Soluble ANPEP Released From Human Astrocytes as a Positive Regulator of Microglial Activation and Neuroinflammation: Brain Renin–Angiotensin System in Astrocyte–Microglia Crosstalk

Astrocytes are major supportive glia and immune modulators in the brain; they are highly secretory in nature and interact with other cell types via their secreted proteomes. To understand how astrocytes communicate during neuroinflammation, we profiled the secretome of human astrocytes following stimulation with proinflammatory factors. A total of 149 proteins were significantly upregulated in stimulated astrocytes, and a bioinformatics analysis of the astrocyte secretome revealed that the brain renin–angiotensin system (RAS) is an important mechanism of astrocyte communication. We observed that the levels of soluble form of aminopeptidase N (sANPEP), an RAS component that converts angiotensin (Ang) III to Ang IV in a neuroinflammatory milieu, significantly increased in the astrocyte secretome. To elucidate the role of sANPEP and Ang IV in neuroinflammation, we first evaluated the expression of Ang IV receptors in human glial cells because Ang IV mediates biological effects through its receptors. The expression of angiotensin type 1 receptor was considerably upregulated in activated human microglial cells but not in human astrocytes. Moreover, interleukin-1β release from human microglial cells was synergistically increased by cotreatment with sANPEP and its substrate, Ang III, suggesting the proinflammatory action of Ang IV generated by sANPEP. In a mouse neuroinflammation model, brain microglial activation and proinflammatory cytokine expression levels were increased by intracerebroventricular injection of sANPEP and attenuated by an enzymatic inhibitor and neutralizing antibody against sANPEP. Collectively, our results indicate that astrocytic sANPEP–induced increase in Ang IV exacerbates neuroinflammation by interacting with microglial proinflammatory receptor angiotensin type 1 receptor, highlighting an important role of indirect crosstalk between astrocytes and microglia through the brain RAS in neuroinflammation.     

A role for endothelial cells in promoting the maturation of astrocytes through the apelin/APJ system in mice

Interactions between astrocytes and endothelial cells (ECs) are crucial for retinal vascular formation. Astrocytes induce migration and proliferation of ECs via their production of vascular endothelial growth factor (VEGF) and, conversely, ECs induce maturation of astrocytes possibly by the secretion of leukemia inhibitory factor (LIF). Together with the maturation of astrocytes, this finalizes angiogenesis. Thus far, the mechanisms triggering LIF production in ECs are unclear. Here we show that apelin, a ligand for the endothelial receptor APJ, induces maturation of astrocytes mediated by the production of LIF from ECs. APJ (Aplnr)- and Apln-deficient mice show delayed angiogenesis; however, aberrant overgrowth of endothelial networks with immature astrocyte overgrowth was induced. When ECs were stimulated with apelin, LIF expression was upregulated and intraocular injection of LIF into APJ-deficient mice suppressed EC and astrocyte overgrowth. These data suggest an involvement of apelin/APJ in the maturation process of retinal angiogenesis.     

Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus

Astrocytes play an important role in initiating and regulating CNS immune responses through the release of proinflammatory cytokines and chemokines. Here we demonstrate that primary astrocytes are capable of recognizing the Gram-positive bacterium Staphylococcus aureus and its cell wall product peptidoglycan (PGN) and respond by producing numerous proinflammatory mediators including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemoattractant protein (MCP-1). Astrocytes have recently been shown to express Toll-like receptor 2 (TLR2), a pattern recognition receptor important for recognizing structural components of various Gram-positive bacteria, fungi, and protozoa. However, the functional significance of TLR2 in mediating astrocyte activation remains unknown. Primary astrocytes from TLR2 knockout mice were used to evaluate the role of TLR2 in astrocyte responses to S. aureus and PGN. The results demonstrate that TLR2 is essential for maximal proinflammatory cytokine and chemokine production, but not phagocytosis, in primary astrocytes following S. aureus and PGN exposure. In addition, both stimuli led to a significant increase in TLR2 mRNA expression in wild-type astrocytes as assessed by real-time quantitative RT-PCR. These findings suggest that astrocytes may play a key role in the initial antibacterial immune response in the CNS through engagement of TLR2.     

Cyclosporine A enhances leukocyte binding by human intestinal microvascular endothelial cells through inhibition of p38 MAPK and iNOS. Paradoxical proinflammatory effect on the microvascular endothelium

The calcineurin inhibitor cyclosporine A (CsA) modulates leukocyte cytokine production but may also effect nonimmune cells, including microvascular endothelial cells, which regulate the inflammatory process through leukocyte recruitment. We hypothesized that CsA would promote a proinflammatory phenotype in human intestinal microvascular endothelial cells (HIMEC), by inhibiting inducible nitric-oxide synthase (iNOS, NOS2)-derived NO, normally an important mechanism in limiting endothelial activation and leukocyte adhesion. Primary cultures of HIMEC were used to assess CsA effects on endothelial activation, leukocyte interaction, and the expression of iNOS as well as cell adhesion molecules. CsA significantly increased leukocyte binding to activated HIMEC, but paradoxically decreased endothelial expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1). In contrast, CsA completely inhibited the expression of iNOS in tumor necrosis factor-alpha/lipopolysaccharide-activated HIMEC. CsA blocked p38 MAPK phosphorylation in activated HIMEC, a key pathway in iNOS expression, but failed to inhibit NFkappaB activation. These studies demonstrate that CsA exerts a proinflammatory effect on HIMEC by blocking iNOS expression. CsA exerts a proinflammatory effect on the microvascular endothelium, and this drug-induced endothelial dysfunction may help explain its lack of efficacy in the long-term treatment of chronically active inflammatory bowel disease.     

Regulation of Astrocyte Morphology by RhoA and Lysophosphatidic Acid

Astrocytes in the CNS undergo morphological changes and start to proliferate after breakdown of the blood–brain barrier. In culture, proliferating astrocytes have a flat, polygonal shape. When treated with cAMP-raising agents, astrocytes adopt a stellate, process-bearing morphology resembling theirin vivoappearance. Stellation is accompanied by loss of actin stress fibers and focal adhesions. Lysophosphatidic acid (LPA), a blood-borne mitogen that signals through its cognate G protein-coupled receptor, stimulates DNA synthesis in astrocytes and causes rapid reversal of cAMP-induced stellation. LPA reversal of stellation is initiated by f-actin reassembly and tyrosine phosphorylation of focal adhesion proteins such as paxillin. Botulinum C3 toxin, which inactivates the Rho GTPase, mimics cAMP-raising agents in inducing stellation, f-actin disassembly, paxillin dephosphorylation, and growth arrest. However, unlike cAMP-induced stellation, C3-induced stellation cannot be reversed by LPA. Conversely, astrocytes expressing activated RhoA fail to undergo cAMP-induced stellation. Thus, RhoA controls astrocyte morphology in that active RhoA directs LPA reversal of stellation, while inactivation of RhoA is sufficient to induce stellation.     

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes.

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.     

Astrocytes from acyclic female rats exhibit lowered capacity for neuronal differentiation

Astrocytes comprise a large proportion of the central nervous system support cells and play a critical role in neural injury and repair. The present study examined the impact of ovarian aging using an ex vivo model system, where astrocytes were derived from the olfactory bulb of young, reproductively competent females and reproductive senescent females. Cellular morphology and the spatial pattern of laminin deposition was altered in astrocyte cultures derived from reproductive senescent females. Young adult astrocytes had a flattened polygonal shape with actin bundles at the cell edges, while reproductive senescent astrocytes had a contractile appearance with thick stress fibers visible throughout the cell. Moreover, in reproductive senescent astrocytes, BDNF was elevated with a concomitant reduction in expression of the BDNF receptor, TrkB. To examine the ability of astrocytes derived from young adult and reproductive senescent females to promote neuronal differentiation, neural progenitor cells (NPCs) were co-cultured with astrocytes derived from these groups. At day 4 in vitro, MAP-2(+) NPCs were located in smaller clusters when co-cultured with young adult astrocytes and in large clusters when co-cultured with older astrocytes. At days 6 and 10, neuronal differentiation was significantly reduced in reproductive senescent astrocyte-NPC co-cultures, as determined by NeuN(+) cell numbers and MAP-2(+) process lengths. Furthermore, estrogen only enhanced neuronal differentiation in young adult-NPC co-cultures. The ovarian age-related astrocyte phenotype thus limits the ability of this cell to promote neuronal differentiation in NPC populations and suggests that the astrocyte-mediated microenvironment in older acyclic females is less conducive to repair following neurovascular injury.     

Astrocyte-synapse interactions during brain development

Bidirectional communication between astrocytes and neurons is essential for proper brain development. Astrocytes, a major glial cell type, are morphologically complex cells that directly interact with neuronal synapses to regulate synapse formation, maturation, and function. Astrocyte-secreted factors bind neuronal receptors to induce synaptogenesis with regional and circuit-level precision. Cell adhesion molecules mediate the direct contact between astrocytes and neurons, which is required for both synaptogenesis and astrocyte morphogenesis. Neuron-derived signals also shape astrocyte development, function, and molecular identity. This review highlights recent findings on the topic of astrocyte-synapse interactions, and discusses the importance of these interactions for synapse and astrocyte development.     

Interleukin-1-β and Tumor Necrosis Factor-α Increase Peripheral-Type Benzodiazepine Binding Sites in Cultured Polygonal Astrocytes

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.     

Secretion of sterols and the NPC2 protein from primary astrocytes

Astrocytes secrete cholesterol in lipoprotein particles. Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1-/-) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1-/- astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors.     

Endothelial activation: intracellular signaling pathways

Tumor necrosis factor (TNF) is the prototypic proinflammatory cytokine and endothelial cells are the principal cellular targets of its actions. Here I review the responses of endothelial cells to TNF, with emphasis on the induction of endothelial leukocyte adhesion molecules. I focus on the biochemistry and cell biology of signal transduction in TNF-treated endothelial cells that lead to the expression of adhesion molecules.     

S100B content and secretion decrease in astrocytes cultured in high-glucose medium

S100B is an astrocyte calcium-binding protein that plays a regulatory role in the cytoskeleton and cell cycle. Moreover, extracellular S100B, a marker of glial activation in several conditions of brain injury, has a trophic or apoptotic effect on neurons, depending on its concentration. Hyperglycemic rats show changes in glial parameters, including S100B expression. Here, we investigated cell density, morphological and biochemical alterations in primary cortical astrocytes from rats and C6 glioma cells cultured in high-glucose medium. Astrocytes and C6 glioma cells have a reduced content of S100B and glial fibrillary acidic protein when cultured in a high-glucose environment, as well as a reduced content of glutathione and cell proliferation rate. Although these cells have been used indistinctly to study S100B secretion, we observed a contrasting profile of S100B secretion in a high-glucose medium: a decrease in primary astrocytes and an increase in C6 glioma cells. Based on the in vitro neurotrophic effects of the S100B protein, our data suggest that chronic elevated glucose levels affect astrocyte activity, reducing extracellular secretion of S100B and that this, in turn, could affect neuronal activity and survival. Such astrocyte alterations could contribute to cognitive deficit and other impairments observed in diabetic patients.     

Targeted Activation of Astrocytes: A Potential Neuroprotective Strategy

Astrocytes are involved in many key physiological processes in the brain, including glutamatergic transmission, energy metabolism, and blood flow control. They become reactive in response to pathological situations, a response that involves well-described morphological alterations and less characterized functional changes. The functional consequences of astrocyte reactivity seem to depend on the molecular pathway involved and may result in the enhancement of several neuroprotective and neurotrophic functions. We propose that a selective and controlled activation of astrocytes may switch these highly pleiotropic cells into therapeutic agents to promote neuron survival and recovery. This may represent a potent therapeutic strategy for many brain diseases in which neurons would benefit from an increased support from activated astrocytes.     

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.     

TLR signaling tailors innate immune responses in human microglia and astrocytes

The specific signals mediating the activation of microglia and astrocytes as a prelude to, or consequence of, CNS inflammation continue to be defined. We investigated TLRs as novel receptors mediating innate immune responses in human glial cells. We find that microglia express mRNA for TLRs 1-9, whereas astrocytes express robust TLR3, low-level TLR 1, 4, 5, and 9, and rare-to-undetectable TLR 2, 6, 7, 8, and 10 mRNA (quantitative real-time PCR). We focused on TLRs 3 and 4, which can signal through both the MyD88-dependent and -independent pathways, and on the MyD88-restricted TLR2. By flow cytometry, we established that microglia strongly express cell surface TLR2; TLR3 is expressed at higher levels intracellularly. Astrocytes express both cell surface and intracellular TLR3. All three TLRs trigger microglial activation upon ligation. TLR3 signaling induces the strongest proinflammatory polarizing response, characterized by secretion of high levels of IL-12, TNF-alpha, IL-6, CXCL-10, and IL-10, and the expression of IFN-beta. CXCL-10 and IL-10 secretion following TLR4 ligation are comparable to that of TLR3; however, other responses were lower or absent. TLR2-mediated responses are dominated by IL-6 and IL-10 secretion. Astrocytes respond to TLR3 ligation, producing IL-6, CXCL-10, and IFN-beta, implicating these cells as contributors to proinflammatory responses. Initial TLR-mediated glial activation also regulates consequent TLR expression; while TLR2 and TLR3 are subject to positive feedback, TLR4 is down-regulated in microglia. Astrocytes up-regulate all three TLRs following TLR3 ligation. Our data indicate that activation of innate immune responses in the CNS is not homogeneous but rather tailored according to cell type and environmental signal.     

中脑星形胶质细胞表达生长抑素mRNA的离体研究

利用细胞培养技术,取胎龄１８天Ｗｉｓｔａｒ大鼠中脑腹侧组织,纯化培养星形胶质细胞,以原位杂交组织化学法检测了体外培养的不同时间的生长抑素（Ｓｏｍａｔｏｓｔａｔｉｎ,ＳＳ）ｍＲＮＡ表达,结果表明中脑星形胶质细胞能合成生长抑素,并且其相对含量随培养时间的延长而逐渐下降。本文并对星形胶质细胞合成神经肽ＳＳ的功能意义进行了初步探讨     

The problem of astrocyte identity

Astrocytes were the original neuroglia of Ramón y Cajal but after 100 years there is no satisfactory definition of what should comprise this class of cells. This essay takes a historical and philosophical approach to the question of astrocytic identity. The classic approach of identification by morphology and location are too limited to determine new members of the astrocyte population. I also critically evaluate the use of protein markers measured by immunoreactivity, as well as the newer technique of marking living cells by using promoters for these same proteins to drive reporter genes. These two latter approaches have yielded an expanded population of astrocytes with diverse functions, but also mark cells that traditionally would not be defined as astrocytes. Thus we need a combination of measures to define an astrocyte but it is not clear what this combination should be. The molecular approach, especially promoter driven fluorescent reporter genes, does have the advantage of pre marking living astrocytes for electrophysiological or imaging recordings. However, lack of sufficient understanding of the behavior of the inserted constructs has led to unclear results. This approach will no doubt be perfected with time but at present an acceptable, practical definition of what constitutes the class of astrocytes remains elusive.