Staurosporine inhibits neutrophil phagocytosis but not iC3b binding mediated by CR3 (CD11b/CD18).

C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data.     

Leishmania promastigotes require opsonic complement to bind to the human leukocyte integrin Mac-1 (CD11b/CD18)

Previous reports have suggested that Leishmania spp. interact with macrophages by binding to Mac-1 (CD1 1b/CD18), a member of the leukocyte integrin family. To better define this interaction, we tested the ability of leishmania promastigotes to bind to purified leukocyte integrins and to cloned integrins expressed in COS cells. We show that leishmania promastigotes bind to cellular or purified Mac-1 but not lymphocyte function-associated antigen-1 in a specific, dose-dependent manner that requires the presence of serum. Binding is inhibited with specific monoclonal antibodies to Mac-1. In the absence of complement opsonization, three different species of leishmania tested fail to bind directly to any of the three leukocyte integrins. We show that binding to Mac-1 requires the third component of complement (C3). Organisms incubated in heat-inactivated serum or serum that has been immunologically depleted of C3 fail to bind to Mac-1. Because the addition of purified C3 to C3-depleted serum restores leishmania binding to Mac-1, we suggest that parasites gain entry into macrophages by fixing complement and subverting a well-characterized adhesive interaction in the immune system between Mac-1 and iC3b.     

Characterization of the human glomerular C3 receptor as the C3b/C4b complement type one (CR1) receptor

The functional and immunochemical characteristics of the human glomerular C3 receptor were investigated by adherence of sheep erythrocytes (Es) coated with defined C3 fragments and by using polyclonal and/or monoclonal antibodies directed against epitopes expressed on complement receptors CR1, CR2, and CR3. C3b-bearing Es (EsC3b) strongly adhered to glomeruli in frozen kidney sections in a reaction that was selectively inhibited by F(ab')2 anti-CR1 antibodies. There was no adherence of EsC3dg, EsC3d, and EsC3bi in the presence or absence of Ca++ and Mg++ under physiologic buffer conditions. The weak glomerular binding of EsC3bi, which was observed in half-isotonic buffer was selectively suppressed by anti-CR1 antibodies. By indirect immunofluorescence, anti-CR1 antibodies stained all podocytes in glomeruli, whereas no staining of kidney sections was seen with OKM1 and anti-Mol antibodies directed against the alpha-chain of CR3 and with anti-CR2 antibodies anti-B2 and BL13. Solubilization of membrane glycoproteins from freshly isolated glomeruli from three human kidneys, in the presence of 0.1% Nonidet P-40, yielded a material that bound to lentil lectin Sepharose and could accelerate the decay of preformed cell-bound amplification C3 convertase sites in a reaction that was inhibited by anti-CR1 antibodies. The material containing CR1 activity was labeled with 125I, immunoprecipitated with anti-CR1, and analyzed by SDS-PAGE and autoradiography. Anti-CR1 immunoprecipitated a form of CR1 of Mr 205,000 in solubilized glomeruli from three donors, and an additional form of Mr 160,000 in glomeruli from two of the donors. Immunoprecipitation of CR1 from surface-labeled erythrocytes from these individuals demonstrated them to be homozygous for the 205,000 Mr form of the receptor. Whether the 160,000 band represents in vitro or in vivo proteolytic cleavage of CR1, or cell specific-modulation of gene expression of glomerular CR1, remains unclear. Thus, CR1 is the only type of C3 receptor expressed in the human kidney. Glomerular CR1 shares the functional antigenic and biochemical properties of the C3b/C4b CR1 receptor of peripheral blood cells.     

Cutting edge: productive HIV-1 infection of dendritic cells via complement receptor type 3 (CR3, CD11b/CD18)

In the present study, we demonstrate that macrophage-tropic HIV-1 opsonized by complement and limited amounts of anti-HIV-IgG causes up to 10-fold higher productive infection of human monocyte-derived dendritic cells than HIV treated with medium or HIV opsonized by Ab only. Enhanced infection is completely abolished by a mAb specific for the ligand-binding site of CD11b (i.e., alpha-chain of complement receptor 3, receptor for iC3b), proving the importance of complement receptor 3 in this process. Inhibition of complement activation by EDTA also prevents enhanced infection, further demonstrating the role of complement in virus uptake and productive infection. Since HIV is, even in the absence of Abs, regularly opsonized by complement, most probably the above-described mechanism plays a role during in vivo primary infection.     

