Susceptibilidad de la cepa murina B10.RIII a padecer enfermedad inflamatoria alérgica pulmonar. Un modelo murino de asma / Susceptibility of B10.RIII mouse strain to develop inflammatory allergic pulmonary disease. A murine asthma model

El asma alérgica (AA) es una enfermedad inflamatoria crónica y compleja con múltiples fenotipos clínicos. Entre otras características destacan una obstrucción al flujo aéreo, una hiperreactividad bronquial y un componente inflamatorio eosinofílico y linfocítico con un patrón de citocinas similar al tipo Th2 murino. El uso de modelos de enfermedad que intentan imitar el AA en diferentes cepas de ratones genera más conocimientos acerca de los mecanismos fisiopatológicos con la finalidad de obtener nuevos y mejores tratamientos. Pero la susceptibilidad a desarrollar cambios pulmonares similares al asma difiere según las cepas de ratón. En este estudio se ha pretendido desarrollar un modelo murino de inflamación pulmonar experimental alérgica utilizando una cepa de ratón empleada frecuentemente en modelos experimentales de otras enfermedades. Métodos: Se inmunizaron ratones de la cepa B10.RIII con ovoalbumina (OVA) intraperitoneal y posteriormente se realizaron exposiciones repetidas de OVA a través de la vía aérea. Un día después de la última provocación se tomaron muestras de suero para la medición de los niveles de IgE total y específica, lavado broncoalveolar para la medición de citocinas, IgE total y la respuesta celular en la vía aérea, histología pulmonar para medición de infiltración eosinofílica parenquimatosa y se obtuvieron células mononucleadas de bazo para medir la capacidad de respuesta ante un estímulo in vitro. Resultados: En ratones B10.RIII se obtuvo una repuesta inflamatoria local en las vías aéreas y parénquima pulmonar similar a la de otras cepas (C57BL/6, C57BL/10) según estudios publicados previamente. Los resultados fueron confirmados con la presencia de títulos elevados de anticuerpos IgE y una respuesta proliferativa de esplenocitos tras la estimulación in vitro con OVA. Conclusiones: Estos resultados demuestran que en la cepa B10.RIII puede inducirse una inflamación pulmonar eosinofílica producida por OVA similar al AA. Este modelo podría ser utilizado para estudiar diferentes aspectos de la enfermedad así como nuevos tratamientos (AU)

Nasal administration of amyloid-beta peptide decreases cerebral amyloid burden in a mouse model of Alzheimer's disease.

Progressive cerebral deposition of amyloid-beta (Abeta) peptide, an early and essential feature of Alzheimer's disease (AD), is accompanied by an inflammatory reaction marked by microgliosis, astrocytosis, and the release of proinflammatory cytokines. Mucosal administration of disease-implicated proteins can induce antigen-specific anti-inflammatory immune responses in mucosal lymphoid tissue which then act systemically. We hypothesized that chronic mucosal administration of Abeta peptide might induce an anti-inflammatory process in AD brain tissue that could beneficially affect the neuropathological findings. To test this hypothesis, we treated PDAPP mice, a transgenic line displaying numerous neuropathological features of AD, between the ages of approximately 5 and approximately 12 months with human Abeta synthetic peptide mucosally each week. We found significant decreases in the cerebral Abeta plaque burden and Abeta42 levels in mice treated intranasally with Abeta peptide versus controls treated with myelin basic protein or left untreated. This lower Abeta burden was associated with decreased local microglial and astrocytic activation, decreased neuritic dystrophy, serum anti-Abeta antibodies of the IgG1 and IgG2b classes, and mononuclear cells in the brain expressing the anti-inflammatory cytokines interleukin-4, interleukin-10, and tumor growth factor-beta. Our results demonstrate that chronic nasal administration of Abeta peptide can induce an immune response to Abeta that decreases cerebral Abeta deposition, suggesting a novel mucosal immunological approach for the treatment and prevention of AD.

