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The Journal of Immunology, 2007, 178, 7395 -7404
Copyright © 2007 by The American Association of Immunologists, Inc.
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Proinflammatory Proteases Liberate a Discrete High-Affinity Functional FPRL1 (CCR12) Ligand from CCL231,2

Zhenhua Miao3,*, Brett A. Premack*, Zheng Wei*, Yu Wang*, Craig Gerard{dagger}, Henry Showell, Maureen Howard*, Thomas J. Schall* and Robert Berahovich*

* ChemoCentryx, Mountain View, CA 94043; and {dagger} Children’s Hospital, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Most chemokines have been found to bind to and signal through single or highly related chemokine receptors. However, a single chemokine protein, a processed form of the alternatively spliced CCL23 (CKbeta8/MPIF-1) gene product, potently engages both the "classical" chemokine receptor CCR1, as well as FPRL1, a type of pattern recognition receptor on innate immune cells. However, the mechanism by which the alternative form of CCL23 is processed is unknown. In this study, we show that proteases associated with inflammation cleave CCL23 immediately N-terminal to the 18-residue domain encoded by the alternatively spliced nucleotides, resulting in potent CCR1 and FPRL1 activity. The proteases also cleave CCL23 immediately C-terminal to the inserted domain, producing a typical CC chemokine "body" containing even further-increased CCR1 potency and a released ~18-aa peptide with full FPRL1 activity but no activity for CCR1. This peptide, which we term SHAAGtide, is by itself an attractant of monocytes and neutrophils in vitro, recruits leukocytes in vivo, and is 50- to 100-fold more potent than all other natural agents posited to act on FPRL1. The appearance of SHAAGtide appears to be transient, however, as the proinflammatory proteases subsequently cleave within the peptide, abolishing its activity for FPRL1. The sequential activation of a transient FPRL1 ligand and a longer-lived CCR1 ligand within a single chemokine may have important consequences for the development of inflammation or the link between innate and adaptive immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In higher organisms, protection from invading pathogens is handled by two distinct yet closely coordinated defense mechanisms—the innate and the adaptive immune responses. Innate immunity is based on receptors that have been selected through evolution to recognize highly conserved and widely distributed features of common pathogens (1, 2). The receptors that recognize these pathogen-associated molecular features are often termed "pattern recognition receptors" or PRRs2 (3).4 These PRRs tend to be expressed by specialized subsets of motile "frontline" innate immune system cells; pathogen recognition by the PRRs generates intracellular signals leading to host cell activation (3, 4). These signals also play a critical role in configuring the subsequent adaptive immune responses (e.g., Th1- vs Th2-regulated effector mechanisms) (5, 6, 7). Certain PRRs lead to Th1 responses against intracellular parasites, viruses and bacteria, while others generate Th2 responses against extracellular parasites and allergens (8, 9, 10).

Chemokines function to orchestrate immune responses by bringing immune system cells to sites of Ag exposure and inflammation. Recently, it has been appreciated that chemokines might function to coordinate innate immune responses to effect successful adaptive immunity (11, 12, 13), although the actual mechanisms have not been defined. Myeloid cells, which include monocytes, macrophages, and certain dendritic cell subsets, are responsive to a number of chemokines (14, 15, 16, 17, 18). Several of these chemokines are absolutely required for myeloid cell homing to appropriate secondary lymphoid organs for Ag presentation to lymphocytes (18, 19).

In addition to chemokine receptors, many myeloid cells also express the unrelated chemotactic peptide receptors, N-formyl peptide receptor (fMLP-R or FPR), and its homologs, the "orphan" receptors FPRL1 and FPRL2 (20, 21, 22, 23, 24). These receptors induce chemotaxis but can also activate myeloid cells and thereby stimulate their Ag-presenting properties (24, 25, 26). FPRL1 was cloned originally from a human phagocyte cDNA library and was characterized by nucleotide homology to FPR, although FPRL1 interacts very weakly with fMLP, the main ligand for FPR (20, 21). FPRL1 has also been reported to act as a functional lipoxin A4 receptor, (27, 28, 29, 30), although there is still some debate over this activity (31, 32). More recently, several groups studying FPRL1 have described a broad spectrum of low-affinity pathogen-related peptide and lipid ligands, as well as several high-affinity, but nonnatural, synthetic peptide ligands (24, 25, 26, 27, 28, 33, 34, 35, 36, 37, 38, 39). The ability of FPRL1 to interact with this broad spectrum of pathogen-related ligands is unusual among G protein-coupled receptors and suggests that FPRL1 may represent a novel type of PRR with the potential for regulating innate immune responses to a number of viral and bacterial pathogens.

In humans, a single gene, CCL23, can give rise to four distinct protein products due to alternative splicing of the third exon and to N-terminal processing. The CCL23 cDNA, encoding a 99-residue protein (herein designated CCL23{alpha}, initially designated CKbeta8, MPIF-1; see review in Ref. 17), was initially isolated from a library derived from human aortic endothelial cells (40). The CCL23{alpha} transcript has been reported to be constitutively expressed in liver, lung, pancreas, and bone marrow (40, 41), and CCL23{alpha} protein has been detected in synovial fluid from rheumatoid arthritis patients (42). In vitro, the CCL23{alpha} protein has chemotactic activity on monocytes and some dendritic cells via the chemokine receptor CCR1 (40, 41). The alternatively spliced form of CCL23, encoding a 116-residue protein termed CKbeta8-1 (herein designated CCL23beta), was isolated from the myeloid cell line THP-1 (43). The CCL23beta mRNA uses a splice acceptor in exon 3, 51 nt upstream of the one used by the CCL23{alpha} mRNA, resulting in the replacement of the CCL23{alpha} Arg25 residue with the 18-residue peptide MLWRRKIGPQMTLSHAAG (43). CCL23beta mRNA expression has been detected in pancreas and skeletal muscle (43), but the protein has heretofore not been detected in human tissue. CCL23{alpha} readily undergoes proteolytic processing by inflammation-associated proteases, which remove the N-terminal domain encoded by exon 2 (42) and thereby increase the protein’s potency for CCR1 (29, 42, 44). Recombinant N-terminally truncated forms of CCL23{alpha} and CCL23beta have been produced and are herein designated CCL23{alpha} {Delta}24 and CCL23beta {Delta}24, respectively.

We previously characterized a number of cell types in the granulocyte, myeloid, and lymphoid lineages for their ability respond to >100 chemotactic stimuli in the chemokine superfamily (our unpublished data). The resulting cell migration array profile revealed unusual migration-inducing activities among the four CCL23 proteins. All four proteins used CCR1 on monocytes, but CCL23beta {Delta}24 was also an extremely potent neutrophil chemoattractant. CCL23beta {Delta}24 was recently shown to be a functional ligand for both CCR1 and FPRL1 (45), which is consistent with our previous observation because neutrophils express FPRL1. However, because CCL23beta protein has not been detected yet in vivo, the identity of the natural processed form is still unknown. Moreover, the process by which the full-length CCL23beta chemokine, which itself is inactive on FPRL1, is processed to the FPRL1-active form is unknown. In this study, we report our investigation into the natural activation of CCL23beta, including defining the mechanism of processing and the determinants responsible for using CCR1 and FPRL1. The implications of these findings on inflammation and immunity are discussed.


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

All chemokines were obtained from R&D Systems. Recombinant CCL23beta was a special order produced according to the published sequence (38). The other three CCL23 variants are R&D Systems regular catalog items (catalog nos. 371-MP, 131-M1, and 508-CK). 125I-CCL3/MIP1{alpha} and 125I-WKYMVm were obtained from PerkinElmer. Lipoxin A4 was obtained from two sources: Calbiochem and BIOMOL. The beta-amyloid protein (1-42) was obtained from American Peptide. Serum amyloid A (SAA)2 was obtained from PeproTech. fMLP, WKYMVm, WKYMVM, SHAAGtide (MLWRRKIGPQMTLSHAAG), scrambled peptide version of SHAAGtide, CCL23beta 1-42 peptide, and SHAAGtide mapping variants were synthesized by either Phoenix Pharmaceuticals or SynPep.

Cells

Human monocytes were isolated from buffy coats (Stanford Blood Center) using CD14 microbeads (Miltenyi Biotec) and magnetic positive selection. Human neutrophils were isolated from fresh peripheral blood of healthy individuals by gradient centrifugation on Ficoll-Hypaque using standard protocols. Transfectants of L1.2 cells expressing human FPRL1 or CCR1 were generated by standard techniques. L1.2 transfectants were maintained in RPMI 1640 medium with 10% FBS with 2 mg/ml G-418 and treated with 8 mM sodium butyrate 16 h before use.

Binding assay

Competition binding studies were conducted using monocytes and L1.2-CCR1 or L1.2-FPRL1 cells. Cells were incubated for 3 h at 4°C with 125I-CCL3/MIP-1{alpha} (final concentration, ~0.05 nM) or 125I-WKYMVm (final concentration, ~0.01 nM) in buffer (25 mM HEPES, 140 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, and 0.2% BSA; adjusted to pH 7.1) in the presence of increasing amounts of unlabeled chemokine. Reactions were aspirated onto polyethyleneimine-treated glass fiber filters using a cell harvester (Packard Instrument). Filters were washed twice (25 mM HEPES, 500 mM NaCl, 1 mM CaCl2, and 5 mM MgCl2; adjusted to pH 7.1). Scintillant (MicroScint-10) was added, and the filters were analyzed in a Packard Topcount scintillation counter. Data were analyzed and plotted using Prism (GraphPad Software).

Receptor signaling assay

For individual measurements, cells were loaded with 2 µM indo-1/AM (Invitrogen Life Technologies) in culture medium for 45 min at room temperature, then washed with HBSS and resuspended at 107 cells/ml in HBSS containing 0.1% BSA. Relative cytosolic calcium levels were determined using a Photon Technology International fluorometer (excitation at 350 nm, ratio of dual emission at 400 and 490 nm). For measuring calcium dose responses, cells were analyzed with a Fluorometric Imaging Plate Reader 384 (FLIPR384) (Molecular Devices). Cells were loaded with 5 µM fluo-4 (Invitrogen Life Technologies) in HBSS with 0.1% BSA for 1 h at 37°C, then washed, transferred to black-wall 96-well plates, and subsequently excited at 505 nm with emission recorded at 530 nm. Data were analyzed and plotted in arbitrary units of fluorescence using Prism. To ensure that responses were specific to FPRL1, a small, molecule antagonist of human FPRL1, identified from a high-throughput compound library screen, was used. This molecule is noncytotoxic and does not interact with any chemokine receptors.

Chemotaxis assay

L1.2-CCR1 and L1.2-FPRL1 transfectants, monocytes, and neutrophils were collected by centrifugation and resuspended in HBSS with 0.1% BSA. The assays were performed in 96-well ChemoTx microplates (NeuroProbe). Chemokines were diluted in HBSS with 0.1% BSA and added to the lower wells (final volume 29 µl), then 20 µl of cell suspension (5 x 106 cells/ml for monocytes; 2.5 x 106 cells/ml for neutrophils) were added to the polycarbonate filters (5-µm pore size for monocytes and FPRL1 transfectants; 3-µm pore size for neutrophils). After incubation of the plates in a humidified chamber at 37°C for 90 min, the filters were removed, and the cells that migrated into the lower chamber were quantified using the CyQuant cell proliferation assay kit (Molecular Probes).

Immunohistochemistry

BALB/c and C57BL/6 mice were injected intradermally with 50 µl of sterile saline with or without 2 µg of synthetic SHAAGtide or a scrambled version of SHAAGtide. Six hours later, mice were euthanized, and the skin surrounding the injection site was excised. After fixation in 10% neutral-buffered formalin and processing and embedding in paraffin wax, 5-µm-wide sections were either stained with H&E (Sigma-Aldrich) or stained immunohistochemically for neutrophils as follows. The slides were immersed in Target Retrieval solution (DakoCytomation) for 20 min at 90°C, rinsed with water, and immersed in TBS containing 2% BSA for 20 min. The sections were then exposed to rat anti-mouse neutrophil mAb (Accurate Chemical and Scientific) or rat IgG2a isotype control Ab (BD Pharmingen) for 1 h, rinsed with TBS, and exposed to biotinylated goat anti-rat Ig (DakoCytomation) for 30 min. The sections were rinsed with TBS, stained with the ABC-AP and fuchsin reagents (both from DakoCytomation), rinsed with water, and counterstained with hematoxylin for 2 min. After rinsing with water, slides were mounted with Crystalmount (Biomedia).

CCL23beta proteolysis

CCL23beta, CCL23beta {Delta}24, or a synthetic peptide corresponding to the N-terminal 42 residues of CCL23beta (containing the N-terminal and SHAAGtide domains) was incubated with proteases at 37°C for varying amounts of time. Proteases included human mast cell chymase (a gift from Dr. N. Schechter, University of Pennsylvania, Philadelphia, PA), Asp-N endopeptidase, and chymotrypsin (both from Sigma-Aldrich). In addition, chemokines were incubated with supernatants collected from a 6-h culture of human neutrophils in serum-free RPMI 1640 medium with or without PMA (0.1 µg/ml) and ionomycin (1 µg/ml; both from Sigma-Aldrich). Control reactions included neutrophil supernatant or protease without chemokine. The reactions were then subjected to SDS-PAGE on a 10–20% gradient tricine acrylamide gel (Invitrogen Life Technologies) and stained with colloidal Coomassie blue (Pierce).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Proinflammatory proteases transform CCL23beta into a FPRL1 ligand

Recently, we published a study showing that serine proteases involved in inflammation perform N-terminal truncations of four alternative CCR1 chemokines in vitro, activating their potency for CCR1 (42). These "NC6" chemokines include human CCL15/MIP-1{delta}/leukotactin-1 and CCL23{alpha}/CKbeta8/MPIF-1 and mouse CCL6/C10 and CCL9/MIP-1{gamma}. Although CCL23beta/CKbeta8-1 contains an 18-aa insertion (termed herein the "SHAAG domain") between the N-terminal inhibitory domain and the C-terminal chemokine domain of CCL23{alpha} (Fig. 1A), we reasoned that the same proteases might also cleave CCL23beta as they cleave CCL23{alpha}. CCL23beta was treated for various times with a panel of human proteases, including chymase, elastase, cathepsin G, chymotrypsin, and Asp-N. In addition, CCL23beta was treated for various times with medium collected from a 6-h culture of freshly prepared neutrophils ("PMN sup") with or without activation by PMA and ionomycin ("act. PMN sup"). By separating the reaction products via SDS-PAGE, processing of CCL23beta into smaller fragments was readily detected (Fig. 1B). Early time points of digestion yielded multiple proteolytic fragments (Fig. 1B, left two panels), with the largest of the fragments exhibiting electrophoretic mobility identical to recombinant CCL23beta {Delta}24 (CCL23beta lacking the N-terminal domain). Because this fragment was the largest fragment, by necessity it must have been produced by one of the initial, if not the initial, cleavage events. N-terminal sequencing of this fragment produced by chymase or the activated neutrophil medium indicated that the latter cleaved CCL23beta after Val21, whereas chymase cleaved CCL23beta after Leu23 (Fig. 1C).


Figure 1
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  		FIGURE 1. Proinflammatory proteases transform CCL23beta into a FPRL1 ligand. A, The CCL23 gene contains two splice acceptors in the third exon and, as a result, gives rise to two gene products, CCL23{alpha} and CCL23beta. The latter contains a novel 18-residue insertion (termed the "SHAAG domain," for its C-terminal 5 aa) in place of Arg25 (Figure 1). Both CCL23{alpha} and CCL23beta contain an inhibitory N-terminal domain of 24 aa (red) and a C-terminal chemokine domain containing 75 aa (blue). B, Digestions of full-length CCL23beta with proteases or with conditioned medium collected from activated neutrophils ("act. PMN sup") were analyzed by SDS-PAGE and Coomassie staining. Left panel, 30 min; middle panel, 10 min; and right panel, 2 h. C, Sequence of the N-terminal 55 aa of CCL23beta, with the SHAAG domain underlined. Sites of cleavage by proteases are indicated. D, Some of the CCL23beta digestions (left panel, 18 h; right panel, 2 h) were added to L1.2-FPRL1 cells at a final concentration of 100 nM input chemokine, and calcium mobilization was monitored over time. E, Digestion of CCL23beta by chymase was analyzed for CCR1 activity by calcium mobilization assay in THP-1 cells. The digestions and control chemokines were used at 5 nM. Chymase-digested CCL23beta was equipotent to CCL23{alpha} {Delta}24, whereas CCL23beta {Delta}24 (containing the SHAAG domain) was poorly active at 5 nM. Right panel, The THP-1 cells were pretreated with 5 µM CCR1-specific compound for 20 s before addition of CCL23{alpha} {Delta}24 or the CCL23beta chymase digestion. FPRL1 signaling was not abrogated by pretreatment of the THP-1 cells with the compound vehicle, DMSO (data not shown).

 
Longer time points of digestion yielded primarily two fragments (Fig. 1B, right panel). The larger of the fragments was similar in mobility to recombinant CCL23{alpha} {Delta}24 (CCL23beta lacking the N-terminal and SHAAG domains), whereas the smaller fragment was similar in mobility to an 18-aa synthetic peptide (termed "SHAAGtide") equivalent to the SHAAG domain. N-terminal sequencing of the larger fragment produced by the proteases indicated that they cleaved the CCL23beta {Delta}24-sized fragment immediately C-terminal to the SHAAG domain, either after Phe43 (chymase, cathepsin G) or Thr46 (elastase or the activated neutrophil medium; Fig. 1C). The CCL23beta {Delta}24-sized fragment and several other fragments present in the short digestions were minor species or undetectable in the longer digestions, suggesting that these fragments were proteolytic intermediates. Indeed, the conversion of the CCL23beta {Delta}24-sized fragment to the CCL23{alpha} {Delta}24-sized fragment indicates that the proteases cleaved CCL23beta sequentially: first, N-terminal to the SHAAG domain and then C-terminal to the SHAAG domain. Digestions of recombinant CCL23beta {Delta}24 by the proteases yielded the same fragments (data not shown).

To determine whether the processed forms of CCL23beta were active for FPRL1, we measured calcium mobilization in L1.2 cells stably expressing human FPRL1. Full-length CCL23beta was unable to induce substantial calcium mobilization in the L1.2-FPRL1 transfectant, similar to control digestions containing protease in the absence of CCL23beta (Fig. 1D). However, the CCL23beta digestions induced calcium mobilization (Fig. 1D). Digestions of CCL23beta with the activated neutrophil medium were particularly potent, perhaps due to the multiplicity of proteases released during neutrophil degranulation. Specificity for FPRL1 was demonstrated by the inability of the digestions to signal in nontransfected L1.2 cells (data not shown).

As expected, the proteolytic fragments produced by cleavage between the SHAAG domain and chemokine body were potent CCR1 ligands (Fig. 1E). Calcium mobilization was detected in THP-1 cells, which express CCR1 but not FPRL1, after exposure to the longer digestions of CCL23beta with chymase. Specificity for CCR1 was demonstrated by the inability of the digestions to signal in THP-1 cells pretreated with the CCR1 antagonist CCX634 (Fig. 1E). The CCR1 activity of the chymase digestions was not unsurprising because the cleavage C-terminal to the SHAAG domain produced a fragment nearly identical to CCL23{alpha} {Delta}24, itself a potent CCR1 ligand (Fig. 1E).

The chemokine domain is not necessary for processed CCL23beta FPRL1 activity

To delineate the cleavage fragment(s) within the digestions that were responsible for FPRL1 activity, similar experiments were performed on a 42-aa peptide containing the N-terminal domain and the SHAAG domain but no sequences from the chemokine body. The 1-42 peptide was digested for 10 or 60 min with the proteases or the neutrophil medium. SDS-PAGE analysis of the digestions revealed that the 1-42 peptide was processed into a form with mobility similar to SHAAGtide (Fig. 2A). The nonactivated neutrophil medium processed the 1-42 peptide with slower kinetics than did the activated neutrophil medium, as some input peptide was still present in the 10-min digestion with the nonactivated neutrophil medium. The increased kinetics of digestion exhibited by the activated neutrophil medium were likely due to increased secretion of proteases upon neutrophil activation. N-terminal sequencing of the chymase and neutrophil medium digestion fragments indicated that they each cleaved the 1-42 peptide at the same sites as full-length CCL23beta (Fig. 2B).


Figure 2
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  		FIGURE 2. Amino acids C-terminal to the inserted domain are not necessary for processed CCL23beta FPRL1 activity. A, Digestions of the CCL23beta 1-42 peptide with proteases were analyzed by SDS-PAGE and Coomassie staining. Left panel, CCL23beta 1-42 peptide was digested for 10 min or 1 h with conditioned medium collected from neutrophils cultured with ("1-42 + Act. PMN sup") or without ("1-42 + PMN sup") PMA and ionomycin. Right panels, 1-42 peptide was digested for 1 h with purified and recombinant proteases. B, Sequence of the 1-42 peptide, containing the N-terminal and SHAAG domains of CCL23beta, with the latter underlined. Sites of cleavage of the 1-42 peptide by chymase and the neutrophil medium are indicated. C–F, Calcium mobilization assays. C, L1.2-FPRL1 cells were exposed to 100 nM 1-42 peptide after 1 h digestion with proteases or neutrophil medium, and calcium mobilization was monitored over time. D, L1.2-FPRL1 cells were exposed to 100 nM 1-42 peptide after 10 min or 1 h digestion with proteases or activated neutrophil medium. Maximal signal levels were plotted in arbitrary units of fluorescence. E, L1.2-FPRL1 cells were exposed to 500 nM SHAAGtide with or without preexposure to 50 nM WKYMVM peptide. F, L1.2-FPRL1 cells were exposed to 100 nM CCL23beta {Delta}24 with or without 2 h pre-exposure to pertussis toxin at 100 ng/ml.

 
To determine whether the processed form(s) of the 1-42 peptide were active for FPRL1, we performed the calcium mobilization assay in the FPRL1 transfectant. The 1-42 peptide was unable to induce substantial calcium mobilization, similar to the control digestions containing protease or medium in the absence of input peptide (Fig. 2C). However, the 1-42 peptide digestions with the proteases and neutrophil medium induced calcium mobilization (Fig. 2C). The SHAAGtide peptide itself was active as well, suggesting that amino acids N-terminal to Met25 were not necessary for FPRL1 function. The pancreatic protease chymotrypsin was not able to render the 1-42 peptide FPRL1 active, nor was a combination of chymotrypsin and Asp-N, suggesting that chymotrypsin cleaved inside the active Asp-N product, rendering it inactive (Fig. 2D). Specificity for FPRL1 was demonstrated by the inability of the digestions to signal in nontransfected L1.2 cells (data not shown) and by the inability of SHAAGtide to signal in L1.2-FPRL1 cells desensitized by WKYMVM peptide (Fig. 2E). As a further control, CCL23beta {Delta}24 was unable to signal in L1.2-FPRL1 cells pretreated with pertussis toxin (Fig. 2F).

SHAAGtide FPRL1 activity mapping

To determine the precise sequence involved in FPRL1 activity, variants of SHAAGtide containing N-terminal or C-terminal truncations were synthesized and tested for potency in receptor signaling in the FPRL1 transfectant (Table I). The data indicate that the N terminus, but not the C terminus, is essential for SHAAGtide activity. After removal of the N-terminal methionine from SHAAGtide, its potency was reduced ~10-fold. Removal of the first two N-terminal amino acids (Met-Leu) totally abolished SHAAGtide’s ability to signal through FPRL1. Conversely, removal of the C-terminal three amino residues (Ala-Ala-Gly) from SHAAGtide had no affect on its activity on FPRL1. Removal of the C-terminal six residues (Lys-Ser-His-Ala-Ala-Gly) reduced the peptide’s potency only 3-fold. We also tested many other naturally occurring FPRL1 ligands, including SAA, beta-amyloid protein 42, lipoxin A4, and the bacterial tripeptide fMLP. However, SHAAGtide was significantly more potent and efficacious in the receptor signaling and chemotaxis assays than these other ligands (Fig. 3). In particular, only SAA and the synthetic, nonnatural peptide WKYMVM exhibited EC50s < 1 µM.


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  		Table I. SHAAGtide activity mapping

 

Figure 3
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  		FIGURE 3. Comparison of FPRL1 ligands. A panel of FPRL1 ligands was tested in calcium mobilization (A) and chemotaxis (B) assays using L1.2-FPRL1 cells. A, Ligands were used at the indicated final concentrations. B, Ligands were used at four different concentrations.

 
SHAAGtide is as potent for FPRL1 as is CCL23beta {Delta}24 but is inactive for CCR1

Because SHAAGtide by itself was functionally active for FPRL1, we compared it to CCL23beta {Delta}24 for potency on L1.2-FPRL1 cells by three methods: receptor signaling, chemotaxis, and receptor binding. Because CCL23beta {Delta}24 is also a ligand for CCR1, we also compared SHAAGtide to CCL23beta {Delta}24 on L1.2 cells stably expressing CCR1. We included CCL23{alpha} {Delta}24 in our analyses because this chemokine is equivalent to CCL23beta {Delta}24 lacking the SHAAG domain. In calcium mobilization assays, L1.2-CCR1 cells responded to CCL23{alpha} {Delta}24 and CCL23beta {Delta}24, as expected; however, SHAAGtide was inactive (Fig. 4A). In the FPRL1 transfectant, SHAAGtide and CCL23beta {Delta}24 induced signaling, whereas CCL23{alpha} {Delta}24 was inactive (Fig. 4B; also see Table I). A control peptide equivalent to SHAAGtide but with the amino acids in a scrambled order was inactive in the assays, indicating that SHAAGtide activity was sequence specific. In chemotaxis assays, L1.2-CCR1 cells migrated to CCL23{alpha} {Delta}24 with an EC50 of 1.3 nM; CCL23beta {Delta}24 was 10-fold less potent and SHAAGtide was inactive (Fig. 4C). In the FPRL1 transfectant, SHAAGtide and CCL23beta {Delta}24 were equipotent, whereas CCL23{alpha} {Delta}24 was inactive (Fig. 4D). In radiolabeled ligand-binding assays, CCL23beta {Delta}24 inhibited the binding of the CCL3/MIP-1{alpha} tracer to the L1.2-CCR1 cells; consistent with the calcium mobilization and chemotaxis assays, SHAAGtide was unable to bind to the L1.2-CCR1 cells (Fig. 4E). To detect FPRL1 binding, competition was performed with radiolabeled WKYMVM, a potent synthetic ligand for FPRL1. SHAAGtide and CCL23beta {Delta}24 were equipotent in their ability to inhibit the binding of the WKYMVM tracer to the FPRL1 transfectant (Fig. 4F). Collectively, these data indicate the FPRL1-using determinant of CCL23beta {Delta}24 is located entirely within the SHAAGtide domain, whereas the CCR1-using determinant lies entirely within the chemokine domain C-terminal to the SHAAGtide domain.


Figure 4
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  		FIGURE 4. Dissection of CCL23beta {Delta}24 and SHAAGtide receptor usage with transfected cell lines. L1.2 cells stably expressing CCR1 (A, C, and E) or FPRL1 (B, D, and F) were analyzed by calcium mobilization (A and B), chemotaxis (C and D), and competitive ligand-binding (E and F) assays. A and B, Ligands were used at 100 nM. C and D, Ligands were used at a range of doses, with EC50s derived from nonlinear curve fitting shown in the adjoining boxes. E, Competition with 125I-labeled CCL3/MIP-1{alpha} tracer was performed with a range of doses of CCL23beta {Delta}24, SHAAGtide, and unlabeled competitor (CCL3/MIP-1{alpha}). F, Competition with 125I-labeled WKYMVm tracer was performed with a range of doses of CCL23beta {Delta}24, SHAAGtide, and unlabeled competitor (WKYMVM). E and F, EC50s derived from nonlinear curve fitting are shown in the adjoining boxes.

 
SHAAGtide is a potent functional ligand for human monocytes and neutrophils

Because SHAAGtide was equipotent with CCL23beta {Delta}24 on the FPRL1 transfectant, the two ligands were compared for their abilities to induce calcium mobilization in freshly isolated human monocytes and neutrophils. Both cell types express FPRL1 and CCR1, but the CCR1 on neutrophils is poorly responsive. As seen with the FPRL1 transfectant, both SHAAGtide and CCL23beta {Delta}24 induced robust receptor signaling in monocytes and neutrophils, whereas the scrambled SHAAGtide was inactive (Fig. 5, A and B). The potent CCR1 ligand CCL15 {Delta}24/leukotactin was unable to desensitize monocytes or neutrophils to SHAAGtide-induced calcium mobilization (data not shown), indicating that SHAAGtide signaling activity was not mediated by CCR1.


Figure 5
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  		FIGURE 5. Functional characterization of CCL23beta {Delta}24 and SHAAGtide. A and B, Receptor signaling assays were performed in monocytes and neutrophils with 100 nM ligand. C and D, Chemotaxis assays were performed in monocytes and neutrophils, with three concentrations of ligand. E and F, Recruitment of leukocytes by SHAAGtide in vivo. E, Micrographs show H&E staining of the subcutis region of mouse skin 6 h after intradermal injection of 2 µg of SHAAGtide or an equivalent volume of saline. Magnification: x100. F, Micrographs show immunohistochemical staining (red color) of the SHAAGtide-injected skin with an Ab specific for mouse neutrophils and an isotype control Ab. Nuclei were counterstained blue with hematoxylin. Magnification: x400.

 
In addition to the receptor signaling assays, chemotaxis assays were performed with both cell types. CCL23beta {Delta}24 and SHAAGtide exhibited identical potencies, both in monocytes and neutrophils, while the scrambled peptide was ineffective (Fig. 5, C and D). CCL23{alpha} {Delta}24, which lacks the SHAAG domain, exhibited potent (but less efficacious) chemoattraction for human monocytes via CCR1 but was unable to induce migration of neutrophils (Fig. 5, C and D).

Both CCL23beta {Delta}24 and SHAAGtide were able to induce calcium mobilization and chemotaxis in murine bone marrow neutrophils (data not shown), suggesting that both ligands can function through a mouse receptor counterpart. To determine whether SHAAGtide was functional in vivo, we injected it intradermally into C57BL/6 and BALB/c mice and analyzed the cells recruited to the site of injection 6 h later by histology. In both strains of mice, a 2-µg dose of SHAAGtide resulted in the consistent (six of six mice) recruitment of leukocytes into the subcutis region of the dermis (Fig. 5E). By immunohistochemistry, a large proportion of the recruited cells were identified to be neutrophils (Fig. 5F). Taken together, these data clearly indicate that the unique SHAAG domain of CCL23beta acts as a full agonist for monocytes and neutrophils in vitro and in vivo.

FPRL1 activity is transient

Interestingly, extended digestions of CCL23beta or the 1-42 peptide with the activated neutrophil medium resulted in less calcium mobilization in the FPRL1 transfectant, compared with shorter digestions (Fig. 6, A and B). SDS-PAGE analysis of these long-term digestions no longer contained a SHAAGtide-sized band, presumably due to protease cleavage within the SHAAGtide. Inspection of the SHAAG sequence revealed putative tryptase cleavage sites near the N terminus critical for FPRL1 activity (Fig. 6C and Table I). Addition of recombinant tryptase to the digestion of the 1-42 peptide with chymase resulted in complete abrogation of the FPRL1 activity (Fig. 6D). N-terminal sequencing of the 1-42 peptide after tryptase exposure indicated that the enzyme indeed cleaved at the putative substrate site (Fig. 6C). Although tryptase is a product of mast cells and not neutrophils, the latter might contain proteases (e.g., matrix metalloproteinases) that cleave inside the SHAAG domain, similar to tryptase.


Figure 6
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  		FIGURE 6. Inactivation of FPRL1 activity. A, The L1.2-FPRL1 calcium mobilization assays were performed with CCL23beta digested with activated neutrophil medium for 4 and 18 h. Note the diminution of calcium mobilization in the 18-h time point compared with the 4-h time point. B, The L1.2-FPRL1 calcium mobilization assay was performed with CCL23beta 1-42 peptide digested with activated neutrophil medium for 1 and 4 h. Note the diminution of calcium mobilization in the 4-h time point compared with the 1-h time point. C, Sequence of the 1-42 peptide, containing the N-terminal and SHAAG domains of CCL23beta, with the latter underlined. Sites of cleavage of the 1-42 peptide by chymase, the neutrophil medium, and tryptase are indicated. D, L1.2-FPRL1 cells were exposed to 100 nM 1-42 peptide after 1-h digestion with chymase, tryptase, or a combination of the two, and calcium mobilization was monitored over time.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this article, we demonstrate that proteases associated with inflammation cleave CCL23beta/CKbeta8-1 (43) into potent CCR1 and FPRL1 functional ligands. The FPRL1-using determinant is located solely within the 18-residue "SHAAG" domain encoded by the alternately spliced nucleotides, whereas the CCR1-using determinant is located solely within the C-terminal chemokine domain. This 74-aa domain is characteristic of the other CC chemokines, including CCL3/MIP-1{alpha}, in that four cysteines are placed in conserved positions and there is no inhibitory N-terminal domain. The role of the extra two cysteines in the chemokine domain of CCL23beta is unknown, but these extra cysteines are found in the other NC6 chemokines (42) and thus may be used to hold the N-terminal inhibitor domain in place until the chemokine is exposed to proteases. In contrast, the FPRL1-using "SHAAG" domain is completely novel within the chemokine family and allows processed CCL23beta to use a chemoattractant receptor outside the chemokine receptor family. Due to the inhibitory N-terminal domain of CCL23beta, both CCR1 and FPRL1 functionality are blocked until the chemokine is processed by proteases associated with inflammation.

Human mast cell chymase, neutrophil cathepsin G and elastase, lysosomal protease Asp-N, and human neutrophil-conditioned medium were all found to remove the N-terminal domain from CCL23beta, activating the chemokine’s FPRL1 activity through exposure of the SHAAG domain. The proteases subsequently cleaved CCL23beta between the SHAAG and chemokine domains, activating the chemokine’s CCR1 activity and also releasing a short peptide ("SHAAGtide") containing the SHAAG domain. Remarkably, SHAAGtide by itself was functional for FPRL1, with potency and efficacy identical to its parent (N-terminally truncated CCL23beta) and to recombinant CCL23beta {Delta}24 (CCL23beta lacking the entire N-terminal domain). With EC50s of 10–30 nM on human monocytes and neutrophils, SHAAGtide is by far the most potent naturally existing ligand for FPRL1 identified to date. SHAAGtide is also the most efficacious (albeit not potent) naturally occurring chemoattractant of monocytes and neutrophils we have tested in vitro, causing more chemotaxis than, for example, CXCL8/IL-8 and CCL2/MCP-1. SHAAGtide not only has potent signaling and chemoattractant properties in vitro, it is functional in vivo, recruiting leukocytes, including neutrophils, to mouse skin after intradermal injection.

Mapping of SHAAGtide indicated that the N terminus is critical for FPRL1 activity. Short N-terminal extensions are tolerated because naturally processed forms contain extensions of one to three amino acids and are active on FPRL1. Deletion of the N-terminal methionine, however, reduces SHAAGtide activity for FRPL1 at least 10-fold. Another group recently produced a SHAAGtide lacking the N-terminal methionine and observed weak activity for FPRL1 (45). In contrast, the C terminus of SHAAGtide is relatively unimportant for FPRL1 activity, as deletion of the C-terminal 3 aa did not affect the peptide’s potency on FPRL1-bearing cells.

The ability of CCL23beta to function through both CCR1 and FPRL1 suggests that this chemokine might play multiple roles in innate and adaptive immunity. CCR1 controls the trafficking of monocytes, dendritic cells, and effector lymphocytes to sites of chronic inflammation, such as seen in rheumatoid arthritis and multiple sclerosis (46, 47, 48, 49). In addition, it is notable that some CCR1 ligands are capable of skewing adaptive immune responses to certain diseases or pathogens (11, 12). FPRL1 functions in the innate immune response by enabling monocytes and neutrophils to recognize with low affinity a variety of proinflammatory and pathogen-associated ligands, including HIV coat proteins, bacterial peptides, Helicobacter coat proteins, serum amyloid-related stress proteins, and host- and pathogen-produced eicosanoids (25, 26, 31, 32, 50, 51, 52, 53, 54, 55, 56, 57, 58). Our discovery that FPRL1 can also function as a high-affinity chemokine receptor for CCL23beta cleavage products suggests that they might function to induce migration of different sets of leukocytes to sites of infection, whereupon the cells would recognize the pathogenic pattern. Depending on the proteases present, CCL23beta could recruit monocytes and neutrophils through FPRL1 (i.e., cleavage N-terminal to the SHAAG domain) or monocytes, dendritic cells, and effector lymphocytes through CCR1 (i.e., cleavage C-terminal to the SHAAG domain). As our data suggest, it is entirely possible that stepwise processing occurs, i.e., cleavage first N-terminal and then C-terminal to the SHAAG domain, producing activity first for FPRL1 and then for CCR1 (Fig. 7).


Figure 7
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  		FIGURE 7. Model for stepwise cleavage of CCL23beta. CCL23beta protein, either present within tissue or generated after resident leukocytes (macrophages, mast cells) encounter foreign Ag, is first cleaved between the N-terminal and SHAAG domains by proteases produced by the resident cells. The truncated CCL23beta {Delta}24 protein (or a variant, based on the specific cleavage site) then recruits blood monocytes and neutrophils via FPRL1. The second cleavage occurs between the SHAAG domain and the C-terminal domain, producing SHAAGtide and CCL23{alpha} {Delta}24 (or a variant, based on the specific cleavage site). It is not known whether the proteases responsible for this cleavage event are produced by the original resident cells or the recruited cells. SHAAGtide continues to recruit monocytes and neutrophils via FPRL1, while CCL23{alpha} {Delta}24 or its variant recruits monocytes, dendritic cells, and effector T cells via CCR1. The third cleavage occurs within SHAAGtide, rendering it inactive for FPRL1. It is not known which cell type produces the proteases responsible for the third cleavage. In contrast to SHAAGtide, CCL23{alpha} {Delta}24 is not cleaved soon after its produced, allowing for long-term trafficking of CCR1-bearing cells.

 
Whatever the case, our results indicate that the FPRL1-using activity might be transient: some time after the N-terminal inhibitor domain was removed from the SHAAG domain, subsequent cleavage events occurred inside SHAAGtide. The liberated fragment became too small to be detected by SDS-PAGE, and the activity on FPRL1 was diminished. This phenomenon occurred when the substrate chemokine was either full-length CCL23beta or a 42-residue peptide containing only the N-terminal and SHAAG domains. Perhaps removal of the N-terminal domain reveals a nearby cryptic substrate site for another protease; indeed, a putative tryptase substrate site (Arg-Arg-Lys) lies near the N-terminal edge of the SHAAG domain in the region critical for FPRL1 potency. Exposure of the 1-42 peptide to recombinant tryptase resulted in cleavage at this site. Furthermore, induction of FPRL1 activity in the 1-42 peptide by chymase was abrogated by tryptase. Exposure of N-terminally truncated CCL23beta or released SHAAGtide to tryptase in vivo might similarly abrogate FPRL1 activity (Fig. 7).

In conclusion, we have provided clear data surrounding the liberation of high-affinity chemokine and chemokine-derived peptide ligands for FPRL1 by naturally occurring proteases. These proteases are apt to constitute a naturally self-limiting regulation of the activation of this important receptor, which we propose be designated CCR12 in all future nomenclature.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health National Institute of Allergy and Infectious Diseases Grant 1 U19 AI056690 (to ChemoCentryx). Back

2 The term CKbeta8 has been used as one of the traditional names for the CCL23 protein, although CKbeta8-1 indicates a variant containing a replacement of Arg25 with an 18-residue peptide, due to alternative splicing of the CCL23 mRNA. Removal of the inhibitory N-terminal domain from CKbeta8 and CKbeta8-1 results in forms beginning with Arg25 (CKbeta8) or with the initial methionine of the inserted peptide (CKbeta8-1). CCL23{alpha} = full-length CKbeta8; CCL23{alpha} {Delta}24 = N-terminally truncated CKbeta8; CCL23beta = full-length CKbeta8-1; and CCL23beta {Delta}24 = N-terminally truncated CKbeta8-1. Back

3 Address correspondence and reprint requests to Dr. Zhenhua Miao, ChemoCentryx, 850 Maude Avenue, Mountain View, CA 94043. E-mail address: zmiao{at}chemocentryx.com Back

4 Abbreviations used in this paper: PRR, pattern recognition receptor; fMLP-R or FPR, N-formyl peptide receptor; SAA, serum amyloid A. Back

Received for publication January 12, 2007. Accepted for publication March 16, 2007.


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 Materials and Methods
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