Immunoneutralization of Endogenous Aminoprocalcitonin Attenuates Sepsis-Induced Acute Lung Injury and Mortality in Rats
2014; Elsevier BV; Volume: 184; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2014.07.025
ISSN1525-2191
AutoresEva Tavares, Rosário Maldonado, F.J. Miñano,
Tópico(s)Neuropeptides and Animal Physiology
ResumoAcute lung injury (ALI) secondary to sepsis is a complex syndrome associated with high morbidity and mortality. We report that aminoprocalcitonin (NPCT), an endogenous peptide derived from the prohormone procalcitonin, plays a critical role in the development of ALI during severe sepsis and is a suggested risk factor for sepsis morbidity and mortality. Lethal sepsis was induced in rats by cecal ligation and puncture (CLP). Two hours after CLP, an i.p. injection of 200 μg/kg of anti-rat NPCT antibody was followed by continuous infusion of anti-NPCT (16 μg per hour) via a minipump for 18 hours. Samples were harvested 20 hours after CLP. High expressions of the CALCA gene, procalcitonin, and NPCT were detected in the lung tissue of rats with severe sepsis. Immunoneutralization of NPCT decreased pulmonary levels of CALCA, procalcitonin, and NPCT; reduced lung inflammation and injury, neutrophil infiltration, and bacterial invasion; and improved survival in sepsis. Anti-NPCT treatment also suppressed sepsis-induced inflammatory cytokine expression, cytoplasmic degradation of the inhibitor of NF-κB, IκBα, and nuclear NF-κB translocation in lung tissues. Therapeutic benefits of anti-NPCT were also associated with increased pulmonary levels of the anti-inflammatory cytokine IL-10. These data support a pathogenic role for NPCT in sepsis and suggest NPCT as a potential new target for clinical prevention and treatment of ALI in severe sepsis. Acute lung injury (ALI) secondary to sepsis is a complex syndrome associated with high morbidity and mortality. We report that aminoprocalcitonin (NPCT), an endogenous peptide derived from the prohormone procalcitonin, plays a critical role in the development of ALI during severe sepsis and is a suggested risk factor for sepsis morbidity and mortality. Lethal sepsis was induced in rats by cecal ligation and puncture (CLP). Two hours after CLP, an i.p. injection of 200 μg/kg of anti-rat NPCT antibody was followed by continuous infusion of anti-NPCT (16 μg per hour) via a minipump for 18 hours. Samples were harvested 20 hours after CLP. High expressions of the CALCA gene, procalcitonin, and NPCT were detected in the lung tissue of rats with severe sepsis. Immunoneutralization of NPCT decreased pulmonary levels of CALCA, procalcitonin, and NPCT; reduced lung inflammation and injury, neutrophil infiltration, and bacterial invasion; and improved survival in sepsis. Anti-NPCT treatment also suppressed sepsis-induced inflammatory cytokine expression, cytoplasmic degradation of the inhibitor of NF-κB, IκBα, and nuclear NF-κB translocation in lung tissues. Therapeutic benefits of anti-NPCT were also associated with increased pulmonary levels of the anti-inflammatory cytokine IL-10. These data support a pathogenic role for NPCT in sepsis and suggest NPCT as a potential new target for clinical prevention and treatment of ALI in severe sepsis. Sepsis and septic shock are among the most common causes of death in critically ill patients.1Riedemann N.C. Guo R.F. Ward P.A. The enigma of sepsis.J Clin Invest. 