Fish cell cultures as in vitro models of inflammatory responses elicited by immunostimulants. Expression of regulatory genes of the innate immune response.

We report the differential expression of various genes related to the regulation of the innate immune responses, including pro-inflammatory (IL-1ß1, IL-8, TNF-α1, TNF-α2) and immune-suppressing (IL-10) cytokines, interferon-induced Mx-1 protein, enzymes regulating nitric oxide (inducible nitric oxide synthase, arginase-2) and eicosanoid (COX-2) production, and Toll-like pathogen pattern-recognition receptors TLR-3, TLR-5 and TLR-9, in two lympho-haematopoietic stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss), as well as in primary cultures of rainbow trout head kidney macrophages, after their exposure to the well-known immunostimulants LPS, levamisole and poly I:C. Although there were differences in the responses between the two stromal cell lines, using reverse transcription followed by real time polymerase chain reaction (RT-qPCR) we demonstrated that exposure to the immunostimulants, particularly poly I:C and LPS, resulted in significant changes in the expression of the immunoregulatory genes in the two stromal cell lines in many cases their responses resembling in fold change magnitudes and in response profiles to those observed in the primary macrophage cultures. Exposure to poly I:C and, with lower fold change values, to LPS produced upregulation of the pro- (IL-1ß, IL-8, TNF-α) and anti-inflammatory (IL-10) cytokine genes, as well as of the Mx-1 gene. Furthermore, the immunostimulation elicited the upregulation of COX-2, iNOS and arginase-2 genes in the cell lines. Likewise, the TSS and TPS-2 cell lines significantly upregulated the expression of TLR-3, TLR-5 and TLR-9 genes after exposure to the immunostimulants, thus explaining the ability of the stromal cells to recognise and respond to the immunostimulants. Such results give support to an important role of lympho-haematopoietic stromal cells in the development and control of pro-inflammatory responses in fish. The upregulation of genes of pro-inflammatory cytokines and of mediators of the innate immune responses correlates well with the previously demonstrated functional capacities, including phagocytosis, microbicidal activity and NO production, exhibited by the TSS and TPS-2 stromal cell lines when exposed to the same immunostimulants. On the other hand, the expression of immunosuppressing genes (IL-10, COX-2 and arginase-2) demonstrate that the lympho-haematopoietic stromal cells are also able to contribute to the control of inflammatory responses. This study reinforce the possibility of using histotypic cell cultures, as those formed by the TSS and TPS-2 cell lines, formed by heterogeneous cell populations that partially replicates the cell-cell and cell-extracellular matrix interactions, to develop cost-effective and repetitive in vitro systems for the screening of immunostimulant candidates for aquaculture, as they are able to replicate in vitro immune regulatory networks occurring in vivo.

Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants.

We have tested the elicitation of innate defence-related responses in two stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss) after they were exposed to different concentrations of lipopolysaccharide (LPS), levamisole, or polyinosinic polycytidylic acid (poly-I:C). For comparison, cultures of rainbow trout head kidney macrophages were also included in the study, and the effect of the immunostimulants on the phagocytic activity, the intracellular and extracellular reactive oxygen species and nitric oxide production were assayed. Although the responses varied depending upon the concentration of the immunostimulants and the particular cell line, our results demonstrate that those activities were enhanced in the TSS and TPS-2 cell lines after exposure to any of the immunostimulants. These results indicate that the stromal cells of the main lympho-haemopoietic organs of O. mykiss develop innate defence responses, which are enhanced by well-known immunostimulants. In addition, such enhancement of the defence responses in the TSS and TPS-2 cell lines could be also elicited when they were exposed to conditioned supernatants from levamisole- or poly I:C-stimulated HK macrophage cultures, thus demonstrating that the haemopoietic stromal cells respond to macrophage-derived factors. Moreover, we demonstrate that the stromal cell lines constitutively expressed the Toll-like receptors TLR3, TLR5 and TLR9 genes. The results are discussed considering the role of the lympho-haemopoietic stromal cells in the innate immune responses, and the possibility of using histiotypic cell cultures of non-leucocyte cells of the haemopoietic organs to develop in vitro methods to select new immunostimulant candidates for aquaculture.

Modulation of the immune response to an Aeromonas hydrophila aroA live vaccine in rainbow trout: effect of culture media on the humoral immune response and complement consumption.

