b2-Integrins mediate stable adhesion in collisional interactions between neutrophils and ICAM-1-expressing cells Eric Lynam,* Larry A. Sklar,* Andrew D. Taylor,† Sriram Neelamegham,† Bruce S. Edwards,* C. Wayne Smith,† and Scott I. Simon† *Cytometry, Cancer Research and Treatment Center, University of New Mexico Health Science Center, Albuquerque; and †Speros P. Martel Section of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine, Houston, Texas
Abstract: The aggregation of human neutrophils in suspension has features that are analogous to their attachment to activated endothelium in that both involve selectin and b2-integrin adhesion receptors. For the collisional interaction that forms neutrophil aggregates in suspension, there is a tethering step in which L-selectin on neutrophils binds PSGL-1. At relatively low shear rates (100– 200 s-1) firm adhesion is mediated in equal measure by LFA-1 binding to ICAM-3, and Mac-1 binding to an as yet undefined ligand. In this report we used a mouse melanoma cell line expressing an estimated 700,000 ICAM-1 (CD54) to examine the relative roles of LFA-1 and Mac-1 over the kinetics of heterotypic cell adhesion in shear mixed suspensions. Neither heterotypic nor homotypic neutrophil aggregates formed with application of shear alone. However, the rate of aggregation peaked within seconds of chemotactic stimulation. In contrast to homotypic aggregation, neither L-selectin nor its O-glycoprotein ligands on neutrophils contributed to heterotypic adhesion. Adhesion was inhibited in a dose-dependent manner as ICAM-1 was titrated with blocking mAb. A direct interaction between LFA-1 and ICAM-1 was preferred over the first minute of stimulation, whereas at later times adhesion was supported equally by Mac-1. Activation with MnCl2 also favored participation of the constitutively expressed LFA-1. Application of defined shear in a cone and plate viscometer showed that adhesion to the ICAM-1 cells decreased from a maximum level to baseline as shear rate increased up to 400 s-1 in a manner typical of integrin adhesion alone. In contrast, homotypic aggregation supported by the transition from selectin to integrin binding exhibited an increase in efficiency up to 800 s-1. The pathophysiological significance of receptor site density and duration of contact in collisional interactions relevant to leukocyte recruitment compared to leukocyte-endothelial cell interactions on surfaces is discussed. J. Leukoc. Biol. 64: 622–630; 1998. Key Words: cell adhesion · flow cytometry 622
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INTRODUCTION The capture of polymorphonuclear neutrophils (PMN) by the vascular endothelium in response to inflammatory stimuli requires transient tethering or rolling via endothelial and leukocyte selectins, and firm attachment via b2-integrins [1–3]. Activated endothelial cells first engage PMN through recognition of glycoproteins by P- and E-selectins (CD62P, CD62E), and by L-selectin (CD62L) on the leukocyte [1, 4–11]. Tethering and rolling of PMN through selectins decreases the velocity and increases the duration of intercellular contact and the membrane contact area on the endothelium [12]. When PMN activation occurs through inflammatory mediators such as intlerleukin-8 (IL-8) and platelet-activating factor (PAF) [1, 4, 7–11], firm attachment occurs via the b2-integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), which recognize ICAM-1 (CD54) and ICAM-2 (CD102) expressed on the endothelium [1, 3, 11, 13]. As in the process of PMN margination on activated endothelium, PMN-PMN adhesion (homotypic aggregation) also involves initial tethering via L-selectin followed by b2-integrindependent firm adhesion [14, 15]. Homotypic aggregation is stimulated by many of the same chemotactic factors that stimulate firm adhesion to vascular sites of inflammation. Although the integrin counter-structure(s) for aggregation remains to be identified, L-selectin tethers cells by transiently binding to PSGL-1 on PMN [16, 17]. Removing L-selectin or blocking its binding to PSGL-1 reduces, but does not eliminate, PMN aggregate formation [15–18]. Transient, homotypic interactions between PMN in the free stream and that adherent to a substrate coated with purified ligand has been shown to amplify the number of cells that marginate and roll under shear flow [19, 20]. This process was inhibited by anti-PSGL-1 or anti-L-selectin. We have recently reported that the requirement for L-selectin tethering in homotypic aggregation depends on
Abbreviations: PMN, polymorphonuclear neutrophils; IL-8, interleukin-8; PAF, platelet-activating factor; fMLF, N-formyl-methionyl-leucyl-phenylalanine; MAA, melanoma-associated antigen; HSA, human serum albumin; LPS, lipopolysaccharide; LBP, LPS-binding protein; PE, phycoerythrin; FITC, fluorescein isothiocyanate. Correspondence: Larry A. Sklar, Ph.D, Cytometry, Cancer Research and Treatment Center, University of New Mexico Health Science Center, Albuquerque, NM 87131. E-mail:
[email protected] Received September 1, 1997; revised July 13, 1998; accepted July 15, 1998.
