Surfactant protein D enhances Pneumocystis infection in immune-suppressed mice.

To further determine the role of surfactant protein (SP)-D in the pathogenesis of Pneumocystis pneumonia, a mouse model of transgenic overexpression (OE) of SP-D was studied. These animals produce roughly 30- to 50-fold greater SP-D than their wild-type (WT) counterparts but show no other differences in lung morphology and function. Animals in both the SP-D OE and WT groups were depleted of CD4 lymphocytes with weekly injections of GK1.5 antibody, before Pneumocystis inoculation, and throughout the subsequent infection period. At various time points, mice were killed and analyzed for inflammatory parameters and organism burden. Proinflammatory cytokines in bronchoalveolar lavage fluid were elevated throughout the period of infection, with OE animals exhibiting significantly higher levels of TNF-alpha and macrophage inflammatory protein-2 compared with WT controls. The total number of cells in the lavage fluid was also increased significantly only in the OE group, whereas the cell differential composition demonstrated lymphocyte and eosinophil infiltration in both groups of animals. Significantly, the organism burden was markedly higher in the SP-D OE animals, whereas the WT mice demonstrated little alteration in organism number over the course of infection. These results further indicate that SP-D facilitates the development of Pneumocystis infection and related lung inflammation in an immunosuppressed mouse model.

Alveolar surfactant protein D content modulates bleomycin-induced lung injury.

RATIONALE: Surfactant protein D (SP-D) is a collectin family member with demonstrated immunomodulatory properties in vitro. We hypothesized that SP-D modulates inflammation during noninfectious lung injury in vivo. OBJECTIVES: To investigate the association of alveolar SP-D and injury, we studied the responses of transgenic mice expressing varying levels of SP-D to intratracheal bleomycin (ITB). METHODS: Eight-week old C57/BL6 SP-D-deficient (-/-) mice and syngeneic wild-type (WT) controls or Swiss Black SP-D-overexpressing (SP-D OE) mice and littermate controls received either ITB or saline and were followed for up to 21 d. MEASUREMENTS AND RESULTS: Kaplan-Meier analysis demonstrated a dose-dependent decrease in survival in ITB SP-D (-/-) mice receiving 2 U/kg bleomycin, with a 14-d mortality of 100% versus 0% mortality for WT receiving 2 U/kg ITB or SP-D (-/-) mice given saline (p < 0.05). At 8 d, ITB SP-D (-/-) mice had greater respiratory distress (frequency/tidal volume) and weight loss than ITB WT mice. Furthermore, bronchoalveolar lavage cellularity, pulmonary parenchymal inflammation, and tissue 3-nitrotyrosine (NO2 Y) were increased to a greater extent in ITB SP-D (-/-) mice. By 21 d, compared with all groups, ITB SP-D (-/-) survivors had increased Trichrome staining and tissue hydroxyproline levels. As proof of principle, SP-D OE mice were highly resistant to bleomycin-induced morbidity and mortality at doses up to 3 U/kg. CONCLUSIONS: These data provide new in vivo evidence for an antiinflammatory role for SP-D in response to noninfectious, subacute lung injury via modulation of oxidative-nitrative stress.

Serum levels of surfactant protein D are increased in mice with lung tumors.

Most murine lung tumors are composed of differentiated epithelial cells. We have reported previously that surfactant protein (SP)-D is expressed in urethane-induced tumors. Serum levels of SP-D are increased in patients with interstitial lung disease and acute respiratory distress syndrome and in rats with acute lung injury but have not been measured in mice. In this study, we sought to determine whether SP-D could be detected in murine serum and discovered that it was increased in mice bearing lung tumors. Serum SP-D concentration was 5.0 +/- 0.2 ng/ml in normal C57BL/6 mice, essentially absent in SP-D nulls, and 63.6 +/- 9.0 ng/ml in SP-D-overexpressing mice. SP-D in serum was verified by immunoblotting. Serum SP-D was increased in mice bearing tumors induced by three different protocols, and the SP-D level correlated with tumor volume. However, in mice with a single adenoma or a few adenomas, SP-D levels were usually within the normal range. SP-D was expressed by the tumor cells, and there was also a field effect whereby type II cells near the tumor expressed more SP-D than type II cells in the remainder of the lung. Serum SP-D was also increased by lung inflammation. In airway inflammation induced by aerosolized ovalbumin in sensitized BALB/c mice, the serum levels were elevated after challenge. In conclusion, serum SP-D concentration is increased in mice bearing lung tumors and generally reflects the tumor burden but is also elevated during lung inflammation.

Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability.

The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A-null mice and was increased in SP-D-overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D-mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane.

Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex.

Removal of cells dying by apoptosis is essential to normal development, maintenance of tissue homeostasis, and resolution of inflammation. Surfactant protein A (SP-A) and surfactant protein D (SP-D) are high abundance pulmonary collectins recently implicated in apoptotic cell clearance in vitro. Other collectins, such as mannose-binding lectin and the collectin-like C1q, have been shown to bind to apoptotic cells and drive ingestion through interaction with calreticulin and CD91 on the phagocyte in vitro. However, only C1q has been shown to enhance apoptotic cell uptake in vivo. We sought to determine the relative importance of SP-A, SP-D, and C1q in pulmonary clearance of apoptotic cells using knockout and overexpressing mice, and to determine the role of calreticulin and CD91 in this process. SP-A, SP-D, and C1q all enhanced apoptotic cell ingestion by resident murine and human alveolar macrophages in vitro. However, only SP-D altered apoptotic cell clearance from the naive murine lung, suggesting that SP-D plays a particularly important role in vivo. Similar to C1q and mannose-binding lectin, SP-A and SP-D bound to apoptotic cells in a localized, patchy pattern and drove apoptotic cell ingestion by phagocytes through a mechanism dependent on calreticulin and CD91. These results suggest that the entire collectin family of innate immune proteins (including C1q) works through a common receptor complex to enhance removal of apoptotic cells, and that collectins are integral, organ-specific components of the clearance machinery.

Targeted expression of the receptor tyrosine kinase RON in distal lung epithelial cells results in multiple tumor formation: oncogenic potential of RON in vivo.

RON, a member of the MET proto-oncogene family, has been implicated in the progression of certain epithelial cancers. The purpose of this study was to determine the oncogenic potential of RON in vivo in lung epithelial cells. Transgenic mice were established using surfactant protein C promoter to express human RON in the distal lung epithelial cells. These mice were born normal but developed multiple lung tumors with distinct morphology and growth patterns. Tumors appeared as a single mass in the lung around 2 months of age and gradually developed into multiple nodules located mostly in the peripheral portions of the lung. A transition from early adenomas to later adenocarcinomas was observed. Morphologically, tumors were characterized as cuboidal epithelial cells with a type II cell phenotype, grew along the alveolar walls, and projected into the alveolar septa. RON was highly expressed and constitutively activated in tumors. These results indicate that overexpression of human wild-type RON causes the formation of lung tumors with unique biological characteristics in vivo. This model provides opportunities to study the role of RON in the pathogenesis of lung tumors and to elucidate the mechanisms underlying this distinct lung tumor.

Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein inhibits inducible cyclooxygenase-2 expression in murine macrophages.

The RON receptor tyrosine kinase is activated by macrophage-stimulating protein, which regulates macrophage migration, phagocytosis, and nitric oxide production. We report here the inhibitory effect of RON on lipopolysaccharide (LPS)-induced cyclooxygenase (Cox)-2 expression in mouse macrophages. In RON-expressing macrophages treated with macrophage stimulating protein, LPS-induced prostaglandin E(2) (PGE(2)) production was significantly reduced. The inhibition was accompanied by reduction of Cox-2 protein and mRNA expression. Transcriptional studies indicated that RON activation inhibits LPS-induced luciferase activity driven by the Cox-2 gene promoter. To determine whether RON activation affects LPS-induced NF-kappa B pathway, which is important for Cox-2 expression. Western blot analyses were performed showing that RON activation inhibits LPS-induced I kappa B alpha degradation. The decreased I kappa B alpha degradation was due to reduced I kappa B alpha phosphorylation at Ser-32 as determined by I kappa B alpha (Ser-32) phosphor-antibody. Moreover, we found that LPS-induced IKK beta activity, an enzyme responsible for phosphorylation of I kappa B alpha, was inhibited upon RON activation. Interestingly, these inhibitory effects were not regulated by RON-mediated phosphatidylinositol-3 kinase. These results suggest that RON activation inhibits LPS-induced macrophage Cox-2 expression. The inhibitory effect is mediated by impairing LPS-activated cascade enzymes that activate NF-kappa B. The inhibition of Cox-2 expression might represent a novel mechanism for the inhibitory functions of RON in vivo against LPS-induced inflammation and septic shock.

Lymphocyte activation in the lungs of SP-D null mice.

Surfactant protein D (SP-D) appears to play an important role in regulating local pulmonary inflammatory responses to pathogens. There is also in vitro evidence that SP-D may suppress local T cell responses. However, the role of SP-D in regulating T cell responses directly in the lung has not been previously evaluated in vivo. SP-D(-)(/-) mice demonstrate peribronchial and perivascular accumulations of lymphocytes. Therefore, we investigated the functional status and abundance of intrapulmonary lymphocytes in SP-D(-)(/-) mice. By morphometric analysis, SP-D(-)(/-) mice demonstrated increased numbers of airway- and vessel-associated lymphocytes without increases in interstitial lymphocytes. There was increased proliferative activity of lymphocytes isolated by enzymatic disassociation of minced lung. Flow cytometry was used to determine the number and functional activation status of intrapulmonary CD4(+) and CD8(+) T cells, as well as B cells and NK cells. Cytokine expression patterns in lung tissues were evaluated using RNase protection assays, reverse transcriptase/polymerase chain reaction, and enzyme-linked immunosorbent assay. There was marked T cell activation in the lungs of SP-D(-)(/-) mice, as reflected by an increased percentage of both CD4(+) and CD8(+) T cells expressing CD69 and CD25. BAL CD4 lymphocytes were increased and the fraction expressing CD69 was also increased. Although there were increases in BAL CD8 lymphocytes, apparent increases in CD69-positive CD8 lymphocytes did not reach statistical significance. In contrast, splenic T cells were not activated in SPD(-)(/-) mice. Of the proinflammatory cytokines evaluated, only interleukin (IL)-12 and IL-6 expression were consistently upregulated in the lungs of SPD(-)(/-) mice. Increased IL-2 expression was apparent but did not reach statistical significance. We conclude that the lack of local pulmonary production of SP-D leads to a state of persistent T cell activation, possibly in response to exogenous antigens. This study therefore provides further evidence of the important local immunoregulatory role of SP-D in vivo.

Three Mile Island : The silent disaster

From Wednesday, March 28, 1979,to Wednesday, April 4, 1979, Dauphin County, Pennsylvania, was in a state of near-panic in response to the Three Mile Island nuclear accident. The Dauphin County Office of Emergency Preparedness quickly attempted to develop a plan to evacuate not only the population of an area 20 miles in radius from the plant but the short-term and long-term care medical facilities as well. For medical evacuation, a system of classification of patients was defined and matched to needed transportation. Furthermore, a critical coordinating link was established with the Hospital Association of Pennsylvania to identify and categorize relocation beds in receiving hospitals far from the incident site in the event of evacuation. Just as this incident was unusual, so too were the planning activities unique since they were never before conceived or accomplished (AU)