2003; 112: 460-467Crossref PubMed Scopus (559) Google Scholar Under those conditions, the ultimate cause of death is usually not due to progression of the infection itself, but rather is secondary to development of multiple organ dysfunctions.2Hotchkiss R.S. Karl I.E. The pathophysiology and treatment of sepsis.N Engl J Med. 2003; 348: 138-150Crossref PubMed Scopus (3199) Google Scholar The respiratory system is the most frequently affected organ system, and diffuse inflammation of lung parenchyma and severe lung dysfunction are the first steps in the development of multiple organ failure and one of the leading causes of death in sepsis. Nearly 50% of patients with severe sepsis will develop acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS).3Rubenfeld G.D. Caldwell E. Peabody E. Weaver J. Martin D.P. Neff M. Stern E.J. Hudson L.D. Incidence and outcomes of acute lung injury.N Engl J Med. 2005; 353: 1685-1693Crossref PubMed Scopus (2847) Google Scholar, 4Maybauer M.O. Maybauer D.M. Herndon D.N. Incidence and outcomes of acute lung injury.N Engl J Med. 2006; 354: 416-417Crossref PubMed Scopus (75) Google Scholar Despite significant advances in our understanding of the pathophysiological features of sepsis, ALI caused primarily by nonpulmonary sepsis remains a major clinical problem, with serious morbidity and mortality.5Matthay M.A. Zemans R.L. 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Immunoneutralization of the aminoprocalcitonin peptide of procalcitonin protects rats from lethal endotoxaemia: neuroendocrine and systemic studies.Clin Sci (Lond). 2010; 119: 519-534Crossref PubMed Scopus (31) Google Scholar ProCT is a 116–amino acid peptide that has an approximate molecular weight of 14.5 kDa and belongs to the calcitonin (CT) superfamily of peptides, including calcitonin gene-related peptides, adrenomedullin, and amylin.14Roos B.A. Okano K. Deftos L.J. Evidence for a pro-calcitonin.Biochem Biophys Res Commun. 1974; 60: 1134-1140Crossref PubMed Scopus (43) Google Scholar These peptides function as ligands for a complex family of G protein–coupled receptors consisting of the calcitonin receptor (CALCR), calcitonin receptor-like receptor (CALCRL), and three receptor-associated modified proteins.15Poyner D.R. Sexton P.M. Marshall I. Smith D.M. Quirion R. Born W. Muff R. Fischer J.A. Foord S.M. International Union of Pharmacology, XXXII: the mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors.Pharmacol Rev. 2002; 54: 233-246Crossref PubMed Scopus (686) Google Scholar ProCT is encoded by the human CALCA gene located on chromosome 11.16Steenbergh P.H. Hoppener J.W. Zandberg J. Visser A. Lips C.J. Jansz H.S. Structure and expression of the human calcitonin/CGRP genes.FEBS Lett. 1986; 209: 97-103Crossref PubMed Scopus (121) Google Scholar The original product of the CALCA gene is a 141–amino acid chain of preprocalcitonin, which is responsible for the production of ProCT. This propeptide is further processed to NPCT, immature CT, and CT carboxyl-terminus peptide-I (or katacalcin).17Le Moullec J.M. Jullienne A. Chenais J. Lasmoles F. Guliana J.M. Milhaud G. Moukhtar M.S. The complete sequence of human preprocalcitonin.FEBS Lett. 1984; 167: 93-97Crossref PubMed Scopus (210) Google Scholar CALCA gene products and their receptors are found throughout the body and are abundantly expressed in nonneuroendocrine cells of the lungs during sepsis.18Goltzman D. Mitchell J. Interaction of calcitonin and calcitonin gene-related peptide at receptor sites in target tissues.Science. 1985; 227: 1343-1345Crossref PubMed Scopus (268) Google Scholar, 19Sexton P.M. Christopoulos G. Christopoulos A. Nylen E.S. Snider R.H. Becker K.L. Procalcitonin has bioactivity at calcitonin receptor family complexes: potential mediator implications in sepsis.Crit Care Med. 2008; 36: 1637-1640Crossref PubMed Scopus (45) Google Scholar, 20Tavares E. Maldonado R. Miñano F.J. Aminoprocalcitonin-mediated suppression of feeding involves the hypothalamic melanocortin system.Am J Physiol Endocrinol Metab. 