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• W2043026025 abstract "Little is known about interactions between endogenous anti-inflammatory paradigms and microvascular thrombosis in lung ischemia/reperfusion (I/R) injury. Interleukin (IL)-10 suppresses macrophage activation and down-regulates proinflammatory cytokine production, but there are no available data to suggest a link between IL-10, thrombosis, and fibrinolysis in the setting of I/R. We hypothesized that hypoxia/ischemia triggers IL-10 production, to dampen proinflammatory cytokine and adhesion receptor cascades and to restore vascular patency by fibrinolytic potentiation. Studies were performed in a mouse lung I/R model. IL-10 mRNA levels in lung were increased 43-fold over base line by 1 h of ischemia/2 h of reperfusion, with a corresponding increase in plasma IL-10. Expression was prominently localized in bronchial epithelial cells and mononuclear phagocytes. To study the link between IL-10 and fibrinolysis in vivo, the induction of plasminogen activator inhibitor-1 (PAI-1) was evaluated. Northern analysis demonstrated exaggerated pulmonary PAI-1 expression in IL-10 (−/−) mice after I/R, with a corresponding increase in plasma PAI/tissue-type plasminogen activator activity. In vivo, IL-10 (−/−) mice showed poor postischemic lung function and survival after I/R compared with IL-10 (+/+) mice. Despite a decrease in infiltration of mononuclear phagocytes in I/R lungs of IL-10 (−/−) mice, an increased intravascular pulmonary fibrin deposition was observed by immunohistochemistry and Western blotting, along with increased IL-1 expression. Recombinant IL-10 given to IL-10 (−/−) mice normalized the PAI/tissue-type plasminogen activator ratio, reduced pulmonary vascular fibrin deposition, and rescued mice from lung injury. Since recombinant hirudin (direct thrombin inhibitor) also sufficed to rescue IL-10 (−/−) mice, these data suggest a preeminent role for microvascular thrombosis in I/R lung injury. Ischemia-driven IL-10 expression confers postischemic pulmonary protection by augmenting endogenous fibrinolytic mechanisms. Little is known about interactions between endogenous anti-inflammatory paradigms and microvascular thrombosis in lung ischemia/reperfusion (I/R) injury. Interleukin (IL)-10 suppresses macrophage activation and down-regulates proinflammatory cytokine production, but there are no available data to suggest a link between IL-10, thrombosis, and fibrinolysis in the setting of I/R. We hypothesized that hypoxia/ischemia triggers IL-10 production, to dampen proinflammatory cytokine and adhesion receptor cascades and to restore vascular patency by fibrinolytic potentiation. Studies were performed in a mouse lung I/R model. IL-10 mRNA levels in lung were increased 43-fold over base line by 1 h of ischemia/2 h of reperfusion, with a corresponding increase in plasma IL-10. Expression was prominently localized in bronchial epithelial cells and mononuclear phagocytes. To study the link between IL-10 and fibrinolysis in vivo, the induction of plasminogen activator inhibitor-1 (PAI-1) was evaluated. Northern analysis demonstrated exaggerated pulmonary PAI-1 expression in IL-10 (−/−) mice after I/R, with a corresponding increase in plasma PAI/tissue-type plasminogen activator activity. In vivo, IL-10 (−/−) mice showed poor postischemic lung function and survival after I/R compared with IL-10 (+/+) mice. Despite a decrease in infiltration of mononuclear phagocytes in I/R lungs of IL-10 (−/−) mice, an increased intravascular pulmonary fibrin deposition was observed by immunohistochemistry and Western blotting, along with increased IL-1 expression. Recombinant IL-10 given to IL-10 (−/−) mice normalized the PAI/tissue-type plasminogen activator ratio, reduced pulmonary vascular