Association of BAP31 with CD11b/CD18. Potential role in intracellular trafficking of CD11b/CD18 in neutrophils

The beta2 integrin CD11b/CD18 is an integral membrane protein that is present in the plasma membrane and secondary granules of neutrophils and functions as a major adhesion molecule. Upon cellular activation, there is translocation of intracellular pools of CD11b/CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defined. Here we report that CD11b/CD18 specifically binds to BAP31, a member of a novel class of sorting proteins regulating cellular anterograde transport. Through experiments aimed at identifying CD11b/CD18-binding proteins, we produced a monoclonal antibody termed E1B2 that recognizes a 28-kDa membrane protein that co-precipitates with CD11b/CD18. Microsequence analysis of the E1B2 antigen revealed that it is BAP31. Co-association of CD11b/CD18 and BAP31 was confirmed in co-immunoprecipitation and protein binding assays. Additional experiments revealed that the binding of BAP31 to CD11b/CD18 was not dependent on divalent cations nor mediated by the I-domain of CD11b. Using glutathione S-transferase fusion chimeras, we determined that binding of CD11b/CD18 to BAP31 is mediated through interactions with the cytoplasmic tail of BAP31. Immunolocalization studies revealed colocalization of BAP31 and CD11b/CD18 within neutrophil secondary granules. Subcellular fractionation studies in polymorphonuclear leukocytes (PMN) revealed similar patterns of redistribution of BAP31 and CD11b/CD18 from fractions enriched in secondary granules to the plasma membrane following stimulation with formylmethionylleucylphenylalanine (fMLP). Given the known sorting properties of BAP31, these findings suggest that BAP31 may play a role in regulating intracellular trafficking of CD11b/CD18 in neutrophils.     

Polymorphonuclear granulocyte expression of CD11a/CD18, CD11b/CD18 and L-selectin in normal individuals

Abstract In this study direct immunofluorescence and flow cytometry with calibration using quantitative bead standards were used to enumerate the cell surface receptors CD11a/CD18, CD11b/CD18 and L-selectin. Holding blood at room temperature and fixation of samples prior to staining induced changes in expression, while immediate staining of polymorphonuclear granulocytes (PMN) in whole blood followed by fixation produced accurate values. The ranges of PMN adhesion molecule expression in 10 normal individuals were CD11a/CD18: 14794–28725, CD11b/CD18: 5300–11939 and L-selectin: 35662–61654 receptors per cell. Differences within individuals over 4 h were also observed. Adhesion molecule expression is used as an index of the adhesive function and state of activation of the cell. The data presented here shows that there is inherent variability in the expression of the PMN adhesion molecules between and within individuals, thus direct comparisons of PMN adhesion molecule expression between patients and “normals” must be interpreted with caution.     

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.     

CD11a/CD18 and CD11b/CD18 modulation by lipoteichoic acid, N-acetyl-muramyl-α-alanyl-d-isoglutamine, muramic acid and protein A from Staphylococcus aureus

Abstract This study investigates the effect of some components of the Staphylococcus aureus cell wall [lipoteichoic acid (LTA), N -acetyl-muramyl-alanyl- d -isoglutamine (MD), muramic acid (MA) and protein A (PA)] in modulating expression of cell-surface adhesion molecules CD11a/CD18, CD11b/CD18 on monocytes qualitatively and quantitatively. Monocytes incubated with bacterial components presented different CD11b/CD18 expressions which were dose-dependent in contrast to controls. The results obtained demonstrated that lymphocytes incubated with bacterial components also increased the expression of CD11a/CD18. The modifications in activation of CD11a/CD18 and CD11b/CD18 expression are probably correlated with modifications of membrane fluidity measured as polarisation fluorescence (P).     

Participation of beta2-integrins CD11b/CD18 and CD11c/CD18 in adhesion and migration of cells on fibrinogen

The role of beta2-integrins CD11b/CD18 and CD 11c/CD 18 in adhesion and migration of leukocytes on fibrinogen was studied. The monoclonal antibodies against CD11b inhibited the spontaneous adhesion of monocytic THP-1 cells on fibrinogen, whereas antibodies to CD11c more effectively inhibited the adhesion stimulated by chemokine MCP-1. By the RNA-interference method the clones of THP-1 with reduced expression of CD11b and general beta2-subunit CD18 were obtained. MCP-I stimulated the adhesion to fibrinogen of THP-1 cells of wild-type and mutant cells with reduced expression of CD11b (THP-1-CD11b-low), but not of cells with low expression of CD18 (THP-1-CD18-low). THP-1-CD18-low cells were also characterized by the impaired chemotaxis in presence of MCP-1. The data obtained suggest that spontaneous cell adhesion to fibrinogen is mediated to a greater extent by CD11b/CD18 integrins, while chemokine-stimulated adhesion and migration is mostly dependent on CD11c/CD18 molecules.     

