Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1

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1 Am J Physiol Cell Physiol 301: C804 C813, First published June 8, 2011; doi: /ajpcell Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1 Ronen Sumagin, Julia M. Kuebel, and Ingrid H. Sarelius Department of Pharmacology and Physiology, University of Rochester, Rochester, New York Submitted 28 April 2011; accepted in final form 3 June 2011 Sumagin R, Kuebel JM, Sarelius IH. Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1. Am J Physiol Cell Physiol 301: C804 C813, First published June 8, 2011; doi: /ajpcell Activated neutrophils interacting with the vessel wall can alter vascular permeability to macromolecules such as albumin via release of various secretion products that induce changes in the endothelial monolayer. In the current work we used cremaster microvessels of anesthetized mice to show that, in addition to this paracrine mechanism, leukocyte ligation of endothelial ICAM-1 directly activates endothelial cell (EC) signaling, altering EC permeability to albumin [i.e., solute permeability (P s )]. We show that antibody cross-linking of surface ICAM-1 in intact microvessels is sufficient to increase P s even in the absence of interacting leukocytes. Unstimulated arterioles do not support leukocyte-ec interactions, but despite this, antibody ligation of ICAM-1 in these vessels induced a twofold increase in P s. Similarly, in venules that were depleted of interacting neutrophils, P s was decreased to below resting levels and was restored by ligation of ICAM-1. Use of function-blocking antibodies to separately block leukocyte rolling or adhesion under unstimulated or TNF- -activated conditions established that both rolling and adhered leukocytes contribute to P s regulation in situ. Both rolling and adhesion activated EC-dependent signaling mechanisms that increased P s. ICAM-1 ligation with primary antibody alone or primary followed by secondary antibodies showed that regulation of P s is directly dependent on the degree of ICAM-1 clustering. Under physiological versus inflamed conditions, respectively, this ICAM-1 clustering-dependent regulation of P s switches from PKC dependent and Src independent to Src dependent and PKC independent. This study thus identifies a new mechanism by which antiadhesion treatment may constitute a potential therapy for tissue edema. leukocyte-endothelial signaling; adhesion molecules; microvessel barrier function INFLAMMATORY DISORDERS are characterized by changes in vascular permeability that encompass both accommodation of leukocyte transmigration (23), and regulation of solute and water exchange (7, 29). The ability of leukocytes to alter vascular permeability to water and key macromolecules such as albumin has come into focus recently and has been elegantly summarized in recent reviews (10, 28). Thus, leukocyte-dependent alterations in barrier function can occur in a paracrine fashion, i.e., result from release of materials by leukocytes upon activation. For example, neutrophil secretion products such as heparin-binding protein (CAP37) (16) and neutrophil elastase (18) can induce changes in permeability. Similarly, other leukocyte-derived products such as leukotrienes (4, 32), reactive oxygen species (52), and TNF- (12, 13, 17, 39) affect vascular permeability under various conditions. However, in Address for reprint requests and other correspondence: I. H. Sarelius, Dept. Pharmacology and Physiology, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY ( ingrid_sarelius@urmc.rochester.edu). addition to release of paracrine products from activated leukocytes, there is emerging evidence indicating that leukocyteendothelial cell (EC) interactions can alter vessel permeability by directly inducing EC-dependent changes. One key molecule that has been implicated in this process is ICAM-1 (39). ICAM-1 is a transmembrane glycoprotein with five extracellular IgG-like domains and a short cytoplasmic tail. While the extracellular domains of ICAM-1 directly bind 2 -integrins on leukocytes, thus physically mediating both leukocyte adhesion in venules (41, 50) and leukocyte rolling in arterioles (42), the intracellular tail of ICAM-1 is associated with cytoskeletal proteins (2, 45) and plays an important role in signal transduction leading to leukocyte transmigration (41, 45, 50). Leukocyte engagement of surface ICAM-1 leads to EC cytoskeletal and junctional reorganization (48), which is mediated by key players such as myosin light chain kinase and Src family kinases (11, 48), Rho family of monomeric GTPases (19), and junctional VE-cadherin (1, 47). Thus, activation of these EC signaling pathways downstream of ICAM-1 engagement by leukocytes implies that there is a direct role for leukocyte adhesive interactions with ECs in regulation of the barrier function of the vascular wall. Indeed, we have recently confirmed this by demonstrating that the TNF- -induced increase in microvascular permeability to albumin [i.e., solute permeability (P s )] is ICAM-1 dependent and is contingent on ICAM-1 interactions with CD18 in both venules and arterioles (39). Moreover, in an earlier study (42) we showed that, in arterioles, the interactions between ICAM-1 and CD18 were directly involved in mediating leukocyte rolling, thus suggesting that leukocyte rolling, in addition to leukocyte adhesion, might also contribute to P s regulation. These studies also emphasize the ability of the arteriolar microcirculation to respond to proinflammatory stimuli, thus contributing, in concert with the venular microcirculation, to the regulation of tissue homeostasis. Engagement of 2 -integrins also triggers intracellular signaling in leukocytes, leading to release of paracrine products that alter vascular permeability. For example, ligation of lymphocyte function associated antigen 1 (LFA-1) induces neutrophil degranulation and secretion of heparin-binding protein (15, 16), which can alter vascular permeability by binding to endothelial ligands (16). In our current study, however, we provide evidence that ligation of endothelial ICAM-1 by interacting leukocytes directly modulates vascular permeability independently of agents released from circulating leukocytes. We thus extend the idea that endothelium actively participates in regulation of vessel barrier function upon leukocyte ligation of adhesion molecules on the EC surface. 1 1 This article is the topic of an Editorial Focus by Marcie R. Williams and Francis W. Luscinskas (49a). C /11 Copyright 2011 the American Physiological Society

