Accommodate Leukocyte Passage Reorganization of These Junctions to Leukocyte Transmigration and Signals for Junctions Is Prefere

Intercellular Adhesion Molecule-1 Enrichment near Tricellular Endothelial Junctions Is Preferentially Associated with Leukocyte Transmigration and Signals for This information is current as Reorganization of These Junctions To of September 30, 2021. Accommodate Leukocyte Passage Ronen Sumagin and Ingrid H. Sarelius

J Immunol 2010; 184:5242-5252; Prepublished online 2 Downloaded from April 2010; doi: 10.4049/jimmunol.0903319 http://www.jimmunol.org/content/184/9/5242 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2010/04/02/jimmunol.090331 Material 9.DC1 References This article cites 44 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/184/9/5242.full#ref-list-1

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Intercellular Adhesion Molecule-1 Enrichment near Tricellular Endothelial Junctions Is Preferentially Associated with Leukocyte Transmigration and Signals for Reorganization of These Junctions To Accommodate Leukocyte Passage

Ronen Sumagin and Ingrid H. Sarelius

Leukocyte transmigration occurs at specific locations (portals) on the endothelium, but the nature of these portals is not clear. Using intravital confocal microscopy of anesthetized mouse cremaster muscle in combination with immunofluorescence labeling, we showed that in microvessels transmigration is mainly junctional and preferentially occurs at tricellular endothelial junctional Downloaded from regions. Our data suggest that enrichment of ICAM-1 near ∼43% of these junctions makes these locations preferred for transmigration by signaling the location of a nearby portal, as well as preparing the endothelial cell (EC) junctions, to accommodate leukocyte passage. Blockade of the extracellular domain of the ICAM-1 significantly reduced transmigration (by 68.8 6 4.5%) by reducing the ability of leukocytes to get to these portals. In contrast, blockade of the cytoplasmic tail of ICAM-1 reduced transmigration (by 71.1 6 7.0%) by disabling VE-cadherin rearrangement. Importantly, venular convergences are optimally equipped to support leukocyte transmigration. Differences in EC morphology result in a significantly higher number of tricellular http://www.jimmunol.org/ junctions in convergences compared with straight venular regions (20.7 6 1.2 versus 12.43 6 1.1/6000 mm2, respectively). Consequently, leukocyte adhesion and transmigration are significantly higher in convergences compared with straight regions (1.6- and 2.6-fold, respectively). Taken together, these data identify an important role for EC morphology and expression patterns of ICAM-1 in leukocyte transmigration. The Journal of Immunology, 2010, 184: 5242–5252.

eukocyte extravasation is a complex multistep process that where transendothelial migration (TEM) occurs. Leukocyte requires overlapping function of both leukocytes and crawling was first identified in isolated cell systems (9) and later by guest on September 30, 2021 L endothelial cells (ECs). Each step has been extensively confirmed in situ (10). Interactions between ICAM-1 and b2 in- studied and is elegantly summarized in recent reviews (1–4). To tegrins are essential for leukocyte crawling (9, 10). The finding effectively exit the blood vessels, leukocytes initiate rolling con- that a leukocyte’s inability to crawl decreases TEM suggests that tacts with endothelium (5), followed by firm adhesion (6). Leu- crawling allows leukocytes to get to specific locations that ac- kocyte rolling is primarily mediated by interactions between commodate TEM and further suggests that some regions of the members of the selectin family and the glycosylated molecules endothelium are better equipped to support leukocyte passage than expressed on the leukocyte surface (7). Leukocyte adhesion is others. Although transcellular transmigration has been described mainly mediated by interactions between members of cell adhe- in vitro (11) and in vivo (12), most agree that leukocyte TEM sion molecule family, such as ICAM-1, and their counterreceptors, primarily occurs via EC junctions (13–15). Numerous proteins the aLb2 integrin (LFA-1) and the aMb2 integrin (MAC-1) (8). associated with EC junctions, including VE-cadherin, CD31, Adhered leukocytes crawl along the EC surface toward locations CD99, and junctional adhesion molecules-1/2/3, are implicated in mediating leukocyte TEM (2, 16, 17). Interestingly, EC surface molecules ICAM-1 and VCAM-1 cluster at regions of leukocyte Department of Pharmacology and Physiology, University of Rochester, Rochester, TEM, suggesting a role for these molecules in TEM as well (11, NY 14642 18, 19). Moreover, these molecules are localized around leuko- Received for publication October 15, 2009. Accepted for publication February 19, 2010. cytes that use either junctional or transcellular routes for emigration (11). This work was supported by National Institutes of Health Grants RO1 HL75186 and PO1 HL18208. Tricellular junctions (intersection of the borders of three adjacent Address correspondence and reprint requests to Dr. Ingrid H. Sarelius, Department ECs) in particular have been suggested to serve as “portals” for of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elm- leukocyte TEM, because of the discontinuity of junctional pro- wood Avenue, Rochester, NY 14642. E-mail address: [email protected]. teins, such as occludin, cadherin, and ZO-1, at these sites (20). edu However, this does not explain transcellular TEM or the finding The online version of this article contains supplemental material. that some junctional molecules, such as VE-cadherin, can undergo Abbreviations used in this paper: bi, bicellular junction; bijunc, bicellular junction; EC, endothelial cell; fps, frames per second; junc, junctional region; non, nonjunc- rearrangement, creating de novo gaps to accommodate leukocyte tional region; nonjunc, nonjunctional region or transcellular route; ROI, region of TEM (21). This report (21) implies that the preferred locations for interest; TEM, transendothelial migration; tri, tricellular junction; trijunc, tricellular leukocyte TEM need not pre-exist but could be created upon re- junction; VE-cadherin, vascular endothelial cadherin. ceiving an appropriate signal. Whether the signal is originated in Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 ECs or the migrating leukocytes is also not clear. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903319 The Journal of Immunology 5243

Thus, the expression levels, distribution patterns, and the overlap Penetratin-ICAM-1 tail peptides of signaling induced by all these molecules can potentially create The peptides used consisted of 16 aa of penetratin (RQIKIWFQNR- a preferred location for leukocyte TEM. RMKWKK), followed by 13 C-terminal amino acids of mouse ICAM-1 Finally, the expression of EC junctional and surface molecules (QRKIRIYKLQQAQ); this peptide was modified for mouse tissue from varies greatly from tissue to tissue (22) and in different locations a previous report (18). The control peptide was an irrelevant sequence from within the vascular network (23), resulting in great variations in rat rodopsin (CKPMSNFRFGENH). In all cases, selected venules were locally perfused with the relevant peptide (100 mg/ml, 15 min) prior to tissue leukocyte-EC interactions (24). Undoubtedly, the variability of stimulation with fMLP. junctional and surface receptors also affects the mechanisms of leukocyte recruitment. Thus, we used an intact system to explore Analyses leukocyte crawling and TEM locations in blood perfused venules in Leukocyte interactions. A venular convergence was defined by measuring situ. Using intravital confocal microscopy combined with immu- 40 mm [an average length of venular EC (23)] in each direction from the nofluorescence labeling, we show that leukocytes, in their innate inner convergence point for each of the inflow vessels at the convergence environment, preferentially use tricellular junctional regions as (Fig. 2A, top panel). This 80-mm length was used to quantify leukocyte p portals for emigration. Moreover, we demonstrate that ICAM-1 adhesion (cells/80-mm vessel wall) in convergences (regions 1, 1 , and 2, white dotted lines; Fig. 1D) and in straight regions at least 40 mm away enrichment near EC junctions likely makes these locations pre- (designated region 3, not illustrated). All leukocytes that remained sta- ferred for leukocyte TEM. EC morphology determines the number tionary or did not exceed a displacement of .8 mm (one leukocyte di- of available portals, making venular converging regions optimally ameter) during 30 s were considered adhered. In all cases, regions 1 and 1p surrounded the smaller of the two inflow vessels. For TEM, leuko- equipped to support leukocyte TEM. p

