Changes in cellular response to mycobacterial antigens and cytokine productionpatterns in leprosy patients during multiple drug therapy V.. In the first year of treatment, the supernatan
Trang 1Changes in cellular response to mycobacterial antigens and cytokine production
patterns in leprosy patients during multiple drug therapy
V T TRAO,* P L T HUONG,* A T THUAN,* D D ANH,* D D TRACH,* G A W ROOK† & E P WRIGHT‡
*National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, †Department of Bacteriology, UCL Medical School, London, UK
and ‡Royal Tropical Institute, Amsterdam, the Netherlands
SUMMARY
Changes in Mycobacterium leprae-induced lymphoproliferative responses and mediator release by
leprosy patients’ lymphocytes were followed during multiple drug therapy (MDT ) At the time
of diagnosis, multibacillary (MB) patients who did not develop reactions responded to both
sonicated M leprae and synthetic disaccharide coupled to bovine serum albumin (ND-BSA)
antigens, but those who would later develop reactions did not respond, even in the presence of
added cytokines The paucibacillary (PB) group initially had high responses to sonicated M.
leprae but no response to ND-BSA, even in the presence of added cytokines In the first year of
treatment, the supernatants of PB patients’ cell cultures contained factors that enhanced the
phytohaemagglutinin (PHA) response of normal cells In contrast, those MB patients who did
not develop reactions at a later stage produced culture supernatants that were inhibitory
Interestingly, the MB patients who later developed reactions during treatment, and did not
initially respond to M leprae, produced supernatants containing enhancing factors, like those of
the PB group Later on in the treatment, all patients had the same patterns: when response to M.
leprae decreased from its highest level, inhibitory factors were produced Further studies revealed
that the supernatants which inhibited the PHA response of normal cells contained the active form
of transforming growth factor-b
1( TGF-b1), whatever the disease type or treatment status of the donor These TGF-b
1levels correlated directly with the degree of inhibition Similarly, supernatants that neither inhibited nor enhanced PHA responses contained the highest levels of interleukin-10
( IL-10), while those from treated patients that enhanced contained the lowest levels of interleukin-4
( IL-4) and interferon-c (IFN-c) These cytokine correlations transcended the conventional disease
classification, and imply that all patients pass through a sequence of patterns of immune response
during treatment These treatment-induced changes may explain occasional reports of response
patterns at variance with the ‘immunological spectrum’ of leprosy
INTRODUCTION associated with neuropathy.1 At the lepromatous pole, patients
show weak cell-mediated immunity (CMI ) or anergy to The variety of immune responses in patients with leprosy leads
Mycobacterium leprae antigens.2 Suppressor mechanisms are
to a broad spectrum of clinical manifestations, from the thought to play an important role in the induction of anergy paucibacillary (PB) forms at the tuberculoid pole to the of these patients, making them susceptible to generalized M. multibacillary (MB) forms at the lepromatous pole, with many leprae infection.
3–5 M leprae antigens have been shown to
gradations in between Patients with tuberculoid leprosy dis- suppress T-cell proliferation in response to mitogens and/or play strong cellular responses to the bacillus, but this is often antigens in both lepromatous and tuberculoid leprosy
patients.6,7 In other studies, however, it was found that the Received 2 October 1997; revised 28 January 1998; accepted ability of M leprae to induce suppressive activity was lower
et al 9 reported that they could detect no evidence for M.
Abbreviations: BB, borderline; BL, borderline lepromatous; BT,
borderline tuberculoid; IFN-c, interferon gamma; IL-4, interleukin-4; leprae antigen-mediated suppression of delayed-type
hypersen-IL-10, interleukin-10; MB, multibacillary; PB, paucibacillary; sitivity in any group of patients Descriptions of the cytokine RT–PCR, reverse transcriptase–polymerase chain reaction; LL, polar profiles in the blood and tissue of leprosy patients of all types lepromatous; TGF-b
1, transforming growth factor-b1; TT, tuberculoid. have been reported;10–16 they are not all in agreement
We have previously reported that the responses we meas-Correspondence: Dr V Tan Trao, Department of Bacteriology
ured in leprosy patients with different disease forms appeared and Immunology, National Institute of Hygiene and Epidemiology
(NIHE ), 1 Yersin Street, Hanoi, Vietnam. to be related less to their clinical classifications than to their
Trang 2treatment status.17,18 The present study was carried out to background control values for cells alone ranged from 100 to follow the changes in the ability of lymphocytes from leprosy 500 counts per minute (c.p.m.) (mean 304±191) throughout
patients to respond to M leprae sonicates and a species- the whole series of experiments
specific disaccharide, as well as the changes in their cytokine Activation, measured by [3H ]thymidine incorporation, was production from the beginning of multiple drug therapy expressed as c.p.m.±standard deviation (SD)
(MDT ) through to its completion Antigens and mitogens The mitogens PHA (10mg/ml in
culture; Sigma type IV, St Louis, MO), Con A (10 mg/ml; Sigma) and PWM (25 mg/ml; Sigma) were used throughout
MATERIALS AND METHODS
the study to monitor the quality of the cells and the adequacy
The 66 leprosy patients were diagnosed and treated in the The antigen preparations used were: sonicated whole M.
