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The HMGCR gene encodes the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the key enzyme for cholesterol synthesis. Mice transgenic for the prokaryotic chloramphenicol acetyl transferase (CAT) reporter gene fused with a 5' Bam H1 fragment including the promoter sequence for murine HMGCR gene have been obtained. Homozygote transgenic mice were derived from a particular line selected for similar regulation of endogenous HMGCR and the transgene expression by nutritional conditions in different tissue. In addition, high expression of the transgene was evidenced in the brain. Cellular expression of the CAT gene in the central nervous system (CNS) was investigated by immunohistochemistry (IHC). This study was performed on frozen sections of the developing and adult brain, using a rabbit anti-CAT antiserum especially raised for that purpose. CAT expression was observed in some rare individuals in different neural cell types including Purkinje cells and astrocytes. But the most outstanding observation was the high level of CAT expression correlated with differentiated pattern of oligodendrocyte (Ol) distribution observed in white-matter tracts. Double and triple labeling for CAT and stage-specific antigens were performed on transgenic Ol-enriched preparations and cultures. This study showed a normal sequence of differentiation in the transgenic oligodendroglial cell lineage and demonstrated a strict correlation between late differentiation and activation of the CAT gene in these cells: CAT expression started in transgenic Ols between galactocerebroside (GC)-positive and myelin basic protein (MBP)-positive stages and was detected in MBP-positive cells during the myelination period. After myelination, the number of CAT-positive Ols decreased in the adult brain. These observations demonstrate a developmental regulation of the CAT transgene in Ols during myelination in CNS and reinforce the hypothesis of endogenous synthesis as major source of cholesterol during myelination.

In the companion paper (J. Rossert et al., Cell. Immunol., 137, 1991), we showed by using limiting dilution analysis that Lewis (LEW) rats injected with HgCl2 and immunized with myelin ( LEWHg MYE ) exhibit anti-basic protein CD4+ T helper cells (Th), at least 10-fold more frequent CD8+ T suppressor cells (Ts), and T contrasuppressor cells (Tcs). These Tcs cells were shown to be CD4+ T cells adhering to Vicia villosa (VV) lectin and allowed Th cells to proliferate despite the presence of Ts cells. The CD8+ Ts cells might be responsible for the protection from experimental allergic encephalomyelitis (EAE) observed in about 70% of LEW rats injected with HgCl2. The concomitant presence of CD4+ Tcs cells might explain that 30% of the rats escaped this protection. The aim of this work is to demonstrate in vivo the roles of CD8+ Ts cells and Tcs cells in mercury-induced protection from EAE. It will be shown that LEWHg/MYE rats depleted of CD8+ cells as well as LEWHg/MYE rats transferred with VV lectin-adherent Tcs cells develop EAE. These data demonstrate that CD8+ Ts cells are responsible for HgCl2-induced protection and that Tcs cells are involved in the control of Ts cells in vivo.

Mercuric chloride (HgCl 2 ) induces in Lewis (LEW) rats a non-antigen-specific immuno-suppression and is able to down-modulate experimental allergic encephalomyelitis in about 70% of the rats. The aim of the present study was to determine the frequencies of lymph node cells involved in the proliferative response to myelin basic protein in rats injected with HgCl 2 and immunized with myelin by using limiting dilution analysis (LDA). Highly frequent CD8+ T suppressor cells and at least 10-fold less frequent protein basic-specific T helper cells were detected in these rats. A third cell type allowing the proliferative response of Th cells in spite of Ts cells was also demonstrated. These cells, which could act as contrasuppressor cells, were CD4+ and adhered to Vicia villosa lectin; their frequency was in the same range as that of T helper cells. These data illustrate the potential role of different levels of T cell immunoregulatory activity in autoimmunity and the major interest of LDA in their analysis.