cytes were counted in the extravascular tissue regions 1, 1 ,2(Fig.1D, Downloaded from black dotted lines), and 3 (not illustrated) adjacent to vessel lengths Materials and Methods where the adhesion was quantified. Regions 1, 1p, and 2 for TEM were Animals defined as follows: region 1 (between the two inflow vessels) was defined by projecting two lines perpendicular to the vessel walls. Regions 1p and Male wild-type C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) 2 were defined by connecting two lines that were projected perpendicular aged 12–15 wk were used. Animals were anesthetized with sodium pen- to each vessel and that were equal in length to the diameter of the vessel tobarbital (65 mg/kg, i.p.) and euthanized by overdose (.100 mg/kg) at they were projected from (illustrated in black). Adhesion and TEM in the completion of the protocols. When indicated, inflammation was in- regions 1 and 1p were averaged and presented as region 1. In straight http://www.jimmunol.org/ duced by locally superfusing the tissue with fMLP (10 mM in 0.01% regions, transmigrated leukocytes were counted in the tissue within DMSO; Sigma-Aldrich, St. Louis, MO) or by treatment with mouse rTNF- 50 mm of the vessel/80-mm length vessel segments. All TEM counts were a (0.5 mg TNF-a in 0.25 ml saline, intrascrotally; Sigma-Aldrich) 4 h prior normalized to 1000 mm2. To quantify leukocyte crawling and TEM to observations. All mice were used according to protocols approved by the routes, leukocytes and ECs were visualized as described above; each site University of Rochester Institutional Review Board. was observed and recorded for 30–40 min at 30 fps. The original movies were time-lapsed to 0.33 fps for offline analysis. Leukocyte crawling Intravital microscopy was defined as a displacement of at least one average leukocyte diameter (8 mm) during 30 min. Only rolling leukocytes that were observed to Mice were prepared for intravital microscopy and maintained throughout firmly adhere and then began to crawl were analyzed. Any crawling the experiment as described elsewhere (25). Observations were made using leukocytes that crawled out of the field of view were not included in the an Olympus BX61WI microscope with an Olympus PlanF1 immersion analysis. Crawling distances were obtained by measuring the path length by guest on September 30, 2021 3 3 objective ( 20, 0.65 numerical aperture or 40, 0.95 numerical aperture). of each crawling leukocyte from the moment it began to move and until it All observed venules ranged from 30–80 mm in diameter. Total leukocyte either detached from the endothelium or underwent TEM. A leukocyte adhesion and TEM (transmigrated leukocytes in the tissue) were quantified was considered to be migrating via a junctionally associated route if using bright-field images acquired via a charge-coupled device camera immediately prior to TEM, it was observed to intersect with the EC (Dage MTI CD72, DageMTI, Michigan City, IN). Leukocyte adhesion, junction and exhibited no measurable lateral displacement as it passed crawling, and TEM with respect to EC junctions were quantified using through the endothelial layer. When unclear, we assigned the trans- confocal fluorescence images that were acquired by illuminating the tissue migrating pathway to “un-assg.” with a 50-mW argon laser and imaging with a Nipkow disk confocal head EC junctions. The number of junctions was quantified in convergences, and (CSU 10; Yokogawa Yokogawa Electric, Tokyo, Japan) attached to an the average area of all convergences (6000 6 187 mm2, n = 11) was used as intensified charge-coupled device camera (XR Mega 10; Stanford Pho- a region of interest (ROI) for comparative analysis of straight regions. tonics Palo Alto, CA). All images were either digitally acquired or re- Vessel diameter in each straight region was used as the width of the ROI, corded to a DVD recorder (SONY DVO100MD) at 30 frames per second and the length was adjusted as needed to achieve an area of 6000 mm2 (Fig. (fps) for offline analysis. 2A, bottom panel). All dimensions were calculated from lengths measured as two-dimensional projections of the three-dimensional length; this In situ immunofluorescence labeling measurement has ,13% projection error (10). All images were acquired as Selected venules were (separately) stained for PECAM-1, VE-cadherin, and “continuous stacks” and presented as summed Z-stack projections for ICAM-1 as described previously (23). Briefly, using microcannulation, measurement of EC junctional length. venules were locally perfused with Abs as follows: anti–PECAM-1 (ER- ICAM-1 enrichment. To determine the level of ICAM-1 enrichment, three MP12 mAb conjugated to Alexa 488, 10 mg/ml, 10 min; Serotec, Oxford, independent measurements on the EC surface were taken. ICAM-1 ex- U.K.); anti–VE-cadherin (BV13 mAb conjugated to Alexa 488, 30 mg/ml, pression near tricellular junctions was measured by placing a 5-mm-di- 10 min; eBioscience, San Diego, CA); and anti–ICAM-1 (YN/1.7.4, 50 ameter ROI at the edges of each EC comprising the junction as close as mg/ml, 15 min; eBioscience), followed by goat anti-rat secondary fluo- possible to each outlined junction. The mean intensity of the three ROIs rescent polyclonal Ab (Alexa 488 anti-rat, 50 mg/ml, 15 min; Molecular represents the ICAM-1 intensity near that junction. These measurements Probes, Eugene, OR) or, alternatively, anti–ICAM-1 (YN/1.7.4 conjugated were taken for all tricellular junctions in the field of view. Similarly, two to Alexa 488, 30 mg/ml, 10 min; BioLegend, San Diego, CA). measurements of ICAM-1 intensity were taken on each side of all bicel- Unless stated differently, to observe leukocyte behavior with respect to lular junctions and averaged. A third measurement was taken randomly in EC junctions, leukocytes were stained for CD11a (anti–CD11a-Alexa 488 the middle of each EC, away from all junctions. Each ROI measurement [M17/4], 3 mg/mouse, i.v.; eBioscience) in addition to the anti–PECAM- was normalized to the mean intensity of the whole EC on which it was 1-Alexa 488 Ab, as above. Because the EC junctions are stationary and located. Intensities that were 2 SDs higher than the mean intensity of the leukocytes are mobile, leukocytes can be easily resolved with respect to ROIs on the EC surface away from junctions were defined as enriched EC junctions despite using the same fluorophore. Although M/17.4 is able regions. Similar analyses were performed on ECs that were labeled using to block integrin function, as shown in Supplemental Figs. 1 and 2, M/17.4 only the primary anti–ICAM-1 Ab (YN-1) conjugated to Alexa 488 as combined with ER-MP12 Abs at the concentrations used in this work controls for possible clustering induced by secondary Ab. No differences did not affect the specified leukocyte behavior. Anti–VE-cadherin (BV13; were found (data not shown). For an additional control, similar analyses 30 mg/ml) Ab left the EC junctions intact, with no visible gaps after 2 h were performed on ECs stained for the unrelated adhesion molecule P- (Supplemental Fig. 3). selectin, using primary followed by secondary Abs (similarly to principal 5244 ICAM-1 IN LEUKOCYTE RECRUITMENT IN MICROVASCULAR NETWORKS approach for labeling of ICAM-1). This showed no P-selectin enrichment respectively; p , 0.01) (Fig. 1B). Likewise, leukocyte TEM in re- in the tricellular junctional regions, thus further supporting our conclusion gion 1 was significantly higher compared with regions 2 and 3 (29.75 that the observed enrichment of ICAM-1 was not an artifact (Fig. 6A). 6 3.1 versus 12.6 6 1.3 and 11.3 6 1.3 leukocytes/10,000 mm2 , Statistics tissue, respectively; p 0.001) (Fig. 1C). Leukocyte TEM localized to the convergence but not the straight venular region is shown in Statistical significance was assessed by one-way ANOVA with Newman- Fig. 1E, Supplemental Movie 1, and Fig. 1F, which displays the Keuls Multiple Comparison Test using GraphPad Prism (version 4.0; origins and paths of transmigrating leukocytes. In this particular GraphPad, San Diego, CA). Statistical significance was set at p , 0.05. vessel, leukocyte TEM is mostly localized to one side of the vessel Results wall (the upper part of the field of view). In different vessels, TEM occurs at specific locations and can primarily occur from either side Leukocyte adhesion and transmigration primarily occur in of the vessel wall or both sides simultaneously and is likely a func- venular convergences tion of the distribution of the “active portals” for TEM. In control We have previously demonstrated increased leukocyte adhesion and (unstimulated) tissue, the number of adhered leukocytes was one to TEM in converging regions of TNF-a–activated venules (26). Simi- two leukocytes per 80-mm vessel length and was not significantly larly, in the current study, fMLP-induced leukocyte adhesion and different in all regions (data not shown). Similarly, the number of TEM were significantly increased in venular convergences (region 1) extravasated leukocytes ranged between one and four leukocytes per compared with the neighboring straight regions (region 3) (Fig. 1A). 10,000 mm2 and was not different in all regions (data not shown). In The number of adhered leukocytes in region 1 of the convergence comparison with the findings for adhesion and TEM, leukocyte was significantly higher compared with regions 2 and 3 (7.0 6 0.4 rolling fluxes (6.6 6 0.5 versus 5.2 6 0.7 and 5.8 6 0.6 leukocytes/ Downloaded from versus 5.1 6 0.4 and 4.3 6 0.5 leukocytes/80-mm vessel length, 40 s for regions 1, 2, and 3, respectively) (Fig. 1A) and rolling http://www.jimmunol.org/

