Acta anat. 100 : 34-43 (1978)
Ultrastructure of reticulum cells in the bone marrow12 A. Biermann and D. Graf von Keyserlingk Institut für Anatomie, Freie Universität Berlin, Berlin
Key words. Reticulum cells • Bone marrow • Ultrastructure Abstract. In this study the attempt was made to classify the reticulum cells of the bone marrow on the basis of electron-microscopic findings. The basis of the differentiation was the ability of the cells to phagocytize substances or not. For two cell types the intracytoplasmic filaments were used as distinctive marks. The following classification resulted: (a) phagocytic reticulum cells, (b) undifferentiated reticulum cells, (c) fibrous reticulum cells of type I, which contain filaments of 4-8 nm diameter and are located near the blood sinus of the bone marrow, (d) fibrous reticu lum cells of type II. which contain intracytoplasmic filaments of 10 nm diameter; since these cells contain neutral fat bodies, the possibility of a reversible conversion to fat cells has to be assumed and (e) fibroblasts, cells which synthesize the substance of the extracellular space. A connexion of reticulum cells to haematopoietic functions or to stem cell functions could not be found.
'Reticulum cell' In the bone marrow or in lymphoreticular tissue is a general expression which is variously applied. It can mean branched cells whose processes build a network (reticulum), or cells which arc con nected to, or even produce, extracellular reticulin (a network of a certain type of collagen), or cells which do not directly serve the haemo- or lymphocytopoiesis. i.e. the stroma cells. It is difficult to show a preference for one of these definitions. Reticulum cells include undifferentiated cells, fibrous reticulum cells, fibro blasts. phagocytes, reticular macrophages, dendritic 1 Dedicated to Prof. W. Schwarz on the occasion of his 70th birthday. 2 We are grateful to Miss Barbara Hass and Miss Monika Dulinski for their skilful assistance.
macrophages, osteoprogenitor cells and others [ Young. 1962; Owen. 1970: Carr. 1973]. The expression 'reti culum cell- causes discomfort not only because of the various definitions [Gall, 1958], but also because, in the past, functions were ascribed to reticulum cells neglect ing the significance of the different cell types. This is due to a general lack of knowledge about the morpho logy and physiology of these cells. Since until now light microscopy has not given a satisfactory differ entiation of the various types of reticulum cells, we have studied the cytological characteristics of these cells with the electron microscope.
Materials and methods Bone marrow specimens from patients with various haematological diseases and from the rat femur were
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Introduction
35
In the current study, all bone marrow cells which had extensive processes were considered first. Endothelium cells, which represent a separate cell group, and megakaryocytes, which can have processes but are not included in the group of reticulum cells, were excepted. The surface foldings of the monocytes, lym phocytes, and granulocytes were not consid ered as cell processes in this connexion.
spondingly many vesicles and lysosomal ele ments. The numerous mitochondria are large and have many cristae. The few cisterns in the endoplasmic reticulum are short and stretched and only partially loaded with ribosomes. After overloading the cells with iron, one finds ferritin distributed diffusely in the cytoplasm; a heavier deposit of ferritin around the Golgi complexes is noticeable (fig. la). After over loading with ferritin, even isolated processes between the parenchymecellscan be associated with the phagocytic reticulum cells (fig. lb). The processes are very long and narrow and follow the contours of other cells which they are often surrounding. They enclose erythroblasts and other cells. In the interior of the processes only homogeneous ground cytoplasm, pinocytotic vesicles, and sometimes ferritin with the same density as in the pericaryon can be seen, but no cell organelles (fig. lc).
Phagocytic reticulum cell The ultrastructure of this cell group has already been well studied, especially in relation to the phagocytized material and its destruc tion [Bessis and Breton-Gorius, 1957: Bessis, 1958,1973; Seki and Shirasa wa, 1965: Berman, 1967; Wetzel et a i, 1967; von Keyserlingk, 1968]. Moreover, it became evident that these phagocytizing reticulum cells have a special function in the maturation of erythroblasts: a phagocytizing reticulum cell becomes en circled by erythroblasts in order to promote maturation of the latter. The origin and further destiny of these reticulum cells have not yet been discovered. The following characteristics distinguish these reticulum cells from others: the nucleus is large, has rounded contours, and can be dented by phagocytized material. It has numerous nuclear pores, and the chromatin is scattered (fig. Ia). The cytoplasm is broad and contains many Golgi complexes and corre
Undifferentiated reticulum cell Caffrey el al. [1966] looked at the undif ferentiated reticulum cell in the light micro scope and described it as a cell with an oval or irregularly shaped nucleus, a weakly coloured chromatin network and a clear nuclear bound ary due to the chromatin's adherence to the membrane. The boundaries of the cytoplasm are not clear. On the basis of these light-micro scopic criteria, the undifferentiated reticulum cell can be located in the electron microscope. Here too, as expected, the contour of the nucleus is wavy and irregular. The nuclear chromatin largely fits well to the nuclear membrane in a narrow dense band (fig. 2a). The nucleolus is ring-shaped. There is little cytoplasm. Since the cell processes are narrow and contain no large inclusion bodies, it is understandable that the cell boundaries can not be seen clearly in the light microscope. The cytoplasm is poor in ribosomes but contains
examined. The specimens were fixed in 1% glutaraldehyde and 1% paraformaldehyde for 2 h, using a 0.05 M cacodilai buffer with pH 7.2. Postfixation was made in 2% OsO, for 2 h. The specimens were dehy drated in acetone up to 100% and embedded in Mikropal (Ferak). Ultrathin sections were contrasted with uranyl acetate and lead citrate and were examined in a Siemens Elmiskop I.