2 To do this, we measured P s in intact blood-perfused microvessels where, before P s measurements, leukocyte-ec interactions could be altered and quantified in the same vessels. We were able to compare P s in unstimulated and TNF- activated arterioles and venules and identify the contribution of both leukocyte rolling and adhesion to P s regulation. We show that differences in the degree of ICAM-1 cross-linking lead to activation of different downstream signaling pathways, and that in unstimulated and activated tissue, respectively, PKCand Src-dependent signaling mechanisms are involved downstream of ICAM-1 in the regulation of vessel permeability to albumin. Importantly, we provide evidence that ligation of endothelial ICAM-1 by interacting leukocytes directly modulates vascular permeability independently of agents released from circulating leukocytes, as we show that antibody ligation of ICAM-1 in the absence of circulating leukocytes and their secretion products is sufficient to induce changes in P s. MATERIALS AND METHODS Animal preparation. All procedures were approved by the Institutional Review Board of the University of Rochester. Male wild-type (WT) (C57BL6J; Jackson Laboratories), or TNF- receptor 1 knockout mice (TNFR1 KO) (Tnfrsf1a tm1imx /J; Jackson Laboratories, or a gift from G. Pryhuber, University of Rochester) aged wk old were initially anesthetized with pentobarbital sodium (65 mg/kg ip) and maintained throughout the experiment as described elsewhere (25, 43). The cremaster muscle was exteriorized and gently pinned over a quartz pedestal for visualization by confocal intravital microscopy (25, 35). During preparation and observation, the tissue was continuously superfused with warmed physiological salt solution (PSS) with the following composition (in mm): NaCl, 4.7 KCl, 2.0 CaCl 2, 1.2 MgSO 4, 18 NaHCO 3, ph 7.4 at 36 C, and equilibrated with gas containing 0% O 2,5%CO 2, and 95% N 2 to maintain tissue PO 2 15 torr (34). A total of 116 mice, weight ranging from g, were used for this study, with groups divided as indicated in figures and figure legends. Upon completion of the protocol, the animal was euthanized by anesthetic overdose. Confocal intravital microscopy. Observations were made using an Olympus BX61WI microscope through an Olympus PlanF1 immersion objective ( 10, 0.65 numerical aperture), allowing spatial resolution of 1.8 m. All observed microvessels ranged from 30 to 80 m in diameter. Leukocytes interacting with ECs were observed using brightfield images that were acquired via a charge-coupled device (CCD) camera (Dade MTI CD72, DageMTI, Michigan City, IN). To measure P s, fluorescence images were acquired by illuminating the tissue with a 50 mw argon laser and imaging with a Nipkow disk confocal head (CSU 10, Yokogawa Yokogawa Electric, Tokyo, Japan) attached to an intensified CCD camera (XR Mega 10, Stanford Photonics, Palo Alto, CA): laser power and camera gain settings were unchanged throughout all the experiments, as previously described (26, 35, 43). All images were recorded using a DVD recorder (SONY DVO100MD) at 30 frames per second for offline analysis. Drug application. To induce local inflammation, recombinant mouse TNF- (0.5 g TNF- in 0.25 ml saline, Sigma-Aldrich) was injected intrascrotally 3 h before the start of the surgical preparation. Observation of the selected microvessels was made between 4 and 5 h after the TNF- injection. Blocking antibodies (P-selectin, RB40.34, 30 g in100 l PBS, BD Pharmingen; CD18, GAME-46, BD Pharmingen, 100 g in100 l PBS and rat IgG control isotype R3 34, BD Pharmingen, 100 g in100 l PBS), as well as ICAM-1 ligating antibody (YN/1.7.4, 100 g in100 l PBS and rat IgG2b control isotype, 100 g in100 l PBS, Ebioscience) were administered intravenously via a second catheter inserted into the jugular vein. Data were collected 30 min after antibody administration. PKC C805 blocker bisindolylmaleimid l (BIM, 1 M in 0.01% DMSO, Calbiochem) or Src blocker PP2 (2 M in 0.01% DMSO, Calbiochem) was added for 10 min to the superfusion solution which was applied onto the tissue. Permeability measurements. Albumin permeability of intact cremaster muscle microvessels was measured using the approach previously published from our laboratory and detailed in references (35, 39). Briefly, fluorescence intensity from the selected microvessel and surrounding tissue was measured and used to calculate the solute flux (J s) per unit surface area (S) and concentration gradient ( C) using the relation: P s J s/s C 1/ I 0(dI f/dt) i(d/4), where I 0 is the fluorescence intensity of the test solute filling the vessel, (di f/dt) is the initial change in fluorescence intensity in the measured conditions as labeled BSA moves across the vessel wall, and D is the microvessel diameter (20). The vessel surface area and volume that act as a source of BSA-488 were corrected from that of a cylindrical blood vessel to the relevant confocal slice (15 m depth for the 10 objective) as described earlier (35). For permeability measurements, a microvessel upstream of the targeted vessels was cannulated with a micropipette and perfused with 10 mg/ml BSA in PSS in which 10% was BSA conjugated with Alexa 488, as detailed previously (35). In each preparation, the targeted vessel (arteriole or venule) was visualized and recorded continuously for 3 6 s (baseline) before the BSA-488 perfusion, during 1 min perfusion, and 3 6 s after the perfusion was stopped and blood flow was reestablished. Recorded sequences were analyzed with NIH Image software (version 6.1; National Institutes of Health, Bethesda, MD). A region of interest (ROI) was identified and the total fluorescence intensity was measured for the ROI starting with baseline and throughout the minute of perfusion with BSA-488. To control for local variations in pressure and flow in the selected regions of intact network, the intensity in the vessel itself was also quantified. Measurements in vessels exhibiting changes in the fluorescence intensity during the measured period were discarded. Leukocyte-EC interactions. The methods to quantify leukocyte-ec interactions in-situ have been previously established in our laboratory (26, 41, 42). Briefly, rolling leukocytes were defined as any leukocytes observed translating along the vessel wall in continuous contact with the endothelium. The number of rolling leukocytes (rolling flux) on the vessel wall was calculated by counting leukocytes rolling past a line perpendicular to the vessel axis per 40-s time interval. All leukocytes that remained stationary or did not exceed a displacement of 8 m (one leukocyte diameter) during 30 s were considered adhered. To deplete neutrophils, mice were injected with anti-gr-1 antibody (Ab), RGB-8C5 (200 g/mouse, ip) as described elsewhere (36) 12 h before observations. This decreased the circulating neutrophil population by 95%, as confirmed by flow cytometry (data not shown). In situ immunofluorescence