Light- and electron microscopical observations on the caecilian spleen a contribution to the evolution of lymphatic organs

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 6, pp. 293-302, 1982 0145-305X/82/020293-I0503.00/0 Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.

LIGHT- AND ELECTRONMICROSCOPICAL OBSERVATIONS ON THE CAECILIAN SPLEEN A CONTRIBUTION TO THE EVOLUTION OF LYMPHATIC ORGANS Ulrich Welsch and Volker Storch Department of Anatomy, University of Kiel, D-2300 Kiel/West Germany Department of Zoology, University of Heidelberg, D-6900 Heidelberg/ West Germany ABSTRACT Red and white pulp were distinguished in the spleen of the caecilian species Ichthyophis paucisulcus and Afrocaecilia taitana. The red pulp was composed of endothelium-lined sinusoids and r e t i cular connective tissue. Between the processes of the reticulum cells, accompanied by fine collagen f i b r i l s , the following cell types were found: lymphocytes, macrophages (frequently containing fragments of erythrocytes), neutrophils, eosinophils, mast cells and/or basophils (metachromatic granules), thrombocytes, plasma cells, pigment cells as well as cells which presumably represent blast cells. Morphological evidence suggested the formation of thrombocytes in the red pulp. Besides sinusoids, ellipsoids and peculiar arteriolar vessels with a high endothelium and a loose layer of muscle cells were observed. Veins were concentrated in the splenic periphery. White pulp consisted of arterioles which were surrounded by a lymphocyte sheath. Follicles were not identified with certainty. Occasionally mitotic figures were associated with lymphocytes. On the basis of our findings, we suggest the following functions of the caecilian spleen: destruction of aged erythrocytes, formation of thrombo- and lymphocytes as well as of plasma cells and, to a marked lesser degree, of other blood cells.

INTRODUCTION This investigation analyses the splenic architecture and cellular composition in a group of primitive amphibians, the caecilians, with improved light" microscopic and fine structural techniques to emphasising those structural details contributing to an understanding of the evolution of lymphatic organs. Studying organs of primitive vertebrates facilitates our understanding of embryonic structures of mammals, including man (16). The spleen of the caecilians (= Gymmophiona) is located in the immediate neighbourhood of the pancreas (28, 14, 26). I t consists of a red and white pulp, a fact which apparently does not apply to all amphibians, especially among certain urodeles in which these two compartments of the spleen can-

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not be clearly separated (11). The white pulp of the caecilian species studied by Weilacher (26) is composed of a large "primary Malpighian corpuscle" and smaller secondary corpuscles, derived from the primary one, and in contrast to anuran amphibians (Xenopus, 22) is pervaded by capillaries. The arteries, supplying the white pulp, contain epithelial cells in their media resembling glandular cells (26). So far, there are only few findings on the function of the caecilian spleen. Weilacher (26) assumes that i t is involved in the formation of lymphocytes and destruction of erythrocytes. New insights into immunity mechanisms operating in caecilians have been detected by studies of lymphatic organs, blood cells and skin transplant rejections by Cooper and coworkers (2, 3, 4, 5, 7).

MATERIALS AND METHODS We have studied the spleens of two Caecilian species: Ichthyophis paucisulcus (5 individuals), collected in Northern Sumatra in April, and Afrocaecilia taitana (4 individuals) from Kenya, collected in December. The spleen was cut into 2 - 3 pieces which were fixed in cold, 3,5 % phosphate-buffered (pH 7,5) glutaraldehyde for 2 hours. After repeated rinses in phosphate buffer (pH 7,5) the pieces were postfixed in 2 % osmic acid, dehydrated in ethanol and embedded in araldite. Sections for light microscopy were stained with methylene blueAzur I I . Thin sections for electron microscopy have been stained with uranylacetate and lead citrate. Electron Microscope: Philips EM 300.

RESULTS The findings as described below apply to the spleens of both caecilian species studied, unless stated otherwise. The spleen appeared to be a particularly small organ (longitudinal diameter 2,5-3 mm, diameter of cross section about 0,7 mm, body length of the animals studied 27-30 cm) which was in striking contrast to the large lobulated l i v e r , which is about 7-8 cm long and 3-4 mm wide (27). The spleen was surrounded by a thin capsule composed of collagen and elastic f i b r i l s . Interspersed between the f i b r i l s were cells with an elongated nucleus, abundant microfilaments, infrequent rough ER-cisternae and no clear peripheral vesicles. Immediately below the capsule, without any bordering structures, the pulp tissue was found into which radiated individual bundles of collagen but no typical trabecular structures (fig la). The area of the red pulp was larger than that of the white pulp which was recognized by its densely packed lymphocytes. The red pulp, which usually borders the capsule, was composed of endothelium-lined sinusoids and cords of reticular connective tissue located between the sinusoids (fig Ib). The sinusoids were lined by a markedly thin endothelium which did not show any interruptions and was underlain by a basement membrane (fig. 2a). The cytoplasm contained bundles of microfilaments, small fields of glycogen, individual pinocytotic vesicles and in occasional more voluminous areas peculiar stacks of smooth ER and accumulations of mitochondria (fig. 2a). Typical veins or venules (wide lumen, individual muscle cells in their wall) were detected mainly in the periphery of the organ (fig. la).

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FIG. la-c Light microscopy of the capsule and red pulp. a: periphery of the spleen (Iohthyophis) with capsule (C) covered by peritoneal epithelium (arrows). Immediately below the capsule, red pulp with two veins (V) and c e l l - r i c h r e t i c u l a r connective tissue, b: red pulp of the Iehthyophis spleen with sinusoids, marked by the dark stained erythrocytes, and small arterioles characterized by t a l l endothelial cells (arrows). M: cells with metachromatic granules, c: e l l i p s o i d in the spleen of Afrooaecilia, note voluminous pale cells around a f l a t endothelium, a - c x 560. The nucleus of the loosely arranged reticulum cells ( f i g . 2b) was of i r r e gular shape and contained up to 4-5 nucleoli as well as fine clumps of heterochromatin concentrated in the peripheral parts. There was a narrow rim of cytoplasm around the nucleus from which extended a number of elongated processes. Main characteristics of the cytoplasm were bundles of m i c r o f i l a ments and small accumulations of mitochondria; the other organelles were inconspicuous. Desmosomal contacts between the reticulum cells were not detected. I , the i n t e r s t i t i a l space, loosely arranged bundles of r e t i c u l a r f i b r i l s occurred which often ran in parallel to the c e l l u l a r processes. The following free cells were recognized in the meshes between the reticulum cells: a) small and medium sized lymphocytes; b) macrophages, large cells with a pale nucleus and lysosomal granules and frequently fragments of ingested erythrocytes ( f i g . 3a); c) neutrophils ( f i g . 4a) with abundant elongated or spindle-shaped granules. These cells have been found more frequently in Ichthyophis than in Afrocaecilia. In Ichtyophis they often are concentrated in the peripheral zones of the organ below the capsule; d) metachromatic, usually voluminous cells with big granules exhibiting a c r y s t a l line core ( f i g . 3b, also on l a ) ; the frequency of these cells is variable, in one specimen of Ichthyophis they were p a r t i c u l a r l y abundant forming small clusters; e) pigment cells with dense granules ( f i g . 3c); f ) cells presumably representing eosinophils (segmented nucleus, two types of electron dense

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FIG. 2a, b

Ichthyophis spleen, red pulp. a: wall of a sinusoid, arrows: basal lamina, note accumulation of mitochondria in thickened part of the endothelial c e l l , L: lumen with erythrocyte, b: reticulum cell (R) and numerous processes of reticulum cells (arrows) pervading the red pulp, i n t e r s t i t i a l space f i l l e d with r e t i c u l a r f i b r i l s , V: small vessel, a x 16000, b x 11000. granules: large oval ones, in part with a c r y s t a l l i n e core, and small roundish ones, f i g . 3d); g) thrombo~yt~which were of variable dimensions and frequently forming small groups, two or three c e l l s of which were close together. The larger cells exhibited a markedly lobulated nucleus ( f i g . 3f) and a cytoplasm which was not yet of the regular structure characterizing the smaller mature thrombocytes ( f i g . 3g): clear vesicles and electron dense granules throughout the c e l l u l a r periphery; h) plasma cells with abundant often s l i g h t l y dilated rough ER-cisternae, an extended Golgi apparatus with electron dense granules (presumably lysosomes) in the neighbourhood. The shape of these cells is variable, frequently they are oval or elongated in o u t l i n e ( f i g . 3e); h) erythrocytes, often near reticulum c e l l s or macrophages; now and then small clusters of immature erythrocytes can be found ( f i g . 4b); j ) c e l l s which so far cannot be i d e n t i f i e d with c e r t a i n t y . Often such cells resemble blast c e l l s with a large pale nucleus and numerous ribosomes. Except for the plasma and pigment cells the above mentioned c e l l s also were found within the lumen of th~ sinusoids or venules. In addition here also lysosome-rich c e l l s , somewhat resembling the macrophages (monocytes ?) occurred. Neutrophils ( f i g . 4a), presumed eosinophils, lymphocytes and thrombocytes migrated through the endothelium of the sinusoids. The red pulp is pervaded by small bundles of nerve fibres which contained microtubules and accumulations of round electron dense granules (diameter about 150 nm). These nerves frequently accompany a r t e r i o l e s but in addition they also occurred at a distance from the vascular smooth muscle cells ( f i g . 4e, also 3e). F i n a l l y , p a r t i c u l a r mention shall be made of two

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FIG. 3a-g spleen, various free c e l l s in the red pulp. a: macrophage w i t h ingested remnants of erythrocytes, b: cell with metachromatic granules, note t h e i r c r y s t a l l i n e core: c: pigment c e l l s , d: cell i n t e r p r e t e d to be an eosoninophil, e: two plasma c e l l s , arrow: nerve f i b r e with dense core ves i c l e s , f : large thrombocyte with features of an immature c e l l . g: mature thrombocytein the lumen of a sinusoid, a - g x 5400.

Ichthyophis

types of blood vessels which have been r e g u l a r l y found in the red pulp. One represents a special a r t e r i o l e w i t h 3-5 t a l l endothelial c e l l s per cross section and one layer of smooth muscle c e l l s . The endothelial c e l l s contained numerous microfilaments and electrondense polymorphic granules which often were concentrated in the c e l l u l a r periphery. The lumen of these a r t e r i o l e s was always closed in our preparations ( f i g . I b ) . The second type of special blood vessels is the e l l i p s o i d , which is lined by a f l a t endothelium which is sourrounded by a layer of large pale c e l l s ( f i g . I c ) . E l l i p soids were of rare occurrance in our material.

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FIG. 4a-e Red (a-c) and white pulp. a: detail of the cytoplasm of a neutrophil, note slender elongated granules, b: immature erythrocytes, c: bundle of nerve fibres with dense core vesicles (arrows) in the red pulp. a x 18000, b x 7200, c x 18000, d x 7200, e and f x 210. d: lymphocytes in the white pulp, note presence of lysosomal granules (arrows) in a least two of the c e l l s , L: lumen of a vessel, presumably sinusoid, M: lymphocyte possibly migrating through the wall of the vessel, e and f: l i g h t microscopy of the white pulp of Ichthyophis (e) and Afrocaecilia ( f ) , note the arteries (arrows) surrounded by lymphocyte sheath.

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The border between red and white pulp is not always clear-cut but usually recognizable ( f i g . 4 e - f ) . Principal constituent of the white pulp are art e r i o l e s surrounded by a sheath of lymphocytes ( f i g . 4e, f ) . Germinal centres or typical f o l l i c l e s have not been observed. Typical a r t e r i o l e s of the white pulp are b u i l t up by 10-15 f l a t endothelial c e l l s per cross-section, an elastica interna, and a media of I-2 layers of smooth muscle c e l l s , which branch extensively. The endothelial c e l l s usually contained electron-dense polymorphic granules. Collagen f i b r i l s concentrate outside the muscle cell layer and from here radiate into the white pulp. The shape of the lymphocytes and t h e i r nuclei was remarkably diverse, so that i t was d i f f i c u l t to c l a s s i f i y lymphocytes on the basis of nuclear morphology. The cytoplasm usually was merely a narrow rim around the nucleus with poorly developed organelles. Not infrequently, however, the cytoplasm was increased l o c a l l y in volume and here contained stacks of annulated lamellae ( f i g . 4d), a few mitochondria, and small groups of dense granules which probably represented lysosomes ( f i g . 4d). The lymphocytes can exhibit m i t o t i c figures. In addition the white pulp was b u i l t up by individual r e t i culum c e l l s , macrophages and plasma c e l l s . Capillaries or extensions of the sinusoids were found mainly in the border zone between red and white pulp.

DISCUSSION The present study has shown that the caecilian spleen undoubtedly is composed of a red and white pulp, thus confirming the results of Weilacher (26). We can say that the caecilian spleen represents among the amphibia an intermediate level of development, since according to Hartmann (10, 11) the spleen of a number of urodeles does not show a separation of the two parts of the pulp at a l l , whereas in anurans the white pulp even can be separated from the red one by a particular layer of reticulum cells (22). In the caecilians, as in urodeles as Megalobatrachus and Hynobius (19, 20, 21), the border between red and white pulp is not sharp but c l e a r l y recognizable. As in a l l tetrapods (24) the main constituents of the caecilian red pulp are sinusoids and cords of r e t i c u l a r connective tissue. As in urodeles (10) sinusoids are not always c l e a r l y separable from venules and especially c a p i l l a r i e s in p a r t i c u l a r in Iehthyophis we observed r e l a t i v e l y narrow sinusoids. The s i nusoidal l i n i n g in urodeles (10) and caecilians seems to e x h i b i t no d i s c o n t i nuations, whereas Sterba (22) found perforations of the sinusoidal wall in the l i g h t microscopic study of the Xenopus spleen. Evidently white blood c e l l s can migrate through t h i s endothelium in caecilians. The presence of numerous erythrocytes outside the vascular lumen suggests that an "open" section exists between terminal c a p i l l a r i e s and sinusoids, as has been described in urodeles and anurans (22). Those small vessels in the red pulp with t a l l endothelium and one layer of smooth muscle c e l l s , which are interpreted to represent a r t e r i o l e s , possibly play a special role in the regulation of the blood flow in the spleen, in p a r t i c u l a r through the sinusoids. As in other a r t e r i a l vessels t h e i r endothelium contained dense granules, ~ndicating secretory a c t i v i t y . The function and exact nature of many of the free c e l l s within the connect i v e tissue of the red pulp remains obscure, e.g. we do not know the task of the c e l l s with brown endogenous pigment in the spleen which in urodeles Nakajima (19) found to vary in number at d i f f e r e n t times of the year. The macrophages, however, evidently destroyed aged red blood c e l l s , the f r a g ments of which were detected within t h e i r cytoplasm. Thus, inagreement with e a r l i e r observations on caecilians (26) and other amphibia ( I ) we can ascribe the function of degrading erythrocytes to the caecilian spleen.

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In lymphatic tissues, i t seems reasonable to interpret cells rich in rough ER cisternae to represent plasma cells. The same type of cells has been described in amphibian lymphatic tissues by Cooper (3) as plasma cells. The electron dense granular inclusions in all probability represent lysosomes. Plasma cells are the expression of the capability to produce antibodies and indicate the presence of B-lymphocytes in the caecilian spleen. For a recent discussion on antibody formation in amphibia see Cooper (3). Our findings on the plasma cells in the spleen of Ichthyophis and Afrocaecilia a r e in good agreement with comparable observations in the anura (3). The plasma cells have been found both in the red and the white pulp and therefore may originate in both compartments of the spleen. Elongated and irregularly shaped plasma cells suggest movements of these cells, such as has been postulated by a number of authors (15, 24). Numerous thrombocytes and the existence of large cells among them, which may represent immature forms (organization of peripheral vacuoles and granules not yet completed), support the view that the amphibian spleen may produce thrombocytes, as has been assumed by Tooze and Davies (25) in urodeles. Evidence for the production of other blood cells (except for lymphocytes, see below) in the caecilian spleen is not yet convincing, since the numerous granulocytes and other free cells may easily have escaped from the blood stream. However, cells with immature organization of the cytoplasm as to be found both in the connective tissue and the blood vessels of the red pulp, indicate a l i mited capacity of granulo- and erythropoesis, a view also put forward by Weilacher (26). The white pulp of the caecilian spleen is composed of a sheath of lymphocytes, surrounding an arteriole. The large number of lymphocytes in the caecilian spleen demonstrates that these cells are of especial importance in this organ; they even multiply here. A considerable number of these and of lymphocytes in the splenic veins contain small groups of granules which in all probability represent lysosomes, thus exhibiting a keycharacter of T-lymphocytes (23). This observation of lymphocytes with characters of T-cells f i t s well with the findings of Cooper and Garcia Herrera (5) and Cooper (2), which demonstrate that caecilians have the capacity for an immune rejection of skin allograft, since this capacity usually is closely connected with T-lymphocytes. Both in caecilians and mammals the splenic T-cells are arranged in periarteriolar sheaths. This finding points to a comparable situation in phylogeny and in the ontogeny of mammals, reminding us of the biogenetic law of Haeckel. Periarteriolar lymphocytic sheaths appear to be present in all primitive tetrapods, especially in the amphibia, which form the stem group of the tetrapods (12, 18, 19, 21). The descriptions of follicles, the B-cell region, e.g. in frogs (12) need confirmation, since images suggestive of follicles, may represent cross-sections through periarteriolar sheaths. Hartmann (11) denies t h e existance of follicles in the urodele spleen, and according to Murata (18) secondary follicles have never been found in amphibia. In their study of embryonic lymphatic tissues of man, MUller-Hermelink and v. Gaudecker (16) observed that the formation of T-cell-regions precedes that of B-cell areas in all lymphatic tissues. Thus, there seems to be a somewhat parallel situation in phylogeny and ontogeny. T-cell areas originate before B-cell areas. In the caecilians as representatives of the amphibia this shall not imply that these animals do not possess B-lymphocytes (see above, plasma cells); they only seem not yet to be concentrated in typical regions of their own. Possibly the B-cell regions, the follicles, phylogenetically are derived from the T-cell regions, the periarteriolar sheaths. This assumption is supported by the observations on radiated animals, in which the T-cell area reappears before the B-cell regions (6). The beforementioned hypothesis has to be tested in future studies. Among others, an analyses of the amphibian reticulum cells seems worthwhile, since in mammals T- and B-cell areas contain different types of reticulum cells (17).

