Attenuation of lung inflammation and fibrosis in CD69-deficient mice after intratracheal bleomycin

Abstract

Background: Cluster of differentiation 69 (CD69), an early activation marker antigen on T and B cells, is also expressed on activated macrophages and neutrophils, suggesting that CD69 may play a role in inflammatory diseases. To determine the effect of CD69 deficiency on bleomycin(BLM)-induced lung injury, we evaluated the inflammatory response following intratracheal BLM administration and the subsequent fibrotic changes in wild type (WT) and CD69-deficient (CD69-/-) mice.

Methods: The mice received a single dose of 3 mg/kg body weight of BLM and were sacrificed at 7 or 14 days post-instillation (dpi). Lung inflammation in the acute phase (7 dpi) was investigated by differential cell counts and cytokine array analyses of bronchoalveolar lavage fluid. In addition, lung fibrotic changes were evaluated at 14 dpi by histopathology and collagen assays. We also used reverse transcription polymerase chain reaction to measure the mRNA expression level of transforming growth factor β1 (TGF-β1) in the lungs of BLM-treated mice.

Results: CD69-/- mice exhibited less lung damage than WT mice, as shown by reductions in the following indices: (1) loss of body weight, (2) wet/dry ratio of lung, (3) cytokine levels in BALF, (4) histological evidence of lung injury, (5) lung collagen deposition, and (6) TGF-β1 mRNA expression in the lung.

Conclusion: The present study clearly demonstrates that CD69 plays an important role in the progression of lung injury induced by BLM.

Figure 1

Effect of bleomycin treatment on body weight in wild-type and CD69-deficient mice. Time course of changes in body weight after bleomycin (BLM) treatment in wild-type (WT) (n = 8) and cluster of differentiation 69 (CD69)-deficient (CD69^-/-) mice (n = 6). Results are expressed as the mean (SEM), *P < 0.05.

Figure 2

Effect of bleomycin treatment on differential cell counts in wild-type and CD69-deficient mice. Differential cell counts in bronchoalveolar fluid (BALF) were determined 7 d after the instillation of BLM or phosphate-buffered saline (sham treatment). Results are expressed as the mean (SEM) (n = 6-8 BLM-treated mice, n = 3 sham-treated mice). *P < 0.05, **P < 0.01.

Figure 3

Effect of bleomycin treatment on lung fluid content in wild-type and CD69-deficient mice. Ratio of wet/dry lung weight 7 d after the instillation of BLM or PBS (sham treatment). Results are expressed as the mean (SEM) (n = 8 WT and 6 CD69^-/- mice). The ratio between wet and dry lung weight is a measure of edema formation. *P < 0.01 vs. sham-treated mice. †P < 0.01 vs. WT mice.

Figure 4

Effect of bleomycin treatment on cytokine expression in wild-type and CD69-deficient mice. (A) Cytokine array analyses of BALF 7 d after the instillation of BLM or PBS (sham treatment) in WT or CD69^-/- mice. Cytokines with increased expression levels are boxed. (B) Expression levels of these cytokines in WT (black bars) and CD69^-/- (white bars) mice. Each expression level was normalized by that of the positive control. The stimulation index is the ratio of the expression level of a cytokine in BLM-treated mice to that in sham-treated mice. Results are expressed as the mean (SEM) (n = 4 mice per group). *P < 0.05, **P < 0.01.

Figure 5

Effect of bleomycin on the lung architecture in wild-type and CD69-deficient mice. Comparison of the lung architecture in WT and CD69^-/- mice after instillation of BLM or PBS (sham treatment), as shown by hematoxylin-eosin (A) and Masson's trichrome (B) staining of representative tissue sections.

Figure 6

Effect of bleomycin on lung fibrotic and biochemical changes in wild-type and CD69-deficient mice. (A) Ashcroft scores, which are a semi-quantitative measure of lung fibrotic changes, were determined 14 d after the instillation of BLM or PBS (sham treatment). Please see the Methods section for an explanation of the scoring criteria. Results are expressed as the mean (SEM) (n = 4 mice per group). *P < 0.01 vs. sham-treated mice. †P < 0.01 vs. WT mice. (B) The lung collagen content was measured 14 d after the instillation of BLM or PBS (sham treatment). Results are expressed as mean (SEM) (n = 6-8 BLM-treated mice, n = 3 sham-treated mice). *P < 0.01 vs. sham-treated mice. †P < 0.01 vs. WT mice. (C) The mRNA expression level of TGF-β1 in the lung was measured 7 d after the instillation of BLM or PBS in WT or CD69KO mice.

Figure 7

Expression of CD69 on macrophages in the lung. The lung from WT mice at 2 dpi with PBS (sham treatment) (A) or BLM (B) were subjected to immunohistochemical staining with an anti-CD69 antibody and an anti-Iba1 antibody, followed by a reaction with Alexa Fluor 594-conjugated and Alexa Fluor 488-conjugated secondary antibodies, respectively. The control lung from WT mice was subjected to immunohistochemical staining with an anti-gp36 antibody and an anti-Iba1 antibody, followed by a reaction with Alexa Fluor 594-conjugated and Alexa Fluor 488-conjugated secondary antibodies, respectively (C). Each arrowhead points to an interstitial macrophage. Each asterisk indicates an alveolar macrophage. All sections were co-stained with DAPI. Each bar represents 50 μm.