Eiji Takayama

Neutrophil elastase, MIP-2, and TLR-4 expression during human and experimental sepsis

Highly activated neutrophils play a critical role in mediating organ injury in sepsis by releasing neutrophil elastase (NE). Toll-like receptors (TLRs) play an important role in the host defense against invading microbes, and their signaling pathway is critical to the activation of the proinflammatory response. However, the relationship between TLR expression and the host defense mechanism during sepsis has not been fully elucidated. In this paper, we investigated the relationships among chemokine (MIP-2), TLR-4, and NE expression in human sepsis and murine peritonitis (CLP). TLR-4 expression on monocytes/macrophages was examined in patients with sepsis and in murine peritonitis and was markedly increased in both populations. LPS-induced MIP-2 production by bronchoalveolar cells and liver mononuclear cells in mice with peritonitis was also significantly increased compared with sham-operated mice. Pretreatment of the macrophage cell line, RAW 264.7 cells, with a NE inhibitor before their exposure to LPS resulted in a significant dose-dependent decrease in MIP-2 production, which was comparable to that seen following pretreatment with TLR-4 antibody. Furthermore, NE and LPS both up-regulated TLR-4 expression on human peripheral blood monocytes. Thus, chemokine-induced recruitment of neutrophils in sepsis may result in further increased chemokine production and increased expression of TLR-4. Neutrophil-derived NE may be associated with increased expression of monocyte/macrophage TLR-4, thereby serving as a positive feedback loop for the inflammatory response among the different cell populations.

The Mechanism of a Defective IFN-γ Response to Bacterial Toxins in an Atopic Dermatitis Model, NC/Nga Mice, and the Therapeutic Effect of IFN-γ, IL-12, or IL-18 on Dermatitis

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-γ levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-γ response to staphylococcal enterotoxin B was correlated to the lack of regular Vβ8+ T cells and Vβ8+ NK T cells, and the low IFN-γ response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-γ, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.

Activation of Monocytes and Endothelial Cells Depends on the Severity of Surgical Stress

Surgical injury not only induces a systemic endocrine-metabolic response but also influences the function of the leukocytes and endothelial cells leading to various systemic responses. These responses appear to depend on the severity of surgical stress, which differs according to the surgical procedures. In this study, we investigated the response of monocytes and endothelial cells, and the development of systemic inflammatory response syndrome (SIRS) in relation to the severity of surgical stress. The postoperative clinical course was evaluated between patients undergoing an esophagectomy (ER group) and a distal gastrectomy (DG group). The tumor necrosis factor α (TNFα) production of monocytes, the serum interleukin 6 (IL-6) levels, the CD11b expression on either monocytes or granulocytes, and the intercellular adhesion molecule-1 (ICAM-1) expression on human umbilical vein endothelial cells (HUVECs) stimulated with culture supernatants of monocytes were compared between the 2 groups. The development of SIRS was observed in all patients in the ER group, whereas no patients demonstrated SIRS in the DG group. The serum IL-6 levels, TNFα production of monocytes, and CD11b intensity on monocytes or granulocytes in the ER group were higher than those in the DG group. In the ER group, the ICAM-1 intensity on HUVECs with monocytes immediately after operation significantly increased compared with before the operation. In conclusion, both the CD11b expression on monocytes and the TNFα production of monocytes are considered to reflect the degree of surgical stress, and the activation of endothelial cells stimulated with these activated leukocytes may therefore lead to both tissue and organ injury.

Autophagy in regulation of Toll-like receptor signaling.

Toll-like receptors (TLRs) serve as the major innate immune sensors for detection of specific molecular patterns on various pathogens. TLRs activate signaling events mainly by utilizing ubiquitin-dependent mechanisms. Recent research advances have provided evidence that TLR signaling is linked to induction of autophagy. Autophagy is currently known to affect both of the immune defense and suppression of inflammatory responses. In TLR-associated immune responses, autophagic lysis of intracellular microbes (called xenophagy) contributes to the former mechanism, while the latter seems to be mediated by the control of the mitochondrial integrity or selective autophagic clearance of aggregated signaling proteins (called aggrephagy). Several autophagy-related ubiquitin-binding proteins, such as SQSTM1/p62 and NDP52, mediate xenophagy and aggrephagy. In this review, we summarize the expanded knowledge regarding TLR signaling and autophagy signaling. After that, we will focus on autophagy-associated signaling downstream of TLRs and the effect of autophagy on TLR signaling, thus highlighting the signaling crosstalk between the TLR-associated innate immune responses and the regulation of innate immunity by xenophagy and aggrephagy.

Preoperative serum interleukin-18 level as a postoperative prognostic marker in patients with gastric carcinoma

Interleukin‐18 (IL‐18), a recently described cytokine produced mainly by macrophages, stimulates interferon‐γ (IFN‐γ) production by natural killer cells and T cells. Although it has been reported that serum IL‐18 levels are higher in patients with advanced tuberculosis and acute graft‐versus‐host disease compared with normal controls, the authors found no reports regarding serum IL‐18 levels in patients with malignant solid tumors. The purpose of this study was to determine serum IL‐18 levels and their clinical significance in patients with gastric carcinoma.

Nuclear translocation of extracellular superoxide dismutase.

Histochemical examination of mouse tissues showed nuclear staining of extracellular superoxide dismutase (EC-SOD), and the nuclear translocation of EC-SOD was also confirmed in cultured cells that had been transfected with its gene, as shown by immunohistochemistry and Western blot analysis. The EC-SOD which was secreted into the medium was incorporated into 3T3-L1 cells and a significant fraction of the material taken up was localized in the nucleus. Site-directed mutagenesis indicated that the heparin-binding domain of EC-SOD functions as the nuclear localization signal. These results suggest that the mechanism of the nuclear transport of EC-SOD involves a series of N-terminal signal peptide- and C-terminal heparin-binding domain-dependent processes of secretion, re-uptake and the subsequent nuclear translocation. The findings herein provide support for the view that the role of EC-SOD is to protect the genome DNA from damage by reactive oxygen species and/or the transcriptional regulation of redox-sensitive gene expression.

Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

The level of serum CCL5, a C‐C chemokine, is reportedly correlated with tumor progression in several cancers. We herein investigated the mechanisms by which CCL5 might contribute to tumor progression in gastric cancer. Serum CCL5 levels significantly correlated with tumor progression and prognosis in patients with gastric cancer. Immunohistochemistry showed that tumor‐infiltrating lymphocytes expressed CCL5, while the tumor cells expressed the CCL5 receptors. Fluorescent double staining showed that tumor‐infiltrating CD4+ cells rather than CD8+ cells preferentially expressed CCL5. Using gastric cancer cell lines (MKN45, KATO III), we examined CCL5 production by coculturing whole peripheral blood mononuclear cells (PBMCs), CD4+ cells, or CD8+ cells, with tumor cells. CD4+ cells cocultured with tumor cells remarkably enhanced CCL5 production in a direct cell–cell contact manner over other cocultured PBMCs, including CD8+ cells. Gastric cancer cell lines expressed CCL5 receptors and augmented their proliferation in response to CCL5 stimulation. Furthermore, we examined the effect of CCL5‐treated cancer cells on the cocultured PBMCs, focusing on the CD4+/CD8+ proportion and apoptosis. Coculture of CCL5‐treated gastric cancer cells with PBMCs resulted in a significant decrease in the proportion of CD8+ cells but not CD4+ cells, suggesting Fas‐FasL‐mediated apoptosis in CD8+ cells. In immunodeficient mice coinjected with KATO III and PBMCs, neutralization of CCL5 significantly suppressed tumor progression, resulting in a favorable outcome. In conclusion, gastric cancer cells might thus induce CD4+ T cells to secrete CCL5 and exploit it for their progression, as well as to aid in the prevention of CD8+ T cell‐involved tumor elimination.

Levels of annexin IV and V in the plasma of pregnant and postpartum women

Annexin (Anx) V is pivotal in the maintenance of pregnancy by preventing the activation of blood coagulation. The homology of the amino acid sequence between Anx IV and Anx V is highest in Anx family proteins. However, little is known about the roles of Anx IV in pregnancy. The aim of this study is to clarify the roles of circulating Anx IV and Anx V in normal pregnancy. Subjects were non-pregnant women (n = 50), 120 pregnant women, and maternal subjects just after delivery (n = 53) or postpartum (n = 67). Anx IV in the plasma of non-pregnant women was at a concentration 20 times that of Anx V. The plasma levels of Anx IV suddenly increase after delivery, but Anx V levels remain low during this period. Anx IV and Anx V exert similar levels of anticoagulant activity. Anx IV protein was expressed on the basal surface of syncytiotrophoblasts; Anx V protein, on the apical surface of syncytiotrophoblasts. These results suggest that Anx IV enters the maternal bloodstream just after delivery and might play a role in preventing disseminated intravascular coagulopathy, and that Anx V helps to prevent clotting in the placenta during pregnancy.

Mouse CD8+ CD122+ T Cells with Intermediate TCR Increasing with Age Provide a Source of Early IFN-γ Production

Although CD8+ IL-2Rβ (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rβ-negative or -low (CD122−) T cells conversely decreased. The IFN-γ production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-γ than did CD8+ CD122− T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-γ production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-γ production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-γ. The CD3-stimulated IFN-γ production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-γ than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-γ production and are suggested to be involved in the immunological changes with aging.

Selective Inhibition of Hepatoma Cells Using Diphtheria Toxin A under the Control of the Promoter/Enhancer Region of the Human α‐Fetoprotein Gene

We constructed a plasmid containing human α‐fetoprotein (AFP) promoter/enhancer to direct the cell type‐specific expression of diphtheria toxin fragment A (DTA), designated as pAF‐DTA, to AFP‐producing hepatocellular carcinoma cells. The transfection was carried out with cationic liposomes (DMRIE‐C) and the expression of the DTA gene was confirmed by a northern blot analysis. When pAF‐DTA was transfected, the growth of AFP‐positive HuH‐7 cells was inhibited, whereas growth inhibition was not observed in AFP‐negative MKN45 cells. In this experiment, the secretion of AFP was similarly suppressed, but the secretion of carcinoembryonic antigen from MKN45 was not altered. pAF‐DTA could also exert its growth inhibitory effect on PLC, a cell line with a low level of AFP. However, no inhibitory effect of pAF‐DTA was observed on the proliferation of primary hepatocyte cells. Furthermore, transfection experiments in which HuH‐7 and splenic stromal cells were co‐cultured revealed the growth inhibition by pAF‐DTA to be selective in HuH‐7 cells. Finally, the growth of HuH‐7 transplanted on BALB/c nu/nu mice was inhibited by the direct injection of pAF‐DTA/liposome complex into a tumor mass. These results suggest that use of pAF‐DTA may be potentially useful as a novel approach for the selective treatment of tumor cells producing AFP even at low levels, without affecting other types of cells.