Abstract
Lymphocyte apoptosis is one reason for immunoparalysis seen in sepsis, although the triggers are unknown. We hypothesized that molecules in plasma, which are up-regulated during sepsis, may be responsible for this. In this study, peripheral lymphocyte apoptosis caused by extracellular histones was confirmed both in mouse and human primary lymphocytes, in which histones induced lymphocyte apoptosis dose-dependently and time-dependently. To identify which intracellular signal pathways were activated, phosphorylation of various mitogen-activated protein kinases (MAPKs) were evaluated during this process, and p38 inhibitor (SB203580) was used to confirm the role of p38 in lymphocyte apoptosis induced by histones. To investigate the mitochondrial injury during these processes, we analyzed Bcl2 degradation and Rhodamine 123 to assess mitochondrial-membrane stability, via cyclosporin A as an inhibitor for mitochondrial permeability transition (MPT). Then, caspase 3 activation was also checked by western-blotting. We found that p38 phosphorylation, mitochondrial injury and caspase 3 activation occurred dose-dependently in histones-mediated lymphocyte apoptosis. We also observed that p38 inhibitor SB203580 decreased lymphocyte apoptotic ratio by 49% (P<0.05), and inhibition of MPT protected lymphocytes from apoptosis. Furthermore, to investigate whether histones are responsible for lymphocyte apoptosis, various concentrations of histone H4 neutralization antibodies were co-cultured with human primary lymphocytes and plasma from cecal ligation and puncture (CLP) mice or sham mice. The results showed that H4 neutralization antibody dose-dependently blocked lymphocyte apoptosis caused by septic plasma in vitro. These data demonstrate for the first time that extracellular histones, especially H4, play a vital role in lymphocyte apoptosis during sepsis which is dependent on p38 phosphorylation and mitochondrial permeability transition. Neutralizing H4 can inhibit lymphocyte apoptosis, indicating that it could be a potential target in clinical interventions for sepsis associated immunoparalysis.
Highlights
Sepsis causes long-term immunosuppression or immunoparalysis, leading to multiple organ failure (MOF) and possibly death [1]
It has been shown that the peripheral lymphocyte apoptotic ratio increases in the cecal ligation and puncture (CLP) mouse model, which was confirmed by the current study (Fig. 1)
Tumor necrosis factor (TNF)-a couldn’t be the reason for the peripheral lymphocyte apoptosis that occurs during sepsis
Summary
Sepsis causes long-term immunosuppression or immunoparalysis, leading to multiple organ failure (MOF) and possibly death [1]. Sepsis has been recognized as one of the top causes of mortality worldwide, its incidence is continuing to rise dramatically, with approximately 1,400 deaths/day worldwide [2]. Severe sepsis or septic shock is one of the leading causes of admissions to intensive care units. There is no specific treatment currently available due to limited understanding of the underlying mechanism behind sepsis [3]. Bundle therapy has been used with barely satisfactory effect, and the costs are high. Further research into the mechanism of sepsis is urgently needed
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