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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 208-217, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4891

Differential Role for T Cells in the Development of Fibrotic Lesions Associated with Reovirus 1/L-Induced Bronchiolitis Obliterans Organizing Pneumonia versus Acute Respiratory Distress Syndrome

Elizabeth I. Majeski, Russell A. Harley, Stephen C. Bellum, Steven D. London and Lucille London

Departments of Microbiology and Immunology and of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina

Address correspondence to: Dr. Lucille London, Ph.D., Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250504, Charleston, South Carolina 29425. E-mail: londonl{at}musc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bronchiolitis obliterans organizing pneumonia (BOOP) and Acute Respiratory Distress Syndrome (ARDS) are two pulmonary diseases with fibrotic components. BOOP is characterized by perivascular/peribronchiolar leukocyte infiltration leading to the development of intra-alveolar fibrosis. ARDS is a biphasic disease that includes an acute phase, consisting of severe leukocyte infiltration, edema, hemorrhage, and the formation of hyaline membranes, and a chronic phase, which is characterized by persistent intra-alveolar and interstitial fibrosis. CBA/J mice infected with 1 x 106 plaque-forming units (pfu) reovirus 1/L develop follicular bronchiolitis and intra-alveolar fibrosis similar to BOOP. In contrast, CBA/J mice infected with 1 x 107 pfu reovirus 1/L develop histologic characteristics of ARDS including diffuse alveolar damage, hyaline membranes, and intra-alveolar fibrosis. In this report, we demonstrate a differential role for T lymphocytes in the development of fibrosis associated with BOOP versus ARDS. Neonatally thymectomized CBA/J mice infected with 1 x 107 pfu (ARDS) reovirus 1/L still develop the hallmark characteristics of ARDS, including a severe viral pneumonia with cellular infiltrates comprised mainly of macrophages and neutrophils, hyaline membrane formation, and hemorrhage during the acute phase of the disease and persistent intra-alveolar fibrosis during the chronic phase of the disease. In contrast, neonatally thymectomized CBA/J mice infected with 1 x 106 pfu (BOOP) reovirus 1/L do not develop intra-alveolar fibrosis associated with BOOP. Therefore, while T cells are necessary for the development of intraluminal fibrosis associated with BOOP, they are not necessary for the development of intraluminal fibrosis associated with ARDS. Furthermore, we suggest that interferon-{gamma} plays a key role in the fibrotic process and that elevated levels of interferon-{gamma} are associated with a continuum from least to more severe fibrosis.

Abbreviations: acute respiratory distress syndrome, ARDS • bronchoalveolar lavage, BAL • bronchoalveolar lavage fluid, BALF • bronchiolitis obliterans organizing pneumonia, BOOP • follicular bronchiolitis, FB • interferon, IFN • IFN-inducible protein, IP • intranasal, i.n. • idiopathic pulmonary fibrosis, IPF • monoclonal antibodies, mAb • monocyte chemoattractant protein, MCP • migration inhibitory factor, MIF • messenger RNA, mRNA • neonatally thymectomized, nTx • plaque-forming units, pfu • regulated on activation normal T cell expressed and secreted, RANTES • RNase protection assay, RPA • transforming growth factor, TGF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic inflammation and tissue fibrosis is a leading cause of morbidity and mortality in granulomatous and interstitial lung disorders as well as in the chronic stage of acute respiratory distress syndrome (ARDS) (1, 2). Lung fibrosis is often the result of aberrant healing mechanisms resulting from infections with viruses or bacteria, inhalation of particulate matter, severe trauma, or unknown causes (1, 2). A number of distinct clinical entities are characterized by a fibrotic component, which may be distinguished by both the location within the lung of the fibrotic lesion and the presence of an interstitial pneumonia (1). Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic fibrosing interstitial pneumonia limited to the lung and associated with the histologic appearance of usual interstitial pneumonia (1). Instillation of bleomycin sulfate in rodents initiates fibrotic lesion development in the lung, which has many of the histologic components of human IPF and thus, has been used to investigate the mechanism of induction of IPF (3–5). Bronchiolitis Obliterans Organizing Pneumonia (BOOP) is a distinct clinical entity that, unlike IPF, is characterized by an excessive proliferation of granulation tissue within small airways (proliferative bronchiolitis) and alveolar ducts associated with chronic inflammation in the surrounding alveoli (6). ARDS is characterized by diffuse alveolar damage usually secondary to an intense host inflammatory response of the lung to an infectious, noninfectious, or extrapulmonary insult (2). Histologically, ARDS progresses rapidly from microvascular injury to widespread epithelial injury, alveolar type II cell proliferation, and the development of fibrotic lesions that are similar in location to the fibrosing alveolitis associated with BOOP (1, 2, 6).

We have described the establishment and characterization of 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 (11–15). This spectrum ranges from a clinical and histopathologically mild pulmonary infection in C3H mice (with mediastinal lymph node hyperplasia as the predominant response) to a severe inflammation of the lung, involving infiltration into both the lung interstitium and the alveolar spaces mainly composed of large and small lymphocytes, macrophages, and, in the later stages, plasma cells in Balb/C and CD-1 mice (11, 13). CBA/J mice infected with 1 x 106 pfu reovirus 1/L develop a severe clinical and histopathologically severe infection with the elicitation of a nonspecific fibrotic response of the lung (BOOP) (12). In contrast, CBA/J mice infected with 1 x 107 pfu reovirus 1/L develop ARDS and provide a model that recapitulates both its acute exudative phase, including the formation of hyaline membranes, as well as its regenerative phase with healing by repair leading to intra-alveolar and interstitial fibrosis similar to the fibrosis observed in BOOP (14, 15). Therefore, reovirus 1/L infection of CBA/J mice can serve as a model to investigate the pathophysiology of fibrotic lesion development associated with BOOP and the late stage of ARDS, including the involvement of cellular and molecular factors.

To investigate the role of T cells in the development of fibrosis associated with BOOP or ARDS, neonatally thymectomized (nTx) CBA/J mice were infected with 1 x 107 pfu (ARDS) or 1 x 106 pfu (BOOP) reovirus 1/L. A differential role for T cells was observed in the development of fibrosis associated with BOOP versus ARDS. While T cells are necessary for the development of intraluminal fibrosis associated with BOOP, they are not necessary for the development of intraluminal fibrosis associated with ARDS. Further, we hypothesize that interferon (IFN)-{gamma} plays a crucial role in the fibrotic process and that elevated levels of IFN-{gamma} are associated with a continuum from least to more severe fibrosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
nTx (3 d after birth) CBA/J mice and their normal littermates were purchased from Jackson Laboratories (Bar Harbor, ME). These mice were received at 4 wk of age and were housed in micro-isolator cages under specific pathogen-free conditions in a BL-2 facility. Experiments were initiated within 1 wk and were performed over a 35-d time course. Cages were housed in a 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 experimental and stock mice. By flow cytometric analysis comparing normal and nTx mice at Day 0, only T cell populations were depleted from the spleen (14 x 106/spleen-normal mice; 2 x 106/spleen-nTx mice) and peripheral lymph nodes (10 x 105 normal mice; 2 x 105 nTx mice), although the total cellularity of both the spleen and peripheral lymph nodes were decreased (data not shown). No alterations in the total number of B cells (16 x 106/spleen-normal mice; 10.5 x 106/spleen-nTx mice) or macrophages (4 x 106/spleen-normal mice; 4 x 106/spleen nTx mice.) were observed.

