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PubMed1986

Decreased expression of the C3b/C4b receptor (CR1) and the C3d receptor (CR2) on B lymphocytes and of CR1 on neutrophils of patients with systemic lupus erythematosus.

Decreased numbers of complement receptor type 1 (CR1) have been observed on erythrocytes of patients with systemic lupus erythematosus (SLE) and on glomerular podocytes of patients having proliferative nephritis of SLE. In the present study, the analysis of the cellular expression of CR1 has been extended to include leukocytes. In addition, expression by B lymphocytes of the C3d receptor (CR2), which also serves as the receptor for the Epstein-Barr virus, was assessed. Receptor expression was measured by 2-color fluorescent flow cytometry of peripheral blood B cells, identified by the presence of the B1 antigen, that had also been stained with anti-CR1 or anti-CR2. B cells from 17 patients with SLE exhibited a mean relative fluorescence for CR1 that was 61% of that found in 17 normal individuals (P less than 0.001). The expression of CR2 by the patients' B cells (n = 14) was 62% of that of the B cells from normal subjects (n = 17) (P less than 0.001). The expression of CR1 correlated with that of CR2 among patients (r = 0.63; P less than 0.01) but not with the expression of CR2 among normal individuals (r = 0.36; P greater than 0.1). The mean CR1 content of the patients' neutrophils was only 59% of the normal mean (P less than 0.001). Thus, abnormalities of complement receptor expression occur on the leukocytes of patients with SLE. These deficiencies may be secondary to interaction of the cells with the products of complement activation, or, in some individuals, the deficiencies may be familial.

Antibodies, Antinuclear↗
PubMed1983

Immune complexes and complement profile in essential mixed cryoglobulinemia before and after plasma exchange.

Five patients affected by essential mixed cryoglobulinemia (EMC) with renal involvement unresponsive to high doses of corticosteroids, have been treated with 16 plasma exchanges (PE). The plasma removed at each apheresis was 1652 +/- 416 ml. The following data were evaluated before and after PE: levels of immune complexes as detected by cryocrit and C1q binding assay; total complement activity (CH50) and alternative pathway complement activity (APCH 50); concentrations of 9 complement components (C1q, C1s, C4, C3, C5, C6, B, I and H) and of the C3 split product C3d. By definition all serum samples had detectable cryoprecipitates and 4 out 5 had C1q binding activity higher than the upper limit of 2 SD. The effect of the PE was the decrease of cryocrit and of C1q binding activity approximately to one half their initial values. The basal complement profile suggested and excessive activation of complement through the classical pathway. Indeed we found low levels of the early complement components (C1q, C1s and C4), reduced CH50, normal levels of C3 and an increase of C3 split product C3d concentrations. APCH50 as well as C5, C6, B, I and H concentrations were found within the normal range. After each PE a significant decrease of the previously normal components was observed. The decrease was independent of the replacement fluid used (5% albumin solution or fresh plasma or cryoglobulin-depleted autologous plasma) and had a short duration. Indeed all these components after the drop following the PE increased rapidly reaching the basal level over a period of 24-48 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-Antibody Complex↗
PubMed2001

Complement deposition in renal allografts with early malfunction.

Among patients with early severe impairment of renal allograft function we have previously identified a group displaying isolated deposition of complement factor C3 in glomeruli. Here we studied the pattern of complement deposition more extensively in allograft biopsies from five patients using an immunofluorescence technique. We found a prominent deposition of C3c, C3d and C4d antigens in the glomerular capillary walls, and a positive reaction to vitronectin (S-protein), but only trace amounts of the complement factor C9 neoepitope. Clq, C4c, C3a, iC3b, factor B, properdin, immunoglobulins IgG, IgA or IgM were not found in glomeruli or in any other cortical structure. These findings indicate that most of the demonstrated glomerular C3 consists of C3b and/or C3c/C3d molecules. By immunoelectron microscopy the C3 antigen was found within the glomerular basement membrane. Our findings indicate that there is a mechanism of complement activation involving the early steps of the classical pathway, despite the lack of demonstrable immunoglobulins in the tissue. In analogy with similar reactions described recently in heart allografts, we suggest that this may be a manifestation of a humoral rejection, possibly mediated by a low titer of circulating antibodies directed against endothelial surface antigens, presumed to be the initial step leading to complement activation.

Adult↗
PubMed1993

Identification of proteins absorbed to hemodialyser membranes from heparinized plasma.

