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Expression of the chemokine receptors CXCR1 and CXCR2 in rat oligodendroglial cells
Developmental Brain Research 128 (2001) 77–81 www.elsevier.com / locate / bres
Short communication
Expression of the chemokine receptors CXCR1 and CXCR2 in rat oligodendroglial cells Dan Nguyen, Martin Stangel* ¨ ¨ Berlin, Hindenburgdamm 30, 12200 Berlin, Germany Benjamin Franklin, Freie Universitat Department of Neurology, Universitatsklinikum Accepted 30 January 2001
Abstract Chemokines are small proteins that act as chemoattractants and activators in leukocytes during physiological and inflammatory processes. In the CNS chemokine receptors have been shown to be expressed on neurons, astrocytes and microglia but their function in the CNS is poorly understood. CXCR1 and CXCR2 are receptors for ELR-positive CXC chemokines which include growth-regulated oncogene alpha (GRO-alpha) and interleukin-8 (IL-8). GRO-alpha is considered to influence proliferation of cultured oligodendrocyte progenitors (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 receptor for GRO-alpha. This finding demonstrates that the chemokine / chemokine receptor system is probably also involved in the regulation of oligodendroglial cells during developmental processes and may even have implications for inflammatory demyelinating diseases like multiple 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 that function as chemoattractants and activators of immune cells [4,16]. Several chemokines are also expressed in the central nervous system (CNS), and functional chemokine receptors are known to be present on neurons, astrocytes and microglia under physiological conditions [3]. Most receptors recognize more than one chemokine, and in general each chemokine can bind to more than one receptor, indicating that redundancy is characteristic for the chemokine / chemokine receptor system [4]. Chemokine receptors are surface bound transmembrane G proteincoupled receptors (GPCRs) that are classified into subfamilies according to the localization of the cyteine residues of the binding chemokines, namely C, CC, CXC, and CX 3 C. In the case of ELR1 -CXC chemokines (socalled, because of the presence of the N-terminal tripeptide motif glutamate-leucine-arginine (ELR) adjacent to the *Corresponding author. Tel.: 149-30-8445-2276; fax: 149-30-84454264. E-mail address: [email protected] (M. Stangel).
cysteine-X-cysteine motif), interleukin-8 (IL-8) binds to both receptors CXCR1 and CXCR2, while growth regulated oncogene alpha (GRO-alpha) is specifically recognized only by CXCR2 [14]. Although the literature about functional expression of chemokine receptors in leukocytes is quite extensive, their significance in the cells of the CNS is poorly understood. A number of functional roles has been proposed in the CNS including neuronal chemotaxis, modulating synaptic transmission, and the control of cell proliferation and survival [2,3]. CXCR2 has been shown to enhance the survival of hippocampal neurones [1,11], and in more recent reports the GRO chemokine has also been found to exhibit trophic and mitotic effects on oligodendrocyte precursors (OLPs) [18,21]. While the source of GRO seems to be astrocytes [18,21], no persuasive evidence has been provided so far that oligodendroglial cells express CXCR2. We therefore studied the expression of this receptor in immature oligodendrocytes both in CG-4 cells, a rat oligodendrocyte precursor cell line [12], and in primary cultures of OLPs. 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. PII: S0165-3806( 01 )00128-6
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D. Nguyen, M. Stangel / Developmental Brain Research 128 (2001) 77 – 81
co’s modified Eagle’s medium (DMEM; Gibco, Germany) containing 30% medium conditioned by the neuroblastoma cell line B104 (B104-CM), 1% ITS1 (Becton-Dickinson, UK), 2 mM glutamine and antibiotics (Biochrom, Germany) as described previously [20]. Primary cultures of glial cells were prepared from 1-day-old Sprague–Dawley rat cerebra. The brains were freed from meninges and mechanically dissociated using DNase and trypsin (Sigma, Germany). Cells were plated into poly-L-lysine-coated culture flasks (2 brains per 75 cm 2 ) and grown in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS, Biochrom), 2 mM glutamine, 50 U / ml penicillin, and 50 mg / ml streptomycin (Biochrom). After 8 days, loosely attached OLPs were detached by vigorous shaking of the cultures. Contaminating microglial cells were removed by adherence to untreated plastic for 30 min. Cells were replated