Size-fractionated heparins have differential effects on human neutrophil function in vitro

Abstract

Background and purpose: Heparin is known to possess a range of activities, other than effects on blood coagulation, many of which are anti-inflammatory. Effects with potential anti-inflammatory applications include the inhibition of elastase release from neutrophils, as well as the adhesion of these cells to vascular endothelium. In the present study we aimed to investigate whether fractionation of heparin may yield molecules with enhanced or specific effects on human neutrophil function.

Experimental approach: Fractions of defined molecular size were obtained from heparin by different methods and assessed for their effects on elastase release induced by formyl Met-Leu-Phe (fMLP), from neutrophils, in some cases following the priming of these cells with tumour necrosis factor-alpha (TNF-alpha). Effects of the fractions on neutrophil adhesion to interleukin-1beta (IL-beta)-stimulated human umbilical vein endothelial cells (HUVECs) were also examined.

Key results: Elastase release was inhibited by very low molecular weight fractions of heparin, with an apparent minimum chain length of 10 saccharides required for full effect. In contrast, neutrophil-endothelial adhesion was unaffected by these fractionated heparins, suggesting that certain non-anticoagulant actions of heparin may be lost by such an approach.

Conclusions and implications: These data suggest that an optimum chain length of heparin possibly exists for certain non-anticoagulant actions of heparin, which may prove to be useful in the design of novel drugs with specific anti-inflammatory actions.

Figure 1

Effects of heparinase-derived LMWH fractions on fMLP-induced elastase release from (a) unprimed and (b) TNF-α-primed neutrophils. Data are expressed as % of control elastase release (fMLP; 10⁻⁷ M), without heparin in (a) saline- and (b) TNF-α (10 ng ml⁻¹)-treated cells, respectively. Values were corrected for basal release under the relevant conditions and represent the mean±s.e.m of experiments with cells from six separate donors, each performed in duplicate. Statistical significance (P<0.05) is indicated (^*) from control release (100%). fMLP, formyl-Met-Leu-Phe; LMWH, low molecular weight heparin.

Figure 2

Effects of UH on fMLP and A23187-induced neutrophil elastase release. (a) A23187 (3 × 10⁻⁸ M) and fMLP (10⁻⁷ M) elicited a similar level of elastase release from neutrophils, both with and without prior priming with TNF-α (10 ng ml⁻¹). UH inhibits this release of elastase (b) in response to fMLP (10⁻⁷ M) but (c) not in response to A23187 (3 × 10⁻⁸ M). Data are expressed as % of control elastase release (fMLP or A23187 without heparin, primed or unprimed cells as appropriate), corrected for basal release under the relevant conditions and represent the mean±s.e.m of experiments with cells from 6 to 8 separate donors, each performed in duplicate. Statistical significance (P<0.05) is indicated (^*) from control release (100%). fMLP, formyl-Met-Leu-Phe; UH, unfractionated heparin.