The protein layers formed during contact of plasma with hemodialysis membranes were studied. Dialysers having membranes of cellulose acetate (CA), saponified cellulose ester (SCE), cuprophane (CUP), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN) were used. Heparinized human plasma was recirculated through the dialysers for four hours. They were then rinsed and the proteins adsorbed to the membranes were eluted with 2% SDS. The yields of protein from the different membranes increased in the order: PMMA < CA < SCE < CUP < PAN. This is the probable order of increasing hydrophilicity. SDS-PAGE and Western blots were performed on the dialyser eluates. The blots were positive for most of the twenty proteins tested for. There were some interesting differences in adsorption patterns among the different membrane materials, notably for high molecular weight kininogen (HMWK), plasminogen and the C3 component of complement. HMWK was intact in the eluates from CA, CUP and SCE, whereas on PMMA and PAN there was evidence of cleavage, suggesting that activation of the contact phase of coagulation was more extensive on the latter two materials. Intact plasminogen was visible on all the blots. However, low molecular weight fragments were visible in the PAN eluates, suggesting activation of the fibrinolytic pathway. Low molecular weight fibrinogen fragments eluted from PAN membranes support this conclusion. C3 was visible in the blots obtained for all membrane materials, and the data suggest that complement is activated by all the membranes. A C3 fragment at about 30 kD (possibly C3d) was seen in the blots for the cellulosic membranes but not for PMMA or PAN.

Adsorption↗
PubMed1980

Three rat monoclonal antibodies to human C3.

Three monoclonal antibodies to human C3 have been obtained from a fusion of the rat myeloma line Y3 Ag 1.2.3. with spleen cells from rats immunized against C3. One, from clone 4, reacts with an antigenic determinant in C3c showing the expected reactivity of the 'C' antigen of C3. The specificity of the other two monoclonal antibodies correspond less clearly with known C3 antigens. By agglutination analysis of complement coated cells the determinant reacting with clone 3 is present in C3d while that for clone 9 appears as a neoantigen on C3bi. In both cases the co-precipitation results are anomalous and more direct studies are needed to define the exact specificity. The possibility that internal sequence duplications in C3 may explain some anomalies is discussed. None of the monoclonal antibodies significantly inhibit C3 functions. The monoclonal antibodies have been found to have unusual properties in co-precipitation assays being able to diffuse through a precipitation line with which they react to react with a further line. One antibody is also able to react strongly with the anodal half of what appears as a single line with a polyclonal antiserum.

Agglutination Tests↗
PubMed1996

Inhibition of human complement by a C3-binding peptide isolated from a phage-displayed random peptide library.

We have screened a phage-displayed random peptide library for binding to C3b, the proteolytically activated form of complement component C3, and have identified a novel peptide that suppresses complement activation. This phage-displayed peptide bound to C3, C3b, and C3c, but not to C3d, indicating that it binds to the C3c region of C3. A synthetic 27-amino acid peptide corresponding to the phage-displayed peptide also bound to C3 and C3 fragments and inhibited both the classical and alternative pathways of complement activation. The inhibition of complement activation was reversible. Studies with overlapping peptides indicated that the functional activity was located in the cyclic 13-amino acid N-terminal region (ICVVQDWGHHRCT) of the parent peptide. Reduction and alkylation of this 13-residue synthetic peptide destroyed its inhibitory activity. Analysis of the mechanism of inhibition revealed that the peptide inhibited C3 cleavage in normal human serum as well as when the alternative pathway was reconstituted with purified complement components, and the observed inhibition was not due to sterically hindered access to the C3a/C3b cleavage site. Further, the peptide did not inhibit the cleavage of factor B, indicating that it did not affect the interaction of CA with factor B or the formation of C3b,Bb. The peptide also had no effect on the binding of properdin to C3, demonstrating that the observed inhibition of C3 cleavage in normal human serum was not due in part to its effect on the properdin-stabilized C3 convertase, C3b,Bb,P. These results indicate that the peptide we have identified interacts with C3 to inhibit its activation.

Amino Acid Sequence↗
PubMed1984

Attachment and phagocytosis by salmon macrophages of agarose beads coated with human C3b and C3bi.

Agarose beads (diameter 5-10 micron) preincubated in human serum became associated (attached and ingested) to 50-60% of the salmon macrophages within 60 minutes. However, Beads preincubated in serum treated with heating (50 degrees C, 20 min) or with EDTA (10 mM) to inhibit the activation of alternative complement pathway, were not associated to the phagocytes. Furthermore, agarose beads coated with human C3b and C3bi after incubation with isolated complement factors (C3, D, B), were associated to 30-40% of the phagocytes. About 80% of the cell-associated agarose beads was intracellularly located. Conversion by trypsin treatment (0.01%) of agarose bound C3bi to C3d, abolished the association of such beads to the macrophages. The results demonstrate that salmon macrophages possess complement receptors that bind human C3b and C3bi. Agarose beads coated with these ligands (C3b and C3bi) are attached and ingested by the phagocytes.

