Jorge Lobo' s immunopathology: cell composition of the inflammatory infiltrate and cytokine quantification in mononuclear cell supernatant and serum

THESIS

Jorge Lobo' s immunopathology: cell composition of the inflammatory infiltrate and cytokine quantification in mononuclear cell supernatant and serum

F. R. Vilani-Moreno

^{Correspondence}

THESIS. F. R. Vilani-Moreno submitted this thesis for her Doctorate in Tropical Diseases at Botucatu School of Medicine, São Paulo State University, UNESP, Botucatu, São Paulo, Brasil, 2002.

Advisor: Professor Diltor Vladimir Araujo Opramolla

ABSTRACT

Jorge Lobo's disease is a cutaneous or subcutaneous mycosis of chronic evolution caused by Lacazia loboi fungus. It occurs predominantly in the Amazon region, affecting mainly rural workers living in close contact with the soil and plants, such as rubber plantation workers. There are few studies on the immunopathological aspects of this disease. So far, cellular components of the granuloma induced by the Lacazia loboi fungus and the role of immune response in granuloma genesis and development are not known. This study aimed to identify the mononuclear cell population in cutaneous lesions and to quantify some macrophage and lymphocyte cytokines in cell culture supernatants and serum. Participants in this study were 15 Jorge Lobo's patients from the Acre State and 15 healthy adults (controls). Blood samples were drawn for serum mononuclear cell isolation. Monocytes were cultivated for 24 hours with or without LPS (10 mg/ml) and L. loboi (5 cells:1 fungus). Lymphocytes were cultivated for 48 hours with or without PHA (8 mg/ml) and L. loboi (5 cells:1 fungus). Supernatants were collected after predetermined times and stored at -70ºC until use. IL-1b, TNF-a, and IL-6 were quantified by ELISA in monocyte culture supernatants and sera. IL-2 and INF-g (Th1 profile) and IL-4 and Il-10 (Th2 profile) were quantified by ELISA in lymphocyte culture supernatants and sera. Histological sections from patients' cutaneous lesions were stained with hematoxylin-eosin and methenamine silver. The following mononuclear cells were identified by immunohistochemical methods: lymphocytes T (CD3⁺); lymphocytes T helper (CD4⁺); lymphocytes T suppressor /cytotoxic (CD8⁺); lymphocytes B (CD20⁺); plasma cells (CD79⁺); NK cells (CD57⁺); histiocytes (CD68⁺); and Langerhans and interdigitating reticular cells (S100⁺). Results showed that the inflammatory infiltrate was mainly composed of histiocytes and CGM and a large number of fungi, many with morphological features of non-viable cells. The number of lymphocytes was low to moderate and neutrophils were rarely found. This mycosis histopathological picture is characteristic of foreign body granulomas. Cell frequency in the inflammatory infiltrate was: histiocytes CD68⁺ (lymphocytes T CD4⁺ > lymphocytes T CD8⁺) > NK cells CD57⁺ > plasma cells CD79⁺ > lymphocytes B CD20⁺ = Langerhans and interdigitating cells S100⁺. Lymphocytes were present close to histiocytes and CGM, forming small foci or grouped around vessels; most were from the helper sub-population (CD4⁺), the ratio CD4⁺:CD8⁺ being approximately 3:2. NK cells were frequently found in the lesion; the 3^rd type was identified, being seen near the histiocytes. The number of plasma cells was higher than lymphocytes B, being found near the lymphocytes T and around vessels. Histopathology of patients with the localized form of the disease (9 patients) and non-localized (6 patients) showed similar features in relation to cell type and distribution. Cytokine quantification from culture supernatants showed higher production of IL-4 and IL-6 and lower levels of IL-2 in patients than in controls. Production of IL-b, TNF-a, IL-10, and INF-g was similar for patients and controls. There was no significant difference in serum cytokine quantification. Mononuclear cells of patients with the non-localized form of the disease produced higher INF-g levels than those with the localized form. These results allow us to suggest that Jorge Lobo's patients show changes in cytokine profile, represented by the predominance of Th2 profile. Further studies are needed to evaluate the in situ role of cytokines in the cell-fungus interaction and possible mechanism involved in L. loboi lysis for a better understanding of Jorge Lobo's disease pathogenesis.

Key words: Jorge Lobo' s disease, Lacazia loboi, cytokines, peripheral blood mononuclear cells, immunopathology, immunohistochemistry.

Publication Dates

• Publication in this collection
  24 Sept 2004
• Date of issue
  2004