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Cytokine profile in Montenegro skin test of patients with localized cutaneous and mucocutaneous leishmaniasis

Perfil de citocinas na intradermorreação de Montenegro em doentes de leishmaniose cutânea localizada e cutâneo-mucosa

Abstracts

American tegumentary leishmaniasis presents as two major clinical forms: localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). The immune response in leishmaniasis is efficiently evaluated by the response to Leishmania antigen through the Montenegro skin test (MST). Both LCL and MCL present positive response to MST, indicating that the patients present cell-mediated immunity against the parasite - Leishmania. In spite of the presence of immunity in MCL, this is not sufficient to stop disease progression and prevent resistance to treatment. In this study we demonstrated interleukin (IL) 2, 4, 5 and interferon (IFN) gamma expression in biopsies of MST of ten patients with American tegumentary leishmaniasis. The obtained results were compared between LCL (n = 5) and MCL (n = 5) patients. The MST of MCL patients displayed a higher expression of IL-2, IL-4 and IL-5, in comparison to LCL. There was no significant difference in IFN-gamma expression between groups. The obtained results suggest the role of IL-4 and IL-5 in the maintenance of the immunopathogenic mechanism of the destructive lesions that characterize MCL.

Human cutaneous leishmaniasis; Immunopathology; Cytokines; Delayed-type hypersensitivity; Skin


A leishmaniose tegumentar americana apresenta duas formas clínicas mais comuns: a leishmaniose cutânea localizada e a leishmaniose cutâneo-mucosa. A imunidade da leishmaniose é avaliada pela resposta ao antígeno Leishmania através da Intradermorreação de Montenegro. Estas duas formas apresentam resposta positiva, indicando que o paciente apresenta imunidade celular contra o parasita Leishmania. Apesar da presença da imunidade celular na leishmaniose cutâneo-mucosa, esta não é suficiente para barrar a progressão da doença e a resistência ao tratamento. Neste estudo, detectamos quatro citocinas por imunohistoquímica, IL-2, IL-4, IL-5 e IFN-gama nas biópsias da intradermorreação de Montenegro de pacientes com leishmaniose tegumentar americana (n = 10), cinco com leishmaniose cutânea e cinco com cutâneo-mucosa. Os resultados mostraram uma alta expressão significativa de IL-2, IL-4, IL-5 na leishmaniose cutâneo-mucosa comparada com a leishmaniose cutânea localizada, mas sem diferença significante na expressão do IFN-γ entre os grupos. Estes resultados sugerem a importância da participação da citocina IL-4 e IL-5 na manutenção do mecanismo imunopatogênico das lesões destrutivas da forma cutâneo-mucosa.


LEISHMANIASIS

Cytokine profile in Montenegro skin test of patients with localized cutaneous and mucocutaneous leishmaniasis

Perfil de citocinas na intradermorreação de Montenegro em doentes de leishmaniose cutânea localizada e cutâneo-mucosa

Marcia Ferraz NogueiraI; Hiro GotoII; Mírian Nacagami SottoIII; Luiz Carlos CucéIV

IDepartamento de Dermatologia, Faculdade de Medicina da Universidade de Santo Amaro (UNISA), São Paulo, Brasil

IILaboratório de Soroepidemiologia e Imunobiologia do Instituto de Medicina Tropical de São Paulo, Faculdade de Medicina da Universidade de São Paulo (USP), São Paulo, SP, Brasil

IIIDepartamento de Dermatologia e Patologia, Faculdade de Medicina da Universidade de São Paulo (USP), São Paulo, SP, Brasil

IVDepartamento de Dermatologia, Faculdade de Medicina da Universidade de Santo Amaro (UNISA), Faculdade de Medicina da Universidade de São Paulo (USP), São Paulo, SP, Brasil

Correspondence to Correspondence to: Marcia Ferraz Nogueira, PHD Rua José Jannarelli 199-cj 71 05615-001 São Paulo, SP, Brasil Tel: +55 11 3721-7166 Fax: +55 11 3722-4332 E-mail: nogueiramf@uol.com.br

SUMMARY

American tegumentary leishmaniasis presents as two major clinical forms: localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). The immune response in leishmaniasis is efficiently evaluated by the response to Leishmania antigen through the Montenegro skin test (MST). Both LCL and MCL present positive response to MST, indicating that the patients present cell-mediated immunity against the parasite - Leishmania. In spite of the presence of immunity in MCL, this is not sufficient to stop disease progression and prevent resistance to treatment. In this study we demonstrated interleukin (IL) 2, 4, 5 and interferon (IFN) gamma expression in biopsies of MST of ten patients with American tegumentary leishmaniasis. The obtained results were compared between LCL (n = 5) and MCL (n = 5) patients. The MST of MCL patients displayed a higher expression of IL-2, IL-4 and IL-5, in comparison to LCL. There was no significant difference in IFN-gamma expression between groups. The obtained results suggest the role of IL-4 and IL-5 in the maintenance of the immunopathogenic mechanism of the destructive lesions that characterize MCL.

Keywords: Human cutaneous leishmaniasis; Immunopathology; Cytokines; Delayed-type hypersensitivity; Skin.

RESUMO

A leishmaniose tegumentar americana apresenta duas formas clínicas mais comuns: a leishmaniose cutânea localizada e a leishmaniose cutâneo-mucosa. A imunidade da leishmaniose é avaliada pela resposta ao antígeno Leishmania através da Intradermorreação de Montenegro. Estas duas formas apresentam resposta positiva, indicando que o paciente apresenta imunidade celular contra o parasita Leishmania. Apesar da presença da imunidade celular na leishmaniose cutâneo-mucosa, esta não é suficiente para barrar a progressão da doença e a resistência ao tratamento. Neste estudo, detectamos quatro citocinas por imunohistoquímica, IL-2, IL-4, IL-5 e IFN-gama nas biópsias da intradermorreação de Montenegro de pacientes com leishmaniose tegumentar americana (n = 10), cinco com leishmaniose cutânea e cinco com cutâneo-mucosa. Os resultados mostraram uma alta expressão significativa de IL-2, IL-4, IL-5 na leishmaniose cutâneo-mucosa comparada com a leishmaniose cutânea localizada, mas sem diferença significante na expressão do IFN-γ entre os grupos. Estes resultados sugerem a importância da participação da citocina IL-4 e IL-5 na manutenção do mecanismo imunopatogênico das lesões destrutivas da forma cutâneo-mucosa.

INTRODUCTION

Leishmaniasis is a disease caused by protozoa of the genus Leishmania, which can occur as the tegumentary or visceral form. It is an endemic anthropozoonosis found in 88 countries around the world, with an estimated 12 million cases worldwide and 1.5 to 2 million new cases occurring annually, 1 to 1.5 million cases are of tegumentary leishmaniasis and 500,000 cases of visceral leishmaniasis33. In Brazil, around 30,000 tegumentary leishmaniasis cases are reported annually3, the majority of them caused by Leishmania (Viannia) braziliensis31.

Clinical manifestations of tegumentary leishmaniasis are diverse, but the most frequent forms in Brazil are localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). LCL is characterized by the presence of a single or few ulcers, usually present in exposed areas of the body, usually healing after therapy. MCL is characterized by the presence of infiltrated lesions in the oral, nasal, pharyngeal and laryngeal mucosa membranes and skin of the surrounding areas. It usually manifests months or years after healing of the localized cutaneous lesion. These lesions are usually resistant to therapy, often disfiguring the face17.

