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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4776-4785
P-Selectin Support of Neonatal Neutrophil Adherence Under Flow:
Contribution of L-Selectin, LFA-1, and Ligand(s) for P-Selectin
By
M. Michele Mariscalco,
M. Hossein Tcharmtchi, and
C. Wayne Smith
From the Sections of Leukocyte Biology and Critical Care Medicine,
Department of Pediatrics, Baylor College of Medicine, Houston, TX.
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ABSTRACT |
To further define the neonatal neutrophil's ability to localize to
inflamed tissue compared with adult cells, we examined the neonatal
neutrophil interactions with P-selectin monolayers under two
conditions: (1) attachment under constant shear stress and flow and (2)
detachment where cells were allowed to attach in the absence of shear
stress and then shear stress is introduced and increased in step-wise
increments. Cord blood and adult neutrophils had minimal interactions
with unstimulated human umbilical vein endothelial cells (HUVECs) at a
constant shear stress of 2 dynes/cm2. There was a marked
increase in the number of both neonatal and adult cells interacting
(interacting cells = rolling + arresting) with HUVECs after
histamine stimulation, although the neonatal value was only 40% of
adult (P < .05). Neonatal neutrophils also had significantly
decreased interaction with monolayers of Chinese hamster ovary (CHO)
cells transfected with human P-selectin (CHO-P-selectin; 60% of adult
values, P < .003). Of the interacting cells, there was a
lower fraction of neonatal cells that rolled compared with adult cells
on both stimulated HUVECs and CHO-P-selectin. That neonatal neutrophil
L-selectin contributes to the diminished attachment to P-selectin is
supported by the following: (1) Neonatal neutrophils had significantly
diminished expression of L-selectin. (2) Anti-L-selectin monoclonal
antibody reduced the number of interacting adult neutrophils to the
level seen with untreated neonatal neutrophils, but had no effect on
neonatal neutrophils. In contrast, L-selectin appeared to play no role
in maintaining the interaction of either neonatal or adult neutrophils
in the detachment assay. Once attachment occurred, the neonatal
neutrophil's interaction with the P-selectin monolayer was dependent
on LFA-1 and to other ligands to a lesser degree based on the
following: (1) Control neonatal neutrophils had decreased rolling
fraction compared with adult neutrophils, although the total number of
interacting neutrophils was equal between groups. (2) Anti-LFA-1
treatment resulted in an increase in the rolling fraction of both
neonatal and adult neutrophils. However, whereas the number of
interacting adult neutrophils remained unchanged, the number of
neonatal neutrophils decreased with increased shear stress. We
speculate that this increased detachment of neonatal cells is due to
differences in neutrophil ligand(s) for P-selectin.
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INTRODUCTION |
THE LOCALIZATION OF neutrophils to
vascular sites of inflammation involves several processes, including
cell capture, rolling, activation, and arrest.1 This
coordinated series of events is mediated by three families of adhesion
receptors: selectins, integrins, and the Ig gene superfamily.
Selectins, consisting of L-selectin (CD62-L) on neutrophils and
E-selectin (CD62-E) and P-selectin (CD62-P) on endothelial cells, have
been shown to mediate capture and rolling but not arrest of neutrophils
on endothelial cells under conditions of flow.2-4 Although
L-selectin is constitutively present on circulating
leukocytes,5,6 P-selectin and E-selectin expression is
induced by several inflammatory cytokines.7-9 The ligands
for the selectins have not been fully identified, although all contain
specific carbohydrate moieties, such as sialylated-fucosylated lactosamines, which are critical components for
binding.10,11 One ligand for P-selectin,
P-selectin-glycoligand-1 (PSGL-1), has been identified as a sialomucin;
ie, it has a large number of O-linked sugar chains clustered together
on the polypeptide backbone.12 It is expressed on
leukocytes, including neutrophils, monocytes, lymphocytes, and
eosinosphils.13-15 PSGL-1 may also serve as a ligand for
E-selectin and L-selectin.16-18 The CD18 integrins, LFA-1
(CD11a/CD18,  L 2) and Mac-1(CD11b/CD18,
M2), mediate arrest and transmigration,
but are unable to mediate capture from the flow stream at shear rates
found in the postcapillary venule (>1
dynes/cm2).19,20 Intercellular adhesion
molecule 1 (ICAM-1; CD54) is a member of the Ig gene superfamily. It is
expressed constitutively on endothelial cells, although its expression
increases when the endothelium is inflamed.21 ICAM-1 serves
as a ligand for LFA-1 and Mac-1 and is required for leukocyte arrest
and transmigration.22
The susceptibility of human neonates to localized soft tissue
infections as well as systemic infections due to bacterial or fungal
agents has prompted extensive investigations of neonatal host defense
mechanisms. Among the most consistently observed functional
abnormalities are those related to leukocyte migration.23 In vivo studies using Rebuck skin windows in human neonates have provided limited data suggesting that inflammatory responses, as
reflected by leukocyte exudation, may differ from those in older
children and adults.24 Studies in experimental animals have
been more extensive. Newborn rabbits, rats, and primates have
diminished leukoctye exudation into inflamed sites compared with adult
animals.25-28 The basis for this appears multifactorial and
includes diminished cell deformability, decreases in f-actin polymerization, abnormalities of microtubule assembly, as well as
qualitative and quantitative defects in the cell surface adhesion receptors.29
Several groups have demonstrated that the expression of Mac-1 on
resting neonatal neutrophils is equal to that of adult
neutrophils30-32; however, the total cell content of Mac-1
is decreased.32 In addition, neonatal neutrophils fail to
upregulate Mac-1 surface expression to the same extent as adult
neutrophils in response to chemotactic factor
stimulation,26,30 and that which is present is functionally
less active.26,30 Recently, Rebuk et al33 have
reported a decrease in baseline expression of neonatal neutrophil Mac-1, but stimulated expression was equal to that of adult
neutrophils. The discrepancies in these studies have not been well
explained to date.33 More consistently reported is that
neonatal neutrophil LFA-1 expression and function is equal to that of
adult neutrophils.30,32-34 In addition, there appears to be
a decreased expression of L-selectin on neonatal
neutrophils.31,35 This decreased expression contributes to
the diminished adherence of neonatal neutrophils to interleukin-1 (IL-1)-stimulated human umbilical vein endothelial cells (HUVECs) and
monolayers of transfected cells expressing E-selectin under conditions
of shear stress.4,35
In the current study, we sought to determine if the impairment in the
neonatal neutrophil's ability to localize to inflamed tissue could
also be due to decreased interaction with P-selectin. Therefore, we
investigated the interaction of neonatal neutrophils with monolayers
expressing P-selectin under defined hydrodynamic shear
stress.35 Using a parallel plate flow system, the
monolayers can support neutrophil attachment, rolling, and arrest at
shear rates of 2 dynes/cm2. We provide evidence here that
neonatal neutrophils have markedly decreased interactions with
P-selectin monolayers compared with adult neutrophils when cells must
be captured from a free-flowing stream at 2 dynes/cm2. This
appears to be due in part to lower levels of neonatal neutrophil L-selectin. We also demonstrate that, of the interacting neonatal neutrophils, a lower fraction is rolling, ie, they are arrested, compared with adult neutrophils. Using a detachment assay in which neutrophils attach in the absence of shear and then shear is introduced and increased in a step-wise fashion, we demonstrate that neonatal and
adult neutrophils are equally able to resist detachment with increasing
shear. However, the interacting neonatal cells have a lower fraction of
rolling cells than adult neutrophils. Decreased rolling (and therefore
increased arrest) of neonatal neutrophils is dependent on LFA-1. If the
rolling fraction is increased by treatment with anti-LFA-1 monoclonal
antibodies (MoAbs), neonatal cells are less able to maintain rolling
interactions compared with adult neutrophils and detach. We speculate
that, under these conditions, the neonatal ligand(s) for P-selectin may
be functionally impaired.
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MATERIALS AND METHODS |
Preparation of isolated neutrophils.
