Journal:

Article Title: Pulmonary Epithelium Is a Prominent Source of Proteinase-activated Receptor-1-inducible CCL2 in Pulmonary Fibrosis

doi: 10.1164/rccm.200712-1827OC

Figure Lengend Snippet: Proteinase-activated receptor-1 (PAR1) and chemokine (CC motif) ligand-2 (CCL2) are coexpressed in bleomycin-induced lung injury. Shown is the immunohistochemical localization of PAR1 (A, C, and E) and CCL2 (B, D, and F) in adjacent lung sections 6 days after instillation of saline (A and B) or bleomycin (C–F). Increased immunoreactivity for both PAR1 and CCL2 was observed on bronchial epithelial cells (BE, arrows) and alveolar epithelial cells (AE) as well as macrophages (M) in both moderate (C and D) and severe (E and F) lesions. Scale bars: (A–F) 50 μm.

Article Snippet: Immunostaining was undertaken by the avidin–biotinylated enzyme complex method (Vector Laboratories, Burlingame, CA) with antibodies against human PAR 1 (anti-TRED; 3 μg/ml), mouse PAR 1 (anti-SFFL; 0.14 μg/ml), human CCL2 (MAB2791; final concentration, 8 μg/ml; R&D Systems), or mouse CCL2 (sc-1784; final concentration, 0.5 μg/ml; Santa Cruz Biotechnology), and equivalent concentrations of polyclonal nonimmune IgG controls.

Techniques: Immunohistochemical staining

Journal:

Article Title: Pulmonary Epithelium Is a Prominent Source of Proteinase-activated Receptor-1-inducible CCL2 in Pulmonary Fibrosis

doi: 10.1164/rccm.200712-1827OC

Figure Lengend Snippet: Proteinase-activated receptor-1 (PAR1) and chemokine (CC motif) ligand-2 (CCL2) immunostaining in idiopathic pulmonary fibrosis (IPF) identifies epithelial cells and macrophages in fibrotic areas as major cell types displaying dual immunoreactivity. Shown is immunohistochemistry for the epithelial cell marker cytokeratin-7 (CK7) (A and B), PAR1 (C and D), CCL2 (E and F), and CD68 (G and H), in serial sections of IPF (right) and control lung (left). Immunostaining for CCL2 (F), observed in discrete areas of the IPF lung, is associated with both the epithelial marker CK7 (B) and PAR1 (D), most prominently on cells morphologically characteristic of hyperplastic alveolar (asterisks) and bronchial epithelium (arrowheads) (B, D, and F). PAR1 and CCL2 staining is also evident in regions of macrophage infiltration demarcated by CD68 immunoreactivity (D, F, and H). Control human lung is immunoreactive for PAR1 (C) in CK7-positive alveolar and bronchial epithelial cells (A). CCL2 staining is absent from all CK7-positive cells in control lung (A and E); however, weak expression is observed in CD68-positive cells morphologically characteristic of macrophages (G), which are positive for PAR1 (C). Scale bars: (A–H) 100 μm.

Article Snippet: Immunostaining was undertaken by the avidin–biotinylated enzyme complex method (Vector Laboratories, Burlingame, CA) with antibodies against human PAR 1 (anti-TRED; 3 μg/ml), mouse PAR 1 (anti-SFFL; 0.14 μg/ml), human CCL2 (MAB2791; final concentration, 8 μg/ml; R&D Systems), or mouse CCL2 (sc-1784; final concentration, 0.5 μg/ml; Santa Cruz Biotechnology), and equivalent concentrations of polyclonal nonimmune IgG controls.

Techniques: Immunostaining, Immunohistochemistry, Marker, Staining, Expressing

Journal:

Article Title: Pulmonary Epithelium Is a Prominent Source of Proteinase-activated Receptor-1-inducible CCL2 in Pulmonary Fibrosis

doi: 10.1164/rccm.200712-1827OC

Figure Lengend Snippet: Dual immunofluorescence reveals coimmunostaining for proteinase-activated receptor-1 (PAR1) and chemokine (CC motif) ligand-2 (CCL2) on epithelial cells overlying fibrotic foci and infiltrating macrophages. Immunohistochemistry for the epithelial cell marker cytokeratin-7 (CK7) (A) and the macrophage marker CD68 (E) in serial sections, directly adjacent to dual immunofluorescence costaining for PAR1 and CCL2 (C). PAR1-positive cells (B) are visualized in red, CCL2 (D) in green, and cells copositive for PAR1 and CCL2 appear orange (C). Cell nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining (F, blue). Colocalization of PAR1 and CCL2 is prominent on CK7-positive epithelial cells (B and A) overlying the fibrotic focus, in addition to infiltrating inflammatory cells positive for the macrophage antigen CD68 (B and E). Scale bars: (A and E) 50 μm.

Article Snippet: Immunostaining was undertaken by the avidin–biotinylated enzyme complex method (Vector Laboratories, Burlingame, CA) with antibodies against human PAR 1 (anti-TRED; 3 μg/ml), mouse PAR 1 (anti-SFFL; 0.14 μg/ml), human CCL2 (MAB2791; final concentration, 8 μg/ml; R&D Systems), or mouse CCL2 (sc-1784; final concentration, 0.5 μg/ml; Santa Cruz Biotechnology), and equivalent concentrations of polyclonal nonimmune IgG controls.

Techniques: Immunofluorescence, Immunohistochemistry, Marker, Staining

Journal:

Article Title: Pulmonary Epithelium Is a Prominent Source of Proteinase-activated Receptor-1-inducible CCL2 in Pulmonary Fibrosis

doi: 10.1164/rccm.200712-1827OC

Figure Lengend Snippet: Proteinase-activated receptor-1 (PAR1) and chemokine (CC motif) ligand-2 (CCL2) mRNA levels are increased in alveolar septae in idiopathic pulmonary fibrosis (IPF). (A) PAR1 and monocyte chemotactic protein (MCP)-1 expression in microdissected alveolar septae from human IPF and donor lungs as analyzed by quantitative real-time polymerase chain reaction. Data are enumerated as the fold increase relative to mean mRNA levels in donors, normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) ± SEM (patients with IPF: MCP-1, n = 8; PAR1, n = 9) (donor subjects: MCP-1, n = 7; PAR1, n = 6). *P < 0.05, **P < 0.005 (Student t test, two-tailed, two sample, equal variance). B and C show a typical example of an alveolar septum (B) before and (C) after microdissection.

Article Snippet: Immunostaining was undertaken by the avidin–biotinylated enzyme complex method (Vector Laboratories, Burlingame, CA) with antibodies against human PAR 1 (anti-TRED; 3 μg/ml), mouse PAR 1 (anti-SFFL; 0.14 μg/ml), human CCL2 (MAB2791; final concentration, 8 μg/ml; R&D Systems), or mouse CCL2 (sc-1784; final concentration, 0.5 μg/ml; Santa Cruz Biotechnology), and equivalent concentrations of polyclonal nonimmune IgG controls.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Laser Capture Microdissection

Journal:

Article Title: Pulmonary Epithelium Is a Prominent Source of Proteinase-activated Receptor-1-inducible CCL2 in Pulmonary Fibrosis