2013; 304: E1251-E1262Crossref PubMed Scopus (8) Google Scholar NPCT, unlike its precursor and CT carboxyl-terminus peptide-I, is a highly evolutionarily conserved peptide in all mammal species studied,21Russwurm S. Wiederhold M. Oberhoffer M. Stonans I. Zipfel P.F. Reinhart K. Molecular aspects and natural source of procalcitonin.Clin Chem Lab Med. 1999; 37: 787-797Crossref Scopus (98) Google Scholar which suggests an important role on a biological level. NPCT is a single-chain 57–amino acid peptide with a molecular weight of approximately 8 kDa. It was first identified in human and medullary thyroid carcinoma cells.22Conlon J.M. Grimelius L. Thim L. Structural characterization of a high molecular-mass form of calcitonin [procalcitonin-(60-116)-peptide] and its corresponding N-terminal flanking peptide [procalcitonin-(1-57)-peptide] in a human medullary thyroid carcinoma.Biochem J. 1988; 256: 245-250Crossref PubMed Scopus (28) Google Scholar, 23Burns D.M. Forstrom J.M. Friday K.E. Howard G.A. Roos B.A. Procalcitonin's amino-terminal cleavage peptide is a bone-cell mitogen.Proc Natl Acad Sci U S A. 1989; 86: 9519-9523Crossref PubMed Scopus (29) Google Scholar Subsequent studies identified NPCT as the most abundant CALCA gene product in the plasma of normal subjects and of medullary thyroid carcinoma and septic patients.24Born W. Beglinger C. Fischer J.A. Diagnostic relevance of the amino-terminal cleavage peptide of procalcitonin (PAS-57), calcitonin and calcitonin gene-related peptide in medullary thyroid carcinoma patients.Regul Pept. 1991; 32: 311-319Crossref PubMed Scopus (20) Google Scholar, 25Snider R.H. Nylén E.S. Becker K.L. Procalcitonin and its component peptides in systemic inflammation: immunochemical characterization.J Investig Med. 1997; 45: 552-560PubMed Google Scholar Recent prospective studies indicate that baseline ProCT and NPCT levels were associated with all-cause and cancer mortality in apparently healthy men with no previous history of cancer.26Cotoi O.S. Manjer J. Hedblad B. Engström G. Melander O. Schiopu A. Plasma procalcitonin is associated with all-cause and cancer mortality in apparently healthy men: a prospective population-based study.BMC Med. 2013; 11: 180Crossref PubMed Scopus (17) Google Scholar However, essentially nothing is currently known regarding the physiological and pathophysiological role of ProCT or its associated peptides as biomediators in carcinogenesis. Under normal metabolic conditions, all of the ProCT is formed in the thyroid C cells and in the pulmonary neuroendocrine cells of the lungs and is converted into CT, so that small amounts of ProCT or its associated peptides are found in the peripheral circulation of healthy subjects.27Becker K.L. Nylén E.S. White J.C. Müller B. Snider R.H. Procalcitonin and the calcitonin gene family of peptides in inflammation, infection, and sepsis: a journey from calcitonin back to its precursors.J Clin Endocrinol Metab. 2004; 89: 1512-1525Crossref PubMed Scopus (411) Google Scholar In the systemic inflammatory response syndrome (SIRS) associated with severe infections and sepsis, nonneuroendocrine tissues also express the CALCA mRNA and high amounts of ProCT and NPCT are produced from all parenchymal tissues and differentiated cells types throughout the body.28Müller B. White J.C. Nylén E.S. Snider R.H. Becker K.L. Habener J.F. Ubiquitous expression of the calcitonin-I gene in multiple tissues in response to sepsis.J Clin Endocrinol Metab. 2001; 86: 396-404Crossref PubMed Scopus (427) Google Scholar Thus, significant concentrations of these peptides can be detected in the blood of patients with severe bacterial infection/sepsis. More important, their levels persist for relatively long periods of time and correlate with sepsis severity and mortality.29Becker K.L. Snider R. Nylen E.S. Procalcitonin in sepsis and systemic inflammation: a harmful biomarker and a therapeutic target.Br J Pharmacol. 