Surfactant protein D binds selectively to Klebsiella pneumoniae lipopolysaccharides containing mannose-rich O-antigens.

Surfactant protein D (SP-D) plays important roles in the regulation of innate immune responses in the lung. We have previously shown that SP-D can agglutinate and enhance the macrophage-dependent killing of specific unencapsulated phase variants of Klebsiella pneumoniae. In the present studies, we used 16 clinical isolates of Klebsiella representing four O-serotypes and examined the interaction of SP-D with their isolated LPSs. Although SP-D bound to the core oligosaccharide of rough LPS from all isolates, it selectively bound to smooth forms of LPS expressed by O-serotypes with mannose-rich repeating units in their O-polysaccharides. SP-D was more potent in agglutinating unencapsulated phase variants of O-serotypes expressing these SP-D "reactive" O-polysaccharides, and more effectively inhibited the adhesion of these serotypes to lung epithelial cells. This novel anti-adhesion activity required the multimerization of trimeric SP-D subunits (dodecamers). Klebsiella serotypes expressing "nonreactive" LPS O-Ags were isolated at a significantly higher frequency from patients with K. pneumoniae. Our findings suggest that SP-D plays important roles in the clearance of opportunistic Gram-negative bacteria and contributes to known serotypic differences in the pathogenicity of Klebsiella through specific interactions with O-polysaccharides.

Surfactant protein B gene variations enhance susceptibility to squamous cell carcinoma of the lung in German patients.

Genetic factors are thought to influence the risk for lung cancer. Since pulmonary surfactant mediates the response to inhaled carcinogenic substances, candidate genes may be among those coding for pulmonary surfactant proteins. In the present matched case-control study a polymorphism within intron 4 of the gene coding for surfactant specific protein B was analysed in 357 individuals. They were divided into 117 patients with lung cancer (40 patients with small cell lung cancer, 77 patients with non small cell lung cancer), matched controls and 123 healthy individuals. Surfactant protein B gene variants were analysed using specific PCR and cloned surfactant protein B sequences as controls. The frequency of the intron 4 variation was similar in both control groups (13.0% and 9.4%), whereas it was increased in the small cell lung cancer group (17.5%) and the non small cell lung cancer group (16.9%). The gene variation was found significantly more frequently in patients with squamous cell carcinoma (25.0%, P=0.016, odds ratio=3.2, 95%CI=1.24-8.28) than in the controls. These results indicate an association of the surfactant protein B intron 4 variants and/or its flanking loci with mechanisms that may enhance lung cancer susceptibility, especially to squamous cell carcinoma of the lung.

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.

The inhibitory effect of C-reactive protein on bacterial phosphorylcholine platelet-activating factor receptor-mediated adherence is blocked by surfactant.

Numerous major bacterial pathogens in the human respiratory tract, including Streptococcus pneumoniae and Haemophilus influenzae, express cell-surface phosphorylcholine (ChoP), a ligand for the receptor for platelet-activating factor (rPAF). ChoP is also bound by C-reactive protein (CRP), which, in the presence of complement, may be bactericidal. This study found that CRP can block the attachment of bacteria expressing cell-surface ChoP to host cells. Concentrations of CRP equivalent to those on the mucosal surface of the human airway blocked most adherence of both S. pneumoniae and H. influenzae to human pharyngeal epithelial cells. ChoP-mediated adherence was also reduced in the presence of an rPAF antagonist. The antiadhesive effects of the rPAF antagonist and CRP were not additive, suggesting that CRP activity is specific to the area of adherence mediated by the receptor. The binding of CRP to ChoP and the effect of CRP on adherence were inhibited by human surfactant (primarily ChoP). The antiadhesive effect of CRP may be diminished in the terminal airway, where surfactant is abundant.

Cutting edge: the immunostimulatory activity of the lung surfactant protein-A involves Toll-like receptor 4.

The collectin surfactant protein-A (SP-A) is involved in the innate host defense and the regulation of inflammatory processes in the lung. In this work we investigated the molecular mechanisms related to the immunostimulatory activity of SP-A using macrophages from C3H/HeJ mice, which carry an inactivating mutation in the Toll-like receptor (TLR)4 gene, and TLR4-transfected Chinese hamster ovary cells. We demonstrate that SP-A-induced activation of the NF-kappaB signaling pathway and up-regulation of cytokine synthesis such as TNF-alpha and IL-10 are critically dependent on the TLR4 functional complex. These findings support the concept that TLR4 is a pattern recognition receptor that signals in response to both foreign pathogens and endogenous host mediators.

[Function of clara cells in lung remodeling].

Clara cells localized in the bronchiole are thought to play a key role in lung remodeling. Clara cell secretory protein (CCSP), which is specifically produced by Clara cells, inhibits pro-inflammatory cytokines and chemotaxis of fibroblasts, and acts as phospholopase A2 inhibitors, and may also inhibit inflammation and fibrosis in the lung. This could be a biomarker of interstitial pneumonitis. Proliferation of Clara cells and neuroendocrine cells at airway epithelial injury may act as progenitor cells during the repair process.

Mechanical ventilation of isolated rat lungs changes the structure and biophysical properties of surfactant.

Mechanical ventilation is an essential but potentially harmful therapeutic intervention for patients with acute lung injury. The objective of this study was to investigate the effects of mechanical ventilation on large-aggregate surfactant (LA) structure and function. Isolated rat lungs were randomized to either a nonventilated control group, a relatively noninjuriously ventilated group [1 h, 10 ml/kg tidal volume, 3 cmH(2)O positive end-expiratory pressure (PEEP)], or an injuriously ventilated group (1 h, 20 ml/kg tidal volume, 0 cmH(2)O PEEP). Injurious ventilation resulted in significantly decreased lung compliance compared with the other two groups. LA structure, as determined by electron microscopy, revealed that LA from the injurious group had significantly lower amounts of organized lipid-protein structures compared with LA obtained from the other groups. Analysis of the biophysical properties by using a captive bubble surfactometer demonstrated that adsorption and surface tension reduction were significantly impaired with LA from the injuriously ventilated lungs. We conclude that the injurious mechanical ventilation impairs LA function and that this impairment is associated with significant morphological alterations.

Absence of SP-A modulates innate and adaptive defense responses to pulmonary influenza infection.

Mice lacking surfactant protein SP-A [SP-A(-/-)] and wild type SP-A(+/+) mice were infected with influenza A virus (IAV) by intranasal instillation. Decreased clearance of IAV was observed in SP-A(-/-) mice and was associated with increased pulmonary inflammation. Treatment of SP-A(-/-) mice with exogenous SP-A enhanced viral clearance and decreased lung inflammation. Uptake of IAV by alveolar macrophages was similar in SP-A(-/-) and SP-A(+/+) mice. Myeloperoxidase activity was reduced in isolated bronchoalveolar lavage neutrophils from SP-A(-/-) mice. B lymphocytes and activated T lymphocytes were increased in the lung and spleen, whereas T helper (Th) 1 responses were increased [interferon-gamma, interleukin (IL)-2, and IgG(2a)] and Th2 responses were decreased (IL-4, and IL-10, and IgG(1)) in the lungs of SP-A(-/-) mice 7 days after IAV infection. In the absence of SP-A, impaired viral clearance was associated with increased lung inflammation, decreased neutrophil myeloperoxidase activity, and increased Th1 responses. Because the airway is the usual portal of entry for IAV and other respiratory pathogens, SP-A is likely to play a role in innate defense and adaptive immune responses to IAV.

Surfactant function in lung transplantation after 24 hours of ischemia: advantage of retrograde flush perfusion for preservation.

OBJECTIVE: Surfactant function was shown to be impaired in clinical and experimental lung transplantation. This study was designed to define the impact of retrograde flush perfusion on graft and surfactant function after an extended period of ischemia. METHODS: Left lung transplantation was performed after 24 hours of graft ischemia in 12 pigs. In half of the grafts antegrade cold flush perfusion (Perfadex) was used for preservation. In the second group grafts were flushed in a retrograde fashion via the left atrium. Graft function was monitored for 7 hours after transplantation. Before transplantation (basal) and after 2 hours of reperfusion, bronchoalveolar lavage fluid was obtained. Minimal surface tension of bronchoalveolar lavage fluid was determined and the ratio of small and large surfactant aggregates was calculated. Lung water content was analyzed online in the reperfusion period. RESULTS: Right-sided heart failure developed in 2 animals of group 1 (antegrade perfusion) within 2 and 4.5 hours of reperfusion, respectively. All other pigs survived the observation period. PO(2)/FIO(2) (P =.001) and dynamic lung compliance (P =.001) were superior in retrogradely flushed grafts. A comparable increase of minimal surface tension was found after reperfusion in both groups. Small/large surfactant aggregate ratio after reperfusion (P =.03), as well as extravascular lung water content, was higher in the antegrade perfusion group. CONCLUSION: Retrograde flush perfusion for 24-hour lung preservation with low-potassium dextran (Perfadex) solution led to better initial graft function than the standard antegrade perfusion technique. A moderate impairment of surfactant function was found in both groups, which was more pronounced in the antegrade perfusion group.

