Human spleen microanatomy: why mice do not suffice

Abstract

The microanatomical structure of the spleen has been primarily described in mice and rats. This leads to terminological problems with respect to humans and their species-specific splenic microstructure. In mice, rats and humans the spleen consists of the white pulp embedded in the red pulp. In the white pulp, T and B lymphocytes form accumulations, the periarteriolar lymphatic sheaths and the follicles, located around intermediate-sized arterial vessels, the central arteries. The red pulp is a reticular connective tissue containing all types of blood cells. The spleen of mice and rats exhibits an additional well-delineated B-cell compartment, the marginal zone, between white and red pulp. This area is, however, absent in human spleen. Human splenic secondary follicles comprise three zones: a germinal centre, a mantle zone and a superficial zone. In humans, arterioles and sheathed capillaries in the red pulp are surrounded by lymphocytes, especially by B cells. Human sheathed capillaries are related to the splenic ellipsoids of most other vertebrates. Such vessels are lacking in rats or mice, which form an evolutionary exception. Capillary sheaths are composed of endothelial cells, pericytes, special stromal sheath cells, macrophages and B lymphocytes. Human spleens most probably host a totally open circulation system, as connections from capillaries to sinuses were not found in the red pulp. Three stromal cell types of different phenotype and location occur in the human white pulp. Splenic white and red pulp structure is reviewed in rats, mice and humans to encourage further investigations on lymphocyte recirculation through the spleen.

Introduction

The spleen is a secondary lymphatic organ of extraordinary histological structure that is primarily occupied with immune surveillance of the blood. The organ is present in all vertebrate species, but has been most thoroughly investigated in mice, rats and humans. In mice and rats, the spleen is the most important organ for lymphocyte recirculation and this is most likely also true for humans.¹ Besides its function in clearing particulate and other antigens, microorganisms and aged red cells from the blood, the spleen is involved in the final steps of B-cell maturation in mice, whereas in humans it is an important reservoir of memory B cells,² platelets^3,4 and most likely also of monocytes.⁵

In mice, rats and humans, the most astonishing feature associated with the spleen is the structure of its microvasculature. First, in contrast to most other organs, medium-sized splenic arteries and veins do not run in parallel. Second, splenic red pulp capillaries feed an open microcirculation. Third, spleens exhibit a peculiar type of microvessels absent in other organs, the venous sinuses. In rats, mice and humans, blood leaves the open ends of splenic red pulp capillaries, freely percolates through the reticular connective tissue and is subsequently re-collected into the sinuses for venous drainage. Hence, entering blood cells do not pass an endothelial barrier in the spleen. In humans, the initial segments of splenic red pulp capillaries are surrounded by special multicellular structures called capillary sheaths.^6,7

In contrast to other secondary lymphatic organs, lymphocytes recirculating through the spleen arrive via the open circulation and are directly attracted to the external surface of medium-sized and smaller arterial vessels so forming the white pulp around central arteries and arterioles. In mice and rats, the white pulp is composed of T-cell zones, the periarterial lymphatic sheaths (PALSs), and B-cell zones, the follicles and the marginal zone (MZ). The surrounding connective tissue containing the smallest arterioles, capillaries, sinuses and venules as well as the open circulation with all types of blood cells is called the red pulp.

The present review gives an overview of human splenic microanatomy in the context of morphological and selected functional data derived from mice and rats to highlight similarities and differences among the species. It is centred on B lymphocytes and stromal cells, because other cell types have not been thoroughly investigated in human spleen.

Microanatomy of mouse and rat splenic white pulp

In mice, rats and humans, blood is transported to the spleen by several branches of the splenic artery entering the hilum of the organ. These branches further divide inside the spleen and feed the trabecular arteries, which are located in strands of myofibroblast-containing dense connective tissue originating from the splenic capsule. The arteries then leave the trabeculae to become central arteries that lack accompanying veins and are surrounded by the white pulp.

The terminology of splenic white pulp regions was first introduced for rats by Snook,⁸ although the regions themselves had been described much earlier. Hence, the microanatomical terms coined in mice and rats need to be explained first.

In both species, the white pulp predominates in the spleen. Its core is formed by central arteries, which are accompanied by a continuous PALS consisting of fibroblastic reticulum cells (FRCs), dendritic cells, macrophages and large numbers of more or less migratory T lymphocytes. It has not been determined around which vessel type the PALS finally ends. In rats, hemispherical follicles are attached to the outside of the PALS at regular but relatively large intervals (Fig.1a,b). Mice exhibit a different arrangement of the white pulp. In cross-sections of mouse spleens the surface of the PALS is studded with follicles that tend to fuse around the T-lymphocyte zone (Fig.1c,d). The number of follicles per given area of splenic tissue is much higher in mice than in rats or humans. (Fig.1a–d). However, it cannot be excluded that the number of follicles per spleen does not differ among both rodent species.

Figure 1.

