Fig. 4.

BM-derived non-B, non-T cells in the lung and fat secrete an IL-4Rα–regulating protein. (A) Flow cytometry analysis of IL-4Rα expression on blood myeloid cells and lymphocytes from TLR4^−/− mice cultured ex vivo for 18 h with 0.5% control or LPS serum. Data are shown as mean and individual mice; n = 5 (control); n = 35 (LPS). Data are representative of >10 experiments. (B Left) Flow cytometry analysis of IL-4Rα expression on TLR4^−/− myeloid blood cells cultured ex vivo with 0.5% control or LPS serum, pretreated (+) or not (−) with Proteinase K. (Right) SDS/PAGE gel of LPS serum pretreated or not with Proteinase K. Data are representative of three experiments. (C) Flow cytometry analysis of IL-4Rα expression on TLR4^−/− myeloid blood cells cultured ex vivo with 0.5% serum from control or LPS-injected WT, TLR4^−/−, and Rag1^−/− mice, as well as irradiated TLR4^−/− mice reconstituted with WT BM cells (WT→TLR4^−/−). Data are representative of two experiments. (D) WT mice were injected with LPS, and organs were harvested, cut into pieces, and cultured ex vivo for 8 h before the cell-free supernatant was collected and added to TLR4^−/− blood cells. Graph shows flow cytometry analysis of IL-4Rα expression on myeloid cells cultured ex vivo with 0.5% LPS serum or 10% (vol/vol) supernatant from organ cultures. Data are representative of three experiments. (E) Flow cytometry analysis of IL-4Rα expression on TLR4^−/− thioglycolate elicited macrophages cultured 20 h with 10% supernatant from lung cultures, as described in D, with control or a neutralizing anti–IL-21 antibody (50 μg/mL). IL-4Rα^−/− thioglycolate-elicited macrophages (−/−) defines the background staining. Data are representative of two experiments. (F) Western blot of phospho-STAT6 (STAT6-p) and actin from TLR4^−/− thioglycolate-elicited macrophages, pretreated with supernatant from lung or control cultures, pulsed with indicated doses of IL-4 for 15 min. Data are representative of three experiments.