Adhesion of monocytes to the arterial endothelium is a major mechanism in the initiation and development of atherosclerotic plaques. It is possible to investigate the mechanisms of this adhesion in vitro by measuring the binding of the U937 monocyte cell line onto unfixed histological frozen transverse sections of human atherosclerotic lesions (Poston & Johnson-Tidey, 1996). HSP60 is a stress protein that is expressed in the arterial endothelium and macrophages of human atherosclerotic plaques, and recently Kol et al. (1999) demonstrated that HSP60 activates human macrophages via the surface receptor CD14. Poston & Johnson-Tidey (1996) have shown that a number of monoclonal antibodies to CD14 inhibited the binding of blood monocytes and U937 cells to the lesions.

Carotid endarterectomy specimens were obtained with the permission of the Ethical Committee of St Thomas’ and Guy’s Hospitals. We now report that two monoclonal antibodies to epitopes of HSP60 expressed on cell surface membranes (ML-30 and II-13, 20 µg ml^-1) significantly reduced (P < 0.05, ANOVA) U937 cell adhesion to the arterial endothelium to respectively 37 ± 16 (S.D.) and 36 ± 8 % of control (4 patients, each artery measured 4 times). Two further antibodies (LK-1 and 4B9/89) to other epitopes similarly significantly reduced adhesion to 59 ± 22 and 57 ± 30 % of control. Adhesion to the intimal connective tissue was also inhibited.

We investigated the binding of fluorescein labelled U937 cells to solid phase recombinant HSP60 to test the idea that the adhesion interaction is between plaque HSP60 and monocyte CD14. U937 cells were incubated with 5-and 6-) carboxyfluorescein diacetate succinimidyl ester (C-1157, Molecular Probes) for 30 min, washed and added for 1 h to black 96-well plates (Corning). These had been previously coated with HSP60 or other proteins by incubation overnight in bicarbonate buffer (pH 9.0). Cell binding was measured, after washing, in an automatic fluorescence spectrophotometer. 37 ± 7 % of the total U937 cells bound to HSP60 (three experiments, triplicate measurements), which was comparable with that induced by fibronectin (30 %) or vitronectin (42 %). Binding to control uncoated wells was < 10 %. The HSP60 interaction was significantly inhibited (P < 0.05, two-tailed t test) by CD14 antibody, to 6.3 ± 5.5 % of cells at 1.6 µg ml^-1. In contrast the same antibody had little effect on binding induced by fibronectin (97 ± 20 % of the controls) or that of vitronectin (100 ± 8 %).

In conclusion, HSP60 behaves as a monocyte binding adhesion molecule through its affinity for CD14, and this interaction can be implicated in the adhesion of monocytes to human atherosclerotic plaques.

We thank the Special Trustees of Guy’s Hospital for financial support, and Ben Mahmud for introducing us to the carboxy-fluorescein cell labelling technique.