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Originally published online as doi:10.1189/jlb.0508322 on August 27, 2008

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(Journal of Leukocyte Biology. 2008;84:1530-1538.)
© 2008 by Society for Leukocyte Biology

Role of neutrophil proteinase 3 and mast cell chymase in chemerin proteolytic regulation

Aude Guillabert*,1, Valérie Wittamer*,1, Benjamin Bondue*, Véronique Godot{dagger}, Virginie Imbault*, Marc Parmentier* and David Communi*,2

* Institute of Interdisciplinary Research in Human and Molecular Biology (I.R.I.B.H.M.), Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium; and
{dagger} Institut National de la Santé et de la Recherche Médicale, Université Paris-Sud 11, Faculté de Médecine Paris Sud, Institut Fédératif de Recherche 13, Clamart, France

2 Correspondence: ULB, I.R.I.B.H.M., 808, route de Lennik Bat C-4 143, Campus Erasme, Brussels 1070, Belgium. E-mail: dcommuni{at}ulb.ac.be


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ABSTRACT
 
Chemerin is a potent chemotactic factor that was identified recently as the ligand of ChemR23, a G protein-coupled receptor expressed by mononuclear phagocytes, dendritic cells (DCs), and NK cells. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active on ChemR23. However, prochemerin can be converted rapidly into a full ChemR23 agonist by proteolytic removal of a carboxy-terminal peptide. This maturation step is mediated by the neutrophil-derived serine proteases elastase and cathepsin G. In the present work, we have investigated proteolytic events that negatively control chemerin activity. We demonstrate here that neutrophil-derived proteinase 3 (PR3) and mast cell (MC) chymase are involved in the generation of specific chemerin variants, which are inactive, as they do not induce calcium release or DC chemotaxis. Mass spectrometry analysis showed that PR3 specifically converts prochemerin into a chemerin form, lacking the last eight carboxy-terminal amino acids, and is inactive on ChemR23. Whereas PR3 had no effect on bioactive chemerin, MC chymase was shown to abolish chemerin activity by the removal of additional amino acids from its C-terminus. This effect was shown to be specific to bioactive chemerin (chemerin-157 and to a lesser extent, chemerin-156), as MC chymase does not use prochemerin as a substrate. These mechanisms, leading to the production of inactive variants of chemerin, starting from the precursor or the active variants, highlight the complex interplay of proteases regulating the bioactivity of this novel mediator during early innate immune responses.

Key Words: inflammation • mast cells • processing


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INTRODUCTION
 
A large set of defense mechanisms has been developed during evolution against infectious agents. Cells taking part to innate immunity, the first line of defense, are mononuclear phagocytes such as dendritic cells (DCs) and macrophages, granulocytes, epithelial cells, mast cells (MCs), and NK lymphocytes. Initial host defense also involves soluble factors, such as the complement system, lysozyme, various antimicrobial peptides, chemokines, and cytokines, which orchestrate the immune response. Proteases also contribute to various aspects of the primary response against injury. Exposed at the surface of cells or released in the extracellular space following activation of neutrophils, MCs, or macrophages, proteases contribute to extracellular matrix (ECM) remodeling. They also allow chemotaxis, as well as generate and mobilize from the matrix a number of inflammatory mediators, growth factors, and angiogenesis regulators. In addition to transcriptional regulation, the post-translational processing by proteases constitutes an important mechanism regulating the biological activity of cytokines involved in inflammatory processes. Most chemokines are processed at their NH2-terminus by CD26/dipeptidyl peptidase IV and other proteases with significant modifications of their bioactivity toward their respective receptors [1 ]. Antimicrobial peptides, such as LL-37 in humans, are derived from the COOH-terminal truncation of their cathelicidin precursors [2 , 3 ].

We previously described chemerin as a novel, natural ligand of the previous orphan receptor ChemR23 [4 ], which exhibits a unique expression pattern among leukocyte populations. It is expressed preferentially by monocyte-derived macrophages and by immature myeloid and plasmacytoid DCs [5 6 7 ]. It was also shown recently to be expressed by the cytotoxic subset of NK cells [8 ]. Chemerin is secreted as a poorly active precursor (prochemerin), present in plasma of nanomolar concentrations. Prochemerin requires the proteolytic removal of a carboxy-terminal peptide to become a full agonist of ChemR23. Prochemerin is expressed by many tissues, including spleen, lymph nodes, and epithelia, and elevated bioactive chemerin was detected in a diverse set of human inflammatory fluids. Characterization of chemerin expression and the regulation of its processing will help to further delineate the role of this novel signaling system in inflammatory processes and pathological situations.

