CD8+ T cells in situ in different clinical forms of human cutaneous leishmaniasis

INTRODUCTION

Leishmaniasis, a disease caused by a protozoan of the genus Leishmania, affects millions of individuals in many regions of the world. Infections by Leishmania braziliensis, the most prevalent species in Brazil, lead to lesions in the skin or mucosa that become cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Cutaneous leishmaniasis lesions start as small papules in the presence or absence of lymphadenopathy, and they progress into one or more skin ulcers. This localized skin ulceration is characterized by a chronic inflammatory response involving lymphocytes, plasma cells and macrophages, with and an eventual granulomatous reaction and necrosis^1,2.

The tissue response in the initial disease manifestation, including early ulcers (≤ 20 days of lesion appearance) and papules from disseminated leishmaniasis (DL), has not been well studied. DL is an emerging form of leishmaniasis that corresponds to 1.9% of all CL cases; it is characterized by the appearance of multiple pleomorphic lesions in more than two noncontiguous areas of the body, starting from a primary ulcer^2–5. The mechanisms involved in DL have not been elucidated. It has been suggested that parasite, host and environmental factors may contribute to the dissemination of the parasite throughout the body⁵.

Cluster of differentiation 8 (CD8)+ T cells are essential in the response against viruses and malignant cells, but few studies report the role of these cells in the defense against Leishmania parasites^6–8. Although the presence of cluster of differentiation 4 (CD4)+ T cells within the inflammatory infiltrate in leishmaniasis lesions has already been shown, cytotoxic T cells in the lesions of CL patients⁶ have also been reported.

The role of CD8+ T cells remains unclear, and contradictory results have been obtained for Leishmaniainfection; these cells may contribute to the killing of Leishmania or to the immunopathology of this parasite. A significant proportion of CD8+ cells in human leishmaniasis tissue fragments has been reported⁹. The CD8+ lymphocytes could participate in parasite growth control through different mechanisms. These cells have been associated with cure after treatment through interferon gamma (IFN-γ production, and they can also interact directly with Leishmania-infected macrophages by killing them through cytotoxic activity via granzyme granules^10–12. Thus, CD8+ T lymphocytes expressing granzyme appear to be important in the immunopathology of leishmaniasis but may also be responsible for the characteristic lesions of CL. Characterization of the cell composition and the localization of granzyme B in all types of cutaneous leishmaniasis lesions, including early events in the immune response, may help to elucidate the mechanism of tissue damage.

METHODS

RESULTS

The clinical profile of the 30 patients is shown in Table 1, and the clinical characteristics of different lesions are shown in Figure 1. Twenty-one men and 9 women were enrolled, with a mean age ± standard deviation (SD) of 31.52 ± 16.4 years (ages ranged from 9 to 76 years). The duration of lesions ranged from 25 to 60 days for L-CL (34 ± 10 days), 15 to 20 days for E-CL (17 ± 2 days), 20 to 90 days for U-DL (43 ± 20 days) and 8 to 90 days for P-DL (30 ± 25 days). Lesion size varied from 56 to 750mm² for L-CL, 9 to 200mm² for E-CL and 56 to 460mm² for U-DL. P-DL lesions were not measured, and the median number of lesions for the 10 patients with DL was 30 (range, 2-200 lesions) for papular lesions and 3 (range, 1-15) for ulcerated lesions. Cutaneous leishmaniasis patients presented only 1 lesion (range, 1-2).

FIGURE 1 – Typical lesions caused by different clinical forms of cutaneous leishmaniasis. A: Early cutaneous leishmaniasis lesion. B: Late cutaneous leishmaniasis lesion. C: Papule lesion, typical non-ulcerated lesion from disseminated leishmaniasis. 

