Deficiency of P-selectin Glycoprotein Ligand-1 is Protective against the Prothrombotic Effects of Interleukin-1β

Abstract

Background

Proinflammatory cytokines are associated with cardiovascular diseases including acute and recurrent myocardial infarction. However, the causal role of cytokines in thrombotic complications of atherosclerosis remains unclear. Interleukin-1β (IL-1β) is currently being targeted in a human clinical trial for prevention of ischemic events.

Objectives

The purpose of the present study was to test the role of IL-1β in arterial thrombosis and a potential protective effect of Psgl-1 deficiency.

Methods and Results

Wild-type and P-selectin glycoprotein ligand-1 (Psgl-1) deficient mice were treated with IL-1β and then subjected to carotid photochemical injury to induce thrombosis. IL-1β shortened the time to thrombosis in wild-type mice while Psgl-1^−/− mice were protected from the prothrombotic effects of IL-1β. A neutralizing antibody to Psgl-1 was also effective in protecting against the prothrombotic effects of IL-1β. The protective effect of Psgl-1 deficiency was associated with reduced plasma levels of soluble P-selectin and collagen-stimulated whole blood aggregation.

Conclusions

Our data demonstrate that Psgl-1 deficiency is protective against the prothrombotic effects of IL-1β and suggest that Psgl-1 inhibition may be a useful treatment strategy for targeting vascular thrombosis associated with enhanced inflammatory states.

Introduction

Thrombotic complications of atherosclerosis such as myocardial infarction and stroke remain the leading cause of mortality in industrialized countries [1]. Thrombosis is precipitated by local plaque rupture which is associated with heightened local plaque inflammatory activity [2]. Local release of cytokines may contribute to the ensuing occlusive thrombosis [3]. Interleukin-1β (IL-1β) is one of many cytokines associated with CV events and may promote local thrombosis via platelet [4] and/or endothelial activation [5]. IL-1β is currently being targeted in a human clinical trial for prevention of CV events [6].

Methods

Animals

Male C57BL6/J and Psgl-1^−/− mice were originally purchased from Jackson Laboratory (Bar Harbor, Maine). Psgl-1^−/− were backcrossed to the C57BL6/J strain >16 generations before use in these experiments. All mice were 8-10 week-old at the time of experiments. All animal use protocols complied with the Principle of Laboratory and Animal Care established by the National Society for Medical Research and were approved by the University of Michigan Committee on Use of and Care of Animals.

IL-1β and Psgl-1 antibody treatment

Recombinant IL-1β (Peprotech, Rocky Hill, NJ) was injected to mice via tail vein (500 ng in 200 µl PBS). Arterial thrombosis studies were performed 4 hours after IL-1β injection. For antibody injection experiments, a rat anti-mouse Psgl-1 antibody 4RA10 or isotype control rat IgG₁ k (100µg in 200µl PBS) (BD Biosciences, San Jose, CA) was injected intraperitoneally the night before thrombosis studies.

Carotid arterial thrombosis

To induce thrombosis, photochemical injury was performed on carotid arteries as previously described [7]. Briefly, mice were anesthetized and secured in the supine position under a dissecting microscope (Nikon SMZ-2T, Mager Scientific, Inc., Dexter, MI). The right common carotid artery was isolated and blood flow was monitored with a doppler flow probe (Transonic, Ithaca, NY). A 1.5-mW green light laser (540 nm) (Melles Griot, Carlsbad, CA) was applied to the mid common carotid artery before injection of Rose Bengal (50mg/kg in PBS) (Fisher, Fair Lawn, NJ) via tail vein. Arterial thrombosis was defined as flow cessation for at least 10 minutes. Flow in the carotid artery was monitored for 90 minutes.

Plasma measurements

Blood samples were collected by retroorbital bleeding with nonheparinized capillary tubes (Fisher Scientific, Pittsburgh, PA). Circulating levels of soluble P-selectin (sP-sel) were measured with commercially available murine ELISA kits (R&D Systems, Minneapolis, MN) according to manufacturer’s instructions.

Whole blood aggregation

Whole blood aggregation was performed as described previously [4, 8]. Briefly, 500μl of whole blood anticoagulated with 3.2% sodium citrate (1: 9) was diluted in an equivalent volume of 0.9% saline and incubated for 5 min at 37°C. Collagen (5μg/ml) - stimulated whole blood aggregation was performed using a Whole Blood / Optical Lumi- Aggregation System (Chrono-Log Corp., Havertown, PA) according to manufacturer’s instruction.

Statistical analysis

All data are presented as mean ± standard error. Results were analyzed using GraphPad Prism. Data were analyzed using unpaired t-test for comparison between two groups and one-way ANOVA followed by Tukey post-test for multiple comparisons. Probability values of p<0.05 were considered statistically significant.