Leprosy Ward of the National Institute for Dermatology in leprae bacilli, isolated from armadillo, and synthetic
disacchar-Hanoi, Vietnam They were clinically classified according to ide coupled to bovine serum albumin (ND-BSA), each at the Ridley-Jopling criteria19 as four tuberculoid (TT ), 13 three concentrations (10, 1 and 0·1 mg/ml in culture) BSA borderline tuberculoid (BT ), one indeterminate leprosy ( I ), alone served as a control for the specificity of response to
17 borderline (BB), 11 borderline lepromatous (BL), four sub- ND-BSA These WHO-IMMLEP antigens were kindly pro-polar lepromatons (LLs) and 16 lepromatous (LL) There vided to us by Dr P Klatser of the Royal Tropical Institute, were 27 females and 39 males; their average age was 30 years Amsterdam and later by Dr R J W Rees
(range 12–60 years) For treatment, the patients were assigned In addition, in some experiments, Mycobacterium
tubercu-either to the PB group, who had a bacterial index of 0 (BI= losis and bacillus Calmette–Gue´rin (BCG) sonicates were also
0) (nine patients: four TT and five BT ) or the multibacillary used, at four concentrations (50, 10, 1 and 0·1 mg/ml ) These group, who had BI≥1 (57 patients: eight BT, 17 BB, one I, were prepared and supplied by the Mycobacteria Laboratory
11 BL, 16 LL and four LLs) The first set of 26 patients (one of the National Institute of Hygiene and Epidemiology,
TT, six BT, eight BB, three BL, three LLs and five LL) were Hanoi, Vietnam.
monitored during treatment by measuring lymphoproliferative All mitogens and antigens were stored at −20° until use.
responses to M leprae antigens at 3-month intervals and the Amplification of the lymphoproliferative response with
cyto-bacterial index (BI ) at 6-month intervals For analysis of the kine-rich supernatants To measure the effect of adding exogen-role of cytokines in the inhibition and enhancement of the ous cytokines, 75 ml of culture supernatant were removed from phytohaemagglutinin (PHA)-induced proliferation of normal target cultures on day 5 and 75 ml of supernatant from PHA-lymphocytes, blood samples were taken at one time point from treated healthy cells was added The target (recipient) cultures
a second set of 40 patients, of whom 15 were untreated (one were then harvested as usual on day 7 Addition of such
TT, five BT, three BB, three BL, three LL) and 25 had been supernatants to control cultures without antigen was a stan-treated for 1–4 years (two TT, three BT, six BB, five BL, nine dard control; these cultures showed increased an increase LL) The samples were processed as described below For each in c.p.m.
group, the treatment schemes were similar to those rec- The control cultures producing cytokine-rich supernatants ommended by the WHO,20 comprising rifampicin and dapsone were of cells from healthy donors, cultured in medium contain-over 6 months or until all clinical symptoms had disappeared ing 10 mg/ml PHA and harvested at 48 hr Control cells treated for the PB group, and rifampicin, clofazimine and dapsone with PHA and used to detect soluble factors produced by over 36 months for the MB group During MDT, 22/66 cultures of patients’ cells were also prepared as described patients from the two sets developed reactions: there were 14 above.
ENL (1 BL, 3 Lls and 8 LL) and 8 RR (1 BT, 4 BB and 3 Enhancing and inhibitory factor(s) production To detect BL) All of the patients were treated with prednisone production of enhancing or inhibitory factors by
antigen-treated patients’ cells, 70 ml of their supernatants were collected
Cell cultures Mononuclear cells were isolated by centrifu- since day 0 After further incubation for 4 days and harvesting,
gation over Ficoll-Hypaque (Pharmacia, Freiburg, Germany), the effect was expressed as follows:
were washed in phosphate-buffered saline (PBS ) and
resus-pended in Iscove’s medium supplemented with 10% heat- %=c.p.m PHA control cells+supernatants
c.p.m PHA control cells−supernatants×100 inactivated human AB serum The cells were cultured in
round-bottom microtitre plates at 5–7·5×104 cells in 100 ml
Cytokine measurements
of medium/well Antigens and pokeweed mitogen (PWM )
Enzyme-linked immunosorbent assay (ELISA) for cytokines.
