Differential expression of suppressors of cytokine signaling-1 and -3 and related cytokines in central nervous system during remitting versus non-remitting forms of experimental autoimmune encephalomyelitis

SJL mice exhibit a relapsing-remitting course of experimental autoimmune encephalomyelitis (EAE), whereas C57BL/6 (B6) mice display a more chronic course without complete remissions. Suppressor of cytokine signaling (SOCS)-1 and SOCS-3 are members of a family of inducible intracellular proteins that negatively regulate cytokine signaling in cells of hematopoietic origin and may influence the Th1 to Th2 balance. SOCS-1 and SOCS-3 are induced by cytokines that are known to be up-regulated during EAE, including IFN-gamma (IFN-g) and IL-6, respectively. To test the hypothesis that the level of induction of SOCS-1 and SOCS-3 correlates with the course of EAE, mRNA levels were compared in spinal cords of SJL and B6 mice during discrete stages of disease. SOCS-1 and SOCS-3 were elevated throughout active disease in both strains. At peak EAE, SOCS-1 was higher and SOCS-3 was lower in B6 cords compared with SJL cords. This correlated with greater expression of the Th1 cytokine, IFN-g, and less of the Th2 cytokine, IL-10, in B6 cords relative to SJL cords during onset and peak disease. SOCS-3 inducers in the IL-6 family were expressed differentially between the strains. IL-6 and leukemia inhibitory factor were higher at onset in B6 cords whereas ciliary neurotrophic factor was increased in SJL cords during peak disease. Expression of fibroblast growth factor-2, which may be involved in remyelination, was higher in SJL cords at peak. Comparison of these models suggests that cytokine autoregulatory mechanisms involving SOCS may play a role in determining the course of EAE.     

SJL and NOD macrophages are uniquely characterized by genetically programmed, elevated expression of the IL-12(p40) gene, suggesting a conserved pathway for the induction of organ-specific autoimmunity

Genetic susceptibility of the SJL mouse to experimental autoimmune encephalomyelitis (EAE) appears, in part, to be a result of genes that promote abnormal development of the pathogenic Type 1 (Th1) phenotype of neuroantigen-specific T-cells. Because antigen-presenting/accessory cells (APCs) produce cytokines that can modulate the development of Th1 and Th2 phenotypes, we addressed whether APCs from SJL mice were genetically programmed for elevated expression of the Th1-promoting cytokine, IL-12. Activated peritoneal macrophages (Mphi; i.e., APC) from na?ve SJL mice produced levels of TNF-alpha, IL-1, IL-6, IL-10, and TGF-beta within the range of six normal strains. In contrast, SJL IL-12p40 (in addition to IL-12p70) production was consistently five- to 20-fold greater than that of any normal strain tested, which arose from elevated expression of the IL-12p40 but not the IL-12p35 gene, because p40 mRNA levels were eight- to 15-fold greater than those of normal strains. This aberrancy in IL-12p40 expression appears identical to that observed in the NOD mouse, another strain prone to organ-specific autoimmunity. A genetically programmed bias toward elevated expression of IL-12 in Mphi from the SJL and NOD strains of autoimmunity provides a conserved mechanism for the dominant Th1 development of naive, autoantigen-specific T-cells in these strains. This study is the first demonstration of a genetically programmed aberrant phenotype that is intrinsically expressed within a cell type in the SJL mouse and provides insight into its predisposition for EAE.     

Pro-inflammatory functions of astrocytes correlate with viral clearance and strain-dependent protection from TMEV-induced demyelinating disease

Intracerebral infection of susceptible strains of mice, e.g. SJL/J, with Theiler's murine encephalomyelitis virus (TMEV) leads to a persistent CNS infection accompanied by development of a chronic-progressive inflammatory CNS autoimmune demyelinating disease which is clinically and pathologically similar to human multiple sclerosis. In contrast, resistant strains of mice, e.g. C57BL/6 (B6), effectively clear TMEV from the CNS and do not develop demyelinating disease. Although CD8(+) T cells are crucial for viral clearance in B6 mice, SJL mice also mount potent CD8(+) T cell responses against virus, thus the reason for the viral persistence in the CNS in these mice is unclear. Here, we examined innate anti-viral responses of CNS-resident astrocytes as a potential determinant of viral persistence and disease susceptibility. We demonstrate that B6 astrocytes produce significantly higher levels of cytokines, chemokines and adhesion molecules in response to TMEV infection, or stimulation with IFN-gamma and TNF-alpha or poly I:C than SJL mice. In addition, TMEV more effectively induces MHC I molecules on B6 astrocytes than SJL, corresponding with an increased ability to activate TMEV-specific CD8(+) T cells directly ex vivo. These results suggest that enhanced anti-viral responses of B6 astrocytes contribute to the ability of these mice to clear TMEV from the CNS and therefore to their resistance to the development of autoimmune demyelinating disease.     

CCR2 regulates development of Theiler's murine encephalomyelitis virus-induced demyelinating disease

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a murine model for multiple sclerosis, involves recruitment of T cells and macrophages to the CNS after infection. We hypothesized that CCR2, the only known receptor for CCL2, would be required for TMEV-induced demyelinating disease development because of its role in macrophage recruitment. TMEV-infected SJL CCR2 knockout (KO) mice showed decreased long-term clinical disease severity and less demyelination compared with controls. Flow cytometric data indicated that macrophages (CD45(high) CD11b(+) ) in the CNS of TMEV-infected CCR2 KO mice were decreased compared with control mice throughout disease. CD4(+) and CD8(+) T cell percentages in the CNS of TMEV-infected control and CCR2 KO mice were similar over the course of disease. There were no apparent differences between CCR2 KO and control peripheral immune responses. The frequency of interferon-gamma-producing T cells in response to proteolipid protein 139-151 in the CNS was also similar during the autoimmunity stage of TMEV-induced demyelinating disease. These data suggest that CCR2 is important for development of clinical disease by regulating macrophage accumulation after TMEV infection.     

TGF-beta-treated microglia induce oligodendrocyte precursor cell chemotaxis through the HGF-c-Met pathway

In acute experimental autoimmune encephalomyelitis (EAE), demyelination is induced by myelin-specific CD4(+) T lymphocytes and myelin-specific antibodies. Recovery from the disease is initiated by cytokines which suppress T cell expansion and the production of myelin-toxic molecules by macrophages. Th2/3 cell-derived signals may also be involved in central nervous system (CNS) repair. Remyelination is thought to be initiated by the recruitment and differentiation of oligodendrocyte precursor cells (OPC) in demyelinated CNS lesions. Here, we report that unlike Th1 cytokines (TNF-alpha, IFN-gamma), the Th2/3 cytokine TGF-beta induces primary microglia from C57BL/6 mice to secrete a chemotactic factor for primary OPC. We identified this factor to be the hepatocyte growth factor (HGF). Our studies show that TGF-beta-1-2-3 as well as IFN-beta induce HGF secretion by microglia and that antibodies to the HGF receptor c-Met abrogate OPC chemotaxis induced by TGF-beta2-treated microglia. In addition we show spinal cord lesions in EAE induced in SJL/J mice to contain both OPC and HGF producing macrophages in the recovery phase, but not in the acute stage of disease. Taken these findings, TGF-beta may play a pivotal role in remyelination by inducing microglia to release HGF which is both a chemotactic and differentiation factor for OPC.     

The central nervous system environment controls effector CD4+ T cell cytokine profile in experimental allergic encephalomyelitis

In experimental allergic encephalomyelitis (EAE), CD4⁺ T cells infiltrate the central nervous system (CNS). We derived CD4⁺ T cell lines from SJL/J mice that were specific for encephalitogenic myelin basic protein (MBP) peptides and produced both Th1 and Th2 cytokines. These lines transferred EAE to naive mice. Peptide-specific cells re-isolated from the CNS only produced Th1 cytokines, whereas T cells in the lymph nodes produced both Th1 and Th2 cytokines. Mononuclear cells isolated from the CNS, the majority of which were microglia, presented antigen to and stimulated MBP-specific T cell lines in vitro. Although CNS antigen-presenting cells (APC) supported increased production of interferon (IFN)-γ mRNA by these T cells, there was no increase in the interleukin (IL)-4 signal, whereas splenic APC induced increases in both IFN-γ and IL-4. mRNA for IL-12 (p40 subunit) was up-regulated in both infiltrating macrophages and resident microglia from mice with EAE. We have thus shown that a Th1 cytokine bias within the CNS can be induced by CNS APC, and that IL-12 is up-regulated in microglial cells within the CNS of mice with EAE. Microglia may therefore control Th1 cytokine responses within the CNS.     

Reduced expression of IL-12 p35 by SJL/J macrophages responding to Theiler's virus infection is associated with constitutive activation of IRF-3

Macrophages responding to viral infections may contribute to autoimmune demyelinating diseases (ADD). Macrophages from ADD-susceptible SJL/J mice responding to Theiler's Virus (TMEV) infection, the TLR7 agonist loxoribine, or the TLR4 agonist-LPS expressed less IL-12 p35 but more IL-12/23 p40 and IFN-beta than macrophages from ADD-resistant B10.S mice. While expression of IRF-1 and -7 was similar between B10.S and SJL/J TMEV-infected macrophages, SJL/J but not B10.S macrophages exhibited constitutively active IRF-3. In contrast to overexpressed IRF-1, IRF-5, and IRF-7, which stimulated p35 promoter reporter activity, overexpressed IRF-3 repressed p35 promoter activity in response to TMEV infection, loxoribine, IFN-gamma/LPS, but not IFN-gamma alone. IRF-3 lessened but did not eliminate IRF-1-stimulated p35 promoter activity. Repression by IRF-3 required bp -172 to -122 of the p35 promoter. The data suggest that pre-activated IRF-3 is a major factor in the differences in IL-12 production between B10.S and SJL/J macrophages responding to TMEV.     

Remission of collagen-induced arthritis is associated with high levels of transforming growth factor-beta expression in the joint

Immunization of genetically susceptible strains of mice with heterologous type II collagen leads to the induction of a self-limiting polyarthritis that begins to subside around 10 days after onset of clinical disease. The aims of this study were to compare pro- and anti-inflammatory cytokine expression in the joints during the course of arthritis in order to identify cytokines involved in spontaneous remission of arthritis. DBA/1 mice were immunized with type II collagen and an immunohistochemical analysis of expression of proinflammatory cytokines [tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6] and anti-inflammatory cytokines [IL-10, IL-1ra, transforming growth factor (TGF)-beta1, TGF-beta2 and TGF-beta3] in joints was carried out over the course of the disease. Both pro- and anti-inflammatory cytokines were found to be expressed in early arthritis. However, around 10 days after onset of arthritis, the level of expression of proinflammatory cytokines declined while the level of expression of anti-inflammatory cytokines, particularly TGF-beta1 and TGF-beta2, increased. Surprisingly, TNF-alpha continued to be expressed at low levels during the period of disease remission (30 days after onset). Blockade of TNF-alpha during the period of disease remission had no effect on TGF-beta expression. This study confirms that the level of inflammation in arthritis correlates strongly with the balance of pro- and anti-inflammatory cytokine expression in the joints. Of the anti-inflammatory cytokines studied, TGF-beta1 and TGF-beta2 predominate during the time of disease remission, suggesting that these cytokines are involved in regulating disease activity.     

Cytokines and Murine Autoimmune Encephalomyelitis: Inhibition or Enhancement of Disease with Antibodies to Select Cytokines, or by Delivery of Exogenous Cytokines Using a Recombinant Vaccinia Virus System

To examine the complex role of cytokines in the pathogenesis of actively induced murine EAE we measured the levels of a number of cytokines (IL-6, IFN7 and TNF) in the spinal cord and CSF of mice with active experimental autoimmune encephalomyelitis (EAE) and found them all to be elevated. We next treated mice with antibodies to these three cytokines, which were over expressed in the CNS, to determine if they would alter disease and found the following: anti-IL-6 had no significant effect on disease, anti-IFNγ exacerbated disease, and anti-TNF either enhanced, had no effect or inhibited EAE depending on the antibody used. We then treated mice with exogenous cytokines, delivered using a recombinant vaccinia virus system, and found that the IL-6 and TNF virus constructs inhibited EAE whereas the IFN1β construct had no effect on disease. Other cytokine recombinant viruses were also tested and it was found that the IL-1β, IL-2 and IL-10 viruses inhibited EAE while an IL-4 virus either had no effect or enhanced disease. We do not know the mechanism of action of the various cytokines in this system, but irrespective of the mechanism(s), this work clearly demonstrates that delivery of select cytokines using recombinant virus-cytokine constructs can provide a powerful means of downregulating experimental organ-specific autoimmune disease.     

Virus expanded regulatory T cells control disease severity in the Theiler's virus mouse model of MS

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) serves as virus-induced model of chronic progressive multiple sclerosis. Infection of susceptible SJL/J mice leads to life-long CNS virus persistence and a progressive autoimmune demyelinating disease mediated by myelin-specific T cells activated via epitope spreading. In contrast, virus is rapidly cleared by a robust CTL response in TMEV-IDD-resistant C57BL/6 mice. We investigated whether differential induction of regulatory T cells (Tregs) controls susceptibility to TMEV-IDD. Infection of disease-susceptible SJL/J, but not B6 mice, leads to rapid activation and expansion of Tregs resulting in an unfavorable CNS ratio of Treg:Teffector cells. In addition, anti-CD25-induced inactivation of Tregs in susceptible SJL/J, but not resistant B6, mice results in significantly decreased clinical disease concomitant with enhanced anti-viral CD4(+), CD8(+) and antibody responses resulting in decreased CNS viral titers. This is the first demonstration that virus-induced Treg activation regulates susceptibility to autoimmune disease differentially in susceptible and resistant strains of mice and provides a new mechanistic explanation for the etiology of infection-induced autoimmunity.     

Quantitative, not qualitative, differences in CD8(+) T cell responses to Theiler's murine encephalomyelitis virus between resistant C57BL/6 and susceptible SJL/J mice

Theiler's murine encephalomyelitis virus (TMEV) infection of the CNS induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis. However, it is not yet clear what immunological parameters determine the susceptibility of SJL/J mice compared to resistant mice. We have here compared the TMEV-specific CD8(+) T cell responses in highly susceptible SJL/J mice with those of highly resistant C57BL/6 mice. Our results clearly indicate that the levels of initial responses of infiltrating CD8(+) T cells to viral capsid proteins are higher in resistant C57BL/6 mice compared to susceptible SJL/J mice. However, the level of virus-specific CD8(+) T cells was much more rapidly reduced in resistant C57BL/6, resulting in a higher CD8(+) T cell level in SJL/J mice later in viral infection. The activation states, cytokine production, as well as the cytolytic function of the CD8(+) T cells were similar to each other in these mice. These results suggest that an initial induction of a vigorous CD8(+) T cell response to TMEV is critically important for the resistance to virally induced demyelinating disease.     

Modulation of susceptibility and resistance to an autoimmune model of multiple sclerosis in prototypically susceptible and resistant strains by neutralization of interleukin-12 and interleukin-4, respectively

Experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, is mediated by Th1 cells. The major Th1 inducer, IL-12, enhances EAE, while its blockade suppresses it. IL-4 suppresses EAE. Here, we determined IFN-gamma and IL-4 production by myelin basic protein-stimulated lymphocytes from prototypically EAE-susceptible SJL/J and EAE-resistant BALB/c mice, 9 days after immunization with spinal cord homogenate. While lymphocytes from SJL/J mice produce IFN-gamma and no IL-4, lymphocytes from BALB/c mice produce IL-4 and no IFN-gamma. Since early endogenous production of IL-12/IFN-gamma or IL-4 is linked to Th1 or Th2 responses, respectively, we determined whether neutralization of IL-12 or IL-4 at immunization modifies susceptibility or resistance to EAE. SJL/J mice given neutralizing anti-IL-12 mAb are protected from EAE. BALB/c mice given neutralizing anti-IL-4 mAb develop EAE, while those treated with control antibody remain resistant. These studies confirm the pivotal role of IL-12 in EAE development and show that endogenous IL-4 is important for determining the genetic resistance to EAE.