From the Unité Propre CNRS 2580, 141 rue de la Cardonille, 34094 Montpellier cedex 5, France
Received for publication, November 25, 2002 , and in revised form, March 26, 2003.
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ABSTRACT |
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INTRODUCTION |
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Many recent studies have established that astrocytes, which are intimately associated with synapses, are active integrators and regulators of neuronal activity and synaptic transmission (26). These astrocytic functions are mediated, at least in part, by the release of various substances, including amino acids and polypeptides. Indeed, glutamate released from synaptic terminals not only binds to glutamate receptors on the post-synaptic neurons but also activates -amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors on the surrounding astrocytes. This activation induces a rapid increase in intracellular Ca2+ and a Ca2+-dependent release of glutamate from astrocytes that in turn activates post-synaptic glutamate receptors on neighboring neurons, thereby enhancing excitatory synaptic transmission (7, 8). A similar modulatory effect involving perisynaptic astrocytes has been demonstrated at inhibitory synapses (9). A new concept of tripartite synapses based on neuronal-astrocytic cooperation for the processing of information has recently emerged from these findings (10). A recent study has demonstrated that perisynaptic glial cells of a molluscan cholinergic synapse respond to acetylcholine by releasing a nicotinic receptor-like protein into the synaptic cleft. This protein can capture acetylcholine released into the synapse, thereby inhibiting cholinergic synaptic transmission (11). Although such a mechanism remains to be established in the mammalian CNS, astrocytic secretion of proteins may be one of the critical determinants involved in the modulation of synaptic transmission. To date, protein secretion by astrocytes has only been investigated in a piecemeal fashion, focusing on one or two protein species (1215).
The present study was carried out to identify the major proteins secreted by murine astrocytes in primary culture, using a proteomic approach based on matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The release of proteins by astrocytes was examined in cultures originating from different brain areas and exposed to pro-inflammatory treatments. Finally, the pattern of proteins secreted by astrocytes was compared with the cerebrospinal fluid (CSF) proteome.
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EXPERIMENTAL PROCEDURES |
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Astrocyte CulturesPrimary cultures of astrocytes from various brain regions were prepared as previously described (16). The brain structures from 18-day-old Swiss mouse embryos were mechanically dissociated in PBS, supplemented with 33 mM glucose (PBS-glucose). Cells were seeded (0.5 x 106 cells/ml) in either 100-mm (15 ml/dish) or 30-mm (3 ml/dish) culture dishes, previously coated with poly-L-ornithine (1.5 µg/ml, molecular weight = 40,000). The culture medium consisted of a 1:1 mixture of Dulbecco's modified Eagle's medium and F12 nutrient, supplemented with glucose (30 mM), glutamine (2 mM), NaHCO3 (13 mM), HEPES buffer (5 mM, pH 7.4), penicillin-streptomycin (100 IU/ml and 100 µg/ml, respectively), and 10% Nu-serum (BD Biosciences, Le Pont de Claix, France). The culture medium was changed every 3 days, and cells were maintained for 21 days at 37 °C in a humidified atmosphere containing 5% CO2. At this stage, cultures were highly enriched in astrocytes. Indeed, more than 98% of the cells were stained by an indirect immunofluorescence technique using a rabbit anti-glial fibrillary acidic protein antibody (nine fields containing 350 cells originating from three independent cultures counted). Fluorescence staining of cultures using fluorescein isothiocyanate-conjugated isolectine B4, a specific marker of microglial and endothelial cells in developing and adult CNS (17), indicated that they contained less than 0.2% of microglial and/or endothelial cells. Cultures were also devoid of neurons, because no immunostaining was observed using a mouse anti-MAP2 antibody. The remaining cells could not be fully differentiated astrocytes, which are unlabeled by glial fibrillary acidic protein antibodies (18).