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.     

Utilization of CD11b knockout mice to characterize the role of complement receptor 3 (CR3, CD11b/CD18) in the growth of Mycobacterium tuberculosis in macrophages

Using CD11b knockout mice as a source of macrophages (Mphi;), we show that complement receptor 3 (CR3) mediates approximately 40-50% of nonopsonic binding and 50-60% of serum-mediated binding of Mycobacterium tuberculosis to resident Mphi;. We demonstrate that opsonic binding of M. tuberculosis to Mphi; is mediated by an immunoglobulin-independent, heat-labile component of serum, in both the presence and the absence of CD11b. The survival and replication of M. tuberculosis in an in vitro Mphi; model and an in vivo mouse model of infection were not significantly affected by the absence of CD11b, indicating that CR3-mediated uptake of M. tuberculosis is not a major factor in controlling the subsequent intracellular survival of the mycobacteria. However, whether a mycobacterium will gain access to the intracellular environment, and the type of Mφ that the bacterium enters, is significantly affected by the presence or absence of CR3.     

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.     

Phagocytosis of Mycobacterium leprae by human monocyte-derived macrophages is mediated by complement receptors CR1 (CD35), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) and IFN-gamma activation inhibits complement receptor function and phagocytosis of this bacterium.

We have examined phagocytosis of Mycobacterium leprae by human monocyte-derived macrophages (MDM). Compared with monocytes, MDM exhibit greatly enhanced adherence of M. leprae (6.5 +/- 2-fold increase). MDM adherence of M. leprae is serum dependent and requires heat-labile serum components because heat inactivation of serum reduces adherence by 70 +/- 3%. mAb against C receptors CR1 (CD35), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) inhibit phagocytosis of M. leprae in fresh nonimmune serum. Single mAb against each receptor inhibit M. leprae adherence by 25 +/- 4% - 33 +/- 6%. Single mAb used in combination against all three receptors inhibit M. leprae adherence by 51 +/- 6%. Most significantly, pairs of mAb used in combination against all three receptors inhibit by 80 +/- 4%. By electron microscopy, MDM ingest all M. leprae that adhere in fresh nonimmune serum. In the presence of mAb against CR1, CR3, and CR4, the percentage of MDM cross-sections that contain intracellular bacteria is reduced 66 +/- 3% and the mean number of bacteria per cross-section is reduced 78 +/- 10%. MDM activated by IFN-gamma exhibit markedly reduced adherence (by light microscopy) and ingestion (by electron microscopy) of M. leprae. MDM in culture for 5 days inhibit M. leprae adherence by 83 +/- 2% and ingestion by 88% when activated for 5 days. Paralleling this, IFN-gamma-activated MDM exhibit markedly reduced C receptor function, reflected by markedly decreased adherence and ingestion of C3b- and C3bi-coated E. Decreased C receptor function by IFN-gamma-activated MDM correlates with decreased surface expression of CR1 but not CR3 or CR4. CR1 expression on MDM in culture for 5 days is reduced by 32 +/- 9% and 75 +/- 3% after IFN-gamma activation for 5 and 2 days, respectively. This study demonstrates that MDM have an enhanced capacity to phagocytize M. leprae, and that in addition to CR1 and CR3, phagocytosis involves CR4, whose expression on MDM is highly maturation-dependent. This study also demonstrates that IFN-gamma activation markedly reduces the capacity of MDM to phagocytize M. leprae, and it provides a molecular mechanism for this phenomenon-decreased C receptor function.     