FIGURE 1. Leukocyte adhesion and transmigration occur primarily in venular convergences. The intact tissue was stimulated by superfusion of fMLP (10 mM, for 10 min). All data were collected 40 min

after the completion of fMLP application. Leukocyte by guest on September 30, 2021 rolling (A), adhesion (B), and TEM (C) were quantified in venular convergences (regions 1 and 2) and compared with straight regions (region 3). For adhesion and TEM (B, C), each data point in region 1 represents an average of leukocytes on both sides of the inflow venule (regions 1 and 1p)(C). Regions 1, 1p, 2, and 3 were defined as described in Materials and Methods. All TEM counts were normalized to 10,000 mm2. D, Image of a representative venular convergence, where leukocyte adhesion and TEM (white and black dotted lines, respectively) were quantified in regions 1, 1p, and 2. To quantify leukocyte adhesion and TEM in the straight region (region 3), the field of view had to be moved away from the convergence, thus it is not illustrated. The arrows show the direction of flow. E, Sequence of images over 20 min showing leukocyte TEM in the converging region (white ellipse) versus straight venular region (white rectangle). F, Time-lapsed images were summed to display the origin and path of migrating leukocytes. Both leukocyte adhesion and leukocyte TEM are significantly enhanced in the regions of venular convergences. For all groups, bars are mean + SE, n = 4 mice, 8 venules. ppp , 0.01, significantly different from other groups. E (all panels) and F: scale bar, 25 mm. The Journal of Immunology 5245

Table I. Dimensions of ECs in straight and converging venular regions

Venular Region EC Length (mm)* EC Width (mm) Area (mm2)* Aspect Ratio** Orientation (Degrees)** n Region 3 (straight) 52.3 6 1.1 16.6 6 0.4 879.9 6 18.2 0.34 6 0.01 10.6 6 1.8 102 Region 1 (convergence) 38.2 6 0.8 16.9 6 0.3 593.8 6 23.6 0.46 6 0.02 17.8 6 1.8 89 Values are means 6 SE. EC junctions in selected venules were immunofluorescently labeled for PECAM-1. The major and minor axes as well as EC area were calculated using ImageJ software. EC aspect ratio is presented as (width/length). EC orientation is the angle of EC major axis relative to the axial direction. pp , 0.01, significantly different from each other; ppp , 0.001. velocities (7.5 6 0.8 versus 8.2 6 0.6 and 9.1 6 0.8 mm/s) were not p , 0.01) compared with straight regions (Table I). The aspect ratio different in converging and straight venular regions. These data (EC width/length, 1 = circle) in convergences was also significantly confirm that proinflammatory stimuli dramatically increase the higher (0.46 6 0.02 versus 0.34 6 0.01; p , 0.001) compared with number of firmly adhered and transmigrating leukocytes, and im- straight regions, indicating that ECs in convergences are less portantly, these data suggest that vessel architecture might play elongated. The alignment of ECs in these regions was significantly a role in leukocyte recruitment in situ. less ordered (17.8 6 1.8 versus 10.6 6 1.8, degrees, major axis relative to axial direction, p , 0.01, n = 219 cells). This irregular EC morphology is different in converging compared with straight alignment of smaller and variously shaped ECs has the potential to

venular regions Downloaded from change the local junctional morphology, because in convergences We have previously explored the differences in flow profiles in two, three, or four adjacent ECs can create an EC junction. Thus, we straight and converging regions (25–27) and concluded that flow is asked whether the number of tricellular EC junctions varies in unlikely to be the main cause for increased leukocyte TEM in straight versus converging venular regions. The number of tricel- venular convergences. EC morphology varies greatly in different lular junctions in convergences was indeed significantly higher vessels (23); thus, we hypothesized that increased leukocyte TEM compared with straight regions (20.7 6 1.2 versus 12.43 6 1.1 2 in venular convergences might result from local differences in EC junctions/6000 mm , respectively), but the total junctional length http://www.jimmunol.org/ morphology (e.g., size and shape), leading to different EC align- per area of vessel wall was not different (623.8 6 54.8 versus ment and junctional arrangement compared with straight regions. 609.0 6 28.3 mm/6000 mm2, respectively) (Fig. 2B). In contrast, Confirming our hypothesis, we found that in 30- to 60-mm-diameter arteriolar ECs, which differ from venular ECs in being significantly venules, ECs in the convergence regions were significantly shorter longer (23), maintain similar morphology in straight and converg- (38.2 6 0.8 versus 52.3 6 1.1 mm; p , 0.01) and had significantly ing regions (data not shown). Consequently, the number of tricel- smaller surface area (593.8 6 23.6 versus 879.9 6 18.2 mm2; lular junctions is not significantly different in straight versus by guest on September 30, 2021