Only one out of 57 A-/A- rabbits immunized with rat or guinea-pig myelin developed clinical signs suggestive of EAE. On the contrary, clinical signs of acute or chronic EAE were found in two thirds of the 102 A+/A+ and A+/A- rabbits immunized in the same way. About one third of the diseased animals had reversible acute EAE, another third died paralysed and the last third developed chronic progressive or relapsing EAE. Incidence and severity of EAE symptoms were positively correlated with age and no significant difference was observed between males and females. Cellular and humoral anti-myelin responses were stronger in A+ than in A- rabbits. Anti-A antibodies, on the contrary, were only detected in A- rabbits. The A+ rabbits did not make Anti-A at any time. Anti-A antibodies increased early, in A- rabbits, after immunization with myelin (11-30 days) and were later replaced by a low, but specific, anti-myelin response (60-90 days). The gene responsible for the susceptibility to EAE is autosomal and dominant over resistance. This gene must be closely linked to the A locus or might be the A gene itself. The low susceptibility of A- rabbits to the disease could be, in this last case, a consequence of the competition between the early anti-A and the normal anti-myelin immune responses, both induced by the injection of myelin.

In this study, we tested the efficiency of an intravitreal injection of tamoxifen, a non-steroidal estrogen receptor modulator, in retinal soluble antigen (S-Ag)-induced experimental autoimmune uveoretinitis (EAU). To increase the bioavailability of tamoxifen, we incorporated tamoxifen into polyethylene glycol (PEG)-coated nanoparticles (NP-PEG-TAM). The localization of the nanoparticles within the eye was investigated using fluorescent-labeled PEG-coated nanoparticles after injection into the vitreous cavity of rats with EAU. Some nanoparticles were distributed extracellularly throughout the ocular tissues, others were concentrated in resident ocular cells and in infiltrating macrophages. Whereas the injection of free tamoxifen did not alter the course of EAU, injection of NP-PEG-TAM performed 1-2 days before the expected onset of the disease in controls resulted in significant inhibition of EAU. NP-PEG-TAM injection significantly reduced EAU compared to injection of NP-PEG-TAM with 17beta-estradiol (E2), suggesting that tamoxifen is acting as a partial antagonist to E2. Diminished infiltration by MHC class II(+) inflammatory cells and low expression of TNF-alpha, IL-1beta, and RANTES mRNA were noted in eyes of NP-PEG-TAM-treated rats. Intravitreal injection of NP-PEG-TAM decreased S-Ag lymphocyte proliferation, IFN-gamma production by inguinal lymph node cells, and specific delayed-type hypersensitivity indicative of a reduced Th1-type response. It increased the anti-S-Ag IgG1 isotype indicating an antibody class switch to Th2 response. These data suggest that NP-PEG-TAM inhibition of EAU could result from a form of immune deviation. Tamoxifen-loaded nanoparticles may represent a new option for the treatment of experimental uveitis.

Relapsing experimental allergic encephalomyelitis (EAE) was induced in DA rats and the ocular pathologic events were examined at the various phases of the illness. About 80% of EAE rats presented anterior uveitis (AU), even after complete EAE recovery. We studied the phenotype and localization of immunocompetent cells, the major histocompatibility complex (MHC) class I and II antigen expression, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1) appearance. In control animals, there were many glial fibrillary acidic protein (GFAP)+ cells and OX42+ cells in the ciliary body, retina, optic nerve and chiasma. Except in retina, we observed constitutive MHC class I and II expression. During the EAE acute phase, there was up-regulation of MHC class II and GFAP antigens in iris, ciliary body, limbus, and optic pathways. MHC class I and ED2 antigens were expressed in meninges and in the prechiasmatic cisterna, by cells which could have a role in immune surveillance. MCP-1 mRNA was highly expressed in optic pathways during the acute phase and the protein was expressed by astrocytes, macrophages, and lymphocytes. During the relapsing phase, MCP-1 was weakly expressed to disappear almost completely during the final recovery phase. The expression of MHC class II on astrocytes was increased during the relapsing and final recovery phase in which the inflammatory lesions persisted. These findings suggest that ocular areas and optic pathways, mainly optic chiasma, are important targets in the relapsing EAE. J. Neurosci. Res. 63:525–535, 2001. © 2001 Wiley-Liss, Inc.