Quantum Proteolytic Activation of CCL15 by Granulocytes Modulates Mononuclear Cell Adhesiveness This information is current as of September 30, 2021. Rudolf Richter, Roxana Bistrian, Sylvia Escher, Wolf-Georg Forssmann, Jalal Vakili, Reinhard Henschler, Nikolaj Spodsberg, Adjoa Frimpong-Boateng and Ulf Forssmann J Immunol 2005; 175:1599-1608; ; doi: 10.4049/jimmunol.175.3.1599 Downloaded from http://www.jimmunol.org/content/175/3/1599

References This article cites 91 articles, 34 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/175/3/1599.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Quantum Proteolytic Activation of Chemokine CCL15 by Neutrophil Granulocytes Modulates Mononuclear Cell Adhesiveness1

Rudolf Richter,2* Roxana Bistrian,† Sylvia Escher,* Wolf-Georg Forssmann,* Jalal Vakili,‡ Reinhard Henschler,† Nikolaj Spodsberg,§ Adjoa Frimpong-Boateng,* and Ulf Forssmann*

Monocyte infiltration into inflammatory sites is generally preceded by . We show here that neutrophils may support this process by activation of CCL15, a human chemokine circulating in blood plasma. Neutrophils were found to release CCL15 proteolytic activity in the course of hemofiltration of blood from renal insufficiency patients. Processing of CCL15 immunoreac- tivity (IR) in the pericellular space is suggested by a lack of proteolytic activity in blood and blood filtrate, but a shift of the retention time (tR) of CCL15-IR, detected by chromatographic separation of CCL15-IR in blood and hemofiltrate. CCL15 mol- Downloaded from ecules with N-terminal deletions of 23 (⌬23) and 26 (⌬26) aa were identified as main proteolytic products in hemofiltrate. Neutrophil cathepsin G was identified as the principal protease to produce ⌬23 and ⌬26 CCL15. Also, elastase displays CCL15 proteolytic activity and produces a ⌬21 isoform. Compared with full-length CCL15, ⌬23 and ⌬26 isoforms displayed a signifi- cantly increased potency to induce calcium fluxes and chemotactic activity on and to induce adhesiveness of mono- nuclear cells to fibronectin. Thus, our findings indicate that activation of monocytes by neutrophils is at least in part induced by quantum proteolytic processing of circulating or endothelium-bound CCL15 by neutrophil cathepsin G. The Journal of Immu- http://www.jimmunol.org/ nology, 2005, 175: 1599–1608.

hemokines are a family of small that are in- CSF, IL-6, and IL-8, by the endothelium (7–16). The subsequent volved in inflammatory processes by inducing migration recruitment of leukocytes to inflamed tissue requires their tethering C and activation of leukocytes. exert their bi- and rolling as well as rapid activation of integrin-dependent arrest ological activities via binding to specific surface receptors, which on the target endothelium as a checkpoint for diapedesis to the belong to seven transmembrane G protein-coupled receptors (1). extravascular tissue (17, 18). Chemokines, present on the endo- CCL15 is a human chemokine which acts predominantly on che- thelial surface under physiologic or pathologic conditions (19–22), mokine receptor CCR1 and to a lesser degree on CCR3 (2). It is a trigger rolling and firm adhesion by activation of Gi protein-cou- by guest on September 30, 2021 potent chemoattractant for monocytes and in vitro pled receptors (23–26). Tethering and rolling of leukocytes at ad- and, after i.p. application, CCL15 induces recruitment neutrophils, hesive contact zones is increased by integrin clustering raising the monocytes, and lymphocytes into mouse peritoneum (3). CCL15 is avidity of integrin-ligand interactions (25, 27–29). Integrin-medi- constitutively expressed in the gut and in the liver (4) and was ated arrest of leukocytes on vascular endothelium is suggested to identified to circulate in human plasma (5). The N terminus of be induced by further interaction partners. Tetraspanins, Cytohe- CCL15 was found to be important to exert its full biological ac- sin-1, pentaspan, CD99/E2, and CD47 are integrin-associated mol- tivity. Lee et al. (6) showed that truncation of the N terminus by at ecules and are suggested either to regulate the affinity of integrins least 24 aa significantly increases the calcium flux and chemotactic to their ligands or to interact with their own endothelial ligand activity. (30–33). One of the first essential steps in inflammation is the activation In this study, we demonstrate a further mechanism for activation of the adhesion between endothelial cells and leukocytes. IL-1 and of migratory behavior of mononuclear cells by chemokines, which TNF-␣ are important mediators leading to increased expression of is associated with the degranulation of neutrophils and proteolytic adhesion molecules E-selectin, VCAM-1, and ICAM-1 as well as activation of chemokine CCL15. Degranulation and release of pro- expression of soluble proinflammatory cytokines, e.g., MCP-1, M- teolytic enzymes by neutrophils is induced by interaction with ac- tivated vascular endothelium, which is stimulated with IL-1 or ␣ *IPF PharmaCeuticals GmbH, Hannover, Germany; †Institute of Transfusion Medi- TNF- (34). Generally, infiltration is preceded by an cine and Immune Hematology, Blood Donation Service of the German Red Cross, initial influx of neutrophils. In cases of clinical cyclic or experi- Frankfurt, Germany; and ‡Institute of Interdisciplinary Research, Universite´Libre de mental neutropenia, complementary influx of mononuclear cells into Bruxelles, Brussels, Belgium; and §Department of Medical Biochemistry and Genet- ics, The Panum Institute, Copenhagen, Denmark inflammatory sites is significantly decreased and delayed. Replenish- Received for publication February 17, 2005. Accepted for publication May 17, 2005. ment of circulating neutrophils reestablishes the normal sequence of events in the development of inflammatory response (35, 36). In our The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance experimental setting, activation of neutrophils is induced on semiper- with 18 U.S.C. Section 1734 solely to indicate this fact. meable hemofilter membranes used for hemofiltration of patients with 1 This work was supported in part by a grant from the German Federal Ministry of chronic renal failure (CRF)3 (37). Immediate filtration of plasma Education and Research (Bundesministerium fu¨r Bildungund Forschung, FKZ 0311815). 2 Address correspondence and reprint requests to Dr. Rudolf Richter, IPF Pharma- 3 Abbreviations used in this paper: CRF, chronic renal failure; IR, immunoreactivity; Ceuticals GmbH, Feodor Lynen Strasse 31, 30625 Hannover, Germany. E-mail ad- tR, retention time; RP, reverse phase; i.d., inner diameter; TFA, trifluoroacetic acid; dress: [email protected] HF, hemofiltrate; MS, mass spectrometry; ESMS, electrospray MS; PTH, phenyl-

