Lung microvascular mitochondria regulate TNFα‐induced TNFR1 shedding

Abstract

Ligation of endothelial (EC) TNFα receptor 1 (TNFR1) by circulating tumor necrosis factor alpha (TNFα) promotes lung inflammation. TNFR1 shedding by inflammatory cells limits inflammation. However, it is not known whether TNFR1 shedding occurs in lung microvessels. To determine shedding mechanisms, we viewed ECs of single microvessels in inflated, isolated, blood‐perfused rat lungs by live confocal microscopy. To quantify cytosolic (Ca2+cyt) and mitochondrial (Ca2+mit) calcium levels, we co‐infused dyes fluo 4 AM and rhod 2 AM respectively. To determine TNFR1 EC expression, we infused vehicle or TNFα then microinjected a fluorophore‐conjugated TNFR1mAb followed with a buffer wash. After infusion of vehicle alone, Ca2+cyt and Ca2+mit oscillation amplitudes and EC TNFR1 expression were stable. By contrast, after TNFα infusion (1 ng/ml, 10 min) oscillation amplitudes increased two fold (p<0.05, n=3) and TNFR1 fluorescence decreased by 68±7% (p<0.05, n=4), indicating TNFR1 shedding. Pre‐infusion of TNFα converting enzyme (TACE) inhibitor TAPI‐1, or mitochondrial SOD mimetic, MnTBAP blocked TNFα‐mediated TNFR1 decrease. These findings imply novel signaling in which TNFR1 ligation enhances Ca2+mit oscillations, leading to increased mitROS triggering TNFR1 shedding. We provide the first evidence that TNFα induces TNFR1 shedding in lung EC in situ. We propose TNFR1 shedding limits lung inflammation.