Purinergic modulation of glutamate transmission: An expanding role in stress-linked neuropathology.

Chronic stress has been extensively linked to disturbances in glutamatergic signalling. Emerging from this field of research is a considerable number of studies identifying the ability of purines at the pre-, post-, and peri-synaptic levels to tune glutamatergic neurotransmission. While the evidence describing purinergic control of glutamate has continued to grow, there has been relatively little attention given to how chronic stress modulates purinergic functions. The available research on this topic has demonstrated that chronic stress can not only disturb purinergic receptors involved in the regulation of glutamate neurotransmission, but also perturb glial-dependent purinergic signalling. This review will provide a detailed examining of the complex literature relating to glutamatergic-purinergic interactions with a focus on both neuronal and glial contributions. Once these detailed interactions have been described and contextualised, we will integrate recent findings from the field of stress research.

In acute experimental autoimmune encephalomyelitis, infiltrating macrophages are immune activated, whereas microglia remain immune suppressed.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) characterized by loss of myelin accompanied by infiltration of T-lymphocytes and monocytes. Although it has been shown that these infiltrates are important for the progression of MS, the role of microglia, the resident macrophages of the CNS, remains ambiguous. Therefore, we have compared the phenotypes of microglia and macrophages in a mouse model for MS, experimental autoimmune encephalomyelitis (EAE). In order to properly discriminate between these two cell types, microglia were defined as CD11b(pos) CD45(int) Ly-6C(neg) , and infiltrated macrophages as CD11b(pos) CD45(high) Ly-6C(pos) . During clinical EAE, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecules (CD80, CD86, and CD40) and proinflammatory genes [interleukin-1ß (IL-1ß) and tumour necrosis factor- α (TNF-α)]. In contrast, CD11b(pos) CD45(high) Ly-6C(pos) infiltrated macrophages were strongly activated and could be divided into two populations Ly-6C(int) and Ly-6C(high) , respectively. Ly-6C(high) macrophages contained less myelin than Ly-6C(int) macrophages and expression levels of the proinflammatory cytokines IL-1ß and TNF-α were higher in Ly-6C(int) macrophages. Together, our data show that during clinical EAE, microglia are only weakly activated whereas infiltrated macrophages are highly immune reactive.

Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR.

BACKGROUND AND PURPOSE: Induction of cellular migration is the primary effect of chemokine receptor activation. However, several chemokine receptor-like proteins bind chemokines without subsequent induction of intracellular signalling and chemotaxis. It has been suggested that they act as chemokine scavengers, which may control local chemokine levels and contribute to the function of chemokines during inflammation. This has been verified for the chemokine-like receptor proteins D6 and DARC as well as CCX-CKR. Here, we provide evidence for an additional biological function of human (h)CCX-CKR. EXPERIMENTAL APPROACH: We used transfection strategies in HEK293 and human T cells. KEY RESULTS: Co-expression of hCCX-CKR completely inhibits hCXCR3-induced chemotaxis. We found that hCCX-CKR forms complexes with hCXCR3, suggesting a relationship between CCX-CKR heteromerization and inhibition of chemotaxis. Moreover, negative binding cooperativity induced by ligands both for hCXCR3 and hCCX-CKR was observed in cells expressing both receptors. This negative cooperativity may also explain the hCCX-CKR-induced inhibition of chemotaxis. CONCLUSIONS AND IMPLICATIONS: These findings suggest that hCCX-CKR prevents hCXCR3-induced chemotaxis by heteromerization thus representing a novel mechanism of regulation of immune cell migration.

Microglia phenotype diversity.

Microglia, the tissue macrophages of the brain, have under healthy conditions a resting phenotype that is characterized by a ramified morphology. With their fine processes microglia are continuously scanning their environment. Upon any homeostatic disturbance microglia rapidly change their phenotype and contribute to processes including inflammation, tissue remodeling, and neurogenesis. In this review, we will address functional phenotypes of microglia in diverse brain regions and phenotypes associated with neuroinflammation, neurogenesis, brain tumor homeostasis, and aging.

The role of CXC chemokine ligand (CXCL)12-CXC chemokine receptor (CXCR)4 signalling in the migration of neural stem cells towards a brain tumour.

