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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2558-2564
The Fc Receptor-Mediated Respiratory Burst of Rolling
Neutrophils to Cytokine-Activated, Immune Complex-Bearing
Endothelial Cells Depends on L-Selectin But Not on E-Selectin
By
Dieter Fröhlich,
Olivier Spertini, and
René Moser
From the Institute of Toxicology, Federal Institute of Technology,
Schwerzenbach; the Division and Central Laboratory of Hematology CHUV,
Lausanne, Switzerland; and the Department of Anesthesiology, University
of Regensburg, Regensburg, Germany.
 |
ABSTRACT |
Intracellular H2O2 generation, as a measure
of the respiratory burst, was determined after stimulation of
neutrophils by immune complex (IC)-bearing human umbilical vein
endothelial cells. Under static conditions, neutrophils basically
responded to the immune deposits on resting endothelial cells. The
rotating shear forces of  0.7 dynes/cm2, corresponding to
the physiological flow in postcapillary venules, completely abolished
this basal H2O2 generation. After activation of
the IC-bearing endothelial layers with interleukin-1 (IL-1) or tumor
necrosis factor (TNF), or both, for 4 hours, rolling adhesion of the
neutrophils was induced, accompanied by considerable H2O2 production. The neutrophil respiratory
burst was prominently inhibited by anti-Fc RIII MoAb 3G8 (72.4%),
and partially by MoAb 2E1 against FcRII (38.5%). Both MoAbs
together inhibited the Fc-mediated H2O2
generation by 93.4%. The respiratory burst and rolling adhesion were
markedly blocked by MoAb LAM1-3 against L-selectin (91.3%),
whereas the nonfunctional anti-L-selectin MoAb LAM1-14 was
ineffective. F(ab)2 fragments of MoAb 7A9 against E-selectin inhibited neutrophil rolling by 98.6%, but not
the respiratory burst. Moreover, rolling adhesion of neutrophils and the related oxidative burst were CD11b/CD18- independent. In summary, L-selectin has a unique auxiliary function in triggering the
FcR-mediated respiratory burst of rolling neutrophils to IC-bearing
endothelial cells, thereby substituting CD11b/CD18 under conditions of
flow.
| |
INTRODUCTION |
IMMUNE COMPLEX deposition and
leukocyte-mediated inflammatory reactions are common to vasculitis
constituting, or accompanying, the different forms of connective tissue
diseases, including systemic lupus erythematosus, rheumatoid arthritis,
progressive systemic sclerosis, allograft rejection, and thrombotic
thrombocytopenic purpura.1-5 Antibodies may bind by their
F(ab) portion directly to unidentified structures of the endothelial
cell lining.6 In addition, circulating immune complexes
(ICs) may be localized in the vascular wall.7 Such
IC-bearing endothelial cells are susceptible to being attacked by
circulating granulocytes and monocytes, expressing Fc receptors.
Activated complement, and many chemotactic mediators, increase the
destroying potential of leukocytes.8
Neutrophils express two different low-affinity Fc receptors (R),
FcRII, a 40-kD protein, and FcRIII, a 50- to 70-kD protein, whereas FcRI, the high-affinity receptor for IgG, is not present on
resting neutrophils.9 FcRII is constitutively present
and is not induced in response to neutrophil activation. By contrast, FcRIII is expressed at low levels on resting neutrophils and is
mobilized from intracellular storage pools during
activation.10 Using static conditions, we have previously
shown that FcRII and FcRIII cooperate in the generation a
respiratory burst in response to IC-bearing resting endothelial
cells.11 Activation of the endothelial monolayers with
proinflammatory cytokines increases the number of interacting
neutrophils and should consequently facilitate the ligation of
neutrophil FcRs. The extravasation of leukocytes, under
physiological shear stress, is a sequential process involving multiple
specific molecular interactions.12,13 L-selectin
mediates the initial tethering of neutrophils to the activated
endothelium and starts a transient and reversible interaction, described as "rolling" adhesion. Leukocyte rolling is mediated by
lectin-carbohydrate interactions, mainly involving the selectin family
of adhesion molecules.14,15 The neutrophil activation, which occurs during rolling, induces L-selectin shedding and
upregulation of CD11b/CD18 (Mac-1) expression.16,17 The
increasing formation of Mac-1-dependent ligations strengthens the
binding forces, decelerates, and finally terminates rolling adhesion.
