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(Journal of Leukocyte Biology. 2000;68:715-722.)
© 2000 by Society for Leukocyte Biology

Fas activation reduces neutrophil adhesion to endothelial cells

Stephanie Greenstein*, Joseph Barnard*,{dagger}, Kairong Zhou{dagger}, Miranda Fong{dagger} and Bill Hendey*

* Department of Pharmacology, Rush University, Chicago, Illinois; and
{dagger} Otsuka America Pharmaceuticals, Rockville, Maryland

Correspondence: Bill Hendey, Department of Pharmacology, Rush University, 2242 W. Harrison, Rm. 264, Chicago, IL 60612. E-mail: bhendey{at}rush.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polymorphonuclear neutrophils (PMN) express apoptotic markers and lose effector functions including adhesion, chemotaxis, and phagocytosis when cultured overnight. Although the loss of function correlates with apoptosis, it is not clear if functions are lost before an early marker of apoptosis, the display of phosphatidylserine (PS), targets PMN for removal by phagocytic cells. To address this question, freshly isolated PMN were treated with Fas-activating antibodies to induce apoptosis rapidly. Early markers of apoptosis and PMA-stimulated adhesion to endothelial cells were measured. After 1 h of Fas exposure, only 16% PMN had externalized PS. In contrast, Fas activation reduced PMA-stimulated adhesion between 68 and 27% depending on PMA concentration. The loss of adhesion was accompanied by a reduction in ß2 integrin expression and receptor clustering. These results indicate that the Fas-induced loss of adhesion may precede PS externalization and could limit participation in the inflammatory response before PS externalization targets PMN for removal.

Key Words: apoptosis • adhesion • integrins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polymorphonuclear neutrophils (PMN) are activated and recruited to sites of infection or inflammation by bacterial peptides and/or chemokines. Activated PMN are able to adhere to the endothelium, extravasate through the vessel, and migrate to the site of infection or inflammation [1 , 2 ]. Once at the site of inflammation or infection, PMN carry out their effector functions including phagocytosis, granule release, and toxic metabolite production [3 ]. Although such functions contribute to the destruction of the invading organisms, they can cause tissue damage [4 ]. PMN have been implicated in the development of various systemic crises including the injury associated with ischemia-reperfusion, adult respiratory distress syndrome, and multiple organ failure [2 , 5 ].

Apoptosis, or programmed cell death, likely plays a role in regulating PMN life span and inflammation [6 , 7 ]. PMN have a limited life span, circulating in the blood stream for 4–10 h before marginating and entering the tissue pool, where they may survive for several days [4 ]. Apoptosis is associated with changes in the composition of the PMN plasma membrane. The externalization of phosphatidylserine (PS) to the cell surface may be a critical apoptotic event because it targets the PMN for phagocytosis by macrophages [7 8 9 ]. The phagocytosis of PMN occurs without the release of PMN cytotoxic contents or the secretion of inflammatory mediators from the phagocytic cell [7 ]. This phagocytic process provides a mechanism for the protection of the surrounding tissue by removing the apoptotic PMN without triggering an inflammatory response [7 ].

The regulation of PMN apoptosis has been studied previously using PMN isolated from peripheral blood. Isolated PMN "spontaneously" undergo apoptosis when cultured for 18–24 h at 37°C [10 , 11 ]. PMN apoptosis can also be initiated in freshly isolated PMN by activation of the Fas receptor, a member of the tumor necrosis factor (TNF) receptor family [12 , 13 ]. Activation of the Fas receptor may be responsible also for the induction of spontaneous apoptosis observed in PMN cultured overnight, because blocking antibodies to the Fas receptor delay spontaneous apoptosis [13 ]. Although it is clear that PMN express the Fas receptor [13 14 15 ], there are conflicting studies concerning the source of Fas ligand. Fas ligand has been detected on PMN, suggesting an autocrine pathway [13 ]. Other investigators have failed to find the ligand on PMN and have suggested that it originates from monocytes [15 ]. In either case, it is clear that PMN express the Fas receptor and that activation results in apoptosis.

Similar to other apoptotic cells, PMN display a stereotypical set of apoptotic changes including membrane composition changes, cell shrinkage, cytoskeletal changes, cytoplasmic vacuolization, and chromatin condensation [6 , 16 ]. These morphological changes result from earlier signaling events including the activation of interleukin-1ß converting enzyme (ICE) or Caspase family of serine proteases [17 , 18 ]. The cellular changes culminate in DNA fragmentation and nuclear collapse, the hallmarks of apoptosis [6 ].

Although apoptosis will result in the eventual removal of PMN via phagocytosis, there is increasing evidence that apoptotic PMN undergo a general loss in cellular functions that could limit their participation in an inflammatory response before their removal from circulation. Specifically, global effector functions such as chemotaxis, phagocytosis, and respiratory burst are impaired in apoptotic PMN [6 , 10 , 19 , 20 ]. Unfortunately, the loss of function has, up until this point, been examined in PMN selected for apoptotic markers following overnight culture. Because many of these cells could be in "end-stage" apoptosis, is unclear when the loss of function occurs within the context of the apoptotic cascade or if it is merely the result of cell death.

