©1991 S. KargerAG. Basel 0301-1569/91 /0536-0349S2.75/0
ORL 1991;53:349-356
Interdigitating Cells in the Péritumoral Infiltrate of Laryngeal Carcinomas: An Immunocytochemical and Ultrastructural Study L. Ruccib, D. Bam3, O. Gallob. R. Arbi Riccardi M.B. Borghi Cirri3, O. Fini-Storchib a ,
■'Department of Human Anatomy and Histology, Section of Histology, hOtorhinolaryngologic Clinic, University of Florence, Florence, Italy
Key Words. Interdigitating cells • Laryngeal carcinomas • Immunocytochemistry • Ultrastructure Abstract. Interdigitating cells (IDCs) have been found in the peritumoral infiltrate of 18 patients with squamous cell carcinoma of the larynx. These cells have a dendritic shape and are characterized by the expression of S-100 protein and CD la antigens. By electron microscopy, these cells are seen to establish intimate contacts with the apposed lymphocytes, which sometimes show signs of functional activation and proliferation. These findings indi cate that IDCs may play a role in setting up a T-cell immune reaction against neoplastic cells, which may influence the biological behaviour and/or local growth of the tumour. Moreover, monocytes and cells with intermediate features between monocytes and IDCs are also found in the peritumoral infiltrate, thus suggesting that IDCs differ entiate in situ from monocytic precursors, possibly under the influence of either tumour-derived factors or the local lymphoid microenvironment.
It is well known that cell-mediated immunity plays a major role in the reaction of the host against a tumour. In recent years, attention has been focused on the den dritic T-zone accessory cells - identified immunocytochemically by the expression of CD la and S-100 anti gens - in the infiltrates of the connective tissue adjacent to adenocarcinomas of the lung [1, 2], stomach [3] and colon [4], nasopharyngeal carcinomas [5], papillary' car cinomas of the thyroid [6] and malignant melanomas of the skin [7], It has been observed that the higher the number of T-zone accessory cells, the more favourable the prognosis was. and it has been suggested that these cells had an important role in setting up an immune response against tumours [5-7], A correlation between a high number of dendritic accessory' cells identified immunocytochemically in the peritumoral infiltrates and favourable prognosis has also been reported for laryngeal carcinomas [8], The purpose of the present study was to provide fur ther insight into the exact nature and immunological role
of S-100- DC la-positive dendritic cells in the peritumo ral infiltrate of laryngeal carcinomas by an ultrastruc tural analysis.
Materials and Methods Patients Eighteen Caucasian patients with laryngeal cancer were studied, staged according to the TNM classification of malignant tumours. None of them underwent any therapy before surgery. In all cases, histopathology was consistent with squamous cell carcinoma. The main clinical data are summarized in table 1. Light Microscopy and Immunocytochemistry Tissue specimens were obtained at surgery from the proximity of the tumour masses. Some fragments were fixed in Bouin’s fluid, embedded in paraffin and cut in 5 pm thick sections. These were either stained with haematoxylin and eosin or immunostained for S-100 protein. Some tissue fragments were snap-frozen and cut with a cryostat in 8 pm thick sections. These were air-dried, fixed in a chloroform-acetone mixture for 5 min and then immunostained for C D la (T6) surface antigen. Immunocytochemistry was performed according to the unlabelled peroxidase anti-peroxidase (PAP) method [9], The primary antisera used were mouse monoclonal
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Introduction
350
Rucci/Bani/Gallo/Riccardi/Borghi Cirri/Fini-Storchi
Fig. 1. Cells showing clear nuclei with prominent nucleoli and wide cytoplasm are seen (arrows) intermingled with lymphocytes. Semi-thin section. X 800. Fig. 2. S-100-positive dendritic cells (ar rows) in the infiltrate underlying the tumour. Section from paraffin-embedded specimen. PAP method. X 320.
Fig. 3. S-100-positive dendritic cells with interconnecting cytoplasmic processes (arrow) contacting surrounding lymphoid cells. Sec tion from paraffin-embedded specimen, PAP method. X 1,200. Fig. 4. CD la-positive dendritic cells sur rounded by lymphoid cells forming rosette like aggregates. Frozen section. PAP method. X 1,200.
Patient
1 2 3 4 5 6 7 8 9
Sex
Age years
TNM
Tumour differentiation
Patient
Sex
Age years
TNM
Tumour differentiation
M M M M M M M M M
50 63 59 62 58 72 69 53 60
T la NO M0 T la NO M0 T la NO M0 T la NO M0 T la N 1 M0 T la N 1 M0 T ib N 1 M0 T ib N 1 M0 T2 NO M0
mild high mild mild mild high mild low mild
10 11 12 13 14 15 16 17 18
M M M M M M M M F
65 63 82 69 61 64 62 71 58
T2 T2 T3 T3 T3 T4 T4 T4 T4
mild low high low mild low mild high mild
NO M0 NI MO NO M0 N 1 M0 NI M0 NO M0 NO MO N 1 MO N2 MO
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Table 1. Clinical data
Interdigitating Cells and Laryngeal Carcinomas
351
anti-CD la (diluted 1:20; Coulter Clone, GB) and rabbit polyclonal anti-S-100 (undiluted; Ortho, USA). Diaminobenzidine was used as chromagen and the immunostained sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted in Permount. Sections incubated without the primary antisera or without any antisera were used as negative controls for the second polyclonal antisera and for the peroxidase enzymatic reaction, respectively. Sections from normal human lymph nodes and skin were used as positive controls. Light microscopical examination was also done on semi-thin sections obtained from specimens processed for electron micros copy and stained with toluidine blue-Na tetraborate. Electron Microscopy Specimens taken from the péritumoral tissue were fixed in cold 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at room tem perature and postfixed in 1% OsOj in 0.1 M phosphate buffer, pH 7.4, at 4°C . They were dehydrated in a graded acetone series, passed through propylene-oxide and embedded in Epon 812. Ultrathin sections were stained with uranyl-acetate and alkaline bismuthsubnitrate [10] and examined under a Siemens Elmiskop 102 elec tron microscope at 80 kV.
