γδ T Cells Attenuate Bleomycin-Induced Fibrosis through the Production of CXCL10
2011; Elsevier BV; Volume: 178; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2010.11.055
ISSN1525-2191
AutoresDerek Pociask, Kong Chen, Sun Mi Choi, Tim D. Oury, Chad Steele, Jay K. Kolls,
Tópico(s)Immune Cell Function and Interaction
Resumoγδ T cells are a subset of T cells associated with epithelial mucosal tissues and play a prominent role in both promoting and dampening inflammatory responses to pathogens; in addition, they strongly mediate epithelial repair. By using a bleomycin model of pulmonary fibrosis, we found that γδ T-cell populations dramatically increased after bleomycin administration. To determine the importance of these cells, we exposed mice lacking the δ chain of the γδ T-cell receptor (γδ knockout [KO]) to bleomycin. Pulmonary fibrosis was more severe in γδ KO mice, as measured by collagen deposition (hydroxyproline) and histopathological features. Furthermore, there was no evidence of resolution of the fibrotic response up to 45 days after bleomycin therapy. In contrast to control mice, γδ KO mice had decreased concentrations of IL-6, granulocyte colony stimulating factor, chemokine CXC ligand (CXCL) 1, and interferon inducible protein 10/CXCL10. In vitro culture of γδ T cells purified from lungs 17 days after bleomycin exposure (a time of peak influx of these cells) demonstrated that γδ T cells produced substantial quantities of all four of these cytokines, suggesting that γδ T cells are a predominant source of these proteins. To demonstrate that γδ T cells are effector cells in the fibrotic response, we performed adoptive transfer experiments with γδ T cells sorted from bleomycin-treated lungs; these cells were sufficient to resolve fibrosis in γδ KO mice and restore CXCL10 levels comparable to wild-type mice. Furthermore, overexpression of CXCL10 in the lung decreased the severity of fibrosis seen in the γδ KO mice. Finally, adoptive transfer of γδ T cells from CXCL10−/− mice failed to reverse the severe fibrosis in γδ KO mice. These results indicate that γδ T cells promote the resolution of fibrosis through the production of CXCL10. γδ T cells are a subset of T cells associated with epithelial mucosal tissues and play a prominent role in both promoting and dampening inflammatory responses to pathogens; in addition, they strongly mediate epithelial repair. By using a bleomycin model of pulmonary fibrosis, we found that γδ T-cell populations dramatically increased after bleomycin administration. To determine the importance of these cells, we exposed mice lacking the δ chain of the γδ T-cell receptor (γδ knockout [KO]) to bleomycin. Pulmonary fibrosis was more severe in γδ KO mice, as measured by collagen deposition (hydroxyproline) and histopathological features. Furthermore, there was no evidence of resolution of the fibrotic response up to 45 days after bleomycin therapy. In contrast to control mice, γδ KO mice had decreased concentrations of IL-6, granulocyte colony stimulating factor, chemokine CXC ligand (CXCL) 1, and interferon inducible protein 10/CXCL10. In vitro culture of γδ T cells purified from lungs 17 days after bleomycin exposure (a time of peak influx of these cells) demonstrated that γδ T cells produced substantial quantities of all four of these cytokines, suggesting that γδ T cells are a predominant source of these proteins. To demonstrate that γδ T cells are effector cells in the fibrotic response, we performed adoptive transfer experiments with γδ T cells sorted from bleomycin-treated lungs; these cells were sufficient to resolve fibrosis in γδ KO mice and restore CXCL10 levels comparable to wild-type mice. Furthermore, overexpression of CXCL10 in the lung