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

TNF is shed from the cell membrane via activity of ADAM-17 26 . In addition to IL-33 induced increases in TNF mRNA expression (Fig. 1B) , we hypothesize that IL-33 further enhances TNF secretion by NK cells (Fig. 2B) via elevated ADAM-17 cleavage of cell-surface TNF. Addition of the ADAM17 inhibitor (TAPI-1) 27 to NK-cell cultures resulted in reduction of secreted TNF from 97.1±10.4 to 45.8±5.2 pg/ml (ELISA) in response to IL-33 and IL-12 stimulation (Fig. 9A) . In contrast, R-1503 more completely ablated the enhancing effect of IL-33 on IL-12-induced TNF secretion. Thus, enhanced TNF secretion in response to IL-12 and IL-33 is at least partially dependent on ADAM-17, which may function downstream of p38 MAPK or be dependent on p38 MAPK induced changes in TNF mRNA expression. While secreted TNF secretion increased in response to the combination of IL-33 and IL-12, cell-associated TNF was reduced 1.9-fold to levels near those induced by IL-12 alone (Fig. 9B) . These flow cytometry measurements were made in the absence of brefeldin-A or other secretion inhibitors, thus reflecting TNF naturally retained by the cells. Importantly, reduced TNF secretion by TAPI-I-treated, IL-12 and IL-33 stimulated cells (Fig. 9A) was associated with a 2.0-fold increase in cellassociated TNF to levels observed with IL-12 alone (Fig. 9B) . Thus, IL-33-stimulated ADAM17 activity contributes to TNF secretion at the expense of cell-associated TNF, likely via cleavage of TNF from the cell surface.