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PubMed1994

Complement receptor 3 (CR3, Mac-1, integrin alpha M beta 2, CD11b/CD18) is required for tyrosine phosphorylation of paxillin in adherent and nonadherent neutrophils.

Expression of the leukocyte (beta 2) integrins is required for many functions of activated neutrophils (PMN), even when there is no recognized ligand for any beta 2 integrin. To investigate the hypothesis that beta 2 integrins may be involved in a signal transduction pathway related to cytoskeletal reorganization, we examined whether beta 2 integrins have a role in tyrosine phosphorylation of the cytoskeletal protein paxillin. Treatment of PMN in suspension with phorbol esters, f-Met-Leu-Phe, and TNF-alpha resulted in paxillin tyrosine phosphorylation. However, treatment of beta 2-deficient (LAD) PMN failed to induce paxillin tyrosine phosphorylation. Normal PMN phosphorylated paxillin in response to adhesion to immune complexes, while the LAD PMN did not. Adhesion of phorbol ester activated-LAD PMN to the extracellular matrix proteins fibronectin, laminin, and vitronectin failed to induce paxillin tyrosine phosphorylation. Treatment of activated normal PMN with mAb directed against the beta 2 integrin alpha chains demonstrated that CR3 (alpha M beta 2) was required for paxillin phosphorylation. Transfection of the cell line K562 with CR3 confirmed that CR3 ligation resulted in paxillin tyrosine phosphorylation. As a control, K562 transfected with CR2 (CD21) which bound equally avidly to the same complement C3-derived ligand (C3bi) as the CR3 transfectants, showed no enhanced tyrosine phosphorylation of paxillin upon receptor ligation. While both CR2 and CR3 transfectants showed efficient adhesion to a C3bi-coated surface, only the CR3 transfectants spread during adhesion and phosphorylated paxillin. Together these data demonstrate that CR3 is required for paxillin phosphorylation during activation of both adherent and nonadherent PMN. Even PMN activated in suspension or by adhesion to immune complexes, when no CR3 ligand is apparent, still require CR3 for a signal transduction pathway leading to paxillin tyrosine phosphorylation. This pathway is likely to be important for PMN function in inflammation and host defense.

Adolescent↗
PubMed1996

Reduction in erythrocyte complement receptor 1 (CR1, CD35) and decay accelerating factor (DAF, CD55) during normal pregnancy.

RBC-membrane CR1 has been assayed in pregnancy and at 48 h postpartum. It has been shown that RBC CR1 is reduced as pregnancy progress, reaching a nadir in the third trimester and that it returns to levels approaching normal within 48 h postpartum. Further, there is a reduced expression of RBC decay accelerating factor (DAF) during pregnancy but no change in expression of RBC CD59. We suggest that the reduction in RBC CR1 and DAF may reflect increased levels of circulating immune complexes and consequent increased complement activation in pregnancy.

Antibodies, Monoclonal↗
PubMed1993

Release of vesicles enriched in complement receptor 1 from human erythrocytes.

Vesicles released from human E by Ca(2+)-loading, ATP-depletion, or storage are enriched in several glycosylphosphatidylinositol-anchored proteins such as acetylcholinesterase (AchE) and decay-accelerating factor (DAF). As a result of this, the remaining E are depleted of these proteins. We analyzed whether vesiculation induced by ATP-depletion in vitro was also responsible for a loss of C receptor 1 (CR1), which is a transmembrane protein arranged predominantly in small clusters. ATP-depleted E had lost 15.4% to 33.9% of their CR1. This loss was similar to that of AchE and DAF. The released vesicles contained CR1. The number of CR1 per band 3 protein was 1.7 to 2.7 that in the original E, indicating an enrichment of CR1 in vesicles. This enrichment was similar to that observed for AchE and DAF (1.83- and 2.6-fold, respectively). The capacity of the vesicles and the ATP-depleted E to bind C3b-coated immune complexes correlated with the CR1 number, suggesting that there was no preferential loss of CR1 clusters. Vesicles released from human E during C attack also contained CR1. In conclusion, in vitro aging induced by ATP-depletion is responsible not only for a loss of glycosylphosphatidylinositol-anchored proteins, but also of CR1. Whether vesiculation explains the loss of CR1 from aging E in vivo and from E of patients with SLE or AIDS remains to be studied.

