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Th1/Th2 immune responses are associated with active cutaneous leishmaniasisand clinical cure is associated with strong interferon- production
LÛcio Roberto Castellano a, Dalmo Correia Filho a, Laurent Argiro b, Helia Dessein b, AluÎzio Prata a,Alain Dessein b, Virmondes Rodrigues a,*a Laboratory of Immunology, Universidade Federal do Triángulo Mineiro, Minas Gerais, Brazilb Laboratory of Parasitology Mycology, Faculty of Medicine, U399 INSERM, Marseille, France
A R T I C L E I N F O
Article history:
Received 4 October 2008
Accepted 15 January 2009
Available online 20 January 2009
Keywords:
Cutaneous leishmaniasis
Cytokines
Antibodies
IFN-gamma
IL-10
A B S T R A C T
In leishmaniasis, Th1-related cytokines production seems to be crucial for hostcontrol of parasiteburden and
clinical cure. Visceral and diffuse cutaneous leishmaniasis are characterizedby negative skin test for parasite
antigens and failure to produce Th1 cytokines, whereas tegumentary leishmaniasis is characterized bypositive skin test andthe ability of peripheral blood mononuclearcells (PBMCs) to produce Th1cytokines.In
this study, specific antibody plasma levels and cytokine production in PBMC culture supernatants were
evaluated by enzyme-linked immunoabsorbent assay in patients withactive or cured cutaneous leishmaniallesions and in subjects without disease history living in the same endemic area. Higher tumor necrosis
factor–, interferon (IFN)– , interleukin (IL)–12, IL-4, and IL-10 levels were observed in patients with active
lesions, whereas cured subjects producedonly IFN- at elevated levels. Analysis of specific antibody isotypescorrelate with cellular immune response observed in vitro, as the production of IgG1 and IgG3 was higher in
patients with active lesions. Our results suggest the presence of a mixed Th1/Th2 response during active
disease and that clinical cure is associated with a sustained Th1 response characterized by elevated IFN-
levels and down-modulation of IL-4 and IL-10 production. 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights
reserved.
1. Introduction
Leishmaniasis is a complex of diseases caused by the intracellu-lar protozoan parasite Leishmania that affects more than 12 millionpersons worldwide. Clinical manifestation is broad, ranging fromsingle cutaneous lesions to lethal visceral infection. Developmentinto different clinical forms is associated with both the immuno-logic statusof thehostand the parasite species. Both Leishmania (L.)
amazonensis and L. (Viannia) braziliensis cause localized cutaneousleishmaniasis (LCL), although subsequent progression to diffusecutaneous leishmaniasis in more susceptible individuals is nor-mallyattributed to L. amazonensis. Mucosal leishmaniasis is usuallyassociated with L. (V.) braziliensis. Whereas diffuse cutaneous leish-maniasis is characterized by large numbers of parasites and a lackof cell-mediated immunity to Leishmania antigens, cutaneous andmucosal leishmaniasis are generally accompanied by strong cellu-lar responses and sparse numbers of parasites in the lesions [1]. Inexperimental models, it is widely accepted that susceptibility of BALB/c mice to L. major infection is associated with interleukin(IL)–4 and IL-10–producing Th2-type T cells, whereas resistance isrelated to early and persistent interferon (IFN)– production (Th1)[2]. However, this dichotomy is not so clear in human infection.
High IFN- production was found to be associated with spontane-ous healing [3]. Simultaneous production of IFN- , tumor necrosisfactor (TNF)–, and IL-10 by antigen-stimulated peripheral bloodmononucleaer cells (PBMCs) from patients with active lesions [4]and IL-2, IL-4, IL-5, IL-10, and IFN- mRNAs were demonstrated inbiopsy samples taken from active lesions [5–8]. IL-10 expressionwas also significantly higher in patients who responded poorly topentamidine treatment [5]. In a previous study, our group showeda major role of IL-10 in the development of skin lesions in human
beings infected with L. (V.) braziliensis [9].Analysis of the antibody response in LCL patients revealed ele-
vated anti-Leishmania IgG antibodies plasma levels, mainly IgG1and IgG3 isotypes [10–12]. Although theprotectiverole of antibod-iesin leishmaniasis is questionable, it is useful for diagnosis andforpredicting in vivo cellular immune responses.
