In vivo allograft rejection in a bony fish Dicentrarchus labrax (L.): characterisation of effector lymphocytes.

Lymphoid cell subpopulations involved in allograft rejection in the teleost Dicentrarchus labrax were characterised at the ultrastructural level and quantified by using monoclonal antibodies against T- and B-lymphocytes. T-cells positive for T-cell receptor beta-chain (TcR beta) were detected by reverse transcription/polymerase chain reaction (RT-PCR) and in situ hybridisation by using RNA probes for TcR beta. Flow cytometry detected a similar percentage of T- and B-lymphocytes (around 17%) in the leucocyte-enriched fraction from allografts. Two different types of T-lymphocytes (DLT 15-immunoreactive) infiltrating the allografts were identified by cytomorphology: small cells with high nuclear/cytoplasmic ratio and cells with a higher cytoplasmic content. RT-PCR revealed a single band (513 bp) corresponding to the TcR beta. In situ hybridisation showed that TcR beta-positive cells in the grafted muscle fibres were less numerous compared with DLT 15-positive cells, as evidenced in parallel sections, suggesting that cytotoxic cells might express different TcR phenotypes. DLIg 3-immunoreactive Ig-producing lymphocytes had: 1) a high nuclear/cytoplasmic ratio or 2) a larger size similar to that of pre-plasma cells (plasma cells lacked any membrane labelling).

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.

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) Hassall's body-like structures localized in the medulla. This study could suggest the occurrence of a wide range of lympho-epithelial interactions throughout thymocytes differentiation.

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.

Immunodetection of lymphocyte subpopulations involved in allograft rejection in a teleost, Dicentrarchus labrax (L.).

Monoclonal antibodies which recognize antigenic determinants expressed by T-cells and Ig-bearing cells, respectively, allowed lymphocyte subpopulations involved in allograft rejection of muscle transplants to be identified in the teleost fish Dicentrarchus labrax (L.). The monoclonal antibody DLT15 first allowed recognizing T-cells involved in an in vivo antigen-driven cellular response in teleosts. Immunohistochemical studies showed a high density of lymphocytes in allografts and provided evidence of predominance of T-cells. The heterogeneity of the cell populations recognized by the antibodies was evidenced by the different size, cytology, and staining patterns of T-cells and Ig-bearing cells.

Distribution of macrophages during fish development: an immunohistochemical study in carp (Cyprinus carpio, L.).

A monoclonal antibody against carp macrophages (WCL15) has been utilised in flow cytometry, immuno-histochemistry and immuno-electron microscopy to assess the distribution of monocytes/macrophages in developing carp lymphoid tissues. In suspensions of living cells WCL15 reacted strongly with cytoplasm and plasmic membrane of macrophages. It also cross-reacted with a subpopulation of thrombocytes, but this reaction could be neglected by double immunostaining in combination with a thrombocyte-specific marker. In Bouin-fixed tissues the antibody distinctly recognised macrophages. Macrophages were found from day 2 post-fertilisation in head kidney and in the dorsal portion of the yolk sac epithelium. From 1 week onwards macrophages were found scattered in thymus and gut and during the second week in spleen. Macrophages increased in number in all lymphoid tissues until the 6-8th week post-fertilisation, but they decreased except in thymus, where they became localised mainly in the cortical-medullary boundary, and in white pulp areas of head kidney. The role of macrophages in allowing an early non-specific defence in young fish and in co-operating during the differentiation processes of T-cells and B-cells is discussed.

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

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 (approximately 80%) and intestine (approximately 55%) and fewer cells in the spleen (approximately 7%), head kidney (approximately 6%) and peripheral blood (approximately 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 (approximately 33%), spleen (approximately 30%) and peripheral blood leukocytes (approximately 21%) and fewer positive cells in the intestine (approximately 3%) and thymus (approximately 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.

Expression of lymphocyte antigenic determinants in developing gut-associated lymphoid tissue of the sea bass Dicentrarchus labrax (L.).

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 degrees 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 -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.

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.

Immunohistochemical and ultrastructural evidence of adrenal chromaffin cell subtypes in sea bass Dicentrarchus labrax (L.).

Histology demonstrated chromaffin cells in the head kidney and opisthonephros of the sea bass. Immunohistochemistry showed the catecholamine-synthesizing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase in these cells. Phenlethanolamine N-methyltransferase was detected in a fraction of the chromaffin cells in the head kidney (59%) and in the opisthonephros (54%). Distinct noradrenaline- and adrenaline-synthesizing cells are therefore suggested. Ultrastructural analysis confirmed the existence of two main chromaffin cell types, distinguished by different types of the secretory granules. Cells that contain vesicles with a round, electron-dense core were interpreted as noradrenaline cells, while cells with electron-lucent vesicles were identified as adrenaline cells. The ultrastructure of these chromaffin cell subtypes does not differ in the head kidney and opisthonephros. A minor population of chromaffin cells was identified, which typically show smaller vesicles with an electron-dense core. This population may account for a limited number of not fully differentiated chromaffin cells.

Ultrastructural study on the plical epithelium of the bursa of Fabricius in chick embryos: influence of partial decerebration and hypophyseal allografts.

The bursa of Fabricius of 18 day normal and partially decerebrated chick embryos, and partially decerebrated embryos bearing a hypophyseal allograft was analysed by scanning and transmission electron microscopy, focusing on the ultrastructural characterisation of the plical epithelium. The plicae of the normal bursa consist of interfollicular (IFE) and follicle associated epithelium (FAE). The FAE is composed of typical polygonal cells and is supported by a layer of epithelial cells which appears as a continuation of the corticomedullary epithelium. Bordering cells lie between the FAE and IFE. The IFE is composed of 4 cell types: (1) undifferentiated, (2) goblet, at various stages of maturity, (3) prismatic, and (4) globular light cells. Partially decerebrated embryos showed a gross impairment of plical epithelium development and the complex of FAE and IFE cells was largely undifferentiated. Partially decerebrated embryos with a hypophyseal allograft displayed the same cellular types as observed in controls, thus indicating a restored differentiation of plical epithelium. These findings suggest that the hypophysis affects the differentiation of plical epithelium during ontogenesis.

Cytological and biochemical studies on chromaffin cells in the head kidney of Gasterosteus aculeatus (Teleostei, Gasterosteidae).

Adrenal chromaffin cells in the head kidney of the stickleback Gasterosteus aculeatus were examined by ultrastructural analysis and determination, by HPLC, of the catecholamine content (adrenaline, noradrenaline, and dopamine). Chromaffin cells are characterized by the presence of membrane-bound vesicles and two cell types were identified: (i) those with vesicles containing a strongly electron-dense core; and (ii) those with vesicles that are completely electron lucent or containing smaller and less-dense granules. HPLC analysis revealed the presence of adrenaline and noradrenaline in both right and left head kidneys. Dopamine was not detected. Cytological and biochemical data suggest that the cells with electron-dense granules contain noradrenaline and those with electron-lucent vesicles contain adrenaline.

Fine localization of the acetylcholinesterase activity in somatic and germ cells during the morphogenesis of chick ovarian cortex.

The ultracytochemical localization of the enzyme acetylcholinesterase (AChE) was studied in the germinal epithelium and cortex during chick ovarian morphogenesis at the 7, 12.5, 14 and 19 day of incubation. The results evidenced at day 7 the presence of the enzyme in some somatic cells, both "dark" and "light" and in some gonocytes. The reaction appears also in some tracts between these types of cells. At day 12.5 the reaction is present in some somatic cells only towards the deepest zone of the cortex, in many oogonia and oocytes and in some tracts between germ and somatic cells. At day 14 and day 19 the various cell categories of the cortex are negative for the reaction. The significance of the presence of the enzyme is discussed in relation to an embryonic cholinergic system active during morphogenesis. Regarding the presence of the enzyme in germ cells, the positivity in endoplasmic reticulum cisternae associated to mitochondria may suggest an implication of the enzymatic activity in the proliferation and transformation of these organelles.

