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Th2 immune responses are associated with active cutaneous leishmaniasis and clinical cure is associated with strong interferon-γ production
Human Immunology 70 (2009) 383-390
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Th1/Th2 immune responses are associated with active cutaneous leishmaniasis and 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 b
Laboratory of Immunology, Universidade Federal do Triángulo Mineiro, Minas Gerais, Brazil 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 host control of parasite burden and clinical cure. Visceral and diffuse cutaneous leishmaniasis are characterized by negative skin test for parasite antigens and failure to produce Th1 cytokines, whereas tegumentary leishmaniasis is characterized by positive skin test and the ability of peripheral blood mononuclear cells (PBMCs) to produce Th1 cytokines. In this study, specific antibody plasma levels and cytokine production in PBMC culture supernatants were evaluated by enzyme-linked immunoabsorbent assay in patients with active or cured cutaneous leishmanial lesions 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 produced only IFN-␥ at elevated levels. Analysis of specific antibody isotypes correlate 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. 䉷 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 intracellular protozoan parasite Leishmania that affects more than 12 million persons worldwide. Clinical manifestation is broad, ranging from single cutaneous lesions to lethal visceral infection. Development into different clinical forms is associated with both the immunologic status of the host and the parasite species. Both Leishmania (L.) amazonensis and L. (Viannia) braziliensis cause localized cutaneous leishmaniasis (LCL), although subsequent progression to diffuse cutaneous leishmaniasis in more susceptible individuals is normally attributed to L. amazonensis. Mucosal leishmaniasis is usually associated with L. (V.) braziliensis. Whereas diffuse cutaneous leishmaniasis is characterized by large numbers of parasites and a lack of cell-mediated immunity to Leishmania antigens, cutaneous and mucosal leishmaniasis are generally accompanied by strong cellular responses and sparse numbers of parasites in the lesions [1]. In experimental 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 is related 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 spontaneous healing [3]. Simultaneous production of IFN-␥, tumor necrosis factor (TNF)–␣, and IL-10 by antigen-stimulated peripheral blood mononucleaer cells (PBMCs) from patients with active lesions [4] and IL-2, IL-4, IL-5, IL-10, and IFN-␥ mRNAs were demonstrated in biopsy samples taken from active lesions [5– 8]. IL-10 expression was also significantly higher in patients who responded poorly to pentamidine treatment [5]. In a previous study, our group showed a 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 elevated anti-Leishmania IgG antibodies plasma levels, mainly IgG1 and IgG3 isotypes [10 –12]. Although the protective role of antibodies in leishmaniasis is questionable, it is useful for diagnosis and for predicting in vivo cellular immune responses. In the present study, we analyzed cytokines involved in T– helper cell balance and specific antibody isotypes in patients with active cutaneous leishmaniasis, individuals with cured leishmaniasis, and individuals without lesions living in the same endemic area. 2. Subjects and methods 2.1. Subjects
* Corresponding author. E-mail address: [email protected] (V. Rodrigues, Jr.).
The study population included 20 patients with active infection characterized by typical lesions and positive Montenegro skin test
0198-8859/09/$32.00 - see front matter 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2009.01.007
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results, and 40 subjects with previous cutaneous leishmaniasis identified in the medical records and characterized by skin scars and positive Montenegro test results, were studied. In addition, 34 asymptomatic individuals living in the same area under similar exposure conditions without either past or present clinical disease manifestation were also included. All subjects lived in the rural area of Buerarema, Bahia State, Brazil. This region is endemic for cutaneous leishmaniasis caused by L. (V.) braziliensis and an epidemic outbreak had occurred in that population between 1995 and 1999. Clinical and epidemiologic surveys were carried out to record the clinical manifestations of the disease and to identify patients with active infection. Montenegro skin test was performed on the ventral face of the left forearm. Test positivity was determined based on at least 4-mm indurations after a 48-hour challenge. After agreement was obtained from the subjects, 15 ml of peripheral blood was collected. The study protocol was approved by the Ethics Committee of the Universidade Federal do TriÃngulo Mineiro in Brazil. 2.2. Cell culture The PBMCs were isolated by Ficoll-Paque centrifugation (Pharmacia, Uppsala, Sweden) at 400 g for 20 minutes at room temperature, 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, 2 mmol/l L-glutamine (Gibco), 40 g/ml gentamicin, and 5% fetal calf serum (Gibco). A total of 2 ⫻ 106 cells per well were cultured in 24-well microplates (Falcon, San Jose, CA) in the presence of medium alone, 5 g/ml PHA (Sigma, St. Louis, MO) or 5 g/ml L. (V.) braziliensis (Lb) antigen. Plates were incubated at 37⬚C in a 5% CO2 atmosphere. Supernatants were collected after 24 and 96 hours, centrifuged, and stored at ⫺70⬚C 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 l of 1 g/ml specific monoclonal antibody (Mabtech, Mariemont, OH, and Pharmingen, San Diego, CA). The plates were then washed four times with PBS containing 0.05% Tween-20 (Sigma), and nonspecific binding was prevented by incubation of the plates with 2% bovine serum albumin (BSA; Sigma) in PBS. Plates were incubated overnight with 100 l 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 appropriate biotinylated anti-cytokine monoclonal antibody (Mabtech and Pharmingen) for 2 hours at 37⬚C, followed by washing and incubation with alkaline phosphatase-conjugated streptavidin for 2 hours at 37⬚C. Finally, plates were washed four times and enzymatic activity was developed by incubation with p-nitrophenyl phosphate (Sigma). Absorbance was read at 405 nm in a microplate reader (BioRad, Hercules, CA). The sensitivity of the assay ranged from 5 to 10 pg/ml. 2.4. Antibody detection Specific anti-Leishmania IgG, IgG1, IgG3, IgG4, and IgE were measured by indirect enzyme-linked immunoabsorbent assay (ELISA) as follows: highly sensitive microplates (Nunc) were sensitized with 100 l of 2 g/ml crude antigen of L. (V.) braziliensis strain Lb2904 at 4⬚C overnight. Nonspecific binding was prevented by incubation with 2% BSA-PBS. Plates were incubated with 100 l of the subject’s plasma in 2% BSA-PBS diluted 1:50 and 1:20 for whole IgG and for IgG isotypes, respectively. Plates were then washed four times with PBS/Tween and incubated with 1 g/ml appropriate biotinylated anti-human antibody class detection monoclonal antibody (Pharmingen) or peroxidase-conjugated protein A for 2 hours at 37⬚C. For the Ig isotypes, the plates were
washed and incubated with alkaline phosphatase-conjugated streptavidin for 2 hours at 37⬚C. Finally, plates were washed four times and enzymatic activity was developed by incubation with ortho-phenyldiamine or p-nitrophenyl phosphate (Sigma). Absorbance 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 Schneider’s medium (Sigma) supplemented with 20% inactivated fetal bovine serum (Sigma), 1% sodium pyruvate, 1% L-glutamine, 750 mg/l calcium chlorate, and 40 g/ml gentamicin. Parasites were cultured in plastic flasks (Corning, Lowell, MA) at 26⬚C for 96 hours. Parasites in the stationary growth phase were collected and centrifuged at 800 g for 30 minutes at 4⬚C; parasites were then washed with RPMI medium (Gibco) by centrifugation. For antibody detection, 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 4000 g for 30 minutes at 4⬚C and the supernatant was filtered through a 0.22-m filter (Millipore, Molsheim, France). Protein concentration was determined by the method of Bradford (Pierce, Rockford, IL) and adjusted to 2 mg/ml and stored at ⫺70⬚C. For in vitro use, L. (V.) braziliensis in the stationary growth phase was resuspended in PBS and submitted to six freeze–thaw cycles. Parasite integrity was determined by light microscopy. Lysed parasites were centrifuged at 1200 g for 30 minutes at 5⬚C, and protein concentration was determined by the method of Bradford and adjusted to 1 mg/ml. The antigen preparation was stored at ⫺70⬚C. 2.6. Statistical analysis Patients were grouped according to the presence or absence of either active or past LCL lesions. The nonparametric Kruskal-Wallis test was applied to compare cytokine and antibody levels between the three groups, with a value of p ⬍ 0.05 being considered significant. The post hoc Dunn test was used to establish the differences indicated by the Kruskal-Wallis test, with the level of significance set at Q ⬎ 2.5. SigmaStat software (Systat, San Jose, CA) was used for statistical analysis. 3. Results 3.1. Specific anti-Leishmania isotypes antibody levels Anti-Leishmania antibodies in sera from endemic area individuals and non– endemic area healthy controls (volunteer donors of the Regional Blood Bank Uberaba) were evaluated. As shown in Figure 1A, endemic area subjects exhibited higher Leishmaniaspecific IgG levels than non-endemic area donors (p ⬍ 0.0001). Two of 47 samples from non– endemic area volunteers strongly recognized Lb antigens, probably because of cross-reactivity with other infections or previous contact with Leishmania. Next, differences among patients with active or healed CL and asymptomatic endemic area subjects were evaluated. Patients with active lesions and with healed cutaneous leishmaniasis presented with higher circulating levels of anti-Leishmania IgG antibodies than did control subjects, although the difference was nonsignificant (Figure 1B). This result indicates a tendency of cutaneous leishmaniasis patients to present with specific antibodies circulating at elevated levels and also suggests that endemic area control subjects were exposed to parasite antigens. Analysis of IgG isotypes showed that IgG1 was significantly higher in patients with active or healed lesions compared with levels in endemic area controls (p ⫽ 0.0004; Q ⫽ 3.54 and Q ⫽ 3.22, respectively). No significant difference was observed between patients with active and healed lesions (Figure 1C). On the other hand, IgG3 levels were significantly higher in patients with active lesions when compared with both healed leishmaniasis and control sub-
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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.
jects (p ⫽ 0.0004; Q ⫽ 2.83 and Q ⫽ 3.96, respectively). No significant difference was observed between patients with healed leishmaniasis and control subjects (Figure 1D). Specific IgG4 was present at low levels in all samples tested, with no significant difference between groups (Figure 1E). Furthermore, specific antiLeishmania IgE antibodies were detected at very low levels, with control subjects presenting with higher plasma levels than patients with active lesions (p ⫽ 0.0161, Q ⫽ 2.876; Figure 1F). When cured subjects were regrouped according to the time after lesion healing of less than 5 years, from 5 to 10 years, and more than 10 years, it was revealed that total IgG and IgG1 plasma titers decreased progressively in cured patients presenting with scars within 5 years after healing (Figure 2A, B), whereas IgG3 plasma titers decreased in healed patients whose scars had more than 10 years after healing (Q ⫽ 2.975; Figure 2C). Anti-Leishmania IgG4 and IgE isotypes did not show any relevance with respect to identification of infected, cured, or noninifected exposed subjects (Figures 2D, 2E). 3.2. Cytokine patterns in patients and asymptomatic controls The cellular immune response was evaluated by the measurement of TNF-␣, IFN-␥, IL-12, IL-4, and IL-10 cytokines in culture supernatants collected after 24 or 96 hours of PHA- or Lb antigenstimulated PBMC.
TNF-␣ levels were analyzed in supernatants after 24 and 96 hours of PBMC culture. In 24-hour supernatants (Figure 3A), significantly 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 difference 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 addition, after stimulation with Lb antigen, PBMCs from patients with active 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-
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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) plasma levels were measured by enzyme immunoassay in inhabitants of 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). 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.
served between patients with cured leishmaniasis and control subjects (Figure 3D). IL-4, a Th2 cytokine, was analyzed in 24-hour supernatants. Significantly higher IL-4 levels were observed in nonstimulated cells from patients with active lesions (p ⬍ 0.001) when compared with subjects with cured leishmaniasis (Q ⫽ 7.16) and the control group (Q ⫽ 7.16). Furthermore, after stimulation with Lb antigen, cells from patients with active lesions produced significantly higher levels of IL-4 (p ⬍ 0.001) compared with 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) were observed for all three culture conditions (medium alone, PHA, and
Lb antigen). PBMCs from patients with active lesions produced higher IL-10 levels than 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), irrespective of the stimuli (Figure 3F). Cytokine production in Lb-stimulated PBMCs also revealed that healed patients with early scars (up to 5 years) presented higher levels of TNF-␣ than asymptomatic endemic area controls (Q ⫽ 2.668) and that after 5 years of healing these levels tended to decrease (Figures 4A, 4B). On the other hand, IFN-␥ levels were constantly higher in all healed patients in comparison to the endemic area asymptomatic subjects, independently of the time of lesion healing (⬍5 years, Q ⫽ 4.539; 5–10 years, Q ⫽ 3.004; ⬎10 years, Q ⫽ 2.649; Figure 4C). IL-12 levels decreased in subjects with
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Fig. 3. Cytokine levels produced by PBMCs from subjects living in a cutaneous leishmaniasis endemic area. Cells were cultured for 24 or 96 hours in the absence (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 according to the presence of active lesions (n ⫽ 20), presence of typical leishmanial 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. Scale bar on the right refers to PHA levels. Statistical analysis is indicated in each graph. *Kruskal-Wallis test, p ⬍ 0.05, post hoc Dunn test statistically significant.
