Receptor binding and internalization of a water-soluble (1-->3)-beta-D-glucan biologic response modifier in two monocyte/macrophage cell lines.

Glucan phosphate, a water-soluble, chemically defined (1-->3)-beta-D-glucan biologic response modifier, has been reported to exert antisepsis activity and accelerate wound healing. In this study we describe the specific binding of glucan phosphate to human and murine monocyte/macrophage cell lines, U937 and J774A.1, respectively. At 37 degrees C, equilibrium binding was rapidly achieved, i.e., within 1 min. In U937 cells, binding occurred with an affinity (Kd) of 37 microM and a Bmax of 65 x 106 binding sites/cell at 37 degrees C. In J774A.1 cells, glucan phosphate bound with an affinity (Kd) of 24 microM and a Bmax of 53 x 106 binding sites/cell at 37 degrees C. In both cases there was insignificant nonspecific binding. We further demonstrated that bound glucan phosphate cannot be displaced by a 50-fold excess of unlabeled ligand, suggesting internalization of glucan phosphate. Transmission electron microscopy showed significantly increased cytoplasmic vacuolization and significantly decreased mitotic activity in glucan phosphate-treated U937 cells compared with that in untreated cells. Pullulan, a random coil alpha-(1-->4)-(1-->6)-linked glucose polymer that served as a control, did not compete for the same binding site as glucan phosphate in either cell line, indicating the specificity of the binding site for (1-->3)-beta-D-glucans. We conclude that water-soluble pharmaceutical grade (1-->3)-beta-D-glucan phosphate specifically binds to and is internalized by U937 and J774A.1 cells.

Involvement of xanthine oxidase in oxidative stress and iron release during hyperthermic rat liver perfusion.

The hepatotoxic effects of hyperthermia have been proposed to be related to lipid peroxidation as a consequence of oxidative stress. This can result from exposure of the cell to "radical oxygen" species such as the superoxide and hydrogen peroxide generated by the activity of the oxidase form (type O) of xanthine oxidase (XO), which is converted to that form by perfusion of the liver at hyperthermic temperatures. These radical species are not reactive enough in themselves to cause cell damage but require the presence of a catalyst such as low molecular weight chelated iron. In these studies, ferritin was shown to be a source of iron for the oxidative stress of hyperthermia. (a) Iron was released from ferritin in vitro by the activity of rat liver XO. The rate of iron release from ferritin in this incubation system was a function of the amount of type O XO present and the temperature. Inclusion of allopurinol or superoxide dismutase in the incubation resulted in significantly lower rates of iron release. (b) Livers from Sprague-Dawley rats were perfused at 42.5 degrees and 37 degrees C for 1 h. During the recirculating perfusion, loss of iron from the liver into the perfusate was significantly greater (P less than 0.05) at 42.5 degrees C than at 37 degrees C. Also, there was a pronounced increase in the lactate dehydrogenase and aspartate aminotransferase enzymes in the perfusate during perfusion at 42.5 degrees C. Furthermore, intrahepatic levels of low molecular weight chelated iron were significantly (P less than 0.05) increased following perfusion at 42.5 degrees C. All these responses were abrogated by the inclusion of allopurinol in the perfusate. (c) Oxidative stress, assessed by the efflux of glutathione and oxided glutathione from the liver at 42.5 degrees and 37 degrees C, was significantly (P less than 0.05) increased at the hyperthermic temperature. This oxidative stress was inhibited by iron chelation and allopurinol. These results demonstrate that there is a causal relationship between the generation of superoxide by type O XO produced by hyperthermic perfusion and mobilization of iron from ferritin to form a pool of low molecular weight chelated iron. This iron pool in combination with active oxygen species leads to oxidative stress and lipid peroxidation.