal Transduction—General Principles Conclusion References

1 3 16 23 30 34 35

Raft Membrane Domains and Immunoreceptor Functions

THOMAS HARDER

I. II. III. IV.

Introduction Lipid Raft Concept: Bridging Biophysics to Biology Immunoreceptor Signaling and Raft Domains Outlook References

45 46 54 79 79

Human Basophils: Mediator Release and Cytokine Production

JOHN T. SCHROEDER, DONALD W. MACGLASHAN, JR., AND LAWRENCE M. LICHTENSTEIN

I. II. III. IV. V.

Introduction Basophil Growth and Maturation Cell Surface Markers Inflammatory Mediators Basophil Activation v

93 94 94 98 101

vi

CONTENTS

VI. Signal Transduction and Pharmacological Control of Secretion VII. Basophils and Allergic Disease References

106 112 114

Btk and BLNK in B Cell Development

SATOSHI TSUKADA, YOSHIHIRO BABA, AND DAI WATANABE

I. II. III. IV. V. VI.

Introduction Btk and B Cell Development Activation of Btk Downstream of Btk BLNK Connects Btk Activity to Downstream Effectors Conclusion References

123 124 129 137 142 150 151

Diversity and Regulatory Functions of Mammalian Secretory Phospholipase A2 s

MAKOTO MURAKAMI AND ICHIRO KUDO

I. II. III. IV. V.

Introduction Structures and Enzymatic Properties of sPLA2 s Expression and Functions of sPLA2 s sPLA2 Receptors Conclusion References

163 164 169 182 183 184

The Antiviral Activity of Antibodies in Vitro and in Vivo

PAUL W. H. I. PARREN AND DENNIS R. BURTON

I. II. III. IV. V. VI. VII. VIII.

Introduction Mechanisms of Neutralization Complement-Mediated Virolysis Antibody-Mediated Phagocytosis Antibody-Mediated Cytotoxicity Intracellular Neutralization Mechanisms of Antibody Protection in Vivo Mechanisms of Antiviral Antibody Activity in Established Infection IX. Observations with Nonviral Pathogens X. Conclusions References

195 196 225 226 226 227 227 241 244 244 248

CONTENTS

vii

Mouse Models of Allergic Airway Disease

CLARE M. LLOYD, JOSE-ANGEL GONZALO, ANTHONY J. COYLE, AND JOSE-CARLOS GUTIERREZ-RAMOS

I. Introduction II. Conclusion References

263 287 287

Selected Comparison of Immune and Nervous System Development

JEROLD CHUN

I. II. III. IV. V. VI. VII.

Introduction Major Cellular Components of the Nervous System Embryonic Divisions of the Nervous System Embryonic Development of the Cerebral Cortex Ventricular Zone Neuroblast Programmed Cell Death Nonhomologous End-Joining and DNA Rearrangement Conclusion References

INDEX CONTENTS OF RECENT VOLUMES

297 297 299 303 309 313 316 317 323 333

This Page Intentionally Left Blank

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Barbara E. Bierer (1), Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, 20892; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 Yoshihiro Baba (123), Department of Molecular Medicine, Osaka University Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan Dennis R. Burton (195), Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 Jerold Chun (297), Department of Pharmacology; Neurosciences Program; Biomedical Sciences Program; School of Medicine; University of California, San Diego, La Jolla, California 92037 Anthony J. Coyle (263), Millennium Pharmaceuticals, Cambridge, Massachusetts 02139 Jose-Angel Gonzalo (263), Millennium Pharmaceuticals, Cambridge, Massachusetts 02139 Jose-Carlos Gutierrez Ramos (263), Millennium Pharmaceuticals, Cambridge, Massachusetts 02139 Thomas Harder (45), Basel Institute for Immunology, CH-4005 Basel, Switzerland Ichiro Kudo (163), Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Lawrence M. Lichtenstein (93), Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224 Clare M. Lloyd (263), Leukocyte Biology Section, Biomedical Sciences Division, Imperial College of Science, Technology, and Medicine, London SW7 2AZ, United Kingdom Donald W. MacGlashan, Jr. (93), Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224 S. Celeste Posey Morley (1), Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, ix

x

CONTRIBUTORS

Maryland, 20892; and Committee on Immunology, Division of Medical Sciences, Harvard Medical School, Boston, Massachusetts 02115 Makoto Murakami (163), Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Paul W. H. I. Parren (195), Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 John T. Schroeder (93), Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224 Satoshi Tsukada (123), Department of Molecular Medicine, Osaka University Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan Dai Watanabe (123), Department of Molecular Medicine, Osaka University Medical School, Yamadaoka, Suita City, Osaka 565-0871, Japan

ADVANCES IN IMMUNOLOGY, VOL. 77

The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction §

S. CELESTE POSEY MORLEY*, AND BARBARA E. BIERER*,# *Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; §Committee on Immunology, Division of Medical Sciences, and #Department of

Pediatrics, Harvard Medical School, Boston, Massachusetts

I. Introduction

The adaptive immune system is regulated in large part by the CD4+ helper T lymphocytes. Antigen-presenting cells (APCs) display peptide antigens in the context of major histocompatibility complex (MHC) molecules on their surface that can bind to the T cell receptor (TCR) on antigen-specific T cells. When bound, the TCR complex generates a complicated array of intracellular signals. The integrated outcome of these signals depends on the cellular context in which the signal is received and may result in T cell activation, anergy, or apoptosis. For instance, a developing thymocyte that binds to a self-antigen too avidly will be deleted in a process known as negative selection, a form of activation-induced cell death that is independent of CD95 (Fas) ligation. A mature T cell that binds to antigen in the periphery in the absence of appropriate cytokines or costimulation may be anergized, or rendered nonresponsive to future stimulation. A mature T cell that binds to a foreign antigen in the presence of appropriate cytokines, such as interleukin (IL)-12, and with the appropriate costimulation (e.g., CD28 ligation by CD80 or CD86), will be activated to mount an immune response appropriate for the eradication of the foreign antigen. A remaining question in immunology is the elucidation of the intracellular mechanisms by which the responding T cells arrive at the outcome of the TCRgenerated signal. How do the proteins within the cell phosphorylate, combine, dissociate, and/or translocate to alter, fundamentally, the physiology of the cell, determining the fate of the T cell? The answer appears to lie, in large part, in the way in which signaling components are spatially organized within the responding T cell (Germain and Stefanova, 1999). Traditionally, the field of T cell signal transduction employed a “billiard ball” model of intracellular signaling. Signaling cascades were, and frequently still are, modeled as linear flow charts from cell surface to cell nucleus. Although important to the early understanding of signaling cascades, this signaling paradigm has numerous (and obvious) limitations. It cannot explain how the same molecule— JNK, for example—can participate in signaling events that have diametrically opposite outcomes, such as T cell activation and T cell death (Dong et al., 1998; 1 C 2001 by Academic Press Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

2

MORLEY AND BIERER

Jacinto et al., 1998). It cannot explain the integration of signals from multiple receptors—anti-CD3 monoclonal antibody (mAb) stimulation alone results in anergy, while that of anti-CD28 and anti-CD3 results in activation. And it does not incorporate what is known about the cytoplasmic and structural organization of a cell. Only by a consideration of the three-dimensional network of proteins involved in signaling does the complexity and plasticity of signaling cascades become apparent. The organization of the cytoplasm by the actin cytoskeleton has long been an operational paradigm in cell biology, and it is becoming clear that dynamic changes in the actin cytoskeleton play a critical role in T cell signaling. The application of confocal microscopy to lymphocyte signaling has allowed the development of a visual image in real time of the supramolecular activation complex of T cells (Monks et al., 1998). Advances in digital video imaging of cellular movement has enabled the measurement of the rate at which the actin cytoskeleton reorients and moves toward the site of TCR engagement (Wulfing and Davis, 1998). Targeted gene disruption by homologous recombination in mice has permitted analysis of the specific contributions by proteins such as Vav (Fischer et al., 1998; Holsinger et al., 1998; Kong et al., 1998) and WASP (Snapper et al., 1998; Zhang et al., 1999a) to the regulation of both actin cytoskeletal dynamics and T cell signal transduction. Finally, the identification and characterization of Rho family proteins as regulators of actin and signaling have revealed new axes of signal transduction pathways. A new paradigm of T cell signaling has evolved in which the spatial and temporal organization of molecules, determined in part by the remodeling of the actin cytoskeleton, is as critical to the effectiveness of signal transduction as the identity of the molecules themselves. The ability to remodel actin, here termed actin dynamicity, is intimately involved in the current paradigm of the initiation of T cell signaling leading to T cell activation. The precise mechanism(s) by which actin dynamicity participates in organizing intracellular signaling components following TCR ligation remains an open question. Actin may play a critical role in the creation of the immunological synapse, the structured interface between the APC and the responding T cell (see below). Movement of actin cytoskeletal elements may recruit actin-bound signaling intermediates, such as CD3 ␨ , to the site of APC–T cell contact. Alternatively, movement of actin may recruit larger-order signaling structures, such as the recently defined lipid membrane microdomains, termed lipid rafts, that serve as platforms for the accumulation of numerous signaling molecules. This discussion reviews the recruitment and activation of tyrosine kinases and adapter proteins during TCR signaling, the structure and function of lipid membrane microdomains, and the regulation of actin cytoskeletal dynamics, focusing on experimental evidence that suggests dynamic, coordinate regulation between these three critical components of T cell signaling.

T CELL SIGNAL TRANSDUCTION

3

II. Overview of T Cell Signaling

A signal is generated when a peptide in the context of an MHC molecule engages the TCR complex (reviewed in Cantrell, 1996; Clements et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999). This binding interaction sets off a cascade of membrane proximal events in T cell signal transduction that include activation (or inactivation) of enzymes (kinases and/or phosphatases), phosphorylation of substrates, and recruitment of adapter proteins that enable the formation of large signaling complexes. These early signaling events of tyrosine phosphorylation and protein–protein interactions enable the propagation of downstream signals that lead to calcium flux and the activation of downstream kinases, such as mitogen-activated protein kinases (MAPK). This in turn stimulates the activation of transcription factors such as nuclear factor of activated T cells (NF-AT) that are required for the up-regulation of IL-2 transcription and subsequent activation of the T cells (Cantrell, 1996; Clements et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999). This cascade of events is discussed in detail in this section (Fig. 1). A. T CELL RECEPTOR COMPLEX The TCR complex expressed by CD4+ T helper lymphocytes contains the ␣/␤ TCR heterodimer noncovalently complexed to CD3 proteins (Clements et al., 1999; Germain and Stefanova, 1999). The CD3 complex itself consists of combinations of five different chains subdivided into two different families. One family consists of CD3 ␦, ε, and ␥ , and the other of CD3 ␨ and/or ␩ (Clements et al., 1999). One CD3 complex contains one ε/␥ pair, one ε/␦ pair, and either a ␨ /␨ homodimer or a ␨ /␩ heterodimer. All chains of the TCR–CD3 complex are transmembrane proteins and therefore contain extracellular, transmembrane, and intracellular regions. The extracellular regions of the TCR ␣/␤ chains contain the peptide–MHC binding site and grant the TCR its antigen specificity. However, the intracellular regions are short, have no intrinsic enzymatic activity, and appear not to serve as docking sites for downstream molecules. In contrast, the extracellular domains of the five CD3 chains are quite small and do not associate with the peptide–MHC complex, but the intracellular regions of these chains are crucial for appropriate signal transduction (Clements et al., 1999). Each of the CD3 ␦, ε, and ␥ chains carries one domain capable of being tyrosine phosphorylated, termed an immune receptor-tyrosine-based activation motif (ITAM), with consensus sequence (D/ExxYxxL/Ix7YxxL/I) (Chu et al., 1998). Each CD3 ␨ chain carries three (for a total of six for the homodimer). Tyrosine phosphorylation of these ITAM motifs upon TCR engagement allows for the downstream propagation of the intracellular signal. The mechanism by which peptide–MHC engagement of the TCR ␣/␤ heterodimer transmits a signal through the CD3 complex is unknown, but appears to depend upon

4

MORLEY AND BIERER

FIG. 1. Recruitment and activation of tyrosine kinases and adapter molecules in T cell receptor (TCR)–mediated signal transduction. Ligation of either TCR-␣/␤ or CD3 results in the recruitment and activation of the tyrosine kinase p56Lck that in turn phosphorylates tyrosines in the intracellular tyrosine activation motifs (IT AMs) of the CD3 chains. Phosphorylation of the ITAMs creates binding sites for the recruitment of the tyrosine kinase ZAP-70. Once recruited, ZAP-70 is phosphorylated by p56Lck and thus activated to phosphorylate downstream adapter proteins, such as the linker of activated T cells (LAT). Phosphorylation of LAT leads to the recruitment and activation of other downstream signaling molecules, such as Grb2 and PLC␥ 1. Other critical T cell signaling molecules are also shown.

conformational and spatial changes within the individual complex and upon the ligation-dependent formation of multi-TCR associations (Clements et al., 1999; Germain and Stefanova, 1999). B. RECRUITMENT OF PROTEIN TYROSINE KINASES AND ADAPTER MOLECULES The phosphorylation of substrates by cytoplasmic protein tyrosine kinases (PTKs) creates docking sites for the binding of other proteins (Clements et al., 1999; Marie-Cardine and Burkhart, 1999). These substrates are frequently adapter proteins that have no intrinsic enzymatic activity but serve to bring together other proteins in large signaling complexes. The protein–protein interactions that hold these signaling complexes together are mediated by binding motifs on the partner proteins. Many of these motifs have been characterized, including Src homology 2 (SH2), Src homology 3 (SH3), phosphotyrosine binding (PTB), and pleckstrin homology (PH) domains (Marie-Cardine and Burkhart,

T CELL SIGNAL TRANSDUCTION

5

1999). SH2 domains bind to a consensus Yxxx motif when the tyrosine residue is phosphorylated and different SH2 domains have different specificities for pYxxx motifs (Marie-Cardine and Burkhart, 1999). For instance, phosphatidylinositol 3-kinase (PI3K) binds preferentially to pYxxM, phospholipase C (PLC)-␥ 1 preferentially to pYVVL motifs, and Src kinase to pYxxV/I/L (reviewed in Fruman et al., 1998; Marie-Cardine and Burkhart, 1999). PTB domains also bind motifs containing phosphorylated tyrosine, but are biased toward the amino acids N terminal to the phosphorylated tyrosine. The PTB domain of Shc is biased toward the consensus motif NPXpY, while the PTB domain of Cbl prefers D(N/D)XpY (Lupher et al., 1996, 1997). SH3 domains bind to proline-rich regions. While PH domains appear to bind preferentially lipids, such as phosphatidylinositol 4,5bisphosphate (PIP2), and may mediate protein–membrane interactions (MarieCardine and Burkhart, 1999). When an appropriate MHC/peptide complex engages the antigen-specific TCR, the tyrosine residues of the CD3 ␨ ITAMs are phosphorylated by the Src family tyrosine kinase p56Lck (Germain and Stefanova, 1999). Constitutively associated with p56Lck, the coreceptor CD4 is engaged by the MHC molecule concurrently with the engagement of the TCR complex. It is believed that the coengagement of CD4 and the TCR complex by the same MHC/peptide complex brings the intracellular kinase p56Lck sufficiently close in proximity to the CD3 ITAMs that the Src kinase can phosphorylate the tyrosine residues contained within the ITAMs (Germain and Stefanova, 1999). Phosphorylation of the ITAMs creates binding sites for the SH2 domains of ZAP-70 (zeta-associated protein of 70 kDa), a member of the Syk family of tyrosine kinases. ZAP-70 is recruited to the signaling complex by binding the partially phosphorylated CD3 ␨ chain; associated with CD3 ␨ , ZAP-70 can then be phosphorylated by p56Lck and thus activated. Activated ZAP-70 itself phosphorylates downstream substrates, such as linker of activated T cells (LAT) and SH2 domain containing leukocyte protein of 76 kDa (SLP-76), that serve as the adaptor proteins necessary for creation of the signaling complex (Cantrell, 1996; Clements et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999). The kinase activities of both Src and Syk family kinases are absolutely required for T cell signal transduction. Mice deficient in p56Lck have a severe block in thymic development, although the kinase p59Fyn can substitute in part for p56Lck in peripheral T lymphocyte function (Groves et al., 1996; van Oers et al., 1996a,b). T cell development is also arrested at an early stage of thymopoiesis in mice doubly deficient for ZAP-70 and Syk (Cheng et al., 1997; van Oers et al., 1996b). CD4/CD8 double negative (DN) thymocytes express an appropriately rearranged V␤ chain and the pre-TCR␣ chain, but cannot receive a signal through this pre-TCR complex to proceed to the CD4+CD8+double positive (DP) stage of development (Cheng et al., 1997; van Oers et al., 1996b).

6

MORLEY AND BIERER

Jurkat T cells deficient in either p56Lck (J.CaM1 cells) or ZAP-70 (P116 cells) have been generated and characterized (Straus and Weiss, 1992; Williams et al., 1998). Both cell lines are deficient in the ability to respond to TCR or CD3 stimulation in that they fail to stimulate calcium influx, NF-AT activation, or IL-2 production. The importance of ZAP-70 in the human immune response has been confirmed by identification of one form of human severe combined immunodeficiency that is caused by a deficiency of this protein (Chan et al., 1994; Elder et al., 1994). 1. Linker of Activated T Cells LAT was originally identified as a heavily phosphorylated doublet (pp36/38) in lysates of stimulated T cells. The gene has recently been cloned (Zhang et al., 1998a) and is predicted to encode a transmembrane protein of 233 amino acids. Four of these amino acids are extracellular, 21 are transmembrane, and the remainder cytoplasmic. The cytoplasmic tail contains 10 different tyrosine amino acids, each of which has the potential for phosphorylation. Phosphorylation of several of these residues by ZAP-70 or Syk upon receptor stimulation creates binding sites for a variety of proteins, such as Grb2, PLC-␥ 1, SLP-76, and the p85 subunit of PI3K (Schraven et al., 1999; Zhang et al., 1998a). Two cysteine residues within LAT, proximal to the intracellular membrane at positions C26 and C29, are palmitoylated (Lin et al., 1999; Zhang et al., 1998b). Palmitoylation is required for the appropriate targeting of LAT to the lipid raft (Lin et al., 1999; Zhang et al., 1998b). Lipid rafts are membrane microdomains that serve as platforms for the recruitment of signaling molecules (discussed in detail below). The requirement for LAT in T cell signal transduction has been shown in a number of experimental systems. Overexpression of a mutated form of LAT in which two tyrosine amino acids had been mutated to phenylalanine (Y171F/ Y191F) inhibited TCR signal transduction, as assayed by transcriptional activation of AP-1 and NF-AT (Zhang et al., 1998a). Inhibition of transcriptional activation correlated with the failure to bind Grb2, PLC-␥ 1, and the p85 subunit of PI3K. The J.CaM2 and ANJ3 cell lines, both derived from Jurkat T cells, lack LAT expression (Finco et al., 1998; Zhang et al., 1999b) and are defective in T cell signal transduction. In J.CaM2, TCR ligation failed to stimulate the phosphorylation of PLC-␥ 1 and SLP-76. There was no calcium flux in response to TCR ligation and downstream MAP kinase activity was not stimulated. In consequence, there was no activation of NF-AT or AP-1 transcriptional activity (Finco et al., 1998). Tyrosine phosphorylation of PLC-␥ 1 and SLP-76 was reduced but not ablated in ANJ3 cells upon TCR stimulation, although calcium flux, extracellular signal-regulated kinase (ERK) activation, and transcriptional activation of NF-AT and AP-1 were completely deficient in this cell line (Zhang et al., 1999b). Reconstitution of both cell lines by overexpression

T CELL SIGNAL TRANSDUCTION

7

of wild-type LAT reversed the signal transduction deficits (Finco et al., 1998; Zhang et al., 1998b). To further confirm the requirement of LAT in T cell signaling, mice with a targeted disruption of LAT were generated (Zhang et al., 1999c). Mice deficient for LAT protein had a complete absence of mature T cells. Thymocyte development was inhibited prior to the DP stage, despite normal rearrangement of the V␤ locus and normal expression of the pre-T␣ chain. The phenotype of the LAT null mice was virtually identical to that of mice deficient either for both Src-family kinases p56Lck and p59Fyn or for both ZAP-70 and Syk tyrosine kinases, consistent with a proximal signaling defect (Zhang et al., 1999c). Finally, LAT mutants that contained C26S/C29S substitutions, and that were therefore not palmitoylated nor localized to lipid rafts, could not reconstitute the signaling deficit of J.CaM2 cells (Lin et al., 1999). Thus, the adapter protein LAT and its appropriate membrane localization were necessary for appropriate T cell signal transduction (Lin et al., 1999; Zhang et al., 1999c). 2. SH2 Domain Containing Leukocyte Protein of 76 kDa (SLP-76) Another adapter protein that, with LAT, coordinately regulates the assembly of large signaling complexes is SLP-76, a cytoplasmic, 533–amino acid protein with a tyrosine-rich N-terminal region, a central proline-rich region, and a C-terminal SH2 binding (reviewed in Clements et al., 1999). The expression of SLP-76 is limited to cells of hematopoietic origin. Like LAT, SLP-76 is a substrate of ZAP-70 and is phosphorylated and recruited to the T cell signaling complex upon TCR or CD3 stimulation (Clements et al., 1999; Schraven et al., 1999). SLP-76 is required, along with LAT, for the appropriate activation of PLC-␥ 1, as PLC-␥ 1 phosphorylation, inositol phosphate production, calcium flux, MAPK activation, and NF-AT transcriptional activation were all ablated in a SLP-76 negative Jurkat T cell derivative (Yablonski et al., 1998). SLP-76 is also required for the appropriate recruitment and activation of Vav, a guanine nucleotide exchange factor (GEF) for Rac (Schraven et al., 1999) that is required for downstream cytoskeletal rearrangement, TCR cap formation, and calcium flux (Fischer et al., 1998; Holsinger et al., 1998). SLP-76, like LAT, is required for thymocyte development, and its absence blocks maturation of CD4/CD8 DN thymocytes at the CD25+CD44−stage of development (Clements et al., 1999). Finally, overexpression of SLP-76 can enhance the response to TCR stimulation (Motto et al., 1996) and can synergize with the overexpression of Vav to enhance NF-AT transcriptional activation in response to TCR engagement (Wu et al., 1996). Thus, early membrane proximal signaling events include the activation of p56Lck and p59Fyn, members of the Src family of PTKs; the recruitment and activation of ZAP-70 and Syk, members of the Syk family of PTKs; and the

8

MORLEY AND BIERER

recruitment and phosphorylation of the adapter proteins LAT and SLP-76. LAT and SLP-76 cooperate to form large signaling complexes by nucleating the association of downstream effectors, such as PLC-␥ 1, Vav, or PI3K, and the association of other adapter molecules, such as Grb2, that in turn can recruit and activate downstream effectors, such as Ras (Cantrell, 1996; Clements et al., 1999; Germain and Stefanova, 1999; Marie-Cardine and Burkhart, 1999). C. Ras/MAPK PATHWAY Ras is the prototype of the Ras superfamily of small GTP-binding proteins (Cantrell, 1996; Clements et al., 1999). When bound to GDP, Ras is inactive. Ras becomes activated when the nucleotide GDP is exchanged for GTP. Ras contains intrinsic GTPase activity, and slowly hydrolyzes the GTP to GDP, thereby becoming inactivated. When LAT is phosphorylated, it can recruit the adapter protein Grb2 to the signaling complex. Grb2 is a 217–amino acid protein that contains one SH2 and two SH3 domains. Grb2 constitutively associates with Sos and thus recruits Sos to the signaling complex. Sos serves as a GEF for Ras that catalyzes the exchange of GDP for GTP on Ras, and thus activates Ras. Ras activation in turn activates the kinase Raf, perhaps by stabilizing the membrane translocation of Raf. Raf is a serine/threonine kinase that phosphorylates MEK1, or MAPKK, that in turn phosphorylates and activates the extracellular signal-regulated kinases ERK1 and ERK2, also called MAPKs (mitogenactivated protein kinases). Activation of ERK1/2 is required for the activation of the transcription factor AP-1 and the downstream consequences of T cell activation such as up-regulation of CD69 and IL-2 production (Cantrell, 1996; Clements et al., 1999; Marie-Cardine and Burkhart, 1999). D. PLC-␥1 PATHWAY Phosphorylation and recruitment of PLC-␥ 1 activates PLC-␥ 1 to cleave its substrate, PIP2, into IP3 and diacylgycerol (DAG) (Cantrell, 1996; Clements et al., 1999; Marie-Cardine and Burkhart, 1999). Production of IP3 stimulates the IP3 receptor that triggers intracellular calcium release. Release of calcium from intracellular stores is sufficient to activate calcium release activated calcium (CRAC) channels in the plasma membrane of the T cell, resulting in oscillations of calcium flux across the cell membrane. Calcium forms a complex with calmodulin that then binds to and activates the serine/threonine phosphatase calcineurin. Calcineurin dephosphorylates the transcription factor NF-AT that, upon dephosphorylation, is activated and translocated to the nucleus. NF-AT and AP-1 form a complex required for the upregulation of IL-2 transcription. DAG and calcium also participate in the activation of various serine/threonine protein kinase C (PKC) isoforms, some of which, such as PKC␪, are critical to appropriate T cell signal transduction (Cantrell, 1996; Clements et al., 1999; Marie-Cardine and Burkhart, 1999).

T CELL SIGNAL TRANSDUCTION

9

E. PI3K AND LIPID METABOLISM PI3K consists of a p85 adapter subunit and a p110 catalytic subunit (reviewed in Fruman et al., 1998). PI3K catalyzes the phosphorylation of the D-3 hydroxyl of the inositol ring, generating PI(3)P, PI(3,4)P2, and/or PI(3,4,5)P3 (Fruman et al., 1998). Generation of these lipids activates downstream effectors such as Akt/PKB, a protein that binds PIP3 by virtue of its PH domain (Franke et al., 1995). Akt is a serine/threonine kinase that phosphorylates and inactivates the pro-apoptotic protein BAD, thus promoting cell survival (Datta et al., 1997). The downstream effect of Akt explained the PI3K dependence of growth factor signaling to cell survival (Datta et al., 1997). Additionally, PI3K-mediated activation of Akt has been demonstrated to stimulate NF-␬B transcriptional activation (Romashkova and Makarov, 1999). Independently of effects on Akt, PI3K has been shown to activate Vav (Han et al., 1998) and the members of the Tec family of tyrosine kinases, such as Itk, Rlk, and Txk, that contain PH domains and bind PIP3 (Bunnell et al., 2000). Tec kinases contribute to the PI3K-mediated activation of certain PLC-␥ isoforms, calcium flux, and MAPK activation (Bunnell et al., 2000). F. Vav/Rac PATHWAY Rho GTPases are members of the Ras superfamily of small GTP-binding proteins (reviewed in Hall, 1998; Mackay and Hall, 1998; Reif and Cantrell, 1998). They are activated when bound to GTP and inactivated when the GTP is hydrolyzed to GDP. Three classes of proteins are known to regulate the nucleotide binding of Rho family proteins. GEFs catalyze the exchange of GDP for GTP on the GTP-binding protein, thus activating the protein. GTPaseactivating proteins (GAP) accelerate the rate at which the GTPase cleaves its bound GTP to GDP, thus inactivating the GTPase. Guanine nucleotide dissociation inhibitors (GDI) stabilize the GDP-bound form of the GTPase, effectively inhibiting nucleotide exchange and thus the activation of the GTPase. The conformation of the GTPase depends on the nucleotide to which it is bound. When bound to GTP, but not to GDP, the GTPase is able to bind downstream effectors and transduce signals (Hall, 1998; Mackay and Hall, 1998; Reif and Cantrell, 1998). Activated by membrane receptors, Rho family members link extracellular signals to cytoskeletal rearrangement (Hall, 1998). Different Rho family members exert strikingly differing effects on cell morphology. Activation of Rho by bombesin or lysophosphatidic acid in fibroblasts generates formation of stress fibers and focal adhesions (Ridley and Hall, 1992), activation of Rac by insulin, PDGF or EGF generates membrane ruffles or lamellopodia (Ridley et al., 1992), and activation of Cdc42 by bradykinin generates filopodia (Nobes and Hall, 1995). There is some interplay between the family members; activation of Rac

10

MORLEY AND BIERER

is sufficient to activate Rho, possibly through the production of arachidonic acid (Peppenlenbosch et al., 1995), and activation of Cdc42 can activate both Rac and Rho (Nobes and Hall, 1995). The effects of the Rho family proteins on cell morphology are thought to be mediated by the ability of the Rho family members to generate de novo actin polymerization at specific locations and in specific formations (Mackay and Hall, 1998). In lymphocytes, Rac and Rho have been demonstrated to regulate, in part, the cytoskeletal alterations required for adhesion, spreading, and motility (D’Souza-Schorey et al., 1998; Verschueren et al., 1997). Rho family members have been implicated in cellular signaling processes beyond that of regulation of cytoskeletal morphology. Rac is required for oncogenic transformation by Ras, and both Rac and Cdc42 have been demonstrated to regulate JNK and p38 MAPK activity (Coso et al., 1995; Minden et al., 1995). Microinjection of constitutively activated forms of Rac, Rho, and Cdc42 can stimulate G1 cell cycle progression (Lamarche et al., 1996). In fibroblasts, the ability of Rac to promote cell cycle progression correlated with downstream actin polymerization, but not with induction of JNK activity (Joneson et al., 1996; Lamarche et al., 1996). Rac can also activate p67PHOX, a component of the NADPH oxidase complex in neutrophils (Diekmann et al., 1994). Other downstream effectors of Rac and Rho include p21-activated kinase (PAK), a Ste20related serine/threonine kinase, and PI(4)P-5 kinase (Reif and Cantrell, 1998). PAK may be a critical intermediate between Rac/Rho and the downstream effects of actin cytoskeletal rearrangement and JNK and MAPK activation (Bagrodia and Cerione, 1999). Regulation of PIP2 synthesis by Rac may be critical for the production of DAG and IP3 during signal transduction (Reif and Cantrell, 1998). In lymphocytes, Rac has been demonstrated to synergize with Syk to activate JNK (Jacinto et al., 1998) and both Rac and Rho have been implicated in lymphocyte apoptosis (Brenner et al., 1997; Lores et al., 1997; Moorman et al., 1996). Whether the participation of Rac/Rho in these lymphocyte systems is dependent on the downstream modification of the actin cytoskeleton is unclear. The proto-oncogene Vav is a GEF for Rac (Crespo et al., 1997) expressed exclusively in hematopoietic cells essential for effective T cell signal transduction (reviewed in Bustelo, 2000). Vav is a 95-kDa protein that contains a PH domain, a calponin homology domain, one SH2 and two SH3 domains, and a Dbl homology (DH) domain. Calponin homology domains are thought to potentially mediate binding to actin, and DH domains contain the GEF catalytic site. Vav is tyrosine phosphorylated in a p56Lck—and ZAP-70-dependent manner upon CD3 and CD28 stimulation, and translocates to the TCR complex upon phosphorylation (Bustelo, 2000; Salojin et al., 1999). SLP-76 and LAT are thought to be adapter molecules critical for the recruitment of Vav into the TCR signaling complex (Salojin et al., 1999; Wu et al., 1996). In turn, Vav is required for the recruitment

T CELL SIGNAL TRANSDUCTION

11

of PKC␪ (Villalba et al., 2000). Recruitment of PKC␪ to the TCR signaling complex is dependent on Vav-stimulated actin polymerization (Villalba et al., 2000). Vav null mice have been generated and are viable, fertile, and appear grossly normal (Fischer et al., 1998; Holsinger et al., 1998). However, there were specific defects in the immune system. Thymic development was impaired by the Vav null mutation in the C57/B16 genetic backgrounds, with an accumulation in CD44−CD25+ DN thymocytes (Fischer et al., 1998). Thymocytes deficient in Vav expression were resistant to in vitro activation-induced cell death (AICD) stimulated by anti-CD3 and anti-CD28 mAb treatment and by peptide-specific TCR stimulation (Kong et al., 1998). Inhibition of AICD seemed to be dependent on defects in proximal TCR signaling events, including calcium flux, actin polymerization, and recruitment and activation of PKC␪. Inhibition of actin polymerization with cytochalasin D prior to stimulation of thymocytes also inhibited the activation of PKC␪ and subsequent AICD, suggesting that downstream effects of Vav are dependent on the actin rearrangement stimulated by Vav (Kong et al., 1998). Mature peripheral T cells exhibited deficits in calcium flux, IL-2 production, and proliferation, although early tyrosine phosphorylation events and the activation of MAPK and JNK were normal (Fischer et al., 1998; Holsinger et al., 1998). Despite the decrease in calcium flux, translocation of NF-ATc1 to the nucleus appeared to be normal, indicating that the decreased calcium flux of Vav null lymphocytes was still sufficient to stimulate NF-AT translocation and that Vav function was not required for nuclear translocation (Holsinger et al., 1998). Vav null lymphocytes were defective in the ability to polymerize actin and to form the TCR cap upon TCR stimulation (Fig. 2) (Fischer et al., 1998; Holsinger et al., 1998). The defects in cap formation, calcium flux, and IL-2 production could be mimicked by treating lymphocytes with cytochalasin D, concordant with the suggestion that downstream signaling of Vav is dependent on the regulation of the actin cytoskeleton by Vav (Fischer et al., 1998; Holsinger et al., 1998). Whether or not the ability of Vav to stimulate rearrangements of actin is dependent on GEF activity toward Rac, on the ability to function as an adapter molecule, or on another as yet unidentified function is unclear. The downstream effectors of Rho family members that trigger de novo polymerization are the subject of current study. The Wiskott–Aldrich syndrome protein (WASP) is thought to be a principle downstream effector of Cdc42 required for modification of the actin cytoskeleton by Cdc42 (Symons et al., 1996). Wiskott–Aldrich syndrome patients suffer from a severe immunodeficiency characterized by thrombocytopenia, impaired immunity, and eczema (Ramesh et al., 1999; Zhang et al., 1999a). T cells from Wiskott–Aldrich syndrome patients fail to proliferate normally in response to anti-CD3 mAb stimulation and have marked cytoskeletal abnormalities (Ramesh et al., 1999). Thymic

