polyclonal goat anti human il 1ra antibody  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions

doi: 10.3389/fimmu.2019.00579

Figure Lengend Snippet: PRRSV-induced IL-1Ra inhibited porcine innate immune functions. PRRSV-induced IL-1Ra inhibited (A) phagocytic activity, (B) SLA-DR and (C) CD86 expression. MoDC were cultured with type 2 PRRSV or mock, in the presence of anti-IL-1Ra Ab. LPS was added into the culture and further incubated for 24 h. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p < 0.05.

Article Snippet: In some experimental conditions, cells were pre-treated with final concentration of 10 ng/mL polyclonal goat anti-porcine IL-1Ra antibody (R&D system, clone AF780) or polyclonal goat IgG isotype control antibody (R&D system) at 2 h post-inoculation to neutralize PRRSV-induced IL-1Ra which was then cultured for another 22 h. To determine the effect of PRRSV-induced IL-1Ra on MoDC phagocytic activity, the antibody pre-treated MoDC (2 × 10 6 cell) were further incubated with inactivated E. coli -FITC (ThermoFisher Scientific) in complete RPMI at a MoDC: E. coli ratio of 1:50 for 10 min at 37°C.

Techniques: Activity Assay, Expressing, Cell Culture, Incubation

Journal: Frontiers in Immunology

Article Title: Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions

doi: 10.3389/fimmu.2019.00579

Figure Lengend Snippet: PRRSV-induced IL-1Ra inhibited (A) IFNA and (B) IL1 , (C) but not IL6 expression. MoDC were cultured with type 2 PRRSV or mock, in the presence of anti-IL-1Ra Ab. LPS was added into the culture and further incubated for 24 h. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p < 0.05.

Article Snippet: In some experimental conditions, cells were pre-treated with final concentration of 10 ng/mL polyclonal goat anti-porcine IL-1Ra antibody (R&D system, clone AF780) or polyclonal goat IgG isotype control antibody (R&D system) at 2 h post-inoculation to neutralize PRRSV-induced IL-1Ra which was then cultured for another 22 h. To determine the effect of PRRSV-induced IL-1Ra on MoDC phagocytic activity, the antibody pre-treated MoDC (2 × 10 6 cell) were further incubated with inactivated E. coli -FITC (ThermoFisher Scientific) in complete RPMI at a MoDC: E. coli ratio of 1:50 for 10 min at 37°C.

Techniques: Expressing, Cell Culture, Incubation

Journal: Frontiers in Immunology

Article Title: Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions

doi: 10.3389/fimmu.2019.00579

Figure Lengend Snippet: PRRSV-induced IL-1Ra altered the expression of helper T lymphocyte transcriptional factors during the recalled antigen test. (A) CSFV had no effect on IL1RA gene expression. (B) Upon activation with the recalled antigen, CSFV, strong upregulation of TBET was observed in the CSFV-primed PBMC. *indicates significant difference levels at p < 0.05. (C–E) The levels of transcriptional factor gene expression in the CSFV-primed PBMC, upon reactivation with the recalled antigen. PRRSV-induced IL-1Ra decreased expressions of (C) TBET and (D) GATA3 , (E) but not FOXP3 gene expressions. The supernatants from type 2 PRRSV- or mock (MARC-145 cell lysate)-infected MoDC were pretreated with anti-IL-1Ra Ab for 2 h prior to addition into the culture system. PBMC obtained from CSFV-immunized pigs were in vitro reactivated with CSFV or mock (SK6 cell lysate), in the presence of pretreated supernatants. Data represents mean ± SD from 5 pigs. Statistical significances were analyzed using t test (A) ANOVA followed by Tukey's test (B–E) . a indicates significant difference between mock-infected cell supernatant (black circle) and PRRSV-infected cell supernatants (close and open light blue squares) at p < 0.05. * indicates significant difference between addition of anti-IL-1Ra Ab (dark blue triangle) and PRRSV-infected cell supernatants (close and open light blue squares) at p < 0.05.

Article Snippet: In some experimental conditions, cells were pre-treated with final concentration of 10 ng/mL polyclonal goat anti-porcine IL-1Ra antibody (R&D system, clone AF780) or polyclonal goat IgG isotype control antibody (R&D system) at 2 h post-inoculation to neutralize PRRSV-induced IL-1Ra which was then cultured for another 22 h. To determine the effect of PRRSV-induced IL-1Ra on MoDC phagocytic activity, the antibody pre-treated MoDC (2 × 10 6 cell) were further incubated with inactivated E. coli -FITC (ThermoFisher Scientific) in complete RPMI at a MoDC: E. coli ratio of 1:50 for 10 min at 37°C.