more than 10 years of healing when compared with endemic area controls (Q ⫽ 2.556), patients with active lesions (Q ⫽ 3.515) and also healed subjects with early scars (Q ⫽ 2.103) (Figure 4D). Moreover, patients with early healed lesions presented with a great decrease in IL-4 and IL-10 production compared with patients with active lesions (Q ⫽ 3.374 and Q ⫽ 3.103, respectively), and these cytokines levels decreased progressively after healing (Figure 4E, 4F). IL-10 levels were even lower as detected in supernatants from patients with more than 10 years of healing compared with patients with other healing times (Q ⫽ 2.050) (Fig. 4F). 4. Discussion The present study analyzed cytokines involved in the activation of an effective anti-Leishmania immune response such as TNF-␣, IFN-␥, and IL-12 and also immunoregulatory cytokines such as IL-4 and IL-10, which seem to be involved in disease progression. Furthermore, the in vivo effects of cytokines observed in cultures were confirmed by their ability to modulate antibody class switches detected in plasma samples. Experimental models of infection with different Leishmania species have been used to establish the importance of the Th1 immune response against intracellular pathogens, whereas the Th2 re-
sponse has been found to be associated with disease susceptibility [2]. More information about immunoregulatory mechanism involved in human leishmaniasis is necessary to allow development of rational strategies of intervention. In this study, subjects from an endemic area presented higher anti-Leishmania IgG circulating plasma levels than healthy blood bank donors from a nonendemic area. Although specific IgG levels did not differ among endemic area patients and controls, this result may suggest a general exposure of the endemic area population to a natural infection with L. (V.) braziliensis. When IgG isotypes were evaluated, IgG1 was able to differentiate endemic area asymptomatic control subjects from patients with active or past CL history. Circulating long-lasting memory B cells present in cured patients might be regulated by the cytokine environment and the persistent antigenic stimulation over the time to switch for IgG1. Alternatively, IgG3 was useful in discriminating patients with active lesions from those with cured disease or no previous history of leishmaniasis. Previously, higher levels of both IgG1 and IgG3 were observed in patients with active lesions compared with cured subjects [12]. Results found in this and other studies regarding the predominance of an IgG1 and IgG3 response over an IgG4 and IgE reflect the tendency of a Th1 immune response during cutaneous
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Fig. 4. Cytokine levels produced by PBMC from subjects living in a cutaneous leishmaniasis endemic area grouped according to time after lesion healing. Cells were cultured for 24 or 96 hours in the absence (medium) or presence of stimuli (PHA or Leishmania braziliensis antigen). Cytokine 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 according to the presence of active lesions (n ⫽ 20), presence of typical leishmanial 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, 10th–90th percentile. Statistical analysis is indicated in each graph. *Kruskal-Wallis test, p ⬍ 0.05, post hoc Dunn test statistically significant.
leishmaniasis course [3,10,12]. Detection of specific anti-Leishmania IgG1 and/or IgG3 isotypes might help in the development of useful diagnostic tools to be routinely applied for the screening of cutaneous leishmaniasis. The involvement of effectors and regulatory cytokines produced by PBMC was also analyzed. Significant production of Th1-, Th2-, and Treg-type cytokines by cells from patients with active lesions, mainly TNF-␣, IFN-␥, IL-12, IL-4, and IL-10, was observed. This mixed pattern of immune response observed in patients with active lesions evolved to a Th1 response characterized by the production of IFN-␥ in cured subjects. Other studies have also reported a mixed Th1/Th2 pattern of cytokine production in cutaneous leishmaniasis [4,9]. It seems that a predominant Th1 response is necessary for parasite control and lesion healing, whereas the concomitant presence of IL-4 and IL-10, and probably other regulatory cytokines such as TGF-, may contribute to parasite escape, sustenance of inflammatory stimulation, and lesion persistence [9,13,14].
Several experimental studies using knockout mice and blockage of cytokines support this hypothesis [15–17]. IFN-␥, a major Th1 cytokine, plays an essential role in the control of Leishmania infection by mediating macrophage killing of intracellular parasites [17]. Studies conducted in human subjects have detected elevated levels of IFN-␥ or its mRNA in biopsy samples of active LCL lesions [5,6,8], whereas spontaneous lesion healing has been associated with high levels of IFN-␥ production [3]. Moreover, low IFN-␥ production was observed to be associated with chronic lesions and therapeutic failure [18]. Our results indicate a high production of IFN-␥ in patients with active lesions, and its persistence at elevated levels after lesion healing also suggest the presence of circulating Leishmania-specific Th1 memory T cells. One of the main factors responsible for the development of a Th1 response and IFN-␥ production is IL-12. Experimental models have shown that IL-12 is necessary for the maintenance of the Th1
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immune response during L. (L.) major infection [2]. Our results showing a high production of IL-12p70 by non-stimulated and Lb antigen-stimulated PBMC from patients with active lesions, suggest that IL-12 is an important factor for the induction and maintenance of IFN-␥ during active LCL. Thus, in human cutaneous leishmaniasis, IL-12 production during the active phase promotes Th1 cell differentiation, whereas after clinical cure Th1 memory cells persist, despite downregulation of IL-12 production. TNF-␣ plays an essential role during LCL development. Both TNF-␣ and IFN-␥ are macrophage activators, especially inducing iNOS to produce nitric oxide in response to intracellular microorganisms [19,20]. In human beings, elevated expression of iNOS in active lesion biopsy samples was correlated with low parasite burdens at these sites [21]. Furthermore, TNF-␣ is a potent proinflammatory mediator related to tissue damage events and inflammatory cell recruitment to local sites of lesions. This cytokine has been shown to be present at elevated levels in patients with severe forms of cutaneous leishmaniasis [22] and in skin biopsy samples [6,8]. In the present study, high levels of TNF-␣ were observed in patients with active lesions under stimulatory conditions. TNF-␣ production might be involved in the attempt of the host to control Leishmania infection, but its continued overproduction may contribute to tissue damage and ulcer formation [23]. The decrease in TNF-␣ production after healing observed in this study supports this idea and reinforces the importance of circulating IFN-␥ ⫹ Th1 memory cells in healed LCL patients. Production of Th1 cytokines in patients with active lesions was accompanied by elevated amounts of IL-4 and IL-10. In experimental models, the administration of anti-IL-4 monoclonal antibody promoted cure of susceptible mouse strains infected with L. (L.) major, with a concomitant increase in IFN-␥–producing cells in lymph nodes [24]. Although the increase on IFN-␥ levels after IL-4 blockade was not responsible for resistance itself, there was a shift to Th1 immunity and the transfer of IL-4-neutralized CD4⫹ T cells to naÐve mice induced resistance to subsequent Leishmania infection [25]. Moreover, patients with active lesions presented with a mixed 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 diminishes IFN-␥ production, promoting the parasite’s escape from the host immune system. IL-10 plays an important role in the regulation of the immune response, mainly by inhibiting IFN-␥ synthesis and macrophage activation. Experimental models of Leishmania infection have shown that initial IL-10 production by susceptible mouse strains inhibited IFN-␥ production and contributed to L. (L.) major escape and maintenance of infection [27]. Recently it was observed that the 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 suggesting that IL-10 is involved both in disease progression, when produced at high levels, and in the development of memory T cells, when produced at low levels [29]. In human beings, a positive correlation has been observed between high intralesional IL-10 mRNA levels and therapeutic failure [5]. It has been demonstrated that a strong IL-10 production was correlated with deficient lymphocyte proliferative response and low IFN-␥ levels in patients with earlier CL lesions. This pattern was completely reversed after chemotherapy or after the addition of IL-12 or monoclonal antibodies to IL-10 to the cultures [30]. Recently it was demonstrated that IL-10 –producing CD4⫹CD25⫹ T cells accumulate at lesion sites and may exert their immunoregulatory functions during infection [31]. Our previous data indicated that functional polymorphisms in positions ⫺819C/T and ⫺592A/C of the IL10 promoter region are associated with higher IL-10 production by CD4⫹CD25⫹Foxp3⫹ T
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cells after Lb stimulation and LCL skin lesion occurrence in endemic area subjects [9]. Our results showed that cells from patients with active lesions produced large amounts of IL-10 under all culture conditions. The decline of IL-4 and IL-10 production after lesion healing suggests that these cytokines may be involved in parasite escape and persistent antigen stimulation, favoring lesion development. Accumulating data obtained from experimental models indicate that susceptibility to Leishmania is a complex event, involving both the Th2 and Treg pathways [2]. We also observed a positive correlation between specific antiLeishmania 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 produced functional effects in vivo. The present results indicate that a mixed pattern of cell-mediated immune response takes place during active disease, and that healing is associated with a decline in the production of IL-4 and IL-10, cytokines able to block IFN-␥ production and macrophage activation, suggesting that the disease is associated with the presence of regulatory cytokines rather than by the absence of IFN-␥ itself. We can also argue for a probable dual role of TNF-␣, first acting on parasite control by its ability to promote macrophage activation and later promoting lesion by its tissue damage potential. The present results suggest that IL-4 and IL-10 are determinant factors in the susceptibility of human beings to L. (V.) braziliensis infection, with both cytokines being interesting as relevant targets for therapeutic strategies. Acknowledgments We are indebted to the Buerarema Municipal Health Council and to the regional branch of Programa de SaÛde da FamÎlia-PSF for logistic 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 the statistical analysis. This work was supported by Conselho Nacional de Desenvolvimento CientÎfico e TecnolÔgico–CNPq, FundaÈÄo de Amparo Á Pesquisa de Minas Gerais-FAPEMIG and CoordenaÈÄo de AperfeiÈoamento de Pessoal de NÎvel Superior (CAPES-DS fellowship to L.R.C.C.). References [1] Convit J, Ulrich M, Fernandez CT, Tapia FJ, Caceres-Dittmar G, Castes M, et al. The clinical and immunological spectrum of American cutaneous leishmaniasis. Trans R Soc Trop Med Hyg 1993;87:444 – 8. [2] Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol 2002;2:845–58. [3] Carvalho EM, Correia Filho D, Bacellar O, Almeida RP, Lessa H, Rocha H. Characterization of the immune response in subjects with self-healing cutaneous leishmaniasis. Am J Trop Med Hyg 1995;53:273–7. [4] Trujillo CM, Robledo SM, Franco JL, Velez ID, Erb KJ, Patino PJ. Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Th1 immune response in comparison to patients with localized cutaneous leishmaniasis. Parasite Immunol 2002;24:455– 62. [5] Bourreau E, Prevot G, Gardon J, Pradinaud R, Launois P. High intralesional interleukin-10 messenger RNA expression in localized cutaneous leishmaniasis is associated with unresponsiveness to treatment. J Infect Dis 2001;184: 1628 –30. [6] Caceres-Dittmar G, Tapia FJ, Sanchez MA, Yamamura M, Uyemura K, Modlin RL, et al. Determination of the cytokine profile in American cutaneous leishmaniasis using the polymerase chain reaction. Clin Exp Immunol 1993;91:500 –5. [7] Louzir H, Melby PC, Ben Salah A, Marrakchi H, Aoun K, Ben Ismail R, et al. Immunologic determinants of disease evolution in localized cutaneous leishmaniasis due to Leishmania major. J Infect Dis 1998;177:1687–95. [8] Pirmez C, Yamamura M, Uyemura K, Paes-Oliveira M, Conceicao-Silva F, Modlin RL. Cytokine patterns in the pathogenesis of human leishmaniasis. J Clin Invest 1993;91:1390 –5. [9] Salhi A, Rodrigues V, Jr., Santoro F, Dessein H, Romano A, Castellano LR, et al. Immunological and genetic evidence for a crucial role of IL-10 in cutaneous lesions in humans infected with Leishmania braziliensis. J Immunol 2008;180: 6139 – 48. [10] Junqueira Pedras M, Orsini M, Castro M, Passos VM, Rabello A. Antibody subclass profile against Leishmania braziliensis and Leishmania amazonensis in the diagnosis and follow-up of mucosal leishmaniasis. Diagn Microbiol Infect Dis 2003;47:477– 85.
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[11] Romero GA, de la Gloria Orge Orge M, de Farias Guerra MV, Paes MG, de Oliveira Macedo V, de Carvalho EM. Antibody response in patients with cutaneous leishmaniasis infected by Leishmania (Viannia) braziliensis or Leishmania (Viannia) guyanensis in Brazil. Acta Trop 2005;93:49 –56. [12] Rodriguez V, Centeno M, Ulrich M. The IgG isotypes of specific antibodies in patients with American cutaneous leishmaniasis; relationship to the cellmediated immune response. Parasite Immunol 1996;18:341–5. [13] Melby PC, Andrade-Narvaez FJ, Darnell BJ, Valencia-Pacheco G, Tryon VV, Palomo-Cetina A. Increased expression of proinflammatory cytokines in chronic lesions of human cutaneous leishmaniasis. Infect Immun 1994; 62:837– 42. [14] Rodrigues V, Jr., Santana da Silva J, Campos-Neto A. Transforming growth factor beta and immunosuppression in experimental visceral leishmaniasis. Infect Immun 1998;66:1233– 6. [15] Anderson CF, Mendez S, Sacks DL. Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice. J Immunol 2005;174:2934 – 41. [16] Chatelain R, Mauze S, Varkila K, Coffman RL. Leishmania major infection in interleukin-4 and interferon-gamma depleted mice. Parasite Immunol 1999; 21:423–31. [17] de Souza-Neto SM, Carneiro CM, Vieira LQ, Afonso LC. Leishmania braziliensis: Partial control of experimental infection by interleukin-12 p40 deficient mice. Mem Inst Oswaldo Cruz 2004;99:289 –94. [18] Ajdary S, Alimohammadian MH, Eslami MB, Kemp K, Kharazmi A. Comparison of the immune profile of nonhealing cutaneous leishmaniasis patients with those with active lesions and those who have recovered from infection. Infect Immun 2000;68:1760 – 4. [19] Stenger S, Thuring H, Rollinghoff M, Bogdan C. Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major. J Exp Med 1994;180:783–93. [20] Diaz NL, Fernandez M, Figueira E, Ramirez R, Monsalve IB, Tapia FJ. Nitric oxide and cellular immunity in experimental cutaneous leishmaniasis. Clin Exp Dermatol 2003;28:288 –93.
[21] Qadoumi M, Becker I, Donhauser N, Rollinghoff M, Bogdan C. Expression of inducible nitric oxide synthase in skin lesions of patients with American cutaneous leishmaniasis. Infect Immun 2002;70:4638 – 42. [22] Cabrera M, Shaw MA, Sharples C, Williams H, Castes M, Convit J, et al. Polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis. J Exp Med 1995;182:1259 – 64. [23] Da-Cruz AM, de Oliveira MP, De Luca PM, Mendonca SC, Coutinho SG. Tumor necrosis factor-alpha in human American tegumentary leishmaniasis. Mem Inst Oswaldo Cruz 1996;91:225–9. [24] Heinzel FP, Sadick MD, Mutha SS, Locksley RM. Production of interferon gamma, interleukin 2, interleukin 4, and interleukin 10 by CD4⫹ lymphocytes in vivo during healing and progressive murine leishmaniasis. Proc Natl Acad Sci U S A 1991;88:7011–5. [25] Sadick MD, Heinzel FP, Holaday BJ, Pu RT, Dawkins RS, Locksley RM. Cure of murine leishmaniasis with anti-interleukin 4 monoclonal antibody. Evidence for a T cell-dependent, interferon gamma-independent mechanism. J Exp Med 1990;171:115–27. [26] Coutinho SG, Da-Cruz AM, Bertho AL, Santiago MA, De-Luca P. Immunologic patterns associated with cure in human American cutaneous leishmaniasis. Braz J Med Biol Res 1998;31:139 – 42. [27] Chatelain R, Mauze S, Coffman RL. Experimental Leishmania major infection in mice: Role of IL-10. Parasite Immunol 1999;21:211– 8. [28] Padigel UM, Alexander J, Farrell JP. The role of interleukin-10 in susceptibility of BALB/c mice to infection with Leishmania mexicana and Leishmania amazonensis. J Immunol 2003;171:3705–10. [29] Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL. CD4⫹CD25⫹ regulatory T cells control Leishmania major persistence and immunity. Nature 2002; 420:502–7. [30] Rocha PN, Almeida RP, Bacellar O, de Jesus AR, Filho DC, Filho AC, et al. Down-regulation of Th1 type of response in early human American cutaneous leishmaniasis. J Infect Dis 1999;180:1731– 4. [31] Campanelli AP, Roselino AM, Cavassani KA, Pereira MS, Mortara RA, Brodskyn CI, et al. CD4⫹CD25⫹ T cells in skin lesions of patients with cutaneous leishmaniasis exhibit phenotypic and functional characteristics of natural regulatory T cells. J Infect Dis 2006;193:1313–22.
Contents lists available at ScienceDirect
Th1/Th2 immune responses are associated with active cutaneous leishmaniasis and 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 b
Laboratory of Immunology, Universidade Federal do Triángulo Mineiro, Minas Gerais, Brazil 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 host control of parasite burden and clinical cure. Visceral and diffuse cutaneous leishmaniasis are characterized by negative skin test for parasite antigens and failure to produce Th1 cytokines, whereas tegumentary leishmaniasis is characterized by positive skin test and the ability of peripheral blood mononuclear cells (PBMCs) to produce Th1 cytokines. In this study, specific antibody plasma levels and cytokine production in PBMC culture supernatants were evaluated by enzyme-linked immunoabsorbent assay in patients with active or cured cutaneous leishmanial lesions 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 produced only IFN-␥ at elevated levels. Analysis of specific antibody isotypes correlate 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. 䉷 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 intracellular protozoan parasite Leishmania that affects more than 12 million persons worldwide. Clinical manifestation is broad, ranging from single cutaneous lesions to lethal visceral infection. Development into different clinical forms is associated with both the immunologic status of the host and the parasite species. Both Leishmania (L.) amazonensis and L. (Viannia) braziliensis cause localized cutaneous leishmaniasis (LCL), although subsequent progression to diffuse cutaneous leishmaniasis in more susceptible individuals is normally attributed to L. amazonensis. Mucosal leishmaniasis is usually associated with L. (V.) braziliensis. Whereas diffuse cutaneous leishmaniasis is characterized by large numbers of parasites and a lack of cell-mediated immunity to Leishmania antigens, cutaneous and mucosal leishmaniasis are generally accompanied by strong cellular responses and sparse numbers of parasites in the lesions [1]. In experimental 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 is related 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 spontaneous healing [3]. Simultaneous production of IFN-␥, tumor necrosis factor (TNF)–␣, and IL-10 by antigen-stimulated peripheral blood mononucleaer cells (PBMCs) from patients with active lesions [4] and IL-2, IL-4, IL-5, IL-10, and IFN-␥ mRNAs were demonstrated in biopsy samples taken from active lesions [5– 8]. IL-10 expression was also significantly higher in patients who responded poorly to pentamidine treatment [5]. In a previous study, our group showed a 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 elevated anti-Leishmania IgG antibodies plasma levels, mainly IgG1 and IgG3 isotypes [10 –12]. Although the protective role of antibodies in leishmaniasis is questionable, it is useful for diagnosis and for predicting in vivo cellular immune responses. In the present study, we analyzed cytokines involved in T– helper cell balance and specific antibody isotypes in patients with active cutaneous leishmaniasis, individuals with cured leishmaniasis, and individuals without lesions living in the same endemic area. 2. Subjects and methods 2.1. Subjects
* Corresponding author. E-mail address: [email protected] (V. Rodrigues, Jr.).
The study population included 20 patients with active infection characterized by typical lesions and positive Montenegro skin test
0198-8859/09/$32.00 - see front matter 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2009.01.007
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results, and 40 subjects with previous cutaneous leishmaniasis identified in the medical records and characterized by skin scars and positive Montenegro test results, were studied. In addition, 34 asymptomatic individuals living in the same area under similar exposure conditions without either past or present clinical disease manifestation were also included. All subjects lived in the rural area of Buerarema, Bahia State, Brazil. This region is endemic for cutaneous leishmaniasis caused by L. (V.) braziliensis and an epidemic outbreak had occurred in that population between 1995 and 1999. Clinical and epidemiologic surveys were carried out to record the clinical manifestations of the disease and to identify patients with active infection. Montenegro skin test was performed on the ventral face of the left forearm. Test positivity was determined based on at least 4-mm indurations after a 48-hour challenge. After agreement was obtained from the subjects, 15 ml of peripheral blood was collected. The study protocol was approved by the Ethics Committee of the Universidade Federal do TriÃngulo Mineiro in Brazil. 2.2. Cell culture The PBMCs were isolated by Ficoll-Paque centrifugation (Pharmacia, Uppsala, Sweden) at 400 g for 20 minutes at room temperature, 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, 2 mmol/l L-glutamine (Gibco), 40 g/ml gentamicin, and 5% fetal calf serum (Gibco). A total of 2 ⫻ 106 cells per well were cultured in 24-well microplates (Falcon, San Jose, CA) in the presence of medium alone, 5 g/ml PHA (Sigma, St. Louis, MO) or 5 g/ml L. (V.) braziliensis (Lb) antigen. Plates were incubated at 37⬚C in a 5% CO2 atmosphere. Supernatants were collected after 24 and 96 hours, centrifuged, and stored at ⫺70⬚C 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 l of 1 g/ml specific monoclonal antibody (Mabtech, Mariemont, OH, and Pharmingen, San Diego, CA). The plates were then washed four times with PBS containing 0.05% Tween-20 (Sigma), and nonspecific binding was prevented by incubation of the plates with 2% bovine serum albumin (BSA; Sigma) in PBS. Plates were incubated overnight with 100 l 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 appropriate biotinylated anti-cytokine monoclonal antibody (Mabtech and Pharmingen) for 2 hours at 37⬚C, followed by washing and incubation with alkaline phosphatase-conjugated streptavidin for 2 hours at 37⬚C. Finally, plates were washed four times and enzymatic activity was developed by incubation with p-nitrophenyl phosphate (Sigma). Absorbance was read at 405 nm in a microplate reader (BioRad, Hercules, CA). The sensitivity of the assay ranged from 5 to 10 pg/ml. 2.4. Antibody detection Specific anti-Leishmania IgG, IgG1, IgG3, IgG4, and IgE were measured by indirect enzyme-linked immunoabsorbent assay (ELISA) as follows: highly sensitive microplates (Nunc) were sensitized with 100 l of 2 g/ml crude antigen of L. (V.) braziliensis strain Lb2904 at 4⬚C overnight. Nonspecific binding was prevented by incubation with 2% BSA-PBS. Plates were incubated with 100 l of the subject’s plasma in 2% BSA-PBS diluted 1:50 and 1:20 for whole IgG and for IgG isotypes, respectively. Plates were then washed four times with PBS/Tween and incubated with 1 g/ml appropriate biotinylated anti-human antibody class detection monoclonal antibody (Pharmingen) or peroxidase-conjugated protein A for 2 hours at 37⬚C. For the Ig isotypes, the plates were
washed and incubated with alkaline phosphatase-conjugated streptavidin for 2 hours at 37⬚C. Finally, plates were washed four times and enzymatic activity was developed by incubation with ortho-phenyldiamine or p-nitrophenyl phosphate (Sigma). Absorbance 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 Schneider’s medium (Sigma) supplemented with 20% inactivated fetal bovine serum (Sigma), 1% sodium pyruvate, 1% L-glutamine, 750 mg/l calcium chlorate, and 40 g/ml gentamicin. Parasites were cultured in plastic flasks (Corning, Lowell, MA) at 26⬚C for 96 hours. Parasites in the stationary growth phase were collected and centrifuged at 800 g for 30 minutes at 4⬚C; parasites were then washed with RPMI medium (Gibco) by centrifugation. For antibody detection, 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 4000 g for 30 minutes at 4⬚C and the supernatant was filtered through a 0.22-m filter (Millipore, Molsheim, France). Protein concentration was determined by the method of Bradford (Pierce, Rockford, IL) and adjusted to 2 mg/ml and stored at ⫺70⬚C. For in vitro use, L. (V.) braziliensis in the stationary growth phase was resuspended in PBS and submitted to six freeze–thaw cycles. Parasite integrity was determined by light microscopy. Lysed parasites were centrifuged at 1200 g for 30 minutes at 5⬚C, and protein concentration was determined by the method of Bradford and adjusted to 1 mg/ml. The antigen preparation was stored at ⫺70⬚C. 2.6. Statistical analysis Patients were grouped according to the presence or absence of either active or past LCL lesions. The nonparametric Kruskal-Wallis test was applied to compare cytokine and antibody levels between the three groups, with a value of p ⬍ 0.05 being considered significant. The post hoc Dunn test was used to establish the differences indicated by the Kruskal-Wallis test, with the level of significance set at Q ⬎ 2.5. SigmaStat software (Systat, San Jose, CA) was used for statistical analysis. 3. Results 3.1. Specific anti-Leishmania isotypes antibody levels Anti-Leishmania antibodies in sera from endemic area individuals and non– endemic area healthy controls (volunteer donors of the Regional Blood Bank Uberaba) were evaluated. As shown in Figure 1A, endemic area subjects exhibited higher Leishmaniaspecific IgG levels than non-endemic area donors (p ⬍ 0.0001). Two of 47 samples from non– endemic area volunteers strongly recognized Lb antigens, probably because of cross-reactivity with other infections or previous contact with Leishmania. Next, differences among patients with active or healed CL and asymptomatic endemic area subjects were evaluated. Patients with active lesions and with healed cutaneous leishmaniasis presented with higher circulating levels of anti-Leishmania IgG antibodies than did control subjects, although the difference was nonsignificant (Figure 1B). This result indicates a tendency of cutaneous leishmaniasis patients to present with specific antibodies circulating at elevated levels and also suggests that endemic area control subjects were exposed to parasite antigens. Analysis of IgG isotypes showed that IgG1 was significantly higher in patients with active or healed lesions compared with levels in endemic area controls (p ⫽ 0.0004; Q ⫽ 3.54 and Q ⫽ 3.22, respectively). No significant difference was observed between patients with active and healed lesions (Figure 1C). On the other hand, IgG3 levels were significantly higher in patients with active lesions when compared with both healed leishmaniasis and control sub-
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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.
jects (p ⫽ 0.0004; Q ⫽ 2.83 and Q ⫽ 3.96, respectively). No significant difference was observed between patients with healed leishmaniasis and control subjects (Figure 1D). Specific IgG4 was present at low levels in all samples tested, with no significant difference between groups (Figure 1E). Furthermore, specific antiLeishmania IgE antibodies were detected at very low levels, with control subjects presenting with higher plasma levels than patients with active lesions (p ⫽ 0.0161, Q ⫽ 2.876; Figure 1F). When cured subjects were regrouped according to the time after lesion healing of less than 5 years, from 5 to 10 years, and more than 10 years, it was revealed that total IgG and IgG1 plasma titers decreased progressively in cured patients presenting with scars within 5 years after healing (Figure 2A, B), whereas IgG3 plasma titers decreased in healed patients whose scars had more than 10 years after healing (Q ⫽ 2.975; Figure 2C). Anti-Leishmania IgG4 and IgE isotypes did not show any relevance with respect to identification of infected, cured, or noninifected exposed subjects (Figures 2D, 2E). 3.2. Cytokine patterns in patients and asymptomatic controls The cellular immune response was evaluated by the measurement of TNF-␣, IFN-␥, IL-12, IL-4, and IL-10 cytokines in culture supernatants collected after 24 or 96 hours of PHA- or Lb antigenstimulated PBMC.
TNF-␣ levels were analyzed in supernatants after 24 and 96 hours of PBMC culture. In 24-hour supernatants (Figure 3A), significantly 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 difference 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 addition, after stimulation with Lb antigen, PBMCs from patients with active 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-
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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) plasma levels were measured by enzyme immunoassay in inhabitants of 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). 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.
served between patients with cured leishmaniasis and control subjects (Figure 3D). IL-4, a Th2 cytokine, was analyzed in 24-hour supernatants. Significantly higher IL-4 levels were observed in nonstimulated cells from patients with active lesions (p ⬍ 0.001) when compared with subjects with cured leishmaniasis (Q ⫽ 7.16) and the control group (Q ⫽ 7.16). Furthermore, after stimulation with Lb antigen, cells from patients with active lesions produced significantly higher levels of IL-4 (p ⬍ 0.001) compared with 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) were observed for all three culture conditions (medium alone, PHA, and
Lb antigen). PBMCs from patients with active lesions produced higher IL-10 levels than 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), irrespective of the stimuli (Figure 3F). Cytokine production in Lb-stimulated PBMCs also revealed that healed patients with early scars (up to 5 years) presented higher levels of TNF-␣ than asymptomatic endemic area controls (Q ⫽ 2.668) and that after 5 years of healing these levels tended to decrease (Figures 4A, 4B). On the other hand, IFN-␥ levels were constantly higher in all healed patients in comparison to the endemic area asymptomatic subjects, independently of the time of lesion healing (⬍5 years, Q ⫽ 4.539; 5–10 years, Q ⫽ 3.004; ⬎10 years, Q ⫽ 2.649; Figure 4C). IL-12 levels decreased in subjects with
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Fig. 3. Cytokine levels produced by PBMCs from subjects living in a cutaneous leishmaniasis endemic area. Cells were cultured for 24 or 96 hours in the absence (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 according to the presence of active lesions (n ⫽ 20), presence of typical leishmanial 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. Scale bar on the right refers to PHA levels. Statistical analysis is indicated in each graph. *Kruskal-Wallis test, p ⬍ 0.05, post hoc Dunn test statistically significant.
more than 10 years of healing when compared with endemic area controls (Q ⫽ 2.556), patients with active lesions (Q ⫽ 3.515) and also healed subjects with early scars (Q ⫽ 2.103) (Figure 4D). Moreover, patients with early healed lesions presented with a great decrease in IL-4 and IL-10 production compared with patients with active lesions (Q ⫽ 3.374 and Q ⫽ 3.103, respectively), and these cytokines levels decreased progressively after healing (Figure 4E, 4F). IL-10 levels were even lower as detected in supernatants from patients with more than 10 years of healing compared with patients with other healing times (Q ⫽ 2.050) (Fig. 4F). 4. Discussion The present study analyzed cytokines involved in the activation of an effective anti-Leishmania immune response such as TNF-␣, IFN-␥, and IL-12 and also immunoregulatory cytokines such as IL-4 and IL-10, which seem to be involved in disease progression. Furthermore, the in vivo effects of cytokines observed in cultures were confirmed by their ability to modulate antibody class switches detected in plasma samples. Experimental models of infection with different Leishmania species have been used to establish the importance of the Th1 immune response against intracellular pathogens, whereas the Th2 re-
sponse has been found to be associated with disease susceptibility [2]. More information about immunoregulatory mechanism involved in human leishmaniasis is necessary to allow development of rational strategies of intervention. In this study, subjects from an endemic area presented higher anti-Leishmania IgG circulating plasma levels than healthy blood bank donors from a nonendemic area. Although specific IgG levels did not differ among endemic area patients and controls, this result may suggest a general exposure of the endemic area population to a natural infection with L. (V.) braziliensis. When IgG isotypes were evaluated, IgG1 was able to differentiate endemic area asymptomatic control subjects from patients with active or past CL history. Circulating long-lasting memory B cells present in cured patients might be regulated by the cytokine environment and the persistent antigenic stimulation over the time to switch for IgG1. Alternatively, IgG3 was useful in discriminating patients with active lesions from those with cured disease or no previous history of leishmaniasis. Previously, higher levels of both IgG1 and IgG3 were observed in patients with active lesions compared with cured subjects [12]. Results found in this and other studies regarding the predominance of an IgG1 and IgG3 response over an IgG4 and IgE reflect the tendency of a Th1 immune response during cutaneous
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Fig. 4. Cytokine levels produced by PBMC from subjects living in a cutaneous leishmaniasis endemic area grouped according to time after lesion healing. Cells were cultured for 24 or 96 hours in the absence (medium) or presence of stimuli (PHA or Leishmania braziliensis antigen). Cytokine 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 according to the presence of active lesions (n ⫽ 20), presence of typical leishmanial 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, 10th–90th percentile. Statistical analysis is indicated in each graph. *Kruskal-Wallis test, p ⬍ 0.05, post hoc Dunn test statistically significant.
leishmaniasis course [3,10,12]. Detection of specific anti-Leishmania IgG1 and/or IgG3 isotypes might help in the development of useful diagnostic tools to be routinely applied for the screening of cutaneous leishmaniasis. The involvement of effectors and regulatory cytokines produced by PBMC was also analyzed. Significant production of Th1-, Th2-, and Treg-type cytokines by cells from patients with active lesions, mainly TNF-␣, IFN-␥, IL-12, IL-4, and IL-10, was observed. This mixed pattern of immune response observed in patients with active lesions evolved to a Th1 response characterized by the production of IFN-␥ in cured subjects. Other studies have also reported a mixed Th1/Th2 pattern of cytokine production in cutaneous leishmaniasis [4,9]. It seems that a predominant Th1 response is necessary for parasite control and lesion healing, whereas the concomitant presence of IL-4 and IL-10, and probably other regulatory cytokines such as TGF-, may contribute to parasite escape, sustenance of inflammatory stimulation, and lesion persistence [9,13,14].
Several experimental studies using knockout mice and blockage of cytokines support this hypothesis [15–17]. IFN-␥, a major Th1 cytokine, plays an essential role in the control of Leishmania infection by mediating macrophage killing of intracellular parasites [17]. Studies conducted in human subjects have detected elevated levels of IFN-␥ or its mRNA in biopsy samples of active LCL lesions [5,6,8], whereas spontaneous lesion healing has been associated with high levels of IFN-␥ production [3]. Moreover, low IFN-␥ production was observed to be associated with chronic lesions and therapeutic failure [18]. Our results indicate a high production of IFN-␥ in patients with active lesions, and its persistence at elevated levels after lesion healing also suggest the presence of circulating Leishmania-specific Th1 memory T cells. One of the main factors responsible for the development of a Th1 response and IFN-␥ production is IL-12. Experimental models have shown that IL-12 is necessary for the maintenance of the Th1
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immune response during L. (L.) major infection [2]. Our results showing a high production of IL-12p70 by non-stimulated and Lb antigen-stimulated PBMC from patients with active lesions, suggest that IL-12 is an important factor for the induction and maintenance of IFN-␥ during active LCL. Thus, in human cutaneous leishmaniasis, IL-12 production during the active phase promotes Th1 cell differentiation, whereas after clinical cure Th1 memory cells persist, despite downregulation of IL-12 production. TNF-␣ plays an essential role during LCL development. Both TNF-␣ and IFN-␥ are macrophage activators, especially inducing iNOS to produce nitric oxide in response to intracellular microorganisms [19,20]. In human beings, elevated expression of iNOS in active lesion biopsy samples was correlated with low parasite burdens at these sites [21]. Furthermore, TNF-␣ is a potent proinflammatory mediator related to tissue damage events and inflammatory cell recruitment to local sites of lesions. This cytokine has been shown to be present at elevated levels in patients with severe forms of cutaneous leishmaniasis [22] and in skin biopsy samples [6,8]. In the present study, high levels of TNF-␣ were observed in patients with active lesions under stimulatory conditions. TNF-␣ production might be involved in the attempt of the host to control Leishmania infection, but its continued overproduction may contribute to tissue damage and ulcer formation [23]. The decrease in TNF-␣ production after healing observed in this study supports this idea and reinforces the importance of circulating IFN-␥ ⫹ Th1 memory cells in healed LCL patients. Production of Th1 cytokines in patients with active lesions was accompanied by elevated amounts of IL-4 and IL-10. In experimental models, the administration of anti-IL-4 monoclonal antibody promoted cure of susceptible mouse strains infected with L. (L.) major, with a concomitant increase in IFN-␥–producing cells in lymph nodes [24]. Although the increase on IFN-␥ levels after IL-4 blockade was not responsible for resistance itself, there was a shift to Th1 immunity and the transfer of IL-4-neutralized CD4⫹ T cells to naÐve mice induced resistance to subsequent Leishmania infection [25]. Moreover, patients with active lesions presented with a mixed 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 diminishes IFN-␥ production, promoting the parasite’s escape from the host immune system. IL-10 plays an important role in the regulation of the immune response, mainly by inhibiting IFN-␥ synthesis and macrophage activation. Experimental models of Leishmania infection have shown that initial IL-10 production by susceptible mouse strains inhibited IFN-␥ production and contributed to L. (L.) major escape and maintenance of infection [27]. Recently it was observed that the 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 suggesting that IL-10 is involved both in disease progression, when produced at high levels, and in the development of memory T cells, when produced at low levels [29]. In human beings, a positive correlation has been observed between high intralesional IL-10 mRNA levels and therapeutic failure [5]. It has been demonstrated that a strong IL-10 production was correlated with deficient lymphocyte proliferative response and low IFN-␥ levels in patients with earlier CL lesions. This pattern was completely reversed after chemotherapy or after the addition of IL-12 or monoclonal antibodies to IL-10 to the cultures [30]. Recently it was demonstrated that IL-10 –producing CD4⫹CD25⫹ T cells accumulate at lesion sites and may exert their immunoregulatory functions during infection [31]. Our previous data indicated that functional polymorphisms in positions ⫺819C/T and ⫺592A/C of the IL10 promoter region are associated with higher IL-10 production by CD4⫹CD25⫹Foxp3⫹ T
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cells after Lb stimulation and LCL skin lesion occurrence in endemic area subjects [9]. Our results showed that cells from patients with active lesions produced large amounts of IL-10 under all culture conditions. The decline of IL-4 and IL-10 production after lesion healing suggests that these cytokines may be involved in parasite escape and persistent antigen stimulation, favoring lesion development. Accumulating data obtained from experimental models indicate that susceptibility to Leishmania is a complex event, involving both the Th2 and Treg pathways [2]. We also observed a positive correlation between specific antiLeishmania 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 produced functional effects in vivo. The present results indicate that a mixed pattern of cell-mediated immune response takes place during active disease, and that healing is associated with a decline in the production of IL-4 and IL-10, cytokines able to block IFN-␥ production and macrophage activation, suggesting that the disease is associated with the presence of regulatory cytokines rather than by the absence of IFN-␥ itself. We can also argue for a probable dual role of TNF-␣, first acting on parasite control by its ability to promote macrophage activation and later promoting lesion by its tissue damage potential. The present results suggest that IL-4 and IL-10 are determinant factors in the susceptibility of human beings to L. (V.) braziliensis infection, with both cytokines being interesting as relevant targets for therapeutic strategies. Acknowledgments We are indebted to the Buerarema Municipal Health Council and to the regional branch of Programa de SaÛde da FamÎlia-PSF for logistic 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 the statistical analysis. This work was supported by Conselho Nacional de Desenvolvimento CientÎfico e TecnolÔgico–CNPq, FundaÈÄo de Amparo Á Pesquisa de Minas Gerais-FAPEMIG and CoordenaÈÄo de AperfeiÈoamento de Pessoal de NÎvel Superior (CAPES-DS fellowship to L.R.C.C.). References [1] Convit J, Ulrich M, Fernandez CT, Tapia FJ, Caceres-Dittmar G, Castes M, et al. The clinical and immunological spectrum of American cutaneous leishmaniasis. Trans R Soc Trop Med Hyg 1993;87:444 – 8. [2] Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol 2002;2:845–58. [3] Carvalho EM, Correia Filho D, Bacellar O, Almeida RP, Lessa H, Rocha H. Characterization of the immune response in subjects with self-healing cutaneous leishmaniasis. Am J Trop Med Hyg 1995;53:273–7. [4] Trujillo CM, Robledo SM, Franco JL, Velez ID, Erb KJ, Patino PJ. Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Th1 immune response in comparison to patients with localized cutaneous leishmaniasis. Parasite Immunol 2002;24:455– 62. [5] Bourreau E, Prevot G, Gardon J, Pradinaud R, Launois P. High intralesional interleukin-10 messenger RNA expression in localized cutaneous leishmaniasis is associated with unresponsiveness to treatment. J Infect Dis 2001;184: 1628 –30. [6] Caceres-Dittmar G, Tapia FJ, Sanchez MA, Yamamura M, Uyemura K, Modlin RL, et al. Determination of the cytokine profile in American cutaneous leishmaniasis using the polymerase chain reaction. Clin Exp Immunol 1993;91:500 –5. [7] Louzir H, Melby PC, Ben Salah A, Marrakchi H, Aoun K, Ben Ismail R, et al. Immunologic determinants of disease evolution in localized cutaneous leishmaniasis due to Leishmania major. J Infect Dis 1998;177:1687–95. [8] Pirmez C, Yamamura M, Uyemura K, Paes-Oliveira M, Conceicao-Silva F, Modlin RL. Cytokine patterns in the pathogenesis of human leishmaniasis. J Clin Invest 1993;91:1390 –5. [9] Salhi A, Rodrigues V, Jr., Santoro F, Dessein H, Romano A, Castellano LR, et al. Immunological and genetic evidence for a crucial role of IL-10 in cutaneous lesions in humans infected with Leishmania braziliensis. J Immunol 2008;180: 6139 – 48. [10] Junqueira Pedras M, Orsini M, Castro M, Passos VM, Rabello A. Antibody subclass profile against Leishmania braziliensis and Leishmania amazonensis in the diagnosis and follow-up of mucosal leishmaniasis. Diagn Microbiol Infect Dis 2003;47:477– 85.
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