labeling of ICAM-1. To quantify the relative expression of ICAM-1 on the EC surface, we utilized an approach that we developed previously (26, 42, 43) to immunofluorescently label surface molecules in intact blood-perfused microvessels. Briefly, microvessels were locally cannulated with glass micropipettes and perfused with anti-icam-1 antibody (YN/1.7.4 conjugated to Alexa 488, BioLegend, 30 g/ml) for 15 min. At the completion of the perfusion, blood flow was reestablished in the targeted microvascular region and the inflow arterioles were checked for tone and blood flow. To account for localized variability in the optical properties of the tissue, a second cannulation was used to perfuse the target vessels with fluorescent standard solution (0.05 mg/ml FITC-dextran in saline, 150 kda molecular mass, Sigma-Aldrich) after the completion of acquisition protocols (26, 43); intensities are expressed relatively to the intensity of the standard solution. The linearity of fluorochrome intensity with concentration in our system was confirmed as previously described (26, 42, 43). Statistical analysis. Statistical tests were performed using Graphpad Prism (version 4.0) to undertake t-tests, ANOVA, linear regres-

3 C806 Fig. 1. Initiation of leukocyte rolling in arterioles results in increased solute permeability (P s). To induce leukocyte rolling in arterioles, mice were treated with TNF- (500 ng intrascrotally) 4 h before observations. Rat IgG control isotype (R3 34, 100 g), anti-p-selectin (RB40.34, 30 g), anti-cd18 (GAME-46, 100 g), or a combination of anti-p-selectin and CD18 blocking antibodies (Abs) was injected intravenously; leukocyte rolling (A) was quantified 30 min following Ab injection, and 20 min later, P s (B) was measured in the same vessels. Blockade of CD18 and P-selectin (separately or together) significantly reduced leukocyte rolling in arterioles and significantly reduced P s. *Significantly different from IgG control group (P 0.05). For all groups the measurements were made in n arterioles from 3 5 mice. sion, or correlation analyses as appropriate. Populations were considered to be significantly different when P RESULTS Initiation of leukocyte rolling in arterioles results in increased P s. Leukocyte rolling is absent in control arterioles but is induced by TNF- treatment (27, 42). TNF- treatment also leads to an in increase in P s in arterioles (39), and a significant upregulation in the surface expression of ICAM-1 in these vessels (43). Thus, here we hypothesized that ICAM-1 ligation by rolling leukocytes in arterioles is directly involved in the observed P s increase. To test this, we used CD18 and P- selectin function-blocking Abs (GAME-46 and RB40.34, 100 g iv), which we have shown previously (42) to attenuate leukocyte rolling in TNF- -treated arterioles. As expected, blockade of either CD18 or P-selectin significantly decreased the number of rolling leukocytes in these arterioles ( and cells/40 s, respectively) compared with untreated vessels ( cells/40 s, Fig. 1A). In the presence of either of these blocking Abs, P s in arterioles was also decreased (Fig. 1B) and was not significantly different from basal (unstimulated) levels ( cm/s, CD18 and cm/s, P-selectin), providing strong evidence that the increased P s in TNF- -treated arterioles was due to leukocyte rolling interactions. In combination, blockade of both CD18 and P-selectin produced a further small, but not significant, decrease in the number of rolling leukocytes and the P s, compared with blockade of P-selectin alone. Leukocyte rolling interactions in unstimulated venules contribute to P s regulation. The majority of interacting leukocytes in unstimulated venules exhibit rolling interactions (42). To test the contribution of leukocyte rolling to P s regulation in unstimulated venules, we used P-selectin function-blocking Ab (RB40.34, 100 g iv) to block leukocyte rolling, and then measured P s in the same vessels. We found that the significant reduction in leukocyte rolling that follows blockade of P- selectin ( 4-fold decrease, Fig. 2) was accompanied by a significant decrease in P s in these vessels (from cm/s, untreated, to cm/s, P-selectin block, Fig. 2B). Interestingly, this venular P s in the absence of leukocyte rolling was not significantly different from P s in unstimulated arterioles [ cm/s; (39)] which also lack leukocyte-ec interactions (42). Our finding that basal P s is similar in both vessel types in the absence of interacting leukocytes suggests that leukocyte rolling in unstimulated venules could account for the differences in P s between unstimulated venules and unstimulated arterioles that we showed previously (39). As expected from earlier work (39), adhesionblocking anti-cd18 Ab had no significant effect on both leukocyte rolling and P s in unstimulated venules (Fig. 2). Leukocyte adhesion in venules is required for the TNF- induced increase in P s. TNF- treatment produces a dramatic increase in leukocyte adhesion in cremaster venules (43) as well as an increase in P s (39). To test the hypothesis that leukocyte adhesion is required for the TNF- -induced increase in venular P s, we measured P s in TNF- -activated venules that were pretreated with CD18 blocking Ab. Confirming our earlier findings (39), blockade of CD18 significantly decreased the number of adhered leukocytes from leukocytes/ 100 m to leukocytes/100 m (Fig. 3), but it had no significant effect on the number of rolling leukocytes under these conditions (Fig. 3). Note that the number of rolling leukocytes in TNF- -activated venules (Fig. 3A) is significantly lower compared with unstimulated venules (Fig. 2A), as the vessel wall under inflamed conditions is occupied primarily by adhered leukocytes (38). The typical TNF- -induced increase in P s was abolished following CD18 block (Fig. 3B), with P s decreased to the level found in unstimulated venules, directly supporting the hypothesis that leukocyte adhesion to endothelial ICAM-1 via 2 -integrins is essential for the TNF- -induced increase in P s. Note that because we show elsewhere in this study that anti-icam-1 antibodies induce signaling in Fig. 2. Leukocyte rolling contributes to P s regulation in venules. Rat IgG control isotype (R3 34, 100 g), anti-p-selectin (RB40.34, 30 g), and anti-cd18 (GAME-46, 100 g) blocking Abs were injected intravenously, and leukocyte rolling in unstimulated venules was quantified 30 min following Ab injection. P s in the same vessels was measured 20 min later. Blockade of P-selectin, which mediates most of the rolling at this time point, but not CD18, significantly reduced leukocyte rolling (A) and P s (B). *Significantly different from IgG control group (P 0.05). For all groups the measurements were made in n venules from 3 5 mice.

4 C807 Fig. 3. Leukocyte adhesion in venules is required for the TNF- -induced increase in P s. To induce inflammation, mice were treated with TNF- (500 ng intrascrotally) 4 h before observations. Either rat IgG control isotype (R3 34, 100 g), anti-p-selectin (RB40.34, 30 g), anti-cd18 (GAME-46, 100 g), or a combination of anti-p-selectin and CD18 blocking Abs was injected intravenously. Leukocyte rolling (A, black bars) and adhesion (A, white bars) were quantified in activated venules 30 min after Ab injection. P s (B) in the same vessels was measured 20 min later. As expected, leukocyte rolling and adhesion were significantly decreased by anti-p-selectin and anti-cd18 Abs, respectively. Ab block of adhesion with CD18 significantly decreased P s to levels observed in unstimulated venules (Fig. 2); blockade of the residual rolling in these vessels using P-selectin Ab in combination with anti-cd18 Ab decreased P s further to a level that is expected for basal P s in ICAM-1 knockout mice (in Ref. 39). *Significantly different from IgG control group (P 0.05). ^Significantly different from each other (P 0.05). For all groups the measurements were made in n venules from 4 6 mice. ECs that leads to changes in P s, we could not use this approach to support the data in Fig. 3. However, in previous work (39) we showed that genetic deletion of ICAM-1 (ICAM-1 KO mice) yielded similar findings to those of Fig. 3. We also asked whether the remaining leukocyte rolling in TNF- -activated venules also contributed to P s regulation. Blockade of P- selectin alone, while significantly decreasing leukocyte rolling as expected (from leukocytes/40 s, in untreated venules, to , with P-selectin block, Fig. 3), did not significantly alter leukocyte adhesion ( leukocytes/ 100 m, untreated venules, vs , P-selectin, Fig. 3). P s in these vessels remained unchanged with P-selectin Ab, consistent with our hypothesis that leukocyte adhesion is essential for the TNF- -induced, ICAM-1-mediated increase in venular P s. Furthermore, we found that blockade of both CD18 and P-selectin together significantly attenuated both leukocyte adhesion and leukocyte rolling in the same vessel (Fig. 3A), effectively eliminating 90% of all leukocyte interactions and leading to a further decrease in P s (from cm/s, TNF- alone, to cm/s, TNF- CD18 P-selectin Abs) compared with that achieved by CD18 block alone ( cm/s, Fig. 3B). P s values under these conditions were again similar to P s in unstimulated arterioles where neither rolling nor adhesion occurs [Fig. 4; (42)]. Thus, taken together, these data show that both rolling and adhered leukocytes contribute to P s regulation in venules; however, leukocyte adhesion is required for the TNF- -induced increase in P s. Antibody ligation of ICAM-1 in the absence of interacting neutrophils is sufficient to alter P s in unstimulated microvessels. In Figs. 1 and 2 we demonstrated that blockade of leukocyte rolling in TNF- -activated arterioles, and in unstimulated venules, was accompanied by a decrease in P s. Based on our previous findings (39) where we showed that surface ICAM-1 is essential for P s regulation in both arterioles and venules, we hypothesized that the observed decrease in P s was due to insufficient engagement of ICAM-1 in the absence of rolling leukocytes. To test this, we depleted circulating neutrophils using anti-gr-1 Ab (RGB-8C5, 200 g/mouse, ip) injected 12 h before the experimental protocols, and measured P s in unstimulated arterioles and venules, as well as in microvessels following ICAM-1 ligation. Neutrophil depletion ( 95%) was confirmed by flow cytometry [using a combination of anti-gr-1 and anti-f4/80 Abs, as described elsewhere Fig. 4. Ligation of ICAM-1 by rolling neutrophils regulates P s in unstimulated microvessels. To test the role of rolling neutrophils in P s regulation, circulating neutrophils were depleted (Depl) using anti-gr-1 Ab (RGB-8C5, 200 g/ mouse ip injection 12 h before microcirculatory measurements), and P s was measured in unstimulated arterioles and venules in the absence of neutrophils. In separate experiments in neutrophil-depleted animals, P s was measured in unstimulated arterioles and venules following ligation (Ligx1) of ICAM-1 with an anti-icam-1 mab (YN/1.7.4, 50 g/ml iv). P s measurements were performed 20 min post-ab ligation. Neutrophil depletion had no effect on baseline P s in unstimulated arterioles (as expected, because unstimulated arterioles do not support leukocyte rolling interactions); however, P s in venules was significantly decreased (below basal level as indicated by dashed line and presented in Fig. 2B). Ligation with ICAM-1 Ab significantly increased P s in both vessel types: to a value not significantly different from P s measured in activated arterioles (Fig. 1) and unactivated venules (Fig. 2), both of which characteristically support rolling interactions. *Significantly different from each other (P 0.05). ^Significantly different from P s value in unstimulated venules (Fig. 2B). For all groups the measurements were made in n 7 10 vessels from 2 4 mice.

5 C808 (5)]. We have previously established (40) that 60% of all interacting leukocytes in cremaster microvessels are neutrophils; after neutrophil depletion the total number of interacting leukocytes was reduced by 60%, confirming that circulating neutrophils had been depleted. As expected, neutrophil depletion had no effect on P s in unstimulated arterioles, since under these conditions arterioles do not support intrinsic leukocyte-ec interactions (42). In contrast, P s in venules in the absence of rolling neutrophils was significantly decreased ( cm/s, Fig. 4), compared with untreated venules ( cm/s, Fig. 2). These findings are consistent with data in Fig. 2A, where a decrease in P s in venules was observed following blockade of leukocyte rolling using P-selectin blocking Ab. Importantly, and further supporting our hypothesis, Ab ligation of ICAM-1 in neutrophil-depleted venules restored P s to normal basal levels ( cm/s, Fig. 4). These findings strongly suggest that direct engagement of ICAM-1 by rolling leukocytes contributes to P s regulation. Consistent with this conclusion, ligation of ICAM-1 in neutrophil-depleted unstimulated arterioles also significantly increased P s ( cm/s, Fig. 4) compared with both untreated arterioles and arterioles that were ligated with a nonspecific IgG. This observation was also consistent with the increase in P s that was observed in arterioles upon initiation of leukocyte rolling by treatment with TNF- (Fig. 1). Together, these observations confirm that indeed, ligation of ICAM-1 by rolling leukocytes contributes to P s regulation. These data also indicate that neutrophils, as the dominant leukocyte subpopulation in microvessels, are primarily responsible for the ECdependent, ICAM-1-mediated changes in P s, independently of their secretion products. The degree of ICAM-1 cross-linking is directly linked to increased P s. While Ab ligation of ICAM-1 in unstimulated arterioles significantly increases P s (Ref. 39 and Fig. 4) thus mimicking the effect on P s produced in arterioles by leukocyte rolling, it had no significant effect in unstimulated venules, which intrinsically support leukocyte rolling (26, 43). To test whether further cross-linking of ICAM-1 has the capacity to increase P s to a level typical of TNF- treatment, we used anti-icam-1 primary Ab followed by a secondary Ab and measured the effect of this additional cross-linking on P s. This approach has been shown to induce ICAM-1 clustering on endothelial cells (49, 51). Additional cross-linking of ICAM-1 with the secondary Ab did not produce a further increase in arteriolar P s ; however, it significantly increased P s in venules from cm/s (Cont, Fig. 5) to cm/s (Ligx2, Fig. 5). As the majority of rolling leukocytes in unstimulated venules become firmly adhered upon TNF- stimulation (38), these results suggest that while ligation of ICAM-1 with primary Ab alone mimics the ICAM-1 engagement typical of leukocyte rolling, the formation of larger clusters of ICAM-1, using the secondary Ab, mimics leukocyte adhesion. These findings also suggest that ICAM-1 clustering by adhered leukocytes is required for the TNF- -mediated increase in P s in venules. In addition, we confirmed that under these conditions, P s regulation was specific for ICAM-1, as ligation of VCAM-1 in unstimulated microvessels had no significant effect on P s (Fig. 5). VCAM-1 was chosen as the cross-linking control because, like ICAM-1, it is a member of the Ig superfamily and is also involved in leukocyte-ec interactions via ligation by interacting leukocytes. Also, again Fig. 5. The degree of ICAM-1 ligation differentially affects P s. Unstimulated arterioles and venules (Cont) were intraluminally perfused with either mouse anti-icam-1 or mouse anti-vcam-1 mabs (50 g/ml iv) and rat anti-mouse secondary Ab (50 g/ml intraluminally) in sequence (Ligx2). P s was measured in selected microvessels 20 min post-ab ligation. Exposure to both the primary and the secondary Abs against ICAM-1 but not VCAM-1 significantly increased P s, suggesting that ICAM-1 but not VCAM-1 clustering is involved in P s regulation. *Significantly different from IgG control group (P 0.05). For all groups the measurements were made in n 7 10 vessels from 4 5 mice. similarly to ICAM-1, VCAM-1 expression can be detected in both resting arterioles and venules, and it is upregulated by treatment with proinflammatory cytokines such as TNF- (42). ICAM-1 expression and ICAM-1-dependent signaling are essential for P s regulation. TNF- treatment results in a significant increase in P s in both arterioles and venules (39). TNF- receptor 1 (TNFR1, also called p55) is known to play a key role in mediating changes in vascular permeability in liver (12) and lungs (3). Thus, we asked whether TNF- induces an increase in P s in cremaster microvessels by acting on TNFR1. To do so, we measured P s in either unstimulated or TNF- -activated cremaster venules in TNFR1 KO mice. We found that, unlike in WT mice, TNF- treatment in TNFR1 KO mice failed to increase P s ( cm/s, Fig. 6A) compared with unstimulated venules ( cm/s, Fig. 6A), confirming that indeed TNFR1 plays an important role in TNF- -mediated changes in P s. Intriguingly, P s values in TNFR1 KO venules under both control and TNF- -activated conditions were significantly lower than those measured in unstimulated venules in WT mice (Fig. 6A) but were not different from P s measured in control arterioles ( cm/s). As we showed in Figs. 1 and 4, rolling leukocytes can contribute to P s regulation by direct ligation of ICAM-1. Either ICAM-1 density on the EC surface or the number of rolling leukocytes, or both, might affect this signal transduction; thus we hypothesized that the lower P s in venules of TNFR1 KO mice (compared with venules of WT mice) could be due either to lower baseline leukocyte rolling in these animals or to lower baseline expression of ICAM-1. We measured leukocyte rolling in control venules in TNFR1 KO mice and found no difference from that in WT (Fig. 6B). Furthermore, the total and differential leukocyte counts in TNFR1 KO mice were not different from those of WT mice (data not shown). TNF- treatment, as expected, had no effect on the number of rolling leukocytes in TNFR1 KO venules ( leukocytes/40 s with TNF- vs in unstimulated conditions, Fig. 6B) and did not induce the significant increase in leukocyte adhe-

6 C809 Fig. 6. ICAM-1 expression and ICAM-1-dependent signaling are essential for P s regulation. P s (A) and leukocyte rolling (black bars) and adhesion (white bars) (B) were quantified in unstimulated (control) and TNF- -treated venules in TNF- receptor 1 (TNFR1) knockout (KO) mice. Both unstimulated and TNF- -activated venules were significantly less permeable than unstimulated venules in wild-type (WT) mice (dashed line and Fig. 2B). Similarly, the increase in adhesion that was observed in TNF- -treated venules in TNFR1 KO mice (B) was significantly less than that in WT mice (dashed line and Fig. 3A). *Significantly different from WT venules (P 0.05). C: the relative expression of ICAM-1 unstimulated (control) and TNF- -activated venules was quantified in TNFR1 KO mice and compared with WT mice. Microvessels were locally cannulated and perfused with anti-icam-1 (YN/1.7.4 conjugated to Alexa 488, 30 g/ml) for 15 min. The data are presented relative to the fluorescent standard solution (0.05 mg/ml FITC-dextran, 150 kda molecular mass) to account for tissue variability. TNFR1 KO mice have significantly lower levels of ICAM-1 under both unstimulated and activated conditions. *,^Significantly different from each other (P 0.05). For all groups the measurements were made in n 9 15 venules from 3 5 mice. sion that occurs in WT venules. Thus it appears unlikely that leukocyte rolling per se in TNFR1 KO mice is responsible for the lower P s in venules of these mice. As the engagement of ICAM-1 by leukocytes is dependent on the availability of ICAM-1 on the EC surface, expression levels of this adhesion molecule might also affect P s ; we thus measured the expression of ICAM-1 in control and TNF- -activated venules in TNFR1 KO mice. The basal expression of ICAM-1 in control venules in TNFR1 KO was significantly lower than in WT mice ( vs , relative intensity gray scale units, Fig. 6C). These findings suggest that the lower P s in TNFR1 KO compared with WT venules is due to decreased ligation of ICAM-1 by rolling leukocytes as a result of the lower availability of surface ICAM-1. The number of rolling leukocytes per se is not affected by the decreased expression of ICAM-1, as ICAM-1 does not directly mediate leukocyte rolling in venules, but only contributes to rolling kinetics (41). As expected, TNF- treatment failed to significantly increase the expression of ICAM-1 in venules in TNFR1 KO mice ( , relative intensity gray scale units), compared with venules of WT mice ( , relative intensity gray scale units, Fig. 6C). This explains why TNF- treatment failed to induce leukocyte adhesion in these mice, and it also suggests that TNF- treatment induces an increase in ICAM-1 expression in WT mice by acting via TNFR1. Importantly, these data again confirm the role for ICAM-1-mediated signaling in P s regulation. To explore in more detail how ICAM-1 triggers signaling that alters P s, we asked whether the cytosolic tail of ICAM-1 plays a role in regulation of P s changes in situ. To do this, we used a cell-permeable mouse ICAM-1 tail peptide, as previously described (41), which inhibits ICAM-1-mediated signaling (50) and hence alters VE-cadherin rearrangement and decreases leukocyte transmigration in WT mice in situ (41). As expected (41, 50), treatment with the ICAM-1 tail peptide had no effect on leukocyte rolling or adhesion; however, the ICAM-1 tail peptide, but not the control peptide, significantly decreased P s in both unstimulated arterioles ( cm/s) and venules ( cm/s) compared with P s in untreated vessels. These data thus confirm that the outside-in signal transduction in ECs that is mediated directly by ICAM-1 regulates P s. ICAM-1 clustering is essential for switching from PKCdependent to Src-dependent regulation of P s. We previously showed (39) that ICAM-1-mediated P s regulation under resting conditions was PKC dependent, whereas the TNF- -induced increase in P s was Src dependent and PKC independent. Here we extend these findings to show that cross-linking of ICAM-1 in the absence of a TNF- stimulus is sufficient to switch the dominant signaling pathway from PKC (as seen in resting tissue) to Src dependent (as seen in activated tissue). We showed in the earlier study (39) that, in unstimulated arterioles, ligation of ICAM-1 with primary Ab produced an increase in P s, similar in magnitude to that induced by TNF- treatment. Here we show that this increase in P s is dependent on Srcmediated signaling and is not dependent on PKC. The Src blocker, PP2 (2 M, 10 min in superfusate), ablated the ligation-induced increase in P s to cm/s (Fig. 7B), in contrast to the PKC blocker, BIM (1 M, 10 min in superfusate), which had no significant effect on the ICAM- 1-ligation-dependent increase in P s ( , 10 6 cm/s, Ab ligation BIM, Fig. 7A). Additional cross-linking of ICAM-1 with a secondary Ab produced no additional increase in P s in arterioles compared with the ligation with primary Ab alone (Fig. 7A) and was, as predicted, blocked by PP2 treatment (Fig. 7B). In venules, P s regulation following ligation with primary Ab was dependent on PKC-mediated signaling and not Src, because in the presence of the PKC blocker BIM, but not with the Src blocker PP2, ligation-dependent P s in these vessels was significantly decreased ( cm/s, Fig. 7A, vs cm/s, Fig. 7B, respectively). This was consistent with the PKC-dependent regulation in unstimulated venules that we have reported previously (39) and which is also dependent on the presence of rolling leukocytes (Fig. 2). Furthermore, and consistent with the Src-dependent P s regulation in TNF- -treated venules that we reported earlier (39), the increase in venular P s that was induced by ligation with both primary followed by the secondary Abs (

7 C810 Fig. 7. ICAM-1 clustering is essential for switching from PKC-dependent to Src-dependent regulation of P s. Unstimulated arterioles and venules were intraluminally perfused with either a mouse anti-icam-1 mab (50 g/ml iv) alone (Ligx1) or followed by rat anti-mouse secondary Ab (50 g/ml intraluminally) (Ligx2). Ten to twenty minutes later, the tissue was superfused with either PKC inhibitor bisindolylmaleimid l (BIM; in 0.01% DMSO, 1 M for 10 min) (A) or Src inhibitor PP2 (in 0.01% DMSO, 2 M for 10 min) (B), and P s was measured in selected microvessels. In arterioles, leukocyte rolling in activated conditions is mimicked by Ligx1, and this Ligx1-dependent increase in P s is not PKC dependent (A) but is Src dependent (B). In venules, rolling in unstimulated conditions is mimicked by Ligx1, and P s is decreased below normal by PKC block (A) and is independent of Src (B). Adhesion in activated venules is mimicked by Ligx2, and the increased P s seen with this ligation is independent of PKC (A) and significantly decreased by Src block (B). Thus, P s changes induced by the maximal ICAM-1 clustering achievable for each vessel type are Src dependent, whereas P s changes induced by nonmaximal ICAM-1 clustering are PKC dependent. ^Significantly different from measurements obtained in the absence of the inhibitors (P 0.05), as shown in Fig. 5 and previously (39). *Significantly different from Ligx1. For all groups, the measurements were made in n 7 10 venules from 3 5 mice. cm/s, Fig. 5) was blocked by the Src inhibitor PP2 ( cm/s, Fig. 7B). PKC block had no effect on P s under these conditions ( cm/s, Fig. 7A). Thus, ICAM-1 cross-linking with primary followed by secondary Abs switched the venule to an inflammatory mode, as is also achieved for P s with TNF- (39). These data indicate that P s changes induced by the maximal ICAM-1 clustering achievable for each vessel type are Src dependent, whereas P s changes induced by nonmaximal ICAM-1 clustering are PKC dependent. Thus, the degree of ICAM-1 cross-linking contributes to determination of which downstream signaling pathway is dominant, and it serves as a cue for the microvessels to switch to the inflammatory state. DISCUSSION In this study we show that leukocyte engagement of EC surface receptors directly activates EC signaling, altering EC permeability. We conclude this from our findings that first, unstimulated arterioles do not support leukocyte-ec interactions; however, ICAM-1 cross-linking in these vessels induced an increase in solute permeability (P s ) (Ref. 39 and Fig. 5); and second, that while depleting the unstimulated venules of interacting neutrophils results in decreased P s (below basal levels), Ab engagement of ICAM-1 was sufficient to restore P s to expected basal levels (Fig. 4). Thus, in addition to the established paracrine regulation of P s by circulating leukocytes, direct engagement of ICAM-1 in microvessels activates a separate, ICAM-1-dependent, mechanism in ECs that regulates P s. Our findings also suggest that both leukocyte rolling and adhesive interactions with the endothelial surface receptor are able to initiate EC signaling leading to alterations in P s, thus directly contributing to P s regulation in both unstimulated and inflamed microvessels. Our study further indicates that these signaling pathways differ under physiological vs. inflamed conditions and are directly dependent on the degree of ICAM-1 clustering. Our results confirm and extend earlier studies that show that permeability is regulated in arterioles under both physiological and inflamed conditions (14, 21, 22, 35, 39). In addition to changes in the expression of surface adhesion molecules and the regulation of leukocyte trafficking in arterioles during inflammation, the arteriolar microcirculation thus also contributes to protein exchange and fluid homeostasis, although clearly, given its characteristically higher permeability and larger surface area, the venular microcirculation will quantitatively dominate regulation of these processes in intact tissues. We speculate that in the arteriolar microcirculation, regulation of permeability will also facilitate delivery of regulatory macromolecules (either directly or via their carriage by albumin) to their target cells outside the vascular compartment. In arterioles, the interactions between 2 -integrins and ICAM-1 can sustain leukocyte rolling (42). Our current data (Figs. 1 and 3) show that in these vessels, ligation of surface ICAM-1 by rolling leukocytes triggers ICAM-1-dependent signaling (via activation of Src kinase, Fig. 7) leading to increased P s. In unstimulated venules, leukocyte rolling also contributes to P s regulation (Fig. 2); however, in contrast to arterioles, where initiation of leukocyte rolling in activated vessels results in increased P s, in venules, intrinsic leukocyte rolling helps maintain basal P s in the unstimulated (physiological) condition. Our data show that in venules, in the absence of leukocyte interactions [either by Ab blockade of rolling interactions (Fig. 2) or by depletion of neutrophils (Fig. 4)], P s is significantly decreased to levels not different from those seen in unstimulated arterioles, where no leukocyte interactions with EC are typically observed (43). We conclude from this that leukocyte-ec interactions are a more important contributor to regulation of P s than are the established differences in structure and EC morphology between arterioles and venules. Our data indicate that, in venules, leukocyte rolling contributes to P s regulation by transiently ligating ICAM-1. We showed previously that leukocyte rolling in venules is not directly mediated by ICAM-1; thus blockade of ICAM-1 does not significantly decrease the number of rolling leukocytes (42). However, there is evidence that ICAM-1 is indeed being ligated by the rolling leukocyte in venules, as it takes part in stabilizing the rolling interactions, and in the absence of ICAM-1, leukocyte rolling velocity is increased (41). In the current work we further show that the degree of

