Hemorrhagic shock induces a sequential dysfunction of electrophysiological response capacity in neutrophil granulocytes

Objectives Hemorrhagic shock (HS) triggers systemic inflammation, thus challenging neutrophil granulocytes (PMN) as first line of defense resulting in a loss of proper cellular function. However, little is known about the dysfunction of PMN during HS and possible markers hereof. Therefore, the present work first evaluates the electrophysiological response of neutrophils being stimulated with chemoattractants and second hypothesizes that these distinct response patterns are impaired during HS.

Methods After ethical approval by the respective authorities (#1341), porcine PMN were isolated before, during HS (target MAP 40±5 mmHg for 3hrs) and 24hrs after resuscitation. Changes in membrane potential (MP) and intracellular pH (pHi) of PMN were determined by flow cytometry after stimulation with platelet-activating factor (PAF, 500 ng/ml), complement factor 5a (C5a, 100 ng/ml), Leukotriene B4 (LTB4, 100 nM), and Interleukin 8 (Il8, 50 ng/ml), respectively. Results are presented in comparison to unstimulated cells as mean±SD with at least five independent experiments.

Results and Conclusion PMN from pigs before hemorrhage responded in a similar manner regarding MP and pHi in comparison to PMN from healthy human volunteers (data not shown). Comparing various chemokines as stimuli for depolarization and alkalization, PAF invoked the largest response in porcine PMN prior to and during shock (PAF > C5a > LTB4 > Interleukin 8). Prior to shock, PAF induced a depolarization of 13,4 mV ± 6,2, p < 0.05. Rapidly, after two hours, depolarization of PMN was significantly reduced (6,0 mV ± 3,6, p < 0.05 vs. pre shock). Likewise, PMN responded with an intracellular alkalization before shock (0,13 ± 0,07, p < 0.05), that remained roughly intact for two hours, but deteriorated after three hours (0,08 ± 0,05, p =0,10 vs. pre shock). [Fig. 1] summarizes the electrophysiological response capacity of PMN stimulated with PAF normalized to before shock = 200 %.

Fig.1 PAF-induced depolarization and intracelluar alkalization normalized to the response of PMN, normalized to before shock = 100%

Despite retransfusion and intensive care therapy, reduction of electrophysiological response capacity of PMN was still evident after 24 hours.

Rapid impairment of first membrane potential and second intracellular alkalization might represent early hallmarks of PMN dysfunction. Cellular response capacity could serve as a potential diagnostic or monitoring marker of immunological (dys-)function during systemic inflammation.

Stichwörter hemorrhagic shock, neutrophil granulocytes, systemic inflammation, platelet-activating factor

Publication History

Article published online:
15 October 2020