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Interleukin 17 (IL-17), produced by T cells, plays an important role in Multiple Sclerosis (MS) and its animal model, Experimental Autoimmune Encephalomyelitis (EAE). In contrast to IL-17-producing CD4+ T cells, the contribution of IL-17-producing CD8+ T cells (Tc17) in CNS autoimmunity has been investigated less intensively. Here we investigate the role of TC17 in EAE. We compare different T cell populations and their cytokine pattern in the MOG35-55- and MOG37-50-induced EAE. We detected a similar cytokine phenotype for both EAE models in the autoimmune process assessed at different stages. Regarding the migratory activity, an involvement of IL-17 and IFN-γ in disease onset was suggested. Furthermore, we show that PAMPs have the ability to drive autoimmune process. To modify the cytokine pattern of different T cell populations, a combination of distinct factors is required (the activation of MyD88 or Syk, the genetic background, the presence of APCs and CD4+ T cells)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Infections with Pseudomonas aeruginosa may cause many different diseases. The spectrum of such infections in general includes inflammation and bacterial sepsis. Hospital-acquired pneumonia, naturally resistant to a wide range of antibiotics, is associated with a particularly high mortality rate in mechanically ventilated patients. The pathogenesis of P. aeruginosa is complex and mediated by several virulence factors, as well as cell-associated factors. We have previously demonstrated that stimulation with different bacteria triggers the cytokine response of thymocytes. In this study, we investigated the effect of P. aeruginosa and its different components on the cytokine production of immature and mature immune cells. We found that the induced cytokine pattern in the thymus and the spleen after infections with P. aeruginosa is primarily mediated by lipopolysaccharide (LPS) of the outer cell membrane, but other components of the bacterium can influence the cytokine secretion as well. Stimulation with heat-killed P. aeruginosa and LPS does not influence the amount of cytokine-producing CD4(+) T cells but instead suppresses the emergence of Th17 cells. However, stimulation with P. aeruginosa or its components triggers the interleukin-17 (IL-17) response both in thymocytes and in splenocytes. We conclude that infections with P. aeruginosa affect the cytokine secretion of immature and mature cells and that IL-17 and Th17 cells play only a minor role in the development of pathological systemic inflammatory disease conditions during P. aeruginosa infections. Therefore, other inflammatory immune responses must be responsible for septic reactions of the host., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The endoplasmic reticulum (ER) serves as the major intracellular Ca(2+) store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca(2+) leak channel also implicated in the response against protein misfolding, thereby connecting the Ca(2+) store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca(2+) levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca(2+) levels, suggesting an exhausted mitochondrial Ca(2+) buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca(2+) homeostasis in lymphocytes.

Interleukin (IL)-17-producing T cells play a critical role in the immune response against microbial pathogens. Traditionally, experimental studies have focused upon understanding the activity of IL-17-producing T cells which differentiate from naive T cells in the peripheral immune system. However, we have demonstrated previously that IL-17-producing T cells are also present in the thymus of naive wild-type mice and can be co-activated there by microbial stimuli. Other studies have supported the concept that IL-17-producing thymocytes have a specific role in the immediate defence against microbial pathogens, which is independent from the development of an adaptive immune response. Given an important role of the thymus in systemic bacterial infection and sepsis, in this study we investigate the effect of a broad spectrum of bacteria and cell wall components on thymocyte cytokine production. Surprisingly, we find that all types of bacteria investigated (including non-pathogenic species) uniformly activate IL-17-producing thymocytes upon α-CD3 stimulation. In contrast, there is a heterogeneous effect on IL-6 and interferon (IFN)-γ-production with Gram-negative bacteria inducing far higher frequencies of IL-6- and IFN-γ-producing thymocytes than Gram-positive bacteria. We conclude that IL-17-producing thymocytes constitute a 'first line of recognition', but not a 'first line of defence' against bacteria in general. Their activity might lead to immune activation, but not necessarily to a pathological inflammatory disease condition. The difference between these two states might be determined by other immunological effector molecules, such as IL-6 and IFN-γ., (© 2014 British Society for Immunology.)

IL-17-producing thymocytes have been recently described and are believed to play a role as an immune cell population which is able to react against microbial components rapidly. For this reason, we here investigated the ability of two microbial stimulants, bacterial flagellin (a ligand for TLR5) and diphtheria toxin from Corynebacterium diphtheriae, to activate or co-activate (together with α-CD3 stimulation) thymocyte cytokine production. We find that both bacterial molecules do not induce cytokine-production by themselves, but co-activate IL-17-producing thymocytes together with α-CD3. Since diphtheria toxin is unlikely to affect mouse cells through the same mechanism as the lethal effect on human cells, our results point to an additional mechanism of diphtheria toxin to act on immune cells. However, there is no additive or super-additive effect after stimulation with diphtheria toxin combined with flagellin and α-CD3 co-activation, which suggests that microbial stimuli used in this study can only activate a limited number of IL-17 producing thymocytes., (Copyright © 2013. Published by Elsevier Ltd.)

