A myelin basic protein fragment induces sexually dimorphic transcriptome signatures of neuropathic pain in mice.

In the peripheral nerve, mechanosensitive axons are insulated by myelin, a multilamellar membrane formed by Schwann cells. Here, we offer first evidence that a myelin degradation product induces mechanical hypersensitivity and global transcriptomics changes in a sex-specific manner. Focusing on downstream signaling events of the functionally active 84-104 myelin basic protein (MBP(84-104)) fragment released after nerve injury, we demonstrate that exposing the sciatic nerve to MBP(84-104) via endoneurial injection produces robust mechanical hypersensitivity in female, but not in male, mice. RNA-seq and systems biology analysis revealed a striking sexual dimorphism in molecular signatures of the dorsal root ganglia (DRG) and spinal cord response, not observed at the nerve injection site. Mechanistically, intra-sciatic MBP(84-104) induced phospholipase C (PLC)-driven (females) and phosphoinositide 3-kinase-driven (males) phospholipid metabolism (tier 1). PLC/inositol trisphosphate receptor (IP3R) and estrogen receptor co-regulation in spinal cord yielded Ca2+-dependent nociceptive signaling induction in females that was suppressed in males (tier 2). IP3R inactivation by intrathecal xestospongin C attenuated the female-specific hypersensitivity induced by MBP(84-104). According to sustained sensitization in tiers 1 and 2, T cell-related signaling spreads to the DRG and spinal cord in females, but remains localized to the sciatic nerve in males (tier 3). These results are consistent with our previous finding that MBP(84-104)-induced pain is T cell-dependent. In summary, an autoantigenic peptide endogenously released in nerve injury triggers multisite, sex-specific transcriptome changes, leading to neuropathic pain only in female mice. MBP(84-104) acts through sustained co-activation of metabolic, estrogen receptor-mediated nociceptive, and autoimmune signaling programs.

Upregulation of ß-1,4-galactosyltransferase I in rat spinal cord with experimental autoimmune encephalomyelitis.

Inflammatory infiltration has been recently emphasized in the demyelinating diseases of the central nervous system including multiple sclerosis. ß-1,4-Galactosyltransferase I (ß-1,4-GalT-I) is a major galactosyltransferase responsible for selectin-ligand biosynthesis, mediating rolling of the inflammatory lymphocytes. In the present study, Western blot showed that expression of ß-1,4-GalT-I was low in normal or complete Freund's adjuvant (CFA) control rats' spinal cords, and it began to increase since early stage and peaked at E4 stage of experimental autoimmune encephalomyelitis (EAE) and restored approximately at normal level in the recovery stage. Immunohistochemisty revealed that upregulation of ß-1,4-GalT-I was predominantly distributed in the white matter of spinal cord , while there was also some increased staining of ß-1,4-GalT-I in the grey matter. Meanwhile, the expression of E-selectin, the substrate of ß-1,4-GalT-I, was significantly increased, with a peak at E4 stage of EAE, and gradually decreased thereafter. Lectin blot showed that the protein bands with molecular weights of 65-25 kDa reacted a remarkable increase at the peak stage of EAE when compared with the normal and CFA control. Ricinus Communis Agglutinin-I (RCA-I) histochemistry revealed that RCA-Ι-positive signals were most intense in white matter of lumbosacral spinal cord at the peak stage of EAE (E4). Immunohistochemistry showed that ß-1,4-GalT-I and CD62E, a marker for E-selectin stainings located in a considerable number of ED1 (+) macrophages in perivascular or in the white matter in EAE lesions, and a good co-localization of ED1 (+) cells with CD62E was observed. All these results suggest that ß-1,4-GalT-I might serve as an inflammatory mediator regulating adhesion and migration of inflammatory cells in EAE, possibly through influencing the modification of galactosylated carbohydrate chains to modulate selectin-ligand biosynthesis and interaction with E-selectin.

Myelin basic protein-reactive T cells persist in an inactive state in the bone marrow of Lewis rats that have recovered from autoimmune encephalomyelitis.

