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Antimicrobial Agents and Chemotherapy, August 1998, p. 1911-1916, Vol. 42, No. 8
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Immunomodulating Properties of the Antibiotic
Novobiocin in Human Monocytes
Anja
Lührmann,1
Jürgen
Thölke,1
Ingrid
Behn,1
Jens
Schumann,2
Gisa
Tiegs,2 and
Sunna
Hauschildt1,*
Department of Immunobiology, Institute of
Zoology, University of Leipzig, Leipzig,1 and
Institute of Experimental and Clinical Pharmacology and
Toxicology, University of Erlangen-Nürnberg,
Erlangen,2 Germany
Received 17 December 1997/Returned for modification 25 February
1998/Accepted 13 May 1998
 |
ABSTRACT |
We show that the coumeromycin antibiotic novobiocin, a potent
inhibitor of ADP ribosylation, prevents lipopolysaccharide
(LPS)-induced tumor necrosis factor alpha (TNF-
), interleukin-1
(IL-1), IL-6, and IL-10 secretion in human peripheral blood mononuclear
cells. It shares these cytokine-suppressing properties with other
inhibitors of ADP ribosylation. We found that novobiocin prevents
TNF- production by inhibiting translation of the TNF- mRNA.
Elevated TNF- levels in mice treated with
D-galactosamine (GalN)-LPS or GalN-TNF were not reduced by
novobiocin; however, the drug exhibited hepatoprotective properties.
Novobiocin causes downregulation of the surface molecules on monocytes,
among which CD14 was the most affected. The diminished expression of
surface molecules was not observed on T and B lymphocytes. Similar to
other inhibitors of ADP ribosylation, novobiocin prevents LPS-induced
phosphate labelling of 
-actins.
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INTRODUCTION |
Antibiotics are widely used as
bacteriostatic or bactericidal drugs in the therapy of bacterial
infections. Whereas the interaction between antibiotics and bacteria
and between bacteria and the immune system has been well studied,
little is known about the effects of antibiotics on the immune system.
In the study described in the present paper we studied the effect of
the antibiotic novobiocin on the immune functions of human monocytes.
The coumarin novobiocin is produced by Streptomyces
(S. niveaus and S. spheroides), and its main
targets are gram-positive bacteria. It acts by inhibiting the bacterial
gyrase activity. Novobiocin interferes with metabolic processes not
only in bacteria but also in eukaryotic cells. It has been shown to
function as a potent inhibitor of ADP ribosylation (1),
i.e., the covalent attachment of multiple or single residues of the ADP
ribose moiety of NAD to various proteins. ADP ribosylation, like
phosphorylation, constitutes an important mechanism in
posttranslational modifications of cellular proteins (10).
By using the inhibitor novobiocin we tried to assess how far this form
of modification might be involved in the regulation of certain monocyte
functions. In the study described here we showed that novobiocin
effectively suppresses the production of proinflammatory cytokines as
well as the anti-inflammatory cytokine interleukin-10 (IL-10). It
induces shedding of CD14 and modulates the expression of other surface antigens. When administered to mice receiving GalN-lipopolysaccharide (LPS) novobiocin prevents an increase in serum transaminase activity. We found that novobiocin inhibits protein biosynthesis and alters the
phosphorylation state of cytosolic proteins, indicating that it may
exert its immunomodulatory properties by interference with these
intracellular events probably via ADP ribosylation-dependent processes.
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MATERIALS AND METHODS |
Reagents.
LPS (Escherichia coli O55:B5),
novobiocin, and nicotinamide were from Sigma (Deisenhofen, Germany).
L-[4,5-3H]leucine (specific activity, 58 Ci/mmol) and [-32P]ATP (specific activity, 5,000 Ci/mmol) were purchased from Amersham Buchler (Braunschweig, Germany).
Cell preparation.
Peripheral blood mononuclear cells (PBMCs)
from healthy donors were isolated by centrifugation over a Ficoll-Paque
(Pharmacia, Freiburg, Germany) density gradient (23). After
repeated washing in phosphate-buffered saline (PBS) the PBMCs were
further separated by counterflow centrifugation with the J6-MC
elutriator system (Beckman Instruments, Palo Alto, Calif.). The
monocyte fraction consisted of >92% monocytes, as determined by
morphology and immunofluorescence staining with a monoclonal antibody
(MAb) against CD14 (BL-M/G14). Peritoneal macrophages were collected
from adult BALB/c mice that immediately after killing had been injected
with 3 ml of RPMI 1640 containing 10% fetal calf serum (FCS).
