The Roles of L-Selectin, β7 Integrins, and P-Selectin in Leukocyte Rolling and Adhesion in High Endothelial Venules of Peyer's Patches This information is current as of September 25, 2021. Eric J. Kunkel, Carroll L. Ramos, Douglas A. Steeber, Werner Müller, Norbert Wagner, Thomas F. Tedder and Klaus Ley J Immunol 1998; 161:2449-2456; ; http://www.jimmunol.org/content/161/5/2449 Downloaded from References This article cites 44 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/161/5/2449.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on September 25, 2021 *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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. b The Roles of L-Selectin, 7 Integrins, and P-Selectin in Leukocyte Rolling and Adhesion in High Endothelial Venules of Peyer’s Patches1 Eric J. Kunkel,* Carroll L. Ramos,* Douglas A. Steeber,† Werner Mu¨ller,‡ Norbert Wagner,‡ Thomas F. Tedder,† and Klaus Ley2* b Lymphocyte trafficking into Peyer’s patches requires 7 integrins and L-selectin. Here, we use intravital microscopy to examine 2/2 b leukocyte rolling and adhesion in Peyer’s patch high endothelial venules (HEV) of wild-type, L-selectin-deficient (L ), 7 b 2/2 b 2/2 integrin-deficient ( 7 ), and 7/L mice. Although the leukocyte rolling flux fraction was reduced by 70%, Peyer’s patches 2/2 b 2/2 in L mice were of normal size and cellularity. In 7 mice, the rolling flux fraction was normal, but the number of adherent 2 2 2 2 / b / Downloaded from leukocytes in HEV was greatly reduced. The median leukocyte rolling velocity was reduced in L mice and increased in 7 b b 2/2 mice, suggesting that 7 integrins and L-selectin mediate rolling in Peyer’s patch HEV at different velocities. 7/L exhibited both a low rolling flux fraction and low adhesion and had severely reduced Peyer’s patch size and cellularity. The residual rolling in these mice was completely blocked by a P-selectin mAb. A significant P-selectin component was also detected in the other genotypes. Twenty-six percent of B and T lymphocytes isolated from Peyer’s patches of wild-type mice expressed functional b 2/2 ligands for P-selectin, and this fraction was increased to 57% in 7/L mice. Peyer’s patch HEV were found to express P-selectin under the conditions of intravital microscopy, but not in situ. Our data suggest a novel P-selectin dependent mechanism of http://www.jimmunol.org/ b lymphocyte homing to Peyer’s patches. In situ, 7 integrins and L-selectin account for all lymphocyte homing to Peyer’s patches, but P-selectin-dependent rolling, as induced by minimal trauma, may support trafficking of effector T lymphocytes to Peyer’s patches. The Journal of Immunology, 1998, 161: 2449–2456. eyer’s patches, one component of the gut-associated lym- response to activating substances, and subsequent emigration out phoid tissue, are located on the wall of the small intestine of the venule (reviewed in Ref. 1). and play an important role in the mucosal immune re- Several molecules have been implicated in mediating lympho- P 21 sponse (particularly an IgA response). Lymphocytes continuously cyte recruitment into Peyer’s patches. L-selectin is a Ca -depen- by guest on September 25, 2021 recirculate through Peyer’s patches to sample sequestered Ag and dent mammalian lectin expressed constitutively on granulocytes further differentiate into mucosal effector T and B lymphocytes. and monocytes as well as on most circulating lymphocytes, in- The first step in lymphocyte recirculation through Peyer’s patches cluding naive and certain effector T cells (2). Although L-selectin- is the movement of these cells from the Peyer’s patch high endo- deficient (L2/2) mice have morphologically normal Peyer’s thelial venules (HEV)3 into the surrounding lymphoid tissue. As in patches, they exhibit a significant reduction in short term homing many other lymphoid and nonlymphoid tissues, lymphocyte re- of exogenous lymphocytes to Peyer’s patches (3). Subsequent data cruitment into Peyer’s patches is thought to require several steps: have shown that this reduction in homing is most likely the result initial attachment to the endothelium from the blood (capture), of a deficit in leukocyte rolling when L-selectin function is rolling along the endothelium, firm adhesion to the endothelium in blocked, because lymphocytes treated with an L-selectin mAb and injected i.v. fail to roll in