Activation of glial cells induces proinflammatory properties in brain capillary endothelial cells

Purpose The blood-brain barrier (BBB), formed by brain endothelial cells (BECs) ensures a stable microenvironment inside the brain by regulating transport of blood-borne molecules to the brain. However, neurodegenerative diseases are often accompanied by neuroinflammation and BBB impairment mediated by activated glial cells through their release of proinflammatory cytokines. To study the effects of glial cells with respect to BECs activation, we aimed to develop an in vitro BBB model with inflammation by preactivating glial cells and subsequently studying their impact on BECs. Methods Primary mixed glial cells (MGCs) mainly containing astrocytes and microglia were lipopolysaccharide (LPS)-stimulated, after which the LPS-containing medium was removed. The glial cells were then co-cultured with differentiated, unstimulated primary mouse BECs in transwells meaning that the BECs were under influence solely from cytokines and other pro-inflammatory molecules released from the activated glial cells. The cytokine expression by MGCs and secretion to the culture medium were quantitated after LPS stimulation using qPCR and Meso Scale analysis. The effects of the inflammatory stimuli from MGCs on the BECs were then measured through changes in BBB integrity, evaluated by trans-endothelial electrical resistance (TEER), passive permeability and tight junction proteins alterations, and possibly altered expression of adhesion molecules. The effects of the indirect stimulation of the MGCs on BECs was further compared to the effects on BECs directly stimulated with LPS. Results LPS stimulation of MGCs significantly upregulated mRNA expression of interleukin 6, interleukin 1β, and tumor necrosis factor α and significantly increased the secretion of several pro-inflammatory cytokines, e.g. IL-6, TNF-α, KC/ GRO (CXCL1) and IL-12p70. Proving that these cytokines influenced BECs, co-culturing BECs with pre-stimulated MGCs significantly affected the barrier integrity similar to direct stimulation with LPS of the BECs leading to lowering of TEER and increased permeability. Tight junction expression was unaltered, but with rearrangements of tight junction proteins. Expression of cell-adhesion molecules was significantly increased in BECs co-cultured with LPS-prestimulated MGCs when compared to that of directly stimulation with LPS. Conclusion Activating MGCs denotes a setting where glial cells influence and transform BECs into a proinflammatory phenotype .