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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 266-274
Blood Polymorphonuclear Leukocytes From the Majority of Sickle Cell
Patients in the Crisis Phase of the Disease Show Enhanced Adhesion to
Vascular Endothelium and Increased Expression of CD64
By
Emma Fadlon,
Susanne Vordermeier,
Thomas C. Pearson,
Anthony R. Mire-Sluis,
Dudley C. Dumonde,
Julia Phillips,
Keith Fishlock, and
K. Alun Brown
From the Departments of Immunology and Haematology, St Thomas'
Hospital, London; and the Department of Immunology, National Institute
for Biological Standards and Control, South Mimms, Hertfordshire, UK.
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ABSTRACT |
There is increasing interest in the role of blood polymorphonuclear
leukocytes (PMNs) in the pathogenesis of sickle cell crisis. We studied
the adherence of PMNs from 18 sickle cell patients in crisis, 25 out of
crisis, and 43 healthy subjects (controls) to monolayers of human
umbilical cord endothelium that were either untreated or pretreated
with tumor necrosis factor  (TNF). Overall, the PMNs from
patients in crisis were more adherent than control PMNs to untreated
endothelial monolayers (mean 53% increase; P < .001) and
TNF-treated monolayers (mean 41% increase; P < .002).
Increased adhesiveness was not associated with an abnormal expression
of CD11a, CD11b, CD11c, CD18, CD62L, or CD15. There was an increase in
the number of PMNs expressing CD64 in patients in crisis (median value,
44%) compared with patients out of crisis (median, 21%; P =
.025) and controls (median, 6.5%; P < .001). Sera from
patients in crisis had normal levels of granulocyte colony-stimulating
factor, granulocyte-macrophage colony-stimulating factor,
interferon- , TNF, interleukin-1 (IL-1), IL-6, or IL-8 and did not
modify the adherence of PMNs or their expression of CD64. Only IFN-
induced CD64 expression on PMNs, but this effect was not associated
with enhanced binding to endothelium. Because PMNs bound to endothelial
monolayers were CD64+ and CD64-enriched PMNs were 7 times
more adherent to endothelial monolayers than CD64-depleted PMNs, it is
likely that CD64 is a marker of adherent PMNs. Two of the three
anti-CD64 antibodies used in our antibody blocking studies (clones 32.2
and 197) partially inhibited the binding of sickle cell PMNs to
untreated endothelium (mean inhibitions of 33% [P = .01]
and 21% [P = .03], respectively), whereas
only one (clone 197) inhibited binding to TNF-treated endothelium
(mean inhibition, 29%; P = .004). In some patients with
sickle cell disease, an enhanced PMN adhesion to vascular endothelium
could contribute to the vascular occlusion that characterizes the acute
crisis of the disease.
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INTRODUCTION |
VASO-OCCLUSIVE crises of sickle cell
disease are a major cause of morbidity and death.1,2 The
pathogenetic mechanisms of vaso-occlusion remain
controversial,3 although it is generally agreed that the
increased binding of sickled erythrocytes to vascular endothelium is an
important contributory factor.4,5 Despite contradictory
reports of whether the percentage of circulating irreversibly sickled
erythrocytes correlates with the severity and frequency of
crisis,6,7 it appears that a combination of adhering,
deformable sickle cells and trapping of nondeformable sickle cells
participate in vaso-occlusion.8,9 The suggestion that
polymorphonuclear leukocytes (PMNs) may play a copathogenic role arises
from the association of sickle cell crises with
infection,10 a polymorphonuclear leukocytosis related to
increased risk of death1,11 and, in severe acute bone pain,
marrow necrosis with PMN infiltration.12
Extravasation of PMNs into tissue is dependent on the cells binding to
endothelial cells before active migration through vessel walls. Several
adhesion molecules (eg, CD11a, CD11b, CD11c/CD18, L-selectin, and CD15)
mediate PMN binding to endothelium through the recognition of
complementary ligands (eg, intercellular adhesion molecule-1
[ICAM-1], E-selectin, and P-selectin) whose expression
is induced or enhanced by the action of cytokines and other
inflammatory factors.13,14 Additional leukocyte adhesion
molecules recognize the R-G-D (arg-gly-asp) sequence within a number of
extracellular matrix proteins.15 In sickle cell disease,
binding of neutrophils to the endothelial surface, particularly at
sites at which inflammatory cytokines are generated, could reduce blood
flow and, in association with sickled erythrocytes, produce
microvascular occlusion and crises. Evidence of an increased
adhesiveness of blood PMNs in this disease comes from the demonstration
that experimental vascular occlusion in patients with sickle disease
produces increased PMN binding to vascular endothelium16
and that laboratory-induced aggregation of PMNs is enhanced during
crisis.17
The high-affinity Fc receptor, CD64, is considered as a marker of PMN
activation,18,19 and increased numbers of CD64+
PMNs are a feature of some bacterial infections.20 In this
study, we report that PMNs from most patients in crisis were highly
adherent to cultures of untreated and tumor necrosis factor-
(TNF)-treated vascular endothelium. We also found that
CD64+ PMNs were increased in the blood of patients with
sickle cell disease, particularly during crisis, and evidence is
presented to show that PMNs expressing CD64 are highly adherent to
endothelial monolayers.
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MATERIALS AND METHODS |
Patients and controls.
A total of 97 homozygous (Hb S/S) patients (18 to 55 years of age) were
studied. A clinical diagnosis of crisis was made on the following
criteria: (1) widespread pain typically involving the limbs, vertebrae,
or ribs that could be ascribed to vascular occlusion and/or
ischaemic tissue damage; and (2) symptomatic relief after analgesia.
Retrospective analysis of the medical records of the patients in crisis
did not show the presence of a specific bacterial infection or
reticulocytopenia. However, this does not exclude the possibility of
viral or covert bacterial infection. Ethical Committee approval for the
study was granted in advance. Age-matched healthy hospital and medical
school staff acted as controls.
Culture of endothelial monolayers.