Venous blood was drawn from the placental cord of normal, full-term
(gestational age, 38 to 41 weeks) neonates and from the peripheral
veins of healthy adult donors. All neonates were products of an
uncomplicated pregnancy delivered by planned caesarean section. Mothers
of these neonates received epidural anesthesia for the delivery. None
of the mothers were in active labor at the time of delivery. Apgar
scores at 1 and 5 minute were  8. Blood samples were drawn immediately
after birth. Informed consent was obtained from healthy adult donors.
The protocol was approved by the Institutional Review Board for Human
Experimentation at Baylor College of Medicine and St Luke's Episcopal
Hospital.
Venous blood samples were anticoagulated with citrate phosphate
dextrose (0.14 mL/mL blood: Abbot, North Chicago, IL) and sedimented in
6% (wt/vol in 0.87% NaCl) dextran (Spectrum Chemical, Gardena, CA)
for 45 minutes at room temperature. Neonatal blood samples were diluted
in Ca2+/Mg2+ free phosphate-buffered saline
(PBS; GIBCO Laboratories, Grand Island, NY), pH 7.4, 1:1, before
sedimentation. Leukocyte-rich plasma was layered on Ficoll-Hypaque
gradients and centrifuged (300g for 20 minutes at room
temperature), as previously described.35 The resulting
granulocyte-erythrocyte pellets were washed and resuspended in PBS
containing 0.2% dextrose (DPBS) at a concentration of 1 × 107 cells/mL.
In some experiments, isolated neutrophils were activated by incubation
with the chemotactic tripeptide
n-formyl-methionine-leucine-phenylalanine (fMLP; 10 nmol/L; Sigma, St
Louis, MO) at room temperature for 15 minutes, as previously
described.4 Activation of the neutrophil results in
cleavage of the portion of L-selectin distal to the cell membrane and a
change in cell shape from round to bipolar.5 However,
bipolar cells are at a disadvantage when compared with unstimulated
spherical neutrophils for adhesion and especially rolling under
conditions of flow. Therefore, at the end of fMLP incubation, the cell
suspension was diluted 10-fold with DPBS, washed to remove stimulant,
and incubated for an additional 15 minutes in DPBS without fMLP. The
abrupt decrease in concentration of chemotactic factor causes a
reversal in neutrophil shape change from bipolar to slightly ruffed and
spherical.4
MoAbs.
For blocking experiments, intact antibody preparations were used. The
anti-L-selectin antibody, DREG 56 (IgG1), was prepared as described
and was the gift of Dr Takashi Kishimoto (Boehringer-Ingleheim Pharmaceuticals, Ridgefield, CT).36 The anti-CD11a, R7.1
(IgG1), and anti-CD18 MoAbs, R15.7 (IgG1), were prepared as described and were the gift of Dr Robert Rothlein (Boehringer-Ingleheim Pharmaceuticals).37,38 The control MoAbs, GAP8.3, an
anti-CD45 (IgG1) and a nonblocking anti-LFA-1, TS2/4, were prepared
from hybridoma supernatant, as was the anti-Mac-1, M1/70 (IgG2a;
American Type Culture Collection [ATCC], Rockville, MD).
All MoAbs directed against leukocyte adhesion markers were titered
using flow cytometry (FACS-Scan; Becton Dickinson & Co, Mountain View,
CA) to determine the concentration that saturated binding sites of
unstimulated and stimulated cells as previously
described.35 Fluorescein isothiocyanate (FITC)-labeled
goat-antimouse antibody was used as second antibody (Jackson
Immuno-Research Laboratories, West Grove, PA). A panel of MoAbs against
ICAM-1 were used in the enzyme-linked immunosorbent assay
(ELISA) and in the static adhesion assay. These included
murine antihuman ICAM-1, R6.5 (IgG2a) and CA7 (IgG1) (both provided by
Dr Robert Rothlein39) murine antirat ICAM-1, 1A29 (IgG1)
(kind gift of M. Miyasaka, Tokyo Metropolitan Institute of Medical
Science, Tokyo, Japan40); rat antimouse ICAM-1, YN-1 (IgG2b) (provided by M. Isobe, University of Tokyo, Tokyo,
Japan41); anticanine ICAM-1, CL18/1 (IgG1) and CL18/6
(IgG1).42 Control antibodies included anti-P-selectin,
Cytel 1747 (PB1.3, IgG143; a gift of Dr J. Paulson, Cytel
Corp, San Diego, CA); antihuman L-selectin, DREG 200 (IgG1; a gift of Dr Takashi Kishimoto36); antihuman VCAM-1,
CL40 (murine IgG1)44; antihuman E-selectin, CL2/6 (murine
IgG2a),45 and 7A9 (murine IgG1) and antihuman HLA-A,B,C,
W6/32 (IgG2a). The latter two MoAbs were produced from hybridomas
purchased from ATCC. Fab fragments of R6.5 were prepared with an
ImmunoPure Fab preparation kit (Pierce, Rockford, IL). Anti-L-selectin
MoAb, FITC-labeled Leu-8 (FITC-Leu-8, IgG2b), and anti-CD11b,
phycoerythrin (PE)-labeled Leu 15 (PE-Leu15, IgG2b), as well as
isotype-matched fluorescent controls were purchased from Becton
Dickinson.
Preparation of monolayers.
Endothelial cells were harvested from five to eight collagenase-treated
umbilical cords, pooled, and plated on fibronectin-coated (1 mL of 5 µg/mL human plasma fibronectin for 30 minutes; GIBCO) 35-mm diameter
tissue culture dishes at sufficiently high density to form a confluent
monolayer without cell division, as previously reported.3
Monolayers were cultured in M199 (GIBCO) supplemented with 15% fetal
bovine serum (GIBCO-defined fetal bovine serum), hydrocortisone (1 µg/mL; Sigma), low molecular weight heparin (1 µg/mL; Sigma),
gentamicin (25 µg/mL; Sigma), and amphotericin B (1.25 µg/mL as
Fungizone; GIBCO). No growth factors were used. Cultures were
maintained for 3 to 5 days at 37°C in a humidified atmosphere with
5% CO2.
CHO cells expressing a phosphatidylinositol-glycan-linked form of
P-selectin (CHO-P-selectin) were a gift of Dr Christine Martens
(Affymax Research Institute, Palo Alto, CA).46 These cells
were plated onto coverdishes or a 96-well microtiter plate and allowed
to reach confluence within 3 days. For the static adhesion assay, cells
were plated onto glass coverslips that had been treated with 0.1%
gelatin (Sigma) for 30 minutes.
Flow cytometry.
The CD11b and L-selectin expression levels of adult and neonatal
neutrophils were determined by flow cytometry using PE-Leu-15 and
FITC-Leu8. As cells were prepared for infusion into the adhesion assay
flow chamber, an aliquot was reserved, immediately cooled to 4°C,
and labeled with the antibodies or fluorescent isotype-matched controls. The cells were washed, and the erythrocytes were lysed and
fixed (BD lysing reagent; Becton Dickinson). The mean fluorescent intensity (MFI) for 5,000 particles/sample was obtained using linear
detection settings. The levels of L-selectin and CD11b for each cell
type for each experiment were normalized against the value of the
isotype-matched control (background).
Cell surface ELISA.
The expression of ICAM-1 or an ICAM-1-like molecule on CHO cells
expressing P-selectin and nontransfected cells was determined by cell
surface ELISA.47 The CHO cells were plated onto a 96-well plate. After confluence was reached, the plate was washed and fixed
with 0.25% paraformaldehyde (Sigma) for 15 minutes at room temperature. The cells were then blocked with 2% bovine serum albumin
(Sigma) for 2 hours at room temperature and labeled in duplicate with
saturating concentrations of anticanine, antihuman, antirat, and
antimurine ICAM-1; antihuman VCAM-1; antihuman L-selectin; antihuman
HLA; and antihuman E-selectin MoAbs for 1 hour at 25°C. Bound
antibody was detected by alkaline phosphatase-conjugated goat antimouse
or antirat IgG (Sigma) with 1 mg/mL p-nitrophenyl phosphate
disodium (Sigma) in 1 mol/L diethanolamine (Sigma; pH 9.8), containing
0.5 mmol/L MgCl2 (Sigma) as the substrate. The plates were
read at 405 nm by an automatic microplate reader (Cambridge Technology,
Waterford, MA).
