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Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, May 1999, p. 420-424, Vol. 6, No. 3
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Gamma Interferon Treatment of Patients with Chronic Granulomatous
Disease Is Associated with Augmented Production of Nitric Oxide by
Polymorphonuclear Neutrophils
Anders
Åhlin,1,2,*
Gerd
Lärfars,2,3
Göran
Elinder,1,2
Jan
Palmblad,2,3 and
Hans
Gyllenhammar2,3
Department of Pediatrics, the Karolinska
Institute at Sachs' Children's Hospital, S-118 95 Stockholm,1 and Department of
Hematology3 and Inflammation and
Hematology Research Laboratory,2 Huddinge
University Hospital, S-141 86 Huddinge, Sweden
Received 26 May 1998/Returned for modification 28 August
1998/Accepted 26 January 1999
 |
ABSTRACT |
Treatment with gamma-interferon (IFN-
) is associated with
reduced frequency and severity of infections in chronic granulomatous disease (CGD), but the mechanism is unknown. Since the inducible nitric
oxide (NO) synthase can be amplified by IFN- in murine macrophages,
for example, we hypothesized that IFN- might modulate NO release
from polymorphonuclear neutrophils (PMNs) in patients with CGD. Eight
patients with CGD and eight healthy controls were studied. Each patient
was given either 50 or 100 µg of IFN- per m2 on two
consecutive days. The production of NO from
N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated
PMNs was assessed as the
NG-monomethyl-L-arginine-inhibitable
oxidation of oxyhemoglobin to methemoglobin in the presence of catalase
and superoxide dismutase. Prior to IFN- treatment, the PMNs from CGD
patients produced 372 ± 27 (mean ± standard error of the
mean) pmol of NO/106 PMNs at 45 min, while the control PMNs
produced 343 ± 44 pmol. On day 1 after IFN- treatment, NO
production increased to 132% ± 25% of that for controls, and on day
3 it reached 360% ± 37% (P < 0.001) of that for
controls. On day 8, the values still remained higher, 280% ± 78%
more than the control values. Likewise, the bactericidal capacity of
PMNs increased on day 3. The present data show that IFN- treatment
of CGD patients is associated with an increased production of NO
from PMNs when activated by fMLP. Since these PMNs lack the capacity to
produce superoxide anions, it is conceivable that this increase in NO
release could be instrumental in augmenting host defense.
| |
INTRODUCTION |
Chronic granulomatous disease (CGD)
is a rare X-linked or autosomal inherited disease characterized by
recurrent life-threatening infections (27). The basic defect
is an inability of phagocytic cells to produce superoxide anions and
hydrogen peroxide, as a result of a defect in one of the subcomponents
of the NADPH oxidase in these cells. In patients with the X-linked form
of CGD, cells lack a membrane-associated part of the oxidase
gp91phox protein (27). Patients with the
autosomal recessive form of CGD fail to demonstrate the presence of
either one of two cytosolic factors, p47phox and
p67phox, or the membrane-bound p22phox
(27).
A multicenter study showed that recombinant human gamma
interferon (IFN-) administered subcutaneously (s.c.) three
times a week significantly reduced serious infections
(17). This regimen has therefore been recommended since 1991 as prophylaxis against infections in patients with CGD. However, the
mechanisms of action of IFN- in CGD are poorly understood. Although
some early reports on variant forms of CGD showed a partially restored
oxidative metabolism in cells of CGD patients after IFN- treatment
(10, 11), studies of more common CGD phenotypes could not
corroborate that observation (17, 25, 28, 30).
Human polymorphonuclear neutrophils (PMNs) produce and release nitric
oxide (NO) spontaneously (31) or following activation (21); both inducible and constitutive isoforms of NO
synthase (NOS) have been purified from human PMNs (6, 29).
