However, after 72 h this effect disappears and cells return to the tolerant state (Fig. has also been pointed out as the principal cause of the non-specific immunosuppression explained in these individuals. In this statement we demonstrate, using a mouse model, that while the maintenance of tolerance is dependent upon GC, the establishment of tolerance by LPS could be inhibited by dexamethasone (Dex), a synthetic GC. Conversely, we shown that mifepristone (RU486), a known GC receptor antagonist, was capable of inducing a transient and reversible disruption of endotoxin tolerance, also permitting partial restoration of the humoral immune response in LPS tolerant/immunosuppressed mice. These results are motivating for the management of immunosuppression in sepsis and/or non-infectious shock, and deserve further investigation in the future. O111:B4, catalogue no. L2630 purified by phenol extraction, were from Sigma-Aldrich (St Louis, MO, USA). Synthetic glucocorticoid dexamethasone (Dex) (Decadrn Shock) was from Sidus S.A. (C.A. Buenos Aires, Argentina). Cytokines and reagents were prepared in sterile pyrogen-free ONO 4817 saline. Corticosterone level was determined by a commercially available radioimmunoassay (RIA) kit from ICN Biomedicals (Costa Mesa, CA, USA). [3H]-dexamethasone ([3H]-Dex) in ethanol was from New England Nuclear (Boston, MA, USA) and experienced a specific activity of 3500 Ci/mM (125400 GBq/mM). Sheep reddish blood cells (SRBC) were from Alfredo Gutierrez? (C.A.). The following anti-mouse antibodies were used: phycoerythrin (PE)-conjugated rat anti-immunoglobulin (Ig)M monoclonal antibody (mAb) (BD-Pharmingen, San Diego, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated goat anti-IgG polyclonal antibody (Jackson ImmunoResearch Laboratories, Western Grove, PA, USA). Mice BALB/c mice were bred in the animal facility of the Division of Experimental Medicine, Academia Nacional de Medicina, Buenos Aires. Female mice aged 12C16 weeks weighing 20C25 g were used throughout the experiments. They were managed under a 12 h lightCdark cycle at 22 2C and fed with standard diet and water observations. The statistical significance of variations between TNF- samples measured from the L-929 bioassay was identified using the non-parametric Friedman test followed by Wilcoxon’s signed-rank test. ELISA and haemagglutination assays were analysed using the MannCWhitney unpaired test. All statistical checks were interpreted inside a two-tailed fashion and 005 was regarded as significant. Results Dexamethasone induces refractoriness to a lethal dose of LPS A daily i.p. injection of LPS (80 g/kg) in mice for 4 days induces the establishment of tolerance to LPS, a trend characterized by low secretion of TNF- in response to subsequent doses of LPS [19,36] and high levels of corticosterone in serum 3 h after the last LPS injection (tolerants: 10996 ng/ml 232 normal: 1637 ng/ml 58; = 5) [15,37C39]. This increase of GC in tolerant animals seems to be important in the refractoriness to LPS, as naive mice (= 6) survived when they were pretreated with Dex 25 mg/kg i.p. between 0 and 3 h before a lethal dose of LPS (8 mg/kg i.p.). However, when LPS was injected 10 h after Dex, the mortality was 572% (= 7) and after 24 h reached ideals of 923% (= 13). This LPS refractoriness induced by Dex correlated with the low amount of TNF- in mice plasma 90 min after the simultaneous injection of Dex and LPS (DexCLPS = 183 67 pg/ml LPS = 8431 1027 pg/ml) (= 6). Related results were acquired when mouse peritoneal macrophages were treated with Dex (40 g/ml) for 30 min, and later on with LPS (20 g/ml) for 6 h. After this period the supernatants were collected and the biological activity of TNF- was identified using the L-929 assay. The LPS-induced secretion of TNF- was reduced significantly by Dex to 67 2% of control (LPS only) (= 6). Taking into account the schedules utilized for these and experiments we investigated if the effect of Dex could be due to a mere connection or blockade of LPS by Dex. For this purpose, LPS and [3H]-Dex were incubated at 37C for 1 h and approved through a Sephadex G-10. The 1st peak eluted from your column (LPS) was devoid of radioactivity, indicating that [3H]-Dex was not certain to LPS. In addition, the capacity of this ONO 4817 maximum of LPS to induce TNF- secretion from mouse macrophages remained intact (not demonstrated). Dexamethasone inhibits Rabbit Polyclonal to LIPB1 the establishment of LPS tolerance Considering that GC are improved in plasma of tolerant mice and that Dex was responsible for animal safety to a lethal dose of LPS, we speculated that Dex would be also capable of inducing tolerance to LPS. However, daily injections of Dex (25 mg/kg) for 4 days ONO 4817 instead of LPS did not induce a tolerant state indicating that, although important for protection, Dex is not involved in the establishment of the tolerant state (not demonstrated). Conversely, ONO 4817 when we tried to tolerize animals through the simultaneous injection.
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