White pulp compartments in the spleen of the turtle Mauremys caspica

Summary The aim of the present study was to analyze the nature of lymphoid and non-lymphoid cellular components occurring in distinct histological compartments of the splenic white pulp of the turtle, Mauremys caspica, in order to define their possible correlations with those of the spleen of higher vertebrates, principally mammals. The white pulp of M.caspica consisted of 3 clearly distinguishable regions: (1) the periateriolar lymphoid sheath, and (2) the inner and (3) the outer zones of the periellipsoidal lymphoid sheath. Reticular cells intimately associated with reticular fibres constituted an extensive meshwork in the periarteriolar lymphoid sheath which housed principally Ig-negative lyphoid cells, mature and immature plasma cells, and interdigitating cells. A few Ig-positive cells were also present in the peripheral region of the periarteriolar lymphoid sheath. The inner and outer zones of the periellipsoidal lymphoid sheath were separated by a discontinuous layer of reticular cell processes. In the inner zone, surface Ig-positive lymphoid cells predominated as well as dendritic cells, resembling ultrastructurally the mammalian follicular dendritic cells, although no germinal centres were found in the turtle spleen. Macrophages, some cytoplasmic Ig-positive cells, and Ig-negative lymphoid cells appeared in the outer zone of the periellipsoidal lymphoid sheath. These results allow us to speculate on a phylogenetic relationship between the periarteriolar lymphoid sheath and the inner and the outer zones of the periellipsoidal lymphoid sheath of the spleen of M. caspica and the periarteriolar lymphoid tissue, the lymphoid follicles and the marginal zone, respectively, of the mammalian splenic white pulp.     

The cellular response of T and B dependent areas of the spleen after focal thermocoagulation of liver and kidney

Using 3H-thymidine autoradiography, the cellular response of the white splenic pulp after single or repeated thermolesions on liver and kidney was studied. Sham operations served as controls, and did not induce any significant changes in the splenic white pulp. After single or repeated thermolesions, the labeling index increased distinctly in the B and T cell areas. The central T-dependent periarteriolar lymphatic sheath (PALS) showed an earlier rise of radioactively labeled lymphocytes after repeated thermolesions than after a single lesion. After a single heat coagulation of liver and kidney a dissociation of the germinal centers occurred with consecutive hyperplasia. However, compared with the splenic reaction after other surgical injuries, especially after cryosurgical operations, the response of the B-dependent splenic areas was weaker. The distinct reaction of the central T-dependent PALS supports the conception of a more prominent cell mediated immune response of the peripheral lymphatic system with temporary T cell dysfunctions. Possibly the inflammatory complications of burned patients and the delayed wound healing of thermosurgical tissue lesions may be caused by the alteration of the lymphatic system with a disturbed cooperation of T and B lymphocytes with macrophages.     

Immunoarchitecture of regenerated splenic transplants: Influence of donor and host age on the regeneration of splenic compartments

Summary Inbred rats were used as a model to determine the influence of the age of the implanted splenic tissue and the age of the host on the structure of transplanted splenic tissue. Monoclonal antibodies against lymphocyte, macrophage and dendritic cell subsets were used to evaluate the different compartments of the spleen. Adult rats received implants from adult, weanling or fetal rats, weanling rats received splenic tissue from adult, weanling or fetal rats and neonatal rats received neonatal or fetal spleens. There were major differences in the structure and cellular composition of the regenerated splenic tissue. The younger the recipients and the donor spleens, the better the normalization of the splenic compartments and the less fibrous tissue was found 3 months after transplantation. The follicles regenerated in all transplants, but the marginal zone was only normally developed in wealing and neonatal hosts. The periarteriolar lymphatic sheath regenerated in a similar manner to the marginal zone. Whenever a compartment developed, its cellular composition was the same as in a normal spleen. The immunhistological techniques enabled splenic regeneration to be characterized revealing a far from normal histological splenic structure in many age groups. These findings suggest that splenic regeneration in children might result in splenic tissue with normal compartments, which would be in contrast to some data in adults.     

The three-dimensional structure of human splenic white pulp compartments.

The precise arrangement of B- and T-lymphocytes in the different compartments of the human splenic white pulp is still largely unknown. We therefore performed a 3D reconstruction of 150 serial sections of a representative adult human spleen alternately stained for CD3 and CD20. The results indicate that the T-cell regions of human spleens may be interrupted by B-cell follicles. Therefore, there is no continuous periarteriolar lymphatic T-cell sheath (PALS) around white pulp arterioles. An arteriole may be surrounded by T-lymphocytes at one level, then run across a follicle without any T-cells around, and finally re-enter a T-cell region. T- and B-cell compartments are intricately interdigitated in the human splenic white pulp. CD4(+) T-lymphocytes and the typical fibroblasts of the T-cell region may extend as a thin shell at the follicular surface within the marginal zone. On the other hand, IgD(++) B-cells continue from the follicular outer marginal zone along the surface of the T-cell region. Our findings indicate that the microanatomy of the splenic white pulp differs between humans and rodents. This may have consequences for the immigration of recirculating lymphocytes and for initial interactions among antigen-specific T- and B-lymphocytes.     

Cellular events during the primary immune response in the spleen

Summary The cellular events during the primary immune response in T and B cell compartments in the splenic white pulp were analysed in germfree mice immunized with sheep erythrocytes. Light, fluorescence and electronmicroscopic studies revealed that the initial formation of lymphoid blast cells occurs in the thymus-dependent area, i.e. the central periarteriolar lymphatic sheath (central PALS), 2 days after immunization. Lymphoblasts were found in close relation with erythrocyte-containing macrophages and with interdigitating cells. With fluorescence microscopy these blast cells were Ig negative. Lymphoblasts in the central PALS showed many polyribosomes in the cytoplasm, but were virtually devoid of endoplasmic reticulum. The ultrastructure of lymphoblasts in the central PALS, and their relation with interdigitating cells, suggests that these cells are the progeny of antigen-activated T cells.Cells with a positive cytoplasmic fluorescence, plasmablasts, appeared 3 days after immunization in the peripheral part of the PALS. During the progress of the immune response these cells accumulated around branches of the central arteriole, and moved along marginal zone bridging channels towards the red pulp. In the electron microscope plasmablasts showed many polyribosomes, short strands of rough endoplasmic reticulum close to mitochondria, and a few electron-dense bodies. The cell organelles of plasmablasts were frequently gathered in a so called uropod, which is a morphological sign of active cell movement.Germinal center formation started within primary follicles, 4 days after immunization. Blast cells in germinal centers did not show cytoplasmic fluorescence. During the course of the immune response, germinal centers extended in diameter, and fluorescent dendritic cells appeared at the periphery of the germinal center.From the present observations we conclude that: (1) cellular cooperation between different lymphoid and non-lymphoid cell types during the immune response against SRBC takes place in the PALS, (2) the cellular cooperation in the PALS results in the differentiation of B cells into immunoglobulin-producing plasmablasts, (3) the cellular cooperation in the PALS preceeds the formation of germinal centers in primary follicles, hence germinal centers are not involved in early T-B cell cooperation.     

Regeneration of splenic tissue after autologous subcutaneous implantation: Homing of T- and B- and Ia-positive cells in the white pulp of the rat spleen

The localization of T- and B lymphocytes and interdigitating cells (IDC) was investigated during the regeneration process of splenic implants. For this purpose a two-step immunoperoxidase technique was used to visualize T-cell antigen, immunoglobulins and Ia-antigen on cryostat sections. The specific localization of the repopulating lymphocytes occurred simultaneously with the development of non-lymphoid elements characteristic for the different compartments of the white pulp, i.e., the periarteriolar lymphocyte sheaths (PALS) and follicles. The marginal zone (MZ) developed after the PALS and primary follicles, but before germinal center reactions were found. During ontogeny, however, the development of a broad MZ precedes the formation of follicles. This difference in sequence of events is discussed.     

Lectin histochemistry of the spleen: a new lectin visualizes the stromal architecture of white pulp and the sinuses of red pulp.

The subcompartmentalization of the white pulp in the spleen is the result of interactions of specific resident stromal cells and migrating subtypes of lymphocytes. Because carbohydrate residues of cell membranes and extracellular matrices are involved in cell-cell and cell-matrix interactions, they were investigated in rat spleen by a broad panel of lectins. Splenic macrophages, which were also demonstrated by Perls' Prussian blue reaction, were labeled selectively by most mannose-specific lectins and gave the characteristic distribution patterns in all splenic (sub)compartments. One recently isolated lectin, Chelidonium majus agglutinin (CMA), visualized predominantly central arterioles, the reticular meshwork (RM) in the periarteriolar lymphatic sheaths (PALS), the circumferential reticulum cells limiting PALS and follicles, and some follicular dendritic cells (FDCs) in white pulp. The endothelial cells of venous sinuses in red pulp were also labeled by CMA and, if frozen sections were used, CMA also labeled the macrophages of the red pulp. Compared to CMA, the monoclonal antibody CD11, which can be used only in frozen sections, stained almost solely the fibrous (extracellular) component of the RM. Because CMA stains the reticulum cells in particular, it is better suited to visualize the stromal architecture of splenic white pulp than the monoclonal antibody. Because CMA can be applied to paraffin-embedded material, it is a particularly useful tool to study the splenic stromal architecture in archival material.     

Ultrastructural study on a variety of non-neural cells immunoreactive for nerve growth factor receptor in developing rats.

The occurrence of nerve growth factor receptor (NGFR) immunoreactivity was newly demonstrated in mesenchymal cells of lung bud and lateral palatine processes. Nerve fibers and enclosing Schwann cells with or without immunoreaction were present only in the periphery of the immunoreactive mesenchymal cell aggregation, but were not found within them. The NGFR-immunoreactive cells in immature skeletal muscles were revealed by immunoelectron microscopy to be perimysial cells without myofilament bundles, but not myoblastic cells. NGFR immunoreactivity was detected in noninnervated epithelial cells performing the invagination such as epithelial cells of the renal glomerulus and the lens placode. The immunoreactivity was also expressed in the innermost cells of the theca cell layer of the postnatal ovary and they contained no lipid droplets. In postnatal lymphatic tissues, NGFR-immunoreactive cells were identified as the interdigitating cells which were located in the internodular and deep cortex of the lymph nodes, in the marginal zone of the splenic white pulp, and in the medulla of the thymus. These findings suggest a potentially more widespread involvement of nerve growth factor and NGFR in the program of ontogeny than expected.     

In vivo effects of lipopolysaccharide on lymphoid and non-lymphoid cells in the mouse spleen

Summary In the present study the effects of lipopolysaccharide (LPS) on the cellular composition and phagocytosis of India ink in the inner parts of the periarteriolar lymphocyte sheaths (PALS) are described.Staining for B-, T-lymphocytes, and reticulin fibers in the spleen of normal and LPS-injected mice shows that the B-dependent follicular area is increased in size after LPS administration. However, the number of T-lymphocytes in the inner PALS is reduced markedly and a relatively high number of B-lymphocytes can be found in this area. The significance of this phenomenon is discussed.In untreated mouse spleen, carbon particles become localized in strongly acid-phosphatase (AP)-positive macrophages of the red pulp, marginal zone and white pulp 24 h after an intravenous injection of India ink. All these macrophages contain numerous carbon particles. After LPS pretreatment, the phagocytosis of carbon particles in the inner PALS is dramatically diminished, although many strongly AP-positive macrophages can be found in this area. The phagocytosis of carbon particles in the other compartments of the spleen did not change. It appears that injection of 2 g LPS or more is sufficient to induce this phenomenon which is most significant when LPS is injected 24 or 48 h before exposure to India ink.Abbreviations LPS lipopolysaccharide - PALS periarteriolar lymphocyte sheath - AP acid phosphatase - IDC interdigitating cells     

Development of antigenic heterogeneity in the splenic meshwork of severe combined immunodeficient (SCID) mice after reconstitution with T and B lymphocytes

Recently, we produced monoclonal antibodies reacting specifically with the reticular meshwork (RM) of lymphoid tissues, and demonstrated that, in the splenic white pulp of normal mouse, the antigenic heterogeneity of RM was associated with the segregation of the T and B lymphocytes. In the present study, we attempted to visualize further the interaction between splenic RM and T and B lymphocytes transferred into severe combined immunodeficient (SCID) mice. The splenic white pulp of naive SCID mice, containing a few T and B cells, showed little tendency for T-B segregation and antigenic diversity of RM. Transfer of spleen or bone marrow cells from normal mice resulted in complete recovery of lymphocyte populations, showing not only a clear segregation of T and B lymphocytes but also a remarkable antigenic diversity of RM. The same results were obtained following the transfer of spleen or bone marrow cells from the nude mouse. Next, we transferred purified T lymphocytes to one group of SCID mice and B cells to another. In mice given T cells, a few B cells were observed in the white puop; T lymphocytes lodged not only in the inner periarterial lymphatic sheath (PALS) but also in the outer PALS and follicles. In the animals to which B cells were transferred, T cells were few and the homing of B cells occurred only into their proper compartments, such as the outer PALS, follicles and marginal zone, but not in the inner PALS. Thus, B cells can home into their proper compartments of the splenic white pulp independently of T lymphocytes.     

Interdigitating cells in the white pulp of the human spleen

Summary Interdigitating cells are demonstrated as a special type of fixed cell in the periarteriolar lymphocytic sheaths of the human spleen. These cells show typical ultrastructural features as well as a characteristic enzyme histochemical pattern that distinguish them from other reticular cells in the splenic white pulp.     

Macrophages and Reticulum Cells in the Spleen of the Dogfish,Scyliorhinus canicula

The presence and ultrastructural features of reticulum cells and macrophages were studied in the spleen of the dogfish Scyliorhinus canicula. Three morphologically distinguishable regions of the spleen were identified: the white pulp, the red pulp and the ellipsoids. In all three, the splenic parenchyma was a meshwork supported by reticulum cells and fibres. Reticulum cells in both the white and the red pulp are irregular elements, the processes of which are joined by cell junctions and embrace developing reticular fibres. The ellipsoids of the dogfish spleen are terminal branches of the splenic arteries of the white pulp, with a sheath consisting of reticulum cells, reticular fibres, ground substance, macrophages and occasional lymphocytes. Isolated melanomacrophages also occur in the ellipsoid walls as well as in the red pulp. In both the white and the red pulp phagocytic reticulum cells, and macrophages appear frequently forming cell associations with surrounding blood cells, mainly lymphocytes. The functional significance of the ellipsoids and the cell-cell clusters of the white and the red pulp is discussed in relation to the immune capacities demonstrated in elasmobranchs.     

Localization of intravenously injected lipopolysaccharide (LPS) in the spleen of the mouse. An immunoperoxidase and histochemical study

In earlier studies we investigated the in vivo effects of lipopolysaccharide (LPS) on lymphoid and non-lymphoid cells in the mouse spleen. In order to find out whether LPS localizes in and/or on cells that are affected by this compound, the aim of the present study was to investigate the localization of intravenously injected LPS in the mouse spleen using an immunoperoxidase technique. At different time points after injection, the localization of LPS is demonstrated and LPS-containing cells are characterized. Most of the injected LPS has been taken up by marginal zone macrophages at 2 h after its administration whereas macrophages in the red pulp and at the periphery of the white pulp (marginal metallophils) have ingested less LPS. In the periarteriolar lymphocyte sheath, LPS is concentrated in a large number of acid phosphatase-negative, Ia-positive, large branched cells which were suggested to represent interdigitating cells. Moreover an extracellular dendritic localization pattern of LPS is demonstrated in the corona and central parts of the follicles at different time intervals after its injection. The significance of the localization pattern of LPS in the mouse spleen is discussed.     

The argentophil reticular cells of the mammalian spleen. II. The argentophil reticular cell arrangement inside the red pulp and its relationship with the endothelial lining cells of the splenic sinuses

The red pulp's argentophil reticular cell network of the spleen is composed by 3 types of fixed cells: 1. the primitive reticular cell, slightly argentophil; 2. the small reticular cell; 3. the larger reticular cell, strongly argentophil and phagocytic. This latter shows the classical morphological characteristics attributed to the reticular cells of the spleen. The large argentophil reticular cell may become free, constituting a 4th cell type, the free macrophage. A 5th reticular cell type is the dendritic cell found into the lymphatic follicles of the white pulp. The argentophil reticular cells of the red pulp assemble together to form the reticular cells' network, that occurs inside the red pulp cords. The primitive and the small reticular cell form the fundamental network on which the large cells are apposed. The reticular cells of this network maitain relationship with the arterial terminal vessels of the red pulp, being responsible by the ellipsoid structure. In those arteriolar segments without ellipsoid and in those mammalian species devoid of ellipsoid, the white pulp reticular cells, that surround the blood vessel as a part of the lymphoid periarteriolar sheath, mix with the red pulp's reticular cells and both can hardly be discriminated. The ellipsoids are formed by large argentophil cells arranged in concentrical layers around its lumen that sometimes appear devoid of endothelial lining cells. The red pulp's argentophil reticular cells, either the small or the large ones, contributed to the structure of the splenic sinuses' wall; its thin processes surround the sinus wall outside the endothelial lining cell as fibrillar structures that cross the back side of the lining cells. Two or more argentophil reticular cells send fibrillar processes to a single sinus. The perisinusal reticular cells may send a process between adjacent endothelial lining, cells that insinuate and attain the sinus lumen; this process becomes thick and eventually, the reticular cell enter the sinus lumen as a free macrophage. The argentophil reticular cells of the red pulp make connection between the capsule or the trabeculae and the reticular cell network. The endothelial lining cells of the splenic sinuses are not argentophil.     

α- and β-receptor control of catecholamine secretion from isolated adrenal medulla cells

Summary The structural characteristics and cellular elements of the boundary zone between the white and red pulp of the human spleen were studied by SEM and TEM. The boundary zone consisted of both the perifollicular region and the region surrounding the periarterial lymphoid sheath. The perifollicular region was further subdivided into two, equally thick layers. The inner half layer of the perifollicular region outside the mantle zone of the lymph follicle was composed of tightly packed medium-sized lymphocytes, interspersed by a small number of reticular cells. The outer half layer was composed of a reticular cell meshwork containing blood cells in vessels, which communicated with the splenic cords of the red pulp. Intermittent rows of reticular cells distinguished the outer from the inner half layer. The region surrounding the periarterial lymphoid sheath revealed the same type of reticular cell meshwork as the outer half layer of the perifollicular region. Capillary ends opened into the reticular cell meshwork, which suggested the presence of an open circulation in the human spleen. A deep lymphatic vessel which communicated with the periarterial lymphoid sheath was noted.     

Characterization of non-lymphoid cells in the white pulp of the mouse spleen: An in vivo and in vitro study

Summary Non-lymphoid cells (marginal metallophils, follicular immunecomplex-retaining cells, interdigitating cells), which are present in certain areas of the white pulp in the mouse spleen were characterized by means of (immuno)enzyme histochemical techniques, carbon uptake and experiments with lethal X-irradiation. Marginal metallophils are clearly present at the inner border of the marginal zone and show a very strong, E-600 sensitive, non-specific esterase (NSE) activity. Follicular immune-complex-retaining cells show a weak and diffuse NSE activity and no carbon uptake as shown by the combined application of an immunohistoperoxidase technique (for the demonstration of immune complexes), enzyme histochemistry (for NSE activity) and carbon uptake (for phagocytosis). Interdigitating cells show a distinct focus of NSE activity in the cytoplasm, weak carbon uptake and high radiation sensitivity. Demonstration of NSE activity is useful for the identification of the different non-lymphoid cells in the white pulp of the mouse spleen. It is suggested that the in vitro observed dendritic cells of Steinman and Cohn (1973) belong to the mononuclear phagocyte system, as transitional cells are encountered with cytological features of both dendritic cells and macrophages. These in vitro dendritic cells (or a portion of them) are probably similar to the interdigitating cells.Abbreviations HRP horseradish peroxidase - IDC interdigitating cells - PALS periarteriolar lymphocytic sheath - NSE non-specific esterase     

Scanning- and transmission electron-microscopic study of lymphatic vessels in the splenic white pulp of the macaque monkey

Summary The fine structure of the lymphatic vessels in splenic white pulp of the macaque monkey was studied by scanning and transmission electron microscopy.Lymphatic vessels were slit-like or widened channels which extended along central arteries and their large branches. The walls of the vessels were very thin in comparison with those of nearby arteries. They were composed only of a layer of endothelium supported by underlying reticular cells. Endothelial cells were mostly ribbon-like and extended along the long axis of the vessels. Perikarya of the endothelial cells were slightly protruded into the lumen. The thin peripheral cytoplasm showed smooth surfaces, except for some tiny processes, especially at boundaries between adjacent cells. The basal surface of the endothelial cells was attached to the lattice of reticular cell processes forming the framework of the white pulp. Basal laminae in strands were intercalated between endothelial cells and reticular cells. Perforations were often seen through the endothelial cell cytoplasm. Lymphocytes or processes of macrophages seen in the perforations were considered to be in migration. Large patent openings through the endothelium were not observed. The wall structure of the lymphatic vessels in the splenic white pulp suggests that lymphocytes in the white pulp may move directly into the lymph flow, in addition to moving into the blood flow via the vascular sinuses.Supported by Research Grant-in Aid from the Ministry of Education, Japan (Grant NO. 56480081).     

Leishmania donovani: evolution and architecture of the splenic cellular immune response related to control of infection.

Infection with the protozoan Leishmania donovani in humans is usually subclinical. Parasites probably persist for the life of the host and the low-level infection is controlled by the cellular immune response. To better understand the mechanisms related to the control of infection, we studied the evolution and architecture of the splenic cellular immune response in a murine model that is most representative of human subclinical infection. Following systemic inoculation with L. donovani, the parasites were primarily localized to the macrophage-rich splenic red pulp. There was an initial increase in the numbers of T cells and dendritic cells in the periarteriolar lymphoid sheath and marginal zone, but the red pulp (where parasitized macrophages were prominent) remained free of these cells until later in the course of infection. Thus, T cells did not colocalize with parasitized red pulp macrophages until later in the course of infection. Early in the course of infection, IL-10 production within the marginal zone and TGF-beta production by cells in the red pulp were prominent. These macrophage-inhibitory cytokines may contribute to the establishment of the infection and early parasite replication. By day 28 of infection, when the visceral parasite burden began to decline, the number of IL-10-producing spleen cells was back to the baseline level, but IFN-gamma production was higher and the number of IL-12-producing cells was increased dramatically. At this time T cells and dendritic cells had moved out of the lymphoid follicle and marginal zone into the red pulp where the parasites were located. These findings therefore suggest that control of infection is associated with IFN-gamma and IL-12 production and migration of T cells and dendritic cells to the site of chronic parasitism.     

The development of IgM- and IgG-containing plasmablasts in the white pulp of the spleen after stimulation with a thymus-independent antigen (LPS) and a thymus-dependent antigen (SRBC)

Summary This study describes the development of IgM and IgG containing plasmablasts in splenic white pulp after a single intravenous injection of the thymus-independent antigen lipopolysaccharide (LPS) or the thymus-dependent antigen sheep erythrocytes (SRBC) using immunohistoperoxidase techniques.Attention has been paid especially to the sites where IgM and IgG blasts develop in the white pulp and their migration route, from the white pulp towards the red pulp. The distribution of IgM and IgG blasts in the different white pulp compartments, i.e. outer periarteriolar lymphocytic sheath (PALS), inner PALS, follicles and the area along the terminal arteriolar branches, has been studied. Our findings indicate that both the thymus-independent IgM response to LPS and the thymus-dependent IgM response to SRBC start in the outer PALS. During the course of the immune response against SRBC the early localization of IgG plasmablasts in the white pulp was dispersed through the whole PALS. Later in the immune response the IgG blasts in the white pulp were localized especially in and at the border of follicle centres. No significant development of IgG blasts was found after LPS administration. The results of the present study suggest that during the immune response the bulk of IgM blasts migrates via the outer PALS and along the terminal arteriolar branches into the red pulp.     

大鼠脾白髓交错突细胞和巨噬细胞的形态计量分析

本文应用形态计量学方法对脾白髓内Ｓ－１００蛋白阳性交错突细胞和ｌｙｓｏｚｙｍｅ阳性巨噬细胞的一些形态计量参数进行比较分析,结果如下：两种细胞相比,交错突细胞和巨噬细胞的平均面积无显著差异,而平均周长和平均形态因子在交错突细胞明显大于巨噬细胞（Ｐ＜０．０１）;在单位面积脾白髓内,交错突细胞的面数密度和面密度明显小于巨噬细胞的相应数值（Ｐ＜０．０１）,提示这两种细胞在形态计量方面也有明显区分。鉴于这些数值与其细胞的形态和数量密切相关,并随细胞的变化而产生相应的改变,因而是有意义的形态计量参数。