Ultrastructure of Human Spleen in Transmission and Scanning Electron Microscope

Home , Red pulp, Trabecular arteries, Trabecular veins

Biologia 64/2: 402—408, 2009 Section Zoology DOI: 10.2478/s11756-009-0046-2

Ultrastructure of human spleen in transmission and scanning electron microscope

Štefan Polák, Paulína Gálfiová &IvanVarga

Department of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4,SK-81108 Bratislava, Slovakia; e-mail: [email protected]

Abstract: Despite new information concerning functional morphology of spleen, there are still some inaccuracies mostly regarding the spleen blood circulation. Billroth’s (splenic) cords are formed from three-dimensional network of fibroblastic reticular cells located among branched sinuses. Results from our study using scanning electron microscopy confirm an intimate contact between adjacent reticular cells and erythrocytes. Arterial terminals can be observed in the Billroth’s cords. The wall of sinuses reminds a sieve and it is lined with a special type of endothelium. In electron microscope, endothelial cells look like rods oriented parallel to the longitudinal axis of sinuses. Based on our observations fibroblastic reticular cells change to fixed phagocytes under no circumstances, hence they do not participate in phagocytosis. They may have a recognition function for cells circulating around them. According to our opinion, the open and the closed blood circulation are present in the human spleen simultaneously. Blood flowing in the closed circulation can help “absorption“ of extra-vascular liquid and the blood elements into the vascular lumen. Due to sporadic occurrence of smooth muscle cells in the capsule and trabeculae, we assume that human spleen is not a blood reservoir, unlike the spleen in some other animals. Key words: human spleen; ultrastructure

Introduction precisely known when exactly the first information concerning the importance of the spleen in the defence Spleen (lien, splen) is the largest encapsulated lym- against infections appeared. The spleen was included phoid organ of humans. It represents the most abun- into the lymphatic system for the first time by Wil- dant accumulation of lymphoid tissue in the human iam Hewson in 1777. Spleen enlargement during febrile body. Based of its structure and function, spleen can diseases and severe infections could have pointed at its be seen as two different organs. On one hand, the white protective role. The question about the importance of pulp of spleen is an important part of the immune sys- the spleen for human life is still emerging ever since the tem involved in the proliferation process of lympho- first splenectomy made by Adrian Zaccareli in 1549. cytes and plasma cells, as well as the production of It is generally known that the spleen surface is antibodies. On the other hand, the red pulp is con- covered by a capsule (capsula fibrosa lienis) forming nected with bloodstream and is engaged in phagocyto- a thick collagen connective tissue with admixture of sis of blood elements (Béder et al. 2005). In the past, elastic fibres and solitary smooth muscle cells. Con- spleen was described as organum plenum mysterii for its nective septa – trabeculas (trabeculae lienis)branch “mystic and mysterious” function. The fact that spleen off connective capsule. Branch of spleen vessels (arte- has no openings might have complicated the investi- riaetvenalienalis) pass through from spleen hilus. gation of its function. First hypotheses described it as Spleen parenchyma, called pulp, forms a tissue with a place of black bile degradation. According to Hip- double composition and arrangement. Their names are pocrates (460–380 BC), the black bile was one of the derived from the colours of the spleen section. White four liquids regulating the human body. It was supposed pulp forms a typical lymphatic tissue. Its stroma is to cause bad mood and sadness, melancholy. Erisistra- formed mostly from reticular connective tissue with in- tus from Alexandria (310–250 BC), who was interested serted B- and T-lymphocytes and other effector cells of in the body symmetry, affirmed that the spleen is an the immune system. White pulp consists of two com- equivalent to liver, localized on the right side. Aris- ponents: the sheath surrounding arteria centralis (pe- toteles (384–322 BC), who was the first to describe riarterial lymphatic sheath, PALS), which is the region anatomical location of spleen, had also similar opin- dependent on thymus (it contains T-lymphocytes). The ion (Wilkins 2002). Attractiveness of this theory could second part of the white pulp consists of lymphatic fol- be explained by prevalent occurrence of enlarged spleen licles (folliculi lymphatici), which are present at places (splenomegaly) in inhabitants of Middle East countries, where arteria centralis branches. In spleen, they are infected with malaria (Polák & Varga 2006). It is not called Malpighian bodies. The borderline between the