Adenosine Triphosphate↗
PubMed1982

Kinetics of interaction of immune complexes with complement receptors on human blood cells: modification of complexes during interaction with red cells.

Antigen-antibody complexes, composed of 125I-BSA and guinea-pig or rabbit antibody, were incubated at 37 degrees C with human blood cells suspended in autologous serum and kinetics of binding analysed. When purified polymorphonuclear (PMN) or mononuclear cells (MNC) were studied, maximum binding was observed within 8 min, and immune complexes (IC) remained associated with cells even after 1 hr. When cells were studied unseparated (in the same amount of serum), maximum binding was observed slightly earlier (within 4 min), but within 15 min most of the IC were found in the serum. Separation of cell types at the time of maximal binding and studies with cell preparations depleted of different elements revealed that binding was principally to red blood cells (RBC). IC recovered in the serum 16 min after addition to unseparated cells bound very slowly to purified PMN or MNC; binding after 30 min was 10-15% of that observed with fresh IC at 8 min. Ultracentrifugal analysis revealed that reduction in binding efficiency correlated with decrease in the size of IC. RBC isolated after binding and release of IC bound newly-formed IC was identical rapidity and capacity as fresh RBC, indicating that receptors were not altered by IC. Kinetics studies with serum in the absence of cells suggested that interaction with RBC accelerated the rate of change in binding properties of IC. Rates of binding and release were independent of antigen/antibody ratio but were slowed and binding to RBC sustained when diluted or hypocomplementaemic (SLE) serum was substituted for neat serum. Our results suggest that competition for IC by RBC is associated with loss of ability of IC to bind to other blood cell types and reduction in size of IC, and that abnormalities of complement can lead to prolonged association of IC with RBC.

Antigen-Antibody Complex↗
PubMed1982

Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes.

Cells expressing a membrane C receptor (CR(3)) specific for C3b-inactivator- cleaved C3b (C3bi) were identified by rosette assay with C3bi-coated sheep erythrocytes (EC3bi) or C3bi-coated fluorescent microspheres (C3bi-ms). C3bi- ms, probably because of their smaller size, bound to a higher proportion of cells than did EC3bi. C3bi-ms bound to greater than 90 percent of mature neutrophils, 85 percent of monocytes, 92 percent of erythrocytes, and 12 percent of peripheral blood lymphocytes. Binding of C3bi-ms to neutrophils, monocytes, and erythrocytes was inhibited by fluid-phase C3bi, Fab anti-C3c, or Fab anti-C3d but was not inhibited by F(ab')(2) anti-CR(1) (C3b receptor) or F(ab')(2) anti-CR(2) (C3d receptor) nor by fluid-phase C3b, C3c, or C3d. This indicated that monocytes, neutrophils, and erythrocytes expressed C3bi receptors (CR(3)) that were separate and distinct from CR(1) and CR(2) and specific for a site in the C3 molecule that was only exposed subsequently to cleavage of C3b by C3b inactivator and that was either destroyed, covered, or liberated by cleavage of C3bi into C3c and C3d fragments. Lymphocytes differed from these other cell types in that they expressed CR2 in addition to CRa. Lymphocyte C3bi-ms rosettes were inhibited from 50 to 84 percent by F(ab')(2)-anti-CR(2) or fluid-phase C3d, whereas C3d-ms rosettes were inhibited completely by F(ab')(2) anti-CR(2), fluid-phase C3bi, or fluid- phase C3d. Thus, with lymphocytes, C3bi was bound to CR(3), and in addition was bound to CR(2) by way of the intact d region of the C3bi molecule. In studies of the acquisition of C receptors occurring during myeloid cell maturation, the ability to rosette with C3bi-coated particles was detected readily with immature low-density cells, whereas this ability was nearly undetectable with high density mature polymorphonuclear cells. This absence of C3bi binding to polymorphs was not due to a loss of the CR(3) but instead was due to the maturation-linked acquisition of the abiity to secrete elastase that cleaved reagent particle-bound C3bi into CR(3)-unreactive C3d. Neither neutrophils nor monocytes bound C3d-coated particles at any stage of maturation. Assay of CR(3) with mature neutrophils required inhibition of neutrophil elastase with either soybean trypsin inhibitor or anti-elastase antibodies, and the amounts of these elastase inhibitors required to allow EC3bi rosette formation increased with neutrophil maturation. Because lymphocytes bound C3bi to CR(2) as well as to CR(3), specific assay of lymphocyte CR(3) required saturation of membrane CR(2) with Fab' anti-CR(2) before assay for rosettes with C3bi-ms. Only 3.5 percent of anti-CR(2)- treated peripheral blood lymphocytes bound C3bi-ms. Therefore, among normal blood lymphocytes the majority of the 12 percent C3bi-ms-binding cells expressed only CR(2) (8.5 percent), and the small proportion of C3bi-ms- binding cells that expressed CR(3) (3.5 percent) represented a distinct subset from the CR2(+) cells. Double-label assay indicated that 3.0 percent out of 3.5 percent of these CR(3)-bearing lymphocytes were B cells because they expressed membrane immunoglobulins. Of the remaining CR(3)(+) cells, 0.2 percent expressed either Leu-1 or 3A1 T cell antigens, and 0.6 percent expressed the OKM-1 monocyte-null lymphocyte determinant.

