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* From the Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, MI.
Correspondence to: Bethany B. Moore, PhD, Division of Pulmonary and Critical Care Medicine, 1150 W. Medical Center Dr, 6301 MSRB III, Ann Arbor, MI 48109-0642; e-mail: Bmoore{at}umich.edu
Intratracheal administration of fluorescein isothiocyanate (FITC) to mice results in an acute lung injury that results in lymphocyte-independent pulmonary fibrosis by day 21.1 We have characterized this model of fibrosis in wild-type (BL/6x129 F2) mice and mice deficient for the monocyte chemoattractant protein (MCP)-1 receptor, chemokine receptor 2 (CCR2) [CCR2 knockout mice]. Following FITC challenge, both protein and messenger RNA levels for MCP-1, a ligand for CCR2, are increased in murine lungs. In response to FITC challenge, wild-type and CCR2 knockout mice experience similar acute lung injury as measured by Evan’s Blue extravasation. Despite this, wild-type mice generate significantly worse pulmonary fibrosis than the CCR2 knockout animals as measured by lung hydroxyproline content (111 ± 18 µg/mL vs 72 ± 6 µg/mL; p = 0.0001) and histology (trichrome staining). The protection from fibrosis was specific to mice with a deletion of the CCR2 gene, as mice with a deletion of the CCR5 gene were not protected from FITC-induced pulmonary fibrosis. Despite the inability to respond to chemotactic signals from MCP-1, CCR2 knockout mice recruit similar numbers of leukocytes to their lungs at days 7, 14, and 21 after FITC challenge as do their wild-type counterparts. Similarly, there were no significant differences between any of the leukocyte subpopulations analyzed in wild-type and CCR2 knockout mice following FITC challenge. To ascertain whether neutrophils played a regulatory role in FITC-induced pulmonary fibrosis, we analyzed the fibrotic response in wild-type mice depleted of neutrophils using anti-granulocyte monoclonal antibody (anti-Ly6G). Wild-type mice treated with anti-Ly6G monoclonal antibodies during the early injury and repair phases (day -1 to day 10 after FITC challenge) showed no differences in development of subsequent pulmonary fibrosis. These data suggest that neutrophils are not involved in regulation of FITC-induced pulmonary fibrosis. Since the differences in FITC-induced fibrosis did not appear to be regulated by the magnitude or types of leukocytes recruited, cytokine expression was analyzed as a measure of leukocyte activation status. Cytokine production is altered in CCR2 knockout mice. The protected CCR2 knockout mice had higher levels of messenger RNA for granulocyte macrophage-colony stimulating factor (GM-CSF) than did wild-type mice at day 7 after FITC challenge. In contrast, the fibrotic wild-type mice had higher levels of messenger RNA for interleukin (IL)-4 at day 7 after FITC challenge than did the CCR2 knockout mice. Thus, pulmonary fibrosis in the wild-type mice is associated with elevated levels of IL-4 and diminished GM-CSF. Diminished GM-CSF is associated with development of more severe pulmonary fibrosis in response to bleomycin injury.2 3 Two findings provide support for a role for MCP-1 in IL-4 regulation. First, anti-MCP-1 antisera reduced levels of IL-4 messenger RNA in cultured wild-type lung cells. Second, recombinant MCP-1 increased the transcriptional activity of the IL-4 promoter in reporter gene assays performed with the MH-S alveolar macrophage cell line. Taken together, these data suggest that MCP-1 regulates IL-4 expression. While the source of IL-4 producing cells is unknown, mast cells (which are CCR2 positive and known to produce IL-4) are present in the fibrotic lesions of wild-type mice. Thus, FITC challenge results in the generation of MCP-1. MCP-1 signaling via the CCR2 receptor results in the generation of profibrotic signals that include increased IL-4 and diminished GM-CSF. These data demonstrate a crucial role for CCR2 and its ligand(s) in generating a fibrotic response to lung injury.
Footnotes
Abbreviations: CCR2 = chemokine receptor 2; FITC = fluorescein isothiocyanate; GM-CSF = granulocyte macrophage-colony stimulating factor; IL = interleukin; MCP = monocyte chemoattractant protein
References
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