Akemi Araki

Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis or necrosis.

To elucidate the relationship between activation of neutrophils and their subsequent death, the effect of phorbol 12‐myristate 13‐acetate (PMA), a potent activator of neutrophils, was examined. PMA‐treated neutrophils showed morphological changes quite different from those of typical apoptosis or necrosis. After fusion of the lobate nucleus, nuclear contents of chromatin uniformly decreased in compactness and soon after the nuclear envelope was broken. Even at this stage, cytoplasmic organelles did not undergo degeneration. Membrane permeability began increasing at 3 h of incubation with PMA, subsequent to nuclear change. Conventional agarose gel electrophoresis and pulsed field gel electrophoresis of DNA from PMA‐treated neutrophils revealed no DNA degradation products smaller than 300 kbp. PKC inhibitors, staurosporine and H‐7, prevented cytotoxicity by PMA. Furthermore, antioxidants, thiourea, dimethylthiourea, pyrrolidinethiocarbamate, and N‐acetyl‐L‐cysteine, but not superoxide dismutase, were also active in preventing PMA cytotoxicity, suggesting that cell suicide resulting from PMA treatment is due to oxygen radicals, especially the hydroxyl radical. A certain population of neutrophils phagocytosing opsonized zymosan also showed changes similar to those observed in PMA‐treated cells.

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.

Nitric oxide is an effector molecule in inhibition of tumor cell growth by rIFN‐γ‐activated rat neutrophils

This study was designed to determine the effector molecule responsible for the tumor‐inhibitory activity of rat neutrophils treated with rat recombinant interferon gamma (rIFN‐γ) in vitro. The results show that nitric oxide (NO) production by neutrophils is dependent on rIFN‐γ concentration, and that neutrophil‐mediated tumor cytostasis is in turn dependent on the amount of NO. NO production and tumor cytostatis by rIFN‐γ‐activated neutrophils were inhibited completely by NG monomethyl‐L‐arginine (NGMMA), a specific competitive NO production inhibitor. Tumor cytostasis was also inhibited by oxyhemoglobin (HbO2), an NO scavenger. An extracellular oxygen radical scavenger, superoxide dismutase (SOD), was found to increase tumor cell inhibition by rIFN‐γ‐activated neutrophils by a factor of 4. This SOD‐enhanced cytostasis was not even inhibited by catalase. Tumor cytostasis was slightly increased by a hydroxyl radical‐(‐OH) scavanger, dimethylthiourea (DMTU), which did not affect NO production by rIFN‐γ‐activated neutrophils. Our findings suggest that tumor cytostasis of neutrophils activated by rIFN‐γ is mediated by L‐arginine‐derived nitrogen oxidation products, and that O2− produced by these neutrophils reduces NO‐mediated tumor cytostasis at low NO concentrations. Int. J. Cancer 71:223‐230, 1997.

rIFN-γ-activated rat neutrophils induce tumor cell apoptosis by nitric oxide

We have previously shown that 1) neutrophils activated with various cytokines, including rat recombinant interferon γ (rIFN‐γ), inhibit tumor cell growth and that 2) nitric oxide (NO) is the effector molecule in tumor inhibition by rIFN‐γ‐stimulated rat peritoneal exudate neutrophils. In this study, we examined the nature of tumor cell death induced by rat peritoneal neutrophils activated by rIFN‐γ in order to clarify the mechanism of apoptosis in neoplastic tumor cell death. DNA of 3 syngeneic rat tumor cell lines was significantly fragmented within 3 hr of incubation in the presence of rIFN‐γ‐activated neutrophils, and this effect was dependent on both the concentration of rIFN‐γ and the number of neutrophils. This DNA fragmentation was inhibited by L‐N‐(I‐iminoethyl)‐ornithine (L‐NIO), a NO synthase inhibitor, but not by superoxide dismutase (SOD). Tumor cells treated with the activated neutrophils were shown by electron microscopy to be apoptotic, exhibiting necrotic features with a longer incubation. On the other hand, cytolysis of tumor cells, as determined by a [3H]‐uridine release assay, was first observed only at 24 hr of incubation with the rIFN‐γ‐activated neutrophils. Taken together, our results suggest that tumor cell apoptosis by activated neutrophils is NO‐dependent and that apoptotic tumor cells undergo necrosis as a secondary process. We suggest that tumor cell apoptosis induced by activated neutrophils plays an important role in regulation of neoplastic tumor cell growth and death in vivo. Int. J. Cancer 71:231–236, 1997.

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.

The neutrophil as an information transmitter in tumor inhibition by a streptococcal biological response modifier, OK-432

Abstract Effective treatment of a rat transplanted ascites tumor by i. p. injection of a streptococcal biological response modifier, OK-432, was abrogated by selective in vivo depletion of neutrophils by a monoclonal antibody, RP-3. The mechanisms by which neutrophils participate in the therapeutic action of OK-432 were studied with Winn’s assay using peritoneal exudate cells periodically obtained from rats i. p. injected with this biological response modifier. Intraperitoneal resident macrophages were first activated with OK-432, and within 3 h, tumor-inhibitory activity had moved to the early exuded neutrophils. However, 6 h after injection, exuded macrophages were the only cells involved in tumor inhibition. Considered together with other findings, it is likely that, in this system, neutrophils may transmit information from resident macrophages to exuded inflammatory macrophages in a series of responses induced by i. p. injection of OK-432.

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.