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• W2021237672 abstract "The oxidation of methionine residues in many proteins, including the serine proteinase inhibitor α1-antitrypsin (AAT), can result in functional inactivation. In this study we investigated the pro-inflammatory properties of oxidized AAT (oxAAT), specifically its ability to activate human monocytes in culture. Monocytes stimulated with oxAAT at concentrations up to 0.2 mg/ml for 24 h showed significant elevation in monocyte chemoattractant protein-1, cytokine interleukin-6, and tumor necrosis factor-α expression and increased NADPH oxidase activity. Monocytes activated with oxAAT showed surprising effects on lipid metabolism. Expression of low density lipoprotein (LDL) receptors increased by up to 76% compared with controls but was not accompanied by any changes in125I-labeled LDL binding and, paradoxically, decreased LDL uptake, degradation, and intracellular cholesterol synthesis. oxAAT also down-regulated the scavenger receptor CD36, which takes up and is up-regulated by oxidized LDL and is down-regulated by cholesterol efflux. As a by-product of oxidative events accompanying inflammation, oxAAT has multiple effects on cytokine expression, generation of reactive oxygen species, and on intracellular lipid metabolism. The up-regulation of monocyte-derived reactive oxygen by oxAAT could potentially result in self-amplification of AAT oxidation and, thereby, the other effects deriving from it. This implies that there are as yet unidentified regulatory processes that control this cycle. The oxidation of methionine residues in many proteins, including the serine proteinase inhibitor α1-antitrypsin (AAT), can result in functional inactivation. In this study we investigated the pro-inflammatory properties of oxidized AAT (oxAAT), specifically its ability to activate human monocytes in culture. Monocytes stimulated with oxAAT at concentrations up to 0.2 mg/ml for 24 h showed significant elevation in monocyte chemoattractant protein-1, cytokine interleukin-6, and tumor necrosis factor-α expression and increased NADPH oxidase activity. Monocytes activated with oxAAT showed surprising effects on lipid metabolism. Expression of low density lipoprotein (LDL) receptors increased by up to 76% compared with controls but was not accompanied by any changes in125I-labeled LDL binding and, paradoxically, decreased LDL uptake, degradation, and intracellular cholesterol synthesis. oxAAT also down-regulated the scavenger receptor CD36, which takes up and is up-regulated by oxidized LDL and is down-regulated by cholesterol efflux. As a by-product of oxidative events accompanying inflammation, oxAAT has multiple effects on cytokine expression, generation of reactive oxygen species, and on intracellular lipid metabolism. The up-regulation of monocyte-derived reactive oxygen by oxAAT could potentially result in self-amplification of AAT oxidation and, thereby, the other effects deriving from it. This implies that there are as yet unidentified regulatory processes that control this cycle. α1-antitrypsin oxidized AAT phosphate-buffered saline monocyte chemoattractant protein-1 polyacrylamide gel electrophoresis interleukin 1 tumor necrosis factor low density lipoprotein limulus amebocyte lysate Proteinases are normally tightly regulated by their naturally occurring inhibitors, but in some pathological conditions, proteinase activity may overwhelm inhibitory capacity as a result of proteolytic or oxidative inactivation of the inhibitor (1.Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1481) Google Scholar, 2.Ossanna P.J. Test S.T. Matheson N.R. Regiani S. Weiss S.J. J. Clin Invest. 1986; 77: 1939-1951Crossref PubMed Scopus (140) Google Scholar, 3.Weiss S.J. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3849) Google Scholar). α1-Antitrypsin (AAT)1 is an acute phase protein and one of the major serine proteinase inhibitors in human plasma. It is synthesized primarily in the liver but also in extra-hepatic tissues and cells including neutrophils, monocytes, and macrophages, alveolar macrophages, intestinal epithelium cells, breast carcinoma cells, and the cornea (4.Perlmutter D.H. Cole F.S. Kilbridge T.H. Rossing T.H. Colten H.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 795-799Crossref PubMed Scopus (155) Google Scholar, 5.Issacson P.D. Jones D.B. Milward-Sadler G.H. Judd M.A. Payne S. J. Clin. Pathol. (Lond.). 1981; 34: 982-990Crossref PubMed Scopus (129) Google Scholar, 6.Ray M.B. Geboes K. Callea F. Desmet V.J. Cell Tissue Res. 1977; 185: 63-68Crossref PubMed Scopus (44) Google Scholar, 7.Geboes K. Ray M.B. Rutgeerts P. Callea F. Desmet V.J Vantrappen G. Histopathology. 1982; 6: 55-60Crossref PubMed Scopus (61) Google Scholar, 8.Boskovic G. Twining S.S. Biochim. Biophys. Acta. 1998; 1403: 37-46Crossref PubMed Scopus (43) Google Scholar). Local regulation of AAT may be important in maintaining the protease-antiprotease balance and preventing tissue damage induced by proteases in the microenvironment of injury or inflammation. For instance, several studies have demonstrated that local release of bacterial endotoxin and/or early production of inflammatory mediators such as interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) in lung tissue may up-regulate AAT expression in monocytes and, thereby, serve an important regulatory role in preventing protease destruction in the lung microenvironment (9.Knoell D.L. Ralston D.R. Coulter K.R. Wewers M.D. Am. J. Respir. Crit. Care Med. 1998; 157: 246-255Crossref PubMed Scopus (70) Google Scholar, 10.Perlmuter D.H. Punsal P.I. J. Biol. Chem. 1988; 263: 16499-16503Abstract Full Text PDF PubMed Google Scholar). Enhanced plasma levels of AAT are also known to be correlated with severity of inflammatory processes associated with coronary atherosclerosis and have been suggested to play an important part in protecting endothelial cells against the degradative effects of proteases released from activated phagocytes (11.Mori T. Sasaki J. Kawaguchi H. Handa K. Takada Y. Matsunaga A. Kono S. Arakawa K. Am. Heart J. 1995; 129: 234-238Crossref PubMed Scopus (77) Google Scholar, 12.Smith E.B. Am. J. Pathol. 1977; 86: 665-674PubMed Google Scholar). It has previously been shown that inflammatory exudates contain AAT in diverse molecular forms including native inhibitory form and several inactive, noninhibitory forms such as complexed with protease, cleaved, polymerized, and oxidized (13.Wawrzos I. Kitagawa Y. Koloczek H. Acta Biochim. Pol. 1996; 43: 481-488Crossref PubMed Scopus (2) Google Scholar, 14.Zhu X.J. Chan S.K. Biochem. J. 1987; 246: 19-23Crossref PubMed Scopus (9) Google Scholar, 15.Wong P.S. Travis J. Biochem. Biophys. Res. Commun. 1980; 96: 1449-1454Crossref PubMed Scopus (95) Google Scholar). AAT is known to be inactivated by cleavage and complex formation with target protease, such as leukocyte elastase, by cleavage by certain nontarget matrix metalloproteases, by oxidation of the reactive site methionine, and by polymerization induced by various factors such as oxidation, low pH, and interactions with other molecules (1.Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1481) Google Scholar, 16.Swaim M.W. Pizzo S.V. J. Leukocyte Biol. 1988; 43: 365-379Crossref PubMed Scopus (85) Google Scholar, 17.Kataoka H. Uchino H. Iwamura T. Seiki M. Nabeshima K. Koono M. Am. J. Pathol. 1999; 154: 457-468Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 18.Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Our understanding of AAT function has been derived primarily from studies of native, functionally active AAT, whereas possible biological roles of oxidized, polymerized, and post-cleavage, noninhibitory forms of AAT have not been thoroughly investigated. Inactivation of AAT with subsequent enhanced proteolysis, particularly by neutrophil elastase, has been invoked in the pathogenesis of lung disease, such as emphysema and lung matrix degradation in adult respiratory distress syndrome as well as in rheumatoid arthritis (19.Koyama H. Geddes D.M. Torax. 1998; 53 Suppl. 2: 510-514Google Scholar, 20.Abbink J.J. Kamp A.M. Nuijens J.H. Swaak T.J.G. Hack C.E. Arthritis Rheum. 1993; 36: 168-180Crossref PubMed Scopus (35) Google Scholar). It has also been proposed that fragmented and complexed AAT promotes an increase in synthesis of AAT in human monocytes and mediates neutrophil chemotaxis (21.Zay K. Loo S. Xie C. Devine D.V. Wright J. Churg A. Am. J. Physiol. 1999; 276: L269-L279PubMed Google Scholar, 22.Michaelis J. Vissers M.C. Winterbourn C.C. Biochem. J. 1990; 270: 809-814Crossref PubMed Scopus (71) Google Scholar), which suggests that proteolytically inactivated AAT may play multiple roles at sites of inflammation. In our previous work, we examined the effects of the proteolytically modified, cleaved form of AAT on HepG2 cells and also the effects of the amyloidogenic C-terminal fragment (C-36, corresponding to amino acid sequence 358–396) of AAT on human monocyte culture. We showed that these forms of AAT induce significant changes in lipid catabolism in both cell types and a remarkable stimulation in pro-inflammatory cytokine and free radical production and also up-regulate scavenger receptor CD36 in primary human monocyte cultures (23.Janciauskiene S. Al Rayyes O. Floren C-H. Eriksson S. Scand. J. Clin. Lab. Invest. 1997; 57: 325-336Crossref PubMed Scopus (19) Google Scholar, 24.Janciauskiene S. Lindgren S. Wright T.H. Eur. J. Biochem. 1998; 254: 460-467Crossref PubMed Scopus (11) Google Scholar, 25.Janciauskiene S. Lindgren S. Hepatology. 1999; 29: 434-442Crossref PubMed Scopus (14) Google Scholar, 26.Janciauskiene S. Wright H.T. Lindgren S. Atherosclerosis. 1999; 147: 263-275Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). This led us to propose that under inflammatory conditions, AAT might play not only a role as an inhibitor of proteases but also as a protease substrate and a reservoir of physiologically active degradation products. Oxidized AAT is a modified form of AAT found in inflammatory exudates at levels of about 5–10% that of total AAT (27.Sepper R. Konttinen Y.T. Ingman T. Sorsa T. J. Clin. Immunol. 1995; 15: 27-34Crossref PubMed Scopus (41) Google Scholar, 28.Zhang Z. Farrell A.J. Blake D.R. Chidwick K. Winyard P.G. FEBS Lett. 1993; 321: 274-278Crossref PubMed Scopus (23) Google Scholar). The amino acid at position P1 in the reactive site of each inhibitory serpin primarily determines the specificity of inhibition and, thereby, its biological activity. P1 in AAT is methionine, the most readily oxidized amino acid of proteins, which is converted by oxidation to methionine sulfoxide. Met can be attacked by various oxidants produced in biological systems, such as peroxide, hydroxyl radicals, hypochloride, chloramines, and peroxynitrite (29.Vogt W. Free Radic. Biol. Med. 1995; 18: 93-105Crossref PubMed Scopus (786) Google Scholar, 30.Wallaert B. Gressier B. Aerts C. Mizon Ch. Voisin C. Mizon J. Am. J. Respir. Cell Mol. Biol. 1991; 5: 437-444Crossref PubMed Scopus (26) Google Scholar). Evidence that this occurs in vivocomes from the observation that inactive AAT purified from inflammatory synovial fluid contains methionine sulfoxide residues (31.Chidwick K. Winyard P.G. Zhang Z. Farrell A.J. Blake D.R. Ann. Rheum. Dis. 1991; 50: 915-916Crossref PubMed Scopus (42) Google Scholar, 32.Johnson D. Travis J. J. Biol. Chem. 1978; 253: 7142-7144Abstract Full Text PDF PubMed Google Scholar). Also, oxidative inactivation of the AAT can be induced in vitro by incubating AAT with purified myeloperoxidase or stimulated phagocytes (33.Carp H. Janoff A. J. Clin. Invest. 