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Chemerin158K Protein Is the Dominant Chemerin Isoform in Synovial and Cerebrospinal Fluids but Not in Plasma

Chemerin158K Protein Is the Dominant Chemerin Isoform in Synovial and Cerebrospinal Fluids but... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 45, pp. 39520 –39527, November 11, 2011 Printed in the U.S.A. Chemerin158K Protein Is the Dominant Chemerin Isoform in □ S Synovial and Cerebrospinal Fluids but Not in Plasma Received for publication, May 6, 2011, and in revised form, September 1, 2011 Published, JBC Papers in Press, September 19, 2011, DOI 10.1074/jbc.M111.258954 ‡ ‡ ‡ ‡ ‡‡ §¶ Lei Zhao , Yasuto Yamaguchi , Shadi Sharif , Xiao-Yan Du , Jason J. Song** , David M. Lee , Lawrence D. Recht , ‡‡ ‡1 ‡ ‡‡2 William H. Robinson** , John Morser , and Lawrence L. K. Leung From the Division of Hematology and **Division of Immunology and Rheumatology, Department of Medicine, and Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California 94305, the Brigham and Women’s Hospital, Boston, Massachusetts 02115, Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland, and the ‡‡ Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94305 Background: Chemerin is a chemokine/adipokine whose activity depends on proteolytic processing. Results: Specific ELISAs demonstrate that in plasma the precursor is dominant, whereas in synovial fluid from arthritis patients and CSF from glioblastoma patients, chem158K dominates. Low levels of active chem157S were found. Conclusion: Chemerin proteolysis occurs during inflammation. Significance: This is the first report about levels of different chemerin isoforms in biological samples. Chemerin is a chemoattractant involved in immunity that The chemerin gene was originally identified as a novel reti- may also function as an adipokine. Chemerin circulates as an noid-responsive gene in psoriatic skin lesions (1). Chemerin is a inactive precursor (chem163S), and its activation requires pro- secreted ligand of the orphan G protein-coupled receptor teolytic cleavages at its C terminus, involving proteases involved chemokine-like receptor 1, chemokine (C-C motif) receptor- in coagulation, fibrinolysis, and inflammation. However, the key like 2, and G protein-coupled receptor 1 (2). Various cell types proteolytic steps in prochemerin activation in vivo remain to be involved in innate and adaptive immunity, including immature established. Previously, we have shown that C-terminal cleavage plasmacytoid dendritic cells, myeloid dendritic cells, macro- of chem163S by plasmin to chem158K, followed by a carboxy- phages, and natural killer cells, express chemokine-like recep- peptidase cleavage, leads to the most active isoform, chem157S. tor 1, and chemerin is a chemoattractant that promotes the To identify and quantify the in vivo chemerin isoforms in bio- recruitment of these cells to lymphoid organs and sites of tissue logical specimens, we developed specific ELISAs for chem163S, injury (3–5). Chemerin is also an adipokine that regulates adi- chem158K, and chem157S, using antibodies raised against pep- pocyte development and metabolic function and may play a tides from the C terminus of the different chemerin isoforms. role in glucose tolerance, obesity, and hyperlipidemia (6). Ele- We found that the mean plasma concentrations of chem163S, vated levels of chemerin have been found in patients with dia- chem158K, and chem157S were 40  7.9, 8.1  2.9, and 0.7 betes (7–10) and fatty liver disease (11, 12), consistent with its 0.8 ng/ml, respectively. The total level of cleaved and non- role as an adipokine whereas its levels are also raised in patients cleaved chemerins in cerebrospinal fluids was 10% of plasma undergoing chronic hemodialysis (13, 14) as well as those with levels whereas it was elevated 2-fold in synovial fluids from Crohn disease (15) and chronic hepatitis C (16), in line with its patients with arthritis. On the other hand, the fraction of cleaved suggested function in inflammation. However, these studies chemerins was much higher in synovial fluid and cerebrospinal have all been carried out using ELISAs that detect total fluid samples than in plasma (75%, 50%, and 18% respectively). chemerin levels. Chem158K was the dominant chemerin isoform, and it was not Chemerin is translated as a 163-amino acid pre-proprotein generated by ex vivo processing, indicating that cleavage of subsequently secreted as a 143-amino acid precursor prochemerin at position Lys-158, whether by plasmin or another (prochemerin, chem163S) following removal of the N-terminal serine protease, represents a major step in prochemerin activation signal peptide (2, 17). Prochemerin chem163S has low biologi- in vivo. Our study provides the first direct evidence that chemerin cal activity and requires further extracellular C-terminal pro- undergoes extensive proteolytic processing in vivo, underlining the teolytic processing to achieve its full biological activity. These importance of measuring individual isoforms. cleavages have been studied in vitro where a variety of enzymes involved in coagulation, fibrinolytic and inflammation such as FXIIa, plasmin, carboxypeptidase B2 (also known as thrombin- activatable fibrinolysis inhibitor), or elastase can proteolyze * This work was supported, in whole or in part, by National Institutes of Health Grants HL057530 and AI085268. chemerin, giving rise to either active or inactive chemerins (2, 4, □ S The on-line version of this article (available at http://www.jbc.org) contains 17–19). Plasmin and elastase cleavage gives rise to chemerin21- supplemental Figs. 1–3. 158 (chem158K) and chemerin21-157 (chem157S), respec- To whom correspondence may be addressed: 269 Campus Dr., CCSR 1155, MC5156, Stanford, CA 94305-5156. Tel.: 1-650-723-7078; Fax: 1-650-721- tively, whereas proteinase 3 and mast cell chymase give rise to 6826; E-mail: [email protected]. inactive chemerin21-155 (chem155A) and chemerin21-154 To whom correspondence may be addressed: 269 Campus Dr., CCSR 1155, (chem154F), respectively (3, 20, 21). However, the catalytic effi- MC5156, Stanford, CA 94305-5156. Tel.: 1-650-723-7078; Fax: 1-650-721- 6826; E-mail: [email protected]. ciencies of most of these proteolytic cleavages of prochemerin 39520 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 This is an Open Access article under the CC BY license. Chemerin158K Is the Dominant Form in Synovial Fluids and CSF have not been characterized, and whether they represent phys- by incubation with 1% BSA in PBS for 1 h. Antibody fractions from iological activation or inactivation steps in vivo remains the cognate peptide affinity chromatography were diluted with 1% unclear. BSA in PBS and incubated for 1 h followed by washing and incu- An alternative approach has been to purify chemerin from bation with peroxidase-conjugated goat anti-rabbit IgG (100 biological fluids. Different C termini were found in the purified ng/ml) in PBS with 1% BSA (Jackson ImmunoResearch Laborato- chemerin dependent on the source from which it was isolated ries, West Grove, PA) for 1 h. After washing, tetramethylbenzidine as well as the methods used to isolate it, e.g. chemerin isoforms substrate (Alpha Diagnostic International, San Antonio, TX) was terminating at Ser-157, Ala-155, and Phe-154 were found in incubated for 20 min followed by the addition of Stop solution ascitic fluids, serum, and hemofiltrate, respectively (15). This (Alpha Diagnostic International) and measurement of absorbance approach allows identification of the chemerin isoforms but at 450 nm. requires a large amount of starting material and is not amenable Preparation of Glutathione S-Transferase (GST)-tagged for characterization of the chemerin isoforms present in rou- Human Chemerins—cDNA (Open Biosystems, Huntsville, AL) tine clinical samples. encoding human chem163S, chem158K, and chem157S lacking In the accompanying paper (26) we show that the most active the 20-amino acid signal peptide were cloned into pGEX 6P-3 form of chemerin is chem157S in both chemotaxis and Ca vector (GE Healthcare) and transformed into BL21 Star (DE3) mobilization assays, whereas chem158K and chem163S have competent cells (Invitrogen). The resultant expression plas- substantially lower activity (26). To identify and quantify the in mids were verified by sequencing. The proteins were produced vivo chemerin isoforms, we have developed specific ELISAs for in 2 YT medium at 37 °C for 4 h. Cells were pelleted before each of these three chemerin isoforms. Here, we communicate suspension in STE buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM the first data on the presence of the different chemerin isoforms EDTA, pH 7.5) containing 1 mM DTT, 5 units/ml rLysozyme in plasma, synovial fluid, and cerebrospinal fluid (CSF) sam- (Novagen, Madison, WI), benzonase nuclease, and Complete ples and demonstrate that in synovial fluid and CSF samples, protease inhibitor (Roche Applied Science). The insoluble pro- extensive proteolytic processing of prochemerin occurs, with teins were solubilized using a rapid GST inclusion body solubi- chem158K representing the dominant isoform, indicating that lization and renaturation kit (Cell Biolabs, San Diego, CA). The this is a key step in the activation of prochemerin in vivo. cleared supernatants were loaded onto a glutathione-Sephar- ose 4B column equilibrated with STE buffer. After exhaustive EXPERIMENTAL PROCEDURES washing with STE buffer, GST-fused chemerins were eluted Antibody Preparation—Rabbit polyclonal antibodies were with 10 mM reduced glutathione. The recovered proteins were raised against peptides derived from the C termini of 90% pure as determined by silver staining following separa- 151 163 the human chemerin sequence, CGQFAFSKALPRS tion on SDS-PAGE. 151 158 (anti-chem163S), CGQFAFSK (anti-chem158K), and Western Blot Analysis of Anti-chemerin IgGs—Purified 149 157 KCGQFAFS (anti-chem157S) that were conjugated to recombinant GST-chem163S, GST-chem158K, and GST- keyhole limpet hemocyanin according to a standard protocol chem157S (280 ng each) were separated by SDS-PAGE under (Covance, Denver, PA). To isolate specific antibodies from the reducing conditions followed by Western blotting