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Chapter 4:

Constitutive expression of CXCL10 in

cultured cortical neurons

Eiko K. de Jong1, Hendrikus W.G.M. Boddeke1, Nieske Brouwer1, Ivica Granic3, Ulrich L.M.

Eisel3, Robert S.B. Liem2, and Knut Biber1* 1 Department of Medical Physiology, University Medical Center Groningen, University of

Groningen, Groningen, The Netherlands 2 Department of Cell Biology, Section Electron Microscopy, University Medical Center

Groningen, University of Groningen, Groningen, The Netherlands 3 Department of Molecular Neurobiology, University of Groningen, Haren, The Netherlands

Re-submitted to Neuroscience

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Abstract

Chemokines are suggested to be involved in the signaling between damaged neurons and

microglia. In line with this, we have recently shown that the chemokine CCL21 is up-

regulated and released by neurons after cellular injury in vitro and in vivo and attracts

microglia via the chemokine receptor CXCR3. Neurons express the chemokine CXCL10

which is also a ligand for microglial CXCR3. Here, we have investigated the expression

pattern of CXCL10 in cultured cortical neurons. In contrast to CCL21, CXCL10 is expressed

constitutively and is not regulated by neuronal injury or stress. Furthermore, we did not find

conditions that induce the release of neuronal CXCL10. These results thus show prominent

differences in the regulation between neuronal chemokines. Whereas neuronal CCL21 might

function as a signal between damaged neurons and microglia, the results presented here do not

support such a role for CXCL10.

Introduction

Microglia are the immune cells of the brain, which under physiological conditions

continuously monitor and sample their environment (Davalos et al., 2005; Nimmerjahn et al.,

2005). When homeostasis in the brain is disturbed, microglia become rapidly activated

(Kreutzberg, 1996; Raivich et al., 1999; van Rossum and Hanisch, 2004). Microglial

activation can either result in a neurotrophic or a neurotoxic response, depending on the input

of the environment (Streit, 2002; van Rossum and Hanisch, 2004; Aschner et al., 1999).

Therefore, a better understanding of the mechanism underlying microglial recruitment and

activation is very important. However the factors which mediate this neuro-glial signaling are

currently unknown.

Chemokines and their receptors have been recognized as important mediators of cell-cell

signaling in both the periphery (Mackay, 2001; Moser et al., 2004; Murphy et al., 2000; Rossi

and Zlotnik, 2000; Rot and von Andrian, 2004) and also in the central nervous system (for

review see (Ambrosini and Aloisi, 2004; Bacon and Harrison, 2000; Bajetto et al., 2002;

Biber et al., 2002; Ransohoff and Tani, 1998)). Chemokines are part of the larger cytokine

family and induce cellular migration in cells expressing the appropriate chemokine receptors.

Four subclasses of chemokines are distinguished based on conserved cysteine residues in their

amino acid sequence. These cysteines are either adjacent (CC), separated by a single amino

acid (CXC), separated by three amino acids (CX3C) or there is only a single cysteine present

(C). The nomenclature for chemokine receptors follows that of the subclass of chemokines

they preferentially bind. CC receptors bind CC chemokines and CXC receptors bind CXC

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chemokines (for review see (Horuk, 2001; Laing and Secombes, 2004; Zlotnik and Yoshie,

2000)). This is a general principle in chemokine biology with only one known exception

which is CCL21 signaling through CXCR3 (Dijkstra et al., 2004; Rappert et al., 2002; Soto et

al., 1998).

The induction of chemokine expression in the brain has been associated with

neurodegenerative events and the infiltration of leukocytes (Fife et al., 2000; Huang et al.,

2001; Izikson et al., 2000; Siebert et al., 2000). However, the expression of chemokines and

their receptors by neurons and glia suggest that they also play a role in local neuro-glial

signaling (Ambrosini and Aloisi, 2004; Biber et al., 2006; Hesselgesser and Horuk, 1999).

Recently we have discovered a possible candidate for neuron-glia signaling, the chemokine

CCL21. Since CCL21 is upregulated in neurons in vivo and in vitro after injury and activates

microglia through the chemokine receptor CXCR3, a role of CCL21-CXCR3 in neuron-

microglia signaling has been proposed (Biber et al., 2001; de Jong et al., 2005; Rappert et al.,

2002). In CXCR3 knockout animals after neuronal injury, induced by entorhinal cortex lesion

(ECL), microglial activation is almost absent, but is unchanged after facial axotomy

indicating an apparent context-specific role for CXCR3 in controlling microglia activity

(Rappert et al., 2004).

CCL21 is not the only neuronal chemokine that binds and activates CXCR3 since CXCL10,

another major CXCR3 ligand is found in neurons as well (Klein et al., 2005; Rappert et al.,

2002; Wang et al., 1998). In order to investigate whether CXCL10 might also serve as a

signal between endangered neurons and microglia we have here examined the expression and

regulation of CXCL10 in cultured neurons in more detail.

It is shown here that CXCL10 is expressed constitutively in cultured neurons with no changes

after several neurotoxic stimuli. Treatment with glutamate, amyloid-beta, chemical hypoxia

and heat shock did not modulate the expression of CXCL10. Furthermore these treatments,

and additional experiments to induce vesicular release (depolarizing the cells with KCl and

calcium influx using ionomycin), did not result in detectable release of CXCL10. These

results indicate that the two CXCR3-activating chemokines CCL21 and CXCL10 are

processed differentially in neurons. Furthermore, these data do not support a role for neuronal

CXCL10 in the signaling between endangered neurons and microglia.

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Materials and Methods

Chemicals

Dulbecco’s Modified Eagle Medium (DMEM), Fetal Calf Serum, MEM Sodium Pyruvaat, L-

glutamin, Penicillin, Streptomycin, Hank’s balanced salt solution (HBSS), Trypsin, PBS,

Neurobasal Medium and B27 supplement from GibcoBRL Life Technologies (Breda, The

Netherlands); Poly-D-Lysin from Sigma-Aldrich Chemie BV (Zwijndrecht, The Netherlands);

DNAse I from Roche (Mannheim, Germany); Recombinant chemokines, CXCL10 antibody

from PeproTech EC Ltd (London, UK); iQ SYBR Green Supermix (Biorad, Hercules CA).

The amyloid-beta peptide 1-42 (Aβ1-42) used in this study was kindly provided by Prof. Dr. B

Penke at the Department of Medical Chemistry, University of Szeged, Szeged, Hungary.

Cell cultures

Cortical Neurons

Cultures of cortical neurons were established as described before (Biber et al., 2001). In brief,

pregnant mice (NMRI) were anesthetized with isoflurane, sacrificed by cervical dislocation

and ED 16 embryos were removed. Cortices were dissected in icecold HBSS supplemented

with 30% glucose. After meninges were removed, cortices were placed in a 0,25% trypsin

solution at 37 degrees Celsius for 20 minutes. Subsequently, tissue was gently dissociated by

trituration and then filtered through a cell strainer (70 µm, Falcon). After one washing step

(100x g for 10 min), cells were seeded out on poly-D-coated glass cover slides and

maintained in complete Neurobasal medium (2% B27-supplement, 0.5 mM glutamine, 1%

penicillin/streptomycin) for at least 7 days in a humidified atmosphere with 5% CO2 at 37

degrees Celsius.

To induce excitotoxicity, cells were treated with glutamate (100 µM/ 30 min) as described

previously (Biber et al., 2001) and mRNA/protein samples were analyzed 0, 2, 4, 6, 12 and

24h after treatment.

To induce cellular hypoxia cells were treated with sodium azide as described elsewhere

(Grammatopoulos et al., 2004). Briefly, after 7 DIV cultured neurons, conditioned medium

was collected and neurons were exposed to 10 mM sodium azide in fresh neurobasal for 5

min. After exposure, cells were rinsed three times with fresh neurobasal to remove sodium

azide residues. After rinsing, original conditioned medium was replaced and the expression of

CXCL10 mRNA and protein was evaluated 24h after treatment.

The solid Aβ1-42 peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; Sigma).

The 1 mM peptide solution was aliquoted and the HFIP was removed by evaporation in a

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SpeedVac (Savant Instruments). The dry peptide films were stored at -20°C until further

processing.

The Aβ1-42 films were dissolved in anhydrous DMSO (Sigma) at 5 mM and subsequently

diluted in phenolred-free neurobasal medium (Gibco) to 100 µM (Stock solution). The stock

solution was incubated at 4°C for 24h to enable Aβ1-42 oligomers forming. Primary cortical

neurons were incubated for 24 h with 50 µM of Aβ1-42 oligomers or phenolred-free neurobasal

media as control. After the incubation with Aβ1-42 oligomers the cell viability was determined

and media samples were collected and snap frozen in liquid nitrogen for further analyses. To

check if appropriate a-beta oligomers had formed, media were checked on western blot using

the the primary anti-Aβ antibody 6E10 (Signet).

Another neurotoxic treatment was the heat shock; putting the cells for 30 min at 42oC and

analyzing after 6h.

To induce release, cells were depolarized with 60 uM KCl for 6h or treated with 1 uM

ionomycin for 6h to raise intracellular calcium concentrations.

Mixed glial cultures

Mixed glial cell cultures were prepared by dissection of mouse cortex from newborn mouse

pups (1-3 days old) on a chilled platform under sterile conditions. Tissue was collected in

medium A (HBSS with 0.6% Glucose, 15 mM HEPES buffer and 1% Pen/Strep). The cortex

was chopped and trypsinized for 20 minutes (0.25% trypsin in medium A and DNase I) at

37̊ C. Trypsin was inhibited with trypsin inhibition medium (medium A with 20% FCS, 1%

DNase I and 1% trypsin inhibitor), and then washed with wash medium (medium A with 10%

FCS and 1% DNase). The cortices were triturated with fire polished glass pipettes. Before

centrifuging, 25 ml of culture medium (DMEM with 4,5 g/l glucose, 2mM Glutamine, 1%

pen/Strep and 1% Sodium Pyruvate) was added. Cells were centrifuged (143 x g for 10

minutes) and resuspended in 1 ml culture medium. Suspended cells of 1,5 mouse brain were

added per culture flask (75 cm2) with 10 ml culture medium. Cultures were maintained at 37

˚C, in a humidified atmosphere (5% CO2). Culture medium was replaced after 2 days and

every 3 days thereafter.

Real-time PCR analysis of CXCL10 mRNA in cultured neurons

Cells were lysed in guanidinium isothyocyanate/mercaptoethanol buffer, total RNA was

extracted and transcribed into cDNA as described previously (Biber et al., 2001). Primers

were designed using Primer Designer version 3.0. CXCL10 primers sequences were: forward

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primer 5’-GCTGCAACTGCATCCATATCG-3’; reverse primer:

5’-CCGGATTCAGACATCTCTGCT-3’. Hydroxymethylbilane synthase (HMBS) primer

sequences were: forward 5’-CCGAGCCAAGGACCAGGATA-3’; reverse primer 5’-

CTCCTTCCAGGTGCCTCAGA-3’. HMBS was used as normalizing gene because its

expression remained stable under various experimental conditions. Experiments were

conducted in 25 ul, using iQ SYBR Green Supermix with primers in a concentration of 250

nmol in 96 wells plates in an i-cycler (Biorad). Reaction conditions were 3 min at 95°C ,

followed by 50 cycles of 10 s at 95°C and 45 s at 58°C, followed by 1 min at 95°C and by 1

min at 55°C. To determine the amount of CXCL10 amplification at various time points after

glutamate stimulation, normalized to the reference gene HMBS and related to a control

sample, the comparative cycle threshold Ct method was used as described previously (Livak

and Schmittgen, 2001).

Western Blotting

Western blot analysis was performed as described previously (Baron et al., 2000). Briefly,

neuronal cultures were harvested in PBS, solubilized in sample buffer and subjected to SDS-

PAGE. Equal amounts of protein were loaded onto 12.5% SDS-polyacrylamide gels and

transferred to Hybond-ECL nitrocellulose membrane (Amersham Biosciences, Arlington

Heights, IL). Membranes were blocked with 5% non fat milk in PBS with 0,1% Tween-20.

