Mu C10

Mu C10 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.2000.11011.

SUMMARY

Alternative names

Murine C10 was isolated from granulocyte±macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cells. Mu C10 is structurally most similar to Mu MIP-1 /MRP-2 in that they have two extra cysteine residues in addition to the usual four cysteine residues. The Mu C10 sequence reveals a 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 induced by IL-4 in macrophages. Mu C10 is a potent ligand for CCR1. Mu C10 and Mu MIP-1 /MRP-2 share the receptor. Recombinant Mu C10 significantly suppressed colony formation by mouse bone marrow, granulocyte±macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors.

Macrophage inflammatory protein-related protein 1 (MRP-1) was identified by a modified differential hybridization method (Youn et al., 1995). Its cDNA sequence is identical to Mu C10. MRP-1 was so named because the overall protein structure was very similar to MIP-1 /MRP-2.

BACKGROUND

Discovery The cDNA encoding Mu C10 was isolated from a cDNA library prepared from myelopoietic mouse bone marrow cultures stimulated by GM-CSF by using differential screening (Orlofsky et al., 1991). The expression of Mu C10 is not normally noted in unstimulated mouse bone marrow cells. Mu C10 begins to be expressed in the course of differentiation of bone marrow stem cells into myeloid lineages.

Structure The cDNA sequence of Mu C10 reveals its closest homology to MIP-1 /MRP-2 (50%), and to a lesser degree, MIP-1 (40%), therefore suggesting that it is a member of the mouse CC chemokine family. Mu C10 is distinguished from other mouse CC chemokine family members by an N-terminal extension containing a cluster of charged residues and by the presence of two extra cysteine residues in addition to the usual conserved four cysteine residues. These characteristics are also conserved in MIP-1 /MRP-2, leukotactin 1 (Lkn-1, MIP-5/HCC-2), MPIF1, and CK 8-1, collectively named the C6 CC chemokine family.

Main activities and pathophysiological roles Recombinant Mu C10 is chemotactic for B cells, CD4+ T cells, monocytes, and natural killer cells (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 11. Mu C10 is most tightly linked to Scya2, the locus of the mouse CC chemokine family member JE, indicating that the Mu C10 gene is located in the same region of mouse chromosome 11 as other members of the CC chemokine superfamily.

Mu C10: M58004

Sequence The cDNA encoding Mu C10 is 1362 bp long, of which 96% bp constitutes 30 UTR with multiple copies of AT-rich sequence thought to mediate rapid turnover of these messages. The open reading frame and 50 UTR comprise 351 bp and 49 bp, respectively (Figure 1).

Cells and tissues that express the gene Bone marrow-derived macrophages, when cultured in M-CSF, express a basal level of Mu C10. When

Figure 1 Nucleotide sequence of a cDNA encoding Mu C10 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. 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 Mu C10 can be significantly augmented. Likewise, peritoneal macrophages stimulated by these cytokines express a high level of Mu C10. Interestingly, a polyclonal mitogen, lipopolysaccharide (LPS), does not induce the Mu C10 message in these macrophages, but significantly induces MIP-1, JE, and RANTES, suggesting that Mu C10 may have distinct functions in vivo (Orlofsky et al., 1994). Murine monocytic and/or macrophage cell lines P38801 and WEH13 constitutively express Mu C10, whereas RAW264.7 does not contain Mu C10 (Youn et al., 1995). It was noted that Mu C10 levels are significantly higher in primary trigeminal ganglion (TG) cell cultures originating from HSV-1-infected mice, compared with uninfected ones (Carr et al., 1998). Levels of C10 were significantly elevated in lung cultures from NG-nitro-L-arginine-methyl ester (L-NAME)-treated mice compared with controls (Hogaboam et al., 1997).

signal peptide. The cleavage of the signal peptide would generate a mature protein consisting of 95 amino acids. When expressed in COS or Sf-21 cells, a single species corresponding to 10 kDa was detected. The N-terminal sequence of the mature protein remains to be determined.

Important homologies The mature Mu C10 harbors 28 amino acids before the dicysteine motifs. This unique N-terminal extension is a common characteristic of the C6 CC chemokine family, including MIP-1 /MRP-2, Lkn-1, CK 8-1, and MPIF1. Mu C10 contains six conserved cysteines that could form three disulfide bonds instead of the two disulfide bonds that are characteristic of the conventional CC chemokines (Figure 2).

RECEPTOR UTILIZATION PROTEIN

Sequence The Mu C10 cDNA sequence reveals a single open reading frame encoding 116 amino acids, of which the first 21 amino acids show characteristics of a putative

Because of its similarity in structure and chemotactic pattern to MIP-1 /MRP-2, it was anticipated that these two chemokines may share a common receptor. Since MIP-1 /MRP-2 exclusively utilizes CCR1 (mCCR1), mCCR1-transfected HEK 293 cells were used for determining receptor usage. As shown in Figure 3a, the mouse MIP-1 (mMIP-1) induces a

Figure 2 Alignment of Mu C10 with other C6 CC chemokines, 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 are denoted by stars. Gaps were introduced for 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 stimulated with the chemokines indicated. Fluorescence was monitored. (We thank Dr P. Murphy for providing the mCCR1 cDNA.)

