Neurovascular unit crosstalk: Pericytes and astrocytes modify cytokine secretion patterns of brain endothelial cells

Abstract

Crosstalk among brain endothelial cells (BECs), pericytes, and astrocytes occurs by way of soluble factors, including cytokines. Here, we studied cytokine secretion from both mouse BEC monocultures and tri-cultured with pericytes and astrocytes. Four cytokines were constitutively secreted by BEC monolayers, 12 by LPS-stimulated BECs, 10 by tri-cultures, and 14 by LPS-stimulated tri-cultures. Cytokine levels were generally higher with either LPS stimulation or tri-culture when compared to monocultures and highest in tri-cultures stimulated by LPS. LPS-stimulated secretions fell into eight patterns as categorized by the polarization of cytokine secretions. To determine the cellular origin of cytokine increases in tri-cultures, we cultured mouse BECs with human pericytes and astrocytes and measured cytokines in species-specific assays. Thus, cytokines detected in the human immunoassay were from pericytes/astrocytes and those detected in the mouse immunoassay were from BECs. Several unique patterns were thus found. For example, TNF-alpha was only of pericyte/astrocyte origin; granulocyte colony-stimulating factor was only of BEC origin; IL-6, MCP-1, and GM-CSF of astrocyte/pericyte origin were found in both the luminal and abluminal chambers, suggesting the presence of brain-to-blood transporters. We conclude that crosstalk influences cytokine secretion under constitutive and stimulated conditions from both BECs and pericytes/astrocytes.

Keywords: Neurovascular unit; astrocyte; blood–brain barrier; cytokines; pericyte.

Figure 1.

Immunohistochemistry of the tight junction proteins claudin-5, ZO-1, and occludin. Upper panels (a–c) are monocultures of mouse brain endothelial cells (EOO) and lower panels (d–f) are tri-cultures of mouse brain endothelial cells, mouse pericytes, and mouse astrocytes (EPA). Both monocultures and tri-cultures expressed tight junction proteins. Tri-cultures tended to have more of the tight junction proteins located at the intercellular interface and less in cytoplasm.

Figure 2.

Effects of astrocytes and pericytes on brain endothelial cell monolayer permeability. Panel A shows that only tri-culture with both pericytes and astrocytes improved TEER; n = 5–6; ANOVA: F(3,18) = 11.6, p < 0.001; Newman–Keuls: ***p < 0.001 EOO vs EPA. Panel B shows that as measured by leakiness to sucrose, astrocytes improved barrier function and pericytes even more so. Adding both pericytes and astrocytes was not more effective than culturing with pericytes only; n = 3–4; ANOVA: F(3,10) = 52.4, p < 0.001; Newman–Keuls: *p < 0.05, ***p < 0.001. Panel C shows that neither astrocytes nor pericytes improved BBB leakiness as measured with albumin. Panel D: Mouse brain endothelial cells were tri-cultured with mouse or human pericytes and astrocytes. Pericytes/astrocytes from either species greatly increased TEER, although mouse pericytes/astrocytes were statistically more effective. LPS dramatically disrupted barrier as evidenced by a decrease in TEER regardless of presence or species of pericytes/astrocytes; ANOVA: F(8,119) = 174, p<<0.001; Newman–Keuls: **p < 0.01; ***p < 0.001. EOO: Mouse brain endothelial cell monocultures; EOA: Mouse brain endothelial cells co-cultured with mouse astrocytes; EPO: Mouse brain endothelial cells co-cultured with mouse pericytes; EPA: Mouse brain endothelial cells tri-cultured with mouse pericytes and mouse astrocytes; E + hPA: Mouse brain endothelial cell tri-cultured with human pericytes and human astrocytes.

Figure 3.

Constitutive secretion of cytokines by monocultures and tri-cultures. Four cytokines (G-CSF, MCP-1, KC, and RANTES) were constitutively secreted by mouse BEC. When tri-cultured with murine pericytes and astrocytes, six other cytokines (IL-6, IL-13, TNF-alpha, eotaxin, MIP-1 alpha, and MIP-1 beta) were also secreted. EOO: Mouse brain endothelial cell monoculutures; EPA: Mouse brain endothelial cells tri-cultured with mouse pericytes and mouse astrocytes; n = 6/group.

Figure 4.