Histopathological examination of tissue fragments from ulcerated or papular lesions revealed chronic inflammatory reactions in the tissue fragment sections from all of the clinical forms. In all stages of CL, inflammation was characterized by the presence of lymphocytes, plasmocytes and macrophages. Other general histopathological patterns were also found in tissue fragments, such as perivascular and papillary edema (45%), necrosis (43%), granuloma (43%), giant cells (30%), rare neutrophils (18%) and pigmentary incontinence (28%).

The localization of cells expressing CD8 and granzyme B in the skin revealed that these cells are mostly in the papillary dermis, next to the epidermis, though the quantity of granzyme B+ cells was lower (Figure 2); the co-localization of the two types of cells was not analyzed. The majority of L-CL lesions (6/10) and U-DL lesions (6/10) were grade 3 (high frequency) for CD8+ frequency in the papillary dermis. Among the recent lesions (E-CL and P-DL), the intensity of stained cells had a greater variation, but a majority of the tissue fragments presented higher numbers of cells in the papillary dermis (Table 2). The granzyme B+ cell frequency was classified as grade 2 in the majority of the groups. Only 4 tissue fragments in the CD8+ T cell analysis and 2 in the granzyme B+ analysis had a greater grade in the reticular dermis than the papillary dermis. Cells also appeared in a homogenous distribution (10 to 40% of cases per group).

FIGURE 2 – Frequency of CD8+ cells in the papillary dermis: A: Tissue fragment graded as low frequency. B: Tissue fragment graded as medium frequency. C: Tissue fragment graded as high frequency. Frequency of granzyme B+ cells in the papillary dermis. D: Tissue fragment graded as low frequency. E: Tissue fragment graded as medium frequency. F: Tissue fragment graded as high frequency. All images have the same magnification (400x). 

Comparing the inflammatory infiltrate percentage between groups, P-DL lesions had the lowest percentage (median=21%; range 6-38%). A higher percentage of inflammation was present in L-CL (median=37%; range 21-52%) and U-DL (median=34%; range 20-56%) patients (Figure 3A).

FIGURE 3 – A: Comparison of the inflammation percentage in different lesions from cutaneous leishmaniasis. B: Comparison of the numbers of CD8+ T cells in different lesions from cutaneous leishmaniasis. C: Comparison of the numbers of granzyme B+ cells in different lesions from cutaneous leishmaniasis. Median ± standard error is represented with bars. The Kruskal-Wallis test was performed. L-CL: late cutaneous lesions; E-CL; early cutaneous lesions; U-DL: primary ulcers of DL; P-DL: secondary papular lesions of DL. 

Analyses of the frequencies of CD8+ and granzyme B+ cells were performed using ImageJ software, which allows manual counting of cells.

CD8 expression, similar to the infiltrate extension, was increased in late ulcerated lesions compared to recent lesions (papules and lesions present < 20 days), which had low absolute numbers of CD8+ lymphocytes (Figure 3B).

To determine the contribution of CD8+ cells in the inflammatory infiltrate, the correlation of these cells and the inflammation percentage was analyzed for each group. Only E-CL (R=0.7; p=0.02) and P-DL (R=0.79; p=0.005) lesions exhibited a correlation between the number of CD8+ T cells and greater inflammation (not shown).

The papular lesions from DL had a higher frequency of granzyme B+ cells than E-CL lesions (p=0.0302) and a frequency similar to those of late and DL ulcers (Figure 3C). Interestingly, only E-CL lesions had a positive correlation between granzyme B+ cells and the inflammatory infiltrate (R=0.7; p=0.0216). The analysis of late (R=-0.29; p<0.05) and DL ulcers (R=-0.22; p<0.05) revealed that the higher the inflammation, the lower the amount of granzyme B+ cells (data not shown).

DISCUSSION

Cutaneous leishmaniasis is the most frequent form of leishmaniasis due to L. braziliensis infection. It is clinically characterized by the presence of single or multiple ulcers with a well-delimited and raised border that can persist for months or heal spontaneously. DL, similar to CL, presents initially with an ulcer and then disseminates throughout the body with multiple acneiform and papular secondary lesions⁵.

