Expression of complement receptors type 1 (CR1) and type 3 (CR3) on circulating granulocytes in experimentally provoked asthma

Neutrophils demonstrate increased complement receptor activity, measured by rosetting of C3b-coated erythrocytes, after asthma that was provoked experimentally. However, it is not clear whether the increased rosetting is due simply to increase in receptor numbers or whether other factors, such as cell adhesiveness, are involved. We have therefore enumerated granulocyte complement receptors, after asthma provoked experimentally, with monoclonal antibodies against the receptors and flow cytometry. There was a maximal 28.2 +/- 7.5% and 33.4 +/- 9.5% (mean +/- SEM; n = 15) increase in granulocyte CR1 and CR3, respectively, at 3 hours after asthma induced by antigen. There was a maximal 32.0 +/- 7.3% (mean +/- SEM; n = 7) increase in granulocyte CR1, but no change in granulocyte CR3, at 1 hour after exercise-induced asthma. No significant changes in granulocyte CR1 or CR3 were observed up to 6 hours after methacholine challenge, or after exercise in subjects who did not develop exercise-induced asthma. There was a maximal 33 +/- 9% (mean +/- SEM; n = 8) increase in granulocyte CR1 at 30 minutes, but no increase in granulocyte CR3, after histamine challenge of subjects with asthma. Incubation of whole blood with histamine in vitro did not lead to any enhancement in expression of granulocyte CR1. This suggests that antigen- and exercise-induced release of histamine may augment granulocyte CR1 expression through an indirect mechanism. These data indicate that there is increase in the numerical expression of CR1 on granulocytes, after asthma provoked experimentally, which is accompanied by increases in granulocyte CR3 after bronchoprovocation with antigen, but not histamine or exercise.     

Human cytomegalovirus downregulates complement receptors (CR3, CR4) and decreases phagocytosis by macrophages

Human cytomegalovirus (HCMV) infection is associated with an increased susceptibility to opportunistic infections. Although the subversion of adaptive immune responses has been extensively studied, the consequences of HCMV infection on natural immune responses are not well documented. A striking selective downmodulation of CD11b/CD18 (CR3) or CD11c/CD18 (CR4) was found upon HCMV infection, on two models, the monocytic THP-1 cell line and monocyte- derived macrophages. HCMV-infected macrophages have an altered adhesion/phagocytic capacity to Candida albicans, a pathogen responsible for some opportunistic infections in immunocompromised patients. These results suggest a new mechanism implicated in the augmentation of opportunistic infections in HCMV patients.     

Presence of serum modulates expression of complement receptor type 1 (CR1) on human granulocytes after quartz exposure

We have investigated the interaction between granulocytes and quartz with respect to the expression of complement receptor type l (CR1) and the presence of normal human serum (NHS). Quartz down-regulates selectively CR1 on activated granulocytes. This down-regulation is abolished in the presence of both NHS and heat-inactivated NHS (NHS₅₆) but not human albumin. When quartz was preincubated with NHS (quartz-NHS) before exposure to activated granulocytes, a down-regulating effect was observed in contrast to preincubation with NHS₅₆, which did not induce a down-regulation. Preincubation with cytochalasin B reduced the down-regulation of quartz-NHS, indicating a cytoskeleton-dependent internalization of the receptor. The serine protease inhibitor PMSF partly reduced this down-regulation. Our results indicate that the presence of NHS in the alveolar space influences the interaction between quartz and recruited granulocytes with respect to CR1 expression. Since CR1 is an important opsonin receptor and soluble CR1 can modulate the inflammatory response, this may be of importance in the inflammation and fibrosing process induced by quartz in the alveolar space and lung interstitium.     

Ontogeny of human complement receptors CR1 and CR3: expression of these molecules on monocytes and neutrophils from maternal, newborn and fetal samples

Higher levels of the expression of CR1 and CR3 molecules were detected on the surface of monocytes and neutrophils from maternal and newborn (cord) blood samples than in adult controls. Chemotactic factors such as formyl-methionyl-leucyl-phenylalanine or leukotriene B4 induced an increase of the expression of CR1 and CR3 which was more pronounced on cells from maternal and cord samples than from nonpregnant adult controls. CR1 and CR3 molecules were detected in monocytes and neutrophils from peripheral blood obtained from fetuses more than 14 weeks old and on subpopulations of cells in bone marrow, spleen and thymus.     

Chemotactic factor-induced low density neutrophils express enhanced complement (CR1 and CR3) receptors and increased complement-dependent cytotoxicity.

