Effects of IL-8, Gro-alpha , and LTB4 on the adhesive kinetics of LFA-1 and Mac-1 on human neutrophils

Scott M. Seo1,2,3, Larry V. McIntire1,3, and C. Wayne Smith2

1 Institute of Biosciences and Bioengineering, Rice University, Houston 77005; 2 Speros P. Martel Section of Leukocyte Biology, Department of Pediatrics, and 3 Structural and Computational Biology and Molecular Biophysics Program, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Firm adhesion of rolling neutrophils on inflamed endothelium is dependent on beta 2 (CD18)-integrins and activating stimuli. LFA-1 (CD11a/CD18) appears to be more important than Mac-1 (CD11b/CD18) in neutrophil emigration at inflammatory sites, but little is known of the relative binding characteristics of these two integrins under conditions thought to regulate firm adhesion. The present study examined the effect of chemoattractants on the kinetics of LFA-1 and Mac-1 adhesion in human neutrophils. We found that subnanomolar concentrations of interleukin-8, Gro-alpha , and leukotriene B4 (LTB4) induced rapid and optimal rates of LFA-1-dependent adhesion of neutrophils to intercellular adhesion molecule (ICAM)-1-coated beads. These optimal rates of LFA-1 adhesion were transient and decayed within 1 min after chemoattractant stimulation. Mac-1 adhesion was equally rapid initially but continued to rise for >= 6 min after stimulation. A fourfold higher density of ICAM-1 on beads markedly increased the rate of binding to LFA-1 but did not change the early and narrow time window for the optimal rate of adhesion. Using well-characterized monoclonal antibodies, we showed that activation of LFA-1 and Mac-1 by Gro-alpha was completely blocked by anti-CXC chemokine receptor R2, but activation of these integrins by interleukin-8 was most effectively blocked by anti-CXC chemokine receptor R1. The topographical distribution of beads also reflected significant differences between LFA-1 and Mac-1. Beads bound to Mac-1 translocated to the cell uropod within 4 min, but beads bound to LFA-1 remained bound to the lamellipodial regions at the same time. These kinetic and topographical differences may indicate distinct functional contributions of LFA-1 and Mac-1 on neutrophils.

intercellular adhesion molecule-1; inflammation; chemokine; emigration; integrin; interleukin-8; leukotriene B4


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ROLLING OF LEUKOCYTES along the walls of small venules is a sensitive and early indicator of inflammation (2) and reflects a necessary step before efficient firm attachment of leukocytes to endothelium can progress to emigration at the site of injury or infection. The transition from rolling to firm adhesion reflects the transition from selectin-dependent adhesion (rolling) to CD18 integrin-dependent arrest. Rolling is not sufficient for firm adhesion, which requires activation of the CD18 integrins LFA-1 and Mac-1 on neutrophils by presumably one or more chemoattractants generated by inflamed endothelium (25, 33). Mechanisms that likely regulate the adhesive function of LFA-1 and Mac-1 on a rolling neutrophil include fluid shear, selectin-mediated signaling, and chemoattractant-mediated activation.

Previous work examined the effects of fluid shear and selectin-mediated signaling on LFA-1 and Mac-1 adhesion in human neutrophils. Taylor et al. (45) studied the effect of defined fluid shear on homotypic neutrophil aggregation in suspension, in which selectins initiated tethering and LFA-1 binding to intercellular adhesion molecule (ICAM)-3 followed. Neelamegham et al. (29) studied the effect of fluid shear on ICAM-1-expressing cells binding to LFA-1 and Mac-1 on neutrophils in suspension in the absence of selectin adhesion. Hentzen et al. (21) further studied the effect of fluid shear on neutrophils binding to high- and low-expressing ICAM-1 cells in suspension in the absence of selectin adhesion. The activating stimulus in these studies was a high concentration of N-formylmethionyl-leucyl-phenylalanine (FMLP). Their general findings were that certain fluid shear conditions favor LFA-1 binding initially within the first few minutes of cell-cell adhesion, but Mac-1 was needed to sustain adhesion. With regard to selectin-mediated signaling of CD18, cross-linking L-selectin (CD62L) on the neutrophil can activate Mac-1 adhesion (16, 38, 42). Two recent studies of neutrophil adhesion in suspension (E. R. Hentzen, unpublished observations) and in a parallel-plate flow chamber (39) indicate that E-selectin (CD62E) binding to neutrophils may prime or activate Mac-1 and possibly LFA-1. Both of these selectin-mediated signaling events appear to involve p38 mitogen-activated protein kinase within neutrophils.

In this study, we address the effect of chemoattractants on the kinetics of LFA-1 and Mac-1 adhesion in human neutrophils. We chose two peptide chemoattractants, interleukin (IL)-8 and Gro-alpha , which are CXC chemokines that are specific stimuli of neutrophils and expressed by inflamed endothelium (3). We chose leukotriene B4 (LTB4), a known lipid chemoattractant of neutrophils, as a comparison. We also examined which CXC chemokine receptors mediate the effects of IL-8 and Gro-alpha on LFA-1 and Mac-1. Finally, we examined the topographical distribution of bound LFA-1 and Mac-1 on neutrophils as a first step in understanding their adhesive differences in response to chemoattractants.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Recombinant human ICAM-1/IgG1 protein [5 extracellular Ig domains (D1-D5) of human ICAM-1 fused to the Fc portion of human IgG1] was generously provided by Pat Hoffman and Don Staunton (ICOS, Bothell, WA). Recombinant human (72-amino acid) IL-8 and Gro-alpha were purchased from R & D Systems (Minneapolis, MN), LTB4 (catalog no. L0517) from Sigma (St. Louis, MO), carboxylate-modified and protein A-labeled yellow-green fluorescent latex beads (both 1 µm diameter) from Molecular Probes (Eugene, OR), anti-CD11a monoclonal antibody (MAb) R3.1 (mouse IgG1) and anti-domain-2 ICAM-1 MAb R6.5 (mouse IgG2a) from Boehringer-Ingelheim (Ridgefield, CT), and anti-CD11b MAb 2LPM19c (mouse IgG1) from Dako. Anti-CXC chemokine receptor R1 (CXCR1) MAb 9H1 (mouse IgG1) (7) and anti-CXC chemokine receptor R2 (CXCR2) MAb 10H2 (mouse IgG2a) (8) were generously provided by Dr. K. Jin Kim (Genentech, San Francisco, CA).

Isolation of human neutrophils. Human peripheral blood was drawn from healthy adult donors into sterile syringes that were preloaded with an amount of anticoagulant heparin (Elkins-Sinn, Cherry Hill, NJ) to give a final concentration of 10 U/ml. Neutrophils were isolated by centrifugation of collected blood on a Ficoll-Hypaque gradient (Mono-Poly resolving medium, ICN Biomedicals, Aurora, OH), as described previously (36). Isolated neutrophils were kept in Ca2+-free HEPES buffer [110 mM NaCl, 30 mM HEPES, 10 mM glucose, 10 mM KCl, 1 mM MgCl2, pH 7.4, and 0.1% human serum albumin (HSA); Centeon, Kankanee, IL]. Neutrophils were maintained at room temperature and used within 3 h after isolation. Greater than 95% of isolated cells were polymorphonuclear leukocytes, and >99% were viable by trypan blue exclusion.

Quantitation of ICAM-1 binding sites on ICAM-1/IgG1-coated microspheres. We used 125I-labeled R6.5 MAb to quantitate the number of ICAM-1 molecules on the ICAM-1/IgG1-coated beads. R6.5 MAb was labeled with Na125I (Perkin Elmer/NEN Life Science Products, Boston, MA) following the protocol outlined in the Iodo-Beads Iodination Reagent instructions (catalog no. 28665, Pierce, Rockford, IL). An Iodo-Bead was added to a 400-µl suspension of 100 µg of R6.5 MAb and 1 mCi of Na125I in phosphate buffer. After the suspension was incubated at room temperature for 15 min, the bead was removed and the solution was spun down on Micro Bio-Spin P-30 Tris chromatography columns (Bio-Rad, Hercules, CA) for removal of unincorporated 125I. Protein concentration of eluted fractions was determined by a modified Lowry method, as described in the protein assay kit (catalog no. P5656, Sigma). The protein-positive fractions were pooled and measured for radioactive activity to obtain a specific activity of 125I-labeled R6.5 MAb (cpm/µg protein). Gamma counts were measured on a model 1272 CliniGamma gamma counter (LKB Wallac).

We prepared ICAM-1/IgG1-coated beads by incubation of soluble ICAM-1/IgG1 at various concentrations (0, 20, and 50 µg/ml) with protein A-coated fluorescent beads (see Preparation of ICAM-1 beads). ICAM-1/IgG1-coated beads were prepared in duplicate or triplicate for each concentration of soluble ICAM-1/IgG1. After preparation of beads, 125I-labeled R6.5 MAb at a final concentration of 2 µg/ml was incubated with the beads for 40 min, then the beads were washed, resuspended, and subjected to gamma counting. Beads that had been prepared with no soluble ICAM-1/IgG1 were used as background controls, and the counts were subtracted from counts for the other bead preparations. Background-corrected counts were converted to number of molecules by specific activity of the 125I-labeled R6.5 MAb, as determined above. ICAM-1 beads prepared with the lower concentration of 20 µg/ml of soluble ICAM-1 had 42 ± 21 binding sites/µm2 (lowICAM-1 beads). With the higher concentration of 50 µg/ml, there were 162 ± 43 binding sites/µm2, which was significantly higher than the binding sites for the lowICAM-1 beads (P < 0.05, n = 4). The site density of the higher-density ICAM-1 beads (highICAM-1 beads) was comparable with that of an unstimulated human umbilical vein endothelial cell (HUVEC; 170 mol/µm2) reported previously; an HUVEC stimulated with inflammatory cytokines was reported to have 630 mol/µm2 (15, 21).

Preparation of albumin-coated latex beads. One-micrometer-diameter, carboxylated-modified, yellow-green fluorescent beads (4 × 1010 ml-1) were washed twice at a volume ratio of 1:10 in Dulbecco's phosphate-buffered saline (PBS; Life Technologies) and incubated at 1.8 × 109 ml-1 in HEPES buffer-0.1% HSA at 37°C for 30 min to coat them with albumin. Beads were centrifuged and resuspended at 1 × 1010 ml-1 in HEPES buffer-0.1% HSA. These albumin-coated latex beads (ACLB) have been shown to be a specific ligand for CD11b-mediated adhesion (22). Beads were dispersed by bath sonication for 30-45 min with an ultrasonic cleaner (model 8845--30, Cole-Parmer, Chicago, IL) and counted before use with a Coulter counter (model ZM, Coulter Electronics, Hialeah, FL).

Preparation of ICAM-1 beads. One-micrometer-diameter, protein A-coated, yellow-green fluorescent beads (1 × 1010 ml-1) were washed once at a volume ratio of 1:10 in blocking solution (BlockAid, Molecular Probes) and resuspended at 2 × 109 ml-1 with ICAM-1/IgG1 (final concentration 20 or 50 µg/ml) for 90 min under bath sonication. Ice was added to the bath sonicator periodically to prevent overheating of protein-bead suspension. To separate bound from unbound soluble ICAM-1/IgG1 (hereafter, ICAM-1), the bead solution was centrifuged and beads were resuspended in PBS to a final concentration of 1 × 1010 ml-1.

Flow cytometric detection of adhesive events. Neutrophils (3 × 105) were preincubated to a final volume of 0.3 ml of HEPES buffer (plus 0.1% HSA and 1.5 mM CaCl2) with 0.04 µg/ml LDS-751 (Molecular Probes), which is a red nucleic acid dye used to identify neutrophils, for 2 min at 37°C in a mixing chamber, which has been previously described (20, 37). For some experimental runs, neutrophils were preincubated at room temperature with MAb for 5 min, which was sufficient for saturation of binding sites, before the incubation at 37°C. For those runs examining LFA-1-dependent adhesion to ICAM-1 beads, neutrophils were preincubated with 2LPM19c (2 µg/ml) to block the small contribution of nonspecific Mac-1 adhesion at later times (after 3-4 min of stimulation). Subsequent to all preincubation(s), beads (stock 1 × 1010 ml-1) were added at a volume ratio of 1:100 to the neutrophil suspension, which was sheared briefly and sampled (15 µl, see below) for the time 0 point. Stimulus was added at a volume ratio of 1:100 to bead-cell suspension, which was immediately sheared by stir bar at a rate of rotation of ~300 rpm, corresponding to shear stresses previously estimated to be <1.0 dyn/cm2 (37). At predetermined time points, 15-µl samples of the suspension were pipetted into 35 µl of ice-cold 0.5% formaldehyde in PBS. These fixed samples were immediately placed on a flow cytometer (3-color FACScan, Becton-Dickinson) to measure bead-cell adhesive events, as previously described (20). Briefly, using CellQuest software (Becton-Dickinson), we quantified the total number of beads bound to a known number of neutrophils (singlets), from which followed the mean number of beads bound per neutrophil, as previously described (36).

Definitions of rates and extent of bead adhesion. To facilitate comparisons between binding curves in a quantitative manner, the following conventions were chosen. We noted consistently three linear phases of binding, 0-1 min, 1-6 min, and 6-11 min, for binding ICAM-1 beads in response to IL-8. We thus chose to perform linear regression analysis on each of these phases for each binding curve. The slopes from these regressions defined the rates of adhesion. The extent of adhesion was calculated by defining a plateau for each binding curve. The plateau was defined as that group of time points (including the 11-min end point) with adhesive values that were not significantly different from each other. For most binding curves, the plateau consisted of three (6, 9, and 11 min) or four (4, 6, 9, and 11 min) time points. The mean of these time points defined the extent of adhesion.

Cell surface distribution of bound beads. Bead-cell suspensions were prepared as described above, except total volume was scaled up to 400 µl. This was done to take larger samples for microscopic analysis. At the 1- and 4-min time points, an 80-µl aliquot of the suspensions was transferred to an equal volume of ice-cold 2% glutaraldehyde in PBS. The concentration of 1 nM of our three chemoattractants was chosen to sufficiently induce the neutrophils to polarize in shape. All fixed samples were centrifuged through a Mono-Poly density gradient for 2 min to separate unbound beads from neutrophils, which were then examined under light microscopy equipped with differential interference contrast optics. The classification of shape changes of neutrophils has been previously described (46). Briefly, neutrophils can assume a variety of shapes that can be broadly classified as spherical or elongated (polarized). On polarized neutrophils, two distinct ends can often be distinguished: the front (anterior) end and the rear (posterior) end, from which a uropod often protrudes. The number and location of beads bound to polarized neutrophils were noted, and means were calculated and averaged over three separate experiments. The mean numbers for each experiment were normalized by the percentage of neutrophils that had bound beads.

Statistics. Values are means ± SE. One-way ANOVA was used with a Newman-Keuls post test; t-tests were used to compare two groups. P < 0.05 was considered significant.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
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A subnanomolar concentration of IL-8 activates LFA-1 to bind to ICAM-1 within seconds. Flow cytometric detection of highICAM-1 beads adhering to neutrophils is illustrated in Fig. 1. Neutrophil-neutrophil adhesion was rarely observed by flow cytometry or by light microscopy. Binding of highICAM-1 beads was predominantly to LFA-1 on neutrophils, as shown by the marked inhibition of neutrophil-associated beads in the presence of a blocking MAb to LFA-1 (Fig. 2). This predominance was most evident during the initial 60 s after exposure of neutrophils to IL-8 and highICAM-1 beads (Fig. 2, inset). Residual binding was Mac-1 dependent, as demonstrated by the addition of blocking MAb to Mac-1. Anti-ICAM-1 MAb inhibited binding to neutrophils to the same extent as MAb to LFA-1 on neutrophils, and the combination of anti-ICAM-1 and anti-LFA-1 MAbs did not further decrease adhesion. The combination of anti-ICAM-1 or anti-LFA-1 MAb with anti-Mac-1 MAb resulted in an additive effect with complete inhibition of bead binding. Together, these data indicate that LFA-1 bound specifically to ICAM-1 on the beads and that the small contribution of Mac-1 appeared to be independent of ICAM-1.


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Fig. 1.   Flow cytometric detection of neutrophils bound to the higher-density intercellular adhesion molecule-1 (highICAM-1) beads. Isolated human neutrophils (1 × 106 ml-1) were preincubated for 5 min at room temperature with anti-CD11b monoclonal antibody (MAb, 2LPM19c), equilibrated at 37°C for 2 min, and then combined with 1-µm-diameter, yellow-green fluorescent highICAM-1 beads (1 × 108 ml-1) and chemoattractant under continuous mixing conditions. At times indicated, small aliquots were fixed in ice-cold 0.5% formaldehyde and then analyzed by flow cytometry. A: representative dot plot displaying the physical characteristics of human neutrophils exposed to 0.10 nM interleukin-8 (IL-8) for 30 s. Unbound highICAM-1 beads were excluded by adjustment of the forward scatter threshold. B: bead fluorescence dot plot corresponding to neutrophils bound to highICAM-1 beads. Dots represent neutrophil-positive events from the preceding dot plot. Distinct groupings of events represent neutrophils bound to integral numbers of highICAM-1 beads. C: histogram of bead fluorescence dot plot that allows quantitation of the mean number of beads bound per neutrophil. M1, unbound neutrophils; M2, M3, M4, M5, and M6, neutrophils bound to 1, 2, 3, 4, and 5 beads, respectively; M7, neutrophils bound to >= 6 beads.



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Fig. 2.   Specificity of neutrophil adhesion to highICAM-1 beads. Neutrophils (1 × 106 ml-1) were preincubated with or without MAbs for 5 min at room temperature, equilibrated at 37°C for 2 min, and then combined with highICAM-1-coated beads (1 × 108 ml-1) in suspension. Stimulus (0.10 nM IL-8) was added, and mixing was initiated. Mean number of beads bound per neutrophil was calculated for each time point using flow cytometric analysis (see Fig. 1). Where indicated, neutrophils were preincubated with blocking MAbs to LFA-1 (R3.1, 8 µg/ml) and/or Mac-1 (2LPM19c, 2 µg/ml). Where indicated, highICAM-1 beads were preincubated for 10 min at room temperature with blocking MAb to ICAM-1 (R6.5, 5 µg/ml). Inset: 1st min of adhesion. black-lozenge , No MAbs; diamond , anti-Mac-1; black-triangle, anti-ICAM-1; triangle , anti-LFA-1; , anti-Mac-1 + anti-LFA-1; , anti-ICAM-1 + anti-LFA-1; *, anti-ICAM-1 + anti-Mac-1; n = 3-4. *P < 0.01, no MAbs and anti-Mac-1 vs. other conditions.

Stimulation of neutrophils with a low concentration of IL-8 rapidly activates LFA-1- and Mac-1-mediated adhesion. We measured bead-cell adhesion from 15 s (the earliest time point at which our assay could achieve appreciable binding above baseline) to 11 min after addition of IL-8 to neutrophils (Fig. 3A). At 0.01 nM IL-8, LFA-1- and Mac-1-dependent adhesion were not significantly different from their time 0 points. At 0.10 nM IL-8, LFA-1- and Mac-1-mediated adhesion was significantly stimulated (Fig. 3A, insets). At 15 s, LFA-1 bound to lowICAM-1 beads 1.6-fold above baseline (P < 0.01). At 30 s, ACLB binding above baseline reached significance (P < 0.05).


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Fig. 3.   Effect of dose and type of chemoattractant on neutrophil adhesion to low-density ICAM-1 (lowICAM-1) beads vs. albumin-coated latex beads (ACLB). Experiments were performed and analyzed by flow cytometry (see Fig. 1). Each experiment was designed to compare neutrophils binding lowICAM-1 beads (left) and ACLB (right) for IL-8 [A; insets: expanded view of 1st min of adhesion for 0.10 vs. 0.01 nM (baseline)], Gro-alpha (B), and leukotriene B4 (LTB4, C). , 1.0 nM; open circle , 0.10 nM; , 0.01 nM; triangle , neutrophils preincubated with anti-LFA-1 (lowICAM-1 beads) or anti-Mac-1 (ACLB) to confirm specificity of adhesion; n = 3-5. *P < 0.05; **P < 0.01, 0.10 mM vs. 0.01 nM. ***P < 0.05, 1.0 nM vs. 0.10 nM.

LFA-1 binding to ICAM-1 has an early time frame for adhesion, which is optimally induced by a subnanomolar concentration of IL-8. On stimulation by 0.10 nM IL-8, the rates of adhesion through LFA-1 and Mac-1 during the 1st min (initial rate) exceeded the initial rates of their baselines (P < 0.001 for ACLB, P < 0.0001 for ICAM-1 beads; Figs. 3A and 4; values for baseline slopes not shown). The initial rates of adhesion between LFA-1 and Mac-1 were not different. However, from 1 to 6 min of adhesion, the rates of adhesion for LFA-1 and Mac-1 were quite different. Although the 1- to 6-min rate of adhesion through Mac-1 was not significantly different from the initial rate, the 1- to 6-min rate for LFA-1 decreased by 67% to baseline (P < 0.01, slope of 1st min vs. slope from 1 to 6 min; Figs. 3A and 4). From 6 to 11 min, the rates of adhesion through LFA-1 and Mac-1 were down to baseline levels (Fig. 4). The sustained rate of adhesion through Mac-1 during the 1- to 6-min time period is reflected in the 2.5-fold greater extent of adhesion vs. LFA-1 (P < 0.05; Fig. 5). Thus the optimal rate of adhesion of LFA-1 to ICAM-1 occurred during the 1st min, while Mac-1 bound at an optimal rate through the first 6 min of stimulation.


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Fig. 4.   Rates of neutrophil-bead adhesion: analysis of data from time courses in Fig. 3. Rates of adhesion were obtained by linear regression for time 0 to 1 min (filled bars), 1 min to 6 min (hatched bars), and 6 min to 11 min (open bars). *P < 0.01 vs. time 0 to 1-min rate, same respective stimulus and concentration. For IL-8 (A): **P < 0.01 vs. lowICAM-1 and ACLB (time 0 to 1-min rate and same concentration of IL-8). For Gro-alpha (B): **P < 0.05 vs. lowICAM-1 and ACLB; ***P < 0.05 vs. lowICAM-1 (comparisons with time 0 to 1-min rate at same respective concentration of Gro-alpha ). For LTB4 (C): **P < 0.01 vs. lowICAM-1; ***P < 0.01 vs. ACLB (comparisons with time 0 to 1-min rate at same respective concentration of LTB4).



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Fig. 5.   Extents of neutrophil-bead adhesion: analysis of data from time courses in Fig. 3. Extents of adhesion were obtained for each time course by analysis described in MATERIALS AND METHODS. *P < 0.05 vs. lowICAM-1 (0.10 nM). For IL-8 (A): **P < 0.05 vs. lowICAM-1 (1.0 nM). For Gro-alpha (B): **P < 0.05 vs. lowICAM-1 (1.0 nM); dagger P < 0.01 vs. ACLB (Gro-alpha , 0.10 nM). For LTB4 (C): **P < 0.05 vs. lowICAM-1 (1.0 nM). dagger P < 0.05 vs. ACLB (0.10 nM).

Two other findings support the early time frame for the optimal rate of adhesion through LFA-1 in response to a subnanomolar concentration of IL-8. One finding addressed the possibility that the time frame for new adhesive events through LFA-1 could be related to ligand density on the beads. A fourfold increase in surface density of ICAM-1 on the beads increased the rate of adhesion during the 1st min by 2.9-fold (P < 0.01, highICAM-1 beads vs. lowICAM-1 beads, 0.10 nM IL-8; Fig. 4A). However, subsequent rates of adhesion, 1-6 and 6-11 min, decreased by 75 and 92%, respectively, to levels that were not different from corresponding rates for lowICAM-1 beads. It is also noteworthy that the rate of adhesion in the 1st min for highICAM-1 beads was 2.4-fold higher than that for ACLB. Yet, even with the greater advantage in initial adhesion, the extents of adhesion plateaus for the two beads were not different (Fig. 5A), which suggests that LFA-1-dependent adhesion decayed more rapidly than Mac-1-dependent adhesion.

Another finding that supported the early time frame for the optimal rate of adhesion through LFA-1 was observed by delaying the addition of beads to IL-8-stimulated neutrophils. When lowICAM-1 beads were added 1, 3, or 6 min after IL-8 stimulation of neutrophils, the rate of adhesion in the initial 60 s was significantly reduced (Fig. 6C). The extent of adhesion was also reduced (P < 0.05, beads added at time 0 vs. at 3 min; Fig. 6B), in marked contrast to extents of adhesion of ACLB added at 1 and 3 min (Fig. 6A). The initial rate of adhesion of ICAM-1 beads added at 1 min was 34% of the initial rate of control (P < 0.001, beads added at 1 min vs. time 0; Fig. 6C). In contrast, the initial rate of ACLB binding was unaffected by delays of 1, 3, or 6 min (Fig. 6C). Together, these data support a time frame of <1 min for an optimal rate of binding of LFA-1 to ICAM-1 beads and >= 6 min for optimal binding of Mac-1 to ACLB.


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Fig. 6.   Effect of delayed addition of beads to stimulated neutrophils. Experiments were performed as described in Fig. 1 legend, except time of addition of beads to neutrophil suspensions was varied as follows: Beads were added coincident with stimulus (black-triangle) and 1 min (triangle ), 3 min (), and 6 min (diamond ) after stimulation of neutrophils with 0.10 nM IL-8. A: ACLB; B: lowICAM-1 beads. C: initial rate of neutrophil-bead adhesion determined over the 1st min after addition of beads. n = 4. *P < 0.001 vs. "add at 0." **P < 0.05, comparison of extents (defined for each n as mean of 9- and 11-min time points).

We tested whether the early time frame for the optimal rate of adhesion through LFA-1 was dependent on the concentration of IL-8. We found that 0.10 nM IL-8 induced optimal levels of LFA-1-dependent adhesion to lowICAM-1 beads. An increase from 0.10 to 1 nM IL-8 did not increase the rates of adhesion through LFA-1 (Fig. 4A). With regard to the extent of adhesion, an increase from 0.10 to 1 nM IL-8 did not change the extent of LFA-1-mediated adhesion to lowICAM-1 beads (Fig. 5A). A higher IL-8 concentration of 5 nM did not induce significant adhesive changes through LFA-1 compared with 1 and 0.10 nM (data not shown). We addressed the possibility that the dose effect of IL-8 on LFA-1-dependent adhesion was limited by the availability of ICAM-1 on the beads. Compared with 0.10 nM IL-8, 1 nM (Figs. 4A and 5A) or 5 nM (data not shown) IL-8 did not induce significant changes in LFA-1-mediated adhesion of the highICAM-1 beads. This suggests that the surface density of ICAM-1 had a greater effect than the concentration of IL-8 on LFA-1-mediated adhesion.

We confirmed that the binding plateaus reflected an absence of binding and not an equilibrium of adhesive and deadhesive events. We diluted the bead-cell suspensions at 6 min by adding buffer, with the same concentration of stimulus, to bring up the volume 10-fold (data not shown). This dilution lowered the probability of adhesive events while allowing the observation of deadhesive events. ACLB and lowICAM-1 bead binding plateaus remained constant on dilution, which supports the belief that plateaus reflect an absence of adhesive events.

Effect of Gro-alpha and LTB4 on LFA-1 binding to ICAM-1. The principal findings for LFA-1 binding to lowICAM-1 and highICAM-1 beads in response to IL-8 were also observed in response to Gro-alpha and LTB4. Whereas either stimulus at 0.01 nM did not induce significant adhesion, we observed rapid activation of LFA-1 and Mac-1 within seconds of stimulation with 0.10 nM Gro-alpha or LTB4 (Fig. 3, B and C). Initial rates of adhesion for ACLB and lowICAM-1 beads in response to Gro-alpha and LTB4 were similar to those in response to IL-8 and were significantly higher than their respective baselines (P < 0.0001 or P < 0.0005 for all 4 comparisons; Fig. 4, B and C; values for baseline slopes not shown).

We observed early time frames for optimal rates of LFA-1-mediated binding of lowICAM-1 beads in response to 0.10 nM Gro-alpha and LTB4. Initial rates of adhesion were higher than baseline levels, with subsequent 1- to 6- and 6- to 11-min rates of adhesion decreasing significantly to baseline levels for their respective time periods (Figs. 3, B and C, and 4, B and C). Compared with 0.10 nM, the early time frame for optimal rates of LFA-1-mediated adhesion of lowICAM-1 and highICAM-1 beads did not change with higher concentrations of 1 and 5 nM (Figs. 3, B and C, and 4, B and C; data not shown for 5 nM). In terms of rates and extents of lowICAM-1 or highICAM-1 beads (except for the extent of adhesion of highICAM-1 beads for LTB4), 1 or 5 nM did not increase adhesion (Fig. 5, B and C). Thus Gro-alpha , LTB4, and IL-8 shared three effects on LFA-1 adhering to ICAM-1: 1) a time frame of ~1 min for optimal rates of adhesion, 2) a subnanomolar optimal dose, and 3) a weaker dependence on dose of chemoattractant than on surface density of ICAM-1.

The more extended time frame for the optimal rate of Mac-1-dependent adhesion seen with IL-8 was also observed with Gro-alpha and LTB4, with one exception. At 0.10 nM Gro-alpha , the initial rate of adhesion of ACLB was not sustained for the 1- to 6-min time frame, as was the case for IL-8 (Fig. 4). Accordingly, the extent of adhesion was comparable to that for lowICAM-1 beads (Fig. 5B). However, stimulation with 1 nM Gro-alpha sustained the rate of adhesion of ACLB until 6 min, which was reflected in a larger extent of adhesion. Thus a higher concentration of 1 nM Gro-alpha induced a higher rate of adhesion through Mac-1 but not through LFA-1. The sustained adhesion rates and corresponding increase in extent of adhesion for Mac-1 were also seen in response to 0.10 and 1 nM LTB4 (Figs. 4C and 5C). Similar to Gro-alpha , 1 nM LTB4 optimally induced Mac-1-mediated adhesion to ACLB. A higher concentration of 5 nM Gro-alpha or LTB4 did not significantly change Mac-1-mediated adhesion in terms of rates or extents of adhesion (data not shown). Hence, under conditions normalized to compare with LFA-1-mediated adhesion, the optimal Mac-1-mediated rates of adhesion had a more sustained time frame and were responsive to a wider range of chemoattractant concentrations.

CXC chemokine receptors R1 and R2 are each capable of activating LFA-1 and Mac-1. IL-8 is known to bind CXCR1 and CXCR2 with nanomolar affinity on the neutrophil surface. Gro-alpha also binds to CXCR2 and with much lower affinity to CXCR1. Recently, Luu et al. (28) found that blocking MAb to CXCR2, but not to CXCR1, was sufficient to prevent a rolling human neutrophil from firmly adhering to cultured inflamed endothelium. Using blocking MAbs previously characterized with respect to human neutrophils (7, 8, 17, 28), we assessed the contribution of CXCR1 and CXCR2 to activation of LFA-1 and Mac-1. We found that 0.10 nM Gro-alpha failed to activate neutrophil adhesion when anti-CXCR2 (10H2) MAb was present. In contrast, neutrophils preincubated with 10H2 MAb and stimulated with 0.10 nM IL-8 exhibited an insignificant decrement in the rate of bead binding in the initial 60 s after stimulation (Fig. 7, B and C). When MAbs to CXCR1 and CXCR2 were present, initial rates for lowICAM-1 bead and ACLB binding dropped to near-baseline levels (Fig. 7, B and C). The use of 9H1 MAb alone to block CXCR1 resulted in a 58% decrease in initial rate of ACLB adhesion and a 51% decrease in initial rate of ICAM-1 bead adhesion [P < 0.05, 9H1 vs. no block (for ACLB); P = 0.06, 9H1 vs. no block (for lowICAM-1 beads); Fig. 7C]. These results suggest that IL-8 binds and mediates activation of neutrophil LFA-1 and Mac-1 via CXCR1 and CXCR2 with a dominant effect through CXCR1.


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Fig. 7.   Contributions of CXC chemokine receptors R1 and R2 (CXCR1 and CXCR2) to neutrophil-bead adhesion. Experiments were performed as described in Fig. 1 legend, except in some cases neutrophils were preincubated with combinations of blocking MAbs to CXCR1 (9H1, 30 µg/ml) and CXCR2 (10H2, 25 µg/ml). CXC chemokines were tested at doses that induce optimal adhesion of lowICAM-1 beads: 0.10 nM Gro-alpha (A) and 0.10 nM IL-8 (B). C: initial rate of adhesion for the corresponding curves in B. , No MAbs to CXCR1 or CXCR2; open circle , 10H2; black-triangle, 9H1; triangle , 10H2 + 9H1; n = 3-5. *P < 0.05; dagger P = 0.06 vs. no block.

Bound LFA-1 distributes differently from bound Mac-1 on the surface of neutrophils. In terms of rate of adhesion to lowICAM-1 beads and ACLB, respectively, LFA-1 and Mac-1 were equivalent at 1 min but divergent from 1 to 6 min after chemoattractant stimulation. In terms of the surface distribution of bound integrin, LFA-1 and Mac-1 were similar at 1 min but widely divergent 4 min after stimulation with all three chemoattractants. We observed that Mac-1-bound ACLB localized to the rear of polarized neutrophils with time. At 1 min after stimulation, ACLB were evenly distributed between the anterior two-thirds and the posterior one-third of neutrophils (Fig. 8). At 4 min, in addition to an expected increase in the total bound ACLB, we found that the distribution greatly favored the posterior one-third vs. the anterior two-thirds by a 2.0-3.8:1 margin for the three stimuli (P < 0.05 for IL-8, Gro-alpha , and LTB4, respectively). These results are consistent with previous studies of Mac-1-bound ACLB redistributing to the rear of polarized neutrophils (13, 40).


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Fig. 8.   Surface distribution of beads bound to neutrophils. Beads and stimulus were added to neutrophil suspensions under mixing conditions described in Fig. 1 legend. Concentration of stimulus was 1 nM, which induces bipolar shape change in neutrophils. Aliquots were taken at 1 and 4 min after stimulation and fixed in ice-cold 2% glutaraldehyde. Fixed aliquots were centrifuged through Mono-Poly density gradients to remove unbound beads. Neutrophils were visualized by differential interference microscopy, and surface distribution of bound beads was determined. n = 3. *P < 0.05 vs. front two-thirds (same time point, bead type, stimulus). dagger P < 0.01 vs. 1 min, rear one-third (same bead type, stimulus). Dagger P < 0.05 vs. 1 min, front two-thirds (same bead type, stimulus).

In contrast to bound Mac-1, we found that bound LFA-1 did not localize to the rear of neutrophils with time. At 1 min, lowICAM-1 beads were fairly evenly distributed between the anterior two-thirds and the posterior one-third, with perhaps a slight preference toward the anterior two-thirds (significantly different for IL-8, P < 0.05; Fig. 8). At 4 min, the total number of bound lowICAM-1 beads significantly increased (P < 0.05 for all 3 stimuli), but the increase in the number of bound beads preferred the anterior two-thirds, with no significant increases on the posterior one-third for any of the three stimuli. In direct contrast with Mac-1, the distribution of lowICAM-1 beads bound to LFA-1 favored the anterior two-thirds vs. the posterior one-third by a 2.4-3.8:1 margin for all three stimuli (P < 0.05 for IL-8 and Gro-alpha ).

Thus, at an early time point that corresponded to similar rates of adhesion, bound Mac-1 and bound LFA-1 had similar surface distributions. However, at a later time that corresponded to very different rates of adhesion, the surface distributions of each bound integrin were concentrated at different ends of polarized neutrophils.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The importance of beta 2 (CD18)-integrins was first recognized in a rare human genetic disease, the underlying cause of which was impaired emigration of neutrophils deficient in CD18 (1). Subsequent work led to the present model of neutrophil emigration from inflamed vessels as a sequence of steps: rolling, activation, firm adhesion, and migration (43). Selectin (CD62)-mediated rolling serves to slow the flowing neutrophil and to keep it close to the vessel wall. This is thought to allow chemoattractants near the vessel wall to engage neutrophil receptors and activate firm adhesion. CD18 integrins have been shown to be the most important mediators of firm adhesion under fluid flow (24). Of the four known CD18 integrins, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) account for all CD18-dependent firm adhesion in neutrophils. Of the two integrins, LFA-1 has been found to have a greater role in overall neutrophil emigration in studies comparing LFA-1-deficient (-/-) and Mac-1-deficient (-/-) mice (10, 27). Recent work in these mice has demonstrated a dominant role for LFA-1 during the firm adhesion phase of emigration (11). Possible mechanisms that regulate the ability of human neutrophils to use LFA-1 and Mac-1 during the firm adhesion phase include selectin-mediated signaling, fluid shear, and chemoattractants. Previous work has focused on the effects of selectins and fluid shear. The present study examined the effects of chemoattractants on LFA-1- and Mac-1-mediated adhesion.

The three chemoattractants that were examined bind to different receptors, and yet all induce rapid (within seconds) and transient (within 1 min) increases in the rates of adhesion through LFA-1. This early time frame for optimal rates of LFA-1 adhesion complements studies examining the role of shear in LFA-1 adhesion. In a study of human neutrophils binding to cultured endothelium under flow, rolling neutrophils that firmly adhered did so within seconds (15). LFA-1 binding ICAM-1 was a necessary component of optimal firm adhesion in that model. In another study of human neutrophils binding to other neutrophils in suspension, LFA-1 bound to ICAM-3 within a 2-min time frame under shear (45). Two recent studies showed that the rate of LFA-1-mediated adhesion of human neutrophils to ICAM-1-expressing cells in suspension was maximal within the 1st min but decayed within 2 min after initiation of shear (21, 29). In these cell-cell studies, adhesion was affected by adhesive decay of LFA-1 and fluid shear forces. In contrast, our relatively shear-resistant model, as evidenced by the stability of LFA-1 binding plateaus, is affected by the decay of the rates of adhesion. This suggests that the rise and decay of the activated state of LFA-1 may be more important than shear or ligand type in the kinetics of LFA-1-mediated neutrophil adhesion.

Our observation that a subnanomolar concentration of chemoattractant optimally induces LFA-1 to bind ICAM-1 suggests a low threshold of activation of LFA-1. To our knowledge, the only other studies that have reported an effect of a given chemoattractant on LFA-1 adhesion in neutrophils are the cell-cell studies mentioned above. Those studies used FMLP at a very high concentration (1 µM). This dose is unlikely to be physiologically relevant to the luminal surface of inflamed vessels. One argument is that higher doses of soluble chemoattractants would be diluted and washed away by the circulation (44). Another reason is that higher doses would inappropriately stimulate neutrophils and inhibit their localization to inflammatory sites, as demonstrated with IL-8, Gro-alpha , and other known chemoattractants (19, 26, 28, 34). It is noteworthy, however, that according to one of those studies, 30% of neutrophils adhered to a cell line expressing a high level of ICAM-1, even in the absence of FMLP stimulation (21). This adhesion was transient, entirely LFA-1 dependent, and considerably higher than negative control. This is consistent with a low threshold of activation of LFA-1. The stimulus in that case may have been the handling of their isolated human neutrophils, which were stored at 4°C and immediately warmed to 37°C before experimentation. We showed recently that this warming of cold neutrophils by itself activates LFA-1 to bind highICAM-1 beads (unpublished observations). This effect was not observed with the lowICAM-1 beads or with Mac-1 binding ACLB.

The density of ICAM-1 on beads had a greater effect on LFA-1 adhesion than the concentration and type of chemoattractant. It is known that the density of ICAM-1 on the surface of cultured endothelium is increased severalfold by inflammatory cytokines (24). Furthermore, the endothelial surface distribution or clustering of ligands has been shown to be important for neutrophil adhesion (4). Taken together with the very minimal dose requirement of three different chemoattractants to optimally activate LFA-1, this may suggest that ligand density for LFA-1 vs. Mac-1 may be a limiting factor in their relative contributions to the overall efficiency of neutrophil adhesion.

In contrast to LFA-1, Mac-1 did not bind to ICAM-1 on the beads. Although previous studies have demonstrated Mac-1 binding to ICAM-1, this interaction appears to be weaker and more variable than LFA-1 binding to ICAM-1 (9, 15, 21, 29, 41). One possible explanation for our observation is a low surface density of ICAM-1, which was comparable to unstimulated HUVEC for the highICAM-1 beads and fourfold less for the lowICAM-1 beads. Another possible reason is differential posttranslational modification of ICAM-1, which has been shown previously to affect the ability of ICAM-1 to bind Mac-1 (9).

Although all CXC chemokines bind CXCR2 on neutrophils, only IL-8 binds CXCR1 with comparable affinity to CXCR2. Little is known, however, about the biological relevance of having two receptors for IL-8. In addition, the specific effects of each receptor on LFA-1 and Mac-1 have not been examined. Using Gro-alpha , we found for the first time that CXCR2 on human neutrophils rapidly activates LFA-1 to bind ICAM-1. In contrast to Gro-alpha , IL-8 preferred to activate LFA-1 through CXCR1, which came as a surprise, since CXCR2 has been reported to have a higher affinity than CXCR1 for IL-8 (6). With regard to functional roles of CXC receptors, IL-8 was reported to act through CXCR1 in in vitro (18, 32) and in vivo (14) chemotaxis models. The adhesive molecular basis for chemotaxis in those studies, however, was not elucidated. CXCR1 also seems to be more important than CXCR2 in the priming and activation of superoxide production (17, 23). Hence, to our knowledge, this is the first demonstration of the ability of CXCR1 to activate LFA-1 to bind ICAM-1, to bind rapidly, and to bind in response to a subnanomolar dose of IL-8. We also found that CXCR2 and CXCR1 had equivalent effects on LFA-1 adhesion in response to Gro-alpha and IL-8, respectively. Although we tested these chemokines at the subnanomolar concentration that was sufficient for optimal activation of LFA-1, we do not exclude the possibility of, for example, Gro-alpha using CXCR1 or IL-8 using both receptors equally to activate integrins at higher concentrations of stimulus.

LTB4 appears to overlap the CXC chemokines with respect to activating LFA-1- and Mac-1-dependent adhesion. However, unlike the CXC chemokines, LTB4 is thought to be an end-target stimulus for neutrophils, which are themselves the primary source of LTB4 (30). One possible role for LTB4 in activating LFA-1 may be to recruit neutrophils from the circulation in the later stages of acute inflammation. This would be consistent with its production by neutrophils that have already emigrated to the extravascular space. Another possible role for LTB4 in activating LFA-1 may be to assist Mac-1 in neutrophil migration. It is noteworthy that beads that bound to LFA-1 or Mac-1 did not release from neutrophils within our observed time frame of 11 min under shear. Although the beads were small (1 µm diameter) and thus fairly shear resistant, this still suggests that force, perhaps generated by a crawling neutrophil, is needed to downregulate adhesion through beta 2-integrins.

In response to 1 nM LTB4, IL-8, and Gro-alpha , the rate of LFA-1-mediated adhesion after 1 min decreased rapidly, while the rate of Mac-1-mediated adhesion was sustained. LFA-1 and Mac-1 are constitutively present on the neutrophil surface and are sufficient to mediate adhesion (5, 31, 47). Additional Mac-1 upregulates to the surface on cell stimulation but does not participate in adhesion without a second, higher dose of stimulus, as demonstrated by Hughes et al. (22) with ACLB binding to neutrophils in suspension. Thus our experiments were designed to measure the adhesive properties of the resident population of LFA-1 and Mac-1 on the cell surface. It has been hypothesized that adhesion through integrins is activated by a combination of mechanisms: receptor conformational changes, receptor distribution on the cell surface, and flattening of apposed membranes (12). The possibility that cell surface distribution of bound integrin might play a role in adhesion led us to test whether this mechanism might explain the adhesive differences between LFA-1 and Mac-1. As a first step toward answering this question, we compared the surface distribution of bound beads. Although the clustering of ACLB at the rear of stimulated neutrophils has been observed previously (13, 40), to our knowledge, the distribution of bound LFA-1 on neutrophils is unknown. The tendency of bound ICAM-1 beads to distribute away from the rear of neutrophils suggests that the regulation or composition of cytoskeletal linkages to LFA-1 and Mac-1 is different. This was a general effect seen in response to all three chemoattractants at 1 nM. Little is known about the relative interactions of alpha L (CD11a)- and alpha M (CD11b)-subunits with the cytoskeleton. The beta 2 (CD18)-subunit, on the other hand, is known to link indirectly to the cytoskeleton to regulate clustering of LFA-1 on lymphocytes (35). It remains to be investigated whether differential surface distribution and, hence, cytoskeletal regulation of LFA-1 vs. Mac-1 can account for their adhesive differences.


    ACKNOWLEDGEMENTS

We thank Pat Hoffman and Don Staunton at ICOS Corporation for providing the ICAM-1/IgG1 protein and Dr. D. K. Jin Kim at Genentech for providing 9H1 and 10H2 MAbs.


    FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-42550 and HL-18672, by Texas Advanced Technology Program Grant 99003604-0018-1999, and by Robert A. Welch Foundation Grant C938.

Address for reprint requests and other correspondence: C. W. Smith, Sect. of Leukocyte Biology, Depts. of Pediatrics and Immunology, Baylor College of Medicine, 1100 Bates, Rm. 6014, Houston, TX 77030-2600 (E-mail: cwsmith{at}bcm.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 May 2001; accepted in final form 28 June 2001.


    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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