Complement receptor type two (CR2,CR21)

Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens. The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells. CR2 is also pivotal for Epstein-Barr virus (EBV) infection. Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized. The potential to target CR2 for clinical therapy or immunization purposes are discussed.     

Human complement receptor 2 (CR2/CD21) as a receptor for DNA: Implications for its roles in the immune response and the pathogenesis of systemic lupus erythematosus (SLE)

Human CR2 is a B cell membrane glycoprotein that plays a central role in autoimmunity. Systemic lupus erythematosus (SLE) patients show reduced CR2 levels, and complete deficiency of CR2 and CR1 promotes the development of anti-DNA antibodies in mouse models of SLE. Here we show that multiple forms of DNA, including bacterial, viral and mammalian DNA, bind to human CR2 with moderately high affinity. Surface plasmon resonance studies showed that methylated DNA bound with high affinity with CR2 at a maximal K(D) of 6nM. DNA was bound to the first two domains of CR2 and this binding was blocked by using a specific inhibitory anti-CR2 mAb. DNA immunization in Cr2(-/-) mice revealed a specific defect in immune responses to bacterial DNA. CR2 can act as a receptor for DNA in the absence of complement C3 fixation to this ligand. These results suggest that CR2 plays a role in the recognition of foreign DNA during host-immune responses. This recognition function of CR2 may be a mechanism that influences the development of autoimmunity to DNA in SLE.     

Complement receptor 1 (CR1) and Alzheimer's disease

Alzheimer's disease (AD) is the most common neurodegenerative disease and it poses an ever-increasing burden to an aging population. Several loci responsible for the rare, autosomal dominant form of AD have been identified (APP, PS1 and PS2), and these have facilitated the development of the amyloid cascade hypothesis of AD aetiology. The late onset form of the disease (LOAD) is poorly defined genetically, and up until recently the only known risk factor was the ε4 allele of APOE. Recent genome-wide association studies (GWAS) have identified common genetic variants that increase risk of LOAD. Two of the genes highlighted in these studies, CLU and CR1, suggest a role for the complement system in the aetiology of AD. In this review we analyse the evidence for an involvement of complement in AD. In particular we focus on one gene, CR1, and its role in the complement cascade. CR1 is a receptor for the complement fragments C3b and C4b and is expressed on many different cell types, particularly in the circulatory system. We look at the evidence for genetic polymorphisms in the gene and the possible physiological effects of these well-documented changes. Finally, we discuss the possible impact of CR1 genetic polymorphisms in relation to the amyloid cascade hypothesis of AD and the way in which CR1 may lead to AD pathogenesis.     

Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.     

Defective B cell ontogeny and immune response in human complement receptor 2 (CR2, CD21) transgenic mice is partially recovered in the absence of C3

Mice prematurely expressing human CR2 (hCR2) in the B cell lineage have a defective B cell ontogeny and immune response. Our recent analysis of this phenotype suggested that signaling through hCR2 and presumably mouse CD19 on the B cell surface, during bone marrow development, could result in the observed changes in B cell function in these mice. To test this hypothesis, we back crossed hCR2(high) transgenic mice onto the CD19(-/-) background. CD19(-/-)hCR2(high) mice were found to possess even fewer mature B cells than their CD19(+/+)hCR2(high) littermates, demonstrating that loss of CD19 exacerbated the effects elicited through hCR2. This data suggests that CD19 provides a survival signal during B cell development in this model. Next, we examined if the removal of the main ligand for CR2, namely C3d, through back-crossing onto the C3(-/-) background could restore normal B cell development. However, we found only minor recovery in peripheral B cell numbers and no obvious change in function. This was despite a three-fold increase in the level of hCR2 expression on B cells isolated from the spleen or bone marrow of C3(-/-)hCR2(high) mice when compared with C3 sufficient littermates. These data demonstrate that hCR2 is integrated in mouse B cell signaling and that the downstream effects of hCR2 expression during early B cell development are partially but not completely due to interaction with C3 fragments and signaling through CD19 in the bone marrow environment.     

