Inducible costimulator ligand regulates bleomycin-induced lung and skin fibrosis in a mouse model independently of the inducible costimulatorinducible costimulator ligand pathway.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 62, No. 6, June 2010, pp 1723–1732 DOI 10.1002/art.27428 © 2010, American College of Rheumatology Inducible Costimulator Ligand Regulates Bleomycin-Induced Lung and Skin Fibrosis in a Mouse Model Independently of the Inducible Costimulator/Inducible Costimulator Ligand Pathway Chihiro Tanaka,1 Manabu Fujimoto,1 Yasuhito Hamaguchi,1 Shinichi Sato,2 Kazuhiko Takehara,1 and Minoru Hasegawa1 Objective. Systemic sclerosis is a connective tissue disease characterized by fibrosis of the skin and internal organs, including the lungs. Inducible costimulator (ICOS), which is expressed on activated T cells, and its ligand ICOSL, which is expressed on antigen-presenting cells, have been considered a single receptor–ligand pair. Although the ICOS/ICOSL pathway is known to play various roles in adaptive immunity, its roles in innate immunity and tissue fibrosis remain unknown. Methods. We assessed the roles of ICOS and ICOSL in tissue fibrosis by administering bleomycin intratracheally or intradermally into mice deficient in ICOS and/or ICOSL. Tissue fibrosis was evaluated by histologic or biochemical examination. Results. ICOS deficiency attenuated the lung and skin fibrosis, whereas ICOSL deficiency aggravated it. Mice deficient in both ICOS and ICOSL exhibited accelerated fibrosis, reflecting a dominant role of ICOSL over ICOS in this model. Interestingly, ICOSL expression on macrophages and B cells derived from bronchoalveolar lavage fluid was significantly elevated in ICOS-deficient mice as compared with wild-type mice during this process. Thus, the levels of ICOSL expres- sion on B cells and macrophages were inversely associated with the severity of tissue fibrosis. Conclusion. Our results indicate that ICOSL expression on antigen-presenting cells plays a previously unknown regulatory role during the development of bleomycin-induced tissue fibrosis that is independent of the ICOS/ICOSL pathway. Further studies will be needed to clarify the roles of ICOS and ICOSL in the development of systemic sclerosis. Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by excessive extracellular matrix deposition in the skin, lungs, and other internal organs (1,2). Interstitial lung disease is a major cause of mortality and morbidity in patients with SSc. A growing body of evidence suggests that in patients with SSc, overproduction of extracellular matrix components by activated fibroblasts results from complex interactions between endothelial cells, lymphocytes, macrophages, and fibroblasts, and via a number of mediators, including cytokines, chemokines, and growth factors (1,2). Bleomycin (BLM)–induced lung fibrosis is widely used as an established animal model of pulmonary fibrosis (3,4). Intratracheal administration of BLM induces acute alveolitis and interstitial inflammation. Yamamoto and colleagues (5) have established a new mouse model of skin fibrosis induced by daily intradermal injections of BLM. In this model, lung fibrosis also develops when sufficient amounts of BLM have been administered (6), thereby making this one of most useful animal models of SSc. Inducible costimulator (ICOS) is the third member of the CD28 family of costimulatory molecules and is induced on the cell surface following T cell activation (7–9). The ligand of ICOS, or ICOSL (also called B7h, Supported by grants-in-aid from the Ministry of Education, Science, and Culture of Japan and by funds for research of intractable diseases from the Ministry of Health, Labor, and Welfare of Japan. 1 Chihiro Tanaka, MD, Manabu Fujimoto, MD, Yasuhito Hamaguchi, MD, PhD, Kazuhiko Takehara, MD, PhD, Minoru Hasegawa, MD, PhD: Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; 2Shinichi Sato, MD, PhD: Tokyo University, Tokyo, Japan. Address correspondence and reprint requests to Minoru Hasegawa, MD, PhD, Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan. E-mail: [email protected] kanazawa-u.ac.jp. Submitted for publication August 19, 2009; accepted in revised form February 18, 2010. 