Leukotactin 1

Leukotactin 1 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.2000.11012.

SUMMARY cDNA for a new member of the human CC chemokine family was isolated and named leukotactin 1 (Lkn-1). Lkn-1, along with murine macrophage inflammatory protein-related proteins 1 and 2 (Mu C10 and MIP-1 /MRP-2), defines a subgroup of CC chemokines based on two conserved cysteines in addition to the four others conserved in all CC chemokines. The putative mature Lkn-1 is composed of 92 amino acids with a calculated molecular weight of 10,160. The Lkn-1 gene was mapped to human chromosome 17, region q12. Recombinant Lkn-1 was a potent chemoattractant for neutrophils, monocytes, and lymphocytes and induced calcium flux in these cells. Lkn-1 specifically induced transient calcium mobilization in CCR1- and CCR3-expressing HOS cells. Lkn-1 suppressed colony formation by human bone marrow, granulocyte±macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors.

calcium flux (Youn et al., 1997). The cDNA encoding Lkn-1 was isolated from the THP1 cDNA library by using a portion of exon 4 as a probe that was derived from the Lkn-1 genomic DNA. The Lkn-1 genomic fragment corresponding to exon 4 was identified by hybridization to MIP-1 /MRP-2 (a presumed mouse ortholog) cDNA probe at low stringency. Therefore, Lkn-1 is likely to be the human counterpart of MIP1 /MRP-2.

Alternative names The identical cDNA clones termed HCC-2, MIP-5, or MIP-1 were independently isolated by the RT-PCR approach, searching the expressed sequence tag (EST) or screening the human spleen cDNA library with a 32 P-labeled cDNA probe representing the full-length coding region of MIP-1 /MRP-2, respectively (Coulin et al., 1997; Pardigol et al., 1998; Wang et al., 1998).

Structure BACKGROUND

Discovery Lkn-1 is a new member of the human CC chemokine family. The name, leukotactin, originated from the observation that major subsets of peripheral blood leukocytes, such as lymphocytes, monocytes, neutrophils, and eosinophils, respond to this chemokine in terms of chemotaxis as well as induction of transient

Two distinct structural features of Lkn-1 (MIP-5/ HCC-2) cause it to diverge from the rest of the human CC chemokine family: first, the long N-terminal extension (31 amino acids) of the predicted mature peptide, and second, the presence of six cysteine motifs, in contrast with most other CC chemokines which have four cysteine motifs. The latter property was also exhibited by Mu C10, MIP-1 /MRP-2, MPIF1, and CK 8-1. Therefore, we propose that these members be named C6 CC chemokines.

1252 Byung-S. Youn and Byoung S. Kwon Figure 1 (a) Nucleotide sequence of the 202 bp Alu I fragment of the Lkn-1 gene. A putative intron sequence is indicated. The putative exon sequence is translated, and the assigned amino acid sequence is shown below the nucleotide sequence. (b) Alignment of the Lkn-1 sequence with the corresponding region of MIP-1 /MRP-2. Conserved amino acids are boxed, and a star indicates the second of the two additional cysteines conserved among other C6 CC chemokine family members. (c) Nucleotide sequence of a cDNA encoding Lkn-1 and the deduced amino acid sequence. The nucleotide sequence of the message strand is numbered in the 50 to 30 direction. The 50 untranslated region sequence is indicated by negative numbers. The predicted amino acid sequence is shown below the nucleotide sequence. The putative signal peptide is underlined. The stop codon is indicated by stars. The portion of cDNA that is boxed corresponds to the exon sequence embedded in the 7.0 kb insert. The region was amplified with PCR and used as a probe for screening the THP1 cDNA library.

Leukotactin 1 1253 Figure 2 Differential cleavage products of recombinant Lkn-1. The amino acid sequence of the mature Lkn-1 is shown. When expressed in heterologous systems, the N-terminal sequences are heterogeneous. The 24 form is a major species expressed in insect cells, whereas the 26 form is a major species expressed in Pichia pastoris. The six conserved cysteine residues are represented by stars. (We thank Ms Eun K. Lee, Mr Seung J. Baek, and Dr Doo H. Park for determining the N-terminal sequence of the 26 Lkn-1.)

