A critical role for complement C3d and the B cell coreceptor (CD19/CD21) complex in the initiation of inflammatory arthritis.
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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. (+info)
Antibody-mediated rejection in human cardiac allografts: evaluation of immunoglobulins and complement activation products C4d and C3d as markers.
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Antibody-mediated rejection (AMR) in human heart transplantation is an immunopathologic process in which injury to the graft is in part the result of activation of complement and it is poorly responsive to conventional therapy. We evaluated by immunofluorescence (IF), 665 consecutive endomyocardial biopsies from 165 patients for deposits of immunoglobulins and complement. Diffuse IF deposits in a linear capillary pattern greater than 2+ were considered significant. Clinical evidence of graft dysfunction was correlated with complement deposits. IF 2+ or higher was positive for IgG, 66%; IgM, 12%; IgA, 0.6%; C1q, 1.8%; C4d, 9% and C3d, 10%. In 3% of patients, concomitant C4d and C3d correlated with graft dysfunction or heart failure. In these 5 patients AMR occurred 56-163 months after transplantation, and they responded well to therapy for AMR but not to treatment with steroids. Systematic evaluation of endomyocardial biopsies is not improved by the use of antibodies for immunoglobulins or C1q. Concomitant use of C4d and C3d is very useful to diagnose AMR, when correlated with clinical parameters of graft function. AMR in heart transplant patients can occur many months or years after transplant. (+info)
Role of complement receptor type 2 and endogenous complement in the humoral immune response to conjugates of complement C3d and pneumococcal serotype 14 capsular polysaccharide.
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Conjugation of the complement fragment C3d to both T-cell-dependent (TD) protein and T-cell-independent type 2 (TI-2) polysaccharide antigens enhances the humoral immune response in mice immunized with either type of antigen. However, the ability of C3d-protein conjugates to enhance the antibody response in mice deficient in complement receptor types 1 and 2 (CR1 and CR2) has raised questions about the role of C3d-CR2 interactions in the adjuvant effect of C3d. In this study, we examined the role of CR2 binding and endogenous complement activation in the antibody response to conjugates of C3d and serotype 14 pneumococcal capsular polysaccharide (PPS14). To block binding of PPS14-C3d conjugates to CR2, mice were immunized with a mixture of vaccine and (CR2)2-immunoglobulin G1 (IgG1). Mice receiving (CR2)2-IgG1 at the time of primary immunization had a marked reduction in the primary anti-PPS14 antibody response but an enhanced secondary anti-PPS14 response, suggesting that C3d-CR2 interactions are required for the primary response but can have negative effects on the memory response. Further, compared with mice receiving PPS14-C3d having a high C3d/PPS14 ratio, mice immunized with PPS14-C3d with low C3d/PPS14 ratios had an enhanced secondary antibody response. Treatment of mice with cobra venom factor to deplete complement had insignificant effects on the antibody response to PPS14-C3d. Experiments with CBA/N xid mice confirmed that PPS14-C3d conjugates retain the characteristics of TI-2 rather than TD antigens. Thus, the adjuvant effect of C3d conjugated to PPS14 requires C3d-CR2 interactions, does not require activation of endogenous complement, and is not mediated by TD carrier effects. (+info)
Factor H and atypical hemolytic uremic syndrome: mutations in the C-terminus cause structural changes and defective recognition functions.
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Atypical hemolytic uremic syndrome is a disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations in the complement regulator factor H are associated with the inherited form of the disease, and >60% of the mutations are located within the C terminus of factor H. The C-terminus of factor H, represented by short consensus repeat 19 (SCR19) and SCR20, harbors multiple functions; consequently, this study aimed to examine the functional effects of clinically reported mutations in these SCR. Mutant factor H proteins (W1157R, W1183L, V1197A, R1210C, R1215G, and P1226S) were recombinantly expressed and functionally characterized. All six mutant proteins showed severely reduced heparin, C3b, C3d, and endothelial cell binding. By peptide spot analyses, four linear regions that are involved in heparin, C3b, and C3d binding were localized in SCR19 and SCR20. A three-dimensional homology model of the two domains suggests that these four regions form a common binding site across both domains. In addition, this structural model identifies two types of residues: Type A residues are positioned on the SCR surface and are represented by mutants W1157R, W1183L, R1210C, and R1215G; and type B residues are buried within the SCR structure and affect mutations V1197A and P1226S. Mutations of both types of residue result in the same functional defects, namely the reduced binding of factor H to surface-attached C3b molecules and reduced complement regulatory activity at the cell surfaces. The buried type B mutations seem to affect ligand interaction of factor H more severely than the surface-exposed mutations. (+info)
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.
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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. (+info)
Disease-associated sequence variations congregate in a polyanion recognition patch on human factor H revealed in three-dimensional structure.
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Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site. (+info)
Deficiency of C4 from donor or recipient mouse fails to prevent renal allograft rejection.
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Complement effector products generated in the transplanted kidney are known to mediate transplant rejection, but which of the three main activation pathways of complement trigger this response is unclear. Here we assessed the role of the classical and lectin pathways by studying the common component C4 in mouse kidney transplant rejection. We transplanted wild-type or C4-null H-2(b) donor kidneys into H-2(k) or H-2(d) recipients, or vice-versa, to assess the roles of donor kidney and recipient expression of complement. Intragraft C4 gene expression rose substantially during rejection. However, we found no significant association between graft acceptance and the presence of C4 in either the donor kidney or recipient mouse. At the time of rejection, we found no significant differences in alloantibody response in the different groups. Tubular deposition of C3 to C9 occurred regardless of the absence or presence of C4 in either the donor or recipient mouse, indicating that C4 was dispensable for complement activation at this site. These data suggest that complement activation and renal allograft rejection are independent of the classical and lectin pathways in these models, implying that in the absence of these pathways the alternative pathway is the main trigger for complement-mediated rejection. (+info)
Decreased capacity of immune cells to cause tissue injury mediates kidney ischemic preconditioning.
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Ischemic preconditioning (IP) is a well-established phenomenon, and the underlying mechanisms of IP are thought to involve adaptive changes within the injured tissue. Because one of the main functions of immune cells is to harbor memory, we hypothesized that circulating immune cells could mediate IP by responding to an initial ischemia reperfusion injury (IRI) and then mediate decreased injury after a second IRI event. C57BL/6 mice underwent 30 min of bilateral renal clamping or sham operation. At 5 days after ischemia, purified leukocytes from spleen were adoptively transferred into T cell-deficient (nu/nu) mice. After 1 wk, these mice underwent 30 min of renal IRI. The nu/nu mice receiving leukocytes from ischemic wild-type mice had significantly reduced renal injury compared with nu/nu mice receiving leukocytes from sham-operated, wild-type mice. Infiltration of neutrophil and macrophage in postischemic kidney did not correlate with the protection. No difference in kidney C3d or IgG deposition was detected between groups. Given that inducible NO synthase (iNOS) has been implicated in IP, leukocytes from ischemic or sham-operated, iNOS-deficient mice were transferred into nu/nu mice. Effects similar to those of wild-type transfer of ischemic leukocytes were demonstrated; thus, iNOS was not mediating the IP effect of leukocytes. This is the first evidence that immune cells are primed after renal IRI and thereby lose the capacity to cause kidney injury during a second episode of IRI. This finding may also be relevant for elucidating the mechanisms underlying cross-talk between injured kidney and distant organs. (+info)