The role of CD154-CD40 versus CD28-B7 costimulatory pathways in regulating allogeneic Th1 and Th2 responses in vivo.

We used signal transducer and activator of transcription 4 (STAT4) and STAT6 gene knockout (-/-) mice as recipients of fully mismatched cardiac allografts to study the role of T-cell costimulatory pathways in regulating allogeneic T-helper 1 (Th1) versus Th2 responses in vivo. STAT4(-/-) mice have impaired Th1 responses, whereas STAT6(-/-) mice do not generate normal Th2 responses. Cardiac allografts from C57BL/6 mice were transplanted into normal wild-type (WT), STAT4(-/-), and STAT6(-/-) BALB/c recipients. STAT4(-/-) and STAT6(-/-) mice rejected their grafts with the same tempo as untreated WT recipients. CD28-B7 blockade by a single injection of CTLA4Ig induced long-term engraftment and donor-specific tolerance in all three groups of recipients. CD154 blockade by a single injection of MR1 was effective in prolonging allograft survival and inducing tolerance in STAT4(-/-) mice but was only marginally effective in STAT6(-/-) recipients and WT controls. In addition, a similar protocol of MR1 was ineffective in prolonging graft survival in CD28(-/-) BALB/c recipients, suggesting that the lack of efficacy seen in WT and STAT6(-/-) mice is not due to the presence of a functional CD28-B7 pathway. Furthermore, there was a similar differential effect of CD28-B7 versus CD154-CD40 blockade in inhibiting immune responses in animals immunized with ovalbumin and complete Freund's adjuvant. These novel data indicate that Th1 and Th2 cells are differentially regulated by CD28-B7 versus CD154-CD40 costimulation pathways in vivo and may have potential implications for the development of therapeutic strategies such as T-cell costimulatory blockade in humans.

Role of passive T-cell death in chronic experimental autoimmune encephalomyelitis.

The mechanisms of chronic disease and recovery from relapses in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, are unknown. Deletion of myelin-specific lymphocytes by apoptosis may play a role in termination of the inflammatory response. One pathway of apoptosis is the passive cell death or "cell death by neglect" pathway, which is under the control of the Bcl family of genes. To investigate the role of passive cell death pathway in EAE, we used mice with transgenic expression of the long form of the bcl-x gene (Bcl-x(L)) targeted to the T-cell lineage. We found that mice transgenic for Bcl-x(L) have an earlier onset and a more chronic form of EAE induced by myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 compared with wild-type littermate mice. This was not due to an expanded autoreactive cell repertoire. Primed peripheral lymphocytes from Bcl-x(L) transgenic mice showed increased proliferation and cytokine production to MOG peptide in vitro compared with lymphocytes from wild-type animals. Immunohistologic studies demonstrated increased cellular infiltrates, immunoglobulin precipitation, and demyelination in the Bcl-x(L) transgenic central nervous system (CNS) compared with controls. There was also a decreased number of apoptotic cells in the CNS of Bcl-x(L) transgenic mice when compared with littermates at all time points tested. This is the first report of an autoimmune disease model in Bcl-x(L) transgenic mice. Our data indicate that the passive cell death pathway is important in the pathogenesis of chronic EAE. These findings have implications for understanding the pathogenesis of multiple sclerosis and other autoimmune diseases.

Costimulatory signal blockade in murine relapsing experimental autoimmune encephalomyelitis.

Blockade of the CD28-B7 or CD40L-CD40 T cell costimulatory signals prevents induction of experimental autoimmune encephalomyelitis (EAE). However, the effect of simultaneous blockade of these signals in EAE is unknown. We show that administration of either MR1 (to block CD40L) or CTLA4Ig (to block B7) after immunization or after the first attack protects from EAE. Treatment with a combination of CTLA4Ig and MR1 provides additive protection, and is associated with complete absence of mononuclear cell infiltrates in the central nervous system, and marked suppression of proliferation of primed T cells in the periphery. Selective B7-1 blockade did not protect from EAE. These observations have implications for therapy of autoimmune diseases.

