Murine Thymus Research Articles

Cutting Edge: Presence of Progenitors Restricted to T, B, or Myeloid Lineage, but Absence of Multipotent Stem Cells, in the Murine Fetal Thymus

AbstractThe most immature population of fetal thymus (FT) cells has been shown to generate not only T but also B and myeloid cells. The present study was undertaken to clarify whether such a multipotent activity of the earliest population of FT cells is attributed to multipotent hemopoietic progenitors or to a mixture of lineage-restricted progenitors. Examination of individual FT progenitors by a recently established clonal assay system, which is able to determine the developmental potential of each progenitor toward T, B, and myeloid lineages, elucidated that a large majority of progenitors in FT were restricted to the T cell lineage. Presence of a small number of B or myeloid lineage-restricted progenitors was also disclosed. No multipotent progenitors, however, were detected in FT. These results are consistent with our recent finding that restriction of hemopoietic stem cells to T, B, and myeloid lineages takes place in the fetal liver.

CDNA Cloning and Functional Characterization of the Mouse Ca2+-gated K+ Channel, mIK1: ROLES IN REGULATORY VOLUME DECREASE AND ERYTHROID DIFFERENTIATION

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.

Pancreatic Hormone Expression in the Murine Thymus: Localization in Dendritic Cells and Macrophages

Pancreatic Hormone Expression in the Murine Thymus: Localization in Dendritic Cells and Macrophages

Pancreatic hormone expression in the murine thymus: localization in dendritic cells and macrophages.

The expression of preproinsulin (ppIns), proglucagon, prosomatostatin, and propancreatic polypeptide was investigated in thymic extracts, thymic cells, and thymic cell lines from C57BL/6 mice by RT-PCR. The expression of pancreatic hormones was similar in thymic extracts taken from neonatal and 2-, 4-, and 8-week-old animals, but was decreased in 20-week-old animals. Pancreatic hormone expression was not observed in mouse liver, salivary gland, or spleen. Analysis of thymic cell populations revealed a 10- to 20-fold enrichment in expression of all hormones in low buoyant density cells. No expression was detected in high buoyant density cells (predominantly thymocytes) or in thymic epithelial cell lines, primary cultures of epithelial cells, or peripheral macrophages. In addition, immunoreactive insulin, measured by specific RIA, was detectable in the low buoyant density population, but not in high buoyant density cells. The enriched cell population was depleted of contaminating lymphocytes and sorted based on reactivity to the cell surface markers F4/80 (macrophage) or N418 (dendritic cells). Cells gated for N418 demonstrated expression for ppIns, but not the other pancreatic hormones. Conversely, expression for proglucagon, prosomatostatin, and propancreatic polypeptide, but not ppIns, was detected in F4/80-gated cells. Our data indicate that pancreatic endocrine hormones are differentially expressed by dendritic cells and macrophages in a normal mice.

Selective regulation of T-cell development and function by calcitonin gene-related peptide in thymus and spleen. An example of differential regional regulation of immunity by the neuroendocrine system.

In the course of our studies, we have shown the presence of calcitonin gene related peptide (CGRP) by immunocytochemistry in cell bodies and nerve fibers of the murine thymus and in a sparse innervation of the spleen. Receptors for CGRP have been characterized within these glands, and their activation by physiological levels of CGRP was found to suppress Con A-stimulated proliferation of thymocytes and splenic T cells as well as antigen-specific T-cell proliferation. This suppression is blocked by the antagonist for CGRP (CGRP 8-37). Within the thymus cultures, the antagonist CGRP (8-37) alone enhanced proliferation of thymocytes during Con A stimulation, most likely by inhibiting the endogenous release of CGRP into the culture medium by resident thymocytes. Some of the CGRP-induced suppression of mitogenic stimulation of thymocytes, but not of splenocytes, was due to apoptosis. The antagonist, CGRP(8-37), did not block apoptosis caused by Con A or CGRP but rather enhanced it. Flow cytometric analysis of CGRP-treated cultures using antibodies to cluster determinates (CD) showed that the majority of thymocytes undergoing apoptosis induced by CGRP were of the CD4/CD8 double-positive type. These data indicate that apoptosis in the thymocytes is mediated by a CGRP receptor not sensitive to the antagonist CGRP(8-37). Because proliferation of thymocytes and splenocytes induced by Con A is blocked by this antagonist and splenocytes are refractory to CGRP induced apoptosis, CGRP appears to mediate at least two separate functions on subpopulations of thymocytes and T cells via two different CGRP receptors within the gland. These effects of a neuropeptide exemplify the phenomenon of differential regional regulation of immunity by the autonomic and neuroendocrine systems.