After rinsing with PBS with 0,1% Tween-20, membranes were incubated overnight with the

primary antibody (CXCL10 at 1:1000 or beta-Actin at 1:2000) in PBS with 1% nonfat milk,

0,1% Tween-20. After rinsing, membranes were incubated with the appropriate horseradish

peroxidase conjugated antibody (Amersham Biosciences). Bands were visualized by ECL.

Digital images of the Western blots were made and ran through ScionImage, a program which

allows quantification of the intensity of the bands. Data of the time points after glutamate

treatment were normalized against beta-actin data and related to control conditions. As a

positive control for the CXCL10 western blot recombinant CXCL10 was used. Blotting of

recombinant CXCL10 resulted in 2 bands one had a molecular weight of 10kDa and the other

less prominent band was approximately 20kDa, which most likely consist of CXCL10 dimers.

The dimerized form of CXCL10 was detected in neuronal cultures only.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde for 30 min then rinsed twice with PBS. Cells were

pre-incubated using PBS with 0.3% Triton X-100 and 5% normal goat serum for 30 min.

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After rinsing twice, cells were incubated with primary antibody (CXCL10/NeuN) in PBS with

0,3% Triton X-100 overnight. After rinsing three times, appropriate secondary antibody either

labeled with Fluorescein Isothiocyanate (FITC) or CY3 was incubated in PBS for 1 hour.

After rinsing three times with PBS glass coverslips were placed on slides and sealed with nail

polish. Analysis was done on an AOBS confocal image system (Leica) or on a Zeiss

Axioskope 2 with Leica FFC300 FX camera and Leica software. Control experiments for

specificity were done in absence of the primary antibody.

Electron Microscopy

EM immunocytochemistry for the ultrastructural detection of CXCL10 was performed on

cultured cortical neurons using a biotinylated secondary antibody and the Vectastain ABC kit

(Vector laboratories, Burlingame, CA) with DAB as chromogen.

Subsequently, intensification of the DAB reaction product was effectuated using the gold-

silver-substituted peroxidase (GSSP) method (Liem et al., 2001). In brief, after completing the

DAB reaction cells were rinsed twice in 2% sodium acetate solution. After destaining in 10%

thioglycolic acid, the cells were immersed for 8 min in a freshly prepared physical developer

containing 2.5% sodium carbonate, 0,1% ammonium nitrate, 0,1% silver nitrate, 0,5%

phosphosilico-tungstid acid and 0,9% paraformaldehyde. After a brief wash with 1% acetic

acid, the deposited silver particles were replaced by gold by immersing the cells in 0,02%

gold chloride for 8 min at RT. Subsequently, they were fixed for two times at 10 min with 3%

sodium thiosulphate, rinsed briefly in 0,1M sodium acetate cacodylate buffer, pH 7,6 and

were then osmicated in 1% OsO4 (w/v) and 1,5% potassium hexacyanoferrate in the same

buffer dehydrated in a graded series of ethanols and embedded in Epon. Semi-thin sections (1

micron) were cut on a Reichert Ultratome and stained with toluidine blue to check the

presence of relevant areas. Ultrathin sections (60nm were then cut, counterstained with uranyl

acetate and lead citrate, and examined using a Philips (Aachen, Germany) CM100

transmission electron microscope.

Enzyme-linked Immunosorbent Assay (ELISA)

CXCL10 release in the supernatant of the cells was measured with an ELISA (R&D,

Minneapolis; USA). Supernatants were spun down briefly to remove cells and then used in a

mouse-CXCL10 specific ELISA as described in the manufacturers’ protocol. Recombinant

murine CXCL10 served as the standard and the detection limit was 15 picogram/mL.

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Statistical Analysis

To compare between groups in the real time PCR, Western and ELISA data for CXCL10

were statistically analysed with Tukey’s ANOVA test for multiple comparisons. A

significance level of 0,05 was used.

Results

Localisation of CXCL10 in cultured neurons

In order to study the distribution of CXCL10 in the cultured neurons more closely, we

performed fluorescent immunocytochemistry.

