C3b Receptors Research Articles

Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts.

Macrophages express high levels of the myristoylated, alanine-rich, C kinase substrate (MARCKS), an actin cross-linking protein. To investigate a possible role of MARCKS in macrophage function, fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos. No differences between the wild-type and MARCKS-deficient macrophages with respect to morphology (Wright's stain) or actin distribution (staining with rhodamine-phalloidin, under basal conditions or after treatment with phorbol esters, lipopolysaccharide, or both) were observed. We then evaluated phagocytosis mediated by different receptors: Fc receptors tested with IgG-coated sheep red blood cells, complement C3b receptors tested with C3b-coated yeast, mannose receptors tested with unopsonized zymosan, and nonspecific phagocytosis tested with latex beads. We also studied fluid phase endocytosis in macrophages and mouse embryo fibroblasts by using FITC-dextran to quantitate this process. In most cases, there were no differences between the cells derived from wild-type and MARCKS-deficient mice. However, a minor but significant and reproducible difference in rates of zymosan phagocytosis at 45-60 min was observed, with lower rates of phagocytosis in the MARCKS-deficient cells. Our data indicate that MARCKS deficiency may lead to slightly decreased rates of zymosan phagocytosis.

A possible role for activated complement component 3 in phagocytic activity exhibited by the mouse trophoblast.

To determine whether any blood plasma factor may play a regulatory role in trophoblast phagocytosis in rodent early pregnancy. The effects of alloplasma on the phagocytosis of cultured mouse trophoblast cells (TCs) were evaluated using erythrocytes as target cells, in the presence of 10% fresh, normal plasma; 10% heat-inactivated plasma; 10% component 3 (C3)-depleted plasma; or medium alone. The possible activation of C3 complement, the phagocytosis of zymosan bound or unbound to C3b, and immunoreactivity to C3b receptor were also estimated. Phagocytic activity was expressed as the percentage of phagocytic TCs, and as the number of phagosomes/TCs. The use of complement sufficient plasma significantly enhanced the phagocytosis of the TCs while the use of heat-inactivated plasma eliminated the erythrophagocytosis. Very low levels of phagocytic activity were seen when the plasma was C3-complement deficient. Phagocytosis of C3b-bound zymosan was remarkable in comparison to zymosan alone, and immunoreactivity to C3b-receptors was seen on the TCs. These results indicate the participation of thermosensitive molecules mediating the phagocytosis of TCs and suggest, as in macrophages, a role for C3-C3b in this process.

Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells.

Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 +/- 0. 6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-alpha also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.

The role of a single N-linked glycosylation site for a functional epitope of herpes simplex virus type 1 envelope glycoprotein gC.

A monoclonal antibody, B1C1, binding to an epitope of antigenic site II of the herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, is a potent inhibitor of two important biological functions of gC-1: its binding to cell surface heparan sulfate and its binding to the receptor for complement factor C3b. Here, we have analyzed a B1C1-resistant HSV-1 variant (HSV-12762/B1C1B4.2), obtained after passage of wild type HSV-1 (HSV-12762) in the presence of high concentrations of B1C1. The transport of newly synthesized mutant gC-1 to the cell surface was comparable to that of wild type glycoprotein, but no binding of surface-associated mutant gC-1 to B1C1 was detected. However, mutant and wild type gC-1 bound equally well to other site II Mabs. Attachment of wild type but not mutant virus was inhibited by B1C1. Sequencing of the mutant gC-1 gene revealed only one nucleotide change, resulting in replacement of Thr150 by an Ile, in turn destroying an N-glycosylation site at Asn148. Loss of one complex type N-linked glycan was confirmed by endoglycosidase digestion and subsequent SDS-polyacrylamide gel electrophoresis. Circular dichroism analysis of purified gC-1 from cells infected with mutant or wild type virus did not reveal any difference in secondary structure between mutant and wild type gC-1. It was not possible to obtain a B1C1-resistant phenotype by nucleotide-directed mutagenesis of gC-1 where Asn148 was changed to a glutamine. These data demonstrated that the threonine of the glycosylation site and not the N-linked glycan in itself was essential for B1C1 binding