doi: 10.1164/rccm.200712-1827OC

Figure Lengend Snippet: Thrombin induces chemokine (CC motif) ligand-2 (CCL2) release by lung epithelial cells via a proteinase-activated receptor-1 (PAR1)-dependent mechanism. (A) Time course for the effect of thrombin on CCL2 protein release by A549 cells. Cells were exposed to serum-free control medium (F12K) or thrombin (10 nM) for incubation times from 3 to 48 hours, supernatants were collected, and CCL2 protein release was measured by ELISA. ***P < 0.001, post-hoc pair-wise comparison of thrombin and medium control groups at each time point (two-way analysis of variance [ANOVA] with Bonferroni post-hoc testing). (B and C) Effect of PAR1 activation on CCL2 protein release by A549 cells. Cells were exposed to serum-free control medium, thrombin (0.1 to 100 nM) (B), the PAR1-activating peptide TFLLR-NH2 (200 μM) (C), or the inactive partial reverse peptide FTLLR-NH2 (C) for 6 hours. Cell supernatants were analyzed for CCL2 protein release by ELISA. Data represent the mean ± SEM of four replicates per group. *P < 0.05, **P < 0.01, comparison of thrombin or TFLLR-NH2 treatment with medium control (one-way ANOVA with Student-Newman-Keuls post-hoc testing). (D) Time course for the effect of thrombin and TFLLR-NH2 on CCL2 mRNA levels. A549 cells were exposed to serum-free control medium (F12K), thrombin (10 nM), or TFLLR-NH2 (200 μM) for incubation times from 1.5 to 12 hours. CCL2 mRNA levels at each time point were assessed by quantitative real-time polymerase chain reaction (qPCR). Data are expressed as fold change relative to time 0 for each time point after normalization to 18S RNA. Data represent the mean ± SEM of four replicates per group at each time point. **P < 0.01, ***P < 0.001, comparison of thrombin-induced change in crossing point (ΔCp) with time point–matched control; ΨΨP < 0.01, ΨΨΨP < 0.001, comparison of TFLLR-induced mRNA levels with time point–matched control (two-way ANOVA with Bonferroni post-hoc testing). (E) Effect of the PAR1-specific antagonist RWJ-58259 on thrombin-induced CCL2 gene expression by A549 cells. Cells were preincubated with RWJ-58259 (2 to 6 μM) or an equivalent dilution of vehicle (DMSO) for 30 minutes before exposure to thrombin for 1.5 hours. CCL2 mRNA levels for each condition were assessed by qPCR. Data are expressed as fold change relative to medium control after normalization to 18S RNA, and represent the mean ± SEM from four replicates per group with each concentration of antagonist. *P < 0.05, comparison of thrombin-induced CCL2 ΔCp with CCL2 ΔCp in the presence of vehicle-alone CCL2 ΔCp; ΨP < 0.05, comparison of thrombin-induced CCL2 ΔCp in the presence of antagonist with thrombin-induced ΔCp in the presence of vehicle alone (one-way ANOVA with Student-Newman-Keuls post-hoc testing). (F) Effect of the PAR1-specific antagonist RWJ-58259 on thrombin-induced CCL2 released by A549 cells (thrombin [10 nM], 6 h). Cell supernatants were analyzed for CCL2 protein release by ELISA. Data represent the mean ± SEM of three replicates per group. ***P < 0.001, comparison of thrombin-induced CCL2 with CCL2 released in the presence of vehicle alone; ΨΨΨP < 0.001, comparison of thrombin-induced CCL2 in the presence of antagonist, with thrombin-induced CCL2 in the presence of vehicle alone (one-way ANOVA with Student-Newman-Keuls post-hoc testing). Similarly, (G) shows the effect of the PAR1–Gαq-specific antagonist, Q94, on thrombin-induced CCL2 release by A549 cells (thrombin [10 nM], 6 h). Cell supernatants were analyzed for CCL2 protein release by ELISA. Data represent the mean ± SEM of three replicates per group. ***P < 0.001, comparison of thrombin-induced CCL2 with CCL2 released in the presence of vehicle alone; ΨΨΨP < 0.001, comparison of thrombin-induced CCL2 in the presence of antagonist with that of thrombin-induced CCL2 in the presence of vehicle alone (one-way ANOVA with Student-Newman-Keuls post-hoc testing). (H) Effect of the PAR1-specific antagonist RWJ-58259 on thrombin-induced protein expression (thrombin [10 nM], 1.5 h and release [6 h] from bronchial epithelial cells [BEAS-2B]). Data are expressed and compared as described in (E) and (F).

