Munehisa Takahashi

Silent Cleanup of Very Early Apoptotic Cells by Macrophages

Apoptotic cells are phagocytosed as soon as they appear in vivo. In this study, we first determined precisely at what stage apoptotic cells are phagocytosed by macrophages, and then examined the subsequent cytokine production. Phagocytosis was confirmed by flow cytometry and confocal laser microscopy, whereas the subsequent response was examined by ELISA and RT-PCR for quantitative and semiquantitative measurement of the protein and mRNA levels of cytokines, respectively. Even the cell populations containing very early apoptotic cells, such as IL-2-dependent CTLL-2 cells cultured in the absence of IL-2 for 4 h and a murine leukemic cell line, P388 cells, treated with etoposide for 5 h, were phagocytosed by macrophages. Although the cell populations containing the very early apoptotic cells used in this study were FITC-Annexin V-negative and did not show a decrease in cell size as compared with untreated cells, they showed a very small increase in phosphatidylserine on the cell surface, as detected with Cy3-Annexin V, and a decrease in mitochondrial membrane potential, indicating that the cell populations had already started the apoptotic process. Phagocytosis of such populations containing very early apoptotic cells was inhibited by phospho-l-serine much more significantly than Arg-Gly-Asp-Ser. In addition, macrophages hardly produced either proinflammatory or anti-inflammatory cytokines after phagocytosis, thus being an almost null response. These results are contrary to the generally accepted concept that the phagocytosis of apoptotic cells leads to the production of anti-inflammatory cytokines, suggesting instead that cells starting to undergo apoptosis are quickly phagocytosed by macrophages without any inflammation in vivo.

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells.

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohns disease and ulcerative colitis.

Activation of extracellular signal-regulated kinase 1/2 is involved in production of CXC-chemokine by macrophages during phagocytosis of late apoptotic cells.

Inefficient clearance of apoptotic cells by macrophages may cause an advanced stage of apoptosis, late apoptosis. Coculturing of macrophages with late apoptotic cells leads to high production of CXC-chemokine, IL-8, or MIP-2, a murine homologue of IL-8. However, the signaling mechanism underlying the production remains largely unknown. In this study, we examined the MAP kinase activation on coculturing of macrophages with late apoptotic cells. Extracellular signal-regulated kinase (ERK)1/2, but not p38 or c-Jun N-terminal kinase, was phosphorylated as early as 5 min after interaction of macrophages with late apoptotic cells. We then examined whether or not ERK activation is involved in the production of MIP-2 by employing selective inhibitors for MAP kinase kinase 1/2, PD98059, and U0126. These inhibitors suppressed the production of MIP-2 by macrophages at the protein and mRNA levels, whereas they did not suppress phagocytosis of late apoptotic cells, as judged on confocal microscopy. These results suggest that activation of ERK is involved in the production of MIP-2 on coculturing of macrophages with late apoptotic cells.

Apolipoprotein A-II Suppressed Concanavalin A-Induced Hepatitis via the Inhibition of CD4 T Cell Function

Con A-induced hepatitis has been used as a model of human autoimmune or viral hepatitis. During the process of identifying immunologically bioactive proteins in human plasma, we found that apolipoprotein A-II (ApoA-II), the second major apolipoprotein of high-density lipoprotein, inhibited the production of IFN-γ by Con A-stimulated mouse and human CD4 T cells. Con A-induced hepatitis was attenuated by the administration of ApoA-II. The beneficial effect of ApoA-II was associated with reduced leukocyte infiltration and decreased production of T cell-related cytokines and chemokines in the liver. ApoA-II inhibited the Con A-induced activation of ERK–MAPK and nuclear translocation of NFAT in CD4 T cells. Interestingly, exacerbated hepatitis was observed in ApoA-II–deficient mice, indicating that ApoA-II plays a suppressive role in Con A-induced hepatitis under physiological conditions. Moreover, the administration of ApoA-II after the onset of Con A-induced hepatitis was sufficient to suppress disease. Thus, the therapeutic effect of ApoA-II could be useful for patients with CD4 T cell-related autoimmune and viral hepatitis.

Second transplantation from HLA 2-loci-mismatched mother for graft failure due to hemophagocytic syndrome after cord blood transplantation

A 7-year-old girl with acute myelogenous leukemia with multilineage dysplasia received unrelated cord blood transplantation but developed hemophagocytic syndrome (HPS) after sepsis with methicillin-resistant coagulase-negative staphylococci before engraftment. Bone marrow aspiration on day 20 revealed a markedly increased number of activated macrophages showing hemophagocytosis. The presence of donor-type chimera in the bone marrow was confirmed at that time. We therefore quickly started immunosuppressive and antibacterial treatment. Although her condition gradually improved, the patient suf-fered graft failure due to HPS. She received peripheral blood stem cell transplantation from her HLA 2-loci-mismatched mother on day 54 and continued in complete remission 12 months after the second transplantation. The results in this case sug-gested that because of fetomaternal microchimerism it may be useful to select an HLA-haploidentical mother as a backup donor for stem cell transplantation.

