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TNF? promotes CAR-dependent migration of leukocytes across epithelial monolayers.

ABSTRACT: Trans-epithelial migration (TEpM) of leukocytes during inflammation requires engagement with receptors expressed on the basolateral surface of the epithelium. One such receptor is Coxsackie and Adenovirus Receptor (CAR) that binds to Junctional Adhesion Molecule-like (JAM-L) expressed on leukocytes. Here we provide the first evidence that efficient TEpM of monocyte-derived THP-1 cells requires and is controlled by phosphorylation of CAR. We show that TNF? acts in a paracrine manner on epithelial cells via a TNFR1-PI3K-PKC? pathway leading to CAR phosphorylation and subsequent transmigration across cell junctions. Moreover, we show that CAR is hyper-phosphorylated in vivo in acute and chronic lung inflammation models and this response is required to facilitate immune cell recruitment. This represents a novel mechanism of feedback between leukocytes and epithelial cells during TEpM and may be important in controlling responses to pro-inflammatory cytokines in pathological settings.

SUBMITTER: Morton PE

PROVIDER: S-EPMC4872059 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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TNFα promotes CAR-dependent migration of leukocytes across epithelial monolayers.

Morton Penny E PE Hicks Alexander A Ortiz-Zapater Elena E Raghavan Swetavalli S Pike Rosemary R Noble Alistair A Woodfin Abigail A Jenkins Gisli G Rayner Emma E Santis George G Parsons Maddy M

Scientific reports 20160519

Trans-epithelial migration (TEpM) of leukocytes during inflammation requires engagement with receptors expressed on the basolateral surface of the epithelium. One such receptor is Coxsackie and Adenovirus Receptor (CAR) that binds to Junctional Adhesion Molecule-like (JAM-L) expressed on leukocytes. Here we provide the first evidence that efficient TEpM of monocyte-derived THP-1 cells requires and is controlled by phosphorylation of CAR. We show that TNFα acts in a paracrine manner on epithelial ...[more]

PMID: 27193388

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Project description:Hyperglycemia is thought to increase production of inflammatory cytokines and permeability of the large intestine. Resulting intestinal inflammation is then often characterized by excess secretion of tumor necrosis factor alpha (TNFα). Thus, hyperglycemia in hospitalized patients suffering from severe trauma or disease is frequently accompanied by TNFα secretion, and the combined impact of these insults on the intestinal epithelium is poorly understood. This study utilized a simple yet elegant model of the intestinal epithelium, comprised of primary human intestinal stem cells and their differentiated progeny, to investigate the impact of hyperglycemia and inflammatory factors on the colonic epithelium. When compared to epithelium cultured under conditions of physiologic glucose, cells under hyperglycemic conditions displayed decreased mucin-2 (MUC2), as well as diminished alkaline phosphatase (ALP) activity. Conditions of 60 mM glucose potentiated secretion of the cytokine IL-8 suggesting that cytokine secretion during hyperglycemia may be a source of tissue inflammation. TNFα measurably increased secretion of IL-8 and IL-1β, which was enhanced at 60 mM glucose. Surprisingly, intestinal permeability and paracellular transport were not altered by even extreme levels of hyperglycemia. The presence of TNFα increased MUC2 presence, decreased ALP activity, and negatively impacted monolayer barrier function. When TNFα hyperglycemia and ≤30 mM glucose and were combined, MUC2 and ALP activity remained similar to that of TNFα alone, although synergistic effects were seen at 60 mM glucose. An automated image analysis pipeline was developed to assay changes in properties of the zonula occludens-1 (ZO-1)-demarcated cell boundaries. While hyperglycemia alone had little impact on cell shape and size, cell morphologic properties were extraordinarily sensitive to soluble TNFα. These results suggest that TNFα acted as the dominant modulator of the epithelium relative to glucose, and that control of inflammation rather than glucose may be key to maintaining intestinal homeostasis.

Project description:The collective migration of vascular endothelial cells plays important roles in homeostasis and angiogenesis. Oxygen concentration in vivo, which is lower than in the atmosphere and changes due to diseases, is a key factor affecting the cellular dynamics of vascular endothelial cells. We previously reported that hypoxic conditions promote the internalization of vascular endothelial (VE)-cadherin, a specific cell-cell adhesion molecule, and increase the velocity of the collective migration of vascular endothelial cells. However, the mechanism through which cells regulate collective migration as affected by oxygen tension is not fully understood. Here, we investigated oxygen-dependent collective migration, focusing on intracellular protein p21-activated kinase (PAK) and hypoxia-inducing factor (HIF)-1α. A monolayer of human umbilical vein vascular endothelial cells (HUVECs) was formed in a microfluidic device with controllability of oxygen tension. The HUVECs were then exposed to various oxygen conditions in a range from 0.8% to 21% O2, with or without PAK inhibition or chemical stabilization of HIF-1α. Collective cell migration was measured by particle image velocimetry with time-lapse phase-contrast microscopic images. Localizations of VE-cadherin and HIF-1α were quantified by immunofluorescent staining. The collective migration of HUVECs varied in an oxygen-dependent fashion; the migration speed was increased by hypoxic exposure down to 1% O2, while it decreased under an extremely low oxygen tension of less than 1% O2. PAK inhibition suppressed the hypoxia-induced increase of the migration speed by preventing VE-cadherin internalization into HUVECs. A decrease in the migration speed was also obtained by chemical stabilization of HIF-1α, suggesting that excessive accumulation of HIF-1α diminishes collective cell migration. These results indicate that the oxygen-dependent variation of the migration speed of vascular endothelial cells is mediated by the regulation of VE-cadherin through the PAK pathway, as well as other mechanisms via HIF-1α, especially under extreme hypoxic conditions.

Dataset Information

TNF? promotes CAR-dependent migration of leukocytes across epithelial monolayers.

Publications

TNFα promotes CAR-dependent migration of leukocytes across epithelial monolayers.

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