(a) Drawing of a small part of the white pulp in a cross-section of an adult rat spleen as visualized by standard haemalum-eosin staining. The follicles are attached to the PALS at relatively large intervals. The MZ primarily covers the T-cell zone. GC, germinal centre; MZ, marginal zone; PALS, periarteriolar lymphocyte sheath; RP, red pulp. (b) Visualization of the marginal zone and certain follicular B lymphocytes in a cross-section of an adult male LEW rat spleen by monoclonal antibody HIS 57. Cryosection, avidin–biotin complex technique with diaminobenzidine chromogen (ABC-DAB) and haemalum nuclear stain. The MZ is interrupted by several MZ bridging channels. Scale bar =100 μm. (c) Drawing of part of the white pulp in a cross-section of an adult mouse spleen as visualized by standard haemalum-eosin staining. The follicles tend to fuse around the PALS (hatched region). Most central arteries are cut transversely. MZ bridging channels are not depicted. (d) Visualization of the marginal zone and follicles by detection of IgM in a cross-section of an adult male C57BL/6 mouse spleen. Paraffin section, ABC-DAB technique with haemalum nuclear stain. Arrows indicate marginal sinus. Scale bar = 100 μm. (e) Drawing of a part of the human splenic white pulp as visualized by standard haemalum-eosin staining. MANZ mantle zone, SZ, superficial zone; PFZ, perifollicular zone. (f) Sequential visualization of IgD (ABC-DAB technique, brown) and CD20 (alkaline phosphatase polymer technique with Fast blue chromogen, blue) in a human splenic PALS (left) and follicle (right). In the most superficial zone of the follicle IgD⁻CD20⁺ B cells prevail. In other experiments most of these cells are CD27⁺. A marginal sinus is absent. Photograph taken from reference 55. Scale bar =100 μm.

Follicles are supported by follicular dendritic cells (FDCs) and may consist of a germinal centre (GC) surrounded by a mantle zone of small recirculating B cells. In rats, PALS and follicles are embedded in a broad marginal zone primarily inhabited by B lymphocytes with relatively pale nuclei and abundant cytoplasm, which are morphologically different from the small mantle zone B lymphocytes (Fig.1a,b). In mice, the MZ surrounds the follicle/PALS associations. Five to ten of these associations are visible per splenic cross-section. The MZ is much thinner in mice than in rats and comprises only about four or five B-lymphocyte layers. Due to the intercalated follicles, it is often not directly adjacent to the PALS as in rats (Fig.1c,d). The outer border of the MZ towards the red pulp is somewhat indistinct in both species, while the inner border of the MZ towards the PALS and the follicles forms a precise line due to a blood-filled cleft, the marginal sinus. In mice, but not in rats,^9,10 mucosal addressin cell adhesion molecule-1-positive (MAdCAM-1⁺) cells line the sinus. MAdCAM-1 expression depends on the presence of lymphotoxin α₁β₂ and the lymphotoxin β receptor.^11–13 In rats and mice, two special macrophage populations are associated with the MZ. Marginal metallophilic macrophages (MMMs) occur between the marginal sinus and the PALS or follicles, while marginal zone macrophages (MZMs) are distributed throughout the MZ.^12,14–16 In mice, both macrophage populations differ in phenotype.^12,15,16

In mice and most probably also in rats and humans, splenic white pulp reticular fibres are not directly covered by fibroblasts but there is an intervening space with basement membrane-like material called a conduit. In mice, the conduits formed around reticular fibres are supposed to permit low-molecular-weight materials direct access from the blood to the interior of the T-cell and B-cell zones of the white pulp, while high-molecular-weight materials are excluded.¹⁷ How this size exclusion is achieved is not entirely clear, because there are no tight barriers at the surface of the splenic white pulp. It is likely that MMM somehow restrict the access of high-molecular-weight substances to the PALS and follicles.¹⁷ Mouse MMMs and MZMs are able to effectively stimulate B-lymphocyte immune responses when targeted via special receptors.¹⁸ A prominent antigen of mouse and rat MMMs and certain MZMs is CD169.¹⁶ In mice, the presence of CD169 is important for normal IgM blood levels¹⁹ and for the uptake of microorganisms carrying sialic acid residues on their surfaces.

The MZ is partially included in the open splenic circulation, because it always contains a certain number of randomly distributed free erythrocytes and may therefore be regarded as part of the red pulp. It is, however, also a B-cell compartment, which may be attributed to the white pulp. Special MZ stromal cells termed marginal reticular cells (MRCs), have been described in mouse spleens.^20,21 MZ B lymphocytes may be a mixture of functionally different cells. In rats, most MZ B cells do not carry hypermutated immunoglobulin genes^22,23 and may therefore belong to an innate type of B lymphocytes predisposed for differentiation into IgM-secreting plasma cells. Rat and mouse MZ B cells were defined as CD21⁺ CD23⁻ IgM⁺ IgD⁻ large lymphocytes,²⁴ although immunohistology also reveals a substantial number of IgD⁺ B cells in the mouse MZ. In rats, a monclonal antibody, His57, has been described, which primarily reacts with MZ B lymphocytes (Fig.1b).^22,25

Whether rat or mouse MZ B cells are related to the B cells found at the surface of human splenic follicles is still unresolved. Rat MZ B cells have long been regarded as sessile cells with a low recirculation potential,²⁴ derived from recirculating precursors.²⁵ More recent findings in mice, however, demonstrated that MZ B cells are motile and locate to the MZ because of the antagonistic consequences of ligand binding to their sphingosine phosphate receptor S1P₁ versus CXCR5.^26,27 Additional chemokines, sphingosine phosphate receptor S1P₃,²⁷ integrins^28,29 and oxysterols³⁰ are also involved in the positioning of mouse MZ B cells. Mouse MZ B cells have been demonstrated to circulate between the MZ and follicles with an exchange rate of about 20% per hour.^31,32 It is supposed that this mechanism permits MZ B cells to transport immune complexes bound by complement receptors on their surface into the follicles and to deposit them on FDCs.^27,31 The systemic migration of rat and mouse MZ B cells needs further study, especially with respect to differences among both rodent species.

Microanatomy of human splenic white pulp

In humans, the splenic white pulp occupies less space than the red pulp. Similar to rats and mice, arteries branching from the trabecular vessels form the decisive innermost structure of the human splenic white pulp. In contrast to rats, follicles are the prevalent white pulp component in humans, whereas PALSs are limited in number and extension. Humans do not possess a marginal zone at the surface of the PALS (Fig.1e,f). Instead, a thin line of potentially recirculating IgM⁺ IgD⁺ CD27⁻ and also of switched B lymphocytes is present in this location (Fig.1f).^33,34

The size of normal splenic follicles is relatively invariant among the different species, which means that it is not related to the size of the spleen. Hence, under specified pathogen-free conditions the average diameters of normal adult splenic follicles are about 300–500 μm in inbred mice (Fig.1d) and rats (Fig.1b). This is also true for healthy adult humans (Fig.1f). In humans, inter-individual variations in the size of follicles are larger than inter-species differences.

Follicle-associated B lymphocytes and stromal cells

In analogy to mice and rats, human splenic follicles may be composed of GCs surrounded by mantle zones with small recirculating B cells. Most normal adult human spleens only exhibit follicles with small non-polarized germinal centres and large mantle zones primarily containing IgM⁺ IgD⁺ CD20⁺ CD27⁻ B cells.³⁵ These GCs are characterized by FDCs, B lymphocytes, several CD27⁺ plasmablasts and/or a few plasma cells and major numbers of CD4⁺ GC T cells, especially at their surface. It cannot be excluded that they represent long-lasting structures involved in permanent low-level plasma cell differentiation. With respect to the immunohistological distribution of lymphocytes and other cells in the human splenic white pulp, there is agreement among various authors.^33,36–42

Up to now, CD27 has been the most widely used surface antigen to characterize human memory B cells.⁴³ A substantial number of CD27⁻ memory cells has, however, also been shown,^44–46 which may be partially organ-specific.⁴⁷ It is supposed that IgM⁺ IgD^+/− CD27⁺ B cells are the human equivalent of rat and mouse MZ B cells.^48,49 As mentioned above, there are, however, several indications that the majority of rat and mouse MZ B cells differ from human CD27⁺ B cells in terms of their function, their immunoglobulin mutation state or their migratory behaviour. Whether human IgM⁺ IgD^+/− CD27⁺ B cells are derived from GCs or whether they represent an innate GC-independent B-cell population,^48–50 is still controversial.

By immunohistology, major numbers of CD27⁺ B cells are detected at the outermost surface of human splenic follicles, but not at the surface of the PALS (Fig.2a).^51–54 These cells are positive for surface IgM, carry somewhat less surface IgD than mantle zone B cells and are larger than these cells. CD27⁺ B cells are the most frequent lymphocytes at the follicular surface, but they are mixed with IgD⁺⁺ B cells, CD23⁺ B cells and CD4⁺ T cells.^51,52 Switched CD27⁺ B cells tend to locate most superficially in follicles, whereas IgM⁺ CD27⁺ B cells occupy a somewhat more central position.⁵⁵ Interestingly, CD27⁺ B cells also occur in the peripheral mantle zone defined by presence of FDCs (Fig2a).⁵⁵ The mantle zone CD27⁺ B cells do not differ from the more superficial CD27⁺ B cells with respect to IgD or IgM expression, if analysed by immunohistology. In contrast to rats and mice, humans do not exhibit a marginal sinus, MMMs or phenotypically distinct MZMs,³³ although macrophages are present at the follicular periphery.

Figure 2.

(a) Schematic drawing of a periarteriolar lymphocyte sheath (PALS) (left), a secondary follicle (centre) and red pulp arterioles (right) in the normal adult human spleen. Only different stromal cells and B-cell types, but not T cells, are depicted. PALS, follicles, vessels and cells are not drawn to scale. The superficial stromal cells and the fibroblastic reticulum cells (FRCs) are sheet-like, when sectioned tangentially. The expression of smooth muscle α-actin (SMA) in FRCs and non-FRC/non-follicular dendritic cell (FDC) stromal cells is individually variable. Follicles may also be traversed by side branches of central arteries. (b) Visualization of CD271 (brown) and SMA (blue) in a paraffin section of an adult human spleen by subtractive double-staining. FRCs in a PALS (left) and FDCs in a follicle (right) are strongly CD271⁺. Capillary sheaths in the surrounding red pulp are also strongly stained. Scale bar = 100 μm. (c) Visualization of CD271 (brown) and MAdCAM-1 (blue) in a cryosection of an adult human spleen by subtractive double-staining. A follicle has CD271⁺ FDCs and is surrounded by MAdCAM-1⁺ non-FRC/non-FDC stromal cells and CD271⁺ capillary sheaths. Scale bar = 100 μm.

In humans, FDCs express CD271, the low-affinity nerve growth factor receptor, in addition to many other antigens (Fig.2b,c).^7,55,56 CD271 is an optimal target for immunohistological detection of FDCs, because it is widely distributed even in the non-polarized follicles of adult human spleens and because it is not expressed by B lymphocytes in amounts detectable by standard immunohistological techniques. CD271⁺ FDCs are present in GCs and in mantle zones (Fig.2a). The innermost follicular CD27⁺ B cells clearly occur within the CD271⁺ mantle zone FDC network. The follicular surface containing the majority of the CD27⁺ B lymphocytes is, however, supported by a different stromal cell type, which is CD271^{− or+/−} and forms shells of differing phenotype. CD90 and MAdCAM-1 are present in the broadest shell of superficial follicular stromal cells, while smaller shells are formed by smooth muscle α-actin (SMA)⁺ and/or CD141⁺ stromal cells (Table1; Fig.2b,c). The diameter of the stromal shells is individually variable. Hence, at least two branched stromal cell types can be distinguished with respect to human follicles, CD271⁺ FDCs and CD271⁻ CD90⁺ MAdCAM-1⁺ superficial stromal cells (Fig.2a–c). In contrast to CD271⁺ FDCs, CD271⁻ MAdCAM-1⁺ superficial stromal cells are sticking to large reticular fibres covered by laminin. These cells are also associated with variable numbers of CD4⁺ T cells, which may form dense ring-like accumulations at the surface of the follicles.^33,36 The superficial stromal cells also continue at the surface of the PALS (Fig.2a,b). They may represent the human equivalent of mouse spleen and lymph node MRCs,²⁰ but their phenotype is most likely organ- and/or species-specific (Table2).⁵⁵ The region positive for MAdCAM-1 also contains scattered IgD⁺⁺ B cells, both at the surface of the follicles and at the surface of the PALS. At the follicular surface, the outer border of the shell of MAdCAM-1⁺ stromal cells coincides with the outer border of the follicular IgM⁺ IgD^+/− CD27⁺ B-cell accumulation, whereas the inner border of the MAdCAM-1⁺ stromal cell accumulation does not correlate with the location of any lymphocyte type investigated so far. It cannot be excluded that dendritic cells,⁵⁷ uncharacterized macrophages or innate lymphoid cells shape this border.

Table 1.

Phenotype of human splenic white pulp stromal cells¹

Area	Cell type	CD271	CD90	MAdCAM-1	CD141	CD105	SMA
PALS	FRCs	+	+	+/−	+	+	+/−
Follicle	FDCs	+	+ (weak or absent in GCs)	−	−	−	−
Most superficial FS	Non-FRCs/non-FDCs (MRCs)	+/− (similar to red pulp)	+	+	+/− or −	+	+/− or − (similar to red pulp)
Intermediate FS	Non-FRCs/non-FDCs (MRCs)	−	+	+	+	+	+
Innermost FS	Non-FRCs/non-FDCs (MRCs)	−	+/− or −	+	+/− or −	−	−

¹Synopsis of subtractive double-staining experiments in normal adult spleens.⁵⁵

The phenotype of superficial non-FRC/non-FDC stromal cells of the PALS is similar to that of superficial and intermediate follicular non-FRC/non-FDC stromal cells, but the MAdCAM-1⁺ area is smaller. An innermost shell of CD105⁻ SMA⁻ stromal cells is not observed around the PALS.

FDC, follicular dendritic cell; FRC, fibroblastic reticulum cell; FS, follicular surface; MRC, marginal reticulum cell; PALS, periarteriolar lymphocyte sheath; SMA, smooth muscle α-actin.

Table 2.

Phenotype of mouse spleen and lymph node marginal reticulum cells (MRCs) and human spleen non-follicular dendritic cell (FDC)/non-fibroblastic reticulum cell (FRC) superficial stromal cells

	MRCs (mouse)20,112	Non-FDC/non-FRC superficial stromal cells (human spleen)55
MAdCAM-1	+	+
CXCL13	+	+2
CR1/CD35	−1	−
podoplanin	+	−
CCL21	−1	+2
RANKL	+	−
CD271	?	−

¹Only described for lymph nodes.¹¹²

²Extra- or intracellular location not analysed.⁵⁵

Conventional and plasmacytoid dendritic cells have been described to occur at the surface of, but not within, human splenic follicles.^39,58–61 CD205⁺ immature dendritic cells, supposed to be distinct from macrophages, were found in the area occupied by MAdCAM-1⁺ stromal cells at the follicular surface.³⁹ Although macrophage and dendritic cell populations of different phenotypes exist in human spleens, consistent functional in vitro studies on these subpopulations are lacking. This may be partially due to the fact that isolation of these cells is rather demanding, because the surface antigens used are expressed by more than one cell type. For example, CD141 is not only present in human spleen dendritic cells,⁶² but also in splenic sinus endothelia⁶³ and in a large number of white pulp fibroblasts.⁵⁵ Hence, in vivo-targeting⁶² of putative human dendritic cell antigens will not be a therapeutic option in the near future, if the immunohistological studies available are taken into account.

Besides macrophages and dendritic cells, innate lymphoid cells may be relevant in the superficial follicular area. Type 3 innate lymphoid cells associated with MAdCAM-1⁺ stromal cells have been described to be decisive for T-cell-independent IgM production of human MZ B cells.⁶⁴ Hence, a refined analysis of non-B/non-T lymphoid cells at the human splenic follicular surface is highly desirable.

In summary, humans do not appear to possess a distinct histological compartment, which represents the equivalent of a rodent MZ. ‘MZ phenotype‘ B cells can be defined by flow cytometry, but their histological distribution differs from that of MZ B cells in mice and rats. It is clear that human IgM⁺ IgD^+/− CD27⁺ B cells are migratory cells, because they can be found in the blood and in other lymphatic organs.^{41,43,51,65,66} The apparent human ‘MZ‘ is neither spleen-specific nor is it supported by a unique stromal cell type. The term ‘MZ‘ should therefore be deleted from the human histological terminology and the follicular area superficial to the mantle zone should be re-named and called the ‘superficial zone‘. This avoids the impractical situation that the apparent ‘MZ‘ needs to be subdivided into an ‘inner MZ‘ which corresponds to the superficial FDC-supported mantle zone and an ‘outer MZ‘ with non-FDC/non-FRC supporting stromal cells.^51,52 The most likely explanation for the special cell arrangement at the surface of human splenic follicles is that CD27⁺ B cells represent migratory cells in transit between two different stromal compartments. CD27⁺ B cells may be observed while either entering or leaving their migration area in the mantle zone or they may even shuttle between the most superficial follicular region and the mantle zone analogous to mouse MZ B cells.^31,32

PALS-associated stromal cells

In normal adult human spleen, the PALS is of limited length. It is primarily found around larger arteries branching from trabecular vessels. Detailed studies on the distribution of T-lymphocyte subpopulations in the PALS other than CD4⁺ or CD8⁺ cells have not been published up to now. In addition, conventional and plasmacytoid dendritic cells were described in the human PALS,^39,58–61 but an in-depth analysis of their phenotype and functions is still lacking. The phenotype of human FRCs differs from that of mice, because podoplanin cannot be demonstrated by immunohistology.⁵⁵ Similar to FDCs, human FRCs are positive for CD271, although they express less of this antigen⁵⁵ (Fig.2a,b). An additional antigen characteristic of FRCs is CD141. CD141 is also present in the superficial stromal cells of the PALS (Table1). These cells lack CD271 and are similar to and continuous with the superficial stromal cells of follicles (Fig.2a). MAdCAM-1 and SMA are only sparsely expressed in FRCs, but strongly occur in the superficial stromal cells of the PALS (Table1). There is some individual variability with respect to expression of the latter two antigens in FRCs. Stromal cells associated with branches of central arteries in the vicinity of follicles express MAdCAM-1 and SMA (Fig.2a). In this location, a large number of T cells occurs, but B cells are also present. Hence, these areas are different from typical PALSs. In some spleens, plasmablasts with high amounts of intracellular IgM are found in the CD271⁻ superficial area of the PALS.⁵⁵

Microanatomy of human, mouse and rat splenic red pulp

The spleen is composed of reticular connective tissue which is continually traversed by bone marrow-derived cells. In contrast to mice and rats, in humans the largest compartment of the spleen is represented by the red pulp. The international histological terminology⁶⁷ lists the following red pulp components: arteries, arterioles, sinuses and veins in addition to the intersinusoidal space, which consists of splenic cords. This compilation is based on the most conspicuous tissue components, but it is incomplete, because it neglects capillaries and venules. It provokes the impression that sinuses are a special form of capillaries, which is not correct, because sinuses exist in addition to capillaries.

Red pulp fibroblasts

Fibroblasts produce the extracellular matrix of the red pulp including reticular connective tissue fibres. Human splenic fibroblasts differ from fibroblasts in non-lymphatic organs by exhibiting an activated phenotype and expressing, among other intracellular or membrane-bound antigens, SMA, CD271 and cytokeratin 8/18.^33,55 Splenic red pulp fibroblasts ensheath reticular fibres and delimit the spaces of the open splenic circulation in humans, mice and rats. As they are directly bathed in blood, red pulp fibroblasts need to either exhibit an anti-coagulatory cell surface or to prevent coagulation by other means. This fact has, however, never been investigated in any species. It is also not clear, whether conduits are present between red pulp fibroblasts and the fibres they cover.

Red pulp macrophages

In mice, rats, humans and other species, the red pulp cords harbour an extensive population of large macrophages. These macrophages are situated directly beneath the venous sinus endothelium. They appear to be able to easily move into the sinus lumen and out again. In humans, the phenotype of red pulp macrophages differs from that of macrophages associated with white pulp follicles or with capillary sheaths.⁷ The open circulation system guarantees that all materials to be removed from the blood have direct access to red pulp macrophages. Loss of the spleen in humans implies the loss of the body′s most active phagocyte compartment.

Red pulp erythrocytes, platelets, leucocytes and plasma cells

The splenic cords contain large numbers of erythrocytes, platelets and white blood cells in connective tissue spaces. It is assumed that about one-third of all mobilizable human platelets are stored in the splenic red pulp.^3,4 Fast running and diving animals pool erythrocytes in the spleen and release these cells on exercise by splenic contraction. In humans it is likely, but still controversial, that mature red cells also form a mobilizable pool in the red pulp. Diving apnoea is reported to provoke an increase in haematocrit that is abolished by splenectomy.⁶⁸ Diving and possibly other types of physical exercise lead to a moderate contraction of the human spleen without reduction of the arterial blood flow.⁶⁹ In the human red pulp, erythrocytes tend to accumulate at the surface of follicles together with granulocytes and monocytes in an area called the ‘perifollicular zone’.^33,70–72 There are also indications that the spleen is a functional reservoir of human⁵ and mouse⁷³ monocytes, which can be mobilized in cases of tissue injury or inflammation. In normal adult human spleens, single B and T lymphocytes are evenly distributed all over the red pulp. Plasma cells occur at larger distances. The immunohistological distribution of human splenic plasma cells expressing different immunoglobulin isotypes has not been investigated up to now.

Red pulp arterioles

In humans, central arteries associated with T-cell zones or with follicles give off red pulp arterioles, which finally feed sheathed and potentially also unsheathed capillaries. In contrast to rats, human central arteries are seldom located in a central position in the white pulp. Human central arteries themselves, or their first-order branches, may also run through the centre of follicles. In this case the artery loses its accompanying T lymphocytes and regains them after having passed the follicle.⁷⁴

Large human red pulp arterioles may form penicilli with a feeding vessel branching into a bunch of smaller arterioles, which all start from one point, but the majority of the small arterioles branch dichotomously. Small red pulp arterioles are covered by mixed accumulations of B and T lymphocytes,^7,55 which neither correspond to PALSs nor to follicles. Sometimes B lymphocytes prevail in this periarteriolar location. The arterioles are surrounded by SMA⁺ MAdCAM-1⁺ stromal cells (Fig.2a).

Sheathed capillaries

Human capillary sheaths most likely correspond to ‘ellipsoids’ found in the red pulp of the majority of vertebrate species. In humans, the sheaths are elongated and often branched structures of relatively small diameter (Fig.3a). Their function is so far unknown. Human capillary sheaths consist of three partially overlapping cell layers surrounding endothelial cells with pericytes.⁷ The innermost sheath layer is formed by specialized CD271⁺⁺ cuboidal stromal cells (Fig.3b). These cells are invaded and surrounded by macrophages of a special phenotype (CD68⁺  CD163⁻), which resembles that of most macrophages associated with B-cell follicles (Fig.3c). The outermost layer consists of individually variable numbers of B lymphocytes (Fig.3b). By immunohistology these B lymphocytes do not appear to differ in phenotype from the migratory B cells in follicular mantle and superficial zones.⁷ The three-layered structure of human capillary sheaths has been ignored up to now, although their composition was partially analysed by electron microscopy.⁶ In addition, the sheaths have been attributed to the wrong vessel type so that an erroneous term (Vagina periarteriolaris macrophagocytica) missing two of the sheath-associated cell types was introduced into the international histological⁶⁷ and embryological⁷⁵ terminologies of the spleen.

Figure 3.

(a) Schematic drawing of a branching arteriole and a sheathed capillary in the adult human splenic red pulp. The vessels are not drawn to scale, the course of the capillary has been shortened and the form of the open capillary end is hypothetical. Whether pericytes also occur in arterioles is not clear. T cells are not depicted. (b) Visualization of a capillary sheath by subtractive double staining for CD20 (brown) and CD271 (blue) in a paraffin section. The CD271⁺ stromal sheath cells are surrounded by CD20⁺ B cells. Scale bar = 50 μm. (c) Visualization of a capillary sheath by subtractive double-staining for CD163 (brown) and CD68 (blue). The majority of the capillary sheath-associated macrophages are CD68⁺CD163⁻. Scale bar = 50 μm.

Capillary sheaths may occur anywhere in the red pulp. They appear to cover the initial capillary segment following arterioles,⁷ although their exact location in the capillary network is not known. The phenotype of sheath-associated macrophages depends on the location of the sheath. Macrophages in sheaths near follicles express much more CD169 than those in sheaths located deeper in the red pulp.⁷ Capillaries with open ends and sheathed capillaries are especially prominent at the surface of follicles.

Ellipsoids are present in most vertebrate species, but do not occur in mice, rats, guinea pigs and rabbits.⁷ In birds, ellipsoids are histologically related to the B-cell zones of the spleen.^76–78 Hence, mice and rats represent evolutionary exceptions with respect to the absence of sheathed capillaries. It may be that human sheath macrophages and stromal sheath cells not only attract migrating B lymphocytes, but also provide decisive survival and differentiation factors. Human stromal sheath cells contain CXCL13 in a peculiar perinuclear position.⁷ It is likely that additional cytokines and chemokines are also present. The fact that CD169 is strongly expressed in human perifollicular sheath macrophages and sometimes also in dispersed perifollicular macrophages, which appear to originate from dissolving sheaths,³³ may indicate that human capillary sheaths exert certain functions associated with the MZ in mice and rats, such as priming of B cells and of cytotoxic T cells or invariant natural killer T cells. Functional studies on human CD169⁺ splenic macrophages are, however, lacking.

Sinuses

It is very likely that human red pulp capillaries have trumpet-shaped ends pouring their blood into the connective tissue of the splenic cords.⁷⁹ A three-dimensional reconstruction of immunostained red pulp serial sections did not reveal connections between splenic capillaries and sinuses in humans.⁷⁹ In the past, much confusion about splenic microcirculation has arisen from misinterpretations of corrosion casts, when the open system of the perifollicular zone was mistaken for the venous sinus system or for a marginal sinus in humans and several animal species.^80–82 Transmission electron microscopy has shown that human erythrocytes re-enter the circulation from the splenic cords by squeezing through openings between the sinus endothelia.⁸³ It is very likely that the majority of recirculating lymphocytes also takes this route for exiting the spleen. However, details of leucocyte exit from the spleen have never been thoroughly investigated in any species.

Human splenic sinuses appear to form a permeable vascular network with functional openings between the endothelial cells, which may be artificially enlarged by fixation for scanning electron microscopy. The phenotype of human splenic sinus endothelia is species-specific when compared to mouse and rat splenic sinus endothelia and it is unique regarding all other human endothelia. Among other antigens, human splenic sinus endothelia express CD8α,^33,84,85 the macrophage mannose receptor (CD206),⁸⁶ CD141,⁶³ TIE-2 (CD202b), VEGFR-3 and LYVE-1.⁸⁶ With the exception of the mannose receptor, macrophage-associated antigens have not been found in these cells.⁷

In humans, mice and rats, sinus endothelia are attached to ‘ring fibres’ of basal membrane-like material containing collagen type IV and laminin, which are covered on their outside by reticular fibres connecting the sinuses to the surrounding splenic cords.⁸³ Sinus endothelia are spindle- or rod-shaped. The cell portion containing the nucleus bulges into the sinus lumen giving the impression of a cuboid endothelium when the nucleus is hit in cross-sections.⁸³ The basal cytoplasm of human sinus endothelia contains rather thick intermediate vimentin filaments, which form several longitudinal stripes per cell visible by light microscopy even without special staining.^83,84 This is similar to rat sinus endothelia, which also contain vimentin filaments associated with actin filaments in their basal cytoplasm.⁸⁷ Both types of filaments appear to be involved in anchoring the sinus endothelia to the extracellular ring fibres. The vimentin filaments in sinus endothelia tend to non-specifically bind certain monoclonal antibodies, especially in paraffin sections.

Sinuses form the beginning of the venous circulation. They gather into smaller and larger red pulp venules which finally form trabecular veins and re-join the arteries.

It is likely that the microcirculation in mouse and rat spleens is similar to that in humans, because cords and sinuses are present in all three species.^87,88 However, in rat spleens the walls of larger veins are directly invaded by lymphocytes and other leukocytes³⁴ and may be involved in recirculation.

Recirculation of lymphocytes through the spleen

The pathways of lymphocyte recirculation through the spleen are still enigmatic in humans. Even in mice and rats, there is uncertainty about the immigration routes of lymphocytes into the spleen. Two different immigration pathways seem to occur in both species. Hence, labelled B and T lymphocytes injected intravenously first arrive in the MZ at the surface of the white pulp travelling via the marginal sinus in rats.^89–91 B cells reach the follicles from the MZ by migrating through the outer PALS, whereas T lymphocytes directly move to the PALS.^89–91 How the marginal sinus is supplied with blood is not known in detail. Nor is it clear, whether the marginal sinus represents a blood vessel or a part of the open splenic circulation. At least 50% of labelled injected lymphocytes are not found in the MZ, but first appear around smaller and subsequently around larger arterioles in the splenic red pulp of mice and rats.^92–94 This second ‘retrograde‘ immigration pathway may be somewhat slower and more long-lasting than the pathway involving the MZ and includes ‘MZ bridging channels’, i.e. small arterioles branching from the central arteries in mice and rats.^95,96