12

MORLEY AND BIERER

FIG. 2. Formation of the T cell receptor (TCR) cap. Upon ligation of the TCR by either antigen or monoclonal antibody, the TCR–CD3 complexes move toward the engaged molecules. This TCR reorganization is dependent on a number of intracellular signaling molecules including Vav and WASP, among others, and on the actin cytoskeleton.

development and mature lymphocyte activation were inhibited in WASP-deficient mice (Zhang et al., 1999a). WASP-deficient thymocytes were delayed at an early stage of progression from CD44−CD25+to CD44−CD25− in the population of DN thymocytes, the same stage at which p56Lck −/− thymocytes were delayed in maturation (Zhang et al., 1999a). In mature WASP-deficient T lymphocytes, calcium flux, proliferation, and up-regulation of CD69 were inhibited in response to anti-TCR mAb stimulation. WASP-deficient lymphocytes were also defective in actin polymerization, cap formation, and receptor internalization following anti-TCR mAb stimulation (Fig. 2) (Snapper et al., 1998; Zhang et al., 1999a). These results support a model in which TCR-stimulated actin polymerization and cap formation generate the supramolecular activation complex (SMAC; reviewed below) required for sustaining the TCR signal (Snapper et al., 1998; Zhang et al., 1999a). Recent work has demonstrated a direct association between WASP and the Arp2/3 complex (Rohatgi et al., 1999). Seven subunits, including the actin-related protein (Arp)2 and Arp3, make up the Arp2/3 complex. This complex is the only known mediator of the nucleation of actin filaments (reviewed below) that can grow at the barbed end (Mullins,

T CELL SIGNAL TRANSDUCTION

13

2000). The association between WASP and the Arp2/3 complex provides a clear mechanism by which Cdc42, via WASP, can trigger de novo actin polymerization and the resulting morphological changes (Rohatgi et al., 1999). In Vav null and WASP null lymphocytes, a deficiency in the ability to transduce the appropriate signal correlated with an inability to remodel the actin cytoskeleton in response to the receptor stimulus (Fischer et al., 1998; Holsinger et al., 1998; Kong et al., 1998; Snapper et al., 1998). Either rearrangement of actin cytoskeletal architecture and transduction of signals are mutually exclusive but parallel events, or the transduction of the signal is dependent on the ability to remodel actin. The latter possibility is suggested by studies in which the addition of exogenous compounds that modulate actin dynamics inhibited T cell signal transduction (Fischer et al., 1998; Holsinger et al., 1998). Dynamic changes in the actin cytoskeleton have been demonstrated to play a role in a variety of signal transduction pathways, but only recently have become the subject of investigation in lymphocytes. While it is now appreciated that actin cytoskeletal morphology is intricately involved in lymphocyte signal transduction, the understanding of the mechanisms by which it does so, and the mechanisms by which actin dynamics are regulated during signal transduction, remains incomplete. G. FORMATION OF THE SUPRAMOLECULAR ACTIVATION COMPLEX The involvement of the actin cytoskeleton in lymphocyte signal transduction was first suggested in 1973 when it was found that treatment of B lymphocytes with cytochalasin D, a fungal metabolite that caps F-actin and induces depolymerization, prevented receptor cap formation in response to anti-IgM (de Petris and Raff, 1973). Upon TCR recognition of specific peptide/MHC complexes, the area of contact between the T cell and the APC becomes enriched with other TCRs, generating a receptor cap (reviewed in Penninger and Crabtree, 1999), so termed because of the appearance of immunofluorescently labeled receptors on responding T cells. This interface is a highly complex structure of surface receptors, costimulatory molecules, and intracellular signaling proteins (Fig. 3). Fluorescent microscopy analysis of this interface revealed that the TCR clustered in a central area of the region of contact, and that this cluster is surrounded by a ring of the adhesion molecule LFA-1 (CD11a/CD18) (Monks et al., 1998). These two areas were mutually exclusive, as no appreciable LFA-1 was found in the central cluster while no TCR was demonstrated in the outer ring (Monks et al., 1998). Segregation of intracellular signaling molecules correlated with receptor segregation: talin, a cytoskeletal protein, was found exclusively in the outer ring while the ␪ isoform of protein kinase C (PKC) colocalized exclusively with the TCR in the central ring (Monks et al., 1997, 1998). This highly organized interface has been termed both the supramolecular activation complex, or SMAC (Monks et al., 1998), and the immunological synapse (Grakoui et al., 1999). Generation of the SMAC correlated with downstream lymphocyte effector function

14

MORLEY AND BIERER

FIG. 3. Formation of the supramolecular activation complex (SMAC). In cross-section, antigen in the context of major histocompatibility complex (MHC) presented by an antigen-presenting cell (below) engages TCR and CD4 co-receptor on the surface of the responding T cell (above) in the central area of the SMAC. The area of TCR engagement is surrounded by a ring of adhesion molecules, such as LFA-1, engaged by ligand, such as CD54. LFA-1 is excluded from the central area of the SMAC, while engaged TCR is exclusively localized to the central area. The organization of some intracellular molecules mirrors the organization of these surface receptors; PKC␪ is exclusively localized to the central area of the SMAC, “beneath” the TCRs, while talin, an actin-binding protein, is exclusively localized in the peripheral ring of the SMAC, beneath LFA-1. Looking down on a SMAC, the organization of these molecules is schematically represented by a central circular area containing TCR and PKC␪ that is surrounded by a ring of LFA-1 and talin, respectively. (Adapted from Monks et al., 1998.)

(Grakoui et al., 1999; Monks et al., 1998), and interference with cap formation inhibited T cell signal transduction (Penninger and Crabtree, 1999). The cytoskeleton is also restructured in response to the TCR/MHC binding event. The microtubule-organizing center and actin microfilaments reorient toward the area of cell–cell contact (Penninger and Crabtree, 1999). Video microscopy has revealed a critical role for actin in costimulatory events (Wulfing and Davis, 1998) (Fig. 4). Briefly, beads coated with anti-CD54 (ICAM-1) mAb were used to monitor T cell cytoskeletal movement in a manner analogous to that in which fibroblast cytoskeletal motility is monitored. CD54 expressed on the surface of the APC can participate in T cell costimulation by binding to its ligand LFA-1, expressed on the responding T cell. However, CD54 on the surface of the T cell is not involved in T cell costimulation. T cell CD54 is linked to the actin cytoskeleton and can therefore be used to track actin cytoskeletal movement within the responding T cell. When a T cell bound to an APC carrying the appropriate peptide–MHC complex, the bead moved toward the region

T CELL SIGNAL TRANSDUCTION

15

FIG. 4. Cytoskeletal movement in costimulation. In T cells, CD54 does not participate in the active T cell signaling complex, but is stably associated with actin cytoskeletal elements and can therefore been used to track cytoskeletal movement. Beads coated with anti-CD54 monoclonal antibody were used to visualize the surface movement of CD54, that should in turn parallel intracellular cytoskeletal movement. When T cells were allowed to adhere to antigen-presenting cells that expressed appropriate MHC with the specific antigen and appropriate co-stimulatory molecules, such as B7 (CD80/CD86), the coated beads were observed to move toward the area of cell–cell contact, implying that effective costimulation of T cells triggered cytoskeletal movement toward the region of contact. In the absence of appropriate co-stimulation, no such movement of the bead was observed. (Adapted from Wulfing and Davis, 1998.)

of contact, indicating that the cytoskeleton was reorienting towards the region of contact (Wulfing and Davis, 1998). Cytoskeletal movement was dependent upon effective costimulation through either CD28 or LFA-1 (Wulfing and Davis, 1998). The intracellular signaling of these costimulatory molecules was dependent upon PI3K activity and calcium. The mechanism by which cytoskeletal movement occurred was dependent on actin filament assembly/disassembly and myosin motor proteins (Wulfing and Davis, 1998). Based on these data and video fluorescence microscopy of T cells adhering to a coverslip coated with peptide-pulsed MHC and CD54, Grakoui and co-workers (1999) proposed a three-step model for SMAC formation (Fig. 5) in which the force created by actin cytoskeletal movement drives the rearrangement of surface receptors. In the first step, CD54 and LFA-1 binding form a junction that creates a fulcrum for cytoskeleton-based protrusive processes that in turn create a ring of T cell membrane that is in close proximity to the APC cell membrane. Close proximity allows for further TCR–peptide–MHC contact and recruitment of additional TCRs. If the TCR recognizes the complex with sufficient affinity,

16

MORLEY AND BIERER

FIG. 5. Formation of the immunological synapse. Engagement of adhension molecules, such as LFA-1, has been hypothesized to be a critical initial step in synapse formation. Adhesion molecules associated with cytoskeletal elements have been proposed to serve as a “fulcrum,” enabling the generation of forced, close approximation of the surface of the T cell and the antigen presenting cell for sufficient duration for the antigen-specific TCR to sample the antigens presented in the context of MHC molecules (1). If an agonistic peptide is encountered, a signal is generated that results in the active movement of TCR to a central area of T cell–APC contact. Adhesion molecules are moved outside this central region (2). The newly formed SMAC, or synapse, is maintained by ongoing signaling processes through as yet undefined mechanisms (3). (Adapted from Grakoui et al., 1999.)

the second step of receptor transport occurs, in which TCRs associated with peptide–MHC complexes and nonengaged TCRs are moved into the central region of contact. The authors hypothesize but do not demonstrate that this is also a cytoskeletally mediated event. In the final step, the synapse is stabilized by an unknown mechanism. At this stage, the synapse is comparable to the previously described SMAC (Grakoui et al., 1999; Monks et al., 1998). The mechanism by which the actin cytoskeleton drives the rearrangements required to create the SMAC is unknown. III. Lipid Rafts

A. STRUCTURE Lateral spatial organization of the lipid membrane is a critical component of appropriate lymphocyte signaling (Germain and Stefanova, 1999). Differential partitioning of the lipids within the cellular plasma membrane has been defined by differential solubility in cold, nonionic detergents such as Triton X-100 (reviewed in Brown, 1998; Brown and London, 1998a,b; Simons and Ikonen,

T CELL SIGNAL TRANSDUCTION

17

1997). The lipids contained within cellular plasma membranes include glycerophospholipids, glycosphingolipids and sterols. Glycerophospholipids contain mostly unsaturated fatty acids and have a low melting temperature. In contrast, sphingolipids contain mostly saturated fatty acids and have a higher melting temperature. Sphingolipids and cholesterol have been hypothesized to stack in a more liquid-ordered (ℓo) phase to form platforms or rafts that move through the glycerophospholipids that in turn exist in a more liquid-disordered (ℓd) phase. These aggregates of (ℓo)-phase lipids have been called rafts and, because of their low buoyant density, can be isolated from Triton X-100 whole cell lysates by sucrose density centrifugation. The fraction in which the rafts are found is referred to as the Triton-insoluble fraction. That which remains in the higher density fractions of the sucrose gradient is referred to as the Triton-soluble fraction. Because of their differential solubility, lipid rafts are sometimes referred to as detergent-insoluble, glycolipid-enriched membrane microdomains (DIGs) or detergent-resistant membranes (DRMs). Because of their lipid constituecy, lipid rafts have also been referred to as glycosphingolipid-enriched membrane microdomains (GEMs) (Brown, 1998; Brown and London, 1998a,b; Simons and Ikonen, 1997). Much work has been devoted to the question of the existence and function of these lipid rafts in physiological cell membranes. As the existence of these rafts was originally suggested by a detergent extraction method, concerns were raised that sphingolipids coalesced into rafts only as an artifact of detergent extraction (Brown and London, 1998b). However, this possiblity was considered unlikely because of studies in which varying ratios of different lipids were mixed and then extracted with Triton X-100. Detergent insoluble lipids were found only under conditions that allowed coalescence of lipids into the ℓd phase prior to addition of Triton X-100. In other words, the coalescence of some lipids into the ℓo phase (and thus into the defined lipid rafts) was not dependent on, and in fact was inhibited by, detergent extraction of other lipids. Further studies have ruled out the possibility that detergent extraction caused mixing of lipids from different phases, or contamination of one lipid phase with components of another (Brown and London, 1998b). However, as the relative detergent insolubility of lipid rafts is dependent upon maintenance of a cold (4◦ C) temperature, there are still concerns that rafts may not exist as such at physiological temperatures. Biophysical evidence from in vitro work using artificially created lipid membranes suggests the possibility of the existence of rafts (Brown, 1998; Brown and London, 1998a,b; Simons and Ikonen, 1997), but biophysical evidence cannot confirm the existence of the raft. B. MICROSCOPIC ANALYSIS OF LIPID RAFTS In the absence of biophysical data, microscopic analysis of lipid membrane morphology has been used in the attempt to demonstrate the existence of lipid

18

MORLEY AND BIERER

rafts (Harder et al., 1998; Varma and Mayor, 1998). One of the more convincing studies used anistropy measurements from homotypic fluorescence resonance energy transfer (FRET) to determine that GPI-linked folate receptor (GPIFR) was nonrandomly distributed on the cell surface, while transmembrane folate receptor was randomly distributed (Varma and Mayor, 1998). The nonrandomly distributed GPI-FR was estimated to be in aggregates approx ≈70nm across (Varma and Mayor, 1998). As this is below the resolution of light fluorescence microscopes (250–300 nm), this measurement offers an explanation of the uniform distribution patterns of GPI-linked proteins and other hypothetical raft markers observed with conventional fluorescence microscopy techniques (Jacobson and Dietrich, 1999). Harder and associates (1998) attempted to circumvent this limitation of microscopy by cross-linking raft markers with mAb, forcing their aggregation into patches large enough to be visualized by light microscopy. In this manner they demonstrated colocalization/copatching of raft markers, as determined by Triton X-100 insolubility, such as the GPI-linked proteins placental alkaline phosphatase (PLAP), Thy-1, and influenza virus hemagglutinin and the ganglioside GM1. Importantly, these patches excluded non-raft markers (proteins that were found in the Triton X-100–soluble fraction) such as transferrin receptor (TfR), the low-density lipoprotein receptor, and the vesicular stomatitis virus glycoprotein. Cross-linking of the non-raft markers also created patches, which were entirely distinct from the patches of cross-linked raft markers. A mosaic pattern of red and green with no overlapping yellow that covered the entire cell membrane was observed when TfR and PLAP were cross-linked and cells stained for these markers. This mosaic pattern indicated that there were no areas of overlap between the membrane domain that contained the GPI-linked PLAP and the membrane domain that contained TfR. The dependence of the existence of these separate domains upon lipid composition of the membrane was demonstrated by cholesterol extraction. When cells were treated with methyl-␤-cyclodextrin, a compound that depletes the membrane of cholesterol, patching of cross-linked TfR and PLAP was inhibited. This study thus offered strong evidence for the existence of two distinct, mutually exclusive membrane microdomains (Harder et al., 1998). C. FUNCTIONS OF LIPID RAFTS Lipid rafts have been implicated in a number of cellular functions, including intracellular trafficking (both biosynthetic and endocytic), apical sorting, regulation of membrane proteases, and signal transduction (Brown and London, 1998a; Jacobson and Dietrich, 1999; Simons and Ikonen, 1997). Two pathways exist for the endocytosis of proteins located on the apical surface, the clathrincoated vesicle pathway and another pathway dependent on lipid rafts. In many cell systems, the lipid rafts are associated with caveolin in membrane depressions

T CELL SIGNAL TRANSDUCTION

19

called caveolae. Not all cell types contain caveolin, but the lipid raft–dependent endocytic pathway seems to function in these cells as well. Lipid rafts also appear to function as apical sorting platforms for the appropriate delivery of GPI-linked and N-glycan–containing proteins to the apical surface (Simons and Ikonen, 1997). Lipid rafts have been implicated in the regulation of both uPA and the coagulation cascade (Brown and London, 1998a). A number of different studies, detailed below, suggest the importance of the integrity of lipid rafts and their associated proteins for the induction and maintenance of appropriate singlaling. 1. Lipid Rafts in Signal Transduction Lipid modification of certain proteins is necessary and sufficient for their targeting to lipid rafts in the absence of activation (Kabouridis et al., 1997; Zhang et al., 1998b). The glycerophosphatidylinositol moiety that anchors GPI-linked proteins in the membrane targets these proteins to rafts. The Src family tyrosine kinases p56Lck and p59Fyn are doubly acylated (one myristoylation and one palmitoylation) at their N termini (Kabouridis et al., 1997). LAT is palmitoylated on two N-terminal cysteine residues (Lin et al., 1999; Zhang et al., 1998b). Importantly, these lipid modifications are required not only for localization to lipid rafts but also for the appropriate function of these molecules in lymphocyte signal transduction (Fig. 6) (Kabouridis et al., 1997; Lin et al., 1999; Zhang et al., 1998b). Xavier and colleagues (1998) demonstrated that the integrity of lipid rafts is required for efficient T cell activation. Using sucrose gradient centrifugation to isolate lipid raft components from T lymphocytes both before and after stimulation through the TCR, they demonstrated that the increase in tyrosine phosphorylation of proteins upon TCR stimulation is most dramatic in the lipid raft, as compared to proteins in the cytoplasm or non-raft plasma membrane. They further demonstrated that proteins critical to TCR signaling are either constitutively localized to lipid rafts, such as Lck, Fyn, Cbl, Syk, Ras, and Grb-2, or translocate to the rafts upon stimulation, as do Vav, Shc, ZAP-70, PLC-␥ 1, and CD3 ␨ (Fig. 6). Some Vav and PLC-␥ 1 are present in the raft prior to stimulation, but the amount is greatly enhanced upon anti-CD3 stimulation (Xavier et al., 1998). Zhang and co-workers (1998b) confirmed the localization patterns of Vav, PLC-␥ 1, Cbl, p56Lck , and Grb-2, though differed on the localization of ZAP-70; they found no evidence of ZAP-70 translocation to the lipid raft upon stimulation. However, this may be due to a difference in detergent extraction conditions (Xavier et al., 1998; Zhang et al., 1998b). The tyrosine phosphorylated forms of these proteins were observed primarily in the lipid raft fraction. Disruption of the detergent-resistant membrane compartment with nystatin and filipin disrupted TCR signaling, as assayed by tyrosine phosphorylation of PLC-␥ 1 and CD3 ␨ and by calcium mobilization (Xavier et al., 1998). Extraction

20

MORLEY AND BIERER

FIG. 6. The T cell signaling complex in the lipid raft. A number of T cell signaling proteins are constitutively localized to lipid membrane microdomains, termed lipid rafts, or translocate to the rafts upon appropriate stimulation. The integrity of the lipid raft is required for efficient T cell signal transduction and activation.

of cholesterol with methyl-␤-cyclodextrin also prevented calcium flux in response to anti-CD3 stimulation. Finally, forced down-modulation of surface lipid raft components by treatment with exogenous gangliosides prevented TCR signal transduction, as assayed by calcium mobilization. These data strongly support the model in which lipid rafts serve as platforms for the association of signaling molecules, and that the integrity of these lipid platforms must be maintained for appropriate signaling (Xavier et al., 1998). Experimental support for a model of rafts serving as signaling platforms comes from the work of Janes and collaborators (1999). These investigators first demonstrated that p56Lck , LAT, and CD3 colocalized to rafts patched by cross-linking GM1 with the B subunit of cholera toxin (CTxB). The association of CD3 with the lipid rafts appeared to be of weaker affinity than that of p56Lck or LAT and was sensitive to extraction in 1% Triton X-100 (Janes et al., 1999), a finding that may explain the discrepancy of these results with others who have found no association of TCR-␣/␤ with lipid rafts (Kosugi et al., 1999). Most intriguingly, cross-linking of GM1 with CTxB was sufficient to stimulate tyrosine phosphorylation of substrates and calcium mobilization in Jurkat T cells, suggesting again

T CELL SIGNAL TRANSDUCTION

21

that lipid rafts serve as platforms for the association of signaling molecules, and that the aggregation or coalescence of these rafts is sufficient to trigger the phosphorylation events necessary for signal transduction (Janes et al., 1999). Debate about the presence or absence of the TCR/CD3 complex in lipid rafts continues. Kosugi and associates (1999) detected CD3 ␨ , but not other CD3 chains or TCR-␣/␤ chains, in lipid rafts following TCR or CD3 stimulation. However, the translocation of CD3 ε, TCR-␣/␤, and CD3 ␨ to the lipid raft compartment was observed in another system (Montixi et al., 1998). The difference in observations may be due to a difference in detergents used to extract the lipid raft components. Although 1% Triton X-100 insolubility has been the defining extraction method for lipid raft constituents, lower concentrations of Triton X-100 (Xavier et al., 1998) and other detergents, such as Brij (Montixi et al., 1998), may maintain the association of proteins with weaker affinities for lipid rafts. The difference in observations could also be explained by the time course of the assays; Montixi and colleagues (1998) stimulated with anti-CD3 ε mAb for 5 min at 37◦ C, while most of the assays performed by Kosugi and associates (1999) stimulated cells with anti-CD3 ε mAb for 45 min at 37◦ C. Given the microscopic evidence (Janes et al., 1999) and the virtually universal observation that TCR/CD3 signaling requires the accumulation of signaling molecules at lipid rafts, it is likely that the TCR/CD3 complex does translocate or associate with the lipid raft, but that this association can be disrupted by extraction with 1% Triton X-100. Viola et al. (1999) recently presented data that suggested the involvement of raft redistribution in effective costimulation. They demonstrated that lipid rafts, as indicated by staining with CTxB–FITC, remained uniformly distributed when a T cell bound to beads coated with anti-CD3 mAb, but redistributed to “cap” at the area of bead–cell contact when the T cell bound to beads coated with both anti-CD3 and anti-CD28 mAbs. Redistribution of the CTxB–FITC– stained patches correlated with downstream activation of the T cell, as indicated by cell proliferation, tyrosine phosphorylation, CD3 down-modulation, and consumption of p56Lck . Passive clustering of the lipid rafts by cross-linking GM1 with CTxB or CD59 (a GPI-linked protein) was sufficient to costimulate T cells in combination with anti-CD3 mAb when immobilized on the surface of plastic tissue culture wells (Viola et al., 1999). These findings suggested that forced coalescence of lipid rafts was sufficient to transduce a signal, possibly by bringing raft constituent components in close proximity such that they become activated (Viola et al., 1999). 2. Lipid Rafts and the Actin Cytoskeleton The fact that the TCR complex and associated signaling proteins form an ordered SMAC at the T cell–APC contact (Monks et al., 1998), that lipid rafts

22

MORLEY AND BIERER

colocalize to this area (Viola et al., 1999), and that the actin cytoskeleton forms a distinct cap at this contact site (Penninger and Crabtree, 1999) leads to the intriguing possibility that tyrosine phosphorylation of substrates, lipid raft coalescence, and actin cytoskeletal rearrangement are all intimately linked in forming the complex necessary for T cell signal transduction. Harder and Simons (1999) have demonstrated the colocalization of polymerized actin, tyrosine phosphorylated substrates, and lipid rafts in Jurkat T cells. Cross-linking of the GPI-linked protein CD59 with anti-CD59 mAb or the ganglioside GM1 with CTx followed by anti-CTx polyclonal antibody resulted in raft patching. These patches accumulated polymerized actin, as visualized by staining with FITC–phalloidin. When a transferrin receptor (TfR) construct that is specifically excluded from lipid rafts was cross-linked, no accumulation of actin was observed. Patching of lipid rafts induced by cross-linking of GM1 or CD59 also induced the recruitment of tyrosine-phosphorylated substrates to the patched lipid rafts, while cross-linking TfR did recruit tyrosine-phosphorylated substrates to the cell membrane. Inhibition of src-dependent tyrosine phosphorylation with the tyrosine kinase inhibitor PP1 prevented not only tyrosine phosphorylation in response to raft patching but also the accumulation of polymerized actin at these sites, suggesting that tyrosine phosphorylation was required for the actin cytoskeletal rearrangement. However, inhibition of actin polymerization by treatment of the cells with latrunculin did not prevent the accumulation of tyrosine-phosphorylated substrates induced by GM1 cross-linking, although the raft patches appeared to be less condensed and the fluorescent signal from the staining of tyrosine phosphorylated substrates was weaker. No specific substrates of tyrosine phosphorylation localized to the lipid rafts were examined, however, and therefore it remains possible that depolymerization of actin by treatment with latrunculin altered the identity of the phosphorylated, raft-associated proteins (Harder and Simons, 1999). The association of lipid rafts with the actin cytoskeleton has been suggested by other reports as well (Holowka et al., 2000; Moran and Miceli, 1998; Oliferenko et al., 1999). Oliferenko and colleagues (1999) demonstrated that CD44containing lipid rafts are anchored by F-actin, as assayed by both sucrose gradient isolation of lipid raft constituents (CD44) and fluorescence recovery after photobleaching (FRAP) in intact cells and in cells treated with the actin-depolymerizing agent latrunculin. Costimulation of T cells through the GPI-linked protein CD48 enhanced the translocation of CD3 ␨ to the insoluble fraction upon stimulation through CD3 (Moran and Miceli, 1998). This translocation correlated with enhanced IL-2 production and could be inhibited by pretreatment with either cytochalasin D or with methyl-␤-cyclodextrin, a compound that extracts membrane cholesterol. Thus, both intact lipid rafts and an intact actin cytoskeleton were required for appropriate signaling through CD3 and CD48 (Moran and Miceli, 1998). Costimulation of T cells through CD28 triggers both actin cytoskeletal redistribution and raft redistribution to the site

T CELL SIGNAL TRANSDUCTION

23

of T cell–APC contact (Kaga et al., 1998; Viola et al., 1999), which again suggests coordination between the actin cytoskeleton and lipid raft regulatory mechanisms. In mast cells, cross-linking of FcεRI, which has been shown to associate with lipid rafts, resulted in the redistribution of the raft-associated proteins Thy-1 and Lyn to large patches coincident with the cross-linked FcεRI (Holowka et al., 2000). At 4◦ C, F-actin also redistributed to these patches but was dispersed when the cells were warmed to 37◦ C. Disruption of F-actin by treatment with cytochalasin D allowed greater association of raft components with cross-linked FcεRI and a prolongation of tyrosine phosphorylation in response to crosslinked FcεRI. These observations support a model in which F-actin regulates the association of proteins such as FcεRI with lipid raft components such as Lyn by segregating lipid rafts (Holowka et al., 2000). In brief, these studies demonstrated that proteins critical to signal transduction must either constitutively localize or translocate upon stimulation to the raft to function in the transduction pathway (Kabouridis et al., 1997; Lin et al., 1999; Zhang et al., 1998b), that cross-linking constitutive protein or lipid components of the lipid rafts is sufficient to transduce a signal (Janes et al., 1999; Viola et al., 1999), that the morphology and constitution of rafts are altered upon signal transduction (Janes et al., 1999; Viola et al., 1999; Xavier et al., 1998; Zhang et al., 1998b) and that these alterations in morphology and constitution are required for signal transduction (Xavier et al., 1998). Furthermore, there is an increasing amount of evidence that regulation of actin polymerization and of lipid rafts are tightly linked, and that alterations in one has profound effects on the other. IV. Actin Cytoskeleton

Microfilaments, microtubules, and intermediate filaments make up the cytoskeleton that maintains the intracellular architecture. Although there is extensive interplay between these three components, the role of microfilaments have been most extensively studied. Actin microfilaments are critical to maintenance of cell shape and adhesion, and are absolutely required for the rapid morphological changes such as ruffling that are required for cell motility. Microfilaments also play an essential role in cell division during cytokinesis, and inhibition of actin dynamics during proliferation generates multinucleate cells. A. STRUCTURE AND REGULATION OF POLYMERIZATION Microfilaments are composed of polymerized actin (Fig. 7) (reviewed in Kabsch and Vandekerckhove, 1992; Mitchison, 1992; Steinmetz et al., 1997). The actin monomer is a 43-kDa protein with a single nucleotide binding site for either ATP or ADP and a cation-binding site, which is thought to be magnesium (Mg2+) (Steinmetz et al., 1997). In its monomeric form, actin is referred

24

MORLEY AND BIERER

FIG. 7. Actin polymerization. Actin exists in either a monomeric (G-actin) or polymerized (Factin) state. F-actin consists of a double-helical linear arrangement of monomers (below), but for simplicity is depicted as a signal array above. Actin contains intrinsic ATPase activity, and polymerization of actin is regulated by nucleotide binding.

to as globular actin, or G-actin. Filamentous actin, F-actin, consists of a parallel, double helical array of linearly assembled actin monomers. The actin filament is polarized; the two “ends” of an actin filament are not identical. These ends are referred to as the barbed end and the pointed end, due to their appearance in electron micrographs. Actin assembly, or polymerization, can occur at either end, but is much faster at the barbed end (Mitchison, 1992; Steinmetz et al., 1997). Polymerization of actin is regulated by ATP binding and hydrolysis (Fig. 7) (Kabsch and Vandekerckhove, 1992; Mitchison, 1992; Steinmetz et al., 1997). G-actin can exist either in an ATP- or ADP-bound form. ATP–G-actin has a higher affinity than ADP-G-actin for the ends of actin filaments, and thus nucleotide exchange of ATP for ADP on G-actin can stimulate actin polymerization. Actin monomers within the actin filament have intrinsic ATPase activity, and bound ATP is slowly hydrolized to ADP. Hydrolysis is slower than new polymerization, so a newly elongating filament will contain both ADP–actin (at the pointed end) and ATP–actin (at the barbed end). ADP-bound actin monomers depolymerize, or dissociate from the filament, from the pointed end. Polymerization can occur at the barbed end while the filament is depolymerizing at the pointed end; this cycle is referred to as treadmilling. Thus, regulating the rate of ADP/ATP exchange on actin monomers and regulating the ATPase activity of actin filaments can regulate the rates of actin polymerization and depolymerization (Kabsch and Vandekerckhove, 1992; Steinmetz et al., 1997).

T CELL SIGNAL TRANSDUCTION

25

B. ACTIN-BINDING PROTEINS Regulation of the structure of the actin cytoskeleton occurs at multiple levels by different classes of actin-binding proteins (reviewed in Puius et al., 1998; Schmidt and Hall, 1998). A few representative proteins are summarized in Table I and in Fig. 8. Regulatory activities include actin monomer sequestration or buffering, nucleotide exchange, nucleation of actin filaments, capping and severing of existing actin filaments, cross-linking and bundling of filaments into higher-order networks, and cross-linking actin filaments to integral membrane proteins (Puius et al., 1998; Schmidt and Hall, 1998). Filament assembly/disassembly is in part regulated by actin-binding proteins that are thought to regulate ATP-related activities and to alter affinities for F-actin (Puius et al., 1998; Schmidt and Hall, 1998). Profilin binds to actin monomers. In addition to serving as a buffering protein, profilin has been

TABLE I BRIEF SUMMARY OF REPRESENTATIVE ACTIN-BINDING PROTEINS Protein Profilin

Activation and regulation Sequestration of actin monomers. Dissociates from G-actin upon PIP2 bindig. Nucleotide exchange.

Thymosin ␤4

Sequestration of actin monomer. Dissociates from G-actin upon PIP2 binding.

Capping protein

Capping of actin filaments. Dissociates from F-actin upon PIP2 binding.

Gelsolin

Severing of actin filaments activated by binding of Ca2+. Capping of actin filaments. Dissociates from F-actin upon PIP2 bidning. Nucleation of actin filaments, enabling elongation at pointed end.

Villin

Severing of actin filaments in high concentrations of Ca2+. Cross-linking and bundling in low concentrations of Ca2+.

Fragmin, adseverin, scinderin

Severing of actin filaments activated by binding of Ca2+. Capping of actin filaments. Dissociates from F-actin upon PIP2 binding.

Cofilin (ADF)

Disassembly of actin filaments, inhibited by serine phosphorylation. Sequestration of actin monomers. Dissociates from G-actin upon PIP2 binding.

␣-Actinin

Cross-linking and bundling of actin filaments. Activity is enhanced by PIP2 binding.

Filamin

Cross-linking and bundling of actin filaments. Activity is inhibited by PIP2 binding.

Spectrin, fimbrin

Cross-linking and bundling of actin filaments.

Talin

Nucleation of actin filaments at membrane.

Arp2/3 complex

Nucleation of actin filaments, enabling elongation at barbed end. Activated by WASP.

Ezrin, radixin, moesin

Cross-linking of F-actin to plasma membrane. Activated by PIP2 and by tyrosine and serine phosphorylation.

ADF, Acting-depolymerizing factor; PIP2, phosphatidylinositol 4,5-bisphosphate; WASP, Wiskott-Aldrich syndrome protein.

26

MORLEY AND BIERER

FIG. 8. Examples of activities of actin-binding proteins. See text for details.

hypothesized to act as an ATP nucleotide exchange factor, enhancing the rate at which ADP is exchanged for ATP on actin monomers (Puius et al., 1998). The complex of ATP–G-actin with profilin has a much higher affinity for F-actin than does ATP–G-actin alone (Didry et al., 1998). Profilin can therefore enhance the rate of actin polymerization by increasing the affinity of actin monomers for actin filaments and by increasing the rate at which ADP–actin is recycled to ATP–actin (Mullins, 2000; Puius et al., 1998). Like many actin-binding proteins, profilin contains a binding site for PIP2 and dissociates from actin monomers when bound to PIP2 (Goldschmidt-Clermont et al., 1991). Cofilin, also called actin-depolymerizing factor (ADF), promotes actin filament disassembly and can, like profilin, buffer actin monomers (Schmidt and Hall, 1998). Cofilin binds to F-actin, preferentially ADP–actin within the filament, and induces a twist that can induce dissociation of the actin monomer from the filament (Bamburg, 1999). Through the promotion of actin assembly at the barbed end and of actin disassembly at the pointed end, profilin and cofilin can work in concert to remodel the actin cytoskeleton (Bamburg, 1999; Didry et al., 1998; Mullins, 2000). PIP2 binding induces dissociation of cofilin from actin (Schmidt and Hall, 1998). Recent work has demonstrated regulation of cofilin by serine phosphorylation by LIM kinase (Arber et al., 1998; Yang et al., 1998). Phosphorylation inactivates cofilin, preventing cofilin-mediated actin filament disassembly. Activation of LIM kinase therefore promotes actin polymerization, and is thought to serve as a downstream effector of Rac (Arber et al., 1998; Yang et al., 1998). Phosphorylation and nuclear translocation of cofilin has been

T CELL SIGNAL TRANSDUCTION

27

demonstrated in activated T lymphocytes (Samstag et al., 1994, 1996), and costimulation of T cells promotes cofilin dephosphorylation and association with actin (Lee et al., 2000). Other activities that control actin dynamics are sequestration of actin monomers and nucleation. Thymosins are small G-actin binding proteins that are thought to “buffer” the pool of G-actin in the cytoplasm and thus regulate how much G-actin is available for polymerization (Mitchison, 1992). As mentioned previously, profilin and cofilin can also serve as buffering proteins (Schmidt and Hall, 1998). New actin filaments are generated from nucleation sites; two G-actin molecules must be brought into close proximity by a nucleating protein to generate a site for actin polymerization. The Arp2/3 complex (described above) is one of the most powerful nucleating complexes known to date (Rohatgi et al., 1999), capable of nucleating actin and generating new barbed ends for rapid polymerization (reviewed in Machesky and Insall, 1999; Schafer and Schroer, 1999). The Arp2/3 complex appears to be recruited into the TCR signaling complex via interactions with the adaptor molecule Fyn-binding protein (Fyb)/SLP-76– associated protein (SLAP) (Krause et al., 2000). Gelsolin (described in detail below) can also nucleate actin filaments, but generates pointed ends for polymerization (Kinosian et al., 1998; Wegner et al., 1994). Severing and capping proteins, such as gelsolin, perform at another level to regulate actin cytoskeletal remodeling. Gelsolin belongs to a family of severing proteins that includes villin, fragmin, adseverin, and scinderin. Severing proteins are generally activated by calcium binding (Puius et al., 1998). When bound to calcium they to bind the side of an actin filament and induce disassembly, generating two shorter actin filaments from one long one. Members of the gelsolin family can also cap severed filaments (Puius et al., 1998). Capping proteins, such as gelsolin and capping protein, bind to the barbed end of an existing actin filament, preventing further elongation (Machesky and Insall, 1999; Puius et al., 1998). PIP2 binding to either gelsolin or capping protein causes them to dissociate from the filament, “uncapping” the filament and allowing further polymerization at the exposed barbed end (Hartwig et al., 1995). Thus, severing and capping proteins control the number and length of actin filaments (Puius et al., 1998). Cross-linking proteins such as fimbrin, spectrin, ␣-actinin, and the ezrin/ radixin/moesin (ERM) family members combine actin filaments into higherorder structures to create the cellular actin architecture (reviewed in Puius et al., 1998; Tsukita and Yonemura, 1999). Fimbrin cross-links actin microfilaments and is inhibited by PIP2 binding. In contrast, PIP2 binding activates the cross-linking activity of ␣-actinin, a protein that also cross-links and bundles actin filaments (Puius et al., 1998). Other cross-linking proteins are listed in Table I (drawn from Schmidt and Hall, 1998). The ERM family members link actin microfilaments to the plasma membrane through interactions with integral membrane proteins (Tsukita and Yonemura, 1999). ERM proteins are localized

28

MORLEY AND BIERER

to areas of distinct cell surface structures, such as microvilli, lamellipodia, and adhesion sites, where cortical actin must interact with the plasma membrane to maintain the surface structure (Tsukita and Yonemura, 1999). The bundles and networks of actin filaments created by these cross-linking proteins are highly stable and resistant to rapid polymerization and depolymerization (Puius et al., 1998). The regulation of actin cytoskeletal dynamics is thus extraordinarily complex, coordinated and modulated by an array of proteins with both overlapping and exclusive functions. While the cortical actin cytoskeleton can be maintained in a highly stable array of actin filaments, other pools of actin are undergoing constant polymerization and depolymerization under the control of a variety of these proteins. The constant flux could provide greater sensitivity to extracellular signals, allowing small perturbations in PIP2 and Ca2+ concentrations to result in rapid remodeling of actin. While the functions of two of the actin binding proteins described here—cofilin and the Arp2/3 complex—have been investigated in the context of T cell signaling (Krause et al., 2000; Lee et al., 2000; Samstag et al., 1994), the potential for the participation of the others—gelsolin, profilin, ERM proteins—remains an open question. C. EXOGENOUS AGENTS THAT MODIFY ACTIN Cell permeant compounds that modify the rates of actin polymerization or depolymerization have been invaluable in the investigation of actin-based pathways. Since genetic manipulation of actin is difficult in many eukaryotic cell systems, and since the molecular mechanisms of actin regulation by many actinregulatory proteins remain unclear, the identification of exogenous compounds with defined activities toward the actin cytoskeleton have been used to define the role of actin dynamics in various cellular processes. Cytochalasins are fungal metabolites that have long been used to induce actin filament destabilization (Carlier et al., 1986). Latrunculin is a more recently identified compound that binds actin monomers, resulting in dramatic depolymerization of actin (Ayscough et al., 1997; Spector et al., 1989). Jasplakinolide has an opposite effect on F-actin, binding to and stablizing existing actin filaments and in some cases driving increased actin polymerization (Bubb et al., 1994, 2000). The identification and mechanisms of these compounds are outlined in greater detail below. 1. Cytochalasin D Cytochalasins are fungal metabolites that have long been used to modify actin dynamics in vivo. Cytochalasin B, but not D or E, has also been demonstrated to inhibit glucose transport (Mookerjee et al., 1981). Cytochalasins B, D, and E cap the barbed end of actin filaments, preventing elongation of the filament (Carlier et al., 1986). Depending on the ionic environment, the cytochalasins can also bind actin monomers and promote ATPase activity, decreasing the pool of

T CELL SIGNAL TRANSDUCTION

29

ATP-bound monomeric actin required for polymerization (Brenner and Korn, 1980). Interestingly, under certain conditions, such as high KCl and substoichiometric concentrations of cytochalasin, cytochalasin D can nucleate actin filaments by forming dimers of actin monomers (Carlier et al., 1986; Goddette and Frieden, 1986). The nucleation effectively promotes actin polymerization, which probably explains observations that under certain conditions cytochalasin D promoted actin polymerization in leukocytes (Rao et al., 1992). Over time, however, the depolymerizing activity of cytochalasin D predominates (Goddette and Frieden, 1986). The activity of cytochalasins toward lymphocyte activation is dependent on cytochalasin concentration; at low concentrations (50 nm. Hence, it was proposed that lateral segregation of these molecules is supported by parallel apposition of T cell and APC plasma membranes in which protein pairs are aligned according to their size in a process that may generate signaling subdomains involved in TCR triggering (Shaw and Dustin, 1997). The enrichment of Lck/Fyn and the TCR complex in the central zone of the immunological synapse implies that raft domains reside in the cSMAC. Indeed, the passive recruitment of GPI-anchored CD48 into the contact zone of a T cell hybridoma and an APC-supported TCR ␨ -chain phosphorylation, the association of TCR with the actin cytoskeleton as well as downstream responses such as IL-2 secretion (Moran and Miceli, 1998). The density of peptide–MHC ligands in the cSMAC was shown to reach up to 350 molecules per square micron for strong agonists. Assuming that densities reached by antibody–cross-linked patches of FcεRI (2000 molecules per micron) resemble those of antibody–cross-linked TCR, this is considerably lower than the density of TCR in antibody–cross-linked patches. Hence, it is important

RAFT MEMBRANE DOMAINS

79

to define whether the densities of engaged TCR molecules reached within the cSMAC are sufficiently high to induce a percolation of the raft into a continuous patch or whether raft domains are dispersed within the immunological synapse. This question may be answered by analyzing bona fide raft markers and their distribution in the immunological synapse. It remains to be defined whether rafts are directly involved in the definition of SMAC–membrane domains and in T lymphocyte activation by an APC or whether the role of rafts is restricted to the initial TCR triggering that precedes formation of the immunological synapse. IV. Outlook

The concept of a lateral compartmentalization of the plasma membrane into membrane domains of different phases has now accumulated a wealth of experimental support. Membrane components are thought to specifically partition into raft domains dictated by inherent partition coefficients. While protein–protein interactions can be characterized by crystal structures, mutational analysis, the study of binding kinetics, and so on, the nature of raft-mediated interactions between membrane proteins are not yet defined. It is not clear whether rafts function by keeping together two proteins in one membrane domain or whether rafts may serve as the meeting point of dynamically exchanging membrane components and proteins mediating stable interactions. Raft dynamics are shifted toward larger, stabilized rafts when proteins are oligomerized. It is an attractive hypothesis that triggering of immunoreceptors involves a change in the raft membrane environment, either by increasing the size of raft domains around IRRs or by a qualitative acquisition of an Lo membrane phase. Future studies will use novel methods that directly address the molecular environment of resting and activated IRRs. We will then need to understand how rafts are involved in the modulation of immune cell activation and how costimulatory or inhibitory signals are perceived and integrated. ACKNOWLEDGMENTS I am grateful to Raul Torres, Klaus Karjalainen, Derek Toomre, and Kai Simons for their critical review of the manuscript. I thank Shasha Tarakhovsky and Burkhard Schraven for communicating results prior to publication. The Basel Institute for Immunology was founded and is supported by Hofmann La Roche Ltd.

REFERENCES Abraham, N., Miceli, M. C., Parnes, J. R., and Veillette, A. (1991). Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature 350, 62–66. Ahmed, S. N., Brown, D. A., and London, E. (1997). On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: Physiological concentrations of cholesterol and sphingolipid

80

THOMAS HARDER

induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 36, 10944–10953. Almeida, P. F., Vaz, W. L., and Thompson, T. E. (1992). Lateral diffusion in the liquid phases of dimyristoylphosphatidylcholine/cholesterol lipid bilayers: A free volume analysis. Biochemistry 31, 6739–6747. Aman, M. J., and Ravichandran, K. S. (2000). A requirement for lipid rafts in B cell receptor induced Ca2+ flux. Curr. Biol. 10, 393–396. Amrein, K. E., and Sefton, B. M. (1988). Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine protein kinase, p56lck, reveals its oncogenic potential in fibroblasts. Proc. Natl. Acad. Sci. USA 85, 4247–4251. Anderson, R. G. (1998). The caveolae membrane system. Annu. Rev. Biochem 67, 199–225. Arni, S., Ilangumaran, S., van Echten-Deckert, G., Sandhoff, K., Poincelet, M., Briol, A., RunggerBrandle, E., and Hoessli, D. C. (1996). Differential regulation of Src-family protein tyrosine kinases in GPI domains of T lymphocyte plasma membranes. Biochem. Biophys. Res. Commun. 225, 801–807. Arni, S., Keilbaugh, S. A., Ostermeyer, A. G., and Brown, D. A. (1998). Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues. J. Biol. Chem. 273, 28478–28485. Arudchandran, R., Brown, M. J., Peirce, M. J., Song, J. S., Zhang, J., Siraganian, R. P., Blank, U., and Rivera, J. (2000). The Src homology 2 domain of Vav is required for its compartmentation to the plasma membrane and activation of c-Jun NH2-terminal kinase 1. J. Exp. Med. 191, 47–60. Bachmann, M. F., Gallimore, A., Linkert, S., Cerundolo, V., Lanzavecchia, A., Kopf, M., and Viola, A. (1999). Developmental regulation of Lck targeting to the CD8 coreceptor controls signaling in naive and memory T cells. J. Exp. Med. 189, 1521–1530. Beaven, M. A., and Metzger, H. (1993). Signal transduction by Fc receptors: The Fc epsilon RI case. Immunol. Today 14, 222–226. Bijlmakers, M. J., and Marsh, M. (1999). Trafficking of an acylated cytosolic protein: Newly synthesized p56(lck) travels to the plasma membrane via the exocytic pathway. J. Cell Biol. 145, 457–468. Bijlmakers, M. J., Isobe-Nakamura, M., Ruddock, L. J., and Marsh, M. (1997). Intrinsic signals in the unique domain target p56(lck) to the plasma membrane independently of CD4. J. Cell Biol. 137, 1029–1040. Bittman, R., Kasireddy, C. R., Mattjus, P., and Slotte, J. P. (1994). Interaction of cholesterol with sphingomyelin in monolayers and vesicles. Biochemistry 33, 11776–11781. Bolard, J. (1986). How do the polyene macrolide antibiotics affect the cellular membrane properties? Biochim. Biophys. Acta 864, 257–304. Boniface, J. J., Rabinowitz, J. D., Wulfing, ¨ C., Hampl, J., Reich, Z., Altman, J. D., Kantor, R. M., Beeson, C., McConnell, H. M., and Davis, M. M. (1998). Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands. Immunity 9, 459–466. Bosselut, R., Zhang, W., Ashe, J. M., Kopacz, J. L., Samelson, L. E., and Singer, A. (1999). Association of the adaptor molecule LAT with CD4 and CD8 coreceptors identifies a new coreceptor function in T cell receptor signal transduction. J. Exp. Med. 190, 1517–1526. Brdicka, T., Cerny, J., and Horejsi, V. (1998). T cell receptor signalling results in rapid tyrosine phosphorylation of the linker protein LAT present in detergent-resistant membrane microdomains. Biochem. Biophys. Res. Commun. 248, 356–360. Brdicka, T., Pavlistova, D., Leo, A., Bruyns, E., Korinek, V., Angelisova, P., Scherer, J., Shevchenko, A., Hilgert, I., Cerny, J., Drbal, K., Kuramitsu, Y., Kornacker, B., Horejsi, V., and Schraven, B. (2000). Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase Csk and is involved in regulation of t cell activation. J. Exp. Med. 191, 1591–1604.

RAFT MEMBRANE DOMAINS

81

Brown, D. (1993). The tyrosine kinase connection: How GPI-anchored proteins activate T cells. Curr. Opin. Immunol. 5, 349–354. Brown, R. E. (1998). Sphingolipid organization in biomembranes: What physical studies of model membranes reveal. J. Cell Sci. 111, 1–9. Brown, D. A., and London, E. (1997). Structure of detergent-resistant membrane domains: Does phase separation occur in biological membranes? Biochem. Biophys. Res. Commun. 240, 1–7. Brown, D. A., and London, E. (1998a). Functions of lipid rafts in biological membranes. Annu. Rev. Cell Dev. Biol. 14, 111–136. Brown, D. A., and London, E. (1998b). Structure and origin of ordered lipid domains in biological membranes. J. Membr. Biol. 164, 103–114. Brown, D. A., and Rose, J. K. (1992). Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533–544. Bubeck Wardenburg, J., Pappu, R., Bu, J. Y., Mayer, B., Chernoff, J., Straus, D., and Chan, A. C. (1998). Regulation of PAK activation and the T cell cytoskeleton by the linker protein SLP-76. Immunity 9, 607–616. Bunnell, S. C., Diehn, M., Yaffe, M. B., Findell, P. R., Cantley, L. C., and Berg, L. J. (2000). Biochemical interactions integrating Itk with the T cell receptor–initiated signaling cascade. J. Biol. Chem. 275, 2219–2130. Byth, K. F., Conroy, L. A., Howlett, S., Smith, A. J., May, J., Alexander, D. R., and Holmes, N. (1996). CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation. J. Exp. Med. 183, 1707–1718. Cantrell, D. (1996). T cell antigen receptor signal transduction pathways. Annu. Rev. Immunol. 14, 259–274. Cantrell, D. (1998). Lymphocyte signalling: A coordinating role for Vav? Curr. Biol. 8, R535–R538. Cerny, J., Stockinger, H., and Horejsi, V. (1996). Noncovalent associations of T lymphocyte surface proteins. Eur. J. Immunol. 26, 2335–2343. Chan, A. C., and Shaw, A. S. (1996). Regulation of antigen receptor signal transduction by protein tyrosine kinases. Curr. Opin. Immunol. 8, 394–401. Cheng, P. C., Dykstra, M. L., Mitchell, R. N., and Pierce, S. K. (1999). A role for lipid rafts in B cell antigen receptor signaling and antigen targeting. J. Exp. Med. 190, 1549–1560. Cinek, T., and Horejsi, V. (1992). The nature of large noncovalent complexes containing glycosylphosphatidylinositol–anchored membrane glycoproteins and protein tyrosine kinases. J. Immunol. 149, 2262–2270. Cochran, J. R., Cameron, T. O., and Stern, L. J. (2000). The relationship of MHC–peptide binding and T cell activation probed using chemically defined MHC class II oligomers. Immunity 12, 241–250. Crespo, P., Schuebel, K. E., Ostrom, A. A., Gutkind, J. S., and Bustelo, X. R. (1997). Phosphotyrosinedependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature 385, 169–172. Curatolo, W. (1987). The physical properties of glycolipids. Biochim. Biophys. Acta 906, 111–136. Czech, M. P. (2000). PIP2 and PIP3: Complex roles at the cell surface. Cell 100, 603–606. Daeron, M. (1997). Fc receptor biology. Annu. Rev. Immunol. 15, 203–234. Davis, M. M., Boniface, J. J., Reich, Z., Lyons, D., Hampl, J., Arden, B., and Chien, Y. (1998). Ligand recognition by alpha beta T cell receptors. Annu. Rev. Immunol. 16, 523–544. De Angelis, D. A., Miesenbock, G., Zemelman, B. V., and Rothman, J. E. (1998). PRIM: Proximity imaging of green fluorescent protein–tagged polypeptides. Proc. Natl. Acad. Sci. USA 95, 12312– 12316. Deans, J. P., Robbins, S. M., Polyak, M. J., and Savage, J. A. (1998). Rapid redistribution of CD20 to a low density detergent-insoluble membrane compartment. J. Biol. Chem. 273, 344– 348.

82

THOMAS HARDER

Deckert, M., Ticchioni, M., Mari, B., Mary, D., and Bernard, A. (1995). The glycosylphosphatidylinositol-anchored CD59 protein stimulates both T cell receptor zeta/ZAP-70–dependent and –independent signaling pathways in T cells. Eur. J. Immunol. 25, 1815–1822. Deckert, M., Ticchioni, M., and Bernard, A. (1996). Endocytosis of GPI-anchored proteins in human lymphocytes: Role of glycolipid-based domains, actin cytoskeleton, and protein kinases. J. Cell Biol. 133, 791–799. DeFranco, A. L. (1997). The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9, 296–308. Delon, J., Gregoire, C., Malissen, B., Darche, S., Lemaitre, F., Kourilsky, P., Abastado, J. P., and Trautmann, A. (1998). CD8 expression allows T cell signaling by monomeric peptide–MHC complexes. Immunity 9, 467–473. D’Oro, U., and Ashwell, J. D. (1999). Cutting edge: The CD45 tyrosine phosphatase is an inhibitor of Lck activity in thymocytes. J. Immunol. 162, 1879–1883. D’Oro, U., Sakaguchi, K., Appella, E., and Ashwell, J. D. (1996). Mutational analysis of Lck in CD45-negative T cells: Dominant role of tyrosine 394 phosphorylation in kinase activity. Mol. Cell. Biol. 16, 4996–5003. Draberova, L., and Draber, P. (1993). Thy-1 glycoprotein and src-like protein-tyrosine kinase p53/p56lyn are associated in large detergent-resistant complexes in rat basophilic leukemia cells. Proc. Natl. Acad. Sci. USA 90, 3611–3615. Field, K. A., Holowka, D., and Baird, B. (1995). Fc epsilon RI–mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling. Proc. Natl. Acad. Sci. USA 92, 9201–9205. Field, K. A., Holowka, D., and Baird, B. (1997). Compartmentalized activation of the high affinity immunoglobulin E receptor within membrane domains. J. Biol. Chem. 272, 4276–4280. Field, K. A., Holowka, D., and Baird, B. (1999). Structural aspects of the association of Fcepsilon RI with detergent-resistant membranes. J. Biol. Chem. 274, 1753–1758. Fischer, K. D., Tedford, K., and Penninger, J. M. (1998). Vav links antigen-receptor signaling to the actin cytoskeleton. Semin. Immunol. 10, 317–327. Flaswinkel, H., Barner, M., and Reth, M. (1995). The tyrosine activation motif as a target of protein tyrosine kinases and SH2 domains. Semin. Immunol. 7, 21–27. Fra, A. M., Williamson, E., Simons, K., and Parton, R. G. (1994). Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae. J. Biol. Chem. 269, 30745–30748. Fra, A. M., Williamson, E., Simons, K., and Parton, R. G. (1995). De novo formation of caveolae in lymphocytes by expression of VIP21–caveolin. Proc. Natl. Acad. Sci. USA 92, 8655–8659. Fridriksson, E. K., Shipkova, P. A., Sheets, E. D., Holowka, D., Baird, B., and McLafferty, F. W. (1999). Quantitative analysis of phospholipids in functionally important membrane domains from RBL–2H3 mast cells using tandem high-resolution mass spectrometry. Biochemistry 38, 8056– 8063. Friedrichson, T., and Kurzchalia, T. V. (1998). Microdomains of GPI-anchored proteins in living cells revealed by crosslinking. Nature 394, 802–805. Frigeri, L., and Apgar, J. R. (1999). The role of actin microfilaments in the down-regulation of the degranulation response in RBL–2H3 mast cells. J. Immunol. 162, 2243–2250. Furuchi, T., and Anderson, R. G. (1998). Cholesterol depletion of caveolae causes hyperactivation of extracellular signal–related kinase (ERK). J. Biol. Chem. 273, 21099–21104. Ge, M., Field, K. A., Aneja, R., Holowka, D., Baird, B., and Freed, J. H. (1999). Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL– 2H3 cells. Biophys. J. 77, 925–933. Germain, R. N. (1997). T-cell signaling: The importance of receptor clustering. Curr. Biol. 7, R640– R644. Gouy, H., Deterre, P., Debre, P., and Bismuth, G. (1994). Cell calcium signaling via GM1 cell surface gangliosides in the human Jurkat T cell line. J. Immunol. 152, 3271–3281.

RAFT MEMBRANE DOMAINS

83

Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M., and Dustin, M. L. (1999). The immunological synapse: A molecular machine controlling T cell activation [see comments]. Science 285, 221–227. Green, J. M., Schreiber, A. D., and Brown, E. J. (1997). Role for a glycan phosphoinositol anchor in Fc gamma receptor synergy. J. Cell Biol. 139, 1209–1217. Gunter, K. C., Germain, R. N., Kroczek, R. A., Saito, T., Yokoyama, W. M., Chan, C., Weiss, A., and Shevach, E. M. (1987). Thy-1–mediated T-cell activation requires co-expression of CD3/Ti complex. Nature 326, 505–507. Hagmann, J., and Fishman, P. H. (1982). Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes. Biochim. Biophys. Acta 720, 181–187. Hakomori, S. (1983). In “Spingolipid Biochemistry” (J. N. Kanfer and S. Hakomori, eds.), pp. 1–164. Plenum, New York. Han, J., Luby-Phelps, K., Das, B., Shu, X., Xia, Y., Mosteller, R. D., Krishna, U. M., Falck, J. R., White, M. A., and Broek, D. (1998). Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 279, 558–560. Hanada, K., Nishijima, M., Akamatsu, Y., and Pagano, R. E. (1995). Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes. J. Biol. Chem. 270, 6254– 6260. Hancock, J. F., Magee, A. I., Childs, J. E., and Marshall, C. J. (1989). All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 57, 1167–1177. Harder, T., and Simons, K. (1997). Caveolae, DIGs, and the dynamics of sphingolipid–cholesterol microdomains. Curr. Opin. Cell Biol. 9, 534–542. Harder, T., and Simons, K. (1999). Clusters of glycolipid and glycosylphosphatidylinositol-anchored proteins in lymphoid cells: Accumulation of actin regulated by local tyrosine phosphorylation. Eur. J. Immunol. 29, 556–562. Harder, T., Scheiffele, P., Verkade, P., and Simons, K. (1998). Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. 141, 929–942. Higgs, H. N., and Pollard, T. D. (1999). Regulation of actin polymerization by Arp2/3 complex and WASp/Scar proteins. J. Biol. Chem. 274, 32531–32534. Hirao, M., Sato, N., Kondo, T., Yonemura, S., Monden, M., Sasaki, T., Takai, Y., and Tsukita, S. (1996). Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: Possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J. Cell Biol. 135, 37–51. Hoessli, D., and Rungger-Brandle, E. (1985). Association of specific cell-surface glycoproteins with a Triton X-100–resistant complex of plasma membrane proteins isolated from T-lymphoma cells (P1798). Exp. Cell Res. 156, 239–250. Holowka, D., and Baird, B. (1996). Antigen-mediated IGE receptor aggregation and signaling: A window on cell surface structure and dynamics. Annu. Rev. Biophys. Biomol. Struct. 25, 79– 112. Holowka, D., Sheets, E. D., and Baird, B. (2000). Interactions between FceRI and lipid raft components are regulated by the actin cytoskeleton. J. Cell Sci. 113, 1009–1019. Honda, Z., Suzuki, T., Kono, H., Okada, M., Yamamoto, T., Ra, C., Morita, Y., and Yamamoto, K. (2000). Sequential requirements of the N-terminal palmitoylation site and SH2 domain of src family kinases in the initiation and progression of FcepsilonRI signaling. Mol. Cell. Biol. 20, 1759–1771. Hope, H. R., and Pike, L. J. (1996). Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains. Mol. Biol. Cell. 7, 843–851. Horejsi, V., Drbal, K., Cebecauer, M., Cerny, J., Brdicka, T., Angelisova, P., and Stockinger, H. (1999). GPI-microdomains: A role in signalling via immunoreceptors. Immunol. Today 20, 356– 361.

84

THOMAS HARDER

Huby, R. D., Dearman, R. J., and Kimber, I. (1999). Intracellular phosphotyrosine induction by major histocompatibility complex class II requires co-aggregation with membrane rafts. J. Biol. Chem. 274, 22591–22596. Ilangumaran, S., Briol, A., and Hoessli, D. C. (1998). CD44 selectively associates with active Src family protein tyrosine kinases Lck and Fyn in glycosphingolipid-rich plasma membrane domains of human peripheral blood lymphocytes. Blood 91, 3901–3908. Ilangumaran, S., Arni, S., van Echten-Deckert, G., Borisch, B., and Hoessli, D. C. (1999). Microdomain-dependent regulation of Lck and Fyn protein-tyrosine kinases in T lymphocyte plasma membranes. Mol. Biol. Cell. 10, 891–905. Ilangumaran, S., He, H. T., and Hoessli, D. C. (2000). Microdomains in lymphocyte signalling: Beyond GPI-anchored proteins. Immunol. Today 21, 2–7. Jacobson, K., Sheets, E. D., and Simson, R. (1995). Revisiting the fluid mosaic model of membranes. Science 268, 1441–1442. Janes, P. W., Ley, S. C., and Magee, A. I. (1999). Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J. Cell Biol. 147, 447–461. Janes, P. W., Ley, S. C., Magee, A. I., and Kabouridis, P. S. (2000). The role of lipid rafts in T cell antigen receptor (TCR) signalling. Semin. Immunol. 12, 23–34. Joazeiro, C. A., Wing, S. S., Huang, H., Leverson, J. D., Hunter, T., and Liu, Y. C. (1999). The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin–protein ligase [see comments]. Science 286, 309–312. Justement, L. B. (1997). The role of CD45 in signal transduction. Adv. Immunol. 66, 1–65. Kabouridis, P. S., Magee, A. I., and Ley, S. C. (1997). S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes. EMBO. J. 16, 4983–4998. Kabouridis, P. S., Janzen, J., Magee, A. L., and Ley, S. C. (2000). Cholesterol depletion disrupts lipid rafts and modulates the activity of multiple signaling pathways in T lymphocytes. Eur. J. Immunol. 30, 954–963. Karnovsky, M. J., Kleinfeld, A. M., Hoover, R. L., and Klausner, R. D. (1982). The concept of lipid domains in membranes. J. Cell Biol. 94, 1–6. Kawabuchi, M., Satomi, Y., Takao, T., Shimonishi, Y., Nada, S., Nagai, K., Tarakhovsky, A., and Okada, M. (2000). Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases. Nature 404, 999–1003. Kenworthy, A. K., Petranova, N., and Edidin, M. (2000). High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Mol. Biol. Cell. 11, 1645–1655. Killeen, N. (1997). T-cell regulation: Thy-1—Hiding in full view. Curr. Biol. 7, R774–R777. Kinet, J. P. (1999). The high-affinity IgE receptor (FcepsilonRI): From physiology to pathology. Annu. Rev. Immunol. 17, 931–972. Kinet, J. P., Quarto, R., Perez-Montfort, R., and Metzger, H. (1985). Noncovalently and covalently bound lipid on the receptor for immunoglobulin E. Biochemistry 24, 7342–7348. Kishihara, K., Penninger, J., Wallace, V. A., Kundig, T. M., Kawai, K., Wakeham, A., Timms, E., Pfeffer, K., Ohashi, P. S., Thomas, M. L., et al. (1993). Normal B lymphocyte development but impaired T cell maturation in CD45-exon6 protein tyrosine phosphatase–deficient mice. Cell 74, 143–156. Klein, U., Gimpl, G., and Fahrenholz, F. (1995). Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry 34, 13784–13793. Koegl, M., Zlatkine, P., Ley, S. C., Courtneidge, S. A., and Magee, A. I. (1994). Palmitoylation of multiple Src-family kinases at a homologous N-terminal motif. Biochem. J. 303, 749–753. Koretzky, G. A., Picus, J., Thomas, M. L., and Weiss, A. (1990). Tyrosine phosphatase CD45 is essential for coupling T-cell antigen receptor to the phosphatidyl inositol pathway. Nature 346, 66–68.

RAFT MEMBRANE DOMAINS

85

Koynova, R., and Caffrey, M. (1995). Phases and phase transitions of the sphingolipids. Biochim. Biophys. Acta 1255, 213–236. Kupfer, A., and Singer, S. J. (1989). Cell biology of cytotoxic and helper T cell functions: immunofluorescence microscopic studies of single cells and cell couples. Annu. Rev. Immunol. 7, 309–337. Kurosaki, T. (1999). Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 17, 555–592. Kurzchalia, T. V., and Parton, R. G. (1999). Membrane microdomains and caveolae. Curr. Opin. Cell Biol. 11, 424–431. Kusumi, A., and Sako, Y. (1996). Cell surface organization by the membrane skeleton. Curr. Opin. Cell Biol. 8, 566–574. Kwong, J., and Lublin, D. M. (1995). Amino-terminal palmitate or polybasic domain can provide required second signal to myristate for membrane binding of p56lck. Biochem. Biophys. Res. Commun. 207, 868–876. Lang, M. L., Shen, L., and Wade, W. F. (1999). Gamma-chain dependent recruitment of tyrosine kinases to membrane rafts by the human IgA receptor Fc alpha R. J. Immunol. 163, 5391– 5398. Leupin, O., Zaru, R., Laroche, T., Muller, S., and Valitutti, S. (2000). Exclusion of CD45 from the T-cell receptor signaling area in antigen-stimulated T lymphocytes. Curr. Biol. 10, 277–280. Lewis, L. A., Chung, C. D., Chen, J., Parnes, J. R., Moran, M., Patel, V. P., and Miceli, M. C. (1997). The Lck SH2 phosphotyrosine binding site is critical for efficient TCR-induced processive tyrosine phosphorylation of the zeta-chain and IL-2 production. J. Immunol. 159, 2292–2300. Ley, S. C., Marsh, M., Bebbington, C. R., Proudfoot, K., and Jordan, P. (1994). Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes. J. Cell Biol. 125, 639–649. Leyton, L., Quest, A. F., and Bron, C. (1999). Thy-1/CD3 coengagement promotes TCR signaling and enhances particularly tyrosine phosphorylation of the raft molecule LAT. Mol. Immunol. 36, 755–768. Lin, J., Weiss, A., and Finco, T. S. (1999). Localization of LAT in glycolipid-enriched microdomains is required for T cell activation. J. Biol. Chem. 274, 28861–28864. Liu, P., Ying, Y., Ko, Y. G., and Anderson, R. G. (1996). Localization of platelet-derived growth factor–stimulated phosphorylation cascade to caveolae. J. Biol. Chem. 271, 10299–10303. Liu, Y., Casey, L., and Pike, L. J. (1998). Compartmentalization of phosphatidylinositol 4,5bisphosphate in low-density membrane domains in the absence of caveolin. Biochem. Biophys. Res. Commun. 245, 684–690. Lobell, R. B. (1998). Prenylation of Ras GTPase superfamily proteins and their function in immunobiology. Adv. Immunol. 68, 145–189. Lou, Z., Jevremovic, D., Billadeau, D. D., and Leibson, P. J. (2000). A balance between positive and negative signals in cytotoxic lymphocytes regulates the polarization of lipid rafts during the development of cell-mediated killing. J. Exp. Med. 191, 347–354. Lowin-Kropf, B., Shapiro, V. S., and Weiss, A. (1998). Cytoskeletal polarization of T cells is regulated by an immunoreceptor tyrosine–based activation motif–dependent mechanism. J. Cell Biol. 140, 861–871. Lupher, M. L., Jr., Rao, N., Eck, M. J., and Band, H. (1999). The Cbl protooncoprotein: A negative regulator of immune receptor signal transduction. Immunol. Today 20, 375–382. Magee, T., and Marshall, C. (1999). New insights into the interaction of Ras with the plasma membrane [comment]. Cell 98, 9–12. Marth, J. D., Cooper, J. A., King, C. S., Ziegler, S. F., Tinker, D. A., Overell, R. W., Krebs, E. G., and Perlmutter, R. M. (1988). Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck). Mol. Cell Biol. 8, 540–550. Mayor, S., and Maxfield, F. R. (1995). Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment. Mol. Biol. Cell. 6, 929–944.