Adhesion assay under static conditions.
A visual static adhesion assay has been described in detail
previously.48 Briefly, CHO cell monolayers grown on 25-mm
round glass coverslips were washed three times in PBS and immediately inserted into a modified Sykes-Moore Chamber. In selected experiments, monolayers were treated for 15 minutes with control or anti-ICAM-1 MoAbs and then rinsed. Untreated neutrophils or neutrophils treated with MoAb and/or chemotactic stimulus (10 nmol/L fMLP for 10 minutes at room temperature) were injected into the chamber and allowed to settle on to the monolayer for 500 seconds. The number
of neutrophils in contact with the monolayer was determined by counting
2 to 3 high-power fields (40× objective). The chamber was then
inverted for an additional 500 seconds so that only adherent cells
remained attached to the monolayer. The number of cells was again
counted in 3 to 10 fields. Results are expressed as the percentage of cells initially in contact with the monolayer that remained adherent per field.
Adherence assay under continuous flow: attachment assay.
Neutrophil interaction with histamine-stimulated HUVECs was assessed
under continuous flow, as previously described.3,4 Briefly,
primary seeded HUVECs were grown to confluence on fibronectin-coated 35-mm tissue culture dishes, rinsed in DPBS (with calcium and magnesium), mounted in parallel plate flow chambers, and perfused for 2 to 3 minutes with DPBS to remove all soluble factors. Histamine in PBS
(final concentration, 10 4 mol/L; Sigma) was perfused
for 10 minutes, at which point neutrophils were added to the feed line
at a final concentration of 1 × 106/mL and perfusion
was continued for an additional 10 minutes. The HUVECs were stimulated
with histamine for the 20-minute duration of the experiment. This
concentration of histamine resulted in maximal adhesion, with no effect
on HUVEC monolayer confluence or neutrophil activation as determined by
assessing neutrophil morphology. Neutrophils were either untreated or
pretreated with MoAbs at saturating concentrations 15 minutes before
being added to the feedline. fMLP-treated neutrophils were prepared as
described earlier. Flow was maintained at a shear stress of
approximately 2 dynes/cm2. A temperature-controlled Lucite
box surrounding the microscope and flow chamber assured that all flow
experiments were performed at 37°C. Interactions between
neutrophils and the endothelial monolayer were observed by
phase-contrast videomicroscopy (Diaphot-TMD microscope [Nikon, Inc,
Garden City, NY] and CCD Video camera [Sony Corp, Park Ridge, NJ])
and quantified with a digital image processing system (Optimas;
BioScan, Edmonds, WA). For each experiment, approximately 6 fields of
view were recorded with a 20× objective at 7.5 minutes at 20 seconds per field. The total number of cells interacting with the
monolayer were determined and referenced per square millimeter of the
monolayer. For the purposes of the present study, interacting cells
were defined as cells rolling at a velocity less than the flow stream
plus those that were arrested. Rolling cells moved more than one cell
diameter during a 1.0-second interval that was determined by
time-lapsed digital subtraction techniques.4 The number of
arrested cells was derived from the arithmetic difference between the
number of interacting and rolling cells. Attachment assays with
monolayers of CHO-P-selectin were performed essentially as described in
the endothelial experiments, except that histamine perfusion was
eliminated.
Adherence assay detachment under increasing shear stress.
To further characterize the neonatal neutrophil's adhesion to
P-selectin, we assayed the strength of the neutrophil interaction with
the monolayer in a detachment assay, as previously
described.49 The number of neutrophils that remained
interacting after a static incubation was quantified as shear stress
was increased, giving a measure of the strength of adhesion to the
monolayer. In this assay, cells were allowed to settle onto the
CHO-P-selectin monolayer for 2 minutes in the absence of shear. The
flow was begun (shear stress of 0.6 dynes/cm2) and then
increased every 20 seconds to achieve stepwise increases in shear
stress (1.4, 2.8, 12.2, and 22.1 dynes/cm2). The number of
interacting cells were determined in three random fields in the final
10 seconds before the next increase in shear stress. Results are
expressed as the percentage of neutrophils remaining interacting at
that shear stress referenced to the number of neutrophils that had
settled onto the monolayer in the absence of shear stress (percentage
of settled cells remaining interacting). As in the attachment assay
described above, interacting neutrophils included those that were
rolling and those arrested (ie, not rolling greater than 1 cell
diameter during a 1-second observation period). The rolling fraction of
neutrophils at each shear stress was determined by dividing the number
of rolling neutrophils by the total number interacting at that shear
stress.
Statistical analyses.
Results are reported as mean ± SEM. Statistical assessments were
performed as follows. The unpaired two-tailed Student's t-test was used to examine expression of adhesion molecules under different treatment conditions; as repeated testing was performed, significance was considered at P < .005. For static adhesion assay and
attachment assays under flow conditions, a one-way analysis of variance
(GraphPad Software, San Diego, CA) was performed. The probability of
statistical significance between interactions of adult and neonatal
neutrophils was determined by the Student-Newmann-Keuls test.
Probability values less than .05 were considered significant. For
detachment assays, a two-way ANOVA was performed to determine the
significance of increasing shear stress on the interactions of adult
and neonatal neutrophils. Significance was set at P < .05.
| |
RESULTS |
Neonatal neutrophils have less interaction with monolayers expressing
P-selectin.
We initially examined the ability of the neonatal neutrophil to be
captured by monolayers expressing P-selectin under hydrodynamic shear
stress of 2 dynes/cm2. Both adult and neonatal neutrophils
had minimal interaction with unstimulated HUVECs (9.8 ± 2.9 v 29.3 ± 18.2 cells/mm2, respectively).
Histamine causes rapid mobilization of P-selectin from the
Weibel-Palade bodies to the surface of the endothelial cells and
markedly increases neutrophil adhesion.3,50 In the present
study, as seen in Fig 1, we also
demonstrated a marked increase in the number of interacting adult
neutrophils (499 ± 133 cells/mm2) to
histamine-stimulated HUVECs. The peak number of interacting neutrophils
occurred 5 to 10 minutes after the addition of cells and 47% ± 11% of the neutrophils exhibited rolling behavior. Arrested neutrophils did not roll for the 1-second observation period. None of
the arrested neutrophils migrated through the monolayer.3 There was also an increase in the number of neonatal neutrophils interacting with histamine-treated HUVECs compared with
nonstimulated HUVECs, although there were significantly fewer neonatal
neutrophils interacting than adult neutrophils (192 ± 63 cells/mm2, P < .05, Fig 1). The percentage of
neonatal neutrophils that rolled during the observation period was 27% ± 6%. Rolling velocity was similar between neonatal and adult
neutrophils (adult, 36.5 ± 5.8 µm/sec; neonate, 31.8 ± 5.4 µm/sec).
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| Fig 1.
Adult ( ) and neonatal ( ) neutrophil adhesion to
HUVECs stimulated for 10 minutes with 104 mol/L
histamine before the addition of neutrophils under shear stress of
approximately 2 dynes/cm2. The total number of interacting
neutrophils per square millimeter of monolayer includes both rolling
and arrested cells. The number of interacting neutrophils was
quantified beginning 7.5 minutes after the neutrophil suspension was
introduced into the chamber. Neutrophils were left untreated (control)
or were preincubated with anti-L-selectin MoAb, DREG 56 (50 µg/mL),
or treated with 10 nmol/L fMLP as described. Data are expressed as the
mean ± SEM from 7 to 11 experiments. *P < .05 compared with
adult control neutrophils.
