Activation of TNFR1 ectodomain shedding by mitochondrial Ca2+ determines the severity of inflammation in mouse lung microvessels - PubMed (original) (raw)

. 2011 May;121(5):1986-99.

doi: 10.1172/JCI43839. Epub 2011 Apr 25.

Mohammad Naimul Islam, Shonit R Das, Alice Huertas, Sadiqa K Quadri, Keisuke Horiuchi, Nilufar Inamdar, Memet T Emin, Jens Lindert, Vadim S Ten, Sunita Bhattacharya, Jahar Bhattacharya

Affiliations

Activation of TNFR1 ectodomain shedding by mitochondrial Ca2+ determines the severity of inflammation in mouse lung microvessels

David J Rowlands et al. J Clin Invest. 2011 May.

Abstract

Shedding of the extracellular domain of cytokine receptors allows the diffusion of soluble receptors into the extracellular space; these then bind and neutralize their cytokine ligands, thus dampening inflammatory responses. The molecular mechanisms that control this process, and the extent to which shedding regulates cytokine-induced microvascular inflammation, are not well defined. Here, we used real-time confocal microscopy of mouse lung microvascular endothelium to demonstrate that mitochondria are key regulators of this process. The proinflammatory cytokine soluble TNF-α (sTNF-α) increased mitochondrial Ca2+, and the purinergic receptor P2Y2 prolonged the response. Concomitantly, the proinflammatory receptor TNF-α receptor-1 (TNFR1) was shed from the endothelial surface. Inhibiting the mitochondrial Ca2+ increase blocked the shedding and augmented inflammation, as denoted by increases in endothelial expression of the leukocyte adhesion receptor E-selectin and in microvascular leukocyte recruitment. The shedding was also blocked in microvessels after knockdown of a complex III component and after mitochondria-targeted catalase overexpression. Endothelial deletion of the TNF-α converting enzyme (TACE) prevented the TNF-α receptor shedding response, which suggests that exposure of microvascular endothelium to sTNF-α induced a Ca2+-dependent increase of mitochondrial H2O2 that caused TNFR1 shedding through TACE activation. These findings provide what we believe to be the first evidence that endothelial mitochondria regulate TNFR1 shedding and thereby determine the severity of sTNF-α-induced microvascular inflammation.

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Figures

Figure 1

Figure 1. Lung microvessels shed TNFR1 ectodomains.

(A) Confocal images showing endothelial fluorescence in microvessels infused with calcein (5 μM, 20 minutes) or the anti-TNFR1 mAb MCA2350 (40 μg/ml, 5 minutes). Images were obtained 30 minutes after infusion with buffer or 0.01% TB. The image at right was obtained from a separate vessel 30 minutes after labeling for TNFR1. Alv, alveolus; Lum, microvascular lumen. Scale bar: 50 μm. (B and C) Endothelial TNFR1 fluorescence in microvessels infused with buffer control or sTNF-α (1 ng/ml) for 10 minutes. Scale bar: 25 μm. (D) Line-scan analyses of endothelial immunofluorescence in vessels given buffer control, goat IgG, sTNF-α, or sTNF-α in the presence of TNFR1 blocking mAb E20 (40 μg/ml). n = 4 per group per time point. *P < 0.05 versus control. (E and F) Effects of TACE (E) and LPS (F) on sTNF-α–induced TNFR1 shedding in microvessels. Data were obtained 1 hour after intratracheal instillation. TAP, TAPI-1 (50 μM); LPS (1 mg/kg). *P < 0.05 versus control; #P < 0.05 versus sTNF-α. (G) IB of lysates of lung tissue (replicated 3 times). (H and I) TNFR1 surface expression in primary isolates of ECs (red boxes denote vWF-positive cells) derived from lungs treated as indicated.

Figure 2

Figure 2. Lung microvascular endothelium does not internalize TNFR1.

Lung microvessels were sequentially infused with red fluorescent calcein red (5 μM, 20 minutes), then (A–C) green fluorescent FITC-conjugated albumin (5%, 10 minutes) or (D–F) green fluorescent sTNF-α–488 (1 ng/ml, 10 minutes). Each infusion was followed by buffer infusion for a further 20 minutes, then by TB. Scale bars: 25 μm. Colocalization of red and green fluorescence is indicated by yellow pseudocolor in the microvessel (B) and in the high-power image of the boxed region showing a single EC (C). Note the lack of colocalization for sTNF-α (F and G). *P < 0.05 versus calcein. n = 4 per group.

Figure 3

Figure 3. sTNF-α–induced TNFR1 shedding in lung microvessels is Ca2+ dependent.

(A) Image shows fluorescence in pseudocolors, indicating Ca2+ levels in the cytosol and mitochondria. Scale bar: 10 μm. (B and C) High-power view of rhod-2–loaded endothelium (B) and tracings (C) show time-dependent fluorescence changes indicative of Ca2+ oscillations. Saponin (0.01%) was infused to confirm loss of cytosolic, but not mitochondrial, fluorescence, indicating spatial selectivity of the fluorophores. BL, baseline. Scale bar: 25 μm. (D–F) Data are for the indicated variables from microvessels showing responses to sTNF-α in the presence of RR (10 μM) and XeC (20 μM). *P < 0.05 versus baseline, #P < 0.05 versus sTNF-α. n as indicated.

Figure 4

Figure 4. Purinergic receptors are required for sustained sTNF-α–induced endothelial Ca2+ oscillations.

Traces from single microvessels and group data show responses in lung microvessels of WT, P2Y2–/–, and P2Y1–/– mice. *P < 0.05 versus baseline, #P < 0.05 versus 5 minutes after sTNF-α. n = 4.

Figure 5

Figure 5. Mitochondrial H2O2 mediates sTNF-α–induced TNFR1 shedding.

DCF (2.5 μM). (A) roGFP fluorescence in mouse lung endothelium. Dashed lines indicate microvascular walls; arrow denotes direction of flow. Scale bar: 25 μm. (B and C) Single experiment (B) and group data (C) showing endothelial roGFP fluorescence responses after infusion of sTNF-α (1 ng/ml, 10 minutes) in the presence of MitoQ (100 nM) or vehicle (Veh; ethanol). H2O2 (10 μM) was administered by microvascular micropuncture. (D) Group data for DCF-loaded microvessels infused with sTNF-α in the presence or absence of MitoQ or in lungs from mice transfected with cCAT or mCAT. Responses in vector-transfected animals were not significantly different from control (n = 3 for each empty vector; not shown). (E) Effect of ROS inhibition on sTNF-α–induced shedding of TNFR1 determined in single microvessels. *P < 0.05 versus baseline or control; #P < 0.05 versus sTNF-α.

Figure 6

Figure 6. Mitochondrial RISP is required for TNFR1 shedding in lung microvessels.

Mice were treated with the indicated siRNAs. (A–D) Lungs were analyzed 2 days after for RISP expression by IB on lysates derived from primary isolates of lung ECs (A; n = 3) and lung homogenates (B; n = 3) in the indicated cell fractions. Determinations on isolated lung mitochondria are shown (C and D; n = 4). *P < 0.05 versus scRNA. (E–G) Microvessels at baseline (E) and 20 minutes after sTNF-α (1 ng/ml, 10 minutes) infusion (F and G). Regions of low (single arrow) and high (double arrow) siRISP uptake are indicated (E). Dashed lines indicate microvascular walls. (H) Group data for microvessels imaged 20 minutes after sTNF-α infusion. *P < 0.05 versus baseline, #P < 0.05 versus sTNF-α.

Figure 7

Figure 7. TNFR1 shedding enhances sTNF-α–induced lung endothelial responses.

(A) Alveolar and vascular immunofluorescence of TACE and the endothelial marker VE-cadherin in lungs from Tacefl/fl and EC-Tace–/– mice. mAbs given as 40 μg/ml. (B–D) Group data for lung endothelial Ca2+ and ROS. DCF (2.5 μM); TAPI-1 (50 μM). *P < 0.05 versus baseline; #P < 0.05 versus WT sTNF-α (B and C) and Tacefl/fl sTNF-α (D).

Figure 8

Figure 8. TNFR1 shedding determines lung microvascular E-selectin expression.

(A) Upper images show live E-selectin immunofluorescence (red) in microvessels coloaded with cytosolic fluorescence of calcein (green). Lungs are from mice transfected with scRNA or siRISP. The microvessels were infused with sTNF-α for 10 minutes, then imaged 2 hours later. E-selectin Ab (40 μg/ml) was microinjected. Unbound antibody was removed by buffer wash. Bar: 25 μm. Lower images show ECs in the boxed regions at high power, indicating increased density of E-selectin in a siRISP-treated mouse. (B) Effect of mitochondrial inhibitors on E-selectin expression, determined by endothelial line-scan analyses. ROT, rotenone (1 μM). (C) IB representative for 3 separate experiments of untreated control or sTNF-α–infused scRNA or siRISP-transfected lungs. (D) Lung E-selectin expression. sTNF-α was infused as indicated in WT, Tacefl/fl, Tace–/–, P2Y1–/–, and P2Y2–/– mice. RR (10 μM); TAPI-1 (50 μM). *P < 0.05 versus control, #P < 0.05 versus sTNF-α.

Figure 9

Figure 9. Mitochondrial mechanisms determine sTNF-α–induced microvascular leukocyte recruitment.

(A and B) Low- (A) and high-magnification (B) confocal images show leukocytes (red) adherent to lung endothelium (green). Lungs were infused as indicated with buffer control or sTNF-α for 10 minutes. Images were obtained after 2 hours. Arrow denotes adherent leukocytes at a microvessel branch-point. Scale bars: 50 μm (A and B, top); 25 μm (B, bottom). (C) Number of adherent leukocytes per lung, determined as the average for 5 regions viewed at low magnification. Rotenone (1 μM). *P < 0.05 versus control; #P < 0.05 versus sTNF-α; †P < 0.05 versus scRNA. n as indicated.

Figure 10

Figure 10. Sequence of events underlying sTNF-α–induced TNFR1 shedding in lung endothelium.

Ligation of TNFR1 by sTNF-α (i) leads to IP3-induced store release of Ca2+ (ii), resulting in increase of cytosolic Ca2+ (iii), increase of mitochondrial Ca2+ (iv), and RISP-dependent ROS generation at complex III (v). Elevation of cytosolic Ca2+ leads to E-selectin expression and leukocyte recruitment (vi). Release of ATP ligates the P2Y2 receptor and potentiates the cytosolic Ca2+ increase (vii). ROS-dependent activation of TACE (viii) limits further sTNF-α signaling by stimulating ectodomain shedding of TNFR1.

Comment in

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