Mechanisms underlying mouse TNF-α stimulated neutrophil derived microparticle generation

https://doi.org/10.1016/j.bbrc.2013.06.118Get rights and content

Highlights

  • TNF-α treatment generates Neutrophil Derived Microparticles (NDMPs).

  • Activation of either TNFr1 or TNFr2 is sufficient for NDMP generation.

  • TNFr1 activation generates NDMPs through Caspase 8.

  • TNFr1 and TNFr2 generate NDMPs through NF-κB.

Abstract

Despite advances in understanding and treatment of sepsis, it remains a disease with high mortality. Neutrophil Derived Microparticles (NDMPs) are present during sepsis and can modulate the immune system. As TNF-α is a cytokine that predominates in the initial stages of sepsis, we evaluated whether and how TNF-α can induce NDMPs in mice. We observed that TNF-α treatment results in increased NDMP numbers. We also determined that the activation of either TNF receptor 1 (TNFr1) or TNF receptor 2 (TNFr2) resulted in increased NDMP numbers and that activation of both resulted in an additive increase. Inhibition of Caspase 8 diminishes NDMPs generated through TNFr1 activation and inhibition of NF-κB abrogates NDMPs generated through activation of both TNFr1 and TNFr2. We conclude that the early production of TNF-α during sepsis can increase NDMP numbers through activation of the Caspase 8 pathway or NF-κB.

Introduction

Sepsis is a serious medical condition characterized by a systemic inflammatory response due to a severe infection. Despite advances in understanding of sepsis, treatment remains supportive and the disease continues to account for a significant number of deaths in the critically ill population [1], [2], with mortality rates ranging between 35 and 50% [1].

Tumor necrosis factor-α (TNF-α) predominates in the initial phases of sepsis and is one of the first cytokines released into the circulation during the inflammatory response to infectious stimuli [3]. It has diverse effects in vivo as a mediator of the immune response, in part, through activation of neutrophils [4]. Observed during sepsis are: impaired neutrophil recruitment to sites of infection, prolonged accumulation of neutrophils to remote sites, and dysregulation of neutrophil function [5]. TNF-α induced multi-organ damage has been shown to be primarily mediated by neutrophils [6], [7], [8], and this damage is ameliorated in neutrophil depleted pigs after TNF-α administration [9]. A better understanding of TNF-α activation of neutrophils is needed to more completely understand sepsis pathophysiology.

Microparticles (MPs) are small, intact vesicles ranging from 0.3–1.0 μm that bleb off the cell membranes, and are generated during cell activation and cell apoptosis, both of which occur during sepsis [10]. Host cytokines such as c5A [11], TNF-α [12], [13], IL-8 [14], as well as bacteria [15] and bacterial byproducts, such as fMLP [14], [16] and endotoxin [17] can increase MP formation. Neutrophils, cytokines, and bacterial products, are present in the septic milieu, and as such Neutrophil Derived Microparticles (NDMPs) may serve as potential mediators in the pathophysiology of this disease.

In vitro studies have demonstrated that NDMPs can have an inflammatory impact on endothelial cells, increasing both IL-6 and IL-8 production [18] and increasing expression of ICAM-1, VCAM-1, and E-selectin [14]. In addition, NDMPs been shown to increase in platelet expression of P-selectin [19]. NDMPs have also been shown to decrease macrophage IL-6, IL-8, IL-10, and TNF-α production[20], [21], while increasing TGF-β1. Recently, we demonstrated that MPs accumulate at infected foci in critically ill patients during sepsis and are predominantly of neutrophil derivation [22]. In addition, we have shown that NDMPs are phagocytosed by monocytes, causing the amplified activation of the ingesting leukocytes and the deactivation of non-ingesting leukocytes, suggesting that they contribute to the disordered immune response during sepsis [22].

To date, the generation of NDMPs following TNF-α administration in vitro has been described in three papers [12], [13], [23], however these papers use TNF-α in conjunction with other agents. In this study, we hypothesized that mouse NDMPs could be generated by administration of TNF-α alone to unactivated neutrophils. To address the mechanisms, not addressed in previous literature, we investigated the role of TNF-α in NDMP generation. We specifically looked at the role of TNF Receptor 1 (TNFr1) and TNF Receptor 2 (TNFr2), as well as MP generation as it relates to pathways involved in apoptosis and cellular fate decisions, namely the caspase and NF-κB pathways. Altogether, increased understanding into the way NDMPs are generated and their effects on cellular function may lead to greater understanding of the inflammatory response and sepsis.

Section snippets

Materials

Male C57BL/6 and TNFr1 KO (stock number: 003242) mice between 6 and 8 weeks of age were obtained from Jackson Labs (Bar Harbor, ME). All experiments involving animals were performed under protocols approved by the Institutional Animal Care and Use Committee of the University of Cincinnati (08-09-19-01). Mouse and human TNF-α Recombinant Protein (rmTNF-α and rhTNF-α, respectively) were purchased from Preprotech (Rocky Hill, NJ). NF-κB inhibitor (BAY 11-7085) and Caspase 8 inhibitor (Z-IETD-FMK)

Mouse TNF-α treatment results in increased neutrophil-derived microparticle generation

Using flow cytometry, forward- and side-channels were set to analyze particles using 0.5-, 0.9-, and 3.0-μm latex beads for calibration as previously described [27] (Fig. 1A). The forward and side scatter gate for microparticles was then set based upon size (Fig. 1B). To enumerate NDMPs, we analyzed neutrophil and NDMP mixture with the neutrophil-specific marker, Ly6G, and the apoptosis marker, annexin V. Vesicles within the 0.3–1.0 μm range that expressed both Ly6G and phosphatidylserine were

Discussion

In this study, we observed that treatment of neutrophils with TNF-α within 30 min (Fig. 1). Additionally we conclude that activation of either TNFr1 or TNFr2 is sufficient for NDMP generation, and that activation of both is additive (Fig. 2). Upon the selective activation of TNFr1, NDMP numbers were sharply decreased but not entirely eliminated with addition of Caspase 8 inhibitor. In contrast, caspase 8 inhibition did not alter NDMP numbers when only TNFr2 was activated (Fig 3). Finally, we

Acknowledgments

Financial Disclosure: The work was funded in part by National Institutes of Health Grants T32 GM08478 and R01 GM100913. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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