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Gene Expression by a Nonlysosomotropic Mechanism1
Department of Microbiology, Evans Memorial Department of Clinical Research, and Department of Medicine, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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release from mononuclear
phagocytes. Because it is unclear how CQ mediates these
immunomodulatory effects, we set out to elucidate its mechanism of
action. CQ exhibited dose-dependent inhibition of LPS-induced TNF-
release from human PBMC at therapeutically attainable concentrations.
Additional studies to determine the specificity of this effect showed
that although CQ reduced IL-1ß and IL-6 release, secretion of RANTES
was unaffected. CQ acted by reducing TNF- mRNA accumulation without
destabilizing its mRNA or interfering with NF-
B nuclear
translocation or p50/p65 isoform composition of DNA-binding complexes.
Intracellular cytokine staining indicated that CQ reduced TNF-
production pretranslationally without interfering with TNF-
processing or release. We utilized bafilomycin A1
pretreatment to block the pH-dependent trapping of CQ in endosomes and
lysosomes. Although bafilomycin A1 alone did not interfere
with TNF- expression, preincubation augmented the ability of CQ to
reduce TNF- mRNA levels, suggesting that CQ did not act by a
lysosomotropic mechanism. Using confocal microscopy, we showed that
bafilomycin A1 pretreatment resulted in a dramatic
redistribution of quinacrine, a fluorescent congener of CQ, from
cytoplasmic vacuoles to the nucleus. These data indicate that CQ
inhibits TNF- gene expression without altering translocation of
NF-B p50/p65 heterodimers. This dose-dependent effect occurs over a
pharmacologically relevant concentration range and does not require
pH-dependent lysosomotropic accumulation of CQ. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A panoply of effects on cellular function have been attributed to CQ. This drug interferes with protein synthesis (4), processing (5), and degradation (6) by mechanisms thought to involve, at least in part, alkalization of endosomes and lysosomes. Other effects appear to be independent of the lysosomotropism of CQ. Thus, CQ can interact with DNA (7), alter its superhelical structure (4), and block DNA synthesis at high concentrations (8). CQ also interferes with generation of reactive oxygen species (9), inositol 1,4,5-triphosphate signaling (10), and protein phosphorylation (11).
CQ is known to inhibit TNF- release from human and murine cells
(5, 12, 13, 14, 15, 16, 17), although the mechanism by which this is
accomplished is unclear. In a murine macrophage cell line (RAW 264.7),
CQ (100 µM) interfered with posttranslational processing of TNF-
(5) by a mechanism postulated to involve alteration of
cellular iron metabolism (13). However, in the same cell
line, other investigators found that CQ (250 µM) primarily reduced
TNF- gene expression (12, 16), an effect possibly
involving CQ cytotoxicity (5, 16). The aim of the present
study was to elucidate the mechanism by which this enigmatic drug
reduces LPS-stimulated TNF- release from human PBMC. We found that
CQ specifically reduced TNF- release at the pretranslational level
by reducing TNF- gene expression without altering mRNA stability or
signaling pathways upstream of NF-B mobilization. Moreover, CQ
appeared to mediate its effects by a nonlysosomotropic mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents were obtained from Sigma (St. Louis, MO) unless stated
otherwise. Experiments were designed to minimize endotoxin
contamination. All plasticware was obtained prepackaged and
endotoxin-free. RPMI 1640 and PBS were obtained from BioWhittaker
(Walkersville, MD) and contained less than 0.005 U/ml endotoxin. RPMI
1640 was supplemented with L-glutamine, penicillin, and
streptomycin. CQ was dissolved in PBS at a stock concentration of 100
mM, syringe-filtered (0.2 µM), aliquoted, and stored at -80° until
use. Bafilomycin A1 was solubilized in methanol
at a stock concentration (100 µM), aliquoted, and stored at -80°
until use. LPS from Escherichia coli O111:B4 was prepared as
a stock solution (100 µg/ml) in PBS, stored in aliquots at -80°C,
and used at a final concentration of 100 ng/ml. Pooled human serum
(PHS) was obtained by combining sera of 10–15 healthy donors under
conditions designed to preserve complement activity (18).
CQ, bafilomycin A1, LPS, and PHS were thawed
immediately before use, and any unused portion was discarded. PBMC
viability was measured 8 h after LPS stimulation of CQ (100
µM)-pretreated PBMC using the Live/Dead Viability/Cytotoxicity Kit
(Molecular Probes, Eugene, OR). At this time point, LPS-induced TNF-
release has already peaked (19).
Isolation of PBMC
After informed consent, peripheral blood was obtained by venipuncture from normal healthy volunteers. Each donor was used no more than once per set of experiments. PBMC were purified using standard methods (19). Blood was anticoagulated with 5 U pyrogen-free heparin (Fujisawa, Deerfield, IL) per ml of blood and centrifuged at 500 x g for 15 min. Leukocyte-rich buffy coats were then subjected to Ficoll-Hypaque density gradient centrifugation before collection of PBMC from the light density fraction. Cells were washed three times with ice-cold PBS before counting by hemocytometer and resuspension in RPMI 1640.
Cytokine release
PBMC (2 x 105–1 x
106) were preincubated in the absence or presence
of inhibitor (CQ or bafilomycin A1) for 2 h
at 37° in 96-well polystyrene plates (Dynatech Laboratories,
Chantilly, VA) containing 200 µl of RPMI 1640. Intracellular
concentrations of CQ (3) and bafilomycin
A1 (20) reach equilibrium within
this time period. Cells were then stimulated for 18 h with LPS
(100 ng/ml) in the presence of 10% PHS. Preliminary experiments
demonstrated that this concentration of LPS induced near-maximal
stimulation of TNF- release from PBMC. Inhibitors were not washed
away before stimulation with LPS. Cell-free supernatant was collected
and analyzed for human TNF-, IL-1ß, IL-6, and RANTES
(21) by ELISA. Briefly, to assay TNF- and RANTES, Ab
pairs and recombinant cytokine (for use as standards) were obtained
from R&D Systems (Minneapolis, MN), and the ELISA was performed
according to the manufacturer’s instructions using HRP as the
detection reagent. IL-1ß and IL-6 concentrations were assayed using
ELISA kits (Biosource International, Camarillo, CA) according to the
manufacturer’s protocol.
Isolation and quantitation of TNF- mRNA by Northern blotting
PBMC (5 x 106) were incubated in
6-well polystyrene plates in RPMI 1640 for 2 h at 37° in the
absence or presence of CQ (10 or 100 µM). Cells were then stimulated
with LPS for 2 h in the presence of 10% PHS. Total cellular RNA
was extracted from PBMC using TRIZOL reagent (Life Technologies, Grand
Island, NY) as previously described (19). Total RNA (5
µg) was separated on 1.2% agarose-formaldehyde gels, transferred to
a nylon membrane (Immobilon-Ny+; Millipore, Bedford, MA), and analyzed
by Northern blotting (22). Briefly, the 633-bp fragment of
the EcoRI and HindIII double digest of plasmid
encoding the full-length cDNA for human TNF- (a gift from Leo Lina,
Cetus, Emeryville, CA) was purified from an agarose gel slice using the
JetSorb Gel Extraction Kit (Genomed, Research Triangle Park, NC). This
fragment (25 ng) was 32P-labeled by the random
primed labeling method (Prime-a-Gene Labeling System; Promega, Madison,
WI). Membranes were incubated in a commercial hybridization solution
(ExpressHyb; Clontech, Palo Alto, CA) containing 
2 x
107 cpm of labeled probe per ml of solution.
Hybridization was quantitated by phosphorimager analysis (Molecular
Dynamics, Sunnyvale, CA). RNA integrity and equal loading were
evaluated by ethidium bromide staining of agarose gels before transfer
as well as by stripping and reprobing the membrane with a G3PDH cDNA
probe (Clontech).
