On-line version ISSN 1806-4841
An. Bras. Dermatol. vol.81 no.6 Rio de Janeiro Nov./Dec. 2006
CLINICAL, EPIDEMIOLOGICAL, LABORATORY AND THERAPEUTIC INVESTIGATION
Histopathological and immunohistochemical profile of the American cutaneous leishmaniasis with emphasis on FXIIIa+ dermal dendrocytes*
Maria Luisa DuarteI; Mayra Carrijo RochaelII
IPhD and master's degree in Pathology,
Adjunct professor of Forensic Medicine and Medical Ethics, Department of Pathology,
Universidade Estadual de Ciências da Saúde de Alagoas - (UNCISAL) -
Maceio - (Al), Brazil
IIPhD in Pathology, Adjunct professor at the Department of Pathology, Coordinator of the Dermatopathology service at the Universidade Federal Fluminense (UFF) - Niteroi - Rio de Janeiro (RJ), Brazil
BACKGROUND: Mucocutaneous leishmaniasis is a parasitic infectious disease
with relevant immunological aspects.
OBJECTIVE: To study the histopathological and immunohistochemical aspects of 21 leishmaniasis tegumentary biopsies.
METHODS: Polyclonal anti-Leishmania antibody was used to confirm the presence of Leishmania amastigotes. Histopathological classification comprised I-V standard groups. The immunopathological pattern was evaluated as to the presence of FXIIIa+ dermal dendrocytes, CD1a+ Langerhans cells, CD68+ macrophages, CD20+ B lymphocytes, and CD3+ T lymphocytes. The FXIIIa+ cells were quantified and compared to specimens of normal skin obtained from unexposed areas. The other cells were counted in a semi-quantitative way. The number of FXIIIa+ cells was statistically analyzed using the Mann-Whitney test.
RESULTS: Among the histopathological groups, I and II standards prevailed. The FXIIIa+ cells were observed for different aspects and compared to normal skin, without significant statistica differences (p = 0.157). There was no relation between the amount of CD1a+, CD68+, CD20+ and CD3+ cells when compared to each other or to FXIIIa+ cells.
CONCLUSION: There was no difference between the number of FXIIIa+ cells in leishmaniasis and in normal skin. However, more studies are needed in order to understand the role of FXIIIa+ cells in leishmaniasis.
BACKGROUND: Mucocutaneous leishmaniasis is a parasitic infectious disease
with relevant immunological aspects.
Keywords: Autoimmunity; Cutaneous leishmaniasis; Dendritic cells; Factor XIIIa
American cutaneous leishmaniasis (ACL) is a parasitic, infectious but non-contagious disease caused by protozoa of several species of genus Leishmania that affects skin and mucosa.
Regardless of the Leishmania species that parasites the individual, the first affected organ is the skin. Patients are infected chronically by the parasite and can later on develop leishmaniasis.
Moros et al.1 stated that the exact mechanism by which the immune system intervenes in the destruction of the parasite is unknown, but cell immunity is considered fundamental in the resolution of the infection.
In a review of 1982, Grimaldi2 observed that the parasites of genus Leishmania induce a variety of complex responses in vertebrate hosts, carried out or modulated by their immune system. The author also pointed out that the fate of Leishmania inside the macrophages depends on particular parasite/host relations, involving not only the parasite intrinsic properties, but also the genetically determined characteristics of the host cell or those of its interactions with other immunocompetent cells.
In this study, tissue immune reaction was investigated by histopathological examination of ACL lesions and immunohistochemical features of dermal dendrocytes (DD), Langerhans cells (LC), macrophages (MO), and B and T lymphocytes, using markers FXIIIa, CD1a, CD68, CD20, and CD3, respectively, with relevance for the FXIIIa+ dermal dendrocytes. These and the Langerhans cells are considered dendritic cells (dC) with antigens, responsible for the cell immune response, and the first cells to get in contact with the invading microorganism (Leishmania).
MATERIAL AND METHODS
Twenty-one ACL biopsies were submitted to this investigation, after patients had been informed about the research and signed the informed consent form, following approval by the Research Ethics Committee.
With hematoxylin-eosin (HE) staining, the five histopathological patterns identified by Magalhães et al.3,4 for the classification of ACL were used and are represented in Table 1.
We used the streptavidin-biotin-peroxidase method. Four-micron thick histological sections were collected on previously prepared slides (Dako Silanized Slides Code No. S3003), deparaffinated in xylene and rehydrated in ethanol and water. The endogenous peroxidase activity was blocked. Antigenic retrieval was done in a 96°C water bath with citrate buffer (Dako target retrieval solution), inhibition of the specific bonds with skimmed milk (30ml TRIS + NaCl + 0.3g of bovine albumin Merck + 0.3g Molico skimmed powdered milk). After draining the milk, the primary antibodies were used at the dilutions described below, then placed in a humid chamber at room temperature for 30 minutes (incubation with specific primary antibody). The biotinylated secondary antibody from an LSAB kit was used. The development (antigen-antibody bond) was visualized with diaminobenzidine, Dako DAB+ kit, the slides counterstained with Harris' hematoxylin, and mounted with cover slips and Entellan.
In the anti-Leishmania polyclonal antibody protocol, the incubation step was modified, bringing it to a whole night in a humid chamber at room temperature, with the antibody diluted at 1/4000 in diluting solution with a Dako unspecific bond reducer. The Leishmania species used was L.(L.) amazonensis (strain MHOM/BR/73/M2269), isolated from a cutaneous leishmaniasis case from the state of Pará, classified by monoclonal antibodies and isozymes, at the Evandro Chagas Institute, in Belém, PA.
The dilution protocols for the antibodies were as follows: anti-FXIIIa, dilution 1/800; anti-CD1a, dilution 1/200; anti-CD68, dilution 1/500; anti-CD20, dilution 1/300; anti-CD3, dilution 1/600; and a special protocol for the anti-Leishmania antibody with a dilution of 1/4000.
An observation was considered as positive if at least one structure was marked in brown (color given by diaminobenzidine, the chromogen used) and was morphologically compatible with an amastigote. In this sense, the size, shape and localization of the structures inside the macrophage vacuoles were considered. The presence of marks in the cytoplasm of mononuclear inflammatory cells, mainly those with morphological characteristics of macrophages and endothelial cells, was also investigated. Such positivity patterns were respectively named "cellular" and "vascular".5,6
Analysis of FXIIIa+ dermal dendrocytes (DDFXIIIa+)
These cells were analyzed as to their localization, distribution and morphological aspect in the dermis and within the inflammatory infiltrate, using the quantitative method. They were examined under an Olympus CBA (Micronal -Brazil) optical microscope, with a 40x lens and a 10x ocular (final magnification of 400x) and adding together five sequential high-magnification fields, starting at the dermal-epidermal junction.7 In the cases in which this procedure was not possible, such as ulceration, cutting fold, etc., the next field in which such alterations were not present was counted. The result of this count was expressed by the sum of the number of cells in the five high-magnification fields, in each lesion examined.
As controls for the number of DDFXIIIa+, we used six normal skin fragments obtained during necropsy from the suprapubic region (unexposed to sun) of cadavers without any skin disease, less than 12 hours from the time of death.
Analysis of Langerhans cells, macrophages, T and B lymphocytes
After immune marking with their respective antibodies, these cells were analyzed for their presence and distribution in the epidermis and dermis, using the semi-quantitative method, with a 40x lens and a 10x ocular of the same microscope, and classified as: absent (0); small amount, up to 5 cells (+); moderate amount, from 5 to 10 cells (++); and great amount, over 10 cells (+++)8.
The amount of DDFXIIIa+ in the leishmaniasis skins was compared with that of normal skins/controls by means of the Mann-Whitney test.
To check the association of the FXIIIa+ cell count, the classification into histopathological groups, and the normal skin control group, the Kruskal-Wallis test was used.
From the 21 cases with a confirmed clinical and epidemiological diagnosis of ACL confirmed by the anti-Leishmania polyclonal antibody, the following results were obtained.
All patients had cutaneous leishmaniasis. The lesions were present predominantly in males, in the third decade of life, on the lower limbs, as shown in Table 2.
Table 3 shows the number of lesions of each patient, the time of progression and the histopathological group to which they belonged. According to the histopathological analysis, the predominant histopathological groups were I and II.
Immunohistochemical test result
Demonstration of Leishmania and of parasite antigens in the ACL lesions was represented by the presence of amastigote forms and by "cell marking". As control material, lesions with amastigote forms evidenced by HE and Giemsa staining and a bone marrow biopsy with a great amount of control amastigote forms were used.
The morphology of the DDFXIIIa+ was fusiform, their localization at the level of the papillary dermis, distributed horizontally under the dermalepidermal junction, around the capillaries or superficial vessels in association with mast cells, monocytes and macrophages, and diffusely in the mid and deep reticular dermis, in loose connective tissue, around the skin appendages, sweat glands and pilosebaceous follicles, as seen in Figure 1.
In the normal skin control group (Figure 2), the results showed a maximum number of DDFXIIIa+ equal to 60 and a minimum number of 42.
Statistical analysis results
Analysis of the data regarding the variable "amount of DDFXIIIa+" obtained in the samples of normal and leishmaniasis skin, using the Mann- Whitney test (U = 36; p-value = 0.157), showed no evidence of a statistically significant difference.
Comparing the amount of FXIIIa+ cells in ACL and the classification into the five histopathological groups, it was demonstrated that the amount of FXIIIa+ cells in group II was significantly larger than in group I (Mann-Whitney test: U = 12.5; p-value = 0.036).
A comparison between the predominant groups I and II and the control skin regarding the number of DDFXIIIa+, using the Kruskal-Wallis test, showed no difference (p = 0.185).
In Leishmania infection, the first target cells of the parasites are dendritic cells - Langerhans cells and dermal dendrocytes - and macrophages.9 In view of the relevant aspects of the DDFXIIIa+, still under study according to the literature, we chose to perform this research using this marker, comparing it in cutaneous leishmaniasis and in normal control skin, in order to observe the behavior of this group of dendritic cells. Other cells also cluster to granulomas, such as lymphocytes, plasmocytes, eosinophils and fibroblasts.10
An analysis of the relation between the progression time of lesions (time of disease) and the histopathological group or immunohistochemical findings was not possible, due to the lack of the first information in 11 cases.
In this study, histiolymphoplasmocytic infiltration was present in all histopathological groups, in agreement with the reports of Magalhães et al.11, appearing to play the role of an unspecific cell response to the presence of a strange agent. Lymphocytes were found in all lesions. Nuclear dust, represented by fragments of neutrophils and eosinophils, was present in most cases examined. In this nuclear dust phase, no plasmocytes were present, as opposed to older lesions. The histiolymphoplasmocytic infiltrate may also play a more aggressive rather than simply reactive role. This assumption should be proven by other studies, using monoclonal antibodies against the various subpopulations of lymphocytes, allowing identification within the tissues of the types of lymphocytes which take part in self-aggression reactions.
In ACL lesions, the FXIIIa+ cells were present at locations similar to those described in the literature. 12-17 These results suggest that the basic pathogenic mechanism in granuloma formation is similar, regardless of its origin, in either sarcoidosis, tuberculosis or leishmaniasis.
DD hypertrophy in inflammatory and granulomatous skin conditions with a clinical and histological aspect of delayed hypersensitivity reaction suggests that they may play the role of skin immune response mediator cells. The localization of these cells, underlying the epidermis, perivascular areas and in the inflammatory cell infiltrate, is compatible with the role of antigen-presenting cells (APC).16,18-20
The examined lesions were from areas exposed to sunlight, and our findings are in contrast with the results of Hoyo et al.13, who reported an increase in the number of these cells, as well as of several cytokines which are present in ACL, stimulated by the ultraviolet rays. Comparing the number of these cells in ACL with the number of cells in normal skin of the suprapubic region, that is unexposed to sunlight and in which the number of DD should be decreased, no statistically significant evidence was found.
Comparing the amount of FXIIIa+ cells in the normal skin control group and in the predominant histopathological groups of ACL (I and II), there was no statistically significant difference. This finding should be analyzed in further studies, correlating the inflammatory cell infiltrate and time of lesion.
Pagliari & Sotto14 investigated the DDFXIIIa+, analyzing their morphological aspects and quantifying them in skin with paracoccidioidomycosis and in normal skin. In the skin with the fungus, the FXIIIa+ cells were observed at locations similar to those found in this study, and the reduced number of these cells in the normal skin was used as control.
In a study on DDFXIIIa+ in 47 ACL cases, Sotto16 also found hypertrophy and an increased number of these cells, as compared to the normal skin control group (five specimens from the thoracic region - an area of little sun exposure - and five from the knee region - an area of relatively greater sun exposure). This DDFXIIIa+ population did not present any difference when compared with the granulomatous or unspecific tissue response. The author further observed that these cells have the capacity of internalizing amastigote forms of Leishmania. Based on these results, he concluded that the DDFXIIIa+ take part in the pathogenesis mechanisms of ACL, probably also acting as APC's, conclusions which are similar to those of a research on chromoblastomycosis. 16,18-20
Natah et al.18 did not rule out that a subgroup of FXIIIa+ cells might represent epithelial Langerhans cells, which do not yet express the CD1a marker, that is determinant in their path to the epithelium.
The locations of the LC observed in this research are consistent with the reports of the literature, since, being located in the epidermis, they would be infected by Leishmanias and would then, some cells in the superficial and deep dermis and amidst the inflammatory infiltrate present in the dermis, be on the migration route towards areas of T cells.21 After skin infection by Leishmania, the LC are responsible for transporting the parasites from the epidermis to the dermis.22 On the other hand, these cells have the unique ability of ingesting the parasites from the infection site in the skin and taking them to T-cell areas drained by the lymph nodes.22
Cerio et al.23 suggested that the dermal dC (DDFXIIIa+) could be a pluripotent cell population capable, under several circumstances, of acting as macrophages, as APC and probably differentiating themselves into Langerhans cells and migrating to the epidermis.
Macrophages, immunomarked with the anti- CD68 antibody, were the cells observed in the greatest amounts as compared to others, both in the epidermis, isolated as exocytoses or in microabscesses, and in the dermis, amidst the inflammatory infiltrate, in focal or diffuse concentrations. Semi-quantitative analysis of these cells did not show any significant quantitative correlation between the FXIIIa+, CD1a and CD20 or CD3 cells.
The B lymphocytes immunomarked with the anti-CD20 antibody varied in marking intensity and semi-quantitative analysis in relation to the histopathological groups. They were not observed in the epidermis, and were present in nests or clusters in the dermis amidst the inflammatory infiltrate. We observed that there is a reverse semi-quantitative relation between the CD20 and the CD3 cells, i.e., the greater the amount of CD20 cells, the smaller or more moderate the amount of CD3 cells, and viceversa.
In relation to the B lymphocytes, the T lymphocytes immunomarked with anti-CD3 antibody were always in a location different from those, or amidst them.
The presence of dermal dendrocytes is related, according to the literature, to antigen presentation to T lymphocytes.24,25 The analysis of T and B lymphocytes, although done in a global manner, had the objective of assessing their relation with dendrocytes.
The similarity of the number of DDFXIIIa+ found in this study with the amount found in normal skin could be a consequence of the alterations observed in the DDFXIIIa+ population, resulting from factors present in the skin environment, related to characteristics of the immune response mediated by ACL cells. It is speculated whether the finding of a non-increased number of DDFXIIIa+, as in other research16,19,20, could result from the migration of LC/DD FXIIIa+ from skin to lymph nodes. A greater number of ACL cases and a comparison of the amount of DDFXIIIa+ with normal skin from sunexposed and unexposed areas are believed to be needed to achieve a statistically significant result.
1)The histopathological groups proposed by Magalhães et al.3,4 that predominate were I (exudative cell reaction) and II (exudative and necrotic reaction), with a similar number of cases. The number of DDFXIIIa+ was statistically higher in group II than in group I.
2) The number of FXIIIa+ dermal dendrocytes showed no statistically significant difference in the ACL lesions when compared to that of normal controls.
3)The CD1a+, CD68+, CD20+ and CD3+ cells did not show any relevant relation, either between each other or with regard to the FXIIIa+ cells.
The authors thank Dr. Cláudia Gomes and Dr. Carlos Corbett, from the Pathology Laboratory for Infectious Diseases of the Faculdade de Medicina da Universidade de Sao Paulo, for the donation of the anti-Leishmania antibody.
Thanks are also due to the director of the Hospital Escola Dr. Hélvio Farias Auto, Dr. Marcelo Constant, and the team of medical collaborators: Dr. Simone D'Andrada Tenório Silva, Dr. Gilberto Salustiano de Lima, Dr. Rosa Rita Gomes dos Santos Vieira, Dr. Mac Dowell Fortes Silveira Cavalcanti and Dr. Mirela Bernardina Borges.
Thanks also to Dr. José Euclides de Oliveira, president of Fapeal during the period of this research.
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Maria Luisa Duarte
Conjunto Jardim Petrópolis II-E, nº 55, Santa Amélia
57063-210 - Maceió - Alagoas
Tel: (82) 3314-1415, (82) 9972-5571
No conflict of interest was declared.
Received on May 03 of 2006.
Approved by the Consultive Council and accepted for publication on Nov. 22 of 2006
* Work done at Department of Pathology, Universidade Estadual de Ciências da Saúde de Alagoas - (UNCISAL), Maceio - (Al), Brazil.