Growth of Fetal Thymocytes in Organ Culture: Effect of Recombinant Lymphokines on Thymocyte Maturation

Abstract

Interleukin 2 (IL2) has a dose-dependent inhibitory effect on the growth and phenotypic maturation of thymocyte populations grown in fetal thymus organ culture. Addition of IL2 (100 U/ml) to 14-day fetal thymus organ cultures induces the appearance of a population of lymphokine-activated killer (LAK) cells which lyse allogeneic, syngeneic, and syngeneic tumor cell targets. The addition of the monoclonal antibody, PC-61, blocks the IL2-dependent growth and activation of LAK cells but does not influence the maturation of CD4⁺ CD8⁺ fetal thymocytes. These data imply that IL2 is not a major regulator of normal fetal thymocyte maturation. The effects of a range of recombinant lymphokines (IL1α, IL1β, IL3, IL4, GM-CSF, G-CSF, M-CSF) on the proliferation and phenotypic maturation of fetal 14-day thymocytes in organ culture has been analysed. Two significant changes were seen. First, IL1α and IL1β inhibited growth and the expression of the CD4 and CD8 antigens in organ culture, and second, GM-CSF increased the expression of Mac-1 cells. IL4, which has known T cell growth-promoting activity, IL3, G-CSF, and M-CSF did not alter either normal growth or surface antigen expression in fetal thymocytes. While some of these lymphokines may function as accessory molecules in fetal thymocyte development, our experiments suggest that they do not have a significant influence on thymocyte maturation when used alone.     

Integrated scRNA-Seq Identifies Human Postnatal Thymus Seeding Progenitors and Regulatory Dynamics of Differentiating Immature Thymocytes

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3,4,3',4'-Tetrachlorobiphenyl distribution and induced effects in the rat adrenal gland. Localization in the zona fasciculata

The distribution of radiolabeled 3,4,3',4'-tetrachlorobiphenyl (TCB) and TCB-induced effects on serum and adrenal gland retinoid content, and adrenal gland morphology was studied by liquid scintillation counting, high performance liquid chromatography, light microscopic autoradiography, and transmission electron microscopy. Adult, female WAG/Rij rats received a single intraperitoneal injection of either vehicle (corn oil), 15 mg TCB/kg, or 200 mg TCB/kg body weight and were sacrificed (N = 3 per group) at 1, 3, 7, and 14 days after treatment. One rat of the high dose group that was sacrificed at each sampling time had received radiolabeled compound (containing 1.85 mCi of 3H-TCB). At day 1, the adrenal gland had the greatest concentration of radioactivity (dpm x 10(-6)/gm wet tissue) of any organ examined. There was a selective distribution of radiolabeled compound to the zona fasciculata accompanied by morphometric evidence of hypertrophy of the zona fasciculata. The vast majority of 3H-TCB present in the adrenal gland was parent compound at all time periods. Serum retinol content was significantly decreased in the high dose group by 61 and 54% at days 3 and 7, respectively. No significant decrease in adrenal gland retinoid content occurred at any time in this study, but in contrast, adrenal gland retinol and retinyl palmitate content was increased. Serum cortisol levels were transiently decreased in the high dose group. Ultrastructural alterations were only observed in cells of the zona fasciculata. Predominant changes included mitochondrial hypertrophy and concentric whorling lamellar arrays of the membranes of the outer mitochondrial compartment and mitochondrial cristae. The results of this study indicate that the rat adrenal gland is an early target organ after TCB intoxication, and that there is an early and selective distribution of TCB in the rat adrenal gland accompanied by morphologic alterations in the sites of compound localization. The results further suggest that the observed morphologic changes did not result from hypovitaminosis A.     

