Journal:

Article Title: Treatment of murine lupus with cDNA encoding IFN-?R/Fc

doi:

Figure Lengend Snippet: IgG deposits and expression of MHC class II, ICAM-1, and MCP-1 proteins in kidney tissues from MRL-Faslpr mice treated from 1 month of age with blank (VR1255) or active (VR1255-IFNγR/Fc) plasmid with electroporation. Active plasmid–treated mice had substantially reduced glomerular IgG deposits, as well as decreased levels of MHC class II, ICAM-1, and MCP-1. Similar reductions were observed in mice treated from 4 months of age with active plasmid (data not shown). IgG deposits were detected with FITC–anti-IgG antibody (×45), and the immunoperoxidase procedure for detection of MHC class II, ICAM-1, and MCP-1 was performed as described in Figure ​Figure55 (×25). Photomicrographs are representative samples from four mice per group.

Article Snippet: Subsequently, sections were incubated with various primary antibodies, including anti–IgG-FITC (Vector Labs), anti-CD3-biotin (PharMingen), anti–F4/80-biotin (Caltag), anti–ICAM-1 (PharMingen), anti–MHC class II (PharMingen), and anti–MCP-1 (Santa Cruz Biotechnology, Santa Cruz, California, USA).

Techniques: Expressing, Plasmid Preparation, Electroporation

Journal:

Article Title: Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6

doi: 10.1073/pnas.0607514104

Figure Lengend Snippet: Chemokines in the LPS model of fetal loss. (A–E) Serum chemokine concentrations after LPS treatment in WT and D6−/− male mice. WT (open symbols) and D6−/− (filled symbols) mice were injected i.p. with 1.35 mg/kg LPS. At the indicated time points, circulating chemokine concentrations were measured by ELISA. Data are from seven mice for each time point. (F–J) Serum chemokine concentrations. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Circulating chemokine concentrations were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. (K–O) Chemokine levels in placenta. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Chemokine concentrations (expressed as nanograms of chemokine per milligram of total proteins of the lysates) were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. Results are reported as mean ± SEM. (A, F, and K) CCL22. (B, G, and L) CCL2. (C, H, and M) CCL11. (D, I, and N) CCL3. (E, J, and O) CXCL2.

Article Snippet: To block inflammatory chemokines, animals were treated with a mixture of goat antibodies to the mouse CC chemokines CCL3L1 (catalog no. AB450NA), CCL4 (catalog no. AB451NA), CCL2 (catalog no. AB479NA) and rat mAb anti-mouse CCL5 (catalog no. MAB478) purchased lyophilized from R&D Systems, resuspended in PBS, and mixed.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

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

Journal: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: Effect of monosodium urate (MSU) crystals on CCL2 production in cultured fibroblast-like synoviocytes (FLS) . (a) FLS were treated with increasing concentrations of MSU crystals for 24 hours. (b) Synoviocytes were incubated in the presence (black circles) or absence (white circles) of 50 μg/ml MSU crystals for the indicated time. CCL2 was measured in culture supernatants with ELISA. Results are presented as mean ± SD of three separate experiments. (c) FLS were stimulated (grey columns) or not (white columns) for 24 hours with 50 μg/ml MSU crystals in the presence or absence of 250 ng/ml IL-1Ra. IL-1Ra was either added with MSU crystals (MSU + IL-1Ra) or added to FLS 1 hour before activation by MSU crystals (IL-1Ra + MSU). (d) FLS were stimulated (black columns) or not (white columns) for 24 hours with 125 pg/ml IL-1β in the presence or absence of 250 ng/ml of IL-1Ra. IL-1Ra was either added with IL-1β (IL-1β + IL-1Ra) or added to FLS 1 hour before activation by IL-1β (IL-1Ra + IL-1β). (c, d) Culture supernatants were analyzed for the production of CCL2. Results are presented as mean ± SD of three separate experiments.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: CCL2 is stored in cytoplasmic vesicles in fibroblast-like synoviocytes (FLS) . FLS were cultured for 24 hours in the absence (a, c) or presence (b) of 50 μg/ml monosodium urate (MSU) crystals. Cells were subjected to immunostaining with anti-CCL2 antibodies (a, b) or incubated with the second antibody only as a negative control (c) , and then analyzed with confocal microscopy, as described in Materials and Methods. Original magnification, ×1,000.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Cell Culture, Immunostaining, Incubation, Negative Control, Confocal Microscopy

Journal: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: High-density lipoproteins (HDL) inhibit CCL2 production induced by monosodium urate (MSU) crystals in fibroblast-like synoviocytes (FLS) . (a, b) FLS were stimulated (grey columns) or not (white columns) for 24 hours with 50 μg/ml MSU crystals in the presence or absence of the indicated concentration of HDL. Alternatively, FLS were pretreated (Ptt.) with the indicated concentration of HDL, washed, and then stimulated for 24 hours with 50 μg/ml MSU crystals. Culture supernatants were analyzed for the production of CCL2 (a) and IL-8 (b). Results are presented as mean ± SD of duplicate determinations and are representative of independent experiments carried out with FLS isolated from three different patients. (c) FLS were stimulated (black columns) or not (white columns) with 10 pg/ml IL-1β for 24 hours. Culture supernatants were analyzed for the production of CCL2. (d) Culture supernatants of FLS, activated as in (a) and (b), were analyzed for their ability to induce mononuclear cell migration, as described in Materials and Methods. Migration induced by culture medium (white column), 10 ng/ml CCL2 (black column), and culture supernatants of FLS activated as in (a) and (b) (grey columns). Four fields were counted for the number of migrated cells. Results represent the mean ± SD of the number of cells/field in four fields. A representative experiment of three is presented.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Concentration Assay, Isolation, Migration

Journal: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: Fibroblast-like synoviocytes (FLS) stimulated by monosodium urate (MSU) crystals in the presence of high-density lipoproteins (HDL) retain intracellular CCL2 . FLS were stimulated or not with MSU crystals (50 μg/ml) in the presence or absence of the indicated concentration of HDL. After 24 hours, cells were fixed and subjected to immunostaining with anti-CCL2 antibodies. (a) Resting FLS; (b, c) FLS stimulated with MSU crystals in the absence (b) or presence of HDL (c) . Original magnification × 600. (d) FLS were treated (white columns) or not (grey columns) with 10 μg/ml cycloheximide (CHX) for 30 minutes and then activated for the indicated time with 50 μg/ml MSU crystals. Results represent mean ± SD of three independent experiments.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Concentration Assay, Immunostaining

Journal: Arthritis Research & Therapy

Article Title: High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

doi: 10.1186/ar2930

Figure Lengend Snippet: High-density lipoproteins (HDL) diminish monosodium urate (MSU) crystal-induced CCL2 transcript levels . Fibroblast-like synoviocytes (FLS) were cultured for 18 hours alone or with MSU crystals (50 μg/ml) in the presence or absence of HDL (100 μg/ml), as indicated. Total RNA was prepared as described in Materials and Methods. CCL2 mRNA levels were determined with duplex quantitative real-time polymerase chain reaction (PCR) analysis of triplicates normalized to the levels of the 18S mRNA. The relative expression levels of CCL2 mRNA are presented as mean ± SD of the percentage of relative CCL2 mRNA expression. The value of mRNA levels in MSU crystal-stimulated FLS (MSU) is arbitrarily considered as 1.0. Results are representative of three distinct experiments.

Article Snippet: After washing and blocking with 2% BSA for 30 minutes at room temperature, cells were incubated for 1 hour with anti-CCL2 mAb (R&D systems, Minneapolis, MN, USA) diluted 1:20 in blocking buffer.

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Expressing