86

THOMAS HARDER

Mayor, S., Rothberg, K. G., and Maxfield, F. R. (1994). Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 264, 1948–1951. McConville, M. J., and Ferguson, M. A. (1993). The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem. J. 294, 305–324. Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G., and Brown, D. A. (1999). Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J. Biol. Chem. 274, 3910–3917. Merritt, E. A., Sarfaty, S., van den Akker, F., L’Hoir, C., Martial, J. A., and Hol, W. G. (1994). Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide. Protein Sci. 3, 166–175. Metzger, H. (1999). It’s spring, and thoughts turn to . . . allergies. Cell 97, 287–290. Michaely, P. A., Mineo, C., Ying, Y. S., and Anderson, R. G. (1999). Polarized distribution of endogenous Rac1 and RhoA at the cell surface. J. Biol. Chem. 274, 21430–21436. Millan, J., Cerny, J., Horejsi, V., and Alonso, M. A. (1999). CD4 segregates into specific detergentresistant T-cell membrane microdomains. Tissue Antigens 53, 33–40. Mineo, C., James, G. L., Smart, E. J., and Anderson, R. G. (1996). Localization of epidermal growth factor–stimulated Ras/Raf-1 interaction to caveolae membrane. J. Biol. Chem. 271, 11930– 11935. Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N., and Kupfer, A. (1998). Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86. Montixi, C., Langlet, C., Bernard, A. M., Thimonier, J., Dubois, C., Wurbel, M. A., Chauvin, J. P., Pierres, M., and He, H. T. (1998). Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17, 5334–5348. Moran, M., and Miceli, M. C. (1998). Engagement of GPI-linked CD48 contributes to TCR signals and cytoskeletal reorganization: A role for lipid rafts in T cell activation. Immunity 9, 787– 796. Muller, S., Demotz, S., Bulliard, C., and Valitutti, S. (1999). Kinetics and extent of protein tyrosine kinase activation in individual T cells upon antigenic stimulation. Immunology 97, 287–293. Mumby, S. M. (1997). Reversible palmitoylation of signaling proteins. Curr. Opin. Cell Biol. 9, 148–154. Neame, S. J., Uff, C. R., Sheikh, H., Wheatley, S. C., and Isacke, C. M. (1995). CD44 exhibits a cell type dependent interaction with Triton X-100 insoluble, lipid rich, plasma membrane domains. J. Cell Sci. 108, 3127–3135. Okamoto, T., Schlegel, A., Scherer, P. E., and Lisanti, M. P. (1998). Caveolins, a family of scaffolding proteins for organizing “preassembled signaling complexes” at the plasma membrane. J. Biol. Chem. 273, 5419–5422. Oliferenko, S., Paiha, K., Harder, T., Gerke, V., Schwarzler, C., Schwarz, H., Beug, H., Gunthert, U., and Huber, L. A. (1999). Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton. J. Cell Biol. 146, 843–854. Ostermeyer, A. G., Beckrich, B. T., Ivarson, K. A., Grove, K. E., and Brown, D. A. (1999). Glycosphingolipids are not essential for formation of detergent–resistant membrane rafts in melanoma cells. Methyl-beta-cyclodextrin does not affect cell surface transport of a GPI-anchored protein. J. Biol. Chem. 274, 34459–34466. Paige, L. A., Nadler, M. J., Harrison, M. L., Cassady, J. M., and Geahlen, R. L. (1993). Reversible palmitoylation of the protein-tyrosine kinase p56lck. J. Biol. Chem. 268, 8669–8674. Parolini, I., Sargiacomo, M., Lisanti, M. P., and Peschle, C. (1996). Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes. Blood 87, 3783–3794.

RAFT MEMBRANE DOMAINS

87

Parolini, I., Topa, S., Sorice, M., Pace, A., Ceddia, P., Montesoro, E., Pavan, A., Lisanti, M. P., Peschle, C., and Sargiacomo, M. (1999). Phorbol ester–induced disruption of the CD4–Lck complex occurs within a detergent-resistant microdomain of the plasma membrane. Involvement of the translocation of activated protein kinase C isoforms. J. Biol. Chem. 274, 14176–14187. Pasquet, J. M., Gross, B., Quek, L., Asazuma, N., Zhang, W., Sommers, C. L., Schweighoffer, E., Tybulewicz, V., Judd, B., Lee, J. R., Koretzky, G., Love, P. E., Samelson, L. E., and Watson, S. P. (1999). LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI. Mol. Cell. Biol. 19, 8326–8334. Paul, W. E., and Seder, R. A. (1994). Lymphocyte responses and cytokines. Cell 76, 241–251. Penninger, J. M., and Crabtree, G. R. (1999). The actin cytoskeleton and lymphocyte activation. Cell 96, 9–12. Pierini, L., Holowka, D., and Baird, B. (1996). FceRI–mediated association of 6-micron beads with RBL–2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling. J. Cell Biol. 134, 1427–1439. Pierini, L., Harris, N. T., Holowka, D., and Baird, B. (1997). Evidence supporting a role for microfilaments in regulating the coupling between poorly dissociable IgE–FceRI aggregates downstream signaling pathways. Biochemistry 36, 7447–7456. Pike, L. J., and Casey, L. (1996). Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains. J. Biol. Chem. 271, 26453–26456. Pike, L. J., and Miller, J. M. (1998). Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover. J. Biol. Chem. 273, 22298–22304. Pingel, J. T., and Thomas, M. L. (1989). Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation. Cell 58, 1055–1065. Polyak, M. J., Tailor, S. H., and Deans, J. P. (1998). Identification of a cytoplasmic region of CD20 required for its redistribution to a detergent-insoluble membrane compartment. J. Immunol. 161, 3242–3248. Poole, A., Gibbins, J. M., Turner, M., van Vugt, M. J., van de Winkel, J. G., Saito, T., Tybulewicz, V. L., and Watson, S. P. (1997). The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen. EMBO J. 16, 2333–2341. Pralle, A., Keller, P., Florin, E., Simons, K., and Horber, J. K. (2000). Sphingolipid–cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J. Cell Biol. 148, 997–1008. Resh, M. D. (1994). Myristylation and palmitylation of Src family members: The fats of the matter. Cell 76, 411–413. Resh, M. D. (1999). Fatty acylation of proteins: New insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451, 1–16. Reth, M., and Wienands, J. (1997). Initiation and processing of signals from the B cell antigen receptor. Annu. Rev. Immunol. 15, 453–479. Rietveld, A., and Simons, K. (1998). The differential miscibility of lipids as the basis for the formation of functional membrane rafts. Biochim. Biophys. Acta 1376, 467–479. Robinson, P. J. (1991). Phosphatidylinositol membrane anchors and T-cell activation. Immunol. Today 12, 35–41. Rodgers, W., and Rose, J. K. (1996). Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains. J. Cell Biol. 135, 1515–1523. Rodgers, W., Crise, B., and Rose, J. K. (1994). Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol–anchored protein targeting to a glycolipid-enriched membrane fraction. Mol. Cell. Biol. 14, 5384–5391. Rohatgi, R., Ma, L., Miki, H., Lopez, M., Kirchhausen, T., Takenawa, T., and Kirschner, M. W. (1999). The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97, 221–231.

88

THOMAS HARDER

Romagnoli, P., and Bron, C. (1997). Phosphatidylinositol-based glycolipid-anchored proteins enhance proximal TCR signaling events. J. Immunol. 158, 5757–5764. Romagnoli, P., and Bron, C. (1999). Defective TCR signaling events in glycosylphosphatidylinositoldeficient T cells derived from paroxysmal nocturnal hemoglobinuria patients. Int. Immunol. 11, 1411–1422. Roy, S., Luetterforst, R., Harding, A., Apolloni, A., Etheridge, M., Stang, E., Rolls, B., Hancock, J. F., and Parton, R. G. (1999). Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains [see comments]. Nature Cell Biol. 1, 98–105. Rozdzial, M. M., Malissen, B., and Finkel, T. H. (1995). Tyrosine-phosphorylated T cell receptor zeta chain associates with the actin cytoskeleton upon activation of mature T lymphocytes. Immunity 3, 623–633. Rozdzial, M. M., Pleiman, C. M., Cambier, J. C., and Finkel, T. H. (1998). pp56Lck mediates TCR zeta-chain binding to the microfilament cytoskeleton. J. Immunol. 161, 5491–5499. Rozelle, A. L., Machesky, L. M., Yamamoto, M., Driessens, M. H., Insall, R. H., Roth, M. G., LubyPhelps, K., Marriott, G., Hall, A., and Yin, H. L. (2000). Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3. Curr. Biol. 10, 311–320. Rudd, C. E., Janssen, O., Prasad, K. V., Raab, M., da Silva, A., Telfer, J. C., and Yamamoto, M. (1993). src-related protein tyrosine kinases and their surface receptors. Biochim. Biophys. Acta 1155, 239–266. Ryan, T. A., Menon, A. K., Holovka, D., Baird, B., and Webb, B. B. (1986). Inter-receptor spacing in IgE receptor aggregates on the surface of RBL cells. Biophys. J. 49, 360a. Sankaram, M. B., and Thompson, T. E. (1990a). Interaction of cholesterol with various glycerophospholipids and sphingomyelin. Biochemistry 29, 10670–10675. Sankaram, M. B., and Thompson, T. E. (1990b). Modulation of phospholipid acyl chain order by cholesterol. A solid-state 2H nuclear magnetic resonance study. Biochemistry 29, 10676–10684. Sankaram, M. B., and Thompson, T. E. (1991). Cholesterol-induced fluid-phase immiscibility in membranes. Proc. Natl. Acad. Sci. USA 88, 8686–8690. Scheek, S., Brown, M. S., and Goldstein, J. L. (1997). Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1. Proc. Natl. Acad. Sci. USA 94, 11179–11183. Scheiffele, P., Roth, M. G., and Simons, K. (1997). Interaction of influenza virus haemagglutinin with sphingolipid–cholesterol membrane domains via its transmembrane domain. EMBO J. 16, 5501–5508. Scherer, P. E., Lewis, R. Y., Volonte, D., Engelman, J. A., Galbiati, F., Couet, J., Kohtz, D. S., van Donselaar, E., Peters, P., and Lisanti, M. P. (1997). Cell-type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J. Biol. Chem. 272, 29337–29346. Schmidt, C. F., Barenholz, Y., Huang, C., and Thompson, T. E. (1978). Monolayer coupling in sphingomyelin bilayer systems. Nature 271, 775–777. Schroeder, R., London, E., and Brown, D. (1994). Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)–anchored proteins: GPIanchored proteins in liposomes and cells show similar behavior. Proc. Natl. Acad. Sci. USA 91, 12130–12134. Schroeder, R. J., Ahmed, S. N., Zhu, Y., London, E., and Brown, D. A. (1998). Cholesterol and sphingolipid enhance the Triton X-100 insolubility of glycosylphosphatidylinositol-anchored proteins by promoting the formation of detergent-insoluble ordered membrane domains. J. Biol. Chem. 273, 1150–1157. Schutz, ¨ G. J., Kada, G., Pastushenko, V. P., and Schindler, H. (2000). Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J. 19, 892–901.

RAFT MEMBRANE DOMAINS

89

Seavitt, J. R., White, L. S., Murphy, K. M., Loh, D. Y., Perlmutter, R. M., and Thomas, M. L. (1999). Expression of the p56(Lck) Y505F mutation in CD45-deficient mice rescues thymocyte development. Mol. Cell. Biol. 19, 4200–4208. Sedwick, C. E., Morgan, M. M., Jusino, L., Cannon, J. L., Miller, J., and Burkhardt, J. K. (1999). TCR, LFA-1, and CD28 play unique and complementary roles in signaling T cell cytoskeletal reorganization. J. Immunol. 162, 1367–1375. Shaw, A. S., and Dustin, M. L. (1997). Making the T cell receptor go the distance: A topological view of T cell activation. Immunity 6, 361–369. Sheets, E. D., Lee, G. M., Simson, R., and Jacobson, K. (1997). Transient confinement of a glycosylphosphatidylinositol-anchored protein in the plasma membrane. Biochemistry 36, 12449– 12458. Sheets, E. D., Holowka, D., and Baird, B. (1999a). Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of FcepsilonRI and their association with detergent-resistant membranes. J. Cell Biol. 145, 877–887. Sheets, E. D., Holowka, D., and Baird, B. (1999b). Membrane organization in immunoglobulin E receptor signaling. Curr. Opin. Chem. Biol. 3, 95–99. Shenoy-Scaria, A. M., Kwong, J., Fujita, T., Olszowy, M. W., Shaw, A. S., and Lublin, D. M. (1992). Signal transduction through decay-accelerating factor. Interaction of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and p59fyn 1. J. Immunol. 149, 3535–3541. Shenoy-Scaria, A. M., Dietzen, D. J., Kwong, J., Link, D. C., and Lublin, D. M. (1994). Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae. J. Cell Biol. 126, 353–363. Sicheri, F., Moarefi, I., and Kuriyan, J. (1997). Crystal structure of the Src family tyrosine kinase Hck [see comments]. Nature 385, 602–609. Silvius, J. R., del Giudice, D., and Lafleur, M. (1996). Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length. Biochemistry 35, 15198–15208. Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569–572. Simons, K., and van Meer, G. (1988). Lipid sorting in epithelial cells. Biochemistry 27, 6197–6202. Simons, M., Friedrichson, T., Schulz, J. B., Pitto, M., Masserini, M., and Kurzchalia, T. V. (1999). Exogenous administration of gangliosides displaces GPI-anchored proteins from lipid microdomains in living cells. Mol. Biol. Cell. 10, 3187–3196. Singer, S. J., and Nicolson, G. L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175, 720–731. Skibbens, J. E., Roth, M. G., and Matlin, K. S. (1989). Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts. J. Cell Biol. 108, 821–832. Slavik, J. M., Hutchcroft, J. E., and Bierer, B. E. (1999). CD28/CTLA-4 and CD80/CD86 families: Signaling and function. Immunol. Res. 19, 1–24. Slotte, J. P., and Bierman, E. L. (1988). Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts. Biochem. J. 250, 653–658. Snapper, S. B., and Rosen, F. S. (1999). The Wiskott–Aldrich syndrome protein (WASP): Roles in signaling and cytoskeletal organization. Annu. Rev. Immunol. 17, 905–929. Snapper, S. B., Rosen, F. S., Mizoguchi, E., Cohen, P., Khan, W., Liu, C. H., Hagemann, T. L., Kwan, S. P., Ferrini, R., Davidson, L., Bhan, A. K., and Alt, F. W. (1998). Wiskott–Aldrich syndrome protein–deficient mice reveal a role for WASP in T but not B cell activation. Immunity 9, 81–91. Sperling, A. I., Sedy, J. R., Manjunath, N., Kupfer, A., Ardman, B., and Burkhardt, J. K. (1998). TCR signaling induces selective exclusion of CD43 from the T cell–antigen–presenting cell contact site. J. Immunol. 161, 6459–6462.

90

THOMAS HARDER

Spiegel, S., Kassis, S., Wilchek, M., and Fishman, P. H. (1984). Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes. J. Cell Biol. 99, 1575–1581. Spink, C. H., Yeager, M. D., and Feigenson, G. W. (1990). Partitioning behavior of indocarbocyanine probes between coexisting gel and fluid phases in model membranes. Biochim. Biophys. Acta 1023, 25–33. Stauffer, T. P., and Meyer, T. (1997). Compartmentalized IgE receptor–mediated signal transduction in living cells. J. Cell Biol. 139, 1447–1454. Stefanova, I., and Horejsi, V. (1991). Association of the CD59 and CD55 cell surface glycoproteins with other membrane molecules. J. Immunol. 147, 1587–1592. Stefanova, I., Horejsi, V., Ansotegui, I. J., Knapp, W., and Stockinger, H. (1991). GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science 254, 1016–1019. Straus, D. B., and Weiss, A. (1992). Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell 70, 585–593. Stulnig, T. M., Berger, M., Sigmund, T., Stockinger, H., Horejsi, V., and Waldhausl, W. (1997). Signal transduction via glycosyl phosphatidylinositol–anchored proteins in T cells is inhibited by lowering cellular cholesterol. J. Biol. Chem. 272, 19242–19247. Stulnig, T. M., Berger, M., Sigmund, T., Raederstorff, D., Stockinger, H., and Waldhausl, W. (1998). Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains. J. Cell Biol. 143, 637–644. Takahama, Y., Ohishi, K., Tokoro, Y., Sugawara, T., Yoshimura, Y., Okabe, M., Kinoshita, T., and Takeda, J. (1998). Functional competence of T cells in the absence of glycosylphosphatidylinositolanchored proteins caused by T cell–specific disruption of the Pig-a gene. Eur. J. Immunol. 28, 2159–2166. Thomas, M. L., and Brown, E. J. (1999). Positive and negative regulation of Src-family membrane kinases by CD45. Immunol. Today 20, 406–411. Thomas, P. M., and Samelson, L. E. (1992). The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells. J. Biol. Chem. 267, 12317–12322. Thomas, J. L., Holowka, D., Baird, B., and Webb, W. W. (1994). Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins. J. Cell Biol. 125, 795–802. Thompson, T. E., Barenholz, Y., Brown, R. E., Correa-Freire, M., Young, W. W., and Tillack, T. W. (1986). Molecular organisation of glycosphingolipids in phosphatidylcholine bilayers and biological membranes. In “Enzymes of Lipid Metabolism II” (L. Freysz, H. Dreyfus, R. Massarelli, and S. Gatt, eds.), pp. 387–396. Plenum, New York. Trowbridge, I. S., and Thomas, M. L. (1994). CD45: An emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu. Rev. Immunol. 12, 85– 116. Turner, J. M., Brodsky, M. H., Irving, B. A., Levin, S. D., Perlmutter, R. M., and Littman, D. R. (1990). Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs. Cell 60, 755–765. Turner, M., Mee, P. J., Walters, A. E., Quinn, M. E., Mellor, A. L., Zamoyska, R., and Tybulewicz, V. L. (1997). A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes. Immunity 7, 451–460. Unkeless, J. C., Shen, Z., Lin, C. W., and DeBeus, E. (1995). Function of human Fc gamma RIIA and Fc gamma RIIIB. Semin. Immunol. 7, 37–44. Valitutti, S., Dessing, M., Aktories, K., Gallati, H., and Lanzavecchia, A. (1995). Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton. J. Exp. Med. 181, 577–584. van den Berg, C. W., Cinek, T., Hallett, M. B., Horejsi, V., and Morgan, B. P. (1995). Exogenous glycosyl phosphatidylinositol–anchored CD59 associates with kinases in membrane clusters on U937 cells and becomes Ca(2+)-signaling competent. J. Cell Biol. 131, 669–677.

RAFT MEMBRANE DOMAINS

91

van der Merwe, P. A., Davis, S. J., Shaw, A. S., and Dustin, M. L. (2000). Cytoskeletal polarization and redistribution of cell-surface molecules during T cell antigen recognition. Semin. Immunol. 12, 5–21. van Leeuwen, J. E., and Samelson, L. E. (1999). T cell antigen–receptor signal transduction. Curr. Opin. Immunol. 11, 242–248. van Leeuwen, J. E., Paik, P. K., and Samelson, L. E. (1999). The oncogenic 70Z Cbl mutation blocks the phosphotyrosine binding domain-dependent negative regulation of ZAP-70 by c-Cbl in Jurkat T cells. Mol. Cell. Biol. 19, 6652–6664. van’t Hof, W., and Resh, M. D. (1997). Rapid plasma membrane anchoring of newly synthesized p59fyn: Selective requirement for NH2-terminal myristoylation and palmitoylation at cysteine-3. J. Cell Biol. 136, 1023–1035. van’t Hof, W., and Resh, M. D. (1999). Dual fatty acylation of p59(Fyn) is required for association with the T cell receptor zeta chain through phosphotyrosine–Src homology domain–2 interactions. J. Cell Biol. 145, 377–389. Varma, R., and Mayor, S. (1998). GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394, 798–801. Verkade, P., Harder, T., Lafont, F., and Simons, K. (2000). Induction of caveolae in the apical plasma membrane of Madin–Darby canine kidney cells. J. Cell Biol. 148, 727–739. Viola, A., Salio, M., Tuosto, L., Linkert, S., Acuto, O., and Lanzavecchia, A. (1997). Quantitative contribution of CD4 and CD8 to T cell antigen receptor serial triggering. J. Exp. Med. 186, 1775–1779. Viola, A., Schroeder, S., Sakakibara, Y., and Lanzavecchia, A. (1999). T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283, 680–682. Wange, R. L., and Samelson, L. E. (1996). Complex complexes: Signaling at the TCR. Immunity 5, 197–205. Webb, Y., Hermida-Matsumoto, L., and Resh, M. D. (2000). Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J. Biol. Chem. 275, 261–270. Weber, J. R., Orstavik, S., Torgersen, K. M., Danbolt, N. C., Berg, S. F., Ryan, J. C., Tasken, K., Imboden, J. B., and Vaage, J. T. (1998). Molecular cloning of the cDNA encoding pp36, a tyrosinephosphorylated adaptor protein selectively expressed by T cells and natural killer cells. J. Exp. Med. 187, 1157–1161. Weintraub, B. C., Jun, J. E., Bishop, A. C., Shokat, K. M., Thomas, M. L., and Goodnow, C. C. (2000). Entry of B cell receptor into signaling domains is inhibited in tolerant B cells. J. Exp. Med. 191, 1443–1448. Weiss, A., and Littman, D. R. (1994). Signal transduction by lymphocyte antigen receptors. Cell 76, 263–274. Welti, R., and Glaser, M. (1994). Lipid domains in model and biological membranes. Chem. Phys. Lipids 73, 121–137. White, D. A. (1973). The phospholipid composition of mammalian tissues. In (G. B. Anseel, J. N. Hawthorne, and R. M. C. Dawson, eds.), pp. 441–482. Elsevier, New York. Williams, J. C., Weijland, A., Gonfloni, S., Thompson, A., Courtneidge, S. A., Superti-Furga, G., and Wierenga, R. K. (1997). The 2.35 A crystal structure of the inactivated form of chicken Src: A dynamic molecule with multiple regulatory interactions. J. Mol. Biol. 274, 757–775. Williams, J. C., Wierenga, R. K., and Saraste, M. (1998). Insights into Src kinase functions: Structural comparisons. Trends Biochem. Sci. 23, 179–184. Wolven, A., Okamura, H., Rosenblatt, Y., and Resh, M. D. (1997). Palmitoylation of p59fyn is reversible and sufficient for plasma membrane association. Mol. Biol. Cell. 8, 1159–1173. Wulfing, ¨ C., and Davis, M. M. (1998). A receptor/cytoskeletal movement triggered by costimulation during T cell activation. Science 282, 2266–2269.

92

THOMAS HARDER

Xavier, R., and Seed, B. (1999). Membrane compartmentation and the response to antigen. Curr. Opin. Immunol. 11, 265–269. Xavier, R., Brennan, T., Li, Q., McCormack, C., and Seed, B. (1998). Membrane compartmentation is required for efficient T cell activation. Immunity 8, 723–732. Xu, W., Harrison, S. C., and Eck, M. J. (1997). Three-dimensional structure of the tyrosine kinase c-Src [see comments]. Nature 385, 595–602. Yamamura, S., Handa, K., and Hakomori, S. (1997). A close association of GM3 with c-Src and Rho in GM3-enriched microdomains at the B16 melanoma cell surface membrane: A preliminary note. Biochem. Biophys. Res. Commun. 236, 218–222. Yang, W. C., Collette, Y., Nunes, J. A., and Olive, D. (2000). Tec kinases: A family with multiple roles in immunity. Immunity 12, 373–382. Yeh, E. T., Reiser, H., Bamezai, A., and Rock, K. L. (1988). TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor. Cell 52, 665–674. Yu, J., Fischman, D. A., and Steck, T. L. (1973). Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J. Supramol. Struct. 1, 233–248. Yurchak, L. K., and Sefton, B. M. (1995). Palmitoylation of either Cys-3 or Cys-5 is required for the biological activity of the Lck tyrosine protein kinase. Mol. Cell. Biol. 15, 6914–6922. Zhang, W., and Samelson, L. E. (2000). The role of membrane-associated adaptors in T cell receptor signalling. Semin. Immunol. 12, 35–41. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P., and Samelson, L. E. (1998a). LAT: The ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92, 83–92. Zhang, W., Trible, R. P., and Samelson, L. E. (1998b). LAT palmitoylation: Its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation. Immunity 9, 239–246. Zhang, W., Irvin, B. J., Trible, R. P., Abraham, R. T., and Samelson, L. E. (1999a). Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11, 943–950. Zhang, W., Sommers, C. L., Burshtyn, D. N., Stebbins, C. C., DeJarnette, J. B., Trible, R. P., Grinberg, A., Tsay, H. C., Jacobs, H. M., Kessler, C. M., Long, E. O., Love, P. E., and Samelson, L. E. (1999b). Essential role of LAT in T cell development. Immunity 10, 323–332. Zhou, M. J., Lublin, D. M., Link, D. C., and Brown, E. J. (1995). Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fc gamma RII or Fc gamma RIIIB ligation in human polymorphonuclear leukocytes. Implications for immune complex activation of the respiratory burst. J. Biol. Chem. 270, 13553–13560. Zlatkine, P., Mehul, B., and Magee, A. I. (1997). Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif. J. Cell Sci. 110, 673–679.

ADVANCES IN IMMUNOLOGY, VOL. 77

Human Basophils: Mediator Release and Cytokine Production JOHN T. SCHROEDER, DONALD W. MACGLASHAN, JR., AND LAWRENCE M. LICHTENSTEIN Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224

I. Introduction

The binding of antigen to receptor-bound IgE on the surface of basophils (and mast cells), resulting in the release of potent inflammatory mediators such as histamine, leukotrienes, and prostaglandins, has long been recognized as the hallmark response contributing to the signs and symptoms associated with allergic disease. The role of the basophil in early studies investigating the mechanisms underlying this reaction was primarily one of a surrogate by which to better understand those of the less accessible tissue mast cell. Indeed, functional similarities between the two cell types were suggested some 30 years ago, when it was shown that in vitro histamine release from basophils to various allergens predicted the severity of the respiratory symptoms experienced by the donor when exposed to that allergen (Lichtenstein et al., 1968). The comparison between basophils and mast cells, however, has since been abandoned, as there is mounting evidence indicating that the basophil, which is far more responsive to a variety of stimuli and cytokines, plays a more significant role in the late responses following allergen exposure rather than the early events that seem most attributed to the mast cell. To extend on this belief, studies have shown that human basophils themselves are cytokine-secreting cells, producing cytokines originally described in a subset of mouse T lymphocytes (Mossman et al., 1986). Although this finding stemmed from work done in murine mast cell lines (Plaut et al., 1989; Wodnar-Filipowicz et al., 1989), there is some doubt that isolated human mast cells possess similar capabilities. Basophils, however, readily generate large quantities of interleukin 4 (IL-4) and IL-13—two of the so-called T helper 2 (Th2) cytokines that are found in tissues during allergic inflammation and are thought to contribute to the overall pathogenesis of disease. This discovery, which is still very much in its infancy, along with the fact that basophils selectively infiltrate allergic lesions along with eosinophils and lymphocytes, raises an important concept: namely, the belief that these cells can contribute to disease by modulating the biological responses of other cell types. Thus, some 130 years since their first description in humans, we are continuing to evaluate the potential of the basophil and its role in immune responses. This chapter focuses on existing as well as more recent information pertaining to the biology of these cells, with particular emphasis on the parameters, pharmacological control, and mechanisms regulating the generation of IL-4 and IL-13 from these cells. 93 C 2001 by Academic Press Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

94

SCHROEDER et al.

II. Basophil Growth and Maturation

Under normal circumstances, basophils develop in the bone marrow and are thought to be released into circulation as mature cells, as which they represent 1010 ). The x-ray crystal structure of the ␣-subunit has recently been resolved to 2.4 A. Within the portion of the subunit that confers specificity for IgE are found four tryptophan residues organized in an unusual loop that provides for a hydrophobic environment and likely accounts for the high affinity for IgE binding (Garman et al., 1998, 1999; Hulett et al., 1999). For basophils, it has been shown that the number of FcεRI receptors can vary between 5000 and 1 million and is very much dependent on the donor (MacGlashan et al., 1983). Studies performed in the late 1970s showed evidence that the expression of FcεRI on circulating basophils correlates with the IgE antibody levels in serum (Malveaux et al., 1978). This led to the hypothesis that the IgE concentration “drove” the number of IgE receptors on basophils and mast cells. Only recently has this concept been confirmed in humans in work made possible with the development of anti-IgE

HUMAN BASOPHILS

97

therapy to prevent allergic disease (Fick, 1999). The intravenous administration of a humanized monoclonal anti-IgE antibody (E25) to subjects allergic to dust mite allergen caused a >90% reduction in their serum IgE levels, which resulted in a dramatic reduction in the ability of antigen to induce histamine release from basophils isolated from these donors (MacGlashan et al., 1997). Most interestingly, the drop in serum IgE levels was accompanied by the loss of FcεRI␣ expression. Upon completion of the anti-IgE therapy, serum IgE levels eventually returned, as did the expression of the receptor and the ability of antigen to induce histamine release (Saini et al., 1999). In vitro studies confirmed these data, elucidating the kinetics for the down-regulation and up-regulation of FcεRI␣ and demonstrating that this regulation could be attributed to the absence or presence of IgE (MacGlashan et al., 1998, 1999). A comprehensive analysis pertaining to the mechanisms and consequences of IgE antibody–dependent regulation of FcεRI expression is beyond the scope of this chapter but has been provided elsewhere (MacGlashan et al., 2000). Although the expression of FcεRI was originally thought to be limited to basophils and mast cells, several studies have indicated that a variety of other cells can express this receptor. Most convincing are those demonstrating that Langerhans cells (Bieber et al., 1992; Wang et al., 1992), monocytes (Maurer et al., 1994), and dendritic cells (Maurer et al., 1996) express FcεRI␣. Interestingly, the ␤-subunit, which is believed to amplify the signaling through FcεRI, has not been identified on these cells. Other reports have suggested that FcεRI␣ is also expressed on eosinophils (Gounni et al., 1994; Rajakulasingam et al., 1998), although there has been intense debate regarding these findings (Kita and Gleich, 1997; Kita et al., 1999). There is evidence, however, that an intracellular pool of the ␣-subunit is found in eosinophils (Seminario et al., 1999). It seems possible that this reservoir may be responsible for the immunodetection of FcεRI␣ in many of the studies reporting cell surface expression. It is important to note that IgE concentration does not appear to up-regulate FcεRI␣ expression on eosinophils, as it does for mast cells and basophils. There is presently no clear explanation, with regard to function, for the expression of FcεRI on cells other than basophils and mast cells, although a role in parasitic immunity has been suggested. Human basophils express several activation-linked markers other than FcεRI. With regard to other Ig receptors, only Fc␥ RII (CD32) has been identified and is responsible for the binding of various subclasses of IgG antibody. Although its role on basophils is not fully understood, there is evidence suggesting that it relays intracellular signals that work to oppose those initiated with FcεRI crosslinking (Daeron et al., 1995). Thus, cross-linking of Fc␥ RII/IgG complexes may prevent basophil activation for mediator release. This is not to be confused with the evidence that anti-IgG antibody can induce histamine secretion by crosslinking IgG–IgE complexes bound to the IgE receptor (Lichtenstein et al., 1992).

98

SCHROEDER et al.

Basophils also express CD40, an activation-linked antigen more commonly found on B cells. CD40 has sequence homology with the tumor necrosis factor (TNF) receptor family, which includes TNF-␣ and nerve growth factor (NGF). Interestingly, human basophils as well as and mast cells have been shown to express the cell surface ligand for CD40 (CD40L) (Gauchat et al., 1993). This antigen is found on a limited number of cell types, including activated T cells, endothelium, and platelets. The interaction between CD40 and CD40L has been shown to constitute an important step for IgE synthesis such that, in the presence of IL-4 or IL-13, it provides a necessary co-stimulus for B cell synthesis of this Ig. In fact, by secreting IL-4 and IL-13 and expressing CD40L, basophils alone can provide the necessary signals for B cell production of IgE, which were classically thought to be provided only by activated T cells (Gauchat et al., 1993; Yanagihara et al., 1998). Three monoclonal antibodies have recently been characterized, and all appear immunologically specific for proteins expressed only by basophils or their progenitors. Antibody 2D7 detects a 72-kDa granule-associated protein that is released upon activation (Kepley et al., 1995). BB1 is also reported to detect a cytoplasmic molecule, which, upon degranulation, is also secreted and thought to represent a novel mediator (McEuen et al., 1999). A third antibody, 97A6, is thought to identify a surface antigen found on basophil progenitors in addition to mature cells (Buhring et al., 1999). While these antibodies detect proteins having no known function, they are currently being used for identifying basophils in sites of allergic inflammation (see below).

