An intense and short-lasting burst of neutrophil activation differentiates early acute myocardial infarction from systemic inflammatory syndromes

Abstract

Background: Neutrophils are involved in thrombus formation. We investigated whether specific features of neutrophil activation characterize patients with acute coronary syndromes (ACS) compared to stable angina and to systemic inflammatory diseases.

Methods and findings: The myeloperoxidase (MPO) content of circulating neutrophils was determined by flow cytometry in 330 subjects: 69 consecutive patients with acute coronary syndromes (ACS), 69 with chronic stable angina (CSA), 50 with inflammation due to either non-infectious (acute bone fracture), infectious (sepsis) or autoimmune diseases (small and large vessel systemic vasculitis, rheumatoid arthritis). Four patients have also been studied before and after sterile acute injury of the myocardium (septal alcoholization). One hundred thirty-eight healthy donors were studied in parallel. Neutrophils with normal MPO content were 96% in controls, >92% in patients undergoing septal alcoholization, 91% in CSA patients, but only 35 and 30% in unstable angina and AMI (STEMI and NSTEMI) patients, compared to 80%, 75% and 2% of patients with giant cell arteritis, acute bone fracture and severe sepsis. In addition, in 32/33 STEMI and 9/21 NSTEMI patients respectively, 20% and 12% of neutrophils had complete MPO depletion during the first 4 hours after the onset of symptoms, a feature not observed in any other group of patients. MPO depletion was associated with platelet activation, indicated by P-selectin expression, activation and transactivation of leukocyte β2-integrins and formation of platelet neutrophil and -monocyte aggregates. The injection of activated platelets in mice produced transient, P-selectin dependent, complete MPO depletion in about 50% of neutrophils.

Conclusions: ACS are characterized by intense neutrophil activation, like other systemic inflammatory syndromes. In the very early phase of acute myocardial infarction only a subpopulation of neutrophils is massively activated, possibly via platelet-P selectin interactions. This paroxysmal activation could contribute to occlusive thrombosis.

Figure 1. Heterogeneity of myeloperoxidase content in circulating neutrophils from patients with acute myocardial infarction is associated to platelet adhesion and activation.

Intracellular MPO content in circulating neutrophils and platelet-neutrophil heterotypic aggregates were evaluated in whole blood samples by four-color flow cytometry. Venous blood samples were obtained within the first 6 hours of onset of symptoms. For confocal determinations, monoclonal antibodies against MPO were labeled with Alexa Fluor 488 (green) and against platelet glycoprotein Ib with Alexa Fluor 546 (red). Hoechst (blue) was used for counterstaining of nuclei. Panel A. Representative confocal images illustrate the MPO (green) content observed in circulating neutrophils of a patient with chronic stable angina. Panel B. Representative confocal images illustrate the heterogeneity in the MPO (green) content observed in circulating neutrophils of a patient with acute myocardial infarction. Panel C-D. The cytofluorimetric histogram shows the trimodal pattern MPO-associated distribution (black) in AMI but not in CSA, vs isotype control (white). Panel E: confocal images of neutrophils with complete MPO depletion, low or normal MPO content. Results are the mean value ± SEM of heterotypic aggregates observed in each neutrophil subpopulation. Panel F: confocal microscopy of aggregates between platelet (red) and degranulated neutrophils from a representative patient with AMI. Panel H. Correlation analysis indicates the association between platelet activation and neutrophil myeloperoxidase content in patients with AMI: platelet P-selectin expression (% of CD61⁺ cells, y axis) are plotted against the neutrophil intracellular MPO content (MFI-within CD66b⁺ cells, x axis; R = 0.6, P<0.0001).

Figure 2. Time course of leukocyte and platelet activation in patients and controls.

36 patients with acute myocardial infarction were studied before any therapeutic treatment and re-studied at different times on PCI. Results (expressed as mean ± SEM) were compared with 69 patients with chronic stable angina candidates (CSA) for PCI and 138 healthy donors. Determinations were performed by flow cytometry, with the exception of troponin I, as described in material and methods. Y axis: serum troponin (Panel A), granulocyte cell counts (panel B), fraction of circulating neutrophils with complete myeloperoxidase (MPO) depletion (Panel C), average of neutrophil MPO content (Panel D), fraction of platelet expressing P-selectin (Panel E), the fraction of platelet-neutrophils heterotypic aggregates (Panel F), fraction of platelet-monocyte heterotypic aggregates (Panel G). * = P<0.05; ** = P<0.01 (both respect to CSA and healthy donors) were determined by ANOVA followed Bonferroni test.

Figure 3. In vivo evidence that circulating activated platelets cause neutrophil degranulation.

Purified platelets from wild type or Psel ^−/− mice were activated (or not) and injected in the tail vein of wild type C57BL/6 mice. Platelet activation was achieved with thrombin. The molecule was then inactivated by addition of hyrudin. Neutrophil myeloperoxidase content, platelet P-selectin expression and platelet-neutrophil heterotypic aggregates were assessed by flow cytometry as described in the Methods. Panel A shows the effect of the in vivo injection of the buffer used for platelet activation, containing both thrombin and hyrudin. Blood was retrieved at different times after injection (hours, x axis) and neutrophils with adherent (filled symbols) or internalized (grey symbols) platelets were identified by flow cytometry. >70% of circulating neutrophils were engaged in the internalization and adhesion of activated, but not resting, platelets after 1 hour. The fraction of circulating neutrophils with internalized and adherent platelets returned to background levels 24 hours after the injection. Results are expressed as mean±S.E.M. 5–7 animals were assessed per each point.