c 2009 Institute of Zoology, Slovak Academy of Sciences Ultrastructure of human spleen 403 white and red pulp is formed by zona marginalis.B- In the regulation of vascular motility of the spleen, lymphocytes are prevalent in the lymphatic nodes as adrenergic nerve components play an important role well as in zona marginalis. The red pulp of the spleen (Schmidtová et al. 1999; Siroťáková et al. 1999, 2002). consists of Billroth’s cords from fibroblastic reticular cells. They are located between branched vascular sinuses. In cords, there are numerous free cells: Material and methods macrophages, lymphocytes, granulocytes, but mainly Samples of the spleen tissue came mostly from the spleens erythrocytes which are responsible for typical colour ablated during splenectomy, performed after accidental in- of this part of the spleen (for rewievs see Jakubovský jury or as a therapy of various diseases, mostly purpura & Hromec 1988; Jakubovský & Porubský 1995; Polák thrombocytopenica and anaemia haemolytica of various ori- & Varga 2006). Up-to-date problem of biopsy examina- gin, liver cirrhosis and other diseases. The set of samples tion is the determination of the borderline between B- consists of 146 bioptic excisions. For examination of the and T-cells dependent regions. Examination of the ex- samples in the transmission electrone microscope (TEM), the excisions were fixed in 3% glutaraldehyde and in 1% tracellular reticulum can help, since this net from retic- OsO4. Fixatives were buffered by phosphates. The tissue ular fibres is typical for periarterial lymphatic sheaths was dehydrated by alcohol and transferred by propylene ox- (region with T-lymphocytes) and is absent in lymphatic ide to Durcupan ACM. Selection of blocks for ultramicro- follicles (typical region with B-lymphocytes). The fluo- tome sectioning was performed from semi thin slides which rescence method for haematoxylin-eosin stained slides were examined by light microscope under phase contrast. can be utilized in diagnostics (Jakubovský et al. 2002). Appropriate samples were cut by ultramicrotome (OmU3 or The problem of blood circulation in the spleen Ultracut E) and stained using uranyl acetate and plumbous has been incessantly discussed. The spleen gets blood citrate contrast dye. The findings were examined under elec- from the spleen artery (arteria lienalis), branching to trone microscopes BS 613, BS 500 (Tesla Brno) and CM100 (Philips Eindhoven). The spleen three dimensional struc- trabecular arteries (arteriae trabeculares) after enter- ture was studied by scanning electrone microscopes DSM- ing the spleen hilus. They change to so-called cen- 950 (Zeiss-Opton Oberkochen), Mira (Tescan Brno) and tral arteries, surrounded by a white pulp after leav- Vega (Tescan Brno). Tissue samples were fixed immedi- ing fibrous trabeculae (arteriae centrales). After leaving ately by double fixation by glutaraldehyde with OsO4 or in the white pulp, the central arteries branch to numer- freshly depolymerised paraformaldehyde (also buffered by ous penicillate arteries (arteriae penicilattae). Before phosphates). The samples were dehydrated by alcohol and the final branching, the arteriae penicilattae surround amyl acetate gradual series. Then the samples were dried by the sheaths from macrophages, so-called Schweigger’s the method of the critical point of CO2 determination and and Seidl’s sheaths (“encapsulated arterioles”). These galvanized with gold. Carbon dioxide has at specific pres- sheaths are discreetly formed in human. The capillar- sure and temperature, called critical pressure and critical temperature, the same volume in both the liquid and the ies running into wide sinuses of the red pulp branch off gas states. There are no changes of the surface tension on encapsulated arterioles. the surface of the dried samples under these conditions and In the closed circulation, the arterial walls are di- the transfer from the liquid into the gas phase is performed rectly connected with the walls of sinuses. In the open without any damage of the surface structure. Without dry- circulation, arteries end blindly in the red pulp, while ing at the critical point of CO2, marked artificial changes the blood freely flows from them into Billroth’s cords. would appear, especially in the structure of Billroth’s cords Consequently, the blood gets into sinuses through three (Polák et al. 1983). The area of section and the refractive dimensional net of fibroblastic reticular cells and subse- area were examined. quently passes through openings in the walls of sinuses. From sinuses the blood runs into veins of the red pulp, Results from there to trabecular veins to vena lienalis.Theopen system of the blood circulation prevails in the human Billroth’s cords of the red pulp are formed from spleen, while the smaller part runs in the closed system a three dimensional net of fibroblastic reticular cells (Brozman et al. 1983; Brozman & Jakubovský 1989; (Fig. 1). They have variously long processes resem- Kashimura 1985; Schmidt et al. 1988). bling the “wings” (Fig. 2), by means of which they Kashimura & Shibata (1989) described even three mutually anastomose. Fibroblastic reticular cells are lo- types of spleen circulation with different functions. cated among branched sinuses. Reticular fibres are sur- Open circulation is engaged in a very sensitive blood rounded by their cytoplasm, what cannot be always no- purge from xenobiotics by numerous macrophages re- ticed in the light microscope. Variously deformed blood siding in Billroth’s cords. For a closed circulation, rapid elements, especially red cells, squeeze through the pro- blood flow as well as the presence of small number of cesses of reticular cells. SEM examinations indicate that macrophages in sinuses is characteristic. The third type there exist a close contact between erythrocytes and of circulation is a microcirculation toward the white reticular cells (Fig. 3). The main functional elements of pulp and marginal zone. This type of circulation helps the spleen’s red pulp cords are macrophages, which are during phagocytosis of xenobiotics from blood and as- able to phagocyte (Fig. 3). sists the contact of white pulp lymphocytes with anti- Arterial terminals can be also seen in some red pulp gens. However, most authors do not mention this third cords. The terminal can have a shape of funnel, ampule, type of circulation. or a sac with circular pores (Fig. 4). We have not found 404 Š. Polák et al.

