Multiple sclerosis: myelin basic protein induced mRNA expression of proinflammatory cytokines in mononuclear cells is suppressed by interferon-β 1b in vitro

Beta-interferon (IFN-β) is a promising treatment in multiple sclerosis (MS), reducing the exacerbation rate and MRI lesion burden, as well as the disease progression in relapsing-remitting MS. IFN-β was originally defined by its antiviral effects, but the interest has recently been focused on its immunomodulatory properties. Myelin basic protein (MBP) is one of several autoantigens considered to be the target for autoaggressive immune responses, which eventually might lead to the development of MS. To study in-vitro effects of IFN-β1b on MBP induced cytokine expression, mRNA for the Th1 cytokines IFN-γ and TNF-α, the Th2 related IL-4 and IL-6, the cytolytic perforin and the immune response downregulating TGF-β was measured with in situ hybridization after culture of blood mononuclear cells (MNC) in the presence and absence of MBP. Numbers of cells expressing IFN-γ, TNF-α, perforin and IL-4 mRNA were significantly suppressed after culture with 10 U/ml IFN-β1b. No such effect was seen on MBP induced IL-6 or TGF-β mRNA expression. These observations suggest that one of the major effects of IFN-β1b is the induction of a shift in the cytokine mRNA profile towards a more immunosuppressive pattern. In parallel in vitro tests, the control substance dexametasone (40 μg/ml) reduced the numbers of cells expressing mRNA for all cytokines under study with the exception of TGF-β, to an extent equal to or even more pronounced than IFN-β1b.     

Increased transforming growth factor-β, interleukin-4, and interferon-γ in multiple sclerosis

The inflammatory nature of multiple sclerosis (MS) implicates the participation of immunoregulatory cytokines, including the T-helper type 1 (Th1) cell–associated interferon-σ (IFN-σ), the Th2 cell–related interleukin-4 (IL-4), and the immune response–downregulating cytokine transforming growth factor-β (TGF-β), but proof for their involvement in MS has been lacking. By adopting in situ hybridization with complementary DNA oligonucleotide probes for human IFN- IL-4, and TGF-β, the expression of mRNA for these cytokines was detected in mononuclear cells (MNC) from blood and cerebrospinal fluids. Strongly elevated levels of MNC expressing all three cytokines were found in peripheral blood and at even higher frequencies in cerebrospinal fluid from untreated patients with MS and optic neuritis, i.e., a common first manifestation of MS, compared with patients with other neurological diseases and healthy subjects. In MS and optic neuritis, IL-4 mRNA expressing cells predominated, followed by TGF-β– and IFN-σ–positive cells. Control patients with myasthenia gravis had similarly elevated levels of IFN-σ and TGF-β mRNA expressing blood MNC but lower numbers of IL-4–positive cells. No or slight disability of MS was associated with high levels of TGF-β mRNA expressing cells, while MS patients with moderate or severe disability had high levels of IFN-σ–positive cells. IFN-σ and TGF-β may have opposing effects in MS, and treatments inhibiting IFN-σ and/or promoting TGF-β might ameliorate MS.     

Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin,tumor necrosis factor α and tumor necrosis factor β

The kinetics of mRNA expression in the central nervous system (CNS) for a series of putatively disease-promoting and disease-limiting cytokines during the course of experimental autoimmune encephalomyelitis (EAE) in Lewis rats were studied. Cytokine mRNA-expressing cells were detected in cryosections of spinal cords using in situ hybridization technique with synthetic oligonucleotide probes. Three stages of cytokine mRNA expression could be distinguished: (i) interleukin (IL)-12, tumor necrosis factor (TNF)-β (=lymphotoxin-α) and cytolysin appeared early and before onset of clinical signs of EAE; (ii) TNF-α peaked at height of clinical signs of EAE; (iii) IL-10 appeared increasingly at and after clinical recovery. The early expression of IL-12 prior to the expression of interferon-γ (IFN-γ) mRNA shown previously is consistent with a role of IL-12 in promoting proliferation and activation of T helper 1 (Th1) type cells producing IFN-γ. The TNF-β mRNA expression prior to onset of clinical signs favours a role for this cytokine in disease initiation. A pathogenic effector role of TNF-α was suggested from these observations that TNF-α mRNA expression roughly paralleled the clinical signs of EAE. This may be the case also for cytolysin. IL-10-expressing cells gradually increased to high levels in the recovery phase of EAE, consistent with a function in down-regulating the CNS inflammation. From these data we conclude that there is an ordered appearance of putative disease-promoting and -limiting cytokines in the CNS during acute monophasic EAE.     