In the present study, we analyzed cytokines involved in T–helpercell balance and specific antibody isotypes in patients with activecutaneous leishmaniasis, individuals with cured leishmaniasis, andindividuals without lesions living in the same endemic area.
2. Subjects and methods
2.1. Subjects
The study population included 20 patients with active infectioncharacterized by typical lesions and positive Montenegro skin test* Corresponding author.E-mail address: [email protected] (V. Rodrigues, Jr.).
Human Immunology 70 (2009) 383-390
Contents lists available at ScienceDirect
0198-8859/09/$32.00 - see front matter 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
results, and 40 subjects with previous cutaneous leishmaniasisidentified in the medical records and characterized by skin scarsand positive Montenegro test results, were studied. In addition, 34asymptomatic individuals living in the same area under similarexposure conditions without either past or present clinical diseasemanifestationwere also included. All subjects livedin the ruralareaof Buerarema, Bahia State, Brazil. This region is endemic for cuta-neous leishmaniasis caused by L. (V.) braziliensis and an epidemicoutbreak had occurred in that population between 1995 and 1999.Clinical and epidemiologic surveys were carried out to record theclinical manifestations of the disease and to identify patients withactive infection. Montenegro skin test was performed on the ven-tral face of the left forearm. Test positivity was determined basedon at least4-mm indurations aftera 48-hour challenge. After agree-ment wasobtained from thesubjects, 15ml of peripheral bloodwascollected. The study protocol was approved by the Ethics Commit-tee of the Universidade Federal do TriÃngulo Mineiro in Brazil.
2.2. Cell culture
The PBMCs were isolated by Ficoll-Paque centrifugation (Phar-macia, Uppsala, Sweden) at 400 g for 20 minutes at room temper-ature, washed three times in RPMI medium (Gibco, Grand Island,NY) and resuspended in Dulbecco’s modified Eagle’s medium(Gibco), supplemented with 50 mol/l 2-mercaptoethanol, 2mmol/l L-glutamine (Gibco), 40 g/ml gentamicin, and 5% fetal calf serum (Gibco). A total of 2 106 cells per well were cultured in24-well microplates (Falcon, San Jose, CA) in the presence of me-dium alone, 5 g/ml PHA (Sigma, St. Louis, MO) or 5 g/ml L. (V.)
braziliensis (Lb) antigen. Plates were incubated at 37C in a 5% CO2
atmosphere. Supernatants were collected after 24 and 96 hours,centrifuged, and stored at70C until analysis.
2.3. Cytokine titration
For TNF-, IFN- , IL-12p70, IL-4, and IL-10 titration, microplates(Nunc, Roskilde, Denmark) were sensitized overnight with 100 lof 1g/ml specific monoclonal antibody (Mabtech,Mariemont, OH,and Pharmingen, SanDiego, CA). The plates were then washedfourtimes with PBS containing 0.05% Tween-20 (Sigma), and nonspe-cific binding was prevented by incubation of the plates with 2%bovine serum albumin (BSA; Sigma) in PBS. Plates were incubatedovernight with 100l of 1:2 dilutions of culture supernatants in 2%BSA-PBS or with recombinant human cytokine (Pharmingen).Plates were washed again and incubated with 1 g/ml of appropri-ate biotinylated anti-cytokine monoclonal antibody (Mabtech andPharmingen) for 2 hours at 37C, followed by washing and incubationwith alkaline phosphatase-conjugated streptavidin for 2 hours at37C. Finally, plates were washedfour times andenzymatic activity
was developed by incubation with p-nitrophenyl phosphate(Sigma). Absorbance was read at 405 nm in a microplate reader(BioRad, Hercules,CA). Thesensitivityof theassay ranged from 5 to10 pg/ml.
2.4. Antibody detection
Specific anti-Leishmania IgG, IgG1, IgG3, IgG4, and IgE weremeasured by indirect enzyme-linked immunoabsorbent assay(ELISA) as follows: highly sensitive microplates (Nunc) were sensi-tized with 100 l of 2 g/ml crude antigen of L. (V.) braziliensis
strain Lb2904 at 4C overnight. Nonspecific binding was preventedby incubation with 2% BSA-PBS. Plates were incubated with 100 lof the subject’s plasma in 2% BSA-PBS diluted 1:50 and 1:20 forwhole IgG and for IgG isotypes, respectively. Plates were then
washed four times with PBS/Tween and incubated with 1 g/mlappropriate biotinylated anti-human antibody class detectionmonoclonal antibody (Pharmingen) or peroxidase-conjugated pro-tein A for 2 hours at 37C. For the Ig isotypes, the plates were
washed and incubated with alkaline phosphatase-conjugatedstreptavidin for 2 hours at 37C. Finally, plates were washed fourtimes and enzymatic activity was developed by incubation withortho-phenyldiamine or p-nitrophenyl phosphate (Sigma). Absor-bance was read at 495 or 405 nm in a microplate reader (BioRad).
2.5. Parasite and antigen preparation
Leishmania (V.) braziliensis strain Lb2904 was grown in Schnei-der’s medium (Sigma) supplemented with 20% inactivated fetalbovine serum (Sigma), 1% sodium pyruvate, 1% L-glutamine, 750mg/l calcium chlorate, and 40 g/ml gentamicin. Parasites werecultured in plastic flasks (Corning, Lowell, MA) at 26C for 96 hours.
Parasites in the stationary growth phase were collected andcentrifuged at 800 g for 30 minutes at 4C; parasites were thenwashed with RPMI medium (Gibco) by centrifugation. For antibodydetection, parasites were resuspended in Tris-HCl buffer, pH 7.2,containing 1% Nonidet P-40 (Sigma) and 0.1 mmol/l TLCK (Sigma).The mixture was centrifuged at 4000g for 30minutesat 4Candthesupernatant was filtered through a 0.22-m filter (Millipore, Mol-sheim, France). Protein concentration was determined by themethod of Bradford (Pierce, Rockford, IL) and adjusted to 2 mg/mland stored at70C.
For in vitro use, L. (V.) braziliensis in thestationary growth phasewas resuspended in PBS and submitted to six freeze–thaw cycles.Parasite integrity was determined by light microscopy. Lysed par-asites were centrifuged at 1200 g for 30 minutes at 5C, and proteinconcentration was determined by the method of Bradford andadjusted to 1 mg/ml. The antigen preparation was stored at70C.
2.6. Statistical analysis
Patients were grouped according to the presence or absence of either active or past LCL lesions. The nonparametric Kruskal-Wallistest was applied to compare cytokine and antibody levels betweenthe three groups, with a value of p 0.05 being considered signif-
icant. The post hoc Dunn test was used to establish the differencesindicated by the Kruskal-Wallis test, with the level of significancesetatQ 2.5. SigmaStatsoftware(Systat, SanJose, CA)was used forstatistical analysis.
3. Results
3.1. Specific anti-Leishmania isotypes antibody levels
Anti-Leishmania antibodies in sera from endemic area individu-als and non–endemic area healthy controls (volunteer donors of the Regional Blood Bank Uberaba) were evaluated. As shown inFigure 1A, endemic area subjects exhibited higher Leishmania-specific IgGlevels than non-endemicarea donors(p0.0001). Twoof 47 samples from non–endemic area volunteers strongly recog-
nized Lb antigens, probably because of cross-reactivity with otherinfections or previous contact with Leishmania.
Next, differences among patients with active or healed CL andasymptomatic endemic areasubjects wereevaluated. Patients withactive lesions and with healed cutaneous leishmaniasis presentedwith higher circulating levels of anti-Leishmania IgG antibodiesthan did control subjects, although the difference was nonsignifi-cant (Figure 1B). This result indicates a tendency of cutaneousleishmaniasis patients to present with specific antibodies circulat-ing at elevated levels and also suggests that endemic area controlsubjects were exposed to parasite antigens.
Analysis of IgG isotypes showed that IgG1 was significantlyhigher in patients with active or healed lesions compared withlevels in endemic area controls (p 0.0004; Q 3.54 and Q 3.22,
respectively). No significant difference was observed between pa-tients with activeand healed lesions (Figure 1C). Onthe other hand,IgG3 levels were significantly higher in patients with active lesionswhen compared with both healed leishmaniasis and control sub-
L.R. Castellano et al. / Human Immunology 70 (2009) 383-390384
jects (p 0.0004; Q 2.83 and Q 3.96, respectively). No signifi-cant difference was observed between patients with healed leish-maniasis and control subjects (Figure 1D). Specific IgG4 waspresent at low levels in all samples tested, with no significantdifference between groups (Figure 1E). Furthermore, specific anti-Leishmania IgE antibodies were detected at very low levels, with
control subjects presenting withhigher plasma levels thanpatientswith active lesions (p 0.0161, Q 2.876; Figure 1F).
When cured subjects were regrouped according to the timeafter lesionhealingof less than 5 years,from 5 to 10years, and morethan 10 years, it was revealed that total IgG and IgG1 plasma titersdecreased progressively in cured patients presenting with scarswithin 5 years after healing (Figure 2A, B), whereas IgG3 plasmatiters decreased in healed patients whose scars had more than 10yearsafter healing (Q 2.975; Figure2C). Anti-Leishmania IgG4andIgE isotypes did not show any relevance with respect to identifica-tion of infected, cured, or noninifected exposed subjects (Figures2D, 2E).
3.2. Cytokine patterns in patients and asymptomatic controls
The cellular immune response was evaluated by the measure-ment of TNF-, IFN- , IL-12, IL-4, and IL-10 cytokines in culturesupernatants collected after 24 or 96 hours of PHA- or Lb antigen-stimulated PBMC.
TNF- levels were analyzed in supernatants after 24 and 96
hours of PBMC culture. In 24-hour supernatants (Figure 3A), signif-
icantly higher TNF- levels were observed in patients with active
lesions compared with cured and control, regardless of stimulation
(PHA or Lb) or basal production (medium alone) (p 0.001). On the
other hand, in 96-hour supernatants (Figure 3B), a significant dif-
ference was only observed for cultures stimulated with Lb antigen(p 0.001).
IFN- levels were analyzed in 96-hour culture supernatants; a
difference between groups was observed only when cells were
stimulated with Lb antigen (p 0.001). PBMCs from patients with
active lesions and from cured subjects produced significantly higher
amounts of IFN- than cells from controls (Q 2.631 and Q 4.903,
respectively). No difference in IFN- levels was observed between
patients with active and cured lesions (Figure 3C).
IL-12 was analyzed in 24-hour supernatants, and was observed
to be significantly higher in nonstimulated PBMC from patients
with active lesions (p 0.015) compared with subjects with cured
leishmaniasis (Q 5.17) and control subjects (Q 5.06). In addi-
tion, after stimulation with Lb antigen, PBMCs from patients withactive lesions produced significantly higher levels of IL-12 (p
0.004) when compared with those from cured subjects (Q 2.51)
and control subjects (Q 3.09). No significant difference was ob-
Fig. 1. Specific anti-L. (V.) braziliensis antibody plasma levels in subjects living in a cutaneous leishmaniasis endemic area. Specific IgG (A) plasma levels were measured by
enzyme immunoassay in inhabitants of a cutaneous leishmaniasis endemic area (n 94) and in blood bank donors from a non-endemic area ( n 47). Each dot represents
individual result. Specific IgG (B), IgG1 (C), IgG3 (D), IgG4 (E), and IgE (F) plasma levels were measured by enzyme immunoassay in inhabitants of a cutaneous leishmaniasis
endemic area grouped according to the presence of active lesions ( n 20), presence of typical scars (n 34) or absence of a history of disease (n 40). Horizontal line
represents the median; bar, the 25th–75th percentile; and vertical line, the 10th–90th percentile. *Kruskal-Wallis test, p 0.05, post hoc Dunn test statistically significant.
**Mann-Whitney test, p 0.0001.
L.R. Castellano et al. / Human Immunology 70 (2009) 383-390 385
served between patients with curedleishmaniasis and control sub-jects (Figure 3D).
IL-4, a Th2 cytokine, was analyzed in 24-hour supernatants.Significantly higher IL-4 levels were observed in nonstimulatedcells from patients with active lesions (p 0.001) when comparedwith subjects with cured leishmaniasis (Q 7.16) and the controlgroup (Q 7.16). Furthermore, after stimulation with Lb antigen,cells from patients with active lesions produced significantlyhigher levelsof IL-4 (p0.001)comparedwith cured subjects (Q 4.84) and to the control group (Q 5.05). No significant difference
was observed after stimulation with PHA (Figure 3E).IL-10, an important immunoregulatory cytokine, was evaluated
in 24-h supernatants. Significant differences (p 0.0001) wereobserved for all three culture conditions (medium alone, PHA, and
Lb antigen). PBMCs from patients with active lesions producedhigherIL-10 levelsthan cells from cured (medium: Q 5.832; PHA:Q 4.687; Lb: Q 5.026) and endemic area control subjects(medium: Q 4.985; PHA: Q 3.747; Lb: Q 4.636), irrespectiveof the stimuli (Figure 3F).
Cytokine production in Lb-stimulated PBMCs also revealed thathealed patients with early scars (up to 5 years) presented higherlevels of TNF- than asymptomatic endemic area controls (Q 2.668) and that after 5 years of healing these levels tended todecrease (Figures 4A, 4B). On the other hand, IFN- levels were
constantly higher in all healed patients in comparison to the en-demic area asymptomatic subjects, independently of the time of lesion healing (5 years, Q 4.539; 5–10 years, Q 3.004; 10years,Q 2.649; Figure4C). IL-12 levelsdecreased in subjects with
Fig. 2. Specific anti-L. (V.) braziliensis antibody plasma levels in subjects living in a cutaneous leishmaniasis endemic area grouped according to time after lesion healing.
Specific IgG(A), IgG1(B), IgG3(C), IgG4(D), and IgE(E) plasmalevelswere measuredby enzyme immunoassay in inhabitantsof cutaneousleishmaniasisendemicarea grouped
according to the presence of active lesions (n 20), presence of typical scars (n 34) or absence of a history of disease ( n 40). Healed patients were grouped according to
time after lesion healing being less than 5 years (n 12), 5–10 years (n 10), and more than 10 years (n 12). Horizontal line represents the median; bar, the 25th–75th
percentile; and vertical line, the 10th–90th percentile. *Kruskal-Wallis test, p 0.05, post hoc Dunn test statistically significant.
L.R. Castellano et al. / Human Immunology 70 (2009) 383-390386
more than 10 years of healing when compared with endemic areacontrols (Q 2.556), patients with active lesions (Q 3.515) andalso healed subjects with early scars (Q 2.103) (Figure4D). More-over, patients with early healed lesions presented with a great
decrease in IL-4 andIL-10production compared with patients withactive lesions (Q 3.374 and Q 3.103, respectively), and thesecytokines levels decreased progressively after healing (Figure 4E,4F). IL-10 levels were even lower as detected in supernatants frompatients with more than 10 years of healing compared with pa-tients with other healing times (Q 2.050) (Fig. 4F).
4. Discussion
The present study analyzed cytokines involved in theactivationof an effective anti-Leishmania immune response such as TNF-,IFN- , and IL-12 and also immunoregulatory cytokines such as IL-4and IL-10, which seem to be involved in disease progression. Fur-thermore, the in vivo effects of cytokines observed in cultures wereconfirmed by their ability to modulate antibody class switches
detected in plasma samples.Experimental models of infection withdifferent Leishmania spe-
cies have been used to establish the importance of the Th1immuneresponse against intracellular pathogens, whereas the Th2 re-
sponse has been found to be associated with disease susceptibility[2]. More information about immunoregulatory mechanism in-volved in human leishmaniasis is necessary to allow developmentof rational strategies of intervention.
In this study, subjects from an endemic area presented higheranti-Leishmania IgG circulating plasma levels than healthy bloodbank donors from a nonendemic area. Although specific IgG levelsdidnot differamong endemic area patients andcontrols, this resultmay suggest a general exposure of the endemic area population toa natural infection with L. (V.) braziliensis. When IgG isotypes wereevaluated, IgG1 was able to differentiate endemic area asymptom-atic control subjects from patients with active or past CL history.Circulating long-lasting memory B cells present in cured patientsmight be regulated by thecytokine environment andthe persistentantigenic stimulation over the time to switch for IgG1.
Alternatively, IgG3 was useful in discriminating patients withactive lesions from those with cured disease or no previous historyof leishmaniasis. Previously, higher levels of both IgG1 and IgG3
were observed in patients with active lesions compared with curedsubjects [12]. Results found in this and other studies regarding thepredominance of an IgG1 and IgG3 response over an IgG4 and IgEreflect the tendency of a Th1 immune response during cutaneous
Fig. 3. Cytokine levelsproducedby PBMCs from subjects livingin a cutaneousleishmaniasis endemic area. Cells were cultured for24 or 96 hours in theabsence (medium) or
presence of stimuli (PHA or Leishmania braziliensis antigen). Cytokines TNF- 24-hour (A), TNF- 96-hour (B), IFN- (C), IL-12p70 (D), IL-4 (E), and IL-10 (F) levels were
measured by ELISA.Patients were grouped accordingto thepresence ofactivelesions(n 20),presenceof typicalleishmanial scars(n 34), orabsence ofa history ofdisease
(n 40). Horizontal line represents themedian; bar, the25th–75th percentile; andvertical line, the10th–90th percentile. Scale baron therightrefers to PHAlevels.Statistical
analysis is indicated in each graph. *Kruskal-Wallis test, p 0.05, post hoc Dunn test statistically significant.
L.R. Castellano et al. / Human Immunology 70 (2009) 383-390 387
immune response during L. (L.) major infection [2]. Our resultsshowing a high production of IL-12p70 by non-stimulated and Lbantigen-stimulated PBMC from patients with active lesions, sug-gest that IL-12 is an important factor for the induction and mainte-nance of IFN- during active LCL. Thus, in human cutaneous leish-maniasis, IL-12 production during the active phase promotes Th1cell differentiation, whereas after clinical cure Th1 memory cellspersist, despite downregulation of IL-12 production.
TNF- plays an essential role during LCL development. BothTNF- and IFN- are macrophage activators, especially inducingiNOS to produce nitric oxide in response to intracellular microor-ganisms [19,20]. In human beings, elevated expression of iNOS inactive lesion biopsy samples was correlated with low parasite bur-dens at these sites [21]. Furthermore, TNF- is a potent proinflam-matory mediator related to tissue damage events and inflamma-tory cell recruitment to local sites of lesions. This cytokine has beenshownto be present at elevatedlevels in patients with severeformsof cutaneous leishmaniasis [22] and in skin biopsy samples [6,8]. Inthe present study, high levels of TNF- were observed in patientswithactive lesions under stimulatory conditions. TNF-production
might be involved in the attempt of the host to control Leishmaniainfection, but its continued overproduction may contribute to tis-sue damage and ulcer formation [23]. The decrease in TNF- pro-duction after healing observed in this study supports this idea andreinforces the importance of circulating IFN- Th1 memory cellsin healed LCL patients.
Production of Th1 cytokines in patients with active lesions wasaccompanied by elevated amounts of IL-4 andIL-10. In experimen-tal models, the administration of anti-IL-4 monoclonal antibodypromoted cure of susceptible mouse strains infected with L. (L.)
major , with a concomitant increase in IFN- –producing cells inlymph nodes [24]. Although the increase on IFN- levels after IL-4blockade was not responsible for resistance itself, there was a shiftto Th1 immunity and the transfer of IL-4-neutralized CD4 T cells
to naÐve mice induced resistance to subsequent Leishmania infec-tion [25]. Moreover, patients with active lesions presented with amixed IFN- – and IL-4–producing CD4 T cells before therapy,whereas cured patients showed an abrogation of IL-4 production[26]. IL-4 inhibits Th1 cell proliferation and consequently dimin-ishes IFN- production, promoting the parasite’s escape from thehost immune system.
IL-10 plays an important role in the regulation of the immuneresponse, mainly by inhibiting IFN- synthesis and macrophageactivation. Experimental models of Leishmania infection haveshown that initial IL-10 production by susceptible mouse strainsinhibited IFN- production and contributed to L. (L.) major escapeand maintenance of infection [27]. Recently it was observed thatthe lack of IL-10 promoted parasite elimination and clinical cure of
animals infected with an extremely virulent L. (L.) major strain[15,28], although complete parasite elimination in the absence of IL-10 activity causes the loss of immunity to reinfection, thus sug-gesting that IL-10 is involved both in disease progression, whenproduced at high levels, and in thedevelopment of memory T cells,when produced at low levels [29]. In human beings, a positivecorrelation has been observed between high intralesional IL-10mRNA levels and therapeutic failure [5]. It has been demonstratedthat a strong IL-10 production was correlated with deficient lym-phocyte proliferative response and low IFN- levels in patientswith earlier CL lesions. This pattern was completely reversed afterchemotherapy or after the addition of IL-12or monoclonal antibod-ies to IL-10 to the cultures [30]. Recently it was demonstrated thatIL-10–producing CD4CD25 T cells accumulate at lesion sites
and may exert their immunoregulatory functions during infection[31]. Our previous data indicated that functional polymorphisms inpositions 819C/T and 592A/C of the IL10 promoter region areassociated with higher IL-10 production by CD4CD25Foxp3 T
cells after Lb stimulation andLCL skinlesion occurrence in endemicarea subjects [9]. Our results showed that cells from patients withactive lesions produced large amounts of IL-10 under all cultureconditions.
The decline of IL-4 and IL-10 production after lesion healingsuggests that these cytokines may be involved in parasite escapeand persistent antigen stimulation, favoring lesion development.Accumulating data obtained from experimental models indicatethat susceptibility to Leishmania is a complex event, involving boththe Th2 and Treg pathways [2].
We also observed a positive correlation between specific anti-Leishmania IgG1 and IgG3 plasma levels and Th1 cytokines (TNF-and IFN- ) produced by PBMC stimulated with Leishmania antigen,suggesting that the cytokine balance observed in vitro producedfunctional effects in vivo.
The present results indicate that a mixed pattern of cell-mediatedimmune response takes place during active disease, and that heal-ing is associated with a decline in the production of IL-4 and IL-10,cytokines able to block IFN- production and macrophage activa-tion, suggesting that the disease is associated with the presence of
regulatory cytokines rather than by the absence of IFN- itself. Wecan also argue for a probable dual role of TNF-, first acting onparasite control by its ability to promote macrophage activationand later promoting lesion by its tissue damage potential. Thepresent results suggest that IL-4 and IL-10 are determinant factorsin the susceptibility of human beings to L. (V.) braziliensis infection,with both cytokines being interesting as relevant targets for thera-peutic strategies.
Acknowledgments
We are indebted to the Buerarema Municipal Health Councilandto the regional branchof Programa de SaÛde da FamÎlia-PSF forlogistic assistance; to Carlos AraÛjo for technical assistance; to Dr.Angela Cruz for providing the parasite strain; and to Dr. Valdo JosÊ
Dias da Silva for helping with thestatistical analysis. This work wassupported by Conselho Nacional de Desenvolvimento CientÎfico eTecnolÔgico–CNPq, FundaÈÄo de Amparo Á Pesquisa de MinasGerais-FAPEMIG and CoordenaÈÄo de AperfeiÈoamento de Pessoalde NÎvel Superior (CAPES-DS fellowship to L.R.C.C.).
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