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PubMed2004

The tetraspanin CD81 is necessary for partitioning of coligated CD19/CD21-B cell antigen receptor complexes into signaling-active lipid rafts.

Tetraspanins have been hypothesized to facilitate the organization of functional multimolecular membrane complexes. In B cells the tetraspanin CD81 is a component of the CD19/CD21 complex. When coligated to the B cell Ag receptor (BCR), the CD19/CD21 complex significantly enhances BCR signaling in part by prolonging the association of the BCR with signaling-active lipid rafts. In this study CD81 is shown to associate with lipid rafts upon coligation of the BCR and the CD19/CD21 complex. Using B cells from CD81-deficient mice we demonstrate that in the absence of CD81, coligated BCR and CD19/CD21 complexes fail to partition into lipid rafts and enhance BCR signaling from rafts. Furthermore, a chimeric CD19 protein that associates only weakly if at all with CD81 fails to promote the association of coligated BCR with lipid rafts. The requirement for CD81 to promote lipid raft association may define a novel mechanism by which tetraspanins function as molecular facilitators of signaling receptors.

Adjuvants, Immunologic↗
PubMed2004

Cutting edge: C3d functions as a molecular adjuvant in the absence of CD21/35 expression.

Complement component C3 covalently attaches to Ags following activation, where the C3d cleavage fragment can function as a molecular adjuvant to augment humoral immune responses. C3d is proposed to exert its adjuvant-like activities by targeting Ags to the C3d receptor (CD21/35) expressed by B cells and follicular dendritic cells. To directly assess the importance of CD21/35 in mediating the immunostimulatory effects of C3d, CD21/35-deficient (CD21/35(-/-)) mice were immunized with streptavidin (SA), SA-C3dg tetramers, recombinant HIV gp120 (gp120), or gp120 fused with linear multimers of C3d. Remarkably, SA- and gp120-specific Ab responses were significantly augmented in CD21/35(-/-) mice when these Ags were complexed with C3d in comparison to Ag alone. In fact, primary and secondary Ab responses and Ab-forming cell responses of CD21/35(-/-) mice approached those of wild-type mice immunized with SA-C3dg and gp120-C3d. Thus, C3d can function as a molecular adjuvant in the absence of CD21/35 expression.

Adjuvants, Immunologic↗
PubMed2004

The electrostatic nature of C3d-complement receptor 2 association.

The association of complement component C3d with B or T cell complement receptor 2 (CR2 or CD21) is a link between innate and adaptive immunity. It has been recognized in experimental studies that the C3d-CR2 association is pH- and ionic strength-dependent. This led us to perform electrostatic calculations to obtain a theoretical understanding of the mechanism of C3d-CR2 association. We used the crystallographic structures of human free C3d, free CR2 (short consensus repeat (SCR)1-2), and the C3d-CR2(SCR1-2) complex, and continuum solvent representation, to obtain a detailed atomic-level picture of the components of the two molecules that contribute to association. Based on the calculation of electrostatic potentials for the free and bound species and apparent pK(a) values for each ionizable residue, we show that C3d-CR2(SCR1-2) recognition is electrostatic in nature and involves not only the association interface, but also the whole molecules. Our results are in qualitative agreement with experimental data that measured the ionic strength and pH dependence of C3d-CR2 association. Also, our results for the native molecules and a number of theoretical mutants of C3d explain experimental mutagenesis studies of amino acid replacements away from the association interface that modulate binding of iC3b with full-length CR2. Finally, we discuss the packing of the two SCR domains. Overall, our data provide global and site-specific explanations of the physical causes that underlie the ionic strength dependence of C3d-CR2 association in a unified model that accounts for all experimental data, some of which were previously thought to be contradictory.

Complement C3d↗
PubMed2004

Distinct sequences in the cytoplasmic domain of complement receptor 2 are involved in antigen internalization and presentation.

B cells express randomly rearranged surface Ig that forms part of a multiprotein complex known as the B cell receptor (BCR). Recognition of Ag via this receptor results in its capture, internalization, proteolysis and presentation to CD4+ T cells. The recognition of Ag by CD4+ T cells is critical for the selection of individual B cells, leading to the eventual secretion of a high affinity version of the BCR as an effective circulating Ab. B cells also express other receptors that recognize Ags associated with components of innate immunity. One of these receptors, CR2, binds Ags coated with activated complement components. Studies have shown that cross-linking CR2 and the BCR with complement-tagged Ags leads to enhanced Ag presentation by B cells. In addition, Ags targeted to B cell CR2 in the absence of BCR coligation are also efficiently presented to T cells. In this report, we identify several distinct sequences within the cytoplasmic domain of mouse CR2 (mCR2) that are essential for mCR2-mediated Ag presentation in both the presence and the absence of BCR cross-linking. The finding that distinct sequences in the cytoplasmic domain of mCR2 are essential for BCR-independent Ag presentation leads us to propose a novel role for CR2.