the magnitude of the hydrodynamic shear. At relatively low shear rates (#100 s-1) homotypic aggregation is L-selectin independent and can be achieved solely by activation of CD11a/CD18 and CD11b/CD18 [21, 22]. At higher shear rates the initial tethering via L-selectin appears to support aggregation by sustaining the duration of intercellular contact enabling integrin receptors to engage. The presence of selectins boosts the efficiency of aggregation by 100%, up to a peak level of 80% successful collisions at shear rates ,800 s-1 [21]. Although selectins and their ligands promote integrindependent adhesion at typical shear stresses in post capillary venules, under low flow condition, leukocyte adhesion to substrates can also become strictly integrin dependent [23, 24]. In rats, lowering blood flow rates by 50% permitted the CD11b/CD18-dependent tethering and firm adhesion of leukocytes in PAF-stimulated mesentery venules under conditions where selectins were blocked [23]. Indeed, b2-integrindeficient leukocytes or normal leukocytes treated with antiCD18 mAbs did not roll on or adhere to surfaces of cultured endothelial cells [25]. Selectin-independent leukocyte recruitment into the inflamed liver microvasculature occurs under conditions of N-formyl-methionyl-leucyl-phenylalanine (fMLF) activation, presumably by b2-integrin binding to ICAM-1, in post-sinusoidal venules [24]. These studies indicate that direct integrin recognition may be important in leukocyte adhesion under conditions of reduced blood flow in vivo or reduced shear in vitro. Recent published data indicate that homotypic neutrophil aggregation mediated by b2-integrin may be limited by the duration of intercellular contact [21, 22]. Two mechanisms that enable integrins to rapidly up-regulate the adhesive function of the cell are a conversion of the individual heterodimers to adopt a high affinity ligand binding state, and clustering of integrins in the plane of the membrane [26]. In this study we address the hypothesis that the limit on integrin bond formation imposed on cells in shear flow may be compensated by an increase in the density of counter-ligand or by the activation state of the integrin. To study the direct b2-integrin-dependent adhesion of neutrophils, we have employed a murine melanoma cell line (Ui11/ E3) that has been transfected to express high levels of human ICAM-1 [27]. This model allows us to examine adhesion over a range of shear rates that typically require a primary tethering step via selectins. Adhesion required a high ICAM-1 surface density and decreased in direct proportion to blocking available ICAM-1 sites. Adhesion via CD11a was favored at early times after stimulation by fMLF or by activation with MnCl2. b2-Integrin-dependent heterotypic adhesion decreased in efficiency as shear was increased, whereas homotypic aggregation mediated by both L-selectin and integrin increased up to a shear rate of 800 s-1. The data indicate that at venous shear rates and at a sufficient ICAM-1 surface density, LFA-1 or Mac-1 can mediate firm neutrophil adhesion provided the contact duration is adequate. The suspension system allows collisional interactions to be formed under contact times up to 25 times longer than those supporting surface adhesion under similar shear rates (see Discussion). Lynam et al.
MATERIALS AND METHODS Cells Human blood was collected by venipuncture into sterile syringes containing 10 U heparin/mL blood. PMN were isolated using Mono-Poly Resolving Medium (Flow Laboratories, McLean, VA) and then washed in HEPES buffer (110 mM NaCl, 10 mM KCl, 10 nM glucose, 1 mM MgCl2, and 30 mM HEPES, pH 7.35) containing 1.5 mM CaCl2. The buffer had been depleted of LPS by affinity chromatography over polymyxin B Sepharose (Detoxigel, Pierce Scientific, Rockford, IL). The isolated cells were kept on ice for up to 3 h until use. The murine melanoma cell line B78H1 [26] was transfected with a melanoma-associated antigen (96-kDa MAA) using chromosomal DNA from human melanoma cells [27]. The transfected cells, designated Ui11, expressed human ICAM-1 as determined by direct immunofluorescence. A subclone of the transfected cells, Ui11/E3, was selected by flow-associated cell sorting on the basis of relatively low expression of the ICAM-1 gene. Ui11/E3 was maintained in RPMI 1640, supplemented with 10–20% fetal bovine serum and in the presence of the selective agent G-418 sulfate (GIBCO, Grand Island, NY).
Reagents and antibodies fMLF (CHO-Met-Leu-Phe) was purchased from Sigma (St. Louis, MO), human serum albumin (HSA) from Armour Pharmaceutical (Kankakee, IL), and LPS Re595 Salmonella minnesota from List Biological (Campbell, CA). LBP, rabbit lipopolysaccharide (LPS) binding protein, was provided by Drs. Richard Ulevitch and Peter Tobias at The Scripps Research Institute (La Jolla, CA) and O-glycoprotein endopeptidase (OSGE), isolated from Pasteurella hemolytica, was provided by Dr. Allan Mellors at the University of Guelph (Guelph, Canada). My-4 (anti-CD14) was purchased from Coulter Immunology (Hialeah, FL) and used at 20 µg/mL. This has been shown to block the shedding of L-selectin and the up-regulation of CD11b in response to LPS/LBP but not the direct effects of OSGE on PMN mucins [16, 18]. Blocking antibodies IB4 (anti-CD18) and DREG-200 Fab (anti L-selectin) were obtained from Dr. David Chambers at La Jolla Institute for Experimental Medicine (La Jolla, CA) and DREG-200 (anti L-selectin) was a gift from Dr. Takashei Kishimoto at Boehringer Ingleheim (Ridgefield, CT). Other blocking antibodies, R6.5 (anti-ICAM-1), R3.1 (anti-CD11a), and R15.7 (anti-CD18) were provided by Dr. Robert Rothlein at Boehringer Ingleheim and 60.1 (anti-CD11b) was obtained from Dr. J. Rusche at Repligen Corp. (Cambridge, MA). IB4 and DREG-200 were either used at 20 µg/mL to block adhesion or at 5 µg/mL for indirect immunofluorescence. mAb R3.1 was used at 20 µg/mL. mAb R15.7 was used at 10 µg/mL, and 60.1 at 20 µg/mL. A Cy3-labeled Fab fragment of R6.5 was used at the doses between 0.5 and 20 µg/mL. To label PMN for detection in aggregation experiments, CD44-FITC or CD45-FITC (Becton Dickinson Immunocytometry Systems, San Jose, CA) was used at 0.3 µg/mL. mAb24, a reporter for the b2-integrin activation epitope, was provided by Dr. Nancy Hogg at the Macrophage Laboratory of the Imperial Cancer Research Fund (London, England) and was used at 5 µg/mL. Leu-8-FITC (Becton Dickinson) was used at 0.3 µg/mL to label cellular L-selectin. Leu-15-PE (Becton Dickinson) was used at 1.25 µg/mL to label CD11b (CD11b/CD18). Anti-sialophorin (CD43) and anti-sialyl Lewis X (Becton Dickinson Cellular Imaging Systems, San Jose, CA) were used at 5 µg/mL as primary probes in indirect immunofluorescence. LDS-751 (Exciton, Dayton, OH), a vital nuclear stain, was dissolved in methanol at 0.2 mg/mL and used at 0.2 µg/mL to label PMN or Ui11/E3 cells.
