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Epithelial ICAM-1 and ICAM-2 regulate the egression of human T cells across the bronchial epithelium.

Porter JC, Hall A - FASEB J. (2008)

Bottom Line: We, therefore, looked for other epithelial ligands for LFA-1 and demonstrate that ICAM-2, but not ICAM-3, is expressed on the bronchial epithelium.Inhibition of LFA-1/ICAM-1 and ICAM-2 interactions on the basolateral epithelium does not prevent egressing T cells from adhering, polarizing, or moving over the basal epithelium, but it does prevent their recognition of the interepithelial junctions.In conclusion, we show that egression of T cells involves three distinct sequential steps: adhesion, junctional recognition, and diapedesis; we further demonstrate that ICAM-2 is expressed on the bronchial epithelium and, together with ICAM-1, has an essential function in the clearance of T cells from the lung.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Cell Biology, University College London, London, UK. joanna.porter@ucl.ac.uk

ABSTRACT
Egression of inflammatory cells from the lung interstitium into the airway lumen is critical for the resolution of inflammation, but the underlying mechanisms of this egression are unclear. Here, we use an in vitro system, in which human T cells migrate across a bronchial epithelial monolayer, to investigate the molecules involved. We show that although inhibition of T-cell LFA-1 blocks egression by 75 +/- 5.6% (P<0.0001), inhibition of the LFA-1-ligand ICAM-1 on the epithelium only inhibits by 52.7 +/- 0.06% (P=0.0001). We, therefore, looked for other epithelial ligands for LFA-1 and demonstrate that ICAM-2, but not ICAM-3, is expressed on the bronchial epithelium. Blocking ICAM-2 inhibits egression by 50.95 +/- 10.79% (P=0.04), and blocking both ICAM-1 and ICAM-2 inhibits egression by 69.6 +/- 5.2% (P< 0.0001). Inhibition of LFA-1/ICAM-1 and ICAM-2 interactions on the basolateral epithelium does not prevent egressing T cells from adhering, polarizing, or moving over the basal epithelium, but it does prevent their recognition of the interepithelial junctions. In conclusion, we show that egression of T cells involves three distinct sequential steps: adhesion, junctional recognition, and diapedesis; we further demonstrate that ICAM-2 is expressed on the bronchial epithelium and, together with ICAM-1, has an essential function in the clearance of T cells from the lung.

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Contribution of Rho-GTPase pathways to epithelial junction integrity and to transepithelial migration. A) TER was measured before and after incubation and washout of inhibitors and is expressed as a percentage of control TER. Preincubation of the monolayer with cytochalasin D was used as a control for junctional disruption; Inhibition of Rho-kinase (Y27632) or N-WASp (wiskostatin) decreased TER. B) Epithelial monolayers were preincubated with inhibitors at doses shown. After 2 h, the inhibitors were extensively washed out, and transepithelial migration was measured. Rho-kinase (Y27632) and cytochalasin D caused an increase, and wiskostatin caused a decrease in transepithelial migration. Data are means ± sd of triplicates; 1 representative experiment of 3 is shown. NS, not significant (P≥0.05). *P < 0.05, **P < 0.005 vs. control; 2-tailed Student’s t test.
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Figure 5: Contribution of Rho-GTPase pathways to epithelial junction integrity and to transepithelial migration. A) TER was measured before and after incubation and washout of inhibitors and is expressed as a percentage of control TER. Preincubation of the monolayer with cytochalasin D was used as a control for junctional disruption; Inhibition of Rho-kinase (Y27632) or N-WASp (wiskostatin) decreased TER. B) Epithelial monolayers were preincubated with inhibitors at doses shown. After 2 h, the inhibitors were extensively washed out, and transepithelial migration was measured. Rho-kinase (Y27632) and cytochalasin D caused an increase, and wiskostatin caused a decrease in transepithelial migration. Data are means ± sd of triplicates; 1 representative experiment of 3 is shown. NS, not significant (P≥0.05). *P < 0.05, **P < 0.005 vs. control; 2-tailed Student’s t test.

Mentions: In an attempt to understand these steps in T-cell egression further, we turned our attention to the epithelial cell. In models of leukocyte extravasation, the engagement and cross linking of endothelial ICAM-1 by adherent lymphocytes triggers Rho GTPase, which may reorganize junctions in preparation for diapedesis (19, 20). We therefore examined epithelial signaling pathways downstream of T-cell adhesion and the role of the Rho family GTPases-Rho, Rac, and Cdc42. Inhibition of Rac with the small molecule NSC23766 (21) had no effect on the permeability of the epithelial monolayer as measured by TER (data not shown), whereas inhibition of the Rho/Rho kinase pathway with the inhibitor Y27632 (22) or inhibition of the Cdc42/N-WASp pathway with wiskostatin (23) caused an increase in epithelial junctional permeability comparable to that seen with cytochalasin D (Fig. 5A; P<0.01 for all 3 conditions). However, although inhibition of epithelial Rho kinase increased transepithelial migration to a similar extent as pretreatment of the epithelium with cytochalasin D (Fig. 5B; P<0.02), pretreatment with wiskostatin inhibited transepithelial migration (Fig. 5B; P=0.014). The effect of wiskostatin was dose dependent, although at high doses (50 μM), the epithelial monolayer became disrupted, and at 100 μM, transepithelial migration increased (data not shown), indicating that wiskostatin was not inhibiting the migration of T cells, but was acting on the epithelial cells. Chlorinated and unhalogenated analogues of wiskostatin that do not inhibit N-WASp had no effect (data not shown).


Epithelial ICAM-1 and ICAM-2 regulate the egression of human T cells across the bronchial epithelium.

Porter JC, Hall A - FASEB J. (2008)

Contribution of Rho-GTPase pathways to epithelial junction integrity and to transepithelial migration. A) TER was measured before and after incubation and washout of inhibitors and is expressed as a percentage of control TER. Preincubation of the monolayer with cytochalasin D was used as a control for junctional disruption; Inhibition of Rho-kinase (Y27632) or N-WASp (wiskostatin) decreased TER. B) Epithelial monolayers were preincubated with inhibitors at doses shown. After 2 h, the inhibitors were extensively washed out, and transepithelial migration was measured. Rho-kinase (Y27632) and cytochalasin D caused an increase, and wiskostatin caused a decrease in transepithelial migration. Data are means ± sd of triplicates; 1 representative experiment of 3 is shown. NS, not significant (P≥0.05). *P < 0.05, **P < 0.005 vs. control; 2-tailed Student’s t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2630786&req=5

Figure 5: Contribution of Rho-GTPase pathways to epithelial junction integrity and to transepithelial migration. A) TER was measured before and after incubation and washout of inhibitors and is expressed as a percentage of control TER. Preincubation of the monolayer with cytochalasin D was used as a control for junctional disruption; Inhibition of Rho-kinase (Y27632) or N-WASp (wiskostatin) decreased TER. B) Epithelial monolayers were preincubated with inhibitors at doses shown. After 2 h, the inhibitors were extensively washed out, and transepithelial migration was measured. Rho-kinase (Y27632) and cytochalasin D caused an increase, and wiskostatin caused a decrease in transepithelial migration. Data are means ± sd of triplicates; 1 representative experiment of 3 is shown. NS, not significant (P≥0.05). *P < 0.05, **P < 0.005 vs. control; 2-tailed Student’s t test.
Mentions: In an attempt to understand these steps in T-cell egression further, we turned our attention to the epithelial cell. In models of leukocyte extravasation, the engagement and cross linking of endothelial ICAM-1 by adherent lymphocytes triggers Rho GTPase, which may reorganize junctions in preparation for diapedesis (19, 20). We therefore examined epithelial signaling pathways downstream of T-cell adhesion and the role of the Rho family GTPases-Rho, Rac, and Cdc42. Inhibition of Rac with the small molecule NSC23766 (21) had no effect on the permeability of the epithelial monolayer as measured by TER (data not shown), whereas inhibition of the Rho/Rho kinase pathway with the inhibitor Y27632 (22) or inhibition of the Cdc42/N-WASp pathway with wiskostatin (23) caused an increase in epithelial junctional permeability comparable to that seen with cytochalasin D (Fig. 5A; P<0.01 for all 3 conditions). However, although inhibition of epithelial Rho kinase increased transepithelial migration to a similar extent as pretreatment of the epithelium with cytochalasin D (Fig. 5B; P<0.02), pretreatment with wiskostatin inhibited transepithelial migration (Fig. 5B; P=0.014). The effect of wiskostatin was dose dependent, although at high doses (50 μM), the epithelial monolayer became disrupted, and at 100 μM, transepithelial migration increased (data not shown), indicating that wiskostatin was not inhibiting the migration of T cells, but was acting on the epithelial cells. Chlorinated and unhalogenated analogues of wiskostatin that do not inhibit N-WASp had no effect (data not shown).

Bottom Line: We, therefore, looked for other epithelial ligands for LFA-1 and demonstrate that ICAM-2, but not ICAM-3, is expressed on the bronchial epithelium.Inhibition of LFA-1/ICAM-1 and ICAM-2 interactions on the basolateral epithelium does not prevent egressing T cells from adhering, polarizing, or moving over the basal epithelium, but it does prevent their recognition of the interepithelial junctions.In conclusion, we show that egression of T cells involves three distinct sequential steps: adhesion, junctional recognition, and diapedesis; we further demonstrate that ICAM-2 is expressed on the bronchial epithelium and, together with ICAM-1, has an essential function in the clearance of T cells from the lung.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Cell Biology, University College London, London, UK. joanna.porter@ucl.ac.uk

ABSTRACT
Egression of inflammatory cells from the lung interstitium into the airway lumen is critical for the resolution of inflammation, but the underlying mechanisms of this egression are unclear. Here, we use an in vitro system, in which human T cells migrate across a bronchial epithelial monolayer, to investigate the molecules involved. We show that although inhibition of T-cell LFA-1 blocks egression by 75 +/- 5.6% (P<0.0001), inhibition of the LFA-1-ligand ICAM-1 on the epithelium only inhibits by 52.7 +/- 0.06% (P=0.0001). We, therefore, looked for other epithelial ligands for LFA-1 and demonstrate that ICAM-2, but not ICAM-3, is expressed on the bronchial epithelium. Blocking ICAM-2 inhibits egression by 50.95 +/- 10.79% (P=0.04), and blocking both ICAM-1 and ICAM-2 inhibits egression by 69.6 +/- 5.2% (P< 0.0001). Inhibition of LFA-1/ICAM-1 and ICAM-2 interactions on the basolateral epithelium does not prevent egressing T cells from adhering, polarizing, or moving over the basal epithelium, but it does prevent their recognition of the interepithelial junctions. In conclusion, we show that egression of T cells involves three distinct sequential steps: adhesion, junctional recognition, and diapedesis; we further demonstrate that ICAM-2 is expressed on the bronchial epithelium and, together with ICAM-1, has an essential function in the clearance of T cells from the lung.

Show MeSH
Related in: MedlinePlus