The neuroendocrine thymus: coexistence of oxytocin and neurophysin in the human thymus.

Immunoreactive oxytocin and neurophysin were identified and measured by radioimmunoassay in human thymus extracts. Serial dilutions of extracts paralleled the appropriate standard curves. Thymus-extracted oxytocin and neurophysin eluted in the same positions as reference preparations on Sephadex G-75. Authenticity of oxytocin was confirmed by biological assay and high-performance liquid chromatography analysis. In most instances, thymus contents of oxytocin and neurophysin were far greater than those expected from known circulating concentrations and declined with increasing age. The molar ratio of oxytocin to neurophysin in thymus was similar to that found in the hypothalamo-neurohypophyseal system, which strongly suggested with the other data a local synthesis of oxytocin. These findings indicate the presence of neurohypophyseal peptides in the human thymus and further support the concept of a neuroendocrine function integrated in an immune structure.

Effects of marrow grafting on preleukemia cells and thymic nurse cells in C57BL/Ka mice after a leukemogenic split-dose irradiation.

A split-dose regimen of whole-body irradiation (4 X 175 rad at weekly intervals) induced thymic lymphomas in C57BL/Ka mice after a latent period of 3-9 months. Meanwhile, preleukemia cells arose in the thymus and bone marrow and persisted until the onset of lymphomas. Simultaneously, thymic lymphopoiesis was impaired; thymocyte numbers were subnormal and thymic nurse cells disappeared in a progressive but irreversible fashion. The depletion of these lymphoepithelial complexes, which are normally involved in the early steps of thymic lymphopoiesis, was related to altered prothymocyte activity in bone marrow and to damaged thymic microenvironment, perhaps as a consequence of the presence of preleukemia cells. The grafting of normal bone marrow cells after irradiation prevented the development of lymphomas. However, marrow reconstitution did not inhibit the induction of preleukemia cells. They disappeared from the thymus during the second part of the latent period. At the same time, thymic lymphopoiesis was restored; thymocytes and nurse cell numbers returned to normal as a consequence of the proliferation of grafted marrow-derived cells within the thymus. The results thus demonstrated an intimate relationship between preleukemia cells and an alteration of thymic lymphopoiesis, which particularly involved the nurse cell microenvironment. Some preleukemia cells in marrow-reconstituted, irradiated mice derived from the unirradiated marrow inoculate. Thus these cells acquired neoplastic potential through a factor present in the irradiated tissues. The nature of this indirect mechanism was briefly discussed.

Prevention of murine radiogenic thymic lymphomas by tumor necrosis factor or by marrow grafting.

BACKGROUND: Split-dose irradiation (1.75 Gy given weekly for 4 weeks) of C57BL/Ka mice induces the emergence of preleukemic cells (PLCs). These cells develop into leukemic cells after a latency period of 3-6 months. The survival and transformation of PLCs are dependent on radiation-induced alterations of the thymic epithelium and of resident lymphocyte (i.e., thymocyte) subpopulations in the thymus. PLCs can be eliminated, concomitantly with the restoration of the thymus, by grafting bone marrow cells immediately after the last irradiation. Our hypothesis was that any agent able to restore the thymus after leukemogenic irradiation would exert the same effects as a bone marrow graft. Tumor necrosis factor-alpha (TNF-alpha) is one such possible agent, since it has been shown to modulate some functions of the thymic epithelium and thymocyte subpopulations. PURPOSE: The goal of this study was to assess the ability of repeated intraperitoneal injections of TNF-alpha to functionally replace bone marrow transplantation in the restoration of normal intrathymic lymphopoiesis and in the prevention of thymic lymphomas in split-dose-irradiated mice. METHODS: We replaced the bone marrow graft with repeated injections of TNF-alpha (25 000 U/injection) in the split-dose-irradiated (4 x 1.75 Gy) C57BL/Ka mouse model. We analyzed the expression of the cell differentiation markers CD4 and CD8 on thymocytes by flow cytometry. We also studied the thymic environment by isolating thymic nurse cells, the bone marrow prothymocyte activity by analyzing thymic repopulation, and the evolution of PLCs by an in vivo transplantation assay. Local production of TNF-alpha after bone marrow grafting was examined by in situ hybridization. Injections of anti-TNF-alpha antibodies were given to split-dose-irradiated mice to test the effect of neutralizing TNF-alpha in vivo. One-way analysis of variance and Newman-Keuls two-tailed tests were used to test statistical significance. RESULTS: Multiple injections of TNF-alpha into split-dose-irradiated mice did not influence bone marrow prothymocyte activity but restored thymocyte subpopulations and thymic epithelium, induced the disappearance of PLCs, and prevented the development of lymphomas. Moreover, a bone marrow graft significantly stimulated intrathymic production of TNF-alpha messenger RNA (P<.01), and anti-TNF-alpha antibodies partially inhibited the antilymphomatous effects of bone marrow graft in split-dose-irradiated mice (P<.05). CONCLUSION: These data strongly suggest that TNF-alpha is a mediator that is involved in the mechanisms by which bone marrow transplantation functions to prevent thymic lymphomas in split-dose-irradiated mice. IMPLICATIONS: Cytokines might be used in some biological systems, particularly in the hemopoietic system, as a therapeutic agent for the secondary prevention of cancer.