Animals↗
PubMed2002

T cell activation induced by cross-linking CD3 and CD28 leads to silencing of Epstein-Barr virus/C3d receptor (CR2/CD21) gene and protein expression.

Complement receptor II (CR2) also known as CD21 is the receptor for C3d on immune complexes. In humans it serves as a receptor for the Epstein-Barr virus (EBV). CR2 is expressed on B cells and in low density in the T cell lineage. EBV can infect T cells and EBV-positive T lymphomas have been described. Although CR2 mRNA is readily detectable in T cells, the function of CR2 in human T lymphocytes remains elusive. Here we have analyzed the expression of CR2 in normal and activated T cells. PCR analyses and immunofluorescence/confocal microscopy of peripheral blood T cells and of activated T cells shows considerable reduction in CR2 mRNA and protein expression upon activation. The downregulation of CR2 expression may modulate life span or immunological reactivity of T cells and the susceptibility of cells to infection by lymphotropic viruses.

B-Lymphocytes↗
PubMed1986

Superinfection of epithelial hybrid cells (D98/HR-1, NPC-KT, and A2L/AH) with Epstein-Barr virus and the relationship to the C3d receptor.

Three Epstein-Barr virus (EBV) genome-positive epithelial/hybrid cell lines (D98/HR-1, NPC-KT, and A2L/AH) were superinfected with EBV derived from P3HR-1 (HR-1), NPC-KT, and B95-8 cells. The HR-1 virus is lytic and induces early antigen in superinfected Raji cells; the virus is not capable of transforming B-lymphocytes. Virus preparations from NPC-KT cells have both transforming and early antigen-inducing properties, while B95-8 virus can only transform B-lymphocytes. It was possible to demonstrate EBV antigens after superinfection of D98/HR-1 cells with both HR-1 virus and NPC-EBV. The NPC-KT hybrid cells, which were originally prepared by fusing human adenoid epithelial cells (Ad-AH) and EBV genome-positive NPC explanted epithelial cells, were susceptible to superinfection with HR-1 virus but not to NPC-EBV. The A2L/AH hybrid cells, which were prepared by fusion between Ad-AH cells and lymphocytes transformed by B95-8 virus, could not be superinfected with any of the virus preparations. In order to further investigate the nature of the EBV receptor as it relates to other cell membrane components, we examined cell surface markers on Ad-AH, NPC-KT, A2L/AH, and D98/HR-1 cells using monoclonal antibodies and by rosette formation. We found that the NPC-KT, A2L/AH, and Ad-AH cell lines express the OKB2 antigen and that the common acute lymphoblastic leukemia antigen is expressed on the A2L/AH cells. We also found that NPC-KT parental cells and a clone of NPC-KT cells express erythrocyte antibody complement b and erythrocyte antibody complement d, as determined by rosette formation, but were negative for C3b and C3d when monoclonal antibodies against these two markers were used. The D98/HR-1 cells were also confirmed to be negative for C3b and C3d. The data suggest that the C3d receptor may be part of the EBV receptor but that the C3d receptor, by itself, is not the only receptor to which EBV can bind.

Antibodies, Monoclonal↗
PubMed1986

Relationship between levels of rheumatoid factor isotypes and complement component C3 conversion in blood from patients with rheumatoid arthritis.

Conversion of complement component C3 in plasma from rheumatoid arthritis patients was measured by two different methods. One of the methods gives an estimation of C3 conversion by ELISA measurement of neodeterminants present on the C3d moiety; the other method measures C3 split products expressing D, but not C, epitopes by rocket immunoelectrophoresis (RIE) with intermediate anti-C3c gel. Results from 20 RA patients obtained by the two methods did not correlate significantly (R = 0.52, 0.02 less than p less than 0.05). The results were compared to the level of rheumatoid factors (RFs) of IgG, IgM, and IgA class in serum. A significant correlation was found between the concentration of C3d measured by RIE and level of IgG RFs, whereas neither IgM nor IgA RFs showed correlation to complement activation. The results of the ELISA estimation of C3 activation showed no correlation with the RF level.

Arthritis, Rheumatoid↗
PubMed1999

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.

Animals↗
PubMed1984

Analysis of C3-receptor activity on human B-lymphocytes and isolation of the complement receptor type 2 (CR2).

