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(Journal of Leukocyte Biology. 2001;70:155-161.)
© 2001 by Society for Leukocyte Biology

Synthetic peptide MMK-1 is a highly specific chemotactic agonist for leukocyte FPRL1

Jin Yue Hu^*,, Yingying Le^*, Wanghua Gong, Nancy M. Dunlop^*, Ji Liang Gao, Philip M. Murphy and Ji Ming Wang^*

^* Laboratory of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, and
The Intramural Research Support Program, SAIC Frederick, Frederick, Maryland;
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; and
Cancer Research Institute, Hunan Medical University, Changsha, China

Correspondence: Dr. Ji Ming Wang, LMI, DBS, NCI-FCRDC, Building 560, Room 31-40, Frederick, MD 21702-1201. E-mail: wangji{at}mail.ncifcrf.gov

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human phagocytic leukocytes express the seven-transmembrane G-protein-coupled receptors formyl peptide receptor (FPR) and FPR-like 1 (FPRL1). MMK-1, a synthetic peptide derived from a random peptide library, is reported to induce calcium mobilization specifically in human FPRL1 gene-transfected cells. However, its actions on human phagocytic leukocytes remain poorly defined. We found that MMK-1 is a potent chemotactic and calcium-mobilizing agonist for human monocytes, neutrophils, and FPRL1-transfected human embryonic kidney (HEK) 293 cells but is inactive in cells transfected with FPR. MMK-1 also activated HEK 293 cells transfected with FPR2, a mouse counterpart of human FPRL1. Furthermore, MMK-1 increased pertussis toxin-sensitive production of inflammatory cytokines in human monocytes. MMK-1 signaling in human phagocytes was completely desensitized by a well-defined FPRL1 agonist, suggesting that FPRL1 is likely a receptor that mediates the action of MMK-1 in primary cells. Since MMK-1 is one of the most potent FPRL1-specific agonists identified so far, it can serve as a modulator of the host defense and a useful agent for further studying the signaling and function of FPRL1.

Key Words: phagocyte • chemotaxis • Ca²⁺ mobilization • cytokines

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Leukocytes respond to a large number of chemoattractants by directional cell movement, activation of integrins, and production of proinflammatory mediators. These chemoattractants include chemokines and the "classical" chemoattractants such as N-formyl methionyl-leucyl-phenylalanine (fMLF), the activated complement fragment 5, leukotriene B₄, and platelet-activating factor [^{1 2 3 4 5 6} ]. Both chemokines and the classical chemotactic factors function by binding and activating G-protein-coupled, seven-transmembrane receptors on leukocytes [^{4 5 6} ]. It is believed that mobilization of phagocytes in response to chemotactic factors constitutes the first line of host defense in inflammation and infection.

fMLF is one of the first "classical" chemoattractants studied. Initially it was tested as part of a series of synthetic peptides under evaluation for chemotactic activity, and later it was purified from Escherichia coli culture supernatant [² , ⁷ ]. In humans, two fMLF receptor genes have been cloned, which encode a high-affinity receptor formyl peptide receptor (FPR) [⁸ ] and a low-affinity receptor FPR-like 1 (FPRL1) [⁹ ]. In addition to the bacterium-derived fMLF, a number of synthetic peptide agonists have been identified for FPR [¹⁰ ]; however, the host-derived agonists for this receptor have not been defined. In contrast, an endogenously derived lipid metabolite lipoxin A4 (LXA4) has been reported to bind FPRL1 with high affinity [¹¹ ]. Furthermore, a proinflammatory acute-phase protein, serum amyloid A, has been shown to induce phagocyte migration and calcium mobilization through FPRL1 [^{12 13 14} ]. These results suggest that by interacting with host-derived agonists, FPRL1 may play important pathophysiological roles in addition to participating in host defense against bacterial invasion.

