LEUKOCYTE-ENDOTHELIAL INTERACTION IS AUGMENTED BY HIGH GLUCOSE-CONCENTRATIONS AND HYPERGLYCEMIA IN A NF-KAPPA-B-DEPENDENT FASHION

We addressed the role of hyperglycemia in leukocyte-endothelium intera ction under flow conditions by exposing human umbilical vein endotheli al cells for 24 h to normal (5 mM), high concentration of glucose (30 mM), advanced glycosylation end product-albumin (100 mu g/ml), or hype rglycemic (174-316 mg/dl) sera from patients with diabetes and abnorma l hemoglobin A(1c) (8.1+/-1.4%). At the end of incubation endothelial cells were perfused with total leukocyte suspension in a parallel plat e flow chamber under laminar flow (1.5 dyn/cm(2)). Rolling and adheren t cells were evaluated by digital image processing. Results showed tha t 30 mM glucose significantly (P < 0.01) increased the number of adher ent leukocytes to endothelial cells in respect to control (5 mM glucos e; 151+/-19 versus 33+/-8 cells/mm(2)). A similar response was induced by endothelial stimulation with IL-1 beta, here used as positive cont rol (195+/-20 cells/mm(2)). The number of rolling cells on endothelial surface was not affected by high glucose level. Stable adhesion of le ukocytes to glucose-treated as well as to IL-1 beta-stimulated endothe lial cells was preceded by short interaction of leukocytes with the en dothelial surface. The distance travelled by leukocytes before arrest on 30 mM glucose, or on IL-1 beta-treated endothelial cells, was signi ficantly (P < 0.01) higher than that observed for leukocytes adhering on control endothelium (30 mM glucose: 76.7+/-3.5; IL1 beta: 69.7+/-4 versus 5 mM glucose: 21.5+/-5 mu m). Functional blocking of E-selectin , intercellular cell adhesion molecule-1, and vascular cell adhesion m olecule-1 on endothelial cells with the corresponding mouse mAb signif icantly inhibited glucose-induced increase in leukocyte adhesion (67+/ -16, 83+/-12, 62+/-8 versus 144+/-21 cells/mm(2)). Confocal fluorescen ce microscopy studies showed that 30 mM glucose induced an increase in endothelial surface expression of E-selectin, intercellular cell adhe sion molecule-1, and vascular cell adhesion molecule-1. Electrophoreti c mobility shift assay of nuclear extracts of human umbilical vein end othelial cells (HUVEC) exposed for 1 h to 30 mM glucose revealed an in tense NF-kB activation. Treatment of HUVEC exposed to high glucose wit h the NF-kB inhibitors pyrrolidinedithiocarbamate (100 mu M) and tosyl -phe-chloromethylketone (25 mu M) significantly reduced (P < 0.05) leu kocyte adhesion in respect to HUVEC treated with glucose alone. A sign ificant (P < 0.01) inhibitory effect on glucose-induced leukocyte adhe sion was observed after blocking protein kinase C activity with stauro sporine (5 nM). When HUVEC were treated with specific antisense oligod esoxynucleotides against PKC alpha and PKC epsilon isoforms before the addition of 30 mM glucose, a significant (P < 0.05) reduction in the adhesion was also seen. Advanced glycosylation end product-albumin sig nificantly increased the number of adhering leukocytes in respect to n ative albumin used as control (110+/-16 versus 66+/-7, P < 0.01). Sera from diabetic patients significantly (P < 0.01) enhanced leukocyte ad hesion as compared with controls, despite normal levels of IL-1 beta a nd TNF alpha in these sera. These data indicate that high glucose conc entration and hyperglycemia promote leukocyte adhesion to the endothel ium through upregulation of cell surface expression of adhesive protei ns, possibly depending on NF-kB activation.