AIMS: It has been shown that neural stem cells (NSCs) migrate towards areas of brain injury or brain tumours and that NSCs have the capacity to track infiltrating tumour cells. The possible mechanism behind the migratory behaviour of NSCs is not yet completely understood. As chemokines are involved in the migration of immune cells in the injured brain, they may also be involved in chemoattraction of NSCs towards a brain tumour. METHODS: The expression profile of various chemokine receptors in NSCs, harvested from the subventricular zone of adult mice, was investigated by reverse transcriptase- polymerase chain reaction analysis. Furthermore, the functionality of the chemokine receptors was assessed in in vitro chemotaxis assays and calcium signalling experiments. To test the in vivo migration of NSCs, a syngeneic mouse model was developed, whereby a B16F10 melanoma cell line was grafted into one hemisphere and later NSCs were grafted in the contralateral hemisphere. Furthermore, the expression of chemokines in this melanoma cell line was investigated. RESULTS AND CONCLUSIONS: Adult mouse NSCs functionally express various chemokine receptors of which CXC chemokine receptor (CXCR)4 shows the highest mRNA levels and most pronounced functional responses in vitro. CXC chemokine ligand (CXCL)12, the ligand for CXCR4, is expressed by the melanoma cell line. In this mouse model for metastatic brain tumours, it is shown that NSCs express CXCR4 at their cell membranes while they migrate towards the tumour, which produces CXCL12. It is therefore suggested that the CXCR4/CXCL12 pathway plays a role in the mechanism underlying tumour-mediated attraction of NSCs.

Neuron-microglia signaling: chemokines as versatile messengers.

Our understanding of microglia biology has significantly changed in the last couple of years. Instead of being predominantly detrimental cells showing a stereotypic activation pattern, microglia today are considered highly adaptive elements with many distinct phenotypes. Microglia activity is aimed to protect and to restore and only in case of uncontrolled or impaired microglia function these cells may have detrimental effects. The control of microglia activity is thus an important issue to understand. The family of chemokines are versatile signals specialized to control cell-cell interactions. Neurons express chemokines in a temporarily and spatially regulated manner and microglia respond to these messengers via the appropriate receptors. Due to these features are chemokines ideal messengers for the communication between neurons and microglia.

Neuronal chemokines: versatile messengers in central nervous system cell interaction.

Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron-astrocyte, neuron-microglia, and neuron-neuron interaction.

Challenge with innate and protein antigens induces CCR7 expression by microglia in vitro and in vivo.

Since activated microglia are able to phagocytose damaged cells and subsequently express major histocompatibility complex class II (MHC-II) and co-stimulatory proteins, they are considered to function as antigen presenting cells (APCs) in the central nervous system. The maturation and migratory potential of professional APCs is associated with the expression of chemokine receptor CCR7. We therefore investigated whether the immunological activation of microglia induces CCR7 expression. We here present that activation of cultured microglia by both the innate antigen lipopolysaccharide and protein antigen ovalbumin rapidly induces CCR7 expression, accompanied by increased MHC-II expression. Moreover, it is shown that CCR7 expression in IBA-1 positive cells is induced during the symptom onset and progression of experimental autoimmune encephalomyelitis, a rodent model for multiple sclerosis. These results suggest that microglia express CCR7 under specific inflammatory conditions, corroborating the idea that microglia develop into APCs with migratory potential toward lymphoid chemokines.

Induction of glial L-CCR mRNA expression in spinal cord and brain in experimental autoimmune encephalomyelitis.

Chemokines and chemokine receptors are important regulators of leukocyte trafficking and immune response. It is well established that chemokines and their receptors are also expressed in the central nervous system (CNS), where their expression has been associated with various neuroinflammatory diseases, such as multiple sclerosis (MS). One of the most important chemokines involved in MS pathology is CCL2 (previously known as MCP-1). CCL2, released by glial cells, activates the chemokine receptor CCR2, causing the infiltration of blood monocytes in tissues affected by MS. There is evidence, however, that CCL2 also has local effects on CNS cells, including induction or modulation of cytokine release and synthesis of matrix metalloproteinases, that might contribute to CNS pathology. These effects are most likely independent of CCR2, since CCR2 expression in glial cells is rarely observed. We have recently provided evidence for the presence of an alternative CCL2 receptor in glial cells called L-CCR and have investigated the expression of L-CCR mRNA in a murine EAE model. It is shown that L-CCR mRNA is expressed in infiltrating macrophages during EAE, but not in infiltrating T cells. Prominent expression of L-CCR mRNA was detected in astrocytes and microglia already at early time points throughout the brain and spinal cord supporting the hypothesis that L-CCR expression in glial cells is related to CNS inflammation.

