Respiratory Reovirus 1/L Induction of Intraluminal Fibrosis, a Model of Bronchiolitis Obliterans Organizing Pneumonia, Is Dependent on T Lymphocytes
2003; Elsevier BV; Volume: 163; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63504-3
ISSN1525-2191
AutoresElizabeth I. Majeski, Manjeet K. Paintlia, Andrea D. Lopez, Russell A. Harley, Steven D. London, Lucille London,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoBronchiolitis obliterans organizing pneumonia (BOOP) is a clinical syndrome characterized by perivascular/peribronchiolar leukocyte infiltration leading to the development of intraalveolar fibrosis. We have developed an animal model of BOOP where CBA/J mice infected with 1 × 106 plaque-forming units (PFU) reovirus 1/L develop follicular bronchiolitis and intraalveolar fibrosis similar to human BOOP. In this report, we demonstrate a role for T cells in the development of intraluminal fibrosis associated with BOOP. Corticosteroid treatment of reovirus 1/L-infected mice both inhibited the development of fibrotic lesions when administered early in the time-course and promoted the resolution of fibrotic lesions when corticosteroid administration was delayed. Further, the depletion of either CD4+ or CD8+ T cells before reovirus 1/L infection also inhibited fibrotic lesion development. Both corticosteroid treatment and depletion of CD4+ or CD8+ T cells also resulted in decreased expression of the proinflammatory and profibrotic cytokines, interferon (IFN)-γ and monocyte chemoattractant protein-1 (MCP-1). Further, treatment of mice with a neutralizing monoclonal antibody to IFN-γ also significantly inhibited the development of fibrosis. Taken together, these results suggest a significant role for T cells in the development of reovirus 1/L-induced BOOP fibrotic lesions in CBA/J mice and suggests that TH1-derived cytokines, especially IFN-γ, may play a key role in fibrotic lesion development. Bronchiolitis obliterans organizing pneumonia (BOOP) is a clinical syndrome characterized by perivascular/peribronchiolar leukocyte infiltration leading to the development of intraalveolar fibrosis. We have developed an animal model of BOOP where CBA/J mice infected with 1 × 106 plaque-forming units (PFU) reovirus 1/L develop follicular bronchiolitis and intraalveolar fibrosis similar to human BOOP. In this report, we demonstrate a role for T cells in the development of intraluminal fibrosis associated with BOOP. Corticosteroid treatment of reovirus 1/L-infected mice both inhibited the development of fibrotic lesions when administered early in the time-course and promoted the resolution of fibrotic lesions when corticosteroid administration was delayed. Further, the depletion of either CD4+ or CD8+ T cells before reovirus 1/L infection also inhibited fibrotic lesion development. Both corticosteroid treatment and depletion of CD4+ or CD8+ T cells also resulted in decreased expression of the proinflammatory and profibrotic cytokines, interferon (IFN)-γ and monocyte chemoattractant protein-1 (MCP-1). Further, treatment of mice with a neutralizing monoclonal antibody to IFN-γ also significantly inhibited the development of fibrosis. Taken together, these results suggest a significant role for T cells in the development of reovirus 1/L-induced BOOP fibrotic lesions in CBA/J mice and suggests that TH1-derived cytokines, especially IFN-γ, may play a key role in fibrotic lesion development. Bronchiolitis obliterans organizing pneumonia (BOOP), first described in 1985, is a pattern of injury characterized histologically as "patchy plugs of fibrous tissue (Masson bodies) filling bronchiolar lumens (bronchiolitis obliterans) and alveolar ducts and spaces (organizing pneumonis)."1Epler GR Colby TV McLoud TC Carrington CB Gaensler EA