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.     

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.     

Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1.

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca(2+), Mg(2+), or Mn(2+) divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (K(d) = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.     

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.     

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.     

Modulation of Mac-1 (CD11b/CD18)-mediated adhesion by the leukocyte-specific protein 1 is key to its role in neutrophil polarization and chemotaxis

Leukocyte-specific protein 1 (LSP1) is an intracellular filamentous-actin binding protein which modulates cell motility. The cellular process in which LSP1 functions to regulate motility is not yet identified. In this study, we show that LSP1 negatively regulates fMLP-induced polarization and chemotaxis of neutrophils through its function on adhesion via specific integrins. Using LSP1-deficient (Lsp1(-/-)) mice, we show increased neutrophil migration into mouse knee joints during zymosan-induced acute inflammation, an inflammatory model in which the number of resident synoviocytes are not affected by LSP1-deficiency. In vitro chemotaxis experiments performed by time-lapse videomicroscopy showed that purified Lsp1(-/-) bone-marrow neutrophils exhibit an increased migration rate toward a gradient of fMLP as compared with wild-type neutrophils. This difference was observed when cells migrated on fibrinogen, but not fibronectin, suggesting a role for LSP1 in modulating neutrophil adhesion by specific integrins. LSP1 is also a negative regulator of fMLP-induced adhesion to fibrinogen or ICAM-1, but not to ICAM-2, VCAM-1, or fibronectin. These results suggest that LSP1 regulates the function of Mac-1 (CD11b/CD18), which binds only to fibrinogen and ICAM-1 among the substrates we tested. fMLP-induced filamentous actin polarization is also increased in the absence of LSP1 when cells were layered on fibrinogen, but not on fibronectin. Our findings suggest that the increased neutrophil recruitment in Lsp1(-/-) mice during acute inflammation derives from the negative regulatory role of LSP1 on neutrophil adhesion, polarization, and migration via specific integrins, such as Mac-1, which mediate neutrophil responses to chemotactic stimuli.     

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.     

CD40 signaling activates CD11a/CD18 (LFA-1)-mediated adhesion in B cells.

Cell-cell adhesion events play critical roles in the sequential migrations and multiple specific cell-cell interactions which B cells undergo during normal development and function. We have observed that mAb to several B cell-associated molecules, including mAb to CD19, CD37, and CD40, induce homotypic aggregation of freshly isolated human B cells. The aggregation of B cells induced by CD40 mAb was due to activation of a cell-cell adhesion system, and not due to agglutination by mAb, because 1) in addition to being energy dependent and cation dependent, the aggregation was blocked by inhibitors of messenger RNA and protein synthesis; and 2) a mouse B cell line transformed with intact human CD40 aggregated in response to CD40 mAb, whereas a line expressing surface CD40, but lacking the cytoplasmic tail and previously shown incapable of transmitting a signal from the cell surface, did not aggregate. The aggregation, although of slow onset, was persistent and of high avidity. In addition, CD40 mAb induced increased surface expression of intercellular adhesion molecule-1 (CD54), a ligand for CD11a/CD18 (LFA-1), and CD18 mAb blocked aggregation. CD40 mAb also augmented the ability of dense B cells to stimulate the proliferation of allogeneic T cells via a CD18-dependent process. We conclude that signaling through CD40, elicited by cross-linking the CD40 protein on the cell surface, activates the CD18/intercellular adhesion molecule adhesion system; in addition, CD40 cross-linking may activate a second adhesion system since CD40 mAb induced aggregation of the B cell line Ramos, which does not express surface CD18. B cell adhesion may be triggered by signaling through multiple surface proteins, thereby lending specificity of activation to adhesion systems which are broadly expressed.     

Endothelial-leukocyte adhesion molecule-1-dependent and leukocyte (CD11/CD18)-dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine-activated human vascular endothelium

We have examined the contributions of endothelial-leukocyte adhesion molecule-1 (ELAM-1) and the complex of leukocyte surface adhesion molecules designated CD11/CD18 to the adhesion of human polymorphonuclear leukocytes (PMN) to cultured human endothelial cells (HEC), activated by rIL-1 beta for 4 or 24 h. Inhibition of PMN attachment to IL-1-activated HEC was measured in a quantitative in vitro monolayer adhesion assay, after treatment with mAb directed to ELAM-1 (mAb H18/17), and to CD11a (mAb L11), CD11b (mAb 44), CD11c (mAb L29), and CD18 (mAb 10F12), alone or in combination. Pretreatment of activated HEC with mAb H18/7 inhibited PMN adhesion by 47 +/- 8% whereas control mAb had no effect. CD11/CD18-directed mAb significantly blocked PMN adhesion to activated HEC (anti-CD11a, 40 +/- 3%; anti-CD11b, 34 +/- 4%; anti-CD18, 78+/- 6% inhibition). The combination of mAb H18/7 and each of the various anti-CD11/CD18 mAb resulted in greater inhibition of PMN adhesion than any Mab alone. After 24 h of rIL-1 beta treatment, when ELAM-1 was markedly decreased but elevated PMN adhesion was still observed, mAb H18/7 had no effect on PMN adhesion. At this time, CD11/CD18-dependent adhesive mechanisms predominated and a CD11c-dependent mechanism became apparent (anti-CD11a, 67 +/- 4% inhibition; anti-CD11b, 45 +/- 9%; anti-CD11c, 26 +/- 6%; anti-CD18, 97 +/- 1%). In summary, PMN adhesion to IL-1-activated HEC involves both CD11/CD18-dependent mechanisms and an ELAM-1-dependent mechanism, and the relative contribution of these varies at different times of IL-1-induced HEC activation. The additive blocking observed at 4 h with mAb H18/7 in combination with CD11/CD18-directed Mab implies that members of the CD11/CD18 complex do not function as an obligate ligand(s) for ELAM-1.     

Role of endothelial leukocyte adhesion molecule-1 and platelet-activating factor in neutrophil adherence to IL-1-prestimulated endothelial cells. Endothelial leukocyte adhesion molecule-1-mediated CD18 activation.

Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta-pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta-pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta-pretreated EC.     

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.     

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.     

A role for beta2 integrin (CD11/CD18)-mediated tyrosine signaling in extravasation of human polymorphonuclear neutrophils

During the recruitment of human polymorphonuclear neutrophils (PMN) to sites of inflammation, leukocyte adhesion molecules of the beta2 integrin (CD11/CD18) family mediate firm adhesion of these cells to the endothelial cell monolayer lining the vessel wall. This process is a prerequisite for shape change and spreading of PMN on the endothelium which eventually allows PMN emigration into the extravascular space. In order to elucidate the molecular mechanisms which mediate this sequence of events, intracellular protein tyrosine signaling was studied subsequent to beta2 integrin-mediated ligand binding. Using western blotting technique, beta2 integrin-mediated adhesion was found to induce tyrosine phosphorylation of different proteins. The effect was absent in PMN derived from CD18-deficient mice which lack any beta2 integrin expression on the cell surface demonstrating the specificity of the observed response. Inhibition of beta2 integrin-mediated tyrosine signaling by herbimycin A almost completely inhibited adhesion, shape change, and subsequent spreading of PMN. Herbimycin A also diminished chemotactic migration of these cells in response to the soluble mediator N-formyl-Met-Leu-Phe (fMLP). In contrast, treatment of PMN with cytochalasin D had no substantial effect on beta2 integrin-mediated signaling or adhesion but inhibited shape change, spreading, and chemotactic migration of PMN. This suggests that the signaling capacity exerted by beta2 integrins upon ligand binding was independent of an intact cytoskeleton. Moreover, the beta2 integrin-mediated activation of intracellular signal transduction pathways was critical for firm adhesion of PMN, the prerequisite subsequent shape change and spreading, which allows emigration of PMN into the extravascular space.     

Interleukin-11 increases cell motility and up-regulates intercellular adhesion molecule-1 expression in human chondrosarcoma cells

Interleukin‐11 (IL‐11) was originally identified as the cytokine that could induce the proliferation of human cells. Recent studies have shown that IL‐11 plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of IL‐11 on human chondrosarcoma cells are largely unknown. Here, we found that IL‐11 increased the migration and expression of intercellular adhesion molecule‐1 (ICAM)‐1 in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significant expression of the IL‐11 which was higher than that in primary chondrocytes. The phosphatidylinositol 3‐kinase (PI3K), Akt, and NF‐κB pathways were activated by IL‐11 treatment, and the IL‐11‐induced expression of ICAM‐1 and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF‐κB cascades. Taken together, our results indicate that IL‐11 enhanced the migration of the chondrosarcoma cells by increasing ICAM‐1 expression through the IL‐11Rα receptor, PI3K, Akt, and NF‐κB signal transduction pathway. J. Cell. Biochem. 113: 3353–3362, 2012. © 2012 Wiley Periodicals, Inc.     

The role of intercellular adhesion molecule-1/LFA-1 interactions in the generation of tumor-specific CD8+ T cell responses

The activation of naive CD4+ T cells requires both TCR engagement and a second costimulatory signal mediated by the interaction of CD28 with CD80/CD86 expressed on professional APC. However, the situation for naive CD8+ T cells is less clear. Although evidence indicates that induction of CD8+ T cell responses is also dependent on professional APC, the ability of some tumors, which do not express CD80/CD86, to induce CTL suggests that other pathways of costimulation exist for the activation of CD8+ T cells. We examined the ability of tumor cells expressing different levels of a tumor-specific Ag to directly prime CD8+ T cells. We demonstrate that CD8+ T cells are directly activated by tumor cells in a CD80/CD86-CD28 independent manner. In this system, costimulation requires ICAM-1/LFA-1 interaction. This results in the generation of CTL capable of inhibiting tumor growth in vivo, and maintaining long-term survival.     

Engagement of the monocyte surface antigen CD14 induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion.

Murine anti-CD14 mAb which recognize different CD14 epitopes induced marked homotypic adhesion of normal human monocytes. Induction of aggregation by anti-CD14 mAb required Mg2+, occurred at an optimal temperature of 37 degrees C, but not at 4 degrees C, and exhibited a kinetics which differed from adhesion triggered by IFN-gamma and anti-CD43 mAb. Monocyte adhesion induced by anti-CD14 mAb required neither Fcy gamma R engagement nor cross-linking of CD14, because adhesion was induced by F(ab)'2 fragments, as well as by monovalent F(ab) fragments of anti-CD14 mAb. mAb to CD11a, CD18, and intercellular adhesion molecule-1 (ICAM-1), but not antibodies to CD11b and CD11c, inhibited monocyte adhesion induced by CD14 engagement. These results indicate that CD14-dependent adhesion is mediated by lymphocyte function-associated Ag-1/ICAM-1 interactions. This was confirmed by the absence of aggregation in anti-CD14-stimulated cells from a patient with leukocyte adhesion deficiency. Monocyte adhesion upon CD14 engagement was blocked by an inhibitor of protein kinases, sphingosine. This suggests that protein kinases play a role in the intracellular signaling pathway(s) which couple CD14 to lymphocyte function-associated Ag-1/ICAM-1.     

Effect of mechanical deformation of neutrophils on their CD18/ICAM-1-dependent adhesion.

Mechanical deformation of polymorphonuclear leukocytes (PMN) changes their expression of the surface adhesion molecule CD11b/CD18. We tested the hypothesis that mechanical deformation of PMN enhances their adhesiveness. Purified human PMN were deformed through either 5- or 3-microm polycarbonate membrane filters and allowed to adhere to 96-well plates coated with human recombinant intercellular adhesion molecule-1 (ICAM-1). Flow cytometric studies showed that deformation of PMN increased CD11b/CD18 expression (P < 0.01). PMN adhesion to ICAM-1-coated plates was dependent on the magnitude of cell deformation (5 microm, 63.8 +/- 8.1%, P < 0.04; 3 microm, 232.4 +/- 20.9%, P < 0.01). Priming of PMN (0.5 nM N-formyl-methionyl-leucyl-phenylalanine) before deformation (5 microm) increased PMN adhesion (63.8 +/- 8.1 vs. 105.3 +/- 16.4%; P < 0.04). Stimulation (5% zymosan-activated plasma) of PMN after deformation resulted in increased adhesion, and the degree of increase was dependent on the magnitude of PMN deformation (stimulation, 50.6 +/- 4%; 5-microm filtration and stimulation, 62.9 +/- 6.6%; 3-microm filtration and stimulation, 249.9 +/- 24.2%; P < 0.01). This study shows that mechanical deformation of PMN causes an increase in PMN adhesiveness to ICAM-1 that was enhanced by both priming of PMN before deformation and stimulation after cell deformation.     

Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins

Human polymorphonuclear leukocytes (PMN) released large quantities of hydrogen peroxide in response to tumor necrosis factor, but only when the cells were adherent to surfaces coated with extracellular matrix proteins. The PMN did not respond when exposed to cytokines and matrix proteins in suspension, or when exposed to cytokines while adherent to surfaces coated with stearic acid. PMN from children with genetic deficiency of the CD11/CD18 integrins underwent a normal respiratory burst upon adherence to uncoated polystyrene, but not in response to tumor necrosis factor when tested on polystyrene that was coated with serum, fibronectin, vitronectin, fibrinogen, thrombospondin, or laminin. Anti-CD18 antibodies, alone of sixteen antibodies tested, induced a similar defect in PMN from normal donors, when the PMN were tested on surfaces coated with serum, fibrinogen, thrombospondin, or laminin; no defect was induced by the anti-CD18 monoclonal antibody IB4 in normal PMN tested on surfaces coated with fibronectin or vitronectin. Thus, for cytokines to induce a respiratory burst in PMN, the cells must be able to use CD11/CD18 integrins and must interact with matrix proteins in the solid phase. CD11/CD18, which is already known to serve as a receptor for fibrinogen, may also be a receptor for thrombospondin and laminin. Finally, receptor(s) exist on PMN for fibronectin and vitronectin which are not blocked by the anti-CD18 antibody IB4 but which are nonetheless CD11/CD18 dependent.