Figure 4. Myeloid cells in PTX-draining lymph nodes produce IL-1β.

(a) Spontaneous production of IL-1β by different myeloid cells sorted from DLN 72 h after immunization with MOG-CFA and injection of PBS or PTX on day 0 and day 2. Sorted cells were cultured in medium for 16 h in the presence of ATP for the last 45 min and protein abundance was measured by ELISA. Data are mean+s.e.m. (n=4) and are representative of two independent experiments with at least 3 mice per group. (b) Pro-IL-1β mRNA abundance in DLN of WT mice injected with Gr1-depleting (RB6-8C5) antibody (left) or Ly6G-depleting (1A8) antibody (centre) or of Ccr2^−/− mice (right) 72 h after immunization. Isotype-matched antibodies were used as control (Ctrl). Data are mean+s.e.m. (n=6–9, n=5–6, n=3–4, respectively), and are representative of three independent experiments with at least 3 mice per group. AU, arbitrary units. (c–e) Absolute number of CD90.1⁺ CD4⁺ 2D2 T cells and percentage of IL-17⁺ and IFN-γ⁺/GM-CSF⁺ 2D2 T cells measured by surface and intracellular cytokine staining in DLN of WT mice injected with Gr1-depleting (RB6-8C5) antibody (c) or Ly6G-depleting (1A8) antibody (d) or of Ccr2^−/− mice (e) on day 5 after immunization. Isotype-matched antibodies were used as control (Ctrl). 2D2 T cells were directly stained or restimulated in vitro for 5 h with PMA and ionomycin, in the presence of BFA for the last 3 h and then stained for intracellular cytokines. Data are mean+s.e.m. (n=3, n=5–7, n=3, respectively) and are representative of more than three independent experiments with at least 3 mice per group. Each symbol represents an individual mouse. (f) Percentage of IL-17⁺ and IFN-γ⁺/GM-CSF⁺ 2D2 T cells in DLN of Ccr2^−/− mice or of Ccr2^−/− mice receiving 3 × 10⁶ WT CCR2⁺ bone marrow-derived inflammatory monocytes on day 0 and 2 (+IMs). Data are mean+s.e.m. (n=3) and are representative of three separate experiments with 3–4 mice per group. *P<0.05, **P<0.01, ***P<0.001, ****, as determined by nonparametric unpaired Mann–Whitney test. NS, not significant.