Bronchiolitis obliterans organizing pneumonia.N Engl J Med. 1985; 312: 152-158Crossref PubMed Scopus (893) Google Scholar, 2Epler GR Bronchiolitis obliterans organizing pneumonia.Arch Intern Med. 2001; 161: 158-164Crossref PubMed Scopus (237) Google Scholar, 3Schlesinger C Koss MN Bronchiolitis: update 2001.Curr Opin Pulm Med. 2002; 8: 112-116Crossref PubMed Scopus (29) Google Scholar This patchy fibrosis may begin as focal lesions within the alveoli and the terminal bronchioles of the lung and progress bilaterally over time.1Epler GR Colby TV McLoud TC Carrington CB Gaensler EA Bronchiolitis obliterans organizing pneumonia.N Engl J Med. 1985; 312: 152-158Crossref PubMed Scopus (893) Google Scholar Other histological features include clusters of mononuclear inflammatory cells, chronic inflammation in the walls of the surrounding alveoli with reactive type II cells, increased numbers of foamy macrophages in the alveoli, and preserved lung architecture.2Epler GR Bronchiolitis obliterans organizing pneumonia.Arch Intern Med. 2001; 161: 158-164Crossref PubMed Scopus (237) Google Scholar, 4Epler GR Bronchiolitis obliterans organizing pneumonia: definition and clinical features.Chest. 1992; 102: 2S-6SCrossref PubMed Google Scholar The development of BOOP is often of unknown etiology (idiopathic BOOP) but BOOP has also been associated as a consequence of lung injury due to environmental toxins, bacterial infections, viral infections, and lung or bone marrow transplantation.3Schlesinger C Koss MN Bronchiolitis: update 2001.Curr Opin Pulm Med. 2002; 8: 112-116Crossref PubMed Scopus (29) Google Scholar, 5Colby TV Pathologic aspects of bronchiolitis obliterans organizing pneumonia.Chest. 1992; 102: 38S-43SCrossref PubMed Google Scholar BOOP is responsive to corticosteroids and treatment with prednisone continues to be the primary treatment for patients with symptomatic and progressive disease.2Epler GR Bronchiolitis obliterans organizing pneumonia.Arch Intern Med. 2001; 161: 158-164Crossref PubMed Scopus (237) Google Scholar, 3Schlesinger C Koss MN Bronchiolitis: update 2001.Curr Opin Pulm Med. 2002; 8: 112-116Crossref PubMed Scopus (29) Google Scholar, 4Epler GR Bronchiolitis obliterans organizing pneumonia: definition and clinical features.Chest. 1992; 102: 2S-6SCrossref PubMed Google Scholar Since infiltrating lymphocytes are associated with the initiation of BOOP lesions,6Costabel U Teschler H Guzman J Bronchiolitis obliterans organizing pneumonia (BOOP): the cytological and immunocytological profile of bronchoalveolar lavage.Eur Respir J. 1992; 5: 791-797PubMed Google Scholar, 7Mukae H Kadota J Kohno S Matsukura S Hara K Increase of activated T cells in BAL fluid of Japanese patients with bronchiolitis obliterans organizing pneumonia and chronic eosinophilic pneumonia.Chest. 1995; 108: 123-128Crossref PubMed Scopus (52) Google Scholar it is possible that these cells play an active role in the progression of inflammatory foci into lesions that are progressively dominated by fibroblasts. In patients with BOOP, there is an increase of activated bronchoalveolar lavage (BAL) lymphocytes with up to 80% to 95% of this cellular infiltrate being comprised of cytotoxic/suppressor CD8+ T cells.6Costabel U Teschler H Guzman J Bronchiolitis obliterans organizing pneumonia (BOOP): the cytological and immunocytological profile of bronchoalveolar lavage.Eur Respir J. 1992; 5: 791-797PubMed Google Scholar, 7Mukae H Kadota J Kohno S Matsukura S Hara K Increase of activated T cells in BAL fluid of Japanese patients with bronchiolitis obliterans organizing pneumonia and chronic eosinophilic pneumonia.Chest. 1995; 108: 123-128Crossref PubMed Scopus (52) Google Scholar, 8Yamamoto M Jna Y Kitaichi M Haraswa M Tamura M Clinical features of BOOP in Japan.Chest. 1992; 102: 21S-25SCrossref PubMed Google Scholar, 9Ogata K Koga T Yagawa K Interferon-related bronchiolitis obliterans organizing pneumonia.Chest. 1994; 106: 612-613Crossref PubMed Scopus (66) Google Scholar These cells may be involved in the inflammation and subsequent fibrosis occurring in BOOP patients.2Epler GR Bronchiolitis obliterans organizing pneumonia.Arch Intern Med. 2001; 161: 158-164Crossref PubMed Scopus (237) Google Scholar, 5Colby TV Pathologic aspects of bronchiolitis obliterans organizing pneumonia.Chest. 1992; 102: 38S-43SCrossref PubMed Google Scholar, 7Mukae H Kadota J Kohno S Matsukura S Hara K Increase of activated T cells in BAL fluid of Japanese patients with bronchiolitis obliterans organizing pneumonia and chronic eosinophilic pneumonia.Chest. 1995; 108: 123-128Crossref PubMed Scopus (52) Google Scholar, 10Ross DJ Jordan SC Nathan SD Kass RM Koerner SK Delayed development of obliterative bronchiolitis syndrome with OKT3 after unilateral lung transplantation: a plea for multicenter immunosuppressive trials.Chest. 1996; 109: 870-873Crossref PubMed Scopus (32) Google Scholar, 11Ross DJ Moudgil A Bagga A Toyoda M Marchevsky AM Kass RM Jordan SC Lung allograft dysfunction correlates with γ-interferon gene expression in bronchoalveolar lavage.J Heart Lung Transplant. 1999; 18: 627-636Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Costabel U King TE International consensus statement on idiopathic pulmonary fibrosis.Eur Respir J. 2001; 17: 163-167Crossref PubMed Scopus (61) Google Scholar Several studies have shown that the infiltration of T lymphocytes may be important in the development of other forms of pulmonary fibrosis, although the data from both animal models and patients has been equivocal.13du Bois RM Wells AU Cryptogenic fibrosing alveolitis/idiopathic pulmonary fibrosis.Eur Respir J. 2001; 18: 43S-55SGoogle Scholar, 14Crystal RG Bitterman PB Mossman B Schwarz MI Sheppard D Almasy L Chapman HA Friedman SL King Jr, TE Leinwand LA Liotta L Martin GR Schwartz DA Schultz GS Wagner CR Musson RA Future research directions in idiopathic pulmonary fibrosis: summary of a National Heart, Lung, and Blood Institute working group.Am J Respir Crit Care Med. 2002; 166: 236-246Crossref PubMed Scopus (165) Google Scholar, 15Ikonen T Uusitalo M Taskinen E Korpela A Salminen US Morris RE Harjula AL Small airway obliteration in a new swine heterotopic lung and bronchial allograft model.J Heart Lung Tranplant. 1998; 17: 945-953PubMed Google Scholar, 16Neuringer IP Mannon RB Coffman TM Parsons M Burns K Yankaskas JR Aris RM Immune cells in a mouse airway model of obliterative bronchiolitis.Am J Respir Cell Mol Biol. 1998; 19: 379-386Crossref PubMed Scopus (84) Google Scholar, 17Lazo JS Hoyt DG Sebti SM Pitt BR Bleomycin: a pharmacologic tool in the study of the pathogenesis of interstitial pulmonary fibrosis.Pharmcol Ther. 1990; 47: 347-358Crossref PubMed Scopus (64) Google Scholar, 18Zhu J Cohen DA Goud SN Kaplan AM Contribution of T lymphocytes to the development of bleomycin-induced pulmonary fibrosis.Ann NY Acad Sci. 1996; 796: 194-202Crossref PubMed Scopus (20) Google Scholar Although these existing models of experimental pulmonary fibrosis have been useful for histopathological and functional investigations of other types of fibrotic events in the lung, the process of fibrotic lesion development in these models may be distinct from BOOP lesion development. Thus, differences in the phenotype of the inflammatory cell infiltrate, expression of soluble mediators, and response to various treatments may be different and, therefore, may fail to accurately reflect the intraluminal and fibroblastic nature of the bronchoalveolar obliteration observed in BOOP lesions.19Piguet PF Collart MA Grau GE Sappino AP Vassalli P Requirement of tumour necrosis factor for development of silica-induced pulmonary fibrosis.Nature. 