The Aeromonas hydrophila aroA is an attenuated strain that has been assessed as a live vaccine in rainbow trout, Oncorhynchus mykiss. In this study the effects of different culture media used to grow the strain on its survival after in vitro exposure to rainbow trout serum, and on its immunogenicity in rainbow trout were compared. Four culture media were tested: Luria broth (LB), Luria broth with 0.25% glucose, trypticase soy broth (TSB), and brain-heart infusion broth (BHIB). Bacteria grown in culture media with glucose (TSB, BHIB and LB with 0.25% glucose) showed reduced complement consumption and a lower serum susceptibility. O. mykiss vaccinated with inocula prepared with BHIB- and LB-grown aroA cells resuspended in phosphate-buffered saline (PBS) showed higher and longer-lasting serum agglutinating antibody titres than those vaccinated with TSB-grown bacteria. Thus, a direct relationship between serum resistance and immunogenicity could not be established, but BHIB and LB culture media were the most effective in increasing the immunogenicity of the A. hydrophila aroA vaccine.

Correlation between production of acyl homoserine lactones and proteases in an Aeromonas hydrophila aroA live vaccine.

Aeromonas hydrophila is a pathogen that causes disease in a wide range of homeothermic and poikilothermic hosts due to its multifactorial virulence. We have previously described the characterisation and use of an auxotrophic aroA mutant of the A. hydrophila AG2 strain as a live attenuated vaccine against A. hydrophila infections in rainbow trout (Oncorhynchus mykiss). In this study we report the expression of extracellular proteolytic activities and of quorum-sensing molecules by this mutant grown under different culture conditions, and in vaccine inocula. The aroA strain expresses extracellular proteases efficiently during in vitro growth and this ability is retained in vaccine inocula that were prepared by washing the bacterial cultures and resuspending the cells in phosphate-buffered saline. Since proteases are considered to be major bacterial antigens, the expression of these enzymes in the live attenuated vaccine may contribute to the superior protection afforded by these kind of vaccines. On the other hand, the production of serine- and metalloprotease activities in A. hydrophila has been described as controlled in a cell density-dependent fashion, through a mechanism known as quorum sensing. A microtiter method was developed that allowed correlation of the production of quorum-sensing molecules and of proteases produced by the aroA strain during in vitro growth and in the vaccine inocula. The production of both products was related to the type of culture medium and conditions used to grow the aroA mutant, whereas there was no correlation between the concentration of acyl homoserine lactones and protease production.

Spontaneous in vitro angiogenesis in a trout pronephric stromal cell line (TPS), and in TPS-haemopoietic co-cultures.

This study describes angiogenic processes taking place in the in vitro micro-environment of a trout pronephric stroma cell line (TPS) under specific culture conditions, in which fetal calf serum, horse serum and hydrocortisone-sodium-21-hemisuccinate were used as supplements to the culture medium. When TPS cultures were kept in the same flask, i.e. without passages, for longer than 7 months, epithelioid cells differentiated into endothelial cells. Early stages of such differentiation were characterised by the presence of intracellular tubular vacuoles in clusters of neighbouring epithelioid cells. Subsequently, the endothelial cells reorganised and gave rise to microvascular structures, which branched over and into the TPS multilayers. The lining cells of the microvasculature showed typical characteristics of endothelial cells, such as ovoid or cubical shape, bundles of microfilaments and microtubules, and particularly numerous small vesicles at the apical pole, some of them fused to the plasma membrane. Similar angiogenic processes were also observed in long-term haemopoietic co-cultures formed by the TPS cell line and trout pronephric cell suspensions. Developing haemopoietic cells were observed at the basal pole of the vessels, and in the vascular lumen, where some immature cells appeared in close contact with the endothelium. These results indicate that the TPS cell line contains endothelial cell precursors, which are able to differentiate under certain culture conditions.

Seasonal changes in the lymphoid organs of wild brown trout, Salmo trutta L: a morphometrical study.