We have studied chemotactic factor-induced 'complement receptor enhancement' to determine whether changes in receptor expression and complement-dependent cytotoxicity were associated with alterations in cell density. Ficoll-Paque separated normal human neutrophils (greater than 90%), when further fractionated on discontinuous metrizamide (MTZ) gradients (18, 19, 20, 21, 22, 23% MTZ), consistently gave two major bands at the 20/21% and 21/22% interfaces. Incubation with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP (10(-8) M)), converted virtually all neutrophils to low density cells sedimenting on MTZ at the 18/19% and 19/20% interfaces. There was a time-dependent change of density after fMLP-stimulation which was maximal at 30 min, with cells reverting towards normal density by 60 min. Control unstimulated cells did not alter their density at any of the time points examined. Activated, low density neutrophils had increased expression of CR1 and CR3 (as shown by flow cytometry and the uptake of 125I-F(ab')2 monoclonal anti-CR1 antibody (E11)). These cells also showed enhanced cytotoxic capacity in vitro for helminthic targets (schistosomula of Schistosoma mansoni) opsonized with autologous complement. There were highly significant correlations between cell density and anti-CR1 uptake (P less than 0.001), and between schistosomular killing and change in density (P less than 0.001). Increased CR1 expression also correlated with enhanced helminthicidal capacity of neutrophils (P less than 0.001). Complement dependent cytotoxicity was partially reduced after treatment of cells with anti-human CR1 and/or CR3 antibodies, but only in the presence of a second antibody. These findings indicate that chemotactic factor-induced complement receptor enhancement of human neutrophils is associated with a decrease in cell density and increased complement-dependent cytotoxicity (CTX).     

Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes.

Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. Two macrophage receptors, the mannose-fucose receptor (MFR) and the receptor for complement component C3bi, CR3, were examined for their roles in the attachment and ingestion of L. donovani by human monocyte-derived macrophages. Two monoclonal antibodies which bind to the human CR3, anti-Mo1 and anti-Mac-1, inhibited both attachment and ingestion of L. donovani promastigotes after preincubation with human monocyte-derived macrophages; attachment was inhibited by 40 and 62% by anti-Mo1 and anti-Mac-1, respectively, and ingestion was inhibited by 34 and 51% by anti-Mo1 and anti-Mac-1, respectively. The interaction between promastigotes and CR3 may not have involved the C3bi-binding site on CR3, however, because a monoclonal antibody which exhibits specificity for this site, OKM10, inhibited promastigote attachment by only 18%. In contrast, OKM1, which is believed to react with the alternate lectinlike binding site on CR3, inhibited ingestion by 65%. MFR activity was inhibited using the soluble MFR ligands, mannan and mannosylated bovine serum albumin, which also inhibited promastigote attachment by 40 and 37%, respectively. The simultaneous inhibition of both CR3 (by anti-Mac-1) and the MFR (by either mannan or mannosylated bovine serum albumin) resulted in a greater decrease in promastigote attachment than inhibition of either receptor alone. Additionally, the reduction of MFR activity by allowing macrophages to adhere to a mannan-coated surface followed by the addition of anti-CR3 antibodies resulted in an 81% inhibition of promastigote ingestion, a greater decrease than was obtained by manipulation of either receptor alone. The results suggest that the MFR and CR3 independently participate in the attachment and ingestion of L. donovani promastigotes by human macrophages.     

Defective complement receptors (CR1 and CR3) on erythrocytes and leukocytes of factor I (C3b-inactivator) deficient patients.

In view of a possible modulation of the C3b receptor (CR1) by its ligand, we studied a situation in vivo in which C3b is constantly present in the serum, i.e. the genetic factor I-deficiency. C3b and iC3b receptors (CR1, CR3) on peripheral blood cells, were analysed in three I-deficient (I-def.) patients, from two unrelated families. CR1 and CR3 were quantified by means of monoclonal antibodies, and functionally tested (phagocytosis of sensitized sheep erythrocytes (EIgG) or rabbit erythrocytes (Er), coated with C3b, and chemiluminescence (CL) induced by serum-opsonized zymosan). Erythrocyte CR1 levels were significantly lower in I-def. patients than in normal individuals. Monocytes and polymorphonuclear neutrophils (PMN) prepared at 4 degrees C, to prevent increase of CR1 expression in vitro, expressed low CR1 numbers. Monocytes prepared at room temperature showed a defective CR1-dependent phagocytosis and an impaired CL response, although their CR1 levels were found normal in these conditions. This discrepancy was also observed on phorbol myristate acetate (PMA)-activated cells. These CR1 abnormalities are likely to result from repeated interactions of CR1 with C3b molecules, which circulated in the serum of I-def. patients and were deposited onto their red cells. Although iC3b, the CR3 ligand, is not produced in I-deficient sera, monocyte CR3-dependent function (phagocytosis of unopsonized Er) was also found to be defective in two out of the three patients.     

