Pavle S. Milutinovic

The Receptor for Advanced Glycation End Products Is a Central Mediator of Asthma Pathogenesis

The receptor for advanced glycation end products (RAGE) is a multiligand receptor that has been shown to contribute to the pathogenesis of diabetes, atherosclerosis, and neurodegeneration. However, its role in asthma and allergic airway disease is largely unknown. These studies use a house dust mite (HDM) mouse model of asthma/allergic airway disease. Respiratory mechanics were assessed and compared between wild-type and RAGE knockout mice. Bronchovascular architecture was assessed with quantitative scoring, and expression of RAGE, immunoglobulins, and relevant cytokines was assessed by standard protein detection methods and/or quantitative RT-PCR. The absence of RAGE abolishes most assessed measures of pathology, including airway hypersensitivity (resistance, tissue damping, and elastance), eosinophilic inflammation, and airway remodeling. IL-4 secretion, isotype class switching, and antigen recognition are intact in the absence of RAGE. In contrast, normal increases in IL-5, IL-13, eotaxin, and eotaxin-2 production are abrogated in the RAGE knockouts. IL-17 indicates complex regulation, with elevated baseline expression in RAGE knockouts, but no induction in response to allergen. Treatment of WT mice with an inhibitor of RAGE markedly reduces inflammation in the HDM model, suggesting that RAGE inhibition may serve as a promising therapeutic strategy. Finally, the results in the HDM model are recapitulated in an ovalbumin model of asthma, suggesting that RAGE plays a role in asthma irrespective of the identity of the allergens involved.

Lack of the Receptor for Advanced Glycation End-Products Attenuates E. coli Pneumonia in Mice

Background The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated. Methodology/Principal Findings C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice. Conclusions/Significance Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.

Pulmonary receptor for advanced glycation end-products promotes asthma pathogenesis through IL-33 and accumulation of group 2 innate lymphoid cells.

BACKGROUND Single nucleotide polymorphisms in the human gene for the receptor for advanced glycation end-products (RAGE) are associated with an increased incidence of asthma. RAGE is highly expressed in the lung and has been reported to play a vital role in the pathogenesis of murine models of asthma/allergic airway inflammation (AAI) by promoting expression of the type 2 cytokines IL-5 and IL-13. IL-5 and IL-13 are prominently secreted by group 2 innate lymphoid cells (ILC2s), which are stimulated by the proallergic cytokine IL-33. OBJECTIVE We sought to test the hypothesis that pulmonary RAGE is necessary for allergen-induced ILC2 accumulation in the lung. METHODS AAI was induced in wild-type and RAGE knockout mice by using IL-33, house dust mite extract, or Alternaria alternata extract. RAGEs lung-specific role in type 2 responses was explored with bone marrow chimeras and induction of gastrointestinal type 2 immune responses. RESULTS RAGE was found to drive AAI by promoting IL-33 expression in response to allergen and by coordinating the inflammatory response downstream of IL-33. Absence of RAGE impedes pulmonary accumulation of ILC2s in models of AAI. Bone marrow chimera studies suggest that pulmonary parenchymal, but not hematopoietic, RAGE has a central role in promoting AAI. In contrast to the lung, the absence of RAGE does not affect IL-33-induced ILC2 influx in the spleen, type 2 cytokine production in the peritoneum, or mucus hypersecretion in the gastrointestinal tract. CONCLUSIONS For the first time, this study demonstrates that a parenchymal factor, RAGE, mediates lung-specific accumulation of ILC2s.

Anesthetic properties of the ketone bodies β-hydroxybutyric acid and acetone

BACKGROUND:We tested the hypothesis that two metabolites that are elevated in ketosis (β-hydroxybutyric acid, and acetone) modulate ion channels in a manner similar to anesthetics and produce anesthesia in animals. METHODS:α1β2&ggr;2s&ggr;-aminobutyric acid type A (GABAA), α1 glycine, NR1/NR2A N-methyl-d-aspartate, and two pore domain TRESK channels were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. The effect of β hydroxybutyric acid and acetone on channel function was measured. The anesthetic effects of these drugs were measured in X. laevis tadpoles. RESULTS:Both β hydroxybutyric acid and acetone enhanced glycine receptor function in the concentration range that is obtained in ketoacidosis in humans. Beta hydroxybutyric acid also enhanced GABAA receptor function at these concentrations. Both acetone and β-hydroxybutyric acid anesthetized tadpoles, with an EC50 for acetone of 264 ± 2 mM (mean ± se) and for β-hydroxybutyric acid of 151 ± 11 mM at pH 7.0. Acetone enhanced GABAA receptors at concentrations of 50 mM and above. Inhibition of TRESK channel function was seen with 100 mM acetone or larger concentration. N-methyl-D-aspartate receptor function was inhibited at concentrations of acetone of 200 mM and larger. CONCLUSIONS:Beta hydroxybutyric acid and acetone are anesthetics. Both ketone bodies enhance inhibitory glycine receptors at concentrations observed clinically in ketoacidosis. In addition, β-hydroxybutyric acid enhances GABAA receptor function at these concentrations. Subanesthetic concentrations of these drugs may contribute to the lethargy and impairment of consciousness seen in ketoacidosis.

Anesthetic-Like Modulation of a -Aminobutyric Acid Type A, Strychnine-Sensitive Glycine, and N-Methyl-D- Aspartate Receptors by Coreleased Neurotransmitters

INTRODUCTION:A mechanism of anesthesia has recently been proposed which predicts that coreleased neurotransmitters may modulate neurotransmitter receptors for which they are not the native agonist in a manner similar to anesthetics. METHODS:We tested this prediction by applying acetylcholine to a NR1/NR2A N-methyl-d-aspartate receptor, glycine to a wild-type &agr;1&bgr;2 and anesthetic-resistant &agr;1(S270I)&bgr;2 &ggr;-amino-butyric acid (GABA) type A receptor, and GABA to a homomeric &agr;1 wild type and anesthetic-resistant &agr;1 S267I glycine receptor. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. RESULTS:We found inhibition of N-methyl-d-aspartate receptor function by acetylcholine, enhancement of glycine receptor function by GABA, and enhancement of GABA type A receptor function by glycine. As expected of compounds with anesthetic activity, GABA showed far less potentiation (enhancement) of the function of the anesthetic-resistant S267I glycine receptor than that of the wild-type receptor. Glycine potentiated the function of wild-type GABA type A receptors but inhibited the function of the anesthetic-resistant S270I GABA type A receptor. CONCLUSIONS:These results show that neurotransmitters that are coreleased onto anesthetic-sensitive receptors may modulate the function of receptors for which they are not the native agonist via an anesthetic-like mechanism. These findings lend support to a recent theory of anesthetic action.

Ammonia has anesthetic properties

BACKGROUND:A recent theory of anesthesia predicts that some endogenous compounds should have anesthetic properties. This theory raises the possibility that metabolites that are profoundly elevated in disease may also exert anesthetic effects. Because in pathophysiologic concentrations, ammonia reversibly impairs memory, consciousness, and responsiveness to noxious stimuli in a manner similar to anesthetics, we investigated whether ammonia had anesthetic properties. METHODS:The effect of ammonia was studied on &agr;1&bgr;2 and &agr;1&bgr;2&ggr;2s &ggr;-amino butyric acid type A, &agr;1 glycine, and NR1/NR2A N-methyl-d-aspartate receptors, and the two-pore domain potassium channel TRESK. Channels were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. The immobilizing effect of ammonia in rats was evaluated by determining the reduction in isoflurane minimum alveolar concentration produced by IV infusion of ammonium chloride. The olive oil-water partition coefficient was measured to determine whether free ammonia (NH3) followed the Meyer-Overton relation. RESULTS:Ammonia positively modulated TRESK channels and glycine receptors. No effect was seen on &agr;1&bgr;2 and &agr;1&bgr;2&ggr;2s &ggr;-amino butyric acid type A receptors or NR1/NR2A N-methyl-d-aspartate receptors. Ammonia reversibly decreased the requirement for isoflurane, with a calculated immobilizing EC50 of 1.6 ± 0.1 mM NH4Cl. The Ostwald olive oil-water partition coefficient for NH3 was 0.018. At a pH of 7.4, and at the anesthetic EC50, the NH3 concentration in bulk olive oil is 0.42 &mgr;M, approximately five orders of magnitude less than observed by anesthetics that follow the Meyer-Overton relation. CONCLUSIONS:These findings support the hypothesis that ammonia has anesthetic properties. Bulk oil concentration did not predict the potency of ammonia.

Clearance kinetics and matrix binding partners of the receptor for advanced glycation end products

Elucidating the sites and mechanisms of sRAGE action in the healthy state is vital to better understand the biological importance of the receptor for advanced glycation end products (RAGE). Previous studies in animal models of disease have demonstrated that exogenous sRAGE has an anti-inflammatory effect, which has been reasoned to arise from sequestration of pro-inflammatory ligands away from membrane-bound RAGE isoforms. We show here that sRAGE exhibits in vitro binding with high affinity and reversibly to extracellular matrix components collagen I, collagen IV, and laminin. Soluble RAGE administered intratracheally, intravenously, or intraperitoneally, does not distribute in a specific fashion to any healthy mouse tissue, suggesting against the existence of accessible sRAGE sinks and receptors in the healthy mouse. Intratracheal administration is the only effective means of delivering exogenous sRAGE to the lung, the organ in which RAGE is most highly expressed; clearance of sRAGE from lung does not differ appreciably from that of albumin.

Tolerance to Isoflurane Does Not Occur in Developing Xenopus laevis Tadpoles

INTRODUCTION: Tolerance is observed for a variety of central nervous system depressants including ethanol, which is an anesthetic, but has not been convincingly demonstrated for a potent halogenated volatile anesthetic. Failure to demonstrate tolerance to these agents may be the result of inadequate exposure to anesthetic. In this study, we exposed Xenopus laevis tadpoles to surgical anesthetic concentrations of isoflurane for 1 wk. METHODS: Xenopus laevis tadpoles were produced by in vitro fertilization, and exposed to isoflurane (0.59%, 0.98%, 1.52%) or oxygen for 1 wk starting from the time of fertilization. RESULTS: Changes in anesthetic EC50 were small and not in a consistent direction. Control animals had an anesthetic EC50 of 0.594% ± 0.003% isoflurane. Tadpoles exposed to 1.52% isoflurane had a lower EC50 than controls (by 16%), whereas tadpoles raised under 0.59% and 0.98% isoflurane had higher EC50s than control (by 4.7% and 7.4%, respectively). CONCLUSION: We provide the first description of week-long exposures of vertebrates to surgical anesthetic concentrations of isoflurane, and the first report of such exposures in developing vertebrates. Tolerance to isoflurane does not occur in developing Xenopus laevis tadpoles. Taken together with studies in other organisms, the development of tolerance to ethanol but not isoflurane suggests that mechanisms shared by these drugs probably do not account for the development of tolerance.