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B Function1
*
Infectious Diseases and Microbiology, Charing Cross Campus, Imperial College, London, United Kingdom;
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; and
Cytokine Biology and Signal Transduction Laboratory, Kennedy Institute of Rheumatology, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation.
Surprisingly, a synergy was observed between the factor and phorbol
ester-directed NF-B activation. The factor had no effect on IL-1- or
LPS-induced NF-B activity, indicating selective activity of the
factor. The factor did not inhibit the degradation of IB
induced
by TNF, indicating that the target for its activity lies within an
undefined part of the TNF signaling mechanism. The data indicate that
the localized immunosuppression associated with Buruli ulcer relates to
the activity of the released factor, and this may provide a target for
future therapeutic strategies for this intractable
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The persistence of the M. ulcerans infection and the ineffectiveness of antibiotics are thought at least in part to relate to a local immunosuppression at the site of infection. The relationship between M. ulcerans, and the immunosuppressive effect is unknown. Therefore, in this study we investigated the activity of a partially purified extract of M. ulcerans factor on a variety of human immune-competent cells and their products. We provide evidence of effects of the factor on T cell and monocyte functions. The factor also had a selective effect on the activity of TNF, a major cytokine in anti-microbial responses. Our data might explain the underlying immunological unresponsiveness and specifically the poor inflammatory reaction during the necrotizing stage of Buruli ulcer disease. These studies would indicate that neutralization of the toxin would relieve the immunosuppressive effect associated with the disease and may provide a new aspect to future treatment of this disabling condition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pathogenic strain (7634) from an original clinical isolate and the nonpathogenic strain (5113) were provided by Dr. J. L. Stanford (Middlesex Hospital, London, U.K.) and Prof. F. Portaels (Institute of Tropical Disease, Antwerp, Belgium), respectively. Mycobacteria were grown in Dubos broth base medium (Difco, Detroit, MI) as previously described (8). Pathogenic activity was determined by mouse footpad inoculation.
Preparation of CF and high density lipid (HDL) fraction
CF were prepared according to the method of Read et al. (9) and Hockmeyer et al. (8) from bacteria in exponential growth phase. HDL fraction was isolated from CF following KBr density gradient ultracentrifugation and dialysis. The final protein concentration (10) of HDL fraction was adjusted to 100 µg/ml, and aliquots were kept at -70°C until use; 1 L typically provided 5 ml of HDL. In this paper the acronyms aHDL and iHDL refers to preparations obtained from CF in which pathogenic and nonpathogenic M. ulcerans were grown, respectively. A chemically defined extracted factor from M. ulcerans CF termed MUPT (M. ulcerans polyketide toxin) was also used in our studies (11).
Preparation of ethanol-extracted fraction, heat and SDS treatments
HDL was treated with cold ethanol (-20°C) at a dilution of 1/5 (v/v) overnight. The samples were centrifuged for 20 min at 13,000 x g in a microfuge at 4°C. The precipitate was washed twice with the same volume of ethanol and further centrifuged at 4°C. The precipitate was dissolved in 10 ml of PBS and then concentrated to the original volume. The ethanol extract was vacuum evaporated. The dried material was then dissolved in 10 ml of PBS and subsequently concentrated to the original volume. Samples were also heat treated in the autoclave for 20 min at 121°C (15 lb/ in2) and stored at -20°C. HDL was treated with 28 mM SDS for 2 h at room temperature. The samples were dialyzed against PBS and concentrated to the original volume by using Centricon-3 concentrator (Amicon, Danvers, MA).
Cell culture and activation
The murine fibroblast cell line L929, murine fibrosarcoma cell
line WEHI 164 clone 13, and murine macrophage cell line RAW 264.7 were
maintained in DMEM (BioWhittaker, Walkersville, MD) supplemented with
10% FCS, 2 mM L-glutamine, penicillin, and
streptomycin at final concentrations of 100 IU/ml and 100 µg/ml,
respectively. The murine IL-2-dependent CT6 T cell line, murine pre-B
cell line 70Z/3, and human T cell line Jurkat subclone 3KB5.2 (NF-B)
containing ß-galactosidase reporter genes (12) were
maintained in RPMI with 5% FCS and antibiotics as described above.
Human PBMC were prepared from blood bags (North London Blood
Transfusion Centre, Edgware, U.K.) using Ficoll (13).
Human peripheral monocytes were isolated from PBMC by elutriation.
Monocyte enrichment was confirmed by flow cytometry using a FACS
(FACScan, Becton Dickinson, Immunocytometry Systems, San Jose,
CA, USA) with forward and side scatter characteristics and staining
with fluorochrome-conjugated anti-CD45 and anti-CD14 Abs
(Leucogate, Becton Dickinson). The human T cell line 3895 was provided
by Dr. S. Cohen (14). Monocytes were activated by 10 ng/ml
LPS extracted from Salmonella abortus equi, provided by Drs.
C. Galanos and M. Feudenberg (Max Plank Institute, Freiburg, Germany).
PBMC or T cell lines were activated with 1 µg/ml PHA from Difco, 50
ng/ml PMA from Sigma (St. Louis, MO), and 1 µg/ml ionomycin (IMN)
from Calbiochem (La Jolla, CA) or solid phase mouse anti-human CD3
mAb (10 ng/ml OKT3). This and subsequent proliferation assays were
performed according to Katsikis et al. (13).
Animal toxicity assay
One-tenth milliliter of concentrated and filter-sterilized CF and HDL was injected intradermally into the right and left flanks of the guinea pigs. The animals were monitored up to 48 h postinjection. Skin samples from inoculated areas of the guinea pigs were removed and fixed in 4% (v/v) neutral buffered formalin. The sections were stained (hematoxylin and eosin) and examined histologically.
Cell viability and adhesion
This was assayed using 1% nigrosin dye in an exclusion technique (9). Cells were collected, centrifuged, resuspended in DMEM, and stained by nigrosin. At least 200 cells for each sample were enumerated, and the percentage of live and dead cells was determined. Cell death was equated with the cytopathic effect (CPE). The CPE was graded from zero (indicating no CPE) to 4+ (indicating >80% cell death). Human monocytes were simultaneously treated with aHDL and LPS for 12 h. Cells were examined microscopically, and the number of cell clumps was counted. A minimum of 10 separate fields were analyzed.
Cytokine immunoassays
Culture supernatants were assayed for IL-2 by ELISA (Biokine IL-2 Test Kit, T Cell Science, Cambridge, U.K.). TNF production was assayed by ELISA (15), and reagents were gifts from Dr. W. Buurman (Rijks Universiteit Limburg, Maastricht, The Netherlands). Recombinant human TNF, a gift from Prof. W. Stec (Center of Molecular and Macromolecular Studies, Polish Academy of Science, Lodz, Poland), was used to construct a standard curve. Alternatively, TNF was assayed by a bioassay technique using WEHI 164 (clone 13) cells (16). Reagents for the IL-10 ELISA were purchased from PharMingen (San Diego, CA) and performed as described previously (17). Recombinant human IL-10, a gift from Dr. S. Smith (Schering Plough, Kenilworth, NJ), was used to construct a standard curve.
Spectrophotometric assay for ß-galactosidase
Jurkat T cells were washed twice and resuspended at 2 x 106 ml in complete RPMI without phenol red. One hundred microliters per well of cell suspension was added to a 96-well plate and treated in the presence or the absence of HDL with stimuli; TNF (10 ng/ml), PMA (50 ng/ml), or a combination of PMA (50 ng/ml) and IMN (1 µg/ml) in triplicate overnight. The cells were lysed by adding 20 µl of Triton X-100 (1% in 25 mM TBS) and then adding 25 µl of enzyme substrate, chlorophenol red-ß-D-galacto-pyranoside (CPRG) (Boehringer Mannheim, Indianapolis, IN; 8 mg/ml in 25 mM TBS). The cell lysates were incubated at 37°C. The absorbance was then measured at 574 nm by a Labsystem Multiskan Bichromatic spectrophotometer (18).
