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PubMed1985

The mouse macrophage receptor for C3bi (CR3) is a major mechanism in the phagocytosis of Leishmania promastigotes.

We examined the role of the macrophage receptor for C3bi, the CR3, in the phagocytosis of Leishmania major promastigotes and report that M1/70, a monoclonal antibody to the CR3, inhibited the binding of leishmania to macrophages both when the assays were performed in the presence of normal serum and in its absence. In serum, leishmania activate complement and fix C3. Fixation and subsequent cleavage to C3bi occurs rapidly, and by as early as 5 min both forms of the molecule can be identified on the parasites' surface. Complement fixation results in an enhanced phagocytosis of leishmania promastigotes by mouse macrophages. In the case of L. major, 63% of this serum-enhanced binding is inhibitable by M1/70. Binding assays were also performed in the absence of serum with the use of thoroughly washed promastigotes. The addition of M1/70 inhibited binding under these conditions by 54%. Two other rat monoclonal antibodies directed against different antigens on the macrophage plasma membrane did not inhibit binding. M1/70 did not inhibit the binding of promastigotes to rat bone marrow cells, nor did it inhibit IgG-SRBC binding to mouse peritoneal macrophages. These data indicate that the inhibition observed in the presence of M1/70 was specific for the CR3 and that the macrophage receptor for C3bi plays a major role in the phagocytosis of Leishmania major promastigotes, even in the absence of serum.

Animals↗
PubMed1984

Use of monoclonal antibodies against factor H to investigate the role of a membrane-associated protein antigenically related to H in C3b-receptor function.

Three murine monoclonal IgG1 kappa-antibodies, MAH-1, MAH-2, and MAH-3, were raised against factor H purified from human plasma. In cross-inhibition studies MAH-3 did not compete with MAH-1 and MAH-2, and vice versa, for the binding to H, whereas MAH-1 and MAH-2 inhibited each other. MAH-1 and MAH-2 inhibited the binding of H to C3b attached to an ELISA plate as well as to C3b bound to sheep erythrocytes by means of the classical pathway convertase and of C3b to H attached to an ELISA plate. The determinant defined by MAH-1 and MAH-2 was no longer accessible on H bound to C3b. In contrast, MAH-3 interfere with the binding of H to C3b or vice versa only to a smaller extent but recognized a determinant still accessible on H bound to C3b and was able to agglutinate EAC14o23b-H in an indirect Coombs test. All three antibodies were shown to bind to tonsil cells and Raji cells in an indirect cell ELISA. The membrane-associated molecule detected by these antibodies had an apparent m.w. of 140,000 D in SDS-PAGE. All three antibodies partially inhibited the binding of EAC14o23b to tonsil lymphocytes and, in the presence of 0.1 mM DFP, to Raji cells; binding of EAC14o23bi and EAC14o23d to tonsil cells was not affected. We conclude that MAH-3 recognizes a determinant distinct from the ones recognized by MAH-1 and MAH-2, the latter possibly defining identical epitopes that are located close to the binding site for C3b. The fact that these two distinct epitopes could be detected on a 140,000-D membrane-associated protein from lymphoid cells strongly suggests that this molecule is at least antigenically related to serum H and shares with H a region carrying the binding site for C3b. The rosette inhibition studies imply that this structure is important for the binding of C3b-coated particles to lymphoid cells.

Animals↗
PubMed1983

Endocytosis of the C3b receptor of complement within coated pits in human polymorphonuclear leukocytes and monocytes.

The distribution and endocytosis of the C3b receptor by human polymorphonuclear leukocytes and monocytes were visualized by both fluorescent and electron microscopic examination of cells that had been labeled with monospecific F(ab')2 anti-C3b receptor and anti-F(ab')2 conjugated with rhodamine or ferritin. When prefixed or unfixed cells that were labeled at 0 to 4 degrees C were examined, the receptor was distributed within clusters on the plasma membrane. After the cells had been warmed to room temperature or to 37 degrees C for 5 minutes, the fluorescently labeled receptors appeared to enter the cells, and the ferritin-tagged receptors often occurred within coated endocytic pits and coated vesicles within the cytoplasm. After incubation at 37 degrees C for 20 minutes, the C3b receptor-antibody complexes were largely cleared from the cell surface, and much of the label was found within lysosomes. These results indicate that C3b receptors may directly mediate endocytosis within coated pits, thus utilizing a mechanism shared by a variety of other receptors for the rapid, efficient, and selective internalization of extracellular ligands.