Recently, construction and screening of random peptide libraries have become a useful approach to developing biologically active agents with pharmaceutical potential. Klein et al. [¹⁵ ] isolated a number of small peptide sequences from a peptide library that could react with FPR and FPRL1. One of these, named MMK-1 (LESIFRSLLFRVM), appears to induce calcium mobilization with high efficacy in human cells transfected with FPRL1 but with very low efficacy on FPR-transfected cells. However, whether MMK-1 can activate native human phagocytic cells or acts as a chemotactic agonist for FPRL1 has not been reported. In this study we demonstrate that MMK-1 is a chemotactic peptide for both human neutrophils and monocytes and is one of the most potent and specific chemotactic agonists identified so far for FPRL1.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents
MMK-1 was synthesized and purified at the Department of Biochemistry, Colorado State University (Fort Collins), according to the published sequence [¹⁵ ]. The purity was >90%, and the amino acid composition was verified by mass spectrometry. The endotoxin levels in the dissolved peptide were undetectable. Synthetic fMLF was purchased from Sigma (St. Louis, MO).

Cells
Human peripheral blood mononuclear cells (PBMCs) were isolated from leukopacks obtained from the Transfusion Medicine Department, National Institutes of Health Clinical Center, Bethesda, MD. Monocytes were further purified by elutriation to yield >90% pure preparations. Human neutrophils were purified from the same leukopacks by dextran sedimentation with a purity of >98%. Rat basophilic leukemia cells stably transfected with epitope-tagged human FPR (ETFR) were a kind gift of H. Ali and R. Snyderman, Duke University (Durham, NC). Human FPRL1 cDNA and mouse FPR2 were cloned and stably transfected into human embryonic kidney (HEK) 293 cells as reported previously [¹⁴ , ¹⁶ ]. All transfected cells were maintained in Dulbecco’s modified eagle’s medium, 10% fetal calf serum, and 0.8 mg/mL of geneticin (G418; Gibco-BRL, Rockville, MD).

Chemotaxis assays
Migration of leukocytes and receptor-transfected cells was assessed using a 48-well microchemotaxis chamber technique as previously described [¹⁴ ]. Different concentrations of stimulants were placed in wells of the lower compartment of the chamber (Neuro Probe, Cabin John, MD). The cell suspension was seeded into wells of the upper compartment which was separated from the lower compartment by a polycarbonate filter (Osmonics, Livermore, CA; 5 µm-diameter pore size for leukocytes, 10-µm-diameter pore-size for ETFR and 293 cells). The filters for migration of receptor-transfected cells were precoated with 50 µg/mL of collagen type I (Collaborative Biomedical Products, Bedford, MA) to favor cell attachment. After incubation at 37°C (90 min for monocytes, 60 min for neutrophils, and 300 min for transfectants), the filters were removed and stained, and the numbers of cells migrating across the filters were counted by light microscopy after the samples were coded. Results are presented as the chemotaxis indexes (CIs) representing the fold increase in the number of migrating cells in response to stimuli over the spontaneous cell migration (in response to control medium).

Calcium mobilization
Calcium mobilization was assayed by incubating 10⁷ cells/mL in loading buffer containing 138 mM NaCl, 6 mM KCl, 1 mM CaCl₂, 10 mM HEPES (pH 7.4), 5 mM glucose, 0.1% bovine serum albumin, and 5 µM Fura-2 (Sigma) at 37°C for 30 min. The dye-loaded cells were washed and resuspended in fresh loading buffer. The cells were then transferred into quartz cuvettes (10⁶/2 mL) which were placed in a luminescence spectrometer LS50 B (Perkin-Elmer Ltd., Beaconsfield, England). Stimulants at different concentrations were added in a volume of 20 µL to the cuvettes at indicated time points. The ratio of fluorescence at 340- and 380-nm wavelengths was calculated using the FL WinLab program (Perkin-Elmer).

Binding assays
The radioiodinated synthetic peptide WKYMVm (¹²⁵I-labeled W peptide) was kindly provided by W. Yieh, NEN Lifesciences (Boston, MA). A single concentration of ¹²⁵I-labeled W peptide was added simultaneously with different concentrations of unlabeled W peptide, fMLF, or MMK-1 to a cell suspension (human monocytes and FPRL1/293 cells or ETFR cells, 1–2 x 10⁶ cells/200µL RPMI 1640 containing 1% bovine serum albumin and 0.05% NaN₃) in duplicate samples in Eppendorf tubes. The samples were incubated under constant rotation for 30 min at room temperature. After incubation, the samples were centrifuged through a 10% sucrose-PBS cushion, and the tips of the tubes containing cell pellets were counted for -ray emissions.

Cytokine production
Monocytes were preincubated with or without 50 ng/mL of pertussis toxin (PT) at 37°C for 4h, then were incubated with stimulants for another 24 h. Supernatants were collected, centrifuged, and measured for IL-1ß and IL-6 by enzyme-linked immunosorbent assay (R & S Systems, Minneapolis, MN).

Statistical analysis
Unless otherwise specified, all experiments were performed three to five times, and the results presented are from representative experiments. The significance of the difference between test and control groups was analyzed with a Student’s t test.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We first verified the capacity of synthetic MMK-1 to induce calcium mobilization in cell lines transfected with either FPR or FPRL1. As shown in Figure 1A , MMK-1 induced calcium mobilization in human FPRL1-transfected HEK 293 (FPRL1/293) cells with a 50% effective concentration (EC50) at 2 nM. This is in agreement with the reported potency of this peptide on FPRL1 [¹⁵ ]. As expected, MMK-1-induced calcium mobilization was attenuated by prior stimulation of the cells with a high concentration (10^-4 M) of fMLF and vice versa (Fig. 1B and 1C) . In addition, the MMK-1 signaling in FPRL1/293 cells was cross-desensitized by a previously defined FPRL1 peptide agonist, F peptide (F pep) [¹⁷ ] (Fig. 1D and 1E) . No calcium mobilization was elicited in FPR-expressing ETFR cells by MMK-1 over a wide concentration range (Fig. 1F) . However, ETFR cells exhibited robust calcium flux responses to fMLF (Fig. 1G) . These results confirmed that the peptide specifically activates FPRL1. Because it is unknown whether MMK-1 also activates human native phagocytic cells, which are known to express FPRL1 [¹⁸ ], we next tested the effect of this peptide on phagocyte functions. Figure 2 shows that MMK1 induced dose-dependent calcium mobilization in both human monocytes (Fig. 2A) and neutrophils (Fig. 2D) with EC50s in the low-nanomolar range. The MMK-1-induced Ca²⁺ flux in human phagocytic cells was also desensitized by pre-exposure of the cells to the defined FPRL1 agonist F pep (Fig. 2B and 2E) but not to the specific FPR agonist peptide T20 [¹⁹ ] (Fig. 2C and 2F) , suggesting that MMK-1 shares with F pep FPRL1 in primary phagocytic leukocytes.

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Figure 1. Calcium mobilization induced by MMK-1 in FPRL1/293 and ETFR cells. (A) Stimulation of FPRL1/293 cells by different concentrations of MMK-1. (B and C) Sequential stimulation of FPRL1/293 cells with fMLF and MMK-1 or vice versa. (D and E) Cross-desensitization of MMK-1 signaling in FPRL1/293 cells by F peptide (F pep). F and G: stimulation of ETFR cells by different concentrations of MMK-1 or fMLF.

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Figure 2. Calcium mobilization induced by MMK-1 in human phagocytes. (A) Signaling of MMK-1 in human monocytes. (B) Cross-desensitization of MMK-1 signaling in monocytes by F pep. (C) Sequential stimulation of monocytes with T20 and MMK-1, and vice versa. (D) Signaling of MMK-1 in human neutrophils. (E) Cross-desensitization of MMK-1 signaling in neutrophils by F pep. (F) Sequential stimulation of neutrophils by T20 and MMK-1, and vice versa.

Because leukocyte migration and accumulation are considered the first steps in host defense against invading pathogens, we investigated whether MMK-1 could induce cell migration through FPRL1. As shown in Figure 3 , MMK-1 induced considerable migration of both human monocytes (Fig. 3A) and neutrophils (Fig. 3B) starting at concentrations of 1 nM (EC50, 10 nM). The dose response of phagocyte migration to MMK-1 was bell shaped and was inhibited by PT but not herbimycin A (HA) (Fig. 3C) , suggesting the involvement of a G-protein-coupled chemotactic receptor. The phagocyte migration induced by MMK-1 was dependent on the concentration gradient of the peptide as examined by checkerboard analysis (Table 1 ), indicating that the effect of MMK-1 is chemotactic rather than chemokinetic. Furthermore, MMK-1 also induced significant migration of FPRL1/293 cells with a maximal response at 10 nM (Fig. 4A ). The parental HEK 293 cells (data not shown) and cells expressing FPR (Fig. 4B) showed no migration in response to a wide range of MMK-1 concentrations. These results suggested that FPRL1 is likely a receptor mediating the leukocyte chemotactic activity of MMK-1.

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Figure 3. Chemotactic activity of MMK-1 for human monocytes and neutrophils. (A and B) Fold increase of monocyte or neutrophil migration in response to MMK-1. *P < 0.05 compared with migration of cells in response to medium alone. (C) Effects of PT or HA on monocyte migration in response to MMK-1. Monocytes were preincubated with 100 ng/mL of PT at 37°C for 30 min or 200 µM HA at 37°C for 2 h, then were washed and examined for migration induced by MMK-1. fMLF at 10-7 M was used as control. *P < 0.05 compared with migration of cells incubated with medium alone.

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Table 1. Checkerboard Analysis of MMK-1 Induced Chemotaxis of Human Monocytes and FPRL1/293 Cellsa

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Figure 4. Chemotactic activity of MMK-1 for FPRL1/293 and ETFR cells. (A) Fold increase of FPRL1/293 cell migration in response to MMK-1 over control medium. (B) Lack of chemotactic activity of MMK-1 for FPR expressing ETFR cells. fMLF at 10-7 M was used as control. *P < 0.05 compared with spontaneous migration to control medium.

To characterize the binding capacity of MMK-1 to its receptor, we used a radioiodinated synthetic peptide—¹²⁵I-labeled W peptide. Our previous study indicated that W peptide activates both human FPR and FPRL1 but activates FPRL1 with higher efficacy [²⁰ ]. Human monocytes showed a high level of binding for ¹²⁵I-labeled W peptide, which was specifically displaced by unlabeled W peptide (Fig. 5A ). Unlabeled fMLF or MMK-1 alone only partially displaced W peptide binding to monocytes. However, the presence of a combination of unlabeled fMLF and MMK-1 resulted in a displacement of ¹²⁵I-labeled W peptide binding on monocytes at the level comparable with that of unlabeled W peptide. Consistent with this, as shown in Figure 5B and 5C , ¹²⁵I-labeled W peptide bound to both FPRL1- and FPR-transfected cells with high efficacy, and the binding was completely displaced by unlabeled W peptide. However, in FPRL1/293 cells, MMK-1 but not fMLF showed a complete inhibition of ¹²⁵I-labeled W peptide binding, and fMLF efficiently competed for ¹²⁵I-labeled W peptide binding only in cells expressing FPR. Thus, ligand-binding results provided additional evidence to support the notion that MMK-1 uses FPRL1 in human cells.

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Figure 5. Displacement of 125I-labeled W peptide binding to FPRL1 by MMK-1. 125I-labeled W peptide was incubated for 30 min at room temperature with human monocytes (A), FPRL1/293 cells (B), or FPR-expressing ETFR cells (C) in the presence of unlabeled W peptide, fMLF, or MMK-1. The cells were then centrifuged through a sucrose-PBS cushion and measured for radioactivity with a -ray counter.