Endothelial cells were isolated from human umbilical cord veins using a
standard method.21 Briefly, endothelial cells were removed
with collagenase II (200 U/mL; Sigma Chemical Co, Poole, UK), washed,
and resuspended in Dulbecco's modified Eagle's medium
(DMEM; Sigma) supplemented with 20% fetal calf serum
(FCS; Myclone; GIBCO, Paisley, UK), 4 mmol/L glutamine (Sigma), 1/50
vol/vol sodium pyruvate (GIBCO), 200 U/mL penicillin (Brittania
Pharmaceuticals, Redhill, UK), 100 U/mL streptomycin (Sigma), and
gentamycin (Roussel, Uxbridge, UK). Cells were grown in gelatin (1%
wt/vol; Sigma)-coated flasks (Costar, Cambridge, MA) in a 10%
CO2-humidified atmosphere at 37°C. When confluent,
cells were detached with trypsin-EDTA (Sigma), seeded onto
gelatin-coated 96-well microtiter plates (Costar), and again grown to
confluence. Identification of endothelial cells was confirmed by
immunofluorescence staining with antibody directed against von
Willebrand factor (Nordic Laboratories, London, UK) and by their
characteristic morphology using light microscopy. Confluent endothelial
monolayers were pretreated for 4 hours at 37°C with either 10 U/mL
recombinant human TNF (Amgen, Thousand Oaks, CA) or with DMEM/FCS only.
Monolayers were washed twice with serum-free DMEM before the addition
of PMNs.
Isolation of PMNs and adherence assay.
Blood samples from both patients and control subjects were processed
within minutes (maximum delay, 35 minutes) of their provision. All
samples were handled in an identical manner and PMNs were isolated by
low-speed centrifugation of lysed blood.22 Briefly, 20 mL
of heparinized blood was diluted with 50 mL of 9 parts 0.16 mol/L
NH4Cl (BDH, Poole, UK):1 part 0.17 mol/L Tris (BDH), pH
7.4, and allowed to stand for 10 minutes at room temperature. Cells
were centrifuged at 400g for 10 minutes, the supernatant was
discarded, and the lysis stage was repeated three times. The PMN pellet
was washed three times (50g for 10 minutes) in Hank's Balanced
Salt Solution without calcium and magnesium (Sigma). The PMNs were
radiolabeled by incubating with 51Cr (sodium chromate;
Amersham International PLC, Amersham, UK) for 45 minutes
at 37°C at a concentration of 3 µCi/106 cells, washed
three times with DMEM-5% FCS, and adjusted to 2 ×
106 cells/mL in DMEM with 10% autologous serum. Cells at 2
× 105 in 100 µL were added to each well for 1 hour
at 37°C. Nonadherent PMNs were removed from the monolayers by
washing five times with serum-free DMEM. Each monolayer was lysed by
200 µL 0.1 N NaOH (BDH) and radioactivity was counted in an
auto-gamma scintillation counter (Wallac, Milton Keynes, UK). Tests
were performed in quadruplicate. The percentage of PMNs adhering to
endothelium was calculated as follows: Adherence = (Mean dpm in
Lysate)/(Mean dpm of Overlaid PMN) × 100%. For each experiment,
the adherent properties of PMNs from 1 patient were compared directly
with PMNs from 1 normal subject using endothelial cells obtained from
the same umbilical cord vein. This procedure ensured that any observed
differences in adherence were the result of the intrinsic properties of
PMNs rather than that of endothelial cell cultures. To ensure that only
confluent monolayers were used for experiment, all wells were inspected
by light microscopy immediately before assay and after incubation of
PMNs with endothelial cells. Neither treatment of endothelial cells
with TNF nor incubation with PMNs resulted in disruption or damage
of the monolayers.
Experiments were also performed to investigate whether cytokines,
F-methionyl-leucyl-phenylalanine (FMLP), and sera modified the
adherence of PMNs from control subjects and patients in and out of
crisis. The PMNs were incubated with either 50 U/mL interferon-
(IFN-), 10 U/mL granulocyte-macrophage colony-stimulating factor
(GM-CSF), 10 U/mL granulocyte colony-stimulating factor (G-CSF), 10
U/mL TNF, 10 U/mL interleukin-1 (IL-1), 10 U/mL IL-6, or
10 11 mol/L IL-8 for 4 or 24 hours before the
adherence assay. The PMNs were incubated with 107
mol/L FMLP for 3 minutes. PMNs were also incubated with 100 µL sera
from control subjects and patients for the same time periods.
Flow cytometric analysis of PMN surface markers.
Cells were prepared using the method of Hamblin et al.23
Briefly, 1 mL of blood was immediately mixed with 1 mL of prewarmed
0.4% paraformaldehyde (BDH)/phosphate-buffered saline (PBS; Sigma) and
incubated for 4 minutes at 37°C. Red blood cells were lysed with 20
mL of 0.16 mol/L NH4Cl/0.17 mol/L Tris buffer, pH 7.4, for
10 minutes at 37°C. The cells were washed twice with DMEM
(400g for 5 minutes) and resuspended in PBS-1% bovine serum
albumin (BSA; Sigma). Aliquots (25 µL) of the leukocyte-rich sample
(1 × 106 cells) were incubated with 10 µL of
unconjugated monoclonal antibodies directed against CD18, CD11a, CD11b,
CD11c, CD15, L-selectin, and CD64 as well as isotype control antibodies
for 30 minutes at 4°C. The cells were washed three times with
PBS-1% BSA and incubated with 25 µL of fluorescein-conjugated rabbit
antimouse Ig (DAKO, High Wycombe, UK) for 30 minutes at 4°C. After
washing in PBS-1% BSA, the cells were resuspended in 0.3 mL
paraformaldehyde/PBS for analysis on a FACScan flow cytometer (Becton
Dickinson, Mountain View, CA). Results were recorded as
the percentage of positive cells and the mean fluorescence intensity
(MFI).
