Knut Biber

Secondary Lymphoid Tissue Chemokine (CCL21) Activates CXCR3 to Trigger a Cl− Current and Chemotaxis in Murine Microglia

Microglial cells represent the major immunocompetent element of the CNS and are activated by any type of brain injury or disease. A candidate for signaling neuronal injury to microglial cells is the CC chemokine ligand CCL21, given that damaged neurons express CCL21. Investigating microglia in acute slices and in culture, we demonstrate that a local application of CCL21 for 30 s triggered a Cl− conductance with lasted for tens of minutes. This response was sensitive to the Cl− channel blockers 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and 4-acetamide-4′-isothiocyanatostilbene, 2,2′-disulfonic acid. Moreover, CCL21 triggered a chemotaxis response, which was sensitive to Cl− channel blockers. In microglial cells cultured from CCR7 knockout mice, CCL21 produced the same type of Cl− current as well as a chemotaxis response. In contrast, in microglial cells from CXCR3 knockout mice, CCL21 triggered neither a Cl− conductance nor a chemotaxis response after CCL21 application. We conclude that the CCL21-induced Cl− current is a prerequisite for the chemotaxis response mediated by the activation of CXCR3 but not CCR7 receptors, indicating that in brain CCL21 acts via a different receptor system than in lymphoid organs.

Ischemia-induced neuronal expression of the microglia attracting chemokine secondary lymphoid-tissue chemokine (SLC)

Recently, it has been demonstrated that secondary lymphoid‐tissue chemokine (SLC) is constitutively expressed in secondary lymphoid organs and controls the homing of naive T‐cells and mature dendritic cells. By screening cDNA isolated from ischemic mouse brain, we found expression of SLC mRNA 6 h up to 4 days after the onset of ischemia. In situ hybridization combined with immunohistochemistry showed neurons expressing SLC mRNA in the ischemic area of the cortex. SLC mRNA expression was also found in cultured neurones after various treatments known to induce neuronal death, but not in cultured glial cells. Stimulation with SLC induced intracellular calcium transients and chemotaxis in cultured microglia. Since mRNA encoding CXCR3, an alternative receptor for SLC, but no CCR7 mRNA was found in microglia, we suggest that the effects of SLC on microglia are mediated by CXCR3. This assumption was corroborated by cross‐desensitization experiments using IP‐10 as a ligand for CXCR3. The inducible expression of SLC in neurones acting on microglia suggests a new and important role of SLC in the neuroimmune system. We propose that SLC is part of a neurone‐microglia signaling system which is related to pathological conditions of the brain like ischemia. GLIA 34:121–133, 2001.

Chemokine action in the nervous system

Inflammatory cytokines are a family of molecules that coordinate inflammatory and immune responses. One important class of inflammatory cytokines are the chemokines (forCHEMOtactic cytoKINES). Chemokines are a large group of proteins that play a central role in regulating the migration of leukocytes

Differential expression of inflammatory mediators in rat microglia cultured from different brain regions

Microglial cells show a rather uniform distribution of cell numbers throughout the brain with only minor prevalences in some brain regions. Their in situ morphologies, however, may vary markedly from elongated forms observed in apposition with neuronal fibers to spherical cell bodies with sometimes extremely elaborated branching. This heterogeneity gave rise to the hypothesis that these cells are differentially conditioned by their microenvironment and, therefore, also display specific patterns of differential gene expression. In this study, microglia were isolated from 2-4 week-old mixed CNS cultures that had been prepared from neonatal rat diencephalon, tegmentum, hippocampus, cerebellum and cerebral cortex, and were investigated 24 h later. Messenger RNA levels of proteins involved in crucial immune functions of this cell type (TNF-alpha, CD4, Fcgamma receptor II, and IL-3 receptor beta-subunit) have been determined by semi-quantitative RT-PCR. The results clearly show, that three of these mRNAs (TNF-alpha, CD4, Fcgamma receptor II) are differentially expressed in microglia with hippocampal microglia displaying the highest levels of these mRNAs. The data strongly support the notion that the status of microglial gene expression depends on their localization in brain and on specific interactions with other neural cell types. Consequently, it is hypothesized that their responsiveness to signals arising in injury or disease may vary from one brain region to another.

Functional expression of the fractalkine (CX3C) receptor and its regulation by lipopolysaccharide in rat microglia

Functional expression of CX3CR1, a recently discovered receptor for the chemokine fractalkine, was investigated in cultured rat microglia. Reverse transcriptase polymerase chain reaction (PCR) experiments show abundant expression of fractalkine receptor mRNA in microglia. mRNA expression of fractalkine was undetectable in astrocytes and microglia but was very strong in cortical neurons. Incubation of microglia with lipopolysaccharide (100 ng/ml) transiently suppressed expression of fractalkine receptor mRNA. Fractalkine induced a concentration-dependent (10(-10)-10(-8) M) and, at high concentrations, oscillatory mobilization of intracellular Ca2+ in microglia The concentration-response curve of fractalkine was shifted to the right after 12 h incubation with lipopolysaccharide. It is concluded that treatment with endotoxin downregulates expression of fractalkine receptor mRNA in rat microglia and suppresses the functional response to fractalkine.

