Clinical Science (1994) 86, 41 1-415 (Printed in Great Britain)
41 I
Nitric oxide production by human peripheral blood
polymorphonuclear leucocytes
Helen F. GOODE, Nigel R. WEBSTER, Peter D. HOWDLE and Barry E. WALKER
Clinical Oxidant Research Group, St lames's University Hospital, Leeds U.K.
(Received 30 June/l2 October 1993; accepted I November 1993)
1. We describe a rapid and reliable technique for the
assessment of basal nitric oxide release in clinical
situations, using peripheral blood polymorphonuclear
leucocytes isolated by a single-step density gradient
procedure. The assay is based on the quantitative
conversion of oxyhaemoglobin to methaemoglobin by
nitric oxide. We have further examined the ability of
these cells to respond to various stimuli.
2. Basal (unstimulated) nitric oxide release occurred,
which was augmented by superoxide dismutase. The
mean value for healthy subjects was 283 f 96.7
pmol min-'
cells.
3. Both phorbol myristate acetate and N-formylmethionyl-leucylphenylalanine induced further release
of nitric oxide, which was increased by preincubation
with lipopolysaccharide, interleukin-6 and interferon-y.
4. Preincubation of cells with NG-monomethyl-Larginine or L-canavanine sulphate inhibited nitric
oxide production.
5. The procedure provides a valuable tool for monitoring nitric oxide up-regulation in clinical situations.
responsiveness of septic shock [13, 141. Neutrophil
infiltration and activation on a large scale with
induction of Ca' +-independent NO synthase is
thought to be a major part of the excessive NO
production seen in such patients (for review, see [151).
Although NO inhibitors such as NG-monomethylL-arginine (L-NMMA) have been used in extreme
clinical circumstances [16], their use has been criticised [17], since the constitutive NO synthase,
necessary for maintaining adequate organ perfusion,
is also inhibited, in addition to the inducible enzyme
C181.
The present study was undertaken to provide a
means of monitoring NO release from intact cells in
conditions as close to those in uivo as possible, and
to investigate the effects of bioactive molecules,
cytokines and inhibitors on cellular NO release.
This methodology could then be applied to clinical
situations to monitor the effects of NO synthase
inhibitors and antioxidants on leucocyte NO
production.
MATERIALS AND METHODS
INTRODUCTION
Nitric oxide (NO) is synthesized from L-arginine
by a constitutive enzyme NO synthase, which is
Ca2+-dependent, and which has an important role
in the physiological maintenance of blood pressure
[13 through guanylate cyclase-induced vascular
smooth muscle relaxation [a]. A second N O synthase, which is Ca' +-independent, is induced in
various cells, including polymorphonuclear cells, in
response to endotoxin [3-51 and cytokines [3, 6, 71.
NO reacts rapidly with superoxide (Oz-') [8] to
form peroxynitrite [9] and eventually highly toxic
hydroxyl radicals [lo], which also have been shown
to cause smooth muscle relaxation via soluble
guanylate cyclase [1 11.
Septic shock during invasive infection is an acute
state of cytokine-mediated cardiovascular derangement [12]. There is growing evidence to suggest
that expression of CaZ+-independent NO synthase
is responsible for the hypotension and hypo-
isolation of polymorphonuclear leucocytes (PMN)
Peripheral venous blood was obtained from a
single subject on several occasions for isolation of
PMN. Informed verbal consent was obtained and
the study had the approval of the local ethics
committee. Blood was collected into lithium heparin
and 5 ml was layered on to 3.5 ml of Polymorphprep
cell separation medium (Nycomed UK Ltd,
Birmingham, U.K.). After centrifugation at 475 g
(30min at 20°C), the upper, mononuclear cell band
was removed and discarded, and the lower band of
PMN was removed and washed (lOOOg, 10min) in
RPMI 1640 medium containing 20 mmol/l Hepes
buffer (ICN Biomedicals, Bucks, U.K.). Any contaminating erythrocytes were lysed by hypotonic
shock, and cells were rewashed twice, counted and
adjusted to l o x 106/ml. PMN from five other
healthy subjects were also isolated for measurement
of basal NO release.
