Table Of ContentMIP-1g/MRP-2
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.2000.11010.
SUMMARY isolated from RAW 264.7, a murine macrophage cell
line cDNA library using different approaches
MurineMIP-1gormacrophageinflammatoryprotein (Poltorak et al., 1995; Youn et al., 1995). MIP-1g
(MIP)-related protein 2 (MRP-2) was isolated from a gene expression was extinguishable by making RAW
murine macrophage cell line, RAW 264.7. Mu MIP- 264.7(cid:2)NIH3T3hybridcells,suggestingthatMIP-1g
1g/MRP-2 is composed of 122 amino acids, of which is a macrophage-specific gene. The partial MRP-2
the first 21 residues constitute a putative signal cDNAwasamplifiedbyRT-PCRwiththedegenerate
sequence.Theputativematureproteiniscomposedof PCR primer set designed from the conserved amino
101 amino acids with a molecular weight of 11,600. acidsequenceamongCCchemokines.Thefull-length
MuMIP-1g/MRP-2isstructurallysimilartoMuC10 MRP-2 cDNA was subsequently isolated from the
and MIP-1(cid:11). Mu MIP-1g/MRP-2 mRNA was same library.
preferentially detected in monocytesand macrophage
celllinesbutnotinTandBcells.Astrikingfeatureof
this chemokine in terms of expression pattern is that Alternative names
the mRNA is ubiquitously found in all tissues and
inflammation is not required for its expression. The
ShortlyafterMIP-1g/MRP-2wasidentified,acDNA
MuMIP-1g/MRP-2gene,termedScya9,wasmapped
termed CCF18 identical to MIP-1g/MRP-2 (Hara
to the central region of mouse chromosome 11 near
et al., 1995) was isolated from a cDNA library of an
Scya1 and Scya2, which are members of the CC
IL-3-dependent murine pro-B cell line (Ba/F3).
chemokine superfamily. Mu MIP-1g/MRP-2 is a
potent ligand for CCR1. Recombinant Mu MIP-1g/
MRP-2 significantly suppressed colony formation by
Structure
mouse bone marrow, granulocyte-macrophage, ery-
throid, and multipotential progenitor cells stimulated
by combinations of growth factors. MIP-1g/MRP-2 is a novel CC chemokine family
member, with similarity to existing members of the
CC chemokine family. MRP-2 shows a 50.8%
sequence identity at the protein level to Mu C10
BACKGROUND
(MRP-1) and a 46.3% identity to MIP-1(cid:11). Unlike
most members of the CC chemokine family, MIP-1g/
Discovery
MRP-2 possesses two extra cysteine residues on top
of the usual four conserved cysteine residues. These
The cDNA encoding murine macrophage inflamma- two extra cysteine residues are also found in Mu C10
toryprotein1g(MIP-1g)ormacrophageinflammatory (MRP-1); thus, MIP-1g/MRP-2 and Mu C10 are
protein-related protein 2 (MRP-2) was independently closely related mouse CC chemokines.
1238 Byung-S. Youn and Byoung S. Kwon
Main activities and Sequence
pathophysiological roles
ThecDNAencodingMIP-1g/MRP-2is1255bplong,
of which 732bp constitutes A 30 UTR with multiple
TherecombinantMIP-1g/MRP-2chemoattractslym-
copies of an AT-rich sequence, which may be
phocytes, monocytes, and neutrophils. When injected
involved in regulating RNA stability. The open
into rats, MIP-1g/MRP-2 promptly induced fever,
reading frame and 50 UTR are composed of 366bp
indicating that MIP-1g/MRP-2 is pyrogenic.
and 156bp, respectively (Figure 1).
GENE AND GENE REGULATION Chromosome location
Accession numbers
ThemurinechromosomallocationofMIP-1g/MRP-2
(designated gene Scya9 for small inducible cyto-
GenBank: kine a9) was determined by interspecific backcross
MRP-2 cDNA: U15209 analysis using progeny derived from matings of
CCF18 cDNA: U19482. ((C57BL/6J(cid:2)M.spretus)F1(cid:2)C57BL/6J)mice(Youn
Figure 1 Nucleotide sequence of a cDNA encoding MIP-1g/MRP-2 and the deduced
aminoacidsequence.Thenucleotidesequenceofthemessagestrandisnumberedinthe
50 to 30 direction. The predicted amino acid sequence is shown below the nucleotide
sequence. The putative signal peptide is underlined. The stop codon is indicated. The
potential mRNA destabilizing sequence (AU-rich sequence) is indicated by heavy
underlining. Two potential polyadenylation signals are boxed.