Stimulation of cells.
PBMCs or monocytes (4 × 106/ml) were suspended in RPMI 1640 supplemented with 10%
(vol/vol) FCS and antibiotics in 24-well culture plates (662160;
Greiner Frickenhausen, Germany) and incubated with novobiocin or
nicotinamide for 10 min prior to the addition of LPS for 6 h. The
supernatants were collected, centrifuged, and analyzed for cytokine
concentrations.
Preparation of cytosolic supernatant and in vitro
phosphorylation.
After monocytes (4 × 106/ml)
suspended in Falcon tubes (Becton Dickinson, Heidelberg, Germany) had
been stimulated as described above, they were washed twice in ice-cold
PBS. To disrupt the cells, the cell pellet was resuspended in ice-cold
permeabilizing buffer containing 10 mM Tris-HCl (pH 7.8), 1 mM EDTA, 4 mM MgCl2, 30 mM 2-mercaptoethanol, and 1 mM vanadate, left
on ice for 10 min, and sonicated (12 strokes; intensity, 1.1; duty
cycle, 80%; Branson Sonifier 250; Danbury, Branson, Conn.). The
cytosol was obtained by ultracentrifugation at 100,000 × g for 1 h.
Aliquots of the cytosolic supernatant containing about 30 µg of
protein in a volume of 50 µl of buffer were added to 25 µl of the
phosphorylation reaction mixture. The mixture contained 100 mM Tris-HCl
(pH 7.8), 120 mM MgCl2, 0.01% leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and [-32P]ATP (5 µCi/aliquot). The reaction mixtures were incubated for 10 min at
37°C. The reactions were terminated by precipitating the proteins
with methanol as described by Wessel and Flügge (27).
The dried pellets were solubilized in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
(14) and boiled for 3 min. The proteins were separated by
SDS-PAGE (12% polyacrylamide gel) (14). The gels were
stained with Coomassie blue (Serva Blue G; Serva, Heidelberg, Germany)
(18) and dried, and autoradiography was performed with
DuPont Cronex film 4 (DuPont, Bad Homburg, Germany) with Kodak X-OMAT
intensifying screens (Eastman Kodak Co., New Haven, Conn.) at 
70°C
for 3 to 5 days.
Soluble CD14 quantitation.
The amount of soluble CD14 in the
supernatants was determined by enzyme-linked immunosorbent assay
(ELISA) as described previously (7). The ELISA kit was
kindly provided by C. Krüger (Immuno Biological Laboratories,
Hamburg, Germany).
Flow cytometry analysis.
The cells (2 × 105/50 µl of PBS) were incubated in the presence of 50 µl of MAb for 45 min at 4°C. The cells were then washed in ice-cold
PBS containing 10% Haemacel and 0.1% sodium azide and were incubated
for 30 min at 4°C with fluorescein isothiocyanate (FITC)-labeled goat
anti-mouse antibody (SIFIN, Berlin, Germany). After washing and
fixation in 1% formaldehyde, the cells were analyzed in a FACScan
analyzer (Becton Dickinson, San Jose, Calif.). The mean fluorescence
intensities of labelled cells were recorded after gating of the cells
by using their forward and side scatter properties. Ten thousand events
were analyzed per sample and are presented as histograms with a
four-decade logarithmic scale.
MAbs.
Fluorescein (FITC)-conjugated anti-CD14 MAbs were from
Biometec (Greifswald, Germany); anti-HLA DR/DQ (MAb BL-Ia1), anti-CD2 (MAb BL-TP2a), anti-CD3 (MAb BL-TP3b), anti-CD4 (MAb BL-TH4), anti-CD5
(MAb BL-TP5), anti-CD6 (MAb BL-TP6a), anti-CD8 (MAb BL-TS8/2), anti-CD11b (MAb BL-M/G1), anti-CD11c (MAb BL-M11c), anti-CD14 (MAb
BL-M/G14), anti-CD21 (MAb BL-B21), anti-CD22 (MAb BL-B22), anti-CD31
(MAb BL-M/G31), anti-CD40 (MAb BL-B40), anti-CD43 (MAb BL-TP43),
anti-CD45 (MAb BL-Leuk45), anti-CD72 (BL-B72), and anti-CD76 (MAb
BL-B76) MAbs were from DiaMAk (Leipzig, Germany); isotype control
antibodies (immunoglobulin G1-FITC and immunoglobulin G2b-FITC) were
from Sigma (Deisenhofen, Germany).
Protein synthesis.
Monocytes suspended at 2 × 105/ml in leucine-deficient RPMI 1640 supplemented with
10% dialyzed FCS were incubated with novobiocin in the presence of
L-[4,5-3H]leucine (4 µCi/ml; specific activity, 58 Ci/mmol). After 3 h the cells were diluted with 2 ml of ice-cold
0.9% NaCl and were centrifuged (at 1,100 × g for 10 min). The washed cells were precipitated with 2 ml of 25% (wt/vol)
trichloroacetic acid after the addition of 100 µl of 0.1% (wt/vol)
bovine serum albumin. After 30 min at 4°C the resulting precipitates
were recovered by centrifugation (3,000 × g for 10 min). The pellets were resuspended in 250 µl of 1 N KOH, precipitated
again, collected on Whatman GF/C glass fiber filters, and washed with
25% trichloroacetic acid. The radioactivity on the filters was counted
by liquid scintillation spectrometry.
Reverse transcription-PCR of TNF mRNA.
Monocytes
(106/ml) were incubated in RPMI 1640 supplemented with 10%
(vol/vol) FCS and antibiotics in Eppendorf vials (Eppendorf, Hamburg,
Germany) in the presence and absence of LPS and novobiocin or
nicotinamide. After 2 h the cells were washed in PBS and RNA was
isolated with the RNeasy Total RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Subsequent reverse transcription was performed as described previously (22).
Genomic DNA was prepared by standard protocols (16). PCR was
carried out in an automatic DNA thermal cycler (Crocodile III;
Appligene, Reutlingen, Germany). To amplify cytokine-specific cDNA
fragments the following gene-specific, intron-spanning primers were
used: tumor necrosis factor alpha (TNF-) sense primer (5'-GAG TGA
CAA GCC TGT AGC-3'), TNF- antisense primer (5'-CCC TTC TCC AGC TGG AAG-3'), 
-actin sense primer (5'-AGC GGG AAA TCG TGC GTG-3'), and
-actin antisense primer (5'-CAG GGT ACA TGG TGG TGG TGC-3'). The
reaction mixture containing 2 µl of cDNA, 1 µM sense and antisense primers, 200 µM deoxynucleoside triphosphates (MWG Biotech,
Ebersberg, Germany), and 1.3 U of Taq polymerase
(Gibco-BRL, Eggenstein, Germany) in a final volume of 35 µl. The
cycle program was set for denaturation at 95°C for 1 min, annealing
at 55°C for 1 min, and extension at 72°C for 1.5 min for a total of
25 cycles (-actin) or 30 cycles (TNF-). Electrophoresis of the
PCR products was performed on 1.5% agarose gels (Gibco-BRL) containing
1 µg of ethidium bromide per ml. The DNA molecular weight marker VI
(Boehringer, Mannheim, Germany) was used.
Detection of cytokines in culture supernatants.
Supernatants
were collected for measurement of IL-1, IL-6, IL-10, and TNF-
concentrations. Determination of IL-1 concentration was based on the
proliferation of IL-1-dependent human fibroblasts (15).
IL-10 concentration was measured by a commercially available ELISA
(Biosource, Ratingen, Germany) according to the manufacturer's instructions. The concentrations of TNF- and IL-6 were determined by
ELISA. The primary anti-IL-6 and anti-TNF- antibodies as well as the
secondary purified rabbit polyclonal anti-IL-6 and TNF- antibodies
were kindly provided by W. Buurman (Maastricht University, Maastricht,
The Netherlands). The peroxidase-labelled goat anti-rabbit antibody was
from SIFIN. Mouse TNF- concentration was measured by a commercially
available ELISA (Genzyme, Cambridge, Mass.).
Shedding of CD14.
The cells (4 × 106/ml)
were incubated with a FITC-labelled anti-CD14 MAb for 20 min at 4°C.
The cells were washed with PBS and incubated in RPMI 1640 containing
10% FCS with LPS (100 ng/ml) or without LPS in the presence or absence
of novobiocin. After washing of the cells, the cells were analyzed by
fluorescence-activated cell sorter (FACS) analysis. A decrease in
fluorescence indicates shedding of CD14; it is not the result of
internalization since internalized CD14 is also detected by this
method. No change in fluorescence could mean either no modulation or
internalization.
GalN-LPS-TNF- challenge in vivo.
BALB/c mice (age, 6 to 8 weeks) were obtained from the internal animal breeding house of the
Institute of Experimental and Clinical Pharmacology and Toxicology
(University of Erlangen-Nürnberg, Erlangen, Germany). The animals
received humane care according to the guidelines of the National
Institutes of Health as well as the legal requirements in Germany and
were maintained under controlled conditions (22°C, 55% humidity,
12-h day and 12-h night rhythm) and were fed a standard laboratory chow
(Altromin 1313) ad libitum. All reagents were injected in a total
volume of 250 µl per 25-g mouse. The mice were pretreated
intraperitoneally with 100 mg of novobiocin (Sigma, Deisenhofen,
Germany) per kg of body weight; control animals received saline instead
of novobiocin. Sixty minutes later the mice were challenged
intraperitoneally either with a combination of 700 mg of
D-galactosamine (GalN; Carl Roth GmbH + Co.,
Karlsruhe, Germany) plus 10 µg of LPS per kg from Salmonella
abortus equi, S form (Metalon GmbH, Ragow, Germany), or with 700 mg of GalN per kg intraperitoneally plus 7.5 µg TNF- (kindly
provided by G. R. Adolf, Bender & Co., Vienna, Austria)
intravenously. For determination of circulating TNF-, blood samples
were taken from the tail vein 60 min after GalN-LPS challenge. Eight
hours after challenge blood was withdrawn by cardiac puncture and
placed into heparinized syringes while the mice were under lethal
pentobarbital sodium (Nembutal 150 mg/kg given intravenously;
Sanofi-Ceva, Wirtschaftsgenossenschaft Deutscher Tierärzte eG,
Hannover, Germany) anesthesia for assessment of liver injury.
Hepatocyte damage in vivo was assessed by measuring the activity of the
liver-specific enzyme alanine aminotransferase (ALT) in plasma as
described by Bergmeyer (3) by an automated procedure.
TNF- concentration was determined by ELISA (Pharmingen, Hamburg,
Germany). A polyclonal sheep anti-mouse TNF- capture antibody,
purified on protein G columns (Pharmacia, Freiburg, Germany), was used
to replace the Pharmingen capture MAb (13).
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RESULTS |
Effect of novobiocin on cytokine production.
As indicated in
Fig. 1, treatment of PBMCs with
novobiocin results in a dose-dependent decrease in TNF-, IL-1, IL-6,
and IL-10 concentrations. At 0.5 mM, cytokine production was nearly completely abolished. Toxic effects can be excluded because cell viability as judged by trypan blue exclusion and by FACS analysis (staining with fluorescein diacetate and propidium iodide) was not
altered by 0.5 mM novobiocin. FACS analysis was used to determine the
percentage of necrotic cells. The cells were incubated with fluorescein
diacetate (green fluorescence indicates living cells) and propidium
iodide (red fluorescence indicates dead cells), and the percentage of
necrotic cells was calculated according to the fluorescence
intensities. Whereas in the absence of novobiocin 1 to 2% of the cells
were necrotic, the percentage of necrotic cells hardly increased (4 to
5%) in the presence of 0.5 mM novobiocin.
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FIG. 1.
Effect of novobiocin on LPS-induced cytokine release.
PBMCs (4 × 106/ml) were incubated in the absence or
presence of different concentrations of novobiocin for 10 min before
the addition of LPS (100 ng/ml) for 6 h. Supernatants were
harvested and analyzed for cytokine content as described in Materials
and Methods. Values are means ± standard deviations of one of
four experiments.  , with LPS;  , without LPS;  , preincubation
with novobiocin.
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To assess the effects of novobiocin at different stages of LPS-induced
TNF-
production we performed a time course study (Table
1). PBMCs were incubated in the presence
of LPS for 6 h, and
novobiocin was added either before the
addition of LPS, at the
same time as the addition of LPS, or after the
addition of LPS.
TNF-
production was almost completely suppressed when novobiocin was
added 10 min before the addition of LPS, simultaneously
with the
addition of LPS, and 1 h after stimulation with LPS.
The
inhibition gradually declined with increasing intervals between
the
time of stimulation with LPS and the subsequent addition of
novobiocin.
After 4 h novobiocin still affected TNF-
production,
presumably
by inhibiting protein synthesis.
Novobiocin inhibits protein synthesis.
The inhibition of
LPS-stimulated cytokine secretion by novobiocin occurs in a setting in
which many proteins are being synthesized by both constitutive and
induced mechanisms. Thus, we assumed that novobiocin should affect
protein biosynthesis. Table 2 indicates that the incubation of monocytes for 3 h with novobiocin leads to
a concentration-dependent inhibition of [3H]leucine
incorporation into proteins. Seventy percent inhibition was observed in
the presence of 0.5 mM novobiocin.
Novobiocin does not inhibit TNF- mRNA expression.
In
previous studies we have shown that inhibitors of ADP ribosylation,
namely, nicotinamide, 3-aminobenzamide, 3-methoxybenzamide, and
meta-iodobenzylguanidine, not only suppress protein
synthesis but also suppress mRNA synthesis (8, 11). To test
whether novobiocin also acts at the transcriptional level, we measured its effects on TNF- mRNA expression. Monocytes were stimulated with
LPS in the presence or in the absence of novobiocin or nicotinamide. Two hours after incubation mRNA expression of TNF- was determined by
reverse transcription-PCR. As seen in Fig.
2, LPS induces TNF- mRNA expression
(lane 2). In contrast to nicotinamide (lane 6), novobiocin (lanes 3 to
5) does not suppress mRNA levels at any novobiocin concentration
tested.
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FIG. 2.
Effect of novobiocin on the expression of TNF- mRNA.
Monocytes (2 × 106/ml) were incubated for 10 min with
various concentrations of novobiocin or nicotinamide before 100 ng of
LPS per ml was added for 2 h. mRNA was isolated and reverse
transcription-PCR was then performed with primers specific for TNF-
and -actin. Data represent the results of one of four representative
experiments. Lanes: 1, unstimulated monocytes; 2, LPS-stimulated
monocytes; 3 to 5, LPS-stimulated monocytes in the presence of 0.1, 0.25, and 0.5 mM novobiocin, respectively; 6, LPS-stimulated monocytes
in the presence of 25 mM nicotinamide. The controls were no cDNA (lane
) and defined cDNA of the concerned protein (lane +). Lane M, marker
(DNA molecular weight marker VI).
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Novobiocin prevents an increase in transaminase activity in
mice.
Having shown that novobiocin inhibits LPS-induced TNF-
production in human monocytes we next assessed whether the drug had similar effects in mice treated with GalN-LPS or GalN-TNF.
Treatment with these compounds has been described to result in
increased serum TNF-
levels and transaminase activities
(
6).
As seen in Fig.
3
novobiocin effectively prevents an increase
in transaminase activity
when it is given prior to the administration
of GalN-LPS. It hardly
affects TNF-
concentrations. The effective
suppression of
transaminase activity by novobiocin was also observed
when LPS was
exchanged for TNF-
in the animal model. The inability
of novobiocin
to reduce the TNF-
concentrations in this model
made us measure the
effect of novobiocin on the TNF-
concentrations
in the supernatants
of mouse peritoneal macrophages stimulated
with LPS. Treatment with
novobiocin does not result in a dose-dependent
decrease in TNF-
concentrations in mouse macrophages, as observed
in human monocytes.
For determination of TNF-
concentrations,
the supernatants of mouse
macrophages (10
6/ml) that were incubated in the absence or
presence of different
concentrations of novobiocin for 10 min before
the addition of
LPS (100 ng/ml) for 6 h were harvested and
analyzed for TNF-
content. For mouse macrophages receiving no
treatment and treatments
with LPS alone, LPS-0.1 mM novobiocin,
LPS-0.25 mM novobiocin,
and LPS-0.5 mM novobiocin, the TNF-
concentrations (means ± standard
deviations of one of two
experiments) were 0, 200 ± 14, 338 ±
14, 796 ± 4, and
168 ± 9 pg/ml, respectively. The values increased
with increasing
concentrations of novobiocin, and only at a novobiocin
concentration of
0.5 mM was a decrease which was not as drastic
as that in human
monocytes observed. This species-dependent response
to novobiocin may
in part account for the failure of novobiocin
to suppress TNF-
concentrations in the animal model.
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FIG. 3.
In vivo protection of mice from GalN-LPS- and
GalN-TNF--induced liver injury due to novobiocin pretreatment.
BALB/c mice received 700 mg of GalN per kg plus 10 µg of LPS per kg
or 700 mg of GalN per kg plus 7.5 µg of TNF- per kg 1 h after
pretreatment with 100 mg of novobiocin per kg. Control animals were
pretreated with saline instead of novobiocin. At 8 h after
challenge blood was withdrawn for an alanine aminotransferase level
determination. The plasma TNF- level was determined 60 min following
LPS treatment. Data are expressed as means ± standard errors of
the means (n = 6 for GalN-LPS or n = 3 for GalN-TNF-). *, P < 0.05 versus
saline-pretreated mice.
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Decreased expression of CD14 is due to shedding.
As shown in
Fig. 4, CD14 expression seems be
extremely susceptible to novobiocin. To verify that the loss of CD14
was due to shedding and not to internalization, the cells were stained with an FITC-labelled anti-CD14 MAb and were incubated in the presence
or absence of 0.5 mM novobiocin (Table
3). After various times, the fluorescence
was measured. Compared to the fluorescence analyzed at time zero, there
was a gradual decrease starting after 4 h of incubation. After
6 h the fluorescence was reduced to 22% of the original level.
This means that CD14 had been shed; if it had been internalized the
fluorescence would not have changed. The shedding is mainly caused by
novobiocin; it is observed only to a minor extent in the absence of
novobiocin (Table 3). To confirm that CD14 has been released, the
amount of soluble CD14 was determined in the supernatants of cells that
had been stimulated with LPS in the presence or absence of novobiocin.
As seen in Table 4, treatment with
novobiocin results in an increase in the soluble CD14 level, and after
6 h the soluble CD14 level is twice as high as that measured in
the absence of novobiocin.
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FIG. 4.
Effect of novobiocin on CD14 expression. PBMCs (4 × 106/ml) were incubated in the presence or absence of
novobiocin (0.5 mM) for 10 min before LPS (100 ng/ml) was added for
6 h. The cells were analyzed by flow cytometry with MAbs against
CD14. The fluorescence of labelled monocytes and lymphocytes was
recorded after gating of the cells by using their forward and side
scatter properties. Curves represent isotype-matched controls for cells
treated with LPS in the presence (B) or absence (A) of novobiocin. Data
represent the results of one of three experiments.
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Novobiocin changes the phosphorylation state of cytosolic
proteins.
To investigate whether the phosphorylation pattern of
activated cells was affected by novobiocin we stimulated monocytes with LPS for different times in the presence and absence of novobiocin and
separated the cytosolic proteins by SDS-PAGE.
As illustrated in Fig.
5 preincubation
with 0.5 mM novobiocin for 10 min resulted in changes in the
phosphorylation states
of several proteins. The changes were more
pronounced after a
16-h incubation time than after a 4-h incubation
time.
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FIG. 5.
Effect of novobiocin on phosphorylation of cytosolic
proteins. Monocytes (4 × 106/ml) were incubated in
the presence or absence of 0.5 mM novobiocin for 10 min before
stimulation with LPS (100 ng/ml). After 4 h, cytosolic
supernatants were prepared and incubated with
[-32P]ATP for 10 min. The proteins were separated by
SDS-PAGE as described in Materials and Methods. The autoradiograph is
representative of three experiments.
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Novobiocin led to a diminished phosphorylation of cytosolic proteins of
36 and 38 kDa. This effect is also seen when other
inhibitors of ADP
ribosylation are used (
11).
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DISCUSSION |
Novobiocin prevents LPS-induced TNF- and IL-6 synthesis. This
effect is shared by two other inhibitors of ADP ribosylation, namely,
meta-iodobenzylguanidine and nicotinamide (11).
In contrast to these two compounds, novobiocin does not reduce the
TNF- activity at the transcriptional level but reduces it at the
translational level. By comparison, the TNF--suppressive drugs
pentoxifylline and thalidomide exert their effects by inhibiting
transcription of the TNF- gene (4) or by enhancing the
degradation of TNF- mRNA (17), respectively. Thus, a
combination of novobiocin with either drug may have synergistic
effects.
Novobiocin prevents not only the production of proinflammatory
cytokines but also that of the anti-inflammatory cytokine IL-10. IL-10
production has been shown to be under the control of TNF- (24) so that the absence of TNF- may be related to the
missing IL-10 synthesis.
The ability of novobiocin to prevent TNF- production in vitro made
us examine its protective effects against an increase in TNF-
production and transaminase activity in GalN-sensitized LPS- or
TNF--challenged mice. Novobiocin led to a drastic decrease in
transaminase activity but did not reduce elevated TNF- levels. The
failure of novobiocin to suppress TNF- production might be species-dependent since the TNF- production of mouse macrophages seems to be less sensitive to novobiocin than the TNF- production of
human monocytes. These findings indicate that novobiocin does not
protect mice from the deleterious effects of GalN-LPS by inhibiting endogenous TNF- production. The beneficial effect of novobiocin becomes evident at the level of apoptosis induction by TNF-, the
central mediator of GalN-LPS-induced liver injury. TNF- destroys liver cells only in GalN-sensitized mice. The mechanism by which novobiocin may inhibit the apoptotic pathway is not known.
Novobiocin not only possesses cytokine-suppressive activity but it also
inhibits expression of MHC class II antigens (HLA-DR/DQ) and molecules
that are involved in the leukocyte-endothelial cell interaction and in
leukocyte migration (CD11b/11c; CD31) (data not shown). Given the role
of MHC II molecules in antigen presentation, one might speculate that
these cells are less effective accessory cells and that they are
reduced in their ability to adhere to the endothelium and to migrate
into tissues. Thus, these cells may be less efficient in encountering a
bacterial challenge.
One major initiator in innate immune responses to bacterial infections
is LPS, the major component of the outer membrane of gram-negative
bacteria (20). LPS complexed with LPS-binding protein
interacts with CD14 receptors on the cell membrane (mCD14) leading to
subsequent cell activation (26, 28). CD14 in its soluble
form (2) also binds to LPS and transmits its action to cells
which are devoid of mCD14, such as endothelial or epithelial cell
(5), and at higher concentrations it blocks the binding of
LPS to monocytes (21). The level of CD14 expression was
found to be drastically reduced in response to novobiocin (Fig. 4). Because the diminished level of expression was due to shedding an
increase in the level of soluble CD14 was observed (Table 4).
The consequences of this effect are difficult to predict. Monocytes may
be prevented from responding to LPS or to other stimuli like soluble
peptidoglycan which also interact with mCD14 (25), or on the
other hand, endothelial cells may be converted to an activation
phenotype expressing both proinflammatory and procoagulant properties
(19). When analyzing the expression of surface molecules on
T and B lymphocytes, neither T-cell-specific (CD2, CD3, CD4, CD5, CD6,
and CD8) nor B-cell-specific (CD21, CD22, CD40, CD72, and CD76)
proteins were downregulated, indicating that the selective suppressive
effects of the drug on monocytes are not secondary to a generalized
mechanism.
The fact that novobiocin inhibits LPS-induced cytokine production
implies that it may interfere with signal transduction events that have
been shown to be involved in cytokine synthesis such as
phosphorylation-dephosphorylation. Treatment of monocytes with novobiocin results in changes in the phosphorylation pattern. It
inhibits phosphorylation of several proteins including -actins (9). The fact that two other inhibitors of ADP ribosylation were also shown to inhibit phosphorylation of the proteins
(11) points to a role of ADP ribosylation in processes
resulting in phosphorylation of - and -actins. The extent of a
correlation between inhibition of cytokine production and inhibition of
phosphorylation of - and -actins remains to be established.
The novobiocin concentrations that we used in the in vitro and in vivo
studies are within the range of concentrations achievable in plasma
(0.16 mM and more), which are reached when novobiocin is used as an
anticancer drug at 4 g/day (12). Thus, application of
novobiocin for the treatment of bacterial infections or in clinical
trials as an anticancer drug may be accompanied by modulated immune
responses such as cytokine production and the expression of surface
molecules on monocytes. It may block these reactions by virtue of its
ability to inhibit endogenous ADP ribosylation.
| |
ACKNOWLEDGMENT |
This work was in part supported by the Deutsche
Forschungsgemeinschaft (grant Ha 2484/1-1).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunobiology, Institute of Zoology, Talstraße 33, D-04103 Leipzig,
Germany. Phone: 49-341-9736747. Fax: 49-341-9736749. E-mail:
shausrz.uni.leipzig.de.
| |
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Antimicrobial Agents and Chemotherapy, August 1998, p. 1911-1916, Vol. 42, No. 8
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
