Steven E. Wilcoxen

Granulocyte-macrophage colony-stimulating factor in the innate immune response to Pneumocystis carinii pneumonia in mice.

Innate immunity plays an important role in pulmonary host defense against Pneumocystis carinii, an important pathogen in individuals with impaired cell-mediated immunity. We investigated the role of GM-CSF in host defense in a model of P. carinii pneumonia induced by intratracheal inoculation of CD4-depleted mice. Lung GM-CSF levels increased progressively during the infection and were significantly greater than those in uninfected controls 3, 4, and 5 wk after inoculation. When GM-CSF gene-targeted mice (GM−/−) depleted of CD4+ cells were inoculated with P. carinii, the intensities of infection and inflammation were increased significantly compared with those in CD4-depleted wild-type mice. In contrast, transgenic expression of GM-CSF directed solely in the lungs of GM−/− mice (using the surfactant protein C promoter) dramatically decreased the intensity of infection and inflammation 4 wk after inoculation. The concentrations of surfactant proteins A and D were greater in both uninfected and infected GM−/− mice compared with those in wild-type controls, suggesting that this component of the innate response was preserved in the GM−/− mice. However, alveolar macrophages (AM) from GM−/− mice demonstrated impaired phagocytosis of purified murine P. carinii organisms in vitro compared with AM from wild-type mice. Similarly, AM production of TNF-α in response to P. carinii in vitro was totally absent in AM from GM−/− mice, while GM-CSF-replete mice produced abundant TNF in this setting. Thus, GM-CSF plays a critical role in the inflammatory response to P. carinii in the setting of impaired cell-mediated immunity through effects on AM activation.

Lysosomal Phospholipase A2 Is Selectively Expressed in Alveolar Macrophages

Lung surfactant is the surface-active agent comprised of phospholipids and proteins that lines pulmonary alveoli. Surfactant stabilizes the alveolar volume by reducing surface tension. Previously, we identified a lysosomal phospholipase A2, termed LPLA2, with specificity toward phosphatidylcholine and phosphatidylethanolamine. The phospholipase is localized to lysosomes, is calcium-independent, has an acidic pH optimum, and transacylates ceramide. Here, we demonstrate that LPLA2 is selectively expressed in alveolar macrophages but not in peritoneal macrophages, peripheral blood monocytes, or other tissues. Other macrophage-associated phospholipase A2s do not show a comparable distribution. LPLA2 is of high specific activity and recognizes disaturated phosphatidylcholine as a substrate. The lysosomal phospholipase A2 activity is six times lower in alveolar macrophages from mice with a targeted deletion of the granulocyte macrophage colony-stimulating factor (GM-CSF), a model of impaired surfactant catabolism, compared with those from wild-type mice. However, LPLA2 activity and protein levels are measured in GM-CSF null mice in which GM-CSF is expressed as a transgene under the control of the surfactant protein C promoter. Thus LPLA2 may be a major enzyme of pulmonary surfactant phospholipid degradation by alveolar macrophages and may be deficient in disorders of surfactant metabolism.

Sublethal Hyperoxia Impairs Pulmonary Innate Immunity

Supplemental oxygen is often required in the treatment of critically ill patients. The impact of hyperoxia on pulmonary host defense is not well-established. We hypothesized that hyperoxia directly impairs pulmonary host defense, beyond effects on alveolar wall barrier function. C57BL/6 mice were kept in an atmosphere of >95% O2 for 4 days followed by return to room air. This exposure does not lead to mortality in mice subsequently returned to room air. Mice kept in room air served as controls. Mice were intratracheally inoculated with Klebsiella pneumoniae and followed for survival. Alveolar macrophages (AM) were harvested by bronchoalveolar lavage after 4 days of in vivo hyperoxia for ex vivo experiments. Mortality from pneumonia increased significantly in mice exposed to hyperoxia compared with infected mice in room air. Burden of organisms in the lung and dissemination of infection were increased in the hyperoxia group whereas accumulation of inflammatory cells in the lung was impaired. Hyperoxia alone had no impact on AM numbers, viability, or ability to phagocytize latex microbeads. However, following in vivo hyperoxia, AM phagocytosis and killing of Gram-negative bacteria and production of TNF-α and IL-6 in response to LPS were significantly reduced. AM surface expression of Toll-like receptor-4 was significantly decreased following in vivo hyperoxia. Thus sublethal hyperoxia increases Gram-negative bacterial pneumonia mortality and has a significant adverse effect on AM host defense function. Impaired AM function due to high concentrations of supplemental oxygen may contribute to the high rate of ventilator-associated pneumonia seen in critically ill patients.

Chemotaxis of alveolar macrophages in response to signals derived from alveolar epithelial cells

We have postulated that alveolar epithelial cells (AEC) play a critical role in local regulation of alveolar macrophage (AM) recruitment and activation for host defense in the lung. The present study explores the effects of conditioned medium from AEC (AEC-CM) on the migration of AM, using a Boyden chamber assay. AEC-CM was chemotactic for AM, with peak activity observed with a 1:10 dilution. We previously showed that rat AEC express the chemokines RANTES (regulated on activation, normal T expressed and secreted) and monocyte chemoattractant protein 1 (MCP-1) as well as granulocyte-macrophage colony-stimulating factor (GM-CSF). Neutralizing antibodies to RANTES and to MCP-1 and immunoprecipitation of GM-CSF decreased the chemotactic activity of AEC-CM by 58%, 29%, and 47%, respectively. Similar levels of chemotaxis were found in response to recombinant RANTES, MCP-1, and GM-CSF. In each instance the optimal dose was very low (0.01 to 0.1 ng/ml), with diminished chemotaxis at higher doses. Peritoneal macrophages (PM) also migrated in response to AEC-CM and each of the recombinant cytokines; however, AM were much more sensitive to AEC-CM, RANTES, and GM-CSF than were PM. AM migrated preferentially from medium conditioned by unstimulated AEC toward supernatants from interleukin 1alpha-stimulated AEC. Therefore, AEC may control the distribution of AM through the creation of local chemotactic gradients and are likely to play a critical role in the host response to low-level antigen entry into the peripheral lung.

Transgenic Overexpression of Granulocyte Macrophage-Colony Stimulating Factor in the Lung Prevents Hyperoxic Lung Injury

Granulocyte macrophage-colony stimulating factor (GM-CSF) plays an important role in pulmonary homeostasis, with effects on both alveolar macrophages and alveolar epithelial cells. We hypothesized that overexpression of GM-CSF in the lung would protect mice from hyperoxic lung injury by limiting alveolar epithelial cell injury. Wild-type C57BL/6 mice and mutant mice in which GM-CSF was overexpressed in the lung under control of the SP-C promoter (SP-C-GM mice) were placed in >95% oxygen. Within 6 days, 100% of the wild-type mice had died, while 70% of the SP-C-GM mice remained alive after 10 days in hyperoxia. Histological assessment of the lungs at day 4 revealed less disruption of the alveolar wall in SP-C-GM mice compared to wild-type mice. The concentration of albumin in bronchoalveolar lavage fluid after 4 days in hyperoxia was significantly lower in SP-C-GM mice than in wild-type mice, indicating preservation of alveolar epithelial barrier properties in the SP-C-GM mice. Alveolar fluid clearance was preserved in SP-C-GM mice in hyperoxia, but decreased significantly in hyperoxia-exposed wild-type mice. Staining of lung tissue for caspase 3 demonstrated increased apoptosis in alveolar wall cells in wild-type mice in hyperoxia compared to mice in room air. In contrast, SP-C-GM mice exposed to hyperoxia demonstrated only modest increase in alveolar wall apoptosis compared to room air. Systemic treatment with GM-CSF (9 micro g/kg/day) during 4 days of hyperoxic exposure resulted in decreased apoptosis in the lungs compared to placebo. In studies using isolated murine type II alveolar epithelial cells, treatment with GM-CSF greatly reduced apoptosis in response to suspension culture. In conclusion, overexpression of GM-CSF enhances survival of mice in hyperoxia; this effect may be explained by preservation of alveolar epithelial barrier function and fluid clearance, at least in part because of reduction in hyperoxia-induced apoptosis of cells in the alveolar wall.

