Inhibition of human neutrophil IL-8 production by hydrogen peroxide and dysregulation in chronic granulomatous disease.
ABSTRACT The innate immune response to bacterial infections includes neutrophil chemotaxis and activation, but regulation of inflammation is less well understood. Formyl peptides, byproducts of bacterial metabolism as well as mitochondrial protein biosynthesis, induce neutrophil chemotaxis, the generation of reactive oxygen intermediates (ROI), and the production of the neutrophil chemoattractant, IL-8. Patients with chronic granulomatous disease (CGD) exhibit deficient generation of ROI and hydrogen peroxide and susceptibility to bacterial and fungal pathogens, with associated dysregulated inflammation and widespread granuloma formation. We show in this study that in CGD cells, fMLF induces a 2- to 4-fold increase in IL-8 production and a sustained IL-8 mRNA response compared with normal neutrophils. Moreover, normal neutrophils treated with catalase (H(2)O(2) scavenger) or diphenyleneiodonium chloride (NADPH oxidase inhibitor) exhibit IL-8 responses comparable to those of CGD neutrophils. Addition of hydrogen peroxide or an H(2)O(2)-generating system suppresses the sustained IL-8 mRNA and increased protein production observed in CGD neutrophils. These results indicate that effectors downstream of the activation of NADPH oxidase negatively regulate IL-8 mRNA in normal neutrophils, and their absence in CGD cells results in prolonged IL-8 mRNA elevation and enhanced IL-8 levels. ROI may play a critical role in regulating inflammation through this mechanism.
- [show abstract] [hide abstract]
ABSTRACT: The effects of chemotactic factors on rabbit neutrophils were evaluated measuring cell migration in modified Boyden chambers and under agarose, in lysosomal enzyme release, leukocyte aggregation, and in vivo neutropenia. Chemotactins employed included the complement-derived C3 and C5 fragments, the bacterial chemotactic factor from culture supernatant fluids of Escherichia coli, and the synthetic chemotactic factors Met-Leu-Phe and formyl-Met-Leu-Phe. A consistent parallelism was found in all the leukocyte responses to a given chemotactic factor. In no instance, with any of the five chemotactic factor preparations, did cells responding in one assay system fail to respond in the four other assay systems, suggesting a common event in all of the cell responses. Boyden chamber chemotaxis was consistently the most sensitive assay; the agarose assay was, in general, less sensitive by a factor of 100 fold. Enzyme release approached, in cell sensitivity to chemotactic factors, that of the Boyden chamber assay. In general, in vitro leukocyte aggregation and in vivo neutropenia were considerably less sensitive assays. Chemotactic factor inactivator (CFI) purified from human serum destroyed in parallel all biological activities of C3 and C5 chemotactic factors but had no effect on the bacterial chemotactic factor and the activities of synthetic chemotactic peptides.Immunopharmacology 01/1979; 1(1):39-47.
- Journal of Experimental Medicine - J EXP MED. 01/2000; 192(3):433-438.
- [show abstract] [hide abstract]
ABSTRACT: The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N-formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC(50)s, approximately 5 microM for calcium flux and chemotaxis, were approximately 100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.Journal of Experimental Medicine 10/1999; 190(5):741-7. · 13.21 Impact Factor
Inhibition of Human Neutrophil IL-8 Production by Hydrogen
Peroxide and Dysregulation in Chronic Granulomatous Disease1
Julie A. Lekstrom-Himes,* Douglas B. Kuhns,†W. Gregory Alvord,‡and John I. Gallin2*
The innate immune response to bacterial infections includes neutrophil chemotaxis and activation, but regulation of inflammation
is less well understood. Formyl peptides, byproducts of bacterial metabolism as well as mitochondrial protein biosynthesis, induce
neutrophil chemotaxis, the generation of reactive oxygen intermediates (ROI), and the production of the neutrophil chemo-
attractant, IL-8. Patients with chronic granulomatous disease (CGD) exhibit deficient generation of ROI and hydrogen peroxide
and susceptibility to bacterial and fungal pathogens, with associated dysregulated inflammation and widespread granuloma
formation. We show in this study that in CGD cells, fMLF induces a 2- to 4-fold increase in IL-8 production and a sustained IL-8
mRNA response compared with normal neutrophils. Moreover, normal neutrophils treated with catalase (H2O2scavenger) or
diphenyleneiodonium chloride (NADPH oxidase inhibitor) exhibit IL-8 responses comparable to those of CGD neutrophils. Ad-
dition of hydrogen peroxide or an H2O2-generating system suppresses the sustained IL-8 mRNA and increased protein production
observed in CGD neutrophils. These results indicate that effectors downstream of the activation of NADPH oxidase negatively
regulate IL-8 mRNA in normal neutrophils, and their absence in CGD cells results in prolonged IL-8 mRNA elevation and
enhanced IL-8 levels. ROI may play a critical role in regulating inflammation through this mechanism. The Journal of Immu-
nology, 2005, 174: 411–417.
