Hisakazu Takatsuka

Liposome‐encapsulated dichloromethylene diphosphonate induces macrophage apoptosis in vivo and in vitro

Dichloromethylene diphosphonate (MDPCl2) encapsulated in multilamellar liposomes was selectively incorporated by macrophages, immediately transferred to lysosomes, then released from liposomes into lysosomes by enzymatic digestion of the liposomal lipid layers. From 4 h after ingesting liposome‐encapsulated MDPCl2 murine macrophages in vivo and in vitro acquired the ultrastructural features of apoptosis, such as condensed nuclear chromatin, nuclear fragmentation, cell shrinkage, and blebbing of the plasma membrane. Murine peritoneal macrophages and isolated rat Kupffer cells incubated in the medium containing liposome‐encapsulated MDPCl2 increased DNA fragmentation in a dose‐dependent manner. Electrophoretic analysis of extracted DNA from the isolated Kupffer cells showed DNA fragmentation. Another diphosphonate, Alendronate (4‐amino‐1‐hydroxybutylidene‐1,1‐diphosphonate) had less potent macrophage cytotoxicity. However, MDPCl2, Alendronate, and gadolinium chloride in solution were not cytotoxic to macrophages. These results implied that the intralysosomal accumulation of MDPCl2 generates signals to induce macrophage apoptosis. J. Leukoc. Biol. 60: 337–344; 1996.

CpG‐DNA‐induced IFN‐α production involves p38 MAPK‐dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors

Human plasmacytoid or CD4+CD11c− type 2 dendritic cell precursors (PDC) were identified as natural type I interferon (IFN)‐producing cells in response to viral and bacterial infection. They represent effector cells of innate immunity and link it to the distinct adaptive immunity by differentiating into mature DC. It has been reported that oligodeoxyribonucleotides containing unmethylated CpG motifs (CpG DNA) stimulate PDC to produce IFN‐α, but the molecular mechanisms involved remain unknown. We found that CpG‐DNA‐induced IFN‐α production in PDC was completely impaired by the inhibitor of the p38 mitogen‐activated protein kinase (MAPK) pathway. Expression of IFN regulatory factor (IRF)‐7 was enhanced by CpG‐DNA treatment, which was preceded by the phosphorylation of signal transducer and activator of transcription (STAT)1 on Tyr‐701, as well as its enhanced phosphorylation on Ser‐727. All of these events were also suppressed by the p38 MAPK inhibitor. STAT1, STAT2, and IRF‐9, components of IFN‐stimulated gene factor 3 (ISGF3), were recognized in the nuclear fraction of CpG‐DNA‐treated cells. Neither anti‐IFN‐α/β antibodies (Ab) nor anti‐IFNAR Ab suppressed STAT1 phosphorylation, enhancement of IRF‐7 expression, or IFN‐α production in the early phase of the culture. These results suggest that CpG DNA induces p38 MAPK‐dependent phosphorylation of STAT1 in a manner independent of IFN‐α/β, which may cause ISGF3 formation to increase the transcription of the IRF‐7 gene, thereby leading to IFN‐α production in human PDC.

Collaborative action of NF-kappaB and p38 MAPK is involved in CpG DNA-induced IFN-alpha and chemokine production in human plasmacytoid dendritic cells.

CpG DNA induces plasmacytoid dendritic cells (pDC) to produce type I IFN and chemokines. However, it has not been fully elucidated how the TLR9 signaling pathway is linked to these gene expressions. We examined the mechanisms involving the TLR9 and type I IFN signaling pathways, in relation to CpG DNA-induced IFN-α, IFN regulatory factor (IRF)-7, and chemokines CXCL10 and CCL3 in human pDC. In pDC, NF-κB subunits p65 and p50 were constitutively activated. pDC also constitutively expressed IRF-7 and CCL3, and the gene expressions seemed to be regulated by NF-κB. CpG DNA enhanced the NF-κB p65/p50 activity, which collaborated with p38 MAPK to up-regulate the expressions of IRF-7, CXCL10, and CCL3 in a manner independent of type I IFN signaling. We then examined the pathway through which IFN-α is expressed. Type I IFN induced the expression of IRF-7, but not of IFN-α, in a NF-κB-independent way. CpG DNA enabled the type I IFN-treated pDC to express IFN-α in the presence of NF-κB/p38 MAPK inhibitor, and chloroquine abrogated this effect. With CpG DNA, IRF-7, both constitutively and newly expressed, moved to the nuclei independently of NF-κB/p38 MAPK. These findings suggest that, in CpG DNA-stimulated human pDC, the induction of IRF-7, CXCL10, and CCL3 is mediated by the NF-κB/p38 MAPK pathway, and that IRF-7 is activated upstream of the activation of NF-κB/p38 MAPK in chloroquine-sensitive regulatory machinery, thereby leading to the expression of IFN-α.

