Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation

We reported an association between the ability of recombinant human keratinocyte growth factor (rHuKGF) to upregulate the expression of surfactant protein A (SP-A) and to downregulate pulmonary inflammation that occurs after allogeneic bone marrow transplantation (BMT). To establish a causal relationship, rHuKGF (5 mg/kg) was administered subcutaneously for three consecutive days before irradiation to SP-A-sufficient and -deficient [SP-A(+/+) and SP-A(-/-), respectively] mice given inflammation-inducing allogeneic spleen T cells at the time of BMT. In contrast with SP-A(+/+) mice, rHuKGF failed to suppress the high levels of TNF-alpha, IFN-gamma, and nitric oxide contained in bronchoalveolar lavage fluids collected on day 7 after BMT from SP-A(-/-) mice. Early post-BMT weight loss was attenuated by rHuKGF in both SP-A(+/+) and SP-A(-/-) recipients. In the absence of supportive respiratory care, however, SP-A deficiency eventually abolished the ability of rHuKGF to prevent weight loss and to improve survival monitored for 1 mo after allogeneic BMT. In further experiments, the addition of cyclophosphamide (which is known to cause severe injury to the alveolar epithelium in donor T cell-recipient mice) to the conditioning regimen prevented rHuKGF-induced upregulation of SP-A and suppression of lung inflammation in both SP-A(+/+) and SP-A(-/-) mice. We conclude that endogenous baseline SP-A levels and optimal upregulation of SP-A are required for the anti-inflammatory protective effects of KGF after allogeneic transplantation.     

Surfactant protein A enhances alveolar macrophage phagocytosis of apoptotic neutrophils

Surfactant protein A (SP-A) is an innate immune molecule that binds foreign organisms that invade the lungs and targets them for phagocytic clearance by the resident pulmonary phagocyte, the alveolar macrophage (AM). We hypothesized that SP-A binds to and enhances macrophage uptake of other nonself particles, specifically apoptotic polymorphonuclear neutrophils (PMNs). PMNs are recruited into the lungs during inflammation, but as inflammation is resolved, PMNs undergo apoptosis and are phagocytosed by AMs. We determined that SP-A increases AM phagocytosis of apoptotic PMNs 280 +/- 62% above the no protein control value. The increase is dose dependent, and heat-treated SP-A still enhanced uptake, whereas deglycosylated SP-A had significantly diminished ability to enhance phagocytosis. Surfactant protein D also increased phagocytosis of apoptotic PMNs by approximately 125%. However, other proteins that are structurally homologous to SP-A, mannose-binding lectin and complement protein 1q, did not. SP-A enhances phagocytosis via an opsonization-dependent mechanism and binds apoptotic PMNs approximately 4-fold more than viable PMNs. Also, binding of SP-A to apoptotic PMNs does not appear to involve SP-A's lectin domain. These data suggest that the pulmonary collectins SP-A and SP-D facilitate the resolution of inflammation by accelerating apoptotic PMN clearance.     

Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages

Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP-A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a (51)Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 +/- 2.5, whereas the addition of rat SP-A (5 microg/ml) significantly reduced the cytotoxic index to 16.6 +/- 1.2 (P < 0. 001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody or D-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica.     

Surfactant protein D reduces alveolar macrophage apoptosis in vivo

Surfactant protein D (SP-D) is a molecule of the innate immune system that recognizes the patterns of surface carbohydrate on pathogens and targets them for phagocytosis and killing. SP-D-deficient mice show an increased number of macrophages in the alveolar space, excess surfactant phospholipid, overproduction of reactive oxygen species, and the development of emphysema. We report here that SP-D-deficient mice have a 5- to 10-fold increase in the number of apoptotic and necrotic alveolar macrophages, as defined by annexin V and propidium iodine staining, respectively. Intrapulmonary administration of a truncated 60-kDa fragment of human recombinant SP-D reduces the number of apoptotic and necrotic alveolar macrophages and partially corrects the lipid accumulation in SP-D-deficient mice. The same SP-D fragment binds preferentially to apoptotic and necrotic alveolar macrophages in vitro, suggesting that SP-D contributes to immune homeostasis in the lung by recognizing and promoting removal of necrotic and apoptotic cells.     

Surfactant protein D is a divalent cation-dependent carbohydrate-binding protein

Surfactant protein D (SP-D, CP4) is a collagenous surfactant-associated glycoprotein synthesized by lung type II epithelial cells. SP-D can be selectively and efficiently eluted from isolated rat surfactant with glucose, maltose, and certain other saccharides. We therefore examined the ability of the purified protein to interact with carbohydrates in vitro. Saccharide-substituted bovine serum albumins (BSA neoglycoproteins) were adsorbed to plastic wells, and binding of purified SP-D was quantified with monospecific antibodies to SP-D using an indirect immunoassay. SP-D showed specific calcium-dependent binding to alpha-D-glucosidophenyl isothiocyanate-BSA and maltosyl-BSA, but negligible binding to beta-D-glucosidophenyl isothiocyanate-BSA or unconjugated BSA. The most efficient inhibitors of SP-D binding were alpha-glucosyl-containing saccharides (e.g. isomaltose, maltose, malotriose). SP-D showed quantitative binding to maltosyl-agarose and was specifically eluted with maltose or EDTA. High affinity binding to maltosyl-BSA was also demonstrated using a solution-phase polyethylene glycol precipitation assay. These studies demonstrate that SP-D is a calcium dependent lectin-like protein and that the association of SP-D with surfactant is mediated by carbohydrate-dependent interactions with specificity for alpha-glucosyl residues.     

Surfactant protein A is defective in abrogating inflammation in asthma

Surfactant protein A (SP-A) regulates a variety of immune cell functions. We determined the ability of SP-A derived from normal and asthmatic subjects to modulate the inflammatory response elicited by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. Fourteen asthmatic and 10 normal control subjects underwent bronchoscopy with airway brushing and bronchoalveolar lavage (BAL). Total SP-A was extracted from BAL. The ratio of SP-A1 to total SP-A (SP-A1/SP-A) and the binding of total SP-A to M. pneumoniae membranes were determined. Airway epithelial cells from subjects were exposed to either normal or asthmatic SP-A before exposure to M. pneumoniae. IL-8 protein and MUC5AC mRNA were measured. Total BAL SP-A concentration did not differ between groups, but the percentage SP-A1 was significantly increased in BAL of asthmatic compared with normal subjects. SP-A1/SP-A significantly correlated with maximum binding of total SP-A to M. pneumoniae, but only in asthma. SP-A derived from asthmatic subjects did not significantly attenuate IL-8 and MUC5AC in the setting of M. pneumoniae infection compared with SP-A derived from normal subjects. We conclude that SP-A derived from asthmatic subjects does not abrogate inflammation effectively, and this dysfunction may be modulated by SP-A1/SP-A.     

Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages

Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles.     

Surfactant protein A inhibits alveolar macrophage cytokine production by CD14-independent pathway

The lung collectin surfactant protein A (SP-A) has both anti-inflammatory and prophagocytic activities. We and others previously showed that SP-A inhibits the macrophage production of tumor necrosis factor (TNF)-alpha stimulated by the gram-negative bacterial component LPS. We propose that SP-A decreases the production of proinflammatory cytokines by alveolar macrophages via a CD14-independent mechanism. SP-A inhibited LPS-simulated TNF-alpha production in rat and mouse macrophages in the presence and absence of serum (72% and 42% inhibition, respectively). In addition, SP-A inhibited LPS-induced mRNA levels for TNF-alpha, IL-1 alpha, and IL-1 beta as well as NF-kappa B DNA binding activity. SP-A also diminished ultrapure LPS-stimulated TNF-alpha produced by wild-type and CD14-null mouse alveolar macrophages by 58% and 88%, respectively. Additionally, SP-A inhibited TNF-alpha stimulated by PMA in both wild-type and TLR4-mutant macrophages. These data suggest that SP-A inhibits inflammatory cytokine production in a CD14-independent manner and also by mechanisms independent of the LPS signaling pathway.     

Surfactant protein D is proatherogenic in mice

Surfactant protein D (SP-D) is an important innate immune defense molecule that mediates clearance of pathogens and modulates the inflammatory response. Moreover, SP-D is involved in lipid homeostasis, and pulmonary accumulation of phospholipids has previously been observed in SP-D-deficient (Spd-/-) mice. Atherogenesis involves both inflammation and lipid deposition, and we investigated the role of SP-D in the development of atherosclerosis. SP-D synthesis was localized to vascular endothelial cells. Atherosclerotic lesion areas were 5.6-fold smaller in the aortic roots in Spd-/- mice compared with wild-type C57BL/6N mice on an atherogenic diet. HDL cholesterol (HDL-C) was significantly elevated in Spd-/- mice. Treatment of Spd-/- mice with a recombinant fragment of human SP-D resulted in decreases of HDL-C (21%) as well as total cholesterol (26%), and LDL cholesterol (28%). Plasma TNF-alpha was reduced in Spd-/- mice (45% difference). SP-D was proatherogenic in the mouse model used. The effect is likely to be due to the observed disturbances of plasma lipid metabolism and alteration of the inflammatory process, which underlie the reduced susceptibility to atherosclerosis in Spd-/- mice.     

