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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 1. DNA vaccination-based anti-IL-27 Abs are highly specific. The Western blot shows that our DNA vaccination-based anti-IL-27 Ab binds mouse IL-27 p28 (lane 1; 27 kDa), but not recombinant mouse IL-18, IL-12, or TNF- (lanes 2, 3, and 4, respectively). A, Coomassie Blue staining verifies the appearance of each cytokine on the loaded gel. B, Western blot showing that of these cytokines, our DNA vaccination-based anti-IL-27 Ab binds only mouse IL-27 p28. These Ab also bound natural mouse IL-27 (verified by sequencing) from supernatant of activated MOGp35–55-specific cultured primary draining lymph node cells (not shown).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Western Blot, Recombinant, Staining, Sequencing, Cell Culture

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 2. Anti-p28 Abs suppresses ongoing severe EAE. A, Four groups of 10 mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g/mouse of anti-p28 Ab (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. The experiment summarized in Fig. 2 shows the results of one of three experiments performed under similar experimental conditions, with similar results. The mean maximal score SE represents six mice per group. The other four mice were killed on day 30 and subjected to histological evaluation (see Fig. 3). B, Five groups of six mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), anti-IL-18 Ab (F), anti-IL-1 Ab (), IgG obtained from Lewis rats previously subjected to an empty plasmid administration (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as the mean maximal score SE of six mice per group. C, Three groups of six mice each were subjected to induction of transferred EAE. Beginning at the onset of disease (day 5), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as mean maximal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Plasmid Preparation

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 4. The beneficial effect of anti-IL-27 is dependent on the con- tinuing administration of protective Abs. Three groups of six mice each were subjected to active induction of EAE. Beginning 1 day after the onset of disease (day 17), these mice were treated with either a single dose of anti-p28 Ab (100 g/mouse; E) or with repeated administration (every other day) of this Ab (Œ) or PBS (f). An observer blind to the experi- mental procedure scored EAE daily. Results are shown as the mean max- imal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 3. Anti-IL-27 therapy reduces the histological score of EAE. Histological evaluation was conducted 30 days after disease induction. Lumbar spinal cord samples from naive mice or from EAE mice treated with PBS, IgG from naive mice, or anti-IL-27 p28 Abs were subjected to histological analysis (nine sections each group). The arrowheads point to the parenchymal mononuclear cell infiltration. The scale for mononuclear cell infiltration used was: 0, no mononuclear cell infiltration; 1, one to five perivascular lesions per section with minimal parenchymal infiltration; 2, five to 10 perivascular lesions per section with parenchymal infiltration; and 3, 10 perivascular lesions per section with extensive parenchymal infiltration. The mean histological score SE was calculated for each group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 5. Protective administration of anti-IL-27 Abs decreases in vivo polarization of CD4 T cells into Th1 and suppresses IFN- production by Ag-specific T cells. C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 12, 14, and 16) of anti-IL-27 p28 Abs (100 g), PBS, or normal rat IgG. On day 17 cervical lymph node cells (that drain the autoimmune site) were subjected to intracellular staining of IL-4 and IFN-. A, FACS analysis of CD4 T cells in this experiment. This experiment represents results obtained in three different independent experiments with very similar data. Subsequently, cervical lymph node T cells from these mice were cultured in the presence of 100 M MOGp35–55. After 72 h of stimulation, supernatants were assayed for the protein level of IFN- (B) and IL-4 (not shown). This experiment represents results obtained in three different independent experiments with very similar data.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: In Vivo, Staining, Cell Culture

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

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 6. Neutralizing the function of IL-27 reduces IFN- produc- tion by IFN--producing T cells. A, C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 3 and 6) of 100 g of anti-IL-27 p28 Abs (group 3), PBS (group 2), or normal rat IgG (group 1). On day 9, spleen cells were subjected to spot ELISA as previously described (38). A, Relative number of positive spots per 107 cultured cells. The average size of positive spots was analyzed. B, The MOGp33–55-specific CD4 T cell line was cultured with or without 100 M MOGp33–55. Cultured cells were supplemented with anti-IL-27 Abs at a final concentration of 10 g/ml (), normal rat IgG (f), or PBS (E). After 60 h of incubation, cells were plates in spot ELISA plates for an additional 24 h for the detection of IFN--positive spots (38). Number of positive spots (y-axis) and spot sizes (x-axis; logarithmic scale) determined as previously described (46).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

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

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 2. C/EBP, NF-B p50, STAT3, c-Rel, and TBP bind the endogenous CRP promoter. Agarose gel of a ChIP assay performed on Hep3B cells treated with cytokines IL-1 and IL-6 for 0–15 h, as described in Materials and Methods. Abs to C/EBP, NF-B p50, STAT3, c-Rel, and TBP were used in the assays with primers flanking the CRP proximal promoter (118 to 115). The mock is a no Ab control. Input is a 1/10 dilution of total chromatin after sonication and preclearing. C/EBP, NF-B p50, and input are shown (top row). STAT3, c-Rel, and mock are shown in the middle, and TBP is shown in the bottom row. Results are representative of four experiments.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Agarose Gel Electrophoresis, Control, Sonication

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 3. C/EBP binds the endogenous CRP promoter in response to cytokines. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were determined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal promoter (118 to 115), as described in Fig. 2. Data show C/EBP occupancy expressed as fold change after subtraction of mock and normalization to input signal (see Materials and Methods). Results are an average of three to four ex- periments, each done in duplicate. Error bar represents SD. Statistical sig- nificance of each time point compared with basal levels was determined by a one-way ANOVA and is defined. , p 0.5.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 4. p50 occupancy of the CRP promoter changes modestly in the presence of cytokines. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were determined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal pro- moter (118 to 115), as described in Fig. 2. Data show NF-B p50 occupancy expressed as fold change after subtraction of mock and nor- malization to input signal (see Materials and Methods). Results are an average of three to four experiments, each done in duplicate. Error bar represents SD. Statistical significance of each time point compared with basal levels was determined by a one-way ANOVA at p 0.5, but the experiment had insufficient statistical power to reliably calculate p values.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 5. STAT3 occupancy of the CRP promoter rises modestly in response to cytokines. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were deter- mined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal pro- moter (118 to 115), as described in Fig. 2. Data show STAT3 occu- pancy expressed as fold change after subtraction of mock and normaliza- tion to input signal (see Materials and Methods). Results are an average of three to four experiments, each done in duplicate. Error bar represents the SD. Statistical significance of each time point compared with basal levels was determined by a one-way ANOVA is defined. , p 0.5.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 6. c-Rel and TBP occupy the CRP promoter in parallel. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were determined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal promoter (118 to 115), as described in Fig. 2. Data show c-Rel (solid line, Œ) and TBP (dashed line, f) occupancy expressed as fold change after subtraction of mock and normalization to input signal (see Materials and Methods). a–c, Average of three to four experiments, each done in duplicate. Error bar represents SD. d and e, Profiles from individual ChIP experiments of c-Rel and TBP promoter occupancy. Statistical significance of each time point compared with basal levels was determined by a one-way ANOVA. , p 0.5 is defined for c-Rel and , p 0.5 is indicated for TBP.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 7. CRP mRNA accumulates in response to cytokines. a, Rep- resentative agarose gel of RT-PCR performed on Hep3B cells treated with cytokines IL-1 and IL-6 for the indicated times (hours). CRP mRNA levels are shown at top and -actin mRNA levels are shown at bottom. b, Average quantification of band intensity measured using ImageQuant of CRP mRNA normalized to -actin mRNA (n 4 measurements). Error bar represents SD.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

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

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 8. Composite graph of transcription factor occupancy on the CRP promoter and CRP mRNA ac- cumulation in response to cytokines. Common time points for transcription factor occupancy from the 12– 36-h data and CRP mRNA accumulation from the 3–24-h data (12, 18, and 24 h) were plotted together. The left y-axis is the fold change above mock for the transcription factor promoter occupancy. The right y- axis is average normalized band intensity for CRP mRNA accumulation. CRP mRNA (dotted dashed gray line F), C/EBP (black line ), STAT3 (light gray line ‚), p50 (light gray line E), c-Rel (short dashed line Œ), and TBP (black dashed line f) are shown.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: