Changes in cellular response to mycobacterial antigens and cytokine production patterns in leprosy patients during multiple drug therapy

Immunology 1998 94 197–206 Changes 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 (TGF-b ), whatever the disease type or treatment status of the 1 donor These TGF-b levels 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 associated with neuropathy.1 At the lepromatous pole, patients show weak cell-mediated immunity (CMI ) or anergy to Mycobacterium leprae antigens.2 Suppressor mechanisms are thought to play an important role in the induction of anergy of these patients, making them susceptible to generalized M leprae infection.3–5 M leprae antigens have been shown to suppress T-cell proliferation in response to mitogens and/or antigens in both lepromatous and tuberculoid leprosy patients.6,7 In other studies, however, it was found that the ability of M leprae to induce suppressive activity was lower in lepromatous than in the intermediate clinical forms.8 Fine et al.9 reported that they could detect no evidence for M leprae antigen-mediated suppression of delayed-type hypersensitivity in any group of patients Descriptions of the cytokine profiles in the blood and tissue of leprosy patients of all types have been reported;10–16 they are not all in agreement We have previously reported that the responses we measured in leprosy patients with different disease forms appeared to be related less to their clinical classifications than to their INTRODUCTION The variety of immune responses in patients with leprosy leads to a broad spectrum of clinical manifestations, from the paucibacillary (PB) forms at the tuberculoid pole to the multibacillary (MB) forms at the lepromatous pole, with many gradations in between Patients with tuberculoid leprosy display strong cellular responses to the bacillus, but this is often Received October 1997; revised 28 January 1998; accepted 31 January 1998 Abbreviations: BB, borderline; BL, borderline lepromatous; BT, borderline tuberculoid; IFN-c, interferon gamma; IL-4, interleukin-4; IL-10, interleukin-10; MB, multibacillary; PB, paucibacillary; RT–PCR, reverse transcriptase–polymerase chain reaction; LL, polar lepromatous; TGF-b , transforming growth factor-b ; TT, tuberculoid 1 Correspondence: Dr V Tan Trao, Department of Bacteriology and Immunology, National Institute of Hygiene and Epidemiology (NIHE ), Yersin Street, Hanoi, Vietnam © 1998 Blackwell Science Ltd 197 198 V Tan Trao et al treatment status.17,18 The present study was carried out to follow the changes in the ability of lymphocytes from leprosy patients to respond to M leprae sonicates and a speciesspecific disaccharide, as well as the changes in their cytokine production from the beginning of multiple drug therapy (MDT ) through to its completion MATERIALS AND METHODS Patients The 66 leprosy patients were diagnosed and treated in the Leprosy Ward of the National Institute for Dermatology in Hanoi, Vietnam They were clinically classified according to the Ridley-Jopling criteria19 as four tuberculoid (TT ), 13 borderline tuberculoid (BT ), one indeterminate leprosy (I ), 17 borderline (BB), 11 borderline lepromatous (BL), four subpolar lepromatons (LLs) and 16 lepromatous (LL) There were 27 females and 39 males; their average age was 30 years (range 12–60 years) For treatment, the patients were assigned either to the PB group, who had a bacterial index of (BI= 0) (nine patients: four TT and five BT ) or the multibacillary group, who had BI≥1 (57 patients: eight BT, 17 BB, one I, 11 BL, 16 LL and four LLs) The first set of 26 patients (one TT, six BT, eight BB, three BL, three LLs and five LL) were monitored during treatment by measuring lymphoproliferative responses to M leprae antigens at 3-month intervals and the bacterial index (BI ) at 6-month intervals For analysis of the role of cytokines in the inhibition and enhancement of the phytohaemagglutinin (PHA)-induced proliferation of normal lymphocytes, blood samples were taken at one time point from a second set of 40 patients, of whom 15 were untreated (one TT, five BT, three BB, three BL, three LL) and 25 had been treated for 1–4 years (two TT, three BT, six BB, five BL, nine LL) The samples were processed as described below For each group, the treatment schemes were similar to those recommended by the WHO,20 comprising rifampicin and dapsone over months or until all clinical symptoms had disappeared for the PB group, and rifampicin, clofazimine and dapsone over 36 months for the MB group During MDT, 22/66 patients from the two sets developed reactions: there were 14 ENL (1 BL, Lls and LL) and RR (1 BT, BB and BL) All of the patients were treated with prednisone Proliferation assay Cell cultures Mononuclear cells were isolated by centrifugation over Ficoll-Hypaque (Pharmacia, Freiburg, Germany), were washed in phosphate-buffered saline (PBS) and resuspended in Iscove’s medium supplemented with 10% heatinactivated human AB serum The cells were cultured in round-bottom microtitre plates at 5–7·5×104 cells in 100 ml of medium/well Antigens and pokeweed mitogen (PWM ) were added on day 0, in 50 ml of culture medium, while PHA and concanavalin A (Con A) were added on day All cultures were set up in triplicate After incubation at 37° for days in a humidified 5% CO atmosphere, 0·15 mCi of methyl-[3H ]thymidine (specific activity 2·0 Ci/mmol; Amersham, UK ) was added to each well and the cultures were incubated for a further 18 hr The cells were then harvested on a Titertek multiple automatic sample harvester and the retained radioactivity was counted in a Beckman liquid scintillation counter ( Tri-carb 1900 CA Liquid Scintillation Analyzer; Beckman, Canberra, Australia) The background control values for cells alone ranged from 100 to 500 counts per minute (c.p.m.) (mean 304±191) throughout the whole series of experiments Activation, measured by [3H ]thymidine incorporation, was expressed as c.p.m.±standard deviation (SD) Antigens and mitogens The mitogens PHA (10 mg/ml in culture; Sigma type IV, St Louis, MO), Con A (10 mg/ml; Sigma) and PWM (25 mg/ml; Sigma) were used throughout the study to monitor the quality of the cells and the adequacy of the culture conditions The antigen preparations used were: sonicated whole M leprae bacilli, isolated from armadillo, and synthetic disaccharide coupled to bovine serum albumin (ND-BSA), each at three concentrations (10, and 0·1 mg/ml in culture) BSA alone served as a control for the specificity of response to ND-BSA These WHO-IMMLEP antigens were kindly provided to us by Dr P Klatser of the Royal Tropical Institute, Amsterdam and later by Dr R J W Rees In addition, in some experiments, Mycobacterium tuberculosis and bacillus Calmette–Gue´rin (BCG) sonicates were also used, at four concentrations (50, 10, and 0·1 mg/ml ) These were prepared and supplied by the Mycobacteria Laboratory of the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam All mitogens and antigens were stored at −20° until use Amplification of the lymphoproliferative response with cytokine-rich supernatants To measure the effect of adding exogenous cytokines, 75 ml of culture supernatant were removed from target cultures on day and 75 ml of supernatant from PHAtreated healthy cells was added The target (recipient) cultures were then harvested as usual on day Addition of such supernatants to control cultures without antigen was a standard control; these cultures showed increased an increase in c.p.m The control cultures producing cytokine-rich supernatants were of cells from healthy donors, cultured in medium containing 10 mg/ml PHA and harvested at 48 hr Control cells treated with PHA and used to detect soluble factors produced by cultures of patients’ cells were also prepared as described above Enhancing and inhibitory factor(s) production To detect production of enhancing or inhibitory factors by antigentreated patients’ cells, 70 ml of their supernatants were collected at 72 hr and added to control cultures incubated with PHA since day After further incubation for days and harvesting, the effect was expressed as follows: %= c.p.m PHA control cells+supernatants ×100 c.p.m PHA control cells−supernatants Cytokine measurements Enzyme-linked immunosorbent assay (ELISA) for cytokines Interleukin-4 (IL-4) and interferon-c (IFN-c) in sera and supernatants were measured with an ELISA (AMS kit, supplied by Cambridge Bioscience, Cambridge, UK ), following the methods recommended by the manufacturer Interleukin-10 (IL-10) was quantified by an ELISA developed in the Immunology Laboratory, Bacteriology Department, UCL Medical School, London, using monoclonal antibodies (mAb) supplied by Pharmingen (San Diego, CA) In brief, plates with 96 flat-bottom wells (Corning Easy Wash) were coated with 50 ml of purified anti-IL-10 capture mAb at © 1998 Blackwell Science Ltd, Immunology, 94, 197–206 199 CMI changes during MDT in leprosy mg/ml in coating buffer (pH 8·2) and incubated overnight at 4° The plates were dried and the wells were filled with 200 ml of PBS/3% BSA, for blocking, at 37° for hr After washing twice with PBS/Tween, standards, undiluted supernatants and sera diluted 154 were added in duplicate and the plates were further incubated for hr at 37° The plates were then washed four times and 100 ml of biotin-conjugated anti-IL-10 detecting mAb was added, diluted to 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) (Sigma, Poole, Dorset, UK ), diluted 151000 in PBS/Tween, was added and the plates were incubated for hr Following this incubation, the plates were washed thoroughly at least four times, 100 ml of substrate solution (p-nitrophenyl phosphate tablet diluted in distilled water, Sigma) was added and the plates were incubated at 37° for 60 The absorbance was measured at 405 nm The results for cytokine levels were calculated as pg/ml in serum and as pg/ml of 2×106 cultured cells Reverse transcriptase–polymerase chain reaction (RT–PCR) The expression of genes for beta-actin, IL-10, transforming growth factor-b (TGF-b ), interleukin-2 (IL-2) 1 and interleukin-12 (IL-12) were measured, using primers obtained from Clontech Laboratories, Palo Alto, CA Total RNA was isolated by the phenol chloroform method Prior to PCR, reverse transcription (RT ) was carried out first at 65° for 10 mins with ml of sample in diethyl-pyro-carbonate (DEPC ) treated water and ml of oligo-dT from a stock of 500 mg/ml, then at 37° for hr, then 95° for in 10 ml of RT mix, containing ml of 5×RT buffer (first-strand buffer), 5·5 ml of DEPC water, 0·5 ml of BSA (1 mg/ml stock, molecular grade acetylated BSA), 0·5 ml of RNAse inhibitor, 0·5 ml of dNTP (stock 10 m) and 0·5 ml MMLVRT (reverse transcriptase) PCR was performed in a Hybaid Omnigene thermocycler The reaction was carried out with ml of sample in a 50-ml final reaction volume with 10 pmol of each oligonucleotide in 10×PCR buffer The PCR conditions were: 30 cycles (95° for 30 seconds, 60° for 30 seconds and 72° for 45 seconds) followed by elongation at 72° for Samples of RT–PCR products were loaded onto a 1·8% agarose gel in TBE and separated by electrophoresis at 83 V for 50 RT–PCR products were visualized under ultraviolet light A DNA ladder (FX174/Hae III; Sigma) was used as DNA size markers The TGF-b bioassay A growth inhibition assay of the mink lung epithelial cell line (ATCC/CCL-64) was used to measure bioactivity of TGF-b 21 In brief, CCL-64 cells were incubated in 96-well plates at a density of 20×103 cells/well for hr in RPMI medium with 14% FCS Then, the TGF-b standard, in serial dilutions in RPMI (rhTGF-b1, R&D Systems, Abingdon, UK ), sera and supernatants from patients were added, including acid-activated supernatants The total volume of the assay was 100 ml The plates were incubated at 37° in a humidified CO incubator until they reached conflu2 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 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 40 Neutralization of TGF-b bioactivity was also performed © 1998 Blackwell Science Ltd, Immunology, 94, 197–206 to check for the specificity of the test The samples and rhTGF-b were incubated with three concentrations of the chicken polyclonal anti-TGF-b (R&D Systems) (10 mg, mg and 2·5 mg) for hr at room temperature, then the CCL-64 cells were added The significance of differences in results between groups or at different times was tested with the Mann–Whitney U-test and Two-tailed t-test RESULTS Changes in response of patients’ mononuclear cells to mycobacterial antigens Four different concentrations of antigens were used to stimulate patients’ cells in vitro; that giving the highest response was used for further comparisons According to their response patterns to these antigens, the patients could be sorted into three groups: the PB group (one TT and five BT ), the MB group without reactions during follow-up (MB – R) (one BT, four BB, two BL and two LL), and the MB group who developed reactions during treatment and follow-up (MB+R) (three BB, one BL, three LL and three LLs) No PB patients developed reversal reactions during this study Figure shows the changes in responses by patients’ cells to M leprae sonicates and ND-BSA antigen for all three groups: PB (Fig 1a), MB without reactions (Fig 1b) and MB with reactions (Fig 1c), with and without addition of cytokinerich supernatants The PB patients tended to have the highest responses to M leprae sonicate at the start of treatment, and they tended to increase by 18 months compared with the first test before treatment began (0·01