An emerging role is postulated for IL-17-producing thymocytes, which in their majority consist of IL-17-producing CD4(+) cells. For these, a specific role in the immediate defense against infectious pathogens is suggested, independent from the development of an adaptive immune response in the immune periphery. Immune response modifiers, like the TLR7 ligands Imiquimod and Gardiquimod™ are effective pharmacological therapeutics applied topically against dermal tumors and virus infections and have been demonstrated to activate immune cells. In this study, we investigated the effect of Imiquimod and Gardiquimod™ on murine thymocyte cytokine production with a particular focus on IL-17. We find that both substances dose-dependently are able to trigger IFN-γ and IL-6 production, but no IL-17 production. Moreover, a strong co-stimulating effect is detected on α-CD3-induced IFN-γ, IL-6 and IL-17 production. We conclude that Imiquimod and Gardiquimod™ are not only modifiers of the adaptive immune response, but might also have additional therapeutic potential by modifying the immune activity in the thymus., (© 2013.)

Antidepressant drugs, in particular those targeting the serotonin (5-HT)-reuptake system via the serotonin transporter (5-HTT), are known to exhibit antiinflammatory properties and have demonstrated therapeutic efficacy in rodent models of autoimmune disease like experimental autoimmune encephalomyelitis or experimental rheumatoid arthritis. A crucial difference between animal models and the actual human autoimmune disease is the fact that in animals predominantly induced T cells are studied after sensitization with autoantigen. In humans, however, naturally occurring cytokine-producing T cells might play a significant role as well. For this reason, we investigated the effect of the selective serotonin reuptake inhibitor citalopram on cytokine-producing cells in the thymus of C57BL/6 mice, focusing on the (predominantly) T-cell-produced cytokines IL-2, IL-4 and IL-17. Citalopram was able to strongly reduce the frequency of IL-4- and IL-2-producing cells triggered by CD3 stimulation, but exhibited a less pronounced effect on IL-17-producing cells. 5-HTT expression was found to be very low in thymocytes in comparison with splenocytes, and the effect of free extracellular serotonin on CD3-induced thymocyte cytokine production did not mimic the effect of citalopram. We conclude that citalopram has a potent suppressive effect on cytokine production in the thymus, and that this effect is unlikely to be mediated by elevation of extracellular serotonin levels via the 5-HTT., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Proinflammatory cytokines are essential mediators of the immunopathology associated with microbial sepsis. The fungal cell wall component zymosan and bacterial DNA are well-studied experimental tools for investigating these processes, simulating the presence of fungal or bacterial infection. Cells of the immune periphery, but also immune cells in the thymus, are affected essentially by the presence of microbes or their immune stimuli in sepsis. For this reason, we investigated the cytokine pattern present in the spleen (containing mature immune cells) and the thymus (containing immature immune cells) upon exposure to zymosan and Escherichia coli DNA. To study the role of T cell activation status, we investigated ex-vivo cultures with and without αCD3 stimulation for changes in their cytokine secretion pattern as measured by cytokine enzyme-linked immunospot (ELISPOT) and flow cytometry analysis. We found that both substances strongly co-stimulate αCD3-induced interferon (IFN)-γ and interleukin (IL)-6 secretion in the thymus and in the spleen, but stimulate IL-17 production only moderately. Moreover, zymosan increases PLP peptide (PLPp)-specific IFN-γ and IL-6 production in experimental autoimmune encephalomyelitis (EAE) induced in Swiss Jim Lambert (SJL)/J mice, confirming that T cell activation status is crucial for the cytokines secreted by an immune cell population encountering a microbial pathogen or immunostimulating parts of it., (© 2012 British Society for Immunology.)

