TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response

Abstract

Leukocyte transendothelial migration (TEM) is a tightly regulated, multistep process that is critical to the inflammatory response. A transient increase in endothelial cytosolic free calcium ion concentration (↑[Ca(2+)]i) is required for TEM. However, the mechanism by which endothelial ↑[Ca(2+)]i regulates TEM and the channels mediating this ↑[Ca(2+)]i are unknown. Buffering ↑[Ca(2+)]i in endothelial cells does not affect leukocyte adhesion or locomotion but selectively blocks TEM, suggesting a role for ↑[Ca(2+)]i specifically for this step. Transient receptor potential canonical 6 (TRPC6), a Ca(2+) channel expressed in endothelial cells, colocalizes with platelet/endothelial cell adhesion molecule-1 (PECAM) to surround leukocytes during TEM and clusters when endothelial PECAM is engaged. Expression of dominant-negative TRPC6 or shRNA knockdown in endothelial cells arrests neutrophils apically over the junction, similar to when PECAM is blocked. Selectively activating endothelial TRPC6 rescues TEM during an ongoing PECAM blockade, indicating that TRPC6 functions downstream of PECAM. Furthermore, endothelial TRPC6 is required for trafficking of lateral border recycling compartment membrane, which facilitates TEM. Finally, mice lacking TRPC6 in the nonmyeloid compartment (i.e., endothelium) exhibit a profound defect in neutrophil TEM with no effect on leukocyte trafficking. Our findings identify endothelial TRPC6 as the calcium channel mediating the ↑[Ca(2+)]i required for TEM at a step downstream of PECAM homophilic interactions.

Figure 1.

Chelation of cytosolic free Ca²⁺ in endothelial cells attenuates leukocyte TEM. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca²⁺ concentration in response to 10 µM histamine were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F₀) of three independent experiments. At least 100 total cells were individually quantified for each condition. (b–d) Freshly isolated human neutrophils (b and c) or monocytes (d) in the presence or absence of anti-PECAM were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. (e) Eluate control experiments were performed as previously described (Mamdouh et al., 2008). In brief, BAPTA-AM eluate collected from BAPTA-AM–treated HUVECs was used to resuspend neutrophils, and their ability to transmigrate was subsequently assessed. (b–e) Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one- or two-way ANOVA).

Figure 2.

Endothelial ↑[Ca²⁺]_i is required for LBRC targeted recycling. (a–e) Freshly isolated neutrophils (a–d) or monocytes (e) were added to HUVEC monolayers that were pretreated with vehicle (DMSO) or 20 µM BAPTA-AM. LBRC trafficking (a, b, and e), neutrophil adhesion (c), and neutrophil crawling (d) were assessed using our targeted recycling assay, as described in Materials and methods. Representative images (a) demonstrate targeted recycling to the site of neutrophil TEM (bars, 10 µm). Arrows denote the site where LBRC is or should be enriched. Insets in a are orthogonal (XZ) projections of the arrested neutrophil with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P < 0.05; ***, P < 0.001 (Student’s t test).

Figure 3.

TRPC6 colocalizes with PECAM at endothelial cell–cell borders and is recruited with PECAM to anti-PECAM–coated polystyrene beads. (a and b) HUVECs were transduced with adenovirus expressing mCherry-tagged TRPC6 for 72 h. Monolayers were then immunostained with anti-PECAM (a) and anti-CD18 (b) and subsequently examined by confocal microscopy. Arrows denote sites in which PECAM and TRPC6 are colocalized. The inset in b is an orthogonal (XZ) projection of the transmigrating monocyte with respect to the endothelial monolayer (dashed line at the position shown in the merged image). Images are representative of at least three independent experiments. (c and d) Polystyrene beads were precoupled with mIgG, anti-PECAM, anti–ICAM-1, or anti–MHC I and were added to HUVECs expressing FLAG-tagged TRPC6. Samples were immunostained with anti-PECAM, anti-FLAG, or anti–VE-cadherin Ab. (e and f) Polystyrene beads precoupled with anti-PECAM were added to TNF-activated HUVECs, which were subsequently fixed and immunostained with one of the following: anti-PECAM, anti-CD99, anti-CD47, or anti–ICAM-1. (c and e) Arrows point to beads. Images are representative of at least three independent experiments. Quantitative analysis of bead experiments represents the mean ± SEM of three independent experiments in which over 800 beads were analyzed per condition. Bars: (a) 50 µm; (b, c, and e) 10 µm. ***, P < 0.001 (one- or two-way ANOVA).

Figure 4.

Activation of endothelial TRPC6 is sufficient to rescue TEM and targeted recycling in neutrophils blocked by anti-PECAM Ab. (a) HUVECs were simultaneously loaded with 2.5 µM Fura-2-AM and either 20 µM BAPTA-AM or vehicle (DMSO). Changes in intracellular Ca²⁺ concentration in response to 10 µM Hyp9 were quantified as described in Materials and methods. The plot displays mean ± SEM of the fluorescence/baseline fluorescence (F/F₀) of three independent experiments. At least 60 total cells were individually quantified for each condition. (b) HUVECs seeded on transwell filter plates were treated with vehicle (DMSO), 10 µM histamine (positive control), Hyp9, or OAG for 10 min, and TEER was assessed at various time points. Data are represented as a percentage of baseline TEER (time 0 min). The plot displays mean of three independent experiments in which each condition was tested in triplicate. (c and d) Freshly isolated human neutrophils in the presence of nonblocking anti–VE-cadherin Ab (control) or anti-PECAM Ab were added to HUVECs that were pretreated with 20 µM BAPTA-AM or vehicle. After allowing 10 min for neutrophils to interact with endothelial monolayers, Hyp9, histamine, or vehicle (−) was added to the co-cultures and TEM was allowed to resume for an additional 5 min. Data for c and d were accumulated from the same experiments, and thus, some data in b were redisplayed in c to illustrate the time dependence of Hyp9-mediated rescue of TEM. (e–h) The effect of Hyp9 treatment on LBRC trafficking (e and f), neutrophil adhesion (g), and neutrophil crawling (h) was assessed using our targeted recycling assay, as described in Materials and methods. Arrows denote LBRC enrichment. Cultures in which neutrophils were arrested by anti-PECAM were treated with Hyp9 or vehicle. Insets in e are orthogonal (XZ) projections of arrested or transmigrating neutrophils with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Images are representative of three independent experiments (bars, 10 µm). Quantitative data displayed in c–h represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P < 0.05; ***, P < 0.001 (two-way ANOVA or Student’s t test).