Figure 1 Distribution pattern of CXCL10 in neurons. (A) When immunocytochemistry was performed without primary antibody as a negative control, no signal could be detected. (B) After the staining procedure, virtually every cell in culture was CXCL10 positive to some extend. (C) Double-labeling experiment with the neuronal nuclear marker NeuN (in green) showed that

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virtually all cells were positive for CXCL10 (in red). (D) Occasionally astrocytes were detected (arrowhead; GFAP in green, < 5%) which also were positive for CXCL10.

Control experiments without the primary antibody or an isotype control antibody with

irrelevant specificity gave virtually no background staining (figure 1A). Our observations

indicate that CXCL10 immunoreactivity was found abundantly throughout the whole cell as

punctuate signals and that most cells (>95%) were positive for CXCL10 (red signal) (figure

1B). To demonstrate that the expression of CXCL10 is specific for neurons a double staining

was performed using NeuN as a neuronal marker. The majority (>95%) of cells in culture that

were positive for CXCL10 (red signal) were also NeuN positive (figure 1C; green signal).

Few cells did not stain positive for NeuN but were positive for CXCL10. GFAP

immunocytochemistry (green signal) revealed that these cells were CXCL10-positive

astrocytes (figure 1D, arrowhead).

The punctuate CXCL10 immunofluorescence in neurons (see figure 1C), suggests the

presence of CXCL10 in vesicle-like structures, similar to what has been reported for the

chemokine CCL21 (de Jong et al., 2005). In order to further investigate the intracellular

location of CXCL10, electron microscopic studies have been performed. At the ultrastructural

level the presence of CXCL10 in vesicles was confirmed. Moreover, CXCL10 was found in

vesicles with particular localisation at pre-synaptic sites as indicated for axo-somatic contacts

(figure 2A), axo-axonal contacts (figure 2B) and axo-dendritic cell contacts (figure 2C).

Figure 2 Ultrastructural localisation of CXCL10 in vesicles. CXCL10 is localised in vesicles in synaptic areas that make an (A) axo-somatic contact, (B) axo-axonal contact and (C) axo-dendritic contact. Arrowheads indicate CXCL10 immunoreactivity.

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Constitutive CXCL10 expression in cultured neurons is not changed after neurotoxic

stimuli.

Using RT-PCR CXCL10 mRNA was detected in cultured neurons under control conditions.

Quantitative real-time PCR showed no significant differences in CXCL10 mRNA expression

levels after treatment with 100 uM glutamate for 0-24h. Also treating the cells for 24h with 50

uM of the amyloid beta Aβ1-42 oligomers or analyzing 24h after inducing a chemical hypoxia

with 10 mM sodium azide did not result in changes of CXCL10 mRNA expression. An

additional trigger for neuronal death, a heat shock (42 oC for 30 min, analyzed after 6h) also

failed to modulate CXCL10 mRNA (figure 3A). At the protein level, Western blot analysis

confirmed the finding that CXCL10 was already present in unstimulated control neurons.

Furthermore, its expression level was unchanged at different time-points after treatment with

glutamate (figure 3B). The treatments with Aβ1-42, sodium azide and heat shock, respectively,

did not yield any change in CXCL10 protein (data not shown). A quantification of the

Western blot of 3 independent experiments revealed no significant changes in neuronal

CXCL10 protein in response to glutamate treatment (figure 3C).

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Figure 3 Expression of CXCL10 mRNA and protein after various neurotoxic treatments. (A) Real-time PCR showed no significant difference in CXCL10 mRNA levels in the neuronal cultures at any of the observed time-points after 100 uM glutamate (2-24h), sodium azide (Az: 10 mM for 24h), amyloid beta (aβ: 50 uM for 24h) and heat shock (HS: 42 oC for 30 min, analyzed after 6h) when compared to the control condition (C; control). (B) Western blot analysis corroborated this finding and demonstrated that CXCL10 protein levels remain at similar levels at any time point after glutamate stimulation. (C) After quantification of the western blots using ScionImage, no significance difference was shown in CXCL10 protein levels at any time points (0-24h) observed after glutamate treatment. Similar results have been obtained in 3 independent experiments and data are represented as mean +/- SEM (n=3).

Cultured neurons do not release CXCL10 in detectable amounts

The vesicular presence of CXCL10 at pre-synaptic sites prompted us to investigate the release

of CXCL10 from neuronal cultures. Enzyme linked immunosorbent assays (ELISA) were

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performed with supernatants of primary cultured cortical neurons that were treated with

glutamate, sodium azide, Aβ1-42 and heat shock (treatment as mentioned in the previous

section). Additionally, two conditions known to induce vesicular release (60 uM KCl for 6h to

induce depolarization and 1 uM ionomycin to induce calcium influx) were tested. None of

these samples contained CXCL10 at a concentration that was significantly above the detection

limit of our ELISA assay (15 pg/ml). This was in contrast with supernatants obtained from

mixed glial cultures, containing astroyctes and microglia, in which CXCL10 was detected

reliably (332,3 +/-8,1 pg/ml). Particularly after lipopolysaccharide treatment (100 ng/ml for

24 h) the concentration of CXCL10 was very high (71168,0 +/- 1341,8 pg/ml) (Figure 4).

Since all of the attempts trying to detect CXCL10 in neuronal supernatants were not

successful, we tested the fate of exogenously applied recombinant CXCL10 in our system.

Cortical neurons were cultured for 6 h in the presence of 100 pM CXCL10 and subsequently

the levels of CXCL10 in the supernatants were evaluated. The results showed that the culture

conditions led to a decrease up to 60-70% in the concentration of exogenously added

CXCL10 (data not shown).

Figure 4 The release pattern of CXCL10. This graph shows that CXCL10 can not be detected in the supernatants of cultured neurons and neurons after stimulation with 100 uM glutamate (glu:

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24h), sodium azide (Az: 10 mM for 24h), amyloid beta (aβ: 50 uM for 24h), KCl (60 uM for 6h), ionomycin (Io: 1 uM for 6h) and heat shock (HS: 42 oC for 30 min, analyzed after 6h). CXCL10 was readily detected in the supernatants of mixed glial cultures especially after treatment with 100 ng/ml LPS for 24h.

Discussion

Recent studies showed induction and release of the neuronal chemokine CCL21 in response to

neuronal injury (Biber et al., 2001; de Jong et al., 2005). Since microglia respond to CCL21 in

a CXCR3-dependent manner (Dijkstra et al., 2004; Rappert et al., 2002) a role of CCL21-

CXCR3 mediated neuron-microglia signaling under neuropathological conditions was

proposed (de Jong et al., 2005). In order to answer whether other CXCR3 ligands might also

serve as a signal from endangered neurons to microglia, we investigated the neuronal

expression of CXCL10. This study shows that cultured cortical neurons constitutively express

CXCL10 which is distributed throughout the whole cell including synaptic regions.

Furthermore it shows that the levels of CXCL10 mRNA and protein could not be induced by

glutamate, sodium azide, amyloid-beta or heatshock. Exposing the neurons to the

aforementioned conditions, including KCl and ionomycin, also did not result in detectable

CXCL10 release in the culture medium. Therefore the data provided here do not support a

role for neuronal CXCL10 in the attraction of microglia after neuronal injury.

Distribution of CXCL10 in neurons

Immunocytochemistry revealed the presence of CXCL10 in virtually every cell in culture in

punctuate structures that were shown to be vesicles . Double-labeling with the neuronal

marker NeuN and morphological observations indicated that the majority of cells in culture

were neurons. Few NeuN negative cells were also found and double-labeling with GFAP

showed that the few astrocytes in the culture also stained for CXCL10. CXCL10 in neurons

could be detected throughout the whole cell. In general, staining for CXCL10 was slightly

more pronounced in the soma compared to the processes and synaptic regions.

Neuronal CXCL10 expression is constitutive in vitro

In contrast to what was described for CCL21, cultured neurons expressed CXCL10

constitutively with little impact of glutamate treatment as a method to induce neuronal death.

A number of other treatments such as amyloid beta, chemical hypoxia and heatshock also did

not influence the expression level of CXCL10 in neurons.

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Controversial results have been published concerning the expression of CXCL10 in

unchallenged neurons. In agreement with our results a constitutive expression of CXCL10 in

cultured neurons (human fetal brain cells) has been described (Sui et al., 2004) whereas others

did not find CXCL10 expression in untreated neurons (Klein et al., 2005; Patterson et al.,

2003). The reason for this contradictory findings is not clear but might reflect small

differences in the culture conditions. The first report that showed CXCL10 expression in

neurons has been published by Wang and colleagues in 1998. They showed an induction of

CXCL10 in neurons under ischemic conditions, however they furthermore reported a low

level of CXCL10 mRNA expression in sham-operated animals also at the contralateral

cortical side of treated animals, suggesting a basal level of neuronal CXCL10 mRNA

expression.

CXCL10 could not be detected in neuronal supernatants

The presence of CXCL10 in synaptic regions suggests that CXCL10 might be released from

neurons. To see if neurons would be able to secrete CXCL10, ELISA analysis was performed

using supernatants of cultured neurons. Stimulation of these neurons with glutamate, sodium

azide, amyloid beta, KCL, ionomycin and heatshock all did not result in any detectable

CXCL10 release using a commercial ELISA protocol. This was in contrast with supernatants

derived from glial cultures where CXCL10 was readily detected under control conditions and

in response to LPS. There are three recent studies that investigated the release of CXCL10

from virus-infected neurons. Measles infection induced both CXCL10 and CCL5 in primary

hippocampal neurons, but it is interesting to note that only CCL5 was detectably released as

measured by ELISA (Patterson et al., 2003). Klein and colleagues investigated CXCL10

expression in West Nile virus infected cerebellar granule neurons by Western blot analysis.

They provide evidence for high expression of CXCL10 within the neurons and a low amount

of CXCL10 in the supernatant of these cells (Klein et al., 2005). Relatively high levels

(around 1000 pg/ml) of CXCL10 were found in the supernatant of simian human

immunodeficiency virus- or gp120-treated human fetal brain cultures (Sui et al., 2004). It

should be noted, however, that the human fetal brain cultures used in this study contained

approximately 30% astrocytes, whereas all other studies concerning CXCL10 expression in

cultured neurons (including ours) used almost pure neuronal cultures (Klein et al., 2005;

Patterson et al., 2003). It is thus apparent from the literature that CXCL10 in neurons (Klein et

al., 2005; Patterson et al., 2003) can be reliably detected, whereas the presence of CXCL10 in

neuronal supernatants is more difficult to show. Our data show that the concentration of

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exogenously added CXCL10 into the supernatant of cultured neurons decreases suggesting

proteolytic degradation or attachment to surface proteoglycans, molecules known for their

chemokine-binding capabilities. This finding indicates that small amounts of CXCL10

released by neurons would be difficult to detect. Even though we show that CXCL10 is

present in vesicles in synaptic areas suggesting that CXCL10 is destined for release, we are

unable to detect its presence in the culture medium. Based on this it is concluded that cultured

neurons do not massively release CXCL10 (like cultured astrocytes), but we can not exclude

the release of neuronal CXCL10 at low levels.

The function of neuronal CXCL10

Recent publications demonstrated a subtle and neuromodulatory role for CXCL10 in neurons.

CXCL10 stimulation was found to induce intracellular Ca2+ transients, enhanced

spontaneous and evoked electrical activity of hippocampal neurons or inhibited long term

potentiation in hippocampal slices (Vlkolinsky et al., 2004); (Nelson and Gruol, 2004). Thus,

CXCL10 stimulation influenced neuronal excitability and signaling, a principle that has also

been described for other chemokines (Guyon et al., 2006; Liu et al., 2003; van Gassen et al.,

2005). It should be noted that in these experiments CXCL10 was applied exogenously. Our

data do not support a role for neuronal CXCL10 in such a neuromodulatory signaling and

further detailed analysis concerning the release of neuronal CXCL10 would be mandatory to

prove this point.

In summary, it is shown here that CXCL10 is constitutively expressed in cultured cortical

neurons and that its expression is not changed by endangering conditions. Since glutamate-

treated neurons are known to release factors that lead to microglia activation, the data

presented here indicate that CXCL10 is not among these signals. In contrast with what was

previously shown for CCL21, these findings do not support a role for CXCL10 in the

communication between excitotoxic neurons and microglia.

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