CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia

Transepithelial migration of neutrophils (PMN) is a defining characteristic of active inflammatory states of mucosal surfaces. The process of PMN transepithelial migration, while dependent on the neutrophil beta 2 integrin CD11b/CD18, remains poorly understood. In these studies, we define a monoclonal antibody, C5/D5, raised against epithelial membrane preparations, which markedly inhibits PMN migration across polarized monolayers of the human intestinal epithelial cell line T84 in a bidirectional fashion. In T84 cells, the antigen defined by C5/D5 is upregulated by epithelial exposure to IFN-gamma, and represents a membrane glycoprotein of approximately 60 kD that is expressed on the basolateral membrane. While transepithelial migration of PMN was markedly inhibited by either C5/D5 IgG or C5/D5 Fab fragments, the antibody failed to inhibit both adhesion of PMN to T84 monolayers and adhesion of isolated T84 cells to the purified PMN integrin, CD11b/CD18. Thus, epithelial-PMN interactions blocked by C5/D5 appear to be downstream from initial CD11b/CD18-mediated adhesion of PMN to epithelial cells. Purification, microsequence analysis, and cross-blotting experiments indicate that the C5/D5 antigen represents CD47, a previously cloned integral membrane glycoprotein with homology to the immunoglobulin superfamily. Expression of the CD47 epitope was confirmed on PMN and was also localized to the basolateral membrane of normal human colonic epithelial cells. While C5/D5 IgG inhibited PMN migration even in the absence of epithelial, preincubation of T84 monolayers with C5/D5 IgG followed by antibody washout also resulted in inhibition of transmigration. These results suggest the presence of both neutrophil and epithelial components to CD47-mediated transepithelial migration. Thus, CD47 represents a potential new therapeutic target for downregulating active inflammatory disease of mucosal surfaces.     

Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion

Neutrophil, or polymorphonuclear leukocyte (PMN), migration across intestinal epithelial barriers, such as occurs in many disease states, appears to result in modifications of epithelial barrier and ion transport functions (Nash, S., J. Stafford, and J. L. Madara. 1987. J. Clin. Invest. 80:1104-1113; Madara, J. L., C. A. Parkos, S. P. Colgan, R. J. MacLeod, S. Nash, J. B. Matthews, C. Delp, and W. I. Lencer. 1992. J. Clin. Invest. 89:1938-1944). Here we investigate the effects of epithelial exposure to IFN-gamma on PMN migration across cultured monolayers of the human intestinal epithelial cell line T84. Transepithelial migration of PMN was initially assessed in the apical- to-basolateral direction, since previous studies indicate general qualitative similarities between PMN migration in the apical-to- basolateral and in the basolateral-to-apical directions. In the apical- to-basolateral direction, epithelial exposure to IFN-gamma markedly upregulated transepithelial migration of PMN in a dose- and time- dependent fashion as measured by both electrical and myeloperoxidase assays. This IFN-gamma-elicited effect on transmigration was specifically due to a IFN-gamma effect on epithelial cells and was not secondary to IFN-gamma effects on epithelial tight junction permeability. Moreover, this IFN-gamma effect was dependent on epithelial protein synthesis, and involved a pathway in which CD11b/18, but not ICAM-1 or CD11a/18, appeared to play a crucial role in PMN- epithelial adhesion. IFN-gamma also substantially modified PMN transepithelial migration in the natural, basolateral-to-apical direction. The IFN-gamma effect on naturally directed transmigration was also specifically due to an IFN-gamma effect on epithelial cells, showed comparable time and dose dependency to that of oppositely directed migration, was CD11b/18 dependent, and required epithelial protein synthesis. Additionally, however, important qualitative differences existed in how IFN-gamma affected transmigration in the two directions. In contrast to apical-to-basolateral directed migration, IFN-gamma markedly downregulated transepithelial migration of PMN in the natural direction. This downregulation of PMN migration in the natural direction, however, was not due to failure of PMN to move across filters and into monolayers. Indeed, IFN-gamma exposure to epithelia increased the number of PMN which had moved into the basolateral space of the epithelium in naturally directed transmigration. These results represent the first detailed report of influences on PMN transepithelial migration by a cytokine, define conditions under which a qualitative difference in PMN transepithelial migration exists, and suggest that migration of PMN across epithelia in the natural direction may involve multiple steps which can be differentially regulated by cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)     

JAM-C is a component of desmosomes and a ligand for CD11b/CD18-mediated neutrophil transepithelial migration

Neutrophil (PMN) transepithelial migration is dependent on the leukocyte beta(2) integrin CD11b/CD18, yet the identity of epithelial counterreceptors remain elusive. Recently, a JAM protein family member termed JAM-C was implicated in leukocyte adhesive interactions; however, its expression in epithelia and role in PMN-epithelial interactions are unknown. Here, we demonstrate that JAM-C is abundantly expressed basolaterally in intestinal epithelia and localizes to desmosomes but not tight junctions. Desmosomal localization of JAM-C was further confirmed by experiments aimed at selective disruption of tight junctions and desmosomes. In assays of PMN transepithelial migration, both JAM-C mAbs and JAM-C/Fc chimeras significantly inhibited the rate of PMN transmigration. Additional experiments revealed specific binding of JAM-C to CD11b/CD18 and provided evidence of other epithelial ligands for CD11b/CD18. These findings represent the first demonstration of direct adhesive interactions between PMN and epithelial intercellular junctions (desmosomes) that regulate PMN transepithelial migration and also suggest that JAM-C may play a role in desmosomal structure/function.     

Impairment of HIV polymorphonuclear leukocyte transmigration across T84 cell monolayers: an alternative mechanisms for increased intestinal bacterial infections in AIDS?

Our objective was to study the influence of HIV infection of polymorphonuclear leukocytes (PMN) on transepithelial migration. To date, reports of functional PMN chemotaxis in AIDS are contradictory. This is the first attempt to assess this function via an in vitro model allowing transmigration of neutrophils through an intestinal epithelial barrier. PMN were isolated from 45 HIV-infected patients and 45 healthy volunteers. PMN transmigration across T84 epithelial cells was initiated by applying either various concentrations of formyl-met-leu-phe peptide (f-MLP) or interleukin-8 and assayed by quantification of myeloperoxidase activity. CD11b, CD18, and CD47 expression on PMN was compared before and after transepithelial migration by flow cytometry analysis. CD11b expression was studied by electron microscopy. Apoptosis of transmigrated HIV PMN and control PMN was investigated by morphology and DNA fragmentation characterization. Compared to control PMN, HIV PMN exhibited a decrease in transepithelial migration that directly correlated with CD4+ counts. Basal and transepithelial migration-mediated expression of CD11b, CD18, and CD47 were unmodified in HIV PMN compared to control PMN. Electron microscopy labeling confirmed no difference in CD11b expression on HIV and control PMN. The index of apoptosis in transmigrated HIV PMN and control PMN was identical. These data provide evidence of a defect in the f-MLP-induced chemotaxis of PMN from HIV-infected patients across an intestinal epithelial barrier. This defective migration is not due to a quantitative modification of CD11b, CD18 and CD47 on HIV PMN suggesting a more subtle alteration. The impairment in the transmigration function may contribute in vivo to an increased susceptibility to intestinal bacterial infection in HIV-infected patients.     

Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils

In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S. typhimurium. We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport. However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued. In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration. Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN. Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells. Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8). However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration. These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium. Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium.     

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.     

The Heparan Sulfate Proteoglycan Form of Epithelial CD44v3 Serves as a CD11b/CD18 Counter-receptor during Polymorphonuclear Leukocyte Transepithelial Migration