1723 1724 TANAKA ET AL B7RP-1, LICOS, and GL50), is weakly expressed on antigen-presenting cells in the steady state and is upregulated after activation of these cells (8,10). The ICOS/ICOSL pathway promotes T cell activation, differentiation, and effector responses, and T cell– dependent B cell responses. ICOS-mediated costimulation of T cells leads predominantly to the production of effector cytokines, such as interleukin-4 (IL-4) and IL-10, and to a lesser extent, IL-2, interferon-␥ (IFN␥), and tumor necrosis factor ␣ (TNF␣) (11), thereby playing a more important role in Th2 responses than in Th1 responses (7,12–14). Furthermore, recent studies demonstrated that ICOS influences the expansion of follicular T cells, Th17 cells, and regulatory T cells (15–17). Since ICOSL–/– and ICOS–/– mice display similar defects in humoral immunity, ICOS and ICOSL have been considered a single receptor–ligand pair (18–20). It is generally accepted that ICOS/ICOSL signaling is critical for adaptive immunity, including autoimmunity, allergy, infectious diseases, and transplantation (20–23). However, ICOSL is constitutively expressed not only on B cells, but also on macrophages and dendritic cells, which contribute to innate immunity (8,10). Therefore, we hypothesized that on these cells, ICOSL may be contributing to innate immunity independently of the ICOS/ICOSL axis. Since tissue fibrosis, including the BLM-induced scleroderma model, involves a strong innate immune component, we applied this model to mice deficient in ICOS and ICOSL to investigate the roles of these molecules in modulating innate and adaptive immunity. MATERIALS AND METHODS Mice. ICOS–/– mice and ICOSL–/– mice were purchased from The Jackson Laboratory. All mice were backcrossed 8–10 generations onto mice of the C57BL/6 genetic background. Mating these ICOS–/– mice with ICOSL–/– mice generated ICOS⫹/–ICOSL⫹/– mice. Then, we generated ICOS–/–ICOSL–/– mice by crossing the ICOS⫹/–ICOSL⫹/– parents. To verify the ICOS or ICOSL genotype, we conducted polymerase chain reaction amplification of each gene using genomic DNA from each mouse. All mice were housed in a specific pathogen–free barrier facility and screened regularly for pathogens. Female mice 8–10 weeks of age were used in the experiments. The Committee on Animal Experimentation at Kanazawa University Graduate School of Medical Science approved all studies and procedures. Intratracheal treatment with BLM. BLM sulfate (Nippon Kayaku) was administered to mice that had been anesthetized by inhalation of diethyl ether. Using aseptic techniques, a single incision was made at the neck, and the muscle covering the trachea was snipped to expose the tracheal rings. A single intratracheal instillation of BLM sulfate (8 mg/kg [7.68 units/ kg]) in 200 l of sterile saline was performed using a 27-gauge needle. Intradermal treatment with BLM. BLM was dissolved in sterile saline at a concentration of 1 mg/ml. Three hundred microliters of BLM or saline was injected intradermally into the shaved backs (the para-midline, lower back region) of the mice with a 27-gauge needle, as described previously (5). Injections were given daily for 4 weeks. Histologic examination of lung and skin fibrosis. Whole lungs were inflated, fixed with 3.5% paraformaldehyde, and embedded in paraffin. Sections (6 m in thickness) were stained with hematoxylin and eosin (H&E) to evaluate alveolitis or with Masson’s trichrome to identify collagen deposition in the lungs. The severity of lung inflammation was determined by a semiquantitative scoring system as previously described (24). Briefly, lung fibrosis in randomly chosen fields of sections from the left middle lobe examined at 100⫻ magnification was graded on a scale of 0 (normal lung) to 8 (total fibrous obliteration of fields). All sections were scored independently by 2 investigators (CT and MH) in a blinded manner. All skin sections were taken from the BLM-injected region of the lower back and were obtained as full-thickness sections extending down to the body wall musculature. Skin samples were fixed in formalin, dehydrated, embedded in paraffin, and used for immunostaining. Six-micrometer sections were stained with H&E or with Azan-Mallory reagents to identify collagen deposition in the skin. Dermal thickness, which was defined as the thickness of skin from the top of the granular layer to the junction between the dermis and intradermal fat, was evaluated independently by 2 investigators (CT and MH) in a blinded manner. Using the free-hand tool in the Photoshop Elements 3.0 software package (Adobe Systems), specific staining with Masson’s trichrome and ␣-smooth muscle actin (␣-SMA) was quantified in the whole lung and skin sections. Hydroxyproline assay. Total lung