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• W2039838662 abstract "CX3CR1 is expressed on monocytes, dendritic cells, macrophages, subsets of T lymphocytes, and natural killer cells and functions in diverse capacities such as leukocyte adhesion, migration, and cell survival on ligand binding. Expression of the CX3CL1 gene, whose expression product is the sole ligand for CX3CR1, is up-regulated in human lungs with chronic cigarette smoke-induced obstructive lung disease. At present, it is unknown whether CX3CL1 up-regulation is associated with the recruitment and accumulation of immune cells that express CX3CR1. We show that mice chronically exposed to cigarette smoke up-regulate CX3CL1 gene expression, which is associated with an influx of CX3CR1+ cells in the lungs. The increase in CX3CR1+ cells is primarily comprised of macrophages and T lymphocytes and is associated with the development of emphysema. In alveolar macrophages, cigarette smoke exposure increased the expression of both CX3CR1 and CX3CL1 genes. The inducibility of CX3CR1 expression was not solely dependent on a chronic stimulus because lipopolysaccharide up-regulated CX3CR1 in RAW264.7 cells in vitro and in mononuclear phagocytes in vivo. Our findings suggest a mechanism by which macrophages amplify and promote CX3CR1+ cell accumulation within the lungs during both acute and chronic inflammatory stress. We suggest that one function of the CX3CR1-CX3CL1 pathway is to recruit and sustain divergent immune cell populations implicated in the pathogenesis of cigarette smoke-induced emphysema. CX3CR1 is expressed on monocytes, dendritic cells, macrophages, subsets of T lymphocytes, and natural killer cells and functions in diverse capacities such as leukocyte adhesion, migration, and cell survival on ligand binding. Expression of the CX3CL1 gene, whose expression product is the sole ligand for CX3CR1, is up-regulated in human lungs with chronic cigarette smoke-induced obstructive lung disease. At present, it is unknown whether CX3CL1 up-regulation is associated with the recruitment and accumulation of immune cells that express CX3CR1. We show that mice chronically exposed to cigarette smoke up-regulate CX3CL1 gene expression, which is associated with an influx of CX3CR1+ cells in the lungs. The increase in CX3CR1+ cells is primarily comprised of macrophages and T lymphocytes and is associated with the development of emphysema. In alveolar macrophages, cigarette smoke exposure increased the expression of both CX3CR1 and CX3CL1 genes. The inducibility of CX3CR1 expression was not solely dependent on a chronic stimulus because lipopolysaccharide up-regulated CX3CR1 in RAW264.7 cells in vitro and in mononuclear phagocytes in vivo. Our findings suggest a mechanism by which macrophages amplify and promote CX3CR1+ cell accumulation within the lungs during both acute and chronic inflammatory stress. We suggest that one function of the CX3CR1-CX3CL1 pathway is to recruit and sustain divergent immune cell populations implicated in the pathogenesis of cigarette smoke-induced emphysema. Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality in the United States and is the only leading cause of death that continues to increase in prevalence.1MacKenzie TD Bartecchi CE Schrier RW The human costs of tobacco use (2).N Engl J Med. 1994; 330: 975-980Crossref PubMed Scopus (187) Google Scholar, 2Murray CJ Lopez AD Alternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study.Lancet. 1997; 349: 1498-1504Abstract Full Text Full Text PDF PubMed Scopus (5640) Google Scholar The disease is characterized by a local inflammatory process that is associated with formation of mucous exudates within the lumens of small airways3Hogg JC Chu F Utokaparch S Woods R Elliott WM Buzatu L Cherniack RM Rogers RM Sciurba FC Coxson HO Pare PD The nature of small-airway obstruction in chronic obstructive pulmonary disease.N Engl J Med. 2004; 350: 2645-2653Crossref PubMed Scopus (2894) Google Scholar and lung parenchymal destruction leading to airspace enlargement.4Shapiro SD Ingenito EP The pathogenesis of chronic obstructive pulmonary disease: advances in the past 100 years.Am J Respir