Evidence of in situ cytotoxicity in American cutaneous leishmaniasis

ARTICLE

American cutaneous leishmaniasis, a major health problem in Central and South America, manifests with several clinical patterns, the most frequent being a single ulcerated lesion; this is occasionally self-healing, but nearly always requires at least one course of systemic treatment with pentavalent antimony in order to cure it [1, 2]. Histopathologically, the lesions of cutaneous leishmaniasis (CL) are characterized by an inflammatory reaction made up of lymphocytes, plasma cells and macrophages, with a granulomatous reaction [3]. Patients with CL respond to leishmania antigen with lymphocyte proliferation and production of type 1 cytokines by peripheral blood mononuclear cells [4]. Previous studies have shown a predominance of cells with helper phenotype over cytotoxic/suppressor phenotype in the cellular infiltrate [5], with a higher number of memory T cells in the lesions [6]. In experimental models of leishmaniasis, it has been shown that cytotoxicity participates in the immune response against leishmania, however little is known concerning the role of tissue cytotoxicity and the involvement of NK and CD8 cells in the pathogenesis of human CL.

In this study we analyzed the local presence of CD8 lymphocytes, T memory/activated cells, NK cells and the expression of TIA-1, a protein associated with cytotoxic activity, in patients with cutaneous leishmaniasis.

Subjects and methods

Patients

Patients (n = 9) were referred to the health post of Corte de Pedra, an endemic area of L. braziliensis infection in Bahia State, Brazil. The diagnosis of cutaneous leishmaniasis was performed by the presence of a suggestive skin lesion, and confirmed by parasite isolation and positive skin test. None of the patients had received prior antileishmanial therapy. The study was approved by the Ethical Committee of the Hospital Universitário Professor Edgard Santos, University of Bahia, and informed consent was obtained from all patients or their guardians. All patients were treated with intravenous meglumine (Glucantime^®; Rhône-Poulenc, Paris) in a conventional schedule (20 mg of Sb^v/kg/day for 20 days).

Laboratorial diagnosis

Leishmania antigen used for the intradermal skin test was obtained from an L. amazonensis strain (MHOM/BR/86/BA-125), and 25 mug in 0.1 ml was injected in the forearm [7]. The larger diameter of the indurated area was measured after 48 hrs, and the reaction was considered positive if the diameter was greater than 5 mm.

Needle aspiration was performed in the skin lesion or in the crural lymph node and cultured in NNN medium overlaid with modified LIT medium. Cultures were kept at 25° C and examined twice weekly. Isolates were characterized by a panel of monoclonal antibodies [8].

Skin biopsies were performed before treatment in all patients, using a 4 mm punch biopsy after local anesthesia with 1% lidocaine, and immediately stored in liquid nitrogen.

Immunostaining

Cryosections (5 to 6 mum) were adhered to silane-coated slides and acetone fixed. After rehydratation with phosphate buffered saline (PBS), sections were incubated with the following monoclonal antibodies: anti-TIA-1 (Coulter Clone), anti-CD56 (Tebu, France), anti-CD45RO (Dako, Denmark), anti-CD8 (Dako, Denmark). Antibody binding was revealed with the EnVision + System (Dako, Denmark), using 3 amino-9-ethylcarbazole as chromogen (Dako EnVision + System) with hematoxylin counterstaining. Cell staining was evaluated independently by two observers using a semiquantitative agreed-upon scale (Table I). Each observer evaluated up to two slides per monoclonal per lesion. Negative controls included sections incubated with non-immune serum.

Results

Nine patients with a diagnosis of CL participated in the study. In six cases the skin lesion had less than 15 days of evolution; three of these cases presented a superficial ulceration and the others a papule with a small central crust, often localized in the lower limbs. The three patients with more than 60 days of disease presented a typical ulcerated lesion with raised borders localized in the lower limbs. All patients also presented a crural lymphadenopathy with more than 2 cm in diameter, on the same side as the lesion. The time of appearance of the lesions, patient's age and the intensity of staining of the different markers are shown in Table I.

