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ABSTRACTPneumocystisinfections increase host susceptibility to additional insults that would be tolerated in the absence of infection, such as hyperoxia. In an in vivo model using CD4-depleted mice, we previously demonstrated that Pneumocystis murinapneumonia causes significant mortality following an otherwise nonlethal hyperoxic insult. Infected mice demonstrated increased pulmonary inflammation and alveolar epithelial cell apoptosis compared to controls. To test the mechanisms underlying these observations, we examined expression of components of the Fas-Fas ligand pathway in P. murina-infected mice exposed to hyperoxia. Hyperoxia alone increased expression of Fas on the surface of type II alveolar epithelial cells; conversely, infection with P. murinaled to increased lung expression of Fas ligand. We hypothesized that inhibition of inflammatory responses or direct inhibition of alveolar epithelial cell apoptosis would improve survival in P. murina-infected mice exposed to hyperoxia. Mice were depleted of CD4+T cells and infected with P. murinaand then were exposed to >95% oxygen for 4 days, followed by return to normoxia. Experimental groups received vehicle, dexamethasone, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Compared with the vehicle-treated group, treatment with dexamethasone reduced Fas ligand expression and significantly improved survival. Similarly, treatment with GM-CSF, an agent we have shown protects alveolar epithelial cells against apoptosis, decreased Fas ligand expression and also improved survival. Our results suggest that the dual stresses of P. murinainfection and hyperoxia induce lung injury via activation of the Fas-Fas ligand pathway and that corticosteroids and GM-CSF reduce mortality in P. murina-infected mice exposed to hyperoxic stress by inhibition of inflammation and apoptosis.

Pneumocystis infections increase host susceptibility to additional insults that would be tolerated in the absence of infection, such as hyperoxia. In an in vivo model using CD4-depleted mice, we previously demonstrated that Pneumocystis murina pneumonia causes significant mortality following an otherwise nonlethal hyperoxic insult. Infected mice demonstrated increased pulmonary inflammation and alveolar epithelial cell apoptosis compared to controls. To test the mechanisms underlying these observations, we examined expression of components of the Fas-Fas ligand pathway in P. murina-infected mice exposed to hyperoxia. Hyperoxia alone increased expression of Fas on the surface of type II alveolar epithelial cells; conversely, infection with P. murina led to increased lung expression of Fas ligand. We hypothesized that inhibition of inflammatory responses or direct inhibition of alveolar epithelial cell apoptosis would improve survival in P. murina-infected mice exposed to hyperoxia. Mice were depleted of CD4+T cells and infected with P. murina and then were exposed to >95% oxygen for 4 days, followed by return to normoxia. Experimental groups received vehicle, dexamethasone, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Compared with the vehicle-treated group, treatment with dexamethasone reduced Fas ligand expression and significantly improved survival. Similarly, treatment with GM-CSF, an agent we have shown protects alveolar epithelial cells against apoptosis, decreased Fas ligand expression and also improved survival. Our results suggest that the dual stresses of P. murina infection and hyperoxia induce lung injury via activation of the Fas-Fas ligand pathway and that corticosteroids and GM-CSF reduce mortality in P. murina-infected mice exposed to hyperoxic stress by inhibition of inflammation and apoptosis.

Reactive oxygen species generated from NADPH oxidase(s) in airway smooth muscle cells and pulmonary artery smooth muscle cells are important signaling intermediates. Nox4 appears to be the predominant gp91 homologue in these cells. However, expression of NADPH oxidase components is dependent on phenotype, and different homologues may be expressed during different functional states of the cell. NADPH oxidase(s) appear to be important not only for mitogenesis by these cells, but also for O2 sensing. The regulation of NADPH oxidase(s) in airway and pulmonary artery smooth muscle cells has important implications for the pathobiochemistry of asthma and pulmonary vascular diseases.

Human proteinase-3 is one of three serine proteinases present in the azurophil granules of polymorphonuclear leukocytes along with elastase and cathepsin G. Proteinase-3 gene expression is confined to the promyelocytic stage of polymorphonuclear leukocyte maturation. The present investigation identifies elements responsible for this highly controlled tissue- and developmental-specific expression of proteinase-3. Within the first 200 base pairs of the proteinase-3 promoter, two elements were identified as important for expression, these elements at −101 and −190 confer the majority of the activity. The element at −101 has a PU.1 consensus. It binds a myeloid nuclear protein of approximately 45 kDa that “supershifts” with PU.1 antibody and is competed by the CD11b PU.1 element. The element at −190 has a core sequence of CCCCGCCC (CG element). The cytidines but not the guanidine are essential for promoter activity. The CG element binds a second nuclear protein with a molecular mass of approximately 40 kDa that is found in cells of myeloid lineage as well as non-myeloid HeLa cells. However, the proteinase-3 promoter is not active in HeLa cells which suggests that the CG element alone is not sufficient for proteinase-3 gene expression. Maturation of promyelocytic cells results in an inhibition of proteinase-3 gene expression and a reduction in nuclear protein binding to the PU.1 and CG elements. Similar elements occur in the elastase and cathepsin G promoters. Using the elastase and cathepsin G PU.1 and CG-like elements as probes results in identical band-shift patterns to that obtained with proteinase-3 PU.1 and CG elements. These data suggest that there is cooperative interaction between a PU.1 and a CG element with a consensus of CCCCXCCC and that they are important control elements for tissue- and developmental-specific expression of azurophil serine proteinases of polymorphonuclear leukocytes.