2010; 159: 253-264Crossref PubMed Scopus (207) Google Scholar Parenchymal cells (including liver, lung, kidney, adipocytes, and muscle) provide the largest tissue mass and principal source of circulating ProCT in sepsis. The inflammatory release of ProCT can be induced either directly, via microbial toxins (eg, endotoxin), or indirectly, via a humoral or cell-mediated host response (eg, IL-1β, TNF-α, and IL-6).30Oberhoffer M. Stonans I. Russwurm S. Stonane E. Vogelsang H. Junker U. Jager L. Reinhart K. Procalcitonin expression in human peripheral blood mononuclear cells and its modulation by lipopolysaccharides and sepsis-related cytokines in vitro.J Lab Clin Med. 1999; 134: 49-55Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 31Linscheid P. Seboek D. Schaer D.J. Zulewski H. Keller U. Müller B. Expression and secretion of procalcitonin and calcitonin gene-related peptide by adherent monocytes and by macrophage-activated adipocyte.Crit Care Med. 2004; 32: 1715-1721Crossref PubMed Scopus (239) Google Scholar However, the function of ProCT synthesized in the nonneuroendocrine tissues under microbial infection is presently unclear. The details of these mechanisms are unknown, and several unanswered questions that merit further studies remain, including the pathophysiological role of NPCT that circulates at high concentrations in sepsis. NPCT was initially described as a neuroendocrine peptide with bone cell mitogen activity.23Burns D.M. Forstrom J.M. Friday K.E. Howard G.A. Roos B.A. Procalcitonin's amino-terminal cleavage peptide is a bone-cell mitogen.Proc Natl Acad Sci U S A. 1989; 86: 9519-9523Crossref PubMed Scopus (29) Google Scholar Subsequent studies suggest that NPCT is also involved in the SIRS. Under normal metabolic conditions, NPCT is constitutively expressed in brain regions involved in the control of energy homeostasis.32Ojeda M.L. Ambrosiani J. Maldonado R. Tavares E. Miñano F.J. Identification and localization of procalcitonin-like immunoreactivity in the rat hypothalamus.Neurosci Lett. 2006; 408: 40-45Crossref PubMed Scopus (9) Google Scholar, 33Tavares E. Maldonado R. Miñano F.J. N-procalcitonin: central effects on feeding and energy homeostasis in rats.Endocrinology. 2007; 148: 1891-1901Crossref PubMed Scopus (10) Google Scholar It was also observed that central administration of NPCT simulates the inflammatory responses that occur in sepsis (fever, anorexia, and weight reduction) via activation of hypothalamic prostaglandin-dependent pathways,34Tavares E. Miñano F.J. Procalcitonin N-terminal peptide causes catabolic effects via the hypothalamus and prostaglandin-dependent pathways.Neuroendocrinology. 2008; 88: 316-326Crossref PubMed Scopus (6) Google Scholar and corticotrophin and melanocortin neurons, where CALCR/CALCRL are abundantly expressed.20Tavares E. Maldonado R. Miñano F.J. Aminoprocalcitonin-mediated suppression of feeding involves the hypothalamic melanocortin system.Am J Physiol Endocrinol Metab. 2013; 304: E1251-E1262Crossref PubMed Scopus (8) Google Scholar, 35Tavares E. Maldonado R. Garcia-Martinez A. Miñano F.J. Central administration of aminoprocalcitonin inhibits food intake and stimulates the hypothalamic-pituitary-adrenal axis in rats via the corticotrophin-releasing factor system.J Neuroendocrinol. 2012; 24: 1040-1054Crossref PubMed Scopus (4) Google Scholar Recent studies suggest that NPCT is actively involved as a mediator of disease progression and severity in sepsis. Prophylactic or therapeutic neutralization of endogenous NPCT with monoclonal or specific polyclonal antibodies raised against a highly conserved amino acid sequence of NPCT decreases the early production of proinflammatory cytokines (IL-1β and TNF-α), increases the late production of protective anti-inflammatory factors (IL-10 and adrenocorticotropic hormone), and reduces the mortality of rats after a lethal dose of endotoxin.13Tavares E. Miñano F.J. Immunoneutralization of the aminoprocalcitonin peptide of procalcitonin protects rats from lethal endotoxaemia: neuroendocrine and systemic studies.Clin Sci (Lond). 