Cell Membrane↗
PubMed1982

Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes.

The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.

Antigen-Antibody Complex↗
PubMed1980

Distribution of complement receptors on human normal and malignant mononuclear cells.

Mononuclear cells from normal human subjects and patients with chronic lymphocytic leukemia (CLL), chronic lymphosarcoma cell leukemia (LCL), and hairy cell leukemia (HCL) were labeled with fluoresceinated, purified human C3b (FI-C3b) and analyzed using the fluorescence-activated cell sorter (FACS). FI-C3b labeled 17.6% +/- 6.0% of peripheral blood mononuclear cells (PBM) from 20 normal subjects, which, when separated by the FACS, consisted of B lymphocytes and approximately 5% monocytes. Analyses in which either monocytes or B lymphcoytes were excluded from consideration demonstrated that both these cell types were labeled by the FI-C3b with a heterogeneous distribution of fluorescence intensity, indicating either heterogeneity of CR density or variable avidity of individual CR for the FI-C3b. FACS profiles of PBM ( < 5% monocytes) from 14 of 15 patients with CLL showed a homogeneous distribution of very low fluorescence intensity, with > 60% of the cells being slightly more fluorescent than unlabeled controls. This low, homogeneous distribution of fluorescence is strikingly similar to profiles of CLL cells labeled with anti-Ig reagents and suggests homogeneity of low CR density and/or avidity. Similarly, CR+ mononuclear cells from five patients with HCL and three patients with LCL displayed more homogeneous FI-C3b labeling than normal CR+ PBM. Homogeneity of FI-C3b binding to CLL, LCL, and HCL cells further supports the concept for a clonal origin for these disorders.

Blood Cells↗
PubMed1989

The murine complement receptor gene family. Analysis of mCRY gene products and their homology to human CR1.

