Is Fas Ligand or Endotoxin Responsible for Mucosal Lymphocyte Apoptosis in Sepsis?

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Abstract

Background

Apoptosis (Ao ) is a normal constitutive process that seems to have pathological effects in diseases of immune deficiency and autoimmune disorders, as well as in certain lymphoid tissues during sepsis. Little is known about this process in mucosal lymphoid tissue, such as intestinal intraepithelial lymphocytes (IELs).

Objectives

To determine whether sepsis induces increased Ao in small intestinal IEL, whether this was associated with functional changes in cytokine gene expression in the IEL, and which mediators control this process and their impact on the survival of the mouse with sepsis.

Design

Male C3H/HeN (endotoxin-sensitive), C3H/HeJ (endotoxin-tolerant), and C3H/HeJ-FasLgld (endotoxin-tolerant/Fas ligand [FasL]-deficient) mice were subjected to sepsis (cecal ligation and puncture [CLP]) and IELs were harvested at 4 (early) or 24 hours (late sepsis). Alterations in the cell phenotype and Ao (TUNEL [terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling] assay) were determined by 3-color flow cytometry. Cytokine gene expression was assessed by multiprobe RNase protection assay.

Results

At 4 hours after CLP, only the frequency of IEL which was CD8+ decreased markedly. By 24 hours after CLP, the number of CD8+ and CD4 (+) cells decreased while the proportion of double-negative cells showed a marked increase when compared with sham-controls. The percentage of Ao positive in CD8+ and CD4+ double-positive and double-negative cells increased markedly 24 hours after CLP concomitant with a significant (P<.05 vs sham-controls, Mann-Whitney U test) increase in expression of the IL-2, IL-10, and IL-15 gene. These data collectively suggest that sepsis causes lymphocyte activation-induced Ao that may be mediated by FasL. Additional studies were done to determine if the increased Ao was due to either endotoxin or FasL. The results of studies with endotoxin-tolerant C3H/HeJ or FasL-deficient C3H/HeJ-FasLgld mice showed an increase in Ao in CD4+ and CD8 (+) cells from septic C3H/HeJ but not C3H/HeJ-FasLgld mice. With regard to septic mortality, our results indicated that there was a marked reduction in mortality in C3H/HeJ-FasLgld vs C3H/HeJ mice.

Conclusions

We conclude that the phenotypic changes associated with increased Ao may be a reflection of localized immune cell activation due to a FasL-mediated process and not endotoxin. Thus, FasL directly and/or indirectly contributes to higher septic mortality.

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