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Inhibition of Pulmonary Fibrosis by the Chemokine IP-10/CXCL10

Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology; Pulmonary and Critical Care Unit; and Immunopathology Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; and Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee

Address correspondence to: Andrew D. Luster, M.D., Ph.D., Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Building 149-8301, 13th Street, Charlestown, MA 02129. E-mail: luster{at}helix.mgh.harvard.edu

	Abstract

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Pulmonary fibrosis is an enigmatic and devastating disease with few treatment options, now thought to result from abnormal wound healing in the lung in response to injury. We have previously noted a role for the chemokine interferon –inducible protein of 10 kD (IP-10)/CXC chemokine ligand 10 in the regulation of cutaneous wound healing, and consequently investigated whether IP-10 regulates pulmonary fibrosis. We found that IP-10 is highly expressed in a mouse model of pulmonary fibrosis induced by bleomycin. IP-10–deficient mice exhibited increased pulmonary fibrosis after administration of bleomycin, suggesting that IP-10 limits the development of fibrosis in this model. Substantial fibroblast chemoattractant and proliferative activities were generated in the lung after bleomycin exposure. IP-10 significantly inhibited fibroblast responses to the chemotactic, but not the proliferative activity generated, suggesting that IP-10 may attenuate fibroblast accumulation in bleomycin-induced pulmonary fibrosis by limiting fibroblast migration. Consistent with this inhibitory activity of IP-10 on fibroblast migration, fibroblast accumulation in the lung after bleomycin exposure was dramatically increased in IP-10–deficient mice compared with wild-type mice. Conversely, transgenic mice overexpressing IP-10 were protected from mortality after bleomycin exposure, and demonstrated decreased fibroblast accumulation in the lung after challenge compared with wild-type mice. Our findings suggest that interruption of fibroblast recruitment may represent a novel therapeutic strategy for pulmonary fibrosis, which could have applicability to a wide range of fibrotic illnesses.

Abbreviations: bronchoalveolar lavage fluid, BALF • basic fibroblast growth factor, bFGF • CXC chemokine ligand, CXCL • CXC chemokine receptor, CXCR • Dulbecco's modified Eagle's medium, DMEM • ethylenediaminetetraacetate, EDTA • epidermal growth factor, EGF • enzyme-linked immunosorbent assay, ELISA • fetal bovine serum, FBS • fibroblast-specific protein, FSP • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • glycosaminoglycan, GAG • interleukin, IL • interferon –inducible protein of 10 kD, IP-10 • interferon-inducible T cell alpha chemoattractant, I-TAC • knockout, KO • monokine induced by interferon , Mig • natural killer, NK • phosphate-buffered saline, PBS • polymerase chain reaction, PCR • phycoerythrin, PE • T helper, Th • von Willebrand Factor, vWF • wild-type, WT

	Introduction

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Pulmonary fibrosis is a progressive fibrosing interstitial pneumonia of unknown etiology and poor prognosis, with a mean survival in the range of 2 to 4 years (1). Recently, pulmonary fibrosis has been proposed to result from abnormal wound healing in the lung in response to injury to the alveolar epithelium (2). The biologic response to injury is extremely complex, requiring the coordinated proliferation and migration of multiple cell types, including leukocytes, epithelial cells, endothelial cells, and fibroblasts. The migration of leukocytes is regulated by members of the chemokine superfamily of chemoattractant cytokines (3). Chemokine expression is tightly regulated after injury (4), with different chemokines being expressed during the sequential phases of wound healing, which are classically described as acute inflammation, granulation tissue formation, and tissue remodeling. Chemokines expressed during the acute inflammatory response perform their well described role of recruiting leukocytes to injured tissue. The expression of the neutrophil chemokines interleukin (IL)-8/CXC chemokine ligand (CXCL) 8 and growth-regulated oncogene (Gro)/CXCL1 peaks during the first day after cutaneous wounding in humans, and correlates temporally and spatially with neutrophil infiltration (5). The monocyte–macrophage chemokine monocyte chemoattractant protein 1/CC chemokine ligand 2 is maximally expressed during the second day after injury, paralleling peak macrophage infiltration (5). Chemokines expressed later after injury, at the time of granulation tissue formation and wound remodeling, are well situated to participate in these processes as well, but this has not been examined in any detail. Expression of the chemokines interferon (IFN) –inducible protein of 10 kD (IP-10)/CXCL10 and monokine-induced by IFN (Mig)/CXCL9, which both bind the same receptor, CXC chemokine receptor (CXCR) 3, and are chemotactic for T cells and natural killer (NK) cells, becomes evident 4 days after cutaneous injury (5), at which time dermal fibroblast migration into the wound clot is occurring (6). We have previously noted a role for IP-10 in the regulation of cutaneous wound healing (7), and consequently investigated whether IP-10 plays a role in the regulation of pulmonary fibrosis in a well characterized animal model of pulmonary fibrosis induced by bleomycin.

	Materials and Methods

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Animals and Bleomycin Administration
Wild-type C57Bl/6 mice used were purchased from Charles River Breeding Laboratories (Wilmington, MA). Experiments comparing IP-10–deficient and wild-type mice used age- and sex-matched groups of IP-10–deficient mice, generated in our laboratory by targeted gene disruption (8), and littermate wild-types. These mice were produced by matings between mice heterozygous for the IP-10 mutant allele that were F1 hybrids of the C57Bl/6 and 129Sv/J genetic backgrounds. Experiments comparing IP-10–transgenic and wild-type mice used mice overexpressing mouse IP-10 driven by the bovine keratin 5 promoter that were generated on the FVB background in our laboratory (7), and age- and sex-matched FVB wild-types purchased from Charles River Breeding Laboratories. A dosage of 0.075 units of bleomycin (Bristol-Myers Squibb Co., Princeton, NJ) in 100 µl sterile saline was injected intratracheally in all experiments except those investigating leukocyte recruitment, angiogenesis, and fibroblast accumulation in IP-10–transgenic and FVB wild-type mice, in which a dosage of 0.020 units was used. All experiments were performed in accordance with National Institutes of Health guidelines and protocols approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.

