Janus faces of microglia in multiple sclerosis

Multiple sclerosis (MS) is the most common cause of neurological disability in young adults. The disease is characterized by inflammatory reactions, demyelination and axonal loss in the brain, spinal cord and optic nerves. Microglia seem to play an important role in the inflammatory processes in MS, since they are found in actively demyelinating lesions. Their role in the differentiation of T cells could led to the expansion of inflammation and tissue destruction. However, microglia are also involved in the termination of an inflammatory response and produce protective factors. To be able to therapeutically manipulate microglia, their exact function in the onset and development of MS needs to be clarified. This review provides an overview of the functions of the most important microglia-associated molecules in MS, being CD40, B7-1 and B7-2, interferon-gamma, tumor necrosis factor-alpha, chemokines, prostanoids, and nitric oxide.     

Peroxisome proliferator-activated receptor gamma agonist, rosiglitazone, suppresses CD40 expression and attenuates inflammatory responses after lithium pilocarpine-induced status epilepticus in rats

Inflammatory responses in the brain are involved in the etiopathogenesis and sequelae of seizures. Ligation of microglial CD40 plays a role in the development of inflammatory responses in the central nervous system (CNS). Our study showed that there was an increased CD40 expression on activated microglia in the brain injury after lithium pilocarpine-induced status epilepticus (SE) in rats. Since peroxisome proliferator-activated receptor gamma (PPARgamma) acts as a regulator of CNS inflammation and a powerful pharmacological target for counteracting CNS diseases, we investigated the role of the PPARgamma agonist, rosiglitazone, in the modulation of CD40 expression and in the pathological processes of inflammation after SE. We found that rosiglitazone inhibited the expression of CD40, tumor necrosis factor (TNF-alpha), and microglial activation in different regions of hippocampus. The results were indicated by immunohistochemistry, Western blot, and ELISA, respectively. Rosiglitazone also prevented neuronal loss in the CA1 area after SE observed by Nissl-staining. These protective effects were significantly reversed by the co-treatment with T0070907, a selective antagonist of the PPARgamma, which clearly demonstrated a PPARgamma-dependent mechanism. Our data provide evidence that rosiglitazone considerably attenuates inflammatory responses after SE by suppressing CD40 expression and microglial activation. Our data also support the idea that rosiglitazone might be a potential neuroprotective agent in epilepsy.     

Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia

Microglial cells are resident cells of the CNS and are implicated as regulators and effectors of immune responses which occur within this compartment. The precise role of parenchymal microglia remains speculative because distinctions between these cells, perivascular “microglia,” and blood-derived monocytes/macrophages are not well defined. The current study describes the phenotype and function of microglia immediately upon isolation from the non-inflamed adult human CNS and the phenotypic changes which occur in these cells when maintained in tissue culture. We find that the characteristic phenotype of immediately ex vivo parenchymal microglia (CD11c^-/CD45^low/CD14^-) corresponds to that found in situ in the “normal” human brain. The phenotype differs from that of perivascular “microglia” in situ and PBDM (both CD45^hi/CD14⁺⁺). The immediately ex vivo microglia express B7-2 and HLA class II molecules and can support alloantigen-induced proliferation by CD4⁺ T cells freshly isolated from peripheral blood. Following in vitro culture, the cells are characterized by a bipolar morphology, continued lower levels of CD45 expression compared to PBDM, and slight upregulation of B7-1 and HLA-DR antigen expression. CD14 becomes expressed at high levels on the cells, suggesting that CD14 can serve as an apparent marker of microglia activation which is not based on changes in morphology or APC capacity. Further, treatment of the cells with IFN-γ and LPS causes further upregulation of HLA-DR and clear expression of B7-1 molecules on the surface. The capacity to characterize phenotypic and functional properties of microglia before and after activation provides an opportunity to determine means to manipulate the immune regulatory and effector properties of this cell type. © 1996 Wiley-Liss, Inc.     

Assessment of melatonin's ability to regulate cytokine production by macrophage and microglia cell types

Evidence in support of melatonin's role as an immunomodulator is incomplete and, in some cases, contradictory. The present studies determined whether melatonin modulates the activity of stimulated macrophages. In vitro lipopolysaccharide (LPS, 10-1000 ng/ml) treatment of alveolar, splenic and peritoneal macrophages isolated from mice and/or rats resulted in a dose-dependent increase in interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF-alpha) secretion. Treatment with melatonin (10(-10)-10(-6) M) prior to the addition of LPS, had no effect on IL-1beta or TNF-alpha release. Additionally, melatonin had no effect on stimulated BV2 microglial cell line cytokine secretion. To determine whether melatonin had an indirect effect on macrophage cytokine release via T cells, melatonin was added to unfractionated mouse spleen cells. Again, melatonin showed no priming effect on LPS-stimulated spleen cells. These results suggest that melatonin has no direct or indirect effect on mouse and rat macrophages. In vivo studies, where melatonin was continuously available in the drinking water, showed that melatonin did not have a priming effect on LPS-stimulated mouse peritoneal macrophages. These findings suggest that melatonin is not an important modulator of macrophage and microglia function.     