CR1(CD35) and CR2(CD21) complement C3 receptors are expressed on normal human thymocytes and mediate infection of thymocytes with opsonized human immunodeficiency virus

The present study demonstrates that the C3b receptor CR1 (CD35) and the C3dg/Epstein-Barr virus receptor CR2 (CD21) are expressed by 25% and 70% of normal human thymocytes, respectively. The expression of CR2 extends to both CD1⁺ and CD1^? cells in the thymus. Two subsets of CR2⁺ thymocytes were defined expressing low and high density of the receptor. The CR2⁺⁺ subset represented 20% of CR2⁺ thymocytes and co-expressed the CR1 receptor. CR2⁺⁺ thymocytes expressed an immature CD1^dull, CD3^?, CD4^dull, CD8^?, CD7⁺⁺ phenotype and included a subpopulation of large cells expressing CD34. Twenty percent of thymocytes expressed the CD21 epitope defined by monoclonal antibody BU32, which is involved in the binding of CD23 to CD21. These observations provide a basis for a role for CD21 in the proliferation and differentiation of thymocytes at early stages of maturation. The functionality of CR1 and CR2 on thymocytes was evidenced by the ability of the receptors to mediate infection of cells with complement-opsonized human immunodeficiency virus (HIV). The results may be relevant to the immunopathogenesis of HIV infection.     

CR1 and CR1-like: the primate immune adherence receptors

Immune adherence describes the phenomenon in which complement‐opsonized substrates, such as immune complexes (IC), viruses, or bacteria, are bound by primate erythrocytes via erythrocyte complement receptors. In vivo studies have shown that this binding allows the erythrocyte to act as an inert shuttle, targeting IC to the monocyte phagocytic system and away from vulnerable tissue. Thus, immune adherence appears to play an integral role in the primate in promoting the safe clearance of circulating IC and preventing IC‐mediated pathologies. The complement receptors that mediate immune adherence comprise two unique but closely related gene products, either the type one complement receptor (CR1) in humans or CR1‐like in non‐human primates. This review focuses on the structure, function, and physiological role of the primate immune adherence receptors.     

Relationship of chemotactic receptors for formyl peptide and C5a to CR1, CR3, and Fc receptors on human neutrophils

The co-expression of C5a and formyl-methionine-leucine-phenylalanine-lysine (FMLPL) receptors with CR1, CR3, and Fc receptors on human neutrophils (PMN) was studied. Fluorescein-conjugated C5a (FL-C5a) and FMLPL (FL-FMLPL) were used to identify C5a and formyl peptide receptors. CR1, CR3, and Fc receptors were identified with monoclonal antibodies and a Texas red-labeled goat anti-mouse immunoglobulin second step reagent. The co-expression of chemotactic receptors with CR1, CR3, or Fc receptors was evaluated using two-color flow cytometry. A direct correlation between the degree of expression of receptors for FL-FMLPL and the expression of CR3, CR1, and Fc receptors on individual PMN was observed. In contrast, no correlation between the degree of C5a receptor expression and CR1, CR3, or Fc receptor expression was found. Similar results were obtained with PMN after up regulation of CR1, CR3, Fc, and FMLPL receptors by incubation at 37 degrees C for 10 min with or without phorbol myristate acetate. These data suggest that the expression of FMLPL, CR1, CR3, and Fc receptors are regulated in a similar manner, whereas C5a receptor expression is regulated independently. Furthermore, these data indicate that within a given population of PMN, a parallel exists between the degree of CR1, CR3, FMLPL, and Fc receptor expression on individual cells.     

Markers of inflammatory activation: upregulation of complement receptors CR1 and CR3 on synovial fluid neutrophils from patients with inflammatory joint disease.