A peptide derived from the intercellular adhesion molecule-2 regulates the avidity of the leukocyte integrins CD11b/CD18 and CD11c/CD18

beta 2 integrin (CD11a,b,c/CD18)-mediated cell adhesion is required for many leukocyte functions. Under normal circumstances, the integrins are nonadhesive, and become adhesive for their cell surface ligands, the intercellular adhesion molecules (ICAMs), or soluble ligands such as fibrinogen and iC3b, when leukocytes are activated. Recently, we defined a peptide derived from ICAM-2, which specifically binds to purified CD11a/CD18. Furthermore, this peptide strongly induces T cell aggregation mainly mediated by CD11a/CD18-ICAM-1 interaction, and natural killer cell cytotoxicity. In the present study, we show that the same ICAM-2 peptide also avidly binds to purified CD11b/CD18, but not to CD11c/CD18. This binding can be blocked by the CD11b antibody OKM10. The peptide strongly stimulates CD11b/CD18-ICAM-1-mediated cell aggregations of the monocytic cell lines THP-1 and U937. The aggregations are energy and divalent cation-dependent. The ICAM-2 peptide also induces CD11b/CD18 and CD11c/CD18-mediated binding of THP- 1 cells to fibrinogen and iC3b coated on plastic. These findings indicate that in addition to induction of CD11a/CD18-mediated cell adhesion, the ICAM-2 peptide may also serve as a "trigger" for high avidity ligand binding of other beta 2 integrins.     

CR3 (CD11b/CD18) activation of nasal neutrophils: a measure of upper airway endotoxin exposure

Inhaled endotoxin (lipopolysaccharide, LPS) initiates an inflammatory response and leads to the expression of CR3 (CD11b/CD18) receptors on polymorphonuclear leukocytes (PMNs). We determined if PMN activation in nasal lavage fluid (NLF) is a possible biomarker of occupational endotoxin exposure. Seven subjects exposed to endotoxin provided NLF samples that were split into three aliquots (negative control – 1?M nicotinamide; sham; positive control – 11 ηg of exogenous LPS) and PMN activation was measured using a chemiluminometer. Differences in mean PMN activation were apparent, negative control: 548?±?15.65 RLU 100 μl^?1; sham: 11469?±?2582 RLU 100 μl^?1; positive control: 42026?±?16659 RLU 100 μl (n?=?7; p <0.05). This technique shows promise as a diagnostic method for measuring upper airway LPS exposure.     

Two leukocyte receptors (CD11a/CD18 and CD11b/CD18) mediate transient adhesion to endothelium by binding to different ligands

Upon stimulation with C5a, TNF, or phorbol dibutyrate (PDB), polymorphonuclear leukocytes (PMN) exhibit first an increase then a decrease in adhesion to unstimulated endothelial cells (EC). Essentially all of this adhesion is mediated by the CD18 family of leukocyte integrins on PMN. To determine the individual roles of CD11a/CD18 (LFA-1), CD11b/CD18 (CR3, Mac-1) and CD11c/CD18 (p150,95) in adhesion of PDB-stimulated PMN to unstimulated EC, mAb against the CD11 chains were used. mAb against CD11a or CD11b each blocked adhesion of PMN to EC by approximately 50%, but mAb against CD11c had no effect. Inasmuch as a combination of anti-CD11a and CD11b mAb completely blocked adhesion, it appears that CD11a/CD18 and CD11b/CD18 make approximately equal contributions to binding, and CD11c does not participate. Anti-CD11a or CD11b each blocked adhesion by about 50% throughout the transient time course of PDB-stimulated adhesion, indicating that the capacity of each of these receptors to bind EC is transiently activated by PDB. We next examined the role of ICAM-1 on EC as a ligand for CD18. Two anti-ICAM-1 mAb (LB-2 and 84H10) each inhibited PMN adhesion in a dose-dependent fashion, reaching a maximal inhibition of approximately 50%. Anti-ICAM-1 mAb blocked the CD11a/CD18-dependent portion of adhesion because concomitant use of anti-CD11a and anti-ICAM-1 did not cause additive inhibition. In contrast, anti-CD11b plus anti-ICAM-1 resulted in complete blockade of adhesion. This result suggests that CD11a/CD18 recognizes ICAM-1 on EC, but CD11b/CD18 recognizes a different ligand(s). To determine if CD11b CD18 has the ability to recognize ICAM-1, human macrophages were plated on culture surfaces coated with purified ICAM-1. Interaction of CD11a/CD18 with the surface-bound ICAM-1 resulted in selective down-modulation of CD11a/CD18 from the apical portion of the macrophages. In contrast, ICAM-1-coated surfaces did not down-modulate CD11b/CD18. The data suggest that CD11b/CD18 does not recognize ICAM-1, and that this receptor functions in adhesion of PMN to EC by recognizing novel ligand(s) on EC.     

