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.

The intrauterine expression of surfactant protein D in the terminal airways of human fetuses compared with surfactant protein A.

UNLABELLED: We investigated the expression of surfactant protein (SP)-D in pulmonary epithelial cells, compared with the expression of SP-A. A total of 15 fetuses aborted at 8, 15, 19, 20, 21 and 23 weeks gestation and four premature babies who were stillborn or died after birth between May 1997 and October 2001 were included in this study. Fetuses showing any findings associated with preterm premature rupture of membranes or infection were excluded. We performed immunohistochemical examinations for SP-D and SP-A. SP-D was not detected by immunostaining at 8, 15 and 19 weeks gestation. At 21 weeks gestation, SP-D was weakly localized, in some cases (5/9), in the epithelial lining of both bronchioles and terminal airways. In contrast at 21 weeks gestation, SP-A was more markedly detected in the epithelial lining of both bronchioles and terminal airways in all cases but not detected in bronchioles and terminal airways at 8, 15 and 19 weeks gestation. CONCLUSION: the findings in this investigation suggests that the production of SP-D in fetal human lungs begins in the bronchiolar and terminal epithelium from about 21 weeks of gestation.

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.

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.

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.

Physiology of airway mucus clearance.

Respiratory tract secretions consist of mucus, surfactant, and periciliary fluid. The airway surface fluid is present as a bilayer, with a superficial gel or mucous layer and a layer of periciliary fluid interposed between the mucous layer and the epithelium. A thin layer of surfactant separates the mucous and periciliary fluid layers. The mucous layer extends from the intermediate airway to the upper airway and is approximately 2-10 microm thick in the trachea. Airway mucus is the secretory product of the goblet cells and the submucosal glands. It is a nonhomogeneous, adhesive, viscoelastic gel composed of water, carbohydrates, proteins, and lipids. In health, the mucous gel is primarily composed of a 3-dimensional tangled polymer network of mucous glycoproteins or mucin. Mucin macromolecules are 70-80% carbohydrate, 20% protein, and 1-2% sulfate bound to oligosaccharide side chains. The protein backbones of mucins are encoded by mucin genes (MUC genes), at least 8 of which are expressed in the respiratory tract, although MUC5AC and MUC5B are the 2 principal gel-forming mucins secreted in the airway. Mucus is transported from the lower respiratory tract into the pharynx by air flow and mucociliary clearance. Expectorated sputum is composed of lower respiratory tract secretions along with nasopharyngeal and oropharyngeal secretions, cellular debris, and microorganisms. Disruption of normal secretion or mucociliary clearance impairs pulmonary function and lung defense and increases risk of infection. When there is extensive ciliary damage and mucus hypersecretion, airflow-dependent mucus clearance such as cough becomes critically important for airway hygiene.

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.

[Effect of perftoran emulsion ventilation of lungs on the pulmonary surfactant system in patients with acute lung injury syndrome]. / Vplyv emul'siinoï ventyliatsiï lehen' "perftoranom" na surfaktantnu systemu lehen' u khvorykh iz syndromom hostroho lehenevoho poshkodzhennia.

The condition was studied of the pulmonary surfactant system in those patients with the syndrome of acute lung injury who had been placed on a partial emulsion ventilation of the lungs. The study of the surface tension of the endobronchial washings was made with the aid of the modified Willihelm balance before the start of the treatment and at post-treatment day 4, 6 and 8. The initial level of surface tension has been found out to be increased by 8.9%. The controlled indices were gradually returning to normal in the wake of the emulsion ventilation of the lungs. Partial emulsion ventilation of the lungs with perftoran makes for a prompt and complete restoration of surface activity of the pulmonary surfactant within 7 days. We consider it essential that a conventional intensive therapy of the acute lung injury syndrome be supplemented by partial emulsion ventilation of the lungs with perftoran.

[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.

Enhanced efficacy of porcine lung surfactant extract by utilization of its aqueous swelling dynamics.

This study investigates the interactions between a porcine lung surfactant (PLS) extract and distilled water, saline solution or Ringer solution. The phases which coexist in equilibrium with water or electrolyte solutions were analysed by X-ray diffraction and cryo transmission electron microscopy (cryo-TEM). A lamellar phase with a structure unit consisting of double bilayers was observed in water, whereas lamellar phases with the usual bilayer structure unit were formed in saline and in Ringer solutions. At 25 degrees C the presence of a 4.2-A peak in the X-ray diffraction wide-angle region of these three maximally swollen phases showed that most of the hydrocarbon chains were organized in a crystalline packing. At 42 degrees C the chains in all three phases were melted which, in combination with the low-angle diffraction, shows that they were liquid-crystalline. Polyhedral-like vesicles and spherically shaped multilamellar vesicles were observed in cryo-TEM. The bilayer unit structures were consistent with the periodicity seen by X-ray diffraction. The dynamic swelling behaviour was followed in the polarizing microscope. A remarkable growth of birefringent networks was seen at the air interface of samples swollen in Ringer solution and saline solution. No such interfacial growth phenomena were observed during swelling in water without electrolytes. Then, these dynamics were analysed in relation to time-dependent pulmonary administration of the surfactant extract in rats. Variation in the time of administration (20 and 60 min) after mixing the extract with saline or Ringer solution showed clear differences in physiological effects. At pulmonary administration when the swelling behaviour in vitro showed a maximum in dynamics, the arterial oxygenation was superior to that of administration at a time after a steady-state had been reached. This means that the clinical performance of mammalian lung surfactant extracts can be significantly improved by taking the time-dependent aqueous swelling of the extract into account.

Molecular interactions in pulmonary surfactant films.

Pulmonary surfactant is a lipid-protein complex which has the essential physiological role of stabilizing the respiratory surface of lungs against collapse. To achieve this function, surfactant forms films at the air-liquid interface that reduce dramatically the surface tension of the thin aqueous layer lining the alveoli. Natural surfactant has optimised two important properties. Once secreted to the alveolar spaces, surfactant adsorbs rapidly to the interface. There, surfactant films reduce surface tension close to 0 mN/m when compressed during expiration. The design and production of efficient artificial surfactants for respiratory therapeutics is critically dependent on the knowledge of the molecular mechanisms governing efficient interfacial adsorption and optimal surface activity, in the context of respiratory cycling. The present review summarizes the data available today on the behaviour of the different molecular components of pulmonary surfactant at air-liquid interfaces. A working model is proposed of how surfactant films could modulate the respiratory dynamics.

[Function of lung surfactant and its deterioration].

Lung surfactant(LS) is a mixture of several lipids and four apolipoproteins(SP-A, -B, -C and -D) and lowers surface tension at air-liquid interface of alveoli. Most of LS is synthesized and secreted by alveolar type II cells. Although lamellar bodies are storage granules of LS, each component appears to take independent intracellular routes to reside in the granules. Patients with infantile respiratory distress syndrome(IRDS) or acute respiratory distress syndrome(ARDS) develop fatal respiratory failure due to lack of LS. In addition, acute phase of interstitial pneumonia also shows deterioration of LS and increased alveolar surface force resulting in decreased lung compliance. SP-A and SP-D are used as serum marker to evaluated activity of interstitial lung diseases. Recently, growing evidences are accumulating that LS plays a role in innate host defense in the lung against large species of bacteria, mycoplasma, and viruses.

Vascular regulation of type II cell exocytosis.