1980; 66: 987-995Crossref PubMed Scopus (178) Google Scholar). This oxidation results in a change in the functional activity of AAT and probably promotes local inflammatory processes, including uncontrolled degradation of connective tissues. Oxidative inactivation of AAT with subsequent enhanced proteolysis, particularly by neutrophil elastase, has been invoked in the pathogenesis of pulmonary emphysema (34.Mohsenin V. J. Appl. Physiol. 1991; 70: 1456-1462Crossref PubMed Scopus (54) Google Scholar) and rheumatoid arthritis (15.Wong P.S. Travis J. Biochem. Biophys. Res. Commun. 1980; 96: 1449-1454Crossref PubMed Scopus (95) Google Scholar). That AAT oxidation and proteolysis occur is supported by findings that, on average, 41% of total AAT in rheumatoid arthritis synovial fluid is inactive (31.Chidwick K. Winyard P.G. Zhang Z. Farrell A.J. Blake D.R. Ann. Rheum. Dis. 1991; 50: 915-916Crossref PubMed Scopus (42) Google Scholar). Recently Scottet al. demonstrate that oxidation of AAT promotes AAT- immunoglobulin A complex formation in vitro. IgA-oxidized AAT complexes isolated form synovial fluid of rheumatoid disease patients were suggested to protect the oxidized AAT molecule from proteolytic cleavage by free elastase (35.Scott L.J. Russell G.I. Nixon N.B. Dawes P.T. Mattey D.L. Biochem. Biophys. Res. Commun. 1999; 255: 562-567Crossref PubMed Scopus (21) Google Scholar). Leukocytes, neutrophils, and macrophages, which secrete large quantities of oxidants at sites of inflammation, were shown to induce oxidative inactivation of AAT in vivo and to result in perturbed protease-antiprotease balance. Although oxidized AAT plays a pro-inflammatory role at sites of inflammation because of its loss of inhibitor activity toward proteases, it cannot be excluded that oxidized AAT may also have other biological activities related to inflammation. In this study, we have examined whether oxidized AAT can stimulate monocyte activation. We show that oxidized AAT induces monocyte chemoattractant protein-1 (MCP-1) and pro-inflammatory cytokine expression, activates NADPH oxidase, decreases LDL uptake and degradation and intracellular cholesterol synthesis, increases LDL receptor number, and decreases scavenger receptor CD36 expression. Native, purified AAT was a gift from Prof. C.-B. Laurell, Department of Clinical Chemistry, MAS, Malmö, Sweden. Native AAT was oxidized with N-chlorosuccinimide (Sigma) as described (32.Johnson D. Travis J. J. Biol. Chem. 1978; 253: 7142-7144Abstract Full Text PDF PubMed Google Scholar). Briefly, a reaction between AAT and N-chlorosuccinimide in a molar ratio 1:25 in a 0.1 m Tris-HCl buffer, pH 8.0, was allowed to proceed at room temperature for 30 min, and oxidized AAT was recovered after passing the reaction mixture through a Sephadex G-25 column (2 cm x 15 cm) that had been equilibrated in 50 mmNH4HCO3 or by using a centrifugal microconcentrator Centricon-30 (Amicon) equilibrated in 0.05m Tris-HCl buffer, pH 7.4, containing 0.15 mNaCl. The quality of oxidized AAT was analyzed by 7.5% SDS-PAGE. The oxidized AAT was also tested for capacity to form covalent complex with pancreatic elastase (EC 3.4.21.36) (Sigma). Samples of oxidized AAT or native AAT were digested with pancreatic elastase at a 1.2:1 molar ratio for 15 min at room temperature. The reaction was stopped by adding SDS sample buffer, and mixtures were analyzed by 7.5% SDS-PAGE without reducing agent and stained with Coomassie Blue. The endotoxin content in the oxAAT preparations used was tested by limulus amebocyte lysate (LAL), Coamatic® Chromo-LAL assay (Chromogenix, AB, Sweden) according to the manufacturer's instructions. Endotoxin standard concentrations (from 50 to 0.005 enzyme units/ml) and tested samples were placed into a microplate (preincubated at 37 °C), mixed with substrate, and incubated in a reader (ThermoMax, Molecular Devices, Inc) at 37 °C for 1 h. Negative controls (endotoxin-free water) were included in every set of assays. Absorbance measurements at 405 nm were collected with time after the addition of chromo-LAL and analyzed by the software program. Assay sensitivity was 0.005 enzyme units/ml. According to this assay, the endotoxin levels ranged between 0.006 and 0.079 enzyme units/ml in all oxAAT preparations used in our experiments. LDL was isolated by sequential preparative Ultracentrifugation using an OptimaTM XL-80K Ultracentrifuge (Beckman) as described previously (25.Janciauskiene S. Lindgren S. Hepatology. 1999; 29: 434-442Crossref PubMed Scopus (14) Google Scholar). A narrow density range (1.034–1.054 kg/liter) was used to prepare LDL for the experiments. LDL was labeled with125I by the iodine monochloride method (36.McFarlane A.S. Nature. 1958; 182: 53Crossref PubMed Scopus (1497) Google Scholar). Unbound125I was removed by chromatography on Sephadex G-25 PD-10 columns (Amersham Pharmacia Biotech) followed by extensive dialysis against 0.15 m NaCl, 1 mm EDTA, and 0.03m KI and further dialysis against 0.15 m NaCl containing 1 mm EDTA. The specific activity of LDL ranged between 229 and 433 cpm/ng of LDL protein. The endotoxin content in the lipoprotein preparations used was tested by LAL assay (E-TOXATE, Sigma). LDL samples were diluted 1:10 in endotoxin-free water, heated at 65 °C for 5 min to inactivate the LAL inhibitor found in plasma, and incubated at 37 °C for 1 h. The positive test performed using endotoxin standard dilutions (0.25, 0.125, and 0.06 enzyme units/ml) was formation of a hard gel, which permits complete inversion of the tube. Absence of hard gel formation was found in all our tested LDL samples, which are endotoxin-free, as assessed by this assay. Human monocytes were isolated from buffy coats obtained from pooled plasma of different donors by the Ficoll-Hypaque procedure as described previously (26.Janciauskiene S. Wright H.T. Lindgren S. Atherosclerosis. 1999; 147: 263-275Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). A monocyte isolation kit (Miltenyi Biotec, Bergisch Glagbach, Germany) was used to obtain a highly pure monocyte population. Cell purity was >90%, as determined on an AC900EO AutoCounter (Swelab Instruments, AB); cell viability was analyzed by 0.4% trypan blue staining. Monocytes were plated at a density of 2 × 106 cells/ml into plastic plates or dishes. After removal of nonadhering cells, the remaining adherent monocytes were cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 2 mm N-acetyl-l-alanyl-l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1% (by volume) nonessential amino acid, 2% (by volume) sodium pyruvate, and 20 mm Hepes (Fluka, Chemie AG) without serum at 37 °C in a 5% CO2. Experiments were performed within 24 h after plating of monocytes. For experiments, monocytes were incubated alone or with the addition of native or oxAAT (0.05, 0.1, and 0.2 mg/ml) or native LDL (100 μg/ml) separately or together. Monocytes seeded into 12-well plates (Nunclon), 2 × 106 cells/well were incubated in 1 ml of RPMI 1640 medium without fetal calf serum (see above) without or with various concentrations of native or oxAAT and with 125I-LDL (3.4 μg of LDL protein/mg of cell