Research report1,25-Dihydroxyvitamin D3 inhibits the expression of inducible nitric oxide synthase in rat central nervous system during experimental allergic encephalomyelitis
Introduction
Initially called EDRF (endothelium-derived relaxing factor), nitric oxide (NO) is a free gaseous radical which mediates a variety of biological functions, including vasodilation, neurotransmission and cytotoxicity [9]. NO is produced from l-arginine by the enzyme NO-synthase (NOS) for which different isoforms have been described in the central nervous system (CNS). Constitutive forms of NOS (cNOS) are Ca2+- and calmodulin-dependent, while inducible forms (iNOS) are Ca2+ and calmodulin-independent and continuously produce NO 16, 26, 28.
During brain inflammation, such as in viral infections or experimental allergic encephalomyelitis (EAE), the iNOS gene is up-regulated [21]. Increased NO production may be responsible, to some extent, for the alteration of brain functions or CNS tissue destruction in EAE-rats 21, 25, 29, 46, 58. In vitro studies suggest that glial cell-derived NO may cause the death of oligodendrocytes and, thus, could participate in the formation of lesions during multiple sclerosis (MS) 3, 37. This hypothesis is supported by the fact that human iNOS mRNA is markedly elevated in demyelinated regions of the brains of MS patients when compared to control brains [6]. However, conflicting results have been reported concerning the effects of pharmacological inhibitors of iNOS on the clinical course of EAE 8, 59, probably because NO can exert both beneficial and harmful effects.
In view of the potential role of NO in the pathogenesis of the CNS, the regulation of iNOS expression has become a matter of intense investigation. Lipopolysaccharide (LPS) and IFNγ, alone or in combination with TNFα or IL1β, are potent inducers of iNOS gene expression in macrophages, astrocytes or microglia [57]. In contrast, anti-inflammatory cytokines, such as interleukin-4 (IL-4), IL-10, IL-13 and TGFβ, have been shown to prevent iNOS induction and/or counteract NO cytotoxicity [11]. Likewise, glucocorticoids and the steroid-like hormone, retinoic acid, have been shown in vitro to antagonize iNOS synthesis in different cell types, including murine macrophages 10, 18, 35, 49, 53.
Like glucocorticoids and retinoic acid, the active hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-D3), exerts profound immunosuppressive effects. However, little is known about its ability to regulate iNOS, especially in CNS cells. 1,25-D3 binds to an intracellular receptor, the vitamin D receptor (VDR), which belongs to the steroid-thyroid hormone receptor superfamily and acts as a ligand-dependent transcription factor [17]. In addition to its role in the control of calcemia and bone metabolism, 1,25-D3 displays anti-inflammatory and immunomodulatory properties [30]. This hormone inhibits B and T lymphocyte proliferation 22, 30, down-regulates the production of IL-2 and IFNγ by T lymphocytes [4], and prevents immunoglobulin secretion by B lymphocytes [24]. In vivo, the immunosuppressive properties of 1,25-D3 have been demonstrated in various animal models of autoimmunity, such as experimental autoimmune thyroiditis or diabetes 15, 31. Moreover, 1,25-D3 administration at the time of immunization with myelin basic protein (MBP), also prevents the development of EAE in SJL mice 7, 23.
There are presently several lines of evidence which indicate that the CNS constitutes a target of 1,25-D3. Binding sites of the radiolabelled hormone have been demonstrated in certain neurons and in non-neuronal structures, such as perivascular organs 5, 55. Moreover, the VDR mRNA was detected in the human hippocampus [56]and in newborn rat astrocyte cultures [42]in which 1,25-D3 was shown to enhance the expression of nerve growth factor (NGF) and neurotrophin-3 (NT3) transcripts 41, 42. In addition, 1,25-D3 can be synthesized from its precursor, 25-hydroxyvitamin D3 (25-D3), by activated microglial cells [43], a finding which suggests the existence of local regulatory loops controlling the action of 1,25-D3 on brain homeostasis. A recent study from our laboratory has shown that 1,25-D3 can exert immunomodulatory effects inside the CNS during an ongoing EAE immune process in the Lewis rat [39]. Treatment of animals after the appearance of clinical signs of EAE resulted in a significant clinical improvement, associated with a down-regulation of CD4 antigen expression by infiltrating macrophages and activated microglial cells in the brainstem and cerebellum.
Since EAE pathology is associated with an excessive production of NO, the aim of the present study was to investigate the regional and cellular expression of the iNOS gene and iNOS immunoreactivity during the time course of chronic relapsing EAE in rats, and to explore the modulatory effects of 1,25-D3 on the iNOS synthesis during the progression of EAE.
Section snippets
Animals and EAE induction
8–9-week-old Lewis female rats were obtained from Charles River France (Cléon) and bred in our animal facilities. Chronic relapsing EAE was induced in rats as previously described 14, 39. Briefly, guinea-pig spinal cords (GPSC) were obtained from female Dunkin-Hartley animals. 1 g GPSC was homogenized in 1 ml saline, then emulsified with 2 ml of complete Freund's adjuvant (CFA) supplemented with 40 mg of Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI, USA). Rats were injected
Cells of monocyte/macrophage lineage
In control rats, no iNOS-immunoreactive cells were detected in CNS sections (Fig. 1C). OX42-staining revealed resting microglia which appeared as cells with a small body and thin elongated processes, either with a bipolar morphology in the white matter or with a stellate morphology in the grey matter (Fig. 1A). A few OX42-positive monocytes, identified on the basis of their small size and rounded morphology, were also detected in the ventricular compartments, but not in the cerebral parenchyma
Discussion
The analysis of iNOS immunoreactivity and mRNA in the rat model of chronic relapsing EAE demonstrated a time- and cell-specific expression of this enzyme. Macrophages and activated microglia appear to express iNOS during all stages of EAE, while astrocytes only express iNOS during the 2nd inductive phase of EAE. The fact that iNOS remains expressed in inflammatory cells during the remission phase of EAE suggests that its role is more complex than expected from other studies, such as in the EAE
Acknowledgements
We would like to thank Dr. C. Montero-Menei for fruitful discussion and Dr. D. Djakiew for critical reading of the manuscript. We thank also Professor N. Passutti from the Laboratory of Experimental Surgery, Faculté de Médecine de Nantes; L. Sindji, A. Leboterff and P. Trelohan for technical assistance; and Dr. S. Carvajal-Gonzalez for computer analysis. This work was supported by INSERM and by a grant from the `Ligue Française contre la Sclérose en Plaques'.
References (59)
- Albina, J.E., Cui, S., Mateo, R.B. and Reichner, J.S., Nitric oxide-mediated apoptosis in murine peritoneal...
- Aquino, D.A., Chiu, F-C., Brosnan, C.F. and Norton, W.T., Glial fibrillary acidic protein increases in the spinal cord...
- Bagasra, O., Michaels, F.H., Zheng, Y.M., Bobroski, L.E., Spitsin, S.V., Fu, Z.F., Tawadros, R. and Koprowski, H.,...
- Bhalla, A.K., Amento, E.P. and Krane, S.M., Differential effects of 1,25-dihydroxyvitamin D3 on human lymphocytes and...
- Bidmon, H.J. and Stumpf, W.E., Choroid plexus, ependyma and arachnoidea express receptor for vitamin D: differences...
- Bö, L., Dawson, T.M., Wesselingh, S., Mork, S., Choi, S., Kong, P.A., Hanley, D. and Trapp, B.D., Induction of nitric...
- Branisteanu, D.D., Waer, M., Sobis, H., Marcelis, S., Vandeputte, M. and Bouillon, R., Prevention of murine...
- Cross, A.H., O'Mara, T. and Raine, C.S., Chronologic localization of myelin-reactive cells in the lesions of relapsing...
- Dawson, T.M. and Snyder, S.H., Gases as biological messengers: nitric oxide and carbon monoxide in the brain, J....
- Di Rosa, M., Radomski, M., Carnuccio, R. and Moncada, S., Glucocorticoids inhibit the induction of nitric oxide...
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