Acquired thymic tolerance: role of CTLA4 in the initiation and maintenance of tolerance in a clinically relevant autoimmune disease model.

Injection of Ag into the thymus of adult animals induces specific systemic tolerance. The mechanisms of acquired thymic tolerance include anergy and the deletion of Ag-specific T cells. Here, we report that anergy to nominal Ag induced via acquired thymic tolerance requires CTL-associated Ag 4 (CTLA4) engagement. The role of CTLA4 in the induction and maintenance of tolerance was then investigated in the murine experimental autoimmune encephalomyelitis model. CTLA4 blockade abrogated the induction but not the maintenance phase of acquired thymic tolerance induced by intrathymic injection of myelin Ags. In addition, CTLA4 blockade had a restricted window of action after priming with Ag, which is consistent with the expression patterns of CTLA4 in vivo. We conclude that: 1) the induction of acquired thymic tolerance requires signaling through CTLA4 and 2) CTLA4 does not appear to be required for the maintenance of acquired thymic tolerance. This is the first report documenting the role of a CTLA4 negative signaling pathway in the induction of tolerance in an autoimmune disease model.

Kinetics of expression of costimulatory molecules and their ligands in murine relapsing experimental autoimmune encephalomyelitis in vivo.

We studied the kinetics of expression of costimulatory molecules and cytokines in the central nervous system (CNS) in murine relapsing experimental autoimmune encephalomyelitis (EAE). During the natural course of EAE, B7-2 expression in the CNS correlated with clinical signs, while B7-1 was exclusively expressed during remissions. Interestingly, B7-1 was expressed on infiltrating mononuclear cells as well as neuronal cells in the CNS. In the periphery, B7-1 expression on APCs peaked with clinical disease but decreased on T cells. CD28 and CTLA4 molecules, the two known ligands for B7-1 and B7-2, had distinct expression patterns in the CNS; CD28 was highly expressed and correlated with B7-2 expression on APCs (macrophages/microglia as well as astrocytes) and with the clinical signs of EAE. CTLA4, on the other hand, was expressed by substantially fewer cells during the effector phase of disease and peaked during remission, which is consistent with the emerging role of this molecule in the termination of immune responses. The expression of CD40 and CD40L in the CNS was increased during clinical attacks. The expression of IL-12, IFN-gamma, and TNF-alpha correlated with disease activity and severity, while TGF-beta was the only factor that was up-regulated during the recovery phase. Interestingly, TGF-beta was also expressed by neurons during remission. This is the first study demonstrating the kinetics of the in vivo expression of costimulatory molecules, their ligands, and cytokines in an autoimmune disease model characterized by remissions and relapses. Our data suggest that the targeting of costimulatory molecules to block an immune response must take into account the expression patterns in the target organ.

Elevated interleukin-12 in progressive multiple sclerosis correlates with disease activity and is normalized by pulse cyclophosphamide therapy.

Multiple sclerosis is postulated to be a Th1-type cell-mediated autoimmune disease. We investigated cytokine profiles in patients with progressive multiple sclerosis by using intracytoplasmic staining. We found increased IL-12 production by monocytes and increased IFN-gamma production by T cells in untreated patients as compared with controls. In patients treated with methotrexate, methylprednisolone, or cyclophosphamide/methylprednisolone (CY/MP), only CY/MP treatment normalized the elevated IL-12 production. Furthermore, CY/MP-treated patients had decreased IFN-gamma and increased IL-4, IL-5, and TGF-beta expression. Patients followed prospectively before and after starting CY/MP treatment showed a gradual decrease in IL-12 and IFN-gamma production and an increase in IL-4 and IL-5. In vitro, addition of 4-hydroperoxycyclophosphamide, a metabolite of cyclophosphamide decreased IL-12 production in mononuclear cell cultures. When patients were classified as having active or stable disease, IL-12 production correlated with disease activity. In summary, our results demonstrate a Th1-type cytokine bias in peripheral blood mononuclear cells of untreated progressive MS patients that is reversed by CY/MP treatment and is associated with Th2 and TGF-beta (Th3) type responses. These findings provide a basis for immune monitoring of patients with MS and suggest that treatments that downregulate IL-12 may prove to be beneficial in progressive MS.