or dorsal skin tissue was freeze-dried overnight, and the hydroxyproline content was determined as previously described (3). Briefly, samples were hydrolyzed in 6N HCl for 20 hours at 105°C, desiccated overnight, and dissolved in 1 ml of citrate/acetate buffer (pH 6.0). Chloramine T solution was added to each optimally diluted sample and left at room temperature for 20 minutes. Next, Ehrlich’s solution was added to each sample, incubated for 15 minutes at 65°C, and the optical density at 550 nm was read. Hydroxyproline standard solutions of 0–20 g/ml were used to construct a standard curve. Determination of collagen contents in lung tissue sections. Approximately 15 m–thick sections were obtained from the paraffin-embedded lung tissue samples. Groups of 10–20 sections were deparaffinized after incubation with xylol, xylol:ethanol (1:1), ethanol, water:ethanol (1:1), and water. Individual samples were placed in small test tubes and covered with 0.2 ml of a saturated solution of picric acid in distilled water that contained 0.1% fast green FCF and 0.1% sirius red F3BA. The samples were rinsed several times with distilled water until the fluid was colorless. One milliliter of 0.1N NaOH in absolute methanol (1:1 [volume/volume]) was added, and the eluted color was read in a spectrophotometer at 540 and 605 nm. This method is based on the selective binding of Sirius ICOSL REGULATION OF BLEOMYCIN-INDUCED MURINE LUNG AND SKIN FIBROSIS 1725 Figure 1. Lung fibrosis induced by intratracheal treatment with bleomycin (BLM). ICOS–/–, ICOSL–/–, and wild-type (WT) mice were treated with a single intratracheal injection of BLM and were evaluated through day 21. A, Survival rates in ICOS–/–, ICOSL–/–, and wild-type mice. Results were compiled from at least 21 mice per group. B–D, Analysis of the lungs for determination of lung fibrosis scores (B), hydroxyproline content (C), and collagen content (semiquantitative) (D). Values are the mean and SEM of 5 mice per group. E, Quantitative results of immunohistochemical stainings shown in F. Values are the mean and SEM area of specific antibody staining with Masson’s trichrome and ␣-smooth muscle actin (␣-SMA) over the total stained area in 5 nonconsecutive whole lung sections from 5 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. F, Representative photomicrographs of lung tissue sections obtained from each BLM-treated mouse strain and from a saline-treated wild-type mouse (day 21), as well as sections obtained from dying mice (2 wild-type, 1 ICOS⫺/⫺, and 6 ICOSL–/– mice examined; days 10–15). Sections were stained with hematoxylin and eosin (H&E) or with Masson’s trichrome (original magnification ⫻ 100). red F3BA and fast green FCF to collagens and noncollagenous proteins, respectively (25). Immunohistochemical staining. Tissues were harvested before and on days 7, 14, 21, and 28 during BLM treatment and were assessed for the numbers of infiltrating T cells, B cells, macrophages, and neutrophils. Deparaffinized sections were treated with endogenous peroxidase blocking reagent and proteinase K (both from DakoCytomation) for 6 minutes at room temperature. Sections were then incubated with rat monoclonal antibodies specific for anti–␣-SMA (Sigma-Aldrich), macrophages (clone F4/80; American Type Culture Collection), myeloperoxidase (MPO; NeoMarkers), CD3 (Dainippon Pharmaceutical), or mouse B220 (BD Biosciences). Rat IgG (SouthernBiotech) was used as a control for nonspecific staining. Sections were then incubated sequentially (for 20 minutes at 37°C) with a biotinylated rabbit anti-rat IgG secondary antibody (Vectastain ABC method; Vector) or a biotinylated goat anti-rabbit IgG secondary antibody (BD Biosciences), followed by horseradish peroxidase–conjugated avidin–biotin complexes. Sections were washed 3 times with phosphate buffered saline (PBS) between incubations, developed with 3,3⬘-diaminobenzidine tetrahydrochloride and hydrogen peroxide, and then counterstained with methyl green. Stained cells were counted in 5 random grids under high magnification (400⫻) using a light microscope. Each section was examined and scored independently by 2 investi- gators (CT and MH) in a blinded manner. The mean score was used for analysis. Determination of cytokine concentrations in tissues. Samples of whole lungs were homogenized in 600 l of lysis buffer (10 mmoles/liter of PBS, 0.1% sodium dodecyl sulfate, 1% Nonidet P40, 5 mmoles/liter of EDTA containing a complete protease inhibitor mixture; Roche Diagnostics) to extract proteins. Homogenates were centrifuged at 15,000 revolutions per minute for 15 minutes at 4°C to remove debris (26). The concentrations of TNF␣, IFN␥, monocyte chemotactic protein 1 (MCP-1), IL-6, IL-10, and IL-12 in supernatants were determined by using a BD Cytometric Bead Array mouse inflammation kit (BD Biosciences) according to the manufacturer’s protocol. Flow cytometry analysis was performed using a FACSCalibur flow cytometer (BD Biosciences). The levels of macrophage inflammatory protein 1␣ (MIP-1␣) (Quantikine Immunoassay) and IL-4 (DuoSet) (both from R&D Systems) in supernatants were measured by enzyme-linked immunosorbent assay according to the manufacturer’s instructions. Total protein in the supernatant was measured with a commercial kit (BCA Protein Assay kit; Thermo Fischer Scientific). Reverse transcription–polymerase chain reaction (RTPCR). Whole lungs were harvested 7 days after intradermal treatment with BLM. Total RNA was isolated from frozen 1726 TANAKA ET AL Figure 2. Skin and lung fibrosis induced by intradermal treatment with bleomycin (BLM). ICOS–/–, ICOSL–/–, and wild-type (WT) mice were treated with daily intradermal injections of BLM for 28 days. A, Lung fibrosis was assessed by determining lung fibrosis scores, hydroxyproline content, and percentage of staining with ␣-smooth muscle actin (␣-SMA). B, Skin fibrosis was assessed by determining dermal thickness, hydroxyproline content, and percentage of staining with ␣-SMA. Values in A and B are the mean and SEM of 5 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. C, Representative photomicrographs of lung tissue sections obtained from each BLM-treated mouse strain and from a saline-treated wild-type mouse. Sections were stained with hematoxylin and eosin (H&E) or with Masson’s trichrome. D, Representative photomicrographs of skin tissue sections obtained from each experimental group. Sections were stained with H&E or with Azan-Mallory stain. Arrows indicate the dermal thickness. (Original magnification ⫻ 100.) lung specimens using RNeasy spin columns (Qiagen) and digested with DNase I (Qiagen) to remove chromosomal DNA in accordance with the manufacturer’s protocols. Total RNA was reverse-transcribed to a complementary DNA using a reverse transcription system with random hexamers (Promega). Cytokine messenger RNAs (mRNA) were analyzed using real-time RT-PCR quantification, according to the manufacturer’s instructions (Applied Biosystems). Sequencespecific primers and probes were designed with predeveloped TaqMan assay reagents (Applied Biosystems). Real-time RTPCR (40 cycles of denaturation at 92°C for 15 seconds and annealing at 60°C for 60 seconds) was performed on an ABI Prism 7000 sequence detector (Applied Biosystems). GAPDH was used to normalize the mRNA. The relative expression of real-time RT-PCR products was determined according to the ⌬⌬Ct method (27) to compare target gene and GAPDH mRNA expression. One of the control samples was chosen as a calibrator sample. Flow cytometric analysis. Splenic macrophages and B cells were purified from untreated mice of each strain as described above. These cells were then used for flow cytometric analysis. Bronchoalveolar lavage (BAL) cells were prepared as described elsewhere (28). Briefly, mice were killed on days 7, 14, and 21 after BLM instillation. BAL fluid was collected as follows: 1 ml of saline was instilled 3 times and withdrawn from the lungs via an intratracheal cannula. For 2-color or 3-color immunofluorescence analyses, BAL cells were stained with the following antibodies: CD4 (RM4-5; BD Biosciences), B220 (RA36B2; BD Biosciences), CD14 (Sa2-8; eBioscience), ICOS (7E17G9; eBioscience), and ICOSL (HK5.3; eBioscience). BAL cells with the forward and side light-scatter properties of lymphocytes were analyzed on a FACScan flow cytometer (BD Biosciences). Positive and negative populations of cells were determined using unreactive isotype-matched monoclonal antibodies (BD Biosciences) as controls for background staining. Statistical analysis. The Mann-Whitney U test was used to determine the level of significance of differences in the sample means. The Bonferroni test was used for multiple comparisons. RESULTS Amelioration of intratracheal BLM–induced lung fibrosis by ICOS deficiency, but aggravation by ICOSL deficiency. To assess whether the ICOS/ICOSL signaling pathway contributed to the development of lung fibrosis, we first assessed intratracheal treatment of BLM in