Cytokine Production and Responsiveness of Fetal T-Cell Receptor Vγ3 Thymocytes

The aim of this study was to examine the cytokine production and cytokine responsiveness of the first T-cell receptor (TcR) positive cells that appear in the murine fetal thymus, namely TcR V gamma 3 cells. It is shown that IL-2-cultured fetal TcR V gamma 3 thymocytes were capable of producing IL-3, GM-CSF, TNF-alpha and IFN-gamma upon TcR triggering. IL-2, IL-4, IL-5 and IL-6 could not be detected. With regard to cytokine responsiveness, TcR V gamma 3 cells proliferated to a high extent when high concentrations of rIL-2 were added. rIL-4 or rIL-7 alone, but not rIL-1 alone, were capable of inducing a modest proliferation of TcR V gamma 3 thymocytes. When combined with low concentrations of IL-2, a synergistic effect could be observed with IL-1, IL-4 or IL-7. It is shown that the synergistic effect of IL-2 with IL-4 was mainly due to induction of IL-2 receptor expression. The synergistic effect of IL-2 and IL-7 on the proliferation of TcR V gamma 3 cells could only be partially inhibited by anti-IL-2 receptor MoAb, and this antibody had no effect on the IL-2 + IL-1 cultures. These observations can explain the extensive proliferation of TcR V gamma 3 thymocytes during fetal life and they indicate that TcR V gamma 3 thymocytes have the potential to play a functional role during fetal thymus development.     

PNA Lectin-Based Separation of Thymocytes into Mature and Immature Subpopulations: CD4-8- Double Negative Cells Display Characteristics of PNAlo Mature Thymocytes

Cortical (immature) thymocytes are widely reported to express intermediate to high levels of receptors for the lectin, peanut agglutinin (PNA). Light-scatter studies of murine fetal thymocytes stained with PNA or anti-mouse CD4 and CD8 monoclonal antibodies indicated, however, that the most immature CD4-8- (DN) thymocyte subpopulation binds levels of PNA commonly described as PNA^lo. Evaluation of the PNA binding characteristics of fetal thymocytes negative for the CD8 antigen confirmed the existence of a major population (approximately 20% of total cells) of CD4^-8^- PNA^lo fetal thymocytes. The majority of these DN thymocytes were subsequently found to bind sub-agglutinating levels of PNA, similar to mature CD4⁺ or CD8⁺ single positive (SP) thymocytes. Given this information, an immunomodulating compound (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) known to produce a maturational delay in murine thymocytes was tested for a possible concurrent effect on thymocyte PNA lectin binding. A TCDD- induced increase in DN thymocytes was found to be paralleled by an increase of equal magnitude in PNA^lo thymocytes. Taken together, these data provide evidence that acquisition of the PNA receptor may be a maturational event occurring during the DN stage of thymocyte ontogeny. Further, these results suggest that separation of thymocytes into mature (medullary) and immature (cortical) subpopulations by PNA agglutination may result in contamination of medullary cells by the most immature (DN) subpopulation of thymocytes.     

Tetrachlorodibenzo-p-Dioxin (TCDD) Inhibits Differentiation and Increases Apoptotic Cell Death of Precursor T-Cells in the Fetal Mouse Thymus

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes thymic atrophy as well as alterations in thymocyte maturity in mice. Multiple mechanisms for thymic hypocellularity have been suggested, and include an increase in thymocyte apoptosis, a maturation arrest of thymocyte development, inhibited thymocyte proliferation, and a diminution of seeding of the thymus by the hematopoietic progenitors in the fetal liver or adult bone marrow. Fetal mice are highly sensitive to hypocellularity induction by TCDD when the chemical is administered during the window of thymic development, between days 10 and 18 of gestation. Treatment of pregnant C57Bl/6 mice in the present experiments with doses of 5 or 10 mu g/kg TCDD by oral gavage on gestation days 14 and 16 severely depressed day 18 thymic cellularity. Histopathologic evaluation of day 18 fetal thymi showed disruption of the normal organ architecture with loss of clear distinction between cortical and medullary regions after TCDD. A decrease in thymocyte density was noted in all regions, and was most dramatic in the cortical zones where pyknotic cells were increased by TCDD treatment. Using day 18 thymocyte suspensions and flow cytometry, the marker 7-AAD showed a decrease in viable thymocytes from TCDD-treated fetal mice, and a concomitant and dose-related increase of thymocytes in early apoptosis. Specifically, relative to control, thymocytes from the 5 and 10 mug/kg TCDD exposure groups displayed 1.9% and 5.3% respective increases in early apoptotic cells. When thymocytes were co-identified by CD4 and CD8 cell surface antigen expression, the enhanced apoptosis occurred in the CD4(+)CD8(+) phenotype with no significant apoptosis seen in the CD4(-)CD8(-), CD4(+)CD8(-), or CD4(-)CD8(+) thymocytes. Given the rapid clearance of apoptotic cells from the thymus, these histopathologic and cytometric data suggest increased thymocyte apoptosis contributes to fetal thymic atrophy after TCDD exposure.     

