Bone marrow-derived cells are known to play important roles in repair/regeneration of injured tissues, but their roles in pathological fibrosis are less clear. Here, we report a critical role for the chemokine receptor CCR2 in the recruitment and activation of lung fibrocytes (CD45 ؉ , CD13 ؉ , collagen 1 ؉ , CD34 ؊ ). Lung fibrocytes were isolated in significantly greater numbers from airspaces of fluorescein isothiocyanate-injured CCR2 ؉/؉ mice than from CCR2 ؊/؊ mice. Transplant of CCR2 ؉/؉ bone marrow into CCR2 ؊/؊ recipients restored recruitment of lung fibrocytes and susceptibility to fibrosis. Ex vivo PKH-26-labeled CCR2 ؉/؉ lung fibrocytes also migrated to injured airspaces of CCR2 ؊/؊ recipients in vivo. Isolated lung fibrocytes expressed CCR2 and migrated to CCL2, and CCL2 stimulated collagen secretion by lung fibrocytes. Fibrocytes could transition into fibroblasts in vitro, and this transition was associated with loss of CCR2 expression and enhanced production of collagen 1. This is the first report describing expression of CCR2 on lung fibrocytes and demonstrating that CCR2 regulates both recruitment and activation of these cells after respiratory injury. Pulmonary fibrosis is characterized by alveolar epithelial cell injury, hyperplasia, inflammatory cell accumulation, fibroblast proliferation, and deposition of extracellular matrix.

Pulmonary fibrosis can be modeled in animals by intratracheal instillation of FITC, which results in acute lung injury, inflammation, and extracellular matrix deposition. We have previously shown that despite chronic inflammation, this model of pulmonary fibrosis is lymphocyte independent. The CC chemokine monocyte-chemoattractant protein-1 is induced following FITC deposition. Therefore, we have investigated the contribution of the main monocyte-chemoattractant protein-1 chemokine receptor, CCR2, to the fibrotic disease process. We demonstrate that CCR2−/− mice are protected from fibrosis in both the FITC and bleomycin pulmonary fibrosis models. The protection is specific for the absence of CCR2, as CCR5−/− mice are not protected. The protection is not explained by differences in acute lung injury, or the magnitude or composition of inflammatory cells. FITC-treated CCR2−/− mice display differential patterns of cellular activation as evidenced by the altered production of cytokines and growth factors following FITC inoculation compared with wild-type controls. CCR2−/− mice have increased levels of GM-CSF and reduced levels of TNF-α compared with FITC-treated CCR2+/+ mice. Thus, CCR2 signaling promotes a profibrotic cytokine cascade following FITC administration.

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Although cells of the innate inflammatory response, such as macrophages and neutrophils, have been extensively studied in the arena of Gram-negative bacterial pneumonia, a role for T cells remains unknown. To study the role of specific T cell populations in bacterial pneumonia, mice deleted of their TCR β- and/or δ-chain were intratracheally inoculated with Klebsiella pneumoniae. γδ T cell knockout mice displayed increased mortality at both early and late time points. In contrast, mice specifically lacking only αβ-T cells were no more susceptible than wild-type mice. Pulmonary bacterial clearance in γδ-T cell knockout mice was unimpaired. Interestingly, these mice displayed increased peripheral blood dissemination. Rapid up-regulation of IFN-γ and TNF-α gene expression, critical during bacterial infections, was markedly impaired in lung and liver tissue from γδ-T cell-deficient mice 24 h postinfection. The increased peripheral blood bacterial dissemination correlated with impaired hepatic bacterial clearance following pulmonary infection and increased hepatic injury as measured by plasma aspartate aminotransferase activity. Combined, these data suggest that mice lacking γδ-T cells have an impaired ability to resolve disseminated bacterial infections subsequent to the initial pulmonary infection. These data indicate that γδ-T cells comprise a critical component of the acute inflammatory response toward extracellular Gram-negative bacterial infections and are vital for the early production of the proinflammatory cytokines IFN-γ and TNF-α.

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4−/−), IL-13 (IL-13−/−), or IL-4 and IL-13 combined (IL-4/13−/−). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4−/− mice developed fibrosis, whereas the IL-13−/− and IL-4/13−/− mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4−/−, IL-13−/−, and IL-4/13−/− mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13−/− mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-β1 production was not reduced in IL-13−/− mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Rα1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.