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Speros Martel Section of Leukocyte Biology, Baylor College of Medicine, Houston, TX 77030; and
Institute of Cell Biology, Zentrum für Molekular Biologie der Entzündung, University of Münster, Münster, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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0.1 dyne/cm2, neutrophils rolled on the E/I. A step
increase to 4.0 dynes/cm2 revealed that 
60% of the
interacting cells remained firmly adherent, as compared with 10% on
L cells expressing E-selectin or ICAM-1 alone. Cell arrest was
dependent on application of shear and activation of Mac-1 and LFA-1 to
bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin,
L-selectin, or PSGL-1 with Abs and by inhibitors to the
mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG
molecule specifically bound sialylated ligands on neutrophils and
activated adhesion that was also inhibited by blocking the
mitogen-activated protein kinases. We conclude that neutrophils rolling
on E-selectin undergo signal transduction leading to activation of cell
arrest through ß2 integrins binding to
ICAM-1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One possible mechanism whereby E-selectin could stimulate neutrophil functions arises from the observation that E-selectin can bind L-selectin (9, 10), a molecule that is predominantly presented on microvillus-like projections from the surface of leukocytes (11). This interaction has been observed experimentally where isolated or recombinant E-selectin binds to the L-selectin obtained from neutrophil lysates but not that obtained from lymphocytes (9, 11). An explanation for the observed distinction is that L-selectin expressed on neutrophils is decorated with sialylated and sulfated carbohydrates that support interactions with the lectin domain of E-selectin (1, 2, 9, 12). If this interaction occurs under physiologic conditions, then some degree of neutrophil activation could occur since L-selectin is now well established as a signaling molecule on the neutrophil (13, 14, 15, 16, 17, 18, 19, 20, 21). Following cross-linking of L-selectin with mAbs, several potentially important events are triggered. There is rapid up-regulation of the adhesive function of members of the ß2 (CD18) integrin family on neutrophils (13, 21, 22). There is also enhancement of the oxidative burst in neutrophils (16), and some elements of a signaling pathway involving the mitogen-activated protein kinases (MAPk)3 have been implicated (14, 17, 19, 20).
In the microcirculation, all three selectins have been shown to mediate the capture of neutrophils from the free stream by binding to a diverse set of sialylated glycoprotein ligands (for a review, see Refs. 23, 24). Subsequent rolling adhesions are mediated by a series of molecular tethering and release events that require and are dependent on the level of fluid shear (25, 26, 27, 28). Selectins exhibit an increase in the efficiency of leukocyte capture on substrates that present purified carbohydrate ligands for each up to a threshold shear stress of between 0.5 and 1 dyne/cm (2, 25). L-Selectin (CD62L) exhibits this behavior in mediating adhesive interactions between a number of cell types (e.g., neutrophil homotypic adhesion (29, 30, 31, 32); lymphocyte interactions with high endothelial venules (33). The physiologic significance of L-selectin has been investigated in experimental animal models. Blocking the functions of L-selectin in vivo with mAbs has resulted in reduced accumulation of neutrophils at sites of inflammation (34, 35), reduced tissue injury (36), and reduced localization of lymphocytes on high endothelial venule (37). Mice with a targeted deletion of L-selectin exhibit a number of immune and inflammatory deficits that may be explained by disruptions of neutrophil and lymphocyte trafficking, and possibly its participation in signal transduction (38, 39, 40). A genetic disruption in the ability to express E-selectin has recently been shown to result in a decrease in the number of firmly adherent leukocytes in cutaneous and peripheral microcirculation, but no difference in the number of transiently interacting leukocytes from normal mice (41). These in vivo models of selectin deficiency provide indirect evidence that leukocyte and endothelial selectins may serve an alternate function in transducing intercellular signals.
In this article, we focus on the functional significance of E-selectin binding to neutrophils under shear flow in the absence of chemotactic stimulation. We utilize two experimental settings. One simulates the potential influence of venular shear stress on intercellular adhesive interactions in a parallel plate flow chamber, and the other employs flow cytometry to examine the specificity in ligand binding and neutrophil activation in suspension.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In accordance with an approved Baylor Institutional Review Board protocol, venous blood from healthy adult donors was collected into a sterile syringe containing 10 U/ml heparin (Elkins-Sinn, Cherry Hill, NJ). Neutrophils were isolated using a one-step Ficoll-Hypaque gradient (Mono-Poly resolving medium; ICN Biomedicals, Aurora, OH) previously as described (13). Cells were kept at 4°C in a Ca2+-free HEPES buffer (110 mM NaCl, 10 mM KCl, 10 mM glucose, 1 mM MgCl2, and 30 mM HEPES (pH 7.35)) containing 0.1% human serum albumin (Armour Pharmaceutical, Kankanee, IL). CaCl2 was added at 1.5 mM final concentration to the cell buffer before addition of cells for analysis of binding and function. Neutrophils viability was >98% as assessed by trypan blue exclusion. Neutrophils isolated in this manner were deemed to remain in a resting state until activated with agonist. The criteria was that incubation of cell suspensions at 37°C for 20 min resulted in <5% of the population to undergo shape change from a spherical geometry or up-regulate the expression of CD11b/CD18, or shed L-selectin.