Monoclonal antibodies to the  2 integrin adhesion molecules were a
gift from Prof A.J. McMichael (Oxford, UK; CD11a and CD11b) as well as
from Dr N. Hogg (London, UK; CD11c) and Boehringer Mannheim (Mannheim,
Germany; CD18). Antibodies against L-selectin (LAMI-3) and CD15 (C3D-1)
were purchased from Coulter (Luton, UK) and Dako
(Glostrup, Denmark), respectively, and an IgG1 monoclonal
antibody to FcR1 (CD64, clone 10.1) was purchased from Serotec
(Oxford, UK). Isotype control antibodies (Dako) were used throughout
the study and fluorescein isothiocyanate (FITC)-conjugated rabbit
antimouse Ig (Dako) was used as the secondary antibody.
To determine whether CD64 expression on PMNs was increased by the
activity of cytokines or patients' sera, the following experiments
were performed. Preparations of PMNs from patients out of crisis and
from healthy control subjects were mixed with either IFN-, GM-CSF,
G-CSF, TNF, IL-1, IL-6, IL-8, or FMLP at the same concentration and
incubation times as outlined in the description of the adherence assay.
Also, PMNs were treated with 100 µL sera from control subjects and
from patients in crisis and out of crisis.
CD64 and PMN adhesion to endothelium.
To identify CD64+ PMNs bound to endothelial monolayers,
adherence assays were performed in 16-well chamber slides (NUNC: Life
Technologies Ltd, Paisley, UK). Formaldehyde-fixed cocultures of PMNs
bound to endothelial cells were treated with the Serotec anti-CD64
antibody (1/50) or isotype control antibody followed by staining with
biotinylated rabbit antimouse Ig (1/500; Dako) and avidin-peroxidase
reagent (1/1,000; Sigma). Between each step, wells were washed with
Tris buffer. The substrate 3,3 -diaminobenzidine (DAB; Sigma) was
added for 5 minutes and washed off with water before counter-staining
with haematoxylin and clearing in water. Slides were dehydrated and
cleared in ethanol:xylene before mounting in DPEX (BDH
Chemicals). The percentage of CD64+ cells in the wells was
calculated by counting at least 100 adherent PMNs under light
microscopy. Experiments were performed in triplicate.
To compare the adherence properties of CD64+ with
CD64 PMNs, 4 × 106 cells from 4
normal subjects were treated with 1:50 dilutions of the anti-CD64
antibody in PBS-1% BSA for 30 minutes at 4°C. After washing, the
cells were resuspended in a 1/500 FITC-conjugated rabbit antimouse IgG1
reagent and processed through a FACSorter (Becton Dickinson) at 300 to
400 cells/s. The CD64+ and CD64
fractions contained 78% and 1% mean CD64+ cells,
respectively, and cell viability was greater than 87%. Enriched cells
were added to the endothelium at 1 × 105 cells/well
(200 µL). The number of PMNs bound to the monolayers after 1 hour of
incubation was determined by counting the number of cells in 5 high
power microscopic fields. All experiments were performed in triplicate
and the results are expressed as the mean number of adherent PMNs per
high power field.
To determine whether PMN adhesion was inhibited by anti-CD64
antibodies, PMNs (4 × 106 in 350 µL medium) were
pretreated with a 1:50 dilution of anti-CD64 monoclonal antibodies from
clones 22, 32.2, and 197 (Cambio, Cambridge, UK) and isotype antibody
controls (IgG1 for clones 22 and 32; IgG2a for clone 197) for 30
minutes at 4°C. The cells were washed before adding to endothelial
monolayers. Parallel blocking experiments were undertaken with
anti-CD18 (R.15.7/H4; Dr R. Rothlein, Boehringer-Ingelheim, Ridgefield,
CT) and anti-L-selectin antibodies (Becton Dickinson,
Oxford, UK). Polymorphonuclear cells were also added to
endothelial cells in the presence of 100 µmol/L of the RGD containing
peptide, 1 Adamantaneacetyl-cys-gly-arg-gly-asp-ser-pro-cys (Sigma),
which inhibits lymphocyte attachment to cytokine-treated
endothelium.24 Blocking studies were also performed with
antibodies against ICAM-1 (RR1/1.1.1; Dr R. Rothlein) and E-selectin
[F(ab)2 fragments of ENA-2; Dr J. Leeuwenberg, Maastricht,
Holland]; for immunocytochemistry, an antibody against P-selectin
(IEA3; Dr A.-K. Ng, University of Maine) was included in
the study.
Measurement of cytokines in sera.
The cytokines G-CSF, GM-CSF, TNF, IL-1, IL-6, and IL-8 were measured
by enzyme-linked immunoassay (ELISA) kits purchased from Boehringer
Mannheim (Lewes, UK) and IFN- by an ELISA developed
within the National Institute for Biological Standards and Control.
Cytokine levels were also assessed by proliferative
bioassays.25 IL-1 was quantitated using the murine T-helper
cell line D10S; IL-6 and GM-CSF with the murine hybridomas B9 and MO7e,
respectively; G-CSF by the murine myeloid leukaemic cell line GNFS-60;
TNF by the human fibrosarcoma WEH1-164; and IFN- by the human
glioblastoma/encephalo-myocarditis virus line, 2DP + EMCV. Briefly,
sera samples and titrations standard of cytokines were added to
suspensions of the above-mentioned cell lines (1 ×
105 cells/mL) for 48 hours at 37°C in a humidified 5%
CO2 atmosphere. Tritiated thymidine (0.5 µCi) was added
and the plates were incubated for 4 hours at 37°C. The contents of
each well were harvested onto filter mats, and the radioactivity
incorporated was determined by liquid scintillation counting.
Statistical analysis.
Differences in PMN adherence between patient and control groups were
evaluated by means of a Wilcoxon paired signed rank test. Where data
were approximately normally distributed, the Student's t-test
was used to assess significance of differences. Prevalence of the CD64
marker in a patient's PMN population was considered exaggerated when
greater than 26%; by analogy,26,27 this
cut-off point was 3 multiples (MoMs) above the median value (6.5%)
obtained for all PMN CD64 values in the 22 normal subjects. The
significance of association between IFN--induced expression and PMN
adherence was examined using Spearman's correlation coefficient.
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RESULTS |
Adherence of PMNs to endothelial monolayers.