Bronchoalveolar Lavage Fluid
Lungs were lavaged with six 0.5 ml aliquots of phosphate-buffered saline (PBS) without calcium and magnesium. Bronchoalveolar lavage fluid (BALF) recovered from the first 1 ml of instilled PBS was collected separately from the rest of the BALF and retained for subsequent analysis of chemoattractant activity and IP-10 concentration. Both BALF fractions were centrifuged at 540 x g at 4°C, and the pelleted cells from both fractions were pooled for analysis.

Recovery of Lung Leukocytes
Lungs were digested for 45 min at 37°C in RPMI with 0.28 U/ml Liberase Blendzyme 3 (Roche, Indianapolis, IN) and 60 U/ml DNase I (Roche), passed through a 70 µm filter, centrifuged at 540 x g at 4°C, and resuspended in PBS with 2 mM EDTA and 0.5% BSA. Cells were incubated with anti-mouse CD45 antibody–linked magnetic beads (Miltenyi, Auburn, CA) for 15 min at 10°C. CD45+ cells were then selected using high-gradient magnetic separation columns (Miltenyi) according to the manufacturer's instructions.

Cytofluorimetry
Cells were incubated for 10 min with 2.4G2 anti-FcIII/II receptor (BD Pharmingen, San Diego, CA). Cells recovered from BALF or lung were then stained with APC-conjugated anti-mouse CD3 and FITC-conjugated anti-mouse CD4 monoclonal antibodies, and either phycoerythrin (PE)-conjugated anti-mouse CD8, PE-conjugated anti-mouse NK-1.1, or PE-conjugated anti-mouse CD49b monoclonal antibodies (BD Pharmingen) at 4°C for 20 min. Cytofluorimetry was performed using a FACS Caliber Cytometer (Becton-Dickinson, San Jose, CA) and analyzed using CellQuest software (Becton-Dickinson).

Histopathologic and Immunohistochemical Examination
Lungs excised for histopathology were fixed with 10% buffered formalin. Multiple paraffin-embedded 5-µm sections of the entire mouse lung were stained with hematoxylin-eosin or Masson's trichrome. Sections for fibroblast-specific protein (FSP) 1 immunostaining were treated with xylene for deparaffinization and rehydrated through a graded series of ethanols. Sections were then pretreated with 0.01% trypsin in PBS for 10 min and incubated with 1% BSA for 20 min. Sections were treated with primary rabbit biotinylated polyclonal antibody to FSP1 (9) for 2 h, and stained by an indirect avidin–biotin immunoperoxidase method (Vectostain ABC kit, Vector Laboratories, Inc., Burlingame, CA). Semiquantitative analysis of FSP1 staining was performed using a subjective grade system. Fields were graded as follows: 0 = no FSP1+ cells in the field; 1 = a few scattered FSP1+ cells; 2 = substantially increased numbers of FSP1+ cells, especially in areas of fibrosis; 3 = numerous clumps of FSP1+ cells, especially in areas of fibrosis; and 4 = diffuse involvement with many FSP1+ cells in the field. Ten nonoverlapping fields were analyzed for each lung section by a pathologist blinded to the mouse genotype.

Hydroxyproline Assay
Lungs were homogenized in 2 ml of PBS, and a 1 ml aliquot was desiccated and then hydrolyzed in 6 N HCl at 110°C for 12 h. Twenty five microliter aliquots were added to 1 ml of 1.4% chloramine T (Sigma, St. Louis, MO), 10% n-propanol, and 0.5 M sodium acetate, pH 6.0. After 20 min of incubation at room temperature, 1 ml of Erlich's solution (1 M p-dimethylaminobenzaldehyde [Sigma] in 70% n-propanol, 20% perchloric acid) was added and a 15 min incubation at 65°C performed. Absorbance was measured at 550 nm and the amount of hydroxyproline was determined against a standard curve.

RNA Analysis
Total cellular RNA was isolated from the lungs and Northern blotting was performed as previously described (8). Signal intensity was determined using a phosphoimager (Molecular Imager System; BioRad, Hercules, CA). Quantitative real-time polymerase chain reaction (PCR) RNA analysis was performed using an Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA) as previously described (10).

Western Blot Analysis
Total cellular protein was isolated from the lungs by homogenizing the tissue in lysis buffer (50 mM Tris, pH 7.5, 1 mM ethylenediaminetetraacetate [EDTA], 1 mM dithiothreitol [DTT], 150 mM sodium chloride, Nonidet P-40 [NP-40] 1%, sodium deoxycholate [DOC] 0.5%, sodium dodecyl sulfate 0.1% and Triton-X100 1%) supplemented with antiproteases (0.1 mg/ml pepstatin A, 0.03 mM leupeptin, 145 mM benzamidine, 0.37 mg/ml aprotinin, and 1 mM phenylmethylsulphonylfluoride [PMSF], all from Sigma). To analyze IP-10 protein expression, 150 µg of total protein of each sample was boiled for 5 min in tricine sample buffer (BioRad) and 4M urea, and fractionated on a 16.5% peptide Criterion precast gel (BioRad) run in electrophoresis buffer containing 0.1M Tris, 0.1M Tricine, and 3.5 mM SDS. Proteins were transferred onto a polyvinylidene difluoride membrane (NEN, Boston, MA) and IP-10 was identified by sequentially incubating the membrane with a 1:10,000 dilution of an affinity-purified polyclonal rabbit anti-mouse IP-10 antibody (11), followed by a 1:5,000 dilution of a horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin antibody (BioRad), and developed using an ECL kit (Amersham Pharmacia, Piscataway, NJ). To analyze von Willebrand Factor (vWF) protein expression, 50 µg of total protein of each sample was boiled for 5 min in Laemmli sample buffer (BioRad) and 4M urea, fractionated on a 5% Tris-HCl Criterion precast gel (BioRad), and run in electrophoresis buffer containing 1.33 mM glycine, 24.8 mM Tris, and 3.5 mM SDS. vWF was identified by incubating the membrane with a 1:200 dilution of rabbit anti-human vWF antiserum (Sigma) followed by horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin antibody.

Enzyme-Linked Immunosorbent Assay Analysis
IP-10 concentrations in the lungs and BALF were assayed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Lungs were homogenized and sonicated in PBS containing antiproteases (2 mM PMSF and 1 µg/ml each of antipain, aprotinin, pepstatin A, and leupeptin). Homogenates were centrifuged at 900 x g for 15 min, and the supernatants were filtered through 1.2 µm pore size Sterile Acrodiscs (Gelman Sciences, Ann Arbor, MI).

BALF Dilution of Epithelial Lining Fluid
The degree to which the process of collecting BALF dilutes epithelial lining fluid was determined using urea as a marker of dilution, as described by Rennard and colleagues (12). Serum and BALF urea concentrations were determined using a commercially available kit (Pointe Scientific, Lincoln Park, MI) according to the manufacturer's instructions.

Matrigel Assay of Angiogenic Activity
Angiogenic activity in the lungs of wild-type, IP-deficient, and IP-10–transgenic mice was assayed by the matrigel bioassay as described by Passaniti and colleagues (13). Lung homogenates were prepared as above, and then concentrated 10-fold by lyophilization (Speed-Vac; Savant, Farmindale, NY). Aliquots of 0.5 ml of liquid matrigel (phenol red–free; BD Bioscience, Bedford, MA) at 4°C were impregnated with one of the following: nothing, 150 ng basic fibroblast growth factor (bFGF, R&D Systems), or wild-type, IP-10–deficient or IP-10–transgenic lung homogenate containing 750 µg total protein, and then injected subcutaneously into the abdominal midline of C57Bl/6 mice. At body temperature, the injected matrigel aliquots solidified rapidly into gel pellets, which were harvested from the mice 7 d after injection. Angiogenesis was quantified by determining the hemoglobin content of the recovered pellets. The pellets were minced and then liquefied by incubation with 300 µl dispase (BD Bioscience) at 37°C for 3 h. Hemoglobin content of the liquefied pellets was determined by the Drabkin method (Hemoglobin Diagnostic Kit; Sigma).

Isolation of Primary Lung Fibroblasts
Lungs from unchallenged C57Bl/6 mice, and mice 5 and 14 d after bleomycin challenge, were digested for 45 min at 37°C in RPMI with 0.28 U/ml liberase blendzyme 3 and 60 U/ml DNase I, passed through a 70 µm filter, centrifuged at 540 x g at 4°C, and plated in tissue culture flasks in Dulbecco's modified Eagle's medium (DMEM) with 15% fetal bovine serum (FBS). Cells were passaged when subconfluent after harvest with trypsin-EDTA (Cellgro, Herndon, VA). Cells were used for experiments at passages 3 and 4.

Fibroblast Chemotaxis
After being serum-starved in DMEM without FBS overnight, subconfluent NIH-3T3 cells or primary lung fibroblasts were harvested with trypsin-EDTA and 50 µl of cells at 5.0 x 10⁵ cells/ml in DMEM were placed in the top of a 48-well modified Boyden microchemotaxis chamber (NeuroProbe, Gaithersburg, MA). 30 µl of 8-fold dilutions of BALF in DMEM were placed in the bottom wells of the chamber and separated from the cells by a 8-µm-pore, polyvinylpyrrolidone-free polycarbonate filter (Osmonics, Westboro, MA) that had been coated with fibronectin (Invitrogen, Carlsbad, CA). For IP-10 inhibition experiments, 10-fold serial dilutions of mouse IP-10 (PeproTech, Rocky Hill, NJ, or produced in our laboratory) were placed in the top and bottom wells of the chamber, with BALF also placed in the bottom wells. The apparatus was incubated at 37°C and 5% carbon dioxide for 3 to 4 h, and cells migrating across the filter and adhering to the bottom side of the filter were stained with Hema 3 stain set (Fisher Diagnostics, Middletown, VA) and counted.

Fibroblast Proliferation
NIH-3T3 cells were seeded into 96-well plates (1 x 10³ cells/well) in DMEM with 10% FBS. After 24 h in culture, cells were serum-starved in DMEM without FBS overnight. 100 µl of 8-fold dilutions of BALF in DMEM were then added to the cells along with increasing concentrations of IP-10 for a total of 48 h. Proliferation was determined by incorporation of [methyl-³H]thymidine (DuPont-NEN, Boston, MA) during the final 18 h of culture.

Statistical Analysis
Differences in hydroxyproline content, leukocyte subsets, gene and protein expression, fibroblast chemotaxis, and fibroblast proliferation were analyzed by Student's t test using Excel software (Microsoft, Redmond, WA). Differences in survival were analyzed by log-rank test. P < 0.05 was considered significant in all comparisons.

	Results

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IP-10 Is Induced in the Lung after Bleomycin Administration
After bleomycin challenge of C57Bl/6 wild-type mice, IP-10 mRNA expression was induced 7-fold over baseline levels at 1 d, 26-fold at 3 d, and 33-fold at 5 d. IP-10 expression then diminished during the second and third weeks after challenge (Figures 1A and 1B). Of the two other ligands for CXCR3, Mig and IFN-inducible T cell chemoattractant (I-TAC), Mig mRNA was induced in a similar time course, but to a lesser extent than IP-10. Mig mRNA expression peaked 5 d after bleomycin administration, when its expression was induced 14-fold over baseline (Figures 1A and 1B). I-TAC mRNA was not induced after bleomycin administration (Figure 1A). In analogous experiments performed with wild-type mice that were mixed F1 hybrids of the C57Bl/6 and S129Sv/J genetic backgrounds, which was the genetic background of the IP-10–deficient and wild-type littermate control animals used in this study, we found IP-10 mRNA expression was induced by bleomycin challenge to a similar extent, and with similar kinetics, as in C57Bl/6 wild-types (data not shown). We then determined by Western blot the expression of IP-10 protein in the lungs of mice at baseline and from 1 d to 2 wk after bleomycin administration. IP-10 protein was induced at 3 and 5 d after bleomycin administration in C57Bl/6 wild-type mice (Figure 1C), corresponding to maximal mRNA expression. IP-10 protein expression was not detectable at baseline or after intratracheal saline administration. The specificity of the Western blot was demonstrated by the absence of IP-10 protein found in the lungs of IP-10–deficient mice 5 d after bleomycin administration, the time of maximal IP-10 protein expression in wild-type mice (Figure 1C).

