Temporin A and Related Frog Antimicrobial Peptides Use Formyl Peptide Receptor-Like 1 as a Receptor to Chemoattract Phagocytes

Reagents

RPMI 1640 and DMEM were purchased from BioWhittaker (Walkersville, MD). FBS was purchased from HyClone Laboratories (Logan, UT). Recombinant human (rh)GM-CSF, rhIL-4, and rhM-CSF were obtained from PeproTech (Rocky Hill, NJ). Pertussis toxin (PTX) was from Sigma-Aldrich (St. Louis, MO). Amyloid β_1–42 (Aβ) peptide was purchased from American Peptide Company (Vista, CA). [¹²⁵I]-Aβ was from Amersham Biosciences (Piscataway, NJ).

MMK-1, an FPRL1-specific ligand of 13 amino acids (LESIFRSLLFRVM) (27), and W peptide, a chemotactic hexapeptide (WKYMVm, m represents a d-Met residue) originally identified from a combinatorial peptide library (28) and later found to be an agonistic ligand for human formyl peptide receptor (29), FPRL1 (29), and FPRL2 (30, 31) were synthesized and purified by the Department of Biochemistry, Colorado State University (Fort Collins, CO). The purity for both peptides was >90%, and the amino acid composition was verified by mass spectrometry. TA, an frog-derived antimicrobial peptide amide of 13 amino acids (FLPLIGRVLSGIL), d-TA (TA composed of d- instead of l-amino acids), Rev-TA (TA with reversed sequence, LIGSLVRGILPLF), Rana-6 (ranateurin 6 from the North American bullfrog R. catesbeiana, FISAIASMLGKFL), T1P (temporin 1P from the North American leopard frog R. pipiens, FLPIVGKLLSGLL), and I4G10-C (FLPIIASLLGKLL) and I4S10-C (FLPIIASLLSKLL), two position 10 isomers of the original consensus sequences derived for 30 temporin-like peptides from frog skin secretions (21), were synthesized as carboxyl-terminal amides (−CONH₂) by solid-phase techniques using 9-fluorenylmethoxycarbonyl chemistry as previously described (22, 24, 25). The peptides were purified (>99%) by reverse-phase HPLC and characterized by amino acid analysis and electrospray ionization mass spectrometry.

Cell preparation and maintenance

Human PBMC were isolated from leukopacks of normal blood donors (courtesy of the Transfusion Medicine Department, Clinic Center, National Institutes of Health, Bethesda, MD) by routine Ficoll-Hypaque density gradient centrifugation. Monocytes were purified from PBMC using of a MACS CD14 monocyte isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s recommendation. Neutrophils (purity ≥97%) were isolated from the same leukopacks by 3% dextran sedimentation as previously described (8). The in vitro generation of monocyte-derived macrophages and immature dendritic cells (DCs) was conducted as previously described (32, 33). Briefly, monocytes were cultured at 1 × 10⁶/ml in complete RPMI 1640 medium (RPMI 1640 plus 10% FBS, 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin) in the presence of either rhM-CSF or a combination of rhGM-CSF and rhIL-4 at 37°C in a humidified CO₂ (5%) incubator for 7 days for the generation of macrophages or immature DCs.

Human embryonic kidney (HEK) 293 cells stably transfected with human FPRL1 receptor (hereafter designated FPRL1/HEK293 cells) or mFPR2 (hereafter designated mFPR2/HEK293 cells) were obtained from Dr. J. M. Wang (National Cancer Institute, Frederick, MD), and were originally generous gifts of Drs. P. M. Murphy and J.-L. Gao (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). The parental HEK 293 cells were maintained in complete DMEM (DMEM containing 10% FBS, 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin). Both FPRL1/HEK293 and mFPR2/HEK293 cells were maintained in complete DMEM in the presence of 1 mg/ml G418 (Invitrogen Life Technologies, Rockville, MD).

