A scanning electron microscopic study of the rat liver sinusoid

Cell Tiss. Res. 164, 371-385 (1975) - �9 by Springer-Verlag 1975

A Scanning Electron Microscopic Study of the Rat Liver Sinusoid Endothelial and Kupffer Cells

Pietro Motta*

Department of Anatomy, Faculty of Medicine, University of Rome, Rome, Italy

Summary. The surface ultrastructure of Kupffer cells in the rat liver has been studied by scanning electron microscopy (SEM). The results demonstrate that Kupffer cells are both significantly different and clearly distinct from endothelial cells. Kupffer cells have neither pores (and/or "sieve plates") nor fenestrations, all of which are present in endothelial cells. They possess a stellate shape, and only indirectly, with slender and irregular evaginations, contribute to the lining of the sinusoidal wall. Furthermore, the luminal surface in some areas contains a large population of short microvilli, microplicae and invaginations. These elements form a kind of microlabyrinth which may correspond to the "worm-like" structures described by transmission electron microscopy (TEM).

In the present study, transition forms between endothelial and Kupffer cells were never found. On the contrary, considering the highly fenestrated nature of the endothelial cells, the Kupffer cells may, by ameboid movements, easily cross the overlapping barrier of the sinusoid and protrude into the lumen. Thus, acting as activated macrophages, the Kupffer cells might function to prevent the entrance of foreign material into the tissues of the liver through the fragile and highly fenestrated endothelium.

Finally, the topographical reconstruction of the sinusoid by correlated SEM and TEM studies demonstrates that Kupffer cells, with their protruding cytoplasm and ability to extend into the lumen of the sinusoid, may actually change the caliber of the vessel, and thus function as a "sphincter" which causes a temporary arrest of the blood flow when the diameter of the sinusoidal lumen is reduced.

Key words: Kupffer cell - Endothelial cell - Sinusoid - Liver - Scanning electron microscopy.

Send offprint requests to: Prof. Pietro Motta, Istituto di Anatomia Umana Normale, Universitfi di Roma, Viale Regina Elena, 289, 00161 Roma, Italy.

* This investigation was supported by a Fulbright grant and by a special fund of the Italian Foreign Office and was partially accomplished at the Department of Molecular, Cellular and Develop- mental Biology, University of Colorado, Boulder, Colorado. The author is indebted to Professor Keith R. Porter for his generosity in making laboratory facilities available and for his useful suggestions and to Dr. Jonathan Van Blerkom for the revision of the manuscript and criticism.

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Introduction

The structure of the liver sinusoidal wall has been a controversial subject in the past (see reviews of Aterman, 1963; Rouiller and Jezequel, 1963; Cossel, 1964; David, 1964; Elias and Sherrick, 1969). Although investigations by transmission electron microscopy (TEM) provided a finer description of this unusual vessel, additional problems became apparent from more detailed analyses (see reviews of Wisse, 1972, 1974). A particular topic of controversy centers around the question whether the so-called Kupffer cells and the endothelial cells of the sinusoid are two separate cell types (Yamagishi, 1959; Carsten, 1961; Schaffner et al., 1963; Rhodin, 1965; Wisse, 1972, 1974) or different morphological expressions of a single cell (Burkel and Low, 1966; Battaglia and Jean, 1967; Nicolescu and Rouiller, 1967; Ito and Shibasaki, 1968; Tanikawa, 1968; Carr, 1970). An additional point of controversy concerns the exact location of the Kupffer cells within the sinusoid (Aterman, 1963; David, 1964; Tanikawa, 1968; Jezequel and Orlandi, 1972).

The application of the scanning electron microscopy (SEM) to the study of the fine architecture of the liver has been undertaken previously (Brooks and Haggis, 1973; Motta, 1973; Grisham e t a l . , 1975; Motta et al., 1975). Also, studies by SEM of the surface ultrastructure of hepatocytes and associated spaces of Disse (Motta and Porter, 1974), sinusoids (Itoshima et al., 1974) and bile canaliculi (Motta and Fumagalli, 1975) have appeared.

