Transplantation studies in C3-deficient animals reveal a novel role of the third complement component (C3) in engraftment of bone marrow cells.

Mice deficient in complement C3 (C3(-/-)) are hematologically normal under steady-state conditions, and yet displayed a significant delay in hematopoietic recovery from either irradiation or transplantation of wild-type (WT) hematopoietic stem/progenitor cells (HSPC). Transplantation of histocompatible WT Sca-1(+) cells into C3(-/-) mice resulted in a (i) decrease in day 12 CFU-S, (ii) 5-7-day delay in platelet and leukocyte recovery, and (iii) reduced number of BM CFU-GM progenitors at day 16 after transplantation. Nevertheless, HSPC from C3(-/-) mice engrafted normally into irradiated WT mice, suggesting that there was a defect in the hematopoietic environment of C3(-/-) mice. Since C3(-/-) mice cannot activate/cleave C3, the C3 fragments C3a, C3a(des-Arg), and iC3b were examined for a role in HSPC engraftment. Liquid-phase C3a and C3a(des-Arg) increased CXCR4 incorporation into membrane lipid rafts (thus potentiating HSPC responses to SDF-1 gradients), whereas iC3b was deposited onto irradiated BM cells and functioned to tether CR3(CD11b/CD18)(+)HSPC to damaged stroma. The activity of C3a(des-Arg) suggested that C3aR(+)HSPC also expressed the C5L2 (receptor for C3a and C3a(des-Arg)) and this was confirmed. In conclusion, a novel mechanism for HSC engraftment was identified, which involves complement activation and specific C3 fragments that promote conditioning for transplantation and enhance HSPC engraftment.

Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/alphaMbeta2-integrin glycoprotein.

Mac-1/CR3 functions as both an adhesion molecule mediating the diapedesis of leukocytes across the endothelium and a receptor for the iC3b fragment of complement responsible for phagocytic/degranulation responses to microorganisms. Mac-1/CR3 has many functional characteristics shared with other integrins, including bidirectional signaling via conformational changes that originate in either the cytoplasmic domain or extracellular region. Another key to its functions is its ability to form membrane complexes with glycosylphosphatidylinositol (GPI)-anchored receptors such as Fc gammaRIIIB (CD16b) or uPAR (CD87), providing a transmembrane signaling mechanism for these outer membrane bound receptors that allows them to mediate cytoskeleton-dependent adhesion or phagocytosis and degranulation. Many functions appear to depend upon a membrane-proximal lectin site responsible for recognition of either microbial surface polysaccharides or GPI-linked signaling partners. Because of the importance of Mac-1/CR3 in promoting neutrophil inflammatory responses, therapeutic strategies to antagonize its functions have shown promise in treating both autoimmune diseases and ischemia/reperfusion injury. Conversely, soluble beta-glucan polysaccharides that bind to its lectin site prime the Mac-1/CR3 of circulating phagocytes and natural killer (NK) cells, permitting cytotoxic degranulation in response to iC3b-opsonized tumor cells that otherwise escape from this mechanism of cell-mediated cytotoxicity.

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.

Beta-glucan, a "specific" biologic response modifier that uses antibodies to target tumors for cytotoxic recognition by leukocyte complement receptor type 3 (CD11b/CD18).

beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.

Therapeutic intervention with complement and beta-glucan in cancer.

Complement (C) has two major effector systems available for host defense. The membrane attack complex (MAC) generated from components C5-C9 can form membrane-penetrating lesions that lead to cell death by causing a rapid loss of cytoplasmic components. The MAC is only effective against pathogens with outer phospholipid membranes, and cannot kill gram-positive bacteria or yeast whose membranes are protected by cell walls. The most important effector mechanism of C is the opsonization of microbial pathogens with the serum protein C3 that leads to their high avidity attachment to the C3-receptors of phagocytic cells. Pathogens that activate complement are first coated with the C3b fragment of C3, which is rapidly proteolyzed into the iC3b fragment by serum factor I. These iC3b fragments serve to promote the high avidity attachment of the 'iC3b-opsonized' pathogens to the iC3b-receptors (CR3, CD11b/CD18) of phagocytic cells and natural killer (NK) cells, stimulating phagocytosis and/or cytotoxic degranulation. Host cells, including neoplastic tumor cells, have been endowed with natural mechanisms for self-protection against both the MAC and the cytotoxic activation of CR3. This review discusses a novel type of immunotherapy for cancer that uses soluble yeast beta-glucan to override the normal resistance of iC3b-opsonized tumor cells to the cytotoxic activation of phagocyte and NK cell CR3, allowing this important effector mechanism of the C system to function against tumor cells in the same way that it normally functions against bacteria and yeast. Moreover, the cytotoxic activation of beta-glucan-primed NK cell CR3 by iC3b-opsonized tumors is shown to be accompanied by a tumor-localized secretion of the cytokines TNFalpha, IFNalpha, IFNgamma, and IL-6.