Chemotaxis assay

Migration of monocytes, neutrophils, DCs, macrophages, and HEK 293, FPRL1/HEK293, and mFPR2/HEK293 cells in response to chemotactic factors was evaluated using a 48-well microchemotaxis chamber (NeuroProbe, Gaithersburg, MD) as previously described (8, 32, 33). Briefly, different concentrations of chemotactic factors diluted in chemotaxis medium (RPMI 1640 containing 1% BSA) were placed in the lower wells of the chamber, and the cells suspended in chemotaxis medium were put in the upper wells. In some experiments, monocytes were pretreated for 30 min at 37°C in the absence or presence of PTX (final concentration = 200 ng/ml) before usage. For checkerboard analysis, TA was simultaneously added into the upper compartments. The lower compartment was separated from the upper compartment by a polycarbonate membrane (NeuroProbe) of different pore size based on the target cells. For neutrophils, monocytes, DCs, and macrophages, 5-μm uncoated membranes were used. For evaluating the migration of HEK 293, FPRL1/HEK293, and mFPR2/HEK293 cells, 10-μm polycarbonate membranes were coated at 37°C for 2 h with type 1 rat tail collagen (BD Biosciences, Bedford, MA) at the final concentration of 50 μg/ml, air-dried, and used. After incubation at 37°C in humidified air with 5% CO₂ for a period of time (60 min for neutrophils, 90 min for monocytes, DCs, and macrophages, 300 min for HEK 293, FPRL1/HEK293 and mFPR2/HEK293 cells), the membranes were removed, scraped and stained, and the cells migrating across the membrane were determined using a BioQuant semiautomatic counting system (Nashville, TN). The results are presented as the number of cells per high power field (HPF).

Measurement of Ca²⁺ flux

Monocytes were loaded by incubating with 5 μM fura 2 (Molecular Probes, Eugene, OR) at 24°C for 30 min in the dark. Subsequently, the cells were washed with and resuspended in 10⁶/ml in saline buffer (138 mM NaCl, 6 mM KCl, 1 mM CaCl₂, 10 mM HEPES, 5 mM glucose, and 1% BSA, pH 7.4). Each 2 ml of loaded monocytes were transferred into a quartz cuvette, which was placed in a luminescence spectrometer LS50 B (PerkinElmer, Wellesley, MA). Ca²⁺ flux of the cells in response to chemotactic factors was measured by recording the ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm.

Detection of MAPK activation

Human monocytes were washed three times with PBS and suspended in serum-free RPMI 1640. Monocytes (2 × 10⁶/sample) were incubated at 37°C in the absence or presence of indicated concentrations of TA, d-TA, or MMK-1 (as a positive control) for a period of time as specified. At the end of incubation, a large amount of ice-cold PBS (10-fold) was added to stop the stimulation, and the monocytes were spun down at 1500 × g for 5 min at 4°C. After complete removal of the supernatant, the monocytes were lysed by adding 50 μl of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8 at 25°C, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% bromphenol blue). The lysate was sonicated for 10 s on ice to shear DNA and boiled for 10 min afterward. The lysates were loaded (20 μl/lane) and separated on a 4–12% NuPAGE Bis-Tris Gel (Invitrogen, Carlsbad, CA) using NuPAGE MES SDS Running buffer (Invitrogen) as the electrode buffer. SeeBlue Plus2 (Invitrogen) was used as the molecular size marker. After the electrophoresis, proteins in the gel were electrotransferred (30 V constant for 1 h) onto a piece of Immobilon membrane (Millipore, Bedford, MA) using NuPAGE transfer buffer (12 mM Tris, 96 mM glycine, pH 8.3, 0.1% v/v antioxidant, 10% v/v methanol). Phosphorylated MAPK was detected by Western blotting as previously described (34). Briefly, the membrane was sequentially washed, blocked for 1 h at room temperature, washed, and incubated at 4°C overnight in the presence of 1/1000 dilution of rabbit anti-phospho-p44/42 (catalogue no. 9101; Cell Signaling Technology, Beverly, MA). On the next day, the membrane was washed and incubated with 1/2000 dilution of HRP-conjugated anti-rabbit IgG (catalogue no. 7074; Cell Signaling Technology) for 1 h. The membrane was washed and incubated with 10 ml working solution of ECL Plus Western Blotting Detection System (Amersham Biosciences) for 5 min at room temperature, and exposed to a piece of BioMax x-ray film (Kodak, Rochester, NY) for 5 s. The x-ray film was developed using an automatic processor (Kodak X-OMAT 200A). The same membrane was stripped and probed for p44/42 protein essentially in the same manner except using rabbit anti-p44/42 (catalogue no. 9102; Cell Signaling Technology) as the primary Ab.

Binding assay

Competitive binding was performed in triplicate by adding constant amount of iodinated Aβ, [¹²⁵I]-Aβ, and increasing amounts of Aβ or TA to individual 1.5-ml microfuge tubes, each containing 2 × 10⁶ FPRL1/HEK293 transfectant cells suspended in chemotaxis medium. After incubation at room temperature with constant mixing for 1 h, the mixture was centrifuged through a 10% sucrose/PBS cushion and the cell-associated radioactivity was measured with a 1227 Wallac gamma counter. The percentage of binding was calculated by the formula: Percentage of binding = [(cpm in the presence of unlabeled ligand)/(cpm in the absence of unlabeled ligand) × 100].

Air pouch experiments

Six to eight-wk-old BALB/c mice were obtained from the Animal Production Facility, National Cancer Institute (Frederick, MD) and maintained under specific pathogen-free conditions in the Experimental Animal Facility at the National Cancer Institute for several weeks to allow acclimation before the experiments. The animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals. Air pouches were raised on the dorsum as previously described (34). Mice with a well-formed air pouch were randomized into two groups. Each air pouch was injected with 1 ml of endotoxin-free PBS alone or PBS containing 1 μM TA. Four hours later, the mice were killed by CO₂ asphyxiation, and cells in the air pouches were washed out and enumerated following trypan blue staining. Differential counting of leukocyte subpopulations migrating into the pouch space was performed after eosin-methylene blue staining of cytospins.

TA is chemotactic for human monocytes and neutrophils

Of the temporins and temporin-like amphibian linear antimicrobial peptides identified so far, TA is one of the very first three temporins identified (18) and has been extensively investigated in terms of antimicrobial activity and the relationship between its structure and antimicrobial activity (21, 22, 23, 24, 26, 35). To investigate whether TA has the capacity to induce chemotaxis, the migration of human peripheral blood monocytes in response to TA was examined using the 48-well microchemotaxis chamber. As shown in Fig. 1⇓A, TA induced monocyte migration with a bell-shaped dose-response curve. The peak response was observed at 250 nM. In the same experiment, two synthetic analogues of TA, d-TA, TA composed of all d-amino acid residues, and Rev-TA, TA with reversed sequence, did not induce the migration of monocytes over the range of concentrations tested, suggesting that TA-induced monocyte migration was based on a chiral interaction. To determine whether TA-induced migration of monocytes was due to chemotaxis or chemokinesis, checkerboard analysis was performed (Table I⇓). Increasing concentrations of TA added simultaneously with the cells to the upper wells of the chamber abrogated monocyte migration induced by TA in the lower wells, indicating that TA-induced migration of monocytes was based on chemotaxis rather than chemokinesis.

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FIGURE 1.

TA-induced migration of human peripheral blood monocytes (A) and neutrophils (B). The migration of monocytes and neutrophils in response to TA, d-TA, and Rev-TA was evaluated by chemotaxis assay. The results are shown as the average cell migration (mean ± SD) for triplicate wells. Similar results were obtained from three separate experiments.

To investigate whether phagocytes other than monocytes could also respond to TA, we tested whether neutrophils could be chemoattracted by TA. Similar to monocytes, neutrophils migrated in response to TA with a bell-shaped dose-response curve (Fig. 1⇑B), indicating that TA is also an agonistic chemoattractant for human neutrophils.