Genetic influence on disease course and cytokine response in relapsing experimental allergic encephalomyelitis.

A protracted and relapsing form of experimental allergic encephalomyelitis (EAE) develops in the DA rat after immunization with rat spinal cord homogenate (SCH) emulsified in incomplete Freund's adjuvant (IFA). The genetic influence on this model has been analyzed by immunizing MHC congenic strains on both LEW and DA genetic backgrounds, and recombinant inbred strains between DA and E3 rats. An in situ hybridization assay was used to examine the expression of mRNA for IFN-gamma, IL-4, IL-10 and transforming growth factor (TGF)-beta both in sections of spinal cords and the antigen-induced expression for these cytokines by splenocytes after in vitro stimulation with encephalitogenic MBP peptides. The susceptibility of relapsing EAE after immunization with SCH in IFA in the DA strain, but not the E3 strain, was correlated with a lack of expression for TGF-beta in the spinal cord. The recombinant inbred DXEB rats developed a severe EAE while surprisingly no signs of disease were observed in the DXEA strain, which shares the MHC region with the DXEB strain, after immunization with the MBP 63-87 peptide. Resistance to relapsing EAE in the DXEA strain correlated with increased non-MHC controlled expression for TGF-beta and lack of IFN-gamma in the spinal cord. The same pattern of cytokine expression was seen in splenocytes after stimulation in vitro with the MBP 63-87 peptide. A spreading of the immune response to the MBP 87-110 peptide was seen. Non-MHC genes controlled the quality of this response: splenocytes from MBP 63-87 immunized DXEB rats responded in vitro towards the MBP 87-110 peptide by expressing mRNA for IFN-gamma, IL-10 and IL-4, whereas in the DXEA strain the corresponding response involved IL-4 and TGF-beta. Taken together these data show that non-MHC controlled expression of mRNA for TGF-beta is associated with resistance to EAE.

Genetic analysis of inflammation, cytokine mRNA expression and disease course of relapsing experimental autoimmune encephalomyelitis in DA rats.

Genetic analysis of experimental autoimmune encephalomyelitis (EAE) can provide clues to the etiology of multiple sclerosis (MS). Identifying the susceptibility genes of DA rats may be particularly rewarding since they are prone to develop a remarkably MS-like chronic and demyelinating disease. As a first step in this direction, we investigated the role of DA genes within and outside the major histocompatibility complex (MHC) for susceptibility to severe protracted and relapsing EAE (SPR-EAE). This form of EAE developed in DA rats but not in LEW. ACI and BN rats after immunization with syngeneic spinal cord and complete Freund's adjuvant. Studies of crosses between DA and BN rats revealed that non-MHC genes determine susceptibility to SPR-EAE. A role for MHC-genes was also established using MHC-congenic rat strains, in which the DA MHC haplotype (av1) associated with relapsing EAE. Again, non-MHC genes were decisive since a high incidence of SPR-EAE only occurred in rats with DA non-MHC genes. Analysis of cytokine mRNA expression and infiltrating cells in the spinal cords of congenic strains revealed that the av1 haplotype associated with a high CD4/CD8 ratio and expression of mRNA for interferon-gamma (IFN-gamma), but not for transforming growth factor-beta (TGF-beta) or interleukin-10 (IL-10). In contrast, the other MHC haplotypes (h, l, u) associated with low CD4/CD8 ratios and mRNA expression for TGF-beta and IL-10, but not for IFN-gamma. DA non-MHC genes determined the intensity of inflammation since the number of cells expressing MHC class II, CD4 and interleukin-2 receptor (IL-2R) was higher in DA rats than in LEW.1AV1 and PVG.1AV1 rats which also carry the av1 haplotype. We conclude that the MHC haplotype of DA rats favors a prolonged proinflammatory autoimmune response associated with relapses, while the DA background intensifies inflammation correlating with a high incidence of relapsing disease.

