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)     

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.     

Neutrophil migration across a cultured epithelial monolayer elicits a biphasic resistance response representing sequential effects on transcellular and paracellular pathways

Migration of polymorphonuclear leukocytes across epithelia is a hallmark of many inflammatory disease states. Neutrophils traverse epithelia by migrating through the paracellular space and crossing intercellular tight junctions. We have previously shown (Nash, S., J. Stafford, and J.L. Madara. 1987. J. Clin. Invest. 80:1104-1113), that leukocyte migration across T84 monolayers, a model human intestinal epithelium, results in enhanced tight junction permeability--an effect quantitated by the use of a simple, standard electrical assay of transepithelial resistance. Here we show that detailed time course studies of the transmigration-elicited decline in resistance has two components, one of which is unrelated to junctional permeability. The initial decrease in resistance, maximal 5-13 min after initiation of transmigration, occurs despite inhibition of transmigration by an antibody to the common beta subunit of neutrophil beta 2 integrins, and is paralleled by an increase in transepithelial short-circuit current. Chloride ion substitution and inhibitor studies indicate that the early-phase resistance decline is not attributable to an increase in tight junction permeability but is due to decreased resistance across epithelial cells resulting from chloride secretion. Since T84 cells are accepted models for studies of the regulation of Cl- and water secretion, our results suggest that neutrophil transmigration across mucosal surfaces (for example, respiratory and intestinal tracts) may initially activate flushing of the surface by salt and water. Equally important, these studies, by providing a concrete example of sequential transcellular and paracellular effects on transepithelial resistance, highlight the fact that this widely used assay cannot simply be viewed as a direct functional probe of tight junction permeability.     

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.     

src family kinases regulate renal epithelial paracellular permeability barrier through an occludin‐independent mechanism

Paracellular permeability is mediated by the epithelial cell tight junction. Studies in intestinal and other epithelia have suggested that the activity of src family kinases (SFKs) increases epithelial paracellular permeability through its action on the tight junction protein, occludin, but the involvement of SFKs and occludin in regulation of renal epithelial paracellular permeability is unclear. In this study, the role of SFKs in regulation of renal epithelial paracellular permeability and the involvement of occludin protein in this regulatory event was examined in two renal epithelial cell lines, LLC‐PK₁ (proximal tubule‐like) and MDCK (distal tubule‐like). The effect of broad spectrum SFK inhibitors on paracellular permeability of calcein and fluorescein‐dextran3000 were examined. SFK inhibitor treatment increased paracellular movement of both compounds in both renal epithelial cell lines. The SFK inhibitor effect was concentration‐dependent and, at low concentrations, was not associated with cell damage/death. Response to SFK inhibitors was acquired progressively after cell populations attained confluence suggesting maturation of the regulatory mechanism. Increased paracellular permeability was not associated with dramatic changes in total cell content of occludin protein, its partitioning between detergent‐soluble and ‐insoluble fractions, or its subcellular localization. Further, the SFK‐induced increase in paracellular permeability was unaffected by either occludin protein overexpression or occludin protein knockdown. These results demonstrate that SFK activity decreases paracellular permeability of renal epithelial cells, as opposed to its effect in intestinal epithelial cells, and that this regulation is not mediated by occludin protein. J. Cell. Physiol. 228: 1210–1220, 2013. © 2012 Wiley Periodicals, Inc.     

