Immortalized human cerebral microvascular endothelial cells maintain the properties of primary cells in an in vitro model of immune migration across the blood brain barrier

Abstract

The immortalized human cerebral microvascular endothelial cell line HCMEC/D3 presents a less expensive and more logistically feasible alternative to primary human brain microvascular endothelial cells (HBMEC's) for use in constructing in vitro models of the blood brain barrier (BBB). However, the fidelity of the HCMEC/D3 cell line to primary HBMEC's in studies of immune transmigration has yet to be established. Flow cytometric analysis of primary human leukocyte migration across in vitro BBB's generated with either HCMEC/D3 or primary HBMEC's revealed that HCMEC/D3 maintains the immune barrier properties of primary HBMEC's. Leukocyte migration responses and inflammatory cytokine production were statistically indistinguishable between both endothelial cell types, and both cell types responded similarly to astrocyte coculture, stimulation of leukocytes with phorbol myristate acetate (PMA) and ionomycin, and inflammatory cytokine treatment. This report is the first to validate the HCMEC/D3 cell line in a neuroimmunological experimental system via direct comparison to primary HBMEC's, demonstrating remarkable fidelity in terms of barrier resistance, immune migration profiles, and responsiveness to inflammatory cytokines. Moreover, we report novel findings demonstrating that interaction effects between immune cells and resident CNS cells are preserved in HCMEC/D3, suggesting that important characteristics of neuroimmune interactions during CNS inflammation are preserved in systems utilizing this cell line. Together, these findings demonstrate that HCMEC/D3 is a valid and powerful tool for less expensive and higher throughput in vitro investigations of immune migration at the BBB.

Fig. 1

HCMEC/D3 maintain the barrier formation characteristics of primary HBMEC’s as assessed by measurement of transendothelial electrical resistance (TEER). (A) Schematic depiction of in vitro BBB’s; see text for details. (B) HCMEC/D3 exhibited virtually identical TEER values for each day of culture in both monoculture and astrocyte coculture systems (p>0.05). (C) Area under the curve (AUC) analysis revealed no significant difference in overall barrier formation kinetics between HCMEC/D3 and HBMEC cultures, with or without astrocyte coculture. Data are mean values of 15 (HCMEC/D3) or 9 (HMBEC) replicates +/− SEM, taken from 3 independent experiments.

Fig. 2

Leukocyte migration patterns are preserved for in vitro BBB’s constructed with HCMEC/D3 compared to primary HBMEC’s. Stimulated (PMA/ionomycin) or unstimulated primary human PBMC’s were used in model BBB transmigration assays as described in the text. TEER values were recorded before and after transmigration experiments (reported as percent change) and cells that migrated to the bottom chamber were identified via flow cytometry (reported as the proportion of total cells added to top chamber). (A) Only stimulated PBMC’s caused a change in barrier resistance; this response was identical for barriers composed of HCMEC/D3 and HBMEC. (B – F) The migration of (B) CD4⁺, (C) CD8⁺, (D) CD11b⁺, (E) CD19⁺, and (F) CD11c⁺ leukocytes was statistically indistinguishable (p>.05) between cultures of HCMEC/D3 (patterned bars) and HBMEC (open bars). These barriers influenced migration identically in every condition tested, including those where significant differences were induced by PBMC stimulation (B, C) or the presence of astrocytes (D, E). (*=p<.05; **=p<.01; ***=p<.001). Data are mean values +/− SEM of 6 replicates taken from 2 independent experiments.

Fig. 3

Inflammatory cytokine production is similar for in vitro BBB’s constructed with HCMEC/D3 compared to primary HBMEC’s. At the end of the 12h PBMC migration experiments, supernatants in both luminal (top) and abluminal (bottom) chambers of each BBB culture were examined for the presence of inflammatory cytokines via BioPlex^™ human cytokine bead array. The expression of (A–B) IL-1β, (C–D) IL-17, (E–F) IFN-γ, and (G–H) TNF-α was statistically indistinguishable (p>.05) between cultures of HCMEC/D3 (patterned bars) and HBMEC (open bars). In both luminal and abluminal chambers, significant changes to cytokine expression due to PBMC stimulation (A–H, p<0.001) or astrocyte coculture (A–B; G–H, p<.01) were similar in HCMEC/D3 and HBMEC cultures. (*=p<.05; **=p<.01; ***=p<.001). Data are mean values of 6 replicates +/− SEM taken from 2 independent experiments.

Fig. 4

Barrier responses to inflammatory cytokine treatment are similar in HCMEC/D3 compared to HBMEC cultures. (A) BBB cultures of both cell types were treated for 6h with either 100ng/ml IFN-γ, TNF-α, or a PBS vehicle. TEER values were taken before and after cytokine treatment (reported as percent change). Both IFN-γ and TNF-α significantly reduced TEER in BBB cultures (p<0.001), while astrocyte coculture enhanced the effect of TNF-α treated cultures only (p<0.01). These effects were statistically indistinguishable between HCMEC/D3 and HBMEC cultures (p>0.05). (B) Confluent monolayers of either HBMEC or HCMEC/D3 were treated for 6h with either 100ng/ml IFN-γ, TNF-α, or a PBS vehicle, then probed via immunocytochemistry for expression of VE-cadherin. Both HBMEC and HCMEC/D3 exhibited discrete staining of VE-cadherin at intercellular junctions, which was diminished by TNF-α but not IFN-γ treatment. (*=p<.05; **=p<.01; ***=p<.001). TEER data are mean values +/− SEM of 6 replicates taken from 2 independent experiments. Immunocytochemistry pictures are representative 63× images of 6 images taken per condition from 2 independent experiments. IC= isotype control.