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Manabu Sugai

Publications and source records attributed to Manabu Sugai.

At least 19 recordsLinked to original sources

Spontaneous large-scale lymphoid neogenesis and balanced autoimmunity versus tolerance in the stomach of H+/K+-ATPase-reactive TCR transgenic mouse.

Autoimmunity is often accompanied by the development of ectopic lymphoid tissues in the target organ, and these tissues have been believed to have close relevance to the severity of the disease. However, the true relationship between the extent of such lymphoid structures and the intensity or type of immune responses mediated by self-reactive T cells has remained unclear. In the present study, we generated transgenic mice expressing TCR from an autoimmune gastritis (AIG)-inducing Th1 cell clone specific for one of the major stomach self-Ags, H(+)/K(+)-ATPase alpha subunit. The transgenic mice spontaneously develop massive lymphoid neogenesis with a highly organized tissue structure in the gastric mucosa, demonstrating Ag-specific, T cell-mediated induction of the lymphoid tissues. Nevertheless, the damage of surrounding tissue and autoantibody production were considerably limited compared with those in typical AIG induced by neonatal thymectomy. Such a moderate pathology is likely due to the locally restricted activation and Th2 skewing of self-reactive T cells, as well as the accumulation of naturally occurring regulatory T cells in the target organ. Altogether, the findings suggest that lymphoid neogenesis in chronic autoimmunity does not simply correlate with the destructive response; rather, the overall activation status of the T cell network, i.e., the balance of self-reactivity and tolerance, in the local environment has an impact.

Adoptive Transfer↗

A transmembrane chemokine, CXC chemokine ligand 16, expressed by lymph node fibroblastic reticular cells has the potential to regulate T cell migration and adhesion.

Stromal cells in lymphoid tissues provide microenvironmental fields required for the triggering of efficient immune responses. Fibroblastic reticular cells (FRCs) are one of the integral constituents of such stromal fields; they construct the reticular network and are considered to regulate immune cells' behavior. However, the factors that mediate the interaction between lymphocytes and FRCs are poorly understood. Here we show that a mouse lymph node (LN)-derived FRC cell line, BLS4, expresses a transmembrane chemokine, CXC chemokine ligand (CXCL) 16, in response to tumor necrosis factor alpha (TNFalpha) and IFNgamma. TNFalpha-induced expression of CXCL16 depends on NFkappaB, p38 MAPK and PKA. Matrix metalloproteinase activity is required for producing soluble CXCL16 in the culture supernatant, likely via shedding at the juxtamembrane region of the extracellular domain. IL-12 enhances the expression of CXCR6 in anti-CD3/CD28-stimulated CD8+ T cells and their adhesion to the BLS4 cell surface in a TNFalpha-dependent fashion. The adherence is significantly inhibited in the presence of both anti-CXCL16 and anti-vascular cell adhesion molecule 1 (VCAM-1) antibodies. CXCL16 expression is also detected in the FRCs in LN sections and in gp38+VCAM-1+ FRCs isolated from LNs. Taken together, these findings suggest that CXCL16 is an important mediator of lymphocyte-stromal interaction within lymphoid tissues.

Animals↗

Enhanced anti-fibrotic activity of plasmid DNA expressing small interference RNA for TGF-beta type II receptor for a mouse model of obstructive nephropathy by cationized gelatin prepared from different amine compounds.

The objective of this study is to increase the transfection efficiency of a plasmid DNA expressing small interference RNA (siRNA) for transforming growth factor-beta receptor (TGF-betaR) by various cationized gelatins of non-viral carrier and evaluate the anti-fibrotic effect with a mouse model of unilateral ureteral obstruction (UUO). Ethylenediamine, putrescine, spermidine or spermine was chemically introduced to the carboxyl groups of gelatin for the cationization. The plasmid DNA of TGF-betaR siRNA expression vector with or without complexation of each cationized gelatin was injected to the left kidney of mice via the ureter to prevent the progression of renal fibrosis of UUO mice. Irrespective of the type of cationized gelatin, the injection of plasmid DNA-cationized gelatin complex significantly decreased the renal level of TGF-betaR over-expression and the collagen content of mice kidney, in marked contrast to free plasmid DNA injection. It is concluded that retrograde injection of TGF-betaR siRNA expression vector plasmid DNA complexed with the cationized gelatin is available to suppress the progression of renal interstitial fibrosis.

Amines↗

Delivery of plasmid DNA expressing small interference RNA for TGF-beta type II receptor by cationized gelatin to prevent interstitial renal fibrosis.

Renal interstitial fibrosis is the common pathway of chronic renal disease, while it causes end-stage renal failure. Transforming growth factor-beta (TGF-beta) is well recognized to be one of the primary mediators to induce accumulation of extracellular matrix (ECM) in the fibrotic area. Therefore, it is expected that local suppression of TGF-beta receptor (TGF-betaR) is one of the crucial strategies for anti-fibrotic therapy. The objective of this study is to investigate feasibility of small interference RNA (siRNA) for TGF-betaR in the selective degradation of TGF-betaR mRNAs, resulting in fibrotic inhibition. A plasmid DNA of TGF-betaR siRNA expression vector with or without complexation of a cationized gelatin was injected to the left kidney of mice via the ureter. Unilateral ureteral obstruction (UUO) was performed for the injected mice to evaluate the anti-fibrotic effect. The injection of plasmid DNA-cationized gelatin complex significantly decreased the level of TGF-betaR and alpha-smooth muscle actin (alpha-SMA) over-expression, the collagen content of mice kidney, and the fibrotic area of renal cortex, in contrast to free plasmid DNA injection. It is concluded that retrograde injection of TGF-betaR siRNA expression vector plasmid DNA complexed with the cationized gelatin is available to suppress progression of renal interstitial fibrosis.

Animals↗

CpG inhibits IgE class switch recombination through suppression of NF kappa B activity, but not through Id2 or Bcl6.

The CpG motif in DNA plays a critical role in immunity via modulating the Th1/Th2 balance. In B cells, CpG-containing oligodeoxynucleotides (CpG ODNs) inhibit IL-4-mediated class switch recombination (CSR) to IgG1 and IgE through inhibition of the germline transcription (GLT) of these isotypes. However, the molecular mechanism of this inhibitory effect remains elusive. We showed here that Id2 and Bcl6, both of which inhibit IgE GLT and CSR, are not involved in this inhibitory pathway. We demonstrated that there is reduced activity of NF kappa B binding to the IgE promoter and a reduction of Irf4 protein in CpG ODN-treated B cells. These data indicate the critical role of NF kappa B and Irf4 in the regulation of IgE CSR through actions downstream of CpG signaling.

Animals↗

Targeting of plasmid DNA to renal interstitial fibroblasts by cationized gelatin.

Renal interstitial fibrosis is the common pathway of chronic renal disease, while it causes end-stage renal failure. A lot of cytokines and biologically active substances are well recognized to be the candidates of primary mediators to induce accumulation of extracelluar matrix (ECM) in the interstitial fibrotic area. Interstitial fibroblasts are played a crucial role in the accumulation of excess ECM during renal interstitial fibrogenesis. Therefore, the targeting of therapeutic drugs and genes to interstitial renal fibroblasts is effective in suppressing the progress of interstitial renal failure. However, despite various approaches and techniques, few successful results have been reported on the in vivo targeting for interstitial fibroblasts. The objective of this study is to deliver an enhanced green fluorescent protein (EGFP) plasmid DNA, as a model plasmid DNA, into renal interstitial space by a cationized gelatin. After the plasmid DNA with or without complexation of the cationized gelatin was injected to the left kidney of mice via the ureter, unilateral ureteral obstruction (UUO) was performed for the mice injected to induce the renal interstitial fibrosis. When the EGFP plasmid DNA complexed with the cationized gelatin was injected, EGFP expression was observed in the fibroblasts in the interstitial area of renal cortex. It is concluded that the retrograde injection of EGFP plasmid DNA complexed with the cationized gelatin is available to target the interstitial renal fibroblasts which are currently considered as the cell source responsible for excessive ECM synthesis.