FIGURE 2. The number of tricellular junctions in venular convergences is significantly higher than in straight regions. A, A representative image of a venular convergence and corresponding straight region (as defined in Materials and Methods), which were lumenally perfused with fluorescently tagged anti–PECAM-1 Ab (10 mg/ml) to visualize EC arrangement. Outlined in white are the regions (6000 mm2) where the number of tricellular junctions and the total junctional length were quantified. All analyses were performed on real-time recordings of each vessel where the focal plane was brought up or downas necessary to focus on the specific, analyzed part of the vessel wall. The squares within the projections outline the tricellular junctions to demonstrate the apparent higher number of these junctions in venular convergences. B and C, Quantification of tricellular junctions in straight and converging/diverging regions of sampled venules (B) and arterioles (C), respectively. For all groups, bars are mean + SE, n = 3–4 mice, 11 straight and converging regions.pp , 0.05, Significantly different from each other; &p , 0.05 (B), significantly different from same region in venules. 5246 ICAM-1 IN LEUKOCYTE RECRUITMENT IN MICROVASCULAR NETWORKS converging regions (8.2 6 0.9 versus 7.9 6 0.7 junctions/6000 mm2, Leukocytes preferentially transmigrate at or near tricellular respectively) (Fig. 2C). Moreover, although the total junctional junctions length per area of vessel wall in arterioles is similar to that in Leukocytes crawl finite distances of up to ∼100 mm (Fig. 4) prior to venules (601.8 6 25.2 mm in arterioles versus 623.8 6 54.8 mmin either undergoing TEM or detaching from the vessel wall. Although venules at the diverging/converging regions and 616.6 6 49.9 only a small fraction of crawling leukocytes eventually undergo versus 609.0 6 28.3 mm in straight regions, respectively), the TEM, this process is significantly more efficient in convergences number of tricellular junctions in arterioles was significantly lower compared with straight regions (32.8 6 6.1 versus 14.6 6 6.2% (∼50% less) compared with venules (Fig. 2). This suggests that EC TEM/adhered) (Fig. 5B). The rest of the crawling leukocytes be- morphology can indeed affect leukocyte TEM by dictating the in- come more rounded and detach from the endothelium. The trajectory of a representative leukocyte that crawled toward the EC cidence and assembly of tricellular junctions. junctional region and transmigrated is shown in Fig. 5D and Sup- The distribution of adhered leukocytes in convergences is plemental Movie 2. different from that in straight venular regions Because it has been suggested that leukocyte TEM occurs at tricellular junctions (20), we hypothesized that the higher TEM in An important question arising from these observed morphological convergences was due to the higher number of tricellular junctions differences in straight and converging regions is whether they impact in these regions. We show that in situ, leukocyte TEM occurs pri- leukocyte recruitment and whether they could explain increased marily via a junctionally associated route in both straight and con- leukocyteTEM in convergences. Wefirst asked whether the different verging regions (91.3 6 0.6 and 87.7 6 1.7%, respectively) (Fig. EC junctional arrangement in straight versus converging regions Downloaded from produced different distributions of adhered leukocytes with respect to EC junctions. Representative images of leukocytes adhered at the different locations are presented in Fig. 3C. Indeed, 55 6 1.8% of firmly adhered leukocytes in convergences were associated with tricellular junctions, leaving 24 6 3.6 and 20 6 3.0% at bicellular

and nonjunctional regions, respectively (Fig. 3A). In contrast, in http://www.jimmunol.org/ straight regions, most adhered leukocytes (58 6 2.3%) were located at bicellular junctions, and only 21 6 2.8% were located at tricellular EC junctional regions (Fig. 3A). To determine whether locations of adhered leukocytes were a direct consequence of EC morphology (different ratio of bi- to tricellular junctions), we randomly generated x-y position coordinates for test “leukocytes” on the mapped EC morphology. The distribution of randomly adherent “virtual” cells was not different from that for actual leukocytes, as shown in Fig. 3B. This implies by guest on September 30, 2021 that leukocyte adhesion on bi- versus tricellular junctions is a direct consequence of EC morphology.

Most leukocytes transmigrate via locations elsewhere from where they originally adhered

To study the behavior of adhered leukocyte prior to TEM, leukocytes FIGURE 3. The distribution of adhered leukocytes is a direct conse- and EC junctions were fluorescently labeled with anti-CD11a and quence of the junctional arrangement. To visualize EC junctions and cir- anti–PECAM-1 Abs, respectively. Confirming previous findings culating leukocytes, venules were locally perfused with anti–PECAM-1 (12), we show that most leukocytes (92.2 6 1.6%) (Fig. 4A)in conjugated to Alexa 488 (10 mg/ml), followed by i.v. injection of anti- straight regions undergo intraluminal crawling. The majority CD11a conjugated to Alexa 488 (3 mg/ml) Abs. A, The number of firmly (89.6 6 1.7%) (Fig. 4A) of adhered leukocytes in convergences also adhered leukocytes at bicellular (bi) and tricellular (tri) junctions, as well as crawled, but average crawling distance was significantly less com- on the nonjunctinal regions (non), were quantified. The number of adhered pared with straight regions (20.1 6 1.1 versus 32.1 6 1.5 mm) (Fig. leukocytes at each location is presented as percentage of the total pop- 4B). In both regions a small subset (#10%) of leukocytes crawled ulation. B, Simulated leukocytes (circles with an 8-mm diameter) were positioned using randomly generated coordinates within the images of distances of up to 100 mm. Leukocyte crawling velocity in con- 6 PECAM-1–stained venules that were previously obtained and used to vergences was 5.4 0.3 mm/min, which was significantly slower quantify leukocyte adhesion as shown in A. Similarly to A, the number of 6 than 11.2 0.5 mm/min in straight regions (velocity distribution) leukocytes that was found at each of the junctional locations was quantified. (Fig. 4C). Interestingly, in both straight and converging regions, For all groups, bars are mean + SE, n = 4 mice, 11 straight and converging leukocyte crawling distances (real-time trajectories) were not sig- regions. ppp , 0.01, significantly different from each other. &p , 0.01, nificantly different from their straight-line displacement from origin significantly different from others in same group. C, For illustrative pur- (20.1 6 1.1 versus 16.7 6 1.8 mm in convergences and 32.1 6 1.5 poses, the endothelium of a blood-perfused venule was stained for PECAM- versus 26.0 6 2.6 mm in straight regions, respectively), suggesting 1 (anti–PECAM-1 conjugated to Alexa 488, 10 mg/ml), and leukocytes were that there was some directionality in the observed crawling. Fur- stained for CD11a (PE anti-CD11a, 3 mg/mouse, red). Representative im- thermore, following fMLP application, most leukocytes crawled in ages (which are Z-stack projections of multiple focal planes) depict adhered leukocytes in straight and converging venular regions. The zoom-in images the direction of, or perpendicular to, blood flow but not against it are a single slice of a Z-stack and show firmly adhered leukocytes at the (Fig. 4D,4E), unlike in control venules, where leukocyte crawling is nonjunctional (arrowhead), tricellular (short arrow), and bicellular junc- independent of the flow direction (10, 28). Taken together, these data tional regions, thus the colocalizing yellow signal. Because of the higher strongly suggest that the locations where leukocytes initially adhere abundance of tricellular junctions in convergences (compared with straight are merely a station where leukocytes pause, subsequently crawling regions), most adhered leukocytes in these regions are found on the tricel- to locations where the environment is right for TEM. lular junctions. Scale bar, 40 mm for both principal images in C. The Journal of Immunology 5247

