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Activation of human monocytes by a formyl peptide receptor 2-derived pepducin
FEBS Letters 584 (2010) 4102–4108
journal homepage: www.FEBSLetters.org
Activation of human monocytes by a formyl peptide receptor 2-derived pepducin Ha Young Lee a,b, Sang Doo Kim a,b, Jae Woong Shim a,b, Hak Jung Kim a,b, Jae Young Kwon a, Jong-Min Kim b, Suk-Hwan Baek c, Joon Seong Park d, Yoe-Sik Bae a,b,⇑ a
Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714, Republic of Korea c Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu 705-717, Republic of Korea d Department of Hematology-Oncology, Ajou University School of Medicine, Suwon 443-721, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 14 June 2010 Revised 25 August 2010 Accepted 25 August 2010 Available online 9 September 2010 Edited by Ned Mantei Keywords: FPR2 Pepducin Monocytes Chemotaxis Pertussis toxin-sensitive G-protein Cytokine
a b s t r a c t We synthesized and investigated the effect of formyl peptide receptor 2 (FPR2)-derived pepducins in human monocytes. The FPR2-based cell-penetrating lipopeptide, ‘‘pepducin” (F2pal-16), stimulated intracellular calcium increase in human monocytes via pertussis toxin (PTX)-sensitive G-protein and phospholipase C (PLC) activity. From a functional aspect, we showed that F2pal-16 stimulated monocyte chemotaxis. F2pal-16 also stimulated the generation of superoxide anion in human monocytes. Moreover, F2pal-16 dramatically increased the production of several kinds of pro-inflammatory cytokines (CXCL8, CCL2, IL-1b and TNF-a) in human monocytes via NF-jB activation. Since FPR2 plays an important role in immune responses, F2pal-16 can serve as a useful reagent for the study of FPR2-mediated immune modulation. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Formyl peptide receptor 2 (FPR2) is a chemoattractant receptor which regulates trafficking of leukocytic cells, such as neutrophils, monocytes, macrophages, and natural killer cells [1–3]. FPR2 has an important immunologic function, including host defense against pathogenic infections by stimulating the generation of reactive oxygen species in human monocytes as well as neutrophils [1,2]. In human monocytes, FPR2 is also involved in the production of some important cytokines, such as CCL2, IL-10, and TNF-a [4,5]. In human natural killer cells, activation of FPR2 stimulates IFN-c production and cytolytic activity, which suggests that FPR2 can be regarded as an important target receptor for cancer therapy [3]. Since human synovial fibroblasts express functional FPR2, past studies have suggested that FPR2 may be involved in the pathogenesis of arthritis [6]. Because FPR2 is expressed in several important leukocytes and inflammatory cells, and the receptor Abbreviations: FPR2, formyl peptide receptor 2; SAA, serum amyloid A; GPCR, Gprotein coupled receptor; PTX, pertussis toxin; MAPKs, mitogen activated protein kinases; [Ca2+]i, intracellular calcium concentration; HPF, high-power fields; WRW4, WRWWWW; PLC, phospholipase C ⇑ Corresponding author at: Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea. Fax: +82 31 290 7015. E-mail addresses: [email protected], [email protected] (Y.-S. Bae).
is regarded as a possible important target for the treatment of inflammatory diseases, the development of various agents that modulate FPR2 activity is required. In terms of ligands for FPR2, many agonists have been identified, including host-derived agonists (LL-37, lipoxin A4, and serum amyloid A [SAA]), and synthetic peptides (WKYMVm and MMK-1) [7– 11]. Cellular responses induced by these FPR agonists have been examined in some leukocytic cells, such as neutrophils and monocytes [1,2,7,9]. The activation of FPR2 by one of the strong synthetic agonists, WKYMVm, induces the activation of NADPH oxidase, resulting in superoxide anion production [12,13]. However, the stimulation of human neutrophils or monocytes with WKYMVm does not induce cytokine production [4]. Another important FPR2 agonist, SAA, stimulates the production of some cytokines, such as TNF-a, IL-10, and CCL2 [4,5]. However, SAA fails to stimulate superoxide production from human monocytes or human neutrophils [13]. We have demonstrated that the activation of FPR2 by different ligands induces differential intracellular signaling and cellular responses, showing a ligand-selective process [4,13]. Although various ligands for FPR2 have been reported, many ligands have failed to stimulate a broad range of activation in phagocytic cells. The G-protein coupled receptors (GPCR), including FPR2, have been shown to induce intracellular signaling via the activation of heterotrimeric G-protein(s) [1,2]. Subsequent mutagenesis studies
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.08.036
H.Y. Lee et al. / FEBS Letters 584 (2010) 4102–4108
have demonstrated that some intracellular loop regions of GPCR, such as the intracellular loop 3 region, are involved in the activation of GPCR [14]. Based on this background, Kuliopulos and coworkers developed several membrane-tethering cell permeable peptides corresponding to the intracellular loop region of PAR as agonists or antagonist for PAR [15,16]. Pepducin is a membranetethering cell permeable peptide [15]. It has been demonstrated that pepducins derived from PAR1 or CXCR1 act as agonists or antagonists for their specific receptors [15–17]. Even though synthesis of pepducin from GPCR is a novel approach by which to develop new ligands for the receptor, nothing is known about FPR2-derived pepducins. In this study, we synthesized a new FPR2-derived pepducin and tested its effect on human monocyte activity and the signaling pathways involved in this process. 2. Materials and methods 2.1. Materials F2pal-16, F2pal-12 and F2pal-7 were synthesized by Anygen (Gwangju, Korea). Pertussis toxin (PTX), PD98059, SB203580, SP600125 and SC-514 were from Calbiochem (San Diego, CA). BAY 11-7082 was purchased from Biomol (Plymouth Meeting, PA). All the antibodies for phospho-mitogen activated protein kinases (MAPKs) were from Cell Signaling Technology, Inc. (Beverly, MA). Rabbit anti-human antibodies against FPR2 were purchased from Gene Tex (San Antonio, TX). 2.2. Isolation of human peripheral blood monocytes Peripheral blood was collected from healthy adult donors. The donors were confirmed not to have taken anti-inflammatory drugs for at least 4 weeks before sampling. Venous blood was collected and peripheral blood mononuclear cells were separated on a Histopaque1077 gradient. After two washings with Hank’s buffered saline solution, without Ca2+ and Mg2+, the peripheral blood mononuclear cells were suspended in RPMI 1640 medium containing 10% FBS and incubated for 60 min at 37 °C to allow the monocytes to attach to the culture dish. The attached monocytes were then collected as described previously [18]. The isolated cells were used immediately. 2.3. Cell culture FPR1- or FPR2-expressing RBL-2H3 cells and vector-transfected RBL-2H3 cells were maintained as previously described [19]. Cells were maintained at about 1 106 cells/ml under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 °C).
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tion of 1 106 cells/ml, and 25 ll of the suspension were placed onto the upper well of a chamber that was separated by a 5 lm polyhydrocarbon filter from the peptide-containing lower well. After incubation for 2 h at 37 °C, non-migrated cells were removed by scraping them out, and cells that migrated across the filter were dehydrated, fixed, and stained with hematoxylin (Sigma, St. Louis, MO). The stained cells in five randomly chosen high-power fields (HPF) (400) in that well were then counted [18]. 2.6. Measurement of superoxide anion generation Superoxide anion generation was determined by measuring cytochrome c reduction using a microtiter 96-well plate ELISA reader (Bio-Tek instruments, EL312e, Winooski, VT) as previously described [12,13]. Human monocytes (2 106 cells in RPMI 1640 medium) were incubated with 5 lM of cytochalasin B at 37 °C for 5 min and then incubated with each peptide in the presence of 50 lM cytochrome c. Superoxide generation was determined by measuring light absorption changes at 550 nm over 10 min at 1 min intervals. 2.7. Cytokine assay Cytokine measurements were performed as previously described [4,5]. Human monocytes (3 105 cells/0.3 ml) were placed in RPMI 1640 medium containing 1% FBS in 24-well plates and kept in a 5% CO2 incubator at 37 °C. After stimulation, the cell-free supernatants were collected, centrifuged, and measured for CCL2 by enzyme-linked immunosorbent assay (ELISA) (BD Biosciences Pharmingen, San Diego, CA) according to the manufacturer’s instruction. 2.8. Measurement of NF-jB activity Nuclear extracts were prepared from monocytes with the Nuclear Extraction kit (Panomics, Inc. Fremont, CA) for use with a transcription factor assay kit. Briefly, monocytes (1 106 cells in RPMI 1640 medium) were incubated with F2pal-16, SAA, or MMK-1 for 30 min. Cells were harvested and washed twice with PBS, and resuspended in 250 ll of lysis buffer (Buffer A). All the cells were incubated on ice for 10 min and then centrifuged at 14 000g for 3 min at 4 °C. The supernatants were discarded and the pellets kept on ice. Pellets were resuspended in 150 ll of extraction buffer (Buffer B) and incubated for 2 h on ice. Nuclear debris was removed by centrifugation (14 000g for 5 min at 4 °C), and the nuclear protein extract was used for NF-jB p65 nuclear transcription factor ELISA kit (Panomics, Inc. Fremont, CA) according to the manufacturer’s instructions.
