Establishment of a mouse primary co-culture of endometrial epithelial cells and peripheral blood leukocytes: effect on epithelial barrier function and leukocyte survival

This study aimed to establish an in vitro co-culture model that would allow us to study the interaction between endometrial epithelial cells and immune cells. Flow cytometry analysis and cell surface marker staining were used to identify suitable immune leukocytes from a range of sources, such as intraepithelial lymphocytes (IEL), thymocytes, splenocytes and peripheral blood leukocytes. Optimizing culture conditions such as cell viabilities, cell seeding ratios and densities and co-culture methods were examined and determined. Results showed that co-culture of mouse endometrial epithelial cells (EEC) with peripheral blood leukocytes (PBL) at seeding densities of 3.0 x 10(6) and 1.0 x 10(6)cells/ml, respectively, appeared to affect both the survival of leukocytes and epithelial barrier function. Cell viability counts of immune cells showed 95% and 72.5% cell survival after isolation and after 4 days in co-culture with EEC, respectively, but only 11% cell survival when cultured alone for 4 days without EEC. Short-circuit current (I(sc)) results also showed that EEC and PBL co-culture exhibited a four-fold increase in the transepithelial resistance (TER) as compared to EEC culture alone, indicating enhanced protective barrier function. Taken together, the currently established in vitro co-culture model of endometrial epithelial cells and immune cells may provide a means to investigate local cellular immune responses upon uterine infections.     

MHC non-restricted, CD95-independent apoptosis of immature thymocytes induced by thymic epithelial cells

The interaction of thymocytes with thymic epithelial cells in the absence of an exogenous antigen was studied in vitro. Thymic, but not splenic epithelial cells induced apoptosis of thymocytes. A thymic epithelial cell line (TEC) induced apoptosis of thymocytes but not of splenic T-cells. The target population for TEC-induced death were immature CD4(+)8(+) (double positive), but not mature single positive thymocytes. TEC also induced DNA fragmentation in day 18 foetal thymocytes, most of which are CD4(+)8(+) cells. Radiation leukemia virus (RadLV)-transformed thymic lymphoma clones expressing various phenotypes reflected this sensitivity, in that a CD4(+)8(+)3(+) clone apoptosed by thymic epithelial cells or TEC. Other, single positive or double negative clones were resistant. Thymocytes from C3H (H-2(k)), C57BL/6 (H-2(b)) and Balb/C (H-2(d)) mice apoptosed equally in response to either C57BL/6 thymic epithelial cells or TEC (H-2(b) x H-2(d)). Likewise, thymocytes from MRLIpr((-/-)) and B6Ipr((-/-)) mice, which do not express CD95 were also apoptosed by TEC.The data suggest that thymic epithelial cells induce MHC non-restricted, Fas-independent apoptosis of immature thymocytes. This response may reflect a mechanism through which thymocytes expressing TcR with no affinity to self MHC/peptide complexes are eliminated.     

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.     

Overexpressed Bcl-x(L) prevents bacterial superantigen-induced apoptosis of thymocytes in vitro

bcl-x, a homologous gene of bcl-2, has an anti-apoptotic function and appears to play a critical role in the development of lymphoid systems. To investigate the effect of overexpressed Bcl-x(L) on the development of T lymphocytes, we established two lines of transgenic mice by using Emu-chicken bcl-x(L) (cbcl-x(L)) transgene, where the cBcl-x(L) protein was expressed mainly in lymphoid cells. Although thymocytes and splenocytes from cbcl-x(L) transgenic mice are resistant to apoptosis in vitro, clonal deletion of thymocytes, recognizing endogenous self-superantigens in the thymus, still normally proceeded and no self-reactive T cells were found in the spleen of the transgenic mice. To dissect clonal deletion, we utilized two in vitro models, thymocytes/antigen presenting cells co-culture system and fetal thymus organ culture system. In both, bacterial superantigen staphylococcus aureus enterotoxin B (SEB) induces apoptosis of T cells with Vbeta8+ T cell receptor (TCR) reacting to SEB, which mimics clonal deletion of self-reactive thymocytes in vivo. SEB-induced depletion of Vbeta8+ T cells from thymocytes when taken from the transgenic mice was effectively inhibited. The data might raise the possibility that cell death process involved in clonal deletion in the thymus is a form of apoptosis inhibited by Bcl-x(L).     