View larger version (44K): [in this window] [in a new window]   Figure 1. Expression of IP-10, Mig, I-TAC, and CXCR3 mRNA, and IP-10 protein after bleomycin administration in C57Bl/6 wild-type mice. (A) Northern blot analysis of 10 µg of total RNA isolated from the lungs of C57Bl/6 mice at the indicated times after bleomycin administration (B) or saline (S). Each lane represents the RNA from a single mouse lung from a representative experiment. The Northern blot was sequentially hybridized with IP-10, Mig, I-TAC, CXCR3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs, and exposed on film for the following times: IP-10 and Mig, 1 wk; I-TAC, 2 wk; CXCR3, 10 wk; and GAPDH, 24 h. The ethidium bromide–stained agarose gel is presented to demonstrate comparability of RNA loading across the time course. (B) Phosphoimager analysis was used to quantify the IP-10 and Mig signals for each sample. Data are presented as the mean level of IP-10 or Mig expression relative to expression at baseline (bleomycin challenge, n = 4 mice/group; saline challenge, n = 2 mice/group. Dark gray bars, IP-10–bleomycin; closed bars, IP-10–saline; light gray bars, Mig–bleomycin; open bars, Mig–saline). (C) Western blot analysis of IP-10 in 150 µg of total protein isolated from the lungs of C57Bl/6 mice at the indicated times after administration of bleomycin or saline. Each lane represents the protein from a single mouse lung from a representative experiment. A total of 200 pg of recombinant mouse IP-10 was used as a positive control, and 150 µg of total protein from the lung of an IP-10–deficient mouse 5 d after bleomycin administration was used as a negative control. (D) ELISA analysis of IP-10 in lung homogenates from unchallenged wild-type (WT) mice (0 d, n = 3 mice/group; 5 d, n = 5 mice/group; and 14 d, n = 4 mice/group) after bleomycin challenge, and IP-10–deficient (IP-10 knockout [KO]) mice (5 and 14 d after bleomycin challenge, n = 3 mice/group). For this analysis, a single homogenate was made for each mouse from both of its lungs harvested together. Data are presented as mean amount of IP-10 (pg) ± SEM. No IP-10 was detected in lungs of IP-10 KO mice at either time after bleomycin administration (none detected [ND]). Significant differences: *P < 0.05, WT 5 and 14 d after bleomycin administration versus WT unchallenged. (E) ELISA analysis of IP-10 in BALF from unchallenged WT mice (n = 5 mice/group), WT mice 5 d (n = 8 mice/group) and 14 d (n = 9 mice/group) after bleomycin challenge, and IP-10 KO mice 5 and 14 d after bleomycin challenge (n = 3 mice/group). Data are presented as mean concentration of IP-10 (pg/ml) ± SEM. No IP-10 was detected in BALF of IP-10 KO mice at either time after bleomycin administration. Significant differences: *P < 0.01, WT 5 and 14 d after bleomycin administration versus WT unchallenged.

Bleomycin-Induced Fibrosis Is Increased in the Absence of IP-10
Fibroblast and extracellular matrix accumulation begins 4 to 14 d after bleomycin administration, such that lungs examined 14 d after challenge typically demonstrate patchy peribronchiolar and parenchymal fibrosis (14). The extent of these changes 14 d after bleomycin administration was substantially increased in IP-10–deficient mice generated in our laboratory in a hybrid C57Bl/6–129Sv/J genetic background, compared with wild-type littermate control animals, as demonstrated by Masson's trichrome staining (Figures 2A and 2B). To quantify the increase in fibrosis that we observed in the IP-10–deficient mice, we compared the amounts of collagen present in the lungs of IP-10–deficient and wild-type mice by determining their hydroxyproline content. Lung hydroxyproline content was comparable in IP-10–deficient and wild-type mice at baseline, but the increase in lung hydroxyproline content induced by bleomycin was significantly greater in IP-10–deficient mice than in wild-type control animals (P = 0.0027; Figure 2C). This increase in fibrosis observed in mice lacking IP-10 demonstrates that IP-10 limits the development of pulmonary fibrosis in this model.

View larger version (35K): [in this window] [in a new window]   Figure 2. Histologic and biochemical analysis of bleomycin-induced pulmonary fibrosis in WT and IP-10 KO mice. Lungs of C57Bl/6–129Sv/J hybrid WT (A) and C57Bl/6–129Sv/J hybrid IP-10 KO mice (B) 14 d after bleomycin administration, stained with Masson's trichrome, demonstrate patchy peribronchiolar and parenchymal fibrosis, greater in lungs of IP-10 KO mice (magnification x150). (C) Hydroxyproline content was measured in the lungs of WT (closed bars) and IP-10 KO (open bars) mice at baseline and 14 d after bleomycin administration (WT baseline, n = 4 mice/group; IP-10 KO baseline, n = 3; WT bleomycin, n = 17 mice/group; IP-10 KO bleomycin, n = 16 mice/group). Data are expressed as mean hydroxyproline content per gram of lung tissue ± SEM. Significant differences: *P < 0.005, IP-10 KO after bleomycin exposure versus WT after bleomycin exposure.

