Abstract
The urokinase-type plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol (GPI) anchored membrane protein, regulates urokinase (uPA) protease activity, chemotaxis, cell-cell interactions, and phagocytosis of apoptotic cells. uPAR expression is increased in cytokine or bacteria activated cell populations, including macrophages and monocytes. However, it is unclear if uPAR has direct involvement in the response of inflammatory cells, such as neutrophils and macrophages, to Toll like receptor (TLR) stimulation. In this study, we found that uPAR is required for optimal neutrophil activation after TLR2, but not TLR4 stimulation. We found that the expression of TNF-α and IL-6 induced by TLR2 engagement in uPAR-/- neutrophils was less than that in uPAR+/+ (WT) neutrophils. Pretreatment of neutrophils with PI-PLC, which cleaves GPI moieties, significantly decreased TLR2 induced expression of TNF-α in WT neutrophils, but demonstrated only marginal effects on TNF-α expression in PAM treated uPAR-/- neutrophils. IκB-α degradation and NF-κB activation were not different in uPAR-/- or WT neutrophils after TLR2 stimulation. However, uPAR is required for optimal p38 MAPK activation after TLR2 engagement. Consistent with the in vitro findings that uPAR modulates TLR2 engagement induced neutrophil activation, we found that pulmonary and systemic inflammation induced by TLR2, but not TLR4 stimulation is reduced in uPAR-/- mice compared to WT counterparts. Therefore, our data suggest that neutrophil associated uPAR could be a potential target for treating acute inflammation, sepsis, and organ injury related to severe bacterial and other microbial infections in which TLR2 engagement plays a major role.
Highlights
Host immune cells, including macrophages and neutrophils, recognize and respond to microbial pathogen invasion using pattern recognition receptors (PRRs), including Toll like receptors (TLRs), Nod-like receptors (NLRs), and RIG-I-like receptors (RLRs) [1,2,3,4,5]
Bone marrow neutrophils isolated from wild type (WT) and urokinase-type plasminogen activator receptor (uPAR) knockout mice were stimulated with the specific synthetic TLR2 ligand, PAM3CSK4 (PAM)
The primary function of uPAR is to serve as the receptor for urokinase-type plasminogen activator (uPA), concentrating the protease activity of uPA in the proximity of cell surface. uPAR has proteaseindependent activities in that it participates in the regulation of cell migration, chemotaxis, cell-cell interaction, and phagocytosis of apoptotic cells [8,9,10,11,12,13,14]
Summary
Host immune cells, including macrophages and neutrophils, recognize and respond to microbial pathogen invasion using pattern recognition receptors (PRRs), including Toll like receptors (TLRs), Nod-like receptors (NLRs), and RIG-I-like receptors (RLRs) [1,2,3,4,5]. Each TLR binds to and is activated by a unique spectrum of molecules, termed pathogen associated molecular patterns (PAMPs), that are present in microbial organisms [1,2,3,4]. A number of intracellular molecules, such as HMGB1 and heat shock proteins, that are released to extracellular milieu by activated cell populations, have been shown to bind and activate specific TLRs [6,7]. These endogenous molecules have been termed as damage associated molecular patterns (DAMPs) [6,7]. Engagement of TLR2 or TLR4 leads to activation of transcription factors, including NF-kB, and enhances the production of pro-inflammatory and immunoregulatory cytokines [1,2,3,4]
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