Role of alveolar epithelial cell intercellular adhesion molecule-1 in host defense against Klebsiella pneumoniae

Intercellular adhesion molecule-1 (ICAM-1) is expressed at high levels on type I alveolar epithelial cells (AEC) in the normal alveolar space. We postulate that AEC ICAM-1 enhances the antimicrobial activity of macrophages and neutrophils in the alveolar space. Wild-type and mutant mice deficient in ICAM-1 were inoculated intratracheally with Klebsiella pneumoniae. After 10 days, 43% of the ICAM-1 mutant mice had died compared with 14% of the wild-type controls (P = 0.003). Significantly more bacteria were isolated from lungs of ICAM-1 mutant mice than controls 24 h after inoculation (log colony-forming units 5.14 +/- 0.21 vs. 3.46 +/- 0. 16, P = 0.001). However, neutrophil recruitment to the lung was not different. In similar experiments in the rat, inhibition of alveolar ICAM-1 by intratracheal administration of antibody resulted in significantly impaired clearance of K. pneumoniae. The role of phagocyte interactions with AEC ICAM-1 for antimicrobial activity was investigated in vitro using primary cultures of rat AEC that express abundant ICAM-1. Alveolar macrophage phagocytosis and killing of K. pneumoniae were increased significantly in the presence of AEC; these effects were inhibited significantly (47.5 and 52%, respectively) when AEC ICAM-1 was blocked. Similarly, neutrophil phagocytic activity for K. pneumoniae in the presence of AEC in vitro was decreased when ICAM-1 on the AEC surface was blocked. Thus in the absence of ICAM-1, there is impaired ability to clear K. pneumoniae from the lungs, resulting in increased mortality. These studies indicate that AEC ICAM-1 plays an important role in host defense against K. pneumoniae by determining the antimicrobial activity of phagocytes within the lung.Intercellular adhesion molecule-1 (ICAM-1) is expressed at high levels on type I alveolar epithelial cells (AEC) in the normal alveolar space. We postulate that AEC ICAM-1 enhances the antimicrobial activity of macrophages and neutrophils in the alveolar space. Wild-type and mutant mice deficient in ICAM-1 were inoculated intratracheally with Klebsiella pneumoniae. After 10 days, 43% of the ICAM-1 mutant mice had died compared with 14% of the wild-type controls ( P = 0.003). Significantly more bacteria were isolated from lungs of ICAM-1 mutant mice than controls 24 h after inoculation (log colony-forming units 5.14 ± 0.21 vs. 3.46 ± 0.16, P = 0.001). However, neutrophil recruitment to the lung was not different. In similar experiments in the rat, inhibition of alveolar ICAM-1 by intratracheal administration of antibody resulted in significantly impaired clearance of K. pneumoniae. The role of phagocyte interactions with AEC ICAM-1 for antimicrobial activity was investigated in vitro using primary cultures of rat AEC that express abundant ICAM-1. Alveolar macrophage phagocytosis and killing of K. pneumoniae were increased significantly in the presence of AEC; these effects were inhibited significantly (47.5 and 52%, respectively) when AEC ICAM-1 was blocked. Similarly, neutrophil phagocytic activity for K. pneumoniae in the presence of AEC in vitro was decreased when ICAM-1 on the AEC surface was blocked. Thus in the absence of ICAM-1, there is impaired ability to clear K. pneumoniae from the lungs, resulting in increased mortality. These studies indicate that AEC ICAM-1 plays an important role in host defense against K. pneumoniae by determining the antimicrobial activity of phagocytes within the lung.

Interaction of rat Pneumocystis carinii and rat alveolar epithelial cells in vitro

During Pneumocystis carinii pneumonia, P. carinii trophic forms adhere tightly to type I alveolar epithelial cells (AECs). However, the manner in which the interaction between P. cariniiorganisms and AECs results in clinical pneumonia has not been explored. To investigate this interaction in vitro, we established a culture system using rat P. carinii and primary cultures of rat AECs. We hypothesized that binding of P. carinii to AECs would alter the metabolic, structural, and barrier functions of confluent AECs. Using fluorescently labeled P. carinii, we demonstrated that P. carinii bound to AECs in a dose-dependent manner. During P. carinii-AEC interaction, both the AECs and the P. carinii organisms remained metabolically active. Immunofluorescent staining demonstrated that AEC expression of the junctional proteins E-cadherin and occludin and the structural protein cytokeratin 8 were unaffected by P. carinii binding. To evaluate the effect of P. carinii on AEC barrier function, transepithelial resistance across AEC monolayers was measured during interaction with organisms. Culture with P. carinii did not result in loss of AEC barrier function but in fact increased AEC transepithelial resistance in a dose- and time-dependent manner. We conclude that the direct interaction of P. carinii with AECs does not disrupt AEC metabolic, structural, or barrier function. Therefore, we speculate that additional inflammatory cells and/or their signals are required to induce the epithelial derangements characteristic of P. carinii pneumonia.During Pneumocystis carinii pneumonia, P. carinii trophic forms adhere tightly to type I alveolar epithelial cells (AECs). However, the manner in which the interaction between P. carinii organisms and AECs results in clinical pneumonia has not been explored. To investigate this interaction in vitro, we established a culture system using rat P. carinii and primary cultures of rat AECs. We hypothesized that binding of P. carinii to AECs would alter the metabolic, structural, and barrier functions of confluent AECs. Using fluorescently labeled P. carinii, we demonstrated that P. carinii bound to AECs in a dose-dependent manner. During P. carinii-AEC interaction, both the AECs and the P. carinii organisms remained metabolically active. Immunofluorescent staining demonstrated that AEC expression of the junctional proteins E-cadherin and occludin and the structural protein cytokeratin 8 were unaffected by P. carinii binding. To evaluate the effect of P. carinii on AEC barrier function, transepithelial resistance across AEC monolayers was measured during interaction with organisms. Culture with P. carinii did not result in loss of AEC barrier function but in fact increased AEC transepithelial resistance in a dose- and time-dependent manner. We conclude that the direct interaction of P. carinii with AECs does not disrupt AEC metabolic, structural, or barrier function. Therefore, we speculate that additional inflammatory cells and/or their signals are required to induce the epithelial derangements characteristic of P. carinii pneumonia.

