Characterization of stromal cells with myoid features in lymph nodes ...

American Journal of Pathology, Vol. 129, No. 1, October 1987 Copyright © American Association of Pathologists

Characterization of Stromal Cells With Myoid Features in Lymph Nodes and Spleen in Normal and Pathologic Conditions

MARIE-FRANCOISE TOCCANIER-PELTE, MD, OMAR SKALLI, PhD, YUSUF KAPANCI, MD, and GIULIO GABBIANI, MD, PhD

From the Department of Pathology, University of Geneva, Geneva, Switzerland

Stromal cells with myoid features were identified in rat or human lymph nodes and spleen in normal and pathologic conditions, using antibodies to desmin, asmooth muscle actin, and smooth muscle myosin. In normal lymph nodes, myoid cells (MCs) were present in the superficial and deep paracortex as well as in the medulla, and absent in lymphoid follicles. In the spleen, they were numerous in the red pulp, less abundant in periarteriolar lymphocyte sheaths of the white pulp, and absent in lymphoid follicles. On double immunostaining, a-smooth muscle actin and smooth muscle myosin were coexpressed with desmin only in the deep paracortex and parafollicular areas of the lymph nodes, as well as in the MCs ofthe periarteriolar lymphocyte sheaths and marginal zone of the spleen; the remaining MCs expressed only desmin. When ex-

amined by means ofelectron microscopy, MCs showed a dendritic shape and cytoplasmic bundles of microfilaments with dense bodies scattered between them. When compared with normal conditions, MCs showed changes of distribution and number in several pathologic situations. Additional findings were 1) staining of pericytes surrounding high endothelium venules of lymph nodes with a-smooth muscle actin antibodies in man and rat and with desmin antibodies in rats; 2) staining of endothelial cells in these venules with desmin antibodies in rats. It is concluded that a subset of reticular cells in lymph nodes and spleen, as well as pericytes and endothelial cells in high endothelium venules display cytoskeletal features suggesting a myoid differentiation and function. (Am J Pathol 1987, 129:109-118)

THE PRESENCE OF smooth muscle cells in lymph node and spleen capsule and trabeculae has been known for a long time."2 However, most authors agree that the stroma of these organs consists of fibroblastic cells which may contain abundant cytoplasmic microfilaments.' 2 The introduction of new differentiation markers, and in particular cytoskeletal markers, has been very useful for the elucidation of the embryologic origin of several normal cells, and has become a routine application in the characterization of tumor biopsies (see review3-5). Smooth muscle cells can be differentiated from other mesenchymal cells, such as fibroblasts or endothelial cells, by the presence ofdesmin, an intermediate filament protein found only in muscle cells,6 a-smooth muscle actin, a specific marker of smooth muscle,7 and smooth muscle myosin.' Cells positive to immunofluorescent staining with a smooth muscle myosin specific antibody have recently been described in the stroma of lymph nodes

and spleen.9 In the present work, we have identified stromal myoid cells (MCs) positive to desmin, asmooth muscle actin, or smooth muscle myosin antibody immunostaining in rat or human lymph nodes and spleen, and correlated their distribution with that of lymphoid cells, characterized by means of specific surface markers. Our results indicate that MCs 1) are a distinct subset of reticular cells, 2) are heterogeneous in both lymph nodes and spleen as far as their cytoskeletal composition is concerned, and 3) behave characteristically in different disease states. Moreover, our results bring new information on the cytoskeletal features of pericytes and endothelial cells of high endothelium venules in lymph nodes.

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Supported in part bv the Swiss National Science Foundation, Grant 3.107-0.85. Address reprint requests to Dr. M.-F. Toccanier-Pelte, University of Geneva, Department of Pathology, CMU, 1 rue Michel-Servet, 121 1 Geneva 4, Switzerland.

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Materials and Methods Human and Animal Specimens The human specimens were collected from our Division of Surgical Pathology. Spleens were obtained from a 5-year-old child and 6 adult patients (with ages ranging from 18 to 71 years) splenectomized because of trauma, spleen enlargement with hypersplenism, or staging laparotomy. Lymph nodes were from 3 children (3-15 years old) and 39 adults (17-72 years old). The diagnoses were as follows: 6 reactive follicular hyperplasias; 4 chronic nonspecific lymphadenitis (which were used as controls); 1 sarcoidosis; 2 chronic granulomatous necrotizing lymphadenitis compatible with cat scratch disease; 7 lymph nodes from homosexuals and/or drug abusers presenting the lymphadenopathy of the acquired immunodeficiency syndrome (AIDS)10"'1 (3 of them had a positive anti-HTLV-Ill serologic test; the Type I histologic pattern '0 was observed in 6 cases and the Type II in one case); 11 Hodgkin's disease (4 mixed cellularity, 4 nodular sclerosis, 3 lymphocytic predominance); 8 non-Hodgkin's malignant lymphomas (2 centroblastic centrocytic follicular, 1 centroblastic centrocytic diffuse, 1 centrocytic diffuse, 1 lymphoplasmocytoid, 1 lymphoblastic Burkitt type, 1 lymphoblastic T cells and 1 T-cell angioimmunoblastic lymphadenopathy type diagnosed according to the Kiel classification'2); and 2 metastases of carcinomas. We studied 5 spleens and 12 axillary lymph nodes from 3 male and 2 female 8-week-old Wistar rats.

Routine Histology and Electron Microscopy Routine histology was performed on tissues fixed in formol or formol-mercuric chloride solutions. For transmission electron microscopy, tissue samples were cut into 1-mm cubes, fixed for 2 hours at room temperature in 2% glutaraldehyde buffered with 0.1 M cacodylate buffer at pH 7.4, and postfixed for 1 hour in 2% osmium tetroxide in the same buffer. The cubes were dehydrated in graded alcohol and propylene-oxide and embedded in Epon 812. Semithin sections were stained with toluidine blue; thin sections were double-stained on copper grids with uranyl acetate and lead citrate and examined in a Philips 400 electron microscope.

Antisera and Immunostaining Procedure Immediately after excision, animal and human tissue samples were embedded in OCT (Miles Laboratories Inc., Naperville, Ill), snap-frozen in liquid nitrogen, and stored at -70C, until use for

immunohistochemistry.

AJP 9 October 1987

Two- to 4-,u-thick cryostat sections were picked up onto slides coated with poly-L-lysin (Sigma Chemical

Co., St. Louis, Mo)'3 and air-dried for 1-4 hours. They were fixed in acetone at -20C for 5 minutes and transferred to phosphate-buffered saline (PBS) before indirect immunofluorescence staining. The different incubations with antibodies were done in moist chambers at room temperature for 30 minutes. Each incubation was followed by three washings of 10 minutes in PBS. As primary antibodies, we used the following affinity-purified polyclonal antibodies, whose specificity and optimal dilutions have been described previously: rabbit antidesmin,14 guinea pig antivimentin,'4 and rabbit antibovine aorta myosin.15 Control immunoglobulins were purified from preimmune sera on a protein A Sepharose 4B column and used at the same protein concentration as the corresponding specific antibody. a-Smooth muscle actin was identified with a specific monoclonal antibody (anti-asm- 1, hybridoma supernatant)"6; another hybridoma supernatant served as control. We also used a commercial monoclonal desmin antibody (Dakopatts AS, Copenhagen, Denmark), the desmin monoclonal antibody P37EH 11 (courtesy of Dr. I. Virtanen, Helsinki, Finland), and a rabbit serum containing antihuman fibronectin antibodies (courtesy of Dr. L. Zardi, IST, Genova, Italy). The phenotypic markers expressed on lymphoid cells were investigated with a panel of previously characterized, 17-23 commercially available monoclonal antibodies (Pan B, IgD, IgM, DRC1, RSCI, EMA, Leu-1, Leu-4, Leu-M1, Leu-M2, Leu-M3, anti-HLA-DR, IgK, IgL, OKT6, and

T29/33) (Dakopatts). As second antibodies, we used FITC-conjugated goat anti-rabbit IgG (Behringwerke AB, Marburg Lahn, FRG), FITC-conjugated goat antimouse IgG (Cappel Laboratories, Cochranville, Pa), FITC-conjugated goat anti-guinea pig IgG (Cappel Laboratories), and TRITC-conjugated swine anti-rabbit IgG (Dakopatts). Double immunostaining was performed with the different monoclonal antibodies against the phenotypic markers expressed on lymphoid cells listed above. Biotinylated horse anti-mouse IgG (Vectastain Kit mouse IgG PK 4002, Vector Laboratories) or anti-mouse IgM (Vector Laboratories, 1: 20) antibodies were then applied and followed by labeling with avidin-TRITC (Sigma Chemical Co., 1: 20). The sections were subsequently stained for desmin immunofluorescence as indicated above. Slides were mounted in polyvinyl alcohol medium.24 Immunotluorescence on cryostat sections of frozen tissues has been used in every case. In addition, se-

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MYOID CELLS IN LYMPH NODES AND SPLEEN

lected cases were examined by means of avidin-biotin complex (ABC) peroxidase method using cryostat sections or on formaldehyde-fixed paraffin-embedded material with the affinity-purified rabbit antidesmin (0.5 ,ug/ml) and the monoclonal anti-asm-l (1: 600). The different steps of the vectastain kit rabbit IgG PK 4001 (Vector Laboratories, Burlingame, Calif) were applied and the revelation of peroxidase activity was done with aminoethyl carbazole (AEC).25 The sections were then counterstained with hematoxylin. Slides were mounted on glycerocol. The results were always similar to those obtained by immunofluorescence on cryostat sections, which suggested that routine material can be studied with these antibodies. Photographs were taken with a Zeiss (Carl Zeiss, Oberkochen, FRG) photomicroscope equipped with epi-illumination and specific excitation filters for FITC (485 nm) or TRITC (546 nm) using Plan Neofluar objectives (1 6X, 25X, 40X) on 640-T color slide films (3M Company, Torino, Italy).

sets ofhuman lymphocytes and macrophages showed that no B- or T-cell markers were detectable on the surface of MCs. Furthermore, no labeling ofMCs was obtained with antibodies specific for follicular dendritic reticulum cells, interdigitating cells, or monocyte-macrophages. Finally, HLA-DR was not expressed by MCs. An unexpected distribution ofimmunofluorescent staining with anti-asm- 1 and antidesmin was observed in high endothelium venules. Here, endothelial cells appeared vimentin-positive in both rat and man, but surprisingly desmin-positive in rat (Figure 2c). They were always anti-asm- 1 negative. Moreover, pericytes of the periendothelial sheath were characteristically anti-asm- 1- and desmin-positive in rats (Figures 2b and c), but anti-asm- 1-positive and desmin-negative in man (Figures 1 f and g). Immunofluorescent staining of human and rat spleen showed a characteristic distribution of MCs (Figure 3). In periarteriolar lymphocyte sheaths around germinal centers and lymphocyte coronas some antidesmin- and anti-asm- 1-positive M( were noted. MCs were numerous in the marginal zone (Figure 3a), but none were present within the lymphoid follicles (Figures 3c and d). Immunofluorescent staining with smooth muscle myosin antibody showed a distribution similar to that of antidesmin and anti-asm- 1 (Figure 3b). In addition, desmin antibody, but not anti-asm- 1 and smooth muscle myosin antibodies, labeled a cellular network in the red pulp (Figure 3e). Antidesmin, anti-asm- 1, and anti-smooth muscle myosin strongly labeled the media of the central arteries in the white pulp (Figures 3a-d) and smooth muscle cells in the capsule and in the septa (Figures 3a, b, and f). Immunostaining with fibronectin antibodies revealed a dense network of extracellular fibrils running between the cells of the white pulp and the red pulp. The number and distribution of MCs were studied in several pathologic conditions of human lymph nodes and spleen (Figure 4). In lymph nodes, MCs were numerous beneath the capsule and around the epithelioid nodules of sarcoidosis. In the two cases of chronic granulomatous necrotizing lymphadenitis compatible with cat scratch disease, MCs were less abundant than in sarcoidosis and situated within the paracortex. All the Type I cases of AIDS lymphadenopathy showed a follicular hyperplasia with irregular lymphoid follicles, mottled mantle zones, and OKT8-labeled T cells crowded in the germinal centers. The Type II case showed an angioproliferative picture with a disappearance of the nodal architecture. In all cases of AIDS lymphadenopathy, MCs were particularly numerous in the paracortical areas

Results Distribution of MCs in Normal and Pathological Conditions Immunostaining with desmin antibodies ofcontrol human lymph nodes (Figure 1) showed many MCs, characterized by their dendritic shape and thin cytoplasmic expansions (Figures c and e), in the deep cortex and in the parafollicular areas; some were situated beneath the capsule and in the medulla (Figure l b and Id). MCs were absent in germinal centers and lymphocyte coronas (Figure ha). A positive desmin staining of MCs was also present (although weaker than with our affinity-purified antibody) after incubation with the commercial desmin antibody and with the monoclonal antibody P37EH 1. In addition, clusters of smooth muscle cells could be seen within the capsule and in the hilum (Figure 1d). Double immunostaining showed that MCs of both lymph nodes and spleen contain vimentin in addition to desmin (data not shown). Moreover, MCs of the deep cortex and parafollicular areas were labeled with both antidesmin and anti-asm- 1, but those beneath the capsule or in the medulla were only desmin-positive. MCs positive to anti-asm- 1 were also positive to anti-smooth muscle myosin. Smooth muscle cells present in the capsule, hilum, and media of arterioles and venules were always stained with desmin, anti-asm- 1, and smooth muscle myosin antibodies. In rat lymph nodes, MC had a distribution similar to that of human lymph nodes (Figure 2). Double immunostaining with desmin antibody and the monoclonal antibodies against various sub-

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Figure 1-Human lymph nodes (reactive follicular hyperplasia). a-c-Indirect immunofluorescent staining with desmin antibodies shows several MCs in the paracortical areas, but none in the germinal center (a); the medulla also contains MC (b). At higher magnification, the desmin positive MCs show a dendritic shape with thin elongated cytoplasmic processes (c). d-e-Immunoperoxidase staining on cryostat sections with desmin antibodies. d-Desmin-positive smooth muscle cells are seen in the capsule; moreover, the subcapsular area of the cortex contains some positive MCs. e-Detail of two MCs showing their elongated shape and cytoplasmic processes. f-g-Double immunofluorescent staining with antidesmin (f) and anti-asm-1 (g) shows that pericytes of high endothelium venules contain a-smooth muscle actin but no desmin and that endothelial celis are negative to both antibodies. gc, germinal center; smc, smooth muscle cells; hev, high endothelium venule. (a, b, d, X200; c, X630; e, Xl 000; f, g, X400)

(Figures 4c and d). MCs were rare in all types of nonHodgkin's malignant lymphomas and absent in metastases. In Hodgkin's disease, they were numerous in the mixed cellularity type (Figure 4e) and less abundant in the lymphocytic predominance type. In the nodular sclerosis type, the elongated cells typically present in sclerotic areas were decorated only with anti-asm- 1 (Figures 4g and h) and vimentin antibodies. Many MCs were observed in the two cases of leukemia (Figure 4f). In every neoplastic condition, the distribution of the MC was anarchic. In human spleen, MCs were constantly present in

cases of trauma and hypersplenism with nonspecific fibrosis; they were similarly distributed, but less numerous than in normal rat spleen. They were absent in cases of myeloid metaplasia with myelofibrosis, Hodgkin's disease, non-Hodgkin's lymphomas, and leukemia. In these conditions, typical smooth muscle cells were still present in the capsule and in the septa.

Electron-Microscopic Features of MCs The ultrastructural features of cells corresponding to MCs were studied essentially in the rat spleen. With

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along the nuclear envelope. No indented, "myofibroblastlike" nuclei26 were noticed. Mitochondria, ribosomes, and rough endoplasmic reticulum were abundant in the perinuclear areas (Figure 5a). The most important ultrastructural feature ofthe MC was the presence of microfilament (6-9 nm in diameter) bundles with focal dense bodies scattered in between (Figure 4b). These could be chiefly observed in the cytoplasmic expansions beneath the plasma membrane and were organized like stress fibers of cultured27 or in vivo26 cells. Occasionally, they could be seen to insert into cell surface condensations (Figure 5b). Finally, some electron-dense areas were present along the cytoplasmic membrane.

Discussion The use of cytoskeletal and contractile proteins as differentiation markers of normal and pathologic cells is presently well accepted (review3-5); the antibodies used in this work identify proteins that represent general differentiation markers of muscular cells (such as desmin36) or specific differentiation markers of smooth muscle cells (such as a-smooth muscle actin7 and smooth muscle myosin8"15). The expression of these proteins may show quantitative variations in different smooth muscle tissues according to anatomic location,7 pathologic situations714'28 and culture conditions.29'30 Our results show that lymph nodes and spleen of both man and rat contain a distinct subset ofreticular cells, for which we suggest the name of MCs, which contain desmin and generally,

a-Indirect immunofluorescent staining Figure 2-Rat lymph node. with desmin antibodies shows numerous positive MCs in a paracortical area; b and c-Double immunofluoMCs are absent from the germinal center. rescent staining with antidesmin (b) and antiasm-1 (c) shows that MCs of the deep cortex and, surprisingly, endothelial cells of the high endothelium venuies express desmin, whereas pericytes of the high endothelium venules (arrowheads) coexpress a-smooth muscle actin and desmin. gc, germinal center. (a, X200; b and c, X400)

conventional electron microscopy, these cells could be distinguished from the other reticular cells by their elongated shape and their long slender cytoplasmic processes closely associated with the extracellular reticulum fibers and neighboring cells (Figure 5). Their nuclei were ovoid, with a regular contour, and showed a finely granular pale chromatin focally accumulated

but not always, a-smooth muscle actin and smooth muscle myosin. Our results on the distribution of smooth muscle myosin in MCs agree in general with those of Pinkus et al,9 with the exception ofthe finding by these authors that contrary to our description, cells positive to smooth muscle myosin antibodies are present in germinal centers. This discrepancy may be due to differences in the selectivity of the antisera, because Pinkus et a19 used uterine myosin as antigen for the production of smooth muscle myosin antibody, whereas we used aortic smooth muscle myosin.-5 The distribution of desmin, a-smooth muscle actin, and smooth muscle myosin in MCs of lymph nodes and spleen is summarized in Table 1. It is noteworthy that MCs of different locations are heterogeneous as far as their cytoskeletal composition is concerned. This may correspond to different functional activities of such cells; further work is necessary in order to test this possibility. In any event, this and other reports9" 6'3'-33 suggest that a proportion ofstromal cells in various organs express proteins that are known as markers of myoid differentiation.

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Figure 3-Normal rat (a-d) and human (e-f) spleen. a-Indirect immunofluorescent staining with desmin antibodies results in a strong staining of the central artery and of smooth muscle cells scattered in the red pulp. MCs are numerous in the red pulp and in the marginal zone and scarce in the white pulp. b- Indirect immunofluorescent staining with smooth muscle myosin antibodies results in a strong staining of the central artery and of smooth muscle cells scattered in the red pulp; MCs of the white pulp and the marginal zone are stained, but not those of the red pulp. c-d-Double indirect immunofluorescent staining with antidesmin (c) and anti-asm-1 (d); the central artery and MCs in the white pulp and marginal zone show a positive staining to both antibodies, whereas the MCs of the red pulp are stained only with desmin antibodies (arrowheads). e-f-lndirect immunofluorescent staining with desmin antibodies of human spleen showing positive MCs in the red pulp (e) and smooth muscle cells in the septa (f). (a, b, and f, Xl 00; c, d, and a, X400)

When examined by means of electron microscopy, MCs contain bundles of microfilaments, similar to those described in "nonphagocytic fibroblastic reticular" cells.34'35 Cells having distribution and shape similar to those of MCs have been recently identified within lymph nodes and spleen by means of a monoclonal antibody directed against a hitherto uncharacterized marker.36 In both spleen and lymph nodes, MCs are characterized by a dendritic shape and elongated cytoplasmic expansions. However, MCs are heterogeneous as

far as their cytoskeletal composition is concerned: those located beneath the capsule and in the medulla of lymph nodes, as well as in the red pulp of the spleen, contain desmin, but no a-smooth muscle actin or smooth muscle myosin. We do not know whether these desmin-positive MCs (or some of them) contain y-smooth muscle actin, thus representing another subtype of MC. In any event, they resemble other desmin-positive anti-asm- 1-negative stromal cells recently described in the intestinal muscularis mucosae, testis interstitium, and uterine stroma.16 Desmin has


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