Masafumi Nakayama

Tim-3 mediates phagocytosis of apoptotic cells and cross-presentation

Phagocytes such as macrophages and dendritic cells (DCs) engulf apoptotic cells to maintain peripheral immune tolerance. However, the mechanism for the recognition of dying cells by phagocytes is not fully understood. Here, we demonstrate that T-cell immunoglobulin mucin-3 (Tim-3) recognizes apoptotic cells through the FG loop in the IgV domain, and is crucial for clearance of apoptotic cells by phagocytes. Whereas Tim-4 is highly expressed on peritoneal resident macrophages, Tim-3 is expressed on peritoneal exudate macrophages, monocytes, and splenic DCs, indicating distinct Tim-mediated phagocytic pathways used by different phagocytes. Furthermore, phagocytosis of apoptotic cells by CD8(+) DCs is inhibited by anti-Tim-3 mAb, resulting in a reduced cross-presentation of dying cell-associated antigens in vitro and in vivo. Administration of anti-Tim-3 as well as anti-Tim-4 mAb induces autoantibody production. These results indicate a crucial role for Tim-3 in phagocytosis of apoptotic cells and cross-presentation, which may be linked to peripheral tolerance.

Multiple pathways of TWEAK-induced cell death.

TWEAK, a recently identified member of the TNF family, is expressed on IFN-γ-stimulated monocytes and induces cell death in certain tumor cell lines. In this study, we characterized the TWEAK-induced cell death in several tumor cell lines that exhibited distinct features. Although the TWEAK-induced cell death in Kym-1 cells was indirectly mediated by TNF-α and was inhibited by cycloheximide, the TWEAK-induced cell death in HSC3 cells or IFN-γ-treated HT-29 cells was not inhibited by anti-TNF-α mAb or cycloheximide, suggesting a direct triggering of cell death via TWEAK receptor in the latter cell lines. The TWEAK-induced apoptosis in HSC3 cells and IFN-γ-treated HT-29 cells was associated with caspase-8 and caspase-3 activation. Although a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, inhibited the TWEAK-induced cell death in HSC3 cells, it rather sensitized HT-29 cells to TWEAK-induced cell death by necrosis. This necrosis was abrogated by lysosomal proteinase inhibitors, particularly a cathepsin B inhibitor, [l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-l-isoleucyl-l-proline methyl ester. During the process of TWEAK-induced necrosis, cathepsin B was released from lysosome to cytosol. Although DR3 has been reported to be a receptor for TWEAK, all TWEAK-sensitive tumor cell lines used in this study did not express DR3 at either protein or mRNA level, but did bind CD8-TWEAK specifically. These results indicated that TWEAK could induce multiple pathways of cell death, including both caspase-dependent apoptosis and cathepsin B-dependent necrosis, in a cell type-specific manner via TWEAK receptor(s) distinct from DR3.

Pro-inflammatory effect of TWEAK/Fn14 interaction on human umbilical vein endothelial cells ☆

TWEAK, a member of the TNF family, induces cell death in some tumor cell lines, but also induces proliferation of endothelial cells and angiogenesis. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which may be responsible for the proliferation of endothelial cells and angiogenesis. In this study, we investigated the pro-inflammatory effect of TWEAK on human umbilical vein endothelial cells (HUVEC). We demonstrated that TWEAK could not only induce the proliferation and migration but also upregulate the cell surface expression of adhesion molecules such as ICAM-1 and E-selectin, and induce the secretion of chemokines such as IL-8 and MCP-1 in HUVEC. Moreover, by using an anti-Fn14 mAb that blocks the TWEAK/Fn14 interaction, we demonstrated that Fn14 was constitutively expressed on HUVEC and totally mediated the biological effects of TWEAK on HUVEC. These results indicated that TWEAK could induce pro-inflammatory reactions via Fn14 on HUVEC.

Fibroblast Growth Factor-Inducible 14 Mediates Multiple Pathways of TWEAK-Induced Cell Death

TWEAK, a TNF family member, is produced by IFN-γ-stimulated monocytes and induces multiple pathways of cell death, including caspase-dependent apoptosis, cathepsin B-dependent necrosis, and endogenous TNF-α-mediated cell death, in a cell type-specific manner. However, the TWEAK receptor(s) that mediates these multiple death pathways remains to be identified. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which was responsible for TWEAK-induced proliferation of endothelial cells and angiogenesis. Because Fn14 lacks the cytoplasmic death domain, it remains unclear whether Fn14 can also mediate the TWEAK-induced cell death. In this study, we demonstrated that TWEAK could induce apoptotic cell death in Fn14 transfectants. A pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, rather sensitized the Fn14 transfectants to TWEAK-induced cell death by necrosis via reactive oxygen intermediates and cathepsin B-dependent pathway. By using newly generated agonistic anti-Fn14 mAbs, we also observed that Fn14 is constitutively expressed on the cell surface of all TWEAK-sensitive tumor cell lines, and can transmit the multiple death signals. Moreover, an anti-Fn14 mAb that blocks TWEAK-Fn14 interaction could totally abrogate TWEAK binding and TWEAK-induced cell death in all TWEAK-sensitive tumor cell lines. These results revealed that the multiple pathways of TWEAK-induced cell death are solely mediated by Fn14.

Effect of silica particle size on macrophage inflammatory responses.

Amorphous silica particles, such as nanoparticles (<100 nm diameter particles), are used in a wide variety of products, including pharmaceuticals, paints, cosmetics, and food. Nevertheless, the immunotoxicity of these particles and the relationship between silica particle size and pro-inflammatory activity are not fully understood. In this study, we addressed the relationship between the size of amorphous silica (particle dose, diameter, number, and surface area) and the inflammatory activity (macrophage phagocytosis, inflammasome activation, IL-1β secretion, cell death and lung inflammation). Irrespective of diameter size, silica particles were efficiently internalized by mouse bone marrow-derived macrophages via an actin cytoskeleton-dependent pathway, and induced caspase-1, but not caspase-11, activation. Of note, 30 nm-1000 nm diameter silica particles induced lysosomal destabilization, cell death, and IL-1β secretion at markedly higher levels than did 3000 nm-10000 nm silica particles. Consistent with in vitro results, intra-tracheal administration of 30 nm silica particles into mice caused more severe lung inflammation than that of 3000 nm silica particles, as assessed by measurement of pro-inflammatory cytokines and neutrophil infiltration in bronchoalveolar lavage fluid of mice, and by the micro-computed tomography analysis. Taken together, these results suggest that silica particle size impacts immune responses, with submicron amorphous silica particles inducing higher inflammatory responses than silica particles over 1000 nm in size, which is ascribed not only to their ability to induce caspase-1 activation but also to their cytotoxicity.

Paired Ig-Like Receptors Bind to Bacteria and Shape TLR-Mediated Cytokine Production

The innate immune system uses a wide variety of pattern recognition receptors including TLRs, scavenger receptors, and lectins to identify potential pathogens. A carefully regulated balance between activation and inhibition must be kept to avoid detrimental and inappropriate inflammatory responses. In this study, we identify murine-paired Ig-like receptor (PIR)-B, and its human orthologs Ig-like transcript 2 and Ig-like transcript 5 as novel receptors for Staphylococcus aureus. PIR-B contains four ITIM motifs and is thought to be an inhibitory receptor. Expression of these receptors enables NIH3T3 cells to bind S. aureus. In mouse bone marrow-derived macrophages, masking of PIR-B by anti-PIR mAb or genetic deletion of PIR-B shows significantly impaired recognition of S. aureus and enhanced TLR-mediated inflammatory responses to the bacteria. These data suggest a novel mechanism for innate immune regulation by paired Ig-like receptor family members.

Characterization of murine TWEAK and its receptor (Fn14) by monoclonal antibodies

In the present study, we characterized murine TWEAK and its receptor (Fn14) by generating cDNA transfectants and specific monoclonal antibodies (mAbs). Recombinant murine TWEAK bound to murine Fn14-transfected L5178Y (mFn14/L5178Y) cells and induced cell death. Some anti-human Fn14 mAbs we previously generated strongly cross-reacted with murine Fn14 and induced cell death in mFn14/L5178Y cells. Murine TWEAK-transfected L5178Y cells expressed murine TWEAK on cell surface and secreted functional TWEAK, which were detected by a newly generated anti-murine TWEAK mAb (MTW-1). Although thioglycolate-elicited murine peritoneal macrophages did not express a detectable level of TWEAK on their surface, they secreted functional TWEAK that was cytotoxic against mFn14/L5178Y cells and neutralized by MTW-1. The anti-murine TWEAK and Fn14 mAbs will be useful for further investigating the physiological and pathological functions of TWEAK and Fn14.

IFN-γ production by lung NK cells is critical for the natural resistance to pulmonary metastasis of B16 melanoma in mice

NK cells are effector lymphocytes playing a critical role in the natural resistance against tumors. However, the precise mechanisms underlying NK cell‐mediated natural resistance against tumor metastasis are still unrevealed. B16 cells, mouse melanoma cells, were resistant to freshly isolated NK cell‐mediated killing; nevertheless, NK cells were critical for natural resistance against experimental lung metastasis of B16 cells. We found that lung metastasis was increased significantly in IFN‐γ–/– mice but not pfp–/–, IFN‐αR–/–, or IL‐12/IL‐18–/– mice. Interestingly, freshly isolated lung NK cells, but not spleen or liver NK cells, displayed augmented IFN‐γ production after B16 inoculation. Adoptive transfer of pfp–/– NK cells, but not IFN‐γ–/– NK cells, significantly decreased B16 lung metastasis in IFN‐γ–/– and pfp/IFN‐γ–/–mice. Lung metastases of IFN‐γRDN B16 was also increased in NK cell‐depleted or IFN‐γ–/– mice, suggesting that the IFN‐γ response of host cells was required in the NK cell and IFN‐γ‐mediated antimetastatic effect. Our results demonstrate that IFN‐γ production from lung resident NK cells is a key response in the natural resistance to the experimental lung metastasis of NK cell‐resistant tumor cells.

Natural killer (NK)–dendritic cell interactions generate MHC class II-dressed NK cells that regulate CD4+ T cells

Natural killer (NK) cells contribute to not only innate but also to adaptive immunity by interacting with dendritic cells (DCs) and T cells. All activated human NK cells express HLA-DR and can initiate MHCII-dependent CD4+ T-cell proliferation; however, the expression of MHCII by mouse NK cells and its functional significance are controversial. In this study, we show that NK–DC interactions result in the emergence of MHCII-positive NK cells. Upon in vitro or in vivo activation, mouse conventional NK cells did not induce MHCII transcripts, but rapidly acquired MHCII protein from DCs. MHCII H2-Ab1–deficient NK cells turned I-Ab-positive when adoptively transferred into wild-type mice or when cultured with WT splenic DCs. NK acquisition of MHCII was mediated by intercellular membrane transfer called “trogocytosis,” but not upon DAP10/12- and MHCI-binding NK cell receptor signaling. MHCII-dressed NK cells concurrently acquired costimulatory molecules such as CD80 and CD86 from DCs; however, their expression did not reach functional levels. Therefore, MHCII-dressed NK cells inhibited DC-induced CD4+ T-cell responses rather than activated CD4+ T cells by competitive antigen presentation. In a mouse model for delayed-type hypersensitivity, adoptive transfer of MHCII-dressed NK cells attenuated footpad swelling. These results suggest that MHCII-dressed NK cells generated through NK–DC interactions regulate T cell-mediated immune responses.

Downregulation of c-FLIP promotes caspase-dependent JNK activation and reactive oxygen species accumulation in tumor cells

Nuclear factor-kappa B (NF-κB) inhibits cell death through suppression of the caspase cascade, the c-Jun N-terminal kinase (JNK) pathway, and reactive oxygen species (ROS) accumulation. To suppress this antiapoptotic function of NF-κB might be a promising strategy to increase susceptibility of tumor cells to stress-induced cell death. We have recently shown that tumor necrosis factor (TNF)α induces caspase-dependent and -independent JNK activation and ROS accumulation in cellular FLICE-inhibitory protein (c-Flip)−/− murine embryonic fibroblasts (MEFs). To apply this observation to tumor therapy, we knocked down c-FLIP by RNA interference in various tumor cells. Consistent with the results using c-Flip−/− MEFs, we found that TNFα stimulation induced caspase-dependent prolonged JNK activation and ROS accumulation, followed by apoptotic and necrotic cell death in various tumor cells. Furthermore, TNFα and Fas induced the cleavage of mitogen-activated protein kinase/ERK kinase kinase (MEKK)1, resulting in generation of a constitutive active form of MEKK1 leading to JNK activation in c-FLIP knockdown cells. Given that ROS accumulation and necrotic cell death enhance inflammation followed by compensatory proliferation of tumor cells, selective suppression of caspase-dependent ROS accumulation will be an alternative strategy to protect cells from ROS-dependent DNA damage and compensatory tumor progression.