The role of Kupffer cells and regulation of neutrophil migration into the liver by macrophage inflammatory protein-2 in primary listeriosis in mice

Depletion of mouse Kupffer cells and splenic macrophages following intravenous administration of liposome‐entrapped clodronate severely reduced host resistance to primary infection with Listeria monocytogenes. Infection of clodronate‐treated mice with a sublethal dose of L. monocytogenes resulted in death of the mice within 3 days. The macrophage depletion resulted in marked increases in bacterial growth in the liver and spleen, but not in other tissues. The proliferation of L. monocytogenes was observed in a large number of hepatocytes that underwent apoptosis. Infiltration of neutrophils in the liver and rapid formation of microabscesses were observed in the control mice after L. monocytogenes infection. However, there was less accumulation of neutrophils in the liver of Kupffer cell‐depleted mice than in the control mice. Expression of macrophage inflammatory protein‐2 (MIP‐2) was enhanced in the livers of both the control and Kupffer cell‐depleted mice after L. monocytogenes infection. MIP‐2 was also induced in a murine hepatocyte cell line following L. monocytogenes infection. The administration of neutralizing anti‐interleukin‐8 receptor homolog antibody severely abrogated neutrophil infiltration into the Listeria‐infected mouse liver. Anti‐MIP‐2 antibody moderately reduced neutrophil infiltration and microabscess formation in the liver. These findings indicate that Kupffer cells protect hepatocytes from L. monocytogenes infection and the resultant apoptosis. Moreover, MIP‐2 and its related molecules produced by the infected hepatocytes regulate neutrophil infiltration and microabscess formation in primary listeriosis.

Macrophage differentiation and expression of macrophage colony-stimulating factor in murine milky spots and omentum after macrophage elimination.

To elucidate the differentiation mechanisms of macrophages in the murine omentum, we studied the repopulation of these cells and the expression of macrophage colony‐stimulating factor (M‐CSF) in the milky spots and omental tissues in mice depleted of macrophages following administration of liposome‐encapsulated dichloromethylene diphosphonate (clodronate). The macrophages in the omentum were spindle or dendritic in shape, expressed several macrophage‐specific antigens and Ia antigen, and phagocytized intraperitoneally injected carbon particles. In the milky spots, macrophages and macrophage precursors were detected, and the number of precursors increased alter elimination of macrophages by intraperitoneal injection of liposome‐encapsulated clodronate. Macrophage precursors in the milky spots proliferated, moved to the omentum, and transformed into dendritic‐shaped macrophages. Expression of M‐CSF mRNA extracted from the milky spots was markedly enhanced at 2 and 3 days after macrophage depletion. Localization of M‐CSF protein and mRNA was observed in the stromal cells of the milky spots. In osteopetrosis (op/op) mutant mice that are defective in the production of functional M‐CSF omental macrophages were absent. These results indicate that M‐CSF locally produced in the milky spots plays an important role in providing a microenvironment for development and differentiation of omental macrophages. J. Leukoc. Biol. 61: 436–444; 1997.

Changes in aortic shape and diameters after death: Comparison of early postmortem computed tomography with antemortem computed tomography

PURPOSE The purpose of this study is to evaluate the postmortem deformation of the aorta on postmortem computed tomography (CT) by comparison with the antemortem CT in the same patient. MATERIALS AND METHODS A total of 58 non-traumatic patients without hemorrhagic events who underwent torso CT before and shortly after death were enrolled. Antemortem chest and abdominal CT were obtained in 44 cases and in 57 cases, respectively. The lengths of the major and minor axes of the ascending and descending thoracic aorta and the abdominal aorta were measured on both antemortem and postmortem CT in the same patient. To evaluate the shape of the aorta, the major axis-minor axis ratio (Ma-MiR) was calculated. Mean values of the diameters of the aorta and Ma-MiRs on postmortem CT were compared with those on antemortem CT using the Wilcoxon signed-rank test. We also evaluated the major and minor axes and Ma-MiRs on both antemortem and postmortem CT in two age groups: 65 years and under (n=13) and over 65 years (n=45). RESULTS At each level tested, the aorta significantly shrank after death (p<0.001) (ascending thoracic aorta, descending thoracic aorta, and abdominal aorta: 38.5 mm × 33.5 mm, 28.0 mm × 25.9 mm, and 24.4 mm × 21.8 mm on antemortem CT, 30.0 mm × 26.2 mm, 24.4 mm × 20.7 mm, and 21.5 mm × 14.5 mm on postmortem CT, respectively). The postmortem Ma-MiRs significantly increased at the descending thoracic aorta and the abdominal aorta (p<0.001). The diameters of the aorta are longer in older cases at all levels on both antemortem and postmortem CT. The reduction rates were larger in younger cases than older cases at all levels. CONCLUSIONS After death, the aorta shrunk at all levels, and became oval in shape in descending thoracic and abdominal aorta. The contraction was greater in younger cases than older cases. Investigators who interpret postmortem imaging should be aware of the postmortem deformation of the aorta.

