Table Of ContentCytokines and
Β Lymphocytes
edited by
R. E. CALLARD
Institute of Child Health,
London, WC1N1EH
ACADEMIC PRESS
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Copyright © 1990 by
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ISBN 0-12-155145-8
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List of contributors
Mark R. Alderson Immunex Corporation, 51 University Street, Seattle,
Washington 98101, USA.
Richard J. Armitage Immunex Corporation, 51 University Street, Seattle,
Washington 98101, USA.
Jacques Bancherau Schering-Plough (UNICET), Laboratory for Im
munological Research, 27 Chemin des Peupliers, 69570 Dardilly, France.
Jean-Yves Bonnefoy Glaxo Institute for Molecular Biology, 46 Route des
Acacias, 1211 Geneva 24, Switzerland.
Robin E. Callard Department of Immunology, Institute of Child Health,
30 Guildford Street, London WC1N 1EH, UK.
Thierry DeFrance Schering-Plough (UNICET), Laboratory for Immuno
logical Research, 27 Chemin des Peupliers, 69570 Dardilly, France.
D. Mark Estes Department of Microbiology, The University of Texas,
Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Anto
nio, Texas 78284-7758, USA.
Andrew J. H. Gearing British Biotechnology, Watlington Road, Cowley
Road, Oxford, OX4 5LY, UK.
John Gordon Department of Immunology, The Medical School, Vincent
Drive, Birmingham, B15 2TJ, UK.
Kenneth H. Grabstein Immunex Corporation, 51 University Street,
Seattle, Washington 98101, USA.
Margaret Harnett Division of Immunology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK.
Kevin Rigley Department of Immunology, Institute of Child Health, 30
Guildford Street, London, WC1N 1EH, UK.
vii
viii List of contributors
Sergio Romagnani Department of Clinical Immunology and Allergology,
University of Florence, Institute di Clinica, Medica 3, Policclinico di
Careggi, 50134 Firenze, Italy.
Virginia M. Sanders NIEHS, mail-drop Cl-04, PO Box 12233, Research
Triangle Park, North Carolina 27709, USA.
John G. Shields Glaxo Institute for Molecular Biology, 46 Route des
Acacias, 1211 Geneva 24, Switzerland.
Judy M. Teale Department of Microbiology, The University of Texas,
Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Anto
nio, Texas 78284-7758, USA.
Ellen S. Vitetta Department of Microbiology, University of Texas, South
western Medical Center, Dallas, Texas 75235, USA.
1
Introduction
ROBIN E. CALLARD
Aims of this book
It is common in books about cytokines to describe in detail the physico-
chemical and biological properties of each one in turn, rather than discuss
the way in which they interact with a particular physiological system or
tissue. This is not the approach taken here. Instead, the emphasis through
out is on how cytokines, in combination with other activation signals,
regulate Β cell growth and subsequent differentiation into antibody-forming
cells. One chapter is devoted to the physico-chemical properties of the
different cytokines and their receptors, as this information is not available
from any other single reference source. The rest of the chapters deal
individually with Β cell activation, proliferation, differentiation, T-B cell
collaboration, isotype selection, autocrine stimulation, the role of cytokines
in disease, and Β cell assays for cytokines. In each case, responses of both
human and murine Β cells are considered so that both their similarities and
their differences are made clear.
For the most part, only cytokines which have been cloned and fully
characterized are included in this book. This is mainly to avoid the uncer
tainties and ambiguities associated with the less well characterized factors,
but also because now is an appropriate time to summarize the many
discoveries made with recombinant cytokines over the past few years.
A brief historical perspective
The regulation of antibody responses has been a major subject of investi
gation for about a hundred years. During this time, there have been a
number of significant milestones that have led to our current understanding
and point to the direction for future research. Before the discovery of Τ cells
and Β cells in the late 1960s, investigations into the regulation of antibody
CYTOKINES AND Β LYMPHOCYTES Copyright © 1990 by Academic Press, London
ISBN 0-12-155145-8 All rights of reproduction in any form reserved.
2 R. Ε. Callard
responses were mostly concerned with the roles of antigen, antibody, and
antigen-antibody complexes. At this time, the animal was treated largely as
a "black box", with little understanding of the cellular mechanisms under
lying immunoregulation. It was not until the small lymphocyte was un
equivocally shown to be the precursor to antibody-secreting cells (Gowans
and McGregor, 1965; Gowans and Uhr, 1966), and the discovery that Τ cell
collaboration was required for optimal antibody responses by Β cells
(Claman etal., 1966; Miller and Mitchell, 1968; Mitchell and Miller, 1968),
that cellular models of immunoregulation came into fashion. These import
ant findings triggered an explosion of cellular regulation studies during the
1970s, culminating in the extremely complex and sometimes bizarre
network models of interacting Τ cells to account for help and suppression of
antibody responses.
The main achievement of this period was to recognize that immuno
regulation depended on complex cellular interactions. However, the way in
which the participating cells communicated was not known, and in the end
the various models failed because they did not address immunoregulation at
a molecular level. With the realization that Τ cells can exert their influence
on Β cells by defined molecular entities (cytokines and cell surface inter
action molecules), immunoregulation entered the molecular era. In the
space of 10 years, more than a dozen cytokines that regulate Β cell growth
and differentiation have been identified. Although, in some ways, the
complexities of cytokine biology have even exceeded the byzantine nature
of the cellular network theories, the application of cDNA and gene cloning
techniques has allowed precise definition of both the molecules concerned
and their cell-surface receptors. These advances have opened the way to
investigations of the transmembrane signalling events, intracellular bio
chemistry and specific gene regulation, which ultimately determine how any
one Β cell will respond to the combination of molecular signals received at
the cell surface.
Cytokine models of Β cell growth and differentiation
In the past, it has been common to think of Β cell differentiation as a linear
transition from a homogeneous population of resting Β cells to antibody-
secreting cells (Figure 1). According to this model, ligand (antigen or anti-
Ig) binding to surface Ig primes the Β cell for subsequent and discrete phases
of proliferation and maturation in response to specific growth and differen
tiation signals. This simple model has been useful, but is now clearly
inadequate to account for the complexity and diversity of Β cell responses. It
is now known that many different signals in various synergistic and/or
Introduction 3
Linear model of Β cell differentiation
antigen
Ν-
resting Β cell antibody forming cell
Τ cells (subsets)
accessory ceils
cytokines
Figure 1 In early models, Β cell differentiation was represented by a linear
transition from a homogeneous population of Β cells stimulated by antigen into
antibody-forming cells, with each discrete step of proliferation and differen
tiation regulated by Τ cells, accessory cells and cytokines.
Regulation of Β cell responses
• antigen presentation
• clonal expansion
• antibody secretion
• isotype switching
• apoptosis - affinity maturation
• memory
• tolerance
• recirculation
Functional surface molecules • tissue localisation
(Slg, CD20, CD19etc.)
Figure 2 It is now known that Β cells can respond in a number of different ways
as illustrated. Each response is regulated by signals received at the cell surface
from regulatory Τ cells, cytokines, and other ligands binding to functional cell-
surface molecules. Cognate Τ cell interactions are known to involve Τ cell-
receptor recognition of antigen, in association with MHC class II antigens, CD4
binding to non-polymorphic determinants on MHC class II, LFA-1 binding to
ICAM-1/ICAM-2, and CD2 binding to LFA-3. In each case binding seems able to
deliver a specific signal to the Β cell. The other functional cell surface antigens
(CD19, CD20, etc.) are known to be signal transducing molecules, but their
natural ligands are so far unknown.
4 R. E. Callard
inhibitory combinations are received at the Β cell surface from interactions
with Τ cells, cytokines and other ligands (Figure 2). It is also important to
appreciate that Β cell responses are far more diverse and complex than had
previously been recognized. In addition to proliferation and differentiation
into antibody-secreting cells, many other functions—such as antigen presen
tation by Β cells, antibody affinity maturation, generation of memory,
tolerance, re-circulation, and microenvironmental localization—must also
be subject to regulation (Figure 2). When this complexity is taken into
account, it is clear why earlier efforts to understand Β cell regulation in
terms of interacting Τ cell subsets were not very successful.
Cytokines play only a part in these diverse Β cell responses and must not
be considered in isolation. Interactions with other signals delivered by
ligand binding to functional cell-surface antigens are also important for
determining Β cell responses (Valentine et al., 1988; Brown et al., 1989).
Moreover, any one cytokine may have different (enhancing and inhibiting)
effects depending on what other signal the cell has received. For example,
interferon γ (IFN-y) can induce proliferation by Β cells stimulated with anti-
IgM (Romagnani et al., 1986; Defrance et al., 1986), or inhibit Β cell
responses to interleukin 4 (Rabin et al., 1986a). Similarly, interleukin 4
(IL-4) activates Β cells and promotes Β cell proliferation (Paul and Ohara,
1987), but inhibits Β cell responses to IL-2 (Defrance etal., 1988; Llorente et
al, 1989).
Most in vitro techniques for investigating cytokine action on Β cell growth
and differentiation require co-stimulation of Β cells with, for example, anti-
Ig, to polyclonally mimic the action of antigen. The rationale behind this
approach is the idea that activation through surface Ig in combination with Τ
cell-derived cytokines is a model for Τ cell help to antigen-stimulated Β cells.
To my mind there are two major problems with this model.
