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We previously demonstrated that injection of myelin basic protein-pulsed (MBP-pulsed)--but not of unpulsed--autologous bone marrow-derived dendritic cells (DC) efficiently prevents experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To define the molecules involved, we used 3 groups of rats pretreated subcutaneously with MBP-DC, or unpulsed DC, or PBS (control EAE). Four weeks later, all rats were immunized with encephalitogenic MBP peptide and adjuvant. Microarray analyses were done to screen for genes that differ among the 3 groups. Based on microarray analysis data, we used real-time PCR to measure expression of six probably involved genes in draining lymph node cells obtained on day 0, day 7 and day 14 post immunization (p.i.). Two of these 6 genes were consistently altered in both microarray analyses and RT-PCR. They are CD24 antigen being persistently low, and myosin light polypeptide 2 (Myl2) being high in the acute immune response in MBP-DC pretreated rats that develop resistance to EAE. These two genes could be targeted to treat EAE and, possibly, multiple sclerosis.

Dendritic cells (DC) can modulate the nature of immune responses in a stimulatory or tolerogenic fashion. Great attention has been given to the induction of immunity to tumour and infection. In this study, bone marrow-derived DC from healthy Lewis rats were pulsed in vitro with encephalitogenic myelin basic protein peptide 68-86 (MBP 68-86), and injected subcutaneously (1 x 106/rat) into normal Lewis rats. Upon observation of the rats pretreated in this way for 4 weeks, when no clinical signs of EAE occurred, these rats were immunized with MBP 68-86 and Freund's complete adjuvant. The pretreated rats failed to develop clinical EAE. This tolerance was associated with augmented proliferative responses, interferon-gamma secretion, inducible nitric oxide synthase (iNOS) expression and NO production. The frequency of apoptotic cells was increased in the rats receiving MBP 68-86-pulsed DC compared with unpulsed control DC. Few infiltrating inflammatory cells were observed in spinal cord sections from rats that had received MBP 68-86-pulsed DC. The data are compatible with the interpretation that MBP 68-86-pulsed DC induce tolerance to EAE possibly through up-regulation of iNOS expression and NO production, which mediate cell apoptosis, thereby reducing infiltration of inflammatory cells within the central nervous system.

The efficacy and mechanism of immunosuppression against experimental autoimmune encephalomyelitis (EAE) by oral low-dose administration of myelin basic protein (MBP) conjugated to cholera toxin B subunit (CTB) were investigated in Lewis rats immunized with MBP together with complete Freund's adjuvant 4 days before the start of treatment. Oral treatment with CTB-MBP conjugate gave almost complete protection against disease, an effect that was totally abrogated by including a low dose of cholera holotoxin (CT). The protection by CTB-MBP was associated with a dramatic reduction in the number of leukocytes staining for CD4, CD8, IL-2R or MHC class II in the spinal cord as examined by immunohistochemistry. The mRNA expressions of T(h)1 cytokines IFN-gamma, IL-12 and tumor necrosis factor-alpha, as well as of chemokines monocyte chemotactic protein (MCP)-1 and RANTES in the spinal cord were also reduced by 76-94%, as assessed by in situ hybridization. In contrast, transforming growth factor (TGF)-beta mRNA-expressing cells were strongly increased in the spinal cord from animals treated orally with the CTB-MBP conjugate. In the draining peripheral lymph nodes, the number of MBP-specific TGF-beta mRNA-expressing cells was also increased, whereas there was a decrease in cells expressing T(h)1 or T(h)2 cytokine mRNA. Protection against EAE could be transferred by injection of cells from the mesenteric lymph nodes of animals fed with CTB-MBP into naive animals exposed to encephalitogenic T cells. The results indicate that the protective anti-inflammatory effect by oral treatment with CTB-MBP conjugate is, to a large extent, due to the induction of TGF-beta-secreting suppressive-regulatory T cells and to local down-regulation of MCP-1 and RANTES in the spinal cord.

Mucosal administration of low doses of myelin basic protein (MBP) peptide 68-86 (MBP 68-86) or anti-inflammatory cytokine IL-10 effectively prevented experimental allergic encephalomyelitis (EAE), but failed to suppress the disease if given after 7 days postimmunization (p.i.), i.e., after T cell priming had occurred. We anticipated that combined administration of autoantigen and IL-10 can treat incipient EAE. Lewis rats with EAE actively induced with MBP 68-86 and complete Freund's adjuvant received 120 microg MBP 68-86 + 200 ng IL-10 per rat per day from day 7 p.i. and for 5 consecutive days. These rats showed later onset, lower clinical scores, less body weight loss, and shorter duration of EAE than rats receiving MBP 68-86 or IL-10 only or PBS. EAE amelioration was associated with decreased infiltration of ED1(+) macrophages and CD4(+) T cells within the central nervous system and with decreased proliferative responses of lymph node cells, indicating that combined administration of MBP 68-86 and IL-10 induced immune hyporesponsiveness. IFN-gamma secretion as well as IFN-gamma, TNF-alpha, IL-4, and IL-10 mRNA expression by lymph node MNC was down-regulated in the treated rats. Immune hyporesponsiveness, rather than immune deviation or regulatory mechanisms, seems to be responsible for the protection of EAE after autoantigen + IL-10 administration by the nasal route., (Copyright 2000 Academic Press.)

