Effects of some biologically active compounds on phagosome-lysosome fusion in peritoneal macrophages of mice.

Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome-lysome (P-L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target. It was found that all three compounds tested enhanced P-L fusion. To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA-DPH incorporated in isolated rat liver lysosomes. In order to characterize the cytoskeleton changes under the action of these biologically active compounds F-actin content in peritoneal macrophages of mice was determined. Our results demonstrate that BR action induces a decrease in DPH and TMA-DPH polarization, FR increases DPH and TMA-DPH polarization, and CR causes only an increase in TMA-DPH polarization in lysosomal membranes. All three compounds tested increase F-actin content in peritoneal macrophages. Thus, the effect of BR on P-L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes. The enhancement of P-L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state.     

Stimulation of macrophages by lipopolysaccharide of Vibrio parahaemolyticus

The effect of lipopolysaccharide (LPS) from Vibrio parahaemolyticus on the biochemical and phagocytic activities of murine peritoneal macrophages was determined. Intraperitoneal treatment with different doses (0.5-25 micrograms) of V. parahaemolyticus LPS markedly increased the cellular RNA content as well as lysosomal enzyme activities of peritoneal macrophages. The treatment also stimulated the phagocytic activities of macrophages. These observations suggest that V. parahaemolyticus LPS causes stimulation of murine peritoneal macrophages.     

Plasma membrane phospholipid translocation in the mouse peritoneal macrophage: differential response to stimulation of eicosanoid production.

The metabolism and translocation of exogenously introduced plasma membrane phosphatidylcholine (PC) having the fluorescent fatty acid analog aminocaproyl NBD (N-nitrobenzo-2-oxa-1,3 diazole) (NBD-PC), in the sn2 position was studied in cultured murine peritoneal macrophages using biochemical and morphological techniques. Following labeling of the cell plasma membrane at 2 degrees C by vesicle lipid exchange, macrophages were warmed in the presence or absence of pharmacological stimuli of eicosanoid production and release. Fluorescence microscopy indicated that the phospholipid was translocated to an internal cellular pool upon stimulation with zymosan. In contrast, the membrane PC analog was primarily metabolized and released after being found diffusely associated with the cytoplasm in macrophages stimulated with the calcium ionophore A23187. Evidence obtained by double labeling zymosan-treated macrophages with NBD-PC and a monoclonal antibody directed against a lysosomal membrane protein demonstrated that the fluorescent lipid is internalized in association with the zymosan particles and both are found in lysosomes. The results suggest that multiple pathways exist in peritoneal macrophages which target plasma membrane PC into different cellular compartments for hydrolysis and conversion to eicosanoid products and release from cells.     

双歧杆菌的完整肽聚糖对小鼠腹腔巨噬细胞膜脂流动性的影响

目的探讨分叉双歧杆菌的完整肽聚糖（WPG）对巨噬细胞膜脂流动性的影响。方法首先分离培养昆明小鼠腹腔巨噬细胞,然后以WPG刺激巨噬细胞,再用细胞膜磷脂荧光探针标记细胞,最后采用激光共聚焦显微镜结合激光漂白后荧光恢复技术检测巨噬细胞的膜脂流动性。结果WPG刺激组反映小鼠腹腔巨噬细胞膜脂流动性的平均荧光恢复率明显高于对照组（P〈0.01）。结论分叉双歧杆菌的完整肽聚糖可提高巨噬细胞膜脂流动性。     

Cholesterol delivered to macrophages by oxidized low density lipoprotein is sequestered in lysosomes and fails to efflux normally

Oxidized low density lipoprotein (LDL) has been found to exhibit numerous potentially atherogenic properties, including transformation of macrophages to foam cells. It is believed that high density lipoprotein (HDL) protects against atherosclerosis by removing excess cholesterol from cells of the artery wall, thereby retarding lipid accumulation by macrophages. In the present study, the relative rates of HDL-mediated cholesterol efflux were measured in murine resident peritoneal macrophages that had been loaded with acetylated LDL or oxidized LDL. Total cholesterol content of macrophages incubated for 24 h with either oxidized LDL or acetylated LDL was increased by 3-fold. However, there was no release of cholesterol to HDL from cells loaded with oxidized LDL under conditions in which cells loaded with acetylated LDL released about one-third of their total cholesterol to HDL. Even mild degrees of oxidation were associated with impairment of cholesterol efflux. Macrophages incubated with vortex-aggregated LDL also displayed impaired cholesterol efflux, but aggregation could not account for the entire effect of oxidized LDL. Resistance of apolipoprotein B (apoB) in oxidized LDL to lysosomal hydrolases and inactivation of hydrolases by aldehydes in oxidized LDL were also implicated. The subcellular distribution of cholesterol in oxidized LDL-loaded cells and acetylated LDL-loaded cells was investigated by density gradient fractionation, and this indicated that cholesterol derived from oxidized LDL accumulates within lysosomes. Thus impairment of cholesterol efflux in oxidized LDL-loaded macrophages appears to be due to lysosomal accumulation of oxidized LDL rather than to impaired transport of cholesterol from a cytosolic compartment to the plasma membrane.     

