Methods
Twenty-eight skin biopsies were obtained from CSU patients older than 18 years, defined by the occurrence of urticaria for more than 6 weeks, without any triggering factor or induced urticaria, according to the criteria established by the EAACI/GA²LEN/EDF/WAO guideline for the definition, classification, diagnosis and management of urticaria, refractory to treatment with second-generation antihistamine drugs (H1 blockers), at optimized doses (four times the doses specified in the leaflet), for 30 days.1 These biopsies were performed on the day of the lesion appearance with more than 6h of onset, according to the follow-up protocol practiced in our outpatient clinic, which includes biopsy of urticaria lesions in patients when they do not achieve control their disease with fourfold doses of second-generation anti-H1 drugs.
After signing the informed consent form, the demographic, clinical, and laboratory data were collected from the patients medical records and the paraffin blocks of the biopsies were retrieved from the database of the Pathology Service to which they were sent. Paraffin blocks of normal skin were obtained from nine individuals undergoing plastic surgery with no history of urticaria or known allergic disease, stored in the same Pathology database.
The project was approved by the Research Ethics Committee of the ABC School of Medicine (Number: 1.545.607 of May 11, 2016).
The clinical and laboratory data collected were: gender, age, time of urticaria evolution, results of complementary tests, such as ASST, CRP values (mg/dL), serum levels of D-dimers (FEU/mL), baseline count in peripheral blood, thorough complete blood count, and serum levels of total IgE (KU/mL).
The paraffin blocks were sectioned onto slides previously prepared with 3% amino-propyltriethoxysilane adhesive solution (Sigma Chemical Co., St. Louis, MO, USA, Product no. A3648) at 2%. The histological sections were dewaxed in two xylol baths, for 20 and 10 min, respectively, at room temperature. Thereafter, the specimens were hydrated in decreasing ethanol solutions (100%, 95%, and 70%) and washed in tap water for 5 min. Subsequently, the slides were incubated in hydrogen peroxide 3% and treated to expose the antigenic sites at 95 °C for 20 min in Target Retrieval Solution pH 9.0 (Product no. S2367, Dako Cytomation, Carpinteria, CA, USA) or Target Retrieval Solution pH 6.0 (Product no. S1699, Dako Cytomation), according to the previous protocols. Next, the slides were washed in tap and distilled water for 5 min each and submerged in buffered saline solution (PBS) at pH 7.4.
Nonspecific tissue proteins were then blocked in a 10% solution of skimmed milk (Molico, Nestle®,Brazil) for 30 min at room temperature. The slides were incubated with the primary antibodies as illustrated in Figure 1. All antibodies were diluted in bovine serum albumin (BSA) fraction V 1% plus 0.1% sodium azide in phosphate-buffered saline (PBS) buffer at pH 7.4, overnight at 4 °C. After washing twice in PBS at pH 7.4 for 5 min each, the slides were incubated with a post primary antibody polymer detection system (Reveal kit, Spring), for 30 min at 37ºC. Thereafter, the slides were washed twice in PBS buffer at pH 7.4, for 5 min each, and incubated with the polymer system (Reveal kit, Spring) in a humid chamber for 30 min at room temperature. The binding sites were rapidly developed with a chromogenic solution of 0.05% diaminobenzidine (3,3-diaminobenzidine, SIGMA Chemical Co., St Louis, MO, USA, Product no. D5637) and 1.2 mL of 3% hydrogen peroxide. The slides were then washed in running water for 5 min, counterstained with Harris hematoxylin for 20 s, and further washed again in running water and dehydrated in ethanol.
The slides were mounted with Permount resin (FISHER Scientific, Fair Lawn, NJ/USA, product no. SP15-100). To verify the presence of the M1 and M2 population of macrophages via immunohistochemistry reactions, the cells were then morphologically classified into macrophages and were divided into two surface receptor markers (cytoplasmic membrane), CD206 mannose receptor and CD163 called hemoglobin scavenger, folate and IL10, and two transcriptional (nuclear) markers, pSTAT-1 and CMAF, to characterize the subpopulations of macrophages (Table 1).7,11
Table 1. Immunohistochemical markers and dilutions.