EFFECT OF VERAPAMIL ON LYMPHOCYTE INFILTRATION

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We read with great interest the report concerning the effect of verapamil on lymphocyte adherence to human vascular endothelial cells by Yamaguchi et al. (Transplantation 1997; 63: 759, 1). They showed that verapamil selectively inhibits cytokine-induced protein and mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and suppresses cell adherence between tumor necrosis factor (TNF)-α-stimulated endothelial cells and mononuclear leukocytes. Based on these results, the authors concluded that verapamil may partly suppress the immune response via inhibition of VCAM-1 expression on endothelial cells and, therefore, might play an important role in the development of an improved immunosuppressive regimen. Findings in our own laboratories also clearly argue for a supporting role of verapamil in cyclosporine-based immunosuppressive therapy. Nevertheless, a significant influence of verapamil on cell adhesion, described by Yamaguchi and coworkers, seems weak and observed only at drug concentrations ≥50μM. Our separate investigations of the lymphocyte adhesion and penetration process showed that about 30% of all lymphocytes attach to endothelial cells. However, of these, less than 10% actually penetrate the endothelial monolayer(2). It should be clearly emphasized that these penetrating lymphocytes, and not the adherent cells, play the predominant role in the initiation of organ rejection. Taking this into account, verapamil does indeed moderately act on lymphocyte adhesion. However, and this is of major importance, verapamil strongly inhibits lymphocyte penetration through allogeneic endothelial cells (3).1 Interestingly, and in accordance with other studies, the efficacy of verapamil can be greatly enhanced when cells have been preincubated with the drug for 24 hr. With this in mind, we calculated the ID50 value to be 11 μM (CD4 T-cell penetration1). Yamaguchi et al. hypothesized that diminished VCAM-1 expression on endothelial cells is responsible for the immunosuppressive effect of verapamil. Surprisingly, the endothelial adhesion receptors intercellular adhesion molecule-1 (ICAM-1), E-selectin, and VCAM-1 were all analyzed 4 hr after cytokine stimulation, although these molecules are characterized by different expression kinetics. ICAM-1 up-regulation is optimal after 12-72 hr, de novo expression of E-selectin after 6-8 hr, and de novo expression of VCAM-1 at 12 hr after interleukin (IL)-1 stimulation (2). Moreover, the authors did not carry out binding experiments of verapamil-treated lymphocytes to untreated endothelial cells to exclude any effects of verapamil on leukocytes. Additionally, taking into account the high verapamil concentrations used (≥50 μM, IL-1-stimulated endothelial cells), their conclusion seems speculative and is not supported by our experiments. Fluorescence-analysis in our laboratory of ICAM-1, E-selectin, and VCAM-1 on IL-1 or TNF-stimulated endothelial cells (cytokine incubation: 6-14 hr) revealed no influence of verapamil on VCAM-1 expression and even an up-regulation of ICAM-1 and E-selectin receptors (4, 5). A slight, but unspecific, down-regulation of VCAM-1 was observed only when endothelial cells were incubated with verapamil concentrations >100μM. On the other hand, verapamil significantly reduced F-actin elements in lymphocytes and effectively suppressed the binding activity of CD4 and CD8 T cells to P-selectin (ID50=4.4 μM; CD4+[3]). Furthermore, separate incubation of CD4 or CD8 T cells, but not of endothelial cells, with verapamil (0-100 μM) blocked the penetration process in a concentration-dependent manner. These results clearly indicate that verapamil predominantly acts on lymphocytes but not on endothelial cells. In good accordance with our data, Bacon et al. have demonstrated an inhibitory effect of verapamil on the chemotactic activity of lymphocytes (6). Potent inhibition of the migration capacity of neutrophils, by verapamil, also reveals that the mode of action of verapamil is directed against leukocytes(7-9). This mode of action highlights the therapeutic value of verapamil (especially when the R-enantiomer is used). The combined use of cyclosporine and verapamil might produce the desired dualistic modulation of the immune system, by interfering in both lymphocyte proliferation and the infiltration cascade. Roman A. Blaheta2 Bernd H. Markus Hospital of the Johann Wolfgang Goethe-University; Department of General Surgery; Frankfurt am Main, Germany

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