Oligodendrocyte lysis by CD4+ T cells independent of tumor necrosis factor

The capacity of human CD4⁺ T cells to lyse heterologous human oligodendrocytes in an 18-hour chromium 51–release assay was compared to that of systemic blood-derived macrophages and central nervous system–derived microglia. CD4⁺ T cells, activated with either phytohemagglutinin, anti-CD3 antibody, or antigen (myelin basic protein), could induce lysis of the oligodendrocytes whereas macrophages and microglia, activated with interferon-γ and lipopolysaccharide, could not. The CD4⁺ T-cell effect was not inhibited with an anti–tumor necrosis factor-α–neutralizing antibody. Both the CD4⁺ T cells and the macrophages could induce lysis of tumor necrosis factor–sensitive rodent cell lines, Wehi 164, and L929; these effects were inhibited with anti–tumor necrosis factor antibody. Pretreatment of the CD4⁺ T cells with cyclosporine or mitomycin C did not inhibit oligodendrocyte lysis. These results indicate that at least in vitro, CD4⁺ T cells can induce a form of oligodendrocyte injury that is not reproduced by macrophages or microglia or by tumor necrosis factor. The non–major histocompatibility complex (MHC)–restricted injury of oligodendrocytes induced by both myelin antigen–reactive and mitogen-stimulated T cells may provide a basis whereby cytotoxic CD4⁺ T cells could interact with a target cell that does not express MHC class II molecules. Our results suggest that immune-mediated oligodendrocyte/myelin injury, as is postulated to occur in the disease multiple sclerosis, may involve multiple effector mechanisms.     

CD8+/perforin+/granzyme B+ effector cells infiltrating cerebellum and inferior olives in gluten ataxia

Up to 8% of patients with gluten sensitivity (GS) develop neurological symptoms such as ataxia, dementia, seizures or peripheral neuropathy. The underlying immunological mechanisms still remain to be elucidated. We here report the case of a 68‐year‐old male patient suffering from progressive ataxia and dementia associated with chronic diarrhea and both elevated IgG and IgA antigliadin‐antibodies. At autopsy, frequent argyrophilic glial and neuronal inclusions within the basal nucleus of Meynert were considered as the structural correlative for the cognitive decline. Significant neuronal loss in the cerebellar cortex and the inferior olives was accompanied by infiltrating CD8⁺/perforin⁺/granzyme B⁺ cells as well as reactive astrogliosis and microglial activation. These CD8⁺ cytotoxic T and NK cells are likely to act as effector cells responsible for neuronal cell death in patients with gluten sensitivity and neurological disease and might therefore at least partly be responsible for cerebellar symptoms in gluten ataxia. In conclusion, our results, showing an absence of B‐ or plasma cells but multiple CD8⁺ as well as granzyme B and perforin expressing cells in ataxia‐associated brain areas, suggest that there are also prominent cytotoxic effects in neuropathogenesis of GS.     

SOX17 is expressed in regenerating oligodendrocytes in experimental models of demyelination and in multiple sclerosis

We have previously demonstrated that Sox17 expression is prominent at developmental stages corresponding to oligodendrocyte progenitor cell (OPC) cycle exit and onset of differentiation, and that Sox17 promotes initiation of OPC differentiation. In this study, we examined Sox17 expression and regulation under pathological conditions, particularly in two animal models of demyelination/remyelination and in post‐mortem multiple sclerosis (MS) brain lesions. We found that the number of Sox17 expressing cells was significantly increased in lysolecithin (LPC)‐induced lesions of the mouse spinal cord between 7 and 30 days post‐injection, as compared with controls. Sox17 immunoreactivity was predominantly detected in Olig2⁺ and CC1⁺ oligodendrocytes and rarely in NG2⁺ OPCs. The highest density of Sox17⁺ oligodendrocytes was observed at 2 weeks after LPC injection, coinciding with OPC differentiation. Consistent with these findings, in cuprizone‐treated mice, Sox17 expression was highest in newly generated and in maturing CC1⁺ oligodendrocytes, but low in NG2⁺ OPCs during the demyelination and remyelination phases. In MS tissue, Sox17 was primarily detected in actively demyelinating lesions and periplaque white matter. Sox17 immunoreactivity was co‐localized with NOGO‐A+ post‐mitotic oligodendrocytes both in active MS lesions and periplaque white matter. Taken together, our data: (i) demonstrate that Sox17 expression is highest in newly generated oligodendrocytes under pathological conditions and could be used as a marker of oligodendrocyte regeneration, and (ii) are suggestive of Sox17 playing a critical role in oligodendrocyte differentiation and lesion repair. GLIA 2013;61:1659–1672     

