Figure 3. Mer regulates IL-23 in vivo and in vitro.

(A) Antibodies (5 μg) to each of the indicated receptors (or isotype controls) were locally microinjected once to the gingiva and the mice were sacrificed 72h later to dissect gingival tissue for cytokine mRNA expression by quantitative real-time PCR. The mRNA expression levels for the indicated cytokines were normalized against Gapdh mRNA and expressed as fold induction relative to the transcript levels of untreated mice, assigned an average value of 1. (B) Mouse BM-derived macrophages from WT (left panel) or CD18^−/− (right panel) mice were allowed to adhere to 24-well culture plates and then incubated for 2h with apoptotic neutrophils (‘apopt’) at a ratio of 3:1 apoptotic neutrophils/macrophages. The wells were then washed with media to remove non-phagocytosed neutrophils, and LPS was added at 100 ng/ml for a 6-h incubation. IL-23p19 mRNA expression was measured by quantitative real-time PCR. Results were normalized against Gapdh mRNA and expressed as fold induction relative to the transcript levels of the untreated group, assigned an average value of 1. In certain groups, Mer-Fc or Fc control (both at 10 μg/ml) were added to the macrophages 30 min prior to adding apoptotic neutrophils. In other groups, prior to adding apoptotic neutrophils, macrophages were incubated for 30 min with cytochalasin D (5 μM) to block phagocytosis. (C) Purified neutrophils (5 × 10⁶ cells) from WT mice were i.v. injected or not into groups of CD18^−/− mice, which were earlier treated or not with anti-Mer or isotype control (local intragingival injection with 5 μg). 72h after adoptive transfer, the mice were euthanized and expression of the indicated cytokines in the gingival tissue was assayed by quantitative real-time PCR, as outlined in panel A. Data are means ± SD and each dot represents the value of an individual mouse or cell culture (A n = 5 mice/group; B n = 6 cultures/group; C n = 6 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 between indicated groups or as compared with untreated control (A). One-way ANOVA with Dunnet’s (A) or Tukey’s (B,C) multiple comparison tests.