Regulation of CD43-induced U937 homotypic aggregation

CD43 (leukosialin, sialophorin), a prominent component of the hemopoietic cell surface, has an enigmatic role in cell-cell interaction. The observation that CD43 ligation triggers homotypic aggregation of monoblastoid U937 cells has permitted analysis of this: CD43-induced aggregation was distinguishable from CD29- (also known as beta1 integrin) or CD98- (also known as 4F2, or fusion-related protein 1) induced aggregation, with different energy requirements and with partial dependence on beta2 integrins. Previous studies have focused on the role of CD43 ligation in tyrosine phosphorylation. However, in the homotypic adhesion assay, although there is initial tyrosine phosphorylation, protein tyrosine kinase inhibitors did not block aggregation. Therefore, other signaling pathways were examined. CD43 ligation induced protein tyrosine dephosphorylation, and protein tyrosine phosphatase inhibitors blocked aggregation. Activation of MAP kinases was not necessary. Cytoskeletal inhibitors amplified aggregation. Protein kinase C (PKC) inhibitors amplified aggregation, implicating PKC as a negative regulator. CD43 ligation up-regulated surface adhesion molecules and enhanced CD29- and CD98-induced aggregation. Thus, CD43 participation in cell-cell adhesion is under stringent control, involving both surface events and several different intracellular signaling pathways, acting together to regulate the process. These mechanisms add a further dimension to the potential role of CD43 in tissue immune responses.     

Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells

We investigated the functional roles of ceramide, an intracellular lipid mediator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP) in HeLa living cells. C(2)-ceramide but not C(2)-dihydroceramide induced translocation of delta PKC-GFP to the Golgi complex, while alpha PKC- and zeta PKC-GFP did not respond to ceramide. The Golgi-associated delta PKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where delta PKC was translocated by ceramide. Gamma interferon also induced the delta PKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg(2+)-dependent neutral sphingomyelinase. Photobleaching studies showed that ceramide does not evoke tight binding of delta PKC-GFP to the Golgi complex but induces the continuous association and dissociation of delta PKC with the Golgi complex. Ceramide inhibited the kinase activity of delta PKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of delta PKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated delta PKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of delta PKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg(2+)-dependent sphingomyelinase induced delta PKC-specific translocation to the Golgi complex and that translocation results in delta PKC activation through tyrosine phosphorylation of the enzyme.     

The cell surface glycoprotein Mac-1 (CD11b/CD18) mediates neutrophil adhesion and modulates degranulation independently of its quantitative cell surface expression

It has previously been shown that during degranulation Mac-1 (CD11b/CD18)--a glycoprotein that plays a central role in neutrophil adhesion-is up-regulated on PMN surfaces. It has been assumed that this quantitative change in adhesion Ag expression on the cell surface would in turn lead to increased cellular adhesiveness. In contrast, we found that at an incubation temperature of 16 degrees C, stimulated neutrophil adhesion to plastic tissue culture dishes in the presence of FMLP (2.5 x 10(-6) M), TNF (10 ng/ml), or PAF (1 x 10(-4) M) occurred without cellular degranulation or Mac-1 surface up-regulation as measured cytofluorometrically. As shown by functional inhibition studies employing monoclonal antibodies 60.3 (anti-CD18) and 60.1 (anti-CD11b), adhesion at 16 degrees C, where no CD11b/CD18 up-regulation was seen, is mediated by CD11b/CD18 just as it is at 37 degrees C, where degranulation and CD11b/CD18 up-regulation could be demonstrated. The physiologic importance of these findings was underscored by experiments done on endothelial monolayers, which showed that PMN association with endothelial cells is absolutely independent from the quantitative up-regulation of Mac-1 on PMN surfaces. When neutrophils were stimulated at 37 degrees C by endotoxin, an agent that does not induce aggregation (a form of intercellular adhesion), Mac-1 surface expression increased only after cells had become adherent, whereas cells held in suspension to prevent cell-substrate adhesion neither degranulated nor up-regulated their Mac-1 surface expression. Thus, not only is adherence independent of degranulation and Mac-1 cell surface up-regulation, but both degranulation and Mac-1 surface up-regulation appear to depend on the process of adhesion. Correspondingly, incubation of neutrophils with antibodies 60.1 and 60.3 inhibited not only adhesion of cells stimulated with FMLP at 37 degrees C but degranulation as well. These results indicate that Mac-1 influences degranulation as well as it controls adhesion not by its mere quantity on the cell surface, but rather by an yet undefined molecular modulation.     

