Masanao Saio

Tumor-infiltrating myeloid-derived suppressor cells are pleiotropic-inflamed monocytes/macrophages that bear M1- and M2-type characteristics

Here, tumor‐infiltrating CD11b+ myelomonocytoid cells in murine colon adenocarcinoma‐38 and GL261 murine glioma were phenotypically characterized. Over 90% were of the CD11b+F4/80+ monocyte/macrophage lineage. They also had a myeloid‐derived suppressor cell (MDSC) phenotype, as they suppressed the proliferation of activated splenic CD8+ T cells and had a CD11b+CD11c+Gr‐1lowIL‐4Rα+ phenotype. In addition, the cells expressed CX3CR1 and CCR2 simultaneously, which are the markers of an inflammatory monocyte. The MDSCs expressed CD206, CXCL10, IL‐1β, and TNF‐α mRNAs. They also simultaneously expressed CXCL10 and CD206 proteins, which are typical, classical (M1) and alternative (M2) macrophage activation markers, respectively. Peritoneal exudate cells (PECs) strongly expressed CD36, CD206, and TGF‐β mRNA, which is characteristic of deactivated monocytes. The MDSCs also secreted TGF‐β, and in vitro culture of MDSCs and PECs with anti‐TGF‐β antibody recovered their ability to secrete NO. However, as a result of secretion of proinflammatory cytokines, MDSCs could not be categorized into deactivated monocyte/macrophages. Thus, tumor‐infiltrating MDSCs bear pleiotropic characteristics of M1 and M2 monocytes/macrophages. Furthermore, CD206 expression by tumor‐infiltrating MDSCs appears to be regulated by an autocrine mechanism that involves TGF‐β.

CD8+ Tumor-Infiltrating T Cells Are Deficient in Perforin-Mediated Cytolytic Activity Due to Defective Microtubule-Organizing Center Mobilization and Lytic Granule Exocytosis

Tumor-infiltrating lymphocytes (TIL) are well known to be functionally impaired typified by the inability to lyse cognate tumor cells in vitro. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8+ TIL are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cytokine mRNAs, and contain perforin and granzyme B proteins which are packaged into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, but not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rapid; mitogen-activated protein/extracellular signal-related kinase kinase, extracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein kinase C are activated; and IL-2 and IFN-γ is secreted. However, on conjugate formation between nonlytic TIL and cognate tumor cells in vitro, the microtubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis of preformed lytic granules and accounting for defective TIL lytic function. Recovery of TCR-mediated, activation-dependent MTOC mobilization and lytic activity requires proteasome function, implying the existence of an inhibitor of MTOC mobilization. Our findings show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction.

Depletion of CD4+CD25+ regulatory T cells enhances interleukin-2-induced antitumor immunity in a mouse model of colon adenocarcinoma

Interleukin 2 (IL)‐2 induces antitumor immunity and clinical responses in melanoma and renal cell carcinoma. However, IL‐2 also increases the number of CD4+CD25+ regulatory T (Treg) cells that suppress antitumor immune responses. The aim of the present study was to elucidate the effect of depletion of Treg cells on IL‐2‐induced antitumor immunity. IL‐2‐transfected mouse colon adenocarcinoma (MC38/IL‐2) cells were implanted subcutaneously or intrahepatically into male C57BL/6 mice, and tumor growth and the proportion of tumor‐infiltrating lymphocytes with Treg‐cell depletion in response to treatment with anti‐CD25 monoclonal antibody (PC61) were determined. In mice treated with phosphate‐buffered saline, 40–60% of MC38/IL‐2 tumors were rejected. In contrast, all MC38/IL‐2 tumors were rejected in mice treated with PC61. The number of tumor‐infiltrating CD8+ T cells in mice treated with PC61 was approximately twice that in mice treated with PBS. The numbers of tumor‐infiltrating CD4+ and natural killer cells were also increased significantly. To test the antimetastatic effects of IL‐2 treatment in combination with Treg‐cell depletion, human recombinant IL‐2 (rIL‐2) and PC61 were administered to mice implanted with MC38/mock cells in the spleen, and hepatic metastasis was investigated. The average liver weight in mice treated with rIL‐2 plus PC61 was 1.04 ± 0.03 g, less than that in mice treated with rIL‐2 (2.04 ± 0.51 g) or PC61 alone (1.81 ± 0.38 g). We conclude that IL‐2‐induced antitumor immunity is enhanced by Treg‐cell depletion and is due to expansion of the tumor‐infiltrating cytotoxic CD8+ T‐cell population. (Cancer Sci 2007; 98: 416–423)

Metallothionein-3 is expressed in the brain and various peripheral organs of the rat

Metallothionein-3 (MT-3), also known as growth inhibitory factor (GIF), was originally identified in the brain. An essential step in elucidating the potential roles of MT-3 is to evaluate its expression levels in organs other than the brain. In this present study, we carried out RT-PCR, Western blot and immunohistochemical analyses to quantify MT-3 mRNA and its protein in the cerebrum, eye, heart, kidney, liver, prostate, testis, tongue, and muscle in male Wistar rats. MT-3 mRNA was detected in the cerebrum, the dorsolateral lobe of the prostate, testis, and tongue. Using a monoclonal anti-MT-3 antibody, we detected MT-3 in the cerebrum, the dorsolateral lobe of the prostate, testis, and tongue as a single band on an immunoblot. Immunohistochemical staining showed MT-3 in some astrocytes in the deep cortex, ependymal cells, and choroidal cells in the cerebrum. MT-3 was also detected in some cells of the glomerulus and the collective tubules in the kidney, some cells in the glandular epithelium of the dorsolateral lobe of the prostate, some Sertoli cells and Lydig cells in the testis, and taste bud cells in the tongue. Although MT-3 immunopositivity was obviously demonstrated in the kidney by the immnunohistochemical method, the expression of MT-3 was not fully detectable by RT-PCR and Western blot analyses. Interestingly, only a subset of cells showed positivity for MT-3, not all cells in all tissues. The localization of MT-3 in peripheral organs outside the brain suggests that MT-3 has roles in these tissues besides its role in growth inhibition of neurites.

Mitotic catastrophe and cell death induced by depletion of centrosomal proteins

Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization.

Expression of survivin and of antigen detected by a novel monoclonal antibody, T332, is associated with outcome of diffuse large B-cell lymphoma and its subtypes

Although diffuse large B‐cell lymphoma (DLBCL) is the most common type of non‐Hodgkin lymphoma, it is both clinically and morphologically heterogenous. The present study investigates the significance of survivin and a novel monoclonal antibody (MAb), T332, immunohistochemically for predicting the prognoses of DLBCL and its subtypes classified as germinal center B‐cell‐like type (GCB) and non‐GCB type (NGCB) based on the expression profiles of CD10, bcl‐6, and MUM1. A total of 60 cases of DLBCL (GCB, n = 22; NGCB, n = 38) were examined for the expression of survivin and T332 antigen. Survivin+ DLBCL had a significantly worse prognosis (P = 0.01) than survivin– cases, as already reported, while survivin+ GCB or NGCB tended to have poor prognoses (P = 0.06 and 0.07, respectively). However, T332+ DLBCL and NGCB had significantly more unfavorable prognoses than T332– cases (P = 0.01 and 0.02, respectively) while there was no significant survival difference between the T332+ and T332– groups of GCB (P = 0.11). Interestingly DLBCL coexpressing survivin and T332 (n = 13) had a significantly worse prognosis (P = 0.009) than the remaining single positive and double negative cases (n = 31). In conclusion, survivin and the novel MAb, T332, might be a good predictor of DLBCL and its subtypes.

Nuclear factor-κB activates dual inhibition sites in the regulation of tumor necrosis factor-α-induced neutrophil apoptosis

Abstract The objective of this study was to elucidate the role of the nuclear factor-κB (NF-κB) pathway in tumor necrosis factor-α (TNF-α)-induced neutrophil apoptosis. A single treatment with TNF-α produced significant caspase-3 activiation in a time- and concentration-dependent manner, while no significant morphological change in neutrophils was observed. After pretreatment of neutrophils with cycloheximide or actinomycin D, TNF-α produced morphologically typical apoptosis in a time- and concentration-dependent manner. Similarly, following pretreatment of neutrophils with the specific NF-κB inhibitors, pyrrolidine dithiocarbamate or SN50, TNF-α also produced neutrophil apoptosis (assessed morphologically). Caspase-3 activation by TNF-α was significantly enhanced by pretreatment with both cycloheximide and pyrrolidine dithiocarbamate. TNF-α-induced a rapid phosphorylation and degradation of IκB-α in neutrophils. Furthermore, TNF-α increased NF-κB DNA binding, which was abolished by pretreatment with pyrrolidine dithiocarbamate. These results indicate that the NF-κB pathway is crucial for neutrophil survival against TNF-α cell toxicity. Furthermore, it is proposed that NF-κB-induced proteins act on dual inhibitory sites, both upstream and downstream of caspase-3, to protect against apoptosis.

Evidence for the Synthesis of Corticosteroid-Binding Globulin in Human Placenta

We demonstrated the expression of corticosteroid-binding globulin (CBG) in human placenta using reverse transcription-polymerase chain reaction-Southern blot analysis and immunohistochemical and immunoblotting studies. In the RT-PCR-Southern blot analysis, only one predicted PCR product was detected without nonspecific products in all samples of human placenta and 3A (tPA-30-1) human placental cells. In Western blot analysis, polyclonal anti-CBG antibodies recognized a protein of approximately 55 kD in the protein extracts prepared from 3A (tPA-30-1) cells. Additionally, CBG mRNA expression was demonstrated by in situ hybridization in the syncytiotrophoblasts. Immunohistochemical studies performed on the placenta demonstrated the presence of specific immunoreactivity in the syncytiotrophoblast layer surrounding the chorionic villi. These findings suggest that CBG is synthesized in human placenta during pregnancy in addition to its synthesis in the liver.

Critical role of CD44 in hepatotoxin-mediated liver injury

BACKGROUND/AIMS Blocking of adhesion molecules is considered to be one of the therapeutic strategies inflammatory diseases, although it remains unclear whether this strategy is beneficial. METHODS We used CD44-deficient mice to assess whether inhibition of CD44 could control liver injury caused by carbon tetrachloride (CCl(4)). RESULTS CD44-deficient mice exhibited suppressed liver inflammation during the early phase (within 6h) after CCl(4) injection due to reduced inflammatory cell infiltration and cytokine production, but showed severe liver inflammation with increased numbers of apoptotic hepatocytes at the late phase (after 12h). The induction of hepatocyte apoptosis was triggered by reduced NF-kappaB activity, which was induced by the low inflammatory cytokine concentrations. Furthermore, macrophages contributed to the induction of hepatocyte apoptosis, since neutralization by an anti-CD11b antibody significantly protected against hepatocyte apoptosis. Finally, we found that blocking of MIP-2 and TNF-alpha reduced hepatocyte apoptosis with decreased numbers of intrahepatic leukocytes and reduced inflammatory cytokine production. CONCLUSIONS These findings suggest that targeting of CD44 as a therapeutic approach for inflammatory liver diseases may require caution for particular immune systems in the liver.

Skewing the Th cell phenotype toward Th1 alters the maturation of tumor-infiltrating mononuclear phagocytes

Mononuclear phagocytes (MPCs) at the tumor site can be divided into subclasses, including monocyte‐lineage myeloid‐derived suppressor cells (MDSCs) and the immunosuppressive tumor‐infiltrating macrophages (TIMs). Cancer growth coincides with the expansion of MDSCs found in the blood, secondary lymphoid organs, and tumor tissue. These MDSCs are thought to mature into macrophages and to promote tumor development by a combination of growth‐enhancing properties and suppression of local antitumor immunoresponses. As little is known about either subset of MPCs, we investigated MPCs infiltrating into murine adenocarcinoma MCA38 tumors. We found that these MPCs displayed immunosuppressive characteristics and a MDSC cell‐surface phenotype. Over 70% of the MPCs were mature (F4/80+Ly6C−) macrophages, and the rest were immature (F480+ Ly6C+) monocytes. MPC maturation was inhibited when the cells infiltrated a tumor variant expressing IL‐2 and soluble TNF type II receptor (sTNFRII). In addition, the IL‐2/sTNFRII MCA38 tumor microenvironment altered the MPC phenotype; these cells did not survive culturing in vitro as a result of Fas‐mediated apoptosis and negligible M‐CSFR expression. Furthermore, CD4+ tumor‐infiltrating lymphocytes (TILs) in wild‐type tumors robustly expressed IL‐13, IFN‐γ, and GM‐CSF, and CD4+ TILs in IL‐2/sTNFRII‐expressing tumors expressed little IL‐13. These data suggest that immunotherapy‐altered Th cell balance in the tumor microenvironment can affect the differentiation and maturation of MPCs in vivo. Furthermore, as neither the designation MDSC nor TIM can sufficiently describe the status of monocytes/macrophages in this tumor microenvironment, we believe these cells are best designated as MPCs.