Effect of a selective adenosine 3 receptor agonist on chemokine, cytokine and nitric oxide production in immunosttmulated macrophages and fibroblasts

Greg Egnaczyk, Gyorgy Haskó, Uszlo Virág, Andrew L. Salzmaa, Csaba Szabo

Research output: Contribution to journalArticle

Abstract

Introduction: Adenosine released from metabolically active cells in response to inflammatory and or ischemic insult or from the sympathetic nervous system has been shown to modulate various immune functions. In this study we investigated the effect of a selective adenosincj receptor (A3) agonist A-{3-iodobeji7yl}-adenosine-5'-/Vmethyluronamide (IB-MECA) on the production of nitric oxide, MIP-1 alpha. interleukin-12 and interleukin-6 by inununostimulaied macrophages and fibroblasts. Methods: The murine monocyte/macrophage cell line RAW 264.7 and marine pulmonary fibroblasts were grown in DMEM supplemented with 10 % fetal calf serum, 100 U/ml penicillin, and 100 (lg/ml streptomycin. Cells were treated with various concentrations (0.1 -300 \iM) of IB-MECA or vehicle 30 min prior to lipoporysaccharide and IFN challenge and thereafter treated with lipopolysaccharide (10 ug/ml) for 24 h. At 24h, MIP-1 alpha (MIP), interleukin-12 (p40 and p70) and interleukin-6 were measured in the supernatant with ELJSA, nitrite (the breakdown product of nitric oxide) was measured by the Griess reaction. Viability was measured by the MTT method. Results: In the RAW 264.7 macrophage cell line, pretreatment of cells with LPS. IFN, or the combination of LPS and IFN-gamma induced production of proinflammatory mediators IL-12 (p40), IL-6, MIP, and NO but not IL-12 (p70). IBMECA (0.1-300 |iM) pretreatment of RAW macrophages decreased IL-12 release from these cells after LPS and IFN-gamma administration. When exposed to LPS and IFN, the macrophages produced more IL-6 than when exposed to LPS alone. Pretreatmenl with ffiMECA suppressed both LPS and LPS+IFN-induced production of this cytokme, although less potently when cells were stimulated with LPS+IFN. Similarly, IB-MECA reduced NO production when macrophages were exposed to LPS alone, LPS+IFN, or IFN alone. Again, IB-MECA was most potent, when NO formation was elicited by LPS alone, less effective when induced by LPS and IFN-gamma, and only moderately potent when induced by IFN gamma alone. IB-MECA also reduced release of MIP from both LPS-sumulated RAW cells and LPS+IFN-gamma stimulated fibroblasts. We detected no change in the viability and mitochondria! respiration by IB-MECA. Conclusions: Thus, stimulation of A3 receptors suppresses the production of multiple pro-inflammatory mediators. Further work is required to elucidate the utility of A3 agonists for the treatment of inflammatory diseases or post-ischémie insults.

Original languageEnglish (US)
JournalCritical Care Medicine
Volume26
Issue number1 SUPPL.
StatePublished - 1998
Externally publishedYes

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Purinergic P1 Receptor Agonists
Chemokines
Interleukin-12
Nitric Oxide
Fibroblasts
Macrophages
Cytokines
Interleukin-6
Adenosine
Sympathetic Nervous System
Streptomycin
Nitrites
Penicillins
Lipopolysaccharides
N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine
Monocytes
Mitochondria
Respiration
Lung
Serum

ASJC Scopus subject areas

  • Critical Care and Intensive Care Medicine

Cite this

Effect of a selective adenosine 3 receptor agonist on chemokine, cytokine and nitric oxide production in immunosttmulated macrophages and fibroblasts. / Egnaczyk, Greg; Haskó, Gyorgy; Virág, Uszlo; Salzmaa, Andrew L.; Szabo, Csaba.

In: Critical Care Medicine, Vol. 26, No. 1 SUPPL., 1998.

Research output: Contribution to journalArticle

Egnaczyk, Greg ; Haskó, Gyorgy ; Virág, Uszlo ; Salzmaa, Andrew L. ; Szabo, Csaba. / Effect of a selective adenosine 3 receptor agonist on chemokine, cytokine and nitric oxide production in immunosttmulated macrophages and fibroblasts. In: Critical Care Medicine. 1998 ; Vol. 26, No. 1 SUPPL.
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abstract = "Introduction: Adenosine released from metabolically active cells in response to inflammatory and or ischemic insult or from the sympathetic nervous system has been shown to modulate various immune functions. In this study we investigated the effect of a selective adenosincj receptor (A3) agonist A-{3-iodobeji7yl}-adenosine-5'-/Vmethyluronamide (IB-MECA) on the production of nitric oxide, MIP-1 alpha. interleukin-12 and interleukin-6 by inununostimulaied macrophages and fibroblasts. Methods: The murine monocyte/macrophage cell line RAW 264.7 and marine pulmonary fibroblasts were grown in DMEM supplemented with 10 {\%} fetal calf serum, 100 U/ml penicillin, and 100 (lg/ml streptomycin. Cells were treated with various concentrations (0.1 -300 \iM) of IB-MECA or vehicle 30 min prior to lipoporysaccharide and IFN challenge and thereafter treated with lipopolysaccharide (10 ug/ml) for 24 h. At 24h, MIP-1 alpha (MIP), interleukin-12 (p40 and p70) and interleukin-6 were measured in the supernatant with ELJSA, nitrite (the breakdown product of nitric oxide) was measured by the Griess reaction. Viability was measured by the MTT method. Results: In the RAW 264.7 macrophage cell line, pretreatment of cells with LPS. IFN, or the combination of LPS and IFN-gamma induced production of proinflammatory mediators IL-12 (p40), IL-6, MIP, and NO but not IL-12 (p70). IBMECA (0.1-300 |iM) pretreatment of RAW macrophages decreased IL-12 release from these cells after LPS and IFN-gamma administration. When exposed to LPS and IFN, the macrophages produced more IL-6 than when exposed to LPS alone. Pretreatmenl with ffiMECA suppressed both LPS and LPS+IFN-induced production of this cytokme, although less potently when cells were stimulated with LPS+IFN. Similarly, IB-MECA reduced NO production when macrophages were exposed to LPS alone, LPS+IFN, or IFN alone. Again, IB-MECA was most potent, when NO formation was elicited by LPS alone, less effective when induced by LPS and IFN-gamma, and only moderately potent when induced by IFN gamma alone. IB-MECA also reduced release of MIP from both LPS-sumulated RAW cells and LPS+IFN-gamma stimulated fibroblasts. We detected no change in the viability and mitochondria! respiration by IB-MECA. Conclusions: Thus, stimulation of A3 receptors suppresses the production of multiple pro-inflammatory mediators. Further work is required to elucidate the utility of A3 agonists for the treatment of inflammatory diseases or post-isch{\'e}mie insults.",
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T1 - Effect of a selective adenosine 3 receptor agonist on chemokine, cytokine and nitric oxide production in immunosttmulated macrophages and fibroblasts

AU - Egnaczyk, Greg

AU - Haskó, Gyorgy

AU - Virág, Uszlo

AU - Salzmaa, Andrew L.

AU - Szabo, Csaba

PY - 1998

Y1 - 1998

N2 - Introduction: Adenosine released from metabolically active cells in response to inflammatory and or ischemic insult or from the sympathetic nervous system has been shown to modulate various immune functions. In this study we investigated the effect of a selective adenosincj receptor (A3) agonist A-{3-iodobeji7yl}-adenosine-5'-/Vmethyluronamide (IB-MECA) on the production of nitric oxide, MIP-1 alpha. interleukin-12 and interleukin-6 by inununostimulaied macrophages and fibroblasts. Methods: The murine monocyte/macrophage cell line RAW 264.7 and marine pulmonary fibroblasts were grown in DMEM supplemented with 10 % fetal calf serum, 100 U/ml penicillin, and 100 (lg/ml streptomycin. Cells were treated with various concentrations (0.1 -300 \iM) of IB-MECA or vehicle 30 min prior to lipoporysaccharide and IFN challenge and thereafter treated with lipopolysaccharide (10 ug/ml) for 24 h. At 24h, MIP-1 alpha (MIP), interleukin-12 (p40 and p70) and interleukin-6 were measured in the supernatant with ELJSA, nitrite (the breakdown product of nitric oxide) was measured by the Griess reaction. Viability was measured by the MTT method. Results: In the RAW 264.7 macrophage cell line, pretreatment of cells with LPS. IFN, or the combination of LPS and IFN-gamma induced production of proinflammatory mediators IL-12 (p40), IL-6, MIP, and NO but not IL-12 (p70). IBMECA (0.1-300 |iM) pretreatment of RAW macrophages decreased IL-12 release from these cells after LPS and IFN-gamma administration. When exposed to LPS and IFN, the macrophages produced more IL-6 than when exposed to LPS alone. Pretreatmenl with ffiMECA suppressed both LPS and LPS+IFN-induced production of this cytokme, although less potently when cells were stimulated with LPS+IFN. Similarly, IB-MECA reduced NO production when macrophages were exposed to LPS alone, LPS+IFN, or IFN alone. Again, IB-MECA was most potent, when NO formation was elicited by LPS alone, less effective when induced by LPS and IFN-gamma, and only moderately potent when induced by IFN gamma alone. IB-MECA also reduced release of MIP from both LPS-sumulated RAW cells and LPS+IFN-gamma stimulated fibroblasts. We detected no change in the viability and mitochondria! respiration by IB-MECA. Conclusions: Thus, stimulation of A3 receptors suppresses the production of multiple pro-inflammatory mediators. Further work is required to elucidate the utility of A3 agonists for the treatment of inflammatory diseases or post-ischémie insults.

AB - Introduction: Adenosine released from metabolically active cells in response to inflammatory and or ischemic insult or from the sympathetic nervous system has been shown to modulate various immune functions. In this study we investigated the effect of a selective adenosincj receptor (A3) agonist A-{3-iodobeji7yl}-adenosine-5'-/Vmethyluronamide (IB-MECA) on the production of nitric oxide, MIP-1 alpha. interleukin-12 and interleukin-6 by inununostimulaied macrophages and fibroblasts. Methods: The murine monocyte/macrophage cell line RAW 264.7 and marine pulmonary fibroblasts were grown in DMEM supplemented with 10 % fetal calf serum, 100 U/ml penicillin, and 100 (lg/ml streptomycin. Cells were treated with various concentrations (0.1 -300 \iM) of IB-MECA or vehicle 30 min prior to lipoporysaccharide and IFN challenge and thereafter treated with lipopolysaccharide (10 ug/ml) for 24 h. At 24h, MIP-1 alpha (MIP), interleukin-12 (p40 and p70) and interleukin-6 were measured in the supernatant with ELJSA, nitrite (the breakdown product of nitric oxide) was measured by the Griess reaction. Viability was measured by the MTT method. Results: In the RAW 264.7 macrophage cell line, pretreatment of cells with LPS. IFN, or the combination of LPS and IFN-gamma induced production of proinflammatory mediators IL-12 (p40), IL-6, MIP, and NO but not IL-12 (p70). IBMECA (0.1-300 |iM) pretreatment of RAW macrophages decreased IL-12 release from these cells after LPS and IFN-gamma administration. When exposed to LPS and IFN, the macrophages produced more IL-6 than when exposed to LPS alone. Pretreatmenl with ffiMECA suppressed both LPS and LPS+IFN-induced production of this cytokme, although less potently when cells were stimulated with LPS+IFN. Similarly, IB-MECA reduced NO production when macrophages were exposed to LPS alone, LPS+IFN, or IFN alone. Again, IB-MECA was most potent, when NO formation was elicited by LPS alone, less effective when induced by LPS and IFN-gamma, and only moderately potent when induced by IFN gamma alone. IB-MECA also reduced release of MIP from both LPS-sumulated RAW cells and LPS+IFN-gamma stimulated fibroblasts. We detected no change in the viability and mitochondria! respiration by IB-MECA. Conclusions: Thus, stimulation of A3 receptors suppresses the production of multiple pro-inflammatory mediators. Further work is required to elucidate the utility of A3 agonists for the treatment of inflammatory diseases or post-ischémie insults.

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