Male rats develop more severe experimental autoimmune encephalomyelitis than female rats: Sexual dimorphism and diergism at the spinal cord level

Compared with females, male Dark Agouti (DA) rats immunized for experimental autoimmune encephalomyelitis (EAE) with rat spinal cord homogenate in complete Freund’s adjuvant (CFA) exhibited lower incidence of the disease, but the maximal neurological deficit was greater in the animals that developed the disease. Consistently, at the peak of the disease greater number of reactivated CD4+CD134+CD45RC− T lymphocytes was retrieved from male rat spinal cord. Their microglia/macrophages were more activated and produced greater amount of prototypic proinflammatory cytokines in vitro. Additionally, oppositely to the expression of mRNAs for IL-12/p35, IL-10 and IL-27/p28, the expression of mRNA for IL-23/p19 was upregulated in male rat spinal cord mononuclear cells. Consequently, the IL-17+:IFN-γ+ cell ratio within T lymphocytes from their spinal cord was skewed towards IL-17+ cells. Within this subpopulation, the IL-17+IFN-γ+:IL-17+IL-10+ cell ratio was shifted towards IL-17+IFN-γ+ cells, which have prominent tissue damaging capacity. This was associated with an upregulated expression of mRNAs for IL-1β and IL-6, but downregulated TGF-β mRNA expression in male rat spinal cord mononuclear cells. The enhanced GM-CSF mRNA expression in these cells supported the greater pathogenicity of IL-17+ T lymphocytes infiltrating male spinal cord. In the inductive phase of the disease, contrary to the draining lymph node, in the spinal cord the frequency of CD134+ cells among CD4+ T lymphocytes and the frequency of IL-17+ cells among T lymphocytes were greater in male than in female rats. This most likely reflected an enhanced transmigration of mononuclear cells into the spinal cord (judging by the lesser spinal cord CXCL12 mRNA expression), the greater frequency of activated microglia/macrophages and the increased expression of mRNAs for Th17 polarizing cytokines in male rat spinal cord mononuclear cells. Collectively, the results showed cellular and molecular mechanisms underlying the target organ specific sexual dimorphism in the T lymphocyte-dependent immune/inflammatory response, and suggested a substantial role for the target organ in shaping the sexually dimorphic clinical outcome of EAE.     

IL-33 blockade suppresses the development of experimental autoimmune encephalomyelitis in C57BL/6 mice

IL-33 is a recently described member of the IL-1 family that has been reported to have a pathogenic role in several inflammatory diseases. In this study, we evaluated the role of IL-33 in a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). We showed that the expression of IL-33 and its receptor, ST2, was markedly elevated in the spinal cord of mice during myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide-induced EAE. Administration of a blocking anti-IL-33 antibody in mice of EAE during the induction phase significantly inhibited the onset and severity of EAE and reduced MOG(35-55)-induced IFN-γ and IL-17 production. In contrast, treatment with recombinant IL-33 worsened the disease course of EAE in association with increased induction of both IFN-γ and IL-17. Furthermore, anti-IL-33 treatment caused a remarkable decrease in expression of IL-17, IFN-γ, T-bet and RORγt, and an upregulation of IL-10 and TGF-β in the spinal cord of EAE mice. These results demonstrate that endogenous IL-33 plays a pivotal role in the pathogenesis of EAE and indicate that blockade of IL-33 has a significant protective effect against EAE.     

Transforming growth factor-β1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis

Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon-γ (IFN-γ) that makes MS worse and transforming growth factor-β (TGF-β), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-β in MS, we examined the effects of recombinant TGF-β1 (rTGF-β1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-β1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-β1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-γ, IL-4, IL-6, tumor necrosis factor-α (TNF-α), TNF-α and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-β itself. rTGF-β1 also suppressed numbers of myelin antigen-reactive IFN-γ- and IL-4-secreting cells in MS and AChR-reactive IFN-γ and IL-4 secreting cells in MG. The selective suppressive effects of TGF-β1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-β1 attractive as a treatment alternative in MS and MG.     

