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. 2003 Sep 15;551(Pt 3):1023-32.
doi: 10.1113/jphysiol.2003.045880. Epub 2003 Jun 26.

Human blood-derived macrophages enhance barrier function of cultured primary bovine and human brain capillary endothelial cells

Dietmar Zenker  1 , David Begley, Hansjürgen Bratzke, Helga Rübsamen-Waigmann, Hagen von Briesen
Affiliations

Human blood-derived macrophages enhance barrier function of cultured primary bovine and human brain capillary endothelial cells

Dietmar Zenker et al. J Physiol. .

Abstract

The characteristic properties of the blood-brain barrier (BBB) forming brain capillary endothelial cells (BCEC) are modulated by their microenvironment, but the cellular sources of the induction signals are still unclear. Apart from astrocytes, another cell type in close contact with cerebral blood vessels is the perivascular macrophages, which are known to be regularly replaced by blood-derived monocytic precursor cells. It is unknown if, and how, these cells may interact with the cerebral endothelium and modulate its BBB-specific functions. In the present study, a cell culture model of the BBB was used to investigate the effect of blood-derived human macrophages on the permeability of cultured bovine and human BCEC, determined by a transendothelial electrical resistance (TEER) measurement. We found that the TEER of postconfluent BCEC was considerably increased by a non-contact coculture with macrophages. After 24 h, we found a TEER augmentation of over 50% compared with the control without coculture, and this effect was comparable to the response of BCEC to a C6 glioma cells coculture. Stimulation or HIV-1 infection of the macrophages did not alter their effect on BCEC monolayer permeability. Investigation of signal transduction pathways showed that TEER increase of BCEC due to macrophage coculture was cAMP-independent and involves neither phospholipase C, protein kinase C nor calmodulin. Our findings demonstrate that macrophages are able to modulate BBB-specific functions in cultured BCEC. Thus, these cells or cerebral cells of monocytic origin (e.g. perivascular macrophages), may be part of the microenvironment of BCEC that modulates their specific properties in vivo.

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Figures

Figure 6
Figure 6. No effect of adenylate cyclase inhibitor on the TEER of BCEC cocultured with macrophages or C6 cells
TEER across postconfluent bovine BCEC monolayers after 24 h and 2 days of coculture with C6 glioma cells or human blood-derived macrophages (MAC) and/or treatment with 100 mm adenylate cyclase inhibitor SQ22536. In the control group, the BCEC remained untreated and without coculture. Data shown are the combined results of two independent BCEC preparations with approximately similar TEER baseline levels before coculture, and each BCEC population was cocultured with macrophages from two different donors and the same C6 cell line (n = 6 for all groups). The mean TEER value before start of the coculture of all inserts used was 1353 ± 39 Ω cm2. The TEER of all untreated coculture groups was found to be statistically different from those of the control group without coculture (*P = 0.001). No statistical differences were found between the TEER of each SQ22536-treated group from those of the corresponding group without †P≥ 0.1).
Figure 5
Figure 5. Treatment of BCEC cocultured with macrophages or C6 cells with adenylate cyclase activator or phosphodiesterase inhibitor resulted in an additional TEER increase
TEER across postconfluent bovine BCEC monolayers after 24 h and 2 days of coculture with C6 glioma cells or human blood-derived macrophages (MAC) and/or treatment with either 50 μM adenylate cyclase activator forskolin (A) or 50 μM phosphodiesterase inhibitor RO 20–1724 (B). In the control group, the BCEC remained untreated and without coculture. Data shown are the combined results of two independent BCEC preparations with approximately similar TEER baseline levels before coculture, and each BCEC population was cocultured with macrophages from two different donors and the same C6 cell line (n = 6 for all groups). The mean TEER value before start of the coculture of all inserts used was 1265 ± 28 Ω cm2. The TEER of all untreated coculture groups was found to be statistically different from those of the control group without coculture (*P = 0.001), and the TEER of each forskolin- or RO 20–1724-treated group was found to be statistically different from those of the corresponding group without †P≤ 0.012).
Figure 1
Figure 1. TEER of bovine BCEC increases due to coculture with human macrophages and is comparable with the coculture with C6 glioma cells
Transendothelial electrical resistance (TEER) across postconfluent bovine brain capillary endothelial cell (BCEC) monolayers after 24 h and 2 days coculture with either C6 glioma cells or human blood-derived macrophages (MAC); in the control group, the BCEC remained without coculture (as described in Methods). Data shown are the combined results of two independent BCEC preparations with approximately similar TEER baseline levels before coculture, and each BCEC population was cocultured with macrophages from two different donors (MAC 1 and 2) and the same C6 cell line (n = 6 for all groups). The mean TEER value before start of the coculture of all inserts used was 1388 ± 17 Ω cm2. The TEER of all coculture groups was found to be statistically different from those of the control group without coculture (P = 0.001).
Figure 2
Figure 2. Delayed increase of the TEER of human BCEC due to coculture with human macrophages
TEER across postconfluent human BCEC monolayers during prolonged coculture with human blood-derived macrophages (MAC); in the control group, the BCEC remained without coculture. In the macrophage group, the data shown are the combined results of two BCEC populations that were cocultured with diversely differentiated macrophages (8 days and 15 days in culture) from two different donors (n = 6 for coculture group, n = 3 for control group). The mean TEER value before start of the coculture of all inserts used was 200 ± 4 Ω cm2. After 2 days of coculture, the TEER of the macrophages coculture group was found to be statistically different from those of the control group without coculture (*P < 0.02).
Figure 3
Figure 3. Proinflammatory stimulation of macrophages did not interfere with their effect on the TEER of the cocultured BCEC
TEER across postconfluent bovine BCEC monolayers after 24 h and 2 days coculture with human blood-derived macrophages (MAC) which were stimulated with lipopolysaccharide (LPS, 100 ng ml−1) or unstimulated. In the control group, the BCEC remained without coculture. In each coculture group, data shown are the combined results for BCEC cocultured with macrophages derived from two different donors (n = 6 for all coculture groups, n = 3 for control group). The mean TEER value before start of the coculture of all inserts used was 1216 ± 12 Ω cm2. TEER of the group in coculture with unstimulated macrophages was found to be statistically different from those of the control group without coculture (P = 0.011). No statistical differences were found between the TEER of the group cocultured with stimulated macrophages with those of the corresponding group cocultured with unstimulated macrophages (P≥ 0.032).
Figure 4
Figure 4. HIV-1 infection of macrophages did not interfere with their effect on the TEER of the cocultured BCEC
TEER across postconfluent bovine BCEC monolayers during prolonged coculture with human blood-derived macrophages (MAC) which were infected with two different macrophagotropic HIV-1 isolates (strain D117III and ADA-M) or uninfected; in the control group, the BCEC remained without coculture. In each coculture group, data shown are the combined results for BCEC cocultured with macrophages from two different donors (n = 6 for all coculture groups, n = 3 for control group). The mean TEER value before start of the coculture of all inserts used was 1304 ± 11 Ω cm2. The TEER of all coculture groups was found to be statistically different from those of the control group without coculture (P < 0.013). No statistical differences were found between the TEER of the groups cocultured with HIV-1-infected macrophages to those of the corresponding group cocultured with uninfected macrophages (*P > 0.1, †P = 0.032).
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