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. 2012 May 1;109(18):7097-102.
doi: 10.1073/pnas.1112519109. Epub 2012 Apr 17.

Dynamic Migration of γδ Intraepithelial Lymphocytes Requires Occludin

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Free PMC article

Dynamic Migration of γδ Intraepithelial Lymphocytes Requires Occludin

Karen L Edelblum et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

γδ intraepithelial lymphocytes (IELs) are located beneath or between adjacent intestinal epithelial cells and are thought to contribute to homeostasis and disease pathogenesis. Using in vivo microscopy to image jejunal mucosa of GFP γδ T-cell transgenic mice, we discovered that γδ IELs migrate actively within the intraepithelial compartment and into the lamina propria. As a result, each γδ IEL contacts multiple epithelial cells. Occludin is concentrated at sites of γδ IEL/epithelial interaction, where it forms a ring surrounding the γδ IEL. In vitro analyses showed that occludin is expressed by epithelial and γδ T cells and that occludin derived from both cell types contributes to these rings and to γδ IEL migration within epithelial monolayers. In vivo TNF administration, which results in epithelial occludin endocytosis, reduces γδ IEL migration. Further in vivo analyses demonstrated that occludin KO γδ T cells are defective in both initial accumulation and migration within the intraepithelial compartment. These data challenge the paradigm that γδ IELs are stationary in the intestinal epithelium and demonstrate that γδ IELs migrate dynamically to make extensive contacts with epithelial cells. The identification of occludin as an essential factor in γδ IEL migration provides insight into the molecular regulation of γδ IEL/epithelial interactions.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
γδ IELs migrate dynamically within the intestinal epithelium. (A) Time-lapse images of a migrating GFP γδ IEL (green) within the jejunal villous epithelium of a transgenic mouse expressing mRFP-ZO-1 (red), to label tight junctions, and injected with Hoechst dye (blue) to label epithelial nuclei. Lateral membranes of adjacent epithelial cells are indicated by dashed white lines and the BM by a dashed yellow line (Movie S3). (Scale bars, 10 μm.) (B) A single time point (Left) and 60 min maximum projection (Right) of γδ IEL migration. GFP γδ IEL (green), the luminal marker Alexa Fluor 633 (red), and Hoechst-labeled nuclei (blue) are shown. The BM is indicated by a dashed white line. For the maximum projection, distance from the lumen is pseudocolored; 0–15 μm, dark blue; 16–30 μm, light blue; >30 μm, yellow. The small region of green signal in the lumen is an artifact of the projection. (Scale bars, 20 μm.) (C) Occludin or ZO-1 (red) were immunolabeled in jejunum from GFP γδ T-cell (green) transgenic mice. Nuclei are labeled with Hoechst (blue). The epithelial tight junction is indicated by white arrows. Punctae of ZO-1 adjacent to the T cell is indicated by yellow arrows. (Scale bars, 10 μm.)
Fig. 2.
Fig. 2.
Occludin forms rings at sites of γδ IEL/epithelial contact and promotes γδ IEL migration into epithelial monolayers. (A) 3D reconstructions, viewed from the lateral membrane, of isolated γδ IELs (CD8α, green) that have migrated into cultured epithelial monolayers. Occludin or ZO-1 is shown in red. (Scale bars, 5 μm.) (B) 3D reconstructions of wild-type γδ IELs (CD8α, green) within occludin KD epithelium or occludin KO γδ IELs within wild-type epithelium. Occludin is shown in red. (Scale bars, 5 μm.) (C) Morphometric analysis of wild-type, occludin, or CD103 KO γδ IEL migration into wild-type, occludin-, or ZO-1–deficient epithelium (n = 3). P < 0.001.
Fig. 3.
Fig. 3.
Occludin is required for γδ IEL migration in vivo. Mixed bone marrow chimeras were generated to express wild-type or occludin KO GFP γδ T cells in T-cell–deficient hosts. (A) Flow cytometric analysis of CD3+ T cells expressing GFP in small intestinal IELs, lamina propria lymphocytes (LP), or splenocytes isolated from wild-type or occludin KO γδ IEL chimeras, as indicated. (B) Jejunum of wild-type or occludin KO GFP γδ chimeras labeled to detect GFP (green), F-actin (blue), laminin (red), and nuclei (cyan). (Scale bars, 20 μm.) (C) A single time point (Left) and 60-min maximum projection (Right) of GFP γδ IEL migration in wild-type and occludin KO γδ IEL chimeras. γδ IEL (green), ZO-1 and intestinal lumen (red), nuclei (blue). The BM is indicated by a dashed yellow line. Distance from the lumen is pseudocolored as described in Fig. 1B. (Scale bars, 20 μm.) (D) Time-lapse images taken from Movies S5 and S6 show wild-type or occludin KO γδ IEL (green) migration over ≈20 min. Intestinal lumen (red) and nuclei (blue) are shown. The BM is indicated by a dashed yellow line. (Scale bar, 10 μm.) (E) Distance of γδ splenocyte tracks in GFP γδ wild-type or GFP γδ occludin KO mice. P < 0.001.

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