the equine spleen: an electron microscopic analysis

THE AMERICAN JOURNAL OF ANATOMY 166:393-416 (1983)

The Equine Spleen: An Electron Microscopic Analysis FERN TABLIN .ANI) LEON WEISS Laboratory ofExperimenta1 Hematology and Cell Biology, University o f Pennsylvania, School of Veterinary Medicine, Philadelphia, Penns,yluania 19104

ABSTRACT The capacity of the equine spleen to store and rapidly release as much as half the circulating blood volume after adrenergic stimulation depends upon the size of the spleen, its muscular capsule, and the distinctive structure of its red pulp. The unit, or lobule, of red pulp is a cylinder of pulp spaces organized in a reticular meshwork, supplied by a peripheral ring of arterial capillaries, and drained by a central venule. Reticular cells, which make up the meshwork of the pulp, contain an extraordinarily large comple- ment of microfilaments and intermediate filaments and are richly innervated by nerves containing both dense and lucent core vesicles typical of adrenergic nerves. The wall of the pulp venule contains large apertures. The capacious red pulp would thus appear capable both of large-scale blood storage and, by the contractile adrenergic innervated reticulum and open venous vasculature, of rapid expression of stored blood into the circulation.

Antigen-presenting cells are present not only in B and T cell zones in white pulp but in the periarterial macrophage sheath of red pulp as well. The periarterial macrophage sheath is one of the first sites of antigen capture, and the presence of these cells confers on it an immunological role.

The horse spleen is important in red-cell storage, capable of holding up to one-half of the animal’s total blood volume at rest and releasing it all upon intensive exercise (Tor- ten and Schalm, 1964; Persson et al., 1973a,b; Archer and Clabby, 1965). Splenectomized horses are incapable of intensive exercise due to the lack of these storage and release functions (Persson et al., 1973a,b). Yet the structure of the equine spleen has received little attention since its stroma was studied by Snook (1950) more than 30 years ago. Using silver impregnation of the fibrillar reticulum he demonstrated a diffuse white pulp, a small marginal zone, and a large well-vascularized nonsinusal red pulp (Snook, 1950). It is the purpose of this paper, through a detailed electron microscopic analysis, to extend his work and present a comprehensive picture of the equine spleen, and to relate its structure to certain major contemporary issues: the homing, sorting, storage, and release of blood cells entering the spleen; and such functions of the spleen as antigen presentation. We have found that the equine spleen is among the simplest of the nonsinusal spleens, and its lobular pattern is among the most marked of mammalian spleens.

MATERIALS AND METHODS

The spleen was removed from four adult horses (one Morgan, one Thoroughbred, and two Grade) sacrificed by intravenous euthan- asia solution (Beuthanasia-D, Burns Bio- tetic). Five-by-ten-millimeter slices of tissue from subcapsular and central regions were hardened in a mixture of glutaraldehyde and paraformaldehyde (Karnovsky, 1965) for 2 hr at 4°C. Each slice was then cut to 2 mm3 and fixed another 24 hr. The tissue was then rinsed and stored in 0.1 M cacodylate buffer, pH 7.2, at 4°C.

The blocks of tissue, cut to 1 mm3, were postfixed in osmium tetroxide 1.4% over- night a t room temperature, dehydrated in graded ethanols, and embedded in Epon 812- Araldite. Sections cut on a Sorval Bloom MT2 microtome were collected on uncoated copper grids, stained with saturated uranyl acetate and lead citrate (Venable and Coggeshall,

Address reprint requests to Leon Weiss, M.D., Department of Animal Biology, University of Pennsylvania, School of Veteri- nary Medicine, 3800 Spruce Street, Philadelphia, PA 19104.

Received June 22, 1982. Accepted November 18, 1982.

0 1983 ALAN R. LISS, INC.

394 F. TABLIN AND L. WEISS

1965), and examined with a Siemens Elm- skop I or JEOL 1OOC.

Karnovsky-fixed tissue postfixed in 1.4% osmium tetroxide was also used for scanning microscopy. The blocks of tissue were critical- point dried in carbon dioxide, mounted on metal stubs, coated with gold-palladium and examined with a JEOL 35.