Amino Acid Sequence↗
PubMed2004

Anti-phospholipid antibodies restore mesenteric ischemia/reperfusion-induced injury in complement receptor 2/complement receptor 1-deficient mice.

Complement receptor 2-deficient (Cr2(-/-)) mice are resistant to mesenteric ischemia/reperfusion (I/R) injury because they lack a component of the natural Ab repertoire. Neither the nature of the Abs that are involved in I/R injury nor the composition of the target Ag, to which recognition is lacking in Cr2(-/-) mice, is known. Because anti-phospholipid Abs have been shown to mediate fetal growth retardation and loss when injected into pregnant mice, we performed experiments to determine whether anti-phospholipid Abs can also reconstitute I/R injury and, therefore, represent members of the injury-inducing repertoire that is missing in Cr2(-/-) mice. We demonstrate that both murine and human monoclonal and polyclonal Abs against negatively charged phospholipids can reconstitute mesenteric I/R-induced intestinal and lung tissue damage in Cr2(-/-) mice. In addition, Abs against beta2 glycoprotein I restore local and remote tissue damage in the Cr2(-/-) mice. Unlike Cr2(-/-) mice, reconstitution of I/R tissue damage in the injury-resistant Rag-1(-/-) mouse required the infusion of both anti-beta2-glycoprotein I and anti-phospholipid Ab. We conclude that anti-phospholipid Abs can bind to tissues subjected to I/R insult and mediate tissue damage.

Animals↗
PubMed2004

The virulence function of Streptococcus pneumoniae surface protein A involves inhibition of complement activation and impairment of complement receptor-mediated protection.

Complement is important for elimination of invasive microbes from the host, an action achieved largely through interaction of complement-decorated pathogens with various complement receptors (CR) on phagocytes. Pneumococcal surface protein A (PspA) has been shown to interfere with complement deposition onto pneumococci, but to date the impact of PspA on CR-mediated host defense is unknown. To gauge the contribution of CRs to host defense against pneumococci and to decipher the impact of PspA on CR-dependent host defense, wild-type C57BL/6J mice and mutant mice lacking CR types 1 and 2 (CR1/2(-/-)), CR3 (CR3(-/-)), or CR4 (CR4(-/-)) were challenged with WU2, a PspA(+) capsular serotype 3 pneumococcus, and its PspA(-) mutant JY1119. Pneumococci also were used to challenge factor D-deficient (FD(-/-)), LFA-1-deficient (LFA-1(-/-)), and CD18-deficient (CD18(-/-)) mice. We found that FD(-/-), CR3(-/-), and CR4(-/-) mice had significantly decreased longevity and survival rate upon infection with WU2. In comparison, PspA(-) pneumococci were virulent only in FD(-/-) and CR1/2(-/-) mice. Normal mouse serum supported more C3 deposition on pneumococci than FD(-/-) serum, and more iC3b was deposited onto the PspA(-) than the PspA(+) strain. The combined results confirm earlier conclusions that the alternative pathway of complement activation is indispensable for innate immunity against pneumococcal infection and that PspA interferes with the protective role of the alternative pathway. Our new results suggest that complement receptors CR1/2, CR3, and CR4 all play important roles in host defense against pneumococcal infection.

Animals↗
PubMed2004

Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice.

Complement plays a critical role in the immune response by opsonizing immune complexes (IC) and thymus-independent type 2 Ags with C3 breakdown product C3dg, a CR2-specific ligand. We used a C3dg-opsonized IC model, anti-CR1/2 mAb 7G6, to investigate how such substrates are processed. We used RIA, whole body imaging, flow cytometry, and fluorescence immunohistochemistry to examine the disposition of 0.1- to 2-microg quantities of mAb 7G6 infused i.v. into BALB/c mice. The mAb is rapidly taken up by the spleen and binds preferentially to marginal zone (MZ) B cells; within 24 h, the MZ B cells relocate and transfer mAb 7G6 to follicular dendritic cells (FDC). Transfer occurs coincident with loss of the extracellular portion of MZ B cell CR2, suggesting that the process may be mediated by proteolysis of CR2. Intravenous infusion of an FDC-specific mAb does not induce comparable splenic localization or cellular reorganization, emphasizing the importance of MZ B cells in intrasplenic trafficking of bound substrates. We propose the following mechanism: binding of C3dg-opsonized IC to noncognate MZ B cells promotes migration of these cells to the white pulp, followed by CR2 proteolysis, which allows transfer of the opsonized IC to FDC, thus facilitating presentation of intact Ags to cognate B cells.