Results
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Bierinann/Graf von Keyserlingk
F ig.l. Phagocytic reticulum cell. After an overloading with iron, occasionally a local artificial clear-
Bierm ann/Graf von Keyserlingk
ingupinthearea oftheGolgicomplexcanbe observed, G = Golgi complex; N = nucleus; I = inclusion
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36
bodies; Fe = ferritin; P = cell processes; Eb = erythroblasts; V = pinocytic vesicles, a x 32,000. b x 90,000. c x 32,000. Fig.2. Undifferentiated reticulum cell. R = Reti-
37
culin. x 18,000. b Filamentous reticulum cell type t (FR I); bundle of cytoplasmic filaments ( = ). S = Blood sinus; En = endothelium; M = myelocyte; L = lymphocyte, x 8,000.
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Ultrastructure of reticulum cells in the bone marrow
Fig.3. Fibrous reticulum cell type II. IP = Interdigitating cell processes; N - nucleus; Fa = fat globule; R = reticulin; f = filaments (0 10 nm)
Bicrmann/Graf von Kcyserlingk
transverse and longitudinal section,
b x 32,000. c x 56.000. d x 32,000.
x 15,000.
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38
Fig. 4. a Cytoplasm of a fibrous reticulum cell type II. ER = Rough endoplasmic reticulum, x 36,000. b Cilium of a fibrous reticulum cell type II (Ci).
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x 30,000. c Fibroblast (Fb). x 1 5,000. d Cisterns of the rough endoplasmic reticulum (C). Vesicles with ribo somes attached to it (V). x 21,000.
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Ultrastruclure of reticulum cells in the bone marrow
some smooth-edged and coated vesicles and a little of a vesicular form of rough endoplasmic reticulum. The Golgi apparatus is little developed (tig. 2a). In the electron microscope a stringy net work of reticulin filaments that covers the surface of the cell can be seen. The reticulin seems to be preferentially attached to the cell processes. The undifferentiated reticulum cells maintain contact with one another through these processes. These cells do not have large amounts ofintracytoplasmic ft laments (fig. 2a). They do not contain phagocytized material either. Filamentous reticulum cell type / Tanaka [1969] described reticulum cells in human bone marrow which contain fibrous structures, i.e. bundles of cytoplasmic fila ments in the processes. The filaments have a diameter of 4-8 nm. At some points they join to form dense bodies. These cells generally do not phagocytize. They characteristically bor der the blood sinus: there they are separated from the endothelium cells by a basal lamina (fig.2b). Fibrous reticulum cell type 11 There is a further type of reticulum cell in bone marrow having not been described on the electron-microscopic level until now. Its most noticeable characteristic is a multiplicity of interdigitating cell processes (fig. 3a. d). These cells contain neither ferritin nor phagocyticmaterial. The nucleus is large and loosely organized, similar to those of the phagocyticreticulum cells, but the chromatin is stored in coarse clumps (fig.3a). In the cytoplasm the cisterns of the rough endoplasmic reticulum are bent and sometimes tubular (fig.4a). Occasionally, there is also a
Biermann Gral' von Keyserling^
small neutral fat globule with no surrounding membrane (fig.3b); consequently, this is not a lysosomal enclosure. In one of the cells a cilium was observed (fig. 4b). The cell processes arc broader and shorter than those of the phagocytic reticulum cells (fig. Ic,3d). They partly contain serpentine filaments with a diameter of 10 nm and a clear but ribosomerich cytoplasm (fig. 3c). Smooth-edged, coated vesicles also appear within the processes. These cells also have connexion to reticulin. The cell processes interlock with each other as well as with processes of other cells (fig. 3d).
Fibroblasts Another stroma cell which differs from the non-phagocytic reticulum cell in several cytological characteristics is the fibroblast or fibrocyte. The nucleus of this cell is long and oval, the chromatin is loosely organized (fig.4c). The mitochondria are large and rich in cristac and possess a generally clear matrix. The mitochondria sometimes lie in clefts in the nucleus. The cell is long and oval and contains much cytoplasm. The cytoplasm is rich in vesicles but contains no phagocytized material. Single ribosomes arc attached to some of the vesicles, whose diameter is 100 nm (fig.4d). The vesicles of the Golgi apparatus are gener ally smaller. The cisterns are not directed toward the nucleus. It cannot be learnt from the electron micrographs what is synthesized in these cells, but the well-developed cisterns and vesicle-rich Golgi apparatus indicate a protein synthesis product which is excreted [Porter, 1961: Mo vat am! Fernando, 1962; Schwärzet ah, 1962: Ross and Benditt, 1965], Since these cells are morphologically similar to fibroblasts in other tissues, it is probable that they synthesize substances for the extracellular space.
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40
Discussion
This study showed that the branched cells of the bone marrow, which cannot be clearly classified as parenchyme (i.e. the reticulum cells), have clear ultrastructural differences. Thus reticulum cells cannot be considered as one class, as is demanded in the concept of the reticuloendothelial or reticulohistiocytic sys tem [Aschoff, 1924], The problems of these sys tems have been repeatedly and thoroughly discussed [Rohr, 1953: Marshall, 1956: llalpern et al., 1957; Akazaki, 1962: Thorhecke and Benecerraf, 1962: Pictet et al., 1969: Bessis, 1973]. The relation between reticulum cells and the stroma, i.e. the identity of reticulum and stroma cells, remains unclear. The stroma is the framework of a tissue with the perma nently fixed cells. There are clues that at least the phagocytic reticulum cells are not neces sarily fixed in position. Like the monocyte may be the precursor of the Kupfier cell, the pulmonal macrophages, and the skin macro phages [Betalanffy, 1964a, b: Carr, 1973], it is considered to be precursors of the phagocytic reticulum cells of the bone marrow [Leder, 1967 ; Gordon and Cohn, 1973]. The experiments which showed such precursor relationships subjected the organism to rather extreme conditions: therefore, it is not certain that all phagocytic reticulum cells are of monocytic origin. Tanaka [1967] was the first to indicate that the non-phagocytic reticulum cells represent a cell group which bears no functional relations to the phagocytic cells: he divided this group into cells with intracytoplasmic filaments and cells with a possible haematopoietic function. He inferred such haematopoietic functions from the observation that there was iron in the mitochondria of reticulum cells during sidero achrestic anaemia [Tanaka, 1967], A transition
41
of reticulum cells to haematopoietic cells has not yet been certified. In literature, too, there are numerous indications that the haemato poietic cell clone arises from rnobil stem cells and not from the bone marrow stroma [Loutit, 1967: Bennett and Cudkowicz, 1968: Metcalf and Moore, 1971: Zucker-Franklinet a!., 1974]. We have not found transitional forms between the phagocytic reticulum cells and the group of non-phagocytic cells either. In contrast to Tanaka's results, we have found two clearly differing cell types which are rich in filaments. The reticulum cell of the type I has filaments of 4 8 nm in diameter: type II has filaments of 10 nm. There are also fibroblasts and un differentiated cells. According to these findings, the following would be classified as fixed and stroma cells: (a) undifferentiated reticulum cells, (b) fibrous reticulum cells of type I, (c) fibrous reticulum cells of type II (interdigitating) and (d) fibro blasts. The undifferentiated reticulum cells con tained not intracytoplasmic filaments and no phagocytized material. Their relation to the other reticulum cells is unclear. There is a certain similarity to such phagocytic reticulum cells which are poor in cytoplasm and contain little phagocytized material, yet are in contact with erythroblasts. But these similarities do not justify the assumption of a transition to phagocytic reticulum cells. The fibroblasts or librocytes of the bone marrow have been known in light microscopy for a long time [Rohr, I960: Burkhardt, 1971], but they have not yet been described at the electron-micro scopic level. The morphological similarity to fibroblasts and fibrocytes in other tissues allows no doubt about the classification of these cells as fibroblasts. They can easily be distinguished from plasma cells, which are also rich in rough endoplasmic reticulum, by the
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Ultrastructure of reticulum cells in the bone m arrow
Bierm ann/G raf von Keyserlingk
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References Akazaki, K.: A concept of reticuloendothelial system. Tohoku J. exp. Med. 76: 107-118 (1962). AschofT. L.: Das reticulo-endotheliale System. Ergebn. inn. Med. Kindcrhcilk. 26: 1-118(1924). Bennett. M. und Cudkowicz, G.: Hemopoietic pro genitor cells with limited potential for dilferenciation. erythropoietic function of mouse marrow 'lymphocytes'. J. cell. comp. Physiol. 72: 129-140 (1968). Berman. J.: The ultrastructure of erythroblastic islands and reticula cells in bone marrow. J. (Jltrastruct. Res. 17: 291-313 (1967). Bertalanffy, F.D.: Respiratory tissue: structure, histophysiology, cytodynamics. Part I. Review and basic cytomorphology. Int. Rev. Cytol. 16: 233 328 (1964a). Bertalanffy, F.D .: Respiratory tissue: structure, histophysiology, cytodynamics. Part II. New ap proaches and interpretations. Int. Rev. Cytol. 17: 2 13-297 (1964b). Bcssis, M.: L'ilol érylhroblastique, unite fonctionnelle de la moelle osseuse. Revue Hémat. 13: 8-12 (1958). Bessis, M.: Living blood cells and their ulirastructurc (Springer, Berlin 1973). Bessis, M. et Breton-Gorius, J.: Etude au microscope élcctronique des granulations ferrugincuses des crythocytes normaux et pathologiques. Anemies hémolytiques. Hemoglobinopathies. Revue He mal. 12: 43-63 (1957). Burkhardt, R.: Bone narrow and bone tissue (Springer, Berlin 1971). Caffrey, R.W.; Everett, N.B., and Rieke, W.O.: Radio-autographic studies of reticular and blast cells in the hemopoietic tissues of the rat. Anal. Rec. 155: 41-57 (1966). Carr. J.: The macrophage: a review of ultrastructure and function (Academic Press, London 1973). Gall. E.A.: The cytological identity and interrelation of mesenchymal cells of lympoid tissue. Ann. N.Y. Acad. Sci. 73: 120-130(1958). Giordano. G .F. and Lichtman, M.A.: The central interaction of barrier pore size and cell maturation. J. clin. Invest. 52: 1154-64 (1973). Gordon, S. and Cohn, Z.A .: The macrophage. Int. Rev. Cytol. 36: 171-214(1973). Giildner, F.H .; Wolff, J.R., and Keyserlingk. D.G. von: Fibroblasts as a part of the contractile system
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organization of the cisterns: in plasma cells the cisterns are partly eccentrically arranged around the nucleus, whereas in the fibroblasts and fibrocytes no particular arrangement with respect to the cell nucleus is visible. Therefore, it can be assumed that these cells synthesized the material of the extracellular space, the material which then aggregates to reticulin filaments and fibrils. In our material we have also found the cells with intracytoplasmic filaments described by Tanaka [1969]. Tanaka lists the filament diam eter as 4-8 nm. These cells lie on or between small blood vessels. Tanaka differentiates them from endothelium cells, pericytes, and fat cells. At irregular intervals the fibrils were joined together in dense bodies. These filamentous structures very much remind of primitive contractile systems, such as occur in fibroblasts in tissue culture or in smooth muscle cells of the small intestive [von Keyserlingk, 1969; Weiss, 1970: Giildner el al., 1972]. If these cells, which lie near to the sinus, were able to contract, they could influence the passage of blood cells from the marrow into the sinus [fTmy, 1970: Giordano and Lichtman, 1973; Meurel el al., in press]. The filaments of the fibrous reticulum cells type II have a diameter of 10 nm and are thus thicker than those of type I. They also differ from the questionably contractile filaments in having a serpentine form. Similar filaments have been described earlier and have been con nected with an elastic function [Prinz et al., 1975], In addition to this, the cells of type II are marked by a number of interdigitated cell processes. These interdigitated cell groups possibly arise by elastic contraction of the cells during the extraction of the bone marrow from the bone. The appearance of neutral fat in these cells indicates that they can convert them selves reversibly into fat cells.