Regulation of Salmonella-induced neutrophil transmigration by epithelial ADP-ribosylation factor 6

Salmonella typhimurium elicits an acute inflammatory response in the host intestinal epithelium, characterized by the movement of polymorphonuclear leukocytes (PMN) across the epithelial monolayer to the intestinal lumen. It was recently shown that SipA, a protein secreted by S. typhimurium, is necessary and sufficient to drive PMN transmigration across model intestinal epithelia (Lee, C. A., Silva, M., Siber, A. M., Kelly, A. J., Galyov, E., and McCormick, B. A. (2000) Proc. Natl. Acad Sci. USA 97, 12283-12288). However, the epithelial factors responsible for this process have not been identified. Here, for the first time, we demonstrate that S. typhimurium-induced PMN transmigration across Madin-Darby canine kidney-polarized monolayers is regulated by the GTPase ARF6. Apically added S. typhimurium promoted the translocation of ARF6 and its exchange factor ARNO to the apical surface. Overexpression of a dominant-negative mutant of ARF6 inhibited Salmonella-induced PMN transmigration, which was due to a reduction in apical release of the PMN chemoattractant PEEC (pathogen-elicited epithelial chemoattractant), without affecting bacterial internalization. Furthermore, ARF6 and its effector phospholipase D (PLD) were both required for bacteria-induced translocation of protein kinase C (PKC) to membranes. These results describe a novel signal transduction pathway, in which Salmonella initiates an ARF6- and PLD-dependent lipid signaling cascade that, in turn, directs activation of PKC, release of PEEC, and subsequent transepithelial PMN movement.     

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.     

Localization of 7H6 Tight Junction-Associated Antigen along the Cell Border of Vascular Endothelial Cells Correlates with Paracellular Barrier Function against Ions, Large Molecules, and Cancer Cells

To study the regulation of the endothelial barrier, we examined the relationship between the paracellular barrier function and the expression of 7H6 antigen localized at tight junctions of endothelial cells by using transendothelial electrical resistance (TER), fluxes of albumin and dextran, transmigration of rat mammary cancer (SST-2) cells across rat lung endothelial (RLE) cells, and immunocytochemical expression of 7H6 antigen as parameters. RLE cells cultured at a confluent cell density did not express immunohistochemically demonstrable 7H6 antigen and had low paracellular barrier functions. However, treatment of the endothelial cells with 0.5 mMdibutyryl–cAMP or 10⁻⁶Mall-trans-retinoic acid for 4 days induced 7H6 antigen preferentially at the cell border and simultaneously enhanced the barrier function twofold, in terms of TER and fluxes of albumin and dextran. Furthermore, RA-treated RLE cell monolayers with the enhanced barrier function significantly inhibited the transmigration of SST-2 cells. These results together with those of our previous study indicate that 7H6 antigen has a crucial role in the regulation of paracellular barrier function not only in epithelial cells but also in vascular endothelial cells. The present study also suggests that tight junctions of vascular endotheliumin vivofunction as a barrier between blood and tissues against metastatic cancer cells.     

Junctional Adhesion Molecule,a Novel Member of the Immunoglobulin Superfamily That Distributes at Intercellular Junctions and Modulates Monocyte Transmigration

Tight junctions are the most apical components of endothelial and epithelial intercellular cleft. In the endothelium these structures play an important role in the control of paracellular permeability to circulating cells and solutes. The only known integral membrane protein localized at sites of membrane–membrane interaction of tight junctions is occludin, which is linked inside the cells to a complex network of cytoskeletal and signaling proteins. We report here the identification of a novel protein (junctional adhesion molecule [JAM]) that is selectively concentrated at intercellular junctions of endothelial and epithelial cells of different origins. Confocal and immunoelectron microscopy shows that JAM codistributes with tight junction components at the apical region of the intercellular cleft. A cDNA clone encoding JAM defines a novel immunoglobulin gene superfamily member that consists of two V-type Ig domains. An mAb directed to JAM (BV11) was found to inhibit spontaneous and chemokine-induced monocyte transmigration through an endothelial cell monolayer in vitro. Systemic treatment of mice with BV11 mAb blocked monocyte infiltration upon chemokine administration in subcutaneous air pouches. Thus, JAM is a new component of endothelial and epithelial junctions that play a role in regulating monocyte transmigration.     

Shear stress affects migration behavior of polymorphonuclear cells arrested on endothelium

Polymorphonuclear cell (PMN) transmigration across the TNF-alpha-stimulated endothelial cell (HUVEC) monolayer in the presence of shear flow was monitored with time-lapse videotapes. More than half of the PMN that arrested on HUVEC transmigrated through endothelial cell junctions within the following 15 min. The kinetics of transmigration was significantly faster than that of PMN placed under static conditions. Once PMN crept into the subendothelial space, they showed random migration beneath the HUVEC monolayer. PMN that did not transmigrate moved on the apical surface of HUVEC in the direction of flow downstream. Anti-beta1 integrin mAb (4B4) and RGD peptide inhibited the transmigration more effectively than anti-beta2 integrin mAb (TS1/18) and almost totally abrogated transmigration. When HUVEC were cultured on fibronectin or laminin, the transmigration was significantly inhibited by anti-alpha5 or alpha6 integrin mAbs, respectively. Our data clearly indicate that shear stress affects the migration behavior of PMN arrested on endothelium and suggest that binding to subendothelial extracellular matrix via beta1 integrins is another essential step in leukocyte extravasation.     

Myosin heavy chain isoforms expression and cyclic AMP concentrations in hypoxia-induced hypertrophied right ventricle in rats

We have previously demonstrated that the relative expression of myosin heavy chain-beta (MHC-β) in both ventricles of rats exposed to long-term hypobaric hypoxia correlated significantly with the relative ventricular mass. In the present study, we investigated whether an increased expression of MHC-β was accompanied by a reduction in cyclic AMP (cAMP) activity in hypoxia-induced hypertrophied right ventricle (RV). We used male Wistar–Kyoto rats born and raised at simulated altitudes (2200 m: H2 group or 4000 m: H4 group) compared to age-matched sea level controls (SC group). There were no significant differences between the groups in basal and forskolin-stimulated adenylyl cyclase (AC) activities. The basal and IBMX-inhibited phosphodiesterase (PDE) activities were slightly higher in both hypoxic groups (p>0.05), except that the H2 group had a higher basal PDE activity than the SC group (p<0.05). The AC/PDE activity ratios were significantly decreased in both hypoxic groups (p<0.05), suggesting that low concentrations of cellular cAMP were maintained in the RV under hypoxic conditions. However, there were no correlations between MHC-β expression and either AC activity, PDE activity, or AC/PDE activity ratio. These results provided evidence against the causal role for cAMP concentration in the expression of MHC-β associated with hypoxia-induced ventricular hypertrophy.     

Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions

Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE- cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE- cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen- reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE-cadherin distribution was affected by PMN adhesion to the vessel wall in vivo too. This work suggests that PMN adhesion could trigger intracellular signals in EC that possibly regulate VE-cadherin /catenin complex disorganization. This effect could increase EC permeability and facilitate PMN transmigration during the acute inflammatory reaction.     

Polymorphonuclear leukocyte migration across model intestinal epithelia enhances Salmonella typhimurium killing via the epithelial derived cytokine,IL-6

The host response to Salmonella typhimurium involves movement of polymorphonuclear leukocytes (PMN) across the epithelium and into the intestinal lumen. Following their arrival in the lumen, the PMN attempt to combat bacterial infection by activating antimicrobial defenses such as granule release, oxidative burst, phagocytosis, and cell signaling. We sought to examine PMN-S. typhimurium interaction following PMN arrival in the lumenal compartment. Here, for the first time, we demonstrate that PMN that have transmigrated across model intestinal epithelia have an enhanced ability to kill S. typhimurium. Our data provide evidence to indicate that the extracellular release of the primary and secondary granules of PMN, myeloperoxidase and lactoferrin, respectively, is correlated with enhanced bacterial killing. Furthermore, epithelial cells, during PMN transmigration, release the cytokine IL-6. IL-6 is known to increase intracellular stores of Ca(2+), and we have determined that this epithelial released cytokine is not only responsible for priming the PMN to release their granules, but also stimulating the PMN to kill S. typhimurium. These results substantiate the pathway in which PMN transmigration activates the epithelial release of IL-6, which in turn increases intracellular Ca(2+) storage. Our results, herein, extend this pathway to include an enhanced PMN granule release and an enhanced killing of S. typhimurium.     

Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1.

rIL-1 beta treatment of cultured human endothelial cells (HEC) promotes polymorphonuclear leukocyte (PMN) adhesion and transmigration. Using in vitro quantitative monolayer adhesion and videomicroscopic transmigration assays, we have examined the contributions of endothelial-leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and the leukocyte adhesion complex, CD11/CD18, to these processes. Maximal enhancement of PMN adhesion and transmigration were observed after 4 h of rIL-1 beta treatment, when surface expression of ELAM-1 had peaked and ICAM-1 was modestly increased. Blocking mAb directed to either ELAM-1 or ICAM-1 inhibited greater than 90% of the up-regulated PMN transmigration. Blocking mAb directed to either CD11a/CD18 (LFA-1, a ICAM-1 counter-receptor), CD11b/CD18 (Mo-1), or CD18 (common beta 2-integrin) also blocked greater than 90% of PMN transmigration. At later time points (24 or 48 h), ELAM-1 surface expression was markedly decreased, whereas ICAM-1 expression was increased over the 4-h level; PMN adhesion remained elevated (approximately 50 to 60% of 4 h level), but transmigration returned to levels seen with unactivated HEC. These data indicate that PMN interaction with at least two distinct HEC adhesion molecules is necessary for transendothelial migration and suggests that PMN adhesion and transmigration, although interrelated, are mechanistically distinct processes.     

Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (claudin-1 and occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ proteins using primary bovine brain microvessel endothelial cells (BBMEC). Hypoxia induced a 2.6-fold increase in [(14)C]sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ protein expression immediately after hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in occludin, ZO-1, and ZO-2 protein localization during hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.     

Protection against hypoxia-induced blood-brain barrier disruption: changes in intracellular calcium

Tissue damage after stroke is partly due to disruption of the blood-brain barrier (BBB). Little is known about the role of calcium in modulating BBB disruption. We investigated the effect of hypoxic and aglycemic stress on BBB function and intracellular calcium levels. Bovine brain microvessel endothelial cells were treated with A-23187 to increase intracellular calcium without hypoxia or treated with a calcium chelator (BAPTA) or calcium channel blockers (nifedipine or SKF-96365) and 6 h of hypoxia. A-23187 alone did not increase paracellular permeability. Hypoxia increased intracellular calcium, and hypoxia or hypoxia-aglycemia increased paracellular permeability. Treatment with nifedipine and SKF-96365 increased intracellular calcium under normoglycemic conditions, instead of blocking calcium influx, and was protective against hypoxia-induced BBB disruption under normoglycemia. Protection by nifedipine and SKF-96365 was not due to antioxidant properties of these compounds. These data indicate that increased intracellular calcium alone is not enough to disrupt the BBB. However, increased intracellular calcium after drug treatment and hypoxia suggests a potential mechanism for these drugs in BBB protection; nifedipine and SKF-96365 plus hypoxic stress may trigger calcium-mediated signaling cascades, altering BBB integrity. nifedipine; SKF-96365; ischemia; permeability; fura 2     

Hypoxia‐induced matrix metalloproteinase‐13 expression in astrocytes enhances permeability of brain endothelial cells

Matrix metalloproteinase‐13 (MMP‐13) is involved in the degradation of extracellular matrix in many kinds of tissues. Here we found that hypoxia increased MMP‐13 protein and mRNA levels in primary rat astrocyte cultures. Hypoxia stimulation also increased the secretion of MMP‐13 from astrocytes, as shown by zymographic analysis. In addition, exposure to hypoxia up‐regulated the expression of c‐Fos and c‐Jun time‐dependently. Hypoxia‐induced MMP‐13 overexpression was antagonized by transfection with antisense oligodeoxynucleotides (AS‐ODN) of c‐Fos or c‐Jun. Furthermore, hypoxic‐conditioned medium (Hx‐CM) collected from astrocytes exposed to hypoxia increased paracellular permeability of adult rat brain endothelial cells (ARBECs). Administration of MMP‐13 neutralizing antibody antagonized Hx‐CM‐induced paracellular permeability of ARBECs. Furthermore, pre‐transfection of astrocytes with AS‐ODN of c‐Fos, c‐Jun or MMP‐13‐shRNA significantly decreased hyperpermeability of ARBECs induced by Hx‐CM. The arrangement of tight junction protein (TJP) zonular occludens‐1 (ZO‐1) of ARBECs disorganized in response to Hx‐CM. Administration of Hx‐CM to ARBECs also resulted in the production of proteolytic fragments of ZO‐1, which was antagonized by transfection of MMP‐13‐shRNA in primary astrocytes. Administration of MMP‐13 recombinant protein to ARBECs led to the disorganization and fragmentation of ZO‐1 protein and also increased paracellular permeability. These results suggest that hypoxia‐induced MMP‐13 expression in astrocytes is regulated by c‐Fos and c‐Jun. MMP‐13 is an important factor leading to the disorganization of ZO‐1 and hyperpermeablility of blood–brain barrier in response to hypoxia. J. Cell. Physiol. 220: 163–173, 2009. © 2009 Wiley‐Liss, Inc.     

Tissue Plasminogen Activator Enhances the Hypoxia/reoxygenation-induced Impairment of the Blood–brain Barrier in a Primary Culture of Rat Brain Endothelial Cells

Hemorrhagic transformation is a major complication associated with tissue plasminogen activator (tPA) therapy for ischemic stroke. We studied the effect of tPA on the blood–brain barrier (BBB) function with our in vitro monolayer model generated using rat brain microvascular endothelial cells subjected either to normoxia or to hypoxia/reoxygenation (H/R) with or without the administration of tPA. The barrier function was evaluated by the transendothelial electrical resistance (TEER), the permeability of sodium fluorescein and Evans’ blue-albumin (EBA), and the uptake of lucifer yellow (LY). The permeability of sodium fluorescein and EBA was used as an index of paracellular and transcellular transport, respectively. The administration of tPA increased the permeability of EBA and the uptake of LY under normoxia. It enhanced the increase in the permeability of both sodium fluorescein and EBA, the decrease in the TEER, and the disruption in the expression of ZO-1 under H/R conditions. Administration of tPA could cause an increase in the transcellular transport under normoxia, and both the transcellular and paracellular transport of the BBB under H/R conditions in vitro. Even in humans, tPA may lead to an opening of the BBB under non-ischemic conditions and have an additional effect on the ischemia-induced BBB disruption.     

Elevations of intracellular cAMP result in a change in cell shape that resembles dome formation in cultured rat glomerular epithelial cells

Summary Cultured glomerular epithelial cells form a continuous monolayer of polyhedral-shaped cells. PGE₂ (1 μg/ml) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX) markedly raises intracellular and medium cyclic AMP (cAMP) levels at 20 min (intracellular: MIX alone, 112 ± 6.6 pmol cAMP/mg protein, MIX plus PGE₂, 2252±63 pmol cAMP/mg protein; medium: MIX, 20.6±2.1 pmol cAMP/mg protein; MIX plus PGE₂, 117±3.8 pmol cAMP/mg protein). By 2 h, when cellular and medium cAMP levels were still elevated, the cells underwent a change in shape that was similar to dome formation (15 to 20% of the monolayer changing shape). Derivatives of cAMP [i. e. dibutyryl and 8-(4-chlorophenylthio)-cAMP], when added to the incubation medium also caused shape change in glomerular epithelial cells at 2 h; cAMP itself did not. The formation of domes has been used as a morphological indicator of the vertorial transport of salt and water in other cultured epithelial cells. This work was supported by grant AM 29787 from the National Institutes of Health, Bethesda, MD.