Neutrophils in galactose-fed dogs: suppressed apoptosis and increased adhesion to retinal capillary endothelial cells

Dogs fed a diet containing 30% galactose develop diabetes-like retinal capillary changes. As retinal capillary occlusion is commonly observed in diabetic retinopathy, neutrophil apoptosis and the interaction of neutrophils with retinal capillary endothelial cells were investigated. Neutrophils were isolated with Ficoll-Hypaque centrifugation from dogs fed a 30% galactose diet and dogs fed a normal, control diet containing 30% non-nutrient filler. Apoptosis of neutrophils was microscopically examined after incubation at 37 degrees C for 3 hours with either 100 U/mL tumor necrosis factor alpha (TNF-alpha), 2 microg/mL cycloheximide or 50 ng/mL phorbol 12-myristate 13-acetate (PMA). Neutrophil adhesion to dog retinal capillary endothelial cells was examined by counting the cells attached to the surface of endothelial cells after the incubation in the presence of either 100 U/mL TNF-alpha or 5 microg/mL lipopolysaccharides (LPS) at 37 degrees C for 3 hours. With all three stimulants TNF-alpha, cycloheximide and PMA, the rate of apoptosis was significantly lower for neutrophils isolated from galactose-fed dogs compared to control dogs fed a normal diet. Preincubation of neutrophils from control dogs in medium containing 30% galactose for 3 hours did not affect the rate of apoptosis. Neutrophil adhesion to retinal capillary endothelial cells induced by incubation in the presence of either 100 U/mL TNF-alpha or 5 microg/ml LPS was significantly higher with neutrophils isolated from galactose-fed dogs than those from control dogs. The data indicate that long-term galactose feeding is essential with development of various neutrophil dysfunctions. These neutrophil changes may contribute to the development of retinal microangiopathy associated with diabetes and galactosemia.