Generation of recombinant fragments of CD11b expressing the functional beta-glucan-binding lectin site of CR3 (CD11b/CD18).

CR3 (Mac-1; alphaMbeta2 integrin) functions as both a receptor for the opsonic iC3b fragment of C3 triggering phagocytosis or cytotoxicity and an adhesion molecule mediating leukocyte diapedesis. Recent reports have suggested that a CR3 lectin site may be required for both cytotoxic responses and adhesion. Cytotoxic responses require dual recognition of iC3b via the I domain of CD11b and specific microbial surface polysaccharides (e.g., beta-glucan) via a separate lectin site. Likewise, adhesion requires a lectin-dependent membrane complex between CR3 and CD87. To characterize the lectin site further, a recombinant baculovirus (rBv) system was developed that allowed high level expression of rCD11b on membranes and in the cytoplasm of Sf21 insect cells. Six rBv were generated that contained truncated cDNA encoding various CD11b domains. Immunoblotting of rBv-infected Sf21 cells showed that some native epitopes were expressed by five of six rCD11b fragments. Lectin activity of rCD11b proteins was evaluated by both flow cytometry with beta-glucan-FITC and radioactive binding assays with [125I]beta-glucan. Sf21 cells expressing rCD11b that included the C-terminal region, with or without the I-domain, exhibited lectin activity that was inhibited by unlabeled beta-glucan or anti-CR3 mAbs. The smallest rCD11b fragment exhibiting lectin activity included the C-terminus and part of the divalent cation binding region. The beta-glucan binding affinities of the three C-terminal region-containing rCD11bs expressed on Sf21 cell membranes were not significantly different from each other and were similar to that of neutrophil CR3. These data suggest that the lectin site may be located entirely within CD11b, although lectin site-dependent signaling through CD18 probably occurs with the heterodimer.

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.

Targeting of natural killer cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and beta-glucan-primed CR3 (CD11b/CD18).

Previous reports have suggested that malignant cells frequently generate a humoral immune response that is ineffective in tumor destruction. Despite coating tumors with IgM and IgG that activate the C system via the classical pathway, normal membrane regulators of C (e.g., membrane cofactor protein and CD59) prevent cytotoxicity. Moreover, C3 deposition on tumors does not result in cytotoxic recognition by phagocytes or NK cells bearing C3 receptors capable of mediating destruction of C3-opsonized bacteria or yeast. The current investigation showed that freshly excised mammary tumors bore IgM, IgG, and C3 detectable by flow cytometry. Normal sera contained natural IgM and IgG Abs reactive with breast tumor cell lines, and IgG Ab titers were increased in patients with breast cancer. Breast tumor cell lines incubated in normal serum from AB+ individuals activated the classical, but not the alternative, pathway of C and became coated with C3. Despite exhibiting membrane-bound C3, serum-opsonized breast tumor cell lines were not killed by CR3 (CD11b/CD18)-bearing NK cells. Priming of NK cell CR3 with small soluble yeast beta-glucan polysaccharides enabled CR3-dependent killing of these same C3-bearing tumor cell lines. Tests of mammary carcinoma cells from freshly excised tumors demonstrated that they also bore sufficient amounts of opsonic C3 for cytotoxic recognition by NK cells bearing polysaccharide-primed CR3, whereas they were largely resistant to NK cells bearing unprimed CR3. This study demonstrates the potential utility of using naturally occurring opsonic C3 on tumor cells for specific immunotherapeutic targeting by NK cells and phagocytes bearing polysaccharide-primed CR3.

Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells.

When phagocyte CR3 binds to iC3b on bacteria or yeast, phagocytosis and degranulation are triggered because of simultaneous recognition of iC3b via a CD11b I-domain binding site and specific microbial polysaccharides via a lectin site located COOH-terminal to the I-domain. By contrast, when phagocyte or natural killer (NK) cell CR3 adheres to iC3b on erythrocytes or tumor cells that lack CR3-binding membrane polysaccharides, neither lysis nor cytotoxicity are stimulated. This investigation showed that soluble CR3-specific polysaccharides such as beta-glucan induced a primed state of CR3 that could trigger killing of iC3b-target cells that were otherwise resistant to cytotoxicity. Anti-CR3 added before sugars prevented priming, whereas anti-CR3 added after sugars blocked primed CR3 attachment to iC3b-targets. Polysaccharide priming required tyrosine kinase(s) and a magnesium-dependent conformational change of the I-domain that exposed the CBRM1/5 activation epitope. Unlike LPS or cytokines, polysaccharides did not up-regulate neutrophil CR3 expression nor expose the mAb 24 reporter epitope representing the high affinity ICAM-1-binding state. The current data apparently explain the mechanism of tumoricidal beta-glucans used for immunotherapy. These polysaccharides function through binding to phagocyte or NK cell CR3, priming the receptor for cytotoxicity of neoplastic tissues that are frequently targeted with iC3b and sparing normal tissues that lack iC3b.

Function of C3 in a humoral response: iC3b/C3dg bound to an immune complex generated with natural antibody and a primary antigen promotes antigen uptake and the expression of co-stimulatory molecules by all B cells, but only stimulates immunoglobulin synthesis by antigen-specific B cells.

Previous studies have shown that an optimal humoral response to a primary protein antigen requires C3 and CR2 (CD21). Sera from non-immunized donors contain natural IgM and IgG antibodies to the primary antigen keyhole limpet haemocyanin (KLH), and these have been previously shown to form immune complexes (IC) that activate the classical pathway of C, fixing iC3b/C3dg onto the KLH antigen. Such KLH IC bind to CR2 on KLH-non-specific B lymphocytes, resulting in antigen processing and MHC class II-dependent presentation to KLH-specific helper T cells. KLH IC also induce B lymphocytes to express the CD80 costimulatory molecule via simultaneous CR2 ligation with C3 and Fc gammaRII (CD32) stimulation by IgG natural antibody. The current study demonstrated that KLH IC ligation to either CR2 or Fc gammaRII resulted in activation of a second co-stimulatory molecule, LFA-1 (CD11a, CD18). The possibility of polyclonal B cell stimulation by the presentation of KLH-iC3b/C3dg by antigen-non-specific B cells was excluded by demonstration that in vitro cultivation of peripheral blood mononuclear cells (PBMC) with KLH-iC3b/C3dg elicited only anti-KLH, and did not stimulate synthesis of antibodies to hepatitis C virus (HCV) or tetanus toxoid (TT). Of greatest significance, a specific anti-KLH response was only detectable in cultures stimulated with KLH-iC3b/C3dg and not in cultures stimulated with KLH alone or KLH-IgG. Thus, iC3b/C3dg that was bound to a primary protein antigen enhanced recognition and specific immunoglobulin synthesis by antigen-specific B cells, even though the antigen was taken up and processed via CR2 by both antigen-specific and non-specific B cells.

Analysis of the sugar specificity and molecular location of the beta-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18).