The structural basis for complement receptor type 2 (CR2, CD21)-mediated alternative pathway activation of complement: studies with CR2 deletion mutants and vaccinia virus complement-control protein-CR2 chimeras

The role of complement receptor 2 (CR2) short consensus repeats (SCR) in binding of hydrolyzed C3 (iC3) to form an alternative pathway (AP) convertase, and promoting C3 fragment deposition following AP activation, was examined. We used (1) K562 cells transfected with CR2 constructs, where the C3d-binding site of CR2 (SCR1+2) was replaced with the four-SCR vaccinia virus complement control protein (VCP), or truncation mutants thereof, and (2) COS cells transfected with wild-type (wt) CR2, or deletion mutants thereof. AP activation required iC3 binding in both systems. Thus, the VCP-CR2 chimera had an iC3 binding efficiency of 11.4 %, compared to wtCR2, and a relative AP activity of 5.5 %, the truncation mutants being inactive. Of the CR2 mutants, only EK (DeltaSCR10 - 11) had AP activity similar to wtCR2. NN (DeltaSCR6 - 8) and NOP (DeltaSCR6-mid14) had reduced AP activity, but near normal iC3 binding. XB (DeltaSCR3 - 6) and PP (DeltaSCR3-mid14) were inactive in both assays. We conclude that, whilst iC3 binding to CR2 via SCR1 - 4 is essential for AP activation, the efficiency of C3 deposition also depends on the midportion of CR2.     

B cell activation leads to shedding of complement receptor type II (CR2/CD21)

Complement receptor type II (CR2/CD21) is the major receptor for C3d fragments on immune complexes. CD21 also serves as the receptor for Epstein-Barr virus in humans. On mature B cells, CD21 reduces the threshold of BCR signaling together with CD81, Leu13 and CD19, but it also occurs on other cells of the immune system where it performs unknown functions. A soluble form of CD21 (sCD21) is shed from the cell surface and is found in human blood plasma. An as-yet-unknown protease is thought to be responsible for this shedding. Altered levels of sCD21 occur in plasma in certain clinical conditions. We show here by mass spectrometry that sCD21 in human plasma of healthy donors is predominantly a short form of CD21 without the exon-11-encoded sequences. Whereas the N terminus of sCD21 was found unmodified, the C terminus is truncated, implying that only the extracellular portion of CD21 is shed. Peripheral blood B cells, but not T cells, contribute to the plasma CD21-pool. CD21 shedding is induced by stimulation with PMA plus Ca(2+) ionophore, or by stimulation of the BCR with anti-IgM+anti-CD40.     

Modulation of murine complement receptor type 2 (CR2/CD21) ectodomain shedding by its cytoplasmic domain

Ectodomain shedding is a mechanism that regulates numerous functions of cell surface proteins. The extracellular domain of the human complement receptor 2 (CR2/CD21) is released by proteolytic cleavage as a soluble protein through a variety of stimuli including the thiol antioxidants N-acetylcysteine (NAC) and glutathione (GSH), and the oxidant pervanadate (PV). In addition, PV mimics B cell antigen receptor (BCR) signaling. Here, we show that murine CD21 is shed upon those stimuli and that the cytoplasmic domain is an important modulator for CD21-shedding. B cells expressing a mutant CD21 cytoplasmic domain with only three amino acids (KHR) showed increased CD21-shedding and required lower stimuli concentrations. At lower PV concentrations, wildtype CD21 was up-regulated on the cell surface, whereas at higher PV concentrations the ectodomain was shed. These findings further indicate that GSH and NAC utilize different pathways than PV to activate CD21-shedding. Altogether, as pre-activated B cells express higher CD21 levels than resting mature B cells or fully activated and antigen-experienced B cells, we suggest CD21-shedding to be a mechanism to fine-tune B cell activation.     

