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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Fig 1. In-vitro interleukin release.

Trypsin-induced release of IL-6 and IL-8 from the epithelial cell culture supernatants. Data is presented as mean ± SD.

Fig 2. Concentration-dependent effect of enoxaparin on interleukin release.

Effect of different concentration of enoxaparin on the release of IL-6 (A) and IL-8 (B) following trypsin-induced in-vitro stimulation epithelial cells. Data is presented as percentage (mean ± SD) of the maximal observed IL-6 and IL-8 concentrations. *p<0.05, and ***p<0.001 versus trypsin-stimulated control.

Fig 3. Effect of enoxaparin on cellular viability and proliferation.

(A) Viability of epithelial cells was determined by trypan blue dye exclusion test and is presented as % of viable cells remaining after 24 hours of incubation with trypsin, enoxaparin or trypsin + enoxaparin. Data is presented as mean ± SD. (B) Proliferation of epithelial cells was determined after 24 hours in the presence of trypsin, enoxaparin or trypsin + enoxaparin. Cells were counted after incubation for 24 hours and expressed as 10⁵ cells/mL. Data is presented as mean ± SD. *p<0.05 versus unstimulated control. (C) Viability of epithelial cells is presented as mean % LDH release ± SD. Cells were incubated for 24 hours in the presence of trypsin, enoxaparin or trypsin + enoxaparin.

Fig 4. Effect of enoxaparin fractions on interleukin release.

Ion-exchange chromatographic (IC) separation of enoxaparin. The separations were performed using a CarboPac PA100 semi-preparative column; optimised NaCl gradient was from 32–74% over 70 minutes with a flow rate of 2 mL/minute and UV detection wavelength of 232 nm. The numbers indicate the area of all the fractions collected. Data represents a typical experiment out of ten independent experiments (A). Inhibition of IL-6 (B) and IL-8 (C) release by IC-derived 14 fractions of enoxaparin (60 μg/mL) after trypsin-induced in-vitro stimulation of epithelial cells. The relative percentile amount of each fraction (fraction 1 to 14) present in 60 μg/mL of intact enoxaparin was: 9.18%, 4.24%, 6.12%, 5.15%, 2.99%, 4.78%, 20.32%, 11.4%, 10.53%, 7.35%, 5.98%, 4.6%, 2.95% and 5.06% respectively. Data is presented as percentage (mean ± SD) of the maximal observed IL-6 and IL-8 concentrations. *p<0.05, and ***p<0.001 versus trypsin-stimulated control.

Fig 5. Concentration-dependent effect of IC-derived fraction 1 on interleukin release.

Inhibition of IL-6 and IL-8 release by fraction 1 of enoxaparin (0 to 25 μg/mL) after in-vitro stimulation of epithelial cells via trypsin. Data is presented as percentage of trypsin-stimulated control (mean ± SD). **p<0.01 and ***p<0.001 versus trypsin-stimulated control.

Fig 6. Effect of sub-fractions of IC-derived fraction 1 on interleukin release.

Inhibition of IL-6 (A) and IL-8 (B) release by enoxaparin fraction 1 and its three different sub-fractions (1A, 1B and 1C) after trypsin-induced in-vitro stimulation of epithelial cells. Data is presented as percentage (mean ± SD) of the maximal observed IL-6 and IL-8 concentrations. ***p<0.001 versus trypsin-stimulated control.

Fig 7. ¹³C-¹H HSQC multiplicity edited spectrum for enoxaparin sub-fraction 1A.

Axis units are chemical shifts in parts per million (ppm). The dark blue contours denote correlations between carbon atoms with one or three attached protons and cyan contours carbons with a single attached proton. The atom nomenclature from Table 1 is applied to the proton and carbon axis projections.

Fig 8. ¹³C-¹H HSQC-TOCSY 120ms spectrum for enoxaparin sub-fraction 1A.

Axis units are chemical shifts in parts per million (ppm). The atom nomenclature from Table 1 is applied to the proton and carbon axis projections.

Fig 9. Anti-factor Xa activity of fourteen enoxaparin fractions derived using IC technique.

The anti-factor Xa assay is described in the experimental section. Data is presented as mean ± SD (n = 3).

Fig 10. Effect of various desulfated fraction 1A on IL-6 release.

Epithelial cells were stimulated with trypsin in the presence of either 2-O-desulfated, N-desulfated or 6-O-desulfated fraction 1A of enoxaparin. Data is presented as mean ± SD. ***p<0.001 versus trypsin-stimulated control.

Fig 11. Binding of proteins with disaccharide of enoxaparin.

Interaction of UFH with thrombin. HP-SEC chromatograms of UFH (10 μM), thrombin (10 μM) and a mixture of UFH/thrombin (1:1 molar ratio) (A). Interaction of IC-derived disaccharide with trypsin. HP-SEC chromatograms of enoxaparin disaccharide (500 μM), trypsin (10 μM) and a mixture of trypsin/disaccharide (1:50 molar ratio) (B). Details of the HP-SEC experimental conditions are given in Methods.