EXPERIMENTAL AND THERAPEUTIC MEDICINE 17: 2053-2060, 2019 B‑cell activating factor receptor expression is associated with germinal center B‑cell maintenance FRANCISCO JOSUÉ CARRILLO-BALLESTEROS1, EDITH OREGON-ROMERO1, RAMON ANTONIO FRANCO-TOPETE2, LUIS HUMBERTO GOVEA-CAMACHO3, ALVARO CRUZ1, JOSÉ FRANCISCO MUÑOZ-VALLE1, FELIPE JESÚS BUSTOS-RODRÍGUEZ2, ANA LAURA PEREIRA-SUÁREZ4 and CLAUDIA AZUCENA PALAFOX-SÁNCHEZ1 1Research Institute of Biomedical Sciences, Department of Medical Clinics; 2Department of Microbiology and Pathology, University Center for Health Sciences, University of Guadalajara; 3Department of Otorhinolaryngology, West National Medical Center, Mexican Institute of Social Security; 4Department of Physiology, University Center for Health Sciences, University of Guadalajara, Guadalajara, Jalisco 44340, México Received April 26, 2018; Accepted November 23, 2018 DOI: 10.3892/etm.2019.7172 Abstract. B-cell activating factor (BAFF) is a major cytokine was observed inside the follicle, mainly in the dark zone. that regulates B-cell survival, maturation and differentia- The present results indicate that BAFF is implicated in the tion through its binding with its receptors: BAFF receptor maintenance of GCs. BAFF-R overexpression in the MZ, (BAFF-R), transmembrane activator and cyclophilin ligand co-localizated with BAFF, suggests that these proteins consti- interactor (TACI) and B-cell maturation antigen (BCMA). tute the principal pathway for the maintenance of the naïve These receptors have been demonstrated to be involved in B-cell population. Furthermore, TACI and BCMA have a role tertiary lymphoid structure formation; however, their role in the GC, where processes of B-cell selection, proliferation in germinal centers (GCs) has remained elusive. The aim of and differentiation into immunoglobulin-secreting plasma the present study was to determine the expression profiles cells occur. of BAFF and its receptors in secondary lymphoid tissues. Tonsils resected due to chronic tonsillitis were used as Introduction lymphoid tissues. To confirm the presence of GCs identified based on their typical structure, CD21 antibody staining was B-cell activating factor (BAFF), also known as B-lymphocyte employed. The expression of BAFF, BAFF-R, TACI and stimulator, is a cytokine belonging to the tumor necrosis factor BCMA was assessed by immunohistochemistry. BAFF was ligand superfamily, existing either as a type 2 transmembrane highly expressed in all regions of the follicle, but the highest protein or in its soluble form (1). BAFF and its homolog A BAFF expression was detected in the mantle zone (MZ). A PRoliferation-Inducing Ligand (APRIL) have an important high expression of BAFF-R was observed on lymphocytes in role in B-cell maturation, survival, selection and differen- the MZ in comparison with the other regions (~80%; P<0.05), tiation (2). The major sources for BAFF cytokine are several which was co-localizated with BAFF (r=0.646; P<0.001), in innate immune cell types, including monocytes, macro- the MZ. TACI and BCMA exhibited similar expression among phages (3), neutrophils (4) and dendritic cells in response the different zones of the GCs, and co-localization with BAFF to Toll-like receptors, type I and II interferons (IFNs) (5), interleukin-10 (6) and granulocyte colony-stimulating factor expression (7). Furthermore, fibroblast-like cells (8) and astrocytes (9) are also able to produce BAFF, and so are B cells (10) and T cells (11) in secondary lymphoid tissues, Correspondence to: Dr Claudia Azucena Palafox-Sánchez, including the spleen, lymph nodes and tonsils. Increased Research Institute of Biomedical Sciences, Department of levels of BAFF have been associated with autoimmune Medical Clinics, University Center for Health Sciences, University diseases (12), including systemic lupus erythematosus (13), of Guadalajara, 950 Sierra Mojada, Edificio Q, Guadalajara, Jalisco 44340, México Sjögren's syndrome (14) and rheumatoid arthritis (15), as well E-mail: [email protected] as with multiple myeloma (16), non-Hodgkin's lymphoma (17), B-lineage lymphomas (18) and Hodgkin's lymphoma (19). Key words: B-cell activating factor, B-cell activating factor A total of 3 BAFF-binding receptors have been estab- receptor, transmembrane activator and cyclophilin ligand interactor, lished, namely BAFF receptor (BAFF‑R), which is specific B-cell maturation antigen, expression, germinal centers for BAFF, and two others that are shared with the homo- logue APRIL, transmembrane activator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen 2054 CARRILLO-BALLESTEROS et al: BAFF AND BAFF RECEPTOR EXPRESSION IN GERMINAL CENTERS (BCMA) (2). BAFF-R is essential for B-cell maturation at zone (MZ), consisting of a lymphocyte fringe