Background: Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate is a promising novel oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. These effects are presumed to originate from a combination of immunomodulatory and neuroprotective mechanisms. We aimed to clarify whether neuroprotective concentrations of dimethyl fumarate have immunomodulatory effects., Findings: We determined time- and concentration-dependent effects of dimethyl fumarate and its metabolite monomethyl fumarate on viability in a model of endogenous neuronal oxidative stress and clarified the mechanism of action by quantitating cellular glutathione content and recycling, nuclear translocation of transcription factors, and the expression of antioxidant genes. We compared this with changes in the cytokine profiles released by stimulated splenocytes measured by ELISPOT technology and analyzed the interactions between neuronal and immune cells and neuronal function and viability in cell death assays and multi-electrode arrays. Our observations show that dimethyl fumarate causes short-lived oxidative stress, which leads to increased levels and nuclear localization of the transcription factor nuclear factor erythroid 2-related factor 2 and a subsequent increase in glutathione synthesis and recycling in neuronal cells. Concentrations that were cytoprotective in neuronal cells had no negative effects on viability of splenocytes but suppressed the production of proinflammatory cytokines in cultures from C57BL/6 and SJL mice and had no effects on neuronal activity in multi-electrode arrays., Conclusions: These results suggest that immunomodulatory concentrations of dimethyl fumarate can reduce oxidative stress without altering neuronal network activity.

Interleukin (IL-)17 is a potent proinflammatory cytokine for which an important role in the immune response against infections and in autoimmune diseases has been demonstrated. Recently, it has been shown that - in addition to mature T cells which are primed in the immune periphery - this cytokine can also be produced by T cells in the thymus, so-called naturally occurring IL-17-producing T cells (nT17 cells). In this study we demonstrate that the generation and activation of nT17 cells in the thymus do not depend on the cytokine IL-6. In addition, nT17 cells are not regulated by IL-2. These properties of nT17 cells significantly differ from induced IL-17-producing T cells primed in the immune periphery (iT17 cells). Given the strong association of IL-17-producing T cells with immune responses against infections and human autoimmune diseases, closer characterization of nT17 cells is warranted., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Experimental autoimmune encephalomyelitis (EAE) is--in certain aspects--regarded as an animal model of the human CNS autoimmune disease multiple sclerosis (MS). While in EAE CNS-autoantigen-specific immunity is induced in a defined way, the initial processes leading to CNS autoimmunity in humans are so far unknown. Despite essential restrictions, which exist regarding the interpretation of EAE data towards MS, EAE might be a useful model to study certain basic aspects of CNS autoimmunity. Studies in MS have demonstrated that established autoimmune pathology can be critically influenced by environmental factors, in particular viral and bacterial infections. To investigate this interaction, EAE as an instrument to study CNS autoimmunity under defined conditions appears to be a suitable experimental tool. For this reason, we here investigated the influence of the Toll-like-receptor (TLR) ligand CpG oligonucleotide (CpG) on already established CNS autoimmunity in murine proteolipid protein (PLP)-induced EAE in SJL mice. CpG were found to co-stimulate PLPp-specific IFN-γ production in the peripheral immune system and in the CNS. However, CpG induced Interleukin (IL)-17 production in the inflamed CNS both alone and in combination with additional PLPp stimulation. These findings might indicate a mechanism by which systemic infections and the microbial stimuli associated with them may influence already existing CNS autoimmune pathology., (© 2011 Smolianov et al; licensee BioMed Central Ltd.)

Systemic administration of Pertussis toxin (PTX) abrogates T cell tolerance mediated by injection of neuroantigens in incomplete Freund's adjuvant (IFA) and causes experimental autoimmune encephalomyelitis (EAE). PTX concomitantly induces high frequencies of neuroantigen-specific IFN-gamma- and IL-17-producing T cells. Both IL-17 and IFN-gamma have been implicated as a key effector cytokines in the pathogenesis of EAE, possibly with different functions. We therefore investigated potential differences in the temporal and spatial kinetics of the PTX-induced neuroantigen-specific IFN-gamma- and IL-17-producing T cell effector populations. IFN-gamma- and IL-17-producing PLPp-specific T cells initially arose in comparable frequencies in the local draining lymph nodes (drLN) after immunization as measured by cytokine ELISPOT. High frequencies of both IFN-gamma- and IL-17-producing T cells were present in the immune periphery before onset of EAE. The highest frequencies of PTX-induced IFN-gamma- and IL-17-producing PLPp-specific cells coincided in the inflamed CNS during acute EAE. During recovery, both IFN-gamma- and IL-17-producing PLPp-specific T cells simultaneously disappeared from the CNS, whereas high frequencies of these cells remained present in the immune periphery. The functional affinity of both IFN-gamma- and IL-17-producing T cells did not change during EAE. Therefore, autoimmune pathology in this model did not correlate with specific PTX effects either on Th1 or Th17 cells regarding their kinetics and CNS migration., (Copyright 2010. Published by Elsevier Ireland Ltd.)

Multiple sclerosis (MS) is regarded as a Tcell mediated autoimmune disease of the central nervous system (CNS). It has been traditionally seen as a Th1-mediated autoimmune disease, a notion which has been largely supported by studies in its animal model, experimental autoimmune encephalomyelitis (EAE). However, evidence has accumulated in recent years that a newly described lineage of (autoantigen-specific) T-cells, which are characterized by the production of the inflammatory cytokine interleukin 17 (Th17) cells, might be the relevant effector cell population instead. This is supported by studies both in MS patients and in the EAE mouse model. Research in this field currently centres on the endogenous factors which are required for the priming and expansion of CNS autoantigen specific Th17 cells as well as on exogenous factors which trigger Th17 differentiation and activation. This review tries to provide an overview of the relevant literature and to summarize the current body of evidence on the role of Th17 cells in CNS autoimmune disease.

Experimental autoimmune encephalomyelitis (EAE) is commonly regarded as an animal model of the human disease multiple sclerosis (MS). Pertussis toxin (PTX) is routinely used for EAE induction in mice. Besides opening the blood-brain barrier, it acts as an adjuvant causing strong expansion of antigen-specific cells after coinjection with neuroantigens in IFA. Using an IL-17 ELISPOT assay we developed previously, we investigated the capability of PTX to induce proteolipid protein peptide 139-151(PLPp)-specific Th-17 cells in the immune periphery and in the thymus after coinjection with PLPp/IFA. PTX was found to induce peripheral PLPp-specific Th-17 cells in the draining lymph node and in the spleen, but not in the thymus. Our study indicates a new mechanism by which microbial agents can initiate or maintain autoimmune reactions and supports the growing role in particular for Th-17 cells in organ-specific autoimmune diseases like multiple sclerosis or EAE.

High serum levels of soluble TRAIL (sTRAIL) before or during the first year of Interferon-beta (IFN-beta) therapy were shown to predict an individual therapeutic response of patients with relapsing-remitting multiple sclerosis (RRMS). Here, we investigated whether sTRAIL plasma levels during long-term IFN-beta treatment correlate with future therapeutic response or adverse effects of treatment. Postinjection short-time bursts of sTRAIL were associated with flu-like symptoms and IP-10/CXCL10 as well as MCP-1/CCL2 induction, and were detected after up to 6 years of continuous IFN-beta therapy. However, neither sTRAIL nor chemokine levels allowed prediction of one- and two-year clinical treatment response in 30 RRMS patients, prospectively followed by blinded investigators.

The neonatal immune system is believed to be biased towards T helper type 2 (Th2) immunity, but under certain conditions neonates can also develop Th1 immune responses. Neonatal Th2 immunity to myelin antigens is not pathogenic and can prevent induction of experimental autoimmune encephalomyelitis (EAE) in adulthood, but the consequences of neonatally induced Th1 immunity to self-antigens have remained unresolved. Here, we show that neonatal injection of mice with myelin antigens emulsified in complete Freund's adjuvant (CFA) induced vigorous production of IFN-gamma and IL-17, but not IL-5, consistent with myelin-specific Th1/Th17 immunity. Importantly, the myelin-specific Th1/Th17 cells persisted in the mice until adulthood without causing symptoms of EAE. Intraperitoneal, but not subcutaneous injection of neonates with myelin antigens protected against induction of EAE as adults. Intraperitoneally injected neonates showed a substantial decrease of the number and avidity of myelin-reactive Th17 cells, suggesting a decrease in IL-17 producing precursor cells as the mechanism of protection from EAE upon re-injection with myelin antigens as adults. The results could provide a rationale for the presence of autoreactive T cells found in healthy human individuals without autoimmune disease.

In experimental autoimmune encephalomyelitis (EAE), the production of proinflammatory cytokines by neuroantigen-specific T cells is thought to initiate and maintain the inflammatory autoimmune pathology. Because gene knockout strategies have shown that IFN-gamma and TNF are not essential for EAE development, there is increasing interest in establishing the role of other proinflammatory cytokines, primarily IL-17 in EAE. We used an IL-17 ELISPOT assay to track the neuroantigen-specific IL-17-producing T cells at single-cell resolution in various organs of SJL mice undergoing PLP 139-151-induced EAE. Overall, the migration patterns and population kinetics of the PLP 139-151-specific IL-17-producing CD4 cells were reminiscent of the IFN-gamma-producing cells, with the exception of IL-17 producers far outnumbering the IFN-gamma and IL-2 producers in the inflamed CNS. The selective enrichment of IL-17-producing CD4 cells in the CNS is suggestive of the pathogenic role of an independent (non-Th1) IL-17-producing proinflammatory effector T cell class in EAE.

The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of multiple sclerosis (MS), however, the mechanisms by which EBV may be involved in MS are unknown. We here have investigated the frequency of EBV-specific cytotoxic T lymphocytes (CTL) in human leukocyte antigen (HLA)-B7(+) patients with MS and healthy controls using enzyme-linked immunospot assays and seven previously characterized HLA-B7-restricted immunogenic EBV peptides. Overall, there were no significant differences in the frequency of EBV-specific CTL between both groups. These data do not support the hypothesis that EBV could play a role in MS by inducing quantitatively altered EBV-specific CTL responses. Other pathogenic mechanisms for EBV in MS remain to be elucidated.

Interleukin (IL)-17A, a recently described novel T cell cytokine, orchestrates inflammation in a variety of immune-mediated diseases. In the present investigation, we analyzed the temporal gene expression pattern of IL-17A and its main regulators IL-23 and IL-15 after chronic constriction injury (CCI) of the sciatic nerve, a lesion paradigm inducing neuropathic pain, by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in mice. IL-17A displayed a monophasic expression in degenerating nerves at day 7 after CCI while transcripts for the IL-17A regulatory cytokines IL-23 and IL-15 peaked earlier. Accordingly, IL-17A positive T cells were detectable within the endoneurium of the injured nerves by immunocytochemistry. In support of a crucial role of T cell inflammation, RAG-1 knockout mice lacking functional T lymphocytes did not express IL-17A mRNA in distal nerve segments following CCI. Interestingly, T cell deficiency was associated with less thermal hyperalgesia and reduced mRNA levels for the macrophage marker molecule F4/80 and the chemokine macrophage chemoattractant protein-1 (MCP-1) after CCI. Our study supports the notion that T cells and T-cell-derived cytokines contribute to the inflammatory response after peripheral nerve injury.

IL-17 is a potent proinflammatory cytokine produced by activated memory T cells. Recent studies in both human autoimmune diseases and in their animal models have indicated that IL-17 rather than IFN-gamma might be the essential T-cell effector cytokine in the T-cell mediated autoimmune process. Since the thymus has a central role in maintaining T-cell self-tolerance and disturbance of thymic self-tolerance is implied in various autoimmune diseases, we here investigated the capability of murine thymocytes to produce IL-17. Our results indicate that thymocytes are a potent source of IL-17 in response to CD3 stimulation and various microbial immune stimuli and thereby show different patterns in the expression of the proinflammatory cytokines IFN-gamma and IL-17. In addition, strong differences between thymocytes and splenocytes were detected. Altered IL-17 production by thymocytes upon contact with foreign pathogens might be a key regulator in the education of adaptive immunity.

Experimental autoimmune encephalomyelitis (EAE) is regarded as an animal model of the human autoimmune disease multiple sclerosis (MS). Autoreactive T cells are present in the peripheral T cell repertoire of healthy mice and mediate clinical autoimmune disease only after activation by immunization or pathogens and migrate into the central nervous system (CNS). Because it is not known whether autoreactive T cells are regulated differentially once entering the CNS we investigated cytokine regulation in T cells from peripheral lymphatic organs and from the inflamed CNS ex vivo obtained from SJL mice after inducing relapsing-remitting EAE with PLP peptide 139-151. We show here that during acute EAE, an interleukin-2 (IL-2) biased T cell response exists in the spleen, while an interferon-gamma (IFN-gamma) biased T cell response prevails in the CNS of mice with acute EAE. The IFN-gamma biased phenotype was stable with optimized costimulation and even after in vitro stimulation with IL-2. After adoptive transfer into naïve syngeneic mice these T cells were only partially reversed to an IL-2 biased phenotype. These findings of our work suggest that a permanent effector phenotype of neuroantigen-specific T cells is finally acquired in the CNS in EAE.

Experimental allergic encephalomyelitis (EAE) is an animal model of multiple sclerosis. While EAE is mediated by the cytokines produced by specific T cells, the cytokine signature of these effector cells is unresolved. We tested CD4 cells from MOG peptide 35-55 immunized C57BL/6 mice for their peptide induced cytokine production on antigen presenting cells of the respective cytokine knockout mice, or wild type mice. IL-4 and IL-6 production was seen on wild type antigen presenting cells, suggesting that IL-4 and IL-6 are not T cell products. In contrast, IFN-gamma, IL-2 and IL-3 were found to be produced by the MOG specific CD4 cells. Understanding the cognate vs. bystander cytokine production in EAE might help dissect the contribution of cytokines to the pathogenesis of the disease.