A new mouse anti-mouse complement receptor type 2 and 1 (CR2/CR1) monoclonal antibody as a tool to study receptor involvement in chronic models of immune responses and disease

Although reagents are available to block mouse complement receptor type 2 and/or type 1 (CR2/CR1, CD21/CD35) function in acute or short term models of human disease, a mouse anti-rat antibody response limits their use in chronic models. We have addressed this problem by generating in Cr2−/− mice a mouse monoclonal antibody (mAb 4B2) to mouse CR2/CR1. The binding of murine mAb 4B2 to CR2/CR1 directly blocked C3dg (C3d) ligand binding. In vivo injection of mAb 4B2 induced substantial down regulation of CR2 and CR1 from the B cell surface, an effect that lasted six weeks after a single injection of 2 mg of mAb. The 4B2 mAb was studied in vivo for the capability to affect immunological responses to model antigens. Pre-injection of mAb 4B2 before immunization of C57BL/6 mice reduced the IgG1 antibody response to the T-dependent antigen sheep red blood cells (SRBC) to a level comparable to that found in Cr2−/− mice. We also used the collagen-induced arthritis (CIA) model, a CR2/CR1-dependent autoimmune disease model, and found that mice pre-injected with mAb 4B2 demonstrated substantially reduced levels of pathogenic IgG2a antibodies to both the bovine type II collagen (CII) used to induce arthritis and to endogenous mouse CII. Consistent with this result, mice pre-injected with mAb 4B2 demonstrated only very mild arthritis. This reduction in disease, together with published data in CII-immunized Cr2−/− mice, confirm both that the arthritis development depends on CR2/CR1 receptors and that mAb 4B2 can be used to induce biologically relevant receptor blockade. Thus mAb 4B2 is an excellent candidate for use in chronic murine models to determine how receptor blockage at different points modifies disease activity and autoantibody responses.     

Complement receptor type 1 gene regulation: retinoic acid and cytosine arabinoside increase CR1 expression

Complement receptor type 1 (CR1) is expressed principally on erythrocytes, monocytes, neutrophils and B cells, where it acts as a negative regulator of the complement cascade and as a clearance mechanism for immune complexes. As CR1 expression occurs in a number of lineages, its regulation may parallel other steps in haematopoiesis. Several leucocyte-cell lines (including K-562, THP-1, U-937 and HL-60) and B-cell lines, as well as peripheral blood cells (PBCs), were tested for the ability of various compounds to up-regulate their CR1 expression. While most of the compounds tested had minimal effects on CR1 message level, retinoic acid induced increases in mRNA levels in nearly all cell lines studied. Furthermore, in K-562 cells, the cytosine analogue Ara-C, an inducer of erythroid differentiation, caused the highest increases in CR1 mRNA levels as compared to untreated cells. Ara-C also induced significant increases in CR1 message in PBCs. These data suggest that induction of specific CR1 expression could be an integral part of blood cell differentiation and that the identified cell induction systems may be useful as models to study the regulation of CR1 gene expression.     

Mucosal type mast cells express complement receptor type 2 (CD21)

Fragments of complement component C3 generated upon activation of the cascade play an important role in the induction and regulation of immune responses. Receptors interacting with various fragments of this versatile complement protein are expressed on a wide variety of cell types, including lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes and consequently, C3-products may influence several biological functions at different sites of the body, where complement activation occurs. Regarding the expression of various C3-receptors on mast cells, mainly rodent serosal type mastocytes have been investigated so far. It has been known for a long time that C3a triggers the release of mediators of immediate type hypersensitivity via binding to serosal-type cells. Complement receptor type 1 (CR1/CD35) and type 2 (CR2/CD21) interacting with the larger activation products, such as C3b and C3d, have so far been shown on serosal type mast cells only. In this study, the expression of CR1/2 on mucosal type mast cells is demonstrated. Using mouse CR1/2 specific single chain antibodies and the natural ligand C3d in cytofluorimetric measurements, we show that the rat mucosal mast cell line RBL-2H3 and mouse bone marrow-derived mast cells (BMMC) express CD21. RT-PCR experiments carried out with mouse CR1 and CR2 specific primers show CD21, but not CD35 specific products in BMMC. It is also demonstrated that, in contrast to serosal type mast cells, mucosal mastocytes do not express CD19. In an attempt to reveal the possible function of CR2 on mucosal type mast cells, the effect of receptor-clustering was tested regarding degranulation, Ca-response and IL-6 production, but no CR2-mediated change was detected in any of these processes.     

