Introduction: Tremendous advances have occured in the technical and anatomic areas of microsurgical transplantation. However, elucidation of the biomolecular events of such reconstructive units remains to be made. Particularly lacking is an understanding of the events that occur at the endothelial cell interface during periods of ischemia and secondary inflammation. Such information would be especially useful in the development of pharmacological and or molecular therapies for low flow and no-flow situations in microsurgical transplantation. The purpose of this study was to evaluate the migration of peripheral blood moncytes across cytokine stimulated vascular endothelium, both with and without nitric oxide donor.

Materials and Methods: Primary cultures of human umbilical vein endothelial cells (HUVEC) were established from cords taken following birth of healthy term newborns. Peripheral blood lymphocytes (PBLC) were obtained via standard Histopaque centrifugation. A single healthy donor was used for each assay. Migration of peripheral blood lymphocytes across a confluent layer of HUVEC was assayed in chemotaxis Transwell cell culture chambers (Costar). Monolayers of HUVEC were stimulated with IL-1B (10 ng per well) or BSA-anti-BSA immune complex (100-200 ug/ml per well), in the presence or absence of the nitric oxide donor L-arginine (100-200 uM), and then seeded with PBMC's (1 x 10 to the 6th cells) and incubated for 48 hours. Cell counts of the supernatant were then made and levels of TNF-a and INF-g determined using standard ELISA kits (Pharmigen). Differences between control and experimental samples were analyzed by a two-tailed Student's t-test and a commercial software package (GraphPad Prism, San Diego, CA).

Results: Transendothelial migration (TEM) of PBLC's was not increased in the absence of IL-1B or BSA-anti-BSA immune complex, with or without L-arginine. Stimulation of HUVEC with cytokines IL-1B or BSA-anti-BSA immune complex did not result in increased TNF-a production, again regardless of the presence of L-arginine or not. However, a significant increase in TEM was noted in HUVEC stimulated with IL-1B or BSA-anti-BSA immune complex. Also, levels of INF-g were increased in this group. This response (TEM and INF-g production) was augmented in an additive manner in the presence of L-arginine.

Conclusions: Lymphocyte transendothelial migration was increased following cytokine and immune complex stimulation. This effect was enhanced by pretreatment with the NO precursor L-arginine. Also, an increase in INF-g production by was noted. These findings suggest a possible role of NO in the transendothelial migration of inflammatory cells and in the secondary production of proinflammatory molecules.