Figure 3 Alignment of Lkn-1 with other C6 -chemokines, MIP-1, and MIP-1 . The putative signal sequence is not shown. The conserved amino sequences of the molecules are boxed. There are four cysteines conserved among the -chemokines. In addition to the four cysteines, Lkn-1 and other C6 chemokines contain two additional conserved cysteines, indicated by asterisks. Gaps were introduced for optimum alignment.

GENE AND GENE REGULATION

Accession numbers GenBank: Lkn-1 (MIP-5/HCC-2) cDNA: U58914 HCC-1 cDNA: Z70292

Sequence cDNA encoding Lkn-1 (MIP-5/HCC-2) is 582 bp, which includes 50 UTR, open reading frame, and 30 UTR. The 30 UTR contains a typical polyadenylation signal (AATAAA) (Figure 1). Wang et al. (1998)

1254 Byung-S. Youn and Byoung S. Kwon reported that the human MIP-1 cDNA has an unusually long 402 bp 50 UTR.

Chromosome location Fluorescence in situ hybridization (FISH) mapping localized the Lkn-1 (MIP-5/HCC-2) gene to the long arm of chromosome 17 (17q12). A yeast artificial chromosome (YAC) encompassing the chromosome region was reported to contain 10 known chemokine genes plus four additional chemokine-like gene sequences. One of the ESTs (NCC-3) presented in the study corresponds to the gene for Lkn-1 (MIP-5/ HCC-2). Further, fine localization of the Lkn-1 (MIP-5/HCC-2) gene was made using a bacterial artificial chromosome (Youn et al., 1998a, 1998b). The Lkn-1 (MIP-5/HCC-2) gene is in proximity to the genes encoding MPIF1/CK 8-1 and LMC (HCC-4/ LEC/NCC-4), within 200 kb.

Most genes encoding -chemokines are comprised of three exons, whereas the Mu C10 (MRP-1), a mouse C6 -chemokine, gene is known to contain four exons. By virtue of structural similarity to Mu C10, the gene for Lkn-1 (MIP-5/HCC-2) would be expected to contain four exons. As expected, Pardigol et al. (1998) reported a high-resolution Lkn-1 (MIP-5/ HCC-2) gene structure which was comprised of four exons. Interestingly, the gene for Lkn-1 (MIP-5/ HCC-2) is separated from the adjacent gene coding for HCC-1 by 12 kbp. This paper also presented evidence that expression of Lkn-1 (MIP-5/HCC-2) and HCC-1, as studied by northern analysis, revealed both monocistronic and bicistronic transcripts, but the expression pattern of these chemokines seemed to be different. The promoter region of Lkn-1 (MIP-5/ HCC-2) was identified by primer extension and RTPCR analysis using total RNA from a variety of tissues.

Figure 4 (a) Chemoattraction of leukocyte subsets by the recombinant Lkn-1 (rLkn-1). Human peripheral blood lymphocytes, monocytes, and neutrophils were exposed to increasing concentrations of the indicated chemokines in a microchamber, and the number of cells that migrated through the membrane was determined. Migrated cells were counted microscopically at 1000 magnification in five randomly selected fields per well. All assays were performed in triplicate. The results shown are expressed at the chemotactic index calculated from the number of cells migrating to the test samples divided by those migrating to the controls. (b) Calcium flux responses of leukocytes subsets to rLkn-1. Fura-2/AM-loaded leukocytes (1  107/mL HBSS with 0.05% BSA) were exposed sequentially to the indicated chemokines. The concentration of chemokines used was 25 nM. Calcium flux assays were performed more than four times for each subset, and a representative result is shown.

Leukotactin 1 1255 Figure 5 rLkn-1 is a potent agonist for CCR1 and CCR3. (a, c) Receptor specificity. Fura-2/AM-loaded CCR1-HOS (a) or CCR3-HOS (c) cells were sequentially exposed to the chemokines indicated; fluorescence was monitored. Arrows indicate the time of addition of the indicated chemokines at 25 nM. (b, d) Potency of the calcium flux. Fura-2/AM-loaded CCR1-HOS (b) or CCR3-HOS (d) cells were stimulated with the indicated concentrations (1±100 nM) of chemokines, and fluorescence was monitored. The peak amplitude of the calcium response was plotted. rLkn-1 and MIP-1 were compared using CCR1-HOS cells (b), and rLkn-1, eotaxin, and RANTES were compared using CCR3-HOS cells (d).

Regulatory sites and corresponding transcription factors The Lkn-1 (MIP-5/HCC-2) promoter contains putative binding sites for transcription factors ZRF-2, ELK-1, and YY-1.

Cells and tissues that express the gene Even if there is some discrepancy regarding the expression pattern and the transcript size of Lkn-1 (MIP-5/HCC-2), liver, intestine, and colon are likely

to be places expressing Lkn-1 (MIP-5/HCC-2). Lkn-1 (MIP-5/HCC-2) was also detected in lung leukocytes. It has been found that Lkn-1 (MIP-5/HCC-2) is expressed in T and B lymphocytes, natural killer cells, monocytes, and monocyte-derived dendritic cells (Wang et al., 1998). One possible drawback of these tissue or cell northern analyses is using the full-length coding region of Lkn-1 (MIP-5/HCC-2) that has 70% identity to CK 8-1 or MPIF1 at amino acid level. Therefore, the crosshybridization pattern between Lkn-1 (MIP-5/HCC-2) and CK 8-1 or MP1F1 was detected. By using the Lkn-1 (MIP-5/HCC-2)-specific oligonucleotide probe, significant expression of Lkn-1 (MIP-5/HCC-2) was detected in adrenal gland, pancreas, heart, and liver (Youn et al., 1998a).

1256 Byung-S. Youn and Byoung S. Kwon

PROTEIN

Accession numbers SwissProt: Lkn-1 (MIP-5/HCC-2): Q16663

Sequence The putative mature Lkn-1 (MIP-5/HCC-2) is composed of 92 amino acids with a calculated molecular weight of 10 kDa. The mature Lkn-1 (MIP-5/ HCC-2) is preceded by the 21 amino acid predicted signal peptide. One interesting aspect of the Nterminal region is that, when expressed in insect cells by using the baculoviral system, the region appears to undergo a site-specific cleavage, causing 24 amino acids to be deleted, thereafter called 24 Lkn-1 (MIP5/HCC-2), and leaving seven amino acid residues (SFHFAAD) in front of the Cys±Cys motif. However, when expressed in Pichia pastoris, two amino acids were further cleaved, resulting in 26 amino acid deletions (26 Lkn-1 (MIP-5/HCC-2)). When 24 Lkn-1 (MIP-5/HCC-2) and 26 Lkn-1 (MIP-5/ HCC-2) were compared with the full-length form, no significant differences in agonistic potential or in the extent of hematopoietic stem cell inhibition were detected. Whether or not these cleaved products exist in vivo is unknown (Figure 2).

Important homologies The signal peptide of Lkn-1 (MIP-5/HCC-2) has 86% amino acid identity to those of CK 8-1 and MPIF1. The mature Lkn-1 (MIP-5/HCC-2) has a fairly extended N-terminal region that consists of 32 amino acids. This region has a 70% and 50% amino acid identity to that of MPIF1 and CK 8-1, respectively (Figure 3). More importantly, six conserved cysteines are found in Lkn-1 (MIP-5/HCC-2) along with CK 8-1, MPIF1, MIP-1 /MRP-2, and Mu C10.

Posttranslational modifications Lkn-1 (MIP-5/HCC-2) does not appear to have putative N- or O-glycosylation sites.

