Concentration-dependent roles for heparin in modifying liopolysaccharide-induced activation of mononuclear cells in whole blood

Abstract

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.

Reduced Frequency of a CD14+CD16+Monocyte Subset with High Toll-Like Receptor 4 Expression in Cord Blood Compared to Adult Blood Contributes to Lipopolysaccharide Hyporesponsiveness in Newborns

“…Some studies have reported lower levels of LPS binding protein (LBP; a soluble acute-phase protein produced by liver in response to infection that binds LPS and markedly enhances its affinity for the TLR4 complex) in cord blood than in adult blood (33,44); however, this observation was not reproduced in another study (45). Lower levels of LBP or other soluble factors in cord blood are unlikely to be a dominant factor, since other authors have shown that LPS can active monocytes under serum-free conditions (46,47). Indeed, a study by Kato and colleagues showed that, in human peripheral blood mononuclear cells (PBMCs), only the interferon regulatory transcription factor 3 (IRF-3)-inducible genes (but not the NF-B-inducible genes that include the TNF-␣ gene) are strictly LBP dependent upon LPS stimulation (48).…”

Global Effect of Interleukin-10 on the Transcriptional Profile Induced by Neisseria meningitidis in Human Monocytes

“…Previous studies have shown that heparin may inhibit the production of cytokines from human monocytes (17,19).…”

Biomimetic sulphated alginate hydrogels suppress IL-1β-induced inflammatory responses in human chondrocytes

“…Preincubation with soluble heparin and chondroitin sulphate has previously been shown to dampen the inflammatory response in cells stimulated with IL-1β, correlated with reduced NF-κB signalling, but the mechanisms of action by the polysaccharides are not clear (Hochart et al, 2006;Jomphe et. al., 2008;Legendre et al, 2008).…”

“…A r t i c u l a r c a r t i l a g e i s r i c h i n s u l p h a t e d glycosaminoglycans (GAGs) which have significant roles in hydration, cell motility and intra-and intercellular communication, and are thus of great interest for incorporation in tissue engineering scaffolds (Chen et al, 2007;Pieper et al, 2000). Heparin, in particular, has been shown to have anti-inflammatory effects by reducing nuclear translocation of NF-κB, thus lowering expression of inflammatory cytokines, and may further inhibit downstream processes by direct association with cytokines, adhesion proteins and the complement and coagulation cascades (Hochart et al, 2006;Parish, 2006;Spillmann et al, 1998;Weiler et al, 1992).…”