protein) for 24 h at 37 °C in 5% CO2. The medium was aspirated for subsequent determinations of LDL degradation, measured as the trichloroacetic acid-soluble noniodine 125I radioactivity in the medium (37.Bierman R.L. Stein O. Stein Y. Circ. Res. 1974; 35: 136-150Crossref PubMed Scopus (307) Google Scholar). Cells were washed 3 times with PBS and scraped into 1 ml of 0.5 m NaOH for125I-LDL uptake measurement (the sum of bound and internalized 125I-LDL) and for cell protein determination. The radioactivity was determined in an LKB 1271 automatic gamma counter (Wallac, Turku, Finland). The results are expressed as ng of LDL protein taken up or degraded per mg of cell protein. The binding of LDL to monocytes was performed at 4 °C. Cells in serum-free medium were incubated with several concentrations of oxAAT (up to 0.2 mg/ml) alone or in the presence of LDL (100 μg/ml). Binding studies at 4 °C were performed by washing the cells three times with 1 ml of ice-cold PBS followed by precooling for 20 min at 4 °C in 1 ml of ice-cold RPMI medium containing 0.5% human serum albumin. After the addition of125I-LDL (3.5 mg/liter), the cells were incubated for 2 h at 4 °C, then washed 3 times with 1 ml of ice-cold PBS. New medium containing 10 g/liter dextran sulfate (M r∼ 500 000, Amersham Pharmacia Biotech) was added (polymer was added for osmotic balance), and the cells were subjected to a second incubation for 1 h at 4 °C in a rotary shaker at 60 rpm. Dextran sulfate is known to release receptor-bound LDL from the surface of the cell (38.Salter A.M. Saxton J. Brindley D.N. Biochem. J. 1986; 240: 549-557Crossref PubMed Scopus (24) Google Scholar). Medium containing dextran sulfate-released125I-LDL was then aspirated, and the radioactivity was measured in a gamma counter. Total RNA from monocytes was isolated as outlined by Davis et al. (39.Davis L.G. Dibner M.D. Battey J.F. Davis L.G. Basic Methods in Molecular Biology. Elsevier Science Publishing Co., Inc., New York1986: 129-156Google Scholar). Cold GT buffer (4m guanidine thiocyanate, 3 m sodium acetate, pH 6, and 7% β-mercaptoethanol) was added directly to the cells in culture dishes. Sarkosyl was added to 2%, and the lysate was layered onto a 4-ml 5.7 m CsCl cushion and centrifuged at 100,000 × g for 16 h. The pellet was washed with ethanol (95%), suspended in diethylpyrocarbonate-treated distilled water (dH2O), precipitated in 95% ethanol at pH 5.0, and stored at −20 °C. LDL receptor mRNA was quantified by reverse transcription polymerase chain reaction as described previously (25.Janciauskiene S. Lindgren S. Hepatology. 1999; 29: 434-442Crossref PubMed Scopus (14) Google Scholar). The oligonucleotides of 5′primer (5′-CAATGTCTCACCAAGCTCTG-3′) and 3′primer (5′-TCTGTCTCGAGGGGTAGCTG-3′) were purchased from Amersham Pharmacia Biotech. The amplification was performed with a Perkin-Elmer thermocycler using the following cycle profile: denaturation at 95 °C for 1 min, then primer annealing and extension at 60 °C for 1 min. The initial denaturation step was prolonged to 3 min, and after 32 cycles, the reaction mixture was incubated at 72 °C for 7 min and then cooled to 4 °C. Each polymerase chain reaction product (20 μl) was electrophoresed along with a DNA molecular weight marker (Amersham Pharmacia Biotech) in a 4% agarose-sieving gel (2:3 (wt/wt) NuSieve-agarose and 1:3 (wt/wt) SeaKem LE-agarose (In Vitro AB, Stockholm, Sweden) in TAE (40 mmTris, 20 mm sodium acetate, 1 mm EDTA, pH 7.4) running buffer at 90 V for 3 to 4 h at 4 °C. The gel was scanned in a FluorImager SI (Molecular Dynamics, Sunnyvale, CA) using an excitation wavelength of 488 nm (argon laser). Images were analyzed using ImageQuaNT software (Molecular Dynamics), and the signal intensity was calculated