Leukocyte β₂-integrin CD11b/CD18 mediates the firm adhesion and subsequent transepithelial migration of polymorphonuclear leukocytes, but the identity of its counter-receptor(s) on epithelia remains elusive. Here we identified a monoclonal antibody, clone C3H7, which strongly bound to the basolateral membranes of epithelial cells and inhibited both the adhesion of epithelial cells to immobilized CD11b/CD8 and the transepithelial migration of PMNs in a physiologically relevant basolateral-to-apical direction. C3H7 antigen expression in epithelial monolayers was significantly increased by treatment with proinflammatory cytokine interferon-γ or a combination of interferon-γ and tumor necrosis factor-α. Up-regulation of C3H7 antigen was also observed in the epithelium of inflamed human colon tissues. Microsequencing and Western blotting of the purified antigen showed it to be CD44 variant 3 (CD44v3), a ∼160-kDa membrane glycoprotein. Further studies demonstrated that this epithelial CD44v3 specifically binds to CD11b/CD18 through its heparan sulfate moieties. In summary, our study demonstrates for the first time that the heparan sulfate proteoglycan form of epithelial CD44v3 plays a critical role in facilitating PMN recruitment during inflammatory episodes via directly binding to CD11b/CD18.A major component of many inflammatory diseases is the migration of large numbers of neutrophils (polymorphonuclear leukocytes, PMNs)² across the epithelium and their accumulation within a lumen. Examples include inflammatory bowel disease (IBD), cholangitis, cholecystitis, bronchial pneumonia, bronchitis, pyelonephritis, and cystitis. Under these pathophysiological conditions, epithelial injury and disease symptoms parallel PMN infiltration of the mucosa (1, 2). The current paradigm for migration of PMN across epithelial monolayers envisions a process consisting of sequential molecularly defined events such as CD11b/CD18-mediated firm adhesion of PMN with epithelia (3) followed by CD47-SIRPα interactions at the post-adhesion stage (4). However, although PMN transepithelial migration (TEM) has been widely demonstrated to be CD11b/CD18-dependent, the epithelial counter-receptor(s) for CD11b/CD18 in mediating PMN-epithelia adhesion has not been identified.Function mapping studies using domain-specific antibodies have demonstrated that the inserted domain (I-domain), a stretch of 200 amino acids of the CD11b subunit, is a major binding domain for CD11b/CD18 ligands (5). The I-domain of CD11b is promiscuous in ligand binding and has many known receptors including ICAM-1 (6, 7), fibrinogen (8), collagen (9), Cyr61 (CCN1), and connective tissue growth factor (CCN2) (10), heparin/heparan sulfate (11, 12), elastase (13), iC3b (14), and platelet glycoprotein Ibα (15). However, none of these ligands appear to mediate the firm adhesion of PMNs to the basolateral surfaces of epithelial monolayers at early stages of transmigration. Thus far, no epithelial basolaterally expressed CD11b/CD18 counter-receptor has been identified. ICAM-1, the best characterized cellular ligand for CD11b/CD18, cannot be the intestinal epithelial CD11b/CD18 ligand that mediates PMN firm adhesion because: (a) ICAM-1 is normally not expressed on intestinal epithelia except under inflammatory conditions (16) and (b) when ICAM-1 expression is induced it is up-regulated on the apical rather than basolateral surface of intestine epithelia. In an effort to understand the mechanisms that govern CD11b/CD18-mediated PMN TEM, previous studies by us and others have found that epithelial surface-sulfated proteoglycans (17) and junction adhesion molecule C (JAM-C) play a significant role in regulating PMN transmigration via interaction with leukocyte CD11b/CD18 (18, 19). However, compared with functional inhibitory anti-CD11b antibodies that completely block PMN TEM, soluble carbohydrates or antibodies against JAM-C create only partial inhibition. These results clearly suggest the existence of unknown epithelial adhesion molecule(s) that bind to leukocyte CD11b/CD18 and regulate PMN TEM. Heparin and heparan sulfate have also been shown to block the adhesion and PMN TEM via binding to CD11b/CD18 (11, 12); thus it is reasonable to suggest that a basolateral membrane glycoprotein decorated with heparan sulfate moieties may serve as a counter-receptor for CD11b/CD18. However, the nature of this epithelial heparan sulfate proteoglycan has not been identified.Here we sought to identify novel epithelial adhesive ligand(s) important in PMN transmigration, in particular, a ligand that can bind to CD11b/CD18 on migrating PMNs and mediate the firm adhesion of PMNs to the epithelial basolateral surfaces. To do this, we screened a panel of monoclonal antibodies generated against epithelial plasma membranes. This screening identified one mAb, termed C3H7, that recognized a basolateral membrane protein and inhibited PMN TEM in a physiologically relevant basolateral-to-apical direction. Further study of these results identified the C3H7 antigen as a v3-type human epithelial CD44 variant, a ∼160-kDa glycoprotein that is decorated with heparan sulfate moieties. Subsequent studies revealed that the C3H7 antigen appears to function as a cellular ligand for CD11b/CD18 in regulating the firm adhesion of PMNs to the epithelium during their transmigration process.     

Salmonella typhimurium SipA-induced neutrophil transepithelial migration: involvement of a PKC-alpha-dependent signal transduction pathway

Salmonella typhimurium elicits an intense proinflammatory response characterized by movement of polymorphonuclear neutrophils (PMN) across the epithelial barrier to the intestinal lumen. We previously showed that S. typhimurium, via the type III secretion system effector protein SipA, initiates an ADP-ribosylation factor-6- and phospholipase D-dependent lipid-signaling cascade that directs activation of protein kinase C (PKC) and subsequent transepithelial movement of PMN. Here we sought to determine the specific PKC isoforms that are induced by the S. typhimurium effector SipA in model intestinal epithelia and to link the functional consequences of these isoforms in the promotion of PMN transepithelial migration. In vitro kinase PKC activation assays performed on polarized monolayers of T84 cells revealed that S. typhimurium and recombinant SipA induced activation of PKC-alpha, -delta, and -epsilon. To elucidate which of these isoforms play a key role in mediating epithelial cell responses that lead to the observed PMN transepithelial migration, we used a variety of PKC inhibitors with different isoform selectivity profiles. Inhibitors selective for PKC-alpha (G?-6976 and 2,2',3,3',4,4'-hexahydroxyl-1,1'-biphenyl-6,6'-dimethanoldimethyl ether) markedly reduced S. typhimurium- and recombinant SipA-induced PMN transepithelial migration, whereas inhibitors to PKC-delta (rottlerin) or PKC-epsilon (V1-2) failed to exhibit a significant decrease in transepithelial movement of PMN. These results were confirmed biochemically and by immunofluorescence coupled to confocal microscopy. Our results are the first to show that the S. typhimurium effector protein SipA can activate multiple PKC isoforms, but only PKC-alpha is involved in the signal transduction cascade leading to PMN transepithelial migration.     

Neutrophil migration across tight junctions is mediated by adhesive interactions between epithelial coxsackie and adenovirus receptor and a junctional adhesion molecule-like protein on neutrophils

Neutrophil (polymorphonuclear leukocytes [PMN]) transepithelial migration during inflammatory episodes involves a complex series of adhesive interactions and signaling events. Previous studies have shown that key adhesive interactions between leukocyte CD11b/CD18 and basally expressed fucosylated glycoproteins followed by binding to desmosomal-associated JAM-C are key elements of the transmigration response. Here we provide the first evidence that PMN-expressed junctional adhesion molecule-like protein (JAML) regulates transmigration via binding interactions with epithelial coxsackie and adenovirus receptor (CAR). Experiments with a JAML fusion protein revealed specific binding of JAML to epithelial CAR expressed at tight junctions in T84 cell monolayers and normal human colonic mucosa. Furthermore, JAML-CAR binding is mediated via the membrane distal immunoglobulin (Ig) loop of CAR and the membrane proximal Ig loop of JAML. PMN bound to immobilized CAR but not JAML in a divalent cation-independent manner. Lastly, in assays of PMN transepithelial migration, JAML/CAR fusion proteins and their antibodies significantly inhibited transmigration in a specific manner. Taken together, these results indicate that JAML and CAR are a novel pair of adhesion molecules that play an important role in modulating PMN migration cross epithelial tight junctions. These findings add a new element to a multistep model of PMN transepithelial migration and may provide new targets for anti-inflammatory therapies.     