Helper CD4 T cells expressing granzyme B cause glial fibrillary acidic protein fragmentation in astrocytes in an MHCII-independent manner

During inflammatory processes of the central nervous system, helper T cells have the capacity to cross the blood–brain barrier and injure or kill neural cells through cytotoxic mechanisms. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is part of the astrocyte cytoskeleton that can become fragmented in neuroinflammatory conditions. The mechanism of action by which helper T cells with cytotoxic properties injure astrocytes is not completely understood. Primary human astrocytes were obtained from fetal brain tissue. Human helper (CD4⁺) T cells were isolated from peripheral blood mononuclear cells and activated with the superantigen staphylococcal enterotoxin E (SEE). Granzyme B was detected by enzyme linked immunosorbent assay and intracellular flow cytometry. GFAP fragmentation was monitored by western blotting. Cell death was monitored by lactic acid dehydrogenase release and terminal biotin-dUTP nick labeling (TUNEL). Astrocyte migration was monitored by scratch assay. Adult human oligodendrocytes were cultured with sublethally injured astrocytes to determine support function. Helper T cells activated with SEE expressed granzyme B but not perforin. Helper T cells released granzyme B upon contact with astrocytes and caused GFAP fragmentation in a caspase-dependent, MHCII-independent manner. Sublethally injured astrocytes were not apoptotic; however, their processes were thin and elongated, their migration was attenuated, and their ability to support oligodendrocytes was reduced in vitro. Helper T cells can release granzyme B causing sublethal injury to astrocytes, which compromises the supportive functions of astrocytes. Blocking these pathways may lead to improved resolution of neuroinflammatory lesions.     

Short‐ and long‐term functional plasticity of white matter induced by oligodendrocyte depolarization in the hippocampus

Plastic changes in white matter have received considerable attention in relation to normal cognitive function and learning. Oligodendrocytes and myelin, which constitute the white matter in the central nervous system, can respond to neuronal activity with prolonged depolarization of membrane potential and/or an increase in the intracellular Ca²⁺ concentration. Depolarization of oligodendrocytes increases the conduction velocity of an action potential along axons myelinated by the depolarized oligodendrocytes, indicating that white matter shows functional plasticity, as well as structural plasticity. However, the properties and mechanism of oligodendrocyte depolarization‐induced functional plastic changes in white matter are largely unknown. Here, we investigated the functional plasticity of white matter in the hippocampus using mice with oligodendrocytes expressing channelrhodopsin‐2. Using extracellular recordings of compound action potentials at the alveus of the hippocampus, we demonstrated that light‐evoked depolarization of oligodendrocytes induced early‐ and late‐onset facilitation of axonal conduction that was dependent on the magnitude of oligodendrocyte depolarization; the former lasted for approximately 10 min, whereas the latter continued for up to 3 h. Using whole‐cell recordings from CA1 pyramidal cells and recordings of antidromic action potentials, we found that the early‐onset short‐lasting component included the synchronization of action potentials. Moreover, pharmacological analysis demonstrated that the activation of Ba²⁺‐sensitive K⁺ channels was involved in early‐ and late‐onset facilitation, whereas 4‐aminopyridine‐sensitive K⁺ channels were only involved in the early‐onset component. These results demonstrate that oligodendrocyte depolarization induces short‐ and long‐term functional plastic changes in the white matter of the hippocampus and plays active roles in brain functions. GLIA 2014;62:1299–1312     

