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. 2010 Aug 15;185(4):2502-15.
doi: 10.4049/jimmunol.0901778. Epub 2010 Jul 12.

Cell adhesion molecules regulate fibrotic process via Th1/Th2/Th17 cell balance in a bleomycin-induced scleroderma model

Ayumi Yoshizaki  1 , Koichi Yanaba, Yohei Iwata, Kazuhiro Komura, Asako Ogawa, Yuichiro Akiyama, Eiji Muroi, Toshihide Hara, Fumihide Ogawa, Motoi Takenaka, Kazuhiro Shimizu, Minoru Hasegawa, Manabu Fujimoto, Thomas F Tedder, Shinichi Sato
Affiliations

Cell adhesion molecules regulate fibrotic process via Th1/Th2/Th17 cell balance in a bleomycin-induced scleroderma model

Ayumi Yoshizaki et al. J Immunol. .

Abstract

Mice s.c. injected with bleomycin, an experimental model for human systemic sclerosis, develop skin and lung fibrosis, which is mediated by inflammatory cell infiltration. This process is highly regulated by multiple adhesion molecules and does not require Ag sensitization. To assess the role of adhesion molecules in this pathogenetic process, bleomycin-induced fibrosis was examined in mice lacking adhesion molecules. L-selectin and/or ICAM-1 deficiency inhibited skin and lung fibrosis with decreased Th2 and Th17 cytokines and increased Th1 cytokines. In contrast, P-selectin deficiency, E-selectin deficiency with or without P-selectin blockade, or P-selectin glycoprotein ligand 1 (PSGL-1) deficiency augmented the fibrosis in parallel with increased Th2 and Th17 cytokines and decreased Th1 cytokines. Furthermore, loss of L-selectin and/or ICAM-1 reduced Th2 and Th17 cell numbers in bronchoalveolar lavage fluid, whereas loss of P-selectin, E-selectin, or PSGL-1 reduced Th1 cell numbers. Moreover, Th1 cells exhibited higher PSGL-1 expression and lower expression of LFA-1, a ligand for ICAM-1, whereas Th2 and Th17 cells showed higher LFA-1 and lower PSGL-1 expression. This study suggests that L-selectin and ICAM-1 regulate Th2 and Th17 cell accumulation into the skin and lung, leading to the development of fibrosis, and that P-selectin, E-selectin, and PSGL-1 regulate Th1 cell infiltration, resulting in the inhibition of fibrosis.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1
Skin fibrosis (A, B) and the numbers of mast cells, macrophages, T cells, and B cells at the bleomycin (BLM) injected site of skin (C) from PBS-treated (white bar) or BLM-treated (black bar) wild type (WT) mice, L-selectin−/− mice (L−/−), ICAM-1−/− mice (ICAM-1−/−), L-selectin/ICAM-1−/− mice (L/ICAM-1−/−), P-selectin−/− mice (P−/−), E-selectin−/− mice (E−/−), E-selectin−/− mice treated with anti-P-selectin mAb (E−/− + anti-P Ab), and PSGL-1−/− mice (PSGL-1−/−). Representative histological sections stained with H&E are shown (original magnification x40, A). Skin fibrosis was assessed by quantitatively measuring dermal thickness and hydroxyproline content 4 weeks after BLM treatment (B). Mast cells were identified by toluidine blue staining, while macrophages, T cells, and B cells were stained with F4/80, anti-CD3 mAb, and anti-B220 mAb, respectively (C). These results represent those obtained with at least 10 mice of each group. The dermal thickness was measured under a light microscope. Cells were counted in 10 random grids under magnification of x400 high power fields (HPF). Each histogram shows the mean (± SD) results obtained for 10 mice of each group. * P < 0.05, ** P < 0.01 versus PBS-treated each group of the mice. † P < 0.05, †† P < 0.01 versus BLM-treated WT mice.
Figure 2
Figure 2
Lung fibrosis (A, B) and the influx numbers of total leukocytes, including neutrophils, macrophages, T cells, and B cells, into BAL fluid (C) from PBS-treated (white bar) or bleomycin (BLM)-treated (black bar) wild type (WT) mice, L-selectin−/− mice (L−/−), ICAM-1−/− mice (ICAM-1−/−), L-selectin/ICAM-1−/− mice (L/ICAM-1−/−), P-selectin−/− mice (P−/−), E-selectin−/− mice (E−/−), E-selectin−/− mice treated with anti-P-selectin mAb (E−/− + anti-P Ab), and PSGL-1−/− mice (PSGL-1−/−). Representative histological sections stained with H&E are shown (original magnification x100, A). Lung fibrosis was assessed by quantitatively measuring lung fibrosis score and hydroxyproline content 4 weeks after BLM treatment (B). The BAL cell counts were as described in the Materials and Methods section. These results were obtained from at least 10 mice in each group. Lung fibrosis score was measured under a light microscope. The differential BAL cells were counted in 10 random grids under magnification of x400 high power fields (HPF). Each histogram shows the mean (± SD) results obtained for 10 mice of each group. * P < 0.05, ** P < 0.01 versus PBS-treated each group. †P < 0.05, ††P < 0.01 versus BLM -treated WT mice.
Figure 3
