Bronchoalveolar lavage (BAL) cells in idiopathic pulmonary fibrosis express a complex pro-inflammatory, pro-repair, angiogenic activation pattern, likely associated with macrophage iron accumulation.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown cause characterized by alveolar epithelial damage, patchy interstitial fibrosis and diffuse microvascular abnormalities. In IPF, alveolar clustering of iron-laden alveolar macrophages-a common sign of microhemorrhage, has been associated with vascular abnormalities and worsening of pulmonary hypertension. As iron-dependent ROS generation has been shown to induce unrestrained macrophage activation in disease models of vascular damage, we explored alveolar macrophage activation phenotype in IPF patients (n = 16) and healthy controls (CTR, n = 7) by RNA sequencing of bronchoalveolar lavage (BAL) cells. The frequencies of macrophages in BAL cells were 86+4% and 83.4+8% in IPF and CTR groups, respectively (p-value = 0.41). In IPF patients, BAL cells showed increased iron-dependent ROS generation (p-value<0.05 vs CTR). Gene expression analysis showed overrepresentation of Gene Ontology processes/functions and KEGG pathways enriched in upregulated M1-type inflammatory (p-value<0.01), M2-type anti-inflammatory/tissue remodeling (p-value<0.0001), and MTPP-type chronic inflammatory/angiogenic (p-value<0.0001) chemokine and cytokine genes. The ex vivo finding was confirmed by the induction of iron-dependent ROS generation and chemokine/cytokine overexpression of Ccl4, Cxcl10 (M1), Il1rn (M2), Cxcl2, and Cxcl7 (MTPP) in MH-S murine immortalized alveolar macrophages exposed to ferric ammonium citrate in culture (p-value<0.05 vs CTR). The data show alveolar macrophage expression of a pro-inflammatory, tissue remodeling and angiogenic complex activation pattern, suggesting that iron accumulation may play a role in macrophage activation.

Development of a sarcoidosis murine lung granuloma model using Mycobacterium superoxide dismutase A peptide.

Sarcoidosis is characterized by noncaseating granulomas containing CD4(+) T cells with a Th1 immunophenotype. Although the causative antigens remain unknown, independent studies noted molecular and immunologic evidence of mycobacterial virulence factors in sarcoidosis specimens. A major limiting factor in discovering new insights into the pathogenesis of sarcoidosis is the lack of an animal model. Using a distinct superoxide dismutase A peptide (sodA) associated with sarcoidosis granulomas, we developed a pulmonary model of sarcoidosis granulomatous inflammation. Mice were sensitized by a subcutaneous injection of sodA, incorporated in incomplete Freund's adjuvant (IFA). Control subjects consisted of mice with no sensitization (ConNS), sensitized with IFA only (ConIFA), or with Schistosoma mansoni eggs. Fourteen days later, sensitized mice were challenged by tail-vein injection of naked beads, covalently coupled to sodA peptides or to schistosome egg antigens (SEA). Histologic analysis revealed hilar lymphadenopathy and noncaseating granulomas in the lungs of sodA-treated or SEA-treated mice. Flow cytometry of bronchoalveolar lavage (BAL) demonstrated CD4(+) T-cell responses against sodA peptide in the sodA-sensitized mice only. Cytometric bead analysis revealed significant differences in IL-2 and IFN-γ secretion in the BAL fluid of sodA-treated mice, compared with mice that received SEA or naked beads (P = 0.008, Wilcoxon rank sum test). ConNS and ConIFA mice demonstrated no significant formation of granuloma, and no Th1 immunophenotype. The use of microbial peptides distinct for sarcoidosis reveals a histologic and immunologic profile in the murine model that correlates well with those profiles noted in human sarcoidosis, providing the framework to investigate the molecular basis for the progression or resolution of sarcoidosis.

Plasminogen is an important regulator in the pathogenesis of a murine model of asthma.