In a previous work, we investigated the nature of the proteolytic enzymes involved in prochemerin maturation, a critical limiting step in the chemerin-mediated recruitment of APCs. We reported that bioactive chemerin generation is mediated by the serine proteases elastase (HLE) and cathepsin G (CG) following activation of neutrophils [9 , 10 ]. These two enzymes specifically generate two distinct forms of bioactive chemerin, lacking, respectively, the last six (chemerin 1–157) or seven (chemerin 1–156) amino acids of the inactive precursor. Both forms were identified as present in vivo in human inflammatory fluids. The pharmacological characterization of synthetic peptides corresponding to the C-terminus of prochemerin and truncated variants thereof identified chemerin-157 and chemerin-156 as the sole bioactive forms of chemerin, the former being more active than the latter [11 ]. Moreover chemerin-157 was the major form observed by mass spectrometry in samples purified from human ascitic fluids [4 ]. Other proteases have been described to mediate chemerin maturation, including proteases from the blood coagulation cascade, and more recently, a cysteine protease secreted by Staphylococcus aureus [10 , 12 , 13 ]. Proteases involved in the negative regulation of chemerin bioactivity have, however, not been described so far. During the original purification of bioactive chemerin from human ascitic fluid, two inactive chemerin variants were copurified (data not shown). Although less abundant, the presence of these two variants, chemerin-155 and chemerin-154, lacking, respectively, the last eight and nine amino acids of the precursor, was also described in human hemofiltrate and serum [6 , 14 , 15 ]. These observations led us to investigate the enzymatic mechanisms involved in the generation of these two inactive and inactivable chemerin forms and consequently, to the identification of the cell types that are implicated in the negative regulation of bioactive chemerin. In the present study, we first describe neutrophil-derived serine protease proteinase 3 (PR3) as a regulator of chemerin activity. By specifically processing prochemerin, this protease directly converts the precursor into chemerin-155, an inactive chemerin variant. In addition, we demonstrate that MC chymase specifically converts active chemerin-157 and to a lesser extent, chemerin-156 (but not prochemerin) into the inactive chemerin-154 form. Our results show that neutrophils and MC stimulation may contribute not only to local inactivation of chemerin but can also influence chemerin generation by modifying the amount of the available precursor, thereby regulating the action of chemerin in immune responses.


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MATERIALS AND METHODS
 
Enzymes and chemicals
Recombinant prochemerin was obtained as described [4 ], and active chemerin and mouse IL-3 were purchased from R&D Systems (Minneapolis, MN, USA). Purified MC tryptase, CG, and chymase were obtained from Calbiochem (San Diego, CA, USA). PR3 was obtained from Elastin Products (Owensville, MO, USA). Cytochalasin B, fMLP, LPS, ionophore A23187, IgE anti-dinitrophenol (anti-DNP; SPE-7), DNP conjugated with human serum albumin (DNP-HSA), and chymotrypsin inhibitor from soybean were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse stem cell factor (SCF) was purchased from Biosource (Camarillo, CA, USA). IgE-biotin, streptavidin-PE, and mouse CD117-FITC antibodies were obtained from BD PharMingen (San Diego, CA, USA).

Aequorin-based calcium release assay
Briefly, a bicistronic eukaryotic expression vector encoding human ChemR23 was used to generate stable transfectants in a Chinese hamster ovary (CHO)-K1 cell line coexpressing a mitochondria-targeted form of apoaequorin and G{alpha}16 (WTA11-ChemR23 cell line). Intracellular Ca2+ release in this CHO-K1 cell line was measured using an aequorin-based assay as described previously [16 ]. Results were expressed as relative light units or as a percentage of the endogenous response to 20 µM ATP.

Assay of prochemerin and chemerin conversion by human proteases
Human recombinant prochemerin (20 ng) or human recombinant chemerin-157 (4 ng) and -156 were incubated for 15 min at 37°C in HBSS medium with 0.05% BSA and various amounts of purified proteases (300 ng/ml–3 pg/ml) before being assayed for activation of ChemR23-expressing CHO-K1 cells. To produce chemerin-156, prochemerin was preincubated for 15 min with CG (150 ng/ml). For chemotaxis assay, human chemerin-457 (50 ng) was incubated for 30 min at 37°C in HBSS medium with 0.05% BSA and 1 ng purified proteases (chymase and PR3). Human prochemerin (250 ng) was incubated for 10 min with 5 ng-purified proteases (HLE and PR3). To stop the enzymatic reaction, the sample was diluted in the chemotaxis medium.

Preparation and stimulation of human neutrophils
Human neutrophils were isolated from healthy donors. Briefly, after sedimentation with 3% Dextran in isotonic NaCl, the leukocyte-rich supernatant was separated on Lymphoprep (AxisShield, Norway), and the remaining erythrocytes were lysed by a hypotonic shock. Cells were washed once and resuspended in the adequate buffer. With the exception of Dextran sedimentation, all steps were carried out at 4°C. Water was checked as endotoxin- and pyrogen-free. For stimulation, polymorphonuclear cells (PMN; 5x105 cells/ml in HBSS without Ca2+ and Mg2+) were incubated for 30 min at 37°C with 5 µg/ml cytochalasin B and 0.5 µM fMLP. Alternatively, the cells were incubated for 5 min at 37°C with 0.1 ng/ml LPS and then for 30 min with 10 nM fMLP.