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We also examined the adhesion of adult and neonatal neutrophils to
confluent monolayers of CHO cells stably transfected with a
phosphatidylinositol-glycan-linked form of human P-selectin (CHO-P-selectin).46 As demonstrated with
histamine-stimulated HUVECs, there were significantly fewer neonatal
neutrophils interacting with the CHO-P-selectin than adult neutrophils
(P < .01; Fig 2). Whereas 63% ± 6% of adult neutrophils rolled, only 32% ± 8% of neonatal
neutrophils did so (P < .01). Rolling velocity was less on
CHO-P-selectin than on histamine-stimulated HUVECs for both neonatal
and adult neutrophils, although there was no difference between the
groups (neonate, 10.9 ± 1.6 µm/sec; adult, 8.6 ± 0.7 µm/sec).
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| Fig 2.
Adult () and neonatal () neutrophil adhesion on CHO
cells stably transfected with human P-selectin under shear stress of approximately 2 dynes/cm2. The total number of neutrophils
per square millimeter includes both rolling and arrested cells.
Neutrophil treatment and analysis were performed as outlined in Fig 1.
Data are expressed as the mean ± SEM from 5 to 13 experiments.
*P < .003 compared with adult control neutrophils.
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Contribution of L-selectin to adult and neonatal neutrophil adhesion
to P-selectin under continuous shear conditions.
We and others have published that L-selectin is important for
attachment of neutrophils to P-selectin-bearing
substrates.3,51 Additionally, we demonstrated that neonatal
neutrophils have decreased expression of L-selectin, resulting in
decreased adhesion of neonatal neutrophils to IL-1-stimulated HUVECs
(which expresses both E-selectin and the L-selectin
ligand35) as well as transfected cell monolayers expressing
E-selectin.4 We hypothesized therefore that the decreased
interaction of neonatal neutrophils with P-selectin monolayers under
continuous shear conditions could at least partly be due to decreases
in expression of L-selectin. Neonatal neutrophils obtained for our
current studies also had significantly less L-selectin than adult
neutrophils (Table 1). Neutrophil
L-selectin function was inhibited by either blocking with the
anti-L-selectin MoAb, DREG 56, or by stimulating with the chemotactic
factor fMLP, which results in the cleavage of the extracellular portion
of L-selectin.5 Incubation of either neonatal or adult
neutrophils with the anti-L-selectin MoAb had no significant effect on
the expression of the L-selectin epitope recognized by the MoAb, Leu8,
or on the expression of Mac-1 (CD11b/CD18; Table 1). Stimulation of
both neonatal and adult neutrophils with fMLP significantly decreased
L-selectin and increased Mac-1 expression (P < .001 v unstimulated controls for both neonatal and adult
neutrophils).
Treatment with anti-L-selectin MoAb resulted in a 64% decrease in the
number of adult neutrophils interacting with histamine-stimulated HUVECs (P < .05; Fig 1) and a 40% decrease in the number on
CHO-P-selectin compared with untreated adult cells (P < .01;
Fig 2). The number of treated adult neutrophils interacting with either
histamine-stimulated HUVECs or CHO-P-selectin was equal to that of
untreated neonatal neutrophils. Treatment of neonatal neutrophils with
the anti-L-selectin MoAb did not decrease the number of interacting
cells (Fig 1). Although there was marked loss of functional L-selectin
from the surface of both neonatal and adult neutrophils with
chemotactic factor stimulation (Table 1), there was no further decrease
in the number of either neonatal or adult neutrophils interacting with
histamine-stimulated HUVECs compared with DREG 56-treated neutrophils
(Fig 1).
Neonatal neutrophils resist detachment from P-selectin with increased
shear stress, but have different rolling behaviors than adult
neutrophils.
We next examined the resistance to detachment of neonatal and adult
neutrophils to P-selectin substrates (see detachment assay in the
Materials and Methods). In this protocol, neutrophils were allowed to
settle onto the CHO-P-selectin monolayer for 2 minutes in the absence
of shear stress. Shear stress was then applied in a stepwise fashion
from 0.6 to 22 dynes/cm2 every 20 seconds. The number of
interacting neutrophils were counted at the end of each 20-second
period in three fields and referenced to the number of neutrophils that
had settled onto the monolayer in the absence of shear stress
(percentage of settled cells remaining interacting;
Fig 3). Interacting neutrophils included those that were rolling and those arrested (ie, not rolling greater than 1 cell diameter during a 1-second observation period). When neonatal cells are allowed to attach to CHO-P-selectin before shear
stress is introduced and are then subjected to increased shear stress,
neonatal neutrophils had an equal percentage of settled cells that
remained interacting compared with adult and were able to resist
detachment to the same extent as adult neutrophils (Fig 3). There was
no contribution of L-selectin to this interaction, because an
anti-L-selectin MoAb had no effect on either adult or neonatal
neutrophils. Thus, it appears that, once neonatal and adult neutrophils
interact with CHO-P-selectin, both are equally able to resist
detachment from the monolayer. This interaction is not dependent on
L-selectin.
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| Fig 3.
The percent of adult ( ,  ) and neonatal (,  )
neutrophils initially attached to CHO cells expressing P-selectin in
the absence of shear stress that remain interacting as shear stress is
then applied and increased. Neutrophils are allowed to settle onto the
monolayer for 2 minutes, at which point the flow is begun and
increasing shear stress is applied every 20 seconds. The number of
neutrophils that remain attached in the last 10 seconds before the next
step up in shear stress is compared with the number of cells that had
originally settled (percentage of interacting cells remaining).
Neutrophils were left untreated (, ) or were preincubated with
anti-L-selectin MoAb, DREG 56 (, ). Data are expressed as the
mean ± SEM from 7 to 14 experiments.
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However, as with neonatal neutrophils that attached under continuous
flow conditions, neonatal neutrophils demonstrated markedly different
rolling behaviors compared with adult neutrophils under increasing
shear conditions. The rolling fraction of neutrophils was determined by
dividing the number of rolling cells by the number of interacting cells
at that shear stress. As seen in Fig 4A,
adult neutrophils had a significantly increased rolling fraction as
shear stress increased from 0.6 dynes/cm2 (32% ± 4.7%) to 22 dynes/cm2 (69% ± 8%, P < .01, one-way ANOVA). In contrast, there was a lower fraction of neonatal
neutrophils that rolled at all shear stresses. This low amount of
rolling did not change with increasing shear stress (Fig 4B) and was
significantly less than adult neutrophils (P < .0001, two-way
ANOVA). Treatment of neonatal neutrophils with the anti-L-selectin
MoAb, DREG 56, had no effect on the rolling behavior (Fig 4B). However,
treatment with the anti-L-selectin MoAb resulted in a small but
significant decrease in the rolling fraction of adult neutrophils
(P < .05, Fig 4A).
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| Fig 4.
Fraction of interacting adult (A) and neonatal (B)
neutrophils that rolled on CHO-P-selectin after a 2-minute stationary
contact period with increasing shear stress. Neutrophils were left
untreated () or were preincubated with anti-CD11a MoAb, R7.1 (10 µg/mL;  ), anti-CD18, R15.7 (10 µg/mL;  ), or anti-L-selectin,
DREG 56 (50 µg/mL; ). Data are expressed as the mean rolling
fraction ± SEM from 7 to 14 experiments. *P < .005 compared
with control neutrophils. +P < .05 compared with control.
**P < .02 versus anti-CD11a-treated neutrophils.
|
|
Adhesion of neonatal and adult neutrophils to nontransfected CHO
monolayers under static conditions is CD18-dependent.
We had shown previously that, under continuous shear stress, adult
neutrophil arrest on histamine-stimulated HUVECs is
CD18/ICAM-1-dependent.3 To avoid CD18/ICAM interactions,
we therefore used CHO cells transfected with P-selectin in the previous
set of experiments. Nonetheless, only 60% to 70% of interacting adult
neutrophils rolled and even fewer neonatal neutrophils did so (22% to
27%) in both the attachment assay under continuous shear and the
detachment assay. We speculated that the arrest of both adult and
neonatal neutrophils on CHO-P-selectin could also be CD18-ICAM-1
dependent.
We sought to determine if CHO cells have an ICAM-1-like molecule using
an ELISA and a panel of MoAbs directed against mouse, human, dog, and
rat ICAM-1. We were unable to detect consistent cross-reactivity
between any of the anti-ICAM, E-selectin, L-selectin, VCAM-1, or HLA
MoAbs on either nontransfected CHO or CHO-P-selectin, although we could
consistently detect increased binding of the anti-P-selectin MoAb
(Cytel 1747) on the transfected cell line. We hypothesized that, if the
interaction between the ICAM-like molecule on CHO and the anti-ICAM
MoAbs were of a low-affinity type, this interaction would be
susceptible to the vigorous washing steps of the ELISA. Therefore, we
performed a static adhesion assay in which fMLP-stimulated adult
neutrophils were allowed to adhere to CHO cell monolayers pretreated
with anti-ICAM or control (W6/32) MoAbs that were then gently washed (3 dips in PBS) before insertion into the Sykes-Moore adhesion chamber.
Treatment with anticanine ICAM (CL18/1, CL18/6) and antihuman ICAM
MoAbs (R6.5 Ig, R6.5 Fab) resulted in a 30% decrease in neutrophil
adhesion compared with PBS- or W6/32-treated monolayers
(Table 2). If we maintained the anti-ICAM
MoAb, R6.5 Fab, or control antibody, W6/32, in the reaction mix in
addition to pretreating the monolayers, adhesion was decreased further
(Table 2). Thus, it appears that CHO cells express a molecule(s) that
can function in cell adhesion and that this adhesion can be blocked by
several anti-ICAM MoAbs.
We performed a static adhesion assay with neonatal neutrophils on
nontransfected CHO cell monolayers. Baseline adhesion of neonatal
neutrophils treated with a control anti-CD45 (GAP8. 3) MoAb was 18% ± 5.4%; treatment with the anti-CD11a MoAb (R7.1) decreased
adhesion significantly (2.7% ± 0.7%, P < .05). In
contrast, adult control neutrophils (treated with anti-LFA-1
nonblocking MoAb, TS2/4) had low baseline adhesion (3.7% ± 1.0%).
Adhesion of TS2/4-treated adult neutrophils was significantly increased to 13.9% ± 1.5% (P < .01) with 10 nmol/L fMLP
stimulation. Stimulated adhesion could be reduced to unstimulated
levels (4.9% ± 3.8%, P < .01 compared with stimulated)
only with coincubation of anti-Mac-1 (M1/70) and anti-LFA-1 (R7. 1)
MoAbs and not with either individually (8.5% ± 4.3% and 8.2% ± 4.2%, respectively). Under static conditions, adult neutrophils
have low baseline adhesion, although adhesion can be increased with
stimulation of the neutrophils. Stimulated adhesion of adult
neutrophils is dependent on LFA-1 and Mac-1 and an ICAM-1-like
molecule. In contrast, unstimulated neonatal neutrophils have increased
adherence to nontransfected CHO cells, and this adhesion is dependent
on LFA-1.
Inhibition of CD11a/CD18 results in increased neonatal neutrophil
rolling fraction and increased detachment with increased shear stress.
To assess the contribution of CD11a/CD18 to neonatal and adult
neutrophil rolling on CHO-P-selectin, neutrophils were treated with
MoAbs against either CD11a (R7.1) or the common CD18 subunit (R15.7) of
LFA-1 and Mac-1. Such treatments had no effect on the expression of
L-selectin or Mac-1 (Table 1). Neutrophils attached to CHO-P-selectin
monolayers in the absence of shear stress, and then shear stress was
initiated and increased every 20 seconds as outlined (detachment assay,
see the Materials and Methods). The number of total interacting cells
and those rolling were counted and the rolling fraction was determined.
The fraction of rolling neutrophils significantly increased when either
adult or neonatal cells were treated with either R7.1 or R15.7 compared
with the age-matched controls (Fig 4A and B). Although the rolling
fraction of adult neutrophils increased with inhibition of LFA-1, the
percentage of cells remaining interacting as shear stress increased did
not change significantly (Fig 5A).
Therefore, with anti-LFA-1 treatment, adult neutrophils had increased
rolling; however, these neutrophils were able to resist detachment with
increasing shear stress and continued to roll. In contrast, as the
rolling fraction of anti-LFA-1-treated neonatal neutrophils increased
with shear stress (Fig 4B), the total number of interacting cells
decreased significantly compared with untreated neonatal cells (Fig
5B). Thus, the rolling neonatal neutrophils were less resistant to
shear stress than adult neutrophils and had increased detachment.
View larger version (20K):
[in this window]
[in a new window]
| Fig 5.
The percentage of adult (A) and neonatal (B) of those
initially attached to CHO-P-selectin in the absence of shear stress that remain interacting as shear stress is applied. Adult and neonatal
neutrophils were treated with the anti-CD11a () or anti-CD18 ()
MoAb or left untreated (). Data are expressed as the mean percentage
of initially interacting cells remaining ± SEM from 7 to 14 experiments. *P < .05 compared with control neutrophils. **P < .005 compared with control neutrophils.
|
|
| |
DISCUSSION |
In the present study, we demonstrate that neonatal neutrophils have
distinct differences compared with adult neutrophils in their
interactions with monolayers expressing P-selectin. Neonatal neutrophils perfused over monolayers of P-selectin at a constant shear
stress of approximately 2 dynes/cm2 demonstrated a decrease
in the total number of cells that interacted with the monolayer
compared with adult neutrophils. Of those cells interacting, there was
a decreased fraction of neonatal neutrophils that rolled during the
1-second observation period compared with adult neutrophils. These two
effects were demonstrated on both histamine-stimulated HUVECs as well
as on CHO cells stably transfected with human P-selectin, although the
differences in the rolling fractions was significant only on the
CHO-P-selectin monolayer. When neonatal and adult neutrophils attached
to CHO-P-selectin monolayers in the absence of shear stress and then
shear stress was introduced, equal numbers of cells interacted with the
monolayer. However, their rolling behavior again was significantly
different in that neonatal neutrophils had a decreased rolling fraction compared with adult neutrophils. Treatment with anti-LFA-1 MoAbs resulted in an increase in the fraction of both neonatal and adult cells that were rolling. However, under these conditions, neonatal cells detached as shear stress increased, whereas adult neutrophils continued to roll and did not detach.
Neutrophil attachment to and rolling along the endothelia under shear
flow is mediated by selectins. We have previously demonstrated that,
under shear flow conditions, neonatal neutrophils have a diminished
ability compared with adult neutrophils to interact with HUVECs
stimulated with IL-1 (which express both E-selectin and an L-selectin
ligand)35 as well as murine L cells transfected with human
E-selectin.4 We and others have shown that neonatal neutrophils have diminished levels of L-selectin compared with adult
neutrophils (Table 1 and previous studies33,35,52,53). This
decrease in neonatal neutrophil L-selectin appears to contribute to
diminished interaction with monolayers expressing the L-selectin ligand
and/or E-selectin.4,35
In the present study, we demonstrate for the first time that neonatal
neutrophils also have a diminished ability to interact with monolayers
expressing P-selectin under continuous shear flow. That this may also
be due to decreased amounts of L-selectin on neonatal neutrophils
(Table 1) is supported by the finding that treatment of adult
neutrophils with the anti-L-selectin MoAb decreases the number of
interacting cells compared with adult control neutrophils. Furthermore,
as demonstrated previously with IL-1-stimulated HUVECs35 and E-selectin monolayers,4 the number of interacting
anti-L-selectin-treated adult neutrophils was equal to that seen with
control neonatal neutrophils (Figs 1 and 2). L-selectin has been
demonstrated to be located at the tips of the microvillus of
adult51 and neonatal neutrophils (M. Mariscalco and A. Burns, unpublished observations). This location of
L-selectin is particularly advantageous for capturing of the
neutrophils from the free-flowing stream. However, L-selectin does not
contribute to continued neutrophil interaction with the P-selectin
monolayer once attachment has already occurred (Fig 3). The requirement
for L-selectin in the initial capture of neutrophils from the
free-flowing stream to E-selectin has been described by Lawrence et
al.49 However, as with this present study, once neutrophils
were attached, L-selectin was not required to maintain the interaction.
