Ultrastructural changes in the thymus of the turtle Mauremys caspica in relation to the seasonal cycle

Summary Changes in the ultrastructure of the thymus of the turtle Mauremys caspica, with special reference to its non-lymphoid components, were studied in relation to the seasonal cycle. The thymic cortex contains framework-forming epithelial-reticular cells and free macrophages, while the medulla includes, in addition, mature and presumptive pro-interdigitating cells. The ultrastructural features of these cells are generally similar to those described for non-lymphoid components of the mammalian thymus. The turtle thymus undergoes cortical involution in spring, with recovery periods in May–June and during autumn. A moderate involution occurs in winter. At the beginning of spring, cortical (but not medullary) epithelial-reticular cells show degenerative changes, probably related to high levels of circulating testosterone. In spring and autumn, mature interdigitating cells are absent, but macrophages, monocytes, and pro-interdigitating cells are found. During May–June, the cortical epithelial-reticular population recovers and macrophages, monocytes, and interdigitating cells are actively phagocytic. In summer, the epithelial-reticular cells in both cortex and medulla display normal ultrastructural features; mature and immature interdigitating cells are absent and some macrophages are detected occasionally. The results suggest that non-lymphoid components of the reptilian thymus can play a role in governing T-lymphocyte differentiation, and that the thymic cortex and medulla exhibit different cycles of seasonal activity.     

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.     

Ultrastructure of splenic white pulp of the turtle,Mauremys caspica

Summary The ultrastructure of splenic tissue of non-immunized turtles, Mauremys caspica, shows two areas, namely, the white pulp which is lymphoid in nature, and the red pulp which is formed by cell cords and sinusoids. Between both areas there is always a marginal zone with gaps through which cells leak. In the white pulp, there are two blood vessel types; one with muscled walls, and the other showing thinner walls sheathed by reticular cells. Reticular cells constitute a network where there occur dendritic macrophages, lymphoblasts and small and medium lymphocytes. Mature plasma cells are scarce in the white pulp.     

In vitro proliferation of blood,spleen and thymus leukocytes from the turtle Mauremys caspica

Cell-mediated immune responses is commonly evaluated by cell proliferation assays. Mitogens are known to induce a vigorous proliferative response in lymphoid cells from mammals but relatively fewer studies have investigated mitogen-mediated lymphoproliferation in non-mammalian animals. In the present work, we incubated spleen, thymus and blood leukocytes with phytohaemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS) and pokeweed mitogen (PWM), by different times of incubation (96 and 120 h) and at different concentrations. Our results show that the optimal mitogen concentrations inducing proliferation on leukocytes from Mauremys caspica were 20 microg/ml PHA, 1 microg/ml Con A, 12.5 microg/ml LPS and 1/150 dilution PWM. The optimal time of incubation was dependent on the type of leukocytes (peripheral blood leukocytes, splenic leukocytes or thymic cells) and the mitogen utilized.     

Distribution and ultrastructure of S-100-immunoreactive cells in the human thymus

Summary The present study deals with the localization and ultrastructure of S-100-immunoreactive cells in the human thymus. These immunoreactive cells are distributed mainly in the medulla with some scattered elements in the cortex. Electron-microscopic observation revealed that the cells are characterized by an irregularly shaped nucleus, tubulovesicular structures in the cytoplasm and characteristic interdigitations of the plasma membrane. The cells often embrace lymphocytes with their branched processes. On the basis of these morphological features, the immunostained elements were identified as interdigitating cells (IDCs). The immunocytochemistry for S-100 visualizes the precise distribution and extension of the IDCs under the light microscope and indicates that the IDCs form no structural networks such as those established by the thymic epithelial cells. Since the IDCs in human lymph nodes have also been reported to contain S-100-like immunoreactivity, S-100 protein can be regarded as a useful marker for identifying the IDCs in the human thymus and other lymphoid organs.     

An immunocytochemical survey of endocrine cells in the gastrointestinal tract of chicks at hatching

Summary The ultrastructure of the micro-environment of the fully functional rat thymus was studied. The thymus consists of two discrete compartments, viz., an epithelial and a mesenchymal compartment. Thymus fibroblasts/fibrocytes, mast cells and granulocytes, are restricted to the mesenchymal compartment. The thymocyte maturation process seems to occur in the epithelial compartment in a network of reticular epithelial cells. The cortex is finely meshed and filled with proliferating thymocytes and some scattered macrophages. Moreover, in the medulla vacuolated epithelial cells form part of a loosely meshed reticulum which is filled with thymocytes and interdigitating cells (IDCs). IDCs frequently contain Birbeck granules and appear to be phagocytic. Together with macrophages, they probably enter the thymus, predominantly in the cortico-medullary region, and cross the separating wall between the two compartments. Some functional aspects of the non-lymphoid cells and in particular the IDCs, which form the micro-environment of the thymus, are discussed with respect to T-cell development.     

The immune response of thymic cells from the turtle Mauremys caspica

In the present work several known mammalian leukocyte functions such as directed mobility, proliferative response to mitogens, antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK)-mediated cytotoxicity were studied in female and male Mauremys caspica turtles. Since the reptilian thymus shows seasonal variations in its structure, we have performed all the assays along the seasonal cycle. Our results show that thymic cells from M. caspica are able to migrate through a chemo-attractant gradient, to proliferate in response to the mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A), and to kill tumoral target cells by both ADCC-mediated and NK-mediated cytotoxicity. Those functions were differentially affected by the seasonal cycle; in general, in autumn the functions studied showed the smallest values for both sexes, while in summer the highest values of cytotoxicity and chemotaxis were found in females. The proliferative responses to PHA and Con A were higher for both sexes in spring and for females in winter than in the other seasons. In summary, thymic cells from M. caspica show a wide range of immune functions, and these are modulated heterogeneously by the seasonal cycle in both sexes.     

Interdigitating reticulum cells in the popliteal lymph node of the rat

Summary Electronmicroscopic and cytochemical studies were performed to localize interdigitating reticulum cells (IDC) in the popliteal lymph node of the rat.The morphological features of the IDC of the rat correspond to those described for other species, but also show similarities to normal macrophages in the rat. This is considered to be an argument in favour of the common origin of IDC's and macrophages.Ultrahistochemical studies with horseradish peroxidase (HRP) reveal no phagocytotic capacity of IDC's. After perfusion fixation containing ruthenium red (RR) the surface coat stains heavily: RR is also found deep in the membrane invaginations of the IDC, indicating the presence of polyanionic sialoglyco-proteins. The post-capillary-venules (PVC) are very permeable to both HRP and RR.The phosphotungstic acid-chromic acid stain (PTA-CrA) also reveals glycoproteins in the surface coat; these glycoproteins are susceptible to -neuraminidase, whereas glycoproteins in the Golgi complexes, lysosomes and in the vesicular complexes of IDC are not. The glycoproteins of the latter are susceptible to 0.1 N NaOH. These findings indicate that IDC produce different kinds of glycoprotein, one of which may be secreted and act as a factor for stimulating peripheral T-lymphocytes.Intimate contact between IDC's and PCV's could be observed. It is therefore conceivable that IDC's play an important role in the homing of T-lymphocytes.     

The axolotl thymus: cell types of the microenvironment

Summary The three-dimensional structure of the axolotl (urodele amphibian) thymus was studied by combined scanning and transmission electron microscopy. The epithelial cell is the major component of the microenvironment forming the meshwork where thymocytes differentiate. Three different types of epithelial cells could be defined by their intracytoplasmic organelles and their localization in the subcapsular or deeper part of the organ. These epithelial cells participate in various types of lymphostromal interactions. Other stromal elements, such as interdigitating reticular cells, macrophages, eosinophil granulocytes and epithelial cysts were also defined. The absence of a true cortico-medullary differentiation in the axolotl thymus, the presence of different stromal elements and the physiological significance of these various microenvironments are discussed.     

Interdigitating cells in the lymphoid tissues of bovine fetuses and calves

Summary Electron-microscopic studies of lymphoid tissues from bovine fetuses and from calves disclosed a non-lymphoid cell type in the thymus-dependent zones of secondary lymphoid tissues and in the thymus that is distinguishable from reticulum cells, epithelial and endothelial cells, and macrophages. Based on morphological and topographical criteria, the cell is identified as the interdigitating cell. In addition, studies of the tissues of normal and virus-challenged fetuses, and of conventionally reared calves, indicated that the interdigitating cells originate from monocytoid cells, which undergo differentiation in the thymus-dependent zones during an immune response.     

Differentiation of epithelial cells in the mouse thymus

Summary The foetal and post-natal development of the mouse thymus was studied with the electron microscope paying particular attention to the differentiation of the epithelial cells. At about 13 days' gestation, the thymus was composed principally of undifferentiated epithelial cells and some lymphoblasts. The latter accumulated rapidly but did not show much evidence of mitotic activity until after the development of differentiated cortical epithelial cells which appeared during the 15th day of gestation. Further differentiation of epithelial cells did not occur until near term when medullary cystic epithelial cells appeared, and post-natally when small Hassall's corpuscles were developed. Undifferentiated and dividing epithelial cells were seen in the medulla and were present in all postnatal animals examined.This is publication number 1400 from the Walter & Eliza Hall Institute of Medical Research.The author is grateful to Prof. G. J. V. Nossal, Dr. J. F. A. P. Miller and Dr. P. J. Russell for their interest and assistance with various aspects of this study. Special thanks are due to Miss Mary Bravington for her skilled technical assistance. This investigation was supported by grants from the Jane Coffin Childs Memorial Fund for Medical Research and the National Health and Medical Research Council of Australia. The Electron Microscope Laboratory was equipped and supported by grants from the Australian Research Grants Committee, J. B. Were and Sons and the Potter Foundation.     

Heterogeneity of epithelial cells in the human thymus

Summary To evaluate interrelationships among epithelial cells, and between morphology and function in the microenvironment, we studied the ultrastructural morphology of epithelial cells in sections of human thymus from donors aged 2 months to 31 years. Six types of epithelial cells were observed: subcapsular-perivascular (type 1); pale (type 2); intermediate (type 3); dark (type 4); undifferentiated (type 5); and large-medullary (type 6). Cells of types 2, 3 and 4 were found throughout the organ. The type-2 to -4 epithelial cells may represent various stages in a differentiation process. In this, type-2 cells are very active and type-4 cells are possibly degenerating elements. Type-4 cells can also contribute to Hassall's corpuscles. Type-5 cells were located mainly in the cortico-medullary region and showed the morphological characteristics of undifferentiated elements. Type-6 cells were located exclusively in the medulla and displayed characteristics of cellular activity. Small Hassall's corpuscles consisted of type-6 epithelial cells; in larger corpuscles many nuclei of type-6 cells were found. Cells of types 2 and 6 contained tubular structures (diameter approximately 20 nm).Concerning the function of thymus epithelial cells, the features associated with protein synthesis observed in cellular types 2 and 6 make them likely candidates for humoral factor-producing and/or secreting elements. In addition, type-2 and -3 cells in the cortex appear to contribute to a special pattern of epithelium-lymphocyte interaction (thymic nurse cells), as demonstrated by the intracytoplasmic location of lymphocytes in the epithelial cells. The various steps in intrathymic T-cell maturation occur at locations in a microenvironment composed of morphologically distinct epithelial cells.     

