Optimal Selectin-Mediated Rolling of Leukocytes During Inflammation in Vivo Requires Intercellular Adhesion Molecule-1 Expression

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 7562–7567, June 1998 Immunology

Optimal selectin-mediated rolling of leukocytes during inflammation in vivo requires intercellular adhesion molecule-1 expression

DOUGLAS A. STEEBER*, MATTHEW A. CAMPBELL†,ABDUL BASIT‡,KLAUS LEY†§, AND THOMAS F. TEDDER*§¶

*Department of Immunology, Duke University Medical Center, Durham, NC 27710; and †Department of Biomedical Engineering and ‡Division of Nephrology, University of Virginia School of Medicine, Charlottesville, VA 22908

Communicated by Gordon G. Hammes, Duke University Medical Center, Durham, NC, April 17, 1998 (received for review March 3, 1998)

ABSTRACT Leukocyte interactions with vascular endo- have shown that, under in vitro conditions of low shear flow, ␣ ␤ ͞ ␣ ␤ thelium during inflammation occur through discrete steps 4 1 (CD49d CD29) and 4 7 integrins, as well as hyaluronan involving selectin-mediated leukocyte rolling and subsequent receptors (CD44) expressed by leukocytes, can mediate rolling firm adhesion mediated by members of the integrin and Ig (5–8). The in vivo relevance of these observations is unknown. families of adhesion molecules. To identify functional synergy The generation of gene-targeted mice deficient in expres- between selectin and Ig family members, mice deficient in both sion of L-, P-, or E-selectin has provided considerable insight L-selectin and intercellular adhesion molecule 1 (ICAM-1) into the molecular interactions that occur during inflammation were generated. Leukocyte rolling velocities in cremaster in vivo (9–11). L-selectin-deficient (L-selectinϪ͞Ϫ) mice have muscle venules were increased significantly in ICAM-1- decreased trauma-induced rolling of leukocytes in the exteri- deficient mice during both trauma- and tumor necrosis factor orized mesentery and have decreased rolling in cremaster ␣-induced inflammation, but rolling leukocyte flux was not muscle venules after tumor necrosis factor (TNF)-␣ treatment reduced. Elimination of ICAM-1 expression in L-selectin- (9, 12, 13). Leukocyte recruitment into sites of inflammation deficient mice resulted in a sharp reduction in the flux of is decreased markedly in L-selectinϪ͞Ϫ mice (9, 14, 15). rolling leukocytes during tumor necrosis factor ␣-induced P-selectinϪ͞Ϫ mice display decreased trauma-induced leuko- inflammation. The observed differences in leukocyte rolling cyte rolling in the exteriorized mesentery at early time points behavior demonstrated that ICAM-1 expression was required and decreased leukocyte entry early during inflammatory for optimal P- and L-selectin-mediated rolling. Increased responses (11–13, 16). E-selectinϪ͞Ϫ mice have increased leukocyte rolling velocities presumably translated into de- leukocyte rolling flux fractions (number of rolling leukocytes creased tissue emigration because circulating neutrophil, expressed as a percent of all leukocytes traveling through each monocyte, and lymphocyte numbers were increased markedly microvessel) and increased leukocyte rolling velocities in in L-selectin͞ICAM-1-deficient mice. Furthermore, neutro- cremaster venules after TNF-␣ treatment (12). Although phil emigration during acute peritonitis was reduced by 80% several inflammatory responses are normal in E-selectinϪ͞Ϫ in the double-deficient mice compared with either L-selectin mice, treatment with a P-selectin function-blocking mAb or ICAM-1-deficient mice. Thus, members of the selectin and dramatically reduces inflammation in these mice when com- Ig families function synergistically to mediate optimal leuko- pared with similarly treated wild-type mice (10). Mice deficient cyte rolling in vivo, which is essential for the generation of in both E- and P-selectin display a virtual absence of leukocyte effective inflammatory responses. rolling for at least 3 hr after TNF-␣ treatment (17) but have normal neutrophil emigration after 24 hr of peritonitis (17, The recruitment of leukocytes into sites of acute and chronic 18). Taken together, these findings demonstrate that, although inflammation involves leukocyte interactions with vascular L-, P-, and E-selectin have distinct roles, the selectins support endothelium under conditions of shear flow. Leukocyte- optimal leukocyte