Critical role of Kupffer cell CR3 (CD11b/CD18) in the clearance of IgM-opsonized erythrocytes or soluble beta-glucan.

Liver macrophages (Kupffer cells) play a major role in blood clearance of both C3-opsonized immune complexes and therapeutic beta-glucan polysaccharides. Human Kupffer cells express three types of C3-receptors: CR1 (C3b-receptor; CD35), CR3 (iC3b- and beta-glucan-receptor), and CR4 (iC3b-receptor; CD11c/CD18). Studies of isolated macrophages have suggested that CR3 is the major receptor mediating capture of either C3-opsonized erythrocytes (E) or beta-glucans. In this investigation, the organ distribution and function of CR3 in the clearance of IgM-opsonized E and soluble CR3-binding polysaccharides were explored in normal vs. CR3-knockout (CR3-KO) mice. Analysis of intravenously (i.v.) injected 125I-anti-CR3 showed that the major vascular reservoir of CR3 was the liver, followed by spleen and lungs. By contrast, clearance of 125I-anti-CR1 appeared to be mediated predominantly by splenic B lymphocytes, as only subsets of splenic macrophages or Kupffer cells were found to express CR1. Clearance of IgM-opsonized 51Cr-E occurred rapidly to the livers of normal mice but was nearly absent in CR3-KO mice. Soluble 125I-beta-glucan exhibited rapid clearance to the liver in normal mice, whereas clearance in CR3-KO mice was significantly reduced. In conclusion, Kupffer cell CR3 plays a crucial role in the clearance of both IgM-opsonized E and beta-glucans.

Regulation of CR3 (CD11b/CD18)-dependent natural killer (NK) cell cytotoxicity by tumour target cell MHC class I molecules.

Phagocyte and NK cell CR3 functions as both an adhesion molecule and an iC3b receptor mediating cytotoxic responses to microorganisms. Cytotoxic activation of iC3b receptor function requires ligation of both a CD11b I-domain site for iC3b and a lectin site located in the C-terminus of CD11b. Because tumours lack the CR3-binding polysaccharides of bacteria and fungi, iC3b-opsonized tumours do not stimulate CR3-dependent cytotoxicity. Previous studies showed that NK cells could be induced to kill iC3b-opsonized tumours with small soluble beta-glucans that bound with high affinity to CR3, bypassing the absence of similar polysaccharides on tumour membranes. Because CR3 signalling requires several tyrosine phosphorylation events, it appeared possible that CR3-dependent killing of autologous tumour cells might be suppressed by NK cell inhibitory receptors for MHC class I (KIR and CD94/NKG2) whose action involves recruitment of SHP-1 and SHP-2 tyrosine phosphatases. In the current study, Epstein-Barr virus (EBV)-transformed B cells were used as targets following opsonization with iC3b. Soluble beta-glucan primed CR3 for killing of iC3b-coated B cells, but autologous class I-bearing targets were 84% more resistant than class I-deficient Daudi cells. Blockade of target cell class I with a MoAb specific for a domain recognized by both KIR and CD94/NKG2 resulted in comparable killing of class I+ B cells. By contrast, another MoAb to class II had no effect on cytotoxicity. These data suggest that NK cell recognition of class I suppresses CR3/tyrosine kinase-dependent cytotoxicity in the same way as it suppresses cytotoxicity mediated by other tyrosine kinase-linked receptors such as FcgammaRIIIA (CD16).

The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells.

Mouse leukocyte CR3 (Mac-1, alphaMbeta2 integrin) was shown to function as a receptor for beta-glucans in the same way as human CR3. Soluble zymosan polysaccharide (SZP) or pure beta-glucans labeled with FITC or 125I bound in a saturable and reversible manner to neutrophils, macrophages, and NK cells. This lectin activity was blocked by anti-CD11b mAb M1/70 or 5C6 and did not occur with leukocytes from CR3-/- (CD11b-deficient) mice. SZP preparations containing primarily mannose or glucose bound to CR3, and the binding of 125I-labeled beta-glucan to CR3 was competitively inhibited by beta-glucans from barley or seaweed, but not by yeast alpha-mannan. Also, as with human CR3, the lectin site of mouse CR3 was inhibited by alpha- or beta-methylglucoside (but not D-glucose), alpha- or beta-methylmannoside, and N-acetyl-D-glucosamine. Phagocytosis of zymosan and serum-opsonized zymosan was partially inhibited by anti-CR3 and was reduced to <40% of normal with leukocytes from CR3-/- mice. As with neutrophils from patients with CD18 deficiency, neutrophils from CR3-/- mice exhibited no phagocytosis of particulate beta-glucan. SZP or beta-glucans primed CR3 of neutrophils, macrophages, and NK cells for cytotoxicity of iC3b-opsonized tumor cells that otherwise did not trigger killing. beta-Glucan priming for cytotoxicity was inhibited by anti-CR3 and did not occur with leukocytes from CR3-/- mice. The primed state of macrophage and NK cell CR3 remained detectable for 18 to 24 h after pulsing with beta-glucans. The similarity of mouse and human CR3 in response to beta-glucans highlights the utility of mouse tumor models for development of therapeutic beta-glucans.

