Il 1α

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, China

IL-1α is a recombinant protein that represents the mature form of the human interleukin-1 alpha (IL-1α) cytokine. IL-1α is a pro-inflammatory cytokine that plays a central role in the regulation of immune and inflammatory responses.

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Recombinant human IL-1α (12 citations) Human IL-1α (5 citations) Recombinant IL-1α (4 citations) Recombinant human interleukin (IL)-1α (2 citations) Anti-IL-1α neutralizing antibodies (2 citations) Recombinant human IL-1α and IL-1β (2 citations) Human IL-1α ELISA Development Kit (2 citations) Recombinant mouse IL-1α (2 citations) Polyclonal IL-1α (2 citations) ALF-161 Armenian hamster anti-mouse IL-1α (2 citations)

94 protocols using il 1α

Pre-CIK Cell Expansion and Activation

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Both unpurified and purified Pre-CIK cells were cultured in serum free aim v medium (AIM-V) complete medium (Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 5×10⁶ cells/mL. Cells were incubated for 2 hours at 37°C with 5% CO₂. Non-adherent cells were collected and cultured at a concentration of 3×10⁶ cells/mL in AIM-V complete medium containing 1,000 U/mL rhIFN-γ and 5% inactivated human serum with 5% CO₂ at 37°C. After 24 hours of incubation, monoclonal antibody against CD3 (OKT-3, 50 ng/mL; PeproTech Inc., Rocky Hill, NJ, USA), IL-1α (100 U/mL; PeproTech Inc.), and rhIL-2 (300 IU/mL; PeproTech Inc.) were added. Medium with IL-2 was changed every 2–3 days.

Shi B., Sun A, & Zhang X. (2018). Influence of different ex vivo cell culture methods on the proliferation and anti-tumor activity of cytokine-induced killer cells from gastric cancer patients. OncoTargets and therapy, 11, 2657-2672.

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Inflammation Signaling Pathway Modulation

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Withaferin A (catalog number S8587), SB203580 (catalog number S1076), and SP600125 (catalog number S1460) were acquired from Selleckchem. Recombinant human TNF-α (catalog number 300-01A), IL-1β (catalog number AF-200-01B), and IL-1α (catalog number 200-01A) were acquired from Peprotech.

McAleavy M., Zhang Q., Ehmann P.J., Xu J., Wipperman M.F., Ajithdoss D., Pan L., Wakai M., Simonson R., Gadi A., Oyejide A., Hamon S.C., Boyapati A., Morton L.G., Shavlakadze T., Kyratsous C.A, & Glass D.J. (2022). The Activin/FLRG Pathway Associates with Poor COVID-19 Outcomes in Hospitalized Patients. Molecular and Cellular Biology, 42(1), e00467-21.

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In vitro expansion of DC-CIK cells

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PBMC from seven health adults were enriched by density gradient centrifugation. Then, PBMC were plated at a density of 5×10⁶ cells/ml in GT-T551 medium (Takara, Dalian, China) with the addition of 1000 U/ml IFN-γ (Peprotech, Rocky Hill, NJ, USA) on the first day. 24 hours later, 50 ng/ml anti-human CD3 monoclonal antibody (eBioscience, San Diego, CA, USA), 500 IE/ml IL-1α (Peprotech, Rocky Hill, NJ, USA) and 500 IE/ml IL-2 (Peprotech, Rocky Hill, NJ, USA) were added. The DC and CIK cells were co-cultured on day 7 at the ratio of 1∶5. Then, the cells were incubated at 37°C with 5% CO₂ and subcultured every 3 days in fresh GT-T551 medium containing 500 IE/ml IL-2. Both CIK and DC-CIK cells were expanded in vitro over 28 days and partly harvested on day 7, 14, 21 and 28 for analysis, respectively.

Pan Y., Tao Q., Wang H., Xiong S., Zhang R., Chen T., Tao L, & Zhai Z. (2014). Dendritic Cells Decreased the Concomitant Expanded Tregs and Tregs Related IL-35 in Cytokine-Induced Killer Cells and Increased Their Cytotoxicity against Leukemia Cells. PLoS ONE, 9(4), e93591.