Cytochrome P450 1A1, glutathione S-transferases M1 and T1 polymorphisms in Ovambos and Mongolians

Cytochrome P450 (CYP) 1A1, glutathione S-transferase (GST) M1, and GSTT1 gene polymorphisms have been shown to be associated with several diseases. In this study, CYP1A1 MspI, GSTM1 and GSTT1 gene polymorphisms were investigated in 134 Ovambo and 207 Mongolians, and the results were compared with those from previous studies. Using polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) the frequency of CYP1A1 MspI mutation was determined. The multiplex PCR was used to determine the GSTM1 and GSTT1 polymorphism. The frequencies of wild-type, heterozygous variant and homozygous variant of the CYP1A1 MspI genotypes were 72.4%, 25.4% and 2.2%, and 22.7%, 55.6% and 21.7% in the Ovambos and Mongolians, respectively. The frequencies of GSTM1 (null) and GSTT1 (null) genotypes were 11.2% and 35.8%, and 46.4% and 25.6% in the Ovambos and Mongolians, respectively. The CYP1A1 MspI and GSTT1 (null) genotype distribution of the Ovambos was similar to that of African-Americans and some Caucasians. In contrast, the GSTM1 (null) genotype distribution was different from that of all other populations. Among Mongolians, the CYP1A1 MspI polymorphism showed the highest mutation frequencies, GSTM1 (null) was similar to that of East Asians, and GSTT1 (null) was different from that of almost all the Asians examined.

Two N-Linked Glycosylation Sites (Asn18 and Asn106) Are Both Required for Full Enzymatic Activity, Thermal Stability, and Resistance to Proteolysis in Mammalian Deoxyribonuclease I

Deoxyribonuclease I (DNase I) is known to be a glycoprotein, and two potential N-linked glycosylation sites (N18 and N106) are known for mammalian enzymes. In the present study, N18 and N106 were mutated in order to investigate the biological role of N-linked glycosylation in three mammalian (human, bovine, and equine) DNases I. The enzyme activities of N18Q and N106Q were lower than that of the wild type, and that of the double mutant (N18Q/N106Q) was lower than those of the single mutants, in accord with the sugar moiety contents in the three mammals. In addition, all mutant enzymes were unstable to heat, suggesting that both sites are required for heat stability. Moreover, in human and equine enzymes, the N18Q and N106Q mutant enzymes were less resistant to trypsin, while N18Q/N106Q was the most sensitive to trypsin. As for bovine DNase I, the trypsin resistance of N18Q and N106Q was similar to that of the wild type, but that of N18Q/N106Q decreased in a time-dependent manner. On the other hand, N-linked glycosylation was not related to pH sensitivity. The results of the present study suggest that N18 and N106 are both necessary for (i) full enzymatic activity, (ii) heat-stability, and (iii) trypsin resistance.

Five age-dependently expressed genes in mouse brain revealed by the fluorescence differential display-PCR technique

We used a fluorescence differential display-PCR (FDD-PCR) technique to analyze the genes expressed in mouse brains collected at nine different developmental stages ranging from 3 days to 15 months after birth, and 5 age-dependently expressed genes were found. Age-dependent expression of each of these 5 genes was confirmed by quantitative real-time PCR analysis. Of the 5 genes, 4 (B1-B4) had high homology with the nucleotide sequences of cDNA clones of known mouse genes (myelin proteolipid protein, transferrin, embryo cDNA from the RIKEN full-length enriched library, and protein tyrosine phosphatase), and the rest (B5) with expressed sequence tags of an unknown gene. Sequencing analysis of the full-length cDNA constructed based on the B5 sequence demonstrated that the gene product of B5 was identical to G-substrate, a specific substrate for cGMP-dependent protein kinase. The expression patterns of known genes obtained in our study may provide a further opportunity to investigate the biological and physiological roles of the proteins they encode.

Cytochrome P450 2J2*7 polymorphisms in Japanese, Mongolians and Ovambos.

Human cytochrome P450 2J2 (CYP2J2) is abundant in cardiovascular tissue and active in the metabolism of arachidonic acid to eicosanoids that have potent vasodilatory properties. Variability of the CYP2J2 gene is highly constrained except for its proximal promoter: there is a relatively common and functionally relevant single nucleotide polymorphism, indicated by −50G > T polymorphism (CYP2J2*7). Although genetic variation is known among ethnic groups, data for allele frequency are limited to a few Caucasian, Asian, and one African populations. In the present study, genotype distribution of CYP2J2*7 polymorphisms was investigated using polymerase chain reaction and restriction fragment length polymorphism assay in Japanese (n = 338), Mongolian (n = 118), and Ovambo (n = 186) populations and the findings compared with other populations. The mutant (CYP2J2*7) frequencies in the Japanese, Mongolians, and Ovambos were 0.0621, 0.0339, and 0.0672, respectively. Except for the Taiwanese, a general uniformity in the polymorphism in the Asian populations was observed. The mutation frequency of Ovambos was relatively lower than that of the African‐American population. This study is the first to investigate the distribution of the CYP2J2*7 gene polymorphisms in Japanese, Mongolians, and Ovambos. These data will be informative and facilitate genetic association studies, in Asian and African populations for CYP2J2‐related diseases such as cardiovascular disorders. Copyright