Molecular mechanisms underlying delayed apoptosis in neutrophils from multiple trauma patients with and without sepsis

Abstract

Delayed neutrophil apoptosis and overshooting neutrophil activity contribute to organ dysfunction and subsequent organ failure in sepsis. Here, we investigated apoptotic signaling pathways that are involved in the inhibition of spontaneous apoptosis in neutrophils isolated from major trauma patients with uneventful outcome as well as in those with sepsis development. DNA fragmentation in peripheral blood neutrophils showed an inverse correlation with the organ dysfunction at d 10 after trauma in all patients, supporting the important role of neutrophil apoptosis regulation for patient's outcome. The expression of the antiapoptotic Bcl-2 protein members A1 and Mcl-1 were found to be diminished in the septic patients at d 5 and d 10 after trauma. This decrease was also linked to an impaired intrinsic apoptosis resistance, which has been previously shown to occur in neutrophils during systemic inflammation. In patients with sepsis development, delayed neutrophil apoptosis was found to be associated with a disturbed extrinsic pathway, as demonstrated by reduced caspase-8 activity and Bid truncation. Notably, the expression of Dad1 protein, which is involved in protein N-glycosylation, was significantly increased in septic patients at d 10 after trauma. Taken together, our data demonstrate that neutrophil apoptosis is regulated by both the intrinsic and extrinsic pathway, depending on patient's outcome. These findings might provide a molecular basis for new strategies targeting cell death pathways in apoptosis-resistant neutrophils during systemic inflammation.

Figure 1

Inhibition of neutrophil spontaneous apoptosis by serum factors. (A) Fold change of DNA fragmentation in neutrophils isolated from patients with (n = 10) or without (n = 10) sepsis development at d 5 and d 10 after major trauma relative to d 1. (B) Correlation of fold change of fragmented DNA with Sequential Organ Failure Assessment (SOFA) score and Multiple Organ Dysfunction Score (MODS) at d 5 and d 10 after major trauma. Spearman rho (ρ) correlation coefficient and P values are indicated. (C) Neutrophils isolated from one healthy control (1 × 10⁶/mL) were incubated with 1% serum from healthy volunteers (control, n = 9) and with patient sera collected at d 1, d 5 and d 10 after major trauma. After 18 h culture, apoptotic neutrophils were quantified by propidium iodide staining and flow cytometry. The white bars indicate apoptosis in cells incubated with sera from patients with uneventful outcome (n = 12); the gray bars indicate apoptosis after treatment with sera from patients with sepsis development (n = 12). *P < 0.05; **P < 0.01; ***P < 0.001 versus control. A, C: □, Nonsepis; , sepsis; ■, control. B: SOFA d 5: ρ = −0.23, P = 0.316; SOFA d 10: ρ = −0.417, P = 0.059; MODS d 5: ρ = −0.24, P = 0.29; MODS d 10: ρ = −0.435, P = 0.04.

Figure 2

mRNA expression of Bcl-2 family members and Dad1. The expression of Mcl-1, A1, DAD1 and Bax mRNA was quantified by real-time PCR in neutrophils isolated from healthy volunteers (n = 10), as well as from patients with (n = 9–10) or without (n = 10–12) sepsis development after major trauma. Gene expression was normalized to that of the 18S RNA gene. □, Nonsepis; , sepsis.

Figure 3

Protein expression of Bcl-2 family members and Dad1. The expression of Mcl-1, A1, Dad1 and Bax protein was determined by Western blot in neutrophils isolated from healthy volunteers (n = 5–13), as well as from patients with (n = 8–9) or without (n = 8–11) sepsis development after major trauma. Blots were analyzed by densitometry and normalized to GAPDH. In each case, representative blots of one healthy control, one patient with and one patient without sepsis development are depicted. *P < 0.05; **P < 0.01. □, Nonsepis; , sepsis; ■, control.

Figure 4

Reduced Mcl-1 protein levels are associated with impaired intrinsic apoptosis resistance during sepsis. (A) Freshly isolated neutrophils from patients (n = 8) at d 1 after trauma were nucleofected with control siRNA (Ctrl) or with mcl-1 siRNA. After 24 h culture, cells were treated with 0.2 μmol/L staurosporine (Sto) or left untreated (Ctrl). Mitochondrial membrane depolarization was quantified by JC-1 staining after 4 h. The percentage of cells with high levels of green (FL1) fluorescence was determined. Fold change versus sample without Sto treatment is depicted (left). After 18 h of culture, apoptotic neutrophils were quantified by propidium iodide staining and flow cytometry (right). Mcl-1 expression was evaluated by Western blot analysis. (B) Freshly isolated neutrophils from one healthy volunteer were preincubated with 1% serum from patients with (n = 12) or without (n = 12) sepsis development after major trauma for 1 h. Then, cells were further incubated with 0.2 μmol/L Sto for 4 h or left untreated. Mitochondrial membrane depolarization was quantified by JC-1 staining (left). Representative dot plots for cells treated with serum isolated at d 10 from one septic or nonseptic patient, respectively, are shown (right). *P < 0.05; **P < 0.01. B: □, Nonsepis; , sepsis.

Figure 5

Reduced caspase-8 activity and diminished Bid truncation in patients with sepsis development. (A) Fold change of caspase-8 activity in neutrophils isolated at d 5 and d 10 from patients with (n = 10) or without sepsis (n = 12) development after major trauma versus d 1. (B) Bid and truncated Bid (tBid) protein expression in neutrophils isolated at d 1, d 5 and d 10 from patients with sepsis development (n = 6) or uneventful recovery (n = 6) was analyzed by Western blot. The ratio of Bid/tBid protein expression is depicted for each patient group. *P < 0.05; **P < 0.01. □, Nonsepis; , sepsis.