Serotonin (5-hydroxytryptamine, 5-HT) is one of the most extensively studied neurotransmitters of the central nervous system. It also has been identified in constituents of the immune system. Therefore serotonin has been suggested to serve as a mediator of bidirectional interactions between the nervous system and the immune system. We investigated this interaction in experimental autoimmune encephalomyelitis (EAE), a well-defined animal model of autoimmune disease of the central nervous system (CNS) mimicking features of the human disease multiple sclerosis. EAE was induced by immunization with the autoantigens myelin basic protein (MBP) or the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG) spanning amino acids 35-55 (MOGp 35-55). We studied EAE in knockout (KO) mice lacking the 5-HT transporter (5-HTT) on a C57.BL/6 background, in comparison with wild-type C57.BL/6 animals. After immunization with MOGp 35-55, or with rat MBP, the disease courses of the 5-HTT knockout mice were attenuated as compared to wildtype control mice. This difference was more pronounced in female animals. To dissect potential immune mechanisms underlying this phenomenon, histological studies of the CNS and cytokine measurements in mononuclear cells from the spleens of 5-HTT KO mice and wild-type controls were performed. We found a reduction of the inflammatory infiltrate in the CNS and of the neuroantigen-specific production of IFN-gamma in splenocytes, again accompanied by a gender difference. These findings suggest a potential role of extracellular 5-HT homeostasis in the fine-tuning of neuroantigen-specific immune responses.

Experimental autoimmune encephalomyelitis (EAE) is widely regarded as an animal model of the human disease multiple sclerosis. A multitude of studies has investigated the neuroantigen-specific T-cell mediated cytokine pattern present in animals with EAE. In particular, the role of the so-called Th1- and Th2-cytokines has been addressed. In a recent study, it has been demonstrated that IL-23 rather than IL-12 is critical for modulating the character of the developing immune response towards a proinflammatory response and leading to EAE. IL-17 is a crucial effector cytokine, whose production is specifically triggered by IL-23, and it has been shown to be an essential inflammatory mediator in other autoimmune diseases and inflammatory conditions. This led us to investigate the role of IL-17 in EAE. Strong antigen-specific production of IL-17 was demonstrated both in peripheral immune organs and in the CNS in acute and chronic EAE, as demonstrated by ELISPOT and RT-PCR analysis. Therapeutic neutralization of IL-17 with IL-17-receptor-Fc-protein in acute EAE ameliorated clinical symptoms. Neutralization of IL-17 with a monoclonal antibody also ameliorated the disease course. We conclude that IL-17 is crucially involved in the cytokine network as an effector cytokine in EAE.

There is accumulating evidence that CD8-positive (CD8+) T-cells and MHC-I expression may also play a role in neurodegeneration associated with multiple sclerosis (MS). We investigated the role of MHC-I and CD8+ T-cells by studying experimental autoimmune encephalomyelitis (EAE) in beta-2 microglobulin knockout mice induced by myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 or whole rat myelin basic protein (rMBP). For both encephalitogens and even after reconstitution of the immune system with MHC-I-positive bone marrow and transfer of mature CD8+ T-cells (iMHC-I+ CD8+ beta2m-/- mice), the disease course in beta2m-/- mice was significantly more severe with a 10-fold increased mortality in the beta2m-/- mice as compared to wild-type C57BL/6 mice. EAE in beta2m-/- mice caused more severe demyelination after immunization with MOG than with rMBP and axonal damage was more marked with rMBP as well as MOG even in iMHC-I+ CD8+ beta2m-/- mice. Immunocytochemical analysis of spinal cord tissue revealed a significant increase in macrophage and microglia infiltration in beta2m-/- and iMHC-I+ CD8+ beta2m-/- mice. The different pattern of T-cell infiltration was underscored by a 2.5-fold increase in CD4-positive (CD4+) T-cells in beta2m-/- mice after induction of MOG 35-55 EAE. We conclude that lack of functional MHC-I molecules and CD8+ T-cells aggravates autoimmune tissue destruction in the CNS. Enhanced axonal damage speaks for pathways of tissue damage independent of CD8+ T-cells and neuronal MHC-I expression.

In humans, studies of autoreactive T cells that mediate multiple sclerosis have been largely confined to testing peripheral blood lymphocytes. Little is known how such measurements reflect the disease-mediating autoreactive T cells in the CNS. This information is also not available for murine experimental allergic encephalomyelitis (EAE); the low number of T cells that can be obtained from the blood or the brain of mice prevented such comparisons. We used single-cell resolution IFN-gamma ELISPOT assays to measure the frequencies and functional avidities of myelin basic protein (MBP:87-99)-specific CD4 cells in SJL mice immunized with this peptide. Functional MBP:87-99-specific IFN-gamma-producing cells were present in the CNS during clinical signs of EAE, but not during phases of recovery. In contrast, MBP:87-99-specific T cells persisted in the blood during all stages of the disease, and were also present in mice that did not develop EAE. Therefore, the increased frequency of MBP:87-99-reactive T cells in the blood reliably reflected the primed state, but not the inflammatory activity of these cells in the brain. The functional avidity of the MBP:87-99-reactive T cells was identical in the brain and blood and did not change over 2 mo as the mice progressed from acute to chronic EAE. Therefore, high-affinity T cells did not become selectively enriched in the target organ, and avidity maturation of the MBP:87-99-specific T cell repertoire did not occur in the observation period. The data may help the interpretation of measurements made with peripheral blood lymphocytes of multiple sclerosis patients.