were added on day 0, in 50 ml of culture medium, while PHA
Interleukin-4 ( IL-4) and interferon-c (IFN-c) in sera and and concanavalin A (Con A) were added on day 3 All cultures
supernatants were measured with an ELISA (AMS kit, sup-were set up in triplicate
plied by Cambridge Bioscience, Cambridge, UK ), following After incubation at 37° for 6 days in a humidified 5% CO
2 the methods recommended by the manufacturer.
atmosphere, 0·15 mCi of methyl-[3H ]thymidine (specific
Interleukin-10 ( IL-10) was quantified by an ELISA devel-activity 2·0 Ci/mmol; Amersham, UK ) was added to each well
oped in the Immunology Laboratory, Bacteriology and the cultures were incubated for a further 18 hr The cells
Department, UCL Medical School, London, using monoclonal were then harvested on a Titertek multiple automatic sample
antibodies (mAb) supplied by Pharmingen (San Diego, CA) harvester and the retained radioactivity was counted in a
In brief, plates with 96 flat-bottom wells (Corning Easy Wash) Beckman liquid scintillation counter ( Tri-carb 1900 CA Liquid
Scintillation Analyzer; Beckman, Canberra, Australia) The were coated with 50 ml of purified anti-IL-10 capture mAb at
© 1998 Blackwell Science Ltd, Immunology, 94, 197–206
Trang 32 mg/ml in coating buffer (pH 8·2) and incubated overnight at to check for the specificity of the test The samples and
rhTGF-b
1 were incubated with three concentrations of the 4° The plates were dried and the wells were filled with 200 ml
of PBS/3% BSA, for blocking, at 37° for 1 hr After washing chicken polyclonal anti-TGF-b
1(R&D Systems) (10 mg, 5 mg and 2·5 mg) for 1 hr at room temperature, then the CCL-64 twice with PBS/Tween, standards, undiluted supernatants and
sera diluted 154 were added in duplicate and the plates were cells were added
The significance of differences in results between groups or further incubated for 3 hr at 37° The plates were then washed
four times and 100 ml of biotin-conjugated anti-IL-10 detecting at different times was tested with the Mann–Whitney U-test
and Two-tailed t-test.
mAb was added, diluted to 1 mg/ml in PBS containing 3%
BSA The plates were incubated at 37° for 90 min, then washed
four times; 100 ml of ExtrAvidin-alkaline phosphatase (ALP)
was added and the plates were incubated for 1 hr Following
Changes in response of patients’ mononuclear cells to
this incubation, the plates were washed thoroughly at least
mycobacterial antigens
four times, 100 ml of substrate solution (p-nitrophenyl
phos-phate tablet diluted in distilled water, Sigma) was added and Four different concentrations of antigens were used to
stimu-late patients’ cells in vitro; that giving the highest response was
the plates were incubated at 37° for 60 min The absorbance
patterns to these antigens, the patients could be sorted into The results for cytokine levels were calculated as pg/ml in
serum and as pg/ml of 2×106 cultured cells three groups: the PB group (one TT and five BT ), the MB
group without reactions during follow-up (MB – R) (one BT,
Reverse transcriptase–polymerase chain reaction
(RT–PCR) The expression of genes for beta-actin, IL-10, four BB, two BL and two LL), and the MB group who
developed reactions during treatment and follow-up (MB+R) transforming growth factor-b
1( TGF-b1), interleukin-2 (IL-2) and interleukin-12 ( IL-12) were measured, using primers (three BB, one BL, three LL and three LLs) No PB patients
developed reversal reactions during this study
obtained from Clontech Laboratories, Palo Alto, CA
Total RNA was isolated by the phenol chloroform method Figure 1 shows the changes in responses by patients’ cells
to M leprae sonicates and ND-BSA antigen for all three
Prior to PCR, reverse transcription ( RT ) was carried out first
at 65° for 10 mins with 8 ml of sample in diethyl-pyro-carbonate groups: PB ( Fig 1a), MB without reactions ( Fig 1b) and MB
with reactions ( Fig 1c), with and without addition of cytokine-(DEPC ) treated water and 2 ml of oligo-dT from a stock of
500 mg/ml, then at 37° for 1 hr, then 95° for 5 min in 10 ml of rich supernatants The PB patients tended to have the highest
responses to M leprae sonicate at the start of treatment, and
RT mix, containing 5 ml of 5×RT buffer (first-strand buffer),
5·5 ml of DEPC water, 0·5 ml of BSA (1 mg/ml stock, molecular they tended to increase by 18 months compared with the first
test before treatment began (0·01<P<0·04) ( Fig 1a) Among
grade acetylated BSA), 0·5 ml of RNAse inhibitor, 0·5 ml of
dNTP (stock 10 m) and 0·5 ml MMLVRT (reverse the MB patients, those who did not later develop reactions
had higher responses at the time of diagnosis (P<0·002) than
transcriptase)
PCR was performed in a Hybaid Omnigene thermocycler did those who would later have reactions The MB – R
patients’ responses decreased over the first 9 months The reaction was carried out with 5 ml of sample in a 50-ml
final reaction volume with 10 pmol of each oligonucleotide in (0·01<P<0·04), after which they increased again until 21
months (P<0·002) and decreased thereafter (P<0·01).
10×PCR buffer The PCR conditions were: 30 cycles (95° for
30 seconds, 60° for 30 seconds and 72° for 45 seconds) followed ( Increase or decrease in these cases is in comparison with the
response at T
0.) In the MB+R group, the low response at the
by elongation at 72° for 7 min
Samples of RT–PCR products were loaded onto a 1·8% start increased over the first 18 months (0·01<P<0·04) to a
peak and then decreased thereafter (P<0·01) The two MB
agarose gel in TBE and separated by electrophoresis at 83 V
for 50 min RT–PCR products were visualized under ultraviolet groups, with and without reactions, thus exhibited opposing
patterns of in vitro proliferative response to M leprae antigens
light A DNA ladder ( FX174/Hae III; Sigma) was used as
To amplify any responses, cytokine-rich supernatants were
The TGF-b
1 bioassay A growth inhibition assay of the
mink lung epithelial cell line (ATCC/CCL-64) was used to added on day 5 to cultures incubated with the mycobacterial
antigens At T
0, the response of the PB and MB – R groups measure bioactivity of TGF-b
1.21 In brief, CCL-64 cells were incubated in 96-well plates at a density of 20×103 cells/well increased significantly (P<0·001) in the presence of added
cytokines, while the lower responses of MB+R were for 2 hr in RPMI medium with 14% FCS Then, the TGF-b
1 standard, in serial dilutions in RPMI (rhTGF-b1, R&D unchanged Later on in the treatment, in the presence of
exogenous cytokines, the responses of PB patients decreased Systems, Abingdon, UK ), sera and supernatants from patients
were added, including acid-activated supernatants The total over the first 6 months (P<0·001), peaked at 1 year (P<0·001)
and then decreased thereafter to 15 months (P<0·01) For
volume of the assay was 100 ml The plates were incubated at
37° in a humidified CO
2incubator until they reached conflu- both MB groups, the responses showed a more gradual change.For MB – R, there was a gradual decrease over the first 9 ence in the untreated control wells (2 days) MTT (10 ml/well
of 0·5% MTT in PBS ) was added to the plates and, following months (P<0·002), which was followed by a gradual increase
until month 30 (P<0·001) The responses of MB+R patients
a 90-min incubation, 50 ml of 10% SDS in 0·02 HCl was
added and the plates were read at 570–590 nm after a further slowly increased during the first 18 months (P<0·001).
PB patients did not respond to ND-BSA (data not shown),
40 min
Neutralization of TGF-b
1bioactivity was also performed while both MB groups responded to this antigen The patterns
© 1998 Blackwell Science Ltd, Immunology, 94, 197–206
Trang 4Time (months)
(a)
(b)
(c)
Time (months)
(a)
(b)
(c)
Figure 2 Changes in production of factors during treatment compared Figure 1 Changes in response to mycobacterial antigens by leprosy
patients’ cells during treatment Responses are shown to whole son- with the response to M leprae antigens When patients’ cells were
cultured with antigen, the supernatants were collected and added to
icated M leprae ($ with or # without added cytokine-rich
super-natants) and to ND-BSA (+ with or6 without added supernatants) cells from healthy controls exposed to PHA The result, inhibitory or
enhancement of proliferative response, is expressed as a percentage of (a) mean±SD of six paucibacillary patients (no response to ND-BSA),
(b) mean±SD of 10 multibacillary patients without reactions (MB–R) response by control cells to PHA in the absence of patients’ culture
supernatants Response to M leprae sonicate is shown (#) as well as
and (c) mean±SD of nine multibacillary patients with reactions
(MB+R) during treatment the effect of the supernatants on control responses ($), expressed as
percentage of PHA response in the absence of the supernatants (a) Mean±SD of six paucibacillary patients, (b) mean±SD of 10 multiba-cillary patients without reactions (MB-R) and (c) mean±SD of nine
of their responses were the same as those to whole M.
multibacillary patients with reactions (MB+R) during treatment.
leprae antigens.
( Fig 2) In the case of PB patients who did not have reactions
Changes in response to M leprae-sonicated antigens: production
during treatment ( Fig 2a), the response to M leprae was high
of enhancing and inhibitory factors
at the start of treatment compared with the MB+R group
(P<0·001), and the cells did not produce inhibitory factors.
Production of factors that could enhance or inhibit the PHA
response of healthy control cells varied throughout the period At 3 months, when the response was lower compared with
that at T
0 (P<0·001), production of enhancing factors
of treatment in a way that was related to proliferative response
© 1998 Blackwell Science Ltd, Immunology, 94, 197–206
Trang 5increased (P<0·001) Only after 1 year of chemotherapy did be detected in the 18 supernatants of the inhibitory groups
( Un- and Tr-), and was present in supernatants from both inhibitory factors begin to appear again in the culture
super-natants, when the response to M leprae had increased to the untreated and treated patients ( Table 1) Again, this apparent
relationship between inhibition of PHA responses and the
original level (P<0·001) One BT patient had a reaction during
treatment; he produced measurable inhibitory factors only presence of active TGF-b
1in the supernatant is confirmed in
a scatterplot (r = 0·60 with P<0·001) ( Fig 5) The inhibition
after 18 months of treatment
The results were different for the two MB groups MB – decreased when the samples were diluted (data not shown) In
order to check the identity of the inhibitory activity, the 17 of
R patients had high antigen responses in the presence of
exogenous cytokines at T
0, and produced inhibitory factors. 18 active supernatants were tested again in the presence ofneutralizing antibody, which completely eliminated the Their responses decreased slowly after 1 year of treatment
(0·01<P<0·04); during that period, their cells began to activity ( Fig 6)
produce enhancing factors They again switched to producing
inhibitory factors at 21 months, when their responses increased
Cytokine patterns in MB patients during treatment
again (P<0·002) Enhancing factors were produced when the
response to antigen was low, and inhibitory factors when the The representative cytokines for the T helper 1 ( Th1) and T
helper 2 ( Th2) subsets, IFN-c and IL-4, were measured in response was high
For MB+R patients, response to antigen at T
0was low sera of the same five groups and in the corresponding super-natants The lowest levels of both IFN-c and IL-4 were found
or non-existent, even in the presence of added cytokines; at
this time, they produced enhancing factors There was a slow in the samples from the treated patients yielding enhancing
supernatants ( Tr+) ( Table 1) For both MB groups, IFN-c change to production of inhibitory factors after 2 years of
treatment (P<0·001), when the proliferative responses had levels were increased in sera and the concentrations reached a
peak when their supernatants contained IL-10 ( Tr0) (P<0·04) increased significantly (P<0·001) This situation remained the
same until at least 3 years after initiation of treatment For the MB – R group, IFN-c levels decreased and were
lowest at about 21 months after treatment ( Tr+) (P=0·049),
then increased again to a peak when IL-10 was found in the
The soluble factors as cytokines
supernatants After that, IFN-c decreased and the supernatants
of both groups contained the active form of TGF-b
1 ( Tr-).
As the presence of M leprae-induced inhibitory or enhancing
factors in cell culture supernatants occurred in all types of For IL-4, the levels in sera of patients in the MB+R group
remained unchanged during treatment and started to decrease patients at different stages in their treatment; we postulated
that these effects might correlate with the pattern of cytokine at the end of treatment, when their supernatants contained
IL-10 For the MB – R group, IL-4 levels were constant in release, rather than with the original patient classification
Therefore, blood samples were taken from a further 40 patients both untreated and treated patients, except for the Tr+ group,
which had significantly lower IL-4 levels (P=0·03) By the
and these were classified, by the methods described above,
1was found in their supernatants, their IL-4 levels were again (1) untreated patients whose cell cultures yielded enhancing
(2) untreated patients yielding inhibitory supernatants
( Un-);
Cytokine patterns in relation to clinical classification
(3) treated patients yielding supernatants with no effect on
normal lymphocytes ( Tr0); Table 2 shows the cytokine levels in TT-BT, BB-BL and LL
groups during treatment The IL-10 levels were significantly (4) treated, enhancing ( Tr+); and
(5) treated, inhibitory ( Tr-) higher in the sera of the LL group than in the TT-BT group
(P=0·03) There was no significant difference between serum
Serum from these donors, and aliquots of these
super-natants, were stored at −20° The cells themselves were used IL-10 levels in TT-BT and BB-BL groups (P=0·22) IL-4
levels were significantly higher in the LL group, compared for the isolation of mRNA
Table 1 shows the concentration of IL-10 in these five with BB-BL and TT groups (P=0·042).
groups of supernatants and sera IL-10 was found at high
levels in the sera of all groups except the Tr+ In the
DISCUSSION
supernatants, IL-10 levels were significantly higher in the Tr0
group, compared with the other four groups (0·028<P<0·04) Leprosy is a chronic disease, and it is sometimes assumed that
the immunological responses associated with the different This relationship between high IL-10 and lack of effect on
PHA responses, is confirmed in the scatterplot ( Fig 3), which forms of leprosy are rather stable Immunological differences
in leprosy patients are known to be associated with the forms shows that the supernatants that markedly inhibited or
enhanced PHA responses were those with low IL-10 content of clinical presentation It is well established that at one pole
of the clinical spectrum, the lepromatous patients have poor The expression of this cytokine gene was found in the mRNA
from all the samples of the Tr0 group that were tested, but in antigen responses in vitro.22–25 At the other pole, in tuberculoid
patients, who seem to be able to clear M leprae from tissues,
only one of seven samples from the other groups ( Fig 4)
The TGF-b
1 gene was found to be expressed in mRNA a strong cellular response to the bacillus can be detected.These differences have been considered to remain constant1 from almost all patients tested ( Fig 4) Similarly, latent
TGF-b
1 was revealed by acidification from all five groups from the time of presentation throughout the course of disease,or of treatment Most previous studies were, however, based ( Table 1) The active form of TGF-b
1 could, however, only
© 1998 Blackwell Science Ltd, Immunology, 94, 197–206
Trang 6Table 1 Relationship between lymphoproliferative responses and cytokine patterns
*Lymphoproliferative response to sonicated M leprae antigens without addition of cytokines was measured as mean±SD in counts per
minute (c.p.m.) of groups of untreated patients: five with enhancing factors ( Un+), six with inhibitory factors ( Un−), and of groups of treated patients: eight with enhancing factors (Tr+), seven with no-effect factors (Tr0) and 12 with inhibitory factors on PHA response of normal cells.
†The cytokine levels were measured (pg/ml ) in sera (/ser) and supernatants (/sup) of the same untreated groups and treated groups The results expressed as mean value of each group±SD.
TGF-b
1was measured in both forms: active (Act) and latent (Lat).
drug therapy The patients’ in vitro lymphocyte proliferative responses to M leprae antigens (including the species-specific
ND antigen) were monitored, considering that the viable antigen loads in untreated, especially MB, patients26 would be changed27 during treatment with anti-leprosy drugs.27,28 These changes could be an important factor influencing immune responses of leprosy patients Furthermore, many studies have found that the dose of antigen affects the type, and not just the intensity, of the response that develops.29,30 During treat-ment, patients of all groups went through changes in bacterial loads, which could potentially lead to changes in activation of different T-cell subpopulations interacting with macrophages During the months or years of treatment, the previously
reported consistent anergy of in vitro lymphoproliferative responses to M leprae antigens was not observed in any of
our patients Antigen response was detected in all patients at
Figure 3 Correlation between IL-10 production and effect on PHA one time or another, especially when low responses were response of normal cells The supernatants were taken from the
amplified by adding cytokine-rich supernatants A lack of enhancing group (>100% PHA response), the no-effect group (100%
detectable response to M leprae antigens was also seen in all
PHA response) and the inhibitory group (<100% PHA response) and
types of patients at different times The same antigen at IL-10 concentration, expressed in pg/ml, was measured The highest
different points in time could induce different responses in one concentration of IL-10 was found in supernatants with no effect on
patient These variations in response to M leprae antigens
PHA response of normal cells, compared with the other two groups
data reported by others who studied, at one time, different patients who were at different points of treatment
on tests of patients’ responses to M leprae antigens at one
There has been disagreement in the literature about whether time point In this study, different types of leprosy patients
were followed during shorter or longer periods of multiple specific responses of leprosy patients could be reconstituted
Table 2 Relationship between clinical classifications and cytokine patterns
Groups IFN/ser IFN/sup IL-4/ser IL-4/sup IL-10/ser IL-10/sup
The number of patients in TT-BT group was 11, in BB-BL was 17 and in LL was 12.
The cytokine levels were measured in pg/ml in sera (/ser) and in supernatants (/sup) of
M leprae-treated cultures from TT-BT, BB-BL and LL groups during treatment.
The results are expressed as mean value±SD in pg/ml.
© 1998 Blackwell Science Ltd, Immunology, 94, 197–206
Trang 7Inhibiting group No effect group
b-actin:
IL-10:
TGF-b1:
Lane 1: Marker, wX 174/HaeIII Lane 2: TT (3 month) Lane 3: BT (0 month) Lane 4: BB (0 month) Lane 5: BT (0 month) Lane 6: BB (0 month) Lane 7: BL (0 month) Lane 8: BL (0 month) Lane 9: LL (24 month)
Lane 1: Marker, wX 174/HaeIII Lane 2: TT (12 month) Lane 3: BB (12 month) Lane 4: BB (32 month) Lane 5: BB (41 month) Lane 6: LL (relapsed)
Figure 4 IL-10 and TGF-b
1gene expressions in cells taken from no-effect and inhibitory groups Beta-actin was used as a control for quality of the cells IL-10 gene expression were found in all six patients tested with no effect on PHA response of normal cells, but only one of the inhibitory group TGF-b
1gene expression was found in 11/12 patients tested from two groups.
by addition of cytokine-rich supernatants and/or purified or exogenous cytokines Two kinds of suppressions were observed
in our study: one was unreconstituted low or undetectable recombinant IL-2 We routinely tried to amplify responses to
M leprae at all time points, by adding cytokine-rich super- response, found only at the start of treatment in the
multibacil-lary group who will proceed to develop reactions (BT to LL natants Responses were increased in PB and MB – R patients
by addition of cytokine-rich supernatants at T
0, while patients) The other was mediated by macrophage activeTGF-b
1, found at the start of treatment in both sera and responses remained low in MB + R patients Later
reconsti-tution was successful in all three groups supernatants of MB – R patients who did not develop
reac-tions, but by the end of treatment only in supernatants of
Kaplan et al 6 have shown that M leprae antigens could
suppress the proliferative responses of cells from LL patients patients of all groups Experimental evidence generated by the
study of autoimmune disease in human as well animal models
as well as those of TT and of healthy individuals never exposed
to M leprae Mehra et al 3–5 proposed that there was high M. strongly implicates endogenous TGF-b
1in the natural suppres-sion of autoimmune disease.33 The presence of this cytokine
leprae-specific suppressor activity in LL, compared with the
other forms of the disease Work by other groups, however, in sera and supernatants of patients who did not have reactions
during treatment was consistent with this suppressive function suggests the contrary.6,26 In our experiments, patients’ cells
were cultured with mycobacterial antigens, with and without The increase in production of this cytokine at the start and
© 1998 Blackwell Science Ltd, Immunology, 94, 197–206
Trang 8monocytes of leprosy patients.16 IL-10 was found in super-natants of 23/40, in sera of 39/40 and in both sera and supernatants of 23/40 patients but in our study this cytokine did not express inhibitory activity, in agreement with Mutis
et al.13 IL-4 could be detected in the supernatants of very few patients (5/20) in the sera of 21/40 and in both sera and supernatants of 2/40 The patients with low supernatant IL-4 values were of all types 2/3 TT, 7/8 BT, 8/9 BB, 8/8 BL and 10/12 LL, as expected from the picture of patterns from separate cell clones.13,14,16 We measured four cytokines in this study, and it was found that only cytokines produced by macrophages, like IL-10 and TGF-b
1, or activating them ( IFN-c), were found at bioactive levels in all patients during treatment This could serve as evidence of continuous macro-phage activation during treatment, producing different factors
in interaction with effector T cells Generally, heterogeneity in
Figure 5 Correlation between TGF-b
1production and effect on PHA the production of cytokines by T cells were observed; there response of normal cells The supernatants were taken from the
was no correlation with clinical status, nor with distinctive enhancing group (>100% PHA response), the no-effect group (100%
T-cell subsets, in contrast to reports from studies on skin PHA response) and the inhibitory group (<100% PHA response),
lesions from leprosy patients10,11 but in agreement with Howe and measured active TGF-b
1 This cytokine was detected only in the et al. 14 In our study, M leprae-sonicated antigens and ND-BSA
supernatants of the inhibitory group (and not the other two groups).
were used, and ND-BSA reacted with the immune system of
the patients in a similar manner, as did whole sonicated M.
end of treatment could reflect its functions in the initiation
leprae antigens, but selectively among MB patients This has
and resolution of inflammation and immunological events.34
also been seen with several recombinant and peptide antigens.15
In our study, TGF-b
1had a non-specific inhibitory effect on To investigate the correlation between the cytokine patterns PHA response of normal cells, and down-regulated
lymphop-and clinical classification (cross-sectional ) or treatment status roliferative responses and production of all cytokines in a
non-( longitudinal ), we studied the cytokine changes in sera and in
specific manner in agreement with Ahuja et al.35
patients’ supernatants, shown in Tables 1 and 2 Table 2 shows
In our study, the levels of cytokines were measured in both
the differences in cytokines related to clinical classification sera and supernatants of patients IFN-c levels within a
Although the variations between individuals were large, the biological activity range were found in the supernatants of
average levels of IFN-c, IL-4 and IL-10 in patients’ sera 29/40 patients of all types, in the sera of 35/40 and in both
tended to increase towards the LL pole, while the levels of sera and supernatants of 29/40 These data were in agreement
with data reported on cell lines and clones,12,13 and on IL-4 and IFN-c in supernatants of all groups remained almost
Figure 6 Neutralization of TGF-b
1 bioactivity in patients’ supernatants by chicken polyclonal anti-TGF-b1 antibody The 17 active supernatants (&) showed inhibition of the growth of ATCC/CCL-64 (% of inhibition >0), but this activity was eliminated completely by addition of neutralizing antibody (%) (approximately 0% inhibition).
© 1998 Blackwell Science Ltd, Immunology, 94, 197–206
Trang 9P M.E. & B P.J (1987) Mycobacterium leprae
the same IL-10 levels were even higher in supernatants of the
antigen-induced suppression of T cell proliferation in vitro.
TT-BT group If compared with Table 1, one could see that
J Immunol 138, 3028.
during treatment, cytokine levels in sera of patients changed
7 S U., S S., R G., L J & I J (1987) from levels of TT group ( low bacterial load ) to LL (higher
Suppression of delayed hypersensitivity skin reaction to tuberculin bacterial loads) and then decreased again For the MB patients
by M leprae antigens in patients with lepromatous and tuberculoid
who did not develop reactions during treatment, the pattern leprosy Clin Exp Immunol 68, 58.
revealed two changes In the second change, from Un+ all 8 S M.C., B S., V R & B L.M (1989) cytokines started lower than TT levels, increased and then Reduced suppressor cell response in lepromatous leprosy Infect
9 F P.E.M., G P.J.K., M N., P J.M., R
An important finding in this study was the difference in
R.J.W. & S J.L (1989) Failure of M leprae soluble
recognition and response to antigens at the start of treatment
antigens to suppress delayed-type hypersensitivity reaction to between MB patients who did not develop reactions (MB-R)
tuberculin Clin Exp Immunol 77, 226.
and MB patients who did develop reactions (MB+R) The
10 Y M., U K., D R.J., W K et al.
MB-R responded to the antigens and the active form of
(1991) Defining protective responses to pathogens: cytokine pro-TGF-b
1 was found in their sera and supernatants, while files in leprosy lesions Science 254, 277.
MB+R did not respond to antigens and produced, as yet 11 S P., A J.S., C C et al (1991) Differing unidentified, enhancing factors These differences could play a lymphokine profiles of functional subsets of human CD4 and role in the occurrence or not of reactions, and warrant further CD8 T cell clones Science 254, 279.
study Another interesting point was the selectivity of response 12 H J.B.A.G., W-M R., R P.C.M et al (1991)
Selection of a human T helper type1-like T cell subset by
myco-to the ND-BSA epimyco-tope of M leprae antigen among MB but
bacteria J Exp Med 174, 583.
not PB patients
13 M T., K E.M., C Y.E., S H., V
In conclusion, during long-term follow-up of leprosy
R.R.P & O T.H.M (1993) Analysis of cytokine
pro-patients of all types, the dynamics of response to M leprae
duction of mycobacterium reactive T cells Failure to explain antigens and the associated cytokine production patterns
Mycobacterium leprae specific nonresponsiveness of peripheral
became obvious This complexity could reflect changes in
blood T cells from lepromatous leprosy patients J Immunol 150,
bacterial load during treatment, the distribution of immunolog- 4641.
ically active cells (the study only concerns cells in the blood ), 14 H R.C., W A., D A & F D (1995) and/or the balance between antigens and the host at the time Functional heterogeneity among CD4+ T-cell clones from blood
of diagnosis and skin lesions of leprosy patients Identification of T-cell clones
distinct from Th0, Th1 and Th2 Immunology 84, 585.
15 M N., M A., W B et al (1995) Cytokine profile
ACKNOWLEDGMENTS
of circulating T cells of leprosy patients reflects both indiscriminate and polarized T-helper subsets: T helper phenotype is stable and
We would like to thank the National Institute of Dermatology, Hanoi
and the Dermatology Centre, Hanoi Health Service for their uninfluenced by related antigens of Mycobacterium leprae.
Immunology 86, 97.
co-operation in supplying the patients’ samples, and the Hanoi Medical
College for providing the control blood samples We would also like 16 D H.M., Y S.K., B K et al (1996) Induction
of Th1 cytokine responses by Mycobacterial antigens in leprosy.
to thank Dr Barry Walker, Elaine Filley and Sahal Alnakhli for their
expert technical assistance We thank Dr Rees and Dr P Klatser for Infect Immun 64, 4385.
17 T V.T., H P.L.T., T A.T., L H.T., T D.D the antigens, Dr A Meager, NIBSC for CCL-64, Dr P Klatser for
assistance in preparing the manuscript, and Dr H F Engers, Dr & W E.P (1988) Response to M leprae by lymphocytes
from new and old leprosy patients: Role of exogenous
lympho-J Louis and Dr G Milon for very helpful discussion We are greatful
to Prof E J Ruitenberg and Prof Rene de Vries for their support. kines Ann Inst Pasteur/Immunol 139, 121.
18 T V.T., L H.T., Q N.D et al (1994) Long-term
The Netherlands Ministry of Development Cooperation through the
University of Amsterdam (project VH-15) and TDR/WHO provided evaluation of immune status in leprosy patients undergoing mul-financial support during the period of the study. tiple drug therapy Int J Leprosy 62, 365.
19 R D.S & J W.H (1966) Classification of leprosy
according to immunity A five-group system Int J Leprosy 34, 255.
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