Differential effects of B7-1 blockade in the rat experimental autoimmune encephalomyelitis model.

Blocking the CD28-B7 T cell costimulatory activation pathway protects animals from developing experimental autoimmune encephalomyelitis (EAE). In the mouse EAE model, selective blockade of B7-1 by specific mAbs has been shown to protect animals from EAE. In the Lewis rat model, we have shown that CD28-B7 blockade by systemic administration of CTLA4Ig prevents actively induced EAE. Since CTLA4Ig binds to both B7-1 and B7-2, we used a mutant form of CTLA4Ig (CTLA4IgY100F) that binds only B7-1, to study the role of B7-1 blockade in this model. Such a reagent avoids the potential of signaling by mAbs. Systemic administration of CTLA4IgY100F in several dosing regimens did not protect from EAE, and in some protocols worsened disease, while CTLA4Ig was always protective. In contrast, systemic injection of APCs preincubated ex vivo with the encephalitogenic peptide of myelin basic protein and either CTLA4Ig or CTLA4IgY100F protected recipients from disease. In vitro studies confirmed the in vivo observations and showed that primed lymph node cells from protected animals had decreased proliferative responses to myelin basic protein as compared with controls, while lymphocytes from animals treated with systemic CTLA4gY100F did not. More importantly, systemic administration of CTLA4IgY100F abrogated the protective effect of ex vivo treated APCs. These data suggest an important regulatory role for B7-1, perhaps through binding to CTLA4, in this model of EAE. Understanding the role and mechanisms of selective blockade of costimulatory molecules has implications for therapy of autoimmune disease.

In vivo mechanisms of acquired thymic tolerance.

Injection of antigen into the thymus of adult animals induces specific systemic tolerance, but the mechanisms of acquired thymic tolerance are not well understood. To investigate these mechanisms we used a model of intrathymic injection of ovalbumin (OVA) in BALB/c mice. We show an antigen-specific decrease in proliferative responses to OVA, as well as a significant decrease in antigen-specific IL-2 secretion and IFN-gamma production by splenocytes and lymph node cells of tolerant mice. Addition of recombinant IL-2 in vitro reversed the defect in IFN-gamma production by cells from OVA-tolerized animals, but did not reverse the proliferation or IL-2 production defects. By using an adoptive transfer system, where a small population of OVA peptide-specific CD4+ TCR transgenic T cells are transferred into syngeneic normal recipients, we show an absence of peripheral antigen-dependent clonal expansion of transferred CD4+ TCR transgenic cells in tolerant mice in vivo. There was an increase in clonotype-positive T cells in the thymus after immunization, confirming that activated T cells circulate through the thymus. Furthermore, thymectomy after intrathymic injection abrogates the effect of acquired thymic tolerance and restores antigen-dependent clonal expansion in vivo. We conclude that intrathymic injection of antigen induces Th1 cell unresponsiveness and prevents the peripheral expansion of antigen-specific CD4(+) T cells in vivo. This is the first demonstration that in acquired thymic tolerance antigen-specific T cells circulate to the thymus where they may be anergized or ultimately deleted.

Major histocompatibility complex-controlled protective influences on experimental autoimmune encephalomyelitis are peptide specific.

