Interleukin-33 promotes type 1 cytokine expression via p38 MAPK in human natural killer cells

David E. Ochayon; Ayad Ali; Pablo C. Alarcon; Durga Krishnamurthy; Leah C. Kottyan; Michael Borchers; Stephen N. Waggoner

Cytokine stimulation. Enriched NK cells (unless mentioned otherwise, 5×10 4 per well in triplicate per condition) were cultured overnight in the presence of 60 U/ml of IL-2. Various concentrations of IL-12, IL-18 or IL-33 or (Peprotech®, Rocky Hill, NJ) were added to cultures for 6 to 16 hours. Cell-free supernatant was collected and analyzed by ELISA for levels of human IFN-, TNF and GM-CSF (Invitrogen, Waltham, MA). For inhibition of mitogenactivated protein kinase (MAPK) activity, 125 nM (measured IC50) of the selective p38 MAPK inhibitor Pamapimod (R-1503, Selleckchem, Houston, TX) was added 150 minutes prior to cytokine stimulation. The inhibition of MK2 (p38/mitogen-activated protein kinaseactivated protein kinase 2) was performed with 5 M MK2 IV (CAYMAN chemical, Ann Arbor, MI). The inhibition of ADAM-17 (a disintegrin and metalloprotease-17) was performed by adding 8 M TAPI-1 (Merck Millipore, Burlington, MA). TAPI-1 and MK2 IV were added 60 minutes before cytokine stimulation. At the tested concentrations, none of the tested inhibitor affected cell viability (data not shown). mRNA stability assay. Primary human NK cells were cultured at 1×10 5 cell per well. Cells were treated with media (0.1% DMSO), IL-12 (2.5 ng/ml) with or without IL-33 (10 ng/ml). Actinomycin D (5 μg/mL, Sigma) was added to cell cultures 4 hours after cytokine treatment, and RNA was isolated before addition of actinomycin D and 2 hours after addition of actinomycin D. RNA was extracted from NK cells with RNeasy kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. The detection of IFNG, TNF, IL5 and IL13 mRNA expression was performed by using TaqMan® probes (Applied Biosystems, Foster City, CA) according to manufacturer's instructions.