Human complement receptor type 2 (CR2/CD21) transgenic mice provide an in vivo model to study immunoregulatory effects of receptor antagonists

We found that transgenic (tg) mice stably expressing a bacterial artificial chromosome (BAC)-derived human complement receptor type 2 (CR2/CD21) gene demonstrate B cell specific hCR2 protein expression, normal B cell development and no changes in B cell subpopulations. To determine whether this BAC-encoded human CR2 (hCR2) can replace mouse CR2/CR1 in Cr2(-/-) mice and restore humoral immune responses to model foreign antigens (Ags), we generated hCR2(+/-)Cr2(-/-) tg mice and immunized them with sheep red blood cells (SRBC). We found that hCR2(+/-)Cr2(-/-) mice demonstrated anti-SRBC antibody (Ab) levels that were initially comparable to Cr2(-/-) mice after a single injection of the Ag, but then showed marked increases in anti-SRBC IgM and IgG1 levels after a second immunization. Identical results were found with a second model Ag, NP-Ficoll. To further confirm that this improvement in Ag-specific Ab production over Cr2(-/-) mice was indeed due to hCR2 expression, as well as to examine the effects of treating hCR2(+/-)Cr2(-/-) mice with an inhibitory anti-hCR2 monoclonal Ab (mAb) in vivo, we used mAb 171, an anti-hCR2 mAb that we have shown directly recognizes the C3d ligand binding site on hCR2. We first found that mAb 171 completely blocked hCR2-dependent co-activation of hCR2-tg B cells by anti-BCR/C3d complexes as measured in vitro by intracellular calcium influx. The i.p. injection of 1mg of mAb 171 was then found to induce for at least three weeks only partial loss of hCR2 surface expression, without modifying B and T cell numbers or the apparent activation status of the cells. Treatment of hCR2(+/-)Cr2(-/-) mice with mAb 171 also substantially suppressed the development of anti-SRBC and anti-NP Abs following immunization with Ags. The development of this model system should allow the study of the effects of manipulating hCR2 function in vivo with potential therapeutic compounds.     

Erythrocyte complement receptor type 1 (CR1) expression and circulating immune complex (CIC) levels in hydralazine-induced SLE.

Family studies were carried out to look at CR1 expression in 24 hydralazine-induced SLE patients (Hz Reactors), who had been off the drug for at least 1 year and were clinically well at the time of the study. Mean expression of CR1 was reduced by 27% in the group of hypertensives who had developed Hz-induced SLE compared with a group of 35 normal individuals. CR1 expression was also slightly reduced in the relatives of the Hz Reactors compared to the normal group. Using a solid-phase Clq binding assay, CIC levels were found to be elevated in the plasma of the Hz reactors and an inverse relationship was found between CR1 levels and CIC levels in this patient group. Both CR1 levels and CIC levels in Hz Reactors and normal individuals were constant over the 36 weeks studied. This study suggests that there is an association between an inability to deal efficiently with CIC and susceptibility to developing Hz-induced SLE.     

The IL-2/IL-2 receptor system: a target for rational immune intervention

The expression of interleukin 2 (IL-2), induction of its multisubunit receptor and subsequent interplay of this ligand with its receptor are pivotal events in T-cell activation. Our present understanding of the normal IL-2/IL-2R system and of disorders in this network in disease opens the possibility for more specific immune intervention. Thomas Waldmann discusses the clinical application of IL-2, agents that inhibit IL-2 synthesis and action, and how the anti-IL-2-receptor-directed therapy provides a novel perspective for prevention of allograft rejection and for treatment of graft versus host disease, select autoimmune disorders and certain neoplastic diseases.     

