Patches in High Endothelial Venules of Peyer's P-Selectin in Leukocyte Rolling and Adhesion Integrins, and 7Β the Roles of L-Se

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

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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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ␤ 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* ␤ Lymphocyte trafficking into Peyer’s patches requires 7 integrins and L-selectin. Here, we use intravital microscopy to examine ␤ ؊/؊ leukocyte rolling and adhesion in Peyer’s patch high endothelial venules (HEV) of wild-type, L-selectin-deficient (L ), 7 ؊/؊ ␤ ؊/؊ ␤ integrin-deficient ( 7 ), and 7/L mice. Although the leukocyte rolling flux fraction was reduced by 70%, Peyer’s patches ؊/؊ ␤ ؊/؊ in L mice were of normal size and cellularity. In 7 mice, the rolling flux fraction was normal, but the number of adherent ؊ ؊ ؊ ؊ / ␤ / Downloaded from leukocytes in HEV was greatly reduced. The median leukocyte rolling velocity was reduced in L mice and increased in 7 ؊/؊ ␤ ␤ 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 ؊/؊ ␤ 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/ ␤ 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 2ϩ 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 (LϪ/Ϫ) 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 LϪ/Ϫ 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 ␣ ␤ , 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- ␣ 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] ␤ ␤ Ϫ/Ϫ Ϫ Ϫ vivo (4). Recently, 7 integrin-deficient ( 7 ) mice have been 3 Abbreviations used in this work: HEV, high endothelial venule; L / , L-selectin ␣ ␤ ␤ Ϫ/Ϫ ␤ ␤ Ϫ/Ϫ ␤ 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

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, ␤ Ϫ/Ϫ 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; ␤ Ϫ/Ϫ Ͼ wild-type and 7 mice was inhibited by 90% with an L- Strobex 236, Chadwick Helmuth, Mountain View, CA) epifluorescence ␤ selectin mAb (5). Here, we examine 7 integrin/L-selectin double illumination (filter block Zeiss 9) with a saline immersion objective (SW mutant (␤ /LϪ/Ϫ) 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-␮l 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. Louis, MO), and analyzed for leukocyte concentration (expressed as Materials and Methods number of leukocytes per microliter of whole blood). Additional blood Animals samples were taken, using the above procedure, before and after adminis- ␤ tration of mAb and at the termination of the experiment. Mice deficient for L-selectin (3) or 7 integrin (5) expression were generated ␤ as described previously. Mice deficient for both L-selectin and 7 integrin Ϫ/Ϫ ␤ Ϫ/Ϫ Paraffin section histology were generated by breeding homozygous L mice and homozygous 7 mice and testing the progeny for both mutated genes by Southern blotting. The Two Peyer’s patches per mouse (one being the observed Peyer’s patch) ␤ absence of cell surface expression of L-selectin and 7 integrins was confirmed were excised from the small intestine and fixed in 4% (w/w) paraformal- Downloaded from 4,5 ␤ Ϫ/Ϫ using two-color immunofluorescence flow cytometry . 7 mice were gen- dehyde (Sigma) for at least 4 h but not Ͼ24 h. The tissues were then Ϫ Ϫ erated on a C57BL/6 background, and L / mice were at least a fifth gener- dehydrated in increasing concentrations of ethanol (75% (v/v), 95% (v/v), ation backcross onto a C57BL/6 background. Control experiments were per- and absolute), cleared in xylene, and soaked in warm (56°C) liquid paraffin formed in age-matched C57BL/6 mice bred at the University of Virginia for several hours to allow saturation. The tissues were embedded by cool- (breeders purchased from Hilltop Farms, Scottdale, PA). All experiments were ing until the paraffin hardened. Ten-micron serial transverse sections were performed on healthy mice that were at least 8 wk of age. All mice were cut (Leitz microtome, Wetzlar, Germany), floated out in a water bath onto housed in a conventional facility. All animal experiments were approved by poly-L-lysine (Sigma)-coated slides, and dried at room temperature. Sec- the institutional animal care and use committee. tions were cleared in xylene, rehydrated with decreasing concentrations of http://www.jimmunol.org/ ethanol (absolute, 95% (v/v), 70% (v/v), and distilled water), stained with Monoclonal Abs hematoxylin and eosin (Sigma), cleared in absolute ethanol and xylene, and The P-selectin mAb RB40.34 (rat IgG1, 30 ␮g/mouse) was a gift from Dr. mounted using Permount (Fisher Scientific, Pittsburgh, PA). Dietmar Vestweber at the University of Munster (Munster, Germany). This Ab blocks P-selectin-dependent adhesion in vitro (8), P-selectin-dependent In vivo labeling of P-selectin leukocyte rolling in vivo (9), and P-selectin-dependent leukocyte recruit- Initial attempts at staining Peyer’s patch HEV with indirect immunofluo- ␮ ment in vivo (8). The L-selectin mAb MEL-14 (rat IgG2a; 100 g/mouse) rescence of peroxidase-conjugated secondary Abs yielded unsatisfactory was purified from hybridoma supernatant (American Type Culture Collec- results, most likely because of low expression of P-selectin (data not tion, Manassas, VA). This Ab blocks L-selectin-dependent lymphocyte shown). Therefore, we developed a new staining technique employing flu- homing to peripheral lymph nodes (10) and L-selectin-dependent leukocyte orescent beads. Recombinant protein G (Sigma) was covalently coupled to by guest on September 25, 2021 ␣ rolling in vivo (9, 11). The mAb PS/2 specific for the 4 integrin chain (rat fluorescent-carboxylated (2-␮m diameter) YG microspheres (Polysciences, ␮ IgG2b; 100 g/mouse) was also purified from hybridoma supernatant Warrington, PA) using a carbodiimide coupling kit according to the man- ␣ ␤ (American Type Culture Collection). This Ab blocks 4 7-dependent bind- ufacturer’s directions (Polysciences). Beads were stored in the storage ing of lymphocytes to recombinant MadCAM-1 in vitro (7) and lympho- buffer provided and used within 1 wk. Thirty minutes before observation of cyte homing to Peyer’s patches in vivo (12). the Peyer’s patch, 200 ␮l of protein G-coupled beads were mixed with 200 ␮ Animal preparation and epifluorescence intravital microscopy g of either a nonbinding rat IgG (Pierce, Rockford, IL) or the P-selectin mAb RB40.34. The mixture was allowed to incubate for 30 min before the Mice were premedicated with an i.p. injection of a mixture of 30 mg/kg beads were washed in isotonic saline with 1% BSA three times. The beads sodium pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL) were resuspended in isotonic saline with 1% BSA and vortexed to break up and 0.1 mg/kg atropine (Elkins-Sinn, Cherry Hill, NJ) in isotonic saline. any aggregates. Animals were injected with three sequential 50-␮l boluses Mice were then anesthetized with an i.p. injection of 100 mg/kg ketamine of the rat IgG-coupled beads over 5 min followed by a 10-min resting hydrochloride (Ketalar, Parke-Davis, Detroit, MI) in isotonic saline. The period to allow for removal of any circulating beads. Subsequently, the trachea, carotid artery, and jugular vein were cannulated, and mice were RB40.34-coupled beads were injected in the same manner, and any bound thermocontrolled at 37°C using a small animal heating pad (model 50- beads were observed by intravital fluorescence microscopy. 7503, Harvard, South Natik, MA). Blood pressure was monitored continuously (model BPMT-2, Stemtech, Menomonee Falls, WI). Determination of P-selectin ligand activity on Peyer’s patch A Peyer’s patch was prepared for intravital microscopy as described lymphocytes previously (4, 5). Briefly, a 1.5-cm incision was made along the linea alba to open the peritoneal cavity and expose the cecum and small intestine. The Indirect immunofluorescence flow cytometry was used to examine binding mouse was then turned on its side, and the intestines were pushed out of the of a recombinant human P-selectin-IgG chimera (gift from S. R. Watson, peritoneal cavity by gently pressing on the back. The intestines were su- Genentech, South San Francisco, CA) to suspensions of Peyer’s patch lym- perfused with thermocontrolled (37°C) bicarbonate-buffered saline as pre- phocytes. Human P-selectin can bind to mouse PSGL-1 (14). Peyer’s ␤ Ϫ/Ϫ Ϫ/Ϫ ␤ Ϫ/Ϫ viously described (13). To immobilize the small intestine and allow for patches were isolated from wild-type, 7 ,L , and 7/L mice and viewing of the Peyer’s patch microvasculature, a plastic coverslip (Baxter, suspended in PBS, pH 7.4, with 2 mM CaCl2, 0.02% sodium azide, and 5% Deerfield, IL) cut to approximately 1 cm2 was overlaid with two parallel goat serum. Lymphocytes from Peyer’s patches were then released by gen- strips of clear silicone, high vacuum grease (Dow Corning, Midland, MI). tle syringe aspiration. A multivalent P-selectin complex (15) was pre- This coverslip was then laid over the preparation, with the small intestine formed by mixing recombinant P-selectin-IgG chimera and FITC-conju- Ј lying parallel between the two rows of grease and the Peyer’s patch flat- gated goat F(ab )2 anti-human IgG (Fc-specific) Ab (Caltag, Burlingame, tened against the coverslip to allow better microscopic viewing. To visu- CA) in a 1/2 (w/w) ratio followed by incubation at 4°C for 30 min. Aliquots of the P-selectin-IgG FITC complex (2 ␮g P-selectin-IgG/4 ␮g goat anti- human IgG FITC) were added to 1 ϫ 106 cells and incubated for 20 min 4 D. A. Steeber, M. L. K. Tang, X.-Q. Zhang, N. Wagner, T. F. Tedder. Lymphocyte on ice. Lymphocytes in the Peyer’s patch preparation were identified by migration across high endothelial venules of mouse Peyer’s patches requires L-se- coincubation with 1 ␮g each of phycoerythrin-conjugated mAb against the ␤ lectin or 7 integrin expression. Submitted for publication. CD3 complex on T cells (clone 17A2, rat anti-mouse IgG2b) and CD45R/ 5 N. Wagner, J. Lohler, T. F. Tedder, K. Rajewsky, W. Mu¨ller, D. A. Steeber. L- B220 on B cells (clone RA3-6B2; rat anti-mouse IgG2a; PharMingen, San ␤ selectin and 7 integrin synergistically mediate lymphocyte migration to mesenteric Diego, CA). Selectin-specific adhesion was confirmed by inhibition of P- lymph nodes. Submitted for publication. selectin complex binding to Peyer’s patch lymphocytes by treating cell The Journal of Immunology 2451