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 1600 QUANTUM PROTEOLYTIC ACTIVATION OF CCL15 BY NEUTROPHIL CATHEPSIN G proteins and peptides subsequent to contact with activated leukocytes min using a linear binary gradient of 100% solvent A (0.1% v/v TFA) to at the filter membrane allows the determination of proteolytically 60% solvent B (100% acetonitrile and 0.1% v/v TFA) in 60 min and from processed molecules in the filtrate. 60% solvent B to 100% solvent B in 5 min. One fraction was collected each minute, starting at minute 10 of the chromatography. The fractions ob- We were able to show that CCL15(1–92) is efficiently processed tained were analyzed by CCL15 RIA. CCL15-IR in human plasma and into highly active molecules by activated neutrophils. We discov- hemofiltrate (HF) was identified by chromatography of 1.5 ml of plasma or ered the neutrophil serine proteinases cathepsin G and elastase 150 ml of HF. After collection from the hemofilter, the sterile filtrate was responsible for this proteolytic processing. A lack of proteolytic immediately frozen to Ϫ80°C to prevent bacterial growth and proteolysis. activity in blood and in the blood filtrate suggest that processing of Generation of an HF peptide library CCL15 is a quantum proteolytic event occurring in the pericellular The generation of a HF peptide library was performed as described previ- space before catalysis is quenched by protease inhibitors. ously (39). Briefly, human HF for large-scale recovery of plasma peptides (40) was obtained from patients with CRF in quantities of 1600–2000 Materials and Methods L/week. Ultrafilters used for hemofiltration had a specified molecular mass Reagents cutoff of 20 kDa. The sterile filtrate was immediately cooled to 4°C and acidified to pH 3 to prevent bacterial growth and proteolysis. For peptide All chemicals were obtained from Sigma-Aldrich unless otherwise stated. extraction, batches of 1000 L of HF were conditioned to pH 2.7 and applied Cell culture medium was purchased from Invitrogen Life Technologies; the onto the strong cation exchanger Fractogel TSK SP 650(M) (100 ϫ 250 CCL15(1–92) variant with the N-terminal pyroglutamic acid was obtained mm; Merck). Batch elution was performed with 10 L of 0.5 M ammonium from Bachem Biochemica; other chemokines were obtained from R&D acetate. The eluate was stored at Ϫ20°C until further use. For production Systems, 125I was obtained from Amersham Pharmacia Biotech, and cell of a peptide library, ammonium acetate extracts of 5000 L of HF were lines were obtained from the American Type Culture Collection. pooled and loaded on a 10-L cation exchange column (Fractogel SP 650(M); Merck). Bound peptides were eluted using seven buffers with Downloaded from Patients increasing pH, resulting in seven pH pools. The seven buffers were com- posed as follows: I, 0.1 M citric acid monohydrate (pH 3.6); II, 0.1 M Hemofiltrate and blood plasma was collected from nine consenting acetic acid plus 0.1 M sodium acetate (pH 4.5); III, 0.1 M malic acid (pH patients with CRF from the Nephrologisches Zentrum Niedersachsen 5.0); IV, 0.1 M succinic acid (pH 5.6); V, 0.1 M sodium dihydrogen phos- (Hannoversch-Mu¨nden, Germany). The patient group had a mean age of phate (pH 6.6); 0.1 M disodium hydrogen phosphate (pH 7.4); and VII, 0.1 Ϯ 67 14 years (range, 49–84 years) and an average time on renal M ammonium carbonate (pH 9.0). pH pool VIII was generated by washing replacement therapy of 77 Ϯ 54 mo. Renal disease was caused by chronic the column with distilled water. The eight pools (pH pools) were collected http://www.jimmunol.org/ glomerulonephritis (one patient), chronic interstitial nephritis (two pa- and each of them was loaded onto a RP column, 125 mm ϫ 100-mm i.d. tients), nephrosclerosis (four patients), polycystic disease (one patient), and (Source RPC, 15 ␮m; Pharmacia) and eluted in an 8-L gradient from 100% diabetic nephropathy (one patient). A (0.01 M HCl in water) to 60% B (0.01 M HCl in 80% acetonitrile). Fractions of 200 ml were collected. Radioimmunoassay Peptide purification from human HF CCL15(27–92) was radio-iodinated using the chloramine-T method. Sep-

Pak C18 cartridges (Waters) were used to separate labeled peptide from free Fractions containing CCL15-IR were detected by CCL15 RIA and Western 125I. Bound peptides were eluted using 0.01 M HCl in 80% acetonitrile. blot analysis. Identified CCL15-IR was further purified to homogeneity by

Fifty micromolar sodium phosphate buffer (pH 7.4), containing 0.025% six consecutive chromatographic steps A–F. Step A: preparative RP-C18 w/v Tween 20, 0.5% w/v BSA, and 0.04% w/v NaN was used as RIA ϫ 3 chromatography using a Vydac PrepPak RP-C18 column, 300 mm by guest on September 30, 2021 buffer. All reagents and samples were dissolved in RIA buffer. For incu- 47-mm i.d., 15–20 ␮m, 300 Å (Vydac). Separations were performed at a bation, affinity-purified goat anti-human CCL15 Ab (0.125 ␮g/ml; R&D flow rate of 40 ml/min using a linear binary gradient of 80% solvent A (10 Systems) and 20,000 cpm/100 ␮lof125I-CCL15 were used. The bound and mM HCl in 20% methanol) to 55% solvent B (10 mM HCl in 100% meth- free Ags were separated by incubation with 100 ␮l of donkey anti-rabbit anol) in 54 min and from 55% solvent B to 100% solvent B in 3 min. IgG ␥-globulin (Immundiagnostik) for 2 h before centrifugation at 2500 ϫ Fractions of 50 ml were collected. Step B: preparative cation exchange g for 20 min. The radioactivity of the precipitate was counted in a gamma chromatography; column Pepkat 1000-7, 150 mm ϫ 20-mm i.d. (Biotek spectrometer (Wallac). Laboratories). Separation was performed at a flow rate of 5 ml/min using a linear binary gradient of 100% solvent A (50 mM sodium phosphate in Western blot analysis water, pH 3.0) to 60% solvent B (50 mM sodium phosphate and 1,5 M NaCl in water, pH 3.0) in 60 min. Fractions of 5 ml were collected. Step For immunoblots, samples were desalted, lyophilized, and resuspended in C: analytical RP-C4 chromatography; column RP-C4, 250 mm ϫ 20-mm sample buffer containing 1% w/v SDS, 7.5 mM Tris-HCl (pH 8.4), 1 mM i.d., 5 ␮m, 300 Å (Biotek Laboratories). Separation was performed at a EDTA, 32.4 mM DTT, 12.5% w/v glycerol, and 0.025% w/v bromphenol flow rate of 7 ml/min using a linear binary gradient from 90% solvent A blue and incubated for 7 min at 95°C (reducing conditions). The samples (0.1% TFA in water) to 60% solvent B (0.1% TFA in 80% acetonitrile) were separated by tricine-SDS-PAGE in 17.5% gels (38). Synthetic within 50 min. Step D: analytical RP-C chromatography; column Protein CCL15(1–92) and CCL15(27–92) were used as external standards. After 18 & Peptide C18, 250 mm ϫ 10-mm i.d., 5 ␮m, 300 Å (Vydac). Separation electrophoresis, proteins were electroblotted onto hydrophobic polyvinyli- was performed at a flow rate of 1.8 ml/min using a linear binary gradient dene difluoride-based membranes (Pall). To minimize nonspecific binding from 85% solvent A (0.1% TFA in water) to 55% solvent B (0.1% TFA in of the Abs, blot strips were incubated with 5% w/v BSA in Tris-buffered 80% acetonitrile) within 50 min. saline (10 mM Tris and 150 mM NaCl, pH 8.0) and 0.05% v/v Triton X-100 (TBST). After washing in TBST, the membranes were incubated Mass spectrometry (MS) overnight at 4°C with affinity-purified goat anti-human CCL15 Ab (1 ␮g/ ml). Immunoreactive proteins were visualized after incubation with alka- Electrospray MS (ESMS) was conducted on an API III Triple stage qua- line phosphatase-blue tetrazolium and 5-bromo-4-chloro-3-indolylphos- druple mass spectrometer equipped with the articulated ion spray phate as chromogens. (PerkinElmer SCIEX). Flow injection was conducted at 5 ␮l/min. The masses were determined in the range from 400 to 2300 Da in the positive

