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PubMed1985

Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils.

In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.

Acetone↗
PubMed1987

Alternative complement pathway activation fragment Ba binds to C3b. Evidence that formation of the factor B-C3b complex involves two discrete points of contact.

Alternative complement pathway C3 convertase formation involves the cleavage of C3b-associated factor B into fragments Ba and Bb. Whereas Bb, in complex with C3b, has proteolytic specificity toward native C3, the function of the Ba moiety in the formation and/or decay of alternative complement pathway C3 convertase is uncertain. Therefore, we have examined the effect of purified Ba fragment on both fluid-phase and surface-bound enzymatic activity and showed that whereas Ba could inhibit the rate of C3 convertase formation, the rate of intrinsic decay remained unaffected. A specific, metal ion-independent interaction between Ba and C3b was subsequently demonstrated by use of the cross-linking reagent dithiobis(succinimidyl propionate). When cell-associated 125I-B was activated by D, the dissociation of Bb fragment displayed simple first-order kinetics with a half-time of 2.4 min, this value being in reasonable agreement with the hemolytically determined decay rate of 1.8 min. In contrast, most of the Ba fragment undergoes rapid dissociation, but there is also evidence to suggest the establishment of a new equilibrium due to the ability of Ba to rebind to C3b. Cumulatively, these data are consistent with a model in which the attachment of intact B to C3b is mediated by two points of contact, one being in the Ba domain and the other in the Bb domain. Due to avidity effects, each of these interactions could be of relatively low intrinsic affinity, and the characteristic unidirectionality of alternative complement pathway C3 convertase decay may simply result from the low intrinsic association of "univalent" Bb for the C3b subunit.

Chemical Phenomena↗
PubMed1989

CR1, the C3b receptor, mediates binding of infective Leishmania major metacyclic promastigotes to human macrophages.

The role of complement receptors on monocyte derived human macrophages in phagocytosis of infective (MP) and noninfective (LP) developmental stages of Leishmania major promastigotes was studied. We compared binding of these specific developmental stages to MO after preincubation in fresh or heat-inactivated serum. Although LP do not require fresh serum for attachment, MP were dependent on serum C opsonization for entry. Inhibition of CR1 substantially abolished binding of the infective MP. In contrast, inhibition of iC3bR (CR3 and p150,95) had no effect on MP binding. Inhibition of both iC3bR, however, did block binding of nonopsonized LP. Attachment of LP to CR3 was blocked by fluid phase addition of mAb OKM1 and M1/70, which inhibit complement-independent binding to CR3, but not by mAb OKM10 which inhibits iC3b binding to this receptor. After fresh serum pretreatment of LP, however, only simultaneous inhibition of CR3 and CR1 effectively blocked their attachment. Addition of mannan did not inhibit attachment of either promastigote stage. Both opsonized and nonopsonized LP trigger a respiratory burst in MO, possibly via the C independent site in CR3, whereas the CR1-mediated uptake of MP does not generate a respiratory burst. The use of this receptor by MP may facilitate their subsequent intracellular survival.

Animals↗
PubMedDate not supplied

Cryopreservation of complement-coated erythrocytes.

Cells coated with complement components (C3b-C4b and C4b cells) were prepared by various methods and stored in liquid nitrogen using a low-glycerol, rapid freeze technique. Freshly coated cells, and cells coated and then frozen were tested versus various antiglobulin and anticomplement reagents to evaluate the reactivity of such frozen, stored, complement-coated human red blood cells. Liquid nitrogen preservation of such coated cells proved to be feasible when such cells were compared with freshly coated control cells.

Animals↗
PubMed1981

Fc and C3b receptors on pulmonary endothelial cells: induction by injury.

Receptors for the activated third component of complement and for the Fc portion of immunoglobulin G are not expressed by apparently normal bovine pulmonary endothelial cells, but are expressed when the cells are exposed to white cell lysates or are infected with influenza or cytomegalovirus. The unmasking of these latent receptors may contribute to the pulmonary inflammatory response characteristic of, for example, anaphylaxis and to those lung diseases characterized by the deposition of immune complexes.

Animals↗
PubMedDate not supplied

The binding of immune complexes by the erythrocyte complement receptor 1 (CR1).

Immune complexes, which have reacted with complement and bear C3b fragments, bind to the complement receptor 1 (CR1) on human erythrocytes. Indeed, CR1 on erythrocyte serves as a transport system for immune complexes in the circulation so as to prevent immune complex deposition outside the fixed macrophage system. A defect in this transport system has been described in several diseases, in which either complement levels or CR1 number on erythrocytes are diminished. Recent studies have shown that the binding of immune complexes to erythrocytes is favored by the multiple C3b binding sites per receptor and the clustered distribution of CR1 on the erythrocyte surface. Only a few immune complexes bind per erythrocyte but these complexes are tightly bound. The other main function of CR1 on erythrocytes is to enhance the inactivation of C3b by factor I present in plasma. This reaction allows the release of immune complexes from the erythrocyte surface and their transfer to fixed macrophages.

Animals↗
PubMed1982

Assay of membrane complement receptors (CR1 and CR2) with C3b- and C3d-coated fluorescent microspheres.

A sensitive and specific fluorescence assay for membrane complement (C) receptors (CR1 and CR2) was developed with purified C3b and C3d fragments coupled to fluorescent microspheres (0.9 mu diameter). C3-microspheres (C3-ms) bound to cells with low numbers of receptors that were undetectable by other assay techniques. Inhibition studies with anti-CR1 and anti-CR2 demonstrated that C3b-ms and C3d-ms bound exclusively to CR1 and CR2, respectively. Preparation of the C3-ms required only small amounts of partially purified C3 and no immunoglobulin or other C components. Once formed, the C3-ms were stable for up to 4 mo at 4 degrees C.

Binding, Competitive↗
PubMed1978

Requirements for beta1H globulin and C3b inactivator in the control of the alternative complement pathway in human serum.

Using beta1H-depleted and C3b inactivator (C3b1NA)-deficient sera we have investigated the regulatory roles of beta1H and C3bINA in the turnover of the alternative pathway. Spontaneous turnover of C3 and factor B occurred in both beta1H-depleted and C3bINA-deficient sera. In neither case was C3d generated. Prevention of activation could be achieved by the addition of the missing protein, but not by increasing the concentration of the remaining protein. Thus both beta1H and C3bINA must be present simultaneously to prevent spontaneous activation of the alternative pathway.

Beta-Globulins↗
PubMed1997

Expression and quantification of the iC3b-binding protein in different Candida albicans strains and their morphological stages.

Expression and quantification of the iC3b-binding protein in yeast, germ-tube, and mycelial forms of several Candida albicans strains were studied. Ten isolates were obtained from patients with recurrent vaginal candidosis. The germ-tubes generated at 37 degrees C and the mycelial forms of all strains grown at 30 degrees C, as well as most of the mycelial forms grown at 37 degrees C, were able to bind complement-coated sheep erythrocytes (EAiC3b). ELISA results revealed that a decrease in the binding of EAiC3b by the mycelial form of strains CBS 5982 and K10 correlated with a decrease of the expression of the iC3b-binding protein, detected by cross-reaction with the monoclonal antibody OKM1, recognising the alpha chain of human CR3. Expression of the iC3b-binding protein in other strains, binding EAiC3b, was higher in the mycelial form or very similar to that of germ-tubes. The dependence of the expression of the iC3b-binding protein on the morphological stages of individual C. albicans isolates suggests a possible association with the virulence of these strains.

Candida albicans↗
PubMed1990

Mechanistic aspects of cellular interactions with artificial dialyzer membrane surfaces.

The platelet adhesion on adhesive protein-coated surfaces was significantly reduced by the addition of the synthetic tetrapeptide, RGDS (Arg-Gly-Asp-Ser), which was identified as the common amino acid sequence of adhesive site of adhesive proteins. The inhibitory effect was also observed for leukocyte(WBC) in complement-inactivated serum. No significant effect was observed for WBC in complement-activated serum. These indicate that the RGDS ligand-receptor mechanism operates on adhesive protein-adsorbed surfaces for both cellular systems and that activated complement factor (C3b)-membrane receptor (CR3) interaction operates for WBC as the complement is activated.

Amino Acid Sequence↗
PubMed1990

Degradation of aggregates of activated C3 (C3b) by monocytes of patients with rheumatoid arthritis is related to vasculitis.

We investigated whether a decreased complement receptor expression or function of monocytes isolated from peripheral blood of 52 patients with rheumatoid arthritis (RA) and rheumatoid vasculitis (RV) could account for the previously observed diminished degradation of immune complexes by monocytes of patients with RA and RV. On average, monocytes from all patients expressed significantly less CR1, and degraded significantly less AC3b when compared with monocytes of healthy controls. In addition, monocytes from RV patients degraded significantly less AC3b when compared with monocytes from patients with RA. The expression of both CR1 and CR3 on monocytes of RV patients was lower compared with RA patients but this difference was only significant for CR3. No differences were found between AC3b degradation and the expression of CR1 and CR3 between patients with active and inactive RA. Using linear discriminant analysis on the variables AC3b, CR1 and CR3, 94% of the patients could be classified correctly as healthy controls, RA or RV, suggesting a true multi-variate relationship between these parameters and patients groups. Our results suggest that the diminished capacity of monocytes from RA patients to degrade AC3b is due partly to a decreased expression of CR1 and CR3.

Adult↗
PubMed1991

Covalent binding of non-proteolysed C3 to Jurkat T cells.

Purified C3 binds covalently to Jurkat T cells upon incubation at neutral pH. This binding does not appear to involve proteolysis of C3; it leads to high-molecular-weight associations, preferentially through ester linkages, which are disrupted upon incubation with hydroxylamine at alkaline pH. Part of the association also appears to involve disulfide links between C3 and Jurkat cells. Similarly, plasma membranes purified from these cells bind C3 with no evidence for proteolysis of C3. Binding of C3 appears to be "catalysed" by Jurkat cells, and is not due to the well-known spontaneous hydrolysis of C3. Binding of C3 involves hydrolysis of its thioester bond, as titratable--SH groups are available in soluble C3 after incubation of purified C3 with Jurkat plasma membranes; loss of C3 haemolytic activity confirms this finding. These observations give evidence for the binding of C3b-like C3 to Jurkat cells, conferring on these cells the potential to interact with other complement receptor-bearing cells such as B cells.

Antigens, Differentiation, B-Lymphocyte↗
PubMed2004

Mutations in CD46, a complement regulatory protein, predispose to atypical HUS.

Membrane cofactor protein (MCP, CD46) is a widely expressed transmembrane complement regulator. As does the soluble regulator factor H, it inhibits complement activation by inactivating the C3b that is deposited on target membranes. Factor H mutations have been described in 15-30% of patients with atypical haemolytic uraemic syndrome (HUS). Recent studies have identified mutations in the MCP gene in four families. In one, a heterozygous deletion resulted in the intracellular retention of the mutant protein. In another, a different heterozygous deletion led to a premature stop codon and the loss of the C-terminus. In the other two, a substitution (S206P) resulted in cell-surface expression but inefficient inactivation of surface-bound C3b. These findings provide further evidence that complement dysregulation predisposes to the development of HUS.

Cell Membrane↗
PubMed1999

The factor H protein family.

The factor H gene family provides a prime example of a multidomain multifunctional protein family whose individual members are defined by conserved common structural elements and display diverse but often overlapping functions. The six identified members of this protein family represent secreted plasma proteins that are primarily synthesized in the liver. Here, we summarize the current understanding of the function of these proteins and suggest a common role in complement control. Factor H is the best characterized member and acts as a complement regulator. The protein displays cofactor activity for factor I in the degradation of the central complement component C3b, acts as a decay accelerating factor for the C3 convertase, C3bBb and is a competitor for factor B binding to C3b. Factor H is a multifunctional protein and displays functions outside the complement system: it binds to the cellular integrin receptor (CD11b/CD18), interacts with cell surface glycosaminoglycans and also binds to the surface of certain pathogenic microorganisms. In addition, factor H has several binding sites for the C3 protein. The factor H-like protein 1 (FHL-1) or reconectin shares the complement regulatory functions with factor H and interacts with heparin. The protein displays cell spreading activity and binds to the N-terminus of the streptococcal M protein. The function of the factor H-related proteins (FHR-1 to FHR-4) is currently under investigation. These proteins are differently distributed. Three proteins (FHR-1, FHR-2 and FHR-4) are constituents of lipoproteins, while FHR-3 interacts with heparin. Binding to C3b and C3d has been demonstrated for FHR-3 and FHR-4 and the two proteins display a cofactor related activity.

Complement Factor H↗
PubMed1988

Quantitative analyses of the relationship between C3 consumption, C3b capture, and immune adherence of complement-fixing antibody/DNA immune complexes.

We have studied the turnover of the third component of C (C3) and capture of the major cleavage fragment of C3 produced during C activation (C3b) that occurs when soluble antibody/DNA immune complexes (IC) active C. We used the Amersham RIA kit for the minor cleavage fragment of C3 produced during C activation (C3a), and a new assay utilizing mAb to C3b to measure the fraction of active C3 in a C source after the IC activate C. These mAb, along with a mAb to human IgG, allowed us to measure IC stoichiometries. The efficiency of C3 turnover by the IC is quite high, and under conditions of Ab excess, the maximum number of IgG bound per dsDNA corresponds to 1 IgG/20 to 30 base pairs. The maximum number of C3b found in the IC corresponds to less than 1 C3b/IgG, and the vast majority of the captured C3b is bound to the IgG, and not to the DNA. We identified several IC that consumed large amounts of C3, and captured large amounts of C3b, but did not bind to human E via C3b receptors (C receptor type 1). This finding suggests that the ability of IC to bind to human E depends upon the number and distribution of captured C3b molecules and the conformation and size of the DNA Ag, which reflects the need for multivalent binding between several properly arrayed C3b and a "cluster" of C receptor type 1 on the human E membrane. IC that activate C3 but do not bind to E would presumably "escape" the E IC clearance mechanism, but could deposit in susceptible organs and tissues and play a role in the pathogenesis of SLE because of their potential to generate the inflammatory products of C activation.

Antigen-Antibody Complex↗
PubMed1984

Characterization of the third component of pig complement and its utilization in a C3b receptor study.

The third component of the pig complement system (C3) was isolated in hemolytically active form and characterized. The C3 component is a beta-globulin with the molecular weight of 191,000 and is composed of 2 non-identical polypeptide chains of Mr 112,000 and 74,000. The isolated C3 can be used for the detection of the C3b receptor on the membranes of heterologous peritoneal macrophages.

Animals↗