Overexpression of sICAM-1 in the alveolar epithelial space results in an exaggerated inflammatory response and early death in Gram negative pneumonia.

BACKGROUND: A sizeable body of data demonstrates that membrane ICAM-1 (mICAM-1) plays a significant role in host defense in a site-specific fashion. On the pulmonary vascular endothelium, mICAM-1 is necessary for normal leukocyte recruitment during acute inflammation. On alveolar epithelial cells (AECs), we have shown previously that the presence of normal mICAM-1 is essential for optimal alveolar macrophage (AM) function. We have also shown that ICAM-1 is present in the alveolar space as a soluble protein that is likely produced through cleavage of mICAM-1. Soluble intercellular adhesion molecule-1 (sICAM-1) is abundantly present in the alveolar lining fluid of the normal lung and could be generated by proteolytic cleavage of mICAM-1, which is highly expressed on type I AECs. Although a growing body of data suggesting that intravascular sICAM-1 has functional effects, little is known about sICAM-1 in the alveolus. We hypothesized that sICAM-1 in the alveolar space modulates the innate immune response and alters the response to pulmonary infection. METHODS: Using the surfactant protein C (SPC) promoter, we developed a transgenic mouse (SPC-sICAM-1) that constitutively overexpresses sICAM-1 in the distal lung, and compared the responses of wild-type and SPC-sICAM-1 mice following intranasal inoculation with K. pneumoniae. RESULTS: SPC-sICAM-1 mice demonstrated increased mortality and increased systemic dissemination of organisms compared with wild-type mice. We also found that inflammatory responses were significantly increased in SPC-sICAM-1 mice compared with wild-type mice but there were no difference in lung CFU between groups. CONCLUSIONS: We conclude that alveolar sICAM-1 modulates pulmonary inflammation. Manipulating ICAM-1 interactions therapeutically may modulate the host response to Gram negative pulmonary infections.

Immune reconstitution during Pneumocystis lung infection: disruption of surfactant component expression and function by S-nitrosylation.

Pneumocystis pneumonia (PCP), the most common opportunistic pulmonary infection associated with HIV infection, is marked by impaired gas exchange and significant hypoxemia. Immune reconstitution disease (IRD) represents a syndrome of paradoxical respiratory failure in patients with active or recently treated PCP subjected to immune reconstitution. To model IRD, C57BL/6 mice were selectively depleted of CD4(+) T cells using mAb GK1.5. Following inoculation with Pneumocystis murina cysts, infection was allowed to progress for 2 wk, GK1.5 was withdrawn, and mice were followed for another 2 or 4 wk. Flow cytometry of spleen cells demonstrated recovery of CD4(+) cells to >65% of nondepleted controls. Lung tissue and bronchoalveolar lavage fluid harvested from IRD mice were analyzed in tandem with samples from CD4-depleted mice that manifested progressive PCP for 6 wks. Despite significantly decreased pathogen burdens, IRD mice had persistent parenchymal lung inflammation, increased bronchoalveolar lavage fluid cellularity, markedly impaired surfactant biophysical function, and decreased amounts of surfactant phospholipid and surfactant protein (SP)-B. Paradoxically, IRD mice also had substantial increases in the lung collectin SP-D, including significant amounts of an S-nitrosylated form. By native PAGE, formation of S-nitrosylated SP-D in vivo resulted in disruption of SP-D multimers. Bronchoalveolar lavage fluid from IRD mice selectively enhanced macrophage chemotaxis in vitro, an effect that was blocked by ascorbate treatment. We conclude that while PCP impairs pulmonary function and produces abnormalities in surfactant components and biophysics, these responses are exacerbated by IRD. This worsening of pulmonary inflammation, in response to persistent Pneumocystis Ags, is mediated by recruitment of effector cells modulated by S-nitrosylated SP-D.

Critical roles of inflammation and apoptosis in improved survival in a model of hyperoxia-induced acute lung injury in Pneumocystis murina-infected mice.

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.

Pneumocystis murina infection and cigarette smoke exposure interact to cause increased organism burden, development of airspace enlargement, and pulmonary inflammation in mice.

Chronic obstructive pulmonary disease (COPD) is characterized by the presence of airflow obstruction and lung destruction with airspace enlargement. In addition to cigarette smoking, respiratory pathogens play a role in pathogenesis, but specific organisms are not always identified. Recent reports demonstrate associations between the detection of Pneumocystis jirovecii DNA in lung specimens or respiratory secretions and the presence of emphysema in COPD patients. Additionally, human immunodeficiency virus-infected individuals who smoke cigarettes develop early emphysema, but a role for P. jirovecii in pathogenesis remains speculative. We developed a new experimental model using immunocompetent mice to test the interaction of cigarette smoke exposure and environmentally acquired Pneumocystis murina infection in vivo. We hypothesized that cigarette smoke and P. murina would interact to cause increases in total lung capacity, airspace enlargement, and pulmonary inflammation. We found that exposure to cigarette smoke significantly increases the lung organism burden of P. murina. Pulmonary infection with P. murina, combined with cigarette smoke exposure, results in changes in pulmonary function and airspace enlargement characteristic of pulmonary emphysema. P. murina and cigarette smoke exposure interact to cause increased lung inflammatory cell accumulation. These findings establish a novel animal model system to explore the role of Pneumocystis species in the pathogenesis of COPD.

Pneumocystis pneumonia increases the susceptibility of mice to sublethal hyperoxia.