Forty-three comparative experiments were undertaken of the adherent
properties of PMNs from 18 patients in crisis, 25 patients out of
crisis, and 43 normal healthy subjects (controls), 38 of whom were of
Afro-Caribbean descent. In each experiment shown in Fig
1, the adherence of PMNs from a patient in
crisis or from a patient out of crisis were compared with PMNs from a
paired control using endothelial monolayers derived from the same
umbilical cord vein (see the Materials and Methods). Variations in the
adherence values of PMNs from healthy subjects are due to the intrinsic
adhesive properties of endothelial cells isolated from different
umbilical cord veins. Endothelial adhesiveness for both patient and
control PMNs was enhanced by pretreating the endothelium with 10 U/mL
TNF. Figure 1A and B shows that, in general, PMNs from patients in
crisis were more adherent than control cells to both untreated
endothelium (mean increase, 53%; P < .001) and endothelium
pretreated with 10 U/mL TNF (mean increase, 41%; P = .002),
although in 6 experiments the adhesive properties of patients' PMNs
were less or similar to that of control PMNs. Comparable adhesion
results were obtained with PMNs isolated by Ficoll hypaque density
gradient centrifugation, showing that the enhanced binding of PMNs from
patients in crisis to endothelial monolayers was not dependent on the
method used to isolate PMNs from blood (data not shown). The PMNs from
patients out of crisis did not differ from those of controls in binding
to either untreated (Fig 1C) or TNF-treated endothelium (Fig 1D).
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| Fig 1.
Increased adherence of blood PMNs to untreated and
TNF-treated cultured endothelium in sickle cell crisis. Summary of
results comparing the adherence of PMNs from 18 control subjects and 18
sickle cell patients in crisis to (A) untreated and (B) TNF-treated
endothelium and of PMNs from 25 control subjects and 25 patients out of
crisis to (C) untreated and (D) TNF-treated endothelium. Each line
represents an experiment in which PMNs from 1 patient and 1 control
subject were incubated with endothelial monolayers prepared from the
same umbilical cord vein. Half of the monolayers had been pretreated
with 10 U/mL TNF for 4 hours. 51Cr-labeled PMNs (2 ×
105/100 µL) suspended in DMEM with 10% autologous serum
were added to confluent monolayers in 96-well microtiter plates. After
coculture for 1 hour at 37°C, the nonadherent PMNs were removed by
washing, the monolayer was lysed by 0.1 N NaOH, and the radioactivity
associated with adherent PMNs was counted. The results, which are the
mean value of quadruplicate measurements, are expressed as the
percentage of adherent PMNs in relation to the total number of PMNs
added to the monolayers. Overall, the PMNs from patients in crisis were
more adherent than control PMNs to untreated endothelial monolayers
(P < .001) and to TNF-treated monolayers (P =
.002).
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Four of the patients in crisis were also studied at subsequent visits
to the clinic (2 attendances in 8 months). Attachment of PMNs from
these patients, who were now out of crisis, to untreated and
TNF-treated monolayers was similar to that of PMNs from age- and
sex-matched healthy subjects. After 10 months, 1 patient was readmitted
with a sickle cell crisis. Analysis of his PMNs showed that the
adherence to resting and TNF-treated endothelial cells was greater than
that of control blood PMNs (65% and 84% increase, respectively;
P < .01).
Expression of CD64 and adhesion molecules on the surface of PMNs.
Figure 2 shows the distribution of PMNs
possessing the high-affinity Fc receptor CD64 in the blood of 16
patients in crisis, 11 patients out of crisis, and 22 normal subjects.
The median values (and 95% confidence intervals) for the prevalence of
CD64+ PMNs in the crisis group was 44% (17% to 88%); in
the out of crisis group was 21% (8% to 68%); and in the normal
subjects was 6.5% (5% to 32%).  2 analysis showed that
patients in crisis had a significantly higher proportion of
CD64+ PMNs than did patients out of crisis (P =
.025). Both groups of patients had a significantly higher percentage of
CD64+ PMNs than did the normal group (patients in crisis,
P < .001; patients out of crisis, P = .02). The mean
fluorescence intensity of CD64 expression on PMNs from patients in
crisis (MFI = 16 ± 8) and out of crisis (MFI = 10 ± 4) did not
differ from that of control PMNs (MFI = 11 ± 5). The distribution
and surface expression of the 2 integrin family (CD11a, CD11b,
CD11c, and CD18), L-selectin, and CD15 were similar for controls and
patients either in or out of crisis.
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| Fig 2.
Increased numbers of CD64+ PMNs in the
blood of sickle cell patients in crisis. Scattergram showing flow
cytometric analysis of the distribution of CD64+ PMNs in
the blood of 16 patients in crisis ( ), 11 patients out of crisis
( ), and 22 normal healthy subjects ( ). The horizontal line (26%)
represents a threefold value above the median percentage
CD64+ PMNs of the 22 normal subjects. The figure shows
that, whereas CD64+ PMNs both in and out of crisis were
supranormal (in crisis, 2 = 23.4, P < .001;
out of crisis, 2 = 5.8, P = .02), their
proportion in crisis was significantly greater than that out of crisis
(2 = 5.53, P = .025). PMNs (1 ×
106 cells) were incubated with anti-CD64 antibody (10 µL)
for 30 minutes at 4°C. Cells were washed and the FITC conjugated Ig
(25 µL) was added for a further 30 minutes at 4°C. Cells were
then washed twice, fixed with 1% paraformaldehyde-PBS, and analyzed
using flow cytometry.
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Effect of patients' serum, cytokines, and FMLP on the distribution
of CD64+ PMNs and on the binding of PMNs to endothelial
monolayers.
All of the above-noted adherence studies were performed in the presence
of autologous serum. Thus, the abnormal adherence properties of PMNs
from patients in crisis and their high prevalence of CD64 positivity
may have arisen from the activity of soluble, adherence-promoting
factors. To test this possibility, PMNs from healthy subjects and
sickle cell patients out of crisis were incubated for 24 hours in serum
from patients in crisis before assay. Such treatments did not modify
CD64 expression or the binding of PMNs to untreated endothelial
monolayers (Fig 3). IFN- is known to
increase the number of PMNs expressing CD6415 and Fig 3A
shows that, after 24 hours of incubation, this cytokine expanded the
CD64 population of PMNs from control subjects and patients out of
crisis. However, IFN- failed to increase the attachment of normal
blood PMNs to endothelium and a dose-dependent increase in CD64
expression by IFN- did not produce a parallel change in PMN
attachment to endothelium (Table 1).