Cytokines in relapsing experimental autoimmune encephalomyelitis in DA rats: persistent mRNA expression of proinflammatory cytokines and absent expression of interleukin-10 and transforming growth factor-β

Experimental autoimmune encephalomyelitis (EAE) in rats is typically a brief and monophasic disease with sparse demyelination. However, inbred DA rats develop a demyelinating, prolonged and relapsing encephalomyelitis after immunization with rat spinal cord in incomplete Freund's adjuvant. This model enables studies of mechanisms related to chronicity and demyelination, two hallmarks of multiple sclerosis (MS). Here we have investigated, in situ, the dynamics of cytokine mRNA expression in the central nervous system (CNS) and peripheral lymphoid organs (lymph node cells and splenocytes) of diseased DA rats. We demonstrate that peripheral lymphoid cells stimulated in vitro with encephalitogenic peptides 69–87 and 87–101 of myelin basic protein responded with high mRNA expression for proinflammatory cytokines; Interferon-7, interleukin-12 (IL-12), tumour necrosis factors α and β, IL-1β and cytolysin. A high expression of mRNA for these proinflammatory cytokines was also observed in the CNS where it was accompanied by classical signs of inflammation such as expression of major histocompatibility complex class I and II, CD4, CDS and IL-2 receptor. The expression of mRNA for proinflammatory cytokines was remarkably long-lasting in DA rats as compared to LEW rats which display a brief and monophasic EAE. Furthermore, mRNAs for putative immunodownmodulatory cytokines i.e. transforming growth factor-β (TGF-/3), IL-10 and IL-4 were almost absent in DA rats, in both the CNS and in vitro stimulated peripheral lymphoid cells, while their levels were elevated in the CNS of LEW rats during the recovery phase. We conclude that the MS-like prolonged and relapsing EAE in DA rats is associated with a prolonged production of proinflammatory cytokines and/or low or absent production of immunodownmodulatory cytokines.     

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP.     

Anti-inflammatory effect of erythropoietin therapy on experimental autoimmune encephalomyelitis

Previous studies report that erythropoietin (EPO) has a neuroprotective role in some neurodegenerative diseases, but the mechanisms are not completely elucidated. The aim of this study was to investigate whether EPO exerts neuroprotective role in experimental autoimmune encephalomyelitis (EAE) via the routes of anti-inflammation. We established an EAE mice model treated intraperitoneally with EPO at the dose of 5,000 IU/kg on schedule, and recorded the clinical score and weight fluctuation. The infiltration of inflammatory cells in the spinal cord of EAE mice was observed with hemotoxylin and eosin (HE) staining, and the levels of IL-10, IFN-γ, IL-17, and MHC-II in central nervous system (CNS)-infiltrating cells and peripheral mononuclear cells were detected by flow cytometry or ELISA. EPO therapy ameliorates clinical signs of EAE mice, inhibits the body weight loss, and decreases the infiltration of inflammatory cells in spinal cords. IL-17 and IFN-γ are reduced, while IL-10 is not increased significantly, in both CNS-infiltrating cells and peripheral mononuclear cells of EPO-treated EAE mice, as compared with EAE control group. EPO also reduces the expression of MHC-II on peripheral antigen presentation cells. Our results indicate that EPO exerts a beneficial role in EAE by inhibiting the levels of IL-17 and IFN-γ in peripheral splenic cells and CNS-infiltrating cells.     

