Hidetoshi Nara

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.

Cloning and characterization of an isoform of interleukin-21

Interleukin‐21 (IL‐21) has pleiotropic functions on the cells, which play roles in both innate and acquired immunity, such as T cells, B cells, natural killer (NK) cells and dendritic cells. In this study we identified a novel isoform of IL‐21, IL‐21iso in human and mouse. IL‐21iso might be an alternative splicing variant form and the C‐terminal region of predicted IL‐21iso amino acid sequences were different from original IL‐21 in both human and mouse. In spite of the differences in C‐terminal amino acid sequences, both human IL‐21 and IL‐21iso showed comparable proliferative effect on anti‐CD40 Ab‐activated primary B cells, anti‐CD3 Ab‐activated primary T cells and human NK cell line, NK0, and upregulated IFN‐γ production from NK0. Furthermore IL‐21 and IL‐21iso similarly activated STAT1 and STAT3. IL‐21iso mRNA was expressed in activated T cells as well as IL‐21 mRNA. However, cycloheximide treatment partially blocked the upregulation of IL‐21iso mRNA in activated T cells while little affected the IL‐21 mRNA expression suggesting that de novo protein synthesis is required for the full expression of IL‐21iso transcript. We also show that the secretion efficiency of hIL‐21iso is much lower than that of hIL‐21. These results may suggest there are some different regulatory mechanisms to produce IL‐21 or IL‐21iso in transcriptional and secretory steps.

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.

Impaired IL-7 signaling may explain a case of atypical JAK3-SCID

Janus kinase 3-severe combined immunodeficiency (JAK3-SCID) is an autosomal recessive immunodeficiency disease caused by various mutations in the JAK3 gene. Typical JAK3-SCID is characterized by a phenotype in which B cells are present but T and NK cells are not, the T(-)B(+)NK(-) phenotype, and by impaired signaling through cytokine receptors that use the common gamma chain (gammac) subunit. An atypical JAK3-SCID case carrying a single glutamate to glycine substitution mutation (E481G) in the JH3 domain of one JAK3 allele, and a deletion mutation (del482-596) in the JH3 and JH2 domains of the other allele was reported previously. Although this patient had CD4(+) T cells and NK cells unlike typical cases, the CD4(+) T cells were functionally impaired. We report here that the JAK3-E481G mutant transduced IL-2-, IL-4-, IL-15-, and IL-21-induced signals as efficiently as wild-type JAK3. However, this mutant failed to respond to IL-7 by phosphorylating JAK1, JAK3, or STAT5. The other mutant JAK3, JAK3-del482-596, was non-functional. Thus, an impaired IL-7 signal may cause SCID and compromise T-cell differentiation, even if the IL-15 signal is preserved and supports NK-cell development, as in this patient.

Regulation of interleukin-21 receptor expression and its signal transduction by WSB-2.

Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates T-cell, B-cell, NK-cell, and myeloid-cell functions. IL-21 binds with its cognate receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain (gammac) receptor subunit. We identified novel IL-21R-binding molecule, WD-40 repeats containing SOCS-box-2, WSB-2. WSB-2 associated with the membrane-proximal intracytoplasmic region of IL-21R, including box1 and box2. Overexpression study of WSB-2 showed the reduction of IL-21R expression and IL-21-induced signal transduction. On the other hand, small interfering RNA for WSB-2 enhanced the expression level of IL-21R and IL-21-induced STAT3 activation, indicating that WSB-2 negatively controls the receptor expression. This report provides the first evidence that WSB-2 is a regulator of IL-21R expression and IL-21-induced signal transduction.

Human Peripheral Neutrophils Express Functional IL-21 Receptors

Neutrophils play key roles in the inflammatory response. The IL-21 cytokine and receptor system is known to be involved in various inflammatory diseases. However, the direct action of IL-21 on neutrophils has not been reported. Here, we show that human neutrophils in peripheral blood express functional IL-21 receptors (IL-21Rs). Expression of the IL-21Rα chain (IL-21Rα) was reduced following various treatments to remove red blood cells, including hypotonic shock, ammonium chloride-mediated lysis, and Percoll density centrifugation. Thus, we utilized whole blood flow cytometric assays to investigate the neutrophil responses to IL-21. IL-21 upregulated the surface expression of CD11b and CD16 on neutrophils and augmented the neutrophils’ phagocytic ability. Our data indicated that IL-21 has the potential to enhance the neutrophil functions.

