Tenascin in Reactive Lymph Nodes and in Malignant Lymphomas

Path. Res. Pract. 188, 1078-1082 (1992)

Tenascin in Reactive Lymph Nodes and in Malignant Lymphomas Y. Soini, M. Alavaikko and V.-P. Lehto Department of Pathology, University of Oulu, Finland

I. Virtanen Department of Anatomy, University of Helsinki, Finland

SUMMARY Tenascin is all extracellular matrix protein which accumulates in the stroma of various malignant and some benign neoplasms. This has bee11 verified ill seueral immunohistochemical studies. The distributio11 of tenascin immunoreactivity in lymphatic tissues and neoplasias, howeuer, has not been thorougly studied. In this investigation we analyzed tenqscin immunoreactivity in seueral benign and maligllant lymphatic lesions, including both Hodgkin's and non-Hodgkin's lymphomas. In benign lymph nodes, fai11t reticular immunoreactivity could be obserued ill the lymphatic tissue. In benign reactiue hyperplasias, a stronger reticular pattern of tenascin immunoreactivity was obserued in the interfollicular and medullary areas, while the lymphoid follicles contained only a few positive fibers. A similar immunoreactivity was obserlled in malignant follicular lymphomas. In diffuse lymphomas, a diffuse meshwork of positively stained fibers was seen. This was also the case for the three cases of Hodgkin's disease of the lymphocyte-predominance nodular subtype. There was no difference in the i11tensity of the immunoreactivity between benign and malignant disorders. However, in Hodgkin's disease of the nodular sclerosis and lymphocyte-depletion subtypes, a much more pronounced immunoreactivity could be observed i11 the fibrous septa and the cords. This suggests that the tumor cells are possibly capable of synthesizing growth factors which stimulate fibroblasts to synthesize tenascin. The results indicate that tenascin does not accumulate in the stroma of malignant lymphoid neoplasms with the exclusion of some subtypes of Hodgkin's disease. The distribution of tenascin immunoreactivity in lymphatic tissue is similar to that of the reticular fibers suggesting that the molecules are associated with these structures. Since lymphoid cells do not adhere to tenascin molecules, it is suggested that tenascin in lymphatic tissues might function as an adhesive protei11 to some other cell types, such as reticular cells. Such adhesiollmight be mediated through a special type of tenascin-bindi11g integrin. Introduction

Tenascin is an extracellular matrix glycoprotein with a molecular weight of approximately 1900 kD4, 10. It constitutes a six-armed structure with a terminal knob at the end of each arm. Three arms are joined together in a structure 0344·0338/92/0188·1078$3.50/0

called T-junction. Two such structures are joined at the central knob to form a six-armed hexabrachion structure, thence also the name hexabrachion lO • Tenascin is synthesized especially by glial cells and fibroblasts 10 . The molecule is found in embryonic tissues 3, 8, 9, especially in areas of epithelial-mesenchymal © 1992 by Gustav Fischer Verlag, Stuttgart

Tenascin in Lymph Nodes· 1079

junctions and in developing brain tissue, and it probably has a role in epithelial-mesenchymal induction and cell migration4 ,6,8-IO. In adult tissues tenascin can be found in healing wounds, in myotendineous and cartilagineous structures and in smooth muscle 4, 7, 10. In neoplastic tissues tenascin immunoreactivity has been found in various malignant mesenchymal tumors and carcinomas, including gliomas, fibrosarcomas, osteosarcomas, melanomas, mammary carcinomas, lung carcinomas and endometrial carcinomas2 , 10, II, 18,26. Reactivity has also been observed in some benign tumors ll ,19. The immunoreactivity is associated with the neoplastic stroma and is probably related to the induction of tenascin synthesis in the stromal fibroblasts by the tumor cells. The mechanism of this induction is unknown, but probably depends on the stimulation by various growth factors synthesized by the tumor cells, such as transforming growth factor B, which has been shown to stimulate fibroblasts to synthesize tenascin4 ,2o. In order to evaluate the putative differential location of tenascin in lymphatic tissue and in tumors of lymphatic origin we have investigated the distribution of tenascin immunoreactivity in benign and malignant lymphatic lesions.