Animals↗
PubMed2005

B cells from mice prematurely expressing human complement receptor type 2 are unresponsive to T-dependent antigens.

Complement receptor type 2 (CR2/CD21), in association with CD19, plays an important role in enhancing mature B cell responses to opsonized Ags. We have shown that mice expressing a human CR2/CD21 (hCR2/CD21) transgene during the CD43(+)/CD25(-) late pro-B cell stage of B cell development demonstrate marked changes in subsequent B cell ontogeny. In the present study, we show that the humoral immune response to the T cell-dependent Ag, sheep RBC, is muted severely in a manner inversely proportional to B cell expression level of hCR2. Individual Ag-specific IgG isotypes vary in the degree to which they are affected but all are reduced while IgM titers are normal. A substantial reduction in germinal centers, both in size and frequency, in the spleens of immunized hCR2 transgenic mice demonstrates a failure to maintain germinal center reaction. However, both IgM expression levels and LPS-proliferative responses appear fully intact in B cells from hCR2-positive mice, suggesting that this alteration in B cell phenotype is different qualitatively from that of specific Ag-defined anergy models. These data suggest that the unresponsiveness to T-dependent Ags displayed by hCR2-positive B cells is linked to an increase in the level of stimulus required to propel the B cell into a fully activated state and thus a normal humoral immune response to Ags. We conclude that this phenotype and these mice may offer an additional means to dissect mechanisms underlying B cell tolerance and Ag responsiveness both in bone marrow and periphery.

Animals↗
PubMed2005

Coligation of the B cell receptor with complement receptor type 2 (CR2/CD21) using its natural ligand C3dg: activation without engagement of an inhibitory signaling pathway.

C3dg is a cleavage product of the C3 component of complement that can facilitate the coligation of the complement receptor 2 (CR2/CD21) with the BCR via C3dg/Ag complexes. This interaction can greatly amplify BCR-mediated signaling events and acts to lower the threshold for B cell activation. Although previous studies have used anti-CR2 Abs or used chimeric Ags in the context of BCR transgenic mice as surrogate C3d-containing ligands, we have used a physiological form of C3d to study signaling in B cells from wild-type C57BL/6 mice. We find that while CR2-enhanced BCR signaling causes intracellular Ca2+ mobilization and total pTyr phosphorylation of an intensity comparable to optimal BCR ligation using anti-IgM Abs, it does so with limited activation of inhibitory effectors (such as CD22, Src homology region 2 domain containing phosphatase 1, and SHIP-1) and without substantial receptor cross-linking. In summary, we demonstrate that CR2-enhanced BCR signaling may proceed not only through the previously described amplification of positive signaling pathways, but is potentially augmented by a lack of normal inhibitory/feedback signaling.

Animals↗
PubMed2005

The influence of immune complex-bearing follicular dendritic cells on the IgM response, Ig class switching, and production of high affinity IgG.

It is believed that Ag in immune complexes (ICs) on follicular dendritic cells (FDCs) selects high affinity B cells and promotes affinity maturation. However, selection has been documented in the absence of readily detectable ICs on FDCs, suggesting that FDC-ICs may not be important. These results prompted experiments to test the hypothesis that IC-bearing murine FDCs can promote high affinity IgG responses by selecting B cells after stimulating naive IgM(+) cells to mature and class switch. Coculturing naive lambda(+) B cells, FDCs, (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (CGG) + anti-CGG ICs, and CGG-primed T cells resulted in FDC-lymphocyte clusters and production of anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl. Class switching was indicated by a shift from IgM to IgG, and affinity maturation was indicated by a change from mostly low affinity IgM and IgG in the first week to virtually all high affinity IgG anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl in the second week. Class switching and affinity maturation were easily detectable in the presence of FDCs bearing appropriate ICs, but not in the absence of FDCs. Free Ag plus FDCs resulted in low affinity IgG, but affinity maturation was only apparent when FDCs bore ICs. Class switching is activation-induced cytidine deaminase (AID) dependent, and blocking FDC-CD21 ligand-B cell CD21 interactions inhibited FDC-IC-mediated enhancement of AID production and the IgG response. In short, these data support the concept that ICs on FDCs can promote AID production, class switching, and maturation of naive IgM(+) B cells, and further suggest that the IC-bearing FDCs help select high affinity B cells that produce high affinity IgG.