Thymic myoid cells protect thymocytes from apoptosis and modulate their differentiation: implication of the ERK and Akt signaling pathways

Thymic myoid cells correspond to a muscle-like cell population present in the thymic medulla. They are well conserved throughout species evolution, but their biological role is not known. We demonstrated that myoid cells protected thymocytes from apoptosis as evidenced by a strong decrease of annexin-V-FITC positive thymocytes. This effect was (1) specific of myoid cells compared to thymic epithelial cells; (2) dependent on direct cell-to-cell contacts and (3) triggered rapidly after 2 h in cocultures. This protective phenomenon was due to the activation of prosurvival mechanisms. Indeed, myoid cells activated extracellular-regulated kinases (ERK1/2) and Akt in thymocytes. Myoid cells also influenced thymocyte maturation. We observed an increase in CD4(+) and a decrease in CD8(+) single positive (SP) thymocytes when cocultured with myoid cells, independently of a CD8(+)SP increased death or a CD4(+)SP overproliferation. Consequently, thymic myoid cells protect thymocytes from apoptosis and could also modulate their differentiation process.     

In vitro enhancing effects of thymic dendritic cells on apoptotic cell death of thymocytes

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Specialized ability of thymic epithelial cells to mediate positive selection does not require expression of the steroidogenic enzyme p450scc

Thymic epithelial cells are uniquely efficient in mediating positive selection, suggesting that in addition to providing peptide/MHC complexes for TCR ligation, they may also provide additional support for this process. Recent studies have shown that although engagement of either the TCR or glucocorticoid (GC) receptors can individually induce apoptosis in thymocytes, together these signals are mutually antagonistic. This had led to the suggestion that local GC production by thymic epithelial cells, by opposing TCR signaling for apoptosis, provides the basis of the ability of these cells to mediate thymocyte positive selection. In this paper we have examined this possibility directly and shown that highly purified cortical epithelial cells, which have the functional ability to mediate positive selection in reaggregate cultures, do not express mRNA for the key steroidogenic enzyme P405scc. Thus we conclude that the ability of thymic epithelial cells to support positive selection does not rely on their ability to produce GC. However, we find that P450scc mRNA is up-regulated in thymocytes on the initiation of positive selection, raising the possibility that any local protective effect of steroid production is mediated at the level of thymocytes themselves.     

IL-6 protects enterocytes from hypoxia-induced apoptosis by induction of bcl-2 mRNA and reduction of fas mRNA

Interleukin-6 (IL-6) has been shown to rescue enterocytes from hypoxia-induced apoptosis when given orally following hemorrhagic shock. In vitro models using an intestinal epithelial cell line (IEC-6) cultured with lipopolysaccharide (LPS) under low O2 conditions, to mimic intestinal conditions, show that these cells also undergo apoptosis, which can be reduced by subsequent culture with IL-6. To examine further the mechanisms of rescue, we cultured normal rat intestinal epithelial cells (IEC-6) under both normoxic and hypoxic conditions and analyzed their responses to LPS and IL-6. We showed that IEC-6 expressed IL-6 receptor on its surface. Further, IEC-6 cells could be rescued from hypoxia-induced apoptosis by co-culture with IL-6. RNase protection assay (RPA) examination revealed that under hypoxic conditions, IEC-6 cells that were resistant to apoptosis showed reduced fas expression and increased bcl-2 expression after co-culture with LPS+IL-6.     

Involvement of Rho GTPases and their effectors in the secretory process of PC12 cells

Intrathymic maturation of thymocytes is essential for the proper formation of T-cell repertoire. This process involves two major biochemical pathways, one initiated by the recognition of MHC/peptide by the T-cell receptor and the other mediated by glucocorticoids. These hormones seem to affect thymocyte maturation by increasing the threshold of TCR-mediated positive and negative selection, and by inducing apoptosis of nonselected thymocytes. We have previously reported that an SV40-immortalized murine thymic epithelial cell line, namely 2BH4, was able to protect thymocytes from dexamethasone-induced apoptosis. Here we show that this protection is independent of cell-to-cell contact and does not seem to involve a Bcl-2-mediated resistance, since incubation of thymocytes with 2BH4 cells or its supernatant does not interfere with the levels of this antiapoptotic molecule. The protection conferred by 2BH4 cells, or by a primary culture of thymic stromal cells, is specific for the CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes, whereas the broad-spectrum caspase inhibitor z-VAD-fmk blocks apoptosis induced by dexamethasone in all thymocyte subpopulations. Our results suggest that positively selected single-positive thymocytes are still susceptible to glucocorticoid-induced apoptosis but are protected from it through the action of a heat-stable protein(s) released by thymic stromal cells.     

Lymphocyte depletion in thymic nurse cells: a tool to identify in situ lympho-epithelial complexes having thymic nurse cell characteristics

Summary In situ pre-existing complexes of epithelial cells and thymocytes having thymic nurse cell characteristics were visualized in the murine thymus cortex using dexamethasone as a potent killer of cortisone-sensitive thymocytes. The degradation and subsequent depletion of cortisone-sensitive thymocytes enclosed within cortical epithelial cells appeared to be paralleled by thymocyte degradation and depletion in thymic nurse cells isolated from thymic tissue fragments from dexamethasone-treated animals. This suggests that thymic nurse cells are derived from pre-existing sealed complexes of cortical epithelial cells and thymocytes. Not all thymocytes situated within in situ epithelial or thymic nurse cells complexes appear to be cortisone-sensitive: a minority of 1–2 thymocytes per complex survives the dexamethasone-treatment, thus constituting a minor subset of cortical cortisone-resistant thymocytes predominantly localized within cortical epithelial cells in situ and within thymic nurse cells derived from such structures. Cortisone resistance in thymocytes thus seems to be acquired within the cortical epithelial cell microenvironment. Cortisone-resistant thymocytes in thymic nurse cells express the phenotype of mature precursors of the T helper lineage, indicating that the in situ correlates of thymic nurse cells may play an important role in T cell maturation and selection.     

The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

Activation of lymphocytes induced by bronchial epithelial cells with prolonged RSV infection

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells,which might be responsible for susceptibility to asthma; however, the underlying mechanism is not clear. This study determined the activation of lymphocytes and drift of helper T (Th) subsets induced by RSV-infected human bronchial epithelial cells (HBECs) in vitro. HBECs had prolonged infection with RSV, and lymphocytes isolated from human peripheral blood were co-cultured with RSV-infected HBECs. Four groups were established, as follows: lymphocytes (group L); lymphocytes infected with RSV (group RL); co-culture of lymphocytes with non-infected HBECs (group HL); and co-culture of lymphocytes with infected HBECs (group HRL). After co-culture with HBECs for 24 hours, lymphocytes were collected and the following were determined in the 4 groups: cell cycle status; apoptosis rate; and concentrations of IL-4, IFN-γ, and IL-17 in the supernatants. Cell cycle analysis for lymphocytes showed a significant increase in S phase cells, a decrease in G1 phase cells, and a higher apoptosis rate in group HRL compared with the other three groups. In group HRL, the levels of IL-4, IFN-γ, and IL-17 in supernatants were also higher than the other three groups. For further study, lymphocytes were individually treated with supernatants from non-infected and RSV-infected HBECs for 24 h. We showed that supernatants from RSV-infected HBECs induced the differentiation of Th2 and Th17 subsets, and suppressed the differentiation of Treg subsets. Our results showed that HBECs with prolonged RSV infection can induce lymphocyte proliferation and apoptosis, and enhance the release of cytokines by lymphocytes. Moreover, subset drift might be caused by RSV-infected HBECs.     

In vitro enhancing effects of thymic dendritic cells on apoptotic cell death of thymocytes

Using terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay and propidium iodide-DNA staining flow cytometry assay, the effects of mouse thymic dendritic cells (MTSC4) on the process of programmed cell death of thymocytesin vitro were investigated. It was noticed that thymocytes bound to MTSC4 used in this study. That the percentages of apoptotic nuclei of the bound thymocytes on MTSC4 were much higher than those of medium-cultured thymocytes, while the bound thymocytes on mouse thymic epithelial cell (MTEC1) showed much lower percentages of apoptosis. FACS analysis quantitatively confirmed the observation. Phenotype analysis showed that MTSC4 induced the deletion of CD4 + CD8 + cells and CD4 + CD8-.cells in 18 h of coculture. The results suggest that the negative selection of medullary thymocytes may be achieved by thymic dendritic cells through their enhancing effects on apoptosis. Project supported by the National Natural Science Foundation of China (Grant No.39670685) and FokYin Tung Education Foundation.     

