In-vitro suppression of IL-6 and IL-8 release from human pulmonary epithelial cells by non-anticoagulant fraction of enoxaparin.

• Main Authors: Madhur D Shastri, Niall Stewart, James Horne, Gregory M Peterson, Nuri Gueven, Sukhwinder S Sohal, Rahul P Patel
• Format: Article
• Language: English
• Published: Public Library of Science (PLoS) 2015-01-01
• Series: PLoS ONE
• Online Access: https://doi.org/10.1371/journal.pone.0126763

<h4>Background</h4>Enoxaparin, a mixture of anticoagulant and non-anticoagulant fractions, is widely used as an anticoagulant agent. However, it is also reported to possess anti-inflammatory properties. Our study indicated that enoxaparin inhibits the release of IL-6 and IL-8 from A549 pulmonary epithelial cells. Their release causes extensive lung tissue damage. The use of enoxaparin as an anti-inflammatory agent is hampered due to the risk of bleeding associated with its anticoagulant fractions. Therefore, we aimed to identify the fraction responsible for the observed anti-inflammatory effect of enoxaparin and to determine the relationship between its structure and biological activities.<h4>Methods</h4>A549 pulmonary epithelial cells were pre-treated in the presence of enoxaparin and its fractions. The levels of IL-6 and IL-8 released from the trypsin-stimulated cells were measured by ELISA. The anticoagulant activity of the fraction responsible for the effect of enoxaparin was determined using an anti-factor-Xa assay. The fraction was structurally characterised using nuclear magnetic resonance. The fraction was 2-O, 6-O or N-desulfated to determine the position of sulfate groups required for the inhibition of interleukins. High-performance size-exclusion chromatography was performed to rule out that the observed effect was due to the interaction between the fraction and trypsin or interleukins.<h4>Results</h4>Enoxaparin (60 μg/mL) inhibited the release of IL-6 and IL-8 by >30%. The fraction responsible for this effect of enoxaparin was found to be a disaccharide composed of α-L-iduronic-acid and α-D-glucosamine-6-sulfate. It (15 μg/mL) inhibited the release of interleukins by >70%. The 6-O sulphate groups were responsible for its anti-inflammatory effect. The fraction did not bind to trypsin or interleukins, suggesting the effect was not due to an artefact of the experimental model.<h4>Conclusion</h4>The identified disaccharide has no anticoagulant activity and therefore eliminates the risk of bleeding associated with enoxaparin. Future in-vivo studies should be designed to validate findings of the current study.