Interleukin-1 and tumor necrosis factor-mediated regulation of C3 gene expression in human astroglioma cells

In this report, we show that in the human astroglioma cell line D54-MG, both interleukin-1 (IL-1β) and tumor necrosis factor-alpha (TNF-α) enhance C3 gene expression in a time- and dose-dependent manner. Kinetic analysis demonstrates that after 96 h, C3 mRNA levels increase approximately 30-fold and 20-fold in response to IL-1β or TNF-α, respectively. C3 protein production increases proportionally, reaching levels 36-fold and 18-fold higher than untreated controls upon exposure to IL-1β or TNF-α, respectively. D54-MG cells require a minimal 1 h exposure to IL-1β in order to enhance C3 gene expression significantly, while 4 to 8 h are required for TNF-α. Simultaneous treatment of D54-MG cells with IL-1β and interferon-gamma (IFN-γ) resulted in an additive increase in both C3 mRNA and protein expression, a finding not seen with the combination of TNF-α and IFN-γ. Primary rat astrocytes also express increased C3 mRNA levels after 48 h in response to IL-1β (5.3-fold increase) and TNF-α (7-fold increase), while an additive effect was observed upon simultaneous treatment with both IL-1β and IFN-γ. In the central nervous system (CNS), endogenous complement and cytokine production by astrocytes, and enhancement by IFN-γ, a product of activated T cells often seen in the CNS in neural autoimmune disease, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis.     

Organ-specific autoantigens induce transforming growth factor-β mRNA expression in mononuclear cells in multiple sclerosis and myasthenia gravis

Multiple sclerosis (MS) is characterized by patchy accumulations of inflammatory cells combined with demyelination. There are mononuclear cells in blood and cerebrospinal fluid of patients with MS that produce interferon-γ and interleukin-4 in response to myelin basic protein (MBP) and proteolipid protein (PLP). Here we describe autoantigen-induced production of transforming growth factor-β (TGF-β). This multifunctional cytokine has inhibitory effects on the growth, differentiation, and effector functions of activated T cells. Blood and cerebrospinal fluid cells were exposed in short-term cultures to MBP and PLP and, after hybridization with complementary DNA oligonucleotide probes, they were evaluated for TGF-β mRNA expression. Patients with MS had higher numbers of MBP- and PLP-responsive TGF-β mRNA expressing cells in blood compared with control patients with other neurological diseases or myasthenia gravis and a five- and threefold further increment in their cerebrospinal fluid. In blood of patients with myasthenia gravis, where the acetylcholine receptor (AChR) is a target for autoaggressive immunity, there were increased levels of AChR-responsive TGF-β mRNA expressing cells. Thymectomized myasthenia gravis patients showed higher levels of TGF-β mRNA expressing cells compared with patients not thymectomized. Numbers of cells responding to AChR in MS and MBP in myasthenia gravis did not differ from numbers found in absence of antigen. Patients with other neurological diseases showed infrequent and low responses to MBP, PLP, and AChR. Diseases with presumed autoimmune pathogenesis are associated with organ-specific autoantigen-induced TGF-β production, which is increased after thymectomy.     

咖啡因慢性干预对小鼠实验性自身免疫性脑脊髓炎影响的观察

目的：探讨不同剂量咖啡因慢性干预对小鼠实验性自身免疫性脑脊髓炎（EAE）的影响及其分子免疫学机制。方法：C57BL／6小鼠随机分为CFA阴性对照组,EAE阳性对照组,咖啡因饮水干预组（1mg·k-1,10mg·kg-1,30mg·kg-1）。使用髓鞘少突胶质细胞糖蛋白35-55（MOG35-55）抗原诱导小鼠EAE模型,咖啡因干预至EAE造模后第20天与对照组小鼠同期处死为止。观察小鼠行为学变化、中枢炎症细胞浸润和损害程度、检测中枢细胞因子IL-17、IFN—γ、TGF—β的mRNA表达含量。结果：咖啡因干预组,特别是30mg·kg-1组的病情严重程度减轻,中枢炎症细胞浸润程度下降,促炎因子表达降低,抑炎因子表达上升。结论：慢性咖啡因干预,可通过降低促炎因子的表达,升高抑炎因子的表达,减轻小鼠EAE的炎症损伤。     

OX-40 antibody enhances for autoantigen specific Vβ8.2+ T cells within the spinal cord of lewis rats with autoimmune encephalomyelitis

The Vβ8.2 T cell receptor (TCR) component is the predominant Vβ 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 Vβ8.2 positive and negative cells were analyzed for IFN-γ mRNA production and OX-40 cell surface expression during the onset of EAE. The Vβ8.2⁺ T cells isolated from the spinal cord produced the majority of mRNA for IFN-γ and also showed a marked enhancement for OX-40 expression compared to Vβ8.2⁺ 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 Vβ8.2 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 Vβ biases and autoantigen specific cells within inflamed tissues even when the antigen specificity is unknown. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

