Characterization of Distinct Macrophage Subpopulations during Nitrogen Mustard-Induced Lung Injury and Fibrosis

Am J Respir Cell Mol Biol. 2016 Mar;54(3):436-46. doi: 10.1165/rcmb.2015-0120OC.


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 with NM (0.125 mg/kg, intratracheally) resulted in an increase in CD11b(+) macrophages in histologic sections. These cells consisted of inducible nitric oxide synthase(+) (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 CD43(+) M1 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 of M1 (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.

Keywords: fibrosis; inflammation; macrophages; nitrogen mustard.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Cytokines / genetics
  • Cytokines / immunology
  • Cytokines / metabolism
  • Disease Models, Animal
  • Gene Expression Regulation
  • Inflammation Mediators / immunology
  • Inflammation Mediators / metabolism
  • Lung / immunology*
  • Lung / metabolism
  • Lung / pathology
  • Lung Injury / chemically induced
  • Lung Injury / genetics
  • Lung Injury / immunology*
  • Lung Injury / metabolism
  • Lung Injury / pathology
  • Macrophages / immunology*
  • Macrophages / metabolism
  • Male
  • Mechlorethamine*
  • Phenotype
  • Pneumonia / chemically induced
  • Pneumonia / genetics
  • Pneumonia / immunology*
  • Pneumonia / metabolism
  • Pneumonia / pathology
  • Pulmonary Fibrosis / chemically induced
  • Pulmonary Fibrosis / genetics
  • Pulmonary Fibrosis / immunology*
  • Pulmonary Fibrosis / metabolism
  • Pulmonary Fibrosis / pathology
  • Rats, Wistar
  • Time Factors


  • Biomarkers
  • Cytokines
  • Inflammation Mediators
  • Mechlorethamine