Changes in the subunit distribution of prosomes (MCP-proteasomes) during the differentiation of human leukemic cells.

The subunit composition of cell-internal and surface prosomes during phorbol myristate acetate (PMA)-induced differentiation of human leukemic T lymphocytes (CCRF-CEM cell line) was studied in relation to clusters of differentiation (CD) markers. PMA inhibited cell growth and decreased the amounts of CD1a and CD4 while CD3, CD8, CD25, CD45, CD57 and MHCI increased it; the p53 anti-oncogene increased while actin levels remained constant. Cells incubated with the inducer PMA for 3 days and placed in fresh inhibitor-free medium resumed growth at a low rate, while the CD values slowly reverted to those of the initial phenotype. The presence and relative amounts of prosome subunits were analyzed by flow cytometry, light and fluorescent microscopy and Western blotting using 3 monoclonal antibodies (p25K, p27K and p30-33K MAbs). The decrease in cytoplasmic antigens on day 3 was remarkable (cells followed for 7 days) while increased surface antigens were observed. Changes in the subcellular distributions of prosome antigens, particularly the p25K and p30-33K subunit, were correlated with a partial arrest of the cell cycle. Interestingly, the composition of cell internal and surface prosomes showed different patterns of change.

Proteasome (prosome) subunit variations during the differentiation of myeloid U937 cells.

20S proteasomes (prosomes/multicatalytic proteinase) are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol-myristate-acetate or retinoic acid plus 1,25-dihydroxy-cholecalciferol by western blot, flow cytometry and immuno-fluoresence. p25K (C3), p27K (IOTA) and p30/33K (C2) subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA + VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno-fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA + VD-induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA-induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non-induced cells; these membrane proteins disappeared when treated with RA + VD, whereas some increased on PMA-induced cells. The differential changes in the distribution and type of proteasomes in RA + VD and PMA-induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.

The oxidative burst triggered by Salmonella typhimurium in differentiated U937 cells requires complement and a complete bacterial lipopolysaccharide.

The bacterial and serum factors involved in the oxidative response triggered by Salmonella typhimurium in differentiated U937 cells were investigated. Complement activation was shown to be required, using sera deficient in complement factors. An original dot-blot technique was developed to study the activation of complement by either bacteria or purified lipopolysaccharide (LPS). Both O-specific and lipid A segments of LPS were found to play a role in the triggering of the oxidative response. Lipid A was responsible for bacterial C3-derived opsonization by inducing an antibody-independent activation of complement classical pathway, whereas O-specific polysaccharide chains (O-Ag) were involved in cellular activation. Inhibition experiments using anti-cell surface marker monoclonal antibodies showed the involvement of the alpha chain of CR3 (CD11b) in the oxidative response developed by differentiated U937 cells in response to S. typhimurium infection. Whether both iC3b and O-Ag interact with different domains of CR3 or whether the binding of O-Ag occurs via a not yet identified receptor remains to be determined.

Dimethyl sulfoxide-induced apoptosis in human leukemic U937 cells.

Dimethyl sulfoxide (DMSO), which induces differentiation of myeloid cells, was found to cause apoptosis in human leukemic U937 cells. Apoptosis was assessed by DNA electrophoresis and flow cytometry. The time needed to induce apoptosis varied from a few hours to 2-3 days, depending on the concentration of DMSO used. The plasma membrane remained intact long after DNA fragmentation had occurred. DMSO-induced apoptosis was inhibited by zinc ions and, to a lesser extent, by the protein kinase C activator: phorbol 12-myristate 13-acetate (PMA). Cycloheximide and actinomycin D did not prevent DMSO-induced apoptosis, showing that U937 cells do not require protein or RNA synthesis to undergo apoptosis. DMSO induced apoptosis despite the expression of the anti-apoptotic Bcl-2 protein in U937 cells. The amount of Bcl-2 remained unchanged during DMSO-induced apoptosis.

Differentiated U937 cells and human monocytes exhibit a differential production of extracellular oxygen species: O2.- excretion versus H2O2 diffusion.

The nature and the localization of the oxidative response triggered by different stimuli in either differentiated U937 cells and peripheral blood-derived human monocytes was investigated using luminometric and cytofluorometric techniques. Differentiated U937 cells essentially produced extracellular superoxide anion (O2.-), whatever the stimulus used. Monocytes, however, responded to Salmonella typhimurium, phorbol esters, and opsonized zymosan by an intracellular, an extracellular, and both an intra- and extracellular production of oxygen species, respectively. Furthermore, H2O2 but not O2.- was detected in the extracellular oxidative response of monocytes. Using differentiated U937 cells, luminol was found to be as efficient as lucigenin in the detection of extracellular O2.-, providing sufficient concentrations of extracellular horseradish peroxidase were present. However, both azide and histidine inhibited the lucigenin-enhanced chemiluminescence, suggesting an initial and transient production of singlet oxygen by differentiated U937 cells. Taken together these results strongly suggest that, when stimulated, differentiated U937 cells directly excrete O2.- in the extracellular medium while, within monocytes, O2.- is rapidly dismutated in H2O2 which can eventually diffuse outside the cell. Such differences in the oxidative response between the two cell types could be explained by the lack of total closure of the phagosome, only observed in differentiated U937 cells.