with each of antisera, positive selection affinity chromatography using the the purified anti-chem163S, anti-chem158K, or anti-chem157S cognate peptide conjugated to Sepharose was performed. Typ- IgG (500 ng/ml). The blot was developed with peroxidase-con- ically, rabbit antiserum (200 ml) was diluted 5-fold with PBS, jugated goat anti-rabbit antibody (100 ng/ml) and detected filtered through a 0.22-m filter, and applied to a 2.5-ml col- using ECL (GE Healthcare) detection. umn with the cognate peptide coupled to amino-link agarose Specific ELISAs for Human Chem163S, Chem158K, and (AminoLink kit; Thermo Scientific, Rockford, IL) equilibrated Chem157S—A commercially available mouse monoclonal anti- with PBS. After extensively washing with PBS, bound antibody human chemerin antibody (R&D Systems, Minneapolis, MN) was eluted with 0.1 M glycine and 250 mM NaCl, pH 3.0, and was used as the capturing antibody. The antibody (4g/ml) was 1-ml fractions were collected in tubes containing 50 lof1 M coated in PBS buffer onto ELISA plates, and nonspecific bind- Tris-HCl, pH 8.5, to neutralize the pH. Fractions containing the ing sites were blocked with BSA as described above for the antibody were identified by their reactivity toward the cognate direct peptide ELISA. Purified recombinant chem163S, peptide in a direct ELISA. The fractions with the highest reac- chem158K, and chem157S, prepared as described in the tivity were pooled and subjected to negative selection affinity accompanying paper (26), were used as standards to construct chromatography by adsorption on amino-link columns cou- the calibration curves. Samples and standards were diluted with pled with the noncognate chemerin peptides to remove cross- 1% BSA in PBS and incubated in the wells for 2 h. After washing reactive antibodies. with 0.05% Tween 20 in PBS, the samples were incubated with Direct Peptide ELISA to Determine Antibody Reactivity— the specific cognate antibodies (500 ng/ml). In the case of anti- Peptide (1 g/ml) corresponding to a chemerin isoform in 0.1 M chem163S, 10 g/ml chem158K peptide was added to remove NaHCO , pH 8.6, was coated directly onto a 96-well ELISA plate at the residual cross-reactivity observed with chem158K, and in room temperature for 2 h. Nonspecific binding sites were blocked the case of anti-chem157S, 10 g/ml chem163S peptide was added to remove the residual cross-reactivity with chem163S. In the peptide competition ELISA, 10 g/ml cognate or non- The abbreviations used are: CSF, cerebrospinal fluid; GBM, glioblastoma; cognate peptides (chem163S, CGQFAFSKALPRS; chem158K, D-Phe-Pro-Arg-chlorom- OA, osteoarthritis; OPN, osteopontin; PPACK, H- ethyl ketone; PsA, psoriatic arthritis; RA, rheumatoid arthritis. CGQFAFSK; chem157S, KKGQFAFS; chem156F, PHSFYF- NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 39521 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF PGQFAF; chem154F, PHSFYFPGQF; chem152G, EDPHSFY- Development of ELISAs Specific for Human Chem163S, Chem158K, and Chem157S—The specific antibodies, anti- FPG) were incubated with specific anti-chemerin antibodies. chem163S, anti-chem158K, and anti-chem157S were used to The plates were then processed as described for the direct pep- develop sandwich ELISAs specific for human chem163S, tide ELISA. The concentrations of chemerin isoforms were cal- chem158K, and chem157S, respectively. A commercially avail- culated from the calibration curves of the purified chemerin able monoclonal antibody that recognizes all three human standards. chemerin isoforms was used as the capture antibody. Recombi- Detection of Chemerin Isoforms in Human Samples—Human nant chemerin proteins chem163S, chem158K, chem157S, and samples were obtained under protocols approved by Stanford chem155A produced as described in the accompanying paper University Medical Center or Partners Healthcare Institutional (26) were used as standards. Recombinant chem163S, but not Review Boards. Informed consent was obtained from donors of recombinant chem158K, chem157S, or chem155A, was plasma CSF samples while synovial fluid samples were obtained detected in a dose-dependent manner by anti-chem163S spe- under a protocol for discarded specimens. Blood was drawn cifically, with a lower limit of detection of 0.2 ng/ml (Fig. 1B, from volunteers into Na-Citrate tubes (BD Biosciences), and left). Likewise, anti-chem158K specifically recognized recom- plasma was prepared. In some experiments H-D-Phe-Pro-Arg- binant chem158K and not chem163S, chem157S, or chem155A chloromethyl ketone (PPACK; Calbiochem) was included in (Fig. 1B, center) whereas anti-chem157S specifically recognized the Na-Citrate collection tubes. Plasma (2 ml) was mixed with recombinant chem157S and not chem163S, chem158K, or 100 l of heparin-agarose (Sigma) and complete protease chem155A. Both of these ELISAs also had similar sensitivity to inhibitor (Roche Applied Science). After incubation at 4 °C for the chem163S ELISA with a lower limit of detection of 0.2 2 h, the heparin-agarose beads were pelleted and washed exten- ng/ml (Fig. 1B, right). sively with PBS, and chemerin was eluted with 0.8 M NaCl in To confirm the specificity of the ELISAs, competitive inhibi- PBS, all in the presence of Complete protease inhibitor. The tion with the cognate peptide was performed. The C-terminal eluates were diluted with 1% BSA in PBS for assay in the specific peptide from human chem163S specifically inhibited the signal ELISAs described above. from recombinant chem163S protein in the ELISA for CSF and synovial joint samples were stored frozen at80 °C chem163S, whereas the C-terminal peptides corresponding to until the time of analysis. After thawing, 400 l of a CSF sample, chem158S and chem157S did not (Fig. 1C, left). In a similar or 30 l of a synovial fluid sample diluted to 400 l with PBS, fashion, the C-terminal peptides of human chem158K and was mixed with 50 l of heparin-agarose and Complete prote- chem157S specifically inhibited the signals from chem158K ase inhibitor before heparin-agarose adsorption was performed and chem157S proteins in the ELISAs for chem158K and as described above and the eluates assayed by the specific chem157S, respectively (Fig. 1C, center and right). ELISAs. Other chemerin isoforms have been reported such as Statistics—Comparison of two samples was by Student’s t chem156F, chem154F, and chem152G (20, 21). We tested test; multigroup comparisons were by Kruskal-Wallis analysis. whether those isoforms would be detected by the specific ELI- The analysis was carried out using Prism v5 (GraphPad, La Jolla, SAs by including the noncognate C-terminal peptides corre- sponding to chem152G, chem154F, and chem156F to the spe- CA). Values of p  0.05 were considered significant. cific ELISAs. They did not inhibit the signal from recombinant chem163S protein in the ELISA for chem163S, nor did they RESULTS inhibit chem158K or chem157S protein signals in the ELISAs Generation and Characterization of Specific Antibodies for for chem158K or chem157S (supplemental Fig. 1). These data Chem163S, Chem158K, and Chem157S—Because the activity show that the ELISAs for the different chemerin isoforms have of the different chemerin isoforms varies significantly, mea- good sensitivity and specificity. surements of total levels of chemerin in biological fluids do not Detection of Chem163S, Chem158K, and Chem157S Levels in give a complete description of the status of the chemerin sys- Human Plasma—Having established the specificity of the ELI- tem. We therefore developed a panel of ELISAs capable of spe- SAs, we used them to determine the levels of chem163S, cifically detecting individual chemerin isoforms in human sam- chem158K, and chem157S in human plasma from normal vol- ples using the methodology we had employed previously to unteers. In the initial experiments to determine levels of develop specific ELISAs for osteopontin (OPN) isoforms (22). chemerin isoforms in plasma, very high values for chem163S Peptide antigens were used to immunize rabbits to raise specific were detected (supplemental Fig. 2). Analysis of plasma by antibodies directed against the different C-terminal sequences Western blotting using the anti-163S antibody showed the of chem163S, chem158K, and chem157S. Specific rabbit anti- presence of a high molecular mass cross-reactive substance chem163S, anti-chem158K, and anti-chem157S IgGs were (supplemental Fig. 3A, lane 3). To circumvent this problem, a purified by positive selection by binding to the cognate peptide- heparin adsorption step was introduced before the ELISA that conjugated Sepharose followed by negative selection to remove separates the interfering substance while allowing quantitative any cross-reacting antibodies using the noncognate peptide- recovery of chemerin isoforms upon elution from the heparin- conjugated Sepharose. The specificity of these anti-chemerin agarose beads (supplemental Fig. 3B). The chemerin detection isoform IgGs was demonstrated by Western blotting in which antibody from R&D did not recognize this high molecular mass each antibody only recognized its cognate protein and not the band (supplemental Fig. 3C, lane 2), but it recognized recom- other two chemerin isoforms (Fig. 1A). binant chem163S, chem158K, chem157S, and chem155A pre- 39522 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF FIGURE 1. Characterization of specific antibodies against recombinant chemerin isoforms. A, purified antibodies specific for different chemerin isoforms were characterized by Western blot analysis of recombinant GST-chem163S (left), GST-chem157S (right), and GST-chem158K (center) with anti-chem163S, anti-chem157S, and anti-chem158K, as described under “Experimental Procedures.” Molecular mass markers are shown on the left of the panels. B, recombi- nant chem163S (), chem158K (ƒ), and chem157S (‚), and chem155A () by anti-chem163S, anti-chem158K, and anti-chem157S were detected using specific ELISAs as described under “Experimental Procedures.” None of the three antibodies detects chem155A (). C, cognate peptide competes in the ELISA, but the noncognate peptides do not. C-terminal chem163S peptide (Œ) specifically inhibited the signal from recombinant chem163S by itself ()inthe chem163S ELISA, but the C-terminal