Keratin 8 is required for the maintenance of architectural structure in thymus epithelium.

Keratins (Ks), the intermediate filament (IF) proteins of epithelia, are coordinately expressed as pairs in a cell-lineage and differentiation manner. Cortical thymic epithelial cells (cTECs) predominantly express the simple epithelium keratin 8/18 (K8/K18) pair, whereas medullary thymic epithelial cells (mTECs) express the stratified epithelium K5/K14 pair, with TECs exhibiting K5 and K8 at the cortico-medullary junction in mature thymus. In the work reported here, we used wild-type (WT) and K8-knockout (K8-null) mice to address the contribution of K8/K18 IFs in the maintenance of the thymic epithelial structure. K8-null thymus maintained the differential cell segregation at the cortex versus the medulla observed in WT thymus, and the distribution of immature thymocytes at the cortex. The K8/K18 loss did not affect thymocyte development. However, it massively perturbed the TEC morphology both at the cortex and the medulla, along with a prominent depletion of cTECs. Such tissue alterations coincided with an increase in apoptosis and a reduced expression of Albatross (Fas-binding factor-1), also known for its capacity to bind K8/18 IFs. In addition, the K8/K18 loss affected the distribution of K5/K14-positive mTECs, but not their differentiation status. Together, the results indicate that K8/K18 IFs constitute key promoters of the thymic epithelium integrity.

TGF-ß type II receptor expression in thymic epithelial cells inhibits the development of Hassall's corpuscles in mice.

Hassall's corpuscles are concentric clusters of keratinized epithelial cells located within the thymic medulla of humans and guinea pigs but are scant in mouse and rat. They are considered to be the terminally differentiated stages of medullary thymic epithelial cells (mTECs) but the mechanisms of their origin are unclear. We have previously deleted the TGF-ß type II receptor (TGFßRII) specifically in mouse TECs and reported that these mice have mitigated thymic involution and exhibit earlier reconstitution post-irradiation. In this study, we analyzed the differentiation of mTECs in the TGFßRII-knockout mice. Interestingly, the TGFßRII-knockout mice display enhanced development of Hassall's corpuscles. The expression of Aire, stromal-cell-derived factor 1 and thymic stromal lymphopoietin in the thymi of the TGFßRII-knockout mice was similar to that previously reported for the human thymus. In addition, the putative epithelial progenitor markers MTS20 and MTS24 labeled Hassall's corpuscles in normal mice, but the extent and intensity of this staining were greatly enhanced in Hassall's corpuscles of the TGFßRII-knockout mice. The phosphorylated forms of ERK and JNK were also found in Hassall's corpuscles of the TGFßRII-knockout mice. Taken together, we suggest that TGFßRII-mediated signaling in TECs inhibits their development into Hassall's corpuscles in mice.

Online reporting system for transfusion-related adverse events to enhance recipient haemovigilance in Japan: a pilot study.

BACKGROUND: A surveillance system for transfusion-related adverse reactions and infectious diseases in Japan was started at a national level in 1993, but current reporting of events in recipients is performed on a voluntary basis. A reporting system which can collect information on all transfusion-related events in recipients is required in Japan. METHODS: We have developed an online reporting system for transfusion-related events and performed a pilot study in 12 hospitals from 2007 to 2010. RESULTS: The overall incidence of adverse events per transfusion bag was 1.47%. Platelet concentrates gave rise to statistically more adverse events (4.16%) than red blood cells (0.66%) and fresh-frozen plasma (0.93%). In addition, we found that the incidence of adverse events varied between hospitals according to their size and patient characteristics. CONCLUSION: This online reporting system is useful for collection and analysis of actual adverse events in recipients of blood transfusions and may contribute to enhancement of the existing surveillance system for recipients in Japan.

RhoB deficiency in thymic medullary epithelium leads to early thymic atrophy.

RhoB, a member of the Rho subfamily of small GTPases, mediates diverse cellular functions, including cytoskeletal organization, cell transformation and vesicle trafficking. The thymus undergoes progressive decline in its structure and function after puberty. We found that RhoB was expressed in thymic medullary epithelium. To investigate a role of RhoB in the regulation of thymic epithelial organization or thymocyte development, we analyzed the thymi of RhoB-deficient mice. RhoB-deficient mice were found to display earlier thymic atrophy. RhoB deficiency showed significant reductions in thymus weight and cellularity, beginning as early as 5 weeks of age. The enhanced expression of TGF-ß receptor type II (TGFßRII) in thymic medullary epithelium was observed in RhoB-null mice. In addition, the expression of fibronectin, which is shown to be regulated by TGF-ß signaling, was accordingly increased in the mutant thymic medulla. Since there is no age-related change of RhoB expression in the thymus, it is unlikely that RhoB in thymic epithelium directly contributes to age-related thymic involution. Nevertheless, our findings strongly support a physiological role of RhoB in regulation of thymus development and maintenance through the inhibition of TGF-ß signaling in thymic medullary epithelium.

Localization of mesenchymal cells in adult mouse thymus: their abnormal distribution in mice with disorganization of thymic medullary epithelium.

Thymic mesenchymal cells are known to be important for the development of the early fetal thymus into a functionally mature organ supporting T cell differentiation. We examined the expression of mesenchymal markers: pan-mesenchymal marker ER-TR7, desmin, alpha-smooth muscle actin (alpha-SMA), and alpha- and beta-chain of platelet-derived growth factor receptor (PDGFRalpha, PDGFRbeta) in thymi of normal adult mice. Desmin and ER-TR7 revealed specific staining in the capsule, septa, and perivascular cells. Most perivascular cells highly expressed PDGFRbeta at the same levels as desmin. Low expression of PDGFRalpha was detected in the capsule, intralobular septa, and some perivascular cells of normal adult thymi. alpha-SMA, used to identify vascular smooth muscle cells, was detectable on arterioles and some large venules but not on capillaries. Thus, desmin, PDGFRalpha, and PDGFRbeta were localized in the capsule, septa, and perivascular cells in thymus of adult mouse, although there were differences in the expression level among these markers. On the other hand, the expression of mesenchymal markers was detectable in the region of the thymic medullary epithelium of lymphotoxin beta receptor-deficient mice and plt/plt mice, indicating that mesenchymal cells were abnormally localized in the region. These results suggest that disorganization of the medullary epithelium may be accompanied by aberrant distribution of mesenchyme in adult mouse thymus.

Distribution of lymphatic vessels in mouse thymus: immunofluorescence analysis.

Thymic blood and lymphatic vessels in humans and laboratory animals have been investigated in morphological studies. However, occasionally a clear distinction between blood vessels and lymphatic vessels cannot be made from morphological characteristics of the vasculature. To visualize thymic lymphatics in normal adult BALB/c mice, we used antibodies against specific markers of lymphatic endothelial cells. Expression of vascular endothelial growth factor receptor-3 (VEGFR-3) was detected throughout the thymus, i.e., the capsule, cortex, and medulla. Most thymic lymphatics were present in capillaries of ~20 mum in caliber. The plexuses of lymphatic capillaries were occasionally detectable. Lymphatic vessels were frequently adjacent to CD31-positive blood vessels, and some lymphatic vessels were seen in the immediate vicinity of or within the perivascular spaces around postcapillary venules. The identity of VEGFR-3-positive vessels as lymphatics was further confirmed by staining with additional markers: LYVE-1, Prox-1, neuropilin-2, and secondary lymphoid tissue chemokine (SLC). The distributions of LYVE-1 were similar to those of VEGFR-3. Most lymphatic vessels were also identified by Prox-1. Neuropilin-2 was restricted to lymphatic vessels in the thymus. The most abundant expression of SLC in the thymus was in medullar epithelial cells; SLC was also expressed in lymphatic vessels and blood vessels. Thus, lymphatic endothelium in mouse thymus was characterized by positive staining with antibodies to VEGFR-3, LYVE-1, Prox-1, neuropilin-2, or SLC, but not with an antibody to CD31. Our results suggest the presence of lymphatic capillary networks throughout the thymus.

Expression of stromelysin-3 (matrix metalloproteinase-11) in macrophages of murine thymus following thymocyte apoptosis.

High expression of stromelysin-3 (ST-3), also known as matrix metalloproteinase-11, has been implicated in tumor progression and intense tissue remodeling. Nonetheless, details of the cell type(s) expressing ST-3 are less well defined. Here, we report that ST-3 expression was elevated in mouse thymus following thymocyte apoptosis after administration of anti-CD3 Ab. TUNEL analysis revealed that many thymocytes in the cortical region were induced to apoptotic cell death 14 h after the injection. After an additional 2-6 h, ST-3 expression in the thymus was more apparent. Co-staining analysis by anti-ST-3 and F4/80 Abs showed that most F4/80-positive macrophages were also positive for ST-3. Murine peritoneal macrophages were found to constitutively express ST-3, and exposure to apoptotic cells hardly affected ST-3 expression in the macrophages. Taken together, our results indicate that ST-3 is not involved in the execution process of thymocyte apoptosis, and the increased levels of ST-3 in the thymus may be due to the presence of macrophages responsible for clearance of apoptotic cells.

Matrix metalloproteinase-9 in macrophages induces thymic neovascularization following thymocyte apoptosis.

Matrix metalloproteinase-9 (MMP-9) has been implicated in the degradation of the extracellular matrix in a variety of physiological and pathological processes. We found that MMP-9 expression in thymuses of BALB/c mice that had been injected with anti-CD3 Ab to induce thymocyte apoptosis was increased both at mRNA and protein levels. Macrophages are shown to be the principal stromal cells responsible for phagocytosis of dying thymocytes, and macrophages were found to constitutively express MMP-9. The activity of plasmin, which is known as one of the activators for MMP-9, was increased in the thymuses with MMP-9 activation. Binding of Ab HUIV26, which recognizes a cryptic epitope on collagen type IV following proteolytic cleavage, was found to be reduced in MMP-9 knockout mice, suggesting that collagen type IV is a substrate of MMP-9. Although the formation of thymic neovessels was found following thymocyte apoptosis, it was diminished in anti-CD3 Ab-injected MMP-9 knockout mice. In vivo administration of Ab HUIV26 resulted in a reduction of thymic neovascularization. After clearance of apoptotic thymocytes, the number of macrophages in the thymuses was decreased, and this decrease was delayed by blocking of HUIV26 epitope. Taken together, our results suggest that MMP-9 expression in macrophages mediates degradation of collagen type IV and facilitates their migration from the thymus after clearance of apoptotic thymocytes. These studies demonstrate a potential role of macrophage MMP-9 in the remodeling of thymic extracellular matrix following thymocyte apoptosis.

Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response.

Macrophage-derived secretory leukocyte protease inhibitor (SLPI) can be induced locally as well as systemically in response to microbial products such as LPS and lipotechoic acid. It is not known whether phagocytosis of apoptotic cells, an essential function of macrophages, can regulate expression and secretion of SLPI. In this study, we report that exposure of peritoneal macrophages of BALB/c mice or murine macrophage cell lines RAW264.7 and J774.1 to apoptotic target cells induced an elevation in SLPI secretion. Secreted SLPI retained its antichymotrypsin activity. SLPI expression in thymuses from BALB/c mice that had been injected with anti-CD3 Ab to induce apoptosis of thymocytes was also elevated both at the mRNA and protein levels. Colchicine, a microtubular inhibitor, blocked the internalization of apoptotic cells by macrophages but not SLPI secretion, suggesting that surface recognition of apoptotic cells is sufficient for the induction of SLPI. Exposure of RAW264.7 cells to apoptotic CTLL-2 cells induced both SLPI and TNF-alpha, and addition of IFN-gamma inhibited SLPI but augmented TNF-alpha production. Transfection of either the secreted or a nonsecreted form of SLPI into RAW264.7 cells led to suppression of TNF-alpha production in response to apoptotic cells. Thus, macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells.

Murine T cells expressing high activity of prolyl endopeptidase are susceptible to activation-induced cell death.

Prolyl endopeptidase (PEP) is widely distributed and thought to play an important role in the degradation of peptide hormones and neuropeptides, but its biological role is totally unknown. In this study, we examined PEP activity in subpopulations of murine T cells and found that PEP activity was significantly higher in immature thymocytes than in mature thymocytes or in peripheral T cells. Stimulation of murine peripheral T cells time-dependently increased PEP activity. Although murine T cell hybridomas exhibited high PEP activity, the PEP activity was fully inhibited by treatment with PEP inhibitor. The pretreated T cells were found to be resistant to activation-induced cell death (AICD). Similar results were obtained in murine thymocytes as well as in activated peripheral T cells. PEP activity in T cell hybridomas remained unchanged during AICD. These results suggest that T cells expressing high PEP activity are susceptible to ACID.