A role for beta(2) integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response.

Growing evidence supports the idea that adhesion via beta(2) integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adherent cells. This study investigates whether beta(2) integrin-mediated adhesion of human polymorphonuclear neutrophils (PMN) has a functional impact on cytokine production. Aggregation of the beta(2) integrin Mac-1 (CD11b/CD18) by antibody cross-linking was found to induce substantial de novo synthesis of IL-8 mRNA as measured by semiquantitative RT-PCR and Northern blotting technique, respectively. Induction of IL-8 mRNA was also observed upon adhesion of PMN to immobilized fibrinogen, a functional equivalent of its clotting product fibrin that serves as a native ligand of Mac-1. Results were confirmed using PMN derived from CD18-deficient mice, which were unable to produce MIP-2 mRNA, a homologue of human IL-8, in the presence of immobilized fibrinogen. In contrast, a substantial increase of MIP-2 mRNA was observed when wild-type PMN were incubated on immobilized fibrinogen. In human PMN, ELISA technique showed that the gene activation that required tyrosine kinase activity resulted in a substantial production and secretion of biologically active IL-8 and IL-1beta. In contrast, no TNF-alpha or IL-6 production was found, revealing that beta(2) integrins mediate differential expression of proinflammatory cytokines. The biological relevance of the present findings was confirmed in an in vivo model of acute inflammation. Altogether, the present findings provide evidence for a functional link between clotting and inflammatory responses that may contribute to the recruitment and/or activation of PMN and other cells at sites of lesion.     

Complement receptor 3 (CR3, Mac-1, integrin alpha M beta 2, CD11b/CD18) is required for tyrosine phosphorylation of paxillin in adherent and nonadherent neutrophils

Expression of the leukocyte (beta 2) integrins is required for many functions of activated neutrophils (PMN), even when there is no recognized ligand for any beta 2 integrin. To investigate the hypothesis that beta 2 integrins may be involved in a signal transduction pathway related to cytoskeletal reorganization, we examined whether beta 2 integrins have a role in tyrosine phosphorylation of the cytoskeletal protein paxillin. Treatment of PMN in suspension with phorbol esters, f-Met-Leu-Phe, and TNF-alpha resulted in paxillin tyrosine phosphorylation. However, treatment of beta 2-deficient (LAD) PMN failed to induce paxillin tyrosine phosphorylation. Normal PMN phosphorylated paxillin in response to adhesion to immune complexes, while the LAD PMN did not. Adhesion of phorbol ester activated-LAD PMN to the extracellular matrix proteins fibronectin, laminin, and vitronectin failed to induce paxillin tyrosine phosphorylation. Treatment of activated normal PMN with mAb directed against the beta 2 integrin alpha chains demonstrated that CR3 (alpha M beta 2) was required for paxillin phosphorylation. Transfection of the cell line K562 with CR3 confirmed that CR3 ligation resulted in paxillin tyrosine phosphorylation. As a control, K562 transfected with CR2 (CD21) which bound equally avidly to the same complement C3-derived ligand (C3bi) as the CR3 transfectants, showed no enhanced tyrosine phosphorylation of paxillin upon receptor ligation. While both CR2 and CR3 transfectants showed efficient adhesion to a C3bi-coated surface, only the CR3 transfectants spread during adhesion and phosphorylated paxillin. Together these data demonstrate that CR3 is required for paxillin phosphorylation during activation of both adherent and nonadherent PMN. Even PMN activated in suspension or by adhesion to immune complexes, when no CR3 ligand is apparent, still require CR3 for a signal transduction pathway leading to paxillin tyrosine phosphorylation. This pathway is likely to be important for PMN function in inflammation and host defense.     

Neutrophil adhesion to TNF alpha-activated endothelial cells potentiates leukotriene B4 production.

Since adhesion of neutrophils (PMN) to endothelial cells may influence PMN activation responses, we examined whether adhesion of PMN to TNF alpha-activated human umbilical vein endothelial cells (HUVEC) stimulates leukotriene B4 (LTB4) production. Endothelial adhesivity towards PMN increased after HUVEC pretreatment with TNF alpha for 4 h. LTB4 production increased markedly in response to stimulation with arachidonic acid (20 microM) when PMN were added to the hyperadhesive HUVEC. In contrast, stimulation of PMN in suspension did not potentiate LTB4 production. LTB4 production persisted when PMN were applied to TNF alpha-pretreated HUVEC fixed with 1% paraformaldehyde excluding the possibility that metabolic activity of endothelium participates in this response. PMN adhesion to plastic and gelatin also enhanced LTB4 indicating that adhesion was a critical event in inducing LTB4 production. We used monoclonal antibodies (mAb) to adhesion molecules on endothelial cells (i.e., endothelial leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1)) or on PMN (CD18) to assess the role of PMN adhesion to the activated endothelium on LTB4 potentiation. Both anti-ELAM-1 mAb and anti-ICAM-1 mAb inhibited PMN adhesion (by 55 and 41%, respectively) as well as LTB4 production (by 65 and 50%, respectively). Anti-CD18 mAb also reduced the adhesion (65%) and the LTB4 production (66%). Furthermore, combination of anti-ELAM-1 mAb (H18/7) and anti-ICAM-1 mAb (RR1/1) or of anti-ELAM-1 mAb (H18/7) and anti-CD18 mAb (IB4) had an additive effect in inhibiting both PMN adhesion as well as LTB4 production. PMN adherence to immobilized recombinant soluble rELAM-1 or rICAM-1 also increased LTB4 production, which was prevented with relevant mAbs. However, neither rELAM-1 nor rICAM-1 stimulated LTB4 production of PMN in suspension. We conclude that PMN adhesion to TNF alpha-stimulated endothelial cells enhances LTB4 production by PMN, a response activated by binding of PMN to expressed endothelial cell surface adhesion molecules.     

Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti- Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12- myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.     

Blockade of alpha 5 beta 1 integrins reverses the inhibitory effect of tenascin on chemotaxis of human monocytes and polymorphonuclear leukocytes through three-dimensional gels of extracellular matrix proteins

Tenascin is an extracellular matrix protein found in adults in T cell-dependent areas of lymphoid tissues, sites of inflammation, and tumors. We report here that it inhibited chemotaxis of chemoattractant-stimulated human monocytes and chemoattractant-stimulated polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of collagen I or Matrigel, and chemotaxis of leukotriene B4-stimulated PMN through fibrin gels. The inhibitory effect of tenascin on monocyte or PMN chemotaxis through these matrices was reversed by Abs directed against alpha5beta1 integrins or by a peptide (GRGDSP) that binds to beta1 integrins. Tenascin did not affect leukotriene B4- or fMLP-stimulated expression of beta1 or beta2 integrins, but did exert a small inhibitory effect on PMN adhesion and closeness of apposition to fibrin(ogen)-containing surfaces. Thus, alpha5beta1 integrins mediate the inhibitory effect of tenascin on monocyte and PMN chemotaxis, without promoting close apposition between these leukocytes and surfaces coated with tenascin alone or with tenascin bound to other matrix proteins. This contrasts with the role played by alpha5beta1 integrins in promoting close apposition between fMLP-stimulated PMN and fibrin containing surfaces, thereby inhibiting chemotaxis of fMLP-stimulated PMN through fibrin gels. Thus, chemoattractants and matrix proteins regulate chemotaxis of phagocytic leukocytes by at least two different mechanisms: one in which specific chemoattractants promote very tight adhesion of leukocytes to specific matrix proteins and another in which specific matrix proteins signal cessation of migration without markedly affecting strength of leukocyte adhesion.     

An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors

CD11b/CD18 is a heterodimeric leukocyte surface receptor which functions in both C3bi-ligand binding and homotypic and heterotypic cell adherence. We have examined the effect of several anti-CD11b/18 mAb on phagocytosis of IgG (EIgG) or complement (EC4b) opsonized erythrocytes by polymorphonuclear leukocytes (PMN) and monocytes. F(ab')2 of two mAb (IB4, an anti-beta-chain mAb and Mo-1 an anti-alpha-chain mAb), inhibited both phagocytosis of EIgG and phorbol ester-stimulated phagocytosis of EC4b by PMN and monocytes. These F(ab')2 inhibited the binding of EIgG to monocytes, but they had no effect on binding of EIgG to PMN, or EC4b to either phagocyte. In addition, IB4 inhibited phorbol-ester stimulated phagocytosis of sheep E opsonized with C component 3bi (EC3bi) without inhibiting rosetting of these same targets. These data separate the anti-phagocytic effect of these mAb from effects on phagocyte-target adherence. When PMN were adherent to an anti-CD11b/CD18 F(ab')2-coated surface, EC3bi binding was abolished, but phagocytosis of EIgG or EC4b was unaffected. Subsequent addition of fluid- phase IB4 or Mo-1 F(ab')2 inhibited phagocytosis of EIgG or EC4b by the adherent cells. This suggested that the CD11b/CD18 involved in C3bi rosetting were mobile in the membrane, whereas those involved in phagocytosis of EIgG or EC4b were not. Cytochalasin treatment of PMN during adherence to F(ab')2-coated plates decreased both apical expression of CD11b/18 and subsequent ingestion of EIgG by 70%, suggesting that microfilaments are important in maintaining immobile CD11b/18 on the apical PMN surface. We conclude that there are functionally distinct populations of CD11b/CD18 on monocytes and PMN: one involved in C3bi rosetting and another involved in the process of phagocytosis mediated via several different receptors. CD11b/18 is not required for optimal target binding in all cases, but is always required for ingestion. As with several other integrins, the CD11b/18 molecules involved in phagocytosis have a functional association with the cell cytoskeleton.     

Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions

Cross-linking of the neutrophil-beta 2- or beta 3-related leukocyte response integrins by extracellular matrix (ECM) proteins or monoclonal antibodies (mAb) stimulates cytoskeletal rearrangement leading to cell spreading and respiratory burst. Tyrosin phosphorylation of a variety of proteins and activation of the Src family kinases within polymorphonuclear leukocytes (PMN) have recently been implicated in the intracellular signaling pathways generated by leukocyte integrins (Yan, S.R., L. Fumagalli, and G Berton. 1995. J. Inflammation. 45:217-311.) To directly test whether these functional responses are dependent on the Src family kinases p59/61hck and p58c-fgr, we examined adhesion- dependent respiratory burst in PMNs isolated from hck -/-, fgr -/-, and hck -/- fgr -/- knockout mice. Purified bone marrow PMNS from wild-type mice released significant amounts of O2- when adherent to fibrinogen-, fibronectin-, or collagen-coated surfaces, in the presence of activating agents such as tumor necrosis factor (TNF) or formyl- methionyl-leucyl-phenylalanine, as described for human PMNs. PMNs from hck-/-fgr-/- double-mutant mic, however, failed to respond. This defect was specific for integrin signaling, since respiratory burst was normal in hck-/-fgr-/-PMNs stimulated by immune complexes or PMA. Stimulation of respiratory burst was observed in TNF-primed wild-type PMN plated on surfaces coated with murine intracellular adhesion molecule-1 (ICAM-1), while hck-/-fgr-/- PMNs, failed to respond. Direct cross-linking of the subunits of beta 2 and beta 2 integrins by surface-bound mAbs was elicited O2- production by wild-type PMNs, while the double-mutant hck- /-fgr-/- cells failed to respond. Photomicroscopy and cell adhesion assays revealed that the impaired functional responses of hck-/-fgr-/- PMNs were caused by defective spreading and tight adhesion on either ECM protein- or mAb-coated surfaces. In contrast, hck-/-or fgr-/-single mutant cells produced O2- at levels equivalent to wild-type cells on ECM protein, murine ICAM-1, and antiintegrin mAb-coated surfaces. Hence, either p59/61 hck and p 58c-fgr is required for signaling through leukocyte beta 2 and beta 3 integrins leading to PMN spreading and respiratory burst. This is the first direct genetic evidence of the importance of Src family kinases in integrin signaling within leukocytes, and it is also the best example of overlapping function between members of this gene family within a defined signal transduction pathway.     

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.     

Recognition of human polymorphonuclear leukocyte receptors for leukotriene B4 by rabbit anti-idiotypic antibodies to a mouse monoclonal antileukotriene B4

Rabbit anti-idiotypic IgG antibodies to the combining site of a mouse monoclonal IgG2b antibody to leukotriene B4 (LTB4) cross-reacted with human polymorphonuclear (PMN) leukocyte receptors for LTB4. Anti-idiotypic IgG and Fab both inhibited the binding of [3H]LTB4, but not [3H]N-formylmethionyl-leucylphenylalanine (fMLP), to PMN leukocytes with similar concentration-effect relationships, whereas neither nonimmune rabbit IgG nor Fab had any inhibitory activity. At a concentration of anti-idiotypic IgG that inhibited by 50% the binding of [3H] LTB4 to PMN leukocytes, the antibodies preferentially recognized high affinity receptors. Anti-idiotypic IgG and Fab inhibited PMN leukocyte chemotactic responses to LTB4, but not fMLP, with concentration-effect relationships resembling those characteristic of the inhibition of binding of [3H] LTB4, without altering the LTB4-induced release of beta-glucuronidase. Chemotaxis and increases in the cytoplasmic concentration of calcium equal in magnitude to those elicited by optimal concentrations of LTB4 were attained at respective concentrations of anti-idiotypic IgG equal to and 1/25 the level required for inhibition of binding of [3H]LTB4 by approximately 50%. Thus, the anti-idiotypic antibodies bound to PMN leukocyte receptors for LTB4 with a specificity, preference for high affinity sites, and capacity to alter PMN leukocyte functions that were similar to LTB4.     

Integrin engagement mediates the human polymorphonuclear leukocyte response to a fungal pathogen-associated molecular pattern

Extravasation of leukocytes from peripheral blood is required for an effective inflammatory response at sites of tissue infection. Integrins help mediate extravasation and navigate the leukocyte to the infectious source. A novel role for integrins in regulating the effector response to a cell wall component of fungal pathogens is the subject of the current study. Although phagocytosis is useful for clearance of unicellular fungi, the immune response against large, noningestible hyphae is not well-understood. Fungal beta-glucan, a pathogen-associated molecular pattern, activates production of superoxide anion in leukocytes without the need for phagocytosis. To model polymorphonuclear leukocyte (PMN) recognition of fungi under conditions in which phagocytosis cannot occur, beta-glucan was covalently immobilized onto tissue culture plastic. Plasma membrane-associated respiratory burst was measured by reduction of ferricytochrome C. Results show that the human PMN oxidative burst response to immobilized beta-glucan is suppressed by addition of beta(1) integrin ligands to the beta-glucan matrix. Suppression was dose dependent and steric hindrance was ruled out. beta(1) integrin ligands did not affect respiratory burst to ingestible beta-glucan-containing particles, phorbol esters or live yeast hyphae. Furthermore, in the absence of matrix, Ab activation of VLA3 or VLA5, but not other beta(1) integrins, also prevented beta-glucan-induced respiratory burst. beta(1)-induced suppression was blocked and burst response restored by treating neutrophils with either the cell-binding fragment of soluble human Fn, cyclic RGD peptide, or Ab specific to VLA3 or VLA5. Together these findings extend the functional role of beta(1) integrins to include modulating PMN respiratory burst to a pathogen-associated molecular pattern.     

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.     

Comparative studies on FcR (FcRII, FcRIII, and FcR alpha) functions of murine B cells

Distribution of FcR II, FcRIII, and FcR alpha on murine splenic B cells was examined by using FITC-labeled heat-aggregated IgG of each subclass and IgA. Almost 60 to 80% of B cells expressed both FcRII and FcRIII. However, FcR alpha was expressed on only a small proportion (6%) of B cells that co-expressed FcRII. By inhibition assays with the use of cold IgG of each subclass and IgA in addition to anti-FcRII mAb (2.4G2), it was found that IgG1, IgG2a, and IgG2b utilized the same receptor (FcRII), whereas IgG3 and IgA bound only to their unique receptors, FcRIII and FcR alpha, respectively. Immune complexes IC prepared by IgG1, IgG2a, IgG2b, and IgA anti-TNP mAb with TNP-coupled SRBC inhibited the polyclonal Ig secretion and proliferative responses of B cells stimulated with either IL-4 or LPS. The inhibition of B cell activation was associated with the blockade of the membrane depolarization. Moreover, IC prepared by these antibodies caused production of suppressive B cell factor (SBF) as is the case with rabbit IgG antibody to SRBC, and SBF thus prepared regulated antibody responses in an isotype-nonspecific manner. In contrast, no inhibition for these responses or production of SBF was attained by the IC of IgG3 antibody. We concluded that FcRII and FcR alpha mediates a suppressive signal for B cells by acting on the initial step of activation, whereas FcRIII lacks this activity.     

Enhancement of macrophage immune and nonimmune receptor-mediated phagocytosis by a low molecular weight soluble factor from resident thymocytes

Macrophage phagocytic activity is regulated in part by products of activated T lymphocytes. We previously reported that a heat-stable soluble factor derived from resident (nonactivated) thymocytes increases murine peritoneal macrophage Fc-dependent phagocytosis. In the present study, we further investigate the effect of the thymocyte factor on immune and nonimmune receptor-mediated phagocytosis, Fc receptor expression, and its approximate m.w. After 4 days of incubation, cellfree thymocyte supernatant produced a mean (three experiments) 2.10-, 2.08-, and 1.97-fold increase in macrophage phagocytosis of C3-, IgG-, and tannic acid-treated erythrocytes, respectively. Macrophage IL 1 production was not enhanced by a similar concentration of thymocyte supernatant. The thymocyte factor(s) increased the number of IgG2a Fc receptors (FcRI) from 2.4 x 10(5) to 3.8 x 10(5) receptor sites per macrophage. The number of Fc receptors that bind IgG1 and IgG2b (FcRII) was not altered. The soluble factor(s) that increased Fc-mediated phagocytosis passed through both 6000- to 8000-dalton and 2000-dalton cutoff dialysis membranes and eluted from a Sephadex G-25 Fine column over a m.w. range of 200 to 1000 daltons, with a peak activity at 450 daltons. These data suggest that resident thymocytes enhance macrophage phagocytosis of opsonized and nonopsonized particles through the elaboration of a low m.w. substance(s).     

Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.     

Unusual expression of IgG Fc receptors on peripheral granulocytes from patients with leukocyte adhesion deficiency (CD11/CD18 deficiency)

Leukocyte adhesion deficiency (LAD) is a hereditary disease characterized by defective expression of leukocyte adhesion glycoproteins; lymphocyte function-associated Ag-1 (CD11a/CD18), CR3 (CD11b/CD18) and p150,95 (CD11c/CD18). Granulocytes, monocytes, and lymphocytes of patients with LAD show profoundly defective in vivo and in vitro adherence-dependent immune functions. We investigated the expression of FcR for IgG on polymorphonuclear cells (PMN) and monocytes from patients with LAD, and their luminol- and lucigenin-enhanced chemiluminescence production in response to SRBC sensitized with murine (m) IgG2a and IgG2b. Unstimulated patient PMN showed an enhanced chemiluminescence in response to mIgG2a-SRBC and an increased phagocytosis of mIgG2a-SRBC. The up-regulated functions were inhibited by monomeric human IgG in a dose-dependent manner, which was attributed to an increase in expression of FcRI on patient PMN, as shown by flow cytometry using monoclonal antibody, 32.2, specific for human FcRI. In contrast, neither the expression of FcR on the monocytes of LAD patients nor their FcR-mediated functions were different from those of controls.     

Leukotriene B4 augments and restores Fc gammaRs-dependent phagocytosis in macrophages

Phagocytosis by macrophages is essential for host defense, i.e. preventing invasion of pathogens and foreign materials. Macrophages engulf immunoglobulin G (IgG)-opsonized particles through the action of the receptors for the Fc of IgG (FcγRs). Leukotriene B(4) (LTB(4)) is a classical lipid chemoattractant derived from arachidonic acid. Leukotriene B(4) receptor 1 (BLT1), a high affinity LTB(4) receptor, is expressed in a variety of immune cells such as neutrophils, macrophages, and dendritic cells. Although LTB(4) has been shown to enhance macrophage phagocytosis, few studies have investigated the intracellular mechanisms involved in this in detail. Furthermore, there have been no reports of the direct cross-talk between LTB(4)-BLT1 and IgG-FcγRs signaling. Here, we show that FcγRs-dependent phagocytosis was attenuated in BLT1-deficient macrophages as compared with wild-type (WT) cells. Moreover, cross-talk between LTB(4)-BLT1 and IgG-FcγRs signaling was identified at the level of phosphatidylinositol 3-OH kinase (PI3K) and Rac, downstream of Syk. In addition, the trimeric G(i) protein (G(i)) was found to be essential for BLT1-dependent phagocytosis. Surprisingly, we found that LTB(4)-BLT1 signaling restores phagocytosis in the absence of FcγRs signaling. These data indicate that LTB(4)-BLT1 signaling plays a pivotal role in macrophage phagocytosis and innate immunity.     

The effects of IL-1, IL-2, and tumor necrosis factor on polymorphonuclear leukocyte Fc gamma receptor-mediated phagocytosis. IL-2 down-regulates the effect of tumor necrosis factor

It has been reported that the Fc gamma R-mediated phagocytic activity of polymorphonuclear leukocytes (PMN) from patients with acute bacterial infections is markedly enhanced when compared with healthy controls. Inasmuch as several potent cytokines are known to be involved in inflammatory and infectious processes, we studied the effects of three such cytokines (IL-1 beta, IL-2, and TNF-alpha) on normal PMN Fc gamma R-mediated phagocytosis. IL-1 beta and TNF alpha both caused a significant increase in the ingestion of EIgG by adherent PMN. In combination, IL-1 beta and TNF-alpha had an additive effect, even when each was used at its optimal concentration. In contrast to the enhancing effects mediated by IL-1 beta and TNF-alpha, IL-2 alone had no significant effect on PMN phagocytosis. Notably, however, IL-2 at a concentration of 10(4) U/ml partially inhibited TNF-alpha-mediated enhancement of phagocytosis by decreasing TNF binding to the PMN cell surface. This inhibitory effect of IL-2 on TNF was reversed by anti-IL-2 antibody and mAb directed against the low affinity IL-2R (anti-Tac), whereas mAb directed against the intermediate affinity receptor (mik-beta 1) had no such effect. These findings may have important physiologic implications, because patients receiving IL-2 therapy have been shown to have increased susceptibility to infection.     

The CD11/CD18 (beta2) integrins modulate neutrophil caspase activation and survival following TNF-alpha or endotoxin induced transendothelial migration

Neutrophils (PMN) are short-lived cells but their survival is often prolonged in inflammation. The beta2 (CD11/CD18) integrins are involved in PMN migration into inflammation but their role in PMN survival is not well understood. We investigated the role of beta2 integrins in PMN caspase activation, a key enzyme cascade in apoptosis. After 20 h, caspase activation (Western blotting) was markedly decreased in PMN cultured on fibrinogen, a ligand for Mac-1 (CD11b/CD18), but not on fibronectin or albumin. In the presence of TNF-alpha or endotoxin (LPS), blockade of CD18 (beta2 chain) with mAb markedly increased caspase activation in PMN on fibrinogen. PMN which migrated through endothelium in vitro in response to TNF-alpha, LPS, IL-1alpha, IL-8 or C5a contained 58% fewer active caspase positive PMN after 20 h than non-migrated PMN remaining on the endothelium. When beta2 (CD18) integrin or lymphocyte function antigen (LFA)-1 (CD11a) plus Mac1 (CD11b) were blocked by mAb (intact or Fab'), the proportion of migrated PMN (but not of non-migrated PMN) with active caspases was significantly increased (2-4-fold) and this was associated with accelerated PMN apoptosis and death. Thus, engagement of ligands on extracellular matrix and endothelium by the beta2 integrins Mac-1 and LFA-1 plays a role in delaying apoptosis in PMN recruited in response to LPS and TNF-alpha. Inhibition of beta2 integrin function may not only inhibit PMN infiltration, but also accelerate PMN clearance from inflamed tissue.     

A crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair

Integrins are ubiquitous transmembrane receptors that play crucial roles in cell-cell and cell-matrix interactions. In this study, we have determined the effects of the loss of beta 1 integrins in keratinocytes in vitro and during cutaneous wound repair. Flow cytometry of cultured beta 1-deficient keratinocytes confirmed the absence of beta 1 integrins and showed downregulation of alpha 6 beta 4 but not of alpha v integrins. beta 1-null keratinocytes were characterised by poor adhesion to various substrates, by a reduced proliferation rate and by a strongly impaired migratory capacity. In vivo, the loss of beta 1 integrins in keratinocytes caused a severe defect in wound healing. beta 1-null keratinocytes showed impaired migration and were more densely packed in the hyperproliferative epithelium. Surprisingly, their proliferation rate was not reduced in early wounds and even increased in late wounds. The failure in re-epithelialisation resulted in a prolonged inflammatory response, leading to dramatic alterations in the expression of important wound-regulated genes. Ultimately, beta 1-deficient epidermis did cover the wound bed, but the epithelial architecture was abnormal. These findings demonstrate a crucial role of beta 1 integrins in keratinocyte migration and wound re-epithelialisation. Movies available on-line     

Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes from a patient with leukocyte adhesion deficiency-1: normal displacement in close quarters via chimneying

The beta2 integrins are known to be important in the motile function of leukocytes in general and in the adhesive response to inflammatory stimuli in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the locomotion of human blood PMN from a patient with Leukocyte Adhesion Deficiency-1 (LAD), a disorder in which beta2 integrins on the cell surface are markedly deficient in number or function. In thin slide preparations such that the leukocytes were somewhat compressed between slide and cover slip, PMNLAD exhibited normal random locomotion and chemotaxis, apparently by using the opposing surfaces to generate the force for locomotion (chimneying). In thicker preparations, an adherence deficit was evident, but chemotaxis still occurred, even by PMNLAD anticoagulated in EDTA. Consistent with the paucity of beta2 integrins on the surface of the PMNLAD was their failure to aggregate in the presence of antibodies to beta2 integrins, even when they had been brought together by chemotaxis. We relate these findings to the reported independence from integrins of PMN in the lung vasculature in LAD, as well as in certain experimental conditions.