Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Temporal expression of growth factors triggered by epiregulin regulates inflammation development.

doi: 10.4049/jimmunol.1400562

Figure Lengend Snippet: FIGURE 1. Areg, BTC, TGF-a, FGF2, PLGF, and TNC are critical for the development of cytokine-induced arthritis. (A) Serum concentrations of Areg, BTC, TGF-a, FGF2, PLGF, and TNC in patients suffering from RA (n = 11) compared with healthy age- and sex-matched subjects (n = 26). (B) IL-17A (0.2 mg) and IL-6 (0.2 mg) on days 0, 1, and 2 were injected into the knee joints of F759 mice. mRNA expressions of Areg, Btc, Tgfa, Fgf2, Plgf2, and TNC in joint synovial tissues were analyzed on day 7. (C) Clinical arthritis scores from the left legs of F759 mice after left ankle joint injections of 0.1 mg IL-6 and IL-17 on days 0, 1, and 2 and joint injections of anti-Areg Abs (1 mg, n = 6), anti-Btc Abs (1 mg, n = 6), anti–TGF-a Abs (1 mg, n = 6), anti-FGF2 Abs (1 mg, n = 6), anti-PLGF2 Abs (1 mg, n = 6), anti-TNC Abs (1 mg, n = 6), anti-IgG (1 mg, n = 6), or PBS with neither IL-6 nor IL-17 (n = 6) once every 2 or 3 d for 0–22 d. (D) Clinical arthritis scores from the left legs of F759 mice after left ankle joint injections of 0.1 mg IL-6 and IL-17 on days 6, 7, and 8 and joint injections of lentivirus encoding shRNA specific for p65 NF-kB (RelA) (1.9 3 105 transducing units [TU], n = 6), Areg (1.9 3 105 TU, n = 6), Btc (1.9 3 105 TU, n = 6), Tgfa (1.9 3 105 TU, n = 6), Fgf2 (1.9 3 105 TU, n = 6), Plgf2 (1.9 3 105 TU, n = 6), TNC (1.9 3 105 TU, n = 6), a nontarget sequence (1.9 3 105 TU, n = 6), or saline with neither IL-6 nor IL-17 (n = 6) on days 0, 2, and 4 during days 0–29. (E and F) IL-17 (0.2 mg) and IL-6 (0.2 mg) on days 0, 1, and 2 were injected into the knee joints of F759 mice in the presence or absence of joint injections of anti-Areg Ab (1 mg, n = 12), anti-BTC Ab (1 mg, n = 12), anti–TGF-a Ab (1 mg, n = 12), anti-FGF2 Ab (1 mg, n = 12), anti-Plgf2 (PLGF) Ab (1 mg, n = 12), anti-TNC Ab (1 mg, n = 12), or control IgG (1 mg, n = 12) on days 0, 1, 2, 4, and 6 followed by analysis of expressions of IL-6 (E) and CCL20 (F) in joint synovial tissues on day 7. Individual values, mean scores (A), and mean scores 6 SEM (B–F) are shown. The p values were calculated using a Wilcoxon test (A, C, and D), Student t test (B), and one- way ANOVA (E and F). *,#p , 0.05, **p , 0.01, ***p , 0.001; *p , 0.05 versus each treatment group in (C) and (D). #p , 0.05 versus sh-Fgf2, sh-Areg, sh-Btc, and sh-Plgf in (D).

Article Snippet: The following Abs were used for in vivo neutralization and immunohistochemistry: monoclonal anti-mouse Areg Ab, anti-mouse BTC Ab, anti- mouse epiregulin Ab, anti-human TGF-a Ab, anti-mouse PLGF2 Ab, anti-human/mouse TNC Ab (R&D Systems, Minneapolis, MN), antimouse FGF2 Ab (Millipore, Tokyo, Japan), polyclonal anti-mouse epiregulin Ab (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse FGF2 Ab (Abcam, Tokyo, Japan), and purified rat IgG (Sigma-Aldrich, Tokyo, Japan).

Techniques: Injection, shRNA, Sequencing, Saline, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Temporal expression of growth factors triggered by epiregulin regulates inflammation development.

doi: 10.4049/jimmunol.1400562

Figure Lengend Snippet: FIGURE 2. Areg, BTC, TGF-a, and FGF2 enhance the expressions of IL-6 and chemokines via the PI3K/NF-kB axis. (A) IL-6 (1 mg) and IL-17 (1 mg) were injected into the left ankle joints of F759 mice on days 0, 1, and 2. Immunohistochemistry of the left ankle joints was performed by using Abs against Areg, epiregulin (Ereg), FGF2, TGF-a, p-STAT3, p-p65, p-EGFR, type 1 collagen, and vimentin on day 7. These experiments were performed at least three times independently. Frequency of cells that showed activation of the inflammation amplifier (p-STAT3+p-p65+), received EGFR signaling (p-EGFR+), or produced growth factors (Areg+Ereg+FGF2+TGF-a+) is indicated. Col1, type 1 collagen; Vim, vimentin. *p , 0.05, **p , 0.01 (Student t test). (B) BC1 cells were stimulated with human IL-6 plus soluble IL-6Ra and/or mouse IL-17 for 24 h with or without Areg, BTC, TGF-a, FGF2, PLGF2, and TNC. Culture supernatants were collected and assessed using ELISA specific for IL-6. Samples without growth factors (filled columns) were compared with samples with each growth factor. xp , 0.05, *p , 0.01, #,†p , 0.001 (one-way ANOVA). (C and D) mRNA expressions of IL-6 (C) and CCL20 (D) in BC1 cells 3 h after stimulation with human IL-6 plus soluble IL-6Ra and mouse IL-17 with or without Areg, BTC, TGF-a, and FGF2 were evaluated using real- time PCR. Samples without growth factors (filled columns) were compared with samples with each growth factor. *p , 0.05, **p , 0.01, ***p , 0.001 (one-way ANOVA). (E) BC1 cells were stimulated with human IL-6 plus soluble IL-6Ra in the presence or absence of Areg, BTC, TGF-a, or FGF2 for 24 h with or without 0.5 h pretreatment of LY294002 (3 mM) or DMSO vehicle control. Culture supernatants were collected and assessed using ELISA specific for mouse IL-6. Cell survival was evaluated based on mitochondrial activity. *p , 0.05, **p , 0.01, ***p , 0.001 (Student t test). (F) BC1 cells were stimulated with human IL-6 plus soluble IL-6Ra and mouse IL-17 in the presence or absence of Areg (A), BTC (B), TGF-a (T), or FGF2 (F) for 30 min and then investigated for the phosphorylation of Akt and p65. (G) IL-6 and IL-17 were injected into the ankle joints of NF-kB reporter Tg/F759 mice with or without 0.2 mg Areg, BTC, TGF-a, or FGF2 followed by analysis of NF-kB reporter activity in the ankle joints on day 7 using the luciferase reporter assay system. *p , 0.05 (one-way ANOVA). Mean scores 6 SD (A–E) and mean scores 6 SEM (G) are shown.

Article Snippet: The following Abs were used for in vivo neutralization and immunohistochemistry: monoclonal anti-mouse Areg Ab, anti-mouse BTC Ab, anti- mouse epiregulin Ab, anti-human TGF-a Ab, anti-mouse PLGF2 Ab, anti-human/mouse TNC Ab (R&D Systems, Minneapolis, MN), antimouse FGF2 Ab (Millipore, Tokyo, Japan), polyclonal anti-mouse epiregulin Ab (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse FGF2 Ab (Abcam, Tokyo, Japan), and purified rat IgG (Sigma-Aldrich, Tokyo, Japan).

Techniques: Injection, Immunohistochemistry, Activation Assay, Produced, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Control, Activity Assay, Phospho-proteomics, Luciferase, Reporter Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Temporal expression of growth factors triggered by epiregulin regulates inflammation development.

doi: 10.4049/jimmunol.1400562

Figure Lengend Snippet: FIGURE 3. Presence of epiregulin-triggered temporal expressions in affected tissues of cytokine-induced arthritis. (A) mRNA expressions of Areg, Btc, Tgfa, epiregulin (Ereg), Fgf2, Plgf2, and Tnc in BC1 cells in the presence or absence of stimulation with IL-17 and IL-6 were evaluated 3 h later using real- time PCR. (B) mRNA expressions of Areg, Btc, Tgfa, Ereg, Fgf2, Plgf2, and Tnc in BC1 cells in the presence or absence of epiregulin stimulation were evaluated 3 h later using real-time PCR. (C) IL-17 (0.2 mg) and IL-6 (0.2 mg) on days 0, 1, and 2 were injected into the knee joints of F759 mice followed by analysis of expressions of Ereg, Areg, and TGF-a in joint synovial tissues on days 0, 1, 3, 5, and 7 (n = 3 for each condition). (D and E) IL-17 (0.2 mg) and IL-6 (0.2 mg) on days 0, 1, and 2 were injected into the knee joints of F759 mice in the presence or absence of joint injections of anti-Ereg Ab (1 mg, n = 12), anti-Areg Ab (1 mg, n = 12), anti-BTC Ab (1 mg, n = 12), anti–TGF-a Ab (1 mg, n = 12), anti-FGF2 Ab (1 mg, n = 12), anti-PLGF2 Ab (1 mg, n = 12), anti-TNC Ab (1 mg, n = 12), or control IgG (1 mg, n = 12) on days 0, 1, 2, 4, and 6 followed by analysis of the expressions of Ereg, Areg, Btc, TGF-a, FGF2, Plgf2, and Tnc (D) and Ereg (E) in joint synovial tissues on day 7. (F–I) IL-6 (1 mg) and IL-17 (1 mg) on days 0, 1, and 2 were injected into the left ankle joints of F759 mice followed staining by using antibodies against Ereg, TGF-a, and FGF2 in paraffin sections of left ankle joints on days 1 (F) and 7 (H) by immunohistochemistry. These experiments were performed at least three times independently; representative data are shown. Arrows indicate cells expressing growth factors in the ankle joint synovial tissues. Scale bars, 100 mm. Quantification of the histological analysis (10 3 0.1 mm2 field) for (F) and (H) is shown (G and I). Mean scores 6 SD (A–E) and mean scores 6 SEM (G and I) are shown. The p values were calculated using a Student t test (A and B) and one-way ANOVA (D and E). *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The following Abs were used for in vivo neutralization and immunohistochemistry: monoclonal anti-mouse Areg Ab, anti-mouse BTC Ab, anti- mouse epiregulin Ab, anti-human TGF-a Ab, anti-mouse PLGF2 Ab, anti-human/mouse TNC Ab (R&D Systems, Minneapolis, MN), antimouse FGF2 Ab (Millipore, Tokyo, Japan), polyclonal anti-mouse epiregulin Ab (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse FGF2 Ab (Abcam, Tokyo, Japan), and purified rat IgG (Sigma-Aldrich, Tokyo, Japan).