Complement receptor CR2/CR1 deficiency protects mice from collagen-induced arthritis and associates with reduced autoantibodies to type II collagen and citrullinated antigens

Collagen-induced arthritis (CIA), a model of autoimmune inflammatory arthritis, depends upon complement activation and effective B cell responses. To determine the importance of complement receptors CR2/CR1 in CIA, the Cr2−/− genotype was backcrossed onto the DBA/1j strain. CIA was induced by immunization with bovine type II collagen in CFA on days 0 and 21. Cr2−/− mice demonstrated a significantly diminished arthritis severity, decreased antibodies to bovine and murine collagen, and a significant reduction in antibodies to citrullinated antigens. Autoantibodies to citrullinated antigens have been shown to amplify anti-type II collagen passive transfer arthritis. To test the hypothesis that that simple replacement of such antibodies might re-establish severe disease in Cr2−/− mice, monoclonal antibodies to citrullinated antigens were administered to mice during the disease course. Although citrullinated antigens targeted by these antibodies were present within the joints of all mice, addition of these monoclonal antibodies increased disease severity only in Cr2+/+ mice. Taken together, these data suggest that CR2/CR1 are required to develop robust autoimmunity in the CIA model and that amplification of arthritis by antibodies to citrullinated antigens depends on factor(s) absent in arthritic Cr2−/− mice.     

The effect of E-N-l-trimethyllysine (TML) on the humoral and cellular immune response

E-N-l-Trimethyllysine glutamate (TML) influences the humoral and cellular immune response of mice. Chronic pre- and post-treatment (100 mg/kg/day, 5 times) transitionally increased the anti-SRBC haemagglutinin titre of female CBA mice. After 400 r whole body irradiation, TML treatment accelerated the normalization of the haemagglutinin level. TML treatment prolonged the life-span of BDF₁ hybrid mice that had first been immunized and then inoculated with L₁₂₁₀ cells. TML diminished the delayed type hypersensitivity mice and dosse-dependently decreased the spontaneous (SLMC) and antibody-dependent (ADCC) cytotoxicity of healthy human lymphocytes,in vitro. As a low molecular weight immunomodulant, TML may also be considered as a therapeutic tool.     

Difference in the clustering of complement receptor type 1 (CR1) on polymorphonuclear leukocytes and erythrocytes: effect on immune adherence

Complement receptor type 1 (CR1) mediates the adherence of complement-reacted immune complexes (IC) to various blood cells. On the erythrocyte, CR1 are clustered, a distribution which favors efficient multivalent binding of C3b-coated IC. IC can also bind to CR1 expressed on polymorphonuclear (PMN) leukocytes. To evaluate the respective importance of these two cell types in immune adherence reactions, functional analysis of IC binding, as well as morphological studies of CR1 distribution at their surface were undertaken. At equal cell concentrations, resting PMN leukocytes bound the same percentage of IC as erythrocytes, despite expressing four times more CR1 at their surface. At equal CR1 concentrations, IC binding to resting or formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN leukocytes was always lower than to erythrocytes. The morphological counterpart of these differences was studied by label-fracture immunoelectron microscopy. On erythrocytes, almost 50% of the CR1 were distributed in clusters of greater than or equal to 3 units, while less than 15% were grouped in such clusters on the surface of PMN leukocytes. Activation of PMN leukocytes by fMLP increased the surface density of CR1, but the proportion of clustered CR1 remained unchanged. These observations suggest that the low responsiveness of PMN leukocytes towards C3b-coated IC may be due to the unaggregated state of CR1. In the circulation, erythrocytes might function as a "buffer" for PMN leukocytes, which would otherwise engage too swiftly in reactions with IC.