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Adhesion Transendothelial Migration Without Affecting VCAM-1 Inhibits An Antibody to the Sixth Ig-like Domain of VCAM-1 Inhibits Leukocyte Transendothelial Migration without Affecting Adhesion This information is current as of September 28, 2021. Sukmook Lee, Il-Hee Yoon, Aerin Yoon, Joan M. Cook-Mills, Chung-Gyu Park and Junho Chung J Immunol published online 1 October 2012 http://www.jimmunol.org/content/early/2012/10/01/jimmun ol.1103803 Downloaded from Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 1, 2012, doi:10.4049/jimmunol.1103803 The Journal of Immunology An Antibody to the Sixth Ig-like Domain of VCAM-1 Inhibits Leukocyte Transendothelial Migration without Affecting Adhesion Sukmook Lee,*,1,2 Il-Hee Yoon,*,†,1 Aerin Yoon,‡ Joan M. Cook-Mills,x Chung-Gyu Park,†,3 and Junho Chung‡,3 VCAM-1 plays a key role in leukocyte trafficking during inflammatory responses. However, molecular mechanisms underlying this function have not been clearly elucidated. In this study, using phage display technology, we developed a rabbit/human chimeric VCAM-1 Ab, termed VCAM-1 domain 6 (VCAM-1-D6), which specifically recognizes aa 511–599 within the sixth Ig-like domain. We report that the VCAM-1-D6 Ab blocked U937 cell transmigration across activated HUVECs but did not alter adhesion of U937 cells to the HUVECs. We also demonstrate that VCAM-1-D6 does not alter TNF-a–stimulated endothelial cell chemokine or Downloaded from cytokine production. Furthermore, through in vivo efficacy testing using a mouse islet allograft model, we demonstrate that VCAM-1-D6 significantly alleviates allograft rejection by blocking leukocyte infiltration to the grafted islets. Taken together, our results suggest that the VCAM-1-D6 Ab may block VCAM-1–mediated inflammation and could be a useful tool in treating inflammatory diseases. The Journal of Immunology, 2012, 189: 000–000. ascular cell adhesion molecule-1 (also known as CD106) cyte arrest on the endothelium. After the high-affinity adhesion, http://www.jimmunol.org/ is a cell adhesion molecule induced in activated endo- VCAM-1, together with PECAM-1, plays a critical role in leu- V thelial cells. During leukocyte recruitment, VCAM-1 kocyte transendothelial migration (14). binds a4b1 integrin, a leukocyte integrin, in its intermediate- VCAM-1 is a member of the Ig superfamily. The extracellular affinity state and, along with selectins and addressins, plays a region of the full-length form of VCAM-1 contains seven ho- key role in the rolling step of leukocytes on the endothelium (1–3). mologous Ig-like domains. Domains 1 and 4, 2 and 5, and 3 and 6 Selectin binding on leukocytes or activation of chemokine re- are homologous to each other (15–17). There are two human forms ceptors stimulates “outside-in” signals in leukocytes, which and two mouse forms of VCAM-1. Human VCAM-1 (hVCAM-1) increases the affinity of the integrin family of receptors to bind has two splice variants that result in a protein with either seven Ig- endothelial cell adhesion molecules, predominantly ICAM-1and like domains or six Ig-like domains (lacking domain 4) (15, 18). by guest on September 28, 2021 VCAM-1 (4–13). This high-affinity interaction results in leuko- Mouse VCAM-1 (mVCAM-1) also has a full-length form with seven Ig-like domains, as well as a truncated form containing only the first three Ig-like domains. *Cancer Research Institute, Xenotransplantation Research Center, College of Medi- cine, Seoul National University, Seoul 110-799, Korea; †Department of Microbiology All variants of VCAM-1 have been shown to bind to a4b1 and Immunology, Seoul National University, Seoul 110-799, Korea; ‡Department of integrin (19–22). In addition to a4b1 integrin, VCAM-1 can bind Biochemistry and Molecular Biology, Seoul National University, Seoul 110-799, Korea; x other integrins, including a4b7, adb2, aMb2, and a9b1 (23–28). and Allergy-Immunology Division, Northwestern University Feinberg School of Med- icine, Chicago, IL 60611 It is known that a4b1 integrin binds Ig-like domains 1 and 4 (29– 1S.L. and I.-H.Y. contributed equally to this work. 32), and that activated integrins are necessary for firm leukocyte adhesion to the endothelium via Ig-like domains 1 and 4 of VCAM- 2Current address: Scripps Korea Antibody Institute, Hyoja-2-dong, Chuncheon-si, Gangwon-do, Korea. 1 (33). 3C.-G.P. and J.C. codirected this work. Leukocyte binding to adhesion molecules, including VCAM-1, Received for publication January 5, 2012. Accepted for publication August 29, 2012. activates signals within endothelial cells that allow the opening of narrow vascular passageways between endothelial cells or This work was supported by Grant 2011K000737 from the Korea Biotechnology Research and Development Group of Next-Generation Growth Engine Project of through an individual endothelial cell for transendothelial mi- the Ministry of Education, Science and Technology, Republic of Korea, and by a grant gration (34–39). When endothelial cell adhesion molecule sig- from Korea Healthcare Technology Research and Development Project, Ministry of Health and Welfare, Republic of Korea (Project A040004 to C.-G.P.). naling is inhibited, leukocytes bind to the endothelium, but transendothelial migration does not occur (40), and leukocytes are Address correspondence and reprint requests to Prof. Junho Chung or Prof. Chung- Gyu Park, Department of Biochemistry and Molecular Biology, Seoul National Uni- often released from the endothelium to continue in the blood flow versity, Seoul, 110-799, Korea (J.C.) or Department of Microbiology and Immunol- (41, 42). ogy, Seoul National University, Seoul 110-799, Korea (C.-G.P.). E-mail addresses: [email protected] and [email protected] (C.-G.P.) In this study, we developed a chimeric rabbit/human VCAM-1 Ab, specific to Ig-like domain 6, using phage display technology. Abbreviations used in this article: HAEC, human aortic endothelial cell; hTNF-a, human TNF-a; hVCAM-1, human VCAM-1; mTNF-a, mouse TNF-a; mVCAM-1, This Ab, VCAM-1 domain 6 (VCAM-1-D6), inhibits the trans- mouse VCAM-1; MVEC, mouse vascular endothelial cell; rhVCAM-1, recombinant endothelial migration of leukocytes without hindering the leuko- human VCAM-1; rhVCAM-1–Fc, recombinant human VCAM-1 Fc fusion protein; rmVCAM-1, recombinant mouse VCAM-1; rmVCAM-1–Fc, recombinant mouse cyte adhesion process on endothelial cells in vitro. In a mouse VCAM-1 Fc fusion protein; SDF-1a, stromal cell-derived factor-1a; STZ, strepto- islet allograft model, this Ab successfully inhibits transmigration of zotocin; TTBS, 10 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.05% (v/v) Tween 20; leukocytes to the graft site and blocks rejection of grafted pan- VCAM-1-D6, VCAM-1 domain 6. creatic b cell islets. Our results suggest that the VCAM-1-D6 Ab Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 may block VCAM-1–mediated inflammation. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103803 2 ROLE OF THE SIXTH Ig-LIKE DOMAIN OF VCAM-1 Materials and Methods Immunocytochemistry Construction of the Ab library and selection of binders Immunocytochemistry was performed as described previously (50). In Four New Zealand white rabbits were immunized and boosted four times brief, coverslips were incubated with 1 mg/ml poly-L-lysine for 1 h; then with recombinant hVCAM-1 (rhVCAM-1; R&D Systems, Minneapolis, HUVECs, HAECs, and MVECs were grown on coverslips in the absence MN) with the approval of the Institutional Animal Care and Use Com- or presence of hTNF-a or mTNF-a at the concentration of 20 ng/ml. After mittee of the Seoul National University Hospital. After the final booster rinsing with 1X PBS two times, the cells were fixed with 3.7% (w/v) injection, total RNA was prepared from the spleen and the bone marrow. paraformaldehyde for 30 min at 37˚C. After washing with 1X PBS and cDNA was synthesized using SuperScript First-Strand cDNA synthesis blocking with the 1X PBS containing 5% (w/v) BSA and 0.1% Triton X- system (Invitrogen, Carlsbad, CA) according to manufacturer’s instruc- 100 for 4 h at 4˚C, the cells were incubated overnight at 4˚C with 10 mg/ml tions. A phage-displayed rabbit/human chimeric Fab Ab library was con- VCAM-1-D6 Fab, control Fab, or the mouse anti–hVCAM-1 Ab 51-10C9. structed using pComb3X phagemid vector system as described previously The cells were washed five times with 1X PBS containing 0.05% Triton X- (43). After the library construction, Fab clones were selected from the 100 and then incubated in the same buffer for 1 h with FITC-labeled goat library through six rounds of biopanning as described previously (2). For anti-human Fab or Alexa Fluor 488-labeled goat anti-mouse IgG Ab each round of biopanning, 2.5 mg recombinant mVCAM-1 Fc fusion (1:100), and 2 mg/ml Hoechst (Invitrogen) for 1 h to visualize VCAM-1 protein (rmVCAM-1–Fc; R&D Systems)-coated magnetic beads (Dyna- and the nucleus, respectively.
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