MIP-1g/MRP-2 Byung-S. Youn1 and Byoung S. Kwon2,3,* 1
Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indiapolis, IN 46202, USA 2 The Immunomodulation Research Center, University of Ulsan, Ulsan, Korea 3 Department 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 Murine MIP-1g or macrophage inflammatory protein (MIP)-related protein 2 (MRP-2) was isolated from a murine macrophage cell line, RAW 264.7. Mu MIP1g/MRP-2 is composed of 122 amino acids, of which the first 21 residues constitute a putative signal sequence. The putative mature protein is composed of 101 amino acids with a molecular weight of 11,600. Mu MIP-1g/MRP-2 is structurally similar to Mu C10 and MIP-1 . Mu MIP-1g/MRP-2 mRNA was preferentially detected in monocytes and macrophage cell lines but not in T and B cells. A striking feature of this chemokine in terms of expression pattern is that the mRNA is ubiquitously found in all tissues and inflammation is not required for its expression. The Mu MIP-1g/MRP-2 gene, termed Scya9, was mapped to the central region of mouse chromosome 11 near Scya1 and Scya2, which are members of the CC chemokine superfamily. Mu MIP-1g/MRP-2 is a potent ligand for CCR1. Recombinant Mu MIP-1g/ MRP-2 significantly suppressed colony formation by mouse bone marrow, granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors.
BACKGROUND
Discovery The cDNA encoding murine macrophage inflammatory protein 1g (MIP-1g) or macrophage inflammatory protein-related protein 2 (MRP-2) was independently
isolated from RAW 264.7, a murine macrophage cell line cDNA library using different approaches (Poltorak et al., 1995; Youn et al., 1995). MIP-1g gene expression was extinguishable by making RAW 264.7 NIH 3T3 hybrid cells, suggesting that MIP-1g is a macrophage-specific gene. The partial MRP-2 cDNA was amplified by RT-PCR with the degenerate PCR primer set designed from the conserved amino acid sequence among CC chemokines. The full-length MRP-2 cDNA was subsequently isolated from the same library.
Alternative names Shortly after MIP-1g/MRP-2 was identified, a cDNA termed CCF18 identical to MIP-1g/MRP-2 (Hara et al., 1995) was isolated from a cDNA library of an IL-3-dependent murine pro-B cell line (Ba/F3).
Structure 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 (MRP-1) and a 46.3% identity to MIP-1 . Unlike most members of the CC chemokine family, MIP-1g/ MRP-2 possesses two extra cysteine residues on top of the usual four conserved cysteine residues. These two extra cysteine residues are also found in Mu C10 (MRP-1); thus, MIP-1g/MRP-2 and Mu C10 are closely related mouse CC chemokines.
1238 Byung-S. Youn and Byoung S. Kwon
Main activities and pathophysiological roles
Sequence
The recombinant MIP-1g/MRP-2 chemoattracts lymphocytes, monocytes, and neutrophils. When injected into rats, MIP-1g/MRP-2 promptly induced fever, indicating that MIP-1g/MRP-2 is pyrogenic.
The cDNA encoding MIP-1g/MRP-2 is 1255 bp long, of which 732 bp constitutes A 30 UTR with multiple copies of an AT-rich sequence, which may be involved in regulating RNA stability. The open reading frame and 50 UTR are composed of 366 bp and 156 bp, respectively (Figure 1).
GENE AND GENE REGULATION
Chromosome location
Accession numbers
The murine chromosomal location of MIP-1g/MRP-2 (designated gene Scya9 for small inducible cytokine a9) was determined by interspecific backcross analysis using progeny derived from matings of ((C57BL/6J M. spretus)F1 C57BL/6J) mice (Youn
GenBank: MRP-2 cDNA: U15209 CCF18 cDNA: U19482.
Figure 1 Nucleotide sequence of a cDNA encoding MIP-1g/MRP-2 and the deduced amino acid sequence. The nucleotide sequence of the message strand is numbered in the 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 panel (designated the Frederick interspecific backcross-mapping panel) has been typed for over 1700 loci that are well distributed among all mouse autosomes and the X chromosome. C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and analyzed by southern blot hybridization for informative RFLPs using a mouse Scya9 cDNA probe. A 2.9 kb M. spretus-specific BamHI fragment was used to follow the segregation of the Scya9 locus in backcross DNAs. The mapping results indicated that Scya9 is located in the middle region of mouse chromosome 11. Although 103 mice were analyzed for all five markers shown in the haplotype analysis, up to 170 mice were analyzed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are: centromere-Nfl-5/134-(Syca1, Scya23, Scya9)-2/119-Mpo. The recombination frequencies (expressed as genetic distances in centimorgans (cM) SE) are: centromere-Nfl-3.7 1.6-(Scya1, Scya2, Scya9)-1.7 1.2-Mpo. No recombinants were detected between Scya1, Scya2, and Scya9 in 161 animals typed in common, suggesting that the three loci are within 1.8 cM of each other (Figure 2 and Table 1).