rolling during inflammation through syn- endothelial cell interactions are envisioned as a series of ergistic and overlapping functions. discrete steps using distinct constitutive or inducible adhesion ICAM-1 is constitutively expressed by endothelial cells and is up-regulated rapidly during inflammation, resulting in in- molecules. Specifically, it is thought that the selectins mediate Ϫ͞Ϫ leukocyte capture and rolling along the vessel wall whereas creased leukocyte–endothelial cell adhesion (19). ICAM-1 interactions between integrins and Ig superfamily members mice have significantly reduced numbers of infiltrating neu- arrest rolling cells and mediate firm adhesion that leads to trophils during some inflammatory responses (14, 20, 21). ␤ migration into sites of inflammation (1–3). L-selectin (CD62L) ICAM-1 interactions with 2 integrins do not support leuko- ␣ is expressed constitutively by most leukocytes whereas P- cyte rolling in vitro (22, 23), and both trauma- and TNF- - selectin (CD62P) and E-selectin (CD62E) are expressed by induced leukocyte rolling flux is normal in exteriorized cremaster muscle venules of ICAM-1Ϫ͞Ϫ mice (24). Although activated endothelial cells and primarily mediate neutrophil Ϫ͞Ϫ and monocyte rolling (4). In addition, leukocytes express the mean leukocyte rolling velocities in ICAM-1 mice are ␤ similar to those of wild-type mice over a 2-hr time period (24), 2 integrins, including lymphocyte function-associated antigen 1 (CD11a͞CD18), which interact with members of the Ig leukocyte rolling velocities have not been examined in detail ͞ Ϫ͞Ϫ superfamily expressed by activated endothelial cells including in these mice. Recent studies in P-selectin ICAM-1 mice suggest a complex role for ICAM-1 in leukocyte rolling when intercellular adhesion molecule 1 (ICAM-1) (CD54). Al- ͞ Ϫ͞Ϫ though lymphocyte function-associated antigen 1͞ICAM-1 P-selectin is absent (24, 25). P-selectin ICAM-1 mice interactions do not support leukocyte rolling, recent studies display a profound decrease in trauma-induced leukocyte

␣ The publication costs of this article were defrayed in part by page charge Abbreviations: ICAM-1, intercellular adhesion molecule 1; TNF- , tumor necrosis factor ␣. payment. This article must therefore be hereby marked ‘‘advertisement’’ in §K.L. and T.F.T. contributed equally to this study. accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed at: Department of © 1998 by The National Academy of Sciences 0027-8424͞98͞957562-6$2.00͞0 Immunology, Box 3010, Duke University Medical Center, Durham, PNAS is available online at http:͞͞www.pnas.org. NC 27710. e-mail: [email protected].

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rolling that persists much longer than in mice deficient in dvessel), where Vb is the mean blood flow velocity, dvessel is the P-selectin alone (24). This results in an almost complete lack in vivo diameter of the vessel, and 2.12 is a median empirical of neutrophil emigration into an inflamed peritoneum at early correction factor obtained from actual velocity profiles mea- time points (25). The significant reduction of rolling in P-se- sured in microvessels in vivo (30). To reduce the influence of lectin͞ICAM-1Ϫ͞Ϫ mice therefore prompted us to determine hemodynamic variables on leukocyte rolling velocities, only the mechanism by which ICAM-1 absence affects leukocyte venules with calculated wall shear rates between 300 and 1000 rolling and inflammation. sϪ1 were analyzed. Rolling velocity varies little over this range To further define the cascade of interactions among families of shear rates (31). of adhesion molecules during leukocyte–endothelial cell in- Microvessel diameter and individual rolling leukocyte ve- teractions in vivo, mice deficient in both L-selectin and locity were measured by using a digital image processing ICAM-1 were generated. The phenotype of these mice dem- system. Freeze frame advancing was used to accurately mon- onstrates a physiologically significant role for ICAM-1 in itor the movements of the individual rolling leukocytes. Each regulating leukocyte rolling flux fractions and rolling velocities rolling leukocyte passing a line perpendicular to the vessel axis at sites of inflammation. was followed for Ϸ150 ␮m downstream. The difference between the end points of the traveled distance was measured MATERIALS AND METHODS either on the monitor with a caliper or by using a custom digital image processing system (28). Rolling velocities for individual Animals. Mice deficient in L-selectin were produced as leukocytes were calculated by dividing this distance by the described (9). ICAM-1Ϫ͞Ϫ mice (20) were obtained from The elapsed time period. Rolling leukocyte flux was determined by Jackson Laboratory. These ICAM-1Ϫ͞Ϫ mice express residual counting the number of leukocytes passing through each amounts of ICAM-1 splice variants in the thymus and spleen venule as described (9, 32, 33). Total leukocyte flux was but not in other organs (26). Mice lacking both L-selectin and estimated as the product of measured systemic leukocyte ICAM-1 were generated by crossing F1 offspring from crosses concentration and blood volume flow. Leukocyte rolling flux of homozygous L-selectinϪ͞Ϫ mice with homozygous ICAM- fraction is defined as the flux of rolling leukocytes as a percent 1Ϫ͞Ϫ mice. Lack of L-selectin surface expression was con- of total leukocyte flux. By definition, leukocyte rolling flux firmed by direct immunofluorescence staining of blood leu- fraction is independent of variations in systemic leukocyte kocytes with the LAM1-116 antibody (27). Presence of the counts. mutated ICAM-1 gene was verified by PCR analysis of DNA Thioglycollate-Induced Peritonitis. Thioglycollate solution from tail biopsies. The L-selectin͞ICAM-1Ϫ͞Ϫ mice were (1 ml; 3% wt͞vol; Sigma) was injected i.p. into mice and the healthy and fertile and did not display any evidence of infection leukocyte infiltrate was recovered by peritoneal lavage as or disease. All mice were backcrossed between 5 and 10 described (15). generations onto the C57BL͞6 background. Mice were 7–12 Statistical Analysis. All data are shown as mean values Ϯ weeks old for all experiments, and age-matched wild-type SEM. ANOVA followed by Student’s t test was used to littermates or C57BL͞6 mice (The Jackson Laboratory) were determine the level of significance of differences in population used as controls. All mice were housed in a specific pathogen- means. Distributions of leukocyte rolling velocities were com- free barrier facility and were screened regularly for pathogens. pared by using a Kruskal–Wallis Multiple-Comparison Z- All studies and procedures were approved by the Animal Care Value test with Bonferroni correction. and Use Committee of Duke University Medical Center and the Animal Research Committee of the University of Virginia. RESULTS Intravital Microscopy. Mice were anesthetized with an 100-mg͞kg i.p. injection of ketamine hydrochloride (Ketalar, Phenotype of L-selectin͞ICAM-1؊͞؊ Mice. Mice deficient in Parke-Davis) after pretreatment with sodium pentobarbital L-selectin and ICAM-1 were bred to generate L-selectin͞ (30 mg͞kg i.p., Nembutal, Abbott) and atropine (0.1 mg͞kg ICAM-1Ϫ͞Ϫ mice homozygous at both loci. There were no i.p., Elkins-Sinn, Cherry Hill, NJ). The mice were kept at 37°C obvious indications of pathology or disease susceptibility for and received Ϸ0.2 ml͞hr diluted pentobarbital in saline i.v. to any of the mice up to 1 year of age. A consistent finding was maintain anesthesia and a neutral fluid balance. For TNF-␣ that both ICAM-1Ϫ͞Ϫ and L-selectin͞ICAM-1Ϫ͞Ϫ mice were studies, mice were pretreated with 0.5 ␮g of TNF-␣ (Genzyme) Ϸ20% larger and heavier than age-matched wild-type or in 0.30 ml of isotonic saline injected intrascrotally 2.5 hr before L-selectinϪ͞Ϫ mice (D.A.S. and T.F.T., unpublished observa- surgery. The cremaster muscle was prepared for intravital tions). microscopy as described (12, 24). In brief, the epididymis and The loss of both L-selectin and ICAM-1 led to elevated testis were gently pinned to the side to expose the well- numbers of circulating neutrophils (580% of wild-type, P Ͻ perfused cremaster microcirculation. The cremaster muscle 0.001), lymphocytes (200%, P Ͻ 0.001), and monocytes (640%, was superfused with thermocontrolled (37°C) bicarbonate- P Ͻ 0.001; Table 1). Mice deficient in L-selectin alone had buffered saline. Blood samples (10 ␮l each) were taken significantly increased numbers of circulating monocytes throughout the experiment from the carotid catheter at Ϸ45 (146% increase) whereas the loss of ICAM-1 significantly min intervals to analyze systemic leukocyte concentrations. increased the numbers of circulating neutrophils by 190%, Differential leukocyte counts were obtained by Kimura stain lymphocytes by 47%, monocytes by 223%, and eosinophils by of the blood samples. 178% relative to wild-type littermates (Table 1) as published Microscopic observations were made on an intravital mi- (20, 34). Eosinophil numbers were increased to a similar extent croscope (Axioskop, Zeiss) with a saline immersion objective in both ICAM-1Ϫ͞Ϫ and L-selectin͞ICAM-1Ϫ͞Ϫ mice. (SW 40͞0.75 numerical aperture). Venules with diameters Leukocyte Rolling. The rolling behavior of leukocytes in between 15 and 50 ␮m were observed, and video recordings L-selectin͞ICAM-1Ϫ͞Ϫ mice was assessed in venules of the were made through a charge-coupled device camera system cremaster muscle after surgical exteriorization. Surgical exte- (model VE-1000CD, Dage–MTI, Michigan City, IN) on a riorization initiates a mild inflammatory response resulting in Panasonic S-VHS recorder (Panasonic, Secausus, NJ). Micro- leukocyte rolling within the venules but limited firm adhesion vascular centerline red blood cell velocity was measured by or transmigration of leukocytes. Estimated wall shear rates, using a dual photodiode and a digital on-line cross-correlation microvessel diameters, and centerline blood flow velocities program (28). Centerline velocities were converted to mean were similar among the four groups of experimental animals blood flow velocities by multiplying with an empirical factor of (Table 2 and data not shown). The properties of 65–365 rolling ͞ 0.625 (29). Wall shear rates were estimated as 2.12 (8Vb leukocytes were assessed in 13–73 venules of each mouse line Downloaded by guest on September 27, 2021 7564 Immunology: Steeber et al. Proc. Natl. Acad. Sci. USA 95 (1998)

Table 1. Numbers of blood leukocytes in L-selectin͞ICAM-1-deficient mice No. of cells͞ml (ϫ10Ϫ5) L-selectin͞ Cell lineage Wild type L-selectinϪ͞Ϫ ICAM-1Ϫ͞Ϫ ICAM-1Ϫ͞Ϫ Total 41 Ϯ 551Ϯ 875Ϯ 7* 115 Ϯ 8*†‡ Neutrophil 6.2 Ϯ 1.1 8.6 Ϯ 2.3 18 Ϯ 3* 36 Ϯ 4*†‡ Lymphocyte 34 Ϯ 439Ϯ 750Ϯ 6* 68 Ϯ 5*†‡ Monocyte 1.3 Ϯ 0.3 3.2 Ϯ 0.9* 4.2 Ϯ 0.7* 8.3 Ϯ 1.1*†‡ Eosinophil 0.9 Ϯ 0.2 0.8 Ϯ 0.5 2.5 Ϯ 0.6* 3.0 Ϯ 0.4*† Differential leukocyte counts were performed on blood smear preparations following Wright–Giemsa staining. Values represent the mean Ϯ SEM results from 13 wild-type, 9 L-selectinϪ͞Ϫ, 23 ICAM- 1Ϫ͞Ϫ, and 37 L-selectin͞ICAM-1Ϫ͞Ϫ mice. FIG. 1. Leukocyte rolling flux fractions (percent of all leukocytes *Differences between test and wild-type mice were significant, P Ͻ rolling) in cremaster venules of adhesion molecule-deficient mice after ␣ 0.05. treatment with TNF- . Mice were pretreated with an intrascrotal Ϫ͞Ϫ Ϫ͞Ϫ ␣ †Differences between L-selectin and L-selectin͞ICAM-1 mice injection of TNF- 2.5 hr before the initiation of surgery. Values Ͻ represent mean rolling flux fractions Ϯ SEM from 14–47 venules. The were significant, P 0.01. Ϫ͞Ϫ ‡Differences between ICAM-1Ϫ͞Ϫ and L-selectin͞ICAM-1Ϫ͞Ϫ mice data from wild-type and L-selectin mice are published (12). Ϫ͞Ϫ ͞ Ϫ͞Ϫ were significant, P Ͻ 0.05. Differences between L-selectin and L-selectin ICAM-1 mice were significant (P Ͻ 0.05). at time points between 10 and 120 min after exteriorization of ␣ the cremaster muscle. The rolling flux fractions of leukocytes kine TNF- before the initiation of surgery. Hemodynamic ͞ Ϫ͞Ϫ Ϯ parameters and estimated wall shear rates were similar among in cremaster venules of L-selectin ICAM-1 mice were 35 ␣ 1% (mean Ϯ SEM, 24 venules in seven mice) at times Ͻ30 min the four groups of experimental animals after TNF- treat- and 41 Ϯ 1% at times Ͼ60 min (68 venules in seven mice). ment (Table 2, and data not shown). There was a significant, 50% (P Ͻ 0.05) decrease in leukocyte rolling flux observed in These values were within the normal range as also reported for Ϫ͞Ϫ Ϫ͞Ϫ L-selectin͞ICAM-1 mice below the level seen in L- ICAM-1 (t Ͻ 60 min, 50 Ϯ 4%; t Ͼ 60 min, 47 Ϯ 4%) and Ϫ͞Ϫ selectin mice (Fig. 1). Consistent with published results (12, wild-type (Ϸ45% for both time periods) mice (24). However, 24), the leukocyte rolling flux fraction was decreased by 34% these results are surprising given the Ϸ50% decrease in Ϫ͞Ϫ in TNF-␣-treated L-selectin mice (P Ͻ 0.05) and was frequency of rolling leukocytes previously observed in L- Ϫ͞Ϫ Ϫ͞Ϫ increased slightly in ICAM-1 mice relative to wild-type selectin mice relative to wild-type mice at time points values (Fig. 1). Therefore, the combined loss of L-selectin and beyond 30 min (13). That the rolling flux fraction of leukocytes ͞ Ϫ͞Ϫ ICAM-1 significantly reduced the fraction of leukocytes roll- in L-selectin ICAM-1 mice was similar to wild-type levels ing in response to TNF-induced inflammation. may result from the 4- to 6-fold increase in numbers of Leukocyte Rolling Velocities. Leukocyte rolling velocities circulating neutrophils observed in L-selectin͞ICAM-1Ϫ͞Ϫ Ϫ͞Ϫ were measured to further investigate rolling behavior in mice relative to wild-type or L-selectin mice (Table 1). ICAM-1Ϫ͞Ϫ and L-selectin͞ICAM-1Ϫ͞Ϫ mice. During trau- Circulating leukocyte counts are factored into the equation ma-induced inflammation, P-selectin predominantly mediates used for determining rolling flux fractions, thus controlling for rolling at early time points whereas L-selectin predominantly higher or lower systemic counts. Neutrophils normally repre- mediates rolling at later time points, as demonstrated by using sent a minority of the circulating leukocytes, but most rolling L- and P-selectin-deficient mice (9, 16). Therefore, we grouped leukocytes under these conditions are neutrophils (16). There- rolling velocity distributions of leukocytes into two time pe- fore, a relative increase in neutrophil numbers in L-selectin͞ riods to reveal these differences: time points Ͻ30 min and time Ϫ͞Ϫ ICAM-1 mice may account for the unaltered flux fraction. points Ͼ60 min. At early time points, leukocytes from ICAM- To determine the effects of L-selectin and ICAM-1 loss on 1Ϫ͞Ϫ mice displayed slightly faster rolling velocities than leukocyte rolling during a more vigorous inflammatory re- wild-type mice, as indicated by a shift to the right of the sponse, adhesion molecule-deficient mice were pretreated cumulative velocity distribution (Fig. 2A, Table 2). Rolling with an intrascrotal injection of the pro-inflammatory cyto- velocities were significantly lower in L-selectinϪ͞Ϫ mice at

Table 2. Leukocyte rolling velocities and wall shear rates in mouse cremaster venules Early (Ͻ30 min after exteriorization) Wild type L-selectinϪ͞Ϫ ICAM-1Ϫ͞Ϫ L-selectin͞ICAM-1Ϫ͞Ϫ Rolling vel. (␮m͞s) 36 Ϯ 0.2 (65) 19 Ϯ 0.1 (105)* 47 Ϯ 0.3 (95) 40 Ϯ 0.2 (120) Wall shear rate (sϪ1) 608 Ϯ 19 (13) 575 Ϯ 8 (19) 551 Ϯ 8 (21) 654 Ϯ 8 (24) Late (Ͼ60 min after exteriorization) Rolling vel. (␮m͞s) 38 Ϯ 0.1 (140) 24 Ϯ 0.1 (365)* 58 Ϯ 0.4 (100)‡ 64 Ϯ 0.1 (340)†§ Wall shear rate (sϪ1) 560 Ϯ 2 (73) 580 Ϯ 8 (28) 564 Ϯ 11 (20) 559 Ϯ 3 (68) TNF-␣ (3 hr) Rolling vel. (␮m͞s) 4.7 Ϯ 0.6 (247) 6.4 Ϯ 0.5 (164) 8.8 Ϯ 0.8 (105)‡¶ 8.2 Ϯ 0.6 (150)‡ Wall shear rate (sϪ1) 526 Ϯ 4 (56) 529 Ϯ 65 (33) 461 Ϯ 42 (14) 491 Ϯ 36 (28) Mean Ϯ SEM of (n) vessels or cells, respectively. vel. ϭ velocity. The TNF-␣ data from wild-type and L-selectinϪ͞Ϫ mice were published previously (12). *Significantly different from all other genotypes at the same time pont (P Ͻ 0.01). †Significantly different from the same genotype at the Ͻ30 min time pont (P Ͻ 0.01). §Significantly different from wild-type at the same time point (P Ͻ 0.01), ‡(P Ͻ 0.05). ¶Significantly different from L-selectinϪ͞Ϫ mice (P Ͻ 0.05). Downloaded by guest on September 27, 2021 Immunology: Steeber et al. Proc. Natl. Acad. Sci. USA 95 (1998) 7565

FIG. 2. Cumulative (Upper) and conventional histograms of the distribution of leukocyte rolling velocities in adhesion molecule-deficient mice. Rolling velocities were measured in hemodynamically similar cremaster venules after surgical exteriorization at times Ͻ30 min (A) and Ͼ60 min (B). (C) Mice also were pretreated with an intrascrotal injection of TNF-␣ 2.5 hr before the initiation of surgery. Cumulative frequency analysis (top row) ranks all individual cells analyzed in each mouse genotype from the cell with the highest rolling velocity (100%) to the lowest (Ͻ1%) on the y axis and plots these values as the percentage of cells that had a velocity lower than the value indicated on the x axis. Cumulative frequency analysis allows direct comparison between data sets and makes the distribution independent of bin size (Low- er; 10-␮m͞s bins in A and B,2-␮m͞s bins in C) used in conventional histograms. For numbers of venules and cells examined and statistical analysis of results see Table 2. The data from wild-type and L- selectinϪ͞Ϫ mice treated with TNF-␣ are published (12).

times Ͻ30 min (Fig. 2A). The molecular basis for this decrease rolling velocities in L-selectinϪ͞Ϫ mice at later time points were in rolling velocity is that leukocyte rolling in L-selectinϪ͞Ϫ slower than in wild-type mice (Fig. 2B), which is consistent Ϫ͞Ϫ mice completely depends on P-selectin under the conditions with the observation that residual rolling in L-selectin mice studied, as described (16). P-selectin mediates rolling at a is mediated by P-selectin under these conditions (16). How- ͞ Ϫ͞Ϫ slower characteristic velocity than L-selectin (16). The finding ever, rolling velocities in L-selectin ICAM-1 mice were ͞ Ϫ͞Ϫ significantly higher (P Ͻ 0.01) than in wild-type or L- that rolling velocities in L-selectin ICAM-1 mice were Ϫ͞Ϫ Ϫ͞Ϫ significantly higher than those of L-selectinϪ͞Ϫ mice (Table 2) selectin mice and higher than in ICAM-1 mice (Fig. 2B, demonstrates that ICAM-1 is a necessary component for Table 2). This finding again indicates that ICAM-1 expression retards the velocity of cells that roll via P-selectin. Moreover, manifestation of the slower velocity of P-selectin-mediated the finding that rolling at later time points was affected more in vivo rolling at early time points . significantly (P Ͻ 0.01) by the combined loss of L-selectin and Leukocyte rolling velocities in ICAM-1Ϫ͞Ϫ mice at later Ͼ ICAM-1 than rolling at early time points indicates that the times (t 60 min) after trauma-induced inflammation were contribution of ICAM-1 to rolling is revealed most readily significantly faster than in wild-type mice, as indicated by a when the relative contribution of P-selectin to rolling is Ͻ shift to the right of the cumulative velocity distribution (P decreased. These differences in leukocyte rolling velocities 0.05; Fig. 2B, Table 2). Because rolling at these time points is verify that ICAM-1 expression is required for optimal P- and predominantly mediated by L-selectin with a contribution by L-selectin-mediated rolling. P-selectin (9, 33), these results suggest that ICAM-1 expression After TNF-␣ treatment, leukocyte rolling velocities were also is required for optimal L-selectin-mediated rolling. Again, reduced 3- to 8-fold compared with untreated mice (Fig. 2C) Downloaded by guest on September 27, 2021 7566 Immunology: Steeber et al. Proc. Natl. Acad. Sci. USA 95 (1998)

Table 3. Emigration of neutrophils during rolling at later time points in vivo (9, 16), the current studies thioglycollate-induced peritonitis indicate a role for ICAM-1 in regulating leukocyte rolling No. of neutrophils, ϫ10Ϫ4 mediated by both P- and L-selectin. These results demonstrate that ICAM-1 expression influences selectin-mediated rolling Genotype 0hr 2hr of leukocytes in vivo, in addition to its well established roles in Wild type 0.9 Ϯ 0.7 99.4 Ϯ 23.5 firm adhesion and transmigration of leukocytes at sites of L-selectinϪ͞Ϫ 0.9 Ϯ 0.9 44.8 Ϯ 15.0* inflammation (35). ICAM-1Ϫ͞Ϫ 0.4 Ϯ 0.4 38.3 Ϯ 5.6* The specific role of ICAM-1 during selectin-mediated roll- Ϫ͞Ϫ L-selectin͞ICAM-1Ϫ͞Ϫ Ͻ0.1 Ϯ 0.1 7.8 Ϯ 3.3*†‡ ing is clarified by comparing the phenotypes of ICAM-1 mice that also lack expression of either L- or P-selectin. Given Values represent the mean Ϯ SEM of results from 4–9 mice of each genotype at each time. that P-selectin primarily mediates rolling in untreated L- selectinϪ͞Ϫ mice (13), the current findings in L-selectin͞ *Differences between test and wild-type mice at 2 hr were significant, Ϫ͞Ϫ P Ͻ 0.05. ICAM-1 mice indicate that leukocyte rolling through †Differences between L-selectinϪ͞Ϫ and L-selectin͞ICAM-1Ϫ͞Ϫ mice P-selectin does not absolutely require ICAM-1 expression. at 2 hr were significant, P Ͻ 0.05. However, ICAM-1 expression was required for P-selectin to Ϫ͞Ϫ Ϫ͞Ϫ ‡Differences between ICAM-1 and L-selectin͞ICAM-1 mice mediate rolling at characteristic velocities (Fig. 2). In P-selec- Ͻ Ϫ͞Ϫ at 2 hr were significant, P 0.05. tin͞ICAM-1 mice, rolling is absent for at least 2 hr after exteriorization of the cremaster muscle (24). Because L- as reported (12, 24). Nonetheless, differences in rolling veloc- Ϫ͞Ϫ ␣ selectin primarily mediates rolling in P-selectin mice (13), ities between groups of mice after TNF- activation were the absence of rolling in P-selectin͞ICAM-1Ϫ͞Ϫ mice indicates similar to those observed for trauma-induced rolling. Among that ICAM-1 expression is essential for leukocyte rolling treated mice, rolling velocities were significantly higher in Ϫ͞Ϫ Ϫ͞Ϫ through L-selectin. These conclusions are reinforced further ICAM-1 mice compared with wild-type or L-selectin by findings that P-selectin function-blocking antibodies inhibit mice (Table 2). As in all other groups, the combined loss of leukocyte rolling flux by 88% in ICAM-1Ϫ͞Ϫ mice after L-selectin and ICAM-1 did not alter the rolling velocity of surgical trauma (24) but have little or no effect at time points leukocytes beyond that observed for ICAM-1 deficiency alone Ͼ60 min in wild-type mice, a time period in which L-selectin (Fig. 2C). These results directly demonstrate a role for ͞ predominantly mediates rolling (13). Conversely, blocking ICAM-1 in the stabilization of leukocyte endothelial cell L-selectin function by a mAb significantly reduces the rolling interactions, which support leukocyte rolling at sites of inflam- flux fraction in wild-type mice (13) but has modest effects in mation. ICAM-1Ϫ͞Ϫ mice (24). Furthermore, L-selectin in combina- Acute Neutrophil Emigration During Peritonitis. The ex- ͞ Ϫ͞Ϫ ͞ tion with E-selectin supports rolling in P-selectin ICAM-1 tent that increased velocity and or decreased rolling flux mice treated with TNF-␣, but blocking E-selectin function in ͞ Ϫ͞Ϫ Ϫ͞Ϫ Ϫ͞Ϫ fractions in L-selectin ICAM-1 mice affected acute neu- P-selectin͞ICAM-1 or P-selectin mice eliminates roll- trophil entry into sites of inflammation was assessed in an ing (17, 24). Thus, L-selectin can only mediate leukocyte experimental model of peritonitis. Small numbers of neutro- rolling in vivo when ICAM-1, P-selectin, E-selectin, or appro- phils were present in the peritoneum of all groups of mice priate combinations of these receptors are expressed. By before thioglycollate injection (Table 3). At 2 hr after thio- contrast, P-selectin can mediate leukocyte rolling in the ab- glycollate injection, the vast majority of neutrophil entry into sence of L-selectin and ICAM-1 expression, albeit at signifi- ͞ Ϫ͞Ϫ the inflamed peritoneum of L-selectin ICAM-1 mice was cantly faster velocities. Ͻ inhibited compared with wild-type (by 92%, P 0.007), Studies (22) under in vitro conditions of shear flow have Ϫ͞Ϫ Ͻ Ϫ͞Ϫ ICAM-1 (by 80%, P 0.001), and L-selectin (by 83%, suggested that L-selectin ligands and ICAM-1 are engaged in Ͻ Ϫ͞Ϫ Ϫ͞Ϫ P 0.05) mice (Table 3). L-selectin and ICAM-1 mice series by leukocytes, rather than in parallel. However, the each had significant reductions (55% and 61%, respectively) in current in vivo results (Fig. 1, Table 2) clearly reveal that the number of infiltrating neutrophils compared with wild-type L-selectin and ICAM-1 function synergistically to mediate Ͻ mice (P 0.05), as reported (9, 20). Thus, a concurrent leukocyte endothelial interactions, including rolling. The re- L-selectin and ICAM-1 loss resulted in a far greater reduction quirement for ICAM-1 expression and function for optimal of acute neutrophil transmigration than would have been selectin-mediated rolling in vivo may relate to the relative predicted by an additive effect from loss of each individual densities of adhesion molecules and their ligands. Under adhesion molecule. physiologic conditions in vivo, relative receptor and͞or ligand densities appropriately displayed on leukocytes or endothelial ͞ DISCUSSION cells may be limiting for individual receptor ligand pairs, thereby requiring cooperative interactions between groups of The generation of mice deficient in both ICAM-1 and L- adhesion molecules. Although adhesion molecule require- selectin has provided a