that surrounds early transitional stages, particularly from T1 to T2 B cells, the GC and exhibits an asymmetric distribution regarding the as well as for B-cell survival (20). TACI is important for central axis to the follicle; and the interfollicular zone (IZ) T-cell-independent responses of B cells, immunoglobulin (Ig) as the remaining area excluding the GCs. Finally, the pres- class switch and it is considered to be a negative regulator of ence of GCs was immunohistochemically confirmed by CD21 B-cell homeostasis (21-23). Whereas, BCMA promotes plasma labeling and as described below. cells survival (24). All three BAFF receptors, BAFF-R, TACI and BCMA, are expressed on B cells; TACI is additionally Immunohistochemical staining. Once tissue sections were expressed on activated T cells, while BAFF-R is also expressed re-hydrated, antigen retrieval was accomplished in one step on follicular helper T cells (TFH ) (21,25). with a water steamer at 96 degrees for 30 min, while slides were Germinal centers (GCs) are important structures in submerged in a cup with 10 mM sodium citrate buffer (pH=6) secondary lymphoid tissues where T cell-dependent immune for later staining with rat monoclonal antibody to BAFF (cat. responses occur, and BAFF has been identified to be impli- no. ab16081; dilution, 1:100), rabbit monoclonal antibodies cated in their formation from lymphoid tissues in murine to TACI (cat. no. ab79023; dilution, 1:200) and BCMA (cat. models (26,27), as well as in tertiary lymphoid structures in no. ab5972; dilution, 1:400), or 1 mM EDTA buffer (pH=9) for autoimmune diseases (28,29). A study reported that BAFF and later staining with mouse monoclonal antibodies to CD21 (cat. APRIL are associated with artery tertiary lymphoid organs in no. ab9492; dilution, 1:10) and BAFF-R (cat. no. ab16232; dilu- giant-cell arteritis, and that BAFF is highly expressed within tion, 1:500) from Abcam (Cambridge, UK). After cooling, slides infiltrating, vascular and endothelial cells, suggesting their were treated with a peroxidase blocking solution composed of involvement in ectopic GCs; however, BAFF receptors were 3% hydrogen peroxide and 10% methanol solution for 15 min not analyzed (28). Likewise, in Hodgkin (30) and non-Hodgkin at room temperature. The slides were then blocked with serum lymphoma (31), the BAFF/BAFF-R pathway has an important depending on the secondary antibody source: Horse serum for role and has been proposed as a predictor of lymphoma devel- CD21 and BAFF-R; rabbit serum for BAFF; and goat serum for opment in primary Sjögren's syndrome (32). However, only TACI and BCMA antibodies (VECTASTAIN Elite ABC-HRP few studies have assessed the expression of BAFF receptors Kit; Vector Laboratories, Inc., Burlingame, CA, USA). The in non-neoplastic lymphoid tissues in humans (30,33,34). samples were incubated with the primary antibodies overnight Therefore, the distribution and expression profiles of BAFF at 4˚C. Detection was performed with the respective provided and their receptors, BAFF-R, TACI and BCMA, in secondary secondary biotinylated antibody (1:200), for 30 min at room follicles from tonsil tissues were analyzed in the present study. temperature and streptavidin (VECTASTAIN Elite ABC-HRP Kit; Vector Laboratories, Inc.) using diaminobenzidine (DAB) Materials and methods until a red-brown color developed. Finally, counterstaining was performed using Harris' hematoxylin. Patients. Tonsils were obtained from nine patients submitted Immunohistochemistry slide staining for CD3 and CD20 to a routine tonsillectomy performed at the Department was performed using the automated Ventana Benchmark of Otorhinolaryngology of the Mexican Institute of Social ULTRA (Roche Diagnostics, Basel, Switzerland) following the Security (Guadalajara, Mexico). The mean age of the patients manufacturer's recommended protocols for paraffin‑embedded was 12 years (range, 4-41 years), and the cohort comprised sections. The primary antibodies anti-CD3 (2GV6) rabbit 7 females and 2 males. The clinical diagnosis for the majority of monoclonal antibody (cat. no. 790-4341) and anti-CD20 the patients was chronic tonsillitis and grade III or IV tonsillar (L26) mouse monoclonal antibody [cat. no. 760-2531; Ventana hypertrophy. All of the patients, or their guardians in the case of iVIEW PATHWAY (Roche Diagnostics) were obtained predi- minors, provided written informed consent in accordance with luted by Roche Diagnostics, and were optimized for use on the Declaration of Helsinki and the current national guidelines Ventana staining platforms. Detection was performed with the and regulations. The ethics committee of the University Center iVIEW DAB detection kit (Roche Diagnostics)]. for Health Sciences, University of Guadalajara (Guadalajara,