Penetration of anti-lysosome serum in macrophages and the localization of anti-lysosome antibodies in their structures]

Penetration of antilysosomal sera into living macrophages, their localization on subcellular structures, and their effects upon the cell, were studied by means of immunofluorescence technique. The rabbit antilysosomal (antimembrane) immune globulins were found to be ingested by the mouse macrophages and to exert obvious effect upon the macrophage function. The specificity of used antisera was confirmed by findings obtained with fixed macrophages.     

Prevention of muscarinic acetylcholine receptor down-regulation by chloroquine: Antilysosomal or antimuscarinic mechanisms

The effect of the antimalarial drug chloroquine on the carbachol-induced down-regulation of muscarinic acetylcholine receptors (mAChRs) was studied in the neuroblastoma-glioma hybrid NG108-15 cells. Chloroquine, which is proposed to have both antilysosomal and antimuscarinic effects (4,11), blocked the loss of both cell surface and total mAChRs as monitored by [³H]N-methyl-scopolamine (NMS) and [³H] quinuclidinyl benzilate (QNB) bindings respectively. To the contrary, NH₄Cl, only an antilysosomal agent, had no effect on the loss of surface receptors, but blocked degradation of internalized receptors following the effect of carbachol. These findings demonstrate that chloroquine prevents the agonist-induced mAChR down-regulation in NG 108-15 cells by both its antilysosomal and antimuscarinic effects.     

Phagocytic inhibitory activity in serum of cats immunized withPseudomonas aeruginosa lipopolysaccharide

Sequentially collected sera from cats chronically immunized withPseudomonas aeruginosa lipopolysaccharide (LPS) serotype 5 were assessed for their effect on phagocytosis by alveolar macrophages from nonimmunized cats. Phagocytosis was measured by incubating macrophage monolayers for 20 min in the presence of³H-labeled bacteria and 5% serum from control or immunized animals. Sustained phagocytic inhibitory activity developed in the sera of eight of 11 immunized cats (mean inhibition ranged from 30% to 73%) after 13 weekly immunizations. The activity was specific because phagocytosis ofStaphylococcus aureus andP. aeruginosa of another serotype (type 6) was unimpaired. An enzyme-linked immunosorbent assay showed an increase in the amount of LPS-specific IgG in postimmunization sera from the eight cats with inhibitory activity. The IgG appeared to be serotype specific because significantly higher titers were obtained against LPS serotype 5 than LPS serotype 6. The results suggest that phagocytic inhibitory activity in sera from LPS-immunized cats may be due to anti-LPS IgG.     

Serum opsonic activity in rodent malaria: functional and immunochemical characteristics in vitro.

The functional and immunochemical characteristics of serum opsonic activity in rodent malaria were examined in the present study. Schizont- and late trophozoite-enriched populations of Plasmodium berghei-infected red blood cells (IRBC) were isolated on a Ficoll density-gradient and used in an in vitro phagocytosis system composed of serum and monolayer cultures of rat peritoneal macrophages. Hyperimmune serum augmented the phagocytosis of IRBC to a greater degree than did nonimmune serum. When either IRBC or macrophages were pre-incubated with serum, the phagocytosis-promoting factors acted on the IRBC rather than on the macrophages in a manner characteristic of serum opsonins. The opsonic activity was specific for IRBC since noninfected red blood cells were rarely phagocytized and were unable to absorb opsonic activity from serum. The opsonic activity of both hyperimmune and nonimmune sera was heat stable, and unaffected by agents known to inactivate or inhibit complement (cobra venom factor and ethylenediaminetetraacetic acid). Finally, the opsonic activity was identified in preparations of purified IgG isolated from both hyperimmune and nonimmune sera.     

