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German Diabetes Research Institute, University of Dusseldorf, Dusseldorf, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, IL-12(p70), and
NO (p < 0.01) in cells of macrophage lines.
Pre-exposure to CTB also suppresses LPS-induced TNF- and IL-12(p70)
formation in human PBMC. Both native and recombinant CTB exhibited
suppressive activity, which was shared by intact cholera toxin. In
cells of the human monocyte line Mono Mac 6, exposure to CTB failed to
suppress the production of IL-10 in response to LPS. Control
experiments excluded a role of possible contamination of CTB by
endotoxin or intact cholera toxin. The suppression of TNF-
production occurred at the level of mRNA formation. Tolerance induction
by CTB was dose and time dependent. The suppression of TNF- and IL-6
production could be counteracted by the addition of Abs to IL-10 and
TGF-
. IFN-
also antagonized the actions of CTB on macrophages. In
contrast to desensitization by low doses of LPS, tolerance induction by
CTB occurred silently, i.e., in the absence of a measurable
proinflammatory response. These findings identify immune-deviating
properties of CTB at the level of innate immune cells and may be
relevant to the use of CTB in modulating immune-mediated
diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The B subunits of both toxins form stable noncovalent pentameric rings, and each monomer expresses a receptor for the GM1 ganglioside present in the membranes of virtually all mammalian cells and with lesser affinity to other galactose-containing glycolipids or glycoproteins devoid of adjacent sialic acid residues. Binding to galactosyl structures on target cells appears to be critical for the immunomodulatory function, because a nonreceptor-binding mutant of EtxB was ineffective (8, 9, 10, 11).
CTB and EtxB appear to induce split tolerance at the T cell level, in that Th1-type responses are suppressed and Th2-type responses are enhanced (1, 2, 3, 8, 11). This effect may be due to the activating effect of CTB on B cells, including the up-regulation of MHC class II molecules (12, 13). Also involved may be the induction of apoptosis in CD8+, but not CD4+, T cells (10). The latter has been suggested to account for the prevention of acute graft-vs-host disease when inocula are pretreated with CTB (14). More recent studies suggested the induction of regulatory Th2-type CD4+ T cells by CTB (15). The polarizing effect on Th cell responses was also found when administering a nontoxic cholera toxin (CT) mutant with retained CTB activity, which was reported to preferentially inhibit Th1-type CD4+ T cell responses (16).
We report here that CTB suppresses proinflammatory macrophage reactivity. CTB was found to suppress the proinflammatory responses to LPS while not affecting IL-10 production. Hence, CTB appears to deviate both adaptive and innate immune responses toward a noninflammatory, Th2-type, regulatory state.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human monocyte cell line Mono Mac 6 (17) was
provided by Dr. H. W. Ziegler-Heitbrock (Institute for Immunology,
University of Munich, Munich, Germany). Mono Mac 6 cells were cultured
in RPMI 1640 medium containing the oxalacetate-pyruvate-insulin (OPI)
supplement (Sigma, Deisenhofen, Germany), 2 mM L-glutamine,
antibiotics (120 mg/L penicillin and 200 mg/L streptomycin) and 10%
FCS (Life Technologies, Eggenstein, Germany) at 37°C and 5%
CO2. The mouse macrophage cell line J774A.1
(18) was cultured in RPMI 1640 medium supplemented as
described above and with 1 mM sodium pyruvate but without the OPI
supplement. Human PBMC were enriched from venous blood freshly drawn
from normal healthy donors. Whole blood was centrifuged on a
Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) gradient
(800 x g, 15 min), followed by washing of the enriched
cells in HBSS (400 x g, 10 min). Cells were
resuspended in RPMI 1640 supplemented with the same additions as
described for Mono Mac 6 cells, except without OPI. PBMC,
isolated according to this method, typically contain 
18% monocytes
as judged from FACS analysis.