First, it seems unlikely that the conditions employed to stimulate Β cells
with polyclonal crosslinking ligands (anti-Ig, dextran sulphate, lipopoly-
saccharide, Staphylococcus aureus Cowan I, etc.) in any way reflects the
nature or local concentrations of most Τ cell-dependent antigens presented
to Β cells in vivo. In fact, concentrations of rabbit anti-Ig up to 1,000-fold
less th-a1n required in co-stimulat-io1n assays with Β cell growth factors (10
ng ml compared with 10//g ml ) have been used successfully with rabbit
Ig-specific Τ cell lines to stimulate Β cell immunoglobulin production (Ton-y1
and Parker, 1985). Interestingly, very low doses of anti-Ig (lOOpgml ),
when coupled to dextran, are also able to stimulate Β cell responses
(Brunswick et al., 1988), but without activation of the phosphoinositide
signalling pathway (Brunswick et al., 1989).
Secondly, it is now known that antigen binding to Β cell surface Ig is
Introduction 5
internalized, processed and then re-expressed on the surface in association
with major histocompatibility complex (MHC) class II for presentation to Τ
cells (Gosselin et al., 1988; Lanzavecchia, 1988). Proliferation and differen
tiation occur after this step in response to signals delivered by Τ cells and Τ
cell-derived cytokines (Figure 3). This model is supported by a recent study,
in which co-stimulation of quiescent Β cells with anti-Ig and IL-4 was shown
to prime Β cells to proliferate in response to subsequent stimulation with
immobilized antibodies to MHC class II plus additional cytokines (IL-4,
IL-5 and mixed lymphocyte reaction (MLR) supernatant) (Cambier and
Lehman, 1989). These results suggest that the combination of signals
delivered by antigen (or anti-Ig) and IL-4 may induce quiescent Β cells to
process antigen for presentation to Τ helper cells, rather than stimulate
proliferation and differentiation. In typical co-stimulation assays with high
dose anti-Ig and cytokines, this important antigen-presenting step is not
taken into account.
Activation of Β cells with IL-4 results in increased MHC class II expres
sion (Noëlle et al., 1984; Rousset, 1988), which may enhance cognate
interactions with Τ cells. Surface IgM expression is also greatly increased
(10-fold) on human Β cells activated with IL-4 (Shields et al., 1989). Of
particular interest is the dose of IL-4 required for this effect. Whereas 50%
maximum expression of Β cell surface CD23, and proliferation of Β cells in-1
co-stimulation assays with anti-IgM, was obtained with about 10 units ml
of IL-4, half maximal stimulation of surface IgM expression -w1as obtained
with less than one-tenth of this IL-4 dose (about 0.2 units ml ) (Callard et
al., 1990). This result shows that IL-4 can have a pronounced effect on Β
cells at doses too low for proliferation. Whether activation by low concen
trations of IL-4 enhances Β cell antigen presentation or whether it serves
some other function has yet to be determined.
Cytokines in specific antibody responses
Most of what we know about the action of cytokines on Β cells has come
from studies with polyclonal activators such as anti-Ig, lipopolysaccharide
(LPS), SAC or phorbol myristate acetate (PMA). As a result, very little is
known about the function of cytokines in specific antibody responses. IL-5 is
a Τ cell replacing factor (TRF) for specific antibody responses in mice
(Takatsu et al., 1988) whereas IL-2 (but not IL-5) is a TRF in man (Callard et
al., 1986; Delfraissy et al., 1988). However, IL-2 is only a TRF for
low/medium density Β cells and is unable to restore antibody production by
high density "resting" Β ceils (Callard and Smith, 1988). Overnight incu-
6 R. Ε. Callard
Antigen Presentation Proliferation Differentiation
Figure 3 In antibody responses to Τ cell-dependent antigens, Β cells first
internalize, process, and re-express antigen in association with MHC class II
antigens for presentation to Τ helper cells. This step may be regulated by IL-4.
Subsequently, interaction with Τ cells and Τ cell derived cytokines (IL-2 in
humans) results in proliferation and maturation into specific antibody-forming
cells (AFC).
bation with antigen and Τ cells converted high-density Β cells to IL-2
responders, suggesting that resting Β cells stimulated with T-dependent
antigens must receive a signal from Τ helper cells before they are able to
respond to cytokines (in this case IL-2) and differentiate into antibody
secreting cells.
Immunoglobulin production by human Β cells stimulated with IL-2 and
SAC was recently reported to be mediated by autocrine production of IL-6
(Xia et al., 1989). Similarly, IL-6 has been shown to be essential for
polyclonal Ig secretion in response to pokeweed mitogen (PWM) (Muragu-
chi et al., 1988). In contrast to these findings, we have shown recently that
although IL-6 sometimes enhances specific antibody responses to influenza
virus by human tonsillar lymphocytes, a blocking IL-6 antibody had no
effect showing that IL-6 is not required for this response (Callard et al.,
1990). In the mouse, IL-6 is required for production of specific antibody to
influenza virus by naïv e (unprimed) Β cells but not by primed Β cells
(Hubert et al., 1989), which is consistent with our findings in human
(primed) responses to influenza virus.
In humans at least, IL-2 seems sufficient for Β cell proliferation and
differentiation into antibody-forming cells after initial activation by antigen
and Τ helper cells. This raises important questions about the function of
