Arch. Histol. Cytol., Vol. 55, No. 4 (1992) p. 351-359 The Enveloping of Intercellular Collagenous Fibrils by Reticular Cell Processes in Postnatal Development of Rat Lymph Nodes Toshiko YOSHIDA and Kenichi TAKAYA Department of Anatomy, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan Received July 20, 1991; revised manuscript June 30, 1992 Summary. In the lymph nodes of adult rats reticular SMIT et al., 1980; HENDRIKS et al., 1981). They are fibers are known to be covered by the processes of similar in their all having a close relation with reti- reticular cells. This study aims to visualize the sequence cular fibers (SORENSEN,1960; HAN, 1961; CLARK, 1962; of the envelopment of reticular fibers by reticular cells MOE, 1963; BAIRATI et al., 1964; MIYATA and TAKAYA, during development. Rat popliteal lymph nodes of one 1981, 1983; HAYAKAWA et al., 1988). Particularly, the to twenty-three days after birth were examined by electron microscopy. reticular cells in the lymph node sinus characteristi- At the earliest stages, collagenous protofibrils were cally enclose the reticular fibers with their own cyto- found in the intercellular space between studded mesen- plasmic processes whith have electron dense areas chymal cells. The protofibrils clustered around the beneath the membrane (MIYATA and TAKAYA, 1981, plasma membrane of immature reticular cells and then 1983, 1984, 1985). In the present study, the sequence of became arranged into microfibrils of 30-40nm in diame- enclosure of the reticular fiber by the reticular cell ter. Bundles of the fibrils which might be called reicular prosess was observed by electron microscopy of rat fibers were surrounded by processes of more than one lymph nodes in postnatal development. reticular cell. Then the reticular fiber came to be en- closed by the cytoplasmic process of a single reticular cell. Finally at 16-23 days after birth, the reticular fiber MATERIALS AND METHODS was completely ensheathed by the thick cytoplasmic process of a single reticular cell closed with a functional complex. Throughout these periods, basal lamina-like Wistar rats of both sexes aged 1-7, 9, 14, 16, 23 days materials existed between the reticular fiber and cyto- and 12 weeks were used for this study. Under Nem- plasmic process. Clumps of fibrils were rarely in contact butal anesthesia, the thorax was opened and the with leukocytes, including lymphocytes. Immature elas- blood was expelled by perfusion of physiological tic fibers appeared among collagenous fibrlis of the saline through the left ventricle. Perfusion fixation reticular fiber when the fiber came to be enclosed by with Karnovsky's fluid followed, and the popliteal processes of some reticular cells. lymph nodes of both sides were removed and refixed It was shown that the enclosure of the reticular fiber in the same fluid at 4°C for 4 h. The tissue blocks were by the reticular cell did not result from physical pres- rinsed in the phosphate buffer with the addition of sure due to the increase of the number of lymphocytes, 10% sucrose, then postfixed in 2% osmium tetroxide, but the reticular cell actively enclosed the reticular fiber. The reticular cell processes enveloping the reticu- and then stained en bloc in 3% aqueous uranyl ace- lar fiber were connected with desmosome-like function- tate solution. They were dehydrated through an al complexes to prevent the fiber from escaping. alcoholic series, cleared in propylene oxide and em- bedded in Quetol 812. Structures were confirmed in the 1, um semithin sections which were stained with Certain varieties of reticular cells have been found toluidine blue. Ultrathin sections were doubly stained in the sinus, cortex and medulla of the lymph node in uranyl acetate and lead citrate. They were (STUART, 1975; HOEFSMIT, 1975; KAMPERDIJKet al., examined under an electron microscope (JEM-1005) 1978; MULLER-HERMELINKand LENNERT, 1978; HOEF- at an acceleration voltage of 80 kV. 351 352 T. YOSHIDA and K. TAKAYA: 1 2 Fig. 1. Popliteal lymph node from a one-day-old rat. A mesenchymal cell (MC) is located near the capillary (C). The cytoplasm contains rough endoplasmic reticulum (rER). Collagenous protofibrils exist in the intercel- lular space (arrow). X4,100 Fig. 2. Popliteal lymph node from a one-day-old rat. Note incontinuous basal lamina (*) present on one side of a mesenchymal cell (MC) and the dense area (D) inside the plasma membrane. Protofibrils (arrows) are located in the intercellular spaces. x11,800 Fig. 3. Popliteal lymph node from a two-day-old rat. Clumps of protofibrils (Pf) in the intercellular spaces are attached to the plasma membrane of a mesenchymal cell in the area with dense material beneath it. In the cytoplasm, bundles of filaments (Fil) and a few mitochondria (mi), multivesicular bodies (mvb) and rough endoplasmic reticulum (rER) filled with flocculent substance are visible. v Vesicle x8,700 Fig. 4. Popliteal lymph node from a four-day-old rat. Several immature reticular cells (RC) are grouped to bridge capillaries (C) and constitute a reticulated network. A cell near the capillary extends its cytoplasmic process to form an arch with another cell process, thus surrounding the reticular fiber (Rf). A high magnification of the area is shown in Fig. 6. En endothelial cell. X3,900 Fig. 5. Popliteal lymph node from a five-day-old rat. The cytoplasm of an immature reticular cell extends its process to cover the reticular fiber (Rf) which consists of collagenous fibrils. A mass of microfibrils (arrows), probably of elastic fibers, is seen in the reticular fiber. No homogeneous substance is recognized yet. Note dilated cistern of rough endoplamic reticulum (rER) filled with flocculent substance. mi mitochondria, Ly lysosome x 10,000 Fig. 6. A higher magnification of the area of Rf in Figure 4. The reticular fiber (Rf) consisting of collagenous fibrils, 30-40 nm in diameter, is covered with the cytoplasmic processes of some immature reticular cells on one side and faced with an endothelial cell (En) on the other side. Elastic fibers (Ef) are constituted only of abundant microfibrils, or those surrounding amorphous materials (arrows). A basal lamina-like substance is seen outside the cell process plasma membrane facing the reticular fiber (arrowheads). X26,000 Reticular Fibers of the Lymph Node in Rat Postnatal Development 353 3 4 5 6 Figs. 3-6. Legends on the opposite page. 354 T. YOSHIDA and K. TAKAYA: 7 8 Fig. 7. Popliteal lymph node from a nine-day-old rat. The reticular fibers (Rf) consisting of collagenous and elastic elements are located among the processes of two or three cells. The fibers show close contact with the plasma membrane with dense areas inside it. RC reticular cell. x6,000 Fig. 8. Popliteal lymph node from a ten-day-old rat. Clumps of protofibrils (Pf) unite to form collagenous fibrils (Cf) in the intercellular space. They closely contact the cell membrane with the dense area (D) beneath it. Note distinct periodicity (arrows) on the collagenous fibrils. The fibrils are 40nm in diameter. mi Mitochondria. x11,000 Fig. 9. Popliteal lymph node from a 16-day-old rat. An immature reticular cell (RC) extends its cytoplasmic process (Cyp) toward the reticular fiber (Rf) to cover it (Cyp and arrows). No other cells-including lymphocyte (L)-come in contact with the reticular fiber. X 10,000 Fig. 10. Popliteal lymph node from a 16-day-old rat. The cytoplasmic process (Cyp and arrows) of a reticular cell (RC) form eaves around the fiber. Rf reticular fiber. x6,600 Fig. 11. Popliteal lymph node from a 23-day-old rat. Note the reticular fiber (Rf) completely surrounded by the cytoplasmic process (Cyp and arrows) of a reticular cell (RC). Dense material is seen inside the plasma membrane of the cell process facing the reticular fiber. The reticular cell attaches to the capillary (C) on one side and extends several cytoplasmic processes on the other. X6,000 Fig. 12. Another example of the reticular fiber (Rf) completely wrapped by its own cytoplasmic process of a single reticular cell seen in the lymph node from a ten-day-old rat. (The feature was quite rarely found in the lymph node at this developmental stage.) The reticular cell possesses junctional complexes (arrowheads) as if in order to prevent the fibers from escaping out of the enclosure. Elastic fibers (Ef) in the reticular fiber consist of only microfibrils and those surrounding amorphous materials. X 16,000 Fig. 13. In the lymph node of a 23-day-old rat, the cytoplasmic processes of the reticular cell are tightly connected with each other by a desmosome-like junctional complex (arrowheads). The reticular fiber (Rf) is completely enclosed. Note the basal lamina-like dense material outside the plasma membrane of the cell process facing the reticular fiber (Rf).x21,000 Reticular Fibers of the Lymph Node in Rat Postnatal Development 355 9 10 12 11 13 Figs. 9-13. Legends on the opposite page. 356 T. YOSHIDA and K. TAKAYA: increased in number and accumulated in the network. RESULTS The reticular cell in primary follicles enclosed the reticular fiber completely by its cytoplasmic process Subcapsular sinuses were present in the popliteal with junctional complexes (Fig. 12). However, in the lymph nodes of rats already on the first postnatal areas surrounding the follicles and in those develop- day. Cells lining the capsule and retothelial cells, ing into follicles, some presumptive reticular cells which were a type of reticular cell, enclosed the were extending their processes to wrap the reticular reticular fibers and made contact with each other by fibers (Figs. 7, 9), while others made eaves around the junctional complexes, as desctibed in the lymph node fibers (Fig. 10). In the area of developing follicles, sinus of the adult rat (MIYATA and TAKAYA, 1981).