The Enveloping of Intercellular Collagenous Fibrils by Reticular Cell Processes in Postnatal Development of Rat Lymph Nodes

Arch. Histol. Cytol., Vol. 55, No. 4 (1992) p. 351-359

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 enclosed 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 intercellular 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

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). clumps of collagenous protofibrils still existed in No distinction was apparent between the cortex and intercellular spaces and were aggregated to fibrils the medulla of the lymph node. They were studded with periodicity (Fig. 8). In these areas, most reticular with numerous capillaries and mesenchymal cells. fibers were surrounded by the cytoplasmic processes Collagenous protofibrils were arranged irregularly in derived from two or three reticular cells. However, the intercellular spaces (Figs. 1, 2). These were below they were rarely found close to mononuclear cells 5 nm in thickness, with no periodicity clearly visible. (Fig. 7). In the cytoplasm of mesenchymal cells, rough endo- At 23 postnatal days, the lymph node was filled plasmic reticulum with dilated cisterns was filled with lymphocytes, other migrating cells and reticular with flocculent materials (Fig. 1). A dense substance cells. Primary follicles and follicles with germinal similar to the dense plaque of smooth muscle cells centers were already visible. Most reticular fibers was formed beneath the plasma membrane, and in- were now recognized either completely enclosed by a continuous basal lamina occurred around some single reticular cell or intervening between the mesenchymal cells (Fig. 2*). processes of two reticular cells. However, they were Two to five days after birth, the distinction be- rarely found close to leukocytes (Fig. 11). The cyto- tween the cortex and the medulla was obvious. In the plasmic processes of a single reticular cell enveloping cortex, interconnected mesenchymal cells bridged the reticular fiber were connected with each other by capillaries (Fig. 4). However, no junctional complexes desmosome-like junctional complexes. It seemed as if were observed between the mesenchymal cells. The they were preventing the fibers from escaping out of cells may be called immature reticular cells, because the enclosure (Fig. 13). Basal lamina-like dense mate- they are progressing toward a close relation with rials were located outside, and punctated dense areas reticular fibers. Lymphocytes and other leukocytes were dispersed inside the plasma membrane of the were few in number. Collagenous protofibrils ac- reticular cell process surrounding the reticular fiber cumulated in the intercellular space outside the (Fig. 13). Elastin in the reticular fibers was not render- plasma membrane near electron dense areas of im- ed dense by the usual uranyl acetate and lead citrate mature reticular cells (Fig. 3). They were united to staining. form fibrils which were 30-40nm in diameter and had In summary, collagenous protofibrils released into a distinct periodicity periodicity (Figs. 5, 6). Clumps the spaces among mesenchymal cells accumulated of collagenous fibrils were covered by the cytoplas- near the dense plaque areas of their plasma mem- mic processes of immature reticular cells. Micro- brane, and were arrayed as fibrils. At the same time fibrils and homogenous elastin surrounded by them they began to be enwrapped by processes of imma- were dispersed among the collagenous fibrils, to form ture reticular cells, which became the mature what can be called a reticular fiber (Figs. 5, 6 arrows). reticular cells enclosing the reticular fiber (Fig. 14). Between the reticular fibers and the plasma membrane of the cytoplasmic processes were basal lamina-like materials. The immature reticular cells DISCUSSION possessed dilated cisterns of endoplasmic reticulum, not great in number, which were filled with amor- The present syudy demonstrated the process of how phous materials (Figs. 3,5). In their cytoplasm a large intercellular collagenous fibrils were enclosed by the number of vesicles, small mitochondria, multivesicu- cytoplasmic processes of reticular cells in the rat lar bodies, lysosomes and bundles of filaments were lymph node during postnatal development. The observed (Fig. 3). sequence of association of reticular fibers and reticu- At 7-16 postnatal days, immature reticular cells lar cells during development seems to suggest the formed a reticulated network with the junctional singificance of the reticular cell-reticular fiber com- complexes. Lymphocytes and other migrating cells plex in the function of the lymph node. They central Reticular Fibers of the Lymph Node in Rat Postnatal Development 357

0-1 day 2-5 day

23 day 7-16 day

Fig. 14. The schema of the process of enclosure of reticular fibers. 0-1 day: Collagenous protofibrils (Pf) localized in intercellular spaces. 2-5 day: Protofibrils clustered around the plasma membrane and arranged into microfibrils (Cf) 30-40nm in diameter. 7-16 day: Bundles of the fibrils (reticular fibers; Rf) are surrounded by the cytoplasmic processes of some reticular cells. Immature elastic fibers (Ef) appear among collagenous fibrils. 23 day: The reticular fibers (Rf) are enclosed by cytoplasmic processes with desmosome-like junctional complexes of a single reticular cell. role of reticular fibers in antigen transport in lymph ment, the reticular fibers were free in the intercel- node has been emphasized by several authors (MOE, lular space, whereas when the lymphocytes were 1963; SAINTE-MARIE and PENG, 1986). They suggested increased in number during development, the reticu- that proteoglycans and glycoproteins among the lar fibers were circumscribed by the reticular cell fibrils assisted the direction of the transport. processes. The colse relation of the reticular cell with the retic- HAYAKAWA et al. (1988) showed by quantitative ular fiber has been documented by many authors electron microscopic observations that 90% of the (SORENSEN,1960; HAN, 1961; CLARK, 1962; MOE, 1963; surface area of the reticular fibers in the paracortex BAIRATIet al., 1964; MIYATA and TAKAYA,1981; SAINT- of both nude and hetero mice was covered by the MARIE and PENG, 1986; HAYAKAWA et al., 1988). It reticular cells, with the rest covered by different has been emphasized that the lymphoid cells are kinds of migratory cells including interdigitating isolated by the reticular cell cytoplasm from the cells, lymphocytes and macrophages. Their observa- reticular fiber (CLARK, 1962). As clarified in this study, tions indicate the possibillity of a key role of the when the lymphocytes were scarce in early develop- reticular fiber in the function of migratory cells. This 358 T. YosHIDA and K. TAKAYA: is contrary to the assumption that the close relation the lymph node has been identified as the mainte- between the reticular cell and the reticular fiber is nance of the integrity of the nodes when they become caused by pressure induced by the accumulation and hypertrophic. The popliteal lymph nodes removed proliferation of lymphocytes. from cortisone-treated animals were hypertrophic, At the earliest stages in the postnatal development and most of them were locally congested when well of the rat lymph node, clumps of protofibrils were developed elastic fibers were found in the reticular released in the intercellular space to assemble into fibers (MIYATA and TAKAYA, 1981, 1983). The func- collagenous fibrils. FUKUDA and MOcHIZUKI (1982) tion of elastic fibers in antigen, nutrition and antibody reported the formation of reticular fibers in the transport needs to be examined, because the authors spleens of chick embryos by the accumulation of (MOE, 1963; SAINTE-MARIE and PENG, 1986) who collagenous microfibrils with periodicity. They also described antigen transport in the reticular fiber did described reticular fibers as being formed a felt-like not mention such a function of the elastic fiber within band made up of amorphous substances in the inter- the fiber. cellular spaces, and continuous with the basement The follicular dendritic cell (FDC) is a representa- membrane. The felt-like band might be equivalent tive of the antigen presenting cell which was demon- to the lamina densa of the basement membrane. strated to be a type of reticular cell enclosing col- Clarification of the relation with the felt-like band lagen fibers and elastic fibers. During their differ- and the basal lamina-like structures recognized in the entiation after antigen stimulation, FDCs are derived rat lymph node in this study awaits further investiga- from cells which were related with reticular fibers tion with regard to its features at earlier stages. and those adhering to the capillary wall (YOSHIDA Incontinuous basal lamina-like structures were and TAKAYA, 1989). The typical FDC posseses a found on the reticular cell at early stages of rat reticular fiber enclosed by the cytoplasmic processes. postnatal development. Also throughout this period, a This study clearly showed both the close association dense basal lamina-like material was found between of reticular cells with reticular fibers, and that the reticular fiber and the process of the reticular cell. reticular cells enclosed reticular fibers actively dur- In the adult lymph nodes, on the other hand, no basal ing development. 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