Early block in maturation is associated with thymic involution in mammary tumor-bearing mice

We previously reported that mice implanted with mammary tumors show a progressive thymic involution that parallels the growth of the tumor. The involution is associated with a severe depletion of CD4+8+ thymocytes. We have investigated three possible mechanisms leading to this thymic atrophy: 1) increased apoptosis, 2) decreased proliferation, and 3) disruption of normal thymic maturation. The levels of thymic apoptosis were determined by propidium iodide and annexin V staining. A statistically significant, but minor, increase in thymic apoptosis in tumor-bearing mice was detected with propidium iodide and annexin V staining. The levels of proliferation were assessed by in vivo labeling with 5'-bromo-2'-deoxyuridine (BrdU). The percentages of total thymocytes labeled 1 day following BrdU injection were similar in control and tumor-bearing mice. Moreover, the percentages of CD4-8- thymocytes that incorporated BrdU during a short term pulse (5 h) of BrdU were similar. Lastly, thymic maturation was evaluated by examining CD44 and CD25 expression among CD4-8- thymocytes. The percentage of CD44+ cells increased, while the percentage of CD25+ cells decreased among CD4-8- thymocytes from tumor-bearing vs control animals. Together, these findings suggest that the thymic hypocellularity seen in mammary tumor bearers is not due to a decreased level of proliferation, but, rather, to an arrest at an early stage of thymic differentiation along with a moderate increase in apoptosis.     

Expression of nerve growth factor is upregulated in the rat thymic epithelial cells during thymus regeneration following acute thymic involution

Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. Nerve growth factor (NGF) is increasingly recognized as a potent immunomodulator, promoting "cross-talk" between various types of immune system cells. The present study describes the expression of NGF during thymus regeneration following acute involution induced by cyclophosphamide in the rat. Immunohistochemical stain demonstrated not only the presence of NGF but also its upregulated expression mainly in the subcapsular, paraseptal, and perivascular epithelial cells, and medullary epithelial cells including Hassall's corpuscles in both the normal and regenerating thymus. Biochemical data obtained using Western blot and RT-PCR supported these results and showed that thymic extracts contain NGF protein and mRNA, at higher levels during thymus regeneration. Thus, our results suggest that NGF expressed in these thymic epithelial cells plays a role in the T lymphopoiesis associated with thymus regeneration during recovery from acute thymic involution.     

Indomethacin inhibits thymic involution in mice with streptozotocin-induced diabetes

Diabetes is chronic disease that is accompanied by a rapid thymus involution. To investigate the factors responsible for thymic involution in a model of STZ-induced diabetes, mice were injected with STZ alone or in combination with the cyclooxygenase 2 inhibitor indomethacin (INDO). Thymus weight, glycemia and serum corticosterone were measured, and apoptosis in thymus and thymocyte cultures was analyzed by flow cytometry. Although earlier studies report that streptozotocin (STZ) is toxic to lymphoid tissues, in our experiments even massive doses of STZ did not negatively affect thymocyte cultures. Cultured thymocytes also seemed unaffected by high glucose concentrations, even after 24 h of exposure. Administration of INDO concomitantly with STZ reduced thymic involution but did not prevent the onset of hyperglycemia or reduce established hyperglycemia. When INDO was given before STZ, the same degree of thymic involution occurred; however, hyperglycemia was reduced, although normoglycemia was not restored. INDO also reduced serum corticosterone. Because thymocytes are known to be sensitive to glucocorticoids, this finding suggests that cyclooxygenase 2 inhibition may retard thymic involution by reducing serum glucocorticoids. In conclusion, our results show that STZ and hyperglycemia are not toxic to thymocytes and that cyclooxygenase 2-mediated mechanisms are involved in thymic involution during diabetes.     

Effect of T-activin on thymus involution in mice

The effect of T-activin on thymus involution in mice was studied. T-activin in a dose of 1.0 micrograms/mouse was injected into young male (CBA X C57BL)F1 mice weighing 24.0 +/- 2.0 g daily for 20 days. Morphometric analysis of the thymus was made 6 months after the treatment with T-activin was completed. It was found that T-activin induced the suppression of physiological involution of the thymus together with the enhancement of the processes of thymocyte transformation and proliferation.     

The human thymic microenvironment: cortical thymic epithelium is an antigenically distinct region of the thymic microenvironment

The thymic microenvironment is a complex tissue essential for normal T cell maturation. Prothymocytes in the subcapsular cortical (SCC) region of the thymus undergo cell division and migrate to the inner cortex. The majority of cortical thymocytes cease dividing and die, but a minority are exported to the periphery. We have previously shown thymic hormones in SCC and medullary thymic epithelium and have identified a monoclonal antibody (TE-4) that defines human endocrine thymic epithelium. However, no marker that selectively defines cortical thymic epithelium has been available. In this study, we have produced two monoclonal antibodies, TE-3A and TE-3B, raised against human thymic stroma that bind to an intracellular antigen in cortical but not medullary thymic epithelium. In double immunofluorescence assays in which we used anti-keratin, anti-thymosin alpha 1, and anti-endocrine thymic epithelium antibodies (TE-4, A2B5), TE-3+ SCC epithelium was TE-4+ and contained keratin and thymosin. alpha 1. In contrast, TE-3+ inner cortical epithelium was TE-4/A2B5 nonreactive and did not contain thymosin alpha 1. An ontogeny study of seven fetal and five neonatal thymuses demonstrated that expression of the TE-3 antigen was acquired at 10 wk fetal gestation. Using TE-3 antibody, we observed sequential stages of separation of cortical and medullary epithelium from 12 to 20 wk fetal gestation. In dysplastic (severe combined immunodeficiency disease) thymuses, strands of TE-3+ nonendocrine cells encircled nests of TE-4+ endocrine epithelium. Thus, human cortical thymic epithelium is antigenically distinct from endocrine medullary epithelium. Antibodies against the TE-3 antigen define an intracellular molecule that may reflect a specialized function of cortical thymic epithelium.     

Analysis of the human neonatal thymus: evidence for a transient thymic involution

The neonatal period is marked by the impairment of the major components of both innate and adaptive immunity. We report a severe depletion of cortical CD4+CD8+ double-positive thymocytes in the human neonatal thymus. This drastic reduction in immature double-positive cells, largely provoked by an increased rate of cell death, could be observed as early as 1 day after birth, delaying the recovery of the normal proportion of this thymocyte subset until the end of the first month of postnatal life. Serum cortisol levels were not increased in newborn donors, indicating that the neonatal thymic involution is a physiological rather than a stress-associated pathological event occurring in the perinatal period. Newborn thymuses also showed increased proportions of both primitive CD34+CD1- precursor cells and mature TCRalphabetahighCD69-CD1-CD45RO+/RAdull and CD45ROdull/RA+ cells, which presumably correspond to recirculating T lymphocytes into the thymus. A notable reinforcement of the subcapsular epithelial cell layer as well as an increase in the intralobular extracellular matrix network accompanied modifications in the thymocyte population. Additionally neonatal thymic dendritic cells were found to be more effective than dendritic cells isolated from children's thymuses at stimulating proliferative responses in allogeneic T cells. All these findings can account for several alterations affecting the peripheral pool of T lymphocytes in the perinatal period.     

Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy

The roles that thymus cytokines might play in regulating thymic atrophy are not known. Reversing thymic atrophy is important for immune reconstitution in adults. We have studied cytokine mRNA steady-state levels in 45 normal human (aged 3 days to 78 years) and 34 myasthenia gravis thymuses (aged 4 to 75 years) during aging, and correlated cytokine mRNA levels with thymic signal joint (sj) TCR delta excision circle (TREC) levels, a molecular marker for active thymopoiesis. LIF, oncostatin M (OSM), IL-6, M-CSF, and stem cell factor (SCF) mRNA were elevated in normal and myasthenia gravis-aged thymuses, and correlated with decreased levels of thymopoiesis, as determined by either decreased keratin-positive thymic epithelial space or decreased thymic sjTRECs. IL-7 is a key cytokine required during the early stages of thymocyte development. Interestingly, IL-7 mRNA expression did not fall with aging in either normal or myasthenia gravis thymuses. In vivo administration of LIF, OSM, IL-6, or SCF, but not M-CSF, i.p. to mice over 3 days induced thymic atrophy with loss of CD4+, CD8+ cortical thymocytes. Taken together, these data suggest a role for thymic cytokines in the process of thymic atrophy.     

Immunological changes in the thymus epithelial tissue of mice during accidental involution

Using sera containing antibodies to antigens of epithelial reticular cells it is possible to detect by immunofluorescence early stages of organ accidental involution and to trace parenchyma demasking that results from lymphocyte migration upon injection of prednisolone, azathioprin or their combinations. Prior to the drug administration only certain short thin processes of epithelial cells masked by a great number of lymphoid elements are detectable in the cortical zone of the thymus. Injection of 10-40 mg/kg prednisolone, 100-200 mg/kg azathioprin or their combinations causes stable different-degree demasking of the epithelial reticulum seen during immunofluorescence as a network of fluorescent processes. Administration of drug combinations also causes marked attenuation of the reaction, probably resulting from dystrophic changes in thymic epithelial cells.     

Rescue of embryonic lethality in hepatocyte growth factor/scatter factor knockout mice

While the targeted disruption of a gene is a powerful tool for investigating the physiological functions of that gene, disruption of a gene essential for embryogenesis leads to embryonic death. Rescue of the defect(s) causing embryonic death should promote survival, thus permitting further evaluation of the roles that the gene plays later in the developmental process. Disruption of the gene for mouse hepatocyte growth factor/scatter factor (HGF/SF) leads to middle-stage embryonic lethality because of a defect in placental development. Here we report that a single injection of HGF/SF at embryonic day 9.5 (E9.5) into the amniotic cavity of HGF/SF(-/-) embryos rescued the placental defect and resulted in the survival of the embryos until term. Histological analysis suggested that HGF/SF is also required at the late stage of development for tissue organogenesis. Thus, injection of a secreted factor can be a useful method to rescue the defects causing embryonic lethality.     

T cell recruitment from the thymus to the spleen in tumor-bearing mice

Summary After inoculation of tumor cells (methylcholanthrene-induced sarcoma), the number of Thy 1⁺ cells and PNA (peanut agglutinin) binding cells, which were shown to be different subpopulations were increased in the spleen of thymus-intact mice, in contrast this increase was not observed in adult thymectomized mice. In experiments performed concurrently with splenic cell analysis, we found that the plasma PGE₂ levels declined in parallel with the tumor growth. Prevention of such a decline of plasma PGE₂ level by replenishment with exogenous PGE₂ inhibited the splenic cell increase in tumor bearers. In the tumorbearing mice, cell traffic systems from the thymus to the periphery was ascertained by injecting fluorescein diacetate (FDA) into the thymus and observing fluorescein positive cells in the periphery. We suggest that increased recruitment of thymic cells to the periphery may be mediated by PGE₂ in the presence of a tumor.Abbreviations PNA⁺ cells, peanut agglutinin binding cells - Ig⁺ cells, surface IgM positive cells - Thy 1⁺ Thy 1.2 antigen positive cells - PGE₂ prostaglandin E₂     

Hepatocyte growth factor activator inhibitor type 1 is a specific cell surface binding protein of hepatocyte growth factor activator (HGFA) and regulates HGFA activity in the pericellular microenvironment

Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor in injured tissues. To date, two specific inhibitors of HGFA have been identified, namely HGFA inhibitor type 1 (HAI-1) and type 2 (HAI-2)/placental bikunin (PB). Both inhibitors are first synthesized as integral membrane proteins having two Kunitz domains and a transmembrane domain, and are subsequently released from cell surface by shedding. Here we show that an active form of HGFA is specifically complexed with membrane-form HAI-1, but not with HAI-2/PB, on the surface of epithelial cells expressing both inhibitors. This binding required the enzyme activity of HGFA. The selective binding of HGFA to the cell surface HAI-1 was further confirmed in an engineered system using Chinese hamster ovary cells, in which only the cells expressing HAI-1 retained exogenous HGFA. The binding of HGFA to HAI-1 was reversible, and no irreversible modifications affecting the enzyme activity occurred during the binding. Importantly, HAI-1 and the HGFA.HAI-1 complex were quickly released from the cell surface by treatment with phorbol 12-myristate 13-acetate or interleukin 1beta accompanying the generation of 58-kDa fragments of HAI-1, which are less potent against HGFA, as well as significant recovery of HGFA activity in the culture supernatant. This regulated shedding was completely inhibited by BB3103, a synthetic zinc-metalloproteinase inhibitor. We conclude that HAI-1 is not only an inhibitor but also a specific acceptor of active HGFA, acting as a reservoir of this enzyme on the cell surface. The latter property appears to ensure the concentrated pericellular HGFA activity in certain cellular conditions, such as tissue injury and inflammation, via the up-regulated shedding of HGFA.HAI-1 complex. These findings shed light on a novel function of the integral membrane Kunitz-type inhibitor in the regulation of pericellular proteinase activity.     

Changes in the electrophoretic mobility of thymus, spleen and lymph node cells in tumor-bearing mice during dynamic tumor growth

Using the analytic microscope "Parmoquant-2" (GDR), histograms were obtained demonstrating electrophoretic mobility (EPM) of lymphoid cells of C57BL/6 mice in the course of growth of the Lewis carcinoma (3LL) and melanoma B16 administered under the skin of the femur. Changes in the average values of EPM of thymic, splenic and lymph nodal cells in the process of tumor growth appeared similar. It is shown that the medium thymocyte EMP is growing towards the terminal stage of tumor growth, at the expense of the decrease in the share of PNA+ cells. Splenic cell bimodal distribution according to EPM became, in the course of tumor growth in intact mice, unimodal with some insignificant decrease in the median EPM values. The median EPM of regional and distant lymph nodes in the process of tumor growth is of phase character. It is supposed that investigation of lymph node EPM could be used for studying tumor growth kinetics.     

Effects of a thymic hormone,THF, on tumor-bearing mice and tumor-resected mice

Summary The administration of THF (thymus humoral factor) to mice bearing a chemically induced fibrosarcoma was followed by a significant improvement of the in vivo anti-tumor reactivity, as measured in the Winn assay, and of the in vitro response to PHA (phytohemagglutinin) of spleen cells. When THF treatment was given to mice after local resection of various metastasizing tumors, the subsequent death rate and survival time were not different from those in controls, and the anti-tumor reactivity of spleen cells of these THF-treated tumor-resected mice was not modified. Moreover, in contrast to tumor-bearing mice, a reduction in the PHA response of spleen cells from tumor-resected mice was noticed. The relevance of the immunologic status before THF treatment is discussed with reference to the present findings.Abbreviations THF thymus humoral factor - PHA phytohemagglutinin - cpm counts per minute - GvH graft-vs-host - MLC mixed lymphocyte culture - BSA bovine serum albumine - FS fibrosarcoma - SE standard error - SD standard deviation The Harold L. Korda Professor of Cancer Research     

Anti-inflammatory effects of hepatocyte growth factor: induction of interleukin-1 receptor antagonist

Hepatocyte growth factor (HGF) prevents liver failure in various animal models including endotoxin-induced acute liver failure. We were interested to find out whether human HGF exerts anti-inflammatory effects by modulation of cytokine synthesis. Therefore, human HepG2 cells were cultured with increasing concentrations of HGF. HGF dose-dependently upregulated the production of interleukin-1 receptor antagonist (IL-1Ra). Incubation of HepG2 cells with interleukin-1beta (IL-1beta) caused an increase in IL-1Ra levels, while interleukin-6 (IL-6) had no effect on IL-1Ra synthesis. Co-stimulation of HepG2 cells with HGF + IL-1beta resulted in a synergistic effect on IL-1Ra mRNA and protein expression. Stimulation of freshly isolated mouse hepatocytes from male C57 BL/6 mice with HGF increased IL-1Ra mRNA and protein synthesis dose-dependently. A co-stimulation with HGF and IL-1beta had a synergistic effect on IL-1Ra mRNA expression but only a partially additive effect on IL-1Ra protein synthesis. HGF-induced IL-1Ra production was significantly decreased by the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Accordingly, HGF stimulation specifically increased MAPK-dependent signalling pathway (p42/44). In contrast, in preactivated PBMC mRNA expression and protein synthesis of IL-1Ra, interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) were unaffected after stimulation with HGF. In conclusion, our data suggest that HGF exerts anti-inflammatory effects by modulating the signal transduction cascade leading to increased expression of IL-1Ra, which might explain the protective and regenerative properties of this cytokine in animal models of liver failure.