Modulation by dantrolene of endotoxin-induced interleukin-10, tumour necrosis factor-alpha and nitric oxide production in vivo and in vitro.

1. Intracellular calcium has been suggested to be an important mediator of the cellular response in endotoxaemia and shock. Dantrolene is an agent that interferes with intracellular calcium fluxes resulting in a decreased availability of calcium in the cytoplasm. Here we have investigated the effect of dantrolene on lipopolysaccharide (LPS)-induced production of interleukin-10 (IL-10), tumour necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) in mice and in cultured RAW 264.7 macrophages in vitro. 2. In BALB/c mice, LPS-induced plasma IL-10 levels were significantly enhanced by pretreatment with dantrolene (20 mg kg(-1), i.p.) (P < 0.005 at the 90 min time-point). On the other hand, dantrolene pretreatment suppressed circulating TNF-alpha and nitrite/nitrate (breakdown products of NO) concentrations. However, dantrolene had no effect on LPS-induced plasma interleukin-6 (IL-6) levels (67.22+/-5.51 ng ml(-1) in vehicle-pretreated mice and 62.22+/-3.66 ng ml(-1) in dantrolene-pretreated mice, n = 9). 3. Dantrolene inhibited TNF-alpha and NO production in C57BL/6 IL-10+/+ mice, as well as in their IL-10 deficient counterparts (C57BL/6 IL-10(0/0)). 4. In RAW 264.7 macrophages, dantrolene (10-300 microM) reduced IL-10, TNF-alpha, and nitrite (breakdown product of NO) production elicited by LPS (10 microg ml(-1)). Dantrolene (300 microM) did not affect the LPS-induced nuclear translocation of transcription factor nuclear factor kappaB in these cells. 5. Although LPS failed to alter the intracellullar concentration of calcium in single macrophages loaded with Fura-2, dantrolene caused a significant decrease of the basal calcium level as determined 30 min after dantrolene treatment (P < 0.005). ATP (1 mM) caused a rapid rise in intracellular calcium levels in both dantrolene-pretreated and vehicle-pretreated cells. 6. These results indicate that unlike the secretion of TNF-alpha and NO, IL-10 production is differentially regulated in vitro and in vivo. The decrease of plasma levels of the pro-inflammatory mediators TNF-alpha and NO, and increase in circulating IL-10 concentrations by dantrolene suggest that this drug might offer a new therapeutic approach in inflammatory diseases and septic shock.