Megumi Takahashi

Breast milk macrophages spontaneously produce granulocyte–macrophage colony‐stimulating factor and differentiate into dendritic cells in the presence of exogenous interleukin‐4 alone

Peripheral blood monocytes extravasate and differentiate into tissue macrophages to mediate effective local defence, but how tissue‐specific stimuli and environments may influence their functions remains unknown. Here, we found that peripheral blood monocytes gained the ability to produce granulocyte–macrophage colony‐stimulating factor (GM‐CSF) upon exposure to breast milk and differentiated into CD1+ dendritic cells (DCs) in the presence of exogenous interleukin‐4 (IL‐4) alone. This in vitro observation appeared physiologically relevant since macrophages that were freshly isolated from breast milk were also found to produce GM‐CSF spontaneously. Furthermore, in contrast to peripheral blood monocytes that differentiated into DCs only in the presence of both exogenous GM‐CSF and IL‐4, differentiation of breast milk macrophages into DCs was induced by incubation with exogenous IL‐4 alone. These IL‐4‐stimulated breast milk macrophages were efficient in stimulating T cells, suggesting their potential role in mediating T‐cell‐dependent immune responses in situ. On the other hand, unexpected expression of DC‐SIGN, a DC‐specific receptor for human immunodeficiency virus (HIV), even in unstimulated breast milk macrophages, may favour HIV infection, resulting in an increased risk of mother‐to‐infant vertical transmission of the virus via breast milk. Thus, tissue‐specific development of macrophages is often linked to effective local immunity, but may potentially provide an opportunity for a pathogen to spread and transmit.

A possible mechanism of intravesical BCG therapy for human bladder carcinoma: involvement of innate effector cells for the inhibition of tumor growth.

Intravesical bacillus Calmette-Guerin (BCG) therapy is considered the most successful immunotherapy against solid tumors of human bladder carcinoma. To determine the actual effector cells activated by intravesical BCG therapy to inhibit the growth of bladder carcinoma, T24 human bladder tumor cells, expressing very low levels of class I MHC, were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) with live BCG. The proliferation of T24 cells was markedly inhibited when BCG-infected dendritic cells (DCs) were added to the culture although the addition of either BCG or uninfected DCs alone did not result in any inhibition. The inhibitory effect was much stronger when the DCs were infected with live BCG rather than with heat-inactivated BCG. The live BCG-infected DCs secreted TNF-α and IL-12 within a day and this secretion continued for at least a week, while the heat-inactivated BCG-infected DCs secreted no IL-12 and little TNF-α. Such secretion of cytokines may activate innate alert cells, and indeed NKT cells expressing IL-12 receptors apparently proliferated and were activated to produce cytocidal perforin among the PBMCs when live BCG-infected DCs were externally added. Moreover, depletion of γδ T-cells from PBMCs significantly reduced the cytotoxic effect on T24 cells, while depletion of CD8β cells did not affect T24 cell growth. Furthermore, the innate effectors seem to recognize MICA/MICB molecules on T24 via NKG2D receptors. These findings suggest the involvement of innate alert cells activated by the live BCG-infected DCs to inhibit the growth of bladder carcinoma and provide a possible mechanism of intravesical BCG therapy.

Rapid Induction of Apoptosis in CD8+ HIV-1 Envelope-Specific Murine CTLs by Short Exposure to Antigenic Peptide

During primary viral infection, in vivo exposure to high doses of virus causes a loss of Ag-specific CD8+ T cells. This phenomenon, termed clonal exhaustion, and other mechanisms by which CTLs are deleted are poorly understood. Here we show evidence for a novel form of cell death in which recently stimulated CD8+ HIV-1 envelope gp160-specific murine CTLs become apoptotic in vitro after brief exposure to free antigenic peptide (P18-I10). Peak apoptosis occurred within 3 h of treatment with peptide, and the level of apoptosis was dependent on both the time after initial stimulation with target cells and the number of targets. Using T cell-specific H-2Dd/P18-I10 tetramers, we observed that the apoptosis was induced by such complexes. Induction of apoptosis was blocked by cyclosporin A, a caspase 3 inhibitor, and a mitogen-activated protein kinase inhibitor, but not by Abs to either Fas ligand or to TNF-α. Thus, these observations suggest the existence of a Fas- or TNF-α-independent pathway initiated by TCR signaling that is involved in the rapid induction of CTL apoptosis. Such a pathway may prove important in the mechanism by which virus-specific CTLs are deleted in the presence of high viral burdens.

Effects of extracellular pH and hypoxia on the function and development of antigen-specific cytotoxic T lymphocytes.

The major effector cells for cellular adaptive immunity are CD8(+) cytotoxic T lymphocytes (CTLs), which can recognize and kill virus-infected cells and tumor cells. Although CTLs exhibit strong cytolytic activity against target cells in vitro, a number of studies have demonstrated that their function is often impaired within tumors. Nevertheless, CTLs can regain their cytotoxic ability after escaping from the tumor environment, suggesting that the milieu created by tumors may affect the function of CTLs. As for the tumor environment, the patho-physiological situation present in vivo has been shown to differ from in vitro experimental conditions. In particular, low pH and hypoxia are the most important microenvironmental factors within growing tumors. In the present study, to determine the effect of these factors on CTL function in vivo, we examined the cytolytic activity of CTLs against their targets using murine CTL lines and the induction of these cells from memory cells under low pH or hypoxic conditions using antigen-primed spleen cells. The results indicated that both cytotoxic activity and the induction of functional CTLs were markedly inhibited under low pH. In contrast, in hypoxic conditions, although cytotoxic activity was almost unchanged, the induction of CTLs in vitro showed a slight enhancement, which was completely abrogated in low pH conditions. Therefore, antigen-specific CTL functions may be more vulnerable to low pH than to the oxygen concentration in vivo. The findings shown here provide new therapeutic approaches for controlling tumor growth by retaining CTL cytotoxicity through the maintenance of higher pH conditions.

Effects of Subminimal Inhibitory Concentrations of Erythromycin, Tetracycline, Clindamycin, and Minocycline on the Neutrophil Chemotactic Factor Production in Propionibacterium acnes Biotypes 1–5

Biotypes 1–5 Propionibacterium acnes (P. acnes) strains were grown in the presence of 1/10 minimal inhibitory concentrations (sub‐MIC) of erythromycin (EM), tetracycline (TC), clindamycin (CLDM), or minocycline (MINO) and their culture filtrates were assayed for human neutrophil chemotactic activity using Boyden chamber methods.

HTLV-I-infected breast milk macrophages inhibit monocyte differentiation to dendritic cells.

Human T-cell leukemia virus type I (HTLV-I), a causative agent of adult T-cell leukemia (ATL), is transmitted from mother to child, predominantly by breastfeeding. Oral HTLV-I infection and infection early in life are associated with a subsequent risk of ATL. Although the pathogenic mechanisms of ATL remain largely unknown, the host immune system seems to play an important role in HTLV-I pathogenesis. Previous studies have shown that monocytes from ATL patients had reduced capacity for dendritic cell (DC) differentiation. Therefore, we performed the present study to clarify the mechanisms responsible for the impairment of DC differentiation using HTLV-I-infected breast milk macrophages (HTLV-BrMMø). We found that when CD14⁺ monocytes were cultured with GM-CSF and IL-4 in the presence of HTLV-BrMMø, they altered the surface phenotype of immature DCs and the stimulatory capacity of T-cell proliferation. The presence of HTLV-BrMMø significantly blocked the increased expression of CD1a, CD1b, CD11b, DC-SIGN, and HLA-DR; however, increased expression of CD1d and CD86 was observed. These effects could be partially replicated by incubation with culture supernatants from HTLV-BrMMø. The impairment of monocyte differentiation might be not due to HTLV-I infection of monocytes, but might be due to unknown soluble factors. Since other HTLV-I-infected cells exhibited similar inhibitory effects on monocyte differentiation to DCs, we speculated that HTLV-I infection might cause the production of some inhibitory cytokines in infected cells. Identifying the factors responsible for the impairment of monocyte differentiation to DCs may be helpful to understand HTLV-I pathogenesis.