Cell Mol Biol. 2005; 32: 367-372Crossref PubMed Scopus (176) Google Scholar Cigarette smoking is the major risk factor for the development of COPD, and COPD severity in smokers is associated with the accumulation of neutrophils,5Finkelstein R Fraser RS Ghezzo H Cosio MG Alveolar inflammation and its relation to emphysema in smokers.Am J Respir Crit Care Med. 1995; 152: 1666-1672Crossref PubMed Scopus (381) Google Scholar macrophages,6Shapiro SD The macrophage in chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 1999; 160: S29-S32Crossref PubMed Scopus (267) Google Scholar natural killer (NK) cells,7Di Stefano A Capelli A Lusuardi M Balbo P Vecchio C Maestrelli P Mapp CE Fabbri LM Donner CF Saetta M Severity of airflow limitation is associated with severity of airway inflammation in smokers.Am J Respir Crit Care Med. 1998; 158: 1277-1285Crossref PubMed Scopus (464) Google Scholar and T lymphocytes8O'Shaughnessy TC Ansari TW Barnes NC Jeffery PK Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1.Am J Respir Crit Care Med. 1997; 155: 852-857Crossref PubMed Scopus (608) Google Scholar, 9Saetta M Di Stefano A Turato G Facchini FM Corbino L Mapp CE Maestrelli P Ciaccia A Fabbri LM CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 1998; 157: 822-826Crossref PubMed Scopus (666) Google Scholar, 10Saetta M Baraldo S Corbino L Turato G Braccioni F Rea F Cavallesco G Tropeano G Mapp CE Maestrelli P Ciaccia A Fabbri LM CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 1999; 160: 711-717Crossref PubMed Scopus (406) Google Scholar in the peripheral airways and lung parenchyma. In particular, T lymphocyte and macrophage accumulation has been associated with worsening obstructive lung disease. These cells can release inflammatory mediators and/or proteinases which are thought to be responsible for the progressive parenchymal destruction in COPD.11Shapiro SD Goldstein NM Houghton AM Kobayashi DK Kelley D Belaaouaj A Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice.Am J Pathol. 2003; 163: 2329-2335Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 12Shapiro SD Senior RM Matrix metalloproteinases. Matrix degradation and more.Am J Respir Cell Mol Biol. 1999; 20: 1100-1102Crossref PubMed Scopus (202) Google Scholar The factors that regulate the recruitment and accumulation of these immune cells into the lungs in response to chronic cigarette smoke exposure remain poorly understood. Recent studies suggest that chemokines and chemokine receptors play important roles in the recruitment of specific immune cell populations to the lungs. In smokers with COPD, increased CCL20 expression in the small airways has been shown to be associated with the accumulation of dendritic cells when compared to never smokers and to smokers without COPD.13Demedts IK Bracke KR Van Pottelberge G Testelmans D Verleden GM Vermassen FE Joos GF Brusselle GG Accumulation of dendritic cells and increased CCL20 levels in the airways of patients with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2007; 175: 998-1005Crossref PubMed Scopus (206) Google Scholar Bracke and colleagues14Bracke KR D'Hulst AI Maes T Moerloose KB Demedts IK Lebecque S Joos GF Brusselle GG Cigarette smoke-induced pulmonary inflammation and emphysema are attenuated in CCR6-deficient mice.J Immunol. 2006; 177: 4350-4359PubMed Google Scholar recently showed that CCR6, the receptor for CCL20, contributes to cigarette smoke-induced inflammation and emphysema in mice. Other studies demonstrated that there is an increase in the number of CXCR3+ T lymphocytes in the peripheral airways of smokers with COPD15Saetta M Mariani M Panina-Bordignon P Turato G Buonsanti C Baraldo S Bellettato CM Papi A Corbetta L Zuin R Sinigaglia F Fabbri LM Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2002; 165: 1404-1409Crossref PubMed Scopus (322) Google Scholar and an increase in the secretion of CXCR3 ligands, CXCL9 and CXCL10, in the lungs.16Grumelli S Corry DB Song LZ Song L Green L Huh J Hacken J Espada R Bag R Lewis DE Kheradmand F An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema.PLoS Med. 2004; 1: e8Crossref PubMed Scopus (391) Google Scholar Although CCR6 and CXCR3 may