The A-domain of beta 2 integrin CR3 (CD11b/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF

The A-domain is a approximately 200-amino acid peptide present within structurally diverse proadhesive proteins including seven integrins. A recombinant form of the A-domain of beta 2 integrins CR3 and LFA-1 has been recently shown to bind divalent cations and to contain binding sites for protein ligands that play essential roles in leukocyte trafficking to inflammatory sites, phagocytosis and target cell killing. In this report we demonstrate that the neutrophil adhesion inhibitor, NIF produced by the hookworm Ancyclostoma caninium is a selective CD11b A-domain binding protein. NIF bound directly, specifically and with high affinity (Kd of approximately 1 nM) to recombinant CD11b A-domain (r11bA). The binding reaction was characterized by rapid association and very slow dissociation, and was blocked by an anti-r11bA monoclonal antibody. No binding was observed to rCD11aA. The NIF-r11bA interaction required divalent cations, and was absent when the mutant r11bA D140GS/AGA (that lacks divalent cation binding capacity) was used. The NIF binding site in r11bA was mapped to four short peptides, one of which being an iC3b binding site. The interaction of NIF with CR3 in intact cells followed similar binding kinetics to those with r11bA, and occurred with similar affinity in resting and activated human neutrophils, suggesting that the NIF epitope is activation independent. Binding of NIF to CR3 blocked its ability to bind to its ligands iC3b, fibrinogen, and CD54, and inhibited the ability of human neutrophils to ingest serum opsonized particles. NIF thus represents the first example of a disintegrin that targets the integrin A-domain, and is likely to be used by the hookworm to evade the host's inflammatory response. The unique structure of NIF, which lacks a disintegrin motif, emphasizes basic structural differences in antagonists targeting A+ and A- integrins, that should be valuable in drug design efforts aimed at generating novel therapeutics. Identification of the region in NIF mediating A-domain binding should also be useful in this regard, and may, as in the case of disintegrins, unravel a new structural motif with cellular counterparts mediating important physiologic functions.     

Characterization of hyaluronan-binding proteins on guinea pig polymorphonuclear leukocytes: possible involvement of complement receptor type 3 (CR3, CD11b/CD18) in the hyaluronan-leukocyte interaction

Hyaluronan (HA), a high-molecular-weight glycosaminoglycan ubiquitously present in the extracellular matrices (ECMs) of animals, plays important roles in ECM organization and cell behavior through binding to hyaluronan-binding proteins (HABPs). We previously reported that HA has anti-inflammatory effects on guinea pig phagocytes, although the nature of guinea pig HABPs was unknown. In this study, we characterized guinea pig HABPs on peritoneal polymorphonuclear leukocytes (PMNs) and blood neutrophils by flow cytometry and affinity chromatography. It was found that PMNs express diverse HABPs with different molecular weights. These HABPs maximally bound with HA over a wide pH range (6-8), and recognized HAs as small as the pentadisaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine. Furthermore, they could be divided into Mg(2+)-dependent and Ca(2+)/Mg(2+)-independent groups. Interestingly, two proteins in the Mg(2+)-dependent group were found to be the two subunits of complement receptor type 3 (CR3, CD11b/CD18). Unlike PMNs, blood neutrophils expressed several functionally inactive HABPs. Among these inactive HABPs, Mg(2+)-dependent proteins including CR3 but not Ca(2+)/Mg(2+)-independent proteins were activated on phorbol ester-stimulation. These results show the existence of diverse HABPs on guinea pig neutrophils and the cell activation-dependent activation of HABPs. It is also suggested that the CR3-HA interaction is possibly involved in the regulation of neutrophil function.     

Analysis of C3-receptor activity on human B-lymphocytes and isolation of the complement receptor type 2 (CR2).