Mouse parenchymal liver cells in culture secrete a growth inhibitor for myeloma cells

METHODS Growth inhibitory activity in the conditioned medium of mouse parenchymal liver cells was examined in three strains of myeloma cells. RESULTS Two strains of myeloma cells were highly sensitive to a low concentration of mouse parenchymal liver cell derived growth inhibitor, whereas one strain was resistant to the same concentration. Interferon-alphabeta and transforming growth factor-beta activity were detected in mouse parenchymal liver cells, while interferon-gamma and tumor necrosis factor-alpha were not. The growth suppression exerted by mouse parenchymal liver cell derived growth inhibitor in the three myeloma strains was distinct from that exerted by transforming growth factor-beta, tumor necrosis factor-alpha, interferon-alphabeta and interferon-gamma. The mouse parenchymal liver cell derived growth inhibitor was eluted with a peak activity in the 18 kDa range and focused into pI values of 3.8-4.0, and it was lost when mouse parenchymal liver cells were treated with heat or trypsin. CONCLUSION These results indicate that mouse parenchymal liver cell derived growth inhibitor differs from the well-characterized growth inhibitors, transforming growth factor-beta, tumor necrosis factor-alpha, interferon-alphabeta and interferon-gamma.

Increased mitochondrial functions in human glioblastoma cells persistently infected with measles virus.

Measles virus (MV) is known for its ability to cause an acute infection with a potential of development of persistent infection. However, knowledge of how viral genes and cellular factors interact to cause or maintain the persistent infection has remained unclear. We have previously reported the possible involvement of mitochondrial short chain enoyl-CoA hydratase (ECHS), which is localized at mitochondria, in the regulation of MV replication. In this study we found increased functions of mitochondria in MV-persistently infected cells compared with uninfected or acutely infected cells. Furthermore, impairment of mitochondrial functions by treatment with mitochondrial inhibitors such as ethidium bromide (EtBr) or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) induced the cytopathic effects of extensive syncytial formation in persistently infected cells. These findings suggest that mitochondria are one of the subcellular organelles contributing to regulate persistent infection of MV. Recent studies showed mitochondria provide an integral platform for retinoic acid-inducible protein (RIG-I)-like cytosolic receptors (RLRs) signaling and participate in cellular innate antiviral immunity. Our findings not only reveal a role of mitochondria in RLR mediated antiviral signaling but also suggest that mitochondria contribute to the regulation of persistent viral infection.

Quantitative analysis of cytokine gene expression in the liver.

The relative levels of cytokine gene expression in the liver were analysed, focusing on IL‐2, IL‐4, IL‐5, IL‐6 and IFN‐γ compared to those in the spleen and Peyers patch by using the reverse transcriptase polymerase chain reaction (RT‐PCR). The levels of expression of cytokines in the liver mononuclear cells (MNC), especially that of IL‐6, were significantly higher than in other organs when mice were reared under specific pathogen‐free (SPF) or conventional conditions. Both the spleen and Peyers patch MNC expressed little of any of the cytokines, except for IL‐4 in Peyers patch MNC. The liver MNC produced significant amounts of IL‐6 in the culture supernatant upon concanavalin A stimulation. These findings suggest that the liver is a potent IL‐6‐ producing organ, which may relate to B cell differentiation, liver regeneration and the induction of acute phase proteins.

Production of B cell differentiation factors by mouse parenchymal liver cells

Previously we reported that most antibody secreting cells secreted IgA in the liver. Here we assessed the possibility that parenchymal liver cells (PLC) produced factors, transforming growth factor (TGF)‐p and IL‐5, which participate in the differentiation of B cells to IgA‐secreting cells. We showed that TGF‐p activity was present in the culture supernatant of PLC. and IL‐5 activity was in the lysate of PLC. Moreover, it was confirmed that IL‐5 protein produced by PLC was mainly localized in the cell membrane by histochemical staining. The findings that both TGF‐P and IL‐5 were produced by PLC should provide useful information concerning the fact that IgA‐secreting cells were dominant in the liver.