Effects of LPS on cytokine secretion patterns in mouse brain endothelial cells in monocultures or tri-cultured with murine pericytes/astrocytes. Patterns for 26 cytokines are shown (Figure 4(a) to (z)). LPS was given either into the luminal or the abluminal chambers and cytokines measured in both the luminal (L) and abluminal (A) chambers. IL-1 alpha was detected in monocultures of BECs, but not in tri-cultures, whereas IL-1 beta, IL-12(p70), and eotaxin were detected in tri-cultures, but not in monocultures of BECs (n = 3–4/group. Newman–Keuls done for cytokines with ANOVA having p < 0.05. single letter (e.g. “a”) p < 0.05; double letter (e.g. “aa”) p < 0.01; triple letter (e.g. “aaa”) p < 0.001); a: luminal LPS increases luminal secretion; b: luminal LPS increases abluminal secretion; c: luminal LPS increases luminal secretion more than abluminal secretion; d: abluminal LPS increases luminal secretion; e: abluminal LPS increases abluminal secretion; f: abluminal LPS results in differences between luminal and abluminal levels; g: luminal secretion is greater with luminal LPS than with abluminal LPS; h: abluminal secretion is greater with abluminal LPS than with luminal LPS. EOO: Mouse brain endothelial cell monocultures; EPA: Mouse brain endothelial cell tri-cultured with mouse pericytes and mouse astrocytes; L-luminal chamber; A-abluminal chamber.

Figure 4.

Effects of LPS on cytokine secretion patterns in mouse brain endothelial cells in monocultures or tri-cultured with murine pericytes/astrocytes. Patterns for 26 cytokines are shown (Figure 4(a) to (z)). LPS was given either into the luminal or the abluminal chambers and cytokines measured in both the luminal (L) and abluminal (A) chambers. IL-1 alpha was detected in monocultures of BECs, but not in tri-cultures, whereas IL-1 beta, IL-12(p70), and eotaxin were detected in tri-cultures, but not in monocultures of BECs (n = 3–4/group. Newman–Keuls done for cytokines with ANOVA having p < 0.05. single letter (e.g. “a”) p < 0.05; double letter (e.g. “aa”) p < 0.01; triple letter (e.g. “aaa”) p < 0.001); a: luminal LPS increases luminal secretion; b: luminal LPS increases abluminal secretion; c: luminal LPS increases luminal secretion more than abluminal secretion; d: abluminal LPS increases luminal secretion; e: abluminal LPS increases abluminal secretion; f: abluminal LPS results in differences between luminal and abluminal levels; g: luminal secretion is greater with luminal LPS than with abluminal LPS; h: abluminal secretion is greater with abluminal LPS than with luminal LPS. EOO: Mouse brain endothelial cell monocultures; EPA: Mouse brain endothelial cell tri-cultured with mouse pericytes and mouse astrocytes; L-luminal chamber; A-abluminal chamber.

Figure 5.

Cell sources of cytokines in tri-cultures. By culturing mouse brain endothelial cells with human astrocytes/pericytes and then using species specific immunoassays, we were able to identify which cytokines were from brain endothelial cells (detected in the murine immunoassay) or pericytes/astrocytes (detected in the human immunoassay). Six of a possible 10 cytokines so detected are shown here; additionally, there was a notable absence of KC. En-Lum: Brain endothelial secretion in luminal chamber; PA-Lum: Pericyte/astrocyte secretion in luminal chamber; En-Abl: Brain endothelial secretion in abluminal chamber; PA-Abl: Pericyte/astrocyte secretion in abluminal chamber; LPS-Lum: LPS added to the luminal chamber; LPS-Abl: LPS added to the abluminal chamber.

Figure 6.

Summarized from Figures 3 and 4. An increase in the number of cytokines secreted occurred with exposure to LPS and with tri-culturing. E: endothelial cells; EPA: endothelial cells, pericytes and astrocytes

Figure 7.

Summarized from Figure 5. Origin and occurrence of six cytokines as determined in tri-cultures of mouse brain endothelial cells and human pericytes/astrocytes. Orange indicates secretion by brain endothelial cells, green indicates pericyte/astrocyte origin. Note several cytokines of pericyte/astrocyte origin appeared in the luminal chamber, indicating passage across the BEC monolayer barrier. Larger font indicates a higher level of cytokine.