The expression of proinflammatory cytokine mRNA in the sciatic–tibial nerve of ischemia–reperfusion injury

We evaluated the proinflammatory cytokines, TNF-α and IL-1β, mRNA expression in the rat sciatic and tibial nerves following ischemia–reperfusion (IR) injury, using competitive RT–PCR, to explore the role of cytokines in IR injury. The expressions of both TNF-α and IL-1β mRNA were related to severity of ischemia and occurred with reperfusion rather than ischemia alone. TNF-α gene expression peaked at 24 h of reperfusion, while that of IL-1β peaked at 12 h. These data support the notion that the proinflammatory cytokines TNF-α and IL-1β are involved in the inflammatory response of IR injury to the peripheral nervous system and may be involved in the pathophysiology of ischemic fiber degeneration.     

Retinoic acid inhibits expression of TNF-alpha and iNOS in activated rat microglia

The release of proinflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide by microglia has been implicated in neurotoxicity in chronic neurodegenerative diseases such as Alzheimer's disease. As all-trans-retinoic acid (RA) has been reported to exert anti-inflammatory actions in various cell types, we have examined its effects on the expression of TNF-alpha and inducible nitric oxide synthase (iNOS) in microglia activated by beta-amyloid peptide (Abeta) and lipopolysaccharide (LPS). Exposure of primary cultures of rat microglial cells to Abeta or LPS stimulated the mRNA expression level of TNF-alpha (6-116-fold) and iNOS (8-500-fold) significantly. RA acted in a dose-dependent manner (0.1-10 microM) by attenuating both TNF-alpha (29-97%) and iNOS (61-96%) mRNA expression in microglia exposed to Abeta or LPS. RA-induced inhibition of TNF-alpha and iNOS mRNA expression in activated microglia was accompanied by the concomitant reduction in release of iNOS and TNF-alpha proteins as revealed by nitrite assay and ELISA, respectively. The anti-inflammatory effects of RA were correlated with the enhanced expression of retinoic acid receptor-beta, and transforming growth factor-beta1 as well as the inhibition of NF-kappaB translocation. These results suggest that RA may inhibit the neurotoxic effect of activated microglia by suppressing the production of inflammatory cytokines and cytotoxic molecules.     

Alzheimer's β-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40

Accumulating evidence suggests that β-amyloid (Aβ)-induced inflammatory reactions may partially drive the pathogenesis of Alzheimer's disease (AD). Recent data also implicate similar inflammatory processes in cerebral amyloid angiopathy (CAA). To evaluate the roles of Aβ in the inflammatory processes in vascular tissues, we have tested the ability of Aβ to trigger inflammatory responses in cultured human vascular cells. We found that stimulation with Aβ dose-dependently increased the expression of CD40, and secretion of interferon-γ (IFN-γ) and interleukin-1β (IL-1β) in endothelial cells. Aβ also induced expression of IFN-γ receptor (IFN-γR) both in endothelial and smooth muscle cells. Characterization of the Aβ-induced inflammatory responses in the vascular cells showed that the ligation of CD40 further increased cytokine production and/or the expression of IFN-γR. Moreover, IL-1β and IFN-γ synergistically increased the Aβ-induced expression of CD40 and IFN-γR. We have recently found that Aβ induces expression of adhesion molecules, and that cytokine production and interaction of CD40–CD40 ligand (CD40L) further increase the Aβ-induced expression of adhesion molecules in these same cells. These results suggest that Aβ can function as an inflammatory stimulator to activate vascular cells and induces an auto-amplified inflammatory molecular cascade, through interactions among adhesion molecules, CD40–CD40L and cytokines. Additionally, Aβ_1–42, the more pathologic form of Aβ, induces much stronger effects in endothelial cells than in smooth muscle cells, while the reverse is true for Aβ_1–40. Collectively, these findings support the hypothesis that the Aβ-induced inflammatory responses in vascular cells may play a significant role in the pathogenesis of CAA and AD.     

Microglia replenished OHSC: A culture system to study in vivo like adult microglia

Recent data suggest that ramified microglia fulfil various tasks in the brain. However, to investigate this unique cell type cultured primary microglia are only a poor model. We here describe a method to deplete and repopulate organotypic hippocampal slice cultures (OHSC) with ramified microglia isolated from adult mouse brain creating microglia‐replenished OHSC (Mrep‐OHSC). Replenished microglia integrate into the tissue and ramify to a degree indistinguishable from their counterparts in the mouse brain. Moreover, wild‐type slices replenished with microglia from TNFα‐deficient animals provide similar results as OHSC prepared from microglia‐specific TNFα‐knockout mice (CX3CR1^cre/TNFα^fl/fl). Furthermore, this study demonstrates that replenished microglia in OHSC maintain original functions and properties acquired in vivo. Microglia from ERCC1^Δ/ko mice, a mouse model of accelerated aging, maintain enhanced Mac2 expression and their activated phenotype after replenishment to wild‐type OHSC tissue. Thus, the present study demonstrates that Mrep‐OHSC are a unique tool to construct chimeric brain slices allowing studying the function of different phenotypes of in vivo like microglia in a tissue culture setting. GLIA 2016 GLIA 2016;64:1285–1297     

