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
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ABSTRACT |
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Firm adhesion of
rolling neutrophils on inflamed endothelium is dependent on
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-
, 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-
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
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INTRODUCTION |
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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-, 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-
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.
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MATERIALS AND METHODS |
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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- 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 ml1) 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 ml1) 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
ml1) 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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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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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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Effect of Gro- 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-
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-
or LTB4 (Fig. 3, B and C).
Initial rates of adhesion for ACLB and lowICAM-1 beads in
response to Gro-
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).
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- 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-
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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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-, 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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DISCUSSION |
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The importance of 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-, 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-, we found for the first time that
CXCR2 on human neutrophils rapidly activates LFA-1 to bind ICAM-1. In
contrast to Gro-
, 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-
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-
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 2-integrins.
In response to 1 nM LTB4, IL-8, and Gro-, 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
L (CD11a)- and
M
(CD11b)-subunits with the cytoskeleton. The
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.
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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.
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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.
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