Figure 6 Chemotactic response of human neutrophils to Lkn-1, MIP-1, or IL-8. (a) Chemotaxis. Human neutrophils were exposed to increasing concentrations of the indicated chemokines in a microchamber, and the number of cells that migrated through the membrane were counted microscopically at 100 magnification. (b) Neutralization experiment. Neutrophil migration in response to Lkn-1 was inhibited by anti-Lkn-1. IL-8 (1000 ng/mL or 500 ng/mL) was preincubated with 10 mg/mL polyclonal antibodies against Lkn-1 or with rabbit Ig fraction as a control for 30 min at 37 C, and then used for chemotaxis. (c) A checkerboard analysis. Lkn-1 was added to lower wells only at 1000 ng/mL for determination of chemotaxis or to both the upper and lower wells at 1000 ng/mL for determination of chemokinesis.

Leukotactin 1 1257 Figure 7 Induction of transient calcium mobilization in human neutrophils stimulated with rLkn-1.Fura-2/AM-loaded neutrophils were sequentially exposed to 50 nM chemokines as indicated and fluorescence was monitored.

Figure 8 (a, b) Impaired calcium mobilization of mouse neutrophils from CCR1-deficient mice in response to Lkn-1and MIP-1. Mouse neutrophils were isolated from CCR1 wildtype or CCR1-deficient mice, loaded with Fura-2/AM, and sequentially stimulated with the chemokines indicated or fMLP, and fluorescence was measured. (We thank Drs Shang M. Zhang and Philip M. Murphy for performing chemotactic assays and calcium flux assays using mouse neutrophils from CCR1-deficient mice, respectively.)

RECEPTOR UTILIZATION Receptor utilization by Lkn-1 (MIP-5/HCC-2) was examined with the use of transfectants expressing a panel of CC chemokine receptors as well as purified subsets of leukocytes. Lkn-1 (MIP-5/HCC-2) induced transient calcium elevation in unstimulated lymphocytes and monocytes. Since the primary responsiveness of lymphocytes or monocytes to RANTES or MIP-1, respectively, significantly reduced the secondary responsiveness of these cells to Lkn-1 (MIP-5/ HCC-2), a common receptor could be used by these chemokines (Figure 4b). When the transfectants expressing a panel of chemokine receptors were tested for receptor usage, Lkn-1 (MIP-5/HCC-2) induced robust calcium flux in the CCR1- and CCR3expressing transfectants (Figure 5). One interesting feature is the ability of Lkn-1 (MIP5/HCC-2) to induce the vigorous migration of neutrophils. This extraordinary property has rarely been ascribed to CC chemokines, although it is noted that human MIP-1 induces a calcium flux in the

human neutrophils, but is unable to chemoattract the cells (Figure 6). When human neutrophils were stimulated with Lkn-1 (MIP-5/HCC-2), it induced a robust calcium flux. IL-8 also induced a robust calcium flux, but did not desensitize the neutrophils to Lkn-1 (MIP-5/HCC-2), suggesting that they utilize distinct receptors. Consistent with the previous observation, MIP-1 induced a weak calcium flux, but did not desensitize neutrophils to Lkn-1 (MIP-5/ HCC-2). However, Lkn-1 (MIP-5/HCC-2) desensitized the cells to MIP-1. This finding suggests that Lkn-1 (MIP-5/HCC-2) and MIP-1 probably share CCR1, but Lkn-1 (MIP-5/HCC-2) is a more potent ligand than MIP-1 (Figure 7). Equilibrium binding assay reveals that the dissociation constants (Kd) for Lkn-1 (MIP-5/HCC-2) and MIP-1 are 1 and 3 nM, respectively. When CCR1-deficient mice were used for the calcium flux assay, Lkn-1 (MIP-5/HCC-2) induced a robust calcium flux only in neutrophils derived from wild-type, not from CCR1-deficient mice (Figure 8). These data suggest that Lkn-1 (MIP5/HCC-2) utilizes CCR1 in vivo.

1258 Byung-S. Youn and Byoung S. Kwon Table 1 Effect of rLkn-1 on colony formation by low-density human bone marrow cellsa Concentration of rLkn-1

Control medium

Agar

Methylcellulose

CFU-GM [GM-CSF]

CFU-GM [GM-CSF+SLF]

CFU-GM

17  6

67  2

66  12 



BFU-E [EPO, SLF, IL-3]

CFU-GEMM

94  2

91 

5  1 (ÿ44)

50 ng/mL

16  4 (ÿ6)

35  6 (ÿ48)

25  3 (ÿ62)

25 ng/mL

18  1 (+6)

40  2 (ÿ40)

33  4 (ÿ50)

50  3 (ÿ47)

6  1 (ÿ33)

6.25 ng/mL

18  3 (+6)



52  6 (ÿ22)

45  6 (ÿ32)





7  2 (ÿ22)

3.125 ng/mL

17  8 (0)

63  5 (ÿ6)

61  10 (ÿ8)

35  3 (ÿ63) 65  2 (ÿ31) 95  6 (+17)

10  1 (+11)

a Low-density human bone 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%) 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. Square brackets, growth factors used to stimulate colony formation. Round brackets, percentage change from control medium.

IN VITRO ACTIVITIES

The Lkn-1 (MIP-5/HCC-2) expression can be upregulated by lipopolysaccharide and IL-4 (Youn et al., 1997) and can be induced by proinflammatory cytokines (IFN and TNF) in monocytes and dendritic cells (Wang et al., 1998).

N. C. (1997). Characterisation of macrophage inflammatory protein-5/human CC cytokine-2, a member of the macrophage-inflammatory-protein family of chemokines. Eur. J. Biochem. 248, 507±515. Pardigol, A., Forssmann, U., Zucht, H.-D., Loetscher, P., SchulzKnappe, P., Baggiolini, M., Forssmann, W.-G., Magert, H.-J. (1998). HCC-2, a human chemokine: gene structure, expression pattern, and biological activity. Proc. Natl Acad. Sci. USA 95, 6308±6313. Wang, W., Bacon, K. B., Oldham, E. R., and Schall, T. J. (1998). Molecular cloning and functional characterization of human MIP-1 , a new C-C chemokine related to mouse CCF-18 and C10. J. Clin. Immunol. 18, 214±222. Youn, B.-S., Zhang, S., Lee, E. K., Park, D. H., Broxmeyer, H. E., Murphy, P. M., Locati, M., Pease, J. E., Kim, K. K., Antol, K., and Kwon, B. S. (1997). Molecular cloning of leukotactin-1: a novel human -chemokine, a chemoattractant for neutrophils, monocytes, and lymphocytes, and a potent agonist at CC chemokine receptors 1 and 3. J. Immunol. (Cutting Edge) 159, 5201±5205. Youn, B.-S., Zhang, S., Broxmeyer, H. E., Cooper, S., Antol, K., Fraser Jr, M. J., and Kwon, B. S. (1998a). Characterization of CK 8 and CK 8-1: two alternatively spliced forms of human chemokine, chemoattractants for neutrophils, monocytes, and lymphocytes, and potent agonists at CC chemokine receptor 1. Blood 91, 3118±3128. Youn, B.-S., Zhang, S., Broxmeyer, H. E., Antol, K., Fraser Jr, M. J., Hangoc, G., and Kwon, B. S. (1998b). Isolation and characterization of LMC, a novel lymphocyte and monocyte chemoattractant human CC chemokine, with myelosuppressive activity. Biochem. Biophys. Res. Commun. 247, 217±222.

References

ACKNOWLEDGEMENTS

Coulin, F., Power, C. A., Alouant, S., Peitsch, M. C., Schroeder, J.-M., Moshizuki, M., Clark-Lewis, I., and Wells,

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

In vitro findings Based on chemotaxis assays, Lkn-1 (MIP-5/HCC-2) exhibited potent chemotactic activity. Consistent with receptor usage, Lkn-1 (MIP-5/HCC-2) was able to chemoattract lymphocytes, monocytes, and neutrophils that express CCR1 and eosinophils that express CCR3 (Figure 4a). One interesting feature of chemokines is their ability to suppress the proliferation of hematopoietic progenitors (Table 1). Lkn-1 (MIP-5/HCC-2) also showed potent hematopoietic cell suppressive activity, comparable to that of MIP1, a potent suppressive cytokine for hematopoietic cells in vitro.

Regulatory molecules: Inhibitors and enhancers