About a half of the lesions (4/9) strongly expressed the TIA-1 antigen (scores 3 and 4), corresponding to 30-50% of immunoreactive cells or more than 50% of cells positive for TIA-1 expression (Fig. 1). A score of 2 was documented in 3 lesions, corresponding to 10-25% of cells positive in the infiltrate. A weak expression (score 1) was found in only 2 lesions. TIA-1 expressing cells were found in the superficial and deep dermis in all patients.

The CD45RO expression was documented with a high score (3 and 4) in 7 out of 9 lesions, and positive cells were found in areas with or without TIA-1 expression. CD56-positive cells in the infiltrate were much rarer than other cell types, therefore the scale adopted was different (Table I). The highest score of positive cells (up to 15%) was observed in two patients, and 3 out of 9 presented a weak expression. NK cells were found mainly in areas with TIA-1 expression, but also in areas lacking TIA-1 staining. Additionally, CD8-positive cells were also found in the infiltrate with an average score of 3.

Discussion

American cutaneous leishmaniasis is characterized by a localized ulceration that may require several months to heal. In the immunological response at tissue level, both subpopulations of T cells have been documented, with predominance of helper over cytotoxic phenotype in patients with the cutaneous and the mucosal form of disease [5]. In chronic lesions of CL an increased expression of inflammatory cytokines such as IL-1alpha, TNFalpha and IFNgamma and also regulatory cytokines such as IL-10 and TGFbeta has been described [9]. Although the TNFalpha and IFNgamma strongly expressed in the tissue of these patients may be related to macrophage activation and leishmania killing, an exuberant and not modulated immune response against parasite antigens may also lead to the pathology of human CL [10]. In fact, a higher level of IL-12 mRNA, a potent inducer of Th1 type cellular immune response, is associated with chronic and non-healing lesions [11]. The early events in the immune response are crucial for the understanding of the pathogenesis of leishmania infection. We have previously shown that in the very initial phase of tissue damage in CL there is a vasculitis with a large number of lymphocytes invading the vascular wall, suggesting an important role for local inflammatory mechanisms in the development of the disease [12]. We show herein that in addition to the presence of cells with cytotoxic phenotype, there is a strong expression of TIA-1, a marker of cytotoxic granules of T and NK cells [13] in the lesions of CL.

A remarkable finding was the presence of CD8⁺ cells, activated T cells and to a lesser extent NK cells in the dermal infiltrate of the lesions. Although both NK and CD8⁺ T cells have been associated with a protective mechanism in leishmaniasis [14, 15], it has also been shown that mice lacking NK cells were capable of mounting an efficient Th1 response and control cutaneous L. major infection [16]. Alternatively, the pathology observed in C57BL infected with L. major is dependent of CD8 T cells [17]. Therefore, the presence of these cells with cytolitic activity in CL argues in favor of an active participation of NK and CD8⁺ T cells in the pathogenesis of the disease.

Taken together our data indicate that both NK cells and activated CD8⁺ T are present within the lesions of CL, and that cytotoxicity is part of the immune response at tissue level. These data show that a cytotoxic activity occurs in human cutaneous leismaniasis, that may be involved in parasite killing but also in the development of the ulceration.

CONCLUSION

Acknowledgements

We are grateful to Dominique Bourchany (Lab. of Pathology, Édouard-Herriot Hospital, Lyon, France) for technical assistance.

We are grateful to Jean-François Nicolas, Sophie Bosset and Hitoshi Akiba (INERM U. 503, Lyon, France) for valuable discussions and suggestions.

Article accepted on 8/7/02

REFERENCES

1. Barral-Netto M, Machado P, Bittencourt A, Barral A. Recent Advances in the Pathophysiology and Treatment of Human Cutaneous Leishmaniasis. Curr Op Dermatol 1997; 4: 51-8.

