Cutting edge: critical role for C5aRs in the development of septic lymphopenia in mice

Abstract

In the early stages of sepsis, lymphocytes undergo apoptosis, resulting in lymphopenia and immunosuppression. The trigger for septic lymphopenia is unknown. Using the polymicrobial model of murine sepsis, we investigated the role of C5a receptors in septic lymphopenia. In wild-type mice, cecal ligation and puncture resulted in splenocyte apoptosis and significant lymphopenia after 3 d, which was not observed in C5aR1(-/-) or C5aR2(-/-) mice. Our data show that mouse neutrophils exposed to recombinant mouse C5a cause release of histones in a dose-dependent and time-dependent manner. Histone levels in spleen were significantly elevated following cecal ligation and puncture but were reduced by the absence of C5aR1. Histones induced significant lymphocyte apoptosis in vitro. Ab-mediated neutralization of histones prevented the development of lymphopenia in sepsis. Together, these results describe a new pathway of septic lymphopenia involving complement and extracellular histones. Targeting of this pathway may have therapeutic benefit for patients with sepsis or other serious illness.

Copyright © 2015 by The American Association of Immunologists, Inc.

Figures

Figure 1

CLP-induced lymphocyte lymphopenia is C5a receptor-dependent. A) Blood leukocyte numbers 3 days after CLP in Wt mice or Wt, C5aR1−/−, and C5aR2−/− mice (n=5–10 mice per group). B) Splenic leukocyte numbers 3 days after CLP in Wt mice or Wt, C5aR1−/−, and C5aR2−/− mice (n=5 mice per group). C) Representative TUNEL labeling of spleen sections from Wt or C5aR1−/− mice 20 hrs after CLP (or sham Wt). The scale bar is for all images. D) Aggregate data from TUNEL labeling expressed as the number of TUNEL+ cells per microscopic field (n=5 mice per group).

Figure 2

Role for extracellular histones in septic lymphopenia. A) Levels of histones in whole spleen homogenates at time points following CLP (n=3–5 mice per group). *p−/− mice (or sham Wt, n=5 mice per group). C) Dose-response (4 hours) and D) time course data demonstrating in vitro release of histones from elicited peritoneal neutrophils exposed to C5a. E) Apoptosis of splenocytes exposed to purified calf thymus histones in vitro for 90 min. Apoptosis was determined by Annexin V binding and use of the vital dye 7-AAD. Representative flow cytometry plots are shown. F) Aggregate data from 3 experiments using splenocytes, or enriched populations of peritoneal macrophages, or peritoneal neutrophils. G) Blood lymphocyte numbers 3 days after CLP or sham surgery. CLP mice received anti-histone (H2A/H4) neutralizing antibody (400 µg i.v.) or a control antibody at the time of CLP (n=6 mice per group).