Developmental Brain Research 128 (2001) 77–81www.elsevier.com/ locate /bres

Short communication

Expression of the chemokine receptors CXCR1 and CXCR2 in ratoligodendroglial cells

*Dan Nguyen, Martin Stangel¨ ¨Department of Neurology, Universitatsklinikum Benjamin Franklin, Freie Universitat Berlin, Hindenburgdamm 30, 12200 Berlin, Germany

Accepted 30 January 2001

Abstract

Chemokines are small proteins that act as chemoattractants and activators in leukocytes during physiological and inflammatoryprocesses. In the CNS chemokine receptors have been shown to be expressed on neurons, astrocytes and microglia but their function inthe CNS is poorly understood. CXCR1 and CXCR2 are receptors for ELR-positive CXC chemokines which include growth-regulatedoncogene alpha (GRO-alpha) and interleukin-8 (IL-8). GRO-alpha is considered to influence proliferation of cultured oligodendrocyteprogenitors (OLPs). Using RT-PCR we show here that the oligodendrocyte precursor cell line CG-4 expresses both CXCR1 and CXCR2.Furthermore we demonstrate that both CG-4 cells and primary cultures of rat OLPs are immunoreactive for CXCR2, the potential receptorfor GRO-alpha. This finding demonstrates that the chemokine /chemokine receptor system is probably also involved in the regulation ofoligodendroglial cells during developmental processes and may even have implications for inflammatory demyelinating diseases likemultiple sclerosis. 2001 Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Glia and other non-neuronal cells

Keywords: Oligodendrocyte; Oligodendrocyte precursor; Chemokines; Chemokine receptor

Chemokines are a large family of small secreted proteins cysteine-X-cysteine motif), interleukin-8 (IL-8) binds tothat function as chemoattractants and activators of immune both receptors CXCR1 and CXCR2, while growth reg-cells [4,16]. Several chemokines are also expressed in the ulated oncogene alpha (GRO-alpha) is specifically recog-central nervous system (CNS), and functional chemokine nized only by CXCR2 [14]. Although the literature aboutreceptors are known to be present on neurons, astrocytes functional expression of chemokine receptors in leukocytesand microglia under physiological conditions [3]. Most is quite extensive, their significance in the cells of the CNSreceptors recognize more than one chemokine, and in is poorly understood. A number of functional roles hasgeneral each chemokine can bind to more than one been proposed in the CNS including neuronal chemotaxis,receptor, indicating that redundancy is characteristic for modulating synaptic transmission, and the control of cellthe chemokine /chemokine receptor system [4]. Chemokine proliferation and survival [2,3]. CXCR2 has been shown toreceptors are surface bound transmembrane G protein- enhance the survival of hippocampal neurones [1,11], andcoupled receptors (GPCRs) that are classified into sub- in more recent reports the GRO chemokine has also beenfamilies according to the localization of the cyteine found to exhibit trophic and mitotic effects on oligoden-residues of the binding chemokines, namely C, CC, CXC, drocyte precursors (OLPs) [18,21]. While the source of

1and CX C. In the case of ELR -CXC chemokines (so- GRO seems to be astrocytes [18,21], no persuasive evi-3

called, because of the presence of the N-terminal tripeptide dence has been provided so far that oligodendroglial cellsmotif glutamate-leucine-arginine (ELR) adjacent to the express CXCR2. We therefore studied the expression of

this receptor in immature oligodendrocytes both in CG-4cells, a rat oligodendrocyte precursor cell line [12], and in*Corresponding author. Tel.: 149-30-8445-2276; fax: 149-30-8445-primary cultures of OLPs.4264.

E-mail address: mstangel@zedat.fu-berlin.de (M. Stangel). The rat OLP cell line CG-4 [12] was grown in Dulbec-

0165-3806/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0165-3806( 01 )00128-6

78 D. Nguyen, M. Stangel / Developmental Brain Research 128 (2001) 77 –81

co’s modified Eagle’s medium (DMEM; Gibco, Germany) sequencing system (ABI PRISM TM 377 DNA Sequencer,containing 30% medium conditioned by the neuroblastoma Perkin Elmer).cell line B104 (B104-CM), 1% ITS1 (Becton-Dickinson, For indirect immunofluorescence staining, OLPs andUK), 2 mM glutamine and antibiotics (Biochrom, Ger- CG-4 cells were grown on glass cover slips coated withmany) as described previously [20]. Primary cultures of poly-L-lysine for 2 days. To maintain a proliferative stateglial cells were prepared from 1-day-old Sprague–Dawley and to prevent further differentiation, both OLPs and CG-4rat cerebra. The brains were freed from meninges and cells were grown in B104-CM. For surface staining cellsmechanically dissociated using DNase and trypsin (Sigma, were incubated with the A2B5 antibody (1:5 hybridomaGermany). Cells were plated into poly-L-lysine-coated supernatant, clone 105, European Collection of Cell Cul-

2culture flasks (2 brains per 75 cm ) and grown in DMEM tures) for 1 h at 378C followed by Cy3-conjugated anti-supplemented with 10% heat-inactivated fetal calf serum mouse IgM (Jackson ImmunoResearch Laboratories, Ger-(FCS, Biochrom), 2 mM glutamine, 50 U/ml penicillin, many). After washing with PBS, cells were fixed withand 50 mg/ml streptomycin (Biochrom). After 8 days, ice-cold methanol for 3 min. and stained for anti glialloosely attached OLPs were detached by vigorous shaking fibrillary acidic protein (anti-GFAP, 1:200, Boehringerof the cultures. Contaminating microglial cells were re- Mannheim, Germany) or CXCR2 (K-19, 1:50, Santa Cruzmoved by adherence to untreated plastic for 30 min. Cells Biotechnology Inc., Germany). The immunoreactivity waswere replated onto poly-L-lysine coated glass cover slips detected by using Cy2 conjugated anti-rabbit IgG (Jackson(20,000 cells per well of a 12-well plate). ImmunoResearch Laboratories) or a complex of

Poly(A)1mRNA was extracted from CG-4 cells using a biotinylated anti-rabbit IgG and dichlorotriazinyl–fluores-modified protocol provided with the Dynabeads mRNA cein-conjugated streptavidin (Dianova, Germany). Nega-DIRECT kit (Dynal, Germany). In brief, fresh cell pellets tive controls were performed following the same pro-

6(1310 cells) were resuspended in lysis buffer containing cedures omitting the primary antibodies. To reveal the100 mM Tris–HCl (pH 8.0), 0.5 M NaCl, 10 mM EDTA specificity for CXCR2, the antibody was neutralized by(pH 8.0), 1% sodium dodecyl sulfate (SDS), and 5 mM preincubation with a blocking CXCR2 peptide used asdithiothreitol (DTT). Poly(A)1mRNAs were isolated di- immunogen (Santa Cruz Biotechnology).rectly by hybridizing to superparamagnetic, oligo(dT)- With reversely-transcribed mRNA isolated from CG-4coated polystyrene beads (Dynal). Following extensive cells, the target sequences with the predicted size of 183washing, the captured mRNA bound to oligo(dT)25 beads and 413 bp were selectively amplified for CXCR1 andwas used immediately to perform solid-phase first strand CXCR2, respectively (Fig. 1). Identity of the sequencescDNA synthesis in 20 ml of a solution containing 50 mM was confirmed by direct sequencing of the PCR products,Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl , 10 mM which revealed 100% homology with both CXCR1 and2

DTT, 40 U RNaseOUT (Gibco, Germany), and 200 U of CXCR2 rat sequences by BLAST searching mode forMoloney Murine Leukemia Virus reverse transcriptase GeneBank data. To confirm that the detected CXCR2(SuperScript II, Gibco). Aliquots (1:6) of the first strand transcript was translated into protein we investigated thecDNA reaction mixture were used to amplify rat CXC presence of CXCR2 in CG-4 cells by indirect immuno-receptor sequences in a PCR approach containing the fluorescence. As shown in Fig. 2, CXCR2 immunoreactivi-following components: 20 mM Tris–HCl (pH 8.4), 50 mM ty and specificity could be revealed in these cells using aKCl, 1.5 mM MgCl , 50 mM tetramethylammonium specific polyclonal antibody and a blocking peptide.2

chloride (TMAC; Serva, Germany), 200 mM dNTP, 20pmol of each primer, and 2.5 U Taq polymerase (Promega,Germany). The PCR conditions were 958C for 1 minfollowed by 35 reaction cycles of 948C for 15 s, 588C for30 s, and 728C for 45 s, with a final 10 min extension at728C. For PCR priming, two primer pairs were designedbased on the published rat cDNA sequences [5,9]: forwardprimers: CXCR1F, 59-CAGGCTTCTCCAGCACACAAG-39; CXCR2F, 59-GCAAACCCTTCTACCGTAG-39; re-verse primers: CXCR1R, 59-TTGGTCATTGGAACCCTC-TTAC-39; CXCR2R, 59-AGAAGTCCATGGCGAAATT-39. Ten microliters from each PCR reaction were run on2% agarose gels and visualized with ethidium bromide. A100-bp ladder (GenSura, Laboratories Inc.) was used as

Fig. 1. Poly A1 RNA isolated from CG-4 cells was reversely transcribedsize standard. Selected PCR products with the expectedand PCR amplified using CXCR1- (lane 1) and CXCR2-specific primers

sizes were isolated and the sequence identity was de- (lane 2) revealing 183 and 413 bp fragments, respectively. To rule outtermined by the dideoxynucleotide chain termination meth- genomic contamination, untranscribed RNA was used in parallel PCRod using the same PCR primers, and an automated experiment (lane 3). St: 100 bp ladder.

D. Nguyen, M. Stangel / Developmental Brain Research 128 (2001) 77 –81 79

Fig. 2. Indirect immunofluorescence staining of CG-4 cells and oligodendrocyte progenitors (OLPs). Cells were double-labelled for A2B5 (B and F) andCXCR2 (C and G), in (A) and (E), the corresponding phase-contrast images are shown. In parallel experiments, the cultures were labelled with polyclonalantibody to CXCR2 preincubated with control CXCR2 peptide (D and H).

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In order to rule out the possibility that the expression of alpha enhances PDGF-induced proliferation of OPLs inCXCR2 in CG-4 cells is due to an artifact of the cell line, vitro [18,21], its influence on other functions like migra-we further investigated the presence of the receptor in tion or its effect in vivo are currently not known. Apartprimary cultures of oligodendroglial cells by immuno- from their possible roles in the developing CNS, thefluorescence. Cells were double-labeled with the OLP studied chemokine receptors in OLPs may have importantmarker A2B5, and antibodies against CXCR2 or GFAP functions in various inflammatory diseases of the CNS(for astrocytes). Cells positively stained for A2B5 were all including multiple sclerosis (MS). CXCR1 and CXCR2 onimmunoreactive to CXCR2 (Fig. 2), whereas all A2B51 OLPs may mediate cell activation leading to OLP prolifer-cells were negative for GFAP (not shown). The specificity ation, migration, differentiation and subsequent regenera-of the staining was confirmed by labeling the cultures with tion of demyelinated lesions. Knowledge about the precisethe antibody to CXCR2 preincubated with the blocking regulatory mechanism of expression and the functions ofpeptide used as immunogen showing a very low level of these receptors in OLPs would be extremely valuable tounspecific fluorescence (Fig. 2). A comparably weak understand the role of chemokines and their receptors inpattern of fluorescence could be observed in negative development and under pathological conditions like in-controls without the CXCR2 antibody (data not shown), flammatory demyelinating diseases of the CNS.further verifying the specificity of the staining. A protocolincluding permeabilization of the cells was required sincethe only available antibody against rat CXCR2 recognizes Acknowledgementsan epitope in the intracellular portion of the receptor. Thus,also intracellular protein was stained. Since the expression We thank Drs. S. Herrmann and H. Funke-Kaiser for thelevel of CXCR1 and CXCR2 is regulated by internaliza- sequencing of the PCR products, E. Lanka and B. Tram-tion and recycling [6,10], this explains the staining pattern. penau for excellent technical assistance, and Prof. Dr. P.Due to the lack of antibodies directed against rat CXCR1 Marx for his continuous support. This study was supportedwe were not able to demonstrate the expression of CXCR1 ¨by the Gemeinnutzige Hertiestiftung.protein.

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