2.4. Ca2+ measurement 2.9. Data analysis Intracellular calcium concentration ([Ca2+]i) was determined by the method of Grynkiewicz using fura-2/AM [20]. Briefly, prepared cells were incubated with 3 lM fura-2/AM at 37 °C for 50 min in serum-free RPMI 1640 medium with continuous stirring. Approximately 2 106 cells were aliquoted for each assay into Ca2+ free Locke’s solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 5 mM HEPES, pH 7.3, 10 mM glucose, and 0.2 mM EGTA). Fluorescence was measured at 500 nm at excitation wavelengths of 340 nm and 380 nm. The fluorescence ratio was then used for calculation of [Ca2+]i..
The results are expressed as mean ± S.E.M. of the number of determinations indicated. Statistical significance of differences was determined by student t-test and statistical significance was considered significant at P < 0.05. 3. Results 3.1. FPR2-pepducin stimulates an increase in intracellular calcium in human monocytes
2.5. Chemotaxis assay Chemotaxis assays were performed using multiwell chambers (Neuroprobe Inc., Gaithersburg, MD) [18]. Briefly, prepared human monocytes were suspended in RPMI 1640 medium at a concentra-
It has been demonstrated that activation of FPR2 elicits an increase in intracellular calcium in human monocytes [21]. In the current study, we synthesized human FPR2 intracellular loop 3based pepducins in a search for an intracellular agonist of FPR2
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(Fig. 1A). We also synthesized non-palmitoylated peptide 16 and its complete reverse sequence palmitoylated peptide for control of active pepducin (Fig. 1A). We examined the effects of three different FPR2 pepducins on the increase in intracellular calcium in human monocytes. Among three FPR2 pepducins tested, F2pal16, but not F2pal-7 or F2pal-12, stimulated an increase in intracellular calcium in human monocytes (Fig. 1B). F2pal-16 stimulated an increase in calcium in monocytes in a concentration-dependent manner, showing maximal activity at approximately 5 lM (Fig. 1C). We also tested the effect of peptide corresponding to F2pal-16 and scrambled pepducin of F2pal-16, neither of which had an effect on calcium signaling in human monocytes (Fig. 1B). The results strongly indicate that F2pal-16 specifically stimulates calcium signaling in human monocytes. 3.2. F2pal-16-stimulated calcium increase is mediated by FPR2 Because F2pal-16 was synthesized based on the FPR2 intracellular loop 3 sequence, we reasoned that F2pal-16 directly activates FPR2 in human monocytes. To confirm this, we examined whether F2pal-16 required the presence of FPR2 to activate signaling. We tested the effect of F2pal-16 on Ca2+ mobilization in FPR1-, or FPR2-expressing RBL-2H3 cells with vector-transfected cells as control. As shown in Fig. 2A, F2pal-16 stimulated Ca2+ release in
FPR2-expressing RBL-2H3 cells, but there was no effect in FPR1expressing or vector-transfected cells. As a control, a well-known FPR1 agonist (WKYMVm) [22] strongly stimulated calcium increase in FPR1-expressing RBL-2H3 cells (Fig. 2A). This finding strongly indicates that F2pal-16 specifically stimulates FPR2, eliciting an increase in calcium. We also tested the effect of F2pal-16 on the FPR2-specific agonist (MMK-1)-induced calcium signaling, and vice versa. MMK-1 could not further increase intracellular calcium levels over those attained by treatment with F2pa1-16, and vice versa (Fig. 2B). Next, we tested the effect of FPR2 antagonist (WRWWWW, WRW4) [19] on the F2pal-16-stimulated increase in intracellular calcium in FPR2-expressing RBL-2H3 cells. Although WRW4 specifically inhibited the MMK-1-stimulated increase in calcium in FPR2-expressing RBL-2H3 cells, the F2pal16-stimulated increase in calcium was not affected by WRW4 (data not shown). It has been demonstrated that FPR2-stimulated signaling is mediated by PTX-sensitive G-protein [23]. We tested the role of PTX-sensitive G-protein in F2pal-16-stimulated calcium signaling. The F2pal-16-stimulated increase in calcium was completely inhibited by PTX (Fig. 2C). Stimulation of calcium signaling by MMK-1, a well-known FPR2 agonist, was also completely inhibited
F2pal-7: Pal-KIHKKGM F2pal-12: Pal-KGMIKSSRPLRV F2pal-16: Pal-KIHKKGMIKSSRPLRV scF2pal-16: Pal-VRLPRSSKIMGKKHIK Pep-16: KIHKKGMIKSSRPLRV
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Pepducin (μM) Fig. 1. F2pal-16 stimulates intracellular calcium increase in primary human monocytes. The sequences of FPR2-based pepducins (A). Freshly isolated human peripheral blood monocytes were loaded with Fura-2/AM. Fura-2 loaded cells were stimulated with 10 lM of each molecule (F2pal-7, F2pal-12, F2pal-16, pep-16, or scPal-16). The changes in 340/380 nm were monitored. The results are representative of three independent experiments (B). Fura-2 loaded cells were stimulated with various concentrations of F2pal-16 (0, 0.01, 0.1, 0.5, 1, 5, or 10 lM), and intracellular [Ca2+]i was measured. The results are presented as the means ± S.E. of three independent experiments. (C). *, P < 0.05, compared with the value obtained from the control (scF2pal-16 treated).
200
-PTX +PTX
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MMK-1 -PTX +PTX
0 1 min Fig. 2. F2pPal-16-induced intracellular calcium increase is mediated by FPR2. Fura2/AM loaded FPR1- or FPR2-expressing RBL2H3 cells, or RBL2H3 cells transfected with empty vector, were stimulated with 5 lM F2pal-16 or 100 nM WKYMVm (A). FPR2-expressing RBL-2H3 cells were sequentially stimulated with 10 lM F2pal-16 or 1 lM MMK-1 as indicated in the figure (B). FPR2-expressing RBL2H3 cells were incubated with vehicle or 100 ng/ml PTX for 24 h, and then the cells were loaded with Fura-2/AM. Fura-2/AM-loaded cells were treated with F2pal-16 (5 lM) or MMK-1 (1 lM) (C) and changes in 340/380 nm were monitored. The results are representative of three independent experiments (A–C).
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by PTX (Fig. 2C). The results strongly suggest that F2pal-16 stimulates calcium signaling via PTX-sensitive G-protein(s). Many GPCRs, including FPR2, induce calcium signaling via the activation of phospholipase C (PLC) [24]. To determine whether F2pal-16 also acts via PLC, we pretreated FPR2-expressing RBL-2H3 cells with a specific PLC inhibitor (U-73122) prior to the addition of F2pal-16. F2pal-16-induced calcium signaling was
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* One of the important roles of monocytes in innate immune responses is the production of reactive oxygen species, such as superoxide anion [25]. We determined the effects of F2pal-16 on
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FPR2 is well-characterized as a classical chemoattractant receptor [1]. Stimulation of FPR2 by its selective agonist induces chemotactic migration of human monocytes [1,2]. In this study, we investigated the effect of F2pal-16 on monocyte chemotaxis. Addition of various concentrations of F2pal-16 induced chemotactic migration of monocytes in a concentration-dependent manner (Fig. 3A). Maximal activity was shown at approximately 5 lM. Preincubation of human monocytes with PTX prior to the chemotaxis assay using F2pal-16 caused complete inhibition of F2pal-16-induced chemotactic migration (Fig. 3B), demonstrating the role of PTX-sensitive G-protein(s) in this process. We also tested the effect of WRW4 on the FPR2 agonist-induced monocyte chemotaxis. Preincubation of human monocytes with 10 lM WRW4 prior to the chemotaxis assay caused dramatic inhibition of monocyte chemotaxis induced by SAA or MMK-1 (Fig. 3C). However, F2pal-16-induced monocyte chemotaxis was not affected by WRW4 (Fig. 3C).