The contribution of L-selectin- to P-selectin-mediated rolled has
been described by us previously.3 There has been
considerable controversy as to whether L-selectin can function as a
ligand for P-selectin. Picker et al51 were able to inhibit
by 40% to 60% neutrophil attachment to P-selectin-transfected COS
cells with the anti-L-selectin MoAb, DREG 56. In contrast, Patel et al16 were unable to inhibit adult neutrophil attachment to
CHO cells expressing P-selectin at continuous shear stress with DREG 56. It is unclear as to why our findings differ from those of Patel et
al,16 because our experimental procedures were remarkably similar. One explanation for our findings may be that the
anti-L-selectin MoAb, DREG 56, inhibits a functional domain of
P-selectin. There are reports of MoAbs which cross-react with one or
more selectin molecules.4,54,55 We were unable to detect
binding of DREG 56 to the transfected cell line expressing P-selectin
using an ELISA, although this does not rule out low-affinity
interactions. Nonetheless, pretreatment of neutrophils resulted in very
low concentrations of DREG 56 in the flow assays itself, making
low-affinity interactions extremely unlikely.
Others have described the contribution of leukocyte-leukocyte
interactions in amplifying the capture of leukocytes from a free-flowing stream.56 This interaction appears to involve
PSGL-1 on one cell and L-selectin on another.17,57 It is
possible that our findings reflect a decreased L-selectin-PSGL-1
interaction due to inhibition of L-selectin (treatment of adult
neutrophils with DREG 56) or decreased amount of L-selectin (neonatal
neutrophils). We were unable to demonstrate leukocyte-leukocyte
recruitment from a review of our videotapes, although our protocol was
not designed specifically to examine these interactions.17
We had demonstrated previously that neutrophils roll on
histamine-stimulated HUVECs expressing P-selectin. In that study, some
interacting neutrophils did not roll. This arrest was dependent on
neutrophil CD18 and the ICAM-1 constitutively present on the HUVECs.3 CHO-P-selectin also supports rolling
interactions.16 Unexpected, however, was the observation
here that CHO cells can also support LFA-1-dependent arrest of both
neonatal and adult neutrophils. Based on our findings, we suggest that
CHO cells express a molecule that can function in a manner similar to
ICAM-1.
Neonatal neutrophils have increased baseline adhesion to nontransfected
CHO cells compared with adult neutrophils in the static adhesion assay.
This adhesion could be blocked by treatment with anti-LFA-1 MoAbs. In
addition, neonatal neutrophils attached to the CHO-P-selectin in the
absence of shear had a significantly decreased rolling fraction
compared with adult neutrophils when shear was introduced and then
increased. Rolling fraction of neonatal neutrophils could be increased
to that seen with adult neutrophils by treatment with anti-LFA-1
MoAbs. These findings suggest that neonatal LFA-1 is functionally more
active than adult LFA-1, because, to date, there have been no reports
of quantitative differences in LFA-1 expression between neonatal and
adult neutrophils.30,32,33 Activation of the integrins
leads to an increase in avidity for their respective
ligands.58 This process has been described in lymphocytes
for LFA-1 and in neutrophils for Mac-1.59-62 Only recently
has there been evidence that activation of the neutrophil may also
result in the affinity modulation of LFA-1.47 Is increased LFA-1 function due to the fact that resting neonatal neutrophils are
activated compared with adult neutrophils? There are several studies
that support this. Kjeldsen et al63 recently demonstrated the augmented release of both gelatinase and specific granules from
neonatal neutrophils isolated from cord blood compared with adult
neutrophils, suggesting that neonatal cord neutrophils appeared primed
compared with control adult cells. Others have reported that neonatal
cord neutrophils had increased oxidative burst activity.64 It is unclear if the increased activity of LFA-1 can compensate for the
decreased ability of neonatal neutrophils to be captured from the
free-flowing stream. Our data suggest that it does not. To answer
whether these observations will ultimately result in neutrophil
emigration defects in vivo will require direct examination of leukocyte
localization in neonatal animal models.
The rolling of neutrophils on purified P-selectin or P-selectin
monolayers (CHO-P-selectin) has been reported to be dependent primarily
on PSGL-1.13,16 Whereas inhibition of LFA-1-mediated arrests results in the increased rolling fraction of adult and neonatal
neutrophils as shear stress increases, the number of interacting
neonatal cells decrease. We propose that the decreased rolling of
neonatal neutrophils is due to quantitative or qualitative differences
in PSGL-1 (or other P-selectin ligands) compared with the adult. These
results remain speculative until neonatal PSGL-1 function is evaluated
directly.
Finally, we demonstrate that there is no statistical difference in the
stimulated expression of Mac-1 on our sample of adult versus neonatal
neutrophils. This may be interpreted as a discrepancy with previous
findings.26,30,34,65 We suggest that this reflects instead
the wide sampling variability in stimulated expression of Mac-1 in
neonatal cord blood and adult samples (Table 1). That this may be so is
supported by our retrospective review of the results of clinical
testing of whole blood from infants (<2 months old) whom we had
examined for the presence of Mac-1 and LFA-1. None of the infants had
leukocyte adhesion deficiency type I or other known neutrophil
defects.66 The average MFI of stimulated neutrophils
stained for Mac-1 ± SD was 1,747 ± 657 for adults versus 1,042 ± 663 for infants (n = 14, P < .01; M. Mariscalco and R.N. Bennett, unpublished observations).
Thus, any individual neonate can have adult levels of Mac-1.
Nonetheless, in such an infant, Mac-1 function may still be depressed,
because our previous study demonstrated that neonatal neutrophil Mac-1
function was decreased comparable to an adult group with equivalent
levels of stimulated Mac-1 expression.34
| |
FOOTNOTES |
Submitted August 25, 1997;
accepted February 10, 1998.
Supported by National Institutes of Health Grant No. NIH-AI-19031.
Address reprint requests to M. Michele Mariscalco, MD, Baylor College
of Medicine, CNRC, 1100 Bates, Room 6014, Houston, TX 77030-2600.
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.
| |
ACKNOWLEDGMENT |
The authors thank the nurses and physicians in the Labor and Delivery
Unit of St Luke's Episcopal Hospital, without whose assistance this
project would not be possible. We also thank Drs Robert Rothlein and
Takashei Kishimoto for supplying antibodies, Dr Christine Martens for
the P-selectin transfected cell line, and Bonnie Hughes, Jia Mei, Carol
Knight, and Michelle Swarthout for their continued technical and
administrative assistance.
| |
REFERENCES |
1.
Albelda SM,
Smith CW,
Ward PA:
Adhesion molecules and inflammatory injury.
FASEB J
8:504,
1994[Abstract]
2.
Smith CW,
Kishimoto TK,
Abbassi O,
Hughes BJ,
Rothlein R,
McIntire LV,
Butcher E,
Anderson DC:
Chemotactic factors regulate lectin adhesion molecule 1 (LECAM-1)-dependent neutrophil adhesion to cytokine-stimulated endothelial cells in vitro.
J Clin Invest
87:609,
1991
3.
Jones DA,
Abbassi O,
McIntire LV,
McEver RP,
Smith CW:
P-selectin mediates neutrophil rolling on histamine-stimulated endothelial cells.
Biophys J
65:1560,
1993[Medline]
[Order article via Infotrieve]
4.
Abbassi O,
Kishimoto TK,
McIntire LV,
Anderson DC,
Smith CW:
E-Selectin supports neutrophil rolling in vitro under conditions of flow.
J Clin Invest
92:2719,
1993
5.
Kishimoto TK,
Jutila MA,
Berg EL,
Butcher EC:
Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors.
Science
245:1238,
1989[Abstract/Free Full Text]
6.
Jung TM,
Dailey MO:
Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C.
J Immunol
144:3130,
1990[Abstract]
7.
Bevilacqua MP,
Nelson RM:
Selectins.
J Clin Invest
91:379,
1993
8.
McEver RP,
Beckstead JH,
Moore KL,
Marshall-Carlson L,
Bainton DF:
GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-palade bodies.
J Clin Invest
84:92,
1989
9.
Bevilacqua MP,
Stengelin S,
Gimbrone,
Jr:
, Seed B: Endothelial leukocyte adhesion molecule 1: An inducible receptor for neutrophils related to complement regulatory proteins and lectins.
Science
243:1160,
1989[Abstract/Free Full Text]
10.
Vestweber D:
Ligand-specificity of the selectins.
J Cell Biochem
61:585,
1996[Medline]
[Order article via Infotrieve]
11.
Varki A:
Perspectives series: Cell adhesion in vascular biology. Selectin ligands: Will the real ones please stand up?
J Clin Invest
99:158,
1997[Medline]
[Order article via Infotrieve]
12.
Norgard KE,
Moore KL,
Diaz S,
Stults NL,
Ushiyama S,
McEver RP,
Cummings RD,
Varki A:
Characterization of a specific ligand for P-selectin on myeloid cells. A minor glycoprotein with sialylated O-linked oligosaccharides.
J Biol Chem
268:12764,
1993[Abstract/Free Full Text]
13.
Moore KL,
Patel KD,
Bruehl RE,
Fugang L,
Johnson DA,
Lichenstein HS,
Cummings RD,
Bainton DF,
McEver RP:
P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on p-selectin.
J Cell Biol
128:661,
1995[Abstract/Free Full Text]
14.
Symon FA,
Lawrence MB,
Williamson ML,
Walsh GM,
Watson SR,
Wardlaw AJ:
Functional and structural characterization of the eosinophil P-selectin ligand.
J Immunol
157:1711,
1996[Abstract]
15.
Alon R,
Rossiter H,
Wang X,
Springer TA,
Kupper TS:
Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P and E selectin under physiological flow.
J Cell Biol
127:1485,
1994[Abstract/Free Full Text]
16.
Patel KD,
Moore KL,
Nollert MU,
McEver RP:
Neutrophils use both shared and distinct mechanisms to adhere to selectins under static and flow conditions.
J Clin Invest
96:1887,
1995
17.
Walcheck B,
Moore KL,
McEver RP,
Kishimoto TK:
Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation on P-selectin in vitro.
J Clin Invest
98:1081,
1996[Medline]
[Order article via Infotrieve]
18.
Spertini O,
Cordey AS,
Monai N,
Giuffrè L,
Schapira M:
P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells.
J Cell Biol
135:523,
1996[Abstract/Free Full Text]
19.
Lawrence MB,
McIntire LV,
Eskin SG,
Smith CW:
Involvement of CD18 in human neutrophil adhesion to endothelium under flow conditions.
FASEB J
2:A1237,
1988
20.
von Andrian UH,
Chambers JD,
McEvoy LM,
Bargatze RF,
Arfors K-E,
Butcher EC:
Two step model of leukocyte-endothelial cell interaction in inflammation: Distinct roles for LECAM-1 and the leukocyte beta-2 integrins in vivo.
Proc Natl Acad Sci USA
88:7538,
1991[Abstract/Free Full Text]
21.
Pober JS,
Lapierre LA,
Stolpen AH,
Brock TA,
Springer TA,
Fiers W,
Bevilacqua MP,
Mendrick DL,
Gimbrone MA Jr:
Activation of cultured human endothelial cells by recombinant lymphotoxin: Comparision with tumor necrosis factor and interleukin 1 species.
J Immunol
138:3319,
1987[Abstract]
22.
Anderson DC:
Role of ICAM-1 in the adherence of human neutrophils to human endothelial cells in vitro
, in Springer TA,
Anderson DC,
Rosenthal AS,
Rothlein R
(eds):
Leukocyte Adhesion Molecules: Structure, Function, and Regulation.
New York, NY, Springer-Verlag
, 1989
, p 170
23.
Anderson DC:
Neonatal neutrophils.
J Lab Clin Med
120:816,
1992[Medline]
[Order article via Infotrieve]
24.
Santos JI,
Shigeoka AO,
Hill HR:
Functional leukocyte administration in protection against experimental neonatal infection.
Pediatr Res
14:1408,
1980[Medline]
[Order article via Infotrieve]
25.
Schuit KE,
Homisch L:
Inefficient in vivo neutrophil migration in neonatal rats.
J Leukoc Biol
35:583,
1984[Abstract]
26.
Fortenberry JD,
Marolda JR,
Anderson DC,
Smith CW,
Mariscalco MM:
CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits.
Blood
84:889,
1994[Abstract/Free Full Text]
27.
Martin TR,
Ruzinski JT,
Wilson CB,
Skerrett SJ:
Effects of endotoxin in the lungs of neonatal rats: Age-dependent impairment of the inflammatory response.
J Infect Dis
171:134,
1995[Medline]
[Order article via Infotrieve]
28.
Cheung ATW,
Kurland G,
Miller ME,
Ford EW,
Avin SA,
Walsh EM:
Host defense deficiency in newborn nonhuman primate lungs.
J Med Primatol
15:37,
1986[Medline]
[Order article via Infotrieve]
29.
Hill HR:
Biochemical, structural, and functional abnormalities of polymorphonuclear leukocytes in the neonate.
Pediatr Res
22:375,
1987[Medline]
[Order article via Infotrieve]
30.
Anderson DC,
Freeman KLB,
Heerdt B,
Hughes BJ,
Jack RM,
Smith CW:
Abnormal stimulated adherence of neonatal granulocytes: Impaired induction of surface Mac-1 by chemotactic factors or secretagogues.
Blood
70:740,
1987[Abstract/Free Full Text]
31.
Torok C,
Lundahl J,
Hed J,
Lagercrantz H:
Diversity in regulation of adhesion molecules (Mac-1 and L-selectin) in monocytes and neutrophils from neonates and adults.
Arch Dis Child
68:561,
1993[Abstract/Free Full Text]
32.
Abughali N,
Berger M,
Tosi M:
Deficient total cell content of CR3 (CD11b) in neonatal neutrophils.
Blood
83:1086,
1994[Abstract/Free Full Text]
33.
Rebuck N,
Gibson A,
Finn A:
Neutrophil adhesion molecules in term and premature infants: Normal or enhanced leukocyte integrins but defective L-selectin expression and shedding.
Clin Exp Immunol
101:183,
1995[Medline]
[Order article via Infotrieve]
34.
Anderson DC,
Rothlein R,
Marlin SD,
Krater SS,
Smith CW:
Impaired transendothelial migration by neonatal neutrophils: Abnormalities of Mac-1 (CD11b/CD18)-dependent adherence reactions.
Blood
78:2613,
1990
35.
Anderson DC,
Abbassi O,
Kishimoto TK,
Koenig JM,
McIntire LV,
Smith CW:
Diminished lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 on neonatal neutrophils underlies their impaired CD18-independent adhesion to endothelial cells in vitro.
J Immunol
146:3372,
1991[Abstract]
36.
Kishimoto TK,
Jutila MA,
Butcher EC:
Identification of a human peripheral lymph node homing receptor: A rapidly down-regulated adhesion molecule.
Proc Natl Acad Sci USA
87:2244,
1990[Abstract/Free Full Text]
37.
Entman ML,
Youker KA,
Shappell SB,
Siegel C,
Rothlein R,
Dreyer WJ,
Schmalstieg FC,
Smith CW:
Neutrophil adherence to isolated adult canine myocytes: Evidence for a CD18-dependent mechanism.
J Clin Invest
85:1497,
1990
38.
Argenbright LW,
Letts LG,
Rothlein R:
Monoclonal antibodies to the leukocyte membrane CD18 glycoprotein complex and to intercellular adhesion molecule-1 inhibit leukocyte-endothelial adhesion in rabbits.
J Leukoc Biol
49:253,
1991[Abstract]
39.
Rothlein R,
Mainolfi EA,
Czajkowski M,
Marlin SD:
A form of circulating ICAM-1 in human serum.
J Immunol
147:3788,
1991[Abstract]
40.
Tamatani T,
Kotani M,
Toshiyuki T,
Miyasaka M:
Molecular mechanisms underlying lymphocyte recirculation. II. Differential regulation of LFA-1 in the interaction between lymphocytes and high endothelial cells.
Eur J Immunol
21:855,
1991[Medline]
[Order article via Infotrieve]
41.
Isobe M,
Yagita H,
Okumura K,
Ihara A:
Specific acceptance of cardiac allograft after treatment with antibodies to ICAM-1 and LFA-1.
Science
255:1125,
1992[Abstract/Free Full Text]
42.
Smith CW,
Entman ML,
Lane CL,
Beaudet AL,
Ty TI,
Youker KA,
Hawkins HK,
Anderson DC:
Adherence of neutrophils to canine cardiac myocytes in vitro is dependent on intercellular adhesion molecule-1.
J Clin Invest
88:1216,
1991
43.
Mulligan MS,
Polley MJ:
Neutrophil dependent acute lung injury.
J Clin Invest
90:1600,
1992
44.
Birdsall HH,
Lane CL,
Ramser MN,
Anderson DC:
Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence.
J Immunol
148:2717,
1992[Abstract]
45.
Kishimoto TK,
Warnock RA,
Jutila MA,
Butcher EC,
Lane CL,
Anderson DC,
Smith CW:
Antibodies against human neutrophil LECAM-1 (LAM-1/Leu-8/DREG-56 antigen) and endothelial cell ELAM-1 inhibit a common CD18-independent adhesion pathway in vitro.
Blood
78:805,
1991[Abstract/Free Full Text]
46.
Martens CL,
Cwirla SE,
Lee RY-W,
Whitehorn E,
Chen EY-F,
Bakker A,
Martin EL,
Wagstrom C,
Gopalan P,
Smith CW,
Tate E,
Kroller KJ,
Schatz PJ,
Dower WJ,
Barrett RW:
Peptides which bind to E-selectin and block neutrophil adhesion.
J Biol Chem
270:21129,
1995[Abstract/Free Full Text]
47.
Gopalan PK,
Smith CW,
Lu H,
Berg EL,
McIntire LV,
Simon SI:
Neutrophil CD18-dependent arrest on ICAM-1 in shear flow can be activated through L-selectin.
J Immunol
158:367,
1997[Abstract]
48.
Smith CW,
Rothlein R,
Hughes BJ,
Mariscalco MM,
Schmalstieg FC,
Anderson DC:
Recognition of an endothelial determinant for CD18-dependent human neutrophil adherence and transendothelial migration.
J Clin Invest
82:1746,
1988
49.
Lawrence MB,
Bainton DF,
Springer TA:
Neutrophil tethering to and rolling on E-selectin are separable by requirement for L-selectin.
Immunity
1:137,
1994[Medline]
[Order article via Infotrieve]
50.
Hattori R,
Hamilton KK,
McEver RP,
Sims PJ:
Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface.
J Biol Chem
264:9053,
1989[Abstract/Free Full Text]
51.
Picker LJ,
Warnock RA,
Burns AR,
Doerschuk CM,
Berg EL,
Butcher EC:
The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140.
Cell
66:921,
1991[Medline]
[Order article via Infotrieve]
52.
Koenig JM,
Simon J,
Anderson DC,
Smith EO,
Smith CW:
Diminished soluble and total cellular L-Selectin in cord blood is associated with its impaired shedding from activated neutrophils.
Pediatr Res
39:616,
1996[Medline]
[Order article via Infotrieve]
53.
Smith JB,
Kunjummen RD,
Kishimoto TD,
Anderson DC:
Expression and regulation of L-selectin on eosinophils from human adults and neonates.
Pediatr Res
32:645,
1992[Medline]
[Order article via Infotrieve]
54.
Abbassi O,
Lane CL,
Krater SS,
Kishimoto TK,
Anderson DC,
McIntire LV,
Smith CW:
Canine neutrophil margination mediated by lectin adhesion molecule-1 (LECAM-1) in vitro.
J Immunol
147:2107,
1991[Abstract]
55.
Jutila MA,
Watts G,
Walcheck B,
Kansas GS:
Characterization of a functionally important and evolutionarily well-conserved epitope mapped to the short consensus repeats of E-selectin and L-selectin.
J Exp Med
175:1565,
1992[Abstract/Free Full Text]
56.
Bargatze RF,
Kurk S,
Butcher EC,
Jutila MA:
Neutrophils roll on adherent neutrophils bound to cytokine-induced endothelial cells via L-selectin on the rolling cells.
J Exp Med
180:1785,
1994[Abstract/Free Full Text]
57.
Bennett TA,
Schammel CMG,
Lynam EB,
Guyer DA,
Mellors A,
Edwards B,
Rogelj S,
Sklar LA:
Evidence for a third component in neutrophil aggregation: Potential roles of O-linked glycoproteins as L-selectin counter-structures.
J Leukoc Biol
58:510,
1995[Abstract]
58.
Hynes RO:
Integrins: Versatility modulations and signaling in cell adhesion.
Cell
69:11,
1992[Medline]
[Order article via Infotrieve]
59.
Van Kooyk Y,
Weder P,
Hogervorst F,
Verhoeven AJ,
Van Seventer GA,
te Velde AA,
Borst J,
Keizer GD,
Figdor CG:
Activation of LFA-1 through a Ca2+-dependent epitope stimulates lymphocyte adhesion.
J Cell Biol
112:345,
1991[Abstract/Free Full Text]
60.
Petruzzelli L,
Maduzia L,
Springer TA:
Activation of lymphocyte function-associated molecule-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) mimicked by an antibody directed against CD18.
J Immunol
155:854,
1995[Abstract]
61.
Dransfield I,
Buckle A-M,
Savill JS,
McDowall A,
Haslett C,
Hogg N:
Neutrophil apoptosis is associated with a reduction in CD16 (Fc RIII) expression.
J Immunol
153:1254,
1994[Abstract]
62.
Simon SI,
Burns AR,
Taylor AD,
Gopalan PK,
Lynam EB,
Sklar LA,
Smith CW:
L-Selectin (CD62L) crosslinking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) 2-integrin.
J Immunol
155:1502,
1995[Abstract]
63.
Kjeldsen L,
Sengelov H,
Lollike K,
Borregaard N:
Granules and secretory vesicles in human neonatal neutrophils.
Pediatr Res
40:120,
1996[Medline]
[Order article via Infotrieve]
64.
Ambruso DR,
Bentwood B,
Henson PM,
Johnston RB Jr:
Oxidative metabolism of cord blood neutrophils: Relationship to content and degranulation of cytoplasmic granules.
Pediatr Res
18:1148,
1984[Medline]
[Order article via Infotrieve]
65.
Graf JM,
Smith CW,
Mariscalco MM:
Contribution of LFA-1 and Mac-1 to CD18-dependent neutrophil emigration in a neonatal rabbit model.
J Appl Physiol
80:1984,
1996[Abstract/Free Full Text]
66.
Anderson DC,
Schmalstieg FC,
Finegold MJ,
Hughes BJ,
Rothlein R,
Miller LJ,
Kohl S,
Tosi MF,
Jacobs RL,
Waldrop TC,
Goldman AS,
Shearer WT,
Springer TA:
The severe and moderate phenotypes of heritable Mac-1, LFA-1, p150,95 deficiency: Their quantitative definition and relation to leukocyte dysfunction and clinical features.
J Infec Dis
152:668,
1985[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract