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(Journal of Leukocyte Biology. 2001;69:639-644.)
© 2001 by Society for Leukocyte Biology

Inflammatory macrophage nuclear factor-{kappa}B and proteasome activity are inhibited following exposure to inhaled isobutyl nitrite

Usha Ponnappan and Lee S. F. Soderberg

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A history of abuse of nitrite inhalants has been correlated with HIV seropositivity and Kaposi’s sarcoma. A series of 14 daily, 45-min exposures of mice to 900-ppm isobutyl nitrite in an inhalation chamber reduced the number of peritoneal exudate macrophages (PEM) by 35% and the number of resident peritoneal macrophages (RPM) by 18%. Although the tumoricidal activity of RPM was not affected by the inhalant, the cytotoxicity of PEM was reduced by 26%. The induction of nitric oxide (NO) and the inducible NO synthase (iNOS) protein in PEM were inhibited by the inhalant to a similar extent. Inhibition of NF-{kappa}B activation in PEM from mice exposed to the inhalant corresponded to reduced degradation of the NF-{kappa}B inhibitor, I{kappa}B{alpha}. 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-{kappa}B inhibitor, I{kappa}B{alpha}.

Key Words: inhalant • signal transduction • immunosuppression • Kaposi’s sarcoma • AIDS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amyl, butyl, cyclohexyl, and isobutyl nitrite inhalants have been abused widely, principally by male homosexuals. Abuse of nitrite inhalants, which was pervasive among male homosexuals in the early 1980s [1 ], has declined with AIDS education but remains high. One-third of a cohort of men who have sex with men was shown recently to abuse nitrite inhalants [2 ]. Epidemiological studies have correlated heavy abuse of these inhalants with HIV seropositivity [3 , 4 ] and with Kaposi’s sarcoma in AIDS patients [5 , 6 ]. Nitrite-inhalant abuse could act as a risk factor for HIV infection or Kaposi’s sarcoma if it impaired immune resistance to virus infection or tumor growth.

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-{gamma} (IFN-{gamma}) 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 (I{kappa}B) of the ubiquitous transcription nuclear factor, NF-{kappa}B [12 , 13 ]. Phosphorylated I{kappa}B is then ubiquitinated and degraded by proteasomes. The liberated NF-{kappa}B then moves into the nucleus and binds to {kappa}B-binding sites, promoting the expression of many genes important in host defense. Genes dependent on activation of NF-{kappa}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-{kappa}B, at least in part, by interfering with proteasomal degradation of I{kappa}B{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal exposure
Isobutyl nitrite (Aldrich Chemical Co., Milwaukee, WI) was stored at 4°C under nitrogen. The inhalant was vaporized in a flask and quantified with a halothane monitor (Puritan-Bennet, Model 222; Datex, Tewkesbury, MA) calibrated for isobutyl nitrite. Groups of five, 6–8-week-old, female C57BL/6 mice were exposed to air or to 900-ppm isobutyl nitrite in an inhalation chamber for 45 min/day for 14 days. Mice were assayed individually for cytotoxicity and NO production 1 day after the last exposure to isobutyl nitrite. Cells were pooled for other assays. Results are representative of at least two separate experiments.

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-{gamma} (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-{gamma} 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-{kappa}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-{kappa}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 I{kappa}B{alpha} (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-{kappa}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 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Because the nitrite inhalant appears to target macrophages, we examined macrophage function and their in vivo responses to inflammatory induction. Groups of mice were exposed to 0- or 900-ppm isobutyl nitrite for 45 min/day for 14 days, and peritoneal cells were collected 1 day after the last exposure. Subchronic exposure of mice to isobutyl nitrite reduced the number of resident peritoneal cells by 18% (P<0.05), and the number of peritoneal-exudate cells responding to thioglycollate was depleted by 35% (P<0.001; Table 1 ). Thus, in control mice, elicitation with thioglycollate doubled the number of peritoneal cells (P<0.001), but in inhalant-exposed mice, peritoneal-cell numbers only increased by 60% (P<0.005) in response to thioglycollate. To measure macrophage function, cell cultures were corrected for cell numbers, activated overnight with IFN-{gamma} 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.


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  		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-{gamma} 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-{gamma} 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.

 
Because the expression of iNOS is regulated by transcription factor NF-{kappa}B, we determined the effect of exposure to isobutyl nitrite on the nuclear induction of NF-{kappa}B. Peritoneal-exudate macrophages from mice exposed to air or isobutyl nitrite were activated with IFN-{gamma} and LPS, and after 4 h, nuclear extracts were prepared. The macrophage nuclear extracts were analyzed using EMSA for NF-{kappa}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-{kappa}B. However, stimulation of cells from inhalant-exposed mice did not increase nuclear NF-{kappa}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-{kappa}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-{kappa}B inhibitor, I{kappa}B, is necessary for NF-{kappa}B nuclear translocation, so we measured macrophage I{kappa}B{alpha} levels by Western blots. As shown in Figure 6 , 34% of cytosolic I{kappa}B{alpha} was degraded in macrophages from control mice following activation with IFN-{gamma} and LPS and in cells from mice exposed to the inhalant, I{kappa}B{alpha}, was only degraded slightly (8%)



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  		Figure 4. Inhalant exposure inhibited macrophage NF-{kappa}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-{gamma} and LPS (S). Nuclear extracts were then prepared and assayed for NF-{kappa}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-{kappa}B subunits p65, p50, and c-Rel. PEM from mice treated as described in Figure 3 were cultured with or without IFN-{gamma} 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 I{kappa}B{alpha} was inhibited by inhalant exposure. Cytosolic extracts were prepared from PEM, cultured overnight with or without IFN-{gamma} and LPS. Western blots were used to measure I{kappa}B{alpha}.

 
Because I{kappa}B{alpha} 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Exposure to inhaled isobutyl nitrite reduced the numbers of resident and inflammatory macrophages. The number of inflammatory cells was reduced by 35%, double the reduction (18%) in resident macrophages. It is likely that there were fewer cells available to respond to thioglycollate, because we found previously that subchronic, inhalant exposure reduced spleen cellularity by 36% and peripheral blood leukocyte counts by 32% [7 ]. Cells resident in the peritoneal cavity were, perhaps, less exposed to the inhalant. In contrast, the numbers of alveolar macrophages actually increased following inhalant exposure [19 ]. This probably reflected a primary inflammatory response, however the increase in cell numbers in the lungs was very small and could account for only a tiny proportion of the cells lost from other sites. We found that mouse erythrocytes exposed to the inhalant in vitro showed increased binding to control macrophages, suggesting that altered cells may be cleared by the liver and spleen [20 ].

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-{kappa}B were blocked essentially in peritoneal exudate macrophages from mice exposed to the inhalant. The reduced NF-{kappa}B activation was consistent with a 75% reduction in activation-induced degradation of its inhibitor, I{kappa}B{alpha}. A cascade of kinases stimulated by LPS results in I{kappa}B{alpha} phosphorylation. Phospho-I{kappa}B{alpha} is then ubiquitinated and degraded by proteasomes, allowing NF-{kappa}B to move into the nucleus [21 ]. The observed decrease in I{kappa}B{alpha} degradation may have been a result of reduced phosphorylation of the I{kappa}B{alpha} or to reduced proteasome activity or both. Preliminary data suggest that the level of activation-induced cytosolic phospho-I{kappa}B{alpha} 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 I{kappa}B{alpha} proteolysis, was impaired severely in activated macrophages from mice exposed to the inhalant. Exogenous NO has been shown to inhibit proteasomal degradation of I{kappa}B{alpha} [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-{kappa}B activation, which is important to HIV replication [25 ]. It has been suggested that inhibition of NF-{kappa}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.


   ACKNOWLEDGEMENTS
 
This work was supported by NIDA grant DA06662. The authors thank Virginia Fitzhugh, Kara Worley, and Jianhui Du for technical assistance.

Received August 3, 2000; revised December 4, 2000; accepted December 5, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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