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.     

Urokinase receptor is necessary for adequate host defense against pneumococcal pneumonia

Cell recruitment is a multistep process regulated by cytokines, chemokines, and growth factors. Previous work has indicated that the urokinase plasminogen activator receptor (uPAR) may also play a role in this mechanism, presumably by an interaction with the beta(2) integrin CD11b/CD18. Indeed, an essential role of uPAR in neutrophil recruitment during pulmonary infection has been demonstrated for beta(2) integrin-dependent respiratory pathogens. We investigated the role of uPAR and urokinase plasminogen activator (uPA) during pneumonia caused by a beta(2) integrin-independent respiratory pathogen, Streptococcus pneumoniae. uPAR-deficient (uPAR(-/-)), uPA-deficient (uPA(-/-)), and wild-type (Wt) mice were intranasally inoculated with 10(5) CFU S. pneumoniae. uPAR(-/-) mice showed reduced granulocyte accumulation in alveoli and lungs when compared with Wt mice, which was associated with more S. pneumoniae CFU in lungs, enhanced dissemination of the infection, and a reduced survival. In contrast, uPA(-/-) mice showed enhanced host defense, with more neutrophil influx and less pneumococci in the lungs compared with Wt mice. These data suggest that uPAR is necessary for adequate recruitment of neutrophils into the alveoli and lungs during pneumonia caused by S. pneumoniae, a pathogen eliciting a beta(2) integrin-independent inflammatory response. This function is even more pronounced when uPAR is unoccupied by uPA.     

Migration of CD18-deficient neutrophils in vitro: evidence for a CD18-independent pathway induced by IL-8

Neutrophils isolated from a child with severe leukocyte adhesion deficiency 1 (LAD1) had a complete absence of expression of the CD11/CD18 beta2 integrin family of adhesion molecules, and were shown to be deficient in the in vitro adhesion and migration properties. However, we found that interleukin-8 (IL8), a potent chemoattractant for neutrophils, and sputum sol phase induced these LAD1 neutrophils to migrate through an endothelial cell layer in vitro, and confirmed that this migration was CD18-independent. These findings add to evidence of CD18-independent mechanisms of neutrophil recruitment, in particular neutrophil infiltration into the lungs, where IL8 may be an important recruitment factor.     

Salmeterol, a beta2-receptor agonist, attenuates lipopolysaccharide-induced lung inflammation in mice

Lipopolysaccharide is ubiquitously present in the environment. To determine the effect of salmeterol, a long-acting beta(2)-receptor agonist, on lipopolysaccharide-induced lung inflammation, mice received lipopolysaccharide (10 microg) intranasally with or without salmeterol intraperitoneally (5 mg/kg) 30 min earlier and 12 h thereafter. Salmeterol dose- and time-dependently inhibited the lipopolysaccharide-induced influx of neutrophils into bronchoalveolar lavage fluid and lung tissue, and these pulmonary neutrophils displayed a reduced expression of CD11b at their surface. To determine the contribution of the salmeterol effect on neutrophil CD11b in the attenuated neutrophil recruitment, we treated mice intranasally exposed to lipopolysaccharide with salmeterol with or without a blocking anti-CD11b antibody. Anti-CD11b profoundly reduced lipopolysaccharide-induced neutrophil influx in bronchoalveolar lavage fluid, an effect that was modestly enhanced by concurrent salmeterol treatment. These data suggest that salmeterol inhibits lipopolysaccharide-induced neutrophil recruitment to the lungs by a mechanism that possibly in part is mediated by an effect on neutrophil CD11b.     

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.     

Alpha(v)beta(6) integrin-A marker for the malignant potential of epithelial ovarian cancer.

The mechanism(s) responsible for the progression of non-metastatic or borderline ovarian cancer to invasive Grade I/III ovarian cancer is still unknown. An epithelium-restricted integrin, alpha(v)beta(6), is present in malignant epithelia but not in normal epithelia. We studied the relative expression and distribution of alpha(v)beta(6) integrin in early and late-stage invasive (Grade I and Grade III) and non-invasive (benign and borderline) ovarian tumors of serous, mucinous, endometrioid, and clear-cell carcinoma subtypes, to assess its potential as a marker for epithelial ovarian cancer progression. Sixty-six specimens, including eight normal, 13 benign, 14 borderline, 13 Grade I, and 18 Grade III tumors were evaluated by immunohistochemistry (IHC) using a monoclonal antibody (MAb) against alpha(v)beta(6) integrin. Normal ovarian surface epithelium was negative for alpha(v)beta(6) integrin expression. All 45 carcinomas studied were positive, and the staining intensity significantly correlated with the grade of the tumor. The Grade III carcinomas of all types showed strong staining intensity. Only mucinous benign tissues were positive, and no reactivity was observed in benign serous neoplasms. On the basis of these observations, we hypothesize that the expression of alpha(v)beta(6) integrin is associated with epithelial ovarian cancer and that a gradual increase in the expression of the molecule may be a correlative index of the progression of this disease.     

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.     

Transgenic overexpression of beta(2)-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.     

Cyclic exposure to ozone alters distal airway development in infant rhesus monkeys

Inner city children exposed to high levels of ozone suffer from an increased prevalence of respiratory diseases. Lung development in children is a long-term process, and there is a significant period of time during development when children growing up in urban areas are exposed to oxidant air pollution. This study was designed to test whether repeating cycles of injury and repair caused by episodes of ozone exposure lead to chronic airway disease and decreased lung function by altering normal lung maturation. We evaluated postnatal lung morphogenesis and function of infant monkeys after 5 mo of episodic exposure of 0.5 parts per million ozone beginning at 1 mo of age. Nonhuman primates were chosen because their airway structure and postnatal lung development is similar to those of humans. Airway morphology and structure were evaluated at the end of the 5-mo exposure period. Compared with control infants, ozone-exposed animals had four fewer nonalveolarized airway generations, hyperplastic bronchiolar epithelium, and altered smooth muscle bundle orientation in terminal and respiratory bronchioles. These results suggest that episodic exposure to environmental ozone compromises postnatal morphogenesis of tracheobronchial airways.     

Inhibition of CD18 or CD11b attenuates acute lung injury after acid instillation in rabbits

Folkesson, Hans G., and Michael A. Matthay. Inhibitionof CD18 or CD11b attenuates acute lung injury after acid instillation in rabbits. J. Appl. Physiol. 82(6):1743-1750, 1997.Acid-induced lung injury is mediatedprimarily by activated neutrophils. Although a prior study demonstratedthat acid-induced neutrophil influx into the air spaces was not CD18dependent, we hypothesized that either a neutralizing anti-CD18monoclonal antibody (MHM23) or a neutrophil inhibitory factor (NIF),NIF (CD11b,18), might attenuate acid-induced lung injury in rabbits byinterfering with neutrophil activation. This hypothesis derived from invitro studies that reported that anti-CD18 therapy prevented tumornecrosis factor--induced neutrophil activation. Hydrochloric acid(pH = 1.5 in one-third normal saline) or one-third normal saline (4 ml/kg) was instilled into the lungs of ventilated, anesthetizedrabbits. The rabbits were studied for 6 h. In acid-instilled rabbitswithout the anti-CD18 monoclonal antibody or NIF (CD11b,18), severelung injury developed. In acid-instilled rabbits, pretreatment (5 minbefore acid) with the anti-CD18 monoclonal antibody (2 mg/kg iv) orpretreatment with the NIF (anti-CD11b,18, 10 mg/kg iv) prevented50-70% of acid-induced abnormalities in oxygenation, the increasein extravascular lung water, and extravascular protein accumulation.The anti-CD18 monoclonal antibody was associated with a significantincrease in air space neutrophils by bronchoalveolar lavage, suggesting that the neutrophils respond normally to chemotactic stimuli but thatthe neutrophils did not injure the lung even though they accumulated inthe air spaces. In summary, neutralization of CD18 attenuates the acutelung injury after acid instillation without reducing the number ofneutrophils in the air spaces, suggesting that anti-CD18 therapy may bebeneficial because of its capacity to reduce neutrophil activation.

