Quantum Proteolytic Activation of Chemokine CCL15 by Neutrophil 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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 neutrophils. 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 monocytes 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 cytokines 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. Chemokines 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 lymphocytes 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, monocyte 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