Cells and tissues that express the gene The MIP-1g/MRP-2 cDNA sequence is highly homologous to Mu C10, so that crosshybridization
between these chemokines takes place. Due to the fact that 50 UTRs of these chemokines are unique to each other, MIP-1g/MRP-2-specific probes can be generated and used for the examination of the transcript. A preferential expression of MIP-1g/MRP-2 is found in murine macrophage cells lines, such as P38801 and RAW 2647 and a monocytic cell line, WEHI3 (Youn et al., 1995). One notable feature of the expression pattern of MIP1g/MRP-2 is that its mRNA was widely expressed in most tissues of normal mice (Poltorak et al., 1995).
Figure 2 Scya9 maps in the middle region of mouse chromosome 11. Scya9 was mapped to mouse chromosome 11 by interspecific backcross analysis. The segregation patterns of Scya9 and flanking genes in 103 backcross animals typed in common are shown at the top of the figure. For some individual pairs of loci, morethan103animalsweretyped.Eachcolumn represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6 M. spretus) F1 parent. The shaded boxes represent the presence of a C57BL/6J allele, while the white boxes represent the presence of an M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 11 linkage is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome and the positions of loci in human chromosomes are shown to the right. Nf1 Scya1 Scya2
Table 1 Map location of human and mouse Scya loci 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
11
17q21.1-q21.3
Scya4
MIP-1 , Act2
11
17q11-q21
Scya5
RANTES
11
17q11.2-q12
Scya6
Mu C10, MRP-1
11
ND
Scya7
MCP-3
ND
17q11.2-q12
Scya8
MCP-2
ND
ND
Scya9
MIP-1g/MRP-2
11
ND
Scya9 Mpo
Nf1
Scya1 Scya2 Scya9 Mpo
1240 Byung-S. Youn and Byoung S. Kwon Although the mRNA is detected in all tissues except the brain, the liver, lung, and thymus are the major sites expressing the MIP-1g/MRP-2 transcript. Most chemokine genes can be induced by mitogens. When lipopolysaccharide was injected into mice, the induction of the MIP-1g/MRP-2 mRNA was detected in the heart and lung, but in no other tissues expressing constant level of the transcript. However, MIP-1 mRNA was detected in these tissues only in the LPSinjected mice.
PROTEIN
Sequence The open reading frame encodes 122 amino acids, of which the first 21 amino acids show characteristics of a putative signal sequence. The putative mature protein consists of 101 amino acids with a calculated molecular weight of 11,600. When the MIP-1g/MRP2 cDNA was expressed in Sf-21 cells by using the baculoviral vector, immunoblot staining exclusively revealed an 8±9 kDa band, indicating that some protein modification may occur. Likewise, Langerhans cells constitutively express MIP-1g/MRP-2, detected as a major 9 kDa band. Therefore, the N-terminal sequence of the processed MIP-1g/MRP-2 remains to be determined.
Important homologies Since MIP-1g/MRP-2 has the highest homologies at the amino acid level to leukotactin 1 (MIP-5/HCC-2) (48%) and CK 8-1 or MPIF1 (45%), and contains six conserved cysteine residues, MIP-1g/MRP-2 could be classified into a subfamily of the mouse C6 CC chemokines. The two additional cysteines may form a third sulfide bond that fixes the C-terminal region 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 is distinct from the rest of the CC chemokine family. Whether or not the third disulfide bond is formed remains to be determined using NMR studies (Figure 3).
Posttranslational modifications MIP-1g/MRP-2 does not appear to have putative N- or O-glycosylation sites.
RECEPTOR UTILIZATION MIP-1g/MRP-2 binds with high affinity (Kd=45 nM) to a receptor on the surface of mouse neutrophils. 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 , and MIP-1 . The putative signal sequences are not shown. The four conserved cysteines are represented by filled circles, whereas the conserved two extra cysteines in C6 CC chemokines are denoted by stars. Gaps were introduced for optimum alignment.
•
•
MIP-1g/MRP-2 1241 a robust calcium flux can be seen. Since murine MIP1 can efficiently displace the binding of MIP-1g/ MRP-2 to mouse neutrophils, and the primary response of the cells stimulated with either chemokine renders the cells insensitive to a subsequent stimulation with the same chemokines, it seems that MIP-1g/ MRP-2 and MIP-1 occupy a common receptor(s).
MIP-1g/MRP-2 induces chemotaxis and calcium flux in CD4+ T cell clones, and prior challenge with MIP1 desensitizes the cells to MIP-1g/MRP-2, suggesting utilization of a common receptor or receptors in CD4+ T cells can be utilized. Mouse neutrophils and CD4+ T cells express CC chemokine receptor 1 (CCR1). Thus, CCR1 could be the common receptor.
Figure 4 Recombinant MIP-1g/MRP-2 is a potent agonist for mouse CCR1 (mCCR1) and human CCR1. mCCR1 and hCCR1 were stably expressed in the human embryonic kidney cells (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 cells, the ligand-dependent calcium flux was tested. As 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 stimulation with MIP-1g/MRP-2 desensitized the cells to the same chemokines. Likewise, initial stimulation of mCCR1 transfectants with mMIP-1 or leukotactin 1 (Lkn-1) desensitized the cells to MIP-1g/MRP-2, whereas initial stimulation with MIP-1g/MRP-2 did not desensitize the cells to MIP-1 or Lkn-1. These data suggest that MIP-1g/MRP-2 utilizes mCCR1, as does mMIP-1 or Lkn-1, but it is a less potent ligand for mCCR1 than mMIP-1 and Lkn-1. mMIP-1 is a potent ligand for the human CC chemokine receptor 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 (hMIP-1 ) or Lkn-1 desensitized the cells to MIP1g/MRP-2. These data also suggest that MIP-1g/ MRP-2 binds to and activates hCCR1, as does mMIP-1 , and could be a mouse homolog of Lkn-1. 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 summary, MIP-1g/MRP-2 exclusively utilize CCR1 in vitro, as does MIP-1 , but its agonistic potential
and binding affinity are a little less than that of MIP-1 .
IN VITRO ACTIVITIES
In vitro findings MIP-1g/MRP-2 chemoattracts CD4+ and CD8+ T cells (Mohamadzadeh et al., 1996). Like MIP-1 , Lkn-1, MPIF1, and CK 8-1, MIP-1g/MRP-2 also shows a potent suppressive activity on the colony formation of different lineages of the hematopoietic progenitor cells (Table 2).
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles Not much information in this regard is currently available, except that MIP-1g/MRP-2 has a pyrogenicity 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
Concentration of MIP-1g/MRP-2
[GM-CSFSLF]
[PWMSCM]
[EPO, SLF, PWMSCM, Human]
Control medium
100 4
68 2
147 7
61 3 (ÿ39)
28 3 (ÿ59)
56 2 (ÿ44)
28 1 (ÿ59)
25 ng/mL
65 4 (ÿ35)
33 1 (ÿ51)
10 ng/mL
78 4 (ÿ22)
42 3 (ÿ38)
1 ng/mL
101 3 (1)
64 3 (ÿ6)
100 ng/mL 50 ng/mL
a
70 6 (ÿ52)
BFU-E
CFU-GEMM
14 1
6 0.6 (ÿ57)
9 0.6
3 0.3 (ÿ67)
5 104 BDF1 mouse bone marrow cells were plated in 0.3% agar culture medium with 10% v/v FBS for CFU-GM stimulated by either rmuGM-CSF (100 U/mL) rmuSLF (50 ng/mL) or 10% PWMSCM and were plated in 1% methylcellulose with 30% FBS for CFU-GM, BFU-E, and CFU-GEMM stimulated by rhuEpo (1 U/mL), rmuSLF (50 ng/mL), 5% PWMSCM and 0.1 mM hemin (Eastman Kodak, Rochester, NY). Colonies were scored after 7 days' incubation in 5% CO2 and 5% O2. Square brackets show growth factors used to stimulate colony formation. Round brackets show percentage change from control medium. Significant percentage change from control medium, P < 0.001; other values are not significantly different from control, P < 0.05.
MIP-1g/MRP-2 1243
References Hara, T., Bacon, K. B., Cho, L. C., Yoshimura, A., Morikawa, Y., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Schall, T. J., and Miyajima, A. (1995). Molecular cloning and functional characterization of a novel member of the C-C chemokine family. J. Immunol. 155, 5352±5358. Mohamadzadeh, M., Poltorak, A. N., Bergstresser, P. R., Beutler, B., and Takashima, A. (1996). Dendritic cells produce macrophage inflammatory protein-1g, a new member of the CC chemokine family. J. Immunol. 156, 3102±3106. Poltorak, A. N., Bazzoni, F., Smirnova, I. I., Alejos, E., Thompson, P., Luheshi, G., Rothwell, N., and Beutler, B. (1995). MIP-1g: molecular cloning, expression, and biological activities of a novel CC chemokine that is constitutively secreted in vivo. J. Inflamm. 45, 207±219. Youn, B.-S., Jang, I.-K., Broxmeyer, H. E., Cooper, S., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Elick, T. A.,
Fraser Jr, M. J., and Kwon, B. S. (1995). A novel chemokine, macrophage inflammatory protein-related protein-2, inhibits colony formation of bone marrow myeloid progenitors. J. Immunol. 155, 2661±2667.
LICENSED PRODUCTS MIP-1g/MRP-2 is sold as recombinant mouse MIP1g by R&D Systems.
ACKNOWLEDGEMENTS SRC funds to IRC from the Korean Ministry of Science and Technology are greatly appreciated.