Ventilatory responses to temperature variation in the fresh water turtle,Mauremys caspica leprosa

A study of lung gas exchange in the fresh water turtle Mauremys caspica leprosa at normal physiological body temperatures (15, 25 and 35 °C) was extended to extreme temperatures (5 and 40 °C) to determine whether the direct relationship between body temperature and ventilatory response found in many lung-breathing ectotherms including other chelonian species was maintained. From 5 to 35 °C the lung ventilation per unit of O₂ uptake and CO₂ removed declined with temperature. Consequently, lung CO₂ partial pressure increased with temperature. Its value was maintained within narrow limits at each thermal constant, suggesting a suitable control throughout the complete ventilatory cycle. At 40 °C the ventilatory response showed the opposite trend. The ratios of ventilation to lung gas exchange increased compared to their values at 35 °C. The impact of this increased breathing-lowering the estimated mean alveolar CO₂ partial pressure-was nevertheless less than expected due to an increase in calculated physiological dead space. This suggests that the relative hyperventilation in response to hyperthermia found in Mauremys caspica leprosa is related to evaporative heat loss.Abbreviations BTPS body temperature, ambient pressure, saturated with water vapour - CTM critical thermal maximum - F_N2 fractional concentration of nitrogen - PA _CO2or PL _CO2 alveolar or lung CO₂ pressure - PA_O2or PL_O2 alveolar or lung O₂ pressure - PI_O2 inspired O₂ pressure - R respiratory exchange ratio - STPD standard temperature, standard pressure, dry - T _a ambient temperature - T _b body temperature - VA alveolar ventilation - VA/VCO₂ relative alveolar ventilation (alveolar ventilation per unit of CO₂ removed) - VO₂ O₂ uptake - VCO₂ CO₂ output - V _D anatomical dead space volume - V _D physiological dead space volume - VE/VO₂ ventilatory equivalent for O₂ - VE pulmonary ventilation or expiratory minute volume - VE/VCO₂ ventilatory equivalent for CO₂ - V _T tidal volume     

Seasonal variations in the activity in vitro of peripheral blood granulocytes in the turtle Mauremys caspica

• 1.1. In the present work we have studied different aspects of the phagocytic process in peripheral blood granulocytes from the turtle Mauremys caspica: (a) tissue adherence capacity, (b) spontaneous mobility and chemotaxis, (c) attachment and ingestion of foreign cells (Candida albicans) or inert particles (latex beads), and (d) capacity to digest ingested material measured by nitroblue tetrazolium (NBT) reduction. These studies were carried out in vitro at 37°C in autumn, winter, spring and summer.
• 2.2. The adherence index showed significantly higher values in autumn, and smaller values in winter. The spontaneous mobility was not affected seasonally, but the chemotaxis was significantly increased in winter and decreased in autumn. The numbers of C. albicans attached and ingested as well as latex beads ingested per 100 granulocytes were higher in autumn and summer, and lower in winter and spring. The digestion capacity was greatly decreased in summer.
• 3.3. The comparison between the results obtained here and those found using the same techniques in blood human granulocytes showed that these animals are endowed with an adequate phagocytic response.

     

Phagocytosis of erythrocytes in the subarachnoid space at spinal nerve exits

Summary Interdigitating cells (IDC) in the thymus of the spotless starling, Sturnus unicolor, were examined by electron microscopy. They occur principally in the thymic medulla and corticomedullary border. They possess an irregular nucleus and a perinuclear area of cytoplasm, containing most of the membranous organelles, surrounded by a peripheral electron-lucent zone. Clusters of smooth Golgi vesicles and complicated labyrinthine membrane-membrane contacts are the most characteristic cytological features. Birbeck granules are absent. Lymphocytes, plasma cells and even myoid cells can be found embedded in the cytoplasm. Immature elements, intermediate between epithelial-reticular cells and interdigitating cells, are tentatively identified as prointerdigitating cells. The functional significance of IDCs, and their phylogenetic significance in the vertebrate immune system, is discussed.     

