Histopathological and ultrastructural studies on ...

Comp Clin Pathol DOI 10.1007/s00580-013-1793-6

ORIGINAL ARTICLE

Histopathological and ultrastructural studies on human cutaneous leishmaniasis Refaat Ali Eid & Mokhtar Taha & Yasmin Omar El-Amir

Received: 13 March 2013 / Accepted: 31 July 2013 # Springer-Verlag London 2013

Abstract Leishmania, a genus of intracellular protozoan parasites of macrophages, is the etiologic agent of cutaneous and visceral disease in man. In our study, localized cutaneous infections with leishmania were studied by light and transmission electron microscopy in 16 patients at phases ranging from onset to a progressive disease. In early infections, epidermal changes could be detected as deep hemorrhagic ulcer characterized by focal massive necrosis of the epidermal layers. Spongiotic vesicles in the epidermis were prominent containing the amastigotes. The dermal changes appeared in the form of diffuse inflammatory infiltrate predominantly composed of macrophages, epithelioid cells, lymphocytes, mast cells, and few plasma cells and eosinophils. Macrophages laden with the parasites were seen dissociating the striated muscle and the collagen bundles which showed degenerative and necrotic changes. In late stages of the disease, multiple granulomas formed predominantly of macrophages containing promastigotes and amastigotes, giant cells, epithelioid cells, and some mast cells were seen in the dermis. Some macrophages appeared vacuolated and loaded with the parasite. The dermal vasculature showed congestion, swelling of the endothelial cells, and fibrinoid necrosis of the wall. Some congested blood vessels demonstrated margination and diapedesis of inflammatory cells. By transmission electron microscopy, intact and degenerated amastigotes were seen phagocytosed inside the macrophagal parasitophorus vacuoles. Erythrophagocytosis and the reaction of other inflammatory cellular components were also described. These results clarified the lesions of leishmania invasion into the skin of the affected patients and its R. A. Eid Department of Pathology, College of Medicine, King Khalid University, PO 3236, Abha, Kingdom of Saudi Arabia e-mail: [email protected] M. Taha (*) : Y. O. El-Amir Department of Pathology & Clinical Pathology, Faculty of Veterinary Medicine, Assiut University, PO 71526, Assiut, Egypt e-mail: [email protected]

defensive mechanism. Moreover, the host macrophagal–parasite relationship was shown on ultrastructural level. Keywords Electron microscopy . Histopathology . Leishmaniasis . Skin

Introduction Leishmaniasis are diseases caused by protozoa of the genera Leishmania. As a result of the multiplicity of agents and of insect vectors and animal reservoirs, this disease occurs in different clinical modalities. In the New World, the etiologic agents of cutaneous and/or mucocutaneous leishmaniasis are a number of Leishmania species (Jose et al. 1993). Among various parasites of the genus Leishmania, Leishmania amazonesis is the causative agent of cutaneous leishmaniasis (Almeida et al. 1996) characterized by the appearance of chronic lesions and disseminated through the skin (Guerra et al. 2010). The severity and the clinical form of the illness are directly related with the parasite as well as with the genetic and immunological factors of the host (Kane and Mosser 2000). Extreme cases of cutaneous infection are characterized by widespread dissemination of highly parasitized macrophages and unresponsiveness (diffuse cutaneous leishmaniasis) or by extensive destruction of nasopharyngeal tissues and hyperresponsiveness to scanty organisms (mucocutaneous leishmaniasis) (Pearson et al. 1983). More commonly, cutaneous leishmaniasis appears as a localized lesion, usually self-healing or responding well to treatment. Recovery is often followed by strong specific immunity and delayed hypersensitivity (Garnham and Humphrey 1969). The course of leishmaniasis depends primarily on macrophage–parasite interaction (Veress et al. 1981; Zaar et al. 1982; Ridley and Ridley 1983). Ridley and Wells (1986) reported that elimination of leishmania is represented by two-stranded immunohistologic response, intracellular killing

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through macrophage activation or killing by macrophage lysis. On ultrastructural level, Bittencourt et al. (1990) and Ramos-Vara et al. (1996) have documented the relationship of the parasite to the inflammatory cellular response. The aim of this paper is to describe the histopathological changes of the observed cutaneous lesions both in early and late infections of the affected patients. In addition, the role of the macrophagal and other inflammatory cellular reaction and their relationship to the parasite are evaluated by transmission electron microscopy.

Materials and methods Patients Sixteen patients with cutaneous leishmaniasis, being of different ages and sex, were diagnosed on the basis of clinical features and history. The samples were collected throughout 2012 from the Department of Dermatology, Assir Central Hospital, Abha, KSA. The clinical data of early and late infection were described in a previous article (data not shown). None of these patients received treatment before biopsies were taken after written consensus. Processing of biopsy material Four millimeter punch biopsies were taken under local anesthesia from the edge of ulcers or nodules and fixed in different fixatives. The biopsy material was divided into two parts, one of which was fixed in 10 % formalin and then processed and embedded in paraffin. Five micron sections were cut and stained with hematoxylin–eosin for light microscopy. The other part was cut into small pieces and fixed in cold 2 % paraformaldehyde and 2 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The tissues were then washed with 0.1 M cacodylate buffer and post-fixed in 2 % osmium tetra oxide for 2 h. After dehydration in different concentrations of alcohol, the specimens were embedded in Epon 812. One micron semi-thin sections were cut with a glass knife on an LKB ultratome and stained with toluidine blue. Ultra-thin sections were cut with a diamond knife, stained with lead citrate and uranyl acetate, and examined under a JEM 1200 EX electron microscope (JEOL, Japan).

Results Light microscopic findings Epidermal changes Light microscopic examination of H and E stained sections revealed prominent focal hemorrhagic ulceration associated with coagulation necrosis of the epidermal layers in some examined cases (Fig. 1a). Along with these changes, intraepidermal spongiotic vesicles were observed in the epidermis. These vesicles characterized by lytic necrosis

and mononuclear cellular infiltrate contained the protozoal organism (Fig. 1b). Free amastigotes were also found between the epidermal layers and in the vesicle. These organisms were ovoid with rounded nucleus surrounded by a clear halo. Neither acanthosis nor hyperplasia of the epidermis was observed. Dermal changes In all examined cases, the small arteries within and around the inflammation revealed congestion, swelling of endothelial cells, subintimal fibrinoid changes (Fig. 1c) as well as heavy perivascular mononuclear cellular infiltrate (Fig. 1d). The presence of congested blood vessels with marginalization and diapedesis of inflammatory cells were a prominent finding (Fig. 2a). Diffuse lymphohistiocytic inflammatory reaction involving the dermis and subcutis was a common finding in the examined cases. The diffuse dermal infiltrate composed of macrophages, multinucleate giant cells, plasma cells, mast cells, and rare eosinophils (Fig. 1d). Large amount of vacuolated macrophages were seen containing many amastigotes (Fig. 2b), while free proamastigotes and amastigotes were detected in the extracellular matrix (Fig. 2c). Moreover, degenerative and necrotic changes were observed in the dermal collagen of these cases (Fig. 2d). A granulomatous pattern of dermal lesions was demonstrated in the deep dermis of some cases. These granulomas composed of macrophages, epithelioid cells, few giant multinucleated cells, lymphocytes, and mast cells (Fig. 3a). Few leishmania amastigotes were identified in the cytoplasm of macrophages. Some granulomas were surrounded by delicate fibroblastic connective tissue and showed newly proliferating small capillaries (Fig. 3b). The observed granulomatous reaction was associated either with moderate, mixed inflammatory infiltrate with few leishmania amastigotes in the macrophages or an extensive inflammation composed of vacuolated macrophages containing large number of the parasites. Electron microscopic observations Epidermal changes The most prominent finding was the presence of mononuclear cells loaded by the amastigotes and containing melanin granules in their cytoplasm (Fig. 4a). Amastigotes demonstrating mitotic division inside these mononuclear cells were also seen (Fig. 4b). Dermal changes In the dermis, ultrastructural finding of macrophages phagocytosing leishmania parasites either intracytoplasmic or in a parasitophorous vacuole was an important finding (Fig. 4c). Some amastigotes appeared undergoing degeneration process inside these vacuoles (Fig. 4d), while others were intact. The intact amastigotes seen were rounded to oval and of almost uniform size. They were surrounded by two layers of membranes and contained a