Zymosan, the cell wall from Saccharomyces cerevisiae, was reported to be a macrophage activator through its beta-glucan over 30 yr ago. Nevertheless, the identity of the beta-glucan receptor has been controversial. This study showed that the alpha M beta 2-integrin, CR3 (Mac-1, CD11b/CD18) served as the beta-glucan receptor through one or more lectin sites located outside of the CD11b I-domain that contains the binding sites for iC3b, ICAM-1, and fibrinogen. Sugar specificity, analyzed with FITC-labeled soluble polysaccharides and flow cytometry, showed CR3-specific staining with several pure beta-glucans but not with alpha-mannan. However, a 10-kDa soluble zymosan polysaccharide (SZP) with high affinity (6.7 x 10(-8) M) for CR3 consisted largely of mannose and approximately 5% glucose. Binding of either SZP-FITC or beta-glucan-FITC to CR3 was blocked not only by pure beta-glucans from yeast, mushroom, seaweed, or barley, but also by N-acetyl-D-glucosamine (NADG), alpha- or beta-methylmannoside, and alpha- or beta-methyl-glucoside. SZP-FITC and beta-glucan-FITC stained all leukocyte types similarly to anti-CR3-FITC, and polysaccharide-FITC staining was inhibited > or = 95% by unlabeled anti-CR3. SZP-FITC staining of cells expressing recombinant chimeras between CR3 and CR4 (p150,95, CD11c/CD18) suggested that both the divalent cation-binding region of CD11b and the region C-terminal to it may regulate binding of polysaccharides to CR3. Unlabeled SZP or beta-glucan also blocked CR3 staining by 11 mAb to C-terminal domain epitopes of CD11b but had no effect on staining by mAb directed to the I-domain. In conclusion, CR3 serves as the leukocyte beta-glucan receptor through a cation-independent lectin site located C-terminal to the I-domain of CD11b. Its sugar specificity is broader than originally appreciated, allowing it to react with certain polysaccharides containing mannose or NADG, as well as glucose.

Natural antibody and complement-mediated antigen processing and presentation by B lymphocytes.

Normal immune responses to primary protein Ags (those not seen previously by the immune system) have been shown to require C3 and the C3 receptor CR2 (CD21). This investigation tested the hypothesis that natural Abs to the primary protein Ag keyhole limpet hemocyanin (KLH) exist in normal serum and will form immune complexes (IC) that activate C and generate bound iC3b/C3dg, thereby promoting B cell CR2-dependent Ag processing. Both IgM and IgG anti-KLH were detectable in sera from 11 normal donors. IC generated with fresh serum bore iC3b and bound to CR1 (CD35), CR2, and CR3 (CD11b/CD18). Further treatment of IC with serum containing rCR1 formed IC-bearing C3dg that bound only to CR2. When ICs were mixed with B lymphoblastoid cell clones, CR2-dependent processing of the KLH occurred that was dependent on bound C3dg and CR2. Processing occurred regardless of whether the B cells bore KLH-specific surface Ig, although the efficiency of processing was greater with KLH-specific B cells. Both KLH-specific and nonspecific B cell clones presented KLH to KLH-specific T cells. The binding of KLH IC by normal B lymphocytes induced expression of the B7/BB1 Ag (CD80), the required co-stimulatory ligand for T cell CD28. Blocking experiments indicated that although bound C3 and CR2 were required to mediate IC binding to B cells, induction of CD80 expression required the secondary ligation of IC-associated IgG to B cell FcRII (CD32). These data support the hypothesis that responses to primary protein Ags involve IgG natural Abs and C3 that mediate Ag processing and presentation via B cell CR2 and FcRII.

Microvascular effects of complement blockade with soluble recombinant CR1 on ischemia/reperfusion injury of skeletal muscle.

Reperfusion of ischemic tissue is associated with tissue injury greater than that resulting from ischemia alone. C activation has been hypothesized to mediate the so-called ischemia/reperfusion injury through both membrane attack and C5a-dependent recruitment of neutrophils to sites of C3 fixation on the endothelium via C3 receptors. Adherence of neutrophils is preconditional to expression of their deleterious effects, which are central to the pathophysiology of ischemia/reperfusion injury. This study was designed to evaluate the effect of inhibition of C activation on ischemia/reperfusion injury using a soluble and truncated recombinant human CR1 (sCR1) molecule, a "tail-less" form of the membrane C3b/C4b receptor (CD35) that functions as a regulator of C activation. Capillary perfusion and leukocyte adherence to venular endothelium were measured after reperfusion in a mouse cremaster muscle model that allowed microscopic video observation of microcirculatory changes. Infusion i.v. with sCR1 before a 4-h period of ischemia and during a 3-h subsequent period of reperfusion prevented the increase in leukocyte adherence to venular endothelium seen in controls, and enhanced the number of reperfusing capillaries by 55%. Trypan blue staining showed an increase in muscle cell viability from 11 to 50% in mice receiving sCR1 as compared to controls. Tests of blood samples from mice infused with sCR1 demonstrated nearly complete inhibition of the mouse alternative pathway of C activation, but no detectable loss of the mouse classical pathway of C activation. It was concluded that C activation in this model of skeletal muscle injury is likely to be due to the alternative pathway, and that inhibition of C activation during reperfusion inhibits leukocyte adherence to blood vessel walls and protects the capillary microcirculation.

CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multiple ligand specificities and functions.

The C3 receptor CR3 is expressed on phagocytic cells, minor subsets of B and T cells, and natural killer (NK) cells. It has important functions both as an adhesion molecule and a membrane receptor mediating recognition of diverse ligands such as intercellular adhesion molecule-1 (ICAM-1) and fixed iC3b. The receptor is capable of undergoing an activation event that regulates both its specificity for various ligands and its ability to mediate phagocytosis or extracellular cytotoxicity. Certain bacteria express carbohydrates or lipopolysaccharides (LPS) that can bind to and activate CR3, allowing the receptor to assume its activated state. Soluble beta-glucan derived from the yeast Saccharomyces cerevisiae is a particularly potent stimulator of CR3, and produces an activated state of the receptor that permits neutrophil phagocytosis of iC3b-coated erythrocytes or NK, cell cytotoxicity of iC3b-coated tumour cells, that are normally resistant to NK cells.

Complement factors H and I synthesized by B cell lines function to generate a growth factor activity from C3.

B lymphocytes and transformed B lymphoblastoid cell lines express CR2 (CD21, C3d/EBV-receptor) that is specific for C3 fragments generated by cleavage of C3b or spontaneously hydrolyzed native C3 (C3i) by the serum enzyme factor I and its cofactor, factor H. It had been shown previously that the Raji B cell line could be cultivated in serum-free medium supplemented with only transferrin and either OKB7 anti-CR2 mAb, C3d, or C3d-derived peptides containing the CR2 binding site. Because these agents appeared to function through ligation of CR2, it was unclear how native C3 could also serve as a growth factor, because C3 does not bind to CR2. It appeared possible that Raji cells might be able to use endogenous factors H and I to generate a CR2 ligand from C3, because previous studies had shown that Raji cells synthesized factor H and probably also synthesized factor I. PCR analysis was used to demonstrate factor I mRNA in Raji cells. Secretion of Raji cell factor I protein was confirmed by a sensitive mAb ELISA. Several B cell lines were examined for C3-dependent growth. Raji cells required both C3 (or OKB7) and transferrin for growth, whereas Wil-2 cells grew with transferrin alone and C3 enhanced the growth-promoting activity of transferrin. Two other B cell lines (Daudi and U698M), the T cell line 8402, and the U937 monocytoid cell line could not be sustained with transferrin plus C3. The C3-dependent growth of Raji cells was inhibited almost completely by either OX-23 anti-factor H or 052.11.3 anti-factor I mAb that also blocked the activity of serum-derived factor H or I, respectively. By contrast, there was no inhibition of growth by either OX-24 anti-factor H or OX-21 anti-factor I mAb that did not block factors H and I activity. After the spontaneous hydrolysis of native C3 to C3i, it is hypothesized that Raji cells convert C3i to iC3i with endogenous factors H and I, and then this iC3i serves as a growth factor by binding to membrane CR2.

CR3 (CD11b/CD18) expressed by cytotoxic T cells and natural killer cells is upregulated in a manner similar to neutrophil CR3 following stimulation with various activating agents.