     

E. coli pneumonia induces CD18-independent airway neutrophil migration in the absence of increased lung vascular permeability

We examined the relationship between neutrophil [polymorphonuclear leukocyte (PMN)] influx and lung vascular injury in response to Escherichia coli pneumonia. We assessed lung tissue PMN uptake by measuring myeloperoxidase and transvascular PMN migration by determining PMN counts in lung interstitium and bronchoalveolar lavage fluid (BALF) in mice challenged intratracheally with E. coli. Lung vascular injury was quantified by determining microvessel filtration coefficient (Kf,c), a measure of vascular permeability. We addressed the role of CD18 integrin in the mechanism of PMN migration and lung vascular injury by inducing the expression of neutrophil inhibitory factor, a CD11/CD18 antagonist. In control animals, we observed a time-dependent sixfold increase in PMN uptake, a fivefold increase in airway PMN migration, and a 20-fold increase in interstitial PMN uptake at 6 h after challenge. Interestingly, Kf,c increased minimally during this period of PMN extravasation. CD11/CD18 blockade reduced lung tissue PMN uptake consistent with the role of CD18 in mediating PMN adhesion to the endothelium but failed to alter PMN migration in the tissue. Moreover, CD11/CD18 blockade did not affect Kf,c. Analysis of BALF leukocytes demonstrated diminished oxidative burst compared with leukocytes from bacteremic mice, suggesting a basis for lack of vascular injury. The massive CD11/CD18-independent airway PMN influx occurring in the absence of lung vascular injury is indicative of an efficient host-defense response elicited by E. coli pneumonia.     

Differential effects of CD18, CD29, and CD49 integrin subunit inhibition on neutrophil migration in pulmonary inflammation

Neutrophil migration to lung alveoli is a characteristic of lung diseases and is thought to occur primarily via capillaries rather than postcapillary venules. The role of adhesion molecules CD18 and CD29 on this migration in a mouse model of lung inflammation has been investigated. The number of neutrophils present in bronchoalveolar lavage fluid was determined 4 h after intratracheal instillation of LPS (0.1-1 microg) or murine recombinant KC (CXC chemokine, 0.03-0.3 microg). Both stimuli produced a dose-related increase in neutrophil accumulation. Intravenous anti-mouse CD18 mAb, 2E6 (0.5 mg/mouse), significantly (p < 0.001) attenuated LPS (0.3 microg)- but not KC (0.3 microg)-induced neutrophil accumulation. The anti-mouse CD29 mAb, HM beta 1-1 (0.02 mg/mouse), significantly (p < 0.05) inhibited both LPS (0.3 microg)- and KC (0.3 microg)-induced neutrophil migration. A second mAb to CD18 (GAME-46) and both F(ab')(2) and Fab of HM beta 1-1 produced similar results to those above, while coadministration of mAbs did not result in greater inhibition. Electron microscopy studies showed that CD29 was involved in the movement of neutrophils from the interstitium into alveoli. The effect of mAbs to CD49 (alpha integrin) subunits of CD29 was also examined. mAbs to CD49e and CD49f inhibited both responses, while anti-CD49b and CD49d significantly inhibited responses to KC only. These data suggest that CD29 plays a critical role in neutrophil migration in pulmonary inflammation and that CD49b and CD49d mediate CD18-independent neutrophil accumulation.     

Urokinase receptor-deficient mice have impaired neutrophil recruitment in response to pulmonary Pseudomonas aeruginosa infection

Leukocytes express both urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). Evidence in vitro has implicated uPAR as a modulator of beta2 integrin function, particularly CR3 (CD11b/CD18, Mac-1). Pseudomonas aeruginosa infection has been demonstrated to recruit neutrophils to the pulmonary parenchyma by a beta2 integrin-dependent mechanism. We demonstrate that mice deficient in uPAR (uPAR-/-) have profoundly diminished neutrophil recruitment in response to P. aeruginosa pneumonia compared with wild-type (WT) mice. The requirement for uPAR in neutrophil recruitment is independent of the serine protease uPA, as neutrophil recruitment in uPA-/- mice is indistinguishable from recruitment in WT mice. uPAR-/- mice have impaired clearance of P. aeruginosa compared with WT mice, as demonstrated by CFU and comparative histology. WT mice have diminished neutrophil recruitment to the lung when an anti-CD11b mAb is given before inoculation with the pathogen, while recruitment of uPAR-/- neutrophils is unaffected. We conclude that uPAR is required for the recruitment of neutrophils to the lung in response to P. aeruginosa pneumonia and that this requirement is independent of uPA. Further, we show that uPAR and CR3 act by a common mechanism during neutrophil recruitment to the lung in response to P. aeruginosa. This is the first report of a requirement for uPAR during cellular recruitment in vivo against a clinically relevant pathogen.     

Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes

Branching epithelial morphogenesis requires interactions between the surrounding mesenchyme and the epithelium, as well as interactions between basement membrane components and the epithelium. Embryonic submandibular gland was used to study the roles of two mesenchymal proteins, epimorphin and tenascin-C, as well as the epithelial protein laminin-1 and one of its integrin receptors on branching morphogenesis. Laminin-1 is a heterotrimer composed of an alpha 1 chain and two smaller chains (beta 1 and gamma 1). Immunofluorescence revealed a transient expression of laminin alpha 1 chain in the epithelial basement membrane during early stages of branching morphogenesis. Other laminin-1 chains and alpha 6, beta 1, and beta 4 integrin subunits seemed to be expressed constitutively. Expression of epimorphin, but not tenascin-C, was seen in the mesenchyme during early developmental stages, but a mAb against epimorphin did not perturb branching morphogenesis of this early epithelium. In contrast, inhibition of branching morphogenesis was seen with a mAb against the carboxy terminus of laminin alpha 1 chain, the E3 domain. An inhibition of branching was also seen with a mAb against the integrin alpha 6 subunit. The antibodies against laminin alpha 1 chain and integrin alpha 6 subunit perturbed development in distinct fashions. Whereas treatment with the anti-E3 resulted in discontinuities of the basement membrane at the tips of the branching epithelium, treatment with the mAb against alpha 6 integrin subunit seemed to leave the basement membrane intact. We suggest that the laminin E3 domain is involved in basement membrane formation, whereas alpha 6 beta 1 integrin binding to laminin-1 may elicit differentiation signals to the epithelial cells.     

Airway epithelial wounds in rhesus monkey generate ionic currents that guide cell migration to promote healing

Damage to the respiratory epithelium is one of the most critical steps to many life-threatening diseases, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. The mechanisms underlying repair of the damaged epithelium have not yet been fully elucidated. Here we provide experimental evidence suggesting a novel mechanism for wound repair: endogenous electric currents. It is known that the airway epithelium maintains a voltage difference referred to as the transepithelial potential. Using a noninvasive vibrating probe, we demonstrate that wounds in the epithelium of trachea from rhesus monkeys generate significant outward electric currents. A small slit wound produced an outward current (1.59 μA/cm(2)), which could be enhanced (nearly doubled) by the ion transport stimulator aminophylline. In addition, inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) with CFTR(Inh)-172 significantly reduced wound currents (0.17 μA/cm(2)), implicating an important role of ion transporters in wound induced electric potentials. Time-lapse video microscopy showed that applied electric fields (EFs) induced robust directional migration of primary tracheobronchial epithelial cells from rhesus monkeys, towards the cathode, with a threshold of <23 mV/mm. Reversal of the field polarity induced cell migration towards the new cathode. We further demonstrate that application of an EF promoted wound healing in a monolayer wound healing assay. Our results suggest that endogenous electric currents at sites of tracheal epithelial injury may direct cell migration, which could benefit restitution of damaged airway mucosa. Manipulation of ion transport may lead to novel therapeutic approaches to repair damaged respiratory epithelium.     

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.     

Exposure to ozone reduces influenza disease severity and alters distribution of influenza viral antigens in murine lungs.

Exposure to ambient levels of ozone (0.5 ppm) was shown to alter the pathogenesis of respiratory infection after aerosol infection of mice with influenza A virus. A semiquantitative method for determination of the sites of virus replication by direct immunofluorescence indicated that exposure to ozone reduced the involvement of respiratory epithelium in the infectious process and resulted in a less widespread infection of the alveolar parenchyma. Furthermore, the ozone-mediated alteration in viral antigen distribution was consistent with significantly reduced influenza disease mortality and prolonged survival time, but only when the oxidant was present during the course of infection. Reduced disease severity in ozone-exposed animals appeared to be independent of peak pulmonary virus titers, pulmonary interferon titers, and pulmonary and serum-neutralizing antibody titers. These studies suggested that the distribution of influenza virus in the murine lung was a key factor in disease severity.     

Expression and polarization of intercellular adhesion molecule-1 on human intestinal epithelia: consequences for CD11b/CD18-mediated interactions with neutrophils.

BACKGROUND: Epithelial dysfunction and patient symptoms in inflammatory intestinal diseases such as ulcerative colitis and Crohn's disease correlate with migration of neutrophils (PMN) across the intestinal epithelium. In vitro modeling of PMN transepithelial migration has revealed distinct differences from transendothelial migration. By using polarized monolayers of human intestinal epithelia (T84), PMN transepithelial migration has been shown to be dependent on the leukocyte integrin CD11b/CD18 (Mac-1), but not on CD11a/CD18 (LFA-1). Since intercellular adhesion molecule-I (ICAM-1) is an important endothelial counterreceptor for these integrins, its expression in intestinal epithelia and role in PMN-intestinal epithelial interactions was investigated. MATERIALS AND METHODS: A panel of antibodies against different domains of ICAM-1, polarized monolayers of human intestinal epithelia (T84), and natural human colonic epithelia were used to examine the polarity of epithelial ICAM-1 surface expression and the functional role of ICAM-1 in neutrophil-intestinal epithelial adhesive interactions. RESULTS: While no surface expression of ICAM-1 was detected on unstimulated T84 cells, interferon-gamma (IFN gamma) elicited a marked expression of ICAM-1 that selectively polarized to the apical epithelial membrane. Similarly, apically restricted surface expression of ICAM-1 was detected in natural human colonic epithelium only in association with active inflammation. With or without IFN gamma pre-exposure, physiologically directed (basolateral-to-apical) transepithelial migration of PMN was unaffected by blocking monoclonal antibodies (mAbs) to ICAM-1. In contrast, PMN migration across IFN gamma-stimulated monolayers in the reverse (apical-to-basolateral) direction was inhibited by anti-ICAM-1 antibodies. Adhesion studies revealed that T84 cells adhered selectively to purified CD11b/CD18 and such adherence, with or without IFN gamma pre-exposure, was unaffected by ICAM-1 mAb. Similarly, freshly isolated epithelial cells from inflamed human intestine bound to CD11b/CD18 in an ICAM-1-independent fashion. CONCLUSIONS: These data indicate that ICAM-1 is strictly polarized in intestinal epithelia and does not represent a counterreceptor for neutrophil CD11b/CD18 during physiologically directed transmigration, but may facilitate apical membrane-PMN interactions after the arrival of PMN in the intestinal lumen.     

Bacterial heat shock protein 60 may increase epithelial cell migration through activation of MAP kinases and inhibition of alpha6beta4 integrin expression

Exogenous heat shock proteins may modify cell behavior of infected epithelium. The effect of heat shock protein 60 (hsp60) of Actinobacillus actinomycetemcomitans and Escherichia coli, and human recombinant hsp60 on migration of HaCaT skin keratinocytes was studied using the Boyden chamber assay. Hsp60 from different species increased cell migration by two- to fivefold and this effect was inhibited by ERK inhibitor PD 98059, p38 inhibitor SB 203580, and a function-blocking epidermal growth factor receptor (EGFR) antibody. Hsp60 reduced the expression of alpha6-integrin mRNA and its protein levels on the cell surface but had no effect on the expression of beta4, beta1, alpha1, alpha5 or alphav integrin subunits. Hsp60 also significantly inhibited cell adhesion to laminin-5, a ligand of alpha6beta4 integrin. These results suggest that exogenous hsp60 released from bacteria or inflammatory cells may promote epithelial cell migration through activation of EGFR and MAP kinases, and inhibition of alpha6beta4 integrin expression.     

Effects of ozone exposure on the lungs and the erythrocytes of rats and monkeys: relative biochemical changes.

Ozone exposure (0.5 p.p.m., 8 hours daily for 7 days) resulted in a 20-26% (P less than 0.05) increase in the level of reduced glutathione (GSH), and the activities of the GSH peroxidase system in rat lungs. The increases were of smaller magnitude (10-15%) in the lungs of ozone-exposed monkeys. No significant changes were observed in these parameters in the erythrocytes of ozone-exposed and control animals of the two species. The results suggest that rats may be more sensitive to ozone than monkeys in terms of biochemical lesions in the lung, and that ozone effects are manifested primarily in the lung.