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     

Structure and histochemical organization of the spleen of Agama stellio (Sauria: Agamidae) and Chalcides ocellatus (Sauria: Scincidae)

The spleen of Agama stellio is composed mainly of red pulp; the white pulp is poorly developed, and its clusters are scattered throughout the organ and contain lymphocytes, reticular cells, and some plasma cells. The red pulp consists of clear reticular cells intermingled with blood cells, sinusoids, and pigment cells. The spleen of Chalcides ocellatus is encapsulated by connective tissue and is composed of white and red pulp. The white pulp consists of lymphoid tissue that surrounds the central arterioles, forming the periarteriolar lymphocyte sheath (PALS). The red pulp is composed of a system of venous sinuses and cords. The results of various histochemical procedures designed to demonstrate mucosubstances, proteins, and nucleic acids indicate that the spleen in these species resembles the mammalian spleen. © 1993 Wiley-Liss, Inc.     

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 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.     

Regeneration of autotransplanted splenic tissue at different implantation sites

Summary Inbred animals (Lewis rats) were used to investigate the regeneration of autologously implanted splenic tissue at intra-omental and subcutaneous sites. Quantitative immunohistology with monoclonal antibodies against lymphocytes and macrophages was performed to analyse the cell density of red pulp (RP), periarteriolar lymphoid sheath (PALS), marginal zone (MZ) and follicle, 7–180 days after transplantation. Antigenic, allogeneic and mitogenic stimulation and Northern blotting were also performed. Transplant groups differed from spleen only in the reduced size of PALS; however, quantitative analysis demonstrated subtle differences between spleen and transplants. The cell density of B-cells and ED-1⁺ macrophages was reduced in the RP, T_supp/cyt-cells were decreased and B-cells increased in PALS, and B-cells and T_helper-cells reduced in the MZ. No differences could be detected between the transplant groups. Flow-cytometric analysis of cell suspensions from spleen and transplants revealed a reduction of T-cells (OX-19⁺), MHC-I and transferrin-receptor-bearing cells in both transplant groups, and a decrease in the number of T_helper-cells and ED-3⁺ macrophages in subcutaneous transplants. Both transplant groups were defective regarding the allogeneic and pokeweed mitogen response. Aberration of the lipopolysaccharide response was restricted to subcutaneous transplants, which additionally showed abnormal expression of interferon-gamma, interleukin-5 and interleukin-6 mRNA. Thus, subtle alterations of the newly developed microenvironment and/or lymphocyte-homing may influence the regeneration of splenic tissue; the implantation site may represent an important parameter in functional reorganisation.     

The significance of the subcompartments of the marginal zone for directing lymphocyte traffic within the splenic pulp of the rat

Summary These studies were designed to gain more detailed information on the sites of lymphocyte migration into the marginal zone, on lymphocyte segregation within this area and on subsequent migration of the cells in individual compartments of the rat spleen. A lymphocyte population enriched in T-lymphocytes was obtained from rat lymph nodes and was labeled with 5-(³H)uridine in vitro. Observations on localization of labeled lymphocytes at short time intervals following their intravenous transfusion into syngeneic recipients, indicate that the sites of emigration from the blood are the internal and external layers of the marginal zone. From here, the cells migrate towards the periarteriolar lymphoid sheath and the red pulp.Fellow of the Alexander-von-Humboldt-Stiftung. On leave from the Department of Histology and Embryology, Institute of Biostructure, Academy of Medicine, ul. Swiecickiego 6, PL-60-781 Pozna, Poland     

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 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.     

Fibroblastic reticular cells guide T lymphocyte entry into and migration within the splenic T cell zone

Although a great deal is known about T cell entry into lymph nodes, much less is understood about how T lymphocytes access the splenic white pulp (WP). We show in this study that, as recently described for lymph nodes, fibroblastic reticular cells (FRCs) form a network in the T cell zone (periarteriolar lymphoid sheath, PALS) of the WP on which T lymphocytes migrate. This network connects the PALS to the marginal zone (MZ), which is the initial site of lymphocyte entry from the blood. T cells do not enter the WP at random locations but instead traffic to that site using the FRC-rich MZ bridging channels (MZBCs). These data reveal that FRCs form a substrate for T cells in the spleen, guiding these lymphocytes from their site of entry in the MZ into the PALS, within which they continue to move on the same network.     

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.     

Marginal zone bridging channels as a pathway for migrating macrophages from the red towards the white pulp in the rat spleen

The spleen of (PvG/c X DA)F1 rats, intravenously injected with carbon, was investigated. Large heavily carbon-laden (LHC) macrophages, which were found only in the red pulp at 30 min, appeared along marginal zone bridging channels (MZBC) from the red pulp towards the white pulp side successively during 1-6 h after carbon injection. After this time, they appeared in the periarterial lymphatic sheaths (PALS) near MZBC and then in the deeper PALS along the arteries by 5-10 days. Frequently, they were found in rows from MZBC into the white pulp. These findings suggest migration of LHC macrophages from the red towards the white pulp trough MZBC. Possible migration of LHC macrophages through MZBC was observed for a long period--at least 3 months examined. LHC macrophages came together preferentially in PALS and in and around the germinal centers consisting of large pyroninophilic lymphoblastoid cells. Occasionally, possible migration of LHC macrophages from regions around sinuses crossing the marginal zone vertically (vertical sinus) was also observed. Sinuses accompanied by LHC macrophages often ran parallel in close association with MZBC, particularly at sites of MZBC near the red pulp.     

The Ultrastructure of Mouse Lung: The Alveolar Macrophage

Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1½ hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small (~6 mµ) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.     