RESULTS

Our results concern a number of stromal cells of the spleen. These include reticular cells, follicular dendritic cells, and interdigitating cells. The definitions of these terms are as follows. Reticular cells are large branched cells which form the meshwork of the pulp. Their cytoplasmic processes enwrap and presumably produce argentophilic reticular fibers. They have the organelles of fibro- blasts: a nucleolus, rough endoplasmic reticulum, and a dilated perinuclear space continuous with rough endoplasmic reticulum. Thin (5-nm) filaments, likely actin, occur in dense patches near the surface of reticular cells processes apposed to reticular fibers. Intermediate (10-nm) filaments, prob- ably vimentin (Franke et al., 1979), lie throughout the cell, but primarily in the peri- karyon.

Follicular dendritic and interdigitating cells represent two major subsets of a re- cently recognized class of cells which bear and present antigen to immunologically com- petent cells. Follicular dendritic cells are large branching cells that lie amongst B lymphocytes in white pulp. Their nucleus dis- plays a continuous peripheral rim of heterochromatin and a small nucleolus. Their cytoplasm is lucent and contains few filaments, dispersed polyribosomes, and clus- tered mitochondria adjacent to short sections of rough endoplasmic reticulum (Steinman and Cohn, 1973; Steinman et al., 1975; Chen et al., 1978a). They can hold antigen-anti- body excess complexes on their cell surface for up to 5 days (Chen et al., 1978b). Interdig- itating cells branch among T lymphocytes and can carry antigen on their surfaces. They closely resemble dendritic cells (Balfour et al., 1981). Their nucleoli, however, are more prominent, their cytoplasm more lucent, their mitochondria fewer, and they contain many multivesicular complexes (Veerman and van Ewijk, 1975; Balfour et al., 1981).

White pulp

The white pulp contains lymphocytes supported by a reticular meshwork (Fig. 1). Mod- erate numbers of platelets and occasional red cells are also present. Follicular dendritic cells, interdigitating cells, and macrophages are also present.

Follicular dendritic cells lie within germinal centers and in the surrounding mantle region, sending their cell processes out among lymphocytes (Fig. 2). Interdigitating or veil cells are periarterial, their cell processes forming invaginations which contain lymphocytes (Fig. 3). Macrophages are present throughout the white pulp and are in highest concentration in germinal centers and at the periphery of the pulp adjacent to the marginal zone.

Each cylinder of white pulp is supplied by an arteriole branch of the central artery. These arterioles and their arterial capillaries possess parallel, rod-shaped endothelial cells with a thin, often discontinuous basement membrane (Fig. 3). The endothelium is densely filamentous, primarily containing patches of intermediate filaments, likely vimentin (Franke et al., 1979). Thin filaments, likely actin (Rostgaard et al., 1972), are present along the lateral and basal cell surfaces.

At the periphery of white pulp, forming its outer boundary, is a single circumferential layer of reticulum.

Marginal zone The marginal zone, junctional tissue be-

tween white and red pulp, occurs in a narrow band. On the white pulp side it is delineated by the same circumferential layer of reticular cells that form the boundary to white pulp. On the red pulp side the marginal zone, less well defined, gradually merges into red pulp. The junction between marginal zone and red pulp is roughly limited by a ring of arterioles. The marginal zone is distin- guished by its arterial terminations, reticular meshwork, and migratory cell population as discussed below.

Fig. 1. White pulp. Numerous lymphocytes (Ly) are supported by the reticular meshwork composed of reticular cells (RC) and their cell processes. Reticular cells may be recognized by their dark cytoplasm and dilated perinuclear space. ~6 ,000 .

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Fig. 2. White pulp. Two follicular dendritic cells (FDC) cytoplasm contains few mitochondria often associated lie among lymphocytes ny). Their large ovoid nucleus with short strips of rough endoplasmic reticulum. exhibits a thin rim of heterochromatin. Their lucent ~7,000.

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Fig. 3. White pulp capillary cut in tangential section. The capillary endothelium (E) is densely filamentous, primarily composed of intermediate ( 1 0-nm) filaments, see insert. A red blood cell (*)and platelet (P) lie in the

lumen of the capillary. Interdigitating cells (IDC) are perlnrtrrinl, their cytoplasmic processes interdigitating with surrounding cells. Numerous platelets are present in the surrounding white pulp. x3,600.