Immune surveillance of the normal human CNS takes place in dependence of the locoregional blood–brain barrier configuration and is mainly performed by CD3+/CD8+ lymphocytes

Despite the blood–brain barrier (BBB) the human CNS is continuously screened by blood‐derived immunological cells. In certain brain areas the local BBB configuration grants passage of large molecules, whereas others are better shielded. We investigated whether these regional BBB compositions are paralleled by differences in the degree of cellular immunosurveillance by investigating tissue from 23 normal human brains for several CD markers, FoxP3, granzyme B, and perforin. Our results provide evidence that immunosurveillance is associated with locoregional BBB configuration and is mainly performed by CD3⁺/CD8⁺/granzyme B^‐/perforin^‐ lymphocytes.     

Oligodendrocyte positioning in cerebral cortex is independent of projection neuron layering

The factors affecting normal oligodendrocyte positioning in the cerebral cortex are unknown. Apart from the white matter, the highest numbers of oligodendrocytes in the rodent cortex are found in Layers V/VI, where the infragranular neurons normally reside. Few, if any, oligodendrocytes are normally found in the superficial cortical layers. To test whether or not this asymmetric positioning of oligodendrocytes is linked to the lamina positions of Layer V/VI projection neurons, mutant mice that cause neuronal layer inversion were examined. In three lines of mutant mice (Reeler, disabled‐1, and p35) examined, representing two different genetic signaling pathways, the oligodendrocyte distribution was altered from an asymmetric to a symmetric distribution pattern. Unlike cortical neurons that are inverted in these mutant mice, the lack of oligodendrocyte inversion suggests a decoupling of the genetic mechanisms governing neuronal versus oligodendrocyte patterning. We conclude that oligodendrocyte positioning is not linked to the layer positions of V/VI projection neurons. © 2008 Wiley‐Liss, Inc.     

Rabies virus‐mediated oligodendrocyte labeling reveals a single oligodendrocyte myelinates axons from distinct brain regions

Oligodendrocytes myelinate neuronal axons during development and increase conduction velocity of neuronal impulses in the central nervous system. Neuronal axons extend from multiple brain regions and pass through the white matter; however, whether oligodendrocytes ensheath a particular set of axons or do so randomly within the mammalian brain remains unclear. We developed a novel method to visualize individual oligodendrocytes and axon derived from a particular brain region in mouse white matter using a combinational injection of attenuated rabies virus and adeno‐associated virus. Using this method, we found that some populations of oligodendrocytes in the corpus callosum predominantly ensheathed axons derived from motor cortex or sensory cortex, while others ensheathed axons from both brain regions, suggesting heterogeneity in preference of myelination toward a particular subtype of neurons. Moreover, our newly established method is a versatile tool for analyzing precise morphology of each oligodendrocyte in animal models for demyelinating disorders and addressing the role of oligodendrocyte in higher brain functions. GLIA 2016. GLIA 2017;65:93–105     