Stimulation of various functions in murine peritoneal macrophages by glucans produced by glucosyltransferases from Streptococcus mutans

The glucan that was produced by glucosyltransferases (GTFs) from Streptococcus mutans was examined for its stimulating functions toward murine peritoneal macrophages. Soluble glucan was obtained by the reaction with cell-free crude GTFs and sucrose, followed by ethanol precipitation, dispersion in water and re-precipitation by ethanol. Soluble glucan, those average molecular weight was about 3 x 10(5), was composed of mixture of alpha-1,6 and alpha-1,3 linkages in a 3:1 ratio. When 30 and 60 microg/ml of the glucan was incubated with peritoneal macrophages, the lysosomal phosphatase activity was increased in a dose-dependent manner, indicating that soluble glucan may activate macrophages. To examine its effects on the various functions of macrophages, soluble glucan was orally administered daily at a level of 100 mg/kg of body weight to C57BL/6 mice. Significant stimulation of the production of H2O2 by the macrophages was observed without any increase in NO production. The production of tumor necrosis factor-alpha (TNF-alpha) by the macrophages was also stimulated from 538.73-555.06 pg/ml to 585.73-596.40 pg/ml during 15 days of oral administration of soluble glucan. The cytotoxicity of peritoneal macrophages against B16 tumor cells was significantly enhanced by 25-38% during 15 days of oral administration. These results may indicate that soluble glucan stimulates the immune functions of macrophages.     

小鼠巨噬细胞膜快速超极化和凋亡

 利用激光扫描共聚焦显微技术、流式细胞术、TUNEL染色技术、FRAP技术等对大剂量地塞米松诱导小鼠腹腔巨噬细胞凋亡过程中膜通透性、膜脂流动性、膜电位等膜生物物理性状改变进行了研究 .结果显示 ,大剂量地塞米松诱导小鼠腹腔巨噬细胞快速凋亡 .凋亡巨噬细胞膜脂流动性升高 ,尤为显著的是 ,膜电位快速超极化 ,胞浆游离 Ca2 + 加速超极化 .结果表明 ,细胞膜电位变化与巨噬细胞这一非兴奋细胞凋亡密切相关     

Leukocyte complement: interleukin-like properties of factor Bb

It has been previously shown that the activated form of Factor B (Factor Bb) of the alternative pathway of complement activation stimulates monocyte spreading and killing of xenogenic erythrocytes and staphylococci. Factor Bb also stimulates lymphocyte blastogenesis in vitro, and native (uncleaved) Factor B is a major constitutive product of murine macrophages. To evaluate the possible "monokine" or "lymphokine"-like properties of Factor Bb, a radioimmunoassay was developed to measure the quantities of Factor B in phytohemagglutinin (PHA)-mitogen-stimulated cultures of human peripheral blood mononuclear cells. Nonstimulated mononuclear cell cultures from human peripheral blood (containing 10-14% monocytes and greater than 85% lymphocytes) at a density of 3 X 10(6) cells/ml (in serum-free medium) released less than 7 X 10(-10) M/liter (60 ng/ml) of Factor B antigen in 24 hr at 37 degrees C, and when mononuclear cells were stimulated with PHA mitogen in serum-free medium, the levels of Factor B antigen in media at 24 hr were significantly higher 1-3 X 10(-8) M/liter (0.9-2.8 micrograms/ml). The molecular size of Factor B in these media was 50-65 kDa by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size appropriate for Factor Bb (60 kDa). Since pathological effects of macrophages in autoimmune disease may result from the release of lysosomal hydrolases, the effects of purified Factor Bb on mononuclear phagocytes were investigated in an in vitro system of murine peritoneal exudate macrophages. Factor Bb induced secretion of marker lysosomal hydrolases N-acetyl-beta-D-glucosaminidase (hexosaminidase) and beta-glucuronidase from thioglycollate-elicited murine peritoneal exudate macrophages in a dose-response and kinetic manner. Hydrolase release was induced in serum-free medium without a known particulate activator at a concentration of 80-200 nM (5-13 micrograms/ml) Factor Bb. Maximal release occurred in 3-5 hr at 37 degrees C and extracellular enzyme activity of hexosaminidase and glucuronidase increased as intracellular enzyme levels decreased, suggesting that Factor Bb triggers release of these enzymes from intracellular lysosomal pools. These results provide an example of a complement protein which is synthesized, released, and activated during mononuclear cell culture and which induces release of lysosomal enzymes from macrophages. In conventional terminology, Factor B or Factor Bb might be termed a "lymphokine," "monokine," or "interleukin".     