Article Snippet: Immunostaining was undertaken by the avidin–biotinylated enzyme complex method (Vector Laboratories, Burlingame, CA) with antibodies against human PAR 1 (anti-TRED; 3 μg/ml), mouse PAR 1 (anti-SFFL; 0.14 μg/ml), human CCL2 (MAB2791; final concentration, 8 μg/ml; R&D Systems), or mouse CCL2 (sc-1784; final concentration, 0.5 μg/ml; Santa Cruz Biotechnology), and equivalent concentrations of polyclonal nonimmune IgG controls.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Concentration Assay

Journal:

Article Title: Pulmonary Epithelium Is a Prominent Source of Proteinase-activated Receptor-1-inducible CCL2 in Pulmonary Fibrosis

doi: 10.1164/rccm.200712-1827OC

Figure Lengend Snippet: Thrombin induces chemokine (CC motif) ligand-2 (CCL2) release by freshly isolated primary alveolar epithelial cells. Confluent monolayers of primary human (A) and murine (B) type II epithelial cells were serum-starved for 24 hours and incubated for 6 hours with either control medium or medium containing thrombin (10 nM). Primary human type II cells (A) were isolated from lungs of grossly normal appearance after resection for lung carcinoma and the data are representative of the trend obtained for three separate donors, although baseline production levels varied for each donor. Data represent the mean ± SEM of three experimental replicates per group. *P < 0.05, ***P < 0.001, comparison of thrombin-induced CCL2 with CCL2 released in the presence of vehicle alone (Student t test).

Article Snippet: Immunostaining was undertaken by the avidin–biotinylated enzyme complex method (Vector Laboratories, Burlingame, CA) with antibodies against human PAR 1 (anti-TRED; 3 μg/ml), mouse PAR 1 (anti-SFFL; 0.14 μg/ml), human CCL2 (MAB2791; final concentration, 8 μg/ml; R&D Systems), or mouse CCL2 (sc-1784; final concentration, 0.5 μg/ml; Santa Cruz Biotechnology), and equivalent concentrations of polyclonal nonimmune IgG controls.

Techniques: Isolation, Incubation

Journal: Arthritis and rheumatism

Article Title: Oncostatin M-induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: an implication for the pathogenesis of arthritis.

doi: 10.1002/art.24452

Figure Lengend Snippet: Figure 1. Oncostatin M (OSM) stimulation of CCL2 expression in human bone marrow–derived osteoblasts and U2OS cells. A and B, Human bone marrow–derived osteoblasts (A) and U2OS cells (B) were incubated for various time periods with 10 ng/ml OSM, and CCL2 mRNA levels were assayed by Northern blotting. C and D, Cells were incubated for 24, 48, and 72 hours with OSM, and CCL2 released into the culture media of human bone marrow–derived osteoblasts (C) and U2OS cells (D) was quantified by enzyme-linked immunosorbent assay. Values are the mean and SD of 3 independent experiments. P 0.05 versus control. E and F, The effect of OSM treatment on the survival of human bone marrow–derived osteoblasts (E) and U2OS cells (F) was examined by MTT assay. Values are the mean and SD. The result at each time point was the average of data from 8 wells.

Article Snippet: Anti-human CCL2 antibody was from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Derivative Assay, Incubation, Northern Blot, Enzyme-linked Immunosorbent Assay, Control, MTT Assay

Journal: Arthritis and rheumatism

Article Title: Oncostatin M-induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: an implication for the pathogenesis of arthritis.

doi: 10.1002/art.24452

Figure Lengend Snippet: Figure 2. Involvement of STATs 1 and 3 in oncostatin M (OSM)–stimulated CCL2 expression. A, U2OS cells were transduced with short hairpin RNA (shRNA) lentivirus vectors targeting STAT-1 and STAT-3. Gene silencing effects were confirmed by Western blotting. B, Cells transduced with shRNA lentivirus targeting STAT-1 and STAT-3 were stimulated with OSM (10 ng/ml) for 72 hours. The amounts of CCL2 released into the culture media were detected by enzyme-linked immunosorbent assay (ELISA). C, U2OS cells were treated with OSM for the indicated time periods. Protein extracts were subjected to immunoblotting with phosphospecific antibodies against S727 of STAT-1 and STAT-3. D and E, Cells were stimulated for 20 minutes with OSM, alone or in combination with 50 M LY294002, 8 M SH-5, 50 ng/ml rapamycin, 50 M PD98059, 10 M SB203580, or 200 M dicumarol (inhibitors were added 3 hours before the addition of OSM), and the amounts of phosphorylated S727 in STAT-1 (D) and STAT-3 (E) were determined. F, CCL2 released into the culture media was quantified by ELISA. Values in B and D–F are the mean and SD of 3 independent experiments. P 0.05 versus control; P 0.05 versus OSM alone.