fibrin deposition, and rescued mice from lung injury. Since recombinant hirudin (direct thrombin inhibitor) also sufficed to rescue IL-10 (−/−) mice, these data suggest a preeminent role for microvascular thrombosis in I/R lung injury. Ischemia-driven IL-10 expression confers postischemic pulmonary protection by augmenting endogenous fibrinolytic mechanisms. ischemia/reperfusion intercellular adhesion molecule-1 plasminogen activator inhibitor-1 interleukin recombinant murine IL-10 tissue-type plasminogen activator urokinase-type plasminogen activator enzyme-linked immunosorbent assay phosphate-buffered saline soluble intercellular adhesion molecule-1 Ischemia/reperfusion (I/R)1 lung injury plays a significant role in clinical situations such as lung transplantation (1.Naka Y. Toda K. Kayano K. Oz M.C. Pinsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 757-761Crossref PubMed Scopus (59) Google Scholar, 2.Pinsky D.J. Thromb. Haemostasis. 1995; 74: 58-65Crossref PubMed Scopus (47) Google Scholar, 3.Snell G.I. Rabinov M. Griffiths A. Williams T. Ugoni A. Salamonsson R. Esmore D. J. Heart Lung Transplant. 1996; 15: 160-168PubMed Google Scholar). Lung failure associated with I/R is characterized by increased microvascular permeability, pulmonary vascular resistance with subsequent edema formation and impairment of gas exchange, and microembolism. The lungs are particularly susceptible to ischemia/reperfusion injury, presumably due to the rich vascularity of the lungs and the relatively large surface area over which blood-borne components can interact with endothelium. The proximate mechanisms of ischemic lung injury are diverse and include leukocyte activation and recruitment (1.Naka Y. Toda K. Kayano K. Oz M.C. Pinsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 757-761Crossref PubMed Scopus (59) Google Scholar), complement activation (4.Naka Y. Marsh H.C. Scesney S.M. Oz M.C. Pinsky D.J. Transplantation. 1997; 64: 1248-1255Crossref PubMed Scopus (44) Google Scholar), abnormalities in pulmonary vascular tone, and increased procoagulant activity, resulting in microcirculatory failure, cellular dysfunction, edema, and cell death. The local production of proinflammatory cytokines, such as IL-1α and tumor necrosis factor-α, is considerably increased in I/R injury (5.Chang D.M. Hsu K. Ding Y.A. Chiang C.H. Am. J. Respir. Crit. Care Med. 1997; 156: 1230-1234Crossref PubMed Scopus (38) Google Scholar,6.Colletti L.M. Remick D.G. Burtch G.D. Kunkel S.L. Strieter R.M. Campbell Jr., D.A. J. Clin. Invest. 1990; 85: 1936-1943Crossref PubMed Scopus (772) Google Scholar), which can also feedback to increase expression of intercellular adhesion molecule (ICAM-1) or P-selectin on pulmonary vascular endothelial cells, the expression of which is likewise deleterious (7.Lo S.K. Everitt J. Gu J. Malik A.B. J. Clin. Invest. 1992; 89: 981-988Crossref PubMed Scopus (155) Google Scholar, 8.Mulligan M.S. Vaporciyan A.A. Miyasaka M. Tamatani T. Ward P.A. Am. J. Pathol. 1993; 142: 1739-1749PubMed Google Scholar, 9.Weller A. Isenmann S. Vestweber D. J. Biol. Chem. 1992; 267: 15176-15183Abstract Full Text PDF PubMed Google Scholar, 10.Ward P.A. Ann. N. Y. Acad. Sci. 1996; 796: 104-112Crossref PubMed Scopus (134) Google Scholar). Although clear roles for proinflammatory cytokines and leukocyte adhesion receptors have been defined in the setting of frank pulmonary I/R (1.Naka Y. Toda K. Kayano K. Oz M.C. Pinsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 757-761Crossref PubMed Scopus (59) Google Scholar, 11.Kapelanski D.P. Iguchi A. Niles S.D. Mao H.Z. J. Heart Lung Transplant. 1993; 12: 294-306PubMed Google Scholar, 12.Horgan M.J. Wright S.D. Malik A.B. Am. J. Physiol. 