Some biochemical and functional characteristics of macrophages activated by Tetrahymena pyriformis

Phagocytosis, enzyme activities and capacity to release hydrogen peroxide (H2O2) and superoxide anion (O2-) of peritoneal macrophages from mice inoculated with Tetrahymena pyriformis, a free-living ciliate, were examined in comparison with resident and BCG-activated macrophages. Fc receptor-mediated phagocytosis of sheep erythrocytes was markedly increased in Tetrahymena-activated macrophages to the same level as that seen in BCG-activated ones. Tetrahymena-activated macrophages showed an increased level of acid phosphatase (lysosomal enzyme) and a reduced level of alkaline phosphodiesterase I (plasma membrane ectoenzyme) as compared with resident macrophages. Similar changes in the activities of the two enzymes were also observed in BCG-activated macrophages. Both Tetrahymena- and BCG-activated macrophages exhibited more enhanced capacity to release H2O2 and O2- than resident macrophages when stimulated with phorbol myristate acetate. In the macrophages from mice inoculated with varying doses of Tetrahymena, a significant correlation was observed between the increased capacity of H2O2 and O2- release as observed in the present study, and the enhanced toxoplasmacidal activity as observed in a previous study, in a dose-dependent fashion.     

Influence of serosal Cl on transport properties and cation activities in frog skin

Summary Treatment of resident peritoneal macrophages of rats with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV) potentiated their activation for tumor cell lysis by endotoxins. The fluorescence polarization of diphenylhexatriene (DPH) embedded in rough endoplasmic reticulum membranes isolated from DPPC SUV-treated macrophages was enhanced. The average fluorescence lifetime of DPH and the rotational correlation time deduced from anisotropy decay were unchanged, whereas the residual anisotropy and hence the order parameter were increased. The measurement of the fluorescence anisotropy of DPH as a function of the temperature showed a phase transition. No phase transition was observed in the rough endoplasmic reticulum membranes of macrophages either treated or not treated with cholesterol/DPPC SUV (1/1; mol/mol). The synergistic effect of DPPC SUV on the tumoricidal activity of macrophages induced by endotoxins appears to be correlated with the changes in the properties of the rough endoplasmic reticulum membranes. Both effects were transient; they had the same kinetics of induction and reversion, and they were both inhibited by cholesterol.     

Ultrastructural cytochemistry of non-specific esterase in murine peritoneal macrophages

Summary Non-specific esterase (NSE) activity was demonstrated in glutaraldehyde-fixed monolayers of murine peritoneal macrophages. Using 2-naphthylthiol acetate (NTA) as substrate and Fast Blue BB as coupling agent a strong osmiophilic reaction product was obtained. The reaction product was observed as electron-dense dots covering cisterns of the rough endoplasmic reticulum, Golgi saccules and vesicles, or as large aggregates in lysosomes. Using -naphthyl butyrate (ANB) as substrate and hexazotized pararosaniline as coupling agent the osmiophilic reaction product was observed extracellularly on the plasma membrane as an electron-dense continuous layer, whereas intracytoplasmic staining of lysosomes was rare. Substitution of the coupling agents in the respective media resulted in a slight reaction with the ANB medium whereas with the NTA medium reaction product was observed only in lysosomal structures. The substrate specificity of the different types of esterases was confirmed after isoelectric focusing on thin-layer polyacrylamide gels. The results indicate that in murine peritoneal macrophages different types on NSE are detected with NTA and ANB, having distinct ultrastructural localizations.     

The influence of stress and methionine-enkephalin on macrophage functions in two inbred rat strains

The aim of our current study was to investigate the effect of acute exposure to electric tail shock stress (ES) and to a stress witnessing procedure (SW), as models for physical and psychological stress paradigms, respectively, on phagocytosis and H(2)O(2) production in peritoneal macrophages isolated from Albino Oxford (AO) and Dark Agouti (DA) rats. In addition, we studied the in vitro effects of methionine-enkephalin (ME) on phagocytosis and H(2)O(2) production in peritoneal macrophages isolated from both AO and DA rats that had been exposed to ES and SW procedures. The results showed that peritoneal macrophages isolated from DA rats were less sensitive to the suppressive effects of ES and SW than macrophages isolated from AO rats. In vitro treatment of macrophages isolated from AO rats with ME mimicked to some extent the suppressive effects of ES and SW on phagocytosis and H(2)O(2) production and additionally diminished H(2)O(2) release in macrophages isolated from AO rats previously exposed to ES or SW. ME did not have any effect on phagocytosis in macrophages isolated from DA rats, but changed H(2)O(2) production in a concentration-dependent manner. In macrophages isolated from DA rats previously exposed to stress the effect of ME was dependent on the macrophage function tested and the particular stress paradigm employed. Our results emphasise the fact that both beneficial and detrimental effects of stress on immune system functions could be attributed to the individual variations in the macrophage's response to stress mediators.     