ICOS–/–, ICOSL–/–, and wild-type mice. Lungs were removed from the animals on day 21 following saline or BLM treatment. As demonstrated by the survival rates, ICOS deficiency resulted in a significantly lower mortality rate than was observed for wild-type mice (Figure 1A). Unexpectedly, ICOSL–/– mice demonstrated significantly higher mortality rates as compared with wild-type mice. ICOSL REGULATION OF BLEOMYCIN-INDUCED MURINE LUNG AND SKIN FIBROSIS 1727 Figure 3. Inflammatory cell infiltration in the skin and lungs induced by daily intradermal injections of bleomycin (BLM). ICOS–/–, ICOSL–/–, and wild-type (WT) mice were treated with daily intradermal injections of BLM for 28 days. The numbers of F4/80⫹ macrophages, B220⫹ B cells, CD3⫹ T cells, and myeloperoxidase (MPO)–positive neutrophils per high-power field (hpf) in the lungs (A) and skin (B) were counted at the indicated times. Values are the mean and SEM of 5 mice per group. ⴱ ⫽ P ⬍ 0.05. Pathologic findings and fibrosis scores in H&Eand Masson’s trichrome–stained section also revealed that the loss of ICOS significantly resolved the lung fibrosis, whereas the loss of ICOSL aggravated it (Figures 1B and F). In addition, visual inspection of fibrotic tissue and myofibroblasts immunostained with Masson’s trichrome and ␣-SMA, respectively, demonstrated a similar tendency in each mouse strain (Figure 1E). Quantification of the hydroxyproline and collagen contents in the lungs confirmed the histologic findings (Figures 1C and D). Since there may be other relevant causes of death in mutant mice, we examined the histologic features of the lungs in dying mice (2 wild-type, 1 ICOS⫺/⫺, and 6 ICOSL–/– mice). In both the wild-type and ICOSL–/– mice, the lungs had a characteristically fibrotic appearance, with disrupted alveolar architecture replaced by continuous cellular interstitial connective tissue (Figure 1F), which indicates that lung injury was the direct cause of death in these mice. Thus, ICOS deficiency reduced the lung fibrosis that was induced by intratracheal treatment with BLM, whereas ICOSL deficiency aggravated it. Opposing effects of ICOS and ICOSL deficiency in skin and lung fibrosis induced by intradermal BLM treatment. As reported previously (5,6), daily intradermal administration of BLM induces skin and lung fibro- sis by day 28 in wild-type mice. In these experiments, we again observed that ICOS loss significantly decreased the intradermal BLM–induced lung fibrosis, but ICOSL loss exacerbated it (Figures 2A and C). In addition, the dermal thickness and histopathologic features noted on H&E-and Azan-Mallory–stained sections, respectively, revealed significantly ameliorated skin fibrosis in mice lacking ICOS (Figures 2B and D). In contrast, ICOSL deficiency significantly exacerbated skin fibrosis. The results of measurements of hydroxyproline content in the skin were consistent with the histologic findings (Figure 2B). Thus, the tissue pathology, fibrosis score, and hydroxyproline content all showed similar patterns in this intradermal BLM–induced skin and lung fibrosis model. Consistent with the results of the tissue fibrosis examination, ␣-SMA⫹ cell numbers were significantly reduced in both the lung and skin of ICOS–/– mice, but were increased in ICOSL–/– mice, as compared with wild-type mice (Figures 2A and B). Thus, ICOS loss ameliorates the lung and skin fibrosis induced by daily intradermal injections of BLM, whereas ICOSL loss aggravates it. Lung and skin inflammation induced by daily intradermal injections of BLM. The numbers of F4/80⫹ macrophages, CD3⫹ T cells, B220⫹ B cells, and MPO⫹ neutrophils were assessed in lung and skin tissues of 1728 mice subjected to daily intradermal injections of BLM. In comparison with wild-type mice, lung tissues from ICOS–/– mice exhibited reduced numbers of macrophages, T cells, and neutrophils throughout the 28-day treatment period (Figure 3A). Conversely, ICOSL–/– mice demonstrated increased numbers of macrophages, T cells, and neutrophils. Similarly, the numbers of macrophages and T cells were reduced in the skin of ICOS–/– mice (Figure 3B), while the number of macrophages, T cells, and neutrophils were increased in the skin of ICOSL–/– mice. Thus, inflammatory cell infiltration was modest in ICOS–/– mice and prominent in ICOSL–/– mice. Cytokine expression in the lungs of mutant mice. To assess the effects of ICOS and ICOSL loss on the production of various cytokines and chemokines, their concentrations in lung lysates were measured by cytometric bead array analysis on day 5 following intratracheal BLM treatment. In addition, levels of expression of mRNA for transforming growth factor ␤ (TGF␤), connective tissue growth factor (CTGF), and FoxP3 in the lungs were assessed by real-time RT-PCR. The concentrations or levels of expression of TNF␣, IFN␥, IL-4, IL-6, IL-10, IL-12, MCP-1, MIP-1␣, TGF␤, CTGF, and FoxP3 in the lungs of BLM-treated wild-type mice were significantly higher than those in the lungs of saline-treated wild-type mice (data not shown). Total protein concentrations in lung lysates were comparable between the BLM-treated strains (mean ⫾ SEM 38.7 ⫾ 3.3 mg/ml in wild-type, 38.0 ⫾ 3.5 mg/ml in ICOS–/–, and 37.1 ⫾ 3.0 mg/ml in ICOSL–/– mice). The concentration of TNF␣, IFN␥, IL-4, IL-6, IL-10, IL-12, MCP-1, and MIP-1␣ in the lungs of BLM-treated mice was not significantly different between groups (Figure 4A). Expression levels of CTGF and FoxP3 were also similar among the mouse strains (Figure 4B). However, the expression levels of TGF␤ were significantly lower in ICOS–/– mice and higher in ICOSL–/– mice relative to the levels in wild-type mice (Figure 4B). Thus, TGF␤ expression was most associated with the severity of lung fibrosis. Phenotype similarity of mice deficient in both ICOS and ICOSL as compared with mice deficient in ICOSL following treatment with BLM. We hypothesized that either ICOS or ICOSL had functions that were independent of the ICOS/ICOSL axis in the BLMinduced fibrosis model. To explore this possibility, we generated mice deficient in both ICOS and ICOSL (ICOS–/–ICOSL–/–) and assessed the severity of BLMinduced lung and skin fibrosis. TANAKA ET AL Figure 4. Levels of cytokines, chemokines, growth factors, and FoxP3 in the lungs of mice treated with intratracheal injections of bleomycin (BLM). ICOS–/–, ICOSL–/–, and wild-type (WT) mice were treated with a single intratracheal injection of BLM. A, Concentrations of tumor necrosis factor ␣ (TNF␣), interferon-␥ (IFN␥), interleukin-4 (IL-4), IL-6, IL-10, IL-12, monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1␣ (MIP-1␣) were measured by cytometric bead array analysis or enzyme-linked immunosorbent assay in supernatants of lung homogenates. B, Expression of transforming growth factor ␤ (TGF␤), connective tissue growth factor (CTGF), and FoxP3 mRNA was measured by real-time quantitative reverse transcription–polymerase chain reaction analysis. Values are the mean and SEM of 5 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. ICOSL REGULATION OF BLEOMYCIN-INDUCED MURINE LUNG AND SKIN FIBROSIS 1729 Figure 5. Skin and lung fibrosis in ICOS–/–ICOSL–/– double-knockout mice as compared with wild-type (WT) mice following treatment with bleomycin (BLM). A, Time course of survival, representative photomicrographs of the lungs, lung fibrosis scores, and percentage of total tissue area in mice treated with intratracheal BLM. H&E ⫽ hematoxylin and eosin. B and C, Inflammation and fibrosis of the lungs (B) and skin (C) following intradermal injection of BLM, assessed as described in Figures 2 and 3. ␣-SMA ⫽ ␣-smooth muscle actin; hpf ⫽ high-power field; MPO ⫽ myeloperoxidase. D, Concentrations of tumor necrosis factor ␣ (TNF␣), interferon-␥ (IFN␥), interleukin-4 (IL-4), IL-6, IL-10, IL-12, monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1␣ (MIP-1␣) in lung lysates, assessed as described in Figure 4. E, Levels of mRNA for transforming growth factor ␤ (TGF␤), connective tissue growth factor (CTGF), and FoxP3 in the lungs, assessed as described in Figure 4. ⴱ ⫽ P ⬍ 0.05. First, we assessed the sensitivity of ICOS–/– ICOSL–/– mice to intratracheal treatment with BLM. Deficiency of both ICOS and ICOSL resulted in high mortality rates, severe fibrosis, and elevated fibrosis scores (Figure 5A). These findings were quite comparable to what we had observed previously in ICOSL–/– mice. Next, we performed daily intradermal injections of BLM. In this model of BLM-induced lung and skin fibrosis, the ICOS–/–ICOSL–/– mice exhibited significantly increased fibrosis scores, ␣-SMA⫹ cell infiltration, and hydroxyproline content, reflecting severe lung fibrosis, as compared with wild-type mice (Figure 5B). ICOS–/–ICOSL–/– mice exhibited significantly greater lung infiltration by macrophages, B cells, T cells, and neutrophils on day 7 as compared with wild-type mice (Figure 5B). Furthermore, deficiency of both ICOS and 1730 TANAKA ET AL Figure 6. Expression of inducible costimulator (ICOS) and ICOS ligand (ICOSL) in mutant mice. A, ICOS expression on CD4⫹ T cells and ICOSL expression on CD14⫹ macrophages and on B220⫹ B cells in the spleens of untreated wild-type (WT), ICOS–/–, and ICOSL–/– mice, as determined by flow cytometry. Histograms are representative of at least 5 experiments. Values at the left are the mean and SEM of 5 mice per group. ⴱⴱ ⫽ P ⬍ 0.01. B, ICOS and ICOSL expression on T cells, macrophages, and B cells in bronchoalveolar lavage fluid prepared in wild-type, ICOS–/–, and ICOSL–/– mice after intratracheal treatment with bleomycin, as determined by flow cytometry. Histograms are representative of at least 5 experiments conducted on day 7. Values at the left are the mean and SEM of 5 mice per group, as determined on the indicated days. ⴱⴱ ⫽ P ⬍ 0.01. ICOSL led to significant increases in dermal thickness, hydroxyproline content, and ␣-SMA⫹ cells in the skin relative to the findings in wild-type mice (Figure 5C). Numbers of infiltrating macrophages, B cells, T cells, and neutrophils were increased in the skin of ICOS–/– ICOSL–/– mice (Figure 5C). The total protein concentration in lung lysates was comparable in ICOS–/–ICOSL–/– mice and in wildtype mice (mean ⫾ SEM 38.7 ⫾ 3.3 mg/ml in wild-type mice and 38.1 ⫾ 2.9 mg/ml in ICOS–/–ICOSL–/– mice). The concentrations of cytokines and chemokines in lysates of ICOS–/–ICOSL–/– mice were not significantly different from those in lysates of wild-type mice (Figure 5D). Levels of mRNA for TGF␤, but not CTGF or FoxP3, were significantly higher in the lung tissues of ICOS–/–ICOSL–/– mice as compared with wild-type mice (Figure 5E). Thus, the results of BLM-induced fibrosis in ICOS–/–ICOSL–/– mice resembled those in ICOSL–/– mice. Inverse association between ICOSL expression levels and the severity of lung fibrosis. In light of the results obtained in ICOS–/–ICOSL–/– mice, we considered that expression of ICOSL, rather than ICOS, played a dominant role in BLM-induced fibrosis. We assessed the expression levels of ICOS and ICOSL by fluorescence-activated cell sorting of splenocytes from mice that had not been treated with BLM (Figure 6A). The expression levels of ICOS on CD4⫹ T cells were significantly elevated by ⬃135% in ICOSL–/– mice than in wild-type mice. In addition, the expression levels of ICOSL on macrophages and B cells were increased ⬃302% and ⬃348%, respectively, in ICOS–/– mice as compared with wild-type mice. Next, we evaluated the association of ICOS and ICOSL expression levels in BAL fluid cells on days 7, 14, and 21 after a single intratracheal treatment with BLM (Figure 6B). The expression levels of ICOS on CD4⫹ T cells and the expression of ICOSL on macrophages and B cells reached a maximum ⬃7 days after the single intratracheal BLM treatment, and then gradually declined. On day 7, the expression level of ICOS on T cells was elevated ⬃233% in ICOSL–/– mice as compared with wild-type mice. In contrast, the expression levels of ICOSL on macrophages and B cells were increased ⬃161% and ⬃290%, respectively, in ICOS–/– mice relative to wild-type mice. Thus, ICOSL expression levels were inversely associated with disease severity in this mouse model. ICOSL REGULATION OF BLEOMYCIN-INDUCED MURINE LUNG AND SKIN FIBROSIS DISCUSSION In the present study, we demonstrated that ICOSL plays roles that are independent of the ICOS/ ICOSL pathway in a mouse model of BLM-induced lung and skin fibrosis. In addition, the severity of tissue fibrosis appeared to correlate with the expression levels of ICOSL on B cells and macrophages. We made the unexpected finding that ICOS–/– mice and ICOSL–/– mice exhibited contrasting phenotypes in the BLM induced lung and skin fibrosis model. Before BLM treatment, no histologic difference was found in the lungs and skin of these mutant mouse strains as compared with wild-type mice (data not shown). However, ICOS deficiency significantly inhibited, whereas ICOSL deficiency exacerbated, BLMinduced lung and skin fibrosis (Figures 1 and 2). It is currently thought that ICOS and ICOSL represent a single receptor/ligand pair with no other known binding partners (19). Consistent with this idea, previous studies have shown that ICOS–/– mice and ICOSL–/– mice have similar phenotypes (18,19). Perhaps because of these previous reports, no simultaneous investigation of the roles of both ICOS and ICOSL in murine disease models has been performed. Furthermore, most studies have investigated the roles of ICOS or ICOSL in disease