Critical Roles of Inflammation and Apoptosis in Improved Survival in a Model of Hyperoxia-Induced Acute Lung Injury in Pneumocystis murina-Infected Mice

ABSTRACT 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.

Disparate mechanisms of sICAM-1 production in the peripheral lung: contrast between alveolar epithelial cells and pulmonary microvascular endothelial cells

Membrane-associated intercellular adhesion molecule-1 (mICAM-1; CD54) is constitutively expressed on the surface of type I alveolar epithelial cells (AEC). Soluble ICAM-1 (sICAM-1) may be produced by proteolytic cleavage of mICAM-1 or by alternative splicing of ICAM-1 mRNA. In contrast to inducible expression seen in most cell types, sICAM-1 is constitutively released by type I AEC and is present in normal alveolar lining fluid. Therefore, we compared the mechanism of sICAM-1 production in primary cultures of two closely juxtaposed cells in the alveolar wall, AEC and pulmonary microvascular endothelial cells (PVEC). AEC, but not PVEC, demonstrated high-level baseline expression of sICAM-1. Stimulation of AEC with TNFalpha or LPS resulted in minimal increase in AEC sICAM-1, whereas PVEC sICAM-1 was briskly induced in response to these signals. AEC sICAM-1 shedding was significantly reduced by treatment with a serine protease inhibitor, but not by cysteine, metalloprotease, or aspartic protease inhibitors. In contrast, none of these inhibitors effected sICAM-1 expression in PVEC. RT-PCR, followed by gel analysis of total RNA, suggests that alternatively spliced fragments are present in both cell types. However, a 16-mer oligopeptide corresponding to the juxtamembrane region of mICAM-1 completely abrogated sICAM-1 shedding in AEC but reduced stimulated PVEC sICAM-1 release by only 20%. Based on these data, we conclude that the predominant mechanism of sICAM-1 production likely differs in the two cell types from opposite sides of the alveolar wall.

REGULATION OF 5-LIPOXYGENASE ACTIVITY IN MONONUCLEAR PHAGOCYTES : CHARACTERIZATION OF AN ENDOGENOUS CYTOSOLIC INHIBITOR

The proinflammatory leukotrienes (LT) play important roles in host defense and disease states. However, no endogenous mechanisms to downregulate 5-lipoxygenase (5-LO), the enzyme catalyzing LT synthesis, have been described. We observed that the cytosolic fraction of rat alveolar macrophages (AMs) and peritoneal macrophages (PMs), and of peripheral blood monocytes (PBMs) contain substantial amounts of 5-LO protein, but little detectable 5-LO activity. We therefore examined these mononuclear phagocyte (MNP) cytosolic fractions for inhibitory activity against 5-LO. MNP cytosol dose-dependently reduced the 5-LO activity in neutrophil (PMN) cytosol and AM membrane. Furthermore, MNP cytosol dose-dependently prolonged the lag phase of soybean lipoxygenase (LO) without affecting the rate of product formation. This effect was overcome by subsequent addition of 13(S)-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HpOD), suggesting that the active factor scavenges hydroperoxides. Inactivation by boiling and roteinase K suggest that is a protein. We speculate that this cytosolic factor(s) may serve as an endogenous means for the down-regulation of 5-LO in macrophages.