High CD30 Ligand Expression by Epithelial Cells and Hassal's Corpuscles in the Medulla of Human Thymus

AbstractCD30 is a member of tumor necrosis factor (TNF) receptor superfamily that is expressed by activated T cells in the presence of interleukin-4 (IL-4). Although CD30 can mediate a variety of signals, CD30-deficient mice have impaired negative selection of T cells, suggesting that at least in the context of murine thymus, CD30 is a cell death–mediating molecule. The ligand for CD30 (CD30L) is a membrane-associated glycoprotein related to TNF, which is known to be expressed mainly by activated T cells and other leukocytes. However, the nature of CD30L-expressing cells involved in the interaction with CD30+ thymocytes is unclear. We report here that in postnatal human thymus the great majority of CD30+ cells are double positive (CD4+CD8+), activated, IL-4 receptor–expressing T cells which selectively localize in the medullary areas. Moreover, many medullary epithelial cells and Hassal's corpuscles in the same thymus specimens showed unusually high expression of CD30L in comparison with other lymphoid or nonlymphoid tissues. These findings provide additional information on the nature and localization of CD30+ thymocytes and show that epithelial cells are the major holder of CD30L in the thymic medulla.

High CD30 Ligand Expression by Epithelial Cells and Hassal's Corpuscles in the Medulla of Human Thymus

CD30 is a member of tumor necrosis factor (TNF) receptor superfamily that is expressed by activated T cells in the presence of interleukin-4 (IL-4). Although CD30 can mediate a variety of signals, CD30-deficient mice have impaired negative selection of T cells, suggesting that at least in the context of murine thymus, CD30 is a cell death–mediating molecule. The ligand for CD30 (CD30L) is a membrane-associated glycoprotein related to TNF, which is known to be expressed mainly by activated T cells and other leukocytes. However, the nature of CD30L-expressing cells involved in the interaction with CD30+ thymocytes is unclear. We report here that in postnatal human thymus the great majority of CD30+ cells are double positive (CD4+CD8+), activated, IL-4 receptor–expressing T cells which selectively localize in the medullary areas. Moreover, many medullary epithelial cells and Hassal's corpuscles in the same thymus specimens showed unusually high expression of CD30L in comparison with other lymphoid or nonlymphoid tissues. These findings provide additional information on the nature and localization of CD30+ thymocytes and show that epithelial cells are the major holder of CD30L in the thymic medulla.

Bax in Murine Thymus Is a Soluble Monomeric Protein That Displays Differential Detergent-induced Conformations