Cell Cycle Regulation and Differentiation in the Human Podocyte Lineage

Mature podocytes are regarded as growth-arrested cells with characteristic phenotypic features that underlie their function. To determine the relationship between cell cycle regulation and differentiation, the spatiotemporal expression of cyclin A, cyclin B1, cyclin D1, the cyclin-dependent kinase inhibitors (CKIs) p27 and p57, and markers of differentiating podocytes in developing human kidneys was investigated by immunohistochemistry. In S-shaped body stage, Ki-67, a cell proliferation marker that labels the G1/S/G2/M phase, was expressed in the majority (more than 80%) of presumptive podocytes, along with cyclin A (approximately 20% of the Ki-67-positive cells) and cyclin B1 (less than 5% of Ki-67-positive cells) expression. Among these cells), cyclin D1 and CKIs were markedly down-regulated. At the capillary-loop stage, by contrast, CKIs and cyclin D1 were intensely positive in podocytes, whereas no Ki-67, cyclin B1, or cyclin A expression was seen. Moreover, double-immunolabeling and serial-section analysis provided evidence that CKIs and markers specific for differentiating podocytes, namely PHM-5 (podocalyxin-like protein in humans), synaptopodin (a foot process-related protein), and C3b receptor, were co-expressed at the capillary-loop stage. Podocytes were the only cells within the glomeruli that expressed CKIs at immunohistochemically detectable levels. Furthermore, bcl-2 (an apoptosis inhibitory protein) showed a reciprocal expression pattern to that of CKI. These results suggest that 1) the cell cycle of podocytes is regulated by cyclin and CKIs, 2) CKIs may act to arrest the cell cycle in podocytes at the capillary-loop stage, and 3) the specific cell cycle system in podocytes may be closely correlated with their terminal differentiation in humans.

Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy

Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy

Phagocytosis of the malarial pigment, hemozoin, impairs expression of major histocompatibility complex class II antigen, CD54, and CD11c in human monocytes.

In Plasmodium falciparum malaria, large proportions of resident macrophages and circulating monocytes and leukocytes contain massive amounts of the malarial pigment, hemozoin. Previous studies have shown that important functions (e.g., the generation of the oxidative burst, the ability to repeat phagocytosis, and protein kinase C activity) were severely impaired in hemozoin-loaded monocytes. Expression of membrane antigens directly involved in the immune response and in the phagocytic process, and/or under protein kinase C control, in hemozoin-loaded human monocytes was studied. Expression of major histocompatibility complex (MHC) class II after gamma interferon stimulation was blocked in hemozoin-loaded monocytes at the protein expression and gene transcription levels but was preserved in control monocytes loaded with opsonized latex beads or anti-D(Rho)-immunoglobulin G (IgG)-opsonized human erythrocytes. Expression of CD54 (intracellular adhesion molecule 1) and CD11c (p150,95 integrin) was also decreased in hemozoin-loaded monocytes. Expression of MHC class I, CD16 (low-affinity Fc receptor for aggregated IgG), CD32 (low-affinity Fc receptor for aggregated IgG), CD64 (high-affinity receptor for IgG), CD11b (receptor for complement component iC3b [CR3]), CD35 (receptor for complement components C3b and C4b [CR1]), and CD36 (non-class-A scavenger receptor) was not specifically affected by hemozoin loading. These results suggest that hemozoin loading may contribute to the impairment of the immune response and the derangement of antigen presentation reported in previous studies of P. falciparum malaria.

Inhibition of phagocytic activity by the N-acetyl-D-galactosamine-specific lectin from Amaranthus leucocarpus.

Amaranthus leucocarpus lectin (ALL), specific for N-acetyl-D-galactosamine, induces inhibition of the erythrophagocytic activity of resident murine peritoneal macrophages and of the macrophage-like cell line J-774. This effect was observed only in macrophages that were Mac-2 (CD11c/CD18 or CR4) negative, indicating that macrophage activation induces important modification to the glycosylation (mainly O-glycosylation) of the membrane. Receptors for IgM and C3b remain unaltered after lectin treatment. Ultrastructural analysis revealed (a) that ALL induced the formation of pinocytic vacuoles, and (b) a regular distribution over the macrophage membrane as well as endosomal vesicles of the gold labeled ALL. Our results suggest that macrophage membrane glycoproteins with constitutive N-acetyl-D-galactosamine residues participate in the regulation of pinocytic-phagocytic vacuole formation.

Relationship between glomerular epithelial cell injury and proteinuria in IgA nephropathy

The present study was designed to elucidate whether glomerular epithelial cell (GEC) injury is associated with the mechanism of proteinuria in biopsy-proven IgA nephropathy (IgAN). Twenty-four IgAN patients were divided into 4 groups based on their grade of proteinuria. Light microscopic examination revealed that GEC injury appeared prior to glomerular lesions such as sclerosis, crescent and adhesion. Reduced glomerular expression of C3b receptor (CR1), an indicator of GEC injury, was detected initially at the portion of damaged GEC and around the lesions of sclerosis, crescent and adhesion. CR1 expression eventually disappeared in the group IV patients who had nephrotic range proteinuria. Moreover, the grade of proteinuria in IgAN patients was associated with GEC injury and reduced glomerular expression of CR1. Taken together, GEC injury might be an important pathological finding which appears initially in the glomeruli, suggesting that GEC injury is a predictor of proteinuria in IgAN patients.

Immunologic Aspects of Lung Diseases and Cystic Fibrosis

Immunologic lung disorders are accompanied by an array of laboratory abnormalities, some of which contribute to disease pathogenesis. Allergic bronchopulmonary aspergillosis, which complicates asthma and cystic fibrosis, causes mucous plugging of airways, eosinophilic pneumonia, and bronchiolitis obliterans. Aspergillus fumigatus, growing saprophytically in bronchial mucus, is responsible for most cases, and prednisone, not antifungal agents, is the drug of choice because it controls the immunologic responses of the lung. In cystic fibrosis, epithelial surface fluid from the lung does not kill Pseudomonas aeruginosa, in part because antibodies to P aeruginosa are plentiful but ineffective in opsonizing bacteria. Neutrophil-derived elastase cleaves immunoglobulins and digests the C3b receptor on neutrophils, which limits phagocytosis of pathogens. In helminth infections and infestations, pulmonary and peripheral blood eosinophilia can be accompanied by increases in total and antiparasite IgE concentrations and generate T(H)2 CD4+ T-lymphocyte responses. Understanding the immunologic abnormalities of lung disorders may lead to more effective therapies.

T lymphocytes and the lack of activated macrophages in wound margin biopsies from chronic leg ulcers.

The objective of this study was to characterize the leucocyte infiltrate which accumulates at the margin of chronic wounds. These leucocytes are a rich source of cytokines and growth factors, and an inappropriate function of these cells may contribute to the maintenance of wound chronicity. The leucocyte populations were stained immunohistochemically with monoclonal antibodies specific for surface receptors which give an indication of cellular function. Wound margin biopsies taken from chronic leg ulcers exhibited a localized infiltrate of CD45+ leucocytes associated with vascularized tissue in the dermis adjacent to the wound margin. Lymphocytes were identified in highest numbers in this area and CD45RO+ T lymphocytes predominated over B lymphocytes, which were either absent or present in very low numbers. In the majority of chronic wounds examined, CD4+ T lymphocytes were present in greater numbers than CD8+ T lymphocytes with a mean (+/-SD) ratio of CD4+:CD8+ of 1.5 +/- 0.6. CD68+ macrophages were identified in all layers of the dermis at the chronic wound margin. In 60% of wounds examined, macrophages were negative for the activation associated markers CD16 (Fc gamma III receptor) and CD35 (C3b receptor). In those biopsies where CD16 and CD35 positive macrophages were observed these were preferentially located in the perivascular regions. These data indicate that as monocytes extravasate into chronic wound tissue they may be subjected to microenvironmental influences which either suppress or do not induce macrophage activation. Suppression of macrophage activation may lead to an inappropriate cytokine/growth factor secretion and contribute to the maintenance of wound chronicity.

Covalent binding of C3b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific B cells.

Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell-mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen-presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT-C3b). The antigen-presenting cells were TT-specific (4.2) or non-specific (BL15) Epstein-Barr virus (EBV)-transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen-specific and antigen non-specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non-specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross-linking between membrane immunoglobulins and complement receptors on antigen-specific cells. The consequences of covalent C3b binding to TT were analysed using antigen-specific and antigen-nonspecific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of nonspecific cells, similar to that observed in antigen-specific cells. Thus, C3b covalently linked to antigen enlarges the array of B-cell types capable of presenting antigen, including non-specific cells.

In vitro and in vivo analysis of human leukocyte binding by the antitumor polysaccharide, lentinan

Lentinan (LNT), a (1→3)- β- d-glucan with (1→6)- β- d-glucopyranoside branches, has marked antitumor effects in syngeneic and autochtonous hosts. Clinically, LNT has proved effective with chemotherapeutic agents in patients with recurrent gastric and colorectal cancer. However, the mechanism that triggers subsequent immunologic reactions remains obscure. We hypothesized that LNT must first bind to host cells. Accordingly, we analyzed LNT binding to host cells in healthy volunteers after incubating their cells under a variety of conditions as well as intravenously injecting LNT then subjecting them to flow cytometry and immunofluorescent staining using monoclonal antibody (anti-LNT mAb). LNT bound to monocytes and neutrophils, but not to lymphocytes in vitro. The most avid LNT binding was to monocytes. The percentage of LNT-bound monocytes after 60 min incubation at 4°C was greater than that at 37°C. The binding of LNT to monocytes was inhibited slightly by anti-iC3h receptor (anti-CR3) mAb, strongly by anti-C3b receptor (anti-CRI) mAb, and completely by anti-CRI and anti-CR3 mAb together. The percentage of LNT-binding monocytes in the peripheral blood increased significantly 3 and 4 h after 2 mg LNT-injection and returned to low levels 5 h later. However, no increase in LNT-binding neutrophils and lymphocytes was observed. We conclude that binding of LNT to human monocytes may initiate the influence of this compound on the immune system and differ between individuals. Its binding site may be similar to the C3b receptor.

Monocyte function associated with intermittent lentinan therapy after resection of gastric cancer

The effect of lentinan administration on monocyte function in the peripheral blood were examined in 33 patients who underwent resection of gastric cancer. As parameters of monocyte function, IL-1 production, C3b receptor, Fc gamma receptor and monocyte ratio were determined every 4 weeks for a maximum of 24 weeks. Among patients who were taking combined therapy with lentinan, an increase in IL-1 beta production of more than 50% was found 8 weeks (2 months) after the initiation of therapy in 8 of 12 patients given 2 mg at 2-week intervals, and in 11 of 16 patients given the same dose at 4-week intervals. This increase was particularly clear in stage II or more advanced cases. There were no significant changes in the other parameters studied. It has been considered that lentinen is effective when administered once a week. The results of the present study, however, suggest that lentinan given every 4 weeks also stimulates monocyte function enough to maintain immunological activity.

Hind III genomic polymorphism of the C3b receptor (CR1) in patients with SLE: Low erythrocyte CR1 expression is an acquired phenomenon

Expression of the human erythrocyte C3b receptor (CR1-CD35) and its Hind III RFLP was studied in a group of 37 patients with SLE, 15 consanguineous relatives of the patients and 48 healthy normal subjects. The CR1 number on erythrocytes was quantitated by ELISA using a mAb to CR1. Serum levels of complement proteins (C3, C4, C3d) and circulating immune complexes (CIC) were estimated simultaneously in controls and relatives. The patients were followed up during the course of the treatment. The CR1/erythrocyte (CR1/E) in patients were found to be significantly low in comparison to controls. The gene frequencies for the alleles H and L (7.4 and 6.9 kb Hind III restriction fragments) in the patients were 0.75 and 0.25, respectively, which did not differ significantly from the controls (0.77 and 0.23 in normal subjects and 0.79 and 0.21 in consanguineous relatives of the patients). However, patients expressed fewer CR1/E within each genotype than their relatives and healthy subjects. CR/E was found to be stable in consecutive samples in controls. In patients, the numbers varied between low and high during the course of the treatment. The variation in the numbers was significantly correlated with C3d and CIC as well as with the severity of the disease. Our results suggest that low levels of CR1 on erythrocytes in SLE patients are required during the course of the disease and that the 6.9 kb restriction fragment does not play a role in causing susceptibility to the disease.

Characterization of rat complement receptors and regulatory proteins. CR2 and Crry are conserved, and the C3b receptor of neutrophils and platelets is distinct from CR1.

In the mouse, CR1 and CR2 on B lymphocytes are encoded by alternatively spliced Cr2 gene transcripts. Immune adherence receptors that bind C3b are present on mouse platelets and unstimulated neutrophils, but they are not CR1. In this study, rabbit anti-mouse CR1/CR2 Ab immunoprecipitated a 145- to 150-kDa CR2 protein from rat platelets, neutrophils, and splenocytes, but not a approximately 200-kDa CR1 protein. By Northern analysis, cDNA for mouse CR2 hybridized to mRNA of 3.7 and 5.2 kb from both mouse and rat splenocytes. The murine decay-accelerating factor and membrane cofactor protein analogue Crry was present in rat platelets, neutrophils, E, and splenocytes as two distinct proteins of 65 to 70 kDa and 75 to 85 kDa. Rat platelets, neutrophils, and splenocytes contained a novel 200-kDa cell membrane protein that specifically bound to a rat C3b-Sepharose column. We have named this protein C3bR-200. C3bR-200 was not identified by anti-mouse CR1/CR2 or anti-human CR1 Ab. Rat E lacked C3bR-200. Rat neutrophils and splenocytes also contained an 80-kDa C3b-binding protein that was distinct from Crry, which we have named C3bR-80. Therefore, CR2 and Crry are present in the rat, and have similar qualities to those from the mouse, except that CR2 is located on rat platelets and neutrophils, which is not the case in the mouse. Rat platelets, neutrophils, and splenocytes have a 200-kDa C3b-binding protein, C3bR-200, that is likely to be the rodent immune adherence receptor.

