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- Materials and methods
Unfractionated 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)-κB. Cultured monocytes were pretreated for 15 min with LMWH or UFH (10 μg and 1 μg/million cells) before stimulation with lipopolysaccharide (LPS) at a dose of 1 ng/million cells. Proinflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-8, IL-6 and IL-1β release were subsequently measured by enzyme-linked immunosorbent assay at 6 h, and nuclear translocation of the proinflammatory NF-κB was assessed at 2 h. Treatment with pharmacological doses of LMWH and UFH significantly attenuated LPS-induced production of TNF-α, IL-8, IL-6 and IL-1β as well as NF-κB translocation. These results indicate equivalent and significant heparin anti-inflammatory properties at low doses on monocyte-mediated immune response. The inhibition of NF-κB 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.
Unfractionated heparin (UFH) and low-molecular weight heparin (LMWH) are highly sulphated proteoglycans that are widely applied as anticoagulant drugs. They represent pivotal agents for the prevention and treatment of thromboembolic disorders, pulmonary embolism, disseminated intravascular coagulation and unstable angina (Hirsh et al, 2001). Their anticoagulant mechanism of action resides in their ability to increase the activity of an endogenous coagulation factor, antithrombin III, which inhibits two serine proteases involved in the coagulation cascade: factor IIa or thrombin and factor Xa (Bourin & Lindahl, 1993). Recently, a number of clinical and experimental studies have suggested that, aside from their anticoagulant capacity, heparins affect inflammatory responses. The accumulation of leucocytes in inflamed brain (Yanaka & Nose, 1996), skin (Teixeira & Hellewell, 1993) and lung (Seeds & Page, 2001) have been reduced by heparin in animal studies, and in several clinical trials heparin appeared to have the potential to treat inflammatory bowel disease (IBD) (Gaffney et al, 1991, 1995; Evans et al, 1997), arthritis (Gaffney & Gaffney, 1996), rhinitis (Vancheri et al, 2001) and human asthma (Page, 1991; Diamant et al, 1996), with an anti-inflammatory effect dissociable from its anticoagulant activity (Lever & Page, 2002). Experimental studies have shown that UFH and a variety of heparin derivatives inhibit poinflammatory cytokine gene expression by lipopolysaccharide (LPS)-stimulated human mononuclear cells (Hogasen & Abrahamsen, 1995; Attanasio et al, 1998; Gori et al, 2004); however, their precise mechanism of action is still unknown. In addition, heparin and o-desulphated heparin have been reported to inhibit the nuclear factor (NF)-κB activation in a tumour necrosis factor (TNF)-α-stimulated human endothelial cell line and in ischaemic-reperfused rat myocardium (Thourani et al, 2000). LMWH also appeared to inhibit the nuclear translocation of NF-κB in high glucose-stimulated human endothelial cells (Manduteanu et al, 2003) and in T cells (Hecht et al, 2004).
The addition of LPS to cells results in activation of the transcription factor NF-κB, which plays crucial roles in regulating the expression of many proinflammatory cytokine genes involved in the inflammatory responses, such as those encoding for TNF-α, interleukin (IL)-8, IL-6 and IL-1β (Baldwin, 1996; Yao et al, 1997). The increased production of TNF-α and IL-1β by NF-κB translocation causes its activation in return, leading to a positive regulatory loop that amplifies local inflammation (Barnes & Karin, 1997). NF-κB activation, a critical phenomenon in host inflammatory response, is implicated in a wide range of diseases, such as sepsis, IBD, asthma and rheumatoid arthritis (Bohrer et al, 1997), and therefore represents an ideal molecular target. The aim of this study was to evaluate and compare the effect of an unfractionated and a LMWH on the production of proinflammatory cytokines and NF-κB activation in LPS-stimulated human monocytes.
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- Materials and methods
Heparin and related glycosaminoglycans are increasingly recognised as modulators of the inflammatory process. Heparin and its derivatives inhibit the activation of inflammatory cells, their cytokine expression and production (Gori et al, 2004) and also their adhesion to vascular endothelial cells (Lever et al, 2000; Smailbegovic et al, 2001). Monocytes play a pivotal role in the host inflammatory response, by producing a wide variety of proinflammatory cytokines. It was therefore of interest to elucidate and understand the mechanism by which heparin exerts its effect on these cells.
