E- and P-selectins synergistically inhibit bleomycin-induced pulmonary fibrosis

Abstract

The development of bleomycin-induced lung injury, which is a model of pulmonary fibrosis, results from inflammatory cell infiltration, a process highly regulated by the expression of multiple adhesion molecules. Therefore, bleomycin-induced lung fibrosis was examined in E-selectin-/- mice, P-selectin-/- mice, and E-selectin-/- mice treated with anti-P-selectin monoclonal antibody (mAb) in comparison of wild-type mice. E-selectin-/- mice treated with anti-P-selectin mAb exhibited augmented lung fibrosis histologically, increased lung collagen deposition, and increased mortality compared to wild-type mice. Furthermore, lung interferon-gamma mRNA expression decreased in E-selectin-/- mice treated with anti-P-selectin mAb relative to wild-type mice, while tumor necrosis factor-alpha and interleukin-6 mRNA expression increased in these mice. Similar changes were observed in E-selectin-/- mice, albeit to a lesser extent than those treated with anti-P-selectin mAb. Remarkably, flow cytometric analysis revealed that the frequency of interferon-gamma-producing natural killer T (NKT) cells in the bronchoalveolar lavage was decreased in E-selectin-/- mice and E-selectin-/- mice treated with anti-P-selectin mAb compared with wild-type mice. Moreover, the majority of NKT cells expressed high levels of CXCR3, suggesting that NKT cell infiltration is also dependent on CXCR3 expression. These results suggest that E- and P-selectins synergistically inhibit lung fibrosis by promoting the recruitment of NKT cells.

Figure 1

Bleomycin-induced lung fibrosis was augmented in E-selectin^−/− mice and E-selectin^−/− mice treated with anti-P-selectin. A: Representative histological sections of lungs from wild-type, P-selectin^−/−, E-selectin^−/−, and E-selectin^−/− mice treated with anti-P-selectin mAb 16 days after intratracheal bleomycin administration. Wild-type mice that received intratracheal saline injection served as controls (control). The lung sections were stained with H&E to evaluate alveolitis and with Azan-Mallory to identify collagen deposition. B: Lung fibrosis was evaluated according to the criteria described in Materials and Methods. The results are indicated as a mean ± SEM of eight mice in each group. Original magnifications, ×100.

Figure 2

Hydroxyproline content of lungs in mutant and wild-type mice 16 days after intratracheal bleomycin administration. Symbols represent results from individual mice with the horizontal line indicating the mean value for each group of mice.

Figure 3

Time course of survival in mutant and wild-type mice after intratracheal bleomycin administration (15 mice in each group).

Figure 4

Time course of neutrophil (A), lymphocyte (B), and macrophage (C) influx into the BAL from mutant and wild-type mice. BAL was collected at 2, 4, 8, 12, and 16 days after intratracheal bleomycin administration. Saline-treated mutant mice or wild-type mice showed no significant increase in leukocyte numbers (data not shown). All values represent the mean ± SEM of results obtained using eight mice in each group. Statistical analysis is presented in the Results section.

Figure 5

mRNA expression of IFN-γ (A), TNF-α (B), IL-6 (C), and TGF-β1 (D) in the lungs of mutant and wild-type mice 8 days after intratracheal bleomycin administration. mRNA expression was assessed by real-time RT-PCR. Values represent the mean ± SEM of results from seven mice in each group.

Figure 6

The reduced NK1.1⁺CD3⁺ cell numbers, decreased IFN-γ and CXCR3 expression levels in E-selectin^−/− mice and E-selectin^−/− mice treated with anti-P-selectin mAb. BAL fluid was analyzed by flow cytometry 4 days after bleomycin administration. Total number of NK1.1⁺CD3⁺ cells (A), intracellular IFN-γ expression level of CD3⁺ cells (B), CXCR3 expression levels of CD3⁺ cells (C), and representative dot plots of CXCR3⁺CD3⁺ cell frequency (D). Percentages of CXCR3⁺CD3⁺ cells are shown in the upper right quadrants. These data are representative of three independent experiments.

Figure 7

Lack of NK1.1⁺ cells augmented bleomycin-induced lung fibrosis. The mean pathological score (A), lung hydroxyproline content (B), and total leukocyte counts from BAL fluid (C) of wild-type mice, wild-type mice injected with anti NK1.1 mAb, E-selectin^−/− mice injected with anti-P-selectin mAb, and E-selectin^−/− mice injected with anti-P-selectin mAb and anti-NK1.1 mAb. Lungs were harvest 16 days after bleomycin administration. The pathological scores, hydroxyproline assay, and BAL cell counts were as described in the Materials and Methods section. The results were obtained from four mice in each group.