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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3423-3429
Raised Neutrophil Phospholipase A2 Activity and Defective
Priming of NADPH Oxidase and Phospholipase A2 in Sickle
Cell Disease
By
Elahe Mollapour,
John B. Porter,
Richard Kaczmarski,
David C. Linch, and
Pamela J. Roberts
From the Departments of Haematology, University College London
Medical School, London; and the North Middlesex Hospital, London,
UK.
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ABSTRACT |
Intermittent painful crises due to vasoocclusion are the major
clinical manifestation of sickle cell disease (SCD), but subclinical episodes may also occur. There is sparse evidence for the involvement of neutrophils in the pathophysiology of SCD, but production of cytokines by the damaged endothelium might influence neutrophil function and modulate responses to subsequent cytokine exposure. In
addition, the activation of neutrophils in the microcirculation could
itself exacerbate vasoocclusion. To test whether neutrophil inflammatory responses were altered in SCD, neutrophil phospholipase A2 and NADPH oxidase activity in response to in vitro
priming by granulocyte-macrophage colony-stimulating factor (GM-CSF)
and tumor necrosis factor- (TNF-) were measured both during and between painful crises. Resting levels of neutrophil phospholipase A2 activity in steady-state SCD (4.0% ± 0.5% of total
cell radioactivity) were raised relative to control values
(2.0% ± 0.2%, n = 10, P = .008). There was no
defect of agonist-stimulated phospholipase A2 or NADPH
oxidase activity in steady-state SCD; however, the ability of
phospholipase A2 to respond to priming with GM-CSF was
attenuated to 63% ± 17% of control values (n = 10,
P = .04). Similarly, neutrophil NADPH oxidase activity
after priming with GM-CSF and TNF- was, respectively, 65% ± 11%
(n = 7, P = .03) and 57% ± 7% of control (n = 10, P = .007) in steady-state disease, and was
further reduced during painful vasoocclusive crises to 34% ± 9% and
25% ± 3% of control for GM-CSF and TNF-, respectively. These
data were not explained by poor splenic function or any racial factor,
as normal cytokine responses were seen in splenectomized patients in
remission from Hodgkin's disease and in healthy Afro-Caribbean
subjects. Abnormal neutrophil cytokine priming responses were not
observed in either patients with rheumatoid arthritis or
iron-deficiency anemia. Our findings are indicative of an ongoing
inflammatory state in SCD between painful crises involving neutrophil
activation and an abnormality of cytokine-regulated neutrophil
function, which may compromise the host defenses against certain
microorganisms.
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INTRODUCTION |
SICKLE CELL DISEASE (SCD) is caused by a
point mutation in the  chain of hemoglobin resulting in
polymerization of hemoglobin S at low oxygen tensions. This leads to
spicule formation, red blood cell (RBC) rigidity, and distortion with
consequent vasoocclusive episodes and hemolytic anaemia. Oxidative
damage occurs to the proteins and lipids of the RBC membrane, with loss
of bilayer asymmetry. This disorganization exposes lipids and protein
adhesion molecules, and induces the binding of plasma adhesion
molecules, thus increasing the tendency for sickled RBC to adhere to
endothelium and platelets. Adherence of sickle RBC stimulates
endothelial cells to upregulate their adhesion molecules, which
accelerates the adhesion cascade.1 Activation of the
endothelial cells results in increased metabolism of endothelial cell
phospholipids, with release of eicosanoids and downstream
proinflammatory products of both lipoxygenase and cyclooxygenase
enzymes.2 Activated endothelium also releases a wide range
of cytokines, including granulocyte-macrophage colony-stimulating
factor (GM-CSF), interleukin (IL)-1, IL-3, IL-6, and tumor necrosis
factor (TNF), and these have been detected in the plasma of patients
with SCD.3,4 Sickled RBC also activate platelets, which
adhere and release their granule contents, initiating the thrombotic
cascade.
