Immortalized human brain endothelial cell line HCMEC/D3 as a model of the blood-brain barrier facilitates in vitro studies of central nervous system infection by Cryptococcus neoformans - PubMed (original) (raw)
Immortalized human brain endothelial cell line HCMEC/D3 as a model of the blood-brain barrier facilitates in vitro studies of central nervous system infection by Cryptococcus neoformans
Kiem Vu et al. Eukaryot Cell. 2009 Nov.
Abstract
Cryptococcus neoformans cells must cross the blood-brain barrier prior to invading the central nervous system. Here we demonstrate that the immortalized human brain endothelial cell line HCMEC/D3 is a useful alternative to primary brain endothelial cells as a model of the blood-brain barrier for studies of central nervous system infection.
Figures
FIG. 1.
C. neoformans crosses the HCMEC/D3 cell line, an in vitro model of the BBB. (A) The in vitro BBB model consists of a transwell apparatus separating the luminal (blood) and abluminal (brain) sides of the BBB. HCMEC/D3 cells were grown in the top transwell chamber on a collagen-coated microporous membrane (8 μm). Fungal cells (1 × 105 cells) were added to the top chamber and collected subsequently from the bottom chamber for CFU determination. (B) A wild-type strain of C. neoformans (H99) readily crossed from the luminal to the abluminal side of the in vitro BBB model. S. cerevisiae was used as a control strain to test the ability of the in vitro barrier to exclude a nonpathogenic yeast. *, results determined by t test (P < 0.05). Data are expressed as means ± standard deviations. (C) The contribution of the capsule of C. neoformans cells to BBB crossing was examined using a wild-type encaspulated strain B3501 and its isogenic acapsular counterpart. The encapsulated strain crossed the endothelial cell monolayer significantly better than the acapsular strain. *, results determined by t test (P < 0.05). Data are expressed as means ± standard deviations. (D) TEER for the in vitro BBB model was monitored over time in the absence (control) or presence of cryptococcal cells or yeast cells. TEER values were not significantly different before and after the addition of fungal cells, indicating that the barrier function of the HCMEC monolayer was not compromised by C. neoformans. Data are expressed as means ± standard deviations. All Cryptococcus neoformans strains (H99 _MAT_α serotype A, B3501, and acapsular B3501 [B3501-acap]) and a Saccharomyces cerevisiae strain (W303) were recovered from 15% glycerol stocks stored at −80°C prior to use. Strains were maintained on YPD (1% yeast extract, 2% peptone, and 2% dextrose) medium. Fungal cells were cultured in YPD medium at 30°C for 24 h. The HCMEC/D3 cell line was obtained under a license from INSERM, France. The original brain endothelial cells used for generating HCMEC/D3 were isolated from the temporal lobe of an adult female with epilepsy (36).
FIG. 2.
The HCMEC/D3 cell line retains features of primary cells including the expression of pecam 1, CD44, claudin 7, and occludin. The transcripts of two key junctional markers, claudin 7 and occludin, and the glycoprotein receptor CD44 were confirmed using reverse transcriptase-PCR (RT-PCR) using RNA isolated from HCMEC/D3 cells alone. Pecam 1 was used here as an endothelial cell marker and represented a positive control for the RT-PCR. RNA was isolated from a culture of HCMEC/D3 (in the absence of C. neoformans) and purified according to the manufacturer's instructions (RNeasy kit; Qiagen, Valencia, CA). RT-PCR was performed with the following primers: for pecam 1, F-atgcagccgaggtgggcccac and R-ctaagttccatcaagggagc; for claudin 7, F-atggccaattcgggcctgca and R-tcacacatactccttggaag; for CD44, F-atggacaagttttggtggca and R-ttacaccccaatcttcatg; and for occludin, F-atgtcatccaggcctcttga and R-ctatgttttctgtctatcat. The expected size of each amplicon was as follows: pecam 1, 2.1 kb; claudin 7, 640 bases; CD44, 1.1 kb; occludin, 1.5 kb. The PCR-generated amplicons were separated by using agarose gel electrophoresis and visualized by ethidium bromide staining. The RT-PCR conditions used were as follows: 30 min at 50°C, 15 min at 95°C, 1 min at 94°C (denaturation), 1 min at 55°C (annealing), and 4 min at 72°C (extension) (30 cycles), followed by 10 min at 72°C (extension).
FIG. 3.
C. neoformans cells can penetrate HCMEC/D3 endothelial cells and induce microvillus formations adjacent to the sites of invasion. (C to F) Endothelial cells grown on a transwell apparatus were infected with C. neoformans for 1 h and prepared for SEM. (A) HCMEC/D3 cells were incubated with glass beads for 1 h under conditions identical to those used with HCMEC/D3 cells in the presence of C. neoformans. (B) SEM images of cryptococcal cells in the absence of endothelial cells (but maintained in the EGM-2 medium) 1 h after they were added to collagen-coated transwell filters. Fibrous structures are likely collagen fibrils. (C) Pronounced microvillus formations on the in vitro barrier can be seen in the vicinity of yeast cells (arrows). (C and E) Cryptococcal cells that have been partially internalized are also shown in the SEM images (arrows). (D to F) Cryptococcal cells can be seen beneath the surface of the HCMEC/D3 monolayer (arrows). The images from SEM were obtained by following standard protocols described above. HCMEC/D3 cells were grown on transwell filters (8 μm; BD Biosciences) under the same conditions used for the transcytosis assays. Approximately 2 × 106 cells of a wild-type strain of C. neoformans (H99) were added to the top chamber for 1 h. The transwell filters were then washed with phosphate-buffered saline to remove nonspecific adhering yeast cells, fixed in Karnovsky's fixative, and sent to an on-campus SEM facility for sample processing (Electron Microscopy Lab, UC Davis). Glass beads (20 to 50 μm) were purchased from Sigma.
References
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