Expression of Toll-like receptors on human platelets

Abstract

Introduction: Platelets play a crucial role in arterial thrombosis, which is the main cause of acute coronary syndrome. Some mycobacteriums, such as Chlamydia pneumoniae, were associated with progression of atherosclerosis and they are interacted with Toll-like receptors (TLRs), which have been defined as pathogen-associated molecular pattern recognition molecules in innate immunity. In the present study, we examined whether human platelets express TLRs. Materials and methods: Human platelets were obtained from healthy volunteers and the mRNA and protein level of TLRs on platelets and Meg-01 cells, megakaryoblastic cell line, were investigated. Results: Reverse transcription–polymerase chain reaction (RT-PCR) demonstrated that TLR1 and TLR6 mRNA were expressed in platelets and Meg-01 cells. Furthermore, interferon-γ up-regulated their mRNA levels in dose and time dependent manners after stimuli. Both TLR1 and TLR6 proteins in platelets were detected by Western blotting, and their expression of platelets was more than that of Meg-01 cells. Flow cytometry analysis revealed the expression of TLR1 and TLR6 on the cell surface of Meg-01 cells. Furthermore, immunohistochemical analysis using human coronary thrombi obtained from patients with acute coronary syndrome confirmed the expression of TLR1 and TLR6 on platelets. Conclusion: In summary, we demonstrated that human platelets and Meg-01 cells expressed a family of TLRs for the first time, and our findings indicated that platelets might recognize antigens directly via TLRs. Our findings suggest a possibility that platelets have the ability to recognize the antigens via TLRs and that there are mechanistic relations between infectious inflammation and atherosclerotic vascular diseases.

Introduction

Atherosclerosis is a chronic inflammatory disease. Inflammation occurs in response to vascular injury induced by oxidative stress and infection. Accumulating evidence indicates a close association of atherosclerotic diseases with chronic infection with ubiquitous pathogens such as Chlamydia pneumoniae. Recent studies have demonstrated a seroepidemiologic association with risk of cardiovascular events. Furthermore, immunohistochemistry has revealed the presence of microorganisms in atherosclerotic lesions. Very recently, Sasu et al. [1] demonstrated that Chlamydial heat shock protein (HSP) 60 stimulated proliferation of vascular smooth muscle cells via Toll-like receptor (TLR).

Inflammation induced by pathogens is a complex process of interaction between various soluble factors and inflammatory cells. It has become evident that a family of TLRs plays a crucial role in innate immunity as the first defense system against microbial infection [2]. TLR has been defined as a pathogen-associated molecular pattern recognition molecule. Microbial antigens, lipopolysaccharide (LPS) and bacterial HSPs interact with the extracellular domain of TLRs and subsequently activate multiple intracellular signaling pathways. TLR1, TLR2 and TLR4 are markedly expressed in human atherosclerotic vessels [3], and TLR4 in macrophages is up-regulated by oxidized low-density lipoprotein [4], strongly suggesting the association between TLRs and atherosclerotic vascular diseases.

Interferon-gamma (IFN-γ) is a proinflammatory cytokine, which contributes to atherogenesis via its various functions such as activation of human peripheral blood monocytes and enhancement of smooth muscle cells proliferation. CD4⁺ T lymphocytes are reported to accumulate in atherosclerotic vessel walls through almost all stages and produce IFN-γ [5], [6]. In the unstable plaques, IFN-γ secreted by T lymphocytes inhibits the collagen synthesis by vascular smooth muscle cells and activates macrophages, which secrete several proteases. These macrophages-derived proteases can break down the extracellular matrix and weaken the fibrous cap, rendering it susceptible to rupture and precipitation of acute coronary syndrome [7].

Platelets play a central role in arterial thrombosis superimposed on disrupted unstable plaques, which is the main cause of acute coronary syndrome. Spontaneous and agonist-induced hyperaggregation and hypersensitivity of platelets have also been implicated in pathogenesis of various cardiovascular disorders. These non-nucleated cells also have several immunomodulatory properties: activated platelets have been shown to induce inflammatory reaction on endothelial cells through the CD40 ligand originally identified on activated CD4⁺ T cells, and to secrete various proinflammatory and anti-inflammatory mediators. Given the close association between platelets and inflammation, this cell type might express a family of TLR. In the present investigation, we examined through various approaches, whether platelets express any of the members of the TLR family. Furthermore, the effect of IFN-γ on their expression was investigated.