Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis
Introduction
Chemoattractant cytokines (chemokines) are structurally related pro-inflammatory peptides (∼70–90 amino acids in length) that elicit leukocyte migration and activation (Baggiolini et al., 1994, Baggiolini et al., 1997). The number of chemokine genes identified continues to expand, and it is now apparent that at least four subfamilies of chemokine peptides exist, a designation which is based principally upon the position of highly conserved cysteine residues. By far, the largest subfamilies of chemokines are the C–X–C and C–C chemokines. In the C–X–C chemokine subfamily, the first two cysteines are separated by an amino acid, while C–C chemokines have these first two cysteines adjacent to one another. Most C–X–C chemokines [e.g., interleukin-8 (IL-8), neutrophil activating peptide-2 (NAP-2) and the GRO peptides] attract and activate neutrophils, while C–C chemokines [e.g., monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory peptide-1α and -1β, and RANTES] target monocytes/macrophages, eosinophils, basophils and T-lymphocytes. Additional chemokines, whose structures are distinguished from members of the other two main classes, have been described recently. Lymphotactin is a lymphocyte chemotactic peptide that possesses only two conserved cysteine residues, corresponding to the cysteine residues in positions 2 and 4 in the C–X–C and C–C chemokine families (Kelner et al., 1994). A human chemokine having a C–X3–C structural motif has also been identified recently (Bazan et al., 1997, Pan et al., 1997). This peptide, termed fractalkine and neurotactin, is expressed by a variety of non-hematopoietic cells and has the functional property of stimulating leukocyte chemotaxis and adhesion.
Receptors for chemokine peptides have been identified by molecular cloning studies, and they are members of the rhodopsin superfamily of G-protein coupled receptors (GCRs) (Baggiolini et al., 1997). Structurally, these receptors are characterized as having seven transmembrane (TM) spanning domains. To date, the primary structures of 13 functional human chemokine receptors have been determined. Four of these receptors bind, and are activated by, C–X–C chemokines (Holmes et al., 1991; Murphy and Tiffany, 1991; Bleul et al., 1996; Loetscher et al., 1996; Oberlin et al., 1996), while the remaining nine receptors are activated by C–C chemokine peptides (Gao et al., 1993; Neote et al., 1993; Charo et al., 1994; Combadiere et al., 1995a, Combadiere et al., 1996; Power et al., 1995; Daugherty et al., 1996; Ponath et al., 1996; Raport et al., 1996; Samson et al., 1996; Baba et al., 1997; Roos et al., 1997; Yoshida et al., 1997; Tiffany et al., 1997); two of the C–C chemokine receptors arise from alternative splicing of the gene encoding CCR2 (Charo et al., 1994). C–C chemokine receptors are approximately 45–60% identical to each other, while the four functional C–X–C receptors share 34–78% amino acid identity. Genes encoding chemokine receptors have also been characterized in rodents (Cerretti et al., 1993; Bozic et al., 1994; Harada et al., 1994; Gao and Murphy, 1995; Post et al., 1995; Boring et al., 1996; Dunstan et al., 1996; Gao et al., 1996; Heesen et al., 1996; Hoogeworf et al., 1996; Kurihara and Bravo, 1996; Meyer et al., 1996; Nagasawa et al., 1996; Wong et al., 1996). Chemokine receptors have also been identified as coreceptors for cellular entry of human immunodeficiency virus (HIV). Human CCR5 and, in some instances both CCR2b and CCR3, serves as the cofactor(s) for entry into cells of primary, macrophage tropic strains of HIV-1 (Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996; Doranz et al., 1996; Dragic et al., 1996), while CXCR4 mediates entry of T-cell and T-cell line adapted strains of HIV-1 (Feng et al., 1996).
Investigations derived from both in vitro and in vivo studies demonstrate that cells of the central nervous system have the capacity to express chemokine peptide genes. In vitro, cultured astrocytes and microglia have been shown to synthesize chemokines in response to application of other cytokines, or in the presence of reactive T-cells (Vanguri and Farber, 1994; Barna et al., 1994; Vanguri, 1995; Hurwitz et al., 1995; Hayashi et al., 1995; Sun et al., 1997). Elevated levels of chemokines can also be detected in the CNS in vivo under certain neurological challenges including EAE/MS (Hulkower et al., 1993; Ransohoff et al., 1993; Godiska et al., 1995; Miyagashi et al., 1995; Berman et al., 1996; Glabinski et al., 1997), ischemia (Liu et al., 1993; Kim et al., 1995; Gourmala et al., 1997), traumatic brain injury (Berman et al., 1996; Ghirnikar et al., 1996; Glabinski et al., 1996), and bacterial infection (Spanaus et al., 1997). The function(s) of chemokines in the CNS is(are) currently not known, although under these neuropathological situations, chemokines likely play the role as principal leukocyte chemotactic factors. Much less is known regarding the expression of chemokine receptors in the CNS; two recent reports indicate that the brain, and cells derived from brain, express chemokine receptors (He et al., 1997; Horuk et al., 1997). Towards our goal of defining chemokine receptor expression in the CNS, we have isolated, by molecular cloning techniques, rat genes encoding the chemokine receptors CCR2, CCR3, CCR5, and CXCR4. We describe expression patterns of these genes in cells and tissues of the CNS, and report regulation of chemokine receptor expression in vitro in cultured rat microglia, and demonstrate in vivo modulation of chemokine receptor expression in EAE rats.
Section snippets
Materials
DNA modifying enzymes and the random primer labeling kit were purchased from Promega (Madison, WI). TRIzol Reagent, DMEM, trypsin, penicillin/streptomycin, LipofectAMINE, and rat recombinant interferon-γ were from Life Technologies (Grand Island, NY). [Alpha-]dCTP (3000 Ci/mmol) and [alpha-]dATP (2000 Ci/mmol) were from DuPont/NEN (Wilmington, DE). [Alpha-]UTP was from Amersham (Arlington Heights, IL). DNA sequencing kit (Sequenase, v. 2.0) was from US Biochemical (Cleveland, OH).
Cloning of rat chemokine receptors
Degenerate oligonucleotide primers were designed based on the highly conserved regions (TM2 and TM7) of known human chemokine receptors (huIL-8RA, huIL-8RB, huCCR1, and huCCR2) and the orphan rat chemokine receptor-like gene, RBS11 (Harrison et al., 1994). DNA fragments encoding potential novel members of the GCR superfamily were generated by PCR using these oligonucleotides and rat genomic DNA as the template. Rat genomic DNA was used because the protein coding sequences of most GCRs in the
Discussion
There is increasing evidence of the CNS expression of both chemokine peptides and receptors. Towards the goal of understanding the functional role of chemokines in the CNS, the present study was undertaken to begin to determine which cells of the rat CNS express receptors for chemokine peptides. In order to achieve this goal, a degenerate PCR-based approach was used to identify DNA sequences encoding fragments of rat chemokine receptors. PCR products containing sequences encoding novel
Acknowledgements
This work was supported in part by grants from the National Multiple Sclerosis Society (PP0470) and the National Institutes of Health (NS34901) to J.K.H. and National Institutes of Health (DK49832) to L.F.
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