Figure 5.

Expression of dominant-negative TRPC6 in endothelial cells attenuates neutrophil TEM and targeted recycling. (a) Nontransduced HUVECs (control) or HUVECs expressing WT FLAG-tagged TRPC6 (WT-T6) or dominant-negative FLAG-tagged TRPC6 (DN-T6) were loaded with 2.5 µM Fura-2-AM. Changes in intracellular Ca²⁺ concentration in response to 10 µM Hyp9 (control n = 2, WT-T6 n = 4, DN-T6 n = 4) or 10 µM histamine (control n = 3, WT-T6 n = 3, DN-T6 n = 3) were quantified as described in Materials and methods. The plots display mean ± SEM of the fluorescence/baseline fluorescence (F/F₀). More than 70 total cells were individually quantified for each condition from three independent experiments. (b) Nontransduced HUVECs (control) or those expressing WT-T6 or DN-T6 were untreated or activated with TNF for 3–4 h and subsequently fixed and immunostained to detect surface ICAM-1. Mean fluorescence intensity of 15 fields was averaged per condition in each experiment. The graph displays mean ± SEM of fold increase in surface ICAM-1 expression in TNF-activated monolayers compared with surface ICAM-1 expression in nonactivated monolayers from three individual experiments. (c and d) Freshly isolated human neutrophils were added to nontransduced HUVECs (control) or those expressing exogenous WT-T6 or DN-T6, and TEM (c) and adhesion (d) were assessed. (e–h) LBRC trafficking (e and f), neutrophil adhesion (g), and neutrophil crawling (h) in WT-T6– and DN-T6–expressing HUVECs were assessed using the targeted recycling assay, as described in Materials and methods. Arrows point to the site where LBRC is or should be enriched (e). Insets in e are orthogonal (XZ) projections of the arrested neutrophil with respect to the endothelial monolayer (dashed line at the position shown in the merged images). Images are representative of three independent experiments (bars, 10 µm). (b–d and f–h) Quantitative data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA).

Figure 6.

shRNA knockdown of TRPC6 in endothelial cells attenuates neutrophil TEM but not apical adhesion. (a and b) TRPC6 protein levels in HUVECs expressing TRPC6 shRNA or scrambled shRNA, with or without the rescue TRPC6 construct were assessed via Western blot (a). Densitometric analysis was performed and is represented by the mean ± SEM. TRPC6 expression was normalized to β-actin expression from three independent experiments (b). (c–g) Freshly isolated human neutrophils were added to HUVECs expressing TRPC6 shRNA or scrambled shRNA with or without the TRPC6 rescue construct. TEM and adhesion (c and d), as well as targeted recycling, adhesion, and leukocyte crawling (targeted recycling assay, as described in Materials and methods; e–g) were assessed. Quantitative data represent the mean ± SEM of at least three independent experiments in which each condition was tested in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA).

Figure 7.

Chimeric mice deficient in endothelial TRPC6 exhibit a profound defect in leukocyte TEM in a model of acute inflammation. (a) WT or TRPC6^−/− mice were treated with croton oil on one ear. After 6 h of stimulation, the mice were euthanized and their ears were harvested for immunohistochemical staining with anti-PECAM and anti–ICAM-1. Images are representative of at least 10 fields per mouse (WT n = 2 mice, TRPC6^−/− n = 2 mice). (b–f) WT or TRPC6^−/− mice were irradiated and injected with either WT or TRPC6^−/− bone marrow. After confirming full reconstitution, they were treated with croton oil on one ear and vehicle on the contralateral ear. After 6 h of stimulation, the mice were euthanized and their ears were harvested for immunohistochemical staining with anti-PECAM (endothelium, red), anti–collagen IV (basement membrane, purple), and anti-MRP14 (neutrophils, green). (b) Representative images of the ears. Insets display an orthogonal view to illustrate the site of neutrophil arrest. Arrows denote the predominating site of neutrophil position relative to the endothelium. (a and b) Bars, 50 µm. (c) Diagram outlining the scoring of neutrophil position relative to the endothelium and basement membrane. (d) Quantification of neutrophil position. (e) Quantification of the percentage neutrophil TEM. (f) Quantification of the number of neutrophils per 200 µm of vessel (i.e., neutrophil trafficking to the site of inflammation). Over 100 neutrophils were scored per mouse (WT→WT n = 8 mice, TRPC6^−/−→TRPC6^−/− n = 4 mice, WT→TRPC6^−/− n = 6 mice) in each of two independent experiments. Quantitative data in d–f are represented as the mean ± SEM. ***, P < 0.001 (one- or two-way ANOVA).