We examined the regulation of LPS-induced proinflammatory cytokines release by LMWH and UFH pretreated monocytes. As expected, the production of TNF-α, IL-8, IL-6 and IL-1β was upregulated by a nanomolar dose of LPS as a result of its binding to the monocyte surface and the subsequent intracellular signalling leading to proinflammatory cytokines gene transcription. The cytokines measured in our experiments are largely involved in the upregulation of inflammatory reactions and thus in numerous inflammatory diseases. The level of cytokine produced by LPS-stimulated monocytes was compared to that of cells pretreated with LMWH and UFH at two different, pharmacologically relevant, concentrations (1 and 0·1 IU/ml) prior to LPS addition [therapeutic heparin levels are equivalent to 0·3–0·7 IU/ml for the treatment of venous thrombosis (Hirsh & Raschke, 2004)]. All cytokine amounts measured were significantly reduced when the cells were treated with heparin, up to 18% on average with 1 IU/ml LMWH, 26% with 0·1 IU/ml LMWH, 24·6% with 1 IU/ml UFH and 29·3% with 0·1 IU/ml UFH. It appeared that the lower heparin concentration (0·1 IU compared with 1 IU) induced a stronger decrease in inflammation. Heparin molecules, either LMWH or UFH, are negatively charged and may aggregate or interact with other components in the medium during incubation, preventing their efficient interaction with monocytes. Conversely, smaller and less numerous molecules possess better access to cell receptors. However, no significant statistical difference was apparent between different heparin treatments.
These results demonstrated an inhibition of LPS induction of proinflammatory cytokines in heparin pretreated cells, and are in agreement with recent similar studies performed on mononuclear cells (Attanasio et al, 1998; Gori et al, 2004). Heparin may therefore represent a potential inhibitory agent in chronic inflammatory diseases, by reducing the release of proinflammatory cytokines.
Lipopolysaccharide activation of monocytes leads to the initiation of proximal signalling events, which result in the increased transcription and stability of proinflammatory cytokine messenger RNA. Increased transcription requires the concerted binding of NF-κB, c-jun and Egr-1 to the promoter region of a wide variety of proinflammatory cytokine genes, such as TNF-α gene (Yao et al, 1997). We showed previously that activated protein C, an anticoagulant that protects against sepsis, inhibited the NF-κB translocation in the LPS-stimulated monocytic cell line THP-1 (White et al, 2000). To further investigate the anti-inflammatory mechanism of heparin action, we therefore examined its effect on monocyte LPS-mediated NF-κB activation. An accurate monitoring of NF-κB activation in cells is crucial for signal transduction pathway analysis, and we demonstrated here that LPS-induced NF-κB translocation from the cytoplasm to the nucleus was significantly reduced in heparin pretreated cells, even at low heparin doses, as shown by the 30% reduction induced with 0·1 IU/ml UFH. These results were further supported by the observation of a downregulation in TNF-α levels expressed by monocytes under the exact experimental conditions. Our findings on the downregulation of NF-κB activation in monocytes by heparin are similar to findings on heparin effects on NF-κB activation in stimulated endothelial cells. Thourani et al (2000) showed that o-desulphated heparin downregulated NF-κB-mediated activation of TNF-α-stimulated human umbilical vascular endothelial cells. Similarly a study by Manduteanu et al (2003) demonstrated a significant inhibition of NF-κB expression by a LMWH in high glucose-stimulated human endothelial cells.
Nuclear factor-κB activation is considered to be an amplifying and perpetuating mechanism of the inflammatory process, and is implicated in a wide range of inflammatory diseases. NF-κB regulates the expression of many genes whose products are chemokines, immune receptors and adhesion molecules, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin, involved in the recruitment of inflammatory cells from the circulation to the site of inflammation. Its activation is also vital for proinflammatory cytokines regulation (Baeuerle & Baichwal, 1997). This transcription factor therefore represents an excellent pharmacological target as it promotes the transcription of the TNF-α gene, among others; and anti-TNF-α therapy has been found to be beneficial in rheumatoid arthritis and Crohn's disease (Feldmann et al, 1994). The inhibition of NF-κB would interrupt the positive inflammatory feedback loop generated by TNF-α and IL-1β, and globally downregulate the production of a wide range of proinflammatory molecules. It is this particular property of NF-κB that makes it such an attractive molecular target for novel anti-inflammatory therapies and offers an explanation for the heparin effects. The ability of heparin to downregulate LPS-induced NF-κB activation may be of considerable clinical significance according to the importance of the latter in inflammatory disorders.
In conclusion, these results demonstrate that pharmacologically relevant doses of heparin possess the ability to inhibit LPS-induced proinflammatory cytokines and nuclear translocation of NF-κB in human monocytes; and indicate a potential mechanism responsible, in part, for the protective effect of the drug in inflammatory disorders. The heparin inhibition of NF-κB translocation identifies a novel and important immunomodulatory pathway. In clinical practice, heparin use as an anti-inflammatory agent is restricted by its potential to induce bleeding complication, though novel, anti-inflammatory non-haemostatically active heparin derivatives are being developed (Gori et al, 2004). Further investigations are required in this area, in order to optimise the development of such inhibitory agents of the inflammatory process and also to elucidate the molecular mechanism of action of heparin and its derivatives.