* Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland; and
SAIC Frederick, Frederick Cancer Research and Development Center, Frederick, Maryland
Correspondence: Steven M. Holland, M.D., Laboratory of Host Defenses, National Institute of Allergy and Infectious Disease, National Institutes of Health, 10 Center Drive, Dr. MSc. 1886, Bethesda, MD 20892. E-mail: smh{at}nih.gov
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Key Words: chronic granulomatous disease • LTB4 • C5a • zileuton • superoxide
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In vitro, reactive oxidants have been shown to inactivate proinflammatory chemotactic factors including leukotrienes, C5a, and N-formyl peptide [6 7 8 ]. Based on these in vitro findings, the lack of reactive oxidant generation by phagocytes from patients with chronic granulomatous disease (CGD)—an inherited disorder of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in which superoxide production is defective—might be thought to lead to impaired degradation of these chemoattractants. Impaired clearance of chemotactic factors at sites of inflammation might in turn lead to increased neutrophil influx. However, to our knowledge, this hypothesis has never been verified in vivo.
In this study, we used naturally occurring C5-deficient (C5-) mice, transgenic 5-lipoxygenase-deficient (5LX-/-) mice, and the transgenic p47phox-/- mouse model of CGD to evaluate the interdependence of these three pathways in thioglycollate-elicited peritonitis.
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5LX-/- mice [10 ] and C57 x 129 wild-type F2 control mice (females 5–7 weeks) were purchased from Jackson Laboratories (Bar Harbor, ME). Congenic C5- and wild-type C57 control mice (females 10-14 weeks) were purchased from Jackson Laboratories.
All mice were housed under specific pathogen-free conditions at the National Institute of Allergy and Infectious Diseases (NIAID) veterinary facility (Bethesda, MD) and received autoclaved chow and acidified water. Only mice that were healthy were used in experiments. All experiments were approved by the NIAID animal care and use committee.
Thioglycollate challenge
Mice were injected intraperitoneally (IP) with 2 ml sterile
thioglycollate (3% wt/vol). Peritoneal exudates were collected at
various times after challenge with cold 10 ml Hanks’ balanced saline
solution (HBSS) or phosphate-buffered saline (PBS). In experiments
involving 5LX-/- mice, 8 ml lavages were used because the
mice were younger and smaller in size than in other experiments. Total
leukocyte count was determined by hemocytometer, and absolute
neutrophil counts were determined by cytospin and differential staining
of the leukocyte preparations.
LTB4 challenge
LTB4 was purchased from Sigma Chemical Co. (St.
Louis, MO). The ethanol solvent was evaporated under N2
flow, and LTB4 was resuspended in dimethyl sulfoxide (DMSO;
10 µg LTB4/ml DMSO). One microgram was injected IP per
mouse. Control mice received an equal volume (100 µl) of DMSO IP.
Peritoneal exudates were collected as described in the thioglycollate
experiments.
LTB4 measurement
Cell-free peritoneal fluid supernatants were frozen at -70°C
for subsequent measurement of LTB4, cytokines, and
chemokines. On the day of analysis, the samples were thawed on ice.
Ethanol was added to each sample to a final concentration (v/v) of
10%. The samples were acidified with 3% formic acid to pH 3.0 and
then adsorbed to a Bond Elut C18 reverse-phase column (Varian, Harbor
City, CA). The column was differentially eluted with H20,
10% ethanol, and n-hexane. Eicosanoids, including LTB4,
were eluted from the column with ethyl acetate. The samples were dried
under a stream of N2 and then reconstituted in PBS
containing 0.1% bovine serum albumin. The LTB4
concentration was determined using a competitive enzyme immunoassay
according to the manufacturer’s recommendations (PerSeptive
Diagnostics, Cambridge, MA). The recovery of mock samples was generally
>80%.
Measurement of the inactive 
-oxidation metabolites of
LTB4, 20-OH-LTB4 and 20-COOH-LTB4,
was determined by UV absorption as described previously with minor
modifications [11
]. The limit of detection of UV
absorption was 1.25 ng/10 µl reconstituted eiconasoid samples,
corresponding to a total recovery of 62.5 ng (1.25x50) product from
the peritoneal lavage.