Article Snippet: Anti-human CCL2 antibody was from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transduction, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Arthritis and rheumatism

Article Title: Oncostatin M-induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: an implication for the pathogenesis of arthritis.

doi: 10.1002/art.24452

Figure Lengend Snippet: Figure 5. Oncostatin M (OSM)–promoted associations between c-Fos, STAT-1, and STAT-3 and their roles in CCL2 promoter activity. A, U2OS cells were incubated with OSM (10 ng/ml) for 30 minutes before lysis. The protein extracts were immunoprecipitated (IP) with anti–c-Fos antibody and subjected to Western blotting with antibodies against STAT-1 and STAT-3. B, U2OS cells (wild-type or transduced with short hairpin RNA [shRNA] lentivirus vector targeting c-Fos) were treated with OSM (10 ng/ml) for 30 minutes. Gene silencing effects were confirmed by Western blotting. C, U2OS cells transduced with shRNA lentivirus vectors targeting STAT-1, STAT-3, and c-Fos, either alone or in combination, were cotransfected with pcDNA3.1- CCL2-Luc and pRL-TK. Cells were subsequently stimulated with OSM (10 ng/ml) for 8 hours, and CCL2 promoter activities were determined by luciferase assay. Values are the mean and SD of 3 independent experiments. P 0.05 versus control; P 0.05 versus OSM alone.

Article Snippet: Anti-human CCL2 antibody was from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Incubation, Lysis, Immunoprecipitation, Western Blot, Transduction, shRNA, Plasmid Preparation, Luciferase, Control

Journal: Arthritis and rheumatism

Article Title: Oncostatin M-induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: an implication for the pathogenesis of arthritis.

doi: 10.1002/art.24452

Figure Lengend Snippet: Figure 6. Effects of AG-490 on STAT activation, CCL2 production, and collagen-induced arthritis (CIA) progression. A–C, U2OS cells were incubated with 10 ng/ml oncostatin M (OSM), alone or in combination with 50 M AG-490 (3 hours before the addition of OSM), for 20 minutes (A), 8 hours (B), or 72 hours (C). A, Protein extracts were subjected to immunoblotting with antibodies against pS727–STAT-1 and pS727–STAT-3. B, CCL2 mRNA levels were assayed by Northern blotting. C, CCL2 protein in the culture media was quantified by enzyme-linked immunosorbent assay. Values are the mean and SD of 3 independent experiments. P 0.05 versus control; P 0.05 versus OSM alone. D–I, Rats with CIA (10 per group) were injected with DMSO alone (D–F) or with AG-490 in DMSO (G–I) on a daily basis until day 21 when they were killed. In the paws of control rats (D–F), there was extensive erosion of bone by pannus (E) and marked expression of CCL2 in osteoblasts (arrowheads) overlying the osteolytic areas and mononuclear round cells (arrows) (F). In the paws of AG-490–treated rats (G–I), there was minimal cartilage/bone erosion and inflammatory cell infiltration (H) and decreased numbers of CCL2 osteoblasts (arrowheads) (I). (Hematoxylin and eosin staining in E and H, avidin–biotin–peroxidase complex staining in F and I; original magnification 400 in E, F, H, and I.) J–L, The severity of arthritis was quantified by the clinical arthritis index (J), the histopathologic score (K), and the percentage of CCL2 osteoblasts (L). Values are the mean and SD. P 0.05 versus control.

Article Snippet: Anti-human CCL2 antibody was from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Incubation, Western Blot, Northern Blot, Enzyme-linked Immunosorbent Assay, Control, Injection, Expressing, Staining, Avidin-Biotin Assay