GM-CSF up-regulates arginase and iNOS in rat alveolar macrophages in a glucocorticoid-sensitive manner

Sonja Matthiesen, Martin Heideking & Kurt Racké, Department of Pharmacology & Toxicology, University of Bonn, Reuterstr. 2b, D-53113 Bonn, Germany.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine involved in the pathogenesis of asthma and development of airway hyperresponsiveness (Yamashita et al., 2002). In bone marrow-derived macrophages, GM-CSF was demonstrated to increase expression and activity of arginase I (Jost et al., 2003), an enzyme linked to cell proliferation and connective tissue production. Arginase I is up-regulated in asthmatic airways (Zimmermann et al., 2003). The present experiments aim to explore the modulatory role of GM-CSF in inducing arginase I and iNOS in primary rat a lveolar macrophages (AM).

AM were obtained by lavage of isolated rat (Sprague-Dawley, either sex ) lungs and cultured in the absence or presence of test substances. After a 20 h incubation period arginase and iNOS activity were determined or RNA was isolated for use in RT-PCR. The amount of active arginase was evaluated in cell lysates by in vitro formation of urea. Nitrite accumulation in culture supernatants was determined to reflect NO synthesis.

Arginase activity in AM cultured under control conditions averaged out at 173 ± 18 mU/mg protein (n=15). Exposure to GM-CSF (1, 10 and 100 ng/ml) caused a concentration-dependent increase of arginase activity to 193 ± 22, 322 ± 33 and 341 ± 40 mU/mg protein, respectively ( mean ± s.e.mean, n=5 each, p<0.05 ). The stimulation was paralleled by enhanced transcription of arginase I as seen in RT-PCR (190 ± 33% of controls in presence of 100 ng/ml GM-CSF, n=5, p=0.04 ). Presence of the glucocorticoid dexamethasone (100 nM) prevented the GM-CSF-mediated stimulation of arginase activity (198 ± 7 mU/mg protein at 100 ng/ml GM-CSF, n=5, p<0.001 ).

A concentration-dependent up-regulation could also be proved for activity of iNOS since s timulation with GM-CSF enhanced formation of nitrite up to 227 ± 21% (100 ng/ml, n=6). RT-PCR confirmed induction of iNOS mRNA up to 193 ± 50 % of controls in presence of GM-CSF (100 ng/ml, n=4, p=0.16). The GM-CSF-induced increase in nitrite accumulation was abolished by addition of 100 nM dexamethasone.

Elucidation of the regulatory function of GM-CSF in stimulating arginase and iNOS activity and the inhibitory effect of glucocorticoids are of particular interest in order to analyse cytokine-modulated immune processes and to find new targets for treatment of chronic or allergic inflammatory diseases.

Jost M M et al. (2003) FASEB J . 17:2281-2283.
Yamashita N et al. (2002) Cell. Immunol. 219:92-97.
Zimmermann N et al. (2003) J. Clin. Invest. 111:1863-1874.

Supported by DFG Ra 400/12-2.