The myelin basic protein (MBP) peptide 63-88-induced experimental autoimmune encephalomyelitis (EAE) and its associated T cell cytokine profile are influenced by the rat major histocompatibility complex (MHC). There is an allele-specific protective influence of the MHC class I region, whereas the MHC class II region display either disease-protective or -promoting effects. To investigate if the MHC-associated protection is dependent on certain combinations of MBP peptide and MHC molecules, we have now used another peptide (MBP 89-101). A broader and different set of rat MHC alleles were associated with EAE induced with MBP 89-101 as compared to MBP 63-88. All EAE-susceptible strains mounted peptide-specific strong T helper (Th) 1-like immune responses in vitro. Immunization of rats with an extended peptide (MBP 87-110) induced EAE associated with the same MHC haplotypes as the 89-101 peptide, except in LEW.1N (RT1 pi) rats which were relatively resistant. Only this strain responded with additional Th2-like and transforming growth factor-beta responses to the peptide in vitro. In vivo depletion of CD8+ cells aggravated the disease in this strain. We conclude that both MHC-controlled promoting and protective influences on EAE are dependent on certain MHC/MBP peptide combinations, and that the 87-110 region of MBP contains a major MHC-associated encephalitogenic epitope in the rat.

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-beta.

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-gamma, interleukin-12 (IL-12), tumour necrosis factors alpha and beta, IL-1 beta 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, CD8 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-beta (TGF-beta), 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.

Dynamics of mRNA expression of interferon-γ, interleukin 4 and transforming growth factor ß1 in sciatic nerves and lymphoid organs in experimental allergic neuritis.

Experimental allergic neuritis (EAN) is a T cell mediated inflammatory demyelinating disorder of the peripheral nervous system (PNS) and an animal model of the Guillain-Barré syndrome. Cytokines including interferon-γ (IFN-γ) have previously been shown to be upmodulated in lymphoid organs and assumed to be involved in the pathogenesis of EAN. Cytokines in the target organ for autoaggressive immunity in EAN, the PNS, could be pivotal for the development of EAN. By adopting in situ hybridization, we studied mRNA expression of the T helper 1 (Th1) cell associated IFN-γ, the Th2 cell related interleukin-4 (IL-4) and the immune response down-regulating TGF-ß1 in the sciatic nerve and, in parallel, in the lymph nodes and the spleen over the course of EAN actively induced by immunization with bovine peripheral nerve myelin (BPM) and Freund's complete adjuvant. The dynamics of IFN-γ mRNA expression in the sciatic nerve followed approximately the clinical course of EAN with peak values around day 14 post immunization (p.i.), whereas IFN-γ was transcribed earlier in the spleen and lymph nodes with maximum on day 7 p.i. In contrast, transcription of IL-4 was only slightly enhanced in EAN, with minor fluctuations in the sciatic nerve peaking on days 11 and 28 p.i. In the lymph nodes, the highest numbers of TGF-ß mRNA positive cells were observed during the clinical improvement of EAN. The data argue for a major proinflammatory role for IFN-γ, and a disease down-regulating function for TGF-ß at the target site in EAN.

Protracted, relapsing and demyelinating experimental autoimmune encephalomyelitis in DA rats immunized with syngeneic spinal cord and incomplete Freund's adjuvant.

Experimental autoimmune encephalomyelitis (EAE) is a model for multiple sclerosis (MS). However, MS is a chronic, relapsing and demyelinating disease, whereas EAE in rats is typically a brief and monophasic disorder showing little demyelination. We demonstrate here that DA rats develop severe, protracted and relapsing EAE (SPR-EAE) after a subcutaneous immunization at the tail base with syngeneic spinal cord and incomplete Freund's adjuvant (IFA). The neurological deficits were accompanied by demyelinating inflammatory lesions in the spinal cord, with infiltrating T lymphocytes and perivascular deposition of immunoglobulins and complement. The induction of SPR-EAE was associated with humoral autoreactivity to myelin oligodendrocyte glycoprotein (MOG) and cellular autoreactivity to the rat myelin basic protein (MBP) peptides 69-87 and 87-101. These two peptides, as well as whole rat MBP, were encephalitogenic. In conclusion, we believe that the presently described demyelinating SPR-EAE represents a useful model for 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 alpha and tumor necrosis factor beta.

