Interleukin 1β il 1β

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Interleukin-1β (IL-1β) is a cytokine that plays a key role in the body's immune response. It is involved in the regulation of inflammatory processes and immune reactions. IL-1β is produced by various cell types, including monocytes, macrophages, and dendritic cells.

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15 protocols using interleukin 1β il 1β

Glucose Metabolism in Cell Cultures

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The cells were plated onto 48-well plates at a density of 20,000 cells per well. After serum-deprivation the cells were incubated for 24 h in DMEM with 0.1 % BSA and different initial concentrations of glucose (5.5, 11, or 22 mmol/L); in some experiments, the cultures were also treated with 1–10 ng/mL interleukin-1β (IL1β; Peprotech, London, UK) or 0.5 mmo/L sodium azide (Sigma). Glucose consumption and lactate release over 8 or 24 h of incubation were determined by measuring the respective levels in the culture medium at time 0 and after 8 or 24 h using commercial kits (Glucose HK Assay kit, Sigma; Lactate Reagent kit, Trinity Biotech, Wicklow, Ireland). Glucose consumption was calculated as the difference between initial and final glucose levels, while lactate production was calculated as the difference between final and initial lactate levels. Results were normalized by cell number. Both glucose consumption and lactate production were expressed as pmol per cell over 8 or 24 h.

Peiró C., Romacho T., Azcutia V., Villalobos L., Fernández E., Bolaños J.P., Moncada S, & Sánchez-Ferrer C.F. (2016). Inflammation, glucose, and vascular cell damage: the role of the pentose phosphate pathway. Cardiovascular Diabetology, 15, 82.

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Redifferentiation and Transduction of Primary Chondrocytes

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Primary normal human articular chondrocytes (Lonza) were redifferentiated in alginate beads per the manufacturer's protocol using chondrogenic differentiation medium (Lonza). Redifferentiated normal human articular chondrocytes were transduced with lentiviruses expressing GFP or PEDF for 48 h, then cultured for 4 days in chondrogenic differentiation medium in the presence or absence of 1 ng/mL of interleukin-1β (IL-1β) (PeproTech). Total RNA from cultured chondrocytes was isolated using the RNeasy Mini Kit (Qiagen) and complementary DNA was generated using Moloney Murine Leukemia Virus Reverse Transcriptase (Invitrogen). Quantitative PCR was performed using an iQ5 Real-Time PCR Detection System (Bio-Rad). All RT-PCR analyses were normalized to TATA box binding protein mRNA expression [33 (link)]. Primer sequences are available from the author upon request.

Nakamura D.S., Hollander J.M., Uchimura T., Nielsen H.C, & Zeng L. (2017). Pigment Epithelium-Derived Factor (PEDF) mediates cartilage matrix loss in an age-dependent manner under inflammatory conditions. BMC Musculoskeletal Disorders, 18, 39.

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Thrombin Inactivation and Neutrophil Extracellular Trap Degradation

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Thrombin inactivation was achieved by reacting of thrombin (50 μL of 1 U/μL) with 1-mM p-amidinophenylmethylsulfonyl fluoride (Millipore Corp) for 2 hours. Inactive thrombin (0.5 μL) was injected intradermally, alone or with 10 ng/μL TNF-α or 10 μg/μL interleukin-1β (IL-1β) (both from Peprotech) into the dorsal ear dermis. To degrade NETs, mice were injected intraperitoneally with 500 IU DNase I (Sigma-Aldrich) in 0.5 mL Iscove's Modified Dulbecco's Medium (IMDM) immediately after intradermal injections of TNF-α.

MacDonald M.E., Weathered R.K., Stewart E.C., Magold A.I., Mukherjee A., Gurbuxani S., Smith H., McMullen P., Mueller J., Husain A.N., Salles C.M., Briquez P.S., Rouhani S.J., Yu J., Trujillo J., Pyzer A.R., Gajewski T.F., Sperling A.I., Kilarski W.W, & Swartz M.A. (2022). Lymphatic coagulation and neutrophil extracellular traps in lung-draining lymph nodes of COVID-19 decedents. Blood Advances, 6(24), 6249-6262.

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Culturing and Stimulating Human NPCs

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Human NPCs were purchased from ScienCell Research Laboratories (Sciencell, Cat. #4800, USA), which were isolated from the NP of human intervertebral disc. NPCs were cultured in 500 ml Nucleus Pulposus Cell Medium (NPCM, Cat. #4801, Sciencell, USA) containing 10 ml fetal bovine serum, 5 ml NPCs growth supplement, and 5 ml penicillin/streptomycin solution, and were then incubated at 37 °C in a humidified environment with 5% CO₂. The medium was changed every two days. The NPCs were passaged once a week, and well-growth NPCs were taken for subsequent experiments. To validate the expression of miR-583 in “degenerative” NPCs, interleukin -1β (IL-1β) and tumour necrosis factor-α (TNF-α) (10 ng/ml, Peprotech, NJ) were used to stimulate NPCs for 24 h.

Li Y., Pan D., Liu S., Xing X., Zhou H., Zhang B., Zhang D., Li B., Li G., Tao B., Ning G, & Feng S. (2020). Identification of circ-FAM169A sponges miR-583 involved in the regulation of intervertebral disc degeneration. Journal of Orthopaedic Translation, 26, 121-131.

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Modulation of Cellular Signaling Pathways

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Cells were treated with mTOR inhibitors- Rapamycin (RAP) (10 μM) (Calbiochem) or Temisirolimus (TEM) (5 μM) (Santacruz biotechnology) or Torin-1 (TOR) (100 nM) (Tocris) or PP242 (100 nM) (Tocris) and cytokines- Tumor Necrosis Factor-α (TNFα) (10 ng/ml) (Peprotech), Interleukin1β (IL1β) (10 ng/ml) (Peprotech) and tumor promoting compound Phorbol 12- myristate 13-acetate (PMA) (100 ng/ml) (Sigma Aldrich). Staurosporin (STS) (50 nM) and UCN01 (500 nM) (Sigma Aldrich) are pan-inhibitors of PKC kinases. Amino salicylic acid (ASA) (20 μM) and BAY11 (10 μM) (Sigma Aldrich) are specific NFκB inhibitors. Experiments were performed on cells treated in complete medium except for gelatinolytic zymography, invasion and migration assays where serum-free medium was used.

Chandrika G., Natesh K., Ranade D., Chugh A, & Shastry P. (2016). Suppression of the invasive potential of Glioblastoma cells by mTOR inhibitors involves modulation of NFκB and PKC-α signaling. Scientific Reports, 6, 22455.

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IL-1β Signaling and COX-2 Regulation

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Interleukin-1β (IL-1β) was obtained from Peprotech (Rocky Hill, NJ). Polyclonal rabbit antibody against COX-2 was from Millipore (Temecula, CA). Polyclonal goat anti-rabbit Immunoglobulins/HRP were from Dako (Glostrup, Denmark). Diaminobenzidine (DAB) was from Vector (Burlingame, CA). Monoclonal antibodies against phospo-NFκB p65 (Ser536), phospho-p38 (Thr180/Tyr182), phospho-ERK1/2 (Thr202/Tyr204), phospho-SAPK/JNK (Thr183/Tyr185), and RelB were from Cell Signaling Technology (Beverly, MA). 12-O-tetradecanoyl phorbol 13-acetate (TPA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other reagents were from Sigma-Aldrich (St. Louis, MO).

Arasa J., Terencio M.C., Andrés R.M., Marín-Castejón A., Valcuende-Cavero F., Payá M, & Montesinos M.C. (2019). Defective Induction of COX-2 Expression by Psoriatic Fibroblasts Promotes Pro-inflammatory Activation of Macrophages. Frontiers in Immunology, 10, 536.

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Organotypic Spinal Cord and DRG Cultures

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Organotypic slice cultures of spinal cord and dorsal root ganglia (DRG) were prepared from adult mice (20g) as previously described.^{17 (link),18 (link)} After decapitation, the spinal cord and bilateral lumbar DRGs from L1 to L6 were aseptically removed and placed into ice-cold Hanks Balanced Salt Solution (HBSS) (Life Technologies, Carlsbad, CA). Lumbar spinal cords were transversely sliced into 1 mm thickness for spinal cord organ culture. The ganglia and spinal cord slices were transferred in serum free DMEM/F12 (Life Technologies, Carlsbad, CA) containing interleukin 1β (IL-1β) (10ng/mL) (PeproTech, Rocky Hill, NJ), TCA in DMSO (1mg/mL) or combination of IL-1β and TCA and incubated for 24 h at 37°C in a humidified 95% air/5% CO₂ incubator. At the end of the culture period the ganglia and spinal cord slices were harvested, quickly frozen on dry ice, and stored at −80°C until they were assayed.

Kroin J.S., Kc R., Li X., Hamilton J.L., Das V., van Wijnen A.J., Dall O.M., Shelly D.A., Kenworth T, & Im H.J. (2016). Intraarticular Slow-release Triamcinolone Acetate Reduces Allodynia in an Experimental Mouse Knee Osteoarthritis Model. Gene, 591(1), 1-5.

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Modulation of Inflammatory Signaling Pathways

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Human interferon β (IFNβ), tumor necrosis factor α (TNFα), and interleukin-1 β (IL-1β) were purchased from Peprotech, resuspended in phosphate buffered saline (PBS) and used at 10 ng/mL unless otherwise specified. AL8810 (PGF_2α inhibitor) was purchased from Santa Cruz Biotechnologies. SP600125 (JNK inhibitor), PD98059 (MEK1/2 inhibitor), and Ruxolitinib (JAK/STAT inhibitor) were purchased from Invivogen. AL8810, SP600125, and PD98095 were reconstituted per the manufacturer’s instructions, used at the indicated concentrations, and were added to cells at the same time as viral inoculation. Ruxolitinib was also reconstituted per the manufacturer’s instructions and used at the specified concentrations, but cells were instead pre-treated for 2h prior to infection. Ionomycin was purchased from Sigma, reconstituted in PBS, and used at the specified concentrations and time points. Mock and untreated controls were “stimulated” with the appropriate vehicle only at the highest volume used for the given experiment and reagent concentration.

Basavappa M.G., Ferretti M., Dittmar M., Stoute J., Sullivan M., Whig K., Shen H., Liu K.F., Schultz D.C., Beiting D., Lynch K., Henao-Mejia J, & Cherry S. (2022). The lncRNA ALPHA specifically targets chikungunya virus to control infection. Molecular cell, 82(19), 3729-3744.e10.

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Neutrophil Activation Assay Protocol

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The bacterial tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLF,) the bacterial membrane components lipopolysaccharide (LPS) and peptidoglycan (PGN), phorbol 12-myristate 13-acetate (PMA) and luminol (97% purity) were obtained from Sigma Aldrich. The cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were bought from Peprotech (Rocky Hill, NJ, USA). The antibodies for flow cytometry anti-CD11a (FITC-labeled; mouse anti-human, clone HI111), anti-CD11b (PE-CY5-labeled; mouse anti-human, clone ICRF44), anti-CD16 (PE-labeled; mouse anti-human, clone 555407) and anti-FPR1 (mouse anti-human, clone 556015) were purchased from BD Biosciences (Franklin Lakes, NJ, USA) and anti-CD62L (APC-labeled; mouse anti-human, clone DREG56) from eBioscience, anti-CD16 (APC-labeled; mouse anti-human, clone 3G8) from Biolegend (San Diego, CA, USA), while anti-FPR2 (mouse anti-human, sc-57141) from Santa Cruz (Dallas, TX, USA). The fluorescent probe SYTOX Green was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Cockx M., Blanter M., Gouwy M., Ruytinx P., Abouelasrar Salama S., Knoops S., Pörtner N., Vanbrabant L., Lorent N., Boon M, & Struyf S. (2021). The Antimicrobial Activity of Peripheral Blood Neutrophils Is Altered in Patients with Primary Ciliary Dyskinesia. International Journal of Molecular Sciences, 22(12), 6172.

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Murine Macrophage Differentiation

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Murine monocyte/macrophage RAW 264.7 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). Cells were cultured in α-minimal essential medium (α-MEM) (Gibco, BRL, Grand Island, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% humidified CO₂ incubator. For stimulation, cells were treated with sRANKL (100 ng/ml) for 4 days followed by cotreatment in the presence of CpG-ODN (1 μM) for an additional 2 days. Soluble recombinant mouse RANKL (purity: 98%) and interleukin-1β (IL-1β) were purchased from PeproTech (Rocky Hill, NJ, USA). Synthetic CpG-ODN 1585 (5′-ggGGTCAACGTTGAgggggg-3′) was purchased from Invitrogen (San Diego, CA, USA).

Kim S.K., Choe J.Y, & Park K.Y. (2022). CpG Oligodeoxynucleotides Inhibit RANKL-Induced Osteoclast Formation by Upregulating A20 Deubiquitinase in RAW 264.7 Cells. Mediators of Inflammation, 2022, 5255935.

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