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.     

Lymphoid and non-lymphoid cells in nasal-associated lymphoid tissue (NALT) in the rat

Summary Lymphocyte and macrophage subpopulations and the stroma of mucosa-associated lymphoid tissue in the nasal cavity of the rat were examined by application of immunohistochemical and enzyme histochemical methods to cryostat sections. Nasal-associated lymphoid tissue was composed of a loose reticular network with lymphocytes and macrophages, covered by epithelium. The epithelium was infiltrated with B cells, T helper (W3/13-positive) and T suppressor/cytotoxic or large granular cells (OX8-positive), ED1-positive macrophages and Ia-positive cells. The B cell areas were populated by B cells, immunopositive for surface IgM or IgG. B cells with surface IgA or IgE were rare. Germinal centres were found infrequently. T helper cells were scattered throughout the B cell area. A few ED1-positive macrophages and ED5-positive follicular dendritic cells were observed. Strong Ia staining (mostly of B cells) was found in this area. The T cell areas contained T helper and T suppressor/cytotoxic cells in about equal amounts, and numerous ED1-positive macrophages. ED1 staining was also found in the subepithelial area. Numerous ED1-, ED2- and ED3-positive macrophages were found in the border between the lymphoid mass and the surrounding connective tissue. A few non-lymphoid cells showed weak acid phosphatase or non-specific esterase activity. The morphological observations suggest that nasal-associated lymphoid tissue plays an important role in the first contact with inhaled antigens.     

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.     

Dendritic cells in the rat spleen follicles

Follicles of peripheral lymphoid organs (rat) contain a type of non-lymphoid cell which is capable of arresting antigen-antibody complexes at the cell surface. These so-called dendritic cells can be visualized in immunized rats by staining antigen-antibody complexes with immunohistoperoxidase techniques. The present study concerns a classification of these cells and comparison with known non-lymphoid cell types such as macrophages, marginal metallophils and tingible body macrophages in the rat spleen follicles. Immunoenzyme histochemical and (enzyme) histochemical techniques have been combined in the same tissue sections to correlate the functional capacity of binding immune complexes with morphological characteristics or phagocytic capacity. Dendritic cells show silver affinity but do not demonstrate a characteristic pattern of hydrolytic enzymes or phagocytosis.     

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     

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     

Distribution of different cell types within the rat thymus in the neonatal period of life

Summary This study attempted to define reciprocal positions of cell types within the thymus. Random or non-random contacts between specific cell types were analyzed by means of graph theory. For analysis, thymus blocks were sectioned serially and, then, thymus cells were categorized into types, based on morphological criteria. The distribution of individual cell types within the cortex, cortico-medullary zone and medulla was presented in form of a map. In the analysis, three types of epithelial cell, characteristic of each thymus zone, macrophages, Langerhans-like cells and lymphocytes were found in non-random relations to one another. Moreover, characteristic groups of cells associated with one another were also demonstrated.This study was supported by the Polish Academy of Sciences     

Regeneration of the arterial wall in microporous,compliant, biodegradable vascular grafts after implantation into the rat abdominal aorta

Summary The ultrastructure of a new type of vascular graft, prepared from a mixture of polyurethane (95 weight %) and poly-L-lactic acid (5 weight %), was examined six weeks after implantation into the abdominal aorta of rats. These microporous, compliant, biodegradable, vascular grafts function as temporary scaffolds for the regeneration of the arterial wall.Smooth muscle cells, covering the grafts, regenerated a neo-media underneath an almost completely regenerated endothelial layer (neo-intima). These smooth muscle cells varied in morphology from normal smooth muscle cells to myofibroblasts. They were surrounded by elastic laminae and collagen fibers.Macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries were present in the disintegrating graft lattices. The epithelioid cells and multinucleated giant cells engulfed polymer particles of the disintegrating grafts.The regeneration of the endothelial and smooth muscle cells is similar to the natural response of arterial tissue upon injury. The presence of macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries in the graft lattices resembles the natural response of tissue against foreign body implants. Both of these responses result in the formation of a neo-artery that possesses sufficient strength, compliance and thromboresistance to function as a small caliber arterial substitute.Supported by Grant nr. 82.042 from the Dutch Heart Foundation     

The postnatal development of cell populations in the rat popliteal lymph node

Summary The postnatal development of the various cell populations in the rat popliteal lymph node was investigated applying enzyme-histochemical and immunohistochemical techniques. From birth, T-lymphocytes and interdigitating cells were demonstrable. During the development of the young lymph node, T-lymphocytes of the helper phenotype outnumbered the T-cells with a suppressor phenotype; they account for approximately 70% and 30% of all T-lymphocytes, respectively. At the very first day of postnatal life, post-capillary venules were already present. B-lymphocytes occurred later than T-cells during ontogeny; they were found on the second day after birth, most of them being IgM- or IgG-bearing lymphocytes. The first primary follicles occurred at day 18 and contained principally membrane-stained IgM cells and, to a lesser extent, membrane-stained IgG cells. The appearance of follicular dendritic cells correlated with the formation of primary follicles. With respect to the macrophages, it appeared that the ED1- and ED3-positive subpopulations were present with a similar distributional pattern as seen in adults, but in considerably lower numbers. The expression of ED2, however, showed a sudden increase in the third week of life. Findings of the present study are discussed in relation to those obtained in other investigations dealing with the ontogenetic development of lymphoid organs.Abbreviations IDC interdigitating cell - cIg cytoplasmic immunoglobulin - sIg surface immunoglobulin - FDC follicular dendritic cell - PBS phosphate-buffered saline - PCV post-capillary venule - PLN popliteal lymph node     

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).     

Distribution of IgM- and IgG-containing cells during the primary immune response in the rat spleen

Summary The morphology and localisation of IgM- and IgG-containing cells in the spleen of rats immunized with sheep red-blood cells (SRBC) were studied by combining immunohistochemical reactions with routine histological and histochemical methods. It was shown that IgM cells occur only in the outer periarteriolar lymphocyte sheath (PALS), whereas IgG cells are present throughout the whole PALS. It has been concluded that these cell types have different routes of migration. Both IgM- and IgG-containing cells were found in the germinal center in close relation with the extracellular immune complexes. The significance of this localisation is discussed.     

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...     

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)     

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.     

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.     

Macrophages in the interstitial tissue of the rat testis

Summary Macrophages were identified in the intertubular tissue of the rat testis by loading animals with a particulate vital dye (trypan blue or India ink) and by localizing immunocytochemically a macrophage membrane antigen (MRC W3/25). Leydig cells were identified by the histochemical staining reaction for 3-hydroxysteroid dehydrogenase activity and by a monoclonal antibody. Macrophages were scattered in the interstitial tissue closely attached to and mixed with the Leydig cells. They were never found in the seminiferous tubules. The macrophages comprised about 25% of all the cells in the interstitium. Double staining with a vital dye and a marker antibody showed that all the phagocytosing cells were macrophages and that the Leydig cells did not take up vital dyes. Double staining for the demonstration of the 3-hydroxysteroid dehydrogenase activity and the macrophage antigen likewise revealed two distinctly different cell populations. Crude Leydig cell preparations obtained by collagenase treatment of the testis contained macrophages (12–14%). Macrophages were present throughout the postnatal prepuberal development of the testis. Their density was increased in the cryptorchid and irradiated testis.     

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.     

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.