chem158K peptide (), chem157S peptide () did not compete (left). C-terminal chem158K peptide () and chem157S peptide () specifically inhibited the signals from chem158K by itself () and chem157S by itself () proteins in the ELISAs for recombinant chem158K and chem157S, respectively (center and right), but the noncognate peptides did not compete. Values are the mean of duplicates. pared as described in the accompanying paper (supplemental Fig. 3D), showing that the high molecular mass band was not related to chemerin. Using the revised method, the levels of the different chemerin isoforms were measured in plasma from normal vol- unteers (Fig. 2). The plasma level for chem163S was 40  7.9 ng/ml (mean  S.D., n  9), chem158K level was 8.1 2.9 ng/ml, whereas chem157S was barely detectable at 0.7 0.8 ng/ml. To exclude the possibility that the chem158K resulted from plasmin cleavage of chem163S occurring during or after the blood draw, blood was drawn into tubes with and without FIGURE 2. Levels of chemerin isoforms in human plasma. Chem163S, 0.1 mM PPACK, an inhibitor of plasmin, thrombin, and other chem158K, and chem157S levels in normal human plasma (n 9) were deter- serine proteases. The levels of chem163S, chem158K, and mined using specific ELISAs as described under “Experimental Procedures.” chem157S were not altered by the presence of PPACK. Because Horizontal lines show the mean. NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 39523 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF FIGURE 3. Levels of chemerin isoforms in CSF of glioblastoma, oligodendrocytoma, and miscellaneous CNS disease. Chem163S, chem158K, and chem157S levels in CSF from GBM (n 12), oligodendrocytoma (Oligo)(n 12), and miscellaneous (noncancer) CNS disease patients (n 7) were determined using specific ELISAs as described under “Experimental Procedures.” Horizontal lines show the mean. plasmin is an enzyme that has been suggested to be responsible with RA, OA, and PsA (Fig. 4). In RA synovial fluid samples, the for chem163S cleavage to chem158K, we measured plasmin– levels of chem163S, chem158K, and chem157S were 12 22.3, anti-plasmin levels in these samples by ELISA. The levels of 56 42.9, and 9.3 10.5 ng/ml (mean S.D., n 23), respec- plasmin–anti-plasmin in all samples were within the normal tively. In OA synovial fluid samples (n 23), the corresponding range for human plasma and no different if PPACK was levels were 9.0  7.2, 74.1  74.4, and 6.1  9.9 ng/ml; and in included during the blood draw (data not shown). Taken PsA samples (n  13), they were 1.1  2.3, 58.9  36.6, and together, these data suggest that the detectable level of 3.2  6.2 ng/ml, respectively. It is striking that the total chem158K in plasma did not result from ex vivo activation of chemerin levels (chem163S plus chem158K plus chem157S) plasminogen and processing of chem163S by plasmin or other found in the synovial fluid samples of all three types of arthritis serine proteases. patients were much higher than in CSF samples from CNS dis- Detection of Chemerin Isoforms in CSF Samples from Patients ease and were approximately 2-fold higher than that of plasma with CNS Disease—Next, we measured the levels of chemerin samples of normal people. The chem158K form was the domi- isoforms in CSF samples of patients with malignant glioblas- nant isoform of chemerin in all disease groups, and there was no toma (GBM), oligodendrocytoma, and miscellaneous CNS dis- significant difference among RA, OA, and PsA. We tested eases that included one sample each from a patient with whether the chem158K had been generated ex vivo in the syno- migraine, pseudotumor cerebi, meningioma (negative cytol- vial fluid samples by inclusion of PPACK in the collecting tubes, ogy), cylindroma necrosis postradiation therapy (negative and we found that the presence of PPACK did not alter the level cytology), malignant hypertension, ascending sensory loss, and of chem158K, suggesting that it had not been generated ex vivo. bilateral VIth nerve palsies (negative cytology) (Fig. 3). In GBM Comparison of Cleaved Chemerins in Human Plasma, CSF, the levels for chem163S, chem158K, and chem157S were Inflammatory CSF, and Synovial Fluid Sample—We compared 3  2.4, 5.1  3.9, and 0.2  0.3 ng/ml, respectively (mean  the fraction of the total chemerin that had been cleaved in the S.D., n  12). In oligodendrocytoma CSF samples (n  12), the different types of human fluid samples. Using chem163S, corresponding levels were 2.9  2.5, 3.8  3.8, and 0.7  1.3 chem158K, and chem157S levels determined by the specific ng/ml, whereas in the miscellaneous CNS disease (noncancer ELISAs, the fraction of cleaved chemerins (chem158K plus CNS disease) CSF samples (n  7), the levels were 5.5  3.8, chem157S) was calculated for each sample. Because the levels 6.3  4.8, and 1.0  0.8 ng/ml, respectively. There were no of chemerin isoforms were not different among the CSF or significant differences among the three disease groups. In all of synovial fluid samples from different disease states, the CSF and these CSF samples, the level of chem163S was much lower synovial fluid samples were analyzed together as two separate (10%) than that in normal plasma. On the other hand, the groups and compared with plasma (Fig. 5). The fraction of fraction of chem163S that had been processed, represented as cleaved chemerin was 0.18 in plasma whereas it was 0.5 in the ratio of chem158K to chem163S, was significantly higher. inflammatory CSF samples, and it approached 0.75 in the syno- The possibility of ex vivo plasmin processing of chem163S was vial fluid samples with the means varying significantly when tested by inclusion of PPACK when a CSF sample was freshly analyzed by the Kruskal-Wallis test (p  0.0001), thus indicat- collected, but again there was no difference noted in the levels ing that significant cleavage and processing of chemerin occur of the chemerin isoforms (data not shown), similar to the situ- in these extravascular compartments in inflammatory diseases. ation in plasma, indicating that chem158K did not arise from ex DISCUSSION vivo proteolytic processing. Detection of Chemerin Isoforms in the Synovial Fluid of Prochemerin (chem163S) can be cleaved in its C-terminal Arthritis Patients—Based on our previous observation that pro- domain by different serine and cysteine proteases in vitro, gen- teolytic cleavage of OPN in synovial fluids was much higher in erating a series of chemerin isoforms with different levels of rheumatoid arthritis (RA) than in either osteoarthritis (OA) or activity (Fig. 6) (23). To investigate which are the relevant forms psoriatic arthritis (PsA) (22), we hypothesized that the inflam- of chemerin in vivo, we have developed ELISAs to measure the matory environment in RA would also lead to higher levels of different chemerin isoforms in biological samples. The ELISAs processing of chemerin. Therefore, we investigated the are based on polyclonal antibodies raised against peptides that chemerin isoform levels in synovial fluid samples of patients represent the C termini of the different isoforms and possess 39524 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF FIGURE 4. Levels of chemerin isoforms in synovial fluid of patients with RA, OA, and PsA. Chem163S, chem158K, and chem157S levels in synovial fluid of patients with RA (n 23), OA (n 23), and PsA (n 13) were determined using specific ELISAs as described under “Experimental Procedures.” Horizontal lines show the mean. On the other hand, CSF samples from patients with a variety of CNS diseases and synovial fluids from arthritis patients showed a completely different picture. The total level of cleaved and noncleaved chemerins was significantly lower (15%) in the CSF than plasma whereas it was 2-fold higher than the plasma level in synovial fluid. Even more striking was the frac- tion of cleaved chemerins present in these samples from patients with inflammatory diseases. In normal plasma, only about 18% of chemerin is cleaved, whereas the fraction was 50% in CSF samples and approached 75% in the arthritis synovial fluid samples. Thus, considerable proteolysis of the intact chemerin is occurring in these two extravascular com- FIGURE 5. Fraction of cleaved chemerin in human plasma, CAF of CNS disease, and synovial fluid of arthritis. Ratio of cleaved chemerin levels partments. The possibility that chemerin is synthesized and (sum of chem158K plus chem157S) to total chemerin (sum of chem163S, processed locally in these compartments is supported by the chem158K, and chem157S) in plasma (●), CSF (Œ), and synovial fluid ()is significant differences between the total amount of chemerin as shown. All samples from Figs. 3, 4, and 5 have been included. Horizontal lines show the mean. well as the extent of its cleavage that was observed in the CSF and synovial fluid samples. It is notable that in both the synovial and CSF compartments, specificity for a single chemerin isoform as demonstrated by chem158K was the dominant form. We have investigated the Western blot analysis and specific competitive inhibition by the possibility of ex vivo plasmin processing by comparison of sam- cognate C-terminal peptide but not the noncognate peptides in ples collected with and without PPACK and found that no the ELISAs (Fig. 1). The lack of competition by the noncognate detectable chemerin processing occurred. It is therefore likely peptides suggests that the key determinants recognized by the that a high fraction of chemerin is cleaved in these compart- antibodies are the C-terminal amino acids, as we have found ments, as a result of the inflammatory milieu in arthritis for the previously in epitope mapping of the specific antibodies for the synovial samples and glioma and other inflammatory CNS dis- different proteolytically cleaved OPN isoforms (18). eases for the CSF samples. In vitro studies have shown that The ELISAs proved to have excellent specificity and sensitiv- either plasmin or tryptase can cleave the five C-terminal amino ity for the different chemerin isoforms allowing for the first acids from chem163S resulting in chem158K (23). Although the time determination of the levels of three key chemerin iso- activity of chem158K is low (26), the levels present