We then investigated whether MMK-1 might also serve as a chemotactic and activating agonist for the murine (m) counterpart of human FPRL1. Several genes encoding putative mfMLF receptors have been identified and cloned. mFPR1 is most homologous to human FPR, whereas mFPR2 is most similar to human FPRL1 [⁵ , ²¹ ]. We therefore tested the effect of MMK-1 on HEK 293 cells transfected to express mFPR1 and mFPR2. As shown in Figure 6 , MMK-1 induced marked calcium mobilization in mFPR2-transfected cells (EC50 =1 nM) (Fig. 6A) . The calcium mobilization induced by MMK-1 in mFPR2/293 cells was attenuated by a high concentration of fMLF (Fig. 6B and 6C) , as well as by the human FPRL1-specific agonist F pep (Fig. 6D and 6E) . No calcium mobilization was induced by MMK-1 in mFPR1-transfected cells at any concentration tested (Fig. 6F) , whereas fMLF induced a dose-dependent Ca²⁺ flux in these cells (Fig. 6G) . In addition, MMK-1 induced a potent chemotactic response of mFPR2-transfected cells at low concentrations with an optimal cell response to 10 nM peptide (Fig. 7 ). MMK-1 did not induce migration of the HEK 293 cells expressing mFPR1. Thus MMK-1 specifically activated mFPR2, the murine homologue of human FPRL1.

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Figure 6. Calcium mobilization induced by MMK-1 in mFPR2/293 cells. (A) Ca2+ flux induced by different concentrations of MMK-1in mFPR2/293 cells. (B and C) Sequential stimulation of mFPR2/293 cells with fMLF and MMK-1 or vice versa. (D and E) Cross-desensitization of MMK-1 signaling in mFPR2/293 cells by F pep. (F and G) Response of mFPR1/293 cells to MMK-1 or fMLF.

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Figure 7. Chemotactic activity of MMK-1 for mFPR2/293 and mFPR1/293 cells. (A) Migration of mFPR2/293 cells in response to MMK-1. (B) Lack of chemotactic activity of MMK-1 for mFPR1/293 cells. fMLF at 10-7 M was used as control. *P < 0.05 compared with spontaneous migration.

The biologic activity of MMK-1 was further tested for its ability to modulate the production of proinflammatory cytokines in human monocytes. Monocytes stimulated with MMK-1 showed a significant increase in production of interleukin (IL)-1ß and IL-6, and the effect of the peptide was inhibited when the cells were pretreated with PT. Another known FPRL1 agonist, F pep [¹⁷ ], also enhanced production of IL-1ß and IL-6 in monocytes, and the effect of F pep was abolished by PT (Table 1) . In contrast, the bacterial lipopolysaccharide (LPS)-induced production of cytokines was resistant to PT. These results indicated that MMK-1 can modulate the host defense and immunological response through the G-protein-coupled receptor FPRL1.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We show that the synthetic peptide MMK-1, derived from a random peptide library, was a chemotactic agonist for phagocytic leukocytes by specifically acting on FPRL1. FPRL1 is cloned by its structure homology to FPR and binds fMLF with low affinity [⁵ , ⁹ , ¹⁰ ]. This receptor has 69% identity at the amino acid level to its prototype FPR [⁵ , ¹⁰ ]. However, in contrast to FPR, FPRL1 mediates calcium flux in response to fMLF only when it is stimulated by high concentrations [¹⁴ , ¹⁶ ], suggesting that high-affinity agonist(s) for FPRL1 differ from those of FPR. This hypothesis is supported by recent identification of various agonists that preferentially bind and activate FPRL1. A peptide domain T21/DP107 derived from HIV-1 envelope protein gp41 can stimulate calcium flux and chemotaxis through both FPR and FPRL1 but has high selectivity for FPRL1 [¹⁶ ]. Two other HIV-1-derived peptides, F pep and V3 peptide, specifically activate only FPRL1 [¹⁷ , ²² ]. In addition, a lipid metabolite, LXA4, has been reported to bind FPRL1 with high affinity and to stimulate GTPase activity through this receptor [¹¹ ]. However, unlike peptide chemotactic agonists, LXA4 is reported to induce an anti-inflammatory signaling cascade that inhibits neutrophil transmigration of epithelial monolayers [²³ ]. In contrast to LXA4, an acute-phase protein serum amyloid A which markedly increases its serum concentration during acute-phase responses and causes tissue or organ amyloidosis in chronic inflammation [²⁴ ] induces chemotaxis of phagocytic leukocytes through FPRL1 [^{12 13 14} ]. Therefore, it is possible that FPRL1 transduces, respectively, both anti-inflammatory and proinflammatory signals in response to lipid agonists versus protein or peptide agonists [¹¹ , ²⁵ ]. In this context, it has recently been suggested that LXA4 and peptide agonists might use divergent domains on FPRL1, and glycosylation of the receptor could be essential for peptide but not for LXA4 signaling. Although FPRL1 was initially identified in phagocytic leukocytes, recent studies have detected the expression of this receptor in a great variety of cell types, including cells of nonhematopoietic origin [²⁶ ]. Identification of specific host-derived agonists for FPRL1 suggests an important role of this receptor in inflammatory responses.

