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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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Role of CAR in transepithelial migration. A) Transepithelial migrations assays were performed as described. Preincubation of T cells with a blocking mAb against LFA-1, or of the epithelial monolayer with a blocking mAb against CAR, caused a significant reduction in transepithelial migration. B–E) LSCM images of basal (B, D) and apical (C, E) epithelium 1 h into a transepithelial migration assay, following preincubation of the epithelial monolayer with an isotype-matched control mAb (B, C) or a blocking mAb against CAR (D, E). Images were taken at 0.2-μm intervals extending 1 μm basal to the ZO-1 staining (B, D) and 1 μm apical to the ZO-1 staining (C, E) and then projected as a composite. In addition, the ZO-1 staining from the same series was analyzed frame by frame, and the composite was imported into Adobe Illlustrator and recreated as an overlay in orange to show the tight junctions. Scale bar = 15 μm. F) Epithelial cell monolayers were untreated (white bars) or pretreated with 5 μM cytochalasin D (black bars) and/or a control mAb or a blocking mAb to CAR. Monolayers were washed and transepithelial migration assays were performed as described above. T cells were untreated or treated with a blocking mAb to LFA-1. Data are means ± sd of triplicates; 1 representative experiment of 3 is shown. NS, not significant (P≥0.05). **P < 0.005, ***P < 0.0005; 2-tailed Student’s t test.
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Figure 4: Role of CAR in transepithelial migration. A) Transepithelial migrations assays were performed as described. Preincubation of T cells with a blocking mAb against LFA-1, or of the epithelial monolayer with a blocking mAb against CAR, caused a significant reduction in transepithelial migration. B–E) LSCM images of basal (B, D) and apical (C, E) epithelium 1 h into a transepithelial migration assay, following preincubation of the epithelial monolayer with an isotype-matched control mAb (B, C) or a blocking mAb against CAR (D, E). Images were taken at 0.2-μm intervals extending 1 μm basal to the ZO-1 staining (B, D) and 1 μm apical to the ZO-1 staining (C, E) and then projected as a composite. In addition, the ZO-1 staining from the same series was analyzed frame by frame, and the composite was imported into Adobe Illlustrator and recreated as an overlay in orange to show the tight junctions. Scale bar = 15 μm. F) Epithelial cell monolayers were untreated (white bars) or pretreated with 5 μM cytochalasin D (black bars) and/or a control mAb or a blocking mAb to CAR. Monolayers were washed and transepithelial migration assays were performed as described above. T cells were untreated or treated with a blocking mAb to LFA-1. Data are means ± sd of triplicates; 1 representative experiment of 3 is shown. NS, not significant (P≥0.05). **P < 0.005, ***P < 0.0005; 2-tailed Student’s t test.

Mentions: It has previously been reported that CAR is expressed at tight junctions in epithelial cells (18). We first confirmed the expression of CAR in bronchial epithelial cells and found it colocalized with the tight junction marker ZO-1 (data not shown). Preincubation of the bronchial epithelial monolayer with a blocking antibody against CAR inhibited transepithelial migration of T cells by 40% (Fig. 4A; P=0.0019). Confocal microscopy at 1 h showed that when CAR was blocked, T cells were still polarized and able to move along the basal epithelium (Fig. 4B). Pretreatment of the epithelial monolayer with cytochalasin D leading to disruption of cell junctions did not significantly increase the baseline transepithelial migration (Fig. 4C; P=0.7), but it did rescue transepithelial migration inhibited by an anti-CAR antibody (Fig. 4C; P=0.0005). Cytochalasin D did not rescue the inhibition of transepithelial migration caused by blocking of LFA-1 (Fig. 4C; P=0.93), indicating that the CAR-dependent and LFA-1-dependent steps are distinct. We conclude that there are at least 3 distinct steps in the movement of T cells across the epithelium: adhesion, LFA-1-dependent movement into the lateral intercellular space to locate the junctions, and CAR-dependent diapedesis.


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

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

Role of CAR in transepithelial migration. A) Transepithelial migrations assays were performed as described. Preincubation of T cells with a blocking mAb against LFA-1, or of the epithelial monolayer with a blocking mAb against CAR, caused a significant reduction in transepithelial migration. B–E) LSCM images of basal (B, D) and apical (C, E) epithelium 1 h into a transepithelial migration assay, following preincubation of the epithelial monolayer with an isotype-matched control mAb (B, C) or a blocking mAb against CAR (D, E). Images were taken at 0.2-μm intervals extending 1 μm basal to the ZO-1 staining (B, D) and 1 μm apical to the ZO-1 staining (C, E) and then projected as a composite. In addition, the ZO-1 staining from the same series was analyzed frame by frame, and the composite was imported into Adobe Illlustrator and recreated as an overlay in orange to show the tight junctions. Scale bar = 15 μm. F) Epithelial cell monolayers were untreated (white bars) or pretreated with 5 μM cytochalasin D (black bars) and/or a control mAb or a blocking mAb to CAR. Monolayers were washed and transepithelial migration assays were performed as described above. T cells were untreated or treated with a blocking mAb to LFA-1. Data are means ± sd of triplicates; 1 representative experiment of 3 is shown. NS, not significant (P≥0.05). **P < 0.005, ***P < 0.0005; 2-tailed Student’s t test.
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Figure 4: Role of CAR in transepithelial migration. A) Transepithelial migrations assays were performed as described. Preincubation of T cells with a blocking mAb against LFA-1, or of the epithelial monolayer with a blocking mAb against CAR, caused a significant reduction in transepithelial migration. B–E) LSCM images of basal (B, D) and apical (C, E) epithelium 1 h into a transepithelial migration assay, following preincubation of the epithelial monolayer with an isotype-matched control mAb (B, C) or a blocking mAb against CAR (D, E). Images were taken at 0.2-μm intervals extending 1 μm basal to the ZO-1 staining (B, D) and 1 μm apical to the ZO-1 staining (C, E) and then projected as a composite. In addition, the ZO-1 staining from the same series was analyzed frame by frame, and the composite was imported into Adobe Illlustrator and recreated as an overlay in orange to show the tight junctions. Scale bar = 15 μm. F) Epithelial cell monolayers were untreated (white bars) or pretreated with 5 μM cytochalasin D (black bars) and/or a control mAb or a blocking mAb to CAR. Monolayers were washed and transepithelial migration assays were performed as described above. T cells were untreated or treated with a blocking mAb to LFA-1. Data are means ± sd of triplicates; 1 representative experiment of 3 is shown. NS, not significant (P≥0.05). **P < 0.005, ***P < 0.0005; 2-tailed Student’s t test.
Mentions: It has previously been reported that CAR is expressed at tight junctions in epithelial cells (18). We first confirmed the expression of CAR in bronchial epithelial cells and found it colocalized with the tight junction marker ZO-1 (data not shown). Preincubation of the bronchial epithelial monolayer with a blocking antibody against CAR inhibited transepithelial migration of T cells by 40% (Fig. 4A; P=0.0019). Confocal microscopy at 1 h showed that when CAR was blocked, T cells were still polarized and able to move along the basal epithelium (Fig. 4B). Pretreatment of the epithelial monolayer with cytochalasin D leading to disruption of cell junctions did not significantly increase the baseline transepithelial migration (Fig. 4C; P=0.7), but it did rescue transepithelial migration inhibited by an anti-CAR antibody (Fig. 4C; P=0.0005). Cytochalasin D did not rescue the inhibition of transepithelial migration caused by blocking of LFA-1 (Fig. 4C; P=0.93), indicating that the CAR-dependent and LFA-1-dependent steps are distinct. We conclude that there are at least 3 distinct steps in the movement of T cells across the epithelium: adhesion, LFA-1-dependent movement into the lateral intercellular space to locate the junctions, and CAR-dependent diapedesis.

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