Expansion of HPCs from cord blood in a novel 3D matrix.

BACKGROUND: An optimal system for the expansion of pluripotent HPCs would ideally eliminate the use of cytokines and animal-derived serum. We have shown previously that a 3D, tantalum-coated porous biomaterial (Cytomatrix) supports the maintenance and expansion of human BM HPCs in the absence of cytokines. METHODS: Umbilical cord blood (UCB) derived HPC were cultured in the Cytomatrix in the absence of exogenous cytokines. Phenotype was determined using FACS. Colony-forming units (CFU) activity was evaluated. Engraftment capacity was evaluated by transplanting the expanded cells into non-obese diabetic (NOD)/SCID mice. RESULTS: We describe the expansion of HPCs from UCB using the Cytomatrix system. When UCB-derived CD34(+) cells were cultured in the Cytomatrix system for 2 weeks we observed an increase in the number of nucleated cells (3-fold) and CFU (2.6-fold). The number of CD45(+) and CD34(+) cells both increased three-fold. Trends demonstrated an increase in the frequency of CD34(+)C38(-) cells, and an increase in both CD34(+)C33(+) cells and CD34(+)C61(+) cells. No expansion of T or B lymphocytes was observed. When expanded UCB cells from the Cytomatrix were injected into sub-lethally irradiated NOD/SCID mice, human cells were detected in the murine peripheral blood and BM 6 weeks post-transplantation. DISCUSSION: This unique approach to the expansion of UCB cells in a serum-free, cytokine-free environment may provide expansion of HPCs with multi-lineage engraftment capability that could be used clinically.

Functional expression of CXCR3 in cultured mouse and human astrocytes and microglia.

It has been established recently that inflammation of the CNS is accompanied by an expression of chemokines within the CNS. Several lines of evidence suggest that chemokines within the CNS initiate and orchestrate the infiltration of the inflamed brain by blood leukocytes. It is also known that endogenous cells of the CNS express functional chemokine receptors, raising the possibility that chemokines may be involved in intercellular signalling between brain cells during brain inflammation. It was shown recently that two chemokine ligands for CXCR3 are induced rapidly in damaged neurons. Little is known yet on the function of neuronal chemokine expression. In order to investigate whether neuronal chemokines contribute to endogenous signalling within the CNS we investigated possible expression of CXCR3 in glial cells. Reverse transcription-polymerase chain reaction experiments and in situ hybridization analysis showed that cultured astrocytes and microglia from both mouse and human sources express CXCR3 mRNA. Protein expression of CXCR3 in both cell types was detected by immunocytochemistry. Moreover, stimulation of cultured glial cells with chemokine ligands for CXCR3 induced intracellular calcium transients and chemotaxis, indicating the functional expression of CXCR3. These results indicate that glial cells in culture functionally express the chemokine receptor CXCR3. Since it has been shown that brain damage rapidly induces expression of neuronal chemokines that activate CXCR3, we suggest that glial CXCR3 might contribute to an intercellular signalling system in the CNS related to pathological conditions.

Expression of interleukin-1 beta in rat dorsal root ganglia.

The expression of interleukin-1beta was examined in dorsal root ganglion (DRG) neurons from adult rats using non-radioactive in situ hybridization and immunocytochemistry. At all spinal levels, approximately 70% of the DRG neurons appeared to express IL-1beta mRNA; about 80% of these DRG neurons actually appeared to produce the IL-1beta protein at markedly varying levels. The expression of IL-1beta was found in large as well as in intermediate diameter sensory neurons but only sporadically in the population of small sensory neurons. The population of IL-1beta immunopositive sensory neurons included most of the large calretinin-positive Ia afferents, but only a few of the small substance P/CGRP positive sensory neurons. In situ hybridization staining for the detection of type 1 IL-1 receptor showed expression of this receptor by most of the sensory neurons as well as by supportive glial-like cells, presumably satellite cells. The functional significance of IL-1beta in the DRG neurons needs to be elucidated, but we speculate that IL-1beta produced by DRG neurons may be an auto/paracrine signalling molecule in sensory transmission.

