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Metallophilic macrophages (MMs) are a distinct cell type of the rodent thymus. Our previous research has focused on the morphological characteristics of MMs, as well as on the molecular mechanisms involved in the development and tissue positioning of these cells. However, the postnatal development of MMs has not been sufficiently studied. In the present study, we investigated the positioning of MMs in the rat thymus between postnatal day 0 (P0) and P30. On P0, MMs were evenly distributed all over the thymic tissue-that is, the cortex, cortico-medullary zone and medulla. From P0 to P15, the number of MMs in the thymic cortex significantly decreased, and after P15, this number did not change. Thus, the present study shows that on P15, MMs almost completely disappear from the thymic cortex and show their adult position in the cortico-medullary zone and in the medulla.

The spleen is the only blood filter in the organism which removes foreign antigens and effete cells from circulation. The significant role in capturing, transporting and presentation of antigens to immune cells is executed by a special subset of splenic macrophages called marginal metallophilic macrophages. Upon stimulation with lipopolysaccharide, these cells promptly migrate from their preferential location at the inner aspect of the splenic marginal sinus into the B-cell lymphoid follicles. This migration is executed via CXC chemokine ligand 13 in a lymphotoxin-dependent fashion. However, the role of tumour necrosis factor-α/tumour necrosis factor receptor-1 signalling axis has not been studied, despite its critical role in the formation of B-cell lymphoid follicles, follicular dendritic cell networks and germinal centres. Here, we show that signalling via tumour necrosis factor receptor-1 is not required for the migration of marginal metallophilic macrophages into the B-cell zone and that the presence of organized B-cell lymphoid follicles is not a prerequisite for their dislocation.

It is well known that bacterial lipopolysaccharide (LPS) induces migration of several cellular populations within the spleen. However, there are no data about the impact of LPS on B and T lymphocytes present in the red pulp. Therefore, we used an experimental model in which we tested the effects of intravenously injected LPS on the molecular, cellular and structural changes of the spleen, with special reference to the red pulp lymphocytes. We discovered that LPS induced a massive relocation of B and T lymphocytes from the splenic red pulp, which was independent of the tumor necrosis factor receptor-1 signaling axis. Early after LPS treatment, quantitative real-time PCR analysis revealed the elevated levels of mRNA encoding numerous chemokines and proinflammatory cytokines (XCL1, CXCL9, CXCL10, CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, TNFα and LTα) which affect the navigation and activities of B and T lymphocytes in the lymphoid tissues. An extreme increase in mRNA levels for CCL20 was detected in the white pulp of the LPS-treated mice. The CCL20-expressing cells were localized in the PALS. Some smaller CCL20-expressing cells were evenly dispersed in the B cell zone. Thus, our study provides new knowledge of how microbial products could be involved in shaping the structure of lymphatic organs.

Metallophilic macrophages hold a strategic position within the thymic tissue and play a considerable function in thymic physiology. The development and positioning of these cells within thymic tissue are regulated by complex molecular mechanisms involving different cytokine/chemokine axes. Herein, we studied the role of XCL1 signaling in these processes. We show that in the XCL1-deficient thymus numerous metallophilic macrophages are aberrantly positioned in the thymic cortex, instead of their normal location in the cortico-medullary zone. Still, these cells retain their normal appearance: very large size with prominent, ramifying cytoplasmic prolongations. This shows that XCL1 signaling is not involved in morphological development, but rather in correct positioning of metallophilic macrophages within the thymic tissue. In contrast to thymic metallophilic macrophages, the positioning of splenic marginal metallophilic macrophages is not affected by XCL1-deficiency. Anat Rec, 297:1472–1477, 2014. © 2014 Wiley Periodicals, Inc.

We have already shown that metallophilic macrophages, which represent an important component in the thymus physiology, are lacking in lymphotoxin-β receptor-deficient mice. However, further molecular requirements for the development and correct tissue positioning of these cells are unknown. To this end, we studied a panel of mice deficient in different chemokine ligand or receptor genes. In contrast to normal mice, which have these cells localized in the thymic cortico-medullary zone (CMZ) as a distinct row positioned between the cortex and medulla, in plt/plt (paucity of lymph node T cells) mice lacking the functional CCL19/CCL21 chemokines, metallophilic macrophages are not present in the thymic tissue. Interestingly, in contrast to the CCL19/21-deficient thymus, metallophilic macrophages are present in the CCR7-deficient thymus. However, these cells are not appropriately located in the CMZ, but are mostly crowded in central parts of thymic medulla. The double staining revealed that these metallophilic macrophages are CCR7-negative and CXCR3-positive. In the CXCL13-deficient thymus the number, morphology and localization of metallophilic macrophages are normal. Thus, our study shows that CCL19/21 and its possible signaling through CXCR3 are required for the development of thymic metallophilic macrophages, whereas the CXCL13-CXCR5 signaling is not necessary.