Ultrastructurc of reticulum cells in the bone marrow
Ross, R. and Benditt, E. R.: Wound healing and col lagen formation. V. Quantitative electron micro scope radioautographic observations of prolincH3 utilisation by fibroblasts. J. Cell Biol. 27:83-106 (1965). Schwarz, W.; Merker, H.J. und Kutzschc, A.: Elek tronenmikroskopische Untersuchungen über die Fibrillogcnese in Fibroblastenkulturen. Z. Zell forsch. mikrosk. Anat. 56: 107 124 (1962). Seki, M. and Shirasawa, H.: Role of reticular cells during maturation process of the erythroblast. Acta path. jap. 15: 387-405(1965). Tanaka. Y.: Iron-laden mitochondria in reticulum cells of hypersidcrotic human bone marrows. Blood 29: 747-753 (1967). Tanaka. Y.: An electron microscopic study of nonphagocytic reticulum cells in human bone marrow. I. Cells with intracytoplasmic fibrils. Acta haemat. jap. 32: 275-286 (1969). Thorbecke, G.J. and Bcnecerraf. B.: The reticulo endothelial system and immunological phenomena. Progr. Allergy, vol.6, pp.559-573 (Karger, Basel 1962). Weiss, L.: Transmural cellular passage in vascular sinuses of rat bone marrow. Blood 36: 189-208 (1970) Wetzel. B. K.; Spicer, S.S., and Horn, R.G.: Fine structural localisation of acid and alkaline phos phatases in cells of rabbit blood and bone marrow. J. Histochem. Cytochem. 15: 311-334(1967). Young, R.W.: Cell proliferation and specialisation during endochondral osteogenesis in young rats. J. Cell Biol. 14: 357-370(1962). Zucker-Franklin, D.; Grusky, G., and L'Esperance, P.: Granulocyte colonies derived from lymphocyte fractions of normal human peripheral blood. Proc. natn. Acad. Sei. USA 71: 2711-14(1974).
Received: October 20. 1976 Dr.med. Alfred Biermann, Institut für Anatomie der Freien Universität Berlin, König n-Luise-Strasse 15, D-1000 Berlin 33 Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 5/31/2018 4:15:59 PM
in duodenal villi of rat. Z. Zellforsch. mikrosk. Anal. 135: 349-360(1972). Halpern. B.N.; Benaccrraf, B., and Delafresnay, J.F. teds): Physiopathology of the reticulo-endothelial system (Blackwell, Oxford 1957). Keyserlingk, D. von: Uber den Abbau von Zellen im Knochenmark des Menschen und der Ratte. Acta haemat. 40: 252-264 (1968). Keyserlingk, D.G. von: Kontraktilität und Ultra struktur glycerin-extrahierter Fibroblasten aus der Gewebekultur. Protoplasma 67: 391-406 (1969). Leder. L. D .: Der Blutmonozyt (Springer, Berlin 1967). Loutit. J. F .: Grafts of haemopoietic tissue: the nature of haemopoietic stem cells. Symp. Tissue Org. Transplant. J. clin. Path. 20: suppl., pp.535-539 (1967). Marshall. A .H .E.: An outline of the cytology and pathology of the reticular tissue (Oliver & Boyd. London 1956). Metcalf, D. and Moore, M.A.S.: Haemopoietic cells. p.304 (North-Holland, Amsterdam 1971). Meuret. G.; Bammert, J.; Heibges, M., and Gessner, U.: Neutrophil marrow release (in press). Movat, H.Z. and Fernando. N .V .P.: The fine struc ture of connective tissue. I. The fibroblast. Expl molec. Path. I: 509-534(1962). Owen. M.: The origin of bone cells. Int. Rev. Cytol. 28: 213-236(1970). Pictet. R.; Orce, L.: Forssmann, W.G., and Girardier, L.: An electron microscope study of the perfusionfixed spleen. I. The splenic circulation and the RES concept. Z. Zellforsch. mikrosk. Anat. 96: 372-399 (1969). Porter. K.R.: The endoplasmic reticulum: some cur rent interpretations of its forms and functions. Biol. Struct. Funct. I: 127-155(1961). Prinz. U.; Kappel, A. und Keyserlingk, D.G. von: Ultrastrukturclle Veränderungen in den Muskel zellen des Rattenuterus während der Schwanger schaft. Verh. Anat. Ges., Jena 69: 749-753 (1975). Rohr. K.: Das retikulohistiozytäre System und seine Erkrankungen vom klinischen Standpunkt. Verh. dt. Ges. Path. 37: 127-139(1953). Rohr. K.: Das menschliche Knochenmark (Thieme, Stuttgart I960).
43
Ultrastructure of reticulum cells in the bone marrow12 A. Biermann and D. Graf von Keyserlingk Institut für Anatomie, Freie Universität Berlin, Berlin
Key words. Reticulum cells • Bone marrow • Ultrastructure Abstract. In this study the attempt was made to classify the reticulum cells of the bone marrow on the basis of electron-microscopic findings. The basis of the differentiation was the ability of the cells to phagocytize substances or not. For two cell types the intracytoplasmic filaments were used as distinctive marks. The following classification resulted: (a) phagocytic reticulum cells, (b) undifferentiated reticulum cells, (c) fibrous reticulum cells of type I, which contain filaments of 4-8 nm diameter and are located near the blood sinus of the bone marrow, (d) fibrous reticu lum cells of type II. which contain intracytoplasmic filaments of 10 nm diameter; since these cells contain neutral fat bodies, the possibility of a reversible conversion to fat cells has to be assumed and (e) fibroblasts, cells which synthesize the substance of the extracellular space. A connexion of reticulum cells to haematopoietic functions or to stem cell functions could not be found.
'Reticulum cell' In the bone marrow or in lymphoreticular tissue is a general expression which is variously applied. It can mean branched cells whose processes build a network (reticulum), or cells which arc con nected to, or even produce, extracellular reticulin (a network of a certain type of collagen), or cells which do not directly serve the haemo- or lymphocytopoiesis. i.e. the stroma cells. It is difficult to show a preference for one of these definitions. Reticulum cells include undifferentiated cells, fibrous reticulum cells, fibro blasts. phagocytes, reticular macrophages, dendritic 1 Dedicated to Prof. W. Schwarz on the occasion of his 70th birthday. 2 We are grateful to Miss Barbara Hass and Miss Monika Dulinski for their skilful assistance.
macrophages, osteoprogenitor cells and others [ Young. 1962; Owen. 1970: Carr. 1973]. The expression 'reti culum cell- causes discomfort not only because of the various definitions [Gall, 1958], but also because, in the past, functions were ascribed to reticulum cells neglect ing the significance of the different cell types. This is due to a general lack of knowledge about the morpho logy and physiology of these cells. Since until now light microscopy has not given a satisfactory differ entiation of the various types of reticulum cells, we have studied the cytological characteristics of these cells with the electron microscope.
Materials and methods Bone marrow specimens from patients with various haematological diseases and from the rat femur were
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Introduction
35
In the current study, all bone marrow cells which had extensive processes were considered first. Endothelium cells, which represent a separate cell group, and megakaryocytes, which can have processes but are not included in the group of reticulum cells, were excepted. The surface foldings of the monocytes, lym phocytes, and granulocytes were not consid ered as cell processes in this connexion.
spondingly many vesicles and lysosomal ele ments. The numerous mitochondria are large and have many cristae. The few cisterns in the endoplasmic reticulum are short and stretched and only partially loaded with ribosomes. After overloading the cells with iron, one finds ferritin distributed diffusely in the cytoplasm; a heavier deposit of ferritin around the Golgi complexes is noticeable (fig. la). After over loading with ferritin, even isolated processes between the parenchymecellscan be associated with the phagocytic reticulum cells (fig. lb). The processes are very long and narrow and follow the contours of other cells which they are often surrounding. They enclose erythroblasts and other cells. In the interior of the processes only homogeneous ground cytoplasm, pinocytotic vesicles, and sometimes ferritin with the same density as in the pericaryon can be seen, but no cell organelles (fig. lc).
Phagocytic reticulum cell The ultrastructure of this cell group has already been well studied, especially in relation to the phagocytized material and its destruc tion [Bessis and Breton-Gorius, 1957: Bessis, 1958,1973; Seki and Shirasa wa, 1965: Berman, 1967; Wetzel et a i, 1967; von Keyserlingk, 1968]. Moreover, it became evident that these phagocytizing reticulum cells have a special function in the maturation of erythroblasts: a phagocytizing reticulum cell becomes en circled by erythroblasts in order to promote maturation of the latter. The origin and further destiny of these reticulum cells have not yet been discovered. The following characteristics distinguish these reticulum cells from others: the nucleus is large, has rounded contours, and can be dented by phagocytized material. It has numerous nuclear pores, and the chromatin is scattered (fig. Ia). The cytoplasm is broad and contains many Golgi complexes and corre
Undifferentiated reticulum cell Caffrey el al. [1966] looked at the undif ferentiated reticulum cell in the light micro scope and described it as a cell with an oval or irregularly shaped nucleus, a weakly coloured chromatin network and a clear nuclear bound ary due to the chromatin's adherence to the membrane. The boundaries of the cytoplasm are not clear. On the basis of these light-micro scopic criteria, the undifferentiated reticulum cell can be located in the electron microscope. Here too, as expected, the contour of the nucleus is wavy and irregular. The nuclear chromatin largely fits well to the nuclear membrane in a narrow dense band (fig. 2a). The nucleolus is ring-shaped. There is little cytoplasm. Since the cell processes are narrow and contain no large inclusion bodies, it is understandable that the cell boundaries can not be seen clearly in the light microscope. The cytoplasm is poor in ribosomes but contains
examined. The specimens were fixed in 1% glutaraldehyde and 1% paraformaldehyde for 2 h, using a 0.05 M cacodilai buffer with pH 7.2. Postfixation was made in 2% OsO, for 2 h. The specimens were dehy drated in acetone up to 100% and embedded in Mikropal (Ferak). Ultrathin sections were contrasted with uranyl acetate and lead citrate and were examined in a Siemens Elmiskop I.
