Masae Furuhata

Positive Modulation of IL-12 Signaling by Sphingosine Kinase 2 Associating with the IL-12 Receptor β1 Cytoplasmic Region

IL-12 is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. IL-12 stimulation results in the activation of Janus kinase 2 and tyrosine kinase 2 and, subsequently, STAT4 and STAT3. In addition, mitogen-activated protein kinase kinase 6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways have been recently demonstrated to be activated by IL-12 and play an important role in IL-12 signaling. To further elucidate the molecular mechanism underlying IL-12 signaling, we have performed a yeast two-hybrid screening and identified mouse sphingosine kinase 2 (SPHK2) as a molecule associating with the mouse IL-12Rβ1 cytoplasmic region. Analyses of various mutants of each molecule revealed that the region including the proline-rich domain in SPHK2 is probably responsible for the binding to IL-12Rβ1, while the regions including the carboxyl terminus and Box II in the IL-12Rβ1 cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-γ production, while that of wild-type SPHK2 enhanced it. In contrast, the expression minimally affected IL-12-induced proliferation. A similar decrease in IL-12-induced IFN-γ production was observed when dominant-negative SPHK2 was expressed in activated primary T cells using a retroviral expression system. These results suggest that SPHK2 associates with the IL-12Rβ1 cytoplasmic region and probably plays a role in modulating IL-12 signaling.

Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis through activation of JNK1 following engagement of membrane immunoglobulin.

Engagement of membrane immunoglobulin (mIg) on WEHI-231 mouse B lymphoma cells results in growth arrest at the G1 phase of the cell cycle, followed by a reduction of mitochondrial membrane potential (DeltaPsim) and apoptosis. WEHI-231 cells resemble immature B cells in terms of the cell surface phenotype and sensitivity to mIg engagement. However, the molecular mechanisms underlying mIg-induced loss of DeltaPsim and apoptosis have not yet been established. In this study, we show that apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase 1 (JNK1) signaling pathway participates in mIg-induced apoptosis through the generation of reactive oxygen species (ROS). Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation immediately (5 min) induces hydrogen peroxide (H2O2) production with a substantial increase during later time points (36-48 h), accompanied by loss of DeltaPsim and an increase in cells with sub-G1 DNA content. The anti-IgM-induced late-phase H2O2 production, loss of DeltaPsim, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone, but enhanced substantially in cells overexpressing a constitutively active form of ASK1. These mIg-mediated events were also partially abrogated by ROS scavenger N-acetyl-L-cysteine (NAC). Taken together, these results suggest that mIg engagement induces H2O2 production leading to activation of ASK1-JNK1 pathway, creating a feedback amplification loop of ROS-ASK/JNK that leads to loss of DeltaPsim and finally apoptosis.

Requirement of Apoptosis-Inducing Kinase 1 for the Induction of Bronchial Asthma following Stimulation with Ovalbumin

Background: Bronchial asthma is a chronic inflammatory disease of the airway. Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, is activated by environmental stress and plays a crucial role in the induction of apoptosis and inflammation. To examine whether ASK1 is involved in the induction of bronchial asthma, we investigated the role of ASK1 using a genetic approach in the production of cytokines, as well as the development of airway hyperreactivity (AHR) and antibody responses using a murine airway inflammation model. Methods: ASK1-deficient (ASK1-/-) and control wild-type (WT) mice were immunized with ovalbumin (OVA) without alum intraperitoneally, followed by intranasal administration of OVA. Airway infiltration of inflammatory cells, cytokine production, AHR and antibody production were assayed. The asthmatic phenotype was assessed following intranasal administration of IL-13 or TNF-α. Results: ASK1-/- mice sensitized with OVA displayed an impaired inflammatory cell infiltration into airways and a decreased AHR relative to WT mice. Moreover, the production of OVA-specific IgE antibodies and proasthmatic cytokines (IL-5, IL-13 and TNF-α) was substantially reduced in OVA-stimulated ASK1-/- mice. Intranasal administration of IL-13 and OVA enhanced the accumulation of inflammatory cells in OVA-primed ASK1-/- mice. The OVA-induced AHR in response to methacholine was enhanced by IL-13 in WT mice but not ASK1-/- mice. Conclusions: The ASK1 signaling pathway regulates the OVA-induced asthmatic phenotype, specifically AHR sensitivity and cytokine production. Therefore, the ASK1 signaling pathway is a promising target for therapeutic intervention in some asthmatic patients.

Requirement of c-Jun N terminal kinase and P38 mitogen-activated protein kinase for anti-IgM-induced reduction in mitochondrial membrane potential in CH31 B lymphoma cells.

CH31 cells have been used for analysis of B cell tolerance, since engagement of membrane immunoglobulin (mIg) results in loss in mitochondrial membrane potential (DeltaPsim), followed by cell death. We have reported that the dominant-negative (dn) form of c-Jun N-terminal kinase (JNK) substantially prevented a loss of DeltaPsim at 24 h, with partial protection around 48 h after anti-IgM stimulation. In this study, we demonstrate that anti-IgM induced a sustained activation of p38 mitogen-activated protein (MAP) kinase. The p38MAP kinase inhibitor SB203580 substantially prevented loss of DeltaPsim at 14 h, with partial prevention (18-24 h) after anti-IgM stimulation. The dnJNK1-mediated prevention of anti-IgM-induced mitochondrial dissipation was enhanced by SB203580 at 42 h, but not 24 h, after stimulation, suggesting that activation of either p38 MAP kinase or JNK may be sufficient for the initiation of early phase of anti-IgM-induced loss of DeltaPsim while both may be necessary in the late phase.

The Multifaceted Role of PD-1 in Health and Disease

Properly functioning T cells should clear invading pathogens and tumors without causing damage to the host. Programmed cell death-1 (PD-1) is a negative costimulatory receptor that is expressed on an effector T cell to regulate its functions; otherwise, uncontrolled activation of T cells might cause collateral tissue damage and autoimmune diseases. However, such a beneficial regulatory tool has been hijacked by several pathogens and tumors to promote chronic infection and evade antitumor immunity, respectively. PD-1 ligands are expressed by a variety of haematopoietic and non-haematopoietic cells all over the body under steady state and their expression is further modulated within different pathological conditions, suggesting that PD-1 is an immune checkpoint that is utilised by the body to regulate T-cell functions in different contexts. Furthermore, blocking PD-1 has recently received attention as a promising therapeutic approach for the treatment of cancer and chronic viral infections. Herein, we shed some light on the current knowledge of PD-1 biology and role in its health and disease.

121 Ovalbumin-induced Bronchial Asthma is Compromised in Apoptosis Signal-Regulating Kinase-Deficient Mice.

Background Apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen –activated protein (MAP) kinase kinase kinases (MAP3Ks) protein family, plays a crucial role in the induction of apoptosis and inflammation in some cell types. Allergic asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR), inflammatory cell infiltration, and airway remodeling. In the present study, we examined whether ASK1 is involved in the induction of bronchial asthma using a mouse model of airway inflammation. Methods ASK1-deficient (ASK1−/−) and wild-type (WT) control mice were sensitized with ovalbumin (OVA) in saline intraperitoneally on consecutive 7 days. Eighteen days later, mice received intranasal administration of OVA aerosol and were assayed for AHR, cytokine production, cell proliferation, antibody (Ab) production, and lung tissue histopathology at 24 hours after the last serial OVA administration. Levels of Ab and cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Results Control WT mice showed inflammatory infiltrates in airways in response to OVA to a greater extent than ASK1−/− mice. The number of cells, especially eosinophils accumulating in airways, was reduced in ASK1−/− mice relative to control mice. OVA-induced AHR is also compromised in ASK1−/− mice. Anti-OVA IgE Ab production in ASK1−/− mice was substantially reduced, although levels of other isotypes were comparable to those in control mice. Levels of some Th2 cytokines (IL-5 and IL-13) and pro-inflammatory cytokine TNF-a in BAL fluid from ASK1−/− mice were substantially diminished relative to control, although a comparable level of a typical Th2 cytokine IL-4 and anti-inflammatory cytokine IL-10 was produced. Although the BAL fluid TNF-a levels from ASK1−/− mice were severely diminished, lymph node cells from ASK1−/− mice produced comparable levels of TNF-a to WT in vitro. Intranasal administration of recombinant TNF-a caused a comparable increase in AHR between ASK1−/− and WT mice, whereas the TNF-a -induced accumulation of inflammatory cells was severely reduced in ASK1−/− mice. Conclusions ASK1 appears to be involved in the induction of OVA-induced bronchial asthma, probably through cytokine production such as TNF-a and IL-13. Moreover, TNF-a sensitivity in response to OVA is also regulated by ASK1.