Cross-talk between B cells, microglia and macrophages, and implications to central nervous system compartmentalized inflammation and progressive multiple sclerosis.

BACKGROUND: B cells can be enriched within meningeal immune-cell aggregates of multiple sclerosis (MS) patients, adjacent to subpial cortical demyelinating lesions now recognized as important contributors to progressive disease. This subpial demyelination is notable for a 'surface-in' gradient of neuronal loss and microglial activation, potentially reflecting the effects of soluble factors secreted into the CSF. We previously demonstrated that MS B-cell secreted products are toxic to oligodendrocytes and neurons. The potential for B-cell-myeloid cell interactions to propagate progressive MS is of considerable interest. METHODS: Secreted products of MS-implicated pro-inflammatory effector B cells or IL-10-expressing B cells with regulatory potential were applied to human brain-derived microglia or monocyte-derived macrophages, with subsequent assessment of myeloid phenotype and function through measurement of their expression of pro-inflammatory, anti-inflammatory and homeostatic/quiescent molecules, and phagocytosis (using flow cytometry, ELISA and fluorescently-labeled myelin). Effects of secreted products of differentially activated microglia on B-cell survival and activation were further studied. FINDINGS: Secreted products of MS-implicated pro-inflammatory B cells (but not IL-10 expressing B cells) substantially induce pro-inflammatory cytokine (IL-12, IL-6, TNFα) expression by both human microglia and macrophage (in a GM-CSF dependent manner), while down-regulating their expression of IL-10 and of quiescence-associated molecules, and suppressing their myelin phagocytosis. In contrast, secreted products of IL-10 expressing B cells upregulate both human microglia and macrophage expression of quiescence-associated molecules and enhance their myelin phagocytosis. Secreted factors from pro-inflammatory microglia enhance B-cell activation. INTERPRETATION: Potential cross-talk between disease-relevant human B-cell subsets and both resident CNS microglia and infiltrating macrophages may propagate CNS-compartmentalized inflammation and injury associated with MS disease progression. These interaction represents an attractive therapeutic target for agents such as Bruton's tyrosine kinase inhibitors (BTKi) that modulate responses of both B cells and myeloid cells. FUNDING: Stated in Acknowledgments section of manuscript.

Sigma-1 receptor agonists as potential protective therapies in multiple sclerosis.

The sigma-1 receptor (σ-1R) is an endoplasmic reticulum (ER) chaperone upregulated during ER stress, and regulates calcium homeostasis. Agonists of σ-1R are neuroprotective. ANAVEX2-73, a new σ-1R agonist, is undergoing several clinical trials. We show that ANAVEX2-73 protects oligodendroglia (OL) and oligodendroglial precursors (OPC) from apoptosis, excitotoxicity, reactive oxygen species (ROS) and quinolinic acid (QA), associated with inflammation. ANAVEX2-73 stimulates OPC proliferation, but does not alter early maturation to OL. We previously reported that dextromethorphan (DM), another σ-1R agonist with a different structure, had similar effects. We now show that both DM and ANAVEX2-73 protect neurons from the four cytotoxic agents.

Exosome-enriched fractions from MS B cells induce oligodendrocyte death.

Objective: To identify whether factors toxic to oligodendrocytes (OLs), released by B cells from patients with MS, are found in extracellular microvesicles enriched in exosomes. Methods: Conditioned medium (Sup) was obtained from cultures of blood B cells of patients with MS and normal controls (NCs). Exosome-enriched (Ex-En) fractions were prepared by solvent precipitation from Sup containing bovine serum and from serum-free Sup by ultracentrifugation (UC) or immunoprecipitation (IP) with antibodies to CD9. Ex-En fractions were diluted 1:4 with OL culture medium and screened for toxic effects on cultured rat OLs as measured by trypan blue uptake. Proteomic analysis was performed on Sup fractions. Results: MS B cell-derived Ex-En fractions prepared from Sup by solvent extraction, UC, or IP induced OL death, whereas corresponding Ex-En fractions from NC showed little toxicity. Proteomic analysis of Sup demonstrated enrichment of proteins characteristic of exosomes from both NC and MS B-cell Sup. Ontology enrichment analysis suggested differences in the types and cargo of exosomes from MS Sup compared with NC, with proteins related to cell surface, extracellular plasma membrane, and gliogenesis enriched in MS. Conclusions: Much of the in vitro toxicity of Sup from B cells of patients with relapsing-remitting MS is found in Ex-En fractions, as confirmed by 3 methods. Proteomic analysis of B-cell Sup indicates multiple differences between MS and NC.

Melanocortin receptor subtypes are expressed on cells in the oligodendroglial lineage and signal ACTH protection.

ACTH, a melanocortin peptide used to treat multiple sclerosis (MS) relapses, acts by stimulating adrenal corticosteroid (CS) production via melanocortin receptor 2 (MC2R), but it may also exert a therapeutic effect independent of CS by stimulating other melanocortin receptors (MCR) distributed in many tissues, including the brain. We reported that oligodendroglia (OL) and oligodendroglial precursor cells (OPC) express MC4R, and that ACTH 1-39 protects OL and OPC in vitro from cell death induced by mechanisms likely involved in white matter damage in MS. This study investigates expression of MC1R, MC2R, MC3R and MC5R in OL and MC4R in OPC using immunocytochemistry with MCR subtype specific antibodies. OL express surface MC1R, MC3R and MC5R, in addition to MC4R. To investigate whether these receptors are functional, we asked if signaling through MCR is involved in ACTH protection of cultured rat OL from apoptosis (staurosporine), or cell death induced by excitotoxicity (glutamate), reactive oxygen species (ROS), or an inflammatory mediator (quinolinic acid). Like ACTH 1-39, MCR subtype specific agonists for MC1R, MC3R, MC4R and MC5R all protected OL from these insults. Conversely, antagonists for MC3R and MC4R blocked ACTH protection of OL. We then investigated the role of MC4R, as a prototype MCR, in protection and proliferation of OPC; MC4R agonists protected OPC and increased their proliferation, while antagonists blocked these effects. Our results demonstrate that MCR on OL and OPC are functional and activate signaling pathways that protect against mechanisms involved in OL damage in MS, suggesting potential beneficial effects in neurologic diseases.

Melanocortins, Melanocortin Receptors and Multiple Sclerosis.

The melanocortins and their receptors have been extensively investigated for their roles in the hypothalamo-pituitary-adrenal axis, but to a lesser extent in immune cells and in the nervous system outside the hypothalamic axis. This review discusses corticosteroid dependent and independent effects of melanocortins on the peripheral immune system, central nervous system (CNS) effects mediated through neuronal regulation of immune system function, and direct effects on endogenous cells in the CNS. We have focused on the expression and function of melanocortin receptors in oligodendroglia (OL), the myelin producing cells of the CNS, with the goal of identifying new therapeutic approaches to decrease CNS damage in multiple sclerosis as well as to promote repair. It is clear that melanocortin signaling through their receptors in the CNS has potential for neuroprotection and repair in diseases like MS. Effects of melanocortins on the immune system by direct effects on the circulating cells (lymphocytes and monocytes) and by signaling through CNS cells in regions lacking a mature blood brain barrier are clear. However, additional studies are needed to develop highly effective MCR targeted therapies that directly affect endogenous cells of the CNS, particularly OL, their progenitors and neurons.

B cells from patients with multiple sclerosis induce cell death via apoptosis in neurons in vitro.

B cells mediate multiple sclerosis (MS) pathogenesis by mechanisms unrelated to immunoglobulin (Ig). We reported that supernatants (Sup) from cultured B cells from blood of relapsing remitting MS (RRMS) patients, but not normal controls (NC), were cytotoxic to rat oligodendrocytes (OL). We now show that RRMS blood B cells, not stimulated in vitro, secrete factor/s toxic to rat and human neurons. Cytotoxicity is independent of Ig and multiple cytokines, not complement-mediated, and involves apoptosis. The factor/s have an apparent mw of >300kDa. B cells could contribute to damage within the central nervous system by secreting molecules toxic to OL and neurons.

Schwann cell differentiation inhibits interferon-gamma induction of expression of major histocompatibility complex class II and intercellular adhesion molecule-1.

Interferon-gamma (IFN-γ) upregulates major histocompatibility complex class II (MHC class II) antigens and intercellular adhesion molecule-1 (ICAM-1) on Schwann cells (SC) in vitro, but in nerves of animals and patients MHC class II is primarily expressed on inflammatory cells. We investigated whether SC maturation influences their expression. IFN-γ induced MHC class II and upregulated ICAM-1; the axolemma-like signal 8-bromo cyclic adenosine monophosphate (8 Br cAMP) with IFN-γ inhibited expression. Delaying addition of 8 Br cAMP to SC already exposed to IFN-γ inhibited ongoing expression; addition of IFN-γ to SC already exposed to 8 Br cAMP resulted in minimal expression. Variability of cytokine-induced MHC class II and ICAM-1 expression by SC in vivo may represent the variability of signals from axolemma.

The melanocortin ACTH 1-39 promotes protection of oligodendrocytes by astroglia.

Damage to myelin and oligodendroglia (OL) in multiple sclerosis (MS) results from a wide array of mechanisms including excitotoxicity, neuroinflammation and oxidative stress. We previously showed that ACTH 1-39, a melanocortin, protects OL in mixed glial cultures and enriched OL cultures, inhibiting OL death induced by staurosporine, ionotropic glutamate receptors, quinolinic acid or reactive oxygen species (ROS), but not nitric oxide (NO) or kynurenic acid. OL express melanocortin receptor 4 (MC4R), suggesting a direct protective effect of ACTH 1-39 on OL. However, these results do not rule out the possibility that astroglia (AS) or microglia (MG) also play roles in protection. To investigate this possibility, we prepared conditioned medium (CM) from AS and MG treated with ACTH, then assessed the protective effects of the CM on OL. CM from AS treated with ACTH protected OL from glutamate, NMDA, AMPA, quinolinic acid and ROS but not from kainate, staurosporine, NO or kynurenic acid. CM from MG treated with ACTH did not protect from any of these molecules, nor did CM from AS or MG not treated with ACTH. While protection of OL by ACTH from several toxic molecules involves direct effects on OL, ACTH can also stimulate AS to produce mediators that protect against some molecules but not others. Thus the cellular mechanisms underlying the protective effects of ACTH for OL are complex, varying with the toxic molecules.

Melanocortin receptor agonist ACTH 1-39 protects rat forebrain neurons from apoptotic, excitotoxic and inflammation-related damage.

Patients with relapsing-remitting multiple sclerosis (RRMS) are commonly treated with high doses of intravenous corticosteroids (CS). ACTH 1-39, a member of the melanocortin family, stimulates production of CS by the adrenals, but melanocortin receptors are also found in the central nervous system (CNS) and on immune cells. ACTH is produced within the CNS and may have direct protective effects on glia and neurons independent of CS. We previously reported that ACTH 1-39 protected oligodendroglia (OL) and their progenitors (OPC) from a panel of excitotoxic and inflammation-related agents. Neurons are the most vulnerable cells in the CNS. They are terminally differentiated, and sensitive to inflammatory and excitotoxic insults. For potential therapeutic protection of gray matter, it is important to investigate the direct effects of ACTH on neurons. Cultures highly enriched in neurons were isolated from 2-3 day old rat brain. After 4-7 days in culture, the neurons were treated for 24h with selected toxic agents with or without ACTH 1-39. ACTH 1-39 protected neurons from death induced by staurosporine, glutamate, NMDA, AMPA, kainate, quinolinic acid, reactive oxygen species and, to a modest extent, from rapidly released NO, but did not protect against kynurenic acid or slowly released nitric oxide. Our results show that ACTH 1-39 protects neurons in vitro from several apoptotic, excitotoxic and inflammation-related insults.

Adrenocorticotropin hormone 1-39 promotes proliferation and differentiation of oligodendroglial progenitor cells and protects from excitotoxic and inflammation-related damage.

Oligodendroglia (OL) are highly susceptible to damage and, like neurons, are terminally differentiated. It is important to protect OL precursors (OPC) because they are reservoirs of differentiating cells capable of myelination following perinatal insult and remyelination in white matter diseases, including multiple sclerosis (MS). Patients with relapsing-remitting MS are commonly treated with high-dose corticosteroids (CS) when experiencing an exacerbation. Adrenocorticotropin hormone (ACTH), a primary component of another approved MS exacerbation treatment, is a melanocortin peptide that stimulates production of CS by the adrenals. Melanocortin receptors are also found in the central nervous system (CNS) and on immune cells. ACTH is produced within the CNS and may have CS-independent effects on glia. We found that ACTH 1-39 stimulated proliferation of OPC, and to a lesser extent astroglia (AS) and microglia (MG), in rat glial cultures. ACTH accelerated differentiation of PDGFRα(+) OPC to a later stage marked by galactolipid expression and caused greater expansion of OL myelin-like sheets compared with untreated cells. Protective effects of ACTH on OPC were assessed by treating cultures with selected toxic agents, with or without ACTH. At 200 nM, ACTH protected OPC from death induced by staurosporine, glutamate, NMDA, AMPA, kainate, quinolinic acid, H2 O2 , and slow NO release, but not against kynurenic acid or rapid NO release. These agents and ACTH were not toxic to AS or MG. Our findings indicate that ACTH 1-39 provides benefits by increasing the number of OPC, accelerating their development into mature OL, and reducing OPC death from toxic insults.

Effects of dextromethorphan on glial cell function: proliferation, maturation, and protection from cytotoxic molecules.

Dextromethorphan (DM), a sigma receptor agonist and NMDA receptor antagonist, protects neurons from glutamate excitotoxicity, hypoxia and ischemia, and inhibits microglial activation, but its effects on differentiation and protection of cells in the oligodendroglial lineage are unknown. It is important to protect oligodendroglia (OL) to prevent demyelination and preserve axons, and to protect oligodendroglial progenitors (OPC) to optimize myelination during development and remyelination following damage. Enriched glial cultures from newborn rat brain were used 1-2 days or 6-8 days after shakeoff for OPC or mature OL. DM had large effects on glial proliferation in less mature cultures in contrast to small variable effects in mature cultures; 1 µM DM stimulated proliferation of OPC by 4-fold, microglia (MG) by 2.5-fold and astroglia (AS) by 2-fold. In agreement with increased OPC proliferation, treatment of OPC with DM for 3 days increased the % of OPC relative to OL, with a smaller difference by 5 days, suggesting that maturation of OPC to OL was "catching up" by 5 days. DM at 2 and 20 µM protected both OL and OPC from killing by glutamate as well as NMDA, AMPA, quinolinic acid, staurosporine, and reactive oxygen species (ROS). DM did not protect against kynurenic acid, and only modestly against NO. These agents and DM were not toxic to AS or MG at the concentrations used. Thus, DM stimulates proliferation of OPC, and protects both OL and OPC against excitotoxic and inflammatory insults.

Direct effects of secretory products of immune cells on neurons and glia.

This review summarizes recent research contributions by Dr. Robert Lisak in collaboration with Dr. Joyce Benjamins on direct effects of secretory products of immune cells on neurons and glia. Highlights from studies analyzing cytokine-induced changes in early gene expression in mixed CNS glial cultures focus on comparison of potential damaging effects of pro-inflammatory cytokines versus protective effects of downregulatory cytokines. The three categories of changes examined include (a) immune-related molecules, (b) neurotrophins, growth factors and structural proteins, and (c) molecules associated with metabolism, signaling and regulation. Subsequent studies in CNS neuronal cultures showed that early responses of neurons to cytokines were fewer in number and lower in magnitude than in glia, consistent with the idea that microglia and astroglia serve as "first responders" to inflammatory signals. To explore the hypothesis that B cells of patients with multiple sclerosis secrete soluble products damaging to oligodendroglia (OL), in collaboration with Dr. Bar-Or at Montreal Neurologic Institute, we compared secretory products of cultured B cells from relapsing remitting multiple sclerosis (RRMS) patients and healthy controls. In support of the hypothesis, 7 supernatants from RRMS B cells induced death of rat OL in vitro, while 3 of 4 control samples did not.

ACTH protects mature oligodendroglia from excitotoxic and inflammation-related damage in vitro.

Corticosteroids (CS) are widely employed to treat relapses in multiple sclerosis (MS). Endogenous ACTH is a 39-amino acid peptide that, among other functions, stimulates CS production. Exogenous ACTH 1-39 is used to treat MS relapses, presumably by stimulating endogenous CS production. However, unlike CS, ACTH binds to melanocortin receptors, found in the central nervous system (CNS) as well as on inflammatory cells. Since glia are implicated in MS and other neurodegenerative diseases, and oligodendroglia (OL) are more sensitive to injury than other glia, we characterized the protective effects of ACTH on OL in vitro without the confounding effects of CS. Rat brain cultures containing OL, astrocytes (AS), and microglia (MG) were incubated for 1 day with potentially cytotoxic agents with or without preincubation with ACTH 1-39. The cytotoxic agents killed 55-70% of mature OL, but caused little or no death of AS or MG at the concentrations used. ACTH protected OL from death induced by staurosporine, AMPA, NMDA, kainate, quinolinic acid, or reactive oxygen species, but did not protect against kynurenic acid or nitric oxide. The protective effects of ACTH were dose dependent, and decreased OL death induced by the different agents by 30-60% at 200 nM ACTH. We show for the first time that melanocortin 4 receptor is expressed on OL in addition to MG and AS. In summary, ACTH 1-39 protects OL in vitro from several excitotoxic and inflammation-related insults. ACTH may be activating melanocortin receptors on OL or alternately on AS or MG to prevent OL death.

Secretory products of multiple sclerosis B cells are cytotoxic to oligodendroglia in vitro.

B cells are important in the pathogenesis of multiple sclerosis (MS) and some of the effects are not dependent on maturation of B cells into immunoglobulin (Ig) producing plasmablasts and plasma cells. B cells present antigen, activate T cells, and are involved in immunoregulation and cytokine secretion. To determine if B cells from MS patients secrete products that have deleterious effects on glial cells not mediated by Ig, and to compare effects with secretory products of normal controls (NC), we isolated B cells from 7 patients with relapsing remitting MS (RRMS) and 4 NC. B cells were cultured alone or after stimulation with CD40 ligand (CD40L), CD40L+cross-linking of the B cell antigen receptor (xBCR) and CD40L+xBCR+stimulation of toll like receptor 9 (TLR9). Supernatants were harvested and incubated with mixed central nervous system (CNS) neonatal rat glial cells. Supernatants from unstimulated NC B cells induced on average death of 7% (range 0-24%) of differentiated oligodendrocytes (OL); in contrast, supernatants from unstimulated B cells from RRMS patients induced death of 57% (range 35-74%) of OL. Supernatants of stimulated B cells from NC did not increase the minimal OL death whereas stimulation of B cells from RRMS had variable results compared to unstimulated B cells. Supernatants from both NC and RRMS induced microglial enlargement and loss of normal resting bipolar morphology. OL death did not correlate with levels of tumor necrosis alpha (TNF-α), lymphotoxin alpha (LT-α), interleukin 6 (IL-6), IL-10, transforming growth factor beta 1 (TGF-ß1) or any combination or ratio of these cytokines. Analysis of 26 supernatants from NC and RRMS patients failed to detect IgM. There were very low levels of IgG in 8 of the 26 supernatants, and no correlation between of OL death and presence or absence of IgG. Sera used in both the B cell and glial cell cultures were heated, which inactivates complement. The effects of B cell supernatants on OL could be direct and/or indirect involving either microglia and/or astrocytes. The identity of the toxic factor(s) is as yet unknown. Thus we have demonstrated that B cells from patients with RRMS but not NC secrete one or more factors toxic to OL. It is possible that such factors produced by peripheral blood B cells when within the CNS could contribute to demyelination in MS patients.

Cytokines regulate neuronal gene expression: differential effects of Th1, Th2 and monocyte/macrophage cytokines.

Inflammatory mediators, including cytokines, contribute to neuronal and axonal dysfunction and cell death. To examine the roles of cytokines in pathogenesis and regeneration in the central nervous system (CNS), we analyzed effects of cytokines on early gene regulation (6h) in neuronal cultures, employing gene arrays. Our hypothesis is that neuronal gene expression is differentially regulated in vitro by cytokine mixtures typical of Th1 and Th2 T cells and monocytes/macrophages (M/M). Th1 and M/M cytokines showed similar patterns for regulation of numerous pathways including cytokine-receptor interactions, MAP kinase, toll like receptors, apoptosis, PPAR signaling, cell adhesion molecules (CAMS), antigen processing, adipocytokine, and JAK-STAT signaling. M/M cytokines uniquely regulated genes in T cell, B cell and ECM receptor signaling pathways. Th2 cytokines had few effects on pathways regulated by Th1 and MM cytokines, but uniquely regulated genes related to neuroactive ligand-receptors and calcium. Th1 and MM cytokines markedly upregulated a wide array of cytokine-related genes. Notably, M/M cytokines uniquely upregulated G-CSF, GM-CSF, CXCL5 and lymphotactin (Xcl1). Th2 cytokines did not upregulate cytokine-related genes, with the exception of CCL11 and FMS-like tyrosine kinase 1, a VEGF receptor. In neuroactive ligand-receptor pathways, Th1 and M/M cytokines upregulated gene expression for tryptophan hydroxylase. Th1 cytokines upregulated gene expression for GABA A receptor, delta, while Th2 cytokines downregulated GABA A receptor, gamma 3. Significant changes occurred in several genes in the wnt and Notch signaling pathways, which are highly conserved and play critical roles in neuronal and glial differentiation. In the ubiquitin-proteasome pathway, proinflammatory cytokine mixtures induced upregulation of several genes, notably ubiquitin D (Ubd/FAT10), ubiquitin ligase and several proteasomal proteins. In agreement with microarray results, QRT-PCR showed marked upregulation of gene expression for Ubd with Th1 and M/M, for transglutaminase 2 with M/M, and for arginase 1 with Th2 cytokines. Expression of Ubd in the nervous system has not been previously reported. Both message and protein for Ubd are expressed in neurons, and upregulated by pro-inflammatory cytokines. Transglutaminase 2 has been implicated in neurodegenerative diseases, and proposed as a therapeutic target. Upregulation of arginase by Th2 cytokines could be potentially neuroprotective by decreasing NO generation and enhancing neurite outgrowth. Our analysis of changes in neuronal gene expression at the time of initial exposure to an abnormal cytokine milieu provides the opportunity to identify early changes that could be reversed to prevent later irreversible neuronal damage and death in multiple sclerosis and other CNS diseases.

Cytokines reduce toxic effects of ethanol on oligodendroglia.

To characterize immunomodulatory mechanisms that affect oligodendroglia (OL) and white matter following ethanol exposure during early CNS development, we investigated the direct effects of ethanol and cytokines on glia. Mixed glial cultures from newborn rat brain were exposed to 6.5-130 mM ethanol for 1-3 days. OL were sensitive to ethanol, with death ranging from 32 to 88% with increasing time and ethanol concentrations. Little cell death occurred in astroglia or microglia. Mixtures of cytokines representative of those produced by pro-inflammatory Th1 and monocyte/macrophage (M/M) cells as well as those produced by anti-inflammatory Th2 cells were all protective. Three of the cytokines in the Th1 mixture, IL-2, TNF-α and IFN-γ, were protective individually, although no single cytokine was as effective as the mixture. The protective effects of the Th1 mixture and of IL-2 were reversed by inhibition of both MAP kinase and PI-3 kinase signaling pathways. We conclude that cytokines can act either directly on OL or indirectly through effects on astroglia or microglia to protect OL from ethanol toxicity.

Cytokines decrease expression of interleukin-6 signal transducer and leptin receptor in central nervous system glia.

Multiple sclerosis (MS) lesion formation is modulated by cytokines secreted within the central nervous system (CNS). Th1 lymphocytes and monocyte/macrophages (MM) likely induce lesion formation, whereas Th2 lymphocytes may inhibit formation. To explore the role of cytokines in MS lesions, we used gene arrays to investigate effects of cytokines representative of Th1 and Th2 cells and M/M on gene expression in cultured CNS glia; at 6 hr, all three increased expression of the interleukin-6 (IL-6) gene and decreased expression of the leptin receptor gene (obr), which mediates IL-6 production and other inflammatory responses. However, expression of a closely related gene, the interleukin-6 signal transducer or gp130 (IL-6st), showed no changes at 6 hr. IL-6st is an essential component of receptor complexes for IL-6 and other cytokines and growth factors that play critical roles in CNS inflammation, protection, and/or regeneration. To analyze expression of IL-6st and leptin receptor over time, we incubated rat CNS glial cultures for 6 hr to 5 days with the cytokines. All three cytokine mixtures down-regulated both IL-6st and leptin receptor mRNA and protein for up to 5 days. Immunocytochemical staining showed expression of both IL-6st and leptin receptor in all three types of glia, with lower IL-6st expression by 3 days. Down-regulation of IL-6st and leptin receptor in glia by cytokines could lead to decreased signaling by the proinflammatory IL-6 and reduced responses to regenerative/protective growth factors such as leukemia inhibitory factor and ciliary neurotrophic factor, potentially affecting the disease course in MS.

Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for molecules associated with metabolism, signaling and regulation in central nervous system mixed glial cell cultures.

BACKGROUND: Cytokines secreted by immune cells and activated glia play central roles in both the pathogenesis of and protection from damage to the central nervous system (CNS) in multiple sclerosis (MS). METHODS: We have used gene array analysis to identify the initial direct effects of cytokines on CNS glia by comparing changes in early gene expression in CNS glial cultures treated for 6 hours with cytokines typical of those secreted by Th1 and Th2 lymphocytes and monocyte/macrophages (M/M). RESULTS: In two previous papers, we summarized effects of these cytokines on immune-related molecules, and on neural and glial related proteins, including neurotrophins, growth factors and structural proteins. In this paper, we present the effects of the cytokines on molecules involved in metabolism, signaling and regulatory mechanisms in CNS glia. Many of the changes in gene expression were similar to those seen in ischemic preconditioning and in early inflammatory lesions in experimental autoimmune encephalomyelitis (EAE), related to ion homeostasis, mitochondrial function, neurotransmission, vitamin D metabolism and a variety of transcription factors and signaling pathways. Among the most prominent changes, all three cytokine mixtures markedly downregulated the dopamine D3 receptor, while Th1 and Th2 cytokines downregulated neuropeptide Y receptor 5. An unexpected finding was the large number of changes related to lipid metabolism, including several suggesting a switch from diacylglycerol to phosphatidyl inositol mediated signaling pathways. Using QRT-PCR we validated the results for regulation of genes for iNOS, arginase and P glycoprotein/multi-drug resistance protein 1 (MDR1) seen at 6 hours with microarray. CONCLUSION: Each of the three cytokine mixtures differentially regulated gene expression related to metabolism and signaling that may play roles in the pathogenesis of MS, most notably with regard to mitochondrial function and neurotransmitter signaling in glia.

Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins.

BACKGROUND: In multiple sclerosis, inflammatory cells are found in both active and chronic lesions, and it is increasingly clear that cytokines are involved directly and indirectly in both formation and inhibition of lesions. We propose that cytokine mixtures typical of Th1 or Th2 lymphocytes, or monocyte/macrophages each induce unique molecular changes in glial cells. METHODS: To examine changes in gene expression that might occur in glial cells exposed to the secreted products of immune cells, we have used gene array analysis to assess the early effects of different cytokine mixtures on mixed CNS glia in culture. We compared the effects of cytokines typical of Th1 and Th2 lymphocytes and monocyte/macrophages (M/M) on CNS glia after 6 hours of treatment. RESULTS: In this paper we focus on changes with potential relevance for neuroprotection and axon/glial interactions. Each mixture of cytokines induced a unique pattern of changes in genes for neurotrophins, growth and maturation factors and related receptors; most notably an alternatively spliced form of trkC was markedly downregulated by Th1 and M/M cytokines, while Th2 cytokines upregulated BDNF. Genes for molecules of potential importance in axon/glial interactions, including cell adhesion molecules, connexins, and some molecules traditionally associated with neurons showed significant changes, while no genes for myelin-associated genes were regulated at this early time point. Unexpectedly, changes occurred in several genes for proteins initially associated with retina, cancer or bone development, and not previously reported in glial cells. CONCLUSION: Each of the three cytokine mixtures induced specific changes in gene expression that could be altered by pharmacologic strategies to promote protection of the central nervous system.