FIGURE 4. Crawling distances and crawling velocities are significantly less in convergences compared with straight regions. EC junctions and interacting leukocytes were immunofluorescently labeled using anti–PECAM-1 conjugated to Alexa 488 (10 mg/ml, local perfusion) and anti-CD11a conjugated to Alexa 488 (3 mg/ml, i.v). Time-lapsed microscopy (390) was used to track crawling leukocytes in straight and in converging regions. A, The fraction of adhered leukocytes that crawled in each region was not different. B and C, Leukocyte displacement (B) and the mean velocity (C, presented as frequency distribution plot) were less in convergences compared with straight regions. pp ,0.05, significantly different from each other. D, Representative leukocyte crawling trajectories (n = 11) from two straight (upper panel) and two converging (lower panel) venular regions. All starting positions were aligned to the same origin. Black arrow indicates flow direction. Axes, distance in micro- meters. E, The direction of leukocyte crawling with respect to blood flow. Directions were defined as fol- Downloaded from lows: parallel to blood flow (within 6 45˚ [parallel]); perpendicular to blood flow (between 6 45 and 90˚ [perpend]); past the origin in the negative direction of the blood flow (against). For all groups in A–C and E, n = 188 leukocytes in nine straight and n = 148 leukocytes in seven converging regions in five mice http://www.jimmunol.org/ were tracked. Bars are mean + SE.

5A). In convergences, most (59.3 6 2.1%) leukocytes used tricel- This again implies a clear preference toward tricellular junctional lular junctions to undergo TEM versus 45.8 6 4.1% using bicellular regions for TEM. junctions. In straight regions, equal fractions of leukocytes un- Consistent with the idea that TEM preferentially occurs at tri- derwent TEM via tricellular and bicellular junctions (45.1 6 4.1 and cellular junctional regions, we also found that in arterioles (where 45.0 6 5.0%, respectively) (Fig. 5C), despite that signiﬁcantly more both leukocyte adhesion and transmigration are rare events) the

adhered leukocytes were originally adhered at bicellular compared number of tricellular junctions was not signiﬁcantly different in by guest on September 30, 2021 with tricellular junctions (58.2 6 2.2 versus 28.4 6 2.1%) (Fig. 3A). straight versus converging regions (Fig. 2B) but was signiﬁcantly

FIGURE 5. Leukocytes preferentially transmigrate at tricellular junctions. EC junctions and interacting leukocytes were immunofluorescently labeled using anti–PECAM-1 conjugated to Alexa 488 (10 mg/ml, local perfusion) and anti-CD11a conjugated to Alexa 488 (3 mg/ml, i.v). Time-lapsed microscopy (390) was used to quantify the number of transmigrating leukocytes at each location. A, Percentage of total leukocytes that underwent TEM at the junctional region (junc) or via transcellular route (nonjunc). When the route could not be clearly assigned to either pathway, it was assigned to “un-assg.” B, The fraction of adhered leukocytes that underwent TEM was determined in straight and converging regions. C, Leukocyte TEM at bicellular (bi) and tricellular (tri) junctions, as well as at nonjunctional (non) regions, as percentage of the total leukocytes that underwent TEM in straight and converging regions. In A–C, white bars represent straight and black bars represent converging regions. For all groups in A–C, n = 188 leukocytes were tracked in nine straight and 148 leukocytes in seven converging regions in five mice. Bars are mean + SE. ppp/^p , 0.001, significantly different from each other or others in same group; ppp , 0.01, significantly different from each other. D, Representative image sequence demonstrating leukocyte initial adhesion location, the crawling route, and TEM during 125-s time period. The white arrows track leukocyte displacement. The black arrow above the images indicates the flow direction. Scale bar, 12 mm. 5248 ICAM-1 IN LEUKOCYTE RECRUITMENT IN MICROVASCULAR NETWORKS lower (∼50% less) compared with venules. Taken together, these representative images Fig. 7C, middle and upper panels). Impor- data suggest that EC morphology and alignment, and in particular tantly, gap formation was significantly attenuated by treatment with the number of tricellular junctions, play a key role in determining the ICAM-1 tail peptide that blocks ICAM-1 signaling (18) (Fig. 7A preferred regions for leukocyte transmigration. and representative image Fig. 7C, bottom panel), consistent with the hypothesis that high ICAM-1 density around EC junctions is im- Proinflammatory activation redistributes ICAM-1 toward the portant for VE-cadherin rearrangement. Approximately 70% of all edges of ECs that form tricellular junctions observed gaps were formed in association with tricellular junctions The distribution of ICAM-1 on ECs is not uniform and under (Fig. 7B), confirming that these are preferred TEM locations. Despite appropriate conditions undergoes redistribution to more localized the overall lower number of gaps formed following treatment with regions within individual ECs (23). Furthermore, ICAM-1 clus- the ICAM-1 tail peptide, the same ∼70% were still seen in associ- tering occurs around transmigrating leukocytes (11, 19, 29), ation with the tricellular junctions (Fig. 7B). suggesting that regions surrounding tricellular junctions could also We also tested the effect of ICAM-1 tail peptide on leukocyte be enriched in ICAM-1. If true, this would explain why these behavior and compared this with the effect of a mAb that blocks junctional regions are preferred locations for TEM. luminal interactions of leukocytes with ICAM-1. As predicted from We show that 50 min following fMPL application, the expression isolated cell systems (18), treatment with the ICAM-1 tail peptide of ICAM-1 on the EC surface significantly increased (Supplemental did not significantly alter the ability of leukocytes to adhere or Fig. 4A). Likewise, ICAM-1 was enriched near EC junctions (Fig. crawl on the endothelium (thus presumably did not affect their 6A,6B). Representative images in Fig. 6A demonstrate enrichment ability to reach the right location for TEM) but reduced leukocyte of ICAM-1 (second panel from the top), near tricellular junctions in TEM by 71% (Fig. 7D), indicating that the inability to open EC Downloaded from venules in fMLP-treated tissue (middle panel), but not under con- junctions is the cause of impaired leukocyte TEM. The direction of trol conditions (upper panel). ICAM-1 enrichment was also ob- crawling and leukocyte crawling velocity also remained unchanged served with TNF-a treatment, which is known to directly activate (Fig. 7E,7G). Interestingly, treatment with the ICAM-1 tail peptide ECs (third panel from the top), but not P-selectin (bottom panel). significantly increased leukocyte crawling distances (41.4 6 2.4 P-selectin (green) exhibits a typical punctate distribution on EC versus 28.9 6 2.7 mm in untreated venules) (Fig. 7F), implying that

surface [as previously determined (27, 30)] but no preferential lo- reduced TEM was due to the inability of leukocytes to find the http://www.jimmunol.org/ calization to the EC junctions (red). Importantly, ∼43% of tricel- portal locations. Furthermore, one of every four leukocytes that lular junctions were associated with enriched ICAM-1 versus only were observed undergoing TEM following blockade of the ICAM-1 23% of bicellular junctions, directly supporting our hypothesis that tail had difficulty in disengaging from the endothelium as it tra- ICAM-1 at least in part contributes to making the tricellular en- versed the vessel wall. Ab blockade of ICAM-1, as expected (32), dothelial junctions a preferred location for leukocyte TEM. This significantly decreased leukocyte adhesion (42%), abrogated leu- enrichment occurred in both converging and straight regions (data kocyte crawling (84%), and resulted in a 68% decrease in leukocyte not shown). The finding that not all tricellular junctions are en- TEM (Fig. 7D). Interestingly, the residual crawling was signifi- riched in ICAM-1 suggests that not all of them will equally support cantly slower than that in untreated venules (6.5 6 0.9 versus TEM. Supporting this, we determined that ∼40% of all crawling 10.5 6 0.8 mm/min) (Fig. 7G), and the direction of crawling was by guest on September 30, 2021 leukocytes that transmigrated, crossed at least one tricellular more random (same fraction in all directions) (Fig. 7E), confirming junction on their way to a transmigratory portal. The trajectory of a role for ICAM-1 in directing leukocyte crawling. Treatment with a typical crawling leukocyte crossing two tricellular junctions is either a nonspecific Ab or a control peptide had no significant effect shown in Fig. 6E and Supplemental Movie 3. Furthermore, we on leukocyte adhesion and TEM and did not prevent the formation found that leukocyte crawling was slowest across tricellular junc- of gaps in VE-cadherin (data not shown). Taken together, these tions (4.2 6 0.3, straight and 4.1 6 0.3 mm/min, converging re- findings suggest a dual role for ICAM-1 in mediating leukocyte gions) (Fig. 6C); velocity across bicellular junctions was also TEM: signaling to leukocytes the location of a portal for TEM, and significantly slower than at nonjunctional regions (7.6 6 0.6 versus preparation of EC junctions to accommodate leukocyte passage. 11.1 6 0.7 mm/min in straight and 7.1 6 0.4 versus 10.4 6 0.6 mm/ min in converging regions, respectively) (Fig. 6C). Moreover, in- Discussion dividual leukocytes that crawled for longer distances (.40mm) and Leukocyte TEM occurs primarily at EC junctions (12, 21, 33), but crossed at least two tricellular junctions predictably slowed down recent studies suggest that changes in EC morphology, and conse- near the EC junction and sped up on the EC surface (Fig. 6D). A quently junctional arrangement, or disruption of b2 integrin-ICAM- representative velocity trace of a leukocyte in relation to EC 1 interactions can lead to transcellular migration (12, 18). Thus, in junctions and its crawling trajectory are shown in Fig. 6D (bottom this study we asked whether differences in EC morphology in panel). The leukocyte slowed down near tricellular junctions, straight and converging venular regions affect leukocyte trafficking consistent with evidence for ICAM-1 enrichment in these regions and TEM in situ. We showed that leukocytes, in their innate envi- (Fig. 6A,6B). Crawling velocity across EC cellular junctions was ronment, preferentially use tricellular junctional regions as “por- not different in straight versus converging regions (Fig. 6C), sug- tals” for TEM. Our data suggest that the “portals” are junctions that gesting that the differences in leukocyte crawling behavior in both are surrounded by enriched regions of ICAM-1. These enriched regions are indeed relatable to local ICAM-1 distribution. ICAM-1 regions mark the location of a nearby portal, as well as preparing the EC junctions to accommodate leukocyte passage. ICAM-1 signaling is required for junctional rearrangement Thus, discontinuities in junctional molecules need not pre-exist but allowing leukocyte passage can be initiated by approaching leukocytes via ICAM-1–mediated ICAM-1engagementleadstophosphorylation ofjunctionalproteins, signaling. Supporting our findings, others have found no evidence such as VE-cadherin (31), likely contributing to its redistribution for pre-existing discontinuities of the endothelial barrier at tricel- away from junctions during TEM (21). We observed redistribution of lular junctions in retinal wholemounts but observed loss of tight VE-cadherin and formation of gaps at the locations of leukocyte junction proteins during leukocyte TEM (34). Although we cannot TEM in situ [as described in monolayers (21)] following fMLP unequivocally demonstrate that all leukocytes transmigrating via treatment of the tissue but not in untreated venules (Fig. 7A and these portals were taking a junctional route (versus a paracellular The Journal of Immunology 5249 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 6. Enrichment of ICAM-1 near tricellular EC junctions is associated with slower leukocyte crawling in these regions. In separate experiments, ICAM-1 distribution and leukocyte crawling velocities near EC junctions were quantified. A and B, Venular ECs were stained for ICAM-1 using anti– ICAM-1 (YN/1.7.4, 50 mg/ml, local perfusion) and fluorescently tagged secondary (anti-rat Alexa 488, 50 mg/ml, local perfusion) Abs in sequence. A, Representative raw and pseudocolored images (on the right) depict ICAM-1 enrichment near tricellular junctions post-fMLP superfusion (second from the top) and following 4-h TNF-a activation (third from the top) but not under control conditions (upper images). Corresponding ICAM-1 fluorescence intensity profiles (on the left) were obtained along the projected white line shown on the raw images. The lines were placed on the ECs that had the best focus in the field of view and that encompassed one or more tricellular junctions. White arrows on the images of venules from fMLP- and TNF-a–activated tissue indicate ICAM-1–enriched regions near tricellular junctions. The broken line on each intensity plot is the mean relative intensity, the patterned areas indicate the width of the junction, and the gray regions represent 5-mm-wide regions extending on both sides of the outlined junction. Similarly, selected venules were stained for P-selectin (in green, RB40.34, 50 mg/ml, followed by anti-rat Alexa 488, 50 mg/ml) and PECAM-1 (in red, ER-MP12-PE, 10 mg/ ml; eBioscience) after fMLP superfusion. Bottom panel (A) depicts a representative image and a corresponding intensity plot (of n = 28 cells), where the relative, corresponding intensities of junctional PECAM-1 (red) and surface P-selectin (green) were obtained along the projected white line. Unlike ICAM- 1, P-selectin is not enriched near EC junctions. Scale bar, 10 mm for all images in A. B, Measurements of relative ICAM-1 expression (as a function of relative fluorescence intensity) near tricellular junctions (tri-junc), bicellular junctions (bi-junc), and random regions on EC surface away from EC junctions (EC-surface) were performed as described in Materials and Methods. Intensities that were 2 SDs higher than the mean intensity of randomly measured ROIs on EC surface (above the dotted line) were defined as enriched regions. The inset shows percentage of enriched tri- and bicellular junctions out of the total number of sampled junctions (n = 58 tri- and 55 bicellular junctions, five venules from three mice). Approximately 43% of all tricellular junctions and only 23% of all bicellular junctions were enriched in ICAM-1. C–E, EC junctions and interacting leukocytes were immunofluorescently labeled using anti– PECAM-1 conjugated to Alexa 488 (10 mg/ml, local perfusion) and anti-CD11a conjugated to Alexa 488 (3 mg/ml, i.v). Time-lapsed microscopy (390) was used to track crawling leukocytes in straight regions and venular convergences 50 min post-fMLP superfusion. C, Leukocyte crawling velocities over tricellular junctions (tri-junc) and bicellular junctions (bi-junc), including the 5-mm-wide region on both sides of the junction, were measured and compared with crawling velocities on the nonjunctional regions (non-junc). pp/^p , 0.01, significantly different from each other. D, In the bottom panel, crawling velocity of a representative leukocyte was tracked in consecutive 5-mm lengths of EC surface, whereas the leukocyte remained in focus. The trajectory of the crawling leukocyte was traced (white dashed line; E , upper left panel), and the intensity plot along that line (left y-axis) was superimposed on the velocity plot (right y-axis). The patterned areas on the intensity plot show the locations of the two tricellular junctions that the leukocyte encountered on this path, and the gray areas are regions of 5-mm width on both sides of the outlined junction; these are regions of the lowest leukocyte crawling velocity. D, Upper panel shows average crawling velocity of all tracked cells (n = 7, five mice) in each of the regions shown in the representative trace (bottom panel). Crawling velocities over tricellular junctions were significantly slower compared with bicellular junctions or nonjunctional regions, consistent with the ICAM-1 enrichment shown in B. E, Image sequence demonstrating the initial location of a representative leukocyte and its crawling route during 863 s. The white arrows track the leukocyte displacement. Long arrow above the image indicates the direction of flow. Scale bar, 10 mm. 5250 ICAM-1 IN LEUKOCYTE RECRUITMENT IN MICROVASCULAR NETWORKS Downloaded from http://www.jimmunol.org/

FIGURE 7. Blockade of either the luminal or the cytoplasmic domains of ICAM-1 reduces leukocyte TEM via different mechanisms. A–C, VE-cadherin distribution. A, Venules were stained for VE-cadherin (anti–VE-cadherin, BV13, 30 mg/ml local intraluminal perfusion). The formation of gaps in VE- cadherin per field of view was quantified with or without fMLP (after 50 min, 10 mM, superfusion) and ICAM-1 tail peptide (100 mg/ml, local cannulation) 2 as indicated. All measurements were normalized to 1000 mm . B, The location of the formed gaps in VE-cadherin was quantified with respect to EC by guest on September 30, 2021 junctions in the presence or absence of ICAM-1 tail peptide. Formation of “gaps” in VE-cadherin was significantly attenuated in the presence of ICAM-1 tail peptide, but did not alter the distribution of those that formed, as the majority (∼70%) remained at tricellular junctions. C, EC junctions stained for VE- cadherin were observed immediately upon completion of the cannulation protocol (a representative image is shown in the upper panel, control conditions) and then again 50 min post-fMLP application (a representative image is shown in the middle panel). Control images of VE-cadherin and 50 min post-fMLP application in the presence of either ICAM-1 tail peptide (a representative image is shown in the bottom panel) or control-penetratin peptide (data not shown) were collected. As shown by white arrows (middle panel), fMLP application to the tissue resulted in formation of a large number of gaps in VE- cadherin. When used, the penetratin peptides were added immediately following control data collection, but prior to fMLP superfusion. For all groupsin A–C, n = 4 mice, seven venules for each condition. Scale bar, 10 mm. To quantify leukocyte-EC interactions (D–G), EC junctions and leukocytes were immunofluorescently labeled using anti–PECAM-1 conjugated to Alexa 488 (10 mg/ml, local perfusion) and anti-CD11a conjugated to Alexa 488 (3 mg/ml, i.v). All observations were made 50 min post-fMLP application (10 mM, superfusion) in the presence of either ICAM-1 blocking Ab (YN/1.7.4, 50 mm/ml), ICAM-1 tail peptide (100 mg/ml), or control peptide (100 mg/ml), which were introduced intraluminally by local cannulation. D, Leukocyte adhesion (leukocytes/80-mm vessel length), crawling (fraction of total adhered), and TEM (leukocytes/10,000 mm2 extravascular tissue). n = 4 mice, seven venules. The residual fraction of crawling leukocytes in ICAM-1 Ab blocking and ICAM-1 tail peptide experiments was quantified for directionality (E), distance (F), and crawling velocity (G). In F and G, the black arrows indicate the average crawling distance and crawling velocity (respectively) as shown in Fig. 4 in the absence of peptide treatment. ppp , 0.001, significantly different from each other or others in the same group. &p , 0.05, significantly different from values shown in Fig. 4 in the absence of peptide treatment. Control peptide did not prevent the formation of gaps in VE-cadherin and did not decrease leukocyte TEM (data not shown), suggesting a role for ICAM-1 signaling in leukocyte TEM. route in very close apposition to the actual junction), our findings assembly of the homologous VE-cadherin interactions is the that the ICAM-1 tail peptide blocked leukocyte TEM and VE- activation of proteins Src and Pyk2 upon engagement of ICAM- cadherin rearrangement at the EC junctions, together with the 1, leading to phosphorylation of VE-cadherin (31). However, the findings that ICAM-1–mediated signaling directly affects VE-cad- ability of leukocytes to exert force on EC contacts during TEM herin phosphorylation and mobility (30), strongly argue that (38) could also contribute to the formation of these gaps and the route taken is indeed junctional. cannot be ruled out. Rearrangement of junctional molecules, such as VE-cadherin, We add another dimension to the complexity of recruitment by and formation of transient gaps during leukocyte TEM have been showing that the arrangement of EC junctions in different venular demonstrated in vitro (21, 35, 36). We confirmed these findings regions greatly contributes to leukocyte recruitment and TEM. ECs in situ and, further, showed that signaling mediated via the invenular convergences are significantly smaller, more rounded, and ICAM-1 cytosolic tail is essential for the formation of observed aligned in a more random fashion than ECs in straight regions (Table gaps in VE-cadherin. As the ICAM-1 tail is a potential binding I). This arrangement produces more tricellular junctions in con- target for Src (37), the likely signaling mechanism for the dis- vergences, apparently making convergences optimally equipped to The Journal of Immunology 5251 support TEM. In previous work, we also showed that in venular gests that not all EC junctions are the same; some EC junctions can convergences the two inlet vessels are predicted to create a region act as “portals” for leukocyte TEM and some apparently cannot. of low velocity, increasing leukocyte adhesion probability (26), as Our study implicates ICAM-1 in making some EC junctions act as was indeed demonstrated in Fig. 1B. Increased leukocyte adhesion portals for TEM. together with a higher number of tricellular junctions in venular Many molecules make up EC junctions, and most have been convergences, resulted in 2.6-fold higher leukocyte TEM compared implicated in leukocyte TEM (14, 16, 41). Importantly, the nature of with straight regions. Thus, although fluid shear likely contributes endothelial cell-cell contacts varies with the need to regulate vessel to leukocyte accumulation in convergences, it is unlikely to affect barrier function. For example, arterioles have a significantly higher leukocyte TEM directly. This is because not only the absolute number of tight junctions compared with venules (42) and are number (higher as a result of more adhered cells in the region) but significantly less permeable (43). Likewise, there are differences in also TEM efficiency (%TEM/all adhered cells) was significantly expression and subcellular localization of junctional adhesion higher in convergences (Fig. 5B). Similarly, we ruled out the dif- molecules (22). In the current work, we suggest that the junctions ferential expression of adhesion molecules as key contributors for that could act as portals for TEM are those junctions that are sur- increased TEM, because the expression of ICAM-1 (Supplemental rounded by high ICAM-1 expression, but an alternative/additional Fig. 4B) and E-selectin (unpublished data) is not different in explanation could be that active portals have a different assembly or straight versus converging regions, confirming local EC morphol- different array of junctional proteins compared with elsewhere. The ogy as a prime candidate. local microanatomy of these junctions might also be different. Previous analysis of leukocyte adhesion in vivo (10), and work As mentioned above, not all junctions are used by leukocytes for in monolayers (39), showed that as a result of the EC morphology, TEM.Infact,onlyasmallportionofthejunctionsactasactiveportals, Downloaded from most adhered leukocytes overlap EC junctions. We have extended and often the same junctional region (bi- or tricellular) is used by these findings to show that differences in leukocyte adhesion multipleleukocytes.SupportingtheideathatportalsareECjunctions distribution with respect to junctional type in convergences versus that are enriched in ICAM-1, only ∼43% of all tri- and 23% of all straight regions are solely due to differences in EC size, shape, and bicellular junctions became enriched in ICAM-1, correlating with alignment, producing a higher ratio of tricellular to bicellular the percentages of leukocytes undergoing TEM in these regions.