onto poly-L-lysine coated glass cover slips (20,000 cells per well of a 12-well plate). Poly(A)1mRNA was extracted from CG-4 cells using a modified protocol provided with the Dynabeads mRNA DIRECT kit (Dynal, Germany). In brief, fresh cell pellets (1310 6 cells) were resuspended in lysis buffer containing 100 mM Tris–HCl (pH 8.0), 0.5 M NaCl, 10 mM EDTA (pH 8.0), 1% sodium dodecyl sulfate (SDS), and 5 mM dithiothreitol (DTT). Poly(A)1mRNAs were isolated directly by hybridizing to superparamagnetic, oligo(dT)coated polystyrene beads (Dynal). Following extensive washing, the captured mRNA bound to oligo(dT)25 beads was used immediately to perform solid-phase first strand cDNA synthesis in 20 ml of a solution containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM DTT, 40 U RNaseOUT (Gibco, Germany), and 200 U of Moloney Murine Leukemia Virus reverse transcriptase (SuperScript II, Gibco). Aliquots (1:6) of the first strand cDNA reaction mixture were used to amplify rat CXC receptor sequences in a PCR approach containing the following components: 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 50 mM tetramethylammonium chloride (TMAC; Serva, Germany), 200 mM dNTP, 20 pmol of each primer, and 2.5 U Taq polymerase (Promega, Germany). The PCR conditions were 958C for 1 min followed by 35 reaction cycles of 948C for 15 s, 588C for 30 s, and 728C for 45 s, with a final 10 min extension at 728C. For PCR priming, two primer pairs were designed based on the published rat cDNA sequences [5,9]: forward primers: CXCR1F, 59-CAGGCTTCTCCAGCACACAAG39; CXCR2F, 59-GCAAACCCTTCTACCGTAG-39; reverse primers: CXCR1R, 59-TTGGTCATTGGAACCCTCTTAC-39; CXCR2R, 59-AGAAGTCCATGGCGAAATT39. Ten microliters from each PCR reaction were run on 2% agarose gels and visualized with ethidium bromide. A 100-bp ladder (GenSura, Laboratories Inc.) was used as size standard. Selected PCR products with the expected sizes were isolated and the sequence identity was determined by the dideoxynucleotide chain termination method using the same PCR primers, and an automated
sequencing system (ABI PRISM TM 377 DNA Sequencer, Perkin Elmer). For indirect immunofluorescence staining, OLPs and CG-4 cells were grown on glass cover slips coated with poly-L-lysine for 2 days. To maintain a proliferative state and to prevent further differentiation, both OLPs and CG-4 cells were grown in B104-CM. For surface staining cells were incubated with the A2B5 antibody (1:5 hybridoma supernatant, clone 105, European Collection of Cell Cultures) for 1 h at 378C followed by Cy3-conjugated antimouse IgM (Jackson ImmunoResearch Laboratories, Germany). After washing with PBS, cells were fixed with ice-cold methanol for 3 min. and stained for anti glial fibrillary acidic protein (anti-GFAP, 1:200, Boehringer Mannheim, Germany) or CXCR2 (K-19, 1:50, Santa Cruz Biotechnology Inc., Germany). The immunoreactivity was detected by using Cy2 conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories) or a complex of biotinylated anti-rabbit IgG and dichlorotriazinyl–fluorescein-conjugated streptavidin (Dianova, Germany). Negative controls were performed following the same procedures omitting the primary antibodies. To reveal the specificity for CXCR2, the antibody was neutralized by preincubation with a blocking CXCR2 peptide used as immunogen (Santa Cruz Biotechnology). With reversely-transcribed mRNA isolated from CG-4 cells, the target sequences with the predicted size of 183 and 413 bp were selectively amplified for CXCR1 and CXCR2, respectively (Fig. 1). Identity of the sequences was confirmed by direct sequencing of the PCR products, which revealed 100% homology with both CXCR1 and CXCR2 rat sequences by BLAST searching mode for GeneBank data. To confirm that the detected CXCR2 transcript was translated into protein we investigated the presence of CXCR2 in CG-4 cells by indirect immunofluorescence. As shown in Fig. 2, CXCR2 immunoreactivity and specificity could be revealed in these cells using a specific polyclonal antibody and a blocking peptide.
Fig. 1. Poly A1 RNA isolated from CG-4 cells was reversely transcribed and PCR amplified using CXCR1- (lane 1) and CXCR2-specific primers (lane 2) revealing 183 and 413 bp fragments, respectively. To rule out genomic contamination, untranscribed RNA was used in parallel PCR 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) and CXCR2 (C and G), in (A) and (E), the corresponding phase-contrast images are shown. In parallel experiments, the cultures were labelled with polyclonal antibody to CXCR2 preincubated with control CXCR2 peptide (D and H).