Ceramide inhibits development and cytokinesis and induces apoptosis in preimplantation bovine embryos

Ceramide is a second messenger induced by various cellular insults that plays a regulatory role in apoptosis. The objective of the present study was to determine whether ceramide signaling can occur in the preimplantation embryo by testing (1) effects of ceramide on development, cytokinesis, and apoptosis and (2) whether heat shock, which can induce apoptosis in embryos, causes activation of neutral or acidic sphingomyelinases responsible for generation of ceramide. Treatment of embryos > or =16 cells collected at Day 5 after insemination with 50 microM C(2)-ceramide increased caspase-9 activity and the proportion of blastomeres undergoing apoptosis but did not increase caspase-8 activity. Induction of apoptosis was more extensive when culture with ceramide was for 24 hr than for 9 hr. Ceramide also reduced the proportion of embryos that developed to the blastocyst stage when exposure was for 24 hr. At the two-cell stage, a period in development when apoptosis responses are blocked, culture of embryos with ceramide did not increase caspase-9 activity or the proportion of blastomeres that were apoptotic. However, culture with ceramide for 24 hr reduced cell proliferation and caused an increase in multinucleated cells because of inhibition of cytokinesis. Exposure of Day 5 embryos to a heat shock of 41 degrees C for 15 hr increased neutral sphingomyelinase activity but did not change acid sphingomyelinase activity. In conclusion, ceramide can regulate embryo development and apoptosis in a time and stage-of-development dependent manner and ceramide generation can be activated by cellular insult. Thus, the ceramide signaling pathway is present in the preimplantation embryo.     

Two-dimensional kinetics of beta 2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta 2-integrin (lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta 2-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta 2-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.     

Transforming growth factor-beta switches the pattern of integrins expressed in MG-63 human osteosarcoma cells and causes a selective loss of cell adhesion to laminin

Transforming growth factor-beta (TGF-beta) induces a marked decrease in adhesion of MG-63 human osteosarcoma cells to laminin-coated surfaces, but does not significantly alter adhesion to fibronectin- or collagen-coated surfaces. We provide evidence that this effect is due to a switch in the repertoire of cell adhesion receptors in response to TGF-beta. MG-63 cells express high levels of alpha 3 beta 1-integrin, which is a polyspecific laminin/collagen/fibronectin receptor, and low levels of alpha 2 beta 1- and alpha 5 beta 1-integrins, which are collagen and fibronectin receptors, respectively. No other integrins of the beta 1-class could be detected in MG-63 cells. Treatment with TGF-beta 1 induces a marked (approximately 60%) decrease in the level of expression of alpha 3-integrin subunit mRNA and protein and a concomitant 8-fold increase in alpha 2-subunit expression. These responses become maximal 7-12 h after addition of TGF-beta 1 to the cells. Expression of alpha 5- and beta 1-integrin subunits also increases in response to TGF-beta 1, but to a lesser extent than alpha 2-subunit expression. Thus, as a result of TGF-beta action, the alpha 2 beta 1-collagen and alpha 5 beta 1-fibronectin receptors replace the alpha 3 beta 1-laminin/collagen/fibronectin receptor as the predominant integrins of the beta 1-class in MG-63 cells. These results suggest that one of the effects of TGF-beta is to modify the adhesive behavior of certain tumor cells by changing the binding specificity of the complement of integrins that they express.     

Cell adhesion and migration properties of beta 2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation

Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.     