in the syno- forms, the precursor, chem163S, and two cleavage products, vial fluid samples, but not the CSF samples, are high enough to chem158K and chem157S. Importantly, the sensitivity of each suggest that they may be sufficient to contribute a significant of the ELISAs is 0.2 ng/ml (0.012 nM) whereas 50% of the amount of chemerin activity without further processing. effective concentration (EC ) for the most active isoform, Although chem158K can be efficiently generated by plasmin chem157S, is 1.17  0.74 nM in a calcium mobilization assay. cleavage in vitro (19), the protease(s) involved in vivo remains to Thus, the range of the ELISAs covers the concentrations at be established. Because prochemerin can be cleaved by a large which we have shown chemerin to be active (26). variety of proteases, the high levels of chem158K present in CSF Use of these ELISAs in our studies showed that in normal and synovial fluids provide in vivo validation of the importance human plasma samples, chem163S, the prochemerin form, of cleavage at lysine 158, by plasmin or other serine proteases, at dominated, whereas chem158K represented only a small per- these extravascular sites, and it is probable that active centage of the total chemerin, and chem157S was barely detect- chemerin, chem157S, is generated via this intermediate (Fig. 6). able. Thus, under normal conditions, most of the chemerin present in plasma was uncleaved and therefore inactive. The Other chemokines such as CCL15 and CCL23 also require low but reproducible level of chem158K suggests that there is a proteolytic processing to generate the isoforms that possess constitutive level of processing of the inactive chem163S in maximum activity (24), although these cleavages are at the N plasma at base line. The protease(s) responsible for this cleav- terminus rather than the C terminus. Analysis of synovial fluids, age remains to be established. some from patients with RA, for cleavage of CCL15 and CCL23 NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 39525 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF FIGURE 6. Schematic of chemerin cleavages. Chem163S (pink box) can be cleaved either by elastase, generating a mixture of chem157S (red box) and chem155A (blue box), by proteinase 3 and tryptase generating chem155A or by plasmin generating chem158K (pink box). Chem158K can then be cleaved by carboxypeptidases, carboxypeptidase B2 (CPB2) or carboxypeptidase N (CPN), to form the active chem157S, which is subsequently inactivated by angiotensin- converting enzyme (ACE) forming inactive chem155A. The heavier arrows represent the cleavage pathway for chem163S described in this paper. The icon for the N-terminal region of chemerin is based on the model in Zabel et al. (25). demonstrated the presence of cleaved active isoforms of CCL15 peptidase N and carboxypeptidase B2 can cleave chem158K to and CCL23. Proteolytic processing of these G protein-coupled chem157S, the fully activated isoform of chemerin (23). There receptor ligands such as chemerin, CCL15, or CCL23 may rep- were elevated levels of OPN-L in synovial fluid samples (18), as resent a general mechanism to localize their effects to the site of well as in GBM CSF samples (data not shown), indicating the inflammation. presence of an active carboxypeptidase that should be capable When the cleavage of OPN in synovial fluids from different of removing the C-terminal lysine from chem158K to generate types of arthritis was compared, we have found previously that chem157S. In addition, we have shown the presence of car- there was far more cleavage of OPN in RA than in OA or PsA to boxypeptidase B2 protein in the synovium (18). Therefore, we the thrombin-cleaved form (OPN-R) as well as the thrombin/ would have expected to detect chem157S in these samples as carboxypeptidase B22 double-cleaved form (OPN-L). How- both the substrate, chem158K, and the enzyme have been ever, there was no difference in cleavage of chemerins when shown to be present. One possible explanation for this is that samples from the different types of arthritis were evaluated, chem157S was rapidly cleaved further, possibly by the angio- suggesting that the regulation of proteolytic processing of OPN tensin-converting enzyme (6), to the inactive chem155A iso- and prochemerin is completely different. Similarly, there was form in vivo (26). This may represent a physiological step to no difference in chemerin levels or in the fraction of cleaved regulate the activity of this potent chemoattractant. Alterna- chemerin when GBM samples were compared with oligoden- tively, chem157S may bind to cells and therefore cannot be drocytoma or noncancer CSF samples. The noncancer samples detected in the fluid phase. We are currently developing a spe- were from patients some of whom had inflammatory diseases cific ELISA for chem155A that will help to resolve this issue. such as cylindroma necrosis and postradiation therapy that Our study provides the first insights into the in vivo levels of may also affect levels and cleavage of chemerin. In the accom- the different chemerin isoforms and suggests that global deter- panying paper (26), analysis of gene expression shows that minations of chemerin levels may be misleading in terms of the chemerin mRNA is increased in grade IV GBM compared with biological potency of chemerin present. In particular, the dif- lower grade gliomas that include oligodendrocytoma or epi- ferent levels of chem158K found in different compartments lepsy samples. The increase in mRNA was not reflected in the emphasize that the generation of fully active chem157S is prob- levels of chemerin present in the CSF, but that may be because ably under tight local regulation. The determination of the dif- the GBM itself is only a minor contributor to the overall protein ferent chemerin isoforms in various disease states should content of the CSF. advance our understanding of this novel chemoattractant that Surprisingly, our study found very low levels of chem157S in acts in the interface between thrombosis, inflammation, and any of the samples. We have shown that both plasma carboxy- immunity. 39526 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF REFERENCES 14. Yamamoto, T., Qureshi, A. R., Anderstam, B., Heimbürger, O., Bárány, P., Lindholm, B., Stenvinkel, P., and Axelsson, J. (2010) Nephrol. Dial. Trans- 1. Nagpal, S., Patel, S., Jacobe, H., DiSepio, D., Ghosn, C., Malhotra, M., plant. 25, 4017–4023 Teng, M., Duvic, M., and Chandraratna, R. A. (1997) J. Invest. Dermatol. 15. Weigert, J., Obermeier, F., Neumeier, M., Wanninger, J., Filarsky, M., 109, 91–95 Bauer, S., Aslanidis, C., Rogler, G., Ott, C., Schäffler, A., Schölmerich, J., 2. Wittamer, V., Franssen, J. D., Vulcano, M., Mirjolet, J. F., Le Poul, E., and Buechler, C. (2010) Inflamm. Bowel Dis. 16, 630–637 Migeotte, I., Brézillon, S., Tyldesley, R., Blanpain, C., Detheux, M., Man- 16. Kukla, M., Zwirska-Korczala, K., Gabriel, A., Waluga, M., Warakomska, I., tovani, A., Sozzani, S., Vassart, G., Parmentier, M., and Communi, D. Szczygiel, B., Berdowska, A., Mazur, W., Wozniak-Grygiel, E., and Kryc- (2003) J. Exp. Med. 198, 977–985 zka, W. (2010) J. Viral Hepat. 17, 661–667 3. Wittamer, V., Bondue, B., Guillabert, A., Vassart, G., Parmentier, M., and 17. Meder, W., Wendland, M., Busmann, A., Kutzleb, C., Spodsberg, N., John, Communi, D. (2005) J. Immunol. 175, 487–493 H., Richter, R., Schleuder, D., Meyer, M., and Forssmann, W. G. (2003) 4. Zabel, B. A., Silverio, A. M., and Butcher, E. C. (2005) J. Immunol. 174, FEBS Lett. 555, 495–499 244–251 18. Zabel, B. A., Nakae, S., Zúñiga, L., Kim, J. Y., Ohyama, T., Alt, C., Pan, J., 5. Vermi, W., Riboldi, E., Wittamer, V., Gentili, F., Luini, W., Marrelli, S., Suto, H., Soler, D., Allen, S. J., Handel, T. M., Song, C. H., Galli, S. J., and Vecchi, A., Franssen, J. D., Communi, D., Massardi, L., Sironi, M., Man- Butcher, E. C. (2008) J. Exp. Med. 205, 2207–2220 tovani, A., Parmentier, M., Facchetti, F., and Sozzani, S. (2005) J. Exp. Med. 19. Zabel, B. A., Ohyama, T., Zuniga, L., Kim, J. Y., Johnston, B., Allen, S. J., 201, 509–515 Guido, D. G., Handel, T. M., and Butcher, E. C. (2006) Exp. Hematol 34, 6. Ernst, M. C., and Sinal, C. J. (2010) Trends Endocrinol. Metab. 21, 1106–1114 660–667 20. Zabel, B. A., Allen, S. J., Kulig, P., Allen, J. A., Cichy, J., Handel, T. M., and 7. Hu, W., and Feng, P. (2011) Diabetes Res. Clin. Pract. 91, 159–163 Butcher, E. C. (2005) J. Biol. Chem. 280, 34661–34666 8. Yang, M., Yang, G., Dong, J., Liu, Y., Zong, H., Liu, H., Boden, G., and Li, L. 21. Guillabert, A., Wittamer, V., Bondue, B., Godot, V., Imbault, V., Parmen- (2010) J. Investig. Med. 58, 883–886 tier, M., and Communi, D. (2008) J. Leukocyte Biol. 84, 1530–1538 9. Pfau, D., Stepan, H., Kratzsch, J., Verlohren, M., Verlohren, H. J., Drynda, K., Lössner, U., Blüher, M., Stumvoll, M., and Fasshauer, M. (2010) Horm. 22. Sharif, S. A., Du, X., Myles, T., Song, J.J., Price, E., Lee, D.M., Goodman, Res. Paediatr. 74, 56–61 S.B., Nagashima, M., Morser, J., Robinson, W.H., and Leung, L.L.K. (2009) 10. Tönjes, A., Fasshauer, M., Kratzsch, J., Stumvoll, M., and Blüher, M. (2010) Arthritis Rheum. 60, 2902–2912 PLoS One 5, e13911 23. Du, X. Y., Zabel, B. A., Myles, T., Allen, S. J., Handel, T. M., Lee, P. P., 11. Dogru, T., Karadurmus, N., Tapan, S., and Genc, H. (2010) Scand J. Gas- Butcher, E. C., and Leung, L. L. (2009) J. Biol. Chem. 284, 751–758 troenterol. 45, 1000–1001; author reply 1001–1002 24. Berahovich, R. D., Miao, Z., Wang, Y., Premack, B., Howard, M. C., and 12. Sell, H., Divoux, A., Poitou, C., Basdevant, A., Bouillot, J. L., Bedossa, P., Schall, T. J. (2005) J. Immunol. 174, 7341–7351 Tordjman, J., Eckel, J., and Clément, K. (2010) J. Clin. Endocrinol. Metab. 25. Zabel, B. A., Zuniga, L., Ohyama, T., Allen, S. J., Cichy, J., Handel, T. M., 95, 2892–2896 and Butcher, E. C. (2006) Exp. Hematol. 34, 1021–1032 13. Pfau, D., Bachmann, A., Lössner, U., Kratzsch, J., Blüher, M., Stumvoll, M., 26. Yamaguchi, Y., Du, X.-Y., Zhao, L., Morser, J., and Leung, L. L. K. (2011) and Fasshauer, M. (2010) Diabetes Care 33, 171–173 J. Biol. Chem. 286, 39510–39519 NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 39527 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Chemerin158K Protein Is the Dominant Chemerin Isoform in Synovial and Cerebrospinal Fluids but Not in Plasma

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 45, pp. 39520 –39527, November 11, 2011 Printed in the U.S.A. Chemerin158K Protein Is the Dominant Chemerin Isoform in □ S Synovial and Cerebrospinal Fluids but Not in Plasma Received for publication, May 6, 2011, and in revised form, September 1, 2011 Published, JBC Papers in Press, September 19, 2011, DOI 10.1074/jbc.M111.258954 ‡ ‡ ‡ ‡ ‡‡ §¶ Lei Zhao , Yasuto Yamaguchi , Shadi Sharif , Xiao-Yan Du , Jason J. Song** , David M. Lee , Lawrence D. Recht , ‡‡ ‡1 ‡ ‡‡2 William H. Robinson** , John Morser , and Lawrence L. K. Leung From the Division of Hematology and **Division of Immunology and Rheumatology, Department of Medicine, and Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California 94305, the Brigham and Women’s Hospital, Boston, Massachusetts 02115, Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland, and the ‡‡ Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94305 Background: Chemerin is a chemokine/adipokine whose activity depends on proteolytic processing. Results: Specific ELISAs demonstrate that in plasma the precursor is dominant, whereas in synovial fluid from arthritis patients and CSF from glioblastoma patients, chem158K dominates. Low levels of active chem157S were found. Conclusion: Chemerin proteolysis occurs during inflammation. Significance: This is the first report about levels of different chemerin isoforms in biological samples. Chemerin is a chemoattractant involved in immunity that The chemerin gene was originally identified as a novel reti- may also function as an adipokine. Chemerin circulates as an noid-responsive gene in psoriatic skin lesions (1). Chemerin is a inactive precursor (chem163S), and its activation requires pro- secreted ligand of the orphan G protein-coupled receptor teolytic cleavages at its C terminus, involving proteases involved chemokine-like receptor 1, chemokine (C-C motif) receptor- in coagulation, fibrinolysis, and inflammation. However, the key like 2, and G protein-coupled receptor 1 (2). Various cell types proteolytic steps in prochemerin activation in vivo remain to be involved in innate and adaptive immunity, including immature established. Previously, we have shown that C-terminal cleavage plasmacytoid dendritic cells, myeloid dendritic cells, macro- of chem163S by plasmin to chem158K, followed by a carboxy- phages, and natural killer cells, express chemokine-like recep- peptidase cleavage, leads to the most active isoform, chem157S. tor 1, and chemerin is a chemoattractant that promotes the To identify and quantify the in vivo chemerin isoforms in bio- recruitment of these cells to lymphoid organs and sites of tissue logical specimens, we developed specific ELISAs for chem163S, injury (3–5). Chemerin is also an adipokine that regulates adi- chem158K, and chem157S, using antibodies raised against pep- pocyte development and metabolic function and may play a tides from the C terminus of the different chemerin isoforms. role in glucose tolerance, obesity, and hyperlipidemia (6). Ele- We found that the mean plasma concentrations of chem163S, vated levels of chemerin have been found in patients with dia- chem158K, and chem157S were 40  7.9, 8.1  2.9, and 0.7 betes (7–10) and fatty liver disease (11, 12), consistent with its 0.8 ng/ml, respectively. The total level of cleaved and non- role as an adipokine whereas its levels are also raised in patients cleaved chemerins in cerebrospinal fluids was 10% of plasma undergoing chronic hemodialysis (13, 14) as well as those with levels whereas it was elevated 2-fold in synovial fluids from Crohn disease (15) and chronic hepatitis C (16), in line with its patients with arthritis. On the other hand, the fraction of cleaved suggested function in inflammation. However, these studies chemerins was much higher in synovial fluid and cerebrospinal have all been carried out using ELISAs that detect total fluid samples than in plasma (75%, 50%, and 18% respectively). chemerin levels. Chem158K was the dominant chemerin isoform, and it was not Chemerin is translated as a 163-amino acid pre-proprotein generated by ex vivo processing, indicating that cleavage of subsequently secreted as a 143-amino acid precursor prochemerin at position Lys-158, whether by plasmin or another (prochemerin, chem163S) following removal of the N-terminal serine protease, represents a major step in prochemerin activation signal peptide (2, 17). Prochemerin chem163S has low biologi- in vivo. Our study provides the first direct evidence that chemerin cal activity and requires further extracellular C-terminal pro- undergoes extensive proteolytic processing in vivo, underlining the teolytic processing to achieve its full biological activity. These importance of measuring individual isoforms. cleavages have been studied in vitro where a variety of enzymes involved in coagulation, fibrinolytic and inflammation such as FXIIa, plasmin, carboxypeptidase B2 (also known as thrombin- activatable fibrinolysis inhibitor), or elastase can proteolyze * This work was supported, in whole or in part, by National Institutes of Health Grants HL057530 and AI085268. chemerin, giving rise to either active or inactive chemerins (2, 4, □ S The on-line version of this article (available at http://www.jbc.org) contains 17–19). Plasmin and elastase cleavage gives rise to chemerin21- supplemental Figs. 1–3. 158 (chem158K) and chemerin21-157 (chem157S), respec- To whom correspondence may be addressed: 269 Campus Dr., CCSR 1155, MC5156, Stanford, CA 94305-5156. Tel.: 1-650-723-7078; Fax: 1-650-721- tively, whereas proteinase 3 and mast cell chymase give rise to 6826; E-mail: [email protected]. inactive chemerin21-155 (chem155A) and chemerin21-154 To whom correspondence may be addressed: 269 Campus Dr., CCSR 1155, (chem154F), respectively (3, 20, 21). However, the catalytic effi- MC5156, Stanford, CA 94305-5156. Tel.: 1-650-723-7078; Fax: 1-650-721- 6826; E-mail: [email protected]. ciencies of most of these proteolytic cleavages of prochemerin 39520 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 This is an Open Access article under the CC BY license. Chemerin158K Is the Dominant Form in Synovial Fluids and CSF have not been characterized, and whether they represent phys- by incubation with 1% BSA in PBS for 1 h. Antibody fractions from iological activation or inactivation steps in vivo remains the cognate peptide affinity chromatography were diluted with 1% unclear. BSA in PBS and incubated for 1 h followed by washing and incu- An alternative approach has been to purify chemerin from bation with peroxidase-conjugated goat anti-rabbit IgG (100 biological fluids. Different C termini were found in the purified ng/ml) in PBS with 1% BSA (Jackson ImmunoResearch Laborato- chemerin dependent on the source from which it was isolated ries, West Grove, PA) for 1 h. After washing, tetramethylbenzidine as well as the methods used to isolate it, e.g. chemerin isoforms substrate (Alpha Diagnostic International, San Antonio, TX) was terminating at Ser-157, Ala-155, and Phe-154 were found in incubated for 20 min followed by the addition of Stop solution ascitic fluids, serum, and hemofiltrate, respectively (15). This (Alpha Diagnostic International) and measurement of absorbance approach allows identification of the chemerin isoforms but at 450 nm. requires a large amount of starting material and is not amenable Preparation of Glutathione S-Transferase (GST)-tagged for characterization of the chemerin isoforms present in rou- Human Chemerins—cDNA (Open Biosystems, Huntsville, AL) tine clinical samples. encoding human chem163S, chem158K, and chem157S lacking In the accompanying paper (26) we show that the most active the 20-amino acid signal peptide were cloned into pGEX 6P-3 form of chemerin is chem157S in both chemotaxis and Ca vector (GE Healthcare) and transformed into BL21 Star (DE3) mobilization assays, whereas chem158K and chem163S have competent cells (Invitrogen). The resultant expression plas- substantially lower activity (26). To identify and quantify the in mids were verified by sequencing. The proteins were produced vivo chemerin isoforms, we have developed specific ELISAs for in 2 YT medium at 37 °C for 4 h. Cells were pelleted before each of these three chemerin isoforms. Here, we communicate suspension in STE buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM the first data on the presence of the different chemerin isoforms EDTA, pH 7.5) containing 1 mM DTT, 5 units/ml rLysozyme in plasma, synovial fluid, and cerebrospinal fluid (CSF) sam- (Novagen, Madison, WI), benzonase nuclease, and Complete ples and demonstrate that in synovial fluid and CSF samples, protease inhibitor (Roche Applied Science). The insoluble pro- extensive proteolytic processing of prochemerin occurs, with teins were solubilized using a rapid GST inclusion body solubi- chem158K representing the dominant isoform, indicating that lization and renaturation kit (Cell Biolabs, San Diego, CA). The this is a key step in the activation of prochemerin in vivo. cleared supernatants were loaded onto a glutathione-Sephar- ose 4B column equilibrated with STE buffer. After exhaustive EXPERIMENTAL PROCEDURES washing with STE buffer, GST-fused chemerins were eluted Antibody Preparation—Rabbit polyclonal antibodies were with 10 mM reduced glutathione. The recovered proteins were raised against peptides derived from the C termini of 90% pure as determined by silver staining following separa- 151 163 the human chemerin sequence, CGQFAFSKALPRS tion on SDS-PAGE. 151 158 (anti-chem163S), CGQFAFSK (anti-chem158K), and Western Blot Analysis of Anti-chemerin IgGs—Purified 149 157 KCGQFAFS (anti-chem157S) that were conjugated to recombinant GST-chem163S, GST-chem158K, and GST- keyhole limpet hemocyanin according to a standard protocol chem157S (280 ng each) were separated by SDS-PAGE under (Covance, Denver, PA). To isolate specific antibodies from the reducing conditions followed by Western blotting with each of antisera, positive selection affinity chromatography using the the purified anti-chem163S, anti-chem158K, or anti-chem157S