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Role of natural and immune IgM antibodies in immune responses
Molecular Immunology 37 (2000) 1141– 1149 www.elsevier.com/locate/molimm
Role of natural and immune IgM antibodies in immune responses Marianne Boes * Department of Pathology, Har6ard Medical School, Building D2, 200 Longwood A6e., Boston, MA 02115, USA
Abstract IgM antibodies constitute the major component of the natural antibodies and is also the first class of antibodies produced during a primary antibody response. The IgM-type antibodies differ from other classes of antibodies in that they are predominantly produced by B1 cells, in the absence of apparent stimulation by specific antigens. In addition, IgM antibodies are mostly encoded by germline V gene segments and have low affinities but broad specificites to both foreign and self structures. New developments regarding the function of both immune IgM antibodies and natural IgM antibodies will be examined here. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Autoimmunity; Bacterial infection; Complement; Immune response; Immunoglobulin M; Viral infection
1. Introduction Interactions between innate immune mechanisms and adaptive immune reactions are now widely viewed as essential for a normal immune response (Fearon and Locksley, 1996; Medzhitov and Janeway, 1997; Carroll and Prodeus, 1998). Most pathogens elicit a humoral immune response that is characterized by an early rise of antigen-specific immunoglobulin (Ig)M, followed by affinity maturation, isotype switching, and the ensuing rise in antigen-specific IgG, IgA and IgE antibodies. The sera of humans and mice also contain ‘natural’ antibodies, which are present prior to the infection. Although the roles of IgM have long been a subject of interest and investigation, their functions had still not been well defined, which changed recently with the generation of mice that completely lack serum IgM.
1.1. Functional properties of IgM antibodies Natural antibodies are mostly of the IgM isotype, and can bind to a particular antigen or pathogen, even if the host has never been exposed to it (Pereira et al., 1986; Avrameas, 1991; Coutinho et al., 1995; Casali and Schettino, 1996). This ‘spontaneously’ produced antibody has also been detected in human cord blood, * Tel.: +1-617-4324789; fax: +1-617-4324775. E-mail address: marianne – [email protected] (M. Boes).
and in ‘antigen-free’ mice (Tlaskalova-Hogenova et al., 1992; Coutinho et al., 1995). Natural antibody appears in the absence of apparent antigenic stimulation, and is secreted by the long-lived, self-renewing B1 subset of B cells (Hamilton et al., 1994). B1 cells differ from the conventional B2 cells by their differentiation during fetal and neonatal development, and their characteristic localization in pleural and peritoneal cavities in the adult (Kantor and Herzenberg, 1993; Hardy and Hayakawa, 1994). Because natural IgM is usually encoded by germline V gene segments, and precursor B cells during early ontogeny lack terminal deoxynucleotidyl transferase activity (Feeney, 1990; Gu et al., 1990), the repertoire of natural antibodies is more restricted than that produced by conventional B cells. A large proportion of the natural antibodies is polyreactive to phylogenetically conserved structures, such as nucleic acids, heat shock proteins, carbohydrates, and phospholipids (Kantor and Herzenberg, 1993; Hardy and Hayakawa, 1994). An additional consequence of the lack of somatic mutations in natural IgM V region segments is that natural IgM tends to have rather low antigen-binding affinities compensated for, to some extent, by the pentameric nature of secreted IgM. Moreover, its multimeric structure makes IgM a strong complement activator; a single bound IgM pentamer can trigger the classical pathway of complement activation and can lyse a red blood cell, while approximately a thousand IgG molecules are
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required to accomplish the same (Cooper, 1985). An immune complex, formed between antigen, IgM and activated complement component C3 (Ag– IgM – C%) can dramatically augment a B cell antibody response (Pepys, 1976; Ahearn and Fearon, 1989; Heyman, 1990). Indeed, the simultaneous administration of antigen and exogenous antigen-specific IgM often (Henry and Jerne, 1968; Heyman et al., 1982; Harte et al., 1983; Lehner et al., 1983; Heyman and Wigzell, 1985), but not always (Klaus, 1979), yields an enhanced antibody response. For the study of IgM under physiological conditions, mice that are specifically deficient in secreted IgM (sIgM) were generated. In both independently generated strains of sIgM-deficient mice, increased B1 cell numbers were reported. B1 cells produce a major portion of sIgM, which indicates a role for IgM in feedback regulation of these cells (Boes et al., 1998a; Ehrenstein et al., 1998).
2. Immediate protection against bacteria and viruses
2.1. Protection by natural IgM to systemic microbial infection Natural IgM displays traits that allow it to bind to invading pathogens as they enter and results in complement activation as a first line of defense: it is present naturally, and it displays polyreactivities with high avidities (Tlaskalova-Hogenova et al., 1992; Coutinho et al., 1995; Fearon and Locksley, 1996). The physiological role of natural IgM was investigated in an infectious disease model that resembles a clinical situation of systemic microbial infection, the cecal ligation and puncture (CLP) model (Wichterman et al., 1980). Mice deficient in sIgM show 70% mortality at 32 h post-CLP, compared to 20% mortality in wild-type control mice. This increased susceptibility is associated with reduced levels of TNFa, decreased neutrophil recruitment and increased bacterial load in the peritoneum. Elevated levels of LPS and proinflammatory cytokines were detected in the circulation, a situation characteristic of sepsis. Moreover, resistance to CLP was fully restored by reconstitution with polyclonal IgM purified from normal mouse serum (Boes et al., 1998b). These observations are most consistent with a role of natural IgM in local confinement of the infection.
2.2. A monoclonal IgM antibody, but not IL-6 induced acute phase reactants protects against systemic microbial infection Natural IgM is required for the protection against systemic bacterial infection, as shown in experiments in
which sIgM-deficient mice were subjected to the CLP model and in other infectious models (Ochsenbein et al., 1999). Interleukin 6 (IL-6) is a major inflammatory cytokine that induces the production of acute phase reactants, such as C-reactive protein (Hirano et al., 1990; Baumann and Gauldie, 1994; Steel and Whitehead, 1994). Similar levels of serum IL-6 in both sIgMdeficient and wild-type mice were found after CLP. Thus, natural IgM appears to have unique functions in the immediate defense to bacterial infection that cannot be compensated for by acute phase reactants induced by IL-6 (Boes et al., 1998b). Monoclonal IgM antibodies can also protect against systemic infection. An obvious candidate was found in IgM specific to phosphatidylcholine (PtC). Anti-PtC IgM is produced by 5–15% of murine B1 cells (Mercolino et al., 1988). PtC is a membrane phospholipid that is ubiquitous in bacterial and mammalian membranes, and is exposed after treatment of murine red blood cells with the protease bromelain (Hayakawa et al., 1984; Mercolino et al., 1988; Hardy et al., 1989). Treatment with an anti-PtC monoclonal IgM proved protective to CLP. In contrast, a second monoclonal IgM directed against phosphorylcholine (PC) did not enhance resistance to CLP. The resistance conferred by anti-PtC IgM, while evident, appeared less effective than polyclonal IgM, probably because polyclonal IgM recognizes many different antigenic determinants. That anti-PtC IgM had a clear protective effect in the CLP model suggests that this natural IgM antibody is involved in the immediate defense against bacterial infection under physiological conditions (Boes et al., 1998b).
2.3. Protection by natural IgM to listeria, 6acciniaand 6esicular stomatitis 6irus infection The notion that natural IgM is essential for immediate protection against pathogens was corroborated by Ochsenbein et al. (1999). Sera from unimmunized mice contain natural IgM antibodies specific for lymphocytic choriomeningitis virus (LCMV), Listeria monocytogenes, vaccinia virus (vacc-WR), and vesicular stomatitis virus (VSV), but such sera lack specific IgG. Injection of antibody-deficient mice (mMT and RAG1 −/−) with Listeria, vacc-WR or VSV, indicated a protective role for IgM in early trapping of viral and bacterial particles in the spleen. Natural IgM thus protects against dissemination to avoid infection of vital target organs. By directing the virus to secondary lymphoid organs, IgM may stimulate the clearance of infection by at least two mechanisms. First, the confinement of IgM-containing immune complexes to lymph nodes and spleen may stimulate their uptake by the macrophages that reside there. Second, guiding the virus to lymphatic organs is expected to stimulate antigen-trapping, for example on follicular dendritic cells and thereby enhance immune responses.
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2.4. Protection by natural and antigen-induced IgM to influenza infection The roles of natural and immune IgM antibodies in immune protection from viral infection are more controversial. Although protection against virus infection is generally considered to be mediated by CD8 T cells, in a recent paper. Baumgarth et al. clearly show that IgM antibody provides protection. In the absence of sIgM, mice showed increased viral loads in the lungs, and produced significantly reduced levels of virus-specific IgG1 and IgG2a. Since IgG2a is the predominant IgG isotype produced in response to virus infection (Coutelier et al., 1987; Hocart et al., 1988), sIgM may protect against influenza virus infection by promoting an efficient neutralizing IgG response (Baumgarth et al., 2000). The possibly individual protective roles of natural IgM and infection-induced IgM were studied separately in influenza virus infection. Survival rates and antibody responses following infection were analyzed in both sIgM− /− mice and in irradiation chimeras that lack sIgM from either B1 or B2 cells. IgM from B1 and B2 cells protects against influenza virus infection, but only when both are present. In the absence of natural IgM, very few anti-viral antibodies are present when infection first occurs. Although a B2 antibody response is induced, it may not be induced sufficiently rapidly to confer full protection. Thus, both types of IgM appear to be necessary for optimal protection against the virus infection (Baumgarth et al., 2000).
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Most of the binding of antigen on FDC occurs via Fc receptors, which bind IgG, or via complement receptors (CD21/CD35 in mice), which bind activated complement C3 fragments. Immune complexes that consist of IgG coupled to antigen are readily bound to FDC through the Fc receptor. IgM– antigen complexes induce deposition of activated C3 on the immune complex and formation of Ag–IgM– C% complex. Accordingly, binding of IgM-containing immune complexes on FDC most likely occurs through binding to CD21/CD35.
3.2. Role of complement receptors in IgM-stimulated IgG responses The complement-dependent ability of IgM to stimulate IgG responses was studied in mice deficient in CD21/CD35. In these complement receptor-deficient mice, antigen-specific IgM, when administered together with antigen, was unable to stimulate IgG responses. IgG has a dual immunoregulatory role: it stimulates antibody responses when administered with soluble antigen and suppresses them when administered with particulate antigen. When mice deficient in CD21/CD35 were treated with immune complexes composed of soluble antigen-specific IgG and antigen, IgG responses were strongly enhanced (Applequist et al., 2000). Thus, CD21/CD35 are needed for IgM-, but not IgG-mediated stimulation of antibody responses.
4. Role of IgM in development of autoreactive IgG and autoimmune disease 3. Role of sIgM in enhancing ensuing IgG responses
3.1. Stimulation of antigen-dri6en IgG responses by IgM antibodies To determine unequivocally the role of endogenous IgM in enhancing an antibody response, sIgM-deficient mice were immunized and the production of antigenspecific IgG was measured. sIgM-deficient mice produced significantly less antigen-specific IgG when immunized with suboptimal doses of a T-cell dependent antigen, (4-hydroxyl-3-nitrophenyl) acetyl-keyhole limpet hemocyanin (NP-KLH). The affinity of the IgG produced was generally lower than that seen in control mice (Boes et al., 1998a; Ehrenstein et al., 1998). The number and size of germinal centers were reduced in mutant mice as compared to wild-type mice. Moreover, antigen bound to follicular dendritic cells (FDC) was clearly detectable in spleen sections of control mice after immunization, whereas no antigen trapping was detected on FDC in spleen sections of similarly immunized mutant mice (Boes et al., 1998a).
4.1. Relationship between IgM antibodies and the de6elopment of autoimmunity Immune complexes may also enhance an autoantibody response, if the bound antigen in the complex is self-derived. The same mechanisms by which immune complexes of antigen, IgM and complement (Ag–IgM– C%) stimulate IgG responses to foreign antigens are likely to be responsible for autoantibody production. Autoreactive B cells are normally present in healthy individuals (Goodnow, 1992; Cornall et al., 1995), and a high proportion of natural IgM is self-reactive (Casali and Notkins, 1989; Kantor and Herzenberg, 1993; Hardy and Hayakawa, 1994). One would expect that the binding of autoreactive IgM to a self-antigen could also result in the activation of complement and the formation of Ag–IgM–C% complex. This immune complex, then, augments the autoantibody response. Consistent with this notion, rheumatoid diseases are often associated with elevated levels of autoantibodies (Smith and Steinberg, 1983; Naparstek and Plotz, 1993). For example, autoantibodies specific for DNA, ribonucle-
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oprotein and the Smith antigen are widely present in patients with systemic lupus erythematosus (SLE) and Sjo¨ gren’s syndrome. The onset of autoimmune diseases correlates with a switch from production of IgM to IgG autoantibodies (Naparstek and Plotz, 1993). Also, the introduction of monoclonal IgG antibody specific to DNA into normal mice, by direct injection of purified antibodies, by implantation of antibody-secreting hybridomas, or by expression from immunoglobulin transgenes, induces lupus-like glomerulonephritis (Tsao et al., 1992; Ohnishi et al., 1994; Ehrenstein et al., 1995; Radic et al., 1995). Still, the exact role of autoreactive IgM in the subsequent autoantibody response and the switch to other Ig isotypes is not known.
4.2. Autoimmune disease models employing sIgM-deficient mice In humans, IgA deficiency is relatively common and is associated with autoimmunity (Liblau and Bach, 1992; Rankin and Isenberg, 1997). It has been postulated that disease might arise as a consequence of the increased number of infections suffered by IgA-deficient patients. A similar logic should apply to the IgM-deficient mice, since they exhibit enhanced sensitivity to microbial challenge (Boes et al., 1998b). The possibility for such a role for IgM antibody in the development of IgG autoantibodies was addressed in two studies (Boes et al., 2000; Ehrenstein et al., 2000). Ehrenstein et al. examined this hypothesis by injecting LPS into sIgMdeficient mice and control mice. Previously, such treatment was shown to stimulate not only serum IgG but also anti-dsDNA antibodies and evoked an earlier onset of autoimmune disease in MLR/lpr mice (Cavallo and Granholm, 1990). Indeed, only in sIgM-deficient mice did repeated injection of bacterial LPS stimulate the production of anti-dsDNA IgG. Therefore, the absence of IgM antibody with its attendant sensitivity to microbial infection may perhaps predispose to autoimmunity (Ehrenstein et al., 2000). The role of IgM antibody in the development of autoimmunity was also investigated by breeding sIgMdeficient mice with lupus-prone lpr mice (Boes et al., 2000). Lpr mice spontaneously develop high levels of anti-dsDNA antibodies and glomerulonephritis (Theofilopoulos and Dixon, 1985). A defective fas (CD95) gene impairs the ability of lpr T cells to undergo activation-induced cell death and prevents proper maintenance of peripheral tolerance (Watanabe-Fukunaga et al., 1992; Singer and Abbas, 1994; Elkon and Marshak-Rothstein, 1996). Compared to regular lpr mice, lpr mice that lack sIgM developed elevated levels of IgG autoantibodies to dsDNA and histones, and had more abundant deposits of immune complexes in the glomeruli. Disease was more severe, and the mice died at an earlier age. Moreover, sIgM-deficient mice on a
normal background developed anti-DNA IgG with age, which was not the case in wild-type mice (Boes et al., 2000; Ehrenstein et al., 2000). Together, these findings suggest that sIgM, including IgM autoantibodies produced naturally or as part of an autoimmune response, may lessen the severity of autoimmune pathology associated with IgG autoantibodies.
4.3. Protection by IgM antibodies to autoimmune disease How does IgM antibody protect against the development of autoreactive IgG and autoimmune disease? The clearance of immune complexes from the circulation may be enhanced through complement-mediated processes. In the course of apoptosis, nuclear components are translocated into membranous blebs (CasciolaRosen et al., 1994). Some of the IgM autoantibodies, such as those reactive to DNA, histones and phosphatidylserine, may bind to self-antigens in the blebs, activate complement, and thereby promote their removal. Alternatively, natural IgM may have a role in protection against development of autoimmunity early in B cell development. Natural IgM may bind non-infectious self-antigens, increase the exposure of immature B cells to these self-antigens and thus induce tolerance. The first mechanism was investigated directly by treating lpr/sIgM-deficient mice with monoclonal IgM specific for dsDNA or histones (Boes et al., 2000). If autoreactive IgM were to protect against the later development of autoimmunity, lower levels of autoreactive IgG would likely be found. Compared to salinetreated mice, IgM-treated lpr/sIgM-deficient mice produced significantly higher levels of IgG1 specific for dsDNA. This result reflects a third possibility for the fate of immune complexes composed of Ag–IgM–C%. Immune complexes are more effective in activating B cells by cross-linking BCR and coreceptors than antigens alone. The timing of exposure either early or late in development may determine which scenario applies (see below). Also, the overall levels and affinity of autoreactive IgM, and/or its local concentration may determine whether IgM may stimulate clearance of self-antigens from the circulation, or activate autoreactive B cells by cross-linking of the BCR and co-receptors. The latter possibility is analogous to the antigen dose-dependent enhancing effect of IgM and complement on an IgG antibody response (Ahearn et al., 1996; Fischer et al., 1996; Boes et al., 1998a).
4.4. Protection by complement component C1q to anti-nuclear autoimmunity Similar to sIgM-deficient mice, mice deficient in the C1q component of complement are predisposed to anti-
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nuclear autoimmunity (Botto et al., 1998; Mitchell et al., 1999). Deficiency in C1q is associated with glomerulonephritis and antinuclear and antihistone antibodies, but not with increases in antibodies against chromatin or DNA. Moreover, C1q-deficient mice accumulate multiple apoptotic bodies in their normal glomeruli, which is not the case in sIgM-deficient mice. To assess the contribution of C3 activation to the development of glomerulonephritis, C1q−/ − mice were crossed with complement factor B- and C2-deficient mice. Factor B is part of the alternative pathway, and C2 is part of the classical pathway of complement; Factor B and C2 act upstream of C3 in the alternative and classical cascade respectively. Sixty-four per cent of these triple deficient mice showed glomerulonephritis, compared to only 8% of the factor B/C2-deficient mice. Therefore, C1q may have a protective role against the development of glomerulonephritis. This role is independent of C3 activation, and thus could help clear apoptotic cells without the necessity for C3 activation. Finally, these results demonstrated that lupus-like disease is independent of C3 activation (Mitchell et al., 1999).
4.5. Protection by IgM against the de6elopment of autoimmunity The sIgM-deficient mice develop severe glomerulonephritis only when injected with LPS (Ehrenstein et al., 2000) or after crossing with lpr mice (Boes et al., 2000). Mice deficient in complement receptors CD21/ CD35 have a similar autoimmune phenotype (Prodeus et al., 1998). Binding of IgM antibody to antigen leads to activation of C1q, and the resulting immune complexes can be cleared by C1q independently of C3 (Mitchell et al., 1999). Moreover, human patients deficient in C1q or C4 develop lupus disease and anti-dsDNA antibodies, and mice deficient in C4 also spontaneously develop anti-dsDNA autoantibodies (E. Paul and M.C. Carroll, personal communication). Therefore, it is likely that efficient clearance of self-antigen containing immune complexes requires IgM, C1q as well as C4. This hypothesis is corroborated by the fact that complement receptors CD21/CD35 bind C1q, C4b and C3 in human and C3b, C3d and C4b in mouse. Alternatively, a role for natural IgM antibodies in maintaining tolerance may be exerted earlier in B cell development, by promoting the exposure of self-antigens to immature, bone marrow resident B cells. A similar role for CD21/CD35 in negative selection of autoreactive B cells has been suggested (Prodeus et al., 1998). Certainly, many natural IgM antibodies are cross-reactive to self-antigens and may bind to these self-antigens within the bone marrow. A role for immune complexes (Ag–IgM – C1q or Ag – IgM – C4) may therefore be to induce tolerance in immature B cells.
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5. Cloning of an Fc receptor that recognizes IgM
5.1. A murine Fch/v receptor Fc receptors have long been regarded as mediators for the interaction of antibody–antigen complexes with cells, resulting in a wide range of immune responses such as phagocytosis, antibody-dependent cytotoxicity, antigen presentation and the production and secretion of many cytokines and chemokines (Daeron, 1997; Ravetch, 1997; Ravetch and Clynes, 1998). With the exception of the polyimmunoglobulin receptor (pIgR), which transports IgM and IgA across epithelia, Fc receptors have been described for IgG and IgE, but not for other Ig isotypes. A screening effort using COS-7 cells transfected with a cDNA library and the use of a monoclonal IgM antibody as a probe have resulted in the characterization of a novel Fca/m receptor (Shibuya et al., 2000). The murine Fca/m receptor is a 70 kDa glycoprotein expressed on B cells and macrophages, but not on granulocytes, T cells or NK cells. The receptor contains a di-leucine motif in its cytoplasmic domain, and mediates endocytosis of IgM-coated beads. Mouse spleen cells incubated with immune complexes composed of FITC-labeled and IgM or IgG- opsonized S. aureus clearly showed endocytosis by B cells. In contrast, mouse spleen cells preincubated with rat anti-mouse Fca/m receptor antibody were unable to endocytose FITC-labeled S. aureus bacteria coated with IgM or IgG antibodies to S. aureus. Thus, the Fca/m receptor appears to mediate endocytosis of IgM-coated microbial pathogens. Moreover, these results indicate that the Fca/m receptor may be involved in protection against bacterial infection by stimulation of phagocytosis, as well as in stimulating antigen processing and presentation for presentation to T helper cells for induction of adaptive immune responses (Shibuya et al., 2000).
6. Synergy between IgM and complement in immune responses
6.1. Immediate protection by IgM and complement to systemic bacterial infection The susceptibility of sIgM-deficient mice to CLP is similar to that of mice lacking mast cells or that are deficient in complement components C3 or C4 (Echtenacher et al., 1996; Malaviya et al., 1996; Prodeus et al., 1997). In the CLP model, mice deficient in complement components C3 or C4 showed 100% mortality at 24 h, compared to less than 25% mortality in wild-type control mice. Because similar results were obtained using C3- and sIgM-deficient mice in CLP-induced infection, part of the protection by IgM is probably afforded
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through a complement-mediated mechanism. Protection by complement is conferred at several levels. Activation of the complement cascade leads to assembly of the C5b –C9 membrane attack complex, which lyses bacteria directly. The smaller cleavage products C3a and C5a function as chemoattractants that recruit leukocytes to the site of infection. Opsonization of bacteria by C3b and iC3b promotes efficient phagocytosis by neutrophils. In addition, activation of complement stimulates efficient activation of mast cells to release TNFa for the initiation of a local inflammatory response (Prodeus et al., 1997). Attachment of multiple copies of C3d to antigen significantly reduces the amount of soluble antigen required to induce a humoral immune response (Dempsey et al., 1996). Moreover, analysis of the humoral immune response of mice deficient in complement component C3 or C4 (Ahearn et al., 1996; Croix et al., 1996; Molina et al., 1996) showed that the humoral immune response is greatly enhanced by the coupling of complement to antigen. It is now firmly established that Ag– IgM – C% immune complexes can augment adaptive responses through two common pathways. First, immune complexes can cross-link B cell antigen– receptor (BCR) and CD19/CD21 complex and reduce the threshold dose of antigen required to activate B cells (Carter and Fearon, 1992; Rickert et al., 1995; Sato et al., 1995; Dempsey et al., 1996). Second, immune complexes are more efficiently trapped on FDC through the expressed complement receptors (CD21 and CD35 in mice), which leads to an effective germinal center reaction during the T-cell dependent antibody response (Pepys, 1976; Ahearn and Fearon, 1989; Szakal et al., 1989).
6.2. Role of complement receptors on either B cells or FDC in enhancing IgG responses In mice, the expression of CD21/CD35 is limited to B cells and FDC (Holers et al., 1992). Lethal irradiation of mice depletes all B cells, but not FDC. In addition, FDC are long-lived and will probably not be replaced by donor cells following bone marrow transfer (Humphrey et al., 1984). These insights were used to compare the roles of CD21/CD35 expressed on B cells versus FDC during an antigen-specific IgG response, by immunization experiments of bone marrow chimeric mice (Fang et al., 1998). For each of two T-cell dependent antigens, expression of CD21/CD35 on both B cells and FDC was necessary to restore the normal phenotype. Mice deficient in CD21/CD35 on B cells had a diminished, but still detectable, IgG response, and germinal centers were comparable to controls. In contrast, mice deficient in CD21/CD35 on FDC alone had a marked decrease in the production of antigenspecific IgG, and germinal centers were smaller in size
compared to normal controls. These results indicate an important role for CD21/CD35 on FDC cells and, to lesser extent, B cells, in stimulating efficient IgG responses. Croix et al. had previously shown that expression of CD21/CD35 on B cells is necessary to generate significant IgG responses to T-cell-dependent antigen, by use of mice generated by injection of homozygous Cr2 -deficient ES cells into RAG2-deficient blastocysts (Croix et al., 1996). In addition, Ahearn et al. reported that reconstitution of Cr2 -deficient mice with wild-type bone marrow resulted in normal IgG responses to T-cell-dependent antigen (Ahearn et al., 1996). Thus, these findings indicate an important role for B cells rather than FDC in generating well-organized IgG responses. These seemingly contradictory findings may be attributable to the use of different antigens, at different dosages. The specific requirement for immune complexes sequestered by FDC was investigated in a transgenic approach (Hannum et al., 2000), in which all B cells produce the membrane-bound, but not the secreted, form of NP-specific IgM. After immunization, the transgenic mice produced virtually no antigen-specific antibody and expressed almost no immune complexes on FDC. Surprisingly, although there was practically no antibody to form immune complexes, germinal center reactions did take place and had an enlarged size and normal frequency and kinetics. Moreover, somatic mutations of the Vl region were found predominantly in the complementarity-determining regions (CDR), which suggests antigen-driven selection in germinal centers (Hannum et al., 2000). Finally, when the transgenic mice were immunized with NP, antigen-specific B cells comprised a large percentage of the splenocytes. Thus, there is no consensus yet about the contribution of immune complex deposition on FDC in enhancing antigen-driven IgG responses. The use of different protocols to determine the involvement of FDC in immune responses may explain the seemingly contradictory observations obtained by the separate groups. Also, antigen trapping can occur in the absence of natural antibodies by acute phase reactants such as C-reactive protein that are thought to have similar functions as natural antibodies (Hirano et al., 1990; Baumann and Gauldie, 1994; Steel and Whitehead, 1994). A reason for not detecting immune complexes that may still be present on FDC could be the large number of antigen-specific B cells that all compete for binding, and thereby may obscure immune complexes during antibody staining. For now, the main message remains that expression of CD21/CD35 is necessary on both B cells and probably FDC, at different time points during a humoral immune response. Initially, cross-linking of BCR and CD19/CD21 coreceptor by immune complexes (Ag–
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IgM – C%) stimulates the intracellular signals leading to B cell activation. Later in the course of a primary immune response, as some antigen-primed B cells migrate to the germinal center, they proliferate and undergo hypermutation and Ig class switching (Kelsoe, 1996). In the germinal center, FDC display immune complexes bound to CD21/CD35 and Fc receptors for presentation to antigen-experienced B cells. Finally, those B cells that express Ig of increased affinity for the antigen, proliferate and mature into plasma cells.
Acknowledgements I thank Drs Jianzhu Chen, Hidde Ploegh and Michael Carroll for critical reading of the manuscript and for helpful discussions.
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Medzhitov, R., Janeway, C.A. Jr., 1997. Innate immunity: the virtues of a nonclonal system of recognition. Cell 91, 295 – 298. Mercolino, T.J., Arnold, L.W., Hawkins, L.A., Haughton, G., 1988. Normal mouse peritoneum contains a large population of Ly-1 + (CD5) B cells that recognize phosphatidyl choline. Relationship to cells that secrete hemolytic antibody specific for autologous erythrocytes. J. Exp. Med. 168, 687 – 698. Mitchell, D.A., Taylor, P.R., Cook, H.T., Moss, J., Bygrave, A.E., Walport, M.J., Botto, M., 1999. Cutting edge: C1q protects against the development of glomerulonephritis independently of C3 activation. J. Immunol. 162, 5676 – 5679. Molina, H., Holers, V.M., Li, B., Fung, Y., Mariathasan, S., Goellner, J., Strauss-Schoenberger, J., Karr, R.W., Chaplin, D.D., 1996. Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2. Proc. Natl. Acad. Sci. USA 93, 3357 – 3361. Naparstek, Y., Plotz, P.H., 1993. The role of autoantibodies in autoimmune disease. Annu. Rev. Immunol. 11, 79 – 104. Ochsenbein, A.F., Fehr, T., Lutz, C., Suter, M., Brombacher, F., Hengartner, H., Zinkernagel, R.M., 1999. Control of early viral and bacterial distribution and disease by natural antibodies. Science 286, 2156 – 2159. Ohnishi, K., Ebling, F.M., Mitchell, B., Singh, R.R., Hahn, B.H., Tsao, B.P., 1994. Comparison of pathogenic and non-pathogenic murine antibodies to DNA: antigen binding and structural characteristics. Int. Immunol. 6, 817 – 830. Pepys, M.B., 1976. Role of complement in the induction of immunological responses. Transplant Rev. 32, 93 – 120. Pereira, P., Forni, L., Larsson, E.L., Cooper, M., Heusser, C., Coutinho, A., 1986. Autonomous activation of B and T cells in antigen-free mice. Eur. J. Immunol. 16, 685 – 688. Prodeus, A.P., Goerg, S., Shen, L.M., Pozdnyakova, O.O., Chu, L., Alicot, E.M., Goodnow, C.C., Carroll, M.C., 1998. A critical role for complement in maintenance of self-tolerance. Immunity 9, 721 – 731. Prodeus, A.P., Zhou, X., Maurer, M., Galli, S.J., Carroll, M.C., 1997. Impaired mast cell-dependent natural immunity in complement C3-deficient mice. Nature 390, 172 – 175. Radic, M.Z., Ibrahim, S.M., Rauch, J., Camper, S.A., Weigert, M., 1995. Constitutive secretion of transgene-encoded IgG2b autoantibodies leads to symptoms of autoimmune disease. J. Immunol. 155, 3213 – 3222. Rankin, E.C., Isenberg, D.A., 1997. IgA deficiency and SLE: prevalence in a clinic population and a review of the literature. Lupus 6, 390 – 394. Ravetch, J.V., 1997. Fc receptors. Curr. Opin. Immunol. 9, 121 –125. Ravetch, J.V., Clynes, R.A., 1998. Divergent roles for Fc receptors and complement in vivo. Annu. Rev. Immunol. 16, 421 –432. Rickert, R.C., Rajewsky, K., Roes, J., 1995. Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice. Nature 376, 352 – 355. Sato, S., Steeber, D.A., Tedder, T.F., 1995. The CD19 signal transduction molecule is a response regulator of B- lymphocyte differentiation. Proc. Natl. Acad. Sci. USA 92, 11558 – 11562. Shibuya, A., Sakamoto, N., Shimizu, Y., Shibuya, K., Osawa, M., Hiroyama, T., Eyre, H.J., Sutherland, G.R., Endo, Y., Fujita, T., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Phillips, J.H., Lanier, L.L., Nakauchi, H., 2000. Fc/ receptor mediates endocytosis of IgM-coated microbes. Nat. Immunol. 1, 441 –446. Singer, G.G., Abbas, A.K., 1994. The fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. Immunity 1, 365 – 371. Smith, H.R., Steinberg, A.D., 1983. Autoimmunity — a perspective. Annu. Rev. Immunol. 1, 175 – 210. Steel, D.M., Whitehead, A.S., 1994. The major acute phase reactants: C-reactive protein, serum amyloid P component and serum amyloid A protein. Immunol. Today 15, 81 – 88.
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Role of natural and immune IgM antibodies in immune responses Marianne Boes * Department of Pathology, Har6ard Medical School, Building D2, 200 Longwood A6e., Boston, MA 02115, USA
Abstract IgM antibodies constitute the major component of the natural antibodies and is also the first class of antibodies produced during a primary antibody response. The IgM-type antibodies differ from other classes of antibodies in that they are predominantly produced by B1 cells, in the absence of apparent stimulation by specific antigens. In addition, IgM antibodies are mostly encoded by germline V gene segments and have low affinities but broad specificites to both foreign and self structures. New developments regarding the function of both immune IgM antibodies and natural IgM antibodies will be examined here. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Autoimmunity; Bacterial infection; Complement; Immune response; Immunoglobulin M; Viral infection
1. Introduction Interactions between innate immune mechanisms and adaptive immune reactions are now widely viewed as essential for a normal immune response (Fearon and Locksley, 1996; Medzhitov and Janeway, 1997; Carroll and Prodeus, 1998). Most pathogens elicit a humoral immune response that is characterized by an early rise of antigen-specific immunoglobulin (Ig)M, followed by affinity maturation, isotype switching, and the ensuing rise in antigen-specific IgG, IgA and IgE antibodies. The sera of humans and mice also contain ‘natural’ antibodies, which are present prior to the infection. Although the roles of IgM have long been a subject of interest and investigation, their functions had still not been well defined, which changed recently with the generation of mice that completely lack serum IgM.
1.1. Functional properties of IgM antibodies Natural antibodies are mostly of the IgM isotype, and can bind to a particular antigen or pathogen, even if the host has never been exposed to it (Pereira et al., 1986; Avrameas, 1991; Coutinho et al., 1995; Casali and Schettino, 1996). This ‘spontaneously’ produced antibody has also been detected in human cord blood, * Tel.: +1-617-4324789; fax: +1-617-4324775. E-mail address: marianne – [email protected] (M. Boes).
and in ‘antigen-free’ mice (Tlaskalova-Hogenova et al., 1992; Coutinho et al., 1995). Natural antibody appears in the absence of apparent antigenic stimulation, and is secreted by the long-lived, self-renewing B1 subset of B cells (Hamilton et al., 1994). B1 cells differ from the conventional B2 cells by their differentiation during fetal and neonatal development, and their characteristic localization in pleural and peritoneal cavities in the adult (Kantor and Herzenberg, 1993; Hardy and Hayakawa, 1994). Because natural IgM is usually encoded by germline V gene segments, and precursor B cells during early ontogeny lack terminal deoxynucleotidyl transferase activity (Feeney, 1990; Gu et al., 1990), the repertoire of natural antibodies is more restricted than that produced by conventional B cells. A large proportion of the natural antibodies is polyreactive to phylogenetically conserved structures, such as nucleic acids, heat shock proteins, carbohydrates, and phospholipids (Kantor and Herzenberg, 1993; Hardy and Hayakawa, 1994). An additional consequence of the lack of somatic mutations in natural IgM V region segments is that natural IgM tends to have rather low antigen-binding affinities compensated for, to some extent, by the pentameric nature of secreted IgM. Moreover, its multimeric structure makes IgM a strong complement activator; a single bound IgM pentamer can trigger the classical pathway of complement activation and can lyse a red blood cell, while approximately a thousand IgG molecules are
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required to accomplish the same (Cooper, 1985). An immune complex, formed between antigen, IgM and activated complement component C3 (Ag– IgM – C%) can dramatically augment a B cell antibody response (Pepys, 1976; Ahearn and Fearon, 1989; Heyman, 1990). Indeed, the simultaneous administration of antigen and exogenous antigen-specific IgM often (Henry and Jerne, 1968; Heyman et al., 1982; Harte et al., 1983; Lehner et al., 1983; Heyman and Wigzell, 1985), but not always (Klaus, 1979), yields an enhanced antibody response. For the study of IgM under physiological conditions, mice that are specifically deficient in secreted IgM (sIgM) were generated. In both independently generated strains of sIgM-deficient mice, increased B1 cell numbers were reported. B1 cells produce a major portion of sIgM, which indicates a role for IgM in feedback regulation of these cells (Boes et al., 1998a; Ehrenstein et al., 1998).
2. Immediate protection against bacteria and viruses
2.1. Protection by natural IgM to systemic microbial infection Natural IgM displays traits that allow it to bind to invading pathogens as they enter and results in complement activation as a first line of defense: it is present naturally, and it displays polyreactivities with high avidities (Tlaskalova-Hogenova et al., 1992; Coutinho et al., 1995; Fearon and Locksley, 1996). The physiological role of natural IgM was investigated in an infectious disease model that resembles a clinical situation of systemic microbial infection, the cecal ligation and puncture (CLP) model (Wichterman et al., 1980). Mice deficient in sIgM show 70% mortality at 32 h post-CLP, compared to 20% mortality in wild-type control mice. This increased susceptibility is associated with reduced levels of TNFa, decreased neutrophil recruitment and increased bacterial load in the peritoneum. Elevated levels of LPS and proinflammatory cytokines were detected in the circulation, a situation characteristic of sepsis. Moreover, resistance to CLP was fully restored by reconstitution with polyclonal IgM purified from normal mouse serum (Boes et al., 1998b). These observations are most consistent with a role of natural IgM in local confinement of the infection.
2.2. A monoclonal IgM antibody, but not IL-6 induced acute phase reactants protects against systemic microbial infection Natural IgM is required for the protection against systemic bacterial infection, as shown in experiments in
which sIgM-deficient mice were subjected to the CLP model and in other infectious models (Ochsenbein et al., 1999). Interleukin 6 (IL-6) is a major inflammatory cytokine that induces the production of acute phase reactants, such as C-reactive protein (Hirano et al., 1990; Baumann and Gauldie, 1994; Steel and Whitehead, 1994). Similar levels of serum IL-6 in both sIgMdeficient and wild-type mice were found after CLP. Thus, natural IgM appears to have unique functions in the immediate defense to bacterial infection that cannot be compensated for by acute phase reactants induced by IL-6 (Boes et al., 1998b). Monoclonal IgM antibodies can also protect against systemic infection. An obvious candidate was found in IgM specific to phosphatidylcholine (PtC). Anti-PtC IgM is produced by 5–15% of murine B1 cells (Mercolino et al., 1988). PtC is a membrane phospholipid that is ubiquitous in bacterial and mammalian membranes, and is exposed after treatment of murine red blood cells with the protease bromelain (Hayakawa et al., 1984; Mercolino et al., 1988; Hardy et al., 1989). Treatment with an anti-PtC monoclonal IgM proved protective to CLP. In contrast, a second monoclonal IgM directed against phosphorylcholine (PC) did not enhance resistance to CLP. The resistance conferred by anti-PtC IgM, while evident, appeared less effective than polyclonal IgM, probably because polyclonal IgM recognizes many different antigenic determinants. That anti-PtC IgM had a clear protective effect in the CLP model suggests that this natural IgM antibody is involved in the immediate defense against bacterial infection under physiological conditions (Boes et al., 1998b).
2.3. Protection by natural IgM to listeria, 6acciniaand 6esicular stomatitis 6irus infection The notion that natural IgM is essential for immediate protection against pathogens was corroborated by Ochsenbein et al. (1999). Sera from unimmunized mice contain natural IgM antibodies specific for lymphocytic choriomeningitis virus (LCMV), Listeria monocytogenes, vaccinia virus (vacc-WR), and vesicular stomatitis virus (VSV), but such sera lack specific IgG. Injection of antibody-deficient mice (mMT and RAG1 −/−) with Listeria, vacc-WR or VSV, indicated a protective role for IgM in early trapping of viral and bacterial particles in the spleen. Natural IgM thus protects against dissemination to avoid infection of vital target organs. By directing the virus to secondary lymphoid organs, IgM may stimulate the clearance of infection by at least two mechanisms. First, the confinement of IgM-containing immune complexes to lymph nodes and spleen may stimulate their uptake by the macrophages that reside there. Second, guiding the virus to lymphatic organs is expected to stimulate antigen-trapping, for example on follicular dendritic cells and thereby enhance immune responses.
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2.4. Protection by natural and antigen-induced IgM to influenza infection The roles of natural and immune IgM antibodies in immune protection from viral infection are more controversial. Although protection against virus infection is generally considered to be mediated by CD8 T cells, in a recent paper. Baumgarth et al. clearly show that IgM antibody provides protection. In the absence of sIgM, mice showed increased viral loads in the lungs, and produced significantly reduced levels of virus-specific IgG1 and IgG2a. Since IgG2a is the predominant IgG isotype produced in response to virus infection (Coutelier et al., 1987; Hocart et al., 1988), sIgM may protect against influenza virus infection by promoting an efficient neutralizing IgG response (Baumgarth et al., 2000). The possibly individual protective roles of natural IgM and infection-induced IgM were studied separately in influenza virus infection. Survival rates and antibody responses following infection were analyzed in both sIgM− /− mice and in irradiation chimeras that lack sIgM from either B1 or B2 cells. IgM from B1 and B2 cells protects against influenza virus infection, but only when both are present. In the absence of natural IgM, very few anti-viral antibodies are present when infection first occurs. Although a B2 antibody response is induced, it may not be induced sufficiently rapidly to confer full protection. Thus, both types of IgM appear to be necessary for optimal protection against the virus infection (Baumgarth et al., 2000).
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Most of the binding of antigen on FDC occurs via Fc receptors, which bind IgG, or via complement receptors (CD21/CD35 in mice), which bind activated complement C3 fragments. Immune complexes that consist of IgG coupled to antigen are readily bound to FDC through the Fc receptor. IgM– antigen complexes induce deposition of activated C3 on the immune complex and formation of Ag–IgM– C% complex. Accordingly, binding of IgM-containing immune complexes on FDC most likely occurs through binding to CD21/CD35.
3.2. Role of complement receptors in IgM-stimulated IgG responses The complement-dependent ability of IgM to stimulate IgG responses was studied in mice deficient in CD21/CD35. In these complement receptor-deficient mice, antigen-specific IgM, when administered together with antigen, was unable to stimulate IgG responses. IgG has a dual immunoregulatory role: it stimulates antibody responses when administered with soluble antigen and suppresses them when administered with particulate antigen. When mice deficient in CD21/CD35 were treated with immune complexes composed of soluble antigen-specific IgG and antigen, IgG responses were strongly enhanced (Applequist et al., 2000). Thus, CD21/CD35 are needed for IgM-, but not IgG-mediated stimulation of antibody responses.
4. Role of IgM in development of autoreactive IgG and autoimmune disease 3. Role of sIgM in enhancing ensuing IgG responses
3.1. Stimulation of antigen-dri6en IgG responses by IgM antibodies To determine unequivocally the role of endogenous IgM in enhancing an antibody response, sIgM-deficient mice were immunized and the production of antigenspecific IgG was measured. sIgM-deficient mice produced significantly less antigen-specific IgG when immunized with suboptimal doses of a T-cell dependent antigen, (4-hydroxyl-3-nitrophenyl) acetyl-keyhole limpet hemocyanin (NP-KLH). The affinity of the IgG produced was generally lower than that seen in control mice (Boes et al., 1998a; Ehrenstein et al., 1998). The number and size of germinal centers were reduced in mutant mice as compared to wild-type mice. Moreover, antigen bound to follicular dendritic cells (FDC) was clearly detectable in spleen sections of control mice after immunization, whereas no antigen trapping was detected on FDC in spleen sections of similarly immunized mutant mice (Boes et al., 1998a).
4.1. Relationship between IgM antibodies and the de6elopment of autoimmunity Immune complexes may also enhance an autoantibody response, if the bound antigen in the complex is self-derived. The same mechanisms by which immune complexes of antigen, IgM and complement (Ag–IgM– C%) stimulate IgG responses to foreign antigens are likely to be responsible for autoantibody production. Autoreactive B cells are normally present in healthy individuals (Goodnow, 1992; Cornall et al., 1995), and a high proportion of natural IgM is self-reactive (Casali and Notkins, 1989; Kantor and Herzenberg, 1993; Hardy and Hayakawa, 1994). One would expect that the binding of autoreactive IgM to a self-antigen could also result in the activation of complement and the formation of Ag–IgM–C% complex. This immune complex, then, augments the autoantibody response. Consistent with this notion, rheumatoid diseases are often associated with elevated levels of autoantibodies (Smith and Steinberg, 1983; Naparstek and Plotz, 1993). For example, autoantibodies specific for DNA, ribonucle-
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oprotein and the Smith antigen are widely present in patients with systemic lupus erythematosus (SLE) and Sjo¨ gren’s syndrome. The onset of autoimmune diseases correlates with a switch from production of IgM to IgG autoantibodies (Naparstek and Plotz, 1993). Also, the introduction of monoclonal IgG antibody specific to DNA into normal mice, by direct injection of purified antibodies, by implantation of antibody-secreting hybridomas, or by expression from immunoglobulin transgenes, induces lupus-like glomerulonephritis (Tsao et al., 1992; Ohnishi et al., 1994; Ehrenstein et al., 1995; Radic et al., 1995). Still, the exact role of autoreactive IgM in the subsequent autoantibody response and the switch to other Ig isotypes is not known.
4.2. Autoimmune disease models employing sIgM-deficient mice In humans, IgA deficiency is relatively common and is associated with autoimmunity (Liblau and Bach, 1992; Rankin and Isenberg, 1997). It has been postulated that disease might arise as a consequence of the increased number of infections suffered by IgA-deficient patients. A similar logic should apply to the IgM-deficient mice, since they exhibit enhanced sensitivity to microbial challenge (Boes et al., 1998b). The possibility for such a role for IgM antibody in the development of IgG autoantibodies was addressed in two studies (Boes et al., 2000; Ehrenstein et al., 2000). Ehrenstein et al. examined this hypothesis by injecting LPS into sIgMdeficient mice and control mice. Previously, such treatment was shown to stimulate not only serum IgG but also anti-dsDNA antibodies and evoked an earlier onset of autoimmune disease in MLR/lpr mice (Cavallo and Granholm, 1990). Indeed, only in sIgM-deficient mice did repeated injection of bacterial LPS stimulate the production of anti-dsDNA IgG. Therefore, the absence of IgM antibody with its attendant sensitivity to microbial infection may perhaps predispose to autoimmunity (Ehrenstein et al., 2000). The role of IgM antibody in the development of autoimmunity was also investigated by breeding sIgMdeficient mice with lupus-prone lpr mice (Boes et al., 2000). Lpr mice spontaneously develop high levels of anti-dsDNA antibodies and glomerulonephritis (Theofilopoulos and Dixon, 1985). A defective fas (CD95) gene impairs the ability of lpr T cells to undergo activation-induced cell death and prevents proper maintenance of peripheral tolerance (Watanabe-Fukunaga et al., 1992; Singer and Abbas, 1994; Elkon and Marshak-Rothstein, 1996). Compared to regular lpr mice, lpr mice that lack sIgM developed elevated levels of IgG autoantibodies to dsDNA and histones, and had more abundant deposits of immune complexes in the glomeruli. Disease was more severe, and the mice died at an earlier age. Moreover, sIgM-deficient mice on a
normal background developed anti-DNA IgG with age, which was not the case in wild-type mice (Boes et al., 2000; Ehrenstein et al., 2000). Together, these findings suggest that sIgM, including IgM autoantibodies produced naturally or as part of an autoimmune response, may lessen the severity of autoimmune pathology associated with IgG autoantibodies.
4.3. Protection by IgM antibodies to autoimmune disease How does IgM antibody protect against the development of autoreactive IgG and autoimmune disease? The clearance of immune complexes from the circulation may be enhanced through complement-mediated processes. In the course of apoptosis, nuclear components are translocated into membranous blebs (CasciolaRosen et al., 1994). Some of the IgM autoantibodies, such as those reactive to DNA, histones and phosphatidylserine, may bind to self-antigens in the blebs, activate complement, and thereby promote their removal. Alternatively, natural IgM may have a role in protection against development of autoimmunity early in B cell development. Natural IgM may bind non-infectious self-antigens, increase the exposure of immature B cells to these self-antigens and thus induce tolerance. The first mechanism was investigated directly by treating lpr/sIgM-deficient mice with monoclonal IgM specific for dsDNA or histones (Boes et al., 2000). If autoreactive IgM were to protect against the later development of autoimmunity, lower levels of autoreactive IgG would likely be found. Compared to salinetreated mice, IgM-treated lpr/sIgM-deficient mice produced significantly higher levels of IgG1 specific for dsDNA. This result reflects a third possibility for the fate of immune complexes composed of Ag–IgM–C%. Immune complexes are more effective in activating B cells by cross-linking BCR and coreceptors than antigens alone. The timing of exposure either early or late in development may determine which scenario applies (see below). Also, the overall levels and affinity of autoreactive IgM, and/or its local concentration may determine whether IgM may stimulate clearance of self-antigens from the circulation, or activate autoreactive B cells by cross-linking of the BCR and co-receptors. The latter possibility is analogous to the antigen dose-dependent enhancing effect of IgM and complement on an IgG antibody response (Ahearn et al., 1996; Fischer et al., 1996; Boes et al., 1998a).
4.4. Protection by complement component C1q to anti-nuclear autoimmunity Similar to sIgM-deficient mice, mice deficient in the C1q component of complement are predisposed to anti-
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nuclear autoimmunity (Botto et al., 1998; Mitchell et al., 1999). Deficiency in C1q is associated with glomerulonephritis and antinuclear and antihistone antibodies, but not with increases in antibodies against chromatin or DNA. Moreover, C1q-deficient mice accumulate multiple apoptotic bodies in their normal glomeruli, which is not the case in sIgM-deficient mice. To assess the contribution of C3 activation to the development of glomerulonephritis, C1q−/ − mice were crossed with complement factor B- and C2-deficient mice. Factor B is part of the alternative pathway, and C2 is part of the classical pathway of complement; Factor B and C2 act upstream of C3 in the alternative and classical cascade respectively. Sixty-four per cent of these triple deficient mice showed glomerulonephritis, compared to only 8% of the factor B/C2-deficient mice. Therefore, C1q may have a protective role against the development of glomerulonephritis. This role is independent of C3 activation, and thus could help clear apoptotic cells without the necessity for C3 activation. Finally, these results demonstrated that lupus-like disease is independent of C3 activation (Mitchell et al., 1999).
4.5. Protection by IgM against the de6elopment of autoimmunity The sIgM-deficient mice develop severe glomerulonephritis only when injected with LPS (Ehrenstein et al., 2000) or after crossing with lpr mice (Boes et al., 2000). Mice deficient in complement receptors CD21/ CD35 have a similar autoimmune phenotype (Prodeus et al., 1998). Binding of IgM antibody to antigen leads to activation of C1q, and the resulting immune complexes can be cleared by C1q independently of C3 (Mitchell et al., 1999). Moreover, human patients deficient in C1q or C4 develop lupus disease and anti-dsDNA antibodies, and mice deficient in C4 also spontaneously develop anti-dsDNA autoantibodies (E. Paul and M.C. Carroll, personal communication). Therefore, it is likely that efficient clearance of self-antigen containing immune complexes requires IgM, C1q as well as C4. This hypothesis is corroborated by the fact that complement receptors CD21/CD35 bind C1q, C4b and C3 in human and C3b, C3d and C4b in mouse. Alternatively, a role for natural IgM antibodies in maintaining tolerance may be exerted earlier in B cell development, by promoting the exposure of self-antigens to immature, bone marrow resident B cells. A similar role for CD21/CD35 in negative selection of autoreactive B cells has been suggested (Prodeus et al., 1998). Certainly, many natural IgM antibodies are cross-reactive to self-antigens and may bind to these self-antigens within the bone marrow. A role for immune complexes (Ag–IgM – C1q or Ag – IgM – C4) may therefore be to induce tolerance in immature B cells.
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5. Cloning of an Fc receptor that recognizes IgM
5.1. A murine Fch/v receptor Fc receptors have long been regarded as mediators for the interaction of antibody–antigen complexes with cells, resulting in a wide range of immune responses such as phagocytosis, antibody-dependent cytotoxicity, antigen presentation and the production and secretion of many cytokines and chemokines (Daeron, 1997; Ravetch, 1997; Ravetch and Clynes, 1998). With the exception of the polyimmunoglobulin receptor (pIgR), which transports IgM and IgA across epithelia, Fc receptors have been described for IgG and IgE, but not for other Ig isotypes. A screening effort using COS-7 cells transfected with a cDNA library and the use of a monoclonal IgM antibody as a probe have resulted in the characterization of a novel Fca/m receptor (Shibuya et al., 2000). The murine Fca/m receptor is a 70 kDa glycoprotein expressed on B cells and macrophages, but not on granulocytes, T cells or NK cells. The receptor contains a di-leucine motif in its cytoplasmic domain, and mediates endocytosis of IgM-coated beads. Mouse spleen cells incubated with immune complexes composed of FITC-labeled and IgM or IgG- opsonized S. aureus clearly showed endocytosis by B cells. In contrast, mouse spleen cells preincubated with rat anti-mouse Fca/m receptor antibody were unable to endocytose FITC-labeled S. aureus bacteria coated with IgM or IgG antibodies to S. aureus. Thus, the Fca/m receptor appears to mediate endocytosis of IgM-coated microbial pathogens. Moreover, these results indicate that the Fca/m receptor may be involved in protection against bacterial infection by stimulation of phagocytosis, as well as in stimulating antigen processing and presentation for presentation to T helper cells for induction of adaptive immune responses (Shibuya et al., 2000).
6. Synergy between IgM and complement in immune responses
6.1. Immediate protection by IgM and complement to systemic bacterial infection The susceptibility of sIgM-deficient mice to CLP is similar to that of mice lacking mast cells or that are deficient in complement components C3 or C4 (Echtenacher et al., 1996; Malaviya et al., 1996; Prodeus et al., 1997). In the CLP model, mice deficient in complement components C3 or C4 showed 100% mortality at 24 h, compared to less than 25% mortality in wild-type control mice. Because similar results were obtained using C3- and sIgM-deficient mice in CLP-induced infection, part of the protection by IgM is probably afforded
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through a complement-mediated mechanism. Protection by complement is conferred at several levels. Activation of the complement cascade leads to assembly of the C5b –C9 membrane attack complex, which lyses bacteria directly. The smaller cleavage products C3a and C5a function as chemoattractants that recruit leukocytes to the site of infection. Opsonization of bacteria by C3b and iC3b promotes efficient phagocytosis by neutrophils. In addition, activation of complement stimulates efficient activation of mast cells to release TNFa for the initiation of a local inflammatory response (Prodeus et al., 1997). Attachment of multiple copies of C3d to antigen significantly reduces the amount of soluble antigen required to induce a humoral immune response (Dempsey et al., 1996). Moreover, analysis of the humoral immune response of mice deficient in complement component C3 or C4 (Ahearn et al., 1996; Croix et al., 1996; Molina et al., 1996) showed that the humoral immune response is greatly enhanced by the coupling of complement to antigen. It is now firmly established that Ag– IgM – C% immune complexes can augment adaptive responses through two common pathways. First, immune complexes can cross-link B cell antigen– receptor (BCR) and CD19/CD21 complex and reduce the threshold dose of antigen required to activate B cells (Carter and Fearon, 1992; Rickert et al., 1995; Sato et al., 1995; Dempsey et al., 1996). Second, immune complexes are more efficiently trapped on FDC through the expressed complement receptors (CD21 and CD35 in mice), which leads to an effective germinal center reaction during the T-cell dependent antibody response (Pepys, 1976; Ahearn and Fearon, 1989; Szakal et al., 1989).
6.2. Role of complement receptors on either B cells or FDC in enhancing IgG responses In mice, the expression of CD21/CD35 is limited to B cells and FDC (Holers et al., 1992). Lethal irradiation of mice depletes all B cells, but not FDC. In addition, FDC are long-lived and will probably not be replaced by donor cells following bone marrow transfer (Humphrey et al., 1984). These insights were used to compare the roles of CD21/CD35 expressed on B cells versus FDC during an antigen-specific IgG response, by immunization experiments of bone marrow chimeric mice (Fang et al., 1998). For each of two T-cell dependent antigens, expression of CD21/CD35 on both B cells and FDC was necessary to restore the normal phenotype. Mice deficient in CD21/CD35 on B cells had a diminished, but still detectable, IgG response, and germinal centers were comparable to controls. In contrast, mice deficient in CD21/CD35 on FDC alone had a marked decrease in the production of antigenspecific IgG, and germinal centers were smaller in size
compared to normal controls. These results indicate an important role for CD21/CD35 on FDC cells and, to lesser extent, B cells, in stimulating efficient IgG responses. Croix et al. had previously shown that expression of CD21/CD35 on B cells is necessary to generate significant IgG responses to T-cell-dependent antigen, by use of mice generated by injection of homozygous Cr2 -deficient ES cells into RAG2-deficient blastocysts (Croix et al., 1996). In addition, Ahearn et al. reported that reconstitution of Cr2 -deficient mice with wild-type bone marrow resulted in normal IgG responses to T-cell-dependent antigen (Ahearn et al., 1996). Thus, these findings indicate an important role for B cells rather than FDC in generating well-organized IgG responses. These seemingly contradictory findings may be attributable to the use of different antigens, at different dosages. The specific requirement for immune complexes sequestered by FDC was investigated in a transgenic approach (Hannum et al., 2000), in which all B cells produce the membrane-bound, but not the secreted, form of NP-specific IgM. After immunization, the transgenic mice produced virtually no antigen-specific antibody and expressed almost no immune complexes on FDC. Surprisingly, although there was practically no antibody to form immune complexes, germinal center reactions did take place and had an enlarged size and normal frequency and kinetics. Moreover, somatic mutations of the Vl region were found predominantly in the complementarity-determining regions (CDR), which suggests antigen-driven selection in germinal centers (Hannum et al., 2000). Finally, when the transgenic mice were immunized with NP, antigen-specific B cells comprised a large percentage of the splenocytes. Thus, there is no consensus yet about the contribution of immune complex deposition on FDC in enhancing antigen-driven IgG responses. The use of different protocols to determine the involvement of FDC in immune responses may explain the seemingly contradictory observations obtained by the separate groups. Also, antigen trapping can occur in the absence of natural antibodies by acute phase reactants such as C-reactive protein that are thought to have similar functions as natural antibodies (Hirano et al., 1990; Baumann and Gauldie, 1994; Steel and Whitehead, 1994). A reason for not detecting immune complexes that may still be present on FDC could be the large number of antigen-specific B cells that all compete for binding, and thereby may obscure immune complexes during antibody staining. For now, the main message remains that expression of CD21/CD35 is necessary on both B cells and probably FDC, at different time points during a humoral immune response. Initially, cross-linking of BCR and CD19/CD21 coreceptor by immune complexes (Ag–
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IgM – C%) stimulates the intracellular signals leading to B cell activation. Later in the course of a primary immune response, as some antigen-primed B cells migrate to the germinal center, they proliferate and undergo hypermutation and Ig class switching (Kelsoe, 1996). In the germinal center, FDC display immune complexes bound to CD21/CD35 and Fc receptors for presentation to antigen-experienced B cells. Finally, those B cells that express Ig of increased affinity for the antigen, proliferate and mature into plasma cells.
Acknowledgements I thank Drs Jianzhu Chen, Hidde Ploegh and Michael Carroll for critical reading of the manuscript and for helpful discussions.
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