Patients with Pneumocystis pneumonia often develop respiratory failure after entry into medical care, and one mechanism for this deterioration may be increased alveolar epithelial cell injury. In vitro, we previously demonstrated that Pneumocystis is not cytotoxic for alveolar epithelial cells. In vivo, however, infection with Pneumocystis could increase susceptibility to injury by stressors that, alone, would be sublethal. We examined transient exposure to hyperoxia as a prototypical stress that does cause mortality in normal mice. Mice were depleted of CD4+ T cells and inoculated intratracheally with Pneumocystis. Control mice were depleted of CD4+ T cells but did not receive Pneumocystis. After 4 weeks, mice were maintained in normoxia, were exposed to hyperoxia for 4 days, or were exposed to hyperoxia for 4 days followed by return to normoxia. CD4-depleted mice with Pneumocystis pneumonia demonstrated significant mortality after transient exposure to hyperoxia, while all uninfected control mice survived this stress. We determined that organism burdens were not different. However, infected mice exposed to hyperoxia and then returned to normoxia demonstrated significant increases in inflammatory cell accumulation and lung cell apoptosis. We conclude that Pneumocystis pneumonia leads to increased mortality following a normally sublethal hyperoxic insult, accompanied by alveolar epithelial cell injury and increased pulmonary inflammation.

T cell costimulatory molecule function determines susceptibility to infection with Pneumocystis carinii in mice.

Loss of T cell number and function during HIV infection or secondary to pharmacologic immunosuppression renders individuals susceptible to opportunistic infections, including Pneumocystis carinii pneumonia. Because costimulatory receptors are critical for optimal T cell function, we hypothesized that these proteins would regulate susceptibility to opportunistic infections. We found that despite normal T cell numbers, mice deficient in the costimulatory molecules CD2 and CD28 spontaneously developed P. carinii pneumonia. In experiments using intratracheal injection of P. carinii organisms to induce infection, the loss of CD28 alone was sufficient to render mice susceptible to acute infection; however, the organism was eventually cleared. Examination of inflammatory responses to P. carinii revealed that mice deficient in both CD2 and CD28 accumulated CD8(+) T cells in their lungs in response to infection and demonstrated markedly reduced specific Ab titers. Analysis of cytokine profiles suggested that regulation of IL-10 and IL-15 may be important elements of the response to this pathogen. Thus, costimulatory molecule function is critical in determining the initial susceptibility to infection with P. carinii. Analysis of immunologic responses in these mice may provide important insights into the defects that render individuals susceptible to opportunistic infection, and provide opportunities for novel immunologically based therapies.

Delayed clearance of pneumocystis carinii infection, increased inflammation, and altered nitric oxide metabolism in lungs of surfactant protein-D knockout mice.

Surfactant protein-D (SP-D), a member of the "collectin" family, has been shown to play a role in innate immunity through modulation of inflammation and clearance of organisms. The role of SP-D in host defense against Pneumocystis carinii pneumonia was assessed using SP-D knockout (KO) mice. When inoculated with P. carinii, both wild-type (wt) and SP-D KO mice required CD4 cell depletion to develop infection. In CD4 cell-depleted models, 2 weeks after infection with P. carinii, SP-D KO mice developed increased intensity of infection, compared with wt mice, despite higher lung-inflammation scores and increased amounts of alveolar inflammatory cells. The increased inflammation seen in SP-D KO mice was accompanied by increases in lung weight, expression of inducible nitric oxide (NO) synthase, total NO levels, and 3-nitrotyrosine levels in lung tissue. These results indicate that SP-D plays a role in host defense against P. carinii in vivo by modulating clearance of organisms, lung inflammation, and metabolism of NO.

Enhanced lung injury and delayed clearance of Pneumocystis carinii in surfactant protein A-deficient mice: attenuation of cytokine responses and reactive oxygen-nitrogen species.

Surfactant protein A (SP-A), a member of the collectin family, selectively binds to Pneumocystis carinii and mediates interactions between pathogen and host alveolar macrophages in vitro. To test the hypothesis that mice lacking SP-A have delayed clearance of Pneumocystis organisms and enhanced lung injury, wild-type C57BL/6 (WT) and SP-A-deficient mice (SP-A(-/-)) with or without selective CD4(+)-T-cell depletion were intratracheally inoculated with Pneumocystis organisms. Four weeks later, CD4-depleted SP-A-deficient mice had developed a more severe Pneumocystis infection than CD4-depleted WT (P. carinii pneumonia [PCP] scores of 3 versus 2, respectively). Whereas all non-CD4-depleted WT mice were free of PCP, intact SP-A(-/-) mice also had evidence of increased organism burden. Pneumocystis infection in SP-A-deficient mice was associated histologically with enhanced peribronchial and/or perivascular cellularity (score of 4 versus 2, SP-A(-/-) versus C57BL/6 mice, respectively) and a corresponding increase in bronchoalveolar lavage (BAL) cell counts. Increases in SP-D content, gamma interferon, interleukin-4, interleukin-5, and tumor necrosis factor alpha in BAL fluid occurred but were attenuated in PCP-infected SP-A(-/-) mice compared to WT mice. There were increases in total BAL NO levels in both infected groups, but nitrite levels were higher in SP-A(-/-) mice, indicating a reduction in production of higher oxides of nitrogen that was also reflected in lower levels of 3-nitrotyrosine staining in the SP-A(-/-) group. We conclude that despite increases in inflammatory cells, SP-A-deficient mice infected with P. carinii exhibit an enhanced susceptibility to the organism and attenuated production of proinflammatory cytokines and reactive oxygen-nitrogen species. These data support the concept that SP-A is a local effector molecule in the lung host defense against P. carinii in vivo.