Mu C10 Byung-S. Youn1 and Byoung S. Kwon2,3,* 1Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indiapolis, IN 46202, USA 2The Immunomodulation Research Center, University of Ulsan, Ulsan, Korea 3Department of Ophthalmology, LSUMC, 2020 Gravier Street Suite B, New Orleans, LA 70112, USA *corresponding author tel: 504-412-1200 ex 1379, fax: 504-412-1315, e-mail: [email protected] DOI: 10.1006/rwcy.2000.11011. SUMMARY Alternative names Murine C10 was isolated from granulocyte–macro- Macrophage inflammatory protein-related protein 1 phage colony-stimulating factor (GM-CSF)-stimu- (MRP-1) was identified by a modified differential lated bone marrow cells. Mu C10 is structurally most hybridization method (Youn et al., 1995). Its cDNA similar to Mu MIP-1(cid:13)/MRP-2 in that they have two sequence is identical to Mu C10. MRP-1 was so extra cysteine residues in addition to the usual four named because the overall protein structure was very cysteine residues. The Mu C10 sequence reveals a similar to MIP-1(cid:13)/MRP-2. single open reading frame encoding 110 amino acids, of which the first 21 amino acids represent a putative signal peptide. Mu C10 mRNA is significantly Structure induced by IL-4 in macrophages. Mu C10 is a potent ligand for CCR1. Mu C10 and Mu MIP-1(cid:13)/MRP-2 The cDNA sequence of Mu C10 reveals its closest share the receptor. Recombinant Mu C10 signifi- homology to MIP-1(cid:13)/MRP-2 (50%), and to a lesser cantly suppressed colony formation by mouse bone degree, MIP-1(cid:11) (40%), therefore suggesting that it is marrow, granulocyte–macrophage, erythroid, and a member of the mouse CC chemokine family. Mu multipotential progenitor cells stimulated by combi- C10isdistinguishedfromothermouseCCchemokine nations of growth factors. family members by an N-terminal extension contain- ing a cluster of charged residues and by the presence of two extra cysteineresidues in addition tothe usual conservedfourcysteineresidues.Thesecharacteristics BACKGROUND are also conserved in MIP-1(cid:13)/MRP-2, leukotactin 1 (Lkn-1, MIP-5/HCC-2), MPIF1, and CK(cid:12)8-1, col- Discovery lectively named the C6 CC chemokine family. The cDNA encoding Mu C10 was isolated from a cDNA library prepared from myelopoietic mouse Main activities and bone marrow cultures stimulated by GM-CSF by pathophysiological roles using differential screening (Orlofsky et al., 1991). The expression of Mu C10 is not normally noted in unstimulated mouse bone marrow cells. Mu C10 Recombinant Mu C10 is chemotactic for B cells, begins to be expressed in the course of differentiation CD4+ T cells, monocytes, and natural killer cells of bone marrow stem cells into myeloid lineages. (Orlofsky et al., 1994). 1246 Byung-S. Youn and Byoung S. Kwon GENE AND GENE REGULATION Chromosome location Accession numbers The Mu C10 gene has been termed Scya6 (small inducible cytokine a6) and located on chromosome Mu C10: M58004 11. Mu C10 is most tightly linked to Scya2, the locus of the mouse CC chemokine family member JE, indicatingthattheMuC10geneislocatedinthesame regionofmousechromosome11asothermembersof Sequence the CC chemokine superfamily. The cDNA encoding Mu C10 is 1362bp long, of Cells and tissues that express which 96% bp constitutes 30 UTR with multiple the gene copies of AT-rich sequence thought to mediate rapid turnover of these messages. The open reading frame and 50 UTR comprise 351bp and 49bp, respectively Bonemarrow-derivedmacrophages,whenculturedin (Figure 1). M-CSF, express a basal level of Mu C10. When Figure 1 Nucleotide sequence of a cDNA encoding Mu C10 and the deduced amino acidsequence.Thenucleotidesequenceofthemessagestrandisnumberedinthe50to30 direction. The predicted amino acid sequence is shown below the nucleotide sequence. The putative signal peptide is underlined. Stop codon is indicated. The potential polyadenylation signal is in a