Lucia Mastrolia

EXPRESSION OF LYMPHOCYTE ANTIGENIC DETERMINANTS IN DEVELOPING GUT-ASSOCIATED LYMPHOID TISSUE OF THE SEA BASS DICENTRARCHUS LABRAX (L.)

Abstract The monoclonal antibodies DLT15 and DLIg3, which recognize antigenic determinants expressed by T cells and Ig-bearing cells, respectively, allowed the development of gut-associated lymphoid tissue of the teleost fish Dicentrarchus labrax (L.) to be studied. DLT15- immunoreactive cells were first detected in the epithelium of the stomach and intestine at day 30 post-hatching of fish maintained at 16° C. At that age, positive cells were found only in the thymus. Between day 44 and day 81 post-hatching, DLT15-immunoreactive cells became numerous, both in and under the gut epithelium. A gradient in the number of lymphocytes was present, concentrating them towards the anus. Until day 81 post-hatching, DLIg3-immunoreactive cells were not found in the gut, although they were present in the kidney, spleen and thymus earlier. Infrequent Ig-bearing cells were found in the gut mucosa of 1-year-old sea bass. This study showed that the gut-associated lymphoid tissue developed earlier than other lymphoid compartments. It also provided evidence of the predominance of T cells in the gut immune system of the sea bass.

Ontogeny of the thymus in a teleost fish, Cyprinus carpio L.: developing thymocytes in the epithelial microenvironment.

A monoclonal antibody, WCL9, specific for membrane molecules of a thymocyte subpopulation was used to detect these cells in situ during the ontogeny of thymus. Cryo-sections revealed WCL9+ cells in the rudiment of the thymus (day 4 post fertilization); thereafter, the positive cells were observed exclusively in the cortex from the first appearance of thymic regionalization (week 4 post fertilization) until adult age. Whole-mount immunostaining of the thymus with WCL9 revealed the three-dimensional structure of the cortex by specific staining. The presence and distribution of apoptotic cells during thymus development was studied by in situ end-labelling of fragmented DNA. From week 4 post fertilization onwards, apoptotic cells were more frequently detected in the cortex than medulla, suggesting a continuous selection of thymocytes in the cortex. Ultrastructural studies confirmed the presence of numerous cortical apoptotic cells inside macrophages. Electron microscopy provided evidence for the existence of epithelial heterogeneity in the thymus. During the ontogeny, the differentiation of epithelial cells was followed from the first weeks until the juvenile age. Cell types were classified on the basis of their localization and cytological characteristics as: i) limiting epithelial cells located in subcapsular, perivascular and peritrabecular zones; ii) reticular epithelial cells situated in medullary and cortical zones; iii) nurse-like cells at the border between the cortex and medulla, iiii) Hassalls body-like structures localized in the medulla. This study could suggest the occurrence of a wide range of lympho-epithelial interactions throughout thymocytes differentiation.

Immunocytochemical detection and cytomorphology of lymphocyte subpopulations in a teleost fish Dicentrarchus labrax

Abstract.The monoclonal antibodies DLT15 and DLIg3 directed against thymocytes and serum immunoglobulins of the sea bass (Dicentrarchus labrax, L.) were used to study cells from the thymus, head kidney, spleen, gut-associated lymphoid tissue and peripheral blood leukocytes of this fish by immunofluorescence and pre-embedding immunoelectron microscopy. Immunofluorescence and flow cytometry of leukocyte fractions revealed a large number of DLT15-positive cells in the thymus (∼80%) and intestine (∼55%) and fewer cells in the spleen (∼7%), head kidney (∼6%) and peripheral blood (∼3%). DLT15-positive cells had two main morphologies, both detectable among thymocytes: a large round heterochromatic nucleus with light sparse cytoplasm (type a) and an irregular and heterocromatic nucleus with cytoplasm rich in polysomes and mitochondria (type b). Type b was most represented in spleen, head kidney, intestine and blood. We suggest that the type b morphology represents more differentiated lymphocytes. Flow cytometry revealed numerous DLIg3-positive cells in the head kidney (∼33%), spleen (∼30%) and peripheral blood leukocytes (∼21%) and fewer positive cells in the intestine (∼3%) and thymus (∼2%). DLIg3-positive cells had the morphology of lymphocytes (with a large round nucleus) or macrophages in all tissues. Plasma cells lacked membrane immunoreactivity. This is the first ultrastructural characterisation of putative T- and B-lymphocyte subpopulations in a fish species; these subpopulations are differentially distributed in teleost lymphoid organs.

