Human Astrocytes: Secretome Profiles of Cytokines and Chemokines

Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (CXCL1), IL-6, IL-8 (CXCL8), MCP-1 (CCL2), MIF and Serpin E1. Following stimulation with IL-1β and TNF-α, activated astrocytes newly produced IL-1β, IL-1ra, TNF-α, IP-10 (CXCL10), MIP-1α (CCL3) and RANTES (CCL5), in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.


Introduction
Astrocytes belong to one of three major types of neuroglia in the central nervous system (CNS) and play active roles in many neuronal functions: maintaining ion and pH homeostasis, promoting the synthesis and removal of neurotransmitters, providing glucose supply and antioxidant defense, and regulating synaptic activity by producing various cytokines, chemokines, growth factors, and metabolites, all of which act as a molecular coordinator of neuron-glia communication [1]. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neurotoxic (inflammatory) and neuroprotective (immunoregulatory) roles in the brains of human neurological diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), and HIV-1 associated dementia (HAD) [2]. However, at present, the exact profile of human astrocytederived cytokines and chemokines during inflammation remain mostly unclear, possibly attributable to the limited availability of non-malignant human astrocyte cell lines that serve as an in vitro model of normal human astrocytes. In the present study, we characterized the comprehensive profiles named ''secretome'' [3] of cytokines and chemokines derived from cultured normal human astrocytes, compared under resting and activated conditions by using a protein microarray.

Ethics statement
Fetal brain tissue was obtained from a 15 weeks human fetus and dissociated cells prepared as described previously [4,5]. Human tissue collected for research purpose was approved by the Chung-Ang University Ethics Committee on Human Subject (Certificate #09-0041). Pregnant women gave written informed consents for clinical procedure and research use of the embryonic tissue in accordance with the declaration of Helsinki.

Human astrocytes in culture
The brain tissue isolated from a fetus of 15 weeks gestation, was dissociated into single cells by incubation with 0.25% trypsin in phosphate-buffered saline (PBS) for 30 min, as described previously [4,5]. Dissociated cells were suspended in the culture medium, composed of the Dulbecco's modified Eagle medium (DMEM) with high glucose (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 20 mg/mL gentamicin (Sigma, St Louis, MO). Dissociated cells were plated at 5610 7 cells/T75 flask and were grown in an incubator with 5% CO 2 atmosphere. In primary brain cell cultures, all microglia freely floating in the medium were removed.
After replating the cultures for three to five times by treatment with trypsin, most of neurons and oligodendrocytes underwent cell death and detached off, while astrocytes were firmly attached onto the flask surface, resulting in enrichment of highly purified astrocytes.

Immunocytochemistry
Human astrocytes cultured on poly-L-lysine-coated Aclar plastic coverslips (9 mm in diameter) were fixed in methanol for 10 min at 220uC. The cells were incubated with primary antibodies specific for GFAP (1:1,000, rabbit; Millipore, Billerica, MA), a cell type specific marker for astrocytes, or tubulin bIII antibody (1:200, mouse, Millipore), a. neuron specific marker, for 48 hrs at 4uC followed by Alexa Fluor594-conjugated anti-rabbit IgG or antimouse IgG for 1 hr at room temperature (RT). For immunostaining for cell type markers for oligodendrocytes and microglia, astrocytes on coverslips were fixed in 4% paraformaldehyde for 2 min, washed in PBS, incubated in anti-galactocerebroside

Molecular network analysis
We imported Entrez Gene IDs corresponding to cytokine and chemokine genes into the Core Analysis tool of Ingenuity Pathways Analysis (IPA) (Ingenuity Systems; www.ingenuity. com). IPA is a commercial knowledgebase that contains information on approximately 3,000,000 biological and chemical interactions with definite scientific evidence. By uploading the list of Gene IDs, the network-generation algorithm identifies focused genes integrated in global molecular networks. IPA calculates the score p-value that reflects the statistical significance of association between the genes and the networks by Fisher's exact test. We considered p-value,0.05 as a significant association. The information on known NF-kB target genes was collected from web accessible databases constructed by Dr. Thomas Gilmore, Boston University (www.bu.edu/nf-kb/gene-resources/target-genes) and by Bonsai Bioinformatics, Laboratoire d'Informatique Fondamentale de Lille (LIFL), Université Lille 1 (bioinfo.lifl.fr/NF-KB), as described previously [6].

