B and proteasome activity are inhibited following exposure to inhaled isobutyl nitrite
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Correspondence: Lee S. F. Soderberg, Ph.D., Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205. E-mail: Soderberglees{at}exchange.uams.edu
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B activation
in PEM from mice exposed to the inhalant corresponded to reduced
degradation of the NF-B inhibitor, IB
. Proteasome-associated,
enzymatic activity was compromised in PEM from inhalant-exposed mice,
suggesting that inhaled isobutyl nitrite compromised macrophage,
tumoricidal activity by inhibiting proteasomal degradation of the
NF-B inhibitor, IB.
Key Words: inhalant • signal transduction • immunosuppression • Kaposi’s sarcoma • AIDS
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To study inhalant-induced immunotoxicity, we have developed a mouse inhalation-exposure model. Abusers expose themselves to very high levels of nitrite inhalants, estimated to be 7000 ppm [7 ]. Such exposures are brief but are repeated frequently by heavy abusers. Some individuals abuse inhalants daily. For our animal model of exposure, we settled on a lower but constant dose of 900-ppm isobutyl nitrite for 45 min. This regimen provides a reproducible exposure, which approximates the overall exposure levels of heavy abusers. We demonstrated previously that inhalation exposure of mice to 900-ppm isobutyl nitrite for 45 min/day for 14 days impaired T-dependent antibody responses [8 ], the induction of cytotoxic T cells [9 ], and macrophage-tumoricidal activity [10 ]. Inhalation exposure to the nitrite inhalant was sufficient to increase the incidence of tumor formation from 21% to 75% in mice injected with limiting numbers of syngeneic tumor cells [11 ]. Macrophages and perhaps other accessory cells appeared to be the primary target of immunotoxicity [9 ].
Resident peritoneal macrophages maintain relatively low levels of
activity. Inflammatory stimuli increase the number and activity of
peritoneal macrophages, and stimulation with lipopolysaccharide (LPS)
and interferon-
(IFN-) boosts the activity of resident and
inflammatory macrophages. The binding of LPS to CD14 and the toll-like
receptor-4 initiate a cascade of protein kinases, which phosphorylates
the inhibitor (IB) of the ubiquitous transcription nuclear factor,
NF-B [12
, 13
]. Phosphorylated IB is
then ubiquitinated and degraded by proteasomes. The liberated NF-B
then moves into the nucleus and binds to B-binding sites, promoting
the expression of many genes important in host defense. Genes dependent
on activation of NF-B include inducible nitric oxide synthase
(iNOS), which encodes the enzyme that oxidizes L-arginine to form
citruline, liberating NO [14
]. The present data suggest
that inhalation exposure to isobutyl nitrite inhibited macrophage
activation of NF-B, at least in part, by interfering with
proteasomal degradation of IB.
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Cell culture
Macrophages were harvested by peritoneal lavage. Peritoneal
exudate macrophages (PEM) were collected 4 days after intraperitoneal
injection with 1 ml 3% thioglycollate. Resident peritoneal macrophages
(RPM) were harvested from mice not receiving thioglycollate.
Macrophages were collected by lavage and cultured at 5 x
105 cells/well in 96-well plates. Cells were maintained in
RPMI 1640 medium, supplemented with 10% fetal calf serum, 50 nM
2-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin.
After 1 h incubation, nonadherent cells were removed by washing.
Macrophages were stimulated with 100 U/ml recombinant IFN- (Genzyme,
Boston, MA) and 1 µg/ml LPS (Escherichia coli 05:B5; Sigma
Chemical Co., St. Louis, MO).
Macrophage tumoricidal activity
Macrophage tumoricidal activity was measured by a standard
51Cr-release assay [10
]. Briefly,
macrophages from control and nitrite-exposed mice were cultured in
medium or activated overnight with IFN- and LPS.
51Cr-labeled, target P815 cells were then added to
triplicate cell cultures at an effector:target cell ratio of 10:1.
After 18 h incubation, the plates were centrifuged at 250 g
for 10 min, and 100 µl supernatant from each well was counted in a
gamma counter (Packard Instrument Co., Meridan, CT). Control
wells contained 51Cr-labeled target cells alone for
spontaneous release or labeled target cells with 0.5% Nonident P-40
(NP-40) for total release. Data are shown as the mean percent
cytotoxicity ± SE using the mean percent lysis of
five individual mice. Significance was determined by t-test.
Nitric oxide assay
Macrophage cultures were prepared and activated as described
above. Supernatants were collected after 24 h and assayed by the
Griess assay for inducible NO production as described
[15
]. Briefly, 100 µl aliquots of supernatant or
sodium nitrite standards was combined with equal volumes of fresh
Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine
dihydrochloride, 2.5% H3PO4) and vortexed.
Tubes were then incubated at room temperature for 10 min, and the
absorbance was measured at 540 nm. The concentration of
NO2- was determined using a sodium nitrite
standard curve.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were obtained, and EMSA was performed as
described previously [16
]. Briefly, levels of nuclear
NF-B were determined by incubating equal amounts of nuclear protein
with a 32P-labeled oligonucleotide probe specific for kappa
binding (Santa Cruz Biotechnology, Santa Cruz, CA), followed by EMSA.
The binding specificity of the NF-B complex was determined by the
inclusion of a 100-fold molar excess of unlabeled probe as a specific
competitor. Similar quantities of a nonbinding mutant probe were also
used in the assay to establish specificity. All fine chemicals, unless
otherwise mentioned, were obtained from Sigma, and electrophoresis
supplies were from Bio-Rad (Hercules, CA). Radioactive bands of EMSA
were detected and quantified using a Phosphoimager (Molecular Dynamics,
Sunnyvale, CA).
Western blotting
Cytosolic extracts for Western blotting were prepared by
homogenization of cells in lysis buffer (1 mM HEPES, 10 mM KCl, 1.5 mM
MgCl2, 1 mM sodium orthovanadate, and 0.5% NP-40).
Protease inhibitors were added prior to use [16
]. Cell
lysates equalized for protein (40 µg) were resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to
nitrocellulose, immunoblotted with antibody specific to iNOS or
IB (Santa Cruz Biotechnology), and detected using
anti-immunoglobulin G (IgG) coupled to horseradish-peroxidase
(Transduction Laboratories, Lexington, KY) followed by enhanced
chemiluminescence (ECL; Amersham, Arlington Heights, IL). Nuclear
extracts were electrophoresed and immunoblotted similarly with
antibodies specific for NF-B subunits p65, p50, and c-Rel (Santa
Cruz Biotechnology).
Determination of proteasome activity
Proteasome-enriched fractions were prepared as described
[17
]. Briefly, 25 µg of this cytosolic fraction was
incubated in 200 µl containing 50 mM Tris-HCl (pH 7.8), 10 mM
MgCl2, 1 mM dithiothreitol (DTT), and 0.5 mM fluorogenic
peptide, Suc-leu-leu-val-tyr-Amido-methyl-coumarin (Sigma), in the
presence or absence of 2 mM adenosine 5'-triphosphate (ATP). After
incubation for 1 h at 37°C, the reaction was quenched with 1 ml
ice-cold ethanol. Hydrolysis of the fluorogenic peptide was determined
by measuring fluorescence on a spectrofluorometer using an excitation
wavelength of 380 nm and emission of 440 nm under linear conditions
[18
].
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and LPS, and tested for tumoricidal
activity using P815 target cells in a standard 18 h
51Cr-release assay. As shown in Figure 1
, the tumoricidal activity of activated, resident macrophages was
not affected significantly by inhalant exposure. However, the
cytotoxicity of activated, inflammatory macrophages was reduced by 26%
(P<0.001) following inhalant exposure. In control
mice, thioglycollate increased the tumoricidal activity of activated,
peritoneal macrophages by 4.3-fold (P<0.001). In
mice exposed to the inhalant, thioglycollate increased (3.5-fold,
P<0.001) macrophage cytotoxicity but by a lesser extent
than with cells from control mice (P<0.05). Because
NO is critical to murine-macrophage-tumoricidal activity when P815
target cells are used [10
], we measured NO production by
RPM and PEM. As shown in Figure 2
, inflammatory-macrophage production of NO was reduced by 28% in
inhalant-exposed mice, and NO production by resident macrophages was
not affected significantly by the inhalant. Thioglycollate elicitation
increased NO production by 5.4-fold in control mice, and in
inhalant-exposed mice, the increase (fourfold) was significantly
(P<0.05) lower. Macrophage production of NO depends
on induction of the NO-generating enzyme, iNOS. Consistent with reduced
NO production, Western blots showed that iNOS was induced by activation
of PEM, but the levels of iNOS protein were 30% lower in cells from
inhalant-exposed mice compared with control cells (Fig. 3
). In vivo, the reductions in tumoricidal activity and NO
production are likely exacerbated by the reductions in cell numbers in
mice exposed to the inhalant. |
View this table: [in a new window] |
Table 1. Effects of Inhaled Isobutyl Nitrite on the in vivo Response of
Macrophages to Thioglycollate
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Figure 1. Inhalant exposure impaired PEM- but not RPM-tumoricidal activity.
Groups of mice were exposed to air or inhaled isobutyl nitrite (900
ppm, 45 min/day for 14 days). PEM elicited with thioglycollate or RPM
were cultured in medium alone or activated with IFN- and LPS
overnight. Tumoricidal activity was measured by a standard 18-h
51Cr-release assay. The data, expressed as percent
cytotoxicity ± SE, represent the results of three
separate experiments.
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Figure 2. Inhalant exposure inhibits inducible NO production by PEM but not RPM.
Macrophages from mice treated as described in Figure 1
were assayed for
NO production with and without activation with IFN- and LPS.
Inducible NO was measured by the Griess assay. * P <
0.001.
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Figure 3. Activation-induced expression of iNOS was inhibited in PEM following
inhalant exposure. Groups of mice were exposed to 0- or 900-ppm
isobutyl nitrite 45 min/day for 14 days. Lysates were prepared from
pooled-activated or nonactivated PEM and assayed for iNOS protein by
Western blots. The results of four experiments showed a mean reduction
of 30% (P<0.05, paired t-test) in the
nitrite group.
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B, we determined the effect of exposure to isobutyl nitrite on
the nuclear induction of NF-B. Peritoneal-exudate macrophages from
mice exposed to air or isobutyl nitrite were activated with IFN- and
LPS, and after 4 h, nuclear extracts were prepared. The macrophage
nuclear extracts were analyzed using EMSA for NF-B binding to a
specific probe. As shown in Figure 4
, stimulation of elicited macrophages from control mice induced a
marked increase in nuclear NF-B. However, stimulation of cells from
inhalant-exposed mice did not increase nuclear NF-B above basal
levels and were much lower than the levels in stimulated control cells.
Binding specificity of the probe was established using a 100-fold
excess of unlabeled probe, which competed successfully with the binding
of the labeled probe. Excess, unlabeled, mutated probe (nonbinding) did
not affect binding of the labeled probe. Western blots of nuclear
extracts showed that the increase in nuclear NF-B upon stimulation
of control cells was largely a result of an increase (4.4-fold) in the
p65 subunit (Fig. 5
). Subunit p50 accumulated in macrophage nuclei to a lesser extent,
and c-Rel was present in the nuclei of unstimulated cells
constitutively. Nuclear p65, p50, and c-Rel proteins were reduced
greatly in cells from nitrite-exposed mice and increased only to low
levels after cell stimulation. Degradation of the NF-B inhibitor,
IB, is necessary for NF-B nuclear translocation, so we measured
macrophage IB levels by Western blots. As shown in Figure 6
, 34% of cytosolic IB was degraded in macrophages from
control mice following activation with IFN- and LPS and in cells
from mice exposed to the inhalant, IB, was only degraded slightly
(8%)
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Figure 4. Inhalant exposure inhibited macrophage NF-B activation. PEM from
mice treated with air (A) or 900-ppm isobutyl nitrite (B) as described
in Figure 3
were cultured for 4 h without activation (U) or
stimulated with IFN- and LPS (S). Nuclear extracts were then
prepared and assayed for NF-B binding to a 32P-labeled
oligonucleotide (B consensus-sequence probe). Binding specificity was
established with 100-fold excess, unlabeled probe (C) or 100-fold
excess of mutant (nonbinding) probe (M).
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Figure 5. Inhalant exposure reduced nuclear NF-B subunits p65, p50, and c-Rel.
PEM from mice treated as described in Figure 3
were cultured with or
without IFN- and LPS. After 4 h, nuclear lysates were prepared
and assayed by Western blot for p65, p50, and c-Rel.
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Figure 6. Activation-induced degradation of IB was inhibited by inhalant
exposure. Cytosolic extracts were prepared from PEM, cultured overnight
with or without IFN- and LPS. Western blots were used to measure
IB.
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B is degraded by proteasomes normally, we next tested
the ability of proteasome-rich fractions of macrophages to hydrolyze
the synthetic peptide, Suc-Leu-Leu-Val-Tyr-AMC, in the presence of ATP.
Basal levels of proteasome activity in macrophages from mice exposed to
the inhalant were similar to the basal levels of control cells
(Fig. 7
). Activation of control macrophages doubled the basal level of
hydrolysis of the peptide. However, unlike control cells, proteasome
activity did not increase following activation in cells from
nitrite-exposed mice.
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Figure 7. Macrophage proteasome-associated enzymatic activity was inhibited
following inhalant exposure. Proteasome-enriched extracts were prepared
following overnight activation of PEM from treated and control mice.
Proteasome activity was measured at 380 nm and 440 nm as hydrolysis of
a chymotrypsin-sensitive fluorogenic peptide.
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Independent of the cellular loss, exposure to the inhalant did not affect the activities of resident peritoneal macrophages, and inflammatory macrophage function was compromised. The tumoricidal and inducible NO activities of activated, resident macrophages are normally at low levels compared with activated, inflammatory cells, and these activities were not reduced significantly following exposure to the inhalant. Inflammatory macrophages, which provide an important early line of defense, produced less NO, consistent with lower iNOS induction, and were less able than control-inflammatory cells to kill tumor cells following exposure to the inhalant. These results extended earlier findings [10 ] of murine PEM activity, which also showed that inducible NO was critical to tumoricidal activity using P815 target cells. The iNOS inhibitor, NGMMA, blocked NO induction and tumoricidal activity totally in murine PEM [10 ]. In the absence of cell activation, neither resident nor elicited peritoneal macrophages produced significant amounts of NO. Thus, the measured nitrite levels can be attributed to inducible NO not to residual isobutyl nitrite. Consistent with impaired macrophage tumoricidal activity, we demonstrated previously [11 ] that mice exposed to inhaled isobutyl nitrite had fourfold increases in the incidence and a growth rate of tumors that developed from injected syngeneic (PYB6) tumor cells.
The reduced responsiveness of inflammatory macrophages from mice
exposed to the inhalant was probably related to interference with the
signaling cascade. We found that activation-induced increases in
nuclear NF-B were blocked essentially in peritoneal exudate
macrophages from mice exposed to the inhalant. The reduced NF-B
activation was consistent with a 75% reduction in activation-induced
degradation of its inhibitor, IB. A cascade of kinases stimulated
by LPS results in IB phosphorylation. Phospho-IB is then
ubiquitinated and degraded by proteasomes, allowing NF-B to move
into the nucleus [21
]. The observed decrease in IB
degradation may have been a result of reduced phosphorylation of the
IB or to reduced proteasome activity or both. Preliminary data
suggest that the level of activation-induced cytosolic phospho-IB
was lower in cells from inhalant-exposed mice, suggesting that inhalant
exposure impaired upsteam kinase activity and/or induction. The
present data show that proteasome-chymotryptic activity, consistent
with IB proteolysis, was impaired severely in activated
macrophages from mice exposed to the inhalant. Exogenous NO has been
shown to inhibit proteasomal degradation of IB
[22
], suggesting that inhaled isobutyl nitrite might act
by a similar mechanism. Although isobutyl nitrite was shown to liberate
NO, inhaled NO at a concentration (115 ppm) equivalent to that produced
by 900-ppm isobutyl nitrite did not alter macrophage tumoricidal
activity [23
]. This suggests that the inhalant, not
released NO, was responsible for inhibiting proteasome activity. In
addition to modulating signal transduction, defective proteasome
activity would be expected to affect cell cycling and the processing of
cytosolic antigens. The reduced proteasome function may play a direct
role in the impaired lymphocyte-proliferative responses and cytolytic
T-lymphocyte (CTL) induction following exposure to inhaled isobutyl
nitrite [9
, 24
].
Although epidemiological studies have identified heavy abuse of nitrite
inhalants as an independent risk-factor for HIV infection
[3
, 4
] and for Kaposi’s sarcoma
[5
, 6
], the mechanisms involved have not
been elucidated. Our data suggest that inhalant exposure impaired
cell-mediated immunity, at least partially, by disrupting a critical
signaling cascade leading to cellular activation. We have demonstrated
that inhalant-induced immunosuppression promoted tumor growth
[11
], and it would be expected to also reduce resistance
to virus infection. In addition, the present study showed that inhalant
exposure impaired macrophage NF-B activation, which is important to
HIV replication [25
]. It has been suggested that
inhibition of NF-B in macrophages could contribute to the extended
asymptomatic state early in HIV infections [22
].
However, we also found that inhalant-exposure inhibited inducible NO.
Inhibition of inducible NO promoted HIV-LTR, transcriptional activation
in human cells [25
], suggesting that conflicting
influences may be induced by inhalant exposure.
Received August 3, 2000; revised December 4, 2000; accepted December 5, 2000.
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and inducible nitric oxide production by alveolar macrophages after exposure to a nitrite inhalant J. Leukoc. Biol. 60,459-464[Abstract] and IkappaB-ß proteolysis by calpain inhibitor I blocks nitric oxide synthesis Arch. Biochem. Biophys. 335,388-395[Medline]This article has been cited by other articles:
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