SummaryUnfractionated heparin (UFH) and low-molecular weight heparin (LMWH) are well defined anticoagulant agents. Recent data suggest that both LMWH and UFH may also have potent anti-inflammatory properties; however, their mechanism of action responsible for the anti-inflammatory effect is not yet fully elucidated. This study was designed to assess the effect of LMWH and UFH on human monocytes production of inflammatory markers and nuclear translocation of nuclear factor (NF)-jB. Cultured monocytes were pretreated for 15 min with LMWH or UFH (10 lg and 1 lg/million cells) before stimulation with lipopolysaccharide (LPS) at a dose of 1 ng/million cells. Proinflammatory cytokines tumour necrosis factor (TNF)-a, interleukin (IL)-8, IL-6 and IL-1b release were subsequently measured by enzyme-linked immunosorbent assay at 6 h, and nuclear translocation of the proinflammatory NF-jB was assessed at 2 h. Treatment with pharmacological doses of LMWH and UFH significantly attenuated LPS-induced production of TNF-a, IL-8, IL-6 and IL-1b as well as NF-jB translocation. These results indicate equivalent and significant heparin antiinflammatory properties at low doses on monocyte-mediated immune response. The inhibition of NF-jB activation certainly represents one of the mechanisms by which heparin exerts its anti-inflammatory effect. LMWH and UFH therefore appear as potential therapeutic inhibitors of inflammation.

In addition to their anticoagulant activity, unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) have important immunomodulatory properties. However, different studies have reported conflicting pro- and anti-inflammatory effects in association with heparin. Moreover, the molecular basis for these heparin effects on inflammation remains unclear. It was the objective of this study to determine how UFH and LMWH regulate lipopolysaccharide (LPS)-induced activation of human mononuclear cells in whole blood, and define the role of lipopolysaccharide-binding protein (LBP) in mediating this effect. Whole blood was pre-treated with UFH or LMWH (0.1-200 IU/ml), prior to stimulation with LPS (10 ng/ml). After six hours, monocyte pro-inflammatory cytokine (interleukin (IL)-1beta, IL-6, IL-8, and TNF-alpha) secretion was determined by plasma ELISA. Parallel experiments using THP-1 cell line and primary monocytes were performed under serum-free conditions, in the presence or absence of LBP (50-100 nM). Under serum-free conditions, heparin demonstrated dose-dependent anti-inflammatory effects, significantly reducing secretion of pro-inflammatory cytokines (IL-1beta, IL-6, IL-8, and TNF-alpha) in response to LPS-stimulation of THP-1 cells and primary monocytes. In contrast, in the presence of LBP, both UFH and LMWH demonstrated dose-dependent pro-inflammatory effects at all heparin concentrations. In ex-vivo whole blood experiments, pro-inflammatory effects (increased IL-1beta and IL-8 following LPS-stimulation) of heparin were also observed, but only at supra-therapeutic doses (10-200 IU/ml). Our data demonstrate that in the absence of LBP, the direct effect of heparin on LPS-stimulated monocytes is anti-inflammatory. However in whole blood, the immunomodulatory effects of heparin are significantly more complex, with either pro- or anti-inflammatory effects dependent upon heparin concentration.

Background: In addition to their etsblished anticoagulant activity, unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are known to possess clinically important immuno-modulatory properties. However different studies have reported conflicting pro- and anti-inflammatory effects in association with heparin. Moreover, the molecular basis for these heparin effects on inflammation remains unclear. In view of the wide and diverse clinical indications for heparin, it is clearly of direct translational relevance to define how UFH and LMWH differentially regulate inflammatory responses to LPS in-vivo. Objectives: To determine how UFH and LMWH regulate lipopolysaccharide (LPS)-induced activation of human mononuclear cells in whole blood, and define the role of lipopolysaccharide binding protein (LBP) in mediating this effect. Methods: Whole blood was pre-treated with UFH or LMWH (0.1–200 IU/ml), prior to stimulation with LPS (10ng/ml). After 6 hours, monocyte pro-inflammatory cytokine (interleukin (IL)-1b, IL-6, IL-8, and TNF-a) secretion was determined by plasma ELISA. Parallel experiments using THP-1 cell line and primary monocytes were performed under serum-free conditions, in the presence or absence of varying doses of recombinant human LBP (range: 50–100nM). Results: Under serum-free conditions, heparin demonstrated dose-dependent anti -inflammatory effects, significantly reducing secretion of pro-inflammatory cytokines (IL-1b, IL-6, IL-8, and TNF-a) in response to LPS-stimulation of THP-1 cells and primary monocytes. In contrast, in the presence of LBP, both UFH and LMWH demonstrated dose-dependent pro-inflammatory effects at all heparin concentrations. In ex-vivo whole blood experiments, pro-inflammatory effects (increased IL-1b and IL-8 following LPS-stimulation) of heparin were also observed, but only at supra-therapeutic doses (10–200IU/ml). Conclusion: In keeping with previous reports, we have demonstrated that both UFH and LMWH can significantly down-regulate cytokine (TNF-a, IL-1b, IL-6 and IL-8) secretion in response to LPS-activation in-vitro. However our novel data demonstrate that the effect of heparin on monocyte activation by LPS is significantly more complex in the setting of whole blood. Firstly, in contrast to the anti-inflammatory effects observed under serum-free conditions, we found that in whole blood, high concentrations of heparin exerted marked pro-inflammatory effects. Secondly we have also demonstrated that the effects of heparin in whole blood are entirely dependent upon heparin concentration and LBP concentration.