grating to sites of infection (1). Upon reaching infected tissues,
neutrophils release toxic proteases, phagocytize pathogens, and
generate reactive oxygen intermediates (ROI)3and hydrogen per-
oxide, promoting the destruction of invading organisms (1).
A neutrophilic response to inflammation is largely dictated by
its initial receptor binding of chemoattractants. In addition to en-
dogenous chemoattractants, including IL-8, C5a, and leukotriene
B4(LTB4) neutrophils detect and respond to the bacterial-derived
formyl peptides (2–5). Formyl peptides are generated by bacterial
endopeptidase cleavage of the first few amino acids, including the
initial formyl-modified methionine group of bacterial proteins (6),
and are also found in mitochondria (7). Neutrophils detect formyl
peptides using either high affinity or low affinity formyl peptide
receptors (FPR and FPRL1) (4). Detection of formyl peptides by
these seven-transmembrane, G protein-coupled receptors elicits a
eutrophils are the principal circulating cellular mediators
of innate immunity, responding to minute concentrations
of exogenous and endogenous chemoattractants and mi-
repertoire of responses, including chemotaxis (4), generation of
reactive oxygen intermediates (8), and degranulation (9).
Patients with chronic granulomatous disease of childhood
(CGD) have genetic mutations in any of four components of the
NADPH oxidase enzyme that is expressed in neutrophils and
monocytes and is necessary for the generation of reactive oxygen
intermediates (ROIs), such as superoxide anion, hydroxyl radical,
and hydrogen peroxide (1). Their profound defect in innate immu-
nity is reflected by their susceptibility to catalase-positive bacteria
and fungi, including the Aspergillus species and Staphylococcus
aureus (1). In addition to this severe immunodeficiency, CGD pa-
tients display signs of dysregulated inflammation, for which the
underlying mechanism is unknown. For example, patients with
CGD develop granulomas throughout their tissues, often resulting
in urinary and gastrointestinal tract obstruction (10).
NADPH oxidase and its redox products induce transcriptional
activation in some models (11–15); however, their potential to
down-regulate the inflammatory response is not known. In this
paper we show in a model of neutrophil activation by formyl pep-
tides using CGD and normal cells that normal IL-8 production is
regulated by hydrogen peroxide resulting from NADPH oxidase
Materials and Methods
The following reagents were purchased from the indicated sources: fMLF,
PMA, diphenyleneiodonium chloride (DPI), catalase, histidine, taurine,
and superoxide dismutase were obtained from Sigma-Aldrich; the Ultra-
spec RNA isolation system was purchased from Biotecx Laboratories;
[?-32P]dATP was obtained from NEN; 4–20% Tris-glycine gels and buff-
ers were purchased from Invitrogen/NOVEX.
Isolation of peripheral blood neutrophils and stimulation
Normal volunteer and CGD patient blood samples were drawn under Na-
tional Institutes of Health protocols 99-CC-0168 and 93-I-0119. Neutro-
phils were harvested by Ficoll-Paque Plus discontinuous gradient centrif-
ugation, RBC sedimentation with dextran, and hypotonic lysis. Harvested
neutrophils were diluted in HBSS (Cambrex) at 1–2 ? 106cells/ml and
aliquoted into polypropylene tubes. Cells were incubated briefly for 10 min
*Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD 20892; and†Clinical Services Program,
SAIC-Frederick, Inc., and‡Data Management Services, National Cancer Institute,
Frederick, MD 21702
Received for publication June 10, 2004. Accepted for publication October 19, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1The content of this publication does not necessarily reflect the views or policies of
the Department of Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsements by the U.S. government.
This work was supported in whole or in part by federal funds from the National
Cancer Institute, National Institutes of Health, under Contract NO1-CO-12400.