Comp Clin Pathol Fig. 1 a Showing epidermal hemorrhagic ulcer and necrosis. b Intraepidermal spongiotic vesicles (stars), phagocytosed amastigotes by macrophage cells in the vesicles or surrounded by clear halo in addition to presence of free parasite between the keratinocytes, c Showing congestion of dermal vessels, endothelial damage, and mild inflammatory cellular reaction. d Showing vascular damage (arrow) and heavy lymphohistiocytic infiltrate (star)

rounded nucleus with a small nucleolus. The flagellum, flagellar pocket, kinetoplast, vacuoles, and electron-dense granules could be distinguished (Fig. 5a). Ultrastructural evidence of erythrophagocytosis was seen where viable extracellular amastigotes had close contact to the membrane of RBCs (Fig. 5b) and monocytes and blood platelets (Fig. 5c). Free amastigotes were also present in the vicinity of other inflammatory cellular reaction of mast cell and eosinophils (Fig. 5d).

Discussion In the present study, light microscopic examination of the epidermis revealed prominent hemorrhagic ulceration associated with coagulation necrosis of the epidermal layers in some examined cases. Similar lesions were described by Pimenta et al. (1987) and De Moura et al. (2005). This may be of ischemic origin resulting from the observed vascular damage of the dermal vasculature and have been considered by Ridley et al. (1980) as one histologic correlate of delayed type hypersensitivity. The associated marginalization and diapedesis of inflammatory cells were also shown by Guerra et al. (2010). Along with these changes, intraepidermal spongiotic vesicles

containing the phagocytosed protozoal organism (amastigotes) were a characteristic finding in the epidermis. We observed amastigotes associated with lymphocytes and other mononuclear cells in spongiotic vesicles within the mid-epidermis. Mononuclear cells that may be macrophages were seen in close contact with amastigotes. The close contact of these antigen-presenting cells with leishmania suggests their capacity to recognize the parasite and facilitate its destruction (Bhutto et al. 1992). Pearson et al. (1983) and Bhutto et al. (1992) recorded similar findings in their studies where amastigotes were detected either free or engulfed by macrophage cells. Amastigotes were also detected in the epidermis between the keratinocytes. The histological studies of Kurban et al. (1966)) stated that keratinocytes appear to play an active part in cellular defense against the parasites. The epidermis is a true participant of immune response through keratinocyte activation as antigen-presenting cell and/or secretion of cytokines and adhesion molecules that modulate local immune response (Luger et al. 1981; Schmitt et al. 1982; Kupper et al. 1986; Tapia et al. 1989; Fine 1990; Oxholm et al. 1991; Boyce 1994). By electron microscopy, melanin pigments were demonstrated in the cytoplasm of some infected macrophages as

Comp Clin Pathol Fig. 2 a Showing necrosis of the vessel wall, marginalization and diapedesis (arrows). b Showing phagocytosed amastigotes by macrophage cells (arrow). c Showing heavy proamastigotes (arrow) and amastigotes in the extracellular matrix. d Showing degeneration and necrosis of dermal collagen with interspersed cellular reaction

observed by Bhutto et al. (1992). The purpose of these melanin granules is unknown, but leukocytes carrying similar bodies have been observed by Wassermann (1965)) in the blood of healthy humans, reptiles, and amphibians. The author added that melanin-containing macrophages are also present within the epithelium of certain groups of fish. Diffuse lymphohistiocytic inflammatory reaction involving the dermis and subcutis was a common finding in the examined cases. The diffuse dermal infiltrate composed mainly of Fig. 3 a Showing dermal granulomas consisting from macrophages, lymphocytes, epitheliod cells, and giant cells. b Granuloma surrounded by delicate fibroblastic connective tissue and newly formed blood capillaries (arrows)

macrophagal reaction, lymphocytes, plasma cells, mast cells, and eosinophils. There have been several ultrastructural studies documenting the identification of leishmania parasitism of inflammatory cells (Pearson and Steigbigel 1981; Ridley and Wells 1986; Pimenta et al. 1987: Bittencourt et al. 1990). By electron microscopic examination, our studies demonstrated the presence of intact and degenerated amastigotes within the macrophages both either inside the parasitophorus vacuoles or free in the cytoplasm. Similar results have also

Comp Clin Pathol Fig. 4 Transmission electron micrographs showing: a infected mononuclear cell with amastigotes (L). Note melanin (arrows) and collagen fibers (F) around amastigotes. m mitochondria. b An amastigote demonstrate mitotic division (arrow) in a macrophage. m damaged mitochondria and N nucleus. c Healthy and damaged phagocytosed amastigotes (L) inside vacuole (V) of macrophage cell (Ma). d Two macrophages (Ma) containing phagocytosed amastigotes undergoing degeneration. L in vacuoles (V). N macrophage nucleus

Fig. 5 Transmission electron micrographs showing: a macrophage showing detailed structure of the parasite (L) inside vacuole. Note debris of degenerated amastigotes (arrows). b Erythrocytes (R) in contact with amastigotes (L). Note dissolving membranes (arrow) between R and L. c Blood platelets (Pb), monocytes (Mo), erythrocytes (R), and amastigotes (L). d Mast cell (MC), eosinophil (Es), and amastigotes (L)

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been presented by many authors who confirmed the multiplicity of mechanisms involved in the destruction of leishmania during the course of a cutaneous lesion (Sandbank 1976; Zaar et al. 1982; El-On et al. 1980; Ridley and Ridley 1983). The authors mentioned that intracellular killing of amastigotes by activated macrophages produced the greatest diversity of macrophage cytologic form. When parasites were numerous and viable, they were situated in cytoplasm and interspersed with small vesicles. These might be delivery vesicles, pinosomes, lysosomes, or due to secretory products of amastigotes. Later, the vesicles disappeared and nonvesiculated macrophages with parasites confined to a phagolysosome increased in number. Once externalized, viable organisms were ingested by undifferentiated macrophages, which became activated to kill the parasites. Ridley et al. (1983) and Nacy et al. (1984) stated that when the parasites are numerous and viable, they result in activation of macrophages and intracellular killing of their parasitic load due to the generation of reactive oxygen species, superoxide, and hydrogen peroxide or to effector lymphokine mediation. In addition, free proamastigotes and amastigotes were seen in the extracellular matrix. De Almeida et al. (2003) suggested that the presence of free parasites in the extracellular matrix leads to activation of resident and inflammatory cells through recognition of parasite surface molecules by receptors present in these cells. Peters et al. (2008) believed that recruitment of these cells participate in the process of tissue repair even in the absence of the parasite. Lymphocytes were seen to be in close contact with parasitized macrophages. This macrophage–lymphocyte combination represents the cooperation of two cells against the parasites. Lymphocytes (probably cytotoxic T cells) have a major role in the immune response to leishmania infection. They may recognize the parasitized macrophage that may finally result in destruction of host macrophage and thereby liberate the parasite (Schurr et al. 1987) or help the macrophage kill the invading leishmania (Mauel et al. 1978). McElrath et al. (1987) has suggested that T cells provide lymphokines that can activate the host macrophage to destroy the parasites intracellularly or that T cells play a cytotoxic role, killing the infected macrophages and helping to destroy the liberated extracellular parasites. Williams and Kupper (1996) and Fuhlbrigge and Kupper (2004) considered that these cells play a crucial role in the initial immune response to the pathogen through the release of cytokines, chemokines, and growth factors. The presence of mast cells in the dermis of some examined cases is related to their direct participation in the initial immune response through the production of several inflammatory mediators (Saha et al. 2004). Eosinophilic reaction was seen in the dermis and between the keratinocytes. Eosinophils recruitment and activation through direct and indirect mechanisms have been described in bacterial and parasitic infections (D’Avila et al. 2007; Driss

et al. 2009). Eosinophils may participate in the process of killing the parasites suggesting an immunomodulatory and protective role in infections (Saha et al. 2004; Akuthota et al. 2008; Blanchard and Rothenberg 2009) and serves as donors of extracellular peroxidase as suggested by Grimaldi and Moriearty (1981)) and Pimenta et al. (1987). Degenerative and necrotic changes were observed in the dermal collagen accompanied by perivascular diffuse inflammatory cellular infiltrate. This collagen changes may be due to ischemia produced by the observed vasculitis. Andrade et al. (1984) described coagulative necrosis of skin lesion in BALB/ c mice infected with L. (L.) amazonensis and considered to be of ischemic origin. Necrosis has been claimed as one of the effective mechanisms eliminating the leishmania (Ridley and Ridley 1986). On the contrary, Gutierrez et al. (1991) related necrosis to the presence of amastigotes and to the necessity for higher total dose of antimonial treatment using extensive human leishmaniasis material. Amastigote morphology was similar to that described for other leishmania species by many authors (Schurr et al. 1987; Barral-Netto et al. 1987: Armijos et al. 1990). A granulomatous pattern of dermal lesions was demonstrated in the deep dermis of some cases where leishmania amastigotes were identified either intact or degenerated in the cytoplasm of macrophages. Granulomatous reaction associated with moderate, mixed inflammatory infiltrate with few leishmania amastigotes in the macrophages to an extensive inflammation composed of vacuolated macrophages containing large number of parasites were described by many authors (Veress et al. 1981; Belosevic et al. 1989: Rojas et al. 1993). Multinucleated giant cells found in granuloma have been related to the effect of interferon-γ (Weinberg et al. 1985; Murray et al. 1987; Belosevic et al. 1989), and interferon-γ is known to be effective in activating macrophages to kill leishmaniasis intracellularly (Murray et al. 1983). The present investigations also showed ultrastructural evidence of erythrophagocytosis where the amastigotes appeared in close attachment to the membrane of RBCs. Similar observations were earlier described in cases of human kala-azar by Woodruff (1973) and Veress et al. (1977). Decker-Jackson and Honigberg (1978) have presented data showing that leishmania parasites have antigens cross-reacting with components of blood cells. This could be the basis of autoimmune destruction of the red blood cells leading to hemolytic anemia, a feature of human visceral leishmaniasis (Woodruff 1973). Additional mechanisms have also been proposed for the anemia in kalaazar. These include dyserythropoiesis and ineffective erythropoiesis attributed to the action of toxins generated by the organisms and hematinic deficiencies (Kasili 1980). This study showed that patients with cutaneous leishmaniasis demonstrated epidermal ulcerative and spongiotic lesions accompanied by macrophagal cellular infiltrate. In the dermis, a characteristic granulomatous lesions associated with chronic

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cellular reaction predominated. The host defensive phagocytic mechanism toward the parasite was expressed by mononuclear cellular macrophages that served to destroy the parasite intracellularly. Other immunogenic cells were also evident that played an active role in controlling the infection. The vascular changes are worthy to mention as it contributed in the pathogenesis of the disease.

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