Mucosal myelin autoantigen administration effectively prevented EAE, but mostly failed to treat ongoing EAE. Patients with multiple sclerosis (MS), for which EAE is considered an animal model, did not benefit from oral treatment with bovine myelin. We anticipated that autoantigen, administered together with a cytokine that counteracts Th1 cell responses, might ameliorate Th1-driven autoimmune disease, and that nasal administration might considerably reduce the amounts of antigen + cytokine needed for treatment purposes. Lewis rats with EAE actively induced with myelin basic protein peptide (MBP 68-86) and Freund's complete adjuvant (FCA), received from day 7 post-immunization, i.e. after T cell priming had occurred, 120 microg MBP 68-86 + 100 ng IL-4 per rat per day for 5 consecutive days. These rats showed later onset, lower clinical scores, less body weight loss and shorter EAE duration compared with rats receiving MBP 68-86 or IL-4 only, or PBS. EAE amelioration was associated with decreased infiltration of ED1+ macrophages and CD4+ T cells within the central nervous system, and with decreased interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) and enhanced IL-4, IL-10 and transforming growth factor-beta (TGF-beta) responses by lymph node cells. Simultaneous administration of encephalitogenic peptide + IL-4 by the nasal route thus suppressed ongoing EAE and induced IL-4, IL-10 and TGF-beta-related regulatory elements.

IL-10 and TGF-beta1 are important immunoregulatory cytokines associated with clinical remissions in multiple sclerosis and amelioration of experimental allergic encephalomyelitis (EAE). IL-10 and TGF-beta1 have previously been shown to prevent the development of EAE. Here, we study effects of IL-10 and TGF-beta1 in ongoing EAE. When IL-10 or TGF-beta1 was administered by the nasal route from day 0 to day 7 postimmunization (pi), both IL-10 and TGF-beta1 prevented the development of acute EAE in Lewis rats. When IL-10 or TGF-beta1 was administered by the nasal route from day 5 to day 12 pi, both IL-10 and TGF-beta1 failed to influence clinical EAE. The inhibition of clinical EAE severity in IL-10-prevented rats was associated with reduced proliferation, IFN-gamma mRNA expression, and IFN-gamma secretion, while proliferation as well as IFN-gamma mRNA expression and secretion were augmented in TGF-beta1-prevented rats. TGF-beta1-prevented rats exhibited high levels of NO production by DC, which may mediate apoptosis of CD4+ T cells and of the DC themselves. For prevention, both IL-10 and TGF-beta1 inhibited infiltration of CD4+ T cells within the CNS, but neither IL-10 nor TGF-beta1 induced immune deviation from Th1 to Th2. Expression of IL-4 mRNA was not altered in IL-10- and TGF-beta1-prevented rats. These results demonstrate that IL-10 and TGF-beta administration by the nasal route can prevent the development of acute EAE, but by different mechanisms. The findings in rats with ongoing EAE have implications for the clinical application of cytokine treatment in autoimmune diseases.

Spontaneous remission of experimental allergic encephalomyelitis (EAE) is usually associated with prominent apoptosis. The mechanisms behind apoptosis are unknown. We examined the functions of dendritic cells (DC) from Lewis rats with EAE induced by immunization with myelin basic protein peptide 68-86 (MBP68 - - 86). Recovery from EAE was associated with three major functional changes of freshly prepared DC: (1) elevated proliferation, (2) increased nitric oxide (NO) production, and (3) augmented IFN-gamma secretion. In Freund's complete adjuvant (FCA)-immunized control rats, no increase of proliferation, NO production or IFN-gamma secretion was observed on day 21 post-immunization (p.i.), i.e., recovery from EAE. In vitro effects of IFN-gamma, TNF-alpha, TGF-beta1, IL-4 and IL-10 on DC were examined. IFN-gamma enhanced proliferation and NO production by DC, while TNF-alpha and IL-4 induced only slight DC proliferation. DC from recovering EAE rats (day 21 p.i.) suppressed MBP68 - - 86-induced T cell proliferation compared to DC obtained at other time points in EAE and FCA-immunized rats. DC-derived NO induced apoptosis of CD4+ T cells, thereby inhibiting autoreactive T cell responses. Besides IFN-gamma stimulation, NO production by DC was mainly induced in an antigen-dependent manner when DC were co-cultured with T cells. The results suggest that spontaneous recovery from EAE is associated with augmented DC functions. Overproduction of NO by DC results in apoptosis of autoreactive CD4+ T cells, thereby decreasing autoreactive T cell reactivities. The existence of such a NO negative feedback loop may contribute to remission of EAE.