The second lymphocyte immigration pathway in mice and rats might correspond to the distribution patterns of B and T lymphocytes found around human splenic red pulp arterioles.^7,55 Hence, the walls of arterial vessels of a certain diameter appear to somehow attract migrating lymphocytes. The special SMA⁺ MAdCAM-1⁺ phenotype of fibroblasts surrounding these vessels may be a cause or consequence of lymphocyte migration. This phenotype is established very early in ontogeny. In human spleens, B lymphocytes are the first migratory cells that cluster around small arterial vessels surrounded by SMA⁺ stromal cells in the centre of vascular lobules long before birth.⁹⁷ It cannot be excluded that arteriolar smooth muscle cells are the first lymphocyte-attracting resident cells in human spleen ontogeny.⁹⁷ The immunohistological findings in adult human spleens provoke the hypothesis that CD27⁺ and CD27⁻ B cells use different migration pathways. CD27⁺ B cells may migrate in association with follicles only, whereas at least a part of the CD27⁻ recirculating B cells may immigrate via red pulp arterioles and the superficial PALS. However, as long as a detailed study of CD27⁺ B cells inside the PALS is lacking, additional pathways cannot be excluded.

How lymphocytes leave the spleen in the steady state is even less clear. There may be a constant efflux of cells back into the open circulation of the red pulp. Antigen-specific T lymphocytes have been described to exit from the PALS to the red pulp via MZ bridging channels in an infection model in mice.⁹⁸ Hence, the channels appear to form migration pathways both into and out of the white pulp. A small number of lymphocytes obviously also emigrates via lymphatics that accompany central arterioles in mice and rats.^99,100 Immunohistological detection of podoplanin in humans demonstrated lymphatic networks only associated with the walls of trabecular vessels and very large central arteries.⁵⁵ Afferent lymphatics are supposed to be absent in the spleen. In mice, the efferent lymphatics collect GFP-transgenic lymphocytes after intravenous injection.⁹⁹ They drain into splenic hilar lymph nodes and the lymph is then transported to coeliac lymph nodes and finally into the thoracic duct. In humans the splenic hilar nodes also receive lymph from parts of the stomach. The proportion and phenotype of recirculating lymphocytes, which do not directly return to the blood but leave the spleen via lymphatics, has never been determined in rodents or humans.

Consequences of splenectomy and gut pathologies in humans

Splenectomized humans are in danger of overwhelming infections especially by bacteria with polysaccharide capsules.¹⁰¹ The incidence of infections depends on the cause of splenectomy and is especially high in children. Susceptibility to encapsulated bacteria may be partially due to a loss of CD27⁺ IgD^+/− IgM⁺ memory B cells and of polysaccharide-specific B cells from the blood, the survival of which appears to depend on the presence of the spleen.^102–106

Interestingly, different gastrointestinal pathologies are often correlated with reduced splenic function and reduced IgM⁺ CD27⁺ B cells in the blood.^104,107,108 This phenomenon may be the cause or consequence of gastrointestinal disease and indicates that spleen and gut are functionally related in humans, at least with respect to this special B-cell population. In rabbits, there is a clearcut reduction of blood CD27⁺ B cells, if major parts of the gut-associated lymphatic tissue are removed.¹⁰⁹

Perspectives

Human spleens exhibit several organ- and/or species-specific traits which merit further investigation as they are of general physiological and immunological importance.

How red pulp fibroblasts prevent blood clotting in the open circulation, should be established. The interaction of capillary stromal sheath cells with migratory B lymphocytes needs clarification. The functional significance of the enormous amount of MAdCAM-1 expressed in human perifollicular splenic stromal cells and the dynamics of CD27⁺ B cells in the follicular periphery should be analysed. In this context, a careful immunohistological documentation of humanized immunodeficient mice may be helpful. Efforts should be undertaken to elucidate whether human IgM⁺ IgD^+/− CD27⁺ B cells recirculate between mucosal sites and spleen. Finally, it might be relevant to look for additional migratory cell types selectively associated either with CD271⁺ mantle zone FDCs or with MAdCAM-1⁺ perifollicular stromal cells. Knowing the in situ distribution of relevant antigens is a prerequisite for the therapeutic application of monoclonal antibodies and for discovering the human equivalents of decisive mouse cell populations such as CD8⁺ splenic dendritic cells^110,111 or MRCs^20,112 (Table2).

Glossary

ABC-DAB

avidin-biotin complex technique with diaminobenzidine chromogen

EC

endothelial cell

FDC

follicular dendritic cell

FRC

fibroblastic reticulum cell

GC

germinal centre

mAb

monoclonal antibody

MAdCAM-1

mucosal addressin cell adhesion molecule-1

MMM

marginal metallophilic macrophage

MRC

marginal reticulum cell

MZ

marginal zone

MZM

marginal zone macrophage

PALS

peri-arterial lymphatic sheath

SMA

smooth muscle alpha-actin

SMC

smooth muscle cell

SZ

superficial zone

Disclosures

There are no conflicting interests influencing this publication.