Material and

Methods~

Tumor Material Twenty-seven cases of malignant non-Hodgkin's and Hodgkin's lymphomas and reactive lymph nodes were collected at the Department of Pathology, University of Oulu, between 1986 and 1990. Additionally, three cases of non-reactive axillary lymph nodes were included. The material was obtained fresh from the operating theatre and stored at -70°C or in liquid nitrogen. The diagnosis of each case was based on a conventional light microscopic examination, supplemented by immunohistochemical investigations when necessary. The diagnosis of the malignant non-Hodgkin's lymphomas was based on the Kiel classification 16. The histopathological diagnoses and the clinical data on the cases are presented in Table 1.

Immunohistological Staining The immunostaining procedure was as follows: Five micrometer thick frozen sections were cut from the specimens. They were air-dried for one hour and were then fixed in acetone for 10 minutes at + 22°C. The endogenous peroxidase was blocked with 0.3% hydrogen peroxidase in methanol for 30 minutes. In the immunostaining the indirect peroxidase method was used. A monoclonal mouse antibody to human tenascin (100 EB 2) with a dilution of 1 : 5 was used as the primary antibody. The characterization of this antibody has been done previously 11. Incubation with the primary antibody (overnight at +4 0c) was followed by incubation with a peroxidase conjugated rabbit anti-mouse secondary antibody (dilution 1: 100). Peroxidase reaction was developed with diaminobenzidine. The sections were counterstained lightly with haematoxylin and mounted in an aqueous medium. Negative -control consisted of substituting PBS (140 ml NaCi, 0.01 M phosphate buffer, pH 7.2) for the primary antibody.

Table 1. Histopathological diagnosis of the cases studied Case

Sex

Location

31 23 65

Female Female Female

56

Female

Axilla Neck Inguinal region Neck

57 54 37

Female Male Male

42

Female

42 35

Female Male

19 70 28

Male Female Female

44

Male

Centrocytic diffuse lymphoma Immunoblastic lymphoma Lymphocytic lymphoma Centroblastic-centrocytic, follicular lymphoma Reactive hyperplasia Atypical hyperplasia

46

Female

64 63 35

Male Male Male

47 17

Female Male

Mb. Hodgkin nodular sclerosis Mb. Hodgkin lymphocyte predominant nodular Mb Hodgkin lymphocyte predominant nodular Mb. Hodgkin nodular sclerosis Mb. Hodgkin lymphocyte depletion Mb. Hodgkin nodular sclerosis Mb. Hodgkin lymphocyte predominant nodular

30

Female

Neck Neck Inguinal region Axilla Axilla Inguinal regIOn Axilla

48

Male

Neck

40

Male

Unknown

38

Female

Neck

54

Male

45

Female

Inguinal region Neck

37

Male

Histopathological diagno- Age SIS

1 2 3 4

5 6 7

8 9 10 11

12 13

14 15 16 17 18 19 20 21 22

23 24 25 26 27

Reactive hyperplasia Reactive hyperplasia Centro blastic diffuse lymphoma Centroblastic-centrocytic, follicular lymphoma Reactive hyperplasia Lymphocytic lymphoma Centroblastic-centrocytic, follicular lymphoma Granulomatous inflammation Reactive hyperplasia Lymphoplasmocytoid lymphoma Toxoplasma hyperplasia Atypical hyperplasia Centroblastic-centrocytic, follicular lymphoma Atypical hyperplasia

Axilla Axilla Clavicular region Clavicular region Axilla A.xilla Neck Neck Inguinal regIOn Inguinal regIOn Neck

Abdominal region

Results Non-reactive Lymph Nodes

In lymph nodes with no hyperplastic reaction, tenascin immunoreactivity could be seen in the walls of small blood vessels. Faint reticular staining could be seen in lymph node tissue. The fibrotic septa and the capsule of lymph nodes stained positive. Hyperplasias

In benign and atypical hyperplasias, a reticular nenvork of positively stained fibers could be observed in the

1080 . Soini et a1.