The method for estimation of opsonic activity in sera of infants

Three methods for the estimation of opsonic activity in the sera of newborn children were tested. Two of them, based on the phagocytosis of opsonised bacteria labelled with radioactive phosphorus³²P as measured byin vivo blood clearance or uptake of bacteria in perfused isolated liver, were found to be unsuitable for long term dynamic study mainly because they do not permit the testing of series of samples. The third method (using isolated phagocytic cellsin vitro) permits the differentiation of the opsonic effect of complement and antibody and, furthermore, the firmness of the bond between microbes and phagocytes (which reflects the degree of opsonization) can be established. It was found that a 2-mercaptoethanol-resistant antibody, probably of the IgG type, was responsible for the opsonic activity of children's sera toEscherichia coli 083. Homologous antibody (toEscherichia coli 083) could be differentiated from beterologous antibody (toEscherichia coli 086) using the opsonic test only at low dilutions of sera. The combination of newborn piglet complement and antibody of children's sera yielded higher values of opsonic activity than either component separately.     

The Inhibitory Effect of Staphylococcus epidermidis Slime on the Phagocytosis of Murine Peritoneal Macrophages Is Interferon-Independent

The extracellular slime produced by Staphylococcus epidermidis has been shown to interfere with several human neutrophil functions in vitro, such as chemotaxis, degranulation and phagocytosis. Slime production has been suggested as a useful marker for clinically significant infections with coagulase-negative Staphylococcus. Since the main role of macrophages in defense mechanisms is phagocytosis, the effect of slime on the phagocytic activity of macrophages was investigated. The phagocytic activity of murine peritoneal macrophages treated with slime in vitro decreased in a dose-dependent fashion. A similar decrease was also observed in macrophages isolated from mice that had previously received intraperitoneal injection of slime. To investigate whether interferon also plays a role in this process, mice were treated with interferon or an interferon inducer, polyinosinic-polycytidylic acid (poly I:C), together with slime before macrophage isolation. The slime-suppressed phagocytic activity of macrophages was partially relieved by both agents, and the recovery effect of poly I:C in slime-suppressed phagocytosis of macrophages in vivo might be attributed to the increased interferon level in peritoneal fluid and sera. However, when slime was given to poly I:C-pretreated mice, the phagocytic activity remained suppressed. Thus, it appears that slime is able to suppress the phagocytic activity of macrophages regardless of the state of macrophage activation by poly I:C. The results suggest that the inhibition of phagocytosis by S. epidermidis slime may be independent from the activation of interferon.     

Neuropeptide Y, an orexigenic hormone, regulates phagocytic activity of lizard splenic phagocytes

Present in vitro study in the wall lizard Hemidactylus flaviviridis, for the first time in ectothermic vertebrates, demonstrated the immunoregulatory role of neuropeptide Y (NPY) and its receptor-coupled downstream signaling cascade. NPY inhibited the percentage phagocytosis and phagocytic index of splenic phagocytes. The inhibitory effect of NPY on phagocytosis was completely antagonized by Y₂ and Y₅ receptor antagonists. This suggests that NPY mediated its effect on phagocytosis through Y₂ and Y₅ receptors. Further, NPY receptor-coupled downstream signaling cascade for NPY effect on phagocytosis was explored using the inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). The SQ 22536/H-89 in a concentration-related manner decreased the inhibitory effect of NPY on phagocytosis. Further, an increase in intracellular cAMP level was observed in response to NPY. Taken together, it can be concluded that NPY via Y₂ and Y₅ receptor-coupled AC-cAMP-PKA pathway downregulated the phagocytic activity of lizard splenic phagocytes.     

Opsonizing activity of sera from animals immunized with pertussis vaccine

Opsonizing activity of guinea pig blood serum containing mercaptoethanol-resistant pertussis antibodies was studied in vitro on a model of microorganism ingestion by the mononuclears of the guinea pig peritoneal exudate. There were revealed distinct differences in the serum activity depending on the phagocytosis object. The blood serum of hyperimmunized rabbits stimulated the ingestion of Bordetella pertussis by mononuclears of guinea pigs--normal and immunized with pertussis vaccine. The blood sera of hyperimmunized guinea pigs and of mice immunized with pertussis vaccine twice displayed opsonins to B. pertussis. The blood sera of animals immunized with pertussis vaccine inhibited the staphylococcus ingestion by the peritoneal exudate mononuclears of guinea pigs, both normal and those immunized with pertussis vaccine.     