The low frequency of islet-cell antigen-reactive T cells in type 1 diabetes makes their direct measurement difficult. Commonly used in vitro expansion could alter in vivo frequencies and Th1/Th2 differentiation states. Using IFN-gamma/IL-4 double color ELISPOT, we tested longitudinally the reactivity of PBMC from HLA-matched diabetic patients and healthy controls to GAD65, IA-2, and proinsulin peptides ex vivo and after in vitro culture. The peptide-reactive T cells showed IFN-gamma bias in the patients' PBMC in the primary assay. During in vitro culture, both IFN-gamma- and IL-4-producing cells were induced in controls, suggesting that the precursor cells were uncommitted naive T cells in vivo. In contrast, in diabetic patients, the ex vivo IFN-gamma response was conserved during culture, suggesting their Th1 commitment. Using CFSE-dye-dilution, we demonstrate that naive T cells expand in vitro at a faster rate than memory cells, which might account for the differences in expansion rates between diabetic patients and controls.

The initial event in the pathogenesis of autoimmune disease is thought to be the priming of naive autoreactive T cells by an infection with a cross-reactive microorganism. Although such cross-reactive priming should be a common event, autoimmune disease does not frequently develop. This situation is reflected after the immunization of C57BL/6 mice with the neuroantigen myelin oligodendrocyte glycoprotein (MOG) with CFA, which primes a type 1 T cell response but does not lead to clinical or histological manifestation of experimental allergic encephalomyelitis unless pertussis toxin is injected in addition. We show in this study that, in MOG:CFA-primed mice, the autoimmune CNS pathology develops after intracerebral deposition of TLR9-activating CpG oligonucleotides, but not following non-CpG oligonucleotide injection or after aseptic cryoinjury of the brain. Thus, access of primed MOG-specific Th1 cells to the uninflamed CNS or to CNS undergoing sterile inflammation did not suffice to elicit autoimmune pathology; only if the APC in the target organ were activated in addition by the TLR9-stimulating microbial product did they exert local effector functions. The data suggest that such licensing of APC in the target organ by microbial stimuli represents a checkpoint for functional self-tolerance. Therefore, microorganisms unrelated to the cross-reactive agent that primes the autoreactive T cells could dictate the onset and exacerbation of autoimmune diseases.

Genetic deficiency or instability of myelin protein zero (P0) results in hereditary motor sensory neuropathy. In view of recent advances in gene therapy, substitution of the molecular defect may become realistic in the near future. Here we investigate the impact of genetic deficiency of P0 on selection of the autoreactive T cell repertoire in the corresponding mouse model. We show that P0 mRNA transcripts are expressed in thymic stroma, similar to other myelin proteins and that expression of intact P0 protein can be detected by Western blot. Using a library of overlapping 20mer peptides spanning the entire length of P0 and applying the ELISPOT technique, we detected a strong immune response toward P0 extracellular domain peptide aa 41-60 in P0(-/-) knockout mice, but not in heterozygous P0(+/-) or wild-type (wt) mice. In addition, one cryptic epitope and two subdominant epitopes of P0 were identified. Using P0(-/-) into wt bone marrow (BM) chimeras we found that P0 expression in the host suffices for full tolerance induction, which is in line with its presence in thymic stroma. However, repopulation of P0(-/-) mice with wt BM led to partial induction of tolerance, suggesting that BM derived cells can also express this protein. Our findings may have implications for secondary autoimmunity developing after gene therapy in hereditary neuropathies and other diseases with genetically determined protein deficiency, because the repaired protein will then represent a foreign, nontolerized Ag.

In general, autoimmune responses are considered harmful to the host. In the best-defined model of autoimmune disease, murine experimental allergic encephalomyelitis (EAE), for example, brain-protein-specific autoimmune responses of both major classes, type-1 and type-2, have been implicated in causing brain pathology. We induced type-1 and type-2 autoimmunity to myelin oligodendrocyte protein (MOG) in C57.BL/6 mice. Instead of using pertussis toxin (PTX) to open the blood-brain barrier (BBB), which is the classic procedure, we set an aseptic cerebral injury (ACI) to see what the consequences of pre-primed, autoreactive type-1 and type-2 memory T cells gaining access to the brain in the course of sterile tissue injury would be. Neither of these autoimmune response types induced pathology; on the contrary, both accelerated re-vascularization and post-traumatic healing. The data suggest that induction of either type-1 or type-2 autoimmune responses is not inherently noxious to the host, but can have beneficial effects on tissue repair. Autoimmune pathology may develop only if molecules of microbial origin such as pertussis toxin additionally induce the "infectious nonself/danger" reaction in the antigen-presenting cells (APC) of the target organ itself.

Pertussis toxin (PT) has been widely used to facilitate the induction of experimental autoimmune encephalomyelitis (EAE) in rodents. It has been suggested that this microbial product promotes EAE by opening up the blood-brain barrier and thereby facilitates the migration of pathogenic T cells to the CNS. However, PT has other biological effects that could contribute to its activity in EAE, such as enhancing the cytokine production by T cells and induction of lymphocytosis. In this work, we investigated the effects of PT on the pathogenicity, cytokine differentiation, and clonal sizes of neuroantigen-reactive T cells in EAE in mice. Our results show that PT prevented the protection from EAE conferred by injection of PLPp139-151 in IFA and induced high frequencies of peptide-specific Th1 cells and disease. Interestingly, the mice developed EAE despite the simultaneous vigorous clonal expansion of PLPp139-151-specific Th2 cells. The data indicate that the Th2 cells in this model neither were protective against EAE nor promoted the disease. Furthermore, the results suggested that the effects of the toxin on neuroantigen-reactive T cells were promoted by the PT-induced activation of APCs in lymphoid tissues and the CNS. Together, the results suggest that microbial products, such as PT, could contribute to the initiation of autoimmune disease by modulating the interaction between the innate and adaptive immune system in the response to self Ags.

Direct measurements of the frequency and the cytokine signature of the neuroantigen-specific effector cells in experimental allergic encephalomyelitis (EAE) are a continuing challenge. This is true for lymphoid tissues, and more importantly, for the CNS itself. Using enzyme-linked immunospot analysis (ELISPOT) assays, we followed proteolipid protein (PLP) 139--151-specific T cells engaged by active immunization of SJL mice. The total numbers of PLP(139--151)-specific CD4 cells were highest before disease onset. At this time, these cells resided in lymphoid and nonlymphoid tissues, but were not detected in the CNS. While the PLP(139--151)-specific cells reached high frequencies in the CNS during clinical EAE, in absolute numbers, less than 20% of them were present in the target organ, with the majority residing in the periphery throughout all stages of the disease. The numbers of PLP(139--151)-specific cells gradually declined in both compartments with time. While eventually this first wave of effector cells completely disappeared from the CNS, PLP(178--191)-specific cells became engaged, being detected first in the CNS. These data suggest that throughout all stages of EAE, the effector cells in the CNS are recruited from a vast peripheral reservoir, and that the second wave of effector cells is engaged while the first wave undergoes exhaustion.

Injection of autoantigens in IFA has been one of the most effective ways of preventing experimental, T cell-mediated, autoimmune disease in mice. The mechanism that underlies this protection has, however, remained controversial, with clonal deletion, induction of suppressor cells or of type 2 immunity being implicated at one time or another. Using high resolution enzyme-linked immunospot (ELISPOT) analysis, we have revisited this paradigm. As models of autoimmunity against sequestered and readily accessible autoantigens, we studied experimental allergic encephalomyelitis, induced by myelin oligodendrocyte glycoprotein, proteolipid protein, myelin basic protein, and renal tubular Ag-induced interstitial nephritis. We showed that the injection of each of these Ags in IFA was immunogenic and CD4 memory cells producing IL-2, IL-4, and IL-5, but essentially no IFN-gamma. IgG1, but not IgG2a, autoantibodies were produced. The engaged T cells were not classic Th2 cells in that IL-4 and IL-5 were produced by different cells. The IFA-induced violation of self tolerance, including the deposition of specific autoantibodies in the respective target organs, occurred in the absence of detectable pathology. Exhaustion of the pool of naive precursor cells was shown to be one mechanism of the IFA-induced tolerance. In addition, while the IFA-primed T cells acted as suppressor cells, in that they adoptively transferred disease protection, they did not interfere with the emergence of a type 1 T cell response in the adoptive host. Both active and passive tolerance mechanisms, therefore, contribute to autoantigen:IFA-induced protection from autoimmune disease.

Several ions in the mass spectra of wax esters were related to the molecular structures. Assigned structures of ions were confirmed by deuterium labeling. A simple, direct method for quantititive analyses of mixtures was developed. The method involved a comparison of sets of three ions, RCO2H(+), RCO2H2 (+) and [R'-1](+) from all compounds in the mixture. The method was found applicable for mixtures of unsaturated wax esters after reduction with tetradeuterio hydrazine.