CR3 (CD11b/CD18) functions both as an iC3b-receptor and as an adhesion molecule for cellular ligands such as ICAM-1. Although CR3 has been well characterized on phagocytic cells, much less is known about CR3 on lymphocytes. In this study, the expression of CR3 was examined on resting and stimulated B, T, and natural killer (NK) cells by three-color flow cytometry. Biotinylated anti-CR3 mAb and streptavidin-FITC were used in combination with anti-CD3 mAb conjugated with peridinin chlorophyll-alpha protein (PerCP) and phycoerythrin-labeled mAbs to CD4, CD8, CD19, or CD56. Among resting lymphocytes, CR3 was expressed on nearly all NK cells (CD56+CD3-), 1% of CD4+CD3+ helper T cells, 7% of CD8+CD3+ cytotoxic T cells, and 20% of B cells (CD19+). Among the 5% of T cells (CD3+) expressing CR3, the majority was CD56+. Incubation of PBMC for 30 min with PMA induced a three- to fivefold increase in CR3 expression on NK cells and a twofold increase on T cells but did not change the expression of CR3 on B cells. This effect of PMA was not blocked by the presence of cycloheximide, suggesting the presence of cytoplasmic (granule) stores of CR3 in these lymphoid cells resembling those previously reported in neutrophils and monocytes. When PBMC were incubated with rIFN-alpha, rIL-2, beta-glucan, or high concentrations of LPS, expression of CR3 on NK cells increased significantly, but > or = 4 hr of stimulation was required. Other cytokines (rIFN-gamma, rIL-1, rIL-4, rIL-6, TNF-alpha) and rC5a had no significant effect on CR3 expression. Among NK cells, both the CD56bright and the CD56dim cells expressed CR3, and the expression of CR3 on both of these NK cell subsets was increased in a similar manner by PMA. However, rIL-2 stimulated a greater increase in CR3 expression on CD56bright cells than on CD56dim cells. These studies suggest that CR3 expressed by NK cells or cytotoxic T cells resembles phagocyte CR3 in that cellular activation stimulates increased surface expression of CR3 derived from cytoplasmic reserves of the receptor.

Macrophage cytoskeleton association with CR3 and CR4 regulates receptor mobility and phagocytosis of iC3b-opsonized erythrocytes.

Alveolar macrophages (AM phi) were examined for CR1 (C3b receptor, CD35), CR3 (iC3b receptor; CD11b/CD18), and CR4 (iC3b receptor; CD11c/CD18) by assays for binding of C3-opsonized sheep erythrocytes (EC3b or EC3bi) and uptake of specific monoclonal antibodies (mAbs). In AM phi isolates from nine normal volunteers, 49% of cells bound EC3b and 71% bound EC3bi. Quantitation of receptors per cell with [125I]mAbs showed 8.5 x 10(4) CR4, 5.1 x 10(4) CR3, and 2.6 x 10(4) CR1. With most AM phi preparations, CR3 was the major receptor mediating attachment of EC3bi, despite the predominance of CR4 antigens. Anti-CR3 inhibited EC3bi rosettes by > or = 50%, whereas anti-CR4 blocked rosettes by < or = 18%. U937 cells differentiated with phorbol myristate acetate resembled AM phi in receptor expression but exhibited almost no CR4-dependent rosetting. Despite the relative inability of CR4 to mediate EC3bi attachment, AM phi ingestion of [51Cr]EC3bi was blocked by either anti-CR3 or anti-CR4. Two lines of evidence indicated that CR3 were more mobile within the membrane than were CR4. Immunofluorescence staining demonstrated patching and occasional capping of CR3, whereas CR4 remained uniformly distributed. This patching and capping of CR3 required the actin cytoskeleton, as it was inhibited by cytochalasin D. Modulation experiments using surfaces coated with anti-CR3 or anti-CR4 also showed that CR3 was more mobile than was CR4. However, there was some variation among AM phi isolates from different donors. In seven isolates, no CR4 modulation was produced with anti-CR4, whereas in six other isolates, CR4 was modulated by 66%. Incubation of cells in cytochalasin D increased modulation of both CR3 and CR4 on mAb-coated surfaces. Cells exhibiting increased mobility of CR4 showed an increased ability to form CR4-dependent EC3bi rosettes. The data are consistent with the hypothesis that CR3 and CR4 exhibit a variable association with the cytoskeleton that regulates their mobility and function. A relatively mobile subset of CR3 and/or CR4 mediates EC3bi attachment, whereas a relatively immobile subset of CR3 and/or CR4 fails to mediate EC3bi attachment but functions to promote ingestion of EC3bi.