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PubMed1996

Complement-dependent binding of C-reactive protein complexes to human erythrocyte CR1.

C-reactive protein (CRP) is an acute phase serum protein that binds to phosphocholine (PC) on phospholipids and polysaccharides and to protein components of chromatin and small nuclear ribonucleoproteins. Complexes between CRP and ligands activate complement and bind to receptors on phagocytic cells. Although complement is required for CRP-mediated clearance or phagocytosis of ligand-coated erythrocytes, the participation of complement and complement receptors in clearance of soluble CRP complexes has not been examined. We have used PC-conjugated BSA to prepare complexes containing either IgG antibody or CRP. We found similar complement-mediated binding of both types of complexes to human erythrocyte complement receptors (CR1, CD35). We also found that serum deficient in C4A or C4B supported binding of CRP and IgG complexes to erythrocytes. These findings indicate that complexes between CRP and soluble ligands may be cleared by the erythrocyte CR1 pathway described for soluble immune complexes.

Antigen-Antibody Complex↗
PubMed1998

Analysis of complement receptor type 1 (CR1) expression on erythrocytes and of CR1 allelic markers in Caucasian and African American populations.

CR1 expression on erythrocytes (E) is regulated by an element that is tightly linked in Caucasians to the site of an RFLP of the CR1 gene. Genomic HindIII fragments of 7.4 and 6.9 kb identify alleles that are expressed in high (H allele) or low (L allele) amounts, respectively. When age-fractionated E of donors heterozygous for both the H and L alleles and for CR1 allotypes of differing molecular weights were analyzed in Western blots, the product of the L allele appeared to have an increased rate of loss during cell aging. A coding sequence polymorphism of CR1 predicted to cause a Pro-->Arg substitution in its proximal extramembranous region was tightly linked in Caucasians to the site of the HindIII RFLP. However, neither this polymorphism nor the HindIII RFLP correlated with CR1 expression among African Americans. Relative instability of CR1 encoded by the L allele thus may derive from another coding sequence polymorphism, or may require both the Pro-->Arg substitution and epistatic effects of another polymorphic gene.

Alleles↗
PubMed1999

Quantification of microglial late reaction to stereotactic irradiation of the rat brain using computer-aided image analysis.

The role of microglial cells in the late delayed reaction following radiotherapy of brain tumors has not been elucidated. To investigate the late delayed response of microglial cells to radiation, we stereotactically irradiated spherical treatment volumes in the right frontal lobe of rat brains. Doses of 20, 30, 40, and 50 Gy were used in combination with two different collimators. The response of microglial cells at 10 and 19 months after irradiation was determined with Anti-CD 11 b/c (Ox 42) as an immunohistochemical marker. For evaluation of immunostaining, we developed a method using computer-aided image analysis in which the ratio of the area of stained cells to that of nonstained brain tissue is calculated. In addition, quantification of Ox-42+ cells per microscopic field was performed. Animals treated with 30 Gy or more had significantly increased total areas of staining at both time points studied. In contrast, the number of stained cells at 10 months increased significantly only in animals treated with 30 or 40 Gy. Likewise, at 19 months, this number increased significantly only in animals treated with 40 Gy or more. These results indicate that computer-aided determination of the area of stained cells is more sensitive than the counting of stained cells. We have demonstrated that microglial cells respond to stereotactic irradiation in a dose-dependent fashion. The image analysis we employed for this purpose is a systematic method to evaluate immunohistochemical staining.

Animals↗
PubMed2000

Expression of complement regulatory proteins-CD 35, CD 46, CD 55, and CD 59-in benign and malignant endometrial tissue.

OBJECTIVE: Complement system plays an important role in host defense mechanisms against microorganisms and tumor cells. To protect themselves from autologous complement-mediated damage, normal host tissues express cell membrane-associated complement regulatory proteins (CRPs). To investigate whether neoplastic endometrial tissues overexpress these proteins to escape complement damage, we examined the distribution of complement receptor type 1 (CR1, CD35), membrane cofactor protein (MCP, CD46), decay-accelerating factor (DAF, CD55), and protectin (MACIF, CD59) on frozen endometrial tissue samples. METHODS: A total of 54 endometrial tissue samples were collected. Cryosections were obtained of 31 benign and 23 malignant tissue specimens. Tissue sections were stained by immunohistochemical staining procedure using specific antibodies and employing the avidin-biotin technique. Quantitation of the protein content of these CRPs was determined using the Samba 4000 image analysis system. RESULTS: For all four of the CRPs studied, a statistically significant difference in protein expression between the benign and malignant endometrial tissue specimens (P < 0.0001) was observed. CONCLUSIONS: Overexpression of all the CRPs studied (CD35, CD46, CD55, CD59) was observed in the malignant as compared with the benign endometrial tissues. The upregulation of these CRPs may promote resistance of the endometrial malignant tissue to complement-mediated damage, thereby allowing the tumor cells to escape from cytolysis and thus promoting carcinogenesis.

Adult↗
PubMed1999

In vivo administration of intravenous immunoglobulin (IVIg) can lead to enhanced erythrocyte sequestration.

Enhanced erythrocyte sequestration is one of the very few major adverse effects of intravenous immunoglobulin (IVIg). IVIg contains high molecular weight IgG complexes ( approximately 300 kDa) which, in the presence of serum, mimic immune complexes by activating complement, binding to CR1 of red blood cells (RBC) (CD35) and mediating erythrophagocytosis. Four of seven patients undergoing IVIg therapy showed significant drops in haematocrit and haemoglobin that were not due to isoantibodies in the IVIg. Prior to treatment, patients' RBC carried IgG and complement (C') 3d that were not bound as immune complexes via CR1 (CD35). The patients whose RBC bound immune complex-like moieties and showed drops in haematocrit and haemoglobin subsequent to IVIg were young adults (22-35 years); older patients (50-69 years) showed no ill effects. In the presence of complement, RBC of young patients bound IVIg complexes in vitro while those of older patients did not. It is not the absolute levels of erythrocyte-associated IgG or C'3 fragments, neither pre- nor post-therapy, which are predictive of IVIg associated decreases in haematocrit and haemoglobin levels. Patient age and RBC inability to bind the IVIg immune complex-like moieties in vitro both appear to be predictors of resistance to sequestration after in vivo treatment with IVIg.

Adult↗
PubMed1996

A possible mechanism for host-specific pathogenesis of Salmonella serovars.

We have identified the complement receptors on human and murine macrophages involved in the recognition of Salmonella serovars, and investigated their relevance to the intracellular survival. S. typhi was capable of surviving within human monocyte-derived macrophages, whereas S. typhimurium was not. Conversely, S. typhimurium, but not S. typhi, resisted intracellular killing by murine macrophages, demonstrating that the intracellular survival of Salmonella serovars is host-dependent. In the presence of serum opsonin, human monocyte-derived macrophages recognized S. typhi and S. typhimurium via complement receptor type 1 (CR1) and type 3 (CR3), respectively. In contrast, murine macrophages recognized S. typhi and S. typhimurium via CR3 and CR1, respectively. These findings demonstrate that the intracellular fate of Salmonella serovars following phagocytosis may depend on the type of complement receptors involved in their recognition, in that CR1-mediated recognition is closely correlated to subsequent intracellular survival. The Tn5 insertion mutant of S. typhimurium which lacks the ability to interact with CR1 was sensitive to intracellular killing by murine macrophages in vitro, and was much less virulent to mice in vivo, confirming the relevance of CR1-mediated bacterial recognition to the pathogenicity of S. typhimurium for mice. These results suggest that selective recognition of Salmonella serovars through CR1 may lead to their subsequent intracellular survival, and is responsible for the host-specific pathogenesis of Salmonella serovars.

Animals↗
PubMed1995

Overexpression in Escherichia coli, folding, purification, and characterization of the first three short consensus repeat modules of human complement receptor type 1.

We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system in Escherichia coli was used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP-Sepharose. The oligomer was folded by one-step dilution in 20 mM ethanolamine/1 mM EDTA supplemented with 1 mM GSH/0.5 mM GSSG. The folded material was processed to a concentrated (> 20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1-3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 x 110 A. SCR(1-3) has an unusual CD spectrum exhibiting a broad maximum at 220-230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.

Amino Acid Sequence↗
PubMed1994

The interaction of glycoprotein C of herpes simplex virus types 1 and 2 with the alternative complement pathway.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) binds the human complement fragment C3b, but the two proteins differ in their ability to bind C3b on infected cell surfaces. In addition, gC-1, but not gC-2, accelerates the decay of the alternative pathway C3 convertase, thereby affecting later steps of the complement cascade. Previously, we constructed linker insertion and deletion mutants of gC-1 and gC-2 and used transient transfection to express mutant proteins in uninfected cells. In spite of the differences between gC-1 and gC-2, C3b binding was localized to residues within the central portion of both proteins, encompassing the first four cysteines. For gC-1, deletion mutants lacking amino acids 33 to 123 or 367 to 469 or lacking both regions still bound C3b. We recombined these deleted forms of gC-1 into gC-39, an HSV-1 strain lacking the gC gene. The altered forms of gC-1 were incorporated into virions, expressed on the surface of infected cells, and bound C3b. We used these proteins to investigate the structural basis for the inhibitory action of gC-1 on the complement cascade. We found that gC-1 does not inhibit formation of the alternative pathway C3 convertase. This convertase is stabilized by the serum protein properdin. Purified gC-1, but not gC-2, inhibits the binding of properdin to C3b, suggesting that this destabilizes the convertase. The mutant lacking amino acids 367 to 449 was able to inhibit properdin binding to a limited extent when present at high concentrations, although it bound to C3b more weakly than wild-type gC. In contrast, the protein lacking amino acids 33 to 123 was unable to inhibit properdin binding to C3b. Thus, gC-1 contains two structural domains, one for C3b binding, residues 124 to 366, and another, residues 33 to 133, which interferes with properdin binding to C3b.

Animals↗
PubMed1996

Complement-activating antibodies in sera from infected individuals and vaccinated volunteers that target human immunodeficiency virus type 1 to complement receptor type 1 (CR1, CD35).

Complement receptor type 1 (CR1) plays a central role in clearing immune complexes from the circulation and probably contributes to the retention of immune complexes on the surface of follicular dendritic cells. Virus-specific, complement-activating antibodies can target human immunodeficiency virus type 1 (HIV-1) to CR1-bearing cells but the potential impact that these antibodies have on HIV-1 pathogenesis is unknown. To study these antibodies, an assay was developed in which immune complexes containing HIV-1, antibody, and complement were formed in vitro and captured on the surface of 96-well immunoplates coated with recombinant soluble human CR1 (rsCR1). Captured virus was detected by p24 immunoassay or by infection of human CD4+ lymphocytes. Two laboratory strains of HIV-1 (IIIB and MN) and primary isolates could be captured using sera from infected individuals or vaccinated volunteers as a source of complement-activating antibodies. HIV-1 immune complexes captured by solid-phase rsCR1 could be transferred to MT-2 cells for productive infection. Antibodies had no activity in this assay when the normal human serum used as a source of complement had been heat-inactivated or depleted of complement component C3, confirming a requirement for complement. These complement-activating antibodies in sera from infected individuals showed strong cross-reactivity with HIV-1 IIIB, MN, and a heterologous primary isolate, but reacted poorly with the autologous isolate obtained at the time of serum collection. Average titers of these antibodies measured with HIV-1 IIIB were moderately lower in HIV-1-infected progressors compared to nonprogressors. In contrast to sera from infected individuals, sera from gp160IIIB-vaccinated volunteers showed specificity for the vaccine strain of virus. These results provide supporting evidence that envelope-specific, complement-activating antibodies induced by infection or gp160 immunization can target HIV-1 immune complexes to CR1. In addition, they demonstrate that such antibodies may sometimes be type-specific and that HIV-1 immune complexes bound to CR1 are infectious.

AIDS Vaccines↗
PubMed1983

The human C3b receptor.

The C3b receptor of human erythrocytes, neutrophils, monocytes, all mature B cells, a subpopulation of T cells, and glomerular podocytes is a single chain glycoprotein that exists in two allotypic forms having Mr's of approximately 250,000 (F) and 260,000 (S). The number of receptors present on erythrocytes varies by eight-fold among different individuals and is genetically regulated by two codominant alleles that are distinct from the alleles determining the structural polymorphism. The number of receptors expressed by neutrophils is subject to rapid increases from 5000 per cell to 40,000 per cell by exposure to nanomolar concentrations of C5adesArg, in vitro, and a similar mechanism is probably the basis for observing increased receptor expression on neutrophils in patients undergoing hemodialysis. Cytoskeletal association of the C3b receptor on monocytes and neutrophils is suggested by experiments demonstrating receptor-mediated phagocytosis, adsorptive endocytosis through coated pits, and restricted lateral diffusion, and by the reciprocal co-redistribution of cross-linked C3b and Fc receptors, and the detergent-insolubility of cross-linked C3b receptors. The factor H-like cofactor activity of the C3b receptor promotes the cleavage of bound C3b to iC3b, C3c and C3d, g, reactions that may enhance the clearance of circulating immune complexes and the generation of ligands for CR2 and CR3. The inherited partial deficiency of erythrocyte C3b receptors in patients with SLE, and the absence of glomerular C3b receptors in these patients with proliferative glomerulonephritis may contribute to systemic and organ-specific abnormalities in the clearance of immune complexes that contribute to the pathogenesis of this disease.

Endocytosis↗
PubMed1988

Absence of complement receptor type 3 and lymphocyte function antigen 1 causing deficient phagocyte and lymphocyte functions.

We describe a patient with delayed umbilical cord detachment, recurrent bacterial infections, and inability to form pus, despite persistent leucocytosis. Immunofluorescence studies with specific monoclonal antibodies showed a severe deficiency in the expression of alpha-chains of the receptor for the C3bi fragment of C3, complement receptor type 3, and the lymphocyte function antigen 1 molecule, found on neutrophil, monocyte and lymphocyte membranes. These membrane antigen defects were responsible for abnormalities in adhesive cell functions. Polymorphonuclear leucocytes demonstrated a markedly reduced chemiluminescence response as well as an impaired nitroblue tetrazolium test and superoxide generation to a particulate stimulus (zymosan), while the responses to a soluble stimulus (phorbol myristate acetate) were normal. In addition, random migration und chemotactic response to zymosan-activated serum were impaired. The lymphocytes demonstrated abolished natural killer cell cytotoxicity as well as abnormal humoral immunity and a lack of antibody response to pertussis and tetanus antigens.

Antigens, Differentiation↗