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Culturing Human Nasal Epithelial Cells for Cytokine and TLR Agonist Stimulation

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Following a previously established protocol, HNECs (Human Nasal Epithelia Cells) isolated from the middle turbinates of CRSsNP patients were cultured [16 (link)]. Briefly, nasal specimens were immersed in DMEM/F12 media (Hyclone) containing 1.4 mg/ml protease K and 0.1 mg/ml DNase for a 1.5 h incubation at 37 °C. Next, all cells were collected and immersed in DMEM/F12 (Hyclone) containing 1% ITS for 2 h at 37 °C before being cultured in BEGM medium (Lonza).
When 80–90% confluence was reached, fresh media without hydrocortisone was added in the presence of the following stimulators or control PBS for 12 h: the recombinant cytokines human IFN-γ (100 ng/mL), IL-4 (100 ng/mL), IL-13 (100 ng/mL), IL-17A (100 ng/mL), TGF-β (10 ng/mL), and IL-1α (100 ng/mL; all purchased from Peprotech); the TLR agonists LPS (500 ng/mL, from Escherichia coli serotype 0111: B4; purchased from Sigma) and the glucocorticoid dexamethasone (Sigma; 10 μg/mL). After stimulation, HNECs were collected for qRT-PCR analysis.

Ma Y., Zheng C, & Shi L. (2018). The kinase LRRK2 is differently expressed in chronic rhinosinusitis with and without nasal polyps. Clinical and Translational Allergy, 8, 8.

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Selectin-Mediated Cancer Cell Adhesion

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The adhesion of UTSCC 2, UTSCC 24A, UTSCC 24B, and Carey 24 cells on rhE-selectin and P-selectin (R&D Systems) and stimulated (10 ng/mL IL-1α, PeproTech, Hamburg, Germany) HUVECs was analyzed under physiological flow conditions in ibidiTreat µ-slide IV0.4 flow chambers, as previously described [51 (link)]. The applied shear rate was 0.5 dyn/cm². Stimulated HUVECs were incubated with an adhesion blocking E-selectin mAb (BioLegend, San Diego, CA, USA) prior to the experiment [51 (link)]. Furthermore, cancer cells were pretreated with pronase, as described in the section above.

Valentiner U., Knips J., Pries R., Clauditz T., Münscher A., Sauter G., Wollenberg B, & Schumacher U. (2019). Selectin Binding Sites Are Involved in Cell Adhesive Properties of Head and Neck Squamous Cell Carcinoma. Cancers, 11(11), 1672.

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Psoriasis-like Keratinocyte Stimulation

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HaCaT and HEKa cells (human keratinocyte cell lines) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂. Cells were stimulated with 10 ng/ml M5 (IL-1α, IL-17A, IL-22, oncostatin M, and TNF-α; PeproTech, USA) for 24 hours at 37°C to mimic a psoriasis-like environment in keratinocytes [17 (link)–20 (link)]. Cells were then either left untreated or pretreated with 1 μg/ml C23 (GRGFSRGGGDRGYGG synthesized from GenScript, Piscataway, NJ; dissolved in phosphate-buffered saline) for 30 minutes and then were stimulated with 1 μg/ml rhCIRP (APG886Hu01, Cloud-Clone Corp, Texas, USA) for 1 hour. After rhCIRP stimulation, cells were collected for qRT-PCR and western blot analysis.

Zhang X., Wang S., Wang W., Song L., Feng S., Wang J., Kang T., Yang P., Wang N., Yang P., Bai R., Shao Y, & Zheng Y. (2022). Extracellular CIRP Upregulates Proinflammatory Cytokine Expression via the NF-kappaB and ERK1/2 Signaling Pathways in Psoriatic Keratinocytes. Mediators of Inflammation, 2022, 5978271.

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Induction and Adoptive Transfer of EAE

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For active immunization, C57Bl/6 mice were subcutaneously immunized over the flanks with 100 μg MOG_35-55 (Biosythesis) in complete Freund’s adjuvant (Difco). Mice were injected intraperitoneally with 300 ng pertussis toxin (List Biological) on days 0 and 2. For adoptive transfer, mice were immunized as described, without pertussis toxin, and the draining lymph nodes (inguinal, brachial, and axillary) were collected at 10–14 days post-immunization. Lymph node cells were cultured for 96 hours in the presence of 50 ug/mL MOG_35-55, 8 ng/ml IL-23 (R&D Systems), 10 ng/ml IL-1α (Peprotech), and 10 μg/mL anti-IFNγ (Clone XMG1.2, BioXcell). At the end of culture, CD4⁺ T cells were purified with CD4 positive selection magnetic beads (Miltenyi), and 3–5x10⁶ CD4⁺ T cells were transferred intraperitoneally into naïve recipients. For induction of relapsing-remitting EAE, SJL mice were subcutaneously immunized over the flanks with 100 μg PLP_139-151 (Biosynthesis) in complete Freund’s adjuvant (Difco) without pertussis toxin. EAE was assessed by a clinical score of disability: 1, limp tail; 2, hind-limb weakness; 3, partial hind-limb paralysis; 4, complete hind-limb paralysis; and 5, moribund state.

Giles D.A., Washnock-Schmid J.M., Duncker P.C., Dahlawi S., Ponath G., Pitt D, & Segal B.M. (2018). Myeloid cell plasticity in the evolution of central nervous system autoimmunity. Annals of neurology, 83(1), 131-141.

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Profiling NF-κB Pathway in Inflammatory MSCs

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Early MSCs until reaching 80% confluence were incubated with 10 ng/mL IL-1α or 10 ng/mL IL-8 recombinants (Peprotech, Rocky Hill, NJ, USA), respectively, in serum-free DMEM supplemented with 1% antibiotics for 24 h. The protein expression analysis of early MSCs and pro-inflammatory activation were performed using a Proteome Profiler Human NF-κB Pathway Kit (ARY029, R&D Systems, Minneapolis, MN, USA). All procedures and data analysis followed the manufacturers’ instructions. Briefly, total protein was extracted using Lysis Buffer. Each sample was run on each array (membrane) under incubation at 4 °C overnight on a shaker. The membrane was washed three times with PBS. Detection Antibody Cocktail was added to each membrane followed by 1 h incubation. The membrane was then incubated with Chemiluminescence Reagent Mix and exposed to generate the profile of spot pixel density image. The density signals of each spot were quantified by Image J software (Rasband W; National Institutes of Health, Bethesda, MD, USA). The relative differences of sample signals were normalized with reference spots.

Chou L.Y., Ho C.T, & Hung S.C. (2022). Paracrine Senescence of Mesenchymal Stromal Cells Involves Inflammatory Cytokines and the NF-κB Pathway. Cells, 11(20), 3324.

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Nucleus Staining and Cell Assays

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DMSO (dimethyl sulfoxide) and Hoechst dye 33,258 for nucleus staining were purchased from Sigma-Aldrich (St. Louis, USA). Daphnetin (7,8-Dihydroxycoumarin, CAS 486–35-1) was obtained from PharmaBlock Sciences (Nanjing, China) and dissolved in DMSO. Carbopol 940 and azone were purchased from Rhawn (Shanghai, China). IMQ cream was obtained from Sichuan Med-Shine Pharmaceutical Co., LTD (Sichuan, China). IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α cytokines were purchased from PeproTech (RockyHill, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). TRIzol™ reagent and primers were obtained from Invitrogen (Carlsbad, USA). RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific (USA). SYBR Green PCR Master Mix kit was purchased from TaKaRa (Japan). Rabbit anti-p65, anti-p-p65 (Ser536), anti-β-actin primary antibodies and Alexa Fluor 555-labeled anti-rabbit secondary antibody were purchased from Cell Signaling Technology (Beverly, USA).

Gao J., Chen F., Fang H., Mi J., Qi Q, & Yang M. (2020). Daphnetin inhibits proliferation and inflammatory response in human HaCaT keratinocytes and ameliorates imiquimod-induced psoriasis-like skin lesion in mice. Biological Research, 53, 48.

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Cytokine Modulation of Primary T Cell Activation

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Human recombinant IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, INF-α, IFN-γ, TGF-β1, and TNF-α were purchased from Peprotech and used at 5, 50, and 500 ng/mL. The data shown in Supplemental Figure 1a are using 500 ng/mL only because the relative effects of cytokines were not different at other doses. Primary human T Cells were treated with neutralizing TGF-β1 antibody (Abcam, 2Ar2) and with small molecule inhibitors, SB431542, Cyclosporin A (Sigma-Aldrich), and SIS3 (Calbiochem) for 1 hr prior to activation at the indicated concentration range.

Park B.V., Freeman Z.T., Ghasemzadeh A., Chattergoon M.A., Rutebemberwa A., Steigner J., Winter M.E., Huynh T.V., Sebald S.M., Lee S.J., Pan F., Pardoll D.M, & Cox A.L. (2016). TGF-β1-mediated Smad3 enhances PD-1 expression on antigen-specific T cells in cancer. Cancer discovery, 6(12), 1366-1381.

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