Animals↗

Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation.

Front-rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton-dependent manner. Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho-ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane "posteriority" in the induction of the uropod in T lymphocytes.

Actins↗

Lymph node fibroblastic reticular cells construct the stromal reticulum via contact with lymphocytes.

The sophisticated microarchitecture of the lymph node, which is largely supported by a reticular network of fibroblastic reticular cells (FRCs) and extracellular matrix, is essential for immune function. How FRCs form the elaborate network and remodel it in response to lymphocyte activation is not understood. In this work, we established ERTR7(+)gp38(+)VCAM-1(+) FRC lines and examined the production of the ER-TR7 antigen. Multiple chemokines produced by FRCs induced T cell and dendritic cell chemotaxis and adhesion to the FRC surface. FRCs can secrete the ER-TR7 antigen as an extracellular matrix component to make a reticular meshwork in response to contact with lymphocytes. The formation of the meshwork is induced by stimulation with tumor necrosis factor-alpha or lymphotoxin-alpha in combination with agonistic antibody to lymphotoxin-beta receptor in a nuclear factor-kappaB (RelA)-dependent manner. These findings suggest that signals from lymphocytes induce FRCs to form the network that supports the movement and interactions of immune effectors within the lymph node.

Animals↗

A novel reticular stromal structure in lymph node cortex: an immuno-platform for interactions among dendritic cells, T cells and B cells.

For efficient adaptive immunity, the lymph nodes (LNs) are equipped with a strategically organized microarchitecture, which is largely supported by the reticular network (RN). The RN can be clearly visualized by fluorescence immunohistochemistry coupled with confocal imaging using a monoclonal antibody, ER-TR7, and can be subdivided into four structurally distinct regions, each of which correlates well with the location of distinct immune cell subsets. In addition, we noticed a characteristic reticular structure designated the 'cortical ridge' at the boundary of the T and B zone, in which dendritic cells are preferentially accumulated. In vitro adhesion assays of frozen sections demonstrated a preference of dendritic cells for the cortical ridge rather than the deeper cortex. Adoptive transfer experiments also demonstrated that antigen-bearing dendritic cells migrated to this region from peripheral tissues, especially in the vicinity of the high endothelial venules, and were anchored on the reticular fibers waiting to interact with the antigen-specific T cells. Taken together, the findings obtained in this study provide new insights into how the LN stromal reticulum works as a specialized 'immuno-platform' for tissue compartmentalization and the immune response.

Animals↗

Role of Id proteins in B lymphocyte activation: new insights from knockout mouse studies.

Id (inhibitor of differentiation) proteins play important roles in cell differentiation, cell cycle control, and apoptosis. They act as negative regulators of basic helix-loop-helix-type transcription factors, which positively regulate differentiation of various cell types. Id proteins work to block B lymphocyte (B cell) maturation at an early differentiation step, as demonstrated by gain-of-function studies. In recent years a series of gene-targeted mice lacking different Ids have been generated. Analyses of these gene-targeted mice provide information useful for understanding the physiological roles of Ids in B cell biology. Id3 is required for proper B cell functions and acts by controlling the cell cycle. Upon B cell activation, Id2 acts as a negative regulator to prevent potentially harmful effects brought about by excessive immunological reactions; one of its special roles is to maintain low serum concentrations of immunoglobulin E (IgE). The Id2 protein does this by antagonizing E2A and Pax5 activities, both of which are required for proper B cell activation. This review presents several new insights into B cell differentiation and activation programs and the physiological role of Id proteins in B cell activation.

Animals↗

In vitro induction of adult hepatic progenitor cells into insulin-producing cells.

Organ-specific stem cells are the natural progenitors in tissue regeneration and possess plasticity to differentiate into specialized cells in adult tissues. Small hepatocytes (SHCs) identified in the adult liver are one such cell type. Here we show that SHCs, which are capable of self-renewal and differentiation into hepatocytes, can be induced to generate insulin-producing cells under appropriate culture conditions. These differentiated cells express pancreatic beta cell differentiation-related transcripts and hepatocyte differentiation-related transcripts, as shown by reverse-transcription PCR/nested PCR. In addition, enforced expression of the homeodomain transcription factor Pdx1 in these cells contributes to enhancement of insulin release in response to insulin secretagogues. These results indicate that the SHCs described here have the ability to differentiate into insulin-producing cells, and further support the idea that engineering to generate insulin-secreting cells could provide a useful resource for future therapies for diabetes mellitus.

Animals↗

Pdx-1 enables insulin secretion by regulating synaptotagmin 1 gene expression.

Pdx-1 plays important roles both in the development of the pancreas and in maintaining pancreatic beta cell function. However, the role of Pdx-1 in the regulation of insulin release is not well established. We previously demonstrated that Pdx-1 overcomes the defect in insulin release from the insulin-producing cells derived from small hepatocytes (SHCs). Insulin secretion is regulated in vivo by the sequential events triggered by the increase of intracellular Ca(2+)-concentration in response to high glucose concentration. In the present study, we identified a new target of Pdx-1 involved in insulin release. Pdx-1 positively regulates the transcription of the gene encoding synaptotagmin 1 (Syt1) (a Ca(2+)-sensor that plays a central role in insulin release) through Pdx-1-binding sites within the 3' regulatory region of the Syt1 gene. We further demonstrated the essential role of Pdx-1 in insulin secretion by the gene knock-down strategy. Small interfering RNA (siRNA) directed against Pdx-1 specifically reduced the levels of Pdx-1 protein and Syt1 transcript in insulinoma lines. Our data indicate that Pdx-1 might contribute to the regulation of insulin release by promoting Syt1 expression in vivo, and provide useful information for future therapy of diabetes mellitus.

Animals↗

Transcription-coupled events associating with immunoglobulin switch region chromatin.

Class switch recombination (CSR) at the antibody immunoglobulin locus is regulated by germline transcription (GLT)-coupled modifications in the accessibility of the switch region, where CSR takes place. Here we show that histone acetylation of switch regions is linked to CSR but that histone acetylation cannot alone promote CSR or GLT. Activation-induced cytidine deaminase (AID) specifically associates with the CSR target chromatin in a GLT-coupled manner, which may occur potentially by means of physical interaction between AID and the transcription machinery. These data indicate an important role of GLT in the regulation of chromatin accessibility, strongly suggesting that the target of AID is chromatin DNA. Our results give insights on the role of AID and the regulatory mechanism of CSR.

Acetylation↗

The balance between Pax5 and Id2 activities is the key to AID gene expression.

Pax5 activity is enhanced in activated B cells and is essential for class switch recombination (CSR). We show that inhibitor of differentiation (Id)2 suppresses CSR by repressing the gene expression of activation-induced cytidine deaminase (AID), which has been shown to be indispensable for CSR. Furthermore, a putative regulatory region of AID contains E2A- and Pax5-binding sites, and the latter site is indispensable for AID gene expression. Moreover, the DNA-binding activity of Pax5 is decreased in Id2-overexpressing B cells and enhanced in Id2(-/-) B cells. The kinetics of Pax5, but not E2A, occupancy to AID locus is the same as AID expression in primary B cells. Finally, enforced expression of Pax5 induces AID transcription in pro-B cell lines. Our results provide evidence that the balance between Pax5 and Id2 activities has a key role in AID gene expression.

Animals↗

Th1-biased tertiary lymphoid tissue supported by CXC chemokine ligand 13-producing stromal network in chronic lesions of autoimmune gastritis.

