Metallothioneins I/II Expression in Rat Strains with Genetically Different Susceptibility to Experimental Autoimmune Encephalomyelitis

Abstract

Objectives: Compared to the Dark Agouti (DA), the Albino Oxford (AO) rat strain exhibits lower susceptibility to the induction of experimental autoimmune encephalomyelitis (EAE). Here, we investigated the potential contribution of the heavy metal-binding proteins metallothioneins (MTs) I/II to these effects. Methods: Rats were immunized with bovine brain homogenate emulsified in complete Freund's adjuvant or only with complete Freund's adjuvant. The expression patterns of MTs mRNA and proteins and tissue concentrations of Zn²⁺ and Cu²⁺ were estimated in the brain and in the liver on days 7 and 12 after immunization, by real-time PCR, immunohistochemistry and inductively coupled plasma spectrometry, respectively. Additionally, the hepatic transforming growth factor beta and nuclear factor kappa B immunoreactivities were tested. Results: Clinical signs of EAE were not induced in AO rats, but they upregulated the expression of MT I/II proteins in the brain (hippocampus and cerebellum) and in the liver, similarly as DA rats. The transcriptional activation of MT-I occurred, however, only in DA rats, which accumulated also more zinc in the brain and in the liver. In contrast, intact AO rats had greater hepatic MT-I mRNA immunoreactivity and more Cu²⁺ in the hippocampus. Besides, in immunized AO rats a high upregulation of transforming growth factor beta and nuclear factor kappa B immunoreactivities was found in several hepatic structures (vascular endothelium, Kupffer cells and hepatocytes). Conclusions: Our data show that AO and DA rats differ in constitutive and inductive MT-I gene expression in the brain and in the liver, as well as in the hepatic cytokine profile, suggesting that these mechanisms may contribute to the discrepancy in the susceptibility to EAE.

Hepatic Expression of Metallothionein I/II, Glycoprotein 96, IL-6, and TGF-βin Rat Strains with Different Susceptibilities to Experimental Autoimmune Encephalomyelitis

“…In our experiments the MT I/II expression correlated more with the peaks of clinical symptoms, in contrast to gp96, which was found in remission phases, suggesting that ER chaperones might be induced to ameliorate the accumulation of misfolded proteins in the CNS and protect neuronal cells against autoimmune attack. The importance of time-course evaluation of the data is emphasized also in the present study, which imply that EAE-resistant rats may more efficiently restrain the autoimmune response, owing to greater constitutive expression of hepatic MT I/II and IL-6 (Figures 1 and 3) and owing to an early upregulation of TGF- β in the liver [12]. …”

“…Supporting the current knowledge about the prominent regulatory functions of metallothioneins in pathogenesis of EAE and other types of brain injuries [35, 45, 47], we previously reported that the expression metallothioneins I/II might be induced in both EAE-prone and EAE-resistant strain of rats, even in the presymptomatic phase of CR-EAE (on the seventh postimmunization day) in cells that form blood-brain and blood-cerebrospinal fluid barriers, in the cerebellar parenchyma and hippocampal dentate gyri, as well as in the liver of rats [12]. However, on the 12th postimmunization day, the levels of hepatic IL-6, TGF-beta, MTs, and gp96 were significantly greater in DA than in AO rats, implying that AO rats might better control the autoimmune reaction [12].…”

“…However, on the 12th postimmunization day, the levels of hepatic IL-6, TGF-beta, MTs, and gp96 were significantly greater in DA than in AO rats, implying that AO rats might better control the autoimmune reaction [12]. Moreover, since in AO rats we noticed an early upregulation of TGF- β on several hepatic structures (vascular endothelium, Kupffer cells, and hepatocytes), we speculated that specific hepatic microenvironment might contribute to the faster recovery of these rats from EAE [12].…”

“…However, on the 12th postimmunization day, the levels of hepatic IL-6, TGF-beta, MTs, and gp96 were significantly greater in DA than in AO rats, implying that AO rats might better control the autoimmune reaction [12]. Moreover, since in AO rats we noticed an early upregulation of TGF- β on several hepatic structures (vascular endothelium, Kupffer cells, and hepatocytes), we speculated that specific hepatic microenvironment might contribute to the faster recovery of these rats from EAE [12]. The new data from current study, also revealed that AO rats have greater basal levels of TGF- β , IL-6, and MT I/II (Figures 4, 3, and 1), implying that the susceptibility in AO and DA rats might depend not only on MHC haplotype, but also on some epigenetic traits that might be responsible for immunomodulation [48].…”

“…Contributing to this field we have recently shown that these rat strains differ also in the activation pattern of metallothioneins I/II [12], which within the mammalian CNS perform essential cytoprotective functions, owing to their metal binding, antioxidative, antiapoptotic, and growth-regulatory activities [13–16]. In these data [12], we have shown that constitutive and induced MT I+II gene expression in EAE-resistant and EAE-prone rats is different both in the organs that were damaged by the autoimmune attack (hippocampus and cerebellum) as well as in the liver, pointing to the high involvement of the central and peripheral MTs-related mechanisms in the induction of EAE. Besides, since in AO rats, early after immunization with encephalitogen (on the seventh postimmunization day), we found a marked upregulation of TGF-beta immunoreactivity on several hepatic structures, we hypothesized that immunosuppressive environment in the liver probably contributed also to the induction of resistance toward EAE [12].…”