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Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4776-4785
By
From the Sections of Leukocyte Biology and Critical Care Medicine,
Department of Pediatrics, Baylor College of Medicine, Houston, TX.
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.
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, 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.
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 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.
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 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.
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).
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).
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.
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.
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.
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.
Submitted August 25, 1997;
accepted February 10, 1998.
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.
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
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
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 C |
Abstract
Introduction
Abstract
L
2) and Mac-1(CD11b/CD18,
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.
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.
) and neonatal (
) neutrophil adhesion to
HUVECs stimulated for 10 minutes with 10
, 
) 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 (
), anti-CD18, R15.7 (10 µg/mL; 
), or anti-L-selectin,
DREG 56 (50 µg/mL; 