Characterization of distinct macrophage subpopulations during nitrogen mustard-induced lung injury and fibrosis

Nitrogen mustard (NM) is an alkylating agent known to cause extensive pulmonary injury progressing to fibrosis. This is accompanied by a persistent macrophage inflammatory response. In these studies, we characterized the phenotype of macrophages accumulating in the lung over time following NM exposure. Treatment of rats withNM(0.125 mg/kg, intratracheally) resulted in an increase in CD11b⁺ macrophages in histologic sections. These cells consisted of inducible nitric oxide synthase1 (iNOS) proinflammatory M1 macrophages, and CD68⁺, CD163⁺, CD206⁺, YM-1⁺, and arginase-II⁺ antiinflammatory M2 macrophages. Although M1 macrophages were prominent 1-3 days after NM, M2 macrophages were most notable at 28 days. At this time, they were enlarged and vacuolated, consistent with a profibrotic phenotype. Flow cytometric analysis of isolated lung macrophages identified three phenotypically distinct subpopulations: mature CD11b^-, CD43^-, and CD68^-, resident macrophages, which decreased in numbers after NM; and two infiltrating (CD11b⁺,) macrophage subsets: immature CD431 M⁺, macrophages and mature CD43^-, M2 macrophages, which increased sequentially. Time-related increases in M1 (iNOS, IL-12α, COX-2, TNF-α, matrix metalloproteinase-9, matrix metalloproteinase-10) and M2 (IL-10, pentraxin-2, connective tissue growth factor, ApoE) genes, as well as chemokines/ chemokine receptors associated with trafficking ofM1(CCR2, CCR5, CCL2, CCL5) and M2 (CX₃CR1, fractalkine) macrophages to sites of injury, were also noted in macrophages isolated from the lung after NM. The appearance of M1 and M2 macrophages in the lung correlated with NM-induced acute injury and the development of fibrosis, suggesting a potential role of these macrophage subpopulations in the pathogenic response to NM.