Chemokine-dependent T cell migration requires aquaporin-3-mediated hydrogen peroxide uptake - PubMed (original) (raw)

Chemokine-dependent T cell migration requires aquaporin-3-mediated hydrogen peroxide uptake

Mariko Hara-Chikuma et al. J Exp Med. 2012.

Abstract

Chemokine-dependent trafficking is indispensable for the effector function of antigen-experienced T cells during immune responses. In this study, we report that the water/glycerol channel aquaporin-3 (AQP3) is expressed on T cells and regulates their trafficking in cutaneous immune reactions. T cell migration toward chemokines is dependent on AQP3-mediated hydrogen peroxide (H(2)O(2)) uptake but not the canonical water/glycerol transport. AQP3-mediated H(2)O(2) transport is essential for the activation of the Rho family GTPase Cdc42 and the subsequent actin dynamics. Coincidentally, AQP3-deficient mice are defective in the development of hapten-induced contact hypersensitivity, which is attributed to the impaired trafficking of antigen-primed T cells to the hapten-challenged skin. We therefore suggest that AQP3-mediated H(2)O(2) uptake is required for chemokine-dependent T cell migration in sufficient immune response.

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Figures

Figure 1.

Figure 1.

Normal cellularity and subpopulations of T cells in AQP3-null mice. (a) The messenger RNA expression levels of AQP3 in sorted CD4+ and CD8+ T cells as well as in kidney and brain tissues were assessed by real-time PCR (SE; n = 4). Data are expressed as the AQP3/GAPDH ratio. (b) Flow cytometric analysis of AQP3 expression in CD4+ (top) or CD8+ (bottom) T cells from WT and AQP3−/− mice. (c) Cell population analysis in the spleen (left) and LN (right). The numbers of total cells, CD4+ and CD8+ cells from WT and AQP3−/− mice (SE; n = 4), are shown. (d) Cell population analysis in the thymus. The numbers of total cells and indicated subsets (SE; n = 4) are shown. (e) [3H]Thymidine incorporation in CD4+ T cells from WT and AQP3−/− mice (SE; n = 4–5). Each experiment was performed three times.

Figure 2.

Figure 2.

Impaired chemotaxis efficiency and F-actin polymerization levels of AQP3-deficient T lymphocytes. (a) Chemotaxis assay. The migration efficiency of CD4+ T cells from WT and AQP3−/− mice toward the ligands CXCL12 (100 ng/ml), CCL19 (100 ng/ml), CCL17 (80 ng/ml), or CCL27 (80 ng/ml) was examined using a transwell chamber with 5-µm pores. Data are expressed as the percentage of WT control migration levels (SE; n = 5; *, P < 0.01). (b) Three-dimensional chemotaxis assay in response to CXCL12 gradient for 60 min. Accumulated distances for each cell (SE; n = 50; *, P < 0.01) are shown. (c) Transendothelial migration of CD4+ T cells through mouse vascular endothelial cells (F-2 cells) in the presence of 100 ng/ml CXCL12 (SE; n = 5; *, P < 0.01). (d) CD4+ WT and AQP3−/− T cells were stimulated with 500 ng/ml CXCL12 and stained with phalloidin-FITC (90 s) or with anti-AQP3 (3 min; cy3). (left) Representative immunofluorescence microscopy. Bars, 10 µm. (right) T cells were stimulated for 60 or 90 s with CXCL12. The aspect ratio of cell shape was quantified by measuring the length of the major axis divided by the width of the minor axis (SE; n = 50; *, P < 0.01). (e) T cells from WT and AQP3−/− mice were stimulated with 500 ng/ml CXCL12 and stained with phalloidin-FITC and CD4–Pacific blue. (left) Flow cytometry analysis of phalloidin-FITC gated on CD4+ cells. One of four representative experiments is shown. (right) The mean fluorescence intensity (MFI) of phalloidin-FITC in the CD4+ cells was analyzed (SE; n = 5; *, P < 0.01, WT vs. AQP3−/− cells). Each experiment was performed three times.

Figure 3.

Figure 3.

Impaired CHS with decreased T cell trafficking to a challenged skin site in AQP3-null mice. (a, left) Mice were sensitized with DNFB, TNCB, or Oxa and challenged 5 d later on the ear. The ear thickness was measured in micrometers 24 h after the challenge (SE; n = 5; *, P < 0.01). (right) Hematoxylin and eosin staining of the ears of sensitized WT and AQP3−/− mice at 24 h after challenge with DNFB. Bars: (left) 100 µm; (right) 20 µm. (b) CHS test using BM cell–transferred mice. C57BL/6 mice received transplants of BM cells from WT and AQP3−/− mice. The CHS test was performed with DNFB 2 mo later (SE; n = 4; *, P < 0.01). (c) Adoptive transfer experiments by intravenous injection. LN cells from sensitized donor WT and AQP3−/− mice (Don) were injected intravenously (3 × 107 cells/head) to recipient mice (Rec). Ear swelling at 24 h after challenge (SE; n = 3–5; *, P < 0.01) is shown. (d) Adoptive transfer experiments by intravenous injection. LN cells from sensitized WT and AQP3−/− donors were stained with CMFDA and injected into recipient WT mice, and these mice were challenged with 0.3% DNFB. The ear skin, which was painted with DNFB, and LNs were excised 24 h after challenge. CD4+ and CMDFA+ cells were analyzed by flow cytometric analysis (SE; n = 4; *, P < 0.01). (e) Adoptive transfer experiments by subcutaneous injection (2 × 105 cells) into the ears. Ear swelling at 24 h after challenge is shown (SE; n = 4). Experiments in a, c, and e were performed in two other independent experiments and in b and d in one other experiment with similar results.

Figure 4.

Figure 4.

Impaired chemokine-induced Cdc42 activation in AQP3-deficient T cells. (a–c) Quantifications of Cdc42, Rac1, and RhoA activation. T cells were incubated with 250 ng/ml CXCL12 for 1 min, and GTP-bound active form of Cdc42 (a), Rac1 (b), and RhoA (c) were assessed using the G-LISA activation assay kit (SE; n = 4–5; *, P < 0.05; **, P < 0.01). (d) Immunoblot analyses to detect the phosphorylation of WASP and Arp2. T cells were incubated with 250 ng/ml CXCL12 for 3 min and were analyzed using antibodies against phospho-WASP (P-WASP), WASP, phospho-Arp2, Arp2, and β-actin. The blots shown are representative of three separate sets of experiments. (e) Phosphorylated Itk in response to CXCL12 (250 ng/ml, 1 min) was quantified with BD cytometric bead array (SE; n = 4–5; **, P < 0.01). (f and g) F-actin polymerization in response to 500 ng/ml CXCL12 in AQP3 knockdown (f) and/or V12-Cdc42–positive human primary T cells (g). One representative experiment of three experiments is shown. Experiments in a–c and e were performed in two other independent experiments.

Figure 5.

Figure 5.

Water and H2O2 permeability depended on AQP3 expression in T cells. (a) The osmotic water permeability of CD4+ T cells isolated from WT and AQP3−/− mice was measured based on the time course of scattered light intensity in response to a 150-mM inwardly directed mannitol gradient generated by stopped flow at 22°C. (left) Representative time course data showing responses to rapid changes in perfusate osmolality between 300 and 450 mOsm. (right) Averaged osmotic water permeability coefficients (_P_f; SE; n = 5; *, P < 0.01). (b) Glycerol permeability was measured in response to a 150-mM inwardly glycerol gradient by stopped flow at 30°C (SE; n = 4). (c) H2O2 uptake into T cells. CD4+ T cells were incubated with 10–300 µM H2O2 for 15 s, and cellular H2O2 levels were detected using CM-H2DCFDA reagent by flow cytometric analysis. The mean fluorescence intensity (MFI) of CM-H2DCFDA fluorescence (SE; n = 4; *, P < 0.01, H2O2 added vs. control cells) is shown. (d) WT CD4+ cells were incubated with 5 µM DPI or 2,000 U/ml catalase for 30 min and followed with 500 ng/ml CXCL12 for 15–180 s at 37°C. CD4+ cellular H2O2 levels were detected using CM-H2DCFDA reagent by flow cytometry analysis (SE; n = 5; *, P < 0.01, CXCL12 treated vs. control cells). (e) Cellular H2O2 levels in WT and AQP3−/− CD4+ cells after CXCL12 stimulation (500 ng/ml, 15 or 30 s; SE; n = 5; *, P < 0.01 vs. control cells). Each experiment was performed three times.

Figure 6.

Figure 6.

Intracellular H2O2 affects CXCL12-induced cell signaling and chemotaxis. (a–c) CD4+ T cells were incubated with 2,000 U/ml catalase (Cat), 5 µM DPI, or vehicle (Veh) for 30 min at 37°C and stimulated with 250 or 500 ng/ml CXCL12. (a) Quantification of Cdc42 active form (GTP bound) with G-LISA activation assay kit (SE; n = 4–5; *, P < 0.01). (b) Cytometric bead array–based quantification of phosphorylated Itk (SE; n = 4–5; *, P < 0.01). (c) Mean fluorescence intensity (MFI) of phalloidin–Alexa Fluor 660 in the CD4+ cells (SE; n = 5; *, P < 0.01). (d) Chemotaxis assay toward 100 ng/ml CXCL12 for 1 h (SE; n = 5; *, P < 0.01). (e and f) CD4+ T cells were incubated with 10–100 µM H2O2 for 1 min at 37°C. (e) Quantification of Cdc42 active form (GTP bound; SE; n = 4; *, P < 0.01). (f) The amount of phosphorylated Itk (SE; n = 4; *, P < 0.01). Experiments in a–d were performed in two other independent experiments and in e and f in one other experiment with similar results.

Figure 7.

Figure 7.

H2O2 supplementation restored the impaired cell signaling and chemotaxis in AQP3-deficient T cells. (a–d) CD4+ T cells from WT and AQP3−/− mice were stimulated by 500 ng/ml CXCL12 together with/without exogenous addition of 100 µM H2O2. (a) Intracellular H2O2 levels for 15 s (SE; n = 4; *, P < 0.01, treated vs. control cells). (b) The amount of phosphorylated Itk (SE; n = 4; *, P < 0.01). (c) Quantification of Cdc42 active form (GTP bound; SE; n = 4; *, P < 0.01). (d) Mean fluorescence intensity (MFI) of phalloidin-FITC in the CD4+ cells was analyzed (SE; n = 4; *, P < 0.01). (e) T cells were incubated with 100 µM H2O2 for 15 s, washed, and assayed for chemotaxis for 30 min toward 200 ng/ml CXCL12 (SE; n = 5; *, P < 0.01). Experiments were performed in one other experiment with similar results.

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