Integrin αvß3 enhances the suppressive effect of interferon-γ on hematopoietic stem cells.

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvß3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin ß3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro-inflammatory cytokine interferon-γ (IFNγ) and ß3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvß3 suppressed HSC function in the presence of IFNγ and impaired integrin ß3 signaling mitigated IFNγ-dependent negative action on HSCs. During IFNγ stimulation, integrin ß3 signaling enhanced STAT1-mediated gene expression via serine phosphorylation. These findings show that integrin ß3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvß3 within the BM niche acts as a context-dependent signal modulator to regulate the HSC function under both steady-state and inflammatory conditions.

Integrin-αvß3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells.

Throughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-ß3 signaling contributes to HSC maintenance. Specific ligation of ß3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvß3-integrin "inside-out" signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent "outside-in" signaling via phosphorylation of Tyr747 in the ß3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between ß3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.

Promotion of mouse ameloblast proliferation by Lgr5 mediated integrin signaling.

Rodent incisors grow throughout the animal's lives, and the tooth-forming cells are provided from proximal ends of the incisors where the tooth epithelium forms a stem cell niche called cervical loop. The committing cells in a cervical loop actively begin to proliferate (pre-ameloblasts), and differentiating into ameloblasts. This study showed that the lower incisors of mice null for CD61 (CD61(-/-) ), also known as integrin ß3, were significantly shorter than those of the wild-type mice at 8-week-old. The protein and mRNA expressions levels of Fgfr2, Lgr5, and Notch1, which are known to be involved in pre-ameloblastic cell proliferation and stem cell maintenance, were reduced in the cervical loop of 2-week-old CD61(-/-) mice. The proliferation of pre-ameloblasts was reduced in CD61(-/-) ameloblasts. The siRNA-mediated suppression of CD61 (siCD61) reduced the proliferation of pre-ameloblastic cell line ALC, and the expression levels of Lgr5 and Notch1 were reduced by the transfection with siCD61. The suppression of Lgr5 by transfection with siLgr5 suppressed the proliferation of the ALC cells. These results suggested that CD61 signaling is required for the proper growth of the cervical loop and for the promotion of the proliferation of pre-ameloblastic cells through Lgr5.

The importance of infiltrating neutrophils in SDF-1 production leading to regeneration of the thymus after whole-body X-irradiation.

Our previous study demonstrated that neutrophils transiently infiltrated into a site where apoptosis had been induced. However, the role of infiltrating neutrophils has not been fully elucidated. In this study, we examined their role in regeneration of the thymus after whole-body X-irradiation by focusing on SDF-1 production. After X-irradiation, the thymus became severely atrophied presumably due to phagocytosis of apoptotic thymocytes. At that time, a significant number of neutrophils were detected in the thymus. The thymus was then partially regenerated on day 7, whereas the level of SDF-1 in it was significantly increased on days 3 and 5. Depletion of neutrophils greatly impaired SDF-1 production and the thymus regeneration. Moreover, administration of a CXCR4 antagonist also greatly suppressed the thymus regeneration. Furthermore, coculturing of a stromal cell line with infiltrating neutrophils increased SDF-1 production. These results suggest that infiltrating neutrophils play an auxiliary role in regeneration of the thymus after whole-body X-irradiation through augmentation of SDF-1 production.

Antimicrobial Activities of Dictyostelium Differentiation-Inducing Factors and Their Derivatives.

At the end of its life cycle, the cellular slime mold Dictyostelium discoideum forms a fruiting body consisting of spores and a multicellular stalk. Originally, the chlorinated alkylphenone differentiation-inducing factors (DIFs) -1 and -3 were isolated as stalk cell inducers in D. discoideum. Later, DIFs and their derivatives were shown to possess several biologic activities including antitumor and anti-Trypanosoma properties. In this study, we examined the antibacterial activities of approximately 30 DIF derivatives by using several bacterial species. Several of the DIF derivatives strongly suppressed the growth of the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis and Enterococcus faecium, at minimum inhibitory concentrations (MICs) in the sub-micromolar to low-micromolar range. In contrast, none of the DIF derivatives evaluated had any noteworthy effect on the growth of the Gram-negative bacterium Escherichia coli (MIC, >100 µM). Most importantly, several of the DIF derivatives strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant E. faecalis and E. faecium. Transmission electron microscopy revealed that treatment with DIF derivatives led to the formation of distinct multilayered structures consisting of cell wall or plasma membrane in S. aureus. The present results suggest that DIF derivatives are good lead compounds for developing novel antimicrobials.

CD61 enriches long-term repopulating hematopoietic stem cells.

Among the subsets that define hematopoietic stem cells (HSCs), CD34- c-kit+ Sca-1+ lineage marker- (CD34-KSL) cells are regarded as one of the populations that have the highest enrichment of HSCs in adult mouse bone marrow. Here, we demonstrate that long-term repopulating hematopoietic stem cells (LTR-HSCs) have high expression of CD61 (integrin beta3) within the CD34-KSL population. Approximately 60% of CD34-KSL cells showed high expression of CD61. CD61HighCD34-KSL populations also exhibited significantly greater properties of HSC, such as expression of HSC markers, the side population (SP) phenotype, and ability for long-term repopulation. In both SP cells and non-SP (NSP) cells, CD61HighCD34-KSL cells also contained significantly more LTR-HSCs than CD61Low/-CD34-KSL cells. Our results indicate that CD61 is exploitable for HSC enrichment as a supportive positive cell surface marker.

Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis.

TNF-alpha, IFN-gamma, IL-4, and MIP-2 are known to be involved in Con A-induced hepatitis. Although Kupffer cells are reportedly involved in TNF-alpha production, it is largely unknown whether or not Kupffer cells also play a role in the production of other cytokines, such as IFN-gamma, IL-4, and MIP-2. In this study we examined the liver injury and the levels of plasma cytokines, including above four cytokines, KC, and IL-10 in Kupffer cell-depleted mice obtained through administration of liposome-encapsulated dichloromethylene bisphosphonate. The liver injury was significantly suppressed in Kupffer cell-depleted mice, as assessed as to the plasma ALT level and histochemistry. The cytokine levels were also significantly suppressed in such mice except for those of IFN-gamma, which was slightly suppressed at 12h, and IL-10, which was not significantly suppressed at any time. Apoptosis was also significantly suppressed in such mice, as found immunohistochemically with anti-ssDNA Ab. Taken together, these results suggest that Kupffer cells are involved in the production of MIP-2, KC, IL-4, and TNF-alpha in Con A-induced hepatitis, thereby contributing to the liver injury either directly or indirectly.

Infiltrating neutrophils induce allospecific CTL in response to immunization with apoptotic cells via MCP-1 production.

Our previous studies demonstrated that i.p. injection of late apoptotic P388 cells caused phagocytosis by macrophages and transient infiltration of neutrophils into the peritoneal cavity. As neutrophils are known to function as effectors as well as regulators in the immune response, we examined the roles of infiltrating neutrophils in alloantigen-specific CTL induction after immunization with late apoptotic P388 cells. The CTL induction and infiltration of CD8(+) T cells into the peritoneal cavity were inhibited by depletion of neutrophils by anti-Gr-1 mAb or inhibition of neutrophil infiltration by anti-MIP-2 antibody, suggesting that neutrophils are involved in CD8(+) T cell infiltration into the peritoneal cavity. It is known that MIP-1alpha, MIP-1beta, and MCP-1 are capable of attracting CD8(+) T cells and that they are produced by neutrophils. These chemokines were detected in the peritoneal cavity, and among them, MCP-1 production was reduced remarkably by suppression of neutrophil infiltration. Moreover, infiltration of CD8(+) T cells into the peritoneal cavity as well as CTL activity was clearly reduced by administering anti-MCP-1 antibody i.p. Furthermore, the CTL induction and infiltration of CD8(+) T cells in neutrophil-depleted mice were restored significantly by administering recombinant murine MCP-1 into the peritoneal cavity. These results indicate that MCP-1 appears to link infiltration of neutrophils with CTL induction.

Computational promoter modeling identifies the modes of transcriptional regulation in hematopoietic stem cells.

