Parallel induction of epithelial surface-associated chemokine and proteoglycan by cellular hypoxia: implications for neutrophil activation.

Neutrophil-induced damage to the protective epithelium has been implicated in mucosal disorders associated with hypoxia, and such damage may be initiated by epithelial-derived chemokines. Because chemokines can bind to membrane proteoglycans, we hypothesized that chemokines may associate with epithelial surfaces and activate polymorphonuclear neutrophils (PMN). Epithelial hypoxia (pO2 20 torr) resulted in a time-dependent induction of interleukin-8 (IL-8) mRNA, soluble protein, as well as surface protein. Such surface IL-8 expression was demonstrated to be dependent on heparinase III expression, and extensions of these experiments indicated that hypoxia induces epithelial perlecan expression in parallel with IL-8. Finally, co-incubation of post-hypoxic epithelia with human PMN induced IL-8-dependent expression of the PMN beta2-integrin CD11b/18. These data indicate that chemokines liberated from epithelia may exist in a surface-bound, bioactive form and that hypoxia may regulate proteoglycan expression.

Endothelial cell injury in cardiac surgery: salicylate may be protective by reducing expression of endothelial adhesion molecules.

OBJECTIVE: Cardiac surgery with cardiopulmonary bypass induces ischemia to the heart, hypoxemia to various tissues and release of endotoxins. The endothelial cell may suffer from hypoxia and trigger cascades of adverse reactions by activation of neutrophils through adhesion molecules. The authors measured expression of intercellular adhesion molecule-1 (ICAM-1), during hypoxia and normoxia and hypothesized that salicylate, which inhibits the nuclear factor-kappaB (NFkappaB), an hypoxia-dependent transmission factor, could reduce this expression. METHODS: Human umbilical vein endothelial cells were cultured and exposed to normoxia and hypoxia in the presence of lipopolysaccharide (LPS). The endothelial cells were thereafter treated with salicylate or indomethacin under the same conditions. The surface expression of ICAM-1 was measured by whole cell enzyme-linked immunosorbent assay (ELISA) and the NFkappaB expression by Western blotting. RESULTS: In the presence of LPS and under hypoxic conditions, the endothelial cells produced a 300 +/- 41% increased expression of ICAM-1 compared with normoxia. The addition of salicylate (0.02-20 mM) completely inhibited the enhanced expression of ICAM-1, the addition of indomethacin at equivalent concentrations did not reduce ICAM-1 expression under either condition. CONCLUSION: ICAM-1 expression is greatly enhanced by the hypoxic endothelial cell in the presence of circulating endotoxin. Pre-treatment with salicylate completely abolishes the enhanced expression. The study suggests that salicylate administered before cardiopulmonary bypass might protect the heart against ischemic/reperfusion injuries and reduce the load of the overall inflammatory reaction.

Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha.

BACKGROUND & AIMS: The intestinal mucosa is lined by a monolayer of protective epithelial cells. This barrier is regulated by immune-derived factors such as interferon gamma (IFN-gamma). Because of the high volume of blood flow, the intestine is a primary target for hypoxic damage. We hypothesize that epithelial cytokine responses are regulated by hypoxia. METHODS: T84 intestinal epithelial cells were used to assess alterations in permeability, major histocompatibility complex class II induction, cytokine receptor expression, and cytokine release in response to combinations of IFN-gamma and cellular hypoxia. RESULTS: Hypoxia potentiated the influence of IFN-gamma on epithelial barrier function. Such responses were conferrable in a >/=10-kilodalton conditioned media fraction from hypoxic epithelia. Subsequent experiments identified this factor as epithelium-derived tumor necrosis factor alpha (TNF-alpha). Add-back of recombinant TNF-alpha in combination with IFN-gamma to normoxic epithelia recapitulated hypoxia and identified basolaterally polarized TNF-alpha receptor types I and II on intestinal epithelia. A similar pattern of TNF-alpha-receptor expression was observed on native intestinal epithelia. Specific inhibition of TNF-alpha using neutralizing antibody or alpha-N-phthalimidoglutarimide (thalidomide) resulted in reversal of the hypoxia-evoked responses. CONCLUSIONS: These studies indicate that during hypoxia, epithelium-derived mediators such as TNF-alpha have the potential to regulate permeability through autocrine pathways.

Hypoxia enhances induction of endothelial ICAM-1: role for metabolic acidosis and proteasomes.

Intercellular adhesion molecule 1 (ICAM-1) is an important molecule in promotion of polymorphonuclear neutrophil transendothelial migration during inflammation. Coincident with many inflammatory diseases is tissue hypoxia. Thus we hypothesized that combinations of hypoxia and inflammatory stimuli may differentially regulate expression of endothelial ICAM-1. Human endothelial cells were exposed to hypoxia in the presence or absence of added lipopolysaccharide (LPS) and examined for expression of functional ICAM-1. Although hypoxia alone did not induce ICAM-1, the combination of LPS and hypoxia enhanced (3 +/- 0.4-fold over normoxia) ICAM-1 expression. Combinations of hypoxia and LPS significantly increased lymphocyte binding, and such increases were inhibited by addition of anti-ICAM-1 antibodies or antisense oligonucleotides. Hypoxic endothelia showed a > 10-fold increase in sensitivity to inhibitors of proteasome activation, and combinations of hypoxia and LPS enhanced proteasome-dependent cytoplasmic-to-nuclear localization of the nuclear transcription factor-kappa B p65 (Rel A) subunit. Such proteasome activation correlated with hypoxia-evoked decreases in both extracellular and intracellular pH. We conclude from these studies that endothelial hypoxia provides a novel, proteasome-dependent stimulus for ICAM-1 induction.

Epithelial exposure to hypoxia modulates neutrophil transepithelial migration.

Polymorphonuclear leukocytes (PMN) are central to the pathogenesis of a number of intestinal diseases. PMN-induced damage to the protective epithelium occurs in hemorrhagic shock, necrotizing enterocolitis and conditions resulting in intestinal reperfusion injury. In such diseases, tissue hypoxia has been implicated as a pathophysiologic mediator. Thus, we hypothesized that exposure of intestinal epithelia to hypoxia may modulate PMN-epithelial interactions. In this study, T84 cell monolayers, a human intestinal crypt cell line, and isolated human PMN were used to examine the influence of hypoxia/reoxygenation (H/R) on PMN transepithelial migration. Confluent T84 cell monolayers were exposed to hypoxia (range 2-21% O2 for 0-72 h) and reoxygenated with buffer containing PMN. Transmigration of PMN (basolateral to apical orientation) was driven by a transepithelial gradient of the chemotactic peptide tMLP. In response to hypoxia/reoxygenation (H/R), transmigration into, and across epithelial monolayer was increased in a dose- (EC50 approximately 7% O2) and time-dependent fashion (3.5 +/- 0.3-fold increase at 2% O2 for 48 h, P < 0.001). Such conditions of H/R were not toxic to epithelia and did not influence epithelial barrier function. The influence of H/R on PMN transmigration was protein synthesis-dependent (> 80% decreased in the presence of cycloheximide) and could be inhibited by addition of functionally inhibitory antibodies to the PMN beta 2 integrin CD11b/18 (> 80% attenuated) and to CD47 (> 90% decreased compared to control). Hypoxia induced epithelial production and basolateral release of the PMN activating chemokine interleukin-8 (IL-8, nearly sixfold increase over normoxic control) which remained avidly associated with the epithelial matrix. Treatment of epithelial cells with IL-8 antisense oligonucleotides resulted in decreased monolayer-associated PMN but did not influence PMN transmigration, suggesting that epithelial IL-8 production may serve as a recruitment signal for PMN to the basal surface of polarized epithelia. The present observations indicate that H/R provides a relevant stimulus for novel biochemical crosstalk between epithelia and PMN.

Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells.

In many diseases, tissue hypoxia occurs in conjunction with other inflammatory processes. Since previous studies have demonstrated a role for leukocytes in ischemia/reperfusion injury, we hypothesized that endothelial hypoxia may "superinduce" expression of an important leukocyte adhesion molecule, E-selectin (ELAM-1, CD62E). Bovine aortic endothelial monolayers were exposed to hypoxia in the presence or absence of tumor-necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS). Cell surface E-selectin was quantitated by whole cell ELISA or by immunoprecipitation using polyclonal anti-E-selectin sera. Endothelial mRNA levels were assessed using ribonuclease protection assays. Hypoxia alone did not induce endothelial E-selectin expression. However, enhanced induction of E-selectin was observed with the combination of hypoxia and TNF-alpha (270% increase over normoxia and TNF-alpha) or hypoxia and LPS (190% increase over normoxia and LPS). These studies revealed that a mechanism for such enhancement may be hypoxia-elicited decrements in endothelial intracellular levels of cAMP (<50% compared with normoxia). Addition of forskolin and isobutyl-methyl-xanthine during hypoxia resulted in reversal of cAMP decreases and a loss of enhanced E-selectin surface expression with the combination of TNF-alpha and hypoxia. We conclude that endothelial hypoxia may provide a novel signal for superinduction of E-selectin during states of inflammation.

Interleukin-4 and interleukin-13 differentially regulate epithelial chloride secretion.

Intestinal epithelia are in intimate contact with subepithelial and intraepithelial lymphocytes. When stimulated, mucosal lymphocytes generate cytokines that act locally and influence functional aspects of many cell types. We have previously defined functional epithelial receptors for interferon-gamma, interleukin (IL)-4, and a recently described IL-4-like cytokine IL-13. In this study, we examine the ion transport properties of T84 cells, a crypt-like epithelial cell line, following exposure to IL-4 and IL-13. Basolateral exposure of epithelial monolayers to both IL-4 and IL-13 attenuated epithelial barrier function and increased paracellular flux of a dextran marker by greater than 65% in a dose- and time-dependent fashion. Stimulated Cl- secretion, as measured by epithelial short circuit current, however, was diminished only by IL-4 and not IL-13, demonstrating cytokine specificity in this epithelial function. Decreased Cl- secretion following IL-4 exposure was associated with diminished Cl- channel activity and IL-4 pretreatment of epithelia decreased expression of the cystic fibrosis transmembrane regulator. Finally, stimulated fluid transport across cultured epithelia was diminished following exposure to IL-4, but not IL-13. These results indicate that while post-receptor signaling events induced by IL-13 and IL-4 may be similar, end point function is cytokine-specific.

Hypoxic stress alone does not modulate endothelial surface expression of bovine E-selectin and intercellular adhesion molecule-1 (ICAM-1).

UNLABELLED: Hypoxemia is a common event in many vascular diseases, especially vascular ischemia. Since endothelial cells of blood vessels are exposed to conditions within the vascular space and leucocytes play a key role in ischemia/reperfusion injury, we hypothesized that endothelial exposure to hypoxia may regulate expression of surface proteins important in leucocyte-endothelial interactions, such as E-selectin and intercellular adhesion molecule (ICAM-1). In this study, we used isolated bovine aortic endothelial monolayers to examine endothelial surface alterations of E-selectin and ICAM-1 induced by tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and hypoxia using a whole cell enzyme-linked immunosorbent assay (ELISA). Bovine endothelial exposure to TNF-alpha (50 ng/mL) induced a time dependent increase (range 0-24h) in specific E-selection surface expression. Endothelial exposure to hypoxia alone (pO2 approximately 3 mmHg, range 0-24 h), however, failed to elicit endothelial E-selectin expression. Endothelial exposure to LPS brought about a dose- and time-dependent (range 0.5 ng/mL and 2-8 h) increase in specific ICAM-1 surface expression (max. 3.5 +/- 0.15-fold increase over no cytokine control at 10 ng/mL, 4 h). Hypoxia (pO2 approximately 3 mmHg, 8h), however, did not induce ICAM-1 surface expression over normoxia levels. IN CONCLUSION: i) bovine endothelial E-selectin and ICAM-1 surface expression are regulated molecules, ii) hypoxia, per se, does not regulate surface expression of either E-selectin or ICAM-1. These results suggest that hypoxic endothelia may require additional external signals for generation of adaptive inflammatory responses.