Infection of differentiated U937 cells by Salmonella typhimurium: absence of correlation between oxidative burst and antimicrobial defence.

The human histiocytic lymphoma cell line U937 can be induced to differentiate along the monocyte/macrophage pathway by either phorbol myristate acetate (PMA) or by the combination of retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (VD). U937 cells treated with either PMA or RA/VD were able to phagocytose Salmonella typhimurium in the presence of non-immune human serum. However, only cells differentiated by RA/VD were capable of developing an oxidative metabolic burst in response to infection. Since the oxidative burst is considered to be a potent antimicrobial mechanism, we investigated its effect on S. typhimurium. The oxidative burst failed to affect either the viability or the multiplication of S. typhimurium suggesting that it plays only a minor role in the host defence against S. typhimurium.

Rapid fluorometric measurement of the intra-cellular concentration of ciprofloxacin in mouse peritoneal macrophages.

A simple fluorometric assay requiring only a single sample of cells to determine the number of cells, from the DNA linked to the fluorochrome 4',6-diamidino-2-phenylindol (DAPI), and the uptake of ciprofloxacin, a natural fluorescent quinolone is described. Mouse peritoneal macrophages were found to concentrate ciprofloxacin up to 12.7 (+/- 1.5)-fold. Combined fluorometry provides a precise, sensitive method for determining the intracellular concentration of fluoroquinolones, as well as that of naturally fluorescent or fluorochrome-linked drugs.

[Use of flow cytometry in the study of a non-specific immunodeficiency in children. A case]. / Utilisation de la cytométrie en flux dans l'étude des déficits de l'immunité non spécifique de l'enfant. Une observation.

Using flow cytometry, we explored a case of nonspecific immunodeficiency in a seven month old girl with repeated infections. This method showed evidence of granulocyte phagocytosis and oxidative metabolism abnormalities suggesting the diagnosis of a variant form of chronic granulomatous disease (CGD). Findings also showed that flow cytometry can be useful to study phagocytic cells during the neonatal period as it allows rapid multiparametric analysis with a very small amount of blood.

Suspended mouse peritoneal macrophages. Preparation and properties.

Since macrophages (MPH) are able to adhere firmly to solid surfaces, the recovery of viable and functional MPH has proven to be extremely difficult. We have developed a simple method using agarose coating for preparing MPH and culturing the cells in suspension. Their properties were tested over 72 h. The oxidative burst declined with time, but could be restored using the lymphokine rich supernatant of pokeweed-stimulated mouse spleen cells. In contrast, phagocytosis and Candida intra-cellular killing remained unchanged.

Cellular responses to "brucelline INRA" during the experimental brucellosis of the mouse.

"Brucelline INRA" is an aqueous extract of a rough strain of Brucella melitensis that is used to test cutaneous delayed hypersensitivity in man and animals. The cellular response of mice (either normal, or sensitized by a previous brucella infection) was investigated with local implanted "cell-traps" and by immunological testing in vitro. Brucelline markedly reduced the number of polymorphonuclear cells in the local infiltrate of sensitized animals during the early, nonspecific phase of the response. In vitro responses were dissociated; while brucelline depressed the incorporation of thymidine into lymphocytes, on controls as well as on sensitized animals, it induced an evident production of macrophage inhibitory factor. Since brucelline is a mixture of several molecular species, and antibodies to some of its components were present in sensitized animals, immunomodulation of these apparently paradoxical responses is probably the result of a combination of different mechanisms. These observations are discussed in the light of physiopathological features of the experimental brucellosis in mice.

Kinetics of the uptake of rifampicin and tetracycline into mouse macrophages. In vitro study of the early stages.

The in vitro uptake by mouse peritoneal macrophages, of chlortetracycline (by fluorescence microscopy) and of tetracycline and rifampicin (by scintillation spectrometry of radioactive antibiotics) has been studied over a six hours period, using various concentrations of the antibiotics, close to the therapeutic concentrations. The incidence of the conditions of the assays, especially that of the use of heterologous serum for the cultivation of cells, has been investigated; a medium supplemented with homologous serum at low concentration has been devised with the technique. The uptake of these antibiotics was a three-phases process suggesting the superposition to a passive diffusion of either an active incorporation, or a restriction of the outflow (perhaps associated). This led to a rather high concentration of the antibiotics into cells, although other studies have shown that this concentration is not as active on intracellular bacteria as one could expect from the in vitro sensitivity.

Ultrastructural localization of NADPH-oxidase activity in murine peritoneal macrophages during phagocytosis of Brucella. Correlation with the production of superoxide anions.