Macrophage-derived chemokine and EBI1-ligand chemokine attract human thymocytes in different stage of development and are produced by distinct subsets of medullary epithelial cells: possible implications for negative selection

The chemoattractant activity of macrophage-derived chemokine (MDC), EBI1-ligand chemokine (ELC), and secondary lymphoid tissue chemokine (SLC) on human thymocytes was analyzed. Both ELC and SLC caused the accumulation of CD4+CD8- or CD4-CD8+ CD45RA+ thymocytes showing high CD3 expression. By contrast, a remarkable proportion of MDC-responsive thymocytes were CD4+CD8+ cells exhibiting reduced levels of CD8 or CD4+CD8- cells showing CD3 and CD45R0, but not CD45RA. MDC-responsive thymocyte suspensions were enriched in cells expressing the MDC receptor, CCR4, selectively localized to the medulla, and in CD30+ cells, whereas ELC-responsive thymocytes never expressed CD30. Reactivity to both MDC and ELC was localized to cells of the medullary areas, but never in the cortex. Double immunostaining showed no reactivity for either MDC or ELC by T cells, macrophages, or mature dendritic cells, whereas many medullary epithelial cells were reactive to MDC or ELC. However, MDC reactivity was consistently localized to the outer wall of Hassal's corpuscles, whereas ELC reactivity was often found in cells surrounding medullary vessels, but not in Hassal's corpuscles. Moreover, while most MDC-producing cells also stained positive for CD30L, this molecule was never found on ELC-producing cells. We suggest therefore that CD30L-expressing MDC-producing medullary epithelial cells attract CCR4-expressing thymocytes, thus favoring the CD30/CD30L interaction, and therefore the apoptosis, of cells that are induced to express CD30 by autoantigen activation. By contrast, ELC production by CD30L-lacking medullary epithelial cells may induce the migration into periphery of mature thymocytes that have survived the process of negative selection.     

Stages of the rat thymic medulla development in foetal period

Development of thymic medulla was examined on consecutive gestational days (GD) in Wistar rats. Medullary thymic epithelial cells (TEC) were identified by immunocytochemical localisation of neuron-specific enolase (NSE). Organisation of thymic medullary architecture was determined by interaction of thymocytes with NSE-positive TEC, that led to formation of lymphoepithelial complexes (GD 19), in which the cells exhibited proliferative activity or traits of apoptosis. The studies indicated that differentiation events and organisation of thymic medulla require stage-specific interactions between TEC and thymocytes.     

Development of mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media

This study investigated the effects of the in vitro co-culture of mouse embryos with non-polarized or polarized uterine epithelial cells, using sequential culture media, on their development to blastocysts, blastocyst quality (blastocyst diameter and cell number), apoptosis, Bcl-2 and Bax gene expression. There were three treatments, all of which used sequential culture media. The treatments were no co-culture (control), non-polarized or polarized epithelial cell monolayer co-culture in 24-well tissue culture plates. Mouse uterine epithelial cells were isolated enzymatically and were seeded either on the surface of the culture plate (non-polarized monolayer) or on a Millipore filter insert coated with extra-cellular matrix extract (polarized monolayer) that was then placed in the culture plate. Two-cell mouse embryos were cultured in G-1 ver3 medium to the eight-cell stage when they were randomly assigned to the treatments. The culture medium was G-2 ver3 during the treatment phase of the study. Significances of differences were evaluated by the one-way analysis of variance for continuous data. The epithelial cells cultured on Millipore filters became polarized and their morphology compared favorably with those cultured on the surface of the culture plate and in vivo uterine epithelial cells. After 96 h on the treatments, the polarized monolayer had supported the development of significantly more hatched blastocysts (80.0%; P<0.05) than the non-polarized monolayer (63.4%) or the control (61.4%) culture treatments. Co-culture resulted in the production of blastocysts with significantly more cells (non-polarized monolayer 56.7+/-2.1, polarized monolayer 61.9+/-2.1) than the control culture (42.8+/-2.6; P<0.05) but the diameter and shape of the blastocysts were not significantly different. The proportion of blastocysts with apoptotic blastomere was higher for the control culture (94.4%) than for the non-polarized (68.2%) or polarized (66.7%) co-culture systems (P<0.05). Moreover, the apoptotic index was significantly higher in control blastocysts (5.6+/-0.9; P<0.05) than in non-polarized (1.7+/-0.3) or polarized (1.5+/-0.3) co-culture. In the control, Bax mRNA was strongly expressed when compared to co-culture treatments (P<0.05), whereas, the relative abundance of Bcl-2 mRNA to the beta-tubulin was lower than co-culture treatments (P<0.05). It is concluded that a co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method of producing mouse embryos.     

