The effects of heparin on the adhesion of human peripheral blood mononuclear cells to human stimulated umbilical vein endothelial cells

Abstract

1. The effects of unfractionated heparin (UH) and a selectively O-desulphated derivative of heparin (ODSH), lacking anticoagulant activity, on the adhesion of human peripheral blood mononuclear cells (HPBMNC) to human stimulated umbilical vein endothelial cells (HUVECs), were investigated. 2. For comparison, the effects of poly-L-glutamic acid (PGA), a large polyanionic molecule without sulphate groups and two different molecular weight sulphated dextrans (DS 5 k and DS 10 k) were studied. 3. UH (50 - 1000 u ml(-1)) significantly (P<0.05) inhibited the adhesion of HPBMNC to HUVECs, stimulated with IL-1beta (100 u ml(-1)), TNF-alpha (1000 u ml(-1)) or LPS (100 microg ml(-1)), when the drugs were added together with stimuli to HUVECs and coincubated for 6 h. Such effects on adhesion occurred with limited influence on expression of relevant endothelial adhesion molecules (ICAM-1 and VCAM-1). 4. UH (100 - 1000 u ml(-1)), when added to prestimulated HUVECs, significantly (P<0.05) increased adhesion of mononuclear cells to endothelium at the higher concentrations tested, without any effect on adhesion molecule expression. In contrast, the opposite effect was observed when human polymorphonuclear leucocyte adhesion was examined, under the same experimental conditions, suggesting that the observed potentiation of HPBMNC adhesion is cell specific. 5. The effects of UH on HPBMNC adhesion were shared by the non-anticoagulant ODSH (600 - 6000 microg ml(-1)) but not by sulphated dextrans or PGA (300 - 6000 microg ml(-1)). 6. Heparin affects the adhesion of HPBMNC to stimulated endothelium, in both an inhibitory and potentiating manner, effects which are unrelated to its anticoagulant activity and not solely dependent on molecular charge characteristics.

Figure 1

Effect of endothelial stimulation on HPBMNC adhesion and expression of adhesion molecules. HUVECs were stimulated with IL-1β (100 u ml⁻¹), TNF-α (1000 u ml⁻¹) or LPS (100 μg ml⁻¹) for 6 h. (a) ⁵¹Cr-labelled HPBMNCs were added for 30 min and adhesion was quantified by γ-counting lysates of adherent cells and calculating the percentage of cells added which had adhered. (b) Endothelial ICAM-1 (open bars) and VCAM-1 (closed bars) were measured by ELISA. Data are expressed as the mean±s.e.mean of six separate experiments, each performed in triplicate. ^*Statistical significance (P<0.05) is indicated.

Figure 2

Effect of heparin on HPBMNC adhesion to HUVECs. HUVECs were stimulated with (a) IL-1β (100 u ml⁻¹), (b) TNF-α (1000 u ml⁻¹) or (c) LPS (100 μg ml⁻¹) for 6 h, in the absence and presence of different concentrations of unfractionated heparin (UH). ⁵¹Cr-labelled HPBMNCs were added for 30 min. Adhesion was quantified by γ-counting lysates of adherent cells and was calculated as a percentage of that seen in control wells that had not been treated with heparin. Data are expressed as the mean±s.e.mean of six separate experiments, each performed in triplicate. ^*Statistical significance (P<0.05) is indicated.

Figure 3

Effect of heparin on endothelial adhesion molecule expression. HUVECs were stimulated with (a) IL-1β (100 u ml⁻¹), (b) TNF-α (1000 u ml⁻¹) or (c) LPS (100 μg ml⁻¹) for 6 h, in the absence and presence of different concentrations of unfractionated heparin (UH). ICAM-1 (open bars) and VCAM-1 (closed bars) were measured by ELISA. Adhesion molecule expression was calculated as a percentage of that seen in control wells that had not been treated with heparin. Data are expressed as the mean±s.e.mean of six separate experiments, each performed in triplicate. ^*Statistical significance (P<0.05) is indicated.

Figure 4

Effect of heparin on HPBMNC and PMN adhesion to pre-stimulated HUVECs. HUVECs were stimulated with IL-1β (100 u ml⁻¹), TNF-α (1000 u ml⁻¹) or LPS (100 μg ml⁻¹) for 6 h. ⁵¹Cr-labelled HPBMNCs (open bars) or PMNs (closed bars) were added for 30 min, in the absence and presence of different concentrations of unfractionated heparin (UH). Adhesion was quantified by γ-counting lysates of adherent cells and was calculated as a percentage of that seen in control wells^† that had not been treated with heparin. Data are expressed as the mean±s.e.mean of six separate experiments, each performed in triplicate. ^*Statistical significance (P<0.05) is indicated. ^†(Control PMN adhesion in wells stimulated with IL-1β, TNF-α or LPS was 19.16±1.56, 17.5±1.28 and 14.5±1.06%, respectively. Basal PMN adhesion was 7.74±1.62%).

Figure 5

Effects of O-desulphated heparin on HPBMNC adhesion to HUVECs under different experimental conditions. HUVECs were stimulated with (a) IL-1β (100 u ml⁻¹), (b) TNF-α (1000 u ml⁻¹) or (c) LPS (100 μg ml⁻¹) for 6 h, in the absence or presence of O-desulphated heparin (ODSH). ⁵¹Cr-labelled HPBMNCs were added for 30 min, either alone, to HUVECs stimulated in the presence of ODSH (open bars) or, in conjunction with different concentrations of ODSH, to HUVECs stimulated in the absence of the drug (closed bars). Adhesion was quantified by γ-counting lysates of adherent cells and was calculated as a percentage of that seen in control wells that had not been treated with ODSH. Data are expressed as the mean±s.e.mean of six separate experiments, each performed in triplicate. ^*Statistical significance (P<0.05) is indicated.

Figure 6

Effect of charged molecules on HPBMNC adhesion to HUVECs. HUVECs were stimulated with (a) IL-1β (100 u ml⁻¹), (b) TNF-α (1000 u ml⁻¹) or (c) LPS (100 μg ml⁻¹) for 6 h. ⁵¹Cr-labelled HPBMNCs were added for 30 min, in the absence and presence of different concentrations of poly-L-glutamic acid (PGA, open bars) or dextran sulphates with molecular weights of 5000 (DS 5 k, closed bars) and 10,000 (DS 10 k, hatched bars), respectively. Adhesion was quantified by γ-counting lysates of adherent cells and was calculated as a percentage of that seen in control wells that had not been treated with PGA or dextrans. Data are expressed as the mean±s.e.mean of six separate experiments, each performed in triplicate. ^*Statistical significance (P<0.05) is indicated.

Figure 7

Effect of charged molecules on HPBMNC adhesion to HUVECs. HUVECs were stimulated with (a) IL-1β (100 u ml⁻¹), (b) TNF-α (1000 u ml⁻¹) or (c) LPS (100 μg ml⁻¹) for 6 h, in the absence and presence of different concentrations of poly-L-glutamic acid (PGA, open bars) or dextran sulphates with molecular weights of 5000 (DS 5 k, closed bars) and 10,000 (DS 10 k, hatched bars), respectively. ⁵¹Cr-labelled HPBMNCs were added for 30 min and adhesion was quantified by γ-counting lysates of adherent cells and was calculated as a percentage of that seen in control wells that had not been treated with PGA or dextrans. Data are expressed as the mean±s.e.mean of six separate experiments, each performed in triplicate. ^*Statistical significance (P<0.05) is indicated.