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.     

The ultrastructure of mouse lung: the alveolar macrophage

Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1(1/2) hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small ( approximately 6 mmicro) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.     

Pathways of lymphocyte migration within the periarterial lymphoid sheath of rat spleen

Summary Male Wistar rats were injected intravenously with 5-(³H)uridine-labeled lymphocytes isolated from lymph nodes of syngeneic donors and enriched in T cells. After short periods of time (3 to 120 min after injection), labeled lymphocytes were localized in spleen compartments using autoradiography to identify routes of lymphocyte movement from blood into splenic parenchyma and to follow migration pathways of recirculating lymphocytes within the periarterial lymphoid sheath (PALS). Topographical analysis of labeled lymphocytes was performed in specific planes of PALS characterized by the diameter of the arterial vessel and termed PALS large, PALS medium, and PALS small (PALS L, PALS M, PALS S, respectively). Attention was also paid to accumulations of labeled lymphocytes close to the arterial vessel wall. Initially, labeled lymphocytes were localized in PALS S and PALS M near the terminal branching of arterial vessels and in the marginal zone (MZ). We conclude that lymphocytes emigrate from blood into splenic parenchyma within two white pulp compartments: in MZ, and directly within PALS through the wall of capillary vessels. The sequential accumulation of labeled cells near arterial vessels of increasing diameter suggests that the recirculating pool of lymphocytes migrates into the central part of PALS L by two routes: from MZ, and along arterial vessels from PALS S and PALS M.R.B. was a fellow of the Alexander von Humboldt-Stiftung, on leave from the Department of Histology and Embryology, Institut of Biostructure, Academy of Medicine, ul. Swiecickiego 6, PL-60-781 Pozna, Poland.     

Topography,ultrastructure and phagocytic capacity of avian lymph nodes

Summary The structure of the avian lymph node (ALN) is characterized by a thin capsule, thin lymphoreticular cords, and an absence of trabeculae. It is not possible to subdivide the ALN into cortex, paracortex and medulla, or to subdivide the system of sinuses into marginal, trabecular and medullary divisions. The lymphoreticular cords contain avian germinal centers (AGC) with B-lymphocytes and the area of T-lymphocytes.Postcapillary venules are responsible for the recirculation of lymphocytes. Sinus reticular cells do not exist in the ALN, but free macrophages are present. The phagocytic capacity of the macrophages was determined by injection of vital dyes (India ink, Berlin blue) and inoculation with Candida cells. Macrophages filled with markers migrate from the lymph sinuses into the lymphoreticular cords and further into the AGC. The mobility of the macrophages is remarkably lower after phagocytosis of Candida cells.This study is dedicated to Prof. Dr. Dr. h.c. Hugo Grau (Weilheim) with sincere appreciation and respect     

Visualization of cholecystokinin receptors on a subset of human monocytes and in rat spleen

Direct radioreceptor binding experiments and Scatchard analysis reveal CCK receptors on elutriator purified human peripheral blood monocytes, but not on purified human T cells. The monocyte receptors have a single class of high (0.1 nM) affinity binding sites. A structure-function analysis of monocyte binding by different CCK analogs correlates well with previously demonstrated chemotactic responses in monocytes and receptors in brain tissue. Biochemical cross-linking indicates that the monocyte CCK recognition molecule is comparable in molecular size to that in brain membranes. Utilizing a novel fluoresceinated Texas Red-CCK conjugate we have visualized that up to 20% of human peripheral monocytes bear receptors for CCK. A discrete and anatomically significant distribution of CCK receptors in rat spleen is shown by film autoradiography of tissue sections. A more detailed microscopic analysis identifies a dendritic population of monocyte-derived cells within the periarteriolar lymphocyte sheath (PALS) of the white pulp as the CCK receptor-bearing cell in spleen. The anatomical localization of receptor-bearing cells within the PALS region suggests a role for CCK in the antigen processing and sensitization phases of the immune response via regulatory effects of this peptide on a specific, local macrophage-related cell population.     

IL-1 gene expression in lymphoid tissues

We examined the expression of IL-1 mRNA in vivo by in situ hybridization. RNA probes for murine IL-1 alpha and IL-1 beta were used to detect IL-1 mRNA in frozen sections of spleen, lymph node, and thymus of mice injected with Salmonella typhi LPS or SRBC. No IL-1 was detected in lymphoid tissues from un-injected mice. This lack of expression correlated with the absence of IL-1 biologic activity. However, after LPS injection, IL-1 alpha and beta mRNA expression was found in macrophages of the red pulp and marginal zone of the spleen. The periarteriolar lymphoid sheath contained cells that only expressed IL-1 beta mRNA. These cells were not lymphocytes and did not stain with the macrophage marker F4/80. A similar cellular response was found after SRBC injection. Scattered macrophages in lymph nodes and thymus were positive, but only after LPS or SRBC injection. The spleens of mice injected with LPS had megakaryocytes containing IL-1 mRNA.     

Macrophage Activation by Muramyl Dipeptide (MDP) without Lymphocyte Participation

An increase in the numbers of spread macrophages caused by macrophage stimulants was found to be a very sensitive measure for macrophage activation. Muramyl dipeptide (MDP), bacterial lipopolysaccharide (LPS), and lymphokines were found to activate macrophages dose-dependently as measured by this parameter. Macrophage activation by MDP was strictly dependent on its adjuvant-active stereochemically specific structures. Macrophage activation by MDP and LPS occurred without lymphocyte participation. It is suggested that LPS also activates macrophages via lymphocytes.