1990; 259: L315-L319Crossref PubMed Google Scholar), the pathophysiological role for localized thrombosis has been ascribed only by inference. Since microvascular thrombosis can impede the return of blood flow even when perfusion pressure is normalized, this can exacerbate and create ongoing tissue damage. In the brain, postischemic microvascular thrombosis and leukocyte recruitment contribute significantly to ischemic cerebral tissue damage (13.Choudhri T.F. Hoh B.L. Zerwes H.-G. Prestigiacomo C.J. Kim S.C. Connolly Jr., E.S. Kottirsch G. Pinsky D.J. J. Clin. Invest. 1998; 102: 1301-1310Crossref PubMed Scopus (213) Google Scholar, 14.Huang J. Kim L.J. Mealey R. Marsh H.C.J. Zhang Y. Tenner A.J. Connolly E.S.J. Pinsky D.J. Science. 1999; 285: 595-599Crossref PubMed Scopus (303) Google Scholar, 15.Choudhri T.F. Hoh B.L. Huang J. Kim L.J. Prestigiacomo C.J. Schmidt A.M. Kisiel W. Connolly Jr., E.S. Pinsky D.J. J. Exp. Med. 1999; 190: 91-99Crossref PubMed Scopus (58) Google Scholar). In the heart, postischemic no reflow has been documented even following relief of the major vascular obstruction. Although the lungs are a particularly vulnerable tissue in terms of their response to I/R injury, and even relatively minor interruptions of blood flow might lead to postischemic hypoperfusion and microvascular dysfunction (16.Gilroy Jr., R.J. Bhatte M.J. Wickersham N.E. Pou N.A. Loyd J.E. Overholser K.A. Am. Rev. Respir. Dis. 1993; 147: 276-282Crossref PubMed Scopus (16) Google Scholar), the contribution of in situ thrombosis to the postischemic no-reflow phenomenon in the lungs remains unclear. The important role of fibrinolysis by the plasminogen activator system has been well studied in the case of large macrovascular thrombotic occlusions, and exogenous tissue-type plasminogen activator (tPA) has been widely used in clinical settings such as acute myocardial infarction or deep vein thrombosis. Plasminogen activator inhibitor-1 (PAI-1) is a 52-kDa serine protease inhibitor that serves as the major plasma inhibitor of tPA and urokinase-type plasminogen activator (uPA) and therefore has been the focus for study as the critical inhibitor of fibrinolysis (17.Fujii S. Sawa H. Saffitz J.E. Lucore C.L. Sobel B.E. Circulation. 1992; 86: 2000-2010Crossref PubMed Google Scholar, 18.Sobel B.E. Woodcock-Mitchell J. Schneider D.J. Holt R.E. Marutsuka K. Gold H. Circulation. 1998; 97: 2213-2221Crossref PubMed Scopus (250) Google Scholar, 19.Handt S. Jerome W.G. Tietze L. Hantgan R.R. Blood. 1996; 87: 4204-4213Crossref PubMed Google Scholar, 20.Abrahamsson T. Nerme V. Stromqvist M. Akerblom B. Legnehed A. Pettersson K. Westin Eriksson A. Thromb. Haemostasis. 1996; 75: 118-126Crossref PubMed Scopus (43) Google Scholar, 21.Fay W.P. Murphy J.G. Owen W.G. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1277-1284Crossref PubMed Scopus (46) Google Scholar, 22.Wiman B. Thromb. Haemostasis. 1995; 74: 71-76Crossref PubMed Scopus (188) Google Scholar). Some studies have suggested a relation between the increased synthesis of PAI-1 and persistence or recurrence of thrombosis (17.Fujii S. Sawa H. Saffitz J.E. Lucore C.L. Sobel B.E. Circulation. 1992; 86: 2000-2010Crossref PubMed Google Scholar, 18.Sobel B.E. Woodcock-Mitchell J. Schneider D.J. Holt R.E. Marutsuka K. Gold H. Circulation. 1998; 97: 2213-2221Crossref PubMed Scopus (250) Google Scholar) even after thrombolytic therapy. We have shown the physiologic relevance of hypoxia-induced modulation of the fibrinolytic response in the pathogenesis of fibrin accumulation in lungs using PAI-1-, tPA-, and uPA-deficient mice (23.Pinsky D.J. Liao H. Lawson C.A. Yan S.F. Chen J. Carmeliet P. Loskutoff D.J. Stern D.M. J. Clin. Invest. 