P-selectin promotes neutrophil extracellular trap formation in mice

Abstract

Neutrophil extracellular traps (NETs) can be released in the vasculature. In addition to trapping microbes, they promote inflammatory and thrombotic diseases. Considering that P-selectin induces prothrombotic and proinflammatory signaling, we studied the role of this selectin in NET formation. NET formation (NETosis) was induced by thrombin-activated platelets rosetting with neutrophils and was inhibited by anti-P-selectin aptamer or anti-P-selectin glycoprotein ligand-1 (PSGL-1) inhibitory antibody but was not induced by platelets from P-selectin(-/-) mice. Moreover, NETosis was also promoted by P-selectin-immunoglobulin fusion protein but not by control immunoglobulin. We isolated neutrophils from mice engineered to overproduce soluble P-selectin (P-selectin(ΔCT/ΔCT) mice). Although the levels of circulating DNA and nucleosomes (indicative of spontaneous NETosis) were normal in these mice, basal neutrophil histone citrullination and presence of P-selectin on circulating neutrophils were elevated. NET formation after stimulation with platelet activating factor, ionomycin, or phorbol 12-myristate 13-acetate was significantly enhanced, indicating that the P-selectin(ΔCT/ΔCT) neutrophils were primed for NETosis. In summary, P-selectin, cellular or soluble, through binding to PSGL-1, promotes NETosis, suggesting that this pathway is a potential therapeutic target for NET-related diseases.

Figure 1

P-selectin is a stimulant of NET formation. (A) Neutrophils were incubated with unstimulated or thrombin (Thr)-stimulated platelets (Plt) in the presence of P-selectin aptamer inhibitor ARC5690 (P-sel ap) or ARC5694 as control aptamer (Control ap). H3Cit⁺ cells and NET formation were evaluated (n = 5; ***P < .001). (B) H3Cit⁺ cells and NET formation were quantified in neutrophils incubated with unstimulated or thrombin-stimulated platelets from WT or P-selectin^−/− mice (P-sel^−/−) (n = 5; **P < .01, ***P < .001). (C) The behavior of platelets in panels A and B was visualized by differential interference contrast microscopy. The images are representative from 5 experiments (scale bar represents 5 μm). (D) NET formation was examined after incubation with unstimulated or thrombin-stimulated WT platelets in the presence of anti-PSGL-1–blocking antibody or its immunoglobulin control (Ig). The percentage of H3Cit⁺ neutrophils was quantified (n = 4; *P < .05). (E) Left: WT neutrophils were stimulated with P-sel-Ig or with the same class of immunoglobulin as a control (n = 4; **P < .01, ***P < .001). Right: Representative fluorescence microscopy images of control immunoglobulin (Control-Ig) or P-sel-Ig treatments showing H3Cit-negative neutrophils (yellow arrow), H3Cit⁺ neutrophils without NET release (red arrow), and H3Cit⁺ neutrophils releasing NETs (white arrowhead). Scale bar represents 20 μm.

Figure 2

Neutrophils of P-selectin^ΔCT/ΔCT mice are primed to release NETs in vitro. Neutrophils isolated from peripheral blood of WT and P-selectin^ΔCT/ΔCT (ΔCT/ΔCT) mice were kept unstimulated (Unst) or stimulated with PAF (A), ionomycin (iono) (B), or PMA (C). The percentage of H3Cit⁺ neutrophils and NET release were quantified (n = 4; *P < .05, **P < .01, ***P < .001). (D) Representative fluorescence microscopy images of unstimulated or ionomycin-stimulated cells showing H3Cit-negative neutrophils (yellow arrow), H3Cit⁺ neutrophils without NET release (red arrow), and H3Cit⁺ neutrophils releasing NETs (white arrowhead). Scale bar represents 20 μm. (E) Left: Neutrophils isolated from peripheral blood of P-selectin^−/−, WT, or P-selectin^ΔCT/ΔCT mice were double stained with APC-conjugated anti-Ly6G and FITC-conjugated anti-P-selectin, and the mean fluorescence intensity (MFI) of P-selectin-positive cells was analyzed in the neutrophil gate (Ly6G-positive cells) by flow cytometry (n = 4-5; *P < .05). Right: Representative fluorescence-activated cell sorter plots for each genotype.