Neutrophil migration across intestinal epithelium: evidence for a role of CD44 in regulating detachment of migrating cells from the luminal surface

The migration of polymorphonuclear leukocytes (PMNs) across the intestinal epithelium is a histopathological hallmark of many mucosal inflammatory diseases including inflammatory bowel disease. The terminal transmigration step is the detachment of PMNs from the apical surface of the epithelium and their subsequent release into the intestinal lumen. The current study sought to identify epithelial proteins involved in the regulation of PMN migration across intestinal epithelium at the stage at which PMNs reach the apical epithelial surface. A panel of Abs reactive with IFN-γ-stimulated T84 intestinal epithelial cells was generated. Screening efforts identified one mAb, GM35, that prevented PMN detachment from the apical epithelial surface. Microsequencing studies identified the GM35 Ag as human CD44. Transfection studies confirmed this result by demonstrating the loss of the functional activity of the GM35 mAb following attenuation of epithelial CD44 protein expression. Immunoblotting and immunofluorescence revealed the GM35 Ag to be an apically expressed v6 variant exon-containing form of human CD44 (CD44v6). ELISA analysis demonstrated the release of soluble CD44v6 by T84 cells during PMN transepithelial migration. In addition, the observed release of CD44v6 was blocked by GM35 treatment, supporting a connection between CD44v6 release and PMN detachment. Increased expression of CD44v6 and the GM35 Ag was detected in inflamed ulcerative colitis tissue. This study demonstrates that epithelial-expressed CD44v6 plays a role in PMN clearance during inflammatory episodes through regulation of the terminal detachment of PMNs from the apical epithelial surface into the lumen of the intestine.     

Enteroaggregative Escherichia coli promotes transepithelial migration of neutrophils through a conserved 12-lipoxygenase pathway

Enteroaggregative Escherichia coli (EAEC) induces release of pro-inflammatory markers and disruption of intestinal epithelial barriers in vitro, suggesting an inflammatory aspect to EAEC infection. However, the mechanisms underlying EAEC-induced mucosal inflammatory responses and the extent to which these events contribute to pathogenesis is not well characterized. Employing an established in vitro model we demonstrated that EAEC prototype strain 042 induces migration of polymorphonuclear neutrophils (PMNs) across polarized T84 cell monolayers. This event was mediated through a conserved host cell signalling cascade involving the 12/15-LOX pathway and led to apical secretion of an arachidonic acid-derived lipid PMN chemoattractant, guiding PMNs across the epithelia to the site of infection. Moreover, supporting the hypothesis that inflammatory responses may contribute to EAEC pathogenesis, we found that PMN transepithelial migration promoted enhanced attachment of EAEC 042 to T84 cells. These findings suggest that EAEC-induced PMN infiltration may favour colonization and thus pathogenesis of EAEC.     

Molecular events in neutrophil transepithelial migration

Neutrophil transepithelial migration is a central component of many inflammatory diseases of the gastrointestinal, respiratory and urinary tracts, and correlates with disease symptoms. In vitro modeling with polarized intestinal epithelial monolayers has shown that neutrophil transepithelial migration can influence crucial epithelial functions, ranging from barrier maintenance to electrolyte secretion. Studies have also demonstrated a dynamic involvement of the epithelium in modulating neutrophil transepithelial migration. Characterization of the molecular interactions between neutrophils and epithelial cells has revealed that transepithelial migration is dependent on the neutrophil β₂ integrin CD11b/CD18, and does not appear to involve adhesive interactions with the selectins or intercellular adhesion molecule-1. Recent studies have implicated another transmembrane glycoprotein, CD47, as a crucial component of the transepithelial migration response. While the precise function of CD47 is not known, current evidence suggests that CD47-dependent events occur after CD11b/CD18-mediated neutrophil adhesion to the epithelium. This review will highlight key features of the current understanding of the molecular events important in neutrophil migration across epithelial surfaces.     

