Regeneration of splenic tissue after autologous subcutaneous implantation: Development of non-lymphoid cells in the white pulp of the rat spleen

Summary Regeneration of splenic tissue after autologous subcutaneous implantation provides a useful model for studying the development of splenic tissue. The development of the various non-lymphoid cells of the white pulp in the rat is described. It appears that regeneration of the implants is initiated by ingrowing vessels and a newly formed reticulum, which forms the microenvironment for the homing lymphocytes. Marginal metallophils are found at their characteristic location at the inner border of the marginal sinus five weeks after implantation. Trapping of antigen-antibody complexes reappears when the first primary follicles can be recognized.     

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 interdigitating cells; their relationship to migrating lymphocytes in the white pulp of rat spleen

Cell and Tissue Research - The pathway of lymphocyte migration through the white pulp of rat spleen and the relationship of migrating cells to the accessory cells (marginal zone macrophages and...     

Ontogeny of macrophage subpopulations and Ia-positive dendritic cells in pulmonary tissue of the rat

Summary The development of macrophage subpopulations and dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gutassociated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.Fellow of the Royal Netherlands Academy of Arts and Sciences     

Ultrastructural changes in cells of the white pulp of the spleen during experimental salmonella infection

The electron microscopic investigation of the immuno-competent cells in various zones of the spleen has revealed certain subcellular bases of the organ's reaction to the experimental salmonellosis. Alterations at early stage of the experiment (3-24 h after infection with Salmonella) are predominantly of destructive character: pyknosis of nuclei, dilated cisterns of the karyotheca, lysis of crests in mitochondria of some cells. On the 3d-14th day hyperplasia and hypertrophy of organelles in the immune-competent cells are revealed; it is connected with increasing processes of immunogenesis. At late stages of the investigation, normalization of the spleen cells ultrastructure and increased functional fibroblastic activity are noted.     

Specific localization of epidermal-type fatty acid binding protein in dendritic cells of splenic white pulp

Dendritic cells in the splenic white pulp of mice were intensely immunoreactive for epidermal-type fatty acid binding protein (E-FABP). This specific immunostaining revealed a clear difference in morphology between the dendritic cells in the periarterial lymphoid sheath (PALS) and follicular dendritic cells in the follicles in terms of cell sizes and process branching. No immunoreactivity was detected in dendritic cells in the marginal zones and the red pulp, although endothelial cells of almost all capillaries in the red pulp were immunoreactive for E-FABP. After peritoneal injection of lipopolysaccharide, the immunoreactive cells in PALS progressively enlarged and became rounded in shape with a peak in size at 24 h postinjection and they eventually resumed the dendritic form at 48 h postinjection. Within each of the enlarged immunoreactive cell perikarya were included small immunonegative apoptotic cells, presumptive lymphocytes. Taken together, E-FABP is useful as a marker for dendritic cells in the splenic white pulp, and may be involved through combination with fatty acids in antigen presentation and retention as well as in cytokine production.     

Comparison of topographical locations of reticular cells with 5'-nucleotidase activity in spleen white pulp and lymph nodes in man and rat]

The cells connections occuring in thymus-dependent areas of the splenic white pulp of rat between 5'nucleotidasic reticular cells and small lymphocytes allowed to research this same associated cells in the rat lymph node and the human splenic white pulp and lymph node. The locations of the thymus- dependent areas seem different in man and rat.     

Antigenic heterogeneity of the reticular meshwork in the white pulp of mouse spleen

Summary Monoclonal antibodies against cellular components of reticular meshworks were produced by immunizing rats with heterogeneous stromal-cell population of mouse spleen. Immunohistochemical screening selected two antibodies, WP-1 and RPSC-2. WP-1 proved to immunostain the meshwork of the B area densely, leaving the marginal zone unstained; it also reacted sparsely with the meshwork of the T-cell region. In contrast, RPSC-2 selectively immunostained the meshwork of the T region. Immuno-electron microscopy clearly visualized, for both antibodies, reaction products being deposited along the cytomembrane of the fibroblastic reticulum cells, along their abundant cytoplasmic processes that were densely intertwined with lymphocytes. Double immunostaining with RPSC-2 followed by WP-1 clearly divided the white pulp into the T and the B domains. The meshwork in the T-cell region proved to be immunostainable with both WP-1 and RPSC-2. Thus, the fibroblastic reticulum cells of the T-and the B-cell areas, while indistinguishable by routine microscopy, are at least partially heterogeneous.Presented at the First International Symposium on Dendritic Cells in Lymphoid Tissues, held in Yamagata, Japan, 7–9 June, 1990     

Selective malformation of the splenic white pulp border in L1-deficient mice

Lymphocytes enter the splenic white pulp by crossing the poorly characterized boundary of the marginal sinus. In this study, we describe the importance of L1, an adhesion molecule of the Ig superfamily, for marginal sinus integrity. We find that germline insertional mutation of L1 is associated with a selective malformation of the splenic marginal sinus. Other splenic structures remain intact. Immunofluorescence analysis of the extracellular framework of the spleen, using an Ab to laminin, reveals that L1 knockout mice have an irregularly shaped, discontinuous white pulp margin. Electron microscopic analysis shows that it is associated with bizarrely shaped marginal sinus lining cells at the periphery of the white pulp. These abnormalities correlate with the localization of L1 in normal mice in that L1 is normally expressed on marginal sinus lining cells at the white pulp border. These L1-immunopositive lining cells coexpress high levels of mucosal addressin cell adhesion molecule-1 and vimentin, indicating that they are of fibroblastic lineage and express a well-characterized addressin. Our findings are the first to implicate L1 in splenic lymphoid architectural development. Moreover, these findings help define the poorly characterized sinusoidal boundary across which mononuclear cells cross to enter the splenic white pulp.     

Electron-microscopic study of the development of the periarteriolar zone in splenic white pulp of rats

Summary To obtain more information concerning the origin of interdigitating cells, the postnatal development and morphology of the periarteriolar lymphatic sheath in splenic white pulp of rats was investigated by light- and electron-microscopy. Special attention was paid to the ontogeny of interdigitating cells. The spleens of the animals were studied in the age range from 1 h to 28 days after birth.The splenic white pulp of neonatal rats consists only of a few reticuloblasts, which are concentrically arranged around central arterioles. After 21 h an increase in promonocytes and monocytes was noted. Between the fifth and seventh postnatal day monocytogenic cells with a light and almost translucent cytoplasm appear, which display long cytoplasmic projections between the adjacent cells. Neighbouring lymphocytes often insert finger-like processes into the invaginated cellular membrane of these transitional forms. This intimate cellular contact is supported by zonulae occludentes. These cells represent transitional forms between monocytes and interdigitating cells.From seven days of age onwards typical interdigitating cells were present as in adult animals. After the differentiation into an inner and outer periarteriolar lymphatic sheath, the T-cell-dependent area of splenic white pulp has attained its adult appearance and further changes are not to be expected.On the basis of these findings, it is highly probable that interdigitating cells develop via transformation of monocytes.This investigation was supported by the Deutsche Forschungsgemeinschaft (^*He 537)     

Lymphocyte traffic through lymph nodes and Peyer's patches of the rat: B- and T-cell-specific migration patterns within the tissue,and their dependence on splenic tissue

The migration routes of lymphocyte subsets through organ compartments are of importance when trying to understand the local events taking place during immune responses. We have therefore studied the traffic of B, T, CD4⁺, and CD8⁺ lymphocytes through lymph nodes and Peyer's patches. At various time points after injection into the rat, labeled lymphocytes were localized, and their phenotype characterized in cryostat sections using immunohistochemistry. Morphometry was also performed, and the recovery of ⁵¹Cr-labeled lymphocytes in these organs was determined. B and T lymphocytes entered the lymph nodes via the high endothelial venules in similar numbers. Most B lymphocytes migrated via the paracortex (T cell area) into the cortex (B cell area), and then back in substantial numbers into the paracortex. In contrast, T lymphocytes predominantly migrated into the paracortex and were rarely seen in the cortex. No obvious differences were seen between various lymph nodes and Peyer's patches and the routes of CD4⁺ and CD8⁺ lymphocytes. After injection of lymphocytes into animals with autotransplanted splenic tissue, the number of B lymphocytes that had migrated into the B cell area of lymph nodes and of Peyer's patches was significantly decreased, whereas CD4⁺ lymphocytes migrated in larger numbers into the T cell area of both organs.This study was supported by the Deutsche Forschungsgemein-schaft (SFB 244, A7).     

Endothelial progenitor cells are integrated in newly formed capillaries and alter adjacent fibrovascular tissue after subcutaneous implantation in a fibrin matrix

Vascularization of bioartificial matrices is crucial for successful tissue engineering. Endothelial progenitor cells (EPC) have shown vascularization potential in ischemic conditions and may also support blood vessel formation in tissue-engineered matrices. The aim of our study was to investigate the impact of a well-characterized murine embryonal EPC line (T17b-EPC) on vascularization and fibrovascular granulation tissue formation after suspension in a fibrine matrix followed by subcutaneous implantation in a separation chamber in rats. EPC were fluorescently labelled in vitro prior to implantation. After 3, 7 or 14 days, animals were killed followed by explantation and histological analysis of the constructs. Before the end of the experiment, Bandeirea Simplicifolia lectin was intravenously injected to mark the vascular ingrowth into the implanted constructs. The transplanted cells were histologically detected at all time-points and located almost exclusively within the fibrin matrix at day 3 but the number of cells in the clot continuously decreased over day 7 to day 14. Conversely, cells were detected within the newly formed granulation tissue in increasing numbers from day 3 over day 7 to day 14. Transplanted cells were also found in the intermuscular septa. Cell viability was confirmed by use of an EPC clone expressing β-galactosidase. Fluorescence microscopy demonstrated integration of the transplanted cells in newly formed blood vessels within the fibrovascular granulation tissue adjacent to the fibrin clot. Presence of cells in the fibrin clot lead to thicker granulation tissue and an increased blood vessel diameter compared to cell-free controls. Organ standard controls showed presence of the transplanted cells in spleens at day 14 after transplantation. In summary, EPC exhibited biological activity after subcutaneous implantation in a fibrin matrix by migration from the fibrin clot into the granulation tissue and along intermuscular septae, undergoing differentiation into mature endothelial cells and integration into newly formed blood vessels and altering fibrovascular granulation tissue development. EPC may hold promise to modulate blood vessel formation in bioartificial matrices.     

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.     

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).