The localization of the enzyme NADPH oxidase in mouse peritoneal macrophages unstimulated or after phagocytosis of Brucella suis was investigated by electron microscopy in normal mice and mice immunized against B. suis. The enzyme was clearly visualized on mitochondrial cristae, smooth endoplasmic reticulum, and the plasma membrane; its activity correlated mainly with the state of the endoplasmic reticulum which itself reflected macrophage activation. The enzyme turnover appeared to be accelerated after phagocytosis; the phagosome membrane was seldom stained by the enzyme reaction. These macrophages were also examined for the production of superoxide anions in vitro, either unstimulated or after phagocytosis. Phagocytosis increased the production of superoxide anions, especially in immunized animals. These results are discussed with regard to the role that the products of oxidative metabolism play in the inactivation of bacteria by phagocytic cells.

Modifying factors on phagocytosis of polymorphonuclear allergic subjects.

Supernatants from pollen-activated cultures of mononuclear (MN) cells obtained from two subjects allergic to gramineae pollen were tested for their effect on phagocytic activity of human polymorphonuclear leucocytes (PMN). An enhancement of phagocytosis was observed. Similarly prepared supernatants from MN cells of non-allergic control subjects did not affect PMN phagocytic activity. On the basis of these findings it is suggested that in the presence of allergen, cultures of MN cells from pollen-sensitized subjects produce a substance that stimulates PMN phagocytic activity. The direct effect of pollen on PMN was also studied. The allergen caused an inhibition of phagocytosis by PMN from allergic subjects. Pollen had no effect on phagocytosis by PMN from non-allergic control subjects.

In vitro immunosuppressive activity of gram-positive (Streptococcus pyogenes) peptidoglycan on mouse lymphocytes.

A peptidoglycan fraction prepared from group-A streptococcus was assayed for in vitro mitogenicity on mouse lymphocytes. This fraction reduced considerably the uptake of radioactive thymidine both on unstimulated cell suspensions and on suspensions stimulated by T(PHA)- or B(LPS)-mitogens. The immunosuppression was induced by relatively moderate doses of the fraction, and was dose-dependent. Several experiments ruled out the possibility that these results could be due to a cytotoxicity of the fraction, or to a non-specific interference with the uptake or metabolism of the radioactive precursor. These results are coherent with the observations made in vivo on the mouse and previously published. It is suggested that the mechanism of the immunosuppression could be in relation with the capacity of this fraction to stimulate the reticulo-macrophagic system.

[Attempts of ultrastructural and biochemical characterisation of cell wall deficient "Brucella" (L forms) (author's transl)]. / Essai de caractérisation ultrastructurale et biochimique de Brucella à paroi déficiente (formes L).

In previous studies the in vivo conversion of Brucella suis to L-form state was put in evidence. The L forms isolated from mouse spleen had original structural aspects in common: the absence of the cell wall layer and the extracellular multilayer "membranous" structures. The biological characterization of these L forms and the preliminary identification of specific chemical markers of the bacterial envelope is reported in the present study, performed with the stable L forms well-growing in the liquid media. The electron microscopy confirmed the absence of cell wall and the presence of numerous dense multilayer membranous structures in the L forms cultivated for a long time on appropriate media. This aspect was changed in the L forms adapted to growth on the ordinary medium for brucella: numerous small dense bodies limited by unit membrane were observed. The chemical analysis of stable L forms showed the absence of diaminopimelic acid, confirming the lack of peptidoglycan. The result of chemical determination in L forms of the Na-2-keto-3-deoxyoctonate was negative. However, biological assays suggested that outer membrane components such as LPS and receptors for the bacteriophage Weybridge remained in the L forms, albeit in reduced amount as compared to parental brucella.

Cellular responses of the mouse to the peptidoglycan of a gram-positive bacterium (Streptococcus pyogenes).

The cellular responses and the stimulation of the reticulo-macrophagic system induced in the mouse by a purified bacterial peptidoglycan (PGL) as previously described, were studied by the changes in the peritoneal cytology, the macrophage-migration-inhibition test and the clearance of colloidal carbon. PGL was submitted to chemical and immunochemical characterization and was shown to be substantially free of contamination by polysaccharides, phospholipids, teichoic acid and nucleic acids, but to contain a detectable amount of peptide contaminants; N-acetylglucosamine and the tetrapeptide (with terminal D-alanine) were shown to be the main antigenic determinants. This substance had no action on polymorphonuclear leucocytes but induced an inhibition of the migration of macrophages. This was due to an immunological reaction rather than to direct cytotoxicity, as shown by the negative cytotoxicity tests and the age and life-environment-dependence of the phenomenon. The reticulomacrophagic system was significantly stimulated after primary inoculation, and still more so after a booster. The possible mechanisms of these activities, which are therefore independent from toxic and/or inflammatory responses, are discussed.