(2011) The pathogenesis of sepsis

(2011) The pathogenesis of sepsis. Annu. as elevated IL-6 levels were associated with decreased numbers of memory-phenotype CD8 T cells in septic mice, and preservation of this subset after administration of anti-LFA-1 mAb conferred improved survival at 7 d. Taken together, these data identify potentially modifiable responses of memory-phenotype CD8 T cells in early sepsis and may be particularly important in the application of immunomodulatory therapies in sepsis. 10 min), and supernatant (serum) was apportioned into 100 l aliquots and stored at ?20C until use. Serum cytokines were evaluated using the Bio-Plex suspension array system and Bio-Plex Mouse Cytokine 23-Plex Panel, according to the manufacturers instructions (both Bio-Rad Laboratories, Marnes-La-Coquette, France). Cytokine assays included antibodies for the following: IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, G-CSF, GM-CSF, IFN-, KC, MCP-1, MIP-1, MIP-1, RANTES, and TNF-. Results were analyzed Rabbit Polyclonal to ATG16L2 using Bio-Plex Manager 3.0 software with 5 parameter logistic (5PL) curve fitting for determination of serum concentrations (pg/ml) of individual cytokines per sample. Statistical analysis Data were analyzed using the statistical software Prism V; all data are reported as means sem. For comparison between sham and CLP groups at individual time points, Students test was used after confirmation of Gaussian distribution. Intragroup comparison of data collected across multiple time points was analyzed using regular two-way ANOVA and Sidaks test to correct for multiple comparisons. For comparison of cytokine concentrations across 3 groups, one-way ANOVA and Tukeys post-test MKT 077 were used. Survival studies were analyzed by 2 analysis. For all those data, a confidence interval of 95% was used to determine significance ( 0.05). RESULTS The first 48 h after sepsis is usually characterized by attrition of memory-phenotype (CD44HICD11aHI) CD8 T cells To evaluate the effect of sepsis on memory-phenotype CD8 T cells, splenocytes from WT B6 mice that had undergone either CLP or sham operations were analyzed by flow cytometry. CD3+ CD8a+ T cells were then subdivided into memory-phenotype (CD44HICD11aHI) and na?ve (CD44LOCD11aLO) subsets (see Fig. 1A for gating strategy). At all time points evaluated, the frequency of CD44HICD11aHI cells in sham-operated mice ranged from 14 to 20%, comparable to that of unmanipulated mice and consistent with reports from other studies of mice raised in pathogen-free conditions [15, 20]. However, septic mice exhibited significantly lower frequencies of CD44HICD11aHI cells, with the most pronounced difference occurring at 24 h (Fig. 1B). To distinguish between the absolute reduction of CD44HICD11aHI cells rather than a proportional increase in the CD44LOCD11aLO (na?ve) populace, absolute cell counts per spleen were calculated. Septic mice had significantly fewer CD44HICD11aHI cells as early as 6 h compared with sham; this reduction was most pronounced at 24 h and was sustained until 48 h (Fig. 1C). By 120 h, the number of CD44HICD11aHI cells was equal between groups, suggesting repletion of this subset. In contrast, numbers of CD44LOCD11aLO cells remained comparable between sham and sepsis at all time points and in fact, underwent growth at 48 and 72 h in sepsis (Fig. 1D). To confirm that our gating strategy was not artificially representing our results by failing to reflect potential increases in the expression of CD44 and CD11a, known to accompany antigen-specific activation [21], CD8 T cell expression of CD44 and CD11a was evaluated by MFI. CD8 T cells from septic mice exhibited significantly lower expression of CD44 at 6, 24, 48, and 72 h and of CD11a at 24, 48, and 72 h (data not shown), corroborating the results of our quantitative analysis. Open in a separate window Physique 1. Sepsis results in attrition of memory-phenotype (CD44HICD11aHI) CD8 T cells from 6 to 72 h after onset.(A) Gating strategy for identification MKT 077 of CD44HICD11aHI and CD44LOCD11aLO CD8 T cells. (B) Percentages of memory-phenotype (CD44HICD11aHI) CD8 T cells are significantly reduced at 24 h after sepsis relative to sham (14.29% in sham vs. 9.91% in CLP; * 0.05), whereas normal (unmanipulated) and sham mice are similar. (C) Septic mice demonstrate decreased numbers of CD44HICD11aHI CD8 T cells at 6, 24, and 48 h (* 0.05 MKT 077 for all those) compared with sham mice. The maximum cell loss in the spleen occurred at 24 h, at which point, the population of memory-phenotype (TM) cells was reduced by 41%. Thereafter, numbers of CD44HICD11aHI CD8 T cells began to recover, with reductions of 38%.