Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function

Abstract

Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/β, CXCL12(3-68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05-0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03-3μM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3-68) was inefficient to activate CXCR4 in Presto-Tango β-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3-68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/β, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3-68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation.

Fig 1. Expression of CXCR4, ACKR3 and CXCR4:ACKR3 heteromers on hPPAEC and effects of CXCR4/ACKR3 ligands on hPPAEC monolayer permeability.

(A) Detection of CXCR4, ACKR3 and CXCR4:ACKR3 heteromers on hPPAEC by PLA. Typical PLA images for the detection of individual receptors and CXCR4:ACKR3 heteromers. Images show merged PLA/4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) signals. Ctrl.: Omission of one secondary antibody. (B) hPPAEC were grown to a confluent monolayer on collagen-coated permeable membranes. Cells were then exposed to vehicle or 50 nM of CXCR4/ACKR3 ligands for 10 minutes, as indicated, followed by the addition of FITC-dextran. Endothelial permeability was assessed by measuring the amount of FITC-dextran that permeated through the cell monolayer. N = 3 in quadruplicate. No cells: 100% permeability, open squares. RFU: Relative fluorescence units. *: p<0.05 vs. vehicle (2-way ANOVA/Bonferroni’s multiple comparison post hoc test).

Fig 2. Impairement of hPPAEC monolayer permeability by thrombin.

(A) hPPAEC were grown to a confluent monolayer on collagen-coated permeable membranes and then exposed to different concentration of thrombin for 10 min, followed by the addition of FITC-dextran. Endothelial permeability was assessed by measuring the amount of FITC-dextran that permeated through the cell monolayer. No cells: 100% permeability. RFU: Relative fluorescence units. N = 3 in quadruplicate. (B) Dose-response curves for thrombin-induced permeability, data from A. 100% permeability = permeability in the absence of hPPAEC. Open squares: Permeability at t = 55 min. Light grey squares: Permeability at t = 135 min. Dark grey squares: Permeability at t = 255 min. Dose-response curves were generated using nonlinear regression analyses.

Fig 3. Effects of CXCL12 and ubiquitin on thrombin-induced impairment of hPPAEC monolayer permeability.

hPPAEC were grown to a confluent monolayer on collagen-coated permeable membranes. (A) hPPAEC were pre-treated with vehicle, 100 nM of CXCL12 or ubiquitin for 10 minutes, as indicated, and then exposed to thrombin (50 nM), followed by the addition of FITC-dextran. Vehicle: no thrombin. Endothelial permeability was assessed by measuring the amount of FITC-dextran that permeated through the cell monolayer. No cells: 100% permeability (open circles). RFU: Relative fluorescence units. N = 3 in quadruplicate. *: p<0.05 vs. vehicle/thrombin. (B-D) hPPAEC were exposed to 35 nM of thrombin or vehicle. After 10 min, thrombin-exposed cells were treated with vehicle, CXCL12 (50 nM) and/or AMD3100 (10 μM) (B), with vehicle, ubiquitin (50 nM) and/or AMD3100 (10 μM) (C) or with various concentrations of ubiquitin (D) followed by the addition of FITC-dextran. The experimental conditions are indicated. Endothelial permeability was assessed by measuring the amount of FITC-dextran that permeated through the cell monolayer. RFU: Relative fluorescence units. N = 3 in quadruplicate. *: p<0.05 vs. thrombin/vehicle (2-way ANOVA/Bonferroni’s multiple comparison post hoc test).

Fig 4. Effects of CXCL12 and ubiquitin on thrombin-induced impairment of HULEC-5a monolayer permeability.

(A) HULEC-5a were grown to a confluent monolayer on collagen-coated permeable membranes and then exposed to different concentration of thrombin for 10 min, followed by the addition of FITC-dextran. Endothelial permeability was assessed by measuring the amount of FITC-dextran that permeated through the cell monolayer. No cells: 100% permeability. RFU: Relative fluorescence units. N = 3 in quadruplicate. (B) Dose-response curves for thrombin-induced permeability, data from A. 100% permeability = permeability in the absence of HULEC-5a. Open squares: Permeability at t = 55 min. Light grey squares: Permeability at t = 135 min. Dark grey squares: Permeability at t = 255 min. (C/D) HULEC-5a were grown to a confluent monolayer on collagen-coated permeable membranes and then exposed to 50 nM of thrombin or vehicle. After 10 min, thrombin-exposed cells were treated with vehicle, CXCL12 (50 nM) (C) or ubiquitin (50 nM) (D), followed by the addition of FITC-dextran. The experimental conditions are indicated. Endothelial permeability was assessed by measuring the amount of FITC-dextran that permeated through the cell monolayer. RFU: Relative fluorescence units. N = 3 in quadruplicate. *: p<0.05 vs. thrombin/vehicle (2-way ANOVA/Bonferroni’s multiple comparison post hoc test).

Fig 5. Electrophoretic mobility of CXCL12α, CXCL12₁ and CXCL12₂.

Per lane 1 μg of protein in 25μL sample buffer (4 μM) were used for SDS-polyacrylamide gel electrophoresis under non-reducing (-) and reducing (+, βME: 0.357 M β-mercaptoethanol) conditions. The position of molecular mass standards is indicated on the left.

Fig 6. Presto-Tango β-arrestin 2 recruitment assays for CXCR4 (A-C) and ACKR3 (D-F).

RLU%: % of the luminescence signal for 1 μM CXCL12α. N = 9 for CXCL12α and n = 3 for all other proteins.

Fig 7. Dose-dependent effects of CXCL12α/β, CXCL12 (3–68) and CXCL12 mutants K27A/R41A/R47A, R47E and S-S4V on thrombin-induced impairment of hPPAEC monolayer permeability.

hPPAEC cells were grown to a confluent monolayer on collagen-coated permeable membranes. hPPAEC were then exposed to 35 nM of thrombin. After 10 min, thrombin-exposed cells were treated with vehicle or 50 nM (A), 5 nM (B), 0.5 nM (C) or 0.05 nM (D) of the various proteins, as indicated. In (D) 5 nM CXCL12α was used as a positive control. N = 3 in quadruplicate.

Fig 8. Dose-dependent effects of CXCL12α, CXCL12₁ and CXCL12₂ on thrombin-induced impairment of hPPAEC monolayer permeability.

hPPAEC cells were grown to a confluent monolayer on collagen-coated permeable membranes. hPPAEC were then exposed to 35 nM of thrombin. After 10 min, thrombin-exposed cells were treated with vehicle or 50 nM (A), 5 nM (B), 0.5 nM (C) or 0.05 nM (D) of the various proteins, as indicated. In (D) 5 nM CXCL12α was used as a positive control. N = 3 in quadruplicate.

Fig 9. Inhibition of thrombin-induced hyper-permeability of hPPAEC by CXCL12/CXCL12 variants.

% inhibiton: % inhibiton of thrombin-induced hyperpermeability. Data from Figs 7 and 8 at t = 255 min. N = 3 inquadruplicate. *: p<0.05 vs. CXCL12α (2-way ANOVA/Bonferroni’s multiple comparison post hoc test).