Critical role for PAR1 in kallikrein 6‐mediated oligodendrogliopathy

Kallikrein 6 (KLK6) is a secreted serine protease preferentially expressed by oligodendroglia in CNS white matter. Elevated levels of KLK6 occur in actively demyelinating multiple sclerosis (MS) lesions and in cases of spinal cord injury (SCI), stroke, and glioblastoma. Taken with recent evidence establishing KLK6 as a CNS‐endogenous activator of protease‐activated receptors (PARs), we hypothesized that KLK6 activates a subset of PARs to regulate oligodendrocyte physiology and potentially pathophysiology. Here, primary oligodendrocyte cultures derived from wild type or PAR1‐deficient mice and the murine oligodendrocyte cell line, Oli‐neu, were used to demonstrate that Klk6 (rodent form) mediates loss of oligodendrocyte processes and impedes morphological differentiation of oligodendrocyte progenitor cells (OPCs) in a PAR1‐dependent fashion. Comparable gliopathy was also elicited by the canonical PAR1 agonist, thrombin, as well as PAR1‐activating peptides (PAR1‐APs). Klk6 also exacerbated ATP‐mediated oligodendrogliopathy in vitro, pointing to a potential role in augmenting excitotoxicity. In addition, Klk6 suppressed the expression of proteolipid protein (PLP) RNA in cultured oligodendrocytes by a mechanism involving PAR1‐mediated Erk1/2 signaling. Microinjection of PAR1 agonists, including Klk6 or PAR1‐APs, into the dorsal column white matter of PAR1^+/+ but not PAR1^?/? mice promoted vacuolating myelopathy and a loss of immunoreactivity for myelin basic protein (MBP) and CC‐1⁺ oligodendrocytes. These results demonstrate a functional role for Klk6‐PAR1 signaling in oligodendroglial pathophysiology and suggest that antagonists of PAR1 or its protease agonists may represent new modalities to moderate demyelination and to promote myelin regeneration in cases of CNS white matter injury or disease.     

Expression of the myelin and oligodendrocyte progenitor marker sulfatide in neurons and astrocytes of adult rat brain

Sulfatide is a myelin component of the central (CNS) and peripheral nervous system (PNS) and is used extensively to identify oligodendrocyte progenitor cells. We have explored sulfatide expression in CNS gray matter (cerebellum, cerebral cortex, and hippocampus) and the PNS in adult rats using an anti-sulfatide antibody (Sulph I) and confocal microscopy. Biochemical analyses revealed two Sulph I antigens, sulfatide and seminolipid; sulfatide was present at about five times higher concentration, and the affinity of Sulph I for sulfatide was 2.5 times higher than that for seminolipid. Thus sulfatide was considered the dominant antigen. We found Sulph I immunostaining, in addition to that in myelinated areas in subpopulations of astrocytes and neurons. Astrocyte Sulph I staining was localized to the cell bodies and in some cases also to the processes. In the cerebellum, some Sulph I-positive astrocytes corresponded to Golgi epithelial cell bodies. We also found Sulph I staining in neuronal cell bodies, which in some neurons was clearly localized to the cytoplasm and in others to the nuclear membrane. Sulph I immunostaining in the PNS was located in the myelin sheath and paranodal end segments. These results demonstrate the expression of sulfatide in cell types other than oligodendrocytes and Schwann cells, showing that sulfatide is not a selective marker for adult oligodendrocyte progenitor cells. Moreover, these findings show that sulfatide is localized also to intracellular compartments and indicate that other roles of sulfatide in astrocytes and neurons, compared to myelin, might be considered.     

Novel NG2‐CreERT2 knock‐in mice demonstrate heterogeneous differentiation potential of NG2 glia during development

NG2 (nerve/glia antigen‐2) is a type I transmembrane glycoprotein and also known as chondroitin sulfate proteoglycan 4. In the parenchyma of the central nervous system, NG2‐expressing (NG2⁺) cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes (OLs) in the developing white matter. However, the differentiation potential of NG2 glia remained controversial, largely attributable to shortcomings of transgenic mouse models used for fate mapping. To minimize these restrictions and to more faithfully mimic the endogenous NG2 expression in vivo, we generated a mouse line in which the open reading frame of the tamoxifen‐inducible form of the Cre DNA recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination. Results from this novel mouse line demonstrate that at different developmental stages of the brain, NG2⁺ cells either stayed as NG2 glia or differentiated into OLs during the whole life span. Interestingly, when Cre activity was induced at embryonic stages, a significant number of reporter⁺ astrocytes could be detected in the gray matter after birth. However, in other brain regions, such as olfactory bulb, brain stem, and cerebellum, all of the NG2 glia was restricted to the OL lineage. In addition, tamoxifen‐sensitive and NG2 gene locus‐dependent gene recombination could be detected in a small, but persistent population of cortical NeuN⁺ neurons starting from the second postnatal week. GLIA 2014;62:896–913     

Cytotoxic CD4+ T cells are correlated with better prognosis in Han Chinese grade II and grade III glioma subjects and are suppressed by PD-1 signaling

Purpose: Malignant gliomas are the most common tumors in the central nervous system with a poor prognosis. Recently, CD4⁺ cytotoxic T cells (CTLs) are being increasingly recognized as possessing antitumor capacity. However, their presence, activity and regulation in glioma have not been investigated in detail. Methods: To examine this, 72 grade II and grade III Han Chinese glioma patients and 30 Han Chinese healthy controls were investigated. Results: We found that compared to healthy controls, glioma patients had significantly upregulated frequencies of circulating CD4⁺ CTLs, identified by the expression of granzyme A (GzmA), granzyme B (GzmB) and/or perforin. The stimulated CD4⁺ CTLs in grade II and grade III glioma patients also had less proliferative ability than those in healthy controls, a feature of suppression that progressed with tumor grade. The frequencies of GzmB-expressing circulating CD4⁺ CTLs were directly associated with prognosis. We hypothesized that the programed death 1 (PD-1)/PD-ligand 1 (L1) interaction possibly contributed to the suppression of CD4⁺ CTLs in grade II and grade III glioma, since an upregulation of PD-1 was observed on CD4⁺ CTLs in glioma compared to those in the healthy individuals. Blockade of the PD-1/PD-L1 interaction with neutralizing antibodies significantly increased the proliferation and granzyme or perforin production by CD4⁺ CTLs in grade II and grade III glioma patients. Conclusions: These data suggest that the CD4⁺ CTLs in grade II and grade III glioma patients contribute to antitumor immunity and could be suppressed by PD-1 signal transduction.     

Cytochemical and cytological properties of perineuronal oligodendrocytes in the mouse cortex

Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase‐positive cells attaching to neuronal perikarya immunostained for microtubule‐associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase‐positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain‐type lipid binding protein and glial fibrillary acidic protein), microglia (Iba‐1) and NG2⁺ glia. However, almost all perineuronal oligodendrocytes expressed glia‐specific or glia‐enriched metabolic enzymes, i.e. the creatine synthetic enzyme S‐adenosylmethionine:guanidinoacetate N‐methyltransferase and l ‐serine biosynthetic enzyme 3‐phosphoglycerate dehydrogenase. As to molecules participating in the glutamate–glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT‐1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV–VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission.     

Pathogenic CD8+ T cells in multiple sclerosis

Traditionally, autoimmune pathogeneses have been attributed to CD4⁺ T lymphocytes, as in multiple sclerosis (MS), rheumatoid arthritis, type 1 diabetes mellitus, and/or to B lymphocytes, as in myasthenia gravis and systemic lupus erythematosus. That is because their primary genetic associations are mostly with certain human leukocyte antigen class II alleles, whose gene products present antigens to CD4⁺ T cells. Because few autoimmune diseases show stronger associations with major histocompatibility complex class I alleles (ankylosing spondylitis, Behçet's disease, and psoriasis), CD8⁺ T cells, which interact with major histocompatibility complex class I molecules, have been largely ignored in autoimmunity research. However, a variety of findings has recently revived interest in this population, particularly in MS. First, it shows associations with major histocompatibility complex class I alleles. Second, its lesions show a predominance of CD8⁺ T cells. Third, these represent effectors that can directly damage central nervous system target cells. Furthermore, several clinical trials of monoclonal antibodies specifically against CD4⁺ T cells, or the polarizing cytokines on which they depend, have failed to show any therapeutic benefit in MS, unlike broader‐spectrum antibodies that deplete all T cells. Here, we review the evidence that CD8⁺ T cells play a role in MS pathogenesis. Ann Neurol 2009;66:132–141     

NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS

Glial progenitor cells of the developing CNS committed to the oligodendrocyte lineage (OPCs) express the chondroitin sulfate proteoglycan, NG2. A proportion of OPCs fail to differentiate past the stage at which they express NG2 and the lipid antigen O4 and persist in the adult CNS in a phenotypically immature form. However, the physiological function of NG2(+) cells in the adult CNS is unknown. Using antibodies against NG2 we show that NG2 is expressed by a distinct cell population in the mature CNS with the homogeneous antigenic phenotype of oligodendrocyte progenitors. The morphology of NG2(+) OPCs varies from region to region, reflecting the different structural environments, but they appear to represent a homogeneous population within any one gray or white matter region. A study of nine CNS regions showed that NG2(+) OPCs are numerous throughout the CNS and numbers in the white matter are only 1.5 times that in the gray. Whereas the ratio of OPCs to myelinating oligodendrocytes in the spinal cord gray and white matter approximates 1:4, gray matter regions of the forebrain have a 1:1 ratio, a phenomenon that will have consequences for oligodendrocyte replacement following demyelination. BrdU incorporation experiments showed that NG2(+) cells are the major dividing cell population of the adult rat CNS. Since very little apoptosis was detected and BrdU became increasingly present in oligodendrocytes after a 10-day pulse chase, with a concomitant decrease in NG2(+) BrdU incorporating cells, we suggest that the size of the oligodendrocyte population may actually increase during adult life.     

NKCC1 Inhibition Attenuates Chronic Cerebral Hypoperfusion-Induced White Matter Lesions by Enhancing Progenitor Cells of Oligodendrocyte Proliferation

Cerebral white matter is vulnerable to ischemic condition. However, no effective treatment to alleviate or restore the myelin damage caused by chronic cerebral hypoperfusion has been found. Na⁺-K⁺-Cl^? cotransporter 1 (NKCC1), a Na⁺-K⁺-Cl^? cotransporter widely expressed in the central nervous system (CNS), involves in regulation of cell swelling, EAA release, cell apoptosis, and proliferation. Nevertheless, the role of NKCC1 in chronic hypoperfusion-induced white matter lesions (WMLs) has not been explored. Here, mice subjected to bilateral common carotid artery stenosis (BCAS) were used as model of chronic cerebral hypoperfusion; density of progenitor cells of oligodendrocyte (OPCs), oligodendrocytes (OLs), astrocytes, and microglia was assessed by immunofluorescent staining and Western blot analysis; working memory was examined by eight-arm radial maze test; expression of MAPK signaling pathway was determined by Western blot analysis. After BCAS, white matter integrity disruption and working memory impairment were observed. NKCC1 inhibition by bumetanide administration enhanced OPC proliferation, attenuated chronic hypoperfusion-induced white matter damage, and promoted recovery of neurological function. However, NKCC1 inhibition caused no significant change in the densities of GFAP- and Iba-1-positive cells in the corpus callosum. Bumetanide administration significantly increased the expression of p-ERK and decreased the expression of p-JNK and p-p38 in comparison to vehicle-BCAS groups. In conclusion, NKCC1 inhibition might significantly ameliorate chronic cerebral hypoperfusion-induced WMLs and cognitive impairment by enhancing progenitor cells of oligodendrocyte proliferation, and this protective function of bumetanide might be mediated by modulation of the MAPK signaling pathway.     

Granzyme‐b mediated cell death in the spinal cord‐injured rat model

Spinal cord injury initiates a complex series of inflammatory and immune responses including the influx of monocytes, macrophages, T‐cells, NK cells and so on, into the injured area. In the present study, we found a significant increase in the levels of granzyme‐b (gra‐b) from the first day after the transection until the third day, with decrease in intensity thereafter. The chemokine IP‐10/CXCL10 was also found to be elevated along with gra‐b correlating with the infiltration of CD‐8⁺ cytotoxic T lymphocytes (CTLs) into the injured spinal cord. We observed an increase in the levels of the 64 kDa poly ADP ribose polymerase fragment, known to be a signature fragment produced by gra‐b. Localization of gra‐b in TUNEL positive neurons indicates that gra‐b might play a crucial role in neuronal death and contributes to the pathophysiology of spinal cord injury.