Figure 3
Levels of IL-4, IL-6, IFN-γ, IL-17, TNF-α, TGF-β1, and IL-10 in serum samples from wild type (WT) mice, L-selectin−/− mice (L−/−), ICAM-1−/− mice (ICAM-1−/−), L-selectin/ICAM-1−/− mice (L/ICAM-1−/−), P-selectin−/− mice (P−/−), E-selectin−/− mice (E−/−), E-selectin−/− mice treated with anti-P-selectin mAb (E−/− + anti-P Ab), and PSGL-1−/− mice (PSGL-1−/−) treated with either PBS (white bar) or bleomycin (black bar). Serum samples were obtained by a cardiac puncture 4 weeks after treatment with either bleomycin or PBS. Serum cytokine levels were assessed using specific ELISA. Each histogram shows the mean (± SD) results obtained for 10 mice of each group. ** P < 0.01 versus PBS-treated each group. †P < 0.05, ††P < 0.01 versus BLM -treated WT mice.
Figure 4
Figure 4
Proliferation and collagen synthesis of dermal fibroblasts obtained from wild type (WT) mice. Cultured fibroblasts were serum starved for 12 hours and then cultured for 24 hours with murine rIL-4 (10 ng/ml), rIFN-γ (10 ng/ml), and rIL-17 (50 ng/ml). Total RNA from fibroblasts was extracted and reverse transcribed to cDNA, and mRNA expression of COL1A2 and TGF-β1 was analyzed by real-time PCR. In proliferation assay, after 24 hour incubation, BrdU (10 μM) was added to each well and incubated for 24 hours. BrdU incorporation in proliferating cells was quantified by ELISA. Each histogram shows the mean (± SD) results obtained for 6 mice of each group. * P < 0.05, ** P < 0.01 versus fibroblasts cultured with media alone.
Figure 5
Figure 5
Th1, Th2, and Th17 cell frequencies of BAL in PBS-treated (white bar) or bleomycin (BLM)-treated (black bar) wild type (WT) mice, L-selectin−/− mice (L−/−), ICAM-1−/− mice (ICAM-1−/−), L-selectin/ICAM-1−/− mice (L/ICAM-1−/−), P-selectin−/− mice (P−/−), E-selectin−/− mice (E−/−), E-selectin−/− mice treated with anti-P-selectin mAb (E−/− + anti-P Ab), and PSGL-1−/− mice (PSGL-1−/−). We determined Th1, Th2, and Th17 cells by surface CD4 expression and intracellular expression of IFN-γ, IL-4, and IL-17 as previously described (36). BAL fluid was analyzed by flow cytometry after 4 weeks PBS or BLM treatment. These data are representative of three independent experiments (A). Percentages of Th1, Th2, and Th17 cells are shown in the each quadrant. We also show summaries of Th1, Th2, and Th17 cell frequencies in each group (B). Each histogram shows the mean (± SD) results obtained for 10 mice of each group. * P < 0.005, ** P < 0.001 versus PBS-treated each group. † P < 0.05, ††P < 0.01 versus BLM -treated WT mice.
Figure 6
Figure 6
The LFA-1 and PSGL-1 expression levels (A and B) and ICAM-1, P-selectin, and E-selectin binding ability (C) in Th0, Th1, Th2, and Th17 cells. We determined Th0, Th1, Th2, and Th17 cells by surface CD4 expression and intracellular expression of IFN-γ, IL-4, and IL-17 as previously described (36). Polarized or nonpolarized splenic CD4+ T cells were analyzed by flow cytometry. These data are representative of three independent experiments. Numbers indicate the percentage of cells in each quadrant. Histograms indicate mean (± SD) fluorescence intensity (MFI).
Figure 7
Figure 7
Serum Ig levels in bleomycin-treated wild type (WT) mice, L-selectin−/− mice (L−/−), ICAM-1−/− mice (ICAM-1−/−), L-selectin/ICAM-1−/− mice (L/ICAM-1−/−), P-selectin−/− mice (P−/−), E-selectin−/− mice (E−/−), E-selectin−/− mice treated with anti-P-selectin mAb (E−/− + anti-P Ab), and PSGL-1−/− mice (PSGL-1−/−). Serum Ig levels in PBS-treated WT mice were used as control Ig levels (CTL). Serum Ig levels were determined by isotype-specific ELISA. Horizontal bars represent mean Ig levels. * P < 0.05, ** P < 0.01 versus each CTL group. † P < 0.05, ††P < 0.01 versus BLM -treated WT mice.
Figure 8
Figure 8
Autoantibody levels in sera from bleomycin-treated wild type (WT) mice, L-selectin−/− mice (L−/−), ICAM-1−/− mice (ICAM-1−/−), L-selectin/ICAM-1−/− mice (L/ICAM-1−/−), P-selectin−/− mice (P−/−), E-selectin−/− mice (E−/−), E-selectin−/− mice treated with anti-P-selectin mAb (E−/− + anti-P Ab), and PSGL-1−/− mice (PSGL-1−/−). Autoantibody levels in sera from PBS-treated WT mice were used as control (CTL). Relative autoantibody levels were determined by Ig subclass-specific ELISA. Values in parentheses represent the dilutions of pooled sera giving half-maximal OD values in autoantigen-specific ELISA, which were determined by linear regression analysis to generate arbitrary units per milliliter that could be directly compared between each group of mice (n = 6 for each). Horizontal bars represent mean Ab levels. * P < 0.05, ** P < 0.01 versus each CTL group. † P < 0.05, †† P < 0.01 versus BLM-treated WT mice.
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