Fig. 1. In this lymph node with reactive follicular hyperplasia (Case 19) a meshwork of tenascin-positive fibers can be seen in the interfollicular area§" while there are only a few tenascinpositive fibers in the reactive follicles (lmmunoperoxidase stain, x 125).

interfollicular areas of the lymph nodes (Fig. 1). The reaction pattern was reminiscent of that of the reticular fibers in lymph nodes. The positively stained reticular meshwork was denser than in the non-reactive lymph nodes. In the germinal centers of the follicles, tenascin immunoreactivity was, however, scarce with only occasional positive fibers seen amongst the proliferating centroblasts and centrocytes (Fig. 1).

Non-Hodgkin 's Lymphomas In various non-Hodgkin's lymphomas a positively stained reticular meshwork could be observed among the neoplastic tumor cells (Fig. 3). The staining was both quantitatively and qualitatively similar to that seen in the reactive lesions, except for the immunoblastic and diffuse centro blastic lymphomas in which the fibers appeared to be somewhat thicker. In follicular lymphomas the neoplastic follicles contained only a few tenascin-positive fibers (Fig. 2) . The interfollicular areas, on the other hand, contained a reticular nerwork of positively stained fibers similar to that seen in hyperplasias (Fig. 2).

Fig. 3. In a case of a weII differentiated lymp hocytic lymphoma (Case 6) a diffuse tenascin-positive reticular staining pattern can be observed (Immunoperoxidase stain, x 260).

Fig. 2. In a centroblastic-centrocytic follicular lymphoma (Case 4) a similar kind of reactivity for tenascin can be seen as in Figure 1; the neoplastic follicles harbour only a few tenascinpositive fib ers, while in the areas between the neoplastic follicles an abundance of tenascin-positive fibers can be seen (Immunoperox idase stain, x 125 ).

Tenascin in Lymph Nodes· 1081

Fig. 4. In a lymphocyte predominant nodular Hodgkin's disease (Case 22) a diffuse reticular staining pattern to tenascin can be seen (Immunoperoxidasc 'stain, x 125 ).

Hodgkin's Lymphomas In the seven cases of Hodgkin's lymphoma, the lymphocytic predominant nodular subtype revealed a similar pattern of reticular staining as was seen in diffuse lymphomas and in the interfollicular areas in hyperplastic lesions (Fig. 4). In the nodular sclerosis subtype, the fibrous stroma was strongly immunoreactive with tenascin. The lymphatic nodules, however, revealed only a few tenascinpositive fibers (Fig. 5). In the lymphocyte depletion subtype, thick tenascin positive fibrous bands could be observed amongst the neoplastic cells. Discussion Tenascin is an extracellular matrix protein which has been shown to accumulate in the stroma of various malignant neoplasias such as gliomas, sarcomas and carcinomas 10 • Recently, immunoreactivity has been shown in some benign lesions, as well ll , 18, 19. Even though immunoreactivity for tenascin in various sarcomas and carcinomas has been studied extensively, no studies have been published on the distribution of this protein in lymphatic neoplasias. In this study we evaluated the distribution of tenascin immunoreactivity in various benign and malignant lymphatic lesions. The results show that tenascin immuno-

Fig. 5. In a nodular sclerosis subtype Hodgkin's disease (Case 21) the fibrotic stroma arou nd the lymphatic nodules stains strongly fo r tenascin while th e nodules contain only a few tenascin positive fibers (Immunoperoxidase sta in, x 125 ).

reactivity can be observed both in benign and malignant disorders. The staining pattern is reticular and resembles reticulin staining in lymphatic tissues. Quantitatively there was no clear difference in the amount of staining between benign and malignant lesions apart from the cases of the nodular sclerosis and lymphocyte depletion variants of Hodgkin's disease, in which strong staining was revealed in fibrotic areas. The pattern of immunoreactivity suggests that tenascin is located in the reticular network of the lymphatic tissues and neoplasms. This is in line with the scarce immunoreactivity observed in both neoplastic and reactive follicles which also contain a low amount of reticular fibers! 5. Of other extracellular matrix proteins fibrone.ctin 25 , lam inin!2,13 and type I, III13, 14, IVIl,1 3 and V collagen! 7 immunoreactivity has been shown to be located in the compartment of reticular fibers. Thus, tenascin appears to have the same location in lymphatic tissues as various other extracellular proteins. Because of the presence of tenascin both in benign and malignant lesions, tenascin cannot be regarded as a strorpal marker for neoplasia or malignancy in lymphatic tissues. An exception to this rule is the nodular sclerosis and lymphocyte depletion variants of Hodgkin's disease in which an excessive amount of immunoreactivity could be observed in the fibrous septa and cords. This suggests that the neoplastic cells in this subtype of Hodgkin's disease are

1082 . Soini et al.

capable, possibly by secreting specific growth factors, of stimulating fibroblasts to synthesize tenascin. The different pattern of immunoreactivity of these two subtypes as compared with the cases of lymphocyte-predominance nodular subtype, might be a reflection of the suggested different histogenesis of the latter entity21,22,24. Unfortunately our material did not contain examples of the mixed subtype of Hodgkin's disease. T enascin receptor belongs to a family of integrin molecules which consist of an alpha and a beta subunit 1 • The beta subunit appears to be identical to that of fibroncctin receptor while the alpha subunit is different 1. It is unlikely that lymphoid cells possess receptors to tenascin since they do not attach to it 1• Thus, tenascin probably does not function as an adhesive molecule for cells of the lymphocytic lineage. However, it may serve as an adhesive substratum to some other cell type, perhaps the reticulum cells which are intimately connected with the reticular fibrous network 23 . The presence of tenascin fibers in lymph nodes also raises the question of their role in the formation of metastatic lesions in lymphatic tissue. It has been shown that various tumor cell lines, including glial, epithelial, endodermal and mesenchymal cell lines, can adhere to tenascin 1. Thus, tenascin might serve as an anchorage point for metastatic cells in lymphatic tissue. On the other hand, tenascin could also affect the entrapment of metastatic cells by virtue of its modulating effect on the interaction between~ other matrix proteins, such as fibronectin, and their receptors 5 . References 1 Bourdon MA, Ruoslahti E (1989) Tcnascin mediates cell attachment through an RGD-dependent receptor. J Cell Bioi 108: 1149-1155 2 Bourdon MA, Wikstrand CJ, Furthmayr H, Matthews TJ, Bigner DD (1983) Human glioma-mesenchymal extracellular matrix antigen defined by monoclonal antibody. Cancer Res 43: 2796-2805 3 Chiquet M, Fambrough DM (1984) Chick myotendineous antigen. 1. A. Monoclonal antibody as a marker for tendon and muscle morphogenesis. J Cell Bioi 98: 1937-1946 4 Chiquet-Ehrismann R (1990) What distinguishes tenascin from fibronectin? FASEB J 4: 2598-2604 5 Chiquet-Ehrismann R (1991) Anti-adhesive molecules of the extracellular matrix. Current Opinion in Cell Biology 3: 800-804 6 Chiquet-Ehrismann R, Mackie EJ, Pearson CA, Sakakura T (1986) Tenascin: an extracellular matrix protein involved in tissue interactions during fetal development and oncogenesis. Cell 47: 131-139 7 Chuong C-M, Chen H-M (1991) Enhanced expression of neural cell adhesion molecules and tenascin (cytotactin) during wound healing. Am J Pathol138: 427-440 R Chuong C-M, Crossin KL, Edelman GM (1987) Sequential expression and differentiation function of multiple adhesion molecules during the formation of cerebellar cortical layers. J Cell Bioi 104: 331-342 9 Crossin KL, Hoffman S, Grumet M, Thiery J-p, Edelman GM

(1986) Site-restricted expression of cytotactin during development of the chicken embryo. J Cell Bioi 102: 1917-1930 10 Erickson HP, Bourdon MA (1989) Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu Rev Cell Bioi 5: 71-92 11 Howeedy AA, Virtanen I, Laitinen L, Gould NS, Koukoulis GK, Gould VE (1991) Differential distribution of tenascin in the normal, hyperplastic and neoplastic breast. Lab Invest 63: 798-806 12 Karttunen T, Alavaikko M, Apaja-Sarkkinen M, AutioHarmainen H (1986) Distribution of basement membrane laminin and type IV collagen in human reactive lymph nodes. Histopathology 10: 841-850 13 Karttunen T, Sormunen R, Risteli L, Risteli J, AutioHarmainen H (1989) Immunoelectron microscopic localization of laminin, type IV collagen and type III pN-collagen in reticular fibers of human lymph nodes. J Histochem Cytochem 37: 279-286 14 Konomi H, Sano J, Nagai Y (1981) Immunohistological localization of types I, III and IV (basement membrane) collagens in the lymph node: co-distribution of types I and III collagens in the reticular fibers. Biomed Res 2: 536-545 15 Lennert K (1978) Malignant lymphomas other than Hodgkin's disease, Springer Verlag, Berlin 16 Lennert K, Feller AC (1990) Histopathologie der NonHodgkin-Lymphome. Springer Verlag, Berlin 17 McCurley TL, Gay RE, Glick AD, Haralson MA, Collins RD (1986) The extracellular matrix in "sclerosing" follicular cell lymphomas: an immunohistochemical and ultrastructural study. Hum Pathol17: 930-938 18 Natali PG, Nicotra MR, Bartolazzi A, Mottolese M, Coscia N, Bigotti A, Zardi L (1990) Expression and production of tenascin in benign and malignant lesions of melanocytic lineage. Int J Cancer 46: 586-590 19 Natali PG, Nicotra MR, Bigotti A, Botti C, Castellani P, Risso AM, Zardi L (1991) Comparative analysis of the expression of the extracellular matrix protein tenascin in normal human fetal, adult and tumor tissues. Int J Cancer 47: 811-816 20 Pearson CA, Pearson D, Shibahara S, Hofsteenge J, Chiquet Ehrismann R (1988) Tenascin: cDNA cloning and induction by TGF-~. EMBO J 7: 2977-2981 21 Pinkus GS, Said JW (1985) Hodgkin's disease, lymphocyte predominance type, nodular-a distinct entity? Unique staining profile for L & H variants of Reed-Sternberg cells defined by monoclonal antibodies to leucocyte common antigen, granulocyte specific antigen, and B-cell specific antigen. Am J Pathol118: 1-6 22 Pinkus GS, Said JW (1988) Hodgkin's disease, lymphocyte predominance type, nodular-further evidence for a B cell derivation. L & H variants of Reed-Sternberg cells express L 26, a pan B cell marker. Am J Pathol133: 211-217 23 Ross MH, Reith EJ (1985) Histology. A Text and Atlas. Harper Row Publishers, J. B. Lippincott Company, New York 24 Stein H, Hannsmann ML, Lennert K, Brandtzaeg P, Gatter KC, Mason DY (1986) Reed-Sternberg and Hodgkin cells in lymphocyte-predominant Hodgkin's disease of nodular subtype contain J chain. Am J Clin Pathol 86: 292-297 25 Stenman S, Vaheri A (1978) Distribution of a major connective tissue proteip, fibronectin, in normal human tissues. J Exp Med 147: 1054-1064 26 Vollmer G, Siegal GP, Chiquet-Ehrismann R, Lightner VA, .Arnholdt H, Knuppen R (1990) Tenascin expression in the human endometrium and in endometrial adenocarcinomas. Lab Invest 62: 725-730

Received October7, 19'91 . Accepted in revised form November 11, 1991

Key words: Tenascin - Lymph node - Lymphoma - Matrix protein - Hodgkin's lymphoma Ylermi Soini, M.D., Department of Pathology, University of Oulu, Kajaanintie 52 D, 90220 Oulu, Finland