Thermal injury-plus-sepsis contributes to a substantial deletion of intestinal mesenteric lymph node CD4 T cell via apoptosis

Abstract

Thermal injury (TI) with septic complications continues to be a serious clinical problem. One of the main concerns in such patients is immunosuppression related to functional derangements in intestinal CD4+ T lymphocytes. Extensive previous studies in thermal injury/septic patients and animal models of thermal injury/sepsis have shown decreased responsiveness of intestinal CD4+ T cells to antigen/mitogen. This hyporesponsiveness could significantly contribute to increase injured host susceptibility to pathogens including those translocating from host's gut lumen. Our previous studies indicated that while thermal injury or sepsis alone lead to suppressed proliferation and IL-2 production of intestinal CD4+ T cells, this study showed a substantial deletion via apoptosis of the Mesenteric Lymph Nodes (MLN) CD4+ T cells. Hence, thermal injury-plus-sepsis contributes not only to suppressed CD4+ T proliferation/IL-2 production but also to a substantial modulation of CD4+ T cell survivability. These findings allow us to conclude that while thermal injury alone can produce attenuated cell mediated responses without an overt change in CD4+ T cell survival, thermal injury with septic complications causes CD4+ T cell death and an irreversible loss of cell-mediated responses. The latter happening could be responsible for high morbidity and mortality in the injured host afflicted with thermal injury plus a critical infection.

Keywords: Burn injury; activation-induced cell death; adaptive immune response; hoescht staining; immune suppression; infection.

Figure 1

Representative cryosections of in situ apoptosis (TUNEL) of mesenteric lymph nodes (MLN) obtained from sham, TI, CLP and TI+CLP animals (Fig. 1A). At least 3 slides with 3 sections per animal for each injury group of animals were examined and data analyzed. Fig. 1B represents a histogram of collective data obtained from 3 experiments representing mean with SE (n=6). Numbers of apoptotic cells were counted per high field of microscope. * p <0.05 between sham vs. TI, ** p<0.05 TI vs. TI+CLP.

Figure 1

Representative cryosections of in situ apoptosis (TUNEL) of mesenteric lymph nodes (MLN) obtained from sham, TI, CLP and TI+CLP animals (Fig. 1A). At least 3 slides with 3 sections per animal for each injury group of animals were examined and data analyzed. Fig. 1B represents a histogram of collective data obtained from 3 experiments representing mean with SE (n=6). Numbers of apoptotic cells were counted per high field of microscope. * p <0.05 between sham vs. TI, ** p<0.05 TI vs. TI+CLP.

Figure 2

Flow cytometric analysis of CD45RC marker surface expression on MLN CD4⁺ T cells. Representative data from four experimental groups i.e. sham, TI, CLP and TI+CLP are shown in the figure (Fig. 2A). CD45RC-positive cells (naïve) and CD45RC-negative (memory/activated) are given in (Fig. 2A). Fig. 2B is a histogram of collective data obtained from 3 experiments showing mean with SE (n=6). * p <0.05 between sham vs. TI, ** p<0.05 TI vs. TI+CLP.

Figure 2

Flow cytometric analysis of CD45RC marker surface expression on MLN CD4⁺ T cells. Representative data from four experimental groups i.e. sham, TI, CLP and TI+CLP are shown in the figure (Fig. 2A). CD45RC-positive cells (naïve) and CD45RC-negative (memory/activated) are given in (Fig. 2A). Fig. 2B is a histogram of collective data obtained from 3 experiments showing mean with SE (n=6). * p <0.05 between sham vs. TI, ** p<0.05 TI vs. TI+CLP.

Figure 3

Apoptosis of MLN CD4⁺ T cells as determined by flow cytometric analysis of Annexin -positive cells. In this series of experiments apoptosis of CD4⁺ T cells was induced by incubation with anti-Fas Antibody (0.5 μg/ml) for 3h. Four experimental groups i.e. sham, TI, CLP and TI+CLP are shown in the figure 3A. Histogram data represents mean with SE (n=6) from 3 different experiments (Fig. 3B), # p >0.05 between sham vs. TI, ** p<0.05 TI vs. TI+CLP.

Figure 3

Apoptosis of MLN CD4⁺ T cells as determined by flow cytometric analysis of Annexin -positive cells. In this series of experiments apoptosis of CD4⁺ T cells was induced by incubation with anti-Fas Antibody (0.5 μg/ml) for 3h. Four experimental groups i.e. sham, TI, CLP and TI+CLP are shown in the figure 3A. Histogram data represents mean with SE (n=6) from 3 different experiments (Fig. 3B), # p >0.05 between sham vs. TI, ** p<0.05 TI vs. TI+CLP.

Figure 4

Representative figures showing apoptosis (Hoescht staining) of MLN CD4⁺ T cells obtained by Microbead separation (MACS) from four experimental groups i.e. sham, TI, CLP and TI+CLP (Figure 2A). The yellow colored arrows indicate apoptotic CD4⁺ T cells with characteristic features of apoptosis (condensation of nuclei and membrane blebbing etc.). Fig. 2B is a histogram of collective data from 3 experiments showing mean with SE (n=6). Number of apoptotic cells as determined by Hoescht stain was counted per high power field (HPF) of microscope. * p <0.05 between sham vs. TI, ** p<0.05 TI vs. TI+CLP.

Figure 4

Representative figures showing apoptosis (Hoescht staining) of MLN CD4⁺ T cells obtained by Microbead separation (MACS) from four experimental groups i.e. sham, TI, CLP and TI+CLP (Figure 2A). The yellow colored arrows indicate apoptotic CD4⁺ T cells with characteristic features of apoptosis (condensation of nuclei and membrane blebbing etc.). Fig. 2B is a histogram of collective data from 3 experiments showing mean with SE (n=6). Number of apoptotic cells as determined by Hoescht stain was counted per high power field (HPF) of microscope. * p <0.05 between sham vs. TI, ** p<0.05 TI vs. TI+CLP.