Role of Vascular Endothelial Growth Factor (VEGF) in Thymus of Mice under Normal Conditions and with Tumor Growth.

In our study, we for the first time investigated a role for VEGF as a factor regulating transendothelial migration of murine thymocytes in vitro. Effects of VEGF were examined in a model of thymocyte migration across a monolayer of EA.hy 926 endothelial cells. We showed that VEGF enhanced transendothelial migration of murine thymocytes and their adhesion to endothelial cells in a dose-dependent manner. VEGF did not influence thymocytes, but rather acted on endothelial cells by upregulating surface expression of adhesion molecule ICAM-1 and downregulating activity of 5'-nucleotidase. Effects from VEGF were comparable with those from TNF-α. Because it is known that administration of VEGF to intact animals results in thymic atrophy, it was assumed that it might play a role in developing thymic involution during tumor growth. Enhanced egress of thymocytes to the periphery was considered as a plausible mechanism underlying effects of VEGF. However, we revealed no difference in parameters of in vitro transendothelial migration for thymocytes from animals bearing a transplantable hepatoma 22a compared to control animals. VEGF mRNA expression in lysates of thymic stroma was found to be upregulated in mice with grafted tumors, whereas at the protein level the amount of VEGF did not differ. While examining expression of VEGF receptors on thymocytes by flow cytometry, both VEGFR-1 and VEGFR-2 were not detected, whereas the percentage of Nrp-1-positive thymocytes in animals with hepatoma 22a was as high as in the control group. Thus, we were unable to confirm a hypothesis regarding participation of VEGF in developing thymic involution during progression of experimental hepatoma. However, a set of novel data concerning a role for VEGF in stimulating transendothelial migration of thymocytes in vitro was obtained, and it may be of significance for understanding mechanisms underlying thymus functioning as well as a role of this cytokine in preparing endothelial cells for egress of thymocytes to the periphery.

REGULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) PRODUCTION BY MOUSE THYMIC EPITHELIAL CELL LINES.

Vascular endothelial growth factor (VEGF) is synthesized in small amounts by thymus epithelial cells in adults and plays a role in supporting vascular homeostasis. However its role becomes dramatically important during the process of thymus reparation after involution caused by chemo-, x-ray or hormonal therapy. The aim of the study was to evaluate the influence of different factors on VEGF production by mouse thymic epithelial cells in vitro. As a model two cell lines were used: cortical cTEC1-2 and medullar mTEC3-10 cells. These cells were characterized by their ability to synthesize VEGF mRNA and protein as well as by their expression of VEGF receptors. VEGFR1 and VEGFR2 mRNA expression in these cells were absent while NRP-1 mRNA revealed low level of expression. It was shown by ELISA that cTEC1-2 cells produced VEGF about 30 times more than mTEC3-10. When cultivated in the presence of cytokines, hormonal factors or thymocytes, both cell lines responded differently. Introduction of keratinocyte growth factor (KGF) induced VEGF mRNA expression as well as VEGF production in medullar cells but simultaneously down-regulated VEGF mRNA expression in cortical cells. Dexamethasone suppressed mRNA VEGF expression and VEGF production in cortical cells while in medullar cells only VEGF production was reduced. Introduction of IL-7, IL-1b or murine thymocytes increased while addition of Semaphorin 3A, SDF-1a or ACTH decreased VEGF production by cortical epithelial cells with no influence on medullar cells. We suggest that our data obtained in vitro can be used for further development of special programs for directed regulation of VEGF synthesis in the thymus epithelial cells in the vivo.

Innate immunity underlies symbiotic relationships.

Here, the modern data regarding interactions between normal microbiota and barrier tissues in plants, humans and animals are reviewed. The main homeostatic mechanisms responsible for interactions between epithelium and innate immune cells with symbiotic bacteria are described. A key step in this process is recognition of soluble microbial products by ligation to pattern-recognition receptors expressed on the host cells. As a result, epithelial cells secrete mucus, antibacterial peptides and immunoregulatory molecules. The main outcomes from immunological reactions towards symbiotic bacteria involve development of conditions for formation and maintenance of microbial biocenosis as well as providing safety for the host. Also, it is considered important to preserve and transfer beneficial bacteria to progeny.

Effects of defensin and lactoferrin on functional activity of endothelial cells in vitro.

We studied the effects of antibacterial peptides and proteins (defensins and lactoferrins) on functional activity of endothelial cells in vitro: proliferative activity and adhesion of human endothelial ECV-304 cells to the matrix were evaluated, alpha-Defensin (NP-2) from rabbit neutrophils, total alpha-defensin (HNP 1-3) from human neutrophils, and lactoferrins from porcine neutrophils and human milk were studied. Defensins stimulated and lactoferrin in doses of 1-10 microg/ml inhibited proliferation and adhesion of endothelial cell. The stimulatory effect of defensins on proliferation and adhesion was reproduced in fibroblast culture. Lactoferrins did not modify proliferation of fibroblasts, but suppressed their adhesion. These data suggest that antibiotic proteins and peptides are prospective objects for the creation of drugs regulating angiogenesis.

Gene expression for VEGF-A, VEGF-C, and their receptors in murine lymphocytes and macrophages.

Vascular endothelial growth factors VEGF-A and VEGF-C are the main angiogenic factors that control growth of new blood and lymphatic vessels in the organism, and they also possess several immunoregulatory activities. Expression of VEGF-A and VEGF-C mRNA as well as mRNA for VEGF receptors in lymphocytes and macrophages of naive mice was investigated. Using reverse transcription and subsequent polymerase chain reaction, we found that peritoneal macrophages, thymocytes, and lymph node cells constitutively expressed VEGF-A and VEGF-C mRNA. In addition, macrophages were positive for VEGFR-1, VEGFR-2, VEGFR-3, NRP-1, and NRP-2 mRNA, whereas thymocytes and lymph node cells expressed mRNA of the same receptors except VEGFR-1. These data expand our knowledge concerning gene distribution of VEGF receptors in the organism, in particular, among the cells of the immune system. This suggests that, along with their major angiogenic properties, VEGF family members additionally might also perform important mediatory functions within the immune system.

Early macrophage and cytokine response during the growth of immunogenic and non-immunogenic murine tumors.

Very little data exist on the mechanisms of innate immunity during the first days after syngeneic tumor inoculation. Nonspecific macrophage reaction precedes the development of specific immune response and is important for further tumor growth and stroma formation. We investigated two lymphoma cell lines of the same origin, differing in immunogenicity: non-immunogenic parental strain P388 and its highly immunogenic subline P388/adria. Early systemic inflammatory response resulted in the enhancement of nitric oxide (NO) and superoxide production by peritoneal macrophages which was at a maximum on the first day after s.c. tumor inoculation and was observed in mice bearing either of these tumors independently of immunogenicity. It was followed by a transient elevation of the serum levels of pro-inflammatory cytokines: TNF-alpha IL-6. In order to evaluate the role of inflammatory response, vaccinations with lethally irradiated lymphoma cells were performed. After two weekly injections, the mice were challenged s.c. with live tumor cells of the same subline. Effective vaccination with P388/adria lymphoma cells induced retardation of tumor growth in parallel with down-regulation of peritoneal macrophage activity and abrogation of serum cytokine release. Non-effective immunization with P388 cells influenced neither tumor growth nor macrophage functions and cytokine level. Thus, a positive correlation was found between down-regulation of the inflammatory response and inhibition of tumor growth. We suppose that, in efficiently immunized mice, special mechanisms exist which are responsible for down-regulation of the inflammatory reaction. Macrophage products may facilitate tumor cell survival by preventing apoptosis or participate in the activation of tumor neoangiogenesis. Suppression of these activities may serve as an important tool for the inhibition of tumor growth at the early stages of malignant transformation.

Involvement of macrophages and cytokines into rejection mechanism of the drug-resistant and immunogenic murine lymphoma P388/adria.

Macrophages and their products may exert either inhibitory or stimulatory effects on malignant cells,thus preventing or supporting tumor growth, however, the mechanisms of this interaction are not fully understood. It was the aim of the present study to elucidate the role of macrophage activation during the growth and rejection of highly immunogenic murine leukemia P388/adria cell line which was made resistant by suboptimal treatment of mice with adriablastin during the serial passaging of parental P388 cells. The functional activity of peritoneal macrophages and the serum level of cytokines IL-1 beta, IL-6 and TNF-alpha were studied in different groups of mice. Mice from group 1 (control) received saline. Mice from group 2 (tumor bearers) with fast subcutaneous (s.c) 100% tumor growth were compared with animals from group 3 that had been twice previously immunized with lethally irradiated P388/adria cells and later inoculated with viable tumor cells. Tumors grew in only 25% of group 3 animals with a significant delay. The activity of peritoneal macrophages was studied by NO2- production and the NBT-test. Both tests revealed the early high systemic activation of macrophages in group 2. This coincided with the elevation of serum TNF-alpha and IL-6 levels. This effect was not dependent on whether alive or lethally irradiated tumor cells were inoculated. The NO2- production by peritoneal macrophages correlated well with the dynamics of serum cytokine levels while the NBT-test did not. Studies on group 3 showed total abrogation of early macrophage and cytokine reactions. The production of inhibitory factors by macrophages in previously immunized mice is suggested. The fact that the early activation of macrophages and increase of serum levels of proinflammatory cytokines occurred in animals with fast growing tumors, which was decreased or absent in animals with tumor delay or rejections, allows us to suppose that this reaction plays more a supporting than a protecting role for tumor growth.

Transcortin does not restrict the transmembrane transfer of cortisol.

We have studied the specific binding of both free and transcortin-bound cortisol to the microvesicles derived from the brush border of the plasma membrane of human placental syncytiotrophoblast. Kinetics of the steroid binding to these microvesicles was found to be independent on cortisol being complexed with transcortin. Both cortisol and transcortin were accumulated in the inner space of the microvesicles. This suggests that transcortin-cortisol complex penetrates the plasma membrane and the transcortin-bound steroid can thus enter syncytiotrophoblast and exert its hormonal effects on this tissue.