1990; 344: 245-247Crossref PubMed Scopus (519) Google Scholar, 20Zhang HY Gharaee-Kermani M Zhang K Karmiol S Phan SH Lung fibroblast α-smooth muscle actin expression and contractile phenotype in bleomycin-induced pulmonary fibrosis.Am J Pathol. 1996; 148: 527-537PubMed Google Scholar, 21Barnes PJ Chronic obstructive pulmonary disease.N Engl J Med. 2000; 343: 269-280Crossref PubMed Scopus (1226) Google Scholar We have described a spectrum of inflammatory lung diseases after respiratory infection with reovirus serotype 1, strain Lang (reovirus 1/L), which is dependent on the strain of mice used.22Bellum SC Hamamdzic DH Thompson AH Harley RA London SD London L Experimental reovirus serotype 1/strain Lang infection of the lung: a model for the study of the lung in the context of mucosal immunity.Lab Invest. 1996; 74: 221-231PubMed Google Scholar, 23Bellum SC Dove D Harley RA Greene WB Judson MA London L London SD Respiratory reovirus 1/L induction of intraluminal fibrosis: a model for the study of bronchiolitis obliterans organizing pneumonia.Am J Pathol. 1997; 150: 2243-2254PubMed Google Scholar, 24Thompson AH London L Bellum SC Hamamdzic D Harley RA London SD Respiratory-mucosal lymphocyte populations induced by reovirus serotype 1 infection.Cell Immunol. 1996; 169: 278-287Crossref PubMed Scopus (18) Google Scholar, 25London L Majeski EI Paintlia MK Harley RA London SD Respiratory reovirus 1/L induction of diffuse alveolar damage: a model of acute respiratory distress syndrome.Exper Mol Pathol. 2002; 72: 24-36Crossref PubMed Scopus (30) Google Scholar, 26London L Majeski EI Altman-Hamamdzic S Enockson C Paintlia MK Harley RA London SD Respiratory reovirus 1/L induction of diffuse alveolar damage: pulmonary fibrosis is not modulated by corticosteroids in acute respiratory distress syndrome in mice.Clin Immunol. 2002; 103: 284-295Crossref PubMed Scopus (22) Google Scholar, 27Majeski EI Harley RS Bellum SC London SD London L Differential role for T cells in the development of fibrotic lesions associated with reovirus 1/L induced BOOP versus ARDS.Am J Respir Cell Mol Bio. 2003; 28: 208-217Crossref PubMed Scopus (34) Google Scholar In this spectrum CBA/J mice infected with 1 × 106 PFU reovirus 1/L develop a histologically severe inflammation characterized by an infiltration of lymphocytes organized adjacent to the pulmonary vasculature of the lung.23Bellum SC Dove D Harley RA Greene WB Judson MA London L London SD Respiratory reovirus 1/L induction of intraluminal fibrosis: a model for the study of bronchiolitis obliterans organizing pneumonia.Am J Pathol. 1997; 150: 2243-2254PubMed Google Scholar This pattern of mononuclear cell organization without the involvement of intraluminal fibrosis results in lesions histopathologically consistent with the non-fibrotic human syndrome termed follicular bronchiolitis (FB). However, accompanying the development of FB in CBA/J mice are the presence of foamy macrophages and the elicitation of a non-specific fibrotic response of the lung characteristic of BOOP fibrotic lesions.23Bellum SC Dove D Harley RA Greene WB Judson MA London L London SD Respiratory reovirus 1/L induction of intraluminal fibrosis: a model for the study of bronchiolitis obliterans organizing pneumonia.Am J Pathol. 