Virus and Inoculation Protocol
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 recloning and amplifying parental stocks on L-929 fibroblast cells (American Type Tissue Collection, Rockville, MD). Following the purification of new stocks, infectious viral titers were obtained by limiting dilution on L-929 monolayers (12). Animals were lightly anesthetized with an intraperitoneal injection of 0.08 cc of 20% Ketamine (Vetalar 100 mg/ml; Fort Dodge Laboratories, Inc., Fort Dodge, IA) and 2% PromAce (Acepromazine Maleate 10 mg/ml, Ayerst Laboratories, NY, NY) prior to immunization. Animals were inoculated by the intranasal (i.n.) application of either 1 x 106 pfu or 1 x 107 pfu 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 time points, animals were killed with an intraperitoneal injection of 0.2 cc sodium nembutal (50 mg/ml; Abbott Laboratories, North Chicago, IL). Viral clearance from the lungs of normal and nTx mice was monitored over time by standard plaque assay as previously described (11, 12). No significant difference in viral clearance was observed between normal and nTx mice, and reovirus was cleared from the lungs between Days 10 and 14 (data not shown).

Histology
Lungs were inflated in situ with 10% neutral buffered formalin (0.5 mls) (Richard-Allan Scientific, Kalamazoo, MI) by intratracheal intubation, removed, and suspended in an additional 10% neutral buffered formalin overnight before being embedded in paraffin. Hematoxylin and Eosin (H and E) stains were performed on 4-µm sections. Mason's trichrome stain was used to visualize collagen deposition. With Mason's trichrome the nuclei stain a dark blue/purple, muscle stains red, and collagen stains blue. The extent of pulmonary fibrosis was determined by estimating total lung collagen as reflected by the measurement of the hydroxyproline (HP) content of the lung as previously described (14, 15).

Antibodies
The following monoclonal antibodies (mAb) 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 {epsilon} chain); FITC-conjugated rat anti-mouse CD8a (53–6.7, Ly-2); R-Phycoerythrin (PE)- conjugated rat anti-mouse CD4 (GK1.5, L3T4); R-PE-conjugated rat anti-mouse Pan-natural killer cells (DX5); FITC-conjugated rat anti-mouse CD45R/B220 (RA3–6B2); R-PE-conjugated rat anti-mouse CD11b (M1/70, integrin {alpha}m chain, Mac-1 {alpha} chain); and FITC-conjugated rat anti-mouse Ly6G (RB6–8C5, Gr-1, neutrophils) (Pharmingen, San Diego, CA). Purified, unlabeled antibodies were also obtained for use in immunohistochemistry.

Immunohistochemistry
Lungs were inflated in situ with OCT compound (0.5 mls) (TissueTek II, Miles Laboratories Inc., Naperville, IL) by intratracheal intubation, removed, suspended in additional OCT compound, and snap-frozen in liquid nitrogen. Ten µm sequential sections were collected on poly-L-lysine (Sigma, St. Louis, MO)-treated slides and immediately fixed for 10 min in 95% ethanol at room temperature. Cell surface antigens were visualized using purified, unlabeled antibodies as described above in conjunction with a biotinylated rabbit anti-rat IgG and avidin-biotin-peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA) (12).

Flow Cytometric Analysis
Bronchoalveolar lavage (BAL) was performed in situ by injecting and withdrawing a 0.5 ml aliquot of Hank's balanced salt solution twice through an intubation needle (21 gauge). BAL fluid (BALF) was frozen at –70°C until use. Cells collected by BAL were washed three times with Hank's balanced salt solution containing 5% fetal calf serum and 0.05% azide, and resuspended at 1 x 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 (13). Isotype-matched controls were run for each sample (Caltag, Co., San Francisco, CA and Pharmingen). Flow cytometric analysis was performed using a dual-laser FACS Caliber flow cytometer and the Cell Quest acquisition and analysis software program (Becton-Dickinson, San Jose, CA).

RNase Protection Assay
Total cellular RNA was isolated from whole lungs by guanidium denaturation utilizing TRI-reagent (Molecular Research Center, Cincinnati, OH). Riboquant multi-probe RNase 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-{gamma}. Template set mCK-2b contained probes for IL-1{alpha}, -1ß, -1Ra, -6, -10, -12, IFN-{gamma} inducing factor, IFN-{gamma}, and migration inhibitory factor (MIF). Template set mCK-3b contained probes for tumor necrosis factor-ß, lymphotoxin-ß, tumor necrosis factor-{alpha}, IL-6, IFN-{gamma}, IFN-ß, transforming growth factor (TGF)-ß1, TGF-ß2, TGF-ß3, and MIF. Template set mCK-5 contained probes for the chemokines, lymphotactin, regulated on activation normal T cells expressed and secreted (RANTES), eotaxin, macrophage inflammatory protein (MIP)-1ß, MIP-1{alpha}, MIP-2, IFN-inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, and T cell activation factor-3. All template sets also contained probes for the control genes glyceraldehyde-3-phosphate dehydrogenase and L32. RPA analysis was performed as previously described (17). Gels were dried and exposed to Fuji RX film (Fuji Medical Systems USA, Inc., Stamford, CT) at -70°C with DuPont Cronex Quanta III intensifying screens (E.I. du Pont de Nemours and Company, Wilmington, DE) for 1–5 d. Band intensities on scanned RPA gels were analyzed using the public domain program NIH Image developed at the U.S. National Institutes of Health. Specific cytokine or chemokine band intensities were normalized to L32 controls to account for differences in total RNA loading in each sample. The mean ± SD of the densitometric measurements from two independent experiments with two mice per time point (four independent autoradiographs) over the indicated time points were determined. Differences in expression level between uninfected control groups and reovirus 1/L-infected groups were examined for statistical significance using a two-tailed Student t test. A value of P < 0.05 was considered significant.

Enzyme-Linked Immunosorbent Assay
A total of 100 µL of BALF was analyzed for mouse IFN-{gamma} and MCP-1 in duplicate using the Quantikine M immunoassay systems (R&D Systems, Minneapolis, MN). Results were expressed as the mean ± SD. Differences between groups were examined for statistical significance using a two-tailed Student t test. A value of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential Development of Fibrosis after Reovirus 1/L Infection of nTx CBA/J mice
To evaluate the potential role for T cells in the development of fibrosis associated with either BOOP or ARDS, normal and nTx CBA/J mice, which lack normal numbers of mature peripheral T cells, were infected i.n. with either 1 x 106 pfu (BOOP) or 1 x 107 pfu (ARDS) reovirus 1/L and lungs were collected for pathologic evaluation over time.

