ABSTRACT
FTY720 is a new oral immunomodulatory therapy for the treatment of multiple sclerosis (MS). There is strong evidence that FTY720 has direct effects on brain resident cells such as astrocytes acting via sphingosine1phosphate (S1P) receptors. In the present study, the mRNA expression of S1P receptors as well as selected cytokines, chemokines and growth factors were investigated in primary murine astrocytes under inflammatory conditions in the presence or absence of the phosphorylated form of FTY720 (FTY720P). Following stimulation with either the proinflammatory cytokine tumor necrosis factorα (TNFα) or with bacterial lipopolysaccharide, there was an increased expression of the receptors S1P1 and S1P3, the cytokines and chemokines interleukin (IL)1ß, chemokine (CCmotif) ligand 2 (CCL2), CCL20 and chemokine (CXCmotif) ligand 12 as well as the growth factors insulinlike growth factor1, ciliary neurotrophic factor and glial cell linederived neurotrophic factor (GDNF). FTY720P led to an increased expression of IL1ß and GDNF at distinct time points following costimulation with TNFα compared with TNFα treatment alone. However, the presence of FTY720P did not have any further significant effects on the expression of S1P receptors, cytokines or growth factors, suggesting that the regulation of these target genes in astrocytes is not likely to be a major mechanism underlying the effect of FTY720P in diseases such as MS.
Subject(s)
Astrocytes/drug effects , Inflammation/metabolism , Organophosphates/pharmacology , Sphingosine/analogs & derivatives , Animals , Astrocytes/cytology , Astrocytes/metabolism , Brain/cytology , Brain/drug effects , Brain/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Chemokine CCL20/genetics , Chemokine CCL20/metabolism , Glial Cell Line-Derived Neurotrophic Factor/genetics , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Inflammation/pathology , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Multiple Sclerosis/drug therapy , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Lysosphingolipid/genetics , Receptors, Lysosphingolipid/metabolism , Sphingosine/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Microglia are resident macrophages in the central nervous system (CNS) and the primary cells that contribute to CNS inflammation in many pathological conditions. Upon any signs of brain damage, microglia become activated and undergo tremendous cellular reorganization to adopt appropriate phenotypes. They migrate to lesion areas, accumulate, phagocytose cells or cellular debris, and produce a large array of inflammatory mediators like cytokines, chemokines, reactive oxygen species, and other mediators. To cope with the extreme cellular rearrangements during activation, microglia have to be highly dynamic. One major component of the cytoskeleton in nonmuscle cells is nonmuscle myosin II (NM II). This study was aimed to examine the functional role of NM II in resting and activated microglia. Using immunohistochemistry, we demonstrate strong expression of NM II isoform B (NM IIB) in microglia during cuprizone-induced demyelination as well as in cultured microglia. Treatment with the NM II inhibitor blebbistatin prevented the morphological shaping of microglial cells, led to functional deficits during chemokine-directed migration and phagocytosis, induced NM IIB redistribution, and affected actin microfilament patterning. In addition, inhibition of NM II led to an attenuated release of nitric oxide (NO), while TNFα secretion was not altered. In conclusion, we propose a pivotal role of NM II in cytoskeleton organization during microglial activation. This is of great importance to understand the mechanisms of microglial action in inflammatory CNS diseases.
Subject(s)
Brain/pathology , Demyelinating Diseases/pathology , Microglia/metabolism , Myosin Heavy Chains/metabolism , Nonmuscle Myosin Type IIB/metabolism , Animals , Animals, Newborn , Brain/cytology , Brain/metabolism , Cell Movement/drug effects , Cells, Cultured , Chemokine CCL2/pharmacology , Cuprizone/toxicity , Demyelinating Diseases/chemically induced , Disease Models, Animal , Dose-Response Relationship, Drug , Heterocyclic Compounds, 4 or More Rings/pharmacology , Male , Mice, Inbred C57BL , Microglia/drug effects , Monoamine Oxidase Inhibitors/toxicity , Phagocytosis/drug effects , Rats , Rats, Sprague-DawleyABSTRACT
For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC) have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE), an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS). In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.