Preparation and stimulation of human and murine MCs
Bone marrow MCs were obtained by culturing mouse bone marrow cells from 6-week-old C57BL/6 mice during 4 weeks in RPMI medium supplemented with 10% heat-inactivated FCS, 10 mM Hepes, 50 µM β-ME, 8 µg/ml gentamycin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 4 mM L-glutamine, 10 ng/ml SCF, and 10 U/ml IL-3. The cell population was consistently composed of >90% MCs, as measured by flow cytometry analysis (CD117+ population, high-affinity IgE receptor). For stimulation, MCs were counted and pooled in HBSS medium and 2 µM ionophore. Cells (100,000/condition) were incubated during 30 min with constant stirring at 37°C and then centrifuged to collect the supernatant. To test enzyme inhibitors, the media were incubated with 2 µM CG I inhibitor (Calbiochem). Alternatively, MCs were sensitized with IgE anti-DNP at 2 µg/ml and then incubated for 1 h at 5% CO2 and 37°C. Cells were washed twice in HBSS and resuspended in HBSS medium containing 10 ng/ml DNP-HSA. Highly purified human cord blood-derived MCs were obtained by means of long-term culture of cord blood progenitor cells, as described previously [17 ]. Cord blood MCs (1x106 cells/mL) were incubated for 4 days at 37°C with 2.5 µg/mL human myeloma IgE and IL-4 (20 ng/mL). Then, cells were harvested, washed in complete IMDM, and incubated with 1 µg/mL goat anti-human IgE (Vector Laboratories, Biovalley, Marne la Vallée, France) for 30 min. Some cells were treated with human myeloma IgE alone to control basal secretion of mediators. The supernatants were collected by means of centrifugation and frozen at –80°C.

Assay of chemerin processing enzyme activity in MC-conditioned medium
Human recombinant chemerin (1 ng) was incubated in murine and human MC-conditioned medium for 30 min and 15 min, respectively. Following stimulation, the sample was diluted in HBSS medium containing 0.05% BSA before being assayed for activation of ChemR23-expressing CHO-K1 cells. To test enzyme inhibitors, the media were preincubated with inhibitors for 30 min at 37°C, after which chemerin was added and incubated at 37°C for 30 min and 15 min for murine and human MCs, respectively, before being assayed. Potential interference of inhibitors with the aequorin assay was evaluated by testing the inhibitors alone and the ability of 0.2 nM chemerin to stimulate the ChemR23-expressing cell line in the presence of inhibitors.

Preparation of monocyte-derived DCs
PBMC were isolated with Lymphoprep density gradient centrifugation of heparinized blood obtained from healthy volunteers. Monocytes were separated by anti-CD14 magnetic beads (Miltenyi Biotec, Auburn, CA, USA) and then cultured for 7 days in a complete RPMI-1600 medium containing 10% FCS, 25 ng/ml GM-CSF, and 10 ng/ml IL-4. The cell population was consistently composed of >90% immature human DCs as measured by flow cytometry analysis (CD14–, CD1a+, HLA-DR+, CD83–).

Mass spectrometry analysis
For in vitro tests, prochemerin or chemerin (30 ng) were incubated for 30 min at 37°C in 25 mM ammonium carbonate buffer, pH 7.4, containing purified PR3, tryptase, or chymase (1 ng/µl). For the tests with PMN- or MC-conditioned media, prochemerin or chemerin (100 ng) were incubated in the media for 1 h at 37°C. The reaction was stopped by heating at 95°C. After trypsin treatment, the digested peptides were processed for mass spectrometry analysis as described previously [9 ]. Mass spectrometry analysis was performed on a Q-TOF Ultima global mass spectrometer equipped with a MALDI source (Micromass). The mass scale of interest was focused on the mass range covering the potential processed COOH-terminal peptides of (pro)chemerin (1590–2100 Da).

Chemotaxis assay
Cell chemotaxis was measured using a 48-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD, USA) and 5.0 µm pore-size cellulose nitrate filters (Neuro Probe). Briefly, cell suspensions and chemotactic factors were prepared and/or diluted in the chemotaxis assay buffer (RPMI supplemented with 10% FBS or 1% BSA). Chemotactic factors or assay buffer alone (30 µl) were added in the lower compartment of the chamber, covered with a 5.0-µm cellulose nitrate filter, and then 50 µl of a cell suspension (2x105 cells/ml) was pipetted into the upper compartment. The chemotaxis chamber was then incubated at 37°C for 90 min. After the incubation period, filters were fixed in 2-propanol and then fixed and stained with Hoechts (dilution 1/2000) for 2 min. The number of migrated cells was enumerated by imageJ 1.36b after acquisition of a picture of each field of the microscope. Statistical significance was determined using the t-test for paired value, and P values <0.05 were considered as significant.


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RESULTS
 
PMN-conditioned medium incubated with prochemerin generates an inactive chemerin form, chemerin-155
As reported previously, the study by mass spectrometry of stimulated or unstimulated neutrophil supernatant incubated with prochemerin has shown the generation of the two sole forms of chemerin that are active on ChemR23 [9 ]. It also revealed the presence of another, unpredicted chemerin form. Indeed, to investigate this further, prochemerin incubated in the presence of purified PMN-conditioned medium during 30 min was analyzed by trypsin digestion and mass spectrometry to determine the different chemerin forms generated by the proteolysis catalyzed by PMN proteases. Figure 1 B and C , shows the generation of a tryptic peptide with a molecular mass of 1669.8 Da, corresponding to the carboxy-terminal sequence of chemerin ending at alanine 155 and lacking the last eight amino acids of prochemerin (Table 1 ). Chemerin-155 is known to be inactive [11 ]. Moreover, the stimulation of PMNs does not affect the extent of this proteolysis event, as this chemerin form was similarly present on mass spectra in unstimulated and stimulated conditions (Fig. 1 B and C) . The presence of this proteolytic variant in an unstimulated supernatant condition is probably a result of a residual degranulation of PMNs. However, the incubation of prochemerin with unstimulated and stimulated entire PMNs generates this chemerin variant equally (data not shown). In contrast, we have shown previously that only stimulated PMNs induce prochemerin maturation into two different active chemerin variants (chemerin-156 and -157). Other peptides detected in mass spectra of Figure 1 B and C , are issued from contaminant proteins present in PMN-conditioned media and do not correspond to prochemerin cleavages.