Construction and screening of random peptide libraries have become important means of identifying biologically active sequences. Hexapeptide sequence WKYMVm (W peptide) isolated from such a library is reported to stimulate the activation of phospholipase D [²⁷ ], phosphoinositide hydrolysis, and Ca²⁺ mobilization in neutrophils and B lymphocytes [²⁸ , ²⁹ ]. We recently identified both FPRL1 and FPR as functional receptors for W peptide [²⁰ ]. MMK-1 was also derived from a random peptide library and was identified by a novel autocrine selection method in yeasts engineered to express human FPRL1 [¹⁵ ]. Several peptides have been found to induce calcium mobilization via interaction with either or both FPRL1 and FPR. MMK-1 was described as a preferential inducer of Ca²⁺ flux through FPRL1 [¹⁵ ]. In our study, we identified this peptide as a highly specific chemotactic factor for FPRL1-transfected HEK 293 cells. In addition, MMK-1 potently activated phagocytic leukocytes and could enhance PT-sensitive production by human monocytes of proinflammatory cytokines IL-1ß and IL-6, which play an important role in the host innate defense and immunological responses [³⁰ ]. Although formal proof could be obtained only by neutralizing anti-FPRL1 antibodies or specific antagonists, which are not available at the present time, the use of FPRL1 by MMK-1 to activate human phagocytes was nevertheless suggested by the observation that MMK-1 signaling in these cells was completely desensitized by well-defined FPRL1 agonist F pep [¹⁷ ]. In addition, in binding studies with ¹²⁵I-labeled W peptide, which is known to activate both FPR and FPRL1, MMK-1 effectively competed with W peptide binding to FPRL1- but not FPR-transfected cells. Furthermore, we demonstrated a highly efficacious and preferential activation of mFPRL2 by MMK-1. In fact, among various chemotactic agonists specific for FPRL1, MMK-1 is one of the most potent [¹⁴ , ¹⁶ , ^{20 21 22} ] and activates the receptor at picomolar and low-nanomolar concentrations. This is supported also by the observation that at equal concentrations, MMK-1 is more effective in stimulating monocyte release of the proinflammatory cytokines IL-1ß and IL-6 (Table 2 ). Thus, in view of the potential importance of FPRL1 in inflammatory and immunological responses, MMK-1 can be a very useful molecule to study the signaling and function of this receptor.

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Table 2. Enhancement of Cytokine Production in Human Monocytes by MMK-1a

 
This project was funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract no. NO1-CO-56000. The authors thank Dr. R. Snyderman, Duke University, Durham, NC, for providing ETFR cells, Dr. J. J. Oppenheim for reviewing the manuscript, and C. Fogle and C. Holan for secretarial assistance. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. The publisher or recipient acknowledges the right of the U.S. government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

Received October 16, 2000; revised January 9, 2001; accepted February 26, 2001.

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