cognate peptide conjugated to Sepharose was performed. Typ- IgG (500 ng/ml). The blot was developed with peroxidase-con- ically, rabbit antiserum (200 ml) was diluted 5-fold with PBS, jugated goat anti-rabbit antibody (100 ng/ml) and detected filtered through a 0.22-m filter, and applied to a 2.5-ml col- using ECL (GE Healthcare) detection. umn with the cognate peptide coupled to amino-link agarose Specific ELISAs for Human Chem163S, Chem158K, and (AminoLink kit; Thermo Scientific, Rockford, IL) equilibrated Chem157S—A commercially available mouse monoclonal anti- with PBS. After extensively washing with PBS, bound antibody human chemerin antibody (R&D Systems, Minneapolis, MN) was eluted with 0.1 M glycine and 250 mM NaCl, pH 3.0, and was used as the capturing antibody. The antibody (4g/ml) was 1-ml fractions were collected in tubes containing 50 lof1 M coated in PBS buffer onto ELISA plates, and nonspecific bind- Tris-HCl, pH 8.5, to neutralize the pH. Fractions containing the ing sites were blocked with BSA as described above for the antibody were identified by their reactivity toward the cognate direct peptide ELISA. Purified recombinant chem163S, peptide in a direct ELISA. The fractions with the highest reac- chem158K, and chem157S, prepared as described in the tivity were pooled and subjected to negative selection affinity accompanying paper (26), were used as standards to construct chromatography by adsorption on amino-link columns cou- the calibration curves. Samples and standards were diluted with pled with the noncognate chemerin peptides to remove cross- 1% BSA in PBS and incubated in the wells for 2 h. After washing reactive antibodies. with 0.05% Tween 20 in PBS, the samples were incubated with Direct Peptide ELISA to Determine Antibody Reactivity— the specific cognate antibodies (500 ng/ml). In the case of anti- Peptide (1 g/ml) corresponding to a chemerin isoform in 0.1 M chem163S, 10 g/ml chem158K peptide was added to remove NaHCO , pH 8.6, was coated directly onto a 96-well ELISA plate at the residual cross-reactivity observed with chem158K, and in room temperature for 2 h. Nonspecific binding sites were blocked the case of anti-chem157S, 10 g/ml chem163S peptide was added to remove the residual cross-reactivity with chem163S. In the peptide competition ELISA, 10 g/ml cognate or non- The abbreviations used are: CSF, cerebrospinal fluid; GBM, glioblastoma; cognate peptides (chem163S, CGQFAFSKALPRS; chem158K, D-Phe-Pro-Arg-chlorom- OA, osteoarthritis; OPN, osteopontin; PPACK, H- ethyl ketone; PsA, psoriatic arthritis; RA, rheumatoid arthritis. CGQFAFSK; chem157S, KKGQFAFS; chem156F, PHSFYF- NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 39521 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF PGQFAF; chem154F, PHSFYFPGQF; chem152G, EDPHSFY- Development of ELISAs Specific for Human Chem163S, Chem158K, and Chem157S—The specific antibodies, anti- FPG) were incubated with specific anti-chemerin antibodies. chem163S, anti-chem158K, and anti-chem157S were used to The plates were then processed as described for the direct pep- develop sandwich ELISAs specific for human chem163S, tide ELISA. The concentrations of chemerin isoforms were cal- chem158K, and chem157S, respectively. A commercially avail- culated from the calibration curves of the purified chemerin able monoclonal antibody that recognizes all three human standards. chemerin isoforms was used as the capture antibody. Recombi- Detection of Chemerin Isoforms in Human Samples—Human nant chemerin proteins chem163S, chem158K, chem157S, and samples were obtained under protocols approved by Stanford chem155A produced as described in the accompanying paper University Medical Center or Partners Healthcare Institutional (26) were used as standards. Recombinant chem163S, but not Review Boards. Informed consent was obtained from donors of recombinant chem158K, chem157S, or chem155A, was plasma CSF samples while synovial fluid samples were obtained detected in a dose-dependent manner by anti-chem163S spe- under a protocol for discarded specimens. Blood was drawn cifically, with a lower limit of detection of 0.2 ng/ml (Fig. 1B, from volunteers into Na-Citrate tubes (BD Biosciences), and left). Likewise, anti-chem158K specifically recognized recom- plasma was prepared. In some experiments H-D-Phe-Pro-Arg- binant chem158K and not chem163S, chem157S, or chem155A chloromethyl ketone (PPACK; Calbiochem) was included in (Fig. 1B, center) whereas anti-chem157S specifically recognized the Na-Citrate collection tubes. Plasma (2 ml) was mixed with recombinant chem157S and not chem163S, chem158K, or 100 l of heparin-agarose (Sigma) and complete protease chem155A. Both of these ELISAs also had similar sensitivity to inhibitor (Roche Applied Science). After incubation at 4 °C for the chem163S ELISA with a lower limit of detection of 0.2 2 h, the heparin-agarose beads were pelleted and washed exten- ng/ml (Fig. 1B, right). sively with PBS, and chemerin was eluted with 0.8 M NaCl in To confirm the specificity of the ELISAs, competitive inhibi- PBS, all in the presence of Complete protease inhibitor. The tion with the cognate peptide was performed. The C-terminal eluates were diluted with 1% BSA in PBS for assay in the specific peptide from human chem163S specifically inhibited the signal ELISAs described above. from recombinant chem163S protein in the ELISA for CSF and synovial joint samples were stored frozen at80 °C chem163S, whereas the C-terminal peptides corresponding to until the time of analysis. After thawing, 400 l of a CSF sample, chem158S and chem157S did not (Fig. 1C, left). In a similar or 30 l of a synovial fluid sample diluted to 400 l with PBS, fashion, the C-terminal peptides of human chem158K and was mixed with 50 l of heparin-agarose and Complete prote- chem157S specifically inhibited the signals from chem158K ase inhibitor before heparin-agarose adsorption was performed and chem157S proteins in the ELISAs for chem158K and as described above and the eluates assayed by the specific chem157S, respectively (Fig. 1C, center and right). ELISAs. Other chemerin isoforms have been reported such as Statistics—Comparison of two samples was by Student’s t chem156F, chem154F, and chem152G (20, 21). We tested test; multigroup comparisons were by Kruskal-Wallis analysis. whether those isoforms would be detected by the specific ELI- The analysis was carried out using Prism v5 (GraphPad, La Jolla, SAs by including the noncognate C-terminal peptides corre- sponding to chem152G, chem154F, and chem156F to the spe- CA). Values of p  0.05 were considered significant. cific ELISAs. They did not inhibit the signal from recombinant chem163S protein in the ELISA for chem163S, nor did they RESULTS inhibit chem158K or chem157S protein signals in the ELISAs Generation and Characterization of Specific Antibodies for for chem158K or chem157S (supplemental Fig. 1). These data Chem163S, Chem158K, and Chem157S—Because the activity show that the ELISAs for the different chemerin isoforms have of the different chemerin isoforms varies significantly, mea- good sensitivity and specificity. surements of total levels of chemerin in biological fluids do not Detection of Chem163S, Chem158K, and Chem157S Levels in give a complete description of the status of the chemerin sys- Human Plasma—Having established the specificity of the ELI- tem. We therefore developed a panel of ELISAs capable of spe- SAs, we used them to determine the levels of chem163S, cifically detecting individual chemerin isoforms in human sam- chem158K, and chem157S in human plasma from normal vol- ples using the methodology we had employed previously to unteers. In the initial experiments to determine levels of develop specific ELISAs for osteopontin (OPN) isoforms (22). chemerin isoforms in plasma, very high values for chem163S Peptide antigens were used to immunize rabbits to raise specific were detected (supplemental Fig. 2). Analysis of plasma by antibodies directed against the different C-terminal sequences Western blotting using the anti-163S antibody showed the of chem163S, chem158K, and chem157S. Specific rabbit anti- presence of a high molecular mass cross-reactive substance chem163S, anti-chem158K, and anti-chem157S IgGs were (supplemental Fig. 3A, lane 3). To circumvent this problem, a purified by positive selection by binding to the cognate peptide- heparin adsorption step was introduced before the ELISA that conjugated Sepharose followed by negative selection to remove separates the interfering substance while allowing quantitative any cross-reacting antibodies using the noncognate peptide- recovery of chemerin isoforms upon elution from the heparin- conjugated Sepharose. The specificity of these anti-chemerin agarose beads (supplemental Fig. 3B). The chemerin detection isoform IgGs was demonstrated by Western blotting in which antibody from R&D did not recognize this high molecular mass each antibody only recognized its cognate protein and not the band (supplemental Fig. 3C, lane 2), but it recognized recom- other two chemerin isoforms (Fig. 1A). binant chem163S, chem158K, chem157S, and chem155A pre- 39522 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF FIGURE 1. Characterization of specific antibodies against recombinant chemerin isoforms. A, purified antibodies specific for different chemerin isoforms were characterized by Western blot analysis of recombinant GST-chem163S (left), GST-chem157S (right), and GST-chem158K (center) with anti-chem163S, anti-chem157S, and anti-chem158K, as described under “Experimental Procedures.” Molecular mass markers are shown on the left of the panels. B, recombi- nant chem163S (), chem158K (ƒ), and chem157S (‚), and chem155A () by anti-chem163S, anti-chem158K, and anti-chem157S were detected using specific ELISAs as described under “Experimental Procedures.” None of the three antibodies detects chem155A (). C, cognate peptide competes in the ELISA, but the noncognate peptides do not. C-terminal chem163S peptide (Œ) specifically inhibited the signal from recombinant chem163S by itself ()inthe chem163S ELISA, but the C-terminal chem158K peptide (), chem157S peptide () did not compete (left). C-terminal chem158K peptide () and chem157S peptide () specifically inhibited the signals from chem158K by itself () and chem157S by itself () proteins in the ELISAs for recombinant chem158K and chem157S, respectively (center and right), but the noncognate peptides did not compete. Values are the mean of duplicates. pared as described in the accompanying paper (supplemental Fig. 3D), showing that the high molecular mass band was not related to chemerin. Using the revised method, the levels of the different chemerin isoforms were measured in plasma from normal vol- unteers (Fig. 2). The plasma level for chem163S was 40  7.9 ng/ml (mean  S.D., n  9), chem158K level was 8.1 2.9 ng/ml, whereas chem157S was barely detectable at 0.7 0.8 ng/ml. To exclude the possibility that the chem158K resulted from plasmin cleavage of chem163S occurring during or after the blood draw, blood was drawn into tubes with and without FIGURE 2. Levels of chemerin isoforms in human plasma. Chem163S, 0.1 mM PPACK, an inhibitor of plasmin, thrombin, and other chem158K, and chem157S levels in normal human plasma (n 9) were deter- serine proteases. The levels of chem163S, chem158K, and mined using specific ELISAs as described under “Experimental Procedures.” chem157S were not altered by the presence of PPACK. Because Horizontal lines show the mean. NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 39523 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF FIGURE 3. Levels of chemerin isoforms in CSF of glioblastoma, oligodendrocytoma, and miscellaneous CNS disease. Chem163S, chem158K, and chem157S levels in CSF from GBM (n 12), oligodendrocytoma (Oligo)(n 12), and miscellaneous (noncancer) CNS disease patients (n 7) were determined using specific ELISAs as described under “Experimental Procedures.” Horizontal lines show the mean. plasmin is an enzyme that has been suggested to be responsible with RA, OA, and PsA (Fig. 4). In RA synovial fluid samples, the for chem163S cleavage to chem158K, we measured plasmin– levels of chem163S, chem158K, and chem157S were 12 22.3, anti-plasmin levels in these samples by ELISA. The levels of 56 42.9, and 9.3 10.5 ng/ml (mean S.D., n 23), respec- plasmin–anti-plasmin in all samples were within the normal tively. In OA synovial fluid samples (n 23), the corresponding range for human plasma and no different if PPACK was levels were 9.0  7.2, 74.1  74.4, and 6.1  9.9 ng/ml; and in included during the blood draw (data not shown). Taken PsA samples (n  13), they were 1.1  2.3, 58.9  36.6, and together, these data suggest that the detectable level of 3.2  6.2 ng/ml, respectively. It is striking that the total chem158K in plasma did not result from ex vivo activation of chemerin levels (chem163S plus chem158K plus chem157S) plasminogen and processing of chem163S by plasmin or other found in the synovial fluid samples of all three types of arthritis serine proteases. patients were much higher than in CSF samples from CNS dis- Detection of Chemerin Isoforms in CSF Samples from Patients ease and were approximately 2-fold higher than that of plasma with CNS Disease—Next, we measured the levels of chemerin samples of normal people. The chem158K form was the domi- isoforms in CSF samples of patients with malignant glioblas- nant isoform of chemerin in all disease groups, and there was no toma (GBM), oligodendrocytoma, and miscellaneous CNS dis- significant difference among RA, OA, and PsA. We tested eases that included one sample each from a patient with whether the chem158K had been generated ex vivo in the syno- migraine, pseudotumor cerebi, meningioma (negative cytol- vial fluid samples by inclusion of PPACK in the collecting tubes, ogy), cylindroma necrosis postradiation therapy (negative and we found that the presence of PPACK did not alter the level cytology), malignant hypertension, ascending sensory loss, and of chem158K, suggesting that it had not been generated ex vivo. bilateral VIth nerve palsies (negative cytology) (Fig. 3). In GBM Comparison of Cleaved Chemerins in Human Plasma, CSF, the levels for chem163S, chem158K, and chem157S were Inflammatory CSF, and Synovial Fluid Sample—We compared 3  2.4, 5.1  3.9, and 0.2  0.3 ng/ml, respectively (mean  the fraction of the total chemerin that had been cleaved in the S.D., n  12). In oligodendrocytoma CSF samples (n  12), the different types of human fluid samples. Using chem163S, corresponding levels were 2.9  2.5, 3.8  3.8, and 0.7  1.3 chem158K, and chem157S levels determined by the specific ng/ml, whereas in the miscellaneous CNS disease (noncancer ELISAs, the fraction of cleaved chemerins (chem158K plus CNS disease) CSF samples (n  7), the levels were 5.5  3.8, chem157S) was calculated for each sample. Because the levels 6.3  4.8, and 1.0  0.8 ng/ml, respectively. There were no of chemerin isoforms were not different among the CSF or significant differences among the three disease groups. In all of synovial fluid samples from different disease states, the CSF and these CSF samples, the level of chem163S was much lower synovial fluid samples were analyzed together as two separate (10%) than that in normal plasma. On the other hand, the groups and compared with plasma (Fig. 5). The fraction of fraction of chem163S that had been processed, represented as cleaved chemerin was 0.18 in plasma whereas it was 0.5 in the ratio of chem158K to chem163S, was significantly higher. inflammatory CSF samples, and it approached 0.75 in the syno- The possibility of ex vivo plasmin processing of chem163S was vial fluid samples with the means varying significantly when tested by inclusion of PPACK when a CSF sample was freshly analyzed by the Kruskal-Wallis test (p  0.0001), thus indicat- collected, but again there was no difference noted in the levels ing that significant cleavage and processing of chemerin occur of the chemerin isoforms (data not shown), similar to the situ- in these extravascular compartments in inflammatory diseases. ation in plasma, indicating that chem158K did not arise from ex DISCUSSION vivo proteolytic processing. Detection of Chemerin Isoforms in the Synovial Fluid of Prochemerin (chem163S) can be cleaved in its C-terminal Arthritis Patients—Based on our previous observation that pro- domain by different serine and cysteine proteases in vitro, gen- teolytic cleavage of OPN in synovial fluids was much higher in erating a series of chemerin isoforms with different levels of rheumatoid arthritis (RA) than in either osteoarthritis (OA) or activity (Fig. 6) (23). To investigate which are the relevant forms psoriatic arthritis (PsA) (22), we hypothesized that the inflam- of chemerin in vivo, we have developed ELISAs to measure the matory environment in RA would also lead to higher levels of different chemerin isoforms in biological samples. The ELISAs processing of chemerin. Therefore, we investigated the are based on polyclonal antibodies raised against peptides that chemerin isoform levels in synovial fluid samples of patients represent the C termini of the different isoforms and possess 39524 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF FIGURE 4. Levels of chemerin isoforms in synovial fluid of patients with RA, OA, and PsA. Chem163S, chem158K, and chem157S levels in synovial fluid of patients with RA (n 23), OA (n 23), and PsA (n 13) were determined using specific ELISAs as described under “Experimental Procedures.” Horizontal lines show the mean. On the other hand, CSF samples from patients with a variety of CNS diseases and synovial fluids from arthritis patients showed a completely different picture. The total level of cleaved and noncleaved chemerins was significantly lower (15%) in the CSF than plasma whereas it was 2-fold higher than the plasma level in synovial fluid. Even more striking was the frac- tion of cleaved chemerins present in these samples from patients with inflammatory diseases. In normal plasma, only about 18% of chemerin is cleaved, whereas the fraction was 50% in CSF samples and approached 75% in the arthritis synovial fluid samples. Thus, considerable proteolysis of the intact chemerin is occurring in these two extravascular com- FIGURE 5. Fraction of cleaved chemerin in human plasma, CAF of CNS disease, and synovial fluid of arthritis. Ratio of cleaved chemerin levels partments. The possibility that chemerin is synthesized and (sum of chem158K plus chem157S) to total chemerin (sum of chem163S, processed locally in these compartments is supported by the chem158K, and chem157S) in plasma (●), CSF (Œ), and synovial fluid ()is significant differences between the total amount of chemerin as shown. All samples from Figs. 3, 4, and 5 have been included. Horizontal lines show the mean. well as the extent of its cleavage that was observed in the CSF and synovial fluid samples. It is notable that in both the synovial and CSF compartments, specificity for a single chemerin isoform as demonstrated by chem158K was the dominant form. We have investigated the Western blot analysis and specific competitive inhibition by the possibility of ex vivo plasmin processing by comparison of sam- cognate C-terminal peptide but not the noncognate peptides in ples collected with and without PPACK and found that no the ELISAs (Fig. 1). The lack of competition by the noncognate detectable chemerin processing occurred. It is therefore likely peptides suggests that the key determinants recognized by the that a high fraction of chemerin is cleaved in these compart- antibodies are the C-terminal amino acids, as we have found ments, as a result of the inflammatory milieu in arthritis for the previously in epitope mapping of the specific antibodies for the synovial samples and glioma and other inflammatory CNS dis- different proteolytically cleaved OPN isoforms (18). eases for the CSF samples. In vitro studies have shown that The ELISAs proved to have excellent specificity and sensitiv- either plasmin or tryptase can cleave the five C-terminal amino ity for the different chemerin isoforms allowing for the first acids from chem163S resulting in chem158K (23). Although the time determination of the levels of three key chemerin iso- activity of chem158K is low (26), the levels present in the syno- forms, the precursor, chem163S, and two cleavage products, vial fluid samples, but not the CSF samples, are high enough to chem158K and chem157S. Importantly, the sensitivity of each suggest that they may be sufficient to contribute a significant of the ELISAs is 0.2 ng/ml (0.012 nM) whereas 50% of the amount of chemerin activity without further processing. effective concentration (EC ) for the most active isoform, Although chem158K can be efficiently generated by plasmin chem157S, is 1.17  0.74 nM in a calcium mobilization assay. cleavage in vitro (19), the protease(s) involved in vivo remains to Thus, the range of the ELISAs covers the concentrations at be established. Because prochemerin can be cleaved by a large which we have shown chemerin to be active (26). variety of proteases, the high levels of chem158K present in CSF Use of these ELISAs in our studies showed that in normal and synovial fluids provide in vivo validation of the importance human plasma samples, chem163S, the prochemerin form, of cleavage at lysine 158, by plasmin or other serine proteases, at dominated, whereas chem158K represented only a small per- these extravascular sites, and it is probable that active centage of the total chemerin, and chem157S was barely detect- chemerin, chem157S, is generated via this intermediate (Fig. 6). able. Thus, under normal conditions, most of the chemerin present in plasma was uncleaved and therefore inactive. The Other chemokines such as CCL15 and CCL23 also require low but reproducible level of chem158K suggests that there is a proteolytic processing to generate the isoforms that possess constitutive level of processing of the inactive chem163S in maximum activity (24), although these cleavages are at the N plasma at base line. The protease(s) responsible for this cleav- terminus rather than the C terminus. Analysis of synovial fluids, age remains to be established. some from patients with RA, for cleavage of CCL15 and CCL23 NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 39525 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF FIGURE 6. Schematic of chemerin cleavages. Chem163S (pink box) can be cleaved either by elastase, generating a mixture of chem157S (red box) and chem155A (blue box), by proteinase 3 and tryptase generating chem155A or by plasmin generating chem158K (pink box). Chem158K can then be cleaved by carboxypeptidases, carboxypeptidase B2 (CPB2) or carboxypeptidase N (CPN), to form the active chem157S, which is subsequently inactivated by angiotensin- converting enzyme (ACE) forming inactive chem155A. The heavier arrows represent the cleavage pathway for chem163S described in this paper. The icon for the N-terminal region of chemerin is based on the model in Zabel et al. (25). demonstrated the presence of cleaved active isoforms of CCL15 peptidase N and carboxypeptidase B2 can cleave chem158K to and CCL23. Proteolytic processing of these G protein-coupled chem157S, the fully activated isoform of chemerin (23). There receptor ligands such as chemerin, CCL15, or CCL23 may rep- were elevated levels of OPN-L in synovial fluid samples (18), as resent a general mechanism to localize their effects to the site of well as in GBM CSF samples (data not shown), indicating the inflammation. presence of an active carboxypeptidase that should be capable When the cleavage of OPN in synovial fluids from different of removing the C-terminal lysine from chem158K to generate types of arthritis was compared, we have found previously that chem157S. In addition, we have shown the presence of car- there was far more cleavage of OPN in RA than in OA or PsA to boxypeptidase B2 protein in the synovium (18). Therefore, we the thrombin-cleaved form (OPN-R) as well as the thrombin/ would have expected to detect chem157S in these samples as carboxypeptidase B22 double-cleaved form (OPN-L). How- both the substrate, chem158K, and the enzyme have been ever, there was no difference in cleavage of chemerins when shown to be present. One possible explanation for this is that samples from the different types of arthritis were evaluated, chem157S was rapidly cleaved further, possibly by the angio- suggesting that the regulation of proteolytic processing of OPN tensin-converting enzyme (6), to the inactive chem155A iso- and prochemerin is completely different. Similarly, there was form in vivo (26). This may represent a physiological step to no difference in chemerin levels or in the fraction of cleaved regulate the activity of this potent chemoattractant. Alterna- chemerin when GBM samples were compared with oligoden- tively, chem157S may bind to cells and therefore cannot be drocytoma or noncancer CSF samples. The noncancer samples detected in the fluid phase. We are currently developing a spe- were from patients some of whom had inflammatory diseases cific ELISA for chem155A that will help to resolve this issue. such as cylindroma necrosis and postradiation therapy that Our study provides the first insights into the in vivo levels of may also affect levels and cleavage of chemerin. In the accom- the different chemerin isoforms and suggests that global deter- panying paper (26), analysis of gene expression shows that minations of chemerin levels may be misleading in terms of the chemerin mRNA is increased in grade IV GBM compared with biological potency of chemerin present. In particular, the dif- lower grade gliomas that include oligodendrocytoma or epi- ferent levels of chem158K found in different compartments lepsy samples. The increase in mRNA was not reflected in the emphasize that the generation of fully active chem157S is prob- levels of chemerin present in the CSF, but that may be because ably under tight local regulation. The determination of the dif- the GBM itself is only a minor contributor to the overall protein ferent chemerin isoforms in various disease states should content of the CSF. advance our understanding of this novel chemoattractant that Surprisingly, our study found very low levels of chem157S in acts in the interface between thrombosis, inflammation, and any of the samples. We have shown that both plasma carboxy- immunity. 39526 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 45 •NOVEMBER 11, 2011 Chemerin158K Is the Dominant Form in Synovial Fluids and CSF REFERENCES 14. Yamamoto, T., Qureshi, A. R., Anderstam, B., Heimbürger, O., Bárány, P., Lindholm, B., Stenvinkel, P., and Axelsson, J. (2010) Nephrol. Dial. Trans- 1. Nagpal, S., Patel, S., Jacobe, H., DiSepio, D., Ghosn, C., Malhotra, M., plant. 25, 4017–4023 Teng, M., Duvic, M., and Chandraratna, R. A. (1997) J. Invest. Dermatol. 15. Weigert, J., Obermeier, F., Neumeier, M., Wanninger, J., Filarsky, M., 109, 91–95 Bauer, S., Aslanidis, C., Rogler, G., Ott, C., Schäffler, A., Schölmerich, J., 2. Wittamer, V., Franssen, J. D., Vulcano, M., Mirjolet, J. F., Le Poul, E., and Buechler, C. (2010) Inflamm. Bowel Dis. 16, 630–637 Migeotte, I., Brézillon, S., Tyldesley, R., Blanpain, C., Detheux, M., Man- 16. Kukla, M., Zwirska-Korczala, K., Gabriel, A., Waluga, M., Warakomska, I., tovani, A., Sozzani, S., Vassart, G., Parmentier, M., and Communi, D. Szczygiel, B., Berdowska, A., Mazur, W., Wozniak-Grygiel, E., and Kryc- (2003) J. Exp. Med. 198, 977–985 zka, W. (2010) J. Viral Hepat. 17, 661–667 3. Wittamer, V., Bondue, B., Guillabert, A., Vassart, G., Parmentier, M., and 17. Meder, W., Wendland, M., Busmann, A., Kutzleb, C., Spodsberg, N., John, Communi, D. (2005) J. Immunol. 175, 487–493 H., Richter, R., Schleuder, D., Meyer, M., and Forssmann, W. G. (2003) 4. Zabel, B. A., Silverio, A. M., and Butcher, E. C. (2005) J. Immunol. 174, FEBS Lett. 555, 495–499 244–251 18. Zabel, B. A., Nakae, S., Zúñiga, L., Kim, J. Y., Ohyama, T., Alt, C., Pan, J., 5. Vermi, W., Riboldi, E., Wittamer, V., Gentili, F., Luini, W., Marrelli, S., Suto, H., Soler, D., Allen, S. J., Handel, T. M., Song, C. H., Galli, S. J., and Vecchi, A., Franssen, J. D., Communi, D., Massardi, L., Sironi, M., Man- Butcher, E. C. (2008) J. Exp. Med. 205, 2207–2220 tovani, A., Parmentier, M., Facchetti, F., and Sozzani, S. (2005) J. Exp. Med. 19. Zabel, B. A., Ohyama, T., Zuniga, L., Kim, J. Y., Johnston, B., Allen, S. J., 201, 509–515 Guido, D. G., Handel, T. M., and Butcher, E. C. (2006) Exp. Hematol 34, 6. Ernst, M. C., and Sinal, C. J. (2010) Trends Endocrinol. Metab. 21, 1106–1114 660–667 20. Zabel, B. A., Allen, S. J., Kulig, P., Allen, J. A., Cichy, J., Handel, T. M., and 7. Hu, W., and Feng, P. (2011) Diabetes Res. Clin. Pract. 91, 159–163 Butcher, E. C. (2005) J. Biol. Chem. 280, 34661–34666 8. Yang, M., Yang, G., Dong, J., Liu, Y., Zong, H., Liu, H., Boden, G., and Li, L. 21. Guillabert, A., Wittamer, V., Bondue, B., Godot, V., Imbault, V., Parmen- (2010) J. Investig. Med. 58, 883–886 tier, M., and Communi, D. (2008) J. Leukocyte Biol. 84, 1530–1538 9. Pfau, D., Stepan, H., Kratzsch, J., Verlohren, M., Verlohren, H. J., Drynda, K., Lössner, U., Blüher, M., Stumvoll, M., and Fasshauer, M. (2010) Horm. 22. Sharif, S. A., Du, X., Myles, T., Song, J.J., Price, E., Lee, D.M., Goodman, Res. Paediatr. 74, 56–61 S.B., Nagashima, M., Morser, J., Robinson, W.H., and Leung, L.L.K. (2009) 10. Tönjes, A., Fasshauer, M., Kratzsch, J., Stumvoll, M., and Blüher, M. (2010) Arthritis Rheum. 60, 2902–2912 PLoS One 5, e13911 23. Du, X. Y., Zabel, B. A., Myles, T., Allen, S. J., Handel, T. M., Lee, P. P., 11. Dogru, T., Karadurmus, N., Tapan, S., and Genc, H. (2010) Scand J. Gas- Butcher, E. C., and Leung, L. L. (2009) J. Biol. Chem. 284, 751–758 troenterol. 45, 1000–1001; author reply 1001–1002 24. Berahovich, R. D., Miao, Z., Wang, Y., Premack, B., Howard, M. C., and 12. Sell, H., Divoux, A., Poitou, C., Basdevant, A., Bouillot, J. L., Bedossa, P., Schall, T. J. (2005) J. Immunol. 174, 7341–7351 Tordjman, J., Eckel, J., and Clément, K. (2010) J. Clin. Endocrinol. Metab. 25. Zabel, B. A., Zuniga, L., Ohyama, T., Allen, S. J., Cichy, J., Handel, T. M., 95, 2892–2896 and Butcher, E. C. (2006) Exp. Hematol. 34, 1021–1032 13. Pfau, D., Bachmann, A., Lössner, U., Kratzsch, J., Blüher, M., Stumvoll, M., 26. Yamaguchi, Y., Du, X.-Y., Zhao, L., Morser, J., and Leung, L. L. K. (2011) and Fasshauer, M. (2010) Diabetes Care 33, 171–173 J. Biol. Chem. 286, 39510–39519 NOVEMBER 11, 2011• VOLUME 286 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 39527

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