JOURNAL OF MORPHOLOGY 20425-32 (1990)

An Ultrastructural Study of the Spleen of the Ranid Frog Rana perezi RAFAEL ALVAREZ Uniuersidad de Leon, Departamento de Biologia Celular y Anatomia, 24071 Leon, Spain

ABSTRACT The spleen of Rana perezi is encapsulated by connective tissue and shows by light microscopy two areas with no obvious border: the white pulp and the red pulp. The white pulplymphoid clusters are scattered throughout the organ and contain lymphocytes, reticular cells, and some plasma cells. The red pulp displays two different portions. The predominant region consists of reticular cells, lympho- cytes, a variety of other leucocytes, and cells undergoing division. This area possibly performs a haemopoietic function. The smaller portion of the red pulp is character- ized by reticular-phagocytic cells and may be haemocaretic in its function. Macroph- ages and pigmented cells occur in both white and red pulp. The organization of the spleen of R. perezi can be considered as a transitional or intermediate state between the primitive condition seen in certain fishes and amphibians and the more complex organ of ammiotes.

The immune system of anuran amphibians can be phylogenetically related to that of am- niotes (Pitchappan, '80). In the family Ranidae the immune system is composed of bone mar- row, thymus, spleen, jugular body, and small lymphoid clusters associated with the digestive duct (Manning and Horton, '82). The spleens of the few anurans examined show both lymphoid and non-lymphoid elements (Baldwin and Co- hen, '81; Barrutia et al., '83). In addition to typical functions of a secondary lymphoid organ (Horton et al., '77), the anuran spleen also plays a haemopoietic role (Carver and Meints, '77).

The vertebrate spleen has an apparently an- cient origin since a primitive but diffuse spleen exists in cyclostomes (Weiss, '85). In elasmo- branches the spleen appears as a net organ (Zap- ata, '80). In amphibians an apparent progression in splenic architecture can be observed, differing from that in some primitive fishes, in which lymphocytes are diffusely distributed. The more advanced amphibian spleen is one in which lym- phocytes aggregate around arteries and form red and white pulp as well-defined regions separated by a distinct marginal zone (Cooper and Wright, '76). Within the amphibians the less organized primitive condition occurs in urodeles, some anurans (Ardavin et al., '84)' and teleost fishes (Secombes and Manning, '80; Zapata, '82). The spleen of reptiles (with the exception of ophidi- ans; Kroese and Van Roijen, '82)' birds, and mammals possesses a clearly defined marginal area and sinus, and these characters are among those marking the advanced condition. Excep-

tional cases are Xenopus laeuis (Baldwin and Sminia, '82) and Bufo calamita (Barrutia et al., '83), in which the spleen shows the same general organizational complexity as amniotes.

In this paper the structure of the spleen of Rana perezi, a poorly known amphibian from histological and cytological points of view, is described, with the goal of increasing the infor- mation available on the ultrastructure of the spleen of diverse amphibians.

MATERIALS AND METHODS

Ninety-six adult frogs (R. perezi) collected in September, 1983 in the vicinity of Le6n (Spain) were kept for 1 year under regulated light and temperature conditions (12 light/l2 dark, 2 G 22OC). Animals were maintained in containers holding a receptacle with water, which was changed on alternate days. Frogs were fed live worms, flies, and grasshoppers every 3 days. Four males and four females were killed (after anaes- thesia with MS 222; Sandoz) each month for 1 year. The spleen was immediately removed, fixed in 2.5% glutaraldehyde in Sorensen buffer at pH 7.4, postfixed in 1% OsO, in the same buffer, dehydrated in acetone, and embedded in Ar- aldite. Semi-thin sections stained with toluidine blue were examined by light microscopy. U1- trathin sections were double-stained with uranyl acetate and lead citrate and examined with a JEOL 100-C electron microscope.

RESULTS

The spleen of R. perezi is covered by a connec- tive tissue capsule that does not project trabecu-

D 1990 WILEY-LISS, INC.

26 R. ALVAREZ

Figures 1 5

SPLEEN STUDY OF RANA PEREZ1 27

lae into the organ. This capsule is slightly thicker at the hilium, through which the splenic vein emerges and the splenic artery enters the organ (Fig. 1). After ramification within the spleen the splenic artery does not constitute the so-called ellipsoids characteristic of some higher and lower vertebrates.

The parenchyma shows two distinct areas, although there is no clear separation in the tran- sitional zone between them: the white and red pulp (Fig. 1). The red pulp is formed of two different portions that also fail to show an obvi- ous border: one portion displays a higher cellular density than the other (Fig. 2). By light micros- copy, the white pulp represents approximately 13% of the total volume, the denser red pulp represents 76 %, and the remaining 11 96 is occu- pied by the less dense red pulp.

The white pulp does not surround arterial vessels and is comprised mainly of lymphocytes (Fig. 3), although it also has some plasma and reticular cells. The reticular cells have character- istic projections that are intermingled with the connective tissue fibers (Fig. 3).

The denser red pulp is associated with the blood sinuses (Fig. 2) and shows a network of reticular cells, similar to those observed in the white pulp (Fig. 4). Cells of the red pulp are also associated with the connective fibres by short projections. The red pulp contains lymphocytes, heterophils, eosinophils, basophils, monocytes, and some cells undergoing division (Fig. 5). Het- erophils and eosinophils (Figs. 6 and 7) display lobate nuclei and two types of cytoplasmic gran- ules. Basophils (Fig. 8) have nonlobated nuclei and a single type of hyaloplasmic granule, some- times containing fibrillar structures. Monocytes

Fig. 1. Semi-thin section a t the hilum, showing the splenic artery (a) and the splenic vein (v). See also the red pulp (RP) and thewhitepulp (WP). x250.

Semi-thin section of the splenic stroma showing the higher cellular density red pulp (RPP) and the lower cellular density red pulp (RPC). WP, white pulp; v, blood vessels. x 250.

Fig. 2.

Fig. 3. Splenic white pulp showing lymphocytes (L), plasma cell (P), and association with both reticular cell pro- cesses and collagenous matrix of connective tissue (arrow- heads). x 8,800.

Fig. 4. Characteristic reticular cell as found both in white pulp and in red pulp of higher cellular density. Note the association with reticulocyte processes and collagenous ma- trix (open arrows). ~20,500.

Fig. 5. Semi-thin section of region of red pulp with higher cellular density. Note some mitotic images (m), some reticu- lar cells (arrows), and a single basophil (b). x 970.

(Fig. 9) show a kidney-shaped nucleus and small hyaloplasmic granules, possibly lysosomal in na- ture. Cells in division (Fig. 10) are possibly matur- ing or developing erythroid cells.

The red pulp with a lower cellular density (Fig. 11) surrounds the blood sinuses and arte- rial vessels, displays a well-developed connective stroma, and contains reticular cells with a phago- cytic appearance. These reticular cells appar- ently act as endothelial cells and contain in their cytoplasm (Fig. 12) rough endoplasmic reticu- lum cisternae in parallel arrays, mitochondria, and abundant dense bodies. Like the other retic- ular cells, they are associated with the connec- tive tissue and establish cellular contacts with connective fibers, sometimes through interdigi- tating processes. However, true cellular junc- tions are never observed.

Distributed among all regions of the spleen of R. perezi are light cells (Fig. 13), which are typi- cally degenerate reticular cells. Likewise, there are scattered macrophages (Fig. 14) having pri- mary and secondary lysosomes and residual bod- ies, and pigmented cells (Fig. 15) with a cyto- plasm completely filled with melanosomes. Macrophage-pigmented cells also exist and are associated in large clusters (Fig. 16) randomly disposed throughout the spleen. The cells in clusters contain a variable quantity of electron- dense bodies, possibly melanosomes (Fig. 17).

DISCUSSION

The spleen is a secondary lymphoid organ in which the reaction against antigens takes place through the actions of lymphoid and non-lym- phoid cells. The spleen of R. perezi, like that of other Ranidae, belongs to the so-called “diffuse pattern” (Cooper and Wright, ’76), in which clus- ters of lymphoid cells are rare or absent. Instead, lymphoid cells are randomly disposed through- out the organ; whatever clusters exist are scarce and poorly defined. Ellipsoids are also absent. Despite the diffuse organizational structure, the white and red pulp are considered homologous to the respective sections of the spleens of other vertebrates. This “diffuse spleen” is characteris- tic of various amphibian species including Asca- phus truei, Scaphiopus conchii, Bufo poweri, and Rana pipiem, whereas the “follicular spleen” pattern is observed in Xenopus laevis and Bufo calamita (Cooper and Wright, ’76).

The spleen is not only a lymphoid organ, but is also haemopoietic and haemocaretic in func- tion. In higher vertebrates the spleen is hae- mopoietic only during embryogenesis (Patt and Patt, ’69), but this function persists throughout life in lower vertebrates, the spleen being the main haemopoietic organ in adult anurans

28 R. ALVAREZ

Figures 6-12

SPLEEN STUDY OF RANA PEREZI

Fig. 6. Heterophil in the red pulp. n, nucleus; g, specific granules. x 11,200.

Fig. 7. Eosinophil in the red pulp. n, nucleus; eg, emino- philic granules. x 11,OOO.

Fig. 8. Basophil in the red pulp. n, nucleus; bg, hasophilic granules. x 23,000.

cleus. ~11,500. Fig. 9. Monocyte in the red pulp. n, kidney-shaped nu-

Fig. 10. Cell in mitosis, showing condensed chromatin masses (c). x 13,500.

Fig. 11. Semi-thin section, in region of red pulp with lower cellular density, showing typical phagocytic-reticular cells (arrows). a, artery. x 1,300.

Fig. 12. Phagocytic-reticular cell. Note the rough endo- plasmic reticulum (arrowheads), mitochondria (large arrows), and electron-dense bodies (arrows). x 16,000.

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30 R. ALVAREZ

Fig. 13. Reticular cells (phagocytic-reticular). One cell Fig. 16. A cluster of macrophage-pigmentary cells (sur- has the more common dense appearance (rc); the adjacent electron-lucent cell (cc) occui-s less frequently. x 7,000.

rounded bylargearrows). ~5,000.

Fig. 17. Possible melanosomes (arrow) in the cytoplasm of one of the cells from a macrophag6pigmentary center. x 10,400.

Fig. 14. Macrophage (large arrows) in the red pulp. x 6,200.

Fig. 15. A pigmented cell. ~7,500.

SPLEEN STUDY

(Carver and Meints, '77). The double role (hae- mopoiesishaemocaresis) is related to the two different areas observed. In the red pulp of R. perezi the denser section is involved with cellu- lar production and haemopoiesis, and the less dense region, having many phagocytic cells, is haemocaretic in function.

The lymphocytes of the white pulp appear to play an important role in splenic immune reac- tions, but a similar role is not as clear in the red pulp, where lymphocytes are scattered through- out. To determine the existence of T- andlor B-lymphocyte subpopulations in the R. perezi spleen, it will be necessary to carry out experi- ments similar to those done in X . laeuis in which both subpopulations have been observed (Man- ning, '71; Nagata and Katagiri, '78; Horton et al., '77; Turner and Manning, '73).

The ultrastructure of reticular cells described here in the white pulp and in the haemopoietic red pulp is similar to that described for B. calam- ita by Barrutia et al. ('83). These "reticular pale cells" are characteristically found in association with the connective fibers, and their cytoplasm is electron-lucent. However, the dense reticular cells Rarrutia et al. ('83) described in the white and red pulp of R. calamita spleen as electron- dense, with electron-dense bodies in their cyto- plasm, are not present in the white pulp of R. perezi, but cells described here as phagocytic- reticular cells in the haemocaretic red pulp may be similar. Therefore, in the spleen of R. perezi, B. calamita, and possibly other species, there are two different reticular cell types, whose disposi- tion in each species is somewhat different.

In the spleen of X . laeuis and B. calamita giant cells, considered initially to be degenerat- ing macro-lymphocytes (Manning, '71), and later called XL cells or dendritic cells (Baldwin and Cohen, '81), have been observed. They have been related to antigen trapping (Baldwin and Sminia, '82; Obara et al., '82), and Barrutia et al. ('83) showed that they are macrophages involved in antigen processing. In the present study, such cells were not observed. To assign to the mac- rophages observed within the spleen of R. perezi a function similar to the dendritic cells is not possible, but macrophages are morphologically similar to cells observed in other lymphoid or- gans of R. perezi and probably have, therefore, no role in splenic antigen processing.

Finally, the macrophage-pigmented cell clus- ters seem to be similar to the melano-macro- phage centers of fishes (Agius and Agbede, '84; Herraez and Zapata, '86), having a role in the erythroid cell catabolism, according to Agius and Roberts ('81).

OF RANA PEREZI 31

In summary, in the spleen of R. perezi lym- phoid clusters are scarce in the whole organ, and the margin between white and red pulp is dif- ficult to recognize. Two ultrastructurally distinct portions exist in the red pulp, presumably hae- mopoietic and haemocaretic in function. The spleen of R. perezi appears to represent the transitional state between the primitive and the more advanced conditions Cooper and Wright ('76) described, as could be expected in an anuran from the family Ranidae.

ACKNOWLEDGMENTS

Thanks to Dr. Paulino de Paz for a critical review of the manuscript, to Dr. Agustin Zapata who initiated my interest in the immune system, and to the two anonymous reviewers who as- sisted me in improving the manuscript.

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