Lewis rats immunized with guinea pig myelin basic protein residues 68-86 develop acute experimental autoimmune encephalomyelitis and recover. The predominant T cell receptor expressed by the encephalitogenic T cells is TCRBV8S2. They persist in bone marrow many weeks after recovery. CD3 is down-regulated, but >90% express CD4. They fail to proliferate to GPMBP68-86 unless a nitric oxide synthase inhibitor is added to the cultures. Perhaps these are memory T cells that are maintained in a suppressed state in BM by a nitric oxide-dependent mechanism.

Protection against experimental autoimmune encephalomyelitis generated by a recombinant adenovirus vector expressing the V beta 8.2 TCR is disrupted by coadministration with vectors expressing either IL-4 or -10.

Adenovirus vectors are increasingly being used for genetic vaccination and may prove highly suitable for intervention in different pathological conditions due to their capacity to generate high level, transient gene expression. In this study, we report the use of a recombinant adenovirus vector to induce regulatory responses for the prevention of autoimmune diseases through transient expression of a TCR beta-chain. Immunization of B10.PL mice with a recombinant adenovirus expressing the TCR Vbeta8.2 chain (Ad5E1 mVbeta8.2), resulted in induction of regulatory type 1 CD4 T cells, directed against the framework region 3 determinant within the B5 peptide (aa 76-101) of the Vbeta8.2 chain. This determinant is readily processed and displayed in an I-A(u) context, on ambient APC. Transient genetic delivery of the TCR Vbeta8.2 chain protected mice from Ag-induced experimental autoimmune encephalomyelitis. However, when the Ad5E1 mVbeta8.2 vector was coadministered with either an IL-4- or IL-10-expressing vector, regulation was disrupted and disease was exacerbated. These results highlight the importance of the Th1-like cytokine requirement necessary for the generation and activity of effective regulatory T cells in this model of experimental autoimmune encephalomyelitis.

Propentofylline and iloprost suppress the production of TNF-alpha by macrophages but fail to ameliorate experimental autoimmune encephalomyelitis in Lewis rats.

Intracellular cAMP levels can be elevated by activation of cAMP-generating adenylate cyclase (AC) or inhibition of cAMP-cleavage by phosphodiesterases. Elevation of intracellular cAMP levels in immune cells inhibits production of some Th1-cytokines, particularly TNF-alpha, and results mainly in downregulation of the immune response. Experimental autoimmune encephalomyelitis (EAE) of Lewis rats is a disease mediated by type 1 T helper lymphocytes and macrophages and serves as a model of multiple sclerosis. In EAE we therefore tested the immunomodulatory potency of an AC-activating, stable prostacyclin analogue, iloprost, and of a potent and non-selective inhibitor of phosphodiesterases, propentofylline, which also has neuroprotective properties. Preventive treatment of Lewis rats with propentofylline (2 x 10 or 12.5 mg/ kg/d), iloprost (2 x 10 or 12.5 micrograms/kg/d), or both did not significantly ameliorate clinical or histological signs of EAE actively induced by immunization with myelin basic protein (MBP) in complete Freund's adjuvant. Furthermore, adoptive transfer EAE (AT-EAE), passively induced by injection of encephalitogenic MBP-specific Th1 lymphocytes, was not altered in its course by the combined application of iloprost (2 x 10 micrograms/kg/d) and propentofylline (2 x 20 mg/kg/d) starting on the day of cell transfer. In vitro assays demonstrated that iloprost strongly and propentofylline moderately inhibited the production of TNF-alpha by macrophages and that iloprost in vivo similarly suppressed TNF-alpha secretion, although this effect was limited to a few hours after a single injection. In contrast to macrophages, TNF-alpha production by antigen-activated encephalitogenic T helper line cells in vitro was completely resistant to modulation by these agents. In addition, the presence of iloprost, propentofylline, or both drugs during activation of the line cells in vitro did not impair their encephalitogenicity in vivo. The findings delineate immunomodulatory effects of both substances, particularly of iloprost, but fail to support a possible therapeutic role of these agents in autoimmune inflammation of the central nervous system.

OX-40 antibody enhances for autoantigen specific V beta 8.2+ T cells within the spinal cord of Lewis rats with autoimmune encephalomyelitis.

The V beta 8.2 T cell receptor (TCR) component is the predominant V beta gene product associated with antigen specific CD4+ T cell response to the major encephalitogenic epitope of myelin basic protein (MBP) in Lewis rats. Lewis rats were actively immunized with MBP in complete Freund's adjuvant and the V beta 8.2 positive and negative cells were analyzed for IFN-gamma mRNA production and OX-40 cell surface expression during the onset of EAE. The V beta 8.2+ T cells isolated from the spinal cord produced the majority of mRNA for IFN-gamma and also showed a marked enhancement for OX-40 expression compared to V beta 8.2+ T cells isolated from the lymph nodes. Only a fraction of IL-2 receptor positive T cells examined ex vivo from the inflammatory compartments co-expressed the OX-40 antigen. These results suggested that OX-40 cell surface expression could be used to identify and isolate the most recently activated T cells ex vivo. OX-40+ T cells isolated from the spinal cord were highly enriched for the V beta 8.2 T cell receptor component compared to OX-40- or unsorted spinal cord lymphocytes. OX-40+ T cells isolated from the spinal cord had an enhanced response to MBP, whereas OX-40+ cells isolated from the lymph nodes responded to both MBP and purified protein derivative. These data suggest that activated T cells can be isolated and characterized with the OX-40 antibody which only respond to the antigens present at the local site. The data also imply that isolation of OX-40+ T cells will be useful in identifying V beta biases and autoantigen specific cells within inflamed tissues even when the antigen specificity is unknown.

Suppression of experimental autoimmune encephalomyelitis in Lewis rats by antibodies against CD2.

The immunotherapeutic potential of three anti-rat CD2 monoclonal antibodies (mAb) (OX34, OX54, OX55) and the combination of OX54 with OX55 was tested in Lewis rat experimental autoimmune encephalomyelitis (EAE). In actively induced EAE, a single injection of OX34 2 days before immunization with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) completely prevented or greatly attenuated EAE in all animals. Injection of OX54 acted moderately suppressive while OX55 or OX54/55 did not affect disease severity. Abrogation of EAE by OX34 was not restricted to its application before immunization. Therapeutic administration of all three mAb and the Ab combination from onset of first clinical signs efficiently blocked progression of disease and prevented all animals from developing hind limb paresis. In adoptive transfer EAE induced with in vitro activated cells of an encephalitogenic T helper line, clinical and histological signs were completely prevented by injection of OX34 on the day of cell transfer and 4 days later, underlining the strong impact of anti-CD2 mAb on the effector phase of disease. Immunocytofluorometric analysis of peripheral blood lymphocytes after a single Ab injection demonstrated that all mAb induced a variable degree of transient reduction in T cell numbers and modulation of CD2 antigens. In contrast to the other mAb, OX34 persisted on lymphocytes for at least 11 days, which may explain its unique suppressive effect on EAE after a single injection before immunization. The assumption that prophylactic administration of OX34 also inhibits MBP-induced EAE, due to persistence into the effector phase, was substantiated by the finding that none of the mAb prevented generation of an antigen-specific cellular response in MBP/CFA-immunized animals. Since none of the Ab induced T cell unresponsiveness or inhibited T cell activation by antigen- or Ab-mediated stimulation of the T cell receptor, we suggest that their marked action on the effector phase of EAE may rely on inhibition of T cell infiltration into the central nervous system. The demonstrated efficacy of these anti-CD2 mAb in EAE suggests a potential therapeutic role that may be equal to that of anti-CD4 or anti-T cell receptor Ab.

B cell responses to acetylcholine receptor in rats orally tolerized against experimental autoimmune myasthenia gravis.

Oral administration of acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freund's adjuvant (CFA) results in the prevention of experimental autoimmune myasthenia gravis (EAMG), and decreased serum levels of anti-AChR antibodies. Using an ELISPOT assay, we have now determined numbers of cells in the popliteal, inguinal and mesenteric lymph nodes, spleen and thymus secreting anti-AChR IgG antibodies. Except for mesenteric lymph nodes, a marked diminution of such cells was detected in these lymphoid organs in rats orally tolerized with AChR compared to buffer-fed or vehicle-fed control rats with EAMG. Of note is that, after AChR feeding, the B cell response to AChR in thymus was diminished to the same low level as in CFA-injected, buffer-fed control rats. The relative affinity of serum anti-AChR IgG antibodies measured by KSCN-ELISA was lower in the orally tolerized rats compared to buffer-fed or vehicle-fed rats. The observations showed that oral administration of AChR, besides preventing clinical EAMG, also counteracts the development of AChR-specific B cells, especially those with high affinity antibody production, in most lymphoid organs.

Inhibition of tumor necrosis factor is protective against neurologic dysfunction after active immunization of Lewis rats with myelin basic protein.

Increasing evidence indicates that the cytokines, tumor necrosis factor (TNF), interleukin-1, and/or interferon-gamma, may play a crucial role in the pathogenesis of multiple sclerosis. Several reports demonstrated that inhibition of TNF is highly protective in experimental allergic encephalomyelitis (EAE) when sensitization is accomplished by the passive transfer of myelin basic protein (MBP) sensitized lymphocytes. However, successful protection has not been reported in EAE that is induced by active immunization with MBP. We examined the effects of a TNF inhibitor, dimeric polyethylene glycol linked form of the type I soluble receptor of TNF, PEG-(rsTNF-RI)2, on actively acquired EAE. Treatment with PEG-(rsTNF-RI)2 at 0.3-3 mg/kg every other day or every third day starting on Day 9 postimmunization with MBP during the effector phase of EAE significantly inhibited clinical signs in a dose-dependent manner. Histological examination of the central nervous system indicated that the administration of PEG-(rsTNF-RI)2 reduced, in part, the cellular infiltrate, particularly in the lumbar and sacral regions of the spinal cord. These studies suggest that TNF is a pivotal mediator of the inflammation resulting from the complete immune response induced by active immunization with MBP.

Monoclonal anti-gamma interferon antibodies enhance experimental allergic encephalomyelitis.

Interferon-gamma (IFN-gamma) is a cytokine with multiple activities on a variety of cells. Under various circumstances, IFN-gamma can exhibit either pro-inflammatory or inhibitory actions. Treatment of SJL/J mice with a monoclonal antibody (Mab) to IFN-gamma during the afferent limb of the immune response to myelin protein produced an enhancement of acute experimental allergic encephalomyelitis (EAE), with increased morbidity, mortality and earlier onset of disease. Systemic administration of IFN-gamma did not improve or worsen clinical outcome, but delayed disease onset. Passive transfer of immune lymph node cells co-activated with MBP and anti-IFN-gamma Mab resulted in more sever disease than that induced by MBP stimulated cells or MBP and IFN-gamma co-stimulated cells. However, in vitro proliferation of an MBP specific T cell line was not influenced by IFN-gamma nor anti-IFN-gamma treatment. Mab to IFN-gamma inhibited suppressor function, in a non-specific assay. These in vivo and in vitro results suggest that systemic IFN-gamma serves as a physiological regulator of a suppressor mechanism in EAE. The abrogation of this regulatory mechanism by anti-IFN-gamma administration contributes to a more severe form of experimental allergic encephalomyelitis.

Shared T-cell receptor gene usage in experimental allergic neuritis and encephalomyelitis.

Experimental allergic neuritis, an autoimmune disease of the peripheral nervous system, is a model for human Guillain-Barré syndrome. Experimental allergic neuritis is mediated by CD4+ T cells reactive with myelin P2 protein. We demonstrate that these T cells use the same members of T-cell receptor V gene families for both their alpha (V alpha 2) and beta (V beta 8) chains as T cells that cause experimental allergic encephalomyelitis, an autoimmune disease of the central nervous system. Furthermore, these T cells appear to be idiotypically related. Therefore, completely different T-cell lines with different antigen specificities, producing entirely different diseases, share common T-cell receptors.

Vaccination against experimental autoimmune encephalomyelitis using a subencephalitogenic dose of autoimmune effector cells (1). Characteristics of vaccination.

We previously reported that rats could be vaccinated against EAE by inoculation with 10(7) anti-basic protein (anti-BP)-activated T cells raised as long-term lines. The activated T lines were irradiated (1,500 rads) to prevent them from causing EAE. We now report that a single inoculation of 10(4) or fewer cells of an activated anti-BP T-cell line did not cause clinical EAE but rather induced marked resistance to EAE produced by adoptive transfer of the anti-BP T cells. Resistance was less effective against EAE induced by active immunization to BP. Vaccination was immunologically specific, long lasting, and could be effected by various routes of administration.

Induction of autoimmune reactions to myelin basic protein in measles virus encephalitis in Lewis rats.

Intracerebral inoculation of weanling Lewis rats with measles virus led to the development of subacute measles encephalomyelitis (SAME) 4-8 weeks after infection. The disease is characterized pathologically by an intense inflammatory infiltration within both the white and grey matter of the central nervous system (CNS) without apparent demyelination. Both during and after SAME splenic lymphocytes from these animals could be restimulated in vitro to proliferate in the presence of myelin base protein (MBP). MBP-specific class II MHC-restricted T cell lines were isolated from this cell population. They were shown to exhibit no cross-reactivity with measles virus and to induce experimental allergic encephalitis (EAE) in naive syngeneic recipients following adoptive transfer. The clinical and histopathological signs of this T cell-mediated disease were identical to that seen in classical T cell-mediated EAE. A humoral immune response to MBP was only detected in a limited number of those rats with SAME. These results indicate that autoimmune reactions to brain antigen can arise during measles virus infection which may contribute to the pathogenesis of measles virus-associated encephalomyelitis.

Inhibition of allergic encephalomyelitis by the iron chelating agent desferrioxamine: differential effect depending on type of sensitizing encephalitogen.

Induction of experimental allergic encephalomyelitis (EAE) in Lewis rats by injection of guinea pig (GP) spinal cord homogenate (SCH) plus adjuvant (SCH-CFA) can be inhibited by treatment with the iron chelating agent desferrioxamine (DFOM). Interestingly, induction of EAE with purified myelin basic protein (BP-CFA) is not inhibited with DFOM. This dichotomy does not appear to be due to any quantitative differences in the two inocula since minimal clinical EAE produced by threshold levels of BP is not inhibited with DFOM. Passive EAE is not inhibited irrespective of the type of encephalitogen used to sensitize the donors. This suggests that the inhibitory effect of DFOM is acting on the afferent limb of the immune response to SCH-CFA. Injection of BP-CFA and SCH-CFA into the same site, mixing BP with central nervous system (CNS) lipids, or incorporating BP into liposomes, all induce EAE which can be partially inhibited by treatment with DFOM. These results support the hypothesis that the close association of lipids with the encephalitogen (i.e. BP) in SCH required extensive lipid breakdown before adequate antigen presentation can occur, and it is at this level that DFOM exerts its inhibitory effect.

A suppressor T-lymphocyte cell line for autoimmune encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is a model for the in vitro and in vivo study of T-cell activation. It is an autoimmune disease mediated by T lymphocytes of the helper T-cell (Th) subset. After sensitization to guinea-pig myelin basic protein in complete Freund's adjuvant, Lewis rats develop an autoimmune response to central nervous system (CNS) myelin basic protein, manifested clinically as paralysis and histologically by a perivascular mononuclear cell infiltrate of the CNS parenchyma. Suppressor cell regulation of EAE has long been suspected because Lewis rats, which spontaneously recover from active disease, are resistant to reinduction of active EAE, even though effector T-cell lines can be rescued from these recovered rats. Using cyclosporin A, an immunosuppressive agent believed to inhibit Th cell function, suppressor T-cell (Ts) lines have now been generated from recovered Lewis rats. These Ts cells, when admixed with guinea pig myelin basic protein-specific Th cells, will prevent the adoptive transfer of EAE. The Ts cells appear to be CD4+, which explains previous observations that CD8+ lymphocytes are not important in the recovery of EAE in the rat. This is the first direct demonstration of Ts-cell regulation of EAE.

Chronic relapsing necrotizing encephalomyelitis produced by myelin basic protein in mice.

Chronic relapsing experimental autoimmune encephalomyelitis is commonly seen in a number of species after a single injection of whole white matter in adjuvant but not after inoculation with myelin basic protein, the major encephalitogen of central myelin. In the present report on large groups of SJL mice, we describe a form of chronic relapsing experimental autoimmune encephalomyelitis with destructive lesions after a single inoculation of myelin basic protein in complete Freund's adjuvant. This condition was studied for up to 19 months postinoculation and was characterized by a relapsing-remitting or a chronic progressive course, usually with a prolonged latent period. Higher doses of 400 and 800 micrograms of myelin basic protein were more effective in inducing this condition than were lower doses of 100 and 200 micrograms. Large lesions were apparent in the white matter. These comprised widespread destruction and Wallerian degeneration with some demyelination towards the margins. Demyelination was an initial, albeit transient, event which was subsequently masked by nerve fiber destruction. Polymorphonuclear leukocytes were early and prominent components of the inflammatory infiltrate and together with macrophages appeared to be involved in the lysis of myelin and axons. Thus, despite the clinical similarities, these features contrast the model with the more purely demyelinative lesions of chronic relapsing experimental autoimmune encephalomyelitis in other species and multiple sclerosis in man.

Experimental allergic encephalomyelitis in the absence of a classical delayed-type hypersensitivity reaction. Severe paralytic disease correlates with the presence of interleukin 2 receptor-positive cells infiltrating the central nervous system.

One characteristic of experimental allergic encephalomyelitis (EAE) in all species is the presence of a considerable leukocyte infiltrate in the central nervous system (CNS). By adoptive transfer of EAE into irradiated or nonirradiated Lewis strain rats we now show that the bulk (greater than 90%) of infiltrating cells in the CNS are superfluous to the induction of disease, as lethally irradiated recipients, despite having very few infiltrating cells in the CNS, acquire severe paralytic EAE. The reduction in the level of infiltration in irradiated recipients is selective, however, as both irradiated and nonirradiated diseased animals have very similar numbers of cells expressing IL-2-R. Disease in irradiated recipient animals is associated with substantial submeningeal hemorrhage in the spinal cord and brain stem and similar hemorrhages are found in recipients rendered leukopenic with cytotoxic drugs. Clinical signs of disease and hemorrhage are preventable, however, by administration to the recipient rats of mAbs specific for the CD4 antigen. Classic delayed-type hypersensitivity (DTH) reactions are transferable with the same cells that produce EAE in both irradiated and nonirradiated recipient rats, but such transfer of DTH is observed only in nonirradiated recipient animals and not in irradiated rats. Collectively, the findings reported herein support the conclusion that the paralysis characteristic of acute EAE is mediated by the direct action of very small numbers of activated CD4+ lymphocytes that infiltrate the CNS and produce their effects by inducing vascular damage. The findings are not consistent with reports that the lesions in EAE are produced by a classic DTH reaction.

Cyclosporin A treatment of an induced attack in a chronic relapsing model of experimental allergic encephalomyelitis.

Chronic relapsing experimental allergic encephalomyelitis (EAE) was induced in 8-week-old SJL/J mice by injecting an encephalitogenic emulsion on Day 0 and Day 7. A third injection was given on Day 70 postinoculation (PI) which precipitated an attack with high mortality (62%) after 7-9 days. Cyclosporin A (CsA) was given at doses of 5, 2, and 0.5 mg per mouse, one or three times per week starting from Day 40 PI and continuing over the next 17 days. High serum levels of CsA were measured by radioimmunoassay. However, gross and microscopic pathological examination showed no indication of hepatic or renal toxicity at these doses. In the CsA-treated mice, there was a dose-dependent shortening of the length and severity of the attack forced by challenge with the third injection. The mortality was significantly (P less than 0.05) reduced when compared with the non-CsA-treated controls. In addition, the data demonstrate a decrease of lymphocyte-derived chemotactic factor produced from phytohemagglutinin-stimulated spleen cells of mice with chronic relapsing EAE treated with CsA when compared to normal mice and mice with chronic relapsing EAE treated with vehicle alone. We conclude that it is possible to effect an induced acute attack in ongoing chronic relapsing EAE with CsA treatment.