Transcriptional pattern analysis of adrenergic immunoregulation in mice. Twelve hours norepinephrine treatment alters the expression of a set of genes involved in monocyte activation and leukocyte trafficking

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Abstract

We investigated in vivo effects of norepinephrine (NE) on the transcription of 200 immunologically relevant genes in the mouse. Balb/c mice were s.c. implanted with NE containing retard tablets. Twelve hours later, splenic mRNA was prepared and hybridized onto cDNA microarrays containing the sequences of the major cytokines, their receptors and all CD-antigens of the mouse. Consistent results were obtained with a set of five genes: in the NE-treated animals four genes (CXCR4, VCAM1, IL-1R2, CD 14) were found 2–8 fold upregulated as compared to sham treated animals, whereas the gene for CCR3 was downregulated (<0.5 fold). The findings were confirmed using quantitative reverse transcriptase Real Time PCR. These first results prove the usefulness of gene microarray technology towards transcription pattern analysis in neuroimmune interactions. Furthermore, they support the relevance of catecholamines in the regulation of leukocyte migration and the inflammatory response.

Introduction

Neuroendocrine immunoregulation is afflicted with a high degree of complexity. The brain, the endocrine and immune systems are all “dissipative systems” with a high degree of freedom in their reaction to a given external or internal stimulus. This can account for contradictory or unpredictable results, particularly in the in vivo situation.

A good example to illustrate the complexity of neuro-immune interactions is adrenergic immunomodulation, where the quantitative and qualitative outcome depends not only on dose and duration of the catecholamine stimulus, but also importantly on the stage of differentiation and/or activation of the different cells of the immune system (Haas and Schauenstein, 2001). Furthermore, catecholamines can act on immune functions in vivo in different ways, e.g. by changing the release of and/or receptivity to cytokines, or by changes in leukocyte trafficking thereby modulating the number of cells that participate in a given immune response (Benschop et al., 1996, Madden, 2001, Murray et al., 1993). The majority of the data so far suggest immunosuppressive effects due to beta-adrenergic stimulation and activation of cyclic AMP in immune cells (Sanders and Straub, 2002, Kohm and Sanders, 2001). However, several reports strongly suggest also immunoregulatory alpha-adrenergic mechanisms (Cupic et al., 2001), particularly in the in vivo situation (Felsner et al., 1995, Rouppe van der Voort et al., 2000). To make things even more confusing, it appears that pronounced species and strain differences are further complicating the picture.

A powerful strategy to tackle the problem of complexity should be to apply molecular multiparameter analysis techniques that allow recognizing changes in patterns of multiple components, instead of putting together single parameter observations, as has mainly been done so far.

Our group is interested in in vivo immunoregulatory effects of long term adrenergic stress. As experimental model we use the subcutaneous implantation of catecholamine containing retard tablets with a defined release to induce a hypercatecholaminemia in experimental animals corresponding to values observed under physiological stress conditions (Stevenson et al., 2001, Felsner et al., 1992, Felsner et al., 1995). In the present study we applied for the first time molecular techniques that allow to define the impact of a 12-h treatment with norepinephrine (NE) on the transcription of multiple, immunologically relevant genes in the mouse.

Section snippets

Animals

Male Balb/c mice were purchased at an age of 4–5 weeks from the Institute for Experimental Animal Breeding, Medical Faculty, University of Vienna, Austria, and maintained three or four animals per plastic cage under a 12/12 h light/dark cycle with laboratory chow and water ad libitum. After arrival they were accommodated to our facilities for at least 1 week before they were used at ages of 6–8 weeks.

Drugs and catecholamine treatment

l-Noaradrenaline bitartrate (NE), Propranolol-hydrochloride, Nadolol and

Results

In accordance with previous data in the rat model (Felsner et al., 1995), the noradrenergic treatment induced a significant loss in body weight of more than 10% and a significantly reduced relative organ weight of the spleen, which served as markers for the increased adrenergic tone (data not shown). Furthermore, also in line with previous experiences (Stevenson et al., 2001), significant changes occurred in WBC. The number of neutrophilic granulocytes significantly increased, whereas the

Discussion

In several previous studies we have used s.c. implanted retard tablets to investigate in vivo effects of long-term enhanced peripheral catecholamines on immune functions of the rat. The main result of this work was that a long term treatment with epinephrine or NE significantly suppressed the responsiveness of T cells only with concomitant blockade of beta-receptors (Felsner et al., 1995, Felsner et al., 1992, Liebmann et al., 1996, Stevenson et al., 2001). Using selective agonists, the

Acknowledgements

The helpful advice of Dr. Eva Roblegg and the technical assistance of Elvira Kloibhofer and Elfgard Heintz is gratefully acknowledged.

This study was supported by the Austrian Science Fund Project P 16060.

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