Immunohistochemical analysis of the distribution of vimentin in human peripheral lymphoid tissues.код для вставкиСкачать
THE ANATOMICAL RECORD 211:43-47(1985) Immunohistochemical Analysis of the Distribution of Vimentin in Human Peripheral Lymphoid Tissues RALPH GIORNO Division of Clinical Immunology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262 ABSTRACT The distribution of the intermediate filament protein vimentin in peripheral lymphoid tissues was determined using a monoclonal antibody. Frozen sections of tissue were stained using a n avidin-biotin immunoperoxidase method. The antibody stained endothelial cells in spleen, lymph node, and tonsil. Unusual rod-like structures were revealed in the sinusoid-lining cells of the spleen. A variety of reticulum cells was detected, including fibroblastic reticulum cells, histiocytic reticulum cells (tingible body macrophages), and splenic marginal zone macrophages. Very few lymphocytes were immunoreactive. Only weak cytoplasmic immunoreactivity was observed in lymphocytes of the periarteriolar lymphocyte sheath of the spleen. The monoclonal antibody employed appears to be of limited usefulness in detecting normal lymphocytes, but is strongly reactive with endothelial structures and some types of reticulum cells. The use of monoclonal antibodies to intermediate filament (IF)proteins is finding widespread application in diagnostic histopatholorn (Osborn and Weber, 1983).Intermediate filaments are filamentous structures (7-11 nm diameter) that are intermediate in size between smaller microfilaments and larger microtubules (Osborn et al., 1977; Hynes and Destree, 1978). These structural proteins represent families of related, though not identical, polypeptides whose expression in various cells and tissues appears to depend on processes of cellular differentiation. Epithelial cells contain I%s related to epidermal tonofilaments which are classified as cytokeratins (Franke et al., 1978).Mesenchymal cells contain only one type of IF protein, vimentin (Franke et al., 1979). Other IF Proteins appear to be rather restricted in their distribution, such as desmin in muscle cells (Gard et al., 19791, m u rofilament in neurons (Liem et al., 1978), and dial fibrillary acidic protein in aStrOCyteS (Goldman et al., 1978). These findings suggest that IF proteins could Serve as differentiation markers and aid in classifying the histogenesis of cells of unknown origin, including those occurring in malignant tumors. Indeed, antibodies to the IF protein vimentin have been used to classify tumors as being Of mesenchYmal origin, sarcomas, 'ymphomas, and melanomas (Gabbiani et al., 1981). Nevertheless, the distribution of IF proteins in normal tissues has often not been well documented. It is particularly important to define the IF protein distribution in normal tissues if one is to apply antibody reagents for diagnostic purposes, since cross reactions With various cell types may be misleading. The purpose of this report is to describe the distribution of vimentin in normal human peripheral lymphoid tissues using immunohistochemical techniques. 0 1985 ALAN R. LISS, INC. MATERIALS AND METHODS ~i~~~~specimens were obtained at Adequate blocks of fresh tissue were embedded in a cryoprotectant (ocT, Ames) in an foil vessel and rapidly frozen by immersion in a dry ice.isopentane bath. F ~ ~ . blocks were stored at - 7 0 0 ~until ready for section. ing. Cryostat sections were cut at 6 pm, air h i e d a t room temperature 1-2 hr, fixed in reagent grade acetone for 20 min at temperature, and air dried again. Sections were rehydrated briefly in modified phosphatebuffered saline (PBS; Wood and Warnke, 1981) and incubated for 20 min at temperaturein the follow. ing avidin.biotin immunoperoxi~ase staining protoco~ (Warnke and Levy, 1980; Giorno, 1984a): 1) unlabeled murine monoclonal antibody to vimentin (obtained from Lab Systemsand diluted in PBS supplemented with 0.2% bovine albumin [ ~ i 1gg4bl); ~ ~ 2) ~ biotinylated ~ , antibody (obtained from Biomeda) to primary antibody; and 3) avidin-peroxidase (obtained from Biomeda). Set. tions were washed with PBS between incubations. After the last incubation and PBS wash, sections were incubated with 3,31-diaminobemidine (DAB, 0.2%) in 0.01% ~~0~ in PBS for 5 min and washed. The DAB staining was intensified by incubation for 5 min in 0.5% cuso4 in o.9% NaCl (Hanker et al., 1979). After washing in water, sections were dehydrated through alcohol, cleared with xylene, and mounted with Permount. proper staining patterns, similar sections T~ were stained with a pan-leukocyte monoc~ona~ antibody HLe-l (pizzolo et al., 1980; obtained from Becton-Dickinson and used a t 1:50) for a positive control or with a n irrelevant monoclonal antibody to keratin (obtained from Lab Systems and used at 1 : l O ) as a negative control. Received May 14,1984;accepted July 31,1984. 44 R. GIORNO Fig. 1 . Cryostat section of spleen immunostained for vimentin. Central arteriolar (open arrow) and sinusoidal (closed arrows) endothelium is immunoreactive. W, white pulp; R, red pulp. Fig. 2. Cryostat section of spleen immunostained for vimentin. Marginal zone macrophages are immunoreactive (arrows). W, white pulp; R, red pulp. Fig. 3.Cryostat section of spleen immunostained for vimentin. Central arteriolar endothelium is strongly immunoreactive (closed arrow). Lymphocytes in surrounding sheath exhibit patchly weak immunoreactivity (open arrow). Fig. 4. Cryostat section of spleen immunostained for vimentin. Unusual rod-like structures are associated with sinusoidal endothelium (arrows). VIMENTIN IN PERIPHERAL LYMPHOID TISSUES 45 Fig. 5. CryoaLaL section of spleen immunostained for vimentin. A longitudinal section of a sinusoid wall shows strongly immunoreactive rod-like structures running parallel to the long axis (arrow). Fig. 7. Cryostat section of lymph node irnmunostained for vimentin. Capillary endothelium is strongly immunoreactive. Surrounding lymphocytes are negative. Fig. 6. Cryostat section of lymph node immunostained for vimentin. Capillary endothelium (open arrow) and mantle zone (closed arrow) are immunoreactive. Fig. 8. Cryostat section of tonsil immunostained for vimentin. The mantle zone (M) appears uniformly immunoreactive. Scattered cells in the germinal center (GC)are positive. 46 R. GIORNO Fig. 9 Cryostat section of tonsil immunostained for vimentin. The mantle zone contains parallel fibers that are strongly immunoreactive (open arrows). Lymphocytes are not stained. Capillary endothelium (closed arrow) is also strongly immunoreactive. Fig. 10. Cryostat section of tonsil immunostained for vimentin. Scattered branching cells in the germinal center are strongly immunoreactive (arrows). Photomicrographs were made using a n interference filter combination which enhances the contrast of the DAB reaction product (Moller et al., 1984). cytes was not appreciated in any regions of the lymph node. The antibody also revealed intense staining of branching cells in the germinal centers of the secondary follicles, which appear to represent histiocytic reticulum cells (see below). The antibody also revealed intense staining of endothelial cells in capillaries and high endothelial venules (Fig. 7). RESULTS Spleen Within the spleen, vimentin was localized in vascular endothelium of central arterioles of the white pulp and the sinusoids of the red pulp (Fig. 1).Vimentin was also present, but less intensely stained, in marginal zone macrophages and reticulum fibers of the mantle zone of secondary lymphoid follicles (Fig. 2). There was very weak reactivity of lymphocytes in the periarteriolar lymphocyte sheath (Fig. 3). The lymphocyte staining could not be localized to the cell membrane and appeared to be cytoplasmic in nature. Staining of sinusoidal endothelium revealed unusual rod-like structures (Figs. 4, 5) which probably correspond to filamentous structures which maintain the rigidity of endothelial slits (Chen and Weiss, 1973; Giorno, 1984~). Palatine Tonsil The distribution of immunoreactivity in the tonsil was similar to that seen in lymph nodes (Fig. 8). The reticulum fibers in the mantle zone (Fig. 9) ran between lymphocytes. The branching cells in the germinal center (Fig. 10) were quite large, irregularly shaped, and intensely immunoreactive. Their distribution was similar to that of tingible body macrophages noted in routine histologic sections. Definitive staining of lymphocytes was not appreciated. DISCUSSION I have described the distribution in peripheral lymLymph Node phoid tissues of material immunoreactive with a monoImmunostaining in the lymph node revealed what ap- clonal antibody to the IF protein vimentin. Rather unpeared to be relatively specific staining in the mantle expectedly, lymphocytes were not immunoreactive. Only zone of the secondary follicle (Fig. 6). On higher power, in the periarteriolar lymphocyte sheath of the spleen the staining was localized to filamentous reticulum fi- was some apparent immunoreactivity noted. This imbers between lymphocytes. Definite staining of lympho- munoreactivity was not membranous in nature, as seen VIMENTIN IN PERIPHERAL LYMPHOID TISSUES with most monoclonal antibodies to lymphocyte surface markers, but rather appeared to be cytoplasmic. The periarteriolar lymphocyte sheath is a T-cell zone without any unique properties, as it is presently understood (Giorno, 1984d). Not all types of reticulum cells (Muller-Hermelink and Kaiserling, 1980)exhibited immunoreactivity with the monoclonal antibody to vimentin. Histiocytic reticulum cells (tingible body macrophages) in germinal centers of secondary follicles were strongly immunoreactive. Macrophages in the marginal zone of the spleen were also immunoreactive. The antibody failed to detect dendritic reticulum cells in germinal centers or interdigitating reticulum cells in T-cell zones (lymph node paracortex). The antibody appears to detect a t least some portions of fibroblastic reticulum cells, i.e., the fibrous reticulum structures in the mantle zone of the secondary follicle which coursed between lymphocytes. These findings suggest either that not all normal cells of mesenchymal origin contain vimentin or that the monoclonal antibody used only recognizes a n epitope of the vimentin polypeptide in certain cell types, but not in others. Further consideration of these possibilities is important in the understanding of the histogenesis of normal lymphoid tissues as well as in attempts to classify malignant tumors based on the presence or absence of vimentin. LITERATURE CITED Chen, L.-T., and L. Weiss (1973) The role of the sinus wall in the passage of erythrocytes through the spleen. Blood, 41:529-537. Franke, W.W., E. Schmid, S. Winter, M. Osborn, and K. Weber (1979) Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates. Exp. 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