Biphasic onset of splenic apoptosis following hemorrhagic shock: critical implications for Bax, Bcl-2, and Mcl-1 proteins

Abstract

Introduction: The innate immune response to trauma hemorrhage involves inflammatory mediators, thus promoting cellular dysfunction as well as cell death in diverse tissues. These effects ultimately bear the risk of post-traumatic complications such as organ dysfunction, multiple organ failure, or adult respiratory distress syndrome. In this study, a murine model of resuscitated hemorrhagic shock (HS) was used to determine the apoptosis in spleen as a marker of cellular injury and reduced immune functions.

Methods: Male C57BL-6 mice were subjected to sham operation or resuscitated HS. At t = 0 hours, t = 24 hours, and t = 72 hours, mice were euthanized and the spleens were removed and evaluated for apoptotic changes via DNA fragmentation, caspase activities, and activation of both extrinsic and intrinsic apoptotic pathways. Spleens from untreated mice were used as control samples.

Results: HS was associated with distinct lymphocytopenia as early as t = 0 hours after hemorrhage without regaining baseline levels within the consecutive 72 hours when compared with sham and control groups. A rapid activation of splenic apoptosis in HS mice was observed at t = 0 hours and t = 72 hours after hemorrhage and predominantly confirmed by increased DNA fragmentation, elevated caspase-3/7, caspase-8, and caspase-9 activities, and enhanced expression of intrinsic mitochondrial proteins. Accordingly, mitochondrial pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were inversely expressed within the 72-hour observation period, thereby supporting significant pro-apoptotic changes. Solely at t = 24 hours, expression of the anti-apoptotic Mcl-1 protein shows a significant increase when compared with sham-operated and control animals. Furthermore, expression of extrinsic death receptors were only slightly increased.

Conclusion: Our data suggest that HS induces apoptotic changes in spleen through a biphasic caspase-dependent mechanism and imply a detrimental imbalance of pro- and anti-apoptotic mitochondrial proteins Bax, Bcl-2, and Mcl-1, thereby promoting post-traumatic immunosuppression.

Figure 1

Total lymphocytes after hemorrhagic shock (HS). HS-induced risk for immunosuppression was confirmed by changes of total lymphocytes in murine peripheral blood. Peripheral blood from HS, sham, and control animals was obtained as described in Materials and methods and analyzed by differential hemogram. *P < 0.05 as determined by analysis of variance (with post hoc Bonferroni/Dunn) test and Mann-Whitney test.

Figure 2

Hemorrhagic shock (HS)-induced apoptosis of murine spleen. (a) DNA fragmentation as shown by TUNEL staining. Splenocytes were isolated from HS and sham animals at t = 0 hours, t = 24 hours, and t = 72 hours after hemorrhage as well as from control animals. Results are representative of at least three animals per group and controls. (b) Quantificational analysis of DNA fragmentation. Results are representative of at least three animals per group and controls. (c) Activity of death-receptor-mediated effector caspase-3/7 and caspase-8 as well as mitochondria-related caspase-9 activity within the entire observation period. *P < 0.05 as determined by analysis of variance (with post hoc Bonferroni/Dunn) test and Mann-Whitney test. Co, control; RFU, relative fluorescent units; RLU, relative light units; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling.

Figure 3

Expression of death receptors after hemorrhagic shock (HS). (a) HS-induced expression of extrinsic CD95 and CD120α death receptors in murine spleen. (b) Gating strategy and percentage of splenic CD3 T cells positive for CD95 as well as for CD120α compared with healthy controls and sham-operated animals (representative dot plots and histograms for at least three experiments, green line = isotype control, red line = specific marker). *P < 0.05 as determined by analysis of variance (with post hoc Bonferroni/Dunn) test and Mann-Whitney test. Co, control; FSC, forward scatter; SSC, side scatter.

Figure 4

Protein expression of mitochondrial proteins after hemorrhagic shock (HS). Western blot analysis of splenic Bax (left), Bcl-2 (middle), and Mcl-1 (right) expression compared to the housekeeping gene β-actin (lower part), detected in splenocytes of sham and controls (a) as well as in HS animals (b). Results are representative of at least three animals per group and controls. *P < 0.05 as determined by analysis of variance (with post hoc Bonferroni/Dunn) test and Mann-Whitney test. Co, control.