Dynamics of neutrophil aggregation in couette flow revealed by videomicroscopy: effect of shear rate on two-body collision efficiency and doublet lifetime

During inflammation, neutrophil capture by vascular endothelial cells is dependent on L-selectin and beta(2)-integrin adhesion receptors. One of us (S.I.S.) previously demonstrated that homotypic neutrophil aggregation is analogous to this process in that it is also mediated by these receptors, thus providing a model for studying the dynamics of neutrophil adhesion. In the present work, we set out to confirm the hypothesis that cell-cell adhesion via selectins serves to increase the lifetimes of neutrophil doublets formed through shear-induced two-body collisions. In turn, this would facilitate the engagement of more stable beta(2)-integrin bonds and thus increase the two-body collision efficiency (fraction of collisions resulting in the formation of nonseparating doublets). To this end, suspensions of unstimulated neutrophils were subjected to a uniform shear field in a transparent counter-rotating cone and plate rheoscope, and the formation of doublets and growth of aggregates recorded using high-speed videomicroscopy. The dependence of neutrophil doublet lifetime and two-body collision-capture efficiency on shear rate, G, from 14 to 220 s(-1) was investigated. Bond formation during a two-body collision was indicated by doublets rotating well past the orientation predicted for break-up of doublets of inert spheres. A striking dependence of doublet lifetime on shear rate was observed. At low shear (G = 14 s(-1)), no collision capture occurred, and doublet lifetimes were no different from those of neutrophils pretreated with a blocking antibody to L-selectin, or in Ca(++)-depleted EDTA buffers. At G > or = 66 s(-1), doublet lifetimes increased, with increasing G reaching values twice those for the L-selectin-blocked controls. This correlated with capture efficiencies in excess of 20%, and, at G > or = 110 s(-1), led to the rapid formation of large aggregates, and this in the absence of exogenous chemotactic stimuli. Moreover, the aggregates almost completely broke up when the shear rate was reduced below 66 s(-1). Partial inhibition of aggregate formation was achieved by blocking beta(2)-integrin receptors with antibody. By direct observation of the shear-induced interactions between neutrophils, these data reveal that steady application of a threshold level of shear rate is sufficient to support homotypic neutrophil aggregation.     

Src-family kinases mediate an outside-in signal necessary for beta2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin-IgG fusion protein, by a mechanism that involved beta2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(beta2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an 'outside-in' signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented beta2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin-IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck-/-fgr-/- and hck-/-fgr-/-lyn-/- mice showed significant impairment of beta2-integrin-mediated adhesion to CHO-E. Moreover, the expression of beta2 integrin activation epitopes at the sites of cell-cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a beta2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for beta2 integrin function in PMN tethered by E-selectin.     

Sialidase treatment exposes the beta1-integrin active ligand binding site on HL60 cells and increases binding to fibronectin

The migration of neutrophils from the circulation to areas of inflammation is the result of the sequential activation of multiple cellular adhesion molecules. beta1-Integrins are cell surface glycoproteins and the class of adhesion molecules responsible for binding to the extracellular matrix. The goal of this study was to determine the contribution of glycosylation, specifically the presence of sialic acid, to beta1-integrin adhesion in a neutrophil model. beta1-Integrins on differentiated HL60 cells were remodeled by treatment with the exoglycosidases, sialidase and beta-galactosidase. beta1-Integrin activity was determined by measuring adherence to the extracellular matrix protein fibronectin. The expression of beta1-integrins, beta2-integrins and activated beta1-integrins was determined by flow cytometry. Remodeling of beta1-integrins by treatment with sialidase increased adhesion by greater than 1,000%. Flow cytometric analysis of remodeled beta1-integrins demonstrated an increased expression of the activated beta1-integrin, but only minor increases in the expression of total beta1- and beta2-integrins. We postulate that glycosidase treatment increases adhesion and expression of activated beta1-integrins by exposure of the normally hidden ligand-binding site. The glycosylation of beta1-integrins on neutrophils may act to hide the ligand-binding site in unstimulated cells thereby contributing to the affinity modulation observed in neutrophil beta1-integrin function.     

Molecular dynamics of the transition from L-selectin- to beta 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.

Homotypic adhesion o2 neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes depend on L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand hydrodynamic stresses. Using cone-plate viscometry to apply a uniform linear shear field to suspensions of neutrophils, we conducted a detailed examination of the effect of shear rate and shear stress on the kinetics of cell aggregation. A collisional analysis based on Smoluchowski's flocculation theory was employed to fit the kinetics of aggregation with an adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency with shear was dependent on L-selectin, and peak efficiency was maintained over a relatively narrow range of shear rates (400-800 s-1) and shear stresses (4-7 dyn/cm2). When L-selectin was blocked with antibody, beta 2-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s-1). The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to stable adhesion.     

Ceramide kinase is a mediator of calcium-dependent degranulation in mast cells

Ceramide kinase (CERK) catalyzes the conversion of ceramide to ceramide 1-phosphate (C1P) and is known to be activated by calcium. Although several groups have examined the functions of CERK and its product C1P, the functions of C1P and CERK are not understood. We studied the RBL-2H3 cell line, a widely used model for mast cells, and found that CERK and C1P are required for activation of the degranulation process in mast cells. We found that C1P formation was enhanced during activation induced by IgE/antigen or by Ca(2+) ionophore A23187. The formation of C1P required the intracellular elevation of Ca(2+). We generated RBL-2H3 cells that stably express CERK, and when these cells were treated with A23187, a concomitant C1P formation was observed and degranulation increased 4-fold, compared with mock transfectants. The cell-permeable N-acetylsphingosine (C(2)-ceramide), a poor substrate of CERK, inhibited both the formation of C1P and degranulation, indicating that C1P formation was necessary for degranulation. Exogenous introduction of CERK into permeabilized RBL-2H3 cells caused degranulation. We identified a cytosolic localization of CERK that provides exposure to cytosolic Ca(2+). Taken together, these results indicate that C1P formation is a necessary step in the degranulation pathway in RBL-2H3 cells.     

Immune complex-stimulated neutrophil LTB4 production is dependent on beta 2 integrins

The beta 2 integrins (LFA-1, Mac-1, and p150,95) are critical for many adhesive functions of leukocytes. Although the binding of the IgG- opsonized particles occurs normally in the absence of beta 2 integrins, phagocytosis of IgG-opsonized particles by activated neutrophils (PMN) requires these integrins. This observation suggests a role for beta 2 integrins in phagocytosis subsequent to particle binding. To investigate the mechanism of involvement of beta 2 integrins in IgG- mediated functions, we examined the role of beta 2 integrins in adhesion to immune complex (IC)-coated surfaces. Initial adhesion and spreading on IC-coated surfaces were equivalent in control and beta 2- deficient phagocytes. However, both genetically beta 2-deficient PMN and PMN treated with the anti-beta 2 mAb IB4 subsequently detached from the IC-coated surfaces. To determine whether biochemical consequences of IgG activation were also affected by beta 2 deficiency, LTB4 production in response to Fc receptor ligation was assessed. LTB4 production by beta 2-deficient PMN adherent to IC-coated surfaces was markedly decreased in comparison with control PMN. Importantly, LTB4 production by PMN stimulated with fluid phase heat-aggregated IgG also required the beta 2 integrins, showing that the defect was not a simple consequence of abnormal adhesion. In contrast, superoxide production by IC-adherent PMN was equivalent in control and beta 2-deficient PMN. The initial rises in intracytoplasmic [Ca2+]i in response to aggregated IgG also were unaffected by inhibition of beta 2 integrins. These data show that lack of beta 2 integrins does not inhibit all FcR-dependent signal transduction. Finally, LTB4 production by normal PMN adherent to ICs was inhibited by antibodies to FcRII, but not FcRIII, showing that FcRII ligation was required for this effect. Together these data identify a role for the beta 2 integrins in a signal transduction pathway leading to sustained adhesion and LTB4 production in response to IC. Since both beta 2 integrins and FcRII are required for these effects, the data further suggest cooperation between these receptors in generating PMN activation in response to IC stimulation.     

Divergence in regulation of nitric-oxide synthase and its cofactor tetrahydrobiopterin by tumor necrosis factor-alpha. Ceramide potentiates nitric oxide synthesis without affecting GTP cyclohydrolase I activity

Synthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH(4)), a required cofactor for inducible nitric-oxide synthase (iNOS) activity, is usually coordinately regulated with iNOS expression. In C6 glioma cells, tumor necrosis factor-alpha (TNF-alpha) concomitantly potentiated the stimulation of nitric oxide (NO) and BH(4) production induced by IFN-gamma and interleukin-1beta. Expression of both iNOS and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the BH(4) biosynthetic pathway, was also markedly increased, as were their activities and protein levels. Ceramide, a sphingolipid metabolite, may mediate some of the actions of TNF-alpha. Indeed, we found that bacterial sphingomyelinase, which hydrolyzes sphingomyelin and increases endogenous ceramide, or the cell permeable ceramide analogue, C(2)-ceramide, but not C(2)-dihydroceramide (N-acetylsphinganine), significantly mimicked the effects of TNF-alpha on NO production and iNOS expression and activity in C6 cells. Surprisingly, although TNF-alpha increased BH(4) synthesis and GTPCH activity, neither BH(4) nor GTPCH expression was affected by C(2)-ceramide or sphingomyelinase in IFN-gamma- and interleukin-1beta-stimulated cells. It is likely that increased BH(4) levels results from increased GTPCH protein and activity in vivo rather than from reduced turnover of BH(4), because the GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, blocked cytokine-stimulated BH(4) accumulation. Moreover, expression of the GTPCH feedback regulatory protein, which if decreased might increase GTPCH activity, was not affected by TNF-alpha or ceramide. Treatment with the antioxidant pyrrolidine dithiocarbamate, which is known to inhibit NF-kappaB and sphingomyelinase in C6 cells, or with the peptide SN-50, which blocks translocation of NF-kappaB to the nucleus, inhibited TNF-alpha-dependent iNOS mRNA expression without affecting GTPCH mRNA levels. This is the first demonstration that cytokine-stimulated iNOS and GTPCH expression, and therefore NO and BH(4) biosynthesis, may be regulated by discrete pathways. As BH(4) is also a cofactor for the aromatic amino acid hydroxylases, discovery of distinct mechanisms for regulation of BH(4) and NO has important implications for its specific functions.     

Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts

CD31 is a member of the immunoglobulin superfamily consisting of six Ig- related domains. It is constitutively expressed by platelets, monocytes, and some lymphocytes, but at tenfold higher levels on vascular endothelial cells. CD31 has both homotypic and heterotypic adhesive properties. We have mapped the homotypic binding sites using a deletion series of CD31-Fc chimeras and a panel of anti-CD31 monoclonal antibodies. An extensive surface of CD31 is involved in homotypic binding with domains 2 and 3 and domains 5 and 6 playing key roles. A model consistent with the experimental data is that CD31 on one cell binds to CD31 on an apposing cell in an antiparallel interdigitating mode requiring full alignment of the six domains of each molecule. In addition to establishing intercellular homotypic contacts. CD31 binding leads to augmented adhesion via beta 1 integrins. The positive cooperation between CD31 and beta 1 integrins can occur in heterologous primate cells (COS cells). The interaction is specific to both CD31 and beta 1 integrins. Neither intercellular adhesion molecule-1 (ICAM- 1)/leukocyte function-associated antigen-1 (LCAM-1) nor neural cell adhesion molecule (NCAM)/NCAM adhesion leads to recruitment of beta 1 integrin adhesion pathways. Establishment of CD31 contacts have effects on the growth and morphology of endothelial cells. CD31(D1-D6)Fc inhibits the growth of endothelial cells in culture. In addition, papain fragments of anti-CD31 antibodies (Fab fragments) disrupt interendothelial contact formation and monolayer integrity when intercellular contacts are being formed. The same reagents are without effect once these contacts have been established, suggesting that CD31- CD31 interactions are critically important only in the initial phases of intercellular adhesion.     

Anti-CD9 monoclonal antibodies induce homotypic adhesion of pre-B cell lines by a novel mechanism

Anti-CD9 mAb are known agonists of platelet aggregation, but have not been implicated in cell-cell adhesion. We show here in an experimental system that the anti-CD9 mAb 50H.19, ALB6, and BA-2 can induce rapid, and irreversible, homotypic aggregation of the CD9-positive pre-B lymphoblastoid cell lines NALM-6 and HOON, but not of the CD9-negative B cell line Raji. The specificity of the response is indicated by the failure to effect aggregation with mAb directed to CD24, or to HLA class I Ag. The initiation of strong homotypic aggregates of lymphoid cells is a property ascribed to lymphocyte function-associated Ag-1 (LFA-1), a member of the beta 2 subfamily of leukocyte integrins. We show that CD9-induced aggregation is an active process which proceeds at 37 degrees C, but not at 4 degrees C, requires the expenditure of metabolic energy, and a functioning cytoskeleton, and is not inhibited by Arg-Gly-Asp-Ser peptide. These are properties described for LFA-1-mediated aggregation. However, because beta 2-integrins are not expressed on NALM-6 or HOON cells, they are not the mediators of CD9-induced aggregation. In contrast to LFA-1-mediated adhesion which is Mg2+ dependent, CD9-induced adhesion has an absolute requirement for Ca2+, but not Mg2+, indicating that a Ca2(+)-dependent event is sufficient for adhesion. However, Mg2+ enhances adhesion even at optimal concentrations of Ca2+, implicating an additional Mg2(+)-dependent event which requires Ca2+ to be effective. These findings suggest that CD9 Ag regulates a novel mechanism for promoting tight cell-cell adhesion which requires both Ca2+ and Mg2+ for optimal expression.     

Involvement of beta 2-integrin in ROS-mediated neutrophil apoptosis induced by Entamoeba histolytica

Cellular adhesion through beta 2-integrin (CD18) is an important step in signal transduction leading to apoptosis of human neutrophils, and NADPH oxidase-derived reactive oxygen species (ROS) are essential for neutrophil apoptosis induced by Entamoeba histolytica. Therefore, we investigated the role of beta 2-integrin-mediated signals in ROS-dependent neutrophil apoptosis induced by E. histolytica. Entamoeba-induced apoptosis was inhibited by pre-incubation of cells with mAb to CD18, but not CD29, suggesting that beta )-integrin plays an important role in this response. Moreover, Entamoeba-induced ROS generation in neutrophils was inhibited by mAbs against CD18 or CD11b, but not by mAbs against CD11a, CD11c, or CD29. A combination of d-galactose plus anti-CD18 mAb had a larger inhibitory effect than d-galactose alone on Entamoeba-induced apoptosis and ROS generation. Furthermore, Entamoeba-induced apoptosis and ROS generation were inhibited by pre-treatment of cells with an inhibitor of phosphatidylinositol-3-kinase (PI-3-kinase). These results indicate that beta 2-integrin and PI-3-kinase are crucial signaling molecules in ROS-dependent apoptosis of neutrophils induced by E. histolytica.     

Adhesion of neutrophils to fibronectin: role of the cd66 antigens

Adhesion of neutrophils to substrate is initiated by receptor-ligand interactions that induce outside-in signaling. Inside-out signals and lateral interactions between surface molecules further fine tune the response. This study investigates the role of CD66 in adhesion of neutrophils to fibronectin, using domain-mapped monoclonal antibodies to CD66. Neutrophils express CD66a, CD66b, and CD66c on their surface. The neutrophil surface molecules that bind to fibronectin are the alpha(4)beta(1) and alpha(5)beta(1) integrins. Our results show that the monoclonal antibody Kat4c, which recognizes the AB domain of CD66a, b, and c and the polyclonal anti-CD66 (anti-carcinoembryonic antigen), augments neutrophil adhesion to fibronectin, while monoclonal antibodies to the individual CD66 antigens, the Fab fragment of Kat4c, and a mixture of the individual antibodies to CD66 antigens were unable to affect the adhesion. Thus heterodimerization of CD66a, b, and c is required for promoting neutrophil adhesion to fibronectin. The increased adhesion in presence of Kat4c was inhibited by antibodies to the beta(1) and beta(2) integrins. Antibody ligation of CD66 antigens causes their clustering and concomitant coclustering of the alpha(M) subunit of the beta(2) integrin, thereby activating the integrin. The sugar alpha-methyl mannoside inhibited anti-CD66-mediated clustering, indicating that a carbohydrate-lectin interaction may exist between CD66 and alpha(M) integrin. It also reduced the increased adhesion of neutrophils to fibronectin, suggesting that beta(2) integrin activation precedes beta(1) integrin activation. Further, the anti-CD66-mediated adhesion to fibronectin is accompanied by increased localization of Src family kinases (lyn and hck) to the cytoskeleton and an increase in their kinase activity. These results suggest that crosslinking of CD66a, CD66b, and CD66c promotes activation of the beta(2) integrin and in turn an alteration in the affinity of the beta(1) integrin, which enhances the adhesion of neutrophils to fibronectin.