Fig. 3. Red pulp of spleen in SEM. Fibroblastic reticular cell in the center (1) with several long and short processes. In the Billroth’s cord macrophages (2) ingesting erythrocytes (3). In the vicinity lymphocyte (4), several erythrocytes (5) and macrophage (6). Scale 5 µm.

Fig. 1. Red pulp of spleen in SEM. Supporting framework is formed by ﬁbroblastic reticular cells (1) with long processes (2), between them lymphocytes (3) and erythrocytes (4). Magnif. 10660×.

Fig. 2. Fibroblastic reticular cell of the asteroid shape in TEM. Reticular fibers located near the cell nucleus (1). In the more dense cytoplasm of the reticular cell three mitochondria (2) with numerous cristas and the rough endoplasmatic reticulum (3). In Fig. 4. Arterial terminus in SEM. Vasal ending with many roud the nucleus with nucleonemats (4), nucleus body (5) and numer- openings (1) ressembles shower rosette. Blood elements (2) inside × ous nuclear pores (6). Around the reticular cell lymphocytes (7) the lumen. Magnif. 7000 . with different amount of cytoplasm. Scale 1 µm. a direct transition of the compact lamina basalis of the macrophages, from where it has to penetrate through terminal arterioles to discontinuous lamina basalis of si- wall structures of sinuses into the closed blood circula- nuses. This finding confirms existence of an open blood tion. The wall of red pulp sinuses is lined with a special circulation. However, we do not exclude a minor partic- endothelial cell type (sinus lining cells) surrounded by ipation of the closed circulation. It is more accessible for ring fibres and processes of fibroblastic reticular cells monitoring by special techniques of the light microscopy (Figs 5–7). Lamina basalis of sinuses is not usually ho- – enzyme histochemistry. Before termination of the ter- mogeneous – it is formed from thick, transversal ring minal arteries in zona marginalis and in the red pulp, fibres (vertically to the longitudinal axis of sinuses) there are created sheaths of macrophages around them. (Fig. 6). In the SEM, the sinuses lining cells can be seen Blood permeates here into red pulp cords filled with as rods oriented in parallel with the longitudinal axis Ultrastructure of human spleen 405

Fig. 7. Sinus in TEM with a large opening in the wall (1) by which it communicates with the Billroth’s cord (2). Macrophage (3) with an ingested erythrocyte (black mater) communicates with the wall of sinus. In the cord erythrocytes (4), polymorphonuclear Fig. 5. Venous sinus in SEM. Nuclei and perinuclear cytoplasms leukocytes (5) and trombocytes (6). Scale 7 µm. of sinus lining cells (1) extrude into the lumen of sinus, ﬁssural openings between sinus lining cells (2) through which blood elements enter the sinus (3). Magnif. 1390×.

Fig. 8. Red pulp of spleen in TEM. Sinus in the center (1), part of sinuses at the edge (2). Flat cells lining sinuses (3). Sinus lining cells sit on the ring fibers (4). In the cytoplasm dense gran- ules around the nucleus (5) of the sinus lining cells. Ring fibers from the side turned away from the lumen of sinus, partly cover Fig. 6. The sinus, outer view, in SEM. Rod shaped sinus lining processes of the fibroblastic reticular cells (6). In the sinus two cells (1) are parallely orientated, surrouned by ring fibers (2). neutrophil granulocytes (7) and platelets (8). In Billroth’s cords Erythrocyte (3) passes through the opening in the wall of sinus lymphocytes (10), macrophages (11), trombocytes (8), erythro- into its lumen. Magnif. 5390×. cytes (9). Scale 1 µm. of sinuses. Parts of cytoplasm around nuclei markedly than platelets and were smaller than 0.5 micrometer. stick up into the lumen of the sinus (Fig. 5). Sinus lin- Definition of zona marginalis has been discussed ing cells are at some places of the side walls intercon- problem up to this day. Its borderlines can be hardly nected by the processes creating irregular openings. If determined via light microscope. For zona marginalis erythrocytes penetrate through these slots, they obtain there is characteristic population of the mixture of dif- variously deformed, even “tail” shapes (Fig. 9). From ferent cells such as lymphocytes, erythrocytes, trombo- sinuses the blood runs into veins of the red pulp, from cytes, granulocytes and monocytes. None of the blood there to trabecular veins (Fig. 10). elements, forming zona marginalis, is characteristic During the examination of two cases of the chronic for it. The basis of the white pulp border and zona autoimmune thrombocytopenia by the scanning elec- marginalis is formed from the circumferential reticulum tron microscope we found during two different time pe- and the outer periarterial lymphatic sheath. Examina- riods a small spherical formation located on the surface tion by SEM shows that there is a canal space between of the sinus lining cells, as well as on the erythrocytes lymphatic follicle and zona marginalis. It has a well- (Fig. 9). These “microparticles” were clearly smaller formed, relatively compact wall at zona marginalis side. 406 Š. Polák et al.

Fig. 11. Zona marginalis in SEM. Lymphocytes of the follicle separated from zona marginalis by the channel (1), having on the zona marginalis side well formed, quite a compact wall (2). From the side of follicle the wall is quite numerously fenestrated (3). Scale 5 µm.

Fig. 9. Erythrocyte penetrating through the wall of sinus in SEM. Microparticles on the surface of erythrocytes in sinus. Scale 2 µm.

Fig. 12. Border between zona marginalis and the spleen red pulp in TEM. On the right edge sinus (1) through the wall of which (2) penetrates an erythrocyte (3). In the neighbouring reticular cells (6), also free macrophages (4), lymphocytes (5) and erythrocytes (7). Phagocyting macrophage (8) appears partly in the lumen of sinus through the crevicular pore. Scale 3 µm.

follicles are mostly accumulations of small and middle- sized B-lymphocytes on the wide side of the proximal PALS. They are often formed at the places where arteria centralis is branched. Lymphocytes in PALS are Fig. 10. Entrance of the vein (1) into the vascular trabecula (2) located between lamellas of circumferential reticulum. in SEM. Endothelium of the vein (3). Magnif 1510×. These lamellas might be some derivatives of fibroblastic reticular cells, what is after all confirmed also by our findings. From the follicle side the wall is numerously fenestrated It was found that the use of SEM is an appropri- (Fig. 11). Zona marginalis ends at the place where si- ate method for analysis of the complex inner architec- nuses of the red pulp start (Fig. 12). tonics of the spleen, which is extraordinary demand- Spleen white pulp consists of the typical lym- ing, regarding the three-dimensional interpretation. In phatic tissue. We can distinguish periarterial lymphatic comparison to transmission electron microscopy, scan- sheaths (Fig. 13) and lymphatic follicles called Mal- ning electron microscopy gives us the possibility of phighi bodies (Fig. 14). PALS is predominant accu- more rapid interpretation of the image; what is more, mulation of T-lymphocytes around arteria centralis, no reconstruction studies of parallel serial sections are branching off from connective trabeculas. Lymphatic needed – as it is in TEM. However, it provides us less Ultrastructure of human spleen 407

nuses to their lumen. These macrophages are important for non-specific protection, during elimination of the body’s own dead cells or pathologically changed cells, as well as exogenous compounds from the body, but they are also important in immune processes where they interact with lymphocytes. Only at pathological conditions with strong phagocytosis, deposits of dense material can be found in cells lining sinuses, reminding of phagocytosis (Hromec & Jakubovský 1977). In accordance to our observations, also Su et al. (1991) described macrophages in red pulp cords. Their processes (filopodes) emerge through the slot openings into lumen of sinuses. In our opinion they can also be processes of fibroblastic reticular cells, which line sinuses. Most contemporary authors agree with our opinion that both, open and closed circulation, are present in human spleen. Both types of circulation were also confirmed in several subhuman animals such as dogs (Alexandre-Pires et al. 2003) or rats (Su et al. 1991). Kashimura (1985) demonstrated the presence of closed Fig. 13. Periarterial lymphatic sheat in SEM. Numerous T- lymphocytes (2) and few erythrocytes (1). Scale 5 µm. circulation in humans by the finding of continuous transition of the flat endothelium of arterioles into endothelium of grilled appearance of the vascular sinuses. How- ever, in humans, the open circulation prevails, i.e. most of the blood from arterioles freely leaks among Bill- roth’ s cords and through the wall of sinuses gets back into the vascular circulation. The red pulp of spleen is a unique place where the blood does not coagulate even though it is outside veins. The proof of the presence of the open circulation is, e.g., the large number of erythrocytes in the network of Billroth’s cords, i.e., extravascularly. Furthermore, in our opinion, the open and the closed blood circulation are present in the human spleen simultaneously. Blood flowing in the closed circulation can help “absorption” of extravascular liquid and the blood elements into the vascular lumen. An interesting finding is the occurrence of microparticles on cells lining sinuses and on erythro- Fig. 14. Lymphatic follicle of the spleen white pulp in TEM. cytes during chronic autoimmune thrombocytopenia. It Several types of lymphocytes can be distinguished differing in is possible that microparticles differ from cytoplasmic the nucleus size and amount of cytoplasm. Scale 4 µm. processes and they are platelet microparticles sticking to the surface of various cells. These particles are too small to be counted during the regular platelet count- information about the inner structure of separate cells. ing under light microscope and they might be too frag- ile for examination in TEM. We consider the hypothesis Discussion of artificial origination of these particles improbable be- cause the demonstrated formations have not occurred Spleen stroma consists of reticular connective tissue. in other pathological states examined. In literature, we According to some Slovak and Czech university text- have found only single morphological information con- books of histology, lining of sinuses are formed from cerning this finding, dealing with platelet microparticles specialized reticular cells – fixed macrophages which sticking to the surface of various cells (Jy et al. 1992). phagocyte old red blood cells. Authors reported that Due to the unique spleen physiology, immunology macrophages and reticular cells in spleen were most ac- and functional morphology, many researches all over tive cells in scavenging bacteria and exogenous material the world have been interested in examination of its in the body. According to our observations, the reticu- functions. In our study we describe ultrastructural mor- lar cells do not change to macrophages in physiological phology of fibroblatic reticular cells, venous sinuses, conditions, so they do not participate in phagocyto- as well as some other structures of the red and white sis. The ability to phagocyte have only macrophages pulp of spleen in accordance with foreign and domes- present in large number among the Billroth’s cords, tic literature. In contrast to this literature, we suppose from where they can pass through the openings of si- that fibroblastic reticular cells do not change to fixed 408 Š. Polák et al. macrophages and they do not participate in phagocyto- Jakubovský J. & Hromec A. 1988. The current state of knowledge sis. But since there is very close contact between reticu- on the functional morphology of human spleen. Bratisl. Lek. 99: lar cells and blood cells, these circulating free cells may Listy. 287–290. Jakubovský J. & Porubský J. 1995. 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