box. Mu C10 1247 stimulated by IL-3, IL-4, or GM-CSF, the level of signal peptide. The cleavage of the signal peptide Mu C10 can be significantly augmented. Likewise, would generate a mature protein consisting of 95 peritonealmacrophagesstimulatedbythesecytokines aminoacids.When expressedinCOSor Sf-21cells, a express a high level of Mu C10. Interestingly, a single species corresponding to 10kDa was detected. polyclonal mitogen, lipopolysaccharide (LPS), does The N-terminal sequence of the mature protein not induce the Mu C10 message in these macro- remains to be determined. phages, but significantly induces MIP-1(cid:11), JE, and RANTES,suggesting thatMu C10 mayhave distinct Important homologies functions in vivo (Orlofsky et al., 1994). Murine monocytic and/or macrophage cell lines P38801 and WEH13 constitutively express Mu C10, whereas The mature Mu C10 harbors 28 amino acids before RAW264.7 does not contain Mu C10 (Youn et al., the dicysteine motifs. This unique N-terminal exten- 1995). It was noted that Mu C10 levels are sion is a common characteristic of the C6 CC significantly higher in primary trigeminal ganglion chemokine family, including MIP-1(cid:13)/MRP-2, Lkn-1, (TG) cell cultures originating from HSV-1-infected CK(cid:12)8-1,andMPIF1.MuC10containssixconserved mice, compared with uninfected ones (Carr et al., cysteines that could form three disulfide bonds 1998). Levels of C10 were significantly elevated in instead of the two disulfide bonds that are character- lung cultures from NG-nitro-L-arginine-methyl ester istic of the conventional CC chemokines (Figure 2). (L-NAME)-treated mice compared with controls (Hogaboam et al., 1997). RECEPTOR UTILIZATION PROTEIN Because of its similarity in structure and chemotactic pattern to MIP-1(cid:13)/MRP-2, it was anticipated that Sequence these two chemokines maysharea commonreceptor. Since MIP-1(cid:13)/MRP-2 exclusively utilizes CCR1 The Mu C10 cDNA sequence reveals a single open (mCCR1), mCCR1-transfected HEK 293 cells were readingframeencoding116aminoacids,ofwhichthe used for determining receptor usage. As shown in first21aminoacidsshowcharacteristicsofaputative Figure 3a, the mouse MIP-1(cid:13) (mMIP-1(cid:11)) induces a Figure 2 Alignment of Mu C10 with other C6 CC chemokines, MIP-1(cid:11), and MIP-1(cid:12). The putative signal sequences are not shown. The four conserved cysteines are represented by filled circleswhereastheconservedtwoextracysteinesaredenotedbystars.Gapswereintroducedfor optimum alignment. 1248 Byung-S. Youn and Byoung S. Kwon Figure 3 Recombinant Mu C10 is a potent agonist for mouse CCR1 (mCCR1) and human CCR1 (hCCR1). cDNAs encoding mCCR1 and hCCR1were stably expressed in the human embryonic kidney cells (HEK 293). These cells were loaded with Fura-2/AM and sequentially stimulatedwiththechemokinesindicated.Fluorescencewasmonitored.(WethankDrP.Murphy for providing the mCCR1 cDNA.) robust calcium flux in these cells. The stimulation of C10becausestimulationofthemCCR1cellswithMu the mCCR1 cells with mMIP-1(cid:11) desensitizes the cells C10 desensitizes the cells to a subsequent challenge toMuC10.Likewise,theinitialresponsivenessofthe with Mu C10. These data clearly show that, like CCR1 cells to Mu C10 renders the cells insensitive to MIP-1(cid:13)/MRP-2, Mu C10 shares mCCR1 with mMIP-1(cid:11). The calcium flux assay is specific to Mu mMIP-1(cid:11). A human C6 CC chemokine, Lkn-1 Mu C10 1249 Table1 Comparativeinfluence ofantibodies againstMuC10andMIP-1(cid:11)onthesuppressiveeffects of Mu C10 and MIP-1(cid:11) on colony formation by human myeloid progenitor cellsa Colony formation (% change from control) CFU-GM BFU-E CFU-GEMM Control medium 50(cid:6)1 62(cid:6)4 19(cid:6)2 Anti-Mu C10 52(cid:6)6 ((cid:135)4) 57(cid:6)1 ((cid:255)8) 21(cid:6)2 ((cid:135)11) Anti-MIP-1(cid:11) 56(cid:6)2 ((cid:135)12) 59(cid:6)3 ((cid:255)5) 22(cid:6)1 ((cid:135)16) Mu C10(cid:135)control medium 36(cid:6)1 ((cid:255)28)(cid:3) 34(cid:6)3 ((cid:255)45)(cid:3) 10(cid:6)1 ((cid:255)47)(cid:3) Mu C10(cid:135)anti-Mu C10 53(cid:6)5 ((cid:135)5) 59(cid:6)5 ((cid:255)5) 21(cid:6)2 ((cid:135)11) Mu C10(cid:135)anti-MIP-1(cid:11) 38(cid:6)1 ((cid:255)24)(cid:3) 30(cid:6)3 ((cid:255)52)(cid:3) 12(cid:6)1 ((cid:255)37)(cid:3) MIP-1(cid:11)(cid:135)control medium 36(cid:6)1 ((cid:255)28)(cid:3) 33(cid:6)1 ((cid:255)47)(cid:3) 10(cid:6)1 ((cid:255)47)(cid:3) MIP-1(cid:11)(cid:135)anti-Mu C10 35(cid:6)1 ((cid:255)30)(cid:3) 31(cid:6)0.3 ((cid:255)50)(cid:3) 12(cid:6)1 ((cid:255)37)(cid:3) MIP-1(cid:11)(cid:135)anti-MIP-1(cid:11) 51(cid:6)3 ((cid:135)2) 59(cid:6)4 ((cid:255)5) 20(cid:6)1 ((cid:135)5) aLow-densitymousemarrowcellswereplatedat5(cid:2)104cells/mLwith10–30%FBSandgrowth factorsina0.3%agaror1%methylcelluloseculturemedium.Colonyformationwasscored14days afterincubationin5%CO andlowered(5%)O .Resultsarerepresentativeofthreeseparatesamples. 2 2 (cid:3)Significantpercentagechangefromcontrolmedium,P<0.001;othervaluesarenotsignificantly differentfromcontrol,P<0.05. MuC10(100ng/mL)andrmuMIP-1(cid:11)(50ng/mL)wereeachpreincubatedfor1houratroom temperaturewitheithercontrol(McCoy’smedium)or200neutralizingunitsofeitheranti-MuC10or anti-MIP-1(cid:11)priortoassessingtheiractivityoncolonyformationby5(cid:2)104low-densityhuman bonemarrowcellsstimulatedasdescribedabove. (MIP-5/HCC-2) has been shown to activate mCCR1 suppressive activity on colony formation by differ- efficiently. Mu C10 efficiently cross-desensitizes the ent lineages of the hematopoietic progenitors mCCR1 cells to Lkn-1. Furthermore, stimulation of (Table 1). themCCR1cellswithMuC10desensitizesthecellsto MIP-1(cid:13)/MRP-2, whereas secondary stimulation of the mCCR1 cells with Mu C10 induces a transient References calciumflux,suggestingthatMuC10isamorepotent agonist for mCCR1 than MIP-1(cid:13)/MRP-2. Just as mMIP-1(cid:13) binds to and activates hCCR1, Mu C10 Carr, D. J. J., Noisakran, S., Halford, W. P., Lukacs, N., Asensio, V.,andCampbell, I.L.(1998).Cytokineandchemo- induces a robust calcium flux in the hCCR1- kineproductioninHSV-1latentlyinfectedtrigeminalganglion transfected cells (Figure 3b). Primary stimulation of cell cultures: effects of hyperthermic stress. J. Neuroimmunol. thehCCR1cellswithMuC10desensitizesthecellsto 85,111–121. hMIP-1(cid:13) or Lkn-1. Likewise, primary stimulation of Hogaboam, C. M., Chensue, S. W., Steinhauser, M. L., the hCCR1 cells with hMIP-1(cid:11) or Lkn-1 desensitizes Huffnagle, G. B., Lukacs, N. W., Strieter, R. M., and Kunkel, S. L. (1997). Alteration of the cytokine phenotype the cells to Mu C10. These data indicate that mMIP- in an experimental lung granuloma model by inhibiting nitric 1(cid:11) and Mu C10 are indistinguishable in terms of oxide.J.Immunol.159,5585–5593. agonistic functions. Orlofsky,A.,Berger,M.S.,andPrystowsky,M.B.(1991).Novel expression pattern of a new member of the MIP-1 family of cytokine-likegenes.CellReg.2,403–412. Orlofsky, A., Lin, E. Y., and Prytowsky, M. B. (1994). Selective IN VITRO ACTIVITIES induction of the (cid:12) chemokine C10 by IL-4 in mouse macro- phages.J.Immunol.152,5084–5091. Youn, B.-S., Jang, I.-K., Broxmeyer, H. E., Cooper, S., In vitro findings Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Elick, T. A., FraserJr.,M.J.,andKwon,B.S.(1995).Anovelchemokine, macrophage inflammatory protein-related protein-2, inhibits As is the case for MIP-1(cid:11), Lkn-1, MPIF1, CK(cid:12)8-1, colony formation of bone marrow myeloid progenitors. and MIP-1(cid:13)/MRP-2, Mu C10 shows a potent J.Immunol.155,2661–2667. 1250 Byung-S. Youn and Byoung S. Kwon LICENSED PRODUCTS ACKNOWLEDGEMENTS Mu C10 is sold under the trademark of recombinant SRC funds to IRC from the Korean Ministry of mouse C10 by R&D Systems. Science and Technology are greatly appreciated.