2010; 119: 519-534Crossref PubMed Scopus (31) Google Scholar These studies indicate that NPCT aggravates inflammatory responses and mediates immune suppression after the induction of sepsis, likely contributing to the increased mortality seen in a rat model of lethal endotoxemia. However, it remains unknown whether therapeutic neutralization of NPCT has any protective effects on severe sepsis-induced ALI and, if so, the potential mechanism(s) responsible for its benefit. We tested the hypothesis that endogenous NPCT plays a role in the pathogenesis of indirect ALI caused primarily by nonpulmonary sepsis, the first step in the development of multiple organ failure and one of the leading causes of death in sepsis. We also postulated that neutralization of endogenous NPCT attenuates severe sepsis-induced ALI. Therefore, we established a rat model of lethal sepsis induced by cecal ligation and puncture (CLP), in which bacterial dissemination and growth leads to organ damage, to assess the role of NPCT and the related molecular mechanisms associated with NPCT in modulating lung inflammatory responses. Male Wistar rats (280 to 300 g; Harlan Iberica, Barcelona, Spain) were individually housed in a temperature-controlled room on a 12-hour light/dark cycle and fed a standard rat chow diet (2014S Teklad; Harlan Iberica). Rats were acclimatized for at least 7 days before experiments. Male animals were starved for 14 hours before any intervention. Severe sepsis was induced by CLP, an animal model that has high clinical relevance to humans, because it reproduces many hallmarks of sepsis that occur in patients.36Hubbard W.J. Choudhry M. Schwacha M.G. Kerby J.D. Rue 3rd, L.W. Bland K.I. Chaudry I.H. Cecal ligation and puncture.Shock. 2005; 24: 52-57Crossref PubMed Scopus (571) Google Scholar, 37Rittirsch D. Huber-Lang M.S. Flierl M.A. Ward P.A. Immunodesign of experimental sepsis by cecal ligation and puncture.Nat Protoc. 2009; 4: 31-36Crossref PubMed Scopus (1308) Google Scholar To perform this procedure, rats were anesthetized with an i.p. mixture of 100 mg/kg ketamine and 5 mg/kg xylazine (Sigma-Aldrich, St. Louis, MO). After shaving the abdominal fur and preparing the abdominal wall with 10% povidone-iodine solution, cecum was exposed via a 2-cm ventral midline abdominal incision and ligated below the ileocecal valve to avoid intestinal obstruction. The ligated part of the cecum was then punctured twice with a 16-gauge needle, and a small amount of cecal contents was expressed through the punctures. After repositioning of the bowel, the incision was closed, and 3 mL/100 g body weight of prewarmed sterile saline was administered s.c. No antibiotics were administered. Sham-operated on animals (ie, control animals) underwent the same procedure with the exception that the cecum was neither ligated nor punctured. To ensure consistency, the same surgeon (R.M.) performed all of the above manipulations. Postoperatively, all rats had unlimited access to food and water. Animals were sacrificed 20 hours after CLP or sham operation for collection of tissue samples. All experiments were performed in accordance with the Council of the European Communities' Directive (86/609/European Economic Community) and Spanish regulations (Boletin Oficial del Estado/67:8509/1988) for the use of experimental animals. The Animal Care and Use Committee of the University of Seville (Seville, Spain) approved the protocol. To examine an involvement of NPCT in sepsis-induced lung inflammation, a rabbit anti-rat antibody raised against a highly conserved terminal region of human and rat NPCT (amino acids 44 to 57) was used. The ability of this antibody to block the in vivo activity of endogenous NPCT in rats and its specificity on rat brain sections by preabsorption with the peptide have been previously reported.20Tavares E. Maldonado R. Miñano F.J. Aminoprocalcitonin-mediated suppression of feeding involves the hypothalamic melanocortin system.Am J Physiol Endocrinol Metab. 2013; 304: E1251-E1262Crossref PubMed Scopus (8) Google Scholar, 35Tavares E. Maldonado R. Garcia-Martinez A. Miñano F.J. Central administration of aminoprocalcitonin inhibits food intake and stimulates the hypothalamic-pituitary-adrenal axis in rats via the corticotrophin-releasing factor system.J Neuroendocrinol. 