Marie-Paule Defresne

The Neurohormonal Thymic Microenvironment: Immunocytochemical Evidence that Thymic Nurse Cells Are Neuroendocrine Cells

Thymic neuroendocrine cells were identified by immunofluorescence in the murine thymus through the use of monoclonal antibody A2B5, and specific polyclonal antisera against neurophysin (NP), oxytocin (OT) and arginine vasopressin (AVP). Two reactive regions were clearly identified: the subcapsular cortex and the medulla. A close correspondence was observed between A2B5-reactive and NP-immunoreactive cells in the medulla. An important epithelial population of the subcapsular cortex, the thymic nurse cells (TNCs), were found to be A2B5-positive and to contain immunoreactive NP, OT and AVP. The neuroendocrine nature of TNCs was further substantiated by their high reactivity with an antiserum against neuron-specific enolase. These observations demonstrate the presence in the thymus gland of an original neuroendocrine microenvironment which could be of functional importance in the mediation of central influences upon T lymphocyte differentiation.

Neighboring adipocytes participate in the bone marrow microenvironment of multiple myeloma cells

Neighboring adipocytes participate in the bone marrow microenvironment of multiple myeloma cells

COLOCALIZATION OF IMMUNOREACTIVE OXYTOCIN, VASOPRESSIN AND INTERLEUKIN-1 IN HUMAN THYMIC EPITHELIAL NEUROENDOCRINE CELLS

Monoclonal antibodies to oxytocin (OT) and vasopressin (VP) revealed some positively staining stromal cells in the subcapsular cortex and in the medulla of the human thymus. We further demonstrated that these cells are a subset of epithelial endocrine cells and also contain immunoreactive interleukin-1 together with the neuropeptides. In addition, the thymic cells stained by monoclonal antibodies directed to the cyclic part of oxytocin or vasopressin also contained some immunoreactive neurophysins. These data support the concept of intrathymic synthesis of neurohypophyseal-like peptides fitting the hypothalamic model. However, we observed that, contrary to the situation in the brain, OT- and VP-like peptides colocalized in the same thymic cells. Furthermore, one monoclonal antibody, specific for the tail part of oxytocin, did not label thymic cells. Therefore, thymic neuropeptide(s) could be related to, but distinct from, authentic OT and VP. These observations suggest some molecular differences between hypothalamic and thymic oxytocin biosynthetic pathways which need to be further investigated.

Neurohypophysial Peptides Stimulate the Phosphorylation of Pre-T Cell Focal Adhesion Kinases

Thymic oxytocin (OT) behaves as a cryptocrine signal targeted at the outer surface of thymic epithelial cell plasma membrane from where OT is able to interact with neurohypophysial peptide receptors expressed by pre-T cells. Immature T cells bear a receptor of the V1 subtype, while OT receptors are predominantly expressed by cytotoxic CD8+ lymphocytes. In both T cell types, neurohypophysial peptide receptors transduce OT via the phosphoinositide pathway. Protein tyrosine phosphorylation is an early event of T cell activation. Western blots of murine pre-T cells (RL12-NP line) proteins probed with anti-phosphotyrosine (PY-20) revealed a great number of proteins the phosphorylation of which increased either with OT or vasopressin treatment. Two were immunoprecipitated with anti-focal adhesion kinase (FAK) mAb 2A7 and were identified one as p125FAK and the other as a coprecipitating 130-kDa protein. The p125FAK is connected to the Ras/MAPK pathway and is also implicated in TCR/CD3 signalling in T cell. Another protein phosphorylated by OT in RL12-NP was identified as paxillin, a 68-kDa protein localised at focal adhesion sites and associated with p125FAK. These results indicate that phosphorylation of focal adhesion kinase may be induced in pre-T cell by thymic OT.

Effect of nicotine on rat gingival fibroblasts in vitro

Tobacco smoking is considered a major risk factor for the development and progression of periodontal diseases (Haber, J. and Wattles, J. (1994). J. Periodontol. 64, 16-23). The purpose of this study was to determine the effects of nicotine on rat gingival fibroblasts (RGF) cultured in vitro. After ether anesthesia, rat gingival tissues were obtained from the attached gingiva of a Wistar rat. Small fragments of gingiva were maintained in culture in Petri dishes. Fibroblasts developing from these explants were collected to obtain monolayer cultures. After the fourth passage (T4), cells were supplemented with nicotine at various concentrations. Control and treated cells were examined under phase contrast or transmission electron microscopy. They were compared as regards their DNA content, mitochondrial activity, collagen and protein synthesis, and cell death by apoptosis or necrosis. Nicotine from 0.05 uM to 1 miVl did not affect the DNA content or protein and collagen synthesis. At concentrations between 3 and 5 niM. growth was significantly diminished and the survival rate reduced. Ultrastructural analysis revealed dilated mitochondria and vacuolization in treated cells, suggestive of necrosis, but increased apoptosis was also revealed by cytometry. On the basis of this in vitro study, it appears that tobacco, through its component nicotine, may directly affect various functions of RGF.

Analysis by in situ hybridization of cells expressing mRNA for tumor-necrosis factor in the developing thymus of mice.

We have used in situ hybridization to investigate the expression of TNF-α genes by thymic cells during fetal development in mice. In 14-day-old fetal thymuses, very scarce cells produce TNF-α mRNA. A second phase of cytokine gene expression starts on day 16. The density of positive cells progressively increases up to day 20. Thymuses at 15 days of gestation and after birth do not express detectable cytokine mRNA. In an attempt to identify the nature of the TNF-α mRNA-producing cells, acid phosphatase activity, which is characteristic of the macrophage lineage, was studied in the same thymuses. Acid phosphatase-positive cells only appear on day 15. Their frequency increases up to birth. However, no correlation can be established between acid phosphatase—and TNFα mRNA— positive cells. The results indicate that a small subset of thymic cells is responsible for TNF-α mRNA production during ontogeny: These cells are not yet identified. The possible role of TNF-α in thymic ontogeny is discussed.

Neuroendocrinology of the thymus

The neuropeptides oxytocin (OT) and vasopressin (VP) are synthesized in the human thymus in a similar way as in the hypothalamo-neurophypophyseal system. Immunocytochemistry with polyclonal and monoclonal antibodies revealed that immunoreactive OT- and VP-producing cells are localized in the subcapsular cortex and medulla of human and murine thymuses. The epithelial nature of the neuroendocrine thymic cells is demonstrated by their immunostaining with a monoclonal antibody against cytokeratin. An original example of a neuroendocrine-immune microenvironment is given by the thymic nurse cells which are composed of a large neuroendocrine epithelial cell enclosing numerous mitotic immature thymocytes. These observations and the previously reported mitogenic and immunomodulatory properties of VP and OT upon mature T cells and thymocytes strongly support the existence of a neuroendocrine thymo-lymphoid axis and an active role of thymic VP and OT in T cell differentiation and activation.

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.

Characterization of Spontaneous Bone Marrow Recovery after Sublethal Total Body Irradiation: Importance of the Osteoblastic/Adipocytic Balance

Many studies have already examined the hematopoietic recovery after irradiation but paid with very little attention to the bone marrow microenvironment. Nonetheless previous studies in a murine model of reversible radio-induced bone marrow aplasia have shown a significant increase in alkaline phosphatase activity (ALP) prior to hematopoietic regeneration. This increase in ALP activity was not due to cell proliferation but could be attributed to modifications of the properties of mesenchymal stem cells (MSC). We thus undertook a study to assess the kinetics of the evolution of MSC correlated to their hematopoietic supportive capacities in mice treated with sub lethal total body irradiation. In our study, colony-forming units – fibroblasts (CFU-Fs) assay showed a significant MSC rate increase in irradiated bone marrows. CFU-Fs colonies still possessed differentiation capacities of MSC but colonies from mice sacrificed 3 days after irradiation displayed high rates of ALP activity and a transient increase in osteoblastic markers expression while pparγ and neuropilin-1 decreased. Hematopoietic supportive capacities of CFU-Fs were also modified: as compared to controls, irradiated CFU-Fs significantly increased the proliferation rate of hematopoietic precursors and accelerated the differentiation toward the granulocytic lineage. Our data provide the first evidence of the key role exerted by the balance between osteoblasts and adipocytes in spontaneous bone marrow regeneration. First, (pre)osteoblast differentiation from MSC stimulated hematopoietic precursors proliferation and granulopoietic regeneration. Then, in a second time (pre)osteoblasts progressively disappeared in favour of adipocytic cells which down regulated the proliferation and granulocytic differentiation and then contributed to a return to pre-irradiation conditions.

Murine Bone Marrow Stromal Cells Sustain In Vivo the Survival of Hematopoietic Stem Cells and the Granulopoietic Differentiation of More Mature Progenitors

The study of the human hematopoietic system would be facilitated by availability of a relevant animal model. Because the medullar microenvironment is made of different types of cells, interactions between hematopoietic cells and stromal cells are difficult to analyze in detail. As an approach for establishing an in vivo model to dissect these interactions, we grafted murine bone marrow fibroblastic cells (MS‐5 cell line) with hematopoietic cells into the kidney capsule of syngenic mice. To identify the origin of cells present in the graft, we used green fluorescent protein–stable transfected MS‐5 cells for the transplantation. To analyze the evolution of stromal cells and identify hematopoietic cells able to develop in these conditions, we performed morphology, histochemistry, and immunohistology on tissue sections at different times after transplantation. When injected alone, MS‐5 cells differentiate into adipocytes. When injected with a bone marrow suspension or with isolated CD45+ cells (leukocytes), the stromal cells keep their fibroblastic morphology and their alkaline phosphatase expression and sustain granulopoiesis. When injected with hematopoietic stem cells called c‐kit+Sca‐1+Lin− suspension, clusters of hematopoietic cells are also observed: They do not present any granulopoietic activity and do not belong to B or T population nor to erythroid lineage. They are quiescent, induce bone marrow recovery and survival of lethally irradiated recipients, are able to form macroscopic colonies in the spleen, and are able to form very few colonies in vitro, suggesting that they are hematopoietic stem cells. In conclusion, our results show that reticular fibroblastic stromal cells MS‐5 sustain the survival of stem cells and are not able to induce their differentiation. However, they can control differentiation, proliferation, and/or survival of hematopoietic cells engaged in myeloid lineage.