TA uses a G protein (G_iα)-coupled receptor

To find out whether TA-induced phagocyte chemotaxis was mediated by a G_iα-coupled receptor, we next examined whether TA-induced monocyte chemotaxis could be inhibited by PTX, a toxin that specifically inhibits G_iα-coupled receptor signaling by ADP-ribosylating G_iα protein (36). Incubation of monocytes at 37°C for 60 min in the presence of PTX (200 ng/ml) before chemotaxis assay completely inhibited the migration of monocytes in response to TA (Table II⇓). Pretreatment with PTX did not affect the absolute motility of monocytes because there was no difference in spontaneous migration (in response to chemotaxis medium) between PTX-pretreated or control monocytes. Furthermore, the inhibition was not due to the preincubation time per se because control incubation of monocytes in the absence of PTX did not inhibit migration in response to TA. As expected, fMLP-induced monocyte migration was also greatly inhibited by PTX. Therefore, TA-induced monocyte chemotaxis seems to be mediated by a G_iα-coupled receptor.

Besides chemotaxis, activation of a G_iα-coupled receptor by its chemotactic agonistic ligands often results in activation of MAPK activation in target cells (37, 38). Therefore, we investigated whether TA could also induce the activation of p44/42 MAPK in monocytes (Fig. 2⇓). PBS-treated monocytes displayed basal level of phospho-p42 with very little phospho-p44 (Fig. 2⇓, lane 1, top). Treatment of monocytes with MMK-1 at 10⁻⁷ M for 5 min (as a positive control) resulted in a marked increase of both phospho-p42 and phospho-p44 as expected (Fig. 2⇓, lane 9, top). Treatment of monocytes with TA for 5 min enhanced the levels of both phospho-p42 and phospho-p44 MAPK in a dose-dependent manner (Fig. 2⇓, lanes 2–4, top). Phosphorylation of p44/42 MAPK in monocytes in response to 1 μM of TA begun to increase upon TA treatment for as short as 1 min (Fig. 2⇓, lanes 5–7, upper panel). Similar to the inability of d-TA to induce monocyte chemotaxis (Fig. 1⇑A), d-TA did not activate p44/42 MAPK (Fig. 2⇓, lane 8, top). Probing the same membrane with anti-p44/42 Ab after stripping revealed identical amounts of p44/42 MAPK on each lane (Fig. 2⇓, bottom), indicating both equal loading and no significant up-regulation of p44/42 MAPK proteins in response to TA. These results provided additional evidence for the notion that TA acts on monocytes through a G_iα-coupled receptor.

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FIGURE 2.

TA activation of p44/42 MAPK in monocytes. Freshly isolated human peripheral monocytes received no treatment or were treated with indicated concentrations of TA, d-TA, and MMK-1 for a period of time as specified before lysis. The cell lysates were separated on a 4–12% Bis-Tris Gel, transferred to polyvinylidene difluoride membrane, and the phosphorylated p44/42 MAPKs were detected by Western blotting with anti-phospho-p44/42 (top). The same membrane was stripped and blotted with anti-p44/42 (bottom). The exposure time for the autoradiographs was 30 s. Shown are the results of one experiment representative of three.

FPRL1 functions as a receptor for TA

As the first step to identify the receptor for TA, we examined whether differentiation of monocytes to either DCs or macrophages (Mφ) would affect their responsiveness to TA. Monocytes purified from the peripheral blood of the same healthy donor were cultured in the presence of M-CSF or a combination of GM-CSF and IL-4 for 7 days for the generation of monocyte-derived Mφ or monocyte-derived immature DCs as previously described (33). Monocyte-derived Mφ migrated chemotactically in response to TA (Fig. 3⇓, upper panel), whereas DCs differentiated from the same starting population of monocytes failed to do so (Fig. 3⇓, lower panel). The failure of monocyte-derived immature DCs to migrate in response to TA was not due to the lack of motility because those immature DCs migrated in response to another chemoattractant, W peptide (Fig. 3⇓, lower panel). Therefore, the G_iα-coupled receptor used by TA must be a receptor that is expressed by monocytes, neutrophils, and monocyte-derived Mφ, but its expression must be down-regulated when monocytes are differentiated to immature DCs in vitro.