We have isolated infiltrating mononuclear cells (inMNC) and lymph node MNC (lnMNC) from Lewis rats with actively induced experimental allergic encephalomyelitis (EAE), and compared their responses to myelin basic protein (MBP). MBP-induced proliferation and MBP-specific IgG secreting cells were lower in inMNC compared to lnMNC, while MBP-reactive IFN-gamma secreting cells in inMNC were higher. Using an in vitro culture system, we observed that astrocytes derived from newborn Lewis rats not only suppressed T cell proliferation and IFN-gamma production but also reduced MBP-specific IgG production by B cells. Astrocyte-derived soluble factor (s), rather than direct cell-to-cell interactions, seems to be responsible for the inhibitory effect. Supernatants from IFN-gamma-stimulated astrocytes exhibited stronger suppressive effects than supernatants from unstimulated astrocytes, whereas supernatants from microglia did not cause downregulation of T and B cell functions. These results indicate that astrocytes are major effector cells in inhibiting functions of MBP-reactive T and B cells. Astrocytes down-regulated expression of ICAM-1 and MHC class II in lnMNC, but did not induce apoptosis of lnMNC. The results in the present study do not exclude the possibility that astrocytes induce T cell apoptosis in a cognate fashion. Taken together, the inhibitory properties of astrocytes may contribute to the confinement of inflammatory lesions in multiple sclerosis and EAE. Our report compares immunoreactivities of inMNC and lnMNC in EAE and elucidates the role of astrocytes in the inactivation of MBP-reactive T and B cells.

Induction of mucosal tolerance by inhalation of soluble peptides with defined T cell epitopes is receiving much attention as a means of specifically down-regulating pathogenic T cell reactivities in autoimmune and allergic disorders. Experimental autoimmune encephalomyelitis (EAE) induced in the Lewis rat by immunization with myelin basic protein (MBP) and Freund's adjuvant (CFA) is mediated by CD4+ T cells specific for the MBP amino acid sequences 68-86 and 87-99. To further define the principles of nasal tolerance induction, we generated three different MBP peptides (MBP 68-86, 87-99 and the non-encephalitogenic peptide 110-128), and evaluated whether their nasal administration on day -11, -10, -9, -8 and -7 prior to immunization with guinea pig MBP (gp-MBP) + CFA confers protection to Lewis rat EAE. Protection was achieved with the encephalitogenic peptides MBP 68-86 and 87-99, MBP 68-86 being more potent, but not with MBP 110-128. Neither MBP 68-86 nor 87-99 at doses used conferred complete protection to gp-MBP-induced EAE. In contrast, nasal administration of a mixture of MBP 68-86 and 87-99 completely blocked gp-MBP-induced EAE even at lower dosage compared to that being used for individual peptides. Rats tolerized with MBP 68-86 + 87-99 nasally showed decreased T cell responses to MBP reflected by lymphocyte proliferation and IFN-gamma ELISPOT assays. Rats tolerized with MBP 68-86 + 87-99 also had abrogated MBP-reactive IFN-gamma and tumor necrosis factor-alpha mRNA expression in lymph node cells compared to rats receiving MBP 110-128 nasally, while similar low levels of MBP-reactive transforming growth factor-beta and IL-4 mRNA expressing cells were observed in the two groups. Nasal administration of MBP 68-86 + 87-99 only slightly inhibited guinea pig spinal cord homogenate-induced EAE, and passive transfer of spleen mononuclear cells from MBP 68-86 + 87-99-tolerized rats did not protect naïve rats from EAE. Finally, we show that nasal administration of MBP 68-86 + 87-99 can reverse ongoing EAE induced with gp-MBP, although higher doses are required compared to the dosage needed for prevention. In conclusion, nasal administration of encephalitogenic MBP peptides can induce antigen-specific T cell tolerance and confer incomplete protection to gp-MBP-induced EAE, and MBP 68-86 and 87-99 have synergistic effects. Non-regulatory mechanisms are proposed to be responsible for tolerance development after nasal peptide administration.