Key words: cells, cytokines, nitric oxide.
Abbreviations: CAN, L-canavanine sulphate; CAT, catalase; FMLP, N-formylmethionyl-leucylphenylalanine; IFN-y, interferon-y IL-la Interleukin-la; 11-6, interleukind; LPS,
lipopolysaccharide; met-Hb, methaemoglobin; L-NMMA, N'-monomethyl-c-arginine; oxy-Hb, oxyhaemaglobin; PMA, phorbol myristate acetate; PMN, polymorphonuclear
leucocytes; SOD, superoxide dismutase; TNF, tumour necrosis factor.
Correspondence: D r H. F. Goode, Clinical Oxidant Research Group, Clinical Sciences Building, St James's University Hospital, Leeds LS9 7TF. U.K.
H. F. G d e et al.
412
Table I. Concentration ranges of cell stimuli tested. Abbreviations:
LPS, lipopolysaccharide; IFN-y, interferon-y; IL-1% interleukin-la; L 2 r ,
recombinant interleukin-2 ILd, interleukind.
Stimulus
Conc.
range (optimum)
FMLP
PMA
LPS
IFN-y
IL-la
IL-2r
IL-6
2>75ng/ml
0.1-0.5pol/l
50-250 ng/ml
100-500units/ml
I 0-5.0 unitsjml
5C-250 unitsjml
5&300pg/rnl
(50ng/ml)
(0.3pol/l)
(100ng/ml)
(300units/ml)
-
(300pg/ml)
Haemoglobin preparation
Human oxyhaemoglobin (oxy-Hb) was prepared
as described by Benesch et al. [19]. Venous blood
was anticoagulated with sodium citrate, and washed
several times in ice-cold saline. Erythrocytes were
then lysed with water and the membranes were
removed using toluene. After centrifugation the
solution was then passed through a Sephadex G25
column and the haemoglobin was eluted with
0.1 mol/l sodium chloride. After a brief period of
oxygenation, the preparation was stored at - 70°C
in small aliquots. The oxy-Hb concentration was
then determined and the purity was confirmed by
scanning spectrophotometry.
NO assay
Oxy-Hb is rapidly and stoichiometrically oxidized
by N O to form methaemoglobin (met-Hb) with a
resulting shift in wavelength in the Soret region. By
measuring the increase in absorbance at 401 nm, the
amount of met-Hb formed, and therefore N O produced, can be quantified. For assay, cells were
incubated in RPMI 1640/Hepes medium. In some
experiments superoxide dismutase (SOD; 50 units/
ml) and/or catalase (CAT; 1000 units/ml) were also
included. In a 1cm semi-micro spectrophotometer
cuvette, 1 x lo6 (0.1 ml) PMN were added to 0.8ml
of the pre-warmed incubation medium, mixed well,
and 0.1 ml (final concentration 1.3 pmol/l) human
oxy-Hb was added before being immediately placed
in the sample compartment of a double-beam spectrophotometer, maintained at 37"C, with medium
and oxy-Hb alone in the reference compartment.
The increase in absorbance was recorded at 401 nm
using a chart recorder set at a full-scale deflection of
0.05 absorbance unit. The optimum wavelength for
the change in absorbance was determined previously
(data not shown). For cytokine stimulation studies,
the cells were incubated in medium for 10min to
allow completion of the basal response, then 0.1 ml
volumes of various concentrations of several stimuli
(Table 1) were added to the cuvettes, and incubated
for 2h. Control cuvettes contained medium only.
After 2 h 0.1 ml of oxy-Hb was added as above, and
the N O release was measured, with and without N -
formylmethionyl-leucylphenylalanine (FMLP). For
FMLP or phorbol myristate acetate (PMA) stimulation, the basal release of N O was measured then
0.1ml of either FMLP or PMA was added to the
cuvette (Table 1).
In some experiments cells were preincubated in
the presence of 2 mmol/l L-canavanine sulphate
(CAN) for 1 h or 0.1 to 0.5mmol/l L-NMMA
(Calbiochem Novabiochem Ltd, Nottingham, U.K.)
for 30min. In these experiments, medium was pretreated with washed AG5-X8 ion-exchange resin
(Bio-Rad Laboratories Ltd, Herts, U.K.) to reduce
the arginine concentration. The molar extinction
coefficient for the quantitative conversion of oxy-Hb
to met-Hb by N O was determined using sodium
nitroprusside as N O donor, and was found to be
37.2 (mmol/l)-' cm-' (data not shown).
Superoxide assay
Measurement of superoxide production by isolated PMN was based on the assay of Curran et al.
[20]. Briefly, 1 x lo6 (0.1ml) cells were added to a
cuvette containing 0.8 ml of pre-warmed RPMI/
Hepes, mixed and 0.1 ml (final concentration
0.1 mmol/l) cytochrome c was added. The increase
in absorbance was recorded as for NO, but at a
wavelength of 550 nm. After completion of the basal
response, 0.1ml of FMLP or PMA was added, and
the increase in superoxide production was measured. In some experiments, cells were pretreated
with CAN or L-NMMA as for NO. The specificity
of the assay was ascertained by the ability of SOD
to completely inhibit the superoxide release. The
molar extinction coefficient for the reduction of
cytochrome c used for calculating the amount of
superoxide produced was that supplied by Sigma
C27.7 (mmol/l) - cm - '3.
Reagents were obtained from Sigma Chemical
Company Ltd, Poole, Dorset, U.K., unless stated
otherwise.
RESULTS
Basal NO and superoxide release
When PMN were incubated in the presence of
oxy-Hb at 37"C, spontaneous basal production of
NO was observed. In the presence of SOD but not
of CAT, the N O release was increased from
182.3k56.0 to 525.0k75.1 pmolmin-'
PMN
(P<O.OOl, Fig. 1). When CAT was also included,
the N O release was 377.9 f83.4 pmol minPMN, which was significantly lower than without
CAT (P<0.05, Fig. 1). SOD and CAT were
included in all further experiments (see the discussion). In the presence of SOD and CAT, the basal
production of N O by PMN from a single individual
measured on several occasions was reproducible,
with a coefficient of variation of 22.1% between-
'
Nitric oxide production by human neutrophils
413
**
5:.
T
*
A
2MM
Z
K
!?
1500 I
c
._
-E
0
lo00
g
a3
1
e
500
s
X
e
n
NoSOD
NoCAT
+SOD
NoCAT
+CAT
+SOD
=
+CAN
v)
0
FMLP
Fig 1. Effect of SOD (50 unitslml), CAT (loo0 unitslml) and preincuand NO (G)
bation with Zrnmol/l CAN on basal superoxide
release from intact human peripheral blood neutrophils. Values are
the means (+SD) of six experiments. *significantly higher than without
enzymes (P<O.Ol, Mann-Whitney U-test); **significantly lower than without enzymes (P <O.WI, Man-Whitney U-test); ***significantly lower than
with SOD alone (P iO.05, Mann-Whitney Utest); ****significantly lower
than without CAN (P <0.05, Mann-Whitney U-test).
PMA
FMLP
+PMA
(a)
Fig. 3. Effect of FMLP and PMA on neutrophil superoxide (0)
and
NO (r)
release after completion of the basal response. Values are
the means (+SD) of six experiments. *significantly lower than FMLP
(P <O.OOI, Mann-Whitney U-test); **significantly higher than with FMLP
alone (P<O.Ol, Mann-Whitney U-test).
Stimulated NO and superoxide release
0.4
1.0
Control 0.2
L-NMMA concn. (mmol/l)
t-NMMA concn. (mmol/l)
Fig. 2. Effect of preincubation of neutrophils with L-NMMA on basal
and FMLP-stimulated NO release, Results are expressed as percentage
of control value (no L-NMMA) and are the means (5SD) of four experiments. *significantly lower than control (P <0.05, Mann-Whitney U-test);
**significantly lower than control (P<O.001, Mann-Whitney U-test).
batch (n=20) and 11.2% within-batch (n=20). The
mean (+SD) basal NO release from six healthy
subjects was 283.3 96.7 pmol min-l lop6 PMN
PMN). Cells which
(range 156-425 pmol minwere preincubated with CAN failed to show basal
release of NO (Fig. 1). L-NMMA quantitatively
inhibited basal NO release (Fig. 2).
Basal superoxide release by PMN was inhibited
by SOD (50 units/ml) and was unaffected by CAT
(Fig. 1). The mean basal superoxide release was
774.9 f 115.9 pmol min- l o p 6 PMN with a coeficient of variation of 15.6% between-batch (n= 10)
and 6.9% within-batch ( n = 10). Neither CAN nor LNMMA had any effect on superoxide release.
'
'
All stimulation experiments were performed after
the basal release was complete, in the presence of
both SOD and CAT (NO release). Dose-response
curves were used to find optimum concentrations of
the various stimuli tested (Table 1). FMLP had a
simultaneous effect on both NO and superoxide
release, inducing N O release after only 1min exposure at a level approximately equal to basal release
(Fig. 3). PMA also induced simultaneous NO and
superoxide production after about 5 min (Fig. 3).
When the cells were exposed to PMA for 3min and
then challenged with FMLP, the NO and superoxide release was increased (P<O.Ol, Fig. 3). Preincubation with L-NMMA quantitatively inhibited
the NO response to FMLP (Fig. 2).
Response to cytokine priming
LPS had no effect on NO production in RPMI/
Hepes medium, but when the medium was supplemented with 5% autologous plasma, NO production
was initiated (Fig. 4). Interleukin-6 and interferon-y
(IFN-y) both induced further NO production after
2 h exposure (Fig. 4), and markedly increased the
subsequent response to FMLP ( P <0.01, Fig. 4).
Neither interleukin-la (IL-la) nor recombinant
interleukin-2 had any stimulatory effect on NO
production at any of the concentrations studied.
Control cuvettes (medium only) also showed no
further NO release after 2 h (Fig. 4).
DISCUSSION
The oxy-Hb assay described was found to be a
reproducible technique, which, in the absence of a
method to determine N O production in uiuo, is
likely to be extremely useful in assessing the degree
414
H. F. Goode et al.
*
T
LPS
IL-6
IFN-y Control
11-6
+
*
r
IFN-y FMLP
+
FMLP FMLP
Fig. 4. Effect of cytokine priming on neutrophil NO release (a) and
(b) FMLP stimulation after completion of the basal response. Values
are the means (+SD) of four experiments. Control: cells incubated
without cytokines; LPS, LPS plus 5% autologues plasma. *significantly higher
than FMLP alone (P <0.05, Mann-Whitney U-test); **significantly higher
than control (P<O.OOI, Mann-Whitney &test).
of upregulation of NO in peripheral neutrophils in
clinical situations. The reaction of N O and oxy-Hb
has been well characterized previously [21, 221, and
has been shown to be a direct reaction and not
resulting from prior conversion of N O to nitrite
[22]. The molar extinction coefficient found in our
study is similar to that obtained previously: 38.6
[4], 45 [23] and 38 (mmol/l)-'cm-' [24].
The isolation of PMN using Polymorphprep is
rapid and reliable, enabling pure and viable (>95%,
data not shown) cell preparations to be obtained
ready for assay within 1 h of venepuncture. Previous
studies have shown that the use of a one-step single
gradient technique to isolate human neutrophils
results in cells which more closely resemble whole
blood than those isolated by conventional density
gradient procedures [25]. Dextran/Ficoll or Percoll
separation techniques cause altered receptor expression [25, 261, reduced oxidase activity [26] and a
muted response to cytokine priming [25] in isolated
neutrophils. The basal release of N O by these cells
is interesting, and could be due to either the
presence of a constitutive N O synthase enzyme in
polymorphonuclear cells, as is found in macrophages, or induction of the inducible enzyme in
response to cell separation.
The effect of SOD on the reaction of N O with
oxy-Hb is due to removal of superoxide, which is
produced simultaneously with NO, and which
avidly reacts with N O [S-lo] preventing the conversion of oxy-Hb to met-Hb. Dismutation of superoxide forms hydrogen peroxide, which is also able
to oxidize oxy-Hb [27]. The increase in met-Hb
formed in the presence of SOD but not CAT is
therefore proportional to the amount of superoxide
produced. Since the response of cells from an individual to any given stimulus may not be the same in
terms of both NO and superoxide release, CAT was
included in all experiments, despite the attenuation
in met-Hb formation that its use caused.
The specificity of the technique was demonstrated
by the inhibitory effect of CAN, an inhibitor of
arginine-utilizing enzymes which has been shown
previously to block PMN N O production [28], and
reduce L-arginine-derived nitrite and nitrate synthesis by a murine macrophage cell line [29]. LNMMA is an analogue of arginine, and reversibly
inhibits NO-induced relaxation [30] and reduces
the activity of N O synthase isolated from brain,
liver and rat PMN [6, 311. We found that LNMMA reduced NO release in v i m from intact
human PMN in a concentration-dependent manner.
FMLP is a bioactive peptide which binds to
specific cell membrane receptors [32] coupled to
intracellular signal transduction pathways responsible for cell activation. Priming with cytokines
results in enhanced functional activity [33]. In the
present study, we found that FMLP initiated N O
release simultaneously with superoxide. In an earlier
study, Wright et al. [27] found no increase in N O
measured by chemiluminescence, nor in nitrite production, by human neutrophils isolated using FicollHypaque, in response to FMLP, confirming the
suggestion that cells separated in this way have a
subdued response to stimulation [25]. FMLP stimulates N O release in porcine endothelial cells [24],
human neutrophils (measured by chemiluminescence) [28] and superoxide release in human alveolar macrophages [34]. PMA has also been shown to
stimulate superoxide production by human neutrophils [20], but there is no previous published work
regarding PMA-induced N O production.
Induction of N O by lipopolysaccharide (LPS) or
cytokines including tumour necrosis factor (TNF),
the interleukins and IFN-y has been described in
glycogen-elicited rat PMN [6], cultured porcine [3],
murine [35] and bovine [36] endothelial cells, and
cultured rabbit vascular smooth muscle cells [7].
When administered in vivo in dogs, TNF-a induced
a fall in blood pressure, which was reversed by
infusing L-NMMA [37], and in rats LPS caused a
loss of vascular responsiveness and shock [29],
which was prevented by L-NMMA [38]. In the
present study, we showed that N O release was
increased by exposure to IFN-y, IL-6 or LPS (plus
autologous plasma), but not by interleukin-2 or ILla, for 2 h. The subsequent response to FMLP was
also augmented by priming with these cytokines.
This is the first report of the effect of cytokine
stimulation on directly measured N O release in
intact human cells. The 2 h exposure to cytokines
used in the present study may be insufficient to
induce the inducible N O synthase, although Rees et
al. [13] showed that aortic rings have an increased
rate and magnitude to N O induced relaxation, and
increased cyclic G M P concentrations after exposure to LPS for 1 h. Others have shown that rat
peritoneal neutrophils contain low levels of a
Ca2+-independent N O synthase, which is enhanced
N i t r i c oxide production
when the cells are cultured ex uiuo in the presence of
IFN-y, but not TNF, for only 4 h [ 6 ] . Rat peripheral blood PMN do not synthesize NO synthase
[39], but there is a report of production by circulating PMN from rabbits and humans [40].
In rabbits, L-NMMA inhibits not only the inducible synthase enzyme, thought to be responsible in
part for cytokine-induced septic shock, but also the
constitutive enzyme necessary for maintaining vascular tone [lS]. If inhibitors of NO are ever to have
therapeutic uses, a technique to assess directly the
NO production by neutrophils is clearly useful, but
requires further validation in the clinical arena. A
large proportion of the excessive NO release
observed in septic shock is likely to come from the
large numbers of activated neutrophils accumulating
as part of the physiological response to infection.
Thus measurement of NO release in intact PMN
isolated using a single-step procedure, which closely
resembles physiological conditions, is likely to be
useful in such patients.
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