decreased the severity of fibrosis seen in the γδ KO mice. Finally, adoptive transfer of γδ T cells from CXCL10−/− mice failed to reverse the severe fibrosis in γδ KO mice. These results indicate that γδ T cells promote the resolution of fibrosis through the production of CXCL10. Both idiopathic pulmonary fibrosis (IPF) and drug-induced pulmonary fibrosis can be progressive life-threatening diseases characterized by fibroblast accumulation and excessive collagen deposition, leading to impaired lung function with decreased gas exchange and ultimately respiratory failure. The etiology of IPF is unknown (and, thus, idiopathic), and it has a high incidence (42.7/100,000),1Mannino D.M. Etzel R.A. Parrish R.G. Pulmonary fibrosis deaths in the United States, 1979–1991: an analysis of multiple-cause mortality data.Am J Respir Crit Care Med. 1996; 153: 1548-1552Crossref PubMed Scopus (119) Google Scholar poor survival (3- to 5-year median), and few limited therapeutic approaches.2Zisman D.A. Keane M.P. Belperio J.A. Strieter R.M. Lynch 3rd, J.P. Pulmonary fibrosis.Methods Mol Med. 2005; 117: 3-44PubMed Google Scholar, 3Raghu G. Weycker D. Edelsberg J. Bradford W.Z. Oster G. Incidence and prevalence of idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2006; 174: 810-816Crossref PubMed Scopus (992) Google Scholar Pulmonary fibrosis is also a complication of autoimmune diseases (eg, systemic lupus erythematosus), rheumatoid arthritis, and drugs (eg, bleomycin). In fact, pulmonary fibrosis is the dose-limiting toxic effect of bleomycin in humans; even with careful dosing, bleomycin can lead to end-stage fibrosis, requiring lung transplantation.4Sleijfer S. Bleomycin-induced pneumonitis.Chest. 2001; 120: 617-624Crossref PubMed Scopus (475) Google Scholar The initial response to injury from a fibrotic agent such as bleomycin is the induction of a strong inflammatory response, including neutrophils, macrophages, and T cells. Several studies have implicated T cells in the development and progression of fibrosis. In humans, T cells are found in high abundance in lung biopsy specimens,5Katzenstein A.L. Zisman D.A. Litzky L.A. Nguyen B.T. Kotloff R.M. Usual interstitial pneumonia: histologic study of biopsy and explant specimens.Am J Surg Pathol. 2002; 26: 1567-1577Crossref PubMed Scopus (184) Google Scholar lavage fluid,6Costabel U. Guzman J. Bronchoalveolar lavage in interstitial lung disease.Curr Opin Pulm Med. 2001; 7: 255-261Crossref PubMed Scopus (149) Google Scholar and circulating blood7Ahmed A. Chang C.C. Chronic idiopathic myelofibrosis: clinicopathologic features, pathogenesis, and prognosis.Arch Pathol Lab Med. 2006; 130: 1133-1143PubMed Google Scholar in patients with IPF. By using mouse models, thymectomized mice8Sharma S.K. MacLean J.A. Pinto C. Kradin R.L. The effect of an anti-CD3 monoclonal antibody on bleomycin-induced lymphokine production and lung injury.Am J Respir Crit Care Med. 1996; 154: 193-200Crossref PubMed Scopus (83) Google Scholar or mice treated with steroids9Lossos I.S. Or R. Ginzburg V. Christensen T.G. Mashriki Y. Breuer R. Cyclosporin A upmodulates bleomycin-induced pulmonary fibrosis in BALB/c mice.Respiration. 2002; 69: 344-349Crossref PubMed Scopus (8) Google Scholar or anti-CD3 antibodies all show reduced fibrosis,8Sharma S.K. MacLean J.A. Pinto C. Kradin R.L. The effect of an anti-CD3 monoclonal antibody on bleomycin-induced lymphokine production and lung injury.Am J Respir Crit Care Med. 1996; 154: 193-200Crossref PubMed Scopus (83) Google Scholar suggesting that T cells are required for fibrosis. However, mice with severe combined immunodeficiency lacking T and B cells and nude mice lacking T cells show no difference in bleomycin-induced disease, causing a debate over the role of T cells.10Helene M. Lake-Bullock V. Zhu J. Hao H. Cohen D.A. Kaplan A.M. T cell independence of bleomycin-induced pulmonary fibrosis.J Leukoc Biol. 1999; 65: 187-195PubMed Google Scholar, 11Corsini E. Luster M.I. Mahler J. Craig W.A. Blazka M.E. Rosenthal G.J. A protective role for T lymphocytes in asbestos-induced pulmonary inflammation and collagen deposition.Am J Respir Cell Mol Biol. 1994; 11: 531-539Crossref PubMed Scopus (44) Google Scholar A limitation of the previously described studies is that the approaches described do not address the specific role of T-cell subsets that may be profibrotic or antifibrotic. Thus, the deletion of both protective and beneficial T-cell populations may reveal no difference in models of fibrosis. In support of this statement, there is evidence that fibrosis may be perpetuated by helper T cell (Th) subtype bias. Generally, a Th2 bias is profibrotic. Tissues from patients with IPF have generally higher levels of IL-4, IL-5, and IL-13.12Furuie H. Yamasaki H. Suga M. Ando M. Altered accessory cell function of alveolar macrophages: a possible mechanism for induction of Th2 secretory profile in idiopathic pulmonary fibrosis.Eur Respir J. 1997; 10: 787-794PubMed Google Scholar, 13Jakubzick C. Choi E.S. Kunkel S.L. Evanoff H. Martinez F.J. Puri R.K. Flaherty K.R. Toews G.B. Colby T.V. Kazerooni E.A. Gross B.H. Travis W.D. Hogaboam C.M. Augmented pulmonary IL-4 and IL-13 receptor subunit expression in idiopathic interstitial pneumonia.J Clin Pathol. 2004; 57: 477-486Crossref PubMed Scopus (51) Google Scholar In mouse models, mice overexpressing GATA-3 (a Th2 transcription factor)14Kimura T. Ishii Y. Yoh K. Morishima Y. Iizuka T. Kiwamoto T. Matsuno Y. Homma S. Nomura A. Sakamoto T. Takahashi S. Sekizawa K. Overexpression of the transcription factor GATA-3 enhances the development of pulmonary fibrosis.Am J Pathol. 2006; 169: 96-104Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar as well as mice in which T-bet (a Th1 transcription factor) is knocked out,15Xu J. Mora A.L. LaVoy J. Brigham K.L. Rojas M. Increased bleomycin-induced lung injury in mice deficient in the transcription factor T-bet.Am J Physiol Lung Cell Mol Physiol. 2006; 291: L658-L667Crossref PubMed Scopus (41) Google Scholar both demonstrate greater sensitivity to fibrotic agents.14Kimura T. Ishii Y. Yoh K. Morishima Y. Iizuka T. Kiwamoto T. Matsuno Y. Homma S. Nomura A. Sakamoto T. Takahashi S. Sekizawa K. Overexpression of the transcription factor GATA-3 enhances the development of pulmonary fibrosis.Am J Pathol. 2006; 169: 96-104Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 15Xu J. Mora A.L. LaVoy J. Brigham K.L. Rojas M. Increased bleomycin-induced lung injury in mice deficient in the transcription factor T-bet.Am J Physiol Lung Cell Mol Physiol. 2006; 291: L658-L667Crossref PubMed Scopus (41) Google Scholar More recently, data suggest that the Th17 response may also be profibrotic because IL-17A was increased in the lavage fluid of patients with IPF16Wilson M.S. Madala S.K. Ramalingam T.R. Gochuico B.R. Rosas I.O. Cheever A.W. Wynn T.A. Bleomycin and IL-1beta-mediated pulmonary fibrosis is IL-17A dependent.J Exp Med. 2010; 207: 535-552Crossref PubMed Scopus (524) Google Scholar and Il-17a−/− mice were less susceptible to bleomycin-induced inflammation16Wilson M.S. Madala S.K. Ramalingam T.R. Gochuico B.R. Rosas I.O. Cheever A.W. Wynn T.A. Bleomycin and IL-1beta-mediated pulmonary fibrosis is IL-17A dependent.J Exp Med. 2010; 207: 535-552Crossref PubMed Scopus (524) Google Scholar, 17Sonnenberg G.F. Nair M.G. Kirn T.J. Zaph C. Fouser L.A. Artis D. Pathological versus protective functions of IL-22 in airway inflammation are regulated by IL-17A.J Exp Med. 2010; 207: 1293-1305Crossref PubMed Scopus (298) Google Scholar and fibrosis.16Wilson M.S. Madala S.K. Ramalingam T.R. Gochuico B.R. Rosas I.O. Cheever A.W. Wynn T.A. Bleomycin and IL-1beta-mediated pulmonary fibrosis is IL-17A dependent.J Exp Med. 2010; 207: 535-552Crossref PubMed Scopus (524) Google Scholar γδ T cells are generally found in low numbers in “normal” lung (approximately 1%) and are found in close proximity to epithelial mucosal cells and alveoli.18Wands J.M. Roark C.L. Aydintug M.K. Jin N. Hahn Y.S. Cook L. Yin X. Dal Porto J. Lahn M. Hyde D.M. Gelfand E.W. Mason R.J. O'Brien R.L. Born W.K. Distribution and leukocyte contacts of gammadelta T cells in the lung.J Leukoc Biol. 2005; 78: 1086-1096Crossref PubMed Scopus (80) Google Scholar Because of their location, γδ T cells are one of the first lines of defense against infection and appear to play a distinctive role in epithelial response to injury, repair, and homeostasis. For example, they are critical for epithelial wound repair in the skin.19Jameson J. Havran W.L. Skin gammadelta T-cell functions in homeostasis and wound healing.Immunol Rev. 2007; 215: 114-122Crossref PubMed Scopus (149) Google Scholar Interestingly, patients with IPF have decreased γδ T cells in their bronchoalveolar lavage (BAL) fluid.20Selman M. Pardo A. Role of epithelial cells in idiopathic pulmonary fibrosis: from innocent targets to serial killers.Proc Am Thorac Soc. 2006; 3: 364-372Crossref PubMed Scopus (402) Google Scholar In lung injury models, such as chlorine gas21Koohsari H. Tamaoka M. Campbell H.R. Martin J.G. The role of gamma delta T cells in airway epithelial injury and bronchial responsiveness after chlorine gas exposure in mice.Respir Res. 2007; 8: 21Crossref PubMed Scopus (34) Google Scholar or ozone,22King D.P. Hyde D.M. Jackson K.A. Novosad D.M. Ellis T.N. Putney L. Stovall M.Y. Van Winkle L.S. Beaman B.L. Ferrick D.A. Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes.J Immunol. 1999; 162: 5033-5036PubMed Google Scholar γδ T cells are required for neutrophil influx and epithelial repair. Furthermore, γδ T-cell populations increase in response to bleomycin injury.23Braun R.K. Sterner-Kock A. Kilshaw P.J. Ferrick D.A. Giri S.N. Integrin alpha E beta 7 expression on BAL CD4+, CD8+, and gamma delta T-cells in bleomycin-induced lung fibrosis in mouse.Eur Respir J. 1996; 9: 673-679Crossref PubMed Scopus (19) Google Scholar For chronic Bacillus subtilis infection, mice lacking γδ T cells demonstrated an enhanced fibrotic phenotype.24Simonian P.L. Roark C.L. Diaz del Valle F. Palmer B.E. Douglas I.S. Ikuta K. Born W.K. O'Brien R.L. Fontenot A.P. Regulatory role of gammadelta T cells in the recruitment of CD4+ and CD8+ T cells to lung and subsequent pulmonary fibrosis.J Immunol. 2006; 177: 4436-4443PubMed Google Scholar Because of the potential role of these cells in epithelial repair (a process that has been hypothesized to be critical for the prevention of fibrosis), we hypothesized that γδ T cells are critical for resolving fibrogenic insults in the lung. The current study demonstrates that γδ T cells play an integral role in the response to bleomycin. Lung γδ T-cell populations increased during the course of injury. In vitro γδ T cells recruited to the lung spontaneously produce large amounts of IL-6, chemokine CXC ligand (CXCL) 1, granulocyte colony stimulating factor (G-CSF), and the antifibrogenic chemokine CXCL10, also known as IP-10. Furthermore, γδ TCR−/− mice showed progressive fibrosis in response to bleomycin and diminished levels of CXCL10 in vivo. Overexpression of CXCL10 resulted in significantly attenuated fibrosis in γδ TCR−/− mice. To confirm that γδ T cells mediate antifibrotic responses via a CXCL10-dependent mechanism, adoptive