EMSA
Nuclear proteins were extracted and assayed for NF-B DNA
binding activity using an oligonucleotide encoding the NF-B binding
sequence according to Clarke et al., (18).
Western immunoblotting
Cells were lysed in buffer containing 1% Triton X-100, 10%
glycerol, 20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM EGTA, 50 mM
ß-glycerophosphate supplemented with 1 mM sodium orthovanadate, 1 mM
PMSF, 1 mM DTT, 3 µg/ml aprotinin, 5 µg/ml,
leupeptin, and 10 mM NaF and left on ice for10 min. The cell suspension
was spun down in a microfuge (10 min, 13,000 x g,
4°C), and the supernatant was collected followed by determination of
the protein concentrations in samples using a bicinchoninic acid
protein assay kit (Pierce, Rockford, IL). Equal protein samples were
resolved by SDS-PAGE followed by transfer to nitrocellulose or
polyvinylidene difluoride membranes. Immunoblotting was performed using
rabbit Abs to IB (Ab C-15, Santa Cruz Biotechnology, Santa Cruz,
CA) and phospho-p38 MAPK (New England Biolabs, Beverley, MA). Blots
were developed with pig anti-rabbit HRP conjugate Ab (Dako,
Carpinteria, CA) in conjunction with the ECL Western blotting system
(Amersham Life Science, Arlington Heights, IL) and further exposure to
Hyperfilm MP (Amersham Life Science, Buckingham Shire, UK) at room
temperature (18).
Cell cycle analysis
The labeling protocol of DNA for flow cytometric analysis has been previously described (19, 20). Briefly, the cell pellet was gently resuspended in 1.5 ml of hypotonic fluorochrome solution (apoptosis study) or the isotonic solution (viability study). The fluorochrome used contained 50 µg/ml propidium iodide (PI) and 0.1% sodium citrate in PBS (viability). Alternatively, the PI and citrate were dissolved in 0.1% (v/v) Triton X-100 (apoptosis). The PI fluorescence of individual nuclei was measured using a FACScan flow cytometer (FACScan, Becton Dickinson, Immunocytometry Systems, San Jose, CA, USA)
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The concept of an exotoxin causing Buruli ulcer when first
proposed (3, 7, 9) employed both animal and tissue culture
systems. Originally, murine fibroblast L929 was the cell line used to
assess the biological activity of M. ulcerans factor, and it
has remained the cornerstone for all further studies. L929 cells were
used in a 48-h assay, and the CPE of the exotoxin was graded from 1+ to
4+ by eye. Using this basic assay we partially purified the exotoxin by
KBr density gradient centrifugation and ultrafiltration. The exotoxin
also caused lesions, reminiscent of Buruli ulcer (Fig. 1
A) when injected
intradermally in guinea pigs; the extracts from the nonpathogenic
strain had no effect (results not shown). Lack of accurate quantitative
analysis led to a search for a more appropriate assay. Different cell
lines either of human or murine origin were tested (data not shown).
Murine 70Z/3 was the most sensitive cell line in a 24-h,
[3H]thymidine/proliferation assay. There is a
marked difference in the activity of extracts on 70Z/3 cells (Fig. 1B) obtained from the culture filtrates of pathogenic and
nonpathogenic M. ulcerans (referred to aHDL and iHDL,
respectively). The aHDL had 50% activity at 1/1280, whereas the iHDL
was >1/40. In contrast, aHDL showed no activity on Jurkat T cells in
the same assay (Fig. 1C). We used the 70Z/3 assay to
determine the physicochemical properties of aHLD. The activity was
totally extractable in organic and polar solvents and was resistant to
extremes of pH and moderate heating (Table I). However, autoclave conditions halved
activity (Fig. 1B and Table I), and the inhibitory effect
was destroyed by SDS treatment.
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We examined the effect of aHDL on cell cycle progression of 70Z/3
cells. After 24-h aHDL (1/400) treatment there was inhibition of cell
cycle progression, as shown by a decrease in the
G2/M peak (Fig. 2A). This was dose dependent.
Also, a broad sub-G1 PI fluorescence indicative
of apoptotic DNA fragmentation was observed (Fig. 2A). This
was not seen in untreated cells or in cells exposed to iHDL. At 48
h, the apoptotic effect was more pronounced, affecting the majority (1
in 400) of cells. Again, this was dose dependent. The iHDL had no
effect on either cell cycle progression or apoptosis. Analysis of cell
viability by PI exclusion flow cytometry supported the above data and
showed an increase in viability with lower dose (Fig. 2B).
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The failure of the host to mount an immune challenge to M.
ulcerans infection (21, 22) suggested that the
infection is associated with an immunosuppressed state. A previous
report showed an inhibitory effect of crude culture filtrate on the Con
A-stimulated proliferation of murine lymphocytes (23).
This suggested that M. ulcerans factor may mediate
immunosuppressive activity. Our own results with human cells showed a
similar inhibition by aHDL (but not iHDL) on PHA- or Ag (tetanus
toxoid)-induced proliferation of human PBMC/T cells (data not shown).
The study was extended by examining the effect of aHDL on IL-2
production by T cells. This was investigated in three systems, an
anti-CD3-activated T cell line (Fig. 3A), PMA/ionomycin-stimulated
PBMC (Fig. 3B), and PMA/ionomycin-stimulated Jurkat T cells
(Fig. 3C). In each case aHDL abolished the production of
IL-2, whereas iHDL had no effect. The aHDL also appears to have an
effect on T cell growth directly, as the addition of exogenous IL-2 to
cultures failed to restore PHA-induced PBMC proliferation (results not
shown).
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Monocytes/macrophages and the monokine TNF are key elements in
anti-mycobacterial responses. LPS-induced TNF production from
purified human peripheral blood monocytes was markedly inhibited by
aHDL, whereas iHDL had no effect (Fig. 4A). The effect was dose
dependent with an IC50 of 1/1600 (Fig. 5B), comparable to that of the
70Z/3 assay. The suppression of TNF production was not due to increased
induction of the potent anti-inflammatory cytokine IL-10, because
the LPS-induced expression of IL-10 was also inhibited by aHDL (Fig. 4C). As a consequence of measuring TNF activity by bioassay
using WEHI 164 as well as by ELISA, we assayed the effect of aHDL on
TNF-induced apoptosis (24). aHDL had no effect on TNF
activity in this assay, although aHDL alone had a marginal, but not
significant, effect on the cell line itself (Fig. 4D). These
data indicate that the effects of aHDL are not indiscriminate.
Regardless of the profound inhibitory effect of aHDL on cytokine
production by monocytes, there was no loss of viability as measured by
dye exclusion (data not shown); however, we observed that the factor
prevented monocytes from adhering to plastic and forming clumps in a
dose-dependent manner following LPS activation (Fig. 5).
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B
activity
The observation that production of the cytokine was
inhibited but TNF-mediated cytotoxicity was not led to examination of
another aspect of this cytokine’s activity, the activation of NF-B.
The importance of NF-B in the transcription of a number of
proinflammatory genes make this transcription factor key to the immune
inflammatory response (reviewed in Ref. 25). We
investigated the effect of aHDL on TNF-induced NF-B activation in
the human Jurkat T cell subline 3KB5.2 that contains an NF-B-linked
ß-galactosidase reporter gene. TNF-induced activation of the reporter
gene was inhibited by aHDL, but not by iHDL (Fig. 6A). In contrast, aHDL had a
synergistic effect on NF-B activation when the cells were stimulated
with PMA (Fig. 6B). No consistent effect on NF-B activity
of aHDL alone was observed (Fig. 6, A and B).
Recently, George et al. (11, 26) also isolated a purified
active component, termed mycolactone, from M. ulcerans by
alternative means. We examined this material for its effects on both
TNF- and PMA-induced activation of NF-B. The purified material, like
aHDL, also reproduced the inhibition of TNF-induced NF-B activation
and the enhancement of PMA signal (Fig. 6, C and
D). The fact that both preparations, aHDL and mycolactone,
behaved similarly and had an identical band on TLC (data not shown)
implies that both activities are exhibited by one molecule. We also
examined the effect of aHDL on NF-B activation using EMSA (Fig. 7A). As in the reporter gene
assay, aHDL inhibited NF-B DNA binding activity. In contrast, aHDL
slightly enhanced (
10%) PMA-induced DNA binding activity (Fig. 7A, detected by phosphorimager analysis). We also examined a
proximal event of NF-B activation, the degradation of IB.
Despite the marked inhibition of nuclear NF-B DNA binding activity,
there was no inhibitory effect on IB degradation (Fig. 7B). These data would suggest that aHDL inhibits NF-B
activity at some undefined stage after nuclear translocation.
Unexpectedly, aHDL does not prevent the resynthesis of IB,
although it is generally thought that this requires NF-B. Previous
studies have suggested (27) that p38 MAPK may have a role
in NF-B activation subsequent to IB degradation. As shown in
Fig. 7C, aHDL had no effect on the TNF-induced stimulation
of this kinase, as measured by Western blot with an Ab specific for the
phosphorylated tyrosine and threonine residues required for kinase
activation. The inhibition of NF-B activation would provide a broad
mechanism by which M. ulcerans could effectively paralyze
the immune response, as many proinflammatory mediators, including TNF,
have an NF-B site in their promoters. We therefore investigated the
effect of aHDL on LPS- and IL-1-induced NF-B activity. The aHDL had
no effect on nuclear NF-B EMSA activity induced by LPS or IL-1 (Fig. 8). This would suggest that the effects
of aHDL are targeted to elements of the TNF signaling pathways to
NF-B that are distinct from pathways used by IL-1 and LPS (see
Discussion below). We did note that unlike Jurkat and HeLa
cells, aHDL induced some NF-B activity in RAW 246.9 cells. This
finding was variable from experiment to experiment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous attempts to elucidate the role of the M. ulcerans toxin have focused solely on the cytotoxic activity described in the L929 murine fibroblast line. We used this as an assay for identifying the active extract during purification. Analysis of the activity of our product, aHDL, showed that the cytotoxic effect was variable between cell lines. in a pre-B cell line, 70Z/3, the most sensitive line we examined, aHDL had a profound inhibitory effect on proliferation, as measured by thymidine incorporation after 24 h. Cell cycle analysis at this time point showed no apparent arrest, but evidence of apoptosis. By 48 h apoptosis was complete, and no viable cells were detected. It is unclear whether aHDL induces apoptosis directly or whether cell death follows inhibition of an undefined earlier event in the cell cycle. This differs from the finding in a recent study, in which L929 cells arrested in G0/G1 (11). However, recent studies (K. George and P. Small, manuscript in preparation) have also reported that longer exposure (72 h) of L929 cells to M. ulcerans factor results in apoptosis. These differences in timing are most likely related to the use of alternative transformed cell lines. We used the exquisite sensitivity of the 70Z/3 assay to examine some of the basic physicochemical properties of aHDL. The heat insensitivity, solubility in organic solvents, and resistance to a range of pH indicated a nonproteinaceous organic-like molecule. This has been confirmed by the very recent structural elucidation of the factor (26).
As localized immunosuppression is key to M. ulcerans infection, we examined whether aHDL could be responsible. The only previous study (23) showed that there was an inhibition of Con A-induced proliferation of murine T cells by crude culture filtrate. Using aHDL, we observed a similar effect on anti-CD3- and PHA-stimulated human T lymphocytes. Further studies showed that the factor had a profound inhibitory effect on IL-2 production from primary T cells and the Jurkat T cell line activated with a range of stimuli. There was also a marked inhibition of proliferation induced by IL-2 and other T cell growth factors. By inhibiting both the production of IL-2 and the growth-inducing effect of this cytokine, the M. ulcerans factor would have a potent inhibitory effect on the adaptive arm of the immune system. The mechanism(s) by which aHDL inhibits the production of IL-2 by T cells is the subject of ongoing work.
Our studies have begun to examine the aspects of innate immune
response, which is key to any anti-microbial challenge. The aHDL
potently inhibited LPS-induced TNF production in human monocytes. The
factor also inhibited the production of IL-10 by LPS-activated
monocytes, showing that suppression of TNF is not secondary to the
production of this potent anti-inflammatory cytokine. Abrogation of
TNF production in the presence of M. ulcerans may go some
way toward explaining why there is little or no immune response in the
necrotizing stage of disease, a finding that distinguishes M.
ulcerans infections from other mycobacterial diseases. TNF is
usually referred to as the inflammatory cytokine, emphasizing its
central role in initiating (along with IL-1) the cytokine cascade and
the release of other factors that make up the immune system’s response
to infection (28, 29). Such a response is critical in the
successful resolution of chronic infectious diseases. In tuberculosis,
TNF synergy with IFN-
increases the production of nitric oxide
metabolites (30), which contributes to mycobacterial
killing (31). The potential to inhibit TNF expression may
explain why in M. ulcerans infections the patient’s general
health is described as normal (3, 32), whereas during
episodes of tuberculosis, the systemic TNF generated results in general
manifestations, such as fever and cachexia (33). We also
noted that although aHDL did not appear to have any cytopathic effect
on monocytes, exposure to the factor did block the ability of the cells
to adhere. The reason for this is unknown, but is likely to relate to
the inhibition of expression/function of adhesion molecules. Further
studies are needed to confirm this.
As TNF is a key player in immuno-inflammatory responses, we chose to
investigate the effect of aHDL on selected functions of this cytokine.
Interestingly, the TNF-induced apoptosis of WEHI 164 cells was not
inhibited by aHDL. In contrast, TNF-induced NF-B activation was
inhibited, indicating selectivity of the factor. The mechanism of this
inhibition appears to lie in some unresolved area of TNF signal
transduction. The aHDL had no effect on IB degradation, which is
thought to be a consequence of triggering a cascade of signaling
molecules involving TNF-R1-associated death domain (34),
TRAF2 (35), NF-B-inducing kinase (36),
receptor-interacting protein (37), and IKK/ß
(reviewed in Ref. 25) that finally results in the
phosphorylation of IB with its subsequent destruction. However,
aHDL had a profound inhibitory effect on nuclear DNA binding activity,
suggesting the existence of other signaling components that control
this aspect of NF-B function. Studies by Ghosh et al.
(38) have suggested that the signaling pathway of NF-B
may involve phosphorylation of the p65 subunit of NF-B by protein
kinase A. The precise details of this are still unclear, and other
studies have indicated the involvement of additional kinases that
affect the regulation of NF-B post-IB degradation
(39, 40, 41, 42, 43, 44, 45). Also, aHDL had no effect on TNF-induced
activation of p38 MAPK, which had been implicated in controlling
NF-B function at the later stage of trans-activation
(27). Activation of this kinase has been linked to TRAF2
(46). The above data may imply that the factor inhibits
unknown components of the TNF signaling mechanism, an observation
compatible with the finding that aHDL does not inhibit TNF-induced
cytotoxicity, which is dependant on TNF-R1-associated death domain,
Fas-associated death domain protein (35), and the caspase
arm of the signaling cascade (47). This is also the
subject of further investigation. It was further observed that aHDL
failed to inhibit LPS- or IL-1-induced NF-B activation. IL-1 and LPS
signal via distinct receptors, IL-1R (reviewed in 60) and toll-like
receptor 2/4 (48, 49), respectively. However, the
cytoplasmic tails of these receptor proteins have homology; both share
toll domains. Both receptors appear to use very similar signaling
mechanisms to activate NF-B involving myeloid differentiation
primary response gene, IL-1R-associated kinase, and TRAF6, leading to
activation of NF-B-inducing kinase and IKK/ß (25, 50, 51, 52, 53, 54). This may explain the insensitivity of LPS and IL-1 to
aHDL. It would also indicate that the inhibition of LPS-induced TNF
release is by a mechanism other than the NF-B pathway.
An intriguing observation was that rather than inhibiting, aHDL
enhanced PMA activation of NF-B. This would indicate yet another
activation pathway for this transcription factor. The differential
effect of the factor on PMA compared with TNF could have been due to
the presence of more than one active principle in our partially pure
extract. Very recently, the M. ulcerans factor was
identified as a polyketide lipid toxin (mycolactone) (26).
Examination of aHDL by TLC has shown that aHDL contains mycolactone as
well as other lipid species (P. Small, unpublished observation).
However, mycolactone in our hands gave identical results as aHDL by
inhibiting TNF-induced NF-B and enhancing PMA-induced NF-B
activation. We also observed that like aHDL mycolactone inhibited TNF
production by LPS-activated monocytes (results not shown).
Nevertheless, the possibility of other immunologically active species
in addition to mycolactone in the aHDL preparation cannot be ruled out.
The mechanism by which PMA induces NF-B is still largely unknown,
and thus, we do not know how the aHDL/mycolactone effect is produced.
However, the data would indicate that the activation of NF-B must
involve multiple mechanisms. The M. ulcerans factor may
therefore provide a useful tool for the future analysis of pathways
that activate NF-B.
Mycolactone is structurally related to the immunosuppressants FK506 and
rapamycin as well as the antibiotic rifampicin, which has
immunosuppression as a well-established side effect
(55, 56, 57, 58). In the context of this study it is interesting
to note that rapamycin has also been reported to inhibit CD28-induced
NF-B activation via inhibiting IB down-regulation
(59). Whether this is related to effects on the target of
rapamycin is unclear. FK506 may also, like cyclosporin A, with which it
shares calcineurin as a common target, inhibit NF-B.
In summary, using the partially purified M. ulcerans soluble
factor, we demonstrated a number of potent inhibitory activities on
monocytes and T cells. These actions are likely to account for the
localized immunosuppressed state that permits the persistence of the
infection. However, the effect of the polyketide factor is restricted.
We found that the inhibition of TNF and IL-2 production,
cytokines that are important to both innate and adaptive immune
systems, may explain the lack of host resistance. Also, the findings of
selective effects on aspects of TNF function and NF-B activation
suggest that the factor has a discrete intracellular target(s). The
indication that the M. ulcerans factor is crucial to the
pathogenesis of disease opens up the possibility that inactivation of
the factor may provide a new therapeutic approach for this disabling
condition.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David J. M. Wright Medical Microbiology Section, Infectious Diseases and Microbiology, Charing Cross Campus, Imperial College, Fulham Palace Road, London, U.K. W6 8RF. E-mail address:
3 Abbreviations used in this paper: CF, culture filtrate; CPE, cytopathic effect; HDL, high density lipid fraction; aHDL, active HDL; iHDL, inactive HDL; IKK/ß, IB kinases and ß; IMN, ionomycin; MAPK, mitogen-activated protein kinases; PI, propidium iodide; TNF-R1, TNF receptor 1; TRAF-2, TNF-R2-associated factor-2.
Received for publication March 31, 1999. Accepted for publication July 19, 1999.
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, aHDL; 
, HTaHDL; 
, iHDL; 
, HTiHDL.
Error bars indicate ±SEM of triplicate samples. This figure is
representative of at least three separate experiments.
