IL-18 induces HMGB1 secretion by NK cells: inhibition by anti-IL-18. rNK cells were cultured for 7 days with 50 IU/mL rhuIL-2 (lane 1) or 1 ng/mL rhuIL-18 (lane 2) or both (lanes 3-5), in the absence (lanes 1-3) or presence of 0.5 μg/mL of a neutralizing anti-IL-18 mAb (lane 4) or of an isotype-matched control mAb (lane 5). At the end of the culture period, culture fluids were replaced and cells were incubated in serum-free medium for an additional 6 hours. These supernatants were then assessed for the presence of HMGB1 by Western blotting (top blot). The bottom blot shows as a control the intracellular content of HMGB1 in all the culture conditions; note that IL-18 is unable to maintain NK cells alive in the absence of IL-2, resulting in an empty lane (lane 2).