View larger version (27K): [in this window] [in a new window]   Figure 3. Lung T cells, NK cells, and NKT cells, and expression of Th1 and Th2 cytokines after bleomycin administration in WT (closed bars) and IP-10 KO (open bars) mice. (A) Percentages of lung leukocytes that were T cells (CD3+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), NK cells (NK-1.1+CD3–) and NKT cells (NK-1.1+CD3+) were determined by flow cytometry in WT and IP-10 KO mice 5 d after bleomycin administration. Data are expressed as mean cell percentages ± SEM (WT, n = 8 mice/group; IP-10 KO, n = 7 mice/group). No differences between WT and IP-10 KO mice were significant. Levels of IFN (B), IL-4 (C), and IL-13 (D) mRNA were measured by real-time quantitative PCR in WT and IP-10 KO mice at 0, 5, and 14 d after bleomycin administration. Data are presented as mean number of copies of cytokine mRNA relative to copies of GAPDH mRNA ± SEM. (WT, n = 3 mice/group; IP-10 KO, n = 3 mice/group). No differences between WT and IP-10 KO mice were significant.

Lung Tissue–Derived Angiogenic Activity and vWF Expression after Bleomycin Administration Is Not Altered by the Absence of IP-10
The growth of new blood vessels has been proposed to be a rate-limiting step in the development of pulmonary fibrosis, such that alterations in the relative expression of pro- and antiangiogenic factors may alter the extent of pulmonary fibrosis that develops after a given fibrogenic stimulus (22). IP-10 can inhibit angiogenesis (11, 23, 24), and consequently its absence could contribute to the increase in bleomycin-induced fibrosis observed in the IP-10–deficient mice by increasing angiogenesis. We investigated this hypothesis by comparing the aggregate lung tissue–derived angiogenic activity present in IP-10–deficient and wild-type mice after bleomycin administration using the matrigel assay of neovascularization (13). In addition, comparisons were made of the expression of the endothelial cell–associated protein vWF (factor VIII–related antigen) in IP-10–deficient and wild-type mice after bleomycin administration using Northern analysis and Western blotting. We found comparable amounts of lung tissue–derived angiogenic activity were present in IP-10–deficient and wild-type mice after bleomycin administration. Lung homogenates of IP-10–deficient and wild-type mice harvested 5 d after bleomycin administration both produced significantly greater amounts of neovascularization in the matrigel assay than matrigel alone, but the angiogenic activities of wild-type and IP-10–deficient lung homogenates were not significantly different from each other (Figure 4A). vWF mRNA was induced from 5 to 14 d after bleomycin administration comparably in IP-10–deficient and wild-type mice, with expression increasing between 1.8- and 2.3-fold over baseline in the wild-type mice, and between 2.2- and 2.6-fold in the IP-10–deficient mice (Figures 4B and 4C). vWF protein expression was also comparable in the lungs of IP-10–deficient and wild-type mice, although expression of the protein was not appreciably induced after bleomycin administration in either genotype (Figure 4D). In separate experiments, we also saw comparable expression of another endothelial cell–associated protein, platelet–endothelial cell adhesion molecule (CD31), by immunostaining in fibrotic areas of the lungs of IP-10–deficient and wild-type mice killed 2 weeks after bleomycin administration (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 4. Lung tissue–derived angiogenic activity and expression of vWF mRNA and protein after bleomycin administration in WT and IP-10 KO mice. (A) The matrigel bioassay was used to quantify the aggregate angiogenic activity contained in lung homogenates (750 µg total protein) from WT and IP-10 KO mice 5 d after bleomycin administration (n = 3 mice/group, both groups). A total of 150 ng bFGF was used as a positive control. Significant differences: *P < 0.05, bFGF (light gray bar), WT lung homogenates (closed bar), and IP-10 KO lung homogenates (open bar) versus matrigel alone (dark gray bar). Angiogenic activities of WT and IP-10 KO lung homogenates were not significantly different (NS). (B) Northern blot analysis of 10 µg of total RNA isolated from the lungs of WT and IP-10 KO mice at the indicated times after bleomycin administration. Each lane represents the RNA from a single mouse lung from a representative experiment. The Northern blot was sequentially hybridized with vWF and GAPDH cDNAs, and exposed on film for the following times: vWF, 16 d; GAPDH, 3 h. (C) Phosphoimager analysis was used to quantify the vWF mRNA signals for each sample. Data are presented as the mean level of vWF expression relative to expression at baseline ± SEM (n = 3 mice/group for each genotype at each time point). No differences between WT (closed bars) and IP-10 KO (open bars) mice were significant. (D) Western blot analysis of vWF in 50 µg of total protein isolated from the lungs of WT and IP-10 KO mice at the indicated times after bleomycin administration. Each lane represents the protein from a single mouse lung from a representative experiment. A total of 50 µg of total protein isolated from mouse pulmonary endothelial cells (EC) was used as a positive control, and 50 µg of mouse NIH-3T3 fibroblast (FB) lysate was used as a negative control.