CD8+ and granzyme B+ lymphocytes were more frequent in the papillary dermis. Histopathological analysis of tissue fragments showed the presence of spongiosis. The aggregation of both cells in the papillary dermis, sometimes also in the epidermis, seems to lead to injury in the basal layer and may contribute to ulcer formation. Recently, it has been suggested that the movement of T cells through the dermis and epidermis and their permanence in one layer or another correlates with the effector or memory phase of the immune response¹³. This finding suggests another mechanism for these cells, which together with cytotoxic activity, may contribute to leishmaniasis immunopathology.

In our study, chronic inflammation was observed in all four types of lesions. DL-P and E-CL together showed less inflammatory infiltrate than L-CL and U-DL (although statistical analysis showed no significance). This suggests that inflammation increases as the lesion progresses. These results are in agreement with those of another study showing that a higher intensity of inflammation is seen in L-CL lesions than in ulcers from CL in a very initial form (not yet ulcerated)¹⁴.

Recent studies have demonstrated an increased frequency of CD8+ T cells over CD4+ T cells in ulcerated lesions as well as correlations between the frequencies of CD8+ T cells and CD8+ T cells that express granzyme and the intensity of the inflammation, which indicate a role for these cells in the pathogenesis of CL^14–16. Santos et al⁸. have also shown the participation of CD8+ granzyme B+ T cells in tissue damage in CL caused by L. braziliensis, data that reinforce the association of the intensity of necrosis and the percentage of these cells⁸.

CD8+ lymphocytes with cytotoxic function seem to execute a cytolytic activity that may be important for tissue destruction with ulcer formation, as is seen in ML, where the same CD8+ T cells seem to be involved in disease exacerbation¹⁷.

Vieira et al¹⁸. showed that both localized CL and papular DL lesions presented a higher frequency of CD8+ T cells than CD4+ T cells in inflammatory infiltrates, suggesting that CD8+ cells participate directly in the immune response against parasites, principally by IFN-γ production, which induces macrophage activation¹⁸. However, CD4+ T cells appear to be the main source of IFN-γ and CD8+ T cells have a principal function of cytotoxicity, contributing to lesion formation⁸. In experimental models, CD8+ T cells have already been shown to be necessary for a protective response and also for the immunopathological development of CL¹⁷.

The evaluation of the presence of cytotoxic activity in inflammatory infiltrates revealed that lesions of less than 20 days had lesser quantities of cells expressing granzyme B than late ulcers and ulcers from DL. This result agrees with a previous study that showed granzyme expression was associated with lesion progression¹⁴. Interestingly, papular lesions from DL showed higher numbers of granzyme B+ cells than early ulcers. The association of DL with mucosal disease due to the rapid dissemination of lesions throughout the body and mucosa and the association of cytotoxic CD8+ T cells to tissue damage in ML have previously been reported^5,19. Furthermore, granzyme B does not seem to participate in parasite growth control⁸. The presence of granzyme B+ cells in DL papular lesions is indicative of their role in pathology. Granzyme B seems to participate in lesion progression in E-CL, as the expression of granzyme B and the inflammation percentage show simultaneous increases in E-CL.

In summary, CD8+ T cells and granzyme B+ cells are present in the inflammatory infiltrates of CL and DL. Further studies are necessary to elucidate the contribution of these cells to the killing or dissemination of the parasite in Leishmania infections. Their cytotoxic function and specific localization may be responsible for tissue destruction and may explain the mechanism of ulceration. Due to the scarcity of patients with the DL form who simultaneously present papules and ulcers, a low number of patients was recruited. Further characterization of the inflammatory reaction in situ in the two clinical forms and the four-lesion spectrum of human leishmaniasis may help elucidate the role of CD8+ cells and their cytotoxic activity in the immunopathogenesis of this disease.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.