The mouse genome contains two sets of gene sequences which are highly homologous to the gene encoding the human C3b/C4b receptor (CR1). These genes, termed murine CRY (mCRY) and murine CRX (mCRX) reside on murine chromosomes 1 and 8, respectively. Analysis of cDNA isolated by using these sequences as probes indicates that there are two related but distinct mRNA which are expressed in a wide variety of murine tissues including spleen, liver, lung, and brain. Both of these transcripts encode proteins which should contain a signal sequence for membrane insertion, a transmembrane/cytoplasmic tail region for membrane anchoring, and five extracellular domains made up of 60 amino acid consensus repeat sequences. The difference between the two is the presence of an additional exon of 129 bp immediately 3' of the signal sequence. This additional exon does not encode a 60 amino acid repeat. The sizes of the mature proteins predicted from the cDNA sequences are 43,998 Mr and 48,680 Mr; however, antisera raised against carboxy-terminal sequences detects a 70,000 Mr protein from murine fibroblasts suggesting a high degree of post-translational modification of the mature protein. A comparison of these murine gene sequences with a partial human CR1 sequence suggests that the human CR1 gene evolved by direct duplication of the ancestral coding sequences contained within these murine genes including those sequences important for membrane anchoring and cytoplasmic protein attachment.

Amino Acid Sequence↗
PubMed2004

The C4A and C4B isotypic forms of human complement fragment C4b have the same intrinsic affinity for complement receptor 1 (CR1/CD35).

Several previous reports concluded that the C4b fragment of human C4A (C4Ab) binds with higher affinity to CR1 than does C4Bb. Because the isotypic residues, (1101)PCPVLD and (1101)LSPVIH in C4A and C4B, respectively, are located within the C4d region, one may have expected a direct binding contribution of C4d to the interaction with CR1. However, using surface plasmon resonance as our analytical tool, with soluble rCR1 immobilized on the biosensor chip, we failed to detect significant binding of C4d of either isotype. By contrast, binding of C4c was readily detectable. C4A and C4B, purified from plasma lacking one of the isotypes, were Cs converted to C4Ab and C4Bb. Spontaneously formed disulfide-linked dimers were separated from monomers and higher oligomers by sequential chromatographic steps. The binding sensorgrams of C4Ab and C4Bb monomers as analytes reached steady state plateaus, and these equilibrium data yielded essentially superimposable saturation curves that were well fit by a one-site binding model. Although a two-site model was required to fit the equilibrium-binding data for the dimeric forms of C4b, once again there was little difference in the K(D) values obtained for each isotype. Independent verification of our surface plasmon resonance studies came from ELISA-based inhibition experiments in which monomers of C4Ab and C4Bb were equipotent in inhibiting the binding of soluble CR1 to plate-bound C4b. Although divergent from previous reports, our results are consistent with recent C4Ad structural data that raised serious doubts about there being a conformational basis for the previously reported isotypic differences in the C4b-CR1 interaction.

Binding, Competitive↗
PubMed1999

Identification of residues within the 727-767 segment of human complement component C3 important for its interaction with factor H and with complement receptor 1 (CR1, CD35).

Mapping approaches employing blocking antibodies and synthetic peptides have implicated the 727-767 segment at the NH2 terminus of C3b alpha'-chain as contributing to the interactions with factor B, factor H, and CR1. Our previous mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C3b to factor B and CR1 but not factor H. We have now extended the charged residue mutagenic scan to cover the remainder of the segment (738-767) and have assessed the ability of the C3b-like C3(H2O) form of the mutant molecules to interact with factor H, CR1, and membrane cofactor protein (MCP) using a cofactor-dependent factor I cleavage assay as a surrogate binding assay. We have found that the negatively charged side chains of Glu-744 and Glu-747 are important for the interaction between C3(H2O) and factor H, a result in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545-552) which implicated residues within the 744-754 segment in H binding. The interactions of the mutants with soluble CR1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of alpha-chain. These are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-744 and the Lys-757/Glu-758 pairing. The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribute basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wider portion of the 727-767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction between soluble MCP and C3(H2O), indicating that despite its functional homology to H and CR1, MCP differs in its mode of binding to C3b/C3(H2O).

Amino Acid Sequence↗
PubMed1997

Complement activation and thromboxane secretion by liposome-encapsulated hemoglobin in rats in vivo: inhibition by soluble complement receptor type 1.