Determination of chromatographic retention time (tR) for ion mode as described by the manufacturer. The peptide masses were cal- CCL15-immunoreactivity (IR) culated using the MacSpec 3.3 software (PerkinElmer SCIEX). MALDI-MS was performed using a LaserTec RBT MALDI-mass spec- To determine tR of CCL15-IR material, a standard chromatography method trometer (Perseptive/Vestec). Isolated CCL15 was applied to a stainless was developed. Chromatography was performed on a preparative reverse steel multiple sample tray as an admixture to sinapinic acid using the dried phase (RP)-HPLC, 250 mm ϫ 10-mm inner diameter (i.d.) (Source RPC drop technique (41). Measurements were performed in linear mode. The 15; Pharmacia). The separations were performed at a flow rate of 2.5 ml/ instrument was equipped with a 1.2-m flight tube and a 337-nm nitrogen laser. Positive ions were accelerated at 30 kV and 64 laser shots were  automatically accumulated per sample position. The time-of-flight data thio-hydantoin; PMN, polymorphonuclear neutrophil; ex, excitation wave length;  were externally calibrated for each sample plate and sample preparation. em, emission wave length; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; E-64 ϭ N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; SLPI, secretory Data acquisition and analysis were performed using GRAMS 386 version leukocyte proteinase inhibitor; FLIPR, fluorometric imaging plate reader. 3.04 software supplied by the manufacturer (Galactic Industries). The Journal of Immunology 1601

Sequence analysis Changes in cellular fluorescence were recorded online after the addition of 50 ␮l of test compounds diluted in wash buffer. Calcium mobilization Sequence analyses of the isolated peptides were performed by stepwise assays on a CCR1-transfected CHO-K1 cell line or CCR1-transfected Edman degradation using a gas-phase automated sequencer (model 473 A; HEK-293 cell line were performed as described previously (44). Applied Biosystems). The resulting phenyl-thio-hydantoin amino acids were identified by integrated HPLC. assay Synthesis CCL15 molecules were tested as potential chemoattractants for monocytes. Chemotaxis was assessed in 48-well chambers (NeuroProbe) by using CCL15 molecules were prepared by F-moc solid-phase peptide synthesis polyvinylpyrrolidone-free polycarbonate membranes (Nucleopore; Neuro- as described elsewhere (42). The purified products were characterized by Probe) with 5-␮m pores. All assays were conducted in triplicate, and the HPLC, capillary zone electrophoresis, ESMS, and sequence analysis. The migrated cells were counted in five randomly selected fields at 1000-fold net peptide content was determined by amino acid analysis. magnification after migration for 1 h (monocytes). Isolation of blood cells Flow chamber experiments PBMC, polymorphonuclear neutrophils (PMNs) (4), and thrombocytes Precoating of glass slides was performed for 2 h with fibronectin from (43) were isolated according to established methods. For isolation of human plasma (Sigma-Aldrich) diluted in PBS (2 ␮g/ml). Subsequently, PBMCs and PMNs, 30 ml of blood was withdrawn and mixed with 10 ml the glass slides were assembled as the lower wall of the flow chamber of hydroxyethyl starch (Plasmasteril; Fresenius). Erythrocytes were sedi- (Circular Parallel Plate Flow Chamber kit; GlycoTech) and were exten- mented for 45 min at room temperature. The supernatant was centrifuged sively washed with RPMI 1640. The flow chamber was mounted on the at 200 ϫ g for 20 min. The cell pellet was resuspended in 20 ml of FCS- stage of an inverted phase-contrast microscope (Zeiss). Cells were perfused free RPMI 1640, layered onto 15 ml of Ficoll-Paque (Pharmacia Biotech), at 106 cells/ml through the chamber at the desired flow rate generated with ϫ and centrifuged at 1000 g for 15 min at room temperature without a an automated syringe pump (Braun). Initial application of the cells was Downloaded from brake. PBMCs were taken from the interface, diluted with two volumes of performed with a shear stress of 0.1 dyn/cm2. After 10 min, the shear stress RPMI 1640, centrifuged at 200 ϫ g for 20 min. The cell pellet was resus- was increased to 2 dyn/cm2 for 20 min. Subsequent to each perfusion 7 pended in RPMI 1640 at a concentration of 1 ϫ 10 /ml. PMNs were iso- period, adherent cells were documented by photographs of three indepen- lated from the cell pellet of the Ficoll-Paque gradient. The pellet was re- dent fields using a gauged grid. To study the influence of CCL15 molecules suspended in RPMI 1640 and centrifuged at 200 ϫ g for 20 min. on the adhesion of mononuclear cells to fibronectin, prestimulation of Erythrocytes were lysed by resuspending the pellet in 24 ml of double- PBMC with CCL15 molecules was performed for 5 min at 37°C in 5% distilled water for 30 s and subsequent addition of 8 ml of 3.6% NaCl CO2. solution. Cells were centrifuged at 500 ϫ g for 8 min and resuspended in http://www.jimmunol.org/ FCS-free RPMI 1640 medium at a concentration of 2 ϫ 107/ml. Statistical analysis Platelets were isolated from 10 ml of blood using sodium citrate as Ͻ anticoagulant. To gain platelet-rich plasma, blood was centrifuged at Data were compared by the Student t test; p values 0.05 were considered 800 ϫ g for 5 min. The supernatant was diluted with 10% of citrate-citric to be significant. acid-dextrose solution containing 2.94 g/100 ml of trisodium citrate, 2.10 g/100 ml of citric acid, and 2.45 g/100 ml of glucose. Subsequently, the Results platelet-rich plasma was centrifuged at 2000 ϫ g for 12 min, the super- Processing of plasma CCL15-IR occurs during hemofiltration of natant was discarded, and the platelet pellet was gently resuspended in blood from patients with renal insufficiency FCS-free RPMI 1640 medium. To differentiate CCL15-IR in blood and HF, a standardized RP by guest on September 30, 2021 Processing of CCL15 chromatographic method was used. The CCL15-IR elution profile 6 6 Thrombocytes, 2 ϫ 10 /100 ␮l of PBMCs, 2 ϫ 10 /100 ␮l of PMNs, or from plasma of renal insufficiency patients reveals a tR of 44–50 Ͼ supernatant of PMNs were incubated at 37°C with 200 nM CCL15(1–92) min with a peak at tR of 50 min, which contains 50% of for 1 h. Subsequently, the cells were sedimented by centrifugation at 400 ϫ CCL15-IR material (Fig. 1). In HF, the CCL15-IR elution profile g for 5 min. The supernatant was withdrawn, acidified with 0.1 M acetic showed a substantial shift with a t of 33–45 min and two main acid, desalted using cartridges packed with Source RPC15 material (Phar- R macia Biotech), lyophilized, and analyzed by Western blotting. For se- peaks at tR of 36 and 38 min. The tR of CCL15(27–92), quence analysis, the proteins were separated by RP chromatography and CCL15(24–92), and CCL15(1–92) were 36, 38, and 51 min, re- fractions were investigated by RIA and MALDI-MS. N-terminal sequenc- spectively (Fig. 1), suggesting that early eluting CCL15-IR mate- ing was performed by automated Edman degradation. rial is proteolytically processed CCL15. The CCL15-IR plasma For inhibition experiments, PMNs were preincubated with inhibitors for 30 min at 37°C. Subsequently, 200 nM CCL15(1–92) were added and incubated for an additional1hat37°C. To test the capacity of granulocyte serine proteases to process CCL15, 1 mU of cathepsin G or elastase was incubated with 200 nM CCL15(1–92) for 1 h. Proteolytic products were analyzed by Western blotting and RP chromatography with subsequent MALDI-MS and amino acid sequencing. Cathepsin G activity Cathepsin G activity in 100 ␮l of plasma or HF was determined by the rate of hydrolysis of the 4-nitroanilide substrate Suc-Ala-Ala-Pro-Phe-pNA (Calbiochem). Samples were diluted with 100 ␮l of buffer to a final con- centration of 0.1 M HEPES at pH 7.4 and 0.4 M NaCl containing 1 mM substrate. In control experiments, 1 mU of human neutrophil cathepsin G (Calbiochem) was added. The absorbance at 405 nm was detected using an MRX II microplate reader (Dynex Technologies). Intracellular Ca2ϩ measurement