A mixed population of immature and mature leucocytes in umbilical cord blood results in a reduced expression and function of CR3 (CD11b/CD18).

Neonatal neutrophils express less membrane and cytoplasmic CR3 (iC3b-receptor, Mac-1, alphaM beta2-integrin) than do adult neutrophils, and it has been suggested that this renders neonatal neutrophils deficient in diapedesis and bactericidal activity. The reason(s) for this deficiency are unknown. In this study, CR3 expression and the CR3-dependent respiratory burst activity of individual neonatal neutrophils are quantified in comparison with adult leucocytes using flow cytometry. Monocytes and neutrophils are defined as CD14highCD15low and CD14lowCD15high, respectively. Although neonatal neutrophils bore less CR3 on average than did adult neutrophils, neonatal neutrophils were more heterogeneous and many neonatal neutrophils expressed adult levels of CR3. Because of higher neutrophil concentrations in cord versus adult blood, the calculated number of neutrophils in cord blood expressing high amounts of CR3 was equivalent to that of adult blood. Similar findings were made with monocytes. The size of the CR3-dependent respiratory burst stimulated by particulate beta-glucan correlated directly with the expression of CR3 by individual neutrophils. With neonatal and adult neutrophils having comparable CR3 densities, the respiratory burst activities were equivalent. Wright-Giemsa differential staining of the subset of neonatal neutrophils with low CR3 levels isolated by fluorescence-activated cell sorting showed a higher proportion of immature cells than the sorted population expressing high CR3 levels. Therefore, higher proportions of immature cells in cord blood probably explain previous reports of deficient CR3 expression and function. The typical neutrophilia of cord blood may compensate for this apparent deficiency by providing adult concentrations of mature neutrophils.

Macrophage nitric oxide synthase (NOS) activation by Nocardia opaca fractions and 15- and 56-kD isolated antigens.

The Gram-positive bacterium, Nocardia opaca, is a source of substances with adjuvant effect, ability to stimulate macrophages and natural killer cells for enhanced cytotoxity and cytokine production and B lymphocytes for polyclonal immunoglobulin secretion. We determined the immunogenicity of isolated N. opaca fractions and prepared MoAbs against immunogenic water-soluble mitogen (NWSM). Two main proteins of molecular mass 15 and 56 kD were detected in western blot analysis and isolated by affinity chromatography using anti-NWSM MoAb B7/7. Both these isolated nocardial antigens were found to stimulate mouse peritoneal macrophage NOS. The effect of 5 micrograms NWSM was comparable to that of 5 micrograms lipopolysaccharide (LPS) or 20 U of interferon-gamma (IFN-gamma) added to cell cultures. The MoAb B7/7 decreased No2- production induced by NWSM or by isolated nocardial antigens, but did not significantly influence the production elicited by LPS or IFN-gamma. On the other hand, NOS activation by NWSM was not affected by anti-IFN-gamma MoAb. The possible independent pathway for IFN-gamma and NWSM macrophage activation is discussed.

Stimulation of human blood lymphocyte by different polyclonal B cell activators of bacterial and plant origin: production of IgM, IgG and IgA estimated by the ELISA method.

Lymphocytes isolated from peripheral blood of healthy donors were stimulated in vitro with pokeweed mitogen, concanavalin A, flagellin, Nocardia delipidated cell mitogen (NDCM) and heat-killed bacteria Escherichia coli and Actinomyces viscosus. A simple and sensitive technique, enzyme-linked immunosorbent assay (ELISA) was used for the detection of nanogram levels of IgM, IgA and IgC in media from lymphocyte cultures after polyclonal stimulation, Pokeweed mitogen, NDCM and E. coli were shown to stimulate a high production of IgM; after stimulation with A. viscosus a higher production of IgA was detected. No immunoglobulin production was observed after stimulation with polymerized flagellin.