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The Journal of Immunology, 2004, 173: 428-436.
Copyright © 2004 by The American Association of Immunologists

Identification of Neutrophil Granule Protein Cathepsin G as a Novel Chemotactic Agonist for the G Protein-Coupled Formyl Peptide Receptor 1,2

Ronghua Sun*, Pablo Iribarren*, Ning Zhang*, Ye Zhou*, Wanghua Gong{dagger}, Edward H. Cho{ddagger}, Stephen Lockett{ddagger}, Oleg Chertov{dagger}, Filip Bednar§, Thomas J. Rogers§, Joost J. Oppenheim* and Ji Ming Wang3,*

* Laboratory of Molecular Immunoregulation, Center for Cancer Research, and {dagger} Basic Research Program and {ddagger} Image Analysis Laboratory, Science Applications International Corp.-Frederick, National Cancer Institute at Frederick, Frederick, MD 21702; and § Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The antimicrobial and proinflammatory neutrophil granule protein cathepsin G (CaG) has been reported as a chemoattractant for human phagocytic leukocytes by using a putative G protein coupled receptor. In an effort to identify potential CaG receptor(s), we found that CaG-induced phagocyte migration was specifically attenuated by the bacterial chemotactic peptide fMLP, suggesting these two chemoattractants might share a receptor. In fact, CaG chemoattracts rat basophilic leukemia cells (RBL cells) expressing the high affinity human fMLP receptor FPR, but not parental RBL cells or cells transfected with other chemoattractant receptors. In addition, a specific FPR Ab and a defined FPR antagonist, cyclosporin H, abolished the chemotactic response of phagocytes and FPR-transfected cells to CaG. Furthermore, CaG down-regulated the cell surface expression of FPR in association with receptor internalization. Unlike fMLP, CaG did not induce potent Ca2+ flux and was a relatively weaker activator of MAPKs through FPR. Yet CaG activated an atypical protein kinase C isozyme, protein kinase C{zeta}, which was essential for FPR to mediate the chemotactic activity of CaG. Thus, our studies identify CaG as a novel, host-derived chemotactic agonist for FPR and expand the functional scope of this receptor in inflammatory and immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils rapidly accumulate at sites of inflammation and infection, and these cells are activated by bacterial and host-derived factors. After phagocytosis of microorganisms or other particulate substances, neutrophils secrete a variety of mediators that possess potent proinflammatory and antimicrobial activities. These mediators include a group of antibiotic peptides and proteases that are stored in neutrophil granules and released during the process of degranulation.

Cathepsin G (CaG) 4 is a member of the serine protease family, largely present in azurophilic granules of neutrophils, and comprises up to 18% of the azurophil granule proteins. It is referred to as a chymotrypsin-like enzyme because it hydrolyzes peptide bonds after leucine, methionine, and phenylalanine residues. In addition to its capacity to proteolytically degrade engulfed cell debris and its microbicidal activity (1, 2, 3), CaG displays a variety of pathophysiological effects, such as degradation of extracellular matrix and regulating bioactivity of cytokines and cytokine receptors (2, 3), induction of proinflammatory cytokines in macrophages, enhancing hemopoietic progenitor cell mobilization by cleaving CD106 (4), disrupting interaction between CXCR4 and stromal cell-derived factor-1{alpha} (5), and regulation of neutrophil migration by modifying the P-selectin receptor P-selectin glycoprotein ligand-1 (6). Human CaG has been shown to promote specific Ab responses when injected in mice (7). In addition, CaG has been implicated in supporting wound healing as shown by decreased wound-breaking strength in mice depleted of CaG gene (8). Recently, CaG purified from human neutrophil granules was reported to be a potent chemoattractant for human phagocytic leukocytes and to increase the random mobility of T lymphocytes in vitro (9, 10). The chemotactic activity of CaG for phagocytic leukocytes was sensitive to pertussis toxin implying the involvement of a G protein coupled seven-transmembrane (STM) receptor (9, 10). Characterization of receptor(s) used by CaG should greatly promote the understanding of the biological role of this neutrophil granule protein in host response in inflammation and microbial infection. In this article, we report the identification of a Gi protein-coupled human STM receptor formyl peptide receptor (FPR), originally defined as a high affinity receptor for the bacterial chemotactic peptide fMLP, as a functional receptor for CaG that is responsible for its chemotactic activity for phagocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and cells