robust calcium flux in these cells. The stimulation of the mCCR1 cells with mMIP-1 desensitizes the cells to Mu C10. Likewise, the initial responsiveness of the CCR1 cells to Mu C10 renders the cells insensitive to mMIP-1. The calcium flux assay is specific to Mu

C10 because stimulation of the mCCR1 cells with Mu C10 desensitizes the cells to a subsequent challenge with Mu C10. These data clearly show that, like MIP-1 /MRP-2, Mu C10 shares mCCR1 with mMIP-1. A human C6 CC chemokine, Lkn-1

Mu C10 1249 Table 1 Comparative influence of antibodies against Mu C10 and MIP-1 on the suppressive effects of Mu C10 and MIP-1 on colony formation by human myeloid progenitor cellsa Colony formation (% change from control) CFU-GM

BFU-E

CFU-GEMM

Control medium

50  1

62  4

19  2

Anti-Mu C10

52  6 (‡4)

57  1 (ÿ8)

21  2 (‡11)

Anti-MIP-1

56  2 (‡12)

59  3 (ÿ5)

Mu C10 ‡ control medium

36  1 (ÿ28)



34  3 (ÿ45)

22  1 (‡16) 

10  1 (ÿ47)

Mu C10 ‡ anti-Mu C10

53  5 (‡5)

59  5 (ÿ5)

21  2 (‡11)

Mu C10 ‡ anti-MIP-1

38  1 (ÿ24)

30  3 (ÿ52)

12  1 (ÿ37)

MIP-1 ‡ control medium

36  1 (ÿ28)

33  1 (ÿ47)

10  1 (ÿ47)

MIP-1 ‡ anti-Mu C10

35  1 (ÿ30)

31  0.3 (ÿ50)

12  1 (ÿ37)

MIP-1 ‡ anti-MIP-1

51  3 (‡2)

59  4 (ÿ5)

20  1 (‡5)

a

Low-density mouse marrow cells were plated at 5  104 cells/mL with 10±30% FBS and growth factors in a 0.3% agar or 1% methylcellulose culture medium. Colony formation was scored 14 days after incubation in 5% CO2 and lowered (5%) O2. Results are representative of three separate samples.  Significant percentage change from control medium, P<0.001; other values are not significantly different from control, P<0.05. Mu C10 (100 ng/mL) and rmu MIP-1 (50 ng/mL) were each preincubated for 1 hour at room temperature with either control (McCoy's medium) or 200 neutralizing units of either anti-Mu C10 or anti-MIP-1 prior to assessing their activity on colony formation by 5  104 low-density human bone marrow cells stimulated as described above.

(MIP-5/HCC-2) has been shown to activate mCCR1 efficiently. Mu C10 efficiently cross-desensitizes the mCCR1 cells to Lkn-1. Furthermore, stimulation of the mCCR1 cells with Mu C10 desensitizes the cells to MIP-1 /MRP-2, whereas secondary stimulation of the mCCR1 cells with Mu C10 induces a transient calcium flux, suggesting that Mu C10 is a more potent agonist for mCCR1 than MIP-1 /MRP-2. Just as mMIP-1 binds to and activates hCCR1, Mu C10 induces a robust calcium flux in the hCCR1transfected cells (Figure 3b). Primary stimulation of the hCCR1 cells with Mu C10 desensitizes the cells to hMIP-1 or Lkn-1. Likewise, primary stimulation of the hCCR1 cells with hMIP-1 or Lkn-1 desensitizes the cells to Mu C10. These data indicate that mMIP1 and Mu C10 are indistinguishable in terms of agonistic functions.

IN VITRO ACTIVITIES

In vitro findings As is the case for MIP-1, Lkn-1, MPIF1, CK 8-1, and MIP-1 /MRP-2, Mu C10 shows a potent

suppressive activity on colony formation by different lineages of the hematopoietic progenitors (Table 1).

References Carr, D. J. J., Noisakran, S., Halford, W. P., Lukacs, N., Asensio, V., and Campbell, I. L. (1998). Cytokine and chemokine production in HSV-1 latently infected trigeminal ganglion cell cultures: effects of hyperthermic stress. J. Neuroimmunol. 85, 111±121. Hogaboam, C. M., Chensue, S. W., Steinhauser, M. L., Huffnagle, G. B., Lukacs, N. W., Strieter, R. M., and Kunkel, S. L. (1997). Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide. J. Immunol. 159, 5585±5593. Orlofsky, A., Berger, M. S., and Prystowsky, M. B. (1991). Novel expression pattern of a new member of the MIP-1 family of cytokine-like genes. Cell Reg. 2, 403±412. Orlofsky, A., Lin, E. Y., and Prytowsky, M. B. (1994). Selective induction of the chemokine C10 by IL-4 in mouse macrophages. J. Immunol. 152, 5084±5091. 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.

1250 Byung-S. Youn and Byoung S. Kwon

LICENSED PRODUCTS

ACKNOWLEDGEMENTS

Mu C10 is sold under the trademark of recombinant mouse C10 by R&D Systems.

SRC funds to IRC from the Korean Ministry of Science and Technology are greatly appreciated.