Increased carbon dioxide levels stimulate neutrophils to produce microparticles and activate the nucleotide-binding domain-like receptor 3 inflammasome

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We hypothesized that elevations of carbon dioxide (CO2) commonly found in modern buildings will stimulate leukocytes to produce microparticles (MPs) and activate the nucleotide-binding domain-like receptor 3 (NLRP3) inflammasome due to mitochondrial oxidative stress. Human and murine neutrophils generate MPs with high interleukin-1β (IL-1β) content when incubated ex vivo in buffer equilibrated with 0.1–0.4% additional CO2. Enhanced MPs production requires mitochondrial reactive oxygen species production, which is mediated by activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase. Subsequent events leading to MPs generation include perturbation of inositol 1,3,5-triphosphate receptors, a transient elevation of intracellular calcium, activation of protein kinase C and NADPH oxidase (Nox). Concomitant activation of type-2 nitric oxide synthase yields secondary oxidants resulting in actin S-nitrosylation and enhanced filamentous actin turnover. Numerous proteins are linked to short filamentous actin including vasodilator-stimulated phosphoprotein, focal adhesion kinase, the membrane phospholipid translocation enzymes flippase and floppase, and the critical inflammasome protein ASC (Apoptosis-associated Speck protein with CARD domain). Elevations of CO2 cause oligomerization of the inflammasome components ASC, NLRP3, caspase 1, thioredoxin interacting protein, and calreticulin - a protein from endoplasmic reticulum, leading to IL-1β synthesis. An increased production rate of MPs containing elevated amounts of IL-1β persists for hours after short-term exposures to elevated CO2.

Graphical abstract

Hypothetical biochemical mechanism for CO2-mediated MPs production: Data suggest that the initial event triggering MPs generation by elevated concentrations of CO2 and/or H2CO3 is carboxylation reactions that enhance mitochondrial production of superoxide (O2.), H2O2 and potentially, a varied array of CO2-derived oxidants. These act on endoplasmic reticulum inositol 1,3,5-triphosphate (IP3) receptors to trigger an elevation of intracellular calcium, followed by activation of protein kinase C isoforms that increase NADPH oxidase (Nox) activity. Nox-derived O2. feeds back to cause further mitochondrial oxidant production and also reacts with nitric oxide (.NO). CO2 and/or H2CO3 are likely to interact at this step to produce agents capable of protein S-nitrosylation (SNO-) leading to formation of cytosolic SNO-actin. Actin turnover is enhanced by linkage of vasodilator stimulated phosphoprotein (VASP) to SNO-actin and accelerated polymerization hastens linkage of Rac 1/2 and focal adhesion kinase (FAK) that are depicted in the figure with dotted lines. FAK links type-2 nitric oxide synthase (iNOS) with actin, enhancing its activity and thus contributing to the auto-catalytic nature of the process. There is also linkage of floppase and flippase to cytosolic actin, which may impact enzyme activity, and which are required for the membrane phospholipid changes ultimately required for MPs formation. Additionally, Apoptosis-associated Speck protein with CARD domain (ASC) links to SNO-actin and secondary association of NLRP3, caspase 1 and calreticulin occur with production of active IL-1β.

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