Secondary lymphoid tissue is developmentally programmed and characterized by well-ordered compartmentalization of lymphocyte subsets and specialized stromal cells supporting the tissue architecture. By contrast, tertiary lymphoid tissue is defined as that induced in ectopic sites by inflammation, although its immunological role is largely unknown. In this study, we characterize the lymphoid tissue induced in the chronic lesion of murine autoimmune gastritis (AIG). Within the lymphoid cluster in the gastric mucosa, there is a clear segregation of T and B cells. Follicle-like B cell areas are always located on the luminal side of the mucosa, while T cells are located in the basal part. A typical lymphoid reticular network and follicular dendritic cells support the structure. Importantly, complement receptor 1(+) follicular dendritic cells within the follicle express a B cell homing chemokine, CXC chemokine ligand 13. The number and size of the clusters correlate with the age of the mice and the serum autoantibody titer, suggesting the functional importance of the clusters in local Ab production, although involvement of the autoantibody in the disease progression is still unclear. AIG gastric lesions are known to constitute a Th1-biased, memory T cell-dependent immunomicroenvironment. The expression pattern of cytokines, including lymphotoxin-beta, and chemokines in the AIG stomach is consistent with this observation. Taken together, these facts suggest that, during the chronic phase of autoimmunity, long-lasting lymphocyte infiltration probably induces a unique tertiary lymphoid tissue that has a function distinct from that of regional lymph nodes. These neolymphoid tissues may maintain the local self reactivity supporting the vicious cycle of Th1-type reaction as well as autoantibody production.

Animals↗

TH2 dominance and defective development of a CD8+ dendritic cell subset in Id2-deficient mice.

BACKGROUND: Although the TH1/TH2 balance is important in many clinical situations, the regulatory mechanisms in vivo have not been well elucidated. OBJECTIVE: We sought to characterize the immunologic status of mice lacking Id2, an inhibitor of basic helix-loop-helix transcription factors. METHODS: We analyzed serum immunoglobulin levels, gene-expression profiles in the spleen, TH1/TH2 balance, and dendritic cell (DC) populations of Id2-/- mice. RESULTS: Serum levels of TH2-mediated IgG1 and IgE were increased more than 10-fold in Id2-/- mice without antigenic stimulation. Gene-expression analysis in Id2-/- splenocytes revealed enhanced expression of TH2-related genes, such as IL-4, and reduced expression of TH1-related genes, including IFN-gamma and IL-12. Intracellular cytokine staining also confirmed that Id2-/- splenic CD4+ T cells are substantially skewed to TH2 cells. However, Id2-/- naive CD4+ T cells differentiated into TH1 cells comparably with wild-type T cells under the appropriate culture conditions. Id2-/- mice displayed a selective and remarkable reduction of the CD8+ DC subset, which is known to induce preferential TH1 differentiation. CONCLUSION: Id2 is an indispensable regulator of the TH1/TH2 balance, possibly through the proper development of CD8alpha+ DCs, and could be a novel target to treat allergic diseases.

Animals↗

Essential role of Id2 in negative regulation of IgE class switching.

Serum concentrations of immunoglobulin E (IgE) in normal circumstances are kept much lower than those of other Ig isotypes to avoid allergic reactions. B cells lacking Id2 have increased E2A activity, which leads to specific enhancement of germline transcription of the immunoglobulin epsilon locus. As a consequence, Id2-deficient B cells undergo class switch recombination (CSR) to IgE at a much higher frequency than wild-type B cells. In contrast, Id2 is induced in wild-type B cells by transforming growth factor-beta1 (TGF-beta1) and suppresses IgE CSR. Our results provide evidence for the inhibitory and selective role of Id2 in IgE CSR in response to TGF-beta1. Id2 might act as molecular safeguard to suppress IgE CSR to prevent serious complications such as allergic hypersensitivity during the normal course of immune responses.

Animals↗