Weon Seo Park

NKT cells promote antibody-induced joint inflammation by suppressing transforming growth factor β1 production

Although NKT cells has been known to exert protective roles in the development of autoimmune diseases, the functional roles of NKT cells in the downstream events of antibody-induced joint inflammation remain unknown. Thus, we explored the functional roles of NKT cells in antibody-induced arthritis using the K/BxN serum transfer model. NKT cell–deficient mice were resistant to the development of arthritis, and wild-type mice administrated with α-galactosyl ceramide, a potent NKT cell activator, aggravated arthritis. In CD1d−/− mice, transforming growth factor (TGF)-β1 was found to be elevated in joint tissues, and the blockade of TGF-β1 using neutralizing monoclonal antibodies restored arthritis. The administration of recombinant TGF-β1 into C57BL/6 mice reduced joint inflammation. Moreover, the adoptive transfer of NKT cells into CD1d−/− mice restored arthritis and reduced TGF-β1 production. In vitro assay demonstrated that interleukin (IL)-4 and interferon (IFN)-γ were involved in suppressing TGF-β1 production in joint cells. The adoptive transfer of NKT cells from IL-4−/− or IFN-γ−/− mice did not reverse arthritis and TGF-β1 production in CD1d−/− mice. In conclusion, NKT cells producing IL-4 and IFN-γ play a role in immune complex–induced joint inflammation by regulating TGF-β1.

Lymphovascular Invasion in Transurethral Resection Specimens as Predictor of Progression and Metastasis in Patients With Newly Diagnosed T1 Bladder Urothelial Cancer

PURPOSE We evaluated the clinical significance of lymphovascular invasion in transurethral resection of bladder tumor specimens in patients with newly diagnosed T1 urothelial carcinoma of the bladder. MATERIALS AND METHODS Enrolled in the study were 118 patients with newly diagnosed T1 urothelial carcinoma of the bladder who underwent transurethral resection of bladder tumor between 2001 and 2007. Patient records were retrieved from a prospectively maintained bladder cancer database. We evaluated the correlation between lymphovascular invasion and other clinicopathological features, and the impact of lymphovascular invasion on disease recurrence, disease progression and metastasis. RESULTS Lymphovascular invasion was histologically confirmed in 33 patients (28.0%). While lymphovascular invasion correlated with tumor grade (p = 0.002), it was not associated with gender, age, bladder tumor history, tumor size, multiplicity or concomitant carcinoma in situ. Recurrence, progression and metastasis developed in 45 (38.1%), 19 (16.1%) and 10 patients (8.5%), respectively. Univariate analysis showed that lymphovascular invasion was marginally associated with recurrence and significantly associated with progression (p = 0.011) and metastasis (p = 0.019). Multivariate Cox proportional hazards analysis revealed that recurrence was significantly associated with lymphovascular invasion (p = 0.029), and with bladder tumor history (p <0.001), tumor size (p = 0.031) and multiplicity (p = 0.043). Lymphovascular invasion was the only independent prognostic factor associated with progression (p = 0.016). CONCLUSIONS In patients with newly diagnosed T1 urothelial carcinoma of the bladder lymphovascular invasion in transurethral resection of bladder tumor specimens predicts disease progression and metastasis.

Natural Killer T (NKT) Cells Attenuate Bleomycin-Induced Pulmonary Fibrosis by Producing Interferon-γ

Pulmonary fibrosis is a progressive illness characterized by interstitial fibrosis. Although the precise mechanism for pulmonary fibrosis is not completely understood, an immune response involving interferon (IFN)-γ appears to play a role. Therefore, we examined the functional roles of natural killer T (NKT) cells, which produce IFN-γ and interleukin-4 on activation, in bleomycin-induced pulmonary fibrosis. In NKT cell-deficient mice, pulmonary fibrosis was worse in terms of histology, hydroxyproline levels, and mortality than in control mice. The transforming growth factor (TGF)-β1 levels were higher in the lung after injecting bleomycin, and blockade of TGF-β1 by neutralizing monoclonal antibody attenuated the pulmonary fibrosis in CD1d−/− mice. In contrast, the production of IFN-γ was reduced in lungs from CD1d−/− mice. Moreover, the adoptive transfer of NKT cells into CD1d−/− mice increased IFN-γ and reduced TGF-β1 production, attenuating pulmonary fibrosis. An in vitro assay demonstrated that IFN-γ was involved in suppressing TGF-β1 production in cells collected from bronchoalveolar lavage. The adoptive transfer of NKT cells from IFN-γ−/− mice did not reverse pulmonary fibrosis or TGF-β1 production in lungs of CD1d−/− mice whereas NKT cells from B6 control mice attenuated fibrosis and reduced TGF-β1 production. In conclusion, IFN-γ-producing NKT cells play a novel anti-fibrotic role in pulmonary fibrosis by regulating TGF-β1 production.

Cloning, genomic organization, alternative transcripts and expression analysis of CD99L2, a novel paralog of human CD99, and identification of evolutionary conserved motifs.

Human CD99 (MIC2) is a 32 kDa cell surface protein and its encoding gene is localized to the pseudoautosomal regions of both Xp and Yp chromosomes. Although sequences of several genes such as human PBDX and MIC2R are known to be related to that of CD99, the murine counterpart of CD99 has not been reported. Here we have identified a novel CD99 mouse paralog, named as CD99L2 (CD99 antigen-like 2), and its human, rat and zebrafish genes. Unlike the rapidly evolved CD99 gene, these CD99L2 genes were highly conserved among those species. However, the genomic organization of human and mouse CD99L2 genes showed a difference in their exon numbers possibly due to exon duplication during evolution. In addition, comparative analysis of the cDNA sequences identified the presence of variants in the region around the exons 3 and 4 even within a species due to a differential splicing event, resulting in species-specific patterns in their transcripts. As determined by in situ hybridization analysis, the CD99L2 gene appeared to be expressed particularly high in neuronal cells despite its ubiquitous distribution. The highly expression on neuronal cells without any variations between species reflects a dominant role of this molecule during neural development. Amino acid sequence alignment revealed five putative functional regions highly conserved between CD99L2 and CD99, indicating a close relationship between the two genes. Moreover, human and mouse CD99L2 were located on their X chromosomes, respectively, whereas the zebrafish mic2l1 gene was in the LG7 chromosome. These observations support the inference that the evolutionary conserved gene, CD99L2, originated from a common ancestor gene of CD99, and its high conservation among species implies at least some essential function.

IL-4-Secreting NKT Cells Prevent Hypersensitivity Pneumonitis by Suppressing IFN-γ-Producing Neutrophils

Hypersensitivity pneumonitis (HP) is mediated by Th1 immune response. NKT cells regulate immune responses by modulating the Th1/Th2 balance. Therefore, we postulated that NKT cells play a critical role in the development of the HP by modulating the Th1/Th2 response. To address this issue, we explored the functional roles of NKT cells in Saccharopolyspora rectivirgula (SR)-induced HP. In CD1d−/− mice, the HP was worse in terms of histological changes, hydroxyproline levels, the CD4:CD8 ratio in bronchoalveolar lavage fluid, and SR-specific immune responses than in control mice. CD1d−/− mice showed elevated IFN-γ production in the lung during the HP, and this was produced mainly by Gr-1+ neutrophils. The blockade of IFN-γ in CD1d−/− mice attenuated the HP, whereas the injection of rIFN-γ aggravated it. Moreover, the depletion of Gr-1+ neutrophils reduced CD8+ T cell numbers in bronchoalveolar lavage fluid during the HP. The adoptive transfer of IL-4−/− mouse NKT cells did not attenuate the HP, whereas wild-type or IFN-γ−/− mouse NKT cells suppressed the HP. In conclusion, NKT cells producing IL-4 play a protective role in SR-induced HP by suppressing IFN-γ-producing neutrophils, which induce the activation and proliferation of CD8+ T cells in the lung.

CD99 Regulates the Transport of MHC Class I Molecules from the Golgi Complex to the Cell Surface

The down-regulation of surface expression of MHC class I molecules has recently been reported in the CD99-deficient lymphoblastoid B cell line displaying the characteristics of Hodgkin’s and Reed-Sternberg phenotype. Here, we demonstrate that the reduction of MHC class I molecules on the cell surface is primarily due to a defect in the transport from the Golgi complex to the plasma membrane. Loss of CD99 did not affect the steady-state expression levels of mRNA and protein of MHC class I molecules. In addition, the assembly of MHC class I molecules and the transport from the endoplasmic reticulum to the cis-Golgi occurred normally in the CD99-deficient cells, and no difference was detected between the CD99-deficient and the control cells in the pattern and degree of endocytosis. Instead, the CD99-deficient cells displayed the delayed transport of newly synthesized MHC class I molecules to the plasma membrane, thus causing accumulation of the molecules within the cells. The accumulated MHC class I molecules in the CD99-deficient cells were colocalized with α-mannosidase II and γ-adaptin in the Golgi compartment. These results suggest that CD99 may be associated with the post-Golgi trafficking machinery by regulating the transport to the plasma membrane rather than the endocytosis of surface MHC class I molecules, providing a novel mechanism of MHC class I down-regulation for immune escape.

BRAF mutations in acute leukemias.

production and consequently stem cell function. There is the possibility that these mtDNA mutations have a subtle effect on mitochondrial function and this increases the risk of a stem cell becoming malignant. While this view is discussed by Gattermann, the only way to test this hypothesis is to directly measure the mutation load (and spectrum) in haematological stem cells from normal adults and to directly compare with the changes seen in leukaemia. The discovery of pathogenic mtDNA mutations in MDS patients is intriguing. In Pearson’s syndrome, sideroblastic anaemia is prominent and thus it is accepted that a respiratory chain abnormality as a consequence of mtDNA mutation (in this case a single, large-scale mtDNA deletion) can affect blood cell maturation. However, in patients with MDS who have a pathogenic mtDNA mutation, the mutation must be acquired and clonally expand through the haematopoeitic tissues. This would seem remarkable for a cell that has a severely compromised respiratory chain function and is in stark contrast to the observed situation in inherited mtDNA abnormalities in which the mutation is lost in blood. The effect of the respiratory chain defect in MDS therefore seems to be more severe on cell maturation than replication. Do our observations have other important implications? As highlighted by Gattermann, the mitochondrial genome is present in multiple copies in individual cells, thus permitting extensive molecular genetic studies at the level of the single cell. On account of this high copy compared to nuclear gene targets, we believe that somatic mtDNA mutations may prove useful in the detection of malignant leukaemic clones and therefore as markers of disease activity. While we do not directly compare mtDNA markers with nuclear markers of mimimal residual disease in our manuscript, such a study may prove important in the future. A much more long-term goal is also to consider the presence of a somatic, clonal mtDNA mutation as a potential target for treatment. The work of our own group focuses predominantly on the detection and characterisation of inherited mtDNA mutations, a common genetic cause of neurological and neuromuscular conditions that affect at least 1 in 10 000 of the population. In spite of our extensive understanding of the pathogenesis of many of these mtDNA mutations, at present there is little we can offer our patients in terms of treatment. Developing molecules and drugs that can specifically target mutated mtDNA is a priority for patients with mtDNA disease, but any such approach, if successful, may also be of value on treating leukaemia or other malignancies in which there are clonal mtDNA mutations. In conclusion, we believe our studies have shown convincingly that acquired mtDNA mutations occur in leukaemia. Our paper documents the phenomenology, but further studies are now required to elucidate the significance of these sequence changes in the development of leukaemia.

PKB/Akt inhibits ceramide-induced apoptosis in neuroblastoma cells by blocking apoptosis-inducing factor (AIF) translocation

Ceramide is a sphingolipid that is abundant in the plasma membrane of neuronal cells and is thought to have regulatory roles in cell differentiation and cell death. Ceramide is known to induce apoptosis in a variety of different cell types, whereas the physiological significance of gangliosides, another class of sphingolipids, in these processes is still unclear. We examined the mechanisms of ceramide‐induced cell death using a human neuroblastoma cell line. Treatment of the human neuroblastoma cell line SH‐SY5Y with ceramide induced dephosphorylation of the PKB/Akt kinase and subsequent mitochondrial dysfunction. In addition, ceramide‐induced neuronal cell death was not completely blocked by inhibition of caspase activity. This incomplete inhibition appeared to be attributable to the translocation of apoptosis‐inducing factor to the nucleus. Furthermore, overexpression of active PKB/Akt or Bcl‐2 successfully blocked ceramide‐induced neuronal cell death through inhibition of the translocation of apoptosis‐inducing factor. J. Cell. Biochem. 102: 1160–1170, 2007.

The CD99 signal enhances Fas-mediated apoptosis in the human leukemic cell line, Jurkat

The CD99 antigen has been implicated in various cellular processes, including apoptosis in T cells. Previously, we reported two monoclonal antibodies that recognize different epitopes of the CD99 molecule, named DN16 and YG32. In this study, we investigated the role of each CD99 epitope in T cell apoptosis. Unlike the DN16 epitope, CD99 ligation via the YG32 epitope failed to induce T cell death. Surprisingly, however, the YG32 signal enhanced Fas‐mediated apoptosis in Jurkat T cells. Augmentation of Fas‐mediated apoptosis by YG32 ligation was inhibited by treatment with either of the caspase inhibitors z‐VAD‐fmk or z‐IETD‐fmk, and YG32 ligation appeared to induce Fas oligomerization. These results suggest that each CD99 epitope plays a distinct role in T cell biology, especially in T cell apoptosis.

Prostate stem cell antigen mRNA in peripheral blood as a potential predictor of biochemical recurrence in high-risk prostate cancer.

To determine whether the presence of prostate stem cell antigen (PSCA) mRNA in peripheral blood can predict biochemical recurrence (BCR) after radical prostatectomy in patients with high‐risk prostate cancer.