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.

Interleukin-6 enhances expression of adenosine A(1) receptor mRNA and signaling in cultured rat cortical astrocytes and brain slices.

The inhibitory neuromodulator adenosine is released in the brain in high concentrations under conditions of exaggerated neuronal activity such as ischemia and seizures, or electroconvulsive treatment. By inhibiting neural overactivity, adenosine counteracts seizure activity and promotes neuronal survival. Since stimulation of adenosine A(2b) receptors on astrocytes induces increased synthesis and release of interleukin-6, which also exerts neuroprotective effects, we hypothesized that the effects of interleukin-6 and of adenosine might be related. We report here that stimulation with interleukin-6 of cultured astrocytes, of cultured organotypic brain slices from newborn rat cortex, and of freshly prepared brain slices from rat cortex induces a concentration- and time-dependent upregulation of adenosine A(1) receptor mRNA. This increased adenosine A(1) receptor mRNA expression is accompanied in astrocytes by an increase in adenosine A(1) receptor-mediated signaling via the phosphoinositide-dependent pathway. Since upregulation of adenosine A(1) receptors leads to increased neuroprotective effects of adenosine, we suggest that the neuroprotective actions of interleukin-6 and adenosine are related and might be mediated at least in part through upregulation of adenosine A(1) receptors. These results may be of relevance for a better understanding of neuroprotection in brain damage but also point to a potential impact of neuroprotection in the mechanisms of the antidepressive effects of chronic carbamazepine, electroconvulsive therapy, and sleep deprivation, which are all accompanied by adenosine A(1) receptor upregulation.

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.

Expression and signaling of group I metabotropic glutamate receptors in astrocytes and microglia.

Stimulation of astrocytes with the excitatory neurotransmitter glutamate leads to the formation of inositol 1,4,5-trisphosphate and the subsequent increase of intracellular calcium content. Astrocytes express both ionotropic receptors and metabotropic glutamate (mGlu) receptors, of which mGlu5 receptors are probably involved in glutamate-induced calcium signaling. The mGlu5 receptor occurs as two splice variants, mGlu5a and mGlu5b, but it was hitherto unknown which splice variant is responsible for the glutamate-induced effects in astrocytes. We report here that both mRNAs encoding mGlu5 receptor splice variants are expressed by cultured astrocytes. The expression of mGlu5a receptor mRNA is much stronger than that of mGlu5b receptor mRNA in these cells. In situ hybridization experiments reveal neuronal expression of mGlu5b receptor mRNA in adult rat forebrain but a strong neuronal expression of mGlu5a mRNA only in olfactory bulb. Signals for mGlu5a receptor mRNA in the rest of the brain were diffuse and weak but consistently above background. Activation of mGlu5 receptors in astrocytes yields increases in inositol phosphate production and transient calcium responses. It is surprising that the rank order of agonist potency [quisqualate > (2S,1 'S,2'S)-2-(carboxycyclopropyl)glycine = trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD) > glutamate] differs from that reported for recombinantly expressed mGlu5a receptors. The expression of mGlu5a receptor mRNA and the occurrence of 1S,3R-ACPD-induced calcium signaling were found also in cultured microglia, indicating for the first time expression of mGlu5a receptors in these macrophage-like cells.

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.

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.

Effects of in utero alcohol exposure on B cell development in neonatal spleen and bone marrow.

The effects of in utero alcohol exposure on neonatal lymphopoiesis were examined in a murine model of fetal alcohol syndrome. At birth, both immature and mature B cells were decreased in the spleens of neonatal animals and these subpopulations of B cells did not recover to normal levels until 3-4 weeks of life. Pre-B cells and total B cells were decreased as well in the bone marrow of ethanol-exposed animals. By 3-4 weeks of life, the number of B cells in the bone marrow recovered to normal levels, but the pre-B cells remained below normal levels through 5 weeks of age. Furthermore, a recently described early B cell progenitor was reduced in frequency in ethanol-exposed neonates. Together, these data suggest that in utero exposure to ethanol can result in abnormalities in B cell development that may initiate at an early stage of B cell development.