Neutrophils and their products are implicated in a number of acute and chronic inflammatory lung diseases. Neutrophil elastase (NE) possesses proteolytic properties thought to play a significant role in the pathogenesis and pathology of pulmonary diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Heparin has been shown to inhibit NE release from activated neutrophils (Brown et al., 2003) and also potently inhibits the enzymatic activity of NE itself (reviewed by Tyrell et al., 1999). In the present study, we investigated the effects of unfractionated heparin (UH) and corresponding concentrations of a low-molecular-weight heparin (LMWH) and the non-anticoagulant O-desulphated heparin (ODH), on NE-induced injury to cultured monolayers of primary human bronchial epithelial cells (HBECs), quantified through the number of cells that detach from the tissue culture plastic.

HBECs were cultured to confluence in 96-well plates and then washed and incubated with NE (0.1
Uml^-1) for 4 hours (at 37°C, 5% CO₂) in the presence of medium alone or different concentrations of UH, LMWH (0.01-100 Uml^-1) or ODH (0.06-600 µgml^-1). The HBEC monolayers were then washed to remove detached cells and the remaining cells fixed, stained and counted. Remaining HBECs were expressed as a percentage of the number of cells present in untreated wells. After counting the cells, all data are reported as means ± standard error of the mean (SEM) from n experiments. Statistical analysis was made by repeated measures ANOVA, followed by Dunnett's test.

UH potently inhibited NE-induced detachment in a concentration-dependent manner. In the presence of NE only, 31.3±3.2% of HBECs remained, whilst co-incubation of NE with UH resulted in a maximum of 97.1±3.8% of HBECS remaining (1Uml^-1, p<0.01, n=6). LMWH had a similar effect (NE only, 28.2±2.6% of HBECs remained; NE plus LMWH, 99.1±2% of HBECs remained (1Uml^-1, p<0.01, n=6)). ODH also potently inhibited the effects of elastase (NE only 24.8±2.7% of HBECs remained; NE plus ODH, 101.9±0.7% of HBECs remained (60µgml^-1, p<0.01, n=6)).

In conclusion therefore, heparin abrogates the cytotoxic effects of NE upon HBECs, in a manner independent of its molecular size and anticoagulant properties. Thus, the ability of heparin to not only inhibit NE release, but also potently inhibit cellular damage mediated by NE, may provide the basis for the design of novel therapeutics for the treatment of airway diseases associated with inappropriate or excessive activity of neutrophils.

Brown et al., 2003. Br. J. Pharmacol. 139; 845-853.
Tyrell et al., 1999. Adv. Pharmacol. 46; 151-208.