Techniques: Expressing, Gene Expression, Activation Assay, Infection, In Vitro

Journal: Frontiers in Immunology

Article Title: Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions

doi: 10.3389/fimmu.2019.00579

Figure Lengend Snippet: PRRSV-induced IL-1Ra inhibited lymphocyte proliferation. PRRSV-induced IL-1Ra inhibited PHA-induced proliferation of the (A) CD4 + , (B) CD8 + , and (C) CD4 + CD8 + subpopulations. PRRSV-induced IL-1Ra inhibited CSFV-specific proliferations of the (D) CD4 + , (E) CD8 + , and (F) CD4 + CD8 + subpopulations. The supernatants obtained from type 2 PRRSV or mock (MARC-145 cell lysate) were pretreated with anti-IL-1Ra Ab for 2 h prior to addition into the culture. PBL or PBMC were culture with PHA, CSFV or controls for 96 h, in the presence of the pretreated supernatants. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p < 0.05.

Article Snippet: In some experimental conditions, cells were pre-treated with final concentration of 10 ng/mL polyclonal goat anti-porcine IL-1Ra antibody (R&D system, clone AF780) or polyclonal goat IgG isotype control antibody (R&D system) at 2 h post-inoculation to neutralize PRRSV-induced IL-1Ra which was then cultured for another 22 h. To determine the effect of PRRSV-induced IL-1Ra on MoDC phagocytic activity, the antibody pre-treated MoDC (2 × 10 6 cell) were further incubated with inactivated E. coli -FITC (ThermoFisher Scientific) in complete RPMI at a MoDC: E. coli ratio of 1:50 for 10 min at 37°C.

Techniques:

Journal: Frontiers in Immunology

Article Title: Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions

doi: 10.3389/fimmu.2019.00579

Figure Lengend Snippet: PRRSV-induced IL-1Ra was not involved in suppression of IFN-γ-producing T lymphocytes in both (A) polyclonal and (B) recalled CSFV responses. The supernatants obtained from type 2 PRRSV or mock (MARC-145 cell lysate) were pretreated with anti-IL-1Ra Ab for 2 h prior to addition into the culture. PBL or PBMC were cultured with PHA, CSFV or controls for 48 h, in the presence of the pretreated supernatants. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p < 0.05.

Article Snippet: In some experimental conditions, cells were pre-treated with final concentration of 10 ng/mL polyclonal goat anti-porcine IL-1Ra antibody (R&D system, clone AF780) or polyclonal goat IgG isotype control antibody (R&D system) at 2 h post-inoculation to neutralize PRRSV-induced IL-1Ra which was then cultured for another 22 h. To determine the effect of PRRSV-induced IL-1Ra on MoDC phagocytic activity, the antibody pre-treated MoDC (2 × 10 6 cell) were further incubated with inactivated E. coli -FITC (ThermoFisher Scientific) in complete RPMI at a MoDC: E. coli ratio of 1:50 for 10 min at 37°C.

Techniques: Cell Culture

Journal: Frontiers in Immunology

Article Title: Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions

doi: 10.3389/fimmu.2019.00579

Figure Lengend Snippet: PRRSV-induced IL-1Ra was partially involved in regulatory T lymphocyte (Treg) induction. PRRSV-induced IL-1Ra decreased numbers of (A) Treg, (B) but not IL-10-producing T lymphocytes. PBMC were cultured in the presence of type 2 PRRSV or mock. Subsequently, anti-IL-1Ra Ab was added into the culture and further incubated for 48 h. ± indicates presence/absence of indicated treatment within the culture. Data represents mean ± SD from 5 pigs. Statistical significance was analyzed using ANOVA followed by Tukey's test. * indicates significant difference at p < 0.05.

Article Snippet: In some experimental conditions, cells were pre-treated with final concentration of 10 ng/mL polyclonal goat anti-porcine IL-1Ra antibody (R&D system, clone AF780) or polyclonal goat IgG isotype control antibody (R&D system) at 2 h post-inoculation to neutralize PRRSV-induced IL-1Ra which was then cultured for another 22 h. To determine the effect of PRRSV-induced IL-1Ra on MoDC phagocytic activity, the antibody pre-treated MoDC (2 × 10 6 cell) were further incubated with inactivated E. coli -FITC (ThermoFisher Scientific) in complete RPMI at a MoDC: E. coli ratio of 1:50 for 10 min at 37°C.

Techniques: Cell Culture, Incubation

Journal: Biomolecules & therapeutics

Article Title: Intranasal Administration of Interleukin-1 Receptor Antagonist in a Transient Focal Cerebral Ischemia Rat Model.

doi: 10.4062/biomolther.2016.050

Figure Lengend Snippet: Fig. 1. Neuroprotective effects of intranasal IL-1RA against tran- sient cerebral ischemia. Brain damage is assessed by using TTC staining 7 days after the ischemic insult (cerebral ischemia for 1 h). The EC, IN, and IP groups received vehicle, intranasal IL-RA, and intraperitoneal IL-1RA, respectively. Representative TTC staining images of brain sections for each group (A). Quantitative compari- sons of infarct volume (B) and edema volume (C) (n=5 in the EC group, n=7 in the IN group, and n=6 in the IP group). Values are shown as mean (SEM). *p<0.005, versus the EC group by one- way analysis of variance with Bonferroni post hoc test.

Article Snippet: Tissue sections were incubated overnight at 4°C with primary antibodies, diluted in 2% BSA with PBS such as monoclonal mouse anti-NeuN (1:50; Millipore, Bedford, MA, USA), monoclonal mouse anti-Iba-1 (1:50; Abcam, Cambridge, UK), polyclonal rabbit anti-IL-1β (1:50, Abcam), and polyclonal goat anti-IL-1ra (R&D Systems).

Techniques: Staining

Journal: Biomolecules & therapeutics

Article Title: Intranasal Administration of Interleukin-1 Receptor Antagonist in a Transient Focal Cerebral Ischemia Rat Model.

doi: 10.4062/biomolther.2016.050

Figure Lengend Snippet: Fig. 4. Distribution of human IL-1RA in the striatum and cortex at 6 h after intranasal administration. A coronal section of the rat brain (A). The square areas represent the location where the following immunostaining images were taken (a, striatum and b, cortex). After intranasal administration, human IL-RA was detected with strong immunoreactivity and largely co-localized with NeuN (B) and Iba-1 (C) in the striatum and cortex of the rats in the IN group. On the other hand, the immunoreactivity in the EC group is minimal.

Article Snippet: Tissue sections were incubated overnight at 4°C with primary antibodies, diluted in 2% BSA with PBS such as monoclonal mouse anti-NeuN (1:50; Millipore, Bedford, MA, USA), monoclonal mouse anti-Iba-1 (1:50; Abcam, Cambridge, UK), polyclonal rabbit anti-IL-1β (1:50, Abcam), and polyclonal goat anti-IL-1ra (R&D Systems).

Techniques: Immunostaining

Journal: Biomolecules & therapeutics

Article Title: Intranasal Administration of Interleukin-1 Receptor Antagonist in a Transient Focal Cerebral Ischemia Rat Model.

doi: 10.4062/biomolther.2016.050

Figure Lengend Snippet: Fig. 5. The effect of intranasal IL-1RA administration on IL-1β expression. In the EC group, IL-1β is detected with strong immunoreactivity and largely co-localized with NeuN (A) and Iba-1(B) in the striatum and cortex. However, the IL-1β expression level is remarkably reduced at 6 h after intranasal administration of IL-1RA in the IN group.

Article Snippet: Tissue sections were incubated overnight at 4°C with primary antibodies, diluted in 2% BSA with PBS such as monoclonal mouse anti-NeuN (1:50; Millipore, Bedford, MA, USA), monoclonal mouse anti-Iba-1 (1:50; Abcam, Cambridge, UK), polyclonal rabbit anti-IL-1β (1:50, Abcam), and polyclonal goat anti-IL-1ra (R&D Systems).

Techniques: Expressing

Journal: Journal of leukocyte biology

Article Title: Mouse neutrophils express the decoy type 2 interleukin-1 receptor (IL-1R2) constitutively and in acute inflammatory conditions.

doi: 10.1189/jlb.0113035

Figure Lengend Snippet: Figure 2. HC up-regulates IL-1R2 expression on BMG. (A) Determina- tion of Il1r2 (upper) and Il1r1 (lower) mRNA levels in myeloid cells upon stimulation with HC. Total mRNA was isolated from not stimu- lated or HC-treated BMG, BMDC, and BMM for analyses by qRT- PCR. Results represent Il1r2 and Il1r1 mRNA expression levels relative to Gapdh mRNA levels. Data are shown as the mean sem of values obtained from three independent cultures. *P 0.05 versus not-stimu- lated samples, as analyzed by unpaired two-tailed Student’s t-test. (B) HC up-regulates the protein expression of IL-1R2 on neutrophils and their precursors but not on other myeloid cells. BMG, BMDC, and BMM were left untreated or stimulated with HC and stained for IL- 1R2 (gray lines) and the matched isotype control (black lines) by ex- tracellular FACS staining. Data are analyzed on total living cells. Data are shown from one of three independent experiments with similar results. (C) HC up-regulates the expression of IL-1R2 in BMG. West- ern blot analysis of lysates of not stimulated and HC-treated BMGs (5106 cell equivalents/lane) using anti-IL-1R2 polyclonal antibody and anti-GAPDH polyclonal antibody as loading control. Arrows indi- cate the specific signal for IL-1R2; *nonspecific band. Data are shown from one of three independent experiments with similar results. (D) IL-1R2 is a highly N-glycosylated protein. Western blot analysis of ly- sates of untreated or PNGase F-treated BMGs prestimulated with HC (2106 cell equivalents/lane) using anti-IL-1R2 polyclonal antibody and anti-GAPDH polyclonal antibody as loading control. Arrows and arrowhead indicate the specific signal for glycosylated or N-deglycosy- lated IL-1R2, respectively. *Nonspecific band. Data are shown from one of three independent experiments with similar results.

Article Snippet: The membrane was blocked in TBS/0.05% Triton X-100, 5% horse serum, and probed with a goat anti-mouse IL-1 polyclonal antibody (detection antibody of IL-1 ELISA; R&D Systems) or a goat anti-mouse IL-1Ra polyclonal antibody (R&D Systems), followed by a HRP-conjugated donkey anti-goat IgG.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Two Tailed Test, Staining, Control, Western Blot

Journal: Journal of leukocyte biology

Article Title: Mouse neutrophils express the decoy type 2 interleukin-1 receptor (IL-1R2) constitutively and in acute inflammatory conditions.

doi: 10.1189/jlb.0113035

Figure Lengend Snippet: Figure 3. IL-1R2 expressed on BMG coprecipitates IL-1 but not IL-1Ra. Coimmunoprecipitation of HC-stimulated BMGs incubated with mouse rIL-1 (A) or mouse rIL-1Ra (B), respectively, using an anti-IL-1R2 pull-down antibody (R2) or the matched isotype control (IgG). Western blot analysis of BMG lysates (5106 cell equivalents/ lane) or 10 ng mouse rIL-1 (A) or mouse rIL-1Ra (B) using anti- IL-1 polyclonal antibody (A) or anti-IL-1Ra polyclonal antibody (B) and anti-IL-1R2 polyclonal antibody (A and B) as control for the effi- ciency of the immunoprecipitation (IP). Arrows indicate IL-1 (A) or IL-1Ra (B). Arrowheads indicate heavy (upper band)- or light (lower band)-chain of the pull-down antibody. Data are shown from one of two independent experiments with similar results.

Article Snippet: The membrane was blocked in TBS/0.05% Triton X-100, 5% horse serum, and probed with a goat anti-mouse IL-1 polyclonal antibody (detection antibody of IL-1 ELISA; R&D Systems) or a goat anti-mouse IL-1Ra polyclonal antibody (R&D Systems), followed by a HRP-conjugated donkey anti-goat IgG.

Techniques: Incubation, Control, Western Blot, Immunoprecipitation

Journal: Journal of leukocyte biology

Article Title: Mouse neutrophils express the decoy type 2 interleukin-1 receptor (IL-1R2) constitutively and in acute inflammatory conditions.

doi: 10.1189/jlb.0113035

Figure Lengend Snippet: Figure 4. IL-1R2 is shed upon LPS stimulation in vitro. (A and B) BMGs were prestimulated with HC or left untreated before shedding was induced by the addition of LPS for 1 h. (A) Samples were stained for IL-1R2 by extracellular FACS staining. Black or gray lines show IL- 1R2 expression, without or after LPS treatment, respectively. Data are analyzed on total living cells. Data are shown from one of three inde- pendent experiments with similar results. (B) Western blot analysis of BMG lysates (4106 cell equivalents/lane) using anti-IL-1R2 polyclonal antibody and anti-GAPDH polyclonal antibody as a loading control. Arrows indicate the specific signal for IL-1R2. *Nonspecific band. Data are shown from one of three independent experiments with similar results. (C) Shed IL-1R2 is found in the supernatant. HC-treated BMGs were stimulated with LPS for 1 h or left untreated. IL-1R2 was immunoprecipitated in lysates or supernatants using an anti-IL-1R2 pull-down antibody (R2) or the matched isotype control (IgG). West- ern blot analysis of BMG lysates (lane 12; 5106 cell equivalents/ lane) or immunoprecipitated samples of lysates (5106 cell equiva- lents/lane) and supernatant using anti-IL-1R2 polyclonal antibody and anti-GAPDH polyclonal antibody as loading control. Arrows indicate the specific signal for IL-1R2. Arrowheads indicate heavy (upper band)- or light (lower band)-chain of the pull-down antibody. *Non- specific band. Data are shown from one of three independent experi- ments with similar results. (D) BMGs were prestimulated with HC for 20 h, and shedding was induced by the addition of LPS (), or cells were left untreated (▫). At indicated time-points, the surface IL-1R2 expression levels were assessed by flow cytometry using mAb for IL- 1R2, and its matched isotype control (upper) or Il1r2 transcripts were analyzed (lower). For protein expression analysis, GMFI was calculated by subtracting the GMFI of the isotype control-stained BMGs from the GMFI of the corresponding IL-1R2-stained BMGs. Data are shown as the mean sem of values obtained from three independent cultures. Data are analyzed on 7-AADneg cells. *P 0.05; ***P 0.001 versus untreated BMG samples, as analyzed by unpaired two-tailed Student’s t-test. For transcript expression analysis, cells were collected, and total mRNA was isolated for analyses by qRT-PCR. Results represent Il1r2 mRNA expression levels relative to Gapdh mRNA levels. Data are

Article Snippet: The membrane was blocked in TBS/0.05% Triton X-100, 5% horse serum, and probed with a goat anti-mouse IL-1 polyclonal antibody (detection antibody of IL-1 ELISA; R&D Systems) or a goat anti-mouse IL-1Ra polyclonal antibody (R&D Systems), followed by a HRP-conjugated donkey anti-goat IgG.

Techniques: In Vitro, Staining, Expressing, Western Blot, Control, Immunoprecipitation, Cytometry, Two Tailed Test, Isolation, Quantitative RT-PCR

Journal: Journal of leukocyte biology

Article Title: Mouse neutrophils express the decoy type 2 interleukin-1 receptor (IL-1R2) constitutively and in acute inflammatory conditions.

doi: 10.1189/jlb.0113035

Figure Lengend Snippet: Figure 6. Effect of HC and LPS on IL-1 ligand and receptor expres- sion in BMG. (A) Determination of Il1r1 (upper left), Il1r2 (upper middle), icIlrn1 (upper right), Il1a (lower left), Il1b (lower middle), and sIl1rn (lower right) mRNA levels in BMGs, which were left un- treated, treated for 1 h with LPS, for 20 h with HC, or for 20 h with HC and subsequently for 1 h with LPS. Total mRNA was isolated from not stimulated, LPS- or HC-treated, or HC- and LPS-treated BMN sam- ples for analyses by qRT-PCR. Results represent Il1r1, Il1r2, icIl1rn1, Il1a, Il1b, and sIl1rn mRNA expression levels relative to Gapdh mRNA levels. Data are shown as the mean sem of values obtained from five independent cultures. *P 0.05; **P 0.01; ***P 0.001, as ana- lyzed by one-way ANOVA with Tukey’s post-test for pairwise compari- son when the ANOVA was statistically significant. (B) Determination of IL-1, IL-1, and IL-1Ra cytokine levels in lysates of BMG. BMG were left untreated, treated for 6 h with LPS, for 20 h with HC, or for 20 h with HC and subsequently for 6 h with LPS. Not stimulated, LPS- or HC-treated, or HC- and LPS-treated BMG samples were lysed, and cytokine levels were measured by ELISA. Data are shown as the mean sem of values obtained from three independent cultures. **P 0.01; ***P 0.001, as analyzed by one-way ANOVA with Tukey’s post-test for pairwise comparison when the ANOVA was statistically significant.

Article Snippet: The membrane was blocked in TBS/0.05% Triton X-100, 5% horse serum, and probed with a goat anti-mouse IL-1 polyclonal antibody (detection antibody of IL-1 ELISA; R&D Systems) or a goat anti-mouse IL-1Ra polyclonal antibody (R&D Systems), followed by a HRP-conjugated donkey anti-goat IgG.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Comparison