Journal: The Journal of Neuroscience

Article Title: Induction of Thermal Hyperalgesia and Synaptic Long-Term Potentiation in the Spinal Cord Lamina I by TNF-α and IL-1β is Mediated by Glial Cells

doi: 10.1523/JNEUROSCI.5087-12.2013

Figure Lengend Snippet: Receptors for the pro-inflammatory cytokines TNF-α and IL-1β are expressed by lamina I neurons and glial cells. Double immunostaining shows colocalization of TNFR1 (Aa,Ba, green) and TNFR2 (Ca,Da,Ea, green) and IL1R (Fa,Ga,Ha, green) with the neuronal marker NeuN (Ab,Cb,Fb, red), the astrocytic marker GFAP (Bb,Db,Gb, red), and the microglial marker Iba1 (Eb,Hb, red) in spinal cord lamina I. Overlays reveal that lamina I neurons express TNFR1, TNFR2, and IL-1R. TNFR1 and IL-1R are also expressed by GFAP-positive cells in the spinal dorsal horn. There is weak costaining for the individual receptors and the microglial marker Iba1.

Article Snippet: Slices were incubated overnight in PBS containing the following primary antibodies: rabbit anti-IL-1R (1:200; Santa Cruz Biotechnology), together either with mouse anti-NeuN (1:500; Millipore) or mouse anti-GFAP (1:800; ThermoScientific) or goat anti-Iba1 (1:2000; Abcam).

Techniques: Double Immunostaining, Marker

Journal: Cureus

Article Title: Melittin Alleviates Oxidative Stress Injury in Schwann Cells by Targeting Interleukin-1 Receptor Type 1 to Downregulate Nuclear Factor Kappa B-Mediated Inflammatory Response In Vitro

doi: 10.7759/cureus.65721

Figure Lengend Snippet: (A) and (B): RT-qPCR analysis of reactive mRNA of IL-1β and TNF-α expression in hypoxic SCs and the intervention effect of melittin. (C) Immunofluorescence assays after staining with anti-Fn and anti-IL-1R1 antibodies (scale bars = 50 μm). ****p < 0.0001 versus the control group. Data are presented as mean ± standard deviation. SCs: Schwann cells; RT-qPCR: Quantitative reverse transcription-polymerase chain reaction; IL-1R1: Interleukin-1 receptor type 1; TNF-α: Tumor necrosis factor-alpha; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: Briefly, cells cultured on coverslips were fixed with 4% paraformaldehyde in PBS for 10 min and then permeabilized with 0.05% Triton X-100 for 10 min. Non-specific binding sites were blocked with 5% goat serum for 1 h. Subsequently, the cells were incubated with a rabbit anti-rat IL-1R1 monoclonal antibody (AF7212, Beyotime, Jiangsu, China) at room temperature for 1.5 h. After washing three times with PBS, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (#7074, Cell Signaling) for 2 h at room temperature.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Control, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction

Journal: Cureus

Article Title: Melittin Alleviates Oxidative Stress Injury in Schwann Cells by Targeting Interleukin-1 Receptor Type 1 to Downregulate Nuclear Factor Kappa B-Mediated Inflammatory Response In Vitro

doi: 10.7759/cureus.65721

Figure Lengend Snippet: (A) RT-qPCR results showing the expression levels of IL-1R1 and IL-1β. (B) Transmission electron microscope images of ultrastructural alterations in CoCl 2 -induced SCs for each group (Left: ×2500, Right: ×7000), with the red arrows indicating mitochondria. (C) Western blot of C-JUN and GDNF protein expression for quantitative analyses. (D) and (E) Western blot and RT-qPCR results showing the expression levels of VEGF, HIF-1α, IL-1R1, ENO1, AR, SOD, NGF, and iNOS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control and other groups. Data are presented as mean ± standard deviation. SCs: Schwann cells; GDNF: Glial cell line-derived neurotrophic factor; RT-qPCR: Quantitative reverse transcription-polymerase chain reaction; VEGF: Vascular endothelial growth factor; HIF-1α: Hypoxia-inducible factor 1-alpha; IL-1R1: Interleukin-1 receptor type 1; ENO1: Enolase 1; AR: Aldose reductase; SOD: Superoxide dismutase; NGF: Nerve growth factor; iNOS: Inducible nitric oxide synthase.

Article Snippet: Briefly, cells cultured on coverslips were fixed with 4% paraformaldehyde in PBS for 10 min and then permeabilized with 0.05% Triton X-100 for 10 min. Non-specific binding sites were blocked with 5% goat serum for 1 h. Subsequently, the cells were incubated with a rabbit anti-rat IL-1R1 monoclonal antibody (AF7212, Beyotime, Jiangsu, China) at room temperature for 1.5 h. After washing three times with PBS, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (#7074, Cell Signaling) for 2 h at room temperature.

Techniques: Quantitative RT-PCR, Expressing, Transmission Assay, Microscopy, Western Blot, Control, Standard Deviation, Derivative Assay, Reverse Transcription, Polymerase Chain Reaction

Journal: Cureus

Article Title: Melittin Alleviates Oxidative Stress Injury in Schwann Cells by Targeting Interleukin-1 Receptor Type 1 to Downregulate Nuclear Factor Kappa B-Mediated Inflammatory Response In Vitro

doi: 10.7759/cureus.65721

Figure Lengend Snippet: (A-E) RT-qPCR results showing mRNA expressions of IKK, IKB-α, p65, p60, and IRAK1. (F) Western blot results of protein expression of IKK, IKB-α, p65, p60, and IRAK1. (G-K) Quantitative analyses of IKK, IKB-α, p65, p60, and IRAK1 protein expression. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control and other groups. Data are presented as mean ± standard deviation. RT-qPCR: Quantitative reverse transcription-polymerase chain reaction; IL-1R1: Interleukin-1 receptor type 1; NF-κB: Nuclear factor kappa B.

Article Snippet: Briefly, cells cultured on coverslips were fixed with 4% paraformaldehyde in PBS for 10 min and then permeabilized with 0.05% Triton X-100 for 10 min. Non-specific binding sites were blocked with 5% goat serum for 1 h. Subsequently, the cells were incubated with a rabbit anti-rat IL-1R1 monoclonal antibody (AF7212, Beyotime, Jiangsu, China) at room temperature for 1.5 h. After washing three times with PBS, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (#7074, Cell Signaling) for 2 h at room temperature.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Astrocyte-Derived Proinflammatory Cytokines Induce Hypomyelination in the Periventricular White Matter in the Hypoxic Neonatal Brain

doi: 10.1371/journal.pone.0087420

Figure Lengend Snippet: A shows TNF-α (30 kDa) and TNF-R 1 (55 kDa), IL-1β (17 kDa), IL-1R 1 (80 kDa) and β-actin (42 kDa) immunoreactive bands, respectively. Bar graphs in B, C show significant increase in the optical density of TNF-α and IL-1β, TNF-R 1 , IL-1R 1 following hypoxic exposure when compared with the corresponding controls (* P <0.01). Panels D and E show the graphical representation of the fold changes in TNF-α and IL-1β, TNF-R 1 , IL-1R 1 mRNA, respectively as quantified by normalization to the β-actin as an internal control. Significant increase in TNF-α and IL-1β, TNF-R 1 , IL-1R 1 mRNA levels in the PWM after the hypoxic exposure is evident when compared with controls (* P <0.01). The panels F show TNF-α (30 kDa) and IL-1β (17 kDa) immunoreactive protein bands in cultured control astrocytes and at 3 h after hypoxic exposure. G is bar graph showing changes in the optical density of TNF-α and IL-1β, respectively, following hypoxic exposure. H (TNF-α and IL-1β mRNA) show the graphical representation of the fold changes quantified by normalization to the β-actin as an internal control. Significant differences in protein and mRNA levels in astrocytes after the hypoxic exposure are evident when compared with controls (* P <0.01).

Article Snippet: Immunofluorescence labeling was carried out using primary antibodies directed against TNF-R1 (rabbit polyclonal IgG 1∶500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat. No.sc-7895), anti-IL-1R 1 (rabbit polyclonal IgG 1∶500) (Santa Cruz Biotechnology; Cat. No.sc-689) and APC monoclonal antibody against adenomatus polyposis coli (APC) (oncogene, mouse monoclonal IgG 1∶20; Cat.No.OP80).

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Astrocyte-Derived Proinflammatory Cytokines Induce Hypomyelination in the Periventricular White Matter in the Hypoxic Neonatal Brain

doi: 10.1371/journal.pone.0087420

Figure Lengend Snippet: The co-localized expression of APC with TNF-R 1 and IL-1R 1 is depicted in C and F, I and L, respectively. Note the expression of TNF-R 1 and IL-1R 1 is upregulated after the hypoxic exposure. Scale bars: A–L, 50 µm.

Article Snippet: Immunofluorescence labeling was carried out using primary antibodies directed against TNF-R1 (rabbit polyclonal IgG 1∶500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat. No.sc-7895), anti-IL-1R 1 (rabbit polyclonal IgG 1∶500) (Santa Cruz Biotechnology; Cat. No.sc-689) and APC monoclonal antibody against adenomatus polyposis coli (APC) (oncogene, mouse monoclonal IgG 1∶20; Cat.No.OP80).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Astrocyte-Derived Proinflammatory Cytokines Induce Hypomyelination in the Periventricular White Matter in the Hypoxic Neonatal Brain

doi: 10.1371/journal.pone.0087420

Figure Lengend Snippet: Confocal images showing TNF-R 1 and IL-1R 1 expression (B, E, H, K, red) in primary cultured oligodendrocytes labeled with APC (A, D, G, J, green) in both control and hypoxia for 3 h. Note TNF-R 1 and IL-1R 1 immunofluroscence intensity is markedly enhanced after hypoxic exposure (E, K) in comparison with the control (B, H). Scale bars: A–L, 50 µm.

Article Snippet: Immunofluorescence labeling was carried out using primary antibodies directed against TNF-R1 (rabbit polyclonal IgG 1∶500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat. No.sc-7895), anti-IL-1R 1 (rabbit polyclonal IgG 1∶500) (Santa Cruz Biotechnology; Cat. No.sc-689) and APC monoclonal antibody against adenomatus polyposis coli (APC) (oncogene, mouse monoclonal IgG 1∶20; Cat.No.OP80).

Techniques: Expressing, Cell Culture, Labeling

Journal: Cellular and Molecular Life Sciences

Article Title: The P2Y 11 receptor of human M2 macrophages activates canonical and IL-1 receptor signaling to translate the extracellular danger signal ATP into anti-inflammatory and pro-angiogenic responses

doi: 10.1007/s00018-022-04548-z

Figure Lengend Snippet: P2Y 11 activation on human M2 macrophages causes IL-1R upregulation and CD39 downregulation as well as VEGF secretion. A–C The regulation of IL-1R1 and the ecto-ATPase CD39 at the protein level was examined by flow cytometry. M2 macrophages were treated for 24 h with the P2Y 11 receptor agonist ATPγS (20 µM) in the presence or absence of the PDE4 inhibitor rolipram (10 µM). The antagonist NF340 (20 µM) was used to confirm that agonist-mediated responses were specific to P2Y 11 receptor stimulation. A Representative FACS histograms of IL-1R1 and CD39 expression from differentially treated M2 macrophages. Numbers represent mean fluorescence intensities (MFIs) of the respective staining after subtraction of isotype control MFIs. B, C Quantification of IL-1R1 ( B ) and CD39 ( C ) expression levels from differentially treated M2 macrophages ( n = 3). * p < 0.05, ** p < 0.01, **** p < 0.0001. D M2 macrophages were treated for 24 h with the P2Y 11 receptor agonist ATPγS (20 µM) in the presence or absence of the PDE4 inhibitor rolipram (10 µM). VEGF levels were measured in cell culture supernatants. NF340 (20 µM) was used to confirm that agonist-mediated responses were specific to P2Y 11 receptor stimulation ( n = 5). **** p < 0.0001

Article Snippet: The following antibodies were used: rabbit polyclonal IgG antihuman P2Y 11 receptor (bs-12071R-A-488; Bioss, Woburn, MA, USA), mouse monoclonal IgG2b antihuman CD14 (clone MϕP9, 345787-APC; BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal IgG1 antihuman CD163 (clone GHI/61, 556018-PE; BD Biosciences), goat polyclonal IgG antihuman IL-1R1 (FAB269P-PE; R&D Systems) and mouse monoclonal IgG2b antihuman CD39 (clone TU66, 561444-FITC; BD Biosciences).

Techniques: Activation Assay, Flow Cytometry, Expressing, Fluorescence, Staining, Control, Cell Culture

Journal: Cellular and Molecular Life Sciences

Article Title: The P2Y 11 receptor of human M2 macrophages activates canonical and IL-1 receptor signaling to translate the extracellular danger signal ATP into anti-inflammatory and pro-angiogenic responses

doi: 10.1007/s00018-022-04548-z

Figure Lengend Snippet: P2Y 11 -mediated sTNFR2 release and VEGF secretion as well as IL-1R upregulation depend on Ca 2+ and protein kinase C, but are differentially regulated by cAMP elevation. A M2 macrophages were treated for 24 h with the P2Y 11 receptor agonist ATPγS (20 µM) in the presence or absence of the PDE4 inhibitor rolipram (10 µM). In addition, cells were treated either with the PDE4 inhibitor rolipram (10 µM), the direct AC activator forskolin (10 µM) or with the combination of both. sTNFR2 and VEGF were measured in cell culture supernatants and IL-1R1 MFIs were determined by flow cytometry. Mean IL-1R1 MFI values are shown ( n = 3). B M2 macrophages were treated for 24 h with the P2Y 11 receptor agonist ATPγS (20 µM) in the presence or absence of the PDE4 inhibitor rolipram (10 µM), either with or without the Ca 2+ chelator BAPTA-AM (10 µM) or the protein kinase C inhibitor calphostin C (250 nM). sTNFR2 and VEGF were measured in cell culture supernatants and IL-1R1 MFIs were determined by flow cytometry. Mean IL-1R1 MFI values are shown ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ## p < 0.01, ### p < 0.001

Article Snippet: The following antibodies were used: rabbit polyclonal IgG antihuman P2Y 11 receptor (bs-12071R-A-488; Bioss, Woburn, MA, USA), mouse monoclonal IgG2b antihuman CD14 (clone MϕP9, 345787-APC; BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal IgG1 antihuman CD163 (clone GHI/61, 556018-PE; BD Biosciences), goat polyclonal IgG antihuman IL-1R1 (FAB269P-PE; R&D Systems) and mouse monoclonal IgG2b antihuman CD39 (clone TU66, 561444-FITC; BD Biosciences).

Techniques: Cell Culture, Flow Cytometry