Leukocyte-endothelial cell interactions, which are mediated by various adhesion molecules, are a crucial event in inflammatory reactions including atherosclerosis. alpha-tocopherol (alpha-Toc) has been used for therapy of vascular diseases because of its antioxidant activity. However, the effect of alpha-Toc on inflammatory reactions has not been investigated very well. In the present study, we examined the effect of alpha-Toc on expression of adhesion molecules on human neutrophils and human umbilical vein endothelial cells (HUVEC). Expression of CD11a, CD11b and CD18 on neutrophils was assessed by immunofluorescence flow cytometry 30 min after the stimulation of neutrophils with 10(-7) M platelet-activating factor (PAF). Surface expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on HUVEC was evaluated by enzyme immunoassay 8 h after the incubation of HUVEC with IL-1 beta (20 U/ml). PAF induced upregulation of CD11b and CD18 on neutrophils and IL-1 beta increased surface expression of ICAM-1 and VCAM-1 on HUVEC. Coincubation of neutrophils with alpha-Toc and pretreatment of HUVEC with alpha-Toc significantly reduced PAF-induced CD11b/CD18 expression and IL-1 beta-induced upregulation of ICAM-1 and VCAM-1, respectively. These findings indicate that alpha-Toc may work as an anti-inflammatory agent through inhibiting neutrophil-endothelial cell adhesive reactions.

We evaluated the effects of ␣-Toc on surface expression of CD11b/CD18 on polymorphonuclear leukocytes (PMN) stimulated with N-formylmethionyl-leucyl-phenylalanine (fMLP) and oxidized low-density lipoprotein (oxLDL). Incubation of PMN with fMLP (1 M) or oxLDL (100 g/mL) increased CD11b/CD18 expression; pretreatment with ␣-Toc reduced in a dose-dependent manner. PMN obtained from healthy adults ingesting 600 mg ␣-Toc per day for 10 days were similarly incubated with fMLP or oxLDL; the surface level of CD11b/CD18 was inversely correlated with serum ␣-Toc concentrations. Adherence of PMN to human umbilical vein endothelial cells was increased by fMLP or oxLDL stimulation but reduced by ␣-Toc pretreatment or anti-CD18 monoclonal antibodies. cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) activity in PMN was also assayed. A PKC inhibitor, but not a PKA inhibitor, suppressed CD11b/CD18 up-regulation, and ␣-Toc slightly decreased fMLP-and oxLDL-induced activation of PKC. These results suggest that ␣-Toc may prevent inflammation by both reducing CD11b/ CD18 up-regulation and decreasing PMN-dependent adherence to EC. J. Leukoc. Biol. 65: 757-763; 1999.

Leukocyte-endothelial cell interactions, which are mediated by various adhesion molecules, are a crucial event in inflammatory reactions including atherosclerosis. Alpha-tocopherol (alpha-Toc) has been used for protection and therapy of vascular diseases because of its antioxidant activity. The objective of the present study was to determine effect of alpha-Toc on endothelial-dependent adhesive interactions with leukocytes elicited by oxidized low density lipoprotein (oxLDL). Incubation of HUVEC with oxLDL (100 microg/mL) increased expression of proteins and messenger RNA of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on enzyme immunoassay and northern blotting assay; pretreatment with alpha-Toc reduced in a dose dependent manner. Adherence of polymorphonuclear leukocytes (PMN) or mononuclear leukocytes (MNC) to oxLDL-activated HUVEC was much increased compared with that to unstimulated HUVEC. Treatment of HUVEC with alpha-Toc, monoclonal antibody to ICAM-1 or VCAM-1 inhibited adherence of PMN or MNC in a dose dependent manner. These results suggest that alpha-Toc works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with leukocytes induced by oxLDL.

Alpha-tocopherol supplementation is reported to protect against cardiovascular disease and to influence cells involved in atherogenesis, such as monocytes. Interactions between monocytes and vascular endothelial cells occur early in atherogenesis, and adhesion is mediated by integrins. We evaluated the effects of alpha-tocopherol on expression of Mac-1 (CD11b/CD18) by monocytes after stimulation with oxidized low-density lipoprotein (LDL), which is implicated as a potent chemotactic agent in atherogenesis. Incubation of whole blood with oxidized LDL (100 microg/ml) increased Mac-1 expression on monocytes, and preincubation with alpha-tocopherol reduced this upregulation in a concentration dependent manner. In another experiment, whole blood was obtained from healthy adult volunteers after 10 days of alpha-tocopherol administration (600 mg/day) and was incubated with oxidized LDL (100 microg/ml). There was a decrease in the upregulation of Mac-1 compared with that measured before administration. Adherence of oxidized LDL-stimulated monocytes to human umbilical vein endothelial cells was reduced by pretreatment with alpha-tocopherol, and was also inhibited by an anti-CD18 monoclonal antibody. Experiments with protein kinase C inhibitors suggested that reduction of Mac-1 upregulation by alpha-tocopherol was secondary to a decrease of protein kinase C activity. In conclusion, alpha-tocopherol suppressed the upregulation of Mac-1 expression on monocytes by oxidized LDL.

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