Techniques: Real-time Polymerase Chain Reaction, Injection, Control, Staining, Immunohistochemistry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Temporal expression of growth factors triggered by epiregulin regulates inflammation development.

doi: 10.4049/jimmunol.1400562

Figure Lengend Snippet: FIGURE 4. Growth factors are critical for the development of an MS model, EAE. (A) Serum concentrations of Areg, BTC, TGF-a, FGF2, PLGF, and TNC in patients suffering from MS (n = 21) compared with healthy age- and sex-matched subjects (n = 15). (B) mRNA expressions of epiregulin (Ereg), Areg, Btc, TGF-a, FGF2, Plgf2, and Tnc in the L5 cord 7 d after transfer of pathogenic CD4+ T cells were evaluated using real-time PCR. (C–E) Pathogenic CD4+ T cells isolated from EAE mice were i.v. transferred into wild-type C57BL/6 mice in the presence or absence of anti–TGF-a Ab administration (i.p., days 0–5 after the pathogenic CD4+ T cell transfer). (C) Clinical EAE scores (n = 5 each) and (D) serum IL-6 concentrations in mice (n = 15). (E) Mononuclear cells from L5 spinal cords of Th17-transferred C57BL/6 mice were isolated on day 10. The resulting cell populations were counted and stimulated in vitro with MOG peptide and bone marrow–derived dendritic cells. Twenty-four hours after in vitro stimulation, intracellular IL-17 and IFN-g levels were examined. The numbers of CD4+IL-17+ and CD4+IFN-g+ T cells in spinal cords were significantly lower in recipients treated with anti–TGF-a Ab (n = 5) than in those treated with control IgG (n = 5). Individual scores, mean scores (A), and mean scores 6 SEM (B–E) are shown. The p values were calculated using Wilcoxon tests (A and C) and Student t test (B, D, and E). *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The following Abs were used for in vivo neutralization and immunohistochemistry: monoclonal anti-mouse Areg Ab, anti-mouse BTC Ab, anti- mouse epiregulin Ab, anti-human TGF-a Ab, anti-mouse PLGF2 Ab, anti-human/mouse TNC Ab (R&D Systems, Minneapolis, MN), antimouse FGF2 Ab (Millipore, Tokyo, Japan), polyclonal anti-mouse epiregulin Ab (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse FGF2 Ab (Abcam, Tokyo, Japan), and purified rat IgG (Sigma-Aldrich, Tokyo, Japan).

Techniques: Real-time Polymerase Chain Reaction, Isolation, In Vitro, Derivative Assay, Control

Journal: Cell

Article Title: PlGF blockade does not inhibit angiogenesis during primary tumor growth.

doi: 10.1016/j.cell.2010.01.033

Figure Lengend Snippet: Figure 1. Characterization of the PlGF-Blocking mAbs C9.V2 and 7A10 (A) Inhibition of PlGF-induced 67NR-VEGFR-1 cell migration by C9.V2 and 7A10 anti-PlGF mAbs. (B) The VEGF-A-blocking abilities of two different anti-VEGF antibodies are shown as a comparison. The average numbers of migrated cells per optical field are indicated. See Experimental Procedures for details.*p < 0.05. (C and D) Inhibition of PlGF-induced VEGFR-1 and MAPK phosphorylation by C9.V2 and 7A10 anti-PlGF mAbs (n = 3, *p < 0.05 versus positive control. Data represent averages of at least four independent experiments). (E) Inhibition of biotinylated NRP-1-Fc binding to mPlGF by anti-PlGF mAbs. Anti-PlGF MAB465 is a commercial mAb from R&D Systems, mFlt1(1-3)-IgG is a soluble VEGFR-1 variant that strongly binds PlGF. Competitive binding studies were performed in PlGF-coated plates (n = 5, data represent the average of three independent experiments). (A–D) Error bars represent standard deviation (SD). See also Figure S1.

Article Snippet: Anti-PlGF MAB465 is a commercial mAb from R&D Systems, mFlt1(1-3)-IgG is a soluble VEGFR-1 variant that strongly binds PlGF.

Techniques: Blocking Assay, Inhibition, Migration, Comparison, Phospho-proteomics, Positive Control, Binding Assay, Variant Assay, Standard Deviation