Inmunohistochemical detection of mastocytes in tissue from patients with actinic prurigo

Background Actinic prurigo (AP) is a type of photodermatosis, the pathophysiology of which has not been determined. AP has been suggested to be a hypersensitivity reaction to the presence of eosinophils and the local production of IgE. Material and Methods Descriptive study, using paraffin blocks of tissue that have been diagnosed with AP from the Dermopathology department, Hospital General Dr. Manuel Gea González. In 66 blocks from 63 patients, eosinophils were identified by hematoxylin and eosin staining, and mastocytes were labeled by immunohistochemistry. Three random microphotographs (40x) were used, and cell counts were calculated as the mean count in the 3 microphotographs. Results Forty cases (63.5%) were female, and 23 (36.5%) were male. The mean age was 26.49 ±14.09 years; regarding the evolution time of the disease, the average was 11.93 years ±11.39. In 38 of 63 cases (60%), the lip, skin, and conjunctiva were affected clinically. In 22 of 63 cases (34%), AP cheilitis was the sole manifestation, and in 4 of 63 cases (6%), there were lesions in the skin and conjunctiva. The mean eosinophil count was 9 per case, the average number of mastocytes/field was 28.48 (range 0 to 66) Kruskal-Wallis p=0.001. Conclusions There are elements in AP that mediate the reaction of hypersensitivity type IV b, necessitating the identification of triggering factors. Key words:Actinic prurigo, eosinophil, hypersensitivity IV b, IgE, mastocytes.

Between 45% and 62% of AP patients present with conjunctivitis and pseudopterygium formation (7,8,10). It evolves chronically, with partial remission (13). By histopathology, epithelial acanthosis, spongiosis and exulceration, and an abundance of eosinophils are seen in the dermis, and there is an infiltrate of nodular lymphoplasmocytes that can form lymphoid follicles, patognomonic image of AP´s chelitis (14,15). Dendritic follicular cells and plasma IgE-producing cells are also present, and IL-2 is produced by B and T lymphocytes (16). Elevated serum IgE has been reported in 10% to 50% of patients with AP and peripheral eosinophilia (17). Ultraviolet radiation in AP, (18)(19)(20)(21)(22) effecting local lymphocyte proliferation through the secretion of soluble compounds by keratinocytes, with no change in serum leucocyte levels (23)(24)(25). Mastocytes participate in inflammatory and allergic reactions. They are activated primarily through the highaffinity IgE receptor (FcεRI), which can bind the IgEantigen complex to initiate a complex transduction of signals that culminate in the secretion of proinflammatory mediators and cytokines. Other mechanisms include anaphylatoxins that generated by activation of the complement pathway, bacteria through Toll-like receptors, the release of TNF-α by mastocytes that amplify a stimulus, and T lymphocyte stimulation (26)(27)(28)(29). The presence of IgE-expressing eosinophils and mastocytes implicates a hypersensitivity reaction in the pathophysiology of AP (30). Generally, hypersensitivity reactions occur on exposure to an antigen through the activation of effector cells. There are 4 types of hypersensitivity reactions according the Gell and Coombs classification (31,32). Hypersensitivity reaction type IV, or retarded reaction, is mediated specifically by T lymphocytes, which produce cytokines that activate various antibodies. The IVa subtype corresponds to a Th1 response, which stimulates macrophages by secreting IFNy in which complementfixing antibodies and complement isotypes are co stimulants of proinflammatory and CD8 cells. Conversely, the subtype IVb initiates a Th2 immune response in which lymphocytes T secrete IL-4, IL-5, and IL-13, which, through B cells that produce IgE and IgG4, stimulate eosinophils and mastocytes and inactivate macrophages. The high production of IL-5 causes eosinophilic inflammation, a characteristic of type IV hypersensitivity reactions (33)(34)(35)(36)(37). The aim of this study was to examine the presence of mast cells and eosinophils in AP to increase our understanding of its pathophysiology.

