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Differential Effects of Leukotactin-1 and Macrophage Inflammatory Protein-1 on Neutrophils Mediated by CCR1¹

Shangming Zhang^2,*, Byung-S. Youn^2,*, Ji-Liang Gao, Philip M. Murphy and Byoung S. Kwon^3,*,

^* Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, and the Walther Cancer Institute, Indianapolis, IN 46202, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and Department of Biological Sciences and the Immunomodulation Research Center, University of Ulsan, Ulsan, Korea

	Abstract

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The human CC chemokine leukotactin-1 (Lkn-1) is both a strong chemoattractant for neutrophils, monocytes, and lymphocytes and a potent agonist for CCR1 and CCR3. However, human neutrophils do not migrate when the cells are stimulated with other human CC chemokines, such as human macrophage inflammatory protein-1 (hMIP-1) and eotaxin, which also use the CCR1 and CCR3 as their receptors. In this report, we demonstrate that while hMIP-1 induced a negligible level of calcium flux and chemotaxis, Lkn-1 produced a high level of calcium flux and chemotaxis in human neutrophils. Lkn-1 cross-desensitized hMIP-1-induced calcium flux, but hMIP-1 had little effect on the Lkn-1-induced response in human neutrophils. The same pattern was observed in peritoneal neutrophils from wild-type mice, whereas neutrophils from CCR1^-/- mice failed to respond to either MIP-1 or Lkn-1. Scatchard analysis revealed a single class of receptor for both hMIP-1 and Lkn-1 on human neutrophils with dissociation constants (K_d) of 3.2 nM and 1.1 nM, respectively. We conclude that CCR1 is a receptor mediating responses to both MIP-1 and Lkn-1 on neutrophils and produces different biological responses depending on the ligand bound.

	Introduction

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Chemokines are a family of small cytokines that are basic, heparin-binding proteins. Classification of chemokines is based on the distance between the first two of four to six conserved cysteine residues. Four subfamilies of chemokines have been discovered to date: CXC(), CC(ß), C(), and CX₃C (1, 2, 3). CXC chemokines act primarily on neutrophils, but show some activity on T lymphocytes (4, 5, 6, 7). CC chemokines function on monocytes, basophils, eosinophils, T lymphocytes, NK cells, and dendritic cells but usually do not affect neutrophils (4, 5, 6, 7, 8, 9, 10). The only known C chemokine is active on T lymphocytes and NK cells (11, 12). Soluble CX₃C chemokine stimulates chemotaxis of T cells and monocytes. The membrane-bound form of CX₃C, which is induced on activated primary endothelial cells, promotes strong adhesion of those leukocytes (3).

Leukotactin-1 (Lkn-1),⁴ a recently described CC chemokine, contains two extra cysteines. Lkn-1 binds to both CCR1 and CCR3 and induces chemotaxis and calcium flux in human neutrophils, monocytes, and lymphocytes (13) as well as eosinophils (S.M.Z. and B.S.K., unpublished observations). Purified rLkn-1 exhibited a site-specific cleavage and was 24 amino acids shorter than intact Lkn-1, which consists of 92 amino acids. Chemotaxis of neutrophils distinguishes Lkn-1 from other CCR1 agonists such as human macrophage inflammatory protein-1 (hMIP-1). MIP-5/HCC2, a synthetic chemokine, has the same amino acid sequence as cleaved Lkn-1. MIP-5/HCC2, a truncated form of Lkn-1, was reported to be an agonist for CCR1 and CCR3 and to stimulate the chemotaxis of monocytes, lymphocytes, and eosinophils but not neutrophils (14). In the present study, we examined the mechanism of neutrophil migration by Lkn-1.

	Materials and Methods

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Production and purification of rLkn-1

His-tagged rLkn-1 containing an additional six His at the C terminus was purified from Escherichia coli using an activated nickel column (Novagen, Madison, WI) followed by a heparin agarose column (Pharmacia, Piscataway, NJ) as previously described (13, 15). The purified rLkn-1 migrated as a single band of 12 kDa on SDS-PAGE. This form of rLkn-1 is called an intact form of rLkn-1 (rLkn-1i) (Fig. 1). rLkn-1 was also produced in the High Five insect cell line (Invitrogen, San Diego, CA) grown in EX-cell 400 medium (JRH Biosciences, Lenexa, KS). rLkn-1 was purified from culture media by heparin and HiTrap-SP columns (Pharmacia) (13). Fractions that showed a single band of 9 kDa on SDS-PAGE were pooled. The N-terminal sequencing of purified rLkn-1 revealed that it was 24 amino acids shorter than intact rLkn-1, which was named cleaved rLkn-1 (rLkn-1c). Purified rLkn-1i and rLkn-1c were dialysed against PBS (Life Technologies, Grand Island, NY) then analyzed by immunoblotting with rabbit polyclonal anti-Lkn-1 (13). Endotoxins in the rLkn-1 preparation were removed by Affi-prep polymyxin matrix (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions.

View larger version (56K): [in this window] [in a new window]   FIGURE 1. Purification of two forms of rLkn-1. His-tagged rLkn-1 produced in E. coli was purified with an activated nickel column and a heparin agarose column followed by Hi-Trap-SP column. rLkn-1 purified from insect cell cultures showed a site-specific cleavage to produce a 9 kDa protein (Lkn-1c, lane 1). His-tagged rLkn-1 migrated as a single band of 12 kDa (rLkn-1i, lane 2). M (in kDa) indicates the protein size marker.

Generation of CCR1^-/- mice was previously described (16).

Cell preparation

Human PBMC and neutrophils were isolated from healthy donors as previously reported (13, 15). The purity of neutrophils was >95% as determined by microscopic examination after Diff-Quick (Baxter Scientific, McGaw Park, IL) staining. The few contaminating cells were mononuclear cells and eosinophils. To obtain enriched mouse neutrophils, the mouse peritoneal cavity was washed with PBS 3 h after peritoneal injection of thioglycollate. The purity of neutrophils was >90% as assessed by light microscopic examination of Diff-Quick-stained cytospin preparations (16).

In vitro migration analysis

Migration of cells was assessed in a 48-well microchamber (Neuroprobe, Cabin John, MD) as previously described (13). Briefly, the lower wells were filled with 27 µl buffer alone or with buffer containing rLkn-1, IL-8, or hMIP-1, and the upper wells were filled with 50 µl of neutrophils in 1x HBSS (Life Technologies) at 1 x 10⁶ cells/ml. The two compartments were separated by a polyvinylpyrrolidone-free filter (Nucleopore; Neuroprobe) with 3-µm pores. After incubation for 1 h at 37°C, the filters were removed from the chamber, washed, fixed, and stained with Diff-Quick. The cells of five randomly selected oil-immersion fields were counted. The chemotactic index (CI) was calculated from the number of cells that migrated to the test chemokines divided by the number of cells that migrated to the control. Significant chemotaxis was defined as CI > 2.

To confirm that all the chemotactic activity for neutrophils was due specifically to rLkn-1, we blocked the activity in vitro with affinity-purified anti-Lkn-1 polyclonal Abs. rLkn-1 (1000 ng/ml) or IL-8 (500 ng/ml) was preincubated with 10 µg/ml affinity-purified rabbit anti-Lkn-1 Abs or purified Ig fraction of rabbit preimmune serum as a control for 30 min at 37°C, then used in the chemotaxis assay described above.

rLkn-1-induced migration may be separated into chemotactic and chemokinetic components. The chemotactic component was determined by the addition of rLkn-1 (1000 ng/ml) to the lower wells only, and the chemokinetic component was determined by the addition of rLkn-1 (1000 ng/ml) to both the upper and lower wells.

Calcium flux assay

The calcium mobilization assay was performed as previously described (13, 15).