Nasal administration of soluble antigens is an exciting means of specifically down-regulating pathogenic T-cell reactivities in autoimmune diseases. The mechanisms by which nasal administration of soluble antigens suppresses autoimmunity are poorly understood. To define further the principles of nasal tolerance induction, we studied the effects of nasal administration of myelin basic protein (MBP) on experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. EAE is a CD4+ T-cell-mediated animal model for human multiple sclerosis. Nasal administration of guinea-pig (gp)-MBP at a dose as low as 30 micrograms/rat can completely prevent gp-MBP-induced EAE, whereas nasal administration of bovine (b)-MBP is not effective even at a much higher dosage. Cellular immune responses, as reflected by T-cell proliferation and interferon-gamma (IFN-gamma)-ELISPOT, were suppressed in rats receiving the two different doses (30 and 600 micrograms/rat) of gp-MBP, but not after administration of b-MBP. Rats tolerized with both doses of gp-MBP had also abrogated MBP-induced IFN-gamma mRNA expression in popliteal and inguinal lymph node mononuclear cells compared with rats receiving phosphate-buffered saline nasally. However, adoptive transfer revealed that only spleen mononuclear cells from rats pretreated with a low dose, but not from those pretreated with a high dose, of gp-MBP transferred protection to actively induced EAE. Low-dose (30 micrograms/rat) gp-MBP-tolerized rats also had high numbers of interleukin-4 (IL-4) mRNA-expressing lymph node cells, while high-dose (600 micrograms/rat) gp-MBP-tolerized rats had low numbers of IL-4 mRNA-expressing lymph node cells. Our data suggest an exquisite specificity of nasal tolerance. Dose-dependent mechanisms also relate to nasal tolerance induction and protection against EAE in the Lewis rat.

EAE is an autoimmune disease of the central nervous system (CNS) that serves as an experimental model for the human inflammatory demyelinating disease multiple sclerosis (MS). Antigen-based immunotherapy including soluble antigen administration via feeding has been shown to be successful in treating EAE in rodents. In the present study, we explore nasal administration of small amounts of myelin basic protein (MBP) as a potential means of treatment of protracted, relapsing EAE (PR-EAE) in a novel DA rat system. We found that nasal administration of MBP prevented EAE induced with whole spinal cord homogenate + Freund's incomplete adjuvant (FIA), and strongly down-regulated levels of MBP-reactive interferon-gamma (IFN-gamma)-secreting Th1-like cells. However, in rats with ongoing PR-EAE receiving the same regimen of MBP, a trend of aggravated disease was recorded, in conjunction with augmented levels of MBP-reactive IFN-gamma-secreting Th1-like splenocytes during the acute phase of EAE. These data have implications for the clinical application of nasal tolerance to autoimmune diseases.

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 micrograms/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 micrograms/rat of MBP. Rats receiving 60 micrograms/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 micrograms/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-gamma secreting Th1-like spleen cells on day 13 and 60 p.i. Rats receiving 60 and 600 micrograms/rat of MBP showed decreased serum anti-MBP IgG2b antibody levels on day 60 p.i., and rats receiving 600 micrograms/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-alpha mRNA expression were observed both in CNS and in splenocytes. Increased IL-4 and TGF-beta mRNA expression were observed in CNS of low (6 micrograms/rat) and median (60 micrograms/rat) dose of MBP tolerized rats and in splenocytes of rats tolerized with 600 micrograms/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-beta mRNA acts as part in the induction of this tolerance.

Oral administration of acetylcholine receptor (AChR) or myelin basic protein (MBP) to Lewis rat prior to immunization with AChr or MBP and complete Freund's adjuvant (CFA) has previously been shown to prevent or delay the onset of experimental autoimmune myasthenia gravis (EAMG) or experimental allergic encephalomyelitis (EAE), which represent animal models of myasthenia gravis and multiple sclerosis, respectively. Here we show that Lewis rats immunized with AChr+MBP+CFA developed both signs of muscular weakness seen in EAMG and paresis characteristic for EAE. This disease was associated with high levels of anti-AChR and anti-MBP antibody secreting cells and of AChR- and MBP-reactive INF-gamma secreting Th1-like cells in lymph nodes. The diseased rats also showed upregulation of AChR- and MBP-induced mRNA expression of IFN-gamma in lymph node cells. Oral tolerization with AChR and MBP in combination prior to immunization with AChR+MBP+CFA alleviated clinical disease as well as AChR- and MBP-specific B cell node cells. The results implicate that oral tolerization simultaneously to more than one autoimmune disease-related autoantigen is feasible, and that suppression of autoantigen-induced IFN-gamma and augmentation of TGF-beta are pivotal in tolerance induction.

Experimental allergic neuritis (EAN) is a T cell mediated disease associated with inflammation and demyelination of peripheral nerves. EAN is an experimental model of Guillain-Barré syndrome. The peripheral nerve myelin components P2 and P0 represent major neuritogens, but the diversity and quantity of B cell responses in EAN are unknown. Lewis rats were immunized with bovine peripheral nerve myelin (BPM), and levels of B cells secreting IgM and IgG antibodies to BPM, P2 and P0, the glycolipid GM1 and five peptides of myelin-associated glycoprotein (MAG) were determined. Already on day 7 post-immunization (p.i.), i.e. before the onset of clinical EAN, lymph nodes contained elevated levels of cells secreting IgM antibodies of all specificities examined. Maximum numbers of IgG antibodies secreting cells were generally reached at the height of clinical disease. The numbers of cells secreting IgG antibodies to BPM, P2, P0, GM1 and MAG peptides were also elevated before disease onset, but they were mostly higher than those of IgM antibodies and they reached their maximum only after recovery. The results imply that EAN is associated with strong B cell responses to all myelin antigens under study without restriction to any immunodominant myelin component or MAG peptides.

Multiple sclerosis (MS) may be an autoimmune disease, partially caused by autoreactivity to myelin basic protein (MBP) and other central nervous system proteins. Acute optic neuritis (ON) is a frequent first symptom of MS. The role of the HLA system in anti-MBP antibody production in ON was investigated employing a restriction fragment length polymorphism system for genomic HLA-DQ and -DR typing and an immunospot assay for the detection of individual cells secreting antibodies to three different synthetic MBP peptides. Thirty-two out of 40 patients (80%) with ON had cells in cerebrospinal fluid secreting anti-MBP peptide antibodies while this occurred in 10/19 patients with other neurological diseases (53%; mainly in patients with inflammatory diseases in the central nervous system). This difference was statistically significant (P = 0.03). None of the three examined peptides were immunodominant in any patient group. It was found, however, that presence of HLA DR15, which is associated with MS, may be associated further with predominant production of antibodies to the MBP amino acids 63-88 in patients with ON (P = 0.002, corrected for multiple comparisons).

Genetic factors influence the susceptibility to multiple sclerosis (MS). This disease is accompanied by augmented T cell responses to CNS myelin components such as myelin basic protein. To evaluate the familial occurrence of such T cell autoreactivity, we have studied 12 MS families including 37 healthy first-degree relatives for occurrence of numbers of interferon-gamma (IFN-gamma) secreting cells among blood mononuclear after culture in presence of myelin basic protein (MBP), eight synthetic MBP peptides and the control antigen acetylcholine receptor (AChR). There were no differences between MS patients and healthy family members regarding frequencies of autoreactive T cells recognizing MBP, the eight different MBP peptides or AChR. None of the MBP peptides predominated as T cell antigen among the MS patients or their unaffected family members. In some families the highest number of MBP peptide reactive T cells were found among unaffected family members. No correlation was observed between numbers of MBP or MBP peptide reactive T cells in various subjects and their HLA-DR-DQ phenotypes. In conclusion, this study has revealed the presence of MBP and MBP peptide reactive T cells of similar frequencies in MS patients and their healthy family members.

Studies on patients with monosymptomatic optic neuritis (ON) should give opportunities to identify features typical for early multiple sclerosis (MS). There are increased T and B cell responses to the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) in both ON and MS, but there is little information on the types of cytokines produced by such cells. We describe the use of in situ hybridization with complementary DNA oligonucleotide probes to detect and enumerate mononuclear cells expressing mRNA for the cytokines interferon-gamma (IFN-gamma) which augments cell-mediated immunity; interleukin-4 (IL-4) which promotes the B cell response; and transforming growth factor beta (TGF-beta) that in many cases downregulates immune responses. Expression of these cytokines was studied in mononuclear cells from peripheral blood and cerebrospinal fluid (CSF) from patients with ON and MS after in vitro exposure to MBP and PLP, and in absence of antigen. There were elevated levels of cells that in response to MBP and PLP expressed IFN-gamma, IL-4 and TGF-beta mRNA in blood and further enriched in CSF in both ON and MS, compared to patients with other neurological diseases. The results suggest that IFN-gamma, IL-4 as well as TGF-beta are involved in both ON and MS, and that the cytokine profile in early MS as reflected by ON is not different from that in clinically definite MS.

Monosymptomatic unilateral optic neuritis (ON) is a common first manifestation of multiple sclerosis (MS) in which increased numbers of autoimmune T and B cells, recognizing different myelin autoantigens including myelin basic protein (MBP) and its peptides, have been implicated in a hypothetical immunopathogenesis. Using an immunospot assay to detect specific antibodies secreted by individual cells, we analyzed the B-cell repertoire to MBP and its amino acid residues 1-20, 63-88, 110-128, and 148-165 in blood and CSF from patients with ON and MS, and from controls. There were cells secreting IgG antibodies to MBP and the four peptides in blood at mean numbers of 0.9 to 4.6 per 10(5) mononuclear cells, without differences between the three patient groups. Mostly, more than 100-fold more B cells with these specificites per 10(5) cells were found in CSF from the patients with ON and MS, without differences between these two groups but with many fewer in CSF from controls. None of the four included MBP peptides represented an immunodominant B-cell epitope in either ON or MS, and the B-cell response was not more restricted in ON than in MS. The autonomy of the autoimmune B-cell response in CSF was further supported by the pronounced asynchrony of the repertoire to the four MBP peptides in CSF compared with blood in individual patients. The large numbers of MBP- and MBP peptide-reactive B cells in CSF in early MS, as manifested by ON, could play a major role in the immunopathogenesis and perpetuation of MS. Alternatively, they could represent myelin breakdown or restoration.

The cause of multiple sclerosis (MS) is unknown. Recently reported abnormal T-cell responses to several myelin proteins and myelin basic protein (MBP) peptides in peripheral blood constitute one line of evidence that autoimmune mechanisms could be involved in the pathogenesis of the disease. Monosymptomatic unilateral optic neuritis (ON) is a common first manifestation of MS and important to examine for a possible restriction of the T-cell repertoire early in the disease. T-cell activities to MBP and the MBP amino acid sequences 63-88, 110-128 and 148-165 were examined by short-term cultures of mononuclear cells from cerebrospinal fluid (CSF) and blood in the presence of these antigens, and subsequent detection and counting of antigen-specific T cells that responded by interferon-gamma (IFN-gamma) secretion. Most patients with MS and ON had MBP and MBP peptide-reactive T cells in CSF, amounting to mean values of between about 1 per 2000 and 1 per 7000 CSF cells and without immunodominance for any of the peptides. Numbers were 10-fold to 100-fold lower in the patients' blood. Values were similar in ON and MS, and no evidence was obtained for a more restricted T-cell repertoire in ON. The MBP peptide-recognizing T-cell repertoire was different in CSF than in blood in individual patients with ON and MS, thereby giving further evidence for an autonomy of the autoimmune T-cell response in the CSF compartment. No relations were observed between numbers of autoreactive T cells and presence of oligoclonal IgG bands in CSF or abnormalities on magnetic resonance imaging of the brain in ON or clinical variables of MS. The high numbers of MBP and MBP peptide-reactive T cells could play a role in the pathogenesis of ON via secretion of effector molecules, one of them being IFN-gamma, as well as in the transfer of ON to MS.

Myelin basic protein (MBP)-autoreactive T cells have a crucial pathogenetic role in experimental allergic encephalomyelitis (EAE) and certain MBP epitopes may be immunodominantly recognized. The heterogeneity and quantity of the T cell response to different epitopes of MBP in multiple sclerosis (MS) and non-MS controls is not so clearly defined. We now study T cell reactivity to six different peptides of MBP in MS compared to controls in short-term cultures of blood mononuclear cells by measuring numbers of T cells that secrete interferon-gamma in response to antigen. In comparison with controls, MS patients showed dramatically increased numbers of MBP peptide-reactive T cells with mean values varying between 10.4 and 22.5 per 10(5) blood mononuclear cells. Among those MBP peptides examined (amino acid 1-20, 63-88, 89-101, 96-118, 110-128 and 148-165), no single peptide is preferentially recognized. Neither is any preferential response apparent after subdivision of the MS patients according to their HLA-DR genotype. Our findings suggest that a quantitative increase of a broad repertoire of myelin-autoreactive T cells with capacity to secrete IFN-gamma can be important for the pathogenesis of MS.

T cell reactivities to the putative autoantigens myelin basic protein (MBP), MBP peptides with amino acid residues 110-128 and 148-165, and myelin proteolipid protein (PLP) were examined in patients with acute ischaemic cerebrovascular disease (CVD) and, for comparison, in patients with inflammatory neurological diseases and other neurological diseases. A quantitative measure of these T cell reactivities was obtained by assessing numbers of T cells among blood and cerebrospinal fluid (CSF) mononuclear cells that secreted IFN-gamma in response to antigen in vitro. Higher numbers of T cells reactive with each of these four antigens were detected in peripheral blood from patients with CVD compared with patients of the two control groups. Among blood cells from the CVD patients, their average number was 2.3-4.2/10(5) mononuclear cells. MBP reactive T cells were several-fold enriched in the CSF of CVD patients. The findings strongly suggest that brain damage in context with acute CVD leads to an in vivo expansion of myelin reactive T cells.

Cells secreting antibodies against guinea pig myelin and synthetic myelin basic protein (MBP) peptides were evaluated in cerebrospinal fluid (CSF) and blood from patients with multiple sclerosis (MS) and a variety of other neurological diseases (OND). The peptides used, reproducing amino acid sequences 1-20, 70-89, 108-126, or 157-166 of MBP, were selected on the basis of their hydrophilic and encephalitogenic properties. Low numbers of cells secreting IgG antibodies against myelin or each of the MBP peptides (about 1 per 50,000) were detected in peripheral blood, with no difference between MS and OND. In CSF, cells secreting IgG antibodies to MBP 70-89 were more frequently (p = 0.007) detected in patients with MS (1/380 IgG-secreting cells on average) than in patients with OND (1/2083 IgG-secreting cells on average). The frequencies of cells secreting antibodies against myelin or the three other MBP peptides were similar in MS and OND. Thus, evaluation of B cell immunity at the cellular level indicates that MBP 70-89 is an immunodominant B cell epitope in MS. It is not clear whether this intrathecal anti-MBP 70-89 IgG antibody response has any pathogenetic relevance in MS or is the result of myelin breakdown.

An autoimmune response to myelin basic protein (MBP) has been proposed to participate in the development of the chronic neurologic manifestations that may accompany Borrelia burgdorferi-induced Lyme disease. Using an immunospot assay, we counted cells secreting antibodies to MBP. Anti-MBP IgG antibody-secreting cells were detected in CSF from eight of 13 consecutive patients with Lyme neuroborreliosis irrespective of stage of disease. The numbers were between 1/370 and 1/5,000 CSF cells (mean, 1/1,250 in the 13 patients). The highest numbers were encountered in two patients with severe signs of CNS involvement. The numbers decreased in parallel with clinical improvement after treatment. Anti-MBP IgG antibody-secreting cells were also observed in the CSF from patients with a variety of other inflammatory diseases of the nervous system, and their role in the development of tissue damage remains unsettled. Anti-MBP IgG antibody-secreting cells were not detected in the patients' blood, reflecting accumulation of this autoantibody response to CSF.

Antibodies to myelin components, such as myelin basic protein (MBP), may play a role in pathogenesis of multiple sclerosis (MS) but results from determinations of anti-MBP antibodies are inconsistent. Enumeration of cells secreting antibodies represents a new approach to evaluate a specific antibody response regarding extent and localization, and reduces effects of e.g. antibody binding to target. Anti-MBP IgG antibody secreting cells were present in MS patients' cerebrospinal fluid (CSF) at a mean value of 1 per 833 cells, and they amounted to a mean value of about 2454 in the whole CSF compartment. Similar numbers were encountered in patients with other inflammatory neurological diseases (OIND). During follow-up, anti-MBP IgG antibody secreting cells persisted regarding frequency and numbers in MS, but decreased in OIND. Such cells were rarely detected in patients with tension headache. No correlations to clinical exacerbation of MS, disability or duration were discernable. In blood from MS and OIND patients, anti-MBP IgG antibody secreting cells were detected infrequently and at low numbers. The anti-MBP antibody response is strongly restricted to the IgG isotype. The anti-MBP IgG antibody response which is persistent and compartmentalized to the diseased organ, may be important for the development of MS.

The B-cell response to myelin and myelin basic protein was studied in patients with multiple sclerosis and in patients with acute aseptic meningoencephalitis by using a nitrocellulose immunospot assay. This method allows detection of single cells producing antibodies. Twenty-seven (79%) of 34 patients with multiple sclerosis had cells producing IgG antibodies against myelin, and 11 (57%) of 19 had cells producing IgG antibodies against myelin basic protein in cerebrospinal fluid (CSF), with mean values of 30 and 14 per 10(4) mononuclear cells, respectively. Total numbers of IgG-producing cells occurred at a mean number of 75 per 10(4) CSF cells. Cells producing antimyelin or anti-myelin basic protein antibodies of IgA or IgM isotypes were rarely found in CSF. Patients with acute aseptic meningoencephalitis less frequently showed CSF cells producing IgG antibodies against myelin and myelin basic protein. No cells producing antibodies against myelin or myelin basic protein were detected in peripheral blood of patients with multiple sclerosis or meningoencephalitis. Thus, a majority of patients with multiple sclerosis had CSF cells that produced IgG antibodies against myelin and myelin basic protein. These cells comprised a large proportion of the total IgG-producing cells. A pronounced B-cell response against autoantigens produced at the target for immune attack might be important in the pathogenesis of multiple sclerosis.

Migration properties and occurrence of antibodies against myelin basic protein (MBP) in paired CSF and serum specimens from patients with multiple sclerosis (MS) were demonstrated after agarose isoelectric focusing, immunoblot transfer, and immunoperoxidase staining. Oligoclonal IgG antibody bands directed against MBP were found in the CSF of 9 of 28 patients with MS (32%), but not in the CSF of any of 34 patients with other neurologic diseases. No serum showed anti-MBP antibody bands. The CSF anti-MBP antibodies migrated to the anodal region of the IgG area in a different fashion from oligoclonal IgG and anti-measles IgG antibodies, which were detected in parallel. The anti-MBP bands were transient in three of seven patients whom we studied consecutively. Enzyme-linked immunosorbent assay (ELISA) of serum and CSF for detection of IgG reactivity against MBP showed absorbance values above 2 standard deviations of controls in 44% of the MS patients and in 21% of those with other neurologic diseases. Results of this assay correlated partly with those of the immunoblot assay. ELISA positive and immunoblot negative results might be due to a broad polyclonal anti-MBP antibody response.

Abnormal immune responses to myelin associated glycoprotein (MAG), a component of myelin of the central and peripheral nervous system, have been suggested to play a role in the pathogenesis of multiple sclerosis (MS) and certain types of inflammatory polyneuropathy. To identify possible immunodominant MAG peptides in neuroinflammation, we examined T and B cell responses to five selected synthetic MAG peptides and myelin proteins in 21 patients with non-inflammatory polyneuropathy, 26 patients with MS, 10 optic neuritis patients and 17 healthy subjects. Enzyme-linked immunosorbent spot-forming cell assays were adopted, allowing the detection and enumeration of individual antigen responsive T and B cells in body fluids. Patients with polyneuropathy as well as those with MS had elevated levels of T and B cells recognizing MAG and its peptides. Any of the five MAG peptides under study functioned as immunodominant T and/or B cell epitope in individual subjects. None of the MAG peptides elicited a specific disease-associated T or B cell response. The enhanced T and B cell response to myelin components like MAG may play some role in initiation and/or progression of these diseases, but they could also represent secondary responses associated with myelin damage and indicate tolerization rather than autoaggressive immunity. [ABSTRACT FROM AUTHOR]

We utilized a model of myelin basic protein (MBP) activation in vivo and MBP-stimulated cultures in vitro to study the influence of TGF-β1 on glial cell proliferation and ICAM-l/leucocyte function-associated antigen-1 (LFA-1) expression, and to observe the antagonistic effects of TGF-β1 and IFN-γ. TGF-β1 inhibited MBP-stimulated and MBP-activated glial cell proliferation, especially in MBP-stimulated separated microglia and astrocytes, and down-regulated the expression of ICAM-1 on MBP-stimulated glial cells and .separated microglia. ICAM-1 expression on MBP-activated glial cells was intensely suppressed, whereas its expression on MBP-stimulated astrocytes was not influenced. TGF-β1 had no effect on LFA-1 expression. In contrast, IFN-γ up-regulated ICAM-1 expression, but inhibited proliferative response on MBP-stimulated glial cells when cultured without TGF-Tbeta;1. Examination of TGF-β1 and IFN-γ interactions revealed that TGF-β1-mediated inhibition of proliferation and down-regulation of ICAM-1 on glial cells were prevented by IFN-γ. The suppressive effect was re-established with high doses of TGF-β1 in cultures, indicating that biological effects of TGF-β1 vary depending on nitric oxide (NO) production, its concentration in the microenvironment and regulation of the cytokine network. [ABSTRACT FROM AUTHOR]

Umbilical cord blood of newborns and peripheral blood of healthy adults were investigated by an immunospot assay for cells secreting IgG, IgA and IgM antibodies against myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) which represent putative antigens for an autoimmune attack in multiple sclerosis (MS) and against acetylcholine receptor (AChR) which is considered an important autoantigen in myasthenia gravis. Cells secreting antibodies against one or more of these autoantigens were detected in 18 out of 24 newborns, and in eight out of 20 adults. Eight of the cord blood samples contained cells secreting antibodies of IgG, IgA and/or IgM isotypes to one antigen, five to two antigens, two to three antigens, two to four antigens, and one to five antigens. Most prominent were anti-MBP IgG antibody secreting cells which were detected in 13 newborns at a mean number of 1/20 000 cord blood cells, and in six adults at a mean number of 1/105 peripheral blood cells. Anti-AChR IgG antibody secreting cells were detected in four out of 12 newborns versus four out of 14 peripheral blood specimens, at mean values of 1/5 cells in both instances. Cells secreting autoantibodies of IgA and IgM isotypes were less frequent both in cord blood and peripheral blood. The occurrence of nervous tissue autoantibody secreting cells in newborns must be related to a possible primary role of such autoantibodies in MS and myasthenia gravis. [ABSTRACT FROM AUTHOR]

We evaluated the B-cell response in cerebrospinal fluid (CSF) and blood by enumerating cells secreting antibodies to myelin-associated glycoprotein (MAG) and, for reference, to myelin basic protein (MBP), two myelin components which may constitute targets for autoimmune attack in multiple sclerosis (MS). Among 25 untreated MS patients, 12 had cells in CSF secreting anti- MAO IgG antibodies (mean value 1 per 1429 CSF cells) and three also had cells secreting anti- MAO antibodies of the IgM isotype but at lower levels. In CSF from 2 out of 10 MS patients examined, anti-MAO and anti-MBP IgO antibody-secreting cells were present concurrently. Antibody-secreting cells were less frequent in blood and bone marrow, reflecting compartmentalization to CSF. Anti-MAG antibody-secreting cells were found in CSF from only 1 out of 27 control patients. The intrathecal production of anti-MAG and anti-MBP antibodies may be important in the pathogenesis of MS. [ABSTRACT FROM AUTHOR]