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry. Effect of silver lactate and hypotonic conditions

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time - a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift - was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonic medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 microM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.     

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time -a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift -was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonie medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 μM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.     

Tumour necrosis factor and the lysosomal enzymes of macrophages or macrophage-like cell line

Summary The relationship between tumour necrosis factor (TNF) and macrophages or macrophage-like cell line, especially the lysosomal enzymes was investigated. The serum lysosomal enzymes and LDH activities were increased in proportion to the TNF production even in different strains of mice. Lysosomal enzymes and TNF activity were released into the supernatant of the culture medium of macrophage-enriched peritoneal exudate cells (PEC) or spleen cells derived from Propionibacterium acnes-primed mice after addition of lipopolysaccharide (LPS). After passage through a Sephadex G-10 column, TNF activity could not be detected in the supernatant of these spleen cells after addition of LPS. Also TNF activity could not be detected in the supernatant following destruction of PEC. These results suggest that TNF producibility is strongly related to the degree of activation of macrophages, especially the lysosomal enzymes. The murine macrophage-like cell line, J 774, also released TNF activity and lysosomal enzymes after addition of LPS.     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E2 and 6-keto-prostaglandin F1 alpha. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 microgram/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 microgram/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolases are independently regulated.     

Changes in the membrane potential, the cytoplasmic Ca2+ concentration and in the transport of fluorescein anions across the macrophage plasma membrane under the influence of O2(-) and H2O2 in small concentrations]

It has been shown that under the influence of superoxide anion (300 nM) and hydrogen peroxide (100 nM) on murine peritoneal macrophages, the depolarization of membrane takes place, and a change in the rate of fluorescein anion efflux from the cells occurs. Hydrogen peroxide (but not superoxide anions) causes a transient increase in the cytoplasmic Ca2+ concentration. These changes are regarded as early signs of macrophage activation. It is assumed that macrophage activation with reactive oxygen intermediates at such a low concentration might be interpreted as follows: a small portion of stimulated phagocytes activates the whole population with the aid of the products of their own oxidative burst.     

Spontaneous lysosomal enzyme secretion by a murine macrophage-like cell line.

Lysosomal enzyme secretion by the murine macrophage-like cell line, P388D1, was compared with that of normal peritoneal macrophages. Unlike macrophages, lysosomal hydrolase secretion by P388D1 cells occurred spontaneously in vitro and was not further stimulated by the presentation of inflammatory agents such as zymosan and asbestos.     

Altered cell-averaged microviscosity of murine peritoneal macrophages undergoing activation in vivo or in vitro

The cell-averaged microviscosity of intact murine peritoneal mononuclear phagocytes in various stages of activation was assessed by quantifying fluorescent depolarization of 1,6-diphenyl-1,3,5-hexatriene. Macrophages activated in vivo with Mycobacterium bovis, strain BCG, were significantly more fluid than resident peritoneal macrophages, responsive macrophages elicited with thioglycollate broth, proteose peptone broth, or fetal bovine serum, or primed macrophages elicited with pyran copolymer, MVE-2. Specifically, the cell-averaged microviscosity decreased from a mean of 3.47 +/- .07 eta 25 degrees C (poise) (range of 3.32 to 3.67 p) to 2.62 eta 25 degrees C. Exposure of responsive macrophages in vitro to bacterial endotoxin plus hybridoma supernatants containing macrophage-activating factor or purified recombinant interferon gamma resulted in decreased microviscosity; the largest effect was seen after 24 hr. Macrophages primed in vivo with MVE-2 and treated in vitro with endotoxin also developed decreased microviscosity. Similar changes in microviscosity were observed in a plasma membrane-enriched fraction isolated from macrophages activated in vitro with interferon gamma and endotoxin, thus suggesting that the cell-averaged measurements reflected changes in membrane viscosity. The optimum concentration of MAF-inducing decreased overall microviscosity was identical to that for inducing tumoricidal capacity. Taken together, the data indicate activation of lytic capacity in murine macrophages is closely associated with decreased cell-averaged microviscosity and that this change reflects, at least in part, decreased microviscosity of the plasma membrane of these cells.     

Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages

The purpose of this study was to elucidate the role of NO and O^-₂ on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time-dependent production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O^-₂), and by increased expression of COX-2. Our results provide evidence that O^-₂ may be involved in the pathways that result in arachidonate release and PGE₂ formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions.