Neutrophils develop rapid proinflammatory response after engulfing Hb-activated platelets under intravascular hemolysis

Abstract

Neutrophils maintain immune homeostasis by engulfing apoptotic cells and debris. We describe the rapid activation of neutrophils after engulfing hemoglobin (Hb)-activated platelets, which are abundant in the circulation of hemolytic patients. Neutrophils from healthy individuals after engulfing Hb-activated platelets express elevated CD11b and secrete significant amounts of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, myeloperoxidase (MPO) and elastase within 4-h platelets, but not with free-Hb only in vitro. These neutrophils exhibit early onset of apoptosis and cell death after engulfing Hb-activated platelets, but not with free-Hb only. Further, our data from mice with phenylhydrazine-induced intravascular hemolysis display a gradual decrease in total neutrophil count, but the number of activated neutrophils and neutrophil-platelet aggregates increases, along with the rise of TNF-α, IL-1β, IL-6 and MPO in circulation. Our data from paroxysmal nocturnal hemoglobinuria (PNH) patients confirmed the observation of decreased total neutrophil counts, but elevated numbers of activated neutrophils, including neutrophil-platelet aggregates, in parallel with elevated expression of TNFA, IL1B and IL6 genes in neutrophils, also increased levels of these cytokines along with MPO in circulation, and this correlated directly with elevated intravascular hemolysis (high free-Hb in plasma). The patients' neutrophils displayed significant localization of intracellular Hb and platelets, unlike the counterparts from healthy individuals. Together, therefore, our observations suggest that Hbactivated platelets, which are abundant in the circulation of patients with hemolytic disorders, including PNH, promotes early onset of neutrophil activation and increases their proinflammatory response and leads to early apoptosis and cell death.

High Neutrophil-to-Platelet Ratio Is Associated With Hemorrhagic Transformation in Patients With Acute Ischemic Stroke

“…The interaction between neutrophil and platelet could enhance the reactive oxygen species generation and exasperate the vascular injury (42), despite of the procoagulant properties of the activated neutrophils (43,44). Moreover, platelet serves as a major contributor of several pro-inflammatory factors like tumor necrosis factor-α (41), which contributed to the increase in activated neutrophils and neutrophil-platelet aggregates (45). An animal study found that the depletion of platelet reduced the neutrophil recruitment and vascular inflammation after the occlusion of the middle cerebral artery (46).…”

α-ketoglutarate augments prolyl hydroxylase-2 mediated inactivation of phosphorylated-Akt to inhibit induced-thrombosis and inflammation

“…Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Hypaque (GE Healthcare, Freiburg, Germany) density gradient centrifugation as mentioned in our earlier work (46). PBMCs were washed twice with PBS, pH 7.4 and seeded in cell culture-treated plates (Corning, NY, USA) in RPMI-1640 medium (Sigma Aldrich, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco Invitrogen, San Diego, CA), 100 U/mL penicillin and 100 µg/mL streptomycin for 2 hrs at 37°C in a humidified atmosphere with 5% CO 2 , to allow monocytes to adhere to the plate.…”

“…At 3hrs and 6hrs, the animals were anaesthetized and peritoneal exudates were harvested in 3 ml of PBS. Different immune cell populations in peritoneal lavage were analysed and counts were determined by flow cytometry using CD45.2, CD11b, CD11c, Ly6G, Ly6C, and CD41 (46, 49). In another set of a similar experiment, BALB/c mice were injected with carrageenan.…”

“…At 3 and 6 hrs, peritoneal inflammation was visualised using bioluminescence based imaging of MPO activity by injecting luminol (i.p. 20 mg/100 g body weight) (Sigma Aldrich) 6 min prior to imaging using an in-vivo imaging system (IVIS; Perkin Elmer, Waltham, MA, USA) as mentioned in our work (46).…”

Misunderstandings Between Platelets and Neutrophils Build in Chronic Inflammation

“…Under intravascular hemolytic conditions, free hemoglobin favors platelet activation. In turn, activated platelet boost neutrophil activation, possibly further feeding the inflammatory cascade (186). In addition, sera from patients with thrombotic microangiopathies fail to degrade NETs, which, in turn, can trigger thrombosis (76, 187).…”