Zileuton
Zileuton (Abbott, Abbott Park, IL), an orally active 5-LX
inhibitor, was purchased commercially as 600 mg tablets, approximately
50% of which is an active ingredient. The tablets were crushed and
suspended in H20 or 10% DMSO/H20. (The latter
solvent is preferred because of increased solubility.) Indicated
concentrations of zileuton were used, and zileuton was administered
subcutaneously (s.c.) or by gavage. A fresh suspension was made for
each experiment.
Dexamethasone and toradol
p47phox-/- and wild-type mice
(n=3–4 mice/group) were intravenously (IV) administered
saline (200 µl), dexamethasone (Elkins-Sinn, Cherry Hill, NJ; 0.2
mg/200 µl saline), or the nonsteroidal anti-inflammatory agent
toradol (Syntex Laboratories, Palo Alto, CA; 0.2 mg/200 µl saline).
Two hours later, mice were administered IP thioglycollate (2 ml). Mice
were sacrificed 4.5 h after thioglycollate, and peritoneal lavage
was performed as described above.
Cytokine and chemokine measurements
Concentrations of tumor necrosis factor-
(TNF-),
interleukin-1ß (IL-1ß), and the chemokines macrophage-inflammatory
protein-2 (MIP-2) and KC in cell-free peritoneal lavage
supernatants were determined by enzyme-linked immunosorbent assay
(ELISA) according to the manufacturer’s instructions (R&D Systems,
Minneapolis, MN).
Statistics
Student’s t-test was used to compare peritoneal
exudate white blood-cell counts. Correlation of peritoneal white
blood-cell counts and LTB4 concentration was assessed by
one-way analysis of variance (ANOVA; JMP software, SAS Institute, Cary,
NC).
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90% neutrophils, and
the remaining 10% was made up of macrophages and lymphocytes. As
expected, LTB4 from peritoneal fluid from
5LX-/- mice was not detectable (unpublished results).
When wild-type mice were pretreated with the 5-LX inhibitor zileuton
(6.25 mg active ingredient suspended in 250 µl 10%
DMSO/H20 administered per mouse by gavage), peritoneal
leukocytosis was reduced by about 50% to 1.2 ± 0.63 x
106 per ml compared with mice pretreated with vehicle
(P<0.005; Fig. 2
).
 View larger version (33K): [in a new window] |
Figure 1. Thioglycollate-elicited peritonitis in 5LX-/- mice. At
4.5 h after thioglycollate challenge, peritoneal white blood-cell
(WBC) exudate was reduced by 50% in 5LX-/-
(n=9) compared with wild-type controls (n=6;
P<0.001). Data are ± SD.
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 View larger version (21K): [in a new window] |
Figure 2. Thioglycollate-elicited peritonitis in C5- mice with and without
zileuton pretreatment. At 4.5 h after thioglycollate challenge,
peritoneal leukocytosis was reduced by 50% in C5- mice and in
wild-type mice pretreated with zileuton compared with wild-type mice
pretreated with vehicle. Zileuton pretreatment of C5- mice led to a
further reduction in peritoneal leukocytosis to almost unstimulated
levels. n = 9–10 mice per genotype per treatment
group. Data are ± SD.
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In earlier work, p47phox-/- mice generated approximately twofold greater neutrophil inflammatory response to thioglycollate compared with wild-type littermates at 5 h after challenge [9 ]. Figure 3 shows that p47phox-/- mice generated a more exuberant inflammatory response than wild-type mice that persisted for at least 15 h following thioglycollate challenge. This exudate was composed of approximately 90% neutrophils at 4.5 and 6 h and 80% neutrophils at 15 h after challenge in p47phox-/- and wild-type mice.
 View larger version (19K): [in a new window] |
Figure 3. Thioglycollate-elicited peritonitis in
p47phox-/- mice. Increased peritoneal white
blood-cell exudate occurred in p47phox-/- mice
(open circles) compared with wild-type controls (solid circles)
following thioglycollate challenge. n = 4–10 mice per
group per time point. Data are ± SD. *,
P < 0.05.
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90%
neutrophils, and the remaining 10% was made up of macrophages and
lymphocytes. Peritoneal leukocytosis correlated with LTB4
concentration in the cell-free peritoneal fluid supernatants
(correlation coefficient=0.8), but there was a broad range of total
recovered IP LTB4 (290–2100 pg/sample). In
p47phox-/- and wild-type mice pretreated with
zileuton, total recovered IP LTB4 was <200 pg/sample.
 View larger version (28K): [in a new window] |
Figure 4. Thioglycollate-elicited peritonitis in
p47phox-/- and wild-type mice pretreated with
subcutaneous zileuton or water. In water-pretreated mice, peritoneal
white blood-cell exudate was greater in
p47phox-/- than wild-type mice
(P<0.005), whereas zileuton eliminated the difference in
peritoneal exudate. n = 4 mice per group. Data are ± SD. *, P < 0.05; **, P < 0.005 relative to untreated, wild-type mice.
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5000/µl) in
wild-type, p47phox-/-, and
5LX-/- mice. Zileuton caused a 50% reduction in
circulating leukocytes in p47phox-/- and
wild-type mice at 6 h but had no effect on circulating leukocytes
in 5LX-/- mice. This argues that the effect of zileuton
is related to inhibition of leukotriene synthesis rather than a
nonspecific toxicity. Next, we evaluated whether zileuton administered by gavage at pharmacologic dosages proportionate by weight to regimens prescribed to humans would reduce thioglycollate-induced peritonitis in p47phox-/- mice. Mice were administered zileuton followed by thioglycollate challenge 1 h later. Peritoneal leukocytosis 4.5 h after thioglycollate challenge was reduced in mice pretreated with zileuton (0.5 mg active ingredient) compared with mice pretreated with an equal volume of solvent alone (10% DMSO/water; Fig. 5 ). A dose-dependent suppression by zileuton of peritoneal white blood-cell exudate was observed (one-way ANOVA; P<0.005), and peritoneal exudate was closely correlated with LTB4 concentration in lavage specimens (correlation coefficient=0.88). In contrast to zileuton, pretreatment with IV dexamethasone or toradol, a nonsteroidal anti-inflammatory agent, had no effect on thioglycollate-elicited peritonitis in p47phox-/- or wild-type mice (unpublished results).
 View larger version (15K): [in a new window] |
Figure 5. Suppression of thioglycollate-induced peritonitis by zileuton in
p47phox-/- mice. At 4.5 h following
thioglycollate challenge, pretreatment with zileuton by gavage
suppressed peritoneal white blood-cell exudate in a dose-dependent
fashion (one-way ANOVA; P<0.005). n = 5–8
mice per group. Data are ± SD.
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 View larger version (15K): [in a new window] |
Figure 6. Peritoneal leukocytosis in p47phox-/- (open
circles) and wild-type mice (solid circles) following IP challenge with
LTB4 (1 µg). At 30 min, the peritoneal white-cell count
remained basal. By 18 h after challenge, peritoneal leukocytosis
was greater in p47phox-/- than wild-type mice.
Data are ± SD. *, P < 0.05.
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 View larger version (16K): [in a new window] |
Figure 7. Serum LTB4 concentration and total recovered IP
LTB4 at 10 and 30 min after IP challenge with
LTB4 (1 µg). The elimination half-life of
LTB4 from the peritoneal cavity was less than 10 min in
both genotypes. A small proportion of LTB4 was recovered in
serum. n = 5–6 mice. Data are ±
SD.
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 View larger version (21K): [in a new window] |
Figure 8. LTB4 concentration in p47phox-/-
and wild-type mice 3 h after IP challenge with LTB4 or
solvent (DMSO). LTB4 recovery was more than
fivefold greater in peritoneal collections from
p47phox-/- than wild-type mice.
n = 4 mice per genotype per treatment group. Data
are ± SD. *, P = 0.01.
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-oxidation to the inactive metabolites
20-OH-LTB4 and 20-COOH-LTB4 by leukotriene
B4
-hydroxylase
[12
13
14
]. Neither metabolite was detected in peritoneal
supernatants from wild-type or p47phox-/- mice
at 30 min or at 3 h after IP challenge with LTB4 (1
µg). The limit of detection of the assay used for the metabolites was
62.5 ng per total peritoneal fluid sample. In p47phox-/- and wild-type mice challenged with thioglycollate, IP production of IL-1ß and neutrophil-chemoattractant CXC chemokines, MIP-2 and KC, was robust and similar at 4.5 h (unpublished results). In contrast to thioglycollate-elicited peritonitis, direct IP challenge with LTB4 resulted in minimal levels of these proinflammatory cytokines in cell-free, peritoneal-lavage supernatants collected at 3 and 18 h after challenge.
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Mechanisms of leukotriene degradation are complex and
incompletely understood. In vitro, leukotriene B4
-hydroxylase converts
LTB4 to the inactive metabolite
-20-OH-LTB4, which can in turn be
converted to
-20-COOH-LTB4
[12
13
14
]. LTB4 is a ligand and activator of
peroxisome proliferator-activated receptors, a group of transcription
factors that up-regulate expression of hepatic enzymes involved in
systemic clearance of LTB4 [17
].
In vitro, leukotrienes undergo rapid enzyme-independent degradation by
reactive oxidants [7
, 8
]. Supernatants
recovered from stimulated neutrophils from CGD and
myeloperoxidase-deficient patients contained higher levels of
LTB4 and LTC4 than those from normal
neutrophils [7
]. By adding radioactive LTC4
to neutrophils and cell-free reactive oxidant-generating systems, it
was shown that reactive oxidants were necessary and sufficient to
degrade LTC4 [7
].
Stimulating normal neutrophils but not CGD neutrophils with phorbol
myristate acetate (PMA), an activator of reduced nicotinamide adenine
dinculeotide phosphate (NADPH) oxidase, led to reduced recovery of
endogenously produced LTB4 [8
].
In fact, PMA stimulation of normal neutrophils led to reduced recovery
of the
-oxidation products, suggesting that
NADPH oxidase-mediated degradation of LTB4 is distinct from
the leukotriene
-hydroxylase pathway. The
chemotactic activity of LTB4 is dependent on its two
hydroxyl groups and its triene portion [18
], potential
sites at which reactive oxidants could alter the activity of
LTB4. The relevance of these findings to leukotriene
degradation in vivo, in particular in the setting of CGD in which
phagocytes are defective in generating reactive oxidants, has remained
uncertain.
Evidence of abnormal neutrophil-inflammatory responses exists in CGD patients and in genetically engineered mouse models. In an experimental skin window model, neutrophil exudate was increased in male CGD patients compared with normal volunteers [19 ]. Consistent with these findings, in the p47phox-/- [9 ] and X-linked [20 ] mouse models of CGD, increased neutrophil peritoneal leukocytosis occurred in CGD mice compared with wild-type littermates following IP challenge with the sterile irritant, thioglycollate. In addition, X-linked CGD mice generate enhanced inflammatory responses to intratracheal challenge with killed Aspergillus fumigatus hyphae [21 ]. These studies indicate that excessive inflammation in CGD is not solely the result of unresolved infection but results from an intrinsic defect in the control of inflammation. CGD mice provide an excellent opportunity to further evaluate mechanisms of regulation of inflammatory responses in vivo.
Recovery of peritoneal LTB4 was similar between p47phox-/- and wild-type mice at 10 and 30 min after IP challenge, but by 180 min, peritoneal LTB4 levels were approximately fivefold greater in p47phox-/- mice (Figs. 7 and 8) . These data are consistent with the role of a NADPH oxidase-mediated breakdown of LTB4. Within the first 30 min after IP LTB4, no significant neutrophil influx occurs (Fig. 6) , but by 3 h, a modest neutrophil accumulation was present in wild-type and p47phox-/- mice, providing a potential source of reactive oxidants in wild-type mice capable of degrading leukotrienes.
Even in p47phox-/- mice, recovered
total peritoneal LTB4 at 180 min after IP challenge was
<1% of the administered dose, indicating that mechanisms independent
of NADPH oxidase contributed to removal of LTB4.
Enzyme-catalyzed
-oxidation is not likely to be
a major factor in inactivating LTB4 because metabolites of
this pathway were not detected in peritoneal supernatants (limit of
detection=62.5 ng/total sample). Because LTB4 is a small,
20-carbon, lipophilic, rapidly diffusible molecule, passive diffusion
of LTB4 from the peritoneal cavity may play an important
role in the removal of LTB4 from inflammatory sites.
Experiments in which LTB4 was administered into joint
spaces of rabbits showed that most of the administered dose was
recovered in the serum 7 min after challenge [3
].
However, as shown in Figure 8 , less than 5% of the administered IP
LTB4 was recovered in the serum at 10 and 30 min after
challenge. It is possible that LTB4 diffused to other
compartments, such as mesenteric fat.
Direct IP administration of LTB4 led to a mild peritoneal
leukocytosis (Fig. 6)
and virtually no elaboration of proinflammatory
cytokines and chemokines. Thus, LTB4 by itself is a weakly
proinflammatory agent in this setting. However, LTB4 is
clearly important in driving the inflammatory response in
thioglycollate-elicited peritonitis based on the following findings:
Peritoneal leukocytosis was reduced by 50% in 5LX-/-
mice (Fig. 1)
, and peritoneal leukocytosis was correlated with
peritoneal LTB4 levels and was suppressed by pharmacologic
inhibition of leukotriene synthesis (Figs. 3
and 4)
. Thus,
LTB4 likely acts with other mediators to generate the full
inflammatory response.
Neither pretreatment with dexamethasone nor toradol affected thioglycollate-elicited peritonitis in p47phox-/- or wild-type mice. Ribeiro et al. [22 ] have shown that pretreatment of rats with dexamethasone but not indomethacin supressed neutrophil migration elicited by IP administration of LTB4. It is possible that corticosteroids may directly suppress the chemoattractant activity of LTB4 without affecting peritoneal leukocytosis following thioglycollate, which elicits a more robust inflammatory response driven by more than a single mediator.
Thioglycollate-elicited peritonitis was reduced in C5- mice by 50%
compared with wild-types, suggesting that peritonitis in this model is
complement-dependent (Fig. 2)
. These observations are intriguing
because reactive oxidants generated by NADPH oxidase in combination
with the myeloperoxidase-halide system have been shown to inactivate
human C5a in vitro [6
]. The lack of C5b activation in
C5- mice may also contribute to reduced thioglycollate-elicited
peritoneal leukocytosis. Peritoneal neutrophils from mice with a
targeted disruption of the LTB4 gene
(BLTR-/-) had normal chemotaxis and calcium
mobilization in response to C5a [23
]. However, in vivo,
LTB4 and C5a may act in concert to elaborate the
inflammatory response [3
]: LTB4 may prime
the inflammatory response by enhancing neutrophil-endothelial cell
adhesion [4
, 5
], and C5a, which is far less
diffusible, may serve to further augment and sustain the response. This
hypothesis is consistent with the observation that C5a but not
LTB4 is elevated in exudates from experimental skin
blisters in humans compared with serum levels [24
]. In
our study, zileuton pretreatment led to a further significant reduction
in thioglycollate-elicited peritonitis in C5- mice, suggesting that
leukotrienes may act independently of C5--dependent pathways in
augmenting inflammation. Reactive oxidants generated by activated
phagocytes may modulate the inflammatory cascade at two levels by
inactivating LTB4 and C5a. Therefore, lack of oxygen
radical formation in CGD may predispose patients to excessive
inflammatory responses.
Taken together, our study suggests that leukotrienes and C5a drive thioglycollate-elicited peritonitis. In C5- mice pretreated with the 5-LX inhibitor zileuton, peritoneal leukocytosis was close to unstimulated levels and was significantly reduced compared with both wild-type mice pretreated with zileuton and C5- mice pretreated with vehicle, suggesting that these two pathways can act independently. In addition, impaired metabolism of LTB4 in the p47phox-/- mouse may lead to enhanced thioglycollate-elicited inflammation. Leukotriene antagonism may be an attractive therapeutic target in controlling abnormal, inflammatory responses in CGD.
Received July 20, 2000; revised September 3, 2001; accepted September 8, 2001.
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