Effects of pentoxifylline on the different steps during adhesion and transendothelial migration of flowing neutrophils

We used a flow system to observe the stepwise adhesion and migration of neu trophils on cultured human umbilical vein endothelial cells (HUVEC) stimula ted with tumour necrosis factor-alpha (TNF) for 4h, and to evaluate the eff ects of pentoxifylline (PTX) at each step. When HUVEC had been stimulated w ith 100 U ml(-1) TNF, treatment of neutrophils with PTX did not reduce the number captured from flow but did cause nearly all adherent cells (> 90%) t o roll, whereas most untreated cells became immobilized and similar to 30% transmigrated within minutes. On washout of the PTX, many rolling cells hal ted and started to migrate. Treatment of the HUVEC with PTX at the same tim e as 100 U ml(-1) TNF did not affect the number of neutrophils adhering, bu t there was a significant increase in the percentage of cells rolling even though PTX was no longer present. Thus PTX reduced presentation of activati ng agents by HUVEC, as well as inhibiting the response by neutrophils to su rface-presented activating agent(s). If HUVEC were stimulated with 10 U ml( -1) TNF with PTX, the adhesion of flowing neutrophils was greatly inhibited compared to TNF alone. Surface ELISA indicated that PTX reduced TNF-induce d upregulation of E-selectin, This reduction was only sufficient to reduce capture of neutrophils at the low dose of TNF. Thus, by using a flow-based model, we have been able to separate the effects of a multipotent agent suc h as pentoxifylline, which acts on leucocytes and endothelial cells, at eac h stage of migration. Copyright (C) 2001 John Wiley & Sons, Ltd.