THE ANATOMICAL RECORD 249:486–494 (1997) Immunohistochemical and Structural Characteristics of the Reticular Framework of the White Pulp and Marginal Zone in the Human Spleen TAKASHI SATOH,* RIKIO TAKEDA, HIROKI OIKAWA, AND RYOICHI SATODATE Department of Pathology, School of Medicine, Iwate Medical University, Morioka, Japan ABSTRACT Background: The reticular framework of the white pulp (WP) and marginal zone (MZ) consists of reticulum cells and reticulin fibers. The antigenic heterogeneity of the reticular framework is well documented in the mouse and rat spleen. The aim of the present study is to characterize the reticular framework of the WP and MZ of the human spleen. Methods: Nine surgically resected human spleens were investigated. Five of the nine spleens were perfused. Formalin-fixed materials were embedded in paraffin and serial sections prepared for hematoxylin-eosin, silver staining, and immunohistochemical examination. Electron and immuno-electron microscopy were also applied. Using confocal laser scanning microscopy, the reticular framework was analyzed threedimensionally. Results: The reticulin fibers of the framework were immunostained for type IV collagen in the WP and MZ. The WP was three-dimensionally delimited by the a-smooth muscle actin (a-SMA)-positive reticulum cells. In the WP, the distribution of a-SMA-positive reticulum cells formed the reticular framework of the periarteriolar lymphoid sheath (PALS). They also ensheathed the reticulin fibers. Interdigitating cells (IDCs) were scattered throughout the framework. A few IDCs attached to the framework. In the lymph follicle (LF), reticulum cells were not a-SMA-positive. The mesh of follicular dendritic cells (FDCs) was found in the germinal center. In places, the reticulin fibers were involved in the mesh of the FDCs and covered by the cytoplasm of FDCs. In the MZ, a-SMA-positive reticulum cells were arranged in a mesh pattern and ensheathed the fine reticulin fibers. Conclusion: The reticular framework of the PALS, LF, and MZ is specialized into heterogeneous components in the human spleen. The heterogeneity of the framework may induce the segregation of T and B lymphocytes. Anat. Rec. 249:486–494, 1997. r 1997 Wiley-Liss, Inc. Key words: reticular framework; human spleen; white pulp; marginal zone; heterogeneity The white pulp (WP) and marginal zone (MZ) constitute the immunologically active compartment of the spleen. In the WP, there are two distinct regions, the periarteriolar lymphoid sheath (PALS) and the lymph follicle (LF). The MZ is located between the WP and the red pulp. The basic framework of the WP and MZ consists of reticulum cells and reticulin fibers (Veerman and Van Ewijk, 1975a; Sasou and Sugai, 1992). However, a distinct microenvironment is formed in the reticular framework of the PALS, LF, and MZ, respectively. The PALS is a thymus-dependent area in which T lymphocytes predominate (Parrott et al., 1996; Goldschneider and McGregor, 1973). The LF and MZ are B lymphocyte areas (Goldschneider and McGregor, 1973; r 1997 WILEY-LISS, INC. Kumararatne et al., 1981). However, the cellular composition of the MZ comprises intermediate-sized B lymphocytes, and the immunophenotype of these lymphocytes is different from the B lymphocytes in the mantle zone of the LF (Kumararatne et al., 1981; Bazin et al., 1982; Van Krieken et al., 1989; Satoh, 1991). In the PALS and LF, interdigitating cells (IDCs) and follicular dendritic cells (FDCs) create a distinct microenvironment and play a role in the homing mechanism of T and B *Correspondence to: Takashi Satoh, M.D., Department of Pathology, School of Medicine, Iwate Medical University, Uchimaru 19-1, Morioka 020, Japan. E-mail: firstname.lastname@example.org Received 12 March 1997; Accepted 1 August 1997 487 RETICULAR FRAMEWORK WHITE PULP/MARGINAL ZONE lymphocytes, respectively (Veerman and Van Ewijk, 1975; Wood et al., 1985). The enzyme histochemical variety of reticulum cells in the WP of the human spleen has been well documented by Müller-Hermelink et al. (1979). A heterogeneous population of reticulum cells was immunohistochemically observed in the WP of the rat spleen (Van den Berg et al., 1989). Yoshida et al. (1991) proved the antigenic heterogeneity of the reticular framework in the WP of the mouse spleen in relation to lymphoid subclasses. They discovered that the reticulum cells are heterogeneous in the PALS and LF. A recent study TABLE 1. Clinical features Case Age (yr) Sex 1 2 3 4 5 59 38 73 65 33 m m m f m 6 7 74 77 m m 8 33 9 2 Splenic weight (g) 701 8501 781 651 5001 Perfusion 1 1 1 1 1 2 2 f 100 not weighed 85 f 40 2 2 Clinical diagnosis Gastric cancer Liver cirrhosis Gastric cancer Gastric cancer Hereditary spherocytosis Gastric cancer Gastric cancer Idiopathic thrombocytopenic purpura Idiopathic thrombocytopenic purpura 1Before perfusion. revealed that the reticular mesh localized between the MZ and WP expresses mucosal addressin cell adhesion molecule-1 in the mouse spleen and plays an essential role in the lymphocyte homing and compartmentalization mechanism in the WP (Tanaka et al., 1996). The aim of the present study is to characterize the reticular framework of the WP and MZ of human spleen, using immunohistochemistry, confocal laser scanning, and electron microscopy. MATERIALS AND METHODS Nine surgically resected human spleens were investigated, and their description is summarized in Table 1. Five spleens were perfused by the arterial and venous pressure-loading method (Suzuki et al., 1989), using Ringer’s solution containing heparin (150 IU/l) to wash out the free cells. The spleens were then fixed by perfusion with 2.8% glutaraldehyde solution in 0.06 M phosphate buffer. They were cut into 10 mm slices and further fixed in 10% buffered formalin solution for 3 days. The other four spleens were not perfused. They were directly cut into 10 mm slices and fixed in 10% buffered formalin solution for 2 days. Tissue blocks, measuring 15 x 10 x 5 mm, were cut from both the perfusion- and nonperfusion-fixed materials, and then embedded in paraffin after dehydration in a graded ethanol series. Serial sections were prepared for hematoxylin-eosin, silver staining (Gomori’s method), and immunohistochemical examination. TABLE 2. Antibodies used for immunohistochemistry Antibodies Type IV collagen a-smooth muscle actin S-100 protein UCHL-1 (CD45RO) L26 (CD20/cy) Ki-M4P Reactivity Working dilution Source Type IV collagen a-smooth muscle isoform of actin Interdigitating cells T lymphocytes B lymphocytes Follicular dendritic cells 1:100 1:100 1:200 1:200 1:100 1:1,000 DAKO DAKO DAKO DAKO DAKO Prof. H.J. Radzun Göttingen, Germany Fig. 1. (a) Silver impregnation. The number and spatial orientation of reticulin fibers are different in the PALS, LF, and MZ. In the PALS, several layers of reticulin fibers run parallel to the central artery. In the LF, reticulin fibers are sparse. Fine reticulin fibers form a mesh structure in the MZ. x 100. (b) Immunostaining of type IV collagen. The reticulin fibers in the PALS, LF, and MZ are immunostained for type IV collage n. x 250. 488 T. SATOH ET AL. Fig. 2. Immunostaining of a-SMA (brown). (a) The WP is clearly bordered by a-SMA-positive spindle-shaped or stellate cells. x 100. (b) A few layers of a-SMA-positive cells (arrowheads) were found at the border of the LF and MZ. In the MZ, a-SMA-positive cells are arranged in a mesh pattern. x 250. Fig. 3. Double immunostaining of a-SMA (brown) and UCHL-1 (blue). a-SMA-positive cells form the reticular framework of the T lymphocyte area (PALS). Fig. 4. Double immunostaining of a-SMA (brown) and L26 (blue). In the LF, no a-SMA-positive cells are found except for the smooth muscle cells of the blood vessels. x 100. Immunohistochemistry for Light Microscopy technique (Shi et al., 1991) was applied for the retrieval of the antigen. For double immunostaining, the first antigen was visualized in red as described above. After washing by PBS, the SABC or APAAP method was applied to the second antigen, which was visualized in blue with the aid of alkaline phosphatase by incubating sections in 1 mg/ml naphthol AS phosphate (Sigma, St. Louis, MO), 1 mg/ml Fast blue BB salt (Sigma), and 1 mM Levamisole (Sigma) in 0.05 M propandiol buffer, pH 9.75. The antibodies used in the present study are summarized in Table 2. Commercially available staining kits (Histofine SAB-PO and SAB-AP, Nichirei, Tokyo, and DAKO APAAP kit, DAKO, Denmark) were used. After deparaffinization or silver impregnation, the sections were immersed in methanol-H2O2 solution for 30 min to block endogenous peroxidase activity. Nonspecific binding of antibodies was blocked by incubation with normal goat or rabbit serum for 15 min at room temperature. The sections were incubated with primary antibodies for 2 hr at room temperature, and then treated using the streptavidin biotin complex (SABC) method. Each step was followed by repeated washing in phosphate-buffered saline (PBS) adjusted to pH 7.4. Coloration was developed in 3-amino-9-ethylcarbazole solution (Graham et al., 1965). The sections were counterstained with hematoxylin. In immunostaining of type IV collagen, the microwave oven heating- Three-dimensional Analysis of the Reticular Framework Formalin-fixed spleens were cut into 50 µm slices with a microslicer (Dosaka, Kyoto, Japan). Sections were immunostained with anti-a-SMA antibodies using the SABC method and then incubated with FITCconjugated streptavidine (DAKO). The sections were attached to glass slides and covered by a coverglass. Images of the reticular framework were obtained using RETICULAR FRAMEWORK WHITE PULP/MARGINAL ZONE 489 Fig. 5. Silver impregnation (black) and immunostaining of a-SMA (brown). (a) The distribution of the a-SMA-positive cells corresponds to that of the reticulin fibers. x 100. (b) a-SMA-positive cells attach to and ensheath the reticulin fibers. x 250. Fig. 6. Double immunostaining of a-SMA (brown) and S-100 protein (blue). IDCs attach to the a-SMA-positive cells. x 500. Fig. 7. Silver impregnation (black) and immunostaining of Ki-M4P (red). In the germinal center of the LF, the reticulin fibers are involved in the mesh of the FDCs. x 500. a confocal laser scanning microscope (LSM-GB 200 type CLSM Olympus, Tokyo, Japan). Fluorescence of FITC was obtained using 488 nm argon/krypton laser excitation. Serial sections were analyzed at 0.8 µm intervals. MICRO VOXEL Ver. 2.2 software (INDEC, Capitola, CA) was used for the three-dimensional reconstruction. Immuno-electron Microscopy Tissue blocks were fixed in 1% glutaraldehyde solution and 4% PLP at 4 °C for 1 day, then cut into 30 µm slices with a microslicer (Dosaka). The immunostaining procedure was the same as used for light microscopy. The sections were incubated with antibodies for 4 hr at room temperature. Coloration was developed in DAB solution (WAKO, Osaka, Japan) to which H2O2 was added at 0.015%. After immunostaining, the sections were postfixed with 1% glutaraldehyde and 1% osmium tetroxide solution, dehydrated in a graded ethanol series, and embedded in Epon. Ultrathin sections were stained with uranyl citrate and examined using an electron microscope (H-7100 Hitachi, Tokyo, Japan). Electron Microscopy For transmission electron microscopy, tissue blocks, measuring 1 x 1 x 1 mm in size, were prepared from the perfusion-fixed spleens. They were postfixed in a 1% osmium tetroxide solution for 2 hr and embedded in Epon after dehydration in a graded ethanol series. Ultrathin sections were stained with uranyl acetate and lead citrate, then examined using a transmission electron microscope (H-7100 Hitachi). RESULTS Light Microscopy The sections treated by silver impregnation for reticulin fibers (Gomori’s method) revealed a basic reticular framework in the WP and MZ (Fig. 1a). The number 490 T. SATOH ET AL. Fig. 8. Three-dimensional analysis of the reticular framework of the WP using a confocal laser scanning microscope. a-SMA-positive cells clearly demarcate the WP (arrows). In the PALS, a wall-like structure is formed by a-SMA-positive cells. x 600. Fig. 9. (a) Ultrastructure of the reticulum cell (R) in the PALS. The reticulum cell has bundles of microfilaments with dense bodies (arrowheads). The cytoplasm ensheathes the reticulin fibers (rf). x 5250. (b) Immuno-electron microscopy of a-SMA. The cytoplasm of the reticulum cell (R) is a-SMA-positive (double arrowheads). x 5000. RETICULAR FRAMEWORK WHITE PULP/MARGINAL ZONE 491 Fig. 10. Ultrastructure of the IDC. The IDC extends cytoplasmic projections between the lymphocytes and entwines cytoplasmic projections around the reticulin fibers (arrowheads). x 4500. and spatial orientation of reticulin fibers were different in the WP and MZ. In the PALS, several layers of reticulin fibers ran parallel to the central artery, which showed a stratiform pattern in longitudinal sections and a circumferential pattern in cross sections. In the LF, reticulin fibers were sparse. A few layers of reticulin fibers bordered the LF and MZ. The reticulin fibers of the MZ were quite fine and anastomosing, forming a mesh structure. Immunohistochemistry The reticulin fibers of the WP and MZ were immunostained for type IV collagen (Fig. lb). The WP was clearly bordered by a-SMA-positive spindle-shaped or stellate cells (Figs. 2a,b). They were connected by their cytoplasmic processes in places. At the border of the LF and MZ, a few layers of a-SMA-positive cells were found (Fig. 2b). The distribution of a-SMA-positive cells in the WP was unique and characteristic. Double immunostaining of a-SMA and UCHL-1 or L26 revealed that a-SMA-positive cells were selectively distributed in the PALS (Figs. 3,4). At the perifollicular region, T lymphocytes were found between a-SMA-positive cells (Fig. 3). Silver impregnation and immunostaining of the a-SMA disclosed the close relation of a-SMA-positive cells and reticulin fibers (Fig. 5a). a-SMA-positive cells attached to and ensheathed the reticulin fibers (Fig. 5b). Interdigitating cells (IDCs) were scattered throughout the reticu- lar framework. Several IDCs attached to the a-SMApositive cells (Fig.6). In the LF, no a-SMA-positive spindle-shaped or stellate cells were found except for the smooth muscle cells of blood vessels (Fig. 4). Immunohistochemistry of Ki-M4P revealed the mesh of follicular dendritic cells (FDCs) in the germinal center (Fig. 7). In places, the reticulin fibers were involved in the mesh of the FDCs and ensheathed by the cytoplasm of the FDCs (Fig. 7). a-SMA-positive cells were arranged in a mesh pattern in the MZ (Fig. 2b). The fine reticulin fibers of the MZ were covered by the cytoplasm of the a-SMApositive cells (Fig. 5b). Three-dimensional Analysis of Reticular Framework a-SMA-positive cells clearly demarcated the WP. In the PALS, they extended their cytoplasm three-dimensionally and formed a wall-like structure (Fig. 8). A mesh structure was formed by a-SMA-positive cells in the MZ. Electron and Immuno-electron Microscopy Free cells remained in the WP after perfusion, whereas they were partly washed away in the MZ. The number and spatial orientation of the reticulin fibers were different as seen by light microscopy. The framework of the WP was constructed of reticulin fibers and reticulum cells. The cytoplasm of the reticulum cells ensheathed the reticulin fibers (Fig. 9a), 492 T. SATOH ET AL. Fig. 11. Immuno-electron microscopy of Ki-M4P. The dendritic processes of the FDCs (arrowheads) extend between the lymphocytes and ensheath the reticulin fibers (rf). x 4500. although some reticulin fibers were not enclosed. Reticulum cells in the PALS had bundles of microfilaments with dense bodies (Fig. 9a). Immmuno-electron microscopy revealed that the reticulum cells in the PALS were a-SMA-positive (Fig. 9b). IDCs extended their cytoplasmic projections between the lymphocytes in the PALS. In places, the cytoplasm of IDCs attached to reticulum cells. Several IDCs directly entwined their cytoplasmic processes around reticulin fibers (Fig. 10). The reticulum cells in the LF were a-SMA-negative. FDCs elongated their slender cytoplasmic processes among the lymphocytes and covered the reticulin fibers (Fig. 11). In the MZ, reticulin fibers and reticulum cells formed the framework of the MZ (Fig. 12a). The reticulin fibers were ensheathed by the cytoplasm of the reticulum cells, which were a-SMA-positive. The cytoplasm of the reticulum cells contained bundles of microfilaments with dense bodies and was connected with that of other reticulum cells. An intermediate junction was observed between their cytoplasms (Fig. 12b). DISCUSSION In the lymphatic tissues, reticulum cells and reticulin fibers form the basic reticular framework. T and B lymphocyte areas are involved in the reticular framework, and a specific microenvironment is prepared for the lymphocytes in each area, respectively. The spleens of human and rodent have an open circulation (Weiss, 1984; Sasou et al., 1986; Satodate et al., 1986a). The splenic arteries terminate in the splenic cord and the marginal zone (MZ). The marginal zone bridging channel was first described by Mitchell (1973), especially as an exit pathway from the PALS to the red pulp. However, this channel also has been described by other authors (Van Ewijk and Van der Kwast, 1980; Brelińska et al., 1984; Pellas and Weiss, 1990; Satoh, 1991) as an entrance pathway of T and B lymphocytes from the red pulp to the PALS. T and B lymphocytes from the blood are segregated during intrasplenic migration and are selectively sorted in the reticular framework according to the distinct microenvironment. Among the various stromal cells of the lymphatic tissues, the dendritic cells are thought to provide a microenvironment specific for lymphoid subclasses. IDCs and FDCs are found in the T and B lymphocyte area, respectively (Veerman and Van Ewijk, 1975a; Wood et al., 1985). Dijkstra et al. (1982) demonstrated that during the regeneration process of heterotropic autotransplanted splenic implants, T and B lymphocytes show a specific homing pattern in the developing WP and that the newly formed reticulum determines the distribution of the homing lymphocytes. Reticulin