Destruction of Hassall's corpuscles by macrophages in the sheep thymus

Summary The dissolution of Hassall's corpuscles by macrophages has been demonstrated in the sheep thymus. The findings indicate that enlarged Hassall's corpuscles are rapidly broken down by macrophages at the end of gestation or immediately after birth and replaced by newly formed corpuscles, and that these cyclic changes in Hassall's corpuscles persist, under normal physiological conditions, throughout life.     

The effect of the seasonal cycle on the splenic leukocyte functions in the turtle Mauremys caspica

The reptile immune system is strikingly affected by seasonal variations, which induce changes in the structure of the lymphoid organs and in the function of the leukocytes. The aim of this work is to study several functions of splenic leukocytes from the turtle Mauremys caspica along its seasonal cycle. The functions assayed were adherence to substrate, mobility directed to a chemoattractant gradient (chemotaxis), lymphoproliferative response to mitogens, antibody-dependent cellular cytotoxicity, and natural killer-like cell-mediated cytotoxicity. Splenic leukocytes showed a positive response in all the assays, and this response was similar to that of mammals. In regard to the effect of the seasonal cycle, we have observed in winter a low adherence to substratum and high chemotaxis and cytotoxic activity, whereas in spring, only lymphoproliferation induced by mitogens showed high values except with lipopolysaccharide, which did not induce any seasonal variation in proliferation percentages. In summer, a high chemotaxis and cytotoxicity were observed, while in autumn, adherence to substratum was increased, but chemotaxis, cytotoxicity, and proliferation were clearly diminished. Our results demonstrate that splenic leukocyte functions are affected by the seasonal cycle, which induces a different pattern of response depending on the function studied.     

Anomalous occurrence of immunoreactive calcitonin cells in the thymus of the rat

Summary In a study of the effect of pinealectomy on thyroid C-cell number, 8 animals out of 66 were found to have thymic tissue in close association with the thyroid. Cells containing immunoreactive calcitonin were found in all of the thyroids but in only one of the 8 pieces of thymus. These cells found in a piece of thymic tissue associated with the right thyroid lobe were located immediately under the capsule and did not form or associate with follicles. Unlike the other animals the rat with thymic calcitonin cells had an unequal distribution of C-cells between the left and right thyroid lobes, but the total number of thyroidal C-cells was the same as that of the other rats. Since the thymus proper was not examined in these 66 animals, ten additional rats were taken for such a study. Thyroid-associated thymic tissue was found in three of these, but none of these thymi showed any immunoreactive cells.Financial support by the Deutsche Forschungsgemeinschaft (grant Vol 35/7) is gratefully acknowledged     

A scanning electron-microscopic study of the rat thymus with special reference to cell types and migration of lymphocytes into the general circulation

Summary The three-dimensional structure of the rat thymus was studied by combined scanning and transmission electron microscopy. The thymus consists mainly of four types of cells: epithelial cells, lymphocytes, macrophages, and interdigitating cells (IDCs).The epithelial cells form a meshwork in the thymus parenchyma. Cortical epithelial cells are stellate in shape, while the medullary cells comprise two types: stellate and large vacuolated elements. A continuous single layer of epithelial cells separates the parenchyma from connective tissue formations of the capsule, septa and vessels. Surrounding the blood vessels, this epithelial sheath is continuous in the cortex, while it is partly interrupted in the medulla, suggesting that the blood-thymus barrier might function more completely in the cortex.Cortical lymphocytes are round and vary in size, whereas medullary lymphocytes are mainly small, although they vary considerably in surface morphology.Two types of large wandering cells, macrophages and IDCs, could be distinguished, as well as intermediate forms. IDCs sometimes embraced or contacted lymphocytes, suggesting their role in the differentiation of the latter cells.Perivascular channels were present around venules and some arterioles in the cortico-medullary region and in the medulla. A few lymphatic vessels were present in extended perivascular spaces.The present study suggests the possible existence of two routes of passage of lymphocytes into the general circulation. One is via the lymphatics, while the other is through the postcapillary venules into the blood circulation. Our SEM images give evidence that lymphocytes use an intracellular route, i.e., the endothelium of venules.