1997; 150: 2243-2254PubMed Google Scholar To investigate the role of the inflammatory cell infiltrate, especially T cells, in the development of reovirus 1/L-induced fibrotic lesions, the effect of either corticosteroid treatment or the depletion of CD4+ or CD8+ T cells before reovirus 1/L infection was determined. Our results indicate that corticosteroid treatment of reovirus 1L-infected mice both inhibited the development of fibrotic lesions when administered early in the time-course and promoted the resolution of fibrotic lesions when corticosteroid administration was delayed. In addition, the depletion of either CD4+ or CD8+ T cells before reovirus 1/L infection also inhibited fibrotic lesion development. Both corticosteroid treatment and depletion of CD4+ or CD8+ T cells also resulted in decreased expression of the proinflammatory and profibrotic cytokines, interferon (IFN)-γ and monocyte chemoattractant protein-1 (MCP-1). Finally, treatment of mice with a neutralizing monoclonal antibody to IFN-γ also significantly inhibited the development of fibrosis. Taken together, the results suggest a significant role for T cells in the development of reovirus 1/L-induced BOOP fibrotic lesions in CBA/J mice and that TH1 derived cytokines, especially IFN-γ, may play a key role in fibrotic lesion development. Four- to 5-week-old female CBA/J mice (The Jackson Laboratory, Bar Harbor, ME) were maintained in microisolator cages under specific pathogen-free conditions in a BL-2 facility. Cages were housed in an HEPA-filtered animal isolator clean room (Nuaire Inc., Plymouth, MN). All animal manipulations were performed in class II biological safety cabinets. Virally primed mice were kept physically isolated from all other mice. Reovirus 1/L was originally obtained from Dr. W. Joklik (Duke University School of Medicine, Durham, NC). Third-passage gradient-purified stocks were obtained by re-cloning and amplifying parental stocks on L-929 fibroblast cells [American Type Culture Collection (ATCC), Rockville, MD] as previously described.23Bellum SC Dove D Harley RA Greene WB Judson MA London L London SD Respiratory reovirus 1/L induction of intraluminal fibrosis: a model for the study of bronchiolitis obliterans organizing pneumonia.Am J Pathol. 1997; 150: 2243-2254PubMed Google Scholar Following the purification of new stocks, infectious viral titers were obtained by limiting dilution on L-929 monolayers.23Bellum SC Dove D Harley RA Greene WB Judson MA London L London SD Respiratory reovirus 1/L induction of intraluminal fibrosis: a model for the study of bronchiolitis obliterans organizing pneumonia.Am J Pathol. 1997; 150: 2243-2254PubMed Google Scholar Animals were lightly anesthetized with an i.p. injection of 0.08 ml of 20% ketamine (Vetalar 100 mg/ml; Fort Dodge Laboratories, Inc., Fort Dodge, IA) and 2% PromAce (acepromazine maleate 10 mg/ml; Ayerst Laboratories, New York, NY) before immunization. Animals were infected by the intranasal (i.n.) application of 1 × 106 PFU of reovirus 1/L in 30 μl (15 μl in each nostril) in sterile injectable grade 0.9% NaCl (Baxter Healthcare Corp., Deerfield, IL). Control animals were inoculated with 30 μl (15 μl in each nostril) of sterile injectable grade 0.9% NaCl. After the indicated timepoints, animals were sacrificed with an i.p. injection of 0.2 ml sodium Nembutal (50 mg/ml; Abbott Laboratories, North Chicago, IL). As an initial dosing regimen either 10 mg/kg or 20 mg/kg methylprednisolone (MPS) (∼0.1 to 0.2 mg/mouse) (Sigma Chemicals, St. Louis, MO) dissolved in PBS was administered i.p. to mice beginning on either days 0, 5, 10, or 14 post-reovirus 1/L infection and given daily until the completion of the time-course. Since these initial studies indicated that treatment with MPS (20 mg/kg) either beginning at day 0 or day 5 post infection or administration of MPS (10 mg/kg) before day 5 post reovirus 1/L infection (beginning at day 0) resulted in an increased mortality rate as compared to that observed in untreated, reovirus 1/L-infected mice (Table 1), all additional studies were performed using MPS at a concentration of 10 mg/kg beginning 5 days post-reovirus 1/L infection unless otherwise noted.Table 1Modulation of Reovirus 1/L-Induced BOOP Fibrotic Lesions in CBA/J MiceDay*Day treatment with methylprednilosone was begun postinfection with 1 × 106 PFU reovirus 1/L.Dose†Dose of methylprednilosone administered daily i.p. in 100 μl PBS.