SHIP negatively regulated IL-6 production caused by FcγR stimulation. (A) Peritoneal macrophages were purified from WT, SHIP^−/−, or γ-chain^−/− mice by adherence and cultured 24 hours. The cells were stimulated with nothing (no stimulus) or with 10 μg/mL of heat-aggregated IgG (ΔIgG) and incubated for an additional 12 hours before supernatants were collected. IL-6 production stimulated by FcγR engagement of peritoneal macrophages was determined by ELISA. Results are from 3 separate experiments and are the mean ± SE of IL-6 in nanograms per milliliter. N.S. indicates not significant. (B) Peritoneal lavage fluid was collected from SHIP^−/− mice and concentrated 10-fold by ultrafiltration (to 250 μL). The concentrated lavage material was incubated with nothing or with Sepharose-conjugated protein A (PA) or GST as indicated. Bone marrow–derived macrophages of SHIP^−/− or γ-chain^−/− mice were then treated with 50 μL of the concentrated lavage fluid and incubated for 12 hours. The supernatants were measured by IL-6 ELISA. Results are from 2 separate experiments and are the mean ± SE of IL-6 in nanograms per milliliter. (Inset) Western blot of 50 μL of lavaged material probed with rabbit anti–mouse Ig.