The rosetting of defined C3-fragment-coated sheep erythrocytes to B-cell-enriched tonsil lymphocytes was measured. The rosetting lymphocytes were homogeneous with respect to expression of C3b, iC3b and C3d receptors. Isolation of receptors for C3 fragments from surface-radioiodinated lymphocytes by affinity chromatography on immobilized C3u, iC3b and C3d,g produced two proteins with partially overlapping specificities. A protein of 240 000 Mr, recognized by the monoclonal antibody To5 and identified as CR1 (complement receptor type 1), had affinity for C3u and iC3b. A protein of 145 000 Mr, recognized by the monoclonal antibody B2, had affinity for all three C3 fragments. Inhibition of rosetting by antibodies to these proteins indicates that CR1 is responsible for C3b-mediated rosetting and that the 145000-Mr receptor (CR2) is responsible for C3d-mediated rosetting. Partial inhibition by both anti-CR1 and anti-CR2 antibodies of iC3b-mediated rosetting indicates that both receptors are involved in iC3b-mediated rosetting. No other protein appears to be involved in tonsil B-cell rosetting to C3-fragment-coated cells. A method for preparing CR2 from tonsil lymphocytes based on affinity chromatography on C3d,g-Sepharose has been developed. Forty tonsil pairs (2 X 10(10) B-cells) yield about 40 micrograms of pure protein equivalent to a purification of 6500-fold from a detergent extract.

Antibodies↗
PubMed2006

Measuring human complement activation by HLA antibodies.

CONTEXT: The clinical significance of HLA antibodies in transplantation depends on their ability to activate complement, which is measured by the standard complement-dependent cytotoxicity test. Recent reports indicate that C3b measurement on target cells may be a better indicator of human complement activation than standard cytotoxicity. OBJECTIVE: To determine the characteristics of the test, the role of other complement components, and the potential influence of the patient's own complement activity. DESIGN: The T-cell deposition of multiple complement components triggered by HLA alloantibodies was evaluated by flow cytometry using normal human serum as the source of complement. Complement activity in patients' sera was measured after activation with a standard antibody. RESULTS: When HLA antibodies activate complement, C3b deposition is usually highest, followed by that of C4b and C5b. Deposition of C1q, C3d, iC3b, or C5b-9 was low or undetectable in this study. The C5 was activated only when relatively high levels of C3b were present. There was a critical concentration of antibody, unique for each patient, below which activation of C3 declined abruptly. The complement activity in the serum of candidates for liver, heart, and lung transplantation that was tested with a standard antibody was similar to normal serum. In contrast, kidney transplant candidates exhibited higher complement activity. Unexpectedly, serum C3 activity was retained for at least 72 hours after collection. CONCLUSIONS: Measurement of C3b, and in some instances C4b or C5b, offers a better definition of the ability of HLA antibodies to activate human complement than tests that are in current use. The results provide a necessary baseline to conduct clinical correlation studies.

Complement Activation↗
PubMedDate not supplied

Serologic comparison of a monoclonal anti-C3d with commercial antiglobulin reagents.

The serologic reactivity of a monoclonal anti-C3d and commercial "monospecific" anti-C3 and polyspecific antiglobulin reagents was compared. The testing consisted of a direct antiglobulin test performed on donor and patient cells and on in vivo and in vitro complement-coated cells; an indirect antiglobulin test was performed with low-ionic-strength solution crossmatches. The monoclonal anti-C3d demonstrated reactivity very similar to that of the commercial heterogeneous rabbit anti-C3d; it gave relatively few weak "false positive" reactions with normal donor and patient red blood cells, and comparably strong reactions with in vitro and in vivo C3d-coated red blood cells. The results suggest a promising future for monoclonal antiglobulin reagents.

Antibodies, Anti-Idiotypic↗
PubMedDate not supplied

Antigenic determinants of C3 and C4 complement components on washed erythrocytes from normal persons.

Well-washed erythrocytes from normal persons were agglutinated by antisera to C3, C3d, and C4, and this agglutination was specifically inhibited by the corresponding C3 or C4 protein. C3 and C4 antigenic determinants were present on the red blood cells of freshly shed blood promptly anticoagulated with EDTA, heparin, ACD, or CPD, and no significant changes in degree of agglutination were observed on storage of EDTA or CPD blood for two weeks at 4 C. Marked differences in degree of agglutination by anti-C3, anti-C3d, and anti-C4 were observed when erythrocytes of 16 normal persons were assayed, and significant correlations were obtained when the quantitative results with any two antisera were compared. Anti-C3c did not agglutinate erythrocytes from normal persons, suggesting that the C3 antigens detected on normal cells are carried by the C3d fragment. To avoid significant agglutination of the erythrocytes from some normal persons, very dilute preparations of anti-C3, -C3d, and -C4 had to be used for instrumented diagnostic direct antiglobulin tests. Stronger reagents could be used for indirect antiglobulin tests when the result of a suitable control could be subtracted.

