Neutrophil Transepithelial Migration: Evidence for Sequential, Contact-Dependent Signaling Events and Enhanced Paracellular Permeability Independent of Transjunctional Migration

Abstract

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.

p120-catenin is essential for maintenance of barrier function and intestinal homeostasis in mice

“…The TER of WT monolayers decreased dramatically upon addition of neutrophils ( Figure 8A), as described previously (42). Interestingly, although respective WT and KD HCA7 monolayers were morphologically similar by light microscopy, TER was severely reduced by p120 KD, irrespective of neutrophil presence ( Figure 8A).…”

“…To clarify the role of the epithelial barrier, as apposed to other events linked to p120 ablation (e.g., neutrophil behavior), we generated p120 knockdown (KD) HCA7 cells and evaluated the in vitro effect of p120 KD on several relevant phenotypes (Figure 8). WT (pLentiLox-GFP [pLL-GFP]) and p120 KD (pLentiLox-hp120i-GFP [pLL-hp120i-GFP]) HCA7 cells were plated on neutrophil permeable (3-μm pore size) filters, as described by Edens et al (42). Neutrophils were then added to the basal side with chemoattractant (fMLP) on the apical side.…”

“…Normal human polymorphonuclear leukocytes (PMNs) were isolated, as described previously by Edens et al (42). PMN transmigration experiments were performed as previously described in detail (68).…”

Regulation of Leukocyte Transmigration: Cell Surface Interactions and Signaling Events

“…We have further observed an increase in epithelial permeability (reduced barrier function) when PMN contact the epithelial basolateral surface in the presence of a transepithelial gradient of fMLP (39). This observation suggests that active signaling events between PMN and epithelial cells at early stages of the transepithelial migration, although independent of ␤ 2 integrins, may serve to facilitate PMN transmigration.…”

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

“…Direct effects of PMNs on epithelial cell function appear to be mediated both by cell-cell contact and by release of factors that initiate subsequent signaling events. For example, we have recently shown that as sub-epithelial PMNs enter the paracellular space in response to a lumenal chemoattractant, epithelial permeability increases through undefined mechanisms that are independent of junctional disruption (Edens et al 2002). In addition, as PMNs migrate across the colonic epithelium, PMN-derived 5'-AMP (Madara et al 1993) is converted to adenosine which activates electrogenic chloride secretion and passive water movement into the lumen (Strohmeier et al 1995(Strohmeier et al , 1997.…”