Cell Membrane↗
PubMed1983

The effect of macrophages on the metabolism of glomerular cells: preliminary studies.

Human glomerular epithelial and mesangial cells were grown in vitro and shown to have distinctive morphologic and functional characteristics. Glomerular epithelial cells or mesangial cells cultured in wells of flat-bottom microtiter plates were treated for 4 hr with dialyzed macrophage supernatants obtained from cultures of mouse peritoneal macrophages or human peripheral monocytes. DNA, RNA, and protein synthesis were evaluated by incorporation of radioactive precursors. Macrophage supernatants stimulated RNA and protein synthesis in epithelial cells but failed to stimulate DNA synthesis. The macrophage factor(s) showed a dose-response activity, was nondialyzable, was destroyed by freezing and thawing, and did not seem to be species specific. In contrast to the results obtained with glomerular epithelial cells, mesangial cell DNA synthesis was stimulated by macrophage supernatants. The observed metabolic effects of macrophage products on glomerular cells in vitro are consistent with observations of in vivo glomerular response to injury in which epithelial cells may be activated to form new basement membrane while mesangial cells may respond by proliferating. These data further support the theory of macrophage involvement in the pathology of glomerulonephritis.

Animals↗
PubMed1983

Expression of C3b receptors on human be cells and myelomonocytic cells but not natural killer cells.

We have examined natural killer (NK) cells, B cells and myelomonocytic cells at different stages of differentiation for the expression of surface C3b receptors (C3bR). Receptor presence was detected using affinity-purified F(ab')2 anti-C3bR antibodies in indirect immunofluorescence assays. NK cells, identified in fetal and adult tissues by the monoclonal antibody HNK-1, were C3bR- except for infrequent C3bR+ HNK-1+ cells in some blood samples. NK cells were not induced to express C3bR by exposure to interferon, target cells, or phorbol myristate acetate. B cells gradually acquired the ability to express C3bR with maturity: 15% of large pre-B cells were C3bR+, 35 to 48% of small pre-B, 60 to 80% of immature B, and 99% of mature B cells. Mature plasma cells were rarely C3bR+. Myelomonocytic cells acquire C3bR relatively late during their development, with neutrophils beginning to express C3bR during the band stage of differentiation. All adult blood myelomonocytic lineage cells, identified by the monoclonal antibody MMA and by morphology, were C3bR+.

Animals↗
PubMed1983

Functional properties of membrane-associated complement receptor CR1.

It was previously shown that membrane receptors for C3b (CR1) purified from human erythrocytes were powerful inhibitors of the complement cascade and that they encompass the regulatory functions of the serum proteins beta 1H (H) and C4-binding protein (C4bp). In the present report we study the functional properties of membrane-associated CR1. When tonsil lymphocytes, which contain between 30 and 60% of CR1-bearing B cells, are incubated with the red cell complement intermediate EAC14oxy2lim or EAC14oxy23lim, they inhibit both C42 and C423 in a dose-dependent manner. These effects are mediated by membrane-associated molecules. Indeed, mild trypsinization of the lymphocytes abolishes their activity, and formaldehyde-fixed cells are as effective as viable cells. The inhibitory effects are in part mediated by CR1. The lymphocyte activities are reversed about 60% if monoclonal antibodies to CR1 or fluid phase C3b are present in the incubation medium. Moreover, upon addition of C3b-inactivator (l), lymphocytes release C3c fragments from EAC14oxy23b. The release of C3c was also abolished by antibodies to CR1. These results support the idea that CR1, as well as other molecules from the lymphocyte membrane, can function as inhibitor(s) of complement activation in their vicinity.

Animals↗
PubMed1983

Induction of granulocyte histaminase release by particle-bound complement C3 cleavage products (C3b, C3bi) and IgG.

The interaction of opsonized particles with human granulocytes promotes a number of important biologic functions, including phagocytosis, superoxide generation, and release of a variety of enzymes, including histaminase. We have previously determined that histaminase release occurs via a C3-dependent process. Although fluid-phase C3b dimers can mediate release, the relative effects of particle-bound C3b and C3bi and of IgG have not been examined. In this report we demonstrate that particle-bound C3 deposited on activators of the alternative C pathway effected histaminase release in the absence of IgG. Particle-bound C3bi and C3b were both effective as mediators of histaminase release. The extent of release varied as a function of the activating surface on which C3 was deposited (zymosan C3b was considerably more potent than C3b bound to rabbit erythrocytes, which was slightly more potent than C3b bound to neuraminidase-treated sheep erythrocytes). In contrast, C3b or C3bi deposited on nonactivating surfaces (such as sheep erythrocytes) at inputs of up to 2,000,000 molecules per granulocyte failed to induce histaminase release unless IgG was also present. The ability of C3b bound to particles that serve as activators of the alternative pathway to induce histaminase release is apparently not the result of decreased susceptibility of C3b to proteolysis or to an increased binding affinity to the C3b receptor, but may relate to the interaction of other surface structures on activating particles with the PMN membrane.

Amine Oxidase (Copper-Containing)↗
PubMed1983

Characterization of human T lymphocytes that express the C3b receptor.

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.

Animals↗
PubMedDate not supplied

[Selective binding in vitro of a modified form of the C3 component of complement to human erythrocytes].

C3 was bound to human erythrocytes from autologous plasma or from serum brought to low ionic strength (mu less than or equal to 0.03) and pH between 4.0 and 5.0, then subsequently incubated with erythrocytes (50/1, v/v) for 20 min at 0 degree C. This capacity was preserved up to 72 h by prolonged incubation at 20, 25 or 37 degrees C, whereas it was quickly lost by incubation at 0 degree C. C3 binding did not require complement activation and was not observed with neuraminidase-treated erythrocytes. Crossed immunoelectrophoretic analysis of the pretreated serum or plasma revealed that a fraction having more cathodal migration than that of native C3 was generated upon incubation in the above-mentioned conditions. This fraction appeared able to selectively bind to the erythrocytes. Cell-bound C3 reacted positively to antisera against C3a, C3c, C3d or native C3; they rosetted positively with EAC3b, clearly showing that this C3 binding was not dependent on the proteolysis of C3 and that it concerned the acceptor sites on the cells, since C3b receptors were free. The functional significance of this C3 binding was also investigated: EC3 were not able to lyse through the alternative pathway, whereas lysis clearly increased when C3 was found to AET-treated erythrocytes. This finding, together with the modulation in the capacity of EC3 or E(AET)C3 to form an alternative pathway convertase by antibodies to C3c or C3d, strongly suggests a contribution of bound C3 to such a convertase. In contrast to "C3b-like" C3, this modified C3 was able to bind to acceptor sites on erythrocytes but, like the former, it retained the capacity to form an alternative pathway convertase. In this light, it may represent an intermediate between C3 and "C3b-like" C3.

Complement Activation↗
PubMed1983

Immune complex alterations occur on the human red blood cell membrane.

Experimental antigen-antibody complexes (Ag-Ab) were incubated at 37 degrees with human red blood cells (RBC) suspended in autologous normal serum and the reaction stopped after progressively increasing times. Bound antigen-antibody-complement complexes (Ag-Ab-C) were eluted from C3b receptors and the eluted Ag-Ab-C re-incubated with different blood cell types suspended in serum, or centrifuged (along with unbound Ag-Ab-C found in the serum) through 20-50% sucrose gradients. Ag-Ab-C recovered from C3b receptors shortly after initial binding to RBC bound efficiently to other RBC, polymorphonuclear and mononuclear cells, and sedimented rapidly. Ag-Ab-C simultaneously present in the serum sedimented with a similar velocity. Ag-Ab-C recovered at a subsequent time during RBC interaction bound less well to each blood cell type, and sedimented less rapidly. Decreased amounts of rapidly sedimenting Ag-Ab-C were present in the serum. Ag-Ab-C recovered from C3b receptors at a still later time in the course of RBC interaction bound poorly to each cell type, and sedimented slowly. Increased amounts of slowly sedimenting Ag-Ab-C were found in the serum. These findings indicate that alterations in properties of immune complexes can occur while they are associated with C3b receptors on RBC membrane in solid phase.

Antigen-Antibody Complex↗
PubMed1983

Complement-mediated binding of monocytes to intermediate filaments in vitro.

An in vitro model for complement (C)-mediated monocyte binding to injured cells is described. This model is based on the property of cytoskeletal intermediate filaments (IMF) to directly and independently activate serum C via the classic pathway. Fixed monolayers of cultured fibroblasts, endothelial cells, and L 132 epithelial cells were each used as targets. The authors first subjected the target cells to limited detergent extraction in order to make permeable the plasma membrane, and then exposed them to normal human serum. Immunohistologic analysis demonstrated that this process permitted binding of clearly demonstrable amounts of C1q, C4, and C3 to cytoplasmic loci containing IMF. The target cells were then incubated for various lengths of time with peripheral blood mononuclear cells containing about 20% monocytes. Preferential binding of monocytes was demonstrated by histochemical and immunologic staining methods. Only 5% of the attached cells were B lymphocytes. By transmission electron microscopy the attached cells exhibited typical monocyte morphology. Attachment to target cells involved contact with areas rich in IMFs and led to signs of activation; both an increased number of cytoplasmic organelles and phagocytosis of target cell material were observed. Attachment of monocytes was clearly dependent on serum treatment of the targets; the ratio was 10:1 when compared with control experiments using target cells that had not been incubated with serum. It is reasonable to consider that bound monocyte-derived macrophages associate with activated C components, most likely C3b fragments or C1q, for which monocyte surface receptors have been established. This mechanism may be triggered in various inflammatory reactions involving cell and tissue injury and as an event in the final stages of macrophage-mediated removal of injured or nonviable cells.

Animals↗
PubMed1983

Comparison of binding characteristics of factors B and H to C3b on normal and paroxysmal nocturnal hemoglobinuria erythrocytes.

To determine if aberrant interactions of the endogenous control proteins with cell-bound C3b contribute to the greater fixation of C3b to paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes when whole serum complement is activated, we compared the characteristics of binding of factors B and H to normal and PNH red cells bearing C3b (EC3b). Factor B binding is homogeneous, there is 1:1 stoichiometry, and the affinity constant at equilibrium for factor B binding is the same for normal and PNH EC3b. In contrast, analysis by Scatchard's method of factor H binding results in a curvilinear plot, the deviation from linearity being exaggerated for the PNH EC3b. The heterogeneity of binding of factor H appears to be a consequence of the nonrandom distribution of C3b about the alternative pathway convertase site. This nonrandom distribution does not induce negative cooperativity but rather effects a biophysical milieu which enhances factor H binding. The greater heterogeneity of binding of factor H to PNH E bearing nonrandomly distributed C3b appears to be due to the presence of a greater proportion of lower affinity binding sites on the PNH EC3b. However, it appears unlikely that this greater heterogeneity of factor H binding contributes to the enhanced fixation of C3b to PNH erythrocytes.

Binding, Competitive↗
PubMed1983

Studies on immune adherence (C3b) receptor activity of human erythrocytes: relationship between receptor activity and membrane osmotic fragility.

Although human erythrocytes (E) possess C3b receptors (C3b-R), their in vivo functions are unknown. We had observed that E from patients with immune complex diseases had defective or impaired C3b-R activity when circulating immune complexes (CIC) could be demonstrated. This phenomenon has been investigated in relation to membrane osmotic fragility of such E by a coil planet centrifugation (CPC) system. Osmotic fragility was defined by the hemolysis starting point (HSP), peak point (HPP) and end point (HEP) using NaCl osmotic gradient (150-30 mOsM) coiled tubes. It was observed that E with low C3b-R activity showed high osmotic fragility. Hemolysis of E with low C3b-R activity started at 104 +/- 8 mOsM (n = 133) compared to 97 +/- 3 mOsM (n = 18) of patients' E with normal C3b-R activity and 96 +/- 5 mOsM of E from normal healthy donors (n = 128). Furthermore, we observed that HSP shifted towards lower osmolarity with clinical and immunological improvement of disease activity after treatment with corticosteroids. When osmotic gradients were lower to 120-50 mOsM, 52 out of 116 E samples with low C3b-R activity separated into 2 E populations. In contrast, none of 18 E samples with normal C3b-R activity separated into 2 E populations. However, we observed broadened fragility patterns in these 18 E samples. Serial studies of C3b-R activity, osmotic fragility and the presence of CIC were performed in 7 patients. Improvement of disease activity was associated with increased C3b-R activity, decreased osmotic fragility and the disappearance of CIC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Hormones↗
PubMed1983

Defective chemotactic factor-induced monocyte complement receptor enhancement in lung cancer.

We have studied monocyte complement (C3b) rosettes, and chemotactic factor (CF)-induced enhancement of rosettes, in 37 patients with bronchial carcinoma (BC), and compared these findings to those obtained from nine age and sex matched patients with non-malignant respiratory disease (NMRD), and 38 normal healthy controls (HC). No differences in monocyte C3b rosettes were found when BC was compared with NMRD or HC. In contrast, there was a highly significant inhibition of CF-induced complement receptor enhancement (CRE) in monocytes from patients with extensive or metastatic BC compared to NMRD or HC. These differences increased with time of incubation and concentrations of CF, and occurred when either f-met-leu-phe, leukotriene B4 (LTB4) or casein were used as the enhancing agent. The defect in monocyte CRE was related to the stage of the disease (with the rank order, metastatic greater than confined to chest greater than localized tumour) but not to the pathohistological type. A similar impairment in CRE was observed when neutrophils from BC were compared to HC. These experiments suggest that, in patients with advanced bronchial carcinoma, monocyte and neutrophil C3b receptors have impaired responsiveness to chemotactic factors.

Adult↗
PubMed1983

The human C3b receptor.

The cellular receptor for the C3b fragment of the third component of complement is a 205,000 molecular weight glycoprotein expressed by erythrocytes, polymorphonuclear leukocytes, monocytes, B lymphocytes, a subset of T lymphocytes and glomerular podocytes. The receptor molecule is a potent inhibitor of complement activation by both the alternative and classical pathways. It serves as a cofactor in the proteolytic degradation of C3b bound to immune complexes. On neutrophils and monocytes, the receptor enhances immunoglobulin-dependent phagocytosis of opsonized particles and triggers internalization of soluble ligands bearing C3b. The number of C3b receptor molecules expressed on erythrocytes is genetically determined and was found to be low in patients with systemic lupus erythematosus: these abnormalities when associated with a low number of receptors in the kidney of patients with non-systemic lupus erythematosus nephritis may predispose to immune complex diseases.

Animals↗
PubMed1984

Immunoglobulin production by human peripheral lymphocytes induced by anti-C3 receptor antibodies.

The exact function of receptors for C3b (CR1), which are present on B lymphocytes, is not clear. The present studies were performed to determine the influence of heterologous anti-CR1 on the production of IgM, IgG, and IgA by human peripheral B lymphocytes in vitro. Anti-CR1, raised in rabbits by immunization with purified erythrocyte CR1, was rendered immunospecific and was converted to F(ab')2 and Fab' by enzyme digestion. Pre-immune F(ab')2 and Fab' served as controls. Normal human blood T lymphocytes and T cell-depleted mononuclear cells were cultured for 6 days in RPMI medium and 10% heated fetal calf serum with a low dose (7 micrograms/ml) of pokeweed mitogen (PWM) alone or with PWM and F(ab')2-anti-CR1. In control experiments the effects of F(ab')2-anti-CR1 without PWM and of pre-immune F(ab')2, Fab'-anti-CR1 and pre-immune Fab' in combination with PWM were also tested. When PWM and F(ab')2-anti-CR1 were present simultaneously, 4.3 X 10(3), 2.5 X 10(3), and 1.8 X 10(3) ng/ml of IgM, IgG, and IgA were synthesized, respectively, in cultures containing 4.5 X 10(5) mononuclear blood cells. All other combinations resulted in synthesis of less than 250 ng/ml of each class of Ig. The presence of purified CR1 in the cultures containing PWM and F(ab')2-anti-CR1 caused a dose-dependent decrease of Ig synthesis. By culturing B cells isolated by E-C3b rosetting with T cells and monocytes, the anti-CR1 responding population was demonstrated to be mainly in the CR1-rich B cell fraction. Fab'-anti-CR1 at high doses was also able to stimulate Ig production in the presence of low concentrations of PWM. These data suggest that triggering of CR1 on B cells results in modulation of antibody production and that triggering of CR1 with more than one antibody is required for an optimal response.

Animals↗
PubMed1984

C3b receptor-negative peripheral blood neutrophils. A study of normal and haematologically abnormal disorders.

Expression of peripheral blood neutrophil (PBN) C3b receptors, as assessed by rosette formation with C3b-coated ox erythrocytes, was examined and compared with neutrophil alkaline phosphatase (NAP) activities in both normal and haematologically abnormal conditions. The results indicate that a small percentage of normal PBN are apparently C3b receptor-negative and that these neutrophils do not appear to differ with respect to age from those with detectable C3b receptors. Examination of PBN C3b receptors from 154 cases of various haematological disorders revealed a significant proportion of cases with increased numbers of C3b receptor-negative neutrophils. These abnormalities did not appear to be related to peripheral leucocyte counts, NAP activities or serum lysozyme concentrations and it is suggested that the increased numbers of C3b receptor-negative PBN may be related to intravascular factors such as immune complexes.

Alkaline Phosphatase↗