The protective action of disodium cromoglycate on alveolar macrophages in phagocytosis of fibrogenic silica.

Treatment of fibrogenic silica (DQ-12) with Disodium Cromoglycate (DSCG) prior to its in-vitro and in-vivo phagocytosis by alveolar macrophages prevents the destruction of the cells and the fibrosis of lung tissue which are a consequence of phagocytosis. However, the treatment of alveolar macrophages with DSCG before phagocytosis of the silica had no, or a negligible, protective effect on the cells. Acid phosphatase activity which was significantly enhanced above the control in cells phagocytosing the silica was returned to the range found in phagocytosis of inert dust when silica treated with DSCG was phagocytized. The inhibitor of DNA- dependent RNA synthesis actinomycin D caused an increase of acid phosphatase activity. DSCG did not depress the phagocytic ability of alveolar macrophages. It appears that the catabolic enzyme process predominant in cells phagocytosing DQ-12 was under control in cells phagocytosing DQ-12 treated with DSCG and that DSCG probably acted as a regulator of the factors permitting catabolism. From the results it is suggested that the equilibrium of the enzyme reactions which accompany phagocytosis was such that the integrity of the phagocytes was preserved.     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method

The ultrastructural localization of NADH oxidase, a possible enzyme in the increased oxidative activity of polymorphonuclear leukocytes (PMN) during phagocytosis, was studied. A new cytochemical technique for the localization of H2O2, a product of NADH oxidase activity, was developed. Cerous ions, in the presence of peroxide, form an electron-dense precipitate. Resting and phagocytically stimulated PMN were exposed to cerous ions at pH 7.5 to demonstrate sites of NADH-dependent, cyanide-insensitive H2O2 production. Resting PMN exhibites slight activity on the plasma membrane; phagocytizing PMN had extensive deposits of reaction product localized within the phagosome and on the plasma membrane. Peroxide involvement was demonstrated by the inhibitory effect of catalase on cerium precipitation; the surface localization of the enzyme responsible was confirmed by using nonpenetrating inhibitors of enzymatic activity. A correlative study was performed with an NADH-dependent, tetrazolium-reduction system. As with cerium, formazan deposition on the surface of the cell was NADH dependent, cyanide insensitive, and stimulated by phagocytosis. Superoxide dismutase did not inhibit tetrazolium reduction, as observed cytochemically, indicating direct enzymatic dye reduction without superoxide interposition. These findings, combined with oxygen consumption studies on resting and stimulated PMN in the presence or absence of NADH, indicate that NADH oxidase is a surface enzyme in human PMN. It is internalized during phagocytosis and retains its peroxide-generating capacity within the phagocytic vacuole.     

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A(2)

Phagocytosis is a hemocytic behavior against bacterial infection. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits immune responses of target insects and causes hemolymph septicemia. This study analyzed how X. nematophila could inhibit phagocytosis to increase its pathogenicity. Granular cells and plasmatocytes were the main phagocytic hemocytes of Spodoptera exigua determined by observing fluorescence-labeled bacteria in the cytosol. X. nematophila significantly inhibited phagocytosis of both hemocytes, while heat-killed X. nematophila lost its inhibitory potency. However, co-injection of X. nematophila with arachidonic acid did not show any significant inhibition of hemocyte phagocytosis. In fact, hemocytes of S. exigua infected with X. nematophila showed significant reduction in phospholipase A(2) (PLA(2)) activity. Dexamethasone, a specific PLA(2) inhibitor, significantly inhibited phagocytosis of both cell types. However, the inhibitory effect of dexamethasone was recovered by addition of arachidonic acid. Incubation of hemocytes with benzylideneacetone, a metabolite of X. nematophila, inhibited phagocytosis in a dose-dependent manner. These results suggest that X. nematophila produces and secretes PLA(2) inhibitor(s), which in turn inhibit the phagocytic response of hemocytes.     

Polymorphonuclear cell derangements in type I diabetes

Polymorphonuclear cell function had been studied in 58 Type I diabetic subjects. Chemotaxis, phagocytosis, adherence, bactericidal activity, NBT reduction capacity were evaluated. We enumerated gamma Fc receptor bearing polymorphonuclear cells and the percentage of immune complexes containing polymorphonuclear cells. These data were studied in accordance with glycemic levels and the presence of infections. All polymorphonuclear functions were decreased compared to non-diabetic subjects with the exception of phagocytosis. The efficiency of the diabetic sera on normal subjects polymorphonuclear cells was decreased (bactericidal activity, chemotactic index and phagocytosis). These abnormalities were independent of the presence of infection. No correlation was found with glycemic level. The percentage of cells bearing an Fc gamma receptor was less in diabetics than in normal (70.1 +/- 17.4 vs 80.2 +/- 7.8%). The percentage of immune complexes containing polymorphonuclear cells was increased (n = 16, 9.06 +/- 4.7 vs 4.75 +/- 2.1% in normals). There again, these data are without correlation with the presence of infections or glycemic level.     

Trifluoperazine inhibits phagocytosis in a macrophagelike cultured cell line

Trifluoperazine, a drug that binds to Ca2+-calmodulin and inhibits its interaction with other proteins, was found to inhibit growth and phagocytosis in a macrophagelike cell line, J774.16. Both effects were reversible and occurred at the same concentrations of drug (25--50 microM) that inhibited the activation of cyclic nucleotide phosphodiesterase by calmodulin in vitro. Fc-mediated phagocytosis was also depressed by W-7, a sulfonamide derivative that inhibits the activity of Ca2+-calmodulin. In contrast, taxol, a drug that stabilizes cellular microtubules, had no effect on Fc-mediated phagocytosis although it inhibited cell growth at nanomolar concentrations. The inhibitory effects of trifluoperazine and W-7 on phagocytosis suggest that calmodulin may be involved in this complex cellular function.     

Effects of fluoxetine on the activity of phagocytosis in stressed mice

We studied the effects of 1, 2, 5, 10 and 20 mg/kg of fluoxetine on the activity of phagocytosis in mice subjected to a chronic auditory stressor. Both the in vitro and in vivo activity of phagocytosis, measured using the zymosan-particle uptake method and the carbon clearance test, respectively, were reduced after 2, 4, 8 and 16 days of stress exposure. A partial recovery on the in vivo activity of phagocytosis was found on day 16th. Daily treatment with fluoxetine partially reversed the adverse effects of stress in a dose-dependent manner on both parameters but did not significantly affect the activity of phagocytosis in unstressed mice. Significant differences appeared when fluoxetine was administered at 2 mg/kg. Maximum effect was reached at 5 mg/kg.     

Synergistic effect of cefepime on the phagocytic killing of Staphylococcus aureus by human polymorphonuclear leucocytes and the determination of this effect by means of nitrite production

In this study the effect of cefepime on the phagocytosis and intracellular killing of Staphylococcus aureus by human polymorphonuclear leucocytes (PMNL) was determined. The opsonophagocytic killing of S. aureus was synergistically enhanced by cefepime at concentrations below 0.5 times the minimal inhibitory concentration (MIC), and four times the MIC at higher concentrations. The effect of cefepime on phagocytosis and the bactericidal activity of PMNL was also investigated by the measurement of nitrite levels using a Sievers analyser. According to the nitrite levels, cefepime enhanced not only the phagocytosis by PMNL 2.1-fold in the 0.5 MIC and 2.8-fold in the four MIC values but also the bactericidal activity of neutrophils 2.5-fold in the 0.5 MIC and 2.8-fold in the four MIC values, respectively. The beneficial cefepime-leucocyte interaction may explain the efficacy of cefepime against intracellular pathogens.     

An extracellular 5'-nucleotidase with both monoesterase and diesterase activity from Micrococcus sodonensis.

Bovine γ-globulin was separated into four fractions by chromatography on cellulose phosphate. The chromatographic distribution was similar to that reported for human and dog γ-globulin. More than 80% of a nonspecific phagocytosis stimulating factor (leucokinin) present in the serum was isolated in γ-globulin fraction IV. Bocine red blood cells and polymorphonuclear leukocytes bind γ-globulin without appreciable selectivity for any of the four chromatographic fractions, but they do selectively bind the phagocytosis stimulating factor. Splenectomy caused no observable change in either the chromatographic distribution or phagocytosis stimulating activity of bovine serum γ-globulin. The tetrapeptide tuftsin stimulates phagocytosis by bovine neutrophiles, but on a molar basis the activity of tuftsin was only 10% that of the phagocytosis stimulating factor. If the factor exerts its effect, as has been proposed, by having a phagocytosis stimulating peptide cleaved from it by an enzyme on the leukocyte membrane, that peptide must differ in structure from tuftsin. This conclusion is supported by the inability of trypsin to liberate an active peptide from bovine serum.     

人参根提取物增强巨噬细胞RAW264.7的自噬水平并提高其增殖和吞噬能力

研究人参根提取物对巨噬细胞RAW264.7的增殖能力、吞噬能力和自噬水平的影响以及其相关性.用细胞计数试剂(CCK-8)检测不同浓度的人参根以及加入对巨噬细胞RAW264.7增殖的影响;采用中性红吞噬实验检测人参根提取物对巨噬细胞吞噬活性的影响;采用吖啶橙染色法(AO染色法)检测自噬体的形成;采用免疫印迹法(Weste...     

Damage-associated molecular pattern S100A9 increases bactericidal activity of human neutrophils by enhancing phagocytosis

The damage-associated molecular-pattern S100A9 is found at inflammatory sites in infections and various autoimmune diseases. It is released at very high concentrations in the extracellular milieu by activated neutrophils and monocytes in response to various agents. This proinflammatory protein is found in infected mucosae and tissue abscesses where it acts notably as a potent neutrophil activator. In this study, we examined the role of S100A9 in the control of infections. S100A9 was found to increase human neutrophil bactericidal activity toward Escherichia coli. Although S100A9 induced the accumulation of reactive oxygen species over time through the activation of NADPH oxidase, its antimicrobial activity was mediated mainly by enhancing the efficiency of neutrophil phagocytosis. Interestingly, S100A9 did not act by increasing cell surface expression of CD16, CD32, or CD64 in neutrophils, indicating that its biological effect in FcR-mediated phagocytosis is independent of upregulation of FcγR levels. However, S100A9-induced phagocytic activity required the phosphorylation of Erk1/2, Akt, and Syk. Taken together, our results demonstrate that S100A9 stimulates neutrophil microbicidal activity by promoting phagocytosis.     

Enhancement of complement activation and opsonophagocytosis by complexes of mannose-binding lectin with mannose-binding lectin-associated serine protease after binding to Staphylococcus aureus

Human mannose-binding lectin (MBL) is a serum protein of the innate immune system that circulates as a complex with a group of so-called MBL-associated serine proteases (MASP-1, MASP-2, and MASP-3). Complexes of MBL-MASP2 are able to activate the complement system in an Ab and C1-independent fashion after binding of the lectin to appropriate microbial sugar arrays. We have evaluated the additive effect of the lectin pathway relative to other complement activation pathways and the subsequent effect on neutrophil phagocytosis. Complement activation in the sera of MBL-deficient individuals was studied with and without the addition of exogenous MBL-MASP. Flow cytometry was used to measure the deposition of C4, factor B, C3b, and iC3b on Staphylococcus aureus. Deposition of the first cleavage product of the lectin pathway, C4b, was increased using the sera of three different MBL-deficient individuals when exogenous MBL-MASP was added. Factor B was deposited in association with C4, but there was no evidence of independent alternative pathway activation. Similar enhancement of C3b deposition was also observed, with evidence of elevated amounts of C3b processed to iC3b. The increase in opsonic C3 fragments mediated by MBL was associated with a significant increase in the uptake of organisms by neutrophils. We also observed significant increases in phagocytosis with MBL-MASPs that were independent of complement activation. We conclude that MBL-MASP makes a major contribution to complement-mediated host defense mechanisms.