EMSA
Nuclear translocation of the transcription factor NF-B was
assayed according to a standard protocol (22). Briefly,
nuclear extracts from PBMC were prepared in the presence of protease
inhibitors after 30 min LPS (100 ng/ml) stimulation of PBMC. Total
protein content of the nuclear extracts was determined using a
commercial kit (Bio-Rad Laboratories, Hercules, CA). An oligonucleotide
containing the NF-B consensus binding sequence was obtained
(Promega) and end-labeled with [-32P]dATP
and [-32P]dCTP using Klenow DNA polymerase
(Promega). Unincorporated nucleotides were removed using a spin column
(Microspin G-25; Amersham Pharmacia Biotech, Piscataway, NJ). Labeled
probe (20,000 cpm) was incubated with nuclear extract (4 µg) for
30 min at room temperature in 1x band shift buffer (10 mM Tris-HCl (pH
7.5), 1 mM EDTA, 40 mM KCl) containing 100 µg/ml poly(dI-dC) and 10%
glycerol. Samples were fractionated by electrophoresis over 4% native
polyacrylamide gels, transferred to 3 MM filter paper (Whatman
Laboratory Products, Clifton, NJ), dried, and analyzed by
phosphorimager. A 250-fold excess of unlabeled probe was used as
specific competitor. To determine the composition of NF-B complexes
by supershift, Ab specific for the p50 or p65 isoforms (Santa Cruz
Biotechnology, Santa Cruz, CA) of NF-B was added immediately after
addition of nuclear extract to labeled oligonucleotide and then
incubated for 30 min at room temperature.
mRNA stability experiments
PBMC (5 x 106) were preincubated for 2 h in the absence or presence of CQ (100 µM) in 6-well polystyrene plates. Cells were then stimulated with LPS (100 ng/ml) in the presence of 10% PHS for 1 h. At this time point, 5,6-dichlororiboside imidazole and actinomycin D were added at 200 µM and 5 mg/ml final concentrations, respectively, to block transcription. Preliminary experiments established that transcription was terminated at these concentrations. At 0, 60, and 120 min after transcriptional blockade, total RNA was extracted, and 5 µg was analyzed by RNase protection assay (RPA) using a commercial kit (PharMingen, San Diego, CA). The mRNA content of specific bands was quantified by phosphorimager analysis.
Localization of quinacrine (QC) within monocytes
PBMC were preincubated in the absence or presence of bafilomycin A1 (100 nM) for 2 h before incubation with a fluorescent congener of CQ, QC (1 µM), for 2 h. Adherent cells were washed once with HEPES and visualized on a Zeiss LSM 510 laser confocal scanning microscope equipped with a x63 oil objective lens.
Flow cytometry
Two-color staining to detect cell surface markers and
intracellular TNF- was performed according to the manufacturer’s
protocol (R&D Systems). PBMC (1 x 106) were
incubated in the absence or presence of CQ (10, 30, or 100 µM) for
2 h at 37° in 24-well polystyrene plates. Cells were then
stimulated for 6 h with LPS (100 ng/ml) in the presence of 10%
PHS, washed twice with cold PBS, and stained with anti-CD14 PE
conjugate (Caltag Laboratories, Burlingame, CA). After an additional
two washes with cold PBS, PBMC were fixed with 2% paraformaldehyde,
permeabilized with saponin, and stained with FITC-conjugated
anti-TNF-, which recognizes intracellular forms of TNF- (R&D
Systems). Stimulation with LPS was performed in the absence or presence
of monensin (2 µM), an inhibitor of protein secretion
(23). PBMC (1–2 x 105) were
counted on a FACScan flow cytometer (Becton Dickinson, San Jose, CA)
and analyzed using CELLQuest software (Becton Dickinson). The monocyte
population was gated on based upon forward and side scatter properties
and CD14 staining (24).
Statistics and presentation of data
Data are expressed as mean ± SE. Data sets were compared by the Student two-tailed, paired t test using a statistical software package (SigmaStat; Jandel Scientific Software, San Rafael, CA). The Bonferroni correction was utilized for multiple comparisons. Significance was considered achieved when the p value multiplied by the number of comparisons was <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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release in a dose-dependent fashion
Initial experiments sought to confirm data by others (5, 12, 13, 14, 15, 16, 17) that CQ inhibits TNF- release. PBMC were pretreated
for 2 h with a micromolar concentration range of CQ before an 18-h
incubation with LPS. CQ inhibited TNF- release, as measured by
ELISA, in a dose-dependent fashion over the 1–100 µM concentration
range tested (Fig. 1
) with a calculated
IC50 value of 8.02 µM. The highest level of CQ
examined, 100 µM, inhibited TNF- release by >98 ± 2%
(p = 0.00002; n = 3).
CQ-induced inhibition of TNF- was not secondary to cell death, in
that viability of the untreated and CQ-treated PBMC was 95.7 ±
1.9% and 92.6 ± 3%, respectively (p =
NS; n = 5).
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To extend these findings and to confirm that CQ was not toxic to
PBMC, levels of the proinflammatory cytokines IL-1ß, IL-6, and RANTES
were measured in the supernatant from LPS-stimulated PBMC pretreated
with CQ (Fig. 2). The highest
concentration of CQ examined (100 µM) nearly completely abrogated
both IL-1ß and IL-6 release. However, IL-6 release was not
significantly affected by 10 µM CQ, unlike IL-1ß secretion. Release
of the ß-chemokine RANTES was unaffected by CQ concentrations up to
100 µM. Thus, there is considerable variation in the
concentrations of CQ required to inhibit proinflammatory cytokine
release from PBMC.
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mRNA accumulation
Having confirmed that CQ inhibited TNF- release without causing
nonspecific cytotoxicity, the next set of experiments sought to
establish at what level of cellular function CQ acted. To determine
whether CQ interfered with TNF- release pretranslationally, we
investigated the effect of this drug on TNF- mRNA accumulation as
measured by Northern blotting and RPA. PBMC were preincubated in the
absence or presence of CQ (10 or 100 µM) and then stimulated for
2 h with LPS. This time point was previously demonstrated to
correspond with peak levels of LPS-induced TNF- gene expression
(25). Compared with unstimulated PBMC, LPS induced TNF-
mRNA levels by 12.0 ± 4.0-fold (p =
0.019; n = 8). CQ pretreatment antagonized TNF- mRNA
accumulation (Fig. 3) with 100 µM CQ
reducing TNF- mRNA levels by 61.2 ± 15.1% (range, 45–99%;
p = 0.03; n = 8). CQ concentrations of
10 µM CQ reduced levels of TNF- mRNA by 31.2 ± 21.5%
(n = 5). CQ did not affect expression of the
housekeeping gene G3PDH. These results demonstrate that CQ interferes
with TNF- release at the pretranslational level.
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mRNA accumulation without reducing mRNA
stability
The mRNA transcripts of numerous proinflammatory cytokines,
including TNF-, contain distinct 3' untranslated regions making them
subject to regulation at the level of mRNA stability (26).
To assess whether CQ reduced levels of TNF- by destabilizing its RNA
transcript, the rate of degradation of its mRNA was determined. PBMC
were preincubated in the absence or presence of CQ (100 µM) and then
stimulated with LPS for 1 h. At this time point, new transcription
was blocked by treatment with 5,6-dichlororiboside imidazole and
actinomycin D. After 0, 60, and 120 min of transcription inhibitor
treatment, total RNA was extracted and analyzed by RPA (Fig. 4, inset). In the absence of
CQ, LPS-stimulated TNF- mRNA had a calculated half-life of 91 min, a
value in close agreement with data from other investigators (27, 28). As expected based on our results using Northern blotting,
CQ treatment of PBMC reduced the intensity of the TNF- mRNA band at
time 0 min. However, the rate of decay of TNF- mRNA (half-life
= 78 min) was not significantly different from that seen in the absence
of CQ. Thus, CQ appears to prevent the initial production of cytokine
transcript, rather than accelerating its degradation.
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B nuclear translocation or the p50/p65
composition of DNA-binding complexes
Nuclear translocation of the transcription factor NF-B is known
to potently induce the expression of numerous genes encoding
proinflammatory cytokines, including TNF- (29, 30).
Because interruption of NF-B activation is a mechanism known to
reduce TNF- gene expression (31), we evaluated the
effect of CQ on NF-B nuclear translocation and p50/p65 isoform
composition in LPS-stimulated PBMC. After a 2-h incubation in the
absence or presence of CQ (100 µM), PBMC were stimulated with LPS for
30 min. Nuclei were harvested from adherent cells, and NF-B was
quantitated in the extracts by EMSA. As described previously,
unstimulated PBMC contained small amounts of NF-B in the nucleus
(Ref. 22 and Fig. 5).
Stimulation with LPS led to 6.2 ± 4.5-fold induction of NF-B
translocation (p = 0.013; n =
3). CQ treatment did not inhibit NF-B translocation. Rather, there
was a trend toward slightly higher levels of NF-B in the nucleus
after CQ treatment of both resting and LPS-stimulated PBMC (Fig. 5).
Although p50/p65 heterodimers potently activate transcription of
TNF- (32), p50 homodimers interfere with NF-B
transactivation by binding the NF-B consensus site and preventing
p50/p65 heterodimer binding (33). Thus, we analyzed the
subunit composition of DNA-binding complexes by Ab supershift. NF-B
oligonucleotide-bound complexes were predominantly p50/p65 heterodimers
(Fig. 5), in that antisera to either or both supershifted the complex.
Bands were completely eliminated by a 250-fold excess of unlabeled
NF-B oligonucleotide probe (data not shown). These findings suggest
that CQ does not reduce TNF- gene expression by perturbing entry of
p50/p65 NF-B heterodimers into the nucleus or by promoting
mobilization of inhibitory p50 homodimers.
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We next sought to determine whether endolysosomal alkalization was
necessary and/or sufficient to inhibit TNF- gene expression. To
mimic the alkalinizing effects of CQ, we treated PBMC with bafilomycin
A1, a specific inhibitor of lysosomal v-ATPase
(20). Bafilomycin A1 treatment alone
had no effect on LPS-stimulated TNF- mRNA levels (Fig. 6), suggesting that alkalization of
lysosomes is not sufficient to reduce TNF- gene expression.
Nevertheless, it still remained possible that CQ accumulation in
endolysosomal compartments could inhibit TNF- gene expression by a
pH-independent mechanism. To exclude this possibility, we preincubated
PBMC with bafilomycin A1 (100 nM) before CQ (100
µM) treatment. The rationale for this approach was that by
alkalinizing endolysosomes, bafilomycin A1
pretreatment would greatly reduce the pH-dependent accumulation of CQ
within this compartment. PBMC sequentially treated with bafilomycin
A1 and CQ before LPS stimulation had less TNF-
mRNA than PBMC treated with CQ alone (Fig. 6).
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, A and B).
However, when added after bafilomycin A1
preincubation, QC was concentrated within the nucleus with a consequent
loss of cytoplasmic staining (Fig. 7, C and D).
Taken together, these results argue that endolysosomal accumulation and
alkalization are neither necessary nor sufficient to reduce TNF-
gene expression. Furthermore, these data suggest that CQ mediates its
effects outside of the endolysosomal compartment.
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release posttranslationally
Although the above experiments demonstrated that isolated
endolysosomal alkalization did not affect TNF- gene expression, it
remained possible that raising the pH of this subcellular compartment
could disrupt TNF- protein processing and/or secretion (34, 35). To address this issue, PBMC were incubated in the absence
or presence of bafilomycin A1 (100 nM) for 2
h and then stimulated with LPS (100 ng/ml). Bafilomycin
A1 treatment reduced TNF- release by 46
± 10.6% (p = 0.0034; n = 5)
relative to untreated, LPS-induced PBMC. The solvent alone (methanol)
had no effect on LPS-induced TNF- release (115 ± 16.8%;
p = NS; n = 3). PBMC viability in the
bafilomycin A1-treated group was 88.5 ±
5.2% (p = NS compared with untreated PBMC;
n = 5).
CQ interferes with TNF- release primarily at the
pretranslational stage
To determine whether CQ similarly interfered with TNF-
processing and secretion, flow cytometry was utilized to measure
cell-associated TNF- in LPS-stimulated PBMC. Initially,
intracellular staining was performed in the absence of monensin, an
inhibitor of secretion. The rationale for this approach was that if CQ
disrupted posttranslational processing and/or secretion of TNF-, it
would be detected as an increase in intracellular TNF-. However, if
TNF- was secreted normally in the presence of CQ, pretreatment would
not be expected to increase intracellular staining above background
levels. Consistent with the latter possibility, CQ did not increase
intracellular TNF- staining in the absence of monensin (Fig. 8, top panels). Subsequent
experiments were performed in the presence of monensin which, by
blocking Golgi apparatus function, causes accumulation of all
translated protein within the cell. In the presence of monensin, CQ
significantly inhibited accumulation of intracellular cytokine in a
dose-dependent fashion (Fig. 8, bottom panels) with 10 and
30 µM CQ reducing cell-associated TNF- levels by 31.8 ± 14.2
and 57.2 ± 7.9%, respectively (p <
0.0015 for both concentrations; n = 3). The highest CQ
concentration tested (100 µM) reduced intracellular TNF- levels by
87 ± 8.3% (p = 0.00021;
n = 3), closely approximating our ELISA findings. These
data argue that CQ primarily acts pretranslationally by reducing
TNF- mRNA levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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release
from human PBMC and begin to dissect the complex intracellular
mechanisms by which CQ exerts its inhibitory effects. CQ interfered
with LPS-induced TNF- release by reducing levels of its mRNA without
affecting the half-life of its RNA transcript or nuclear translocation
of p50/p65 NF-B heterodimers. Moreover, the inhibitory effect of CQ
on TNF- gene expression appeared to be independent of its ability to
alkalinize endolysosomal compartments. Bafilomycin
A1, which alkalinizes endosomes and lysosomes by
a mechanism distinct from CQ, had no significant effect on LPS-induced
TNF- mRNA levels. Moreover, potent inhibition of TNF- gene
expression was observed when PBMC were sequentially treated with
bafilomycin A1 and then CQ. Such treatment would
be expected to greatly reduce entry of CQ into endolysosomal
compartments, as is evidenced by the confocal imaging studies using QC,
a fluorescent congener of CQ.
Interference with LPS signaling by CQ is one possible explanation for
the reduction of TNF- mRNA levels. LPS forms a complex with
LPS-binding protein and the glycosyl-phosphatidylinositol-linked
receptor, CD14. The transmembrane LPS signal is transduced when this
complex interacts with its signaling partner, a member of the Toll-like
family of receptors (36). Once ligated, the cytoplasmic
domain of Toll-like receptor interacts with the adapter protein MyD88
(37), which then interacts with and activates the
IL-1R-associated kinase (38). IL-1R-associated kinase, via
interaction with the adapter protein TNFR-associated factor-6,
activates the NF-B-inducing kinase to phosphorylate two I-B
(inhibitory protein that dissociates from NF-kB) kinases, IK and
IKß (38, 39). These kinases phosphorylate cytoplasmic
I-B, targeting it for ubiquitination and proteasomal destruction
(40). Free NF-B is now able to translocate to the
nucleus.
Our results demonstrating that CQ does not inhibit nuclear
translocation of NF-B argue that this LPS signal transduction
pathway likely remains intact. Furthermore, by supershift analysis, we
did not see an increase in inhibitory p50 homodimers (33).
On the contrary, there was a trend toward increased nuclear p50/p65
heterodimers after CQ treatment. This increase is consistent with the
capacity of CQ to inhibit cytosolic protease activity (41, 42). It is important to note that our gel shift findings do not
reveal whether the p50/p65 NF-B heterodimers appearing in the
nucleus were capable of activating transcription. Additional proteins
such as CREB-binding protein and p300, coactivators that bridge
transcription factors with the transcriptional apparatus
(43), are also required for transcriptional activation and
could conceivably be inhibited by CQ.
NF-B activation is necessary but not sufficient for TNF- gene
expression (44). Thus, it is possible that CQ interferes
with the mobilization or activity of an essential transcription factor
other than NF-B. In support of this concept, CQ has been shown to
interfere with binding of certain transcription factors to consensus
binding sites on DNA (45). However, the interaction of
NF-B with DNA is insensitive to the presence of millimolar
concentrations of CQ (46). Finally, CQ has been shown to
bind to DNA (7), altering its structure (4).
This could conceivably interfere with accessibility of chromatin or
transcriptional activity, resulting in reduced levels of certain
transcripts. Future studies will be directed at evaluating the
molecular basis by which CQ inhibits TNF- gene expression.
In addition to control at the transcriptional level, TNF- is known
to undergo extensive posttranslational regulation. After translation,
the 26-kDa membrane-bound pro-TNF- is cleaved at the cell surface by
a matrix metaloproteinase, TNF- converting enzyme (ADAM-17),
releasing a soluble 17-kDa form of the cytokine (47). In
our study, when PBMC were treated with bafilomycin
A1, LPS-induced TNF- release was reduced
without altering levels of its mRNA, suggesting that endolysosomal
alkalization interfered with secretion or processing of TNF-.
However, we were unable to detect a similar pH-dependent effect after
CQ treatment. Thus, flow cytometry in the absence of monensin did not
reveal an accumulation of cell-associated TNF- after CQ treatment.
In support of this finding, the reduction in levels of TNF- mRNA and
release were comparable, arguing that CQ acts primarily by reducing
TNF- gene expression. However, these data must be interpreted
cautiously because flow cytometry might lack the sensitivity to detect
an effect on protein processing if the amount of protein present within
human cells is already reduced by the pretranslational effects of CQ.
Therefore, posttranslational inhibitory effects of CQ on LPS-stimulated
TNF- release from human PBMC cannot be totally excluded.
In contrast to our findings, a previous study using murine monocytic
cell lines (RAW 264.7 and P388D1) demonstrated that CQ (100 µM)
inhibited LPS-stimulated TNF- release by interfering with TNF-
processing and release (5), causing pro-TNF- to become
lodged in the secretory apparatus. Only at toxic concentrations of CQ
(250 µM) were reduced levels of TNF- mRNA observed (5, 16). These findings are contrasted by our data and by a previous
study evaluating the effect of CQ (100 µM) on TNF- release from
human whole blood (15), showing that CQ interferes with
TNF- gene expression in human cells at physiologic concentrations.
Furthermore, we were unable to demonstrate effects of CQ on protein
processing or secretion. Thus, it appears that CQ might differentially
affect TNF- gene expression in human and murine monocytic cells.
This hypothesis is supported by the observation that transcriptional
induction of TNF- appears to differ in humans and mice
(48). For example, NF-B is very important for murine
TNF- gene expression, whereas its role in humans is controversial.
It is possible that CQ interferes with the action of a transcription
factor other than NF-B that is involved in human, but not murine,
TNF- expression.
The inhibitory effects of CQ on LPS-stimulated TNF- gene expression
and release are highly unlikely to result from nonspecific cellular
toxicity. First, although CQ also inhibited release of IL-1ß and
IL-6, numerous other cellular functions were unaffected by CQ,
including nuclear translocation of NF-B, expression of the
housekeeping genes L32 and GAPDH, and release of the proinflammatory
ß-chemokine RANTES. Second, PBMC viability was not affected after
treatment with the highest concentration of CQ studied (100 µM). This
lack of toxicity is consistent with results from other investigators
(13, 14, 15, 17) and is not surprising considering that in
vitro treatment of leukocytes with 100 µM CQ results in intracellular
concentrations of CQ comparable to those obtained in vivo during CQ
therapy (3).
The clinical implications of our data are speculative. TNF- is
critical for the development of both the innate and adaptive immune
response (49, 50). On the other hand, it has been argued
that the pathogenesis of septic shock involves dysregulated production
of this and other proinflammatory mediators. Therefore, numerous
studies have postulated that immunomodulation of the proinflammatory
cytokines TNF- and IL-1ß would reduce sepsis-related morbidity and
mortality. Although certain animal models of LPS-induced septic shock
argue that blockade of proinflammatory cytokine signaling is
beneficial, clinical trials utilizing anti-cytokine strategies to
block TNF- or IL-1ß function have failed to show any benefit and
have occasionally worsened clinical outcomes in sepsis (51, 52). Nevertheless, in certain infectious and autoimmune
diseases, neutralization of TNF- with mAbs or soluble TNF-
receptors has improved clinical outcomes (53, 54). Despite
their potentially deleterious properties, CQ and its congeners remain
safe and affordable therapies for malaria and rheumatologic disorders.
Interestingly, in severe falciparum malaria, the capacity of CQ to
inhibit TNF- release might contribute to its clinical efficacy
(55, 56). Clearly then, caution must be exercised when
utilizing drugs known to modulate the proinflammatory cytokine
response, a "double-edged sword" at best. By further dissecting the
mechanism by which CQ reduces TNF- release, it might be possible to
design congeners with either greater or lesser anti-inflammatory
properties. Such drugs could prove more efficacious in certain disease
states.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stuart M. Levitz, Section of Infectious Diseases, 650 Albany Street, Boston, MA 02118.
3 Abbreviations used in this paper: CQ, chloroquine; PHS, pooled human serum; RPA, RNase protection assay; QC, quinacrine.
Received for publication March 9, 2000. Accepted for publication May 11, 2000.
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) or presence (
) of CQ (100 µM) and stimulated
with LPS for 1 h. At this time point, further transcription was
blocked. At the indicated time after transcriptional blockade, total
RNA was extracted and 5 µg was analyzed by RPA. The mRNA content of
specific bands was quantified by phosphorimager analysis. Data are
expressed as a percent of LPS-induced densitometry units at time 0 min.
Data represent mean ± SE for three experiments performed in
duplicate. Inset shows a representative gel.