IV. Inflammatory Mediators

A. HISTAMINE Basophils and mast cells synthesize significant amounts of histamine in a reaction resulting from the decarboxylation of L-histidine. It is stored in the cytoplasmic granules of basophils complexed with the highly charged proteoglycan, chondroitin sulfate, as opposed to heparin in the mast cell. The amount of histamine stored in basophils is remarkably consistent among donor populations, amounting to ∼1 pg per cell, and both IL-3 and GM-CSF have been shown to have an important role in its increased synthesis during the latter stages of basophil maturation. Its release from activated cells has been well characterized ultrastructurally for several different modes of activation (Dvorak, 1998). The physiological consequences of histamine release result from its role as a potent smooth muscle spasmogen and its ability to cause vascular leakage by dilating terminal aterioles and constricting postcapillary venules. There are data that histamine can modulate specific immune responses. In particular, cytotoxic T lymphocyte (CTL) responses are down-regulated by histamine binding to H2

HUMAN BASOPHILS

99

receptors on CD8+ T cells, which is accompanied by increases in intracellular cAMP levels (Plaut et al., 1973; Rocklin and Habarek-Davidson, 1984). H2 antagonists have been shown to prevent increases in intracellular cAMP and to reverse the inhibitory effects on CTL activity (Griswold et al., 1986; White and Ballow, 1985). More recently, the production of IL-12 and interferon ␥ (IFN-␥ ) by monocytes has been shown to be inhibited by histamine, while IL-10 secretion is augmented by this amine (Elenkov et al., 1998; Lagier et al., 1997). In light of this evidence, it seems possible that histamine may function to down-regulate Th1-like activity while promoting Th2-like responses. This hypothesis, in fact, seems consistent with the knowledge that basophils secrete IL-4—a cytokine that is known to promote the development of Th2-like responses (see below). B. LEUKOTRIENE C4 Unlike histamine, leukotriene C4 (LTC4) is not stored in basophils but is synthesized within minutes after activation by the metabolism of arachidonic acid through the lipoxygenase pathway. Both phosphatidycholine and phosphatidylinositol likely provide for the arachidonic acid in this reaction, and these phospholipids are themselves thought to be metabolized through the enzymatic activity of phospholipase A2. It has been known for some time that the amount of LTC4 generated per basophil (10–100 fg) is far less than the picograms-per-cell quantities of histamine found in these cells. On a molar basis, however, LTC4 is some 6000 times more potent than histamine in contracting smooth muscle (Bochner, 1995). Along with its metabolites, LTD4 and LTE4, the three leukotrienes have a profound bronchoconstrictive and mucus-producing effect when released in the respiratory system and induce a prolonged wheal-and-flare reaction when secreted in the skin. In fact, there is the belief that the pathophysiology of asthma is mediated, to a large extent, by the actions of these mediators. The recent clinical introduction of LT inhibitors demonstrates this to be the case (Calhoun et al., 1998; Kane et al., 1996). C. CYTOKINES For many years, basophils were thought to release only preformed histamine and newly synthesized LTC4 after activation by a variety of stimuli. As noted above, there is now firm evidence from many laboratories that basophils are also a major source of IL-4 and IL-13 (Brunner et al., 1993; Gibbs et al., 1996; Li et al., 1996a; MacGlashan et al., 1994; Ochensberger et al., 1996; Schroeder et al., 1994b). Both cytokines are found at sites of allergic inflammation, and the immunomodulatory properties mediated by each are recognized as pivotal in the pathogenesis of allergic inflammation and disease. In particular, IL-4 and IL-13 are the only two known cytokines that are capable of inducing an Ig isotype switch in B lymphocytes from IgM to IgE (Defrance et al., 1994;

100

SCHROEDER et al.

de Vries and Zurawski, 1995; Punnonen et al., 1993; Vercelli and Geha, 1993). For both cytokines, this is initiated through receptor-mediated activation of the transcription factor STAT6, which produces germ-line cepsilon transcription (Hill et al., 1999; Kaplan et al., 1996). The subsequent interaction of CD40 on the B cell with its ligand, CD40L, found on several cell types including the basophil, results in transcriptional signals followed by the secretion of IgE. As noted, both IL-4 and IL-13 activate the endothelium for increased expression of VCAM-1 and this adhesion molecule promotes the selective transendothelial migration of eosinophils, basophils, and T lymphocytes (Bochner et al., 1995; Hemler, 1990; Moser and Fehr, 1992; Schleimer et al., 1992; Thornhill et al., 1990). The expression of major histocompatibility complex (MHC) class II antigens (i.e., HLA-DR) is also up-regulated on antigen-presenting cells following exposure to IL-4 or IL-13 (Defrance et al., 1994). Most significantly, the development of the Th2 phenotype in so-called Th0 lymphocytes has been shown to be dependent on IL-4 exposure (Abehsira-Amar et al., 1992; Swain et al., 1990). Thus, in order for T cells to produce IL-4 and other Th2 cytokines (e.g., IL-13, IL-5, and IL-6), they must first be exposed to IL-4. This has spawned a number of theories as to what is responsible for the initial secretion of IL-4, and a variety of cell types have been suspected, including specific T cell populations (i.e., NK1.1 cells in the mouse), mast cells, basophils, and eosinophils) (reviewed in Romagnani, 1998). Whereas IL-13 does not share this Th2-promoting property of IL-4 (IL13–specific receptors have not been identified on T cells), this cytokine does appear to have a novel role in collagen deposition (Chiaramonte et al., 1999) and mucus production (Grunig et al., 1998; Wills-Karp et al., 1998), both of which are prominent features in chronic inflammatory diseases such as asthma. To date, there is no evidence showing that basophils secrete Th1-like cytokines or any other Th2-like cytokines. One report has shown evidence that the chemokine macrophage inflammatory protein 1␣ is secreted by basophils, suggesting an additional role in cell recruitment (Li et al., 1996b). The production of other chemokines by basophils has not been reported, although there are unpublished studies suggesting that they might. While the seminal studies linking FcεRI activation with IL-4 and IL-13 generation were performed with mouse mast cell lines (Plaut et al., 1989), in humans there is strong evidence that basophils are perhaps the predominant source of these cytokines. We first demonstrated this in blood by showing that both protein and mRNA for IL-4 correlated with the presence of basophils (MacGlashan et al., 1994). Others have since shown that basophils are the predominant source of IL-4 and IL-13, even in mixed leukocyte cultures (1–2% basophils) receiving specific allergen as stimulus (Devouassoux et al., 1999a; Kasaian et al., 1996). This has been a novel finding, since antigen-specific T cells were commonly thought to be the primary source of these cytokines in response to allergen. However, the explanation for these findings is rather straightforward: the frequency of

HUMAN BASOPHILS

101

antigen-specific basophils (i.e., those expressing antigen-specific IgE) far outnumbers that of allergen-specific T cells, which, at best, is on the order of 1 in 1000–5000 naive T cells. Thus, it has been shown that even a small percentage of basophils can secrete up to 10-fold greater levels of IL-4 and IL-13 compared to those produced by mixed lymphocytes (Devouassoux et al., 1999a). This may mean that basophils represent the major source of these cytokines in late-phase allergic reactions (see below), despite reports that they constitute a relatively small percentage (∼2–5%) of the cellular infiltrate. The technical problems of identifying and isolating basophils infiltrating allergic lesions have made it difficult to determine whether these cells contribute to the production of IL-4 and IL-13 at these sites. As noted below, we have recently shown that basophils obtained by bronchoalveolar lavage following segmental allergen challenge in the lungs are capable of producing IL-4, thus providing the first evidence that these cells secrete this cytokine at sites of allergic inflammation. In contrast, studies using immunohistochemical staining techniques have suggested that T cells, mast cells, and eosinophils produce IL-4 and other cytokines in biopsies taken from allergic lesions (Moller et al., 1996; Pawanker et al., 1997; Ying et al., 1997). However, the in vitro evidence supporting the secretion of IL-4 by isolated tissue mast cells has not been confirmed since its original description (Bradding et al., 1992). Thus, it seems possible that mast cells require a tissue component in order to generate IL-4 or that the techniques utilized in their isolation render them nonproductive. Alternatively, this discrepancy may reflect technical issues between the assays utilized to detect IL-4 (i.e., immunohistochemistry versus enzyme-linked immunosorbent assay). Nonetheless, the parameters and requirements for the in vitro generation of IL-4 and IL-13 by basophils are well established. In contrast, little information is available regarding how these cytokines are made by mast cells (or by eosinophils), despite numerous reports describing their expression in these cells (Ebisawa et al., 1995; Gibbs et al., 1997; Jaffe et al., 1995; Moqbel et al., 1995; Nakajima et al., 1996). V. Basophil Activation

A. IgE DEPENDENT As noted above, the IgE-dependent secretion of mediators and cytokines from basophils and mast cells, in its simplest form, is initiated when IgE antibody bound to FcεRI is cross-linked by specific antigen. In actuality, this interaction is quite complex, with the overall response being very much dependent on the sensitivity of the cell (i.e., the number of receptors needed for aggregation in order to achieve 50% of maximal secretion) (MacGlashan, 1993; MacGlashan et al., 1986). In fact, the sensitivity among donor basophils has been determined for

102

SCHROEDER et al.

mediator release and is quite variable, requiring as few as 200 and up to 30,000 receptor aggregations in order to induce a half-maximal response (MacGlashan et al., 1983). Because of the technical difficulties involved in their isolation, the sensitivity of mediator release from mast cells is not known but is generally thought to be similar to the basophil response. Since basophils and mast cells can be sensitized with IgE to a multitude of antigens, the consequences of cell sensitivity become complex, yet very important (reviewed in MacGlashan et al., 2000). Furthermore, much of the information pertaining to sensitivity has been derived from studies investigating basophil histamine release; thus, it is difficult to say whether similar parameters will apply for the generation of LTC4 and cytokines. Studies have suggested, however, that there are only subtle differences with regard to sensitivity for the release of these three classes of mediators (MacGlashan and Schroeder, 2000). It seems, however, that the number of receptor aggregates necessary for a half-maximal response of histamine will also produce a 50% response in cytokine and LTC4 secretion. The basophil response follows a classic bell-shaped curve when cells are challenged with a wide range of antigen concentrations. The same is true when crosslinking is induced by anti-IgE antibody, which is commonly used as an in vitro stimulus of basophils because of its ability to mimic, to some extent, the stimulation mediated by antigen and its ability to induce release from cells obtained from most donors, both allergic and nonallergic. Under optimal conditions, the IgE-mediated release of preformed histamine is nearly complete by 20 min. The generation and release of LTC4 follow a time course similar to those seen with histamine. In contrast to mediator release, cytokine secretion is considerably slower. Induced levels of IL-4 are first detected by 60–90 min, the response is half-maximal by 120 min, and it is essentially complete by 240 min after activation (MacGlashan et al., 1994; Schroeder et al., 1994b, 1998a). There is evidence that small quantities of IL-4 protein (100 ng/mL), IL-3 has been shown to induce histamine release from the cells of selected allergic donors, particularly those also reacting to HRF (MacDonald et al., 1989). Most significantly, IL-3 has been shown to be a potent stimulus of IL-13, inducing the secretion of this cytokine from most donor cells even in the absence of histamine and IL-4 release (Ochensberger et al., 1996; Redrup et al., 1998). This latter activity of IL-3 is of biological importance because it implies that basophils need not express IgE in order to make IL-13. Thus, IL-13 secreted from basophils exposed to IL-3 may have a role in inflammatory processes not directly linked to immediate hypersensitivity. Finally, it is well known that IL-3, like HRF, will enhance IgE-mediated responses, resulting in the increased secretion of histamine, LTC4, IL-4, and IL-13 induced by a wide range of anti-IgE or antigen concentrations. However, unlike HRF, IL-3 will also prime basophils for increased histamine and LTC4 release when activated with IgE-independent stimuli, such as C5a and FMLP (Kurimoto et al., 1989; MacGlashan and Warner, 1991). This priming effect occurs within minutes of IL-3 exposure, and we are just now beginning to understand some of the intracellular mechanisms involved (see below). While IL-3 and HRF are the predominant cytokines modulating basophil secretion, there is evidence that other cytokines may have similar capabilities. Both IL-1 and NGF are reported to enhance IgE-mediated secretion of histamine and LTC4 (Bischoff and Dahinden, 1992; Massey et al., 1989). For NGF, this is apparently mediated through the trk A rather than trk B, trk C, or the low-affinity NGF receptors, suggesting that other neurotrophins lack priming capabilities (Burgi et al., 1996). It is not presently known whether these cytokines have similar effects on basophil cytokine production. VI. Signal Transduction and Pharmacological Control of Secretion

A great deal of information concerning the intracellular signals regulating FcεRI-mediated signaling has come from studies investigating various rodent cell lines, including transformed rat basophilic leukemia cells and murine (IL-3– dependent) mast cell lines (Okazaki and Siraganian, 1999). It remains uncertain, however, whether the signal transduction mechanisms described in these models have relevance to human cells. Both the HMC-1 and KU812 cell lines (Butterfield et al., 1988; Fukuda et al., 1987), which resemble immature human mast cells and basophils, respectively, lack the expression of functional FcεRI receptors, therefore making it difficult to perform studies investigating IgEmediated signaling in these cells. Additional complications exist in that most subclones of these human cell lines are generally poor producers of IL-4 and

HUMAN BASOPHILS

107

IL-13 and, in many instances, fail to secrete these cytokines. As noted earlier, the same seems true for tissue-derived human mast cells, not to mention that these cells are extremely difficult to purify from tissue. In contrast, human basophils secrete large quantities of mediators and cytokines, making it possible to detect these products from relatively few cells. Furthermore, it has become possible to routinely purify basophils from blood to >99% purity and in numbers exceeding tens of millions of cells, which has led to marked progress in further delineating the signal transduction processes that account for the production and release of histamine, LTC4, IL-4, and IL-13. As a result, this section focuses primarily on the developments pertaining to the signals regulating the release of these three classes of mediators from human basophils, with particular emphasis on cytokine generation. The aggregation of FcεRI on human basophils results in the recruitment and activation of receptor-associated tyrosine kinases, namely, p53/56lyn (a member of the src family of kinases) and p72syk (Kepley et al., 1998b; Lavens-Phillips and MacGlashan, 2000). The expression and phosphorylation of these kinases in basophil lysates can be detected within minutes following IgE-mediated activation. Inhibitors of tyrosine kinases (e.g., PP1 and PP2) that appear selective for src kinases have been shown to reduce the phosphorylation of lyn and the subsequent activation of several proteins, including syk (Lavens-Phillips and MacGlashan, 2000). Most significantly, this inhibition causes a marked reduction in the secretion of all classes of mediators, suggesting that lyn and syk activation constitute early signals regulating IgE-mediated secretion. Studies suggest, in fact, that the so-called “nonreleaser”phenotype, which is characterized by basophils completely unresponsive to cross-linking stimuli, is the result of a deficiency in syk expression (Kepley et al., 1999). Furthermore, nonreleaser basophils have been shown to convert into “releaser” basophils following 4 days’ incubation in IL-3 (Yamaguchi et al., 1996), which implies that IL-3 signaling likely modulates syk expression. It is not known whether other components in the signaling cascade are also deficient. It is important to note that tyrosine kinase inhibitors fail to inhibit basophil histamine and LTC4 rapidly released through GTP-binding protein–coupled receptors, such as that occurring with FMLP activation. Maximal degranulation with this univalent stimulus is extremely rapid (∼1 min), or some 5- to 10-fold faster than that occurring with IgE-mediated stimulation, and is sensitive to pertussis toxin, which causes ADP ribosylation of GTP-binding proteins (Saito et al., 1987; Warner et al., 1987). Taking into account that FMLP also does not readily induce IL-4 and IL-13 secretion, the phosphorylation of lyn and syk associated with FcεRI activation appears to play a critical role in regulating downstream signals important for cytokine generation in basophils. While pharmacological studies indicate a commonality in the early events (i.e., lyn and syk activation) regulating the three classes of mediators generated in response to cross-linking, there is the belief that their intracellular

108

SCHROEDER et al.

mechanisms diverge from one another in downstream events (Schroeder and MacGlashan, 1997). The approach taken in identifying such divergent pathways in human basophils has been to measure specific intracellular events and their kinetics of activation and to determine whether specific inhibitors of these events also prevent the secretion of one or all of the classes of mediators. Using this approach, we have recently gained an understanding of the intracellular signaling involved in LTC4 formation. As noted above, the generation of this lipid mediator is very much dependent on cytosolic phospholipase A2 (cPLA2) activity. The so-called extracellular signal–regulated kinases (ERKs, such as ERK1 and ERK2), which are downstream of p21ras, are thought to target cPLA2 for activation. In recent studies, the sequential phosphorylation of the ERKs and cPLA2 correlated with the IgE-dependent (and -independent) release of LTC4 from basophils. By reducing the phosphorylation of ERK1 and ERK2 with the kinase inhibitor, PD098059, the subsequent phosphorylation of cPLA2 was also interrupted and the release of LTC4 was inhibited (Gibbs and Grabbe, 1999; Miura et al., 1999). Most interestingly, this inhibitor had little effect, if any, on the secretion of histamine and IL-4, suggesting that the intracellular events regulating the release of these mediators are upstream of the ERK1/ERK2 pathway. One potential pathway involved in both cytokine generation and mediator release may involve phosphatidylinositol 3-kinase, which is downstream of lyn and syk. This enzyme initiates a variety of signaling pathways that are important for ribosomal activity in the translation of some proteins. Both wortmannin and LY294002 inhibit phosphatidylinositol 3-kinase, and both compounds also prevent the secretion of all three classes of mediators released in response to the IgE-mediated activation (Gibbs and Grabbe, 1999). There is a large amount of data, derived mostly from studies using rodent cell models, that cytosolic calcium and protein kinase C (PKC) play pivitol roles in the secretory responses of basophils and mast cells. The basic features for their generation are as follows: both univalent (e.g., FMLP and C5a) and FcεRI cross-linking stimuli are thought to activate a phospholipase C enzyme via a GTP-binding protein or via a tyrosine kinase (i.e., syk), respectively. One of the consequences of both pathways is the metabolism of phospholipids resulting in the formation of two important messengers that relay the initiating signal further along. Diacylglycerol constitutes one of these messengers and functions as an important regulator of PKC activity. The other is triphosphates (derived from phosphoinositol metabolism), which have a role in regulating free calcium levels by causing the release of this ion from intracellular stores into the cytosol. Whereas the activation of PKC is well documented in the pathways leading to mediator release in rat basophilic leukemia cells, its role in the prodegranulatory events in human basophils following IgE-mediated activation has been challenged (Miura et al., 1998). Several PKC isozymes have been identified

HUMAN BASOPHILS

109

in basophils, including ␤1, ␤2, and ␦. However, the activation of these isozymes, as assessed by their translocation to the membrane fraction, has been observed only after stimulation either with phorbol myristate acetate (PMA)—a direct activator of PKC activity— or with FMLP. In contrast, activation with anti-IgE has not produced evidence of PKC translocation. In fact, inhibitors of PKC activity, such as the bisindolylmaleimides (BIS I and II), have actually been found to enhance anti-IgE–induced histamine release, suggesting that PKC activation plays more of an inhibitory role during IgE-mediated degranulation. Furthermore, the BIS compounds have no effect on histamine released by FMLP. It is important to note that staurosporine, which was used in early studies as an inhibitor of PKC, does prevent IgE-mediated but not FMLP-mediated histamine release (Warner and MacGlashan, 1990). However, the results with staurosporine are less clear in light of more recent information that this compound also inhibits the early and late tyrosine kinase activity associated with FcεRI activation. PKC activation plays an even more complex role in the generation of cytokines in basophils by possessing both anti- and pro-secretory activity. Studies show that PMA is a potent activator of basophils, causing nearly 100% histamine release at concentrations beginning at 1 ng/mL (Schleimer et al., 1981a). However, little, if any, IL-4 protein is secreted by cells cultured with this amount, or higher concentrations, of PMA even after 48 hr of incubation. In fact, the large quantities of IL-4 produced by cells cultured with calcium ionophores (e.g., ionomycin) are, remarkably, down-regulated some 70% with the simultaneous addition of PMA. This inhibitory effect of PMA is reversed with BIS II, suggesting that PKC activity negatively affects IL-4 generation in basophils (Schroeder et al., 1998a). In sharp contrast, PMA exerts quite an opposite effect on the secretion of IL-13 by basophils. In this instance, the phorbol ester will directly stimulate the secretion of this cytokine, and this response, as expected, is prevented by PKC inhibitors (Redrup et al., 1998). Thus, PKC activation appears to play a dual role in the production of cytokines by basophils by negatively regulating IL-4 while promoting the secretion of IL-13. Cytosolic calcium responses play a critical role in the pro-secretory events occurring in basophils, and both IgE-mediated and univalent stimuli are capable of inducing a calcium response resulting in degranulation. Although several studies have noted a good correlation between elevations in free cytosolic calcium and histamine release from basophils (Knol et al., 1992; Warner and MacGlashan, 1990), causal testing of this linkage indicates that the relationship is more complex than was first anticipated (MacGlashan and Botana, 1993). In particular, the linkage is better for secretion of LTC4 and IL-4 than for histamine release. The tight linkage for LTC4 secretion comes about because of the absolute requirement for cytosolic calcium elevations and the activity of cPLA2 and 5-lipoxygenase. Based on the current understanding of signaling elements needed for cytokine generation, secretion of IL-4 may have similar absolute

110

SCHROEDER et al.

requirements. First, early studies showed that IgE-mediated secretion of IL-4 is very much dependent on adequate elevations in cytosolic free calcium (Schroeder et al., 1994b). Second, during low-level cross-linking of FcεRI, which, as noted above, is optimal for the secretion of IL-4 (and IL-13), the cytosolic calcium responses are sustained compared to the short-lived responses seen during activation with the 10-fold greater concentrations of cross-linking stimuli that are optimal for histamine release (MacGlashan and Botana, 1993). Finally, the lack of IL-4 and IL-13 secreted in response to FMLP and C5a may reflect the fact that these univalent stimuli induce cytosolic calcium responses in basophils that are also short-lived, despite being sufficient for histamine and LTC4 release (MacGlashan and Warner, 1991; Warner and MacGlashan, 1990). At this time, there is reason to believe that a calcium/calcineurin pathway has a role in the transcription of cytokine induced with FcεRI activation (Schroeder and MacGlashan, 1997). This belief is founded, in part, by several observations that are relevant to the importance of free cytosolic calcium in this response. First, the addition of a chelator of calcium (e.g., EGTA) will immediately halt cytokine secretion induced by anti-IgE by preventing the accumulation of mRNA for this cytokine. Second, calcium ionophores, which sustain intracellular calcium levels, are the most potent activators of IL-4 and IL-13, inducing the secretion of up to 1000 pg/106 basophils for either cytokine. Most significant is the fact that FK-506 and cyclosporine A are the most potent inhibitors of IL-4 and IL-13 secreted in response to IgE-mediated activation (Redrup et al., 1998; Schroeder et al., 1999). Well known as selective inhibitors of calcineurin phosphatase activity, these immunophilins prevent IL-4 secretion in basophils at subpicomolar concentrations and are some 50- to 100-fold more effective at preventing the secretion of this cytokine than they are at inhibiting histamine release (De Paulis et al., 1991). It is important to note that while these drugs similarly inhibit IL-13 induced by anti-IgE, they have little to no effect on the secretion of this cytokine induced by PMA or by IL-3, both of which do not directly induce cytosolic calcium changes in basophils. Finally, the translocation of specific members of the nuclear factor of activated T cell (NFAT) family of transcription factors from the cytoplasm to the nucleus of activated T cells has been associated with the generation of cytokines, including IL-4 (Rao et al., 1997). Calcineurin initiates this subcellular localization to the nucleus by removing phosphates on inactive cytosolic NFAT. Thus, FK-506 and cyclosporine A are well-known selective inhibitors of this nuclear translocation. In data not yet published, we have evidence that antibodies specific for the NFAT2 and -4 isoforms, but not for NFAT1, detect cytosolic proteins in basophils that translocate to the nucleus within a time (1 hr) that is consistent with the generation of IL-4 (J. T. Schroeder, 2000). At this time, it is not known whether these antibodies detect both isoforms or if they cross-react with a unique NFAT isoform found in basophils.

HUMAN BASOPHILS

111

Whereas similarities exist between the generation of IL-4 and IL-13, particularly following IgE-mediated activation, there are clear differences in the stimuli and mechanisms regulating their generation in basophils. As suggested above, IL-13 is somewhat unique in that its release is not dependent chiefly on IgE– FcεRI interactions. In other words, its release is linked to neither histamine nor IL-4. It is also the case that a variety of IgE-independent stimuli can induce IL-13, a major difference from IL-4, suggesting that this cytokine has a more significant role in the pathogenesis of chronic allergic disease—a concept that has recently gained considerable attention in murine models of asthma (Grunig et al., 1998; Wills-Karp et al., 1998). There is pharmacological evidence to support the existence of at least three distinct pathways resulting in the generation of IL-13, all of which are dependent on the specific stimuli that induce its secretion (Schroeder et al., 1999). As noted, the production of cytokines such as IL-4 and IL-13 in response to stimuli that induce a sustained calcium response (e.g., calcium ionophores or anti-IgE) is inhibited by FK-506 and cyclosporine A. However, the generation of IL-13 mRNA or protein that occurs following induction by IL-3 is completely resistant to these drugs, and only marginal inhibition is observed for the IL-13 induced by PMA. As predicted, inhibitors of PKC prevent the secretion of IL-13 made in response to PMA, but not to IL-3. Glucocorticoids, which have proven efficacy in the treatment of allergic disease, have been shown to inhibit both IL-4 and IL-13 (Schroeder et al., 1997b, 1998c; Shimitzu et al., 1998) induced by all the stimuli tested thus far. In fact, these drugs require little exposure time (1 : 32 given i.p.) were required to inhibit the development of skin lesions and to prolong survival of lethally infected mice. Purified F(ab′ )2 fragments were ineffective even when given repeatedly to maintain neutralizing titer. IgG was effective in C5-deficient mice. The results are consistent with the notion that ADCC becomes crucial in protection against HSV if sterile protection is not provided at challenge. Many studies have looked at the ability of mAbs to protect against HSV infection. Early papers showed that both neutralizing anti-gD and gC mAbs and nonneutralizing anti-gD, gC, gB, gD, and gE mAbs could protect against lethal footpad challenge in mice (Balachandran et al., 1982; Dix et al., 1981). The latter study found that a mixture of mAbs had an efficacy corresponding roughly to the sum of that of the individual mAbs and that protection was equally apparent in C5-deficient mice. A good correlation was reported between protection and the titers of the nonneutralizing mAbs in an ADCC assay. It was suggested that ADCC could provide protection against lethal HSV infection. In the murine zosteriform spread model, infection is initiated at a cutaneous site and spreads to the peripheral nervous system, from which the virus (HSV-1) reemerges and infects regions of the epithelium remote from the inoculation

ANTIVIRAL ACTIVITY OF ANTIBODIES

237

site. The model mimics the course of human herpetic disease. High doses (0.2–5 mg/mouse) of anti-gD, gC, and one gB mAb offered partial protection in the model (Mester et al., 1991). It was reported that protection correlated better with in vitro ADCC than neutralization activity. In addition, F(ab′ )2 and Fab fragments of two protective anti-gC mAbs were nonprotective. Investigation of the ability of a panel of anti-gD mAbs to protect against HSV-2 challenge showed that a mAb, neutralizing virus even in the absence of complement, was the most effective (Ishizaka et al., 1995). Of a series of mAbs that neutralized in the presence of complement, the order of efficacy of a series of switch variants was IgG2a>IgG2b>IgG1. A human neutralizing antigD mAb was shown to completely protect nude mice against lethal challenge with HSV-1 using a high iv dose (450 ␮g/mouse) (Sanna et al., 1996). The same mAb applied topically could protect C57Bl/6 mice against vaginal challenge with much lower doses of mAb (400 ng/mouse) (Zeitlin et al., 1996). IgG and F(ab′ )2 fragment were approximately equivalent in topical protective ability, with the Fab fragment being somewhat less effective. J. ORTHOMYXOVIRUSES—INFLUENZA VIRUS A detailed study (Mozdzanowska et al., 1997) has compared the in vitro neutralization and in vivo protective activities of a number of mAbs to influenza virus HA. The mAbs were chosen to have a range of neutralizing activities. In the presence of complement (1.6% serum), neutralizing ability was differentially enhanced from 2- to 75-fold. The serum antibody concentration conferring protection was two to three orders of magnitude higher than the concentration required for 50% neutralization in vitro in the presence of complement. However, the rank order of protective ability was not well predicted by the order of neutralizing ability. K. PICORNAVIRUSES McCullough et al. (1986) have shown in passive transfer studies that a good correlation exists between neutralization and protection against FDMV, as only antibodies which neutralized a certain FDMV challenge isolate strongly or moderately provided protection. Interestingly, however, these antibodies protected at concentrations 10- to 60-fold below those required for in vitro neutralization of FDMV. The enhanced protective ability of these antibodies appeared Fc-mediated, as conversion of the mAb into F(ab′ )2 fragments reduced protective ability, whereas their neutralizing ability was mostly retained (McCullough et al., 1986). Neutralization titers were not affected by the addition of complement, suggesting that CMC did not play a significant role (McCullough et al., 1988). The effect rather appeared due to the induction of phagocytosis of sensitized virus, as impairment of phagocytosis by silica treatment abrogated the enhanced protection. In silica-treated mice, neutralizing antibody concentrations

238

PARREN AND BURTON

well over the 90% neutralization titers were required for protection, indicating that in the absence of phagocytosis, protection correlates with neutralization of all the challenge virus. It is likely that efficient protection against FDMV infection by neutralizing antibody at low occupancy is provided by an efficient phagocytosis of sensitized virions, as has been demonstrated for FDMV in vitro (McCullough et al., 1988, 1992 ). L. PARAMYXOVIRUSES Many studies have looked at the activities of antibodies against RSV, and one anti-RSV mAb is in clinical use for the prophylaxis of RSV disease. Passively transferred polyclonal antibody administered i.p. to produce a serum neutralizing titer of 1:380 or greater provided sterile protection in the lungs of cotton rats challenged with RSV (Prince et al., 1985b). Similarly, high doses of anti-F or anti-G glycoprotein mAbs reduced the virus titers in the lungs of cotton rats challenged with RSV to undetectable levels (Walsh et al., 1984). From 15 antiRSV mAbs, including 10 to the F glycoprotein, only 2 were neutralizing in vitro, and these were the only mAbs that were completely protective in mice (Stott et al., 1984). In contrast, a nonneutralizing anti-G mAb that protects SCID mice against intranasal RSV challenge at a dose of 5 mg/kg has been described (Corbeil et al., 1996). Protection is reduced in decomplemented or C5-deficient mice, suggesting complement is important in the antiviral activity. In bovine RSV, two neutralizing anti-F mAbs administered by the intratracheal route at moderate dose (0.4 mg/kg) protected against infection, whereas a nonneutralizing mAb was not protective (Thomas et al., 1998). An anti-F IgA at 0.5 mg/kg introduced intranasally to mice prior to RSV challenge greatly reduces virus titers in the lungs (Weltzin et al., 1994). In human phase III trials, the enhanced activity of one mAb relative to another has been attributed at least in part to its better neutralizing ability in vitro (Johnson et al., 1999). The antiviral activity of a panel of nine mAbs to the F protein of Sendai virus has been compared in vitro and in vivo (Mochizuki et al., 1990). None of the mAbs were neutralizing in standard assays, but two were potently neutralizing in the presence of complement. These mAbs protected very young mice against lethal infection and allowed them to thrive in terms of body weight gain, when given at high dose. Three other mAbs showing weak or no neutralization in the presence of complement were also protective. Two of these mAbs were of the IgG1 isotype, which lead to the suggestion that ADCC rather than complement might be important in protection. M. REOVIRUSES Passive transfer of a neutralizing mAb against bluetongue virus (BTV) has been shown to protect mice and sheep from disease, whereas nonneutralizing mAb did not protect. A neutralizing antibody titer of 1:20 protected the

ANTIVIRAL ACTIVITY OF ANTIBODIES

239

sheep from disease, but did not provide sterile immunity, as an increase in neutralization titers was observed 8 to 9 days postchallenge. The results are in strong contrast with the inability of 100-fold higher neutralizing antibody titers to clear virus from sheep with existing infection (Letchworth and Appleton, 1983). The impact of antibody on established infection is discussed in detail below. N. NEUROPATHOGENIC LACTATE DEHYDROGENASE-ELEVATING VIRUS AND HIV-1: CAUTIONARY NOTES ON THE DIFFICULTIES OF COMPARING IN VITRO AND IN VIVO ANTIBODY ACTIVITIES Infection of mice of certain strains with neuropathogenic LDV results in a fatal paralytic disease through interaction with endogenous retroviruses. The infection of anterior horn neurons by LDV and the development of disease are prevented by anti-LDV antibodies. The mechanism by which motor neurons were protected from infection by LDV was unclear as in addition to neutralizing antibodies also nonneutralizing (polyclonal) antibodies prevented neuron destruction and disease. In addition, protection occurred in the absence of any apparent effect of antibody on LDV replication in a subpopulation of macrophages known to be the primary permissive host cells. The resolution to this paradox is a lesson with regard to the ability of viral quasispecies to mislead (Chen et al., 1999). It appears that neuropathogenic LDV isolates contain both neuropathogenic and nonneuropathogenic quasispecies. Using biological clones, it was shown that the nonneuropathogenic species were about 100 times more resistant to in vitro neutralization than the neuropathogenic species. Some antibodies therefore do not neutralize nonneuropathogenic viruses and are scored as “nonneutralizing” by in vitro assays. These antibodies do, however, neutralize neuropathogenic viruses and therefore are protective in vivo. The paradox described above appears to be due to LDV heterogeneity. It is of interest that mixed virus populations with distinct neutralization properties have also been described for another arterivirus: PRRSV (Weiland et al., 1999). T-cell line adapted strains of HIV-1 represent a striking example of how studying neutralization of viral variants selected in vitro may lead to aberrant conclusions with respect to the neutralizing responses in infection. The adaptation of HIV-1 to growth in CD4+ T cell lines selects for variants that are readily neutralized by soluble CD4 and a large spectrum of different mAbs. By contrast, plasma virus or viruses which have only been passaged in primary cultures of activated peripheral blood mononuclear cells (PBMC) are mostly resistant to neutralization by these same ligands (reviewed in Moore and Ho, 1995; Parren et al., 1999). An explanation for this phenomenon may be the high expression levels of heparan sulfate proteoglycans on the surface of T cell lines, which through an interaction with gp120 may select for HIV-1 viruses with unusual

240

PARREN AND BURTON

strongly basic gp120 V3 loops (Moulard et al., 2000). The envelope spike of these TCLA viruses adopts a much more open configuration, which provides access to a range of epitopes which are inaccessible on naturally occurring HIV1 isolates. Consequently, sera from HIV-1 infected individuals, as well as sera from vaccinees immunized with recombinant HV-1 envelope subunits, typically contain high titers of neutralizing antibodies against TCLA strains of HIV-1, whereas the more relevant titers against HIV-1 primary isolates are usually very poor. O. FURTHER CAUTIONARY NOTES ON COMPARING IN VITRO AND IN VIVO ANTIBODY ACTIVITIES: THE POSSIBLE EFFECTS OF VIRUS CHALLENGE DOSE AND ANATOMICAL CONSIDERATIONS It is in the nature of passive transfer studies that typical virus challenge doses are relatively large to ensure that all control animals become infected. It is sometimes argued that, in some human infections, challenge doses could be smaller and protection achieved at lower antibody concentrations than indicated from animal studies. For example, SIV or SHIV experiments in macaques usually involve virus challenge doses of 10 AID50 (50% animal infectious dose) or more. However, the typical human challenge dose with HIV-1 is probably 0.01 AID50 or less, since the frequency of infection is about 1:100 or less, depending upon the nature of exposure. This has been interpreted to indicate that relatively low vaccine-induced serum antibody concentrations compared to in vitro neutralization titers may offer protective benefit against HIV-1 infection. From a thermodynamic standpoint, this argument appears to have little merit, since the amount of serum antibody will be in vast molar excess over the challenge virus in most scenarios, and the extent of antibody coating of virus (and therefore neutralization) should be determined by the binding constant of antibody to virus rather than the number of challenge particles. However, there are no studies of which we are aware that have directly addressed the ability of antibody to protect at very low challenge doses. For a given virus, the existence of factors during low dose natural infection processes outside the laboratory that complicate the extrapolation from animal studies to humans cannot be categorically excluded. Anatomical considerations may also complicate the interpretation of in vivo protection data. Antibody serum concentrations can be readily measured. For the most part, tissue antibody concentrations are not measured, and the concentration of antibody that may in fact be responsible for blocking infection at a tissue site is not known. It is possible that the high serum antibody concentrations required for protection in some instances reflect the difficulty of achieving protective antibody concentrations at a critical tissue site. If some antibodies are better able than others to diffuse to tissue sites, they may therefore show enhanced in vivo relative to in vitro activity.