Immunoglobulin protein and gene transcripts in ovarian follicles throughout oogenesis in the teleost Dicentrarchus labrax

Transfer of immunoglobulins (IgM-like) from the female to the teleost embryo has been demonstrated but mechanisms of uptake into and storage within the eggs remain to be clarified. The monoclonal antibody DLIg3 against Dicentrarchus labrax Ig light chain revealed an active role of both follicle cells and oocytes in the Ig uptake. The primordial follicular cells showed DLIg3 immunoreactivity even at a pre-vitellogenetic stage. Early vitellogenetic oocytes (lipid vesicle stages) had DLIg3 staining of pore canals, plasmalemma and outer cortex and of their follicular cells. In protein yolk granule oocytes, DLIg3 staining was also detected within vesicles of the outer-mid cortex and juxtanuclear yolk granules; therefore, a centripetal transport of Ig throughout oocyte development is apparently carried out. Immunoelectron microscopy confirmed the presence of Ig within thecal and granulosa cells (and in the interposed basement membrane) of pre-vitellogenic and vitellogenic follicles. Thus, the transport of Ig to the egg apparently occurs also by transcytosis across the follicle cells. Igs were localised in the pore canals surronding the microvilli and in vesicles of outer-mid cortex of vitellogenic oocytes. Reverse transcription/polymerase chain reaction with primers designed for the constant region of sea bass Ig light chain detected Ig mRNA in hydrated oocytes, a smaller content in released eggs and no signal in larvae at day two post-hatching. These findings show that a significant level of Ig gene transcription in the oocyte and/or a transfer of transcripts may also occur.

Cytology of lymphomyeloid head kidney of Antarctic fishes Trematomus bernacchii (Nototheniidae) and Chionodraco hamatus (Channicthyidae).

Species that live in extreme conditions have specially adapted physiology and tissue/organ organisation. The adaptation of lymphoid organs to low temperatures in polar species could be an original field of study, indicating how the immune system works under extreme conditions. In fishes, the head kidney is a key organ for immunity and here the cytology of this organ is studied in two common Antarctic species: Trematomus bernacchii and Chionodraco hamatus. Ultrastructural analysis revealed heterogeneity of epithelial cells, with reticular cells, subcapsular- and perivascular-limiting cells. Differences in the size and morphology of epithelial cells were observed between the polar species and warm water species of fish. Intermingled with epithelial cell leucocytes, such as lymphocytes, thrombocytes and macrophages, had comparable morphology in both species, contrary to sharp differences observed in the morphology of erythrocytes and granulocytes. The functional adaptation of the head kidney to the low temperatures of polar water is discussed.

Majority of TcRβ+ T-lymphocytes located in thymus and midgut of the bony fish, Dicentrarchus labrax (L.)

Real-time polymerase chain reaction (PCR) and in situ hybridization analyses were performed to investigate the occurrence and distribution of T-lymphocytes expressing TcRβ in intestine and lymphoid tissues of the bony fish, Dicentrarchus labrax (sea bass). Immunohistochemistry with the monoclonal antibody DLT15 (pan-T-cell marker) was carried out to compare the cytology, distribution and number of T-cells and TcRβ+ cells in the various sampled lymphoid organs. The highest TcRβ expression was revealed by real-time PCR in the thymus, with high levels also being found in the gut. In the thymus, DLT15+ and TcRβ+ cell populations were concentrated in the cortex and TcRβ+ cells were notably reactive at the cortical-medullary border, suggesting a specialized role of this region in thymocyte selection. The density of DLT15+ T-cells increased from the anterior to posterior intestine, whereas TcRβ+ lymphocytes were more numerous in the middle intestine compared with other segments. The existence, in fish thymus, of a medulla and a cortex comparable with those of mammals is revealed by this study. The concentration of TcRβ+ cells in the sea bass midgut also strongly suggests a special role of this intestinal segment in antigen-specific cellular immunity. The large population of TcRβ-/DLT15+ T-cells in the posterior gut can probably be ascribed to the TcRγδ phenotype fraction.