Capillaries, originating from the central artery, run to the periphery of white pulp, penetrate the circumferential reticulum, and terminate, opening into the marginal zone. Before termination, interendothelial slits appear, permitting egress of blood. As the capillaries terminate, their endothelial cells may flare to form ampullae. Endothelial cells re- main densely filamentous and may not be as high as those in white pulp. The basement membrane remains discontinuous.

The reticulum of the marginal zone is ar- ranged in a rather loose meshwork. Filamen- tous reticular cells branch out from the circumferential reticulum, their branches forming scaffolding for the migratory cells. These reticular cells meet those of the red pulp spaces. Unmyelinated nerve endings, containing both lucent and dense-core vesicles, may be present within reticular cell processes. These reticular cells have been designated axon-bearing reticular cells (Blue and Weiss, 1981a).

Macrophages lie both on the circumferential reticulum and in the reticular meshwork, their phagolysosomes often containing portions of granulocytes and erythrocytes (Figs. 4, 5).

A dense population of migratory cells, presumably deposited by the terminal capillaries, is supported by the reticular meshwork. Lymphocytes, red cells, platelets, and granulocytes are all present. A gradient exists in which lymphocytes predominate closest to white pulp, progressively giving way to other blood cells closer to the red pulp spaces (Fig. 5).

Red pulp The red pulp of the horse is markedly lob-

ular in character. The lobule consists of a cylindrical structure of reticulum drained by a central venule and fed by a peripheral ring of fine arterial vessels.

The arterioles of the lobule commonly occur in sprays of three or four. Their endothelium is rod-shaped and densely filamentous. Intermediate filaments predominate throughout the cytoplasm, with patches of thin filaments present a t basal and lateral borders.

A prominent, continuous, basement membrane is present, apposed by smooth muscle cells of the media. Nerve terminals, containing both lucent and dense-core vesicles, lie within axon-bearing reticular cells surrounding the arteriole.

Arterioles branch extensively, extending into the lobule to form arterial capillaries. Virtually all arterial capillaries bear periarterial macrophage sheaths (ellipsoids). Cap- illaries occur in sprays of two or three, each with its own periarterial macrophage sheath. The capillaries possess interendothelial slits through which blood cells can pass out into the pulp spaces (Blue and Weiss, 1981a,b). Nonmyelinated nerve terminals within axon- bearing reticular cells lie adjacent to the capillaries (Fig. 6 and 11B).

The periarterial macrophage sheath (PAMS) contains a reticular cell stroma. Mac- rophages lie within the stroma, their cell processes interdigitating extensively with those of the reticular cells. Further, cells morpho- logically similar to interdigitating cells of the white pulp are located at the periphery of the PAMS. Plasma cells are often associated with them. Platelets and red cells may be found within the PAMS, presumably leaving the capillary and entering the pulp spaces (Fig. 6).

As the capillaries leave the PAMS and terminate in the pulp spaces, they form “terminal ampullae” (Robinson, 1926; Snook, 1950). Endothelial cells, still densely filamentous, flare apart, and interendothelial slits en- large. Macrophages and plasma cells lie in the pulp space adjacent to the terminal endothelium. Nerve terminals within axon- bearing reticular cells accompany the terminal capillaries, suggesting that these vessels are under nervous control (Fig. 7).

As blood cells flow out of the terminal capillaries, they enter the pulp spaces. These are the interstices comprised of branching reticular cells and their associated reticular f i - bers (Fig. 8). The reticular cells of horse red pulp are notable for their cytoplasm rich in

Fig. 4. Circumferential reticulum (CR) separating white pulp, on the left, from marginal zone on the right. An arterial capillary (likely of white-pulp origin) rests on the circumferential reticulum, its red blood cells adjacent to interendothelial slits in a pattern suggestive of movement into the marginal zone. A macrophage (M) lies adjacent to the capillary. On the marginal-zone side, lymphocytes (Ly), red blood cells, and platelets (PI are present. ~3,600.

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intermediate filaments. In addition, thin filaments occur in dense patches at the plasmalemma (Fig. 9).