Several lines of evidence imply that this function is of importance for host defense (22), possibly in CGD and furthermore in the
inflammatory response as reviewed previously (14, 24). NO
has been shown to possess cytotoxic (5) and bactericidal
actions, particularly against intracellular pathogens (4,
13). In healthy individuals, NO released from PMNs reacts rapidly
with superoxide anion-forming peroxynitrite (3). However, in
CGD this reaction is not possible, and thus, NO by itself may be of a
relatively larger importance. In murine systems, IFN- is a potent
inducer of NOS (24). Inducible NOS activity in vivo has been
demonstrated in PMNs from the urine of patients with urinary tract
infections (29), a situation where PMNs are exposed to not
only bacterial products, such as chemotactic oligopeptides, but also
bacteria and a considerable presence of cytokines. Also, PMNs from CGD
patients (herein referred to as CGD PMNs) have been shown to produce NO
in vitro (7). Thus, we asked if the mechanism for improved
host defense in CGD induced by IFN- could be associated with effects
on PMN NO release, activated by a synthetic analogue to naturally
occurring bacterial oligopeptides, and compared these data with those
of a simultaneously run PMN microbicidal assay.
| |
MATERIALS AND METHODS |
Percoll and Sephadex G-25 were from Pharmacia Fine Chemicals
(Uppsala, Sweden). Endotoxin-free water and Hanks' balanced salt solution (HBSS) were from Gibco (Paisley, Scotland).
L-Arginine, catalase, bovine hemoglobin, and
N-formyl-methionyl-leucyl-phenylalanine (fMLP)
were from Sigma (St. Louis, Mo.). Superoxide dismutase (SOD) was
from Boehringer Mannheim (Mannheim, Germany).
NG-Monomethyl-L-arginine
(L-NMMA) was a kind gift from Wellcome Research
Laboratories (Beckenham, United Kingdom).
Tetrakis(3-methoxy-4-hydroxyphenyl)nickel(II)-porphyrin was from
Interchim (Montluçon, France). Nafion and pure NO gas were from
Aldrich (Milwaukee, Wis.).
PMNs were prepared from venous blood by Percoll density centrifugation
essentially as described previously (15). The cells (purity
was >99%; viability by trypan blue exclusion was >98%) were
resuspended in HBSS at 2 × 106/ml. After suspension
in HBSS, the cells were kept in aliquots at 4°C until 60 min prior to
analysis. At that time, SOD, catalase, and either
L-arginine or the NOS inhibitor L-NMMA were
added and the cells were placed in a shaking water bath at 37°C until use.
Nitroblue tetrazolium reduction and chemiluminescence augmented by
luminol were assessed as previously described (15, 23).
Patients.
The eight CGD patients tested (Table
1) had a history of recurrent infections
since childhood. They had a negative nitroblue tetrazolium test and
chemiluminescence reaction. All patients were free from infection at
the time of the study. Details about the patients, e.g., mutations, are
published in reference 1. Prior to this study, they
had not been treated with IFN-. Some were on treatment with a
prophylactic antibiotic; however, the same antibiotic dosage regimen
was used throughout the study. Healthy members of the laboratory staff
served as controls; however, they did not receive IFN-.
Study design.
After informed consent was obtained, patients
were randomized to receive either 50 or 100 µg of IFN- (Imukin;
Boehringer Ingelheim, Ingelheim, Germany) per m2 on two
consecutive days, assuming that this regimen would induce a rapid and
transient increase of the cell functions studied and yet have tolerable
side effects. IFN- was administered s.c. by a nurse, and the doses
were given in a double-blinded manner. Venous blood samples were
obtained prior to IFN- administration and on days 1, 3, and 8 after
the last dose, between 8 and 9 a.m. Cells from the controls were
consistently assessed simultaneously. The randomization code was broken
after all results were registered. CGD patients were divided into
different subgroups to determine whether there were any differences in
the responses of the PMNs.
The study was approved by the local ethical committee and the Swedish
Board for Pharmaceutical Trials.
Preparation of oxyhemoglobin (HbO2) was performed
essentially as described previously (21). A solution of
bovine hemoglobin in distilled water was made at a concentration of 1 mM. The hemoglobin solution was oxygenated by bubbling with
O2 for 5 min. Subsequently, the hemoglobin was reduced with
1.5 mM sodium dithionite, prepared in deoxygenated doubly
distilled water, and reoxygenated by bubbling with O2 for
20 min. The sodium dithionite was removed on a Sephadex G-25
column. Fresh oxyhemoglobin was prepared for each day of the
experiment, and the concentration was determined spectrophotometrically at 415 nm (12).
Measurement of methemoglobin was performed essentially as described
previously (21) but with some important differences. Briefly, this was performed spectrophotometrically at 401 versus 411 nm
(12, 21) at 37°C in a Perkin-Elmer Lambda 7 spectrophotometer. Cells were treated with L-arginine or
L-NMMA for 45 min at 37°C in a shaking water bath. During
the incubation, SOD (30 µg/ml) and/or catalase (15 µg/ml) was
present. Oxyhemoglobin was added immediately prior to analysis. After
the background absorbance was read, the stimulus was added and the
reaction was monitored continuously for up to 4 h. All samples
were assessed as the difference between cells with
L-arginine and cells with L-NMMA, thus directly giving the amount of NO formed. The following equation was used to
calculate the amount of methemoglobin formed: 
= 19.7 mM
1 cm1 (12). The viability of
the cells was repeatedly determined with trypan blue exclusion and was
not affected by methemoglobin, SOD, catalase, or the arginines.
Pretrial experiments had shown that the major part of NO release was
over after 25 min, and at 45 min no further net NO generation was detected.
Electrochemical detection of NO release was performed with a
three-electrode potentiostatic Biopulse system (Tacussel, Lyon, France). The working electrode was a carbon fiber (8 µm in diameter and approximately 1 mm in length) coated with
tetrakis(3-methoxy-4-hydroxyphenyl)nickel(II)-porphyrin and Nafion
films, and NO was produced as described previously (19, 20).
The suspension of PMNs (1.75 × 106 to 3.5 × 106/ml in HBSS with Ca2+) was incubated for 20 min at 37°C with L-arginine or L-NMMA (both at 1 mM) and SOD (15 µg/ml). fMLP at 100 nM was added when a stable current baseline was reached, and NO production was monitored for 5 min. There were no changes in baseline current when L-NMMA, L-arginine, or SOD was added to unstimulated PMNs, either
for different concentrations of H2O2 or upon
preincubation with catalase (data not shown). The electrode was
calibrated with standard NO solutions as described previously (19,
20), and a standard curve with PMNs present was made for each
experiment and at the end of each measurement. All samples were
assessed as the difference between samples with L-arginine
and identical samples with L-NMMA. Thus, only the
L-NMMA-inhibitable response was assessed and taken to represent the net
amount of NO released from PMNs.
Mini-bactericidal assay.
A miniaturized bactericidal assay
was used as described previously (9). Staphylococcus
aureus was cultured in soy broth at 37°C and then washed. PMNs
(1.4 × 106) in 70 µl of NaCl with 0.1% bovine
serum albumin, 106 bacteria in 10 µl of HBSS with 0.1%
bovine serum albumin, and 20 µl of human AB plasma were
mixed in sterile 96-well plates. The plates were incubated in a shaking
water bath for 90 min at 37°C, and samples were taken at 0, 45, and
90 min. Two ml of sterile water was added to 10 µl of test solution
to lyse PMNs; 100 µl of this solution was added to 1 ml of sterile
water. This solution (100 µl) was then put on blood agar plates and
incubated overnight in 37°C; subsequently, CFU were counted. All
samples were run in duplicate. For comparison, the control PMNs from
the laboratory staff were tested for bactericidal capacity, and
bacterial samples in HBSS without PMN, as well as PMN samples without
bacteria, were run on agar plates to study normal growth and to exclude contamination.
Statistical assessment was performed with Student's t test
for paired samples where appropriate.
| |
RESULTS |
Three patients received 100 µg of IFN- per m2,
and five patients received 50 µg/m2 (Table 1). Few
adverse effects were reported, and IFN- was well tolerated, even in
the group receiving 100 µg/m2.
PMN NO production.
First, we assessed if the conditions
that the PMNs were subjected to in this study affected our
ability to detect the release of NO. To this end, we applied both
an electrochemical method, based on an NO-sensitive porphyrinic
electrode, and the spectrophotometric detection of NO-dependent, i.e.,
L-NMMA-inhibitable, oxidation of HbO2 to
methemoglobin. When activated with fMLP (1 µM), the production of NO
from control PMNs over 5 min (the reliable life span of the electrode
under these conditions) was 55.7 ± 18.3 pmol of
NO/106 PMNs/min (n = 6). This corresponded
well with similar measurements by the HbO2 method, as
applied in this study, on other aliquots of cells from the same cell
preparations producing 60.9 ± 15.2 pmol of NO/106
PMNs/min (n = 6). The release of NO from buffer-exposed
cells was not detectable with the electrode or with the
HbO2 method. Due to the paucity of cells and the limited
measurement times possible with the electrochemical method, the
HbO2 method was preferred for the measurements of the NO
production of the IFN--treated patients.
Prior to IFN- treatment, the CGD PMNs produced slightly more NO
following stimulation with fMLP than the controls did, 372 ± 27 (mean ± standard error of the mean) pmol of NO/106
PMNs at 45 min, compared with 343 ± 44 pmol of NO/106
PMNs for control PMNs (i.e., 108% ± 16% of the control value) (Fig.
1). On day 1 after IFN- treatment,
there was a slight increase of this function in the CGD PMNs,
to 132% ± 25% of the control value. On day 3 there was a
maximal enhancement of NO production, to 360% ± 37% (P < 0.001) of the control value. On day 8, the values were still
elevated, i.e., they were 280% ± 78% of the control value.
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FIG. 1.
NO production in CGD PMNs following in vivo IFN-
administration and stimulation with fMLP. Results are individual values
for CGD patients on each day of the experiment.  , patient 1;  ,
patient 2;  , patient 3; ×, patient 4;
 , patient 5;  , patient
6; +, patient 7;  , patient 8.
|
|
There was no significant difference in the increase of NO production
between the two different doses, since the patients receiving the
higher dose (n = 3), 100 µg/m2, increased
their PMN NO production on day 3 by a mean of 337% and the low-dose
group (n = 5) increased their NO production by a mean
of 350% after fMLP stimulation. There was no significant difference
between the improvement of the patients with X-linked disease and the
improvement of the patients with autosomal recessive disease.
We also tested whether IFN- might facilitate NO release from PMNs in
vitro. NO production following in vitro incubation of CGD PMNs with
1,000 U of IFN- (Fig. 2) revealed a
173% increase following fMLP stimulation (compared to samples without
IFN-), while control PMNs increased their NO release only 130%
(Fig. 2) following incubation with IFN- and fMLP stimulation.
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|
FIG. 2.
Effect of IFN- treatment of PMNs for 45 min in vitro
on activated NO release from PMNs. Cells were obtained from CGD
patients (n = 3) or healthy volunteers (controls)
(n = 2) and activated with fMLP at 1 µM. The release
of NO was monitored continuously for 45 min by the HbO2
method, as described in Materials and Methods. For each experimental
condition, samples were run in duplicate. All values given are the
L-NMMA-inhibitable responses and are expressed as
means ± standard errors of the means. **, P < 0.001.
|
|
PMN bactericidal capacity.
Bacterial growth without PMNs
present resulted in a 249% ± 122% (mean ± standard deviation
[SD]) increase in CFU after 90 min (compared to the starting sample,
with 100%). There was no contamination of bacteria in PMN preparations
or in HBSS in any of the samples. Control PMNs killed bacteria
efficiently, leaving 35.5% ± 11% residual bacteria alive after 90 min of incubation (Fig. 3). In contrast,
CGD PMNs showed no bactericidal capacity prior to IFN- treatment, as
evidenced by a growth level of 159% ± 80% CFU after 90 min, a figure
close to the bacterial growth in the absence of PMNs. In the
IFN--treated CGD patients, there was an improvement in the
bactericidal capacity of the PMNs on day 1 after IFN- treatment,
with 93% ± 45% residual bacteria after 90 min (Fig. 3).
However, the highest bactericidal capacity of the PMNs was on day
3, with 75% ± 14% residual bacteria (Fig. 3). On day 8 the
bactericidal capacity of the CGD PMNs was back to pretreatment values.
There was no significant difference between the improvement of the
patients with X-linked disease and the improvement of the patients with
autosomal recessive disease. Neither was there any significant
difference between low- and high-dose treatments.
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|
FIG. 3.
Bactericidal capacity of CGD PMNs after IFN-
treatment in vivo. CFU were counted after 90 min of incubation with
S. aureus. The shaded area illustrates the bactericidal
capacity of control PMNs (± SD); controls were not treated with
IFN-. Pretreatment there was no killing capacity of CGD PMNs at all,
leaving 159% residual bacteria, i.e., a bacterial growth. However, on
day 3 a maximal killing capacity, leaving 75% residual bacteria,
was noted. Error bars indicate SDs.
|
|
| |
DISCUSSION |
In this study we have evaluated the effects of IFN-, given s.c.
on two consecutive days, on the NO production and on the bactericidal
capacity of CGD PMNs. Our finding of enhanced NO production after this
treatment is contradictory to the conclusions of another study
(8), where no such effect could be found. However, profound
differences in study design and methods used for NO detection may
explain the differing results. In our study, we measured the release of
NO per se, whereas the other study used the measurement of the
metabolites NO2 and
NO3. Furthermore, our study evaluated the
effects of IFN- on NO production and microbicidal capacity after two
consecutive doses, and three patients received a higher dose than
normally used (100 µg/m2). The idea with that dose
regimen was to elicit a more powerful response and yet have tolerable
side effects. It has previously been shown that PMNs do not produce
NO2 or NO3 in the
absence of azide (18). However, a question which cannot be
resolved by the present data is the effect of IFN- treatment over a
prolonged period of time on PMN NOS activity.
Since CGD is mainly a defect in oxidative functions, initial reports on
oxidative functions of CGD patients after IFN- treatment suggested
enhanced superoxide production and increased gene expression for the
oxidase components, i.e., cytochrome b558
(10, 11). Some of these early studies were performed on
variant forms of CGD (defined as CGD PMNs with some NADPH rest
activity), revealing for some patients a normalization of superoxide
anion production (11). No such patients were included in our
study. In the placebo-controlled multicenter study of IFN- treatment
of CGD patients (17) and also in a follow-up study
(28), neither superoxide anion production nor bactericidal
capacity was improved in the IFN- group after a prolonged time of
treatment. In this study there was an effect on the PMN bactericidal
capacity that coincided timewise with the peak in NO production on day
3; however, we made evaluations only of two consecutive doses to
previously untreated patients. Publications on the
Aspergillus-damaging capacity of CGD PMNs after IFN-
treatment report that this function is increased (26). Furthermore, a previous study performed by some of our group
(2) revealed that the maximal
Aspergillus-damaging capacity seems to peak timewise with
the production of NO, as shown in this study on day 3 after IFN-
treatment. Other investigators have looked at other possible
explanations for the beneficial effect of IFN- on these patients,
for instance, antimicrobial proteins in the PMNs, but have found none
(25). Also, Fc receptor I expression is increased after
this treatment, as previously shown (2, 16).
Thus, we conclude that the present data strongly support the concept
that IFN- treatment of CGD patients is associated with an increased
production of NO from PMNs. Since these PMNs lack the capacity to
produce superoxide anions, it is conceivable that this increase in NO
release is one function of PMNs which could be considered to be, at
least in part, instrumental in augmenting host defense.
| |
ACKNOWLEDGMENTS |
This study was supported by grants from the Swedish Children's
Cancer Association, Stiftelsen Samariten, the Swedish Medical Research
Council (grants 19X-05991 and 19P-8884), the Funds of the Karolinska
Institute, Tore Nilson's Fund, the Funds of the Swedish Medical
Association, the Swedish Heart and Lung Fund, the Funds for Medical
Development in Southern Stockholm, the Swedish Association Against
Rheumatism, and Boehringer Ingelheim AB, Stockholm, Sweden.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Pediatrics, Sachs' Children's Hospital, Box 179 12, S-118 95 Stockholm, Sweden. Phone: 46-8-6164074. Fax: 46-8-6164014. E-mail:
anders.ahlin{at}sachsska.sos.sll.se.
| |
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Clinical and Diagnostic Laboratory Immunology, May 1999, p. 420-424, Vol. 6, No. 3
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