We have previously shown that CBA/J mice infected with 1 x 107 pfu (ARDS) reovirus 1/L become acutely ill and ~ 60% of these mice die of the acute infection between Day 7 and Day 10 after infection (14, 15). nTx CBA/J mice infected with 1 x 107 pfu (ARDS) reovirus 1/L also became acutely ill and a similar mortality rate was observed (data not shown). Histologically, both normal CBA/J mice (Figure 1A) (14) and nTx CBA/J mice (Figure 1B) infected with 1 x 107 pfu (ARDS) reovirus 1/L demonstrate signs of an acute viral pneumonia. Accompanying this pneumonia are edema, capillary dilation and hemorrhage, and the formation of hyaline membranes (Figure 1A and 1B, data not shown) (14). Similarly, both normal CBA/J mice (Figure 1C) (14) and nTx CBA/J mice (Figure 1D) that survive the acute phase of the disease begin to resolve the inflammatory features of ARDS and develop both intra-alveolar and interstitial fibrosis.



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  		Figure 1. Inhibition of fibrosis associated with BOOP but not ARDS in nTx CBA/J mice infected with reovirus 1/L. Normal (A, C, E, G) or nTx (B, D, F, H) CBA/J mice were i.n. infected with either 1 x 107 pfu (ARDS) (A–D) or 1 x 106 pfu (BOOP) (E–H) reovirus 1/L and paraffin-embedded lung sections were stained with H and E on the indicated time points as follows: (A) reovirus 1/L infected lung (1 x 107 pfu) from CBA/J mouse at Day 10; (B) reovirus 1/L infected lung (1 x 107 pfu) from nTx CBA/J mouse at Day 10; (C) reovirus 1/L infected lung (1 x 107 pfu) from CBA/J mouse at Day 12; (D) reovirus 1/L infected lung (1 x 107 pfu) from nTx CBA/J mouse at Day 12; (E) reovirus 1/L infected lung (1 x 106 pfu) from CBA/J mouse at Day 21; (F) reovirus 1/L infected lung (1 x 106 pfu) from nTx CBA/J mouse at Day 21; (G) reovirus 1/L infected lung (1 x 106 pfu) from CBA/J mouse at Day 35; (H) reovirus 1/L infected lung (1 x 106 pfu) from nTx CBA/J mouse at Day 35. Images are representative of four independent experiments containing two mice per time point. Objective magnification: 20x.

 
In addition, we have previously shown that CBA/J mice infected with 1 x 106 pfu (BOOP) reovirus 1/L become severely ill and ~ 20% of these mice die by Day 14 after infection (12). nTx CBA/J mice infected with 1 x 106 pfu (BOOP) reovirus 1/L also became severely ill and a similar mortality rate was observed (data not shown). In contrast to normal CBA/J mice that develop characteristic BOOP fibrotic lesions between Days 14 and 21 after infection with 1 x 106 pfu (BOOP) reovirus 1/L (Figure 1E) (12), the development of significant fibrotic lesions was not observed in nTx CBA/J mice at any time over a time course of 35 d (Figure 1F and 1H). Similar to normal CBA/J mice (Figure 1G), nTx CBA/J mice (Figure 1H) still develop follicular bronchiolitis (FB) that persists at least through Day 35 after infection.

Mason's trichrome stain was used to visualize collagen deposition in normal and nTx mice inoculated with reovirus 1/L (Figure 2) . Uninfected, normal (Figure 2A), and nTx (Figure 2B) CBA/J mice only display collagen staining within the vasculature. Collagen staining was not detected within the alveolar epithelium or airspaces. In contrast, after infection with 1 x 106 pfu (BOOP) reovirus 1/L, while significant collagen staining is observed in normal CBA/J mice (Figure 2C), collagen staining is not observed in nTx CBA/J mice (Figure 2D). However, after infection with 1 x 107 pfu (ARDS) reovirus 1/L, collagen staining was observed in both normal (Figure 2E) and nTx (Figure 2F) CBA/J mice.



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  		Figure 2. Differential collagen expression after reovirus 1/L-induced BOOP but not ARDS in normal versus nTx CBA/J mice. Normal (A, C, E) or nTx (B, D, F) CBA/J mice were i.n. inoculated with saline (A, B) or i.n. infected with 1 x 106 pfu (BOOP) (C, D) or 1 x 107 pfu (ARDS) (E, F) reovirus 1/L and paraffin-embedded lung sections were stained with Mason's trichrome for determination of collagen deposition. (A) control, saline Day 7, normal CBA/J mice; (B) control, saline Day 7, nTx CBA/J mice; (C) reovirus 1/L infected lung (1 x 106 pfu) from normal CBA/J mouse at Day 21; (D) reovirus 1/L infected lung (1 x 106 pfu) from nTx CBA/J mouse at Day 21; (E) reovirus 1/L infected lung (1 x 107 pfu) from normal CBA/J mouse at Day 14; (F) reovirus 1/L infected lung (1 x 107 pfu) from nTx CBA/J mouse at Day 14. Images are representative of four independent experiments containing two mice per time point. Objective magnification: 20x.

 
Cellular Infiltration is Modulated in nTx CBA/J Mice Receiving 1 x 106 pfu (BOOP) Reovirus 1/L but not in Mice Receiving 1 x 107 pfu (ARDS) Reovirus 1/L
The phenotype of the inflammatory infiltrate in normal and nTx CBA/J mice infected i.n. with either 1 x 106 pfu (BOOP) or 1 x 107 pfu (ARDS) reovirus 1/L cells was determined either by immunohistochemical analysis of frozen lung tissue sections or by flow cytometric analysis of cells collected by BAL. The cellular infiltrate was analyzed using mAb specific for T cell subsets (CD3, CD4, CD8), B cells (B220), macrophages (CD11b), neutrophils (GR-1), and NK cells (pan-NK).