Histological and cytological studies on the developing thymus of sharpsnout seabream, Diplodus puntazzo

The structure of the developing thymus of the marine teleost, Diplodus puntazzo, was studied by light and transmission electron microscopy. The first anlage of the thymus developed by d 20 postfertilisation (p.f.) as a group of undifferentiated cells dorsal to the epithelium of the branchial chamber. The organ increased significantly in size around d 51–66 p.f. and differentiation of cortex and medulla occurred concomitantly. On the basis of their localisation, 4 main types of epithelial cell were distinguished: (1) limiting, adjacent to the connective capsule; (2) medullary and cortical reticular cells; (3) nurse cells, located in the corticomedullary boundary; (4) Hassall‐like corpuscles. The majority of medium to large blast‐like lymphoid cells were localised in the medulla, while small lymphocytes were housed in the cortical region. These morphological features were maintained at later stages. However, in juveniles in the medulla we observed reticular epithelial cells with cysts and rare Hassall‐like corpuscles. The study was designed to obtain more information concerning the histology of the developing thymus of sharpsnout seabream and give a concise description of the differentiation of epithelial cells and lymphoid cells in the thymic parenchyma.

Histological observations on lymphomyeloid organs of the Antarctic fish Trematomus bernacchii (Teleostei: Nototheniidae)

Abstract Lymphomyeloid organs of the Antarctic fish Trematomus bernacchii were studied with the aim of analysing some morphological aspects related to adaptation to low environmental temperature. The thymus of T. bernacchii was flattened, incompletely lobated and scarcely regionalised. It was filled by lymphoid elements intermingled with stromal elements. The head kidney appeared highly vascularised and mainly lymphopoietic. The spleen appeared mainly erythropoietic, with scarcely developed areas of white pulp.

Lymphomyeloid organs of the Antarctic fish Trematomus nicolai and Chionodraco hamatus (Teleostei: Notothenioidea): a comparative histological study

Abstract Lymphomyeloid organs of two common species of Antarctic fish, Trematomus nicolai and Chionodraco hamatus, were studied with the aim of analysing some morphological aspects of these organs in relation to adaptation to low environmental temperature. The thymuses of T. nicolai and C. hamatus were flattened, incompletely lobated, with numerous Hassall-like bodies, which were mainly located in the central part of the organ in C. hamatus. In T. nicolai, thymocytes, erythroid and reticular epithelial cells filled the organ. In C. hamatus, the thymocytes intermingled with reticular epithelial cells were often close to groups of melano-macrophages. In both species, the thymus did not show distinct compartmentalisation; however, the thymocytes had significantly different sizes in the outer and inner portions of the thymus. The head kidney of both species was completely filled by haematopoietic tissue, highly vascularised and mainly lymphopoietic in T. nicolai, while both erythropoietic and lymphopoietic in C. hamatus. The spleen appeared mainly erythropoietic in T. nicolai and mainly lymphopoietic in C. hamatus. Solitary melano-macrophages in T. nicolai were close to numerous small vascular ellipsoids where erythroid and lymphoid cells were intermingled without the formation of red and white pulp areas. In C. hamatus, large lymphoid areas were organised around the capillaries. The possible adaptation of lymphoid organs to the low temperature of polar water is discussed.

Influence of partial decerebration and hypophyseal allograft on differentiation of thymic epithelial cells in chick embryos: an ultrastructural study

The thymus of 18-day-old normal-chick embryos, partially decerebrated chick embryos, and partially decerebrated embryos bearing hypophyseal allografts were analysed by light and transmission electron microscopy. The hypophyseal influence on the cytological differentiation of epithelial components has been studied. The thymus of partially decerebrated embryos showed a delayed differentiation of some types of epithelial cells and a marked decrease in number of lymphoid cells. Partially decerebrated embryos with hypophyseal implants showed a consistent recovery in the degree of differentiation of epithelial components. These findings indicate the influence of the hypophysis in establishing a correct environment for stromal cell differentiation.