fibers were commonly positive for type IV collagen in the WP and MZ in the present study. Type IV collagen is the major protein in basement membranes (Martinetz-Hernandez and Amenta, 1983). Foidart et al. (1980) suggest that laminin is an ubiquitous component of basement membranes in addition to type IV collagen, although the distribution of laminin was not examined in the present study. Type III collagen, RETICULAR FRAMEWORK WHITE PULP/MARGINAL ZONE 493 Fig. 12. Ultrastructure of the reticular framework in the MZ. (a) Reticulum cells (R) and reticulin fibers (rf) form the mesh. The cytoplasm of each reticulum cell has bundles of microfilaments with dense bodies and ensheathes the reticulin fibers (small arrowheads). x 6000. (b) An intermediate junction is observed between the cytoplasms of the reticulum cells (large arrowheads). x 25500. which is mainly localized in the capsule and trabeculae, is also contained in matrix proteins of the reticulin fibers of the WP and MZ, although it is absent in the early stage of the neonatal development of the WP and MZ (Macarak et al., 1986; Liakka et al., 1991). The present study demonstrated that heterogeneous reticulum cells formed the reticular framework of the WP and MZ. a-SMA-positive reticulum cells delimited the WP from the surrounding MZ and were arranged similar to the marginal metallophil cells of the rat spleen (Snook, 1964; Satodate et al., 1971) in the perifollicular region. In the rat spleen, marginal sinuses, formed by the arterial terminals of the WP, also envelop the WP and bound the inner edge of the MZ (Snook, 1964; Sasou et al., 1976), although these structures are not found in the human spleen (Sasou et al., 1986). In the WP, the distribution of a-SMA-positive reticulum cells corresponded to the types of lymphoid subclasses and formed the reticular framework of the T-lymphocyte area. Actin is an ubiquitous cytoskeletal protein in microfilaments. a-SMA is the isoform of actin and routinely used as a reliable probe for smooth muscle cell differentiation (Skalli et al., 1986). The ultrastructure and immunoreactivity of the reticulum cells to a-SMA in the T lymphocyte area suggest the myofibroblastic differentiation of these cells. In the LF, the reticulum cells showed no positive reactivity for a-SMA. The mesh of FDCs involved the reticular framework of the germinal center. Although the ontogeny of FDCs remains contro- versial (Parwaresch et al., 1983; Wood et al.,1985; Imai and Yamakawa, 1996), FDCs seem to play a role in the formation of the reticular framework of the germinal center. The MZ is a discrete compartment and functions as an immunological filter where blood-borne antigens are trapped (Veerman and Van Rooijen, 1975b; Satodate et al., 1977; Satodate et al., 1986b). The major cell population of the MZ is medium-sized B lymphocytes (Van Krieken et al., 1989). MZ B lymphocytes are CD23-, KiB3- , IgD-, and enzyme histochemistry reveals the alkaline phosphatase activity of MZ B lymphocytes (Kumararatne et al., 1981; Bazin et al., 1982; Van Krieken et al., 1989; Satoh, 1991). The immunophenotype and enzyme phenotype distinguish them from the B lymphocytes in the mantle zone of the LF. The reticulum cells in the MZ were a-SMA-positive and their immunohistochemical character was different from those in the LF. Immunostaining of the a-SMA well characterizes the reticular framework of the WP and MZ in the human spleen and is applicable to the further examination of the reticular framework in other human lymphoid tissues. The results of the present study demonstrate that the reticular framework of the PALS and LF in the human spleen is specialized into heterogeneous components as in the rat and mouse spleen (Van den Berg et al., 1989; Yoshida et al., 1991). Although the present study has not yet proceeded to the functional analysis of the reticular framework regarding the adhesion mol- 494 T. SATOH ET AL. ecules or their ligand in lymphocyte homing and segregation, the antigenic heterogeneity of the reticular framework of the PALS and LF may induce the segregation of the lymphoid subclasses. ACKNOWLEDGMENTS The authors thank Mr. S. Hayashi, Laboratory of Electron Microscopy, School of Medicine, Iwate Medical University, for his technical assistance in the threedimensional analysis. LITERATURE CITED Bazin, H., D. Gray, B. Platteau, and I.C.M. MacLennan 1982 Distinct d1 and d-B-lymphocyte lineages in the rat. Ann. NY Acad. Sci., 399:157-174. Brelińska, R., C. Pilgrim, and I. 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