Results Light microscopical examination of both paraffin and semi-thin sections showed that the péritumoral infiltrate was made up of varying amounts of cells, most of which were lymphocytes. Non-lymphoid cells were also present having a clear cytoplasm and a nucleus with dispersed chromatin and prominent nucleolus, usually seen to be in contact with surrounding lymphocytes (fig. 1).
By immunocytochemistry, dendritic cells were found in the péritumoral infiltrate in all the cases studied. They showed positive labelling with either anti-S-100 or antiCD 1a antisera and were seen frequently close to lympho cytes (fig. 2-4). In some areas where the péritumoral infiltrate was particularly scarce, there were mainly den dritic cells present, and only a few, or no lymphoid cells were found in the connective tissue close to the tumour masses. By electron microscopy, varying numbers of dendritic cells were found in the péritumoral infiltrate in all the cases examined. These cells had long cytoplasmic pro cesses by which they linked with each other forming a cellular network, and showed intimate contacts with lym phocytes (fig. 5). Their nuclei were large and lobated or ovoid-shaped with an undulating outline. The chromatin was dispersed or aggregated in small clumps, and there were one or two large nucleoli present. The cytoplasm contained several profiles of rough and smooth endoplas mic reticulum and a well-developed Golgi apparatus. There were dendritic cells present with multiple Golgi stacks and unusually numerous mitochondria and cisternae of rough endoplasmic reticulum, the latter filling both the cell body and the cytoplasmic processes (fig. 6,7). These cells showing morphological signs of enhanced met abolic activity were scanty, apart from patients 2 and 18, where they predominated and formed a dense meshwork with many lymphocytes embedded in it. Long tracts of the plasma membranes of the lymphocytes adhered closely to the surface of dendritic cells. Some lymphocytes showed
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Fig. 5. An interdigitating cell (upper) with long cytoplasmic processes which at some points have established intimate contacts with the surrounding lymphocytes (arrow). EM. X 6.800.
Fig. 6. Interdigitating cells (IDC) with nu merous organelles from a cellular network with enclosed lymphocytes (L), which have irregularly shaped nuclei, large amounts of polyribosomes and several mitochondria. EM. X 6.000.
Fig. 8. An interdigitating cell (IDC) tightly adhering to a lymphocyte (LY) undergoing mi tosis. EM. X 11.000.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Fig. 7. Two interdigitating cells (IDC) closely apposed to a lymphocyte with an irreg ular nucleus and containing many polyribo somes and mitochondria and some RER cisternae. EM. X 7,700.
Interdigitating Cells and Laryngeal Carcinomas
353
Fig. 9. A typical monocyte (left), contain ing free polyribosomes and primary lyso somes, apposed to an interdigitating cell (right), close to the tumoral tissue (below right). EM. X 6,000. Fig. 10. A typical monocyte (below) in close contact with lymphocytes (upper). EM. X 6.000.
blast-like features, i.e. abundant cytoplasm filled with free polyribosomes, numerous mitochondria and irregularly shaped nuclei with more dispersed chromatin compared with resting lymphocytes (fig. 6,7). Lymphocytes undergoing mitosis and tightly adhering to dendritic cells were also observed (fig. 8). Monocytes were also encountered interspersed among the dendritic and lymphoid cells, showing the
well-known ultrastructural features: clear nucleus with small chromatin clumps, cytoplasm containing many free polyribosomes, moderate amounts of mitochondria, short cisternae of rough endoplasmic reticulum, sparse cytofilaments and a poorly-developed Golgi apparatus with primary lysosomes nearby. These cells appeared to be in close contact with either dendritic of lymphoid cells (fig. 9-11).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Fig. 11. A typical monocyte (MO) adhering to a dendritic cell (DC) with intermediate fea tures between monocyte and interdigitating cell. Both cells are in close contact with a lym phocyte (LY). EM. X 7,000.
Rucci/Bani/Gallo/Riccardi/Borghi Cirri/Fini-Storchi
Besides typcial monocytes and dendritic cells, other non-lymphoid cells were also present showing interme diate features. They were characterized by stumpy cyto plasmic processes giving the cells a dendritic shape. Their nucleus was clear with regular contours, and their cytoplasm contained more numerous mitochondria and cisternae of rough endoplasmic reticulum and a larger Golgi apparatus compared to typical monocytes, and usually there were clustered primary lysosomes and bun dles of cytofilaments running beneath the plasma mem brane. These cells laid close to the lymphoid cells (fig. 11). It should be stressed that no Langerhans cells (LCs), identifiable by the presence of Birbeck granules in their cytoplasm, or phagocytic macrophages were ever found in the péritumoral infiltrate.