Monocytes were isolated as previously described by Pardigol et al. (4) and FIGURE 1. Elution profile of CCL15-IR in blood plasma and HF of were seeded in 96-black well plates (Costar) at 200,000 cells/well. After 30 patients with CRF, using the standardized RP-chromatography described in min at 37°C in a loading medium (HEPES-dissolved HBSS (pH 7.4) con- Materials and Methods f ‚ taining 2.5 mM probenecid and 1 ␮M Fluo-4 AM; Molecular Probes), cells . CCL15-IR detected in plasma and HF ( ). Data were washed in a loading medium without Fluo-4 AM. The cells were represent the mean of three independent experiments. The determined elu- placed in the fluorometric imaging plate reader (FLIPR), and basal fluo- tion positions of CCL15(27–92), CCL15(24–92), and CCL15(1–92) are  rescence was determined before agonist addition at room temperature ( ex indicated by arrows. Data from one of three representative experiments are ϭ  ϭ 488 nm, excitation wavelength; em 540 nm, emission wavelength). shown. 1602 QUANTUM PROTEOLYTIC ACTIVATION OF CCL15 BY NEUTROPHIL CATHEPSIN G

Table I. CCL15 molecules isolated from HFa

N-Terminal Detected Mr Theoretical Average C-Terminal Peptide Sequence ESMS Mr Sequence CCL15(24–92) NSFHFAADXXTS 7552.8 7554.8 KKLKPYSI CCL15(24–91) NSFHFAADXXTS 7441.6 7441.9 KKLKPYS CCL15(27–92) HFAADXXTSYIS 7206.6 7206.5 KKLKPYSI CCL15(27–91) HFAADXXTSYIS 7091.8 7093.3 KKLKPYS CCL15(27–90) HFAADXXTSYIS 7004.3 7006.2 KKLKPY

a Analysis of the molecules was performed by N-terminal Edman sequencing and ESMS. The C-terminal amino acid sequence was deduced from the published amino acid sequence and the MS results. Cysteine residues are not sequenced by Edman degradation (X). concentration in CRF patients was 1.48 Ϯ 0.32 nM, whereas in chromatographic steps using a preparative RP-HPLC method, a HF, the CCL15-IR concentration was 0.33 Ϯ 0.08 nM, which cor- preparative cation exchange chromatographic method and two relates with an average sieving coefficient for CCL15-IR of ϳ0.2. subsequent analytical RP-HPLC methods. Purity of the material To exclude hydrolytic degradation of CCL15 during collection of was determined by ESMS. Using this strategy, CCL15(24–92), HF, the stability of CCL15(1–92) at pH 2.0 was determined. Under CCL15(24–91), CCL15(27–92), CCL15(27–91), and CCL15(27– these conditions, CCL15(1–92) was not degraded within the in- 90) were identified by ESMS and Edman degradation (Table I). Downloaded from vestigation period of 3 days. PMNs process CCL15(1–92) to ⌬23 and ⌬26 CCL15 isoforms. CCL15-IR in human HF are CCL15 molecules with N-terminal Cathepsin G is the principal protease ⌬ ⌬ deletions of 23 ( 23) and 26 ( 26) aa Purified PBMCs, PMNs, and platelets were incubated with The peptide library was produced from peptide extracts of 10,000 CCL15(1–92) for 1 h. Western blot analysis of cell supernatants

L of human HF. Fractionation of the extracts by cation exchange suggested a processing of CCL15 by PMNs (Fig. 2A). This was http://www.jimmunol.org/ chromatography and RP-HPLC resulted in 8 different pH pools also found in experiments using 125I-CCL15(1–92). Standardized containing 448 fractions, which were screened for CCL15-IR by a RP chromatography of the PMN supernatant revealed a shift of the CCL15-specific RIA. CCL15-IR was predominantly detected in radioactive peak to an early retention position, whereas no shift the fractions 16–22 of pH pools 5 and 6. Western blot analysis of was seen in the chromatography of PBMC and platelet superna- the IR fractions revealed CCL15-IR bands with apparent molecu- tants (results not shown). The acellular, serum-free conditioned lar masses of 7–9 kDa. medium of PMNs also contained the proteolytic activity for For characterization of the molecular structure of the identified CCL15. To identify the processing sites, PMNs were incubated CCL15-IR in pH pools 5 and 6, the material was purified in four with CCL15(1–92) and the supernatant was chromatographed in by guest on September 30, 2021