Interferon-beta-1a treatment increases CD56 natural killer cells and CD4+CD25+ Foxp3 expression in subjects with multiple sclerosis

Disease modifying effects of interferon (IFN)-β therapy in patients with multiple sclerosis (MS) may be mediated in part through enhanced immunoregulation by the CD56^bright subpopulation of natural killer (NK) cells and by Foxp3+ (not italicized) CD4+CD25+ regulatory T cells (Treg). We found that IFN-β-1a(IM) treatment of relapsing–remitting (RR)MS subjects over 12 months significantly increased both percentage of CD56^bright NK cells and Foxp3 mRNA expression compared to baseline values, untreated RRMS subjects and healthy controls (HC). This striking enhancement of two prominent immunoregulatory pathways lends support to the idea that beneficial effects of IFN-β-1a in MS include control of pernicious autoimmunity.     

Effects of malonate C60 derivatives on activated microglia

Activated microglia in acute and chronic neurodegenerative disease of the central nervous system (CNS) can produce large amounts of free radicals, such as reactive oxygen species (ROS), which subsequently contribute to neuropathogenesis. Thus, it is believed that the induction of microglial deactivation can reduce neuronal injury. Buckminsterfullerene (C60) derivatives that possess free radical scavenging properties have been demonstrated to prevent neuronal cell death caused by excitotoxic insult. In this study, we investigated the biological role of two malonic acid C60 derivatives referred as trans-2 and trans-3 on microglia in the presence of the endotoxin lipopolysaccharide (LPS). Treatment of LPS-activated microglia with trans-2 and trans-3 induced a significant degree of transformation of amoeboid microglia to the ramified phenotype. To understand the mechanism underlying this C60 mediated microglial morphological transformation, we examined the production of proinflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), as well as the final NO products (nitrate and nitrite) in the microglial culture supernatant. Although inducible nitric oxide (iNOS) mRNA and protein expression in LPS-activated microglia were slightly decreased by trans-2 and trans-3, levels of nitrate and nitrite were unaffected. Paradoxically, trans-2 and trans-3 were found to increase the release of IL-1beta in the activated microglial culture. However, trans-2 and trans-3 improved the activity of the antioxidant enzyme, superoxide dismutase (SOD) in LPS-treated microglia. Therefore, our results suggest that the C60 derivatives might increase microglial SOD enzymatic activity which causes microglial morphological transformation from the activated amoeboid phenotype to the resting ramified form.     

Parathyroid hormone-related protein (PTHrP) induction in reactive astrocytes following brain injury: a possible mediator of CNS inflammation

PTHrP, a peptide induced in parenchymal organs during endotoxemia and in the synovium in rheumatoid arthritis, has recently been shown to be expressed in immature or transformed human astrocytes, but not in normal cells. This finding has led us to postulate that PTHrP might also be induced in reactive astrocytes in inflamed brain and, thus, act as a mediator of CNS inflammation. To test this hypothesis, PTHrP expression was examined following cortical stab wound injury in rats, a classical model of reactive gliosis. To determine whether PTHrP was induced in glia by TNF-alpha, a known mediator of inflammation in brain and of PTHrP induction in peripheral tissues, and to determine whether PTHrP, in turn, mediated inflammatory changes in glia, in vitro studies with rat astrocytes and glial-enriched mixed brain cells were also undertaken. Consistent with previous reports of PTHrP expression in normal brain, neurons were the primary site of immunoreactive PTHrP expression in the injured cortex 1 day after stab wound injury. Over the subsequent 3 days, specific immunostaining for PTHrP and for GFAP, a marker of reactive astrocytes, appeared in reactive astrocytes at the wound edge and in perivascular astrocytes, reaching a maximum level of expression at the last time point examined (day 4). TNF-alpha induced PTHrP expression in astrocyte and glial-enriched brain cells in vitro, suggesting that this pro-inflammatory peptide was a possible mediator of PTHrP expression in CNS inflammation. PTHrP(1-34) acted in an additive fashion with TNF-alpha to induced astrocyte expression of IL-6, a cytokine with demonstrated neuroprotective effects. Astrocyte proliferation was inhibited by PTHrP(1-34) and PTHrP(1-141), acting via a PTH/PTHrP receptor cAMP signaling pathway. These studies suggest that PTHrP, analogous to its regulatory functions in other non-CNS models of inflammation, may be an important mediator of the inflammatory response in brain.