Peyer’s patch HEV (4), and functional blockade of L-selectin with a mAb in vivo reduces the number of rolling leukocytes in Peyer’s patch HEV by up to 90% (5). How- *Department of Biomedical Engineering, University of Virginia School of Medicine, ever, the normal size and cellularity of Peyer’s patches in L2/2 Charlottesville, VA 22908; †Department of Immunology, Duke University Medical Center, Durham, NC 27710; and ‡Institute for Genetics, University of Cologne, Co- mice (3) suggest that other important adhesion molecules can by- logne, Germany pass the requirement for L-selectin in lymphocyte recruitment to Received for publication February 2, 1998. Accepted for publication April 23, 1998. Peyer’s patches. In fact, the absence of L-selectin delays, but does The costs of publication of this article were defrayed in part by the payment of page not prevent, lymphocyte homing to Peyer’s patches (3). charges. This article must therefore be hereby marked advertisement in accordance The most important adhesion molecule in lymphocyte recruit- with 18 U.S.C. Section 1734 solely to indicate this fact. ment to Peyer’s patches is a b , an integrin expressed at low levels 1 This work was supported by National Institutes of Health Grants HL54136 and 4 7 HL58108 (to K.L.) and Grants HL50985, CA54464, and AI26872 (to T.F.T.). E.J.K. on naive T and B cells and at high levels on effector and memory is a predoctoral trainee on National Heart Lung and Blood Institute Grant T cells within the gut (6). This integrin can mediate rolling inter- T32HL07284 (to B. R. Duling). C.L.R. is supported by U.S. Public Health Service National Research Scientist Award Postdoctoral Fellowship HL09578-02. N.W. is actions in vitro independent of L-selectin (7). Treatment of lym- a supported by a grant from the Deutsche Forschungsgemeinschaft (WA 1127/1-1). phocytes with a mAb specific for the 4 chain of this integrin 2 Address correspondence and reprint requests to Dr. Klaus Ley, Department of Bio- results in a twofold increase in the rolling velocity of exogenous medical Engineering, University of Virginia School of Medicine, Box 377, Health lymphocytes passing through HEV of wild-type Peyer’s patches in Sciences Center, Charlottesville, VA 22908. E-mail address: kfl[email protected] b b 2/2 2 2 vivo (4). Recently, 7 integrin-deficient ( 7 ) mice have been 3 Abbreviations used in this work: HEV, high endothelial venule; L / , L-selectin a b b 2/2 b b 2/2 b generated (5) to address the role of 4 7 under physiologic con- deficient; 7 , 7 integrin deficient; 7/L , 7 integrin/L-selectin double knock- out. ditions. These mice have severely reduced Peyer’s patch cellularity Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 2450 LEUKOCYTE ROLLING IN PEYER’S PATCH VENULES and decreased lymphocyte adhesion to Peyer’s patch HEV. How- alize intravascular leukocytes by epifluorescent microscopy, each mouse ever, the number of leukocytes rolling in Peyer’s patch HEV of was given a 0.15-ml venous injection of 1 mg/ml acridine red (Chroma, b 2/2 mice was normal, although the leukocyte rolling velocity Stuttgart, Germany) 30 min before intravital observation. 7 Microscopic observations were made using an intravital microscope was increased. Lymphocyte rolling in Peyer’s patch HEV of both (Axioskop, Carl Zeiss, Thornwood, NY) modified for stroboscopic (60/s; b 2/2 . wild-type and 7 mice was inhibited by 90% with an L- Strobex 236, Chadwick Helmuth, Mountain View, CA) epifluorescence b selectin mAb (5). Here, we examine 7 integrin/L-selectin double illumination (filter block Zeiss 9) with a saline immersion objective (SW mutant (b /L2/2) mice that lack both adhesion molecules known 40, 0.75 numerical aperture). HEV were observed and recorded (S-VHS 7 recorder, Panasonic, Osaka, Japan) through a CCD camera system (model to be important in lymphocyte homing to Peyer’s patches. We VE-1000CD, Dage-MTI, Michigan City, IN). Throughout the experiment, hypothesize that in the absence of these two adhesion molecules, 10-ml blood samples were withdrawn at 45-min intervals from the carotid other, previously unknown, adhesion mechanisms in Peyer’s patch catheter, diluted 1/9 with Kimura (11 ml of 5% (w/w) toludine blue, 0.8 ml HEV may be unmasked. of 0.03% light green SF yellowish, 0.5 ml of saturated saponin, and 5 ml of 0.07 M phosphate buffer, pH 6.4; all reagents were obtained from Sigma, St.