Loss of function during apoptosis may be a result of changes in plasma membrane composition, such as the loss of the Fc{gamma}RIIIb receptor (CD16) [10 , 21 ]. CD16 is a low-affinity receptor for immunoglobulin G (IgG) that is expressed on PMN in a glycosylphosphatidylinositol (GPI)-anchored form. Loss of CD16 from the PMN cell surface correlates with other measures of apoptosis [10 , 11 ]. CD16 mediates the binding of Ig-opsonized particles, so the loss of CD16 reduces PMN response to such inflammatory stimuli [10 , 21 ].

Changes in receptor expression may affect the adhesion of apoptotic PMN. Apoptosis correlates with a reduced expression of the L-selectin ligand [16 , 20 ] that is necessary for the slowing and rolling of PMN on endothelium [2 ]. In addition, there is a decrease in the ability of spontaneously apoptotic PMN to adhere to fibrinogen [16 ]. The loss of the fibrinogen binding is important because the ß2 or CD18 integrin receptor that mediates the adhesion to fibrinogen also mediates the firm adhesion to the endothelium. These adhesion deficiencies suggests that apoptotic PMN will be unable to adhere to the endothelium, a necessary first step in their movement to sites of inflammation.

To determine if apoptosis would result in the decreased PMN adhesion to endothelial cells, we initiated apoptosis by Fas stimulation of freshly isolated PMN. The effect of Fas stimulation on PMN adhesion to endothelial cells and the induction of apoptotic markers were measured. We determined that 1 h Fas stimulation reduced the stimulated adhesion of PMN to endothelial cells and inhibited ß2-receptor expression and clustering. In addition, the reduced adhesion of Fas-stimulated PMN occurred in a greater percentage of cells than the surface expression of PS or loss of CD16 at the same time point. These results are consistent with the idea that early changes associated with the initiation apoptosis can impair PMN function, and perhaps more significantly, such changes occur before the PMN could be recognized as apoptotic and removed by macrophages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Monoclonal antibodies (mAbs) DX-2 and G155-228 were purchased from Pharmingen (San Diego, CA). Ch-11 was purchased from PanVera (Madison, WI). 3G8 and SZ21 were purchased from Coulter (Westbrook, ME). Anti-Erk 2 (IgG2b) and Anti-Erk 3 (IgM) were purchased from Transduction Laboratories (Lexington, KY). IB4 was purchased from Alexis Biochemicals (San Diego, CA). Goat F(ab')2 antimouse IgG-fluorescein isothiocyanate (FITC) was purchased from Leinco Technologies (St. Louis, MO). Phorbol-12-myristate-13-acetate (PMA) was purchased from Sigma (St. Louis, MO). Annexin-V and propidium iodide were purchased from Clontech (Palo Alto, CA). CD16-FITC (Fc{gamma}RIIIb antibody) was purchased from Immunotech (Marseilles, France). Vitronectin was purchased from Collaborative Biomedical Products (Bedford, MA). Fibronectin was purchased from Becton Dickinson (Bedford, MA). Rat pulmonary microvascular endothelial cells (RLEC) were isolated as in Kelly et al. [22 ] and were a gift from W. Joseph Thompson, Ph.D., at the University of South Alabama (Mobile, AL).

RLEC culture
RLEC were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal calf serum (FCS), 50 U/ml penicillin G, and 50 µg/ml streptomycin sulfate on 100 mm dishes. Cell passage was performed as described in Kelly et al. [22 ]. For use in the adhesion assays, confluent cells were trypsinized, centrifuged, counted, and resuspended in DMEM at a concentration of 5 x 103/100 µl. Falcon or Costar 96-well plates coated with human fibronectin (Becton Dickinson) were used for seeding the RLEC at a density of 5 x 103 cells/well. RLEC were grown to confluence, which was assessed by microscopy before being used in an adhesion assay.

PMN isolation
Human whole blood was obtained from healthy volunteers via venipuncture. Blood was collected in sodium-heparin tubes, and PMN were isolated using a single-step gradient (Polymorphprep; Nycomed, Oslo, Norway) [23 , 24 ]. Cells were washed with phosphate-buffered saline (PBS; Gibco/Life Technologies, Rockville, MD), red cells were lysed using hypotonic shock, and following a final rinse in PBS, the PMN were resuspended in incubation media (150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2,20 mM HEPES, pH 7.4) or Hanks’ balanced salt solution (HBSS; Gibco/Life Technologies). The final cell preparation consisted of >97% PMN [25 ]. Concern that any contamination of incubation media with endotoxin may affect onset of apoptosis led us to repeat and confirm all results using endotoxin-free HBSS with Mg2+, Ca2+, and glucose (Gibco/Life Technologies) in place of incubation media. All of the results were unchanged by the buffer substitution.

PMN labeling and treatment
Calcein-labeled cells were used to allow for fluorescent detection of the PMN. Previous work indicates that calcine labeling does not interfere with PMN function [23 ], and calcine-labeled cells have been used in adhesion assays [26 ]. PMN were fluorescently labeled using the acetyloxy methyl ester of calcein, calcein-AM (Molecular Probes, Eugene, OR), as described by Everitt et al. [23 ]. Briefly, 50 µg calcein was suspended in 5 µl dimethyl sulfoxide (DMSO), 5 µl 250 mg/ml plurionic, and 60 µl heat-inactivated FCS. The labeling solution was diluted to 5 ml with the addition of 4.0 x 107 PMN in incubation media. The tube was covered in a foil sleeve to protect it from light and was rocked for 1 h at room temperature. Some of the PMN were treated with anti-Fas antibody DX-2 or Ch-11 during the incubation period to induce apoptosis or with isotype-matched control antibodies. After 1 h, the labeled PMN were centrifuged and washed twice with HBSS (Gibco/Life Technologies). The PMN were resuspended in a 4% bovine serum albumin (BSA) incubation-media buffer at a density of 4 x 106 PMN/ml and incubated at 37°C for 10 min to allow cleavage of the acetyloxy methyl ester from calcein. To verify that the relationship between cell number and cell fluorescence was linear, varying numbers of fluorescently labeled PMN were plated in wells of a 96-well plate. Fluorescence readings were made using a Cytofluor II (PerSeptive Biosystems, Framingham, MA). Fluorescence and cell number were linear between 2.5 x 104 and 8 x 105 cells (n=4).

Adhesion assay
An adhesion assay was performed essentially as in refs. 27 28 29 30 except that calcine labeling was used in place of Cr51, and the rinse protocol was modified to eliminate inadvertent cell shearing caused by aspiration. Briefly, endothelial cells were grown to confluence in a 96-well plate, and eight wells were used for each experimental condition. The media was removed from the wells, and 100 µl calcine-labeled PMN was added for a final concentration of 4 x 105 PMN/well. The indicated concentration of PMA or H2O2 was added to stimulate PMN adhesion. An initial fluorescence reading was done to assess baseline fluorescence. The PMN were allowed to attach for 30 min at 37°C. The unbound PMN were then removed by flicking the plate, washing the plate in a bath of incubation media, and flicking again. The wells were then filled with 100 µl of warm 4% BSA incubation-media buffer. A second fluorescence reading was taken to measure the residual fluorescence in each well. The residual fluorescence represents the PMN that remained attached to the endothelial cells.

The percent adhesion for each condition of the experiment was calculated by dividing the mean residual fluorescence by the mean baseline fluorescence for each condition. Calculating the percent adhesion for each experiment allowed for the comparison of replicate experiments. Each experiment was repeated on different days using fresh preparations of PMN. The data from replicate experiments were averaged to determine the mean percent adhesion and the SE of the mean for each condition. The actual number of replicates (N) for each set of experiments is indicated in Results and the figure legends.

To measure the effect of variable-length Fas treatments, it was necessary to stagger the starting point of Fas antibody exposure in the time-course assay. The PMN were calcine-labeled and one treatment group was incubated at 37°C immediately with Fas-activating antibody Ch11 (1 µg/ml). After 2 h, Fas was added to another group of PMN, which were incubated for 1 h. Fifteen minutes before addition to the endothelial cell monolayers, one treatment group was treated with anti-ß2 antibody, IB4 (10 µg/ml). After three hours had elapsed, all of the treated PMN and the untreated control cells were plated on a single microwell plate, treated ± 5 nM PMN, and the adhesion assay was performed. By staggering the exposure to Fas, eight wells of same 96-well dish could be used for each treatment time point, eliminating any dish-to-dish variation in the rinse procedure.

Annexin-V assay
Externalized phosphatidylserine (PS) was detected using FITC-conjugated Annexin-V. Annexin is a 35.8 kD protein that binds to PS. Annexin-V FITC was obtained from Clontech and used according to the direction of the manufacturer. Freshly isolated PMN were treated with anti-Fas antibody or isotype-matched control antibodies for the indicated times (0 min, 1 h, 2 h, and 3 h). After the incubation period, the Fas antibody was removed by centrifugation, and the PMN were washed with PBS and resuspended in 200 µl binding buffer with the 10 µg/ml Annexin-V FITC, according to the manufacturer’s protocol. The samples were then incubated in the dark for 15 min at room temperature. PMN from each group were then plated in a minimum of three wells of a 96-well plate at a concentration of 1 x 105 cells/well. The labeled cells were observed using fluorescence microscopy. Fields from each of the three wells were selected randomly, and cells displaying distinctive halo of fluorescence were counted as positive for PS externalization. Bright-field microscopy was used to count the total number of cells in a given field. In each experiment, at least 100 cells were counted per treatment condition. The percentage of labeled cells was determined by dividing the number of labeled cells per field by the total number of cells. Each experiment was repeated with fresh preparations of PMN. Results were averaged across multiple experiments and are shown as the mean %-labeled cells ± the SE, N = number of experiments. A time course and an antibody comparison assay were performed.

To confirm microscopy results, externalized PS was also measured via flow cytometry. Annexin-V binding was measured at baseline, 1 h, and 3 h after Fas stimulation. Fluorescence was measured using a FACScan (Becton Dickinson) flow cytometer. Cells were analyzed via quadrant analysis of two fluorescent detectors. For each condition, 1.5 x 104 cells were measured. Unstained PMN were used as negative controls at each time point. In a separate set of experiments, PMN were double-labeled with Annexin-V FITC and propidium iodide to measure apoptosis vs. necrosis.

Fc{gamma}RIIIb (CD16) receptor assay
Freshly isolated PMN were treated with anti-Fas antibody or isotype-matched control antibodies for the indicated times. After the incubation period, 5 x 105 cells were removed from each experimental group, and 20 µl FITC-conjugated CD16 was added in incubation media as per the manufacturer’s instructions. PMN were incubated with the CD16 for 30 min in the dark at room temperature. After the 30 min, cells were centrifuged, and the supernatant was removed. The PMN were then washed with incubation media and resuspended in 100 µl. PMN were then plated in an eight-chambered coverglass tray (Nunc, Naperville, IL). The percentage of apoptotic cells was determined via fluorescent microscopy by counting the number of labeled cells per field and dividing that sum by the total number of cells in each field. Each experiment was repeated with fresh preparations of PMN. Results were averaged across multiple experiments and are shown as the mean %-labeled cells ± the SE, N = number of experiments. A time course and an antibody-comparison assay were performed.

Measurement of ß2-receptor expression
Freshly isolated PMN (5x105-1x106/condition) were treated ± Fas for 1 h and treated ± 5 nM PMA for 30 min. The cells were then centrifuged 400 g for 7 min at 4°C. Cells were then resuspended in PBS. For quantification of integrin expression, cells were incubated with a 1° antibody to ß2 (IB4; 1 µg/ml) for 30 min on ice in the dark. Two rinses in 500 µl PBS gel azide (1 mg/ml NaN3, 1 mg/ml gelatin) were followed by 2° antibody treatment. PMN resuspended in 200 µl PBS were incubated with 5 µg/ml goat F(ab')2 antimouse IgG-fluorescein secondary antibody for 30 min on ice in the dark. Some PMN, control and Fas-treated, were labeled with 2° antibody only as a control. After two final washes in PBS gel azide, the PMN were fixed in PBS gel azide with 1% formaldehyde. For all flow cytometry measurements, PMN were resuspended at 5 x 105 cells/ml, and fluorescence was measured using a FACScan (Becton Dickinson) flow cytometer. Cell analysis was gated on a forward- and side-scatter. For each condition, 104 cells were measured. Controls were performed for each experiment to rule out nonspecific loss of surface receptors.

Immunofluorescence
Freshly isolated PMN (1x107) were diluted to 4 ml in M2 and divided into the following groups: unstimulated, +PMA, Fas, and Fas + PMA. PMN were treated ± Fas for 1 h at 37°C, followed by treatment with 5 nM PMA for 30 min. During the PMN incubation period, an eight-chambered dish was coated with vitronectin (10 µg/ml) for 40 min. PMN (5x105) were plated per well, and100 µl incubation media was added to cover the cells. The dish was covered, and cells were allowed to attach on a warm tray (37°C) for 5 min. Cells were checked on a microscope to verify that the appropriate number had been added to each well. The cells were then fixed in 4% paraformaldehyde in PBS for 4 min at room temperature and incubated with blocking buffer [PBS with 2 µg/ml BSA, 10% fetal bovine serum (FBS), 1 mM MgCl, 0.5 mg/ml NaN3, and the protease inhibitors: 200 µM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 10 µM leupeptin, and 10 µg/ml aprotinin) for 30 min, as in Lawson and Maxfield [31 ]. Fluid was removed, and the cells were incubated with 1 µg/ml mAb IB4 for 1 h. Controls for these experiments included a 2° antibody-only treatment as well as treatment with an irrelevant, isotype-matched antibody, anti-erk2, an anti-IgG2b for IB4. Cells were rinsed gently with blocking buffer three times and then treated with 1 µg/ml goat F(ab')2 antimouse IgG-FITC secondary antibody for 1 h in the dark. Following secondary antibody treatment, PMN were rinsed 3x with blocking buffer, and the wells were filled with 200 µl blocking buffer to cover the bottom of the well completely. Cells were visualized via fluorescent microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adhesion assay
PMN show a low basal adhesion to RLEC (4.5±0.3%; mean±SE). When stimulated with increasing doses of PMA (1 nM, 2 nM, 5 nM, 10 nM), adhesion increased (Fig. 1A ). These findings were expected because PMN adhere to endothelial cells poorly, but PMA stimulation results in a profound increase in adhesion [27 , 32 ].



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  		Figure 1. Reduction in PMA-stimulated adhesion following Fas stimulation. PMN were pretreated with anti-Fas-activating antibody Ch-11 (1 µg/ml) and plated on confluent monolayers of endothelial cells. Adhesion was measured in response to 30 min PMA stimulation as in Materials and Methods. (A) PMN were pretreated with Ch-11, an isotype-matched control (IgM), mAb G155, or no antibody for 1 h at 37°C. Endothelial cell adhesion was stimulated for 30 min with the indicated concentration of PMA. Plotted are the mean ± SE for four experiments. Treatment with Fas-activating antibody reduced PMA-stimulated adhesion to the endothelial cells (significant at p<.001 using ANOVA), and the isotype-matched control antibody had no effect. (B) PMN were pretreated with Ch-11 for 0, 1, or 3 h, or with the ß2-blocking antibody, IB4 (10 µg/ml) for 15 min before plating. PMN-EC adhesion was measured in response to 5 nM PMA. Plotted are the mean ± SE for four experiments. Treatment with Ch-11 for 1 or 3 h reduced PMA-stimulated adhesion to the endothelial cells significantly as compared with control. The smaller difference between the 1- and 3-h Fas incubations was not statistically significant. Treatment with IB4 inhibited PMA-stimulated adhesion significantly as compared with control and Fas-treated PMN. *Significance at p < .05 using ANOVA with Sheffe F test for comparison between individual treatments.