MIP-1g/MRP-2 1239
et al., 1995). This interspecific backcross-mapping betweenthesechemokinestakesplace.Duetothefact
panel (designated the Frederick interspecific back- that 50 UTRs of these chemokines are unique to each
cross-mapping panel) has been typed for over 1700 other, MIP-1g/MRP-2-specific probes can be gener-
loci that are well distributed among all mouse auto- atedandusedfortheexaminationofthetranscript.A
somes and the X chromosome. C57BL/6J and M. preferential expression of MIP-1g/MRP-2 is found in
spretus DNAs were digested with several restriction murine macrophage cells lines, such as P38801 and
enzymesand analyzedby southernblothybridization RAW 2647 and a monocytic cell line, WEHI3 (Youn
for informative RFLPs using a mouse Scya9 cDNA et al., 1995).
probe. A 2.9kb M. spretus-specific BamHI fragment OnenotablefeatureoftheexpressionpatternofMIP-
was used to follow the segregation of the Scya9 locus 1g/MRP-2 is that its mRNA was widely expressed in
in backcross DNAs. The mapping results indicated most tissues of normal mice (Poltorak et al., 1995).
that Scya9 is located in the middle region of mouse
chromosome11.Although103micewereanalyzedfor
all five markers shown in the haplotype analysis, up
to 170 mice were analyzed for somepairs ofmarkers. Figure2 Scya9mapsinthemiddleregionof
mousechromosome11.Scya9wasmappedto
Eachlocuswasanalyzedinpairwisecombinationsfor
mouse chromosome 11 by interspecific back-
recombination frequencies using the additional data.
cross analysis. The segregation patterns of
The ratios of the total number of mice exhibiting
Scya9 and flanking genes in 103 backcross
recombinant chromosomes to the total number of
animalstypedincommonareshownatthetop
miceanalyzedforeachpairoflociandthemostlikely
ofthefigure.Forsomeindividualpairsofloci,
geneorderare:centromere-Nfl-5/134-(Syca1,Scya23, morethan103animalsweretyped.Eachcolumn
Scya9)-2/119-Mpo. The recombination frequencies represents the chromosome identified in the
(expressed as genetic distances in centimorgans backcrossprogenythatwasinheritedfromthe
(cM)(cid:6)SE)are:centromere-Nfl-3.71.6-(Scya1,Scya2, (C57BL/6(cid:2)M.spretus)F1parent.Theshaded
Scya9)-1.7(cid:6)1.2-Mpo.Norecombinantsweredetected boxes represent the presence of a C57BL/6J
betweenScya1,Scya2,andScya9in161animalstyped allele, while the white boxes represent the
presence of an M. spretus allele. The number
in common, suggesting that the three loci are within
of offspring inheriting each type of chromo-
1.8cM of each other (Figure 2 and Table 1).
someislistedatthebottomofeachcolumn.A
partialchromosome11linkageisshownatthe
bottomofthefigure.Recombinationdistances
Cells and tissues that express
betweenlociincentimorgansareshowntothe
the gene leftofthechromosomeandthepositionsofloci
inhumanchromosomesareshowntotheright.
The MIP-1g/MRP-2 cDNA sequence is highly
homologous to Mu C10, so that crosshybridization Nf1
Scya1
Scya2
Table 1 Map location of human and mouse Scya loci
Scya9
Mpo
Map location
Locus Other gene names Mouse Human
Scya1 I-309, Tca3 11 17q12
Scya2 MCP-1, Je, Sigie 11 17q11.2-q12
Scya3 MIP-1(cid:11) 11 17q21.1-q21.3
Nf1
Scya4 MIP-1(cid:12), Act2 11 17q11-q21
Scya5 RANTES 11 17q11.2-q12
Scya6 Mu C10, MRP-1 11 ND
Scya1
Scya2
Scya7 MCP-3 ND 17q11.2-q12
Scya9
Scya8 MCP-2 ND ND Mpo
Scya9 MIP-1g/MRP-2 11 ND
1240 Byung-S. Youn and Byoung S. Kwon
Although the mRNA is detected in all tissues except Important homologies
thebrain,theliver,lung,andthymusarethemajorsites
expressing the MIP-1g/MRP-2 transcript. Most che-
Since MIP-1g/MRP-2 has the highest homologies at
mokinegenescanbeinducedbymitogens.Whenlipo-
the amino acid level to leukotactin 1 (MIP-5/HCC-2)
polysaccharide was injected into mice, the induction
(48%) and CK(cid:12)8-1 or MPIF1 (45%), and contains
of the MIP-1g/MRP-2 mRNA was detected in the
sixconservedcysteineresidues,MIP-1g/MRP-2could
heart and lung, but in no other tissues expressing
be classified into a subfamily of the mouse C6 CC
constant level of the transcript. However, MIP-1(cid:11)
chemokines.Thetwoadditionalcysteinesmayforma
mRNA was detected in these tissues only in the LPS-
third sulfide bond that fixes the C-terminal region
injectedmice.