Bcl-2, Bcl-XL, and Bax are members of the Bcl-2 family that play important roles in apoptosis regulation. These proteins are believed to be membrane-bound and to regulate apoptosis through formation of homo- and heterodimers. However, we recently found by subcellular fractionation that whereas Bcl-2 is predominantly a membrane protein as previously reported, Bax and a significant fraction of Bcl-XL are soluble in thymocyte and splenocyte extracts. In addition, we have demonstrated that the ability of Bax to form dimers appears to be a detergent-induced phenomenon that coincides with a detergent-induced conformational change. We have further investigated the tertiary and quaternary states of Bax in the presence of various detergents. Detergents such as Triton X-100 and Triton X-114 readily enable Bax hetero- and homodimerization. However, other detergents such as polydocanol, W-1, octyl glucoside, dodecyl maltoside, Tween 20, and sodium cholate allow varying degrees of Bax hetero- and homodimerization. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Brij 35 allow neither hetero- nor homodimer formation. Immunoprecipitation analysis with the conformation-sensitive antibody uBax 6A7 revealed that whereas Triton X-100 readily exposes the N-terminal Bax epitope (amino acid 13-19), only limited exposure of the epitope occurs in Triton X-114, polydocanol, dodecyl maltoside, and sodium cholate, and no exposure of this epitope was observed in W-1, Chaps, octyl glucoside, Tween 20, and Brij 35. Moreover, we could not detect any proteins associated with the cytosolic form of Bax based on immunopurification of this protein. Sephacryl S-100 gel filtration chromatography analysis of the cytosolic Bax indicated that this protein is monomeric and displays an apparent molecular mass of 25 kDa. Induction of apo-ptosis which causes the insertion of the soluble form of Bax into membranes did not result in appreciable Bax/Bcl-XL, Bax/Bcl-2 or Bax/Bax dimer formation as determined by cross-linking studies. Further analysis of Bax after apoptosis induction by immunoprecipitation in the presence of Chaps also revealed no significant heterodimer formation. In conclusion, Bax displays several distinct states in different detergents that expose defined regions of the protein. In addition, these results suggest that mechanisms other than the simple dimerization among members of the Bcl-2 family may be required for the regulation of apoptosis.

Fetal and neonatal NK1.1+ Ly-49- cells can distinguish between major histocompatibility complex class I(hi) and class I(lo) target cells: evidence for a Ly-49-independent negative signaling receptor.

Natural killer (NK) cell function is regulated by both positive and negative signaling receptors. In adult splenic NK cells, negative signaling has been shown to be mediated by the Ly-49 family receptors. NK1.1- 2B4+ CD3- cells that are phenotypically and functionally similar to adult splenic NK cells can be derived from murine fetal liver and thymus. These cells do not express any known Ly-49 molecules on their surface nor do they contain the known Ly-49 transcripts. Surface expression of Ly-49 molecules is first detected on splenic NK1.1+ cells 4-6 days after birth. Despite the absence of these negative signaling receptors, fetal and neonatal Ly-49- NK cells lyse TAP-/- , major histocompatibility complex (MHC) class I(lo) but not TAP+/+, MHC class I(hi) target cells. This suggests that fetal and neonatal NK cells express negative signaling receptors other than Ly-49 molecules.

Calmodulin-Dependent Protein Kinase IV during T-Cell Development

In a primary cell culture system of fetal rat brain, the calmodulin-dependent protein-kinase IV (CaMKIV) could be induced by the thyroid hormone T3in a time- and concentration-dependent manner, provided the tissue was excised not later than day 15 of gestation (E15) (Krebset al., J. Biol. Chem.271, 11055, 1996). We report here that in the fetal thymus CaMKIV could not be detected earlier than day 16 of gestation and that the expression of this enzyme was fully upregulated at day 18. In mouse fetal thymus organ culture (FTOC) of day 14 embryonic thymus, CaMKIV could not be detected, even after several days of culture if a minimal culture medium lacking fetal calf serum was used. However, after addition of fetal calf serum to the culture medium the expression of CaMKIV could be specifically induced. Furthermore, it could also be shown that during T-cell development in the adult murine thymus the expression of CaMKIV was tightly regulated. Taken together, these results demonstrate that the expression of CaMKIV, an enzyme involved in the regulation of Ca2+-dependent gene expression, is itself under stringent regulatory control during tissue development.

Dexamethasone Modulates IL-13 and IL-10 Expression

Interleukin (IL-)-10 and IL-13 are Th2-cell-associated cytokines with a variety of biologic activities in immune and inflammatory responses. It is known that glucocorticoids (GCs) modulate inflammatory and immune functions. In fact, GCs are involved to regulate the transcription of cytokines which are relevant in chronic inflammation and cell-mediated immune response. In the present study we analyzed, in vitro, the effects of DEX on the expression of the IL-10 and IL-13 lymphokines in murine spleen and thymus cells. DEX-stimulation induced down-regulation of the expression of IL-10 and IL-13 mRNA. This effect was already evident 0.5 hr after treatment and persisted in time, in both resting and activated lymphocytes. These results suggest that GCs could have inhibitory effect on Th2 cytokine production.

Cytokine gene expression during ontogeny in murine thymus on activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds and activates the aryl hydrocarbon receptor (Ah-R), an endogenous transcription factor that is expressed in the thymus. TCDD exposure leads, among other effects, to thymus atrophy and immunosuppression. We previously analyzed the interference of TCDD with differentiation processes in fetal thymus organ cultures and found that in the presence of TCDD, the proliferation rate of immature (CD4- CD8- and CD4- CD8+ HSA+) thymocytes is inhibited, whereas the maturation along the CD4/CD8 path is accelerated. Moreover, the differentiation of thymocytes is skewed by TCDD at < or = 40% (compared with approximately 15% without TCDD) of the CD8 single-positive subset of future cytotoxic T cells, and apparently more cells audition for and pass positive selection. The fetal murine thymus expresses functional Ah-R mRNA, as shown by reverse transcription-polymerase chain reaction and TCDD-inducible CYP1A1 and CYP1B1 expression. Because the differentiation of thymocytes is to a considerable extent controlled by cytokines and many cytokine genes are potential targets of the Ah-R due to Ah-R-binding elements (xenobiotic response elements) in their promoters, we analyzed the cytokine expression in fetal thymus organ culture exposed to TCDD. Fetal thymi were cultured from gestation day 15 for < or = 8 days, thus covering ex vivo the period after population of the thymus anlage until birth. We show with semiquantitative reverse transcription-polymerase chain reaction that more interleukin (IL)-1beta, IL-2, IL-6, tumor growth factor (TGF)-beta3, and tumor necrosis factor-alpha are produced in TCDD-exposed thymi, whereas other cytokines (e.g., TGF-beta1, PAI-2, or IL-4) are only slightly up- and down-modulated during the culture period or not modulated at all (e.g., IL-1beta, IL-7, interferon-gamma, and TGF-beta2).

Molecular analysis of mouse T cell receptor expression using PCR.

This unit describes the use of the polymerase chain reaction (PCR) to characterize rearranged murine T cell receptor (TCR) transcripts present in primary lymphoid tissues, peripheral lymphoid tissues, and extra-lymphoid sites of inflammation, and to quantify them either relatively or absolutely. This methodology has been used extensively to characterize TCR variability in murine thymus and liver in T cell clones, and in extralymphoid tissues where the absolute number of T cells is limited. A procedure for cloning and sequencing PCR-amplified cDNA transcripts is provided to examine the junctional diversity of expressed genes. Also included is a method for reverse transcription of RNA into cDNA that is optimal for analysis of tissue samples where the number of T cells is limited. The detailed protocols are followed by a commentary that discusses strategies and artifacts as well as providing troubleshooting suggestions for PCR amplification and TCR analysis.

The winged-helix transcription factor Trident is expressed in cycling cells.

We describe the cloning and characterization of Trident , a novel member of the fork head/winged-helix family, from murine thymus. In the mouse embryo, the gene was expressed in all tissues, whereas in adult mice expression was only detected in the thymus. Further analysis revealed that Trident expression strictly correlated with cell cycling, independent of cell type. Timing of [3H]thymidine incorporation showed that mRNA and protein expression were strongly upregulated upon entry into the S phase of the cell cycle. Moreover, the protein was phosphorylated in M phase. PCR-mediated selection of optimal binding sites yielded a consensus motif resembling that of other family members. These results identify Trident as a transcription factor, which is likely involved in cell cycle-specific gene regulation.

Studies on fibroblasts and matrix molecules in the cortex of murine thymus

Studies on fibroblasts and matrix molecules in the cortex of murine thymus

Studies on fibroblasts and matrix molecules in the cortex of murine thymus

Studies on fibroblasts and matrix molecules in the cortex of murine thymus

Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments.

In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho-stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cell-specific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4-4, TB4-20, and TB4-28. While TB4-4 and TB4-20 are both epithelium specific, TB4-28 detects an epitope expressed on both epithelial- and mesenchymal-derived stromal cells. TB4-4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4-20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.

Glucocorticoid-Induced Thymocyte Death in the Murine Thymus: The Effect at Later Stages.

Although glucocorticoid has been considered to cause thymocyte apoptosis in vitro, few studies have presented its in vivo effect. We report here on kinetics of glucocorticoid-induced murine thymocyte death in vivo by the TUNEL method. TUNEL-positive cells were observed as early as at 2 h after intraperitoneal injection of glucocorticoid. Most TUNEL-positive thymocytes were phagocytosed by acid phosphatase positive macrophages. "Free" (not phagocytosed) TUNEL-positive cells were not detected at early stages (by 4 h). At 6 to 8 h after the injection, the number of phagocytosed thymocytes per individual macrophage had reached its maximum, and at 8 to 12 h many ruptured macrophages ingesting too many dying thymocytes became noticeable. During the process, no additional macrophages appeared to be mobilized to the thymus. At 6 to 8 h after the injection, however, coincidentally with the fact that macrophages had become unable to further ingest dying lymphocytes, dead cells were left unphagocytosed, and ultimately became "free" positive cells, probably due to some proteolytic process ongoing within the thymus. As late as at 12 h, morphological examination revealed that epithelial cells seemed to begin engulfing thymocytes, almost simultaneously with the start of rupture of the macrophages due to the ingestion of too many thymocytes. Epithelial cells were readily identified by desmosomes and tonofilaments, in addition to euchromatic nuclei. Altogether, these results suggest that: 1) even though thymocytes were exposed to glucocorticoid in vivo, most of them were not TUNEL-positive unless they were phagocytosed; 2) even after most macrophages had ingested too many cells at later stages, macrophages in other locations did not migrate to the thymus; and finally, 3) deletion of damaged thymocytes was also carried out by thymic epithelial cells, though not frequently, at around 12 h and later.

Thymic overexpression of CD40 ligand disrupts normal thymic epithelial organization.

We characterized the distribution of CD40 and CD40 ligand (CD40-L) in the adult and developing murine thymus. Before birth, CD40 was almost exclusively localized to scattered foci of medullary cells. By birth there was a dramatic upregulation of CD40 expression by cortical epithelial cells, which was accompanied by a consolidation of medullary epithelial foci. CD40-L+ thymocytes displayed a medullary location. Analysis of mice deficient in CD40-L expression indicated that CD40-L/CD40 interactions were not required for development of the medullary compartment. Overexpression of CD40-L targeted to thymocytes altered thymic architecture, as reflected by a dramatic loss of cortical epithelial cells, expansion of the medullary compartment, and extensive infiltration of the capsule with a mixture of CD3+ cells, B-cells, and macrophages/dendritic cells. Reconstitution of lethally irradiated normal mice with lck CD40-L bone marrow cells also resulted in loss of cortical epithelium and expansion of the medullary compartment. Disruption of the normal pattern of thymic architecture and epithelial differentiation as a consequence of increased intrathymic levels of CD40-L expression points to a role for CD40-L/CD40 interactions in the normal pattern of epithelial compartmentalization/differentiation within the thymic environment.

Analysis of fluorescence lifetime and quenching of FITC-conjugated antibodies on cells by phase-sensitive flow cytometry.

Fluorescent antibodies are often used to measure the number of receptor sites on cells. The quantitative estimate of the number of receptor sites using this procedure assumes that the fluorescence intensity on a cell is proportional to the number of bound antibodies. Quenching may invalidate this assumption. For many fluorophores, intermolecular interactions and energy transfer between molecules in close proximity to one another results in self-quenching. This effect can occur in antibody probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity antibodies relative to one another on a highly labeled cell surface. Since self-quenching is accompanied by a change in the fluorescence decay and a decrease in the fluorescence lifetime, it may be conveniently identified using fluorescence lifetime spectroscopy. In this paper we apply the phase-sensitive detection method to investigate the impact of self-quenching on fluorescence lifetimes by flow cytometry, using a model system consisting of FITC conjugated anti-mouse Thy1.2 antibodies bound to murine thymus cells. We show that in addition to the expected variation of lifetimes as a function of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labeling concentration on the cell surface. This is consistent with self-quenching effects expected at high densities of FITC molecules.