powerful tool to further unravel the ments for optimal rolling may vary significantly between complex interactions that occur between the selectins and different vascular beds, the consequences from loss of both members of the Ig superfamily during leukocyte interactions L-selectin and ICAM-1 were more than additive in the accu- with endothelial cells at sites of inflammation. Of primary mulation of neutrophils during acute peritonitis (Table 3). importance are two findings. First, the frequency of rolling Thus, L-selectin interactions with a vascular ligand(s) may be Ϫ͞Ϫ leukocytes in L-selectin mice treated with TNF-␣ was insufficient to mediate stable leukocyte rolling under shear decreased significantly by the additional loss of ICAM-1 flow unless leukocyte interactions with vascular endothelial expression (Fig. 1). This demonstrates a direct role for cells are supported by other adhesion molecules, such as ICAM-1 in leukocyte rolling in inflamed vessels. Second, ICAM-1 or P-selectin. leukocyte rolling velocities were significantly greater in The generally accepted model of leukocyte-endothelial in- Ϫ͞Ϫ ICAM-1 mice than in wild-type mice (Fig. 2, Table 2). teractions suggests a multistep process, with each step medi- Ϫ͞Ϫ Greater leukocyte rolling velocities for ICAM-1 mice were ated via different families of adhesion molecules. The current notable at early time points (t Ͻ 30 min) and were quite study demonstrates cooperation between the adhesion mole- significant at later time points (t Ͼ 60 min) after trauma- cules that mediate leukocyte rolling and those that previously induced inflammation (Fig. 2 A and B, Table 2). Because were presumed to mediate firm adhesion. Although the selec- ͞␤ P-selectin predominantly mediates trauma-induced rolling at tins are functionally dominant during rolling, ICAM-1 2 early time points and both L-selectin and P-selectin mediate integrin interactions stabilize selectin-mediated interactions Downloaded by guest on September 27, 2021 Immunology: Steeber et al. Proc. Natl. Acad. Sci. USA 95 (1998) 7567

between leukocytes and endothelial cells in vivo, allowing 14. Tang, M. L. K., Hale, L. P., Steeber, D. A. & Tedder, T. F. (1997) effective rolling and leukocyte entry into sites of inflamma- J. Immunol. 158, 5191–5199. tion. A requirement for ICAM-1 in stabilizing rolling may 15. Tedder, T. F., Steeber, D. A. & Pizcueta, P. (1995) J. Exp. Med. explain why anti-CD18 antibodies reduce leukocyte rolling in 181, 2259–2264. vivo at low shear rates (36). ICAM-1-mediated decreases in 16. Jung, U., Bullard, D. C., Tedder, T. F. & Ley, K. (1996) Am. J. leukocyte rolling velocities are likely to increase the frequency Physiol. 271, H2740–H2747. 17. Bullard, D. C., Kunkel, E. J., Kubo, H., Hicks, M. J., Lorenzo, I., of firm adhesions between leukocytes and endothelial cells in ␣ ␤ ␣ ␤ Doyle, N. A., Koerschuk, C. M., Ley, K. & Beaudet, A. L. (1996) vivo (37, 38). Because the 4 7 and 4 1 integrins and CD44 J. Exp. Med. 183, 2329–2336. can each support rolling in vitro under low shear flow condi- 18. Frenette, P. S., Mayadas, T. N., Rayburn, H., Hynes, R. O. & tions (6–8, 39), they also may contribute to leukocyte rolling Wagner, D. D. (1996) Cell 84, 563–574. in vivo and may regulate rolling velocities. Although integrin 19. Dustin, M. L., Rothlein, R., Bhan, A. K., Dinarello, C. A. & function predominates during firm adhesive interactions be- Springer, T. A. (1986) J. Immunol. 137, 245–253. tween cells, the selectins also may contribute to this process 20. Sligh, J. E., Jr., Ballantyne, C. M., Rich, S. S., Hawkins, H. K., (40). Therefore, instead of rolling and firm adhesion repre- Smith, C. W., Bradley, A. & Beaudet, A. L. (1993) Proc. Natl. senting separate processes mediated by different molecular Acad. Sci. USA 90, 8529–8533. mechanisms, rolling and firm adhesion are interrelated events 21. Xu, H., Gonzalo, J. A., St. Pierre, Y., Williams, I. 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