to the core of the molecule, and is likely to provide
a great degree of rigidity to the molecule. Therefore,
it may be expected that the binding mode or
receptor activation by the C6 CC chemokine family
PROTEIN
is distinct from the rest of the CC chemokine
family. Whether or not the third disulfide bond is
Sequence
formedremains to bedetermined using NMR studies
(Figure 3).
The open reading frame encodes 122 amino acids, of
which the first 21 amino acids show characteristics of
Posttranslational modifications
a putative signal sequence. The putative mature pro-
tein consists of 101 amino acids with a calculated
molecularweightof11,600. WhentheMIP-1g/MRP- MIP-1g/MRP-2 does not appear to have putative
2 cDNA was expressed in Sf-21 cells by using the N- or O-glycosylation sites.
baculoviral vector, immunoblot staining exclusively
revealed an 8–9kDa band, indicating that some pro-
RECEPTOR UTILIZATION
tein modification may occur. Likewise, Langerhans
cells constitutively express MIP-1g/MRP-2, detected
as a major 9kDa band. Therefore, the N-terminal MIP-1g/MRP-2bindswithhighaffinity(K =45nM)
d
sequenceoftheprocessedMIP-1g/MRP-2remainsto to a receptor on the surface of mouse neutrophils.
be determined. When the cells are stimulated with MIP-1g/MRP-2,
Figure 3 Alignment of MIP-g/MRP-2 with other C6 CC chemokines, murine MIP-1(cid:11), and
MIP-1(cid:12). The putative signal sequences are not shown. The four conserved cysteines are
representedbyfilledcircles,whereastheconservedtwoextracysteinesinC6CCchemokinesare
denoted by stars. Gaps were introduced for optimum alignment.
• •
MIP-1g/MRP-2 1241
arobustcalciumfluxcanbeseen.SincemurineMIP- MIP-1g/MRP-2 induces chemotaxis and calcium flux
1(cid:11) can efficiently displace the binding of MIP-1g/ inCD4+Tcellclones,andpriorchallengewithMIP-
MRP-2 to mouse neutrophils, and the primary 1(cid:11) desensitizes the cells to MIP-1g/MRP-2, suggest-
responseofthecellsstimulatedwitheitherchemokine ing utilization of a common receptor or receptors in
renders the cells insensitive to a subsequent stimula- CD4+T cells can beutilized. Mouse neutrophilsand
tionwiththesamechemokines,itseemsthatMIP-1g/ CD4+ T cells express CC chemokine receptor 1
MRP-2 and MIP-1(cid:11) occupy a common receptor(s). (CCR1).Thus,CCR1couldbethecommonreceptor.
Figure 4 Recombinant MIP-1g/MRP-2 is a potent agonist for mouse CCR1 (mCCR1) and
humanCCR1.mCCR1andhCCR1werestablyexpressedinthehumanembryonickidneycells
(HEK 293). These cells were loaded with Fura-2/AM and sequentially stimulated with the
indicated chemokines. Fluorescence was monitored. (We thank Dr P. Murphy for providing
the mCCR1 cDNA.)
1242 Byung-S. Youn and Byoung S. Kwon
Using mouse CCR1 (mCCR1)-expressing HEK 293 and binding affinity are a little less than that of
cells,theligand-dependentcalciumfluxwastested.As MIP-1(cid:11).
noted in Figure 4a, MIP-1g/MRP-2 induces a robust
calcium flux in mCCR1 transfectants. The calcium
flux was ligand-specific, because the primary stimula-
tion with MIP-1g/MRP-2 desensitized the cells to the IN VITRO ACTIVITIES
same chemokines. Likewise, initial stimulation of
mCCR1transfectantswithmMIP-1(cid:11)orleukotactin1 In vitro findings
(Lkn-1) desensitized the cells to MIP-1g/MRP-2,
whereas initial stimulation with MIP-1g/MRP-2 did MIP-1g/MRP-2 chemoattracts CD4+ and CD8+ T
not desensitize the cells to MIP-1(cid:11) or Lkn-1. These cells (Mohamadzadeh et al., 1996). Like MIP-1(cid:11),
datasuggestthatMIP-1g/MRP-2utilizes mCCR1,as Lkn-1, MPIF1, and CK(cid:12)8-1, MIP-1g/MRP-2 also
doesmMIP-1(cid:11)orLkn-1,butitisalesspotentligand shows a potent suppressive activity on the colony
formCCR1thanmMIP-1(cid:11)andLkn-1.mMIP-1(cid:11)isa formation of different lineages of the hematopoietic
potent ligand for the human CC chemokine receptor progenitor cells (Table 2).
1 (hCCR1). When the hCCR1 transfectants were
stimulated with MIP-1g/MRP-2, a robust calcium
flux was seen (Figure 4b). Initial stimulation of the
hCCR1 transfectants with the human MIP-1(cid:11) IN VIVO BIOLOGICAL
(hMIP-1(cid:11)) or Lkn-1 desensitized the cells to MIP-
ACTIVITIES OF LIGANDS IN
1g/MRP-2. These data also suggest that MIP-1g/
ANIMAL MODELS
MRP-2 binds to and activates hCCR1, as does
mMIP-1(cid:11), and could be a mouse homolog of Lkn-1.
Normal physiological roles
Since Lkn-1 is a ligand for CCR3, MIP-1g/MRP-2
was tested to determine whether it activates mCCR3.
This yielded no ligand-dependent calcium flux. In Not much information in this regard is currently
summary, MIP-1g/MRP-2 exclusively utilize CCR1 available, except that MIP-1g/MRP-2 has a pyro-
in vitro, as does MIP-1(cid:11), but its agonistic potential genicity when injected into rats.
Table 2 Influence of purified recombinant MIP-1g/MRP-2 on colony formation by murine bone marrow myeloid
progenitor cellsa
Agar Methylcellulose
CFU-GM CFU-GM CFU-GM BFU-E CFU-GEMM
Concentration of [GM-CSF(cid:135)SLF] [PWMSCM] [EPO, SLF, PWMSCM, Human]
MIP-1g/MRP-2
Control medium 100(cid:6)4 68(cid:6)2 147(cid:6)7 14(cid:6)1 9(cid:6)0.6
100ng/mL 61(cid:6)3 ((cid:255)39)(cid:3) 28(cid:6)3 ((cid:255)59)(cid:3) 70(cid:6)6 ((cid:255)52)(cid:3) 6(cid:6)0.6 ((cid:255)57)(cid:3) 3(cid:6)0.3 ((cid:255)67)(cid:3)
50ng/mL 56(cid:6)2 ((cid:255)44)(cid:3) 28(cid:6)1 ((cid:255)59)(cid:3)
25ng/mL 65(cid:6)4 ((cid:255)35)(cid:3) 33(cid:6)1 ((cid:255)51)(cid:3)
10ng/mL 78(cid:6)4 ((cid:255)22)(cid:3) 42(cid:6)3 ((cid:255)38)(cid:3)
1ng/mL 101(cid:6)3 ((cid:135)1) 64(cid:6)3 ((cid:255)6)
a5(cid:2)104BDF1mousebonemarrowcellswereplatedin0.3%agarculturemediumwith10%v/vFBSforCFU-GMstimulated
byeitherrmuGM-CSF(100U/mL)(cid:135)rmuSLF(50ng/mL)or10%PWMSCMandwereplatedin1%methylcellulosewith
30%FBSforCFU-GM,BFU-E,andCFU-GEMMstimulatedbyrhuEpo(1U/mL),rmuSLF(50ng/mL),5%PWMSCMand
0.1mMhemin(EastmanKodak,Rochester,NY).Colonieswerescoredafter7days’incubationin5%CO and5%O .
2 2
Squarebracketsshowgrowthfactorsusedtostimulatecolonyformation.
Roundbracketsshowpercentagechangefromcontrolmedium.
(cid:3)Significantpercentagechangefromcontrolmedium,P<0.001;othervaluesarenotsignificantlydifferentfromcontrol,P<0.05.
MIP-1g/MRP-2 1243
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