Results and Discussion

To determine the in vivo capacity of IL-1β to promote arterial thrombosis, recombinant IL-1β or PBS was administered to C57BL6/J or Psgl-1^−/− mice via tail vein infusion 4 hours prior to induction of arterial thrombosis. Thrombosis in the right common carotid artery was induced via photochemical injury with Rose Bengal. Time to occlusive carotid thrombosis was significantly shorter in mice treated with IL-1β compared to vehicle treated mice (Fig. 1A). IL-1β treatment was also associated with elevated plasma levels of soluble P-selectin (sP-sel) (Fig. 1B). Since inhibition of Psgl-1 has been shown to attenuate responses to IL-1β [9], and may therefore serve as a therapeutic target, mice with genetic deficiency of Psgl-1 were studied in the carotid thrombosis model. At baseline, in the absence of inflammatory stimuli, the time to occlusive thrombosis in mice deficient in Psgl-1 was not different than occlusion times in wild-type mice (Fig.1A). However, following IL-1β challenge, the time to occlusive thrombosis was similar in Psgl-1 deficient mice to wild-type mice treated with only PBS (Fig. 1A). This protection from thrombosis was associated with reduced levels of sPsel (Fig. 1B). To test the potential therapeutic strategy of targeting Psgl-1 to attenuate the prothrombotic effect of IL-1β, a Psgl-1 blocking antibody or isotype control antibody was given to wild-type mice 12 hours prior to IL-1β challenge. After IL-1β treatment, the time to thrombosis was longer in Psgl-1 antibody-treated mice compared with isotype control antibody-treated mice (Fig.1C). After IL-1β treatment, the levels of sPsel were significantly lower in wild-type mice treated with Psgl-1 antibody compared with those treated with control antibody (Fig. 1D).

Figure 1.

A: Time to occlusive carotid thrombosis in wild-type (WT) mice and Psgl-1^−/− mice with or without IL-1β treatment (n=7 mice per group). B: Plasma levels of soluble P-selectin (sP-selectin) in WT and Psgl-1^−/− mice with or without IL-1β treatment (n=7 mice per group). C: Time to occlusive carotid thrombosis in IL-1β -treated WT mice after Psgl-1 antibody or isotype control antibody treatment (n=7 mice per group). D: Plasma levels of sP-selectin in IL-1β -treated WT after Psgl-1 antibody or isotype control antibody treatment (n=7 mice per group). *P<0.05, **P<0.01.

To determine potential mechanisms by which Psgl-1 deficiency may protect against the prothrombotic effects of IL-1β, whole blood aggregation studies were performed on blood from wild-type and Psgl-1^−/− mice. Whole blood from wild-type mice treated with IL-1β (4 hours) demonstrated enhanced collagen (5μg/ml)-stimulated aggregation compared with whole blood from control mice treated with PBS (Fig. 2A). After treatment with IL-1β, whole blood aggregation was significantly reduced in Psgl-1^−/− mice compared with wild-type mice (Fig. 2A). No difference was detected between wild-type and Psgl-1^−/− mice in the absence of cytokine stimulation. The effect of in vitro treatment of whole blood with the Psgl-1 blocking antibody was also tested. Whole blood from wild-type mice treated with PBS or IL-1β (4 hours) was incubated with the Psgl-1 antibody or isotype control antibody (10ug/ml) at 37°C for one hour prior to aggregation measurements. Collagen-stimulated whole blood aggregation was significantly reduced in blood from IL-1β-treated mice when the blood was then treated with the Psgl-1 antibody compared to blood from mice treated with a control antibody (Fig. 2B). Aggregation was similar between IL-1β-treated mice and subsequent in vitro Psgl-1 antibody treatment, and mice treated with only PBS (Fig. 2B).

Figure 2.

A: Amplitude of whole blood aggregation in whole blood from PBS or IL-1β-treated WT and Psgl-1^−/− mice (n=4 mice per group). B: Amplitude of whole blood aggregation in whole blood from PBS or IL-1β-treated WT mice after in vitro treatment with Psgl-1 or isotype control antibody (n=5 mice per group). *P<0.05, **P<0.01.

Inflammatory cytokines are upregulated locally and systemically in the setting of acute coronary syndromes. Il-1β may be causally related to atherosclerotic complications and is being investigated in a clinical trial. Following inhibition of IL-1β, with the antibody canakinumab, in humans at high risk for acute coronary syndromes, there are reductions in downstream inflammatory biomarkers, including IL-6, c-reactive protein, and fibrinogen [10]. IL-1β was also recently shown to activate platelets and to promote platelet adhesion [4].

The current study was performed to investigate the in vivo effect of elevated IL-1β on arterial thrombosis following injury. IL-1β promoted arterial thrombosis indicating that in the setting of acute or chronic inflammation, local or systemic elevations of IL-1β may be a mediator of enhanced thrombosis. This effect is associated with elevations of a platelet-associated biomarker (sP-sel) and enhanced whole blood aggregation. Both Psgl-1 deficiency and antibody-mediated Psgl-1 blockade were associated with protection from the prothrombotic effects of IL-1β while no effect of Psgl-1 deficiency was observed in the absence of IL-1β stimulation. An intervention that specifically attenuates a thrombotic response to cytokine stimulation without affecting baseline hemostasis could be clinically useful. It has been shown that platelet P-selectin is involved in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates [11]. Our data indicate that Psgl-1 may also play an important role in the setting of arterial thrombosis promoted by inflammatory cytokines. In the present study, we demonstrated the neutralizing effect of Psgl-1 deficiency toward the prothrombotic effect of IL-1β. We cannot assume that Psgl-1 deficiency will also be protective from other inflammatory stimuli as each cytokine will need to be tested individually, however it has been shown previously that leukocyte-endothelial interactions and inflammatory responses triggered by tumor necrosis factor-α are reduced in the setting of Psgl-1 deficiency [12]. Although the leukocyte appears to be the relevant Psgl-1 cellular pool for regulation of adhesive interactions [13], Psgl-1 expressed by platelets [14] and/or endothelial cells [15] may contribute to the prothrombotic effects of IL-1β. Whether Psgl-1 neutralization would be more efficacious or safer than directly inhibiting IL-1β in atherothrombotic diseases is unknown, however these findings provide additional insight into the regulatory role of Psgl-1 in thrombosis. Psgl-1 inhibition may be a useful treatment strategy for targeting vascular thrombosis associated with enhanced inflammatory states, although this will need to be confirmed in humans.