 
Treatment of PMN with anti-Fas-activating mAb Ch-11, an IgM (100 ng/ml), decreased PMA-stimulated adhesion to endothelial cells [p<.001 using analysis of variance (ANOVA); Fig. 1A ]. An isotype-matched control antibody (IgM), G155-228, which does not bind to a specific receptor on PMN, had no effect on PMN adhesion to the endothelial cells, demonstrating that the Fas response is not a nonspecific antibody effect. Similar results of decreased adhesion were seen with another anti-Fas-activating mAb, DX-2 (200 ng/ml). DX-2 treatment reduced PMA-stimulated adhesion by 43 ± 9% (mean±SE; n=6). An antibody that binds to an irrelevant epitope was used as an isotype-matched control for DX-2. SZ21, an IgG1 antibody, binds to the ß3 receptor on human PMN [24 ]. In three matched experiments, SZ21 treatment had no effect on PMA-stimulated EC adhesion of control PMN (96±5% of PMA-stimulated adhesion). In the same group of experiments, treatment with DX-2 resulted in a 41 ± 6% loss of adhesion.

The adhesion experiments were performed using H2O2 as a stimulus also. H2O2 has been shown to induce adhesion of PMN to endothelial cells but not to the same extent as PMA [30 ]. The H2O2-stimulated adhesion is also decreased by pretreatment of the PMN with an anti-Fas-activating antibody (Fig. 2 ). The decreased adhesion was statistically significant (p<.05 using ANOVA with Scheffe F test for individual comparisons). These results indicate that treatment with anti-Fas-activating antibody reduced the PMA- and H2O2-stimulated adhesion of human PMN.



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  		Figure 2. Reduction in H2O2-stimulated adhesion following Fas stimulation. Adhesion of calcine-labeled PMN to endothelial cells was measured in the response to H2O2 stimulation (see Materials and Methods). The effect of anti-Fas-activating antibody DX-2 pretreatment on PMN or endothelial cells was assessed in six experiments. PMN show significantly reduced H2O2-stimulated adhesion to endothelial cells after 1 h exposure with the anti-Fas antibody (significant at p<.05 using an ANOVA with Sheffe F test for individual comparisons). Treatment of the endothelial cells with Fas had no effect on their H2O2-stimulated adhesion to PMN.

 
To verify that the loss of adhesion of the PMN to the RLEC was a result of Fas effects on the PMN and not to an effect on the endothelium, the RLEC were pretreated with the anti-Fas mAb, DX-2 (200 ng/ml). Pretreatment of the endothelium with DX-2 had no effect on H2O2-stimulated adhesion (Fig. 2) . Even when the endothelial cells are treated with a tenfold increase in DX-2 concentration, there was no reduction in H2O2-stimulated adhesion (10±4% with 2 µg DX-2 treatment of the endothelium vs. 11±3% without any pretreatment, n=6). These results indicate that the loss of adhesion caused by Fas stimulation is a result of an effect of the Fas antibodies on the PMN and not on the endothelial cells (Fig. 2) .

PMA-stimulated adhesion was measured after variable periods of Fas stimulation to determine the time course of Fas effects. In four matched experiments, 30 min of Fas treatment did not affect adhesion. Treatment with 5 nM PMA stimulated the adhesion of 57 ± 10% of the 30 min Fas-treated PMN as compared with 46 ± 8% of the control cells. Longer periods of Fas treatment were examined in another set of four experiments. Three hours of Fas treatment decreased adhesion significantly (Fig. 1B) . The decrease in adhesion observed at 3 h was not statistically greater then that observed at 1 h.

Because our adhesion model is a heterologous system, it was necessary to confirm that the PMN-EC adhesion was mediated by the ß2 receptor. mAb IB4, a ß2 receptor-blocking antibody, recognizes the common ß2 (CD18) subunit of the {alpha}Mß2 and {alpha}Lß2 receptors. Both of these receptors are capable of mediating the tight adhesion of PMN to the intercellular adhesion molecule-1 (ICAM-1) counter receptor on the endothelial cells [3 , 33 , 34 ]. Treatment of the PMN with IB4 (10 µg/ml) for 15 min before addition to the EC reduced PMA-stimulated adhesion significantly, indicating that the PMN-EC adhesion was ß2-dependent (Fig. 1B) . Treatment with IB4 blocked PMN adhesion to a greater extent than did either time of Fas treatment (Fig. 1B) . These results indicate that Fas activation partially inhibits the adhesion of the PMN in our assay.

Annexin-V assay
An early event in apoptosis is the translocation of the membrane phospholipid PS from the inner surface of the plasma membrane to the outer cell surface [6 , 8 ]. Once externalized, the PS can be detected by binding to Annexin-V FITC, a 35.8 kD protein conjugated to a fluorophore [35 ]. The exposure of PMN to anti-Fas-activating antibodies DX-2 and Ch-11 causes a time-dependent increase in the number of cells labeled by Annexin-V FITC as assessed by fluorescent microscopy (Fig. 3 ). We confirmed the results of the fluorescent microscopy with four additional experiments in which we measured Annexin-V binding via flow cytometry and fluorescence microscopy in parallel. In agreement with the fluorescence microscopy results, flow cytometry indicates that Annexin-V bound to <10% of control cells when measured at baseline and after 1 or 3 h of incubation. After 1 and 3 h of Fas treatment, 13 ± 1% and 23 ± 4% of the cells were Annexin-V-positive as measured by flow cytometry. Analysis of the same experiments using fluorescence microscopy gave slightly higher estimates of 17 ± 2% for 1 h and 29 ± 8% for 3 h of Fas treatment. The higher microscopy estimates likely represent differences in the sensitivity of the assays. The flow cytometry experiments used a basal fluorescence cutoff determined by nonlabeled cells. In contrast, fluorescence microscopy relied on a morphologic criteria for Annexin-V-positive cells. Specifically, cells were scored as positive if the characteristic membrane "halo" of Annexin-V FITC labeling was present, and cells showing a uniform or granular fluorescence characteristic of autofluorescence were not counted as positive. The difference between these criteria means that weakly labeled cells showing the characteristic membrane halo of Annexin-V labeling would be included in the fluorescent microscopy count but might fall below the autofluorescence threshold used in flow cytometry.