Role of Thromboxane A2 in the Induction of Apoptosis of Immature Thymocytes by Lipopolysaccharide

Lipopolysaccharide (LPS) causes apoptotic deletion of CD4⁺ CD8⁺ thymocytes, a phenomenon that has been linked to immune dysfunction and poor survival during sepsis. Given the abundance of thromboxane-prostanoid (TP) receptors in CD4⁺ CD8⁺ thymocytes and in vitro evidence that thromboxane A₂ (TXA₂) causes apoptosis of these cells, we tested whether enhanced generation of TXA₂ plays a role in LPS-induced thymocyte apoptosis. Mice injected with 50 μg of LPS intraperitoneally displayed a marked increase in generation of TXA₂ and prostaglandin E₂ in the thymus as well as apoptotic deletion of CD4⁺ CD8⁺ thymocytes. Administration of indomethacin or rofecoxib inhibited prostanoid synthesis but did not affect thymocyte death. In contrast, thymocyte apoptosis in response to LPS was significantly attenuated in TP-deficient mice. These studies indicate that TXA₂ mediates a portion of apoptotic thymocyte death caused by LPS. The absence of an effect of global inhibition of prostanoid synthesis suggests a complex role for prostanoids in this model.     

Semaphorin 3A Negatively Affects Proliferation of Mouse Thymus Epithelial Cells In Vitro

Bulletin of Experimental Biology and Medicine - We studied effects of semaphorin 3A, keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and their combinations on the proliferative...     

$3,3',4,4',5$ -Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), the most toxic ofall coplanar polychlorinated biphenyls (Co-PCBs), were examinedin KB-3 cells expressing human wild-type and mutantP-glycoprotein in which the 61st amino acid was substituted forserine or phenylalanine (KB3-Phe⁶¹). In the cellsexpressing P-glycoproteins, accumulations of vinblastine andcolchicine decreased form 85% to 92% and from 62% to 91%,respectively, and the drug tolerances for these chemicalswere increased. In KB3-Phe⁶¹, the decreases in drugaccumulation were inhibited by adding PCB-126 in a way similar tothat with cyclosporine A: by adding 1μM PCB-126, theaccumulations of vinblastine and colchicine increased up to 3.3-and 2.3-fold, respectively. It is suggested that PCB-126decreased the drug efflux by inhibiting the P-glycoprotein inKB3-Phe⁶¹. Since there were various P-glycoproteins andmany congeners of Co-PCBs, this inhibition has to be considered anew cause of the toxic effects of Co-PCBs.     

Reconstitution Properties of Thymus Stem Cells in Murine Fetal Liver

Injection of day-12 murine fetal liver cells into thymus lobes of Thy-1 congenic adult recipients results in a wave of thymocyte development. The kinetics of repopulation by donor cells reaches a peak after 20–25 days. The frequency of thymic stem cells (TSC) in day-12 fetal liver was estimated, by limit dilution, as 1 in 4x10⁴ cells. Within 8 hr of injection into a thymus lobe, fetal liver TSC commit to T-cell development, losing stem-cell activity. When fetal liver cells are maintained in culture for 7 days, with no exogenous cytokines added, and then injected intra-thymically (I.T.), thymus recolonization is not observed. However, TSC can be maintained in culture for 7 days with IL-1β, IL-3, IL-6, or LIF added, alone or in combination, with steel factor (SLF). Poisson analysis of fetal liver cells cultured with SLF and IL-3 together revealed a precursor frequency of 1 in 1.8x 10⁵ cells. In contrast, the frequency of TSC in adult bone marrow was estimated by limit dilution as 1 in 12,000 cells.     

Studies on the In Vitro Culture of Murine Fetal Thymus: Effect of Factors Promoting Epithelial Growth*

ABSTRACT: Fourteen-day fetal thymus was grown in organ culture under conditions reportedly optimizing epidermal/epithelial cell growth. While the fibroblastoid component is stimulated by epidermal growth factor and fibroblastoid growth factor, embryonic thymic epithelial cells do not grow significantly under any conditions, although they persist in organ explants. The major cell types cultured are fibroblastoid and macrophage. In organ explant cultures, epithelial cells appear to be oriented by the presence of a cell coat demonstrated by electron microscopy. This coat is absent from certain ciliated cells consistently found in deeper regions of the explant. Using amniotic fluid as a growth factor, almost pure cultures of thymic macrophages can be obtained.     

