Falko R. Fischer

Developmental plasticity of CNS microglia

Microglia arise from CD45+ bone marrow precursors that colonize the fetal brain and play a key role in central nervous system inflammatory conditions. We report that parenchymal microglia are uncommitted myeloid progenitors of immature dendritic cells and macrophages by several criteria, including surface expression of “empty” class II MHC protein and their cysteine protease (cathepsin) profile. Microglia express receptors for stem cell factor and can be skewed toward more dendritic cell or macrophage-like profiles in response to the lineage growth factors granulocyte/macrophage colony-stimulating factor or macrophage colony-stimulating factor. Thus, in contrast to other organs, where terminally differentiated populations of resident dendritic cells and/or macrophages outnumber colonizing precursors, the majority of microglia within the brain remain in an undifferentiated state.

Modulation of experimental autoimmune encephalomyelitis: effect of altered peptide ligand on chemokine and chemokine receptor expression.

Experimental autoimmune encephalomyelitis (EAE) is a T helper 1 (Th1) cell mediated demyelinating disease and the principal animal model for multiple sclerosis. Spinal cords from SJL mice primed with proteolipid protein peptide 139-151 (pPLP) expressed the chemokines RANTES, MCP-1, MIP-2, KC, MIP-1alpha, MIP-1beta, Mig, and fractalkine. We also identified IP-10 in these samples and described a sequence polymorphism in this transcript. Chemokine expression was specific for tissues of the central nervous system. MCP-1, IP-10, and MIP-2 RNA expression significantly correlated with clinical score. Chemokine receptor expression generally correlated with ligand expression. pPLP-primed mice expressed the Th1-associated markers CCR5 and CXCR3 on mononuclear cells. In addition, cells expressing CCR1, CCR2, CCR3, CCR4, CCR8, and CXCR2 were detected. Here we demonstrate that altered peptide ligand (APL)-induced protection from EAE was accompanied by modulation of chemokine and chemokine receptor expression. Spinal cord tissue sections from APL-protected mice showed greatly reduced levels of all chemokines and of CCR1, CCR5, CCR8, CXCR2 and CXCR3. The Th2-associated chemokine receptors CCR3 and CCR4 were found in protected mice, supporting the hypothesis that Th1 but not Th2 cells are down-regulated by APL treatment. This report concludes that chemokines and chemokine receptors can be useful tools to follow modulation of autoimmune disease.

Granulocyte-Macrophage Colony-Stimulating Factor Induces an Expression Program in Neonatal Microglia That Primes Them for Antigen Presentation

Neonatal microglial cells respond to GM-CSF and M-CSF by acquiring different morphologies and phenotypes. To investigate the extent and consequences of this process, a global gene expression analysis was performed, with significant changes in transcript levels confirmed by biochemical analyses. Primary murine microglial cells underwent substantial expression reprogramming after treatment with GM-CSF or M-CSF with many differentially expressed transcripts important in innate and adaptive immunity. In particular, many gene products involved in Ag presentation were induced by GM-CSF, but not M-CSF, thus potentially priming relatively quiescent microglia cells for Ag presentation. This function of GM-CSF is distinct from its primary function in cell proliferation and survival.

Macrophage inflammatory protein-2 and KC induce chemokine production by mouse astrocytes.

Astrocytes are specialized cells of the CNS that are implicated in the pathogenesis of multiple sclerosis and experimental allergic encephalomyelitis. In acute and relapsing-remitting experimental allergic encephalomyelitis, the neutrophil chemoattractant CXC chemokines macrophage-inflammatory protein (MIP)-2 and KC are associated with reactive astrocytes in the parenchyma. In vitro treatment of primary astrocyte cultures with nanomolar concentrations of MIP-2 or KC markedly up-regulated expression of the monocyte/T cell chemoattractants monocyte chemoattractant protein-1, inflammatory protein-10, and RANTES by a mechanism that includes stabilization of mRNA. Production of TNF-α and IL-6 transcripts were also noted, as was autocrine induction of MIP-2 and KC message. In addition, low levels of MIP-1α and MIP-1β were induced following treatment with MIP-2 or KC. These effects are specific to astrocytes as MIP-2 treatment of microglial cells failed to elicit chemokine production. The astrocyte chemokine receptor for MIP-2 has 2.5 nM affinity for ligand. Astrocytes from CXCR2-deficient mice still respond to KC and MIP-2, indicating the presence of an alternative or novel high affinity receptor for these ligands. We propose that this KC/MIP-2 chemokine cascade may contribute to the persistence of mononuclear cell infiltration in demyelinating autoimmune diseases.

