A recent paper published in Nature reports sensory nerve fibers in the skin that give local immune cells important instructions for the organization of an immune response; in this particular case the cooperation between the nervous and immune systems had disastrous consequences, namely an auto-destruction of the skin.
Glucocorticoids (GCs) are the standard therapy for treating multiple sclerosis (MS) patients suffering from an acute relapse. One of the main mechanisms of GC action is held to be the induction of T cell apoptosis leading to reduced lymphocyte infiltration into the CNS, yet our analysis of experimental autoimmune encephalomyelitis (EAE) in three different strains of genetically manipulated mice has revealed that the induction of T cell apoptosis is not essential for the therapeutic efficacy of GCs. Instead, we identified the redirection of T cell migration in response to chemokines as a new therapeutic principle of GC action. GCs inhibited the migration of T cells towards CCL19 while they enhanced their responsiveness towards CXCL12. Importantly, blocking CXCR4 signaling in vivo by applying Plerixafor(®) strongly impaired the capacity of GCs to interfere with EAE, as revealed by an aggravated disease course, more pronounced CNS infiltration and a more dispersed distribution of the infiltrating T cells throughout the parenchyma. Our observation that T cells lacking the GC receptor were refractory to CXCL12 further underscores the importance of this pathway for the treatment of EAE by GCs. Importantly, methylprednisolone pulse therapy strongly increased the capacity of peripheral blood T cells from MS patients of different subtypes to migrate towards CXCL12. This indicates that modulation of T cell migration is an important mechanistic principle responsible for the efficacy of high-dose GC therapy not only of EAE but also of MS.
Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) that is initiated when self-reactive T cells enter the brain and become locally activated after encountering their specific nervous antigens. When and where the disease-relevant antigen encounters occur is unclear. Here we combined fluorescently labeled nuclear factor of activated T cells (NFAT) with histone protein H2B to create a broadly applicable molecular sensor for intravital imaging of T cell activation. In experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, we report that effector T cells entering the CNS become activated after short contacts with leptomeningeal phagocytes. During established disease, the activation process is extended to the depth of the CNS parenchyma, where the cells form contacts with microglia and recruited phagocytes. We show that it is the activation processes during the preclinical phase rather than during established disease that are essential for the intensity and duration of the disease bout.
The concept of neuroprotective autoimmunity implies that immune cells, especially autoantigen-specific T cells, infiltrate the central nervous system (CNS) after injury and contribute to neuroregeneration and repair by secreting soluble factors. Amongst others, neurotrophic factors and neurotrophins such as brain-derived neurotropic factor (BDNF) are considered to play an important role in this process. New data raise the possibility that this concept could also be extended to neuroinflammatory diseases such as multiple sclerosis (MS) where autoantigen-specific T cells infiltrate the CNS, causing axonal/neuronal damage on the one hand, but also providing neuroprotective support on the other hand. In this review, we summarize the current knowledge on BDNF levels analyzed in MS patients in different compartments and its correlation with clinical parameters. Furthermore, new approaches in experimental animal models are discussed that attempt to decipher the functional relevance of BDNF in autoimmune demyelination.
Current concepts attribute an early and central role for auto-aggressive, myelin-specific T-lymphocytes in the pathogenesis of multiple sclerosis. This view emerged from immunological and pathological findings in experimental autoimmune encephalitis, an animal model characterised by pathological lesions closely resembling the ones found in multiple sclerosis. Furthermore, therapeutic strategies targeting the functions of these encephalitogenic T cells which attenuate their pathogenicity such as glatiramer acetate or anti-VLA4 antibody treatments represent proven approaches in multiple sclerosis. Nonetheless, all therapies evaluated to date either insufficiently dampen down inflammation or completely block immune processes. For this reason, there is a need to identify new therapeutic targets. We have employed live intravital two-photon microscopy to learn more about the behaviour of T cells during the preclinical phase of EAE, when T cells acquire the properties required to invade their target organ. Furthermore, we were able to identify an unexpected locomotive behaviour of T cells at the blood-brain barrier, which occurs immediately before diapedesis and the induction of paralytic disease. Such studies might open new avenues for the treatment of CNS autoimmune diseases. Multiple sclerosis is considered to be an autoimmune disease in which self-reactive T cells enter the central nervous system (CNS) and create an inflammatory milieu that destroys myelin and neurons. Immunomodulatory strategies for the treatment of multiple sclerosis target this process by attempting to inactivate these auto-aggressive T cells. However, so far, these strategies have failed to extinguish disease activity completely. For this reason, there is a need to understand in more detail the mechanisms by which T cells become encephalitogenic, how they enter the nervous system, and what the signals are that guide them along this path. If these processes could be better understood, it may be possible to design more effective and specific therapies for multiple sclerosis. This article will give a brief overview about our recent findings obtained using intravital imaging of autoaggressive effector T cells in an experimental model of multiple sclerosis. This new technological approach might help to fill some gaps in the understanding of autoimmune pathogenesis of multiple sclerosis.