2012; 24: 1040-1054Crossref PubMed Scopus (4) Google Scholar Control rabbit nonimmune IgG was used as control (Sigma-Aldrich). Anti-NPCT and control IgG were dissolved in phosphate-buffered saline (PBS; pH 7.4) to a final concentration of 2.0 mg/mL. All solutions were passed through 0.22-μm pore-size Millipore filters and stored at −80°C. The same batches and solutions were used for all experiments and contained an endotoxin level <0.1 ng/mL of protein (ie, lower than the detection limit of endotoxin assay) measured by the Limulus amebocyte lysate method, as previously described.13Tavares E. Miñano F.J. Immunoneutralization of the aminoprocalcitonin peptide of procalcitonin protects rats from lethal endotoxaemia: neuroendocrine and systemic studies.Clin Sci (Lond). 2010; 119: 519-534Crossref PubMed Scopus (31) Google Scholar A 200-μL Alzet osmotic minipump (8 μL per hour delivery rate; model 2001D; Durect, Cupertino, CA) was placed intradermally in the peritoneal skin and a polyethylene catheter of the pump was inserted into the peritoneum at the time of surgery. Before implantation, pumps were prefilled (2.0 μg protein/μL) with anti-NPCT antibody or control IgG solution and primed in sterile PBS for 2 hours at 37°C. After an i.p. injection of 200 μg/kg of the appropriate antibody 2 hours after CLP, the minipump was then connected to the catheter inserted into the peritoneum. The total dose of anti-NPCT or control IgG each rat received was approximately 1200 μg/kg body weight. The concentration of anti-NPCT was determined by performing a dose-response curve for the antibody in a separate set of animals and by selecting the concentration that significantly improved survival 24 hours after CLP (Supplemental Figure S1). We randomly divided animals into three groups (n = 10 per group): i) control sham-operated on animals, ii) control IgG antibody plus CLP, and iii) anti-NPCT plus CLP. Because severe sepsis and multiple organ dysfunctions occurred at 20 hours after CLP,38Baker C.C. Chaudry I.H. Gaines H.O. Baue A.E. Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model.Surgery. 1983; 94: 331-335PubMed Google Scholar we chose this time point to assess lung injury. Twenty hours after CLP (ie, 18 hours after implantation of the minipump), the rats were anesthetized. Cardiac puncture was used for collecting blood samples, and peritoneal fluid was collected as described below. Then, the lungs were surgically exposed and allowed to collapse freely, after which the trachea was carefully intubated. The tracheal cannula was connected to a syringe and a water-graded column, and the lungs were inflated with air at a pressure of 15 to 20 cm H2O. Care was taken to prevent overinflation and alveolar damage. The main bronchus was tied and the cardiopulmonary tract, including the lower part of the trachea, was carefully extracted from the rib cage and placed in a sterile ice-cold Ringer's lactate solution. The heart was withdrawn, and the entire lung was carefully removed to avoid pleural rupture and blood contamination of the airways. The upper and lower lobes of the left lung were then divided. The lower lobes of the left lung were snap frozen in liquid nitrogen and stored at −80°C for gene expression analysis, protein extraction, cytokine determination, and myeloperoxidase (MPO) activity measurement. The left upper lobes were used to obtain the wet/dry (W/D) ratio. Right lungs were used for histological analysis. The survival rate was also investigated in separate experiments. The lung water content was assessed by the W/D weight ratio. Freshly harvested upper lobe of left lung was washed three times with PBS at 4°C to remove residual blood, gently dried using blotting paper, and weighed to obtain the wet lung weight (W). The wet lungs were then dried in a drying oven at 70°C for 48 hours, after which they were weighed again to obtain the dry lung weight (D). The W/D ratio was calculated as follows:W/Dratio(%)=[(wetweight−dryweight)/dryweight]×100.