Synergistic induction of IL-10 by hypertonic saline solution and lipopolysaccharides in murine peritoneal macrophages

Background. Liver injury after ischemia/reperfusion is an important cause o f morbidity in surgical patients. We have shown that the preconditioning of animals that were subjected to liver ischemia/reperfusion with hypertonic saline solution (HTS) prevented injury by inhibiting Kupffer cell tumor nec rosis factor (TNF) production. We postulated that the induction of anti-inf lammatory interleukin-10 (IL-10) by HTS might contribute to protection. Methods. Murine thioglycolate-elicited peritoneal exudative macrophages (PE Ms) were used to model the effects of HTS on IL-10 release from Kupffer cel ls. Cells were preconditioned with 500 mOsm HTS (or isotonic saline medium) for 2 hours and then stimulated with lipopolysaccharide (LPS; 1 mug/mL) or vehicle for 4 hours under isotonic conditions. TNF-alpha and IL-10 were me asured in the culture supernatant by enzyme-linked immunosorbent assay; TNF , IL-10, and SOCS-3 messenger RNA expression were assessed by Northern blot . NF-kappaB activation was examined by electrophoretic mobility shift assay and Western blot for I kappaB degradation. Results. In the absence of LPS, isotonic medium-and HTS-pretreated PEMs pro duced little IL-10 (24.9 +/- 66.0 and 0 pg/mL, respectively); however. stim ulation of PEMs with LPS increased IL-10 (134.9 +/- 72.2 pg/mL). Preconditi oning with HTS significantly augmented LPS-induced IL-10 production, result ing in a 2-fold increase in IL-10 compared with the isotonic solution LPS g roup (270.7 +/- 106.8 pg/mL; P<.01). HTS alone increased IL-10 mRNA levels and markedly augmented levels induced by LPS alone. To determine whether IL -10 accounted for HTS-induced TNF inhibition, cells from IL-10 knockout ani mals were studied. A lack of IL-10 did not reverse the inhibitory effect of HTS on LPS-induced TNF. NF-<kappa>B activation was the same in HTS-and iso tonic solution-Pretreated groups after LPS. Conclusions, HTS augments IL-10 induction by LPS at the gene level. Althoug h TNF is reduced, it is not causally related to increased IL-10 or altered NF-kappaB signaling. HTS might exert its beneficial effects ky independentl y modulating pro- and anti-inflammatory molecules, accounting for the poten t immunomodulation exerted by HTS in vivo.