Consequently, leukocytes firmly adhere to the activated endothelium and
immediately start directed transendothelial migration. It is unknown
how circulating neutrophils may recognize endothelial immune deposits.
Therefore, we have studied the conditions leading to initiation of the
respiratory burst to endothelial immune deposits with regard to
neutrophil tethering and rolling. Purified IgG antibody against human
fibronectin (FN) were bound to the extracellular fibronectin of HUVEC
monolayers. The oxidative response of neutrophils to these IC-bearing
endothelial cells was studied with respect to the endothelial
activation with proinflammatory cytokines, using rotating shear forces
in the range of 0.7 dynes/cm2.
| |
MATERIALS AND METHODS |
Cultures of human umbilical vein endothelial cells (HUVEC).
Endothelial cells from human umbilical cord veins were harvested by
collagenase digestion and seeded on fibronectin-coated culture flasks.
The cultures were grown in Medium 199 enriched with sodium heparin (90 µg/mL; Novo Industries, Copenhagen, Denmark), endothelial cell growth
supplement (15 µg/mL; Collaborative Research, Waltham, MA), and 20%
human serum as described.18 Final monolayers, in their
second to fourth passage, were grown on 30-mm petri dishes (Falcon,
Becton Dickinson, Oxnard, CA) after a concentric area of 20 mm in
diameter was bordered with a cotton bud, wetted with nontoxic
dimethylpolysiloxane (Sigma, St Louis, MO), and precoated with human
fibronectin. Exhibition of cytoplasmic factor VIII activity was tested
by indirect immunofluorescence with rabbit anti-human factor VIII von
Willebrand antibody.18
Preparation of neutrophil suspension.
Venous blood from healthy human donors was drawn into 20-mL syringes
containing lithium heparin. A total of 20 mL of blood was layered onto
15-mL Ficoll-Paque (Pharmacia, Uppsala, Sweden), and the erythrocytes
were allowed to sediment spontaneously at an ambient temperature.
Subsequent enrichment by buoyant density centrifugation over
Ficoll-Paque, and short-time hypotonic lysis to remove residual
erythrocytes was performed as described.18 The neutrophils
were washed twice with Gey's buffer (GIBCO, Glasgow, Scotland) and
were resuspended in Hank's buffered salt solution (HBSS), supplemented
with 0.1 mg/mL of human serum albumin (HBSS-A) (OHRA 20/21,
Behringwerke AG, Marburg, Germany). The cell suspension contained more
than 95% CD13+ neutrophils, as assessed by flow
cytofluorometry.
Pretreatment of HUVEC monolayers.
First, the culture medium was discarded, and the endothelial monolayers
were washed three times with Medium 199 to completely remove
fibronectin-containing serum. Thereafter, the monolayers were
preincubated with the purified Ig fraction of a polyclonal goat
antiserum against human fibronectin (Cappel, Organon Teknika, West
Chester, PA), at a protein concentration of 20 µg/cm2 in
fresh Medium 199 for 1 hour at 4°C. No endothelial detachment was
observed under these conditions. To remove unbound fibronectin antibody, the monolayers were washed twice with HBSS. Subsequently, the
HUVEC monolayers were preincubated with culture medium containing 30 ng/mL of human recombinant interleukin-1 (IL-1) (Roche, Nutley, NJ) or
10 ng/mL TNF (Cetus, Emeryville, CA) for 4 hours at 37°C. Before
co-incubation with neutrophils, the monolayers were washed twice with 1 mL of HBSS-A.
Quantification of neutrophil adherence under rotation.
Rolling adhesion was assayed using a modified Stamper-Woodruff assay.
Pretreated HUVEC monolayers were washed twice with HBSS, overlayered
with 2 × 106 neutrophils in 100 µL HBSS-A, and
immediately placed onto the 37°C prewarmed platform of a horizontal
shaker-incubator (Lab-Shaker, A. Kühner AG, Birsfelden,
Switzerland) for 10 minutes at 64 rpm. The experiments were stopped by
aspiration of the leukocyte suspension. The remaining sticky leukocytes
were carefully overlayered with 250 µL 2% paraformaldehyde in PBS
and fixed for 15 minutes. The monolayers were washed once and protected
by a glass coverslip. Adherent leukocytes were assessed in four
randomly chosen fields of 1 mm2 at a magnification of
400×, using phase contrast microscopy. The fields were located at a
half-radius distance from the center of the concentric HUVEC monolayer.