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D. Nguyen, M. Stangel / Developmental Brain Research 128 (2001) 77 – 81
In order to rule out the possibility that the expression of CXCR2 in CG-4 cells is due to an artifact of the cell line, we further investigated the presence of the receptor in primary cultures of oligodendroglial cells by immunofluorescence. Cells were double-labeled with the OLP marker A2B5, and antibodies against CXCR2 or GFAP (for astrocytes). Cells positively stained for A2B5 were all immunoreactive to CXCR2 (Fig. 2), whereas all A2B51 cells were negative for GFAP (not shown). The specificity of the staining was confirmed by labeling the cultures with the antibody to CXCR2 preincubated with the blocking peptide used as immunogen showing a very low level of unspecific fluorescence (Fig. 2). A comparably weak pattern of fluorescence could be observed in negative controls without the CXCR2 antibody (data not shown), further verifying the specificity of the staining. A protocol including permeabilization of the cells was required since the only available antibody against rat CXCR2 recognizes an epitope in the intracellular portion of the receptor. Thus, also intracellular protein was stained. Since the expression level of CXCR1 and CXCR2 is regulated by internalization and recycling [6,10], this explains the staining pattern. Due to the lack of antibodies directed against rat CXCR1 we were not able to demonstrate the expression of CXCR1 protein. Although a number of chemokine receptors has been found to be expressed in the CNS [3], little is known about their functional roles. While the expression of the CXCR2 has been detected in neurons of the brain and spinal cord [11], we demonstrate in the present study that immature oligodendrocytes express genes for both CXCR1 and CXCR2. To the best of our knowledge, this is the first report to identify chemokine receptors on cells of the oligodendroglial lineage. Among various receptors for chemokines in the CNS, the neuronal CXCR2, upon binding to IL-8, is considered to be involved in several physiological functions including modulation of synaptic functions and increase in survival of neurons [1,8,17]. Robinson et al. [18] have demonstrated that the CXCR2 ligand GRO-alpha can promote oligodendrocyte progenitor proliferation in the presence of platelet-derived growth factor (PDGF). Therefore, our finding that genes for both CXCR1 and CXCR2 are expressed in immature oligodendrocytes, raises the possibility that GRO and IL-8 might play an important role in chemokine / chemokine receptor mediated regulation of development of these cells. In the CNS, OLPs arise in specific regions (ventricular zones), then proliferate and migrate through the CNS before differentiating into myelinating mature oligodendrocytes [7,13,19]. Since both GRO-alpha and PDGF are synthesized by astrocytes [15,18], it is possible that these processes of embryonic and early development are at least partly controled by astrocytes in a chemokine / chemokine receptor pathway. Although the oligodendrocyte expansion has been found to correlate developmentally with the levels of GRO-alpha expressed by astrocytes, and GRO-
alpha enhances PDGF-induced proliferation of OPLs in vitro [18,21], its influence on other functions like migration or its effect in vivo are currently not known. Apart from their possible roles in the developing CNS, the studied chemokine receptors in OLPs may have important functions in various inflammatory diseases of the CNS including multiple sclerosis (MS). CXCR1 and CXCR2 on OLPs may mediate cell activation leading to OLP proliferation, migration, differentiation and subsequent regeneration of demyelinated lesions. Knowledge about the precise regulatory mechanism of expression and the functions of these receptors in OLPs would be extremely valuable to understand the role of chemokines and their receptors in development and under pathological conditions like inflammatory demyelinating diseases of the CNS.
Acknowledgements We thank Drs. S. Herrmann and H. Funke-Kaiser for the sequencing of the PCR products, E. Lanka and B. Trampenau for excellent technical assistance, and Prof. Dr. P. Marx for his continuous support. This study was supported ¨ by the Gemeinnutzige Hertiestiftung.
References [1] D.M. Araujo, C.W. Cotman, Trophic effects of interleukin-4, -7 and -8 on hippocampal neuronal cultures: potential involvement of glial-derived factors, Brain Res. 600 (1993) 49–55. [2] V.C. Asensio, I.L. Campbell, Chemokines in the CNS: plurifunctional mediators in diverse states, Trends Neurosci. 22 (1999) 504–512. [3] K.B. Bacon, J.K. Harrison, Chemokines and their receptors in neurobiology: perspectives in physiology and homeostasis, J. Neuroimmunol. 104 (2000) 92–97. [4] M. Baggiolini, Chemokines and leucocyte traffic, Nature 392 (1998) 565–568. [5] C.A.N. Dunstan, M.N. Salafranca, S. Adhikari, Y. Xia, L. Feng, J.K. Harrison, Identification of two rat genes orthologous to the human interleukin-8 receptors, J. Biol. Chem. 271 (1996) 32770–32776. [6] R. Feniger-Barish, M. Ran, A. Zaslaver, A. Ben-Baruch, Differential modes of regulation of CXC chemokine-induced internalization and recycling of human CXCR1 and CXCR2, Cytokine 11 (1999) 996–1009. [7] S.A. Gilmore, Neuroglial population in the spinal white matter of neonatal and early postnatal rats: an autoradiographic study of numbers of neuroglia and changes in their proliferative activity, Anat Rec. 171 (1971) 283–291. [8] A. Giovannelli, C. Limatola, D. Ragozzino, A.M. Mileo, A. Ruggieri, M.T. Ciotti, D. Mercanti, A. Santoni, F. Eusebi, CXC chemokines interleukin-8 (IL-8) and growth-related gene product alpha (GROalpha) modulate Purkinje neuron activity in mouse cerebellum, J. Neuroimmunol. 92 (1998) 122–132. [9] A.E. Gobl, M.R. Huang, S. Wang, Y. Zhou, K. Oberg, Molecular cloning and characterization of a cDNA encoding the rat interleukin8 receptor, Biochim. Biophys. Acta 1326 (1997) 171–177. [10] C.J. Hauser, Z. Fekete, E.R. Goodman, E. Kleinstein, D.H. Livingston, E.A. Deitch, CXCR2 stimulation primes CXCR1 [Ca 21 ]i responses to IL-8 in human neutrophils, Shock 12 (1999) 428–437. [11] R. Horuk, A.W. Martin, Z. Wang, L. Schweitzer, A. Gerassimides,
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H. Guo, Z. Lu, J. Hesselgesser, H.D. Perez, J. Kim, J. Parker, T.J. Hadley, S.C. Peiper, Expression of chemokine receptors by subsets of neurons in the central nervous system, J. Immunol. 158 (1997) 2882–2890. J.C. Louis, E. Magal, D. Muir, M. Manthorpe, S. Varon, CG-4, a new bipotential glial cell line from rat brain, is capable of differentiating in vitro into either mature oligodendrocytes or type-2 astrocytes, J. Neurosci. Res. 31 (1992) 193–204. R.H. Miller, J. Payne, L. Milner, H. Zhang, D.M. Orentas, Spinal cord oligodendrocytes develop from a limited number of migratory highly proliferative precursors, J. Neurosci. Res. 50 (1997) 157– 168. P.M. Murphy, M. Baggiolini, I.F. Charo, C.A. Hebert, R. Horuk, K. Matsushima, L.H. Miller, J.J. Oppenheim, C.A. Power, International union of pharmacology. XXII. Nomenclature for chemokine receptors, Pharmacol. Rev. 52 (2000) 145–176. N. Pringle, E.J. Collarini, M.J. Mosley, C.H. Heldin, B. Westermark, W.D. Richardson, PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve, EMBO J. 8 (1989) 1049–1056.
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[16] A. Proost, A. Wuyts, J.V. Damme, The role of chemokines in inflammation, Int. J. Clin. Lab. Res. 26 (1996) 211–223. [17] D. Ragozzino, A. Giovannelli, A.M. Mileo, C. Limatola, A. Santoni, F. Eusebi, Modulation of the neurotransmitter release in rat cerebellar neurons by GRO beta, Neuroreport 9 (1998) 3601–3606. [18] S. Robinson, M. Tani, R.M. Strieter, R.M. Ransohoff, R.H. Miller, The chemokine growth-regulated oncogene-alpha promotes spinal cord oligodendrocyte precursor proliferation, J. Neurosci. 18 (1998) 10457–10463. [19] M.E. Schwab, L. Schnell, Region-specific appearance of myelin constituents in the developing rat spinal cord, J. Neurocytol. 18 (1989) 161–169. [20] M. Stangel, A. Compston, N.J. Scolding, Oligodendroglia are protected from antibody-mediated complement injury by normal immunoglobulins (‘IVIg’), J. Neuroimmunol. 103 (2000) 195–201. [21] Q. Wu, R.H. Miller, R.M. Ransohoff, S. Robinson, J. Bu, A. Nishiyama, Elevated levels of the chemokine GRO-1 correlate with elevated oligodendrocyte progenitor proliferation in the jimpy mutant, J. Neurosci. 20 (2000) 2609–2617.
Short communication
Expression of the chemokine receptors CXCR1 and CXCR2 in rat oligodendroglial cells Dan Nguyen, Martin Stangel* ¨ ¨ Berlin, Hindenburgdamm 30, 12200 Berlin, Germany Benjamin Franklin, Freie Universitat Department of Neurology, Universitatsklinikum Accepted 30 January 2001
Abstract Chemokines are small proteins that act as chemoattractants and activators in leukocytes during physiological and inflammatory processes. In the CNS chemokine receptors have been shown to be expressed on neurons, astrocytes and microglia but their function in the CNS is poorly understood. CXCR1 and CXCR2 are receptors for ELR-positive CXC chemokines which include growth-regulated oncogene alpha (GRO-alpha) and interleukin-8 (IL-8). GRO-alpha is considered to influence proliferation of cultured oligodendrocyte progenitors (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 receptor for GRO-alpha. This finding demonstrates that the chemokine / chemokine receptor system is probably also involved in the regulation of oligodendroglial cells during developmental processes and may even have implications for inflammatory demyelinating diseases like multiple 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 that function as chemoattractants and activators of immune cells [4,16]. Several chemokines are also expressed in the central nervous system (CNS), and functional chemokine receptors are known to be present on neurons, astrocytes and microglia under physiological conditions [3]. Most receptors recognize more than one chemokine, and in general each chemokine can bind to more than one receptor, indicating that redundancy is characteristic for the chemokine / chemokine receptor system [4]. Chemokine receptors are surface bound transmembrane G proteincoupled receptors (GPCRs) that are classified into subfamilies according to the localization of the cyteine residues of the binding chemokines, namely C, CC, CXC, and CX 3 C. In the case of ELR1 -CXC chemokines (socalled, because of the presence of the N-terminal tripeptide motif glutamate-leucine-arginine (ELR) adjacent to the *Corresponding author. Tel.: 149-30-8445-2276; fax: 149-30-84454264. E-mail address: [email protected] (M. Stangel).
cysteine-X-cysteine motif), interleukin-8 (IL-8) binds to both receptors CXCR1 and CXCR2, while growth regulated oncogene alpha (GRO-alpha) is specifically recognized only by CXCR2 [14]. Although the literature about functional expression of chemokine receptors in leukocytes is quite extensive, their significance in the cells of the CNS is poorly understood. A number of functional roles has been proposed in the CNS including neuronal chemotaxis, modulating synaptic transmission, and the control of cell proliferation and survival [2,3]. CXCR2 has been shown to enhance the survival of hippocampal neurones [1,11], and in more recent reports the GRO chemokine has also been found to exhibit trophic and mitotic effects on oligodendrocyte precursors (OLPs) [18,21]. While the source of GRO seems to be astrocytes [18,21], no persuasive evidence has been provided so far that oligodendroglial cells express CXCR2. We therefore studied the expression of this receptor in immature oligodendrocytes both in CG-4 cells, a rat oligodendrocyte precursor cell line [12], and in primary cultures of OLPs. 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. PII: 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) containing 30% medium conditioned by the neuroblastoma cell line B104 (B104-CM), 1% ITS1 (Becton-Dickinson, UK), 2 mM glutamine and antibiotics (Biochrom, Germany) as described previously [20]. Primary cultures of glial cells were prepared from 1-day-old Sprague–Dawley rat cerebra. The brains were freed from meninges and mechanically dissociated using DNase and trypsin (Sigma, Germany). Cells were plated into poly-L-lysine-coated culture flasks (2 brains per 75 cm 2 ) and grown in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS, Biochrom), 2 mM glutamine, 50 U / ml penicillin, and 50 mg / ml streptomycin (Biochrom). After 8 days, loosely attached OLPs were detached by vigorous shaking of the cultures. Contaminating microglial cells were removed by adherence to untreated plastic for 30 min. Cells were replated onto poly-L-lysine coated glass cover slips (20,000 cells per well of a 12-well plate). Poly(A)1mRNA was extracted from CG-4 cells using a modified protocol provided with the Dynabeads mRNA DIRECT kit (Dynal, Germany). In brief, fresh cell pellets (1310 6 cells) were resuspended in lysis buffer containing 100 mM Tris–HCl (pH 8.0), 0.5 M NaCl, 10 mM EDTA (pH 8.0), 1% sodium dodecyl sulfate (SDS), and 5 mM dithiothreitol (DTT). Poly(A)1mRNAs were isolated directly by hybridizing to superparamagnetic, oligo(dT)coated polystyrene beads (Dynal). Following extensive washing, the captured mRNA bound to oligo(dT)25 beads was used immediately to perform solid-phase first strand cDNA synthesis in 20 ml of a solution containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM DTT, 40 U RNaseOUT (Gibco, Germany), and 200 U of Moloney Murine Leukemia Virus reverse transcriptase (SuperScript II, Gibco). Aliquots (1:6) of the first strand cDNA reaction mixture were used to amplify rat CXC receptor sequences in a PCR approach containing the following components: 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 50 mM tetramethylammonium chloride (TMAC; Serva, Germany), 200 mM dNTP, 20 pmol of each primer, and 2.5 U Taq polymerase (Promega, Germany). The PCR conditions were 958C for 1 min followed by 35 reaction cycles of 948C for 15 s, 588C for 30 s, and 728C for 45 s, with a final 10 min extension at 728C. For PCR priming, two primer pairs were designed based on the published rat cDNA sequences [5,9]: forward primers: CXCR1F, 59-CAGGCTTCTCCAGCACACAAG39; CXCR2F, 59-GCAAACCCTTCTACCGTAG-39; reverse primers: CXCR1R, 59-TTGGTCATTGGAACCCTCTTAC-39; CXCR2R, 59-AGAAGTCCATGGCGAAATT39. Ten microliters from each PCR reaction were run on 2% agarose gels and visualized with ethidium bromide. A 100-bp ladder (GenSura, Laboratories Inc.) was used as size standard. Selected PCR products with the expected sizes were isolated and the sequence identity was determined by the dideoxynucleotide chain termination method using the same PCR primers, and an automated
sequencing system (ABI PRISM TM 377 DNA Sequencer, Perkin Elmer). For indirect immunofluorescence staining, OLPs and CG-4 cells were grown on glass cover slips coated with poly-L-lysine for 2 days. To maintain a proliferative state and to prevent further differentiation, both OLPs and CG-4 cells were grown in B104-CM. For surface staining cells were incubated with the A2B5 antibody (1:5 hybridoma supernatant, clone 105, European Collection of Cell Cultures) for 1 h at 378C followed by Cy3-conjugated antimouse IgM (Jackson ImmunoResearch Laboratories, Germany). After washing with PBS, cells were fixed with ice-cold methanol for 3 min. and stained for anti glial fibrillary acidic protein (anti-GFAP, 1:200, Boehringer Mannheim, Germany) or CXCR2 (K-19, 1:50, Santa Cruz Biotechnology Inc., Germany). The immunoreactivity was detected by using Cy2 conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories) or a complex of biotinylated anti-rabbit IgG and dichlorotriazinyl–fluorescein-conjugated streptavidin (Dianova, Germany). Negative controls were performed following the same procedures omitting the primary antibodies. To reveal the specificity for CXCR2, the antibody was neutralized by preincubation with a blocking CXCR2 peptide used as immunogen (Santa Cruz Biotechnology). With reversely-transcribed mRNA isolated from CG-4 cells, the target sequences with the predicted size of 183 and 413 bp were selectively amplified for CXCR1 and CXCR2, respectively (Fig. 1). Identity of the sequences was confirmed by direct sequencing of the PCR products, which revealed 100% homology with both CXCR1 and CXCR2 rat sequences by BLAST searching mode for GeneBank data. To confirm that the detected CXCR2 transcript was translated into protein we investigated the presence of CXCR2 in CG-4 cells by indirect immunofluorescence. As shown in Fig. 2, CXCR2 immunoreactivity and specificity could be revealed in these cells using a specific polyclonal antibody and a blocking peptide.
Fig. 1. Poly A1 RNA isolated from CG-4 cells was reversely transcribed and PCR amplified using CXCR1- (lane 1) and CXCR2-specific primers (lane 2) revealing 183 and 413 bp fragments, respectively. To rule out genomic contamination, untranscribed RNA was used in parallel PCR experiment (lane 3). St: 100 bp ladder.