Identification of novel agonists of the integrin CD11b/CD18

We report the identification of novel small molecule agonists of integrin CD11b/CD18, which increased, in a dose-dependent manner, the adhesion of the integrin CD11b/CD18 expressing cells to two physiologically relevant ligands: Fibrinogen and iC3b. Compound 6 showed an ex vivo EC₅₀ of 10.5 μM and in vitro selectivity for binding to the recombinant αA-domain of CD11b/CD18. In silico docking experiments suggest that the compounds recognized a hydrophobic cleft in the ligand-binding αA-domain, implying an allosteric mechanism of modulation of integrin affinity by this novel compound.     

Modulation of CD11b/CD18 adhesive activity by its extracellular,membrane-proximal regions

The integrin receptor CD11b/CD18 is normally kept in a low adhesive state and can be activated by many different agents. However, the mechanism underlying receptor activation is not yet fully understood. We hypothesized that the extracellular, membrane-proximal regions of CD11b/CD18 are critically involved in modulation of its adhesive functions. To test our hypothesis, we perturbed the extracellular, membrane-proximal regions of individual CD11b and CD18 subunits and studied their effect on ligand binding, receptor clustering, and lipid raft association. We report here three major findings: 1) perturbation of the extracellular, membrane-proximal region of either subunit leads to enhanced adhesion, caused by changes in receptor conformation, but not the state of receptor clustering or lipid raft association; 2) the CD11b subunit plays a more important role in confining the receptor in an inactive state; and 3) upon modification of the extracellular, membrane-proximal region, the mutant CD11b/CD18 acquires the ability to respond to stimulation by "inside-out" signaling. Our results suggest that the extracellular, membrane-proximal region of the receptor plays an important role in integrin activation and therefore could be targeted by certain cell surface proteins as a conduit to control the integrin "inside-out" signaling process.     

Mac-1 (CD11b/CD18) mediates adherence-dependent hydrogen peroxide production by human and canine neutrophils

Human neutrophils exposed to protein-coated polystyrene or cultured endothelial monolayers produce large quantities of H2O2 in response to soluble stimuli that elicit little or no secretion of reactive oxygen species from cells in suspension. To characterize the mechanisms involved in this adherence-dependent respiratory burst, we have investigated the possible role of one integrin known to participate in the adhesion of neutrophils to endothelial cells, CD11b/CD18 (Mac-1). H2O2 production was examined with chemotactic factor-stimulated human and canine neutrophils exposed to protein-coated surfaces and cultured human and canine endothelial cells. The two protein-coated surfaces used were type I collagen-coated glass or plastic, a surface to which neither human nor canine neutrophils adhered, and keyhole limpet hemocyanin (KLH)-coated glass or plastic, a surface to which human and canine neutrophils adhered only after chemotactic stimulation. FMLP-stimulated human neutrophils and platelet activating factor-stimulated canine neutrophils failed to produce detectable H2O2 when in contact with type I collagen, but secreted large amounts of H2O2 when adherent to KLH or endothelial cell monolayers. FMLP-stimulated neutrophils from patients with CD18-deficiency failed to adhere to any of these surfaces and failed to produce H2O2 under these conditions. mAb reactive with CD18 and CD11b were equally effective in markedly inhibiting the adhesion of normal human neutrophils to these surfaces and markedly inhibited the production of H2O2. A mAb reactive with CD18 blocked adhesion of stimulated canine neutrophils, and mAb directed against both CD18 and CD11b blocked H2O2 production by canine neutrophils on KLH and endothelium. A nonbinding mAb and a mAb reactive with CD11a did not inhibit H2O2 production of human cells on KLH or endothelial monolayers, and nonbinding and binding control mAb did not inhibit H2O2 production by canine neutrophils. These results indicate that Mac-1 (CD11b/CD18) can mediate adhesion-dependent H2O2 production by human and canine neutrophils exposed to chemotactic factors.     

Lipopolysaccharide-coated erythrocytes activate human neutrophils via CD14 while subsequent binding is through CD11b/CD18

Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enhanced by LPS-binding protein (LBP). Integrins as well as CD14 play a role in the interaction of erythrocytes (E) coated with LPS or whole Gram-negative bacteria with phagocytes. We reasoned that the density of LPS on a particle is an important determinant in these interactions. Therefore, E were coated with different concentrations of LPS (ELPS). The binding of these ELPS to neutrophils was evaluated by flow cytometry. Simultaneously, we measured fMLP receptor expression to evaluate neutrophil activation. ELPS only bound to neutrophils in the presence of LBP. Blocking CD14 inhibited both activation and binding, whereas blocking complement (C) receptor 3 (CR3) inhibited binding but not activation. TNF activation restored ELPS binding in CD14-blocked cells but not in cells in which CR3 was blocked. Salmonella minnesota did bind to neutrophils independent of CR3 or CD14. The addition of LBP enhanced binding twofold, and this surplus was dependent upon CD14 but not on CR3. We conclude that ELPS interact with neutrophils via CD14, initially giving rise to cell activation; subsequently, binding is solely mediated by activated CR3.     

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.     

CD157 is part of a supramolecular complex with CD11b/CD18 on the human neutrophil cell surface

CD157 is a GPI-anchored cell surface glycoprotein expressed by human peripheral blood neutrophils. Cross-linking of CD157 induces intracellular Ca2+ mobilization and re-shaping in neutrophils, thus regulating their adhesive and migratory properties. Results obtained by immunolocalization and confocal microscopy indicate that CD157 lies in close proximity to the CD11b/CD18 complex which is strongly expressed on the activated neutrophil cell membrane where it plays a predominant role in adhesion. This study analyses the physical association between CD157 and CD18 in human neutrophils by co-immunoprecipitation experiments. The anti-CD157 monoclonal antibody RF3 co-precipitates CD18, and the anti-CD18 antibody TS1/18 co-precipitates CD157 from human neutrophil lysates. These results confirm that CD157 physically interacts with CD11b/CD18 complex in human neutrophils.     

H2O2 activates CD11b/CD18-dependent cell adhesion

Treatment of monoblastoid U-937 cells with low concentrations of H2O2 caused adhesion of the cells to plastic. The H2O2 induced adhesion was rapid with a t1/2 of congruent to 6 min and was optimally stimulated by 100 microM H2O2 with an ED50 of congruent to 50 microM. The response to H2O2 closely resembled the adhesive response of U-937 cells to phorbol esters in its time dependency, requirement for extracellular Mg2+ and inhibition by cytochalasin B as well as inhibition by monoclonal antibodies against the leucocyte adhesion molecules CD11b and CD18. Phorbol ester treatment of U-937 cells stimulated the phosphorylation of at least three endogenous substrates, pp28, pp34 and pp43, of which pp28 and pp43 also responded to H2O2-treatment with increased 32P-incorporation. The results suggest that H2O2 might be a physiological modulator of leucocyte adhesion, possibly operating by activating protein kinase C.     

CD11b/CD18-dependent interactions of neutrophils with intestinal epithelium are mediated by fucosylated proteoglycans

CD11b/CD18-mediated adhesive interactions play a key role in regulating polymorphonuclear leukocytes (PMN)) migration across intestinal epithelium. However, the identity of epithelial ligands for migrating PMN remains obscure. In this study we investigated the role of carbohydrates in mediating adhesive interactions between T84 intestinal epithelial cells and CD11b/CD18 purified from PMN. Fucoidin, heparin/heparin sulfate, N-acetyl-D-glucosamine, mannose-6-phosphate, and laminarin were found to inhibit adhesion of T84 cells to CD11b/CD18. The most potent inhibitory effects were observed with fucoidin (50% inhibition at 1-5 x 10(-8) M). Binding assays demonstrated that fucoidin directly bound to CD11b/CD18 in a divalent cation- and sulfation-dependent fashion that was blocked by anti-CD11b mAbs. Experiments employing CD11b/CD18 as a probe to blot T84 cell fucosylated proteins purified via fucose-specific lectin column revealed several candidate CD11b/CD18 binding proteins with molecular masses of 95, 50, 30, 25, and 20 kDa. Fucosidase treatment of T84 cells resulted in significantly reduced cell adhesion to CD11b/CD18, while no inhibition was observed after neuraminidase treatment. Finally, significant inhibition of T84 cell adhesion to CD11b/CD18 was observed after blocking cell proteoglycan synthesis with p-nitrophenyl-beta-D-xylopyranoside. These findings implicate epithelial cell surface proteoglycans decorated with sulfated fucose moieties as ligands for CD11b/CD18 during PMN migration across mucosal surfaces.     

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.