Figure 1
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  		Figure 1. Structural analysis of prochemerin proteolytic products generated by stimulated neutrophils. Human recombinant prochemerin (100 ng) was incubated with conditioned media from PMNs stimulated with cytochalasin B and fMLP in the absence of BSA before trypsin digestion. (A) Mass spectrum of trypsin-digested human recombinant prochemerin. The COOH-terminal tryptic peptide corresponds to a peak with a molecular mass of 2032.01 Da. (B and C) Mass spectrometry analysis of the samples resulting from the incubation with conditioned media of unstimulated and stimulated PMNs, respectively. An additional COOH-terminal peptide with a mass of 1669.8 Da is observed, which corresponds to prochemerin devoid of the last eight amino acids (chemerin-155). The mass scale of interest is focused between 1650 and 2050 Da, and analysis of the entire mass spectrum did not reveal any additional cleavage products of prochemerin.


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  		Table 1. List of the Carboxy-Terminal Tryptic Fragments Derived from the Chemerin Variants

PR3 generates inactive and inactivable chemerin-155
We determined which protease was responsible for chemerin-155 formation. A third serine protease known to be abundant in PMN granules (together with HLE and CG) is PR3, which was described to be present in PMN azurophil granules but also at the plasma membrane. Its substrate specificity is similar to that of HLE, with valine or alanine accepted in the P1 position [18 ]. To determine whether PR3 could be responsible for the generation of this new inactive chemerin form, prochemerin was incubated with PR3, and the products were analyzed by mass spectrometry after trypsin treatment, with an emphasis on the COOH-terminal end. Incubation of prochemerin without PR3 generated the tryptic COOH-terminal peptide of prochemerin with a molecular mass of 2031.9 Da (Fig. 2A and Table 1 ). After PR3 incubation, an additional signal appeared on the spectrum, with a molecular mass of 1669.8 Da, which corresponds to the tryptic COOH-terminal peptide of chemerin-155 (Fig. 2B) . This is consistent with the previous description of the PR3 preferred cleavage site [18 ]. Surprisingly, bioactive chemerin did not appear to be a substrate for PR3, as it was not processed into the inactive chemerin-155 form (data not shown).


Figure 2
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  		Figure 2. Identification by mass spectrometry of prochemerin proteolytic products generated by purified PR3. Human recombinant prochemerin (20 ng) was incubated in buffer or with purified PR3 (1 ng/µl) for 30 min at 37°C, digested by trypsin, and analyzed by mass spectrometry, focusing on the mass range covering the peptides generated from the COOH-terminus of prochemerin (1600–2100 Da). (A) Mass spectrum of human trypsin-digested prochemerin. The carboxy-terminal tryptic peptide corresponds to a peak with a molecular mass of 2031.91 Da. (B) Following treatment with PR3, a COOH-terminal peptide of mass 1669.74 Da is observed, which corresponds to prochemerin, devoid of the last eight amino acids. Analysis of the entire mass spectrum did not reveal any additional cleavage products of prochemerin.

Competition among HLE, CG, and PR3 for chemerin processing
As HLE, CG, and PR3 act on the same substrate but generate products with distinct bioactivities, we evaluated the competitive, inhibitory role of PR3 toward CG and HLE action in vitro. When prochemerin was preincubated with PR3 (10 ng) before addition of CG or HLE (at the same concentration), an important decrease of the resulting functional activity on ChemR23 was observed (Fig. 3 A and B ). In both cases, 80% of the functional activity generated in the absence of PR3 was lost. When prochemerin was incubated simultaneously with physiological concentrations of PR3 (1 or 10 ng) and HLE (10 ng), the functional activity was decreased by 50% as compared with the control condition without PR3 (Fig. 3C) . Similar results were obtained following coincubation with PR3 and CG (Fig. 3D) . These results confirm that prochemerin constitutes a good substrate for PR3. By consuming prochemerin, PR3 could therefore negatively control the prochemerin maturation catalyzed by CG and HLE.


Figure 3
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  		Figure 3. PR3-mediated effect on prochemerin maturation in competition with HLE and CG action. (A and B) Prochemerin (20 ng) was incubated in the presence or absence of PR3 at 300 ng/ml and then incubated with HLE (A) or CG (B) at 300 ng/ml. (C and D) Prochemerin was incubated simultaneously with PR3 (30 or 300 ng/ml) and 30 ng/ml HLE (C) or CG (D), and the generation of bioactive chemerin was monitored. The controls correspond to the biological response obtained in the aequorin-based assay, and prochemerin was incubated in the presence of CG or HLE at the tested concentrations. *, If P < 0.05; **, if P < 0.01; ***, if P < 0.001.

Chemerin is processed by MC chymase
In a second step, we focused on a set of serine proteases involved in inflammatory processes to determine in vitro their potential proteolytic action on prochemerin or on bioactive chemerin, i.e., MC proteases. It allowed us to highlight an unknown activity of chymase on the most bioactive chemerin (chemerin-157). Incubation of bioactive chemerin with increasing concentrations of chymase resulted in a drastic reduction of its functional activity as an agonist of ChemR23. This activity was abolished completely at the highest concentration of chymase (Fig. 4A ). Such inactivation of chemerin was not observed with MC tryptase, and a modest activation of prochemerin into bioactive chemerin was obtained with this enzyme [10 ]. Functional assay of the degradation of chemerin-156 by chymase showed that chymase was eightfold less efficient in proteolyzing chemerin-156 as compared with chemerin-157 (Fig. 4B) .


Figure 4
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  		Figure 4. Screening of serine proteases for their potential converting activity on human prochemerin and chemerin. (A) Following incubation of prochemerin (20 ng) or chemerin-157 (4 ng) for 15 min with various concentrations of PR3, tryptase (TRY), or chymase (CHY) or (B) following preincubation of prochemerin (20 ng) in the presence of CG (5 ng) for 15 min (Chem-156) or chemerin-157 alone (Chem-157; 1 ng), incubated with chymase for 15 min with various concentrations, the activity on ChemR23-expressing cells was measured in the aequorin-based assay. A weak basal activity of the prochemerin preparation, corresponding to the presence of contaminating, active chemerin, was subtracted. The data are representative of three independent experiments.

Chemerin variants generated by PR3 and chymase do not induce DC chemotaxis
In addition to the absence of functional activity of chemerin-154 and chemerin-155 on ChemR23 measured by calcium release assay, we tested their capacity to chemoattract monocyte-derived DCs (Fig. 5 ). These primary cells highly express ChemR23 at the surface, ~80% for different donors [4 ]. The incubation of chemerin in the presence of chymase abolishes the chemotactic effect of chemerin alone, whereas PR3 has no effect (Fig. 5B) accordingly with the fact that PR3 selectively cleaves the inactive precursor prochemerin and not bioactive chemerin. In contrast, prochemerin does not chemoattract DCs, and it remains inactive after incubation with PR3, whereas HLE produces bioactive chemerin and induces chemotaxis of DCs (Fig. 5A) .


Figure 5
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  		Figure 5. Chemotaxis assay of chemerin proteolytic products. After incubation of prochemerin (250 ng; A) or chemerin (50 ng; B) for 30 min and 10 min, respectively, with a fixed concentration of PR3 and HLE in case of prochemerin (A) and of PR3 and CHY in case of chemerin (B), the chemotactic activity of different dilutions of these samples was tested on monocyte-derived DCs. RANTES (10 nM) was added in each experiment as control. The data are representative of three independent experiments on three distinct donors. *, If P < 0.05; **, if P < 0.01; ***, if P < 0.001.

Human chymase generates the inactive chemerin-154 variant from human active chemerin
MC chymase presents a chymotrypsin-like substrate specificity, cleaving substrates with an aromatic P1 amino acid residue [19 ]. As shown in Figure 6A , the carboxy-terminal tryptic peptide of recombinant chemerin-157 presents a molecular mass of 1903.8 Da (Fig. 6 and Table 1 ). The in vitro processing of bioactive chemerin (chemerin-157) by chymase revealed the production of a new chemerin variant, which is inactive on ChemR23 (Fig. 4) , characterized by the generation of a tryptic peptide of 1598.7 Da (Fig. 6B and Table 1 ). The fragmentation of this peptide demonstrated that it corresponds to the COOH-terminal end of another truncated form of chemerin, chemerin-154. Thus, human chymase cleaves recombinant chemerin-157 between Phe154 and Ala155 at its carboxy-terminal end. In contrast, the incubation of prochemerin with chymase did not reveal any carboxy-terminal cleavage (Fig. 6 C and D) . Mass spectrometry analysis of samples issued from the proteolysis of chemerin-156 by chymase has revealed that chymase is less efficient on this substrate to produce chemerin-154 (data not shown).


Figure 6
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  		Figure 6. Identification by mass spectrometry of proteolytic products generated by purified chymase from bioactive chemerin or prochemerin. Human chemerin-157 (A and B) or prochemerin (C and D; 30 ng) was incubated in the absence or presence of purified chymase (1 ng/µl) for 30 min at 37°C, and samples were analyzed by mass spectrometry after trypsin digestion (focusing on the mass range covering the carboxy-terminal peptides). (A) The carboxy-terminal tryptic peptide of chemerin-157 corresponds to a peak with a molecular mass of 1903.84 Da. (B) After treatment with chymase, a carboxy-terminal peptide with a mass of 1598.69 Da appears. (C) The carboxy-terminal tryptic peptide of prochemerin corresponds to a peak with a molecular mass of 2032.02 Da. (D) After treatment with human chymase, no additional COOH-terminal peptide is observed in the spectrum. Analysis of the entire mass spectrum did not reveal any other cleavage products of (pro)chemerin.

