1201-1206 10/10/08 13:52 Page 1201 ONCOLOGY REPORTS 20: 1201-1206, 2008 Neurotrophins and their receptors in human lingual tonsil: An immunohistochemical analysis MARCO ARTICO1, ELENA BRONZETTI2, LAURA M. FELICI2, VALENTINA ALICINO1, BRUNELLA IONTA1, BENEDETTO BRONZETTI3, GIUSEPPE MAGLIULO1, CLAUDIA GRANDE2, LORIS ZAMAI4, GUIDO PASQUANTONIO5 and MARCO DE VINCENTIIS1 1Giorgio Ferreri Department of Otorhinolaryngology, Audiology, and Phoniatry, University of Rome ‘La Sapienza’, Piazzale A. Moro 5, I-00185 Rome; 2Department of Cardiovascular, Respiratory and Morphological Sciences, University of Rome ‘La Sapienza’, Via A. Borelli 50, I-00161 Rome; 3Nuovo Presidio Ospedaliero Cutroni-Zodda, Barcellona Pozzo di Gotto, Messina; 4Institute of Histology and Laboratory Analysis and Center of Cytometry and Cytomorphology, University of Urbino, Urbino; 5Department of Dental Sciences, University of Tor Vergata, Rome, Italy Received May 14, 2008; Accepted June 13, 2008 DOI: 10.3892/or_00000130 Abstract. Lymphoid organs are supplied by many nerve Introduction endings associated with different kinds of cells and macro- phages. The role of this innervation on the release of locally The nasopharyngeal tonsil (adenoids) and palatine and lingual active molecules is still unclear. Lingual tonsils belong to tonsils constitute the major part of Waldeyer's Ring, with the Waldeyer's Ring, in close association with palatine tonsils tubal tonsils and lateral pharyngeal bands as less prominent and nasopharyngeal (adenoids) tonsils, thus constituting part components. All parts of Waldeyer's Ring are located to of NALT (nasal-associated lymphoid tissue) together with perform regional immune functions because they are exposed the tubal tonsils and lateral pharyngeal bands. In this study, to both airborne and alimentary antigens. Although tonsils we focused our attention on the expression of some neuro- and adenoids play an important immune-inductive role as trophins (NTs) and their high- and low-affinity receptors in components of MALT (mucosa-associated lymphoid tissue), human lingual tonsils. Light immunohistochemistry showed these structures also present similarities with lymph nodes that human tonsillar samples were generally positive for all and may participate as effector organs of local systemic-type the NTs investigated (NGF, BDNF, NT-3, NT-4) and their adaptive immunity (1). Tonsils and adenoids contain four receptors (TrKA, TrKB, TrKC and p75) with some different specialized lymphoid compartments (2), namely the reticular expression levels. NGF and TrKC were strongly expressed in crypt epithelium, the extra-follicular area (T cell-area), the macrophages, but weakly in lymphocytes. However, BDNF mantle zones of lymphoid follicles and the follicular germinal and TrKB was highly expressed in lymphocytes and weaker centres (B cell-inductive compartment). in macrophages. The low-affinity receptor for NGF, p75, Both the nervous and immune systems are functionally was mainly moderately expressed in the analysed samples. related and the lymphoid tissues of the human mucosa are These results suggest the presence of a pattern of neurotrophin considerably innervated (3). As a part of the immune system, innervation in the human lingual tonsil which may play a role tonsils are supposed to closely interact with the nervous in sustaining inflammatory conditions and in modulating a system by an extensive network, involving many signalling close interaction between the nervous system and the different molecules. In this context, there is still a scarcity of infor- immune cellular subtypes. mation regarding neurotrophin expression and innervation of the human lingual tonsil. Neurotrophins (NTs), also known as neurotrophic factors, represent a family of dimeric proteins _________________________________________ working as polypeptidic growth factors which include NGF, BDNF, NT-3, NT-4/5 and NT-6, the latter being apparently specific for fish (4). Biological actions of NTs are mediated Correspondence to: Professor Elena Bronzetti, Department of by the binding with two families of membrane receptors, the Cardiovascular, Respiratory and Morphological Sciences, University of Rome ‘La Sapienza’, Via Alfonso Borelli 50, I-00161 Rome, high-affinity tyrosine kinase (TrK) and the low-affinity Italy p75NTR (5,6). The TrK family includes TrKA, TrKB and E-mail: [email protected] TrKC receptors, whereas p75NTR belongs to the trans- membrane molecules serving as receptors for tumor necrosis Key words: human lingual tonsil, immunohistochemistry, neuro- factor and cytokines (6). TrKA is specifically activated by trophins, receptors NGF, whereas TrKB and TrKC are primarily receptors for BDNF and NT-3 respectively (7,8). NTs are involved in vertebrate neuronal cell