FIGURE 2. Proteolytic processing of synthetic CCL15 by neutrophil cathepsin G. A, 200 nM CCL15(1–92) was incubated with 2 ϫ 106/100 ␮lof PMNs, 2 ϫ 106/100 ␮l of PBMCs, or 100 ␮l of thrombocyte suspension for1hat37°C. Western blot analysis of CCL15-IR revealed proteolytic processing by PMNs. B, Inhibition of CCL15(1–92) processing by PMNs in the presence of different protease inhibitors; lane 2, AEBSF (4 mM); lane 3, EDTA (1 mM); lane 5, leupeptin (20 ␮M); lane 6, pepstatin (10 ␮M); lane 7, 1,10-phenanthroline (1 mM); lane 8, E-64 (2.5 ␮M). Supernatant of PMNs without CCL15 or protease inhibitors was applied to lane 1. Supernatant of PMNs incubated with CCL15(1–92) was applied to lane 10. C, Identification of specific neutrophil serine proteases: CCL15(1–92) was incubated with PMNs with or without specific serine protease inhibitor SLPI (2.5 ␮M), AEBSF (4 mM), and elastatinal (50 ␮M). D, Time course of proteolytic processing of CCL15 on coincubation with PMN. Two ϫ 106/100 ␮l of PMNs were coincubated with 2 ␮M CCL15(1–92) for the time period indicated. Supernatants were subsequently acidified with 1 M acetic acid and desalted using a RP column (Source RPC15; Pharmacia) and lyophilized. Aliquots of the extracts were separated by SDS-PAGE and CCL15-IR was detected by Western blot analysis. One hundred nanograms of CCL15(1–92) and CCL15(27–92) was run in parallel for comparison. Data from one of three representative experiments are shown. The Journal of Immunology 1603

Table II. CCL15 molecules generated from CCL15(1–92) by proteolytic activity of purified PMNsa

Determined N-Terminal Detected Mr Theoretical Average C-Terminal Peptide Sequence MALDI-MS Mr Sequence CCL15(22–92) VLNSFHFAADX 7768.0 7767.1 KKLKPYSI CCL15(24–92) NSFHFAADXXT 7556.6 7554.8 KKLKPYSI CCL15(24–91) 7442.4 7441.7 CCL15(25–92) 7442.4 7440.7 CCL15(27–92) HFAADXXTSYI 7207.1 7206.6 KKLKPYSI CCL15(29–92) 6921.1 6922.1

a Identification was performed by MALDI-MS. CCL15(22–92), CCL15(24–92), and CCL15(27–92) were also identified by N-terminal Edman sequencing. The C-terminal amino acid sequence was deduced from the published amino acid sequence and the MS results. two subsequent chromatographic steps. Processed CCL15 was that granulocytes process full-length CCL15 by cathepsin G and identified by RIA, MALDI-MS, and amino acid sequencing. These elastase. Therefore, we investigated whether cathepsin G and elas- investigations revealed that PMNs are able to process CCL15(1– tase were able to process full-length CCL15. Western blot analysis 92) to N-terminally truncated molecules CCL15(22–92), of the processed CCL15 products suggests that these enzymes are

CCL15(24–92), CCL15(27–92), and CCL15 (29–92). Further- able to process CCL15. RP-chromatography of the processed mol- Downloaded from more, within the IR fractions, a Mr 7442.4 was detected which ecules with subsequent MALDI-MS of the collected fractions, and correlates with the theoretical Mr CCL15(25–92) or CCL15(24– amino acid sequencing of the identified molecules revealed that ca- 91) (Table II). The ratio of the processed CCL15 molecules was thepsin G processed synthetic CCL15(1–92) predominantly to determined by CCL15-IR concentrations in the fractions of the CCL15(24–92), CCL15(27–92), and CCL15(29–92), whereas elas- first chromatographic step and their respective MALDI-mass spec- tase produces CCL15(22–92) (Table III). To analyze the time course tra. The percentages of CCL15(22–92), CCL15(24–92), on the processing of CCL15(1–92), 2 ϫ 106/100 ␮l of PMNs were http://www.jimmunol.org/ CCL15(27–92), CCL15(29–92), and CCL15(25–92) or coincubated with 2 ␮M CCL15(1–92). Separation by SDS-PAGE CCL15(24–91) in the PMN supernatant was 41.7, 27.9, 12.4, 6.3, revealed that CCL15(1–92) was completely converted to a molecule and 11.8%, respectively. similar in size to CCL15(27–92) within 15 to 30 min (Fig. 2D). To identify the protease(s) responsible for the conversion of CCL15, a range of protease inhibitors on the CCL15-processing Cathepsin G proteolytic activity is not detectable in blood activity of PMNs was tested. First, class-specific inhibitors active plasma and HF on serine, cysteine, aspartate, and metalloproteases were used. The For detection of cathepsin G activity in plasma and HF, the sub- active inhibitors, identified by Western blot analysis, were among strate Suc-Ala-Ala-Pro-Phe-pNA was used. This substrate is not

the serine protease inhibitors. The serine protease inhibitors hydrolyzed by leukocyte elastase (48). The absorbance of hydro- by guest on September 30, 2021 ␣ AEBSF (Pefabloc) and 1-antitrypsin, as well as the serine- and lytic substrate products at 405 nm was suppressed in plasma, com- cysteine protease inhibitor chymostatin and the endoprotease in- pared with the control medium, by ϳ15%. In plasma of healthy ␣ hibitor 2-macroglobulin, blocked processing of CCL15(1–92). donors and in plasma and HF of CRF patients, no intrinsic cathep- Another serine protease inhibitor, leupeptin, did not inhibit the sin G activity was detectable. Plasma compounds significantly in- processing of CCL15. Inhibitors of metalloproteases (EDTA, 1,10- hibited the activity of 1 mU of cathepsin G added to 100 ␮lof phenanthroline), cysteine proteases (pepstatin), and aspartic pro- plasma, whereas 100 ␮l of HF did not contain the capacity to teases (E-64) had no activity in this assay (Fig. 2B). The concen- inhibit this proteolytic activity (Fig. 3A). In experiments with 100 tration of the potent serine protease inhibitor leupeptin (0.02 mM) ␮l of HF, 0.062 mU of cathepsin G displayed residual proteolytic used in this assay is expected to inhibit most serine-like proteases, activity, whereas 100 ␮l of plasma totally inhibited this activity. In with the exception of a few enzymes, e.g., cathepsin G and elastase incubation experiments, HF revealed no proteolytic activity for (45, 46), that are known to be relatively resistant to this inhibitor. CCL15(1–92) (Fig. 3B). This led us to hypothesize that one or more granulocyte serine ⌬ ⌬ proteases, elastase, cathepsin G, and proteinase 3 are involved in Compared to CCL15(1–92,) 23 and 26 CCL15 isoforms the processing of CCL15. To test this hypothesis, the specific elas- reveal increased calcium flux-inducing activity, chemotactic tase inhibitor elastatinal and secretory leukocyte proteinase inhib- activity, and adhesiveness to fibronectin itor (SLPI), which is known to inhibit cathepsin G and elastase, but The calcium flux-inducing activity of CCL15(1–92), CCL15(24– not proteinase 3, were tested (47). Whereas elastatinal only par- 92), and CCL15(27–92) were tested on the CHO-K1 cell line ex- tially inhibited the processing of CCL15, SLPI inhibited the pro- pressing CCR1, monocytes, and THP-1 cells. On CHO-K1 cells, cessing of CCL15 almost totally (Fig. 2C). These results suggest the analysis revealed that N-terminal processing of CCL15(1–

