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*
Department of Pediatrics, Section of Leukocyte Biology, Baylor College of Medicine, Houston, TX 77030;
Department of Medicine, Division of Respirology, University of Toronto, Toronto, Ontario, Canada; and
Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two distinct mechanisms contribute to the localization of neutrophils at sites of inflammation: a decrease in deformability and an increase in adhesive avidity (13, 14, 15). At shear forces >2 dyne/cm2, selectins are required to initiate leukocyte capture and rolling adhesive interactions. Although all three selectins can mediate rolling behavior, L-selectin appears to be the major receptor mediating the initial cell capture (12, 16, 17, 18, 19, 20, 21, 22). In response to inflammatory stimulation, the endothelium expresses a distinct ligand for L-selectin that is sufficient for capture of leukocytes (16).
In the multistep paradigm of leukocyte adhesion, the initial phase of selectin-mediated rolling facilitates subsequent integrin-mediated firm adherence (1, 23). It has been proposed that during rolling, the leukocyte integrins are activated by a soluble or membrane-bound chemoattractant such as IL-8 or platelet-activating factor (24). A second mechanism that we and others have described is through L-selectin-mediated signaling that may directly initiate or amplify ("cosignal") neutrophil activation. Examples of cellular responses induced by L-selectin include an increase in intracellular calcium ([Ca2+]i), tyrosine phosphorylation, activation of extracellular signal-regulated kinases (3), Ras activation, plasma membrane ruffling, and cytoskeletal reorganization (2, 25, 26, 27, 28, 29). Exocytosis of granules containing CD11b/CD18 accompanied by an increase in the adhesive function of the ß2 integrins has also been observed (27). These events occur within seconds of Ab-mediated cross-linking of L-selectin, a process that is believed to simulate the clustering of L-selectin by its cognate ligand expressed on inflamed endothelium (30).
ß2 integrins are constitutively expressed in a
low-avidity state and require activation to mediate cell adhesion
(15, 31). A variety of soluble stimuli, such as fMLP and
TNF-
, can activate ß2 integrin binding and
neutrophil chemokinesis. Once activated, Mac-1 is capable of binding to
diverse ligands including ICAM-1, iC3b, and albumin-coated substrates
(32). Enhanced adhesiveness may result from de novo
expression of active integrin, clustering of integrin receptors, or
alterations in the affinity of individual receptors for ligand
(33). We have recently reported that activation of
neutrophils through chemotactic receptors is synergistic with
signaling through L-selectin for a variety of responses that
are associated with emigration and transmigration on inflamed
endothelium (30, 34). We observed that costimulation of
neutrophils by cross-linking L-selectin in conjunction with exposure to
a chemotactic peptide resulted in increased adhesivity of CD11b/CD18
receptors, as measured by binding of Ab-coated latex beads
(30). Interestingly, this costimulation was associated
with a significant decrease in cell motility. An unanswered question
was whether the decrease in cell mobility was due to supraoptimal
activation of CD11b/CD18 resulting in avid adhesion, or whether
cross-linking L-selectin effectively thwarted mobility of activated
CD11b/CD18, which is required for effective cell motility.
In the current study, we utilized Ab-mediated cross-linking in vitro as a model system to examine potential cellular alterations in neutrophils induced by ligation of L-selectin that might contribute to their localization at sites of inflammation. We observed that clustering of L-selectin by Ab cross-linking led to a rapid (in seconds) induction of actin assembly that correlated with a decrease in cell deformability. In addition, L-selectin clustering induced an enhanced surface expression of the ß2 integrin, CD18, that became spatially colocalized with L-selectin in the plasma membrane. These observations have important implications for our understanding of the synergistic mechanisms leading to initial sequestration and activation of neutrophils in the microvasculature.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Krebs-Ringer phosphate buffer with glucose (KRPD) included the following: 122 mM NaCl, 4.8 mM KCl, 3.1 mM NaH2PO4, 12.5 mM Na2HPO4, 1.2 mM MgSO4, and 11 mM glucose. All reagents were obtained from Mallinckrodt (Paris, KY) and were endotoxin free. Percoll and dextran T500 were obtained from Pharmacia Biotech (Baie D’Urfe, Quebec, Canada). Rhodamine phalloidin and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phallicidin were from Molecular Probes (Eugene, OR). Lysophosphatidylcholine was obtained from Avanti Polar Lipids (Pelham, AL). Paraformaldehyde was from J. B. EM Services (Point Claire-Dorval, Quebec, Canada). HEPES, KCl, DMSO, cytochalasin D, Triton X-100, and Ficoll-Hypaque were from Sigma (St. Louis, MO). Formalin was from Fisher Scientific (Unionville, Ontario, Canada). Genistein and herbimycin A were from Calbiochem (La Jolla, CA). Pertussis toxin was from List Biological Laboratories (Campbell, CA). Sodium citrate was purchased from Fisher Scientific (Fair Lawn, NJ), 0.9% NaCl and sterile water were purchased from Abbot Laboratories (Saint-Laurent, Quebec, Canada), CaCl2 and NaCl were purchased from BDH Chemicals (Toronto, Ontario, Canada), sodium bicarbonate was obtained from J. T. Baker (Phillipsburg, NJ), and albumin fraction V was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). fMLP (Sigma) was diluted in buffer to 1 and 5 nM, which are the activating concentrations. All of the reagents were of molecular biology grade unless otherwise noted.
Antibodies
Humanized DREG 200 and DREG 55 (HuD200/55) have been described
previously (35). They are murine anti-CD62L IgG1
spliced to human Fc
2, which has low affinity
for human FcR. Both were used at 10 µg/ml. Anti-HLA IgG2a, W6/32, was
used as a control nonactivating mAb (36). Goat
anti-mouse (GAM) F(ab')2 and goat
anti-human F(ab')2 were used to cross-link
W6/32 and HuD200/55, respectively. The cross-linking Abs were used at a
concentration of 20 µg/ml. Abs to L-selectin, including DREG 55, DREG
56, DREG 200, DREG 56 F(ab')2, and LAM1-14, were
developed as previously described (37). FMC 46
(anti-L-selectin), anti-HLA class I, including W6/32 (mouse
IgG2a) (36) and FITC-conjugated B-H9 (mouse IgG1),
FITC-conjugated B-E16 (mouse IgG2a) against human FcRIII, and
FITC-conjugated B-Z1 (mouse IgG1), an isotype-specific negative
control, were from Serotec Canada (Toronto, Ontario, Canada). GAM Abs
used for cross-linking were from two sources. Affinity-purified
F(ab')2 fragments of GAM directed against
F(ab')2 fragments of mouse IgG were from Bio/Can
Scientific (Mississauga, Ontario, Canada). GAM directed against whole
mouse Ig was from Cappel (Organon-Teknika, Durham, NC). Labeled
secondary Abs used to stain unconjugated primary Abs included ST-AR 9,
which is a F(ab')2 fragment of rabbit
anti-mouse, labeled with FITC; ST-AR 10, a
F(ab')2 fragment of rabbit anti-mouse labeled
with tetramethylrhodamine isothiocyanate (TRITC) from Serotec (Toronto,
Ontario, Canada); and Texas Red and Cy3 F(ab')2
fragments of GAM from The Jackson Laboratory (Bar Harbor, ME).
Neutrophil isolation
Healthy adult volunteer whole blood (5–20 cc) was drawn into heparinized syringes (10 U/ml). Neutrophils were isolated either by dextran sedimentation followed by plasma-Percoll gradient centrifugation as previously described (38) or by centrifugation through a Ficoll-Hypaque solution (Mono-Poly resolving medium; ICN Pharmaceuticals, Costa Mesa, CA) for 30 min as previously described (30). Neutrophils were suspended in a 0.1% human serum albumin and HEPES buffer (30 mM HEPES, 110 mM NaCl, 10 mM KCl, 1 mM MgCl2, and 10 mM glucose) in the absence of calcium. This buffer was sterile filtered, autoclaved, and found to be endotoxin free. Neutrophils were stored at 4°C until use, at which time they were warmed in buffer containing 1.5 mM CaCl2.
Neutrophil adhesion to albumin-coated latex beads (ACLB)3
Yellow-green fluorescent, carboxylate-modified latex beads (Molecular Probes) were washed twice in Dulbecco’s PBS (no calcium, no magnesium) and coated with albumin by suspension in 0.1% human serum albumin for 30 min at 37°C as previously described (27). These ACLB were sonicated for 1 h before experiments to minimize bead aggregation.
Neutrophils suspended in HEPES buffer at 106/ml were incubated at room temperature with either HuD200/55 or W6/32 for 10 min. Cell suspensions were centrifuged to remove unbound mAb, and polymorphonuclear leukocytes (PMNs) were resuspended in buffer. PMN suspensions were activated (t = 0) by addition of polyclonal Ab cross-linker and/or fMLP (1 nM) and ACLB and allowed to incubate at 37°C in a thermomixer. At 5 min, 100 µl of sample was fixed with an equal volume of 4% glutaraldehyde. Cell suspensions were also stimulated for an additional 5 min with another pulse of fMLP at 5 nM and then fixed at 10 min. All fixed samples were centrifuged through a Mono-Poly density gradient for 2 min to separate unbound ACLB from the PMNs, which were then examined under a phase-contrast microscope. The classification of shape changes of PMNs has been previously described (30). Briefly, cells were assigned a classification from I to IV on the basis of the extent of shape change, as follows: I, spherical; II, spherical and ruffled plasma membrane; III, bipolar elongated; and IV, uropod formation at one end. In addition, the location at which ACLB were adherent to the PMN was recorded. The mean number of ACLB were computed from three separate experiments. One-way ANOVA was calculated using the commercial statistical software Prism (Graphpad Software, San Diego, CA). Probability of statistical significance between means was determined by the Newman-Keuls test (p < 0.05 was taken to be significant).
Confocal microscopy
To examine the distribution of L-selectin in resting neutrophils, cells were incubated with saturating amounts of primary Abs in KRPD for 30 min on ice followed by incubation with FITC- or TRITC-labeled secondary GAM Ab for an additional 30 min on ice followed by fixation with 1.5% paraformaldehyde for 15 min at room temperature. For cross-linking experiments, neutrophils were treated with saturating amounts of primary Abs in KRPD at 37°C for 20 min, washed, and resuspended at 37°C. Results were not different when the cells were incubated with primary Ab on ice. Primary Abs were cross-linked with secondary GAM F(ab')2 Ab at 37°C. After the specified time, the cells were fixed with 1.5% paraformaldehyde for 15 min at room temperature. The cells were washed and resuspended in PBS with 0.5% BSA and 20 mM glucose. Similar results were obtained when L-selectin was cross-linked using nonfluorescent GAM secondary Ab followed by fixation and addition of FITC-labeled tertiary goat anti-rabbit Abs. The distribution of CD18, CD16, or HLA class I was examined using directly conjugated FITC-labeled anti-human Abs in the same buffer. For dual labeling, the cells were incubated with primary anti-L-selectin Abs, followed by TRITC, Cy3, or Texas Red-GAM Ab (cross-absorbed against mouse IgG) as indicated, fixed, and washed twice before incubation with rat anti-human CD18, CD16, or HLA Abs.
The distribution of F-actin within cells was examined using rhodamine phalloidin as previously described (39). Briefly, after cross-linking, fixation, and/or staining for L-selectin, the cells were allowed to settle on coverslips that were previously coated with 1 mg/ml poly-L-lysine. After 20 min, the coverslips were rinsed in PBS, and the neutrophils were permeabilized by incubation in 0.1% Triton X-100 in KRPD for 15 min. The cells were stained with 1.65 x 10-7 M rhodamine phalloidin for 10 min at 37°C and then washed with several rinses in PBS. The coverslips were mounted with Immumount (Shandon, Pittsburgh, PA). The slides were viewed using a Zeiss laser scanning confocal microscope and digital images captured as TIFF files using the Zeiss software. These images were imported into Adobe Photoshop, labeled, and printed on a Kodak digital printer.
Flow cytometry
Surface expression of L-selectin, CD18, CD16, and HLA class I was measured using direct immunofluorescence and flow cytometry. Cells were stained using the same techniques as described for confocal fluorescence microscopy. Stained cells were analyzed on a FACScan (Becton Dickinson, Palo Alto, CA) using FL1 detector (488-nm excitation and 530-nm emission wavelengths). Cells were gated on the forward and right-angle light scatter to remove debris and cell clumps. Typically, 1 x 105 cells were analyzed per condition, and all values are expressed as specific fluorescence by subtracting the fluorescence after staining with secondary Ab in the absence of primary Ab. The quantity of polymerized F-actin within neutrophils was measured as previously described (40). Briefly, after experimental manipulation, cells were simultaneously fixed and permeabilized with lysophosphatidylcholine (0.1 mg/ml final concentration) in buffered formalin. After a 5-min incubation at 37°C, NBD-phallacidin (evaporated down from methanol and resuspended in KRPD) was added to a final concentration of 1.65 x 107 M. Cells were examined on a FACScan, and values are expressed as relative fluorescence index by dividing the linearized fluorescence of the experimental group by the value for the unstimulated control cells. This method has been shown to correlate with biochemical measurements of F-actin.
Fluorescence resonance energy transfer (FRET)
Buffers. HEPES-NaCl medium contained 140 mM NaCl, 4 mM KCl, 10 mM glucose, 10 mM HEPES, 1 mM MgCl2, and 1 mM CaCl2, and 0.5% BSA (pH 7.4). All cell incubations and washes were in this medium.
Abs. Mouse mAb to human CD11a (clone MEM-25) IgG1 conjugated with FITC and mouse mAb to human CD11b (clone CR3) IgG1 conjugated with FITC were purchased from Caltag Laboratories (Burlingame, CA); mouse anti-human CD18 (clone IB4) was obtained from Dr. David Chambers (San Diego Regional Cancer Center). Goat anti-human F(ab')2 fragment to cross-link to human IgG was obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). A humanized Ab to human L-selectin (HuDREG 200 to CD62L) was generously provided by Dr. Ellen Berg at Protein Design Labs (Mountain View, CA) (35). The Ab was conjugated with TRITC from Molecular Probes, as described below.
Fluorescence conjugation to Abs. Tetramethylrhodamine 10 mg/ml in DMSO was added to the HuDREG 200 Ab in sodium bicarbonate buffer 0.2 M (pH 8.3) and incubated for 2 h at room temperature. The separation of conjugate from unreacted dye was done by gel filtration (NICK columns, Pharmacia Biotech, Uppsala, Sweden) equilibrated with PBS according to the manufacturer’s instructions.
Experimental procedures. Neutrophils in suspension (108 cells/ml) were incubated at 4°C for 1 h with the following Abs: anti-L-selectin HuDREG 200-TRITC (acceptor) and mouse anti-human CD18 conjugated with FITC (donor). Cells were washed twice, and the cell suspension was divided into two parts. One part was fixed immediately, and the other part was warmed to 37°C for 10 min and then fixed. This set of experiments included several controls. Cells were incubated with HuDREG 200-TRITC (acceptor alone) as above and then warmed to 37°C for 10 min and fixed with 1.6% paraformaldehyde. Additionally, quiescent cells were incubated with anti-CD18-FITC (donor) and unlabeled HuDREG 200 as above, warmed to 37°C for 10 min, and fixed with 1.6% paraformaldehyde. As a third control, the cells were incubated with unlabeled anti-CD18 and HuDREG 200-TRITC as above, warmed to 37°C for 10 min, and fixed with 1.6% paraformaldehyde. As a fourth control, the fluorophores were reversed with anti-L-selectin (FMC 46)-FITC as the donor and anti-CD18 (IB4)-TRITC as the acceptor. To cross-link the L-selectin, cells were incubated at 4°C for 1 h with HuDREG 200-TRITC and anti-CD18-FITC and then incubated at 4°C for 1 h with a secondary F(ab')2 fragment to human IgG. After two washes, the cell suspension was divided into two parts. One part was fixed immediately, and the other part was warmed to 37°C for 10 min and subsequently fixed.