Pulmonary surfactant and inflammation in septic adult mice: role of surfactant protein A.

Surfactant alterations, alveolar cytokine changes, and the role of surfactant protein (SP)-A in septic mice were investigated. Sepsis was induced via cecal ligation and perforation (CLP). Septic and sham mice were euthanized at 0, 3, 6, 9, 12, 15, and 18 h after surgery. Mice deficient in SP-A and mice that overexpressed SP-A were euthanized 18 h after surgery. In wild-type, sham-operated mice, surfactant pool sizes were similar at all time points, whereas in the CLP groups there was a significant decrease in small-aggregate surfactant pool sizes beginning 6 h after CLP. Interleukin-6 concentrations in bronchoalveolar lavage fluid from septic animals increased from 6 to 18 h after surgery. Identical surfactant alterations and concentrations of cytokines were observed in septic mice that were SP-A deficient or that overexpressed SP-A. In conclusion, alterations of pulmonary surfactant and alveolar cytokines occur simultaneously, 6 h after a systemic insult. In addition, we did not detect a role for SP-A in regulating surfactant phospholipid pool sizes or pulmonary inflammation in septic mice.

Surfactant protein D inhibition of human macrophage uptake of Mycobacterium tuberculosis is independent of bacterial agglutination.

The innate immune system in the lung is essential for controlling infections due to inhaled pathogens. Mycobacterium tuberculosis (M.tb) encounters components of the innate immune system when inhaled into the lung, but the consequences of these interactions are poorly understood. Surfactant protein D (SP-D) binds to and agglutinates M.tb bacilli, and reduces the uptake of the bacteria by human macrophages. In the current studies, we utilized a recombinant SP-D variant (CDM) that lacks the collagen domain to further characterize the interaction of SP-D with M.tb, and determine the effects of agglutination on bacterial uptake by human monocyte-derived macrophages. These studies demonstrate that the binding of SP-D and CDM to M.tb is saturable and inhibited by carbohydrate competition and Ca(2+) chelation, implicating the carbohydrate recognition domain in the interaction. Fluorescence microscopy reveals that dodecameric SP-D leads to agglutination of the bacilli, whereas the trimeric CDM does not, demonstrating that the multivalent nature of SP-D is essential for agglutination of M.tb. However, preincubation of M.tb with increasing concentrations of SP-D or CDM leads to a concentration-dependent reduction in the uptake of the bacteria by macrophages, indicating that agglutination does not play a direct role in this observation. Finally, the reduced uptake of M.tb by SP-D is associated with reduced growth of M.tb in monocyte-derived macrophages. These studies provide direct evidence that the inhibition of phagocytosis of M.tb effected by SP-D occurs independently of the aggregation process.

Dose and time response after intraamniotic endotoxin in preterm lambs.

Intraamniotic endotoxin causes chorioamnionitis, which is followed by improved fetal lung function after 4 d in fetal sheep. We evaluated 0.1 mg, 1 mg, 4 mg, and 10 mg endotoxin for inflammation and lung maturation effects after 7 d. Four and 10 mg endotoxin caused similar lung maturation and inflammation in the lung and chorioamnion. The number of neutrophils in cord blood and the inflammatory cells in alveolar lavage and fetal lung tissue increased in a dose-dependent manner. Lower endotoxin doses induced indicators of chorioamnionitis, lung and systemic inflammation without inducing lung maturation. Therefore, some degree of inflammation can occur without subsequent lung maturation. The inflammatory changes caused by 4 mg endotoxin were assessed after 5 h, 24 h, 72 h, and 7 d to discern local versus systemic inflammation after intraamniotic endotoxin. At 5 h active inflammatory cells were in the airways producing hydrogen peroxide, and interleukin-6 and -8 were increased in the cord blood indicating both lung and systemic responses. Cells recruited into the amniotic fluid produced proinflammatory cytokine mRNA for 7 d with no cytokine mRNA in chorioamnion, lung, or spleen after 72 h. The cells in the amniotic fluid may be a source of prolonged fetal exposure to proinflammatory cytokines.