Agonists, inhibitors, and mAb
FMLP chemotactic peptide and pertussis toxin were purchased from
Sigma (St. Louis, MO). IL-8 was obtained from R&D Systems (Minneapolis,
MN). The specific inhibitors of the MAPk p44/42 extracellular response
kinase (ERK) (PD098059) and p38 MAPk (SB203580) were purchased from
Alexis (San Diego, CA). In collaboration with Merck Research
Laboratories (Rahway, NJ), we obtained an imidazole-based inhibitor
of p38 MAPk (42, 43) denoted Merck-C,
(S)-5-[2-(1-phenylethylamino)pryimidin-4-yl]-1-methyl-4-(3-trifluoromethylphenyl)-2-(4-piperidinyl)imidazole,
and an inactive control Merck-A,
(S)-4-[2-(1-phenylethylamino)pryimidin-4-yl]-1-methyl-5-(3-trifluoromethylphenyl)-2-(4-piperidinyl)imidazole,
which differs only slightly in molecular charge. Neuraminidase, which
cleaves sialyl Lewisx from the neutrophil
surface, was obtained from Boehringer Mannheim (Indianapolis, IN).
Chymotrypsin, which cleaves the L-selectin receptor, was
purchased from Sigma. Anti-L-selectins (HuDreg55 and HuDreg200)
(14) and anti-E-selectin (HuEP5C7
2)
(44) are all humanized IgG2 mAbs that were generously
provided by Dr. Ellen Berg (Protein Design Labs, Mountain View, CA).
Recombinant fusion proteins of E-selectin IgG (E-sel Ig) and
VE-cadherin IgG (VE-cad Ig) are chimeric proteins containing mouse
adhesion domains fused to a human IgG1 that were generated and purified
as described previously (45). The mAb 9A9 that blocks the
lectin domain of mouse E-selectin was kindly supplied by Dr. Michael
Lawrence (University of Virginia, Charlottesville, VA). A blocking
anti-CD18, IB4(46), was provided by Dr. David Chambers
(Sidney Kimmel Cancer Center, San Diego, CA). A humanized IgG2 form of
a blocking anti-CD11b mAb, Hu60.1 (47), was kindly
supplied by Lora Whitehouse (Repligen, Cambridge, MA). R3.1
(anti-CD11a, IgG1) and R6.5 (anti-ICAM, IgG1) were generously
provided by Dr. Robert Rothlein (Boehringer-Ingelheim Pharmaceuticals,
Ridgefield, CT). The blocking ability of the aforementioned mAbs has
been previously documented (47). LAM1-3, a blocking mAb to
L-selectin, and LAM1-14, a nonblocking mAb, were provided by Cell
Genesys (Foster City, CA) (15). Anti-PSGL-1 KPL-1 was
generously provided by Dr. Geoffrey Kansas (Northwestern Unviersity,
Chicago, IL) and prepared as a Fab fragment using an ImmunoPure Fab
preparation kit (Pierce, Rockford, IL). HECA452 is an IgM that
recognizes sialyl Lewisx (PharMingen, San Jose,
CA). All mAbs were titrated by flow cytometry to determine saturating
concentrations. For experiments requiring the cross-linking or
detection of primary mAbs to the neutrophil surface,
fluorescein-labeled and unlabeled goat anti-human and mouse IgG(H +
L) F(ab')2 fragments were purchased from
Kirkegaard & Perry Laboratories (Gaithersburg, MD). For quantitation of
receptor expression, Leu-8 FITC (anti-L-selectin) was purchased
from Becton Dickinson Immunocytometry Systems (San Jose, CA) and
2LPM19c PE (anti-CD11b) from Dako (Carpinteria, CA).
Flow cytometric detection of adhesion receptor expression and neutrophil activation and adhesion
For the detection of fusion protein binding, neutrophils (106/ml) were incubated with E-sel Ig (2 µg/ml) or VE-cad Ig (10 µg/ml) in HEPES buffer/0.1% human serum albumin (HSA) at 37°C for 10 min. Excess IgG was removed by brief centrifugation and a FITC-conjugated secondary goat anti-human IgG(H + L) was added at 20 µg/ml for an additional 10 min before excess was washed out and samples were read on a FACScan flow cytometer on linear amplifier settings (Becton Dickinson). Cross-linking of the Ig-fusion proteins or L-selectin was performed by preincubating cells with LAM1.3Fab or LAM1.14Fab (10 µg/ml) washing out excess and cross-linking with goat anti-mouse or anti-human F(ab')2 IgG(H + L) for an additional 7 min. For kinase inhibition, cells were preincubated in the presence or absence of PD098059 (50 µM), SB203580 (10 µM), or Merck-C at 37°C for 45 min and left in excess before activating cells by cross-linking L-selectin. The expression of L-selectin and CD11b/CD18 was then detected by flow cytometry using Leu-8 FITC and 2LPM19cPE as described previously (14).
Neutrophils (106/ml) were incubated with 1U/ml chymotrypsin (37°C for 25 min) or 0.1U/ml neuraminidase at 37°C for 1 h to cleave L-selectin and PSGL-1 or sialyl Lewisx, respectively (48). Following enzymatic treatment, we measured the binding of 2 µg/ml E-sel Ig, 10 µg/ml VE-cad Ig, 10 µg/ml KPL-1Fab, 10 µg/ml LAM1-3Fab, or 10 µg/ml HECA452 by addition at room temperature for 20 min and secondary goat anti-human (or mouse)-FITC Ab detection as described above. To block E-sel Ig, we added mouse anti-E-selectin (9A9) at 30 µg/ml at room temperature for 30 min before addition of neutrophils (106/ml).
To measure CD11b/CD18 adhesion (13, 49), carboxylated
fluorescent latex beads (diameter, 2 µm; Molecular Probes, Eugene OR)
were washed three times with Dulbecco’s PBS (Life Technologies,
Rockville, MD) and incubated in HEPES buffer/0.1% HSA at 37°C for 45
min with sonication to coat them with albumin. Beads were then counted
and used at a 40:1 ratio of beads to cells (2 x
107/ml). Neutrophils (5 x
105) were mixed with 1 x
107 albumin-coated beads and LDS-751 (0.04
µg/ml), a red nucleic acid dye added to identify neutrophils
(Molecular Probes), in a final volume of 0.5 ml of HEPES buffer (0.1%
HSA and 1.5 mM CaCl2) in a mixing tube containing
a small magnetic stir bar. The sample was then placed for 2 min in a
specially designed mixing chamber maintained at 37°C as described
previously (14, 50). To stimulate neutrophil adhesion,
L-selectin or E-sel Ig bound to the neutrophil was cross-linked as
described above and cells were incubated at 37°C for 2 min. To
promote collision and adhesion, cell-bead suspensions were then sheared
in a test tube at a rate of rotation of 300 rpm corresponding to
shear stresses estimated at <1.0 dyne/cm (2, 50). The
mixing assembly of magnetic stirrer and test tube was placed upstream
from the sample inlet of a FACScan flow cytometer to measure the
kinetics of neutrophil-bead adhesion in real time. To quantitate
PMN-bead adhesion over time the nonadherent single neutrophil
population and each distinct population of neutrophil-bead conjugates
were gated on two-parameter fluorescence histograms as described
previously (13, 14). The mean number of beads/neutrophil
was computed at maximum activation (3 min of shear) by summing the
number of cell-bound beads and dividing by the total number of adherent
and nonadherent neutrophils. We have previously determined that
adhesion was dependent on activation of CD11b/CD18 and the level of
binding was proportional to the extent of cell activation (13, 14). Data are presented as the mean number of beads adherent per
neutrophil normalized by the number adherent to unstimulated sheared
cell-bead suspensions (e.g., typically one bead per two
neutrophils).
Cell culture and reagents
Transfected L cells expressing human E-selectin were prepared by using standard techniques as described previously (1) and maintained in mycophenolic-containing media. Human E-selectin and ICAM-1 (E/I) double transfectants were made by introducing ICAM-1 cDNA into E-selectin L cells as described previously (15). Briefly, 10 µg of human ICAM-1 in a mammalian expression vector CDM8 was cotransfected with 1 µg of pPol2-neor DNA into E-selectin-stable transfectants using the liposomal transfection reagent N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (Boehringer Mannheim). Transfectants were selected for resistance to both mycophenolic acid and neomycin by growing them in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS (HyClone, Logan, UT), 1% penicillin-streptomycin (Life Technologies), medium supplement (Sigma), 5 µg/ml mycophenolic acid (Sigma), 250 5-hydroxytryptamine µg/ml xanthine (Sigma), and 500 µg/ml geneticin (Sigma). The colony expressing the highest levels of E-selectin and ICAM-1 (L-E/I) was selected by flow cytometry using mAbs HuEP5c7 and R6.5, respectively. The expression levels were found to be comparable to that expressed on IL-1-stimulated human umbilical endothelial cells (15). L cells for the adhesion assays were suspended in 0.05% trypsin/0.53 mM EDTA and then passaged onto uncoated 35-mm tissue culture dishes (Life Technologies). Monolayers were allowed to form over 2–3 days and used at confluence in the flow and static chamber assays.
Adhesion assay under flow conditions
Neutrophil adhesion was measured in a parallel plate flow
chamber that produces a uniform laminar flow field as previously
described (1, 15, 51). Briefly, confluent L cell
monolayers were washed three times with Dulbecco’s PBS and mounted in
the flow chamber. We used an experimental protocol in which neutrophils
were introduced at an initial low flow to facilitate interaction and
then stepped up to high shear to detach nonadherent cells and
quantitate cell arrest (Fig. 1
). The
isolated neutrophils (1.2 x 106) diluted in
HEPES with Ca 2+ and Mg2+
were introduced all at once into the chamber at a flow rate
corresponding to a shear stress of between 0.1 and 2
dynes/cm2 for an initial 3-min interaction
period. To determine the number of neutrophils that were firmly
adherent, the shear stress was stepped up to 4
dynes/cm2 for an additional 3 min and then up to
a shear stress of 20 dynes/cm2 for a final 3 min.
Interaction of neutrophils with the L cell monolayers was observed on a
phase-contrast video microscope with a x20 objective (Diaphot-TMD;
Nikon, Garden City, NY and charge-coupled device video camera; Sony,
Park Ridge, NJ). A temperature-controlled Lucite box surrounding the
microscope and flow chamber assured that all flow experiments were done
at 37°C. A continuous record was obtained on videotape from the time
when the neutrophils first passed over the monolayer up to 9 min. For
each experiment, 3–5 fields of view were recorded over for the first 3
min, and 8–10 additional fields of view during the last 6 min for at
least 10 s/field. Quantification of cell rolling and firm adhesion was
performed using Optimas image analysis software (BioScan, Edmonds, WA).
In some experiments, E-selectin and/or ICAM-1 were blocked on L cell
monolayers by incubation with EP5C72 or
R6.5Fab, respectively, for 10 min in a 37°C/5%
CO2 environment. Adhesion molecules on
neutrophils were blocked by preincubation with anti-L-selectin
(HuDreg55 and HuDreg200), anti-PSGL-1 (KPL-1Fab), anti-CD18
(IB4), anti-CD11a (R3.1), or anti-CD11b (Hu60.1) for 5 min at
37°C. In some experiments, neutrophils were incubated with pertussis
toxin at 37°C for 2 h. Neutrophils were also treated with MAPk
inhibitors to p42/44 (PD098059, ERK at 50 µM) and/or p38 (SB203580,
MAPk at 10 µM) or (Merck-C at 10 nM) at 37°C for 45 min. These
reagents were in general left in excess upon injection of the
neutrophil suspensions. However, control experiments in which the L
cells were treated with inhibitors and then washed did not result in
diminished adhesion of neutrophils. E-Selectin ligands on the
neutrophil were enzymatically removed with chymotrypsin or
neuraminidase at 37°C for 1 h.
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The total number of neutrophils in contact with the L cell
monolayers were readily identified by their phase-bright appearance
when in the same focal plane as the monolayer. The total number of
rolling and arrested cells from three to five fields of view were
enumerated over the final 1 min of flow during the interaction period.
Cell arrest was defined as those cells that remained stationary or that
moved less than one cell diameter in a 5-s interval, whereas rolling
cells were defined as having moved more than one cell diameter. The
percentage of arrested neutrophils was assessed over the high shear
periods (II and III in Fig. 1
) between 4 and 6 min at 4
dynes/cm2 and 7 and 9 min at 20
dynes/cm2. The arrested fraction was computed by
dividing the average number of arrested cells by the average number of
interacting cells (tethering and arrested). The rolling velocity was
computed by dividing the distance rolled until a cell transversed the
field of view (15).
Adhesion assay under static conditions
Cell suspensions (volume, 800 µl) were injected into Smith-Hollers chambers as previously described (52). A 25-mm glass coverslip containing a monolayer of L cells formed the substrate. A second coverslip was placed above this and sandwiched between is a rubber O ring that forms a watertight seal by securing both coverslips in a thin stainless steel slide chamber. After incubation at 37°C for a period of 500 s, the number of cells that settled and contacted the monolayer were counted on three microscopic fields (x40 objective). Nonadherent cells were detached by inverting the chamber for an additional 500 s, at which time the number of cells remaining adherent on the L cells were again enumerated for three fields. The percentage of neutrophils that remained adherent following inversion was computed by dividing this value by the total number of cells in contact at the 500-s time point. The adhesive molecules on the neutrophils and on the ICAM-1- or E/I-transfected L cells that supported static adhesion were determined by preincubation with blocking mAbs for 10 min at 37°C as described above.
Statistical analysis
Data were collected for separate conditions in each experiment and are plotted as means ± SEM. For the receptor expression levels and cell arrest analyses, a one-way ANOVA was performed using GraphPad Software Prism (San Diego, CA). The probability of statistical significance between two interventions was determined by the Student-Newman-Keuls test. The p values <0.05 were considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the initial low flow phase (see Fig. 1), the majority of
neutrophils were observed to roll on the L cell monolayer before
becoming arrested. Relatively few neutrophil-neutrophil interactions
were observed during this phase (13 ± 2 of the total interacting
neutrophils per field were observed in homotypic collisions). Moreover,
<5% of the total number of arrested neutrophils per field were
adherent as aggregates, as shown in the representative micrograph of
cells sheared at 4 dynes/cm2 (Fig. 1).
The rolling velocities of neutrophils not firmly adherent during the
initial 3 min of flow varied with the shear in a manner consistent with
previous reports of interactions on E-selectin (3, 53)
(Fig. 2a). At shears between
0.1 and 0.5 dyne/cm2, the velocity plateaued at
5 µm/s. We examined the molecules supporting neutrophil-adhesive
interactions at 0.1 and 2.0 dynes/cm2 on
E-selectin-expressing monolayers (Fig. 2b; Table I). At the low shear stress, virtually
all of the neutrophils observed in the flow chamber sedimented to the
substrate and were observed to roll relatively smoothly on E-selectin
in the presence or absence of ICAM-1 at a velocity of 4 µm/s. This
indicated that ICAM-1 was having no detectable effect on the velocity
of rolling. In contrast, on untransfected L cells or those expressing
only ICAM-1, neutrophils translated down the chamber at >12 µm/s and
cells exhibited an inconsistent tumbling along the substrate.
Paraformaldehyde-fixed neutrophils exhibited little tumbling and moved
at a velocity of 25 µm/s, a speed consistent with that calculated
for nonadherent particles near the surface of the monolayer at this
flow rate. Latex beads (8 µm in diameter) also moved at a velocity of
25 µm/s at this wall shear stress.
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7 µm/s, and the number
of cells observed rolling was influenced only by treatment of the
neutrophils with anti-L-selectin (Table I). Only at the low shear
stress, a small but significant velocity increase was seen in the
presence of anti-CD18 (Fig. 2b). Control binding Ab
W6/32 to the HLA receptor was without effect on rolling. The ability of
neutrophils to bind E-selectin at these low shear stresses was
demonstrated by the increase in rolling velocity seen in the presence
of anti-E-selectin mAb, and by the finding that even under static
conditions, E-selectin will support neutrophil adhesion to E/I
monolayers (Table II). In contrast,
static or flow adhesion to ICAM-1 was low (Table II and Fig. 5). On
monolayers expressing both E-selectin and ICAM-1, the contribution of
ICAM-1 under static conditions was again low, provided that E-selectin
was blocked by mAb (Table II). The contribution of CD18 to adhesion
under static conditions was also confirmed by Ab blocking. A
contribution of 10–15% due to CD18 integrin in the absence of shear
stress on either monolayer was detected. Thus, in the absence of added
stimulus, ICAM-1-dependent adhesion was quite low in these
experiments.
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). The overall strength of adhesion was
assessed by a second step to 20 dynes/cm2,
revealing that 30% of the neutrophils remained arrested (Fig. 4a). Conversion from rolling
to firm adhesion was evident within 1 min of interaction at the low
shear, whereas the fraction arrested was greatest between the 4- and
6-min time points. Activation of firm adhesion was transient,
decreasing by 40% within 10 min of shear.
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). If the initial shear was increased to
0.3 or 0.5 dyne/cm2, this high level of firm
adhesion was not significantly affected (Fig. 3). However, the fraction
of arrested neutrophils significantly decreased if the initial shear
stress was 1.0 or 2.0 dynes/cm2 for 3 min before
the step up in shear (Fig. 3). We examined the influence of the contact
duration on the substrate, which decreases with an increase in shear
stress, by applying a variation of this protocol. An initial shear
stress of 1.0 dyne/cm2 was maintained for 2.5
min, and then the flow was abruptly stopped for 30 s to prolong
contact of interacting neutrophils with the monolayer before the shear
was stepped up to 4.0 dynes/cm2. This variation
resulted in >60% converting to firm adhesion, a value significantly
greater than results without interruption in flow during the initial 3
min of interaction. Adhesion molecules involved in neutrophil arrest under shear flow
Preincubation of the L cell monolayers expressing both E-selectin
and ICAM-1 with Abs to ICAM-1 or the neutrophils with
anti-ß2 integrins significantly reduced the
fraction of neutrophils that were arrested (Fig. 4a). Abs to
either CD11b or CD11a were each less effective than anti-CD18 in
reducing stable adhesion. It appears that neither Mac-1 (CD11b/CD18)
nor LFA-1 (CD11a/CD18) is sufficient to completely support stable
adhesion under these experimental conditions. Blocking CD18 and ICAM-1
together inhibited firm adhesion to the baseline level seen on
untransfected parent L cells.
In the presence of anti-E-selectin, the arrested fraction of
neutrophils was no greater than that on parent L cells (Fig. 4b). In addition, mAbs to either L-selectin or PSGL-1, but
not a binding control Ab to HLA, resulted in partial inhibition of
stable adhesion as did exposure of the neutrophils to enzymes
(O-sialoglycoprotease) that alter the neutrophil surface so
as to inhibit E-selectin binding (Fig. 4b). Simultaneous
addition of mAbs to L-selectin and PSGL-1 resulted in no more
inhibition than either mAb separately, and the combination was less
effective than anti-E-selectin in preventing stable adhesion.
E-selectin-dependent activation of neutrophil adhesion
The high level of firm adhesion of neutrophils to L cell
monolayers depended on the presence of both E-selectin and ICAM-1
expressed simultaneously on the L cells (Fig. 5). We examined whether
this apparent synergy between E-selectin and ICAM-1 was simply the
result of sequential tethering or whether an intracellular signaling
event was involved. Neutrophils were preincubated with pertussis toxin
using a protocol previously devised to block Gi
protein-mediated signal transduction (54). This
pretreatment failed to reduce neutrophil arrest under flow on L cell
monolayers expressing both E-selectin and ICAM-1 (Fig. 5). To assess
the competence of this pretreatment to block Gi
protein-mediated signaling, firm adhesion induced by addition of IL-8
to the neutrophil suspension before flow over the monolayer was
evaluated (Fig. 5). Under these experimental conditions, firm adhesion
to ICAM-1 alone was reduced to baseline, and on monolayers expressing
both E-selectin and ICAM-1 arrest was reduced to the levels seen
without IL-8 stimulation (Fig. 5). Thus, pertussis toxin inhibited
activation by IL-8, but potentiated E-selectin-dependent activation of
firm adhesion.
Previous studies have shown that activation of neutrophils through
cross-linking of either L-selectin or PSGL-1 is accompanied by tyrosine
phosphorylation of several cytoplasmic proteins including the MAPk
(17, 19, 55 . Inhibitors of the p38 MAPk and p42/44 (ERK)
MAPk were used in the current flow protocols, and levels of neutrophil
firm adhesion were determined. Inhibitors to these two MAPk had
different effects on firm adhesion of neutrophils to monolayers
expressing both E-selectin and ICAM-1. In contrast to pertussis toxin,
either inhibitor significantly reduced neutrophil arrest (Fig. 6). Neutrophil suspensions pretreated
with the high-affinity imidazole-based inhibitor of p38 MAPk denoted
Merck-C (10 nM) reduced cell arrest to background, whereas the PD098059
inhibitor of p42/44 ERK at 10 µM inhibited <50%. A chemical
analogue Merck-A, which exhibits a 10,000-fold lower affinity for
binding to p38 MAPk as compared with Merck-C, was without inhibitory
effect. In contrast to these results, cell arrest when IL-8 was added
to the neutrophil suspension was inhibited only partially by the p42/44
inhibitor and not at all by the p38 inhibitor (Fig. 6b).
When arrest induced by IL-8 stimulation was evaluated on L cells
expressing only ICAM-1, the p42/44 inhibitor was completely effective
whereas the p38 was much less active (Fig. 6c).
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To assess the potential for L-selectin to participate in this signaling
event, we assessed the effect of cross-linking L-selectin- on
Mac-1-dependent adhesion, since Mac-1 is at least one component of the
stationary adhesion under flow. We confirmed our previous studies
showing an increase in ACLB binding following cross-linking, and
demonstrated that inhibition of p38 MAPk reduced Mac-1-dependent
adhesion by 76 ± 6% (Fig. 7).
These observations are consistent with results shown in Fig. 6
regarding adhesion in the flow chamber. The ability of PSGL-1 to
participate was also assessed by cross-linking the Ab KPL-1. No
significant increase over background levels due to treatment with
20 cross-linker alone was detected in either
Mac-1 surface expression (0.4 ± 0.2-fold) or adhesion to ACLB
(0.5 ± 0.1-fold).
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). We confirmed that binding of the mAb
HECA-452 that binds to glycoprotein receptors expressing sialyl
LewisX was markedly reduced following treatment
with neuraminidase (data not shown). Chymotrypsin was also found to
markedly reduce binding of Abs to PSGL-1 and L-selectin on
neutrophils.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The requirement of shear stress to detect this signaling function was
apparent in our experiments. In the absence of shear, a low level of
CD18 integrin-dependent adhesion was detected on L cells expressing
ICAM-1 (
10%) and on L cells expressing both E-selectin and ICAM-1
in the presence of a blocking Ab to E-selectin (15%). Only under
flow conditions in which neutrophils were observed to roll on
E-selectin was it possible to discern activation of cell arrest through
binding to ICAM-1. E-selectin alone was insufficient to support arrest
under flow. Activation of firm adhesion was detected over a range of
shear stress found in the microcirculation from 0.1 to 2 dynes/cm
(2, 41). This arrest was entirely accounted for by the
adhesive functions of Mac-1 and LFA-1, with ICAM-1 appearing to play a
major role as ligand. A similar pattern of neutrophil adhesion has been
reported on IL-1-stimulated umbilical vein endothelial cell monolayers
in which Mac-1 and LFA-1 contributed equally to cell arrest and
transmigration under shear flow conditions (56). The
apparent efficiency of E-selectin-dependent activation of firm adhesion
was greatest when neutrophils were interacting with the substratum at
wall shear stresses of 0.1–0.5 dyne/cm2. This
shear coincided with a rolling velocity of 5 µm/s, whereas at
higher shears and corresponding rolling velocities the efficiency of
firm adhesion was reduced by 40%. However, at the shear stresses of
1–2 dynes/cm2, a similar pattern of inhibition
through blocking of L-selectin or PSGL-1 was observed. Previous studies
on the adhesion between neutrophils and ICAM-1-expressing transfectants
under defined shear indicated that the efficiency of firm adhesion
through activated CD18 integrins decreased as shear rate was increased,
requiring a minimum collisional contact duration of 5 ms (47, 57). In the current system, the efficiency of cell arrest was
also found to decrease from a maximum as the applied shear was
increased. Transiently stopping the flow from the high shear and
allowing a brief period of prolonged contact between the rolling
neutrophils and the substrate recovered the maximum arrested fraction.
These observations indicate that the formation of a sufficient number
of ß2 integrin bonds and the transition to
stable adhesion may be regulated by the fluid shear rate. The magnitude
of shear in part determines the duration that a portion of cell
membrane remains in contact with the substrate (53).
Ab blocking studies indicated that both L-selectin and PSGL-1
contributed to the conversion from rolling to arrest. Blocking each
with Ab or enzymatically cleaving these receptors or the sialyl
LewisX-decorated ligands recognized by E-selectin
on the neutrophils significantly inhibited firm adhesion. The
functional contribution of these ligands on neutrophils was consistent
with the binding specificity of the recombinant soluble E-sel Ig as
determined by flow cytometry here and affinity isolation of L-selectin
from cell lysates as previously reported using the identical
recombinant human Ig construct (9). A greater level of
inhibition obtained with anti-E-selectin than with the combination
of anti-L-selectin and anti-PSGL-1 provided evidence for
additional ligands on the neutrophil (Fig. 4b). Although the
existence of a novel E-selectin ligand on human neutrophils has not
been established, one has been cloned that is expressed on mouse
leukocytes (58).
As noted in the introduction, there is evidence indicating that at least in human neutrophils, L-selectin and PSGL-1 are decorated with appropriate carbohydrates for recognition by E-selectin (1, 2, 12). Together with previous observations of signaling adhesion functions through cross-linking of L-selectin with mAbs (13, 14, 15), or carbohydrate ligands (59), the current data implicate a mechanism in which E-selectin may activate neutrophils through direct interaction with L-selectin. Supporting this was the observation that anti-L-selectin Abs partially block the transition to stable adhesion on L cells expressing both E-selectin and ICAM-1, as well as the up-regulation of Mac-1 in experiments where E-sel Ig was cross-linked on the neutrophil. Additional evidence for the ability of L-selectin to signal the conversion to firm adhesion of neutrophils was provided in an earlier report (15), where cross-linking L-selectin boosted the conversion to firm adhesion from 20 to 70% of rolling neutrophils in the flow chamber on a substrate of E-selectin/ICAM-1-expressing L cells.
One potential concern with the interpretation that direct
L-selectin–E-selectin interactions accounted for firm adhesion is the
well-known ability of L-selectin to support homotypic interactions
between neutrophils (29, 60). In parallel plate flow
experiments, others have observed that homotypic interactions can
account for some of the capture of flowing neutrophils by neutrophils
already rolling on the substratum (30, 31, 32). However,
previous studies of homotypic adhesion both in linear and rotational
shear configurations have not provided evidence that collisional
interactions alone result in activation of CD18-dependent firm adhesion
without addition of a chemotactic stimulus (61, 62). If
this occurred in the experimental setting in this report, and thereby
permitted the activation of CD18 by some yet undefined mechanism, then
blocking L-selectin would appear to block the E-selectin-mediated
activation. Evidence that argues against homotypic interactions
contributing significantly to the activation of stable adhesion is the
finding that on average only 13% of neutrophils rolling into a field
of view were observed to collide with other neutrophils. Furthermore,
of the 60% of neutrophils that were activated to arrest on the
substrate, the vast majority were individually attached to the L cell
monolayers and only a small fraction (<5%) were adherent as
aggregates (Fig. 1).
It was recently reported that PSGL-1 on mouse neutrophils when bound to a P-selectin fusion protein triggered the stimulation of ß2 integrin-dependent adhesion (63). However, evidence that PSGL-1 can signal activation of human neutrophils is not consistent in the literature. Early reports on the binding of PSGL-1 by purified P-selectin failed to show integrin activation on human neutrophils (63, 64, 65). In a recent report, homotypic neutrophil aggregation was elicited when cell suspensions were mixed with P-selectin expressed on Chinese hamster ovary cells or with a P-selectin-IgG fusion protein (66). Subsequent activation of ß2 integrin adhesion correlated with tyrosine phosphorylation; however, the experimental conditions did not allow differentiation between signaling due to P-selectin-induced homotypic aggregation and the binding of PSGL-1. In the current study, the cooperativity of PSGL-1 in neutrophil activation on E-selectin was not reflected by its ability to induce significant up-regulation of CD18 expression or signal-adhesive function upon cross-linking with mAb as shown here and recently published (63). In the absence of evidence here for direct signaling through bound PSGL-1 on individual neutrophils, its role in facilitating stable adhesion remains unknown. PSGL-1 and L-selectin are both expressed on the tips of membrane surface ruffles, and this preferential presentation correlates with their functional role in cell capture (67). One hypothesis is that coordinate expression and close topographic distribution of these receptors could enable PSGL-1 to serve as an anchor facilitating tight binding to E-selectin and promoting the clustering and signaling through L-selectin and other sialylated glycoprotein receptors.
We have previously reported that costimulation of neutrophils through
IL-8 and cross-linking L-selectin resulted in a synergistic increase in
CD18 integrin-dependent adhesion (14, 15). In the current
study, we also observed a significant increase in firm adhesion by the
addition of IL-8 (1 nM). We discriminated between the signaling through
E-selectin and chemotactic receptors by the addition of pertussis
toxin, which reduced activation to the level on L cells alone. This
indicated that Gi protein-mediated signal
transduction was not involved in signaling through selectins, although
other G proteins not blocked by pertusis toxin might be involved.
Rather, inhibition of selectin-mediated activation was obtained with
specific inhibitors to the MAPk. Pretreatment of neutrophils with the
Merck-C inhibitor that is reported to block phosphorylation of p38 MAPk
with an IC50 of 0.24 nM (42, 43)
brought Mac-1 up-regulation and activation of cell arrest down to
baseline levels. Inhibition of p38 MAPk has previously been reported to
inhibit chemotaxis and superoxide generation induced by TNF-
and
FMLP (68, 69), but this is the first observation of its
effectiveness in blocking selectin-mediated signaling. Less effective
at blocking selectin-mediated activation was the inhibitor PD98059 that
blocks activation of p42/44 (ERK) at a concentration (10 µM)
similar to the SB20358 inhibitor of p38 MAPk. Further evidence for two
distinct signaling pathways was found with the ERK inhibitor, which
abolished IL-8-stimulated adhesion on ICAM-1. Blocking ERK with PD98059
only significantly inhibited neutrophil adhesion on L cells in the
presence of IL-8. We have recently observed the phosphorylation of p38
MAPk by Western blot analysis within 1 min of cross-linking L-selectin
with Abs (70). This phosphorylation was completely
inhibited by pretreatment with Merck-C at 10 nM, but not the
control Merck-A compound. Taken together, the data suggest that the
signaling pathway by which selectin activation occurs is via
serine-threonine-p38 MAPk and distinct from that of IL-8
(69).
The physiologic importance of signaling through selectins may be related to its ability to potentiate neutrophil activation (i.e., increase in cell shape change and rigidity, ß2 integrin adhesion, and endothelial transmigration) at a very low concentration of chemotactic stimuli, on the order 10–25 pM for IL-8 or platelet-activating factor (21). Signaling through L-selectin could serve to amplify neutrophil recruitment in the microvasculature at the onset of inflammatory disease or in chronic states in which low levels of chemotactic stimuli and E-selectin are presented on inflamed endothelium. However, our data also indicate that treatment of inflammation by inhibition of p38 may not significantly influence neutrophil extravasation when conditions favor robust activation through ligation of IL-8.
Participation of selectins in leukocyte trafficking, distinct from their requirement for capture and rolling in the microcirculation, has recently been implicated in murine gene knockout models of adhesion molecule deficiency. E-Selectin-deficient mice have normal numbers of circulating leukocytes that roll on the endothelium in numbers equivalent to those in wild-type mice as a result of P-selectin-dependent mechanisms (41). However, in the E-selectin-deficient mice, there is a significant decrease in the fraction of arrested leukocytes in inflamed dermal endothelium that are perfused at relatively low shear and in isolated vessels in the mesenteric microvasculature (41). It may be that these observations are related to our findings in the flow chamber model where binding of leukocytes to E-selectin under flow conditions results not only in tethering as evidenced by rolling, but signaling of CD18-dependent arrest. The specific molecules recognized by E-selectin and subsequent alterations in the conformation and topography of the ligands on neutrophils associated with signal transduction remain areas of continued study.
In summary, the current data provide evidence for the activation of neutrophils rolling on E-selectin that was independent of G proteins known to signal neutrophil activation. A cooperative mechanism was found in which both L-selectin and PSGL-1 were required for optimal activation, although no direct evidence was found for signaling through PSGL-1. Under conditions in which the prolonged expression of E-selectin is elicited such as in cutaneous inflammatory stimulation, selectin signaling may provide an important accessory signal to optimize leukocyte localization.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Scott I. Simon, University of California, Davis, Biomedical Engineering, One Shields Avenue, Davis, CA 95616.
3 Abbreviations used in this paper: MAPk, mitogen-activated protein kinase; E-sel, E-selectin; VE-cad, VE-cadherin; HSA, human serum albumin; E/I, E-selectin/ICAM-1; PMN, polymorphonuclear neutrophil.
Received for publication November 4, 1999. Accepted for publication February 7, 2000.
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