The Epidermal Growth Factor-Seven Transmembrane (EGF-TM7) Receptor CD97 Is Required for Neutrophil Migration and Host Defense This information is current as of September 23, 2021. Jaklien C. Leemans, Anje A. te Velde, Sandrine Florquin, Roelof J. Bennink, Kora de Bruin, René A. W. van Lier, Tom van der Poll and Jörg Hamann J Immunol 2004; 172:1125-1131; ; doi: 10.4049/jimmunol.172.2.1125 Downloaded from http://www.jimmunol.org/content/172/2/1125 References This article cites 52 articles, 19 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/172/2/1125.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Epidermal Growth Factor-Seven Transmembrane (EGF-TM7) Receptor CD97 Is Required for Neutrophil Migration and Host Defense1 Jaklien C. Leemans,* Anje A. te Velde,* Sandrine Florquin,† Roelof J. Bennink,‡ Kora de Bruin,‡ Rene´A. W. van Lier,§ Tom van der Poll,* and Jo¨rg Hamann2§ The epidermal growth factor-seven transmembrane (EGF-TM7) family is a group of seven-span transmembrane receptors pre- dominantly expressed by cells of the immune system. Family members CD97, EGF module-containing mucin-like receptor (EMR) 1, EMR2, EMR3, EMR4, and EGF-TM7-latrophilin-related protein are characterized by an extended extracellular region with a variable number of N-terminal EGF-like domains. EGF-TM7 receptors bind cellular ligands as demonstrated by the interaction of CD97 with decay accelerating factor (CD55) and dermatan sulfate. Investigating the effect of newly generated mAb on the Downloaded from migration of neutrophilic granulocytes, we here report for the first time in vivo data on the function of CD97. In dextran sulfate sodium-induced experimental colitis, we show that homing of adoptively transferred neutrophils to the colon was significantly delayed when cells were preincubated with CD97 mAb. The consequences of this defect in neutrophil migration for host defense are demonstrated in a murine model of Streptococcus pneumoniae-induced pneumonia. Mice treated with CD97 mAb to EGF domain 1 (1B2) and EGF domain 3 (1C5) displayed a reduced granulocytic inflammatory infiltrate at 20 h after inoculation. This was associated with a significantly enhanced outgrowth of bacteria in the lungs at 44 h and a strongly diminished survival. http://www.jimmunol.org/ Together, these findings indicate an essential role for CD97 in the migration of neutrophils. The Journal of Immunology, 2004, 172: 1125–1131. D97 (1–7) is a defining member of the epidermal growth joined by a noncovalent linkage with the TM7/cytoplasmic ␤ factor seven transmembrane (EGF-TM7)3 family (8). subunit. C Molecules of this family, comprising also EGF module- Due to alternative RNA splicing, the number of EGF domains in containing mucin-like related protein (EMR)1 (F4/80) (9–11), EGF-TM7 receptors is variable (8). For human CD97 (hCD97), iso- EMR2 (12), EMR3 (13), EMR4 (FIRE) (14–16), and EGF-TM7- forms with three, four, and five EGF domains have been identified (4), by guest on September 23, 2021 latrophilin-related protein (17, 18), are characterized by an ex- designated hereafter as hCD97(EGF1,2,5), hCD97(EGF1,2,3,5), and tended extracellular region. They possess N-terminal EGF-like do- hCD97(EGF1,2,3,4,5). Mouse CD97 (mCD97) also exists in three mains, which are connected by a stalk region to TM7 segments isoforms (5, 6). Next to isoforms with three and four EGF domains, homologous to those found in peptide hormone-binding class B G indicated as mCD97(EGF1,2,4) and mCD97(EGF1,2,3,4), a third iso- protein-coupled receptors (12, 13, 17, 19). First discovered for form mCD97(EGF1,2,X,3,4) was detected. This isoform has a se- CD97 (4), there is growing evidence that most if not all EGF-TM7 quence of 45 aa between the second and third EGF domain that does receptors are expressed at the cell surface as heterodimers (5, 15, not correspond to known protein modules. EGF domains 3 and 4 in 17, 20, 21). After cleavage of the polypeptide at a G protein-cou- mCD97 are the homologues of EGF domains 4 and 5 in hCD97, pled receptor-proteolytic site (22) immediately proximal to the first respectively (6). Schematic structures of CD97 in humans and mice ␣ transmembrane segment, the large extracellular subunit is re- are depicted in Fig. 1. Receptors of the EGF-TM7 family are expressed by cells of the immune system and by smooth muscle cells. In contrast to the *Laboratory for Experimental Internal Medicine, Departments of †Pathology and rather restricted expression profile of other family members (Ref. ‡Nuclear Medicine, and §Laboratory for Experimental Immunology, Academic Med- ical Center, University of Amsterdam, Amsterdam, The Netherlands 8 and references therein; Refs. 14, 17, 20), CD97 is found on a Received for publication August 8, 2003. Accepted for publication October 21, 2003. broad array of hematopoietic cells including activated lympho- The costs of publication of this article were defrayed in part by the payment of page cytes, granulocytes, monocytes, macrophages, and dendritic cells charges. This article must therefore be hereby marked advertisement in accordance (1, 2, 23, 24). High expression and elevated levels of soluble CD97 with 18 U.S.C. Section 1734 solely to indicate this fact. at sites of inflammation suggest a role for CD97 in the onset of 1 This work has been supported by a grant from The Netherlands Organization for protective as well as destructive immune responses (4, 25, 26). In Scientific Research (to J.C.L). J.H. is a fellow of the Royal Netherlands Academy of Arts and Sciences. addition, smooth muscle cells and malignant cells in various epi- 2 Address correspondence and reprint requests to Dr. Jo¨rg Hamann, Laboratory for thelial tumors express CD97 (23, 27–29). Experimental Immunology, G1-106, Academic Medical Center, University of Am- We previously identified decay accelerating factor (CD55) as a sterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail address: cellular ligand of CD97 (30). CD55 is a GPI-linked molecule that [email protected] prevents complement deposition on self cells by inhibiting 3 Abbreviations used in this paper: EGF, epidermal growth factor; TM7, seven trans- membrane; EMR, EGF module-containing mucin-like receptor; hCD97, human C3/C5 convertases (31, 32). The binding site for CD55 is formed by CD97; mCD97, mouse CD97; GAG, glycosaminoglycan; i.n., intranasal; DSS, dex- the EGF domain region (33). Lin et al. (34) showed that tran sulphate sodium; HMPAO, hexamethylpropylene amine oxime; MIP-2, mac- hCD97(EGF1,2,5) binds CD55 with low affinity (86 ␮M) and a rapid rophage-inflammatory protein-2; SPECT, single photon emission computed Ϫ tomography. off-rate (0.6 s 1). Affinity for CD55 differs between CD97 isoforms Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 1126 REQUIREMENT OF CD97 FOR NEUTROPHIL MIGRATION AND HOST DEFENSE mCD97(EGF1,2,3,4) cDNA in pcDNA3.1/Zeo(ϩ) (6). Cells were selected with zeocin (Invitrogen, Leek, The Netherlands) at 500 mg/ml in culture medium. Resulting stably transfected clones were tested for mCD97 ex- pression by flow cytometry with the earlier generated mCD97 mAb 1A2 (6). One clone was selected and used for immunization. Three i.p. injec- tions with 10 ϫ 106 irradiated (50 Gy) cells in PBS were given at weekly intervals. Eight weeks after the third injection, the hamster was boosted i.p. with 10 ϫ 106 cells. Three days later, hamster spleen cells were fused with mouse myeloma SP2/0 cells by standard hybridoma technology. Binding of hybridoma supernatants to the ARHO12 clone stably expressing mCD97(EGF1,2,3,4) was tested by flow cytometry. Flow cytometry of COS cells expressing mCD97 isoforms or chimeras between mCD97 and hCD97 was used to identify hybridomas that recognize different regions of mCD97. Selected hybridomas were subcloned until they were monoclonal and stable. The hybridomas 1B2, 1C5, and 1D2 were grown at large amounts and Ig was purified using protein A-Sepharose CL-4B (Sigma- Aldrich, St. Louis, MO). Generation of chimeric m-hCD97 constructs Generation of an expression construct encoding mCD97(EGF1h,2,4) by overlap extension-PCR has been described earlier (6). In a similar manner, constructs in which EGF domain 1 or EGF domains 1 and 2 of mCD97 Downloaded from have been exchanged by homologues EGF domains of hCD97 were made. In a first step, two overlapping PCR fragments were generated in separate FIGURE 1. Schematic structure of CD97. Depicted are the three differ- PCRs. For hCD97(EGF1m,2,5), the N-terminal part of mCD97 including ent isoforms that have been identified in both human and mouse. For de- EGF domain 1 was amplified from mCD97 cDNA (6) using a standard T7 ϩ Ј tails, see text. primer (( ) strand) and the specific primer 5 -CGTTGATGTCTTCA CAGCTCTCTGCAGGGTTAG-3Ј (nt 214–237 of the (Ϫ) strand; the se- quence overlapping with EGF domain 2 of hCD97 is underlined).