Intravenous administration of liposome-encapsulated hemoglobin (LEH) in rats led to an early (within 15 min) decline of hemolytic complement (C) activity in the plasma along with a significant, parallel rise in thromboxane B2 (TXB2) levels. The TXB2 response was inhibited by co-administration of soluble C receptor type 1 (sCR1) with LEH, as well as by C depletion with cobra venom factor. These observations provide evidence for a causal relationship between LEH-induced C activation and TXB2 release, and suggest that sCR1 could be useful in attenuating the acute respiratory, hematological and hemodynamic side effects of LEH described earlier in the rat.

Animals↗
PubMed1982

Acute infection of mice with lactate dehydrogenase-elevating virus enhances Fc and complement receptor activity of peritoneal macrophages.

Peritoneal macrophages isolated from Balb/c mice 1 day after infection with lactate dehydrogenase-elevating virus (LDV) exhibited a 5- to 10-fold enhancement of attachment and ingestion of sheep red blood cells coated with immunoglobulin (EAIgG) or immunoglobulin plus complement (EAIgMC). Macrophages isolated from mice 7 days after LDV infection or macrophages infected with LDV in culture were also slightly more active than macrophages from uninfected mice, but the differences were not significant. The results indicate that a specific increase in the number of Fc and C3 receptors on macrophages occurs during the acute phase of infection. This increase correlates with the transient appearance of interferon in acutely infected mice. We postulate that during the acute phase the productive infection of a subpopulation of macrophages that is permissive for LDV results in the synthesis of sufficient interferon to cause activation of the remaining non-permissive macrophages in the animal.

Animals↗
PubMed1975

Specificity of human lymphocyte complement receptors.

Erythrocytes, bone marrow-derived lymphocytes, monocytes, and granulocytes were shown to have a receptor activity for C4. Theis C4 receptor activity was studied in relation to the previously identified C3b and C3d receptors. By assay for inhibition of rosette formation by fluid-phase complement (C), only two different lymphocyte C receptors were demonstrated. The immune adherence receptor, the only one of the two shared in common with erythrocytes, was specific for C4 or the C3c region of C3b, but was unreactive with C3d. The other lymphocyte receptor, the C3d receptor, was specific for C3d fragments, but would also react to a lesser extent with the C3d region of uncleaved C3b. ThC3d receptor did not react with either C3c or C4. This specificity of the C3d receptor allowed certain cells which contained only C3d receptors to form rosettes with EAC1-3b and EAC1-3d, but not with EAC14. However, because C3d receptors bound EAC1-3d or C3d fragments more firmly than they did EAC1-3b or C3b fragments, many other types of cells containing only C3d receptors, formed rosettes with EAC1-3d but not with EAC1-3b. Erythrocytes and those lymphocytes which contained only immune adherence receptors, formed rosettes with EAC14 and EAC1-3D but not with EAC1-3d. A double-label assay was devised for the simultaneous detection of both types of C receptors on individual lymphocytes. This assay involved fluorescence labeling of one of the two C receptors with soluble C fragments in combination with the usual rosette method for labeling the other type of C receptor. With this double-label assay, it was observed that the two different lymphocyte C receptors capped independently and thus were located on different molecules which could each move through the fluid membrane matrix independently of the other.

Animals↗
PubMed1979

Complement receptors in human peripheral nerve tissue.

Cryostat sections of human peripheral nerve tissue adsorbed sheep erythrocytes sensitized with antibody and human complement (EAC). Activated complement was essential for the reaction to occur. The receptor had specificity for the C3b fragment. In order to obrain binding to nerve tissue, the indicator cells had to be coated with more complement than that required for binding to C3b receptors in spleen and renal tissue. The receptors are located within the nerve fascicles and are probably of glycoprotein nature. The receptors for C3b in peripheral nerve tissus may be of significance in the deposition of immune complexes, which may play a role in acute polyradiculoneuritis.

Complement Activation↗
PubMed1977

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. II. Is the complement receptor necessarily present on the killer cells?

