Activation of Neutrophil Granulocytes by Platelet-Activating Factor Is Impaired During Experimental Sepsis

Abstract

Platelet-activating factor (PAF) is an important mediator of the systemic inflammatory response. In the case of sepsis, proper activation and function of neutrophils as the first line of cellular defense are based on a well-balanced physiological response. However, little is known about the role of PAF in cellular changes of neutrophils during sepsis. Therefore, this study investigates the reaction patterns of neutrophils induced by PAF with a focus on membrane potential (MP), intracellular pH, and cellular swelling under physiological and pathophysiological conditions and hypothesizes that the PAF-mediated response of granulocytes is altered during sepsis. The cellular response of granulocytes including MP, intracellular pH, cellular swelling, and other activation markers were analyzed by multiparametric flow cytometry. In addition, the chemotactic activity and the formation of platelet–neutrophil complexes after exposure to PAF were investigated. The changes of the (electro-)physiological response features were translationally verified in a human ex vivo whole blood model of endotoxemia as well as during polymicrobial porcine sepsis. In neutrophils from healthy human donors, PAF elicited a rapid depolarization, an intracellular alkalization, and an increase in cell size in a time- and dose-dependent manner. Mechanistically, the alkalization was dependent on sodium-proton exchanger 1 (NHE1) activity, while the change in cellular shape was sodium flux- but only partially NHE1-dependent. In a pathophysiological altered environment, the PAF-induced response of neutrophils was modulated. Acidifying the extracellular pH in vitro enhanced PAF-mediated depolarization, whereas the increases in cell size and intracellular pH were largely unaffected. Ex vivo exposure of human whole blood to lipopolysaccharide diminished the PAF-induced intracellular alkalization and the change in neutrophil size. During experimental porcine sepsis, depolarization of the MP was significantly impaired. Additionally, there was a trend for increased cellular swelling, whereas intracellular alkalization remained stable. Overall, an impaired (electro-)physiological response of neutrophils to PAF stimulation represents a cellular hallmark of those cells challenged during systemic inflammation. Furthermore, this altered response may be indicative of and causative for the development of neutrophil dysfunction during sepsis.

Simultaneous Measurement of Changes in Neutrophil Granulocyte Membrane Potential, Intracellular pH, and Cell Size by Multiparametric Flow Cytometry

“…Additionally, C5a-induced changes in neutrophil pH i and FSC after pre-exposure to LPS and other proinflammatory mediators were reduced, which further exemplifies an alteration in the neutrophil response pattern that can be detected by this method in agreement with results from previous studies [17,18]. This is of special interest and potential translational value, because these alterations have been described earlier in the context of systemic inflammation, as outlined in the introduction [14,17,18,23]. It is tempting to speculate that further improvements and/or applications of the method described in this manuscript may have diagnostic and/or prognostic value in systemic inflammation, including possibly in severe injuries, hemorrhage, or sepsis.…”

“…Neutrophil activation by the above-mentioned chemoattractants typically induces a certain response pattern, including depolarization of the membrane potential (MP), alkalization of the intracellular pH (pH i ), and a change in cellular shape as reported by our group [4,[14][15][16][17][18] and many others [19][20][21][22][23][24]. These distinct alterations are closely associated with crucial neutrophil functions.…”

“…It is of note that patients with defects in NOX activity respond to stimulation with considerably reduced depolarization [ 28 , 30 ]. Neutrophil alkalization is largely mediated by the activity of the sodium-proton-exchanger 1 and of the voltage-sensitive proton channel 1 [ 4 , 17 , 18 , 31 , 32 ]. These variations in pH i influence neutrophil activity.…”

“…Even though there is no possibility to measure the pH i directly, the underlying current through proton channels could be assessed. Similar to the MP, to measure changes in pH i , fluorescent dyes are used to circumvent this issue [ 15 , 17 , 18 , 22 , 40 , 41 , 42 ]. As a surrogate for cell volume, membrane capacity could be measured by patch-clamp techniques.…”

Deep Learning Tissue Analysis Diagnoses and Predicts Treatment Response in Eosinophilic Esophagitis

“…To our knowledge, this is the first study that has investigated Eo size in EoE. In other inflammatory cell types such as neutrophils, larger cell size is related to cellular activation during an inflammatory response; 22 however, we found Eo size was poorly correlated with other Eo metrics such as PEC or average Eo count (r=0.41). There were no significant associations with Eo size at remission and treatment groups or symptoms.…”