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3.3. F2pal-16 stimulates chemotactic migration of human monocytes
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Fig. 3. F2pal-16 stimulates monocyte migration through a PTX-sensitive G-protein dependent signaling pathway. For all experiments, freshly isolated human peripheral blood monocytes (1 106 cells/ml of serum-free RPMI 1640 medium) were added to the upper well of a 96-well chemotaxis chamber and assessed for migration across a membrane with a 5 lm pore size after incubation at 37 °C for 2 h. The number of cells which migrated across the membrane was determined by counting the cells in five microscope high-power fields (400; HPF). Various concentrations of F2pal-16 were used in the lower chamber for the chemotaxis assay (A). The cells were incubated in the presence or absence of 100 ng/ml PTX for 24 h before the chemotaxis assay. Vehicle (DMSO), 5 lM F2pal-16, 2 lM SAA, and 1 lM MMK-1 were added to the lower chamber for the chemotaxis assay (B). Human monocytes were incubated in the presence or absence of 10 lM WRW4 for 30 min, and then subjected to the chemotaxis assay. Vehicle (DMSO), 5 lM F2pal16, 2 lM SAA, and 1 lM MMK-1 were used added to the lower chamber (C). The results are presented as the means ± S.E. of three independent experiments (A–C). *P < 0.05, compared with the value obtained from the control (scF2pal-16 treated) (A); #P < 0.05, significantly different from the control (–PTX or –WRW4) (B and C).
O2- generation (nmole/106 cells)
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completely inhibited by U-73122, indicating that F2pal-16 stimulates calcium signaling by activation of PLC (data not shown). Stimulation by the FPR2 agonist MMK-1 was also completely inhibited by U-73122 in FPR2-expressing RBL-2H3 cells (data not shown).
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Fig. 4. F2pal-16 stimulates superoxide generation in human primary monocytes. The superoxide generation was measured using a cytochrome c reduction assay (A and B). Human monocytes were stimulated with various concentrations of F2pal-16 (0.01, 0.1, 1, 5, and 10 lM) (A). Vehicle (DMSO), 5 lM F2pal-16, 2 lM SAA, or 1 lM MMK-1 was added to freshly isolated human monocytes (B). The results are presented as the means ± S.E. of three independent experiments. *P < 0.05, compared with the value obtained from the control (scF2pal-16 treated) (A) or (vehicle treated) (B).
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* CCL2 (ng/ml)
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Fig. 5. F2pal-16 stimulates the production of pro-inflammatory cytokines from human monocytes. Freshly isolated human peripheral blood monocytes were treated with 5 lM F2pal-16 for various times (0, 1, 3, 6, 12, or 24 h) (A and B); with various concentrations of F2pal-16 (0.01, 0.1, 1, 5, and 10 lM) for 12 h (C); with vehicle or 100 ng/ml of PTX for 24 h before treatment with vehicle, F2pal-16 (5 lM), SAA (2 lM), or MMK-1 (1 lM) (D); or incubated in the presence or absence of 10 lM WRW4 for 30 min before treatment with vehicle, F2pal-16 (5 lM) or SAA (2 lM) (E). Secreted CXCL8 (A) and CCL2 (B–E) levels were determined by ELISA. The results are presented as the means ± S.E. of three independent experiments. *P < 0.05, compared with the value obtained from the control (0 h for A, B; scF2pal-16 treated for C; vehicle treated for D and E); #P < 0.05, significantly different from the control (-PTX or -WRW4) (D and E).
the production of superoxide anion using the cytochrome c reduction assay. F2pal-16 stimulated superoxide anion production in a concentration-dependent manner, showing maximal activity at 5 lM (Fig. 4A). The superoxide anion-producing activity of F2pal16 was compared with that of two other FPR2 agonists, SAA and MMK-1. Stimulation of human monocytes with MMK-1 (1 lM) enhanced superoxide anion production (Fig. 4B). The MMK-1-induced superoxide anion production was about twofold more potent than that observed with F2pal-16. However, stimulation of human monocytes with SAA (2 lM) failed to enhance superoxide anion production (Fig. 4B). 3.5. F2pal-16 stimulates production of several inflammatory cytokines in human monocytes The effect of F2pal-16 on the production of several cytokines was investigated in human monocytes. When the cells were stim-
ulated with 5 lM F2pal-16, F2pal-16 induced IL-8 production was assayed at various times, and showed maximal activity within 12 h of stimulation (Fig. 5A). F2pal-16 also stimulated the production of TNF-a, IL-1b, and CCL2 in human monocytes (Fig. 5B and data not shown). F2pal-16-induced TNF-a production was maximal 6 h after stimulation. IL-1b and CCL2 production by F2pal-16 was maximal approximately 12 h after stimulation (Fig. 5B and data not shown). Stimulation of human monocytes with various concentrations of F2pal-16 induced CCL2 production in a concentrationdependent manner (Fig. 5C). We also compared the effect of F2pal-16, MMK-1, or SAA on the production of CCL2 in human monocytes. Stimulation of human monocytes with F2pal-16 (5 lM), or SAA (2 lM) for 24 h elicited CCL2 production (Fig. 5D). However, MMK-1 (5 lM) failed to stimulate CCL2 production from the cells (Fig. 5D). We then tested the role of PTX-sensitive G-protein on the agonist-stimulated CCL2 production from human monocytes. Preincubation of human
H.Y. Lee et al. / FEBS Letters 584 (2010) 4102–4108
4. Discussion
NF−κB binding activity (fold of basal)
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Inhibitor (μM) 0
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F2pal-16 (μM) Fig. 6. F2pal-16-stimulated CCL2 production is mediated by NF-jB activity. Human monocytes were stimulated with vehicle F2pal-16 (5 lM), SAA (2 lM), or MMK-1 (5 lM) for 30 min. Nuclear protein was extracted and NF-jB activity was measured using a NF-jB activity measuring kit (A). Human monocytes were pre-incubated with several concentrations of BAY 11-7082 or SC-514 (0, 1, 5, 10, and 20 lM) at 37 °C for 1 h, followed by stimulation with 5 lM of F2pal-16 for 12 h (B). Secreted CCL2 levels were determined by ELISA (B). The results are presented as the means ± S.E. of three independent experiments. *P < 0.05, compared with the value obtained from the control (vehicle treated); #P < 0.05, significantly different from the control (F2pal-16 only treated) (B).
monocytes with PTX prior to the addition of F2pal-16 or SAA inhibited CCL2 production by these two agonists (Fig. 5D), indicating that F2pal-16 or SAA-induced CCL2 production is mediated by PTX-sensitive G-protein(s). Preincubation of human monocytes with 10 lM WRW4 prior to the addition of SAA or F2pa1-16 led to dramatic inhibition of CCL2 production by SAA (Fig. 5E), whereas F2pal-16-stimulated CCL2 production was not inhibited by WRW4 (Fig. 5E). 3.6. F2pal-16 stimulates NF-jB activity, resulting in CCL2 production in human monocytes Past studies have demonstrated that the expression of the CCL2 gene requires the activation of NF-jB [26,27]. We also tested the effect of F2pal-16 on NF-jB activity in human monocytes using an ELISA kit to detect activated NF-jB. The NF-jB activity was enhanced after exposing monocytes to F2pal-16 for 30 min (Fig. 6A). We further investigated the role of NF-jB on F2pal-16-induced CCL2 production by using two NF-jB inhibitors (BAY 11-7082 and SC-514). CCL2 production by F2pal-16 was almost completely inhibited by two inhibitors (Fig. 6B). The results indicate that NFjB is required for F2pal-16-induced CCL2 production in human monocytes. Another FPR2 agonist, SAA, but not MMK-1, stimulated NF-jB activity in human monocytes (Fig. 6A). The result was correlated with the data that SAA, but not MMK-1, stimulated production of pro-inflammatory cytokines such as CCL2 from human monocytes.