Two fluorescent signals were measured by flow cytometry (FACSCalibur, Becton Dickinson, Mountain View, CA) using Cellquest software (Becton Dickinson). Live cells were gated on the basis of forward and side scatter. Fluorescence excitation was 488 nm; emission wavelength of the anti-CD18-FITC was 520 nm, and that of the HuDREG 200-TRITC L-selectin was 580 nm. Energy transfer between the donor-acceptor pair of the anti-CD18-FITC and anti-L-selectin-TRITC was analyzed.
Filtration assay of cell deformability
Purified human neutrophils were subjected to filtration through microporous membranes as previously described (13, 41, 42). In brief, polycarbonate filters (Poretics Corp., Livermore, CA) with a mean pore size of 6.5 µm (range, 6.0–7.0 µm; coefficient of variation, <10%) in diameter, polypropylene chambers, and siliconized plastic i.v. tubing were protein coated by incubation in 20% heat-inactivated human plasma at 37°C for 2 h to minimize cell adhesion to the tubing and chambers. A multichannel infusion pump (Harvard Apparatus, Millis, MA) was used to provide a constant flow rate of the buffer across the filters. Immediately upstream of each filter chamber, a pressure transducer connected to a strip chart recorder continuously measured pressure. This apparatus allowed three filtration systems to be run and monitored simultaneously.
Neutrophils were labeled with 111In (New England
Nuclear, Boston, MA) by incubating 20 µCi of
111InCl/106 neutrophils
with 5 x 10-4 M tropolonate (Fluka,
Ronkonkoma, NY) for 5 min in KRPD followed by a wash. For the
experiments, 1 x 105 neutrophils were used
per filter, which represents a ratio of 
1 leukocyte/4 pores (the
number of pores in the filter was taken from the manufacturer’s
specifications). 111In-labeled neutrophils in
suspension were injected as a bolus over 2 s into the port of the
filtration system, and the effluent was collected into 50-ml
polypropylene containers and subsequently transferred into plastic
scintillation vials for counting. The cells in suspension were allowed
to flow through the filtration system until a total of 5 ml was
collected. At this time the flow was interrupted, the tubing was
disconnected from the filter chambers, and the filters were removed.
The filters, proximal and distal chambers, were also placed in plastic
scintillation vials and counted, along with the effluent, in a gamma
well counter ( 7000, Beckman Coulter, Fullerton, CA). All values
were expressed as a percentage of total radioactivity recovered. All
filtration experiments were conducted at room temperature.
Magnetic twisting cytometry
Ferromagnetic beads coated with GAM IgG (Fc) (Spherotech, Libertyville, IL) were incubated with mouse anti-human CD45 or anti-CD11b (Dako, Carpinteria, CA) at a concentration of 1 µg/106 beads for 30 min at 4°C and washed three times with PBS. Isolated human neutrophils were treated with 20 µg/ml mouse anti-human L-selectin Ab (clone DREG 55, Biosource International, Camarillo, CA) or mouse IgG for 20 min at room temperature. After one wash, neutrophils (2 x 105/well) and beads coated with anti-CD45 or anti-CD11b Ab (3.5 x 105/well) were combined in plastic wells (Immulon 2, Dynatech Laboratories, Chantilly, VA) and gently centrifuged at 150 x g for 4 min. The unbound beads were washed off, and each well was placed in the magnetic twisting cytometer. The biomechanical properties of neutrophils were measured as previously described (43, 44). Briefly, the beads were exposed to a brief (10-µs) but strong (1000-gauss) magnetic field, which magnetized the beads in the horizontal direction. Twenty seconds later, the beads were twisted by a much weaker (30-gauss) but continuous (1-min) vertical magnetic field. This twisting field was not strong enough to remagnetize the beads, but it caused the beads to rotate. The magnitude of magnetic vector in the horizontal direction (remnant magnetic field) was measured by an in-line magnetometer. From this value, the average bead rotation (angular stain) was calculated (43, 44). The rotational stress was calculated by rotating the beads in a viscous standard. For these beads, a twisting field of 10 gauss corresponds to an applied torque at the start of the twist (initial stress) of 7 dyne/cm2, and the stress is proportional to the magnitude of this twisting field. The specific torque (stress) on the beads at the end of the 1-min twist (stress1 min) was calculated using the initial stress times the ratio of remnant field at the end of 1-min twist and the remnant field at time 0. The apparent stiffness was measured at 1 min of twist and is defined as the ratio of stress1 min to the angular strain at this time point. To evaluate the effect of L-selectin cross-linking on the biomechanical properties of neutrophils, 50 µg/ml goat IgG F(ab)2 fragment directed against mouse IgG Fc fragment along with 2 mM Ca2+ was added to the wells, and the stiffness of neutrophils was measured before or 2, 5, 10, and 15 min later.
Statistics
All data are reported as mean ± SD unless otherwise specified. Results were analyzed by ANOVA with corrections for multiple comparisons (Sheffé test) or using the Newman-Keuls test as indicated with p < 0.05 considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of neutrophils by ligation and clustering of L-selectin
is associated with a rapid change in cell shape. We examined the
kinetics of F-actin formation following addition of an
anti-L-selectin mAb and a secondary polyclonal Ab to cross-link
L-selectin. Addition of DREG 200 alone, an Ab that recognizes the
lectin domain of L-selectin, resulted in a small but significant
increase in F-actin as measured by the increase in fluorescence of
NBD-phallacidin (Fig. 1
A).
Importantly, subsequent addition of a polyclonal GAM secondary
(cross-linking) Ab induced an 80% increase in the amount of F-actin
compared with baseline. A similar and sustained increase in F-actin was
also elicited by cross-linking LAM1-14, an Ab that binds to the EGF
domain of the L-selectin receptor (Fig. 1B). We confirmed
that the increase in F-actin was not mediated by binding of the Fc
domains of the anti-L-selectin Abs to the FcII or FcIII
receptors on the neutrophil, given that pretreatment with blocking Fab
or F(ab')2 fragments to both Fc receptors did not
inhibit the increase in F-actin in response to LAM1-14 (Fig. 1B) (29).
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C, under these
conditions, L-selectin-mediated actin assembly remained intact,
indicating that pertussis toxin-sensitive GTP-binding proteins such as
Gi-1 were not involved in this pathway. In
contrast, treatment with pertussis toxin largely abrogated the increase
in F-actin induced by exposure to the chemoattractant peptide fMLP, as
previously reported (45, 46), confirming that the toxin
was active under these conditions.
We have previously reported that ligation of L-selectin resulted in
increased tyrosine phosphorylation of several cytosolic proteins
(26). To determine whether this enhanced tyrosine
phosphorylation was involved in L-selectin-mediated actin assembly,
cells were pretreated with two chemically distinct tyrosine kinase
inhibitors (genistein 100 µM for 30 min at 37°C and herbimycin A 5
µM for 4 h at 37°C) before cross-linking L-selectin. We have
previously demonstrated that these conditions abrogate increases in
cellular tyrosine phosphorylation and calcium transients in response to
stimulation via either L-selectin or Fc receptors (26, 47). In contrast, Fig. 1D illustrates that
L-selectin-mediated actin assembly was unaffected by either of these
inhibitors, indicating that tyrosine kinases were likely not involved
in L-selectin-mediated alterations in the actin cytoskeleton.
Cross-linking L-selectin induces spatial colocalization of CD18
We have previously reported that signaling through L-selectin
results in a rapid increase in the expression of CD11b/CD18 and the
avidity of neutrophil adhesion to CD18 ligands, including ICAM-1,
expressed on activated endothelium and cell lines (27, 48). We next examined the effect of L-selectin cross-linking on
the plasma membrane distribution of CD18 on neutrophils in suspension.
The distribution of CD18 and L-selectin on the surface of resting cells
was detected with fluorescence-conjugated Abs using confocal
microscopy. L-selectin was labeled with a FITC-conjugated mAb and CD18
with a Texas Red- or TRITC-conjugated Ab as indicated. Each Ab emitted
at a distinct wavelength. Discrete and simultaneous detection of each
adhesion receptor required that we first confirm that very little
spectral crossover was detected between the emission wavelengths
measured on the respective fluorescence channels. As illustrated in
Fig. 2a, L-selectin was evenly
distributed over the entire neutrophil surface with some small clusters
or patches, in agreement with previously published reports (18, 49). Cells labeled with Ab to CD18 also showed a similar surface
distribution (Fig. 2b). These two panels also illustrate the
lack of crossover of fluorescence signal from either fluorophore into
the other channel. Dual-labeled cells, stained for both CD18 and
L-selectin, demonstrated a similar peripheral "ring" of membrane
staining with some small clusters (Fig. 2c).
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d).
Redistribution of CD18 was also observed after cross-linking L-selectin
(Fig. 2e) and, in cells that were stained for both
L-selectin and CD18, it was apparent that the patches of each receptor
colocalized to identical regions (Fig. 2e). These patches of
adhesion receptors also colocalized with subjacent aggregates of
F-actin (Fig. 2f). Cross-linking other neutrophil surface
Ags such as HLA or CD45 led to patching of the cross-linked Ag but not
to copatching of CD18 (not illustrated). Additionally, cross-linking of
L-selectin did not lead to patching of HLA (not illustrated). These
results indicate the specificity of the interaction between L-selectin
and CD18. Cross-linking L-selectin potentiates chemoattractant-induced increases in CD18 adhesive avidity
We next examined whether stimulation through cross-linking of
L-selectin, in the presence of pulse stimulation with fMLP (hereafter
denoted as costimulation), would alter the binding and mobility of
CD11b/CD18 compared with either stimulus alone. Quiescent
(unstimulated) neutrophils retained a spherical shape with numerous
surface folds and few adherent ACLB (Fig. 3, top left). Exposure to a
low concentration of fMLP (1 nM) initiated cell shape change
(characterized by a ruffled or polarized shape) and led to activation
of CD11b/CD18 sites in a random pattern along the cell body
(50). Cross-linking L-selectin increased the fraction of
cells that adopted a ruffled membrane (45% of the population)
relative to unstimulated cells (Fig. 3a, top
right). However, when compared with cells stimulated with a low
concentration of fMLP alone, cross-linking L-selectin elicited far less
uropod formation (<10%). Stimulation with 1 nM fMLP induced a
significant amount of cell shape change, including bipolarization and
the formation of a distinct uropod in >90% of cells within 5 min. At
this time point in samples stimulated with fMLP alone, one-half of the
ACLB were bound to the cell body, and an equal number were transported
to the uropod (Fig. 3b). Pulse stimulation of neutrophils
with a low dose of fMLP (1 nM) followed in 5 min by a second higher
concentration of stimulus (5 nM) induced movement of ACLB to the
trailing end of the neutrophil (e.g., defined as the uropod in
chemotaxing neutrophils) (Fig. 3a, bottom left,
and Fig. 3b), indicating enhanced mobility of the integrin
receptor. Cross-linking L-selectin also induced CD11b/CD18 activation
as indicated by a 5-fold increase in adhesion of beads relative to
control (unstimulated) cells (Fig. 3a, top right,
and Fig. 3b) or cells subjected to cross-linking of the HLA
receptor. Stimulation through L-selectin alone did not induce mobility
of adherent ACLB, clearly distinguishing the effects of chemoattractant
from those of cross-linking L-selectin. Costimulation by the
combination of fMLP and cross-linking L-selectin (or the control HLA
receptor) did not significantly increase the extent of ACLB bound after
5 min. In contrast, an additional pulse of 5 nM fMLP resulted in a
significant increase in ACLB bound and an enhanced transport from the
body to the uropod by 10 min in costimulated samples (Fig. 3a, bottom right, and Fig. 3b). Taken
together, the data indicate that cross-linking L-selectin by itself
induced a significant increase in CD11b/CD18 avidity but little
receptor mobility. Furthermore, in the presence of costimulation with
fMLP, mobility of ACLB to the uropod was not inhibited, indicating that
the decrease in chemokinesis after cross-linking L-selectin observed in
our previous study was likely due to supraoptimal adhesivity of
CD11b/CD18.
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The confocal fluorescence microscopic observations illustrated in
Fig. 2 suggested that a portion of the CD18 and L-selectin clustered
together on the plasma membrane in patches following cross-linking. A
technique that affords much greater spatial resolution of
intermolecular distance is FRET. This has previously been used to
demonstrate association of CD18 with the FcRIII receptor on
neutrophils (51). The strategy used in the present study
was to ligate CD18 with an Ab conjugated to FITC that would register a
decrease in its emission fluorescence (quenching) on donating a photon
to an acceptor fluorophore (TRITC) bound to an anti-L-selectin Ab.
This quenching of the CD18 fluorescence signal by the L-selectin-bound
fluorophore occurs when the two surface receptors come within 150 Å
of each other on the plasma membrane. Several controls were performed,
including switching the donor and acceptor fluorophores on CD18 and
L-selectin to ensure that modulation in expression of either receptor
did not bias the data. We also used Abs to different -chains (CD11a
and CD11b) of CD11/CD18, as well as a control Ab to another surface
receptor (HLA), in place of CD18 to examine the specificity of the
signal. Additionally, we used cross-linking Abs specific to different
epitopes (e.g., human vs mouse) on the donor and acceptor
molecules.
A humanized form of DREG 200, engineered to present a low-affinity
IgG2 Fc domain, was conjugated with TRITC, and
a murine anti-CD18 (IB4) was conjugated with FITC. The fluorescence
intensity of the acceptor (DREG 200-TRITC)- and donor
(IB4-FITC)-labeled neutrophils was measured on a flow cytometer and
compared between resting cells and those in which L-selectin had been
cross-linked. Neutrophils were labeled on ice and then washed free of
excess Ab before being warmed to 37°C for 10 min in the absence
(resting control) and presence of goat anti-human
F(ab')2 to specifically cross-link L-selectin.
The data are presented as the mean fluorescence intensity (MFI) as a
percentage of control of dual-labeled fluorescent CD18 donor and
L-selectin acceptor compared with cells labeled with fluorescent CD18
donor and nonfluorescent L-selectin (Fig. 4). In resting cells incubated at 37°C
with the labeled Abs but in the absence of a secondary (cross-linking)
Ab, a negligible decrease in MFI was measured (Fig. 4A).
However, following L-selectin cross-linking, the CD18-FITC
fluorescence was quenched by 25%, indicating the close
association and energy transfer between the fluorophores bound to the
two adhesion molecules (Fig. 4A). A comparable response was
observed when the fluorophores were reversed on the anti-selectin
and anti-ß2 integrin Abs and when alternate
anti-L-selectin and anti-ß2 integrin
Abs were used (data not shown). These data indicate that true energy
transfer occurred between Abs bound to CD18 and L-selectin and that
there was a negligible contribution from potential shedding of
L-selectin on neutrophil activation. As an additional control, we
assessed the potential for energy transfer between L-selectin and
another surface receptor HLA class I under similar conditions of
cross-linking L-selectin. As illustrated in Fig. 4B, no FRET
occurred between L-selectin and HLA class I, demonstrating the
specificity of interaction between L-selectin and the
ß2 integrin.
|
-chains of the
ß2 integrin, CD11a and CD11b, to L-selectin by
measurement of the FRET between FITC-conjugated Abs to each integrin
subunit, respectively, and anti-L-selectin DREG 200-TRITC (Fig. 4, C and D). A smaller but significant decrease in
fluorescence was observed on cross-linking L-selectin in cells labeled
with anti-CD11a-FITC or anti-CD11b-FITC, indicating that FRET
occurred for either integrin subunit following cross-linking of
L-selectin. We confirmed that the fluorescence quenching was associated
with the clustering of surface receptors, by performing the identical
experiments entirely at 4°C where diffusion of surface receptors is
very low. Under these conditions, no fluorescence quenching on
cross-linking of L-selectin was observed (not illustrated), indicating
that in resting neutrophils at 37°C there is no detectable
association between the ß2 integrin and
L-selectin. Taken together, these observations demonstrate that within
minutes of cross-linking of L-selectin at 37°C, a fraction of CD18
comes into molecular proximity with L-selectin. Cross-linking L-selectin increases neutrophil retention in filters
After chemotactic stimulation in the microcirculation, neutrophils
exhibit the ability to rapidly modulate not only their adhesion
receptor avidity, but also their rheologic properties. Both of these
properties have been found to contribute to the microvascular
sequestration of the cells. To assess potential alterations in cell
deformability as a consequence of L-selectin clustering, we examined
the filterability of neutrophils across a porous membrane
(diameter = 6.5 µm) driven by a defined pressure gradient. The
measured parameters included the driving pressure immediately upstream
of the filter required to maintain a constant flow rate of 1 ml/min
over time and the fraction of neutrophils that were retained in the
membrane at the final time point following ligation and cross-linking
of L-selectin. In untreated (control) cell suspensions or in those
treated with secondary cross-linking Ab in the absence of primary
anti-L-selectin Ab, an increase in pressure from 0.4 to 1 cm
H2O was required to maintain the flow rate (Fig. 5A). This correlated with
25% of the neutrophils becoming trapped in the membrane (Fig. 5B). In response to addition of anti-L-selectin
(LAM1-14), a small increase in the driving pressure was noted that
correlated with a small increase in the neutrophil retention. In
contrast, on addition of cross-linking GAM Abs to LAM1-14-decorated
neutrophils, a 3-fold increase in perfusion pressure was observed
(1.02 ± 0.15 (control) vs 3.3 ± 0.3 cm
H2O (LAM1-14 + GAM
F(ab')2); p < 0.01) that
directly correlated with a similar increase in neutrophil retention in
the filters. No significant aggregation of neutrophils was observed in
the perfusion buffer or on the filters after L-selectin cross-linking
(not illustrated). Taken together, the data indicate that cell
stimulation by clustering L-selectin with Ab results in a rapid
decrease in cell deformability and trapping of cells within the
filters.
|
As an alternative approach to gauge the effect of cross-linking
L-selectin on the biomechanical properties of neutrophils, we measured
the angular rotation of ferromagnetic beads bound to surface receptors
on human neutrophils in response to an applied torque (stress). We
measured the rotational mobility of ferromagnetic beads bound to the
membrane via CD11b or CD45, a transmembrane tyrosine phosphatase that
also is associated with cytoskeletal elements (52).
Cross-linking of L-selectin led to changes in the biomechanical
properties of neutrophils as evaluated by magnetic twisting cytometry
using ferromagnetic beads coated with either anti-CD45 or
anti-CD11b Ab (Fig. 6). Binding
L-selectin with anti-L-selectin Ab alone did not alter the baseline
stiffness of neutrophils. However, cross-linking L-selectin with
GAM Abs increased neutrophil stiffness within 2 min. This increase
persisted for at least 15 min. This cross-linking-induced stiffness
required pretreatment with anti-L-selectin Ab, as pretreatment with
nonimmune mouse IgG had no effect. Treatment with 1 µM fMLP alone
also induced an increase in neutrophil stiffness that lasted for at
least 15 min and was similar in magnitude.
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils circulate as spherical cells containing numerous small surface folds that constitute a pool of excess membrane area that can be rapidly mobilized within seconds to minutes of stimulation. Measurement of cell shape change is a sensitive indicator of the extent of activation both in vitro (30) and in the microcirculation (54). Low doses of chemotactic factor or cross-linking of L-selectin resulted in the formation of numerous surface folds (e.g., membrane ruffling) that correlated with a rapid increase in F-actin formation. Higher doses of chemotactic stimulation, rather than cross-linking of L-selectin, induced more pronounced shape change, including cell polarization, characterized by formation of pseudopodia at the leading edge, and a uropod at the rear. The main consequence of cross-linking L-selectin was to enhance the avidity of CD18 receptors rather than alter the lateral mobility of CD18. The lateral mobility of the ß2 integrin may have important physiologic consequences, as the flow of CD18 from lamellipodia/cell body to the uropod has been shown to be essential for locomotion (55).
The rheologic consequence of cross-linking L-selectin was the retention of neutrophils in filters containing pores of a diameter similar to that of capillaries. The inability of these cells to transit the pores in the filter was indicated in two ways: by the increase in driving pressure required to maintain flow across the filter and by an increase in the percentage of cells trapped within the filter. This increase in the mechanical rigidity of neutrophils that in the current studies was correlated with the L-selectin-mediated increase in F-actin formation and membrane ruffling has also been observed in neutrophils stimulated with chemotactic factors (56, 57). Importantly, from a physiologic perspective, such an increase in neutrophil stiffness following L-selectin ligation on inflamed endothelium may contribute to the mechanical trapping of cells in the microcirculation of the lung and other microvascular beds (13, 14, 15, 58, 59, 60).
In the current study, we used several experimental approaches to
visualize the changes in adhesion receptor distribution that
accompanied activation through cross-linking L-selectin. Observations
of resting neutrophils by confocal microscopy revealed a uniform
circumferential distribution of fluorescently labeled CD18 and
L-selectin. ß2 Integrin and L-selectin appeared
to be closely associated spatially, because cross-linking the latter
induced colocalization of both adhesion receptors to a patch of
membrane emitting two-color fluorescence. Using FRET, we measured the
proximity between CD18 and L-selectin. Cross-linking L-selectin
conjugated to TRITC resulted in the quenching of FITC conjugated to an
Ab bound to the common ß-chain CD18, as well as to the -chains
CD11a and CD11b. These data indicate that L-selectin and the
ß2 integrin came into molecular proximity (<90
Å) when L-selectin was cross-linked.
CD18 detected by immunogold electron microscopy appears to be uniformly
distributed on the plasma membrane of neutrophils obtained from the
venous circulation in a quiescent state by drawing blood into a syringe
containing fixative (49). While the levels of surface
expression of the two -subunits CD11a and CD11b are equivalent on
unstimulated cells, their surface distribution is not. The CD11b
-subunit is predominantly localized to the cell body and largely
excluded from the numerous surface folds, whereas the CD11a -subunit
is primarily observed along the length of the surface folds (P. Hentzen
et al., manuscript in preparation). With chemotactic
stimulation, dynamic changes occur in CD11b; its expression is
up-regulated on both the cell body and the surface folds. In contrast,
the expression of CD11a does not change with stimulation, and it
remains on the surface folds. Using the FRET signal, which provides
molecular scale resolution, we observed that a fraction of the
ß2 integrin subunits, including the common
ß-chain and CD11a and CD11b, were spatially associated with
L-selectin at distances on the order of that previously observed for
clustered L-selectin on the tips of microvilli (18).
We have previously reported that cross-linking L-selectin results in the clustering of the receptor on the plasma membrane and transduction of signals that lead to the up-regulation of CD11b expression and adhesion to ACLB (27, 30, 34). Costimulation through agonist and L-selectin-potentiated cell shape change was observed to enhance ß2 integrin-dependent adhesion and transmigration on IL-1-stimulated endothelial cells (30). Moreover, costimulation with IL-8 and L-selectin cross-linking was observed to increase the strength of cell-substrate adhesion and hence decrease cell mobility. In the current study a low dose of fMLP (1 nM) induced bead binding and transport of a portion of these beads rearward, toward the uropod of polarized cells, within 5 min. A second, higher, dose of fMLP significantly increased the number of beads transported to the uropod, which was not affected by costimulation with L-selectin cross-linking. In contrast, activation through L-selectin resulted in enhanced bead binding, but few neutrophils had formed uropods or transported beads. Taken together, the data indicate that signaling through L-selectin enhances CD11b avidity, but it does not influence its subsequent mobility in response to chemotactic stimuli.
The physiologic significance of L-selectin has been largely attributed
to its ability to enable the capture and rolling of cells on inflamed
endothelium that present counterligands at sufficiently high density.
In this report, we examined the hypothesis that ligation and clustering
of L-selectin at these vascular sites results in activation of
transmembrane signaling cascades, leading to a series of cell responses
that promote the microvascular sequestration of neutrophils. Our
results document that L-selectin cross-linking alters the biomechanical
properties of neutrophils both at the cellular level as measured by
pore filtration and at the receptor level as detected by magnetic
twisting cytometry. Within minutes of L-selectin ligation and
cross-linking, neutrophils became less deformable, and
ß2 integrins adhered to beads more efficiently
and exhibited an increased resistance to an induced stress. Clustering
of L-selectin by Ab had the effect of increasing the local site density
of ß2 integrin, and its localization with
F-actin aggregated to the subjacent cytoplasm. Mac-1 has been
previously shown to become laterally associated with other receptors,
such as FcIIIRb, plasminogen receptor, and the
LPS/lipopolysaccharide binding protein (CD14) receptor
(61). This association is vital for functions such as
adhesion and migration, phagocytosis of microorganisms, and superoxide
production (62, 63, 64). We speculate that the colocalization
of L-selectin and CD18 at the site of membrane contact on the
endothelium prepares the neutrophil for firm adhesion and
transmigration.
In conclusion, we have demonstrated that activation of neutrophils by signals initiated by ligation of L-selectin leads to alterations in the actin cytoskeleton and decreased cell deformability, as well as to increased adhesive affinity of the ß2 integrin. These alterations would favor localization of neutrophils selectively at sites of inflammation. We observed that clustering of L-selectin induced an enhanced surface expression of the ß2 integrin, CD18, that came into molecular proximity with L-selectin in the plasma membrane. These observations have important implications for our understanding of the earliest alterations that occur in the microvasculature leading to sequestration and activation of neutrophils in these vessels.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Gregory P. Downey, Clinical Sciences Division, Room 6264 Medical Sciences Building, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail address:
3 Abbreviations used in this paper: ACLB, albumin-coated latex beads; TRITC, tetramethylrhodamine isothiocyanate; KRPD, Krebs-Ringer phosphate buffer with glucose; NBD-phallicidin, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); GAM, goat anti-mouse; PMN, polymorphonuclear leukocyte; FRET, fluorescence resonance energy transfer; MFI, mean fluorescence intensity.
Received for publication February 22, 1999. Accepted for publication June 23, 1999.
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Mß2; CD11b/CD18) restores IgG-dependent phagocytosis in transfectants expressing a phagocytosis-defective Fc RIIA (CD32) tail-minus mutant. J. Immunol. 157:5660.[Abstract]
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