Lung granulomas are associated with numerous conditions, including inflammatory disorders, exposure to environmental pollutants, and infection. Osteopontin is a chemotactic cytokine produced by macrophages, and is implicated in extracellular matrix remodeling. Furthermore, osteopontin is up-regulated in granulomatous disease, and osteopontin null mice exhibit reduced granuloma formation. Animal models currently used to investigate chronic lung granulomatous inflammation bear a pathological resemblance, but lack the chronic nature of human granulomatous disease. Carbon nanoparticles are generated as byproducts of combustion. Interestingly, experimental exposures to carbon nanoparticles induce pulmonary granuloma-like lesions. However, the recruited cellular populations and extracellular matrix gene expression profiles within these lesions have not been explored. Because of the rapid resolution of granulomas in current animal models, the mechanisms responsible for persistence have been elusive. To overcome the limitations of previous models, we investigated whether a model using multiwall carbon nanoparticles would resemble chronic human lung granulomatous inflammation. We hypothesized that pulmonary exposure to multiwall carbon nanoparticles would induce granulomas, elicit a macrophage and T-cell response, and mimic other granulomatous disorders with an up-regulation of osteopontin. This model demonstrates: (1) granulomatous inflammation, with macrophage and T-cell infiltration; (2) resemblance to the chronicity of human granulomas, with persistence up to 90 days; and (3) a marked elevation of osteopontin, metalloproteinases, and cell adhesion molecules in granulomatous foci isolated by laser-capture microdissection and in alveolar macrophages from bronchoalveolar lavage. The establishment of such a model provides an important platform for mechanistic studies on the persistence of granuloma.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-activated, nuclear transcription factor that regulates genes involved in lipid and glucose metabolism, inflammation, and other pathways. The hematopoietic growth factor, granulocyte macrophage colony-stimulating factor (GM-CSF), is essential for lung homeostasis and is thought to regulate surfactant clearance, but mechanisms involved are unknown. GM-CSF is reported to stimulate PPAR-gamma, but the activation status of PPAR-gamma in human alveolar macrophages has not been defined. In pulmonary alveolar proteinosis (PAP), a rare interstitial lung disease, surfactant accumulates in alveolar airspaces, resident macrophages become engorged with lipoproteinaceous material, and GM-CSF deficiency is strongly implicated in pathogenesis. Here we show that PPAR-gamma mRNA and protein are highly expressed in alveolar macrophages of healthy control subjects but severely deficient in PAP in a cell-specific manner. Further, we show that the PPAR-gamma-regulated lipid scavenger receptor, CD36, is also deficient in PAP. PPAR-gamma and CD36 deficiency are not intrinsic to PAP alveolar macrophages, but can be upregulated by GM-CSF therapy. Moreover, GM-CSF treatment of patients with PAP fully restores PPAR-gamma to healthy control levels. Based upon these novel findings, we hypothesize that GM-CSF regulates lung homeostasis via PPAR-gamma-dependent pathways.

Peroxisome proliferator-activated receptor γ (PPARγ) is constitutively expressed at high levels in healthy alveolar macrophages, in contrast to other tissue macrophages and blood monocytes. PPARγ ligands have been shown to down-regulate IFN-γ-stimulated inducible NO synthase (iNOS) in macrophages. Because NO is an important inflammatory mediator in the lung, we hypothesized that deletion of alveolar macrophage PPARγ in vivo would result in up-regulation of iNOS and other inflammatory mediators. The loss of PPARγ in macrophages was achieved by crossing floxed (+/+) PPARγ mice and a transgenic mouse containing the CRE recombinase gene under the control of the murine M lysozyme promoter (PPARγKO). Alveolar macrophages were harvested by bronchoalveolar lavage (BAL). Lymphocytes (CD8:CD4 ratio = 2.8) were increased in BAL of PPARγKO vs wild-type C57BL6; p ≤ 0.0001. Both iNOS and IFN-γ expression were significantly elevated (p ≤ 0.05) in BAL cells. Th-1 associated cytokines including IL-12 (p40), MIP-1α (CCL3), and IFN inducible protein-10 (IP-10, CXCL10) were also elevated. IL-4 and IL-17A were not detected. To test whether these alterations were due to the lack of PPARγ, PPARγ KO mice were intratracheally inoculated with a PPARγ lentivirus construct. PPARγ transduction resulted in significantly decreased iNOS and IFN-γ mRNA expression, as well as reduced BAL lymphocytes. These results suggest that lack of PPARγ in alveolar macrophages disrupts lung homeostasis and results in a Th1-like inflammatory response.