Induction of Apoptosis and p53 Expression in Immature Thymocytes by Direct Interaction with Thymic Epithelial Cells

Apoptosis of normal thymocytes was shown to be triggered by several mechanisms (e.g. glucocorticoids, γ-irradiation). In the present study the authors report on thymocyte apoptosis that is induced by thymic epithelial cells. The thymocytes undergo a massive apoptotic death within 24 h of cocultivation with thymic epithelial cell monolayers derived from primary cultures (PTEC) or from a thymic epithelial cell line (TEC). Non-thymic monolayers were inactive. Apoptosis induction in this experimental model requires direct contact between the thymocytes and the thymic epithelial monolayer and can be blocked by anti-CD2 and anti-LFA-1 antibodies. The immature CD3^−/+dull CD4⁺CD8⁺ thymocytes were the cells which undergo apoptosis. The fact that the authors are dealing with a massive apoptotic process of immature cells in the absence of exogenous antigen suggests that it involves the nonselected thymocytes. The apoptotic pathway selected by thymocytes following their culturing on TEC involves p53 expression. Indeed it was found that TEC-induced apoptosis, led to the accumulation of p53 protein that preceded the step of DNA fragmentation in freshly isolated thymocytes as well as in a glucocorticoid resistant thymoma cell line. Since glucocorticoid-induced thymocyte apoptosis is p53-independent, glucocorticoids are conceivably not involved in TEC-induced thymocyte death. The in vitro experimental model presented here may reflect the physiological sequence of events leading to thymocyte death in the thymus.     

Peptidergic Regulation of Thymocyte Differentiation, Proliferation, and Apoptosis during Aging of the Thymus

The effects of T-31, AB-17, and AB-9 peptides on old (passage 8) thymocyte culture were studied. Only AB-9 peptide exhibited a complex geroprotective effect on thymocytes during their aging. Peptide AB-9 stimulated proliferative activity and differentiation of thymocytes and inhibited their apoptosis.     

Interrelation of Cell Apoptosis and Proliferation in the Thymus during Its Involution

In people over 60 years, thymocytes contain proliferation protein Ki67 and proapoptotic protein P53. During aging, apoptosis of thymus cell prevails over proliferation, though thymocytes retain proliferative capacity even in long-livers.     

Pathogenesis of Human Parainfluenza Type 3 Virus Infection in Hamster Tracheal Organ Culture

Hamster tracheal organ culture was employed as a model for the study of the pathogenesis of human parainfluenza type 3 virus infection. It clearly supported replication of the virus over a 2-week period of time. Infected tracheal explants were examined with light, electron, and immunofluorescence microscopy. They exhibited specific cytopathologic alterations including nuclear swelling and chromatin margination, multinucleated syncytia and binucleated epithelial cells, and fibroblasts and chondrocytes. Focal destruction and denudation of the respiratory epithelium occurred in later stages of infection. Virus was detected in close association with cilia and was observed budding off the unit membrane of epithelial cells.     

In Vivo Effects of TRH,T3 and cGH on Antibody Production and T- and B-Lymphocytes Proliferation in Immature Male Chickens

Newly hatched White Leghorn male chicks were used in this study. Different doses of T₃ (O.1 or 1 ppm) or TRH (1 or 5 ppm) were administered in the feed for an 8-week period. Chicken growth hormone (cGH) (10 μg/kg BW) was injected (i.v.) into a different group of chicks twice daily for 1 week starting at 7 weeks of age. A different group received both T₃ (0.1 and 1 ppm) and cGH. Serum concentrations of T₄, T₃ and GH, antibody production against sheep red blood cells (SRBC) and Brucella Abortus (BA), and in vitro proliferative response of both T- and B-lymphocytes to mitogenic stimulation were measured. Supplementation of T₃ (1 ppm) significantly lowered T₄ and increased T₃ concentrations. No effect of any hormone treatment on antibody production was observed. T₃ supplementation and cGH injection alone or with T₃ (0.1 ppm) significantly increased blastogenic response of lymphocytes to either Con-A or LPS mitogenic stimulation. It was concluded that T₃ and GH are involved in lymphocyte activity of chickens.