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Gene Expression in Activated Alveolar Macrophages: Decreased NF-
B Activation and IB Kinase Activity1
Department of Developmental Biochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in NR8383, a cloned cell line derived from
rat alveolar macrophages (RAM), and in primary cultures of RAM.
Production of NO and TNF-, and expression of inducible NO synthase
was inhibited by Tau-Cl in activated NR8383 cells as well as in RAM.
Temporal (2, 4, 8, 24 h) expression of inducible NO synthase and
TNF- mRNAs was reduced by Tau-Cl in NR8383 cells. Tau-Cl depressed
NF-
B migration into the nucleus of activated NR8383 cells and caused
a more sustained presence of IB in the cytoplasm. Stabilization of
cytoplasmic IB- in Tau-Cl-treated cells resulted from decreased
phosphorylation of IB- serine-32 and a lower activity of IB
kinase (IKK). Additional experiments demonstrated that Tau-Cl does not
directly inhibit IKK activity. These results suggest that Tau-Cl exerts
its effects at some level upstream of IKK in the signaling pathway and
inhibits production of inflammatory mediators through a mechanism that,
at least in part, involves inhibition of NF-B
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the mechanism of taurine protection is uncertain, the ability
of taurine to attenuate the highly toxic effects of
HOCl/OCl
produced by activated
polymorphonuclear leukocytes, eosinophils, and basophils is thought to
be important. Taurine reacts with HOCl/OCl to
form taurine chloramine
(Tau-Cl),4 an
oxidant that is far less reactive and more stable than
HOCl/OCl (8, 9, 10, 11). Although the
detoxification of HOCl/OCI by Tau-Cl formation
has been postulated to account for the protective effects of taurine,
more recent studies suggest that Tau-Cl may be a significant biological
effector molecule. This is supported by reports that Tau-Cl inhibits
production of NO, TNF-, and other proinflammatory mediators by
activated cells from a variety of different tissues (1, 12, 13, 14, 15, 16, 17). However, the mechanism of Tau-Cl action has not
been determined and the effects of Tau-Cl on activated alveolar
macrophages have not been reported, except in preliminary form
(16).
Production of proinflammatory mediators is primarily regulated at the
level of gene transcription through the activity of several
transcription factors. Of prominent importance in this regard is the
nuclear transcription factor NF-B because NF-B is ubiquitously
involved in regulating the expression of inducible NO synthase (iNOS),
TNF-, and several other proinflammatory genes (18, 19, 20).
NF-B is a hetero- or homodimeric protein consisting of various
combinations of subunits belonging to the Rel family: p65 (Rel A), p50,
p52, Rel B, and cRel. The p65/p50 dimer is the prototypical form of
NF-B in the nucleus of most activated cells and is the most potent
trans activator of proinflammatory gene expression
(18, 21). In unstimulated cells, NF-B is sequestered in
the cytoplasm as a complex with a member of the IB family; IB-
is the most well characterized. Upon stimulation, IB- is
phosphorylated at serine residues 32 and 36, ubiquitinated, and
degraded by the 26S proteasome (22, 23, 24). Degradation of
IB- unmasks the nuclear localization sequence of NF-B,
allowing translocation into the nucleus and binding to the promoter
region of target genes (25).
Phosphorylation of IB is catalyzed by IB kinase (IKK)- and
IKK-
, which are components of a large (800 kDa) multiprotein complex
referred to as IKK. Other components of IKK include NF-B essential
modifier and IKK complex-associated protein (22, 26, 27). The IKK complex is a common mediator of several upstream
signaling cascades that regulates IKK- and IKK- phosphorylation
of IB. In the present study, Tau-Cl is demonstrated to inhibit
production of NO and TNF- by activated rat alveolar macrophages
(RAM) in culture. Our results show that Tau-Cl inhibits the expression
of iNOS and TNF- genes by attenuating the NF-B signal
transduction pathway at a point that is above the level of IKK
activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monoclonal Ab against iNOS was purchased from Transduction
Laboratories (Lexington, KY). Polyclonal Ab to p65, p50, cRel,
IB-, IB-, IKK-, and actin were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA), as was mAb against IKK-. Ab against
phosphorylated IB- and GST-IB- were obtained from New
England Biolabs (Beverly, MA). Culture medium and rat recombinant
IFN-
were purchased from Life Technologies (Grand Island, NY). LPS W
(Escherichia coli 0111:B4) was purchased from Difco
(Detroit, MI) and heat-inactivated FBS was purchased from Gemini
Bio-Products (Calabasas, CA). Plasmids containing cDNA probe for iNOS
(murine), TNF- (murine), and GAPDH (rat) were graciously provided by
Dr. C. Nathan (Cornell University, New York, NY), Chiron
(Emeryville, CA), and Dr. R. Dong (London University, London, U.K.),
respectively. NaOCl was purchased from Fisher Scientific (Fairlawn,
NJ). Taurine, MG-132 (N-CBZ-Leu-Leu-Leu-A1) and other chemicals were
obtained from Sigma (St. Louis, MO). Tau-Cl was freshly synthesized on
the day of use by adding equimolar amounts of NaOC1 dropwise to taurine
at pH 8.3 and was authenticated by measuring its UV absorption spectra
(190–350 nm) which assured monochloramine formation and the absence of
dichloramine, NH2Cl, and unreacted
HOCl/OCl (15, 28).
Alveolar macrophage cultures
Bronchoalveolar cells were obtained as described previously (29), with some modifications. Briefly, adult female Sprague Dawley rats (Taconic Farms, Germantown, NY) were anesthetized by an injection (i.p.) of sodium pentobarbital. The thoracic cavity was opened, the trachea was cannulated, and the lungs were lavaged with calcium and magnesium-free HBSS. The lungs were massaged with each lavage and were sequentially washed until 60 ml of lavage fluid was accumulated. Cells were collected by centrifugation (250 x g), washed in HBSS, and a differential cell count was performed. Cells were resuspended in RPMI 1640 medium supplemented with 2 mM glutamine, 10% FCS, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cells were placed into tissue culture plates and incubated at 37°C in 5% CO2 for 1 h before nonadherent cells were removed by washing with medium. Adherent macrophages (RAM) continued in culture until used.
NR8383 cells, a clonal cell line derived from RAM, were obtained from
the American Type Culture Collection (Manassas, VA) and were grown in
flasks containing Ham’s F-12 nutrient mixture supplemented with 15%
FBS, 2 mM glutamine, and antibiotics. Experiments were conducted in
DMEM supplemented with heat-inactivated 2% FBS, 1% penicillin, and
1% streptomycin at a density of 1 x 107
cells/100-mm diameter culture dish or 1 x
106/well in six-well plates for both cell types.
Cells were activated with LPS (1 µg/ml) and IFN- (10 U/ml), and
Tau-Cl was added immediately thereafter.
Nitrite and TNF- measurements
Samples (100 µl) of conditioned medium were mixed with equal
volumes of Griess reagent (1% sulfanilamide, 0.1% naphthalene diamine
dihydrochloride, and 2.5% phosphoric acid) and incubated at room
temperature for 10 min before measuring OD at 550 nm using sodium
nitrite as standard (16). Concentrations of TNF- were
determined by ELISA using an immunoassay kit specific for rat TNF-
according to the manufacturer’s instructions (BioSource International,
Camarillo, CA).
Northern blot analyses
Northern blot analyses were conducted as previously described (16). Briefly, total RNA was extracted from NR8383 cells using Tri-Reagent (Molecular Research Center, Cincinnati, OH), size fractioned by electrophoresis in 1% agarose-formaldehyde gel, transferred to Nytran membrane, and cross-linked to the membrane by UV irradiation. Blots were prehybridized in ExpressHyb Hybridization Solution (Clontech Laboratories, Palo Alto, CA) for 1 h (68°C) before hybridization with [32P]dCTP random prime-labeled cDNA at 68°C for 16–18 h. Blots were washed three times at room temperature in 2x SSC containing 0.5% SDS followed by two washes at 50°C in 0.1x SSC containing 0.1% SDS. Membranes were stripped of cDNA probe between sequential hybridizations. RNA hybridized with cDNA probe was visualized by phosphor imager analyses and by autoradiography using Kodak XAR-5 film. Autoradiograms were analyzed by computer-assisted densitometry.
Western blot analyses
Cells were collected, centrifuged, washed in PBS, and lysed in radioimmunoprecipitation assay buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing aprotinin (5 U/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), PMSF (1 mM), sodium orthovanadate (1 mM), and sodium fluoride (1 mM). After centrifugation, cell lysates were diluted in SDS containing sample preparation buffer, subjected to SDS-PAGE, and transferred to nitrocellulose filters. Filters were blocked in a solution of PBS containing 5–10% nonfat dry milk or BSA and 0.05% Tween 20. After incubations with the indicated primary and secondary Abs, reactive bands were visualized by ECL (Amersham, Arlington Heights, IL). Protein concentrations were determined by bicinchoninic acid with BSA as standard (Pierce, Rockford, IL).
Cytoplasmic and nuclear protein preparations
Cytoplasmic (postnuclear) and nuclear protein fractions were prepared according to Schreiber et al. (30), with modifications. Cells were washed twice in PBS and suspended in buffer A. After incubating for 15 min on ice, Nonidet P-40 was added to a final concentration of 0.6% (v/v) and the samples were vigorously vortexed before centrifuging at 16,000 x g for 30 s at 4°C. The supernatant cytoplasmic fraction was removed and stored at -80°C until used. The nuclear pellet was suspended in buffer C (30) and vigorously vortexed intermittently for 15 min before centrifuging at 16,000 x g for 20 min at 4°C. The supernatant nuclear protein extract was stored at -80°C until used.
NF-B EMSAs
Protein-DNA binding interactions were performed by incubating
(22°C, 15 min) 3–5 µg of nuclear protein with 1 ng of
32P-labeled (50,000 cpm) double-stranded
oligonucleotide probe (custom synthesized by Bioserve
Biotechnologics, Laurel, MD) in 20 mM Tris-HC1 (pH 7.9)
containing 50 mM NaC1, 2 mM MgC12, 1 mM EDTA,
10% glycerol, 0.1% Nonidet P-40, 1 mM DTT, 0.5% BSA, and 2 µg of
poly(dI-dC) in a final volume of 20 µl. For gel supershift analyses,
Abs were included in the above reaction mixture and incubated at 4°C
for 90 min before addition of the 32P-labeled
oligonucleotide probe, followed by incubation at 22°C for 15 min. The
protein-DNA complexes were fractionated on 6% native polyacrylamide
gels run in 0.5x TBE buffer (Tris-borate-EDTA). Probes were
labeled using the Klenow fragment of DNA polymerase and
[32P]dCTP. The consensus NF-B sequence
5'-AGTTGAGGGGACTTTCCCAGGC-3' was used as probe for EMSA.
The mutant NF-B probe used for competition in EMSA had the same
sequence with the exception of a single base (underlined) that was
changed to C. Gels were dried and bands were visualized by
autoradiography.
Immunoprecipitation and kinase assays
Immunoprecipitation of IKK from cell lysates (800 µg of
protein) was accomplished using anti-mouse IKK- mAb and
ultralink immobilized protein A/G (Pierce) incubated overnight at
4°C. Immunoprecipitates were washed five times with
radioimmunoprecipitation assay buffer and twice with kinase reaction
buffer (50 mM Tris-HCl (pH 8.00) containing 50 mM magnesium chloride, 3
µM okadaic acid, and 300 nM ATP). Enzyme assays were performed by
adding 1 µg of GST-IKB- and 6.8 µCi of
[-32P]ATP (7000Ci/mmol) to the kinase
reaction buffer containing the immunoprecipitates and incubating at
30°C for 30 min. The reaction was stopped by the addition of 2x SDS
sample preparation buffer followed by boiling for 5 min. The samples
were subjected to SDS-PAGE, transferred to nitrocellulose filters,
analyzed by autoradiography, and finally subjected to
immunoblotting.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(10 U/ml) resulted in a dose-dependent
inhibition of NO production, as measured by
NO2 medium accumulation over
24 h (Fig. 1
). Tau-Cl inhibited NO
production by NR8383 and by primary cultures of RAM with similar
potency (IC50 
0.5 mM) and efficacy. Western
blot analyses of cell lysates revealed that iNOS protein was reduced in
both cell types by Tau-Cl in a concentration-dependent manner (Fig. 1).
Whereas unactivated NR8383 cells expressed low basal levels of iNOS
protein, unactivated primary cultures of RAM did not. In addition,
unlike unactivated primary RAM cultures, unactivated NR8383 cells
produced low concentrations (4–6 µM) of
NO2. Production of TNF- by
both cell types was also dose-dependently inhibited by Tau-Cl (Fig. 2). Unactivated NR8383 and RAM did not
secrete detectable amounts of TNF- into the medium. Cell viability
was unaffected by the range of Tau-Cl concentrations used in these
studies (data not shown) as measured by trypan blue exclusion and by
conversion of the tetrazolium salt MTS into a formazan product using a
kit from Promega (Madison, WI). Further studies of the molecular
mechanism of Tau-Cl inhibition of NO and TNF- production were
conducted using the clonal cell line NR8383.
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mRNAs was evaluated by
Northern blots (Fig. 3). Neither iNOS nor
TNF- mRNAs were detected in preparations from unactivated cells.
Transcripts for TNF- were expressed earlier than iNOS transcripts in
activated NR8383 cells and Tau-Cl concentration-dependently inhibited
expression of both mRNAs. Tau-Cl appeared to exert its most profound
effects during early phases of message expression, regardless of the
relatively different temporal patterns, i.e., at 2–4 h for TNF- and
4–8 h for iNOS mRNA. These results suggested that early events in gene
transcription may be affected by Tau-Cl.
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B appears to be obligatory in most cell types. Since iNOS and
TNF- gene expression is regulated by NF-B, the effects of Tau-Cl
on NF-B-binding activity were examined in nuclear protein extracts
of NR8383 cells by EMSA (Fig. 4). The
binding activity of NF-B recovered from the nucleus increased within
30 min of activation, relative to that of unactivated cells, and
cumulatively increased over the remaining 24 h of activation.
Nuclear protein extracts from cells activated in the presence of Tau-Cl
(1.0 mM) exhibited greatly reduced NF-B-binding activity relative to
that of activated cells at all times examined. In addition, a second
band of NF-B-binding activity was evident after 3, 6, and 24 h
of activation, but was not recovered from the nuclear protein fraction
of Tau-Cl-treated cells. Supershift EMSA analyses of nuclear protein
extracts prepared from NR8383 cells activated for 3 h demonstrated
that the upper NF-B band consisted of p50/p65 (Fig. 5). This was evident because Ab against
p50 and Ab against p65, but not cRel, supershifted the upper band. The
lower NF-B band is a homodimer consisting of p50 subunits because
p50 Ab selectively supershifted the entire complex while p65 and cRel
Abs were ineffective. Specificity of NF-B binding was determined by
preincubating nuclear protein extracts with excess (50-fold) unlabeled
NF-B probe or with a one-base mutant NF-B probe as competitor
before incubating with radiolabeled probe and conducting the EMSA.
Similar results were obtained with Tau-Cl-treated cells, i.e., the
identity and specificity of p50/p65 was verified (data not shown) but
the lower band (p50/p50) was not observed (Fig. 4). These results
indicated that Tau-Cl was either inhibiting NF-B binding to its
nuclear recognition sites or was inhibiting some earlier event in the
process of NF-B activation.
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B into the
nucleus, Western blot analyses of nuclear protein fractions were
conducted (Fig. 6). Accumulation of p50
and p65 NF-B subunits in the nuclear protein fraction was greatly
reduced in Tau-Cl-treated cells relative to control NR8383 cells over
the entire 24 h of activation. No changes in p50 or p65 were
detected in cytosolic fractions when measured over time or as a result
of Tau-Cl treatment (data not shown). These results suggested that
Tau-Cl was not inhibiting the binding of NF-B to its nuclear
recognition site per se, but was inhibiting some aspect of NF-B
activation in the cytoplasm. This was evaluated by determining the
presence of cytosolic IB- in the same cell preparations as used
for Fig. 6. Control cells exhibited a rapid disappearance of cytosolic
IB- during the first 1 h of activation with gradual
reappearance occurring between 3 and 24 h of activation (Fig. 7). Cells activated in the presence of
Tau-Cl (1.0 mM) did not exhibit this transient change in cytoplasmic
IB-, but demonstrated increased IB- content 24 h after
activation similar to that of control activated cells. These results
suggest that in Tau-Cl-treated cells IB was not being degraded and
resynthesized, but was being retained as a complex with NF-B in the
cytoplasm.
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B translocation to the nucleus, cytosolic
IB must be phosphorylated, ubiquitinated, and degraded by the 26S
proteasome (22, 23, 24). To address the effects of Tau-Cl on
this event, we used MG-132, a peptide that blocks proteasome activity,
thus allowing phosphorylated IB to accumulate in the cytoplasm
(31, 32). Preliminary studies demonstrated that a 60-min
preincubation (37°C, 5% CO2) of NR8383 cells
with 10 µM MG-132 before activation was adequate for inhibiting
degradation of phosphorylated IB. Treatment with MG-132 also
inhibited NO production, measured 24 h later by activated cells
(data not shown). The effects of Tau-Cl on phosphorylation of IB-
were determined by using cells that were preincubated (1 h, 37°C, 5%
CO2) with 10 µM MG-132 or vehicle (DMSO, 0.25%
(v/v) final concentration) before activation with LPS + IFN-. Tau-Cl
was added at the time of activation. Cell lysates were prepared 1
h later and analyzed by Western blot using Abs specific for IB-
phosphorylated at serine residue 32 (Fig. 8). Preparations from activated cells
contained reduced amounts of unphosphorylated IB- and increased
p-IB- relative to unactivated cells. Preincubation with MG-132
(10 µM) for 1 h before activation greatly enhanced the
p-IB- signal, whereas cells activated in the presence of Tau-Cl
(1 mM) accumulated only low amounts of p-IB. Cells activated in the
presence of Tau-Cl maintained unphosphorylated IB- levels that
were similar to those of unactivated controls.
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B-, cells were activated in the presence of various
concentrations of Tau-Cl for 30 and for 60 min (Fig. 9). Preincubation of cells in the
presence of 10 µM MG-132 greatly enhanced the p-IB- signal in
activated cells relative to controls, with more p-IB-
accumulating after 60 min of activation than after 30 min. Tau-Cl
dose-dependently inhibited accumulation of p-IB- after 30 min of
activation and inhibition persisted for up to 60 min. These results
(Figs. 8
and 9) suggested that Tau-Cl attenuated NF-B activation
primarily by inhibiting the phosphorylation of IB- rather than by
affecting ubiquitination or 26S proteasome activity.
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B- is phosphorylated by the IKK multiprotein complex, the
effect of Tau-Cl on the status of intrinsic cellular IKK activation was
determined (Fig. 10A).
NR8383 cells were activated in the presence of various concentrations
of Tau-Cl for 1 h. The IKK complex was immunoprecipitated from
cell lysates and analyzed for IKK activity using GST-IB- as
substrate. Blots used for autoradiograms were subsequently probed for
IKK- and IKK- by Western blot (Fig. 10A). Cells
activated in the presence of Tau-Cl had greatly reduced IKK activity as
compared with that of activated control cells (A,
second lane) even though the relative amounts of
immunoprecipitated IKK- and IKK- were similar among the
conditions used in this experiment. Immunoprecipitated IKK also
contained IKK- in equivalent amounts across treatment conditions
(M. R. Quinn, M Barua, and V. Serban, unpublished
observations). Additional experiments were conducted to more
stringently test the effect of Tau-Cl on IKK activity. IKK was
immunoprecipitated from cell lysates of activated cells and Tau-Cl was
added at the initiation of the assay for IKK activity (Fig. 10B). The activity of IKK was unimpaired by the presence of
Tau-Cl. These results suggest that Tau-Cl exerts its effect by
inhibiting upstream signaling pathways that activate IKK, rather than
by directly inhibiting IKK activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present studies demonstrate that Tau-Cl dose-dependently inhibits
production of NO and TNF- and expression of iNOS protein in
activated RAM. Tau-Cl inhibits production of these proinflammatory
mediators primarily through depressing iNOS and TNF- gene
transcription as evidenced by decreased expression of iNOS and TNF-
mRNAs in activated NR8383 cells. Studies using other cell types also
suggest that Tau-Cl inhibits expression of inflammatory genes (1, 12, 13, 14, 15, 16, 17) and this appears to occur without affecting protein
synthesis in general (1, 17). Although the effects of
Tau-Cl on protein synthesis were not directly evaluated in the present
study, Tau-Cl elicited increased production of inducible heat shock
protein 70 in NR8383 cells 3–6 h after activation (M. R. Quinn,
Y. Liu, and M. Barua, unpublished observations). This suggests that the
present results cannot be accounted for by a general suppression of
gene transcription or protein synthesis by Tau-Cl.
Further studies demonstrated that the transcriptional effects of Tau-Cl
on genes regulating expression of iNOS and TNF- result, in part,
from decreased translocation of NF-B into the nucleus of activated
cells. Transcription of iNOS and TNF- genes is critically dependent
on the NF-B transcription factor signaling pathway
(41, 42, 43). Decreased p50/p65 nuclear binding in
Tau-Cl-treated cells is of particular interest because NF-B with
this subunit composition potently trans-activates target
genes, whereas the p50 homodimer is thought to suppress or to exert
relatively low trans activation (18, 21).
Inhibition of NF-B activation was accounted for by stabilization of
cytosolic IB in cells activated in the presence of Tau-Cl. The
release of cytosolic NF-B from the NF-B-IB complex requires
phosphorylation of IB by the IKK multiprotein complex, followed by
ubiquitination and degradation by the 26S proteasome
(22, 23, 24, 25). Our results suggest that the kinase activity of
IKK was attenuated in cells that were activated in the presence of
Tau-Cl through a mechanism that most likely involves upstream signaling
pathways. This is substantiated by the lack of effects of Tau-Cl on IKK
activity when Tau-Cl is directly added to the IKK assay mixture
containing IKK immunoprecipitated from activated NR8383 cells. Although
it is possible that Tau-Cl interferes with the regulatory interaction
between NF-B essential modifier (IKK-) and IKK- (26, 44), it seems more likely that Tau-Cl interferes with one of the
key upstream kinases in the signaling pathway, e.g., NF-B-inducing
kinase or mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase kinase 1 (23, 27), or some
as yet to be identified kinase (45).
The present results are consistent with reports (1, 17)
that Tau-Cl exerts its most potent inhibitory effects on production of
proinflammatory mediators when present around (±2 h) the time of
activation, but is relatively ineffective if added 16–14 h later.
Recently, it was reported that taurine administration, in combination
with niacin, protected mice from bleomycin-induced lung damage
(5, 34, 35). The protective effects were associated with
diminished NO production, accompanied by decreased iNOS mRNA and iNOS
protein expression in extracts of whole lung. In addition, nuclear
protein extracts prepared from whole lung exhibited decreased NF-B
binding (EMSA) accompanied by increased IB- in mice treated with
taurine and niacin (35). Although these changes were not
localized to a specific cell type, alveolar macrophages and neutrophils
would be expected to be present and to contribute to the pathology
observed in this model. It is intriguing to speculate that the
mechanism of Tau-Cl action described in the present communication may
explain, in part, the reported protective effects of taurine (34, 35). Although we have not evaluated the combined effects of
taurine and niacin on any of the parameters measured in the present
study, taurine alone has been demonstrated to be without effect (Refs.
1, 12, and 17; M. R. Quinn, unpublished observations).
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Acknowledgments |
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, and GAPDH, respectively. The technical assistance of
Maria Tonna-DeMasi and the secretarial services of Janis Kay are
greatly appreciated. |
Footnotes |
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2 M.B and Y.L. contributed equally to the conduct of this work.
3 Address correspondence and reprint requests to Dr. Michael R. Quinn, Department of Developmental Biochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314. E-mail address: drmrquinn{at}hotmail.com
4 Abbreviations used in this paper: Tau-Cl, taurine chloramine; iNOS, inducible NO synthase; IKK, IB kinase; MG-132, N-CBZ-Leu-Leu-Leu-A1; RAM, rat alveolar macrophage.
Received for publication March 8, 2001. Accepted for publication June 8, 2001.
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