Cutting Edge: Activity of Human Adult Microglia in Response to CC Chemokine Ligand 21

The approximately 50 known chemokines are classified in distinct subfamilies: CXC, CC, CX3C, and C. Although the signaling of chemokines often is promiscuous, signaling events between members of these distinct chemokine classes are hardly observed. The only known exception so far is the murine CC chemokine ligand (CCL)21 (secondary lymphoid tissue chemokine, Exodus-2, 6Ckine), which binds and activates the murine CXC chemokine receptor CXCR3. However, this exception has not been found in humans. In this study, we provide evidence that human CCL21 is a functional ligand for endogenously expressed CXCR3 in human adult microglia. In absence of CCR7 expression, CCL21 induced chemotaxis of human microglia with efficiency similar to the CXCR3 ligands CXC chemokine ligand 9 (monokine induced by IFN-γ) and CXC chemokine ligand 10 (IFN-γ-inducible protein-10). Because human CCL21 did not show any effects in CXCR3-transfected HEK293 cells, it is indicated that CXCR3 signaling depends on the cellular background in which the CXCR3 is expressed.

LPS-induced expression of a novel chemokine receptor (L-CCR) in mouse glial cells in vitro and in vivo

There is increasing evidence that chemokines, specialized regulators of the peripheral immune system, are also involved in the physiology and pathology of the CNS. It is known that glial cells (astrocytes and microglia) express various chemokine receptors like CCR1, ‐3, ‐5, and CXCR4. We have investigated the possible expression of the known CC chemokine receptors (CCR1–8 and D6) in murine glial cells. In addition, we examined possible glial expression of the orphan CC chemokine receptor L‐CCR that has been identified previously in murine macrophages. We report here expression of L‐CCR mRNA in murine astrocytes and microglia. Furthermore, L‐CCR mRNA expression was strongly induced after application of bacterial lipopolysaccharide (LPS), both in vitro and in vivo. Functional studies and binding experiments using biotinylated monocyte chemoattractant protein (MCP)‐1 (CCL2) indicate that CCL2 could be a candidate chemokine ligand for glial L‐CCR. Based on the data presented, it is suggested that L‐CCR is a functional glial chemokine receptor that is important in neuroimmunology. GLIA 41:327–336, 2003.

Carbamazepine-induced upregulation of adenosine A1-receptors in astrocyte cultures affects coupling to the phosphoinositol signaling pathway.

The anticonvulsant and antibipolar drug carbamazepine (CBZ) is known to act as a specific antagonist at adenosine A1-receptors. After a 3-week application of CBZ, A1-receptors are upregulated in the rat brain. We have investigated the consequences of this upregulation for the A1-receptor-mediated signal transduction in primary astrocyte cultures from different regions of the rat brain. CBZ treatment for 10 days had no effect on adenosine A1-receptor mRNA expression in cultures with high basal A1-receptor mRNA levels, but increased A1-receptor mRNA in cultures exhibiting low basal A1-receptor mRNA levels. This upregulation of A1-receptor mRNA was accompanied by an upregulation or induction of A1-receptor-mediated potentiation of PLC activity, a property that was not found in these cultures before CBZ treatment. Thus, CBZ treatment for 10 days induces a new quality of adenosine A1-receptor-mediated signal transduction in cells that express low basal A1-receptor numbers.

Expression of L-CCR in HEK 293 cells reveals functional responses to CCL2, CCL5, CCL7, and CCL8

It has become clear in the past years that chemokines and chemokine receptors are pivotal regulators of cellular communication and trafficking. In addition to the ∼20 chemokine receptors that have been cloned and described, various orphan receptors with a chemokine receptor‐like structure are known. We have investigated the orphan mouse chemokine receptor (L‐CCR) in HEK 293 cells, a receptor that was originally described in a mouse macrophage cell line. Cells expressing this receptor show pertussis toxin‐sensitive chemotaxis and small intracellular calcium transients in response to the chemokines CCL2, CCL7, CCL8, and CCL5. Biotinylated CCL2 binds to L‐CCR‐expressing cells, and transfection experiments with an L‐CCR–green fluorescent protein fusion protein showed L‐CCR expression in the membranes of recombinant HEK 293 cells. Although radioligand binding was not detected, it is suggested that L‐CCR is a functional chemokine receptor.

IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C

Adenosine binds to a class of G-protein coupled receptors, which are further distinguished as A(1), A(2a), A(2b) and A(3) adenosine receptors. As we have shown earlier, the stable adenosine analogue NECA (N6-(R)-phenylisopropyladenosine) stimulates IL-6 expression in the human astrocytoma cell line U373 MG via the A(2b) receptor. The mechanism by which NECA promotes astrocytic IL-6 expression has not been identified. By using various inhibitors of signal transduction, we found that p38 mitogen-activated protein kinases (MAPK) activation (inhibitor SB202190), but not extracellular signal-regulated kinase (ERK) (PD98059) and c-jun N-terminal kinase (JNK)(SP600125), is essential in the NECA-induced signalling cascade that leads to the increase in IL-6 synthesis in U373 MG cells. Results obtained with protein kinase C (PKC) inhibitors that have different substrate specificities, indicated that the PKC delta and epsilon isoforms are also involved in adenosine receptor A(2b) dependent upregulation of IL-6 expression. This is supported by the fact that NECA induced the activation of PKC delta and epsilon in U373 MG cells.