Discussion
Due to the limited availability of human brain tissues, only a small number of studies have previously reported the cytokine production profiles of normal human astrocytes [9][10][11]. In the present study, we characterized the more comprehensive profile of cytokine and chemokine named ''secretome'' derived from nonstimulated resting and activated human astrocytes by using a protein microarray. We found that both non-stimulated and IL-1b/TNFa-activated astrocytes produce distinct sets of cytokines and chemokines, nearly all of which represent direct targets of transcription factor NF-kB. One exception is MCP-1 (CCL2), a direct target of NF-kB [12], was down-regulated in activated astrocytes following exposure to IL-1b and TNF-a. In contrast, a previous study has indicated that the activated adult human astrocytes in culture produced increased amounts of MCP-1 [11]. The discrepancy between our results and previous findings is attributable to a difference in maturation of cultured cells, i.e. fetal versus adult astrocytes employed. These results suggest that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory environments.
NF-kB acts as a central regulator of innate and adaptive immune response, stress response, cell proliferation, and apoptosis [13]. The NF-kB family proteins consist of five members, such as RelA (p65), RelB, c-Rel, NF-kB1 (p105), and NF-kB2 (p100). The latter two are processed proteolytically into p50 and p52, respectively. The NF-kB family proteins constitute either homodimers or heterodimers, except for RelB that exclusively forms heterodimers. The NF-kB dimers interact with consensus DNA sequences termed the kB site located on promoters to activate or repress transcription of target genes. Only p65 and c-Rel act as a potent transcriptional activator, whereas p50 and p52 homodimers generally repress transcription, leading to differential regulation of gene expression of NF-kB targets [14]. We found that MCP-1 (CCL2), a target of NF-kB, is down-regulated in NF-kB-activated human astrocytes. Importantly, NF-kB target genes often activate NF-kB itself, providing a positive regulatory loop that amplifies and perpetuates inflammatory responses [15]. IL-1 b and TNF-a are the prototypes of NF-kB activators for the canonical NF-kB signaling pathway. We found that non-stimulated human astrocytes do not constitutively produce IL-1b or TNF-a, while activated human astrocytes could produce both, as described previously [9,11]. In contrast, a previous study has shown that IL-1b is undetectable at both mRNA and protein levels in nonstimulated or cytokine-stimulated cultured human astrocytes [10]. A different study from the same group showed that IL-8 (CXCL8) is undetectable in non-stimulated human fetal astrocytes in culture [16], being inconsistent with our results. In our study, nonstimulated astrocytes expressed a panel of NF-kB targets, such as G-CSF, GM-CSF, IL-6, GROa (CXCL1), IL-8 (CXCL8), MCP-1 (CCL2), MIF and serpin E1, suggesting that the NF-kB signaling pathway is constitutively active to a certain extent in normal human astrocytes in culture. Notably, GM-CSF serves as an antiapoptotic and neurotrophic factor [17].  Chemokines constitutes a group of structurally related proteins that play a pivotal role in regulation of immune cell trafficking involved in inflammatory and immunoregulatory processes in the CNS [18]. In the present study, molecular network of cytokine and chemokine secretome of activated human astrocytes strongly supported the view that these soluble factors are critically involved in regulation of the cellular interaction and trafficking of immune cells. Various chemokine receptors, such as CCR2 with the ligand MCP-1 and CXCL2 with multiple ligands, such as Groa, Grob and IL-8, are up-regulated in brain lesions of trauma, ischemia, and multiple sclerosis (MS) [19]. MIP-1 belongs to a family of C-C chemokines with a potent chemotactic activity for neutrophils and other leukocytes, composed of two members MIP-1a (CCL3) and MIP-1b (CCL4), both of which exhibit very similar but not identical proinflammatory properties through binding differentially to the receptors CCR1, CCR4 or CCR5. The upregulated expression of MIP-1a and MIP-1b was identified in acute MS lesions [20]. However, we identified MIP-1a (CCL3) but did not detect the expression of MIP-1 b (CCL4) in non-stimulated or activated human astrocytes in culture, suggesting that the gene regulatory mechanism is different between MIP-1a and MIP-1 b, although both are directly regulated by NF-kB.
RANTES is a member of C-C chemokines involved in the pathogenesis of MS and HIV-1 encephalitis by binding to the receptors CCR1, CCR3 or CCR5 [21,22]. A previous study has shown that fetal human astrocytes upon activation by IL-1b secretes a large amount of RANTES (CCL5) protein [23], supporting our observation that RANTES is the most prominently up-regulated chemokine in activated human astrocytes in culture. Importantly, RANTES plays a neuroprotective role in ischemic brain injury [24]. The expression of IP-10 (CXCL10), secreted by monocytes, endothelial cells, and fibroblasts in response to IFN-c, shows a chemotactic activity for T cells, NK cells, dendritic cells and monocytes/macrophages through binding to the receptor CXCR3. We found that IP-10 production is greatly enhanced in activated human astrocytes in culture. Notably, the expression of IP-10 and CXCR3 is up-regulated in the brains of AD, where CXCR3 is expressed constitutively on neurons, while IP-10 expression is enhanced in a subset of reactive astrocytes surrounding senile plaques [25].
In conclusion, the comprehensive cytokine and chemokine secretome of activated human astrocytes, closely linked to NF-kB activation, suggested that astrocyte-derived cytokines and chemokines play a central role in proinflammatory (neurotoxic) and immunoregulatory (neuroprotective) responses in the CNS.  Functional networks were studied by importing Entrez Gene IDs of 14 up-regulated and 2 down-regulated cytokines in IL-1b/TNF-a-activated human astrocytes into the core analysis tool of IPA. They are listed with functional networks, focused molecules, and p-value of the Fisher's exact test. The first rank network is illustrated in Fig. 4