Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin 6 mediates production of interleukin 10 in metastatic melanoma

doi: 10.1007/s00262-011-1084-5

Figure Lengend Snippet: Inhibitions of IL-6 pathways to block IL-10 production by CM005 melanoma cells. a Inhibition of IL-6-induced IL-10 productions by various kinase inhibitors. CM005 cells were treated with various kinase inhibitors 30 min prior to rhIL-6 (2 ng/ml) stimulation. After 24 h of incubation, the production of IL-10 in the supernatant was analyzed by ELISA assay. The data were shown as a fraction in comparison with IL-10 production in control (Ctl, no inhibitor). *P < 0.05. b Suppression of STAT3 and ERK1/2 phosphorylation by kinase inhibitors. After treatment with individual kinase inhibitors followed by IL-6 stimulation, CM005 cells were lysed and phosphorylation of STAT3 (left) and ERK1/2 (right) was measured by Western blot. The intensity of bands for phosphorylated STAT and ERK1/2 was compared to that of control (Ctl, no inhibitor). *P < 0.05, **P < 0.01

Article Snippet: Neutralizing rat anti-human IL-6 antibody and isotype-matched rat IgG1 were purchased from BD Biosciences (San Jose, CA).

Techniques: Blocking Assay, Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Comparison, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin 6 mediates production of interleukin 10 in metastatic melanoma

doi: 10.1007/s00262-011-1084-5

Figure Lengend Snippet: Relationship of IL-6 and IL-10 productions in metastatic melanoma cells. a IL-6 production by fresh metastatic melanoma cell suspensions. Three fresh metastatic melanoma cell suspensions produced high levels of IL-6 (>1,000 pg/ml, high IL-6 producers, solid bars), while seven suspensions produced small or no quantity of IL-6 (low IL-6 producers, open bars) in 18-h culture. b Increased IL-10 production by fresh metastatic melanoma cell suspensions in response to exogenously added IL-6. Fresh metastatic melanoma cell suspensions were cultured with various concentration of rhIL-6. “Control, 0 ng/ml” indicates constitutive production of IL-10 without IL-6 stimulation. Two of 7 low IL-6 producers (DIL and Ho88) produced IL-10 constitutively and increased IL-10 production in response to exogenously added IL-6 in a dose-dependent manner. The rest of low IL-6 producers had minimum or no constitutive production of IL-10 and did not responded to exogenously added IL-6. All three high IL-6 producers (Cla, Beu, Ho91) constitutively secreted IL-10 with mild increase in IL-10 production in response to high dose of IL-6. *P < 0.05, **P < 0.01. c Maintaining IL-6-induced IL-10 production in long-term cultured metastatic melanoma cell lines. Two cell lines (DIL and Ho88) established from low IL-6 producers maintained their capability of producing IL-10 in response to exogenously added IL-6 in a dose-dependent manner. The cell line, Beu, established from a high IL-6 producer continued to produce the high amount of IL-6 and responded to exogenous IL-6 only at the highest dose (20 ng/ml). In contrast, the two cell lines (Cr and Cla) did not produce IL-10 and did not respond to IL-6. *P < 0.05, **P < 0.01

Article Snippet: Neutralizing rat anti-human IL-6 antibody and isotype-matched rat IgG1 were purchased from BD Biosciences (San Jose, CA).

Techniques: Produced, Cell Culture, Concentration Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin 6 mediates production of interleukin 10 in metastatic melanoma

doi: 10.1007/s00262-011-1084-5

Figure Lengend Snippet: Expression of IL-6 receptors in CM005 cells. a Expression of mRNA for gp80 and gp130. b Expression of CD126 (gp80) and CD130 (gp130) on the surface of CM005 melanoma cells. Thick line: specific antibody, thin line: isotype-matched control antibody

Article Snippet: Neutralizing rat anti-human IL-6 antibody and isotype-matched rat IgG1 were purchased from BD Biosciences (San Jose, CA).

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin 6 mediates production of interleukin 10 in metastatic melanoma

doi: 10.1007/s00262-011-1084-5

Figure Lengend Snippet: Involvement of STAT3 in IL-6-induced production of IL-10. a STAT3 and ERK phosphorylation after stimulation of CM005 melanoma cells with IL-6. The CM005 cells were harvested after 16 h in the absence or presence of 10% FBS and then stimulated with 2 ng/mol of rhIL-6 for 24 h. Phosphorylation of STAT3 (Y705) and subsequent production of IL-10 after IL-6 stimulation were more dominant in CM005 cells cultured in serum-free medium. ERK1/2 was spontaneously phosphorylated without IL-6 stimulation. b Kinetics of IL-6-induced IL-10 production and its association with STAT3 and ERK activation. CM005 cells were stimulated with rhIL-6 (2 ng/ml) for 30 min, 1 h, 3 h, 5 h, and 24 h. The IL-10 levels in supernatant were analyzed by ELISA. Intensities of phosphorylated STAT3 (STAT3Y705) and ERK1/2 bands were compared to those of GAPDH control by the Western blot. *P < 0.05. c Blocking of STAT 3 by siRNA and subsequent decrease in IL-6-induced IL-10 production. After a 2-day knockdown with siRNA fragments of control (Ctl) and STAT3, quiescent CM005 cells were stimulated with 2 ng/ml of IL-6 for 24 h. The STAT3 transcription was decreased by STAT3 siRNA and IL-10 production after IL-6 stimulation was also decreased. **P < 0.01

Article Snippet: Neutralizing rat anti-human IL-6 antibody and isotype-matched rat IgG1 were purchased from BD Biosciences (San Jose, CA).

Techniques: Cell Culture, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Blocking Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin 6 mediates production of interleukin 10 in metastatic melanoma

doi: 10.1007/s00262-011-1084-5

Figure Lengend Snippet: LPS-stimulated PBMC media increased the production of IL-10 by melanoma cells. a IL-10 levels after co-culture with PBMC-derived media. CM005 melanoma cells were cultured with culture supernatants from unfractionated PBMC without LPS (control) or non-adherent, adherent, and unfractionated PBMC with 10 ng/ml of LPS for 2 days, and then, the supernatants were collected. The IL-10 productions in supernatants were analyzed by the ELISA assay. The average of duplicates in a representative experiment was shown. b Cytokine levels in conditional culture media. The average of duplicates in a representative experiment was shown

Article Snippet: Neutralizing rat anti-human IL-6 antibody and isotype-matched rat IgG1 were purchased from BD Biosciences (San Jose, CA).

Techniques: Co-Culture Assay, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Interleukin 6 mediates production of interleukin 10 in metastatic melanoma

doi: 10.1007/s00262-011-1084-5

Figure Lengend Snippet: IL-6 stimulates IL-10 production by CM005 melanoma cells. a Increase in IL-10 in response to exogenously added cytokines. The levels of IL-10 produced by CM005 cells were measured after 2-day incubation with various concentrations (1, 10, and 100 folds of levels in medium of LPS-stimulated adherent PBMC) of individual cytokines (IL-1β, IL-6, IL-12, and TNF-α). Only IL-6 promoted IL-10 production by CM005 cells. b Anti-IL-6 antibody abrogated stimulative effects of LPS-activated PBMC supernatant. c IL-10 production in response to exogenously added IL-6. Exogenously added IL-6 promoted the production of IL-10 by melanoma cells in a dose-dependent manner (P < 0.01)

Article Snippet: Neutralizing rat anti-human IL-6 antibody and isotype-matched rat IgG1 were purchased from BD Biosciences (San Jose, CA).

Techniques: Produced, Incubation

Journal: Journal of Neuroinflammation

Article Title: Murine astrocytes produce IL-24 and are susceptible to the immunosuppressive effects of this cytokine

doi: 10.1186/s12974-019-1444-1

Figure Lengend Snippet: IL-24 augments the expression of suppressive cytokine signaling components by murine astrocytes and limits inflammatory cytokine release by these cells. a Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 30 min and the presence of phosphorylated STAT3 and STAT1 was determined by immunoblot analysis. Expression of β-actin is shown as a loading control and these immunoblots are representative of two separate experiments. b Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 2 or 4 h, and SOCS3 mRNA expression was determined by semi-quantitative RT-PCR. Expression of the housekeeping gene product GAPDH is shown, and relative SOCS3 expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean ± the SEM of three independent experiments, and an asterisk indicates a statistically significant difference from unchallenged cells at each time point ( p < 0.05). c Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 8 h prior to immunoblot analysis for SOCS3 protein expression. Expression of the housekeeping gene β-actin is shown and relative SOCS3 protein expression was determined by densitometric analysis normalized to untreated cells. Asterisks indicate statistically significant differences from unchallenged cells (p < 0.05). d Astrocytes were untreated or treated with IL-24 (0.5, 3, 10, 30, or 100 ng/mL) for 4 h prior to challenge with bacterial LPS (5 ng/mL) or vehicle control for 12 h, and IL-6 secretion was determined by specific capture ELISA. Asterisks indicate a statistically significant difference ( p < 0.05) from similarly challenged cells in the absence of IL-24 ( n = 3). e Primary astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 4 h prior to being uninfected or infected with Nm for 48 h before cell viability analysis via MTS assay. Data is presented as the mean absorbance ± SEM for three experiments. 0.1% Triton X-100 was used as a positive control, and an asterisk indicates a statistically significant difference from unchallenged cells in the absence of IL-24 ( p < 0.05)

Article Snippet: Commercially available Duoset® ELISA kits were used to measure IL-10 and TNF-α secretion (R&D Systems), while murine IL-6 secretion was measured using a rat anti-mouse IL-6 capture antibody (Clone MP5-20F3) and a biotinylated rat anti-mouse IL-6 detection antibody (Clone MP5-C2311) (BD Biosciences).

Techniques: Expressing, Recombinant, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Infection, MTS Assay, Positive Control