Inhibition of ozone-induced SP-A oxidation by plant polyphenols

Surfactant protein-A (SP-A) is the best studied and most abundant of the protein components of lung surfactant and plays an important role in host defense of the lung. It has been shown that ozone-induced oxidation of SP-A protein changes its functional and biochemical properties. In the present study, eight plant polyphenols (three flavonoids, three hydroxycinnamic acids, and two hydroxybenzoic acids) known as strong antioxidants, were tested for their ability to inhibit ozone-induced SP-A oxidation as a mechanism for chemoprevention against lung damage. SP-A isolated from alveolar proteinosis patients was exposed to ozone (1 ppm) for 4 h. The flavonoids protected SP-A from oxidation in a dose dependent manner. ( - )-Epicatechin was the most potent flavonoid and exhibited inhibition of ozone-induced formation of carbonyls by 35% at a concentration as low as 5 μM. Hydroxybenzoic acids inhibited SP-A oxidation in a dose-dependent manner although they were less potent than flavonoids. On the other hand, hydroxycinnamic acids exhibited a different inhibitory pattern. Inhibition was observed only at medium concentrations. The results indicate that inhibition of SP-A oxidation by plant polyphenols may be a mechanism accounting for the protective activity of natural antioxidants against the effects of ozone exposure on lungs.     

Differences in biochemical properties and in biological function between human SP-A1 and SP-A2 variants, and the impact of ozone-induced oxidation

The human surfactant protein A (SP-A) locus consists of two functional genes, SP-A1 and SP-A2, with several alleles characterized for each gene. Functional variations between SP-A1 and SP-A2 variants either before or after ozone exposure have been observed. To understand the basis of these differences, we studied SP-A1 and SP-A2 variants by comparing coding sequences, oligomerization patterns under various conditions, composition of oligomers with regard to amino terminal sequence isoforms, biological activity (regulation of phosphatidylcholine (PC) secretion by alveolar type II cells), and the impact of ozone-induced oxidation. We found that (i) the SP-A1 (6A(4)) allele is the most divergent from all SP-A2 alleles, particularly from the SP-A2 (1A(1)). (ii) Differences exist in oligomerization among SP-A1, SP-A2, and coexpressed SP-A1/SP-A2, with higher order multimers (i.e., consisting of more subunits) observed for SP-A1 than for SP-A2 variants. Differences among SP-A1 or SP-A2 gene products are minimal. (iii) Amino acid variants in the amino terminal sequences are observed after signal peptide removal, including variants with an extra cysteine. (iv) Oxidation is observed after ozone exposure, involving several SP-A residues that include cysteine, methionine, and tryptophan. (v) The SP-A2 variant (1A(0)) and the coexpressed protein 1A(0)/6A(2) inhibit ATP-stimulated PC secretion from alveolar type II cells to a greater extent than SP-A1 (6A(2)), a biologic activity that was susceptible to ozone treatment.     

Human SP-A protein variants derived from one or both genes stimulate TNF-alpha production in the THP-1 cell line

In humans, two functional genes of surfactant protein (SP) A, SP-A1 and SP-A2, and several alleles of each functional gene have been characterized. SP-A is a multimeric molecule consisting of six trimers. Each trimer contains two SP-A1 molecules and one SP-A2 molecule. Until now, it has been unclear whether a single SP-A gene product is functional or whether there are functional differences either among alleles or between single-gene SP-A products and SP-A products derived from both genes. We tested the ability of in vitro expressed SP-A variants to stimulate tumor necrosis factor (TNF)-alpha production by THP-1 cells. We observed that 1) single-gene products and products derived from both genes stimulate TNF-alpha production, 2) there are differences among SP-A1 and SP-A2 alleles in their ability to stimulate TNF-alpha production, and 3) the increases in TNF-alpha production are lower after treatment with the SP-A1 alleles than after treatment with the SP-A2 alleles. Furthermore, coexpressed SP-As from SP-A1 and SP-A2 genes have a higher activity compared with SP-As from individual alleles or mixed SP-As from SP-A1 and SP-A2 genes. These data suggest that the SP-A-induced increases in TNF-alpha levels differ among SP-A variants and appear to be affected by SP-A genotype and whether SP-A is derived from one or both genes.     

Modulatory effects of ozone on THP-1 cells in response to SP-A stimulation

Ozone (O(3)), a major component of air pollution and a strong oxidizing agent, can lead to lung injury associated with edema, inflammation, and epithelial cell damage. The effects of O(3) on pulmonary immune cells have been studied in various in vivo and in vitro systems. We have shown previously that O(3) exposure of surfactant protein (SP)-A decreases its ability to modulate proinflammatory cytokine production by cells of monocyte/macrophage lineage (THP-1 cells). In this report, we exposed THP-1 cells and/or native SP-A obtained from bronchoalveolar lavage of patients with alveolar proteinosis to O(3) and studied cytokine production and NF-kappaB signaling. The results showed 1) exposure of THP-1 cells to O(3) significantly decreased their ability to express TNF-alpha in response to SP-A; TNF-alpha production, under these conditions, was still significantly higher than basal (unstimulated) levels in filtered air-exposed THP-1 cells; 2) exposure of both THP-1 cells and SP-A to O(3) did not result in any significant differences in TNF-alpha expression compared with basal levels; 3) O(3) exposure of SP-A resulted in a decreased ability of SP-A to activate the NF-kappaB pathway, as assessed by the lack of significant increase and decrease of the nuclear p65 subunit of NF-kappaB and cytoplasmic IkappaBalpha, respectively; and 4) O(3) exposure of THP-1 cells resulted in a decrease in SP-A-mediated THP-1 cell responsiveness, which did not seem to be mediated via the classic NF-kappaB pathway. These findings indicate that O(3) exposure may mediate its effect on macrophage function both directly and indirectly (via SP-A oxidation) and by involving different mechanisms.     

Amyloid beta and amylin fibrils induce increases in proinflammatory cytokine and chemokine production by THP-1 cells and murine microglia

Activated microglia surrounding amyloid beta-containing senile plaques synthesize interleukin-1, an inflammatory cytokine that has been postulated to contribute to Alzheimer's disease pathology. Studies have demonstrated that amyloid beta treatment causes increased cytokine release in microglia and related cell cultures. The present work evaluates the specificity of this cellular response by comparing the effects of amyloid beta to that of amylin, another amyloidotic peptide. Both lipopolysaccharide-treated THP-1 monocytes and mouse microglia showed significant increases in mature interleukin-1beta release 48 h following amyloid beta or human amylin treatment, whereas nonfibrillar rat amylin had no effect on interleukin-1beta production by THP-1 cells. Lipopolysaccharide-stimulated THP-1 cells treated with amyloid beta or amylin also showed increased release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6, as well as the chemokines interleukin-8 and macrophage inflammatory protein-1alpha and -1beta. THP-1 cells incubated with fibrillar amyloid beta or amylin in the absence of lipopolysaccharide also showed significant increases of both interleukin-1beta and tumor necrosis factor-alpha mRNA. Furthermore, treatment of THP-1 cells with amyloid fibrils resulted in an elevated expression of the immediate-early genes c-fos and junB. These studies provide further evidence that fibrillar amyloid peptides can induce signal transduction pathways that initiate an inflammatory response that is likely to contribute to Alzheimer's disease pathology.     

SP-A1 and SP-A2 variants differentially enhance association of Pseudomonas aeruginosa with rat alveolar macrophages

Chronic airway inflammation caused by Pseudomonas aeruginosa is an important feature of cystic fibrosis (CF). Surfactant protein A (SP-A) enhances phagocytosis of P. aeruginosa. Two genes, SP-A1 and SP-A2, encode human SP-A. We hypothesized that genetically determined differences in the activity of SP-A1 and SP-A2 gene products exist. To test this, we studied association of a nonmucoid P. aeruginosa strain (ATCC 39018) with rat alveolar macrophages in the presence or absence of insect cell-expressed human SP-A variants. We used two trios, each consisting of SP-A1, SP-A2, and their coexpressed SP-A1/SP-A2 variants. We tested the 6A(2) and 6A(4) alleles (for SP-A1), the 1A(0) and 1A alleles (for SP-A2), and their respective coexpressed SP-A1/SP-A2 gene products. After incubation of alveolar macrophages with P. aeruginosa in the presence of the SP-A variants at 37 degrees C for 1 h, the cell association of bacteria was assessed by light microscopy analysis. We found 1) depending on SP-A concentration and variant, SP-A2 variants significantly increased the cell association more than the SP-A1 variants (the phagocytic index for SP-A1 was approximately 52-95% of the SP-A2 activity); 2) coexpressed variants at certain concentrations were more active than single gene products; and 3) the phagocytic index for SP-A variants was approximately 18-41% of the human SP-A from bronchoalveolar lavage. We conclude that human SP-A variants in vitro enhance association of P. aeruginosa with rat alveolar macrophages differentially and in a concentration-dependent manner, with SP-A2 variants having a higher activity compared with SP-A1 variants.     

Surfactant protein A increases matrix metalloproteinase-9 production by THP-1 cells

Matrix metalloproteinase (MMP)-9 from alveolar macrophages is a major source of elastolytic activity in the lung. It is increased in the bronchoalveolar lavage fluid of patients with emphysema. Although the importance of macrophage-derived elastolytic activity in the pathogenesis of emphysema is well established, questions remain about MMP-9 regulation and activity. Because surfactant protein A (SP-A) is capable of modulating other functions of human monocytic cells, we hypothesized that SP-A may regulate MMP-9 expression. Vitamin D3-differentiated THP-1 cells and peripheral blood mononuclear cells were stimulated in vitro with several concentrations of SP-A for different incubation times. MMP-9 mRNA expression was measured by dot-blot analysis, gelatinolytic activity in the medium was determined by gel zymography, protein expression was determined by ELISA, and a specific MMP-9 activity assay was used to measure the state of activation of this enzyme in the cell supernatants. SP-A induced the expression of MMP-9 in both cell types, the effect was time and dose dependent, and MMP-9 was released in its zymogen form. On the basis of results of neutralizing antibody studies, we believe that SP-A action is mediated through Toll-like receptor-2. Even though the biological meaning of these findings remains to be elucidated, these observations suggest the presence of a novel, locally controlled mechanism by which MMP-9 levels may be regulated in alveolar macrophages. We speculate that SP-A may influence the protease/antiprotease balance in the lungs of patients with quantitative and/or qualitative changes in surfactant constituents favoring an abnormal breakdown of extracellular matrix components.     

Comparison of SP-A and LPS effects on the THP-1 monocytic cell line

Surfactant protein A (SP-A) increases production of proinflammatory cytokines by monocytic cells, including THP-1 cells, as does lipopolysaccharide (LPS). Herein we report differences in responses to these agents. First, polymyxin B inhibits the LPS response but not the SP-A response. Second, SP-A-induced increases in tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-8 are reduced by >60% if SP-A is preincubated with Survanta (200 microgram/ml) for 15 min before addition to THP-1 cells. However, the LPS effects on TNF-alpha and IL-8 are inhibited by <20% and the effect on IL-1beta by <50%. Third, at Survanta levels of 1 mg/ml, SP-A-induced responses are reduced by >90%, and although the inhibitory effects on LPS action increase, they still do not reach those seen with SP-A. Finally, we tested whether SP-A could induce tolerance as LPS does. Pretreatment of THP-1 cells with LPS inhibits their response to subsequent LPS treatment 24 h later, including TNF-alpha, IL-1beta, and IL-8. Similar treatment with SP-A reduces TNF-alpha, but IL-1beta and IL-8 are further increased by the second treatment with SP-A rather than inhibited as with LPS. Thus, whereas both SP-A and LPS stimulate cytokine production, their mechanisms differ with respect to inhibition by surfactant lipids and in ability to induce tolerance.     

Combined effect of testosterone and apocynin on nitric oxide and superoxide production in PMA-differentiated THP-1 cells

Human inducible nitric oxide synthase (iNOS) is most readily observed in macrophages from patients with inflammatory diseases like atherosclerosis. The aim of the present study was to find out the combined effect of male sex hormone; testosterone and apocynin (NADPH oxidase inhibitor) on cytokine-induced iNOS production. THP-1 cells were differentiated into macrophages by phorbol myristate acetate (PMA). Expression of iNOS was induced by the addition of cytokine mixture? Testosterone was added at different concentrations (10(-6)-10(-12) M) with apocynin (1 mM). Testosterone (10(-8), 10(-10) M) inhibited NOx production in cytokine-added THP-1 cells which was further confirmed by quantikine assay of iNOS protein and RT-PCR analysis. Testosterone treatment decreased 40% of superoxide anion production. Testosterone showed inhibition of NADPH oxidase, especially expression of p67phox and p47phox (cytosol subunits). Addition of testosterone with apocynin further decreased the expression of p67phox and p47phox subunits of NADPH oxidase. The findings of the present study suggest that, testosterone; the male androgen plays an important role in the prevention of atherogenesis. Even though apocynin does not have any role on NO production, addition of apocynin together with testosterone is effective in suppressing iNOS 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 (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.     

Combined SP-A-bleomycin effect on cytokines by THP-1 cells: impact of surfactant lipids on this effect

Surfactant protein A (SP-A) plays a role in host defense and inflammation in the lung. In the present study, we investigated the hypothesis that SP-A is involved in bleomycin-induced pulmonary fibrosis. We studied the effects of human SP-A on bleomycin-induced cytokine production and mRNA expression in THP-1 macrophage-like cells and obtained the following results. 1) Bleomycin-treated THP-1 cells increased tumor necrosis factor (TNF)-alpha, interleukin (IL)-8, and IL-1beta production in dose- and time-dependent patterns, as we have observed with SP-A. TNF-alpha levels were unaffected by treatment with cytosine arabinoside. 2) The combined bleomycin-SP-A effect on cytokine production is additive by RNase protection assay and synergistic by enzyme-linked immunosorbent assay. 3) Although the bleomycin effect on cytokine production was not significantly affected by the presence of surfactant lipid, the additive and synergistic effect of SP-A-bleomycin on cytokine production was significantly reduced. We speculate that the elevated cytokine levels resulting from the bleomycin-SP-A synergism are responsible for bleomycin-induced pulmonary fibrosis and that surfactant lipids can help ameliorate pulmonary complications observed during bleomycin chemotherapy.     

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.     

Differences in the translation efficiency and mRNA stability mediated by 5'-UTR splice variants of human SP-A1 and SP-A2 genes

Surfactant protein A (SP-A) plays an important role in host defense, modulation of inflammatory processes, and surfactant-related functions of the lung. The human SP-A (hSP-A) locus consists of two functional genes, SP-A1 and SP-A2. Several hSP-A 5'-untranslated region (UTR) splice variants for each gene have been characterized and shown to be translated in vitro and in vivo. In this report, we investigated the role of hSP-A 5'-UTR splice variants on SP-A production and molecular mechanisms involved. We used in vitro transient expression of hSP-A 5'-UTR constructs containing luciferase as the reporter gene and quantitative real-time PCR to study hSP-A 5'-UTR-mediated gene expression. We found that 1) the four (A'D', ABD, AB'D', and A'CD') 5'-UTR splice variants under study enhanced gene expression, by increasing luciferase activity from 2.5- to 19.5-fold and luciferase mRNA from 4.3- to 8.8-fold compared with the control vector that lacked hSP-A 5'-UTR; 2) all four 5'-UTR splice variants studied regulated mRNA stability. The ABD variant exhibited the lowest rate of mRNA decay compared with the other three constructs (A'D', AB'D', and A'CD'). These three constructs also exhibited significantly lower rate of mRNA decay compared with the control vector; 3) based on the indexes of translational efficiency (luciferase activity/mRNA), ABD and AB'D' exhibited higher translational efficiency compared with the control vector, whereas the translational efficiency of each A'D' and A'CD' was lower than that of the control vector. These findings indicate that the hSP-A 5'-UTR splice variants play an important role in both SP-A translation and mRNA stability.     

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.     

Suppression of cytokine production and cell adhesion molecule expression in human monocytic cell line THP-1 by Tripterygium wilfordii polysaccharide moiety

Extracts of the vine-like plant Tripterygium wilfordii (TW) have been widely used in China as an immunosuppressant and anti-inflammatory drug for the treatments of rheumatoid arthritis, lupus erythematosus and other inflammatory disorders. In this study the molecular mechanisms of action of three TW extracts (ethanol, aqueous, polysaccharide) on the expression of inflammatory cytokines and adhesion molecules were investigated by RT-PCR and immunofluorescence binding techniques. The lipopolysaccharide (LPS)-mediated stimulatory effects of tumor necrosis factor-alpha (TNF-alpha) cytokine production and cell adhesion molecule (CD11c, CD18, CD14, CD54) expression in human monocytic THP-1 cells were modulated by treatments of the TW extracts or tacrolimus (FK506). The TW polysaccharide moiety exhibited more profound immunosuppressive properties than the aqueous and ethanol extracts. Biochemical characterization of the polysaccharide moiety revealed a major molecular weight of 22 kDa (viz. PSP22). The PSP22 was found to be a potential immunosuppressant that manifests the necessary immunomodulating properties.     

Structural and functional differences among human surfactant proteins SP-A1, SP-A2 and co-expressed SP-A1/SP-A2: role of supratrimeric oligomerization

SP-A (surfactant protein A) is a membrane-associated SP that helps to maintain the lung in a sterile and non-inflamed state. Unlike SP-As from other mammalian species, human SP-A consists of two functional gene products: SP-A1 and SP-A2. In all the functions examined, recombinant human SP-A1 invariably exhibits lower biological activity than SP-A2. The objective of the present study was to investigate why SP-A2 possesses greater biological activity than SP-A1 and what advantage accrues to having two polypeptide chains instead of one. We analysed structural and functional characteristics of recombinant baculovirus-derived SP-A1, SP-A2 and co-expressed SP-A1/SP-A2 using a wide array of experimental approaches such as analytical ultracentrifugation, DSC (differential scanning calorimetry) and fluorescence. We found that the extent of supratrimeric assembly is much lower in SP-A1 than SP-A2. However, the resistance to proteolysis is greater for SP-A1 than for SP-A2. Co-expressed SP-A1/SP-A2 had greater thermal stability than SP-A1 and SP-A2 and exhibited properties of each protein. On the one hand, SP-A1/SP-A2, like SP-A2, had a higher degree of oligomerization than SP-A1, and consequently had lower K(d) for binding to bacterial Re-LPS (rough lipopolysaccharide), higher self-association in the presence of calcium and greater capability to aggregate Re-LPS and phospholipids than SP-A1. On the other hand, SP-A1/SP-A2, like SP-A1, was more resistant to trypsin degradation than SP-A2. Finally, the importance of the supratrimeric assembly for SP-A immunomodulatory function is discussed.