小鼠胸腺皮质外位腺苷三磷酸酶的细胞化学定位

对探索细胞之间相互作用的机制,对ＢＡＬＢ／ｃ小鼠胸腺皮质内腺苷三磷酸酶进行了细胞化学定位。结果表明该酶活性主要位于毛细血管基膜,内此细胞皮膜和吞饮小泡;上皮性网状细胞和胸腺细胞的质膜外层,特别是二者的相邻面。巨噬细胞及上皮性网状细胞的溶酶和囊泡也具有此酶活性。本文对外位腺苷三磷酸酶有抑制腺苷三磷酸诱发胸腺细胞凋落死亡的可能性进行了讨论。     

Fas is expressed early in human thymocyte development but does not transmit an apoptotic signal.

We investigated the expression and function of Fas on human thymocytes prepared from fetal and pediatric tissue specimens and from SCID-hu Thy/Liv grafts. Unlike mouse thymocytes, human thymocytes exhibited a pattern of Fas expression skewed to immature cells, in that the highest expression was seen on double negative thymocytes and on intrathymic T progenitor cells. Fas expression was intermediate on double positive human thymocytes, and low or negative on mature single positive CD4 and CD8 medullary thymocytes. In spite of this relatively abundant surface expression, cross-linking of Fas with agonist mAb was incapable of triggering an apoptotic signal in human thymocytes. Apoptotic signaling was not enhanced by treatment with cycloheximide, nor by restoring a cosignaling milieu by addition of thymic stromal cells. Mouse thymocytes were induced to apoptosis by cross-linked recombinant soluble human Fas ligand both in vitro and in vivo, though human thymocytes were also resistant to this mode of receptor ligation. Membrane-bound Fas ligand also induced apoptotic death in murine thymocytes but not in human thymocytes. Human thymocytes were as sensitive as Jurkat cells, however, to apoptosis induced by TNF-alpha, suggesting that these cells have a signaling defect before activation of the earliest caspases. These data demonstrate a durable and specific resistance of human thymocytes to apoptosis induced through Fas receptor engagement, and reveal significant species-specific differences in the biology of thymocyte-programmed cell death.     

Lysosomal-mediated degradation of apoptotic thymocytes within thymic nurse cells

A thymic epithelial cell line (tsTNC-1) that maintains the ability to selectively bind and internalize immature alphabetaTCR(lo)CD4(+)CD8(+) thymocytes in vitro was used in long-term coincubation experiments to determine the ultimate fate of thymocytes that remained within intracytoplasmic vacuoles of thymic nurse cells (TNCs). In an earlier report, a subset of the population released from the TNC interaction was shown to mature to the alphabetaTCR(hi)CD69(hi) stage of development, while thymocytes that bided within the TNC cytoplasm died through the process of apoptosis. Here, we show the presence of both apoptotic and nonapoptotic thymocytes within the cytoplasm of freshly isolated TNCs as well as in tsTNC-1 cells in culture. A microscopic analysis revealed total degradation of the cytoplasmic apoptotic thymocyte population that remained in tsTNC-1 cells after an 8- to 10-h incubation period. A quantitative analysis showed an increase of cytoplasmic thymocyte degradation over time to almost 80% after 9 h of incubation. However, in the presence of bafilomycin A1, which is used to inhibit acidification of lysosomal vesicles, degradation of apoptotic thymocytes never reached 10%. These data suggest that lysosomes within TNCs play a role in the degradation of apoptotic thymocytes. We examined tsTNC-1 cells before the addition of thymocytes to cultures and found lysosomes to be clustered around the nucleus in the cytoplasm of TNCs. Shortly after the internalization event, apoptotic thymocytes move to the area of the cytoplasm containing lysosomes. Using the confocal microscope, we obtained evidence that shows the degradation event to be facilitated through the fusion of lysosomes with the specialized vacuoles within TNCs containing apoptotic cells.