In nTx CBA/J mice infected with 1 x 107 pfu (ARDS) reovirus 1/L, the predominant cell types found to infiltrate the lungs were macrophages (Figure 3A) and polymorphonuclear leukocytes (Figures 3B) as determined by immunohistochemistry. No significant infiltration of T cells was observed in nTx CBA/J mice over a time course of 14 d (data not shown). In contrast, a differential pattern of cellular infiltration was observed in normal versus nTx CBA/J mice infected with 1 x 106 pfu (BOOP) reovirus 1/L. In nTx CBA/J mice infected with 1 x 106 pfu (BOOP) reovirus 1/L, macrophages were found to be the predominant cell type infiltrating the lungs (Figure 3C). Little to no infiltration of polymorphonuclear leukocytes was observed (Figure 3D). Flow cytometric analysis revealed that in normal CBA/J mice, CD3+ T cells comprised 42% of the cellular infiltrate in the BALF on Day 7 after infection (0.21 x 106 CD3+ cells; 0.5 x 106 total cells) (Figure 4A) , with 28% of these cells being CD4+ (0.14 x 106) and 15% being CD8+ (0.075 x 106) (Figure 4B). In contrast, nTx CBA/J mice infected with 1 x 106 pfu (BOOP) reovirus 1/L, the percentage of infiltrating T cells was decreased to ~ 9% (0.06 x 106; 0.7 x 106 total cells) (Figure 4D) with ~ 4% of these cells being CD4+ and ~ 9% being CD8+ (Figure 4E). The percentage of infiltrating macrophages was increased from 17% (0.085 x 106) (Figure 4C) to 34% (0.24 x 106) (Figure 4F), and the percentage of infiltrating NK cells was increased from 11% (0.055 x 106) (Figure 4A) to 32% (0.22 x 106) (Figure 4D) in normal CBA/J versus nTx CBA/J mice. In addition, B220+ cells accounted for 33% of the infiltrating cells (0.165 x 106) in normal CBA/J mice (Figure 4C) and 23% of the infiltrating cells in nTx mice (0.16 x 106) (Figure 4F). Thus, the alterations in the phenotype of the cellular infiltrate from normal as compared with nTx mice can be accounted for by the 33% decrease in the CD3 and in the increase in the NK (21%) and macrophage (17%) populations, respectively.



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  		Figure 3. Presence of PMNs in nTx CBA/J mice infected with 1 x 107 pfu (ARDS) but not 1 x 106 pfu (BOOP) reovirus 1/L. nTx CBA/J mice were i.n. infected with either 1 x 107 pfu (ARDS) (A and B) or 1 x 106 pfu (BOOP) (C and D) reovirus 1/L and frozen lung sections were analyzed by immunohistochemistry and immunoperoxidase staining on Day 7. Sections were stained with (A and C) rat anti-mouse CD11b (M1/70, integrin {alpha}m chain, Mac-1 {alpha} chain); and (B and D) rat anti-mouse Ly6G (RB6–8C5, Gr-1, neutrophils). Cells were counter-stained with Hematoxylin. Images are representative of two independent experiments containing two mice per time point. Objective magnification: 20x.

 


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  		Figure 4. Lack of T lymphocytes in the BALF of nTx CBA/J mice infected with reovirus 1/L. Normal (A–C) and nTx (D–F) CBA/J mice were i.n. infected with 1 x 106 pfu (BOOP) reovirus 1/L and two-color flow cytometric analysis of the infiltrating cell populations in the BAL were analyzed on Day 7 after infection. (A and D) hamster anti-mouse CD3 (145–2C11, CD3 {epsilon} chain) versus rat anti-mouse Pan-NK cells (DX5); (B and E) rat anti-mouse CD8a (53–6.7, Ly-2) versus rat anti-mouse CD4 (GK1.5, L3T4); (C and F) rat anti-mouse CD45R/B220 (RA3–6B2) versus rat anti-mouse CD11b (M1/70, integrin{alpha}m chain, Mac-1 {alpha} chain). Data are representative of two independent experiments containing two mice per time point.

 
Cytokine and Chemokine Expression Are Depressed in the Lungs of nTx CBA/J Receiving 1 x 106 pfu (BOOP) Reovirus 1/L but not in the Lungs of nTx CBA/J Receiving 1 x 107 pfu (ARDS) Reovirus 1/L
Cytokine and chemokine messenger RNA (mRNA) expression in total lung tissue was evaluated by RPA. Three cytokine RPA template sets and one chemokine template set (Figure 5) were evaluated in nTx CBA/J mice after i.n. infection with 1 x 106 pfu (BOOP) or 1 x 107 pfu (ARDS) reovirus 1/L over a time course of 12 or 14 d.



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  		Figure 5. Differential cytokine and chemokine mRNA expression from the lungs of nTx CBA/J mice following i.n. infection with 1 x 107 pfu (ARDS) or 1 x 106 pfu (BOOP) reovirus 1/L. nTx CBA/J mice were i.n. infected with either 1 x 107 pfu (ARDS) or 1 x 106 pfu (BOOP) reovirus 1/L, RNA was harvested from the whole lungs at the indicated time points, and probed by RPA analysis using the following multi-probe template sets: (A) mCK-1b (T cell derived cytokine panel); (B) mCK-2b (inflammatory cytokine panel); (C) mCK-3b (cytokines associated with fibrosis); and (D) mCK-5 (chemokine panel). Individual cytokine or chemokine mRNA expression is indicated on the right. Gels are representative of two independent experiments containing two mice per time point.

 
Substantial expression of mRNA for a number of cytokines and chemokines was observed in the lungs of nTx CBA/J mice infected i.n. with 1 x 107 pfu (ARDS) reovirus 1/L (Figure 5). These include IL-15 (Figure 5A), IL-1 ß, IL-1 Ra, IL-6 (Figure 5B), IFN-{gamma}, (Figures 5A, 5B, and 5C), MIF (Figures 5B and 5C), MCP-1, RANTES, and IP-10 (Figure 5D). In comparison to nTx CBA/J mice infected with 1 x 107 pfu (ARDS) reovirus 1/L, after i.n. infection of nTx CBA/J mice with 1 x 106 pfu (BOOP) reovirus 1/L, significantly less expression of the following cytokines and chemokines was observed: IL-15 (Figure 5A), IL-1 ß, IL-1 Ra, IL-6 (Figure 5B), IFN-{gamma}, (Figures 5A, 5B, and 5C), MIF (Figures 5B and 5C), MCP-1, RANTES, and IP-10 (Figure 5D).

Because both IFN-{gamma} and MCP-1 have been implicated in the fibrotic process, we quantitated the autoradiographs and found a significant upregulation of both IFN-{gamma} and MCP-1 mRNA in nTx CBA/J mice infected with 1 x 107 pfu (ARDS) reovirus 1/L (Figure 6A , Table 1). However, in nTx CBA/J mice infected with 1 x 106 pfu (BOOP) reovirus 1/L, the mRNA expression of IFN-{gamma} and MCP-1 was not significantly different from that in control mice (Figure 6A, Table 1). In a similar manner, the protein expression for both IFN-{gamma} and MCP-1 in the BALF of nTx CBA/J mice infected with 1 x 106 pfu (BOOP) reovirus 1/L was significantly less than that observed in the BALF of normal CBA/J mice infected with the same dose (Figure 6B, Table 1). Table 1 presents a comparison of reovirus 1/L-induced models of BOOP (CBA/J; 1 x 106 pfu) (12) and ARDS (CBA/J; 1 x 107 pfu) (14, 15) and quantitates both the protein concentration in the BALF and the relative level of expression of mRNA in the lungs for IFN-{gamma} and MCP-1 as well as HP content which is a measure of the extent of fibrotic development. As the severity of the inflammatory and fibrotic response increases (HP) there is a corresponding increase in the level of IFN-{gamma} protein detected in the BALF (ARDS > BOOP > FB). When the fibrotic process in BOOP is inhibited (in nTx mice or by treatment with a corticosteroid, such as methylprednisolone), the concentration of IFN-{gamma} in the BALF decreases (Table 1). A similar response is observed with MCP-1 expression.