L-selectin shedding protocols LPS Re595 Salmonella minnesota was suspended at 40 µg/mL by repeated brief sonication into an LPS buffer (10 mM EDTA, 50 mM HEPES, and 0.1% HSA, pH 7.4) and stored at 4°C until use. Complexes of LPS/LBP were produced by combining LPS and LBP in a mass ratio of 1:3, respectively, at 37°C for 10 min as described earlier [28]. The resulting stock solution of LPS/LBP contained LPS at 10 µg/mL. Neutrophil treatment with LPS/LBP was performed at a concentration of 100 ng/mL. OSGE was used at 25 µg/106 cells to deplete PMN of O-linked glycoproteins [16, 29]. To prevent the effects of LPS in the OSGE preparation, PMN were pretreated with 20 µg/mL My-4 at 37°C for 10 min before exposure to OSGE.
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Receptor expression studies For direct immunofluorescence, cells (106/mL) were suspended in HEPES buffer containing 0.1% HSA and incubated at 4°C with fluorophore-conjugated antibodies for 1 h. Receptor expression was analyzed using a FACScan flow cytometer, equipped with FACScan Research software (Becton Dickinson). The specificity of labeling for direct immunofluorescence was determined by subtracting the fluorescence of the samples with their respective matched isotype controls. During indirect immunofluorescence measurements, samples were incubated with the primary antibody at 4°C for 30 min, washed, and resuspended in fresh buffer, then labeled with (10 µg/mL) phycoerythrin (PE)-conjugated polyclonal goat-anti-mouse F(ab’)2 (DAKO, Carpinteria, CA) at 4°C for 30 min. In indirect immunofluorescence, specific labeling was determined by subtracting the fluorescence of samples labeled with secondary antibody alone and validated by comparison with matched isotype controls.
in HEPES buffer containing 0.1% HSA at 37°C. Likewise, Ui11/E3 cells were labeled with LDS-751, washed, and in some cases preincubated with adhesion blocking mAbs at 22°C for 15 min and combined with PMN (3 3 106/mL each). This mixture was equilibrated for 2 min at 37°C and stirred at ,500 rpm using a 7 3 2-mm magnet in the sample tube. The device was directly connected to the sampling port of the flow cytometer and the initial particle size distribution was measured. Two thousand cellular events were acquired before stimulation and at intervals following the addition of fMLF (100 nM) or MnCl2 (3 mM). Parallel, sham-treated controls were run for each variable tested. Aggregation was resolved on the basis of homogenous fluorescence as singlets and aggregates (Fig. 1). The mean percentages of PMN aggregates and E3-ICAM-1 cells in heterotypic aggregates with PMN were calculated as follows: % PMN homotypic aggregation 5 [(2 3 G2 1 3 3 G3 1 4 3 G41)/(G1 1 2 3 G2 1 3 3 G3 1 4 3 G41)] 3 100. %E3-ICAM-1 heterotypic aggregation 5 [(R1G1 1 R1G2 1 R1G3)/(R1 1 R1G1 1 R1G2 1 R1G3)] 3 100.
Measurement of the mAb24 epitope on PMN
Cone and plate shear studies
mAb24 was directly conjugated with Cy3 using a fluorolink antibody-labeling kit from Amersham Life Sciences (Pittsburgh, PA). On excitation at 488 nm Cy3 is detected on the red flow cytometer channel. Isolated PMN were preincubated with mAb24-Cy3 (5 µg/mL) for 10 min at 22°C at a low cell density (105/mL) in order to minimize aggregate formation after stimulation. The suspension was then warmed to 37°C for 2 min and the initial mAb24-Cy3 fluorescence was measured by gating on the singlet PMN population. Shear was then initiated either in the presence or absence of 3 mM MnCl2 and the fluorescence emitted by the Cy3-conjugated antibody measured for 5 min. After this, fMLF (100 nM) was added and measurements were continued for another 10 min.
The cone-plate viscometer consists of a stationary plate beneath a rotating cone, which enables a uniform shear rate to be applied over the entire sample [21, 22]. The shear rate (G) is independent of distance from the cone center and angular velocity of the cone, and is given by G 5 wsinq, where w is the rotation rate and q is the cone angle. A truncated cone with an angle of 1° was used, and the gap between the cone and plate ranged from 90 µm in the center to 610 µm at the outside edge. At a defined shear rate the shear stress (t) for a Newtonian fluid is t 5 µG, where µ is the fluid viscosity, ,0.007 poise for buffer at 37°C. In cone and plate aggregometry, PMN were labeled with 5 µg/mL anti-CD45fluorescein isothiocyanate (FITC; Becton Dickinson), and E3-ICAM-1 cells were stained with LDS-751. Like the stirred system, these spectrally distinct labels allowed the quantitation of homotypic PMN aggregates, as well as PMN-Ui11/E3 heterotypic aggregates. Excess label was removed by centrifugation, and the two cell populations (each at 3 3 106 cells/mL) were combined in aggregation buffer and equilibrated at 37°C for 2 min. The combined sample was then stimulated with fMLF and exposed to fluid shear as described above. Aliquots of 50 µL were taken at desired time points and fixed in 50 µL of 0.5% cold paraformaldehyde. Paraformaldehyde does not fluoresce with 488 nm excitation and does not interfere with dual-color fluorescence detection. In the shear dependence study, heterotypic adhesion was expressed as the number of PMN adherent to Ui11/E3 cells normalized by the total PMN.
Heterotypic aggregation Two types of mixing devices were used to initiate neutrophil-Uill/E3 collisions in suspension and consequently heterotypic aggregation. The first induced shear in a stirring device with the use of a 7 3 2-mm magnet in the polypropylene flow cytometer sample tube [30]. Although this stirred aggregometry technique allowed live sampling of the aggregating sample, its vigorous mixing sytem caused non-uniform and ill-defined shear fields. Therefore, the shear dependence was also studied in a cone-plate viscometer (Ferranti Electric, Inc., Commack, NY).
Shear mixing using a stir bar The measurement of aggregation of live cells by flow cytometry has been described previously [14–18, 30, 31]. Briefly, PMN were untreated or pretreated with LPS/LBP (100 ng/mL [18] or OSGE (25 µg/106 cells, in the presence of My4) at 37°C for 25 min. Alternatively, PMN were preincubated with adhesion blocking mAbs at 37°C for 5 min. The cells were labeled with anti-CD44-FITC for 5 min, quickly pelleted (2–3 s at 3000 g), and resuspended
RESULTS Expression of adhesive epitopes on ICAM-1 We examined the expression of molecules on E3-ICAM-1 target cells that could potentially serve as adhesive ligands for PMN.
Fig. 1. Flow cytometric analysis of the aggregation of PMN with E3-ICAM-1 cells. Isolated PMN (3 3 106/mL) were labeled with anti-CD44-FITC and E3-ICAM-1 cells (3 3 106/mL) were labeled with LDS-751 as described in Materials and Methods. After labeling, cells were stirred together for 2 min at 37°C before acquisition of a baseline measurement. Aggregation was initiated by addition of fMLF (100 nM). Resolution of cells and aggregate types within the mixed sample is depicted in representative dot plots of: (A) light scatter properties in unstimulated cells; (B) two-color detection, E3-ICAM-1 vs. PMN fluorescence, unstimulated; (C) dual population aggregation of E3-ICAM-1 and PMN, stimulated (G1 5 single PMN, G2 5 doublets, G3 5 triplets and larger aggregates, R1 5 single E3-ICAM-1, R1G1 5 heterotypic aggregate with 1 PMN and 1 E3-ICAM-1, R1G2 5 PMN doublet with 1 E3-ICAM-1, etc.). Each dot represents one particle event as detected on the flow cytometer. Representative dot-plots are from experiments performed at least 10 times in duplicate.
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Expression of human ICAM-1 was quantitated by comparing the fluorescence of mAb R6.5-FITC on target cells with mAb binding to Simply Cellular calibration beads. The transfectants were found to bind ,340,000 mAb molecules. The transfectants did not express human antigens LFA-3 (CD58), CD11a, CD18, CD2, CD45, CD3, CD5, or CD16 [32]. We verified that other classes of adhesive ligands that support PMN aggregation were also not detected: b2-integrins, L-selectin, and mucins with derivatives of sialyl Lewis-X as terminal carbohydrates.
PMN adhesion analyzed by flow cytometry Equal concentrations of PMN and the transfectants were added to a cytometry test tube and sheared with a magnetic stir bar at a level corresponding to ,1–2 dynes/cm2. Light scatter on the flow cytometer (Fig. 1) resolved the two populations. The transfectants exhibit higher forward angle light scatter and lower side scatter as compared to the PMN due to their larger size (,14 µm diameter vs. ,8 µm diameter for PMN) and lower granularity (Fig. 1A). Unstimulated PMN labeled green with CD45-FITC and transfectants stained red with the LDS-751 fluorescent dye remained as discrete populations under shear as depicted on the two-color dot-plot (Fig. 1B). After chemotactic stimulation, PMN engaged in both homotypic aggregation and heterotypic adhesion with the transfectants (Fig. 1C). Homotypic PMN aggregates were resolved along the green fluorescence axis. Heterotypic aggregates of PMN with transfectants exhibited both red and green fluorescence. Neither the transfectants nor the parental cell line B78H1 aggregated in response to stimulation. Microscopic observation confirmed that gluteraldehyde-fixed transfectants were in aggregates with one to four PMN. The kinetics of aggregate formation were measured for sheared samples, mixed, and directly injected into the flow cytometer (Fig. 2). Unstimulated PMN exhibited little tendency to adhere in the absence of chemotactic activation (,5% of cells in homotypic aggregates) [15–18, 30, 31]. On addition
Fig. 2. Kinetics of PMN adhesion to E3-ICAM-1 and homotypic aggregation. PMN and E3-ICAM-1 combined at 3 x 106/mL each were sheared and stimulated as described in Figure 1. After the pre-stimulation data point was acquired, aggregation was induced with fMLF (100 nM). Subsequent data points were acquired at the times indicated. Data are presented as the mean percent of PMN in aggregates (closed symbols) and the mean percent of total E3-ICAM-1 in aggregates with PMN (open symbols) 6 SEM.
Lynam et al.
Fig. 3. Selectin dependence of PMN adhesion to E3-ICAM-1. PMN were stimulated in suspension with E3-ICAM-1cells in the presence or absence of anti-L-selectin (DREG 200, 20 µg/mL), LPS (100 ng/mL) complexed with LBP, and OSGE (25 µg/106 PMN) as detailed in Materials and Methods. Data represent the percent of peak heterotypic aggregation 6 SEM in response to fMLF (100 nM). Where indicated the untransfected parent cell line B78H1 was used as the target cells.
of 100 nM fMLF, homotypic and heterotypic aggregates formed rapidly. Homotypic aggregation reached a maximum near 30 s, whereas heterotypic aggregation continued to increase over the 1st min after stimulation. Both adhesive interactions were reversible with time after maximum adhesion.
Role of L-selectin receptors in PMN adhesion In the absence of stimulus a low baseline level of adhesion was measured for neutrophil adhesion with ICAM-1 transfectants (9 6 2% of PMN in heterotypic aggregates or about 20% of the maximum response, Fig. 3). This level was comparable to fMLF-stimulated adhesion to the untransfected parental cell line B78H1 (Fig. 3). After addition of fMLF, adhesion increased four- to fivefold over unstimulated levels. Homotypic aggregation has been shown to depend on the expression of several classes of adhesion molecules, including the b2-integrin, L-selectin [14, 15], and more recently PSGL-1, a mucin-like glycoprotein expressed on the PMN [16, 17]. We evaluated the effect of agents that block or shed L-selectin and its Oglycosylated ligands on PMN adhesion. DREG-200, a mAb that binds the lectin domain of L-selectin inhibited homotypic aggregation but did not affect the fraction of transfectants bound to PMN. Incubation of PMN with LPS/LBP induces the shedding of L-selectin and the up-regulation of CD11b [28, 33, 34]. As previously observed, this treatment inhibited homotypic aggregation in response to fMLF [15, 16, 18]. However, despite a threefold increase in CD18 expression the extent of adhesion between PMN and transfectants was not significantly different from the stimulated control. An OSGE isolated from Pasteurella hemolytica has been shown to deplete surface expression of CD43, CD44, CD45, and PSGL-1 [7, 29], and also inhibit homotypic adhesion [16, 17, 19, 20]. Pretreatment of PMN with
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OSGE abolished homotypic (not shown) [see ref. 16] but not heterotypic aggregation. Taken together, neither cellular Lselectin nor the OSGE-sensitive glycoproteins including PSGL-1 were required for PMN adhesion to the ICAM-1 transfectants.
Numeric dependence on ICAM-1 for heterotypic adhesion We explored the dependence of adhesion on ICAM-1 expression level by preblocking the transfectants with increasing concentrations of mAb R6.5-Fab (Fig. 4). The binding of R6.5 to transfectants yielded an equilibrium dissociation constant, Kd ,94 µg/mL. At this concentration of anti-ICAM-1, approximately 50% of the heterotypic adhesion was inhibited. Adhesion was reduced in proportion to the fraction of ICAM-1 blocked by mAb. Homotypic aggregation was not affected at any dose of R6.5 tested (data not shown).
Requirement for CD11a and CD11b in heterotypic adhesion It is well established that both CD11a and CD11b can bind to ICAM-1 and mediate PMN adhesion [13]. We examined the requirement for b2-integrins on PMN in mediating adhesion to transfectants under hydrodynamic shear in response to stimulation by fMLF. Pretreatment of PMN with an anti-CD18 mAb R15.7 (not shown) or a combination of anti-CD11a and anti-CD11b inhibited the heterotypic adhesion to baseline levels (Fig. 5). Blocking CD11a (R3.1) or CD11b (60.1) separately inhibited the maximal heterotypic aggregation by 30 and 40%, respectively. However, at the 30-s time point, blocking CD11a inhibited considerably better than blocking CD11b (Fig. 5, P , 0.05 in matched t test). These data indicate that CD11a and CD11b account for PMN adhesion to ICAM-1 target cells. Moreover, CD11a is favored at early time points .
Integrin activation and adhesion to ICAM-1 Divalent cations such as Mn21 induce and stabilize integrin adhesive functions independent of signaling through chemotac-
Fig. 4. ICAM-1 dependence of PMN-E3-ICAM-1 aggregation. PMN and E3-ICAM-1 were incubated with R6.5 Fab for 15 min at room temperature at concentrations from 0.5 to 20 µg/mL. The fraction of occupancy relative to saturation was determined by flow cytometry for the binding of R6.5-Fab to E3-ICAM-1 cells (Kd ,0.94 µg/mL). The percent of heterotypic aggregation relative to control unblocked at 1 min after addition of fMLF is plotted vs. the fraction of ICAM-1 sites bound with mAb.