View larger version (46K): [in this window] [in a new window]   Figure 5. Fibroblast chemotactic activity generated in the lung after bleomycin administration and inhibition by IP-10. (A–C) Light microscope analysis of the migration of NIH-3T3 mouse fibroblasts in a modified Boyden microchemotaxis chamber toward (A) BALF from an unchallenged mouse, (B) BALF from a mouse 5 d after bleomycin administration, and (C) the same BALF as in (B) with 1 µg/ml IP-10 added. Data shown are from a representative experiment (n = 3, magnification x400). (D) Time course of NIH-3T3 fibroblast chemotactic activity induced after bleomycin administration and inhibition by IP-10. Data shown are from a representative experiment (n = 3) and are presented as mean chemotactic index (cells per four high-power fields counted in duplicate wells moving in response to BALF [gray bar] or BALF + IP-10 [closed bar] relative to cells moving in response to media control) ± SEM. BALF, n = 3 mice/group; BALF + 1 µg/ml IP-10, n = 3 mice/group for each time point. Significant differences: *P < 0.005 BALF Days 5 and 14 versus BALF Day 0; **P < 0.05 BALF versus BALF + IP-10, Days 5 and 14. (E) Dose-dependent inhibition by IP-10 at the indicated concentrations of primary mouse lung fibroblast chemotaxis induced by BALF from a mouse 14 d after bleomycin administration. Data shown are from a representative experiment (n = 2) and are presented as chemotactic index ± SEM. Significant differences: *P < 0.05 BALF versus BALF + 1 ng/ml IP-10, BALF + 10 ng/ml IP-10, BALF + 100 ng/ml IP-10, and BALF + 1,000 ng/ml IP-10. (F) Lack of inhibition by IP-10 at the indicated concentrations of Day 5 after bleomycin BALF–induced NIH-3T3 fibroblast proliferation. Data shown are from a representative experiment (n = 3) and are presented as proliferative index (counts per minute [CPM] incorporated into cells proliferating in response to BALF or BALF + IP-10 counted in triplicate wells relative to CPM incorporated into cells proliferating in media control) ± SEM. No differences between BALF and BALF + IP-10 were significant. (E and F) Open bars, media + IP-10; closed bars, BALF + IP-10.

BALF from mice either 5 or 14 d after bleomycin administration also potently induced proliferation of NIH-3T3 mouse fibroblasts. In contrast to chemotaxis, however, IP-10 had no effect on BALF-induced fibroblast proliferation, even at an IP-10 concentration 10-fold higher than the highest concentration used in our chemotaxis assays (Figure 5F). This absence of an effect on proliferation indicates that the ability of IP-10 to inhibit fibroblast chemotaxis was not due to cell toxicity.

Given the ability of IP-10 to inhibit fibroblast motility, we hypothesized that fibroblast recruitment into sites of lung injury after bleomycin administration would be increased in the absence of IP-10. To investigate this hypothesis, we performed immunoperoxidase staining of the lungs of IP-10–deficient and wild-type mice after bleomycin administration with anti-FSP1 antibody (Figures 6A and 6B). FSP1 is a fibroblast-specific protein in the S100 class of cytoplasmic calcium-binding proteins that was identified by subtractive hybridization of mouse fibroblasts and isogenic epithelium (9). Semiquantitative analysis of anti-FSP1 staining revealed that the numbers of FSP1+ cells increased 14 d after bleomycin administration in the lungs of both IP-10–deficient and wild-type mice, but that the increase in FSP1+ cells was dramatically greater in the IP-10–deficient mice (P = 0.0013; Figure 6C), consistent with greater numbers of fibroblasts being recruited to the injured lung after bleomycin administration in the absence of IP-10. In separate experiments, we found similar results after staining with ER-TR7 (27), another fibroblast-specific antibody (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 6. Accumulation of FSP1+ fibroblasts after bleomycin challenge in WT and IP-10 KO mice. Lungs of WT (A) and IP-10 KO mice (B) 14 d after bleomycin administration, stained with anti-FSP1 antibody/peroxidase (magnification x400), demonstrate increased fibroblasts in lungs of IP-10 KO mice. Sections shown are from a representative experiment. (C) The numbers of FSP1+ cells were scored semiquantitatively in 10 nonoverlapping fields in lung sections of WT (closed bars) and IP-10 KO (open bars) mice at baseline and 14 d after bleomycin administration (n = 3 mice/group, all groups). Data are expressed as mean FSP1+ staining score ± SEM. Significant differences: *P < 0.005, IP-10 KO after bleomycin challenge versus WT after bleomycin challenge.

Transgenic Mice Overexpressing IP-10 Are Protected from Mortality after Bleomycin Administration
To further confirm the role of IP-10 in limiting bleomycin-induced pulmonary fibrosis in vivo, we have performed experiments with IP-10 transgenic mice generated in our laboratory to overexpress IP-10 in epithelial cells using the bovine keratin 5 promoter (7). We have previously demonstrated that these mice overexpress IP-10 protein in the lungs at baseline and in a model of allergic pulmonary inflammation (28), and confirmed by quantitative real-time PCR mRNA analysis that they overexpress IP-10 in the lungs after bleomycin administration ( 20-fold compared with wild-type mice, data not shown). These mice are in the FVB genetic background, which we found to have substantially greater mortality after bleomycin challenge than mice in either C57Bl/6 or C57Bl/6–129Sv/J hybrid backgrounds. However, the survival after bleomycin administration of the IP-10–transgenic mice was significantly increased compared with FVB wild-type mice (P = 0.041; Figure 7A). This result is consistent with IP-10 having a protective role in this model, limiting the pulmonary pathology occurring after bleomycin administration.

View larger version (50K): [in this window] [in a new window]   Figure 7. Survival and lung leukocytes, angiogenic activity, and fibroblast accumulation in WT and IP-10–transgenic mice after bleomycin challenge. (A) FVB WT and FVB IP-10–transgenic mice were followed for 25 d after challenge with 0.075 units of bleomycin (solid line, WT, n = 13 mice/group; dashed line, IP-10–transgenic, n = 14 mice/group). Significant difference: P < 0.05, IP-10–transgenic versus WT. (B) Percentages of lung leukocytes that were T cells (CD3+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), and NK cells (CD49b+) were determined by flow cytometry in WT (closed bars) and IP-10–transgenic (open bars) mice 5 d after 0.020 units bleomycin. Data are expressed as mean cell percentages ± SEM (n = 7 mice/group, both groups). No differences between WT and IP-10–transgenic mice were significant. (C) Angiogenic activities contained in lung homogenates (750 µg total protein) from WT and IP-10–transgenic mice 5 d after 0.020 units bleomycin were quantified by the matrigel bioassay (n = 3 mice/group, both groups). A total of 150 ng bFGF (light gray bar) was used as a positive control. Angiogenic activities of WT (closed bar) and IP-10–transgenic (open bar) lung homogenates were not significantly different (NS) (dark gray bar, matrigel only). (D) The numbers of FSP1+ cells were scored semiquantitatively in 10 nonoverlapping fields in lung sections of WT (closed bar) and IP-10–transgenic (open bar) mice 14 d after administration of 0.020 units bleomycin (n = 3 mice/group, all groups). Data are expressed as mean FSP1+ staining score ± SEM. Significant difference: *P < 0.02, IP-10–transgenic versus WT mice. Examples of lung sections stained with anti-FSP1 antibody/peroxidase (magnification x400) from WT (E) and IP-10–transgenic mice (F) 14 d after administration of 0.020 units bleomycin demonstrate decreased fibroblasts in lungs of IP-10–transgenic mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our study demonstrates that IP-10 expression is induced dramatically in the lung after exposure to bleomycin. Functionally, IP-10 limits the extent of pulmonary fibrosis that develops in this model, as indicated by the increase in bleomycin-induced fibrosis that we observed in mice genetically deficient for IP-10. Consistent with this observation, we found mice genetically overexpressing IP-10 had the opposite phenotype: these mice were protected from mortality after bleomycin administration. Similar to our finding of increased survival in these transgenic mice overexpressing IP-10, intraperitoneal administration of exogenous IP-10 has previously been shown to limit the development of bleomycin-induced pulmonary fibrosis, although these investigators presented evidence that exogenous IP-10 functioned to inhibit fibrosis-associated angiogenesis (29).

In our investigation of IP-10's mechanism of action in this model, we were surprised to find no effect of IP-10 deficiency on either of the activities traditionally described as being regulated by IP-10: leukocyte recruitment or angiogenesis. Unexpectedly, we found no difference between IP-10–deficient and wild-type mice in the recruitment of leukocytes known to express CXCR3, including T cells, NK cells, and NKT cells, into the lung after bleomycin challenge. Leukocyte recruitment was comparable between IP-10–deficient and wild-type mice both 5 days after bleomycin exposure, the time of peak lung IP-10 expression, and 14 days after bleomycin exposure, the time at which fibrosis was analyzed in our experiments, though it is possible that leukocyte recruitment differed at other time points. However, we saw no significant increase in CXCR3 expression in the lungs of either wild-type or IP-10–deficient mice at any of multiple time points examined after bleomycin administration. Further, in keeping with our observation of preserved lung leukocyte recruitment in the absence of IP-10, we also found no effect of the presence of increased amounts of IP-10 on leukocyte recruitment into the lung after bleomycin exposure in the transgenic mice overexpressing IP-10. We were also surprised to find no effect of IP-10 deficiency on the aggregate angiogenic activity contained in the lungs 5 days after bleomycin challenge, with this time point again chosen to coincide with peak IP-10 expression in wild-type mice. Consistent with this result however, we also observed no effect of the presence of increased amounts of IP-10 on angiogenic activity in the lung after bleomycin exposure in the transgenic mice over-expressing IP-10.

Despite the absence of differences in leukocyte recruitment or angiogenesis, we did find dramatic differences in the accumulation of fibroblasts in the lungs of IP-10–deficient and IP-10–overexpressing mice after bleomycin challenge, suggesting to us that IP-10 may have a direct effect on fibroblast recruitment. We found that fibroblast accumulation in the lung after bleomycin administration is dramatically increased in the absence of IP-10 in IP-10–deficient mice, and dramatically decreased in the presence of increased amounts of IP-10 in IP-10–overexpressing mice. As a potential mechanism for this observation, we found that IP-10 inhibits the substantial fibroblast chemotactic activity that is generated endogenously in the lung after bleomycin exposure, and that inhibitory concentrations of IP-10 are present in the injured lung. Consequently, we hypothesize that IP-10 may limit the development of bleomycin-induced pulmonary fibrosis through a direct effect on fibroblasts by inhibiting their migration. In support of this hypothesis, IP-10 has been shown to inhibit fibroblast motility induced by multiple growth factor chemoattractants, as previously noted (26).

The pathogenesis of pulmonary fibrosis involves fibroblast migration into the airspaces in addition to excessive fibroblast proliferation and deposition of extracellular matrix. In the normal lung parenchyma, fibroblasts reside in the interstitial compartment of alveolar walls, which consists of the space between the alveolar and capillary basal laminae (30). Examination of lung biopsies of pulmonary fibrosis patients, however, has demonstrated that the fibroblasts that actively synthesize collagen in this disease are not located in the interstitium, but rather have migrated into the fibrinous exudates present in the airspaces (30). In the model of pulmonary fibrosis investigated, we have demonstrated that substantial fibroblast chemoattractant activity is generated in the lung endogenously after bleomycin exposure that is capable of inducing fibroblast recruitment. Studies of patient BALF indicate that fibroblast chemoattractant activity is generated similarly in the lungs of patients developing pulmonary fibrosis. BALF from patients with idiopathic pulmonary fibrosis contains elevated chemoattractant activity for fibroblasts compared with BALF from control subjects (31). Likewise, BALF from patients developing acute respiratory distress syndrome (32) and from patients developing obliterative bronchiolitis (33), a fibroproliferative process that occurs after lung transplantation, significantly stimulated fibroblast migration, whereas fluid from controls did not.

The fibroblast chemoattractant activity generated in the lung after bleomycin administration was significantly inhibited by IP-10 in a dose-dependent manner. The concentrations of IP-10 determined to be present in the epithelial lining fluid after bleomycin challenge in vivo were within the range found to inhibit fibroblast chemotaxis in vitro. Additionally, the IP-10 concentrations in the epithelial lining fluid at focal sites of lung injury and repair would be expected to be considerably higher than the values determined, which represent averages of the IP-10 concentrations present in the epithelial lining fluid throughout the entire lung. The concentrations of IP-10 we found to inhibit fibroblast chemotaxis are also within the range of IP-10 concentrations that have been documented to be present in other biological fluids under inflammatory conditions (34, 35), and are comparable to IP-10 concentrations that direct lymphocyte migration (36). IP-10's inhibitory activity on fibroblast migration was not due to a nonspecific toxic effect on fibroblasts, as IP-10 had no effect on BALF-induced fibroblast proliferation. Because IP-10 did not affect BALF-induced fibroblast proliferation, we hypothesize that IP-10 regulates the accumulation of fibroblasts in the lung in this model through the inhibition of fibroblast recruitment.

