Figure 4

Inhibition of Neisseria meningitidis as well as Streptococcus pneumoniae -induced Camp (rat) expression by the FPRL1 antagonist WRW4 and the G-protein inhibitor pertussis toxin in glial cells. Bacterial supernatants from Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP) were added to astrocytes (A) and microglia (B) with the addition of 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation) and with PTX (16 h preincubation) and WRW4 (30 min preincubation) alone to analyze the effect on Camp mRNA expression after 24 h (astrocytes; a) or 6 h (microglia; b) of treatment. The induction was analyzed and compared to an untreated sample (also with DMSO in equivalent amount). GAPDH (housekeeping gene) was used as an internal control. The data from three independent experiments performed in triplicate were assessed. An asterisk (*, p < 0.05; **, p < 0.01) indicates a significant difference between Camp expression after treatment and control (as determined by ANOVA followed by the Bonferroni test). Astrocytes or microglia were incubated with NM (C) or SP (D) with or without 1, 5 or 10 μM WRW4 (30 min preincubation) and WRW4 alone for 24 h or 12 h, respectively. Glial cells were fixed and labelled with anti-rCRAMP antibodies and protein expression was examined by immunofluorescence microscopy. Bisbenzimide was used for nuclear counter-staining (blue). The figures show representative results from one of three independent experiments. Scale bar: 20 μm.