Palmitoylated peptide sequences of some GPCRs are known to act as ligands or modulators of GPCRs [16,17]. In this study we developed a novel FPR2 agonist, F2pal-16, which stimulates FPR2-induced G-protein activation. The pepducin stimulated a broad range of cellular responses in human monocytes, including superoxide anion production, chemotactic migration, and cytokine production. We demonstrated that one palmitoylated peptide (F2pal-16) can act a novel agonist for FPR2. Characterization of F2pal-16 as an agonist for FPR2 revealed that F2pal-16 stimulates a broad range of cellular responses, including superoxide anion production, chemotactic migration, and cytokine production. Some agonists for FPR2 stimulated a limited range of cellular responses. For example, WKYMVm potently stimulates chemotactic migration and superoxide anion production, but fails to stimulate cytokine (CXCL8 and CCL2) production in human neutrophils [13,28]. SAA strongly stimulates chemotactic migration and cytokine production, but does not stimulate superoxide anion production in human neutrophils [13]. In contrast, we have demonstrated that F2pal-16 stimulates all tested cellular responses (chemotactic migration, superoxide anion production, and cytokine production) in human monocytes. WKYMVm and SAA are peptide ligands that act on the receptor without membrane-tethering, whereas F2pal-16 stimulates FPR2 after membrane-tethering. It has been shown in other studies that pepducins act on the juxtaposed region of GPCR and G-protein(s) [16,17,29]. We suggest that F2pal-16 stimulates a broad range of cellular responses because it can act on FPR2 with a unique mechanism of action. In terms of the site of action of F2pal-16 on FPR2, it may bind to a site distinct from that of extracellular ligands. In this study we examined the effect of WRW4 (an FPR2 antagonist) on F2pal-16-induced signaling. Pretreatment of WRW4 prior to F2pal-16 treatment did not block F2pal-16-induced calcium signaling (data not shown). However, WRW4 pretreatment completely blocked calcium signaling induced by another FPR2 agonist, MMK-1 (data not shown). Previous work has demonstrated that pepducin, which is synthesized from the intracellular loop of GPCR, acts intracellularly by regulating the coupling of heterotrimeric G-protein and target GPCR [16,17]. In our study, we synthesized a pepducin peptide based on the intracellular loop 3 region of FPR2, which is expected to act on an intracellular target, but not on the cell surface. Keeping in mind that WRW4 is an antagonist which acts on the cell surface receptor in the extracellular milieu, it makes sense that F2pal-16-induced calcium signaling was not inhibited by WRW4. As shown in Figs. 3 and 5, WRW4 only blocked monocyte chemotaxis or CCL2 production induced by SAA or MMK-1, but not that induced by F2pal-16. These results suggest that F2pal-16 has a different binding site from that of WRW4 on FPR2. Past study already clearly demonstrated that palmitoylated peptides act on the intracellular region of specific GPCRs such as PAR1 [30]. Taking together the past study and our data, we can suggest that our F2pal-16 also acts on the intracellular region of FPR2. In terms of receptor specificity of F2pal-16 in the FPR family, we clearly demonstrated that F2pal-16 stimulates FPR2 but not FPR1 (Fig. 2). Since human primary monocytes express all 3 FPRs (FPR1, FPR2 and FPR3), at this point we could not clearly rule out an involvement of FPR3 on the F2pal-16-induced responses in human monocytes. Several previous reports have demonstrated that NF-jB is required for the induction of CCL2 in some cell types [26,27]. We demonstrated that F2pal-16 stimulates NF-jB (Fig. 6A). Regarding the role of NF-jB on F2pal-16-induced CCL2 induction, we observed that BAY 11-7082 completely inhibited F2pal-16-induced CCL2 production from human monocytes (Fig. 6B), suggesting that
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the NF-jB pathway is required for the induction of CCL2 by F2pal16 in monocytes. Taken together, the results suggest that NF-jB signaling is a prerequisite for the proper induction of CCL2 by F2pal-16 in monocytes. Keeping in mind that F2pal-16 permeabilizes and stimulates FPR2, thus eliciting G-protein activation, it is reasonable to suggest that F2pal-16 induces the activation of a broad range of signaling down-stream of FPR2, resulting in the activation of the NF-jB pathway. In conclusion, we have developed a novel FPR2-specific membrane-tethering cell permeable peptide, F2pal-16. Since F2pal-16 stimulated a broad range of FPR2 down-stream signaling resulting in superoxide anion production and cytokine production, this molecule can be regarded as an important ligand for the study of the mechanism of activation and immunologic role of FPR2.
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5. Disclosure statement [15]
Some of the authors (Ha Young Lee and Yoe-Sik Bae) have pending patent applications.
[16]
Acknowledgements
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This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20100020940); National Research Foundation of Korea grant funded by the Korea government (2009 0093198); and by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (0920220). References [1] Le, Y., Yang, Y., Cui, Y., Yazawa, H., Gong, W., Qiu, C. and Wang, J.M. (2002) Receptors for chemotactic formyl peptides as pharmacological targets. Int. Immunopharmacol. 2, 1–13. [2] Migeotte, I., Communi, D. and Parmentier, M. (2006) Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses. Cytokine Growth Factor Rev. 17, 501–519. [3] Kim, S.D., Kim, J.M., Jo, S.H., Lee, H.Y., Lee, S.Y., Shim, J.W., Seo, S.K., Yun, J. and Bae, Y.S. (2009) Functional expression of formyl peptide receptor family in human NK cells. J. Immunol. 183, 5511–5517. [4] Lee, H.Y., Kim, S.D., Shim, J.W., Lee, S.Y., Lee, H., Cho, K.H., Yun, J. and Bae, Y.S. (2008) Serum amyloid A induces CCL2 production via formyl peptide receptorlike 1-mediated signaling in human monocytes. J. Immunol. 181, 4332–4339. [5] Lee, H.Y., Kim, M.K., Park, K.S., Shin, E.H., Jo, S.H., Kim, S.D., Jo, E.J., Lee, Y.N., Lee, C., Baek, S.H. and Bae, Y.S. (2006) Serum amyloid A induces contrary immune responses via formyl peptide receptor-like 1 in human monocytes. Mol. Pharmacol. 70, 241–248. [6] O’Hara, R., Murphy, E.P., Whitehead, A.S., FitzGerald, O. and Bresnihan, B. (2004) Local expression of the serum amyloid A and formyl peptide receptor-like 1 genes in synovial tissue is associated with matrix metalloproteinase production in patients with inflammatory arthritis. Arthritis Rheum. 50, 1788–1799. [7] Yang, De., Chen, Q., Schmidt, A.P., Anderson, G.M., Wang, J.M., Wooters, J., Oppenheim, J.J. and Chertov, O. (2000) LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J. Exp. Med. 192, 1069–1074. [8] Maddox, J.F., Hachicha, M., Takano, T., Petasis, N.A., Fokin, V.V. and Serhan, C.N. (1997) Lipoxin A4 stable analogs are potent mimetics that stimulate human monocytes and THP-1 cells via a G-protein-linked lipoxin A4 receptor. J. Biol. Chem. 272, 6972–6978. [9] Su, S.B., Gong, W., Gao, J.L., Shen, W., Murphy, P.M., Oppenheim, J.J. and Wang, J.M. (1999) A seven-transmembrane, G protein-coupled receptor, FPRL1,
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journal homepage: www.FEBSLetters.org
Activation of human monocytes by a formyl peptide receptor 2-derived pepducin Ha Young Lee a,b, Sang Doo Kim a,b, Jae Woong Shim a,b, Hak Jung Kim a,b, Jae Young Kwon a, Jong-Min Kim b, Suk-Hwan Baek c, Joon Seong Park d, Yoe-Sik Bae a,b,⇑ a
Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714, Republic of Korea c Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu 705-717, Republic of Korea d Department of Hematology-Oncology, Ajou University School of Medicine, Suwon 443-721, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 14 June 2010 Revised 25 August 2010 Accepted 25 August 2010 Available online 9 September 2010 Edited by Ned Mantei Keywords: FPR2 Pepducin Monocytes Chemotaxis Pertussis toxin-sensitive G-protein Cytokine
a b s t r a c t We synthesized and investigated the effect of formyl peptide receptor 2 (FPR2)-derived pepducins in human monocytes. The FPR2-based cell-penetrating lipopeptide, ‘‘pepducin” (F2pal-16), stimulated intracellular calcium increase in human monocytes via pertussis toxin (PTX)-sensitive G-protein and phospholipase C (PLC) activity. From a functional aspect, we showed that F2pal-16 stimulated monocyte chemotaxis. F2pal-16 also stimulated the generation of superoxide anion in human monocytes. Moreover, F2pal-16 dramatically increased the production of several kinds of pro-inflammatory cytokines (CXCL8, CCL2, IL-1b and TNF-a) in human monocytes via NF-jB activation. Since FPR2 plays an important role in immune responses, F2pal-16 can serve as a useful reagent for the study of FPR2-mediated immune modulation. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Formyl peptide receptor 2 (FPR2) is a chemoattractant receptor which regulates trafficking of leukocytic cells, such as neutrophils, monocytes, macrophages, and natural killer cells [1–3]. FPR2 has an important immunologic function, including host defense against pathogenic infections by stimulating the generation of reactive oxygen species in human monocytes as well as neutrophils [1,2]. In human monocytes, FPR2 is also involved in the production of some important cytokines, such as CCL2, IL-10, and TNF-a [4,5]. In human natural killer cells, activation of FPR2 stimulates IFN-c production and cytolytic activity, which suggests that FPR2 can be regarded as an important target receptor for cancer therapy [3]. Since human synovial fibroblasts express functional FPR2, past studies have suggested that FPR2 may be involved in the pathogenesis of arthritis [6]. Because FPR2 is expressed in several important leukocytes and inflammatory cells, and the receptor Abbreviations: FPR2, formyl peptide receptor 2; SAA, serum amyloid A; GPCR, Gprotein coupled receptor; PTX, pertussis toxin; MAPKs, mitogen activated protein kinases; [Ca2+]i, intracellular calcium concentration; HPF, high-power fields; WRW4, WRWWWW; PLC, phospholipase C ⇑ Corresponding author at: Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea. Fax: +82 31 290 7015. E-mail addresses: [email protected], [email protected] (Y.-S. Bae).
is regarded as a possible important target for the treatment of inflammatory diseases, the development of various agents that modulate FPR2 activity is required. In terms of ligands for FPR2, many agonists have been identified, including host-derived agonists (LL-37, lipoxin A4, and serum amyloid A [SAA]), and synthetic peptides (WKYMVm and MMK-1) [7– 11]. Cellular responses induced by these FPR agonists have been examined in some leukocytic cells, such as neutrophils and monocytes [1,2,7,9]. The activation of FPR2 by one of the strong synthetic agonists, WKYMVm, induces the activation of NADPH oxidase, resulting in superoxide anion production [12,13]. However, the stimulation of human neutrophils or monocytes with WKYMVm does not induce cytokine production [4]. Another important FPR2 agonist, SAA, stimulates the production of some cytokines, such as TNF-a, IL-10, and CCL2 [4,5]. However, SAA fails to stimulate superoxide production from human monocytes or human neutrophils [13]. We have demonstrated that the activation of FPR2 by different ligands induces differential intracellular signaling and cellular responses, showing a ligand-selective process [4,13]. Although various ligands for FPR2 have been reported, many ligands have failed to stimulate a broad range of activation in phagocytic cells. The G-protein coupled receptors (GPCR), including FPR2, have been shown to induce intracellular signaling via the activation of heterotrimeric G-protein(s) [1,2]. Subsequent mutagenesis studies
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.08.036
H.Y. Lee et al. / FEBS Letters 584 (2010) 4102–4108
have demonstrated that some intracellular loop regions of GPCR, such as the intracellular loop 3 region, are involved in the activation of GPCR [14]. Based on this background, Kuliopulos and coworkers developed several membrane-tethering cell permeable peptides corresponding to the intracellular loop region of PAR as agonists or antagonist for PAR [15,16]. Pepducin is a membranetethering cell permeable peptide [15]. It has been demonstrated that pepducins derived from PAR1 or CXCR1 act as agonists or antagonists for their specific receptors [15–17]. Even though synthesis of pepducin from GPCR is a novel approach by which to develop new ligands for the receptor, nothing is known about FPR2-derived pepducins. In this study, we synthesized a new FPR2-derived pepducin and tested its effect on human monocyte activity and the signaling pathways involved in this process. 2. Materials and methods 2.1. Materials F2pal-16, F2pal-12 and F2pal-7 were synthesized by Anygen (Gwangju, Korea). Pertussis toxin (PTX), PD98059, SB203580, SP600125 and SC-514 were from Calbiochem (San Diego, CA). BAY 11-7082 was purchased from Biomol (Plymouth Meeting, PA). All the antibodies for phospho-mitogen activated protein kinases (MAPKs) were from Cell Signaling Technology, Inc. (Beverly, MA). Rabbit anti-human antibodies against FPR2 were purchased from Gene Tex (San Antonio, TX). 2.2. Isolation of human peripheral blood monocytes Peripheral blood was collected from healthy adult donors. The donors were confirmed not to have taken anti-inflammatory drugs for at least 4 weeks before sampling. Venous blood was collected and peripheral blood mononuclear cells were separated on a Histopaque1077 gradient. After two washings with Hank’s buffered saline solution, without Ca2+ and Mg2+, the peripheral blood mononuclear cells were suspended in RPMI 1640 medium containing 10% FBS and incubated for 60 min at 37 °C to allow the monocytes to attach to the culture dish. The attached monocytes were then collected as described previously [18]. The isolated cells were used immediately. 2.3. Cell culture FPR1- or FPR2-expressing RBL-2H3 cells and vector-transfected RBL-2H3 cells were maintained as previously described [19]. Cells were maintained at about 1 106 cells/ml under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 °C).
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tion of 1 106 cells/ml, and 25 ll of the suspension were placed onto the upper well of a chamber that was separated by a 5 lm polyhydrocarbon filter from the peptide-containing lower well. After incubation for 2 h at 37 °C, non-migrated cells were removed by scraping them out, and cells that migrated across the filter were dehydrated, fixed, and stained with hematoxylin (Sigma, St. Louis, MO). The stained cells in five randomly chosen high-power fields (HPF) (400) in that well were then counted [18]. 2.6. Measurement of superoxide anion generation Superoxide anion generation was determined by measuring cytochrome c reduction using a microtiter 96-well plate ELISA reader (Bio-Tek instruments, EL312e, Winooski, VT) as previously described [12,13]. Human monocytes (2 106 cells in RPMI 1640 medium) were incubated with 5 lM of cytochalasin B at 37 °C for 5 min and then incubated with each peptide in the presence of 50 lM cytochrome c. Superoxide generation was determined by measuring light absorption changes at 550 nm over 10 min at 1 min intervals. 2.7. Cytokine assay Cytokine measurements were performed as previously described [4,5]. Human monocytes (3 105 cells/0.3 ml) were placed in RPMI 1640 medium containing 1% FBS in 24-well plates and kept in a 5% CO2 incubator at 37 °C. After stimulation, the cell-free supernatants were collected, centrifuged, and measured for CCL2 by enzyme-linked immunosorbent assay (ELISA) (BD Biosciences Pharmingen, San Diego, CA) according to the manufacturer’s instruction. 2.8. Measurement of NF-jB activity Nuclear extracts were prepared from monocytes with the Nuclear Extraction kit (Panomics, Inc. Fremont, CA) for use with a transcription factor assay kit. Briefly, monocytes (1 106 cells in RPMI 1640 medium) were incubated with F2pal-16, SAA, or MMK-1 for 30 min. Cells were harvested and washed twice with PBS, and resuspended in 250 ll of lysis buffer (Buffer A). All the cells were incubated on ice for 10 min and then centrifuged at 14 000g for 3 min at 4 °C. The supernatants were discarded and the pellets kept on ice. Pellets were resuspended in 150 ll of extraction buffer (Buffer B) and incubated for 2 h on ice. Nuclear debris was removed by centrifugation (14 000g for 5 min at 4 °C), and the nuclear protein extract was used for NF-jB p65 nuclear transcription factor ELISA kit (Panomics, Inc. Fremont, CA) according to the manufacturer’s instructions.
2.4. Ca2+ measurement 2.9. Data analysis Intracellular calcium concentration ([Ca2+]i) was determined by the method of Grynkiewicz using fura-2/AM [20]. Briefly, prepared cells were incubated with 3 lM fura-2/AM at 37 °C for 50 min in serum-free RPMI 1640 medium with continuous stirring. Approximately 2 106 cells were aliquoted for each assay into Ca2+ free Locke’s solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 5 mM HEPES, pH 7.3, 10 mM glucose, and 0.2 mM EGTA). Fluorescence was measured at 500 nm at excitation wavelengths of 340 nm and 380 nm. The fluorescence ratio was then used for calculation of [Ca2+]i..
The results are expressed as mean ± S.E.M. of the number of determinations indicated. Statistical significance of differences was determined by student t-test and statistical significance was considered significant at P < 0.05. 3. Results 3.1. FPR2-pepducin stimulates an increase in intracellular calcium in human monocytes
2.5. Chemotaxis assay Chemotaxis assays were performed using multiwell chambers (Neuroprobe Inc., Gaithersburg, MD) [18]. Briefly, prepared human monocytes were suspended in RPMI 1640 medium at a concentra-
It has been demonstrated that activation of FPR2 elicits an increase in intracellular calcium in human monocytes [21]. In the current study, we synthesized human FPR2 intracellular loop 3based pepducins in a search for an intracellular agonist of FPR2
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(Fig. 1A). We also synthesized non-palmitoylated peptide 16 and its complete reverse sequence palmitoylated peptide for control of active pepducin (Fig. 1A). We examined the effects of three different FPR2 pepducins on the increase in intracellular calcium in human monocytes. Among three FPR2 pepducins tested, F2pal16, but not F2pal-7 or F2pal-12, stimulated an increase in intracellular calcium in human monocytes (Fig. 1B). F2pal-16 stimulated an increase in calcium in monocytes in a concentration-dependent manner, showing maximal activity at approximately 5 lM (Fig. 1C). We also tested the effect of peptide corresponding to F2pal-16 and scrambled pepducin of F2pal-16, neither of which had an effect on calcium signaling in human monocytes (Fig. 1B). The results strongly indicate that F2pal-16 specifically stimulates calcium signaling in human monocytes. 3.2. F2pal-16-stimulated calcium increase is mediated by FPR2 Because F2pal-16 was synthesized based on the FPR2 intracellular loop 3 sequence, we reasoned that F2pal-16 directly activates FPR2 in human monocytes. To confirm this, we examined whether F2pal-16 required the presence of FPR2 to activate signaling. We tested the effect of F2pal-16 on Ca2+ mobilization in FPR1-, or FPR2-expressing RBL-2H3 cells with vector-transfected cells as control. As shown in Fig. 2A, F2pal-16 stimulated Ca2+ release in
FPR2-expressing RBL-2H3 cells, but there was no effect in FPR1expressing or vector-transfected cells. As a control, a well-known FPR1 agonist (WKYMVm) [22] strongly stimulated calcium increase in FPR1-expressing RBL-2H3 cells (Fig. 2A). This finding strongly indicates that F2pal-16 specifically stimulates FPR2, eliciting an increase in calcium. We also tested the effect of F2pal-16 on the FPR2-specific agonist (MMK-1)-induced calcium signaling, and vice versa. MMK-1 could not further increase intracellular calcium levels over those attained by treatment with F2pa1-16, and vice versa (Fig. 2B). Next, we tested the effect of FPR2 antagonist (WRWWWW, WRW4) [19] on the F2pal-16-stimulated increase in intracellular calcium in FPR2-expressing RBL-2H3 cells. Although WRW4 specifically inhibited the MMK-1-stimulated increase in calcium in FPR2-expressing RBL-2H3 cells, the F2pal16-stimulated increase in calcium was not affected by WRW4 (data not shown). It has been demonstrated that FPR2-stimulated signaling is mediated by PTX-sensitive G-protein [23]. We tested the role of PTX-sensitive G-protein in F2pal-16-stimulated calcium signaling. The F2pal-16-stimulated increase in calcium was completely inhibited by PTX (Fig. 2C). Stimulation of calcium signaling by MMK-1, a well-known FPR2 agonist, was also completely inhibited
F2pal-7: Pal-KIHKKGM F2pal-12: Pal-KGMIKSSRPLRV F2pal-16: Pal-KIHKKGMIKSSRPLRV scF2pal-16: Pal-VRLPRSSKIMGKKHIK Pep-16: KIHKKGMIKSSRPLRV
Ca2+ (nM)
Vector /RBL2H3
FPR1 /RBL2H3
F2pal-16
WKYMVm
200 100
FPR2 /RBL2H3
F2pal-16 F2pal-16
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200
F2pal-16 100
F2pal-7 F2pal-12
0
1 min 200 F2pal-16 scF2pal-16
*
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F2pal-16 F2pal-16 MMK-1 MMK-1 F2pal-16 MMK-1
100 0
*
1 min
*
FPR2 /RBL2H3 F2pal-16
*
100
Ca2+ (nM)
Ca2+ (nM)
150
FPR2 /RBL2H3
scF2pal-16 Pep-16
Ca2+ (nM)
Ca2+ (nM)
0
50 0 1E-3 0.01 0.1
0.5
1
5
10
Pepducin (μM) Fig. 1. F2pal-16 stimulates intracellular calcium increase in primary human monocytes. The sequences of FPR2-based pepducins (A). Freshly isolated human peripheral blood monocytes were loaded with Fura-2/AM. Fura-2 loaded cells were stimulated with 10 lM of each molecule (F2pal-7, F2pal-12, F2pal-16, pep-16, or scPal-16). The changes in 340/380 nm were monitored. The results are representative of three independent experiments (B). Fura-2 loaded cells were stimulated with various concentrations of F2pal-16 (0, 0.01, 0.1, 0.5, 1, 5, or 10 lM), and intracellular [Ca2+]i was measured. The results are presented as the means ± S.E. of three independent experiments. (C). *, P < 0.05, compared with the value obtained from the control (scF2pal-16 treated).
200
-PTX +PTX
100
MMK-1 -PTX +PTX
0 1 min Fig. 2. F2pPal-16-induced intracellular calcium increase is mediated by FPR2. Fura2/AM loaded FPR1- or FPR2-expressing RBL2H3 cells, or RBL2H3 cells transfected with empty vector, were stimulated with 5 lM F2pal-16 or 100 nM WKYMVm (A). FPR2-expressing RBL-2H3 cells were sequentially stimulated with 10 lM F2pal-16 or 1 lM MMK-1 as indicated in the figure (B). FPR2-expressing RBL2H3 cells were incubated with vehicle or 100 ng/ml PTX for 24 h, and then the cells were loaded with Fura-2/AM. Fura-2/AM-loaded cells were treated with F2pal-16 (5 lM) or MMK-1 (1 lM) (C) and changes in 340/380 nm were monitored. The results are representative of three independent experiments (A–C).
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by PTX (Fig. 2C). The results strongly suggest that F2pal-16 stimulates calcium signaling via PTX-sensitive G-protein(s). Many GPCRs, including FPR2, induce calcium signaling via the activation of phospholipase C (PLC) [24]. To determine whether F2pal-16 also acts via PLC, we pretreated FPR2-expressing RBL-2H3 cells with a specific PLC inhibitor (U-73122) prior to the addition of F2pal-16. F2pal-16-induced calcium signaling was
*
F2pal-16 scF2pal-16
60
*
40
* 20
0
0
0.01 0.1
1
5
10
Pepducin (μM)
100 80
3.4. F2pal-16. stimulates superoxide anion production in human monocytes
*
-PTX +PTX
* One of the important roles of monocytes in innate immune responses is the production of reactive oxygen species, such as superoxide anion [25]. We determined the effects of F2pal-16 on
*
60 8 F2pal-16
40 20
#
#
#
0
Vehicle F2pal-16 SAA
Migrated cells / 5HPF
FPR2 is well-characterized as a classical chemoattractant receptor [1]. Stimulation of FPR2 by its selective agonist induces chemotactic migration of human monocytes [1,2]. In this study, we investigated the effect of F2pal-16 on monocyte chemotaxis. Addition of various concentrations of F2pal-16 induced chemotactic migration of monocytes in a concentration-dependent manner (Fig. 3A). Maximal activity was shown at approximately 5 lM. Preincubation of human monocytes with PTX prior to the chemotaxis assay using F2pal-16 caused complete inhibition of F2pal-16-induced chemotactic migration (Fig. 3B), demonstrating the role of PTX-sensitive G-protein(s) in this process. We also tested the effect of WRW4 on the FPR2 agonist-induced monocyte chemotaxis. Preincubation of human monocytes with 10 lM WRW4 prior to the chemotaxis assay caused dramatic inhibition of monocyte chemotaxis induced by SAA or MMK-1 (Fig. 3C). However, F2pal-16-induced monocyte chemotaxis was not affected by WRW4 (Fig. 3C).
120
MMK-1
*
-WRW4
O2- generation (nmole/106 cells)
Migrated cells / 5HPF
120
3.3. F2pal-16 stimulates chemotactic migration of human monocytes
100
40
*
6
* 4
2
0
*
80 60
*
scF2pal-16
+WRW4
0
*
0.01
0.1
1
#
10
16
#
*
14
20 0 Vehicle F2pal-16 SAA
5
Pepducin ( μM)
MMK-1
Fig. 3. F2pal-16 stimulates monocyte migration through a PTX-sensitive G-protein dependent signaling pathway. For all experiments, freshly isolated human peripheral blood monocytes (1 106 cells/ml of serum-free RPMI 1640 medium) were added to the upper well of a 96-well chemotaxis chamber and assessed for migration across a membrane with a 5 lm pore size after incubation at 37 °C for 2 h. The number of cells which migrated across the membrane was determined by counting the cells in five microscope high-power fields (400; HPF). Various concentrations of F2pal-16 were used in the lower chamber for the chemotaxis assay (A). The cells were incubated in the presence or absence of 100 ng/ml PTX for 24 h before the chemotaxis assay. Vehicle (DMSO), 5 lM F2pal-16, 2 lM SAA, and 1 lM MMK-1 were added to the lower chamber for the chemotaxis assay (B). Human monocytes were incubated in the presence or absence of 10 lM WRW4 for 30 min, and then subjected to the chemotaxis assay. Vehicle (DMSO), 5 lM F2pal16, 2 lM SAA, and 1 lM MMK-1 were used added to the lower chamber (C). The results are presented as the means ± S.E. of three independent experiments (A–C). *P < 0.05, compared with the value obtained from the control (scF2pal-16 treated) (A); #P < 0.05, significantly different from the control (–PTX or –WRW4) (B and C).
O2- generation (nmole/106 cells)
Migrated cells / 5HPF
80
completely inhibited by U-73122, indicating that F2pal-16 stimulates calcium signaling by activation of PLC (data not shown). Stimulation by the FPR2 agonist MMK-1 was also completely inhibited by U-73122 in FPR2-expressing RBL-2H3 cells (data not shown).
12 10 8
*
6 4 2 0
Vehicle F2pal-16
SAA
MMK-1
Fig. 4. F2pal-16 stimulates superoxide generation in human primary monocytes. The superoxide generation was measured using a cytochrome c reduction assay (A and B). Human monocytes were stimulated with various concentrations of F2pal-16 (0.01, 0.1, 1, 5, and 10 lM) (A). Vehicle (DMSO), 5 lM F2pal-16, 2 lM SAA, or 1 lM MMK-1 was added to freshly isolated human monocytes (B). The results are presented as the means ± S.E. of three independent experiments. *P < 0.05, compared with the value obtained from the control (scF2pal-16 treated) (A) or (vehicle treated) (B).
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* CCL2 (ng/ml)
CXCL8 (ng/ml)
*
6
*
4
25
*
*
8
2
*
20 15 10 5 0
0 0
1
3
6
12
24
0
1
3
Time (h)
12
24
35
30
*
F2pal-16 scF2pal-16
25 20
*
*
15 10
30
CCL2 (ng/ml)
CCL2 (ng/ml)
6
Time (h)
*
5
25
* *
20 15 10 5
0
-PTX +PTX
#
#
0
0
0.01
0.1
1
5
10
Vehicle F2pal-16
SAA
MMK-1
Pepducin (μM) 30
*
-WRW 4
CCL2 (ng/ml)
25
+WRW 4
*
20 15
#
10 5 0 Vehicle
F2pal-16
SAA
Fig. 5. F2pal-16 stimulates the production of pro-inflammatory cytokines from human monocytes. Freshly isolated human peripheral blood monocytes were treated with 5 lM F2pal-16 for various times (0, 1, 3, 6, 12, or 24 h) (A and B); with various concentrations of F2pal-16 (0.01, 0.1, 1, 5, and 10 lM) for 12 h (C); with vehicle or 100 ng/ml of PTX for 24 h before treatment with vehicle, F2pal-16 (5 lM), SAA (2 lM), or MMK-1 (1 lM) (D); or incubated in the presence or absence of 10 lM WRW4 for 30 min before treatment with vehicle, F2pal-16 (5 lM) or SAA (2 lM) (E). Secreted CXCL8 (A) and CCL2 (B–E) levels were determined by ELISA. The results are presented as the means ± S.E. of three independent experiments. *P < 0.05, compared with the value obtained from the control (0 h for A, B; scF2pal-16 treated for C; vehicle treated for D and E); #P < 0.05, significantly different from the control (-PTX or -WRW4) (D and E).
the production of superoxide anion using the cytochrome c reduction assay. F2pal-16 stimulated superoxide anion production in a concentration-dependent manner, showing maximal activity at 5 lM (Fig. 4A). The superoxide anion-producing activity of F2pal16 was compared with that of two other FPR2 agonists, SAA and MMK-1. Stimulation of human monocytes with MMK-1 (1 lM) enhanced superoxide anion production (Fig. 4B). The MMK-1-induced superoxide anion production was about twofold more potent than that observed with F2pal-16. However, stimulation of human monocytes with SAA (2 lM) failed to enhance superoxide anion production (Fig. 4B). 3.5. F2pal-16 stimulates production of several inflammatory cytokines in human monocytes The effect of F2pal-16 on the production of several cytokines was investigated in human monocytes. When the cells were stim-
ulated with 5 lM F2pal-16, F2pal-16 induced IL-8 production was assayed at various times, and showed maximal activity within 12 h of stimulation (Fig. 5A). F2pal-16 also stimulated the production of TNF-a, IL-1b, and CCL2 in human monocytes (Fig. 5B and data not shown). F2pal-16-induced TNF-a production was maximal 6 h after stimulation. IL-1b and CCL2 production by F2pal-16 was maximal approximately 12 h after stimulation (Fig. 5B and data not shown). Stimulation of human monocytes with various concentrations of F2pal-16 induced CCL2 production in a concentrationdependent manner (Fig. 5C). We also compared the effect of F2pal-16, MMK-1, or SAA on the production of CCL2 in human monocytes. Stimulation of human monocytes with F2pal-16 (5 lM), or SAA (2 lM) for 24 h elicited CCL2 production (Fig. 5D). However, MMK-1 (5 lM) failed to stimulate CCL2 production from the cells (Fig. 5D). We then tested the role of PTX-sensitive G-protein on the agonist-stimulated CCL2 production from human monocytes. Preincubation of human
H.Y. Lee et al. / FEBS Letters 584 (2010) 4102–4108
4. Discussion
NF−κB binding activity (fold of basal)
1.8
*
1.6 1.2 1.0 0.8 0.6 0.4 0.2
Vehicle F2pal-16
25
CCL2 (ng/ml)
*
1.4
0.0
4107
*
SAA
MMK-1
BAY 11-7082 SC-514
*
20 #
15 #
10
# #
#
#
5 0
Inhibitor (μM) 0
0
1
5
10
20
F2pal-16 (μM) Fig. 6. F2pal-16-stimulated CCL2 production is mediated by NF-jB activity. Human monocytes were stimulated with vehicle F2pal-16 (5 lM), SAA (2 lM), or MMK-1 (5 lM) for 30 min. Nuclear protein was extracted and NF-jB activity was measured using a NF-jB activity measuring kit (A). Human monocytes were pre-incubated with several concentrations of BAY 11-7082 or SC-514 (0, 1, 5, 10, and 20 lM) at 37 °C for 1 h, followed by stimulation with 5 lM of F2pal-16 for 12 h (B). Secreted CCL2 levels were determined by ELISA (B). The results are presented as the means ± S.E. of three independent experiments. *P < 0.05, compared with the value obtained from the control (vehicle treated); #P < 0.05, significantly different from the control (F2pal-16 only treated) (B).
monocytes with PTX prior to the addition of F2pal-16 or SAA inhibited CCL2 production by these two agonists (Fig. 5D), indicating that F2pal-16 or SAA-induced CCL2 production is mediated by PTX-sensitive G-protein(s). Preincubation of human monocytes with 10 lM WRW4 prior to the addition of SAA or F2pa1-16 led to dramatic inhibition of CCL2 production by SAA (Fig. 5E), whereas F2pal-16-stimulated CCL2 production was not inhibited by WRW4 (Fig. 5E). 3.6. F2pal-16 stimulates NF-jB activity, resulting in CCL2 production in human monocytes Past studies have demonstrated that the expression of the CCL2 gene requires the activation of NF-jB [26,27]. We also tested the effect of F2pal-16 on NF-jB activity in human monocytes using an ELISA kit to detect activated NF-jB. The NF-jB activity was enhanced after exposing monocytes to F2pal-16 for 30 min (Fig. 6A). We further investigated the role of NF-jB on F2pal-16-induced CCL2 production by using two NF-jB inhibitors (BAY 11-7082 and SC-514). CCL2 production by F2pal-16 was almost completely inhibited by two inhibitors (Fig. 6B). The results indicate that NFjB is required for F2pal-16-induced CCL2 production in human monocytes. Another FPR2 agonist, SAA, but not MMK-1, stimulated NF-jB activity in human monocytes (Fig. 6A). The result was correlated with the data that SAA, but not MMK-1, stimulated production of pro-inflammatory cytokines such as CCL2 from human monocytes.
Palmitoylated peptide sequences of some GPCRs are known to act as ligands or modulators of GPCRs [16,17]. In this study we developed a novel FPR2 agonist, F2pal-16, which stimulates FPR2-induced G-protein activation. The pepducin stimulated a broad range of cellular responses in human monocytes, including superoxide anion production, chemotactic migration, and cytokine production. We demonstrated that one palmitoylated peptide (F2pal-16) can act a novel agonist for FPR2. Characterization of F2pal-16 as an agonist for FPR2 revealed that F2pal-16 stimulates a broad range of cellular responses, including superoxide anion production, chemotactic migration, and cytokine production. Some agonists for FPR2 stimulated a limited range of cellular responses. For example, WKYMVm potently stimulates chemotactic migration and superoxide anion production, but fails to stimulate cytokine (CXCL8 and CCL2) production in human neutrophils [13,28]. SAA strongly stimulates chemotactic migration and cytokine production, but does not stimulate superoxide anion production in human neutrophils [13]. In contrast, we have demonstrated that F2pal-16 stimulates all tested cellular responses (chemotactic migration, superoxide anion production, and cytokine production) in human monocytes. WKYMVm and SAA are peptide ligands that act on the receptor without membrane-tethering, whereas F2pal-16 stimulates FPR2 after membrane-tethering. It has been shown in other studies that pepducins act on the juxtaposed region of GPCR and G-protein(s) [16,17,29]. We suggest that F2pal-16 stimulates a broad range of cellular responses because it can act on FPR2 with a unique mechanism of action. In terms of the site of action of F2pal-16 on FPR2, it may bind to a site distinct from that of extracellular ligands. In this study we examined the effect of WRW4 (an FPR2 antagonist) on F2pal-16-induced signaling. Pretreatment of WRW4 prior to F2pal-16 treatment did not block F2pal-16-induced calcium signaling (data not shown). However, WRW4 pretreatment completely blocked calcium signaling induced by another FPR2 agonist, MMK-1 (data not shown). Previous work has demonstrated that pepducin, which is synthesized from the intracellular loop of GPCR, acts intracellularly by regulating the coupling of heterotrimeric G-protein and target GPCR [16,17]. In our study, we synthesized a pepducin peptide based on the intracellular loop 3 region of FPR2, which is expected to act on an intracellular target, but not on the cell surface. Keeping in mind that WRW4 is an antagonist which acts on the cell surface receptor in the extracellular milieu, it makes sense that F2pal-16-induced calcium signaling was not inhibited by WRW4. As shown in Figs. 3 and 5, WRW4 only blocked monocyte chemotaxis or CCL2 production induced by SAA or MMK-1, but not that induced by F2pal-16. These results suggest that F2pal-16 has a different binding site from that of WRW4 on FPR2. Past study already clearly demonstrated that palmitoylated peptides act on the intracellular region of specific GPCRs such as PAR1 [30]. Taking together the past study and our data, we can suggest that our F2pal-16 also acts on the intracellular region of FPR2. In terms of receptor specificity of F2pal-16 in the FPR family, we clearly demonstrated that F2pal-16 stimulates FPR2 but not FPR1 (Fig. 2). Since human primary monocytes express all 3 FPRs (FPR1, FPR2 and FPR3), at this point we could not clearly rule out an involvement of FPR3 on the F2pal-16-induced responses in human monocytes. Several previous reports have demonstrated that NF-jB is required for the induction of CCL2 in some cell types [26,27]. We demonstrated that F2pal-16 stimulates NF-jB (Fig. 6A). Regarding the role of NF-jB on F2pal-16-induced CCL2 induction, we observed that BAY 11-7082 completely inhibited F2pal-16-induced CCL2 production from human monocytes (Fig. 6B), suggesting that
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the NF-jB pathway is required for the induction of CCL2 by F2pal16 in monocytes. Taken together, the results suggest that NF-jB signaling is a prerequisite for the proper induction of CCL2 by F2pal-16 in monocytes. Keeping in mind that F2pal-16 permeabilizes and stimulates FPR2, thus eliciting G-protein activation, it is reasonable to suggest that F2pal-16 induces the activation of a broad range of signaling down-stream of FPR2, resulting in the activation of the NF-jB pathway. In conclusion, we have developed a novel FPR2-specific membrane-tethering cell permeable peptide, F2pal-16. Since F2pal-16 stimulated a broad range of FPR2 down-stream signaling resulting in superoxide anion production and cytokine production, this molecule can be regarded as an important ligand for the study of the mechanism of activation and immunologic role of FPR2.
[10]
[11]
[12]
[13]
[14]
5. Disclosure statement [15]
Some of the authors (Ha Young Lee and Yoe-Sik Bae) have pending patent applications.
[16]
Acknowledgements
[17]
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20100020940); National Research Foundation of Korea grant funded by the Korea government (2009 0093198); and by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (0920220). References [1] Le, Y., Yang, Y., Cui, Y., Yazawa, H., Gong, W., Qiu, C. and Wang, J.M. (2002) Receptors for chemotactic formyl peptides as pharmacological targets. Int. Immunopharmacol. 2, 1–13. [2] Migeotte, I., Communi, D. and Parmentier, M. (2006) Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses. Cytokine Growth Factor Rev. 17, 501–519. [3] Kim, S.D., Kim, J.M., Jo, S.H., Lee, H.Y., Lee, S.Y., Shim, J.W., Seo, S.K., Yun, J. and Bae, Y.S. (2009) Functional expression of formyl peptide receptor family in human NK cells. J. Immunol. 183, 5511–5517. [4] Lee, H.Y., Kim, S.D., Shim, J.W., Lee, S.Y., Lee, H., Cho, K.H., Yun, J. and Bae, Y.S. (2008) Serum amyloid A induces CCL2 production via formyl peptide receptorlike 1-mediated signaling in human monocytes. J. Immunol. 181, 4332–4339. [5] Lee, H.Y., Kim, M.K., Park, K.S., Shin, E.H., Jo, S.H., Kim, S.D., Jo, E.J., Lee, Y.N., Lee, C., Baek, S.H. and Bae, Y.S. (2006) Serum amyloid A induces contrary immune responses via formyl peptide receptor-like 1 in human monocytes. Mol. Pharmacol. 70, 241–248. [6] O’Hara, R., Murphy, E.P., Whitehead, A.S., FitzGerald, O. and Bresnihan, B. (2004) Local expression of the serum amyloid A and formyl peptide receptor-like 1 genes in synovial tissue is associated with matrix metalloproteinase production in patients with inflammatory arthritis. Arthritis Rheum. 50, 1788–1799. [7] Yang, De., Chen, Q., Schmidt, A.P., Anderson, G.M., Wang, J.M., Wooters, J., Oppenheim, J.J. and Chertov, O. (2000) LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J. Exp. Med. 192, 1069–1074. [8] Maddox, J.F., Hachicha, M., Takano, T., Petasis, N.A., Fokin, V.V. and Serhan, C.N. (1997) Lipoxin A4 stable analogs are potent mimetics that stimulate human monocytes and THP-1 cells via a G-protein-linked lipoxin A4 receptor. J. Biol. Chem. 272, 6972–6978. [9] Su, S.B., Gong, W., Gao, J.L., Shen, W., Murphy, P.M., Oppenheim, J.J. and Wang, J.M. (1999) A seven-transmembrane, G protein-coupled receptor, FPRL1,
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