A morphometrical evaluation was made of the seasonal changes affecting the numbers of lymphocytes in the thymus, spleen and pronephros of wild brown trout, Salmo trutta, while the size of the thymus and the three thymic zones were also determined. Results reveal statistically significant changes throughout the year in the number of lymphocytes in the lymphoid organs studied. The spleen and pronephros have similar annual patterns of lymphocyte distribution with high numbers in two seasons, spring and autumn, and two periods of lymphoid involution in winter and summer. The highest numbers of thymocytes occur in trout caught in May and August, and the lowest in winter. In addition to normal lymphocytes, degenerated lymphoid cells that show pale cytoplasm devoid of cell organelles, also occurred in all the lymphoid organs. A negative correlation exists between the numbers of normal lymphocytes and that of degenerated lymphoid cells. The thymic size, as well as that of the subcapsular, inner and outer thymic zones, undergo very significant changes over the year. We discuss the relevance of cell proliferation, cell migration and in situ cell death for the circannual variations observed in the cell content of trout lymphoid organs, together with the possible causes.

Tissue distribution and structure of barrier cells in the hematopoietic and lymphoid organs of salmonids.

BACKGROUND: Barrier cells have been recognized as a discrete group of fibroblastic- or myofibroblastic-like cells located in the lymphoid and hematopoietic organs of mammals. This paper reports the results of a morphological study of the main lymphoid organs of three salmonid species, in which cells structurally similar to the mammalian barrier cells were observed in healthy animals. METHODS: The spleen, kidney, and thymus of fingerlings of rainbow trout, Oncorhynchus mykiss, and Coho salmon, Oncorhynchus kisutch, and of adult brown trout, Salmo trutta fario, were processed for electron microscopy study using various fixation methods. Semithin sections were used for the Periodic Acid-Schiff (PAS) staining technique, and for the demonstration of the endogenous peroxidase activity. RESULTS: The kidney and spleen of all the species contained a variable, but usually low, number of electron-dense, elongated, and branched cells, ultrastructurally similar to the mammalian barrier cells. They also occurred in the thymus of some brown trout and Coho salmon, but not of rainbow trout. The electron density of this cell type was present after the various types of fixation procedures. They show numerous ribosomes, well-developed secretory organelles, electron-clear vesicles, large granules, and microfilaments. In all the salmonid species, barriers cells were positive for PAS staining and for endogenous peroxidase activity. A small number of barrier cells were in mitosis. In the different organs barrier cells appeared as isolated cells, or forming syncytial networks. They were found lining the blood sinusoids of the splenic red pulp and of the renal hematopoietic tissue, in the periellipsoidal sheaths, and closely associated with erythropoietic and plasmacytopoietic foci. CONCLUSIONS: Our results demonstrate the presence of cells closely resembling mammalian barrier cells in the hematopoietic and lymphoid organs of salmonids. The structure and tissue distribution of the salmonid barrier cells are discussed in relation to the functional roles described for this cell type in mammals.

In vitro and in situ characterization of fish thymic nurse cells.

We present an enzyme- and immuno-cytochemical, and ultrastructural characterization of trout thymic nurse cells (TNCs). Our data suggest that isolated trout thymic multicellular complexes are epithelial cells with acidic compartments that may be involved in the processing of antigens and in the generation of the MHC-II proteins that these cell express, and also that isolated TNCs are the in vitro equivalent of the pale and intermediate electronlucent epithelial cells located in the inner zone of the trout thymus, constituting indirect evidence of the phylogenetical relationships of the inner zone of the teleost thymus with the thymic cortex of higher vertebrates.

Histopathology of the thymus in Saprolegnia-infected wild brown trout, Salmo trutta L.

A comparative analysis was made of the thymic cytoarchitecture of healthy and Saprolegnia-infected wild brown trout. In Saprolegnia-infected fish, even when the thymus was not invaded by fungal hyphae, the thymic architecture was lost without signs of regionalization. Intercellular oedema, some thymocytes, hypertrophic and degenerated epithelial cells and increased phagocytic activity were also observed. In thymi invaded by the fungus, hyphae occurred isolated or in close apposition to epithelial cells, and the thymic disorganization was more noticeable. Most thymocytes were pyknotic and both epithelial cells and macrophages contained engulfed dead cells. However, no inflammatory response to the fungal invasion was observed. These results confirm the immunodepressed condition of Saprolegnia-infected wild brown trout previously observed in peripheral lymphoid organs. We discuss the secretion of fungal products and/or the high levels of corticosteroids observed in these fish as possible origins of the condition.

[Fulminant pneumonia caused by Streptococcus milleri: rare or not diagnosed?]. / Neumonía de curso fulminante causada por Streptococcus milleri: infrecuente o no diagnosticada?

Streptococcus milleri is increasingly isolated in laboratory samples and is worthy of consideration as a differential diagnosis in pyogenic infections, particularly in adults with underlying infections. We describe a well-documented case of pleuropulmonary infection in a young man with no known risk factors. We analyze the diversity of microbiological features of these bacteria in culture, as well as their clinical importance as pathogens.

Seasonal intrathymic erythropoietic activity in trout.

Thymus glands of two salmonid species, Salmo trutta and Oncorhynchus mykiss, caught monthly throughout the year, were examined by light and transmission electron microscopy. Erythropoietic foci, consisting of both developing and mature erythroid cells, occurred in the subcapsular, inner, and outer thymic zones from April to November. We discuss the possible physiological significance of this seasonal erythropoietic activity, together with the role played by the thymic cell microenvironments and endocrine factors.

Long-term myelopoietic cultures from the renal hematopoietic tissue of the rainbow trout, Oncorhynchus mykiss W.: phenotypic characterization of the stromal cells.

This study describes the conditions for the long-term culture of the renal hematopoietic tissue of the rainbow trout, Oncorhynchus mykiss, the characterization of the stromal cells, and their relationships with myelopoietic cells. The long-term cultures consisted of stromal cells, which supported active myelopoiesis. Stromal cells were analyzed by enzyme-cytochemical techniques and electron microscopy. Major stromal cell types in long-term cultures consisted of fibroblastic reticular cells and epithelioid cells. Myelopoietic cells differentiated in close association with the fibroblastic reticular cells, and mature granulocytes were released into the culture medium.

Effects of early partial decapitation on the ontogenic development of chicken lymphoid organs. I. Thymus.

For the present study we used the classic model of early partial decapitation (DCx) of chicken embryos (Fugo, J. Exp. Zool., 85: 271-298, 1940; Betz, Gen. Comp. Endocrinol., 9: 172-186, 1967) in an attempt to analyze the neuroendocrine immune relationships during ontogeny. The elimination of the prosencephalon in chickens at 33-38 hr of incubation induced profound structural, histochemical, and morphometric changes in the embryonic development of the thymus gland. These included a greater development of the cortex than of the medulla, an increased mitotic index, high numbers of pyknotic cells, and enlarged connective tissue trabeculae containing numerous large lymphoid cells; hypertrophied reticular-epithelial cells; delayed appearance of medullary epithelial cysts; and intrathymic granulopoiesis. Furthermore, preliminary radioimmunoassays revealed a sharp increase in the values of circulating thymic hormones, mainly thymosin beta 4 in 17-day-old embryos. The results are discussed with regard to the possible role of prolactin, thyroxine, testosterone, and thymic hormones in the ontogenic development of the chicken thymus.

Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri: an ultrastructural study.

This study reports the ultrastructure of subpopulations of epithelial cells of the thymic parenchyma during the post-hatching development of the rainbow trout, Salmo gairdner, kept at 14 degrees C. At hatching, the thymus contained a small number of medium and large thymocytes interspersed among three different types of epithelial cells: (1) epithelial cells adjacent to the connective tissue capsule; (2) ramified dark epithelial cells with electron-dense cytoplasm; and (3) pale electron-lucent epithelial cells displaying secretory-like features. All these cells types were anchored to one another by desmosomes and had apparently differentiated from the pharyngeal epithelium. At 4 days after hatching, the thymus enlarged, and numerous gaps occurred between the cell processes of contiguous epithelial cells adjacent to the capsular connective tissue. In 21-day-old trout, thymic trabeculae developed carrying blood vessels, and a subcapsular zone became evident containing lymphoblasts and large subcapsular epithelial cells. In 30-day-old trout, an outer thymic zone developed consisting of spindle-shaped epithelial cells which formed a dense network. At this stage, scattered cystic cells, which apparently differentiated from the pale epithelial cells, were present.

Enzyme- and immuno-histochemical study of the thymic stroma in the rainbow trout, Salmo gairdneri, Richardson.

Owing to the lack of data about thymic non-lymphoid cells in fish we decided to perform a histochemical characterization of these cells in order to ascertain their relationships to other thymic components. In the present study we analyze the enzyme-histochemical patterns for acid phosphatase, alkaline phosphatase, non-specific sigma-naphthyl acetate esterase and 5' nucleotidase activities, as well as the presence of keratin demonstrated by immunoperoxidase staining, in the non-lymphoid cell populations of the thymus of the rainbow trout, Salmo gairdneri. According to their location in the organ, morphology and histochemical reactivities, we were able to define seven different subpopulations of keratin-positive epithelial cells: 1) Epithelial cells limiting with the capsular and septal connective tissues; 2) Subcapsular epithelial cells; 3) Stellate epithelial cells of the inner thymic zone; 4) Large, ovoid epithelial cells of the inner thymic zone; 5) Acidophilic epithelial cells of the outer thymic zone; 6) Cystic cells; and 7) Goblet cells. The significance of the heterogeneity of the epithelial cell (EC) population, its specific distribution in the organ, which apparently conforms distinct cell microenvironments, as well as the possible phylogenetical relationships between these microenvironments and the classical cortex and medulla of the mammalian thymus, are discussed.

Alterations in the peripheral lymphoid organs and differential leukocyte counts in Saprolegnia-infected brown trout, Salmo trutta fario.

The number of circulating leukocytes and the structure of splenic and renal lymphoid tissue were comparatively analysed in healthy and Saprolegnia-infected wild brown trout, Salmo trutta fario. Sick trout showed lymphopenia, mainly due to decreased numbers of circulating small lymphocytes, and heterophilia. The splenic and renal lymphoid tissue of infected trout exhibited similar changes, consisting of cellular depletion, lymphoid cell degeneration, and vascular alterations with blood vessel enlargement and hypertrophy of sinusoidal endothelial cells. Furthermore, the endothelial cells in the spleen and kidney of the infected trout contained cytoplasmic vesicles filled with material of possible fungal origin. The absence of a reticular sheath was also evident in the splenic ellipsoids. These results suggest some immunodepression in Saprolegnia-infected trout which might favour the course of the disease.

Ultrastructural changes in the spleen of the natterjack, Bufo calamita, after antigenic stimulation.

In the present study comparative aspects of the ultrastructure of the spleen were analyzed in non-immunized and T-dependent antigen-challenged natterjacks, Bufo calamita. Special attention is focused on the role of the non-lymphoid components in the splenic immunoreactivity. Ten days after primary immunization with sheep erythrocytes, splenic lymphoid follicles increase considerably in number and size. By that time, lymphoblasts, medium and large lymphocytes abound in the periphery of the white pulp near the marginal zone. Meanwhile, in the red pulp numerous monocytes migrating across the sinusoidal walls apparently transform into giant, dendritic-like cells. Twenty days after immunization the splenic lymphoid follicles decrease in number, although certain reactivity persists and numerous plasma cells occur in the cell cords and sinusoids of the red pulp. These results are discussed comparatively with those reported in other lower vertebrates.

Interdigitating cells in the thymus of the turtle Mauremys caspica. Possible relationships to macrophages.

Interdigitating cells are non-lymphoid elements in the thymus and peripheral, secondary lymphoid organs of higher vertebrates. Their origin and functional significance are a matter of controversy. In the present investigation we analyze, for the first time, the nature of presumptive interdigitating cells of the thymus of an ectothermic vertebrate, the turtle Mauremys caspica. This model is specially useful because of the seasonal variations that affect the reptilian lymphoid organs. Immature pro-interdigitating cells and phagocytosing mature interdigitating cells are described with special emphasis on their ultrastructural characteristics and possible relationships with monocytes and macrophages.

Postnatal development of the non-lymphoid elements in the rat lymph node. Connective reticulum cells, macrophages and postcapillary venules.

The present study analyzes ultrastructurally the non-lymphoid elements occurring in the diffuse deep cortex and medulla of the postnatal developing lymph nodes of the rat. The supporting meshwork of the organ consist of connective reticulum cells joined together themselves by intercellular junctions. Two morphologically different macrophage cell types can be described in the developing lymph nodes. The first one consists of free-migrating macrophages homing the cortical parenchyma, apparently related with phagocytosis of necrotic cells. In contrast, macrophages located in the lumen of the medullary lymphatic sinuses are anchored to connective reticulum cell processes. They form lymphocyte-macrophage clusters and are involved also in erythrophagocytosis phenomena. Postcapillary venules lined by a low cuboidal endothelium, which allow lymphocyte diapedesis, appear in the diffuse deep cortex of the neonatal lymph nodes. The possible significance of all these cellular elements is discussed with respect to their role as constituents of the local lymph node microenvironments.