ANTIVIRAL ACTIVITY OF ANTIBODIES

241

VIII. Mechanisms of Antiviral Antibody Activity in Established Infection

Although there is evidence that antibody can impact upon a number of established viral infections (Chanock et al., 1993), there are little quantitative data relating in vivo and in vitro activities. In the data reviewed below, we consider antibody given 1 day or more after virus as having activity against “established” infection as opposed to the prophylactic activity considered above. This is clearly a somewhat artificial distinction, and antibody generally appears to have diminished activity the further infection progresses. Elegant studies show that SCID mice infected with influenza A for 1 day can be cured using neutralizing mAbs to viral HA with a close correlation between prophylactic and curative activity (Mozdzanowska et al., 1997). The data suggest that the curative effect is mainly due to the neutralizing activity of antibody against free virus, with some contribution from activity against infected cells. This conclusion is supported by investigation of two nonneutralizing mAbs, one to viral NA and one to M2 (Mozdzanowska et al., 1999). Both mAbs reduced pulmonary viral titers in established infection by 100- to 1000-fold, but they failed to clear infection even at high dose in combination. It might be predicted that neutralizing antibody would have activity against a virus such as influenza that does not propagate via cell-to-cell spread and is cytopathic for infected cells. The presence of high levels of neutralizing antibody should eventually terminate infection. However, for viruses that do propagate via cell-to-cell spread, antibody would be expected to be less effective, since higher concentrations of neutralizing antibody are generally required to inhibit infection by this route than are required to inhibit infection by free virions (Hooks et al., 1976; Pantaleo et al., 1995). Indeed, for HIV-1, hu-PBL-SCID mouse studies suggest that the virus replicates unhindered in a significant proportion of cases in the presence of serum concentrations of a single mAb that are largely protective if administered prior to virus challenge (Poignard et al., 1999). In the remainder of cases, neutralization escape occurs rapidly, showing that the mAb does exert some pressure on virus replication in those animals. If a cocktail of mAbs is administered at high dose (50 mg/kg) in established infection, then neutralization escape is rapidly apparent. However, as the serum mAb concentrations wane to around 10 times 90% in vitro neutralization levels, the wild-type neutralizationsensitive virus reemerges. This suggests that the escape variants are less fit than the wild type, and establishes a high threshold of serum antibody to impact upon propagation of infection. Another study shows that passive transfer of neutralizing polyclonal anti-SIV antibody has a very modest effect upon SIV replication in macaques (Binley et al., 2000). The effect of antibody on established retrovirus infection has also been investigated in the F-MuLV system (Hasenkrug et al., 1995). High doses of neutralizing mAb could induce recovery, but success was dependent upon the presence of

242

PARREN AND BURTON

both CD4+ and CD8+ T lymphocytes. The results strikingly demonstrate the ability of antibody and two populations of T cells to work together in a way which is not fully understood at this time. RSV is a virus that, from its name, one might expect to propagate via cellto-cell spread. However, although syncytia are observed in vitro, they have not been observed in vivo, and the mode of infection propagation is unclear. Because the tracheal epithelium is only sparsely infected at any point in time, however, it is unlikely that cell-to-cell spread is a major mechanism of spread in vivo (McIntosh and Chanock, 1990). In any case, a number of studies have shown that neutralizing antibody can have a therapeutic effect. Early work in the cotton rat model showed that passively administered polyclonal immune antibody resulting in serum neutralizing titers in the range 1:400–1:1000 could drastically reduce the virus titers in the lungs by as much as a factor of 104 (Prince et al., 1985a). Later work focused more on intranasal inoculation when relatively small quantities of neutralizing antibody were found to be effective in cotton rats, mice, and monkeys (Crowe et al., 1994; Hemming et al., 1985; Prince et al., 1987, 1990; Weltzin et al., 1996). Therapeutic effect could be achieved with F(ab′ )2 (Prince et al., 1990) and Fab fragments (Crowe et al., 1994) of neutralizing antibody, suggesting that it was a direct result of interaction between antibody and virus. In the case of Fab fragments, viral loads in the lungs of infected mice could be reduced by a factor of almost 104 by as little as 13 ␮g of protein introduced intranasally. A number of studies show that antibody can be highly effective against established CNS infection in rodent models, preventing disease or death. Administration of large amounts of hyperimmune anti-HSV-1 serum (0.5 ml/mouse) completely protected na¨ıve animals from illness when given up to 24 hr following footpad challenge (Lubinski et al., 1998). As the time interval from challenge to antibody administration was increased, the incidence of illness increased to 25% at 24 hr, 62% at 72 hr, and 86% at 96 hr. MAbs at high dose given 24 hr postexposure were similarly shown to protect against HSV-1-induced ocular disease in mice, with protection occurring for nonneutralizing as well as neutralizing mAbs, suggesting, as expected, the importance of activity against infected cells. Protection against intracerebral challenge of mice with YF virus was shown to occur even when mAbs were given i.p. several (3–5) days after virus inoculation when peak infectious virus titers and histopathological evidence of infection were present in brains. Nearly complete protection (eight of nine animals) was noted for one mAb given at a dose of approximately 1.5 mg/kg 4 days after cerebral challenge. Protection was apparent for neutralizing and nonneutralizing mAbs. Furthermore, some of the nonneutralizing mAbs were shown to inhibit viral replication in vitro in a neuroblastoma cell line, hinting at a novel mechanism of antibody protection.

ANTIVIRAL ACTIVITY OF ANTIBODIES

243

A thorough study showed that both polyclonal antibodies and mAbs can protect against neurally spreading reovirus type 3 (Dearing ) in mice (Virgin et al., 1988) even when given several days after cerebral or footpad challenge. For instance, a potent neutralizing mAb given at approximately 8 mg/kg 1 week postchallenge led to the survival of roughly half of the animals infected. Protection was apparent with both neutralizing and nonneutralizing polyclonal antibodies, but a nonneutralizing mAb was far less effective than the neutralizing mAb. Serum complement was not required for antiviral activity. Attempts to investigate the activity of F(ab′ )2 fragments in comparable experiments were thwarted because of the short half-life of F(ab′ )2, so that even daily administrations did not maintain serum levels. This careful work emphasizes that, unless F(ab′ )2 levels are specifically monitored, conclusions drawn from comparing IgG and F(ab′ )2 should be treated with caution. Further studies show that high doses of neutralizing mAbs can protect mice against lethal challenge with Theiler’s murine encephalomyelitis virus (Fujinami et al., 1989) and neurotropic measles virus (Liebert et al., 1990) when given 2 days and 5–8 hr postexposure, respectively. In the latter case, nonneutralizing mAbs are ineffective. Further, the latter study again provides evidence of the ability of antibody to restrict viral replication inside an infected cell by binding to viral antigen. This is an interesting and potentially very important phenomenon. It was first described by Fujinami and Oldstone (1979, 1980) for measles virus-infected cells, and has been extensively investigated by Griffin and co-workers for Sindbis virus infection of neurons (Levine et al., 1991). Treatment of persistently infected SCID mice with mAbs to the E2 glycoprotein of Sindbis virus results in the gradual noncytopathic removal of viral RNA by a process which is independent of complement and T cells. Antibodies can also clear Sindbis virus from persistently infected neuronal cell cultures. The isotype of antibody is not important, but bivalency is required (Ubol et al., 1995). It is suggested that clearance involves a novel mechanism triggered when antibody cross-links viral protein expressed on the surface of infected cells. A similar mechanism has been proposed for antibody activity against rabies-infected neural cells (Dietzschold et al., 1992). This mechanism is distinct from earlier studies that described the ability of antibody to modulate antigen expression at the surface of infected cells and thereby reduce the efficiency of virus budding (e.g., Chesebro et al., 1979). Finally, nonneutralizing IgA mAbs can resolve an ongoing rotavirus infection, apparently by interaction between antibody and virus during transcytosis (Burns et al., 1996). SCID mice, infected with rotavirus for at least 2 months, were transplanted subcutaneously with hybridomas secreting mAbs to VP4 (an outer capsid viral protein) and VP6 (a major inner capsid viral protein). Only two nonneutralizing IgA mAbs to VP6 were capable of resolving chronic infection. These mAbs were not, however, active when presented directly to the luminal

244

PARREN AND BURTON

side of the intestinal tract, suggesting their mode of action is during transcytosis as described in vitro (Bomsel et al., 1998; Manzanec et al., 1992). Neutralizing IgA mAbs to VP4 did not resolve infection. IX. Observations with Nonviral Pathogens

We note that a correlation between inactivation of infectivity of a pathogen and antibody coating extends beyond the viruses on which we have focused in this review. For example, as discussed above, a correlation exists between neutralization of the intracellular bacterium Chlamydia trachomatis and coating of the infectious elementary bodies with antibody (Peeling and Brunham, 1991). Complement may enhance neutralization of opsonized C. trachomatis (Megran et al., 1988). Passive transfer of an antibody against an outer surface protein of Borrelia burgdorferi, which strongly stained nonpermeabilized B. burgdorferi cells, completely protected mice from challenge (Mbow et al., 1999). Protection against the parasite Trypanosoma cruzi by antibody, furthermore, has been correlated with antibodies that bind to living trypomastigotes in immunofluorescence (Heath et al., 1990). X. Conclusions

We began with an assertion that some general rules describing the in vitro and in vivo activities of antibodies against viruses can be discerned from the literature. We now review our interpretation of the data as a whole and its significance for these rules. There has been much discussion of the mechanisms of neutralization of viruses in vitro. A prominent opinion has been that antibodies can act at many different stages of the infectious process, including post-viral entry to the target cell, that several different mechanisms may operate in concert, and that critical sites on the virion surface must be occupied for neutralization (Dimmock, 1993). We disagree. We believe that the data are consistent, in the vast majority of cases, with a simple occupancy model essentially as initially proposed by Macfarlane Burnet in 1937. According to this model, neutralization occurs when a sizable fraction of available sites on the virion are occupied by antibody, leading to inhibition of virus attachment or interference with the entry (fusion) process. The relatively large bulk of the antibody molecule, very roughly similar to that of a typical envelope spike for an enveloped virus, is suggested to be critical (Fig. 6). The model is consistent with a number of observations. First, we have shown here that there is a roughly linear relationship between the surface area of a virus and the number of antibody molecules bound at neutralization. This number is approximately that predicted to effectively coat the virion particle, given the

FIG. 6. Model for proposed interactions between envelope spikes and neutralizing IgG. The molecules depicted are drawn to scale assuming that an IgG molecule has roughly the same molecular weight as the monomer of a typical trimeric envelope spike. The model explains how antibody coating of the virus surface may interrupt infection without occupying all available binding sites. (a) Envelope spike with an IgG molecule bound. Additional binding sites on the envelope spike are still available and the binding of additional antibodies to distinct epitopes or recombinant soluble receptor molecules may not be inhibited. An antibody to the V3 loop on HIV-1 gp120, for example, will not inhibit binding of soluble CD4. (b) Envelope spike with various numbers of IgG molecules associated. Coating of virion spikes with antibody, irrespective of the epitope(s) recognized, interferes with the initiation of a productive infection, as the establishment of multiple critical contacts with membrane receptors required for infection is inhibited by steric hindrance and geometric constraints (see also Parren et al. (1998)).

246

PARREN AND BURTON

size of the antibody molecule. Second, any antibody that binds well to and coats the virion surface should neutralize the virus, as is almost universally found. Nonneutralizing antibodies should not coat the virus, although they may bind at lower levels of occupancy. Heterogeneous envelope spikes provide an opportunity for binding to virions without neutralization because of low occupancy, as discussed above for rabies virus and the FPV/VSV mixed virions. Third, the model predicts that the precise epitope recognized by the antibody on the virion surface should not be crucial. Rather, the number of antibody molecules bound per unit area, which will be determined by the affinity of antibody for virionexpressed antigen, will be most important. This is precisely that which is found for HIV-1, as discussed above. Antibody enhancement of viral infection in vitro receives much attention, although its significance in vivo has only been demonstrated for dengue virus infection. It appears to occur in the presence of subneutralizing concentrations of neutralizing antibodies. The model described above suggests that this is a phenomenon arising from low occupancy of virion sites, as discussed earlier. The question whether some antibodies bind well to the virus without neutralization is often raised. In particular, the concern is expressed that nonneutralizing antibodies might compete with the binding of neutralizing antibodies to virus, thereby interfering with neutralization. First, we strongly question the evidence for binding but nonneutralizing antibodies. Generally, there is a very strong correlation between occupancy and neutralization, and most examples of such nonneutralizing antibodies can indeed be explained by poor virus binding, or even a failure to appreciate that antibodies which bind well to isolated envelope or capsid molecules do not necessarily bind well to the virus particle. Some convincing but isolated examples exist, such as the rabies mutant virus, which merit further investigation. Second, to our knowledge, there is no convincing and confirmed evidence for a nonneutralizing antibody interfering with neutralization. Virion coating but non-neutralizing antibodies therefore do not appear to play a significant role (if any) in the humoral response against viruses. Antibody activity in vivo can arise through binding to virions or virion products on infected cells. Protection by antibody at the level of sterile immunity requires activity against free virions, i.e., neutralizing antibodies. Several studies with mAbs show that sterile immunity is only achieved when serum concentrations of the challenged animal are of the order of two to three orders of magnitude higher than in vitro neutralization titers, that is, serum concentrations capable of neutralizing essentially all of the challenge virus. Expressed in another way, serum neutralizing titers of 1:100–1:1000 are required for sterile protection. In a number of cases (discussed above), even such high levels of neutralizing antibody do not provide sterile protection, although they do prevent disease. Most vaccines do not elicit very high neutralizing titers, especially over an extended period, but then they probably do not provide sterile protection. Rather, it seems

ANTIVIRAL ACTIVITY OF ANTIBODIES

247

likely that they reduce the effective challenge to such a level that infected cells can be controlled by cellular immunity and possibly by antibodies. There are a number of cases (discussed above) where in vivo protection by neutralizing antibodies is independent of the presence of the Fc part of the molecule. In these cases, it seems likely that the mechanism of protection in vivo is essentially equivalent to neutralization in vitro. In a number of other cases, it appears that F(ab′ )2 fragments, that are equally active as whole IgG molecules at neutralization in vitro, are ineffective at protection. Further, neutralizing mouse IgG1 switch variants are ineffective at protection, whereas IgG2a molecules are effective. In many of these examples, protection is independent of complement, suggesting that protection by neutralizing antibodies requires activity against infected cells as well as free virions. This requirement, in some cases, for activity against infected cells as well as virions may contribute to observations of incomplete correlation between neutralization and protection for neutralizing antibodies, although other factors such as the use of different cell types in vitro and in vivo should be considered. It is worth emphasizing, though, that most neutralizing antibodies protect at appropriate concentrations, and antibodies assessed as the most potent in neutralization assays in vitro generally are the most effective at protection in vivo. There are numerous examples of protective activity exhibited by nonneutralizing antibodies. This activity appears to be directed at infected cells, and generally appears to be somewhat less potent than that of neutralizing antibodies. For instance, cases are described above where neutralizing antibodies are protective against higher challenge doses or more pathogenic viruses than nonneutralizing antibodies. In many cases, protection by nonneutralizing antibodies is shown to depend critically on the Fc part of the antibody molecule and to occur in complement-deficient mice, suggesting that ADCC (or phagocytosis) may be crucial in clearing antibody-complexed infected cells. It should be noted that protection with nonneutralizing antibodies is mostly restricted to protection against enveloped viruses. What is the significance of these conclusions for vaccine design? In the first case, the time-honored focus on eliciting neutralizing antibodies is well justified. Serum neutralizing antibody titers of the order of, or greater than, 1:100 provide the greatest likelihood that antibody alone can protect against viral challenge. In many cases, antibody and cellular responses may cooperate to protect, although, with notable exceptions (Dittmer et al., 1999a), this is an underexplored area. The model described above asserts that neutralization is determined by the extent of coating of virus by antibody. At a given antibody concentration, this is in turn determined by the affinity of antibody for the antigen on the virion surface. Hence, the model predicts that a vaccine should simply aim to elicit antibodies of the highest affinity for the virion surface antigen. This is most directly achieved by immunization with molecules identical to or as similar to the viral surface

248

PARREN AND BURTON

antigen as possible. This may not be easy. We have argued that a number of enveloped viruses have evolved surface proteins of low immunogenicity (Burton and Parren, 2000). Subunit envelope proteins appear, in a number of instances, to elicit antibody responses that show little reactivity with the form of the envelope (usually oligomeric) expressed on the virion surface (Parren et al., 1998; Roben et al., 1994; Sakurai et al., 1999; Sattentau and Moore, 1995). Another interesting question is the value of eliciting antibodies targeted to infected cells rather than virions. There are examples, such as arenaviruses, where complete passive protection appears to require antibodies to infected cells as well as virions. Of course, neutralizing antibodies could fulfill both roles by binding, for instance, to envelope molecules on virions and infected cells. However, it is also possible that epitopes expressed on virions are not expressed optimally on infected cells, for ADCC for example, and then the induction of nonneutralizing antibodies may be beneficial. This is a factor worthy of consideration in vaccine design, particularly using subunit proteins to elicit antibodies. Finally, could passively administered antibodies be used in the treatment of acute viral diseases? The studies discussed above suggest high doses would be required to have any reasonable chance of efficacy. However, given the availability of human antibodies from new technologies such as transgenic mice and phage display (Burton and Barbas, 1994; Green et al., 1994; Lonberg et al., 1994; Winter et al., 1994), and the ability to produce large amounts of such antibodies relatively cheaply in culture systems (e.g., Verma et al., 1998), larger animals (e.g., Pollock et al., 1999), or plants (e.g., Fischer et al., 1999), antibody intervention in acute viral disease may become increasingly realistic. Antibodies are already used in certain situations in a postexposure mode to prevent, e.g., disease due to Junin virus (Argentine hemorrhagic fever), rabies virus, and TBEV. However, the evaluation of antibodies in humans following the appearance of symptoms in infections due to viruses such as RSV, dengue, and hanta would be of great interest. ACKNOWLEDGMENTS We thank Drs. Kim Hasenkrug and Michael Buchmeier for reviewing the manuscript. We thank Erica Ollmann Saphire for preparing the molecular models of poliovirus. We are grateful to Drs. Linda M. Stannard and H. W. Ackermann for providing electron microscopic images. We acknowledge the financial support of the National Institutes of Health under Grant numbers AI33292 and HL59727 (to DRB); AI40377 and AI48494 (to PWHIP).

REFERENCES Arthos, J., Deen, K. C., Chaikin, M. A., Fornwald, J. A., Sathe, G., Sattentau, Q. J., Clapham, P. R., Weiss, R. A., McDougal, J. S., Pietropaolo, C., Axel, R., Truneh, A., Maddon, P. J., and Sweet, R. W. (1989). Identification of the residues in human CD4 critical for the binding of HIV. Cell 57, 469–481.

ANTIVIRAL ACTIVITY OF ANTIBODIES

249

Arthur, L. O., Bess, J. W., Jr., Sowder, R. C. II, Benveniste, R. E., Mann, D. L., Chermann, J.-C., and Henderson, L. E. (1992). Cellular proteins bound to immunodeficiency viruses: Implications for pathogenesis and vaccines. Science 258, 1935–1938. Arthur, L. O., Bess, J .W., Jr., Urban, R. G., Strominger, J. L., Morton, W. R., Mann, D. L., Henderson, L. E., and Benveniste, R. E. (1995). Macaques immunized with HLA-DR are protected from challenge with simian immunodeficiency virus. J. Virol. 69, 3117–3124. Ashe, W. K., and Notkins, A. L. (1967). Kinetics of sensitization of herpes simplex virus and its relationship to the reduction in the neutralization rate constant. Virology 33, 613–617. Ashe, W. K., Mage, M., and Notkins, A. L. (1969). Kinetics of neutralization of sensitized herpes simplex virus with antibody fragments. Virology 37, 290–293. Baba, T. W., Liska, V., Hofmann-Lehmann, R., Vlasak, J., Xu, W., Ayehunie, S., Cavacini, L. A., Posner, M. R., Katinger, H., Stiegler, G., Bernacky, B. J., Rizvi, T. A., Schmidt, R., Hill, L. R., Keeling, M. E., Lu, Y., Wright, J. E., Chou, T.-C., and Ruprecht, R. M. (2000). Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian–human immunodeficiency virus infection. Nat. Med. 6, 200–206. Bachmann, M. F., Kalinke, U., Althage, A., Freer, G., Burkhart, C., Roost, H.-P., Aguet, M., Hengartner, H., and Zinkernagel, R. M. (1997). The role of antibody concentration and avidity in antiviral protection. Science 276, 2024–2027. Balachandran, N., Bacchetti, S., and Rawls, W. E. (1982). Protection against lethal challenge of balb/c mice by passive transfer of monoclonal antibodies to five glycoproteins of herpes simplex type 2. Infect. Immun. 37, 1132–1137. Baldridge, J. R., and Buchmeier, M. J. (1992). Mechanisms of antibody-mediated protection against lymphocytic choriomeningitis virus infection: Mother-to-baby transfer of humoral protection. J. Virol. 66, 4252–4257. Bedinger, P., Moriarty, A., von Borstel, R. C., Donovan, N. J., Steimer, K. S., and Littman, D. R. (1988). Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4. Nature 334, 162–165. Beijerinck, M. W. (1898). Over een Contagium vivum fluidum als oorzaak van de vlekziekte der tabaksbladen. Versl. Gew. Verg. Wis en Natuurk. Afd., Kon. Acad. Wetensch. Amsterdam VII, 229–235. Belnap, D. M., McDermott, B. M., Jr., Filman, D. J., Cheng, N., Trus, B. L., Zuccola, H. J., Racaniello, V. R., Hogle, J. M., and Steven, A. C. (2000). Three-dimensional structure of poliovirus receptor bound to poliovirus. Proc. Natl. Acad. Sci. 97, 73–78. Berger, E. A. (1997). HIV entry and tropism: The chemokine receptor connection. AIDS 11 (Suppl A), S3–S16. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000). The protein data bank. Nucleic Acids Res. 28, 235–242. Binley, J. M., Clas, B., Gettie, A., Vesanen, M., Montefiori, D. C., Sawyer, L., Booth, J., Lewis, M., Marx, P. A., Bonhoeffer, S., and Moore, J. P. (2000). Passive infusion of immune serum into simian immunodeficiency virus-infected rhesus macaques undergoing a rapid disease course has minimal effect on plasma viremia. Virology 270, 237–249. Blasco, R., and Moss, B. (1992). Role of cell-associated enveloped vaccinia virus in cell-to-cell spread. J. Virol. 66, 4170–4179. Blondel, B., Crainic, R., Fichot, O., Dufraisse, G., Candrea, A., Diamond, D., Girard, M., and Horaud, F. (1986). Mutations conferring resistance to neutralization with monoclonal antibodies in type 1 poliovirus can be located outside or inside the antibody-binding site. J. Virol. 57, 81–90. Boere, W. A. M., Benaissa-Trouw, B. J., Harmsen, T., Erich, T., Kraaijeveld, C. A., and Snippe, H. (1985). Mechanisms of monoclonal antibody-mediated protection against virulent Semliki Forest virus. J. Virol. 54, 546–551.

250

PARREN AND BURTON

Bomsel, M., Heyman, M., Hocini, H., Lagaye, S., Belec, L., Dupont, C., and Desgranges, C. (1998). Intracellular neutralization of HIV transcytosis across tight epithelial barriers by anti-HIV envelope protein dlgA or IgM. Immunity 9, 277–287. Booy, F. P., Roden, R. B. S., Greenstone, H. L., Schiller, J. T., and Trus, B. L. (1998). Two antibodies◦ that neutralize papillomavirus by different mechanisms show distinct binding patterns at 13 A resolution. J. Mol. Biol. 281, 95–106. Boyer, V., Delibrias, C., Noraz, N., Fischer, E., Kazatchkine, M. D., and Desgranges, C. (1992). Complement receptor type 2 mediates infection of the human CD4-negative Raji B-cell line with opsonized HIV. Scand. J. Immunol. 36, 879–883. Brioen, P., Thomas, A. A. M., and Boeye, A. (1985). Lack of quantitative correlation between the neutralization of poliovirus and the antibody-mediated pI shift of the virions. J. Gen. Virol. 66, 609–613. Britt, W. J., and Chesebro, B. (1983). Use of monoclonal anti-gp70 antibodies to mimic the effects of the Rfv-3 gene in mice with Friend virus-induced leukemia. J. Immunol. 130, 2363– 2367. Buchmeier, M. J., Lewicki, H. A., Talbot, P. J., and Knobler, R. L. (1984). Murine hepatitis virus-4 (strain JHM)-induced neurologic disease is modulated in vivo by monoclonal antibody. Virology 132, 261–270. Burnet, F. M., Keogh, E. V., and Lush, D. (1937). The immunological reactions of the filterable viruses. Austral. J. Exp. Biol. Med. Sci. 15, 227–368. Burns, J. W., Siadat-Pajouh, M., Krishnaney, A. A., and Greenberg, H. G. (1996). Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity. Science 272, 104–107. Burton, D. R., and Barbas, C. F. (1994). Human antibodies from combinatorial libraries. Adv. Immunol. 57, 191–280. Burton, D. R., and Parren, P. W. H. I. (2000). Vaccines and the induction of functional antibodies: Time to look beyond the molecules of natural infection? Nat. Med. 6, 123–125. Burton, D. R., Williamson, R. A., and Parren, P. W. H. I. (2000). Antibody and virus: Binding and neutralization. Virology 270, 1–3. Burton, D. R., and Woof, J. M. (1992). Human antibody effector function. Adv. Immunol. 51, 1–84. Capobianchi, M. R., Fais, S., Castilletti, C., Gentile, M., Ameglio, F., and Dianzani, F. (1994). A simple and reliable method to detect cell membrane proteins on infectious human immunodeficiency virus type 1 particles. J. Infect. Dis. 169, 886–889. Chanock, R. M., Crowe, J. E., Jr., Murphy, B. R., and Burton, D. R. (1993). Human monoclonal antibody Fab fragments cloned from combinatorial libraries: Potential usefulness in prevention and/or treatment of major human viral diseases. Infect. Agents Dis. 2, 118–131. Che, Z., Olson, N. H., Leippe, D., Lee, W.-M., Mosser, A. G., Rueckert, R. R., Baker, T. S., and Smith, T. J. (1998). Antibody-mediated neutralization of human rhinovirus 14 explored by means of cryoelectron microscopy and X-ray crystallography of virus-Fab complexes. J.Virol. 72, 4610– 4622. Chen, Z., Li, K., Rowland, R. R., and Plagemann, P. G. (1999). Selective antibody neutralization prevents neuropathogenic lactate dehydrogenase-elevating virus from causing paralytic disease in immunocompetent mice. J. Neurovirol. 5, 200–208. Chesebro, B., Wehrly, K., Doig, D., and Nishio, J. (1979). Antibody-induced modulation of Friend virus cell surface antigens decreases virus production by persistent erythroleukemia cells: Influnce of the Rfv-3 gene. Proc. Natl. Acad. Sci. USA 76, 5784–5788. Collins, J. J., Sackie, D. M., and Johnson, G. R. (1983). Immunotherapy of murine leukemia. IX. The requirement for the Fc portion of antibody for successful passive serum therapy of Friend leukemia virus-induced disease. Virology 126, 259–266. Colonno, R. J., Callahan, P. L., Leippe, D. M., Rueckert, R. R., and Tomassini, J. E. (1989). Inhibition

ANTIVIRAL ACTIVITY OF ANTIBODIES

251

of rhinovirus attachment by neutralizing monoclonal antibodies and their Fab fragments. J. Virol. 63, 36–42. Connor, R. I., Dinces, N. B., Howell, A. L., Romet-Lemonne, J.-L., Pasquali, J.-L., and Fanger, M. W. (1991). Fc receptors for IgG (Fc␥ Rs) on human monocytes and macrophages are not infectivity receptors for human immunodeficiency virus type 1 (HIV-1): Studies using bispecific antibodies to target HIV-1 to various myeloid cell surface molecules, including the Fc␥ R. Proc. Natl. Acad. Sci. USA 88, 9593–9597. Corbeil, S., Seguin, C., and Trudel, M. (1996). Involvement of the complement system in the protection of mice from challenge with respiratory syncytial virus Long strain following passive immunization with monoclonal antibody 18A2B2. Vaccine 14, 521–525. Corbin, A., Prats, A. C., Darlix, J. L., and Sitbon, M. (1994). A nonstructural gag-encoded glycoprotein precursor is necessary for efficient spreading and pathogenesis of murine leukemia viruses. J. Virol. 68, 3857–3867. Crainic, R., Couillin, P., Blondel, B., Cabau, N., Boue, A., and Horodniceanu, F. (1983). Natural variation of poliovirus neutralization epitopes. Infect. Immun. 41, 1217–1225. Crowe, J. E., Jr., Murphy, B. R., Chanock, R. M., Williamson, R. A., Barbas, C. F., and Burton, D. R. (1994). Human RSV monoclonal antibody Fab is effective therapeutically when introduced directly into the lungs of RSV-infected mice. Proc. Natl. Acad. Sci. USA 91, 1386–1390. Dalziel, R. G., Lampert, P. W., Talbot, P. J., and Buchmeier, M. J. (1986). Site-specific alteration of murine hepatitis virus type 4 peplomer glycoprotein E2 results in reduced neurovirulence. J. Virol. 59, 463–471. Daniels, C. A (1975). Mechanisms of viral neutralization. In Viral Immunology and Immunopathology. New York: Academic Press, pp. 79–97. Della-Porte, A. J., and Westaway, E. G. (1977). A multi-hit model for the neutralization of animal viruses. J. Gen. Virol. 38, 1–19. Diamond, D. C., Jameson, B. A., Bonin, J., Kohara, M., Abe, S., Itoh, H., Komatsu, T., Arita, M., Kuge, S., Nomoto, A., Osterhaus, A. D. M. E., Crainic, R., and Wimmer, E. (1985). Antigenic variation and resistance to neutralization in poliovirus Type 1. Science 229, 1090–1093. Dietzschold, B., Kao, M., Zhen, Y. M., Chen, Z. Y., Maul, G., Fu, Z. F., Rupprecht, C. E., and Koprowski, H. (1992). Delineation of putative mechanisms involved in antibody-mediated clearance of rabies virus from the central nervous system. Proc. Natl. Acad. Sci. USA 89, 7252–7256. Dimmock, N. J. (1993). Neutralization of animal viruses. Curr. Top. Microbiol. Immunol. 183, 1–149. Dimmock, N. J. (1995). Update on the neutralization of animal viruses. Rev. Med. Virol. 5, 165–179. Dittmer, U., Brooks, D. M., and Hasenkrug, K. J. (1998). Characterization of a live-attenuated retroviral vaccine demonstrates protection via immune mechanisms. J. Virol. 72, 6554– 6558. Dittmer, U., Brooks, D. M., and Hasenkrug, K. J. (1999a). Requirement for multiple lymphocyte subsets in protection by a live-attenuated vaccine against retroviral infection. Nat. Med. 5, 189– 193. Dittmer, U., Race, B., and Hasenkrug, K. J. (1999b). Kinetics of the development of protective immunity in mice vaccinated with a live-attenuated retrovirus. J. Virol. 73, 8435–8440. Dix, R. D., Pereira, L., and Baringer, J. R. (1981). Use of monoclonal antibody directed against herpes simplex virus glycoproteins to protect mice against acute virus-induced neurological disease. Infect. Immun. 34, 192–199. Dubay, J. W., Dubay, S. R., Shin, H.-J., and Hunter, E. (1995). Analysis of the cleavage site of the human immunodeficiency virus type 1 glycoprotein: Requirement of precursor cleavage for glycoprotein incorporation. J. Virol. 69, 4675–4682. Dulbecco, R., Vogt, M., and Strickland, A. G. R. (1956). A study of the basic aspects of neutralization of two animal viruses, Western equine encephalitis virus and poliomyelitis virus. Virology 2, 162–205.