% Mortality‡Percent mortality on day 14 postreovirus 1/L infection.FB§Follicular bronchiolitis severity on day 14 postreovirus 1/L infection.Fibrosis¶Fibrotic lesion severity on day 21 postreovirus 1/L infection.Untreated0.0 mg/kg20%+++++++Day 0‖Experiment performed once with two mice per timepoint.20 mg/kg60%+++NDDay 0‖Experiment performed once with two mice per timepoint.10 mg/kg40%+++NDDay 5‖Experiment performed once with two mice per timepoint.20 mg/kg40%+++Day 5**Experiment performed five times with two mice per timepoint.10 mg/kg20%+++Day 10Experiment performed twice with two to four mice per timepoint.10 mg/kg20%+++++Day 14Experiment performed twice with two to four mice per timepoint.10 mg/kg20%+++++UntreatedMice were depleted of CD4+ or CD8+ cells prior to reovirus 1/L infection as described in Materials and Methods. CD4−0.0 mg/kg0%+Follicular bronchiolitis severity on day 21 post-reovirus 1/L infection.+UntreatedMice were depleted of CD4+ or CD8+ cells prior to reovirus 1/L infection as described in Materials and Methods. CD8−0.0 mg/kg0%++Follicular bronchiolitis severity on day 21 post-reovirus 1/L infection.+ND, not determined.* Day treatment with methylprednilosone was begun postinfection with 1 × 106 PFU reovirus 1/L.† Dose of methylprednilosone administered daily i.p. in 100 μl PBS.‡ Percent mortality on day 14 postreovirus 1/L infection.§ Follicular bronchiolitis severity on day 14 postreovirus 1/L infection.¶ Fibrotic lesion severity on day 21 postreovirus 1/L infection.‖ Experiment performed once with two mice per timepoint.** Experiment performed five times with two mice per timepoint.†† Experiment performed twice with two to four mice per timepoint.‡‡ Mice were depleted of CD4+ or CD8+ cells prior to reovirus 1/L infection as described in Materials and Methods.§§ Follicular bronchiolitis severity on day 21 post-reovirus 1/L infection. Open table in a new tab ND, not determined. Adult CBA/J mice were treated i.p. with either 0.5 mg of purified GK1.5 monoclonal antibody (mAb)28Dialynas DP Quan ZS Wall KA Pierres A Quintans J Loken MR Pierre M Fitch FW Characterization of the murine T cell surface molecule designated L3T4, identified by monoclonal antibody GK1.5: similarities to the human Leu3/T4 molecule.J Immunol. 1983; 131: 2445-2451PubMed Google Scholar for depletion of CD4+ lymphocytes or 0.25 mg of purified 53–6.72 mAb29Ledbetter JA Herzenberg LA Xenogeneic antibodies to mouse lymphoid differentiation antigens.Immuno Rev. 1979; 47: 63-90Crossref PubMed Scopus (1666) Google Scholar for depletion of CD8+ lymphocytes for three consecutive days. Depleted mice were then infected i.n. with 1 × 106 PFU of reovirus 1/L in 30 μl (15 μl in each nostril) in sterile injectable grade 0.9% NaCl. Control, depleted animals were inoculated with 30 μl (15 μl in each nostril) of sterile injectable grade 0.9% NaCl. The depleted state was maintained by treating with either 0.5 mg purified GK1.5 or 0.25 mg of purified 53–6.72 mAb every 6 days. Depletion of the appropriate subset of T cells was verified by flow cytometry of cells obtained from the lymph node and spleen before infection with reovirus 1/L on day 0 and on days 7 and 14 postinfection. Depleted mice were evaluated for the development of BOOP fibrotic lesions at day 21 post-reovirus 1/L infection. An anti-IFN-γ mAb (R4–6A2, rat IgG1, ATCC HB170) was obtained from ATCC30Spitalny GL Havell EA Monoclonal antibody to murine γ interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities.J Exp Med. 1984; 159: 1560-1565Crossref PubMed Scopus (318) Google Scholar, 31Tay CH Welsh RM Distinct organ-dependent mechanisms for the control of murine cytomegalovirus infection by natural killer cells.J Virol. 1997; 71: 267-275PubMed Google Scholar, 32Sayles PC Johnson LL Intact immune defenses are required for mice to resist the ts-4 vaccine strain of Toxoplasma gondii.Infect Immun. 1996; 64: 3088-3092PubMed Google Scholar and ascites fluid was generated for in vivo use (Strategic Biosolutions, Newark, NJ). Reovirus 1/L-infected (106 PFU BOOP) CBA/J mice were treated i.p. every 3 days beginning on day 3 postinfection with either 100 μg anti-IFN-γ antibody in PBS or 100 μg normal rat IgG (Sigma) in PBS. Mice were evaluated on days 14 and 21 postinfection for the development of fibrotic lesions by hematoxylin and eosin (H&E) and Mason's trichrome stain. BAL was performed in situ by injecting and withdrawing a 0.5 ml aliquot of Hank's balanced salt solution (HBSS) twice through an intubation needle (21 gauge). A total of 1.5 ml of HBSS was used. BAL fluid was frozen at −70°C until use. Cells collected by BAL were washed three times with HBSS containing 5% fetal calf serum (FCS) and 0.05% azide, and resuspended at 1 × 106 cells/ml. Lungs were inflated in situ with 10% neutral buffered formalin (0.5 mls) (Richard-Allan Scientific, Kalamazoo, MI) by intratracheal (i.t.) intubation, removed, and suspended in an additional 10% neutral buffered formalin overnight before being embedded in paraffin. H&E stain and Mason's trichrome stain, which was used to visualize collagen deposition, were performed on 4-μm sections. Inflammatory infiltration with the development of FB, which is defined as a mononuclear cell infiltrate that condenses into prominent peribronchiolar lymphoid accumulations, was blindly evaluated. FB was scored on a scale of 0 to 3: 0, normal; 1, mild (< 4 follicles per lobe); 2, moderate (between 5 and 8 follicles per lobe); 3, severe (> 8 follicles per lobe). Fibrosis was scored on a scale of 0 to 4: 0, normal; 1, mild; 2, moderate; 3, severe; 4, very severe. The extent of pulmonary fibrosis was also determined by estimating total lung collagen as reflected by the measurement of the HP content of the lung as previously described.25London L Majeski EI Paintlia MK Harley RA London SD Respiratory reovirus 1/L induction of diffuse alveolar damage: a model of acute respiratory distress syndrome.Exper Mol Pathol. 2002; 72: 24-36Crossref PubMed Scopus (30) Google Scholar, 26London L Majeski EI Altman-Hamamdzic S Enockson C Paintlia MK Harley RA London SD Respiratory reovirus 1/L induction of diffuse alveolar damage: pulmonary fibrosis is not modulated by corticosteroids in acute respiratory distress syndrome in mice.Clin Immunol. 2002; 103: 284-295Crossref PubMed Scopus (22) Google Scholar, 33Reddy GK Enwemeka CS A simplified method for the analysis of hydroxyproline in biological tissues.Clin Biochem. 1996; 29: 225-229Crossref PubMed Scopus (1091) Google Scholar Mice were sacrificed at various intervals after infection with reovirus 1/L and the lungs were removed, lyophilized, and weighed. Differences between groups were examined for statistical significance using two-tailed Student's t-test. A P value less than 0.05 was considered significant. The following monoclonal antibodies were used in this study: Cy-Chrome-conjugated rat anti-mouse CD45 (30-F11, leukocyte common antigen, Ly-5); fluorescein isothiocyanate (FITC)-conjugated hamster anti-mouse CD3 (145–2C11, CD3 ε chain); FITC-conjugated rat anti-mouse CD8a (53–6.7, Ly-2); R-phycoerythrin (PE)-conjugated rat anti-mouse CD4 (GK1.5, L3T4) (Caltag, Burlingame, CA); R-PE-conjugated rat anti-mouse Pan-NK cells (DX5); FITC-conjugated rat anti-mouse CD45R/B220 (RA3–6B2); R-PE-conjugated rat anti-mouse CD11b (M1/70, integrinαm chain, Mac-1 α chain); and FITC-conjugated rat anti-mouse Ly6G (RB6–8C5, Gr-1, neutrophils) (Pharmingen, San Diego, CA); hamster anti-rat CD3 (ε-chain, 48–2B) (Santa Cruz Biotechnology, Santa Cruz, CA); rat anti-mouse CD11b (Mac-1 α chain) (Serotec, Westbury, NY); and rat anti-mouse neutrophil (MCA 771F) (Serotec). Cells collected by BAL were washed three times with HBSS containing 5% FCS and 0.05% azide, and resuspended at 1 × 106 cells/ml. Cells were stained for cell surface marker expression as previously described except that all cells were also stained with anti-CD45 (30-F11), leukocyte common antigen Ly-5, and only anti-CD45-positive cells were acquired for analysis.24Thompson AH London L Bellum SC Hamamdzic D Harley RA London SD Respiratory-mucosal lymphocyte populations induced by reovirus serotype 1 infection.Cell Immunol. 1996; 169: 278-287Crossref PubMed Scopus (18) Google Scholar, 26London L Majeski EI Altman-Hamamdzic S Enockson C Paintlia MK Harley RA London SD Respiratory reovirus 1/L induction of diffuse alveolar damage: pulmonary fibrosis is not modulated by corticosteroids in acute respiratory distress syndrome in mice.Clin Immunol. 2002; 103: 284-295Crossref PubMed Scopus (22) Google Scholar Isotype-matched controls were run for each sample (Caltag and Pharmingen). The total number of PMNs was obtained by adding the anti-Gr-1 (Ly6G) single-positive cells and the anti-Gr-1/anti-Mac-1 (integrinαm chain) double-positive cells. The total number of B cells was obtained by adding the anti-B220 (CD45R) single-positive cells plus the anti-B220/anti-Mac-1 double-positive cells. The total number of macrophages was obtained by enumerating those cells stained only with anti-Mac-1. Flow cytometric analysis was performed using a dual-laser FACS Caliber flow cytometer and the Cell Quest acquisition and analysis software program (BD Biosciences, San Jose, CA). Total cellular RNA was isolated from whole lungs by guanidium denaturation using TRI-reagent (Molecular Research Center, Cincinnati, OH). Riboquant multiprobe ribonuclease protection assay (RPA) mouse template sets mCK-1b, mCK-2b, mCK-3b and mCK-5 were purchased from Pharmingen. Template set mCK-1b contained probes for Interleukin (IL)-2 - 5, -9, -10, -13, -15, and IFN-γ. Template set mCK-2b contained probes for IL-1α, -1β, -1Ra, -6, -10, -12, IFN-γ inducing factor (IGIF), IFN-γ, and migration inhibitory factor (MIF). Template set mCK-3b contained probes for tumor necrosis factor (TNF)-β, lymphotoxin (LT)-β, TNF-α, IL-6, IFN-γ, IFN-β, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, and MIF. Template set mCK-5 contained probes for the chemokines, lymphotactin (Ltn), regulated on activation normal T cells expressed and secreted (RANTES), eotaxin, macrophage inflammatory protein (MIP)-1β, MIP-1α, MIP-2, interferon inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, and T-cell activation factor (TCA)-3. All template sets also contained probes for the control genes GAPDH and L32. RPA analysis was performed as previously described26London L Majeski EI Altman-Hamamdzic S Enockson C Paintlia MK Harley RA London SD Respiratory reovirus 1/L induction of diffuse alveolar damage: pulmonary fibrosis is not modulated by corticosteroids in acute respiratory distress syndrome in mice.Clin Immunol. 2002; 103: 284-295Crossref PubMed Scopus (22) Google Scholar using radiolabeled RNA transcripts from the multiprobe sets generated by in vitro transcription (Pharmingen) following the manufacturer's instructions. Gels were dried and exposed to Fuji RX film at −70°C with DuPont Cronex Quanta III intensifying screens for 1 to 5 days. Band intensities on scanned RPA gels were analyzed using the public doma
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