8 ICAM-1 cross-linking is reflected in its ability to signal changes in P s. Ligation of ICAM-1 by rolling leukocytes, or ligation with primary Ab alone, could increase P s ; however, to achieve an increase in P s comparable to that induced by TNF- (and the consequent presence of adhered leukocytes), a higher degree of ICAM-1 clustering (cross-linking with both primary and secondary antibodies) was needed. This suggests that, similarly to the different degree of Ab-induced clustering, engagement of ICAM-1 by rolling vs. adhered leukocytes might also result in a different degree of ICAM-1 clustering, with greater ICAM-1 clustering being induced by leukocyte adhesion. As apparent expression density of ICAM-1 in resting and inflamed arterioles and venules is highly heterogeneous (43), it will be of great importance in future studies to determine the degree of ICAM-1 clustering that is needed to induce changes in P s under these various conditions. As described above, our data show that the interactions between 2 -integrins and ICAM-1 directly trigger alterations in barrier function. ICAM-1 is known to bind both CD11b (Mac-1) and CD11a (LFA-1) on leukocytes (3, 8). Mac-1 (9) and LFA-1 (37) bind ICAM-1 at different extracellular domains. Moreover, it is established that LFA-1 and Mac-1 have different roles in mediating leukocyte-ec interactions (31); thus it is possible that binding of each of these integrins to ICAM-1 will have different clustering effects, leading to differential effects on P s. This raises the important question as to which of the 2 -integrins, LFA-1 or Mac-1 (or both), is primarily responsible for these changes in P s. There is evidence that ICAM-1 valence and its distribution on EC-surface is important for ICAM-1 signal transduction. For example, ICAM-1 dimers, compared with monomers, have been shown to more efficiently bind LFA-1 (24, 30). Similarly, ICAM-1 dimers, but not monomers, activate Src in neutrophils (33). Leukocyte adhesion to endothelium induces formation of ICAM-1 clusters (44) and assembly of cytoskeletal machinery (46), suggesting that when clustered, ICAM-1 might trigger either different, or at the least a higher magnitude, of signaling. Confirming this idea, we show that cross-linking of ICAM-1 (using primary followed by secondary Abs, Fig. 5) indeed increased P s in unstimulated venules similarly to the increase in P s induced by leukocyte adhesion following TNF- treatment (Fig. 3). Note that P s in TNF- -treated venules was still significantly higher than that induced by ICAM-1 clustering alone, in the absence of adhesion of activated leukocytes, consistent with the expectation that secretion products from activated leukocytes are also involved in this process. Overexpression of ICAM-1 in monolayers results in increased permeability (6), strongly arguing that ICAM-1 plays an important role in P s regulation. However, we show that in in situ microvessels, ICAM-1 expression density alone is insufficient to mediate increases in P s ; this requires ligation of ICAM-1 by rolling leukocytes. The expression of ICAM-1 in unstimulated venules is at least twofold higher compared with unstimulated arterioles (43); however, in the absence of leukocyte rolling, P s in unstimulated venules (Fig. 2) was not significantly different from that measured in unstimulated arterioles (39). Note that basally expressed ICAM-1 also contributes to basal P s regulation independently of leukocytes, via a PKC-dependent signaling mechanism, as in the absence of ICAM-1 (in ICAM-1 KO mice) P s in both arterioles and venules is significantly lower than basal P s in WT mice (39). We recently identified two distinct ICAM-1-mediated signaling pathways, PKC-dependent in unstimulated vessels, and Src-dependent in TNF- -activated microvessels (39). Based on the current data, we hypothesize that ligation of ICAM-1 by rolling leukocytes leads to dimerization of ICAM-1, which turns on a PKC-dependent pathway, versus leukocyte adhesion, which leads to ICAM-1 clustering and a resultant switch to Src-dependent signaling: this hypothesis remains to be tested directly in future studies. As evident from data presented in Fig. 7, ICAM-1 clustering is indeed essential to switch from PKC-dependent to Srcdependent regulation of P s. In summary, in this study we demonstrate clearly that leukocyte engagement of ECs via ICAM-1 can lead to alteration in vessel permeability independently of effects on permeability produced by leukocyte secretion products. We also show that both leukocyte rolling and leukocyte adhesion play a role in this P s regulation, presumably by inducing different degrees of ICAM-1 clustering. In turn, these events regulate P s changes via different EC signaling mechanisms. ACKNOWLEDGMENTS We thank Dr. V. H. Huxley and Dr. K. Fujiwara for critical reading of the manuscript. GRANTS This work was supported by National Heart, Lung, and Blood Institute Grants HL and HL DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). REFERENCES C Allingham MJ, van Buul JD, Burridge K. ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration. J Immunol 179: , Amos C, Romero IA, Schultze C, Rousell J, Pearson JD, Greenwood J, Adamson P. Cross-linking of brain endothelial intercellular adhesion molecule (ICAM)-1 induces association of ICAM-1 with detergent-insoluble cytoskeletal fraction. 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