Cytokine phenotype of human autoreactive T cell clones specific for the immunodominant myelin basic protein peptide (83–99)

Experimental allergic encephalomyelitis (EAE), an animal model resembling multiple sclerosis (MS), is mediated by myelin antigen-specific CD4+ T cells secreting cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-β (TNF-β), and the proinflammatory cytokine TNF-α—all associated with the T-helper-1 (Th1) T cell subset. Based on numerous similarities between MS and EAE, it has been postulated that Th1-like T cells are involved in the pathogenesis of MS. Production of proinflammatory cytokines such as IFN-γ and, in particular, TNF-α/β by autoreactive T cells is considered crucial for the initiation and amplification of inflammatory brain lesions and possibly also for direct myelin damage. In contrast, regulatory cytokines such as interleukin-4 (IL-4), IL-10, and IL-13, which are associated with the Th2-like phenotype, may play a role in the resolution of relapses. Although the human T cell response to myelin basic protein (MBP) is well characterized in terms of antigen specificity, HLA restriction, and T cell-receptor (TCR) usage, little is known about the cytokine pattern of these autoreactive T cells. To gain such information, conditions for studying cytokine secretion by human autoreactive T cell clones (TCC) were established. The cytokine secretion profile of human autoreactive CD4+ TCC, specific for myelin basic protein peptide (83–89) [MBP(83–99)], a candidate autoantigen in MS, was investigated. Our results show that TCC cytokine production in long-term culture was stable. In addition, the correlation of various cytokines within specific TCC revealed differences compared to murine T cells. The comparison of 30 human MBP(83–99)-specific TCC demonstrated heterogeneity in cytokine secretion, with a continuum between Th1- and Th2-like cells rather than distinct Th1 or Th2 subsets. These data are important for further investigation of the potential role of cytokines in the inflammatory process of MS, and provide a powerful tool to investigate therapeutic interventions with respect to their influence on cytokine secretion of autoreactive T cells. © 1996 Wiley-Liss, Inc.     

Lymphokine mRNA expression in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis is associated with a host recruited CD45R hi/CD4 population during recovery

To evaluate CD4⁺ T cell subpopulations involved in the induction and recovery from experimental autoimmune encephalomyelitis (EAE), the CD45R phenotype and lymphokine mRNA profile was evaluated for encephalitogenic CD4⁺ T cell lines in vitro and compared to CD4^* T cells islated from the spinal cord of Lewis rats with EAE were > 90% of the myelin basic protein (MBP)-specific T cell lines and clones that adoptively transferred EAE were > 90% CD4⁺ and > 90% CD45R lo. A time course of EAE disease progression was monitored as a function of the percentage of CD45R hi/CD4⁺ T cells isolated from the spinal cords of diseased animals. The majority of CD4⁺ T cells found in the central nervous system during the early phase of passive EAE were CD45R lo (the same as the encephalitogenic lines/clones). A large increase of the CD45R hi/CD4⁺ T cells (up to 45%) was observed during the peak and recovery phases of EAE. Lymphokine mRNA production was analyzed from antigen-stimulated MBP-specific lines, and from spinal cord lymphocytes isolated from rats with EAE. The BP-specific lines produced Th1 lymphokines (IL-2, IFN-γ, and TNF-α), while the spinal cord lymphocytes produced the same Th1 lymphokines as well as IL-4 and IL-10. The CD45R hi/CD4⁺ T cells isolated from the spinal cords were larger and expressed more lymphokine RNA per cell than the CD45R lo/CD4⁺ T cells. The encephalitogenic cells (CD45R hi/CD4⁺ T detected in the spinal cords of rats with a fluorescent dye and by allelic transfers and all of the CD45R hi/CD4⁺ lymphocytes found in the spinal cells were found to be host recruited. Thus it appears that the CD45R hi/CD4⁺ lymphocytes found in the spinal cord represent a host-recruited, activated cellular infiltrate that increased in number in the recovery phase of EAE and synthesized both Th1 and Th2 lymphokines.     

Dibutyryl cyclic AMP and inflammatory cytokines mediate C3 expression in schwann cells

Schwann cells (SchC), the myelinating glia of the peripheral nervous system, are immunocompetent cells and secrete a variety of immune and inflammatory mediators. In this report, we show that rat SchC in vitro express both C3 mRNA and protein in response to dibutyryl cyclic AMP (dbcAMP) and the cytokines IFN-γ, TNF-α, and IL-1β. SchC in culture constitutively expressed low levels of C3 which were significantly upregulated upon stimulation with 1mM dbcAMP by 24 hours, and persisted up to 120 hours. This response was minimally enhanced by costimulation with 100 U/ml IFN-γ, whereas costimulation with 100 U/ml IFN-γ together with 150–450 ng/ml TNF-α induced a greatly increased C3 response. TNF-α alone did not induce C3 expression in SchC. Cycloheximide inhibited this dbcAMP-dependent delayed C3 production, thus implying an intermediary signal in the induction pathway requiring protein synthesis. Treatment with 0.1–10 ng/ml IL-1β for 0–72 hours induced C3 mRNA and protein in a dose-dependent manner. C3 mRNA was detectable at 1 hour and mRNA and protein peaked by 6–12 hours on stimulation with 10 ng/ml IL-1β, or at 48 hours with 1.0 ng/ml IL-1β. Furthermore, IL-1β mRNA was detected at 6 hours in dbcAMP-treated SchC, preceding the dbcAMP-induced C3 expression by 18 hours. Induction of C3 mRNA and protein by dbcAMP at 24 hours was inhibited >85% by a neutralizing anti-IL-1β antibody and 76% with an IL-1 receptor antagonist. This suggests that dbcAMP-induced synthesis of IL-1β mediates the C3 production by SchC in an autocrine/paracrine fashion by binding to a functional IL-1 receptor expressed on the surface of SchC. Endoneurial IL-1 and C3 production by SchC may therefore contribute to the inflammatory events associated with peripheral nerve demyelination. GLIA 20:308–321, 1997. © 1997 Wiley-Liss, Inc.     

Nasal administration of myelin basic protein prevents relapsing experimental autoimmune encephalomyelitis in DA rats by activating regulatory cells expressing IL-4 and TGF-β mRNA

This study explores nasal administration of myelin basic protein (MBP) as a potential means of inducing tolerance to relapsing experimental autoimmune encephalomyelitis (PR-EAE), an experimental multiple sclerosis (MS) model that was induced in DA rats by immunization with rat spinal cord homogenate and incomplete Freund's adjuvant. DA rats received a total dosage of 0, 6, 60, 600 μg/rat of bovine MBP on ten consecutive days prior to immunization. EAE with typical course was observed in control rats receiving only PBS nasally, and in rats receiving 6 μg/rat of MBP. Rats receiving 60 μg/rat of MBP developed acute EAE but no relapse during 60 days of observation post immunization (p.i.). Only one of eight rats receiving 600 μg/rat of MBP developed slight, transient EAE. This protection was confirmed at the histology level and was associated with decreased levels of MBP-reactive IFN-γ secreting Th1-like spleen cells on day 13 and 60 p.i. Rats receiving 60 and 600 μg/rat of MBP showed decreased serum anti-MBP IgG2b antibody levels on day 60 p.i., and rats receiving 600 μg/rat of MBP had marginally increased anti-MBP IgG1 antibody levels in serum compared to control EAE rats. Cytokine mRNA profiles in central nervous system (CNS) and spleen mononuclear cells were evaluated. Dose-dependent reduction of TNF- mRNA expression were observed both in CNS and in splenocytes. Increased IL-4 and TGF-β mRNA expression were observed in CNS of low (6 μg/rat) and median (60 μg/rat) dose of MBP tolerized rats and in splenocytes of rats tolerized with 600 μg/rat of MBP. We conclude that nasal administration of MBP in DA rat prevents EAE induced by immunization with whole rat spinal cord homogenate that, besides MBP, contains multiple antigenic myelin proteins. A mechanism involving MBP-reactive regulatory cells expressing IL-4 and TGF-β mRNA acts as part in the induction of this tolerance.