be involved in immune cell accumulation in the peripheral airways of individuals with COPD, we do not have a complete understanding of the factors that regulate the recruitment of specific cell populations in COPD lungs. Recent comprehensive gene expression profiling has shown an increase in CX3CL1 (also known as fractalkine or mouse neurotactin) expression in the lung tissues of smokers with COPD when compared to smokers without COPD.17Ning W Li CJ Kaminski N Feghali-Bostwick CA Alber SM Di YP Otterbein SL Song R Hayashi S Zhou Z Pinsky DJ Watkins SC Pilewski JM Sciurba FC Peters DG Hogg JC Choi AM Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease.Proc Natl Acad Sci USA. 2004; 101: 14895-14900Crossref PubMed Scopus (285) Google Scholar Among the 327 differentially expressed genes, CX3CL1 was the only chemokine found to be up-regulated.17Ning W Li CJ Kaminski N Feghali-Bostwick CA Alber SM Di YP Otterbein SL Song R Hayashi S Zhou Z Pinsky DJ Watkins SC Pilewski JM Sciurba FC Peters DG Hogg JC Choi AM Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease.Proc Natl Acad Sci USA. 2004; 101: 14895-14900Crossref PubMed Scopus (285) Google Scholar CX3CL1 is a unique molecule that exists as both a membrane-bound protein and a soluble chemokine.18Bazan JF Bacon KB Hardiman G Wang W Soo K Rossi D Greaves DR Zlotnik A Schall TJ A new class of membrane-bound chemokine with a CX3C motif.Nature. 1997; 385: 640-644Crossref PubMed Scopus (1697) Google Scholar The membrane-anchored protein is expressed on inflamed endothelium,19Imai T Hieshima K Haskell C Baba M Nagira M Nishimura M Kakizaki M Takagi S Nomiyama H Schall TJ Yoshie O Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1160) Google Scholar, 20Ancuta P Rao R Moses A Mehle A Shaw SK Luscinskas FW Gabuzda D Fractalkine preferentially mediates arrest and migration of CD16+ monocytes.J Exp Med. 2003; 197: 1701-1707Crossref PubMed Scopus (451) Google Scholar epithelial cells,21Lucas AD Chadwick N Warren BF Jewell DP Gordon S Powrie F Greaves DR The transmembrane form of the CX3CL1 chemokine fractalkine is expressed predominantly by epithelial cells in vivo.Am J Pathol. 2001; 158: 855-866Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar dendritic cells,22Papadopoulos EJ Sassetti C Saeki H Yamada N Kawamura T Fitzhugh DJ Saraf MA Schall T Blauvelt A Rosen SD Hwang ST Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is up-regulated upon dendritic cell maturation.Eur J Immunol. 1999; 29: 2551-2559Crossref PubMed Scopus (147) Google Scholar and neurons23Harrison JK Jiang Y Chen S Xia Y Maciejewski D McNamara RK Streit WJ Salafranca MN Adhikari S Thompson DA Botti P Bacon KB Feng L Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia.Proc Natl Acad Sci USA. 1998; 95: 10896-10901Crossref PubMed Scopus (907) Google Scholar and can mediate leukocyte adhesion.19Imai T Hieshima K Haskell C Baba M Nagira M Nishimura M Kakizaki M Takagi S Nomiyama H Schall TJ Yoshie O Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.Cell. 1997; 91: 521-530Abstract Full Text Full Text PDF PubMed Scopus (1160) Google Scholar Soluble CX3CL1 is released from the cell membrane through proteolytic cleavage and can function as an effective chemoattractant.24Yoshikawa M Nakajima T Matsumoto K Okada N Tsukidate T Iida M Otori N Haruna S Moriyama H Imai T Saito H TNF-alpha and IL-4 regulate expression of fractalkine (CX3CL1) as a membrane-anchored proadhesive protein and soluble chemotactic peptide on human fibroblasts.FEBS Lett. 2004; 561: 105-110Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 25Kanazawa N Nakamura T Tashiro K Muramatsu M Morita K Yoneda K Inaba K Imamura S Honjo T Fractalkine and macrophage-derived chemokine: T cell-attracting chemokines expressed in T cell area dendritic cells.Eur J Immunol. 1999; 29: 1925-1932Crossref PubMed Scopus (91) Google Scholar CX3CR1, the sole receptor for CX3CL1, is a seven-transmembrane, G-protein-coupled receptor that is expressed by cytotoxic effector CD8+ and CD4+ T lymphocytes in addition to γδ T lymphocytes, NK cells, dendritic cells, and monocytes.26Niess JH Brand S Gu X Landsman L Jung S McCormick BA Vyas JM Boes M Ploegh HL Fox JG Littman DR Reinecker HC CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance.Science. 