Incubation of PMNs with the cytokines G-CSF, GM-CSF, TNF, IL-1,
IL-6, or IL-8 for 24 hours had no effect on CD64 expression or
adherence (Fig 3B). When the above-mentioned experiments were repeated
using 4 hours of incubation, neither the cytokines, including IFN-,
nor the sera modified CD64 expression or PMN binding to endothelium
(data not shown). An increase in binding was only recorded after
treatment with the PMN agonist FMLP, which itself did not modify the
distribution of CD64. Ten sera from patients in crisis and 10 from
patients out of crisis and controls were also screened for their levels
of G-CSF, GM-CSF, IFN-, TNF, IL-1, IL-6, and IL-8. Analysis by
immunoassay and bioassay showed that all of the patient sera samples
had levels of cytokines within normal limits.
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| Fig 3.
Effects of cytokines and serum (A) on the expression of
CD64 on PMNs and (B) on the binding of PMNs to untreated endothelium.
(A) Blood PMNs (1 × 106 cells) from 5 patients out of
crisis ( ) and from 5 normal healthy subjects ( ) were incubated
with cytokines (10 U/mL except for IFN-, which was 50 U/mL) or 100
µL patient and control serum for 24 hours and with FMLP for 3 minutes
before flow cytometric analysis. Only IFN- significantly increased
the number of CD64+ PMNs (9 experiments) by comparison
with the untreated samples (normal controls, P = .02;
patients, P = .03). (B) PMNs from the 5 control subjects were
also examined in the adherence assay. The cells were incubated with
cytokines or serum for 24 hours or with FMLP for 3 minutes and overlaid
onto untreated endothelial cells. A significant increase of PMN
adherence was only seen after FMLP treatment (P = .008). In
both (A) and (B), data are expressed as the mean result ± SD.
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CD64 expression and PMN adhesion to endothelium.
To determine whether PMNs bound to endothelial monolayers were
CD64+, adherent PMNs from patients in crisis and normal
healthy controls were stained with the anti-CD64 (10.1) antibody (see
the Materials and Methods). The anti-CD64 antibody did not stain
cultured endothelium. Table 2 shows that,
before introduction into the adherence assay, the mean percentages of
CD64+ PMNs in the patient and control samples were 46% and
10%, respectively. Examination of the monolayers with bound PMNs
showed that the majority of adherent PMNs from both patients (mean,
71%) and controls (mean, 68%) expressed the CD64 phenotype.
Further adhesion experiments were undertaken with PMN fractions
enriched for CD64+ and CD64 cells from
normal blood by preparative flow cytometry. Because fewer cells were
available for experiment, in comparison with the above-mentioned
studies, the adherence assay was modified and results expressed as the
number of PMNs bound per high power field (see the Materials and
Methods). Table 3 shows that, in four
experiments, the mean number of CD64+ PMNs bound to
endothelium was 61 cells/high power field in contrast to only 8
cells/high power field with the CD64-depleted fractions (P =
.004).
Finally, to ascertain whether CD64 had a role in promoting adhesion,
PMNs from sickle cell patients were treated with anti-CD64 antibodies
of three different clones (22, 32.2, and 197) for 30 minutes at 4°C
before overlaying onto endothelial monolayers. Aliquots of PMNs were
also incubated with anti-CD18 and anti-L-selectin antibodies and the
RGD peptide. Table 4 shows that PMN
adhesion to untreated endothelium was significantly impaired by
anti-CD64 antibodies of clone 32.2 (mean inhibition, 33%; P =
.01) and clone 197 (mean inhibition, 21%; P = .03) but not by
those of clone 22. Only antibodies of clone 197 inhibited the
attachment of PMNs to TNF-treated endothelium (mean inhibition, 29%;
P = .004). Binding of PMNs from 6 healthy subjects to untreated
and TNF-treated endothelial monolayers was not modified by any of the
anti-CD64 antibodies. The anti-CD18 antibody inhibited the adhesion of
patients' PMNs to untreated (mean inhibition, 45%; P = .02)
and TNF-treated endothelium (mean inhibition, 43%; P = .01),
and similar results were obtained with PMNs from normal healthy
subjects. The adherence of PMNs from sickle cell patients to resting
and activated endothelium was not impeded by isotype control
antibodies, an RGD containing peptide, or anti-L-selectin antibodies
(Table 4). When PMNs from 4 patients in crisis and 4 healthy controls
were suspended in normal serum and added to TNF-activated endothelial
cells that were pretreated for 1 hour with anti-ICAM-1 antibody, there
was a mean 27% ± 6% (P < .05) and 19% ± 15%
(P < .05) inhibition of adhesion, respectively.
Anti-E-selectin antibodies did not significantly alter the adhesion of
PMNs from patients in crisis or control subjects to TNF-treated
endothelium, and immunocytochemical analysis28 showed that
neither sera nor PMNs from 6 patients in crisis induced the expression
of E- or P-selectin on endothelial monolayers.
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Table 4.
Effect of Antibodies Against CD64, CD18, L-Selectin, and
an RGD-Containing Peptide on the Binding of Sickle Cell PMNs to
Endothelial Monolayers
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DISCUSSION |
The results demonstrate that peripheral blood PMNs from the majority of
sickle cell patients in crisis were more adherent to cultured
endothelial monolayers than PMNs from patients out of crisis and
healthy control subjects. We also found that CD64 was more prevalent on
PMNs from patients in crisis. An association of CD64 with adherence was
suggested by the finding that anti-CD64 antibodies partially inhibited
the binding of PMNs to endothelial cells and that most of the PMNs from
the sickle cell patients and control subjects that bound to endothelial
monolayers expressed the CD64 antigen. It is unlikely that endothelial
interaction accounts for CD64 induction, because the adherence to
endothelial monolayers of CD64-enriched PMNs, prepared by flow
cytometry, was several fold greater than that of CD64-depleted PMNs.
