Journal: Arthritis and rheumatism

Article Title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

doi: 10.1002/art.23937

Figure Lengend Snippet: Figure 1. Effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM)–stimulated CCL2 production and MEK/ERK phosphorylation. A, Human bone marrow–derived osteoblasts were incubated for various periods of time with 10 ng/ml OSM, and the CCL2 mRNA levels were assayed by Northern blotting. B and C, Human bone marrow–derived osteoblasts (B) and MG-63 cells (C) were incubated for 8 hours with OSM, alone or in combination with 10 g/ml EGCG (3 hours before the addition of OSM), and CCL2 mRNA levels were subjected to Northern blotting. D and E, Cells were incubated for 24, 48, or 72 hours with OSM, alone or in combination with 10 g/ml EGCG (3 hours before the addition of OSM). CCL2 released into the culture media of human bone marrow–derived osteoblasts (D) and MG-63 (E) was quantified by enzyme-linked immunosorbent assay. Open bars indicate OSM; solid bars indicate OSM combined with EGCG. Values are the mean and SD results from 3 independent experiments. P 0.05 versus OSM at 0 hour; P 0.05. F, MG-63 cells were incubated for 20 minutes with OSM, alone or in combination with EGCG, and were assessed for phosphorylation at MEK-1/2 (Ser217/221) and ERK-1/2 (Thr202/Tyr204).

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

Techniques: Phospho-proteomics, Derivative Assay, Incubation, Northern Blot, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

doi: 10.1002/art.23937

Figure Lengend Snippet: Figure 4. Effects of EGCG on OSM-induced activator protein 1 (AP-1)–DNA binding and AP-1–CCL2 promoter interaction in MG-63 cells. A–C, Cells were incubated for 30 minutes with 10 ng/ml OSM, alone or in combination with 10 g/ml EGCG (3 hours before the addition of OSM). The expression of c-Fos (A) and c-Jun (B) mRNA was determined by Northern blot analysis, and the respective protein levels were examined by Western blotting (C). D, Nuclear extracts from cells were subjected to electrophoretic mobility shift assay with supershift, using anti–c-Fos antibody. E, Cells were also subjected to chromatin immunoprecipitation (IP) assay with the indicated antibodies or with normal rabbit serum as control (mock). Immunoprecipitates from each sample, including input and mock, were analyzed by polymerase chain reaction using primers specific for the AP-1 binding region of human CCL2 promoter. Ac-H3 acetylated histone 3 (see Figure 1 for other definitions).

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

Techniques: Binding Assay, Incubation, Expressing, Northern Blot, Western Blot, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Control, Polymerase Chain Reaction

Journal: Arthritis and rheumatism

Article Title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

doi: 10.1002/art.23937

Figure Lengend Snippet: Figure 6. Immunolocalization of CCL2 and CD68 in the ankle joints of rats with collagen-induced arthritis. A–D, Sections from the control group. A, Pannus (P) formation and obliteration of joint space (JS). B, High-power view of A, showing erosion of cartilage and bone by pannus. C, Marked expression of CCL2 in osteoblasts (arrowheads) overlying osteolytic areas and mononuclear round cells (arrows). D, Clearly visible CD68 in macrophages (arrowheads) and osteoclasts (arrows) adjacent to resorption lacunae. E–H, Sections from the group treated with epigallocatechin- 3-gallate. E, Preservation of joint space, cartilage, and bone. F, High-power view of E, showing minimal inflammatory cell infiltration and cartilage erosion. G, Diminished numbers of CCL2 osteoblasts (arrowheads). H, Decreased infiltration of CD68 macrophages (arrowheads) and osteoclasts (arrows). (Hematoxylin and eosin staining in A, B, E, and F; avidinbiotinperoxidase staining in C, D, G, and H. (Original magnification 25 in A and E; 200 in B, C, D, F, G, and H.)

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

Techniques: Control, Expressing, Preserving, Staining

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Whole-lung murine IL-13, CCL6, and CCL21 levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Lung samples were removed at days 35 and 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice, and all soluble proteins were measured by specific ELISA. Data shown are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with appropriate the C.B-17SCID/bg group that received normal fibroblasts. ττP ≤ 0.01, τττP ≤ 0.001 compared with whole-lung cytokine and chemokine levels at the day 35 time point in the C.B-17SCID/bg groups with either IPF/UIP or NSIP fibroblasts The whole-lung cytokine and chemokine levels in control C.B-17SCID/bg group that did not receive fibroblasts were as follows: IL-13, 0.18 ± 0.014 ng/mg protein; CCL6, 0.37 ± 0.05 ng/mg protein; and CCL21, 2.0 ± 0.1 ng/mg protein.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: SuperArray Analysis of Human CCR7 and CCL21 and TaqMan Analysis of Human CCR7 in Whole-Lung Samples from C.B-17SCID/bg Mice at Day 35 after i.v. Human Fibroblast Injection

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Injection

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Representative Mason trichrome-stained histological sections from C.B-17SCID/bg mice that received normal (A–C), NSIP (D–F), or IPF/UIP (G–I) fibroblasts. No interstitial remodeling was apparent in C.B-17SCID/bg mice that received normal fibroblasts, but vascular anomalies were observed in this group (B), and the IgG (A), anti-CCL21 monoclonal antibody (B), and anti-CCR7 monoclonal antibody (C) therapies did not alter the lung histological appearance in this group. Pulmonary remodeling was apparent in C.B-17SCID/bg mice that received NSIP fibroblasts, and this pattern was not altered by IgG (D), whereas the anti-CCL21 antibody (E) or anti-CCR7 antibody (F) therapies markedly reduced the interstitial remodeling in whole-lung samples. Interstitial pulmonary fibrosis was apparent in C.B-17SCID/bg mice that received IPF/UIP fibroblasts, and this pattern was not altered by IgG (G), whereas the anti-CCL21 antibody (H) or anti-CCR7 antibody (I) therapies markedly reduced the interstitial remodeling in whole-lung samples. Monoclonal antibody therapies began at day 35 and continued to day 63, and lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Original magnification, ×400.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Staining

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Quantitative TaqMan PCR analysis of extracellular matrix-associated genes MMP-2 (top) and MMP-19 (bottom) in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Changes in gene expression are expressed as mean ± SEM of the fold increase in transcript expression above a group of C.B-17SCID/bg mice that received PBS, PKH26, and one of IgG, anti-CCL21 antibody, and anti-CCR7 antibody. *P ≤ 0.05, ***P ≤ 0.001 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgG treatment.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Expressing

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Whole-lung hydroxyproline levels in C.B-17SCID/bg mice that received no fibroblasts (ie, control) or received normal, NSIP, or IPF/UIP fibroblasts. All groups of mice received either IgG or monoclonal antibody therapy. IgG, anti-CCL21, and anti-CCR7 monoclonal antibody therapies began in separate groups of C.B-17SCID/bg mice at day 35 and continued to day 63. Lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Data shown are mean ± SEM. *P ≤ 0.05 compared with the control C.B-17SCID/bg group, which did not receive any human fibroblasts; τττP ≤ 0.001 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgG treatment; τP ≤ 0.05 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgC treatment.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Control

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Whole-lung murine IL-13, CCL6, and CCL21 levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts and IgG or monoclonal antibody therapies. Anti-CCL21 and anti-CCR7 monoclonal antibody therapies began at day 35 and continued to day 63. Lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Specific ELISA was used to measure all soluble proteins. Data shown are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01 compared with indicated protein levels measured in whole-lung samples from C.B-17SCID/bg mice that received human fibroblasts.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Enzyme-linked Immunosorbent Assay