The evolution of the disease depends on the species of the protozoa and the host's immune response10,28. In human and experimental leishmaniasis, immunity is predominantly mediated by lymphocytes. In experimental cutaneous leishmaniasis, using isogenic mouse strains infected with Leishmania (Leishmania) major, a paradigm was established associating a subpopulation of CD4+ T cells producing mainly IFN-gamma (T helper 1 cells = Th1 cells) to the resistance, and CD4+ T cells producing mainly IL-4 and IL-10 (T helper 2 cells = Th2 cells) to susceptibility18,27. However, in the patients with LCL and MCL, the pathogenesis does not seem to be related only to the difference in the host's susceptibility, as not many parasites are seen in the lesions, even in the most severe mucocutaneous form of the disease. This condition differs from what is observed in the lesions of the susceptible mouse, with diffuse cutaneous leishmaniasis, where there is a high expression of IL-4 and lots of parasites. Furthermore, the delayed-type hypersensitivity reaction to MST, counterpart of the T-cell mediated immunity, is positive in both forms of the disease. Indeed, in MCL this immune response does not prevent the aggressive evolution of the disease28. Differently, in this context, cytokines may play different roles in the pathogenesis, being more related to the maintenance of the inflammatory reaction in the tissue. Studies in this context have identified both Th1 and Th2-type cytokines in Leishmania antigen-stimulated peripheral blood mononuclear cell supernatant in LCL11,13,15 and also in MCL13. PCR studies of lesion samples have shown Th1 profile LCL and both Th1 and Th2 in MCL26. On the other hand, immunohistochemistry analysis demonstrated both Th1 and Th2 -cytokine expression in LCL lesions14. These apparently controversial observations might be due to the different assays used and to the comparison of peripheral blood mononuclear cells and in situ inflammatory cell results.

A positive MST is an indication of the presence of cell-mediated immune response of the host against Leishmania. In the present study, we demonstrated the expression of IFN-gamma, interleukin (IL) 2, 4 and 5, in MST biopsies of LCL and MCL in order to address the immunopathogenic mechanism in these different forms of leishmaniasis.

MATERIAL AND METHOD

The sample study consisted of ten patients (eight males and two females) aged 19 to 82 years (mean age of 49.5 years), with confirmed diagnosis of American tegumentary leishmaniasis. The five LCL and five MCL patients had just started the specific treatment for the disease. The study was approved by the Ethics Research Board of the Institutions (University of São Paulo and Santo Amaro Medical School). Informed consent was obtained from all patients. Nine patients were from different regions of Brazil and one patient was from Bolivia (patient 3). The mean ± standard deviation (SD) time of disease was 8 ± 6.2 months in LCL and 20.1 ± 19.9 years in MCL.

The diagnosis of leishmaniasis was confirmed by: the direct parasitological test, histopathological assessment including the demonstration of Leishmania antigen by immunohistochemistry in the lesions32, positive MST, and detection of anti-Leishmania antibody in the serum (by indirect immunofluorescence technique and ELISA).

Montenegro skin test: A sample of 100 mL of dead Leishmania promastigote suspension, in phenolic and sodium fluoride solution (two to three millions microorganisms per mL, called Montenegro antigen, produced at Adolfo Lutz Institute, in São Paulo, Brazil) was injected intradermally in the arm of each patient21.

MST biopsies were obtained before specific treatment in eight patients, and 15 and 18 months after treatment in two patients with LCL.

The MST evaluation was carried out 72 hours after antigen inoculation, by measuring two diameters of the resulting papule. The mean of the two diameters was calculated for statistical analysis. MST with papule = 5 mm, was considered positive4.

The biopsy samples were taken from the center of the erythematous papule using a 4-mm punch, under local anesthesia with 2% xylocaine, after the MST clinical evaluation. The control skin sample of the assessed cases consisted of normal skin biopsies from the contralateral arm.

Detection of cytokines in the skin by immunohistochemical technique: Skin fragments were placed in sterile saline solution, later pre-frozen in isopentane and finally frozen in liquid nitrogen at -180 ºC. Fragments were kept at -80 ºC and later sectioned in cryostat.

Cytokines were detected by the immunohistochemical method, using anti-human IL-2, anti-human IL-4, anti-human IL-5 and anti-human IFN-gamma purified monoclonal goat IgG, purchased from R&D Systems (MN, USA). Four-µm cryostat sections were immersed in acetone for 10 minutes, dried at room temperature, followed by immersion in water for five minutes. After serial incubation to prevent nonspecific staining with 0.03% hydrogen peroxide in Tris solution, pH 7.4, and afterward with bovine serum albumin for one hour at 37 ºC, the sections were incubated with the primary antibody for 12 hours at 8 ºC. That was followed by incubation with goat biotinylated antibody IgG (R&D Systems, MN, USA) and with peroxidase-conjugated avidin for 30 minutes at 37 ºC. The slides were washed with 0.01 M phosphate-buffered saline, pH 7.2 (PBS) after each incubation step. The reaction was developed using 0.06% hydrogen peroxide and 0.3 mg/mL 3,3'-diaminobenzidine (Sigma Chemical, USA) in PBS. Counterstaining was performed with Harry's hematoxylin (Sigma Chemical, USA). The cytokines were identified in a light brown color.

Semi quantification of the cytokine staining: Immunostaining of the cytokines in the dermis was analyzed and semi-quantified by two independent investigators, by attributing scores as follows: zero for negative immunolabeling; 1+ for up to one third of positive stained mononuclear cells in the dermal cell infiltrate; 2+ for more than one third up to two thirds of positive stained mononuclear cells in the dermal infiltrate; 3+ for more than two thirds of positive stained mononuclear cells in the dermal infiltrate. In case of discordance between the two independent investigators, an independent third investigator provided consensus about the final score.

Statistical analysis: We analyzed the arithmetic mean and median of the results from the two groups of patients and analyzed them by Friedman's and Fisher's tests. The level of significance was set at 0.05.

RESULTS

Intensity of the Montenegro skin test: The results of the MST in LCL and MCL are shown in Table 1.

The mean value of the MST intensity, evaluated at the time of the patients' diagnoses, was significantly higher in the MCL when compared to the LCL group (p < 0.01). On the other hand, there was no significant difference regarding the MST evaluation when the groups were tested again before the biopsy procedure. In addition, we did not find any statistical difference between the intensity of the MST, at the diagnostic procedures as well as at the time of the study, in both LCL and MCL patients.

Detection of cytokines in Montenegro skin test: The median values of the cytokines in LCL and MCL are shown in Table 2.

There was a predominance of Th1 cytokine profile in the Montenegro skin tests in LCL. The statistical analyses showed a predominance of IFN-γ in comparison to the other three cytokines (p < 0.05).

Both types of cytokine profiles (Th1 and Th2) were observed in MCL patients.

No cytokine expression was observed in the normal control skin of all patients studied.

The statistical analyses showed a higher expression of IL-2, IL-4 and IL-5 in the dermal infiltrate of MCL when compared with LCL patients (p < 0.01).

DISCUSSION

The immune response in cutaneous leishmaniasis is characterized by a cell-mediated immune response, with a wide participation of humoral immunity6. The cellular immune response in leishmaniasis is clinically evidenced by Montenegro skin test23,28.

In this study, the results of the intensity of the MST were different at the diagnosis and at the time of the present study, showing that the MST can change clinically with a second inoculation, as it has been demonstrated by other investigators1,24. In spite of that, the MST remains positive before and after treatment, and therefore, the MST response is independent from the treatment8, although we cannot be certain about the changes of the MST intensity after treatment, as this has not been measured by the authors. In comparison with in vitro studies, the treatment does not change the immunological response of the patients with LCL, as demonstrated by some authors1,13,30. DA-CRUZ et al.13 demonstrated in peripheral blood of patients with localized cutaneous leishmaniasis caused by L. braziliensis, that the type of cytokine remains the same, i.e., the Th1 type, before and after the treatment. This fact supports our findings in the MST that was performed after treatment in two patients of LCL. However, in MCL patients, a change in cytokine profile after treatment has been observed in vitro, as the observed Th1 and Th2 types, changed to Th1 type11,13.

The population of T lymphocytes present in the granulomatous infiltrate of the lesion of tegumentary leishmaniasis was mainly composed by CD4+ cells, and a lower proportion of CD8+ cells6,9,22,25. The subtypes of CD4+ T cells (Th1 and Th2 profile) have been characterized by many investigators in all forms of tegumentary leishmaniasis.

The immune response of the New World LCL caused by L. braziliensis, is characterized by a Th1 response, with a predominance of IFN-gamma expression in vitro and in situ and a very low or sometimes absent expression of IL-4, IL-5 and IL-101,2,5,11-15,20. In relation to other cytokines, such as IL-2, they have been demonstrated in vitro and in situ, with a low expression. Additionally, the immune response of the Old World LCL caused by L. major, L. tropica and L. aethiopica, is characterized by this same cytokine profile (Th1), also observed by many investigators19,29. However, in acute and chronic lesions of LCL, studied in situ and with stimulation of peripheral blood mononuclear cells by Leishmania antigen, the cytokines in acute lesions show a tendency to the Th1 type, whereas, in chronic lesions, there is a tendency to a mixed cytokine type19,29.

MCL is characterized by a mixture of Th1 and Th2 responses, which has been well established by two investigators using different techniques, both in situ and in vitro26. The cytokines observed in MCL are IL-4, IL-5, IL-10 at a higher proportion in comparison to LCL11,13,14, but lower than in diffuse cutaneous leishmaniasis4,22.

The Th1 response in MCL is more intense than in LCL, as demonstrated by the IFN-gamma and IL-2 that are observed at high proportions in vitro and in situ, and an exacerbated lymphoproliferative response to Leishmania antigen in vitro4,6,31.

In the present study, we demonstrated a difference in the cytokine profile identified in the MST of patients with LCL and MCL. There was a predominance of Th1 response in the MST of LCL patients and both types, Th1 and Th2, in the MST of MCL patients. We observed a significantly higher expression of IL-2 (p < 0.05), IL-4 and IL-5 in MCL in comparison to LCL (p < 0.01). Initially, we thought that a low expression of IL-2 in the LCL group was due to a technical problem, but the literature shows how weakly this cytokine is expressed in this group; its expression is a little higher in MCL, although at a very low frequency. Perhaps this cytokine is important for the initiation of the immune response, but not for its maintenance. Finally, the results showed no difference in IFN-gamma expression in both groups studied.

The predominance of IL-4 and IL-5 in Montenegro skin test of MCL may suggest that these cytokines participate in the modulation of the inflammatory reaction caused by the high amount of IFN-gamma. The destructive lesions may occur when the high quantities of IFN-gamma are not controlled by the Th2 cytokines7.

In this study, the identification of the immunological aspects demonstrated in the MST is similar to those seen in ATL lesions. We suggest that the MST can be used as a mirror of the disease itself. These aspects have also been demonstrated by other authors in studies about the inflammatory response in the MST16,22,26. On the other hand, these studies did not compare the in situ MST immunopathological characteristics in the two forms of the disease. Further studies are needed to compare the expression of cytokines in leishmaniotic lesions and MST biopsies simultaneously. Perhaps, we will be able to study the immune response of these two clinical forms during a phase of the disease when there are no lesions, but only the positive response of the MST. This reaction has 83.7 to 100% of sensitivity, it is very specific for the Leishmania genus and it stays positive for a long time after the cure or even throughout life17,23,28. We must remember that the blood lymphocytes represent 5% of all lymphocytes and that these cells stay in circulation for only one hour, so the study using these techniques sometimes do not actually show the reality of the disease.

The Montenegro skin test may show earlier changes, before the symptoms appear in the mucosa of the patients, so it is the only test able to assess the patient's immunological reaction to the Leishmania antigen, before the mucocutaneous form appears. The MST might be able to answer a question that is so often formulated: When does the localized form change to the mucocutaneous form?

ACKNOWLEDGMENTS

We thank Dr. Neusa S. Valente for assistance in the evaluation of immunohistochemical results and Mr. Cleiton Alves for technical procedures.

Received: 4 March 2008

Accepted: 7 July 2008

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  • Correspondence to:
    Marcia Ferraz Nogueira, PHD
    Rua José Jannarelli 199-cj 71
    05615-001 São Paulo, SP, Brasil
    Tel: +55 11 3721-7166
    Fax: +55 11 3722-4332
    E-mail:
  • Publication Dates

    • Publication in this collection
      09 Dec 2008
    • Date of issue
      Dec 2008

    History

    • Accepted
      07 July 2008
    • Received
      04 Apr 2008
    Instituto de Medicina Tropical de São Paulo Av. Dr. Enéas de Carvalho Aguiar, 470, 05403-000 - São Paulo - SP - Brazil, Tel. +55 11 3061-7005 - São Paulo - SP - Brazil
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