Monocyte migration through the alveolar epithelial barrier: adhesion molecule mechanisms and impact of chemokines

Alveolar monocyte influx requires adherence and transmigration through the vascular endothelium, extracellular matrix, and alveolar epithelium. For investigating the monocyte migratory process across the epithelial barrier, we employed both the A549 cell line and isolated human alveolar epithelial cells. Under baseline conditions, spontaneous bidirectional transepithelial monocyte migration was noted, which was dose-dependently increased in the presence of the monocyte chemoattractant protein-1. TNF-alpha stimulation of the alveolar epithelium provoked the polarized apical secretion of monocyte chemoattractant protein-1 and RANTES and up-regulation of ICAM-1 and VCAM-1 expression, accompanied by markedly enhanced transepithelial monocyte traffic in the basal-to-apical direction. Multiple adhesive interactions were noted to contribute to the enhanced monocyte traffic across the TNF-alpha-stimulated alveolar epithelium: these included the beta 2 integrins CD11a, CD11b, CD11c/CD18, the beta 1 integrins very late Ag (VLA)-4, -5, and -6, and the integrin-associated protein CD47 on monocytes, as well as ICAM-1, VCAM-1, CD47, and matrix components on the epithelial side. In contrast, spontaneous monocyte migration through unstimulated epithelium depended predominantly on CD11b/CD18 and CD47, with some additional contribution of VLA-4, -5, and -6. In summary, unlike transendothelial monocyte traffic, for which beta 1 and beta 2 integrins are alternative mechanisms, monocyte migration across the alveolar epithelium largely depends on CD11b/CD18 and CD47 but required the additional engagement of the beta 1 integrins for optimal migration. In response to inflammatory challenge, the alveolar epithelium orchestrates enhanced monocyte traffic to the apical side by polarized chemokine secretion and up-regulation of ICAM-1 and VCAM-1.     

Negative regulation of epithelium-neutrophil interactions via activation of CD44

Polymorphonuclear neutrophil (PMN) migration across epithelia is a common feature of active inflammation. Given the suggested role of carbohydrates in this process, we examined the receptor CD44. The standard CD44 isoform was expressed at the cell surface of PMN. PMN migration across model polarized intestinal epithelia was reduced (by 60%) if the CD44 receptor was activated by either a specific antibody (clone IM7) or the natural soluble ligand, hyaluronic acid. This inhibitory effect following receptor activation occurred with both basolateral-to-apical- and apical-to-basolateral-directed migration. The anti-CD44 antibody similarly reduced PMN migration through filters in the absence of epithelia, while preincubation of the antibody with the epithelium did not alter subsequent PMN transepithelial migration. These data suggest that PMN, rather than epithelial, CD44 is responsible for these effects. A similar inhibitory effect of anti-CD44 antibody was also observed on migration of intraepithelial lymphocytes. The molecular mechanism involved in such negative signaling following CD44 activation may include modulation of outside-in cell signaling. While neither the anti-CD44 antibody nor CD44 ligand affected PMN mobilization of intracellular Ca(2+), both led to increased adenylate cyclase activity, an inhibitory signal for PMN migration. Together, these results suggest that CD44 of PMN may potentially serve as a negative regulator of leukocyte migration across biological surfaces such as columnar epithelia.     

Mechanisms of neutrophil transmigration across renal proximal tubular HK-2 cells.

BACKGROUND: Adhesion of intratubular leukocytes to proximal tubules in biopsies of patients with rapidly progressive glomerulonephritis and the appearance of leukocytes in the urine in interstitial nephritis suggest interactions between leukocytes and tubular epithelia in renal diseases. The aim of this study was to investigate the effect of cytokines and endotoxin on leukocyte migration through proximal tubular epithelial cells and also to determine the role of the transmembrane adhesion molecules ICAM-1 and CD47 in this process. METHODS: Experiments determined transepithelial migration (TEM) of PMN (polymorphonuclear) leukocytes through monolayers of HK-2. Expression of ICAM-1 and CD47 was assessed via confocal immunofluorescence, FACS analysis and western blotting. The effect of antibodies against ICAM-1 and CD47 on TEM was examined. Furthermore measurements of cytokine release (IL- 6 and IL-8) were performed. RESULTS: Preincubation of HK-2 cells with either TNFalpha or LPS resulted in stimulation of PMN migration through monolayers of HK-2 cells. There was no preferred direction of transmigration. ICAM-1 was expressed by HK-2 cells and expression was increased after 4 h stimulation with TNFalpha or LPS. Application of ICAM-1 antibodies inhibited TEM. CD47 was expressed in both HK-2 cells and PMN. CD47 antibodies inhibited predominantly basolateral-to-apical TEM. HK-2 cells released IL-8 and IL-6 preferably into the apical compartment. Additionally, we showed that fMLP induced transmigration through monolayers of HK-2 cells was associated with significant increased CD47 expression on PMN cell surfaces. CONCLUSIONS: Inflammatory mediators stimulate TEM of PMN through monolayers of HK-2 cells without a clearly discernible preference of direction. Mechanisms involved in TEM stimulated by cytokines or endotoxin appear to be mainly changes in surface receptor densities of HK-2 cells with ICAM-1 and CD47 playing an essential role.     

Receptor for advanced glycation endproducts mediates neutrophil migration across intestinal epithelium

Receptor for advanced glycation endproducts (RAGE) is an Ig superfamily cell surface receptor that interacts with a diverse array of ligands associated with inflammatory responses. In this study, we provide evidence demonstrating that RAGE is involved in inflammatory responses in the intestines. We showed that RAGE is expressed in intestinal epithelial cells, primarily concentrated at the lateral membranes close to the apical cell junction complexes. Although RAGE expression was low in epithelium under normal conditions, this protein was up-regulated after treatment with the inflammatory cytokines IFN-gamma and/or TNF-alpha. RAGE expression was also elevated in colon tissue samples from patients with inflammatory bowel diseases. Using in vitro transmigration assays, we found that RAGE mediates neutrophil (polymorphonuclear leukocytes (PMN)) adhesion to, and subsequent migration across, intestinal epithelial monolayers. This activity appears to be mediated by the binding of RAGE to the PMN-specific beta(2) integrin CD11b/CD18. Thus, these results provide a novel mechanism for the regulation of PMN transepithelial migration and may suggest a new therapeutic target for intestinal inflammation.     

Neutrophil transepithelial migration: evidence for sequential,contact-dependent signaling events and enhanced paracellular permeability independent of transjunctional migration

Active migration of polymorphonuclear leukocytes (PMN) through the intestinal crypt epithelium is a hallmark of inflammatory bowel disease and correlates with patient symptoms. Previous in vitro studies have shown that PMN transepithelial migration results in increased epithelial permeability. In this study, we modeled PMN transepithelial migration across T84 monolayers and demonstrated that enhanced paracellular permeability to small solutes occurred in the absence of transepithelial migration but required both PMN contact with the epithelial cell basolateral membrane and a transepithelial chemotactic gradient. Early events that occurred before PMN entering the paracellular space included increased permeability to small solutes (<500 Da), enhanced phosphorylation of regulatory myosin L chain, and other as yet undefined proteins at the level of the tight junction. No redistribution or loss of tight junction proteins was detected in these monolayers. Late events, occurring during actual PMN transepithelial migration, included redistribution of epithelial serine-phosphorylated proteins from the cytoplasm to the nucleus in cells adjacent to migrating PMN. Changes in phosphorylation of multiple proteins were observed in whole cell lysates prepared from PMN-stimulated epithelial cells. We propose that regulation of PMN transepithelial migration is mediated, in part, by sequential signaling events between migrating PMN and the epithelium.     

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

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

Human neutrophils facilitate tumor cell transendothelial migration

Tumor cell extravasation plays a key role in tumor metastasis.However, the precise mechanisms by which tumor cells migrate throughnormal vascular endothelium remain unclear. In this study, using an invitro transendothelial migration model, we show that humanpolymorphonuclear neutrophils (PMN) assist the human breast tumor cellline MDA-MB-231 to cross the endothelial barrier. We found thattumor-conditioned medium (TCM) downregulated PMN cytocidal function,delayed PMN apoptosis, and concomitantly upregulated PMNadhesion molecule expression. These PMN treated with TCM attached totumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did nottransmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) butdid not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 onPMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells stillpossessed the ability to proliferate after PMN-assisted transmigration.These results indicate that TCM-treated PMN may serve as a carrier toassist tumor cell transendothelial migration and suggest that tumorcells can exploit PMN and alter their function to facilitate their extravasation.