Discussion The péritumoral infiltrate from all the patients with laryngeal carcinomas examined contained cells identi fied as IDCs on the basis of their immunophenotypical and ultrastructural features, resembling those described in previous immunocytochemical, ultrastructural and immunoelectronmicroscopical studies [11-16]. IDCs are considered as the major class of T-lymphocyte accessory cells and have been described as typical components of T-dependent areas of secondary lymphoid organs and of the thymic medulla in physiological and pathological states [15-22]. Moreover, these cells seem to be present in all conditions in which a T-cell immune response takes place [23]. Since Birbeck granules have never been found in any of the dendritic cells present in the péritumoral infil trates studied, it can be excluded that LCs - character ized by Birbeck granules in their cytoplasm [24] - form a substantial part of the dendritic cell population in the péritumoral infiltrates of laryngeal carcinomas. This is not surprising, because it is widely accepted that LCs play a role in the afferent phase of the cell-mediated immune response by taking up antigens and carrying them to the regional lymph nodes [25-27], On the other hand, it is well established that, as a rule, antigen presen tation leading to lymphocyte activation is exerted by IDCs and not by Langerhans cells [28]. This requires the transmission of a series of signals through intimate con tacts between the plasma membrane of IDCs and lym phocytes [28, 29], As a matter of fact, images of close
apposition between these two cell types accompanied by cytological signs of activation or even proliferation of lymphocytes have been observed. This was especially frequent in those cases in which IDCs also showed fea tures of enhanced activity in the péritumoral infiltrates of the laryngeal carcinomas studied here. This suggests a major role of IDCs in setting up a cell-mediated immune reaction, probably directed against tumour cells. In such a view, previous reports of a relationship between high numbers of dendritic cells with IDC-inimunophenotype in the péritumoral infiltrates and a favourable prognosis of different kinds of neoplasms [1-6], including laryn geal carcinomas [8], can be better understood. In fact, the presence of numerous IDCs in the lymphoid infil trates surrounding tumours might evoke and maintain an appropriate immune response, capable of influencing the local growth and/or the spreading ability of the neo plasm. The only patient in which IDCs were not accompa nied by a substantial lymphocytic infiltrate was 82-yearold (case 12). This uncommon finding may be related to the advanced age of the patient, reflecting depression of immunologic activity [30, 31]. In all the cases examined IDCs coexisted with varying numbers of monocytes and cells showing intermediate features which can be considered as sequential stages of the differentiation of monocytes into mature IDCs. This led us to hypothesize that IDCs originate in situ from monocytic precursors, possibly attracted from the blood by tumour-derived factors [32] and induced to differen tiate into IDCs under the influence of the local microen vironment, similar to that of T-dependent areas of lym phoid organs, as also proposed for other physiological and pathological conditions [33-36]. The possibility that IDCs may also derive from LCs or phagocytic macro phages. as reported previously [25. 26, 37-39] cannot be ruled out a priori. However, it should be pointed out that LCs and macrophages were never found in the péritumo ral infiltrates of the cases examined here, thus leading us to the exclusion of a substantial contribution of these cell types to form the péritumoral pool of IDCs.
References 1 Watanabe S, Sato Y, Kodama T, Shimosato Y: Immunohisto chemical study with monoclonal antibodies on immune re sponse in human lung cancer. Cancer Res 1983:43:5883-5889. 2 Furukawa T, Watanabe S. Kodama T. Sato Y, Shimosato Y, Suemasu K: T-zone histioyctes in adenocarcinoma of the lung in
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
354
relation to post-operative prognosis. Cancer 1985:56:2651— 2656. 3 Tsujitani S. Furukawa T. Tamede R. Okamura T. Yasumoto K. Sugimachi K: Langerhans cells and prognosis in patients with gastric carcinoma. Cancer 1987;59:501-505. 4 Ambe K, Mori M. Enjoji M: S-100 protein-positive dendritic cells in colorectal adenocarcinomas. Distribution and relation to the clinical prognosis. Cancer 1989;63:496-503. 5 Nomori H, Watanabe S. Nakajima T. Shimosato Y. Kameya T: Histiocytes in nasopharyngeal carcinoma in relation to progno sis. Cancer 1986;57:100-105. 6 Schroeder S, Schwarz W, Rehpenning W. Loening T, Boecker W: Dcndritic/Langcrhans cells and prognosis in patients with papillary thyroid carcinomas. Immunocytochemical study of 106 thyroid neoplasms correlated to follow-up data. Am J Clin Pathol 1988;89:295-300. 7 Ralfkiaer E. Hou-Jensen K, Gaiter K. Drzewiecki KT. Mason D: Immunohistological analysis of the lymphoid infiltrate in cuta neous malignant melanomas. Virchows Arch A (Pathol Anat) 1987;410:355-361. 8 Gallo O, Asprella-Libonati G. Gallina E, Giannini A, Urso C, Bondi R: Langerhans cells related to prognosis in patients with laryngeal squamous cell carcinoma. Acta Otorhinolaryngol Head Neck Surg 1991 ; 1:in press. 9 Sternberger LA: Immunocytochemistry; 2nd ed. New York, Wi ley, 1979. 10 Riva A: A simple and rapid staining method for enhancing the contrast of tissues previously treated with uranyl acetate. J Microscopic 1974;19:105-108. 11 Takahashi K. Yamaguchi H, Ishizeki J, Nakajima T. Nakazato Y: Immunohistochemical and immunoelectronmicroscopical lo calization of S-100 protein in the interdigitating reticulum cells of the human lymph node. Virchows Arch B (Cell Pathol) 1981; 37:125-135. 12 Ralfkiaer E, Stein H, PlesnerT. Hou-Jensen K, Mason D: In situ immunological characterization of Langerhans cells with mono clonal antibodies. Comparison with other dendritic cells in skin and lymph nodes. Virchows Arch A (Pathol Anat) 1984:403: 401-411. 13 Goordyal P. Isaacson PG: Immunocytochemical characteriza tion of monocyte colonies of human bone marrow: A clue to the origin of Langerhans cells and interdigitating reticulum cells. J Pathol 1985:146:189-195. 14 Bani D, Moretti S, Pimpinelli N, Giannotti B: Interdigitating reticulum cells in the dermal infiltrate of mycosis fungoides. An immunohistochemical and ultrastructural study. Virchows Arch A (Pathol Anat Histopathol) 1988;412:451-458. 15 Yamamoto Y, Okato S, Takahashi H, Takeda K, Magari S: Dis tribution and morphology of macrophages in palatine tonsils. Acta Otolarnygol (Stockh) 1988;(suppl 454):83—95. 16 Okato S, Magari S, Yamamoto Y. Sakanaka H, Takahashi H: An immuno-electron microscopic study on interaction among den dritic cells, macrophages and lymphocytes in the human palatine tonsils. Arch Histol Cytol 1989;52:231-240. 17 Veldman JE: Histophysiology and electron microscopy of the immune response. University of Groningen, 1970, PhD disserta tion. 18 Heusermann U, Stutte HJ, Muellcr-Hcrmclink HK: Interdigitat ing cells in the white pulp of the human spleen. Cell Tiss Res 1974;153:415-417.