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Fig. 5. Dependence of heterotypic aggregation on CD11a and CD11b. PMN were pretreated with 60.1 F(ab)2 (anti-CD11b, 20 µg/mL), R3.1-Fab (antiCD11a, 20 µg/mL), or the combination of 60.1 and R3.1 as denoted. PMN were mixed with E3-ICAM-1 and stimulated with fMLF. The fraction of E3-ICAM-1 taken up into heterotypic aggregates with time is plotted. Shown is a representative plot from at least four experiments.
tic or chemokine receptors [35–37]. Mn21 induced aggregate formation at levels 30–50% of that observed with optimal fMLF stimulation (Fig. 6A). Subsequent activation with fMLF resulted in a second phase of heterotypic aggregation. Aggregation was dependent on LFA-1, and to a lesser extent Mac-1; blocking with the combination of anti-CD11a and anti-CD11b, or anti-CD18 (data not shown) resulted in inhibition to baseline. Pretreatment with mAb R3.1-Fab to block CD11a suggested that CD11a was somewhat favored over CD11b in both Mn21and fMLF-stimulated adhesion. Antibody studies suggest that Mn21 can induce an active ligand binding state in CD11a, and CD11b [36–38]. To understand the effects of Mn21 and chemotactic stimulation in terms of b2-integrin activation, we examined the expression of the epitope for mAb 24 that is associated with ligand binding and cell adhesion [38]. In the absence of activation, isolated PMN express very low levels of the mAb 24 epitope. Mn21 increased expression of the mAb 24 epitope to only a small fraction of the level induced by fMLF (Fig. 6B). This increase was apparently due to activation of constitutively expressed b2-integrin because a mAb to a common epitope of CD18 did not up-regulate with Mn21. Moreover, the appearance of the mAb 24 epitope occurred before the up-regulation of CD18. After activation by fMLP, both mAb 24 and anti-CD18 increased with comparable rates. The correlation between the kinetics and extent of mAb 24 binding and adhesion indicated that Mn21 induced expression of an activation epitope that was associated with adhesion to ICAM-1.
Adhesion under defined hydrodynamic shear Adhesion to ICAM-1 transfectants and homotypic neutrophil aggregation occurs simultaneously in a cone and plate viscometer (Fig. 7). As reported in isolated PMN suspensions [21], a two-step process involving selectin tethering and integrin firm adhesion resulted in increasing PMN aggregation as shear rates
DISCUSSION A hallmark of the versatility of PMN in inflammation is the repertoire of adhesive interactions that enable them to emigrate at specific sites in the vasculature. PMN have been shown to transiently adhere and roll on endothelial cells [13, 25, 39], platelets [40], and other PMN [19, 20]. Current models predict a shift in the molecular dependence from selectin-mediated cell capture and rolling, to firm adhesion mediated by integrins and ICAMs [1–3, 39, 40–43]. This transition has been observed experimentally as an increased resistance to the shear forces of blood or fluid flow [21, 25]. Under experimental conditions in which both selectin- and integrin-dependent adhesion mechanisms appear to occur simultaneously, it is difficult to perform a direct analysis of integrin-mediated adhesion. In this report we present an experimental model to study the integrin receptor dynamics that support adhesion between PMN and target cells
Fig. 6. Effects of sequential stimulation with MnCl2 and fMLF on PMN adhesion to ICAM-1. (A) Heterotypic adhesion between PMN and E3-ICAM-1 stimulated with MnCl2 (3 mM) at t . 0 s followed by addition of fMLF (100 nM) at t 5 5 min. The mean 6 SEM (n 5 4) percent heterotypic aggregation was measured for cell suspensions pretreated with 60.1 F(ab)2 (anti-CD11b, 20 µg/mL), R3.1-Fab (anti-CD11a, 20 µg/mL), or the combination (*significant difference in the means of anti-CD11a/CD18 vs. anti-CD11b/CD18 at P , 0.05). (B) b2-integrin expression was quantitated with mAb24-Cy3 (5 µg/mL) to an activation epitope on CD18 or with MHM23-FITC to a common epitope. PMN were incubated for 10 min at 22°C, then warmed to 37°C. After measuring the baseline fluorescence, MnCl2 (3 mM) was added followed by fMLF (100 nM) as denoted. Data depict the increase in the mean fluorescence intensity of bound mAb relative to that at baseline before activation and is a representative plot from at least three separate determinations.
increase from 100 to 800 s-1 (Fig. 7A). As in the stir bar mixing system, shear alone did not induce significant heterotypic adhesion of the ICAM-1 transfectants (data not shown). After fMLF stimulation, aggregation peaked within 1 min and cells disaggregated over a period of several minutes, particularly at shear rates $400 s-1 (Fig. 7B). The extent of heterotypic aggregation in the viscometer decreased as shear increased in a manner comparable to PMN with L-selectin shed or blocked by mAb [21]. Adhesion at a shear rate of 300 s-1 was comparable to that in suspensions mixed with the magnetic stir bar. Heterotypic aggregation peaked by ,90 s after stimulation. The rate of disaggregation was markedly less than for homotypic aggregates and was somewhat resistant to a ramp increase in shear from 100 to 800 s-1. Lynam et al.
Fig. 7. Kinetics of aggregation under defined hydrodynamic shear. Isolated PMN and E3-ICAM-1 cells were labeled and combined for the aggregation assay as described in Materials and Methods for cone and plate shear studies. Shear rates in the viscometer are defined by the first equation in Materials and Methods. At the designated time points before and after activation with fMLF (100 nM), small aliquots were removed and fixed in 0.5% cold paraformaldehyde. Aggregation was determined by flow cytometry and is depicted for (A) homotypic aggregation and (B) PMN-E3-ICAM-1 adhesion. Data are expressed for the mean aggregation 6 SEM (n 5 3).
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expressing levels of ICAM-1 typical of cytokine-activated endothelium. This cell line (1) expresses adequate numbers of counter-structure for b2-integrin-dependent adhesion in sheared suspensions; (2) lacks counter-structures such as selectins, which would augment integrin-dependent adhesion; and (3) possesses adhesive properties that are not altered by compounds that activate PMN. To characterize the ICAM-1 transfectant model, we verified that PMN aggregated with the target cells via b2-integrin binding to ICAM-1. This was demonstrated by the dosedependent inhibition of aggregation by anti-ICAM-1 mAb, and by the low level of PMN adhesion to the untransfected parent cell line B78H1. Also, antibodies directed at CD18, or both CD11a and CD11b, blocked heterotypic adhesion, whereas anti-CD11a or anti-CD11b alone resulted in partial inhibition. The dependence of heterotypic aggregation on L-selectin and O-glycoproteins was ruled out. Consistent with previous studies [15–18, 21, 22], the blockade or shedding of L-selectin sites or the proteolytic cleavage of O-glycoproteins inhibited homotypic, but did not inhibit PMN-ICAM-1 transfectant adhesion. It appears that the process of L-selectin ligation and subsequent signal transduction previously reported to potentiate the activation of b2-integrin adhesion is not requisite for the equivalent avidity increase exhibited by b2-integrin binding to the ICAM-1 transfectants. Together with the apparent lack of other relevant adhesion molecules on the transfectants, the inhibition studies support the conclusion that direct adhesion involving b2-integrin and ICAM-1 accounts for heterotypic aggregation at levels of hydrodynamic shear that also support homotypic aggregation. Although recognition occurred when there was a large excess of ICAM-1 sites relative to b2-integrins, we do not yet know how the interaction depends on the relative density of the two classes of receptors. It is conceivable that when activated b2-integrins and their counter-structures are both numerous, direct recognition could entirely supplant selectin-dependent mechanisms. Conversely, if cognate integrin ligands exist at comparatively low densities, then the sequential adhesion pathway may afford precise control of leukocyte trafficking by limiting leukocyte adhesion when cellular L-selectin is diminished, unless shear rates are also reduced [23, 24].
Comparison of homotypic and heterotypic adhesion Because leukocyte recruitment or secondary adhesion is intrinsically a collisional process, the ability of adhesion molecules to function on suspended cells where the encounter geometry differs than on a surface must be experimentally verified. In circulating blood, collisions occur between cells flowing in free suspension and between freely flowing cells and immobile endothelial cells lining vessel walls. Mathematical models of these two types of collisions suggest important differences that impact adhesion receptor operation. First, the average contact angle for cell collisions in free suspensions is ,49°, as compared to ,22° for cells colliding with planar monolayers analogous to vessel walls [21]. Second, at a fixed shear rate, the average duration of cell contact, independent of receptor 628
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engagement, is estimated to be ,26 times longer for cells colliding in suspension [45, 46]. The simultaneous aggregation of PMN with the ICAM-1 transfectants and other PMN permitted an evaluation of the relationship between integrin activation and efficiency in one-step and two-step processes of adhesion by collision. At the lower shear rates produced in the test tube by the rotating stir bar, the one-step and two-step mechanisms produce comparable aggregate formation (Fig. 2). However, in the cone and plate viscometer, the processes were clearly distinguishable with homotypic adhesion increasing in efficiency and heterotypic adhesion decreasing in efficiency as the shear rate increased (Fig. 7). Taylor et al. [21] have calculated that 30% of the collisions between activated PMNs result in aggregate formation at a shear rate of 100 s-1, which increased to 80% at 800 s-1. Calculations (not shown) indicate that PMN adhesion to the ICAM-1 transfectants and homotypic aggregation occurs at similar efficiencies at 100 s-1. Adhesion efficiency falls off with increased shear rate in the same manner for b2-integrindependent adhesion to ICAM-1 transfectants and homotypic PMN aggregation when the L-selectin-mediated step was blocked by mAb or shedding [21]. The interpretation is that it is the brief intercellular contact duration that limits aggregate formation mediated by integrin binding alone. At shear rates that correspond to optimal homotypic aggregation (400 s-1), the estimated duration over which two cells were in compression was ,6 ms [21]. This interval was insufficient for firm adhesion mediated through binding of Mac-1 and LFA-1 alone. However, at 100 s-1 and an estimated encounter duration of ,25 ms, adhesion through integrin binding alone was detected. At shear rates greater than 400 s-1 the encounter duration should still be well within the molecular association rate reported for selectins (,107 s-1). Therefore, a critical function of the L-selectin bond, which has been reported to have a lifetime of ,150 ms [20], is to enable engagement of sufficient Mac-1 and LFA-1 bonds to form. Although chemotactic stimulation produces a profound upregulation of integrin expression and binding activity, integrin function can also be induced without cellular activation. Mn21 binding at millimolar concentrations has been shown to induce up to a fivefold increase in CD11b-dependent cellular adhesion to substrates compared with extracellular Ca21 or the combination of Ca21 and Mg21 [35, 36]. We have recently shown that MnCl2 prolongs the interaction between b2-integrins and their counter-structure in stimulated homotypic aggregation, where it has no effect on the resting levels or shedding of L-selectin after fMLF activation [31]. In this study, MnCl2 (3 mM) promoted binding of an antibody (mAb 24) that recognizes an activationinduced epitope of b2-integrin (Figs. 5 and 6). The direct recognition of ICAM-1 could be induced by cellular activation with fMLF or by integrin activation with Mn21. In a previous study using GlyCAM-1 to activate lymphocytes through L-selectin, binding of mAb 24 was initiated within seconds and was accompanied by adhesion to the same ICAM-1 transfectants used here [44]. The current data indicate that the constitutively expressed LFA-1 early in the kinetics of adhesion contributed to a greater degree than Mac-1 in binding to ICAM-1 after stimulation with Mn21. Sequential stimulation of
PMN with Mn21 followed by fMLF correlated with slow and fast phases of adhesion between PMN and ICAM-1 transfectants. The slow aggregation was associated with Mn21-activated b2-integrin as detected by mAb 24, which did not require up-regulation of additional CD18 as detected by MHM23 binding to the common epitope. After fMLF stimulation, both epitopes of CD18 were up-regulated at comparable rates. These results indicate that, although LFA-1 and Mac-1 can contribute equally to adhesion with maximal chemotactic stimulation, their avidity appears to be modulated differently over time and in response to the specific stimuli. These studies performed on neutrophils under defined shear rates support a model for integrin binding and anchoring that is dependent on contact duration and the relative activation state of LFA-1 and Mac-1. The time course of the contributions of Mac-1 and LFA-1 to the capture of E3-ICAM-1 cells has been studied recently in more detail (47). The implications for neutrophil recruitment in vivo are that hydrodynamics and collisional geometry can influence cell targeting to sites of inflammation. ICAM-1 may be up-regulated by cytokines not only on endothelium but also on parenchymal cells, including hepatocytes [48]. One of us (C. W. S.) has recently reported on the molecules involved in the adherence of neutrophils to hepatocytes in vitro. In the absence of exogenous chemotactic activation, neutrophils adhered to cytokine-treated hepatocytes via LFA-1 and ICAM-1 [49]. Adhesion via activated Mac-1 is subsequently required for the release of proteolytic enzymes and reactive oxygen species by neutrophils that are cytotoxic to parenchymal cells in vitro (e.g., cardiac myocytes or hepatocytes). The pathophysiological importance of activated CD18 binding to ICAM-1 is underscored by the reduction in neutrophil-induced tissue damage in vivo after the addition of blocking antibodies to these adhesion receptors [50].
Summary The findings of this study are consistent with the idea that PMN can attach to other cells through distinct mechanisms, some of which may be better suited to rolling or collisional adhesion geometries. With respect to cell surfaces or already adherent cells, the predominant mechanism of interaction at normal shear rates involves sequential participation of selectins and integrins, whereas at low shear rates, adhesion can be supported solely through integrin and ICAM-1 [23, 24]. This latter circumstance may arise when integrins and/or counterstructures are expressed at relatively high levels. Such a condition might occur in a venule when the endothelium has been activated or in systemic inflammation when the activated PMN express elevated integrin and depressed selectin levels. Collisional aggregation of suspended cells also includes the potential for both direct and sequential adhesive recognition. Homotypic aggregation is characterized by a simultaneous participation of selectin- and integrin-dependent interactions to evolve high-avidity intercellular contacts, which are then maintained by the integrins [14, 31]. In contrast, we demonstrate here that when ICAM-1 sites are plentiful (at levels comparable to those on activated endothelial cells) and when b2-integrins become activated, PMN can engage cellular targets via direct b2-integrin-ICAM-1 recognition even under Lynam et al.
venous levels of hydrodynamic shear as defined in the cone and plate viscometer.
ACKNOWLEDGMENTS This work was supported by grants ACS CB 162, HL 56384, AI31652, RR 01315, E506091, HL42550, AI19031 and a Whitaker Foundation Grant to S. I. S., who is an Established Investigator of the American Heart Association and by the UNM Cancer Research and Treatment Center. E3-ICAM-1 studies were made possible by the generous material contributions of Dr. Lloyd Graf, Univ. of Illinois, Chicago IL, B78H1 and Ui11 cell lines; Drs. Richard Ulevitch and Peter Tobias, TSRI, La Jolla CA, LBP; Dr. David Chambers, Salk Institute, La Jolla, CA, IB4; Dr. Takashei Kishimoto, Boehringer Inglehiem, Ridgefield, CT, Dreg-200; Dr. Robert Rothlein, Boehringer Ingleheim, Ridgefield, CT, R3.1, R6.5, and R15.7; Dr. J. Rasche, Repligen, Cambridge, MA, 60.1; Dr. Nancy Hogg, Macrophage Laboratory, Imperial Cancer Research Fund, London, England, mAb 24; Dr. Alan Mellors, University of Guelph, Ontario, Canada, OSGE.
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