transfer experiments were performed with γδ T cells from wild-type (WT) or Cxcl10−/− mice. Adoptive transfer of WT T cells into γδ TCR−/− mice significantly reduced pulmonary fibrosis in response to bleomycin, whereas γδ T cells from Cxcl10−/− mice were unable to protect against fibrosis. Taken together, these data demonstrate the critical importance of γδ T-cell production of CXCL10 and its role in the attenuation of the fibrotic response. Specific pathogen-free mice were used in all experiments and housed in specific pathogen-free conditions within animal care facilities at Children's Hospital of Pittsburgh, Pittsburgh, PA, or Louisiana State University Health Sciences Center, New Orleans, LA. All mouse experiments were approved by the University of Pittsburgh or the Louisiana State University Institutional Animal Care and Use Committee. B6.129S4-Cxcl10tm1Adl/J [CXCL10 knockout (KO) mice], B6.129P2-Tcrbtm1Mom/J (Tcrb KO mice) and appropriate age- and sex-matched C57BL/6 controls were purchased from Jackson Laboratory (Bar Harbor, ME). B6.129P2-Tcrdtm1Mom (γδ KO) mice were bred in-house and backcrossed over 10 generations with C57BL/6 mice. For all experiments, male mice (aged 6 to 8 weeks) were used. All treatments were performed using the oropharyngeal aspiration-tongue pull technique on isoflurane-anesthetized mice, as previously described.25McKinley L. Alcorn J.F. Peterson A. Dupont R.B. Kapadia S. Logar A. Henry A. Irvin C.G. Piganelli J.D. Ray A. Kolls J.K. TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice.J Immunol. 2008; 181: 4089-4097PubMed Google Scholar, 26McKinley L. Logar A.J. McAllister F. Zheng M. Steele C. Kolls J.K. Regulatory T cells dampen pulmonary inflammation and lung injury in an animal model of pneumocystis pneumonia.J Immunol. 2006; 177: 6215-6226PubMed Google Scholar Bleomycin USP (Hospira Pharmaceuticals Lake Forest, IL) was administered (2-U/kg body weight) in sterile PBS (100 μL). Adenovirus encoding CXCL10 or green fluorescent protein (adenovirus control) was administered in 100 μL of sterile saline at a concentration of 108 plaque-forming units.27Zeng X. Moore T.A. Newstead M.W. Deng J.C. Lukacs N.W. Standiford T.J. IP-10 mediates selective mononuclear cell accumulation and activation in response to intrapulmonary transgenic expression and during adenovirus-induced pulmonary inflammation.J Interferon Cytokine Res. 2005; 25: 103-112Crossref PubMed Scopus (33) Google Scholar Lungs were harvested at days 3 to 45 for evaluation of pulmonary fibrosis. Animals were sacrificed by i.p. injection of 0.9-ml/kg body weight ketamine, followed by exsanguination through the renal artery. After exposing the chest cavity, the right main bronchus was sutured at the base of the main stem and the right lung was excised and snap frozen in liquid nitrogen and stored at −70°C for protein analysis. The left lung was inflated with 10% neutral-buffered formalin (Sigma Chemical Co, St Louis, MO) at a pressure of 25 cm H2O for 15 to 20 minutes, removed from the animal, and placed in fresh 10% neutral-buffered formalin for 16 to 20 hours at 4°C before processing and paraffin embedding. For histological analysis, 5-μm sections were cut and stained with hematoxylin-eosin. The severity of disease was scored by a pathologist (T.D.O) blinded to the groups, as previously described.28Fattman C.L. Tan R.J. Tobolewski J.M. Oury T.D. Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase.Free Radic Biol Med. 2006; 40: 601-607Crossref PubMed Scopus (77) Google Scholar Individual fields were scored at ×400 magnification, with a minimum of 20 fields per slide. To be included, each field had to contain greater than 50% alveoli. Scoring in each field was based on the percentage of alveolar tissue with interstitial fibrosis, according to the following scale: 0 indicates no fibrosis; 1, up to 25% fibrosis; 2, 25% to 50% fibrosis; 3, 50% to 75% fibrosis; and 4, 75% to 100% fibrosis. To visualize collagen, Gomori's trichrome staining (Sigma Chemical Co) was performed according to manufacturer's instructions. Tracheas were exposed and cannulated. Bronchoalveolar lavage fluid was collected in 10 × 1-ml aliquots of sterile PBS supplemented with 0.6-mmol/L EDTA. Aliquots were combined and centrifuged for 10 minutes at 500 × g. Cells were resuspended in 1 ml of PBS-EDTA and counted. A total of 1 × 105 cells were spun onto a glass slide and stained for differential counting. All protein analysis was performed on lung homogenate. Briefly, lungs were homogenized in 1 ml of PBS with complete miniprotease inhibitor cocktail (Roche Scientific, Indianapolis IN) using a Polytron handheld homogenizer (Daigger, Vernon Hills, IL). Lung homogenate was centrifuged at 12,000 × g for 15 minutes, and supernatant was stored at −80°C for later cytokine analysis. G-CSF, granulocyte macrophage stimulating factor, interferon-γ, IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 p70, IL-13, IL-15, IL-17, CXCL10/IP-10, CXCL1/CXCL1 chemokine (c-c motif) ligand 5 (CCL5), macrophage inflammatory protein 1, and tumor necrosis factor α were analyzed using Luminex (Millipore, Billerica, MA) on a reader (Bioplex, Bio-Rad, Hercules, CA). Data were analyzed using computer software (Bioplex manager software). To examine whole lung T-cell populations, lungs were isolated, minced, and placed in RPMI 1640 medium with 5% bovine serum albumin. Collagenase was added, and lungs were digested for 30 minutes at 37°C in an orbital shaker. Digests were passed through 70- and 40-μm filters and centrifuged (for 10 minutes at 500 × g). Cells were resuspended in 9 ml of RPMI 1640 medium, layered over 5 ml of Ficoll (GE Healthcare, Waukesha, WI), and spun for 15 minutes at 1350 × g. The interphase was removed, placed in RPMI 1640 media, and centrifuged for 10 minutes at 500 × g. For flow sorting, cell pellets were resuspended in PBS containing 2% fetal bovine serum. Cells were stained with fluorochrome-conjugated primary antibodies for 60 minutes on ice and washed twice with PBS-fetal bovine serum. Analysis was performed by gating on the predominantly lymphocytic population based on size using forward-and-side scatter. Autofluorescence was accounted for by running unstained cells and subtracting the fluorescence as background. Nonspecific binding was controlled by running isotype controls. For cell sorting, the delta chain of the TCR was detected using the GL3 antibody (BD Pharmingen, San Diego, CA). γδ T cells were sorted as GL3+/TCRβ− cells. Purity was checked by reanalyzing sorted cells and gating on GL3+ cells. For adoptive transfer, cells were sorted from whole lung 17 days after bleomycin treatment and verified to be greater than 95% pure based on the sorting. Non-γδ T cells were all considered to be GL3− cells. The RNA was collected from sorted cells using a modified Trizol (Invitrogen, Carlsbad, CA) RNA extraction protocol to extract RNA from a few cells. Briefly, cells were placed in Trizol, and chloroform was added. After centrifugation (12,000 x g, 15 minutes) the aqueous layer was removed. Five µg of glycogen was added, and the RNA was precipitated after the addition of isopropyl alcohol and centrifugation (12,000 x g, 10 minutes). The RNA was washed twice in 75% ethanol. The RNA was quantified by nanodrop, reverse transcribed using SuperScript III (Invitrogen), and verified by RT-PCR amplification of the β-actin housekeeping gene. The subtype of γδ T cell was determined by RT-PCR amplification with reagents (Clonetech) using published primers29Takada H. Matsuzaki G. Yoshida H. Sumichika H. Nomoto K. Functional difference between Thy-1-positive and Thy-1-negative gamma delta T cells induced by Escherichia coli infection in mice.Immunology. 1996; 88: 261-268Crossref PubMed Scopus (7) Google Scholar: Cγ, 5’-CTTATGGAGATTTGTTTCAGC-3’; Vγ1/2, 5’-ACACAGCTATACATTGGTAC-3’; Vγ2, 5’-CGGCAAAAAACAAATCAACAG-3’; Vγ4, 5’-TGTCCTTGCAACCCCTACCC-3’; Vγ5, 5’-TGTGCACTGGTACCAACTGA-3’; Vγ6, 5’-GGAATTCAAAAGAAAACATTGTCT-3’; and Vγ7, 5’-AAGCTAGAGGGGTCCTCTGC-3’. The PCR conditions were verified by observing the expected Vγ5 subset in mouse skin cells. To estimate the total amount of collagen in the lungs, hydroxyproline was measured as previously described.30Woessner Jr, J.F. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid.Arch Biochem Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3371) Google Scholar Briefly, right lungs were removed and homogenized in 6N HCl at 110°C overnight. Precipitates were removed by centrifugation. Supernatants were dried overnight and dissolved in distilled water. Each sample was tested in duplicate; 50 μL of chloramine T in acetate buffer (pH, 6.0), 50 μL of perchloric acid, and 50 μL of Erlich's reagent were added sequentially to each sample, as previously described, and absorbance was measured at 570 nm. All data are represented as the mean ± SD. Significance was determined using either an unpaired t-test when comparing two groups or a one-way analysis of variance with Tukey's post hoc test when comparing multiple groups. All statistics were calculated using computer software (GraphPad Prism 4). γδ T cells have been strongly implicated in epithelial wound repair in skin.19Jameson J. Havran W.L. Skin gammadelta T-cell functions in homeostasis and wound healing.Immunol Rev. 2007; 215: 114-122Crossref PubMed Scopus (149) Google Scholar To understand their role in bleomycin-induced lung injury, we examined the kinetics of infiltrating interstitial γδ T cells in lung tissue after administration of bleomycin. γδ T cells were identified as GL3+ CD3+ cells (Figure 1A), and staining specificity was determined by running Armenian hamster isotype control (see Supplemental Figure S1 at http://ajp.amjpathol.org). In WT C57BL/6 mice, interstitial γδ T-cell populations substantially increased from day 1 to 17 from less than 104 cells in naive mice to 2 × 105 cells per mouse lung, as shown in Figure 1B. To identify which subsets of γδ T cells were found in the lung after bleomycin exposure, PCR was performed on flow-sorted cells from day 14. As seen in Figure 1C, bleomycin induced a polyclonal γδ T-cell response, with Vγ1, Vγ2, Vγ4, Vγ5, and Vγ6 populations all being identified. To understand the biological significance of these cells in bleomycin-induced injury and fibrosis, mice deficient in γδ T cells (γδ KO) mice were administered bleomycin. As previously shown in this model, WT C57BL/6 mice showed a time-dependent increase in hydroxyproline, a measurement of collagen deposition in the lung with some degree of resolution by day 45 (Figure 2A). In contrast to this response, γδ KO mice showed similar levels of hydroxyproline in lung tissue as WT mice at day 7, followed by substantially greater increases up to day 45 (Figure 2A). Furthermore, there was no evidence of resolution of fibrosis in the γδ KO mice. In comparison, TCRβ−/− mice did not show any perturbations in fibrosis and were not different from WT mice 21 days after bleomycin exposure (see Supplemental Figure S2 at http://ajp.amjpathol.org). The increases in hydroxyproline were also supported by histological analysis, which demonstrated that mice deficient in γδ T cells had thickened alveolar septa and ablation of alveolar spaces on days 14 (Figure 2, B and C) and 28 after bleomycin treatment compared with WT mice (Figure 2, E and F); in addition, γδ KO mice demonstrated a significantly reduced inflammatory infiltrate. Blinded scoring28Fattman C.L. Tan R.J. Tobolewski J.M. Oury T.D. Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase.Free Radic Biol Med. 2006; 40: 601-607Crossref PubMed Scopus (77) Google Scholar of the severity of fibrotic lesions revealed that γδ KO mice had significantly higher histological scores at 14 and 28 days after instillation of bleomycin (Figure 2, D and G). To examine if γδ T cells regulate early inflammatory events in the lung after bleomycin treatment, WT and γδ KO mice were treated with bleomycin and sacrificed at 3 and 7 days after instillation for BAL fluid and cytokine analysis. The WT mice showed significant increases in total inflammatory cells in the BAL fluid at both 3 and 7 days (Figure 3, A and B), with both increases in neutrophil and macrophage recruitment compared with vehicle controls, which showed 0.92 ± 0.1 × 105 macrophages. In stark contrast, bleomycin-exposed γδ KO mice showed significantly fewer total cells in the BAL fluid at both 3 (0.95 ± 0.12 × 106) and 7 (1.2 ± 0.25 × 106) days, with significantly fewer (P < 0.001) neutrophils and macrophages at these same points (Figure 3, A and B). Because the cellular analysis of BAL fluid demonstrated that γδ KO mice have decreased inflammatory cell recruitment 3 to 7 days after bleomycin treatment compared with WT mice, we evaluated chemokine and cytokine production using whole lung homogenate, as previously described.25McKinley L. Alcorn J.F. Peterson A. Dupont R.B. Kapadia S. Logar A. Henry A. Irvin C.G. Piganelli J.D. Ray A. Kolls J.K. TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice.J Immunol. 2008; 181: 4089-4097PubMed Google Scholar, 26McKinley L. Logar A.J. McAllister F. Zheng M. Steele C. Kolls J.K. Regulatory T cells dampen pulmonary inflammation and lung injury in an animal model of pneumocystis pneumonia.J Immunol. 2006; 177: 6215-6226PubMed Google Scholar Compared with WT mice, γδ KO mice had significantly reduced levels of CXCL1, G-CSF, IL-6, and CXCL10 in BAL fluid (Figure 3C). Of the four cytokines/chemokines observed to be significantly different, CXCL10 was the only one still elevated by 14 days. We did not observe induction of IL-17 protein during this study. Our data show that γδ T cells are increased in the lung in response to bleomycin, and ablation of the γδ TCR results in reduced acute inflammation but substantially worse fibrosis. To determine whether these cells are effector cells in the fibrotic response to bleomycin, we performed adoptive transfer experiments. Given the abundance and properties of γδ T cells at 17 days after bleomycin treatment, we chose to use this point to collect and purify pulmonary γδ T cells for transfer. As previously stated, these cells are polyclonal and express TCRδ chains 1 through 6. Cells were gated as described in the Flow Sorting and Cytology subsection of Materials and Methods, and representative plots are found in Supplemental Figure S1 and Supplemental Figure S3 (at http://ajp.amjpathol.org). The γδ KO mice were instilled with bleomycin, followed 1 day later by oropharyngeal administration of purified γδ T cells or non-γδ T (GL3−) cells (50,000 cells per mouse). Adoptive transfer of polyclonal γδ T cells significantly attenuated bleomycin-induced weight loss of γδ KO mice (Figure 4A). Furthermore, adoptive transfer of γδ T cells significantly attenuated collagen deposition, whereas adoptive transfer of the non-γδ population had no effect on attenuating bleomycin-induced increases in hydroxyproline (Figure
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