Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells.

Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs(flox/flox);mb1(cre/)(+):Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

Role of interleukin-21 isoform in dextran sulfate sodium (DSS)-induced colitis.

Interleukin-21 (IL-21) is overproduced in human intestines affected by inflammatory bowel disease (IBD) and in the gut of mice with DSS-induced colitis. IL-21-deficient mice are largely protected against DSS-induced colitis, indicating that IL-21 plays a key role in the development of IBD. We previously identified a novel IL-21 isoform named IL-21iso. In this study, we found that in addition to the conventional IL-21, IL-21iso mRNA was also expressed in the colon with DSS-induced colitis. To investigate whether IL-21iso plays a role in DSS-induced colitis, we established transgenic mice (mIL-21iso-Tg mice) that expressed mouse IL-21iso under the control of the lck proximal promoter. Although mIL-21iso-Tg mice did not have any gross physical abnormalities, their peripheral lymphocytes counts were higher than those in wild-type littermates. Notably, their CD8(+) T cell and CD4(+) effector memory T-cell populations were elevated. DSS-induced colitis was far more severe in the mIL-21iso-Tg mice than in wild-type mice, and was accompanied by a marked loss of body weight and by colon inflammation with increased cellular infiltration. In DSS-treated mice, colon tissues from mIL-21iso-Tg mice had significantly higher gene activation levels for cytokines such as IL-17A, TNF-α, IL-6, IL-10, and IL-4, and for transcription factors such as T-bet, GATA-3, RORγt, and Foxp3, than were found in wild-type mice. These results indicate that besides IL-21, IL-21iso may be another regulator of gut inflammation.

Loss of hrs in the central nervous system causes accumulation of ubiquitinated proteins and neurodegeneration.

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs(flox/flox);SynI-cre mice. Notably, the hrs(flox/flox);SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs(flox/flox);SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.

Inhibition of tumor growth and metastasis by depletion of vesicular sorting protein Hrs: its regulatory role on E-cadherin and beta-catenin.

Abnormally high signals from receptor tyrosine kinases (RTK) are associated with carcinogenesis, and impaired deactivation of RTKs may also be a mechanism in cancer. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is one of the master regulators that sort activated receptors toward lysosomes and shut down their signals. Hrs contains a ubiquitin-interacting motif and is involved in the endosomal sorting of monoubiquitinated membrane proteins, such as growth factor receptor and E-cadherin. Here, we investigated the role of Hrs in determining the malignancy of cancer cells and discovered that the targeted disruption of Hrs by small interfering RNA effectively attenuated the proliferation, anchorage-independent growth, tumorigenesis, and metastatic potential of HeLa cells in vitro and in vivo. The restoration of Hrs expression increased cell proliferation and anchorage-independent growth in a mouse embryonic fibroblast line established from a Hrs knockout mouse. Further analysis revealed that Hrs depletion was associated with the up-regulation of E-cadherin and reduced beta-catenin signaling. The aberrant accumulation of E-cadherin most likely resulted from impaired E-cadherin degradation in lysosomes. These results suggest that Hrs may play a critical role in determining the malignancy of cancer cells by regulating the degradation of E-cadherin.

Signal-transducing adaptor molecules STAM1 and STAM2 are required for T-cell development and survival.

We previously reported that the STAM family members STAM1 and STAM2 are phosphorylated on tyrosine upon stimulation with cytokines through the gammac-Jak3 signaling pathway, which is essential for T-cell development. Mice with targeted mutations in either STAM1 or STAM2 show no abnormality in T-cell development, and mice with double mutations for STAM1 and STAM2 are embryonically lethal; therefore, here we generated mice with T-cell-specific double mutations for STAM1 and STAM2 using the Cre/loxP system. These STAM1(-/-) STAM2(-/-) mice showed a significant reduction in thymocytes and a profound reduction in peripheral mature T cells. In proliferation assays, thymocytes derived from the double mutant mice showed a defective response to T-cell-receptor (TCR) stimulation by antibodies and/or cytokines, interleukin-2 (IL-2) and IL-7. However, signaling events downstream of receptors for IL-2 and IL-7, such as activations of STAT5, extracellular signal-regulated kinase (ERK), and protein kinase B (PKB)/Akt, and c-myc induction, were normal in the double mutant thymocytes. Upon TCR-mediated stimulation, prolonged activations of p38 mitogen-activated protein kinase and Jun N-terminal protein kinase were seen, but activations of ERK, PKB/Akt, and intracellular calcium flux were normal in the double mutant thymocytes. When the cell viability of cultured thymocytes was assessed, the double mutant thymocytes died more quickly than controls. These results demonstrate that the STAMs are indispensably involved in T-cell development and survival in the thymus through the prevention of apoptosis but are dispensable for the proximal signaling of TCR and cytokine receptors.

Relation between blood pressure control, body mass index, and intensity of medical treatment.

A cross-sectional study was performed to assess the rates of blood pressure (BP) control to a systolic goal (< 140 mmHg), to a diastolic goal (< 90 mmHg), and to both in a sample of 226 treated hypertensive patients. We also examined the association between obesity, BP control, intensity of treatment defined as dose score (summed ranks of doses of all antihypertensive drugs taken), and number of drugs. Of the 226 treated patients (mean age, 60 years; 20.4% women), 67.7% were controlled to the systolic goal, 72.1% were controlled to the diastolic goal, and 53.5% were controlled to both. Patients were divided into four groups according to body mass index (BMI): less than 23, A; 23-24.9, B; 25-26.9, C; and 27 or more, D. There were no differences in the rates of BP control to the systolic goal, to the diastolic goal, or to both among the four groups; however, the dose score and number of drugs administered were higher in patients of group D than in those of the other groups. There was a tendency toward a higher intensity of drug treatment in obese patients with either controlled or uncontrolled BP. The dose score and number of drugs correlated positively with BMI. In representative subgroups (n = 62), serum insulin and the homeostasis model assessment value of insulin resistance were higher in the patients in group D than in the other groups. These findings indicate that hypertensive patients managed in a hypertension clinic had satisfactory BP control in 53.5% of cases. A higher intensity of medical treatment is needed to achieve BP control in obese hypertensive patients characterized by insulin resistance.

Co-inhibitory roles for glucocorticoid-induced TNF receptor in CD1d-dependent natural killer T cells.

Invariant natural killer T (iNKT) cells are a special subset of alphabeta T cells with invariant TCR, which recognize alpha-galactosylceramide (alpha-GalCer) presented by CD1d. In addition to signals through the invariant TCR upon stimulation with alpha-GalCer, costimulatory signals, such as signals through CD28 and OX40, are indispensable for full activation of iNKT cells. In this study, we investigated the functions of a well-known costimulatory molecule, glucocorticoid-induced TNF receptor (GITR), on Ag-induced iNKT cell activation. Unexpectedly, engagement of GITR by agonistic mAb DTA-1 suppressed proliferation and cytokine production of iNKT cells upon alpha-GalCer stimulation. In addition, GITR signals in iNKT cells during only the Ag-priming phase was sufficient to inhibit the iNKT cell activation. Consistent with these results, the GITR-deficient iNKT cells showed enhanced proliferation and increased cytokine production upon alpha-GalCer stimulation both in vitro and in vivo. Furthermore, the in vivo administration of alpha-GalCer suppressed tumor metastasis more efficiently in GITR-deficient mice than in wild-type mice. Collectively, GITR plays a co-inhibitory role in Ag-induced iNKT cell activation.

OX40 (CD134) and OX40 ligand interaction plays an adjuvant role during in vivo Th2 responses.

The role of OX40-OX40 ligand (OX40L) interaction in Th cell differentiation remains contentious. In vitro studies have revealed a Th2-biased effect by OX40 signals in T cells. However, in vivo studies demonstrated that OX40-OX40L interaction is involved in responses either Th1 or Th2, or both, which appears to be dependent on the experimental conditions used. We document in our report Th cell differentiation in OX40L-deficient and OX40L-transgenic (Tg) mice in response to protein antigens (Ag) and to Leishmania major (L. major) infection. Upon immunization with protein Ag, we demonstrate the adjuvant effect of OX40 signals during in vivo Th2 responses. However, adjuvant treatment to mice ameliorates the Th2-specific effect of OX40-OX40L interaction and rather induces concurrent promotion of both Th1 and Th2 responses via OX40 signals. Thus, previous reports showing promotion of Th1 response by OX40-OX40L interaction may in actual fact be affected by the adjuvant effects mediated by the various experimental conditions. Indeed, constitutive OX40-OX40L interactions in OX40L-Tg mice converted the normally resistant C57BL/6 strain, into a susceptible status following L. major infection due to an extraordinary elevated Th2 response. These results provide convincing evidence demonstrating that the OX40-OX40L interaction is paramount in the development of Th2 responses in vivo.

Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases.

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.

A transgenic mouse model for non-immune hydrops fetalis induced by the NS1 gene of human parvovirus B19.

Human parvovirus B19 (B19) infection during pregnancy is associated with the adverse foetal outcome known as non-immune hydrops fetalis (NIHF). Although B19 is known to infect erythroid-lineage cells in vivo as well as in vitro, the mechanism leading to the occurrence of NIHF is not clear. To investigate the possible involvement of the B19 non-structural protein NS1 in NIHF, three independent lines of transgenic mice were generated that expressed NS1 under the control of the Cre-loxP system and the GATA1 promoter. Two of the three lines expressed NS1 in erythroid-lineage cells. Most of the transgenic mice died at the embryonic stage, some of which developed hydropic changes caused by severe anaemia at embryonic day 15.5 (E15.5). Histological examination of embryos at E15.5 showed significantly fewer erythropoietic islands in the liver parenchyma, whereas their hearts showed no abnormal signs, such as cardiomegaly and apoptotic cells. The NS1-transgenic mouse lines established here provide an animal model for human NIHF and suggest that NS1 plays a crucial role in the adverse outcome associated with intrauterine B19 infection in humans.

Distinct roles for the OX40-OX40 ligand interaction in regulatory and nonregulatory T cells.

The OX40 (CD134) molecule is induced primarily during T cell activation and, as we show in this study, is also expressed on CD25+CD4+ regulatory T (Treg) cells. A necessary role for OX40 in the development and homeostasis of Treg cells can be inferred from the reduced numbers of the cells present in the spleens of OX40-deficient mice, and their elevated numbers in the spleens of mice that overexpress the OX40 ligand (OX40L). The homeostatic proliferation of Treg cells following transfer into lymphopenic mice was also found to be potentiated by the OX40-OX40L interaction. Suppression of T cell responses by Treg cells was significantly impaired in the absence of OX40, indicating that, in addition to its homeostatic functions, OX40 contributes to efficient Treg-mediated suppression. However, despite this, we found that CD25-CD4+ T cells became insensitive to Treg-mediated suppression when they were exposed to OX40L-expressing cells, or when they were treated with an agonistic OX40-specific mAb. OX40 signaling could also abrogate the disease-preventing activity of Treg cells in an experimental model of inflammatory bowel disease. Thus, although the data reveal important roles for OX40 signaling in Treg cell development, homeostasis, and suppressive activity, they also show that OX40 signals can oppose Treg-mediated suppression when they are delivered directly to Ag-engaged naive T cells.

Consequences of OX40-OX40 ligand interactions in langerhans cell function: enhanced contact hypersensitivity responses in OX40L-transgenic mice.

Langerhans cells (LC) represent the dominant antigen-presenting cells (APC) in the epidermis and thus play an important role in cutaneous immune responses to approaching pathogens. These responses are mediated by several costimulatory molecules after antigenic challenge. OX40 ligand (OX40L), a member of TNF superfamily, is expressed on several APC such as splenic dendritic cells (DC) and activated B cells. This molecule has been reported to provide potent costimulation in APC-T cell interactions upon binding to its cognate receptor, OX40, on activated T cells. Little is known, however, regarding OX40L expression and function on LC. In the present study, we report the expression of both OX40L and OX40 on differentiated LC derived from draining lymph nodes in the FITC-sensitized mice. During contact hypersensitivity responses, OX40L-deficient mice demonstrated a significant reduction in both hapten-induced ear swelling and hapten-specific T cell responses despite intact migratory responses. Conversely, these responses were markedly increased in two different OX40L-transgenic strains with variations in OX40L overexpression. In the LC-induced MLR, OX40L-deficient and OX40L-overexpressing LC were capable of reducing and elevating the responses of allogeneic CD4+ T cells, respectively. Thus the requirement of OX40L during the antigen presentation function of LC in T cell priming is here demonstrated.