The inhibition of fibroblast migration by IP-10 was not mediated by its identified receptor, CXCR3, because fibroblasts did not express this receptor. We can envision several plausible mechanisms by which IP-10 could inhibit fibroblast migration that do not involve its interaction with CXCR3. First, IP-10 could inhibit signaling through other chemokine receptors expressed by fibroblasts by acting as a competitive antagonist. For example, IP-10 has been demonstrated to act as an antagonist for CCR3 by competing with CCR3 ligands for binding to this receptor (37). IP-10 could also inhibit signaling through other chemokine receptors expressed by fibroblasts by competing with the ligands of these receptors for binding to fibroblast cell surface glycosaminoglycans (GAGs), thereby denying these ligands access to their receptors. Accumulating evidence indicates that chemokine interactions with GAGs are critical for chemokine function in vivo (38), and may be required for maximizing chemokine binding to target cells, thereby facilitating chemokine signaling through their specific receptors. IP-10 has been demonstrated to inhibit CXCR2 by interfering with the GAG binding of CXCR2 ligands, such as GRO/CXCL1 (39).

Alternatively, IP-10 could act by inhibiting fibroblast migration induced by chemotactic growth factors rather than by inhibiting chemokines and/or chemokine receptors. IP-10 has been demonstrated to inhibit fibroblast migration in response to growth factors such as EGF, heparin-binding EGF-like growth factor, and platelet-derived growth factor by inhibiting growth factor–induced fibroblast uropod detachment (26). In this study, IP-10 was noted to increase cellular cAMP levels in HS68 normal human diploid fibroblasts (26). IP-10–induced signaling in these fibroblasts may be mediated by interaction with fibroblast surface GAGs, as has been suggested for RANTES-induced signaling in HeLa-CD4 and CHO cells, which lack any of the chemokine receptors bound by RANTES (40). IP-10 signaling induced by interaction with fibroblast cell surface GAGs therefore represents another possible mechanism for IP-10–mediated inhibition of fibroblast migration that would not require fibroblast expression of CXCR3. We favor a mechanism for IP-10 inhibition of fibroblast migration that involves the inhibition of other chemokines and/or their receptors, however, because we have found that the chemoattractant activity of BALF after bleomycin challenge appears to be mediated by pertussis toxin–sensitive chemoattractant receptors (data not shown). Elucidating the mechanism by which IP-10 inhibits fibroblast migration induced by BALF chemoattractants will be the subject of future investigations.

Consistent with the hypothesis that IP-10 limits the development of fibrosis by inhibiting fibroblast migration, we have previously demonstrated delayed wound healing in the IP-10–transgenic mice used in the present study (7). After mechanical wounding, increased expression of IP-10 resulted in disorganized migration of smooth muscle actin–positive cells in the wound. Many of the fibroblastic cells accumulating in wounded tissue are myofibroblasts, with features of both fibroblasts and smooth muscle cells, including the expression of smooth muscle actin. These previous findings in the IP-10–transgenic mice, therefore, also are consistent with our hypothesis that IP-10 inhibits fibroblast recruitment after tissue injury.

Although chemokines have been classically identified as cytokines directing leukocyte migration, their ability to regulate the migration, proliferation, and/or activation of nonleukocytes, including epithelial cells (41), endothelial cells (22, 42), smooth muscle cells (43), and fibroblasts (26), has also been demonstrated. In this study, we have demonstrated that IP-10, which is markedly induced after bleomycin challenge, inhibits the migration of fibroblasts in vitro to chemoattractants generated in the lung endogenously after injury. Although we have not formally demonstrated that IP-10 inhibits fibroblast migration into sites of lung injury in vivo as we hypothesize, this hypothesis is consistent with our observations of dramatically increased accumulation of fibroblasts after bleomycin injury in the lungs of IP-10–deficient mice, and decreased accumulation of fibroblasts after bleomycin administration in the lungs of trangenic mice overexpressing IP-10. Alternatively, we acknowledge that inhibition of fibroblast accumulation by IP-10 in vivo could be an indirect effect of IP-10, resulting from actions of IP-10 on leukocyte recruitment prior to the time points we investigated, or on CXCR3-expressing cells that we have not yet identified, leading to reduction or inhibition of other mediators of fibroblast migration. In either case, we believe interruption of fibroblast recruitment has the potential to be a novel therapeutic strategy for pulmonary fibrosis, which may have broad applicability to a wide range of fibrotic illnesses, most of which are not amenable to current therapies. Our results suggest that IP-10 has potential to make this an effective treatment approach.

	Acknowledgments

 
The authors thank Mr. Edward Rogers for technical assistance, Dr. Terry Means for assistance with quantitative PCR, Dr. Klaus Elenius for advice on fibroblast chemotaxis, and Dr. Claudia Jakubzick for advice on primary lung fibroblast isolation and culture. These studies were supported by National Institutes of Health grants K08-HL04087 to A.M.T., R01-AI39054 to R.L.K., R01-HL61419 to T.S.B., and R01-CA69212 to A.D.L.

	Footnotes

 
Conflict of Interest Statement: A.M.T. has no declared conflicts of interest; R.L.K. has no declared conflicts of interest; P.L. has no declared conflicts of interest; S.D.B. has no declared conflicts of interest; G.S.V.C. has no declared conflicts of interest; C.P.L. has no declared conflicts of interest; V.P. has no declared conflicts of interest; L-H.Z. has no declared conflicts of interest; H.S. has no declared conflicts of interest; T.S.B. has no declared conflicts of interest; and A.D.L. has no declared conflicts of interest.

^* These authors contributed equally to this work.

Received in original form May 28, 2004

Received in final form June 17, 2004

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