Animals↗
PubMed2005

Complement component C3d-antigen complexes can either augment or inhibit B lymphocyte activation and humoral immunity in mice depending on the degree of CD21/CD19 complex engagement.

C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 microg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes.

Animals↗
PubMed2005

Several genes contribute to the production of autoreactive B and T cells in the murine lupus susceptibility locus Sle1c.

The systemic lupus erythematosus 1 (Sle1) locus mediates the loss of tolerance to nuclear Ags in the NZM2410 mouse model of lupus through intrinsic defects in both B and T cells. Congenic analysis has shown that Sle1 corresponds to at least three genetic loci, Sle1a, Sle1b, and Sle1c. Telomeric Sle1c is associated with abnormal B cell responses to subthreshold stimulation with anti-IgM and C3d and with decreased T-dependent humoral immune responses. We have proposed that these phenotypes resulted from polymorphisms in the C3 complement receptor Cr2 gene. We have also found that Sle1c was associated with the production of histone-specific autoreactive CD4(+) T cells, which correlated with higher activation and proliferative responses, and a reduction in the CD4(+)CD25(+)CD62L(+)forkhead/winged helix transcription factor gene (Foxp3(+)) compartment. In this study we showed, using congenic recombinants, that the decreased humoral immune response and impaired GC formation map to the NZM2410 Cr2 allele. A chronic graft-vs-host disease model also showed that Sle1c produces significantly more autoreactive B cells than B6 controls, and that this phenotype maps to two regions excluding the Cr2 gene. Mixed bone marrow chimera demonstrated that the increased activation, proliferative response, and reduced regulatory T cell compartment were intrinsic to Sle1c-expressing CD4(+) T cells. These phenotypes mapped to the same two loci identified with the chronic graft-vs-host disease model, excluding the Cr2 region. Overall, these results show that Sle1c results in the production of autoreactive B and T cells through the expression of three different genes, one of which is consistent with Cr2, based on the phenotypes of the Cr2-deficient mice, and the other two corresponding to as yet unidentified genes.

Animals↗
PubMed2005

A critical role for complement C3d and the B cell coreceptor (CD19/CD21) complex in the initiation of inflammatory arthritis.

Complement C3 cleavage products mediate the recognition and clearance of toxic or infectious agents. In addition, binding of the C3d fragment to Ag promotes B lymphocyte activation through coengagment of the BCR and complement receptor 2 (CD21). Signal augmentation is thought to be achieved through enhanced recruitment and activation of CD21-associated CD19. In this study we show, using the DBA/1 collagen-induced arthritis (CIA) model, that conjugation of C3d to heterologous type II collagen is sufficient to cause disease in the absence of the mycobacterial components of CFA. Transient depletion of C3 during the inductive phase of CIA delays and lessens the severity of disease, and DBA/1 mice deficient for coreceptor components CD19 or CD21 are not susceptible to CIA. Adoptive transfer experiments revealed that CD21 expression on either B cells or follicular dendritic cells is sufficient to acquire disease susceptibility. Although CD19(-/-) and CD21(-/-) mice produce primary Ab responses to heterologous and autologous type II collagen, they are impaired in the ability to activate T cells, form germinal centers, and produce secondary autoantibody responses. These findings indicate that binding of C3d to self-Ags can promote autoimmunity through enhanced Ag retention and presentation by follicular dendritic cells and B cells, respectively.

Animals↗
PubMed2006

Complement-dependent P-selectin expression and injury following ischemic stroke.

The mechanisms that contribute to inflammatory damage following ischemic stroke are poorly characterized, but studies indicate a role for both complement and P-selectin. In this study, we show that compared with wild-type mice, C3-deficient mice showed significant improvement in survival, neurological deficit, and infarct size at 24 h after middle cerebral artery occlusion and reperfusion. Furthermore, P-selectin protein expression was undetectable in the cerebral microvasculature of C3-deficient mice following reperfusion, and there was reduced neutrophil influx, reduced microthrombus formation, and increased blood flow postreperfusion in C3-deficient mice. We further investigated the use of a novel complement inhibitory protein in a therapeutic paradigm. Complement receptor 2 (CR2)-Crry inhibits complement activation at the C3 stage and targets to sites of complement activation. Treatment of normal mice with CR2-Crry at 30 min postreperfusion resulted in a similar level of protection to that seen in C3-deficient mice in all of the above-measured parameters. The data demonstrate an important role for complement in cerebrovascular thrombosis, inflammation, and injury following ischemic stroke. P-selectin expression in the cerebrovasculature, which is also implicated in cerebral ischemia and reperfusion injury, was shown to be distal to and dependent on complement activation. Data also show that a CR2-targeted approach of complement inhibition provides appropriate bioavailability in cerebral injury to enable complement inhibition at a dose that does not significantly affect systemic levels of serum complement activity, a potential benefit for stroke patients where immunosuppression would be undesirable due to significantly increased susceptibility to lung infection.

Animals↗
PubMed2006

Cutting edge: Epstein-Barr virus transactivates the HERV-K18 superantigen by docking to the human complement receptor 2 (CD21) on primary B cells.

EBV, a ubiquitous human herpesvirus, is the causative agent of infectious mononucleosis and is associated with many carcinomas. We have previously shown that the EBV latent genes LMP-1 and LMP-2A (for latent membrane proteins 1 and 2A), transactivate a human endogenous retrovirus (HERV), HERV-K18, in infected B lymphocytes. The envelope (Env) protein of HERV-K18 encodes a superantigen that strongly stimulates a large number of T cells. In this study we report that HERV-K18 env is transactivated even earlier in the infection process, before the establishment of latency; namely, we found that EBV, through its interaction with its cellular receptor CD21, induces the HERV-K18 env gene in resting B lymphocytes. This transactivation is direct and immediate, as up-regulation of transcripts can be detected within 30 min after EBV exposure. Thus, EBV binding to human CD21 on resting B cells triggers the expression of an endogenous superantigen. The biological significance of this superantigen expression for the EBV life cycle is discussed.

Animals↗
PubMed2006

Factor I-mediated processing of complement fragments on HIV immune complexes targets HIV to CR2-expressing B cells and facilitates B cell-mediated transmission of opsonized HIV to T cells.

Our study demonstrates that binding of complement-opsonized HIV to complement receptor type 1 on human erythrocytes (E) via C3b fragments is followed by a rapid normal human serum-mediated detachment of HIV from E. The release was dependent on the presence of factor I indicating a conversion of C3b fragments to iC3b and C3d on the viral surface. This in turn resulted in an efficient binding of opsonized HIV to CR2-expressing B cells, thus facilitating B cell-mediated transmission of HIV to T cells. These data provide a new dynamic view of complement opsonization of HIV, suggesting that association of virus with E might be a transient phenomenon and the factor I-mediated processing of C3b to iC3b and C3d on HIV targets the virus to complement receptor type 2-expressing cells. Thus, factor I in concert with CR1 on E and factor H in serum due to their cofactor activity are likely to be important contributors for the generation of C3d-opsonized infectious HIV reservoirs on follicular dendritic cells and/or B cells in HIV-infected individuals.

B-Lymphocytes↗
PubMed2006

Brain edema after intracerebral hemorrhage in rats: the role of inflammation.

BACKGROUND: Intracerebral hemorrhage (ICH) results in secondary brain edema and injury that may lead to death and disability. ICH also causes inflammation. It is unclear whether inflammation contributes to brain edema and neuron injury or functions in repairing the brain tissue. AIMS: To understand the effect of inflammation in ICH, we have carried out an investigation on the various aspects and the dynamic changes of inflammation. SETTINGS AND DESIGN: An ICH model was generated by injecting 50 microl autologous tail artery blood stereotactically into the right caudate nucleus of 30 rats, which were randomly divided into five ICH groups. Similarly, five Sham control groups were generated by inserting the needle to the right caudate nucleus of rats. MATERIALS AND METHODS: Rat behavior was evaluated over the time course (6 h, 24 h, 48 h, 72 h and 7 d) in each group. The rats were then killed by administering an overdose of pentobarbital. Following the euthanasia, the brain water content, neuronal loss, glia proliferation, inflammatory infiltration and brain morphology of the rats were measured. Additionally, the expression of TNF-alpha, IL-6, ICAM-1, VEGF, NF-kappaB, C3 and CR2 was analyzed by immunohistochemistry. STATISTICAL ANALYSIS: The data were analyzed by student's t test. RESULTS: Rat brain water content increased progressively over the time course and reached its peak at 48 h followed ICH. The maximum of inflammatory infiltrate (especially neutrophils) and immunopositive cells of TNF-alpha, IL-6 and NF-kappaB, were at 48 h. The expression of C3 and CR2 reached their peaks at 48-72 h, while the expression ICAM-1 and VEGF were at maximum at 72 h followed ICH. CONCLUSIONS: The results suggested that the inflammatory cytokines, complement system and VEGF may have a function in the development of the brain edema and neuron injury followed ICH.

Animals↗