Titus A. Reaves

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.

Neutrophil transepithelial migration: role of toll-like receptors in mucosal inflammation

The symptomatic phases of many inflammatory diseases are characterized by migration of large numbers of neutrophils (PMN) across a polarized epithelium and accumulation within a lumen. For example, acute PMN influx is common in diseases of the gastrointestinal system (ulcerative colitis, Crohns disease, bacterial enterocolitis, gastritis), hepatobiliary system (cholangitis, acute cholecystitis), respiratory tract (bronchial pneumonia, bronchitis, cystic fibrosis, bronchiectasis), and urinary tract (pyelonephritis, cystitis). Despite these observations, the molecular basis of leukocyte interactions with epithelial cells is incompletely understood. In vitro models of PMN transepithelial migration typically use N-formylated bacterial peptides such as fMLP in isolation to drive human PMNs across epithelial monolayers. However, other microbial products such as lipopolysaccharide (LPS) are major constituents of the intestinal lumen and have potent effects on the immune system. In the absence of LPS, we have shown that transepithelial migration requires sequential adhesive interactions between the PMN beta2 integrin CD11b/CD18 and JAM protein family members. Other epithelial ligands appear to be abundantly represented as fucosylated proteoglycans. Further studies indicate that the rate of PMN migration across mucosal surfaces can be regulated by the ubiquitously expressed transmembrane protein CD47 and microbial-derived factors, although many of the details remain unclear. Current data suggests that Toll-like receptors (TLR), which recognize specific pathogen-associated molecular patterns (PAMPs), are differentially expressed on both leukocytes and mucosal epithelial cells while serving to modulate leukocyte-epithelial interactions. Exposure of epithelial TLRs to microbial ligands has been shown to result in transcriptional upregulation of inflammatory mediators whereas ligation of leukocyte TLRs modulate specific antimicrobial responses. A better understanding of these events will hopefully provide new insights into the mechanisms of epithelial responses to microorganisms and ideas for therapies aimed at inhibiting the deleterious consequences of mucosal inflammation.

CD47 and TLR-2 Cross-Talk Regulates Neutrophil Transmigration

Neutrophil (PMN) infiltration into tissues is a hallmark of acute inflammation and is crucial for the rapid removal of microbial pathogens. Previous studies have shown that PMN transmigration is regulated by the cell surface protein CD47. However this phenomenon in the context of microbial invasion and subsequent TLR signaling is poorly understood. In this study, we assessed the role of TLR2 and CD47 costimulation in regulating PMN transmigration. Human PMN transmigration across acellular collagen-coated filters toward the bacterial chemoattractant fMLP was more significantly inhibited by MALP-2 (TLR2/6 agonist) than Pam3CSK4 (TLR2/1 agonist). Subsequent experiments demonstrated that treatment with MALP-2 or anti-human CD47 mAbs delayed human PMN transfilter migration, while combined treatment led to further delayed inhibition. Interestingly, stimulation of PMNs with MALP-2 resulted in an increase in surface expression of CD11b, but not CD47. In experiments addressing the role of TLR agonists in regulating CD47-mediated PMN transmigration, incubation with MALP-2 or with anti-mouse CD47 mAbs did not inhibit transfilter migration of TLR2−/− or MyD88−/−-deficient murine bone marrow-derived PMNs. Similarly, inhibition of MyD88 homodimerization reversed the attenuation of human PMN transmigration induced by MALP-2 or anti-human CD47 mAbs. Separate experiments demonstrated that CD47−/− murine bone marrow-derived PMNs exhibited 4-fold decreased sensitivity toward MALP-2. Collectively, these findings suggest that activation of CD47 signaling enhances PMN sensitivity toward TLR2 activation which, in turn, signals their arrival at a site of invasion and may facilitate antimicrobial function.