Table III. CCL15 molecules generated from CCL15(1–92) by proteolytic processing with purified granulocyte elastase and cathepsin Ga

Determined N- Detected Mr Theoretical Average Enzyme Peptide Terminal Sequence MALDI-MS Mr Elastase CCL15(22–92) VLNSFHFAADX 7764.5 7767.1 Cathepsin G CCL15(24–92) NSFHFAADX 7553.4 7554.8 CCL15(27–92) HFAADX 7203.6 7206.5 CCL15(29–92) AADX 6918.2 6922.1

a The N terminus, determined by Edman sequencing, and the Mr, determined by MALDI-MS, are presented. 1604 QUANTUM PROTEOLYTIC ACTIVATION OF CCL15 BY NEUTROPHIL CATHEPSIN G

FIGURE 3. Proteolytic activity of cathepsin G in plasma and HF. A, Detection of proteolytic activity of cathepsin G in plasma of healthy donors and in plasma and HF of CRF patients with substrate Suc-Ala-Ala-Pro-Phe-pNA. Regulation of cathepsin G activity by protease inhibitors was estimated by addition of 1 mU of purified enzyme to plasma and HF. Proteolytic activity was detected after3hofincubation in three different plasma or HF samples. B, Proteolytic activity for CCL15 processing in HF. One microgram of CCL15(1–92) was applied to 1 ml of HF. After 0 min ࡗ,30minf,60minछ, and 120 min ϫ 100 ␮l of HF was withdrawn, acidified to pH 2.0, and chromatographed with the standardized RP method. Downloaded from

92) to CCL15(24–92) and CCL15(27–92) is correlated with an CCL15(27–92) for 5 min significantly increased the number of 2 Ϯ increase in the activity (Fig. 4A). The EC50 values for adhesive cells to fibronectin at 0.1 dyn/cm from 53.0 7,4 to CCL15(1–92), CCL15(24–92), and CCL15(27–92) are Ͼ1000, 132.3 Ϯ 26.6 and 175.3 Ϯ 34.2, respectively ( p Ͻ 0.000001; n ϭ 2 5.4 Ϯ 3.0, and 3.6 Ϯ 1.9 nM, respectively. According to the 9), and at 2.0 dyn/cm from 43.2 Ϯ 12.2 to 119,6 Ϯ 31.3 and http://www.jimmunol.org/ Ϯ Ͻ ϭ EC50 values, CCL15(24–92) and CCL15(27–92) were more po- 158.6 33.0, respectively ( p 0.00001; n 9). tent in inducing calcium flux than RANTES in the CCR1-trans- In summary, proteolytic activation of CCL15(1–92) to fected cells (Table IV). CCL15(24–92) and CCL15(27–92) reveals a strong induction of The calcium flux-inducing activity of the different isoforms on intracellular calcium flux and adhesive capacity, whereas the ca- monocytes also demonstrates that the N-terminal-processed pacity to induce chemotactic migration of monocytes is only mod- CCL15(24–92), and CCL15(27–92) induced robust calcium fluxes erately activated, indicating a differential activation of distinct sig- Ϯ Ϯ with EC50 values of 0.84 0.15 and 0.62 0.12 nM, respec- nal transduction pathways responsible for adhesiveness and tively, whereas CCL15(1–92) was 1000 times less potent in in- chemotactic activity by the different CCL15 isoforms (see also ϳ ␮ by guest on September 30, 2021 ducing calcium fluxes, with an estimated EC50 value of 1 M Table IV). (Fig. 4B and Table IV). In THP-1 cells, CCL15(24–92) and Ϯ CCL15(27–92) induced calcium fluxes with EC50 values of 35.2 Discussion 13.8 and 17.44 Ϯ 2.4 nM, respectively, whereas the CCL15(1–92) The present study demonstrates an efficient processing of the che- and the CCL15(1–92) variant with the N-terminal pyrrolidone car- mokine CCL15 by neutrophils and consequences for the activation boxylic acid did not induce a calcium flux using concentrations up of adhesive properties of PBMCs. Processing of CCL15 was in- to 333 nM (Table IV). duced in two different models: 1) by incubation of CCL15 with To investigate whether elastase digestion has a significant im- isolated PMNs and 2) by hemofiltration of blood from renal in- pact on activation of CCL15(1–92), the molecule was digested sufficiency patients, a treatment in which substantial activation of with elastase for 15 min. Subsequently, the reaction was stopped PMNs and monocytes by interaction with the filter membrane has with elastatinal. Using the FLIPR assay, we could show that the been described previously (49, 50). Proteolytic processing of elastase treatment of CCL15(1–92) significantly increases the cal- plasma CCL15 during hemofiltration is suggested by different cium flux-inducing activity on CCR1-transfected HEK-293 cells compositions of CCL15-IR material in plasma and HF of CRF (Fig. 4C). patients. The compositions were analyzed by the use of a highly In chemotaxis assays with monocytes, CCL15(24–92) and sensitive and reproducible two-dimensional method, which links CCL15(27–92) showed similar potency to induce chemotactic mi- chromatographic separation of CCL15-IR molecules and their IR. gration (maximal migration observed at 1 nM), whereas full-size This method was essential for the study since it allowed us to CCL15(1–92) was 10-fold less potent (maximal migration ob- differentiate CCL15 isoforms in 250 ␮l of plasma containing ϳ300 served at 10 nM) (Fig. 4C). CCL15(27–92) and CCL15(24–92) fmol of CCL15-IR. Such a level of sensitivity for analysis of a showed a significantly higher efficacy to induce monocyte migra- molecule and its isoforms in the complex mixture of plasma mol- tion (177 Ϯ 7 and 166 Ϯ 9 migrated cells per high-power field, ecules has not been achieved with other methods until now. Using respectively; detected at concentrations of 1 nM) compared to this method, a significant shift of CCL15-IR retention position tR Ϯ CCL15(1–92) (126 11 migrated cells per high-power field; de- in blood to earlier tR in HF was found. tR of CCL15(27–92) and tected at concentrations of 10 nM; mean Ϯ SEM; p Ͻ 0.01). CCL15(24–92) isolated from HF correspond to the predominant

Adhesiveness of PBMCs to plasma fibronectin was investigated CCL15-IR peaks in HF. The shift of CCL15-IR tR is not attribut- in flow chamber experiments. Preincubation of PBMCs with 150 able to accumulation of CCL15-IR with early tR in HF and reten- nM CCL15(1–92) for 5 min did not significantly modify their ad- tion of CCL15 with late tR in blood, since under these supposed 2 hesiveness to plasma fibronectin at 0.1 dyn/cm , whereas at 2.0 conditions the high concentrations of CCL15-IR with early tR de- dyn/cm2 a slight but significant increase in the number of adhesive tected in HF should preexist and should be detectable in blood cells from 43.22 Ϯ 12.20 to 53.78 Ϯ 5.93 was found ( p Ͻ 0.05; plasma. In contrast, isolated CCL15-IR materials from HF were n ϭ 9). Preincubation of PBMCs with 150 nM CCL15(24–92) or always identified as CCL15 molecules by amino acid sequencing The Journal of Immunology 1605

and mass spectrometric analysis, indicating that CCL15-IR in HF and blood represents CCL15 molecules. A sieving coefficient of 0.2 for CCL15-IR does not completely exclude an entire retention

of the CCL15-IR peak at tR of 50 min, but a sieving coefficient of 0.0 for proteins and peptides was rarely found (51–53), indicating that this peak also represents CCL15. Proteolytic degradation of CCL15-IR in HF by bacterial con- tamination or low pH was excluded by immediate freezing of the samples at Ϫ80°C and by the evidence of the hydrolytic stability of CCL15(1–92) at pH 2.0. Therefore, we investigated the pro- cessing of CCL15(1–92) by isolated PBMCs, PMNs, and platelets. Our results indicate that at least one activity for proteolytic pro- cessing of CCL15(1–92) is released by activated PMNs (54). Ex- periments with proteinase inhibitors indicate conclusively that this activity is constituted by the neutrophil granule serine proteases cathepsin G and elastase. These two proteinases are known to be liberated from PMNs during blood filtration into the plasma (49, 55) and play a central role during inflammation by processing in-

flammatory modulators (56–61). Cathepsin G was shown to pro- Downloaded from cess CCL15(1–92) to CCL15(24–92), CCL15(27–92) and CCL15(29–92) and elastase processes CCL15(1–92) predomi- nantly to CCL15(22–92). Amino acids Leu-23, Phe-26, and Phe-28 at the P1 site indicate a chymotrypsin-like processing, which is common for cathepsin G (62). The amino acid Val-21 at

P1 is a common processing site for elastase (63). The predominant http://www.jimmunol.org/ N-terminal processing sites Leu-23/Asn-24 and Phe-26/His-27 for the isolated CCL15 molecules from blood filtrate support the hy- pothesis of a cathepsin G-like processing in the course of hemofiltration.

Processing of CCL15 by PMN resulted in a molecule with a Mr 7442.4, which fits to CCL15(25–92) or CCL15(24–91). Recently Youn et al. (64) described CCL15(25–92), which resulted from proteolytic processing between Asn-24 and Ser-25 during expres- sion in a cabbage looper insect cell line. For this unusual process- by guest on September 30, 2021 ing site, legumain is a putative protease which has a highly re- stricted specificity requiring an asparagine at the P1 site (65). Legumain is a lysosomal protease, appears to be expressed in re- sponse to stress (66), and may be secreted from cells under some conditions (67), and like cathepsin L it may be active in the peri- cellular environment (68). Legumain is expressed in various or- gans (69) and was found in (70), dendritic cells (71), and in vivo in tumors with high invasive and metastatic potential (66). Under these conditions, legumain may be responsible for processing of CCL15 to the molecule CCL15(25–92) and may than support the immune response or the malignant character of a tumor. Quantum proteolytic processing (72) of CCL15 in the close proximity of activated neutrophils by millimolar concentrations of cathepsin G and elastase, when released from single azurophil granules of neutrophils before catalysis is quenched by pericellular inhibitors, is suggested by two different facts: 1) In HF, neither proteolytic activity for processing of additionally added CCL15 nor intrinsic cathepsin G activity was detected. 2) In blood plasma, no proteolytic activity for processing of additionally added FIGURE 4. Calcium mobilization and chemotactic activity of CCL15 CCL15, no proteolytic products CCL15(24–92) and CCL15(27– isoforms. Calcium mobilization and chemotaxis assays were performed 92), and no intrinsic cathepsin G activity were detected. Also, with CCL15(1–92) Œ, CCL15(24–92) ࡗ, and CCL15(27–92) f. A, CCR- 1-transfected CHO-K1 cells. A, Calcium mobilization was also detected for RANTES F. B, Calcium mobilization in monocytes. C, Comparison of calcium flux induced by untreated and human neutrophil elastase-digested described in Materials and Methods. Each panel is representative of at least CCL15(1–92). In brief, 0.4 nmol/ml of CCL15(1–92) was digested with or three independent experiments. The chemotaxis experiments represent without 10 mU of elastase for 15 min. The reaction was stopped with 200 mean Ϯ SEM for triplicate wells. E, Adhesion rate of PBMC to plasma ␮M elastatinal. Samples resulting in a final concentration of 100 nM were fibronectin induced by CCL15(1–92), CCL15(24–92), and CCL15(27–92) .(p Ͻ 0.0001 ,ءء ;p Ͻ 0.05 ,ء) subjected to the FLIPR performed with CCR1-transfected HEK-293 cells. at shear stresses of 0.1 and 2.0 dyn/cm2 D, Chemotaxis of freshly isolated human monocytes were conducted as Values are the mean for at least nine independent determinations. 1606 QUANTUM PROTEOLYTIC ACTIVATION OF CCL15 BY NEUTROPHIL CATHEPSIN G

Table IV. Functional parameters of PBMCs, monocytes, CCR1-transfected CHO-K1 cells, and THP-1 cells for CCL15(1–92), N-terminally truncated CCL15 molecules, and the reference chemokine RANTESa

Calcium Mobilization Adhesion to Fibronectin: PBMCs Maximal Migration in Efficacy of Migration: % of control Monocytes CCR1 THP-1 Chemotaxis Assay: Monocytes (cells/high- Ϯ Ϯ Ϯ Ϯ 2 2 Ligand (EC50 SEM) (EC50 SEM) (EC50 SEM) Monocytes power field SEM) 0.1 dyn/cm 2.0 dyn/cm CCL15(1–92) Ն1000 nM Ͼ1000 nM Ͼ333 nM 10 nM 126 Ϯ 11 106.6 Ϯ 22.6 126.0 Ϯ 18.2 CCL15(24–92) 0.84 Ϯ 0.15nM 4.7 Ϯ 3.0 nM 35.2 Ϯ 13.9nM 1 nM 166 Ϯ 9 247.9 Ϯ 29.6 273.1 Ϯ 36.0 CCL15(27–92) 0.62 Ϯ 0.12nM 3.4 Ϯ 2.1 nM 17.5 Ϯ 2.4 nM 1 nM 177 Ϯ 8 331.9 Ϯ 64.3 371.4 Ϯ 87.3 RANTES ND 34.9 Ϯ 25.0nM ND ND ND ND ND

a For detection of maximal chemotactic activity, four different concentrations of the CCL15 molecules were tested. Efficacy of migration was detected at concentrations of 10 nM for CCL15(1–92) and at 1 nM for CCL15(24–92) and CCL15(27–92). Adhesion of PBMCs to plasma fibronectin is given as a percentage of the control for shear stresses of 0.1 and 2.0 dyn/cm2. Values are the mean for at least three independent determinations. blood plasma from healthy volunteers and CRF patients shows a 92), and CCL15(29–92). Additionally, for CCL15(30–92), a sig- high capacity to inhibit the activity of additionally added cathepsin nificantly reduced chemotactic activity was found (6, 77). Thus, G, suggesting that granulocyte cathepsin G liberated into blood the results demonstrate that truncation of CCL15 N-terminally to plasma during blood filtration is immediately inactivated by high aa 22–29 results in a significant increase of the biological potency Downloaded from concentrations of protease inhibitors in plasma (49, 55, 73–75). of the molecule. A fast rate of activation of CCL15 is also suggested by entire The findings indicate a dissociation of the dose-response curves processing of 2 nmol/ml CCL15(1–92) by 2 ϫ 107/ml neutrophils for chemotaxis and calcium mobilization for the investigated within 15–30 min, indicating significantly faster kinetics than for CCL15 molecules. Whereas CCL15(1–92) induces chemotaxis, the processing of other chemokines by neutrophils (76). In addi- reaching a maximum at a concentration of 10 nM, it does not 6 tion, an increased ratio of neutrophils (2–5 ϫ 10 /ml) to CCL15-IR induce an efficient calcium mobilization on monocytes, the mono- http://www.jimmunol.org/ concentrations (1.5 pmol/ml) in blood by a factor of 100–300 may cytic cell line THP-1, or on CCR1-transfected cells. In contrast, result in a much faster processing rate for filtered CCL15, which CCL15(24–92) and CCL15(27–92) induce maximum chemotaxis then occurs within seconds before millimolar concentrations of at concentrations of 1 nM as well as a robust calcium flux. It is not cathepsin G and elastase are diluted and inhibited by protease clarified in detail how chemokine-mediated cell migration is re- inhibitors. lated to the transient rise in intracellular free Ca2ϩ, which is nec- To evaluate the functional relevance of N-terminal processing, essary for the cytoskeletal remodeling events that control cell mo- CCL15(1–92) and the identified N-terminally truncated CCL15 tility and contractility. Different studies on chemokines suggest molecules were synthesized and investigated for their chemotactic, that there is no strict correlation of calcium influx and migratory calcium-mobilizing, and adhesive properties. Calcium mobiliza- activity (78–81). by guest on September 30, 2021 tion assays with monocytes, CCR1-transfected CHO-K1 cells, and Since calcium mobilization is associated with detachment, ar- THP-1 cells displayed a remarkable increase in agonistic potency rest, and locomotion of leukocytes on vascular endothelium (78), of the N-terminal truncated CCL15(24–92) or CCL15(27–92) we investigated the influence of the different CCL15 molecules on compared to full-length CCL15. For monocytes, EC50 values for the adhesion of PBMCs to fibronectin in flow chamber experi- full-length CCL15 are above 1 ␮M and decrease below 1 nM for ments. Fibronectin was chosen as a substrate for the adhesion as- CCL15(24–92) and CCL15(27–92). Chemotaxis assays with the says since fibronectin ligands on leukocytes are indispensable for different CCL15 molecules on monocytes do not show such a re- the coordinated interaction of leukocytes with endothelial cells and markable difference of their chemotactic potency. CCL15(1–92) can be functionally assessed this way in a reliable fashion (17, 82). induced a maximum chemotactic activity at 10 nM, whereas Compared with controls, CCL15(1–92) only modestly increased CCL15(24–92) and CCL15(27–92) had a potency of 1 nM. The the adhesiveness of PBMCs, whereas the N-terminally truncated efficacy to induce chemotaxis was ϳ30% higher for CCL15(24– CCL15(24–92) and CCL15(27–92) significantly increased their 92) and CCL15(27–92) compared with CCL15(1–92). To assess adhesiveness to fibronectin. This finding suggests that N-termi- the relevance of human neutrophil elastase in activation of CCL15, nally truncated CCL15 molecules activate at least one of the in- ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ CCL15(1–92) was digested with the protease and subjected to cal- tegrins 5 1, 4 7, v 1,or v 3, which are expressed on PBMCs cium mobilization assays. Our results show that also elastase di- and are ligands for plasma fibronectin (82–88). The chemokines gestion significantly increases the potency of CCL15 to induce an MIP-1␣ and RANTES, which act also via the chemokine receptors ␣ ␤ intracellular calcium flux via CCR1. In contrast, preliminary re- CCR1 and CCR3, are known to activate the integrin 5 1 (VLA- ␣ ␤ sults indicate that CCL15(22–92), the predominant molecule pro- 5), suggesting that CCL15 also activates 5 1 (82). VLA-4, an- duced by elastase digestion, in comparison to CCL15(24–92) and other integrin activated by MIP-1␣ and RANTES, is a weak ligand CCL15(27–92), has an reduced calcium flux-inducing activity. for plasma fibronectin due to limited exposure of the VLA-4-spe- The results are consistent with the findings of Lee et al. (6), who cific binding sequence CS-1 (89) excluding this integrin as a can- compared calcium flux-inducing and chemotactic activity of didate for increased adhesiveness to fibronectin. CCL15(1–92) and eight different N-terminally truncated CCL15 Our findings lead us to the assumption that cathepsin G and forms on CCR1-transfected human osteosarcoma cells. They elastase, released from activated PMNs, e.g., during their adhesion found that N-terminally truncated forms CCL15(25–92), to endothelium (90, 91) at sites of tissue trauma, infection, or in- CCL15(28–92), and CCL15(29–92) are strong inducers of cal- flammation, proteolytically activate circulating or endothelium- cium flux and chemotaxis. In their investigations, CCL15(1–92) bound CCL15 and support monocyte infiltration into inflammatory and CCL15(30–92) carry at least a 100-fold lower potency to in- sites. This activation might also support the recruitment of neu- duce calcium flux and CCL15(1–92) a 10-fold lower potency to trophils to sites of tissue injury, since neutrophils, exposed to induce chemotactic migration than CCL15(25–92), CCL15(28– IFN-␥ and GM-CSF, were found to up-regulate CCR1 and CCR3 The Journal of Immunology 1607 and increase their receptiveness to CCR1 and CCR3 ligands (2, 92, 21. Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, and 93). Processing of CCL15 by neutrophils is locally limited to the L. T. Williams. 1998. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. close proximity of the cells by immediate inactivation of cathepsin Natl. Acad. Sci. USA 95: 258–263. G and elastase by blood-derived protease inhibitors. Given that 22. Kasama, T., M. Muramatsu, K. Kobayashi, N. Yajima, F. Shiozawa, R. Hanaoka, Y. Miwa, M. Negishi, H. Ide, and M. Adachi. 2002. Interaction of monocytes N-terminally truncated CCL15 molecules are not detectable in with vascular endothelial cells synergistically induces ␥-inducible pro- blood plasma, it might be possible that these molecules immedi- tein 10 expression through activation of specific cell surface molecules and cy- ately bind to and activate monocytes. This might support the de- tokines. Cell. Immunol. 219: 131–139. 23. Broxmeyer, H. E., and C. H. Kim. 1999. 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