Abstract
Heparanase is a β-D-endoglucuronidase that cleaves heparan sulfate (HS), facilitating degradation of the extracellular matrix (ECM) and the release of HS-bound biomolecules including cytokines. The remodeling of the ECM by heparanase is important for various physiological and pathological processes, including inflammation, wound healing, tumour angiogenesis and metastasis. Although heparanase has been proposed to facilitate leukocyte migration through degradation of the ECM, its role in inflammation by regulating the expression and release of cytokines has not been fully defined. In this study, the role of heparanase in regulating the expression and release of cytokines from human and murine immune cells was examined. Human peripheral blood mononuclear cells treated ex vivo with heparanase resulted in the release of a range of pro-inflammatory cytokines including IL-1β, IL-6, IL-8, IL-10 and TNF. In addition, mouse splenocytes treated ex vivo with heparanase resulted in the release of IL-6, MCP-1 and TNF. A similar pattern of cytokine release was also observed when cells were treated with soluble HS. Furthermore, heparanase-induced cytokine release was abolished by enzymatic-inhibitors of heparanase, suggesting this process is mediated via the enzymatic release of cell surface HS fragments. As soluble HS can signal through the Toll-like receptor (TLR) pathway, heparanase may promote the upregulation of cytokines through the generation of heparanase-cleaved fragments of HS. In support of this hypothesis, mouse spleen cells lacking the key TLR adaptor molecule MyD88 demonstrated an abolition of cytokine release after heparanase stimulation. Furthermore, TLR4-deficient spleen cells showed reduced cytokine release in response to heparanase treatment, suggesting that TLR4 is involved in this response. Consistent with these observations, the pathway involved in cytokine upregulation was identified as being NF-κB-dependent. These data identify a new mechanism for heparanase in promoting the release of pro-inflammatory cytokines that is likely to be important in regulating cell migration and inflammation.
Highlights
Inflammation is an important innate immune response to remove injurious stimuli and is mediated by a complex array of soluble molecules and leukocytes that are resident and/or attracted to the site of inflammation
Mass spectrometry and SDS-PAGE analysis confirmed the identity of HPSE (Fig. S1A-B) and Heparan sulfate (HS)-cleaving enzyme activity was confirmed by a time-resolved fluorescence energy transfer (TR-FRET) activity assay (Fig. S1C) [39]
To investigate the ability of HPSE to modulate the release of pro-inflammatory cytokines, human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and treated with exogenous endotrap-purified HPSE, upon which the levels of a panel of pro-inflammatory secreted cytokines (IL-8, IL-6, IL-1b, TNF, IL-10 and IL-12p70) were determined using a cytometric bead array (CBA) assay
Summary
Inflammation is an important innate immune response to remove injurious stimuli and is mediated by a complex array of soluble molecules and leukocytes that are resident and/or attracted to the site of inflammation. It is well described that inflammation contributes to many diseases including arthritis, atherosclerosis, diabetic nephropathy and cancer [1,2,3,4,5,6]. The migration and activation of leukocytes in inflammation are regulated by the action of chemokines and cytokines. Leukocytes express enzymes that degrade the extracellular matrix (ECM) to aid their migration to sites of inflammation [7,8]. Heparan sulfate (HS) is a key structural component of the ECM and basement membrane (BM) which binds an array of growth factors, chemokines and cytokines [9,10]. The only known mammalian endoglycosidase to cleave HS is the b-D-endoglucuronidase heparanase (HPSE) [11,12]
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