2. Llanos-Cuentas EA, Marsden PD, Lago EL, Barreto AC, Cuba CC, Johnson WD. Human mucocutaneous leishmaniasis in Três Braços, Bahia-Brazil. An area of Leishmania braziliensis infection. II Cutaneous disease presentation and evolution. Rev Soc Bras Med Trop 1984; 17: 169-77.

3. Bittencourt AL, Barral-Netto M. Leishmaniasis. In: Doerr W, Seifert G, eds. Tropical Pathology. 2nd ed. Heidelberg: Springer, 1995: 597-651.

4. Carvalho EM, Johnson WD, Barreto E, Marsden PD, Costa JML, Reed S, Rocha H. Cell mediated immunity in American cutaneous and mucosal leishmaniasis. J Immunol 1985; 135: 4144-8.

5. Barral A, Jesus AR, Almeida RP, Carvalho EM, Barral-Netto M, Costa JML, Badaró R, Rocha H, Johnson WD. Evaluation of T-cell subsets in the lesion infiltrates of human cutaneous and mucocutaneous leishmaniasis. Parasite Immunol 1987; 9: 487-97.

6. Pirmez C, Cooper C, Paes-Oliveira M, Schubach A, Torigian VK, Modlin RL. Immunologic responsiveness in American cutaneous leishmaniasis lesions. J Immunol 1990; 145 (9): 3100-4.

7. Reed SG, Badaró R, Masur H, Carvalho EM, Lorenço R, Lisboa A, Teixeira A, Johnson WD, Jones TC. Selection of a skin test antigen for American cutaneous leishmaniasis. Am J Trop Med Hyg 1986; 35: 79-85.

8. Barral A, Almeida RP, de Jesus AR, Neto EM, Santos IA, Johnson Jr W. The relevance of characterizing leishmania from cutaneous lesions. A simple approach for isolation. Mem Inst Oswaldo Cruz 1987; 82: 579.

9. 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 (3): 837-42.

10. Ribeiro-de-Jesus A, Almeida RP, Lessa H, Bacellar O, Carvalho EM. Cytokine profile and pathology in human leishmaniasis. Braz J Med Biol Res 1998; 31: 143-8.

11. Melby PC, Andrade-Narvaez FJ, Darnell BJ, Valencia-Pacheco G. In situ expression of interleukin-10 and interleukin-12 in active human cutaneous leishmaniasis. FEMS Immunol Med Microbiol 1996; 15 (2-3): 101-7.

12. Machado P, Araújo C, da Silva AT, Almeida RP, D'Oliveira Jr A, Bittencourt A, Carvalho EM. Early cutaneous leishmaniasis: failure of early therapy in preventing the development of an ulcer. Clin Infect Dis 2002; 34(12): E69-73.

13. Anderson P, Nagler-Anderson C, O'Brien C, Levine H, Watkins S, Slayter HS, Blue ML, Schlossman SF. A monoclonal antibody reactive with a 15-kDa cytoplasmatic granule-associated protein defines a subpopulation of CD8⁺ T lymphocytes. J Immunol 1990; 144: 574-82.

14. Maasho K, Sanchez F, Schurr E, Hailu A, Akuffo H. Indications of the protective role of natural killer cells in human cutaneous leishmaniasis in an area of endemicity. Infect Immun 1998; 66: 2698-704.

15. Muller I, Kropf P, Louis JA, Milon G. Expansion of gamma interferon-producing CD8⁺ T cells following secondary infection of mice immune to Leishmania major. Infect Immun 1994; 62(6): 2575-81.

16. Satoskar AR, Stamm LM, Zhang X, Satoskar AA, Okano M, Terhorst C, David JR, Wang B. Mice lacking NK cells develop an efficient Th1 response and control cutaneous Leishmania major infection. J Immunol 1999; 162: 6747-54.

17. Belkaid Y, Von Stebut E, Mendez S, Lira R, Udey MC, Sacks DL. CD8⁺ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major. J Immunol 2002; 168 (8): 3992-4000.