252

PARREN AND BURTON

Emini, E. A., Kao, S.-Y., Lewis, A. J., Crainic, R., and Wimmer, E. (1983a). Functional basis of poliovirus neutralization determined with monospecific neutralizing antibodies. J. Virol. 46, 466–474. Emini, E. A., Ostapchuk, P., and Wimmer, E. (1983b). Bivalent attachment of antibody onto poliovirus leads to conformational alteration and neutralization. J. Virol. 48, 547–550. Engelstad, M., Howard, S. T., and Smith, G. L. (1992). A constitutively expressed vaccinia gene encodes a 42-kDa glycoprotein related to complement control factors that forms part of the extracellular virus envelope. Virology 188, 801–810. Fazekas de St.Groth, S. (1962). The neutralization of viruses. Adv. Virus Res. 9, 1–125. Fischer, R., Liao, Y. C., Hoffman, K., Schillberg, S., and Emans, N. (1999). Molecular farming of recombinant antibodies in plants. Biol. Chem. 380, 825–839. Flamand, A., Raux, H., Gaudin, Y., and Ruigrok, R. W. (1993). Mechanisms of rabies virus neutralization. Virology 194, 302–313. Flynn, D. C., Olmsted, R. A., Mackenzie, J. M. J., and Johnston, R. E. (1988). Antibody-mediated activation of Sindbis virus. Virology 166, 82–90. Fodor, W. L., Rollins, S. A., Bianco-Caron, S., Rother, R. P., Guilmette, E. R., Burton, W. V., Albrecht, J. C., Fleckenstein, B., and Squinto, S. P. (1995). The complement control protein homolog of herpesvirus saimiri regulates serum complement by inhibiting C3 convertase activity. J. Virol. 69, 3889–3892. Forthal, D. N., Landucci, G., Katz, J., and Tilles, J. G. (1993). Comparison of measles virus-specific antibodies with antibody-dependent cellular cytotoxicity and neutralizing functions. J. Infect. Dis. 168, 1020–1023. Fujinami, R. S., and Oldstone, M. B. (1980). Alterations in expression of measles virus polypeptides by antibody: Molecular events in antibody-induced antigenic modulation. J. Immunol. 125, 78–85. Fujinami, R. S., and Oldstone, M. B. A. (1979). Antiviral antibody reacting on the plasma membrane alters measles virus expression inside the cell. Nature 279, 529–530. Fujinami, R. S., Rosenthal, A., Lampert, P. W., Zurbriggen, A., and Yamada, M. (1989). Survival of athymic (nu/nu) mice after Theiler’s murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody. J. Virol. 63, 2081–2087. Fujioka, H., Emancipator, S. N., Aikawa, M., Huang, D. S., Blatnik, F., Karban, T., DeFife, J., and Manzanec, M. B. (1998). Immunocytochemical colocalization of specific immunoglobulin A with sendai virus protein in infected polarized epithelium. J. Exp. Med. 188, 1223–1229. Gauduin, M.-C., Parren, P. W. H. I., Weir, R., Barbas, C. F., Burton, D. R., and Koup, R. A. (1997). Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1. Nature Med. 3, 1389–1393. Gelderblom, H., Reupke, H., Winkel, T., Kunze, R., and Pauli, G. (1987). MHC-antigens: Constituents of the envelopes of human and simian immunodeficiency viruses. Z. Naturforsch 42c, 1328–1334. Gelderblom, H. R. (1991). Assembly and morphology of HIV: Potential effect of structure on viral function. AIDS 5, 617–637. Gould, E. A., Buckley, A., Barrett, A. D., and Cammack, N. (1986). Neutralizing (54K) and nonneutralizing (54K and 48K) monoclonal antibodies against structural and non-structural yellow fever virus proteins confer immunity in mice. J. Gen. Virol. 67, 591–595. Green, L. L., Hardy, M. C., Maynard-Currie, C. E., Tsuda, H., Louie, D. M., Mendez, M. J., Abderrahim, H., Noguchi, M., Smith, D. H., Zeng, Y., David, N. E., Sasai, H., Garza, D., Brenner, D. G., Hales, J. F., McGuinness, R. P., Capon, D. J., Klapholz, S., and Jakobovits, A. (1994). Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nature Genetics 7, 13–21. Halstead, S. B. (1979). In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody. J. Infect. Dis. 140, 527–533. Halstead, S. B. (1982). Immune enhancement of viral infection. Progr. Allergy 31, 301–364.

ANTIVIRAL ACTIVITY OF ANTIBODIES

253

Harris, L. J., Larson, S. B., Hasel, K. W., and McPherson, A. (1997). Refined structure of an intact IgG2a monoclonal antibody. Biochemistry 36, 1581–1597. Hasenkrug, K. J., Brooks, D. M., and Chesebro, B. (1995). Passive immunotherapy for retroviral disease: Influence of major histocompatibility complex type and T-cell responsiveness. Proc. Natl. Acad. Sci. USA 92, 10492–10495. Hawkes, R. A., and Lafferty, K. J. (1967). The enhancement of virus infectivity by antibody. Virology 33, 250–261. Hayashida, I., Nagafuchi, S., Hayashi, Y., Kino, Y., Mori, R., Oda, H., Ohtomo, N., and Tashiro, A. (1982). Mechanism of antibody-mediated protection against herpes simplex virus infection in athymic nude mice: Requirement of Fc portion of antibody. Microbiol. Immunol. 26, 497–509. He, Y., Bowman, V. D., Mueller, S., Bator, C. M., Bella, J., Peng, X., Baker, T. S., Wimmer, E., Kuhn, R. J., and Rossman, M. G. (2000). Interaction of the poliovirus receptor with poliovirus. Proc. Natl. Acad. Sci. USA 97, 79–84. Healey, D., Dianda, L., Moore, J. P., McDougal, J. S., Moore, M. J., Estess, P., Buck, D., Kwong, P. D., Beverley, P. C., and Sattentau, Q. J. (1990). Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding. J. Exp. Med. 172, 1233–1242. Heath, A. W., Martins, M. S., and Hudson, L. (1990). Monoclonal antibodies mediating viable immunofluorescence and protection against Trypanosoma cruzi infection. Trop. Med. Parasitol. 41, 425–428. Hemming, V. G., Prince, G. A., Horswood, R. L., London, W. T., Murphy, B. R., Walsh, E. E., Fischer, G. W., Weisman, L. E., Baron, P. A., and Chanock, R. M. (1985). Studies of passive immunotherapy for infections of respiratory syncytial virus in the respiratory tract of a primate model. J. Infect. Dis. 152, 1083–1087. Hewat, E., and Blaas, D. (1996). Structure of a neutralizing antibody bound bivalently to human rhinovirus 2. EMBO J. 15, 1515–1523. Hewat, E., and Blaas, D. (2001). Structural studies on antibody interacting with viruses. Curr. Top. Microbiol. Immunol. (in press) Homsy, J., Meyer, M., Tateno, M., Clarkson, S., and Levy, J. (1989). The Fc and not CD4 receptor mediates antibody enhancement of HIV infection in human cells. Science 244, 1357–1360. Hooks, J. J., Burns, W., Hayashi, K., Geis, S., and Notkins, A. L. (1976). Viral spread in the presence of neutralization antibody: Mechanisms of persistence in foamy virus infection. Infect. Immun. 14, 1172–1178. Hoxie, J. A., Fitzharris, T. P., Youngbar, P. R., Matthews, D. M., Rackowski, J. L., and Radka, S. F. (1987). Nonrandom association of cellular antigens with HTLV-III virions. Hum. Immunol. 18, 39–52. Icenogle, J., Shiwen, H., Duke, G., Gilbert, S., Rueckert, R., and Anderegg, J. (1983). Neutralization of poliovirus by a monoclonal antibody: Kinetics and stoichiometry. Virology 127, 412–425. Ichihashi, Y. (1996). Extracellular enveloped vaccinia virus escapes neutralization. Virology 217, 478–485. Iorio, R. M., and Bratt, M. A. (1983). Monoclonal antibodies to newcastle disease virus: Delineation of four epitopes on the HN Glycoprotein. J. Virol. 48, 440–450. Iorio, R. M., and Bratt, M. A. (1984). Neutralization of Newcastle disease virus by monoclonal antibodies to the Hemagglutinin-Neuraminidase Glycoprotein: Requirement for antibodies to four sites for complete neutralization. J. Virol. 51, 445–451. Isaacs, S., Wolffe, E. J., Payne, L. G., and Moss, B. (1992). Characterization of a vaccinia virusencoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope. J. Virol. 66, 7217–7224. Ishizaka, S. T., Piacente, P., Silva, J., and Mishkin, E. M. (1995). IgG subtype is correlated with efficiency of passive protection and effector function of anti-herpes simplex virus glycoprotein D monoclonal antibodies. J. Infect. Dis. 172, 1108–1111.

254

PARREN AND BURTON

Jerne, N. K., and Avegno, P. (1956). The development of the phage-inactivating properties of serum during the course of specific immunization of an animal: Reversible and irreversible inactivation. J. Immunol. 76, 200–208. Johnson, S., Griego, S. D., Pfarr, D. S., Doyle, M. L., Woods, R., Carlin, D., Prince, G. A., Koenig, S., Young, J. F., and Dillon, S. B. (1999). A direct comparison of the activities of two humanized respiratory syncytial virus monoclonal antibodies: MEDI-493 and RSHZ19. J. Infect. Dis. 180, 35–40. Karlsson, G. B., Gao, F., Robinson, J., Hahn, B., and Sodroski, J. (1996). Increased envelope spike density and stability are not required for the neutralization resistance of primary human immunodeficiency viruses. J. Virol. 70, 6136–6142. Kato, H., Kato, R., Fujihashi, K., and McGhee, J. R. (2000). Role of mucosal antibodies in viral infections. Curr. Top. Microbiol. Immunol. (in press). Kilbourne, E. D., Laver, W. G., Schulman, J. L., and Webster, R. G. (1968). Antiviral activity of antiserum specific for an influeza virus neuraminidase. J. Virol. 2, 281–288. Kimura, Y. (1973). Phenotypic mixing of vesicular stomatitis virus with HVJ (Sendai Virus). Japan. J. Microbiol. 17, 373–381. Kingsford, L. (1984). Enhanced neutralization of La Crosse Virus by the binding of specific pairs of monoclonal antibodies to the G1 glycoprotein. Virology 136, 265–273. Klasse, P. J., and Moore, J. P. (1996). Quantitative model of antibody- and soluble CD4-mediated neutralization of primary isolates and T-cell line-adapted strains of human immunodeficiency virus type 1. J. Virol. 70, 3668–3677. Kotwal, G. J., Isaacs, S. N., McKenzie, R., Frank, M. M., and Moss, B. (1990). Inhibition of the complement cascade by the major secretory protein of vaccinia virus. Science 250, 827–830. Kreil, T. R., and Eibl, M. M. (1997). Pre- and postexposure protection by passive immunoglobulin but no enhancement of infection with a flavivirus in a mouse model. J. Virol. 71, 2921–2927. Krummel, W. M., and Uhr, J. W. (1969). A mathematical and experimental study of the kinetics of neutralization of bacteriophage ␾X 174 by antibodies. J. Immunol. 102, 772–785. Lafferty, K. J. (1963a). The interaction between virus and antibody. Virology 21, 61–75. Lafferty, K. J. (1963b). The interaction between virus and antibody II. Mechanism of the reaction. Virology 21, 76–90. Lamarre, A., and Talbot, P. J. (1995). Protection from lethal coronavirus infection by immunoglobulin fragments. J. Immunol. 154, 3975–3984. Lamarre, A., Yu, M. W., Chagnon, F., and Talbot, P. J. (1997). A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice. Eur. J. Immunol. 27, 3447–3455. Lamb, R. A., and Krug, R. M. (1996). Orthomyxoviridiae: The viruses and their replication. In Fields Virology (Fields, B. N., Knipe, D. M., Howley, P. M., Chanock, R. M., Melnick, J. L., Monath, T. P., Roizman, B., and Straus, S. E., Eds.). Philadelphia, PA: Lippincott-Raven Publishers), pp. 1353–1395. Lamon, E. W., Powell, T. J., Jr., Walia, A. S., Lidin, B. I., Srinivas, R. V., Baskin, J. G., and Kearney, J. F. (1987). Monoclonal IgM antibodies that inhibit primary Moloney murine sarcoma growth. J. Natl. Cancer. Inst. 78, 547–556. Lecomte, J., Cainelli-Gebara, V., Mercier, G., Mansour, S., Talbot, P. J., Lussier, G., and Oth, D. (1987). Protection from mouse hepatitis virus type 3-induced acute disease by an antinucleoprotein monoclonal antibody. Arch. Virol. 97, 123–130. Ledbetter, J., and Nowinski, R. C. (1977). Identification of the Gross cell surface antigen associated with murine leukemia virus-infected cells. J. Virol. 23, 315–322. Lederman, M. M., Purvis, S. F., Walter, E. I., Carey, J. T., and Medof, M. E. (1989). Heightened complement sensitivity of acquired immunodeficiency syndrome lymphocytes related to diminished expression of decay-accelerating factor. Proc. Natl. Acad. Sci. USA 86, 4205–4209.

ANTIVIRAL ACTIVITY OF ANTIBODIES

255

Lee, B., Sharron, M., Blanpain, C., Doranz, B. J., Vakili, J., Setoh, P., Berg, E., Liu, G., Guy, H. R., Durell, S. R., Parmentier, M., Nan Chang, C., Price, K., Tsang, M., and Doms, R. W. (2000). Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function. J. Biol. Chem. 274, 9617–9626. Lee, T. H., Essex, M., De Noronha, F., and Azocar, J. (1982). Neutralization of feline leukemia virus with feline antisera to leukocyte alloantigens. Cancer. Res. 42, 3995–3999. Lefrancois, L. (1984). Protection against lethal viral infection by neutralizing and nonneutralizing monoclonal antibodies: Distinct mechanisms of action in vivo. J. Virol. 51, 208–214. Lemon, S. M., and Binn, L. N. (1985). Incomplete neutralization of hepatitis A virus in vitro due to lipid-associated virions. J. Gen. Virol. 66, 2501–2505. Letchworth, G. J. I., and Appleton, J. A. (1983). Passive protection of mice and sheep against bluetongue virus by a neutralizing monoclonal antibody. Infect. Immun. 39, 208–212. Levine, B., Hardwick, J. M., Trapp, B. D., Crawford, T. O., Bollinger, R. C., and Griffins, D. E. (1991). Antibody mediated clearance of Sindbis encephalomyelitis in SCID mice: A novel mechanism for recovery from viral infection. FASEB J. 5 (Abstract). Li, L., Coelingh, K. L., and Britt, W. J. (1995). Human cytomegalovirus neutralizing antibodyresistant phenotype is associated with reduced expression of glycoprotein. H. J. Virol. 69, 6047– 6053. Liebert, U. G., Schneider-Schaulies, S., Baczko, K., and Ter Meulen, V. (1990). Antibody-induced restriction of viral gene expression in measles encephalitis in rats. J. Virol. 64, 706–713. Lonberg, N., Taylor, L. D., Harding, F. A., Trounstine, M., Higgins, K. M., Schramm, S. R., Kuo, C.-C., Mashayekh, R., Wymore, K., McCabe, J. G., Munoz-O’Regan, D., O’Donnell, S. L., Lapachet, E. S. G., Bengoechea, T., Fishwild, D. M., Carmack, C. E., Kay, R. M., and Huszar, D. (1994). Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 368, 856–859. Lubinski, J., Nagashunmugam, T., and Fridman, H. M. (1998). Viral interference with antibody and complement. Semin. Cell. Dev. Biol. 9, 329–337. Lund, O., Hansen, J., Sørensen, A. M., Mosekilde, E., Nielsen, J. O., and Hansen, J. E. (1995). Increased adhesion as a mechanism of antibody-dependent and antibody-independent complementmediated enhancement of human immunodeficiency virus infection. J. Virol. 69, 2393–2400. Mandel, B. (1961). Reversibility of the reaction between poliovirus and neutralizing antibody of rabbit origin. Virology 14, 316–328. Mandel, B. (1976). Neutralization of poliovirus: A hypothesis to explain the mechanism and the one-hit character of the neutralization reaction. Virology 69, 500–510. Manzanec, M. B., Coudret, C. L., and Fletcher, D. R. (1995). Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies. J. Virol. 69, 1339– 1343. Manzanec, M. B., Lamm, M. E., Lyn, D., Porter, A., and Bedrud, J. G. (1992). Comparison of IgA versus IgG monoclonal antibodies for passive immunization of the murine respiratory tract. Virus. Res. 23, 1–12. Maruyama, T., Rodriguez, L. L., Jahrling, P. B., Sanchez, A., Khan, A., Nichol, S. T., Peters, C. J., Parren, P. W. H. I., and Burton, D. R. (1999). Ebola virus can be effectively neutralized by antibody produced in natural human infection. J. Virol. 73, 6024–6030. Mascola, J. R., Lewis, M. G., Stiegler, G., Harris, D., Van Cott, T. C., Hayes, D., Louder, M. K., Brown, C., Sapan, C. V., Frankel, S. S., Lu, Y., Robb, M. L., Katinger, H., and Birx, D. L. (1999). Protection of macaques against pathogenic SHIV-89.6PD by passive transfer of neutralizing antibodies. J. Virol. 73, 4009–4018. Mascola, J. R., Mathieson, B. J., Zack, P. M., Walker, M. C., Halstead, S. B., and Burke, D. S. (1993). Summary report: Workshop on the potential risks of antibody-dependent enhancement in human HIV vaccine trials. AIDS Res. Hum. Retroviruses 9, 1175–1184.

256

PARREN AND BURTON

Mascola, J. R., Stiegler, G., VanCott, T. C., Katinger, H., Carpenter, C. B., Hanson, C. E., Beary, H., Hayes, D., Frankel, S. S., Birx, D. L., and Lewis, M. G. (2000). Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat. Med. 6, 207–210. Massey, R. J., and Schochetman, G. (1981). Viral epitopes and monoclonal antibodies: Isolation of blocking antibodies that inhibit virus neutralization. Science 213, 447–449. Mathews, J. H., Roehrig, J. T., and Trent, D. W. (1985). Role of complement and the Fc portion of immunoglobulin G in immunity to Venezuelan equine encephalomyelitis virus infection with glycoprotein-specific monoclonal antibodies. J. Virol. 55, 594–600. Mbow, M. L., Gilmore, R. D., Jr., and Titus, R. G. (1999). An OspC-specific monoclonal antibody passively protects mice from tick-transmitted infection by Borrelia burgdorferi B31. Infect. Immun. 67, 5470–5472. McCullough, K. C., Crowther, J. R., Butcher, R. N., Carpenter, W. C., Brocchi, E., Cappucci, L., and De Simone, F. (1986). Immune protection against foot-and-mouth disease virus studied using virus neutralizing and non neutralizing concentrations of monoclonal antibodies. Immunology 58, 421–428. McCullough, K. C., De Simone, F., Brocchi, E., Capucci, L., Crowther, J. R., and Kihm, U. (1992). Protective immune response against foot-and-mouth disease. J. Virol. 66, 1835–1840. McCullough, K. C., Parkinson, D., and Crowther, J. R. (1988). Opsonization-enhanced phagocytosis of foot-and-mouth disease virus. Immunology 65, 187–191. McIntosh, K., and Chanock, R. M. (1990). Respiratory syncytial viruses. In Fields Virology. New York: Raven Press, pp. 1045–1072. McKendall, R. R. (1985). IgG-mediated viral clearance in experimental infection with herpes simplex virus type 1: Role for neutralization and Fc-dependent functions but not C′ cytolysis and C5 chemotaxis. J. Infect. Dis. 151, 464–470. McLain, L., and Dimmock, N. J. (1994). Single- and multi-hit kinetics of immunoglobulin G neutralization of human immunodeficiency virus type 1 by monoclonal antibodies. J. Gen. Virol. 75, 1457–1460. Megran, D. W., Stiver, G., Peeling, R., Maclean, I. W., and Brunham, R. C. (1988). Complement enhancement of neutralizing antibody to the structural proteins of Chlamydia trachomatis. J. Infect. Dis. 158, 661–663. Merkenschlager, M., Buck, D., Beverley, P. C., and Sattentau, Q. J. (1990). Functional epitope analysis of the human CD4 molecule. The MHC class II-dependent activation of resting T cells is inhibited by monoclonal antibodies to CD4 regardless of whether or not they recognize epitopes involved in the binding of MHC class II or HIV gp120. J. Immunol. 145, 2839–2845. Merz, D. C., Scheid, A., and Choppin, P. W. (1981). Immunological studies of the functions of paramyxovirus glycoproteins. Virology 109, 94–105. Mester, J. C., Glorioso, J. C., and Rouse, B. T. (1991). Protection against zosteriform spread of herpes simplex virus by monoclonal antibodies. J. Infect. Dis. 163, 263–269. Mochizuki, Y., de Ming, T., Hayashi, T., Itoh, M., Hotta, H., and Homma, M. (1990). Protection of mice against Sendai virus pneumonia by non-neutralizing anti-F monoclonal antibodies. Microbiol. Imunol. 34, 171–183. Moore, J. P., Cao, Y., Qing, L., Sattentau, Q. J., Pyati, J., Koduri, R., Robinson, J., Barbas, C. F., Burton, D. R., and Ho, D. D. (1995). Primary isolates of human immunodeficiency virus type 1 are relatively resistant to neutralization by monoclonal antibodies to gp120, and their neutralization is not predicted by studies with monomeric gp120. J. Virol. 69, 101–109. Moore, J. P., and Ho, D. D. (1995). HIV-1 neutralization: The consequences of viral adaptation to growth on transformed T cells. AIDS 9 (Suppl A), S117–S136. Morens, D. M., Halstead, S. B., and Marchette, N. J. (1987). Profiles of antibody-dependent enhancement of dengue virus type 2 infection. Microb. Pathog. 3, 231–237. Moulard, M., Lortat-Jacob, H., Mondor, I., Roca, G., Wyatt, R., Sodroski, J., Zhao, L., Olson, W.,

ANTIVIRAL ACTIVITY OF ANTIBODIES

257

Kwong, P. D., and Sattentau, Q. J. (2000). Selective interactions of polyanions with basic surfaces on human immunodeficiency virus type 1 gp120. J. Virol. 74, 1948–1960. Mozdzanowska, K., Furchner, M., Washko, G., Mozdzanowski, J., and Gerhard, W. (1997). A pulmonary influenza virus infection in SCID mice can be cured by treatment with hemagglutininspecific antibodies that display very low virus-neutralizing activity in vitro. J. Virol. 71, 4347–4355. Mozdzanowska, K., Maiese, K., Furchner, M., and Gerhard, W. (1999). Treatment of influenza virusinfected SCID mice with nonneutralizing antibodies specific for the transmembrane proteins matrix 2 and neuraminidase reduces the pulmonary virus titer but fails to clear the infection. Virology 254, 138–146. Murphy, B. R., Kasel, J. A., and Chanock, R. M. (1972). Association of serum anti-neuraminidase antibody with resistance to influenza in man. N. Engl. J. Med. 286, 1329–1332. Murphy, B. R., and Webster, R. G. (1996). Orthomyxoviruses. In Fields Virology (Fields, B. N., Knipe, D. M., Howley, P. M., Chanock, R. M., Melnick, J. L., Monath, T. P., Roizman, B., and Straus, S. E., Eds.). Philadelphia, PA: Lippincott-Raven Publishers, pp. 1397–1446. Nakanaga, K., Yamanouchi, K., and Fujiwara, K. (1986). Protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice. J. Virol. 59, 168–171. Oakes, J. E., and Lausch, R. N. (1981). Role of Fc fragments in antibody-mediated recovery from ocular and subcutaneous herpes simplex virus infections. Infect. Immun. 33, 109–114. Old, L. J., Stockert, E., Boyse, E. A., and Geering, G. (1967). A study of passive immunization against a transplanted G+ leukemia with specific antiserum. Proc. Soc. Exp. Biol. Med. 124, 63–68. Oldstone, M. B. A. (1975). Virus neutralization and virus-induced immune complex disease. Progr. Med. Virol. 19, 84–119. Ollmann Saphire, E., Parren, P. W. H. I., Barbas, C. F., III, Burton, D. R., and Wilson, I. A. (2001). Crystallization and preliminary structure determination of an intact human immunoglobulin b12: An antibody that broadly neutralizes primary isolates of HIV-1. Acta Crystallogr. (in press). Orentas, R. J., and Hildreth, J. E. K. (1993). Association of host cell surface adhesion receptors and other membrane proteins with HIV and SIV. AIDS Res. Hum. Retroviruses 9, 1157–1165. Pantaleo, G., Demarest, J. F., Vaccarezza, M., Graziosi, C., Bansal, G. P., Koenig, S., and Fauci, A. S. (1995). Effect of anti-V3 antibodies on cell-free and cell-to-cell human immunodeficiency virus transmission. Eur. J. Immunol. 25, 226–231. Parren, P. W. H. I. (1992). Preparation of genetically engineered monoclonal antibodies for human immunotherapy. Hum. Antibod. Hybridomas 3, 137–145. Parren, P. W. H. I., and Burton, D. R. (1997). Antibodies against HIV-1 from phage display libraries: Mapping of an immune response and progress towards anti-viral immunotherapy. Chem. Immunol. 65, 18–56. Parren, P. W. H. I., Ditzel, H. J., Gulizia, R. J., Binley, J. M., Barbas, C. F., Burton, D. R., and Mosier, D. E. (1995). Protection against HIV-1 infection in hu-PBL-SCID mice by passive immunization with a neutralizing human monoclonal antibody against the gp120 CD4-binding site. AIDS 9, F1–F6. Parren, P. W. H. I., Gauduin, M.-C., Koup, R. A., Poignard, P., Sattentau, Q. J., Fisicaro, P., and Burton, D. R. (1997a). Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design. Immunol. Lett. 58, 125–132. Parren, P. W. H. I., Maruyama, T., Burton, D. R., and Jahrling, P. B. (2001a). A human neutralizing antibody protects against challenge with Ebola Zaire in an animal model. Manuscript in preparation. Parren, P. W. H. I., Marx, P., Cheng-Mayer, C., Harouse, J., Moore, J. P., and Burton, D. R. (2001b). A neutralizing human monoclonal antibody protects macaques against challenge with a pathogenic R5 SHIV. Manuscript in preparation. Parren, P. W. H. I., Mondor, I., Naniche, D., Ditzel, H. J., Klasse, P. J., Burton, D. R., and Sattentau, Q. J. (1998). Neutralization of HIV-1 by antibody to gp120 is determined primarily by occupancy of sites on the virion irrespective of epitope specificity. J. Virol. 72, 3512–3519.

258

PARREN AND BURTON

Parren, P. W. H. I., Moore, J. P., Burton, D. R., and Sattentau, Q. J. (1999). The neutralizing antibody response to HIV-1: Viral evasion and escape from humoral immunity. AIDS 13 (Suppl A), S137–S162. Parren, P. W. H. I., Sattentau, Q. J., and Burton, D. R. (1997b). HIV-1 antibody—Debris or virion? Nature. Med. 3, 366–367. Peeling, R. W., and Brunham, R. C. (1991). Neutralization of Chlamydia trachomatis: Kinetics and stoichiometry. Infect. Immun. 59, 2624–2630. Philipson, L. (1966). Interaction betwen poliovirus and immunoglobulins. II. Basic aspects of virus– antibody interaction. Virology 28, 35–46. Philipson, L., Killander, J., and Albertsson, P.-A. (1966). Interaction between poliovirus and immunoglobulins I. Detection of virus antibodies by partition in aqueous polymer phase systems. Virology 28, 22–34. Pincus, S. H., Cole, R., Ireland, R., McAtee, F., Fujisawa, R., and Portis, J. (1995). Protective efficacy of nonneutralizing monoclonal antibodies in acute infection with murine leukemia virus. J. Virol. 69, 7152–7158. Poignard, P., Sabbe, S., Picchio, G. R., Wang, M., Gulizia, R. J., Katinger, H., Parren, P. W. H. I., Mosier, D. E., and Burton, D. R. (1999). Neutralizing antibodies have limited effects on the control of established HIV-1 infection in vivo. Immunity 10, 431–438. Pollock, D. P., Kutzko, J. P., Birck-Wilson, E., Williams, J. L., Echelard, Y., and Meade, H. M. (1999). Transgenic milk as a method for the production of recombinant antibodies. J. Immunol. Meth. 231, 147–157. Portis, J. L., Spangrude, G. J., and McAtee, F. J. (1994). Identification of a sequence in the unique 5′ open reading frame of the gene encoding glycosylated Gag which influences the incubation period of neurodegenerative disease induced by a murine retrovirus. J. Virol. 68, 3879–3887. Possee, R. D., Schild, G. C., and Dimmock, N. J. (1982). Studies on the mechanism of neutralization of influenza virus by antibody: Evidence that neutralizing antibody (anti-haemagglutinin) inactivates influenza virus in vivo by inhibiting virions transcriptase activity. J. Gen. Virol. 58, 373–386. Prince, A. M. (1994). Challenges for development of hepatitis C virus vaccines. FEMS Microbiol. Rev. 14, 273–277. Prince, G. A., Hemming, V. G., Horswood, R. L., Baron, P. A., and Chanock, R. M. (1987). Effectiveness of topically administered neutralizing antibodies in experimental immunotherapy of respiratory syncytial virus infection in cotton rats. J. Virol. 61, 1851–1854. Prince, G. A., Hemming, V. G., Horswood, R. L., Baron, P. A., Murphy, B. R., and Chanock, R. M. (1990). Mechanism of antibody-mediated viral clearance in immunotherapy of respiratory syncytial virus infection of cotton rats. J.Virol. 64, 3091–3092. Prince, G. A., Hemming, V. G., Horswood, R. L., and Chanock, R. M. (1985a). Immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in the cotton rat. Virus Res. 3, 193– 206. Prince, G. A., Horswood, R. L., and Chanock, R. M. (1985b). Quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats. J. Virol. 55, 517–520. Prohaszka, Z., Nemes, J., Hidvegi, T., Toth, F. C., Kerekes, K., Erdei, A., Szabo, J., Ujhelyi, E., Thielens, N., Dierich, M. R., Spath, P., Ghebrehiwet, B., Hampl, H., Kiss, J., Arlaud, G., and Fust, G. (1997). Two parallel routes of the complement-mediated antibody-dependent enhancement of HIV-1 infection. AIDS 11, 949–958. Rappaport, I. (1970). An analysis of the inactivation of MS2 bacteriophage with antiserum. J. Gen. Virol. 6, 25–32. Raux, H., Coulon, P., Lafay, F., and Flamand, A. (1995). Monoclonal antibodies which recognize the acidic configuration of the rabies glycoprotein at the surface of the virion can be neutralizing. Virology 210, 400–408.