2005; 307: 254-258Crossref PubMed Scopus (1287) Google Scholar, 27Combadiere C Salzwedel K Smith ED Tiffany HL Berger EA Murphy PM Identification of CX3CR1. A chemotactic receptor for the human CX3C chemokine fractalkine and a fusion coreceptor for HIV-1.J Biol Chem. 1998; 273: 23799-23804Crossref PubMed Scopus (255) Google Scholar, 28Combadiere C Gao J Tiffany HL Murphy PM Gene cloning, RNA distribution, and functional expression of mCX3CR1, a mouse chemotactic receptor for the CX3C chemokine fractalkine.Biochem Biophys Res Commun. 1998; 253: 728-732Crossref PubMed Scopus (58) Google Scholar Although the role of the CX3CL1-CX3CR1 pathway in the inflammatory process of COPD has never been evaluated, this ligand-receptor pair has been investigated in other inflammatory disease states. Local and systemic inflammation play critical roles in the development of atherosclerotic plaques, and macrophages are prominent in these lesions. The deletion of cx3cl1 on an apoE−/− background decreased the size of brachiocephalic artery atherosclerotic lesions by 85% when compared to the wild-type mice,29Teupser D Pavlides S Tan M Gutierrez-Ramos JC Kolbeck R Breslow JL Major reduction of atherosclerosis in fractalkine (CX3CL1)-deficient mice is at the brachiocephalic artery, not the aortic root.Proc Natl Acad Sci USA. 2004; 101: 17795-17800Crossref PubMed Scopus (170) Google Scholar and the deletion of cx3cr1 on an apoE−/− background afforded significant protection from macrophage recruitment and atherosclerotic plaque formation.30Lesnik P Haskell CA Charo IF Decreased atherosclerosis in CX3CR1−/− mice reveals a role for fractalkine in atherogenesis.J Clin Invest. 2003; 111: 333-340Crossref PubMed Scopus (414) Google Scholar These results suggest that CX3CR1+ macrophages are playing a vital role in the development of early atherosclerotic lesions. Bjerkeli and colleagues31Bjerkeli V Damas JK Fevang B Holter JC Aukrust P Froland SS Increased expression of fractalkine (CX3CL1) and its receptor. CX3CR1, in Wegener's granulomatosis possible role in vascular inflammation.Rheumatology (Oxford). 2007; 46: 1422-1427Crossref PubMed Scopus (36) Google Scholar showed that patients with Wegener's granulomatosis have increased levels of plasma CX3CL1 as well as enhanced gene expression of CX3CR1 in peripheral blood mononuclear cells. In addition, CX3CR1+ peripheral blood mononuclear cells and T lymphocytes from patients with Wegener's granulomatosis secreted more inflammatory chemokines and showed increased chemotactic and adhesive responses to CX3CL1.31Bjerkeli V Damas JK Fevang B Holter JC Aukrust P Froland SS Increased expression of fractalkine (CX3CL1) and its receptor. CX3CR1, in Wegener's granulomatosis possible role in vascular inflammation.Rheumatology (Oxford). 2007; 46: 1422-1427Crossref PubMed Scopus (36) Google Scholar Yano and colleagues32Yano R Yamamura M Sunahori K Takasugi K Yamana J Kawashima M Makino H Recruitment of CD16+ monocytes into synovial tissues is mediated by fractalkine and CX3CR1 in rheumatoid arthritis patients.Acta Med Okayama. 2007; 61: 89-98PubMed Google Scholar found high levels of soluble CX3CL1 in the synovial fluid of patients with rheumatoid arthritis, and CD16+ monocytes, expressing high levels of CX3CR1, were localized to the synovial lining and sublining layers. These authors suggest that CX3CL1 may promote the migration of CD16+/CX3CR1+ monocytes into the synovial tissues of patients with rheumatoid arthritis and may contribute to the tissue injury and joint destruction.32Yano R Yamamura M Sunahori K Takasugi K Yamana J Kawashima M Makino H Recruitment of CD16+ monocytes into synovial tissues is mediated by fractalkine and CX3CR1 in rheumatoid arthritis patients.Acta Med Okayama. 2007; 61: 89-98PubMed Google Scholar Given these findings, we hypothesized that CX3CR1+ cells comprise the inflammatory response in the lungs to chronic cigarette smoke exposure. We used a murine model of environmental tobacco smoke exposure and show increases in CX3CR1 expression in the lungs of animals with emphysema. We immunophenotyped CX3CR1+ cells to the T-lymphocyte and macrophage populations and demonstrate the inducibility of CX3CR1 expression by resident mononuclear phagocytes of the lungs during both acute and chronic inflammatory stimuli. A detailed method of our chronic cigarette exposure method has been previously described.33Slebos DJ Ryter SW van der Toorn M Liu F Guo F Baty CJ Karlsson JM Watkins SC Kim HP Wang X Lee JS Postma DS Kauffman HF Choi AM Mitochondrial localization and function of heme oxygenase-1 in cigarette smoke-induced cell death.Am J Respir Cell Mol Biol. 2007; 36: 409-417Crossref PubMed Scopus (191) Google Scholar Briefly, total body cigarette smoke exposure was performed in a stainless steel chamber using a smoking machine (model TE-10; Teague Enterprises, Woodland, CA) similar to that reported by others.34Rangasamy T Cho CY Thimmulappa RK Zhen L Srisuma SS Kensler TW Yamamoto M Petrache I Tuder RM Biswal S Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice.J Clin Invest. 2004; 114: 1248-1259Crossref PubMed Scopus (810) Google Scholar, 35Witschi H Espiritu I Maronpot RR Pinkerton KE Jones AD The carcinogenic potential of the gas phase of environmental tobacco smoke.Carcinogenesis. 1997; 18: 2035-2042Crossref PubMed Scopus (131) Google Scholar, 36Witschi H Espiritu I Peake JL Wu K Maronpot RR Pinkerton KE The carcinogenicity of environmental tobacco smoke.Carcinogenesis. 1997; 18: 575-586Crossref PubMed Scopus (141) Google Scholar The cigarette smoking machine puffed each 1R3F research cigarette for 2 seconds, for a total of nine puffs before ejection, at a flow rate of 1.05 L/minute, providing a standard puff of 35 cm3. The smoke machine was adjusted to deliver five cigarettes at one time. The cigarette smoke from 80 cigarettes was delivered to the mice each day, 5 days/week for up to 24 weeks. The daily exposure time was ∼3 to 4 hours per day. Age-, gender-, and strain-matched mice served as controls and were exposed to only filtered air in an identical body exposure chamber for a similar period of time. The smoking chamber atmosphere was periodically measured for total particulate matter concentrations of ∼100 to 120 mg/m3. After 2 weeks of cigarette exposure, carboxyhemoglobin levels in mice, immediately after exposure, were less than 8%. The body weights of the mice were measured weekly. The animals were sacrificed at 12- and 24-week time points. Animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee at the University of Pittsburgh. At 8 weeks of age, male AKR/J mice (The Jackson Laboratory, Bar Harbor, ME) were divided into air-exposed control animals and cigarette-exposed animals. The animals underwent the chronic exposure model as described above. Age-, gender-, and strain-matched male B6.129P-cx3cr1tm1Litt/J mice (The Jackson Laboratory) were also used for immunofluorescence studies of the lungs. B6.129P-cx3cr1tm1Litt/J mice were homozygous for a targeted mutation at the cx3cr1 loci and expressed the enhanced green fluorescent protein (EGFP) instead of the endogenous gene.37Jung S Aliberti J Graemmel P Sunshine MJ Kreutzberg GW Sher A Littman DR Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.Mol Cell Biol. 2000; 20: 4106-4114Crossref PubMed Scopus (1857) Google Scholar The method of mouse necropsy has been previously described.38Lee JS Wurfel MM Matute-Bello G Frevert CW Rosengart MR Ranganathan M Wong VW Holden T Sutlief S Richmond A Peiper S Martin TR The Duffy antigen modifies systemic and local tissue chemokine responses following lipopolysaccharide stimulation.J Immunol. 2006; 177: 8086-8094PubMed Google Scholar Briefly, animals were euthanized with an intraperitoneal, overdose injection of pentobarbital of 100 mg/kg. The thorax was opened, and the trachea was identified and cannulated with a 20-gauge angiocatheter. The left hilum was identified and ligated with a 2-0 silk suture. The left lung was removed and immediately frozen in liquid nitrogen for subsequent RNA studies. The right lung was inflated by gravity with 4% paraformaldehyde in phosphate-buffered saline (PBS) and was held at a pressure of 30 cm H2O for 15 minutes. The right lung was gently dissected from the thorax and placed in 4% paraformaldehyde in PBS for 6 to 8 hours. The samples were cut para-sagittally and embedded in paraffin. Paraffin-embedded sections were cut to ∼5 μm thickness. To obtain whole lungs for frozen tissue sections, the trachea was cannulated with an 18-gauge angiocatheter and up to 2.5 ml of Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA) was slowly instilled into both lungs. The trachea was ligated with a 2-0 silk suture, and the lungs were carefully dissected from the thorax. The instilled lungs were mounted in O.C.T. compound, and cryomolds were slowly frozen in liquid nitrogen. Frozen sections were cryosectioned to ∼5 μm thickness. The animals were euthanized, and the thorax was opened as described above. The trachea was identified and cannulated with an 18-gauge angiocatheter, and sequential lavages of 1.2 ml, 1.0 ml, 1.0 ml, and 1.0 ml of BAL fluid (0.9% saline with 0.6 mmol/L ethylenediaminetetraacetic acid warmed to 37°C) were instilled into the lungs. The fluid was allowed to sit for ∼30 seconds before it was slowly removed from the lungs. The four aliquots were pooled for analysis. Cytospin slides were created using 150-μl aliquots and the Shandon Cytospin 3 centrifuge (Thermo Fisher Scientific, Inc., Waltham, MA). Manual cell counts and differentials were performed and confirmed that 97 to 99% of the BAL cells were macrophages. Human lung tissue samples were obtained through the University of Pittsburgh Lung Tissue Center and were approved by University of Pittsburgh Institution Review Board. Sections of uninflated human lung tissue were mounted in O.C.T. compound and cryomolds were slowly frozen in liquid nitrogen. The specimens were stored at −80°C until needed. The frozen samples were cryosectioned to ∼5 μm thickness. Paraffin-embedded mouse lung sections were used for quantitative morphometric measurements. The paraffin was melted at 55°C for 20 minutes, and the slide was washed several times in xylene. The samples were rehydrated slowly in graded ethanol washes and then stained overnight in equal amounts of Gills solution (GHS-332; Sigma-Aldrich, St. Louis, MO) and Harris hematoxylin solution (HHS-32, Sigma-Aldrich). After several washes with water and ammonium hydroxide and with increasing concentrations of ethanol, the slides were allowed to air-dry. After the coverslips were applied, digital images were obtained with the use of a light microscope (Zeiss Axiophot; Carl Zeiss MicroImaging, Thornwood, NY) equipped with a digital camera (Zeiss Axiocam HRc, Carl Zeiss MicroImaging). Twelve random images were taken at ×200 from seven smoke-exposed and five air-exposed animals and saved as tagged image file format (TIFF) images. Mean linear intercept measurements were taken using modified Image J software available from the National Institutes of Health website (http://rsb.info.nih.gov/ij/). Twelve black and white pictures for each animal were saved as 1300 × 1030 pixel digital images. Large airways, blood vessels, and other nonalveolar debris were manually removed from the image. The Image J software program automatically thresholded the images, and a median filter, set to a 2 pixel radius, smoothed the image edges. The program laid a line grid composed of 1353 lines (each line measuring 21 pixels) over the individual images. The software then counted the number of lines that ended on or intercepted alveolar tissue. These data were used to calculate the volume of air, the volume of tissue, the surface area, the surface area to tissue volume ratio. The mean linear intercept, which assesses the degree of alveolar airspace enlargement, was calculated using the equation: (4/surface area to volume tissue ratio) according to the methods adapted from Dunhill.39Dunnill M Quantitative methods in the study of pulmonary pathology.Thorax. 1962; 17: 320-328Crossref Scopus (388) Google Scholar The mean linear intercept increases with increasing airspace enlargement. We examined airway thickening morphometrically based on our previously published methods40Lee JS Frevert CW Matute-Bello G Wurfel MM Wong VA Lin SM Ruzinski J Mongovin S Goodman RB Martin TR TLR-4 pathway mediates the inflammatory response but not bacterial elimination in E. coli pneumonia.Am J Physiol. 2005; 289: L731-L738Google Scholar and those of George and colleagues41George CL Jin H Wohlford-Lenane CL O'Neill ME Phipps JC O'Shaughnessy P Kline JN Thorne PS Schwartz DA Endotoxin responsiveness and subchronic grain dust-induced airway disease.Am J Physiol. 2001; 280: L203-L213Google Scholar Briefly, paraffin-embedded lung sections prepared as described above were stained with hematoxylin and eosin. Airways were identified in sections at ×400, and images were captured digitally as described above. The analysis was performed with Metamorph Offline software, version 7.1.0.0 (Molecular Devices Corp., Sunnyvale, CA). Obliquely cut airways showing a length to width ratio >2.0 were excluded from analysis. Airways were divided into three groups based on diameter: 1) small airways 90 μm or less in diameter; 2) medium airways 90 to 129 μm in diameter; and 3) large airways >129 μm in diameter. Airway epithelial area and subepithelial area were calculated for each airway by defining the internal perimeter, external perimeter, and basement perimeter. Immunostaining was performed using rabbit polyclonal anti-CX3CR1, rabbit polyclonal anti-prosurfactant protein C (Abcam, Cambridge, MA); mouse monoclonal anti-CD68 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); rat anti-mouse MAC3 and Armenian hamster anti-mouse CD3ε (BD Biosciences Pharmingen, San Diego, CA); rat monoclonal anti-Gr1 (Cedarlane, Ontario, Canada); Armenian hamster anti-mouse CD11c (eBioscience, San Diego, CA); rat anti-mouse CD11b (clone M1/70.15; MorphoSys US Inc., Raleigh, NC). Immunostaining using control antibodies rabbit IgG, mouse IgG, rat IgG, and/or Armenian hamster IgG was performed on serial lung sections. Secondary antibodies to rabbit, goat, mouse, rat, and Armenian hamster were conjugated with Cy3 or Alexa488. Paraffin-embedded sections were deparaffinized by warming the slides to 55°C on a slide warmer for 10 minutes and then washing the slides several times in xylene. Graded ethanol washes were used to rehydrate the samples, and the antigen retrieval step was performed by heating the samples on high in the microwave in an acidic citrate bath. Samples were blocked with 5% serum for 1 hour at room temperature. The primary antibody was allowed to incubate for 18 hours at 4°C. After several washes in PBS, the samples were incubated with the secondary antibody for 1 hour at room temperature. After several washes in PBS, Hoechst nucleic acid stain, diluted 1:1000 (Invitrogen Corp., Carlsbad, CA), was applied for 30 seconds. After several more washes in PBS, the samples were allowed to air-dry, and a coverslip was applied. Frozen, O.C.T.-inflated samples were cut to 5-μm sections and fixed in acetone at −20°C for 2 minutes. After drying for 30 minutes at room temperature, the samples were rehydrated in a PBS wash. The blocking step and the incubation of the primary and secondary antibodies were completed as described for the paraffin-embedded samples. Immunofluorescence imaging was performed using an Olympus Provis Ax-70 microscope and digital camera (Olympus America, Center Valley, PA) with the assistance of MagnaFire 2.1B (Indigo Scientific, Ltd., Baldock, Hertfordshire, UK) imaging software. Images were acquired at ×400 in the individual red, blue, and green channels, and a color, 24-bit, merged TIFF image was created and saved for analysis. A minimum of 10 random images of alveolar tissue was acquired from each lung sample. If a random high-powered field contained large airways or blood vessels, the image was not captured and the next random field was examined. The images of the alveolar parenchyma were analyzed using Metamorph Offline software, version 7." @default.
• W2039838662 created "2016-06-24" @default.
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• W2039838662 date "2008-10-01" @default.
• W2039838662 modified "2023-10-15" @default.
• W2039838662 title "CX3CL1 Up-Regulation Is Associated with Recruitment of CX3CR1+ Mononuclear Phagocytes and T Lymphocytes in the Lungs during Cigarette Smoke-Induced Emphysema" @default.
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