Catalase plays a critical role in the CSF-independent survival of human macrophages via regulation of the expression of BCL-2 family.

M-colony-stimulating factor (M-CSF)-induced monocyte-derived macrophages (M-Mphi) required continuous presence of M-CSF for their survival, and depletion of M-CSF from the culture induced apoptosis, whereas human alveolar macrophages (A-Mphi) and granulocyte-macrophage (GM)-CSF-induced monocyte-derived macrophages (GM-Mphi) survived even in the absence of CSF. The expression of BCL-2 was higher in M-Mphi, and M-CSF withdrawal down-regulated the expression. The expression of BCL-X(L) was higher in A-Mphi and GM-Mphi, and the expression was CSF-independent. The expression of MCL-1 and BAX were not different between M-Mphi and GM-Mphi and were CSF-independent. Down-regulation of the expression of BCL-2 and BCL-X(L) by RNA interference showed the important role of BCL-2 and BCL-X(L) in the survival of M-Mphi and GM-Mphi, respectively. Human erythrocyte catalase (HEC) and conditioned medium obtained from GM-Mphi or A-Mphi cultured in the absence of GM-CSF prevented the M-Mphi from apoptosis and restored the expression of BCL-2. The activity of the conditioned medium was abrogated by pretreatment with anti-HEC antibody. Anti-HEC antibody also induced the apoptosis of M-Mphi cultured in the presence of M-CSF and GM-Mphi and A-Mphi cultured in the presence or absence of GM-CSF and down-regulated the expression of BCL-2 and BCL-X(L) in these Mphis. GM-Mphi and A-Mphi, but not M-Mphi, can produce both extracellular catalase and cell-associated catalase in a CSF-independent manner. Intracellular glutathione levels were kept equivalent in these Mphis, both in the presence or absence of CSF. These results indicate a critical role of extracellular catalase in the survival of human macrophages via regulation of the expression of BCL-2 family genes.

Differences in morphology of phagosomes and kinetics of acidification and degradation in phagosomes between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar.

Phagocytosis plays an important role in the pathogenicity of the intestinal protozoan parasite Entamoeba histolytica. We compared the morphology of phagosomes and the kinetics of phagosome maturation using conventional light and electron microscopy and live imaging with video microscopy between the virulent E. histolytica and the closely-related, but non-virulent E. dispar species. Electron micrographs showed that axenically cultivated trophozoites of the two Entamoeba species revealed morphological differences in the number of bacteria contained in a single phagosome and the size of phagosomes. Video microscopy using pH-sensitive fluorescein isothiocynate-conjugated yeasts showed that phagosome acidification occurs within 2 min and persists for >12 h in both species. The acidity of phagosomes significantly differed between two species (4.58 +/- 0.36 or 5.83 +/- 0.38 in E. histolytica or E. dispar, respectively), which correlated well with the differences in the kinetics of degradation of promastigotes of GFP-expressing Leishmania amazonensis. The acidification of phagosomes was significantly inhibited by a myosin inhibitor, whereas it was only marginally inhibited by microtubules or actin inhibitors. A specific inhibitor of vacuolar ATPase, concanamycin A, interrupted both the acidification and degradation in phagosomes in both species, suggesting the ubiquitous role of vacuolar ATPase in the acidification and degradation in Entamoeba. In contrast, inhibitors against microtubules or cysteine proteases (CP) showed distinct effects on degradation in phagosomes between these two species. Although depolymerization of microtubules severely inhibited degradation in phagosomes of E. histolytica, it did not affect degradation in E. dispar. Similarly, the inhibition of CP significantly reduced degradation in phagosomes of E. histolytica, but not in E. dispar. These data suggest the presence of biochemical or functional differences in the involvement of microtubules and proteases in phagosome maturation and degradation between the two species.

Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.