ANTIVIRAL ACTIVITY OF ANTIBODIES

259

Rizzuto, C. D., and Sodroski, J. G. (1997). Contribution of virion ICAM-1 to human immunodeficiency virus infectivity and sensitivity to neutralization. J. Virol. 71, 4847–4851. Roben, P., Moore, J. P., Thali, M., Sodroski, J., Barbas, C. F., and Burton, D. R. (1994). Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J. Virol. 68, 4821–4828. Robinson, W. E., Montefiori, D. C., and Mitchell, W. M. (1988). Antibody-dependent enhancement of human immunodeficiency virus type 1 infection. Lancet. i, 790–794. Roden, R. S., Weissinger, E. M., Henderson, D. W., Booy, F., Kirnbauer, R., Mushinski, J. F., Lowy, D. R., and Schiller, J. T. (1994). Neutralization of bovine papillomavirus by antibodies to L1 and L2 capsid proteins. J. Virol. 68, 7570–7574. Rosengard, A. M., Alonso, L. C., Korb, L. C., Baldwin, W. M., Sanfilippo, F., Turka, L. A., and Ahearn, J. M. (1999). Functional characterization of soluble and membrane-bound forms of vaccinia complement control protein. Mol. Immunol. 36, 685–697. Rother, R. P., Rollins, S. A., Fodor, W. L., Albrecht, J. C., Setter, E., Fleckenstein, B., and Squinto, S. P. (1994). Inhibition of complement-mediated cytolysis by the terminal complement-inhibitor of herpesvirus saimiri. J. Virol. 68, 730–737. Saarlos, M. N., Sullivan, B. L., Czerniewski, M. A., Parameswar, K. D., and Spear, G. T. (1997). Detection of HLA-DR associated with monocytotropic, primary, and plasma isolates of human immunodeficiency virus type 1. J. Virol. 71, 1640–1643. Saifuddin, M., Ghassemi, M., Patki, C., Parker, C. J., and Spear, G. T. (1994). Host cell components affect the sensitivity of HIV type 1 to complement-mediated virolysis. AIDS Res. Hum. Retroviruses 10, 829–837. Saifuddin, M., Parker, C. J., Peeples, M. E., Gorny, M. K., Zolla-Pazner, S., Ghassemi, M., Rooney, I. A., Atkinson, J. P., and Spear, G. T. (1995). Role of virion-associated glycosylphosphatidylinositollinked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-1. J. Exp. Med. 182, 501–509. Sakurai, H., Williamson, R. A., Crowe, J. E., Beeler, J. A., Poignard, P., Bastidas, R. B., Chanock, R. M., and Burton, D. R. (1999). Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: Significance for subunit vaccines. J. Virol. 73, 2956–2962. Sanna, P. P., De Logu, A., Williamson, R. A., Hom, Y.-L., Straus, S. E., Bloom, F. E., and Burton, D. R. (1996). Protection of nude mice by passive immunization with a type-common human recombinant monoclonal antibody against HSV. Virology 215, 101–106. Sanner, M. F. (1999). Python: A programming language for software integration and development. J. Mol. Graphics. Mod. 17, 57–61. Sanner, M. F., Duncan, B. S., Carrillo, C. J., and Olson, A. J. (1999). Integrating computation and visualization for biomolecular analysis: An example using python and AVS. Proc. Pacific. Symp. Biocomputing 99, 401–412. Sanner, M. F., Spehner, J.-C., and Olson, A. J. (1996). Reduced surface: An efficient way to compute molecular surfaces. Biopolymers 38, 305–320. Sattentau, Q. J., Dalgleish, A. G., Weiss, R. A., and Beverley, P. C. (1986). Epitopes of the CD4 antigen and HIV infection. Science 234, 1120–1123. Sattentau, Q. J., and Moore, J. P. (1995). Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer. J. Exp. Med. 182, 185–196. Sattentau, Q. J., Zolla-Pazner, S., and Poignard, P. (1995). Epitope exposure on functional, oligomeric HIV-1 gp41 molecules. Virology 206, 713–717. Schlesinger, J. J., and Chapman, S. (1995). Neutralizing F(ab′ )2 fragments of protective monoclonal antibodies to yellow fever virus (YF) envelope protein fails to protect mice against lethal YF encephalitis. J. Gen. Virol. 76, 217–220.

260

PARREN AND BURTON

Schmaljohn, A. L., Johnson, E. D., Dalrymple, J. M., and Cole, G. A. (1982). Non-neutralizing monoclonal antibodies can prevent lethal alphavirus encephalitis. Nature 297, 70–72. Schmaljohn, A. L., Kokubun, K. M., and Cole, G. A. (1983). Protective monoclonal antibodies define maturational and pH-dependent antigenic changes in Sindbis virus E1 glycoprotein. Virology 130, 144–154. Scho /nning, K., Lund, O., Lund, O. S., and Hansen, J. E. S. (1999). Stoichiometry of monoclonal anitbody neutralization of T-cell line-adapted human immunodeficiency virus type 1. J. Virol. 73, 8364–8370. Schutten, M., Andeweg, A. C., Bosch, M. L., and Osterhaus, A. D. (1995). Enhancement of infectivity of a non-syncytium inducing HIV-1 by sCD4 and by human antibodies that neutralize syncytium inducing HIV-1. Scand. J. Immunol. 41, 18–22. Scicluna, L. A., and McCullough, K. C. (1999). Rapidity of specific antibody–antigen interactions. Scand. J. Immunol. 50, 167–176. Shibata, R., Igarashi, T., Haigwood, N., Buckler-White, A., Ogert, R., Ross, W., Willey, R., Cho, M. W., and Martin, M. A. (1999). Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nat. Med. 5, 204–210. Smith, G. L. (1999). Vaccinia virus immune evasion. Immunol. Lett. 65, 55–62. Smith, T. J., Chase, E. S., Schmidt, T. J., Olson, N. H., and Baker, T. S. (1996). Neutralising antibody to human rhinovirus 14 penetrates the receptor-binding canyon. Nature 383, 350–354. Smith, T. J., Olson, N. H., Cheng, R. H., Liu, H., Chase, E. S., Lee, W. M., Leippe, D. M., Mosser, A. G., Rueckert, R. R., and Baker, T. S. (1993). Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody. J. Virol. 67, 1148–1158. Smith, T. J. (2001). Antibody interactions with rhinovirus; lessons for mechanisms of neutralization and the role of immunity in viral evolution. Curr. Top. Microbiol. Immunol. (in press). Spear, G. T., Sullivan, B. L., Landay, A. L., and Lint, T. F. (1990). Neutralization of human immunodeficiency virus type 1 by complement occurs by viral lysis. J.Virol. 64, 5869–5873. Spiller, O. B., Hanna, S. M., Devine, D. V., and Tufaro, F. (1997). Neutralization of cytomegalovirus virions: The role of complement. J. Infect. Dis. 176, 339–347. Stewart, P. L., Chiu, C., Huang, S., Muir, T., Zhao, Y., Chait, B., Mathias, P., and Nemerow, G. R. (1997). Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization. EMBO J. 16, 1189–1198. Stewart, P. L., and Nemerow, G. R. (1997). Recent structural solutions for antibody neutralization of viruses. Trends Microbiol 5, 229–233. Stoiber, H., Clivio, A., and Dierich, M. P. (1997). Role of complement in HIV infection. Annu. Rev. Immunol. 15, 649–674. Stott, E. J. (1994). Towards a vaccine against AIDS: Lessons from simian immunodeficiency virus vaccines. Curr. Top. Microbiol. Immunol. 188, 221–237. Stott, E. J., Bew, M. H., Taylor, G., Jebbett, J., and Collins, A. P. (1984). The characterization and uses of monoclonal antibodies to respiratory syncytial virus. Dev. Biol. Stand. 57, 237–244. Sullivan, B. L., Knopoff, E. J., Saifuddin, M., Takefman, D. M., Saarloos, M.-N, Sha, B. E., and Spear, G. T. (1996). Susceptibility of HIV-1 plasma virus to complement-mediated lysis. J. Immunol. 157, 1791–1798. Sullivan, B. L., Takefman, D. M., and Spear, G. T. (1998a). Complement can neutralize HIV-1 plasma virus by a C5-independent mechanism. Virology 248, 173–181. Sullivan, N., Sun, Y., Binley, J., Lee, J., Barbas, C. F., III, Parren, P. W. H. I., Burton, D. R., and Sodroski, J. (1998b). Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies. J. Virol. 72, 6332–6338. Sullivan, N., Sun, Y., Li, J., Hofmann, W., and Sodroski, J. (1995). Replicative function and neutralization sensitivity of envelope glycoproteins from primary and T-cell line-passaged human immunodeficiency virus type 1 isolates. J.Virol. 69, 4413–4422.

ANTIVIRAL ACTIVITY OF ANTIBODIES

261

Takeda, A., Tuazon, C. U., and Ennis, F. A. (1988). Antibody-enhanced infection by HIV-1 via Fc receptor-mediated entry. Science 242, 580–583. Takefman, D. M., Sullivan, B. L., Sha, B. E., and Spear, G. T. (1998). Mechanisms of resistance of HIV-1 primary isolates to complement-mediated lysis. Virology 246, 370–378. Taylor, H. P., Armstrong, S. J., and Dimmock, N. J. (1987). Quantitative relationships between an influenza virus and neutralizing antibody. Virology 159, 288–298. Thomas, L. H., Cook, R. S., Wyld, S. G., Furze, J. M., and Taylor, G. (1998). Passive protection of gnotobiotic calves using monoclonal antibodies directed at different epitopes on the fusion protein of bovine respiratory syncytial virus. J. Infect. Dis. 177, 874–880. Tortorella, D., Gewurz, B. E., Furman, M. H., Schust, D. J., and Ploegh, H. L. (2000). Viral subversion of the immune system. Annu. Rev. Immunol. 18, 861–926. Truneh, A., Buck, D., Cassatt, D. R., Juszczak, R., Kassis, S., Ryu, S. E., Healey, D., Sweet, R., and Sattentau, Q. (1991). A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion. J Biol. Chem. 266, 5942–5948. Ubol, S., Levine, B., Lee, S.-H., Greenspan, N. S., and Griffin, G. E. (1995). Roles of immunoglobulin valency and the heavy-chain constant domain in antibody-medicated downregulation of sindbis virus replication in persistently infected neurons. J.Virol. 69, 1990–1993. Ugolini, S., Mondor, I., Parren, P. W. H. I., Burton, D. R., Tilley, S. A., Klasse, P. J., and Sattentau, Q. J. (1997). Inhibition of virus attachment to CD4+ target cells is a major mechanism of T cell line-adapted HIV-1 neutralization. J. Exp. Med. 186, 1287–1298. Vanderplasschen, A., Hollinshead, M., and Smith, G. L. (1997). Antibodies against vaccinia virus do not neutralize extracellular enveloped virus but prevent virus release from infected cells and comet formation. J. Gen. Virol. 78, 2041–2048. Vanderplasschen, A., Matthew, E., Hollinshead, M., Sim, R. B., and Smith, G. L. (1998). Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope. Proc. Natl. Acad. Sci. USA 95, 7544–7549. Van Kammen, A. (1999). Beijerinck’s contribution to the virus concept–An introduction. Arch. Virol. (Suppl) 15, 1–8. Verma, R., Boleti, E., and George, A. J. (1998). Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems. J. Immunol. Meth. 216, 165–181. Vernon, S. K., Neurath, A. R., and Rubin, B. A. (1972). Electron microscopic studies on the structure of rabies virus. J. Ultrastruct. Res. 41, 29–42. Virgin, H. W., IV, Bassel-Duby, R., and Fields, B. N. (1988). Antibody protects against lethal infection with the neurally spreading reovirus type 3 (Dearing). J. Virol. 62, 4594–4604. Vzorov, A. N., and Compans, R. W. (2000). Effect of the cytoplasmic domain of the simian immunodeficiency virus envelope protein on incorporation of heterologous envelope proteins and sensitivity to neutralization. J.Virol. 74, 8219–8225. Wallis, C., and Melnick, J. L. (1967). Virus aggregation as the cause of the non-neutralizable persistent fraction. J. Virol. 1, 478–488. Wallis, C., and Melnick, J. L. (1970). Herpesvirus neutralization: Induction of the persistent fraction by insufficient antibody. Virology 42, 128–137. Walsh, E. E., Schlesinger, J. J., and Brandriss, M. W. (1984). Protection from respiratory syncytial virus infection in cotton rats by passive transfer of monoclonal antibodies. Infect. Immun. 43, 756–758. Webster, R. G., and Laver, W. G. (1967). Preparation and properties of antibody directed specifically against the neuraminidase of influenza virus. J. Immunol. 99, 49–55. Wege, H., Dorries, R., and Wege, H. (1984). Hybridoma antibodies to the murine coronavirus JHM: Characterization of epitopes on the peplomer protein. J. Gen. Virol. 65, 1931–1942. Weiland, E., Wieczorek-Krohmer, M., Kohl, D., Conzelmann, K. K., and Weiland, F. (1999). Monoclonal antibodies to GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to GP4. Vet. Microbiol. 66, 171–186.

262

PARREN AND BURTON

Weiss, L., Okada, N., Haeffner-Cavaillon, N., Hattori, C., Faucher, C., Kazatchkine, M. D., and Okada, H. (1992). Decreased expression of the membrane inhibitor of complement-mediated cytolysis CD59 on T-lymphocytes of HIV-infected patients. AIDS 6, 379–385. Weltzin, R., Hsu, S. A., Mittler, E. S., Georgakopoulos, K., and Monath, T. P. (1994). Intranasal monoclonal immunoglobulin A against respiratory syncytial virus protects against upper and lower respiratory tract infections in mice. Antimicrobial Agents Chemother. 38, 2785–2791. Weltzin, R., Traina-Dorge, V., Soike, K., Zhang, J. Y., Mack, P., Soman, G., Drabik, G., and Monath, T. P. (1996). Intranasal monoclonal IgA antibody to respiratory syncytial virus protects rhesus monkeys against upper and lower respiratory tract infection. J. Infect. Dis. 174, 256–261. Westaway, E. G. (1965). The neutralization of arboviruses. I. Neutralization in homologous virusserum mixtures with two group B arboviruses. Virology 26, 517–527. Wild, F., Cathala, F., and Huppert, J. (1975–1976). Vesicular stomatitis virus (measles) pseudotypes: Tool for demonstrating defective measles infections. Intervirology 6, 185–189. Wilson, J. A., Hevey, M., Bakken, R., Guest, S., Bray, M., Schmaljohn, A. L., and Hart, M. K. (2000). Epitopes involved in antibody-mediated protection from Ebola Virus. Science 287, 1664–1666. Winter, G., Griffiths, A. D., Hawkins, R. E., and Hoogenboom, H. R. (1994). Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455. Wright, K. E., and Buchmeier, M. J. (1991). Antiviral antibodies attenuate T-cell-mediated immunopathology following acute lymphocytic choriomeningitis virus infection. J. Virol. 65, 3001– 3006. Wu, H., Kwong, P. D., and Hendrickson, W. A. (1997). Dimeric association and segmental variability in the structure of human CD4. Nature 387, 527–530. Zavada, J., and Rosenbergova, M. (1972). Phenotypic mixing of vesicular stomatitis virus with fowl plague virus. Acta Virol. 16, 103–114. Zebedee, S. L., and Lamb, R. A. (1988). Influenza A virus M2 protein: Monoclonal antibody restriction of virus growth and detection of M2 in virions. J. Virol. 62, 2762–2772. Zeitlin, L., Whaley, K. J., Sanna, P. P., Moench, T. R., Bastidas, R., De Logu, A., Williamson, R. A., Burton, D. R., and Cone, R. A. (1996). Topically applied human recombinant monoclonal IgG antibody and its Fab and F(ab′ ) fragments protect mice form vaginal transmission of HSV-2. Virology 225, 213–215.

ADVANCES IN IMMUNOLOGY, VOL. 77

Mouse Models of Allergic Airway Disease CLARE M. LLOYD,* JOSE-ANGEL GONZALO,† ANTHONY J. COYLE,† AND JOSE-CARLOS GUTIERREZ-RAMOS† *Leukocyte Biology Section, Biomedical Sciences Division, Imperial College of Science,

Technology, and Medicine, London SW7 2AZ, United Kingdom; and †Millennium Pharmaceuticals, Cambridge, Massachusetts 02139

I. Introduction

Asthma is a clinical syndrome characterized by intermittent episodes of wheezing and coughing. The diagnosis is confirmed by abnormal lung physiology, including reversible airway obstruction and airway hyperresponsiveness (AHR) to spasmogenic stimuli. The fact that the clinical and biological manifestations of asthma are extremely heterogeneous reflects the multitude of causative and aggravating factors, as well as the presence of many underlying pathophysiological mechanisms. Pathological manifestations have been found to include airway inflammation, remodeling, and mucus hypersecretion. The airways of patients with even mild asthma are inflamed, and some data suggest that the severity of asthma parallels the degree of this inflammation (Broide et al., 1991; Pare and Bai, 1995; Peters, 1990). In addition, the localization and activation of specialized leukocytes correlate with the temporal phases of airway obstruction and enhanced bronchial responsiveness to spasmogenic stimuli. Among the leukocytes considered causative players in the development of bronchial inflammation, eosinophils are thought to be critical, since eosinophilia is a common feature of asthmatic airways and eosinophils have been demonstrated to cause mucosal injury. However, evidence points to the fact that although eosinophils are largely responsible for asthmatic symptoms, their function is largely under the control of specialized subsets of chronically activated memory T cells sensitized against an array of allergenic, occupational, and viral antigens that home to the lungs after appropriate antigen exposure or viral infection. In asthmatics, CD4+ T cells producing interleukin 4 (IL-4), IL-5, and IL-13 have been identified in bronchoalveolar lavage (BAL) and airway biopsies. Evidence for their functional involvement stems from the fact that T helper 2 (Th2) cells are present in the airways and that Th2-derived cytokines are required for the development of airway eosinophilia and immunoglobulin E (IgE) production. Despite the fact that particular leukocytes and mediators have been implicated as causative agents in asthma, the mechanisms responsible for the initiation and maintenance of allergic inflammation remain poorly defined. Animal models, including guinea pigs, monkeys, rats, and mice, have been used to study the pathogenesis of asthma. Mouse models of allergic lung disease 263 C 2001 by Academic Press Copyright  All rights of reproduction in any form reserved. 0065-2776/01 $35.00

264

LLOYD et al.

have been utilized to dissect the complex pathophysiological mechanisms underlying the asthma phenotype. Mice are increasingly attractive for such studies for a number of reasons. The progress in outlining pathways and mediators in murine immunology has been substantive, and a plethora of technologies and reagents to manipulate these pathways have been developed. Moreover, technology has been developed that gives us the ability to manipulate the mouse genome by production of transgenic lines, as well as ablation of specific genes and thus their protein products, through homologous recombination. Therefore, it is possible to dissect inflammatory pathways to investigate the functional roles of particular mediators or cells. A wealth of research activity has shown that mice can be induced to display a range of the pathophysiological features that are hallmarks of the human disease. Mice have been shown to develop inflammatory infiltrates in the lungs, both in peribronchiolar tissues (as shown in lung sections) and in the airway lumen (collected in bronchiolar lavage). Although eosinophils are generally the most prolific cell type within these infiltrates, lymphocytes are also present in significant numbers. Lung sections show, too, that there is an increase in mucus secretion from the bronchoepithelial surface. Analysis of serum reveals that mice show an increase in both total and allergen-specific IgE, as well as increased IgG2a titers. This Th2-type profile is reflected in the cytokines generated within the lungs, IL-4 and IL-5 being present in significantly greater quantities than interferon ␥ (IFN-␥ ). Many investigators have also documented changes in lung function following allergen provocation, using a variety of techniques. The physical properties of the lungs can be assessed after their removal from the host, mice can be anesthetized and attached to instruments to allow recording of airway mechanics, or alternatively, mice can remain unanesthetized and unrestrained while function is measured by plethysmography. These techniques have been used to determine airway hyperreactivity to ␤2 -agonists before and after provocation with allergen. The variety of protocols that have been used to induce pulmonary eosinophilia, bronchial hyperreactivity, and mucus hypersecretion is tremendous. In this chapter, we review the models that have been used, in an attempt to identify the immunological and pathophysiological mechanisms underlying the asthma phenotype. The use of animal models has enabled us to highlight specific pathways and has given us the opportunity to study the function of these pathways in vivo. The challenge is to connect these pathways observed and identified in animal models to the equivalent in human airway disease. A. ACTIVE IMMUNIZATION MODELS Active immunization models rely on the delivery of an antigen to replicate a sensitization and challenge phase, in order to mimic the allergic response to exogenous or innocuous stimuli. This protocol involves preimmunization with the allergen before a sensitization phase in which the allergen is introduced to the target organ, in this case the lungs—intranasally, as an aerosol,

ALLERGIC AIRWAY DISEASE IN MICE

265

or via the trachea. This basic protocol induces a pulmonary eosinophilia, generally in conjunction with an increase in circulating IgE levels. However, there are countless variations to this protocol that have profound consequences for the range of pathophysiological features developed. This method has been used with a variety of antigens and protocols, varying from complex microorganisms to simple proteins and chemicals. Although all of these methods ultimately produce a pulmonary inflammation, we have observed that the type and degree of inflammation are widely variable. In this section, we review the relative merits of different antigens, as well as critical parameters of various immunization protocols. 1. Selection of Antigen Antigens used to initiate pulmonary eosinophilia range from simple protein antigens to complex microorganisms. The immune response to these different allergens is likely to be very different and will affect the ensuing tissue inflammation. For example, complex microorganisms such as parasites will have a much higher number and range of antigenic epitopes available during T cell receptor priming compared to those for a soluble protein antigen. These and other factors are important when comparing the effects of particular antigens in a protocol. a. Complex Microorganisms. i. Fungi. Allergic airway inflammation may occur after sensitization from spores from fungi such as Aspergillus or Candida (Kauffman et al., 1995; Pacheco et al., 1998). Patients may develop an allergic eosinophilia or, in the case of Aspergillus fumigatus, two different forms—as asthma with increased serum IgE titers or hypersensitivity pneumonitis with increased serum IgG and low IgE titers (Kurup and Kumar, 1991). In mice, sensitization and intratracheal challenge with A. fumigatus induce pulmonary eosinophilia, lavage IL-4 and IL-5 production, and increased serum IgE titers in conjunction with heightened AHR (Grunig et al., 1997; Lukacs et al., 1999; Shibuya et al., 1999). ii. Parasites. Due to the specialized structure of the lung, it is a target for the trapping of parasites during phases of the life cycle. Some parasite infections are associated with tropical pulmonary eosinophilia caused when larval stages of the life cycle pass through or get trapped in the lungs. The immunological reaction to the worm (in the case of filarial infection) or the egg (in the case of schistosomal infection) bears some resemblance to the pathophysiological reaction to allergen, and this has been exploited in animal models. iii. Schistosoma models. The immunological response to surface antigens of the Schistosoma mansoni egg stage has been exploited to establish an animal model of some facets of asthma. Intratracheal delivery of parasite antigen to presensitized mice was found to elicit a Th2 response and pulmonary inflammation that resolved after 3–4 days (Lukacs et al., 1994). Soluble egg antigens induce an antigen-specific eosinophil recruitment to the lungs—in conjunction

266

LLOYD et al.

with IL-4, tumor necrosis factor ␣ (TNF-␣), and C–C chemokines macrophage inflammatory protein 1␣ and RANTES—as well as AHR (Lukacs et al., 1996; Padrid et al., 1998; Wynn and Cheever, 1995). This model has been exploited to characterize Th2 and Th1 responses in the lungs. Lung granulomas were elicited by beads coated with purified protein derivative of Mycobacterium bovis to induce a Th1-type response or with soluble egg antigens for a Th2-type response (Chensue et al., 1995). The type 1 granuloma was composed mostly of mononuclear cells and is largely dependent on IFN-␥ and TNF-␣, whereas the type 2 granuloma is eosinophil rich and is largely dependent on IL-4. This model has proved useful in determining the role of cytokines and chemokines in granuloma formation (Chensue et al., 1997; Lukacs et al., 1996). iv. Worm models. Adult filarial parasites reside in the lymphatic organs of the host and chronically release large numbers of larvae into the bloodstream, some of which become trapped in the lungs. In humans, this can lead to a pulmonary eosinophilia associated with symptoms that resemble the pathophysiological features of chronic asthma (Ong and Doyle, 1998; Ottesen and Nutman, 1992). Mouse models have been used to try to replicate this condition, and a variety of studies have shown that infection with worms such as Nippostrongylus, Angiostrongylus and Necator, Trichinella, Taenia, and Trichuris leads to eosinophilic lung inflammation (Hall et al., 1998; Wilkinson et al., 1990). In some cases, this is also associated with an increase in bronchial hyperreactivity and a rise in Th2-type cytokines. b. Protein Antigens. Soluble protein antigens are widely used to elicit allergic pulmonary inflammation and range from simple proteins such as ovalbumin (OVA) to complex, environmentally relevant antigens such as cockroach or house dust mite proteins. The immune response to these antigens is much more controlled and reproducible, since a defined amount of antigen can be delivered at a particular site. Thus, it is perhaps easier to establish a more stable model than if using an intact biological organism. The most commonly used protein antigen is chicken egg OVA, the use of which models late-phase events such as eosinophilia, and in some protocols, AHR in vivo. OVA is an important human allergen and has the advantage of reliably inducing in mice antigen-specific IgE responses that are largely dependent on IL-4. The majority of investigators have found that sensitization and subsequent challenge with OVA result in a significant increase in the number of eosinophils and lymphocytes to both the peribronchiolar tissue and the BAL. These increases occur in conjunction with a significant increase in levels of Th2-type cytokines (IL-4 and IL-5) as well as serum IgE levels. The use of this type of model by a range of investigators has been successful in defining the role that individual cells and immune pathways play in mediating the eosinophilic response. A comparison of several of the models currently in use in the literature

ALLERGIC AIRWAY DISEASE IN MICE

267

using OVA as the allergen is depicted in Table I. Although all of the protocols use the same allergen, they vary in terms of route of administration, the use of adjuvant, and the frequency of challenge. These variations give rise to very different pathophysiological symptoms. Other investigators have tried to establish models using more environmentally relevant antigens, such as those proteins derived from cockroaches or house dust mites. Sensitivity to cockroach antigen is a common problem in inner cities and for those living in crowded, lower socioeconomic areas (Rosenstreich et al., 1997). A model has been established to determine the specific responses associated with sensitivity to cockroach antigens. In this model, the allergic responses to intratracheal cockroach antigens in sensitized mice included allergen-specific airway eosinophilia and significantly altered airway physiology concomitant with eosinophilic airway inflammation (Campbell et al., 1998, 1999). Similarly, sensitization and challenge of mice with the house dust mite Dermatophagoides farinae have been found to elicit pulmonary inflammation (Coyle et al., 1996b; Yu et al., 1996; Yasue et al., 1998). Intranasal challenge with D. farinae in previously sensitized mice induces pulmonary edema, inflammatory cell recruitment to the lungs, eosinophilia, production of cytokines, and AHR (Yu et al., 1996). This eosinophilia was also found to be CD4+ T cell dependent. c. Chemical Compounds. A model of industrial asthma has also been established using toluene diisothiocyanate (TDI), a low-molecular-weight compound known to cause occupational asthma in 5–10% of exposed workers. Mice sensitized subcutaneously and challenged intranasally with TDI show tracheal hyperreactivity to carbachol. This tracheal hyperreactivity was found to be lymphocyte dependent but IgE independent and was not associated with leukocyte infiltration of the airways (Scheerens et al., 1996). Exposure over a prolonged period elicited TDI-specific IgE antibodies and in vivo AHR (Scheerens et al., 1999). 2. Parameters of Immunization Protocol a. Route of Immunization. There is a wealth of data to show that sensitization/challenge models reproduce facets of the human asthmatic condition; however, subtle differences in the basic protocol can have drastic effects on the development of pathophysiology, and the interpretation of results becomes critical. This is especially important when inhibitory reagents or genetically modified animals are used to outline the potential functional importance of selected molecules. Of particular importance in this context is the choice or route of administration of antigen for either sensitization or challenge. The basic active immunization protocol relies on a sensitization phase to induce peripheral priming of the immune system followed by antigenic challenge directly to the target organ. In the case of pulmonary inflammation, the lungs can be targeted by intranasal inoculation, aerosolization of the antigen, or installation by intratracheal

268

269

270

LLOYD et al.

injection. This route of antigen administration and the combination of challenges have been shown to be critical to the final pathological outcome. In an analysis of the role of systemic versus local administration of OVA in inducing pulmonary allergic responses, Zhang and colleagues (1997) found that a combination of systemic and local exposure to OVA resulted in a maximal and reproducible induction of responses. These responses included AHR in vivo, production of allergen-specific IgE, peri-airway infiltration with eosinophils and their appearance in BAL, and increased expression of Th2 cytokines in the local lymphoid. A protocol using subcutaneous sensitization with alum-precipitated OVA combined with multiple intranasal doses of OVA in normal saline is also effective in inducing AHR, eosinophilic inflammation of the lung parenchyma, and increased total IgE (Eum et al., 1995). One report suggests that the site of antigen delivery is critical, in that the pulmonary environment promotes preferential Th2-type differentiation (Constant et al., 2000). In this study, an antigen/mouse combination was used that, in almost all conditions of immunization previously examined, is strongly biased toward priming for Th1 CD4+ T cells. However, administration of Leishmania major parasites to B6 mice via the intranasal route preferentially induced a Th2-dominated response. These included an influx of lymphocytes and eosinophils into alveoli, as well as the induction of Th2-type foci of inflammation around pulmonary blood vessels and airways. In addition, high levels of Th2-associated cytokines (IL-4 and IL-5) were generated when lung-draining lymph node and tissue cells were restimulated with L. major lysate. Although this study demonstrated that the lung environment favors Th differentiation using antigen given solely via the airways, we and others (Stampfli et al., 1998; Zhang et al., 1997) have found that mice given OVA solely by the intranasal route produced neither local responses (e.g., AHR or BAL eosinophilia) nor systemic responses (e.g., plasma IgE). These results contrast with those of a study in which OVA delivered repeatedly by nebulization generated circulating levels of IgE and increased AHR but not pulmonary eosinophilia (Renz et al., 1992). These differences probably reflect differences in the vehicle, dose, or frequency of allergen challenge and illustrate once more the diverse outcome possible using various protocols. b. Adjuvant. One other major difference in protocols is the use of an adjuvant during the priming phase to boost the immune response to the allergen in use. This seems to be particularly important when using OVA, since this is a relatively simple protein structure and is not particularly antigenic when used alone. Specifically, the use of aluminum compounds (alum) is associated with the induction of Th2 responses (Brewer et al., 1996; Grun and Maurer, 1989). OVA alum was found to induce IL-4 and IL-5 production in the absence of IL-4 signaling in mice deficient in IL-4R␣–and Stat6-deficient mice (Brewer et al., 1999a).

ALLERGIC AIRWAY DISEASE IN MICE

271

c. Dose. A major factor in the elicitation of an effective T cell response is in the dose of antigen used in the priming stages. As shown in Table I, the choice of antigen dose for the sensitization step varies 10-fold. A thorough review of the process of induction of Th1 and Th2 effector T cells described a conflict in findings relating to whether Th1- or Th2-type responses are elicited by high versus low doses of antigen (Constant and Bottomly, 1997). There are reports suggesting that priming with high-dose antigen leads to humoral responses, whereas low-dose antigen precipitates cell-mediated immunity (Parish and Liew, 1972). However, the reverse has also been documented, whereby mice given repeated low doses of antigen develop Th2-like responses with high IgE production (Wang et al., 1996). Constant and Bottomly (1997) concluded that there is no clear-cut conclusion regarding dose and the type of immunity developed but pointed out that the type of antigen used is critical. They remarked that the studies in which low-dose Th1 responses were elicited used parasite antigens, whereas low doses of soluble proteins tended to elicit a Th2-type response. Thus, it may be that the antigen itself can influence the nature of the immune reaction. Antigen dose is also an important issue in the context of immunotherapy. It has been suggested that a suitable immunotherapy for T cell–mediated disease may be immunization with immunodominant T cell epitopes (Yssel et al., 1994). Immunotherapy with suboptimal doses of OVA has been found to down-regulate AHR and BAL eosinophilia with concomitant decreased production of Th2 cytokines (Janssen et al., 1999). However, the same study found that immunotherapy with an immunodominant epitope of OVA aggravates AHR and increases BAL eosinophilia (Janssen et al., 1999). d. Genetic Background. Perhaps one of the most striking differences in the development of a complete set of features of the asthmatic syndrome rests in the genetic background of the mouse strain used. One particular facet of the pulmonary allergic response that seems to be genetically restricted is AHR. Not only does allergen-induced AHR vary among different strains of mouse, but it seems that native AHR is also dependent on the background strain of the mouse, consistent with the hypothesis that AHR is a heritable trait (reviewed in Drazen et al., 1999). Levitt and associates (1990) measured AHR in nine different strains of commonly used laboratory mice and found that the AKR/J and A/J strains showed the greatest degree of airway responsiveness, while the C57BL/6J, SJL/J, and C3H/HeJ strains were the least responsive. Moreover, there was a 6-fold difference in AHR for acetylcholine between the most divergent strains (A/J and C3H/HeJ). Subsequent studies have confirmed these data (Chiba et al., 1995). Further studies have been carried out to determine the genetic variability in AHR as well as cellular and antibody production following antigen challenge in multiple strains of mice. After choosing two strains in which they found widely differing responses to acetylcholine stimulation under naive conditions,

272

LLOYD et al.

Wills-Karp and Ewart (1997) measured responses in A/J and C3H/HeJ mice after antigen challenge. They determined that inbred strains of mice are genetically predisposed to be susceptible (A/J) or resistant (C3H/HeJ) to the bronchoconstrictor effects of cholinergic agonists under inflammatory and noninflammatory conditions. Interestingly, these data conflict with a study by Brewer and colleagues (1999b) that measured pulmonary pathophysiology and serum IgE responses in mice of 12 different inbred strains following allergen challenge. The results of the study showed that the intravenous methacholine dose required to reduce lung conductance by 50% varied by 3-fold depending on the strain used. Moreover, BAL eosinophils ranged from 3% to 91% of total cells, while tissue eosinophilia varied from being not detectable to being widespread and severe. OVA-specific IgE concentrations ranged from