IL-1Ra and cytokine production. (A) IL-1Ra mRNA expression in WT T cells. Purified T cells from WT mice were stimulated with 10 μg/ml of plate-coated anti-CD3 mAb for 0, 6, 12, or 24 hours, and IL-1Ra mRNA levels were determined by RT-PCR. T cell_depleted splenocytes (Non T) were stimulated with 5 μg/ml LPS for 0 and 12 hours as controls. β₂m is used as an internal control. (B) Intracellular staining for IL-1Ra in PECs. PECs from WT and IL-1Ra^–/– mice were stimulated with LPS, and IL-1Ra⁺ cells in the CD11b⁺ population are shown as histograms. Black-lined histograms: staining with IL-1Ra Ab. Gray-lined histograms: staining with isotype control Ig. Percentages of IL-1Ra⁺ cells are indicated. (C) Intracellular staining for IL-1Ra in splenocytes (Spl) and CD4⁺ T cells from WT mice after stimulation with anti-CD3 mAb. Upper panels show staining with isotype control Ig and lower panels show staining with anti_IL-1Ra Ab. (D) Expression of cell lineage_specific surface molecules on cells from ankle joints of WT and IL-1Ra^–/– (KO) mice. B220, Gr-1, and CD11b are markers for B cells, granulocytes (including neutrophils), and macrophages, respectively. (E) Intracellular staining for cytokines in cells from the ankle joints of WT and IL-1Ra^–/– mice. Joint-derived cells were stimulated with 10 ng/ml PMA and 400 ng/ml ionomycin for 6 hours with 2 μM monensin. Similar results were obtained in 3 independent experiments.