Receptor binding studies

Purified rLkn-1 and hMIP-1 (carrier-free, R & D Systems, Minneapolis, MN) were labeled with ¹²⁵I using Iodo-beads iodination reagent (Pierce, Rockford, IL) according to the manufacturer’s instructions. The specific activities of the labeled rLkn-1 and hMIP-1 were 2 x 10⁷ cpm/µg protein. Human neutrophils, prepared as described above, were suspended in DMEM supplemented with 10% FCS at a concentration of 2 x 10⁷ cells/ml; 100 µl of the suspension was added to each tube. The cells were incubated in the presence of various concentrations of labeled rLkn-1 or hMIP-1 with 100-fold excess of the corresponding unlabeled ligand or with medium (200 µl of total reaction volume) at 4°C for 1 h with continuous rotation. Incubation was terminated by centrifuging the cell suspension over 1 ml of 10% sucrose (Sigma, St. Louis, MO) cushion. Cell pellets were cut from the tubes, and cpm were counted.

In vivo activity

To test the in vivo chemotactic effects of rLkn-1, rLkn-1i was injected into the peritoneum of C3H mice at 0.5 µg in 0.5 ml PBS per mouse. Human serum albumin (HSA; Sigma), or PBS, was applied to Affi-prep polymyxin matrix columns to remove endotoxin, and then injected i.p. as a control (six mice/group). Two hours after injection, peritoneal exudate was collected and the total number of cells counted by hemocytometer. Leukocytes in three randomly selected oil-immersion fields were counted differentially after Diff-Quick staining.

	Results

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rLkn-1, but not hMIP-1 or eotaxin, is a potent neutrophil chemoattractant

We examined chemotaxis stimulated by the two forms of rLkn-1, i.e., the intact form (rLkn-1i) and the cleaved form (rLkn-1c), and compared the chemotaxis with hMIP-1, eotaxin, and IL-8. Both forms of rLkn-1 were chemotactic for neutrophils at concentrations from 100 to 1000 ng/ml. Maximum migration was reached with 1000 ng/ml Lkn-1i (CI, 7.87 ± 0.18) or Lkn-1c (CI, 6.8 ± 0.12) and 100 ng/ml IL-8 (CI, 8.43 ± 0.39) (Fig. 2A). hMIP-1 failed to induce significant chemotaxis of neutrophils (maximal CI of 1.97 ± 0.20 at 1000 ng/ml, Fig. 2A). Eotaxin was not able to induce chemotaxis in neutrophils (data not shown). Anti-Lkn-1 Abs neutralized almost all neutrophil chemotactic activity but did not affect the chemotaxis of IL-8. Control rabbit Ig failed to block the activity of rLkn-1 (Fig. 2B). Moreover, a checkerboard analysis (17) revealed that the migration-stimulating effect of rLkn-1 was chemotactic in nature but not chemokinetic for neutrophils (Fig. 2C).

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Chemotactic response of human neutrophils to rLkn-1, hMIP-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 x1000 magnification in five randomly selected fields per well. All assays were performed in triplicate. The results were expressed as the CI, calculated from the number of cells migrating to the test chemokines divided by that migrating to the control. These results represent a single experiment among five performed with similar results. B, Neutralization experiment. Neutrophil migration in response to rLkn-1 was inhibited by specific Abs. A total of 1000 ng/ml or 500 ng/ml IL-8 were preincubated with 10 mg/ml of polyclonal Abs against rLkn-1 or with rabbit Ig fraction as a control for 30 min at 37°C and then used for chemotaxis. C, A checkerboard-type analysis. rLkn-1 was added to only lower wells at 1000 ng/ml for determination of chemotaxis or to both the upper and lower wells at 1000 ng/ml for determination of chemokinesis. Assays were performed in triplicate.

rLkn-1 and hMIP-1 share a neutrophil signaling pathway to calcium mobilization

While Lkn-1 and hMIP-1 share certain properties such as binding to CCR1 and suppression of colony formation by bone marrow stem/progenitor cells, only Lkn-1 is chemotactic for neutrophils. We then examined their receptor usage using a calcium flux assay. The two forms of Lkn-1 induced calcium flux in neutrophils and fully desensitized each other, which is consistent with the chemotaxis data (Fig. 2). Importantly, Lkn-1i completely desensitized the response of neutrophils to 50 nM hMIP-1. However, hMIP-1 failed to desensitize the response of neutrophils to the same amount of rLkn-1i (Fig. 3A). The rLkn-1-induced calcium flux in neutrophils was dose-dependent with a threshold < 5 nM (Fig. 3B). hMIP-1 strongly induced calcium mobilization in PBMC, demonstrating that the chemokine was active (data not shown). These data suggest that rLkn-1 shares a signaling pathway with hMIP-1, but that rLkn-1 was a more potent inducer of calcium mobilization than was hMIP-1 in human neutrophils.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Calcium flux response of neutrophils to rLkn-1. Fura-2/AM-loaded neutrophils were exposed sequentially to 50 nM of the indicated chemokines. Calcium flux assays were performed at least three times, and representative data is shown here. A, Cross-desensitization of neutrophils between the indicated chemokines at 50 nM. B, Dose-response curve of calcium flux induced by rLkn-1. The result was expressed as peak amplitude of relative fluorescence.

CCR1 mediates Lkn-1 action in mouse neutrophils

Lkn-1 is an agonist for human CCR1 and CCR3 (13), but as stated above, neutrophils do not express CCR3 (19). We used neutrophils from CCR1^+/+ and CCR1^-/- mice to assess the role of CCR1 in Lkn-1 signaling. In neutrophils from CCR1^+/+ mice, rLkn-1i was a potent inducer of calcium mobilization. Cross-desensitization experiments demonstrated that rLkn-1i completely desensitized the response of neutrophils to hMIP-1, while hMIP-1 at 200 nM failed to abolish the calcium flux response to Lkn-1i in mouse neutrophils (Fig. 4A). Notably, rLkn-1i did not affect FMLP-induced calcium flux (Sigma) in neutrophils. In neutrophils from CCR1^-/- mice, the FMLP-induced signal was observed, but responses to hMIP-1 and rLkn-1i were absent (Fig. 4B). These results demonstrate that CCR1 is a receptor for Lkn-1 and MIP-1 on mouse neutrophils.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Calcium mobilization response of mouse neutrophils to rLkn-1. Mouse neutrophils were isolated from thioglycollate-injected mouse peritoneal cavities and stimulated with the indicated chemokines or with FMLP at the indicated concentrations. A, Calcium flux of neutrophils derived from CCR1 wild-type mice by rLkn-1 or hMIP-1. B, Calcium flux of neutrophils derived from CCR1-/- mice by rLkn-1 or hMIP-1.

We next compared the binding properties of hMIP-1 and rLkn-1 to human neutrophils. Scatchard analysis revealed a single class of receptors for hMIP-1 and for rLkn-1 on human neutrophils with K_d of 3.2 nM and 1.1 nM, respectively. These data indicate that the receptor(s) shows different affinity for hMIP-1 and rLkn-1 and that human neutrophils expressed receptors for both Lkn-1 and hMIP-1 at a density of 2 x 10⁴ binding sites per cell (Fig. 5).

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Binding characteristics of 125I-Lkn-1 or hMIP-1 to human neutrophils. A and C, Saturable binding of 125I-Lkn-1 or hMIP-1 to human neutrophils. A total of 2 x 106 cells were incubated for 1 h at 4°C with indicated concentrations of 125I-Lkn-1 or hMIP-1. Nonspecific binding determined by the addition of a 100-fold excess of the corresponding unlabeled ligand was subtracted. Representative results from three separate experiments are shown here. B and D, Scatchard analysis of the binding data. A single class of receptors for both Lkn-1 and hMIP-1 with a calculated Kd of 1.1 nM and 3.2 nM, respectively. Human neutrophils expressed receptor for both Lkn-1 and hMIP-1 at a density of 2 x 104 binding sites per cell.