Table I. Systemic blood leukocyte counts and differential counts a

Differential Count

Systemic Leukocyte % % Genotype No. Count (per ␮l) Neutrophils Mononuclear

Wild-type 6 3239 Ϯ 503b 37 Ϯ 562Ϯ 5 LϪ/Ϫ 5 6254 Ϯ 800 38 Ϯ 661Ϯ 6 ␤ Ϫ/Ϫ Ϯ Ϯ Ϯ 7 5 7094 1147 35 5645 ␤ Ϫ/Ϫ Ϯ Ϯ Ϯ 7/L 7 5262 541 29 5715 a Percent mononuclear includes both lymphocytes and monocytes. b Significantly lower than systemic counts in all other genotypes (p Ͻ 0.05). suspensions with 5 mM EDTA. Cell suspensions were analyzed on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using CellQuest software (version 1.2). Data analysis Microvessel diameters were measured using a digital image processing Downloaded from system (16). Centerline blood flow velocity was determined after i.v. injection of 2-␮m diameter fluorescent YG microspheres (Polysciences, Warrington, PA) by measuring frame-to-frame displacement (three microspheres per venule). Average blood flow velocity was determined by di- viding the measured centerline velocity by a factor of 2. The rolling leukocyte flux fraction was determined as the number of rolling leukocytes expressed as a percentage of all leukocytes passing through the venule per unit time. In Peyer’s patch HEV, the number of rolling leukocytes was http://www.jimmunol.org/ consistently higher (by ϳ60%) than the product of the flow rate and the systemic leukocyte concentration, a parameter used in previous studies (9, 11) as the denominator for the rolling flux fraction. An increased concentration of leukocytes in microvessels can occur as a consequence of multiple flow partition at bifurcations in the microvascular network (17, 18), leading to leukocyte accumulation in the most distal branches of the arteriolar tree. Consequently, venules fed by these branches also carry blood containing a higher leukocyte concentration. Therefore, the flux fraction in wild-type mice was set at 100%, and the flux fractions in the other genotypes were adjusted accordingly. The leukocyte rolling flux fraction was also corrected for variations in centerline velocity and venular diameter as by guest on September 25, 2021 described previously (9). Individual leukocyte rolling velocities were measured from video recordings by analyzing 5 to 15 leukocytes/venule and measuring the time necessary to travel a fixed distance (ϳ50–80 ␮m) using a digital image-processing system (16). Statistical analysis All statistical comparisons were conducted using an analysis of variance with a post-hoc Student-Newman-Keuls multiple comparison procedure. FIGURE 1. Low magnification histology of Peyer’s patches from wild- SPSS software (SPSS, Chicago, IL) was used for all statistical analyses. type, LϪ/Ϫ, and ␤ /LϪ/Ϫ mice. Peyer’s patches were excised from the small Statistical significance was set at p Ͻ 0.05. 7 intestine, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. A, A representative Peyer’s patch Results from a wild-type mouse showing several follicles, well populated with General observations lymphocytes, protruding both into the lumen of the small intestine and into All mice used in this work appeared healthy and of normal size and the peritoneum. B, As reported previously (3), a Peyer’s patch from an Ϫ/Ϫ weight for their ages. During the intubation surgery, no enlarged L mouse looks similar to that from a wild-type mouse, including similar follicular density and cellularity. C, In contrast, a Peyer’s patch from a cervical lymph nodes were observed in any mice. Similar to what ␤ Ϫ/Ϫ 7/L mouse is extremely small, protrudes neither luminally nor peri- has been previously reported, the systemic leukocyte counts in toneally, and is noticeably hypocellular. ϫ4 objective. Bar ϭ 500 ␮m. LϪ/Ϫ mice (3) were slightly higher than those in wild-type mice (Table I). Although previous work has not identified an increase in ␤ Ϫ/Ϫ circulating counts in 7 mice (5), we found that circulating counts in these mice were also higher than those in wild-type mice Ϫ Ϫ using a dissecting microscope on the wall of the small intestine in (Table I). Similarly, the circulating leukocyte counts in ␤ /L / 7 ␤ /LϪ/Ϫ mice were small and hypocellular (Fig. 1C) compared mice were elevated (Table I). The percentages of granulocytes and 7 mononuclear leukocytes were similar in each genotype (Table I). with wild-type Peyer’s patches (Fig. 1A). The number of Peyer’s patches in LϪ/Ϫ mice was not different from that in wild-type Peyer’s patch histology mice, and they were similar in size and cellularity to those in their We first examined the morphology of Peyer’s patches from wild- wild-type counterparts (Fig. 1B). Concomitant with reduced Pey- Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ ␤ ␤ er’s patch size in 7 mice, the number of HEV observed type, L , and 7/L mice (Fig. 1). The number of Peyer’s patches visible along the small intestine was reduced from ϳ8to through intravital observation was reduced compared with that in ϳ ␤ Ϫ/Ϫ ␤ Ϫ/Ϫ wild-type mice (data not shown) and the average diameter of those 10 in wild-type mice to 1 to 2 in both the 7 and 7/L mice (data not shown). The Peyer’s patches that could be located seen was slightly smaller (Table II). 2452 LEUKOCYTE ROLLING IN PEYER’S PATCH VENULES