Macrophage activation
Mono Mac 6 cells or PBMC were cultured at a density of 1 x 106 cells/ml in 24-well flat-bottom tissue culture plates at a volume of 2 ml or in 96-well flat-bottom plates (Falcon; BD Biosciences, Heidelberg, Germany) and challenged with LPS from E. coli serotype 026 B6 (Sigma) at 1 µg/ml. At the end of the experiments, culture supernatants were collected and stored at -20°C until further analyses. Pretreatment of cells with purified CT or CTB (Sigma), with recombinant CTB (gift from Dr. C. Cerkinsky), or with 10 ng/ml LPS was performed in 24-well plates, followed by two washes at 800 x g and 4°C for 5 min and resuspension at 1 x 106 cells/ml. To neutralize for endotoxin, 0.1 µg/ml polymyxin B sulfate (Sigma) was added to cell cultures. Heat treatment was performed by keeping samples for 10 min at 100°C, followed by rapid cooling in iced water.
J774A.1 macrophages exhibit adherent growth and were seeded at 1 x 106 cells/ml in tissue culture dishes (Falcon; BD Biosciences) in a total volume of 5 ml. After pretreatment with medium or CTB, cells were detached by incubation with ice-cold Ca2+, Mg2+-free HBSS (Life Technologies) for 7 min. Cells were washed once at 300 x g and 4°C for 5 min and resuspended at 1 x 106 cells/ml. Challenge with LPS was performed in 96-well plates (2 x 105 cells in a total volume of 200 µl/well as described for Mono Mac 6 cells.
To exclude a possible effect of contaminations with cholera toxin A (CTA subunit) or the holotoxin that causes a rise in intracellular cAMP concentrations, 2',3'-dideoxyadenosine (Sigma), an adenylate cyclase inhibitor, was together added with 10 µg/ml CTB to the cell culture. After 5 h, the Mono Mac 6 cells were pooled, centrifuged, washed with culture medium, and seeded at a cell density of 1 x 106 cells/ml in a 24-well plate. Then the cells were stimulated with 1 µg/ml LPS for the next 5 h, and supernatants were collected for analysis.
In an attempt to prevent macrophage tolerance induction, human Mono Mac
6 cells were precultured for 24 h without or with 10 ng/ml LPS or
10 µg/ml CTB in the presence of 1200 U/ml human IFN- (Biozol,
Eching, Germany) or 50 ng/ml GM-CSF (Pharma Biotechnologie, Hannover,
Germany). In a second approach to preventing macrophage tolerance,
human Mono Mac 6 cells were pretreated for 24 h with 10 µg/ml
CTB in the presence of 1 µg/ml anti-IL-10 or 10 µg/ml
anti-TGF1,2,3 (BD PharMingen, San Diego, CA) mAbs. To reverse
CTB-induced macrophage tolerance, human Mono Mac 6 cells were
pretreated for 5 h with 10 µg/ml CTB. After washing with culture
medium the cells were cultured without or with 1200 U/ml IFN- or 50
ng/ml GM-CSF. After 24 h the cells were washed and stimulated with
1 µg/ml LPS for the next 5 h.
Determination of TNF-, IL-6, IL-10, and IL-12(p70)
The amounts of the cytokines TNF-, IL-6, IL-10, and
IL-12(p70) in the culture supernatants were determined by ELISA
(19). For TNF- determination, microtiter plates (Nunc,
Wiesbaden, Germany) were coated with rat anti-mouse or mouse
anti-human mAb (BD PharMingen). TNF- bound to plates was
determined by complexing with biotinylated TNF- Ab of matching
specificity, subsequent binding of avidin-HRP (Dianova, Hamburg,
Germany), and addition of
2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)diammonium salt
as substrate.
Concentrations of human IL-6 were determined using the PeliKine Compact kit (CLB, Amsterdam, The Netherlands). Microtiter plates were coated with an anti-human IL-6 mAb before the samples were added. Bound IL-6 was determined by a biotinylated sheep Ab to human IL-6, followed by the addition of HRP-conjugated streptavidin and the substrate 3,5,3',5'-tetramethylbenzidine (TMB).
IL-10 concentrations were determined by coating microtiter plates with rat anti-human IL-10 mAb (BD PharMingen). Samples from culture supernatants were added, and bound IL-10 was determined by a biotinylated rat mAb directed against human IL-10 (BD PharMingen). Detection was performed by HRP-conjugated streptavidin and TMB.
For the determination of IL-12(p70) in the culture supernatants, an OptEIA kit (BD PharMingen) was used. A mouse anti-human IL-12 mAb was coated on microtiter plates as the capture Ab. The samples were added, and bound IL-12 was detected by a biotinylated mouse anti-human IL-12 mAb, followed by the addition of HRP-streptavidin conjugate and TMB.
In all cytokine ELISAs, the ODs of the samples were determined by
spectrometry on a microplate reader at 405 nm (TNF-) and 450 nm
(IL-6, IL-10, IL-12(p70)), and the cytokine concentrations were
quantified from standard curves, which were obtained from sequential
dilutions of the corresponding recombinant cytokines.
Measurement of nitrite production
The amount of nitrite (NO2-) released by macrophages was detected in cell-free supernatants by the colorimetric Griess reaction as described previously (20). Briefly, 50 µl of supernatant and serial dilutions of NaNO2 standard solution were placed in 96-well plastic plates and then mixed with an equal volume of Griess reagent containing 1% sulfanylamide, 0.1% naphthylethylene-diamide-dihydrochloride, and 2.5% H3PO4. After incubation for 10 min at room temperature, the OD of reaction products reflecting the concentration of NO2- was assessed at 550 nm on a microplate reader. The results were expressed as micromoles of NO2- per milliliter.
TNF- mRNA analysis
Cell cultures for RNA studies were performed at cell densities
as described above, but in 25-cm2 culture flasks
(Falcon; BD Biosciences) and a volume of 5 ml of RNA was isolated by
Tri-Reagent (Sigma) essentially as suggested by the manufacturer. After
spectrophotometric determination of the concentration, an aliquot of
the RNA was electrophoresed for quality control on an ethidium
bromide-stained formaldehyde agarose gel to check for RNA degradation
(data not shown). Before PCR, 10 µg of the total RNA was reverse
transcribed with Superscript II (Life Technologies) in a total volume
of 40 µl, and 2 µl of each sample was used as a template for the
quantitative determination of mRNA molecules by TaqMan PCR (Applied
Biosystems PRISM 7700 Sequence Detector System; PE Applied Biosystems,
Foster City, CA). This analysis was performed as previously described
(21) with minor modifications. Briefly, the standard curve
for TaqMan PCR used 1 x 107–1 x
101 molecules of a cloned cDNA standard for each
parameter to be determined. All samples were measured in triplicate and
added in a volume of 10–40 µl Master Mix. As an internal reference
dye, 6-carboxy-tetramethyl-rhodamine was used instead of
6-carboxy-X-rhodamine, and uracil-N-glycosylase Amperase was
omitted. The reaction conditions were 2 min at 50°C, 10 min at
95°C, 40 cycles with 15 s at 95°C and 1 min at 60°C, and a
final hold at 25°C. The efficiency of the PCR was monitored by the
slope of the standard curve and was near 100%. For analysis of the
mRNA levels, the ratio of the molecule numbers of TNF-/-actin was
determined and plotted as the means of triplicate determinations in
four independent experiments.
Statistical analysis
Data were expressed as the mean ± SD. Statistical analysis was performed using Student’s two-sided t test. Differences were considered statistically significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Incubation of mouse macrophages with CTB rendered the cells
refractory to subsequent activation by treatment with LPS. As shown in
Fig. 1
A, pretreatment with CTB
suppressed the TNF- response to LPS by >80%. CTB itself did not
elicit TNF- production. The same result was obtained with a human
monocyte line. Again, CTB pretreatment suppressed the TNF- response
to LPS (Fig. 1B).
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). CTB alone did not induce nitrite
formation.
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A). A contribution of CTA
chain was tried by adding increasing amounts of 2',3'-dideoxyadenosine.
Such treatment did not prevent the suppressive action of CTB (Fig. 3B).
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, IL-12, TNF-, and NO, from bone
marrow-derived macrophages (22). We therefore repeated the
experiment with recombinant CTB. As shown in Fig. 4, identical results were obtained when
recombinant CTB was used instead of purified CTB. For comparison, we
also pretreated macrophages with the holotoxin CT, which is able to
exert potent proinflammatory activities, and observed suppression of
LPS-induced TNF- production to a similar extent as with CTB (Fig. 4). A final control was to test for eventual endotoxin contamination of
the recombinant CTB preparation. Following a detailed analysis, boiling
of recombinant proteins is the only safe way to check for such
contamination (23). Boiling for 10 min completely
abolished the suppressive action of recombinant CTB as well as that of
CT, whereas LPS retained its biological activity (Fig. 4).
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Mono Mac 6 cells were pretreated with varying concentrations of
CTB. As shown in Fig. 5A, the
suppressive action of CTB was dose dependent and no more recognizable
at 1 µg/ml or less. Next we analyzed whether CTB-induced
desensitization is a permanent or transient state of monocyte function.
We therefore exposed Mono Mac 6 cells to CTB for 5 h and
challenged the cells with LPS after different periods of time. Our data
show that after only 5 h of pretreatment with CTB maximal
suppression of TNF- production was reached (Fig. 5B).
Monocyte resistance toward LPS persisted at the same level for 24
h and started to fade by 48 h of culture.
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Next we examined whether CTB desensitizes not only for the
LPS-induced synthesis of the proinflammatory mediators TNF- and NO,
but also for other cytokines involved in the regulation of inflammatory
reactions. Fig. 6A shows that
CTB pretreatment of monocytes is able to suppress the production of
IL-12(p70), another proinflammatory cytokine. As shown in Fig. 6B the formation of IL-6 was suppressed by about 60%
(p < 0.05). By contrast, pretreatment of Mono
Mac 6 cells with CTB did not impair the IL-10 response to LPS; rather,
cells were primed for significantly enhanced, but not lasting,
production of IL-10 (Fig. 6C). CTB alone did not induce
measurable IL-12(p70), IL-6, and IL-10 secretion. CTB also did not
induce cytokine secretion if cells were washed after 5 h, and new
medium was added for another 24 h to mimic the pretreatment
protocol (data not shown).
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and IL-12
production
To investigate whether the desensitizing effect of CTB is limited
to macrophage cell lines we pretreated freshly isolated human PBMC with
medium or recombinant CTB (10 µg/ml) for 5 h and challenged the
cells with LPS (1 µg/ml). In control samples without CTB
preincubation, LPS induced TNF- production of 11.6 ng/ml and IL-12
production of 246 pg/ml (Fig. 7), which
is about 40-fold higher than the amount of IL-12 released by
LPS-exposed Mono Mac 6 cells (Fig. 6
A). Pre-exposure to CTB
significantly inhibited LPS-induced formation of TNF- (Fig. 7A) and IL-12 (Fig. 7B).
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To determine whether the suppressive action of CTB occurred at the
level of gene expression, RNA was isolated from monocytes, and the
level of TNF- mRNA was determined by quantitative RT-PCR. Incubation
of Mono Mac 6 cells with LPS led to a 19.2-fold increase in TNF-
mRNA levels. Pretreatment with CTB almost abolished TNF- mRNA levels
in response to LPS (p < 0.01; Fig. 8A). CTB alone did not
increase TNF- mRNA levels. For comparison, macrophage tolerance was
induced by pretreatment with low dose LPS (10 ng/ml), which also
significantly suppressed TNF- mRNA levels after challenge with high
dose LPS (Fig. 8A). As a consequence, significantly less
TNF- was released into the supernatant after either CTB or LPS
pretreatment and challenge with LPS (p < 0.01;
Fig. 8B). TNF- secretion in these experiments occurred in
25-cm2 flasks and was 3.2 times lower than
that described above for assays in 96-well tissue culture plates.
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were involved in CTB suppression. Mono Mac 6 cells
were incubated with a combination of neutralizing Abs to IL-10 and
TGF- during pretreatment with CTB. As shown in Fig. 9A such treatment resulted in
a partial and weakly significant (p < 0.05)
restoration of the TNF- response to subsequent LPS challenge. The
presence of the individual anti-IL-10 and anti-TGF- Abs
during CTB pretreatment did not significantly counteract CTB-mediated
desensitization (data not shown). Pretreatment with Abs directed
against IL-10 and TGF- successfully counteracted CTB-mediated
desensitization of the LPS-induced production of the cytokine IL-6. As
shown in Fig. 9B the IL-6 response was restored to about
60–80% of the value achieved without CTB preincubation
(p < 0.01).
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and/or GM-CSF in addition to CTB.
As shown in Fig. 10A,
IFN- completely abolished the suppressive action of CTB. The
addition of GM-CSF did not counteract CTB action, nor did GM-CSF
antagonize or synergize with IFN-. We determined whether IFN-
would directly interfere with CTB signal transduction or would yield an
independent signal antagonizing CTB signals. Monocytes were first
treated with CTB for 5 h, washed, and then cultured in the
presence of cytokines for 24 h. IFN-, when given after CTB,
reversed suppression and restored normal reactivity to LPS (Fig. 10B). As before, GM-CSF was neutral and did not modulate the
actions of CTB or IFN-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or nitrite. In
control experiments possible endotoxin contaminations were neutralized
by the addition of polymyxin B, and this did not prevent suppressive
effects of CTB. Since the CTB preparation might contain traces of the
intact toxin or of the A subunit, an inhibitor of toxin action was
added, but this did not interfere with CTB activity over a wide
concentration range. More importantly, recombinant CTB, which is devoid
of any CT contamination, exerted suppressive activity to a similar
degree as the native CTB preparation, although CTB does not activate
adenylate cyclase (24). These findings clearly establish
that the lectin CTB exhibits immunosuppressive activities and that such
properties of CTB are not due to trace contamination with CT as has
been suggested previously (25).
The suppressive action of CTB was dose dependent and required 0.1
µM of the CTB pentamer for a significant biological effect. CTB
induces not an irreversible but, rather, a transient state of
unresponsiveness in the monocytes. The desensitizing effect of CTB on
the macrophage response to LPS lasted for at least 48 h and was
already fully established within 5 h of incubation.
CTB-induced LPS tolerance is reminiscent of desensitization of
macrophages to LPS challenge by pretreatment of cells with small
endotoxin doses (26, 27, 28, 29). In the latter case macrophage
function is not completely down-regulated, but cells acquire an
alternate state of activation upon LPS rechallenge, characterized by
the suppression of proinflammatory mediators such as TNF- or NO. Our
findings show that CTB pretreatment suppresses the formation of the
proinflammatory mediators TNF-, IL-6, NO, and IL-12, a major
Th1-associated cytokine. In primary human PBMC pre-exposure to CTB
also resulted in an inhibition of LPS-induced TNF- and IL-12
formation. Although the desensitization of PBMC was not as pronounced
as in Mono Mac 6 cells, these results indicate that the sensitivity to
CTB-mediated desensitization is not restricted to monocytic cell lines,
but represents a general property of monocytes. The desensitizing
effect of CTB may also be relevant in murine experimental colitis,
which resembles human Crohn’s disease (30). In this
animal model of a Th1-driven mucosal inflammation the administration of
CTB resulted in an inhibition of the disease associated with a
reduction of IFN- secretion and an inhibition of IL-12 production.
In contrast to the strong suppression of proinflammatory mediators by
LPS desensitization the production of antagonistic mediators such as
IL-10 is still inducible and may even be potentiated (28, 31). We therefore also analyzed for IL-10 production in
CTB-pretreated monocytes and found no suppression of cytokine
formation. Rather, elevated peak levels of IL-10 were observed after
5 h of LPS exposure, thereby coinciding with the strong
suppression of the proinflammatory mediator TNF-. Subsequently, the
high IL-10 levels declined more rapidly than in control cells without
CTB pretreatment, which may be explained by the macrophage-deactivating
properties of IL-10. From these observations it may be concluded that
CTB induces split tolerance in macrophages by suppressing
proinflammatory responses while preserving the inducibility of
antagonistic mediators, i.e., IL-10. Therefore, LPS receptor function
is still present in CTB-pretreated macrophages, but the outcome of
signaling is different.
Since CTB transiently shifted macrophage reactivity toward IL-10
secretion during tolerance induction, we wondered whether tolerance
induction could be overcome by Th1-associated cytokines. Indeed,
pretreatment with IFN- completely prevented tolerance induction by
CTB. Pretreatment with GM-CSF did not prevent macrophage tolerance
induction. When the cytokines were added to macrophages after their
exposure to CTB, macrophage reactivity to LPS could largely be restored
by IFN-, but not by GM-CSF. It appears that macrophages can be
shifted from the proinflammatory to the antagonistic state and vice
versa by the corresponding mediators.
LPS-induced macrophage tolerance is prevented in the presence of Abs to
IL-10 and TGF-, suggesting that these two cytokines are involved in
the desensitization process (32). We found that the
cytokine Abs also counteract CTB-induced macrophage tolerance. The
preventive effect of the Abs was much pronounced for IL-6 than for the
proinflammatory mediator TNF-. This observation indicates that IL-10
and TGF- only partially account for the suppression of TNF-
production. The preferential activity of CTB to suppress
proinflammatory mediators may be important in weakening the defense
response of the innate immune system against cholera bacteria. Since
IL-10 and TGF- are key mediators of oral tolerance responses
(33, 34), our findings may explain the mechanism by which
CTB potentiates oral tolerance responses when physically linked to Ags
(1).
An important characteristic of CTB is that it shares with LPS the
ability of inducing macrophage tolerance, but, in contrast to LPS, is
devoid of any proinflammatory properties. CTB did not elicit a TNF-
or nitrite response while deviating macrophage reactivity. In contrast,
tolerizing doses of LPS usually elicit a substantial proinflammatory
response before rendering cells refractory to a second challenge with
LPS (28). Very low doses of LPS may result in an enhanced
secondary response (35, 36). Interestingly, preincubation
with CT holotoxin, which is known to be a potent proinflammatory and
macrophage-activating agent (37, 38, 39, 40), also was found to
desensitize Mono Mac 6 cells to a similar extent as after pre-exposure
to LPS. The observed macrophage-desensitizing capacity of CT
corresponds to previous studies demonstrating tolerizing properties of
the holotoxin in models of allograft transplantation (41, 42), delayed-type hypersensitivity (43), and
experimental autoimmune neuritis (44). Hence, similar to
LPS, CT shares pro- and anti-inflammatory properties.
Because of the lack of a proinflammatory response, macrophage tolerance induction by CTB is a silent process, which could also be relevant when CTB is used for the treatment of autoimmune diseases. In these situations CTB may desensitize APC to release proinflammatory mediators in response to inflammatory signals, thereby down-regulating the progression of the autoaggressive process. Synthetic compounds mimicking the immunomodulatory effects of CTB on macrophages may provide a new approach for deviating the proinflammatory activity of innate immune cells.
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Acknowledgments |
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Footnotes |
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2 V.B. and Y.-E.K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Hubert Kolb, German Diabetes Research Institute, Auf’m Hennekamp 65, D-40225 Dusseldorf, Germany. E-mail address: kolb{at}dfi.uni-duesseldorf.de
4 Abbreviations used in this paper: CTB, cholera toxin B chain; CT, cholera toxin; CTA, cholera toxin A; EtxB, labile enterotoxin B chain of E. coli; OPI, oxalacetate-pyruvate-insulin; TMB, 3,5,3',5'-tetramethylbenzidine; NOD, nonobese diabetic.
Received for publication April 14, 2001. Accepted for publication December 12, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting cells and suppressing chemokine expression. Int. Immunol. 12:1449.1 and 2 chain expression. J. Exp. Med. 189:541.. J. Exp. Med. 181:1887. and TGF- responses regulate the occurrence of mucosal inflammation. Immunol. Today 18:61.[Medline]
and nitric oxide production in mouse peritoneal macrophages. J. Exp. Med. 177:511.This article has been cited by other articles:
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LPS). In
parallel, samples were pretreated with medium and challenged with LPS
or CTB at time zero. The concentrations of IL-12(p70)
(A) and IL-6 (B) were determined in the
culture supernatants after 24 h. Shown are mean cytokine
concentrations from six experiments ± SD. *,
p < 0.05; **, p < 0.01.
For IL-10 concentrations (C) the kinetics are shown in
the culture supernatants (mean of three experiments performed in
triplicate ± SD). At 5 h the difference between CTB
