Cross-Regulation of iNOS and COX-2 by its Products in Murine Macrophages Under Stress Conditions

Abstract

Exposure of macrophages to heat shock induces rapid synthesis of heat shock proteins (HSPs) which are important for cell homeostasis. Prostaglandins (PGs) and nitric oxide (NO) are important cell regulatory molecules. We have therefore investigated the interactions between these molecules in the LPSinduced expression of iNOS and COX-2 and in the mitochondrial activity of macrophages. Cultures of the murine macrophage cell line, J774, were exposed to heat shock (43°C, 30min) and stimulated with LPS (1 µg/ml), concomitantly or after 8h of cell recovery. NO production was measured by Griess reaction; PGE 2 by ELISA; HSP70, iNOS and COX-2 by immunobloting; mitochondrial activity by MTT assay. Heat shock induced HSP70, but not iNOS or COX-2 whereas LPS induced iNOS and COX-2 but not HSP70. When heat shock and LPS were given concomitantly, iNOS but not COX-2 expression was reduced. When a period of 8h was given between heat shock and LPS stimulation, iNOS, COX-2, PGE 2 and NO levels were significantly increased. Under these conditions, the expression of COX-2 was reduced by L-NAME (NOsynthesis inhibitor) and of iNOS by nimesulide (PGssynthesis inhibitor). Such cross-regulation was not observed in cells at 37°C. These treatments significantly reduced MTT levels in cells at 37°C but not in cells submitted to heat shock. These results suggest that HSPs and cross-regulation of iNOS and COX-2 by their products might be of relevance in the control of cell homeostasis during stress conditions.

Leukotrienes Produced in Allergic Lung Inflammation Activate Alveolar Macrophages

“…To evaluate nitric oxide (NO) production, the nitrite concentrations in the supernatants of the AM cultures were measured using the standard Griess reaction [21]. Briefly, 50 μl of supernatant culture medium was added to 50 μl of Griess reagent [equal volumes of 1% (w/v) sulfanilamide in 2.5% (v/v) phosphoric acid and 0.1% (w/v) naphthylenediamine-HCl] and incubated at room temperature for 10 min.…”

Insulin Inhibits LPS-Induced Signaling Pathways in Alveolar Macrophages

“…Then, AMs were stimulated for 30 minutes with 100 ng/mL LPS and were placed on ice for 10 minutes to stop the reaction. After that, AMs were washed three times in ice-cold PBS and lysed by sonication in ice-cold lyses buffer containing 150 mM TrisHCl (pH 8.0), 100 mM NaCl, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 1 µg/mL leupeptin, followed by ultracentrifugation at 100 000 g for 20 minutes at 4°C, the supernatant was frozen -70ºC for immunobloting [15,17].…”

Insulin Suppresses LPS-induced iNOS and COX-2 Expression and NF-κB Activation in Alveolar Macrophages and

“…Then, AMs were stimulated for 30 minutes with 100 ng/mL LPS and were placed on ice for 10 minutes to stop the reaction. After that, AMs were washed three times in ice-cold PBS and lysed by sonication in ice-cold lyses buffer containing 150 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 1 µg/mL leupeptin, followed by ultracentrifugation at 100 000 g for 20 minutes at 4°C, the supernatant was frozen -70ºC for immunobloting and measuring of NO and PGE 2 production [15,16]. 2 To evaluate NO production, nitrite concentration in the supernatants of AMs cultures was measured using the standard Griess reaction.…”