Lymphotoxin β-receptor (LTβR) and TNF receptor-1 (TNFR1) are important for the development of secondary lymphoid organs during embryonic life. The significance of LTβR and TNFR1 for the formation of lymphoid tissue during adult life is not well understood. Immunohistochemistry, morphometry, flow cytometry, and laser microdissection were used to compare wild-type, LTβR−/−, TNFR1−/− spleens with splenic tissue that has been newly formed 8 wk after avascular implantation into adult mice. During ontogeny, LTβR is sufficient to induce formation of the marginal zone, similar-sized T and B cell zones, and a mixed T/B cell zone that completely surrounded the T cell zone. Strikingly, in adult mice, the formation of splenic compartments required both LTβR and TNFR1 expression, demonstrating that the molecular requirements for lymphoid tissue formation are different during embryonic and adult life. Thus, interfering with the TNFR1 pathway offers the possibility to selectively block the formation of ectopic lymphoid tissue and at the same time to spare secondary lymphoid organs such as spleen and lymph nodes. This opens a new perspective for the treatment of autoimmune and inflammatory diseases.

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are small lipid molecules with a variety of physiological roles. Additionally, their involvement in the initiation and progression of malignant tumors has been increasingly recognized in recent years. However, the data on the expression of S1P and LPA receptors on different cancer cells are very few. Real-time polymerase chain reaction was used for the analysis of mRNA expression of five S1P((1-5)) and three LPA((1-3)) receptors on a large panel of 13 colon, breast, melanoma, and lung cancer cell lines. Furthermore, the modulation of S1P and LPA receptor mRNA expression was studied upon xenotransplantation of tumor cells into severe combined immunodeficient (SCID) mice. The S1P and LPA receptors were expressed to a variable degree on all tumor cell lines tested (with exception of colon cancer SW480). Most notably, tumor cell lines in vitro expressed S1P(2) mRNA that was down-regulated upon xenotransplantation, whereas LPA(2) receptor mRNA was strongly expressed both in vitro and in vivo (except by breast cancer cells). The latter was especially distinctive for small cell lung tumor cells. The S1P and LPA receptors are differentially expressed on tumor cell lines in vitro. Their expression is modulated upon xenografting into SCID mice in vivo.

Until recently, B lymphocyte maturation was considered to be independent of the thymus and T lymphocytes. However, using nude animals, which lack the functional thymus, we have shown that T lymphocytes are required for the peripheral phase of B lymphocyte maturation. We showed that the proportion of immature B lymphocyte subsets (CD90highIgMhigh and CD90highIgMlow) was significantly increased, whereas that of mature B lymphocyte subsets (CD90−IgMlow and CD90−IgMhigh) was decreased in the peripheral blood and lymph nodes of nude rats. In addition, the expression of functionally important surface molecules MHC class II, ICAM-1, CD44 and L-selectin was significantly down-regulated both on immature and mature B lymphocyte subsets. The implantation of thymic tissue under the kidney capsule of nude rats alleviated the block in B lymphocyte maturation and normalized of the defective expression of surface molecules. Comparable effects were seen after the adoptive transfer of T lymphocytes. This shows that in nude rats B lymphocytes do not mature properly due to the lack of T cell help and that T lymphocytes are required for the peripheral phase of B lymphocyte maturation, as well as for the appropriate expression of surface molecules. This should be considered when treating patients with T lymphocyte deficiencies.

Microfibrillar-associated protein 4 (MFAP4) is an extracellular protein belonging to the fibrinogen-related protein superfamily and is recognized as an integrin ligand with suggested functions in pulmonary and vascular tissue homeostasis. MFAP4 expression in the spleen is increased during infections; however, the significance of MFAP4 for the function of the spleen is unknown. Immunohistochemistry, morphometry and real-time RT-PCR were used to analyze wild-type and MFAP4-deficient spleens. In addition, they were compared with splenic tissue, which was newly formed 8 weeks after avascular implantation into adult mice in order to obtain information about the role of MFAP4 in the formation of splenic tissue during ontogeny and adult life. The present study shows that MFAP4 is co-localized with laminin in the B- and T-cell zones of the spleen, in addition to capsular and trabecular expression. MFAP4 is most likely produced by fibroblastic reticulum cells and follicular dendritic cells of the spleen but can also be imported via the blood from other tissues. The development of splenic tissue is not disturbed in MFAP4-deficient mice. However, in splenic tissue regenerating under MFAP4-deficient conditions, the number of FDCs is significantly decreased but is corrected by MFAP4 imported from other tissues. No differences were observed for lymphocyte numbers or splenic structure. The data indicate that MFAP4 promotes FDC development in regenerating splenic tissue and warrant further investigations regarding the MFAP4 dependency of splenic B-cell maturation.

Lymph nodes are the sentinels of the immune system that control the surfaces of the body to the outside (skin, lung, gut) and the tissues, whereas the spleen monitors the inside surface (vasculature) and the blood. Lymph nodes are both matchmakers and copy shops. They are matchmakers because they bring together antigens with antigen-specific B lymphocytes and T lymphocytes. This is a major challenge since antigens can enter and develop at any site of the body while the average frequency of antigen-specific B cells and T cells is in the range of only 1 in 100 000. Lymph nodes solve this problem by concentrating antigen-containing lymph and attracting huge numbers of B cells and T cells from the blood. Lymph nodes are copy shops because they provide a microenvironment that facilitates an enormous expansion of B cells and T cells bearing receptors specifically directed against the cognate antigen. Due to their huge number, these antigen-specific B cells and T cells are now able to successfully protect the body. This article describes the anatomical structures allowing the lymph node to be a matchmaker and a copy shop.

Lymphotoxin-beta receptor (LTbetaR) axis plays a crucial role in development and compartmentalization of peripheral lymphatic organs. But, it is also required for the appropriate function and maintenance of structural integrity of the thymus: in LTbetaR-deficient animals the clonal deletion of autoreactive lymphocytes is impaired and differentiation of thymic medullary epithelial cells is disturbed. In this study, using several markers, we showed that thymic metallophilic macrophages were lacking in LTbetaR-deficient mice. In tumor necrosis factor receptor-I (p55)-deficient mice (which we used as positive control) thymic metallophilic cells were located, similarly as in normal mice, in the thymic cortico-medullary zone at the junction of cortex and medulla. These findings show that LTbetaR is necessary for maintenance of metallophilic macrophages in the thymus and provide further evidence that these cells may represent a factor involved in thymic negative selection.

B-lymphocyte maturation is considered to be independent of the thymus. However, there is circumstantial evidence suggesting that it may be impaired in nude animals that lack the thymus. Our study shows that the proportion of immature B-lymphocyte subsets (CD90(high) IgM(high) and CD90(high) IgM(low)) was significantly increased, whereas that of mature B-lymphocyte subsets (CD90- IgM(low) and CD90- IgM(high)) was decreased in the blood and lymph nodes of nude rats. In addition, the expression of major histocompatibility complex class II, intercellular adhesion molecule-1, CD44 and l-selectin was significantly down-regulated both on immature and mature B-lymphocyte subsets. After implantation of thymic tissue under the kidney capsule of nude rats the block in B-lymphocyte maturation was alleviated and the expression of surface molecules was normalized. Comparable effects were seen after the adoptive transfer of T lymphocytes. Thus, we show that in nude rats B cells do not mature properly because of the lack of T-cell help and that T lymphocytes are required for the peripheral phase of B-lymphocyte maturation, as well as for the appropriate expression of surface molecules. This should be considered when treating patients with T-cell deficiencies.

There is an abundance of data dealing with recirculation of T cells in the rats, but relatively little is known about the traffic of B cells. The adhesion molecules expressed on the surface membrane are of great significance for recirculation of lymphocytes. However, very little is known about the expression of various adhesion molecules on B-cell subsets. Here we show that in normal rats various adhesion molecules are differentially expressed on B-cell subsets and that the level of their expression changes after the entry of B lymphocytes from the blood into the lymphoid tissues. In splenectomized rats, the surface expression of LFA-1 and ICAM-1 is selectively reduced on B-cell subsets in blood and lymph node, which is accompanied by a selective increase in the number of all B-cell subsets in the blood. The decreased surface expression of adhesion molecules results in faster migration of B lymphocytes through lymph nodes with subsequent accumulation of these cells in the blood.

Summary In this study transmission electron microscopy was used to investigate the ultrastructural features of macrophages which are strategically positioned in the thymic cortico-medullary zone and the dark zone of germinal centers of peripheral lymphatic organs in adult Wistar rats. We show that this, morphologically distinct, type of macrophage displays the entire range of cytoplasmic inclusions, which structurally closely correspond to those of endosomal/MHC-II-enriched compartments of antigen presenting cells. The macrophages of the cortico-medullary zone and germinal centers contain numerous multivesicular bodies, as well as various kinds of cytoplasmic inclusions ranging from single to aggregated multivesciular/multilamellar bodies to large vacuoles. These multilamellar inclusions are composed of elongated, irregularly shaped cisternae, with abundance of internal membrane profiles and dense bodies. Often, cortico-medullary zone and germinal center macrophages contain the typical multiamellar bodies. Poly-saccharides are detected by the thiocarbohydrazidesilver proteinate method within the dense bodies of these macrophages. The functional significance of cortico-medullary zone and germinal center macrophages is briefly discussed.

Recently, many details of the interplay between tumor cells and tumor-associated stromal elements leading to the progression of malignant disease were elucidated. In contrast, little is known about the role of uninvolved stromal tissue in the remote surrounding of the malignant tumor. Therefore, we performed a computer-aided morphometric study of rectal mucosa in samples taken 10 cm and 20 cm away from the malignant tumor during endoscopic examination of 23 patients older than 60 years. The samples of rectal mucosa from 10 healthy persons of corresponding age subjected to diagnostic rectoscopy during active screening for asymptomatic cancer were used as control. All structural elements of the rectal mucosa were studied and the number of nucleated cells in the lamina propria per 0.1 mm² of tissue was assessed. Our study revealed a reduced number of cells in the lamina propria of the rectal mucosa 10 cm and 20 cm away from the tumor lesion in both male and female patients. The decreased mucosal height and increased crypt number were registered in female patients 10 cm away from the tumor. The connective tissue of lamina propria showed a disorderly organization: the collagen fibers were frail, loosely arranged and signs of tissue edema were present. Small blood vessels and capillaries were much more frequently seen than in healthy tissue. Our results demonstrate the complex interactions between the cancer and remote mucosal tissue of the affected organ.

Metallophilic macrophages hold a strategic position within the thymic tissue and play a considerable function in thymic physiology. The development and positioning of these cells within thymic tissue are regulated by complex molecular mechanisms involving different cytokine/chemokine axes. Herein, we studied the role of XCL1 signaling in these processes. We show that in the XCL1-deficient thymus numerous metallophilic macrophages are aberrantly positioned in the thymic cortex, instead of their normal location in the cortico-medullary zone. Still, these cells retain their normal appearance: very large size with prominent, ramifying cytoplasmic prolongations. This shows that XCL1 signaling is not involved in morphological development, but rather in correct positioning of metallophilic macrophages within the thymic tissue. In contrast to thymic metallophilic macrophages, the positioning of splenic marginal metallophilic macrophages is not affected by XCL1-deficiency.

For a very long time, we studied the metallophilic macrophages of the rodent thymus and in this review our results on morphological, histochemical, enzymehistochemical, immunohistochemical, ultrastructural and functional features of these cells, as well as the molecular regulation of their development, will be presented. Furthermore, the differences between species will also be discussed and the comparisons with similar/related cell types (metallophilic macrophages in the marginal sinus of the spleen, subcapsular sinus of the lymph nodes and germinal centers of secondary lymphoid follicles) will be made. Metallophilic macrophages are strategically positioned in the thymic cortico-medullary zone and are very likely to be involved in: (i) the metabolism, synthesis and production of bioactive lipids, most likely arachidonic acid metabolites, based on their histochemical and enzymehistochemical features, and (ii) the process of negative selection that occurs in the thymus, based on their ultrastructural features and their reactivity after the application of toxic or immunosuppressive/immunomodulatory agents. Taken together, their phenotypic and functional features strongly suggest that metallophilic macrophages play a significant role in the thymic physiology.

As revealed with ED1 and ED2 monoclonal antibodies, macrophages are scattered throughout the thymic tissue. However, in contrast to the cortex and medulla, in the cortico-medullary zone macrophages are large and show strong reactivity with rabbit polyclonal antisera to cyclooxygenase. Only few smaller cortical macrophages also show weaker presence of prostaglandin synthase. After cyclosporin treatment cortical macrophages become strikingly similar to the macrophages of the cortico-medullary zone of the normal thymus. Cortical macrophages become enlarged and develop the strong expression of prostaglandin synthase. Our results show that a specific type of macrophages (with distinct histochemical characteristics, enzyme profile and ultrastructural organization, which is strategically positioned within the thymic tissue - as we demonstrated earlier) possesses the enzyme capacity required for prostaglandin synthesis. After cyclosporin treatment, which interferes with the maturation of thymocytes, cortical macrophages thoroughly change and develop the strong prostaglandin synthase expression, similar to that of normal cortico-medullary zone macrophages.

It is well recognized that tumor necrosis factor receptor-1 (TNFR1) signaling pathway (with lymphotoxin-β receptor) is of critical importance for the development, activation, and clustering of follicular dendritic cells (FDCs) within the lymphoid follicles. However, further information on the molecular control of these processes is very sparse. Here, we show that intravenous application of lipopolysaccharide induces the clear and prominent morphological signs of FDC development and activation in vivo, which is independent of TNFR1 pathway.

Summary The close topographical correlation between naphthol AS-D chloroacetate esterasepositive macrophages and prostaglandin synthase-rich macrophages in the thymus of normal and cyclosporin-treated rats was observed. It seems possible that chloroacetate esterase is an enzyme related to metabolism of arachidonic acid and/or production of its active metabolites.

Small intestine mucosa is often affected with malabsorptive, autoimmune and inflammatory pathological processes. However, morphometric data on the healthy human small intestine mucosa, especially ileum, are scarce. We aimed to obtain histoquantitative data on the healthy jejunal and ileal mucosa and assess the effects of gender and ageing on these parameters. Computer-aided morphometric analysis was performed on 24 jejunal and 25 ileal biopsy samples collected upon routine endoscopy screening of healthy persons with a family history of intestinal malignancy. Subjects were distributed in four groups according to age and sex: adult (60 years) and elderly (60 years) males, and adult (60 years) and elderly (60 years) females. Results were statistically analyzed with Mann-Whitney U test. Jejunal mucosal thickness was significantly reduced in elderly subjects (p0.05), especially in elderly females compared to adult ones (p0.05). Jejunal villi were significantly wider in adult than in the elderly subjects (p0.05), whereas ileal villi were significantly wider in elderly compared to adult subjects (p0.01) and in male compared to female subjects (p0.05). No statistically significant differences were found in other histoquantitative parameters (mucosa epithelium height, crypt numerical density, villous height, crypts and villous perimeter, diameter and epithelium height) of jejunal and ileal mucosa. This study provides complete morphometric data on the healthy human jejunum and the first relevant data on the healthy ileal mucosa, thus representing a valuable morphometric reference for future histoquantitative studies of human small bowel mucosa in both healthy and disease affected individuals.

usc The significance of the autoimmune regulator (Aire) transcription regulator in establishing central tolerance has recently been elucidated in great detail. Still, the role of Aire in medullary thymic epithelial cell (mTEC) physiology is not fully understood. To shed more light on this issue, we studied the ultrastructure of mTECs in Aire‐deficient thymus. We show that all types of mTECs show ultrastructural signs of activation and increased intracellular traffic, which suggests that in the absence of Aire their physiology is impaired. Type 6 ‘large’ mTECs are fully developed in Aire‐deficient mice and more frequent than in the normal thymus. The frequency of type 5 ‘undifferentiated’ mTECs is also increased. Collectively, our results suggest that the role of Aire in the physiology of mTECs could be more profound and not restricted only to the presentation of self‐tissue‐restricted antigens and/or apoptosis of end‐stage fully mature cell types. Refereed/Peer-reviewed

The spleen is composed of several tissue compartments and the respective histoquantitative data are essential for complete understanding of immune or pathological processes in this organ. The aim of our study was to determine and compare the stereologic parameters of all tissue compartments of the gunshot-injured and blunt-injured human spleen. The model-based stereology with point-counting method was utilized to study the volume densities of red pulp, perifollicular zone, marginal zone, white pulp (follicles and periarteriolar lymphoid sheath), and connective tissue. The areal numerical density (the number of follicles per mm(2) of tissue section), the numerical density (the number of follicles per mm(3) of tissue) of lymphoid follicles and the mean follicle diameter were also determined. Our study provides stereological parameters for all tissue compartments of the human spleen. No morphometric differences were registered between tissue compartments of the blunt-injured and gunshot-injured spleen. As the gunshot-injured spleen was taken as presumably unstimulated in immunological regard, our results suggest that both gunshot-injured and blunt-injured organs may be used as models of the normal human spleen.

The recent advances in molecular biology and genetics, as well as the progress of in vitro techniques, have provided a more coherent image of the thymic function on the molecular level. But they have shifted the attention away from studies on the cellular level, which are necessary to clarify the biological roles of different cell types of the thymic microenvironment. The structure and function of the normal thymus depend on mutual interactions between thymocytes and nonlymphocyte cells. In this review a detailed description of morphological and phenotypic features of both maturing thymocytes and nonlymphocyte cells is given. The recent genetic and biochemical data are presented in conjunction with cytological results to enlighten the thymus cell-cell interactions during thymopoiesis and organization of thymic microstructure. Special emphasis is put on the experimental approaches, which may be used to study the interactions between thymocytes and nonlymphocyte cells in vivo.

The recent advances in molecular biology and genetics, as well as the progress of in vitro techniques, have provided a more coherent image of the thymic function on the molecular level. But they have shifted the attention away from studies on the cellular level, which are necessary to clarify the biological roles of different cell types of the thymic microenvironment. The structure and function of the normal thymus depend on mutual interactions between thymocytes and nonlymphocyte cells. In this review a detailed description of morphological and phenotypic features of both maturing thymocytes and nonlymphocyte cells is given. The recent genetic and biochemical data are presented in conjunction with cytological results to enlighten the thymus cell-cell interactions during thymopoiesis and organization of thymic microstructure. Special emphasis is put on the experimental approaches, which may be used to study the interactions between thymocytes and nonlymphocyte cells in vivo.

Splenectomy increases the number of B cells in the blood of humans and animals. It is unknown whether this is due to changes in migration, proliferation, or both. The numbers of naı̈ve (IgD+IgM+), memory (IgD−IgMhigh), newly formed (IgMhighCD90high), early recirculating follicular (IgMlowCD90high), recirculating follicular (IgMlowCD90−), and marginal zone (IgMhighCD90−) phenotype B cells were determined in control and splenectomized rats by flow cytometry. All subsets increased significantly in the blood after splenectomy. Because surface molecules are involved in the regulation of migration and proliferation, their expression (lymphocyte function-associated antigen 1 [LFA-1], intercellular adhesion molecule 1 (ICAM-1), L-selectin, α4-integrins, CD44, major histocompatability complex class II, interleukin 2 receptor-α chain) was determined on B- and T-cell subsets of both groups. B cells, but not T cells, showed a significantly reduced LFA-1 and ICAM-1 expression in blood and lymph nodes, whereas the expression of the other surface molecules analyzed remained unchanged. The down-regulation of these molecules did not influence the adherence of B cells to high endothelial venules in vitro. In vivo, however, ICAM-1low–expressing B cells migrated significantly faster through lymph nodes (ICAM-1low 41 ± 5 hours versus ICAM-1high58 ± 3 hours), whereas proliferation of B cells in bone marrow, lymph node, and blood remained unchanged. Thus, the presence of one organ is necessary for appropriate expression of LFA-1 and ICAM-1 on B cells in other, distant organs. The more rapid transit of ICAM-1low B cells through lymph nodes may be responsible for the increased B-cell number in the blood after splenectomy.

Transmission electron microscopy was used to study the ultrastructural features of thymic epithelial cells of intact and cyclosporin-A-treated adult male Wistar rats. The animals received a peroral daily dose of 30 mg of cyclosporin A per kg body weight for 21 consecutive days. On the basis of ultrastructural features, seven subsets of epithelial cells were distinguished within the intact rat thymus. Four subsets were predominantly situated in the cortex (type 4 - "dark" cells - occasionally penetrated into the medullar region) and three were positioned within the thymic medulla. All subsets of thymic epithelial cells were markedly changed after the application of cyclosporin A. The prominent enlargement of cytokeratin contents was registered in type 1 - "subcapsular/paraseptal/perivascular" - epithelial cells. At the same time these cells acquired the morpho-functional aspect of resting cells. All other subsets of cortical epithelial cells were enlarged (though in different ways) in comparison to the corresponding subsets of the control thymus, whereby their subcellular organization was suggestive of increased synthetic and secretory activity. The decrease in the number of epithelial cells, with the prevalence of phenotypically immature subsets, was evident within the residual islands of thymic medullary tissue.

Publisher Summary This chapter presents a comprehensive study to elucidate the changes of all types of thymic nonlymphoid cells induced by cyclosporin (CS) and the interplay between thymic lymphoid and nonlymphoid cells. For this study, male 6-week-old Wistar rats with an average body weight of 145 g at the beginning of the experiment were used. The changes of all types of thymic epithelial cells were studied after the application of CS by using the immunohistochemical methods with various monoclonal antibodies such as OX-6 (anti-Ia), KII, K8, K8.13 (panepithelial), K19, R-MC 20 (subcapsular/medullary epithelium), and KL1 (subset of medullary epithelial cells). The study found that the application of CS to rats induced prominent changes of all types of thymic nonlymphoid cells. Thymic cortical epithelial network became denser and coarser, and cortical epithelial cells became stocky with coarse cellular prolongations. The subcapsular epithelial cells, although phenotypically dissimilar from cortical epithelium, changed in a very similar manner. Cortical macrophages became enlarged and rounded, but their number did not increase. Phenotypically, they become similar to macrophages of the corticomedullary zone. Considering that after CS treatment the expression of prostaglandin synthase is increased, it is very likely that cortical macrophages become engaged in the production of arachidonic acid metabolites, similarly to macrophages of the corticomedullary zone of the normal thymus.

Summary The structure of the bursa of Fabricius and the immune response to Brucella abortus in Prelux chickens were studied three and six months after neonatal gonadectomy. The different structural changes and immune response were revealed in male and female chickens. In six-months-old gonadectomized males an increased volume density of lymphoid follicles and follicular medulla was found. The follicular number and the follicular diameter were increased. In three- and six-months-old orchidectomized animals, the immune response exceeded that of the nonoperated males of the same age. In ovariectomized chickens, antibody levels were lower than (three months) or equivalent to (six months) to those of nonoperated females. Histoquantitative analysis showed that the volume density of lymhoid follicles and follicular cortex decreased. The follicular number and the mean follicular diameter also decreased. Our results demonstrate that sex steroids have an immunomodulatory role and influence the size and structure of the bursa.

Summary Quantitative stereological study of the bursa of Fabricius in Prelux chickens was performed 3 and 6 months after neonatal gonadectomy. The different structural changes were revealed in bursal follicles, epithelium and stroma of male and female chickens. In orchidectomized animals, the weight of bursa was increased compared to the nonoperated males. Stereological analysis revealed that the absolute amount of bursal follicles was increased due to the increased mass of follicular medulla. The number of areas of follicle-associated epithelium at the bursal mucosa, which in a mature bursa corresponds to the follicle number, was decreased. The height of follicle-associated epithelium (3 months) was decreased and the diameter of the follicle-associated epithelium (6 months) was increased. No changes in the surface density of bursa] epithelium was registered 3 and 6 months after neonatal gonadectomy. In ovariectomized chickens the weight of bursa was decreased in comparison to the nonoperated females. Histoquantitative analysis showed that the bursal follicular mass was decreased due to the decreased absolute amount of follicular medulla (3 and 6 months) and cortex (6 months), whereas the number and diameter of follicles were not changed. The height of interfollicular and follicle-associated epithelium was decreased, but no changes in the surface density of bursal epithelium was registered. Relative proportion of bursal stroma was increased. The studies presented further support of the concept that the functional relation between gonads and bursa of Fabricii exists during the postnatal period in the chicken.

A histoquantitative study of the bursa of Fabricius in 3- and 6-month-old male Prelux Bro chickens (domestic selection) was made. All compartments of normal bursal tissue were examined. At 3 months of age, there were no significant differences in bursal weight and structure between males and females. The only difference observed was the higher volume density of follicular cortex and the lower proportion of follicular medulla in males compared to females. In 6-month-old male and female chickens, a different degree of bursal involution was observed. In the bursa of males, an advanced stage of involution was observed, whereas only the initial signs of bursal involution were found in females.

Naphthol AS D chloroacetate esterase (NASDCE)-positive macrophages are positioned in the cortico-medullary zone (CMZ) of the normal rat thymus. These cells contain the very strongly NASDCE-positive granules of varying size in the cytoplasm. An identical distribution within the thymic parenchyma and an identical morphological appearance is observed in CMZ macrophages after staining with aldehyde fuchsin. The incubation of thymic sections in 10% formalin at pH 7.0 for 48 h does not inhibit the activity of NASDCE in CMZ macrophages. The activity of nonspecific esterase is almost totally abolished by this treatment: only the single, largest globular inclusion within some of the cells remains weakly or moderately positive. The granular content of the CMZ macrophages does not stain metachromatically with toluidine blue and these cells are endogenous peroxidase-negative. NASDCE-positive thymic macrophages are easily distinguished from: a) NASDCE-positive mast-cells, which are confined to the capsular and septal connective tissue and contain densely packed metachromatic granules, and b) NASDCE-positive neutrophilic granulocytes, which have a specific morphological appearance and show very strong endogeneous peroxidatic activity.

Summary A specific type of macrophage, located exclusively in the cortico-medullary zone of the normal rat thymus, contains intracellular material of heterogeneous composition, which at least in part represents partially oxidized unsaturated fats, according to its positive histochemical reactions for ceroid. This globular material is Periodic Acid-Schiff—positive and Sudan Black B—positive. It shows acid-fastness by the long Ziehl-Neelsen method and displays autofluorescence. The histochemical reaction for late lipofuscin is negative. The possible significance of the presented results is discussed.

Secondary lymphoid follicles in peripheral lymphoid organs (parathymic, mesenteric and inguinal lymph nodes and spleen) from young adult Wistar rats of both sexes were studied. Different numbers of tingible body macrophages containing aldehyde fuchsin-positive cytoplasmic granules of varying size, were present in the germinal centers. An identical staining pattern to that obtained with aldehyde fuchsin in terms of the number, distribution and size of positive cells was seen after staining for succinic dehydrogenase.

Young adult Wistar rats received 40 mg/kg of cyclosporin perorally for 21 days. Cyclosporin induced almost total disappearance of thymic medulla, whereas the cortex remained preserved. Although the density of cortical macrophages did not change significantly, their characteristics altered markedly and they became enlarged and rounded. In addition to an increase in acid phosphatase and nonspecific esterase activities, cortical macrophages developed very strong succinic dehydrogenase and chloroacetate esterase activities and a fine, granular, aldehyde fuchsin-positive cytoplasmic content. However, these cytoplasmic granules were PAS-negative and were not sudanophilic. Cortical macrophages retained their normal antigenic properties (which were studied by the use of ED1, ED2 and R-MC 41 monoclonal antibodies). Phagocytic cells in the remaining medullary islands retained their usual characteristics. The changes in cortical macrophages after cyclosporin treatment are discussed, especially in relation to the characteristics of macrophages of the cortico-medullary zone in the normal rat thymus.

The topographical distribution, number and histological properties of metallophilic and PAS-positive cells were investigated during the process of spontaneous regeneration of the thymic tissue after the application of consecutive doses of Cyclophosphamide (5 ± 60 mg/kg). The aim of the study was to elucidate the relationship between these cell types which are both localized in the cortico-medullary zone of the normal rat thymus.

The ultrastructural stereological analysis of prolactin, growth hormone, and gonadotroph cells in the hypophysis of neonatally gonadectomized, 3-month-old male and female chickens was performed. The observed ultrastructural changes (especially of granular endoplasmic reticulum and content of secretory granules) demonstrated reduced biosynthetic activity of prolactin cells in neonatally ovariectomized females and increased activity of prolactin cells in neonatally orchidectomized male chickens.