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  		Figure 6. Differential expression of IFN-{gamma} and MCP-1 in nTx CBA/J mice infected with 1 x 107 pfu (ARDS) or 1 x 106 pfu (BOOP) reovirus 1/L. (A) Relative differences in mRNA expression of either IFN-{gamma} or MCP-1 over time were determined by comparing the ratio of cytokine/chemokine mRNA with the housekeeping gene L32 from RPA autoradiograms. (open bars) control, uninfected mice; (closed bars) nTx CBA/J mice i.n. infected with 1 x 107 pfu (ARDS) or 1 x 106 pfu (BOOP) reovirus 1/L on Days 5–8 after infection; and (hatched bars) nTx CBA/J mice i.n. infected with 1 x 107 pfu (ARDS) or 1 x 106 pfu (BOOP) reovirus 1/L on Days 9–12/14. *P < 0.05 reovirus 1/L infected animals compared with saline-inoculated, control mice. The mean ± SD of the densitometric measurements from two independent experiments with two mice per time point (four independent autoradiographs) over the above-indicated time points are presented. (B) Detection of IFN-{gamma} or MCP-1 proteins by enzyme-linked immunosorbent assay in the BALF from (closed symbols) normal CBA/J mice i.n. infected with 1 x 106 pfu (BOOP) reovirus 1/L or (open symbols) nTx CBA/J mice i.n. infected with 1 x 106 pfu (BOOP) reovirus 1/L. The mean ± SD of four independent experiments evaluated in duplicate is shown. *P < 0.05 reovirus 1/L-infected animals significantly different from saline-inoculated, control mice.

 

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  		TABLE 1 Interferon-{gamma} and monocyte chemoattractant protein-1 expression in the spectrum of inflammatory lung diseases induced by reovirus 1/L infection

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have utilized a spectrum of inflammatory lung diseases elicited by reovirus 1/L infection to study cellular and molecular events important in the development of pulmonary fibrosis. Using T cell–deficient, neonatally thymectomized (nTx) CBA/J mice, we found that T cells played a major role in the development of intraluminal fibrosis associated with BOOP, but were not involved in the development of intraluminal fibrosis associated with ARDS. T cell–deficient nTx CBA/J mice do not develop intraluminal fibrosis after infection with 1 x 106 pfu (BOOP) reovirus 1/L, unlike their normal littermates. In addition, both the phenotype of the infiltrating cells and the expression of a number of cytokines and chemokines were significantly altered in these nTx CBA/J mice as compared with normal CBA/J mice. In contrast, both normal and nTx CBA/J mice develop intraluminal fibrosis after infection with 1 x 107 pfu (ARDS) reovirus 1/L. A severe pneumonia, characterized primarily by the infiltration of neutrophils and macrophages, the presence of hyaline membranes, and diffuse alveolar damage was observed. In addition, the expression profile for a number of cytokines and chemokines was not altered in nTx CBA/J mice as compared with normal CBA/J mice after infection with 1 x 107 pfu (ARDS) reovirus 1/L. These results suggest that whereas T cells and/or T cell–derived products may play an important role in the development of intraluminal fibrosis associated with BOOP, other cell types and/or their products may initiate the development of intraluminal fibrotic lesions after intense injury, as seen in reovirus 1/L-induced ARDS. Further evaluation of cytokine/chemokine expression after infection of normal or nTx CBA/J mice with either 1 x 107 pfu (ARDS) or 1 x 106 pfu (BOOP) reovirus 1/L suggests that the hierarchy of display of IFN-{gamma} may account for this difference. Thus, we hypothesize that IFN-{gamma} plays a crucial role in the fibrotic process and that elevated levels of IFN-{gamma} are associated with a continuum from least to more severe fibrosis. While our data suggest a key role of IFN-{gamma} in this process, they also suggest that T lymphocytes are not the only population of inflammatory cells involved in the development of pulmonary fibrosis, and that other infiltrating or resident cellular populations may play a key role in this process as well.

Several studies have shown that the infiltration of T lymphocytes may be important in the development of some forms of pulmonary fibrosis, although the data from both animal models and patients has been equivocal. Intratracheal administration of bleomycin in rodents, a process that has been used as a model for IPF, results in the development of an interstitial fibrosis accompanied by a significant infiltration of both T and B lymphocytes (5, 16). In this model, some reports have demonstrated that the inhibition or depletion of lymphocytes by antilymphocyte antibody, mAb to T cell subsets, or treatment with steroids inhibited the development of bleomycin-induced fibrosis (17, 18). However, others have reported no effect of T cell depletion on fibrotic lesions induced by bleomycin instillation (19–21). Similarly, experiments evaluating bleomycin-induced fibrosis in either nude mice lacking T cells or severe combined immunodeficient mice deficient in both T and B cells have also produced conflicting results, demonstrating either a reduction or no effect on the development of interstitial fibrosis (22–24). Currently, few small animal models of BOOP exist. These models typically examine the development of fibrosis as a result of lung transplant rejection where increases in CD4+ and CD8+ T cells, B cells, and macrophages were observed after allograft transplantation (25). In SCID allograft models, no influx of lymphocytes or fibrosis was observed, suggesting that lymphocytes may play an important role in the development of BOOP in a model of chronic graft rejection (26). Our data clearly support a role for T cells in the development of intraluminal fibrosis associated with BOOP because we observed both the lack of fibrotic lesion development in reovirus 1/L-infected nTx CBA/J mice and the ability of corticosteroid treatment of reovirus 1/L-induced BOOP to either inhibit the development or promote the resolution of fibrotic lesions (personal communication). In addition, depletion of T cells with mAb to either CD4 or CD8 also resulted in the inhibition of fibrosis after inoculation with 1 x 106 pfu reovirus 1/L (BOOP) (personal communication), further supporting a role for T cells in the development of BOOP-like fibrotic lesions (personal communication). These results are consistent with clinical findings that two-thirds of the patients with characteristic BOOP fibrosis who are treated with corticosteroids exhibit a clinical recovery (27). In addition, increases in activated T cells have been found in the BALF of patients with clinical cases of BOOP, further suggesting a role for T cells in BOOP pathogenesis (6, 28–31). In contrast to BOOP, a definitive role for T cells in the development of ARDS or ARDS-induced fibrosis has not been established. While treatment with corticosteroids has been shown to be ineffective in preventing or treating ARDS in its early stages, some benefits have been observed when used to treat the later, fibrosing alveolitis phase of the disease (2, 7–10). In reovirus 1/L-induced ARDS, both infection of nTx mice and corticosteroid treatment have shown little effect in either the early inflammatory response or the later fibrotic response (15). Therefore, our models support a role for T lymphocytes in the development of fibrosis associated with BOOP but not with fibrosis associated with ARDS.