(1) For histological evaluation, the right lung was dissected and fixed immediately in 10% neutral-buffered formalin, embedded in sucrose, frozen in dry ice using optimum cutting temperature medium, cryosectioned (5 μm thick), and stained with hematoxylin and eosin. All of the procedures were performed according to previously published methods.39Gonzalez-Rey E. Chorny A. Robledo G. Delgado M. Cortistatin, a new antiinflammatory peptide with therapeutic effect on lethal endotoxemia.J Exp Med. 2006; 203: 563-571Crossref PubMed Scopus (104) Google Scholar A pathologist who was blinded to the experimental conditions examined all of the sections. The histopathological evaluation was performed under a light microscope using a grid system. A scoring system to grade the degree of lung injury was used, on the basis of the following histological features: edema, hyperemia and congestion, neutrophil margination and tissue infiltration, intra-alveolar hemorrhage and debris, and cellular hyperplasia. Each feature was graded from 0 to 3 on the basis of its absence (0) or presence to a mild (1), moderate (2), or severe (3) degree.40Bachofen M. Weibel E.R. Structural alterations of lung parenchyma in the adult respiratory distress syndrome.Clin Chest Med. 1982; 3: 35-56Abstract Full Text PDF PubMed Google Scholar Overall lung injury was categorized according to the sum of the score (0 to 3: normal to minimal injury; 4 to 6: mild injury; 7 to 9: moderate injury; 10 to 12: severe injury). The maximum possible score per animal was 12. PMN accumulation in lungs was quantitated by counting PMNs at the alveolar septal walls in five randomly selected nonoverlapping fields from each stained section of the individual lungs. PMNs were identified as cells with multilobed nuclei exhibiting lighter-red nuclear staining than surrounding the cells. PMN entrapment was expressed as the mean number of PMNs per 10 high-powered fields at ×1000 magnification. MPO activity, an indicator of PMN accumulation in lungs, was determined as previously described.39Gonzalez-Rey E. Chorny A. Robledo G. Delgado M. Cortistatin, a new antiinflammatory peptide with therapeutic effect on lethal endotoxemia.J Exp Med. 2006; 203: 563-571Crossref PubMed Scopus (104) Google Scholar Lung tissues were weighed and homogenized (50 mg/mL) in 50 mmol/L potassium phosphate (pH 6) containing 5 mmol/L hexadecyltrimethylammonium bromide (Sigma-Aldrich) and subjected to three cycles of freezing and thawing, followed by centrifugation at 30,000 × g for 20 minutes at 4°C to obtain the supernatant. MPO activity was determined by combining 20 μL tissue supernatant with 60 μL assay buffer containing 0.167 mg/mL O-dianisidine dihydrochloride (Sigma-Aldrich) and 0.0005% hydrogen peroxide. The MPO activity was measured using a biophotometer spectrophotometer at 450 nm (Thomas Scientific, Swedesboro, NJ). The MPO activity was calculated as the change in absorbance (ΔA) at 450 nm over 1 minute and expressed as ΔA450/minute per gram of protein. Blood and peritoneal fluid were obtained as described above for bacterial load. Whole blood for PMN count was collected into sterile tubes containing potassium EDTA and counted on an automated hematology cell counter (Cell-Dyn 3500; Abbott Laboratories, Abbott Park, IL), as previously described.41Tavares E. Miñano F.J. Maldonado R. Dascombe M.J. Endotoxin fever in granulocytopenic rats: evidence that brain cyclooxygenase-2 is more important than circulating prostaglandin E(2).J Leukoc Biol. 2006; 80: 1375-1387Crossref PubMed Scopus (15) Google Scholar Peritoneal fluid was mixed with Turks solution (0.2 mg methylene blue in 1 mL glacial acetic acid, 6.25% v/v) in a 1:10 dilution. PMNs
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