D. Nguyen, M. Stangel / Developmental Brain Research 128 (2001) 77 – 81
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Fig. 2. Indirect immunofluorescence staining of CG-4 cells and oligodendrocyte progenitors (OLPs). Cells were double-labelled for A2B5 (B and F) and CXCR2 (C and G), in (A) and (E), the corresponding phase-contrast images are shown. In parallel experiments, the cultures were labelled with polyclonal antibody to CXCR2 preincubated with control CXCR2 peptide (D and H).
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D. Nguyen, M. Stangel / Developmental Brain Research 128 (2001) 77 – 81
In order to rule out the possibility that the expression of CXCR2 in CG-4 cells is due to an artifact of the cell line, we further investigated the presence of the receptor in primary cultures of oligodendroglial cells by immunofluorescence. Cells were double-labeled with the OLP marker A2B5, and antibodies against CXCR2 or GFAP (for astrocytes). Cells positively stained for A2B5 were all immunoreactive to CXCR2 (Fig. 2), whereas all A2B51 cells were negative for GFAP (not shown). The specificity of the staining was confirmed by labeling the cultures with the antibody to CXCR2 preincubated with the blocking peptide used as immunogen showing a very low level of unspecific fluorescence (Fig. 2). A comparably weak pattern of fluorescence could be observed in negative controls without the CXCR2 antibody (data not shown), further verifying the specificity of the staining. A protocol including permeabilization of the cells was required since the only available antibody against rat CXCR2 recognizes an epitope in the intracellular portion of the receptor. Thus, also intracellular protein was stained. Since the expression level of CXCR1 and CXCR2 is regulated by internalization and recycling [6,10], this explains the staining pattern. Due to the lack of antibodies directed against rat CXCR1 we were not able to demonstrate the expression of CXCR1 protein. Although a number of chemokine receptors has been found to be expressed in the CNS [3], little is known about their functional roles. While the expression of the CXCR2 has been detected in neurons of the brain and spinal cord [11], we demonstrate in the present study that immature oligodendrocytes express genes for both CXCR1 and CXCR2. To the best of our knowledge, this is the first report to identify chemokine receptors on cells of the oligodendroglial lineage. Among various receptors for chemokines in the CNS, the neuronal CXCR2, upon binding to IL-8, is considered to be involved in several physiological functions including modulation of synaptic functions and increase in survival of neurons [1,8,17]. Robinson et al. [18] have demonstrated that the CXCR2 ligand GRO-alpha can promote oligodendrocyte progenitor proliferation in the presence of platelet-derived growth factor (PDGF). Therefore, our finding that genes for both CXCR1 and CXCR2 are expressed in immature oligodendrocytes, raises the possibility that GRO and IL-8 might play an important role in chemokine / chemokine receptor mediated regulation of development of these cells. In the CNS, OLPs arise in specific regions (ventricular zones), then proliferate and migrate through the CNS before differentiating into myelinating mature oligodendrocytes [7,13,19]. Since both GRO-alpha and PDGF are synthesized by astrocytes [15,18], it is possible that these processes of embryonic and early development are at least partly controled by astrocytes in a chemokine / chemokine receptor pathway. Although the oligodendrocyte expansion has been found to correlate developmentally with the levels of GRO-alpha expressed by astrocytes, and GRO-
alpha enhances PDGF-induced proliferation of OPLs in vitro [18,21], its influence on other functions like migration or its effect in vivo are currently not known. Apart from their possible roles in the developing CNS, the studied chemokine receptors in OLPs may have important functions in various inflammatory diseases of the CNS including multiple sclerosis (MS). CXCR1 and CXCR2 on OLPs may mediate cell activation leading to OLP proliferation, migration, differentiation and subsequent regeneration of demyelinated lesions. Knowledge about the precise regulatory mechanism of expression and the functions of these receptors in OLPs would be extremely valuable to understand the role of chemokines and their receptors in development and under pathological conditions like inflammatory demyelinating diseases of the CNS.
Acknowledgements We thank Drs. S. Herrmann and H. Funke-Kaiser for the sequencing of the PCR products, E. Lanka and B. Trampenau for excellent technical assistance, and Prof. Dr. P. Marx for his continuous support. This study was supported ¨ by the Gemeinnutzige Hertiestiftung.
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