Complement component C3b and immunoglobulin Fc receptors on neutrophils from calves with leukocyte adhesion deficiency

SUMMARY Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence- related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (lad) on ligand binding and receptor expression. We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from lad, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (cl) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of cl was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1. A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from lad- affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and lad-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between lad-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from lad-affected calves. Receptor expression for aIgG was greater on neutrophils from lad-affected calves than on those from normal calves. Luminol-enhanced cl of neutrophils in response to IgG2 opsonized zymosan was not different between lad-affected and normal calves. Results indicate increased binding and expression of Fc receptors for aIgG and decreased binding and expression for C3b and IgM on neutrophils from calves with lad. Leukocyte adhesion deficiency may be compounded by added defects in the expression and binding of receptors for opsonins, such as C3b and IgM.

Increased expression of complement receptor type 1 (CR1, CD35) on human peripheral blood T lymphocytes after polyclonal activation in vitro.

The receptor for C3b and C4b--complement receptor type 1 (CR1, CD35)--is present on a variety of cell types including erythrocytes, phagocytic cells, B lymphocytes and a small subpopulation of T lymphocytes. The function of the receptor varies according to the different cell types, but on T lymphocytes the function is as yet not known. The present study concerns the influence of polyclonal stimulation on CR1-expressing T lymphocytes. Incubation with PHA resulted in a dose-dependent increase in the number of CR1-positive T lymphocytes. The CR1-expression T lymphocytes were found in both the CD4- and the CD8-positive subpopulation, but a significant stimulatory increase was only found in the CD4-positive population. A significant increase in the number of CR1-expressing T lymphocytes was found when monocytes were present during stimulation, indicating an importance of monocytes and/or monocyte products. However, the increase was not regulated by arachidonic acid metabolites of the cyclo-oxygenase pathway as indomethacin failed to inhibit the increase. Neither did rIL-1 alpha, rIL-1 beta, rTNF alpha nor rIL-6 alter the number of CR1-expressing T lymphocytes. The results of this study indicate a role for CR1 on T lymphocytes in the regulation of the immune system.

Glomerular antigens in severe hereditary nephrosis.

In search of the basic defect and cell type responsible for the massive treatment-resistant proteinuria of congenital nephrotic syndrome of the Finnish type (CNF), we examined tissue samples of CNF kidneys using established antibody and lectin markers of various glomerular cell types. Markers of vascular endothelium (antibodies to factor VIII and a human homologue of podocalyxin (anti-PHM5) and UEA I lectin) showed no qualitative changes in the endothelial cells of glomeruli or peritubular areas in CNF as compared with controls. Markers of glomerular mesangial cells (antibodies to desmin, smooth muscle actin, RCA I lectin) revealed a secondary increase in mesangial reactivity reflecting the sclerosis and expansion of the mesangial areas in CNF. Markers of visceral epithelial cells (antibodies to a human homologue of podocalyxin, C3b receptor, vimentin, common lymphocytic leukemia antigen, gp44, and the WGA, LFA and, after neuraminidase treatment, PNA lectin) failed to show appreciable qualitative changes in CNF kidney samples. Interestingly, the alpha 2 beta 1 integrins appeared greatly reduced in all CNF samples studied, possibly explaining the mechanisms of CNF-associated proteinuria.

Expression of Complement Regulatory Proteins on Human Eggs and Preimplantation Embryos

To investigate the relation between the complement system and reproduction, expression of complement regulatory proteins (C3b receptors and inhibitor of the membrane attack complex) were screened on unfixed human eggs and preimplantation embryos. Unfixed unfertilized oocytes and preimplantation embryos obtained from an in vitro fertilization program were stained by indirect immunofluorescence using monoclonal antibodies raised against membrane cofactor protein, (MCP or CD46), decay accelerating factor (DAF or CD55), protectin (CD59), human C3b/C4b receptor (CR1 or CD35), and major histocompatibility complex class I antigen (MHC class I). CD55 and CD59 were both expressed by the plasma membrane of unfertilized oocytes and pre-implantation embryos. CD46 was not expressed by unfertilized oocytes but appeared at the 6-to-8 cell stage embryo when human gene expression first occurs. CD35 and MHC class I antigens were not expressed at all on oocytes and preimplantation embryos. Selective expression of complement regulatory proteins (DAF and protectin) associated with the lack of MHC class I antigens may represent an immune protective mechanism by which human oocytes and preimplantation embryos escape complement-mediated damage during their travel through the female genital tract. Furthermore, participation of these complement regulatory proteins including MCP in cell to cell interaction during fertilization and/or implantation cannot be excluded.