Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Real-time PCR probes and details of primers

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Real-time Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Examples of imunohistochemical staining for the IL-1 family. IL-1β (row A), IL-1Ra (row B), and IL-1 receptor, type I (row C) in grade-1 non-degenerate discs (column 1) and grade-12 degenerate discs (column 2), IgG controls (row D) were all negative. Immunopositivity is revealed by brown staining. N.B In non-degenerate discs, no cell clusters were seen and little immunopositivity was observed in the single cells. In degenerate discs, a large number of cell clusters were observed, which were predominately immunopositive. Bars = 570 μm.

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Immunopositive staining for the IL-1 family in human intervertebral discs. Numbers of cells with immunopositivity for IL-1α (a) , IL-1β (b) , IL-1 receptor antagonist (c) , IL-1 receptor, type I (d) , and IL-1β-converting enzyme (e) , according to place of origin in the disc and grade of intervertebral disc degeneration ( n = 30). Data are presented as means ± 2 standard errors (as a representative of 95%CI). * P < 0.1,; ** P < 0.05

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Effects of IL-1β on gene expression in cells from non- degenerate or degenerate intervertebral discs

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Gene Expression

Journal: Arthritis Research & Therapy

Article Title: The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

doi: 10.1186/ar1732

Figure Lengend Snippet: Effect of IL-1 treatment on the IL-1 family gene expression in human intervertebral disc cells. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene and untreated controls (hence control is graphed at 1 on the log scale) for IL-1α (a) , IL-1β (b) , IL-1 receptor antagonist (IL-1Ra) (c) , and IL-1 receptor, type I (IL-RI) (d) following IL-1β treatment of disc cells from two regions of non-degenerate (non-deg) ( n = 6) and degenerate ( n = 24) discs. ** P < 0.05.

Article Snippet: Sections were incubated overnight at 4°C with mouse monoclonal primary antibodies against human IL-1Ra (1:200 dilution, R&D Systems, Abingdon, UK), IL-1RI (1:50 dilution, R&D Systems), and goat polyclonal primary antibodies against human IL-1α (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:300 dilution, SantaCruz), and ICE (1:10 dilution, SantaCruz).

Techniques: Gene Expression, Control

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

Article Title: IL-1β induces the rapid secretion of the antimicrobial protein IL-26 from Th17 cells

doi: 10.4049/jimmunol.1900318

Figure Lengend Snippet: (A) Sorting scheme used to isolate helper T cell subsets, a representative donor is shown, n = 3. (B) Isolated Th1, Th2, and Th17 cells were stimulated with IL-1β for 48 hours and cell free supernatants were analyzed for IL-26 secretion by ELISA, n = 3. (C) A representative sorting scheme for isolating IL-1RI+ and IL-1RI- Th17 cells, n= 3. (D, E) IL-1RI+ and IL-1RI- Th17 cells were stimulated with 100 ng/ml IL-1β or 5 µl/ml anti-CD3/CD28 for 9 hours before IL-26 (D) and IL-17A (E) was measured in cell free supernatants by ELISA, n = 3. (B,D) Data are represented as mean ± SEM, statistics calculated using one-way ANOVA with Tukey’s post-test (B, D, E). **p ≤ 0.01. *** p ≤ 0.001

Article Snippet: In some experiments, memory CD4 + T cells were incubated overnight with 1 nM recombinant human IL-2 and stained additionally with antibodies against APC-IL-1RI (R&D Systems, polyclonal goat anti-human) to sort IL-1RI + Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI + ) and IL-1RI - Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI - ).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

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

Article Title: IL-1β induces the rapid secretion of the antimicrobial protein IL-26 from Th17 cells

doi: 10.4049/jimmunol.1900318

Figure Lengend Snippet: (A) Unsupervised principal component analysis of IL-1RI+ and IL-1RI- Th17 cells from 3 donors treated with IL-1β, anti-CD3/CD28, or media as a control. Numbers within symbols indicate the donor. (B) Hierarchical clustering of the samples. (C) Differential gene expression in untreated IL-1RI+ Th17 cells vs. IL-1RI- Th17 cells is represented in a volcano plot and some of the most differentially expressed genes are named and highlighted with black dots. (D) Genes with a (FC) ≥ 1.5 and p-adj ≤0.05 after treatment of IL-1RI+ Th17 cells with IL-1β vs media are plotted based on their normalized counts after IL-1β or anti-CD3/CD28 treatment. The line y = x was plotted on the graph to show genes with greater relative counts in each condition. Genes in the IPA “Th17 cell activation” canonical pathway and CXCL8 are highlighted with black dots. (E) Memory CD4+ T cells were treated with 100 ng/ml IL-1β and 5 µl/ml anti-CD3/CD28 for 48 hours before supernatants were collected and IL-8 protein concentrations were measured by ELISA, n = 4. Data are represented as mean ± SEM, statistics calculated using one-way ANOVA with Tukey’s post-test. *** p ≤ 0.001.

Article Snippet: In some experiments, memory CD4 + T cells were incubated overnight with 1 nM recombinant human IL-2 and stained additionally with antibodies against APC-IL-1RI (R&D Systems, polyclonal goat anti-human) to sort IL-1RI + Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI + ) and IL-1RI - Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI - ).

Techniques: Control, Gene Expression, Activation Assay, Enzyme-linked Immunosorbent Assay

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

Article Title: IL-1β induces the rapid secretion of the antimicrobial protein IL-26 from Th17 cells

doi: 10.4049/jimmunol.1900318

Figure Lengend Snippet: “Th17 cell activation” pathway genes significantly upregulated by IL-1β treatment of IL-1RI + Th17 cells. The IPA canonical pathway “Th17 cell activation” was the most significantly enriched canonical pathway within our dataset (p = 2.51 × 10 −17 ). This pathway contained 90 genes, 19 of which, or 21%, were contained in our dataset and are displayed in the table.

Article Snippet: In some experiments, memory CD4 + T cells were incubated overnight with 1 nM recombinant human IL-2 and stained additionally with antibodies against APC-IL-1RI (R&D Systems, polyclonal goat anti-human) to sort IL-1RI + Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI + ) and IL-1RI - Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI - ).

Techniques: Activation Assay

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

Article Title: IL-1β induces the rapid secretion of the antimicrobial protein IL-26 from Th17 cells

doi: 10.4049/jimmunol.1900318

Figure Lengend Snippet: (A, B) Genes with a log2(FC)≥ 0.58 and p-adj ≤0.05 after treatment of IL-1RI+ Th17 cells with IL-1β (A) or anti-CD3/CD28 (B) were analyzed for enrichment of upstream transcription factors using Enrichr and plotted with their -log10(p-adj). C. Memory CD4+ T cells were stimulated with either 20 ng/ml IL-1β or 5 µl/ml anti-CD3/CD28 for 24 hours, with or without 1 hour pretreatment with 10µM Bay 11–7082, an NF-κB activation inhibitor. IL-26 secretion into cell supernatants was measured by ELISA, n = 3. Data are represented as mean ± SEM, statistics calculated using one-way ANOVA with Tukey’s post-test. *p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: In some experiments, memory CD4 + T cells were incubated overnight with 1 nM recombinant human IL-2 and stained additionally with antibodies against APC-IL-1RI (R&D Systems, polyclonal goat anti-human) to sort IL-1RI + Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI + ) and IL-1RI - Th17 cells (CCR6 + CD161 + CCR4 + CXCR3 - IL-1RI - ).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroscience

Article Title: System xc Activity and Astrocytes Are Necessary for Interleukin-1 -Mediated Hypoxic Neuronal Injury

doi: 10.1523/jneurosci.2459-07.2007

Figure Lengend Snippet: Figure1. Comparisonofischemicbraindamagebetweenwild-typeandIL-1RInullmutant animals. A, Representative TTC staining of coronal brain sections (2 mm) 70.5 h after a 90 min reversible MCAO for wild-type (IL-1RI/; left) and IL-1RI-deficient (IL-1RI/; right) male mice.ThelackofTTCstainingindicatesaninfarctedarea.B,Infarctvolumesinthehemisphere (hemi), cerebral cortex (cortex), and caudate–putamen (CP) of wild-type (IL-1RI/; gray bars) and IL-1RI knock-out (IL-1RI/; hatched bars) male mice were measured over five coronalsections.InfarctvolumesarereportedasthemeanSEMpercentageofnon-ischemic hemisphere, corrected for edema. Actual infarct volumes for wild-type versus null mutant ani- mals were as follows: hemisphere, 177.3 18.0 versus 65 16.4 mm 3; cortex, 99.8 8.2 versus29.711.3mm 3;caudate–putamen,31.22.0versus18.12.9mm 3.Anasterisk indicates a value significantly different from wild-type animals, as assessed via an unpaired t test (n 9 animals per group). Significance was assessed at p 0.05. C, Post-ischemic neu- rologicaldeficitswerescoredasdescribedinMaterialsandMethodsat24and72hafterMCAO in wild-type (IL-1RI/; squares; gray bars) and IL-1RI null mutant (IL-1RI/; circles; hatched bars) male mice. Neurological scores between groups were different at 24 and 72 h after MCAO, as determined by a Mann–Whitney U test (n 9 animals per group, statistical significance was assessed at p 0.05).

Article Snippet: In experiments using recombinant mouse IL-1ra (rIL-1ra) (R & D Systems) or monoclonal anti-mouse IL-1RI antibody directed against the extracellular domain of mouse IL-1RI (MAB7711; R & D Systems), the respective solutions were prepared with IL-1 (1.5 ng/ml; 1.5 ) and added as described above.

Techniques: Staining, Knock-Out, Mutagenesis, MANN-WHITNEY

Journal: Journal of Neuroscience

Article Title: System xc Activity and Astrocytes Are Necessary for Interleukin-1 -Mediated Hypoxic Neuronal Injury

doi: 10.1523/jneurosci.2459-07.2007

Figure Lengend Snippet: Figure 2. Role of IL-1RI signaling in the potentiation of hypoxic neuronal injury by IL-1 in vitro. A, B, Mixed cortical cell cultures were treated with 1 ng/ml IL-1 for 20–24 h in the presenceorabsenceofrIL-1ra(10–1000ng/ml;A)oranti-IL-1RI(0.1–100g/ml;B),washed, andthendeprivedofoxygen(5h).Thepercentageoftotalneuronalcelldeathwasdetermined 20–24hlater.Anasteriskindicatesvaluessignificantlygreaterthanhypoxiaalone,whereas# denotes a significant diminution of the IL-1-mediated increase in injury (IL-1) as deter- mined by one-way ANOVA followed by a Student–Newman–Keuls t test. Significance was assessed at p 0.05 (n 3–9 cultures pooled from 2–3 different experiments).

Article Snippet: In experiments using recombinant mouse IL-1ra (rIL-1ra) (R & D Systems) or monoclonal anti-mouse IL-1RI antibody directed against the extracellular domain of mouse IL-1RI (MAB7711; R & D Systems), the respective solutions were prepared with IL-1 (1.5 ng/ml; 1.5 ) and added as described above.

Techniques: In Vitro

Journal: Journal of Neuroscience

Article Title: System xc Activity and Astrocytes Are Necessary for Interleukin-1 -Mediated Hypoxic Neuronal Injury

doi: 10.1523/jneurosci.2459-07.2007

Figure Lengend Snippet: Figure9. AstrocyteIL-1RIsignalingisrequiredforthemediationofIL-1-inducedhypoxic neuronal injury. Chimeric mixed cortical cell cultures were obtained by plating wild-type (IL- 1RI/) or IL-1RI-deficient (IL-1RI/) neurons on either wild-type (IL-1RI/) or IL-1RI- nullmutant(IL-1RI/)astrocytesTheseculturesweretreatedwith1ng/mlIL-1for20–24 h (black bars) or vehicle (hatched bars), washed, and then deprived of oxygen. A, The percent- age of total neuronal cell death was determined 20–24 h later as described in Materials and Methods(n18–24culturespooledfrom4differentexperiments).B,After60min,cellswere washedwithHBSS,and 14C-L-cystinewasaddedfor5minasdescribedinMaterialsandMeth- ods. Data are expressed as 14C-L-cystine uptake in picomoles per minute per milligram protein (n 6–12 cultures pooled from 2–4 different experiments). C, After 120 min of hypoxia, supernatant was collected to measure accumulation of glutamate in the bathing medium via HPLC. Data are expressed as the mean SEM glutamate accumulation in micromolar (n 6 culturespooledfrom3differentexperiments).Anasteriskindicatesavaluesignificantlygreater than oxygen deprivation alone (IL-1), as determined by two-way ANOVA followed by a Bonferroni’s t test for multiple comparisons. Significance was assessed at p 0.05.

Article Snippet: In experiments using recombinant mouse IL-1ra (rIL-1ra) (R & D Systems) or monoclonal anti-mouse IL-1RI antibody directed against the extracellular domain of mouse IL-1RI (MAB7711; R & D Systems), the respective solutions were prepared with IL-1 (1.5 ng/ml; 1.5 ) and added as described above.

Techniques: