Mitochondrial Reactive Oxygen Species Regulate Spatial Profile of Proinflammatory Responses in Lung Venular Capillaries

Abstract

Cytokine-induced lung expression of the endothelial cell (EC) leukocyte receptor P-selectin initiates leukocyte rolling. To understand the early EC signaling that induces the expression, we conducted real-time digital imaging studies in lung venular capillaries. To compare receptor- vs nonreceptor-mediated effects, we infused capillaries with respectively, TNF-α and arachidonate. At concentrations adjusted to give equipotent increases in the cytosolic Ca2+, both agents increased reactive oxygen species (ROS) production and EC P-selectin expression. Blocking the cytosolic Ca2+ increases abolished ROS production; blocking ROS production abrogated P-selectin expression. TNF-α, but not arachidonate, released Ca2+ from endoplasmic stores and increased mitochondrial Ca2+. Furthermore, Ca2+ depletion abrogated TNF-α responses partially, but arachidonate responses completely. These differences in Ca2+ mobilization by TNF-α and arachidonate were reflected in spatial patterning in the capillary in that the TNF-α effects were localized at branch points, while the arachidonate effects were nonlocalized and extensive. Furthermore, mitochondrial blockers inhibited the TNF-α- but not the arachidonate-induced responses. These findings indicate that the different modes of Ca2+ mobilization determined the spatial patterning of the proinflammatory response in lung capillaries. Responses to TNF-α revealed that EC mitochondria regulate the proinflammatory process by generating ROS that activate P-selectin expression.

Hyperosmolarity enhances the lung capillary barrier

“…To test this hypothesis in the context of inflammation, we considered the secretion of the proinflammatory leukocyte adhesion receptor P-selectin. Previously, we reported that P-selectin secretion is a characteristic TNF-α-induced response (17). However, here the effect was completely blocked by hyperosmolar treatment, although it was rescued by inhibition of the associated actin polymerization with latrunculin B.…”

“…We also determined the increase in filtration rate at a fixed capillary pressure of 3 cmH 2 O (∆Jv), as ∆Jv denotes barrier deterioration (15). In addition, we determined expression of the leukocyte adhesion receptor P-selectin, which in lung venular capillaries marks an endothelial proinflammatory response (17). We report here our novel findings that hyperosmolar infusions enhanced capillary barrier properties and blocked P-selectin expression.…”

“…All data were obtained from venular capillaries (20-30 µm in diameter) that lie immediately downstream of the alveolar septum (14,15). To avoid leukocyte effects, we maintained bloodfree conditions in the experimental capillaries by infusing isosmolar Ringer's buffer containing 4% albumin through a wedged venous microcatheter (17). This catheter was also used for infusing reagents and dyes.…”

“…Our methods of preparing and imaging isolated blood-perfused rat lungs have been described (17)(18)(19). Briefly, lungs excised from adult male Sprague-Dawley rats were continuously pump-perfused (10-12 ml/min) with autologous rat blood warmed to 37°C.…”

Mechanisms of endotoxin-induced NO, IL-6, and TNF-α production in activated rat hepatic stellate cells: Role of p38 MAPK

“…[46][47][48] Mitochondrial ROS and p38 have been implicated in the elicitation of gene transcription responses to a variety of stimuli. 49,50 In cardiac fibroblasts, ROS-induced IL-6 expression was shown to be p38-and ERK-dependent. 46 However, our results show that exogenous H 2 O 2 does not stimulate p38.…”