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)-beta (= lymphotoxin-alpha) and cytolysin appeared early and before onset of clinical signs of EAE; (ii) TNF-alpha 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-gamma (IFN-gamma) 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-gamma. The TNF-beta mRNA expression prior to onset of clinical signs favours a role for this cytokine in disease initiation. A pathogenic effector role of TNF-alpha was suggested from these observations that TNF-alpha 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.

Interferon gamma, interleukin 4 and transforming growth factor beta in experimental autoimmune encephalomyelitis in Lewis rats: dynamics of cellular mRNA expression in the central nervous system and lymphoid cells.

The potential role of certain important immunoregulatory and effector cytokines in autoimmune neuroinflammation have been studied. We have examined the expression of mRNA, with in situ hybridization, of interferon gamma (IFN-gamma), interleukin 4 (IL-4) and transforming growth factor beta (TGF-beta) both in sections of spinal cords and the antigen-induced expression of these cytokines by lymphoid cells after stimulation with a dominant encephalitogenic peptide of MBP (MBP 63-88) during the course of actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In spinal cords, the target organ in EAE, cells expressing mRNA for IFN-gamma, first appeared at the onset of clinical signs, i.e., day 10 postimmunization (p.i.), peaked at the height of disease (day 13 p.i.) and then gradually decreased concomitant with recovery. Very few IL-4 mRNA-expressing cells appeared in the spinal cord with no clear relation to clinical signs or histopathology. In contrast, expression of mRNA for TGF-beta did not increase until day 13 p.i., at height of the disease, shortly preceding recovery. These data are consistent with a disease upregulating role of IFN-gamma, while TGF-beta may act to limit central nervous system (CNS) inflammation. In lymphoid organs, primed MBP 63-88 reactive T cells showed an interesting time-dependent evolution of their cytokine production in vitro. Thus, early after immunization there was a conspicuous MBP 63-88-induced production of both IFN-gamma and IL-4. Such cells may act in the initiation and promotion of the disease. Later, in the recovery phase, MBP 63-88 induced lymphoid cells to TGF-beta production. Thus, an autoantigen-specific production of TGF-beta occurred during EAE and hypothetically such a mechanism may serve to downregulate aggressive autoimmunity systemically.

Protective influences on experimental autoimmune encephalomyelitis by MHC class I and class II alleles.

Experimental autoimmune encephalomyelitis (EAE) is influenced by polymorphism of the MHC. We have previously found that Lewis rats with certain MHC haplotypes are susceptible to disease induced with the myelin basic protein (MBP) peptide 63-88, whereas Lewis rats with other MHC haplotypes are resistant. Interestingly, rats with the MHC u haplotype develop an immune response to the MBP 63-88, but do not get EAE. In this study we have used intra-MHC recombinant rat strains to compare the influences of the MHC u with the a haplotype. We discovered the following: 1) The class II region of the MHC a haplotype permits EAE and a Th1 type of immune response as measured by IFN-gamma production after in vitro challenge of in vivo-primed T cells with MBP 63-88. 2) The class II region of the u haplotype is associated with a disease-protective immune response characterized by production of not only IFN-gamma, but also of IL-4 mRNA expression by the MBP 63-88-activated cells. 3) The class I region upstream of the class II region of the u haplotype is associated with a disease-protective effect and the expression of mRNA for TGF-beta after MBP 63-88-induced activation. Thus, such a TGF-beta response occurs in all strains expressing the class I Au allele. Treatment with Abs to CD8+ cells abrogates peptide-induced TGF-beta mRNA expression, and aggravates disease in strains with the class I Au allele.