Correlation of alkaline phosphatase activity to normal T-cell differentiation and to radiation leukemia virus-induced preleukemic cells in the C57BL mouse thymus.

Cytochemical methods at the light and electron microscopic level were used to define the pattern of alkaline phosphatase (APase) activity in normal thymus and to study its modifications after inoculation with the thymotropic leukemogenic radiation leukemia virus in correlation with the emergence of preleukemic cells and their thymus dependency. APase was found in numerous lymphoblasts of the fetal thymus. The enzyme was also detected in a few lymphoid blast cells of the normal young adult thymus, which were closely associated with thymic nurse cells. The observed distribution of APase in normal thymus suggests that its expression could be limited to an early stage of the T-cell differentiation pathway. After inoculation with radiation leukemia virus, APase activity remained normal for almost the entire latency period, during which virus replication spread to the cortex and thymus-dependent preleukemic cells appeared. An important increase in the number of APase-positive cells occurred later, i.e., at the end of the latency period, in nontumoral thymus, which displayed lymphocytic depletion and contained autonomous thymus-independent preleukemic cells. These latter features obviously reflected the malignant transformation of thymus lymphoblasts, which eventually led to the development of the thymic lymphomas. The results raise the question of the possible filiation between the thymic nurse cell-associated APase-positive lymphoid cells of the normal thymus and the target cells susceptible to productive infection and to neoplastic transformation after radiation leukemia virus infection.

Nuclear localization of a new c-cbl related protein, CARP 90, during in vivo thymic apoptosis in mice.

This study investigates the involvement of the c-cbl protooncogene in thymocyte apoptosis occurring in vivo after hydrocortisone treatment. In the thymus of untreated mice, a few medullary and cortical thymocytes expressed p120cbl, mainly in the cytoplasm. In the cortex, their number and distribution resemble that of apoptotic cells evidenced by TUNEL staining. The expression of Cbl is rapidly increased when apoptosis is triggered by hydrocortisone. This Cbl-specific immunostaining was detected in the nucleus and is due to a Cbl-related 90 kDa protein (CARP 90). These results show that a c-cbl product could localize in the nucleus and suggest that it could be involved as a regulator of thymic apoptosis.

Tumor necrosis factor and interferon gamma inhibit the development of radiation-induced thymic lymphomas in C57BL/Ka mice.

Recombinant tumor necrosis factor and/or gamma-interferon were injected into C57BL/Ka mice after completion of a whole body split dose irradiation, which usually induces thymic lymphomas in more than 90% of the animals. The survival and the incidence of thymic lymphomas were significantly reduced in the cytokine-injected irradiated mice. The protective effect was similar to that obtained by grafting normal bone marrow cells after irradiation. The mechanisms of lymphoma inhibition by TNF or IFN-gamma are discussed.

Further studies on the mechanism of radiation induced thymic lymphoma prevention by bone marrow transplantation in C57BL mice.

Whole body fractionated irradiation induces thymic lymphomas in C57BL/Ka mice after a latent period during which intrathymic lymphopoiesis is modified; thymocyte numbers are subnormal and the epithelial component of thymic nurse cells (TNCs) is altered as estimated by the number of TNCs in vivo and by its ability to interact with immature thymocytes in vitro. A graft of normal bone marrow cells immediately after the last irradiation prevents the development of lymphomas; but when such a graft is performed 1 month later, it does not inhibit the emergence of tumors. In both cases the grafted precursors home and repopulate the thymus. However, the delayed graft does not exert any effect upon the altered epithelial component of TNCs, whereas the early one restores the numbers of TNCs and the function of their epithelial component. The results thus demonstrate that lymphoid thymic repopulation by a bone marrow graft is not sufficient to prevent the development of lymphomas and that there is an intimate relationship between tumor development and alterations of nurse cells microenvironment.

Mixed phenotype murine leukemias.

Cell lines were derived from eight individual leukemias induced by X-rays in NFS mice. First typed as null cells (surface immunoglobulin negative, Thy-1 negative), they turned out to have a mixed phenotype with myeloid cytochemical markers, pre-B surface antigens and molecular markers of pro-B lymphocytes. They represent murine models for mixed phenotype (pro-pre-B-myeloid) leukemias.

Involvement of insulin-like growth factors in early T cell development: a study using fetal thymic organ cultures.

The expression of insulin-like growth factor (IGF) and IGF receptor genes was investigated by RT-PCR during ontogeny of the murine thymus. IGF-1, IGF-1R, M6P/IGF-2R genes are expressed in the thymus both in fetal and postnatal life, whereas IGF-2 messenger RNAs (mRNAs) decline after birth but are still detectable on the seventh week. By in situ hybridization, IGF-2 transcripts were located in the outer cortex and medulla of the postnatal thymus, and on the whole surface ofthe epithelial-like network in the fetal thymus. The effects of anti-IGFs and IGF-receptors neutralizing Abs on the generation of pre-T cell subpopulations were then investigated using fetal thymic organ cultures (FTOC). FTOC treatment with an anti-IGF-2 mAb, an anti-IGF-1R mAb, or an anti-M6P/IGF-2R polyclonal Ab induced a blockade of T cell differentiation at the CD4-CD8- stage, as shown by a significant increase in the percentage of CD4-CD8- cells and a decrease in the percentage of CD4+CD8+ cells. Moreover, anti-IGF-2 Ab treatment induced an increase in CD8+ cells suggesting that thymic IGF-2 might have a role in determining differentiation into the CD4 or CD8 lineage. Anti-IGF-1 Ab treatment decreased the proportion in CD4-CD8- cells and increased the frequency in CD4+CD8+. FTOC treatment with anti-(pro)insulin did not exert any significant effect on T cell development. These data indicate that the intrathymic IGF-mediated signaling plays an active role in the early steps of T cell differentiation during fetal development.

Identification of nuclear triiodothyronine receptors in the thymic epithelium.

Thymic epithelial cell physiology is known to be under neuroendocrine control. In particular, thyroid hormones modulate thymic hormone secretion by thymic epithelial cells in vivo and in vitro, thus suggesting the existence of specific receptors for those hormones in this component of the thymic microenvironment. Yet, thyroid hormone-binding sites have previously been detected only in crude thymus fractions and lymphocytes. We, thus, decided to search for T3 receptors in the thymic epithelium, by using an antinuclear T3 receptor monoclonal antibody. In situ immunohistochemical analysis of thymic frozen sections showed nuclear labeling of both lymphoid and nonlymphoid cells in the cortex and medulla. Moreover, in vitro studies using thymic epithelial cell lines and the so-called thymic nurse cells revealed a positive reaction in the chromatin, with nucleoli remaining negative. Immunoblot data clearly showed a single protein band of 57K reactive with the antinuclear T3 receptor antibody in murine thymus extracts as well as in the thymic epithelial cell lines. Lastly, in vitro treatment of these cells with T3 resulted in a transient, yet profound, down-modulation of the receptor. In conclusion, our findings provide molecular evidence that the action of thyroid hormones on thymic epithelium occurs via the typical 57K nuclear T3 receptors.

Isolation of follicular dendritic cells from human tonsils and adenoids. I. Procedure and morphological characterization.

Follicular dendritic cells have been isolated from human tonsils and adenoids and characterized at the ultrastructural level. Follicles were dissected and digested with different hydrolytic enzymes. The cells were separated by sedimentation at unit gravity. By this procedure we obtained follicular dendritic cells enveloping lymphocytes with their cytoplasmic extensions in a way analogous to that described for isolated thymic nurse cells. The ultrastructural features of isolated follicular dendritic cells are similar to those observed in situ. Prolonged enzymatic action caused loss of the enveloped lymphocytes.

Target cells and thymus microenvironment in the pathogenesis of thymic lymphomas in C57BL/Ka mice.

In C57BL/Ka mice, the induction of thymic lymphomas either by inoculation of radiation leukemia virus (RadLV) or by a split dose irradiation requires complex cellular events: Target cells are found among the population of thymic subcapsular blast cells, or, alternatively, of marrow or spleen prothymocytes; Progression of target cells to lymphoma growth requires a multi-step process, which occurs only within thymic microenvironment; Target cells are rapidly induced as "preleukemic" cells; After inoculation of RadLV, the initial events occur when target cells are in close association with cells of a specialized component of thymic epithelium, i.e., the so-called "nurse cells"; The leukemogenic agents induce damages to the thymic microenvironment itself; Lymphoma prevention by marrow grafting after irradiation results from mechanisms still unknown which inhibit the progression of "preleukemic" cells to neoplastic growth.

Cellular aspects of radiation leukemogenesis in C57 BL/Ka mice: alterations to thymic microenvironment and lymphopoiesis.

After a leukemogenic split dose course of irradiation, thymic nurse cells (TNCs) disappear. We have correlated this with the loss of an epithelial cell surface antigen (recognized by monoclonal antibody ER-TR3 and tentatively identified as Ia). In addition, epithelial cells have lost their capacity to interact with fetal thymocytes in vitro. Marrow grafting early after irradiation, that prevents the development of lymphomas, restores thymic nurse cells and thymocyte population. Such reconstitution and lymphoma prevention were not observed when marrow grafting was performed later (1 month after irradiation) during the preleukemic period.

Thymic nurse cells account for the thymus dependency of preleukemic cells in mice after inoculation of radiation leukemia virus.

Inoculation of Radiation Leukemia Virus (RadLV) into C57BL/Ka mice induces thymic lymphomas after a 3-6 month latent period. The leukemogenic process requires a sequence of events from the productive infection of susceptible target cells and induction of preleukemic cells to irreversible neoplastic transformation. Preleukemic cells were detected in the thymus during the first week following virus injection. The thymus dependency of these cells was shown to depend transiently upon peculiar lymphoepithelial complexes called "Thymic Nurse Cells" (TNCs). Indeed, the first preleukemic cells appearing in the RadLV-inoculated thymuses were observed selectively within TNCs. They remained closely associated with these complexes during the first 2 or 4 weeks. Later on, TNCs disappeared almost completely whereas non-TNCs associated preleukemic cells were found. Lymphoepithelial interactions within TNCs were thus required for the initial events of RadLV-induced lymphomagenesis. The subsequent TNCs depletion expressed a disturbance of thymic lymphopoiesis in relation with the neoplastic process.

Phenotype of thymic lymphomas in the mouse.

The MP2 cell line was established from a murine leukemia virus-induced thymic lymphoma. Half of the cells were consistently L3T4 positive and less than 5% of the cells were Lyt-2 positive. Single cell cloning on the basis of the presence or absence of Lyt-2 allowed the isolation of four clones with stable phenotypes: (1) Lyt-2-, L3T4-; (2) Lyt-2+, L3T4+; (3) Lyt-2-, L3T4+; (4) Lyt2+, L3T4-. These data are discussed in relation to tumour cell heterogeneity and to normal T-cell differentiation pathways.

Thymic nurse cells are the first site of virus replication after inoculation of the radiation leukemia virus.

The induction of thymic lymphomas by inoculation of the Radiation Leukemia Virus (RadLV) requires interactions between RadLV, lymphoid cells and thymus microenvironment. The possible localization of this interaction within the peculiar lymphoepithelial complexes called 'thymic nurse cells' (TNCs) has been investigated. Electron microscopic studies, as well as in vitro experiments using a very sensitive infectious centre detection assay demonstrated that most of the first virus producing cells after RadLV inoculation are located within the TNCs. Most of these structures belong to the thymus subcapsular zone. They contain lymphoid cells with the phenotype of the major (cortical) thymocyte population. Data support the view that a limited subpopulation of subcapsular immature thymocyte can act as specific targets for productive infection with RadLV. Furthermore, the initiation of virus replication appears related to the interaction between the immature thymocyte and the 'nurse cells' microenvironment.

Evidence of recombinant ecotropic provirus integration in thymic lymphomas induced by direct or indirect radiation effects.

Several investigators described the occurrence of ecotropic recombinant proviruses in the DNA of in-vivo or in-vitro propagated radio-induced lymphomas, but such proviruses were never detected in primary tumors. To assess their biological significance in the tumorigenic process, we reinvestigated the presence of new proviruses chiefly in primary radio-induced tumors and in models of radioleukemogenesis which could give additional support for their role. Such models included thymic lymphomas originating after (i) graft of non-irradiated thymuses in thymectomized irradiated mice and (ii) the injection of a B-ecotropic retrovirus (T1223/B) in association with a subleukemogenic dose of irradiation. We report for the first time that new ecotropic proviral sequences are encountered in a significant number (30%) of primary lymphomas induced directly by irradiation or indirectly in non-irradiated thymuses grafted in irradiated hosts. The existence of a 3.5-kbp Kpn1 restriction fragment with ecotropic sequences in the digested DNA of these tumor cells indicates that these new sequences belong to an ecotropic provirus recombinant in the gag-pol region. We observed that most of the primary radio-induced tumors in which novel recombinant provirus could be detected, displayed the integration at a single or at a few sites, demonstrating their clonality with respect to viral integration. The same was observed in thymic lymphomas arising after T1223/B virus injection and irradiation and in in-vivo or in-vitro propagated tumors. Altogether, these data bring the first evidence of the integration of ecotropic recombinant proviral genomes in a significant number of primary radiation induced thymic lymphomas and of their possible role in view of their frequent occurrence in grafted thymomas.

p53, Bax and Bcl-2 in vivo expression in the murine thymus after apoptogenic treatments.

BACKGROUND: Neoplasia can results from a lack of cell elimination by apoptosis. In order to determine if mechanisms controlling apoptosis are disturbed during neoplastic transformation in a model of murine radio-induced thymic lymphomas, we have assessed the kinetics of p53, Bax and Bcl-2 in situ expression after induction of thymic apoptosis by irradiation or glucocorticoids at first in normal mice. MATERIALS AND METHODS: TUNEL method was used for in situ detection of apoptosis and protein expression was determined by indirect immunohistochemistry. RESULTS: After hydrocortisone injection, levels of p53 and Bax, but not Bcl-2, expression were raised. A whole body sublethal irradiation led to an increase of p53 and Bcl-2, but not Bax, expression. CONCLUSIONS: This is the first in vivo report of in situ protein expression in the thymus after apoptogenic treatments of mice. The results suggest that Bax could be involved in glucocorticoid-mediated apoptosis. The increased levels of Bcl-2 expression are discussed.

Apoptosis during the development of radiogenic thymic lymphomas: effects of treatments inhibiting lymphoma development.

INTRODUCTION: Whole body fractionated irradiation induces thymic lymphomas in C57BL/Ka mice after a latent period during which preleukemic cells progressively transform into leukemic cells within an abnormal thymic microenvironment. A bone marrow graft or repeated cytokine injections prevent lymphoma development. We think that these treatments restore altered mechanisms controlling apoptosis. MATERIALS AND METHODS: Apoptosis was analyzed by flow cytometry in thymocytes from different groups of mice (control, preleukemic, prevented mice). RESULTS: The apoptotic rates did not change in freshly isolated thymocytes from different experimental groups. However, after culture, the level of apoptosis increased in preleukemic thymuses; and returned to normal value in cultured thymocytes from irradiated mice after lymphoma preventing treatments. Furthermore, thymic microenvironmental factors can control thymocyte apoptosis. CONCLUSION: We propose that after leukemogenic irradiation, there is an increase of cells with an activated suicide program, but that alterations of thymic environmental factors rescue them from apoptosis, allowing their further neoplastic transformation.

Marrow stromal cell recovery after radiation-induced aplasia in mice.

PURPOSE: The identification of fibroblast-like cells of the marrow stroma by means of alkaline phosphatase (ALP) cytochemistry reveals delicate ALP-positive structures interspersed among haematopoietic cells and arranged in a loosely meshed network. These cells are often referred to as 'reticular' cells and the network they form is known as the 'ALP network'. The purpose was to analyse the evolution of this ALP network in relation to haemopoietic regeneration after whole-body irradiation. MATERIALS AND METHODS: The total surface occupied by ALP-positive processes revealed by means of ALP cytochemistry was expressed as a ratio of the total marrow area. ALP-positive cells were counted using nuclei as the defining unit. Cell proliferation was analysed by the detection of bromodeoxyuridine (BrdU) incorporation. Fat cells were identified by oil red O staining and alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity. RESULTS: The ALP network and ALP-positive cell number began to increase 24 h after 4-Gy irradiation to reach a maximum after 72 h, when the bone marrow was almost completely empty of haemopoietic cells. This increase was in advance of haemopoietic recovery and was not due to cell proliferation. A decrease in the ALP network occurred in parallel with an increase in haemopoiesis and was accompanied by a transient increase in fat cells on day 7. CONCLUSIONS: These data indicate that the recovery of the ALP network, which is partially due to the recruitment of ALP- positive cells, occurs in advance of the haemopoietic recovery and that the equilibrium between fat cells and ALP-positive cells seems to be controlled by haemopoietic cells.