Paraoxonase-1 Suppresses Experimental Colitis via the Inhibition of IFN-γ Production from CD4 T Cells

Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract, where excessive Th1 cell responses are observed. We performed experiments to identify immunologically bioactive proteins in human plasma and found that paraoxonase (PON)-1, which has esterase activity and is associated with high-density lipoproteins, inhibited the IFN-γ production by both murine and human differentiating Th1 cells. Trinitrobenzene sulfonic acid–induced colitis was attenuated by the administration of PON-1. The beneficial effects of PON-1 were associated with a reduced ratio of IFN-γ–producing CD4 T cells in the mesenteric lymph nodes and decreased production of T cell–related cytokines in the colon. PON-1 inhibited the TCR-induced activation of ERK-MAPK signaling and the nuclear translocation of NF-κB in CD4 T cells. Interestingly, an excessive CD4 T cell response was observed in PON-1–deficient mice under physiological and pathological conditions. Additionally, the efficacy of PON-1 or G3C9-C284A (G3C9), which shows a higher esterase activity than PON-1, on colitis was similar to that of an anti–TNF-α mAb, which is a clinically used CD treatment. Moreover, G3C9 more effectively suppressed CD4+CD45RBhigh cell transfer–induced chronic colitis in mice than did PON-1, and the efficacy of G3C9 against the colitis was similar to that of the anti–TNF-α mAb. Therefore, PON-1 (or G3C9) administration may be clinically beneficial for CD patients.

Immature dendritic cells reduce proinflammatory cytokine production by a coculture of macrophages and apoptotic cells in a cell-to-cell contact-dependent manner

We have demonstrated that phagocytosis of late apoptotic cells by mouse macrophages leads to the production of proinflammatory cytokines, notably macrophage‐inflammatory protein (MIP‐2), and therefore, a yet‐unknown mechanism(s) should keep our body free of inflammation. In this study, we examined the effect of the addition of immature dendritic cells (iDCs) to a coculture of macrophages and apoptotic cells on MIP‐2 production and phagocytosis by macrophages. The addition of iDCs to the coculture reduced MIP‐2 production significantly but unexpectedly enhanced the phagocytosis by macrophages. Further study revealed that the reduction of MIP‐2 production was dependent on cell‐to‐cell contact partly involving the β2 integrin family Mac‐1. In addition, anti‐inflammatory cytokines, interleukin‐10 and transforming growth factor‐β, were involved in the reduction of MIP‐2 production, as antibodies against these cytokines recovered MIP‐2 production. Both cytokines were expressed by iDCs more sigificantly than macrophages at the mRNA levels, although they were hardly detected in the supernatant at the protein levels, suggesting that minute amounts of these anti‐inflammatory cytokines were produced mainly by iDCs to block MIP‐2 production in a cell‐to‐cell contact‐dependent manner. Thus, this study reveals a new mechanism by which MIP‐2 production by macrophages phagocytosing apoptotic cells is prevented.

Murine Schnurri-2 controls natural killer cell function and lymphoma development.

Abstract Schnurri (Shn)-2 is a large zinc finger-containing protein implicated in cell growth, signal transduction and lymphocyte development. Here, we report that Shn-2-deficient (Shn-2−/−) mice develop CD3-positive lymphoma spontaneously. In Shn-2−/− mice, we observed decreased cytotoxicity of natural killer (NK) cells accompanied by decreased expression of perforin and granzyme-B. In addition, phosphorylation of signal transducer and activator of transcription (STAT) 5 was reduced in Shn-2−/− NK cells, while phosphorylation of STAT3 and protein expression of nuclear factor-κB p65 subunit were enhanced in Shn-2−/− NK cells. Moreover, cell-surface expression of activation molecules such as CD27, CD69 and CD122 were decreased on Shn-2−/− NK cells. Thus, Shn-2 is considered to play an important role in the activation and function of NK cells and the development of T cell lymphoma in vivo.

Non-myeloid cells are major contributors to innate immune responses via production of monocyte chemoattractant protein-1/CCL2

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 is a chemokine regulating the recruitment of monocytes into sites of inflammation and cancer. MCP-1 can be produced by a variety of cell types, such as macrophages, neutrophils, fibroblasts, endothelial cells, and epithelial cells. Notably, macrophages produce high levels of MCP-1 in response to proinflammatory stimuli in vitro, leading to the hypothesis that macrophages are the major source of MCP-1 during inflammatory responses in vivo. In stark contrast to the hypothesis, however, there was no significant reduction in MCP-1 protein or the number of infiltrating macrophages in the peritoneal inflammatory exudates of myeloid cell-specific MCP-1-deficient mice in response to i.p injection of thioglycollate or zymosan A. Furthermore, injection of LPS into skin air pouch also had no effect on local MCP-1 production in myeloid-specific MCP-1-deficient mice. Finally, myeloid-specific MCP-1-deficiency did not reduce MCP-1 mRNA expression or macrophage infiltration in LPS-induced lung injury. These results indicate that non-myeloid cells, in response to a variety of stimulants, play a previously unappreciated role in innate immune responses as the primary source of MCP-1.