Activated leukocytes, endothelial cells, and effects of pentoxifylline: observations by VEC-DIC microscopy

Using video-enhanced contrast (VEC)-differential interference contrast (DIC) microscopy, ultrastructural observations were made of the activation of polymorphonuclear leukocytes (PMNLs), the interaction between activated PMNLs and endothelial cells (ECs), and the effects of pentoxifylline (PTX). The ECs were obtained from a commercial source as human umbilical cord vein endothelial cells (HUVECs) or were obtained from pig or rat brains. They were cultured on a coverglass with DMEM for about 1 week. The human PMNLs were obtained from the authors' venous blood. The control appearance of the PMNLs resembled an elastic ball covered with fine villi. The PMNL was activated spontaneously and became flattened on the glass surface within 10 min in the observation chamber. The activation of the PMNLs was estimated arbitrarily from the polymorphous changes in cell shape, agitation of the intracellular granules, and apparent increase in adhesiveness. Preadministered PTX prevented such PMNL activation, and the PMNLs remained round for more than 15 min. PMNL activation was accelerated by chemoattractants (PAF, fMLP, and PMA). In one case, a PMNL that had been activated by PMA tended to recover its round shape with PTX, but finally ended by swelling and bursting. When PMNLs were introduced into the EC-containing chamber, they became entrapped by the ECs and activated, with degranulation followed by release of a smoke-like material. After about 3 h, the EC with an attached PMNL shrank and fell into a state of coagulation necrosis. When PTX was introduced at the time of adhesion of the flattened PMNL, the PMNL appeared to be deactivated, becoming smaller and assuming its previous round shape, and detached from the EC. PTX prevented the spontaneous activation of PMNLs, and of deactivated PMNLs even after their adherence to the endothelium.