The effect of various lymphocyte depletion techniques on SLMC effector cell activity against the cell line K562 could be attributed to the effects that these procedures had on the proportions of Fc/C3 receptor-bearing cells in the preparations. depletion of cytotoxic activity by removal of Fc receptor-bearing cells could be augmented by the presence of complement on the 7S-EA indicator cells used for rosette depletion. Two methods of assessing the presence of surface receptors on cytotoxic cells are presented: (1) linear regression analysis of spontaneous cytotoxicity plotted as a function of the proportions of receptor-bearing cells remaining after various depletion techniques; and (2) the use of a mathmatical formula to estimate the proportion of cytotoxic cells with any particular receptor, based on the effects of receptor-bearing cell depletion. Linear regression analysis of cytotoxicity vs the proportions of SRBC, Fc or C3 receptor-bearing cells demonstrated that all cytotoxic cells had the Fc receptor, but that cytotoxicity could still occur in the absence of cells with the C3 receptor. This indicated that the C3 receptor is not necessary for the demonstration of SLMC activity. The mathematical formula to predict cytotoxicity after depletion of C3 receptor-bearing lymphocytes showed, however, that a significant proportion of the Fc receptor-positive killer cells were also C3 receptor-positive. No direct evidence was found to support a role for T cells in SLMC, although E rosette depletion resulted in lower than expected cytotoxicity in view of the number of Fc receptor-bearing cells remaining in the preparation.

Antibodies, Neoplasm↗
PubMed1996

Recombinant soluble human complement receptor type 1 inhibits antisperm antibody- and neutrophil-mediated injury to human sperm.

The pathogenesis of antisperm antibody (ASA)-mediated infertility is postulated to be related in part to complement (C)-dependent sperm dysfunction in the female genital tract. We have previously demonstrated that C can be involved in ASA-mediated sperm injury by the deposition of activated C3 fragments and the assembly of terminal membrane attack complex (C5b-9) leading to C3-mediated sperm binding to neutrophils or C5b-9-mediated sperm motility loss. This study evaluated the protective effect of recombinant soluble C receptor type 1 (sCR1) on ASA-and C-mediated neutrophil/sperm interaction, neutrophil aggregation, and sperm motility loss. Motile sperm with or without neutrophils were incubated in the presence of 10% C-fixing ASA+ serum or ASA- control sera in the presence or absence of sCR1. After defined incubation periods, the following neutrophil and sperm parameters were evaluated: 1) neutrophil aggregation (by the flow cytometric pulse processing method), 2) sperm phagocytosis (by light microscopy), 3) the deposition of C3 cleavage fragments (C3b, iC3b, and C3d) on motile sperm (by immunofluorescence flow cytometry), and 4) the relation between sperm motility loss and sperm-bound C3d. Only the coincubation of neutrophils with sperm in the presence of C-fixing ASA+ sera resulted in marked neutrophil aggregation (20.5 +/- 0.26% vs. 2.4% +/- 1.6; p < 0.0001) and a concomitant increase in neutrophils containing ingested sperm (71 +/- 5.8% vs. 3.5%; p < 0.0001). Soluble CR1 inhibited ASA- and C-mediated neutrophil aggregation by 46% and sperm phagocytosis by 57%. Motile sperm incubated with C-fixing ASA- sera showed a time-dependent increase in the binding of C3 fragments as detected by flow cytometry using anti-iC3b neoantigen, anti-C3c, and anti-C3d monoclonal antibodies (mAbs). A negative correlation (r2 = -0.930; p < 0.001) was found between the increase in sperm-associated C3d fluorescence and the percentage motile sperm in the presence of ASA- sera. Soluble CR1 (200 micrograms/ml) maximally inhibited the binding of anti-C3b, anti-C3c, and anti-C3d mAbs to sperm by 96%, 83%, and 72%, respectively. Thus, sCR1 abrogated the binding of C3 fragments to human sperm and fully protected sperm from C5b-9-mediated sperm immobilization. These findings suggested the therapeutic potential of sCR1 as an intravaginal pharmacophore to prevent C-dependent sperm dysfunction and related inflammatory events in the female genital tract.

Acrosome↗