SP-D and GM-CSF regulate surfactant homeostasis via distinct mechanisms.

Both surfactant protein (SP) D and granulocyte-macrophage colony-stimulating factor (GM-CSF) influence pulmonary surfactant homeostasis, with the deficiency of either protein causing marked accumulation of surfactant phospholipids in lung tissues and in the alveoli. To assess whether the effects of each gene were mediated by distinct or shared mechanisms, surfactant homeostasis and lung morphology were assessed in 1) double-transgenic mice in which both SP-D and GM-CSF genes were ablated [SP-D(-/-),GM(-/-)] and 2) transgenic mice deficient in both SP-D and GM-CSF in which the expression of GM-CSF was increased in the lung. Saturated phosphatidylcholine (Sat PC) pool sizes were markedly increased in SP-D(-/-),GM(-/-) mice, with the effects of each gene deletion on surfactant Sat PC pool sizes being approximately additive. Expression of GM-CSF in lungs of SP-D(-/-),GM(-/-) mice corrected GM-CSF-dependent abnormalities in surfactant catabolism but did not correct lung pathology characteristic of SP-D deletion. In contrast to findings in GM(-/-) mice, degradation of [(3)H]dipalmitoylphosphatidylcholine by alveolar macrophages from the SP-D(-/-) mice was normal. The emphysema and foamy macrophage infiltrates characteristic of SP-D(-/-) mice were similar in the presence or absence of GM-CSF. Taken together, these findings demonstrate the distinct roles of SP-D and GM-CSF in the regulation of surfactant homeostasis and lung structure.

Localization and functions of SP-A and SP-D at mucosal surfaces.

Pulmonary surfactant protein A (SP-A) and D (SP-D), members of the collectin family, are implicated in innate host defense of the lung. Collectins consist of a collagen-like domain and a carbohydrate recognition domain. SP-A and SP-D recognize and interact with glycoconjugates on the surface of microorganisms. They protect the lung by interacting with a wide variety of potential pathogens, including viruses, bacteria, and fungi. This may result in enhanced killing and/or clearance by phagocytes. Although most extensively studied in the lung, both SP-A and SP-D, or proteins closely resembling SP-A and SP-D, are found in a number of other sites in the body and therefore may play a protective role at other sites than the lung. SP-A and SP-D protein and/or mRNA have been detected at various sites of the body: the respiratory tract, the gastrointestinal tract, the middle ear, and in the peritoneal cavity. The presence of SP-A and SP-D at these mucosal surfaces, in close contact with numerous potentially harmful microorganisms, supports a role for these "lung"-collectins in innate mucosal defense. SP-A and SP-D may be important molecules in a threefold innate defense, particularly in the neonatal period between maternally acquired immunity and a fully developed adaptive immune system; the time interval between first exposure to a pathogen and generation of specific antibodies; and states of impaired immune function.

Decreased surfactant protein-B expression and surfactant dysfunction in a murine model of acute lung injury.

This study examines the relationships between inflammation, surfactant protein (SP) expression, surfactant function, and lung physiology in a murine model of acute lung injury (ALI). 129/J mice received aerosolized endotoxin lipopolysaccharide [LPS] daily for up to 96 h to simulate the cytokine release and acute inflammation of ALI. Lung elastance (E(L)) and resistance, lavage fluid cell counts, cytokine levels, phospholipid and protein content, and surfactant function were measured. Lavage and lung tissue SP content were determined by Western blot and immunohistochemistry, and tissue messenger RNA (mRNA) levels were assessed by Northern blot and in situ hybridization. Tumor necrosis factor-alpha and neutrophil counts in bronchoalveolar lavage fluid increased within 2 h of LPS exposure, followed by increases in total protein, interleukin (IL)-1beta, IL-6, and interferon-gamma. E(L) increased within 24 h of LPS exposure and remained abnormal up to 96 h. SP-B protein and mRNA levels were decreased at 24, 48, and 96 h. By contrast, SP-A protein and mRNA levels and SP-C mRNA levels were not reduced. Surfactant dysfunction occurred coincident with changes in SP-B levels. This study demonstrates that lung dysfunction in mice with LPS-ALI corresponds closely with abnormal surfactant function and reduced SP-B expression.