Neutrophils may also become activated during this cascade of
vasoocclusive events and neutrophil adherence may contribute to
vasoocclusion,4 as well as endothelial cell
damage.5 Activation of endothelial cells and platelets by
sickle RBC may affect neutrophil function, and a recent in vitro study
demonstrated that neutrophils can be directly activated by sickle
RBC.6 Patients with SCD have an increased tendency to
infection,7 especially with encapsulated organisms, which
is due in part to the poor splenic function,8 but might
also be a feature of altered neutrophil function.9
Exposure of neutrophils to cytokines in vitro greatly increases the
capacity of the phagocyte NADPH oxidase and phospholipase A2 to respond to activation via cell-surface receptors for
chemotactic peptide10,11 or IgG12 a process
referred to as "priming." Priming of neutrophils by GM-CSF and
G-CSF has been demonstrated in vivo.13,14 To participate in
the host-defense response, neutrophils must adhere to the vascular endothelium in an infective focus, transmigrate into the tissues, be
primed by inflammatory cytokines, and then partake in the processes of
phagocytosis and killing of microorganisms. This is largely dependent
on the activation of the NADPH-dependent oxidase in a respiratory
burst, which generates oxygen metabolites and free radicals. Activation
of primed neutrophils also results in the stimulation of phospholipase
A2, with the generation of a range of arachidonic acid
derivatives, including the leukotrienes, which have potent chemotactic
properties and recruit further phagocytes to the inflammatory site. The
priming events induced by the inflammatory cytokines are central to
these processes. The aim of this study was to look for neutrophil
activation and altered neutrophil inflammatory responses in SCD. We
therefore examined the activation state of the neutrophil respiratory
burst and phospholipase A2 both in steady-state SCD and
during painful vasoocclusive crises, and studied the effects of
cytokine-mediated priming on these enzymes.
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MATERIALS AND METHODS |
Patients and Controls
Twenty-three patients from University College London Hospital and the
North Middlesex Hospital who were homozygous for the sickle gene (HbSS)
were studied: (a) 17 patients (age 17 to 50 years, 12 males) in
steady-state between painful vasoocclusive crises were tested on 20 occasions (10 for NADPH oxidase and 10 for phospholipase
A2). The mean interval since the previous crisis that
required hospital admission was 40 ± 10 weeks (range, 3 to 132). All
of these patients were asymptomatic at the time of study and were
receiving prophylactic antibiotic therapy (penicillin V 250 mg twice
daily) and folic acid 5 mg daily. None were receiving repeated blood
transfusions, opiate analgesics, or hydroxyurea; (b) six hospitalized
HbSS patients (age 17 to 31 years, three males) during vasoocclusive
crisis treated by prophylactic antibiotics and analgesia. Two of these
patients had received a blood transfusion within 6 days of being tested
and their levels of HbS were 47% and 24%, respectively, but their
neutrophil functional responses were not different from those of the
nontransfused patients in crisis. Non-SCD control groups were: (a)
three splenectomized patients in long-term remission from Hodgkin's
disease (age 35 to 50 years, two males); (b) six healthy
Afro-Caribbeans (age 25 to 36 years, two males); (c) six female
patients with rheumatoid arthritis (age 41 to 62 years) who were
receiving medication (nonsteroidal antiinflammatory drugs [dichlofenac
sodium, salazopyrine, or meloxicam, n = 4], gold injections
[n = 1], hydroxychloroquine [n = 1]); (d) six female patients
with iron-deficiency anemia (age 27 to 60 years); and (e) 41 healthy
laboratory personnel (age 24 to 62 years, 14 males, tested on 48 occasions), one of whom was tested each time that blood from the above
categories was tested (same-day controls). In all cases, peripheral
blood was obtained with the informed consent of the donor.
Neutrophil Function Tests
For assays of phospholipase A2, venous blood was taken into
2 mmol/L EDTA and neutrophils were purified by centrifugation (1,400g for 30 minutes at room temperature) through a
discontinuous gradient of Histopaque (Sigma Chemical, Poole, Dorset,
UK) (densities, 1.077 and 1.119 g/L). The neutrophil suspension was
washed twice in Dulbecco's phosphate-buffered saline (PBS) (without
calcium and magnesium; GIBCO-BRL, Paisley, UK) by centrifugation
(170g for 7 minutes at room temperature). Purified cells were
resuspended to 2 × 106/mL PBS supplemented with 0.9 mmol/L calcium, 0.5 mmol/L magnesium, and 5 mmol/L
D-glucose (PBSG). The cellular composition of the final
suspension was 92% ± 1% neutrophils, 2.0% ± 0.2% monocytes, 2.7% ± 0.3% lymphocytes, and 3.2% ± 0.4%
erythrocytes, as assessed by Leishman's stain. The purity of patients
and control neutrophil suspensions were similar. Neutrophils
(1 × 106 in 0.5-mL samples) were incubated for 15 minutes with either 1 µmol/L calcium ionophore, A23187 (Sigma), or
PBSG diluent, and phospholipase A2 activity was measured by
the release of tritiated arachidonic acid from radiolabeled
intracellular phospholipid stores, as previously
described.10 For priming experiments, neutrophils were
preincubated with either recombinant human (rh)GM-CSF (a kind gift of
Behringwerke, Marburg, Germany) at the concentrations indicated in the
text or fetal calf serum (FCS; GIBCO-BRL; final concentration, 0.01%
vol/vol) for 20 minutes at 37°C before stimulation with ionophore.
