Role Of TNF-α In Vascular Endothelial Cell Injury Mediated By Frozen/Thawed PMN

Objective: Role of TNF-a in vascular endothelial cell injury mediated by frozen/thawed PMN was investgated in order to furnish new evidence for clarifying the pathogenesis of frostbite. Methods: Freezing/thawing cells model was founded using PMN of rat isolated by dextran sedimentation technique and VEC of rat cultured in vitro as experimental material. TNF-α concentration in medium was measured with TNF-α sandwich ELIS A. The injury level of VEC was indicated by measuring activity of LDH in medium. The number of frozen/thawed PMN adhering to VEC was counted with phagocytizing reactive dyes to measure the degree of frozen/thawed PMN and VEC adhesion. Expression of LFA-1 on the surface of frozen/thawed PMN was analyzed with flow cytometry. TUNEL and fluorescence staining were used to moniter apoptosis of normal VEC and frozen/thawed PMN respectively. Results: 1. Level of TNF-α secretion in frozen/thawed VEC increases, adherence of frozen/thawed PMN to normal VEC could effectively increase quantity of TNF-α secretion. 2. TNF-α could obviously upregulate expression of LFA-1 on surface of frozen/thawed PMN, upregulation of LFA-1 expression promotes adhesion of frozen/thawed PMN and normal VEC, and aggravates VEC injury. Monoclonal antibody against LFA-1 could partly block adhesion of frozen/thawed PMN and normal VEC, and attenuate VEC injury. 3. TNF-α could induce apoptosis of frozen/thawed PMN and normal VEC, and apoptosis exhibits a dose-dependence tendency at TNF-α concentration from 250U/ml to 3000U/ml. TNF-α at concentration above 2000U/ml might effect adhering of frozen/thawed PMN to normal VEC and VEC injury to a certain extent. Conclusion: TNF-α produced from process of VEC freezing/thawing and PMN-VEC adhesion could promote expression of LFA-1 on surface of frozen/thawed PMN, then adhering of frozen/thawed PMN to normal VEC and VEC injury increase. Monoclonal antibody against LFA-1 could partly block PMN-VEC adhesion and attenuate VEC injury. TNF-α could induce apoptosis of frozen/thawed PMN and normal VEC, and cause the cells death.