Although the process and underlying mechanisms of fibrosis have not been clearly elucidated, the release of cytokines and chemokines from inflammatory cells has been implicated in the development and regulation of fibrosis. In the mouse model of bleomycin-induced interstitial fibrosis, IFN-{gamma} is one cytokine that has been suggested to be important in the fibrotic process. This is supported by the observations that susceptible mouse strains produce high amounts of IFN-{gamma} (20) compared with nonsusceptible mouse strains, depletion of T cells downregulates both IFN-{gamma} expression and fibrosis, (17, 32), and high levels of IFN-{gamma} expression and fibrosis are observed in SCID mice which are susceptible to bleomycin-induced fibrosis (20). Bleomycin treatment of IFN-{gamma} knockout mice (IFN-{gamma} -/-) on a susceptible strain background resulted in both a significant inhibition of pulmonary inflammation and fibrosis (33). IFN-{gamma} has also been implicated in the fibrotic process associated with the tracheal transplant model of BOOP (26, 29, 34) and in lung fibrosis that occurs in patients or animal models with fibrosing alveolitis, IPF, sarcoidosis, chronic beryllium disease, silicosis, and lung allograft fibrosis (35–39). We believe that our data support a role for IFN-{gamma} in the fibrotic process and that it acts as a profibrotic agent in the spectrum of fibrosis induced in reoviurs 1/L-infected mice. In Table 1, we have related the expression of IFN-{gamma} as reported in this study to our previous and ongoing studies involving the spectrum of pathology induced by reovirus 1/L infection of mice. Table 1 shows both the protein concentration in the BALF and the relative level of expression of mRNA in the lungs for IFN-{gamma} in reovirus 1/L-induced models of BOOP (CBA/J; 1 x 106 pfu), and ARDS (CBA/J; 1 x 107 pfu). When infected with 1 x 107 pfu reovirus 1/L, CD-1 mice develop FB, which is characterized by an inflammation of the lung, involving infiltration into both the lung interstitium and the alveolar spaces, mainly composed of large and small lymphocytes and macrophages which organized over time around the bronchioles (11). This model does not have a fibrotic component and the concentration of IFN-{gamma} in the BALF is minimal (unpublished data, 50 pg/ml). As the severity of the inflammatory and fibrotic response increases, there is a corresponding increase in the level of IFN-{gamma} protein detected in the BALF (ARDS > BOOP > FB). Taken together, these data show a direct correlation of IFN-{gamma} expression and the development of fibrosis. Not only are these results consistent with other models of fibrosis (e.g., bleomycin, silica) that propose a profibrotic role for IFN-{gamma}, but they also directly demonstrate that at least a tenfold increase in IFN-{gamma} expression is observed in mice that develop fibrotic lesions as a consequence of reovirus 1/L infection (ARDS, BOOP). This is in contrast to lower levels of IFN-{gamma} in mice that only respond to reovirus 1/L infection with a viral pneumonia without a fibrotic component (FB). A direct evaluation of the role of IFN-{gamma} in the development of fibrosis in reovirus 1/L-induced BOOP or ARDS using neutralizing mAb to IFN-{gamma} is currently underway. Our data also demonstrate an increased expression of mRNA for MCP-1 in both normal (1 x 106 pfu or 1 x 107 pfu reovirus 1/L) and nTx CBA/J mice that develop fibrosis (1 x 107 pfu reovirus 1/L). MCP-1 is expressed in a number of inflammatory conditions in patients that demonstrate a fibrotic component, including ARDS, IPF, systemic sclerosis, and BOOP (5, 40–44), as well as in a number of mouse models of fibrosis (4, 44, 45). In addition, depletion of MCP-1 by treatment with anti–MCP-1 antibodies or by loss of CC chemokine receptor 2 signaling results in a significant reduction in fibro-obliteration (4, 44).

Taken together, our data support the following model of events that may explain the differential role of T lymphocytes in the development of fibrosis associated with BOOP versus the fibrosis associated with ARDS. We propose that a positive feedback loop exists between the expression of MCP-1 and IFN-{gamma} in the fibrotic response associated with reovirus 1/L-induced BOOP and ARDS. We suggest that while the initial infection of resident epithelial cells by reovirus 1/L leads to MCP-1 expression and cellular infiltration, the sustained production of MCP-1 is the result of an autocrine or paracrine mechanism driven by the secretion of IFN-{gamma}. This sustained production of MCP-1 leads to the continued recruitment of inflammatory cells, eventually leading to fibrotic development. If IFN-{gamma} expression is inhibited, as was observed in nTx mice or corticosteroid treatment of normal mice in reovirus 1/L-induced BOOP, decreased expression of MCP-1 is observed, ultimately leading to an inhibition of fibrosis. However, in reovirus 1/L-induced ARDS, where IFN-{gamma} induction is not inhibited, MCP-1 expression is sustained and the fibrotic process ensues. In support of this model, IFN-{gamma} has been shown to induce the expression of MCP-1 from a number of cell types, including macrophages, epithelial cells, vascular endothelial cells, and fibroblasts (45–49). Whereas T cells may be the predominant cell type producing IFN-{gamma} in BOOP that ultimately leads to fibrosis, non-T cells, such as NK cells, may be responsible for the production of IFN-{gamma} in ARDS that ultimately leads to fibrosis. An examination of the differential role of individual cytokines/chemokines, especially IFN-{gamma} and MCP-1 in the spectrum of histopathology observed after reovirus 1/L infection, may lead to a better understanding of the pathogenic process underlying these distinct but related clinical syndromes.


   Acknowledgments
 
The authors thank Ms. Sanja Altman-Hamamdzic for technical assistance, Mr. Bruce H. Yarnell for animal care, Ms. Margaret Romano for the preparation of histologic sections, and the Medical University of South Carolina and the Hollings Cancer Center for their support of the Medical University of South Carolina Analytical Flow Cytometry Facility. This work was supported by U.S. Public Health Service grants AI R01 40175 (L.L.), AI R21 A40175 (L.L.), DE00378 (S.D.L.), and a grant from the American Lung Association (L.L.).