Preparation of Astrocyte-conditioned Media and Cell ExtractsCells, grown in 100-mm culture dishes, were washed six times with the serum-free culture medium. This washing procedure efficiently eliminated all serum proteins, because we did not detect any trace of bovine albumin, the major serum protein, in the astrocyte-conditioned medium. Cells were then covered with a minimal volume of the serum-free medium (6 ml per dish) for 18 h at 37 °C and 5% CO2 in the presence of the indicated treatments. Astrocyte-conditioned media were harvested and centrifuged successively at 200 x g (5 min), 1,000 x g (10 min), and 20,000 x g (25 min) to remove non-adherent cells and debris. Samples (about 40 µg of protein/dish) were precipitated for 2 h with 10% ice-cold trichloroacetic acid. The cells were scraped off in 5 ml of PBS-glucose and centrifuged at 200 x g for 5 min. They were then resuspended in ice-cold lysis buffer containing Tris-HCl (50 mM, pH 7.4), EDTA (1 mM), SDS (1%), and a mixture of protease inhibitors (Roche Applied Science) and homogenized 20 times with a glass-Teflon homogenizer. Samples were centrifuged for 30 min at 10,000 x g, and the supernatants (whole cell extracts) were precipitated with 10% trichloroacetic acid. Protein concentration in conditioned media and cell extracts was determined with the bicinchoninic acid method (19).
Preparation of Cerebrospinal FluidThe cerebrospinal fluid (CSF) was collected from the cisterna magna (subcerebellar cisterna) of 2-month-old male Swiss mice previously anesthetized with an intraperitoneal injection of pentobarbital (60 µg/g of body weight). The CSF samples were centrifuged successively at 200 x g (5 min), 1,000 x g (10 min), and 20,000 x g (25 min) to eliminate cells and other insoluble material. The final supernatants were precipitated with 10% trichloroacetic acid.
High Resolution Two-dimensional Gel ElectrophoresisTrichloroacetic acid precipitates were washed three times with diethyl ether and resuspended in 350 µl of isoelectrofocusing medium containing urea (7 M), thiourea (2 M), CHAPS (4%), ampholines (preblended, pI 3.59.5, 8 mg/ml, Amersham Biosciences, Uppsala, Sweden), dithiothreitol (DTT, 100 mM), tergitol NP7 (0.2%, Sigma), and traces of bromphenol blue (20). Proteins (30 µg/gel) were first separated according to their isoelectric point along linear immobilized pH-gradient (IPG) strips (pH 310, 18 cm long) using the IPGphor apparatus (Amersham Biosciences). Sample loading for the first dimension was performed by passive in-gel re-swelling. After the first dimension, the IPG strips were equilibrated for 10 min in a buffer containing urea (6 M), Tris-HCl (50 mM, pH 6.8), glycerol (30%), SDS (2%), DTT (10 mg/ml), and bromphenol blue and then for 15 min in the same buffer containing 15 mg/ml iodoacetamide instead of DTT. For the second dimension, the strips were loaded onto vertical 12.5% SDS-polyacrylamide gels, unless otherwise indicated. The gels were silver-stained according to the procedure of Shevshenko et al. (21).
Image Acquisition and Two-dimensional Gel Spot Pattern Analysis Gels to be compared were always processed and stained in parallel. Gels were scanned using a computer-assisted densitometer. Spot detection, gel alignment, and spot quantification were performed using the Image Master 2D Elite software (Amersham Biosciences). Quantification of proteins was expressed as volumes of spots. To correct for variability resulting from silver staining, results were expressed as relative volumes of all spots in each gel. Data are the means of values from four gels originating from experiments performed on different sets of cultured astrocytes.
MALDI-TOF Mass Spectrometry and Protein IdentificationProteins of interest were excised and digested in gel using trypsin (sequencing grade, Promega, Charbonnières, France), as previously described (21). Digest products were completely dehydrated in a vacuum centrifuge and resuspended in 10 µl of formic acid (2%), desalted using Zip Tips C18 (Millipore, Bedford, MA), eluted with 10 µl of acetonitrile-trifluoroacetic acid (800.1%), and concentrated to 2 µl. Aliquots (0.5 µl) were mixed with the same volume of -cyano-4-hydroxy-trans-cinnamic acid (Sigma, 10 mg/ml in acetonitrile-trifluoroacetic acid, 500.1%) and loaded on the target of a BIFLEX III MALDI-TOF mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany). Analysis was performed in reflectron mode with an accelerating voltage of 20 kV and a delayed extraction of 400 ns. Spectra were analyzed using the XTOF software (Bruker-Franzen Analytik) and auto-proteolysis products of trypsin (molecular weights: 842.51, 1045.56, and 2211.10) were used as internal calibrates. Identification of proteins was performed using both Mascot and PeptIdent software (available at www.matrixscience.com and www.expasy.org/tools/peptident.html, respectively). A mass deviation of 100 ppm was allowed for data base interrogation, but the mass accuracy of our analyses was usually better than 50 ppm. Coverage of the full-length protein exceeding 15% was considered to be sufficient unless there were some obvious conflicts between the experimental molecular weight or isoelectric point, and those of the identified protein. Matching peptides with one missed cleavage were considered as pertinent only when there were two consecutive basic residues or when arginine and lysine residues were followed by a proline or acidic residues inside the peptide amino acid sequence (22, 23).
ImmunoblottingProteins, resolved on one- or two-dimensional (1-D or 2-D) gels, were transferred electrophoretically onto nitrocellulose membranes (Hybond-C, Amersham Biosciences). Membranes were incubated overnight with primary antibodies. Immunoreactivity was detected with an enhanced chemiluminescence method (Renaissance Plus, PerkinElmer Life Sciences, Boston, MA).
Measurement of Astrocyte ViabilityThe viability of astrocyte cultures exposed to pro-inflammatory treatments was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay (24). Cells were incubated with MTT (60 µg/ml, directly added to the serum-free medium) for 45 min at 37 °C. The blue formazan derivative was solubilized in Me2SO, and optical density was read at 560 nm. Lack of membrane integrity of astrocytes was also estimated by measuring propidium iodide (PI) incorporation (3 µg/ml, 10 min at room temperature). Cells were then fixed with 4% paraformaldehyde in PBS (30 min at 4 °C), and nuclei were stained with Hoechst 33258 (1 µg/ml for 10 min at room temperature). They were then washed successively with PBS and distilled water and mounted in Mowiol under coverslips. Nuclear DNA staining was examined by digital fluorescence imaging microscopy (Axiophot 2 microscope, Zeiss). Necrosis was estimated by counting PI-positive nuclei versus total nuclei (stained with Hoechst 33258) in at least nine different fields (about 350 cells per field) originating from three independent cultures.
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RESULTS |
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Regional Specificity of Astrocytic Protein ReleaseTo examine the regional specificity of astrocytic protein release, we compared the pattern of proteins in the medium conditioned by striatal astrocytes with the pattern of proteins released by cultures of astrocytes isolated from other CNS regions. A similar profile of extracellular proteins was found in the medium conditioned by astrocytes from the striatum, the cerebral cortex, the hippocampus, and the cerebellum, with a few remarkable differences, which are depicted in Fig. 4. Striatal astrocytes released larger amounts of apolipoprotein E than did astrocytes originating from the other brain regions examined. In contrast, striatal astrocytes secreted the lowest amounts of IBP-2. A regional specificity was also observed for the release of metalloproteinase inhibitors. Indeed, matrix metalloproteinase inhibitor-2 was only detectable in the supernatant of striatal astrocytes, whereas TIM-1 was specifically released by astrocytes from the cerebellum. AOP2, a protein that is released by a BFA-insensitive pathway, was greatly enriched in the conditioned medium of striatal, cortical, and hippocampal astrocytes (about 2% of total extracellular protein content), compared with that measured in the supernatant of cerebellar cultures (0.28 ± 0.07% of total extracellular protein content, n = 4) (Fig. 4).
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Effects of Pro-inflammatory Treatments on Astrocytic Protein ReleaseIt is well documented that astrocytes play a pivotal role in the type and extent of CNS immune and inflammatory response, in part by their ability to release various cytokines and chemokines in response to inflammatory stimuli (31). We thus examined the effects of pro-inflammatory treatments on the pattern of proteins secreted by cultured striatal astrocytes. Treating cells for 18 h with either LPS (1 µg/ml) or two pro-inflammatory cytokines, IL-1 (200 pg/ml) and TNF-
(10 ng/ml), respectively, increased the amount of four protein species (B2MG, ceruloplasmin, complement C3
and
chains) in the medium conditioned by striatal astrocytes (Fig. 5A). In contrast, their intracellular level remained unchanged (data not shown). Surprisingly, the astrocytic secretion of pentraxin-related protein-3 (PTX3), a protein known to be highly induced by IL-1
or TNF-
in several cell types (3234), was not increased by the pro-inflammatory treatments performed in the present study (data not shown). Two additional proteins appeared in the supernatant of cells exposed to these treatments (Fig. 5A). The first one is chitinase-3 like protein-1 (C3L1), a glycoprotein of the chitinase protein family, which is thought to contribute to the capacity of cells to respond to changes in their environment (35) and is increased in the serum of patients with inflammatory diseases such as osteoarthritis and rheumatoid arthritis (36). The second one is neutrophil gelatinase-associated lipocalin (NGAL), a protein that was originally identified to be associated with 92-kDa gelatinase/matrix metalloproteinase 9 (MMP9) and secreted in specific granules from activated neutrophils (37). Lipocalins are a family of mainly extracellular proteins involved in the binding, transport, and presentation of small lipophilic molecules, including retinoids, steroids, and fatty acids (38). NGAL was one of the major proteins identified in the medium conditioned by striatal astrocytes treated with LPS or IL-1
(the NGAL isoforms represent 10.4 ± 2.5 and 6.6 ± 1.7% of the total amount of extracellular proteins in cultures treated with LPS and IL-1
, respectively). We have verified that pro-inflammatory treatments did not significantly decrease cell viability, as assessed by the MTT assay (93 ± 5, 97 ± 2, and 97 ± 3% of cell survival measured in cultures treated with LPS, IL-1
, and TNF-
, respectively, n = 3). Moreover, these treatments did not alter membrane integrity of astrocytes, a process that could be responsible for the increased expression of some proteins in culture supernatant, as indicated by the lack of PI incorporation in cell nuclei (less than 0.1% of PI-positive cells in cultures exposed to sham or pro-inflammatory treatments).
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Next, we have examined whether astrocytes isolated from other brain regions exhibited differences in reactivity to pro-inflammatory treatments. We focused on LPS, because this treatment was the most efficient in stimulating the secretion of the aforementioned proteins in striatal astrocyte cultures, when compared with the other pro-inflammatory treatments used. Treating astrocytes isolated from the cerebral cortex, the hippocampus, and the cerebellum with LPS increased the secretion of B2MG, ceruloplasmin, complement C3 ( and
chains), and NGAL to the same extent as that observed in striatal cultures (Fig. 5B). LPS also markedly stimulated the secretion of C3L1 in cortical cultures. However, a much lower expression of C3L1 was detected in supernatants of cerebellar astrocytes exposed to LPS (Fig. 5B). C3L1 was already detectable in the supernatant of untreated astrocytes from the hippocampus, and its secretion was strongly stimulated by LPS (Fig. 5B).
Detection in the Cerebrospinal Fluid of Proteins Released by AstrocytesSeveral proteins identified in the medium conditioned by astrocytes, such as B2MG, complement C3 and
chains, apolipoprotein E, and cystatin C, are known as major protein constituents in the human CSF (39). This suggests that astrocytes may be an important source of proteins in the CSF. To our knowledge, no proteomic map of mouse CSF is available. To determine which proteins released by astrocytes are present in the CSF, a proteomic analysis of mouse CSF proteins was performed. The mouse CSF 2-D gel map was compared with the 2-D gel map of proteins released by astrocytes (Fig. 6). As observed in human CSF (40), the mouse CSF showed an enrichment in plasma proteins such as albumin, apolipoprotein A1, transferrin,
1-antitrypsin,
2-macroglobulin, hemopexin, and transthyretin. In addition, more than two thirds of the proteins identified in the astrocyte-conditioned medium were also detectable in the mouse CSF (Fig. 6). They include proteins secreted through a BFA-sensitive pathway such as complement chains and ceruloplasmin, and proteins released through a BFA-insensitive mechanism, including PEBP, 14-3-3 proteins, antioxidant proteins (thioredoxin peroxidases 1 and 2, AOP2, and superoxide dismutase) and several cytosolic enzymes.
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DISCUSSION |
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Our procedure based on silver staining allowed the detection of proteins secreted in relatively low amounts (a few nanograms) by astrocytic cultures. Silver staining also allows full use of the sensitivity of our MALDI-TOF mass spectrometer (femtomole range). However, despite its relatively high sensitivity, our approach only provides insight into a restrictive population of proteins, presumably the major proteins secreted by astrocytes. Indeed, we detected no growth factor or cytokine, which are known to be produced by astrocytes, even in cultures exposed to pro-inflammatory treatments.
The comparative analysis between proteins released by astrocyte cultures and the CSF proteome indicated that more than two thirds of the proteins identified in the astrocyte-conditioned medium were also detected in the CSF. If the profile of protein secreted by astrocytes in vitro is reflective of proteins released by astrocytes in situ, our results suggest that astrocytes constitute one of the major sources of CSF proteins. However, we must point out that the cultured astrocytes used in the present study were isolated from mouse embryos, whereas the CSF was from 2-month-old animals. Moreover, other cell types can contribute to the CSF protein content. A previous study has shown that cultured leptomeningeal cells secrete various CSF proteins (41). Many CSF proteins, such as 14-3-3 proteins, can also be released by neurons, in particular following neurotoxic processes. In this regard, the isoform pattern of 14-3-3 proteins in the human CSF has been considered as a diagnostic marker of Creutzfeldt-Jakob disease (42).
The comparative analysis of proteins secreted by astrocytes isolated from various CNS regions showed a similar profile of protein secretion, even though a regional specificity was observed for the release of a few protein species. These disparities in secretion profiles may reflect regional differences in vivo, although one cannot exclude that they result from the proliferation of a small subset of the isolated cells in our cultures.
Our results indicate that astrocytes can secrete proteins from the biosynthetic pathway by exocytosis of secretory vesicles. This conclusion is supported by 1) the inhibition of the secretion of several proteins by BFA, a specific inhibitor of exocytotic vesicle assembly, and 2) the accumulation of these proteins in the extracellular medium compared with their low amounts in whole cell extracts. We also provide evidence that an alternative pathway contributes to the astrocytic secretion of proteins. Indeed, a differential 2-D gel analysis of astrocyte-conditioned medium against whole-cell extract indicated an extracellular enrichment of several proteins lacking any classic signal sequence for transport into the endoplasmic reticulum (18 proteins showing an enrichment ≥ 2.5-fold in cell-conditioned medium versus whole cell extract were identified). Moreover, the accumulation of these proteins in the extracellular medium was not inhibited by BFA. Several observations further support that this accumulation results from an active secretion process: 1) some of these proteins have already been identified to be secretion products of various cell types. In this regard, cyclophilin A was detected in the conditioned medium of immune cells stimulated by LPS as well as the synovial fluid of patients with rheumatoid arthritis (27, 43). Similarly, the immunodetection of PEBP on the cell surface and the presence of recombinant His-tagged PEBP in the supernatant of transfected cells indicate that the localization of this protein is not restricted to the cytosol or the inner leaflet of the plasma membrane (26). 2) Most of these proteins have already been found in biological fluids and were identified as major protein components of the mouse CSF in the present study.
In addition to the classic exocytotic pathway, the endocytic pathway is an alternative mechanism that may account for the secretion of cytosolic proteins in the extracellular medium (44). This secretion process results from the fusion of late multivesicular endosomes with the plasma membrane and the release of intraluminal vesicles in the extracellular medium. These vesicles, dubbed exosomes, are generated by inward budding from the limiting membrane into the lumen of endosomes. This creates a membrane-enclosed compartment in which the lumen is topologically equivalent to the cytosol (44). To date, the release of exosomes has been described for various cell types, including reticulocytes, B- and T-lymphocytes, macrophages, dendritic cells, and intestinal epithelial cells (4446). These cells are mainly of hematopoietic origin and/or antigen-presenting cells. Although astrocytes cannot function as fully competent antigen-presenting cells, they were the first CNS cell type shown to express major histocompatibility complex antigens and some co-stimulatory molecules. Moreover, the overall protein composition of exosomes of dendritic and epithelial cells, as determined by proteomic analyses (45, 46), showed many similarities with the protein pattern of the astrocyte-conditioned medium. These observations suggest that the accumulation of some cytosolic proteins in astrocyte-conditioned medium may result from their release through the exosomal secretory pathway. Another unconventional secretion process, which requires specific export and/or internalization sequences, has been involved in the release of homeoproteins such as Engrailed 2, HIV-transactivator protein, and other "messenger proteins" (47). This secretion process is probably not involved in the astrocytic release of proteins lacking signal peptides. Indeed, none of them bear strong sequence similarities to either the leucine-rich nuclear export sequence of Engrailed 2 or the translocation sequence identified in Antennapedia and the HIV-transactivator protein.
With regard to function, antioxidant proteins constitute the largest group of proteins identified in astrocyte-conditioned medium. These proteins may contribute to the well documented neuroprotective effect of astrocytes against oxidative stress (48). GST class-mu, the unique GST identified in the astrocyte-conditioned medium, shows a high detoxifying activity toward 4-hydroxy-2-nonenal, which is the major hydroxy-alkenal formed during the peroxidation of polyunsaturated fatty acid and is highly cytotoxic to neuronal cells (49, 50). Astrocytes also release 3 peroxiredoxins (peroxiredoxin 1 and 2, AOP2), which belong to a family of enzymes that reduce hydroperoxides and might play a major role in the clearance of low concentrations of H2O2, such as those measured in the CNS (51). All peroxiredoxins except AOP2 share two conserved reactive cysteine residues in the active site and use thioredoxin as a physiological electron donor (28). A recent study (51) has demonstrated that AOP2, which contains only one of the conserved cysteine residues, binds to cyclophilin A and that this protein supports its peroxidase activity as an immediate electron donor. This suggests that cyclophilin A released by astrocytes also contributes to extracellular peroxidase activity. Finally, ceruloplasmin, which is essential for brain iron metabolism, may also participate in the antioxidant function of astrocytes (52).
Proteases and protease inhibitors constitute another major group of proteins secreted by astrocytes that may modulate vulnerability to neurotoxicity. In this regard, both beneficial and detrimental effects on neuronal survival have been attributed to extracellular proteases, including matrix metalloproteinases (MMPs) (5355). Our study suggests that astrocytes are an important source of MMP inhibitors rather than MMPs. We also provide evidence that astrocytes release substantial amounts of the multifunctional protein PEBP, which was recently identified as a major inhibitor of serine proteases, including thrombin, neuropsin, and trypsin (26).
Extracellular proteases also remodel the pericellular micro-environment, primarily through the cleavage of extracellular matrix components. They participate in structural changes in neuronal architecture and in the pattern and number of neuronal connections associated with long lasting forms of synaptic plasticity (56, 57). SPARC is another major protein constitutively released by astrocytes that may contribute to synaptic plasticity (58). Finally, the astrocytic secretion of the octadecaneuropeptide DBI, a negative allosteric modulator of GABAA receptor channels, may participate in the regulation of GABAergic synaptic transmission (25, 59).
In conclusion, the present study provides the first unbiased characterization of the major proteins secreted by astrocytes in vitro. If astrocytes in situ produce the same protein species, the secretion of proteins may be one critical mechanism by which astrocytes modulate neuronal survival and function.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 33-4-67-14-29-83; Fax: 33-4-67-14-29-10; E-mail: marin{at}montp.inserm.fr.
1 The abbreviations used are: CNS, central nervous system; AOP2, antioxidant protein 2; BFA, brefeldin A; B2MG, 2-microglobulin; C3L1, chitinase-3-like protein-1; CSF, cerebrospinal fluid; CYSC, cystatin C; DBI, diazepam binding inhibitor; GST1, glutathione S-transferase class-Mu; IBP2, insulin-like growth factor binding protein 2; IL-1
, recombinant murine interleukin 1
; IPG, immobilized pH gradient; LPS, lipopolysaccharide; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; MMP, matrix metalloproteinase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; NGAL, neutrophil gelatinase-associated lipocalin protein; PEBP, phosphatidylethanolamine-binding protein; PI, propidium iodide; PTX3, pentraxin-related protein chain 1; SPARC, secreted protein acid and rich in cysteine; TIM1, and 2, metalloproteinase inhibitors 1 and 2; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; 1-D, one-dimensional; 2-D, two-dimensional; HIV, human immunodeficiency virus.
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REFERENCES |
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