MC-conditioned medium abolishes the activity of chemerin on ChemR23
Mouse MC were differentiated from bone marrow multipotential stem cells by culturing them for 4 weeks in the presence of mouse SCF and IL-3. The cleavage of human bioactive chemerin (chemerin-157) generated by conditioned media from MCs was tested after two stimulation pathways, resulting in MC degranulation. Using the aequorin-based calcium-mobilization assay, incubation of bioactive chemerin for 30 min with conditioned medium from unstimulated MCs resulted in a mild decrease of its activity on ChemR23 (Fig. 7A ). Stimulation by 2 µM A23187 ionophore promoted a strong degranulation of MCs and the release of serine proteases from secretory granules (Fig. 7A1) . Following incubation with the conditioned medium of these activated MCs, the chemerin activity was decreased by ~80%. A set of protease inhibitors was tested for their ability to inhibit the degradation of chemerin, but only β-ketophosphonic acid, a CG I inhibitor also known to inhibit human chymase [20 ], was able to completely inhibit the degradation of bioactive chemerin by MC proteases. As a second activation procedure, MCs were first treated with IgE anti-DNP and then with DNP-HSA. Incubation of human chemerin with the conditioned medium of these activated MCs gave results similar to those obtained after ionophore stimulation (Fig. 7A2) . The reduction in functional activity was, however, weaker in these conditions, reaching ~60%. HPLC fractionation, after incubation of human bioactive chemerin with the stimulated MC-conditioned medium, followed by mass spectrometry analysis, permitted to determine the cleavage site of murine chymase MC protease 5 (mMCP-5) on bioactive chemerin. A single tryptic COOH-terminal fragment was found, corresponding to an inactive chemerin-155 form (data not shown), in accordance with the cleavage specificity of murine MC chymase, which is described to differ in terms of cleavage specificity from its human ortholog [21 ]. As the murine model is not completely transposable with the human one as a result of the diversity of murine chymases, we tested supernatants of human MCs (Fig. 7B) , which, when issued from cord blood differentiation, were stimulated with IgE, followed by anti-IgE stimulation. We observed a complete degradation of bioactive chemerin in stimulated MC-conditioned medium. This proteolytic action was abolished in the presence of a chymotrypsin inhibitor.


Figure 7
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  		Figure 7. Chemerin degradation by MC-derived proteases. (A) Bioactive chemerin (1 ng) was incubated with the conditioned medium of murine MCs (105 cells/100 µl), degranulated (or not) in the presence of 2 µM A23187 ionophore (A1) or in the presence of 2 µg/ml IgE-anti-DNP and then 10 ng/ml DNP-HSA (A2). The inhibition of protease activity was performed with CG I inhibitor at 1 µM, added in the conditioned medium during the stimulation (A1) or added together with DNP-HSA (A2). (B) Bioactive chemerin was incubated with the conditioned medium of human MCs (5x104 cells/100 µl), degranulated (or not) in the presence of 1 µg/ml anti-IgE. The inhibition of protease activity was performed with the chymotrypsin inhibitor (6 µg) after a preincubation with the conditioned medium. The activity of the media in an aequorin-based assay on ChemR23-expressing cells is displayed for the different experimental conditions as the percentage of the response to 20 µM ATP. The data are representative of three independent experiments. *, If P < 0.05; **, if P < 0.01; ***, if P < 0.001.


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DISCUSSION
 
Chemerin may represent an important recruitment signal for selected leukocyte populations in inflammatory situations and human diseases. ChemR23 is expressed in immature plasmacytoid and myeloid DCs, as well as NK cells [5 , 8 ]. Expression of chemerin and recruitment of these ChemR23-expressing cell populations were described recently in human chronic inflammatory diseases of skin and mucosa, namely, systemic lupus erythematosus and oral lichen planus [5 ]. To understand the precise role of chemerin in the physiopathology of these and other diseases, it is crucial to determine precisely the mechanisms of chemerin proteolytic regulation. An early event in the host defense against pathogens and in the pathogenesis of inflammatory diseases is the recruitment of PMNs, which upon local activation, degranulate and release a set of proteases. PMN serine proteases are known to regulate the activity of diverse cytokines and chemokines positively or negatively. We have shown here that the three main proteases of PMNs use prochemerin as a substrate with opposite functional consequences for HLE and CG on one side and PR3 on the other side. Upon degranulation, PMNs were shown previously to promote the proteolytic maturation of prochemerin into active chemerin [9 ]. Serine protease inhibitors inhibited this processing, and HLE and CG were shown to mediate this conversion. HLE and CG remove, respectively, six or seven amino acids from the prochemerin C terminus, generating active chemerin forms ending, respectively, at serine 157 and phenylalanine 156. Chemerin-157 is the most bioactive form and is the major form purified from human ascitic fluids (chemerin-156 was nearly undetectable) [4 ]. We reported previously that only chemerin-157 and chemerin-156 are able to activate ChemR23 with significant potency using peptides corresponding to the carboxy-terminal end of (pro)chemerin and deletion variants. Addition or removal of one additional amino acid resulted in a drastic loss of activity. Precise processing of prochemerin is therefore required to generate a bioactive ChemR23 ligand [11 ].

We describe here for the first time a negative regulation of prochemerin maturation. Neutrophil PR3 generates from prochemerin an inactive variant, chemerin-155, observed previously in human samples, including ovarian cancer ascitis (data not shown) and human serum [4 , 6 ]. Two functional tests, i.e., intracellular calcium release measurement by an aequorin-based assay and monocyte-derived DC chemotaxis assay, confirmed the absence of biological activity of chemerin-155 on ChemR23. Thus, the two activatory proteases, HLE and CG, and the inhibitory protease PR3 are in competition for the same substrate, prochemerin, to control bioactive chemerin production (Fig. 8 ).


Figure 8
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  		Figure 8. Proposed model for (pro)chemerin maturation and degradation. Neutrophils and MCs recruited in the early stages of an inflammatory reaction degranulate and release CG, HLE, and PR3 (neutrophils) and chymase (MCs). Among other actions, CG and HLE maturate inactive prochemerin into bioactive chemerin, which contributes, together with a number of chemokines, to the recruitment of DCs and macrophages. PR3 can act as a down-regulating protease by processing prochemerin into an inactive and inactivable chemerin variant. Chymase cleaves bioactive chemerin (chemerin-157 and to a lesser extent, chemerin-156), generating an inactive chemerin form as well.

These three neutrophil serine proteases act as intracellular microbicidial agents and participate in the remodeling of the ECM, but they also regulate the activity of extracellular mediators such as chemokines. HLE and CG were shown to generate shorter CCL15 variants, displaying higher chemotactic activities for monocytes [22 ]. Conversely, proteolysis of CCL3/MIP-1{alpha} by HLE, CG, and PR3 inhibits its chemoattractant activity [23 ]. PR3 was described to process CXCL8/IL-8 [24 ] and TNF-{alpha} [25 ], to potentiate the consequences of platelet activation [26 ], and to promote CXCL8/IL-8 synthesis by endothelial cells [27 ], as well as myeloid differentiation [28 , 29 ]. PR3 is also a major autoantigen in Wegener’s granulomatosis, a systemic, necrotizing granulomatous vasculitis characterized by high titers of antineutrophil cytoplasmic antibodies [30 31 32 ], which gain access to the autoantigen PR3 following neutrophil stimulation by LPS, IL-8, or TNF-{alpha}, inducing the translocation of PR3 from the azurophil granules to the plasma membrane [33 ].

However, recent studies have shown the presence of PR3 at the plasma membrane of neutrophils in the absence of stimulating agents [34 , 35 ]. Such membrane expression can vary according to individuals from 0 to 95% of neutrophils, and a high proportion of PR3 at the plasma membrane was characterized as a risk factor for vasculitis and rheumatoid arthritis [36 , 37 ]. The presence of PR3 at the membrane of neutrophils suggests potential roles of this enzyme before degranulation, in contrast with HLE and CG, which are essentially active after the release of azurophil granules. Similar amounts of these three proteases are present in PMNs: ~3 pg per cell for PR3, 1.1 pg per cell for HLE, and 0.85 pg per cell for CG [38 , 39 ]. As bioactive chemerin is generated by PMNs upon following degranulation [9 ], the combined action of HLE and CG predominates over that of PR3 in these circumstances. As active chemerin is also found in human inflammatory fluids [4 ], the same situation likely prevails in vivo. However, PR3 generates an inactive and inactivable chemerin form from prochemerin and thereby competes with HLE and CG by consuming their common substrate. Therefore, the action of PR3 may predominate in noninflammatory conditions, when PR3 is partially exposed at the surface of PMNs, and HLE and CG are kept in granules. Circulating prochemerin levels might thus be regulated by sentinel neutrophils, a hypothesis supported by the presence of chemerin-155 in human serum [6 ]. Recently, a chemerin-155-derived peptide has been described as a potential anti-inflammatory effector in a dextran-induced inflammation mice model; however, its chemotactic activity was nearly undetectable [40 ]. It reinforces the hypothesis of potential in vivo roles of these different chemerin variants, independently of their chemotactic activity.

Besides neutrophil CG and HLE, other serine proteases have been described as triggering chemerin activation, including proteases of the coagulation and fibrinolytic cascades [10 ]. MC tryptase is also able to generate a weak activation of prochemerin into chemerin in vitro (Fig. 4) . MCs act as sentinel cells as a result of their localization in connective and mucosal tissues. They have important roles in innate immunity and are key effectors of allergic inflammation. MC precursors circulate in blood and require SCF to maturate and migrate into tissues. Upon activation, MCs release a variety of proinflammatory mediators, such as cytokines, chemokines, histamine, and PGD2, as well as proteases, including tryptase, chymase, and carboxypeptidase A. These mediators recruit and activate other inflammatory cells such as eosinophils [41 ], basophils [42 ], neutrophils [43 ], T cells, and macrophages [44 ].

We highlighted here a new proteolytic action of MC chymase onto the bioactive chemerin-157. Human chymase cleaves preferentially after an aromatic amino acid [19 ], and we hypothesized that such activity might generate chemerin-154, another inactive chemerin form ending by a phenylalanine (Table 1) . Chemerin-154 exists in vivo, as it was copurified with active chemerin from human ascitic fluid [4 ] (data not shown) and by others from human hemofiltrate [14 ]. No protease was described so far to generate this variant. Recently, John et al. [12 ] described the degradation process of the synthetic COOH-terminal peptide of chemerin-154 by the angiotensin-converting enzyme. As for chemerin-155, the absence of functionality of the chemerin-154 variant was confirmed by intracellular calcium release measurement by an aequorin-based assay and monocyte-derived DC chemotaxis assay.

Mass spectrometry analysis showed that incubation of bioactive chemerin in the presence of purified human MC chymase generated the chemerin-154 variant, whereas prochemerin appears as a poor substrate of this enzyme. So, chymase generates an inactive variant only from bioactive chemerin (chemerin-157). The other active chemerin variant (chemerin-156) is a poorer substrate for chymase in comparison with chemerin-157, but chemerin-156 is much less abundant in human purified inflammatory fluids [4 ]. So, in physiopathological conditions, where the most active chemerin-157 is mainly produced, chymase tends to suppress the major part of chemerin activity. As other granule proteases, chymase was described as a link between innate and adaptive immunity, promoting the accumulation of neutrophils and others inflammatory cells in vivo [45 ]. Human chymase is known for its proteolytic activation of the potent inflammatory cytokine IL-1β [46 ] and the release of latent TGF-β1 associated with the ECM [47 ]. Furthermore, chymase was described to cleave the precursor connective tissue-activating peptide III into biologically active CXCL7/neutrophil-activating peptide 2 [48 ].

We also showed in ex vivo experiments that stimulated human and murine MCs promote the degradation of bioactive chemerin. Although there are functional differences between murine and human leukocyte populations, especially MCs, the same down-regulating effect on chemerin was observed for human and mouse MCs. Only one chymase gene, belonging to the {alpha}-family, has been identified in humans. In contrast, rodents were shown to express a number of β-chymase genes: mMCP-1, mMCP-2, mMCP-4, and mMCP-9, in addition to an {alpha}-chymase mMCP-5, which is the most similar to human {alpha}-chymase. Bone marrow MCs contain essentially this {alpha}-chymase [49 , 50 ]. As a result of the difficulty to transpose this model in mice, human MCs were generated and induced, upon their stimulation, the same effect on bioactive chemerin. We demonstrated here in vitro and ex vivo that chymase can inactivate bioactive chemerin. In ex vivo experiments, this proteolytic processing occurred only on MC activation, a situation in which chymase is released in the extracellular medium. This processing was also blocked by an inhibitor of chymotrypsin-like proteases. We expect that in vivo, in situ activation of MCs in inflammatory conditions may result in chymase release and in the inactivation of bioactive chemerin generated by neutrophil HLE and CG. MCs may therefore play an important role in the regulation of chemerin levels in inflammatory conditions.

In conclusion, we propose a more complete and complex regulation model for the maturation and degradation of prochemerin and bioactive chemerin. Prochemerin is synthesized by many tissues, and recent investigations have shown the presence of immunoreactive chemerin in endothelial cells lining blood vessels from lesions of lupus and oral lichen planus, whereas it is not seen in endothelial cells in normal conditions [5 ]. In contrast to chemokines, which are essentially regulated at the transcriptional level, chemerin production is regulated strongly by extracellular proteases from immune sentinel cells, positively (HLE and CG) or negatively (PR3 and chymase). HLE and CG trigger the maturation of inactive prochemerin into two bioactive chemerin variants, respectively: chemerin-157 and chemerin-156. We identified here two additional serine proteases, PR3 and chymase, as negative regulators of this maturation process. PR3 acts on prochemerin and generates an inactive and inactivable chemerin form, chemerin-155, whereas human chymase acts on bioactive chemerin and inactivates it into chemerin-154 (Fig. 8) . This suggests a complex, spatio-temporal regulation of chemerin activity in vivo. The regulation of chemerin activity by MCs and neutrophils, which are key sentinel cells of innate immunity, supports further a pivotal role of chemerin as a regulator of the subsequent adaptive immune responses.


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ACKNOWLEDGEMENTS
 
This work was supported by the Actions de Recherche Concertées of the Communauté Française de Belgique, the Interuniversity Attraction Poles Program (P6-14), Belgian State, Belgian Science Policy, the European Union (grant LSHB-CT-2005-518167/INNOCHEM), the Fédération Belge contre le Cancer, The Walloon Region (Program d’excellence "CIBLES"), the Fonds de la Recherche Scientifique Médicale of Belgium, and the Fondation Médicale Reine Elisabeth. D. C. is senior research associate of the Belgian Fonds National de la Recherche Scientifique (F.R.S-F.N.R.S.). V. W. was supported by First Grant of the Walloon Region, Télévie, and a grant from the Fédération Belge contre le Cancer. The authors have no financial conflicts of interest. We thank Drs. Barbara Dessars and Stéphane Schurmans for their expert assistance in MC culture.


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FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received May 27, 2008; revised August 6, 2008; accepted August 6, 2008.


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Acta Biochim Biophys Sin, December 1, 2009; 41(12): 973 - 979.
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S. Luangsay, V. Wittamer, B. Bondue, O. De Henau, L. Rouger, M. Brait, J.-D. Franssen, P. de Nadai, F. Huaux, and M. Parmentier
Mouse ChemR23 Is Expressed in Dendritic Cell Subsets and Macrophages, and Mediates an Anti-Inflammatory Activity of Chemerin in a Lung Disease Model
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F. Schiemann, E. Brandt, R. Gross, B. Lindner, J. Mittelstadt, C. P. Sommerhoff, J. Schulmistrat, and F. Petersen
The Cathelicidin LL-37 Activates Human Mast Cells and Is Degraded by Mast Cell Tryptase: Counter-Regulation by CXCL4
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