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FIGURE 3.

TA induction of chemotaxis of monocyte-derived macrophages. The chemotaxis of macrophages (Mφ) and immature dendritic cells (iDCs) differentiated from monocytes of the same blood donor in response to TA was evaluated by chemotaxis assay. The results are shown as the average cell migration (mean ± SD) of triplicate wells. Similar results were obtained from two separate experiments.

Analysis of the expression patterns of all known G_iα-coupled receptors based on published reports revealed one possible candidate, FPRL1, which is expressed by monocytes (29, 33, 39, 40), neutrophils (29, 39, 40), and monocyte-derived Mφ (33), but down-regulated on immature DCs differentiated from monocytes (33). To test whether TA indeed acts on FPRL1 in monocytes, we investigated whether TA could induce Ca²⁺ flux in monocytes, and if so, whether its capacity to induce Ca²⁺ flux in monocytes could be homologously desensitized by an FPRL1-specific agonistic ligand, MMK-1 (27). As expected, both MMK-1 and TA induced a dose-dependent Ca²⁺ flux in monocytes (Fig. 4⇓, A and B). In complete agreement with the results showing that d-TA and Rev-TA could not induce chemotaxis of monocytes, neither d-TA nor r-TA could induce Ca²⁺ flux in monocytes (Fig. 4⇓, C and D). Interestingly, the Ca²⁺ flux induced by TA at 1 μM was completely desensitized by pretreatment of monocytes with 100 nM MMK-1 (Fig. 4⇓E). Conversely, TA at 3 μM also completely desensitized the Ca²⁺ flux in response to 5 nM MMK-1 (Fig. 4⇓F). Because homologous cross-desensitization of Ca²⁺ flux is often due to two agonistic ligands acting on the same receptor, these data indicate that TA uses FPRL1 as a functional receptor.

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FIGURE 4.

TA stimulation of Ca²⁺ flux in monocytes and cross-desensitization. Ca²⁺ flux of fura 2-loaded human monocytes in response to MMK-1 (A), TA (B), d-TA (C), and Rev-TA (D) was measured by recording the ratio of emission at λ₅₁₀ after simultaneous excitation at λ₃₄₀ and λ₃₈₀. Cross-desensitization of Ca²⁺ flux was determined by sequential adding MMK-1 and TA at indicated concentrations (E) or vice versa (F).

To directly test whether TA uses FPRL1 as a receptor, we examined whether TA could chemoattract HEK 293 cells stably transfected to express FPRL1. Indeed, TA dose-dependently induced the migration of FPRL1/HEK293 cells, but not the parental HEK 293 cells lacking FPRL1 expression (Fig. 5⇓A) or HEK 293 stably transfected to express several other G_iα-coupled receptors (data not shown). To confirm whether TA binds to FPRL1, we investigated whether TA could inhibit the binding of iodinated Aβ, a selective agonistic ligand for FPRL1 (41), to FPRL1/HEK293 cells (Fig. 5⇓B). As expected, unlabeled Aβ dose-dependently inhibited the binding of iodinated Aβ to FPRL1/HEK293 cells. TA, although less potent than Aβ, also displaced the binding of iodinated Aβ to FPRL1/HEK293 cells in a dose-dependent manner with an EC₅₀ of ∼2 μM. These data collectively confirm that TA uses FPRL1 as a receptor.

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FIGURE 5.

TA usage of FPRL1. A, TA induction of FPRL1/HEK293 cell chemotaxis is shown. The migration of FPRL1/HEK293 and parental HEK 293 cells in response to TA was examined and the average cell migration (mean ± SD) of triplicate well is shown. B, TA inhibition of [¹²⁵I]-Aβ binding to FPRL1/HEK293 cells. The binding of [¹²⁵I]-Aβ in the presence of increasing concentration of Aβ (•) or TA (▴) was determined using binding assay and shown as the average percentage binding (mean ± SD) of triplicate tubes. The error bar is not evident if smaller than the symbol.