Received in original form May 3, 2002

Received in final form July 22, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Costabel, U., and T. E. King. 2001. International consensus statement on idiopathic pulmonary fibrosis. Eur. Respir. J. 17:163–167.[Free Full Text]
  2. Ware, L. B., and M. A. Matthay. 2000. The acute respiratory distress syndrome. N. Engl. J. Med. 342:1334–1349.[Free Full Text]
  3. Chandler, D. B. 1990. Possible mechanisms of bleomycin-induced fibrosis. Clin. Chest Med. 11:21–30.[Medline]
  4. Smith, R. E., R. M. Strieter, S. H. Phan, and S. L. Kunkel. 1996. C–C chemokines: novel mediators of the profibrotic inflammatory response to bleomycin challenge. Am. J. Respir. Cell Mol. Biol. 15:693–702.[Medline]
  5. Lazo, J. S., Hoyt, D. G., Sebti, S. M., and B. R. Pitt. 1990. Bleomycin: a pharmacologic tool in the study of the pathogenesis of interstitial pulmonary fibrosis. Pharmacol. Ther. 47:347–358.[CrossRef][Medline]
  6. Epler, G. R. 2001. Bronchiolitis Obliterans Organizing Pneumonia. Arch. Intern. Med. 161:158–164.[Abstract/Free Full Text]
  7. Marras, T., M. Herridge, and S. Mehta. 1999. Corticosteroid therapy in acute respiratory distress syndrome. Intensive Care Med. 25:1191–1193.[CrossRef][Medline]
  8. Meduri, G. U., A. S. Headley, E. Golden, S. J. Carson, R. A. Umberger, T. Kelso, and E. A. Tolley. 1998. Effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial. JAMA 280:159–165.[Abstract/Free Full Text]
  9. Bernard, G. R., J. M. Luce, C. L. Sprung, J. E. Rinaldo, R. M. Tate, W. J. Sibbald, K. Kariman, S. Higgins, R. Bradley, C. A. Metz, T. R. Harris, and K. L. Brigham. 1987. High-dose corticosteroids in patients with the adult respiratory distress syndrome. N. Engl. J. Med. 317:1565–1570.[Abstract]
  10. Bone, R. C., C. J. Fisher, Jr., T. P. Clemmer, G. J. Slotman, and C. A. Metz. 1987. Early methylprednisolone treatment for septic syndrome and the adult respiratory distress syndrome. Chest 92:1032–1036.[Abstract/Free Full Text]
  11. Bellum, S. C., D. H. Hamamdzic, A. H. Thompson, R. A. Harley, S. D. London, and L. London. 1996. 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. 74:221–231.[Medline]
  12. Bellum, S. C., D. Dove, R. A. Harley, W. B. Greene, M. A. Judson, L. London, and S. D. London. 1997. Respiratory reovirus 1/L induction of intraluminal fibrosis: a model for the study of bronchiolitis obliterans organizing pneumonia. Am. J. Pathol. 150:2243–2254.[Abstract]
  13. Thompson, A. H., L. London, S. C. Bellum, D. Hamamdzic, R. A. Harley, and S. D. London. 1996. Respiratory-mucosal lymphocyte populations induced by reovirus serotype 1 infection. Cell. Immunol. 169:278–287.[CrossRef][Medline]
  14. London, L., E. I. Majeski, M. K. Paintlia, R. A. Harley, and S. D. London. 2002. Respiratory reovirus 1/L induction of diffuse alveolar damage: a model of acute respiratory distress syndrome. Experimental and Molecular Pathology 72:24–36.[CrossRef][Medline]
  15. London, L., E. I. Majeski, S. Altman-Hamamdzic, C. Enockson, M. K. Paintlia, R. A. Harley, and S. D. London. 2002. 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. 103:284–295.[CrossRef][Medline]
  16. Zhu, J., D. A. Cohen, S. N. Goud, and A. M. Kaplan. 1996. Contribution of T lymphocytes to the development of bleomycin-induced pulmonary fibrosis. Ann. N.Y. Acad. Sci. 796:194–202.[Medline]
  17. Sharma, S. K., J. A. MacLean, C. Pinto, and R. L. Kradin. 1996. The effect of an anti-CD3 monoclonal antibody on bleomycin-induced lymphokine production and lung injury. Am. J. Respir. Crit. Care Med. 154:193–200.[Abstract]
  18. Phan, S. H., R. S. Thrall, and C. Williams. 1981. Bleomycin-induced pulmonary fibrosis: effects of steroid on lung collagen metabolism. Am. Rev. Respir. Dis. 124:428–434.[Medline]
  19. Janick-Buckner, D., G. E. Ranges, and M. P. Hacker. 1989. Effect of cytotoxic monoclonal antibody depletion of T-lymphocyte subpopulations on bleomycin-induced lung damage in C57BL/6J mice. Toxicol. Appl. Pharmacol. 100:474–484.[CrossRef][Medline]
  20. Khalil, N., C. Whitman, L. Zuo, D. Danielpour, and A. Greenberg. 1993. Regulation of alveolar macrophage transforming growth factor-beta secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat. J. Clin. Invest. 92:1812–1818.
  21. Sterling, K. M., T. DiPetrillo, K. R. Cutroneo, and A. Prestayko. 1982. Inhibition of collagen accumulation by glucocorticoids in rat lung after intratracheal bleomycin instillation. Cancer Res. 42:405–408.[Abstract/Free Full Text]
  22. Helene, M., V. Lake-Bullock, J. Zhu, H. Hao, D. A. Cohen, and A. M. Kaplan. 1999. T cell independence of bleomycin-induced pulmonary fibrosis. J. Leukoc. Biol 65:187–195.[Abstract]
  23. Schrier, D. J., S. H. Phan, and B. M. McGarry. 1983. The effects of the nude (nu/nu) mutation on bleomycin-induced pulmonary fibrosis: a biochemical evaluation. Am. Rev. Respir. Dis. 127:614–617.[Medline]
  24. Szapiel, S. V., N. A. Elson, J. D. Fulmer, G. W. Hunninghake, and R. G. Crystal. 1979. Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse. Am. Rev. Respir. Dis. 120:893–899.[Medline]
  25. Ikonen, T., M. Uusitalo, E. Taskinen, A. Korpela, U.S. Salminen, R. E. Morris, and A. L. Harjula. 1998. Small airway obliteration in a new swine heterotopic lung and bronchial allograft model. J. Heart Lung Tranplant. 17:945–953.[Medline]
  26. Neuringer, I. P., R. B. Mannon, T. M. Coffman, M. Parsons, K. Burns, J. R. Yankaskas, and R. M. Aris. 1998. Immune cells in a mouse airway model of obliterative bronchiolitis. Am. J. Respir. Cell Mol. Biol. 19:379–386.[Abstract/Free Full Text]
  27. Cooper, J. D., G. A. Patterson, and E. P. Trulock. 1994. Results of single and bilateral lung transplantation in 131 consecutive recipients. Washington University Lung Transplant Group. J Thorac Cardiovas. Surg. 107:460–470.[Abstract/Free Full Text]
  28. Snell, G. I., D. S. Esmore, and T. J. Williams. 1996. Cytolytic therapy for the bronchiolitis obliterans syndrome complicating lung transplantation. Chest 109:874–878.[Abstract/Free Full Text]
  29. Ross, D. J., A. Moudgil, A. Bagga, M. Toyoda, A. M. Marchevsky, R. M. Kass, and S. C. Jordan. 1999. Lung allograft dysfunction correlates with gamma-interferon gene expression in bronchoalveolar lavage. J. Heart Lung Transplant. 18:627–636.[CrossRef][Medline]
  30. Ross, D. J., S. C. Jordan, S. D. Nathan, R. M. Kass, and S. K. Koerner. 1996. Delayed development of obliterative bronchiolitis syndrome with OKT3 after unilateral lung transplantation: a plea for multicenter immunosuppressive trials. Chest 109:870–873.[Abstract/Free Full Text]
  31. Costabel, U., H. Teschler, and J. Guzman. 1992. Bronchiolitis obliterans organizing pneumonia (BOOP): the cytological and immunocytological profile of bronchoalveolar lavage. Eur. Respir. J. 5:791–797.[Abstract]
  32. Tager, A., A. Luster, and R. Kradin. 1999. T-cell chemokines interferon-inducible protein-10 and monokine induced by interferon-gamma are upregulated in bleomycin-induced lung injury. Chest 116:90S.[Free Full Text]
  33. Chen, E. S., B. M. Greenlee, M. Wills-Karp, and D. R. Moller. 2001. Attenuation of lung inflammation and fibrosis in interferon-gamma-deficient mice after intratracheal bleomycin. Am. J. Respir. Cell Mol. Biol. 24:545–555.[Abstract/Free Full Text]
  34. Boehler, A., X. H. Bai, M. Liu, S. Cassivi, D. Chamberlain, A. S. Slutsky, and S. Keshavjee. 1999. Upregulation of T-helper 1 cytokines and chemokine expression in post-transplant airway obliteration. Am. J. Respir. Crit. Care Med. 159:1910–1917.[Abstract/Free Full Text]
  35. Davis, G. S., L. M. Pfeiffer, and D. R. Hemenway. 2000. Interferon-gamma production by specific lung lymphocyte phenotypes in silicosis in mice. Am. J. Respir. Cell Mol. Biol. 22:491–501.[Abstract/Free Full Text]
  36. Robinson, B. W., and A. H. Rose. 1990. Pulmonary gamma interferon production in patients with fibrosing alveolitis. Thorax 45:105–108.[Abstract/Free Full Text]
  37. Shaw, R. J., S. H. Benedict, R. A. Clark, and T. E. King, Jr. 1991. Pathogenesis of pulmonary fibrosis in interstitial lung disease. Alveolar macrophage PDGF(B) gene activation and up-regulation by interferon gamma. Am. Rev. Respir. Dis. 143:167–173.[Medline]
  38. Awad, M., V. Pravica, C. Perrey, A. El Gamel, N. Yonan, P. J. Sinnott, and I. V. Hutchinson. 1999. CA repeat allele polymorphism in the first intron of the human interferon-gamma gene is associated with lung allograft fibrosis. Hum. Immunol. 60:343–346.[CrossRef][Medline]
  39. Shigehara, K., N. Shijubo, M. Ohmichi, R. Takahashi, S. Kon, H. Okamura, M. Kurimoto, Y. Hiraga, T. Tatsuno, S. Abe, and N. Sato. 2001. IL-12 and IL-18 are increased and stimulate IFN-gamma production in sarcoid lungs. J. Immunol. 166:642–649.[Abstract/Free Full Text]
  40. Iyonaga, K., M. Takeya, N. Saita, O. Sakamoto, T. Yoshimura, M. Ando, and K. Takahashi. 1994. Monocyte chemoattractant protein-1 in idiopathic pulmonary fibrosis and other interstitial lung diseases. Hum. Pathol. 25:455–463.[CrossRef][Medline]
  41. Suga, M., K. Iyonaga, H. Ichiyasu, N. Saita, H. Yamasaki, and M. Ando. 1999. Clinical significance of MCP-1 levels in BALF and serum in patients with interstitial lung diseases. Eur. Respir. J. 14:376–382.[Abstract]
  42. Hasegawa, M., S. Sato, and K. Takehara. 1999. Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis. Clin. Exp. Immunol. 117:159–165.[CrossRef][Medline]
  43. Car, B. D., F. Meloni, M. Luisetti, G. Semenzato, G. Gialdroni-Grassi, and A. Walz. 1994. Elevated IL-8 and MCP-1 in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis. Am. J. Respir. Crit. Care Med. 149:655–659.[Abstract]
  44. Belperio, J. A., M. P. Keane, M. D. Burdick, J. P. Lynch, III, Y. Y. Xue, A. Berlin, D. J. Ross, S. L. Kunkel, I. F. Charo, and R. M. Strieter. 2001. Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome. J. Clin. Investig. 108:547–556.[CrossRef][Medline]
  45. Moore, B. B., R. Paine, III, P. J. Christensen, T. A. Moore, S. Sitterding, R. Ngan, C. A. Wilke, W. A. Kuziel, and G. B. Toews. 2001. Protection from pulmonary fibrosis in the absence of CCR2 signaling. J. Immunol. 167:4368–4377.[Abstract/Free Full Text]
  46. Andoh, A., H. Takaya, J. Makino, H. Sato, S. Bamba, Y. Araki,, K. Hata, M. Shimada, T. Okuno, Y. Fujiyama, and T. Bamba. 2001. Cooperation of interleukin-17 and interferon-gamma on chemokine secretion in human fetal intestinal epithelial cells. Clin. Exp. Immunol. 125:56–63.[CrossRef][Medline]
  47. van der Velden, V. H., M. M. Verheggen, S. Bernasconi, S. Sozzani, B. A. Naber, C. A. van der Linden-van Beurden, H. C. Hoogsteden, A. Mantovani, and M. Versnel. 1998. Interleukin-1 beta and interferon-gamma differentially regulate release of monocyte chemotactic protein-1 and interleukin-8 by human bronchial epithelial cells. Eur. Cytokine Netw. 9:269–277.[Medline]
  48. Bauermeister, K., M. Burger, N. Almanasreh, H. P. Knopf, R. R. Schumann, P. Schollmeyer, and G. J. Dobos. 1998. Distinct regulation of IL-8 and MCP-1 by LPS and interferon-gamma-treated human peritoneal macrophages. Nephrol. Dial. Transplant 13:1412–1419.[Abstract/Free Full Text]
  49. Brown, Z., M. E. Gerritsen, W. W. Carley, R. M. Strieter, S. L. Kunkel, and J. Westwick. 1994. Chemokine gene expression and secretion by cytokine-activated human microvascular endothelial cells: differential regulation of monocyte chemoattractant protein-1 and interleukin-8 in response to interferon-gamma. Am. J. Pathol. 145:913–921.[Abstract]




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