1998; 102: 919-928Crossref PubMed Scopus (161) Google Scholar). Since hypoxia is an important component of the ischemic vascular milieu, these data suggest that I/R injury might involve not only induction of the inflammatory response but also abnormalities in the fibrinolytic system that lead to clot formation. In most biological systems, when one set of pathways is triggered, countervailing forces are activated to modulate the effects of uncontrolled activation of the primary pathway. The current studies were undertaken to elucidate the potential negative regulatory effects of IL-10 on critically relevant issues of cytokine induction and thrombosis in lung I/R injury. IL-10 is one of the Th2 type cytokines that is believed to exert anti-inflammatory effects in different systems by its ability to suppress macrophage activation and down-regulate proinflammatory cytokine production (24.Abbas A.K. Murphy K.M. Sher A. Nature. 1996; 383: 787-793Crossref PubMed Scopus (3883) Google Scholar). IL-10 inhibits several macrophage functions, including antigen presentation to T cells, synthesis of several proinflammatory cytokines (such as IL-1α and -β, IL-6, IL-8, tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor, and granulocyte colony-stimulating factor), and production of reactive oxygen intermediates and nitric oxide (25.Fiorentino D.F. Bond M.W. Mosmann T.R. J. Exp. Med. 1989; 170: 2081-2095Crossref PubMed Scopus (2499) Google Scholar, 26.de Waal Malefyt R. Abrams J. Bennett B. Figdor C.G. de Vries J.E. J. Exp. Med. 1991; 174: 1209-1220Crossref PubMed Scopus (3398) Google Scholar, 27.Cunha F.Q. Moncada S. Liew F.Y. Biochem. Biophys. Res. Commun. 1992; 182: 1155-1159Crossref PubMed Scopus (366) Google Scholar). With regard to the lung, several studies have shown that IL-10 reduces the intensity of cellular recruitment in pulmonary inflammation and is an inhibitor of the induced release of several proinflammatory cytokines such as TNF-α and macrophage inflammatory proteins 1 and 2, supporting an anti-inflammatory role of IL-10 in the lung (28.Greenberger M.J. Strieter R.M. Kunkel S.L. Danforth J.M. Goodman R.E. Standiford T.J. J. Immunol. 1995; 155: 722-729PubMed Google Scholar). Furthermore, some studies have shown that IL-10 has significant protective effects in lung inflammatory injury by suppressing the expression ICAM-1 (29.Shanley T.P. Schmal H. Friedl H.P. Jones M.L. Ward P.A. J. Immunol. 1995; 154: 3454-3460PubMed Google Scholar, 30.Mulligan M.S. Jones M.L. Vaporciyan A.A. Howard M.C. Ward P.A. J. Immunol. 1993; 151: 5666-5674PubMed Google Scholar). Although a recent report shows that IL-10 may inhibit coagulation and potentiate the fibrinolytic system in human endotoxemia (31.Pajkrt D. van der Poll T. Levi M. Cutler D.L. Affrime M.B. van den Ende A. ten Cate J.W. van Deventer S.J. Blood. 1997; 89: 2701-2705Crossref PubMed Google Scholar), no data are available with respect to its effects on the coagulant/fibrinolytic mechanism in I/R. Therefore, the current studies were driven by a 2-fold hypothesis: 1) that microvascular thrombosis represents a significant component of lung I/R injury and 2) that endogenous IL-10 plays a pivotal role in regulating the fibrinolytic system in lung I/R injury. IL-10-deficient mice (IL-10 (−/−), C57/6-IL-10tm1cgn, male, 10 weeks old) (32.Kuhn R. Lohler J. Rennick D. Rajewsky K. Muller W. Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3686) Google Scholar) and their wild-type controls (IL-10 (+/+), C57/6J, male, 10 weeks old), which were purchased from Jackson Laboratories (Bar Harbor, ME), were used in these experiments according to a protocol approved by the Institutional Animal Care and Use Committee at Columbia University, in accordance with guidelines of the American Association for the Accreditation of Laboratory Animal Care. Animals were initially anesthetized intraperitoneally with 0.1 mg/g of mouse weight of ketamine and 0.01 mg/g of mouse weight of xylazine, followed by intraperitoneal continuous infusion of one-third of the initial dose per hour using a syringe pump (model 100 series, KD Scientific Inc.). After ensuring appropriate depth of anesthesia, mice were intubated via tracheostomy and placed on a Harvard ventilator (tidal volume = 0.75 ml, respiratory rate = 120/min) with room air, followed by bilateral thoracotomy. The left hilum was cross-clamped for a period of 1 h, after which the cross-clamp was released. Reperfusion proceeded from 1 to 3 h according to the following groups: untreated lung in sham operation; group I, 1-h ischemia without reperfusion; and R-1, R-2, or R-3 groups, consisting of 1-h ischemia followed by 1-, 2-, and 3-h reperfusion, respectively. After observation, blood samples were obtained for ELISA (IL-10), and lung specimens were taken for Northern blot analysis. For all experiments, the surgical operator was blinded by a colleague in the laboratory as to either the strain of mice being used (all mice were black in appearance) or to the specific substance being injected. Four groups were studied: 1) IL-10 (+/+) mice (received 300 μl of PBS without additive); 2) IL-10 (−/−) mice (received 300 μl of PBS without additive); 3) IL-10 (−/−) mice given 1 μg of recombinant murine IL-10 (R & D Systems) after thoracotomy but before pulmonary ischemia. rmIL-10 was prepared as 1 μg/300 μl in PBS; 4) IL-10 (−/−) mice given 1.0 mg/kg recombinant hirudin (direct and specific thrombin inhibitor; Sigma) after thoracotomy but before pulmonary ischemia. Recombinant hirudin was prepared for a 1.0 mg/kg injection in 300 μl in PBS. For all four groups, the experimental procedures were as follows. After 1-h ischemia followed by 2-h reperfusion, the contralateral (right) hilum was permanently ligated, so that the animal's survival and gas exchange depended solely upon the reperfused lung, and observation continued for 1 h among these four groups. As the mouse continued to be ventilated, death of the mouse was defined as a combination of 1) cessation of regular cardiac activity; 2) the apparent collapse of the left atrium; and 3) brief clonic activity indicating cessation of cerebral blood flow. At the time of death, blood samples were obtained for ELISA (IL-1α, sICAM-1) or PAI/tPA activity assays, and lung specimens were taken for the measurement of wet/dry ratios or Northern blot analyses . In a separate series of survival experiments, lung function was ascertained by arterial blood gas analysis (sampled from the left ventricle) in mice that survived for 30 min after right hilar ligation. Immediately after determination of lung function, mice were heparinized, and lung specimens were taken for Western blot or immunohistochemical analysis for fibrin. These experiments were performed as a separate group so that obtaining the left ventricular sample of blood did not impact on mouse survival. When mice were sacrificed after the survival experiments, the left hilum was ligated, and then the left lung (including residual blood) was taken and weighed as a wet weight. The lung specimen was desiccated at 80 °C for 24 h and weighed again as dry weight. Wet weight was divided by dry weight for the calculation of wet/dry ratio. IL-10 (+/+) mice were divided into untreated, I, R-1, R-2, and R-3 groups. In each group, blood was drawn from the heart, kept at 4 °C overnight, and centrifuged at 13,000 rpm for 20 min to obtain serum, which was then divided into aliquots and frozen at −80 °C until the time of use. The serum IL-10 level was assayed by ELISA kits (R & D Systems), and IL-1α and sICAM-1 levels were assayed by an ELISA kit (Endogen). The lower limits of detection for IL-10, IL-1 α, and sICAM-1 assays are 4 pg/ml, 6 pg/ml, and 5 ng/ml, respectively. Values are expressed as the mean ± S.E. of duplicate