Extrinsic and intrinsic regulators are responsible for the tight control of hematopoietic stem cells (HSCs), which differentiate into all blood cell lineages. To understand the fundamental basis of HSC biology, we focused on differentially expressed genes (DEGs) in long-term and short-term HSCs, which are closely related in terms of cell development but substantially differ in their stem cell capacity. To analyze the transcriptional regulation of the DEGs identified in the novel transcriptome profiles obtained by our RNA-seq analysis, we developed a computational method to model the linear relationship between gene expression and the features of putative regulatory elements. The transcriptional regulation modes characterized here suggest the importance of transcription factors (TFs) that are expressed at steady state or at low levels. Remarkably, we found that 24 differentially expressed TFs targeting 21 putative TF-binding sites contributed significantly to transcriptional regulation. These TFs tended to be modulated by other nondifferentially expressed TFs, suggesting that HSCs can achieve flexible and rapid responses via the control of nondifferentially expressed TFs through a highly complex regulatory network. Our novel transcriptome profiles and new method are powerful tools for studying the mechanistic basis of cell fate decisions.

Nov/CCN3 regulates long-term repopulating activity of murine hematopoietic stem cells via integrin αvß3.

Throughout life, hematopoietic stem cells (HSCs) sustain the blood cell supply through their capacities for self-renewal and multilineage differentiation. These processes are regulated within a specialized microenvironment termed the 'niche'. Here, we show a novel mechanism for regulating HSC function that is mediated by nephroblastoma overexpressed (Nov/CCN3), a matricellular protein member of the CCN family. We found that Nov contributes to the maintenance of long-term repopulating (LTR) activity through association with integrin αvß3 on HSCs. The resultant ß3 integrin outside-in signaling is dependent on thrombopoietin (TPO), a crucial cytokine involved in HSC maintenance. TPO was required for Nov binding to integrin αvß3, and stimulated Nov expression in HSCs. However, in the presence of IFNγ, a cytokine known to impair HSC function, not only was TPO-induced expression of Nov suppressed, but the LTR activity was conversely impaired by TPO-mediated ligation of integrin αvß3 with exogenous ligands, including Nov, as well. Thus, Nov/integrin αvß3-mediated maintenance of HSCs appears to be modulated by simultaneous stimulation by other cytokines. Our finding suggests that this system contributes to the regulation of HSCs within the bone marrow niche.

Expression of Integrin beta3 is correlated to the properties of quiescent hemopoietic stem cells possessing the side population phenotype.

With significant attention paid to the field of tissue-specific stem cells, the identification of stem cell-specific markers is of considerable importance. Previously, the side population (SP) phenotype, with the capacity to efflux the DNA-binding dye Hoechst 33342, has been recognized as a common feature of adult tissue-specific stem cells. In this study, we show that high expression of integrin beta(3) (CD61) is an attribute of SP cells isolated from mouse bone marrow. Additionally, we confirmed that the expression of integrin beta(3) is correlated with properties of quiescent hemopoietic stem cells (HSCs) including the strength of the SP phenotype, cell cycle arrest, expression of HSC markers, and long-term hemopoiesis. Importantly, Lineage(-) (Lin(-))/integrin beta(3)(high) (beta(3)(high)) SP cells have as strong a capacity for long-term hemopoiesis as c-Kit(+)/Sca-1(+)/Lin(-) SP cells, which are regarded as one of the most highly enriched HSC populations. Finally, the integrin beta(3) subunit that is present in SP cells having the properties of HSCs, is associated with integrin alpha(v) (CD51). Therefore, our results demonstrate that high expression of integrin beta(3) is correlated to the properties of quiescent HSCs and suggest that the integrin beta(3) subunit is available as a common surface marker of tissue-specific stem cells.

A novel accessory role of neutrophils in concanavalin A-induced hepatitis.

Concanavalin A (Con A)-induced hepatitis has been investigated as a model of T cell-mediated liver injury, in which IFN-gamma plays an essential role by inducing apoptosis of liver cells. Since a large number of neutrophils infiltrate into the liver in the model, the role of neutrophils was investigated in this study. Con A hardly caused liver injury in neutrophil-depleted mice, as assessed as to the plasma alanine aminotransferase level as well as histochemistry. Neutrophil-depleted mice also failed to produce IFN-gamma. Intracellular IFN-gamma staining revealed that, among liver leukocytes, T and NK cells but not neutrophils are the main producers of IFN-gamma. Nylon wool-purified "T cells", however, failed to produce IFN-gamma in response to Con A in vitro, while the production was restored by the addition of neutrophils. Overall, this study suggests that neutrophils play a novel accessory role in IFN-gamma production in Con A-induced hepatitis.