CaG, purified from neutrophil granules, was purchased from Athens Research and Technology (Athens, GA). The synthetic N-formyl peptide fMLP, {alpha}1-antichymotrypsin ({alpha}1-ACT), PMA, staurosporine, and wortmannin were purchased from Sigma-Aldrich (St. Louis, MO). MMK-1 peptide was synthesized and purified by the Department of Biochemistry, Colorado State University (Fort Collins, CO) according to the published sequence (11). Gö6850 and chelerythrine chloride were from Calbiochem (San Diego, CA). Fura 2-AM was obtained from Molecular Probes (Eugene, OR). Anti-human FPR Ab was from BD PharMingen (San Diego, CA). Cyclosporin H (CsH) was a gift from Novartis (Basel, Switzerland). Anti-total and phosphorylated (p) ERK1/2, p38 MAPK, and Akt Abs were products of Cell Signaling Technology (Beverly, MA). Anti-protein kinase C (PKC) {alpha}{beta}, {delta}, and {zeta} polyclonal Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Human PBMC were isolated from leukopacks obtained through the courtesy of Transfusion Medicine Department, National Institutes of Health Clinical Center (Bethesda, MD) by Ficoll-Hypaque (Sigma-Aldrich) density gradient centrifugation. Monocytes were purified from human PBMC by Percoll gradient (Amersham Biosciences, Little Chalfont, U.K.) to yield >90% pure preparations. Rat basophilic leukemia cells (RBL cells) stably transfected with epitope-tagged high affinity fMLP receptor FPR (designated ETFR cells) were a gift from Drs. H. Ali and R. Snyderman, Duke University (Durham, NC) and maintained in the presence of 0.8 mg/ml geneticin (G418; Life Technologies, Gaithersburg, MD) in DMEM supplemented with 10% FCS.

Chemotaxis assay

Migration of phagocytes and ETFR cells was assessed using a 48-well microchemotaxis chamber (Neuroprobe, Cabin John, MD) as previously described (12). Briefly, different concentrations of stimulants were placed in the wells of the lower compartment of the chamber, and 50 µl of cells (1.5 x 106 cells/ml for monocytes and neutrophils, 1 x 106 ETFR cells/ml) were added to the wells of the upper compartment. The lower and upper compartments were separated by either a 5-µm (for monocytes) or a 10-µm pore size, collagen-coated (for ETFR cells) polycarbonate filter (Osmonics, Livermore, CA). After incubation at 37°C in humidified air with 5% CO2 (60 min for neutrophils, 90 min for monocytes, 4.5 h for ETFR cells), the filter was removed and stained, and the cells migrating across the filter were counted with light microscopy. The results are presented as the mean number of cells (± SD) in three high power fields. For analysis of cross-desensitization, cells were pretreated with indicated reagents for 1 h at 37°C. After thorough washing with cold medium (RPMI 1640, 1% BSA), the cells were resuspended and assayed. To measure the effect of various signaling inhibitors, unless otherwise indicated, the cells were preincubated at 37°C for 60 min with the inhibitors and were then added to the upper wells of the chemotaxis chamber.

Calcium flux

Monocytes or ETFR cells (107 cells/ml in RPMI 1640 containing 10% FBS) were loaded with 5 µM fura-2 at room temperature (30 min for monocytes, 60 min for ETFR cells) in the dark. The cells were washed and resuspended (0.5 x 106 cells/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). Two milliliters of the cell suspension were transferred to a quartz cuvette, which was placed in a luminescence spectrometer LS55 (PerkinElmer, Beaconsfield, U.K.). Calcium mobilization was measured by recording the ratio of the fluorescence excited at 340 and 380 nm in response to stimulants.

Phosphorylation of MAPKs and Akt

ETFR cells were plated in petri dishes and cultured in DMEM at 37°C in humidified 5% CO2 atmosphere for 48 h. After further culture in serum-free DMEM overnight, the cells were treated with stimulants for indicated time periods. The reaction was stopped by adding 1 ml of ice-cold PBS, and the cells were lysed in 1x SDS lysis buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol, and 50 mM DTT, pH 6.8) and harvested into tubes for sonication. Proteins (50 µg for each sample) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were blocked for 2 h at room temperature in 3% nonfat milk prepared in Tris-buffered saline-T (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20) and probed with anti-p-ERK1/2, pp38 MAPK, or p-Akt (1/1000) at room temperature for 1–2 h or at 4°C overnight. The membranes were then washed and incubated with HRP-conjugated secondary Abs diluted in Tris-buffered saline-T for 1 h at room temperature. The immune complexes on the membranes were visualized using ECL reagents. Total ERK1/2, p38 MAPK, and Akt were visualized by stripping the same membrane and probing with Abs against total ERK1/2, p38 MAPK, and Akt.

Measurement of FPR cell surface expression

Cell surface expression of FPR was monitored by a flow cytometer (FACS). Monocytes and ETFR cells (1 x 106 cells/sample) were pretreated with medium or stimulants at 37°C for 30 min. After two washes with cold FACS buffer (Dulbecco’s PBS (DPBS), 1% FCS, 0.02% sodium azide, and 5 mM EDTA, pH 7.4), the cells were blocked with 5% normal rat serum followed by incubation with a monoclonal anti-human FPR Ab or isotype matched IgG on ice for 30 min. After three washes with FACS buffer, FITC-labeled rat anti-mouse Ab (PharMingen) was applied and incubated on ice for 30 min. The cells were then washed twice with FACS buffer and analyzed immediately by flow cytometry (BD Biosciences, San Jose, CA).

Binding assays with [3H]fMLP and 125I-CaG

3[H]fMLP was purchased from Amersham Biosciences (Piscataway, NJ). Human monocytes or ETFR cells (2 x 106 cells in 100 µl of RPMI 1640 containing 1% BSA and 0.5% sodium azide) in duplicate samples were incubated with 3 nM [3H]fMLP in the presence of increasing concentrations of unlabeled fMLP or CaG at room temperature for 30 min. The cells were then filtered onto Whatman paper discs, which were air-dried and measured for {beta} emission. The level of nonspecific [3H]fMLP binding to the cells was determined by the residual radioactivity on cells in the presence of 10 µM unlabeled fMLP (<20% of total binding in the absence of unlabeled fMLP). The results were presented as the capacity of CaG to displace specific [3H]fMLP binding to the cells (percent inhibition).

CaG was iodinated with a chloramine-T method (Lofstrand Laboratories, Gaithersburg, MD) with a specific activity of 15.8 pCi/µg protein. Duplicate samples of ETFR cells or the parental RBL cells (2 x 106 cells in 200 µl of RPMI 1640 containing 1% BSA, 25 mM HEPES, and 0.5% NaN3) were incubated with 25 nM 125I-CaG in the presence of increasing concentrations of unlabeled ligands. After incubation and rotation at room temperature for 1 h, the cells were pelleted through a 10% sucrose-PBS cushion for 1 min at 10,000 x g. The supernatant was removed, and the radioactivity associated with cell pellets was measured in a gamma counter (CliniGamma; Pharmacia Biotech, Uppsala, Sweden). The binding data were analyzed with a Macintosh computer-aided program LIGAND (Dr. P. Munson, Division of Computer Research and Technology, National Institutes of Health, Bethesda, MD).

Measurement of PKC translocation and FPR internalization

ETFR cells were seeded on coverslips and cultured overnight in DMEM containing 1% FCS at 37°C. After further culture in FCS-free DMEM for 3 h, cells were treated with stimulants for indicated time periods at 37°C and were then fixed in 4% paraformaldehyde for 5 min at room temperature. The cells were permeabilized with 0.2% Tween 20 for 30 min at 4°C and the reaction was blocked with 5% goat normal serum for 15 min at room temperature. The rabbit anti-PKC{alpha}{beta}, {delta}, and {zeta} Abs (1:1000 in DPBS) were applied separately for 1 h at room temperature. After three rinses with DPBS, the cells were stained with FITC-labeled goat anti-rabbit secondary Ab (BD PharMingen, 1:1000 in DPBS) for 1 h at room temperature. The coverslips were mounted with an antifade, water-based mounting medium with propidium iodide (Vector Laboratories, Burlingame, CA) and analyzed under a laser scanning confocal fluorescence microscope (Zeiss LSM510; NLO Meta, Jena, Germany). Excitation wavelengths 488 (for FITC) and 568 (for propidium iodide) nm were used to generate fluorescence emission in green and red, respectively.

For detection of FPR internalization, ETFR cells were seeded on coverslips and cultured overnight in DMEM containing 10% FCS at 37°C. After pretreatment with medium or stimulants for 30 min at 37°C, the cells were fixed in 0.25% paraformaldehyde for 15 min at 4°C. The cells were then permeabilized with 0.2% Tween 20 for 30 min at 4°C, followed by incubation with a monoclonal anti-human FPR Ab or isotype-matched IgG for 30 min at 4°C. After three washes with 0.1% Tween 20 in DPBS, a FITC-labeled rat anti-mouse Ab (BD PharMingen) was applied to the cells for 30 min at 4°C. The coverslips were covered with 4',6'-diamidino-2-phenylindole, then mounted with an antifade, water-based mounting medium, and analyzed under a laser scanning Zeiss LSM410 fluorescence confocal microscope.

Statistical analysis

All results shown were representatives from at least three experiments. The statistical significance of the differences in cell migration between testing and control groups was analyzed using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cathepsin G induces phagocyte migration mediated by FPR

To define the nature of the putative receptor(s) used by CaG on phagocytes, we examined the capacity of a number of chemoattractants to desensitize phagocyte response to CaG, an approach widely used to identify shared or unique G protein-coupled STM receptors. Although cross-desensitization of Ca2+ mobilization is a reliable and commonly used strategy, unfortunately CaG at the concentrations up to 20 µg/ml (800 nM) lacked significant Ca2+ mobilizing activity in either monocytes or neutrophils (data not shown). Therefore, we resorted to cross-attenuation of chemotaxis. CaG dose-dependently induced monocyte migration (Fig. 1A), which reached maximal activity at a concentration of 5 µg/ml of the protein (200 nM). Of the many chemoattractants tested, the bacterial chemotactic peptide fMLP attenuated monocyte response to CaG (Fig. 1B). Because fMLP has been reported to activate at least two human FPRs, the high affinity receptor FPR and the low affinity FPRL1 (13), we tested the capacity of agonists specific for these two receptors to attenuate the monocyte response to CaG. MMK-1, a specific and highly efficacious peptide agonist for FPRL1 (Ref. 12 and Fig. 1A), did not affect CaG-induced monocyte chemotaxis (Fig. 1B). In contrast, fMLP at nanomolar concentrations effectively attenuated monocyte migration induced by CaG. CaG was also able to attenuate monocyte chemotaxis to low concentrations of fMLP but not MMK-1 (Fig. 1C and data not shown). Because at low concentrations fMLP specifically activates FPR (13), we postulated that CaG shared FPR with fMLP for its phagocyte chemotactic activity. This hypothesis was tested with a mAb against FPR, which when preincubated with monocytes inhibited cell migration to either CaG or fMLP, but not to the FPRL1 agonist MMK-1 (Fig. 1D). In addition, CsH, an antagonist that specifically disrupts FPR signaling and inhibits fMLP-induced phagocyte activation (14), abolished monocyte chemotaxis to CaG and fMLP but not to MMK-1 (Fig. 1E). Thus, CaG appears to use FPR to induce chemotaxis of monocytes.



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  		FIGURE 1. CaG induces human monocyte migration by interacting with FPR. A, Comparison of migration of monocytes in response to CaG, the bacterial peptide fMLP, and MMK-1, a synthetic peptide specific for FPRL1. The results are expressed as the mean number of migrating cells (±SD) in three high power fields (HPF). *, Statistically significant increase in cell migration induced by stimulants vs medium control (p < 0.01). B, Attenuation of monocyte chemotaxis. Monocytes were preincubated with medium alone, fMLP (100 nM), medium containing 0.05% DMSO (as a solvent for MMK-1), or MMK-1 (5 µM) at 37°C for 30 min. After extensive washing, the chemotactic responses of the cells to fMLP (10 nM), MMK-1 (100 nM), or CaG (10 µg/ml, 400 nM) were measured. *, Significantly reduced cell migration in response to stimulants as compared with cells treated with medium alone or with medium containing 0.05% DMSO (p < 0.001). C, Attenuation of monocyte chemotaxis to fMLP by CaG. Monocytes preincubated with 10 µg/ml for 30 min at 37°C were tested for chemotactic response to different concentrations (Log molar) of fMLP. *, Significantly reduced migration as compared with cells treated with medium alone. D, Blockade of CaG-induced monocyte chemotaxis by anti-FPR Ab. Monocytes were preincubated with different concentrations of anti-FPR at room temperature for 15 min and then evaluated for chemotactic response to CaG (10 µg/ml), fMLP (10 nM), or MMK-1 (100 nM). *, Significantly reduced chemotactic response as compared with cells treated with normal mouse IgG. E, Inhibition of CaG-induced monocyte migration by CsH. Monocytes mixed with different concentrations of CsH were assessed for their chemotactic response to CaG (10 µg/ml), fMLP (10 nM), or MMK-1 (100 nM). *, Significantly decreased chemotactic response as compared with cells treated with medium alone (p < 0.01).

 
CaG induces chemotaxis of RBL cells stably transfected with human FPR

To confirm that CaG uses FPR, we used RBL cells transfected to solely express this receptor (ETFR cells). ETFR cells showed a marked and dose-dependent chemotactic response to CaG and fMLP (Fig. 2A). The same cells failed to respond chemotactically to MMK-1, a ligand for FPRL1. Similar to its activity on monocytes, the optimal concentration for CaG to induce ETFR cell migration was 5 µg/ml. Neither CaG nor fMLP induced migration of parental RBL cells (data not shown) suggesting the presence of FPR is required for CaG- and fMLP-induced chemotaxis. Furthermore, anti-FPR Ab (Fig. 2B) and CsH (Fig. 2C) abolished ETFR cell response to CaG. These results confirm CaG as a chemotactic ligand for FPR.



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  		FIGURE 2. CaG is chemotactic for RBL cells transfected with FPR (ETFR cells). A, Migration of ETFR cells in response to different concentrations of CaG, fMLP, or MMK-1. *, Significantly increased chemotactic response as compared with response to medium alone (p < 0.01). B, Inhibition of CaG-induced ETFR cell migration by anti-FPR antibody. ETFR cells were preincubated with different concentrations of anti-FPR Ab or control IgG at room temperature for 15 min and then examined for their chemotactic response to CaG (10 µg/ml). *, Significantly reduced migration of anti-FPR Ab treated cells in comparison with IgG-treated cells (p < 0.01). C, Inhibition of ETFR cell chemotaxis by CsH. ETFR cells were mixed with designated concentrations of CsH and their chemotactic response to CaG (10 µg/ml) or fMLP (100 nM) was assessed. *, Significantly reduced migration of CsH-treated cells compared with cells treated with medium alone (p < 0.01). HPF, High power field.

 
Characteristics of CaG-induced FPR signaling

The signaling events induced by CaG through FPR exhibited several unique characteristics. Although CaG at low micrograms per milliliter (in 4–400 nM range) was potently chemotactic for monocytes, neutrophils, and RBL cells transfected to express FPR (ETFR cells), it was a weak inducer of Ca2+ flux only in ETFR cells (Fig. 3), presumably due to a markedly higher level expression of transfected FPR. Pretreatment of the cells with 10 µg/ml CaG attenuated cell response induced by low concentrations of fMLP (Fig. 3), suggesting that CaG exhibits lower affinity for FPR as compared with fMLP. This notion was supported by a weaker, but significant ability of CaG to down-regulate FPR from the surface of ETFR cells and monocytes (Fig. 4A) as compared with a marked effect of fMLP. The down-regulation of FPR from the cell surface induced by CaG was associated with receptor internalization as assessed by confocal microscopy. Fig. 4B shows that FPR expressed on ETFR cells was rapidly internalized into the cytoplasmic region of the cells on treatment with fMLP. CaG, albeit exhibiting a lower efficacy than fMLP, also significantly and dose-dependently induced FPR internalization. In receptor binding competition experiments, unlabeled fMLP displaced [3H]fMLP binding to monocytes and ETFR cells with an IC50 of ~3 nM, whereas CaG exhibited an IC50 of 100 nM (2.5 µg/ml; Fig. 4C). 125I-CaG was used to directly measure its capacity to bind to RBL cells with or without FPR. ETFR cells exhibited a substantial number of binding sites for 125I-CaG with an estimated Kd of 85 nM (~2 µg/ml; Fig. 4D). The binding of 125I-CaG to ETFR cells was potently inhibited by fMLP (Fig. 4D, left). In contrast, there was no measurable levels of specific binding of 125I-CaG to parental RBL cells (Fig. 4D, right). These results provide additional supporting evidence for CaG to directly interact with FPR. CaG also positively and rapidly (within 1–5 min) activated ERK1/2 (Fig. 5A), p38 MAPK (Fig. 5 C), and Akt (Fig. 5D), molecules downstream of the FPR signaling cascade in ETFR cells in both a time- and dose-dependent manner. The parental RBL cells did not show any increased phosphorylation of ERK1/2 in response to CaG (Fig. 5B). In parallel experiments to measure p-ERK, fMLP at 100 nM showed a maximal effect at 1 min (Fig. 5A). Although these results suggest that compared with fMLP, CaG appears to be a less efficacious ligand for FPR, nevertheless whether a slightly slower rate of CaG in inducing p-ERK in ETFR cells implies the generation of new FPR agonist(s) by this neutrophil protein merits further investigation.



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  		FIGURE 3. Effect of CaG on Ca2+ mobilization in phagocytic leukocytes and ETFR cells in response to fMLP. Cells loaded with fura-2 were examined for Ca2+ mobilization in response to CaG (10 µg/ml) and fMLP. CaG failed to induce significant Ca2+ flux in monocytes (A) or neutrophils (B) but induced a measurable level of Ca flux in ETFR cells (C). CaG (10 µg/ml) significantly attenuated cell response to 0.1 nM fMLP, and in ETFR cells the cell response to CaG (10 µg/ml) was attenuated by 0.1 nM fMLP (C). RFU, Relative fluorescence units.

 


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  		FIGURE 4. Effect of CaG on the cell surface expression of FPR and on binding of [3H]fMLP. ETFR cells or monocytes (A; 1 x 106) were pretreated with medium alone, 5 µg/ml CaG, or 1 µM fMLP at 37°C for 30 min. The cells were washed and stained with a FITC-conjugated mouse anti-human FPR Ab ({alpha}-FPR) or with an isotype-matched control IgG (IgG). Cell surface-expressed FPR was then detected by FACS. The internalization of FPR induced by fMLP (1 µM) or different concentrations of CaG was assessed by incubating ETFR cells on chamber slides with the ligands for 30 min at 37°C. The cells were then washed, stained with anti-FPR Ab followed by a FITC-conjugated secondary Ab, and examined for fluorescence distribution with confocal microscopy (B). ETFR cells and monocytes (C) were also measured for binding of [3H]fMLP and competition by unlabeled fMLP or CaG. The total binding was determined by using 3 nM (3 )[3H]fMLP on 2 x 106 cells. The nonspecific binding was assessed by residual radioactivity (counts per minute) on cells in the presence of 10 µM unlabeled fMLP. The results are expressed as the percent inhibition of specific binding by different concentrations of unlabeled fMLP or CaG. The IC50 values for fMLP and CaG were estimated at 3 and 100 nM, respectively. ETFR cells (D, left) or the parental RBL cells (D, right) were measured for binding of 125I-CaG (25 nM) in the presence or absence of unlabeled CaG or fMLP. The number of binding sites per cell and Kd were estimated with a LIGAND computer program.

 


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  		FIGURE 5. Activation of MAPKs and Akt by CaG. ETFR cells (A, C, and D) or parental RBL cells (B) were plated in petri dishes and grown for 48 h. After culture in serum-free medium overnight, the cells were stimulated with 10 µg/ml CaG for different time periods or with different concentrations of CaG for 10 min at 37°C. Cell lysates were subject to SDS-PAGE and Western blotting with specific anti-p-ERK1/2 (A and B), p-p38 MAPK (C), or p-Akt (D) Abs. The stripped membranes were subsequently analyzed to determine total ERK1/2, p38 MAPK, or Akt protein levels. fMLP at 100 nM was used in control experiments.

 
FPR-dependent activation of PKC{zeta} by CaG

Translocation of PKC isozymes to different cellular compartments is a hallmark of selective PKC activation (15). Among the PKC isozymes, PKC{xi} has been reported as a critical signal transducer in promoting cell migration induced by a variety of stimulants (16, 17). In unstimulated ETFR cells, PKC{zeta} was mainly detected in the cytoplasm. After stimulation with CaG, a substantial proportion of PKC{zeta} translocated to the cell membrane region as assessed by confocal microscopy (Fig. 6A). The induction of PKC{zeta} translocation by CaG was dependent on the presence of FPR because CaG did not cause any changes in the pattern of PKC{zeta} distribution in the parental RBL cells (Fig. 6B). CaG did not induce translocation of PKC{alpha} and {beta} in ETFR cells, two members of the classical PKC subfamily that are dependent on the generation of diacyl glycerol and Ca2+, two second messengers coupled to many cell surface receptors (18). This is in agreement with a weaker capacity for CaG to trigger FPR-dependent Ca2+ mobilization. In contrast, fMLP, which elicits both FPR-dependent chemotaxis and Ca2+ flux, promoted the translocation of not only PKC{zeta}, but also PKC{alpha}{beta} (Fig. 6A). Neither CaG nor fMLP stimulated PKC translocation in parental RBL cells, whereas PMA activated these PKC isozymes in both ETFR and RBL cells (Fig. 6). CaG and fMLP did not induce translocation of PKC{delta}, a member of the novel PKC subfamily, suggesting that activation of this PKC isozyme may not be involved in the signaling of FPR on ligation with CaG or fMLP. Furthermore, both staurosporine, a general PKC inhibitor, and wortmannin, a PI3K inhibitor, abrogated chemotaxis of ETFR cells induced by either CaG or fMLP (Fig. 7, A and B), and chelerythrine chloride, a specific PKC{zeta} inhibitor markedly inhibited the cell migration to CaG and fMLP (Fig. 7C). In contrast, Gö 6850 (bisindolylmaleimide I), an inhibitor of classical ({alpha}, {beta}1, and {beta}2) and novel ({delta}, {epsilon}, {eta}, and {theta}) PKC (19) had a limited effect on fMLP- and CaG-induced ETFR cell migration (our unpublished data). These results were also confirmed in human neutrophils where the PKC{zeta} inhibitor chelerythrine chloride abolished the cell chemotaxis to CaG, fMLP as well as a chemokine IL-8, whereas Gö 6850 had no effect (Fig. 7, D and E).



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  		FIGURE 6. Translocation of PKC{zeta} induced by CaG in ETFR cells. ETFR cells (A) or parental RBL cells (B) were treated with medium, 100 nM fMLP, or 10 µg/ml CaG at 37°C for 10 min. The cells were fixed with 4% PFA and were separately stained with anti-PKC{alpha}{beta}, {delta}, or {zeta} Ab. PMA (500 nM), an activator of diacyl glycerol-calcium-dependent PKC isozymes, was used as a positive control, which induced translocation of PKC{zeta}, PKC{alpha}{beta}, and PKC{delta} in ETFR and RBL cells.

 


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  		FIGURE 7. Effect of protein kinase inhibitors on CaG-induced cell migration. ETFR cells (A–C) or neutrophils (D and E) were incubated with different concentrations of chelerythrine (C and D), Gö6850 (E), staurosporine (A), or wortmannin (B) at 37°C for 1 h. The cells were then examined for chemotaxis in response to CaG (10 µg/ml) or fMLP (100 nM). *, Significantly reduced cell migration compared with cells treated with medium alone (p < 0.01). None of the inhibitors at concentrations used in the studies affected spontaneous cell migration. HPF, High power field.

 
CaG does not generate new and soluble FPR agonist(s)

Cleavage by proteases is required for regulation of the function of a number of molecules in triggering proper physiological processes (20). Modification of the enzymatic site of CaG with PMSF and incubation with {alpha}1-ACT inhibited its chemotactic activity (9), implying that an enzymatically intact form of CaG is important for its phagocyte chemotactic activity. Therefore, we examined whether interaction of FPR with CaG might cleave FPR to generate a new de facto agonist for this receptor, such as in the model of thrombin activation of its receptors (21, 22, 23). ETFR cells were cultured with CaG under the conditions sufficient for cleavage of other proteins by CaG (24). The supernatant was collected and examined for its chemotactic activity for FPR. Fig. 8 shows that although the supernatant from CaG-digested ETFR cells contained a low level of chemotactic activity, such activity was completely abolished by additional treatment with the CaG inhibitor {alpha}1-ACT, suggesting that the chemotactic activity contained in the supernatant of CaG-digested ETFR cells is due to the residual active CaG but not a newly generated FPR agonist. Supernatants from CaG-treated RBL cells also exhibited some chemotactic activity for ETFR cells, which was inhibited by {alpha}1-ACT, suggesting that CaG also did not generate any new FPR agonists from RBL cells (Fig. 8). {alpha}1-ACT did not affect the chemotactic activity of fMLP for FPR (Fig. 8), supporting the notion that CaG is unlikely to cause the release of small and soluble peptide agonist(s) during its interaction with FPR-expressing cells. Also, detection of CaG-induced FPR internalization as shown in Fig. 4B by confocal microscopy and direct binding of 125I-CaG to ETFR cells argue against the potential cleavage of FPR by CaG. Nevertheless, whether CaG may generate chemotactic molecules that could tightly associate with the membrane on the leading edge of migrating cells remains to be clarified.



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  		FIGURE 8. CaG treatment of ETFR cells did not generate soluble chemotactic activity. ETFR and RBL cells (10 x 106/ml) were incubated with medium (med) or 10 µg/ml CaG at 37°C for 1 h. Supernatants were collected and measured for chemotactic activity for untreated ETFR cells. The supernatants were also treated for 15 min at room temperature with {alpha}1-ACT (0.75 µM) before being tested for chemotactic activity for ETFR cells. {alpha}1-ACT-treated fMLP (100 nM) was tested in parallel. ETFR + med, supernatant from ETFR cells treated with medium; ETFR + CaG, supernatant from ETFR cells treated with CaG; RBL + med and RBL + CaG, supernatants from RBL cells treated with medium or CaG. fMLP (100 nM) and CaG (10 µg/ml) cultured in the absence (ACT–) or presence (ACT+) of {alpha}1-ACT were used as positive controls. *, Significantly reduced chemotactic activity shown by {alpha}1-ACT-treated CaG and supernatants compared with medium treatment. HPF, High power field.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study identifies the neutrophil granule protein CaG as a novel chemotactic agonist for the human Gi protein-coupled STM receptor FPR. FPR was originally identified as a high affinity receptor on human monocytes and neutrophils for the bacterial and synthetic chemotactic formyl peptide fMLP (25). Activation of FPR by fMLP results in a G protein-mediated intracellular signaling cascade that leads to increased leukocyte chemotaxis, phagocytosis, and production of reactive oxygen intermediates (25, 26, 27). These properties of FPR led to the assumption that the receptor might play a significant role in host defense against microbial infection by promoting phagocyte recruitment and secretion of bactericidal mediators. Evidence to support this assumption was provided by reduced resistance to Listeria monocytogenes infection by mice devoid of murine FPR1, a mouse orthologue of human FPR (28). In humans, dysfunctional variant FPR alleles are associated with localized juvenile periodontitis caused by Actinobacillus actinomycetescomitans, and the FPR variants encoded by mutated genes exhibited severe or complete deficiency in Gi protein coupling in transfected cells, consistent with a direct causal role of dysfunctional FPR in the disease (29). Thus, both human and mouse models of diseases imply active participation of FPR in anti-microbial responses.

Over the past few years, the functional expression of FPR has been detected in cells of the nonhemopoietic origin, such as hepatocytes (30) and astrocytoma cells (31). This, in conjunction with the findings that FPR interacts not only with bacterial products but also with host-derived polypeptide agonists (25), suggests that FPR may have a broader pathophysiological role. For instance, FPR was reported to mediate the chemotactic activity of formyl peptides released by mitochondria of ruptured cells (32). These peptides may mobilize phagocytic leukocytes to mediate the clearance and repair of damaged tissue. Another newly identified, host-derived FPR agonist is annexin I, also termed lipocortin, a glucocorticoid-regulated protein with both pro- and anti-inflammatory properties (33). At low concentrations, the annexin I holoprotein and its N-terminal peptides elicit Ca2+ transients through FPR in neutrophils without fully activating the MAPK pathway. Annexin I also induces neutrophil desensitization, resulting in reduced transendothelial migration in response to other unrelated chemoattractants, such as the chemokine IL-8. At high concentrations, annexin I peptides fully activate neutrophils in vitro and become potent proinflammatory stimulants (33). Thus, FPR also participates in the fine-tuning of neutrophil extravasation when multiple chemoattractants are present. Consistent with this notion, spinorphin (LVVYPWT), a host-derived heptapeptide of the opioid hemorphin family, has been identified as a specific antagonist of FPR (34). This discovery suggests a direct mechanism by which an FPR ligand may negatively regulate the inflammatory response.

CaG has been reported to bind to a variety of cells including alveolar macrophages (35), T cells, and NK cells (36). On platelets, 2 x 107 binding sites have been detected for CaG with maximal binding at 35 µg/ml of the protein (37). In fact, two G protein-coupled receptors, protease-activated receptors (PAR) 1 and 4, which were originally identified as thrombin receptors, have been reported to mediate CaG-induced Ca2+ mobilization and aggregation in platelets (38). However, other studies presented evidence that CaG and thrombin may use different receptors on platelets, because Ab against thrombin receptor, although inhibiting thrombin-induced platelet responses, had no effect on the activity of CaG (39). Nevertheless, it is not known whether platelets also express FPR and are able to respond chemotactically to CaG and the bacterial peptide fMLP. A recent study reported activation by CaG of cardiomyocytes, including increased phosphorylation of MAPKs and Akt, from mice lacking PAR1 or PAR4, indicating these receptors are not involved in the effect of CaG on cardiomyocytes (40). In our studies of human phagocytic leukocytes and FPR-transfected RBL cells, preincubation of the cells with thrombin had no effect on subsequent cell migration induced by CaG or fMLP (R. Sun, unpublished data). Thus, although we cannot rule out that CaG may have the capacity to interact with multiple receptors to exert its diverse activities on different cell types, its chemotactic activity for human phagocytes is clearly mediated by FPR.

Compared with bacterial chemotactic peptide fMLP, CaG appears to possess lower affinity for FPR, as evidenced by the requirement of 5–10 µg/ml (200–400 nM) of the protein to elicit optimal cell migration. Unlike fMLP, CaG did not trigger potent Ca2+ transients in phagocytic leukocytes or FPR-transfected RBL cells and, despite its rapid effect on MAPK phosphorylation, there is a certain degree of delay (10–15 min) for CaG to reach its maximal activity as compared with fMLP. Furthermore, fMLP more potently desensitized CaG-induced cell chemotaxis as compared with the capacity of CaG to attenuate cell response to fMLP. A lower capacity of CaG to activate FPR as compared with fMLP was also reflected in the results where fMLP induced translocation of PKC{alpha}{beta} and {zeta}, members of the classical and atypical PKC subfamilies, respectively, and CaG induced the translocation only of PKC{zeta}, an atypical PKC crucial for neutrophil chemotaxis (17). Despite its apparently lower affinity for FPR as compared with fMLP, CaG may be one of the most potent chemotactic factors released by neutrophils during degranulation (9), and micromolar concentrations of CaG could potentially be secreted at sites of acute inflammation and infection where a large quantity of neutrophils accumulate. In addition, low affinity interaction has been reported between a number of pathophysiologically important chemoattractants and their G protein-coupled STM receptors, including the interaction of the FPR variant receptor FPRL1 with Alzheimer’s disease-associated amyloid {beta} peptide 1–42 (41) and another amyloidogenic protein serum amyloid A (42). These chemoattractants are usually present at high concentrations together with significant leukocyte infiltration in the lesion (25). Thus, low affinity agonist-receptor interaction may contribute to the recruitment of leukocytes to sites where high concentrations of the chemoattractant ligands are generated.

The identification of FPR as a chemotactic receptor for CaG may have pathophysiological relevance. Neutrophils represent the first line of host defense against inflammation and bacterial infection by rapidly infiltrating diseased sites in response to chemoattractants produced by invading microorganisms and damaged tissues. On activation, neutrophils release a number of mediators with antimicrobial activity. Among these mediators are granule proteins that act as proteolytic enzymes and as endogenous polypeptide antibiotics such as defensins and cathelicidins (43). Such enzymes and polypeptides may play much broader roles in innate and adaptive host immune responses, because in addition to CaG, a number of other neutrophil granule proteins exhibit chemotactic activity for various cell types by interacting with specific G protein-coupled receptors (9, 44). LL-37/CAP18, a 37-aa peptide naturally cleaved from the C terminus of the neutrophil granule antibacterial protein cathelicidin, is microbicidal, binds and neutralizes the activity of bacterial endotoxin, and is chemotactic for phagocytic leukocytes and resting T cells by interacting with an FPR variant receptor FPRL1 (45). Two other types of neutrophil antimicrobial proteins, the {alpha}-defensins (46) and azurocidin/CAP37, are also leukocyte chemoattractants that use cell surface pertussis toxin-inhibitable G protein-coupled receptors (44, 9). CaG joins the list of antimicrobial chemoattractants released from neutrophil granules with a defined functional receptor FPR as demonstrated in the present studies. The release of chemoattractant peptides by neutrophils has been postulated as an important step in the chain of events galvanizing the host immune system during inflammation, infection (47), and wound healing (3). Because FPR is expressed by a variety of cell types including immature dendritic cells (48, 49), CaG may also act as a link between innate and adaptive immunity by recruiting DC, and CaG-FPR interaction may modulate the function of nonhemopoietic cells that express functional FPR. Thus, identification of CaG as a host-derived chemotactic agonist for FPR provides unique opportunities for further studies of the pathophysiological role of this ligand-receptor pair in inflammation and immunity.


   Acknowledgments
 
We thank Y. Le, G. Ying, and N. M. Dunlop for technical assistance and discussion. The secretarial assistance of C. Fogle and C. Nolan is gratefully appreciated.


   Footnotes
 
1 This work was supported in part by National Cancer Institute, National Institutes of Health Contract N01-C0-12400. F.B. and T.J.R. are supported by National Institutes of Health Grants DA-14230, DA-11130, and DA-06650. Back

2 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 right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. Back

3 Address correspondence and reprint requests to Dr. Ji Ming Wang, Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Building 560, Room 31-40, Frederick, MD 21702. E-mail address: wangji{at}mail.ncifcrf.gov Back

4 Abbreviations used in this paper: CaG, cathepsin G; STM, seven-transmembrane; FPR, formyl peptide receptor; {alpha}1-ACT, {alpha}1-antichymotrypsin; CsH, cyclosporin H; RBL cells, rat basophilic leukemia cells; PAR, protease-activated receptor; PKC, protein kinase C; DPBS, Dulbecco’s PBS. Back

Received for publication December 23, 2003. Accepted for publication April 22, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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