Activation of C6 glioblastoma cell ceruloplasmin expression by neighboring human brain endothelia-derived interleukins in an in vitro blood-brain barrier model system

Background: Iron transport across the blood-brain barrier (BBB) involves the cooperation of brain microvascular endothelial cells (BMVEC) and their neighboring astrocytes. Astrocytes secrete a soluble form of ceruloplasmin (sCp) which, in turn, acts to export iron from ferroportin (Fpn) on the basolateral surface of BMVEC. Although regulation of astrocyte sCp gene expression has been demonstrated to be influenced by interleukin-1 beta (IL-1β) and interleukin-6 (IL-6), the role of neighboring BMVEC in this regulation has yet to be determined and is the basis for this work. Results: We provide evidence that human BMVEC (hBMVEC) IL-1β and IL-6 positively influence the expression of sCp transcript by neighboring C6 glioma cells (astrocytes). The effect of hBMVEC on C6 glioma sCp expression at the level of transcript and protein was repressed via the addition of IL-1β and IL-6 pathway inhibitors (IL-1 receptor antagonist protein and SC144, respectively). Stimulation of hBMVEC interleukin gene expression by apical exposure to bacterial endotoxin lipopolysaccharide significantly enhanced hBMVEC-mediated C6 glioma sCp gene expression. Conclusion: hBMVEC influence the gene expression of neighboring C6 glioma sCp. This change in gene expression is mediated by the secretion of IL-1β and IL-6 from hBMVEC. Furthermore, the hBMVEC-induced increase in neighboring C6 glioma sCp gene expression leads to an increased rate of hBMVEC iron efflux. Taken together, our results indicate that hBMVEC-secreted cytokine activity increases the gene expression of neighboring C6 glioma sCp, which reciprocally acts on basolateral hBMVEC Fpn to enhance brain iron import.