Pulmonary Surfactant protein-A Research Articles

The Immune Change of the Lung and Bowel in an Ulcerative Colitis Rat Model and the Protective Effect of Sodium Houttuyfonate Combined With Matrine.

ObjectiveTo explore the immune change of lung injury of Ulcerative colitis (UC) by observing the changes of inherent immunity and adaptive immunity of the lung and bowel in UC rat models after the treatment of Sodium Houttuyfonate combined with Matrine. MethodUC rat models were established with the mucous membrane of colon allergize combined with TNBS-alcohol enteroclysis for 1 week and 5 weeks. 1-week experimental rats were divided into normal group and model group, 5/each group. 5-weeks experimental rats were divided into normal group, model group, Sodium Houttuyfonate (2.9mg/ml) combined with Matrine (1.47mg/ml), and positive control sulfasalazine (10mg/ml), 5/each group. All rats were administered by gavage for 5 weeks. The histopathological and fibrotic changes in the lung and bowel were observed, and the expressions of Tumor Necrosis Factor (TNF)- α, interleukin (IL)-8 in the lung, bowel, and serum were detected by radio-immunity and immunohistochemistry, and the mRNA expressions of Toll-like receptor (TLR)-4, nuclear factor kappa (NF-κB), Macrophage migration inhibitory factor (MIF), Mucosal addressing cell adhesion molecule-1 (MadCAM1) and Pulmonary surfactant protein-A (SP-A) in the lung and bowel were detected by Real time-PCR.ResultCompared with the normal group, the model rats had significant histopathological and fibrotic changes both in the lung and bowel, and all treatment groups were improved. After treatment, TLR4, IL-8, MIF, and TNF-α in the lung decreased (P<0.05); NF-KB, IL-8, and MIF in the bowel increased (P<0.05); MadCAM1 both in lung and bowel decreased (P<0.05); SP-A decreased in bowel and increased in the lung (P<0.05).ConclusionThe cause of lung injury in this model was found to be related to inherent immunity and adaptive immunity, while the cause of bowel injury in this model was found to be mainly related to adaptive immunity. Sodium Houttuyfonate combined with Matrine could improve bowel and lung injury.

An internal amino-terminal FLAG-tag octapeptide alters oligomerization of expressed surfactant protein-A

Pulmonary surfactant protein-A (SP-A) is expressed by lung alveolar and bronchiolar epithelial cells and plays a critical role in innate immunity of the lung. Exposure of the lung to various environmental insults alters SP-A homeostasis. To investigate the cellular mechanisms involved in these alterations, we added the FLAG octapeptide (DYKDDDDK) to the carboxy-terminus (SP-A/C-FLAG) or near the amino-terminus (SP-A/N-FLAG) of mouse SP-A (WT-SP-A) to tag specific pools of protein. We hypothesized that addition of FLAG would have negligible effects on SP-A expression, oligomerization and secretion. Analysis of Chinese hamster ovary cells expressing these proteins indicated that tagged SP-A mRNA could be distinguished from WT-SP-A by northern analysis and RT-PCR using sequence-specific oligonucleotides. Tagged SP-A protein could be differentiated from WT-SP-A by western analysis using antibodies specific for the FLAG epitope. Subcellular fractionation and immunocytochemistry indicated the majority of each protein was present in punctuate (presumably endocytic) vesicles, and all forms of SP-A protein were secreted. These results suggest that a FLAG epitope added to the carboxy-terminus or inserted into the amino-terminus of the mature SP-A protein has little effect on its expression and cellular processing. However, disruptions of the amino-terminal end of SP-A prevents proper oligomerization, suggesting that this region of mature SP-A is critical in proper oligomeric assembly and is not useful for studies intended to define mechanisms underlying SP-A homeostasis.

Modified Da-chai-hu Decoction regulates the expression of occludin and NF-κB to alleviate organ injury in severe acute pancreatitis rats

Modified Da-chai-hu Decoction (MDD), a traditional Chinese medicinal formulation, which was empirically generated from Da-chai-hu decoction, has been utilized to treat severe acute pancreatitis (SAP) for decades. The aim of the present study was to explore its potential organprotective mechanism in SAP. In the present study, rat SAP model was induced by retrograde injection of 3.5% sodium taurocholate into the biliopancreatic duct, MDD (23.35 g/kg body weight, twelve times the clinical dose) were orally given at 2 h before and 10 h after injection. At 12 h after model induction, blood was taken from vena cava for analysis of amylase, diamine oxidase (DAO), pulmonary surfactant protein-A (SP-A), and C-reactive protein (CRP). Histopathological change of pancreas, ileum and lung was assayed by H&E staining, myeloperoxidase (MPO) activity were determinated using colorimetric assay, and the expressions of occludin and nuclear factor-κB (NF-κB) were detected by real-time RT-PCR and western blot, respectively. In addition, the tissue concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that in SAP rats, MDD significantly alleviated histopathological damage, depressed the MPO activity and the concentrations of TNF-α, IL-1β, and MCP-1 of pancreas, ileum and lung, and reduced the serum levels of amylase [(3283.4 ± 585.5) U·L-1vs (5626.4 ± 795.1)U·L-1], DAO [(1100.1 ± 334.3) U·L-1vs (1666.4 ± 525.3) U·L-1] and CRP [(7.6 ± 1.2) μg·mL-1vs (17.8 ± 3.8) μg·mL-1]. However, the serum SP-A concentration [(106.1 ± 16.6) pg·mL-1vs (90.1 ± 14.9) pg·mL-1] was elevated when treated SAP rats with MDD. Furthermore, MDD increased the occludin expression and reduced the NF-κB expression in pancreas, ileum and lung of SAP rats. Our findings suggested that MDD administration was an effective therapeutic approach for SAP treatment. It could up-regulate occludin expression to protect intercellular tight junction and down-regulate NF-κB expression to inhibit inflammatory reaction of pancreas, ileum and lung.

The protective effect of different airway humidification liquids to lung after tracheotomy in traumatic brain injury: The role of pulmonary surfactant protein-A (SP-A)

The purpose of this study was to establish a rat model of a brain injury with tracheotomy and compared the wetting effects of different airway humidification liquids, afterward, the best airway humidification liquid was selected for the clinical trial, thus providing a theoretical basis for selecting a proper airway humidification liquid in a clinical setting. Rats were divided into a sham group, group A (0.9% NaCl), group B (0.45% NaCl), group C (0.9% NaCl+ambroxol) and group D (0.9% NaCl+Pulmicort). An established rat model of traumatic brain injury with tracheotomy was used. Brain tissue samples were taken to determine water content, while lung tissue samples were taken to determine wet/dry weight ratio (W/D), histological changes and expression levels of SP-A mRNA and SP-A protein. 30 patients with brain injury and tracheotomy were selected and divided into two groups based on the airway humidification liquid instilled in the trachea tube, 0.45% NaCl and 0.9% NaCl+ambroxol. Blood was then extracted from the patients to measure the levels of SP-A, interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α). The difference between group C and other groups in lung W/D and expression levels of SP-A mRNA and SP-A protein was significant (P<0.05). In comparison, the histological changes showed that the lung tissue damage was smallest in group C compared to the three other groups. Aspect of patients, 0.45% NaCl group and 0.9% NaCl+ambroxol group were significantly different in the levels of SP-A, IL-6, IL-8 and TNF-α (P<0.01). In the present study, 0.9% NaCl+ambroxol promote the synthesis and secretion of pulmonary surfactant, and has anti-inflammatory and antioxidant effects, which inhibit the release of inflammatory factors and cytokines, making it an ideal airway humidification liquid.

Oral administration of surfactant protein-a reduces pathology in an experimental model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) frequently results in significant morbidity and mortality in premature infants. Others reported that mice deficient in pulmonary surfactant protein-A (SP-A) born and raised in a nonhygienic environment succumb to significant gastrointestinal tract pathology, and enteral administration of purified SP-A significantly reduced mortality. We hypothesized that oral administration of purified SP-A can ameliorate pathology in an experimental model of neonatal NEC. Experimental NEC was induced in newborn Sprague-Dawley rat pups by daily formula gavage and intermittent exposure to hypoxia. Purified human SP-A (5 μg/day) was administered by oral gavage. After 4 days, surviving pups were sacrificed, and intestinal pathology was assessed by histological examination of distal terminal ileal sections. Intestinal levels of inflammatory cytokines (IL-1β, IFN-γ, and TNF-α) were assessed by enzyme-linked immunosorbent assay and levels of Toll-like receptor 4 (TLR4) by Western analysis. Sixty-one percent of the gavaged rat pups that survived to day 4 met the criteria for experimental NEC after hypoxia, whereas treatment with SP-A significantly reduced mortality and assessment of NEC. Intestinal levels of proinflammatory cytokines were significantly increased in pups exposed to hypoxia. Administration of SP-A to pups exposed to hypoxia significantly reduced IL-1β and TNF-α levels, but had little effect on elevated levels of IFN-γ. SP-A treatment of hypoxia-exposed pups significantly reduced expression of intestinal TLR4, key in NEC pathogenesis. In a rat model of experimental neonatal NEC, oral administration of SP-A reduces intestinal levels of proinflammatory cytokines and TLR4 protein and ameliorates adverse outcomes associated with gastrointestinal pathologies.

A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting.

Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA) is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies’ potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high affinity of the nanobody for the lung. Studying acute and chronic toxicity of Nb17 revealed its safety in rats without causing apparent histological alterations. Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.

Staphylococcus aureus Proteases Degrade Lung Surfactant Protein A Potentially Impairing Innate Immunity of the Lung

The pulmonary surfactant is a complex mixture of lipids and proteins that is important for respiratory lung functions, which also provides the first line of innate immune defense. Pulmonary surfactant protein-A (SP-A) is a major surfactant component with immune functions with importance during Staphylococcus aureus infections that has been demonstrated in numerous studies. The current study showed that S. aureus can efficiently cleave the SP-A protein using its arsenal of proteolytic enzymes. This degradation appears to be mediated by cysteine proteases, in particular staphopain A (ScpA). The staphopain-mediated proteolysis of SP-A resulted in a decrease or complete abolishment of SP-A biological activity, including the promotion of S. aureus phagocytosis by neutrophils, aggregation of Gram-negative bacteria and bacterial cell adherence to epithelium. Significantly, ScpA has also efficiently degraded SP-A in complete bronchi-alveolar lavage fluid from human lungs. This indicates that staphopain activity in the lungs is resistant to protease inhibitors, thus suggesting that SP-A can be cleaved in vivo. Collectively, this study showed that the S. aureus protease ScpA is an important virulence factor that may impair innate immunity of the lungs.

Pseudomonas aeruginosa elastase provides an escape from phagocytosis by degrading the pulmonary surfactant protein-A.

Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-A-/- mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitro and in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin.

Engineering Novel Complement Activity into a Pulmonary Surfactant Protein

Complement neutralizes invading pathogens, stimulates inflammatory and adaptive immune responses, and targets non- or altered-self structures for clearance. In the classical and lectin activation pathways, it is initiated when complexes composed of separate recognition and activation subcomponents bind to a pathogen surface. Despite its apparent complexity, recognition-mediated activation has evolved independently in three separate protein families, C1q, mannose-binding lectins (MBLs), and serum ficolins. Although unrelated, all have bouquet-like architectures and associate with complement-specific serine proteases: MBLs and ficolins with MBL-associated serine protease-2 (MASP-2) and C1q with C1r and C1s. To examine the structural requirements for complement activation, we have created a number of novel recombinant rat MBLs in which the position and orientation of the MASP-binding sites have been changed. We have also engineered MASP binding into a pulmonary surfactant protein (SP-A), which has the same domain structure and architecture as MBL but lacks any intrinsic complement activity. The data reveal that complement activity is remarkably tolerant to changes in the size and orientation of the collagenous stalks of MBL, implying considerable rotational and conformational flexibility in unbound MBL. Furthermore, novel complement activity is introduced concurrently with MASP binding in SP-A but is uncontrolled and occurs even in the absence of a carbohydrate target. Thus, the active rather than the zymogen state is default in lectin·MASP complexes and must be inhibited through additional regions in circulating MBLs until triggered by pathogen recognition.

Immunohistochemical Detection of Pulmonary Surfactant Proteins and Retroviral Antigens in the Lungs of Sheep with Pulmonary Adenomatosis

The lungs and mediastinal and bronchial lymph nodes from 26 sheep with ovine pulmonary adenomatosis (OPA) were examined. Microscopically, the tumour was disseminated throughout the lungs and displayed acinar or papillary growth. The neoplastic cells were cuboidal or columnar with clear cytoplasm and a low mitotic rate. Retrovirus antigen (Jaagsiekte Sheep Retrovirus Capsid Protein, JSRV CA) was demonstrated in the cytoplasm of tumour cells in the lung and lymph nodes by immunohistochemistry. The neoplastic cells had more diffuse and intense expression of pulmonary surfactant protein-A (SP-A) compared with the expression of SP-B or SP-C. SP-A and SP-B expression was localized to the apical cytoplasm of the neoplastic cells, whereas SP-C was most strongly expressed in the perinuclear area of the tumour cells. In the lungs of two sheep, low numbers of tumour cells expressed Clara cell secretory protein (CCSP). The nuclei of the neoplastic epithelial cells and of the germinal centre lymphocytes within the peribronchiolar lymphoid tissue expressed the proliferating cell nuclear antigen (PCNA). CD3(+) T lymphocytes infiltrated the pulmonary tissue and surrounded the neoplastic foci. The results of this study demonstrate that JSRV continues to replicate in neoplastic cells after they have been transformed, and that the neoplastic cells produce pulmonary surfactant proteins. A local T-cell response occurs within affected lungs.

The Mycobacterium tuberculosis Cell-surface Glycoprotein Apa as a Potential Adhesin to Colonize Target Cells via the Innate Immune System Pulmonary C-type Lectin Surfactant Protein A

Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional domains.

Differential allele expression of host defense genes, pulmonary surfactant protein-A and Osteopontin, in rat

Differential allele-specific expression has been observed in several genes involved in immunity. SP-A and OPN play a role in innate host defense. To determine whether SP-A and OPN are subject to differential allele-specific regulation, we investigated their gene or allele-specific expression in various tissues. The results showed: (1) Tissue-specific expression with high levels in lung ( SP-A) and kidney ( OPN). (2) Differences in allele-specific expression among individuals and tissues. SP-A showed an exclusively balanced biallelic expression (BB) in lung, but both BB and imbalanced biallelic (IB) expression in colon. Allele expression of OPN was more heterogeneous, e.g. in colon BB (22%), IB (64%), and monoallelic expression (MO) (14%). (3) Differential allele-specific expression was observed in all tissues studied ( OPN) or in all extrapulmonary tissues ( SP-A). (4) Family studies indicated that inheritable factor(s) may be involved in the regulation of allele-specific expression. (5) Analysis of co-expression of gene-specific alleles from double heterozygous rats revealed lack of coordinate allele expression among SP-A, SP-D, and OPN. We conclude that allele-specific expression occurs among genes of innate host defense. This may yet provide another level of regulatory complexity for molecules involved in the first line of defense.

Pulmonary Surfactant Protein-A (SP-A) Restores the Surface Properties of Surfactant after Oxidation by a Mechanism That Requires the Cys6 Interchain Disulfide Bond and the Phospholipid Binding Domain

Reactive oxygen species produced by activated leuko-cytes in the alveolar epithelial lining fluid have been implicated in the inactivation of pulmonary surfactant and the impairment of lung function. Oxidation of bovine lipid extract surfactant (BLES), a therapeutic surfactant, with hypochlorous acid (H-BLES) or the Fenton reaction (F-BLES) led to temporary increases in conjugated dienes and formation of malondialdehyde and 4-hydroxy-2-nonenal. Electrospray ionization mass spectrometry revealed the appearance of lipid hydroperoxides, peroxides, lysophospholipids, and free fatty acids. Captive bubble tensiometer studies of H-BLES demonstrated prolonged adsorption times, film instability at low surface tensions during film compression, and reduced respreadability during film expansion. F-BLES exhibited prolonged adsorption times, a marked effect on increasing compressibility during compression, and a lesser effect on reducing respreadability on expansion. Addition of native bovine or rat surfactant-associated protein A (SP-A) reversed the effects of oxidation on surfactant biophysical properties. Studies using mutant recombinant rat SP-As indicated that an intact carbohydrate recognition domain and disulfide-dependent oligomeric assembly are critical for these effects, but the collagen-like region is not required. We conclude that SP-A can reverse the detrimental effects of surfactant oxidation on the biophysical properties of surfactant, by a mechanism that is dependent on interchain disulfide bond formation and the C-terminal domains of the protein.

Role of CBP/p300 and SRC-1 in Transcriptional Regulation of the Pulmonary Surfactant Protein-A (SP-A) Gene by Thyroid Transcription Factor-1 (TTF-1)

Surfactant protein-A (SP-A) gene expression is developmentally regulated in fetal lung type II cells and is enhanced by cAMP. cAMP stimulation of SP-A gene expression is mediated by protein kinase A (PKA) phosphorylation of thyroid transcription factor 1 (TTF-1), expressed selectively in developing lung epithelium. In this study, we analyzed roles of CREB-binding protein (CBP) and steroid receptor coactivator-1 (SRC-1) in TTF-1 regulation of SP-A expression. Upon differentiation of human fetal lung in culture, nuclear localization of CBP, SRC-1, and TTF-1 increased in ductular epithelium in association with type II cell differentiation and induction of SP-A expression. In transient transfections, CBP and SRC-1 acted synergistically with TTF-1 to increase SP-A promoter activity. Overexpression of PKA catalytic subunit enhanced hSP-A promoter activation by SRC-1 plus TTF-1. Adenoviral E1A overexpression reduced TTF-1 +/- SRC-1 induction of SP-A promoter activity, suggesting a role of endogenous CBP/p300. TTF-1 interacted with SRC-1 and CBP in vitro. SRC-1 immunodepletion from type II cell nuclear extracts reduced binding to the TTF-1 binding element upstream of SP-A gene. In cultured type II cells, cAMP increased TTF-1 acetylation. This suggests that cAMP-mediated TTF-1 phosphorylation facilitates interaction with CBP and SRC-1, resulting in its hyperacetylation, further enhancing TTF-1 DNA-binding and transcriptional activity.

Normal surfactant pool sizes and inhibition-resistant surfactant from mice that overexpress surfactant protein A.

Pulmonary surfactant protein-A (SP-A) has been reported to regulate the uptake and secretion of surfactant by alveolar type II cells, to stabilize large surfactant aggregates including tubular myelin, and to protect the surface activity of surfactant from protein inhibitors. In this study we investigated the consequences of overexpression of SP-A on pulmonary homeostasis and surfactant function in transgenic mice. The human SP-C promoter was used to direct synthesis of rat surfactant protein A (rSP-A) in alveolar type II cells and nonciliated bronchiolar cells of the distal respiratory epithelium. Levels of SP-A measured through enzyme-linked immunosorbent assay were 7- to 8-fold higher in lung homogenates and alveolar lavage fluid of the rSP-A mice than in those of transgene-negative littermates. The swimming exercise tolerance and lung compliance of mice bearing the transgene were unchanged. Mean air space sizes seen in randomly selected light-microscopic fields were not significantly different in the transgene-positive and -negative mice by morphometric analysis, but 15% of transgenic animals had scattered foci containing dilated alveoli and alveolar ducts without evidence of inflammation or fibrosis. Some alveolar macrophages contained bar-shaped osmophilic inclusions that had a highly ordered ultrastructure. There were no differences between the transgene-positive and -negative mice in the tissue or alveolar pool sizes of saturated phosphatidylcholine or in the large-aggregate composition of alveolar surfactant. The surface activity of surfactant isolated from the rSP-A mice was similar to that of the controls, but in the presence of protein inhibitors, the surface tension-reducing properties of the rSP-A surfactant were better preserved (P < 0.05). We conclude that overexpression of SP-A does not affect resting surfactant phospholipid levels, but that it enhances the resistance of surfactant to protein inhibition.

Pulmonary Surfactant Protein A (SP-A) Is Expressed by Epithelial Cells of Small and Large Intestine

Surfactant protein A (SP-A) is the most abundant protein associated with phospholipids in pulmonary surfactant. There are several lines of evidence that pulmonary and gastrointestinal epithelium produce closely related surface-active materials, although the presence of SP-A in gastrointestinal tract has so far not been reported. Indirect immunofluorescence experiments using different antibodies raised against rat pulmonary SP-A showed that some jejunal and colonic but not gastric epithelial cells positively stained for SP-A. Analysis of the proteins in cell lysates from rat small intestine and colon studied by Western blot revealed several immuno-reactive bands, including the characteristic triplet of 26-, 32-, and 38-kDa monomeric proteins, less strongly labeled than in lung cells, and higher molecular mass forms of 66 and 120 kDa also present in lung cells. The 66- and 120-kDa bands displayed the expected isoelectric pH of SP-A after two-dimensional electrophoresis. Alkylation induced conversion of the 120-kDa form (almost completely) and the 66-kDa form (partly) into the 26-38-kDa monomeric species. The presence of SP-A mRNA in rat stomach, small intestine, and colon was then searched for by conventional cDNA/reverse transcriptase-polymerase chain reaction. Products of appropriate size (372 base pairs) identical to that of pulmonary tissue were amplified in small intestine and colon but not in stomach or in other tissues used as controls. Cloning and sequencing of rat colon SP-A cDNA revealed the same sequence as the one reported for rat lung SP-A. Furthermore, analysis of the transcriptional initiation site of SP-A gene in colon by anchored-polymerase chain reaction showed that transcription was initiated at the same site in both colon and lung. These data, which demonstrate that small intestine and colon express SP-A constitutively and that this protein is present in some epithelial cells, extend the concept of intestinal surfactant and underline its close relationships to pulmonary surfactant.

Pulmonary surfactant protein-A (SP-A) modulates human PBMC proliferation and cytokine production in vitro

Pulmonary surfactant protein-A (SP-A) modulates human PBMC proliferation and cytokine production in vitro

Biphasic glucocorticoid regulation of pulmonary SP-A: characterization of inhibitory process.

Pulmonary surfactant, which is necessary for normal lung function, is under both developmental and hormonal regulation. Glucocorticoids induce all components of surfactant and have a unique biphasic effect on surfactant protein A (SP-A), either stimulating or inhibiting accumulation in cultured fetal lung depending on dose and time of exposure. In this study we further characterized glucocorticoid inhibition of SP-A in cultured explants of human fetal lung. Decreased content of SP-A mRNA was the dominant response to dexamethasone added either early or later during culture. Inhibition occurred at < or = 1 nM dexamethasone on prolonged exposure, was blocked by RU 486, and was observed with other glucocorticoids but not sex steroids. When cortisol was removed from the culture medium, inhibition was rapidly reversed. The immediate inhibitory effect of 100 nM dexamethasone on SP-A mRNA content was completely blocked in the presence of cycloheximide. SP-A gene transcription, measured by nuclear elongation assay, was decreased by 60% after 4- to 8-h exposure to 100 nM dexamethasone. Stability of SP-A mRNA, determined both by addition of actinomycin D and by label-chase experiments, was transiently decreased immediately after adding dexamethasone (t1/2 approximately 3 h). In tissue treated with dexamethasone for > or = 8 h the stability of SP-A mRNA in control and treated explants was not different (t1/2 approximately 8 h). Our findings indicate that inhibition of SP-A is the dominant response to glucocorticoid. This effect is receptor mediated and apparently involves induction of a labile protein(s) that decreases gene transcription and transiently reduces mRNA stability.