RATIONALE: Asthma is a syndrome whose common pathogenic expression is inflammation of the airways. Plasminogen plays an important role in cell migration and is also implicated in tissue remodeling, but its role in asthma has not been defined. OBJECTIVES: To test whether plasminogen is a critical component in the development of asthma. METHODS: We used a mouse model of ovalbumin-induced pulmonary inflammation in Plg(+/+), Plg(+/-), and Plg(-/-) mice. MEASUREMENTS AND MAIN RESULTS: The host responses measured included lung morphometry, and inflammatory mediators and cell counts were assessed in bronchoalveolar lavage fluid. Bronchoalveolar lavage demonstrated a marked increase in eosinophils and lymphocytes in ovalbumin-treated Plg(+/+) mice, which were reduced to phosphate-buffered saline-treated control levels in Plg(+/-) or Plg(-/-) mice. Lung histology revealed peribronchial and perivascular leukocytosis, mucus production, and increased collagen deposition in ovalbumin-treated Plg(+/+) but not in Plg(+/-) or Plg(-/-) mice. IL-5, tumor necrosis factor-alpha, and gelatinases, known mediators of asthma, were detected in bronchoalveolar lavage fluid of ovalbumin-treated Plg(+/+) mice, yet were reduced in Plg(-/-) mice. Administration of the plasminogen inhibitor, tranexamic acid, reduced eosinophil and lymphocyte numbers, mucus production, and collagen deposition in the lungs of ovalbumin-treated Plg(+/+) mice. CONCLUSIONS: The decreased inflammation in the lungs of Plg(-/-) mice and its blockade with a plasminogen inhibitor indicate that plasminogen plays an important role in orchestrating the asthmatic response and suggests that plasminogen may be a therapeutic target for the treatment of asthma.

Elevated gelatinase activity in pulmonary alveolar proteinosis: role of macrophage-colony stimulating factor.

Pulmonary alveolar proteinosis (PAP) is an anti-granulocyte macrophage-colony stimulating factor (GM-CSF) autoimmune disease resulting in the accumulation of phospholipids in the alveoli. GM-CSF knockout (KO) mice exhibit a strikingly similar lung pathology to patients with PAP. The lack of functionally active GM-CSF correlates with highly elevated concentrations of M-CSF in the lungs of PAP patients and GM-CSF KO mice. M-CSF has been associated with alternative macrophage activation, and in models of pulmonary fibrosis, M-CSF also contributes to tissue resorption and fibrosis. Matrix metalloproteinase-2 (MMP-2) and MMP-9 have been implicated in extracellular matrix degradation in animal models of fibrosis and asthma. We show for the first time that the lungs of PAP patients contain highly elevated levels of MMP-2 and MMP-9. PAP broncholaveolar lavage (BAL) cells but not bronchial epithelial cells expressed increased MMP-2 and MMP-9 mRNA relative to healthy controls. Both MMPs were detectable as pro and active proteins by gelatin zymography; and by fluorometric global assay, PAP-MMP activity was elevated. BAL cells/fluids from GM-CSF KO mice also demonstrated significantly elevated MMP-2 and MMP-9 gene expression, protein, and activity. Finally, PAP patients undergoing GM-CSF therapy exhibited significantly reduced MMPs and M-CSF. These data suggest that in the absence of GM-CSF, excess M-CSF in PAP may redirect alveolar macrophage activation, thus potentially contributing to elevated MMP expression in the lung.

In vivo regulation of plasminogen function by plasma carboxypeptidase B.

The major functions of plasminogen (Plg) in fibrinolysis and cell migration depend on its binding to carboxy-terminal lysyl residues. The ability of plasma carboxypeptidase B (pCPB) to remove these residues suggests that it may act as a suppressor of these Plg functions. To evaluate this role of pCPB in vivo, homozygote pCPB-deficient mice were generated by homologous recombination, and the resulting pCPB(-/-) mice, which were viable and healthy, were mated to Plg(-/-) mice. Plg(+/-) mice show intermediate levels of fibrinolysis and cell migration compared with Plg wild-type and deficient mice, reflecting the intermediate levels of the Plg antigen in their plasma. Differences in Plg-dependent functions between pCPB(+/+), pCPB(+/-), and pCPB(-/-) mice were then analyzed in a Plg(+/-) background. In a pulmonary clot lysis model, fibrinolysis was significantly increased in mice with partial (pCPB(+/-)) or total absence (pCPB(-/-)) of pCPB compared with their wild-type counterparts (pCPB(+/+)). In a thioglycollate model of peritoneal inflammation, leukocyte migration at 72 hours increased significantly in Plg(+/-)/pCPB(+/-) and Plg(+/-)/pCPB(-/-) compared with their wild-type counterparts. These studies demonstrate a definitive role of pCPB as a modulator of the pivotal functions of Plg in fibrinolysis and cell migration in vivo.