For H2O2 assays, venous blood was
anticoagulated with 10 IU preservative-free heparin/mL (Monoparin; CP
Pharmaceuticals, Wrexham, Clwyd, UK). Care was taken to ensure that the
patients and their same-day controls were venesected within 30 minutes
of each other, as the capacity of neutrophils to produce
H2O2 diminished if blood was stored for more
than 90 minutes at room temperature. Aliquots of blood were incubated
at 37°C for 15 minutes with 100 µmol/L dichlorodihydrofluorescein
diacetate (DCF; Molecular Probes, Eugene, OR) and then for 30 minutes
with either rhGM-CSF or rhTNF (at the doses stated in the text), or
FCS diluent (final concentration, 0.01% vol/vol). Stimulation of NADPH
oxidase was with either 1 µmol/L
formyl-methionyl-leucyl-phenylalanine (FMLP; Sigma) or 20 µmol/L
phorbol-myristate-acetate (PMA; Sigma) for 15 minutes. Intracellular
H2O2 production was measured in a whole-blood
flow-cytometric assay of the oxidation of DCF, as previously
described.13 H2O2 production by
resting cells was determined from the mean cell fluorescence (MCF) of
the ungated neutrophil population measured on a linear scale. From this
distribution, a "positive" gate was set to include 5% of the
brightest cells in the control population. H2O2
production stimulated by FMLP and PMA was determined as the product of
the percentage and MCF of cells entering the preset positive gate. In
the cytokine-priming experiments, these MCF values were normalized by
expressing the MCF of cytokine-treated cells as a percentage of the MCF
of the diluent control. An estimate of the total
H2O2 production in a given sample was made by
multiplying the percentage of positive cells by the normalized MCF, and
the data expressed in arbitrary fluorescence units.
Data Analysis
Unless otherwise stated, the data are the mean ± 1 SE of the number
of experiments given in the text. Statistical analysis of the data was
performed by Wilcoxon's matched-pairs signed-ranks test.15
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RESULTS |
Phospholipase A2
Resting levels of neutrophil phospholipase A2 activity, as
measured by the release of arachidonate from radiolabeled phospholipid stores, were twofold greater in patients with steady-state SCD than
same-day controls (Fig 1A). The mean
resting rate of arachidonate release in neutrophils from steady-state
patients was 4.0% ± 0.5% of total cell radioactivity, and in
controls was 2.0% ± 0.2% (n = 10, P = .008). Resting
levels of phospholipase A2 activity did not alter when
neutrophils were incubated with either 1 or 10 ng/mL of rhGM-CSF and
the differential between patient and control values remained
significantly different (Fig 1A).
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| Fig 1.
Neutrophil phospholipase A2 activity in
patients with SCD is shown. Purified neutrophils labeled with tritiated
arachidonate were incubated with either rhGM-CSF or FCS for 20 minutes
at 37°C, followed by stimulation for 15 minutes with either (A) PBS
or (B) 1 µmol/L calcium ionophore, A23187. Phospholipase
A2 activity was determined from the release of arachidonate
into the supernatant as described in Materials and Methods, and
expressed as the percentage of total cellular radioactivity. The data
shown are the mean ±1 SE for 10 patients, compared with controls
tested on the same day. The absolute values for the resting levels of
arachidonate release shown in A are 3,204 ± 116 cpm/106
neutrophils and 1,404 ± 211 cpm/106 neutrophils for SCD
patients and their same-day controls, respectively. (B) Increments in
arachidonate release above resting level after stimulation with calcium
ionophore. Significant differences between patients and controls
(Wilcoxon's matched pairs signed-ranks test) are shown: *P < .05,  P < .01. ( ), Patient; ( ), control.
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Stimulation of neutrophils with calcium ionophore (A23187) resulted in
the activation of phospholipase A2 above resting levels (Fig 1B). The increment due to calcium-dependent stimulation was determined by subtracting the background values for control samples stimulated with PBSG alone (given in the legend to Fig 1).
A23187-stimulated arachidonate release was not significantly different
in the steady-state patients compared with their respective same-day
controls (SCD patients, 2.6% ± 0.5% of total cell radioactivity;
controls, 3.9% ± 0.8%; P = .06). Priming of neutrophils
with rhGM-CSF (1 and 10 ng/mL) for 20 minutes before stimulation with
A23187 resulted in a dose-dependent increase in phospholipase
A2 activity (Fig 1B), which was significantly smaller in
neutrophils from patients with steady-state SCD than their respective
controls (Fig 1B). The primed phospholipase A2 responses of
SCD patients was only 63% ± 17% of control values (P < .05, n = 10) when 10 ng/mL GM-CSF was used.
NADPH Oxidase
Activation of the neutrophil NADPH-dependent oxidase was measured by a
fluorescent assay of H2O2 production. There was
no measurable difference in the resting rate of neutrophil
H2O2 production between SCD patients in
steady-state (MCF of the ungated neutrophil population, 59 ± 10;
n = 9) and their respective same-day controls (MCF, 53 ± 8;
n = 9; P > .05). Six patients in painful crisis of SCD
were also studied, and again the resting values for neutrophil H2O2 production were not different from control
(MCF for patients in crisis, 62 ± 6; same-day controls, 60 ± 8;
n = 6, P > .05).
Stimulation of neutrophil NADPH oxidase with the chemotactic peptide,
FMLP, increased H2O2 production to between two
and four times the resting level (Table 1).The mean FMLP responses in both steady-state and crisis SCD patients
were greater than control, but the values were not significantly
different (in all cases, P > .05). PMA was a more potent
agonist than FMLP and stimulated a 30-fold to 80-fold increase in
H2O2 production above resting level (Table 1).
The PMA responses of neutrophils from patients in the steady-state were
the same magnitude as control, but the PMA responses of neutrophils
from patients in crisis were on average only 89% of their same-day
controls. This small difference was statistically significant (n = 6,
P = .014), but unlikely to be of major physiologic
importance. Preincubation with varying concentrations of GM-CSF (0 to
10 ng/mL) and TNF- (0 to 500 U/mL) before stimulation with FMLP
caused a dose-dependent enhancement of neutrophil FMLP-stimulated H2O2 responses in both patient and control
samples (Fig 2). Neutrophils from SCD
patients in steady-state produced significantly less H2O2 after priming with either cytokine than
their same-day controls (Fig 2A and B). At the maximal doses of GM-CSF
and TNF- used, H2O2 production was 65% ± 11% (n = 7, P = .03) and 57% ± 7% (n = 10, P = .007) of same-day controls, respectively. Similar
experiments with SCD patients in crisis (Fig 2C and D) showed a more
marked defect in neutrophil H2O2 production
after priming with cytokines. At maximal doses of GM-CSF and TNF-,
H2O2 production was 34% ± 9% (n = 6,
P = .03) and 25% ± 3% (n = 6, P = .03) of
same-day controls, respectively.
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| Fig 2.
Cytokine-mediated priming of neutrophil NADPH oxidase
activity in patients in steady-state (A and B) or crisis (C and D) of SCD compared with controls tested on the same day. Whole-blood samples
were incubated with either rhTNF- (A and C), rhGM-CSF (B and D), at
the doses indicated, or FCS diluent for 30 minutes at 37°C, followed
by stimulation for 15 minutes with 1 µmol/L FMLP or PBS. Neutrophil
H2O2 production was measured by the oxidation of dichlorodihydrofluorescein (DCF) using flow cytometry, as described in Materials and Methods. Total H2O2 production
was calculated as the product of the percent of positive cells and
their standardized MCF. The data shown are the mean ± 1 SE of (A) 10 experiments, (B) 7 experiments, and (C and D) six experiments.
Significant differences between patients and controls (Wilcoxon's
matched pairs signed-ranks test) are shown: *P < .01, P < .005. (- -), Patient + FMLP; (- -), control + FMLP; (- -), patient + PBS; (--), control + PBS.
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Representative fluorescence distributions of neutrophils from patients
and controls obtained from the fluorescence activated cell sorter are
shown in Fig 3. In confirmation of previous
studies of cytokine-mediated priming of the respiratory burst measured in whole blood,13,14 the increase in FMLP-stimulated
fluorescence after TNF and GM-CSF priming was due to an increase in
both the percentage of positive cells and their MCF. Table
2 shows that in patients with SCD, there
were fewer cells recruited into the positive population and the MCF of
these positive cells was also lower than that of controls.
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| Fig 3.
Representative flow-cytometric histograms show the
effects of priming on the FMLP-stimulated neutrophil respiratory burst. Shown are data from a healthy control (histograms A, B, and C), a SCD
patient in steady-state (histograms D, E, and F), and a SCD patient in
crisis (histograms G, H, and I). Whole-blood samples were incubated
with either growth-factor diluent (histograms A, D, and G), 100 U/mL
TNF (histograms B, E, and H), or 10 ng/mL GM-CSF (histograms C, F,
and I) for 30 minutes, followed by stimulation for 15 minutes with 1 µmol/L FMLP. Neutrophil H2O2 production was
measured by the oxidation of DCF as described in Materials and Methods.
Shown for comparison in histograms A, D, and G are the overlaid
distributions of nonprimed samples stimulated with and without FMLP. In
histograms A and G, the distributions ± FMLP were identical; in D,
the stimulated distribution is marked with an arrow.
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Table 2.
Effect of Cytokine-Mediated Priming of FMLP-Stimulated
H2O2 Production in Patients With SCD
Compared With Healthy Controls
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Studies With Control Groups
Asplenic controls.
Adult patients with SCD have poor or absent splenic function. To
investigate whether this accounted for the defect seen after priming,
neutrophil H2O2 production and cytokine priming
were measured in three patients in long-term remission from Hodgkin's disease who had been splenectomized many years previously. No defect in
cytokine-mediated priming was observed compared with nonsplenectomized
controls tested on the same day. The total H2O2 production for patients expressed as percentage of the value for the
same-day control was 104% ± 8% and 90% ± 5% (n = 3,
P > .05), respectively, for samples primed with 1 and 10 ng/mL GM-CSF; and 101% ± 19% and 103% ± 17% (n = 3,
P > .05), respectively, for samples primed with 50 and 500 U/mL TNF-.
Ethnic controls.
All but two of the SCD patients studied were of African or
Afro-Caribbean descent, whereas the same-day controls were
non-Afro-Caribbeans. To examine whether any racial factor may have
contributed to the results of the priming experiments, blood from six
healthy non-SCD Afro-Caribbeans was studied in parallel with samples
from healthy non-Afro-Caribbeans, and no significant differences were
seen between the values for these two groups in their priming response to either GM-CSF or TNF-. The total H2O2
production for Afro-Caribbeans expressed as the percentage of the value
for non-Afro-Caribbean controls was 105% ± 27% and 100% ± 24%
(n = 6, P > .05), respectively, for samples primed with 1 and 10 ng/mL GM-CSF; and 104% ± 17% and 108% ± 23% (n = 6,
P > .05), respectively, for samples primed with 50 and 100 U/mL TNF-.
Patients with arthritis.
Six patients with seropositive erosive rheumatoid arthritis, classified
according to the American Rheumatism Association criteria, were studied
to determine whether the defect in cytokine-mediated priming could be
observed in patients with ongoing inflammation. They were receiving a
variety of medications (see Methods and Materials). There was no
difference in the resting rate of neutrophil H2O2 production or unprimed neutrophil
responses to FMLP between patients and sex-matched controls (data not
shown) and, as shown in Fig 4A and B, there
was no significant difference in neutrophil H2O2 production after priming with either
GM-CSF or TNF-, between the patient and control groups
(P > .05, n = 6).
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| Fig 4.
Cytokine-mediated priming of neutrophil NADPH oxidase
activity in patients with acute arthritis (A and B) and iron-deficiency anemia (C and D). Treatment with cytokines and measurement of neutrophil H2O2 production were as described in
the legend to Fig 2. Data shown are the mean ± 1 SE of six
experiments with each patient category. ( ) Patient + PBS; (),
control + PBS; (), patient + FMLP; (), control + FMLP.
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Anemic controls.
To investigate whether the priming defect was associated with the
anemia of SCD, six females with iron-deficiency anemia were studied.
The mean hemoglobin value for these patients was 76 ± 5 g/L
(mean ± 1 SE), compared with 81 ± 13 g/L (n = 8) for patients with steady-state SCD and 90 ± 16 g/L (n = 6) for SCD patients in
crisis. Iron-deficient patients were selected who were not infected, or
recovering from surgery, or suffering from any form of malignancy, as
this might affect their neutrophil responses. As shown in Fig 4C and D,
there was no significant difference in FMLP-stimulated neutrophil
H2O2 production between patients with
iron-deficiency anemia and their sex-matched controls either with or
without prior priming by TNF- or GM-CSF (in all analyses, P > .05, n = 6).
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DISCUSSION |
Activation of neutrophils as evidenced by enhanced neutrophil
aggregation was previously noted during episodes of vasoocclusive crisis in SCD,16 but there is little published evidence of
neutrophil activation during the steady-state. If there is ongoing
interaction between damaged endothelium and activated neutrophils in
the steady-state, it is conceivable that this may contribute to the
initiation of vasoocclusive crises. Agents that decrease such
interactions might therefore be of value to reduce the frequency of
painful crises. This study shows that neutrophil phospholipase
A2 activity in steady-state SCD is increased approximately
twofold compared with control, demonstrating that neutrophils are
activated in steady-state disease. The mechanism of this activation is
unclear at present. A recent study showed that the neutrophil NADPH
oxidase could be activated by contact with sickled RBC in
vitro,6 and neutrophil phospholipase A2 may
also be activated in this way. However, we found no evidence for
parallel basal activation of NADPH oxidase activity of neutrophils in
SCD when tested in the whole-blood mileau. Release of phospholipase
A2 from activated neutrophils might contribute to the
threefold increase above control in the plasma levels of the secreted
isoform of phospholipase A2 that were recently detected in
patients with steady-state SCD.17
Our study also demonstrates that neutrophils from patients in the
steady-state of SCD have reduced NADPH oxidase and phospholipase A2 responses to the agonists FMLP and calcium ionophore
after in vitro priming with the cytokines GM-CSF and TNF-. The
amounts of H2O2 and arachidonate produced were
approximately 50% of that produced by control cells and, in addition,
the defect in oxidase activity was more severe in patients in crisis.
One consequence of suboptimal oxygen radical production after priming
would be the failure to kill microorganisms that require high
concentrations of H2O2 for their destruction.
Such organisms are likely to be those that have developed protective
mechanisms against the phagocyte respiratory burst, such as
encapsulation, catalase production, or oxidase
inhibition.18 Thus, the neutrophil defect could potentially exacerbate the infective risks with Streptococcus pneumoniae
and Haemophilus influenzae that accompany the hyposplenic state
in sickle cell syndromes.8
Priming of the NADPH oxidase by GM-CSF and endotoxin has been
attributed to increased production of arachidonate10,19;
thus, the inability of SCD neutrophils to fully upregulate NADPH oxidase activity could be a sequela of the suboptimal activation of
phospholipase A2 by agonist after cytokine-mediated
priming. Unprimed neutrophils from steady-state SCD patients produced
equivalent amounts of H2O2 as control cells in
response to stimulation with both FMLP and the more potent stimulus,
PMA (elicits maximal production of H2O2 in
control cells), suggesting that the defect in priming was not explained
by an intrinsic problem with the NADPH oxidase itself. This is in
accord with previous studies demonstrating a normal respiratory burst
in unprimed phagocytes from patients with SCD.20,21 Assays
of phospholipase A2 used washed and purified neutrophils,
unlike the NADPH oxidase assay, which was performed in whole blood,
demonstrating that the reduced responses to cytokine-mediated priming
were an inherent cellular defect.
Spicule formation in sickled RBC with concomitant destabilization of
membrane organization promotes adherence of RBC to endothelium resulting in stimulation of endothelial cells. These cells when activated can secrete a wide range of biologic response modifiers, which may contribute to the increased levels of GM-CSF, IL-1, IL-3,
IL-6, and TNF- that have been detected in the peripheral blood of
some SCD patients.3,4,22,23 This raises the possibility that the defective in vitro cytokine-mediated priming of neutrophils might be due to prior exposure to cytokines in the circulation. This
would leave the cells refactory to further priming, either because of
downregulation of cytokine receptors or postreceptor signal
transduction mechanisms. Previous work has shown that neutrophils primed in vivo by infusion of GM-CSF are not responsive to subsequent in vitro priming with GM-CSF, and after a period of 2 hours have a
priming capacity that is reduced to approximately 50% of preinfusion values,13,14 confirming that this is a possible mechanism.
Surprisingly, there was little evidence of recent or sustained in vivo
priming of neutrophils as markedly elevated responses to agonist were
not observed in either the NADPH oxidase or PLA2 assays
when cells were stimulated in the absence of in vitro cytokines. This
might have been expected if cells had been recently primed in vivo, as
demonstrated by our previous studies,13,14 but there are
two explanations why evidence of recent priming was not observed.
First, activation of neutrophils by interaction with damaged
endothelium may only be transient. For example, the primed state of the
neutrophil NADPH oxidase can be short-lived when induced by
platelet-activating factor (1 to 2 hours)24 or by cytokines
such as IL-8 (30 minutes) and TNF- (1 hour), whereas the response to
GM-CSF can last for several hours.25 It is possible that
there had been in vivo priming with such a transient priming agent and
we were testing the cells during a postpriming refractory period.
Second, inflammatory events in SCD may produce a range of activated
states in the neutrophil. Fully activated phagocytes are lost from the
circulation, as evidenced by a reduction in levels of circulating
neutrophils after in vivo infusions of cytokines26 due to
margination or migration into the tissues.27 In our study,
neutrophils primed in vivo may similarly have left the circulating
pool, leaving the less activated cells to be collected from the
peripheral blood for in vitro assays.
In an attempt to further understand the mechanism of altered neutrophil
function in SCD, we studied four groups of people who did not have SCD.
Racial differences in neutrophil function and poor splenic function
were excluded as mechanisms, as no defects in neutrophil
H2O2 production were seen in either healthy
Afro-Caribbean subjects or splenectomized patients when tested against
control subjects. The effects we observed in SCD were also not
reproduced in a group of patients with active rheumatoid arthritis. Our
study appears to be the first to test neutrophil function in whole
blood from patients with arthritis, whereas previous studies using
purified cells or diluted whole blood have produced contradictory
results, some showing evidence of activated28-30 or
defective31,32 neutrophils in the circulation with reduced
responses after cytokine-mediated priming,29,31 with others
showing no difference from control.33,34 Our findings with
rheumatoid arthritis patients emphasize that the defective priming seen
in SCD is likely to be specifically associated with events in the
circulation, rather than being a general feature of inflammatory
disorders. Finally, we investigated whether the priming defect could be
attributed to the marked anemia of the patients with SCD, who had
hemoglobin levels of 80 to 90 g/L. However, we could find no evidence
for reduced H2O2 production or
cytokine-mediated priming in a series of patients with iron-deficiency anemia whose mean hemoglobin level was 76 ± 5 g/L (n = 6).
In conclusion, we have shown definitive evidence of neutrophil
activation with increased phospholipase A2 activity in
steady-state SCD, which may contribute to the triggering of
vasoocclusive crises. This observation may provide a rationale for
beneficial therapeutic intervention with antiinflammatory drugs. In
addition, we demonstrated that neutrophils in the peripheral blood of
SCD patients have a limited capacity to respond to priming with
cytokines, and this may contribute to the susceptibility of these
patients to infection.
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FOOTNOTES |
Submitted March 13, 1997;
accepted December 15, 1997.
Supported by the Kay Kendall Leukaemia Research Fund (P.J.R.).
Address reprint requests to Pamela J. Roberts, PhD, Department of
Haematology, University College London Medical School, 98 Chenies Mews,
London WC1E6HX, UK.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We thank Dr Anne Yardumian, Department of Haematology, The North
Middlesex Hospital, London, UK, and Prof David Isenberg, Department of
Rheumatology, The Middlesex Hospital, London, UK, for allowing us to
study patients in their care.
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REFERENCES |
1.
Natarajan M,
Udden MM,
McIntire LV:
Adhesion of sickle red blood cells and damage to interleukin-1 stimulated endothelial cells under flow in vitro.
Blood
87:4845,
1996[Abstract/Free Full Text]
2.
Setty B,
Chen D,
Stuart MJ:
Sickle red blood cells stimulate endothelial cell production of eicosanoids and diacylglycerol.
J Lab Clin Med
128:313,
1996[Medline]
[Order article via Infotrieve]
3.
Croziat H:
Circulating cytokines in sickle cell patients during steady state.
Br J Haematol
87:592,
1994[Medline]
[Order article via Infotrieve]
4.
Kasschau MR,
Barabino A,
Bridges KR,
Golan DE:
Adhesion of sickle neutrophils and erythrocytes to fibronectin.
Blood
87:771,
1996[Abstract/Free Full Text]
5.
Varani J,
Ginsburg I,
Schuger L,
Gibbs DF,
Bromberg J,
Johnson KJ,
Ryan US,
Ward PA:
Endothelial cell killing by neutrophils: Synergistic interaction of oxygen products and proteases.
Am J Pathol
135:435,
1989[Abstract]
6.
Hofstra TC,
Kalra VK,
Meiselman HJ,
Coates TD:
Sickle erythrocytes adhere to polymorphonuclear neutrophils and activate the neutrophil respiratory burst.
Blood
87:4440,
1996[Abstract/Free Full Text]
7.
Francis RB,
Johnson CS:
Vascular occlusion in sickle cell disease: Current concepts and unanswered questions.
Blood
77:1405,
1991[Free Full Text]
8.
Pearson H:
Sickle cell anaemia and severe infection due to encapsulated bacteria.
J Infect Dis
136:S25,
1977
9.
Boghossian SH,
Wright G,
Webster DB,
Segal AW:
Investigations of host defence in patients with sickle cell disease.
Br J Haematol
59:523,
1985[Medline]
[Order article via Infotrieve]
10.
Roberts PJ,
Williams SL,
Linch DC:
The regulation of neutrophil phospholipase A2 by GM-CSF and its role in priming superoxide production.
Br J Haematol
92:804,
1996[Medline]
[Order article via Infotrieve]
11.
Gasson JC:
Molecular physiology of granulocyte-macrophage colony-stimulating factor.
Blood
77:1131,
1991[Free Full Text]
12.
Roberts PJ,
Devereux S,
Pilkington GR,
Linch DC:
Fc RII-mediated superoxide production by phagocytes is augmented by GM-CSF without a change in FcRII expression.
J Leuk Biol
48:247,
1990[Abstract]
13.
Jaswon MS,
Khwaja A,
Roberts PJ,
Jones HM,
Linch DC:
The effects of rhGM-CSF on the neutrophil respiratory burst when studied in whole blood.
Br J Haematol
75:181,
1990[Medline]
[Order article via Infotrieve]
14.
Khwaja A,
Carver JE,
Linch DC:
Interactions of GM-CSF, G-CSF and TNF in the priming of the neutrophil respiratory burst.
Blood
79:745,
1992[Abstract/Free Full Text]
15. Siegel S: Non-parametric Statistics for the Behavioural
Sciences. Tokyo, Japan, McGraw-Hill Kogakusha, 1956
16.
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]
17.
Styles LA,
Schalkwijk CG,
Aarsman AJ,
Vichinsky EP,
Lubin BH,
Kuypers FA:
Phospholipase A2 levels in acute chest syndrome of sickle cell disease.
Blood
87:2573,
1996[Abstract/Free Full Text]
18.
Perry FE,
Elson CJ,
Mitchell TJ,
Andrew PW,
Catterall TJ:
Characterisation of an oxidative response inhibitor produced by Streptococcus pneumoniae.
Thorax
49:676,
1994[Abstract/Free Full Text]
19.
Forehand JR,
Johnston RB,
Bomalaski JS:
Phospholipase A2 activity in human neutrophils: stimulation by lipopolysaccharide and possible involvement in priming for an enhanced respiratory burst.
J Immunol
151:4918,
1993[Abstract]
20.
Strauss R,
Johnston R,
Asbrock T,
Moreno H,
Lehmeyer J:
Neutrophil oxidative metabolism in sickle cell disease.
J Pediatrics
89:391,
1976[Medline]
[Order article via Infotrieve]
21.
Dias-Da-Motta PM,
Arruda UR,
Muscara MN,
Saad STO:
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[Medline]
[Order article via Infotrieve]
22.
Thomson AP,
Dick M:
Endotoxinaemia in sickle cell disease.
Clin Lab Haematol
10:397,
1988[Medline]
[Order article via Infotrieve]
23.
Francis RB Jr,
Haywood LJ:
Elevated immunoreactive tumour necrosis factor and interleukin-1 in sickle cell disease.
J Natl Med Assoc
84:611,
1992[Medline]
[Order article via Infotrieve]
24.
Kitchen E,
Rossi AG,
Condliffe AM,
Haslett C,
Chilvers ER:
Demonstration of reversible priming of human neutrophils using platelet activating factor.
Blood
88:4330,
1996[Abstract/Free Full Text]
25.
Roberts PJ,
Pizzey AR,
Khwaja A,
Carver JE,
Mire-Sluis AR,
Linch DC:
The effects of interleukin-8 on neutrophil fMetLeuPhe receptors, CD11b expression and metabolic activity, in comparison and combination with other cytokines.
Br J Haematol
84:586,
1993[Medline]
[Order article via Infotrieve]
26.
Devereux S,
Linch DC,
Campos Costa D,
Spittle MF,
Jelliffe AM:
Transient leucopaenia induced by granulocyte-macrophage colony-stimulating factor.
Lancet
2:1523,
1987[Medline]
[Order article via Infotrieve]
27.
Devereux S,
Bull HA,
Campos-Costa D,
Saib R,
Linch DC:
Granulocyte macrophage colony stimulating factor induces changes in cellular adhesion endothelium: In-vitro and in-vivo studies in man.
Br J Haematol
71:323,
1989[Medline]
[Order article via Infotrieve]
28.
Laurindo IMM,
Mello SBV,
Cossermelli W:
Influence of low doses of methotrexate on superoxide anion production by polymorphonuclear leukocytes from patients with rheumatoid arthritis.
J Rheumatol
22:633,
1995[Medline]
[Order article via Infotrieve]
29.
Eggleton P,
Wang L,
Penhallow J,
Crawford,
Brown KA:
Differences in oxidative response of subpopulations of neutrophils from healthy subjects and patients with rheumatoid arthritis.
Ann Rheum Dis
54:916,
1995[Abstract/Free Full Text]
30.
Miesel R,
Hartung R,
Kroeger H:
Priming of NADPH oxidase by tumor necrosis factor alpha in patients with inflammatory and autoimmune rheumatic diseases.
Inflammation
20:427,
1996[Medline]
[Order article via Infotrieve]
31.
Al-Balla S,
Johnston C,
Davis P:
The in vivo effect of non-steroidal antiinflammatory drugs, gold sodium thiomalate and methotrexate on neutrophil superoxide radical generation.
Clin Exp Rheumatol
8:41,
1990[Medline]
[Order article via Infotrieve]
32.
Mur E,
Zabernigg A,
Hilbe W,
Eisterer W,
Halder W,
Thaler J:
Oxidative burst of neutrophils in patients with rheumatoid arthritis: Influence of various cytokines and medication.
Clin Exp Rheumatol
15:233,
1997[Medline]
[Order article via Infotrieve]
33.
Kowanko IC,
Ferrante A,
Clemente G,
Youssef PP,
Smith M:
Tumor necrosis factor priming of peripheral blood neutrophils from rheumatoid arthritis patients.
J Clin Immunol
16:216,
1996[Medline]
[Order article via Infotrieve]
34.
Storgaard M,
Jensen MP,
Stengaard-Pedersen K,
Moller BK,
Andersen PL,
Obel N:
Effects of methotrexate, sulphasalazine and aurothiomalate on polymorphonuclear leucocytes in rheumatoid arthritis.
Scand J Rheumatol
25:168,
1996[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract