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 A 3 (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 hepA 3 is synthesized by epithelial cells and secreted from their apical surface in response to conditions that stimulate inflammatory events. Our data further establish that hepA 3 acts to draw PMNs, via the establishment of a gradient across the epithelial tight junction complex. The functional significance of hepA 3 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 hepA 3 production) could dramatically reduce PMN-mediated tissue trauma, demonstrating that hepA 3 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 mediat...

Lung inflammation resulting from bacterial infection of the respiratory mucosal surface in diseases such as cystic fibrosis and pneumonia contributes significantly to the pathology. A major consequence of the inflammatory response is the recruitment and accumulation of polymorphonuclear cells (PMNs) at the infection site. It is currently unclear what bacterial factors trigger this response and exactly how PMNs are directed across the epithelial barrier to the airway lumen. An in vitro model consisting of human PMNs and alveolar epithelial cells (A549) grown on inverted Transwell filters was used to determine whether bacteria are capable of inducing PMN migration across these epithelial barriers. A variety of lung pathogenic bacteria, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa are indeed capable of inducing PMN migration across A549 monolayers. This phenomenon is not mediated by LPS, but requires live bacteria infecting the apical surface. Bacterial interaction with the apical surface of A549 monolayers results in activation of epithelial responses, including the phosphorylation of ERK1/2 and secretion of the PMN chemokine IL-8. However, secretion of IL-8 in response to bacterial infection is neither necessary nor sufficient to mediate PMN transepithelial migration. Instead, PMN transepithelial migration is mediated by the eicosanoid hepoxilin A3, which is a PMN chemoattractant secreted by A549 cells in response to bacterial infection in a protein kinase C-dependent manner. These data suggest that bacterial-induced hepoxilin A3 secretion may represent a previously unrecognized inflammatory mechanism occurring within the lung epithelium during bacterial infections.

Acute pulmonary infection by Streptococcus pneumoniae is characterized by high bacterial numbers in the lung, a robust alveolar influx of polymorphonuclear cells (PMNs) and a risk of systemic spread of the bacterium. We investigated host-mediators of S. pneumoniae-induced PMN migration and the role of inflammation in septicemia following pneumococcal lung infection. Hepoxilin A3 (HXA3) is a PMN chemoattractant and a metabolite of the 12-lipoxygenase (12-LOX) pathway. We observed that S. pneumoniae infection induced the production of 12-lipoxygenase in cultured pulmonary epithelium and in the lungs of infected mice. Inhibition of the 12- LOX pathway prevented pathogen-induced PMN transepithelial migration in vitro and dramatically reduced lung inflammation upon high-dose pulmonary challenge with S. pneumoniae in vivo, thus implicating HXA3 in pneumococcus-induced pulmonary inflammation. PMN basolateral-to-apical transmigration in vitro significantly increased apical-to-basolateral transepithelial migration of bacteria. Mice suppressed in the expression of 12-lipoxygenase exhibited little or no bacteremia and survived an otherwise lethal pulmonary challenge. Our data suggest that pneumococcal pulmonary inflammation is required for high level bacteremia and systemic infection, partly by disrupting lung epithelium through 12-LOX-dependent HXA3 production and subsequent PMN transepithelial migration.

Shiga toxin-producing E. coli (STEC) is a food-borne pathogen that causes serious illness, including hemolytic-uremic syndrome (HUS). STEC colonizes the lower intestine and produces Shiga toxins (Stxs

Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). BecauseStx-producing Escherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS. We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response. We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-␣, GRO-␤, GRO-␥, and ENA-78. Like that of IL-8, GRO-␣ and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-␣ protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-␣ mRNA. We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins. Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection.

The establishment of tight junctions (TJ) between columnar epithelial cells defines the functional barrier, which enteroinvasive pathogens have to overcome. Salmonella enterica serovar Typhimurium (S. typhimurium) directly invades intestinal epithelial cells but it is not well understood how the pathogen is able to overcome the intestinal barrier and gains access to the circulation. Therefore, we sought to determine whether infection with S. typhimurium could regulate the molecular composition of the TJ and, if so, whether these modifications would influence bacterial translocation and polymorphonuclear leukocyte (PMN) movement across model intestinal epithelium. We found that infection of a model intestinal epithelium with S. typhimurium over 2 h resulted in an approximately 80% loss of transepithelial electrical resistance. Western blot analysis of epithelial cell lysates demonstrated that S. typhimurium regulated the distribution of the TJ complex proteins claudin-1, zonula occludens (ZO)-2, and E-cadherin in Triton X-100-soluble and insoluble fractions. In addition, S. typhimurium was specifically able to dephosphorylate occludin and degrade ZO-1. This TJ alteration in the epithelial monolayer resulted in 10-fold increase in bacterial translocation and a 75% increase in N-formylmethionin-leucyl-phenyalanine-induced PMN transepithelial migration. Our data demonstrate that infection with S. typhimurium is associated with the rapid targeting of the tight junctional complex and loss of barrier function. This results in enhanced bacterial translocation and initiation of PMN migration across the intestinal barrier. Therefore, the ability to regulate the molecular composition of TJs facilitates the pathogenicity of S. typhimurium by aiding its uptake and distribution within the host.

Inflammatory mediators including chemokines play a critical role in acute pancreatitis. The precise nature of early inflammatory signals within the pancreas remains, however, unclear. We examined the ability of isolated pancreatic acini to synthesize CC chemokine monocyte chemotactic protein-1 (MCP-1) and CXC chemokine cytokine-induced neutrophil chemoattractant (CINC) and the response to the secretagogue cerulein at physiological and supraphysiological concentrations. Isolated rat pancreatic acini maintained in short-term (< or =48 h) primary culture constitutively synthesized MCP-1 and CINC. Cerulein (10(-7) M; supramaximal dose) increased production of MCP-1 but not CINC. Cerulein-induced increase in MCP-1 synthesis was accompanied by increase in nuclear factor (NF)-kappaB activation shown by EMSA. Pretreatment with NF-kappaB inhibitors N-acetylcysteine (NAC) and N-tosylphenyalanine chloromethyl ketone (TPCK) blocked cerulein-induced NF-kappaB activation and abolished cerulein's effect on MCP-1 synthesis. Pretreatment with calcium antagonist BAPTA-AM also blocked cerulein's effect on MCP-1 synthesis. These results indicate that isolated acini synthesize MCP-1 and CINC and support the idea of acinar-derived chemokines as early mediators of inflammatory response in acute pancreatitis. Although cerulein hyperstimulation increased MCP-1 synthesis by a calcium-dependent mechanism involving NF-kappaB activation, CINC synthesis was not affected. This suggests that regulation of CC and CXC chemokines within acinar cells may be quite different.

In the 1980s, Shiga toxin (Stx)-producing Escherichia coli O157:H7 (STEC) was identified as a cause of hemorrhagic colitis in the United States and was found to be associated with hemolytic uremic syndrome (HUS), a microangiopathic hemolytic anemia characterized by thrombocytopenia and renal failure. The precise way that Stxs cause hemorrhagic colitis and HUS is unclear. Stxs have been thought to cause disease by killing or irreversibly harming sensitive cells through a nonspecific blockade of mRNA translation, eventually resulting in cytotoxicity by preventing synthesis of critical molecules needed to maintain cell integrity. Because STEC is noninvasive, we have been exploring the host-toxin response at the level of the gastrointestinal mucosa, where STEC infection begins. We have found that Stx is capable of interleukin-8 (IL-8) superinduction in a human colonic epithelial cell line. Despite a general blockade of mRNA translation, Stx treatment results in increased IL-8 mRNA as well as increased synthesis and secretion of IL-8 protein. Our data suggest that an active Stx A subunit is required for this activity. Ricin, which has the same enzymatic activity and trafficking pathway as Stx, has similar effects. Exploration of the effects of other protein synthesis inhibitors (cycloheximide, anisomycin) suggests a mechanism of gene regulation that is distinct from a general translational blockade. Use of the specific p38/RK inhibitor SB202190 showed that blocking of this pathway results in decreased Stx-mediated IL-8 secretion. Furthermore, Stxs induced mRNA of the primary response gene c-jun, which was subsequently partially blocked by SB202190. These data suggest a novel model of how Stxs contribute to disease, namely that Stxs may alter regulation of host cell processes in sensitive cells via activation of at least one member of the mitogen-activated protein kinase family in the p38/RK cascade and induction of c-jun mRNA. Stx-induced increases in chemokine synthesis from intestinal epithelial cells could be important in augmenting the host mucosal inflammatory response to STEC infection.