2Address correspondence and reprint requests to Dr. John I. Gallin, National Insti-
tutes of Health, Building 10, Room 2C146, 10 Center Drive, Bethesda, MD 20892.
E-mail address: email@example.com
3Abbreviations used in this paper: ROI, reactive oxygen intermediates; LTB4, leu-
kotriene B4; CGD, chronic granulomatous disease; DPI, diphenyleneiodonium chlo-
ride; GRO-?; growth-related oncogene ?.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
at 37°C, followed by stimulation with the indicated doses of fMLF and
incubation at 37°C for 2–3 h. Experiments using reactive oxygen species
scavengers or hydrogen peroxide were performed using neutrophils treated
with the indicated mediator for 10 min before the addition of fMLF unless
otherwise indicated. Reactive oxygen scavengers were used at the recom-
mended concentrations (16).
Neutrophil cell protein harvest and chemokine/cytokine
Neutrophils were lysed in protein lysis buffer (0.1% Igepal CA 630 (Sig-
ma-Aldrich), 50 mM Tris-HCl (pH 8.0), 0.15 M sodium chloride, 10 mM
sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodium orthovana-
date, Complete inhibitor minitablet (Roche)/10 ml buffer, and 100 ?g/ml
4-(2-aminoethyl)benzenesulfonyl fluoride (Pefabloc; Roche)). After a 1-h
incubation on ice, debris was pelleted, and supernatant proteins were stored
at ?80°C. Alternatively, neutrophils were solubilized in 0.2% Triton
X-100 and IL-8, IL-1?, TNF-?, IL-6, IL-1 receptor antagonist, growth-
related oncogene ? (GRO-?), MIP-1?, and MCP-1 were quantitated using
commercial ELISA kits (R&D Systems), according to the manufacturer’s
Messenger RNA analysis
Neutrophil total RNA was extracted using the Ultraspec RNA isolation kit
according to the manufacturer’s instructions. After quantitation by spec-
trophotometer, RNA was separated by electrophoresis through a denaturing
gel as previously described (18). RNA was transferred to a Nytran nylon
membrane (Schleicher & Schuell), and hybridized to32P-dATP labeled
IL-8 probe (R&D Systems human IL-8 probe mixture) as described pre-
viously (18), with subsequent washing and autoradiography. In addition,
levels of IL-8, IL-1?, and GAPDH mRNA were determined using com-
mercial Quantikine mRNA quantitation kits (R&D Systems) according to
the manufacturer’s instructions.
Hydrogen peroxide production
H2O2was measured using the Amplex Red Hydrogen Peroxide/Peroxidase
Assay kit (Molecular Probes). Briefly, neutrophils (2 ? 106cells/ml
HBSS) were incubated in a mixture containing 50 ?M Amplex Red reagent
and 0.1 U/ml HRP. Either fMLF (5 ? 10?9M) or PMA (100 ng/ml) was
added, and the fluorescence was monitored using a fluorescence microplate
reader (CytoFluor II; PerSeptive Biosystems) with a ?excitationof 530 nm
and a ?emissionof 590 nm. Unknowns were calculated from an H2O2stan-
dard curve that ranged from 0.2–20 ?M.
Data in this study were analyzed using standard univariate ANOVA, lon-
gitudinal repeated measures generalized least squares and linear effects
mixed models, graphical techniques, and post-hoc tests (Tukey’s, Dun-
nett’s, and t tests). Longitudinal mixed models account for within-subject
correlations over time. All tests were two-sided; a value of p ? 0.05 was
considered significant. In many cases, data were transformed to their com-
mon logarithm to satisfy homogeneity of variance requirements. The data
presented represent the mean ? SEM. To determine the t1/2of IL-8 mRNA,
the data point representing the peak response and the remainder of the
points through the end of the incubation period were connected using a
first-order exponential decay curve, and the decay constant, ?, was deter-
mined. The t1/2was calculated using the formula: t1/2? 0.693/?.
Differential expression of neutrophil IL-8 in normal and CGD
Previous observations of the effects of formyl peptide stimulation
on peripheral blood neutrophils from normal subjects were con-
firmed using a chemotactic dose of fMLF (5 ? 10?9M) in vitro,
resulting in an increase in neutrophil IL-8 production that was
detected within 30 min and continued through 120 min (19) (Fig.
1, top). In contrast, treatment of neutrophils from CGD patients
with fMLF resulted in an increase in neutrophil IL-8 production
that was detected within 30 min and continued through 240 min.
The response was independent of the CGD genotype (not shown)
and was significantly greater than that seen in normal neutrophils
(p ? 0.0382).
The average rate of IL-8 accumulation in CGD and normal neu-
trophils did not differ through 90 min; subsequently, however, the
average rate of IL-8 production in normal neutrophils dropped in
the 90- to 120-min interval whereas IL-8 production in CGD neu-
trophils remained significantly elevated (0.016 ? 0.007 ng of IL-
8/106/min in normal cells vs 0.049 ? 0.010 ng of IL-8/106/min in
CGD cells; p ? 0.0119). By 120–480 min, the average rate of IL-8
accumulation in CGD neutrophils dropped to the level observed in
normal neutrophils (0.000 ? 0.001 ng of IL-8/106/min in normal cells
vs 0.002 ? 0.001 ng of IL-8/106/min in CGD cells). The prolonged
rate of production of IL-8 in CGD neutrophils was associated with a
50% or greater increase in IL-8 protein in CGD neutrophils compared
with normal neutrophils, confirming our previous report (19).
The difference in IL-8 production in normal vs CGD neutrophils
was not a general effect on all cytokine production, because no
significant differences in the production of IL-1? were observed
between normal and CGD neutrophils (Fig. 1, bottom). It should be
noted that the level of IL-8 found in neutrophils was nearly 1000-
fold greater than that of IL-1?. In addition, the IL-1? response was
transient, suggesting that IL-1? is used and/or cleared comparably
by normal and CGD neutrophils.
Neutrophils from both normal volunteers and CGD patients re-
sponded to fMLF stimulation in a dose-responsive manner, with
significant increases (p ? 0.01) in IL-8 production at doses of 5 ?
10?9and 1 ? 10?7M fMLF and with maximal stimulation at a
dose of 5 ? 10?9M; however, IL-8 protein levels in CGD neu-
trophils were 2- to 4-fold higher than levels measured in normal
volunteer neutrophils (Fig. 2). At higher doses of fMLF (0.1 ?M),
production of neutrophil IL-8 returned to baseline levels in both
normal and CGD neutrophils (Fig. 2). The loss of IL-8 synthesis at
higher doses of fMLF is not without precedent; neutrophil chemo-
taxis toward formyl peptides is also reduced at 1- to 5-?M doses
and CGD neutrophils. Neutrophils (1 ? 106/0.5 ml HBSS with HEPES, pH
7.3) from either normal subjects or patients with CGD were incubated in
the absence (E and ?) or the presence (F and f) of fMLF (5 ? 10?9M).
At the indicated times, the incubation was halted by the addition of cold
0.2% Triton X-100, and total IL-8 and IL-1? were determined as described
in Materials and Methods. The data represent the mean ? SEM of neu-
trophils isolated from 13 normal subjects and 13 patients with CGD. The
difference in IL-8 expression between normal and CGD neutrophils was
significant (p ? 0.0382).
Differential expression of IL-8 and IL-1? in normal (NL)
412H2O2INHIBITION OF IL-8 SYNTHESIS
of fMLF and is attributed in part to the differing affinities of the
two human formyl peptide receptors expressed on neutrophils (4).
Differential expression of IL-8 mRNA in normal and CGD cells
The increased levels of IL-8 protein detected in CGD neutrophils
may be due to alterations in IL-8 mRNA synthesis or destruction
or in IL-8 protein translation or release, or in some combination of
these processes. To investigate the varied contributions of these
events to our observations, IL-8 mRNA in normal and CGD neu-
trophils was examined and quantitated in response to both dose of
formyl peptides and time of stimulation.
mRNA blotting of total RNA harvested from normal and CGD
neutrophils after 3 h of stimulation with different doses of formyl
peptide showed increased levels of IL-8 transcripts in CGD cells
compared with normal cells (Fig. 3). Interestingly, an analysis of
the effect of increasing concentrations of fMLF on IL-8 mRNA at
45 min after stimulation revealed similar responses in normal and
CGD neutrophils (Fig. 4, left panel), suggesting that early after
stimulation, IL-8 transcription was similar in normal and CGD
cells. However, 4 h after fMLF stimulation, normal cells showed
little or no increase in IL-8 mRNA, whereas CGD cells continued
to show sustained IL-8 mRNA (Fig. 4, right panel).
Time-course analysis of IL-8 mRNA levels in normal and CGD
neutrophils showed that the kinetics of IL-8 transcription in CGD
cells were significantly altered compared with those in normal
cells. In the first 90 min after fMLF stimulation, IL-8 mRNA levels
in CGD cells and normal cells were very similar, corroborating the
dose-response studies after 45 min of formyl peptide stimulation
(Fig. 4). Subsequently, IL-8 mRNA levels observed in CGD cells
from 90 min through 480 min were significantly elevated (p ?
0.01; Fig. 5A) compared with those in normal cells. Integration of
the areas under the curves revealed an average 2.5-fold increase in
the IL-8 mRNA of CGD neutrophils compared with normal neu-
trophils (p ? 0.01). Analysis of the mRNA decay curve (starting
at the peak level through the return to basal level) yielded similar
t1/2values for IL-8 mRNA in normal and CGD neutrophils (t1/2?
99 ? 7 min for normal cells vs 113 ? 5 min for CGD cells; p ?
0.127), suggesting that the prolonged IL-8 mRNA response in
CGD neutrophils was due to increased transcription and not to a
change in mRNA stability. Additional studies using actinomycin D
to block RNA synthesis indicated that the decay of synthesized
IL-8 mRNA was not different in normal and CGD neutrophils
treated with buffer or fMLF; therefore, the regulation of IL-8
mRNA in fMLF-treated neutrophils occurred at the level of
mRNA synthesis, not at the level of RNA stability (our unpub-
lished observations). Hence, it is likely that the elevated levels of
IL-8 transcripts detected in CGD neutrophils were the result of
prolonged transcription of the IL-8 gene in response to formyl
peptide stimulation and not the attenuation of IL-8 transcript
In contrast to the processes regulating IL-8 synthesis, treatment
of neutrophils with fMLF resulted in similar transient increases in
IL-1? mRNA in normal and CGD neutrophils, in agreement with
the IL-1? protein response (Fig. 5B). Interestingly, a small, but
significant, increase in GAPDH mRNA was observed after treat-
ment with fMLF, but these responses were not different in normal
and CGD neutrophils (Fig. 5C).
Effects of scavengers and inhibitors of ROI in normal cells (Fig. 6)
The functional NADPH oxidase enzyme complex generates H2O2
in addition to other free radical oxygen molecules. In CGD cells,
mutation or deletion of any one of four components of NADPH
oxidase will result in a nonfunctional enzyme with little or no
generation of ROI. NADPH oxidase catalyzes a single electron
reduction of O2to superoxide anion, O2., with subsequent conver-
sion to H2O2by superoxide dismutase. Neutrophil-derived myelo-
peroxidase, in turn, produces hypochlorous acid from H2O2and
CGD neutrophils. Neutrophils were treated with increasing doses of fMLF
as described in Fig. 1. The data represent the mean ? SEM of 12 normal
subjects and four CGD patients. ?, p ? 0.01.
Dose response of IL-8 accumulation in normal (NL) and
mRNA in CGD neutrophils. The top panels represent Northern blot anal-
ysis of RNA isolated from both normal (NL; left) and CGD (right) neu-
trophils after 3-h incubation with the indicated doses of fMLF. The blots
were hybridized with oligonucleotide probes encoding antisense IL-8.
RNA loading was verified by ethidium staining of the membrane before
hybridization (bottom panels).
Northern blot analysis of prolonged expression of IL-8
Neutrophils isolated from two normal subjects (NL; F) and a CGD patient
(f) were incubated with the indicated doses of fMLF for 45 min (left
panel) and 4 h (right panel). IL-8 mRNA was determined as described in
Prolonged expression of IL-8 mRNA in CGD neutrophils.
413 The Journal of Immunology
chloride. DPI inhibits NADPH oxidase and other flavoenzymes by
electron transfer and phenylation of the FAD component of the
oxidase enzyme apparatus (20). Using scavengers and enzyme in-
hibitors of the ROI cascade, we examined the effects of individual
components of the NADPH oxidase-driven respiratory burst on
IL-8 production in normal volunteer neutrophils. Normal neutro-
phils treated with catalase (1000 U/ml), which degrades extracel-
lular H2O2, produced significantly higher levels of IL-8 compared
with PBS-treated cells (5.50 ? 0.82 vs 1.06 ? 0.31; p ? 0.001).
Addition of superoxide dismutase, which catalyzes the conversion
of O2.to H2O2, had no effect on neutrophil IL-8 production com-
pared with that by control PBS-treated cells. Other oxygen inter-
mediate scavengers, such as histidine, taurine, and DMSO, which
reduce levels of singlet oxygen (1O2), hypochlorous acid, and hy-
droxyl radical ( ? OH), respectively, had no effect on neutrophil
IL-8 compared with PBS-treated cells. Thus, elimination of H2O2
from the buffer with catalase or prevention of its synthesis with
DPI resulted in elevated IL-8 protein levels in normal neutrophils,
analogous to observations made with CGD neutrophils, suggesting
that H2O2may be the causative agent in modulating IL-8 synthe-
sis. We next examined the effects of adding H2O2to normal neu-
trophils to determine whether formyl peptide-stimulated IL-8 pro-
duction could be suppressed.
Effects of hydrogen peroxide and hypoxanthine plus xanthine
To test the hypothesis that exogenous H2O2would suppress the
synthesis of IL-8 in neutrophils, we examined the effects of the ad-
dition of H2O2directly to cells in buffer as well as using the hy-
poxanthine/xanthine oxidase H2O2-generating system. Addition of
physiologic doses of H2O2(10–100 nmol/ml, levels comparable to
those produced by 1 ? 106normal neutrophils activated with 100
ng/ml PMA) to normal neutrophils immediately before the addi-
tion of fMLF resulted in a dose-dependent inhibition of IL-8 pro-
duction, but had no effect on untreated cells (Fig. 7, top). This
inhibition was completely abrogated by the addition of catalase.
Moreover, the addition of a H2O2-generating system, hypoxan-
thine plus xanthine oxidase, resulted in a dose-dependent inhibi-
tion of fMLF-induced IL-8 production, but had no effect on IL-8
production in untreated cells (Fig. 7, bottom). Maximum inhibition
was achieved at a dose of 0.3 mU of xanthine oxidase activity/ml.
This level of hypoxanthine plus xanthine oxidase activity resulted
in the production of 10–15 nmol of H2O2during a 2-h incubation
compared with 100–150 nmol of H2O2produced after treatment of
normal neutrophils with 100 ng/ml phorbol ester. Interestingly,
neutrophils appeared to be more sensitive to a lower level of H2O2
produced in a sustained fashion with the hypoxanthine plus xan-
thine oxidase-generating system than the same level of H2O2
(10?4M; Fig. 7) given as a bolus. Heat treatment of the xanthine
oxidase resulted in abrogation of its inhibitory effect, indicating
that the activity of the enzyme was responsible for the inhibition.
Effect of ROI on cytokine production in normal neutrophils
To determine whether the enhancement of IL-8 levels with
NADPH oxidase inhibition could be generalized to other cytokines
and chemokines produced by neutrophils, immunoassays were
conducted on normal neutrophils treated with buffer or fMLF (5 ?
10?9M) or with concurrent NADPH oxidase inhibition with either
DPI (2 ?M) or scavenging of hydrogen peroxide with catalase
(1000 U/ml; Table I). Differences in chemokine production with
NADPH oxidase inhibition reached statistical significance only
with IL-8 and IL-?. Other cytokines and chemokines, including
TNF-?, IL-6, IL-1R antagonist, GRO-?, MIP-1?, and MCP-1,
showed no enhanced production with NADPH oxidase inhibition.
GAPDH mRNA. Neutrophils (2 ? 106/ml) isolated from both 15 normal
subjects (NL; F and E) and 14 CGD patients (f and ?) were treated with
fMLF (5 ? 10?9M) for the indicated times, harvested by centrifugation,
and dissolved in the cell lysis buffer. Levels of IL-8, IL-1?, and GAPDH
mRNA were determined as described in Materials and Methods. Error bars
not shown are buried within the symbol. ?, p ? 0.01.
fMLF induces transient alterations in IL-8, IL-1?, and
mal neutrophils. Neutrophils (1 ? 106/0.5 ml) were treated with DPI (2
?M), catalase (1000 U/ml), superoxide dismutase (SOD; 100 ?g/ml), his-
tidine (100 ?M), taurine (10 mM), or DMSO (10 mM) for 10 min at 37°C
before the addition of fMLF (5 ? 10?9M). Neutrophil IL-8 was deter-
mined as described in Fig. 1. The data represent the mean ? SEM of five
Effects of scavengers of ROI on IL-8 accumulation in nor-
414H2O2INHIBITION OF IL-8 SYNTHESIS
Effect of catalase and hypoxanthine/xanthine oxidase on normal
and CGD IL-8 protein and mRNA (Fig. 8)
Because CGD neutrophils fail to produce ROI, it was postulated
that ROI could regulate IL-8 mRNA. Addition of catalase (1000
U/ml; a scavenger of hydrogen peroxide) to normal neutrophils
resulted in a prolonged IL-8 mRNA response and increased IL-8
protein (p ? 0.008 and p ? 0.0465, respectively, vs fMLF alone)
comparable to the response observed in CGD neutrophils (Fig. 8).
Catalase had no effect on the IL-8 mRNA and IL-8 protein re-
sponse of CGD neutrophils treated with fMLF. In contrast, the
addition of an O2./H2O2-generating system, hypoxanthine/xanthine
oxidase (0.3 mU/ml) inhibited the IL-8 mRNA and IL-8 protein
response in both normal and CGD neutrophils (p ? 0.05 vs fMLF
alone). Control experiments using labeled recombinant human
IL-8 protein or mRNA demonstrated that H2O2(10?7–10?3M)
did not degrade IL-8 protein or mRNA.
Redox responsive transcriptional regulation has been shown in a
number of studies to induce IL-8 expression in epithelial and en-
dothelial cell lines (11–15). In human neutrophils, we show that
inhibition of NADPH oxidase by a variety of chemical means and
a genetic defect significantly increases IL-8 production in response
to formyl peptide stimulation. Exposure of CGD neutrophils to an
H2O2-generating system, hypoxanthine plus xanthine oxidase, un-
der conditions that generate amounts of H2O2comparable to those
Table I. Cytokine specificity in normal neutrophilsa
AnalyteBuffer fMLF (5 ? 10?9M)
340 ? 60
0.34 ? 0.10
0.45 ? 0.23
0.04 ? 0.03
1150 ? 270
70.6 ? 15.3
5.17 ? 1.49
2.11 ? 1.60
2030 ? 640b
1.73 ? 0.18b
2.98 ? 1.63
0.13 ? 0.09
1310 ? 360
62.1 ? 13.2
7.45 ? 1.78
2.04 ? 1.47
2810 ? 330c
8.91 ? 3.35c
2.25 ? 1.26
0.12 ? 0.07
1200 ? 340
78.8 ? 13.5
6.73 ? 1.34
2.26 ? 1.31
3270 ? 500c
15.66 ? 7.66c
1.73 ? 0.54
0.36 ? 0.14
1580 ? 410
84.7 ? 15.0
17.38 ? 4.56
3.65 ? 2.11
aNormal neutrophils (2 ? 106/ml) were incubated for 10 min at 37°C with either DPI (2 ?M) or catalase (1000 U/ml). fMLF
(5 ? 10?9M) was then added, and the incubation was continued for 8 h. The incubation was terminated with the addition of
ice-cold Triton X-100 (0.2%). The cellular lysates were analyzed for the indicated analytes. Values are expressed as picograms
bp ? 0.05 vs buffer.
cp ? 0.05 vs fMLF.
production in normal neutrophils treated with fMLF. Normal neutrophils
were incubated in the presence of increasing doses of either H2O2(top
panel; n ? 3) or xanthine oxidase plus 50 ?M hypoxanthine (bottom panel;
n ? 10) in the absence (E) or the presence (F) of fMLF (5 ? 10?9M).
IL-8 accumulation was determined as described in Fig. 1. Addition of
catalase (1000 U/ml) to 10?4M H2O2(?) reversed the effect of H2O2. ?,
p ? 0.05, comparing cells treated with H2O2or hypoxanthine plus xanthine
oxidase with cells treated with buffer alone.
Effects of H2O2and hypoxanthine/xanthine oxidase on IL-8
normal (NL) and CGD IL-8 mRNA and protein synthesis. Neutrophils
isolated from normal subjects (top panel; n ? 3) and patients with CGD
(bottom panel; n ? 3) were treated with fMLF (5 ? 10?9M) in the
presence of buffer, catalase (1000 U/ml), or hypoxanthine (50 ?M)/xan-
thine oxidase (0.3 mU/ml). Neutrophil IL-8 mRNA and protein were de-
termined as described in Figs. 1 and 3.
Effects of catalase and hypoxanthine/xanthine oxidase on
415 The Journal of Immunology