according to the vendor's instructions. The amount of LDL receptor polymerase chain reaction fragment was normalized to that of the internal standard. Values are expressed as percent of levels of LDL receptor mRNA in control monocytes. Monocytes were grown on coverslips in the presence of oxAAT (up to 0.25 mg/ml) and/or LDL (100 μg/ml) for 24 h. At the end of the incubation period cells were washed with PBS and fixed with 4% PBS-buffered formaldehyde for 15 min. In the next step, cells were rinsed with water, dipped for a few seconds in 60% isopropanol, stained in the Oil Red O for 10–15 min and rinsed again in 60% isopropanol to remove excess of stain. Cell nuclei were stained for a few seconds in the hematoxylin solution, washed with water, and mounted with commercially available mounting medium (DAKO). Samples were analyzed by microscope (Olympus Bx60) using the PC program Olympus MicroImage. Images were taken by digital camera (Sony, DKC-5000) at magnification 40×. Cellular synthesis of cholesterol was estimated by measuring [14C]acetate incorporation into sterols from cell extracts as described (25.Janciauskiene S. Lindgren S. Hepatology. 1999; 29: 434-442Crossref PubMed Scopus (14) Google Scholar). Cells were grown in 60-mm Petri dishes for 48 h. Native or oxAAT alone, AAT together with LDL, or LDL (100 μg/liter) alone was added together with [14C]acetate (1 μCi/ml media in 1.8 mmsodium acetate) and incubated for 24 h. After the medium was aspirated, cells were washed once with PBS and harvested in 1 ml of cold medium containing 2 mm sodium acetate. Cells were centrifuged at 500 rpm for 5 min, resuspended in 1 ml of 20 mm Tris buffer, pH 7.5 (cold), and 9 ml of acetone:ethanol (1:1), vortexed, and precipitated on ice for 15 min and centrifuged (1000 rpm, 5 min). Supernatant (5-ml aliquots) was collected, and 100 μl of cholesterol carrier (1 mg/ml cholesterol in acetone) and 2 ml of digitonin (5 mg/ml in 50% ethanol) were added and precipitated overnight. Precipitates were washed twice with acetone:ether (1:1) and once with ether, dissolved in methanol, and counted in a β-counter (Liquid Scintillation System TRI-CARB 300C). Monocytes incubated without and with oxAAT and native LDL separately or together were lysed on ice in PBS containing 1% (v/v) Triton X-100 and 10 mmol/liter benzamidine for 15 min or were sonicated with repeated freeze-thaw cycles and centrifuged at 13,000 × g for 15 min. The aliquots were collected, and the protein concentration was determined. 50 μg of cell protein was separated on a 7.5% SDS-polyacrylamide gel. Gels were calibrated by high range molecular weight markers (MultiMarkTM, Multi-Colored Standard, Novex, San Diego, CA). Proteins were transferred to a polyvinylidene difluoride membrane. A monoclonal antibody against CD36 receptor (SMO, Santa Cruz Biotechnology) or antibody against human LDL receptor (Amersham Pharmacia Biotech) (1:5000) was used for Western blot analysis. The antibody was visualized with horseradish peroxidase-conjugated secondary antibodies (1:10,000) using the enhanced chemiluminescence (ECL+Plus) Western blotting detection system kit (Amersham Pharmacia Biotech). Polyvinylidene difluoride membranes were exposed to high performance autoradiography Hyperfilm™ MP (Amersham Pharmacia Biotech) for the indicated times. Immunoblots were quantified by scanning densitometry (model Personal Densitometer SI, Molecular Dynamics). The ImageQuaNT software (Molecular Dynamics, Ins.) was used to display images and to quantitate the result. Cell culture supernatants from monocytes treated with oxAAT (up to 0.25 mg/ml) and/or LDL (100 μg/ml) for 24 h were analyzed to determine human IL-1 and IL-6 and TNFα. A quantitative sandwich enzyme immunoassay (QuantikineTM, R&D Systems, Minneapolis, MN) technique sensitive to pg/ml assay levels was used according to manufacturer's instructions. Monocytes were cultured for various time points alone or with the addition of oxAAT and/or LDL as described above. Culture medium was collected, and MCP-1 expression was assayed by a quantitative sandwich immunoassay technique according to manufacturer's instructions (R&D Systems Europe Ltd, Abingdon, UK). The optical density was determined using a microplate reader at 450 nm. The readings at 570 nm were subtracted from the readings at 450 nm for wavelength correction. The duplicate readings for each standard, control, and samples were averaged, and the average zero standard optical density was subtracted. Superoxide produced from the NADPH oxidase was monitored by the superoxide dismutase-inhibitable rate of cytochrome c reduction (40.Li Y. Trush M.A. Biochem. Biophys. Res. Commun. 1998; 253: 295-299Crossref PubMed Scopus (399) Google Scholar). Monocytes (2 × 106/ml) were incubated for various time points up to 3 h at 37 °C, with 1.5 mg/ml cytochrome c and oxAAT in 2 ml of air-saturated PBS containing 0.5 mm MgCl2, 0.7 mm CaCl2, and 0.1% glucose in the presence or absence of 300 units/ml superoxide dismutase. After incubation, the cells were removed by centrifugation at 400 × g for 5 min, and the reduced cytochrome c in the supernatant was measured at 550 nm. The rate of superoxide production is given by the difference in the rate of cytochrome c reduction in the absence and presence of superoxide dismutase. Cells were incubated with and without added oxidized or native AAT for 20 h. [3H]Thymidine (Amersham Pharmacia Biotech) was then added to the cells (0.2 μCi/ml) for a further 4-h incubation at 37 °C. After the medium was aspirated, the cells were washed twice with 0.5m NaCl and incubated for 5 min with 5% trichloroacetic acid. Cells were then washed with water, dissolved in 1 ml 0.5m NaOH, and neutralized with 200 μl of HCl, and radioactivity was determined in a β-counter (Packard 300CD liquid scintillation spectrometer; Packard Instrument Co.). The differences in the means in experimental results were analyzed for their statistical significance with independent sample two-sided t test and/or one-way analysis of variance combined with a multiple comparisons procedure (Scheffé multiple range test) with the overall significance level of α = .05. Statistical Package for Social Sciences (SPSS for Windows, Version 6.0) was used for the calculations (41.Norusis M.J. SPSS for Windows, Version 6.0. SPSS Inc., Chicago1993Google Scholar). AAT can be rendered inactive toward elastase by at least two known mechanisms: either by oxidation of the reactive center Met-358 or by protease cleavage of peptide bonds close to the reactive center (1.Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1481) Google Scholar, 31.Chidwick K. Winyard P.G. Zhang Z. Farrell A.J. Blake D.R. Ann. Rheum. Dis. 1991; 50: 915-916Crossref PubMed Scopus (42) Google Scholar). In our experimental model, purified native AAT was oxidized with N-chlorosuccinimide and characterized for its ability to interact with pancreatic elastase. Samples of native and oxAAT alone or incubated with pancreatic elastase were subjected to 7.5% SDS-PAGE. In Fig.1 a single band with similar molecular mass is seen for both native and oxAAT. Interaction betw" @default.
• W2021237672 created "2016-06-24" @default.
• W2021237672 creator A5001538826 @default.
• W2021237672 creator A5062552750 @default.
• W2021237672 date "2000-03-01" @default.
• W2021237672 modified "2023-10-16" @default.
• W2021237672 title "Activation of Primary Human Monocytes by the Oxidized Form of α1-Antitrypsin" @default.
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