WSB-1, a novel IL-21 receptor binding molecule, enhances the maturation of IL-21 receptor

Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates T-cell, B-cell, NK-cell, and myeloid-cell functions. IL-21 binds with its cognate receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain. We identified a novel IL-21R-binding molecule, WSB-1, which contains WD-40 repeats and a SOCS-box domain. WSB-1 associates with the middle part of intracytoplasmic region of IL-21R and enhances the maturation of IL-21R from N-linked glycosylated form to fully glycosylated mature form. Furthermore, WSB-1 moderates IL-21R degradation. Taken together, our present study suggests that WSB-1 has a role in the tuning of the maturation and degradation of IL-21R.

IL-21 isoform is a membrane-bound ligand and activates directly interacted cells.

IL-21 is a pleiotropic cytokine that regulates the function of T cells, B cells, natural killer cells, and myeloid cells. We previously identified an IL-21 isoform, IL-21iso, in humans and mice, and found that IL-21iso was secreted in much smaller amounts than conventional IL-21. In this study, we determined that secreted IL-21iso also has less signaling activity than IL-21. However, the amounts of intracellular IL-21 or IL-21iso, and the level of STAT3 phosphorylation induced by the two IL-21 forms, were similar. IL-21-sensitive reporter cells co-cultured with cells producing IL-21iso showed STAT3 activation, apoptosis, and proliferation. However, when IL-21iso-producing cells were cultured in a transwell chamber, which prevented direct contact with the IL-21-sensitive cells, no IL-21iso-induced signaling was observed. Though IL-21iso is secreted in smaller amounts and has less potent signaling activity than IL-21, IL-21iso acts both on IL-21iso-bearing cells and other IL-21-sensitive cells through direct interactions probably without being secreted. Thus, IL-21isos regulation of immune cells may be limited to the immediate proximity around the IL-21iso-producing cells, in regions such as immune organs or inflammation sites.

Apurinic/apyrimidinic endonuclease1/redox factor-1 (Ape1/Ref-1) is essential for IL-21-induced signal transduction through ERK1/2 pathway

IL-21 is a pleiotropic cytokine that regulates T-cell and B-cell differentiation, NK-cell activation, and dendritic cell functions. IL-21 activates the JAK-STAT, ERK, and PI3K pathways. We report here that Ape1/Ref-1 has an essential role in IL-21-induced cell growth signal transduction. Overexpression of Ape1/Ref-1 enhances IL-21-induced cell proliferation, but it is suppressed by overexpressing an N-terminal deletion mutant of Ape1/Ref-1 that lacks the redox domain. Furthermore, knockdown of the Ape1/Ref-1 mRNA dramatically compromises IL-21-induced ERK1/2 activation and cell proliferation with increasing cell death. These impaired activities are recovered by the re-expression of Ape1/Ref-1 in the knockdown cells. Our findings are the first demonstration that Ape1/Ref-1 is an indispensable molecule for the IL-21-mediated signal transduction through ERK1/2 activation.

The pattern of GPI-80 expression is a useful marker for unusual myeloid maturation in peripheral blood

Myeloid‐derived suppressor cells (MDSCs) have a wide spectrum of immunosuppressive activity; control of these cells is a new target for improving clinical outcomes in cancer patients. MDSCs originate from unusual differentiation of neutrophils or monocytes induced by inflammatory cytokines, including granulocyte‐colony stimulating factor (G‐CSF) and granulocyte–macrophage (GM)‐CSF. However, MDSCs are difficult to detect in neutrophil or monocyte populations because they are not uniform cells, resembling both neutrophils and monocytes; thus, they exist in a heterogeneous population. In this study, we investigated GPI‐80, a known regulator of Mac‐1 (CD11b/CD18) and associated closely with neutrophil maturation, to clarify this unusual differentiation. First, we demonstrated that the mean fluorescence intensity (MFI) of GPI‐80 and coefficient of variation (CV) of GPI‐80 were increased by treatment with G‐CSF and GM‐CSF, respectively, using a human promyelocytic leukaemia (HL60) cell differentiation model. To confirm the value of GPI‐80 as a marker of unusual differentiation, we measured GPI‐80 expression and MDSC functions using peripheral blood cells from metastatic renal cell carcinoma patients. The GPI‐80 CV was augmented significantly in the CD16hi neutrophil cell population, and GPI‐80 MFI was increased significantly in the CD33hi monocyte cell population. Furthermore, the GPI‐80 CV in the CD16hi population was correlated inversely with the proliferative ability of T cells and the GPI‐80 MFI of the CD33hi population was correlated with reactive oxygen species production. These results led us to propose that the pattern of GPI‐80 expression in these populations is a simple and useful marker for unusual differentiation, which is related to MDSC functions.