355
19 Kaiserling E. Lennert K: Die interdigitierende Reticulumzelle im menschlichen Lymphoknoten. Virchows Arch (B) 1974:16: 51-61. 20 Kaiserling E, Stein H. Muciler-Hermelink HK: Interdigitating cells in the human thymus. Cell Tiss Res 1974;155:47-55. 21 Groscurth P: Non-lymphatic cells in the lymph node cortex of the mouse. I. Morphology and distribution of the interdigitating cells and the dendritic reticular cells in the mesenteric lymph node of the adult IRC mouse. Path Res Pract 1980; 169:212— 234. 22 Veldman JE, Kaiserling E: Interdigitating cells; in Carr I, Daems WT (eds): The Reticuloendothelial System. New York, Plenum, 1980, vol 1, pp 381-416. 23 Austyn JM: Lymphoid dendritic cells. Immunology 1987;62: 161-170. 24 Birbeck MS, Breathnach AS, Everall JD: An electron micro scopic study of basal melanocytes and high-level clear cells (Langerhans cells) in vitiligo. J Invest Dermatol 1961:37:51 — 64. 25 Balfour BM. Drexhage HA. Kamperdijk EWA. Hoefsmith ECM: Antigen presenting cells, including Langerhans cells, veiled cells and interdigitating cells; in Microenvironments in Haemopoictic and Lymphoid Differentiation (Ciba Found. Symp.). London, Pitman Medical, 1981, pp 281-301. 26 Hoefsmith ECM. Duijvestijn AM, Kamperdijk EWA: Relation between Langerhans cells, veiled cells and interdigitating cells. Immunobiology 1982;161:255-265. 27 Macotonia SE. Knight SC, Edwards AJ. Griffiths S. Fryer P: Localization of antigen on lymph node dendritic cells after expo sure to the contact sensitizer fluorescein isothiocyanate. Func tional and morphological studies. J Exp Med 1987; 166:1654— 1667. 28 King PD, Katz DR: Mechanisms of dendritic cell function. Immunol Today 1990:11:206-211. 29 Unanuc ER, Beller DI, Lu CY, Allen PM: Antigen presentation: Comments on its regulation and mechanism. J Immunol 1984; 132:1-5. 30 Gross L: Immunologic defeci in aged population and its relation to cancer. Cancer 1965; 18:201 —204. 31 Waldorf DS. Willkens RF. Decker JL: Impaired delayed hyper sensitivity in an aging population. Association with antinuclear reactivity and rheumatoid fever. J Am Med Ass 1968:203:111114. 32 Bottazzi B, Polentarutti N, Acero R. Balsari A, Boraschi D, Ghezzi P, Salmona M. Mantovani A: Regulation of the macro phage content of neoplasms by chemoattractants. Science 1983; 220 :210- 212. 33 Veerman AJP: On the interdigitating cells in the thymus depen dent area of the rat spleen: A relation between the mononuclear phagocyte system and T lymphocytes. Cell Tiss Res 1974:148: 247-257. 34 Schrader JW, Clarkc-Lewis 1, Wong GHW: T-cell lymphokines and the production and function of accessory cells. Immuno biology 1984;168:425-440. 35 Mueller-Hermelink HK, Kaiserling E: Different reticulum cells of the lymph node: Microecological concept of lymphoid tissue organization; in van der Tweel JG et al (eds): Malignant Lymphoproliferative Diseases. The Hague. Nijhoff, 1980, pp 57705783.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Interdigitating Cells and Laryngeal Carcinomas
36 Bani D, Giannolli B: Differentiation of interdigitating reticulum cells and Langerhans cells in the human skin with T-lymphoid infiltrate. An immunocytochemical and ultrastructural study. Arch Histol Cytol 1989;52:361-372. 37 Rausch E, Kaiserling E, Goos M: Langerhans cells and interdigi tating reticulum cells in the thymus-dependent region in human dermatopathic lymphadenitis. Virchows Arch B (Cell Pathol) 1977;25:327-343. 38 Kamperdijk EWA, Raaymakers EM, De Leeuw JHS, Hoefsmith ECM: Lymph node macrophages and reticulum cells in the immune response. 1. The primary response to parathyphoid vac cine. Cell Tiss Res 1978;192:1-23.
Rucci/Bani/Gallo/Riccardi/Borghi Cirri/Fini-Storchi
39 Schuler G, Steinman RM: Murine epidermal Langerhans cells mature into potent ¡mmunostimulatory dendritic cells in vitro. J Exp Med 1985:161:526-546. Received: October 12, 1990 Accepted after revision: January 7, 1991 Dr. Lucio Rucci Istituto di Clinica Otorinolaringoiatrica Policlinico di Careggi Viale G.B. Morgagni. 85 1-50139 Firenze (Italia)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
356
ORL 1991;53:349-356
Interdigitating Cells in the Péritumoral Infiltrate of Laryngeal Carcinomas: An Immunocytochemical and Ultrastructural Study L. Ruccib, D. Bam3, O. Gallob. R. Arbi Riccardi M.B. Borghi Cirri3, O. Fini-Storchib a ,
■'Department of Human Anatomy and Histology, Section of Histology, hOtorhinolaryngologic Clinic, University of Florence, Florence, Italy
Key Words. Interdigitating cells • Laryngeal carcinomas • Immunocytochemistry • Ultrastructure Abstract. Interdigitating cells (IDCs) have been found in the peritumoral infiltrate of 18 patients with squamous cell carcinoma of the larynx. These cells have a dendritic shape and are characterized by the expression of S-100 protein and CD la antigens. By electron microscopy, these cells are seen to establish intimate contacts with the apposed lymphocytes, which sometimes show signs of functional activation and proliferation. These findings indi cate that IDCs may play a role in setting up a T-cell immune reaction against neoplastic cells, which may influence the biological behaviour and/or local growth of the tumour. Moreover, monocytes and cells with intermediate features between monocytes and IDCs are also found in the peritumoral infiltrate, thus suggesting that IDCs differ entiate in situ from monocytic precursors, possibly under the influence of either tumour-derived factors or the local lymphoid microenvironment.
It is well known that cell-mediated immunity plays a major role in the reaction of the host against a tumour. In recent years, attention has been focused on the den dritic T-zone accessory cells - identified immunocytochemically by the expression of CD la and S-100 anti gens - in the infiltrates of the connective tissue adjacent to adenocarcinomas of the lung [1, 2], stomach [3] and colon [4], nasopharyngeal carcinomas [5], papillary' car cinomas of the thyroid [6] and malignant melanomas of the skin [7], It has been observed that the higher the number of T-zone accessory cells, the more favourable the prognosis was. and it has been suggested that these cells had an important role in setting up an immune response against tumours [5-7], A correlation between a high number of dendritic accessory' cells identified immunocytochemically in the peritumoral infiltrates and favourable prognosis has also been reported for laryngeal carcinomas [8], The purpose of the present study was to provide fur ther insight into the exact nature and immunological role
of S-100- DC la-positive dendritic cells in the peritumo ral infiltrate of laryngeal carcinomas by an ultrastruc tural analysis.
Materials and Methods Patients Eighteen Caucasian patients with laryngeal cancer were studied, staged according to the TNM classification of malignant tumours. None of them underwent any therapy before surgery. In all cases, histopathology was consistent with squamous cell carcinoma. The main clinical data are summarized in table 1. Light Microscopy and Immunocytochemistry Tissue specimens were obtained at surgery from the proximity of the tumour masses. Some fragments were fixed in Bouin’s fluid, embedded in paraffin and cut in 5 pm thick sections. These were either stained with haematoxylin and eosin or immunostained for S-100 protein. Some tissue fragments were snap-frozen and cut with a cryostat in 8 pm thick sections. These were air-dried, fixed in a chloroform-acetone mixture for 5 min and then immunostained for C D la (T6) surface antigen. Immunocytochemistry was performed according to the unlabelled peroxidase anti-peroxidase (PAP) method [9], The primary antisera used were mouse monoclonal
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Introduction
350
Rucci/Bani/Gallo/Riccardi/Borghi Cirri/Fini-Storchi
Fig. 1. Cells showing clear nuclei with prominent nucleoli and wide cytoplasm are seen (arrows) intermingled with lymphocytes. Semi-thin section. X 800. Fig. 2. S-100-positive dendritic cells (ar rows) in the infiltrate underlying the tumour. Section from paraffin-embedded specimen. PAP method. X 320.
Fig. 3. S-100-positive dendritic cells with interconnecting cytoplasmic processes (arrow) contacting surrounding lymphoid cells. Sec tion from paraffin-embedded specimen, PAP method. X 1,200. Fig. 4. CD la-positive dendritic cells sur rounded by lymphoid cells forming rosette like aggregates. Frozen section. PAP method. X 1,200.
Patient
1 2 3 4 5 6 7 8 9
Sex
Age years
TNM
Tumour differentiation
Patient
Sex
Age years
TNM
Tumour differentiation
M M M M M M M M M
50 63 59 62 58 72 69 53 60
T la NO M0 T la NO M0 T la NO M0 T la NO M0 T la N 1 M0 T la N 1 M0 T ib N 1 M0 T ib N 1 M0 T2 NO M0
mild high mild mild mild high mild low mild
10 11 12 13 14 15 16 17 18
M M M M M M M M F
65 63 82 69 61 64 62 71 58
T2 T2 T3 T3 T3 T4 T4 T4 T4
mild low high low mild low mild high mild
NO M0 NI MO NO M0 N 1 M0 NI M0 NO M0 NO MO N 1 MO N2 MO
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Table 1. Clinical data
Interdigitating Cells and Laryngeal Carcinomas
351
anti-CD la (diluted 1:20; Coulter Clone, GB) and rabbit polyclonal anti-S-100 (undiluted; Ortho, USA). Diaminobenzidine was used as chromagen and the immunostained sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted in Permount. Sections incubated without the primary antisera or without any antisera were used as negative controls for the second polyclonal antisera and for the peroxidase enzymatic reaction, respectively. Sections from normal human lymph nodes and skin were used as positive controls. Light microscopical examination was also done on semi-thin sections obtained from specimens processed for electron micros copy and stained with toluidine blue-Na tetraborate. Electron Microscopy Specimens taken from the péritumoral tissue were fixed in cold 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at room tem perature and postfixed in 1% OsOj in 0.1 M phosphate buffer, pH 7.4, at 4°C . They were dehydrated in a graded acetone series, passed through propylene-oxide and embedded in Epon 812. Ultrathin sections were stained with uranyl-acetate and alkaline bismuthsubnitrate [10] and examined under a Siemens Elmiskop 102 elec tron microscope at 80 kV.
Results Light microscopical examination of both paraffin and semi-thin sections showed that the péritumoral infiltrate was made up of varying amounts of cells, most of which were lymphocytes. Non-lymphoid cells were also present having a clear cytoplasm and a nucleus with dispersed chromatin and prominent nucleolus, usually seen to be in contact with surrounding lymphocytes (fig. 1).
By immunocytochemistry, dendritic cells were found in the péritumoral infiltrate in all the cases studied. They showed positive labelling with either anti-S-100 or antiCD 1a antisera and were seen frequently close to lympho cytes (fig. 2-4). In some areas where the péritumoral infiltrate was particularly scarce, there were mainly den dritic cells present, and only a few, or no lymphoid cells were found in the connective tissue close to the tumour masses. By electron microscopy, varying numbers of dendritic cells were found in the péritumoral infiltrate in all the cases examined. These cells had long cytoplasmic pro cesses by which they linked with each other forming a cellular network, and showed intimate contacts with lym phocytes (fig. 5). Their nuclei were large and lobated or ovoid-shaped with an undulating outline. The chromatin was dispersed or aggregated in small clumps, and there were one or two large nucleoli present. The cytoplasm contained several profiles of rough and smooth endoplas mic reticulum and a well-developed Golgi apparatus. There were dendritic cells present with multiple Golgi stacks and unusually numerous mitochondria and cisternae of rough endoplasmic reticulum, the latter filling both the cell body and the cytoplasmic processes (fig. 6,7). These cells showing morphological signs of enhanced met abolic activity were scanty, apart from patients 2 and 18, where they predominated and formed a dense meshwork with many lymphocytes embedded in it. Long tracts of the plasma membranes of the lymphocytes adhered closely to the surface of dendritic cells. Some lymphocytes showed
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Fig. 5. An interdigitating cell (upper) with long cytoplasmic processes which at some points have established intimate contacts with the surrounding lymphocytes (arrow). EM. X 6.800.
Fig. 6. Interdigitating cells (IDC) with nu merous organelles from a cellular network with enclosed lymphocytes (L), which have irregularly shaped nuclei, large amounts of polyribosomes and several mitochondria. EM. X 6.000.
Fig. 8. An interdigitating cell (IDC) tightly adhering to a lymphocyte (LY) undergoing mi tosis. EM. X 11.000.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Fig. 7. Two interdigitating cells (IDC) closely apposed to a lymphocyte with an irreg ular nucleus and containing many polyribo somes and mitochondria and some RER cisternae. EM. X 7,700.
Interdigitating Cells and Laryngeal Carcinomas
353
Fig. 9. A typical monocyte (left), contain ing free polyribosomes and primary lyso somes, apposed to an interdigitating cell (right), close to the tumoral tissue (below right). EM. X 6,000. Fig. 10. A typical monocyte (below) in close contact with lymphocytes (upper). EM. X 6.000.
blast-like features, i.e. abundant cytoplasm filled with free polyribosomes, numerous mitochondria and irregularly shaped nuclei with more dispersed chromatin compared with resting lymphocytes (fig. 6,7). Lymphocytes undergoing mitosis and tightly adhering to dendritic cells were also observed (fig. 8). Monocytes were also encountered interspersed among the dendritic and lymphoid cells, showing the
well-known ultrastructural features: clear nucleus with small chromatin clumps, cytoplasm containing many free polyribosomes, moderate amounts of mitochondria, short cisternae of rough endoplasmic reticulum, sparse cytofilaments and a poorly-developed Golgi apparatus with primary lysosomes nearby. These cells appeared to be in close contact with either dendritic of lymphoid cells (fig. 9-11).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Fig. 11. A typical monocyte (MO) adhering to a dendritic cell (DC) with intermediate fea tures between monocyte and interdigitating cell. Both cells are in close contact with a lym phocyte (LY). EM. X 7,000.
Rucci/Bani/Gallo/Riccardi/Borghi Cirri/Fini-Storchi
Besides typcial monocytes and dendritic cells, other non-lymphoid cells were also present showing interme diate features. They were characterized by stumpy cyto plasmic processes giving the cells a dendritic shape. Their nucleus was clear with regular contours, and their cytoplasm contained more numerous mitochondria and cisternae of rough endoplasmic reticulum and a larger Golgi apparatus compared to typical monocytes, and usually there were clustered primary lysosomes and bun dles of cytofilaments running beneath the plasma mem brane. These cells laid close to the lymphoid cells (fig. 11). It should be stressed that no Langerhans cells (LCs), identifiable by the presence of Birbeck granules in their cytoplasm, or phagocytic macrophages were ever found in the péritumoral infiltrate.
Discussion The péritumoral infiltrate from all the patients with laryngeal carcinomas examined contained cells identi fied as IDCs on the basis of their immunophenotypical and ultrastructural features, resembling those described in previous immunocytochemical, ultrastructural and immunoelectronmicroscopical studies [11-16]. IDCs are considered as the major class of T-lymphocyte accessory cells and have been described as typical components of T-dependent areas of secondary lymphoid organs and of the thymic medulla in physiological and pathological states [15-22]. Moreover, these cells seem to be present in all conditions in which a T-cell immune response takes place [23]. Since Birbeck granules have never been found in any of the dendritic cells present in the péritumoral infil trates studied, it can be excluded that LCs - character ized by Birbeck granules in their cytoplasm [24] - form a substantial part of the dendritic cell population in the péritumoral infiltrates of laryngeal carcinomas. This is not surprising, because it is widely accepted that LCs play a role in the afferent phase of the cell-mediated immune response by taking up antigens and carrying them to the regional lymph nodes [25-27], On the other hand, it is well established that, as a rule, antigen presen tation leading to lymphocyte activation is exerted by IDCs and not by Langerhans cells [28]. This requires the transmission of a series of signals through intimate con tacts between the plasma membrane of IDCs and lym phocytes [28, 29], As a matter of fact, images of close
apposition between these two cell types accompanied by cytological signs of activation or even proliferation of lymphocytes have been observed. This was especially frequent in those cases in which IDCs also showed fea tures of enhanced activity in the péritumoral infiltrates of the laryngeal carcinomas studied here. This suggests a major role of IDCs in setting up a cell-mediated immune reaction, probably directed against tumour cells. In such a view, previous reports of a relationship between high numbers of dendritic cells with IDC-inimunophenotype in the péritumoral infiltrates and a favourable prognosis of different kinds of neoplasms [1-6], including laryn geal carcinomas [8], can be better understood. In fact, the presence of numerous IDCs in the lymphoid infil trates surrounding tumours might evoke and maintain an appropriate immune response, capable of influencing the local growth and/or the spreading ability of the neo plasm. The only patient in which IDCs were not accompa nied by a substantial lymphocytic infiltrate was 82-yearold (case 12). This uncommon finding may be related to the advanced age of the patient, reflecting depression of immunologic activity [30, 31]. In all the cases examined IDCs coexisted with varying numbers of monocytes and cells showing intermediate features which can be considered as sequential stages of the differentiation of monocytes into mature IDCs. This led us to hypothesize that IDCs originate in situ from monocytic precursors, possibly attracted from the blood by tumour-derived factors [32] and induced to differen tiate into IDCs under the influence of the local microen vironment, similar to that of T-dependent areas of lym phoid organs, as also proposed for other physiological and pathological conditions [33-36]. The possibility that IDCs may also derive from LCs or phagocytic macro phages. as reported previously [25. 26, 37-39] cannot be ruled out a priori. However, it should be pointed out that LCs and macrophages were never found in the péritumo ral infiltrates of the cases examined here, thus leading us to the exclusion of a substantial contribution of these cell types to form the péritumoral pool of IDCs.
References 1 Watanabe S, Sato Y, Kodama T, Shimosato Y: Immunohisto chemical study with monoclonal antibodies on immune re sponse in human lung cancer. Cancer Res 1983:43:5883-5889. 2 Furukawa T, Watanabe S. Kodama T. Sato Y, Shimosato Y, Suemasu K: T-zone histioyctes in adenocarcinoma of the lung in
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
354
relation to post-operative prognosis. Cancer 1985:56:2651— 2656. 3 Tsujitani S. Furukawa T. Tamede R. Okamura T. Yasumoto K. Sugimachi K: Langerhans cells and prognosis in patients with gastric carcinoma. Cancer 1987;59:501-505. 4 Ambe K, Mori M. Enjoji M: S-100 protein-positive dendritic cells in colorectal adenocarcinomas. Distribution and relation to the clinical prognosis. Cancer 1989;63:496-503. 5 Nomori H, Watanabe S. Nakajima T. Shimosato Y. Kameya T: Histiocytes in nasopharyngeal carcinoma in relation to progno sis. Cancer 1986;57:100-105. 6 Schroeder S, Schwarz W, Rehpenning W. Loening T, Boecker W: Dcndritic/Langcrhans cells and prognosis in patients with papillary thyroid carcinomas. Immunocytochemical study of 106 thyroid neoplasms correlated to follow-up data. Am J Clin Pathol 1988;89:295-300. 7 Ralfkiaer E. Hou-Jensen K, Gaiter K. Drzewiecki KT. Mason D: Immunohistological analysis of the lymphoid infiltrate in cuta neous malignant melanomas. Virchows Arch A (Pathol Anat) 1987;410:355-361. 8 Gallo O, Asprella-Libonati G. Gallina E, Giannini A, Urso C, Bondi R: Langerhans cells related to prognosis in patients with laryngeal squamous cell carcinoma. Acta Otorhinolaryngol Head Neck Surg 1991 ; 1:in press. 9 Sternberger LA: Immunocytochemistry; 2nd ed. New York, Wi ley, 1979. 10 Riva A: A simple and rapid staining method for enhancing the contrast of tissues previously treated with uranyl acetate. J Microscopic 1974;19:105-108. 11 Takahashi K. Yamaguchi H, Ishizeki J, Nakajima T. Nakazato Y: Immunohistochemical and immunoelectronmicroscopical lo calization of S-100 protein in the interdigitating reticulum cells of the human lymph node. Virchows Arch B (Cell Pathol) 1981; 37:125-135. 12 Ralfkiaer E, Stein H, PlesnerT. Hou-Jensen K, Mason D: In situ immunological characterization of Langerhans cells with mono clonal antibodies. Comparison with other dendritic cells in skin and lymph nodes. Virchows Arch A (Pathol Anat) 1984:403: 401-411. 13 Goordyal P. Isaacson PG: Immunocytochemical characteriza tion of monocyte colonies of human bone marrow: A clue to the origin of Langerhans cells and interdigitating reticulum cells. J Pathol 1985:146:189-195. 14 Bani D, Moretti S, Pimpinelli N, Giannotti B: Interdigitating reticulum cells in the dermal infiltrate of mycosis fungoides. An immunohistochemical and ultrastructural study. Virchows Arch A (Pathol Anat Histopathol) 1988;412:451-458. 15 Yamamoto Y, Okato S, Takahashi H, Takeda K, Magari S: Dis tribution and morphology of macrophages in palatine tonsils. Acta Otolarnygol (Stockh) 1988;(suppl 454):83—95. 16 Okato S, Magari S, Yamamoto Y. Sakanaka H, Takahashi H: An immuno-electron microscopic study on interaction among den dritic cells, macrophages and lymphocytes in the human palatine tonsils. Arch Histol Cytol 1989;52:231-240. 17 Veldman JE: Histophysiology and electron microscopy of the immune response. University of Groningen, 1970, PhD disserta tion. 18 Heusermann U, Stutte HJ, Muellcr-Hcrmclink HK: Interdigitat ing cells in the white pulp of the human spleen. Cell Tiss Res 1974;153:415-417.
355
19 Kaiserling E. Lennert K: Die interdigitierende Reticulumzelle im menschlichen Lymphoknoten. Virchows Arch (B) 1974:16: 51-61. 20 Kaiserling E, Stein H. Muciler-Hermelink HK: Interdigitating cells in the human thymus. Cell Tiss Res 1974;155:47-55. 21 Groscurth P: Non-lymphatic cells in the lymph node cortex of the mouse. I. Morphology and distribution of the interdigitating cells and the dendritic reticular cells in the mesenteric lymph node of the adult IRC mouse. Path Res Pract 1980; 169:212— 234. 22 Veldman JE, Kaiserling E: Interdigitating cells; in Carr I, Daems WT (eds): The Reticuloendothelial System. New York, Plenum, 1980, vol 1, pp 381-416. 23 Austyn JM: Lymphoid dendritic cells. Immunology 1987;62: 161-170. 24 Birbeck MS, Breathnach AS, Everall JD: An electron micro scopic study of basal melanocytes and high-level clear cells (Langerhans cells) in vitiligo. J Invest Dermatol 1961:37:51 — 64. 25 Balfour BM. Drexhage HA. Kamperdijk EWA. Hoefsmith ECM: Antigen presenting cells, including Langerhans cells, veiled cells and interdigitating cells; in Microenvironments in Haemopoictic and Lymphoid Differentiation (Ciba Found. Symp.). London, Pitman Medical, 1981, pp 281-301. 26 Hoefsmith ECM. Duijvestijn AM, Kamperdijk EWA: Relation between Langerhans cells, veiled cells and interdigitating cells. Immunobiology 1982;161:255-265. 27 Macotonia SE. Knight SC, Edwards AJ. Griffiths S. Fryer P: Localization of antigen on lymph node dendritic cells after expo sure to the contact sensitizer fluorescein isothiocyanate. Func tional and morphological studies. J Exp Med 1987; 166:1654— 1667. 28 King PD, Katz DR: Mechanisms of dendritic cell function. Immunol Today 1990:11:206-211. 29 Unanuc ER, Beller DI, Lu CY, Allen PM: Antigen presentation: Comments on its regulation and mechanism. J Immunol 1984; 132:1-5. 30 Gross L: Immunologic defeci in aged population and its relation to cancer. Cancer 1965; 18:201 —204. 31 Waldorf DS. Willkens RF. Decker JL: Impaired delayed hyper sensitivity in an aging population. Association with antinuclear reactivity and rheumatoid fever. J Am Med Ass 1968:203:111114. 32 Bottazzi B, Polentarutti N, Acero R. Balsari A, Boraschi D, Ghezzi P, Salmona M. Mantovani A: Regulation of the macro phage content of neoplasms by chemoattractants. Science 1983; 220 :210- 212. 33 Veerman AJP: On the interdigitating cells in the thymus depen dent area of the rat spleen: A relation between the mononuclear phagocyte system and T lymphocytes. Cell Tiss Res 1974:148: 247-257. 34 Schrader JW, Clarkc-Lewis 1, Wong GHW: T-cell lymphokines and the production and function of accessory cells. Immuno biology 1984;168:425-440. 35 Mueller-Hermelink HK, Kaiserling E: Different reticulum cells of the lymph node: Microecological concept of lymphoid tissue organization; in van der Tweel JG et al (eds): Malignant Lymphoproliferative Diseases. The Hague. Nijhoff, 1980, pp 57705783.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
Interdigitating Cells and Laryngeal Carcinomas
36 Bani D, Giannolli B: Differentiation of interdigitating reticulum cells and Langerhans cells in the human skin with T-lymphoid infiltrate. An immunocytochemical and ultrastructural study. Arch Histol Cytol 1989;52:361-372. 37 Rausch E, Kaiserling E, Goos M: Langerhans cells and interdigi tating reticulum cells in the thymus-dependent region in human dermatopathic lymphadenitis. Virchows Arch B (Cell Pathol) 1977;25:327-343. 38 Kamperdijk EWA, Raaymakers EM, De Leeuw JHS, Hoefsmith ECM: Lymph node macrophages and reticulum cells in the immune response. 1. The primary response to parathyphoid vac cine. Cell Tiss Res 1978;192:1-23.
Rucci/Bani/Gallo/Riccardi/Borghi Cirri/Fini-Storchi
39 Schuler G, Steinman RM: Murine epidermal Langerhans cells mature into potent ¡mmunostimulatory dendritic cells in vitro. J Exp Med 1985:161:526-546. Received: October 12, 1990 Accepted after revision: January 7, 1991 Dr. Lucio Rucci Istituto di Clinica Otorinolaringoiatrica Policlinico di Careggi Viale G.B. Morgagni. 85 1-50139 Firenze (Italia)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 2:51:11 AM
356