models that were related to antigen-specific adaptive immunity (20–23). While ICOS is expressed on effector and memory T cells, ICOSL is constitutively expressed on macrophages and dendritic cells, which contribute to innate immunity, in addition to its expression on B cells. Several studies have shown that ICOSL is expressed on endothelial cells and some epidermal cells, although we did not detect expression on these cell types, at least by immunohistochemical staining (results not shown). Our findings therefore suggest that ICOSL contributes to innate immunity in BLM-induced tissue fibrosis independently of the ICOS/ ICOSL pathway. Since ICOS–/–ICOSL–/– mice displayed similar phenotypes as ICOSL–/– mice after BLM treatment (Figure 5), this suggests that the ICOS/ICOSLindependent functions of ICOSL dominate over its ICOS-dependent functions in this model system. Our study also revealed that the expression levels of ICOS and ICOSL were related to each other. That is, ICOS expression was markedly up-regulated by ICOSL loss, and ICOSL expression was dramatically increased by the lack of ICOS before and during BLM treatment (Figure 6). These results reflect the previously reported increase in ICOSL expression on B cells from ICOSdeficient patients (29). Although these findings may 1731 arise from feedback regulation resulting from interactions between ICOS and ICOSL, the underlying mechanism remains unclear. A recent study demonstrated that ICOSL expression on B cells was reduced in ICOS-transgenic mice, resulting in a phenotype resembling that of ICOS–/– mice (30). These findings suggested that ICOSL expression on B cells is tightly regulated by ICOS expression on T cells, which interacted with these B cells. In our current study, ICOSL expression on macrophages and B cells from BAL fluid was markedly elevated during BLM treatment in ICOS-deficient mice as compared with wild-type mice (Figure 6). Since ICOSL loss exacerbated tissue fibrosis in spite of the presence of ICOS, the levels of ICOSL expression on macrophages or B cells were found to be inversely associated with the severity of fibrosis in this mouse model. Therefore, ICOSL expression on antigenpresenting cells may play regulatory roles that are independent of the ICOS/ICOSL pathway during development of BLM-induced tissue fibrosis. As described above, the mechanism by which ICOSL expression levels regulate tissue inflammation and fibrosis remains unclear. However, the severity of lung involvement was found to be correlated with lung TGF␤ expression levels in each strain we studied (Figures 4B and 5E). That is, TGF␤ was significantly reduced in ICOS–/– mice and significantly increased in ICOSL–/– or ICOS–/–ICOSL–/– mice. Since TGF␤ has been considered the central player in tissue fibrosis, we proposed that ICOSL regulation of TGF␤ might be affecting the development of lung fibrosis. Our findings suggest that independently of its interactions with ICOS, ICOSL plays a dominant role in regulating TGF␤ induction and the development of fibrosis in BLM-induced murine fibrosis models. A limitation of this study is that there are alternative explanations for the findings, among which is the possibility that the procedure we followed to obtain ICOSL–/– mice could have generated additional unwanted gene-encoded mutations that represent a more relevant explanation of the findings. In addition, the findings in the BLM-induced mouse model of fibrosis are not simply translatable to the pathogenesis and the therapeutic approach to SSc in humans. A recent article proposed some criteria to use in selecting the most promising molecular targets for trials in SSc (31). One of those criteria is that the antifibrotic effects should be confirmed in at least 2 complementary animal models of SSc. Further studies that include assessments in additional animal models will be needed to clarify the roles of ICOS and ICOSL in the development of SSc. 1732 TANAKA ET AL AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Hasegawa had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Fujimoto, Hasegawa. Acquisition of data. Tanaka. Analysis and interpretation of data. Tanaka, Fujimoto, Hamaguchi, Sato, Takehara, Hasegawa. REFERENCES 1. Varga J, Abraham D. Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest 2007;117:557–67. 2. Gabrielli A, Avvedimento EV, Krieg T. Scleroderma. N Engl J Med 2009;360:1989–2003. 3. Schrier DJ, Phan SH, McGarry BM. 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