We further tested whether rLkn-1 can attract neutrophils in vivo because it induced chemotaxis in human neutrophils and calcium mobilization in neutrophils from wild-type mice. Intraperitoneal injection of rLkn-1i produced a remarkable increase in the total number of peritoneal cells within 2 h, increasing in the number of neutrophils from (0.23 ± 0.01) x 10⁶ cells per mouse injected with PBS or (0.46 ± 0.07) x 10⁶ cells per mouse injected with HSA, to (1.13 ± 0.14) x 10⁶ cells per rLkn-1-injected mouse (Fig. 6) (p < 0.006, compared with HSA). The increase in the number of neutrophils seen with HSA injection may indicate a nonspecific recruitment of neutrophils in response to the foreign protein. rLkn-1 injection also increases the number of monocytes and lymphocytes (data not shown). These in vivo results support our findings with human neutrophils in vitro.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. rLkn-1 induced recruitment of neutrophils to the peritoneum. Peritoneal exudate was collected from C3H mice 2 h after injection of Lkn-1 or control PBS or HSA (six mice/group), and the total number of cells were counted by hemocytometer. The subtypes of leukocytes in three randomly selected oil-immersion fields per section were differentially determined by microscopic examination after Diff-Quick staining. Data shown are representative of three experiments with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Both chemotaxis and calcium mobilization data (Figs. 2 and 3) indicated that an unidentified receptor specific for Lkn-1 may exist on neutrophils or that Lkn-1 may have higher binding affinity than hMIP-1 to CCR1 on human neutrophils. To determine whether there is an unidentified receptor specific for Lkn-1 on neutrophils, we used CCR1-deficient mice to examine whether Lkn-1 induced calcium mobilization in CCR1^-/- neutrophils. As shown in Fig. 4B, CCR1 is the only class of receptor used to mediate responses of murine neutrophils to hMIP-1 and Lkn-1. We next compared the binding affinities between hMIP-1 and rLkn-1 in human neutrophils. Scatchard analysis demonstrates that Lkn-1 has a higher binding affinity than hMIP-1 to its receptor on human neutrophils, but that both chemokines bind to receptors with a single affinity (Fig. 5).

The structural difference between hMIP-1 and Lkn-1 may be responsible for the difference of their biological effects. Lkn-1 contains two extra cysteines, which may form a third disulfide bond. How this structural change may effect receptor interactions is unclear. We are unable to explain the discrepancy between our data (13 and this report) and the report where MIP-5/HCC2 was shown to be inactive on human neutrophils (14). The observed differences in neutrophil chemotaxis may be a result of different assay conditions for chemotaxis or alternate methods of neutrophil isolation. In summary, we have demonstrated that Lkn-1 is a strong chemoattractant for neutrophils by both in vivo and in vitro assays. Our data supports that even though hMIP-1 and Lkn-1 signal through CCR1 in neutrophils, only Lkn-1 is chemotactic for neutrophils. The factors responsible for differential sorting of receptor signals to effector functions remains to be determined.

	Acknowledgments

 
We thank Dr. Mark H. Kaplan for help in writing this manuscript; Ihn-Kyung Jang for helping making the graphs; Patricia Mantel, Sister Mary Etta Kiefer, and Dr. Young-J. Kim for editing; and Audrey Carson for typing this manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI 28125 and DE 12156 (to B.S.K.) and research funds from the University of Ulsan and Molecular Medicine Program (98-MM-02-01-A-04), Ministry of Science and Technology, Korea.

² S.M.Z. and B.S.Y. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Byoung S. Kwon, Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202-5120. E-mail address:

⁴ Abbreviations used in this paper: Lkn-1, leukotactin-1; hMIP-1, human macrophage inflammatory protein 1; rLkn-1i, intact rLkn-1; rLkn-1c; cleaved rLkn-1; CI, chemotactic index.

Received for publication May 26, 1998. Accepted for publication January 29, 1999.

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