Animals↗
PubMedDate not supplied

A rat model of pregnancy-induced sensitization to transplants.

Previous pregnancy is a known risk factor for alloantibody production and graft rejection in clinical transplantation. However, in previous rat models, immune responses to RT1.A antigens induced by allogeneic pregnancy resulted in prolonged survival of subsequent allografts. This study was designed to investigate the effects of a previous pregnancy on alloantibody response, complement activation, and allograft survival in a highly immunogenic rat strain combination. C6-sufficient and -deficient female PVG.1U (RT1.A(u)B(u)) rats were mated with allogeneic PVG.R8 (RT1.A(a)B(u)) males or control isogeneic PVG.1U (RT1.A(u)B(u)) males. Three weeks after parturition, experimental and control females received cardiac allografts from female PVG.R8 donors. A low dose of cyclosporine (CsA, 5 mg/kg on alternate days) was used for immunosuppression after transplantation. Allogeneic, but not control isogeneic, pregnancy elicited a weak, transient IgG alloantibody response that declined before transplantation. Experimental female recipients produced a rapid, vigorous IgM and IgG alloantibody response to the transplant despite CsA treatment. C6-sufficient recipients rejected their transplants at an accelerated rate (5 days, n = 6) compared with control animals (7 days, n = 5). In contrast, allografts to C6-deficient recipients functioned until sacrifice at 90 days in both the experimental group (n = 7) and control group (n = 4). Most experimental C6-deficient recipients continued to produce strong IgG alloantibodies for 90 days. Complement activation resulting from the alloantibody response was evidenced by the diffuse deposition of C3d on the vascular endothelium of the grafts. In summary, previous pregnancy leads to memory alloantibody responses that accelerate allograft rejection even with immunosuppression. Membrane attack complex is required for accelerated rejection induced by previous pregnancy.

Animals↗
PubMed1982

Breakdown of C3 after complement activation. Identification of a new fragment C3g, using monoclonal antibodies.

The physiological breakdown of C3 has been studied using monoclonal anti-C3 antibodies, and it has been found that the later stages of this process--the breakdown of C3bi--is more complex than had previously been recognized. C3bi is the reaction product produced from C3b by the action of factor I which, in the presence of factor H, produces a double cleavage in the alpha chain of C3b. It is here reported that, both on cells and in the fluid phase, the breakdown of C3bi in serum gives rise to two products: C3c and the product previously described as alpha 2D, which we now propose to designate C3d,g. Alpha 2D differs from C3d in that it contains an additional fragment of approximately 8,000 mol wt that carries the antigenic determinant for the clone 9 monoclonal anti-C3 antibody. C3g cannot be precipitated by anti-C3 antisera and therefore behaves as a uni- or bideterminant antigen. The cleavage of C3d,g to C3d and C3g does not occur in sterile serum. It is also still uncertain what enzyme cleaves C3bi to C3c and C3d,g in plasma. Plasmin can do so in vitro, but plasminogen-depleted serum can still produce the cleavage. The antigenic determinant recognized by clone 9 in C3 is not exposed in C3 or C3b, but appears as a neoantigen in C3bi (and in C3d,g). Anti-C3g therefore is a potentially useful ligand for detecting complement-activation products. C3g represents a new, highly anionic C3 fragment and seems not to be identical with the C3e fragment described by others.

Animals↗
PubMed1991

Atypical structured glomerular deposits: an immunohistochemical study.

Atypical structured glomerular deposits were identified in sub-epithelial, sub-endothelial and mesangial areas in biopsy tissue from a female, aged 33, who presented with anaemia and was found to have proteinuria and microscopic haematuria. Histological examination showed that the deposits were periodic acid Schiff positive and silver negative whilst stains for amyloid were negative. Immunofluorescent staining for all immunoglobulins was negative, and only C3 showed moderate labelling. Conventional electron microscopy revealed that all deposits contained microtubular structures of variable length but with an average diameter of 25 nm and a periodicity of approximately 16 nm. The glomerular basement membrane was interrupted in many areas by deposits, and also contained 'myelin-like' structures. Free microtubular structures were also seen in the urinary space. Immunoelectron microscopy using protein-A-gold confirmed the immunofluorescent findings with immunoglobulins and fibrinogen, showed marked positive labelling of deposits with C1s and C3d and also intense labelling of coiled microtubular structures with C9. Other complement components C1q, C1r, C3c, C4 and C5 showed weak or negative results. Although these organised glomerular deposits contain complement components, their pathogenesis remains uncertain.

Adult↗