Brain pericytes increase the lipopolysaccharide-enhanced transcytosis of HIV-1 free virus across the in vitro blood–brain barrier: evidence for cytokine-mediated pericyte-endothelial cell crosstalk

HIV-1 (MN) CL4/CEMX174 (T1) prepared and rendered non-infective by aldrithiol-2 treatment as previously described [51, 52] was a kind gift of the National Cancer Institute, NIH. The virus was radioactively labeled by the chloramine-T method, a method which preserves vial coat glycoprotein activity [53, 54]. Two mCi of ¹³¹I-Na (Perkin Elmer, Boston, MA), 10 μg of chloramine-T (Sigma) and 5.0 μg of the virus were incubated together for 60s. The radioactively-labeled virus was purified on a column of Sephadex G-10 (Sigma, St. Louis, MO). Human serum albumin (5 μg) was labeled by the chloramine-T method with 1 mCi of ¹²⁵I-Na (Perkin Elmer) and purified on a G-10 column.

Primary cultures of MBECS and mouse brain pericytes were prepared from 8-week-old CD-1 mice. All experiments were approved by the local (VA St Louis) animal use committee. BMECs were isolated by a modified method of Szabó et al. [55] and Nakagawa et al. [56]. In brief, the cerebral cortices from 8-week-old CD-1 mice were cleaned of meninges and minced. The homogenate was digested with collagenase type II (200 U/mL; Invitrogen, Carlsbad, CA) and DNase I (30 U/mL; Sigma,) in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) containing 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamicin and 2 mM GlutaMAX™-I (Invitrogen) at 37°C for 40 min. The pellet was separated by centrifugation in 20% bovine serum albumin (BSA)-DMEM (1,000 × g for 20 min) to remove neuron and glial cells. The microvessels obtained in the pellet were further digested with collagenase/dispase (1 mg/mL; Roche, Mannheim, Germany) and DNase I (30 U/mL) in DMEM at 37°C for 30 min. Microvessel fragments containing pericytes and endothelial cells were separated by a 33% continuous Percoll (Amersham Biosciences, Piscataway, NJ) gradient centrifugation (1,000 × g for 10 min) collected and washed in DMEM (1,000 × g for 10 min). To obtain BMECs, the obtained microvessel fragments were seeded on 60 mm culture dishes (BD FALCON™, BD Biosciences, Franklin Lakes, NJ) coated with 0.05 mg/mL fibronectin (Sigma), 0.05 mg/mL collagen I (Sigma) and 0.1 mg/mL collagen IV (Sigma). They were incubated in DMEM/Nutrient mixture F-12 HAM (DMEM/F-12) (Invitrogen) supplemented with 20% plasma derived bovine serum (PDS, Animal Technologies, Tyler, TX), 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamicin, 2 mM GlutaMAX™-I and 1 ng/mL basic fibroblast growth factor (bFGF; Sigma) at 37°C with a humidified atmosphere of 5% CO₂/95% air. The next day, the BMECs migrated from the isolated capillaries and started to form a continuous monolayer. To eliminate contaminating cells (mainly pericytes), BMECs were treated with 4 μg/mL puromycin (Sigma) for the first 2 days [57]. After 2 days of the treatment, puromycin was removed from the culture medium. Culture medium was changed every other day. After 7 days in culture, BMECs typically reached 80-90% confluence.

Mouse brain pericytes were obtained by a prolonged, 2-week culture of isolated brain microvessel fragments under selective culture conditions because brain microvessel fragments contain pericytes [56, 58, 59]. Brain microvessel fragments were isolated as described above for microvessels, and then the obtained fragments were seeded on uncoated 60 mm culture dishes (BD FALCON™) in DMEM containing 20% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamicin, 2 mM GlutaMAX™-I and incubated at 37°C with a humidified atmosphere of 5% CO₂/95% air. The culture medium was changed twice a week. After 14 days in culture, pericytes typically reached 80-90% confluence. Brain pericytes were characterized by positive immunostaining for a-smooth muscle actin and negative for von Willebrand factor and were used at passage 2–3.

Monocultures of BMEC were used to produce conditioned media and to study I-HIV permeability. Monocultures of pericytes were used in cytokine assays, and co-cultures of BMECs with pericytes were used in I-HIV permeability studies (Table 1, Figure 1). BMEC monolayers were obtained by seeding BMECs (4 × 10⁴ cells/well) on the inside of the fibronectin-collagen IV (0.1 and 0.5 mg/mL, respectively)-coated polyester membrane (0.33 cm², 0.4 μm pore size) of a Transwell®-Clear insert (Costar) and cultured in DMEM/F-12 supplemented with 20% PDS, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamicin, 2 mM GlutaMAX™-I, 1 ng/mL bFGF and 500 nM hydrocortisone (Sigma) at 37°C with a humidified atmosphere of 5% CO₂/95% air until the BMECs reached confluence (3 days). For monocultures of brain pericytes, cells were seeded (4 × 10⁴ cells/well) into 24-well culture plates (Costar, Corning, NY) and used after a few days.

Co-culture of BMECs and brain pericytes was as described previously [58]; after a few days of pericyte culture as outlined above, BMECs (4 × 10⁴ cells/well) were seeded on the inside of the fibronectin-collagen IV (0.1 and 0.5 mg/mL, respectively)-coated polyester membrane (0.33 cm², 0.4 mm pore size) of a Transwell®-Clear insert (Costar) placed in the well of a 24-well culture plate containing layers of brain pericytes (pericyte co-culture) and cultured for 3 days. To check the barrier integrity of BMECs in the BMEC monolayer and pericyte co-culture systems, transendothelial electrical resistance (TEER in Ω × cm²) was measured before the experiments and after an exposure of LPS using an EVOM voltohmmeter equipped with STX-2 electrode (World Precision Instruments, Sarasota, FL). The TEER of cell-free Transwell®-Clear inserts were subtracted from the obtained values.

Lipopolysaccharide from Salmonella typhimurium (LPS; Sigma) was dissolved in serum-free DMEM/F-12 (DMEM/F-12 containing 1 ng/ml bFGF and 500 nM hydrocortisone) to concentrations of 10, 50, and 100 μg/ml. To initiate exposure of in vitro BBB models prepared as above to LPS, the culture medium containing serum in both the luminal and abluminal chambers was removed and then serum-free DMEM/F-12 was added to the abluminal chamber. Immediately, medium containing various concentrations of LPS (10, 50, 100 μg/ml) was added to the luminal chamber of the co-culture. Serum-free DMEM/F-12 without LPS was used as the control medium. TEER was measured at the end of 4 h incubation at 37°C. To initiate transport experiments, the medium was removed and BMECs were washed with physiological buffer containing 1% BSA (141 mM NaCl, 4.0 mM KCl, 2.8 mM CaCl₂, 1.0 mM MgSO₄, 1.0 mM NaH₂PO₄, 10 mM HEPES, 10 mM D-glucose and 1% BSA, pH 7.4). The physiological buffer containing 1% BSA was added to the outside (abluminal chamber; 0.6 mL) of the Transwell® insert and ¹³¹I-HIV-1 (3 × 10⁶ cpm/mL) was loaded into the luminal chamber. Samples were removed from the abluminal chamber at 15, 30, 60 and 90 min and immediately replaced with an equal volume of fresh 1% BSA/physiological buffer. The sampling volume from the abluminal chamber was 0.5 mL. All samples were mixed with 30% trichloroacetic acid (TCA; final concentration 15%) and centrifuged at 5,400 × g for 15 min at 4°C. Radioactivity in the TCA precipitate was determined in a gamma counter. The permeability coefficient and clearance of TCA-precipitable ¹³¹I-HIV-1 was calculated according to the method described by Dehouck et al. [60]. Clearance was expressed as microliters (μL) of radioactive tracer diffusing from the luminal to abluminal (influx) chamber and was calculated from the initial level of radioactivity in the loading chamber and final level of radioactivity in the collecting chamber:

where [C]_L is the initial radioactivity in a μL of loading chamber (in cpm/μL), C_C is the radioactivity in a μL of collecting chamber (in cpm/μL), and V_C is the volume of collecting chamber (in μL). During a 90-min period of the experiment, the clearance volume increased linearly with time. The volume cleared was plotted versus time, and the slope was estimated by linear regression analysis. The slope of clearance curves for the BMEC monolayer plus Transwell® membrane was denoted by PS_app, where PS is the permeability × surface area product (in μL/min). The slope of the clearance curve with a Transwell® membrane without BMECs was denoted by PS_membrane. The real PS value for the BMEC monolayer (PS_e) was calculated from 1 / PS_app = 1 / PS_membrane + 1 / PS_e. The PS_e values were divided by the surface area of the Transwell® inserts (0.33 cm²) to generate the endothelial permeability coefficient (P_e, in cm/min).

To obtain BMEC-conditioned media, the abluminal chamber of the BMEC monolayer was filled with 600 μL of serum-free DMEM/F-12. At the same time 100 μL of serum-free DMEM/F-12 (DMEM/F-12 containing 1 ng/ml bFGF and 500 nM hydrocortisone) without or with 100 μg/ml LPS was added to the luminal chamber of the BMEC monolayer as described above. After 4 h of exposure, the fluid in the abluminal chamber of the BMEC monolayer (600 μL) was collected as the BMEC conditioned media, sterilized by passing it through a 0.45 μm filter syringe, and stored at −80°C until use. Brain pericytes (4 × 10⁴ cells/well) were seeded on the wells of 24-well culture plate (Costar). We divided the pericyte cultures into 4 groups: pericytes treated with (1) BMEC conditioned medium, (2) LPS-treated BMEC-conditioned medium, (3) serum-free DMEM/F-12 containing no LPS (control), and (4) serum-free DMEM/F-12 containing LPS (100 μg/mL) where these media are from above. Pericytes were washed with serum-free DMEM/F-12, and then exposed to 200 μL of BMEC-conditioned medium, LPS-treated BMEC conditioned medium, or serum-free DMEM/F-12 with or without LPS (100 μg/mL) for 4 hr at 37°C. Culture supernatant (200 μL) was collected and stored at −80°C until use. The 22 cytokines [granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-g), interleukin-1 alpha (IL-1a), IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12(p70), IL-13, IL-15, IL-17, interferon-inducible protein-10 (IP-10), keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1a), regulated upon activation, normal T-cell expressed and secreted (RANTES), and tumor necrosis factor-alpha (TNF-a)] in the collected supernatants, BMEC conditioned medium, and LPS-treated BMEC conditioned medium were measured with the mouse cytokine/chemokine Lincoplex® kit (Linco Research, St. Charles, MO) by following the manufacturer’s instructions.

Table 1 shows that co-culturing with pericytes improved brain endothelial cell monolayer TEER but had no effect on albumin permeability. I-HIV crossed the BMEC monolayers with or without pericyte co-cultures faster than I-Alb despite the much larger size of I-HIV. Pericytes reduced the permeation of I-HIV across the BBB to a statistically significant degree (t = 2.60, df = 27, p<0.05). These results show that monolayers with or without pericytes are more permeable to HIV-1 than the much smaller albumin, demonstrating that HIV-1 is crossing BMEC by a process other than leakage. The results also show that pericytes have differing effects on the three mechanisms of permeability illustrated in this table: increasing TEER (decreasing paracellular permeability) and decreasing HIV permeability (adsorptive transcytosis), but not affecting albumin permeability (macropinocytosis).

Addition of LPS produced a dose-dependent increase in I-HIV-1 transport across the BMEC monolayer. Results (Figure 2) were expressed as percent of control in order to reduce statistical variance and to allow comparison across treatments and trials. For I-HIV, two-way ANOVA showed significant effects for culture conditions [with or without pericytes: F(1,76) = 29.5, p<0.001] and treatment [LPS concentration: F(3,76) = 27.5, p<0.001] with no effect for interaction. Tukey’s multiple comparison test showed that 50 and 100 μg/ml of LPS increased I-HIV transport across the monolayer in the absence of pericytes and that 10, 50, and 100 μg /ml of LPS increased I-HIV transport across the monolayer in the presence of pericyte co-culture. Additionally, any given concentration of LPS produced a statistically greater transport of HIV-1 in the monolayers co-cultured with pericytes when compared to the monocultures (Figure 2 upper panel).

LPS also decreased TEER (Figure 2, lower panel) as shown by the two-way ANOVA that found effects for culture conditions [F(1, 76) = 14.8, p<0.001] and LPS treatment [F(3,76) = 111.5, p<0.001]. However, Tukey’s multiple comparisons test found no effect of pericytes on the LPS-induced decrease in TEER.

To further investigate the mechanism by which pericytes, LPS, and brain endothelial cells interact, we determined whether culture media obtained from brain endothelial cells could induce cytokine release from pericytes. We determined the ability of pericytes to release cytokines either constitutively or when stimulated by LPS. Of 22 cytokines measured, seven (G-CSF, GM-CSF, IL-1 alpha, IL-6, IP-1-, KC, and MCP-1) were constitutively released by pericytes and two others (IL-5 and RANTES) were induced when pericytes were exposed to LPS for 4 h (Figure 3). LPS also caused a statistically significant increase in the release of each of the seven constitutively- released cytokines, p<0.01 (Figure 3).

We then incubated pericytes with culture media obtained from brain endothelial cells; in some cases, the endothelial cells had been exposed to LPS. Table 2 shows that four cytokines were detectable in the endothelial cell-conditioned culture media. Of the nine cytokines released by pericytes (Figure 3), endothelial culture media only affected the release of two of them (Figure 4): KC [F(2,14) = 58.8, p<0.001] and MCP-1 [F(2,14) = 137, p<0.001]. In both cases, culture media from endothelial cells that had been exposed to LPS, but not culture media from cells that were unexposed, produced the increase.