The rosetting of defined C3-fragment-coated sheep erythrocytes to B-cell-enriched tonsil lymphocytes was measured. The rosetting lymphocytes were homogeneous with respect to expression of C3b, iC3b and C3d receptors. Isolation of receptors for C3 fragments from surface-radioiodinated lymphocytes by affinity chromatography on immobilized C3u, iC3b and C3d,g produced two proteins with partially overlapping specificities. A protein of 240 000 Mr, recognized by the monoclonal antibody To5 and identified as CR1 (complement receptor type 1), had affinity for C3u and iC3b. A protein of 145 000 Mr, recognized by the monoclonal antibody B2, had affinity for all three C3 fragments. Inhibition of rosetting by antibodies to these proteins indicates that CR1 is responsible for C3b-mediated rosetting and that the 145000-Mr receptor (CR2) is responsible for C3d-mediated rosetting. Partial inhibition by both anti-CR1 and anti-CR2 antibodies of iC3b-mediated rosetting indicates that both receptors are involved in iC3b-mediated rosetting. No other protein appears to be involved in tonsil B-cell rosetting to C3-fragment-coated cells. A method for preparing CR2 from tonsil lymphocytes based on affinity chromatography on C3d,g-Sepharose has been developed. Forty tonsil pairs (2 X 10(10) B-cells) yield about 40 micrograms of pure protein equivalent to a purification of 6500-fold from a detergent extract.     

Calcium signaling capacity of the CD11b/CD18 integrin on human neutrophils.

The CD11b/CD18 integrin is a major cell adhesion molecule of myelomonocytic cells. Exposure of human neutrophils in suspension to CD11b or CD18 monoclonal antibodies (mAbs)2 does not affect the resting level of cytosolic free Ca2+ in these cells; however, a subsequent cross-linking of either of these antibodies triggers a prompt and significant cytosolic-free Ca2+ transient lasting about 10 min. The rise in cytosolic-free Ca2+ (from 130 +/- 2 to 414 +/- 12 nM or 111 +/- 12 to 331 +/- 22 nM caused by cross-linking of CD11b or CD18 subunits, respectively) is due to both mobilization of Ca2+ from intracellular stores and influx of Ca2+ across the plasma membrane. Cross-linking of the common leukocyte antigen (CD45) did not alter the basal level of cytosolic free Ca2+. In accordance with other adherence-induced phenomena and with CD11/CD18-mediated phagocytosis, these Ca2+ signals were only modestly affected by pertussis toxin. Thus, the present data clearly indicate that the CD11b/CD18 integrin on human neutrophils is capable of inducing a prompt cytosolic-free Ca2+ signal. These findings directly support the recent suggestion that the CD11b/CD18 integrin is responsible for the "spontaneous oscillations" of cytosolic-free Ca2+ observed in adherent neutrophils and, at least partially, also explain how integrin-mediated adherence can modify the functional responsiveness of neutrophils to a subsequent agonist stimulation.     

Factor I-dependent inactivation of human complement C4b of the classical pathway by C3b/C4b receptor (CR1, CD35) and membrane cofactor protein (MCP, CD46).

Proteolytic inactivation of C4b is a crucial step for regulation of the classical complement pathway. A plasma protease factor I and membrane cofactors, C3b/C4b receptor (CR1) and membrane cofactor protein (MCP), participate in the regulation of cell-bound C4b although the physiological potency of these cofactors remains unknown. We have examined the optimal conditions of the factor I-mediated C4b regulatory system using purified cofactors. CR1 being a cofactor at a cofactor/C4b ratio less than 0.1 (w/w), fluid phase C4b, and methylamine-treated C4 (C4ma) were degraded by factor I into C4bi: minimal Cd4 was generated in the fluid phase. Liposome-bound C4b (LAC4b), on the other hand, was degraded into C4c and C4d. CR1 showed two optimal pHs (6.0 and 7.5) for fluid phase C4b, but one (6.0) for LAC4b, and in both cases low conductivity conditions enhanced the C4bi generation. CR1 cofactor activity was barely influenced by the NP-40 concentration. On the other hand, MCP degraded C4b and C4ma, as a factor I-cofactor, more efficiently into C4c and C4d. Though MCP cofactor activity, like that of CR1, was enhanced under low conductivity conditions, it has only one optimal pH, 6.0, in both fluid and solid phases. Furthermore, as in the case of C3b cleavage, a sufficient NP-40 concentration to solubilize membrane was needed for MCP to express full cofactor activity for C4b, in contrast to CR1. MCP was less potent for C4b inactivation than for C3b inactivation, while CR1 acted as a slightly more effective cofactor for C4b cleavage than for C3b cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Native C3 does not bind to the C3b receptor (CR1) of human blood B lymphocytes or alter immunoglobulin synthesis

Complement receptors on lymphocytes were first described more than 12 yr ago (1-3) and have come to be used as a common marker for the identification of B cells (4). The function of these receptors on the lymphocyte and their possible role in induction and/or regulation of the immune response remain unclear. In particular, there continues to be controversy as to whether native C3 can bind to the C3b receptor of these cells without cleavage to C3b (5-10). The resolution of this question is critical in order to clarify the expected state of availability of the receptor in vivo, because in plasma, the C3 concentration is relatively high (1.1 to 1.5 mg/ml), whereas there is little or no circulating C3b due to efficient degradation by factor H and the C3-inactivator (11). With the recent development of an improved method for the isolation of C3 from human plasma, it has been possible to obtain biochemically and functionally pure C3 that has not undergone structural or conformational alteration during processing and fully retains the specific hemolytic activity of C3 in fresh serum (12). Berger et al. (13) were able to demonstrate that C3 prepared in this way failed to bind to the C3b receptor of human polymorphonuclear leukocytes or erythrocytes. Similar observations were made by Schreiber et al. (14), also with phagocytic cells and erythrocytes, and by Dixit et al. (15) with an isolated membrane receptor preparation from rabbit macrophages. In the present communication, we extend these observations to human peripheral blood B lymphocytes. Purified C3 in its native state fails to block B lymphocyte-EA (IgM) C4b3b rosettes, whereas C3b causes 50% inhibition at 5 to 6 micrograms/ml. Furthermore, C3 failed to alter polyclonal immunoglobulin (Ig) production by human B cells, whereas C3b inhibited this B cell function. These data suggest that native C3 does not bind to the C3b receptors of B lymphocytes, and thus they are not occupied under normal conditions in vivo.     

Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins.

Intercellular adhesion molecule-4 (ICAM-4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins. The location of the ICAM-4 binding sites on CD11a/CD18 and CD11b/CD18 are not known. CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding. ICAM-4 lacks such a residue, which is replaced by an arginine. However, we demonstrate here that ICAM-4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins. The binding was inhibited by anti-I domain and anti-ICAM-4 antibodies and it was dependent on divalent cations. Interestingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced. Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM-4 in a cation dependent manner. Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different. However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized by the ICAM-1 and ICAM-2. Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins.     

Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by beta-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcgammaRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) beta(2) integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by beta-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by beta-glucan. A single CD11b lectin site for beta-glucan and uPAR was suggested because the binding of either beta-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of (125)I-labeled beta-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of (125)I-labeled beta-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.     

Human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) belong to Fc gamma receptor-positive CD4-positive T cells

The phenotypic characteristics of human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) were investigated using dual-color surface immunofluorescence and cytofluorometric analysis of stained peripheral blood mononuclear cells (PBMC) from normal individuals. Two to ten percent of PBMC coexpressed CR1 and the CD5, CD2, or CD3 antigen. CR1 was detected on a subset of CD4+ T lymphocytes but not on CD8+ or on Leu-7+ lymphocytes. Costaining for CR1 and for the CD4 subpopulation markers anti-Leu-8, TQ1, OKT17, 2H4, and 4B4 indicated that CR1 on lymphocytes may be coexpressed with any of these phenotypic determinants. All CR1+ lymphocytes expressed Fc gamma receptors (Fc gamma Rs) as assessed by their ability to bind biotinylated dimeric human IgG. The expression of CR1 was increased in mixed lymphocyte reaction with kinetics similar to those of HLA-DR antigen expression. Coexpression of CR1 and Fc gamma R+ may provide a subset of CD4+ lymphocytes with an enhanced ability to bind and respond to C3-bearing complexes of IgG and antigen.