Myelinated axons carried by axon-bearing reticular cells occur in larger numbers than in other species previously reported (Blue and Weiss, 1981a) and ramify deep into the pulp spaces (Fig. 10 and 11A). As a result, the equine reticular meshwork is highly innervated.

Many markedly phagocytic macrophages lie in the pulp spaces, their cell processes in close association with red cells and granulocytes (Fig. 10). The pulp spaces are an expan- sive storage space, primarily for red cells, but also for platelets and granulocytes (Fig. 10). Unlike the dog and cat pulp spaces examined by Blue and Weiss (1981a,d), neither reticu- locytes nor megakaryocytes were found in the equine red pulp spaces.

As red cells, granulocytes, platelets, and plasma cells move through the pulp spaces, they exit through the central venule. These venules, similar to those described by Blue and Weiss (1981d), course through pulp meshwork and drain into trabecular veins. Prior to entering trabeculae, veins often run alongside trabeculae supported by their muscle branches. Trabecular veins drain into branches of the splenic vein, leaving the spleen through the hilus.

The pulp venules of the horse are very large, consisting of a trilaminar wall composed of endothelium, basement membrane, and adventitital reticular cells. The venular endothelium is squamous and less filamentous than arterial endothelium, but it still possesses both intermediate and thin filaments. Venule endothelial cells contain both coated and uncoated vesicles. The endothelium appears moderately phagocytic, as shown by the presence of heterolysosomes. Endothelial cells overlap one another, and their luminal surface may be thrown into folds. The basement membrane is discontinuous. Adventitial reticular cells form the outer layer of the venule. Their cell processes extend into the surrounding pulp, meeting processes of other reticular cells (Fig. 12).

Apertures into the venule wall are created by separation of endothelial cells (Fig. 12). These openings vary considerably in size and distribution, suggesting they are transient, not permanent; and they resemble those in the cat (Blue and Weiss, 1981d). In the horse, reticular adventitial cells regulated by ten.

sion in red-pulp reticular meshwork pull an edge of an endothelial cell away from its lumen. Such apertures are of sufficient width to admit blood cells without deforming them (Figs. 12, 13).

Trabecular veins resemble venules except that there are no apertures, and the basement membrane is continuous.

DISCUSSION Antigen-presenting cells

Horse white pulp, like that of other species, consists of periarterial lymphatic sheaths (T cell zones) and lymphatic nodules (B cell zones) (Veerman and van Ewijk., 1975). An- tigen-presenting cells are well represented in both white pulp and periarterial macrophage sheaths (ellipsoids) of red pulp. Two types of antigen-presenting cells have been identified in murine spleens: interdigitating-veil cells and follicular dendritic cells.

Interdigitating cells are large branched cells whose processes extend amongst T cells, They likely develop in bone marrow and travel in the blood as veil ceIls. Veil cells, so designated because they possess broad veil- like cytoplasmic processes, are best seen by scanning electron microscopy (Balfour et al., 1981). In blood or lymph stained by Roma- novsky-type stains, veil cells may appear as monocytes (Kelly et al., 1978). Veil cells which leave the blood and enter the epidermis become Langerhans celIs. Langerhans cells may carry markers, the Birbeck granules (BG). Indeed, Langerhans cells high in the epidermis typically contain BG while those near the dermis may not, suggesting these granules may be an index of cell ma- turity (Friedman, 1981). Veil cells and interdigitating cells may contain BG. Langerhans cells may leave the epidermis and through lymphatics reach the cortex of lymph nodes and the white pulp and periarterial macro-

Fig. 5. Marginal zone. A sparse reticular meshwork composed of reticular cells (RC) and their reticular fibers support a migratory cell population. A gradient exists between lymphocytes (Ly) at the top of the field and red blood cells (*) and granulocytes (G) at the bottom of the field. ~4,800.

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phage sheath (PAMS)of spleen. Once in lymphatic tissue they are termed interdigitating cells. These cells bear la antigen on their surface, as well as receptors for Fc and C3. Veil cells and interdigitating cells can cluster lymphocytes in culture and promote T-cell response to mitogens (Balfour et al., 1981).

The splenic dendritic cell identified by Steinman and co-workers (Steinman and Cohn, 1973; Steinman et al., 1975)is similar

Fig. 6. Red-pulp capillary and its periarterial macrophage sheath (PAMS) (tranverse section). (Consult labelled tracing.) Many platelets (PI are present within the interstices of the PAMS, presumably having left the capillary through interendothelial slits. Several large cells (IDC) whose morphology is that of the interdigitating cells of white pulp surround the capillary; extensive cytoplasmic interdigitations are present. At left, is a portion of a reticular cell (RC) with reticular fibers (RF). A macrophage (M2) lies adjacent to the basement membrane of the capillary and in the upper right. The nerve terminal (n) of an axon bearing reticular cell is present at the top center. A higher magnification of this nerve is presented in Figure 11B. - 4 , 5 0 0 .

to the interdigitating-veil cell morphologi- cally and in its surface la antigen. Both in vivo and in vitro, moreover, the dendritic cell has been shown to be an obligate accessory cell for T-cell immune functions. Further, in the syngeneic mixed leukocyte reaction, dendritic cells and T cells cluster (Nussenzweig and Steinman, 1980). Dendritic cells can also function as Langerhans cells in the presentation of antigen and in the contact hyper- sensitivity reaction (Britz et al., 1982). Because of these similarities, Humphrey (1 98 1)postulated that the interdigitating-veil cell and dendritic cell are the same cell type.

As the interdigitating-veil dendritic cell functions in T-cell zones, another antigen- presenting cell type, the follicular dendritic cell, functions in B-cell zones. It resembles the interdigitating-dendritic cell but lacks surface la determinants. Follicular dendritic cells have been postulated by Chen et al. (1978a) to develop from dendritic cells at the time of germinal-center formation.

We found antigen-presenting cells in both T-cell (periarterial lymphatic sheath) and B- cell (lymphatic follicles) zones, and we be- lieve a third group of antigen-presenting cells is present within the periarterial macrophage sheath of equine red pulp. These cells resemble the interdigitating-dendritic cells and lie outside the arterial capillary endothe- hum. Their cytoplasmic processes form extensive interdigitations amongst the cells of the periarterial macrophage sheath. These cells likely cooperate with the macrophages present within the sheath and confer on them the capacity to prepare antigen for an immune response.

Thus there appears to be a system of antigen-presenting cells in which the veil cell represents the traveling form and the interdigitating-dendritic and follicular dendritic cells the tissue representatives, functioning to present antigen and cluster their respec- tive lymphocytes, facilitating selection and clonal expansion essential to the immune response.

Marginal zone The marginal zone is the junctional tissue

between red pulp and white pulp. At its periphery, its junction with red pulp is indis- tinct; centrally, its junction with white pulp is marked by the circumferential reticulum.

The largest number of fine arterial vessels empty into the marginal zone, and it there-

EQUINE SPLEEN 405

fore becomes the primary site of entrance of blood cells into the spleen. These cells subse- quently migrate either into white pulp, through a perforate circumferential reticulum (Niewenhuis and Ford, 19761, or into red pulp, through the reticular meshwork (Mitchell, 1973; Clark and Weiss, 1972). Mar- ginal-zone macrophages may modulate migration of B lymphocytes into the white pulp (Humphrey, 1980).

The marginal zone acts as a filter. India ink and lead oxide gelatin particles injected into the abdominal aorta of rats by Sasou et al. (1976) and Pereira (1978) first accumu- lated in the marginal zone. The fibrillar reticulum of the marginal zone of rats was preferentially stained by silver nitrate (Jeli- nek and Pereira, 1980), highlighting the large volumes of fluid filtered in this region of the spleen. Because many granulocytes, platelets, and erythrocytes are carried into the marginal zone by terminating arterial vessels, and macrophages and particulate material are also held there, the marginal zone contains a distinctive population of such migratory cells. Indeed, the location of the marginal zone may be defined by its population of migratory cells. In cats, the marginal zone lies within the circumferential reticulum (Blue and Weiss, 1981~). In rodents (Snook, 1964), human beings, dogs, and horses, the site of the marginal zone is more typically outside the outermost layer of circumferential reticulum. The horse is distinctive in that there is but a single layer of circumferential reticulum. This layer therefore serves as the outer limit of white pulp and the inner limit of the marginal zone. This circumferential ring is perforate, moreover, allowing for cellular traffic between the marginal zone and white pulp. Thus lymphocytes, macrophages and antigen-presenting cells released into the marginal zone may migrate into white pulp and may move from white pulp out to marginal zone and red pulp,

Fig. 7. Red-pulp terminal capillary. Densely filamentous endothelial cells (E) flare apart, dividing to form a dilated ampulla on the left. A process of an axon-bearing reticular cell (Ax) is present in the upper right, its nerve terminal in close association with the capillary endothelium (E). ~5,000.

perhaps through the preferential channels of Mitchell (1973).

Unmyelinated nerve fibers travel within axon-bearing reticular cells of the marginal zone, their nerve terminals containing both dense and lucent core vesicles. Similar axons have been noted by Reilly et al. (1979) in the periarterial lymphatic sheath of mouse spleen and were characterized both ultra- structurally and histochemically as purely adrenergic fibers.

Red pulp The horse is remarkable in its capacity for

sustained physical exercise. After splenectomy this capacity is severely compromised (Persson et al., 1973a,b). While storage capacity is present in both sinusal and nonsinusal spleen of other species, it is most highly de- veloped in the horse. The equine spleen is capable of storing up to one-half of the circulating red cell mass. Excitation of normal horses resulted in a 40% increase in packed cell volume (PCV), while splenectomized horses showed no increase in PCV with excitation (Torten and Schalm, 1964). This storage capacity is particularly evident upon morphologic examination. Large numbers of red cells supported by a contractile axon- bearing reticular meshwork are found in all regions of the red pulp.

The lobular structure of the equine spleen is quite clearly laid out. It is a vascular unit consisting of a capacious pulp space organized by reticular cells and fibers. Blood is brought into this space by numerous peripheral arterial capillaries and is drained by a central perforate venule. The lobule appears well suited to storing and expressing blood, and thereby underlies the remarkable capacity for exercise that horses possess. Further characteristics of the red pulp lobule discussed below make it appear that the vasculature and reticular meshwork of the pulp spaces are contractile and highly innervated.

The arterial capillary endothelium is densely filamentous. Intermediate filaments predominate, with thin filaments present near the plasmalemma. This distribution of intermediate and thin filaments is similar to those of other arterial endothelial cells described by Chen and Weiss (19721, Yohro and Burnstock (19731, Lauweryns et al. (19751, and Blue and Weiss (1981a,b). Intermediate filaments have been shown by Franke et al. (1979) to be vimentin, the major protein of


Fig, 8. Red pulp. At low magnification (A) the extensive reticular meshwork of the pulp is apparent. X640 The meshwork formed by reticular cells and their cyto-

plasmic process (*I is evident a t higher magnification 03). x 1,650.

EQUINE SPLEEN 407

Fig, 9. Red-pulp reticular cell process. The process contains many intermediate filaments (io. Thin fila-

ments (ti3 occur in dense patches along the plasmalemma. X50.000.

EQUINE SPLEEN 409

Fig. 10. Red pulp. (Consult the labelled tracing.) Re- ticular cells (RC) and associated reticular fibers (RF) form the meshwork that supports a migratory cell population of red blood cells and granulocytes. Many macrophages (M) with prominent phagolysosomes are present.

An axon-bearing reticular cell (N) is in the lower left- hand corner. Its nerve terminal containing both dense and lucent core vesicles is demonstrated in Figure 11A. x3,600.

EQUINE SPLEEN 411

intemediate filaments in cells of mesenchy- ma1 origin. These noncontractile filaments function as the endothelial cytoskeleton. Thin filaments have been demonstrated by arrowhead decoration with heavy meromyosin to be f-actin (Sanger 1975; Rostgaard et al., 1972). Their intracelluIar association with myosin in nonmuscle cells has been demonstrated by Toh et al. (1976) and Clarke and Spudich (1979). The presence of thin filaments may be partially responsible for control of the patency of the interendothelial slits and thus cell passage from the arterial capillary out into the reticular meshwork.

The pulp venules possess a flat endothelium whose cells overlap slightly. Their basement membrane is thin and the adventitial layer sparse. These venules are perforate, open to the pulp spaces a t their distal ends where they originate. They are different from the imperforate venules of the mouse (Chen and Weiss, 1972) and similar to those of the cat (Blue and Weiss, 198ldkother animals with nonsinusal spleens. Migratory cells from the pulp have easy access to the venous lumen. The mural apertures are not only large but may be kept open by attachments of adventitial reticular cells to the basal surface of endothelial cells. Because erythrocytes are not squeezed through slits as in passage through the walls of venous sinuses of sinusal spleens, equine spleen does not pit or clear Heinz bodies. The failure to clear Heinz bodies (and, likely, intraerythrocytic para- sites) is shared by the nonsinusal cat spleen (Jain, 1973; Harvey and Gaskin, 1977).

The reticular cells on the adventitial surface of the venule branch out into the pulp spaces which surround and support the venule, joining other reticular cells to form the meshwork. These reticular cells possess large numbers of both intermediate and thin filaments. Dense patches of thin filaments located a t the plasmalemma and within cell branches are likely composed of actin. We presume that these contractile reticular cells

Fig. 11 A. A higher magnification of the axon (N) in Figure 10. Several axons (Ax) are present, surrounded by cytoplasm of an axon-bearing reticular cell (RC). These axons and accompanying reticular cell lie among other filamentous reticular cells (RC2) and reticular fibers (*). ~33,000. B. The nerve terminals (n) of Figure 6 contain both dense (D) and lucent a) core vesicles, characteristic of adrenergic nerves, surrounded by the cytoplasm of an axon-bearing reticular cell (RC). X55,OOO.

have the capacity to divert cell passage, increase or decrease the volume of the pulp spaces-and therefore the volume for cell storage-and form pathways from arterial capillary termination to venule which may be as efficient as a conventional blood vessel in carrying blood.

The axons and terminals of both myelinated and nonmyelinated nerves present within axon-bearing reticular cells contain both dense and lucent core vesicles. They are purely sympathetic (adrenergic) in both the mouse (Reilly et al., 1979) and the cat (Fil- lenz, 1970) spleens, as shown by fluorescence and electron microscopy. In the horse, sympathetic innervation to the spleen has been demonstrated by physiological studies (Tor- ten and Schalm, 1964; Persson et al., 1973a,b). Axon-bearing reticular cells are present in the pulp spaces, in the periarterial macropahge sheath (PAMS), adjacent to arterioles, and within the marginal zone and white pulp. Release of catecholamines in a filamentous contractile meshwork is suggestive of a neural influence upon contractile reticular cells in the equine spleen and places blood tlow, cell storage, and release under adrenergic control.

Arterial pulsatile flow in the transilluminated cat spleen was observed to be mediated by mural smooth muscle cells (MacKenzie et al., 1941). Regulation of blood flow at the arterial capillary terminations is likely con- trolled at the endothelial level by the opening and closing of interendothelial slits in response to blood pressure (Blue and Weiss, 1981b) and also by the contractile innervated reticular cells surrounding the capillary. These contractile reticular cells may cause shunts, allowing plasma and cells to flow through a more direct meshwork toward the readily accessible central venule or, alterna- tively, to store large numbers of red cells and platelets away from an area of cell traffic.

ACKNOWLEDGMENTS

This work was supported by the United States Public Health Service, NIH, Institute National Research Service Award AM-07185- 06 (F.T) and research grant AM-19920-06 (L. W.), and by the Biomedical Research Sup- port Grant Program, Division of Research Resources, NIH, BRSG 507-RRO5464 (F.T.).

We wish to thank Ms. Barbara Rowe for her assistance in the preparation of this manuscript.

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Fig. 12. Red pulp venule. (Consult the labelled tracing.) Endothelial cells (E) often overlap, and intercellu- lar junctions may also be present. These cells often contain heterolysosomes. The adventitial layer (R) is

quite incomplete and in the upper right is withdrawn from the endothelial resulting in a passageway for red- cell entry into the venule. X4,OOO.


Fig. 13. The endothelium (E) appears to protrude into the endothelium has been lost, revealing the cell process the venule lumen, presumably due to underlying cells of an underlying adventitial reticular cell (arrow). about to enter the venule. At the top of the micrograph, ~5,400.

EQUINE SPLEEN 415

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