Endothelial TNFR1 shedding by mitochondria

Abstract

Lung inflammation critically results from the binding of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα) to TNFα receptor 1 (TNFR1). Although the binding sheds TNFR1 in leukocytes, responses in lung endothelial cells (EC) are not known. To determine TNFR1 dynamics in lung EC, we considered the role of the mitochondrial Rieske Iron‐Sulphur protein (RISP). We gave tail‐vein injections of liposome‐conjugated, fluorescent siRNA that were specific (siRISP) or non‐specific (siNS) for RISP. To quantify responses in real‐time, after 48 hours we imaged single microvessels of inflated isolated, blood‐perfused mouse lungs by live confocal microscopy. By micropuncture, we infused microvessels with a fluorophore‐conjugated TNFR1 mAb followed by buffer wash. The residual fluorescence denoted EC TNFR1 expression. TNFR1 expression was stable for >1 hr. Line scan analyses revealed no correlation between expressions of TNFR1 and siRISP. In siNS‐treated lungs, TNFα infusion (1 ng/ml) globally decreased TNFR1 fluorescence by >50% within 30 minutes (P<0.05, n=3). By contrast, siRISP expression blocked the decrease (P<0.05). We conclude that in lung EC, TNFα causes TNFR1 shedding, thereby possibly limiting inflammation. Our findings implicating RISP are the first evidence that a mitochondrial protein determines lung expression of a critical proinflammatory receptor (HL57556).