IFN- selectively increased the number of CD64+ PMNs from
healthy subjects as well as from sickle cell patients, extending
published work.18,29 Although IFN- induction of CD64
expression on PMNs is associated with enhanced phagocytic
capacity,29 the cytokine did not further increase PMN
attachment to endothelial cells.
The expression of CD64 is increased on PMNs from subjects of
Afro-Caribbean descent30 and such individuals were almost
exclusively the main population of our control group. Administration of
G-CSF to neutropenic patients leads to an increase in the number of
circulating CD64+ PMNs by an effect on myeloid precursors
in the bone marrow.31,32 An increase in CD64 expression has
recently been proposed to be a reliable indicator of a systemic
inflammatory response.33 Circulating CD64+ PMNs
are raised during the leukocytosis accompanying bacterial
infections,20 and some of these patients have high serum
levels of G-CSF.34 In the present study, sera from patients
in crisis and out of crisis were found to contain normal levels of
G-CSF, GM-CSF, IFN-, and other cytokines. High titers of
anti-endothelial cell antibodies as measured by ELISA35
were not detected in any of the patient samples and incubation of PMNs
in patients sera did not enhance adherence or increase CD64 expression.
Because the abnormal adhesiveness and increased CD64 distribution of
PMNs from patients in crisis does not appear to be due to the activity
of soluble factors in the circulation, they could be induced by contact
of the cells with blood vessel walls in distinct areas of the
vasculature or by the microenvironment of the bone marrow.
Alternatively, PMN activation could result from interaction with
erythrocytes since sickle erythrocytes are capable of binding to and
stimulating the respiratory burst of PMNs.36
The relationship between CD64 expression and adherence was investigated
further by blocking studies using three clones of anti-CD64 antibodies.
Antibodies of clone 197 block Ig binding to CD64 and recognize a non-Fc
binding domain of the molecule, whereas clones 22 and 32.2 do not
inhibit IgG binding but are directed against CD64 epitopes that are
distinct from one another and those recognized by clone
197.37 Clones 32.2 and 197 partially impaired the
attachment of PMNs from sickle cell patients to untreated endothelium,
but only clone 197 inhibited adhesion to TNF-treated endothelium. These
findings introduce the consideration that, on sickle cell PMNs,
determinants on the CD64 molecule might be recognizing unidentified
endothelial ligands or that the 32.2 and 197 clones are interfering
with the recognition of adhesion-promoting determinants associated with
CD64. Although labeling of sickle cell PMNs with anti-CD64 antibodies
(clone 10.1) did not impair CD11a or CD18 expression, these
observations do not preclude the possibility that anti-CD64 antibodies
are modifying the expression of active forms of these or other adhesion
molecules present on the PMN surface. Occupancy of the high-affinity
receptors by monomeric IgG could promote binding to Fc receptors
expressed on endothelium, but this seems improbable because the
introduction of monomeric IgG into the assay did not modify adherence
and our studies with radiolabeled IgG aggregates showed that Fc
receptors were not present on cultures of untreated or TNF-treated
endothelium (unpublished data). It is most likely that
CD64 has only a passive role in adhesion and that it is a marker for
adherent PMNs. Binding of normal blood PMNs to endothelial monolayers
was not impaired by anti-CD64 antibodies and increasing the percentage
of CD64+ cells in samples of normal blood PMNs activated by
IFN- did not enhance binding to endothelial cells. Further
investigations are warranted to establish whether the CD64 antigen is
contributing in some way to the adhesion of PMNs in the crisis phase of
sickle cell disease or whether it is simply a surrogate marker for a
subpopulation of PMNs that preferentially bind to endothelium.
The demonstration that PMNs from some patients in crisis did not
exhibit a supernormal binding for endothelial monolayers shows the
variable nature of PMN adhesiveness. Such heterogeneity in adhesion
could reflect the number and functional status of circulating PMNs
available for attachment to vessel walls, which, in turn, depends on
the stage and/or intensity of the painful crisis. Binding of
PMNs to endothelial cells is controlled by the expression of surface
adhesion molecules, which include the CD11/CD18 family and
L-selectin,13 and our antibody blocking studies showed that
CD18 was responsible for promoting the endothelial binding of nearly
one half of the PMNs derived from patients and control subjects. Flow
cytometric analysis showed that the adhesion molecules (CD11a, CD11b,
CD11c, CD18, CD15, and CD62L) were normally expressed on PMNs of sickle
cell patients in or out of crisis. Further investigations will clarify
whether changes in conformation or phosphorylation of these molecules
underlie the increased PMN adhesion in sickle cell crisis. The
RGD-containing peptide did not impede PMN interaction, possibly because
several endothelial adhesion molecules, including ICAM-1, do not
contain or recognize an RGD sequence.15
The highest levels of PMN adherence occurred when cells from some of
the patients in crisis were added to TNF-treated endothelium.
Inflammatory cytokines such as TNF are generated by stress, by
trauma, and at sites of infection and, because crises may be
precipitated by these events,10 a role for such cytokines
is implicated in the pathogenesis of vaso-occlusion. TNF
acts on endothelial cells to enhance the expression of ICAM-1, which is
recognized by members of the 2 leukocyte integrins. Our finding that
antibodies directed against the chain of this integrin family and
against ICAM-1 inhibited to a similar extent the binding of PMNs from
patients in crisis and controls to TNF-treated endothelium suggests
that adhesion molecules other than the 2 integrins are responsible
for the increased adhesiveness of sickle cell PMNs. Before margination,
PMNs roll along vessel walls by binding to E- and P-selectins on the
endothelial surface, and these vascular adhesion molecules are involved
in the initial tethering of the cells to endothelium rather than their
firm adhesion.13,38 Indirect support for this view comes
from the demonstration that, in the static adherence assay of the
current investigation, which measures strong leukocyte attachment,
antibodies against E-selectin did not inhibit PMN adhesion and that
incubation of endothelial cells with sera or PMNs from sickle cell
patients in crisis did not generate either E- or P-selectin expression.
However, it is conceivable that, under conditions of physiologic
stress, these selectins could preferentially promote the rolling of
sickle cell PMNs to inflammatory endothelium. Accordingly, experiments
are planned to examine this consideration by introducing PMNs from
sickle cell patients in crisis into a flow adhesion assay.
An enhanced adhesiveness of patient PMNs may relate to an increased
susceptibility to FMLP-induced aggregation during crisis16
and binding to blood vessel walls after experimentally induced vascular
occlusion.17 Neutrophils from sickle cell patients were
recently shown to exhibit an increased adherence to
fibronectin39; it was postulated that this effect would
promote adhesion to vascular endothelium or to the subendothelial
matrix. Activation of PMNs enhances endothelial
attachment40 and evidence of PMN activation in sickle cell
disease is provided by low Fc receptor III expression and high
intracellular levels of F-actin.41 The adherence of sickle
PMNs to serum-coated glass was reported to be reduced during the crisis
stage of the disease.42 Discrepancies between that study
and the present investigation could arise from differences in
experimental design or, alternatively, that during crisis PMNs are
predisposed to endothelial attachment and downregulate surface moieties
that govern interactions with serum components.
In conclusion, this study suggests that, in certain patients, PMNs may
contribute to the vaso-occlusive crises of sickle cell disease. This
could be envisaged in terms of aberrant PMN:endothelial interactions or
of aberrant PMN interactions operating via the cytokine
network.2 Unravelling the events governing pathophysiologic
interactions of PMNs with endothelial cells in this disease may yield a
more selective approach to the understanding and management of painful
crises.
| |
FOOTNOTES |
Submitted November 30, 1994;
accepted August 20, 1997.
Supported by The Medical Research Council (UK), The Dunhill Medical
Trust, and the Special Trustees of St Thomas' Hospital.
Address reprint requests to K. Alun Brown, PhD, Department
of Immunology, The Rayne Institute, St Thomas' Hospital, London SE1
7EH, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
REFERENCES |
1.
Davies SC,
Brozovic M:
The presentation, management and prophylaxis of sickle cell disease.
Blood Rev
3:29,
1989[Medline]
[Order article via Infotrieve]
2.
Platt OS,
Brambilla DJ,
Rosse WF,
Milner PF,
Castro O,
Steinberg MH,
Klug PP:
Mortality in sickle cell disease.
N Engl J Med
330:1639,
1994[Abstract/Free Full Text]
3.
Kaul DK,
Nagel RL:
Sickle cell vaso-occlusion: Many issues and some answers.
Experientia
49:5,
1993[Medline]
[Order article via Infotrieve]
4.
Hebbel R:
Beyond hemoglobin polymerization: The red blood cell membrane and sickle cell disease pathophysiology.
Blood
77:214,
1991[Free Full Text]
5.
Vordermeier S,
Singh S,
Biggerstaff J,
Harrison P,
Grech H,
Pearson TC,
Dumonde DC,
Brown KA:
Red blood cells from patients with sickle cell disease exhibit an increased adherence to cultured endothelium pretreated with tumour necrosis factor (TNF).
Br J Haematol
81:591,
1992[Medline]
[Order article via Infotrieve]
6.
Billet HH,
Kim K,
Fabry ME,
Nagel RL:
The percentage of dense red cells does not predict incidence of sickle cell painful crisis.
Blood
68:301,
1986[Abstract/Free Full Text]
7.
Ballas SK,
Larner J,
Smith ED,
Surrey S,
Schwartz E,
Rappaport E:
Rheologic predictors of the severity of the painful sickle cell crisis.
Blood
72:1216,
1988[Abstract/Free Full Text]
8.
Kaul DK,
Fabry ME,
Nagel RL:
Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: Pathophysiological implications.
Proc Natl Acad Sci USA
86:3356,
1989[Abstract/Free Full Text]
9.
Kaul DK,
Chen D,
Zhan J:
Adhesion of sickle cells to vascular endothelium is critically dependent on changes in density and shape of the cells.
Blood
83:3006,
1994[Abstract/Free Full Text]
10.
Kaul DK,
Fabry ME,
Nagel RL:
The pathophysiology of vascular obstruction in the sickle syndromes.
Blood Rev
10:29,
1996[Medline]
[Order article via Infotrieve]
11. Serjeant GR: Sickle Cell Disease. New York, NY, Oxford, 1985, p
296
12.
Platt OS:
Easing the suffering caused by sickle cell disease.
N Engl J Med
30:783,
1994
13.
Bevilacqua MP:
Endothelial-leukocyte adhesion molecules.
Annu Rev Immunol
11:767,
1993[Medline]
[Order article via Infotrieve]
14.
Cronstein BN,
Weissmann G:
The adhesion molecules of inflammation.
Arthritis Rheum
36:147,
1993[Medline]
[Order article via Infotrieve]
15.
Ruoslahti E,
Pierschbacker MD:
New perspectives in cell adhesion: RGD and integrins.
Science
238:491,
1987[Abstract/Free Full Text]
16.
Lipowsky MG,
Sheikh NA,
Katz DM:
Intravital microscopy of capillary haemodynamics in sickle cell disease.
J Clin Invest
80:117,
1987
17.
Lachant NA,
Oseas RS:
Case report: Vaso occlusive crisis-associated neutrophil dysfunction in patients with sickle cell disease.
Am J Med Sci
294:253,
1987[Medline]
[Order article via Infotrieve]
18.
Buckle AM,
Hogg N:
The effect of IFN and colony-stimulating factors on the expression of neutrophil cell membrane receptors.
J Immunol
143:2295,
1989[Abstract]
19.
Akerley WL,
Guyre PM,
Davis BH:
Neutrophil activation through high-affinity Fc receptor using a monomeric antibody with unique properties.
Blood
77:607,
1991[Abstract/Free Full Text]
20.
Guyre PM,
Campbell AS,
Kniffin WD,
Fanger MW:
Monocytes and polymorphonuclear neutrophils of patients with streptococcal pharyngitis express increased numbers of type I IgG Fc receptors.
J Clin Invest
86:1892,
1990
21.
LeRoy F,
Brown KA,
Greaves MW,
Vora AJ,
Slavin B,
Robinson M,
Ellis BA,
Dowd PM,
Dumonde DC:
Blood mononuclear cells from patients with psoriasis exhibit an enhanced adherence to cultured vascular endothelium.
J Invest Dermatol
97:511,
1991[Medline]
[Order article via Infotrieve]
22.
Brown KA,
Vora A,
Biggerstaff J,
Edgell C-JS,
Oikle S,
Mazure G,
Taub N,
Meager A,
Hill T,
Watson C,
Dumonde DC:
Application of an immortalized human endothelial cell line to the leucocyte:endothelial adherence assay.
J Immunol Methods
163:13,
1993[Medline]
[Order article via Infotrieve]
23.
Hamblin A,
Taylor M,
Bernhagen J,
Shakoor Z,
Mayall S,
Noble G,
McCarthy D:
A method of preparing blood leucocytes for flow cytometry which prevents upregulation of leucocyte integrins.
J Immunol Methods
146:219,
1992[Medline]
[Order article via Infotrieve]
24.
Cardavelli PM,
Cobb RR,
Nowlin DM,
Scholz W,
Gorcsan F,
Moscinski M,
Yashura M,
Chiang S-L,
Lobl TJ:
Cyclic RGD peptide inhibits 41 interacting with connecting segment 1 and vascular cell adhesion molecule.
J Biol Chem
169:18668,
1994
25.
Mire-Sluis AR,
Page L,
Thorpe R:
Quantitative cell line based bioassay for human cytokines. Review article.
J Immunol Methods
187:191,
1995[Medline]
[Order article via Infotrieve]
26.
Westgard JO,
Groth T,
Avonsson T,
Falk H,
de Verdier C-H:
Performance characteristics of roles for internal quality control: Problems of false rejection and error detection.
Clin Chem
23:1857,
1977[Abstract/Free Full Text]
27.
Wald N,
Stone R,
Cuckle HS,
Grudzinskas JG,
Barkai G,
Brambati B,
Teisner B,
Fuhrmann W:
First trimester concentrations of pregnancy associated plasma protein A and placental protein 14 in Down's syndrome.
Br Med J
305:28,
1992
28.
Watson C,
Whittaker S,
Smith N,
Vora AJ,
Dumonde DC,
Brown KA:
IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes.
Clin Exp Immunol
105:112,
1996[Medline]
[Order article via Infotrieve]
29.
Ruiz P,
Gomez F,
Lopez R,
Chien P,
Rossman MD,
Schreiber AD:
Granulocyte Fc receptor recognition of cell bound and aggregated IgG: Effect of -interferon.
Am J Hematol
39:257,
1992[Medline]
[Order article via Infotrieve]
30.
Moxey-Mimms MM,
Frank MM,
Lin EY,
Francis C,
Gaither TA:
Increased expression of FcRI on isolated PMN from individuals of African descent.
Clin Immunol Immunopathol
69:117,
1993[Medline]
[Order article via Infotrieve]
31.
Repp R,
Valerius TH,
Sendler A,
Gramatzki M,
Iro H,
Kalden R,
Platzer E:
Neutrophils express the high affinity receptor for IgG (Fc RI, CD64) after in vivo application of recombinant human granulocyte colony-stimulating factor.
Blood
78:885,
1991[Abstract/Free Full Text]
32.
Kerst JM,
de Haas M,
van der Schoot CE,
Slaper-Cortenbach ICM,
Kleijer M,
Von dem Borne AEGK,
Van Oers RHJ:
Recombinant granulocyte colony-stimulating factor administration to healthy volunteers: Induction of immunophenotypically and functionally altered neutrophils via an effect on myeloid progenitor cells.
Blood
82:3265,
1993[Abstract/Free Full Text]
33.
Davis BH,
Bigelow NW,
Curnutte JT,
Ornvold K:
Neutrophil CD64 expression: Potential diagnostic indicator of acute inflammation and therapeutic monitor of interferon- therapy.
Lab Haematol
1:3,
1995
34.
Kawakami M,
Tsutsumi H,
Kumakawa T,
Abe H,
Hatai M,
Kurosawa S,
Mori M,
Fukushima M:
Levels of serum granulocyte colony-stimulating factor in patients with infections.
Blood
76:1962,
1990[Abstract/Free Full Text]
35. (suppl)
Baguley E,
Brown KA,
Haskard D,
Harris EN,
Hughes GRV:
Anti-endothelial cell antibodies in the connective tissue diseases.
Br J Rheumatol
26:95,
1987
36. Dias-Da-Motta PM, Arruda VR, Muscara MN, Saad ST, De Nucci G,
Costa FF, Condino-Neto A: The release of nitric oxide and
superoxide anion by neutrophils and mononuclear cells from patients
with sickle cell anaemia. Br J Haematol 93:333, 1996
37.
Guyre PM,
Graziano RF,
Vance BA,
Morganelli PM,
Fanger W:
Monoclonal antibodies that bind to distinct epitopes on FcR1 are able to trigger receptor function.
J Immunol
143:1650,
1989[Abstract]
38.
Springer TA,
Lasky LA:
Sticky sugars for selectins.
Nature
349:196,
1993
39.
Kasschau MR,
Barabino GA,
Bridges KR,
Golan DE:
Adhesion of sickle neutrophils and erythrocytes to fibronectin.
Blood
87:771,
1996[Abstract/Free Full Text]
40.
Webster RO,
Wysolmerski RB,
Lagunoff D:
Enhancement of human polymorphonuclear leukocyte adherence to plastic and endothelium by phorbol myristate acetate.
Am J Physiol
125:369,
1986
41. (abstr, suppl 1)
Coates TD,
Fisher TC,
Pecsvarady Z,
Fabok A,
Deemer K,
Hiti AL,
Johnson CS,
Meiselman HJ:
Neutrophil deformability and FcRIII expression correlate with clinical severity (b globin haplotype) in sickle cell disease.
Blood
80:109a,
1992
42.
Boghossian SH,
Nash G,
Dormandy J,
Bevan DH:
Abnormal neutrophil adhesion in sickle cell anaemia and crisis: Relationship to blood rheology.
Br J Haematol
78:437,
1991[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract