Identification of hepoxilin A3 in inflammatory events: a required role in neutrophil migration across intestinal epithelia

The mechanism by which neutrophils [polymorphonuclear leukocyte (PMNs)] are stimulated to move across epithelial barriers at mucosal surfaces has been basically unknown in biology. IL-8 has been shown to stimulate PMNs to leave the bloodstream at a local site of mucosal inflammation, but the chemical gradient used by PMNs to move between adjacent epithelial cells and traverse the tight junction at the apical neck of these mucosal barriers has eluded identification. Our studies not only identify this factor, previously termed pathogen-elicited epithelial chemoattractant, as the eicosanoid hepoxilin A3 (hepA3) but also demonstrate that it is a key factor promoting the final step in PMN recruitment to sites of mucosal inflammation. We show that hepA3 is synthesized by epithelial cells and secreted from their apical surface in response to conditions that stimulate inflammatory events. Our data further establish that hepA3 acts to draw PMNs, via the establishment of a gradient across the epithelial tight junction complex. The functional significance of hepA3 to target PMNs to the lumen of the gut at sites of inflammation was demonstrated by the finding that disruption of the 12-lipoxygenase pathway (required for hepA3 production) could dramatically reduce PMN-mediated tissue trauma, demonstrating that hepA3 is a key regulator of mucosal inflammation. Bacterial pathogens continually confront epithelial barriers of the body, such as those of the gastrointestinal, respiratory, and reproductive tracts. Polymorphonuclear leukocyte (PMNs) represent a class of white cells critical to defend the host from such pathogens. Previous studies have identified factors such as IL-8, secreted from the basolateral surface of epithelial barriers that establish chemical gradients essential for PMN activation and recruitment from the bloodstream (1). After this IL-8 gradient, PMNs are drawn to the lateral surfaces of epithelial cells. Migration to the actual site of bacterial infection, however, i.e., within the intestinal lumen, requires the action(s) of an additional chemical gradient established across a final barrier present at the apical neck of epithelia, the tight junction (TJ) complex. Any molecule that could function to establish a gradient across such a barrier would have unique properties: selective secretion from the apical rather than basolateral epithelial cell surface, capacity to permeate the TJ to establish a chemical gradient, and a labile nature that would prevent excessive PMN migration. Identification of such a factor has been an important unanswered question of epithelial pathobiology. Salmonellosis, a frequent cause of diarrhea worldwide, represents one example of epithelial pathobiology where extensive PMN transmigration into the lumen is observed, in this case into small intestine crypts in response to apical infection by Salmonella typhimurium. PMN actions on the epithelium, culminating in the formation of intestinal crypt abscesses and subsequent loss of barrier function, underline the key events in mediating not only the clinical manifestations of S. typhimurium-induced enteritis (2, 3) but also idiopathic diseases associated with inflammatory bowel disease (4). To model such inflammatory events occurring at the intestinal mucosa, we have used an in vitro model of S. typhimurium infection of human intestinal epithelial cells, T84, grown as monolayers to detect the presence and obtain an initial characterization of pathogen-elicited epithelial chemoattractant (PEEC). PEEC is a small molecule (M r < 1,500) that stimulates pertussis-toxin-sensitive G protein-coupled Ca2+ mobilization; however, in contrast to other known PMN chemoattractants, it was shown to induce no degranulation or oxidative burst even at saturating concentrations and in the presence of “primers” such as cytochalasins (5), thus distinguishing PEEC from all other factors known to affect PMN chemotaxis. Using the T84-S. typhimurium infection model (6), we have now purified and identified a molecule secreted from the apical surface of T84 cell monolayers that is stimulated by pathogenic, but not nonpathogenic, strains of S. typhimurium and demonstrated this molecule, hepoxilin A3 (hepA3), to recapitulate previously established characteristics for PEEC both in vitro and in vivo.