Brain-derived neurotrophic factor (BDNF) is involved in neuronal and glial development and survival. While neurons and astrocytes are its main cellular source in the central nervous system (CNS), bioactive BDNF is also expressed in immune cells and in lesions of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Previous data revealed that BDNF exerts neuroprotective effects in myelin oligodendrocyte glycoprotein-induced EAE. Using a conditional knock-out model with inducible deletion of BDNF, we here show that clinical symptoms and structural damage are increased when BDNF is absent during the initiation phase of clinical EAE. In contrast, deletion of BDNF later in the disease course of EAE did not result in significant changes, either in the disease course or in axonal integrity. Bone marrow chimeras revealed that the deletion of BDNF in the CNS alone, with no deletion of BDNF in the infiltrating immune cells, was sufficient for the observed effects. Finally, the therapeutic effect of glatiramer acetate, a well-characterized disease-modifying drug with the potential to modulate BDNF expression, was partially reversed in mice in which BDNF was deleted shortly before the onset of disease. In summary, our data argue for an early window of therapeutic opportunity where modulation of BDNF may exert neuroprotective effects in experimental autoimmune demyelination.
Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis, a chronic inflammatory and demyelinating disease of the CNS. Albino Oxford (AO) rats are resistant to the induction of EAE, while the disease can be readily induced in Dark Agouti (DA) rats. Here we investigated a potential contribution of the CNS milieu in the limitation of the encephalitogenic autoimmune response. EAE was induced by immunization of the respective rat strains with spinal cord homogenate emulsified in complete Freunds adjuvant. AO rats did not exhibit clinical signs after immunization while DA rats developed severe neurologic deficits. Infiltration of immune cells into spinal cords (SC) was evident in both strains 12-14 days after the immunization. EAE lesions of AO rats contained substantially lower numbers of CD4+ T cells and CD11b+ cells compared to those in DA rats. This went together with lower levels of interferon (IFN)-? and interleukin (IL)-17 in the cells isolated from SC. We found a dramatic increase of CXCL12 expression in SC tissue and microvessels of AO rats, whereas DA rats markedly decreased the expression of this chemokine within their CNS. Administration of the CXCL12 antagonist AMD3100 to a substrain of AO rats that developed a weak EAE led to earlier onset and exacerbation of the disease. These results suggest a role of CXCL12 in down-regulating autoimmune processes in AO rats during EAE. Therapeutic modulation of CXCL12 could be a promising strategy for the treatment of CNS autoimmunity.
Nicotinic acid adenine dinucleotide phosphate represents a newly identified second messenger in T cells involved in antigen receptor-mediated calcium signalling. Its function in vivo is, however, unknown due to the lack of biocompatible inhibitors. Using a recently developed inhibitor, we explored the role of nicotinic acid adenine dinucleotide phosphate in autoreactive effector T cells during experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis. We provide in vitro and in vivo evidence that calcium signalling controlled by nicotinic acid adenine dinucleotide phosphate is relevant for the pathogenic potential of autoimmune effector T cells. Live two photon imaging and molecular analyses revealed that nicotinic acid adenine dinucleotide phosphate signalling regulates T cell motility and re-activation upon arrival in the nervous tissues. Treatment with the nicotinic acid adenine dinucleotide phosphate inhibitor significantly reduced both the number of stable arrests of effector T cells and their invasive capacity. The levels of pro-inflammatory cytokines interferon-gamma and interleukin-17 were strongly diminished. Consecutively, the clinical symptoms of experimental autoimmune encephalomyelitis were ameliorated. In vitro, antigen-triggered T cell proliferation and cytokine production were evenly suppressed. These inhibitory effects were reversible: after wash-out of the nicotinic acid adenine dinucleotide phosphate antagonist, the effector T cells fully regained their functions. The nicotinic acid derivative BZ194 induced this transient state of non-responsiveness specifically in post-activated effector T cells. Naïve and long-lived memory T cells, which express lower levels of the putative nicotinic acid adenine dinucleotide phosphate receptor, type 1 ryanodine receptor, were not targeted. T cell priming and recall responses in vivo were not reduced. These data indicate that the nicotinic acid adenine dinucleotide phosphate/calcium signalling pathway is essential for the recruitment and the activation of autoaggressive effector T cells within their target organ. Interference with this signalling pathway suppresses the formation of autoimmune inflammatory lesions and thus might qualify as a novel strategy for the treatment of T cell mediated autoimmune diseases.
T cell vaccination (TCV) with irradiated encephalitogenic T cells induces resistance to EAE. However, the fate of the encephalitogenic T cells in vivo following TCV has yet to be studied. Here we used anti-MBP encephalitogenic T cells that were transduced to express GFP to study the effects of TCV on these cells. In naïve rats or in control-vaccinated (Ova-GFP) rats injected i.v. with GFP-labeled effector cells, high numbers of effector T cells were found along with macrophages, CD8 T cells and Non-GFP CD4 cells in the spleens, parathymic lymph nodes (PTLN) and spinal cords. In contrast, the recipients that had been treated with TCV (anti-MBP T-cell lines) showed few if any GFP-labeled effector T cells throughout the disease (day 1-8) and their spinal cords were almost clear of macrophages, CD4 and CD8 cells. Splenocytes in the control groups secreted IFNgamma in response to MBP and showed high numbers of IFNgamma secreting CD4 and CD8 cells in their spinal cords at the disease peak. In the TCV-protected groups, splenocytes showed no reactivity to MBP but secreted IFNgamma in response to irradiated encephalitogenic T cells--an anti-idiotypic response. Thus, TCV leads to a marked decrease in the numbers of effector T cells in the CNS and lymphoid organs, to a marked reduction in the Th1 cytokine producing cells in the CNS, and to the appearance of T cells responsive to the anti-MBP effector T cells.
Since the first applications of two-photon microscopy in immunology 10 years ago, the number of studies using this advanced technology has increased dramatically. The two-photon microscope allows long-term visualization of cell motility in the living tissue with minimal phototoxicity. Using this technique, we examined brain autoantigen-specific T cell behavior in experimental autoimmune encephalitomyelitis, the animal model of human multiple sclerosis. Even before disease symptoms appear, the autoreactive T cells arrive at their target organ. There they crawl along the intraluminal surface of central nervous system (CNS) blood vessels before they extravasate. In the perivascular environment, the T cells meet phagocytes that present autoantigens. This contact activates the T cells to penetrate deep into the CNS parenchyma, where the infiltrated T cells again can find antigen, be further activated, and produce cytokines, resulting in massive immune cell recruitment and clinical disease.
The tissues of the central nervous system are effectively shielded from the blood circulation by specialized vessels that are impermeable not only to cells, but also to most macromolecules circulating in the blood. Despite this seemingly absolute seclusion, central nervous system tissues are subject to immune surveillance and are vulnerable to autoimmune attacks. Using intravital two-photon imaging in a Lewis rat model of experimental autoimmune encephalomyelitis, here we present in real-time the interactive processes between effector T cells and cerebral structures from their first arrival to manifest autoimmune disease. We observed that incoming effector T cells successively scanned three planes. The T cells got arrested to leptomeningeal vessels and immediately monitored the luminal surface, crawling preferentially against the blood flow. After diapedesis, the cells continued their scan on the abluminal vascular surface and the underlying leptomeningeal (pial) membrane. There, the T cells encountered phagocytes that effectively present antigens, foreign as well as myelin proteins. These contacts stimulated the effector T cells to produce pro-inflammatory mediators, and provided a trigger to tissue invasion and the formation of inflammatory infiltrations.
The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca(2+) signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. BZ194 neither interfered with Ca(2+) mobilization by d-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose nor with capacitative Ca(2+) entry. BZ194 specifically and effectively blocked NAADP-stimulated [(3)H]ryanodine binding to the purified type 1 ryanodine receptor. Further, in intact T cells, Ca(2+) mobilization evoked by NAADP or by formation of the immunological synapse between primary effector T cells and astrocytes was inhibited by BZ194. Downstream events of Ca(2+) mobilization, such as nuclear translocation of "nuclear factor of activated T cells" (NFAT), T cell receptor-driven interleukin-2 production, and proliferation in antigen-experienced CD4(+) effector T cells, were attenuated by the NAADP antagonist. Taken together, specific inhibition of the NAADP signaling pathway constitutes a way to specifically and effectively modulate T-cell activation and has potential in the therapy of autoimmune diseases.
cADPR (cyclic ADP-ribose) is a universal Ca(2+) mobilizing second messenger. In T-cells cADPR is involved in sustained Ca(2+) release and also in Ca(2+) entry. Potential mechanisms for the latter include either capacitative Ca(2+) entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N(1)-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N(1)-cIDPR evoked Ca(2+) signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca(2+) signalling induced by 8-Br-N(1)-cIDPR consisted of Ca(2+) release and Ca(2+) entry. Whereas Ca(2+) release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca(2+) entry was inhibited by the Ca(2+) entry blockers Gd(3+) (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca(2+) entry evoked by 8-Br-N(1)-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H(2)O(2) was enhanced dramatically in those cells, Ca(2+) signalling induced by 8-Br-N(1)-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N(1)-cIDPR. In summary, the sensitivity to the Ca(2+) entry blockers Gd(3+) and SKF-96365 is in favour of the concept of capacitative Ca(2+) entry, secondary to store depletion by 8-Br-N(1)-cIDPR. Taken together, 8-Br-N(1)-cIDPR appears to be the first cADPR agonist affecting Ca(2+) release and secondary Ca(2+) entry, but without effect on TRPM2.
The aim of this study was to assess the feasibility of transplanting mesenchymal stem cells (MSCs), genetically modified to express glial-derived neurotrophic factor (GDNF), to the contused rat spinal cord, and to subsequently assess their neural differentiation potential. MSCs expressing green fluorescent protein were transduced with a retroviral vector to express the neurotrophin GDNF. The transduction protocol was optimized by using green fluorescent protein-expressing retroviral constructs; approximately 90% of MSCs were transduced successfully after G418 selection. GDNF-transduced MSCs expressed the transgene and secreted growth factor into the media (approximately 12 ng/500,000 cells secreted into the supernatant 2 weeks after transduction). Injuries were established using an impactor device, which applied a given, reproducible force to the exposed spinal cord. GDNF-expressing MSCs were transplanted rostral and caudal to the site of injury. Spinal cord sections were analyzed 2 and 6 weeks after transplantation. We demonstrate that GDNF-transduced MSCs engraft, survive, and express the therapeutic gene up to 6 weeks posttransplantation, while maintaining an undifferentiated phenotype. In conclusion, transplanted MSCs have limited capacity for the replacement of neural cells lost as a result of a spinal cord trauma. However, they provide excellent opportunities for local delivery of neurotrophic factors into the injured tissue. This study underlines the therapeutic benefits associated with cell transplantation and provides a good example of the use of MSCs for gene delivery.
The blood–brain barrier (BBB) and the environment of the central nervous system (CNS) guard the nervous tissue from peripheral immune cells. In the autoimmune disease multiple sclerosis, myelin-reactive T-cell blasts are thought to transgress the BBB and create a pro-inflammatory environment in the CNS, thereby making possible a second autoimmune attack that starts from the leptomeningeal vessels and progresses into the parenchyma. Using a Lewis rat model of experimental autoimmune encephalomyelitis, we show here that contrary to the expectations of this concept, T-cell blasts do not efficiently enter the CNS and are not required to prepare the BBB for immune-cell recruitment. Instead, intravenously transferred T-cell blasts gain the capacity to enter the CNS after residing transiently within the lung tissues. Inside the lung tissues, they move along and within the airways to bronchus-associated lymphoid tissues and lung-draining mediastinal lymph nodes before they enter the blood circulation from where they reach the CNS. Effector T cells transferred directly into the airways showed a similar migratory pattern and retained their full pathogenicity. On their way the T cells fundamentally reprogrammed their gene-expression profile, characterized by downregulation of their activation program and upregulation of cellular locomotion molecules together with chemokine and adhesion receptors. The adhesion receptors include ninjurin 1, which participates in T-cell intravascular crawling on cerebral blood vessels. We detected that the lung constitutes a niche not only for activated T cells but also for resting myelin-reactive memory T cells. After local stimulation in the lung, these cells strongly proliferate and, after assuming migratory properties, enter the CNS and induce paralytic disease. The lung could therefore contribute to the activation of potentially autoaggressive T cells and their transition to a migratory mode as a prerequisite to entering their target tissues and inducing autoimmune disease.
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