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A c k n o w l e d g e m e n t . W e t h a n k Prof. E.L. C o o p e r who read the manuscript.

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17. MOLLER-HERMELINK, H.K., HEUSERMANN, U., KAISERLING, E., STUTTE, H.J. Human lymphatic microecologyspecificity, characterization, and ontogeny of different reticulum cells in the B-cell and T-cell regions. In: Immune reactivity of lymphocytes. Hrsg.: Feldmann, M., Bloberson, A., Adv. in exp. Med. Biol. 66, 117-182, 1976. 18. MURATA, H. Comparative studies of the spleen in submammalian vertebrates. I I . Minute structure of the spleen, with special reference to the periarterial lymphoid sheath. Bull. yamaguchi med. school 6, 83-106, 1959. 19. NAKAjIMA, A. Ober die Morphogenese der Milz von Megalobatrachus japonicus. Folia anat. jap. 7, 93-112, 1929. 20. NAKAJIMA, A. Zur Morphologie und Entwicklungsgeschichte der Milz von Hynobius fuscus. Folia ant. jap. 7, 305-323, 1929. 21. OHUYE, T. Hemocytopoietic effect of splenectomy in the newt. Sci. Rep. Tohoku Imp. Univ. Sendai (4) 7, 49-63, 1932. 22. STERBA, G. Untersuchungen an der Milz des Krallenfrosches (Xenopu8 laevis Daudin). Morph. Jb. 90, 221-248, 1950. 23. STUTTE, H.J., MOLLER-HER~IELINK, H.K. Lysosomen in Blutzellen als diagnostischer Parameter. Verh. dtsch. Ges. Path. 60, 155-175, 1976. 24. TlSCHENDORF, F. Die Milz. In: Handbuch der mikroskopischen Anatomie des Menschen. BegrUndet von v. M~llendorff, fortgefUhrt von W. Bargmann, herausgegeben von A. Oksche und L. Vollrath. Bd V l / I . Berlin, Heidelberg, New York, Springer, 1969. 25. TOOZE, J., DAVIES, H.G. Light- and electron ,microscopic studies on the spleen of the newt Trituru8 cristatus: the fine structure of the erythropoietic cells. J. Cell Sci. 2, 617-640, 1967. 26. WEILACHER, S. Die Milz der Gymnophionen. Beitrag zur Kenntnis der Gymnophionen Nr. XVlI. Morph. Jb. 72, 469-498, 1933. 27. WELSCH, U., STORCH, V. Elektronenmikroskopische Untersuchungen an der Leber von Ichthyophis kohtaoensi8 (Gymnophiona). Zool. Jb. Anat. 89, 621-635, 1972. 28. WIEDERSHEIM, R. Vergleichende Anatomie der Wirbeltiere. Jena, G. Fischer, 1909. Received lday, 1981 Accepted February, 1982