Adult Astrocytes Research Articles

In vivo reprogramming of astrocytes to neuroblasts in the adult brain

Adult differentiated cells can be reprogrammed into pluripotent stem cells or lineage-restricted proliferating precursors in culture; however, this has not been demonstrated in vivo. Here, we show that the single transcription factor SOX2 is sufficient to reprogram resident astrocytes into proliferative neuroblasts in the adult mouse brain. These induced adult neuroblasts (iANBs) persist for months and can be generated even in aged brains. When supplied with BDNF and noggin or when the mice are treated with a histone deacetylase inhibitor, iANBs develop into electrophysiologically mature neurons, which functionally integrate into the local neural network. Our results demonstrate that adult astrocytes exhibit remarkable plasticity in vivo, a feature that might have important implications in regeneration of the central nervous system using endogenous patient-specific glial cells.

Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aβ42 prompted by exogenous fibrillary or soluble Aβ25–35 in human cortical astrocytes and neurons—Therapeutic relevance to Alzheimer's disease

The “amyloid-β (Aβ) hypothesis” posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ25–35, an Aβ42 proxy, evokes a surplus endogenous Aβ42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAβ25–35 and soluble (s)Aβ25–35 on Aβ42 and Aβ40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aβs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAβ25–35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aβ42, while Aβ40 also accrued but its secretion was unchanged. Accordingly, secreted Aβ42/Aβ40 ratio values rose for astrocytes and neurons. While slightly enhancing Aβ40 secretion by fAβ25–35-treated astrocytes, NPS 2143 specifically suppressed the fAβ25–35-elicited surges of endogenous Aβ42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aβ42/Aβ40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/β-secretase and γ-secretase activity in fAβ25–35-treated astrocytes. Compared to fAβ25–35, sAβ25–35 also stimulated Aβ42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aβ/CaSR-induced surplus secretion of endogenous Aβ42 and hence further vicious cycles of Aβ self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.

Characterization of Adult Rat Astrocyte Cultures

Astrocytes, a major class of glial cells, regulate neurotransmitter systems, synaptic processing, ion homeostasis, antioxidant defenses and energy metabolism. Astrocyte cultures derived from rodent brains have been extensively used to characterize astrocytes' biochemical, pharmacological and morphological properties. The aims of this study were to develop a protocol for routine preparation and to characterize a primary astrocyte culture from the brains of adult (90 days old) Wistar rats. For this we used enzymatic digestion (trypsin and papain) and mechanical dissociation. Medium exchange occurred from 24 h after obtaining a culture and after, twice a week up to reach the confluence (around the 4th to 5th week). Under basal conditions, adult astrocytes presented a polygonal to fusiform and flat morphology. Furthermore, approximately 95% the cells were positive for the main glial markers, including GFAP, glutamate transporters, glutamine synthetase and S100B. Moreover, the astrocytes were able to take up glucose and glutamate. Adult astrocytes were also able to respond to acute H2O2 exposure, which led to an increase in reactive oxygen species (ROS) levels and a decrease in glutamate uptake. The antioxidant compound resveratrol was able to protect adult astrocytes from oxidative damage. A response of adult astrocytes to an inflammatory stimulus with LPS was also observed. Changes in the actin cytoskeleton were induced in stimulated astrocytes, most likely by a mechanism dependent on MAPK and Rho A signaling pathways. Taken together, these findings indicate that the culture model described in this study exhibits the biochemical and physiological properties of astrocytes and may be useful for elucidating the mechanisms related to the adult brain, exploring changes between neonatal and adult astrocytes, as well as investigating compounds involved in cytotoxicity and cytoprotection.

The Adult Astrocyte Is Different

The expression of metabotropic glutamate receptors in brain astrocytes is down-regulated in early postnatal development.

The Adult Astrocyte Is Different

The Adult Astrocyte Is Different

Glutamate-Dependent Neuroglial Calcium Signaling Differs Between Young and Adult Brain

An extensive literature shows that astrocytes exhibit metabotropic glutamate receptor 5 (mGluR5)-dependent increases in cytosolic calcium ions (Ca(2+)) in response to glutamatergic transmission and, in turn, modulate neuronal activity by their Ca(2+)-dependent release of gliotransmitters. These findings, based on studies of young rodents, have led to the concept of the tripartite synapse, in which astrocytes actively participate in neurotransmission. Using genomic analysis, immunoelectron microscopy, and two-photon microscopy of astrocytic Ca(2+) signaling in vivo, we found that astrocytic expression of mGluR5 is developmentally regulated and is undetectable after postnatal week 3. In contrast, mGluR3, whose activation inhibits adenylate cyclase but not calcium signaling, was expressed in astrocytes at all developmental stages. Neuroglial signaling in the adult brain may therefore occur in a manner fundamentally distinct from that exhibited during development.

