Inhibition of TNF-alpha-induced sickle RBC retention in retina by a VLA-4 antagonist

PURPOSE. Patients with sickle cell disease have elevated circulating levels of cytokines including tumor necrosis factor (TNF) alpha. TNF-alpha stimul ates expression by endothelial cells of adhesion molecules, including vascu lar cell adhesion molecule (VCAM) 1. Others have demonstrated that VLC-4 (a lpha (4)beta (1)), a ligand for VCAM-1 or fibronectin, is present on a frac tion of sickle reticulocytes. The intent of this study was to determine, us ing a rat model, if TNF-alpha increases retention of sickle erythrocytes in retina and if that retention can be inhibited. METHODS. TNF-alpha was given intraperitoneally to rats 5 hours before TV ad ministration of FITC-labeled, density-separated sickle erythrocytes. After 5 minutes, rats were exsanguinated, and retinas were excised and incubated for ADPase activity, permitting the determination of the number and locatio n of retained cells. RESULTS. TNF-alpha caused a three- to fourfold increase in retention of sic kle erythrocytes in retinal capillaries (P < 0.05) but nut of normal human erythrocytes. Preincubation of sickle erythrocytes with TBC772, a peptide t hat blocks the binding of <alpha>(4)beta (1) and alpha (4)beta (7), or a mo noclonal antibody against VLA-4 (19H8), significantly inhibited the TNF-alp ha -induced retention (P less than or equal to 0.02), whereas a control cyc lic peptide and antibody had no effect. IV TBC772 also inhibited sickle ery throcyte retention (P = 0.01). Two intravenously administered anti-fibronec tin antibodies inhibited sickle cell retention as well, but an anti-rat VCA M-1 antibody did not inhibit retention. CONCLUSIONS. The authors conclude that TNF-alpha stimulates retention of si ckle erythrocytes in the retinal vasculature. This increased retention can be blocked by a VLA-4 antagonist, suggesting that the cells retained after cytokine stimulation are reticulocytes. The counter-receptor for VLA-4 in t his rat retina model appears to be fibronectin and not VCAM-1, based on dat a obtained using antibodies against these molecules.