Acetylcholinesterase activity in the adrenal chromaffin vesicles of Urodela.

The presence of acetylcholinesterase (AChE) activity in the adrenal chromaffin cells of Necturus maculosus and Ambystoma maculatum (Amphibia, Urodela) has been demonstrated by cytochemical method at the electron microscope level. The enzymatic activity is localized in RER and perinuclear cisternae, on the plasma membrane and within the chromaffin vesicles, both in adrenaline (A) and noradrenaline (N) cells. Moreover N cells appear to be more reactive than A cells and Necturus more reactive than Ambystoma. The possible function of the AChE activity inside the vesicles is discussed as a mechanism of protons donor or as peptidasic activity acting on various peptides present in the vesicle.

Comparative data on acetylcholinesterase cytochemistry in various chromaffin cell types of Discoglossus pictus (Amphibia, Anura).

The cytochemical localization of acetylcholinesterase (AChE) activity was studied in the adrenal chromaffin cells of Discoglossus pictus. Reaction end-products were associated with all types of chromaffin cells, i.e. adrenaline (A), noradrenaline (N) and small granule chromaffin (SGC-A, SGC-N) cells, and nervous elements present in the gland. The SGC-A and SGC-N showed the same intensity of AChE reaction in A and N cells, respectively. On the whole, the A and SGC-A cells were more reactive than the N and SGC-N cells. The functional role of the SGC cells is discussed on the basis of the cytochemical results.

Acetylcholinesterase and pseudocholinesterases in developing chick adrenal.

The distribution of acetylcholinesterase (AChE) and pseudocholinesterases (PsChE) in chick adrenal gland during the first phases of organogenesis was studied. Acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates for the two enzymes, respectively, whereas BW284c51 (1,5 bis (4-allyldimethylammonium-phenyl)pentan-3-one-dibromide) and ISO-OMPA (tetraisopropylpyrophosphoramide) were used as respective inhibitors of AchE and PsChE. AchE was present on the plasma membrane, in the perinuclear cisterna and in some cisternae of the rough endoplasmic reticulum of both interrenal and chromaffin cells; moreover enzymatic activity was found in the same sites of ganglion cells and mesenchymatic undifferentiated cells, i.e. on the inside and in the proximity of the glandular anlage. PsChE activity was localized in the perinuclear space and in the rough endoplasmic reticulum of all types of cells in the anlage. It is suggested that these enzymatic activities may be implicated in morphogenetic mechanisms.

Ultrastructural localization of acid phosphatase in germ cells of chick embryo left ovary.

The ultracytochemical localization of acid phosphatase was studied in oogonia and oocytes of the chick embryo left ovary. The reaction products are evident in lysosomes of various types and, in some cells, in the GERL as well. Furthermore, from the onset of the meiotic prophase, the enzymatic reaction also appears in the rough endoplasmic reticulum. Non-incubated sections of the same stages were observed, with the aim of identifying and describing the structure of the organelles, in particular lysosomes which appeared positive in incubated sections. The significance of the presence of the enzyme is discussed.

The adrenal chromaffin cells of Salmo gairdneri Richardson (Teleostei, Salmonidae).

The chromaffin cells of the adrenal homologue of Salmo gairdneri R. have been studied by light and electron microscopy. The chromaffin tissue was localised in the head kidney adjacent to the wall of the proximal part of the cardinal veins and their main branches. Specific histochemical techniques failed to demonstrate different types of chromaffin cells. With the electron microscope two different types of chromaffin cells were observed. The first type, characterised by numerous dense cytoplasmic granules of average diameter 90 nm, was interpreted as a noradrenalin cell; the second type was characterised by the presence of moderately electron-dense granules of average diameter of 85 nm, and was interpreted as an adrenalin cell. The reaction for acetylcholinesterase activity was present on nerve terminals and sometimes in the vacuolar membrane systems of both chromaffin cells.