Macrophages respond to CpG secreting cytokines and small molecules that mediate an inflammatory response. The aim of this work was to investigate whether CpG modulate nitric oxide (NO) production. We focused on arginase, enzime which shares the L-arginine substrate with iNOS. Bone marrow-derived macrophages (BMMØ) were cultured with medium, CpG, IFNg or CpG+IFNg for 48h. Arginase activity (AA) was not induce by CpG alone but it was by CpG+IFNg (medium:78±13; CpG:83±7; CpG+IFNg:187±5 mU/mg protein). This effect was correlated with increased expression of arginase II isoform. Specific inhibitors were used to examine AA pharmacological sensitivity. CpG+IFNg-mediated AA depends on p38 and ERK MAPK activation but it is independent of JNK or NFkB. A time course essay corroborated the p38 and ERK phosphorilation. Since IL10 has been implied in AA regulation, IL10 was dosed in culture supernants. CpG alone augmented IL-10 secretion, but IL10 level dramatically diminished in presence of IFNg (medium: 272±113;CpG: 583±76;CpG+IFNg:70±30 pg/mL). Upon preincubation of BMMØ with IFNg, washing and subsequent stimulation with CpG, arginase activity increased (medium: 56±2;CpG:183±59 mU/mg protein). However, preincubation with CpG and stimulation with IFNg did not exhibit this effect. A novel and interesting observation of this study is that the AA induction with CpG is absolutely dependent on IFNg priming. Finally, we investigated the relationship between arginase and iNOS. Accumulation of NO in supernatant of BMMØ with CpG+IFNg peaked at 48h and then remained stable while arginase activity increased over 72h. The increasing AA in time might be a regulatory mechanism of NO production. Then BMMØs inflammatory response to CpG is regulated by IFNg, one of the mayor cytoquine produced in vivo in response to this stimulus.
