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Inhibition of TNF-alpha-induced sickle RBC retention in retina by a VLA-4 antagonist
1Wilmer Ophthalmological Institute, Johns Hopkins Hospital, 170 Woods Research Building, 600 N. Wolfe Street, Baltimore, MD 21287-9115, USA. glutty@jhmi.edu
Purpose:
Patients with sickle cell disease have elevated circulating levels of cytokines including tumor necrosis factor (TNF) alpha. TNF-alpha stimulates expression by endothelial cells of adhesion molecules, including vascular cell adhesion molecule (VCAM) 1. Others have demonstrated that VLA-4 (alpha(4)beta(1)), a ligand for VCAM-1 or fibronectin, is present on a fraction of sickle reticulocytes. The intent of this study was to determine, using 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 IV administration 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 location of retained cells.
Results:
TNF-alpha caused a three- to fourfold increase in retention of sickle erythrocytes in retinal capillaries (P < 0.05) but not of normal human erythrocytes. Preincubation of sickle erythrocytes with TBC772, a peptide that blocks the binding of alpha(4)beta(1) and alpha(4)beta(7), or a monoclonal antibody against VLA-4 (19H8), significantly inhibited the TNF-alpha-induced retention (P < or = 0.02), whereas a control cyclic peptide and antibody had no effect. IV TBC772 also inhibited sickle erythrocyte retention (P = 0.01). Two intravenously administered anti-fibronectin antibodies inhibited sickle cell retention as well, but an anti-rat VCAM-1 antibody did not inhibit retention.
Conclusions:
The authors conclude that TNF-alpha stimulates retention of sickle 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 this rat retina model appears to be fibronectin and not VCAM-1, based on data obtained using antibodies against these molecules.