Brain microvascular pericytes are immunoactive in culture: cytokine, chemokine, nitric oxide, and LRP-1 expression in response to lipopolysaccharide

Abstract

Background: Brain microvascular pericytes are important constituents of the neurovascular unit. These cells are physically the closest cells to the microvascular endothelial cells in brain capillaries. They significantly contribute to the induction and maintenance of the barrier functions of the blood-brain barrier. However, very little is known about their immune activities or their roles in neuroinflammation. Here, we focused on the immunological profile of brain pericytes in culture in the quiescent and immune-challenged state by studying their production of immune mediators such as nitric oxide (NO), cytokines, and chemokines. We also examined the effects of immune challenge on pericyte expression of low density lipoprotein receptor-related protein-1 (LRP-1), a protein involved in the processing of amyloid precursor protein and the brain-to-blood efflux of amyloid-β peptide.

Methods: Supernatants were collected from primary cultures of mouse brain pericytes. Release of nitric oxide (NO) was measured by the Griess reaction and the level of S-nitrosylation of pericyte proteins measured with a modified "biotin-switch" method. Specific mitogen-activated protein kinase (MAPK) pathway inhibitors were used to determine involvement of these pathways on NO production. Cytokines and chemokines were analyzed by multianalyte technology. The expression of both subunits of LRP-1 was analyzed by western blot.

Results: Lipopolysaccharide (LPS) induced release of NO by pericytes in a dose-dependent manner that was mediated through MAPK pathways. Nitrative stress resulted in S-nitrosylation of cellular proteins. Eighteen of twenty-three cytokines measured were released constitutively by pericytes or with stimulation by LPS, including interleukin (IL)-12, IL-13, IL-9, IL-10, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, eotaxin, chemokine (C-C motif) ligand (CCL)-3, and CCL-4. Pericyte expressions of both subunits of LRP-1 were upregulated by LPS.

Conclusions: Our results show that cultured mouse brain microvascular pericytes secrete cytokines, chemokines, and nitric oxide and respond to the innate immune system stimulator LPS. These immune properties of pericytes are likely important in their communication within the neurovascular unit and provide a mechanism by which they participate in neuroinflammatory processes in brain infections and neurodegenerative diseases.

Figure 1

Determination of the purity of the pericyte culture. A primary culture of pericytes isolated from mouse brain microvessels was labeled with anti-α smooth muscle actin antibody (pericyte marker; red) (Panel A), anti-CD13 antibody (pericyte marker; green) (Panel B), anti-GFAP antibody (astrocytes marker; green) (Panel C), Griffonia simplicifolia lectin (microglial marker; green) (Panel D) or anti-factor VIII antibody (endothelial cell marker; green) (Panel E) and counterstained with nuclear stain DAPI (blue). Visual observation of immunostained cells in pericyte cultures demonstrates that they primarily consist of a α-smooth muscle actin/CD13 positive pericytes. No contamination with microglia, astrocytes or endothelial cells was detected. Scale bar: 40 μm.

Figure 2

Release of nitric oxide and nitrosative stress in primary brain pericytes after LPS stimulation. Brain pericytes were stimulated for 4, 8, and 24 h with LPS (0.1 and 1 ug/ml), media collected, and analyzed for NO production by the Griess reaction. LPS (0.1 ug/ml and 1 μg/ml) induced a significant NO release from cells after 8 and 24 hours (A). Nitrative stress was accompanied by massive S-nitrosylation of cellular proteins (B). Values of nitrite accumulation from treated cells represent the mean ± SEM of two independent experiments conducted in tetraplicates. *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated cells.

Figure 3

Involvement of MAPK pathways in nitric oxide production by pericytes after LPS stimulation. Brain pericytes were stimulated for 4, 8, and 24 h with LPS (0.1 and 1 ug/ml). MAPK pathway inhibitors were added to the culture medium 1 h before LPS treatment. Media was collected and analyzed for NO production by Griess reaction. Addition of MAPK pathways inhibitors significantly reduced NO production by LPS treated pericytes. Values represent the mean ± SEM of two independent experiments conducted in tetraplicates. *P < 0.05, ***P < 0.001 vs. untreated cells.

Figure 4

Release of cytokines and chemokines from primary brain pericytes constitutively and after LPS stimulation. Brain pericytes were stimulated for 4, 8, and 24 h with LPS (0.1 and 1 ug/ml). Media was collected and cytokine and chemokine concentrations were determined via commercial magnetic bead immunoassay. Addition of LPS at 0.1 ug/ml concentration induced significant changes in production of several pro-inflammatory cytokines and chemokines from brain pericytes. Values of cytokine production represent the mean ± SEM of two independent experiments conducted in triplicates *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated cells.

Figure 5

LPS induce up-regulation of LRP-1 expression in brain pericytes. Primary brain pericytes were stimulated for 24 h with LPS (0.1 and 1 ug/ml). After 24 h, expression of both LRP-1 subunits was analyzed by western blot as described in the Material and methods. LPS at 1 ug/ml concentration induced significant increases in expression of the large (515 kDa) and small (85 kDa) subunits of LRP-1. A representative western blot (A) and density quantification (B) based on ratios between the antibody signal (LRP-1 85 or 515 kDa) and total protein loading per lane (SYPRO) is shown. Lane designation: 1-PEA13 (LRP-1 knockout as negative control), 2-MEF1 (LRP-1 wild type as positive control), 3-CTRL, 4-LPS 0.1 ug/ml, 5-LPS 1 ug/ml. Values represent the mean ± SEM of two independent experiments * P < 0.05 vs. untreated cells, n = 5.