The maximal wall shear stress at the bottom with the medium used was
0.7 dynes/cm2, corresponding to the lower range of shear in
post capillary venules in vivo. The value was computed according to the
formula given by Ley et al19 for a similar shaking
incubator.
Flow cytometric determination of H2O2
production under static and shear conditions.
Intracellular H2O2, generated following
stimulation on IC-bearing HUVEC, was assessed by quantifying the
intracellular oxidation of the indicator dye dihydrorhodamine 123, a
nonfluorescent and membrane-permeable fluorogenic substrate (DHR)
(Molecular Probes, Eugene, OR), which is a sensitive probe for
detection of the respiratory burst activity in neutrophils. DHR is
oxidized mainly by H2O2 to the intracellularly
accumulating fluorescent rhodamine-123.20,21 The dishes
were prepared as described for the assessment of adherence. 5 × 105 nontreated neutrophils, suspended in 100 µL HBSS-A
containing 1 µmol/L dihydrorhodamine-123, were allowed to interact at
37°C with IC-bearing nonactivated HUVEC or IC-bearing
cytokine-activated HUVEC, respectively. Shear was applied as described
above. The assay was stopped after 60 minutes on ice. The cells were
scraped off the dishes and suspended in ice-cold HBSS. Dead cells were counterstained with 30 µmol/L propidium iodide (Serva, Heidelberg, Germany). A FACScan cytofluorometer (Becton Dickinson, San Jose, CA)
with argon ion laser excitation at 488 nm was used to measure 10,000 cells of each stained sample. Data were acquired and processed using
LYSIS-II software. Neutrophils were identified by their typical side
scatter (SSC) and forward scatter (FSC) light patterns, allowing the
formation of a gate for analysis of the neutrophil DHR fluorescence
(Fig 1). The neutrophils positive for
propidium iodide were excluded from analysis.
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| Fig 1.
Flow cytometric record of the respiratory burst of
neutrophils (10,000 cells counted) interacting with IC-bearing
endothelial cells. Forward scatter (FSC), as a measure of the cell
size, is presented on the x-axis, whereas side scatter (SSC), as a
measure of granularity, is presented on the y-axis. The elliptic gate depicts neutrophils. The difference between cell populations in FSC was
used to establish gates for the analysis of the dihydrorhodamine-123 oxidation in neutrophils.
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Calculation of the inhibition of neutrophil
H2O2 production.
Inhibition of the neutrophil H2O2 production
was calculated according to the estimation 100  [(a c)/(b c)] × 100 where a is neutrophil
H2O2 production in the presence of MoAb on
cytokine-activated HUVEC-bearing IC, b is neutrophil
H2O2 production without MoAb on
cytokine-activated HUVEC bearing IC, and c is neutrophil
H2O2 production on nonactivated IC-bearing
HUVEC. The same type of equation was used to calculate the inhibition
of neutrophil adhesion.
Antibodies.
The MoAbs LAM1-3 and LAM1-14, directed against human
L-selectin, are of the IgG1 isotype.22 The MoAb
LAM1-3 recognizes a functional epitope of L-selectin
interfering with neutrophil rolling on cytokine-activated endothelial
cells, whereas the anti-L-selectin MoAb LAM1-14 does not functionally
block L-selectin ligations.23 F(ab)2 fragments of MoAb 7A924 against
E-selectin and the MoAb W6/32 (IgG2a, HLA class I  chain) were generously supplied by Dr F. W. Luscinskas (Department of Pathology, Brigham and Women's Hospital, Boston, MA). MoAb IB-4 is of
the IgG2a class and was a gift of Dr M. Patarroyo
(Karolinska Institute, Stockholm, Sweden). MoAb 3G8 (anti FcRIII,
Immunotech, Marseille, France) and 2E1 (anti FcRII, Immunotech) were
used for functional inhibition of the IC-mediated respiratory burst of
the neutrophils. All these antibodies were used at a saturating concentration of 10 µg/mL.
Statistical analysis.
Statistical validation was performed using Student's two-tailed
t-test for unpaired observations.
| |
RESULTS |
The respiratory burst of neutrophils in response to IC-bearing HUVEC
monolayers with and without shear.
In a previous study we showed that pretreatment of HUVEC monolayers
with antibodies against extracellular matrix proteins, may serve as a
model to study in vitro the response of neutrophils to deposited
IC.11 Antibody to FN were found to be most suitable, because their deposition did not interfere with the integrity of the
HUVEC monolayers. By direct immunofluorescence using FITC-labeled FN
antibody, extracellular FN was detected on the surface of endothelial cells and the fibrous structure of the subendothelial matrix. After 1 hour of incubation, the FITC-labeled FN antibody accumulated at the
intercellular regions of adjacent endothelial cells, from which it
continued to diffuse into the subendothelial matrix. Regardless of
whether IC deposition was performed before or after cytokine
activation, there was no difference in terms of neutrophil respiratory
burst induction (data not shown).
Consideration of the physiological shear stress is necessary to
investigate the physiological conditions, leading circulating neutrophils to recognize immune deposits at the vascular barrier. Therefore, adhesion and respiratory burst were studied under nonstatic conditions, using a rotating adhesion assay that was initially introduced by Stamper and Woodruff25 to assess lymphocyte
attachment to high endothelial venules under physiological flow. The
assay was modified by Spertini et al23 to study in vitro
the adhesion of leukocytes to endothelial cells in culture. In our
study, the assay was adapted to assess the adherence and respiratory
burst of neutrophils to IC-bearing endothelial monolayers. HUVEC
monolayers were grown in the central area of culture dishes within the
circular limits of a nontoxic silicon oil coat. The resulting
monolayers of 20 mm in diameter were pretreated with cytokines or
MoAbs, or both, washed, and overlayered with 2 × 105
neutrophils suspended in 100 µL medium, before the dishes were incubated under flow or static conditions (for details see Materials and Methods).
The respiratory burst of neutrophils gaining access to the immobilized
IC was quantified by assessing oxidation of the indicator dye
dihydrorhodamine-123 to rhodamine-123 by flow cytofluorometry, a
measure of the intracellular H2O2
production.20,21 Endothelial cells and neutrophils could be
clearly distinguished by the different forward- and side-scatter
patterns (Fig 1). Because of the scraping dispersion procedure using
cold phosphate-buffered saline (PBS), barely any HUVEC-neutrophil
aggregates were detectable.
The spontaneous H2O2 generation of freshly
isolated neutrophils in suspension was low (6.4 ± 0.3 U;
mean ± SEM of three experiments). After activation with 100 nmol/L
phorbol myristate acetate (PMA), H2O2
generation markedly increased (172.3 ± 5.0 U; mean ± SEM of
three experiments). Under static conditions, spontaneous settling of
neutrophils on resting IC-bearing endothelial cells led to impressive
respiratory burst induction, which was significantly enhanced after
preincubation of the endothelial cells for 4 hours with IL-1 or tumor
necrosis factor (TNF), or both (P  .001; Fig 2). Control
experiments with F(ab)2 fragments of anti-FN Ab
did not induce the respiratory burst (data not shown).
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| Fig 2.
Respiratory burst of neutrophils on endothelial cells
bearing ICs (black bars) or untreated (white bars) under static
conditions (static) or rotating shear stress (nonstatic). The
endothelial monolayers were preincubated for 2 hours with the indicated
stimuli. Values are expressed as mean ± SEM of three experiments;
P < .001 between experiments with untreated and IC-bearing
HUVECs; *nonsignificant experiment. Particular experiments were
performed using the same batch of neutrophils and endothelial cells.
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Under rotating shear, the respiratory burst of neutrophils to
nonactivated HUVECs was very low. Even in the presence of endothelial immune deposits, the neutrophil response did not increase and was
comparable to the spontaneous respiratory burst of freshly isolated
neutrophils in suspension. Similarly, no significant respiratory burst
was observed after cytokine activation of HUVECs without immune
deposits. Significant H2O2 generation was only detected in the presence of immune deposits and after preactivation of
the endothelial cells with TNF or IL-1. It reached about 50% of the
amount obtained under static conditions (Fig
2). Thus, activated endothelial cells
provide adhesive conditions, which are indispensable for the
Fc-mediated activation of the respiratory burst of rolling neutrophils.
Effect of MoAbs against FcRII and
FcRIII.
Under static conditions, both low-affinity FcRs similarly
participated in binding to endothelial immune deposits.11
However, the Fc-mediated respiratory burst of rolling neutrophils was
predominantly inhibited by the MoAb 3G8 against FcRIII, whereas MoAb
2E1 against FcRII was less effective. Both MoAbs together almost
completely blocked the FcR-mediated respiratory burst (Fig
3).
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| Fig 3.
Inhibition of the neutrophil respiratory burst to
IC-bearing endothelial cells by the MoAbs 2E1 against anti-FcRII and
3G8 against FcRIII. The IC-bearing endothelial cells were activated for 4 hours with IL-1 and TNF before the respiratory burst was estimated with the indicated MoAbs at 10 µg/mL under shear (black bars) and static conditions (white bars). For calculation of inhibition refer to Materials and Methods. Values are expressed as means ± SEM
of three experiments; *P < .001 for the effect of
anti-FcRII 2E1 versus anti-FcRIII 3G8 MoAbs.
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The role of  2 integrins and selectins.
The neutrophil respiratory burst, generated under static conditions,
was significantly blocked by MoAb IB-4 against CD18 (inhibition: 76.0% ± 2.3%; mean ± SEM of three experiments;
P < .001; Fig 4). So far, the
data correspond to the function of CD11b/CD18 in mediating adhesion
under static conditions. By contrast, the anti-L-selectin MoAb LAM1-3 (inhibition, 8.4% ± 2.4%; mean ± SEM of three
experiments) or F(ab)2 fragments of MoAb 7A9
against E-selectin did not inhibit (inhibition,
1.5% ± .5%; mean ± SEM of three experiments; Fig 4).
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| Fig 4.
Inhibitory effects of MoAbs against L-selectin
(LAM1-3), E-selectin (7A9), and CD18 (IB-4) on the
respiratory burst of neutrophils under shear stress (left histograms)
and static conditions (right histograms). Control experiments were
performed in the presence of buffer instead of MoAbs (control), or the
MoAb LAM1-14, recognizing a nonfunctional epitope of
L-selectin (LAM1-14). Endothelial cells were preactivated
with IL-1 (30 ng/mL) for 4 hours. Neutrophils on resting endothelial
cells in the absence of ICs, were used as control (filled histograms).
The data are representative of three experiments.
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Under rotating shear, MoAb IB-4 lost its effect (inhibition:
9.0% ± 3.2%; mean ± SEM of three experiments; Fig 4),
indicating that the respiratory burst of rolling neutrophils to
immobilized IC is CD18 independent. However, the functionally blocking
MoAb LAM1-3 was almost completely inhibiting (91.3% ± 2.6%;
mean ± SEM of three experiments; P < .001), whereas
MoAb LAM1-14, recognizing a nonfunctional epitope of
L-selectin, did not block the Fc-mediated H2O2 generation of neutrophils. Thus, adhesive
ligation by L-selectin is crucial to the induction of the
respiratory burst in our assay. By flow cytofluorometry, saturating
concentrations of MoAb LAM1-3 and LAM1-14 did not compete with the
binding of MoAb 2E1 and MoAb 3G8 (data not shown), thus excluding
artifactual steric competition of FcRII and FcRIII by the
anti-L-selectin MoAbs.
By contrast, F(ab)2 fragments of the adhesion
blocking MoAb 7A9 against E-selectin did not inhibit the
H2O2 generation of rolling neutrophils
(5.6% ± 3.7%; mean ± SEM of three experiments; Fig 4). The
optimal expression of E-selectin in our experimental setup
and its function in providing rolling adhesion (see below) indicates
that E-selectin does not support the FcR-mediated
respiratory burst of rolling neutrophils to immobilized IC.
P-selectin, by its ligation to PSGL-1, is another candidate to support
the response of rolling neutrophils to endothelial immune deposits.
P-selectin is constitutively expressed on HUVECs and decreases with the
number of passages. By flow cytofluorometry and laser scan microscopy,
early passages of HUVEC cultures, expressed detectable levels of
P-selectin (data not shown). We previously showed by transmission
electron microscopy that early passage HUVECs contain Weibel-Palade
bodies.26 Nevertheless, the neutrophil respiratory burst
induced by nonactivated IC-bearing HUVECs was not significantly
different from that induced by control HUVEC monolayers (Fig 2). Hence,
an auxiliary role of the P-selectin-PSGL-1 ligand pair in this model
is unlikely. In summary, the data demonstrate that functional epitopes
of L-selectin are crucial to the recognition of endothelial
immune deposits under fluid shear.
The rolling adherence of neutrophils to IC-bearing HUVEC monolayers
under rotating shear.
The spontaneous rolling adherence of neutrophils to resting HUVECs was
relatively low, regardless of whether IC were present (Fig
5). By contrast, the adhesion of
neutrophils greatly increased after activation of the endothelial
monolayers with IL-1 (Fig 5) or TNF (data not shown). The MoAb LAM 1-3 against L-selectin (inhibition: 80.3% ± 0.2%;
mean ± SEM of three experiments; P < .001), markedly
decreased neutrophil adhesion to cytokine-activated IC-bearing HUVECs,
whereas MoAb LAM1-14 and W6/32 showed no significant inhibition. Of
note, F(ab)2 fragments of the
anti-E-selectin MoAb 7A9 (inhibition, 98.6% ± .1%;
mean ± SEM of three experiments; P < .001) were even more potent in inhibiting neutrophil rolling adhesion. This predominant role of E-selectin is consistent with its ability to maintain the lowest rolling velocity,15 which initiates stable
adhesion.
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| Fig 5.
Adhesion of neutrophils to IC-bearing and IL-1 activated
(30 ng/mL for 4 hours) or resting endothelial cells under nonstatic conditions. The role of L-selectin and E-selectin
in mediating neutrophil adhesion to IC-bearing, IL-1-activated
endothelial cells was determined by preincubating neutrophils and
endothelial cells for 30 minutes at 4°C with the indicated MoAbs
diluted at 10 µg/mL. The adhesion assay was performed under rotation
(64 rpm) for 10 minutes at 37°C, keeping the concentration of MoAbs at 10 µg/mL. Experiments with resting, IC-bearing, and IL-1-activated endothelial cells (first three bars from the bottom) were performed. Control experiments contained buffer instead of MoAbs (Buffer). Values
are expressed as mean ± SEM of three experiments; *P < .0001 for MoAbs v buffer.
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| |
DISCUSSION |
We have previously shown that, under static conditions, neutrophils
basically adhere to nonactivated, IC-bearing endothelial cells,
generating a CD11b/CD18-dependent respiratory burst by ligation of
FcRII and FcRIII.11 The data presented in this report
show that, under shear conditions, neutrophils do not adhere to
nonactivated, IC-bearing endothelial cells. Accordingly, no respiratory
burst was induced, suggesting that the FcRs of circulating neutrophils cannot bind to immobilized IC. Activation of the
endothelial cells with proinflammatory cytokines caused neutrophil
rolling adhesion, which was sufficient to induce the IC-mediated
respiratory burst. Unlike spontaneously settling neutrophils, the
respiratory burst of rolling neutrophils was CD11b/CD18 independent.
Under static conditions, cooperation between CD11b/CD18 and FcRIII is a prerequisite for the phosphorylation of FcRII, initiation of
downstream signaling, and oxidase assembly in
neutrophils.27 The fact that ligation by
2-integrins is lost under fluid shear stress23 may explain these data and rises the question
whether a similar receptor cooperation is working under fluid shear.
The process of rolling has extensively been analyzed as a coordinated
action of different members of the selectin family of adhesion
molecules sustaining leukocyte rolling within a broad range of fluid
shear forces.15 L-Selectin is known to predominate tethering of neutrophils at higher fluid shear forces. On high endothelial venules, leukocyte adhesion through L-selectin to peripheral lymph node addressin has been shown to require a minimum level of fluid shear stress to sustain rolling
interactions.28 Lawrence et al29 showed that
fluid shear above a threshold of 0.5 dynes/cm2 wall shear
stress significantly enhances HL-60 myelocyte rolling on P- and
E-selectin. As a result, a rank order in terms of rolling velocity has been defined for neutrophils, with L-selectin > P-selectin > E-selectin15 representing an
overlapping functional cascade dedicated to decelerate circulating
leukocytes. The selected flow rate in our assay is well beyond the
critical threshold and covers the shear requirements to study the
different selectins. Accordingly, rolling adhesion on activated HUVECs
was markedly inhibited by the MoAb LAM1-3 against L-selectin,
and by F(ab)2 fragments of the MoAb 7A9 against
E-selectin, supporting the previous findings of Spertini et
al.23 The data presented here show that rolling neutrophils
specifically use L-selectin to respond to endothelial immune
deposits. The observation that L-selectin is uniquely
localized at microvillous sites of initial anchoring neutrophils may
explain the key role of L-selectin.30-32
Similarly, nonactivated neutrophils, preferentially express FcRIII
at membrane protrusions,33 whereas 2-integrins and CD44 are exclusively localized on the
cell body.34,35 The respiratory burst of rolling
neutrophils was mainly FcRIII dependent, whereas in arrested
adhesion FcRII and FcRIII equally contributed to the neutrophil
H2O2 generation.11 These data further support a concept in which spatial proximity at microvillous protrusions favors ligation of FcRs during initial capture.
Rolling adhesion of neutrophils is a self-limited process during which
continuous shedding of L-selectin and gradual upregulation of
functionally competent CD11b/CD18 is paralleled by a decrease in
rolling velocity, ultimately leading to arrested
adhesion.12,36 Several studies suggest that juxtacrine
signals, exchanged by engaged adhesion molecules, induce these
phenotypic changes.36-41 In particular,
L-selectin ligation by anti-L-selectin
MoAbs40 and sulfatides42 generates
intracellular signals leading to potentiation of the neutrophil
respiratory burst induced by soluble mediators.43 In this
context, the rapid phosphorylation of downstream signaling proteins,
including the 42-kD mitogen-activated protein kinase, corresponded to
priming rather than direct induction of reactive oxidative
intermediates.40,43 These data support our finding that the
MoAbs LAM1-3 and LAM1-14 did not increase the Fc-mediated respiratory
burst under static conditions. Thus, in our experimental setup, the
outside-in signaling function of L-selectin likely primes for
Fc-mediated respiratory burst induction, although the adhesive function
of L-selectin was indispensable.
Its constitutive expression makes P-selectin the earliest mediator of
leukocyte rolling during an inflammatory response and might therefore
be another candidate contributing to ligation of FcRs in our assay.
The main ligand of P-selectin is P-selectin glycoprotein ligand
(PSGL-1), which is expressed on leukocytes and platelets. Corresponding
with the microvillous expression of L-selectin, PSGL-1
confers rolling on P-selectin.44 Despite the fact
that early passages of resting HUVECs in our experiments expressed
P-selectin, no Fc-mediated respiratory burst was detectable. This
indicates that P-selectin does not support the recognition of
endothelial immune deposits. An auxiliary role for PSGL-1 as a ligand
of E-selectin45-48 is excluded by the lack of an
active function of E-selectin. Future studies have to focus
on the possibility that L-selectin can also interact with
PSGL-1 to mediate neutrophil rolling on adherent
neutrophils,48,49 in order to contribute to the
L-selectin-supported initiation of the respiratory burst.
In summary, the study demonstrates that L-selectin has a
unique auxiliary function in triggering the FcR-mediated respiratory burst of rolling neutrophils to IC-bearing endothelial cells. Hence,
L-selectin covers the function of CD11b/CD18, which is restricted to static conditions.
| |
FOOTNOTES |
Submitted July 1, 1997;
accepted November 20, 1997.
Supported by the Swiss National Science Foundation Grant No.
3100-40796.94 (R.M.); by the Müller-Hartmann Stiftung, Zurich, Switzerland; and by Deutsche Forschungsgemeinschaft, Grant No. FR1165/1-1 (D.F.).
Address reprint requests to René Moser, IBR GmbH, PO Box, CH-9545
Waengi, Switzerland.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract