Kazuya Shimoda

Enhanced Th2 Cell-Mediated Allergic Inflammation in Tyk2-Deficient Mice

Allergic inflammation is mediated by Th2 cell-derived cytokines, including IL-4, IL-5, and IL-13, and down-regulated by IFN-γ and IL-12. Tyk2 is a member of the Janus family of protein tyrosine kinases and is activated by a variety of cytokines: IFN-αβ, IL-6, IL-10, IL-12, and IL-13. In this study, we investigated the role of Tyk2 in the regulation of Ag-induced Th cell differentiation and Ag-induced allergic inflammation in the airways using Tyk2-deficient (Tyk2−/−) mice. When splenocytes were stimulated with antigenic peptide, IL-12-mediated Th1 cell differentiation was decreased, but IL-4-mediated Th2 cell differentiation was increased in Tyk2−/− mice. In vivo, Ag-specific IgE and IgG1 production was increased, but Ag-specific IgG2a production was decreased in Tyk2−/− mice as compared with those in control mice. In addition, Ag-induced eosinophil and CD4+ T cell recruitment, as well as the production of Th2 cytokines in the airways, was increased in Tyk2−/− mice. Adoptive transfer experiments revealed that CD4+ T cells were responsible for the enhanced Ag-induced eosinophil recruitment in Tyk2−/− mice. In contrast, although the level of IL-13 was increased in the airways of Tyk2−/− mice after Ag inhalation, the number of goblet cells, as well as Muc5ac mRNA expression, was decreased in Tyk2−/− mice. Together, these results indicate that Tyk2 plays a bilateral role in the regulation of allergic inflammation in the airways: Tyk2 plays a role in the down-regulation of Th2 cell-mediated Ab production and eosinophil recruitment in the airways by regulating Th1/Th2 balance toward Th1-type, while Tyk2 is necessary for the induction of IL-13-mediated goblet cell hyperplasia in the airways.

Human cord blood--derived cells generate insulin-producing cells in vivo.

Here we report the capacity of human cord blood (CB)–derived cells to generate insulin‐producing cells. To investigate in vivo capacity of human CB–derived cells, T cell–depleted mononuclear cells were intravenously transplanted into nonobese diabetic/severe combined immunodeficient/β2‐microglobulinnull mice within 48 hours of birth. At 1–2 months post‐transplantation, immunofluorescence staining for insulin and fluorescence in situ hybridization (FISH) analysis using a human chromosome probe indicated that human CB–derived cells generated insulin‐producing cells at a frequency of 0.65% ± 0.64% in xenogeneic hosts. Reverse transcription–polymerase chain reaction analysis confirmed the transcription of human insulin in the pancreatic tissue of the recipient mice. To clarify the mechanism underlying CB‐derived insulin‐producing cells, double FISH analysis using species‐specific probes was performed. Almost equal proportions of human chromosome+ murine chromosome− insulin+ cells and human chromosome+ murine chromosome+ insulin+ cells were present in recipient pancreatic islets. Taken together, human CB contains progenitor cells, which can generate insulin‐producing cells in recipient pancreatic tissues across a xenogeneic histocompatibility barrier by fusion‐dependent and ‐independent mechanisms.

Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion

To obtain insights into the cardiomyogenic potential of hematopoietic tissue, we intravenously (i.v.) injected purified hematopoietic stem/progenitor cells into newborn recipients that may fully potentiate the developmental plasticity of stem cells. Transplantation of mouse bone marrow (BM) lineage antigen‐negative (Lin−) cells resulted in the generation of the cells that displayed cardiomyocyte‐specific antigenic profiles and contractile function when transplanted into syngeneic newborn recipients. To clarify the mechanism underlying the cardiomyogenic potential, green fluorescent protein (GFP)‐labeled BM Lin−ScaI+ hematopoietic progenitors were transplanted into neonatal mice constitutively expressing cyan fluorescence protein (CFP). Lambda image acquisition and linear unmixing analysis using confocal microscopy successfully separated GFP and CFP, and revealed that donor GFP+ cardiomyocytes coexpressed host‐derived CFP. We further reconstituted human hemopoietic‐ and immune systems in mice by injecting human cord blood (CB)‐derived Lin−CD34+CD38− hematopoietic stem cells (HSCs) into neonatal T cell−B cell−NK cell− immune‐deficient NOD/SCID/IL2rγnull mice. Fluoroescence in situ hybridization analysis of recipient cardiac tissues demonstrated that human and murine chromosomes were colocalized in the same cardiomyocytes, indicating that cell fusion occurred between human hematopoietic progeny and mouse cardiomyocytes. These syngeneic‐ and xenogeneic neonatal transplantations provide compelling evidence that hematopoietic stem/progenitor cells contribute to the postnatal generation of cardiomyocytes through cell fusion, not through transdifferentiation.— Ishikawa, F., Shimazu, H., Shultz, L. D., Fukata, M., Nakamura, R., Lyons, B., Shimoda, K., Shimoda, S., Kanemaru, T., Nakamura, K‐i., Ito, H., Kaji, Y., Perry, A. C. F., Harada, M. Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion. FASEB J. 20, E11–E17 (2006)

Physical and Functional Interactions between Daxx and DNA Methyltransferase 1-Associated Protein, DMAP1

Daxx has been shown to play an essential role in type I IFN-αβ-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify how Daxx regulates B cell development, we examined Daxx interacting partners by yeast two-hybrid screening. DNA methyltransferase 1 (DNMT1)-associated protein (DMAP1) was identified and demonstrated to interact with Daxx. The interaction regions in both proteins were mapped, and the cellular localization of the interaction was examined. Both Daxx and DMAP1 formed a complex with DNMT1 and colocalized in the nucleus. DMAP1 enhanced Daxx-mediated repression of glucocorticoid receptor transcriptional activity. Furthermore, Daxx protected protein degradation of DMAP1 in vivo. These results provide the novel molecular link between Daxx and DNMT1, which establishes a repressive transcription complex in the nucleus.

Monitoring of cytomegalovirus reactivation after allogeneic stem cell transplantation: comparison of an antigenemia assay and quantitative real-time polymerase chain reaction.

Cytomegalovirus (CMV) antigenemia and quantitative real-time polymerase chain reaction (PCR) were compared for monitoring of CMV reactivation after allogeneic stem cell transplantation. The number of CMV antigen-positive cells by the antigenemia assay and the level of CMV DNA by real-time PCR correlated well. The sensitivity and specificity of the antigenemia assay was 55.4% and 95.5%, respectively, using real-time PCR as the reference standard. The probability of positive antigenemia at day 100 was 76.5%, with a median of first detection at day 37 in 51 patients, compared with a positive PCR of 84.3% and day 33, respectively. When HLA-identical sibling donor transplant recipients and other donor transplant recipients were analyzed separately, there was no difference between the two tests. However, temporal patterns of first detection of CMV antigen-positive cells and CMV DNA differed between HLA-identical and alternative recipients; patients without CMV (29%) or with sporadic positive PCR results (14%) were more common in HLA-identical sibling transplants, whereas patients with simultaneous antigenemia and positive PCR occurred more in alternative transplants (48%). Two of 51 patients (4%) developed CMV colitis despite antigenemia-guided prophylaxis, but both were successfully treated with ganciclovir. Although PCR is more sensitive than antigenemia, both tests are useful in the early detection of CMV after allogeneic stem cell transplantation.

Constitutive production of granulocyte colony-stimulating factor and interleukin-6 by a human lung cancer cell line, KSNY: Gene amplification and increased mRNA stability

Abstract: A human lung squamous cell carcinoma cell line designated KSNY was established from a patient suffering from marked neutrophilia and polyclonal hyper‐γ‐globulinemia. In our previous report, we demonstrated colony‐stimulating activities in the culture supernatant of this cell line. To determine the exact molecules for the activities, we investigated the gene expression of various cytokines in KSNY cells and showed the mRNA expression of both granulocyte colony‐stimulating factor (G‐CSF) and interleukin‐6 (IL‐6). We also detected substantial amounts of G‐CSF and IL‐6 in the culture supernatant with sensitive enzyme‐linked immunosorbent assays (ELISA). The amplification of the gene locus for G‐CSF, but not for IL‐6 was shown by Southern blot analysis. Furthermore, we also showed that the mRNAs for G‐CSF and IL‐6 were relatively stable in KSNY cells. These findings are thought to be related to the constitutive production of the cytokines in KSNY cells.

Signal Transducers and Activators of Transcription 3 Augments the Transcriptional Activity of CCAAT/Enhancer-binding Protein α in Granulocyte Colony-stimulating Factor Signaling Pathway

The Janus kinase (Jak)-Stat pathway plays an essential role in cytokine signaling. Granulocyte colony-stimulating factor (G-CSF) promotes granulopoiesis and granulocytic differentiation, and Stat3 is the principle Stat protein activated by G-CSF. Upon treatment with G-CSF, the interleukin-3-dependent cell line 32D clone 3(32Dcl3) differentiates into neutrophils, and 32Dcl3 cells expressing dominant-negative Stat3 (32Dcl3/DNStat3) proliferate in G-CSF without differentiation. Gene expression profile and quantitative PCR analysis of G-CSF-stimulated cell lines revealed that the expression of C/EBPα was up-regulated by the activation of Stat3. In addition, activated Stat3 bound to CCAAT/enhancer-binding protein (C/EBP)α, leading to the enhancement of the transcription activity of C/EBPα. Conditional expression of C/EBPα in 32Dcl3/DNStat3 cells after G-CSF stimulation abolishes the G-CSF-dependent cell proliferation and induces granulocytic differentiation. Although granulocyte-specific genes, such as the G-CSF receptor, lysozyme M, and neutrophil gelatinase-associated lipocalin precursor (NGAL) are regulated by Stat3, only NGAL was induced by the restoration of C/EBPα after stimulation with G-CSF in 32Dcl3/DNStat3 cells. These results show that one of the major roles of Stat3 in the G-CSF signaling pathway is to augment the function of C/EBPα, which is essential for myeloid differentiation. Additionally, cooperation of C/EBPα with other Stat3-activated proteins are required for the induction of some G-CSF responsive genes including lysozyme M and the G-CSF receptor.

Levels of human serum granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor under pathological conditions

Levels of serum granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in patients with various leukocyte disorders were estimated by enzyme linked immunosorbent assay (ELISA). Some cases of acute myelogenous leukemia and aplastic anemia showed elevated serum levels of G-CSF and/or GM-CSF, whereas almost all of 23 healthy controls showed G-CSF and GM-CSF levels lower than 100 pg/ml. High levels of both types of CSF were noted in patients with granulocytosis due to infection. These levels became lower after resolution of the infection. Daily changes in serum CSF levels were also examined in a patient with autoimmune neutropenia, and it was found that the peripheral neutrophilic granulocyte count changed almost in parallel with the serum G-CSF level but not with GM-CSF, following the pattern with a delay of about 4–5 h, suggesting the possibility that G-CSF mainly regulates peripheral neutrophil circulation.

Immunosuppressive therapy for patients with refractory anemia.

Abstract. Trials of immunosuppressive therapy have been reported in some case reports of hypoplastic myelodysplastic syndrome (MDS). In this study, we gave immunosuppressive therapies to eight patients with normo- or hyperplastic MDS of refractory anemia subtype without karyotypic abnormalities and analyzed the HLA-DRB1 type or the presence of paroxysmal nocturnal hemoglobinuria (PNH) neutrophils in these patients. Cyclosporin A (CyA) therapy was effective for improving cytopenia in four of the eight MDS patients. While the side effects of CyA were mostly mild and transient, one patient demonstrated karyotypic abnormality following CyA therapy and accelerated to refractory anemia with an excess of blasts. Additional antithymocyte globulin (ATG) therapy was effective in one of three nonresponders to CyA therapy. One patient died due to leukemic transformation after ATG therapy. When we analyzed the correlation between the response to CyA therapy and the HLA-DRB1 type, there were more responders with DRB1*1501 (three of four patients) than without (one of four patients), but a statistically significant difference was not evident between the two groups. In addition, the presence of PNH neutrophils was not correlated with the response to CyA and/or ATG therapy. These results indicate the usefulness of immunosuppressive therapies even for normo- or hyperplastic MDS patients. Further trials using more patients with a long follow-up period would be worthwhile in order to clarify the possibility of disease progression and in order to predict the response of patients.

Cytokine production by peripheral blood monocytes and T cells during haemopoietic recovery after intensive chemotherapy.

Summary We studied the production of cytokines by peripheral blood monocytes and T cells during the period of haematological recovery following intensive chemotherapy. Twelve adults with haematological malignancies received consolidation chemotherapy of complete remission. Monocytes and T cells were collected during the phase of recovery from intensive chemotherapy, and were incubated for 24 h in a culture medium with 10% FCS. Concentrations of cytokines in the culture supernatant were measured with an enzymelinked immunosorbent assay. During the recovery phase, concentrations of IL‐6, G‐CSF and IL‐1β in the culture supernatant of the collected monocytes significantly exceeded those of the monocytes obtained from normal healthy subjects. Similarly, the concentrations of GM‐CSF and IFN‐γ in the supernatant of recovery phase T cells significantly exceeded those of normal T cells. Plasma levels of these cytokines were also elevated. These data suggest that the monocytes and T cells may be activated in vivo to produce haemopoietic cytolines during haematological recovery, and that, during haematological recovery, the monocytes and T cells may be actively involved in the induction of haematopoiesis following the myelosuppression induced by chemotherapy.