Table II. Hemodynamic parameters of observed HEV Leukocyte rolling in vivo is known to be affected by geometric and hemodynamic conditions such as venular surface-to-volume ratio and Venules Diameter Centerline Velocity Wall Shear Rate average blood flow velocity (9, 22). Therefore, we measured several Ϫ Genotype No. (n) (␮m) (␮m/s) (s 1) parameters that could affect leukocyte rolling in these experiments Wild-type 6 25 19.2 Ϯ 0.9 1907 Ϯ 103 1065 Ϯ 65 (Table II). Fluorescent beads were used to measure the centerline LϪ/Ϫ 5 27 22.1 Ϯ 1.5 2110 Ϯ 188 1020 Ϯ 79 blood flow velocity in each observed venule. The data from all ge- ␤ Ϫ/Ϫ Ϯ a Ϯ Ϯ 7 5 34 17.9 0.8 1918 146 1162 95 notypes were stratified for venules with similar average wall shear ␤ Ϫ/Ϫ Ϯ a Ϯ Ϯ 7/L 7 33 16.0 0.6 2096 197 1378 122 rates. a Significantly smaller diameter than in wild-type and LϪ/Ϫ mice (p Ͻ 0.05). Leukocyte rolling velocities Adhesion molecules involved in leukocyte rolling may change the Leukocyte rolling in Peyer’s patch HEV total number of rolling leukocytes, their rolling velocity, or both We used blocking mAb to determine which adhesion molecules were parameters (11). Therefore, we also analyzed the velocities of roll- mediating the leukocyte rolling in each genotype (Fig. 2). As shown ing leukocytes in each genotype (Fig. 3). In HEV of wild-type previously (4, 5), leukocyte rolling in Peyer’s patches from wild-type mice, leukocyte rolling velocities ranged from Ͻ10 to Ͼ100 ␮m/s mice was predominantly mediated by L-selectin, since the L-selectin (Fig. 3A) with a median velocity of 43 ␮m/s (Fig. 3E and Table function-blocking mAb MEL-14 blocked most of the observed rolling III). In LϪ/Ϫ mice, the population of faster rolling leukocytes was (Fig. 2A). However, injection of the mAb RB40.34 significantly re- missing (Fig. 3B), which significantly reduced ( p Ͻ 0.05 vs wild- Downloaded from duced the rolling flux fraction by about 29% in wild-type mice (Fig. type mice) the median rolling velocity to 30 ␮m/s (Fig. 3E and 2A). Interestingly, the percentage of granulocytes in the wild-type Table III). Conversely, slow rolling leukocytes were missing in ␤ Ϫ/Ϫ Ͻ mice investigated here was about 29 to 38% of the total circulating 7 mice (Fig. 3C), which significantly increased ( p 0.05 vs leukocyte count (Table I), suggesting that many of the cells rolling wild-type and LϪ/Ϫ mice) the median velocity to 85 ␮m/s (Fig. 3E using P-selectin may be granulocytes. Neutrophils are known to ex- and Table III). Taken together, these findings indicate that the press a constitutively functional ligand for P-selectin called PSGL-1 leukocyte rolling velocity histogram in wild-type mice is com- ␤ http://www.jimmunol.org/ (19). However, effector T cells also express functional PSGL-1 (20, posed of two distinct populations: slower 7 integrin-dependent 21) and may be rolling via P-selectin in Peyer’s patches. rolling and more rapid L-selectin-dependent rolling. It should be Consistent with the mAb blocking data, the leukocyte rolling noted, however, that the rolling flux fraction in LϪ/Ϫ mice is much flux fraction in LϪ/Ϫ mice was only about one-third of that found lower (Fig. 2). This suggests that L-selectin may be important in in wild-type mice (Fig. 2B). Residual rolling in these mice was capturing leukocytes from the flowing blood in Peyer’s patches in largely mediated by P-selectin (the mAb RB40.34 to P-selectin addition to mediating rolling. It therefore appears that efficient leu- ␤ blocked almost all leukocyte rolling), but the rolling flux was un- kocyte attachment requires L-selectin even in the presence of 7 ␣ ␤ Ϫ/Ϫ affected by the mAb PS/2 to the 4 integrin chain (Fig. 2B). Sim- integrin. In 7/L mice (Fig. 3D), only a residual population of

ilar to wild-type mice and consistent with previous results (5), rolling leukocytes was detected (Fig. 2), which rolled at an inter- by guest on September 25, 2021 ␤ Ϫ/Ϫ ␮ leukocyte rolling in 7 mice was mediated by L-selectin (Fig. mediate velocity with a median of 56 m/s (Fig. 3E and Table III), ␤ Ϫ/Ϫ 2C). P-selectin blockade with the mAb RB40.34 in 7 mice led not significantly different from the velocity distribution in wild- ϳ ␤ Ϫ/Ϫ to a slight ( 10%), but not significant, reduction in the rolling flux type mice. Rolling in 7/L mice was mediated by P-selectin ␤ Ϫ/Ϫ fraction (Fig. 2C). 7/L mice showed a low rolling flux fraction (Fig. 2D). The observed velocity distribution is similar to that seen similar to that found in LϪ/Ϫ mice. This residual leukocyte rolling in venules of the mouse cremaster muscle where rolling is domi- was totally P-selectin dependent (Fig. 2D). nated by P-selectin (23).

FIGURE 2. Leukocyte rolling flux fractions in Ϫ/Ϫ ␤ Ϫ/Ϫ Peyer’s patch HEV from wild-type, L , 7 , and ␤ Ϫ/Ϫ 7/L mice. The number of leukocytes rolling through Peyer’s patch HEV was counted, and the rolling flux fraction was calculated in wild-type (A), LϪ/Ϫ ␤ Ϫ/Ϫ ␤ Ϫ/Ϫ (B), 7 (C), and 7/L (D) mice. mAb to P- ␣ selectin (RB40.34), L-selectin (MEL-14), or 4 integrin (PS/2) were injected i.v. * indicates significantly lower than no mAb in same genotype (p Ͻ 0.05). # indicates significantly lower than wild-type with no mAb (p Ͻ 0.05). ¶ indicates significantly lower than both no mAb and plus RB40.34 in the same genotype (p Ͻ 0.05). ‡ indicates significantly lower than LϪ/Ϫ mice with RB40.34 (p Ͻ 0.05). Data shown are the mean Ϯ SEM of 2 to 8 mice and 8 to 34 venules. The Journal of Immunology 2453

FIGURE 3. Leukocyte rolling velocity histograms and cumulative distributions. Five to ten leukocytes in each observed HEV were picked at random, and the time necessary to travel a ﬁxed distance (ϳ50–80 ␮m) was determined. A, Wild- type mice exhibited a bimodal leukocyte rolling velocity distribution with peaks around 30 and 80 ␮m/s. B,LϪ/Ϫ mice had Downloaded from a rolling velocity distribution with one peak around 30 ␮m/s containing only slower ␤ Ϫ/Ϫ rolling leukocytes. C,In 7 mice, the rolling velocity distribution was shifted to higher velocities (peak at ϳ75 ␮m/s). D, ␤ Ϫ/Ϫ 7/L mice had a rolling velocity distri-

bution similar to that seen in wild-type http://www.jimmunol.org/ mice. E, The cumulative distribution of leukocyte rolling velocities for each genotype shows the median velocity (line at 50%). The number of leukocytes analyzed is shown (n) for each genotype. Statistical analysis is shown in Table III. by guest on September 25, 2021

The velocities of the residual rolling leukocytes was also exam- to the lack of a population of leukocytes that rolls more slowly ␤ Ϫ/Ϫ ined after several blocking mAb treatments. Functional blockade using P-selectin. In 7 mice, addition of RB40.34 did not sig- of P-selectin with the mAb RB40.34 in wild-type mice caused a nificantly alter the rolling flux fraction (Fig. 2B) and also did not significant increase ( p Ͻ 0.05) in the median leukocyte rolling affect the median rolling velocity (Table III). As shown above, velocity from about 43 to about 96 ␮m/s (Table III). Since block- P-selectin mediated almost all the rolling in LϪ/Ϫ mice (Fig. 2C). ing P-selectin in wild-type mice also causes an ϳ30% reduction in Interestingly, the median velocity of the few remaining rolling the rolling flux fraction (Fig. 2A), the velocity increase may be due leukocytes in these mice was very low (Table III).

P-selectin expression in Peyer’s patch HEV Table III. Median leukocyte rolling velocities by genotype and mAb treatment mAb blocking data (Fig. 2) and rolling velocity data (Fig. 3) showed that P-selectin contributes to leukocyte rolling in Peyer’s patches. To Leukocytes Median Rolling Velocity directly visualize P-selectin expression, we attempted to use standard ␮ Genotype No. (n) ( m/s) immunoperoxidase staining using biotinylated secondary Ab and Wild type 6 146 43 streptavidin-peroxidase conjugate as described previously (24). This Wild type ϩ RB40.34 2 32 96a method was not sensitive enough to detect P-selectin in Peyer’s patch Ϫ/Ϫ b L 58530 HEV. To increase the sensitivity, we developed a bead-based assay. LϪ/Ϫ ϩ RB40.34 2 20 20b ␤ Ϫ/Ϫ a Fluorescent beads were coated with the P-selectin mAb RB40.34 and 7 5 165 76 ␤ Ϫ/Ϫ ϩ a injected i.v. (Fig. 4). Injection of control beads coated with an irrel- 7 RB40.34 2 49 84 ␤ Ϫ/Ϫ 7/L 78056 evant rat IgG Ab showed low, but detectable, background binding to a Signiﬁcantly higher median velocity than wild-type and LϪ/Ϫ mice (p Ͻ 0.05). the Peyer’s patch venular endothelium of wild-type mice (Fig. 4A), b ␤ Ϫ/Ϫ Ͻ Signiﬁcantly lower median velocity than wild-type and 7 mice (p 0.05). whereas the RB40.34-coated beads bound in much greater quantities 2454 LEUKOCYTE ROLLING IN PEYER’S PATCH VENULES

tin (24). RB40.34-coated beads bound avidly to the venular (Fig. 4D), but not arteriolar (data not shown), endothelium in the mouse cre- Ϫ/Ϫ ␤ Ϫ/Ϫ master muscle. HEV in Peyer’s patches of L and 7/L mice also bound RB40.34-coated beads (data not shown), consistent with the observed P-selectin-dependent rolling in these vessels (Fig. 2). P-selectin expression is known to be induced by surgical trauma in several models of inﬂammation (9, 24–26). We injected both control and RB40.34-coated beads into intact mice before surgery. After wait- ing 20 min (the time necessary after an injection to clear any circulating beads), the Peyer’s patch was exteriorized and observed with intravital microscopy. Very few beads were bound to the endothelium (data not shown). In contrast, when the same number of beads was injected i.v. 5 to 10 min later (after exteriorization), extensive binding was observed. These data suggest that the P-selectin expression was increased as a consequence of the manipulation of the small intestine during intravital microscopic viewing.

Peyer’s patch lymphocytes express P-selectin binding activity Downloaded from Because we observed that P-selectin was involved in leukocyte rolling in all genotypes (Figs. 2 and 3) and was expressed on the endothelium of Peyer’s patch HEV (Fig. 4), we determined whether purified Pey- er’s patch lymphocytes from each genotype exhibited P-selectin ligand activity. A multivalent P-selectin complex was formed by cross- linking a P-selectin-IgG chimera with a FITC-conjugated secondary Ab (15) and was used to investigate P-selectin binding activity of http://www.jimmunol.org/ Ϫ/Ϫ ␤ Ϫ/Ϫ purified Peyer’s patch lymphocytes from wild-type, L , 7 , and ␤ Ϫ/Ϫ 7/L mice by flow cytometry (Fig. 5). Approximately 26% of wild-type Peyer’s patch lymphocytes bound multivalent P-selectin (Fig. 5A). A similar number (ϳ32%) of Peyer’s patch lymphocytes from LϪ/Ϫ mice also stained positively (Fig. 5C). In contrast, 45% of ␤ Ϫ/Ϫ lymphocytes from 7 mice (Fig. 5B) and 57% of lymphocytes ␤ Ϫ/Ϫ from 7/L mice (Fig. 5D) stained positively for P-selectin ligand

activity. Binding was selectin speciﬁc, as shown by complete inhibi- by guest on September 25, 2021 tion in the presence of 5 mM EDTA (dotted lines in Fig. 5). As a positive control, we used HL-60 cells, a myeloid precursor cell line known to bind to P-selectin (27, 28). All HL-60 cells stained positively with the P-selectin complex (data not shown).

Discussion We have analyzed leukocyte-endothelial interactions in mice deficient for two molecules deemed very important for lymphocyte recruitment ␤ to Peyer’s patches: L-selectin and 7 integrin. Using epifluorescence intravital microscopy of Peyer’s patches in these mice, we have de- FIGURE 4. P-selectin expression in Peyer’s patch HEV. Two-micron ␤ termined that in the absence of L-selectin and 7 integrin, endothelial fluorescent beads coupled to rat IgG (control mAb) or RB40.34 (P-selectin P-selectin can mediate a low level of leukocyte rolling. We show mAb) were injected i.v. and allowed to bind to the venular endothelium in expression of P-selectin on the endothelium of Peyer’s patch HEV the Peyer’s patch. Very few control beads bound to Peyer’s patch HEV (A), whereas the number of RB40.34-coated beads that bound to the endothe- after surgical manipulation and P-selectin binding activity of lympho- lium was approximately 10-fold greater (B). Binding was specific, since cytes isolated from Peyer’s patches. We hypothesize that although pretreatment of the mouse with an i.v. injection of RB40.34 prevented the P-selectin is probably not involved in recruitment of naive lympho- binding of RB40.34-coated beads to the endothelium (C). As a positive cytes to Peyer’s patches, this molecule may be very important for control, RB40.34-coated bead binding to the venular endothelium in the recruitment of effector T cells to sites of mucosal inflammation. acutely exposed mouse cremaster muscle is shown (D). Free flowing beads Although we observed P-selectin-dependent leukocyte rolling in all (double exposure due to the stroboscopic illumination) are shown by ar- Ϫ/Ϫ ␤ Ϫ/Ϫ mouse genotypes studied in this work (i.e., wild-type, L , 7 , rows. Bar ϭ 50 ␮m. ␤ Ϫ/Ϫ and 7/L ), P-selectin has never before been implicated in partic- ipating in the recruitment of lymphocytes to Peyer’s patches. In fact, no evidence exists that suggests that P-selectin is even expressed on (Fig. 4B). Binding was specific for P-selectin on the endothelium be- Peyer’s patch HEV. P-selectin has, however, been shown to be ex- cause i.v. pretreatment of the mouse with RB40.34 reduced the bind- pressed on HEV in inflamed as well as noninflamed palantine and ing of the RB40.34-coated beads to levels comparable to that of the adenoid tonsils (29, 30). Additionally, P-selectin expression has been control beads (Fig. 4C). As a positive control, the binding of detected in the small intestine using 125I-labeled RB40.34 (31), but RB40.34-coated beads to the venular endothelium of the acutely ex- was not localized to any specific region (e.g., Peyer’s patches or lam- teriorized mouse cremaster muscle was investigated. Under these con- ina propria). By using fluorescent beads coupled to the P-selectin ditions, cremaster muscle venules, but not arterioles, express P-selec- mAb RB40.34, we show that P-selectin is indeed expressed on the The Journal of Immunology 2455

FIGURE 5. P-selectin ligand activity on Peyer’s patch lymphocytes. Suspensions of Peyer’s patch lymphocytes identified by expression of CD3 (T cell) or CD45R (B cell) Ϫ/Ϫ ␤ Ϫ/Ϫ from wild-type (A), L (B), 7 (C), and ␤ Ϫ/Ϫ 7/L (D) mice were incubated with a multivalent P-selectin complex. Immunofluores- cence flow cytometry was used to detect P- selectin complex binding to lymphocytes. Compared with incubation of cell suspensions with secondary Ab alone (thin solid lines), positive staining with P-selectin complex (bold solid lines) was detected on lymphocytes from each mouse genotype. The percentage of positive staining in wild-type mice (26%) was similar to that in LϪ/Ϫ mice (32%). A greater level of P-selectin ligand activity was found ␤ Ϫ/Ϫ ␤ on lymphocytes from 7 (45%) and 7/ Ϫ Ϫ L / (57%) mice. Selectin-dependent binding Downloaded from was demonstrated by inhibition of P-selectin complex binding by EDTA (5 mM; dotted lines). Data show pooled lymphocyte samples from two to five mice. http://www.jimmunol.org/ vascular endothelium of Peyer’s patch HEV under the conditions phenotypic changes. We were interested in observing leukocyte- studied. Our data represent the first evidence for P-selectin expression endothelial interactions under relatively unperturbed conditions, so in Peyer’s patch HEV. More importantly, we also show that P-selectin isolated, labeled lymphocytes were not used in this study. Never- is functional in these vessels and can mediate rolling after minimal theless, we found that a significant number of isolated Peyer’s trauma (exteriorization). P-selectin expression is known to occur in patch lymphocytes from all genotypes express P-selectin ligand response to mediators such as histamine (26, 32) or during inflam- activity. Even though P-selectin expression in Peyer’s patch HEV mation in response to cytokines such as TNF-␣ and IL-1 (33, 34). is below the detection limit of our current methods under in situ Since RB40.34-coated beads did not bind to the vascular endothelium conditions, significant numbers of T and B lymphocytes in the by guest on September 25, 2021 in Peyer’s patches before surgery was initiated, we conclude that this Peyer’s patch can bind to this molecule (26–57%). Naive lympho- expression is due to up-regulation of surface expression of this mol- cytes may enter a Peyer’s patch through the use of L-selectin and ␤ ecule after surgical manipulation of the small intestine. Therefore, 7 integrin and may acquire P-selectin binding activity after dif- P-selectin may not be involved in the recruitment of lymphocytes to ferentiation into Ag-specific effector lymphocytes. These T lym- Peyer’s patches under in situ conditions, but may play a role in traf- phocytes would then be able to traffic to mucosal inflammatory ficking of effector T lymphocytes during trauma or inflammation. sites using P-selectin, similar to what has been observed for Th1 Many types of leukocytes express P-selectin ligand activity. cell recruitment to inflamed skin (42). The increased fraction of Neutrophils (19), monocytes (35), and eosinophils (36) express a P-selectin-binding lymphocytes found in Peyer’s patch prepara- ␤ Ϫ/Ϫ ␤ Ϫ/Ϫ constitutively functional P-selectin ligand (PSGL-1) that mediates tions from 7 mice and 7/L mice may be due to an en- at least 90% of the P-selectin binding activity during inflammation richment in the number of effector T lymphocytes (which can bind ␤ in vivo (37). Although all lymphocytes express PSGL-1 (38), only to P-selectin) in Peyer’s patches of mice lacking 7 integrin, since certain subsets, including both effector ␣␤ and ␥␦ T-cells (20) and fewer naive lymphocytes can enter this tissue when this molecule memory T cells (39), can bind to P-selectin. PSGL-1 functionality is absent. A similar enrichment in effector T lymphocytes has been is regulated by an inducible fucosyl transferase called FucT-VII observed in peripheral lymph nodes of LϪ/Ϫ mice, since few naive (40). Thus, for a T cell to bind to P-selectin, PSGL-1 must have the lymphocytes can traffic through this tissue in the absence of L- proper post-translational modifications (41). P-selectin binding to selectin (44). This interpretation is consistent with the recent ob- ␤ Ϫ/Ϫ 4 PSGL-1 appears to mediate Th1 cell recruitment into inflamed skin servation of a shift in lymphocyte subsets in 7/L mice . ␤ in vivo (42). Of the leukocyte subsets present in the circulation Our data support a role for 7 integrin in mediating leukocyte roll- under normal conditions, neutrophils, eosinophils, and effector T ing. This evidence comes from the shift in the rolling velocity distri- ␤ cells all can bind to and roll on P-selectin. We therefore suspect bution observed in the absence of 7 integrin. An increase in rolling ␣ ␤ ␤ that neutrophils, eosinophils, monocytes, and effector T cells are velocity when 4 7 integrin has been blocked (4) or when 7 integrin rolling through Peyer’s patch HEV using P-selectin expressed on is knocked out (5) has been reported. Our present analysis confirms the HEV endothelium after surgical manipulation. and expands these data to show that at least three molecules are in- In vivo differentiation of leukocyte subsets in Peyer’s patch volved in modulating leukocyte rolling velocity in Peyer’s patches: ␤ HEV is not achievable with current intravital microscopic tools, so L-selectin, 7 integrin, and P-selectin. The rolling velocity distribu- we are unable to determine which types of leukocytes are rolling tion in wild-type mice appears to be a superposition of three distri- via P-selectin in this model. Ex vivo labeling of isolated lympho- butions: a population of leukocytes that rolls rapidly (ϳ80 ␮m/s) ␤ Ϫ/Ϫ cytes would allow differentiation of lymphocytes from granulo- mainly via L-selectin (seen in the 7 mice), a population of leu- ϳ ␮ ␤ cytes, as shown previously (43). However, excessive handling and kocytes that rolls more slowly ( 30 m/s) mainly using 7 integrin manipulation of isolated cells may lead to partial activation and (seen in the LϪ/Ϫ mice), and a smaller population of leukocytes that 2456 LEUKOCYTE ROLLING IN PEYER’S PATCH VENULES rolls with an intermediate range of velocities via P-selectin (seen in 17. Ley, K., A. R. Pries, and P. Gaehtgens. 1988. Preferential distribution of leuko- ␤ Ϫ/Ϫ cytes in rat mesentery microvessel networks. Pflugers Arch. 412:93. the 7/L mice). These three distributions superimposed can rep- ␤ 18. Ley, K., J. U. Meyer, M. Intaglietta, and K. E. Arfors. 1989. Shunting of leuko- licate the wild-type distribution measured here. Pure 7 integrin-de- cytes in rabbit tenuissimus muscle. Am. J. Physiol. 256:H85. Ϫ Ϫ pendent leukocyte rolling can be seen in L / mice treated with a 19. Moore, K. L., K. D. Patel, R. E. Bruehl, F. Li, D. A. Johnson, H. S. Lichenstein, P-selectin mAb. In these mice, the residual rolling flux is only 5% of R. D. Cummings, D. F. Bainton, and R. P. McEver. 1995. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. J. Cell Biol. 128: that in wild-type mice, suggesting that L-selectin is important for ef- 661. ficient leukocyte capture in Peyer’s patch HEV. The median rolling 20. Diacovo, T. G., S. J. Roth, C. T. Morita, J. P. Rosat, M. B. Brenner, and Ϫ Ϫ ␣ ␤ ␥ ␦ velocity in L / mice treated with a blocking P-selectin mAb is on T. A. Springer. 1996. Interactions of human / and / T lymphocyte subsets ␮ in shear flow with E-selectin and P-selectin. J. Exp. Med. 183:1193. the order of 20 m/s. A small rolling flux fraction in conjugation with 21. Vachino, G., X. J. Chang, G. M. Veldman, R. Kumar, D. Sako, L. A. Fouser, ␤ a low rolling velocity suggests that 7 integrin mainly functions to M. C. Berndt, and D. A. Cumming. 1995. P-selectin glycoprotein ligand-1 is the strengthen rolling interactions (i.e., reduce the rolling velocity), but is major counter-receptor for P-selectin on stimulated T cells and is widely distrib- uted in non-functional form on many lymphocytic cells. J. Biol. Chem. 270: unable to efficiently mediate leukocyte capture and initiate rolling. 21966. In conclusion, we present evidence that P-selectin can be in- 22. Firrell, J. C., and H. H. Lipowsky. 1989. Leukocyte margination and deformation duced on Peyer’s patch venular endothelium but is probably not in mesenteric venules of rat. Am. J. Physiol. 256:H1667. 23. Jung, U., D. C. Bullard, A. L. Beaudet, T. F. Tedder, and K. Ley. 1996. Velocity involved in the recruitment of naive lymphocytes to Peyer’s differences between L-selectin and P-selectin dependent neutrophil rolling in patches. Our data showing P-selectin ligand activity in lympho- venules of the mouse cremaster muscle in vivo. Am. J. Physiol. 271:H2740. cytes isolated from Peyer’s patches suggests that effector lympho- 24. Jung, U., and K. Ley. 1997. Regulation of E-selectin, P-selectin, and intercellular adhesion molecule 1 expression in mouse cremaster muscle vasculature. Micro- cytes can bind to P-selectin and may use this molecule for recruit- circulation 4:311. ment to mucosal sites of inflammation. Under physiologic 25. Fiebig, E., K. Ley, and K. E. Arfors. 1991. Rapid leukocyte accumulation by Downloaded from conditions, L-selectin and ␤ integrins appear to account for all “spontaneous” rolling and adhesion in the exteriorized rabbit mesentery. Int. 7 J. Microcirc. Clin. Exp. 10:127. lymphocyte rolling and adhesion in Peyer’s patch HEV. 26. Kubes, P., and S. Kanwar. 1994. Histamine induces leukocyte rolling in post- capillary venules: a P-selectin-mediated event. J. Immunol. 152:3570. Acknowledgments 27. Moore, K. L., N. L. Stults, S. Diaz, D. F. Smith, R. D. Cummings, A. Varki, and R. P. McEver. 1992. 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