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  		Figure 3. Time course of Annexin-V labeling of PMN following Fas stimulation. Freshly isolated PMN were exposed to anti-Fas-activating antibodies DX-2 or Ch-11 for varying lengths of time (0, 1, 2, and 3 h) and treated with FITC-conjugated Annexin-V (see Materials and Methods). Annexin-V is a 35.8 kD protein that binds to PS on the PMN cell surface. Fas-treated PMN show a gradual increase in the percentage of Annexin-V-labeled cells. Control PMN have a very low percentage of cells bound by Annexin-V at baseline, 1 h, and after 3 h of incubation.

 
Flow cytometry was used also to measure double-labeling of PMN with Annexin-V FITC and propidium iodide. Greater than 99% of control and Fas-treated PMN excluded propidium iodide, indicating that the PMN have intact membranes and are not necrotic.

Four separate experiments were performed at the 3 h time point comparing the effect of DX-2, Ch-11, and isotype-matched control antibodies. DX-2 and Ch-11 treatments resulted in 34 ± .01% and 34 ± .04% Annexin-V binding, respectively. The labeling observed on the Fas-treated PMN was statistically greater than that observed on isotype-matched controls or no antibody controls. Statistical significance was determined using an ANOVA and a Scheffe F test (p<.01) for individual comparisons.

Fc{gamma}RIIIb (CD16) receptor assay
CD16 is a low-affinity receptor for IgG (Fc{gamma}RIIIb), which is expressed on the surface of PMN. Loss of this receptor correlates with other measures of apoptosis in PMN cultured overnight [10 , 11 ], but a time course for the loss of this receptor has not been established. Granulocytes isolated using standard methods are >95% PMN and contain a small percentage of eosinophils typically that do not express CD16 [21 ]. In line with these observations, 96 ± 1% (mean±SE; n=4) of control PMN expressed CD16 after 1 h of buffer incubation following isolation (Fig. 4 ). In contrast, initiation of apoptosis with anti-Fas-activating antibody Ch-11 results in a decrease in CD16 binding (Fig. 4) . After 1 h of Fas exposure, Fc{gamma}RIIIb surface expression is observed on only 81 ± 4% (mean±SE; n=4) of the cells. After 3 h of Fas stimulation, the loss of CD16 receptor expression is 50 ± 7% (mean±SE; n=4). Untreated PMN show a more gradual loss of the Fc{gamma}RIIIb receptor, with a loss of 20 ± 5% (mean±SE; n=4) occurring only after 3 h. Five additional experiments were done at the 3 h time point, comparing the effect of Ch-11 and DX-2, and isotype-matched control antibodies. PMN treated with Ch-11 and DX-2 show significant reductions in CD16 surface expression: 55 ± 4% and 71 ± 3%, respectively. The labeling observed in the isotype-matched control antibody-treated PMN was greater statistically than the Fas-treated PMN and not different from no antibody-treated controls. Statistical significance was determined using an ANOVA and a Scheffe F test (p<.01) for individual comparisons.



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  		Figure 4. Time course of Fc{gamma}RIIIb receptor surface-expression reduction on Fas-treated PMN. Freshly isolated PMN were exposed to anti-Fas-activating antibody Ch-11 for varying lengths of time (1, 2, and 3 h) and treated with an FITC-conjugated CD16 antibody (see Materials and Methods). Fas-treated PMN show a decrease in the percentage of cells bound by CD16-FITC. Although Fc{gamma}RIIIb receptor-surface expression decreases with time in control PMN, it remains higher than with Fas stimulation.

 
ß2 (CD18) expression
ß2 integrin expression was measured using antibody IB4, a mAb that recognizes the common ß2 (CD18) subunit of the {alpha}Mß2 and {alpha}Lß2 receptors that mediate the tight adhesion of PMN to the ICAM-1 counter receptor on the endothelial cells [3 , 33 , 34 ]. Flow cytometry was used to monitor receptor expression. In these experiments, the cells were pretreated (±) the Fas antibody Ch-11 (IgM) for 1 h, rinsed, and stimulated (±) PMA for 30 min. Cells were then fixed and incubated with IB4 (IgG2b) or an isotype-matched antibody (anti-Erk 2) and counter-stained with a 2° antibody [goat F(ab')2 antimouse IgG-fluorescein]. Figure 5A and B , shows representative flow cytometry histograms. Figure 5A shows ß2 expression on unstimulated control cells and the decrease in expression associated with Fas antibody exposure. Figure 5B compares the PMA-stimulated ß2 expression on control and Fas-treated PMN. These histograms indicate that Fas activation reduced but did not abrogate the increased expression of ß2 integrin receptors caused by PMA. In Figure 6 , the data from eight matched experiments are summarized as the mean fluorescence for each treatment condition (mean±SE). PMA treatment increased the mean fluorescence, a measure of receptor expression, of the unstimulated PMN. In contrast, Fas stimulation significantly reduced baseline and the PMA-stimulated increases in ß2-receptor expression (p>.05 using an ANOVA with Scheffe F test for means comparison).



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  		Figure 5. Reduced ß2 integrin expression following Fas activation. ß2 integrin expression on freshly isolated PMN was measured by flow cytometry using a mAb to the ß2 receptor (IB4; 1 µg/ml) or an isotype-matched control antibody (anti-Erk2 mAb; 1 µg/ml). The effect of 1-h Fas activation and 5 nM PMA stimulation on ß2 surface expression was assessed. (A) PMN treated with Fas (bold line) have reduced surface expression of ß2 receptors compared with untreated control PMN (solid line). Binding of the isotype-matched control antibody is shown (broken line). (B) A 30-min incubation with 5 nM PMA increases ß2 expression on control PMN (solid line). Fas-treated cells show a reduced response to PMA (bold line). Binding of the isotype-matched control antibody is shown (broken line). Histograms are representative tracings of eight comparisons each using freshly isolated PMN.

 


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  		Figure 6. Effect of Fas stimulation on ß2 integrin-receptor expression. Freshly isolated PMN were stimulated with Fas for 1 h followed by 5 nM PMA for 30 min (see Materials and Methods). Integrin expression was measured by flow cytometry using a primary antibody to ß2 (IB4; 1 µg/ml). Antibody binding, as measured by mean fluorescence, indicates receptor-surface expression. Data are shown in summary form (N=8). *Significant (p<.05) using an ANOVA with Scheffe F test for means comparison.

 
Because the Ch-11 antibody was used to activate the Fas receptor, steps were taken to assure that changes in ß2 antibody reactivity were not an artifact of the prior Fas antibody stimulation. Ch-11 was used for Fas stimulation because it is an IgM and should not react with the goat F(ab')2 antimouse IgG-fluorescein 2° antibody used in the above experiments. This was verified by comparing the fluorescence of Fas (Ch-11)-treated or untreated cells. In these experiments, PMN were treated ± Ch-11 for 1 h, rinsed, and then treated with no primary antibody or isotype-matched primary antibodies and then stained with the 2° fluorescent antibody. There was no difference in fluorescence of Fas (Ch-11)-treated or untreated cells in any of these conditions (p<.05 ANOVA).

Integrin clustering
PMA stimulation has been shown to cause the aggregation of the ß2 integrins [36 ] and this aggregation or clustering of the receptors is correlated with cell adhesion [36 ]. Immunofluorescence studies were performed using mAb IB4 to assess the effects of Fas activation on receptor clustering. Unstimulated PMN show a uniform staining on rounded cells or a membrane "ring" pattern of ß2 receptors on more spread cells (Fig. 7A ). Fas activation alone does not disturb the staining pattern (Fig. 7B) . Exposure of control PMN to PMA results in discrete areas of clustered receptors (Fig. 7C) . In contrast, Fas activation reduces PMA-induced receptor aggregation (Fig. 7D) . These observations were repeated in six separate experiments. There was a consistent reduction of receptor aggregation after Fas activation. Although this was not an all or none response, as seen in Figure 7 , the Fas-treated PMN consistently displayed less clustered receptors in response to PMA stimulation than those treated with PMA alone.



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  		Figure 7. PMA-induced ß2-receptor clustering is inhibited by Fas activation. Fas-receptor stimulation inhibits PMA-induced ß2-receptor clustering. A shows the surface expression of the ß2-integrin receptor on unstimulated PMN. B displays Fas-treated PMN. These cells show little or no receptor clustering and look very similar to control PMN. C shows the effect of PMA stimulation on control cells. (Note the aggregation of the receptor.) D shows that treatment with Fas reduces PMA-induced receptor clustering. All panels are to the same scale, and the bar length is 10 µM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although unactivated PMN adhere poorly to endothelial cells, PMN can be stimulated to adhere by a variety of activating agents [27 ]. These observations were repeated in an in vitro assay of PMN adhesion to endothelial cells (Fig. 1A) . PMA and H2O2 stimulated the adhesion of PMN to the endothelial cells (Figs. 1 and 2) . However, when an activating antibody to the Fas receptor was used to induce apoptosis, a decrease in the PMA or H2O2-stimulated adhesion was observed (Figs. 1 and 2) . As mentioned in Results, isotype-matched control antibodies failed to inhibit adhesion, indicating that the loss of adhesion was likely a result of the induction of apoptosis rather than a nonspecific effect of antibody stimulation.

The loss of stimulated adhesion has been observed previously in PMN made apoptotic by culturing overnight and in PMN selected for apoptotic markers after 24 h incubation [16 ]. Because these were end-stage apoptotic PMN, it is unclear from these studies if the loss of adhesion is a step in the apoptotic cascade or merely a consequence of cell death. To address this issue, Fas stimulation was used to initiate rapid apoptosis in freshly isolated PMN. As mentioned in Results, 30 min of Fas activation did not inhibit adhesion. After 1 h of Fas activation, there was a decrease in adhesion (Fig. 1A) . After 3 h of Fas stimulation, the loss of adhesion was slightly greater but not statistically different from that observed at 1 h (Fig. 1B) .

To place the reduction in adhesion within the context of the apoptotic cascade, the expression of two markers of apoptosis, externalization of PS and loss of CD16, was examined. Externalization of PS, as measured by Annexin-V binding, was seen on 16–17% of the Fas-activated cells at 1 h and increased to 32–33% of the cells by 3 h (Fig. 3) . In contrast, externalized PS remained below 10% for control cells even after 3 h in media (Fig. 3) . This time course for Fas-induced PS externalization parallels that seen in other cell types [6 ].

Annexin-V binding was assessed using microscopy because it allowed confirmation of normal morphology and allowed the scoring of weakly labeled cells showing the characteristic membrane halo of Annexin-V binding. Flow cytometric measurement of Annexin-V binding was also used to confirm the microscopy results. As detailed in the results, the flow cytometry estimates of Annexin-V-positive cells were slightly lower than that observed in the immunofluorescence assays. For example, 1 h of Fas exposure resulted in flow cytometry estimates of 14 ± 1% and fluorescence microscopy estimates of 17 ± 2% of Annexin-V-positive cells. Whether one accepts microscopy or the flow cytometry estimates, both methods indicate that only a small percentage of PMN is Annexin-V-positive after 1 h of Fas activation.

A marker associated with PMN apoptosis, the loss of CD16 (Fc{gamma}RIIIb), was also measured. Within 1 h of Fas stimulation, there was a 19% reduction in CD16 surface expression, and surface expression was further decreased to 50% at 3 h (Fig. 4) . These results indicate that the reduction of adhesion occurs early in the apoptotic cascade and in a greater percentage of cells relative to cell-surface markers of apoptosis. After only 1 h of Fas activation, there was a 27–68% reduction in adhesion depending on PMA dose (Fig. 1A and 1B) , and <20% of the cells showed any change in outward signs of apoptosis (Figs. 3 and 4) . Given this timing, it is likely that early events in the Fas-induced apoptotic cascade interfere with the intracellular signaling necessary for the stimulated adhesion of PMN to endothelial cells.

To determine if Fas activation disrupts the events necessary for adhesion, we examined the effect of Fas stimulation on the ß2 integrins that are necessary for tight adhesion to endothelial cells. PMN have two receptors, {alpha}Lß2 (CD11a/CD18) and {alpha}Mß2 (CD11b/CD18), which can mediate tight adhesion to ICAM-1 on endothelial cells [1 , 33 , 34 ]. IB4, a blocking antibody to the common ß subunit of the ß2 receptors, inhibited PMN adhesion to the endothelial cells used in this assay (Fig. 1B) . Flow cytometry was used to determine if Fas activation had an effect on the expression of the common ß2 subunit by measuring IB4 labeling. In line with previous studies [37 ], PMA treatment led to a dramatic increase in ß2 expression (Figs. 5B and 6) . Fas activation led to a decrease in ß2 expression in unstimulated cells (Figs. 5A and 6) . PMA treatment of the Fas-activated PMN led to some increase in ß2 expression relative to cells treated with Fas alone (Figs. 5A and 5B and 6) , but the ß2 expression of the Fas-activated PMA-stimulated cells remained below that of the PMA-stimulated control cells (Figs. 5B and 6) .

Although Fas stimulation reduced ß2 surface expression relative to controls, it is unlikely that the simple reduction in ß2 integrin surface expression could totally account for the reduction in adhesion. Unstimulated PMN have many ß2 receptors, and changes in ß2-receptor aggregation and activation are more closely tied to stimulated adhesion than is receptor number [32 , 38 ]. PMA-stimulated adhesion to the endothelium correlates with an aggregation of ß2 receptors [36 ]. Immunofluorescence microscopy was used to determine if the Fas-stimulated loss of adhesion is related to changes in the clustering of the ß2 integrin. In line with previous findings of others, stimulation of control cells with PMA changes the uniform membrane pattern of ß2 receptors (Fig. 7A) to one with discrete areas of clustered receptors (Fig. 7C) . In contrast, Fas activation inhibits PMA-induced receptor aggregation (Fig. 7D) . Thus, activation of Fas results in a decrease in receptor number and a decrease in receptor aggregation.

In summary, Fas activation results in a reduction in ß2-receptor expression and aggregation, with a concomitant reduction in PMN adhesion. Further, the loss of adhesion occurs after 1 h of Fas stimulation and precedes the full manifestation of traditional markers of apoptosis, including the display of PS, as measured by Annexin-V binding and the reduction of CD16 surface expression. The rapidity of the onset of the loss of adhesion was unexpected because the externalization of PS is generally considered to be an early event in the apoptotic pathway, and most cells did not express PS until 3 h after Fas stimulation. The early loss of stimulated adhesion suggests that it is not a nonspecific consequence of cell death. Rather, it occurs early in the apoptotic cascade and could play a role in limiting inflammation before the display of PS would target the PMN for removal by phagocytic cells. Specifically, the induction of the apoptotic cascade by Fas-receptor stimulation might keep circulating PMN from engaging in an inflammatory response by preventing endothelial adhesion, a necessary first step for PMN extravasation and migration to sites of inflammation or infection.


   ACKNOWLEDGEMENTS
 
This work supported by National Institute of Health Grant #AI40253 to B. H. and American Heart Association Grant #96012700 to J. B.

Received January 20, 2000; revised May 22, 2000; accepted May 23, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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