Results
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Bierinann/Graf von Keyserlingk
F ig.l. Phagocytic reticulum cell. After an overloading with iron, occasionally a local artificial clear-
Bierm ann/Graf von Keyserlingk
ingupinthearea oftheGolgicomplexcanbe observed, G = Golgi complex; N = nucleus; I = inclusion
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36
bodies; Fe = ferritin; P = cell processes; Eb = erythroblasts; V = pinocytic vesicles, a x 32,000. b x 90,000. c x 32,000. Fig.2. Undifferentiated reticulum cell. R = Reti-
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culin. x 18,000. b Filamentous reticulum cell type t (FR I); bundle of cytoplasmic filaments ( = ). S = Blood sinus; En = endothelium; M = myelocyte; L = lymphocyte, x 8,000.
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Ultrastructure of reticulum cells in the bone marrow
Fig.3. Fibrous reticulum cell type II. IP = Interdigitating cell processes; N - nucleus; Fa = fat globule; R = reticulin; f = filaments (0 10 nm)
Bicrmann/Graf von Kcyserlingk
transverse and longitudinal section,
b x 32,000. c x 56.000. d x 32,000.
x 15,000.
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38
Fig. 4. a Cytoplasm of a fibrous reticulum cell type II. ER = Rough endoplasmic reticulum, x 36,000. b Cilium of a fibrous reticulum cell type II (Ci).
.39
x 30,000. c Fibroblast (Fb). x 1 5,000. d Cisterns of the rough endoplasmic reticulum (C). Vesicles with ribo somes attached to it (V). x 21,000.
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Ultrastruclure of reticulum cells in the bone marrow
some smooth-edged and coated vesicles and a little of a vesicular form of rough endoplasmic reticulum. The Golgi apparatus is little developed (tig. 2a). In the electron microscope a stringy net work of reticulin filaments that covers the surface of the cell can be seen. The reticulin seems to be preferentially attached to the cell processes. The undifferentiated reticulum cells maintain contact with one another through these processes. These cells do not have large amounts ofintracytoplasmic ft laments (fig. 2a). They do not contain phagocytized material either. Filamentous reticulum cell type / Tanaka [1969] described reticulum cells in human bone marrow which contain fibrous structures, i.e. bundles of cytoplasmic fila ments in the processes. The filaments have a diameter of 4-8 nm. At some points they join to form dense bodies. These cells generally do not phagocytize. They characteristically bor der the blood sinus: there they are separated from the endothelium cells by a basal lamina (fig.2b). Fibrous reticulum cell type 11 There is a further type of reticulum cell in bone marrow having not been described on the electron-microscopic level until now. Its most noticeable characteristic is a multiplicity of interdigitating cell processes (fig. 3a. d). These cells contain neither ferritin nor phagocyticmaterial. The nucleus is large and loosely organized, similar to those of the phagocyticreticulum cells, but the chromatin is stored in coarse clumps (fig.3a). In the cytoplasm the cisterns of the rough endoplasmic reticulum are bent and sometimes tubular (fig.4a). Occasionally, there is also a
Biermann Gral' von Keyserling^
small neutral fat globule with no surrounding membrane (fig.3b); consequently, this is not a lysosomal enclosure. In one of the cells a cilium was observed (fig. 4b). The cell processes arc broader and shorter than those of the phagocytic reticulum cells (fig. Ic,3d). They partly contain serpentine filaments with a diameter of 10 nm and a clear but ribosomerich cytoplasm (fig. 3c). Smooth-edged, coated vesicles also appear within the processes. These cells also have connexion to reticulin. The cell processes interlock with each other as well as with processes of other cells (fig. 3d).
Fibroblasts Another stroma cell which differs from the non-phagocytic reticulum cell in several cytological characteristics is the fibroblast or fibrocyte. The nucleus of this cell is long and oval, the chromatin is loosely organized (fig.4c). The mitochondria are large and rich in cristac and possess a generally clear matrix. The mitochondria sometimes lie in clefts in the nucleus. The cell is long and oval and contains much cytoplasm. The cytoplasm is rich in vesicles but contains no phagocytized material. Single ribosomes arc attached to some of the vesicles, whose diameter is 100 nm (fig.4d). The vesicles of the Golgi apparatus are gener ally smaller. The cisterns are not directed toward the nucleus. It cannot be learnt from the electron micrographs what is synthesized in these cells, but the well-developed cisterns and vesicle-rich Golgi apparatus indicate a protein synthesis product which is excreted [Porter, 1961: Mo vat am! Fernando, 1962; Schwärzet ah, 1962: Ross and Benditt, 1965], Since these cells are morphologically similar to fibroblasts in other tissues, it is probable that they synthesize substances for the extracellular space.
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Discussion
This study showed that the branched cells of the bone marrow, which cannot be clearly classified as parenchyme (i.e. the reticulum cells), have clear ultrastructural differences. Thus reticulum cells cannot be considered as one class, as is demanded in the concept of the reticuloendothelial or reticulohistiocytic sys tem [Aschoff, 1924], The problems of these sys tems have been repeatedly and thoroughly discussed [Rohr, 1953: Marshall, 1956: llalpern et al., 1957; Akazaki, 1962: Thorhecke and Benecerraf, 1962: Pictet et al., 1969: Bessis, 1973]. The relation between reticulum cells and the stroma, i.e. the identity of reticulum and stroma cells, remains unclear. The stroma is the framework of a tissue with the perma nently fixed cells. There are clues that at least the phagocytic reticulum cells are not neces sarily fixed in position. Like the monocyte may be the precursor of the Kupfier cell, the pulmonal macrophages, and the skin macro phages [Betalanffy, 1964a, b: Carr, 1973], it is considered to be precursors of the phagocytic reticulum cells of the bone marrow [Leder, 1967 ; Gordon and Cohn, 1973]. The experiments which showed such precursor relationships subjected the organism to rather extreme conditions: therefore, it is not certain that all phagocytic reticulum cells are of monocytic origin. Tanaka [1967] was the first to indicate that the non-phagocytic reticulum cells represent a cell group which bears no functional relations to the phagocytic cells: he divided this group into cells with intracytoplasmic filaments and cells with a possible haematopoietic function. He inferred such haematopoietic functions from the observation that there was iron in the mitochondria of reticulum cells during sidero achrestic anaemia [Tanaka, 1967], A transition
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of reticulum cells to haematopoietic cells has not yet been certified. In literature, too, there are numerous indications that the haemato poietic cell clone arises from rnobil stem cells and not from the bone marrow stroma [Loutit, 1967: Bennett and Cudkowicz, 1968: Metcalf and Moore, 1971: Zucker-Franklinet a!., 1974]. We have not found transitional forms between the phagocytic reticulum cells and the group of non-phagocytic cells either. In contrast to Tanaka's results, we have found two clearly differing cell types which are rich in filaments. The reticulum cell of the type I has filaments of 4 8 nm in diameter: type II has filaments of 10 nm. There are also fibroblasts and un differentiated cells. According to these findings, the following would be classified as fixed and stroma cells: (a) undifferentiated reticulum cells, (b) fibrous reticulum cells of type I, (c) fibrous reticulum cells of type II (interdigitating) and (d) fibro blasts. The undifferentiated reticulum cells con tained not intracytoplasmic filaments and no phagocytized material. Their relation to the other reticulum cells is unclear. There is a certain similarity to such phagocytic reticulum cells which are poor in cytoplasm and contain little phagocytized material, yet are in contact with erythroblasts. But these similarities do not justify the assumption of a transition to phagocytic reticulum cells. The fibroblasts or librocytes of the bone marrow have been known in light microscopy for a long time [Rohr, I960: Burkhardt, 1971], but they have not yet been described at the electron-micro scopic level. The morphological similarity to fibroblasts and fibrocytes in other tissues allows no doubt about the classification of these cells as fibroblasts. They can easily be distinguished from plasma cells, which are also rich in rough endoplasmic reticulum, by the
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Ultrastructure of reticulum cells in the bone m arrow
Bierm ann/G raf von Keyserlingk
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References Akazaki, K.: A concept of reticuloendothelial system. Tohoku J. exp. Med. 76: 107-118 (1962). AschofT. L.: Das reticulo-endotheliale System. Ergebn. inn. Med. Kindcrhcilk. 26: 1-118(1924). Bennett. M. und Cudkowicz, G.: Hemopoietic pro genitor cells with limited potential for dilferenciation. erythropoietic function of mouse marrow 'lymphocytes'. J. cell. comp. Physiol. 72: 129-140 (1968). Berman. J.: The ultrastructure of erythroblastic islands and reticula cells in bone marrow. J. (Jltrastruct. Res. 17: 291-313 (1967). Bertalanffy, F.D.: Respiratory tissue: structure, histophysiology, cytodynamics. Part I. Review and basic cytomorphology. Int. Rev. Cytol. 16: 233 328 (1964a). Bertalanffy, F.D .: Respiratory tissue: structure, histophysiology, cytodynamics. Part II. New ap proaches and interpretations. Int. Rev. Cytol. 17: 2 13-297 (1964b). Bcssis, M.: L'ilol érylhroblastique, unite fonctionnelle de la moelle osseuse. Revue Hémat. 13: 8-12 (1958). Bessis, M.: Living blood cells and their ulirastructurc (Springer, Berlin 1973). Bessis, M. et Breton-Gorius, J.: Etude au microscope élcctronique des granulations ferrugincuses des crythocytes normaux et pathologiques. Anemies hémolytiques. Hemoglobinopathies. Revue He mal. 12: 43-63 (1957). Burkhardt, R.: Bone narrow and bone tissue (Springer, Berlin 1971). Caffrey, R.W.; Everett, N.B., and Rieke, W.O.: Radio-autographic studies of reticular and blast cells in the hemopoietic tissues of the rat. Anal. Rec. 155: 41-57 (1966). Carr. J.: The macrophage: a review of ultrastructure and function (Academic Press, London 1973). Gall. E.A.: The cytological identity and interrelation of mesenchymal cells of lympoid tissue. Ann. N.Y. Acad. Sci. 73: 120-130(1958). Giordano. G .F. and Lichtman, M.A.: The central interaction of barrier pore size and cell maturation. J. clin. Invest. 52: 1154-64 (1973). Gordon, S. and Cohn, Z.A .: The macrophage. Int. Rev. Cytol. 36: 171-214(1973). Giildner, F.H .; Wolff, J.R., and Keyserlingk. D.G. von: Fibroblasts as a part of the contractile system
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organization of the cisterns: in plasma cells the cisterns are partly eccentrically arranged around the nucleus, whereas in the fibroblasts and fibrocytes no particular arrangement with respect to the cell nucleus is visible. Therefore, it can be assumed that these cells synthesized the material of the extracellular space, the material which then aggregates to reticulin filaments and fibrils. In our material we have also found the cells with intracytoplasmic filaments described by Tanaka [1969]. Tanaka lists the filament diam eter as 4-8 nm. These cells lie on or between small blood vessels. Tanaka differentiates them from endothelium cells, pericytes, and fat cells. At irregular intervals the fibrils were joined together in dense bodies. These filamentous structures very much remind of primitive contractile systems, such as occur in fibroblasts in tissue culture or in smooth muscle cells of the small intestive [von Keyserlingk, 1969; Weiss, 1970: Giildner el al., 1972]. If these cells, which lie near to the sinus, were able to contract, they could influence the passage of blood cells from the marrow into the sinus [fTmy, 1970: Giordano and Lichtman, 1973; Meurel el al., in press]. The filaments of the fibrous reticulum cells type II have a diameter of 10 nm and are thus thicker than those of type I. They also differ from the questionably contractile filaments in having a serpentine form. Similar filaments have been described earlier and have been con nected with an elastic function [Prinz et al., 1975], In addition to this, the cells of type II are marked by a number of interdigitated cell processes. These interdigitated cell groups possibly arise by elastic contraction of the cells during the extraction of the bone marrow from the bone. The appearance of neutral fat in these cells indicates that they can convert them selves reversibly into fat cells.
Ultrastructurc of reticulum cells in the bone marrow
Ross, R. and Benditt, E. R.: Wound healing and col lagen formation. V. Quantitative electron micro scope radioautographic observations of prolincH3 utilisation by fibroblasts. J. Cell Biol. 27:83-106 (1965). Schwarz, W.; Merker, H.J. und Kutzschc, A.: Elek tronenmikroskopische Untersuchungen über die Fibrillogcnese in Fibroblastenkulturen. Z. Zell forsch. mikrosk. Anat. 56: 107 124 (1962). Seki, M. and Shirasawa, H.: Role of reticular cells during maturation process of the erythroblast. Acta path. jap. 15: 387-405(1965). Tanaka. Y.: Iron-laden mitochondria in reticulum cells of hypersidcrotic human bone marrows. Blood 29: 747-753 (1967). Tanaka. Y.: An electron microscopic study of nonphagocytic reticulum cells in human bone marrow. I. Cells with intracytoplasmic fibrils. Acta haemat. jap. 32: 275-286 (1969). Thorbecke, G.J. and Bcnecerraf. B.: The reticulo endothelial system and immunological phenomena. Progr. Allergy, vol.6, pp.559-573 (Karger, Basel 1962). Weiss, L.: Transmural cellular passage in vascular sinuses of rat bone marrow. Blood 36: 189-208 (1970) Wetzel. B. K.; Spicer, S.S., and Horn, R.G.: Fine structural localisation of acid and alkaline phos phatases in cells of rabbit blood and bone marrow. J. Histochem. Cytochem. 15: 311-334(1967). Young, R.W.: Cell proliferation and specialisation during endochondral osteogenesis in young rats. J. Cell Biol. 14: 357-370(1962). Zucker-Franklin, D.; Grusky, G., and L'Esperance, P.: Granulocyte colonies derived from lymphocyte fractions of normal human peripheral blood. Proc. natn. Acad. Sei. USA 71: 2711-14(1974).
Received: October 20. 1976 Dr.med. Alfred Biermann, Institut für Anatomie der Freien Universität Berlin, König n-Luise-Strasse 15, D-1000 Berlin 33 Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 5/31/2018 4:15:59 PM
in duodenal villi of rat. Z. Zellforsch. mikrosk. Anal. 135: 349-360(1972). Halpern. B.N.; Benaccrraf, B., and Delafresnay, J.F. teds): Physiopathology of the reticulo-endothelial system (Blackwell, Oxford 1957). Keyserlingk, D. von: Uber den Abbau von Zellen im Knochenmark des Menschen und der Ratte. Acta haemat. 40: 252-264 (1968). Keyserlingk, D.G. von: Kontraktilität und Ultra struktur glycerin-extrahierter Fibroblasten aus der Gewebekultur. Protoplasma 67: 391-406 (1969). Leder. L. D .: Der Blutmonozyt (Springer, Berlin 1967). Loutit. J. F .: Grafts of haemopoietic tissue: the nature of haemopoietic stem cells. Symp. Tissue Org. Transplant. J. clin. Path. 20: suppl., pp.535-539 (1967). Marshall. A .H .E.: An outline of the cytology and pathology of the reticular tissue (Oliver & Boyd. London 1956). Metcalf, D. and Moore, M.A.S.: Haemopoietic cells. p.304 (North-Holland, Amsterdam 1971). Meuret. G.; Bammert, J.; Heibges, M., and Gessner, U.: Neutrophil marrow release (in press). Movat, H.Z. and Fernando. N .V .P.: The fine struc ture of connective tissue. I. The fibroblast. Expl molec. Path. I: 509-534(1962). Owen. M.: The origin of bone cells. Int. Rev. Cytol. 28: 213-236(1970). Pictet. R.; Orce, L.: Forssmann, W.G., and Girardier, L.: An electron microscope study of the perfusionfixed spleen. I. The splenic circulation and the RES concept. Z. Zellforsch. mikrosk. Anat. 96: 372-399 (1969). Porter. K.R.: The endoplasmic reticulum: some cur rent interpretations of its forms and functions. Biol. Struct. Funct. I: 127-155(1961). Prinz. U.; Kappel, A. und Keyserlingk, D.G. von: Ultrastrukturclle Veränderungen in den Muskel zellen des Rattenuterus während der Schwanger schaft. Verh. Anat. Ges., Jena 69: 749-753 (1975). Rohr. K.: Das retikulohistiozytäre System und seine Erkrankungen vom klinischen Standpunkt. Verh. dt. Ges. Path. 37: 127-139(1953). Rohr. K.: Das menschliche Knochenmark (Thieme, Stuttgart I960).
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