Expression of the C3 receptors CR1 and CR3 was investigated on neutrophils from paired peripheral blood and synovial fluid samples from 34 patients with inflammatory joint disease (21 patients with rheumatoid arthritis (RA) and 13 patients with other articular diseases (OAD)). Using monoclonal antibodies (anti-CD35, anti-CD11b) and immunofluorescence flow cytometric analyses the percentages of positively labeled cells and the relative fluorescence intensities (as a measure of receptor number) were determined. CR1 and CR3 were found to be present on the majority (> 85%) of circulating neutrophils from normal subjects, RA and OAD patients, and on synovial fluid neutrophils from both patient groups. A strong correlation between neutrophil CR1 and CR3 expression was observed in peripheral blood samples from normal subjects (r = 0.81; P = 0.001), RA (r = 0.79; P = 0.001), and OAD patients (r = 0.83; P = 0.001); in each case the levels of CR3 expression were approximately twice those recorded for CR1. Both CR1 and CR3 expression was upregulated on synovial fluid neutrophils compared with that observed on the corresponding peripheral blood cells. Mean percentage increases observed were: RA patients: CR1, 16.5% (P < 0.001) and CR3, 28.7% (P < 0.001); and OAD patients: CR1, 4.1% and CR3, 26.9% (P = 0.001). Correlation of serum and synovial fluid IL-6, IL-8, and immune complex levels with neutrophil CR1 and CR3 expression failed to demonstrate any significant relationship between the concentrations of these soluble factors and receptor expression. Upregulation of CR1 and CR3 receptors, reflecting neutrophil activation within the inflamed joint, is a consistent finding in patients with inflammatory arthropathies.     

Selective up-regulation of human granulocyte integrins and complement receptor 1 by cytokines.

The percentage of human granulocytes expressing the integrins CD11b and CD11c as well as complement receptor 1 (CD35) was increased by short-term incubation of whole blood with interleukin-2 (IL-2), interleukin-4 (IL-4) and tumour necrosis factors alpha and beta (TNF-alpha and TNF-beta). The mean fluorescence intensity of granulocyte CD18 was also increased by the above cytokines, whilst that of CD11b was only increased by TNF-alpha. Up-regulation of granulocyte CD18 expression was seen with 1 U/ml of IL-2, TNF-alpha or TNF-beta, in contrast to the effect of IL-4 which was only observed with 100 U/ml. Similarly, enhanced expression of CD35 was induced by 1 U/ml of IL-2 or TNF-alpha but not by concentrations of IL-4 or TNF-beta lower than 100 U/ml. Cytokine effects on the CD11/CD18 complex and CD35 molecules were not modified by cycloheximide, suggesting that their increased expression was not due simply to synthesis de novo. None of the granulocyte surface determinants investigated was altered upon short-term incubation of blood with either IL-1, IL-6 or interferon-gamma (IFN-gamma). The demonstration in vitro that cytokines selectively up-regulate granulocyte integrins and complement receptor 1, suggests that similar mechanisms may be operating during the control of granulocyte-mediated inflammatory processes.     

Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.     

Characterization of the human erythrocyte complement receptor CR1 (C3b receptor) by epitope mapping

Monoclonal antibodies and an anti-idiotypic serum against human complement receptor CR1 (C3b receptor, immune adherence receptor) were used to identify CR1 and some of its proteolytic fragments by an immunoblotting technique. The anti-idiotypic serum had a specificity for the C3b-binding site, as could be shown by its cross-reactivity with complement factor H. The monoclonal antibodies GARP-4 and GARP-37 were specific for epitopes located nearby the ligand-binding site, because they blocked the immune adherence reaction. For the immunoblotting technique, it was essential to use non-reducing conditions, since reduction of CR1 destroyed the epitopes. Therefore, mainly large (disulphide-linked) fragments of CR1 were obtained. A chymotryptic fragment of Mr 56,000 identified by GARP-4, was the smallest cleavage product to be associated with the C3b-binding domain. Different proteases gave CR1 degradation products of similar Mr, indicating the presence of distinct domains, three of which had a Mr approximately 38,000. A schematic model of CR1 substructure was deduced from the epitope mapping data.     

Interleukin-3-induced up-regulation of CR3 expression on human eosinophils is inhibited by dexamethasone.

Eosinophil function is regulated by several cytokines, including interleukin-3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Culture of human eosinophils with IL-3 produced a marked, dose-dependent up-regulation of CR3 expression. This was maximal after 1 day in culture and dependent on protein and RNA synthesis, as demonstrated by inhibition with cycloheximide and actinomycin D, respectively. IL-5 and GM-CSF had a similar effect on eosinophil complement receptor type 3 (CR3) expression, but the maximal response to IL-5 was always less than to IL-3 or GM-CSF. Dexamethasone inhibited the Il-3-induced up-regulation of CR3 expression in a dose-dependent manner, with an IC50 of 5 x 10(-8) M. This study demonstrates the effect of IL-3, IL-5 and GM-CSF on eosinophil CR3 expression and confirms the capacity of eosinophils to modify their phenotype through de novo protein synthesis. This process could be inhibited by physiological concentrations of glucocorticoids, thus providing an additional mechanism for their mode of action in allergic disease.     