Material and Methods
This descriptive study we performed in the Dermopathology Department of Hospital General Dr. Manuel Gea González. The study samples were obtained from a pool of 40 tissue samples in skin with prurigo through a search of paraffin blocks using the clinical and histological criteria of AP. From this database, we drew the demographics and characteristics of the patients whose tissues were included (sex, age, clinical localization, family records, and evolution time of the disease). Using H&E slides for all cases, the diagnosis of AP was confirmed, and the presence of eosinophils and mastocytes was examined. The tissue block were, then slice and processed by immunohistochemistry. The tissues were deparaffinized, rehydrated, and subjected to antigenic recovery with 0.1% sodium citrate, pH 6.2, endogen peroxidase was inactivated (0.9% hydrogen peroxide), and the slides were incubated with 1% bovine serum albumin to block nonspecific sites. Washes were performed with PBS. Tissues were incubated for 45 minutes with polyclonal anti-CD 117 (Dako1:50), after which anti-mouse/antirabbit secondary antibody was applied. Streptavidin-peroxidase complex was added to the slides for 30 minutes, and the reactions were visualized with diaminobenzidine (Dako) and counterstained with Hills´ hematoxylin. The positive controls comprised 8 paraffin blocks of biopsies from various patients-2 healthy mucosal samples and 1 of each of the following: mucocele, venous lake, fibrous hyperplasia, traumatic cheilitis, sclerotic lichen, and granulomatous cheilitis. Eosinophil counts and immunohistochemistry evaluation were performed semi-quantitative, based on positivity to anti-CD 117 (Dako1:50) in the membrane and cytoplasm of mastocytes. Three 40x microphotographs were selected randomly for H&E staining for eosinophils and immunohistochemistry for mastocytes, all of them with and cells of 5x4, which include a surface of 0.3mm 2 . Counting of the cells present in each of the cells was performed and it was added respectively so the number of the total positive cells was able to be known in the whole image. Average counts were calculated over the 3 microphotographs.

Results
Sixty-six blocks of tissue with a diagnosis of AP or AP cheilitis were selected. They had been diagnosed between 1995 and 2006 and contained enough biological material to be included in the study. Forty cases (63.5%) were female, and 23 (36.5%) were male. The mean age was 26.49±14.09 (range 7-63 years). The mean time in which the AP developed was 11.93 years ±11.39; time from diagnosis of AP ranged from 6 months to 45 years. Twenty-three patients (36.5%) had any immediate family member with a history of AP. In 38 of 63 (60%) cases, the lip, skin, and conjuntiva were clinically affected; in 22 cases (34%), AP cheilitis was the sole manifestation (only used for the study tissue of AP located in skin). Four cases (6%) had lesions in the skin and conjunctiva but not in the lip. In the 66 tissue samples that were diagnosed with AP and stained with H&E, the average number of eosinophils was 9 per case (Fig. 1). The average number of mastocytes in the mucosa of patients with AP was 28.48/ field, ranging from 0 to 66 (Table 1), versus 9.75/field in the control groups (range 5 to 16) and 7 mastocytes/field in healthy mucosa (Kruskal-Wallis p=0.001). Figure 2 shows representative immunohistochemistry staining of a randomly selected field. There were no significant differences in evolution time of disease or mean number of mastocytes per field. By calculation of Pearson´s correlation coefficient, we did not observe any linear relationship between mastocytes number and the evolution time of the disease ( Table 2) or between eosinophil number and the number of positive mastocytes.

Discussion
Actinic prurigo occurs predominantly in women.
Although the first signs of which develop in childhood, patients are usually diagnosed late. At the time that the clinical history is taken, it is common to record data on any relatives with the disease (7,(12)(13)(14)(15)38). The characteristics of our study sample-a predominance of female patients, early age of onset (mean 14.35 years), and 36.3% of patients with a family history of PA-are consistent with these rates. The number of mastocytes in normal skin increases from 44 to 75 mastocytes/mm 2 and has an irregular distribution throughout the body, concentrating in the acral areas (39). In contrast, our AP patients had 189 mastocytes/mm 2 at an enlargement of 40x and with a surface of 0.3 mm 2 .
The density of mastocytes per field was greater in mucosa of patients with AP versus healthy mucosa and other lesions, including those with an inflammatory origin, implicating these cells in the pathophysiology of AP.
In contrast, there were no significant differences in the quantity of mastocytes or time of evolution. Previous studies have reported the predominance of a Th2 response in AP, leading to the hypothesis that AP was a type I hypersensitivity reaction. In recent studies, however, when taking into account the presence of eosinophils, local production of IgE by plasma cells, increase in mastocytes number, detection of B and T cells, and participation of IL-2, an updated model suggests that the pathophysiology of AP is based on a type IV hypersensitivity reaction-specifically, subtype b-although subtype a can not be dismissed completely, based on these findings (16,24,36,(39)(40)(41). Future studies should attempt to identify an antigen that, with exposure to ultraviolet light, triggers an inflammatory reaction and leads to the clinical manifestations of AP.

Conclusions
The higher density of mastocytes in samples from patients with actinic prurigo confirms the presence of all of the principal elements that mediate the reaction of late hypersensitivity type IV b, prompting us to hypothesize about the existence of a triggering allergen. More studies are required to resolve the pathophysiology of AP-specifically with regard to IL-2, IL-4, IL-5, and NK cells-and determine if and which apoptotic pathways are involved.