Kikumi Hata

A Role for IL-27 in Early Regulation of Th1 Differentiation

IL-27 is a novel IL-6/IL-12 family cytokine that is considered to play a role in Th1 differentiation, whereas the exact role of IL-27 in Th1 differentiation and its molecular mechanism remain unclear. In this study we demonstrate a role for IL-27 in the early regulation of Th1 differentiation and its possible molecular mechanism. The ability of IL-27 to induce Th1 differentiation was most prominent under Th1-polarizing conditions, but without IL-12 in a STAT4- and IFN-γ-independent manner, and was overruled by IL-12 dose dependently. IL-27 rapidly up-regulated the expression of ICAM-1 on naive CD4+ T cells, but not on APCs, and blocking Abs against ICAM-1 and LFA-1 inhibited the IL-27-induced Th1 differentiation. Although IL-27 augmented T-bet expression in naive CD4+ T cells as previously reported, T-bet was not necessary for the IL-27-induced rapid up-regulation of ICAM-1 expression and Th1 differentiation. In contrast, STAT1 was revealed to be required for the rapid up-regulation of ICAM-1 expression and Th1 differentiation by directly mediating the transcriptional enhancement of ICAM-1 gene expression. These results indicate that IL-27 efficiently induces Th1 differentiation under Th1-polarizing conditions, but without IL-12, and that the rapid up-regulation of ICAM-1 expression on naive CD4+ T cells is important for the IL-27-induced Th1 differentiation. Considering that IL-27 is produced from macrophages and DCs earlier than IL-12, the present results suggest that IL-27 may play a pivotal role in early efficient induction of Th1 differentiation until sufficient IL-12 is produced.

CD40 Ligand Rescues Inhibitor of Differentiation 3-Mediated G1 Arrest Induced by Anti-IgM in WEHI-231 B Lymphoma Cells

The engagement of membrane-bound Igs (mIgs) results in growth arrest, accompanied by apoptosis, in the WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells. Inhibitor of differentiation (Id) proteins, members of the helix-loop-helix protein family, functions in proliferation, differentiation, and apoptosis in a variety of cell types. In this study, we analyzed the involvement of Id protein in mIg-induced growth arrest and apoptosis in WEHI-231 cells. Following stimulation with anti-IgM, expression of Id3 was up-regulated at both the mRNA and protein levels; this up-regulation could be reversed by CD40L treatment. Retrovirus-mediated transduction of the Id3 gene into WEHI-231 cells resulted in an accumulation of the cells in G1 phase, but did not induce apoptosis. E box-binding activity decreased in response to anti-IgM administration, but increased after stimulation with either CD40L alone or anti-IgM plus CD40L, suggesting that E box-binding activity correlates with cell cycle progression. WEHI-231 cells overexpressing Id3 accumulated in G1 phase, which was accompanied by reduced levels of cyclin D2, cyclin E, and cyclin A, and a reciprocal up-regulation of p27Kip1. Both the helix-loop-helix and the C-terminal regions of Id3 were required for growth-suppressive activity. These data suggest that Id3 mimics mIg-mediated G1 arrest in WEHI-231 cells.

OVA-bound nanoparticles induce OVA-specific IgG1, IgG2a, and IgG2b responses with low IgE synthesis

There is an urgent requirement for a novel vaccine that can stimulate immune responses without unwanted toxicity, including IgE elevation. We examined whether antigen ovalbumin (OVA) conjugated to the surface of nanoparticles (NPs) (OVA-NPs) with average diameter of 110nm would serve as an immune adjuvant. When BALB/c mice were immunized with OVA-NPs, they developed sufficient levels of OVA-specific IgG1 antibody responses with low levels of IgE synthesis, representing helper T (Th)2-mediated humoral immunity. OVA-specific IgG2a and IgG2b responses (i.e., Th1-mediated immunity) were also induced by secondary immunization with OVA-NPs. As expected, immunization with OVA in alum (OVA-alum) stimulated humoral immune responses, including IgG1 and IgE antibodies, with only low levels of IgG2a/IgG2b antibodies. CD4-positive T cells from mice primed with OVA-NPs produced substantial levels of IL-21 and IL-4, comparable to those from OVA-alum group. The irradiated mice receiving OVA-NPs-primed B cells together with OVA-alum-primed T cells exhibited enhanced anti-OVA IgG2b responses relative to OVA-alum-primed B cells and T cells following stimulation with OVA-NPs. Moreover, when OVA-NPs-primed, but not OVA-alum-primed, B cells were cultured in the presence of anti-CD40 monoclonal antibody, IL-4, and IL-21, or LPS plus TGF-β in vitro, OVA-specific IgG1 or IgG2b antibody responses were elicited, suggesting that immunization with OVA-NPs modulates B cells to generate IgG1 and IgG2b responses. Thus, OVA-NPs might exert their adjuvant action on B cells, and they represent a promising potential vaccine for generating both IgG1 and IgG2a/IgG2b antibody responses with low IgE synthesis.

Engagement of membrane immunoglobulin enhances Id3 promoter activity in WEHI-231 B lymphoma cells

AbstractAim:We have recently shown that engagement of membrane immunoglobulin (mIg) induced upregulation of inhibitor of differentiation 3 (Id3) mRNA, resulting in growth arrest at G1 phase in WEHI-231 cells. In the present study, we examined whether engagement of mIg will affect promoter activity of the Id3 gene in WEHI-231 cells.Methods:DNA fragments corresponding to the 5′-flanking region of mId3 gene were amplified by polymerase chain reaction (PCR) using genomic DNA as the template. Three DNA fragments upstream of the transcription start site (+1) of the mId3 gene were subcloned into the luciferase reporter vector PGV-B2. The recombinant constructs were transiently transfected into WEHI-231 cells by an electroporation method. After incubation for 24 h, WEHI-231 cells were stimulated with 10 mg/L anti-IgM or irradiated CD40L-expressing NIH3T3 cells or control NIH3T3 cells for further 24 h, followed by assay for luciferase activity.Results:The luciferase analysis demonstrated that basal promoter activity of the Id3 gene was found in the region between -200 and +54. The Id3 promoter activity was increased 2-fold following stimulation with anti-IgM, but not CD40L, compared with medium alone.Conclusion:The mIg-mediated upregulation of Id3 expression is controlled, at least in part, through transcriptional regulation, as assessed by luciferase assay.

c-Jun-NH2-terminal kinase potentiates apoptotic cell death in response to carboplatin in B lymphoma cells

PurposeExposure to carboplatin (CBDCA) has been demonstrated to result in apoptotic and/or necrotic cell death, but molecular mechanisms underlying CBDCA-induced apoptosis or necrosis remain largely unclear. Here, we examined whether activation of c-Jun NH2-terminal kinase (JNK) modulates the mode of cell death induced by CBDCA in CD31 B lymphoma cells.MethodsThe mode of cell death (apoptosis versus necrosis) was investigated by flow cytometry using 7-amino-actinomycin D (7-AAD) and annexin-FITC probes. To evaluate the role of JNK1 in CBDCA-induced cell death, CH31 B lymphoma cells overexpressing dominant-negative form of JNK1 (dnJNK1) or constitutively active form of JNK1 (MKK7-JNK1) were established. Intracellular accumulation of superoxide anion (O2−) was determined by flow cytometry using the fluorescent probe dihydroethidium (DHE).ResultsThe CBDCA-induced primary apoptosis and secondary necrosis were abrogated in the dnJNK1-overexpressing CH31 cells, while it was somewhat enhanced in the MKK7-JNK1-overexpressing cells. In contrast, the CBDCA-induced primary necrosis was reduced by MKK7-JNK1, with a concurrent decrease in production of O2−. The superoxide anion scavenger for butylated hydroxyanisol (BHA) partially reduced the CBDCA-induced O2− production and necrotic, but not apoptotic, death in both wild type and dnJNK1-overexpressing CH31 cells.ConclusionsProlonged activation of JNK1 appears to be involved in CBDCA-induced apoptosis with prevention of necrosis induction, and the induction of necrosis appears to correlate with CBDCA-induced O2− production, which is partially blocked by co-culture with BHA. These observations provide valuable information for understanding molecular mechanisms underlying CBDCA-induced cell death, and hopefully for the design of novel treatment modalities for patients with tumors.

c‐Jun NH2‐terminal kinase (JNK)‐dependent nuclear translocation of apoptosis‐inducing factor (AIF) following engagement of membrane immunoglobulin on WEHI‐231 B lymphoma cells

WEHI‐231 B lymphoma cells have been employed for analysis of antigen‐induced B cell unresponsiveness because these cells undergo cell cycle arrest in G1, accompanied by induction of apoptosis. In the present study, we examined the requirement for toxic small molecules apoptosis‐inducing factor (AIF) and cytochrome c, and subsequent caspase activation in apoptotic cell death in WEHI‐231 and CH31 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Pan‐caspase inhibitor BD‐fmk blocked mIg‐mediated increase in cells with sub‐G1 DNA content, whereas it did not affect mIg‐mediated loss of mitochondrial membrane potential and phosphatidylserine exposure on B cell membrane. Dominant‐negative form of c‐Jun NH2‐terminal kinase1 (JNK1) blocked the translocation of AIF into the nuclei and cytosol from the mitochondria in the WEHI‐231 and CH31 cells following mIg engagement, whereas constitutively active form of JNK1 enhanced it. This AIF translocation was also blocked by Bcl‐xL, but not by BD‐fmk. Moreover, AIF‐deficient clones via small interfering RNA (siRNA)‐mediated method showed small increase in loss of mitochondrial membrane potential. After mIg engagement, the AIF‐deficient clones displayed an enhanced sensitivity to mIg‐mediated apoptosis, concomitant with translocation of a residual AIF into the nuclei, compared with control clone. Our findings are compatible with the notion that AIF has dual role, with a proapoptotic function in the nuclei and a distinct anti‐apoptotic function in the mitochondria. These observations would be valuable for analysis of B cell unresponsiveness and hopefully for treatment of diseases involving B cell dysfunction. J. Cell. Biochem. 104: 1927–1936, 2008.

Differential regulation of T-cell dependent and T-cell independent antibody responses through arginine methyltransferase PRMT1 in vivo.

Protein arginine methyltransferase 1 (PRMT1), a major PRMT in mammalian cells, has been shown to play a crucial role in multiple biological functions in vitro. To explore the role of PRMT1 in B cells in vivo, we generated B cell‐specific PRMT1‐deficient (Prmt1−/−) mice using a Cre‐loxP system. Prmt1−/− mice showed a defect in B‐cell development with diminished levels of serum antibodies. Antibody responses in Prmt1−/− mice were absent after stimulation with the type 2 T cell‐independent antigen NP‐Ficoll but intact after stimulation with the T cell‐dependent antigen NP‐OVA. Our findings comprise the first evidence showing that PRMT1 is necessary for lymphocyte functions in vivo.

Arginine methylation regulates antibody responses through modulating cell division and isotype switching in B cells.

Protein arginine methylation plays crucial roles, including signal transduction, transcriptional control, cell proliferation and/or differentiation. B cells undergo clonal division, isotype switching and differentiate into antibody forming cells following stimulation with Toll‐like receptor‐ligand, lipopolysaccharide (LPS) and T cell‐derived signals, including CD40‐ligand (CD40‐L) and interleukin 4 (IL‐4). Whether protein arginine methylation affects B cell division and/or isotype switching to IgG1 in response to LPS, IL‐4, and CD40‐L was examined using the arginine methyl transferase inhibitor adenosine‐2′,3′‐dialdehyde (AdOx). Addition of AdOx substantially reduced the number of division cycles of stimulated B cells, whereas cell viability remained intact. Upon stimulation with LPS/IL‐4/CD40‐L, the proportion of surface IgG1 positive cells in each division cycle was slightly diminished by AdOx. However, the degree of expression of γ1 germ line transcript and activation‐induced cytidine deaminase (AID) in response to LPS/IL‐4/CD40‐L were unaffected by addition of AdOx, suggesting that AdOx influences class switch recombination independent of AID expression through transcriptional control. Taken together, arginine methylation appears to be involved in B cell isotype switching, as well as in clonal expansion of B cells in response to LPS/IL‐4/CD40‐L.

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.