Surfactant protein A enhances apoptotic cell uptake and TGF-beta1 release by inflammatory alveolar macrophages

The phagocytosis of apoptotic inflammatory cells by alveolar macrophages (AMs) is a key component of inflammation resolution within the air space. Surfactant protein A (SP-A) has been shown to stimulate the phagocytosis of apoptotic neutrophils (PMNs) by normal AMs. We hypothesized that SP-A promotes the resolution of alveolar inflammation by enhancing apoptotic PMN phagocytosis and anti-inflammatory cytokine release by inflammatory AMs. Using an LPS lung inflammation model, we determined that SP-A stimulates the phagocytosis of apoptotic PMNs threefold by normal AMs and AMs isolated after LPS injury. Furthermore, SP-A enhances transforming growth factor-beta1 (TGF-beta1) release from both AM populations. Inflammatory AMs release twofold more TGF-beta1 in culture than do normal AMs. SP-A and apoptotic PMNs together stimulate TGF-beta1 release equivalently from normal and inflammatory cultured AMs (330% of unstimulated release by normal AMs). In summary, SP-A enhances apoptotic PMN uptake, stimulates AM TGF-beta1 release, and modulates the amount of TGF-beta1 released when AMs phagocytose apoptotic PMNs. These findings support the hypothesis that SP-A promotes the resolution of alveolar inflammation.     

Platelet activating factor (PAF-acether) is released into rat pulmonary alveolar fluid as a consequence of hypoxia

Hypoxia provokes pulmonary constriction and because PAF-acether is a very strong pulmonary constrictor, we looked for PAF-acether in lung alveolar lavage (LAL) with a biological method based on the measurement of rabbit platelet aggregation. We first demonstrated a PAF-acether secretion during bronchoalveolar lavage with sterile isotonic NaCl (pH 7.2). PAF-acether secretion was completely suppressed with isotonic NaCl containing 5 mM EDTA but lyso-PAF-acether was still present (1.9 +/- 0.55 nmoles). Upon hypobaric hypoxia, PAF-acether was detected in LAL (1.05 +/- 0.25 10(-2)nmoles). The amount of lyso-PAF-acether increased by 6 times (12.1 +/- 4.1 nmoles). These results are given for 10(4) nmoles phospholipids of LAL. They indicate that alveolar macrophages might be activated by hypobaric hypoxia, so they produce PAF-acether in the alveole. Such a process could be involved in the well-known bronchoconstriction accompanying hypoxia.     

Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells

Surfactant protein D (SP-D) is a member of the collectin family of the innate host defense proteins. In the lung, SP-D is expressed primarily by type II cells. Gene-targeted SP-D-deficient [SP-D(-/-)] mice have three- to fivefold higher surfactant lipid pool sizes. However, surfactant synthesis and secretion by type II cells and catabolism by alveolar macrophages are normal in SP-D(-/-) mice. Therefore, we hypothesized that SP-D might regulate surfactant homeostasis by influencing surfactant structure, thereby altering its uptake by type II cells. Large (LA) and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) from SP-D(-/-), wild-type [SP-D(+/+)], and transgenic mice in which SP-D was expressed under conditional control of doxycycline in alveolar type II cells. Uptake of both LA and SA isolated from SP-D(-/-) mice by normal type II cells was decreased. Abnormally dense lipid forms were observed by electron microscopy of LA from SP-D(-/-) mice. SA from SP-D(-/-) mice consisted of atypical multilamellated small vesicles. Abnormalities in surfactant uptake by type II cells and in surfactant ultrastructure were corrected by conditional expression of SP-D in vivo. Preincubation of BALF from SP-D(-/-) mice with SP-D changed surfactant ultrastructure to be similar to that of SP-D(+/+) mice in vitro. The rapid changes in surfactant structure, increased uptake by type II cells, and decreased pool sizes normally occurring in the postnatal period were not seen in SP-D(-/-) mice. SP-D regulates uptake and catabolism by type II cells and influences the ultrastructure of surfactant in the alveolus.     

Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.     

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.     

Surfactant replacement attenuates the increase in alveolar permeability in hyperoxia

Rabbits exposed to hyperoxia develop surfactant deficiency, abnormal lung mechanics, and increased permeability to solute. We investigated whether replenishment of depleted alveolar surfactant by the intratracheal instillation of calf lung surfactant extract (CLSE) would mitigate the increase in alveolar permeability to solute. Twenty-eight rabbits were exposed to 100% O2 for 72 h and received intratracheal instillations of 125 mg CLSE (approximately 170 mumol dipalmitoyl phosphatidylcholine) at 24 and 48 h. The interlobar and intralobar distribution of CLSE was quantified by adding [14C]dipalmitoyl phosphatidylcholine liposes into the instillate and measuring the levels of activity in lung tissue. CLSE was nonuniformly distributed in the different lung lobes, the right lower lobe receiving more CLSE than the rest. Alveolar epithelial permeability to solute was assessed by instilling 10 ml isotonic saline, which contained a trace amount of [57Co]cyanocobalamin, in the right lower lobe and measuring the disappearance of the tracer from the alveolar saline and its appearance in the arterial blood during a 1-h period. CLSE treatment was associated with significantly increased 72-h survival in hyperoxia compared with saline-treated controls (number of survivors: 16/17 vs. 5/11, P less than 0.01). CLSE treatment significantly reduced the rate constant for the movement of cyanocobalamin out of the alveolar space (24 +/- 5 vs. 42 +/- 6 min-1 x 10(-3), P less than 0.01) and tracer appearance in the blood at the end of the study (7 +/- 1 vs. 34 +/- 13%, P less than 0.01) when compared with values in saline controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surfactant protein D regulates NF-kappa B and matrix metalloproteinase production in alveolar macrophages via oxidant-sensitive pathways

Targeted ablation of the surfactant protein D (SP-D) gene caused progressive pulmonary emphysema associated with pulmonary infiltration by foamy alveolar macrophages (AMs), increased hydrogen peroxide production, and matrix metalloproteinase (MMP)-2, -9, and -12 expression. In the present study, the mechanisms by which SP-D influences macrophage MMP activity were assessed in AMs from SP-D(-/-) mice. Tissue lipid peroxides and reactive carbonyls were increased in lungs of SP-D(-/-) mice, indicating oxidative stress. Immunohistochemical staining of AMs from SP-D(-/-) mice demonstrated that NF-kappaB was highly expressed and translocated to the nucleus. Increased NF-kappaB binding was detected by EMSA in nuclear extracts of AMs isolated from SP-D(-/-) mice. Antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited MMP production by AMs from SP-D(-/-) mice. To assess whether increased oxidant production influenced NF-kappaB activation and production of MMP-2 and -9, AMs from SP-D(-/-) mice were treated with the NADPH oxidase inhibitors diphenylene iodonium chloride and apocynin. Inhibition of NADPH oxidase suppressed NF-kappaB binding by nuclear extracts and decreased production of MMP-2 and 9 in AMs from SP-D(-/-) mice. SN-50, a synthetic NF-kappaB-inhibitory peptide, decreased MMP production by AMs from SP-D(-/-) mice. Oxidant production and reactive oxygen species were increased in lungs of SP-D(-/-) mice, in turn activating NF-kappaB and MMP expression. SP-D plays an unexpected inhibitory role in the regulation of NF-kappaB in AMs.     

A human polyadenylation factor is a G protein beta-subunit homologue.

Cleavage stimulation factor (CstF) is one of the multiple factors required for polyadenylation of mammalian pre-mRNAs in vitro. We have shown previously that this factor is composed of three distinct subunits of 77, 64, and 50 kDa, and that the 64-kDa subunit can be UV-cross-linked to RNA in a polyadenylation signal (AAUAAA)-dependent manner. By molecular cloning, the 64-kDa subunit was shown to contain a ribonucleoprotein-type RNA binding domain and a novel repeat structure. To study the functions of the other subunits, we have now isolated cDNAs encoding the 50-kDa subunit of human CstF. This subunit shares extensive homology with mammalian G protein beta-subunits and has a characteristic repeat structure (transducin repeat), in which an approximately 44-amino acid-long sequence is repeated seven times. To our knowledge, the 50-kDa subunit is the first example of a functional beta-subunit-like protein in vertebrates. Possible roles of the transducin repeat, both in CstF function specifically and in other beta-subunit homologues more generally, are discussed.