Hepatic metallothioneins I+II, gp96, IL-6 and TGF- as potential regulators of strain-dependent susceptibility to experimental autoimmune encephalomyelitis in rats

Background: Albino Oxford (AO) rats in comparison to the Dark Agouti (DA) strain exhibit lower susceptibility to the induction of EAE. Mechanisms include the differences in peripheral response to immunization and those linked with the CNS milieu, which contribute to initiation and limitation of the injury. In a search for peripheral factors related to these differences, herein we estimated the role of the liver in the pathogenesis of EAE, analyzing the expression pattern of cysteine rich proteins-metallothioneins (MT) I+II that maintain the metal ion homeostasis and of endoplasmic reticulum (ER)-resident heat shock protein (HSP)-gp96, which have marked anti-inflammatory, immunoregulatory and cytoprotective properties. Besides, since the differentiation of naive T cells into the regulatory T or into the pathogenic Th17 cells may be regulated particularly by the transforming growth factor (TGF)-s and its combination with IL-6, in this study we compared also the hepatic levels of these two cytokines in EAE prone and EAE-resistant rats. Methods: AO and DA rats were immunized with bovine brain homogenate (BBH) emulsified in complete Freund adjuvant (CFA) or only with CFA. The expression pattern of hepatic MT-I mRNA and MT I+II proteins, gp96 and cytokines were estimated on day 12 after immunization, as well as in intact AO and DA rats, by immunohistochemistry, real time PCR and Western blot analysis. Results: AO rats, injected with BBH+CFA have not shown any clinical sign of EAE, in contrast to DA rats, which developed a typical chronic-relapsing form of disease, characterized by two peaks of clinical symptoms (on the 12th and the 22nd postimmunization day). Herein, we show that these rats significantly differ also in the constitutive and inducible expression of MTs, gp96 and cytokines in the liver. Thus, during the first attack of EAE the significant upregulation of MTs and gp96 was found only in EAE-prone DA rats, where the levels of MT I+II and Gp96 proteins were three times greater that those in the intact liver (p<0, 001). Moreover, in DA rats simultaneously increased the hepatic expression of IL-6, and its expressions positively correlated with the upregulation of MTs and gp96 in the liver. In contrast, AO rats reacted on immunization with BBH+CFA by marked upregulation of TGF- immunoreactivities in several hepatic structures (vascular endothelium, Kupffer cells and hepatocytes) in the absence of IL-6. Besides, in the liver of intact AO rats significantly greater levels of MT-I+II and IL-6 were found than in EAE-prone DA rats (p<0, 001), suggesting that the greater constitutive expression of these proteins has contributed to the resistance of this strain to EAE. Conclusions: The data show that during the acute phase reaction the hepatic MTs, ER-resident HSPs and cytokines participate to the creation of the specific hepatic microenvironment that governs the local balance between tolerance and immunity in EAE. Moreover, the data imply that critical influences in these events may have hepatic TGF-, which may contribute to the generation of Treg cells that by cell contact-dependent and independent mechanisms may suppress the function of MBP-specific effector cells, participating in the induction of the antigen-specific peripheral tolerance.