In mammals, Rab5 and Rab7 play a specific and coordinated role in a sequential process during phagosome maturation. Here, we report that Rab5 and Rab7 in the enteric protozoan parasite Entamoeba histolytica, EhRab5 and EhRab7A, are involved in steps that are distinct from those known for mammals. EhRab5 and EhRab7A were localized to independent small vesicular structures at steady state. Priming with red blood cells induced the formation of large vacuoles associated with both EhRab5 and EhRab7A ("prephagosomal vacuoles (PPV)") in the amoeba within an incubation period of 5-10 min. PPV emerged de novo physically and distinct from phagosomes. PPV were gradually acidified and matured by fusion with lysosomes containing a digestive hydrolase, cysteine proteinase, and a membrane-permeabilizing peptide amoebapore. After EhRab5 dissociated from PPV, 5-10 min later, the EhRab7A-PPV fused with phagosomes, and EhRab7A finally dissociated from the phagosomes. Immunoelectron and light micrographs showed that PPV contained small vesicle-like structures containing fluid-phase markers and amoebapores, which were not evenly distributed within PPV, suggesting that the mechanism was similar to multivesicular body formation in PPV generation. In contrast to Rab5 from other organisms, EhRab5 was involved exclusively in phagocytosis, but not in endocytosis. Overexpression of wild-type EhRab5 enhanced phagocytosis and the transport of amoebapore to phagosomes. Conversely, expression of an EhRab5Q67L GTP form mutant impaired the formation of PPV and phagocytosis. Altogether, we propose that the amoebic Rab5 plays an important role in the formation of unique vacuoles, which is essential for engulfment of erythrocytes and important for packaging of lysosomal hydrolases, prior to the targeting to phagosomes.

Innate apoptosis of human B lymphoblasts transformed by Epstein-Barr virus: modulation by cellular immortalization and senescence.

B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus have a phenotype corresponding to activated B-lymphoblasts. Although they are widely used as models in various biological and medical studies, their innate morphological differentiation and apoptosis has been little studied. We report here that a large proportion of LCL cells spontaneously differentiate into smaller lymphoid cells which ultimately undergo apoptosis during conventional cell culture. Two distinct types of apoptosis with some intermediate types exist: type 1 apoptosis in small and medium-size cells with shrunken nuclei having heavily condensed chromatin in the whole nucleus region accompanied by relatively large internucleosomally fragmented DNA (above 2 kbp); type 2 apoptosis in large lymphoblasts with extremely lobulated nuclei having chromatin condensation beneath the nuclear membrane alone accompanied by smaller internucleosomally fragmented DNA (below 2 kbp). Type 1 apoptotic cells were far more numerous than type 2 apoptotic cells. The incidence of type 1 apoptosis was suppressed by cellular immortalization and was extremely stimulated at the end of the lifespan (crisis). These results provide essential information for us to use LCLs for various biological and medical studies including cellular immortalization, tumorigenesis and senescence.

A Cell Kinetic and Cytological Study on the Asymmetric Cell Division of Thymic Lymphoblasts of the Embryonic Rat.

Cell division of thymus lymphoid cells from embrynonic and young rats was investigated cytologically on cell smears, focusing attention on asymmetric cell division. Some of thymic lymphoblasts displayed features implicating asymmetric cell division. At the telophase of such cells, two immature daughter cells looked dissimilar: one of them was smaller in size and possessed a more condensed nucleus, compared with the counterpart cell. Furthrmore, in most cases the cytoplasm of the smaller daughter cell was stained with Giemsa more deeply. It was suggested that the asymmetry of the nucleus emerges at anaphase and telophase probably due to some polarized situation of the cytoplasm. Asymmetrically-dividing cells were relatively frequently observed during the developmental period when large lymphoblasts actively transform into smaller lymphocytes :16% to 17% of whole dividing cells were under asymmetric cell division on days 16 and 17 of gestation, while less than 5% on day 19 or thereafter. In correlation with this observation, asymmetrically-dividing cells were more frequently observed among large lymphoblasts than among other smaller cell fractions. These results support the view that the asymmetric cell division may play some essential role in the transformation of large lymphoblasts into smaller lymphocytes.

DEVELOPMENT OF THE EMBRYONIC CHICKEN THYMUS III. UNEQUAL DIVISION OF LYMPHOID CELLS.

Cell division of thymus lymphoid cells from 11- to 17-day old embryonic chickens, as well as chickens just after hatch was investigated on cell smears stained with Giemsa. Unequally dividing cells were observed in the developmental stage of thymocytes. At the telophase of such cells, the cytoplasm of one of two future daughter cells was apparently larger in amount and was sometimes stained deeper than the cytoplasm of its counterpart. Unequal division was also observed in pro-, meta- and anaphase; sometimes a dividing cell had a large cytoplasmic process belonging to one hemisphere, suggesting that only one of the two daughter cells would receive the cytoplasmic process through cell division. The incidence of unequal division calculated by a rough estimation was around 10% of the total cell division between 11 and 13 days of embryonic development, and decreased progressively thereafter.