development, differentiation, survival and functional activities (4,9). NTs are also involved in the 1201-1206 10/10/08 13:52 Page 1202 1202 ARTICO et al: HUMAN LINGUAL TONSIL INNERVATION modulation of adult central nervous system functions and p75 precursor of human origin and is not cross-reactive with organization, as well as in the vegetative innervation of other growth factor receptors. Incubation with primary anti- several organs (7,9). Moreover, detailed studies have revealed bodies was performed overnight at 4˚C at a final concentration significant actions of neurotrophins in a wide variety of tissues of 2-5 μg/ml. Optimal antisera dilutions and incubation outside the nervous system, especially in the immune system times were assessed in a series of preliminary experiments. (10-14). In particular, it is thought that immune tissue is After exposure to the primary antibodies, slides were rinsed capable of concentrating NGF, which in turn may modulate twice in phosphate-buffer and incubated (1 h and 30 min at the level of innervation via the sympathetic nervous system room temperature) with the appropriate secondary antibody (15,16). conjugated to horseradish peroxidase (HRP) (final dilution In order to gain information on this matter, in the present 1:100). The secondary antibody-HRP linked against rabbit report we studied the innervation and the expression of NTs immunoglobulins was purchased from Boehringer (Boehringer and their receptors in different cellular subtypes (lympho- Mannheim GmbH, Mannheim, Germany), while secondary cytes, macrophages, endothelium of blood vessels) of human antibodies-HRP linked against mouse and goat immuno- lingual tonsils. globulins were from Sigma (Sigma Chemicals Co, St. Louis, MO, USA). After a further wash with phosphate- Materials and methods buffer, slides were treated with 0.05% 3,3-diaminobenzidine and 0.1% H2O2. Finally, sections were counterstained with Patients. Small specimens of human lingual tonsil tissue Mayer's hematoxylin and observed by using a light micro- were surgically removed during a biopsy (after written scope. To block endogenous peroxidase activity, slides were informed consent of the patients) in the course of operations pre-treated with 3% H2O2, whereas the non-specific binding performed for palatine tonsil removal from ten patients and of immunoglobulins was prevented by adding 3% fetal calf subjected to immunohistochemical analysis. Experiments serum to the incubation medium. Negative control experiments were performed in compliance with the Italian laws and were done: i) by omitting the primary antibody; ii) by guidelines concerning the informed consent of the patients. substituting the primary antibody with equivalent amount of non-specific immunoglobulins; and iii) by pre-incubating Immunohistochemistry. The following molecules were the primary antibody with the specific blocking peptide investigated: nerve growth factor (NGF), brain derived (antigen/antibody = 5 according to customer's instructions). neurotrophic factor (BDNF), NT-3, NT-4, TrKA, TrKB, In preliminary experiments, immunohistochemistry was also TrKC and p75NTR. performed on frozen sections of human tonsil tissue. We found For light microscope immunohistochemical analysis, no differences in the intensity or distribution of immuno- small fragments of tonsil tissue were washed in PBS, fixed staining using the two types of sections, but we preferred in 10% formalin and embedded in paraffin according to a paraffin-embedded material because microanatomical details standard procedure. Serial 10 μm thick sections were cut were better preserved. The intensity of immune reaction using a rotatory microtome, mounted on gelatine-coated was assessed microdensitometrically by an IAS 2000 image slides and processed for immunohistochemistry. To study the analyzer (Delta Sistemi, Rome, Italy) connected via a TV immunolocalization of neurotrophins and their receptors, the camera to the microscope. The system was calibrated taking antibodies we used were: i) rabbit anti-NGF polyclonal anti- as zero the background obtained in sections exposed to non- body (Santa Cruz Biotechnology, CA, USA), which display immune serum. Ten 100 μm2 areas were delineated in each <1% cross-reactivity against recombinant human NT-3, NT-4 section by a measuring diaphragm. Quantitative data of the and BDNF; ii) rabbit anti-BDNF polyclonal antibody (Santa intensity of the immune staining were analyzed statistically Cruz), which recognizes the amino-terminus of mouse BDNF by analysis of variance (ANOVA) followed by Duncan's and do not cross-react with NT-3 or NGF; iii) rabbit anti- multiple range test as a post hoc test. NT3 polyclonal antibody (Santa Cruz Biotechnology), raised against the amino-terminus of mouse