Alzheimer’s disease: An update of the roles of receptors, astrocytes and primary cilia (Review)

The pathophysiological mechanisms underlying the onset and inexorable progression of the late‑onset form of Alzheimer's disease (AD) are still the object of controversy. This review takes stock of some most recent advancements of this field concerning the complex roles played by the amyloid‑β (Aβ)‑binding p75 neurotrophin receptor (p75NTR) and calcium‑sensing receptor (CaSR) and by the primary cilia in AD. Apart from their physiological roles, p75NTR is more intensely expressed in the hippocampus of human AD brains and Aβ‑bound p75NTR triggers cell death, whereas Aβ‑bound CaSR signalling induces the denovo synthesis and release of nitric oxide (NO), vascular endothelial growth factor (VEGF)‑A and Aβ peptides (Aβs), particularly on the part of normal adult human astrocytes. The latter effect could significantly increase the pool of Aβ‑ and NO‑producing nerve cells favouring the progressive spread of a self‑sustaining and self‑reinforcing 'infectious' mechanism of neural and vascular (i.e. blood-brain barrier) cell damage. Interestingly, primary cilia concentrate p75NTR receptors in their membranes and are abnormally structured/damaged in transgenic (Tg) AD‑model mice, which could impact on the adult neurogenesis occurring in the dentate gyrus's subgranular zone (SGZ) that is necessary for new memory encoding, thereby favouring typical AD cognitive decline. Altogether, these findings may pave the way to novel therapeutic approaches to AD, particularly in its mild cognitive impairment (MCI) and pre‑MCI stages of development.

Role of Gap Junction Protein Connexin43 in Astrogliosis Induced by Brain Injury

Astrogliosis is a process that involves morphological and biochemical changes associated with astrocyte activation in response to cell damage in the brain. The upregulation of intermediate filament proteins including glial fibrillary acidic protein (GFAP), nestin and vimentin are often used as indicators for astrogliosis. Although connexin43 (Cx43), a channel protein widely expressed in adult astrocytes, exhibits enhanced immunoreactivity in the peri-lesion region, its role in astrogliosis is still unclear. Here, we correlated the temporal and spatial expression of Cx43 to the activation of astrocytes and microglia in response to an acute needle stab wound in vivo. We found large numbers of microglia devoid of Cx43 in the needle wound at 3 days post injury (dpi) while reactive astrocytes expressing Cx43 were present in the peripheral zone surrounding the injury site. A redistribution of Cx43 to the needle site, corresponding to the increased presence of GFAP-positive reactive astrocytes in the region, was only apparent from 6 dpi and sustained until at least 15 dpi. Interestingly, the extent of microglial activation and subsequent astrogliosis in the brain of Cx43 knockout mice was significantly larger than those of wild type, suggesting that Cx43 expression limits the degree of microgliosis. Although Cx43 is not essential for astrogliosis and microglial activation induced by a needle injury, our results demonstrate that Cx43 is a useful marker for injury induced astrogliosis due to its enhanced expression specifically within a small region of the lesion for an extended period. As a channel protein, Cx43 is a potential in vivo diagnostic tool of asymptomatic brain injury.

Neurotoxins released from interferon-gamma-stimulated human astrocytes

Astrocytes become activated in degenerative neurological diseases. In order to gain a greater understanding of the inflammatory factors released upon activation, we stimulated adult human astrocytes with interferon-gamma and examined the resultant conditioned medium (CM) for toxicity against differentiated human neuroblastoma SH-SY5Y cells. Cell death was measured by lactate dehydrogenase release assay. We then used various treatments of the media to determine the distribution and nature of the toxic components.Removal of interleukin-6 by a specific antibody reduced the toxicity by 22%. Blockade of proteases with an inhibitor cocktail reduced it by a further 22%. When oxygen-free radical production was blocked with NADPH oxidase inhibitors, the toxicity was reduced by 15.4%. When prostaglandin production was blocked by cyclooxygenase inhibitors, the toxicity of the CM was reduced by 14.5%. When glutamate was removed by treatment with glutamate decarboxylase, the toxicity was reduced by 10.3%. When the inhibitors were added together to the astrocyte culture, the total toxicity of the CM was reduced by 91%. This was in reasonable agreement with the 85.37% total obtained by adding the individual components. The data show that activated astrocytes release a specific combination of neurotoxic compounds. They suggest that effective anti-inflammatory treatment of such neurodegenerative diseases as Alzheimer’s disease, Parkinson’s disease and Amyotrophic lateral sclerosis could be improved by using an appropriate combination of anti-inflammatory agents instead of relying on any single agent.

Aralar mRNA and protein levels in neurons and astrocytes freshly isolated from young and adult mouse brain and in maturing cultured astrocytes

Intense glucose-based energy metabolism and glutamate synthesis by astrocytes require malate–aspartate-shuttle (MAS) activity to regenerate NAD+ from NADH formed during glycolysis, since brain lacks significant glycerophosphate shuttle activity. Aralar is a necessary aspartate/glutamate exchanger for MAS function in brain. Based on cytochemical immunoassays the absence of aralar in adult astrocytes was repeatedly reported. This would mean that adult astrocytes must regenerate NAD+ by producing lactate from pyruvate, eliminating its use by oxidative and biosynthetic pathways. We alternatively used astrocytes and neurons from adult brain, freshly isolated by fluorescence-activated cell sorting, to determine aralar protein by a specific antibody and its mRNA by real-time PCR. Both protein and mRNA expressions were identical in adult neurons and astrocytes and similar to whole brain levels. The same level of aralar expression was reached in well-differentiated astrocyte cultures, but not until late development, coinciding with the late-maturing brain capability for glutamate formation and degradation.

Purification and characterization of adult optic nerve head astrocytes

To establish a method of purifying and characterizing adult astrocytes from optic nerve head (ONH). Experimental study. The lamina cribrosa tissue from ONH of human eye was isolated under anatomic microscopy, and then 4 to 6 little explants were incubated in each culture plate containing culture medium DMEM/F12. After 8 to 10 weeks, the cells were removed by digesting cells with 0.25% trypsogen. Selective astrocyte culture medium is subsequently used. After two passages, astrocytes were identified by the observation of cell morphology and immunofluorescent staining of GFAP and NCAM. After 2 to 3 weeks of explants planting, cells showed an obvious migration procession by crawling in succession from the verge of the explants and rapidly splitting. Most cells displayed a flat star shape or polygon after digested with trypsogen. Several cells are long fusiformis. Almost all cells presented a flat star shape and simultaneously expressed GFAP and NCAM when the cells cultured with selective astrocyte culture medium. Cultured human ONH astrocytes can be obtained by precisely separating lamina cribrosa and placing the explants on the margin of culture medium, a method that promotes cell adherence. Using selective astrocyte culture medium is very effective and convenient in purifying primary astrocytes.

P4‐016: Differences in pathways used for beta‐amyloid uptake by adult human astrocytes and microglia?

P4‐016: Differences in pathways used for beta‐amyloid uptake by adult human astrocytes and microglia?

The Increased Activity of TRPV4 Channel in the Astrocytes of the Adult Rat Hippocampus after Cerebral Hypoxia/Ischemia

The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult.

Molecular and kinetic characterization of histamine transport into adult rat cultured astrocytes

Astrocytes have a key role in the clearance and inactivation of histamine in the adult central nervous system, but transporters which mediate histamine uptake into astrocytes have not been fully characterized. We therefore investigated the kinetic and molecular characteristics of histamine uptake into cultured adult rat astrocytes. [3H]-histamine was taken up by astrocytes in a temperature-, time- and concentration-dependent manner and was inhibited up to 60–70% by 1mM ouabain or by substitution of NaCl with choline chloride. Specific [3H]-histamine uptake, determined as the difference between transport at 37 and 4°C, displayed saturation kinetics with the apparent Michaelis–Menten constant (Km) of 141 and 101μM and the apparent maximal uptake rate (Vmax) of 22.5 and 17.8pmol/min/mg protein, as estimated from the Woolf and the Eadie–Hofstee plots, respectively. Since our data suggested the presence of a carrier-operated histamine uptake system, we assessed the possible involvement of the organic cation transporters (OCT) 1, 2 and 3, which have been previously described to play a role in histamine transport in the central nervous system. Low level mRNA expression of all OCT isoforms was detected, but in contrast to rat brain cortex homogenate, where OCT3 was the most prominently expressed OCT isoform, OCT2 mRNA was the predominant OCT species in cultured astrocytes. However, OCT inhibitors corticosterone and decynium 22 (D22) had no effect or only modestly reduced [3H]-histamine uptake. Thus, our data indicate that adult rat astrocytes possess an efficient high-capacity, low-affinity carrier-operated histamine uptake system, which does not seem to involve OCTs.

Caspase activation contributes to astrogliosis

Caspases, a family of cysteine proteases, are widely activated in neurons and glia in the injured brain, a response thought to induce apoptosis. However, caspase activation in astrocytes following injury is not strongly associated with apoptosis. The present study investigates the potential role of caspase activation in astrocytes with another characteristic response to neural injury, astrogliosis. Caspase activity and morphological and biochemical indices of astrogliosis and apoptosis were assessed in (i) cultured neonatal rat astrocytes treated with astrogliosis-inducing stimuli (dibutryl cAMP, β-amyloid peptide), and (ii) cultures of adult rat hippocampal astrocytes generated from control and kainate-lesioned rats. The effects of broad spectrum and specific pharmacological caspase inhibitors were assessed on indicators of astrogliosis, including stellate morphology and expression of glutamine synthetase and fibroblast growth factor-2. Reactive neonatal and adult astrocytes demonstrated an increase in total caspase activity with a corresponding increase in the expression of active caspase-3 in the absence of cell death. Broad spectrum caspase inhibition with zVAD significantly attenuated increases in glutamine synthetase and fibroblast growth factor-2 in the reactive astrocytes. In the reactive neonatal astrocyte cultures, specific inhibition of caspases-3 and -11 also attenuated glutamine synthetase and fibroblast growth factor-2 expression, but did not reverse the morphological reactive phenotype. Astrogliosis is observed in all forms of brain injury and despite extensive study, its molecular triggers remain largely unknown. While previous studies have demonstrated active caspases in astrocytes following acute brain injury, here we present evidence functionally implicating the caspases in astrogliosis.

Adult and neonatal astrocytes exhibit diverse gene expression profiles in response to beta amyloid <i>ex vivo</i>

Astrocytes are implicated in the neuropathology of Alzheimer’s disease (AD) by clustering with other activated inflammatory cells at the sites of amyloid beta (Aβ) deposits formed in the cortex and hippocampus. Astrocytes are known to contribute to the clearance of Aβ in the AD brain. Also, adult but not neonatal mouse astrocytes are able to clear Aβ deposits from the tissue sections of transgenic AD mice and human brain ex vivo. Because these findings suggest that cultured neonatal astrocytes may not represent a relevant cell for modeling the function of astrocytes in neurodegenerative diseases, we studied whether neonatal and adult astrocytes show different responses in gene expression when exposed to brain sections burdened by deposits of human Aβ. Whole genome microbarrays demonstrated greater alteration of gene expression in adult astrocytes than in neonatal astrocytes. When exposed to Aβ burdened brain sections adult but not neonatal astrocytes up-regulated genes related to peptidase (such as MMP13, MMP12, Phex, Htra1), scavenger receptor (Scara5, Enpp2) and glutathioine transferase (Gsta1, Gsta2, Gclm) activity, suggesting increased ability to degrade and endocytose Aβ peptides and protect against oxidative bursts. Quantitative RT-PCR analysis confirmed the significant alteration in gene expression of key peptidases, scavenger receptors and cholesterol synthesis. Our data suggest that adult astrocytes in culture are more sensitive to disease-relevant stress showing more extensive genetic response compared to neonatal astrocytes. In addition, the identified peptidases and scavenger receptors which increase expression selectively in adult astrocytes suggest their major role in astrocyte-mediated clearance of Aβ deposits in AD.

CCL2 binding is CCR2 independent in primary adult human astrocytes

Chemokines are low relative molecular mass proteins, which have chemoattractant actions on many cell types. The chemokine, CCL2, has been shown to play a major role in the recruitment of monocytes in central nervous system (CNS) lesions in multiple sclerosis (MS). Since resident astrocytes constitute a major source of chemokine synthesis including CCL2, we were interested to assess the regulation of CCL2 by astrocytes. We showed that CCL2 bound to the cell surface of astrocytes and binding was not modulated by inflammatory conditions. However, CCR2 protein was not detected nor was activation of the classical CCR2 downstream signaling pathways. Recent studies have shown that non-signaling decoy chemokine receptors bind and modulate the expression of chemokines at site of inflammation. Here, we show that the D6 chemokine decoy receptor is constitutively expressed by primary human adult astrocytes at both mRNA and protein level. In addition, CCL3, which binds to D6, but not CCL19, which does not bind to D6, displaced CCL2 binding to astrocytes; indicating that CCL2 may bind to this cell type via the D6 receptor. Our results suggest that CCL2 binding to primary adult human astrocytes is CCR2-independent and is likely to be mediated via the D6 decoy chemokine receptor. Therefore we propose that astrocytes are implicated in both the establishment of chemokine gradients for the migration of leukocytes into and within the CNS and in the regulation of CCL2 levels at inflammatory sites in the CNS.

The effect of amyloid associated proteins on the expression of genes involved in amyloid-β clearance by adult human astrocytes

Astrocytes appear to be important mediators in the clearance of amyloid beta1-42 (Aβ), the key component of senile plaques characteristic of Alzheimer's disease (AD). Recently, we found the amyloid associated proteins (AAPs) α1-antichymotrypsin (ACT), apolipoprotein J and E (ApoJ and ApoE) and a mixture of serum amyloid P (SAP) and C1q (SAP–C1q) to modify Aβ-uptake by human astrocytes. Here we investigated the effect of oligomeric (Aβoligo) and fibrillar Aβ (Aβfib), alone and in combination with a panel of AAPs on the astrocytic expression of genes proposed to be involved in Aβ-uptake and degradation. Primary human astrocytes (isolated from non-demented control (n=4) and AD patient (n=4) brain specimens) were exposed to either Aβoligo or Aβfib preparations with or without the above mentioned AAPs. Quantitative gene expression analysis of Aβ-receptors Scavenger receptor B1 (SCARB1), macrophage receptor with collagenous structure (MARCO) and low density lipoprotein receptor related protein-2 (LRP2 or megalin) as well as of Aβ-degrading enzymes neprilysin (NEP), insulin-degrading enzyme (IDE) and metalloproteinase-9 (MMP-9) was performed by real-time PCR. Basal expression of NEP, IDE and SCARB1 was easily detected whereas expression of MARCO, LRP2 and MMP-9 could only be detected upon pre-amplification. Basal expression of NEP, IDE and SCARB1 did not change upon exposure to Aβoligo or Aβfib alone in any of the investigated astrocyte cultures. Interestingly NEP expression was increased upon exposure to ApoE in combination with both Aβ-preparations, and also SCARB1 expression was induced upon treatment with ApoE in combination with Aβfib in astrocytes from non-demented controls. Further, SAP–C1q increased SCARB1 expression in control astrocytes when combined with Aβoligo. These alterations were not found in astrocytes from AD patients. Thus, we conclude that Aβ alone apparently does not affect the astrocytic expression of IDE, NEP or SCARB1. However, NEP and SCARB1 expression is increased in astrocytes from non-demented subjects when exposed to Aβ combined with AAPs like ApoE. These astrocytic gene expression-regulatory mechanisms appear to be defective in AD and thus might contribute to the development and progression of AD pathology.

Immunogold Detection of L-glutamate and D-serine in Small Synaptic-Like Microvesicles in Adult Hippocampal Astrocytes

Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

Detailed analysis of inflammatory and neuromodulatory cytokine secretion from human NT2 astrocytes using multiplex bead array

Astrocytes are a very important cell type in the brain fulfilling roles in both neuroimmunology and neurotransmission. We have conducted the most comprehensive analysis of secreted cytokines conducted to date (astrocytes of any source) to determine whether astrocytes derived from the human Ntera2 (NT2) cell-line are a good model of human primary astrocytes. We have compared the secretion of cytokines from NT2 astrocytes with those produced in astrocyte enriched human brain cultures and additional cytokines implicated in brain injury or known to be expressed in the human brain. The concentration of cytokines was measured in astrocyte conditioned media using multiplex bead array (MBA), where 18 cytokines were measured simultaneously. Resting NT2 astrocytes produced low levels (∼1–30pg/ml) of MIP1α, IL-6 and GM-CSF and higher levels of MCP-1, IP-10 and IL-8 (1–11ng/ml) under non-inflammatory conditions. All of these in addition to IL-1β, TNFα, and IL-13, were increased by pro-inflammatory activation (TNFα or IL-1β stimulation). In contrast, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, LTα, and IFNγ were not detected in astrocyte conditioned media under any of the culture conditions tested. NT2 astrocytes were unresponsive to IL-2 and the adenyl cyclase agonist, forskolin. Interestingly, IFNγ stimulation selectively increased IP-10 secretion only. As astrocytes stimulated with IL-1β or TNFα produced several chemokines in the ng/ml range, we next assessed the chemoattractant properties of these cells. Conditioned media from TNFα-stimulated astrocytes significantly chemoattracted leukocytes from human blood. This study provides the most comprehensive analysis of cytokine production by human astrocytes thus far, and shows that NT2 astrocytes are highly responsive to pro-inflammatory mediators including TNFα and IL-1β, producing cytokines and chemokines capable of attracting leukocytes from human blood. We conclude that in the absence of adult human primary astrocytes that NT2-astrocytes may provide a valuable alternative to study the immunological behaviour of human astrocytes.