RANTES-Induced Chemokine Cascade in Dendritic Cells

Dendritic cells (DC) are the most potent APCs and the principal activators of naive T cells. We now report that chemokines can serve as activating agents for immature DC. Murine bone marrow-derived DC respond to the CC chemokine RANTES (10–100 ng/ml) by production of proinflammatory mediators. RANTES induces rapid expression of transcripts for the CXC chemokines KC and macrophage inflammatory protein (MIP)-2, the CC chemokines MIP-1β and MIP-1α, and the cytokines TNF-α and IL-6. Synthesis of KC, IL-6, and TNF-α proteins were also demonstrated. After 4 h, autoinduction of RANTES transcripts was observed. These responses are chemokine specific. Although DC demonstrated weak responses to eotaxin, DC failed to respond to other chemokines including KC, MIP-2, stromal-derived factor-1α, MIP-1β, MIP-1α, monocyte chemoattractant protein-1, T cell activation gene 3, or thymus-derived chemotactic agent 4. In addition, RANTES treatment up-regulated expression of an orphan chemokine receptor termed Eo1. Chemokine induction was also observed after treatment of splenic DC and neonatal microglia with RANTES, but not after treatment of thymocytes or splenocytes depleted of adherent cells. TNF-α-treated DC lose responsiveness to RANTES. DC from mice deficient for CCR1, CCR3, and CCR5 respond to RANTES, indicating that none of these receptors are exclusively used to initiate the chemokine cascade. RANTES-mediated chemokine amplification in DC may prolong inflammatory responses and shape the microenvironment, potentially enhancing acquired and innate immune responses.

RANTES stimulates inflammatory cascades and receptor modulation in murine astrocytes.

Cultured mouse astrocytes respond to the CC chemokine RANTES by production of chemokine and cytokine transcripts. Stimulation of astrocytes with 1 nM RANTES or 3–10 nM of the structurally related chemokines (eotaxin, macrophage inflammatory protein‐1α and ‐β [MIP‐1α, MIP‐1β]) induced transcripts for KC, monocyte chemoattractant protein‐1 (MCP‐1), tumor necrosis factor‐α (TNF‐α), MIP‐1α, MIP‐2, and RANTES in a chemokine and cell‐specific fashion. Synthesis of chemokine (KC and MCP‐1) and cytokine (TNF‐α) proteins was also demonstrated. RANTES‐mediated chemokine synthesis was specifically inhibited by pertussis toxin, indicating that G‐protein‐coupled chemokine receptors participated in astrocyte signaling. Astrocytes expressed CCR1 and CCR5 (the redundant RANTES receptors). Astrocytes derived from mice with targeted mutations of either CCR1 or CCR5 respond after RANTES stimulation, suggesting multiple chemokine receptors may separately mediate RANTES responsiveness in astrocytes. Preliminary data suggest activation of the MAP kinase pathway is also critical for RANTES‐mediated signaling in astrocytes. Treatment with RANTES specifically modulated astrocyte receptors upregulating intercellular adhesion molecule 1 (ICAM‐1) and downregulating CX3CR1 expression. Thus, after chemokine treatment, astrocytes release proinflammatory mediators and reprogram their surface molecules. The combined effects of RANTES may serve to amplify inflammatory responses within the central nervous system. GLIA 39:19–30, 2002.

Chapter 4.3 – Chemokine Interactions with Astrocytes

Publisher Summary This chapter summarizes the effects of chemokines on astrocytes and the potential receptors responsible for these interactions. Chemokines stimulate a variety of astrocyte responses in vitro, including cell migration, cytokine and chemokine production, and modulation of heterologous receptors. In vivo evidence supporting each type of response was reported using genetically engineered mouse models in which chemokine expression was targeted to the central nervous system (CNS) or in inflammatory diseases affecting the CNS. Accumulating data suggest several additional chemokine and orphan receptors are also present on astrocytes. The sources and biological function of chemokines along with their cognate receptors on leukocytes are well described. Although there is ample evidence at the RNA and protein level that chemokine receptors are expressed on astrocytes, the functional significance of these astrocyte receptors remains an item of conjecture. Most astrocyte chemokine receptors are expressed in both primates and rodents, but species differences may regulate receptor distribution for some chemokine receptors. The signaling mechanisms responsible for triggering astrocyte responses are under investigation, with preliminary data hinting that distinct signaling pathways may differentially control astrocyte responsiveness. The receptor modulation that accompanies chemokine stimulation further contributes to the dynamic nature of astrocytes.

Torticollis under cyclobenzaprine.

The muscle-relaxing 5-HT2 receptor antagonist cyclobenzaprine is structurally closely related to amitriptyline. It is widely used to treat patients presenting with back pain and fibromyalgia. Very rarely cyclobenzaprine toxicity can result in extrapyramidal symptoms, but occurrence of torticollis has not been reported so far. We report on a patient presenting with torticollis and myoclonic movements after treatment with cyclobenzaprine, who was successfully treated with intravenous biperiden. This case might be additional evidence for the necessity of appropriate dosage in case of liver impairment. Secondly there are possibly consequences as regards the therapy of motor side effects.