determinations. In dedicated experiments, the left lung was rapidly excised and snap-frozen in liquid nitrogen until the time of mRNA extraction. After tissue homogenization using a Brinkmann Polytron homogenizer, total RNA from the lung tissues was isolated by the Tryzol method (Life Technologies, Inc.), and then poly(A) mRNA were purified using Poly(A)Ttract® mRNA Isolation Systems (Promega, Madison, WI). To detect IL-1α, IL-10, PAI-1, and tPA transcripts, equal amounts of poly(A) mRNA (2.5 μg/lane) or total RNA (25 μg/lane) were loaded onto an 0.8% agarose gel containing 2.2 mformaldehyde for size fractionation and then transferred overnight to nylon membranes (Duralon-UVTM membranes; Strategene) with 20× SSC buffer. A murine IL-10 (1.5 kilobases; American Type Culture Collection), IL-1α (789 bp), PAI-1 (900 bp; the plasmid, containing a pBS vector and a 3014-bp insert, was generously provided by M. Cole), and tPA (800 bp; composed of a 2.5-kb insert from a pKS +/− plasmid vector (33.Rickles R.J. Darrow A.L. Strickland S. J. Biol. Chem. 1988; 263: 1563-1569Abstract Full Text PDF PubMed Google Scholar)) cDNAs were purified using a Qiagen II gel extraction kit (QIAGEN Inc.). These fragments were used as cDNA probes after32P-random primer labeling (Prime-It RmT; Strategene) with [α-32P]dCTP. After prehybridization and hybridization using QuikHyb hybridization solution (Strategene) at 68 °C for 1 h, the blots were washed twice for 15 min with 2× SSC, 0.1% SDS at room temperature, followed by one wash for 30 min with 0.1× SSC, 0.1% SDS at 60 °C. Blots were developed with X-Omat AR film exposed with an intensifying light screen at −80 °C for 3 days. Normalized absorption values were obtained by densitometry scanning (Molecular Imager® System; Bio-Rad) of cDNAs including β-actin bands. In order to make RNA probes forin situ hybridization, the polymerase chain reaction was first performed using total RNA from the lung tissue after 1-h ischemia followed by 2-h reperfusion. Reverse transcription was performed on total RNA with oligo(dT) primers, and amplification was carried out for 35 cycles by polymerase chain reaction with specific primers for IL-10 (CLONTECH): 5′ primer, 5′-ATGCAGGACTTTAAGGGTTACTTGGGTT-3′; 3′ primer, 5′-ATTTCGGAGAGAGGTACAAACGAGGTTT-3′. An aliquot of the polymerase chain reaction product mixture was run in a 1% agarose gel stained with ethidium bromide. The polymerase chain reaction products (455 bp) were recovered using a Qiagen II gel extraction kit (QIAGEN) and inserted to pGEM-T® Easy Vector using the T4 ligation method (Promega). The RNA expression plasmid was linearized withNcoI and SalI enzymes to allow in vitro run-off synthesis of both sense- and antisense-oriented RNA probes. Both sense and antisense probes were labeled by transcription with a digoxigenin RNA labeling kit (Roche Molecular Biochemicals), and the labeled probes were then purified. Both untreated lungs and left lungs after 1-h ischemia/2-h reperfusion were snap-frozen embedded in OCT compound (Miles Scientific) in a cryomold in liquid nitrogen. The frozen sections were cut at 5 μm thick and placed on glass slides precoated with opaque (VWR Scientific Products). Briefly, slides were prefixed in 4% paraformaldehyde for 20 min and then digested with 14 μg/ml proteinase K in Tris-EDTA (pH 8.0) for 15 min at 37 °C, fixed in 4% paraformaldehyde for 10 min. Sections were acetylated with 0.1 mol/liter triethanolamine (pH 8.0) with 0.25% (v/v) acetic anhydride. Sections were then equilibrated for 60 min in hybridization buffer consisting of 4× SSC, 50% formamide, 5% dextran sulfate, 0.1 mg/ml yeast tRNA, and 0.05 mg/ml salmon sperm DNA. Hybridization was carried out overnight at 45 °C with either IL-10 sense or antisense probe (1:25 dilution in prehybridization