The purpose of the present report is to: 1) describe the three-dimensional surface ultrastructure of Kupffer cells by correlated SEM and TEM; 2) provide a more well-defined characterization of these cells and 3) ultimately clarify their location in the sinusoidal wall.

Material and Methods

Liver tissues f rom healthy, young, adult albino rats were perfused through the left ventricle with Tyrode 's solution. The perfusion solution was at room temperature, contained oxygen, 0.1% procaine and a drop of heparin. A perfusion flow rate of about 60 mil/min was obtained by gravity. After about 30 seconds, the flow of Tyrode's solution was interrupted and 2.5% glutaraldehyde, in 0.18 M cacodylate buffer solution at pH 7.3 (Sabatini et al., 1963) was perfused for an additional 10 minutes. Subsequently small pieces of liver were excised and immersed in the same fixative. After one or two days, the tissues were washed for 45 minutes in the same buffer and then either cut into small blocks with a razor blade, or carefully fragmented (pulled apart) with jeweler's forceps.

For S EM the fragments were rapidly dehydrated in a graded series of acetone and then transferred to liquid CO2 for critical point drying (Porter et al., 1972). The dried samples were mounted on a luminum studs using conducting silver paint and coated with a layer of carbon and gold in a high vacuum evaporator (DV-502, Denton Vacuum). All the specimens were examined and photo- graphed using a Cambridge Stereoscan Model $4 operated at 10 to 20 kV.

For t ransmission electron microscopy the specimens were postfixed in 1.2% osmium tetroxide in 0.18 M cacodylate buffer at pH 7.3 for 30 minutes, washed in the same buffer and dehydrated through a graded series of ethanols. The blocks were embedded in Epon 812 (Luft, 1961), sectioned with glass kinves in a Porter-Blum MT-1 ultramicrotome and then stained with uranyl acetate (Watson, 1958) followed by lead citrate (Reynolds, 1963). Sections were examined with a Zeiss EM 9 electron microscope.

Scanning Electron Microscopy of Kupffer and Endothelial Cells 373

Fig. 1. Part of a liver lobule with sinusoids (S), sheets ofhepatocytes (E) with bile canaliculi (arrowheads). Numerous erythrocytes are present in this preparation. Note a Kupffer cell (K and arrow). • 1050

Observations

The endothelial wall o f the sinusoids was smooth (Fig. 1). A few short microvilli, small pits and occasional slender cytoplasmic processes were r andomly distributed on the luminal surface. The bulbous por t ion o f the endothelial cell, which repre- sents the cytoplasmic area containing the nucleus, was generally the only region o f the cell clearly p ro t rud ing into the lumen (Fig. 2). This region was also clearly demonst ra ted by T E M (Fig. 3). In other areas, the cytoplasm was reduced to

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Figs. 2--5. Scanning and transmission electron micrographs of an endothelial cell body (b) with laminar extensions containing several pores (arrowheads) (frequently arranged in "sieve-plates", st, and arrows in Fig. 5 and fenestrations, F). E hepatocytes; D space of Disse. Fig. 2, x 2800; Fig. 3, x4800 , Fig. 4, x2160; Fig. 5, x5760

very thin and flattened cellular expansions which reached a maximum thickness of about 500-800 A. These slender and flattened expansions of the endothelial cells were frequently provided with a variable number of perforations (Figs. 1 5). Some of these perforations were similar to " p o r e s " and frequently, when arranged in clusters, formed sieve plate-like structures (Figs. 5, 7); larger perforations, with a maximum diameter of about 1-2 ~tm, could be considered true "fenestrat ions". The number and arrangement of these fenestrations was highly variable. In some zones they appeared rather sparse and were present along with the pores (Fig. 5, 7), whereas in other areas, they were so numerous that


Figs. 6 - 8 . Endothelial wall showing pores (arrows) which are isolated and/or contained in "sieve- plates" (sv), and large fenestrations (F). In Fig. 8, the overlapping laminar extensions of adjacent endothelial cells are evident. E hepatocytes. Fig. 6, • 10800; Fig. 7, • 8800; Fig. 8, x 4000

the delicate endothelial barrier was actually reduced to a series of thin, flattened and largely fenestrated cytoplasmic expansions (Figs. 8, 9). In regions where the fenestrations were both more abundant and larger, the endothelial wall was clearly doubled owing to the overlapping of large, flattened portions of adjacent endothelial cells (Figs. 8, 9).

Through the larger fenestrations numerous microvilli, arising from the surface of hepatocytes facing the space of Disse, were often distinguishable (Fig. 5).

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Figs. 9 - 1 1 . In Fig. 9 a large fenestrated sinusoid is shown (S). Figs. 10 and 11 are, respectively, scanning and transmission electron micrographs of periluminal cells (*) located within an intercellular space of Disse and demonstrating large cellular extensions (arrows) projecting into the endothelial wall. E hepatocytes, R red blood cell. Fig. 9, x 4 500; Fig. 10, x 6120; Fig. 1 l, x 11 250


Fig. 12. Kupffer cell protruding into the lumen of the sinusoid and connected to the endothelial wall by ameboid evaginations. R erythrocytes, x 11270

Occasionally a few bundles of reticular fibers were also evident in these spaces (Figs. 4, 7, 8). In the larger spaces between the hepatocytes and the sinusoidal wall, isolated, spindle-shaped cells provided with a few slender cytoplasmic extensions were evident. The surface of these cells was smooth and the few cytoplasmic extensions arising from these cells reached the spaces between adjacent hepatocytes and crossed the larger fenestrations present on the endothelial wall

378 P. Motta

Figs. 13 and 14. Kupffer cells (K) bulging into the lumen of the sinusoid and connected by large branching arms with fenestrated endothelial walls. Note the rough surface of the cells. Fig. 13, x4860; Fig. 14, x6120

(Figs. 10, 11). Considering the location and morpho logy of these cells, it is likely that they correspond to other peri luminar cells (generally termed pericytes), or fibrocytes.


F i g s . 1 5 - 17. Figs. 15 and 16 are, respectively, scanning and transmission electron micrographs showing areas of the sinusoids with a reduced lumen and erythrocytes (R) attached to the branching process (arrows) and/or to the cell body of the Kupffer cells (K). Fig. 17 is a high magnification micrograph ofa Kupffer cell. Note the labyrinthine surface arrangement composed of a heterogeneous population of short microvilli, microplicae and pits. Fig. 15, x4860; Fig. 16, • Fig. 17, • 13 140

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Figs. 18 -20 . Figs. 18 and 19 are transmission electron micrographs to be correlated with the Kupffer cells (K) shown in Figs. 13 and 14. Large ameboid extensions (arrows) of these cells are bulging conspicuously into the sinusoidal lumen (SL) and are related to the space of Disse (D). Fig. 20 shows a spindle-shaped cell containing a large population of small, spherical surface-blebs (" mulberry- like cells") (M). These cells are quite different from Kupffer cells. Fig. 18, x 11520; Fig. 19, • 11520; Fig. 20, x 10080


The sinusoida! lumen occasionally contained rather voluminous cells which had an irregular surface. As substantiated by correlated SEM and TEM observations, they were Kupffer cells. In most cases these elements possessed a large cellular body which protruded to a varying extent into the capillary lumen. From these cells, large irregular ameboid protrusions arose in a stellate fashion and occupied part of the sinusoid. The ameboid processes of these cells with their branching arms, were occasionally connected to the endothelial wall and thus interrupted the fenestrated continuity of the sinusoid (Fig. 12).

With SEM the Kupffer cells were found to be significantly different from the thin flattened endothelial cells described above. In some instances, processes of these cells extended from one side of the sinusoid to the other and consequently greatly reduced the diameter of the vessel (Figs. 13, 14). Erythrocytes and/or various types of white blood cells were occasionally aggregated with the branching processes of the Kupffer cells, thus reducing the capillary lumen even further (Figs. 15 and 16). The surface of the Kupffer cells was rough owing to the presence of a number of irregular, short, polymorphic processes (Figs. 12 and 14). In particular, the plasma membrane facing the sinusoidal lumen showed numerous small, flattened, microvilli and microplicae, about 1 000-1 500A thick. These microvilli when associated with holes and]or pits, formed a sort of labyrinth (Fig. 17). The cellular expansions of the Kupffer cells were ameboid-like evaginations but did not contain either pores or fenestrations as was observed on the flattened expansions of endothelial cells.

With SEM, the Kupffer cells, in comparison to endothelial cells, were found to have different location in relation to the sinusoidal lining. In some cases, they protruded into the lumen of the sinusoid, but only with the distal ends of their processes. These cells often overlapped or contacted the endothelial wall (Figs. 12, 13 and 18). In other instances, Kupffer cells were observed: 1) with varying portions of the cell body located within gaps between adjacent endothelial cells and 2) contained within large endothelial fenestrations by means of slender cytoplasmic extensions (Figs. 14 and 19). In the latter case, the Kupffer cells were usually partially located within the space of Disse.

Occasionally, enlarged cells with an irregular, spindle-shape appearance were observed. These cells were distinguishable only by SEM, were morphologically different from the Kupffer cells and possessed an extraordinarily high number of spherical, rough blebs on their surfaces. These cells were termed "mulberry-like cells" (Fig. 20) and were noted in the areas of the sinusoidal vessel which possessed only isolated pores or sieve plates. Unfortunately, the location of these cells in relation to the endothelial wall was not evident with SEM alone, as they appeared to be somewhat integrated into the endothelium.

Discussion

The present scanning electron microscopic (SEM) findings demonstrate that the Kupffer cells of the rat liver are very different and clearly distinguishable from endothelial cells. The endothelial cells are flattened and expanded elements which limit the sinusoidal lumina and form an open network which results from their numerous pores, sieve plates and, in particular, fenestrations. By contrast, the Kupffer cells have no pores and/or fenestrations, possess an irregular, stellate

382 P. Motta

shape, are frequently observed bulging into the lumen of the sinusoids and are occasionally in contact with the subendothelial space of Disse. Furthermore, while their extensions may appear to overlap endothelial cells, they only indirectly contribute to the lining of the endothelial wall. The Kupffer cells, as observed by SEM, possess several typical surface characteristics. The slender processes which originate from all sides of the cell give these cells an appearance which is similar to SEM descriptions of macrophages (Carr and Carr, 1970; Albrecht et al., 1972; Hosoya and Fujita, 1973; Motta et al., 1975). The luminal surface of the Kupffer cells is, in some areas, provided with a large number of very small, irregular microvilli, microplicae and invaginations which collectively form a microlabyrinth. These findings are in agreement with previous TEM studies of Kupffer cells and the microlabyrinthine arrangement most likely corresponds to "worm-like structures" (Orci et al., 1967; Matter et al., 1968; Wisse, 1974) located on the luminal surface of the Kupffer cells but not on the endothelial cells. In addition, the presence of particulate material on the surface of the Kupffer cells where numerous pits and holes are located suggests that the labyrinthine, membranous, evaginations of the plasma membrane might be a mani- festation of phagocytosis (as suggested by Wisse, 1974). Although in some areas the endothelial cells appeared to be highly fenestrated, they never assumed a morphological arrangement similar to that displayed by the Kupffer cells. Thus, transition forms between endothelial and Kupffer cells were never found. In contrast, the present findings support the hypothesis, recently strengthened by elegant TEM and cytochemical studies (Wisse, 1972, 1974) that endothelial cells canno t -a t least under normal physiological conditions-develop into Kupffer cells.

Considering the location of the Kupffer cells in the sinusoidal wall and the fenestrated nature of the endothelial cells, these elements are probably associated with the tissue side of the endothelium and are dynamically transformed into activated macrophages, such that, by ameboid movements, these cells can cross the overlapping barrier of the sinusoid and protrude into the lumen. As suggested by SEM observations, some of these cells are located primarily in the intersinusoi- dal spaces and, thus, can extend long processes through the large fenestrations of the endothelial layer. On the other hand, Kupffer cells can be considered simply as macrophages which arise through the transformation of blood mono- cytes (Carr, 1970) and which freely move into and out of the lumen of the perisinusoidal spaces. The fact that these macrophages are so frequently and readily encountered in the sinusoidal wall might be related to the highly fenestrated nature of these vessels which act as a sort of net and possibly serve to facilitate their attachment to the wall. Similar to other macrophages, the Kupffer cells most likely represent "cleaning cells" which prevent the entrance of foreign material into the liver tissue. In particular, the present considerations arising from the study of the Kupffer cells do not differ greatly from those regarding the existence of macrophages in the alveolar wall (Carr, 1970). In both liver and lung, as well as in other tissues and organs (such as spleen: Miyoshi and Fujita, 1971; lymph node: Fujita et al., 1972; peritoneal cavity: Carr and Carr, 1970; Albrecht et al., 1972 ; Motta and Barberini, 1975 ; choroid plexus: Hosoya and Fujita, 1973), very fragile and delicate cellular barriers (endothelium, mesothe-


l ium and epi the l ium) mus t be p ro t ec t ed f rom the en t rance o f fore ign bodies which could interfere wi th the n o r m a l func t ion o f t issues and organs .

A n add i t i ona l po in t which shou ld be emphas i zed regards the f requent in t ra lumina l loca t ion o f the Kupf fe r cells. The t opog ra ph i c a l r econs t ruc t ion o f several s inusoids , by co r re l a t ed S E M and T E M , clear ly demons t r a t e s tha t the K upf f e r cells, wi th thei r p r o t r u d i n g cell bod ies and cel lular extensions , ac tua l ly reduce the ca l iber o f the vessel. In some cases, a n u m b e r o f red and whi te b l o o d cells were aggrega ted with the cell b o d y and processes o f the Kupf fe r cells, which were p r o t r u d i n g into the lumen. These f indings should be subjec ted to fur ther analysis in the l ight o f phys io log ica l s tudies dea l ing with the mic roc i rcu la - t ion o f the l iver in which the r educ t ion o f c i rcu la t ion has been no ted and ten ta t ive ly a t t r i bu t ed to the Kupf f e r cells (McCuskey , 1966). F r o m the present observa- t ions, it is evident tha t Kupf fe r cells, because o f their loca t ion and movemen t s , might be capab le o f ac t ing as a " s p h i n c t e r " reduc ing the s inuso ida l lumen. A final p o i n t which was no t c lar i f ied by the present s tudy regards the na tu re and exact loca t ion o f the " m u l b e r r y - l i k e ce l l s" which were occas iona l ly observed in the s inusoida l a reas o f p o o r l y fenes t ra ted vessels. Poss ib ly they are a t r ans i t iona l phase o f the Kupf f e r cell re la ted to phagocy t i c act ivi ty or even to thei r ac tua l d i f ferent ia t ion. I t is equal ly poss ib le tha t they are s imply the " f a t - s t o r i n g ce l l s" which have been prev ious ly descr ibed by I to and Sh ibasak i (1968).

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Received July 28, 1975


Note Added in Proof Further information on the same subject are contained in a paper published by Muto in Arch. histol, japon. 37, 369 386, 1975 and appeared when the present article was already in press.

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A scanning electron microscopic study of the rat liver sinusoid