Antigen localization within the splenic marginal zone restores humoral immune response and IgG class switch in complement C4-deficient mice
Berit Zachrau1,
Doreen Finke1,
Katja Kropf1,
Hans-Juergen Gosink2,
Holger Kirchner1 and
Siegfried Goerg1
1 Institute for Immunology and Transfusion Medicine and 2 Department of Radiotherapy and Nuclear Medicine, Medical University of Luebeck, Ratzeburger Allee 160, D-23560 Luebeck, Germany
Correspondence to: S. Goerg; E-mail: goerg{at}immu.mu-luebeck.de
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Abstract
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Defects of early complement components (C1, C4 and C2) are associated with the development of systemic lupus erythematosus (SLE). The availability of complement knockout mice has increased our knowledge on the role of complement in the regulation of adaptive immunity. An impaired removal of apoptotic bodies, a disturbed clearance of IgG immune complexes (ICs) and an insufficient B-cell regulation via complement receptors CD21/CD35 have been discussed as explanations for the induction of autoimmunity; however, a unifying hypothesis for the loss of B-cell tolerance in the absence of C1 or C4 is still lacking. Using IgM-containing ICs, we observed a significant accumulation of antigen within the splenic marginal zone (MZ) of C4-deficient mice but not in C3-deficient or complement receptors CD21/CD35-deficient mice. The targeting of antigen toward the MZ restored adaptive immunity (antibody response and class switch) in C4-deficient animals. A new explanation for the association of SLE and complement C4 deficiency would be that in the absence of C4, natural antibodies (IgM type) localize more self-antigen toward the MZ so that the auto-antibody response is increased and autoimmune disease ensues. As such, an inadequate localization of self-antigens might be responsible for the annulment of peripheral B-cell tolerance in the absence of C4.
Keywords: B cells, complement, immune complexes, marginal zone
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Introduction
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Deficiency of factors involved in the classical complement activation pathway (C1, C4 and C2) is associated with an impaired humoral immune response in mammals (1). Complement C1, C4 and C2 deficiency is also a predisposing factor for autoimmune diseases such as systemic lupus erythematosus (SLE) (2). The first observation might be explained by the fact that immune complexes (ICs) consisting of antigen and complement C3d induce positive regulation of B cells via synergistic effects on B-cell receptors and complement receptor CD21 (3, 4), whereas in the absence of C1 or C4 the classical activation pathway and integration of C3d in ICs are inhibited so that co-stimulation and humoral immune response are both impaired (5). Moreover, an improved humoral immune response is observed if specific IgM is injected prior to immunization with antigen, and this phenomenon seems to be dependent on CD21 (6).
Regarding the development of SLE in case of C4 deficiency, it was possible in two independent tolerance models (lprlpr and sHELantiHEL) to demonstrate that the deficiency of both complement receptors CD21/CD35 and complement C4 promotes the development of autoimmune phenomena or inhibits the development of anergy (7). These results suggested that the association of C4 deficiency with impaired humoral immune response on the one hand and increased auto-antibody production on the other could be explained by an insufficient co-stimulation of CD21/CD35.
In contrast to C1q- (8) or C4-deficient mice (9), CD21/CD35-deficient animals do not spontaneously develop autoimmune disease (9), while SLE is rarely observed in cases of C3 deficiency in mice or humans (2, 7, 10). As such, the induction of SLE seems to be influenced by the early complement components C1q and C4 and is independent of C3 and the complement receptors CD21/CD35. One explanation could be that apoptotic cell remnants acting as a source of auto-antigens could not be phagocytosed in C1q- (8) and C4-deficient mice (11). Since complement-mediated phagocytosis (opsonization) is in part dependent on C3b and because C3-deficient animals rarely develop SLE, we asked whether another function of early complement components might explain this problem. The facilitation and clearance of ICs is influenced by complement, an idea supported by a large amount of in vivo (9, 12) and in vitro data (13, 14). However, most IC clearance studies used IgG-containing ICs (9, 1517) that interfere with Fc receptors. The good complement activation capabilities of IgM and the fact that the administration of specific IgM improves humoral immune responses (4) led us to hypothesize that a disturbed facilitation of IgM-containing ICs in states of C1 or C4 deficiency is involved in the induction of humoral immune responses that lead to autoimmunity.
We therefore determined the distribution of IgG-ICs and IgM-ICs by using complement C4-deficient (C4null), C3-deficient (C3null) and CD21/CD35-deficient mice (Cr2null) in comparison to C57Bl6 wild-type (wt) mice and measured the humoral immune response over a period of 24 days in wt, C4null as well as C4C3null (deficient in both C4 and C3) mice. ICs were formed in vivo by intravenous injection of a murine anti-4-hydroxy-3-nitrophenylacetyl (Np) antibody of either a polyclonal IgG type or a monoclonal IgM type 15 min prior to injection of the corresponding antigen. The sequential injection of antibody and antigen was chosen because preformed IC behaved differently regarding their ability to activate complement or to bind to certain cell types (1820).
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Methods
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Mice
Mice with targeted disruption for complement gene C3 (C3null) and complement gene C4 (C4null), and normal mice (wt) were used. In addition, mice deficient for complement receptors CD21/CD35 (Cr2null) were also included. The genetic background of wt and C4null mice was C57BL/6, and that of Cr2null and C3null was mixed 129/C57BL/6 (breeding pairs of knockout mice were kindly donated by M. C. Carroll).
Antibodies
Monoclonal murine IgM anti-Np antibody (B1-8 hybridoma, kindly donated by H. Wagner, Cologne, Germany) was purified from culture supernatant by ammonium sulfate precipitation followed by affinity purification (HiTrap IgM purification column, Amersham Pharmacia Biotech, Freiburg, Germany), micro-concentration (Amicon Millipore, San Jose, CA, USA) and dialysis against PBS. Polyclonal murine IgG anti-Np was prepared from the serum of normal mice (C57Bl6) immunized twice with Np-chicken gamma globulin [Np(37)-CgG] on occasions set 2 weeks apart. Serum was harvested after 4 weeks before it was ammonium sulfate precipitated and purified using a Protein G column (Boehringer Mannheim, Mannheim, Germany). The preparation ultimately consisted of IgG1, 2a and 2b and IgA, while only trace amounts of IgM or IgG3 were detected in dot blots. Antibodies were incubated at 55°C for 15 min before use in order to inactivate any remaining complement.
Antigen
BSA was labeled with (4-hydroxy-3-nitrophenyl)acetic acid (Biosearch Technologies, Novato, CA, USA). Np-BSA was radiolabeled with iodine-125 (Amersham Pharmacia Biotech) (Np-BSA-125I) or biotinylated (Np-BSA-biotin) with biotinamidocaproate-N-hydroxysuccinimide ester (Sigma, Deisenhofen, Germany). Concentrated antibodies and antigens were diluted with HBSS (Sigma).
Experiments and histology
Anesthetized mice were injected intravenously with 20 µg antibody followed 15 min later by 1.5 µg of the corresponding antigen Np-BSA-125I or Np-BSA-biotin. The determined ratio of antigen and antibody resulted in an antibody excess. After 30 min, mice were bled and sacrificed, and their livers, spleens and kidneys were harvested. Radioactivity within the organs was determined using a gamma-counter and the results obtained were normalized with respect to organ weights. With the Np-BSA-biotin experiments spleens were snap-frozen in embedding medium and cryosectioned, and the localization of ICs was monitored in situ using an avidinalkaline phosphatase conjugate followed by a specific substrate (Fast Blue) (Sigma). Alternatively, staining was performed using a rat anti-mouse C1q antibody (7H8-6) or C3 antibody (II-3-2) (kindly donated by E. Kremmer, Munich, Germany) and peanut agglutininHRP as a counterstain.
ELISA
After injections of antibodies and antigen (Np-BSA), mice were bled at days 0, 1, 2, 9 and 24 and sera were stored at 40°C until ELISAs were performed. Microtiter plates (Pharmacia CAT RPN 4905, Freiburg, Germany) were coated overnight at 4°C with Np-BSA (Biosearch Technologies) in ELISA coating buffer (Sigma). They were then washed and blocked with PBS containing 0.25% Tween 20 and 1% cytochrome c. Mouse sera at various dilutions [and aliquots of polyclonal IgG or IgM (B-18) as standards] were incubated at 37°C for 1 h before secondary antibodies (monoclonal rat anti-mouse IgG1, X56), IgG2a (R19-15), IgG2b (R12-3), IgG3 (R40-82) (all from Pharmingen) and polyclonal anti-IgM (Sigma) conjugated with alkaline phosphatase were added. p-Nitrophenyl phosphate (Sigma) was used as a substrate and reading was performed at 405 nm.
Formation of ICs in vitro
The ability of the IgM and IgG antibodies to form ICs with the corresponding antigen Np-BSA was demonstrated using a 0.7% agarose gel double-immune diffusion technique (Ouchterlony) and staining with Coomassie blue. Monoclonal IgM (B1-8) and polyclonal IgG were able to activate human complement as demonstrated by ELISA. Briefly, ELISA plates were coated with Np-BSA and incubated with antibodies diluted in fresh human serum. After washing, deposited C3 was detected using goat anti-human C3 antibody (Atlantic Antibodies, Windam, ME, USA). IgM resulted in an expected (21) complement activation at about one-tenth of the concentration at which polyclonal IgG caused activation.
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Results
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If radiolabeled antigen is injected intravenously without a specific antibody, no statistically significant difference is observed in any of the four different groups of animals. After 30 min, there was no specific uptake in kidney, liver or spleen (Fig. 1a). When polyclonal IgG anti-Np was injected 15 min prior to the injection of antigen, a rapid clearance from the intra-vascular circulation was observed in all groups of mice with a preferential uptake into the liver, but no difference between wt and complement-deficient animals was observed (Fig. 1b). The uptake in spleen and kidney was not affected compared with mice receiving antigen only, indicating that these organs do not participate in the elimination of IgG-ICs in mice. Despite the fact that polyclonal IgG anti-Np is able to activate complement (data not shown), the clearance mechanisms in vivo seem to be relatively independent of complement C3 and C4 and of the complement receptors CD21/CD35.

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Fig. 1. Organ distribution of radiolabeled antigen. (a) Without prior injection of specific antibody, the distribution is similar in all four groups of mice. (b) Prior injection of polyclonal IgG antibody leads to a preferential rapid and higher uptake in the liver, but no difference between wt and complement-deficient animals was observed. (c) Injection of monoclonal IgM antibody produced an increase of antigen uptake in the spleens of wt (P = 0.088), C3null (P = 0.019) and Cr2null (P = 0.02) mice compared with animals receiving antigen alone. In C4null mice the splenic uptake was significantly higher compared with C4null antigen-only mice, and compared with all other groups of mice that received IgM-IC.
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Since it might be argued that uptake of IgG-ICs into the liver is Fc receptor mediated, we tested the processing of ICs by using IgM anti-Np antibodies where the activation of complement is pronounced compared with IgG (21). As a first result, there was no higher specific uptake of in vivo-formed IgM-ICs into liver or kidney of all tested animals within 30 min, compared with mice receiving antigen only (Fig. 1c). Wt, Cr2null and C3null mice showed a slight increase in splenic uptake, increasing from <43 cpm mg1 in antigen-only mice to >63 cpm mg1 with prior injection of antibody; this was statistically significant for C3null (P = 0.019) and Cr2null (P = 0.02) mice, while wt mice only revealed a tendential increase (P = 0.088). This increase in splenic uptake was not effective at eliminating IgM-ICs since comparable amounts of antigen remained in serum as were seen in mice receiving antigen only, and the mass of the spleens (between 60 and 120 mg) did not suffice to extract ICs from an estimated distribution volume of at least 2 ml blood. A surprising difference was observed in C4null mice where the splenic uptake was increased 7-fold (243 cpm mg1) compared with C4null antigen-only mice (34 cpm mg1) (P = 0.004), and was also significantly higher compared with that seen in all other groups of mice receiving IgM-ICs (P = 0.011 for wt, P = 0.020 for Cr2null and P = 0.019 for C3null mice).
Immunohistochemistry of splenic sections revealed deposition of biotinylated antigen within the marginal zone (MZ) of C4null animals (Fig. 2a), whereas no specific uptake was observed in wt animals (Fig. 2b). Within the MZ are several types of immune-competent cells like marginal zone macrophages (MZMs) and marginal zone B cells (MZBs) which interact with each other. MZMs are involved in the processing of T-independent antigens such as polysaccharides or carbohydrates; the deposition of colloidal carbon particles (ink) after intravenous injection shows a pattern similar to that displayed by IgM-IC (data not shown). Staining of splenic sections with anti-C1q antibodies revealed co-localization of ICs and C1q, indicating activation and binding of the first components of the classical pathway (Fig. 2c), whereas wt mice showed no specific C1q deposition within the MZ (Fig. 2d). Staining of C1q within an incidental germinal center (GC) seems to be independent of antibody treatment as we generally observe C1q staining in GC. Surprisingly, an increased distinct MZM deposition of complement C3 was observed in the absence of C4 (Fig. 2e) as well as in the presence of C4 (Fig. 2f), indicating an alternative complement activation mechanism.

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Fig. 2. Immunohistochemistry of splenic sections taken from mice receiving IgM. (a) Biotinylated antigen (blue) was detected in the MZ of splenic follicles only in C4null mice with prior injection of IgM whereas (b) wt mice showed no specific deposition of antigen. The marginal sinus is demonstrated with peanut agglutinin (red). (c) Deposition of C1q (red) in the MZ of C4null mice demonstrates classical complement activation whereas (d) wt mice showed no specific C1q deposition within the MZ but apparently in GCs. Deposition of C3 (blue) on MZM indicates an alternative mechanism of complement activation in the absence of C4 (e) as well as in the presence of C4 (f). (C4null mice in a, c and e, wt mice in b, d and f.)
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Next we investigated whether the remarkable difference in the MZ deposition of IgM-ICs in C4-deficient mice had an effect on the humoral immune response. To this end, antibody responses of IgG sub-classes were measured by ELISA over a period of 24 days after injection of wt or C4null mice with IgM plus antigen or antigen only. By measuring IgM anti-Np, it was possible to recover injected antibodies on the first day after the injection (data not shown). As expected, the IgG response in C4null and C4C3null animals was decreased compared with wt mice if antigen only was injected (Fig. 3). The administration of IgM increased the IgG1, IgG2a and IgG2b antibody response in wt mice. Although the underlying mechanism (CD21-dependent B-cell activation) was only available in C4null mice through an alternative complement activation mechanism, but not present at all in C4C3null animals, these mice showed an increase in all IgG sub-classes on day 9 (Fig. 3) and at further measured points of time [days 16 and 24 (data not shown)], leading to an almost normal immune response.

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Fig. 3. Humoral immune response in C4null, C3C4null and wt mice receiving IgM. Anti-IgG ELISA demonstrates restoration of the humoral immune response and IgG class switch in complement C4- and C3C4-deficient mice (mean on day 9 after injections compared with day 0; bars depict standard errors of the mean, n > 8, *P < 0.05).
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Discussion
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It was long believed that an impaired clearance of ICs consisting of auto-antibodies and auto-antigen was responsible for their deposition and a subsequent inflammatory reaction in SLE patients with C4 deficiency. Here we demonstrate that intra-vascular clearance of IgG-IC does not depend for the most part on complement. The pre-injection of specific polyclonal IgG is efficient at clearing (although not completely) IgG-ICs from the circulation within 30 min, although all four groups of mice (wt, C4null, C3null and Cr2null) revealed a similar reduction in serum counts and liver uptake. Since we only measured at one time point, we cannot exclude the possibility that the rate of clearance may have been slightly different in complement-deficient mice. Other investigators did find an influence of complement on the rate of clearance; however, the findings are difficult to compare since most of the IC clearance studies used in vitro heat-aggregated IgG as a surrogate (12) or in vitro-preformed ICs (9, 15); in vitro-generated ICs have different cell-binding properties, clearance kinetics and organ distributions compared with in vivo-generated ICs (1820). This is supported by the fact that in our model of in vivo-formed IgG-IC, platelets or red cells did not participate in clearance since the remaining counts in serum revealed no avidity toward blood cells (radioactivity remained in the supernatant after whole-blood centrifugation, results not shown).
The preferential uptake of ICs in the liver is most likely explained by interaction with Fc receptors on hepatic von Kupffer cells, although recent publications suggested that Fc receptors are not involved in the clearance of soluble ICs (22). However, these studies used a model of lupus mice and concluded that from the same amounts of endogenous ICs the rate of production and clearance was also similar in Fc
-chain-deficient mice compared with mice with a heterozygous
-chain deficiency. Our data support those of others who found that clearance of IgG-IC depends on Fc receptors located on residential monocytes and macrophages (12); however, this interaction seems to be independent of complement, as is the clearance of IgG-sensitized erythrocytes (23).
Regarding IgM, we observed a heavy deposition of IgM-ICs within the splenic MZ of C4-deficient animals. Our data suggest that, in mice, complement C4 is more important than C3 for keeping IgM-IC in circulation and preventing their precipitation (13), since in C3null mice splenic uptake is only slightly increased compared with that seen in wt mice. In the absence of C4 the solubility of IgM-IC is reduced and ICs are filtered by an additional barrier of splenic MZM. The MZ represents a location in which several types of immune-competent cells reside permanently or temporarily; interaction of MZM with MZB can result in altered antibody responses, although conflicting results have been reported regarding this (24, 25), and the mechanisms involved are not completely understood. Despite the fact that C1q and C3 co-localize with ICs on MZM in C4null mice, the receptor responsible for IC binding may act independently of complement since MZMs express membrane receptors that take up and phagocytose particles even in the absence of prior opsonization (24) or independent of complement (26) or might be a result of IgM-IC binding to a newly described IgM Fc receptor (27).
There is a large body of evidence suggesting that both pre-injected specific IgM (6) as well as natural IgM have antibody-promoting effects (28, 29). In wt mice the IgM-enhanced immune response is dependent on a cascade of classical complement activation, C3 and CD21/CD35 on B cells (6) and may preferentially take place in GCs within a network consisting of follicular dendritic cells, T and B cells. Accordingly, we observed an increased humoral immune response in wt mice when IgM was pre-injected (Fig. 3).
In contrast to wt mice, co-stimulation of B cells via CD21/CD35 (5) is attenuated in the absence of C4 unless there is an alternative complement activation mechanism, and completely inhibited in the absence of both C4 and C3 (C4C3null mice). Accordingly, the immune response in C4null and C4C3null mice receiving antigen only is dramatically diminished. Surprisingly, pre-injection of IgM increased the localization of IgMantigenIC toward the splenic MZ and restored adaptive immunity (antibody response and class switch) in the absence of C4 alone or C3 and C4 combined.
Although the activation of MZB is increased if complement delivers a co-stimulatory signal via CD21/CD35 (29, 30), the observed increased antibody response in C4-deficient and C4C3null mice suggests that this mechanism bypasses the complement system altogether and therefore appears to be influenced initially and primarily by the localization of the antigen within the MZ. The involvement of MZMs and MZBs, which apparently have differing repertoires, suggests that this process is relatively independent of T-cell help (24) and might instead be explained by a Toll-like receptor-mediated mechanism (31) involving the production of IFN
by MZMs (D. Finke et al., in preparation).
In wt mice the MZ seems not to be involved in the IgM-mediated enhancement of antibody production since the splenic uptake of IgM-IC over 30 min was negligible compared with mice receiving antigen only (Fig. 1). It should be pointed out, however, that the dose of soluble antigen used was relatively small (1 µg). Injection of higher doses of IgM antibody (100 µg) and antigen (7.5 µg) resulted in similar increased splenic uptakes in C4null and wt mice (data not shown). It was also then possible to visualize MZ deposition, suggesting that higher doses of antigen lead to a consumption of complement so that an overriding of C4's precipitation prevention limit occurs. We therefore propose that an early step of IgM enhancement of antibody responses involves MZ localization especially in states of complement deficiency or in cases of complement consumption when higher doses of antigen or IgM-IC are applied (32).
One explanation for the association of SLE with complement C1, C4 and C2 deficiency, and to a lesser extent with C3 deficiency (2), originates from an impaired IC solubility, an IC deposition in certain tissues and the inflammatory responses that might result from this. Our observation that ICs are targeted toward splenic MZ suggests that the influence of C4 deficiency on immune-regulatory functions is more important than its influence on inflammatory responses. The protective role of C4 against lupus erythematosus is independent of CD21/CD35 (9) and C3 (10) and might be reflected by a differential processing of low amounts of natural IgM self-antibodies (29) and self-antigens, resulting in a localization of IgM-IC in the splenic MZ during early immune events.
In general, T-dependent humoral immune responses are executed by follicular B cells within GCs. These depend on follicular localization of antigen and are increased if the co-receptor CD21 is triggered; the latter effect is facilitated if antigen-specific IgM activates complement. Without C4 and with IgM, ICs are filtered by a barrier of MZMs and activate MZBs without the involvement of CD21. A remarkable immune response is nevertheless observed, a fact which demonstrates the importance of the site at which antigen is deposited (33).
Our result demonstrating the ability of the IgM-dependent localization of antigen within the splenic MZ to overcome an impaired immune response in C4-deficient animals provides a novel explanation for the association between SLE and complement C4 deficiency.
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Acknowledgements
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This paper is dedicated to the memory of Susanne Otte. We are grateful to M. C. Carroll for providing the complement-deficient animals and for his advice. We would also like to offer our thanks to M. Ma, A. P. Prodeus and R. R. Reid for undertaking initial experiments. Supported by DFG SFB 367.
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Abbreviations
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GC | germinal center |
IC | immune complex |
MZ | marginal zone |
MZB | marginal zone B cell |
MZM | marginal zone macrophage |
Np | 4-hydroxy-3-nitrophenylacetyl |
SLE | systemic lupus erythematosus |
wt | wild-type |
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Notes
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Transmitting editor: D. T. Fearon
Received 17 December 2003,
accepted 20 August 2004.
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