junctions in venular convergences. Moreover, we showed that gaps in VE-cadherin staining were pri- http://www.jimmunol.org/ Leukocyte crawling is presumably a tool to get adhered leu- marily formed at tricellular junctions (Fig. 7) and were mediated via kocytes to the nearest EC junction, where TEM occurs. Confirming ICAM-1 signaling, again suggesting that the observed leukocyte previous observations (10, 12), we showed that ∼90% of leuko- behavior is directly connected to ICAM-1–enriched regions. cytes undergo intraluminal crawling (Fig. 4). This suggests that Some regions within the venular wall express lower levels of key the initial location of leukocyte adhesion is simply a way station extracellular matrix proteins that make up the basal lamina than where leukocytes pause before crawling to a “portal” to undergo other regions in the same vessel (44). These regions are also as- TEM. Leukocyte crawling, in control venules and monolayers (28, sociated with gaps between the pericytes and are preferentially 39) or in MIP-2–stimulated venules (12), is random (independent used by migrating leukocytes (44). We speculate that localization of blood flow). However, in our study, leukocyte crawling in the of these regions with EC junctions could also make these locations by guest on September 30, 2021 presence of fMLP was parallel or perpendicular to blood flow but optimal for leukocyte TEM. very rarely against it, suggesting some degree of directionality. The In summary, we show that leukocytes in their innate environment finding that blockade of the luminal portion of the ICAM-1 mol- preferentially target tricellular EC junctions to traverse the vessel ecule, but not blockade of the cytoplasmic tail, resulted in loss of wall. We show that EC morphology plays an important role in the observed directionality (Fig. 7E) argues that ICAM-1 might determining these portals, making venular converging regions play a role in directing leukocyte crawling. optimally equipped to support leukocyte TEM. Moreover, in ex- We showed that 50 min after fMLP application ICAM-1 ex- ploring the nature of the active portals, we suggest that enrichment pression was significantly increased (Supplemental Fig. 4A) com- of ICAM-1 surrounding some EC junctions makes these locations pared with control conditions, indicating endothelial activation. The preferred for TEM by signaling to leukocytes the location of the fMLP-evoked response in this time frame is surprising as fMLP is portal and further by mediating rearrangement of junctional considered to be a leukocyte activator, and there is very little evi- molecules at these locations. dence for its ability to affect ECs. Whether the increase in ICAM-1 expression was directly induced by fMLP treatment or whether it was a consequence of fMLP-induced leukocyte activation and ad- Acknowledgments hesion is not clear and will require new studies. Importantly, upon We thank J.M. Kuebel for expert technical assistance and Hen Drori for exposure of the tissue to fMLP, otherwise homogenously expressed thoughtful discussion. ICAM-1 on individual ECs became enriched near EC junctions (primarily tricellular) (Fig. 6A,6B). The presence of ICAM-1–en- Disclosures riched regions near tricellular junctions could explain the shorter The authors have no financial conflicts of interest. crawling distances that leukocytes exhibit in venular convergences, as well as their lower crawling velocities (Fig. 4D). References In our preparation, fMLP-induced TEM occurred mainly at or 1. Ley, K., C. Laudanna, M. I. Cybulsky, and S. Nourshargh. 2007. Getting to the very close to EC junctions. Our results differ from those previously site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Im- reported (40), possibly because of the route of fMLP administration munol. 7: 678–689. or the different tissue that was investigated. Intriguingly, leukocytes 2. Muller, W. A. 2003. Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends Immunol. 24: 327–334. that originally adhered at bi- or tricellular junctions were often 3. Nourshargh, S., and F. M. Marelli-Berg. 2005. Transmigration through venular observed to crawl away and either detach from the endothelium or walls: a key regulator of leukocyte phenotype and function. Trends Immunol. 26: transmigrate elsewhere. This argues that initial adhesion doesn’t 157–165. 4. Alcaide, P., S. Auerbach, and F. W. Luscinskas. 2009. Neutrophil recruitment determine whether the leukocyte will undergo TEM or affect the under shear flow: it’s all about endothelial cell rings and gaps. Microcirculation route it will use, although this requires future studies. It also sug- 16: 43–57. 5252 ICAM-1 IN LEUKOCYTE RECRUITMENT IN MICROVASCULAR NETWORKS

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