Binding and uptake of pulmonary surfactant protein (SP-A) by pulmonary type II epithelial cells

A glycoprotein of Mr 26-36,000 (SP-A) is an abundant phospholipid-associated protein in pulmonary surfactant. SP-A enhances phospholipid reuptake and inhibits secretion by Type II epithelial cells in vitro. We have used two electron microscopic cytochemical methods to demonstrate selective binding and uptake of SP-A by rat pulmonary Type II epithelial cells. Using an immunogold bridging technique, we showed that SP-A binding was selective for Type II cell surfaces. Binding was dose dependent and saturable, reaching maximal binding at approximately 10 ng/ml. On warming to 23 degrees C, SP-A binding sites were clustered in coated pits on the cell surface. To characterize the internalization and intracellular routing of SP-A, we used the biotinyl ligand-avidin-gold technique. Biotinyl SP-A was bound by rat Type II epithelial cells as described above. On warming, biotinyl SP-A was seen in association with coated vesicles and was subsequently located in endosomes and multivesicular bodies. Biotinyl SP-A-gold complexes were seen in close approximation to lamellar bodies 10-60 min after warming. Binding of biotinyl SP-A was inhibited by competition with unlabeled SP-A. These results support the concept that Type II epithelial cells bind and internalize SP-A by receptor-mediated endocytosis. This newly described uptake system may play a role in the recycling of surfactant components or mediate the actions of SP-A on surfactant phospholipid secretion.     

Internalization of pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells

We investigated the uptake of surfactant by isolated alveolar type II cells by using native pulmonary surfactant complexed with colloidal gold. Internalization to lamellar bodies (LB) occurred via vesicles (mainly coated) and the endosomal system. The highest labeling density was found in the endosomal system: vacuoles and the electron-lucent multivesicular bodies (MVB), which were labeled within 10 min. The labeling of electron-dense MVB (D-MVB) and LB was time dependent, reaching a plateau after 120 min, at which time approximately 30% and 70% of the LB and D-MVB were labeled, respectively. Internalization of surfactant-gold was inhibited by the addition of native surfactant or treatment of the gold complex with antibody against surfactant apoproteins. The internalization pathway of lectin from Macula pomifera (MPA) complexed with gold was compared to that of surfactant. Both pathways were found to be similar, except that mainly smooth vesicles rather than coated ones were involved in the process of MPA-G internalization. The partial labeling of the LB, the possible routing to lysosomes, and the endosomes as junction between the biosynthetic and endocytic pathways are discussed.     

Synthesis of surfactant components by cultured type II cells from human lung

We examined the effect of monolayer culture on surfactant phospholipids and proteins of type II cells isolated from human adult and fetal lung. Type II cells were prepared from cultured explants of fetal lung (16-24 weeks gestation) and from adult surgical specimens. Cells were maintained for up to 6 days on plastic tissue culture dishes. Although incorporation of [methyl-3H]choline into phosphatidylcholine (PC) by fetal cells was similar on day 1 and day 5 of culture, saturation of PC fell from 35 to 26%. In addition, there was decreased distribution of labeled acetate into PC, whereas distribution into other phospholipids increased or did not change. The decrease in saturation of newly synthesized PC was not altered by triiodothyronine (T3) and dexamethasone treatment or by culture as mixed type II cell/fibroblast monolayers. The content of surfactant protein SP-A (28-36 kDa) in fetal cells, as measured by ELISA and immunofluorescence microscopy, rose during the first day and then fell to undetectable levels by the fifth. Synthesis of SP-A, as measured by [35S]methionine labeling and immunoprecipitation, was detectable on day 1 but not thereafter. Levels of mRNAs for SP-A and for the two lipophilic surfactant proteins SP-B (18 kDa) and SP-C (5 kDa) fell with half-times of maximally 24 h. In contrast, total protein synthesis measured by [35S]methionine incorporation increased and then plateaued. In adult cells, the content of SP-A and its mRNA decreased during culture, with time-courses similar to those for fetal cells. We conclude that in monolayer culture on plastic culture dishes, human type II cells lose their ability to synthesize both phospholipids and proteins of surfactant. The control of type II cell differentiation under these conditions appears to be at a pretranslational level.     

GM130 regulates pulmonary surfactant protein secretion in alveolar type II cells

Science China Life Sciences - Pulmonary surfactant is a lipid-protein complex secreted by alveolar type II epithelial cells and is essential for the maintenance of the delicate structure of...     

Interactions of pulmonary surfactant protein SP-A with monolayers of dipalmitoylphosphatidylcholine and cholesterol: roles of SP-A domains

Pulmonary surfactant protein A (SP-A) is an oligomeric glycoprotein that binds dipalmitoylphosphatidylcholine (DPPC). Interactions of rat SP-A and recombinant SP-As with pure and binary monolayers of DPPC and cholesterol were studied using a rhomboid surface balance at 37 degrees C. A marked inflection at equilibrium surface tension (23 mN/m) in surface tension-area isotherm of a pure DPPC film was abolished by rat SP-A. The inflection was decreased and shifted to 18 mN/m with wild-type recombinant SP-A (SP-Ahyp). Both rat SP-A and SP-Ahyp decreased surface area reduction required for pure DPPC films to reach near zero surface tension from 30 to 25%. SP-Ahyp, E195Q,R197D, mutated in carbohydrate recognition domain (CRD) known to be essential for SP-A-vesicle interactions, conveyed a detrimental effect on DPPC surface activity. SP-ADeltaG8-P80, with deletion of collagen-like domain, had little effect. Both SP-Ahyp, C6S (Ser substitution for Cys6) and SP-Ahyp,DeltaN1-A7 (N-terminal segment deletion) which appear mainly as monomers on non-reducing SDS-PAGE analysis, increased required surface area reduction for minimal surface tension. All SP-As reduced collapse surface tension of a pure cholesterol film from 27 to 23 mN/m in the presence of Ca2+. When mixed films were formed by successive spreading of DPPC/SP-A/cholesterol, rat SP-A, SP-Ahyp, or SP-ADeltaG8-P80 blocked the interaction of cholesterol with DPPC; SP-Ahyp,E195Q,R197D could not impede the interaction; SP-Ahyp,C6S or SP-Ahyp,DeltaN1-A7 only partially blocked the interaction, and cholesterol appeared to stabilize SP-Ahyp,C6S-DPPC association. These results demonstrate the importance of CRD and N-terminal dependent oligomerization in SP-A-phospholipid associations. The findings further indicate that SP-A-cholesterol interactions differ from SP-A-DPPC interactions and may be nonspecific.     

Pulmonary surfactant secretion in briefly cultured mouse type II cells

There is little information on the regulation of surfactant secretion in mouse type II cells. We isolated type II cells from C57BL/6 and FVB mice, cultured them overnight, and then examined their response to known surfactant secretagogues. Secretion of phosphatidylcholine, surfactant protein (SP)-B and SP-C was stimulated by terbutaline, 5'-N-ethylcarboxyamidoadenosine (NECA), ATP, UTP, TPA, and ionomycin. Phosphatidylcholine secretion was increased approximately twofold by all agonists in both strains of mice. The response to terbutaline and NECA is the same as in rat type II cells, whereas the response to ATP, UTP, TPA, and ionomycin is considerably less. Secretion of SP-B and SP-C was increased sevenfold by terbutaline and threefold by ATP, effects similar to those in rat type II cells. The response to terbutaline was significantly decreased in type II cells from beta(2)-adrenergic receptor null mice. These data establish that briefly cultured type II cells provide a suitable model for investigation of surfactant secretion in normal and genetically altered mice.     

Roflumilast-N-oxide induces surfactant protein expression in human alveolar epithelial cells type II

Surfactant proteins (SPs) are important lipoprotein complex components, expressed in alveolar epithelial cells type II (AEC-II), and playing an essential role in maintenance of alveolar integrity and host defence. Because expressions of SPs are regulated by cyclic adenosine monophosphate (cAMP), we hypothesized that phosphodiesterase (PDE) inhibitors, influence SP expression and release. Analysis of PDE activity of our AEC-II preparations revealed that PDE4 is the major cAMP hydrolysing PDE in human adult AEC-II. Thus, freshly isolated human AEC-II were stimulated with two different concentrations of the PDE4 inhibitor roflumilast-N-oxide (3 nM and 1 μM) to investigate the effect on SP expression. SP mRNA levels disclosed a large inter-individual variation. Therefore, the experiments were grouped by the basal SP expression in low and high expressing donors. AEC-II stimulated with Roflumilast-N-oxide showed a minor increase in SP-A1, SP-C and SP-D mRNA mainly in low expressing preparations. To overcome the effects of different basal levels of intracellular cAMP, cyclooxygenase was blocked by indomethacin and cAMP production was reconstituted by prostaglandin E2 (PGE2). Under these conditions SP-A1, SP-A2, SP-B and SP-D are increased by roflumilast-N-oxide in low expressing preparations. Roflumilast-N-oxide fosters the expression of SPs in human AEC-II via increase of intracellular cAMP levels potentially contributing to improved alveolar host defence and enhanced resolution of inflammation.     

Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells

The purpose of this study is to clarify the involvement of protein kinase C in pulmonary surfactant secretion from adult rat alveolar type II cells in primary culture. Surfactant secretion in vitro is stimulated by at least two classes of compounds. One class, (e.g. terbutaline) increases intracellular cyclic AMP, whereas the other class (e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA] does not. TPA has been shown to activate protein kinase C in other cell systems. In our studies, 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is a direct activator of protein kinase C, stimulated [3H] phosphatidylcholine secretion by alveolar type II cells in a dose- and time-dependent manner. Tetracaine, which is an inhibitor of protein kinase C, inhibited the TPA-induced secretion of [3H]phosphatidylcholine from alveolar type II cells in a dose-dependent manner. However, tetracaine had no effect on terbutaline-induced secretion. The effects of terbutaline and OAG upon surfactant secretion were significantly more than additive, but those of TPA and OAG were less than additive. The specific activity of protein kinase C was 6-fold higher than cyclic AMP-dependent protein kinase found in type II cells when both kinases were assayed using lysine-rich histone as a common phosphate acceptor. Ninety-four per cent of protein kinase C activity was recovered in the cytosolic fraction of unstimulated type II cells, and 40% of activity in cytosolic fraction was translocated to particulate fraction upon treatment with TPA. As observed in other tissues, protein kinase C of alveolar type II cells was highly activated by 1,2-dioleoyl-sn-glycerol or TPA in the presence of Ca2+ and phosphatidylserine. These results suggest that pulmonary surfactant secretion in vitro is stimulated by both protein kinase C and cyclic AMP-dependent protein kinase.     

Incorporation of biotinylated SP-A into rat lung surfactant layer, type II cells, and clara cells

The goal of this study was to compare the functions of Clara and type II cells during alveolar clearance and recycling of surfactant protein (SP) A, a secretory product of both cell types. We examined the incorporation of instilled biotinylated SP-A (bSP-A) into rat lung type II and Clara cells as a measure of clearance and recycling of the protein. Ultrastructural localization of bSP-A was accomplished by an electron-microscopic immunogold technique at 7, 30, and 120 min after intratracheal instillation. Localization of bSP-A was quantitatively evaluated within extracellular surfactant components (lipid-rich forms: myelin figures, vesicles, and tubular myelin; and lipid-poor hypophase) and in compartments of type II and Clara cells. bSP-A was incorporated into myelinic and vesicular forms of extracellular surfactant, but tubular myelin and hypophase had little bSP-A. Lamellar bodies of type II cells demonstrated a significant time-dependent increase in their incorporation of bSP-A. There was a concentration of bSP-A in the secretory granules and mitochondria of Clara cells, but no Clara cell compartment showed a pattern of time-dependent change in immunolabeling. Our immunolabeling data demonstrated a time-dependent movement of exogenous SP-A from extracellular components into type II cells and their secretory granules. Clara cells did not demonstrate a time-dependent incorporation of bSP-A into their secretory granules during the period of this study. If Clara cells recycle SP-A, they must reach a steady state very quickly or very slowly.     

Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A²) and SP-A2 (1A⁰), and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A⁰) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A²) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.     

Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A(2)) and SP-A2 (1A(0)), and the coexpressed SP-A1/SP-A2 (6A(2)/1A(0)) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A(0)) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A(2)) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A(2)/1A(0)) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.     

Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films, II: albumin-inhibited pulmonary surfactant films and the effect of SP-A

Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.     

Pollen grains bind to lung alveolar type II cells (A549) via lung surfactant protein A (SP-A)

Lung surfactant protein A (SP-A) is the most abundant surfactant-associated protein present in the lung. A receptor for SP-A has been shown to be present on A549 alveolar type II cells and on other cell types, including alveolar macrophage. The SP-A receptor on A549 cells has been identified as the collectin receptor, or C1q receptor, which binds several structurally-related ligands. SP-A contains C-type lectin domains, but the role of carbohydrate binding by SP-A in physiological and pathological phenomena is not yet established. In this paper we report the binding of SP-A to pollen from Populus nigra italica (Lombardy Poplar), Poa pratensis (Kentucky blue grass),Secale cerale (cultivated rye) and Ambrosia elatior (short ragweed). Saturable and concentration dependent binding of SP-A to pollen grains was observed. Interaction of SP-A with pollen grains takes place through waterextractable components, in which the major species present, in Lombardy poplar pollen,are 57 kD and 7 kD (glyco)proteins. The binding of SP-A to pollen grains and their aqueous extracts was calcium ion dependent and was inhibited by mannose, and is therefore mediated by the lectin domain. Binding of SP-A to pollen grains was found to mediate adhesion of pollen grains to A549 cells. The results suggest that pollen grains or other carbohydrate-bearing particles (e. g. microorganisms) could potentially interact with different cell types via the collectin receptor (C1q Receptor) in the presence of SP-A.     

Impact of ozone exposure on the phagocytic activity of human surfactant protein A (SP-A) and SP-A variants

Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. SP-A1 and SP-A2 encode human (h) SP-A; SP-A2 products enhance phagocytosis more than SP-A1. Oxidation can affect SP-A function. We hypothesized that in vivo and in vitro ozone-induced oxidation of SP-A (as assessed by its carbonylation level) negatively affects its function in phagocytosis (as assessed by bacteria cell association). To test this, we used P. aeruginosa, rat alveolar macrophages (AMs), hSP-As with varying levels of in vivo (natural) oxidation, and ozone-exposed SP-A2 (1A, 1A0) and SP-A1 (6A2, 6A4) variants. SP-A oxidation levels (carbonylation) were measured; AMs were incubated with bacteria in the presence of SP-A, and the phagocytic index was calculated. We found: 1) the phagocytic activity of hSP-A is reduced with increasing levels of in vivo SP-A carbonylation; 2) in vitro ozone exposure of hSP-A decreases its function in a dose-dependent manner as well as its ability to enhance phagocytosis of either gram-negative or gram-positive bacteria; 3) the activity of both SP-A1 and SP-A2 decreases in response to in vitro ozone exposure of proteins with SP-A2 being affected more than SP-A1. We conclude that both in vivo and in vitro oxidative modifications of SP-A by carbonylation reduce its ability to enhance phagocytosis of bacteria and that the activity of SP-A2 is affected more by in vitro ozone-induced oxidation. We speculate that functional differences between SP-A1 and SP-A2 exist in vivo and that the redox status of the lung microenvironment differentially affects function of SP-A1 and SP-A2.     

c-fos基因在内皮素-1促肺泡Ⅱ型细胞表面活性物质合成中的作用

应用反义技术探讨ｃ－ｆｏｓ基因ＥＴ－１调控肺泡Ⅱ型细胞（ＡＴⅡ）表面活性物质（ＰＳ）合成的胞内信号转导中的作用,结果显示：（１）内皮素－１（ＥＴ－１）可提高ＡＴⅡ细胞的＾３Ｈ－胆碱掺入。（２）蛋白激酶Ｃ（ＰＫＣ）激活剂ＰＭＡ可使ＡＴⅡ细胞的＾３Ｈ－胆碱掺入量增加,ＰＫＣ抑制剂Ｈ７可抑制ＥＴ－１的促ＰＳ合成效应。（３）ＥＴ－１和ＰＭＡ可显著提高Ｆｏｓ蛋白表达量,Ｈ７和ｃ－ｆｏｓ反义寡核苷酸（ＯＤＮ）     

Effects of inhibiting protein synthesis on the secretion of surfactant by type II cells in primary culture

Pulmonary surfactant is isolated from the alveolar lumen as a complex of lipid and protein (King, R.J., Martin, H., Mitts, D. and Holmstrom, F.M. (1977) J. Appl. Physiol. 42, 483-491). We wished to determine whether the secretion of this complex was dependent upon cellular activities associated with the synthesis of protein, and whether the pre-formed lipids of surfactant would be released from the cell even though the synthesis of newly-formed protein was inhibited. Alveolar epithelial Type II cells were isolated from adult rat lung and grown to confluency in primary culture. The synthesis and secretion of the apolipoprotein of surfactant and its principal lipid, dipalmitoyl phosphatidyl-choline, were followed by isotopic precursor techniques. The synthesis of the apolipoprotein was reduced to 14% of control by 1 . 10(-4) M cycloheximide and to 2.5% of control by 4 . 10(-4) M cycloheximide. These concentrations of cycloheximide, however, had no effect on the rate of synthesis or release of DPPC. The secretion of the apolipoprotein which had been synthesized before the addition of 1 . 10(-4) M cycloheximide was not inhibited by this compound. Cells maintained at 5 degrees C neither synthesized nor released surfactant. We conclude, therefore, that the synthesis of cellular protein is not required for the secretion of surfactant, but that the continuous generation of metabolic energy may be essential. These results, together with those of previous kinetic studies (see above references), suggest that the lipid and protein constituents of surfactant may be contained within lamellar bodies prior to their release into the extracellular environment.     

Adsorption of pulmonary surfactant protein SP-A to monolayers of phospholipids containing hydrophobic surfactant protein SP-B or SP-C: potential differential role for tertiary interaction of lipids, hydrophobic proteins, and SP-A

Surface balance techniques were used to study the interactions of surfactant protein SP-A with monolayers of surfactant components preformed at the air-water interface. SP-A adsorption into the monolayers was followed by monitoring the increase in the surface pressure Deltapi after injection of SP-A beneath the films. Monolayers of dipalmitoylphosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (8:2, mol/mol) spread at initial surface pressure pi(i) = 5 mN/m did not promote the adsorption of SP-A at a subphase concentration of 0.68 microg/mL as compared to its adsorption to the monolayer-free surface. Surfactant proteins, SP-B or SP-C, when present in the films of DPPC:PG spread at pi(i) = 5 mN/m, enhanced the incorporation of SP-A in the monolayers to a similar extent; the Deltapi values being dependent on the levels of SP-B or SP-C, 3-17 wt %, in the lipid films. Calcium in the subphase did not affect the intrinsic surface activity of SP-A but reduced the Deltapi values produced by the adsorption of the protein to all the preformed films independently of their compositions and charges. The divalent ions likely modified the interaction of SP-A with the monolayers through their effects on the conformation, self-association, and charge state of SP-A. Values of Deltapi produced by adsorption of SP-A to the films of DPPC:PG with or without SP-B or SP-C were a function of the initial surface pressure of the films, pi(i). In the range of pressures 5 相似文献   

Lamellar bodies of cultured human fetal lung: content of surfactant protein A (SP-A), surface film formation and structural transformation in vitro

Lamellar bodies were isolated from dexamethasone and T3-treated explant cultures of human fetal lung, using sucrose density-gradient centrifugation. We examined their content of surfactant apoprotein A (SP-A), and their ability to form surface films and to undergo structural transformation in vitro. SP-A measured by ELISA composed less than 2% of total protein within lamellar bodies; this represented, as a minimum estimate, a 2-12-fold enrichment over homogenate. One- and two-dimensional gel electrophoresis also suggested that SP-A was a minor protein component of lamellar bodies. Adsorption of lamellar bodies to an air/water interface was moderately rapid, but accelerated dramatically upon addition of exogenous SP-A in ratios of 1:2-16 (SP-A:phospholipid, w/w). Similar adsorption patterns were seen for lamellar bodies from fresh adult rat and rabbit lung. Lamellar bodies incubated under conditions that promote formation of tubular myelin underwent structural rearrangement only in the presence of exogenous SP-A, with extensive formation of multilamellate whorls of lipid bilayers (but no classical tubular myelin lattices). We conclude that lamellar bodies are enriched in SP-A, but have insufficient content of SP-A for structural transformation to tubular myelin and rapid surface film formation in vitro.     

Surfactant protein A regulates pulmonary surfactant secretion via activation of phosphatidylinositol 3-kinase in type II alveolar cells

Pulmonary surfactant is secreted by the type II alveolar cells of the lung, and this secretion is induced by secretagogues of several types (e.g., ionomycin, phorbol esters, and terbutaline). Secretagogue-induced secretion is inhibited by surfactant-associated protein A (SP-A), which binds to a specific receptor (SPAR) on the surface of type II cells. The mechanism of SP-A-activated SPAR signaling is completely unknown. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 rescued surfactant secretion from inhibition by SP-A. In order to directly demonstrate a role for PI3K in SPAR signaling, PI3K activity was immunoprecipitated from type II cell extracts. PI3K activity increased rapidly after SP-A addition to type II cells. Since many receptors that activate PI3K do so through tyrosine-specific protein phosphorylation, antisera to phosphotyrosine, insulin-receptor substrate-1 (IRS-1), or SPAR were also examined. These antisera coimmunoprecipitated PI3K activity that was stimulated by SP-A. In addition, the tyrosine-specific protein kinase inhibitors genistein and herbimycin A blocked the action of SP-A on surfactant secretion. We conclude that SP-A signals to regulate surfactant secretion through SPAR, via pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of PI3K. This activation leads to inhibition of secretagogue-induced secretion of pulmonary surfactant.     

Binding of surfactant protein A (SP-A) to herpes simplex virus type 1-infected cells is mediated by the carbohydrate moiety of SP-A.

Pulmonary surfactant protein A (SP-A) has been shown to act as an opsonin in the phagocytosis of viruses by alveolar macrophages. To determine whether SP-A binds to viral proteins and which part of the SP-A molecule is involved in this interaction, binding studies were undertaken. SP-A was labeled with fluorescein isothiocyanate, and its binding to herpes simplex virus type 1-infected HEp-2 cells, as a model for virus-infected cells in general, was studied using flow cytometry. The binding of SP-A to virus-infected cells was saturable, reversible, and both time- and concentration-dependent, reaching a maximal level after 30 min at an SP-A concentration of 10 micrograms/ml. An approximately 4-fold increase in binding of SP-A to infected cells over control cells was observed. Yeast mannan, a mannose homopolysaccharide, did not influence the binding. However, heparin inhibited binding of SP-A in a concentration-dependent manner. In addition, heparin could also dissociate cell-bound SP-A, indicating that polyanionic oligosaccharides are involved in the binding of SP-A to virus-infected cells. Deglycosylated SP-A, obtained by digestion with N-glycosidase F, did not bind to infected cells. Heparin or deglycosylation of SP-A had no effect on the stimulation of alveolar macrophages by SP-A. It is concluded that the carbohydrate moiety of SP-A is involved in the recognition of viruses by SP-A and may play a role in the antiviral defenses of the lung.