buffer). Sections were subjected to stringent washes consisting of a single wash with 2× SSC, two 30-min washes with 1 × SSC at room temperature, two 30-min washes with 0.1× SSC at 37 °, and two 20-min washes with Tris buffer (100 mmol/liter Tris-HCl, 150 mmol/liter NaCl). After blocking with blocking buffer (0.1% Triton X-100, 4% sheep serum, 100 mmol/liter Tris-HCl, and 150 mmol/liter NaCl), sections were incubated with a 1:100 dilution of anti-digoxigenin antibody (Roche Molecular Biochemicals) for 2 h at room temperature. After four washes, color was allowed to develop for 4 h, and development was stopped by dipping the slides briefly in Tris-EDTA buffer (pH 8.0) and then rinsing. Sections were covered with coverslips with water-soluble mounting medium. PAI/tPA activity was determined by a functional rate assay described by Ranby et al. (34.Ranby M. Norrman B. Wallen P. Thromb. Res. 1982; 27: 743-749Abstract Full Text PDF PubMed Scopus (166) Google Scholar) and its adaptation to plasma samples, as described by Wiman et al. (35.Wiman B. Mellbring G. Ranby M. Clin. Chim. Acta. 1983; 127: 279-288Crossref PubMed Scopus (201) Google Scholar). Blood samples (F, n = 9; IL-10 (+/+),n = 9; IL-10 (−/−), n = 9; and IL-10 (−/−) plus rmIL-10, n = 9) were drawn at the end of survival experiments and acidified by acetate buffer immediately. The samples were centrifuged at 2000 × g for 5 min. Equal volumes of acetate buffer and Tris buffer were added to acidified plasma and incubated at 37 °C for 20 min. The activity was assayed by Spectrolyse® tPA/PAI activity assay kits (American Diagnostica). In brief, each sample was added to reaction mixture containing a known quantity of tPA, soluble fibrin (Desafib; American Diagnostica), and a plasmin substrate (Spectrozyme PL; American Diagnostica). Plasmin generated by the reaction of tPA and fibrin cleaves the Spectrozyme substrate to generate a yellow color, which can be measured at an OD of 405 nm. PAI activity is expressed as the amount of PAI that inhibits 1 IU of tPA. Lung tissues were harvested following systemic heparinization and snap-frozen in liquid nitrogen until the time of fibrin extraction. These tissues were placed in buffer (0.05 m Tris, 0.15 m NaCl, 500 units/ml heparin, final pH 7.6) on ice and homogenized. Plasmin digestion was performed by a modification of the methods of Francis (36.Francis C.W. Marder V.J. Martin S.E. Blood. 1980; 56: 456-464Crossref PubMed Google Scholar), as described previously (34.Ranby M. Norrman B. Wallen P. Thromb. Res. 1982; 27: 743-749Abstract Full Text PDF PubMed Scopus (166) Google Scholar). Human plasmin (0.32 units/ml; Sigma) was added to the tissue homogenate, followed by agitation at 37 °C for 6 h. More plasmin (0.32 units/ml) was then added, and samples were agitated for an additional 2 h, and then the mixture was centrifuged at 2300 × g for 15 min, and the supernatant was aspirated. As a positive control, mouse fibrinogen (2.5 mg in 0.25 ml; Sigma) was clotted with human thrombin (4 units; Sigma) in Tris-buffered saline (1.75 ml) in the presence of calcium chloride (0.013 ml of 2.5 m) for 4 h at room temperature. Clotted fibrinogen was centrifuged for 5 min, and the pellet was suspended in Tris-buffered saline (1.0 ml) containing human plasmin (0.32 units/ml) and agitated at 37 °C. Additional plasmin (0.32 unit/ml) was added after 6 h, and samples were agitated for an additional 2 h. As a negative control, unclotted mouse fibrinogen was processed in an identical manner. Protein concentration of plasmin-treated lung supernatants and plasmin-treated unclotted and clotted fibrinogen solutions was measured by the Bradford method (37.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217390) Google Scholar) before loading the gel. Samples were boiled for 3 min under reducing conditions, loaded onto a SDS-polyacrylamide gel (7.5% reduced gel; 10 μg of protein/lane), and subjected to electrophoresis. Samples were electrophoretically transferred to nitrocellulose, and blots were reacted with a monoclonal anti-fibrin IgG1 (Biodesign International) that had been prepared with human fibrin-like β peptide as immunogen (38.Hui K.Y. Haber E. Matsueda G.R. Science. 1983; 222: 1129-1132Crossref PubMed Scopus (199) Google Scholar). The cross-reactivity of this antibody with murine fibrin was confirmed by blotting with the positive (murine fibrin) and negative (murine fibrinogen) controls prepared as described above. Secondary detection of sites of primary antibody localization was accomplished using a horseradish peroxidase-conjugated goat anti-mouse IgG (Fc) (Sigma). Final detection of bands was performed using the enhanced chemiluminescence Western blotting system (Amersham Pharmacia Biotech). In addition to Western blot analyses performed as described above, fibrin accumulation was determined by immunohistochemistry. Left lung tissue from untreated and IL-10 (−/−) groups, harvested in survival experiments (with antemortem heparinization to limit postmortem thrombosis) was used to identify the fibrin accumulation by immunostaining. The left lung was snap-frozen embedded in OCT compound, and sections were cut at 5 μm thick, air-dried, and acetone-fixed. Endogenous peroxidase activity was blocked by incubation for 20 min in PBS containing 0.3% hydrogen peroxide. Sections were immunostained using the same primary antibody (1:50) as that used for Western blotting, which is reactive to murine fibrin. Sites of primary antibody binding were visualized with mouse ExtraAvidin® alkaline phosphatase staining kit (Sigma) and Sigma FAST 228 FAST RED (Sigma). In order to more specifically localize fibrin deposits, a double immunostaining technique was employed on these same sections. Sections were overlaid with 20% goat serum for 30 min, washed, and then incubated for 1 h at room temperature with a rabbit polyclonal antihuman von Willebrand's antibody (Cortex Biochem, San Leandro CA). Detection of the primary antibody was accomplished using a biotinylated goat anti-rabbit IgG and the peroxidase avidin-biotin staining procedure. Immunostaining for mononuclear phagocytes was accomplished using a primary rat monoclonal anti-mouse panmacrophage marker (MOMA-2; BIOSOURCEInternational, Camarillo, CA) (39.Lloyd C.M. Minto A.W. Dorf M.E. Proudfoot A. Wells T.N. Salant D.J. Gutierrez-Ramos J.C. J. Exp. Med. 1997; 185: 1371-1380Crossref PubMed Scopus (440) Google Scholar). Development and visualization were accomplished as described above with the exception that slides were counterstained with methyl green. The number of positively stained macrophages was determined in 10 random high power fields (× 400 magnification), and the average number of macrophages/field was calculated for each group. The data were expressed as mean ± S.E. All statistical comparisons were performed using a commercially available statistical package for the Macintosh personal computer (STAT VIEW-J 5.0; Abacus Concepts). Analysis of variance was used to compare different conditions among the groups of mice. The product limit (Kaplan-Meier) estimate of the cumulative survival was assessed with the log-rank test to evaluate significa" @default.
• W2043026025 created "2016-06-24" @default.
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• W2043026025 date "2000-07-01" @default.
• W2043026025 modified "2023-10-16" @default.
• W2043026025 title "Potentiation of Endogenous Fibrinolysis and Rescue from Lung Ischemia/Reperfusion Injury in Interleukin (IL)-10-reconstituted IL-10 Null Mice" @default.
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