TA acts as a phagocyte chemoattractant in vivo

To investigate whether TA could chemoattract phagocytes in vivo in mice, we first examined whether mFPR2, the mouse ortholog for human FPRL1 (39, 41, 42, 43, 44), could host TA as an agonistic ligand. As shown by Fig. 6⇓A, TA dose-dependently induced the migration of mFPR2/HEK293 cells, indicating that TA can function as an agonistic ligand for mFPR2. Accordingly, we reasoned that TA should also be active in mice because mouse leukocytes express mFPR2 mRNA (42), and mouse phagocytes can be activated by agonistic ligands for FPRL1 (43, 44, 45). Therefore, the potential in vivo chemoattracting activity of TA was studied by the use of mouse air pouch model. One milliliter of PBS or TA diluted in PBS at 1 μM was injected into each air pouch on the back of mice and the cells recruited into the pouches were lavaged out and evaluated 4 h after the injection. As shown by Fig. 6⇓B, TA markedly increased the number of leukocytes in the air pouches when compared with PBS control group, indicating that TA functions as a leukocyte chemoattractant in vivo. Based on the microscopic morphologies and differential counting, ∼95% of the leukocytes recruited by TA into the air pouches were phagocytes, including neutrophils (73%) and monocytes (22%), which was in agreement with its in vitro chemotactic activities for both neutrophils and monocytes and macrophages (Fig. 7⇓C). Thus, TA is chemotactic for phagocytes both in vitro and in vivo and uses FPRL1 or its ortholog (mFPR2 in mice) as its receptor.

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FIGURE 6.

In vivo recruitment of mouse phagocytes by TA. A, TA induced chemotaxis of mFPR2/HEK293 cells. B, One milliliter of PBS or TA diluted in PBS at 1 μM was injected into the air pouch of mice (n = 4) and leukocytes recruited into the pouch were washed out, enumerated, and shown as the average cell number (mean ± SD) per pouch. ∗, p < 0.001 by Mann-Whitney U test. C, The percentage of neutrophils, monocytes, and lymphocytes recruited by TA into the air pouch was determined by differential counting after staining.

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FIGURE 7.

Induction of chemotaxis of human phagocytes and FPRL1/HEK293 cells by temporin and its analogues. A, Migration of human monocytes (left) or neutrophils (right) in response to temporins or related AMPs at concentrations as specified. B, Chemotaxis of FPRL1/HEK293 cells in response to TA and six related AMPs at indicated concentrations. Chemotaxis assay was performed as described in Materials and Methods. The results shown were the average cell migration (mean ± SD) of triplicate wells. MMK-1, a selective ligand for FPRL1, was used for a positive control.

Chemoattraction of human phagocytes by other temporins or synthetic analogues and receptor use

To address whether the phagocyte-attracting activity was unique for TA, we investigated whether two additional temporins, Rana-6 and T1P, and two consensus synthetic analogues deduced from all temporins identified so far (21) could also induce the in vitro migration of human phagocytes. Of the four AMPs tested, Rana-6, I4S10-C, and I4G10-C induced the migration of human monocytes and neutrophils with an optimal dose from 0.5–5 μM (Fig. 7⇑A). T1P chemoattracted neutrophils, but not monocytes (Fig. 7⇑A). To investigate whether these AMPs also use FPRL1, we examined whether they could induce the migration of FPRL1/HEK293 cells using MMK-1, TA, d-TA, and Rev-TA for positive and negative controls (Fig. 7⇑B). Similar to TA, I4S10-C, I4G10-C, and Rana-6 induced the migration of FPRL1/HEK293 cells, suggesting that these AMPs also use FPRL1 as the chemotactic receptor. d-TA and Rev-TA, as negative controls did not induce the migration of FPRL1/HEK293 cells. T1P, although chemotactic for neutrophils (Fig. 7⇑A), did not induce the migration of FPRL1/HEK293 cells, suggesting that it uses a receptor other than FPRL1 on neutrophils.