To determine whether lung capillary pressure regulates surfactant secretion, we viewed alveoli of the constantly inflated, isolated blood-perfused rat lung by fluorescence microscopy. By alveolar micropuncture we infused fura 2 and lamellar body (LB)-localizing dyes for fluorescence detection of, respectively, the alveolar cytosolic Ca(2+) concentration ([Ca(2+)](i)) and type II cell exocytosis. Increasing left atrial pressure (Pla) from 5 to 10 cmH(2)O increased septal capillary diameter by 26% and induced marked alveolar [Ca(2+)](i) oscillations that abated on relief of pressure elevation. The rate of loss of LB fluorescence that reflects the LB exocytosis rate increased fourfold after the pressure elevation and continued at the same rate even after pressure and [Ca(2+)](i) oscillations had returned to baseline. In alveoli pretreated with either 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, the intracellular Ca(2+) chelator, or heptanol, the gap junctional blocker, the pressure-induced exocytosis was completely inhibited. We conclude that capillary pressure and surfactant secretion are mechanically coupled. The secretion initiates in a Ca(2+)-dependent manner but is sustained by Ca(2+)-independent mechanisms.

Surface-active phospholipid: a Pandora's box of clinical applications. Part I. The lung and air spaces.

Almost everywhere in the body there are phospholipids, not only comprising the lipid bilayer of membranes, but also in the free state. What is seldom appreciated, except in respirology, is that these 'free' phospholipids are unusual in that many are highly surface active. Surface activity is a property of certain substances (surfactants), conferred by their molecular constitution and configuration, which predisposes them to locate at interfaces because, in doing so, they reduce interfacial energy. When adsorbed (reversibly bound) to solid surfaces, surfactants can impart many highly desirable properties that have been widely studied and long accepted in the physical sciences, while their commercial applications have withstood the test of time. These desirable properties include lubricity (boundary lubrication), release (antistick) and dewatering, while providing a barrier to corrosion, abrasion, solute transmission and to biological microorganisms. Many of these offer obvious roles for surface-active phospholipid (SAPL), ranging from a corrosion inhibitor in the stomach to a load-bearing lubricant in the joints. This opens a veritable 'Pandora's box' of potential clinical applications. Part I of this review challenges traditional beliefs in respirology that 'surfactant' is unique to the lung and, moreover, that its actions are confined to the liquid-air interface. Evidence is discussed that, by binding to alveolar epithelium, SAPL imparts semi-permeability needed before channels pumping ions can also pump water vital for maintaining fluid balance. Evidence is also reviewed for a lining to upper airways, sinuses and Eustachian tube where it can act like a standard release agent.

Complementation of pulmonary abnormalities in SP-D(-/-) mice with an SP-D/conglutinin fusion protein.

Surfactant protein D (SP-D) and serum conglutinin are closely related members of the collectin family of host defense lectins. Although normally synthesized at different anatomic sites, both proteins participate in the innate immune response to microbial challenge. To discern the roles of specific domains in the function of SP-D in vivo, a fusion protein (SP-D/Cong(neck+CRD)) consisting of the NH(2)-terminal and collagenous domains of rat SP-D (rSP-D) and the neck and carbohydrate recognition domains (CRDs) of bovine conglutinin (Cong) was expressed in the respiratory epithelium of SP-D gene-targeted (SP-D(-/-)) mice. While SP-D/Cong(neck+CRD) fusion protein did not affect lung morphology and surfactant phospholipid levels in the lungs of wild type mice, the chimeric protein substantially corrected the increased lung phospholipids in SP-D(-/-) mice. The SP-D/Cong(neck+CRD) fusion protein also completely corrected defects in influenza A clearance and inhibited the exaggerated inflammatory response that occurs following viral infection. However, the chimeric protein did not ameliorate the ongoing lung inflammation, enhanced metalloproteinase expression, and alveolar destruction that characterize this model of SP-D deficiency. By contrast, a single arm mutant (RrSP-D(Ser15,20)) partially restored antiviral activity but otherwise failed to rescue the deficient phenotype. Our findings directly implicate the CRDs of both SP-D and conglutinin in host defense in vivo. Our findings also strongly suggest that the molecular mechanisms underlying impaired pulmonary host defense and abnormal lipid metabolism are distinct from those that promote ongoing inflammation and the development of emphysema.