Human liver Kupffer cells express CR1, CR3, and CR4 complement receptor antigens. An immunohistochemical study

The expression of complement receptor antigens by human Kupffer cells (KC) was investigated by immunohistochemical techniques in seven normal human liver biopsies. Polyclonal and monoclonal antibodies were revealed by double labeling of cells using indirect immunofluorescence and immunoenzymatic techniques or by using double immunoenzymatic techniques. In most experiments, one antigen was revealed by streptavidin-biotin-peroxidase complexes whose reaction product was examined by light microscopy and the second antigen stained using the alkaline phosphatase antialkaline phosphatase method visualized by fluorescence microscopy using fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate filters. KC were identified using monoclonal antibody EBM11 that recognizes 100% of KC in hepatic lobules and was paired with each antibody directed against complement receptors. CR1 and CR3 (alpha- and beta-chains) were found to be the predominant receptor antigens expressed by human KC. CR4 (p150,95) was expressed on all KC, but staining with anti-CR4 monoclonal antibodies was consistently weaker than that observed with anti-CR3 antibodies. No staining of KC was observed with anti-CR2 (CD 21) antibodies. Expression of CR1, CR3, and CR4 complement receptors on KC provides the cells with an optimal capacity to bind and phagocytize particles or immune complexes coated with any type of ligands for C3 receptors.     

Phagocytosis by receptors for C3b (CR1), iC3b (CR3), and IgG (Fc) on human peritoneal macrophages

Human peritoneal macrophages (HPM) obtained via laparoscopy were examined for the presence and functional capacity of complement and Fc receptors. Between 5 and 20 ml of peritoneal fluid containing 1-2 X 10(6) macrophages/ml was available for each study. Macrophages made up 80-95% of the cells in the fluid. Fc and C3 receptors on HPM were characterized by rosette formation with, and phagocytosis of, IgG- and C3-coated sheep erythrocytes (E). ElgG were bound by 82% and ingested by 63% of HPM, with 4-15 E ingested/HPM. The HPM formed rosettes with EC3b (56%) and EC3bi (71%) but not EC3d,g or EC3d. Antibodies to complement receptors type 1 (CR1) and type 3 (CR3) inhibited rosette formation with EC3b and EC3bi, respectively, indicating that HPM possessed separate and distinct receptors for the C3b and iC3b ligands. In 60% of the samples studied, HPM demonstrated the ability to ingest both EC3b and EC3bi, as well as ElgG. Because of the heterogeneous nature of the cells obtained in peritoneal fluid, due to their progressive change from monocytelike cells into mature macrophages, HPM were separated by 1 g velocity sedimentation into fractions of increasing maturity. They were then examined for phagocytosis via Fc and complement receptors. Fc receptor mediated phagocytosis occurred throughout the monocyte-to-macrophage maturation sequence, while the ability of HPM to ingest via CR1 and CR3 was maturation dependent, with ingestion via CR3 occurring before CR1, in a manner analogous to in vitro differentiation of monocyte-derived macrophages.     

Immunocytochemical localization of CR3 complement receptors with OX-42 in amoeboid microglia in postnatal rats

Summary The present study described the labelling of amoeboid microglial cells in the postnatal rat brain with OX-42, an antibody that recognizes type 3 complement receptors CR3 in mononuclear phagocytes. Of the diverse morphological forms of amoeboid microglia present in the corpus callosum in early postnatal (2–5 days) rats, cells with a round regular outline, or showing short stout processes, were the most intensely stained. When traced from the main cell colony into the borderline zone with the cortex, the immunoreactivity of amoeboid microglia that assumed a ramified form was drastically reduced. Examination of materials from the late postnatal (8–12 days) age group showed that the majority of the OX-42 positive cells in the corpus callosum became oval, elongated and ramified. Immunoelectron microscopy confirmed the above observations, and also showed that the immunoreactivity in the round amoeboid microglia was localized in their plasma membrane, surface projections and invaginations, as well as in some of the subsurface vacuoles. The immunoreactivity was reduced in the oval cells, and diminished in the elongated or ramified form. It is proposed that the presence of CR3 membrane receptors in amoeboid microglial cells is related to their active role in endocytosis. These, however, diminish with the growth of the brain.