Multimodal monitoring of neutrophil activity during cardiac surgery

Abstract

Cardiac surgery and the associated ischemia-reperfusion injury trigger an inflammatory response, which, in turn, can contribute to organ damage, prolonged hospitalization, and mortality. Therefore, the present study performed comprehensive monitoring of neutrophil-related inflammation in patients who underwent aortic valve surgery, including extracorporeal circulation. Neutrophil-related inflammation, as well as alterations in cellular physiology, phenotype, and function, were analyzed by flow cytometry, ELISA, and microscopy. Neutrophil activation occurred intraoperatively and preceded the upregulation of conventional inflammatory markers such as C-reactive protein and interleukin-6. Perioperatively, neutrophils maintained a stable response to platelet-activating factor (PAF) with regard to CD11b and CD66b expression but showed a decreased response in CD10. Postoperatively, neutrophils exhibited marked alterations in PAF-induced depolarization, while reactive oxygen species generation and phagocytic activity remained largely stable. Surprisingly, platelet-neutrophil complex formation was severely impaired intraoperatively but returned to normal levels postoperatively. Further studies are needed to elucidate the implications of these intraoperative and postoperative changes in neutrophil and platelet activity with respect to a potential immune dysfunction that temporarily increases susceptibility to infectious or hemostatic complications.

Keywords: cardiac surgery; inflammation; ischemia-reperfusion injury; neutrophil granulocytes; platelet-activating factor; platelet-neutrophil complexes; platelets; thrombocytes.

Figure 1

Cohort description and general parameters. (A) Summary of the experimental approach analyzing patients with cardiac surgery on admission (A), 45 min after the initiation of extracorporeal circulation in the operation room (OR), and 24, 48, and 120 h after the end of surgery in comparison to age- and sex-matched healthy volunteers (HVs). (B) General characteristics of the HVs and patients. Laboratory data for (C) neutrophil and thrombocyte count, (D) C-reactive protein (CRP) and creatine phosphokinase-MB (CK-MB), (E) matrix metalloproteinase 9 (MMP9) and myeloperoxidase (MPO), and (F) interleukin-6 (IL6) and procalcitonin (PCT). Median with interquartile range, n = 14. HVs vs. A: unpaired t test; A vs. OR, 24, 48, and 120 h post OR: ordinary one-way ANOVA in conjunction with a Dunnett’s multiple comparison test with *, **, ***, and **** indicating a p value of < 0.05, < 0.01, < 0.001, and <0.0001, respectively.

Figure 2

Analysis of neutrophil phenotype for (A) CD10, (B) CD11b, and (C) CD66b from patients with cardiac surgery on admission (A), 45 min after the initiation of extracorporeal circulation in the operation room (OR), and 24, 48, and 120 h after the end of surgery compared to healthy volunteers (HVs). Blood samples were stimulated with buffer control (control, left) or 1 µM platelet-activating factor (PAF, right). Median with interquartile range, n = 14. HVs vs. A: Unpaired t test; A vs. OR, 24, 48, and 120 h post OR: ordinary one-way ANOVA with *, **, ***, and **** indicating a p value of < 0.05, < 0.01, < 0.001, and <0.0001, respectively. MFI = median fluorescence intensity.

Figure 3

Analysis of neutrophil properties in patients with cardiac surgery on admission (A), 45 min after the initiation of extracorporeal circulation in the operation room (OR), and 24, 48, and 120 h after the end of surgery compared to healthy volunteers (HVs). (A) PAF-induced depolarization, (B) PAF-induced generation of reactive oxygen species (ROS), (C) phagocytic activity in samples exposed to buffer control (ctrl), and (D) phagocytic activity in blood exposed to 1 µM platelet-activating factor (PAF). Median with interquartile range, n = 14. HV vs. A: unpaired t test; A vs. OR, 24, 48, and 120 h post OR: ordinary one-way ANOVA with ** indicating a p < 0.01.

Figure 4

Analysis of platelet-neutrophil complex (PNC) formation and platelet activation in blood from patients with cardiac surgery on admission (A), 45 min after the initiation of extracorporeal circulation in the operation room (OR), and 24, 48, and 120 h after the end of surgery compared to healthy volunteers (HV). (A) Representative distribution of CD61 as a platelet-lineage marker on neutrophils from a HV in blood exposed to buffer control (ctrl) or 1 µM platelet-activating factor (PAF). (B) Representative PNC as detected by light microscopy. Flow cytometric analysis of PNC formation in samples stimulated with (C) Ctrl or (D) PAF. Confirmation of PNC formation by light microscopy in samples stimulated with (E) Ctrl or (F) PAF. Analysis of platelet activation indicated by CD62P expression in platelets exposed to (G) buffer control or (H) PAF. Median with interquartile range, n = 14. HV vs. A: unpaired t test; A vs. OR, 24, 48, and 120 hours post OR: ordinary one-way ANOVA with *, **, or **** indicating a p value of < 0.05, <0.01, and <0.0001, respectively. AU, arbitrary units.

Figure 5

Analysis of CD11b expression on neutrophils without (PMN) or with platelet-neutrophil complex (PNC) formation in samples from patients with cardiac surgery on admission (A), 45 min after the initiation of extracorporeal circulation in the operation room (OR), and 24, 48, and 120 h after the end of surgery compared to healthy volunteers (HV). Blood samples were stimulated with (A) buffer control or (B) 1 µM platelet-activating factor (PAF). Frequency of PMN and PNCs are shown in Figure 4 C) + D) PMN vs. PNC: unpaired t test; HV vs. A: unpaired t test; A vs. OR, 24, 48, and 120 h post OR: ordinary one-way ANOVA. * and **** indicating a p value of < 0.05 and <0.0001, respectively. MFI, median fluorescence intensity.

Figure 6

Analysis of neutrophils without (PMN) or with platelet-neutrophil complex (PNC) formation in samples from patients with cardiac surgery on admission (A), 45 min after the initiation of extracorporeal circulation in the operation room (OR), and 24, 48, and 120 h after the end of surgery compared to healthy volunteers (HV). Phagocytic activity in samples exposed to (A) buffer control or (B) 1 µM platelet-activating factor (PAF). Generation of reactive oxygen species (ROS) by neutrophils exposed to (C) buffer control or (D) 1 µM PAF. Percentage values below the bar chart indicate the frequency of PMN and PNCs as shown in Figure 4 C) + D) PMN vs. PNC: A) + B) + D) unpaired t test, C) One sample Wilcoxon test with **, ***, and **** indicating a p value of < 0.01, < 0.001, and <0.0001, respectively.