The effect of phagocytosis of Candida albicans blastospores on alkaline phosphatase levels in rat polymorphonuclear leucocytes

Summary The phagocytic function of young polymorphonuclear leucocytes with high levels of leucocyte alkaline phosphatase, which are present in the peripheral blood during an inflammatory response, was compared with that of normal polymorphonuclear leucocytes.Candida albicans blastospores were used as phagocytic targets. The phagocytic index of polymorphonuclear leucocytes with low levels of leucocyte alkaline phosphatase was higher than that of cells with high levels of enzyme.Monitoring of leucocyte alkaline phosphatase levels with increasing times of incubation of leucocytes with the blastospores showed a progressive decline in the level of the enzyme. Thus loss of the enzyme is linked to the phagocytic function, although the mechanism of this dynamic process is unclear.     

Fluorescence assay to monitor phagocytosis by bloodclot derived polymorphonuclear leucocytes. 2. Study of patients with psoriasis and the effect of exposing leucocytes to psoriatic skin scales

Comparisons of phagocytic parameters were carried out by a recently developed fluorescence test which is reproducible, simple and fast. Phagocytosis by polymorphonuclear leucocytes (PMNs) obtained from patients with psoriasis was compared with that of healthy individuals. Psoriatic skin scales, non-sterile and sterile, were tested for stimulatory effect on PMNs and compared with the effect of normal skin scrapings. Results confirm enhanced phagocytosis of bacteria by PMNs from patients with psoriasis over that of PMNs from healthy volunteers. Furthermore, the supernatant fluid from suspensions of psoriatic skin scales, non-sterile and sterile, stimulated PMNs activity.     

Inhibition of phagocytosis of polymorphonuclear leucocytes by adenosine and HoCl3 in vitro

Phagocytosis of polymorphonuclear leucocytes treated with NaF, HoCl3 and adenosine were studied. The highest concentration used was 25 mM of NaF, 25 mM of adenosine and 5 mM of HoCl3. It was ascertained that these substances, inhibitors of erythrocyte contractile protein, inhibit both phagocytosis and ability of polymorphonuclear leucocytes to change their shape. These unfavourable effects may be induced by the chemicals interfering with polymorphonuclear leucocytes contractile protein. NaF, HoCl3 and adenosine are also responsible for morphological changes in the cell nucleus.     

Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied. Attached and interiorized bacteria were distinguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phagocytosis. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes. Our ultrastructural results demonstrate that V. anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.     

Chemiluminescence induced by phagocytosis of Escherichia coli by polymorphonuclear leucocytes

Chemiluminescence emitted by phagocytosing human polymorphonuclear leucocytes stimulated by Escherichia coli was measured using a liquid scintillation counter equipped with a multichannel analyser. In the presence of the amplifying agent luminol, light emission can be divided into two channels, one of which ('high energy') appears to correlate directly with phagocytic activity of the PMNL, and the other ('low energy') with the background luminol dioxygenation by the cells. Measuring in the 'high energy' window also eliminates the normal 'out of coincidence' background. The method is applicable to measuring opsonizing capacity of different sera, and responds to PMNL number, age, composition of assay medium and the integrity of the stimulating bacteria. Other bacterial strains produce a similar response, as does the artificial stimulator zymosan. Low temperature and anaerobiosis, which inhibit phagocytic killing, also suppress light emission.     

NADPH-dependent reduction of ubiquinone-1 associated with the superoxide-forming oxidase of pig polymorphonuclear leucocytes

NADPH-dependent ubiquinone-1 reductase activity was present in the phagocytic vesicles of pig polymorphonuclear leucocytes. The apparent Km-value of the reductase for NADPH was 29 microM which is similar to that of the NADPH-dependent superoxide formation. Increase of the quinone-reductase activity by increasing the concentrations of ubiquinone-1 was associated with the decrease of the superoxide forming activity, the rate of the NADPH oxidation being constant independent of the quinone concentration. p-Chloromercuribenzoate inhibited both superoxide formation and reduction of the quinone, whereas low concentrations of cetyltrimethylammonium bromide which inhibit the superoxide formation did not inhibit the reduction of the quinone. The reduction of 2,6-dichlorophenolindophenol which has been shown not to be inhibited by both inhibitors. The quinone-reductase activity could be extracted with a mixture of deoxycholate and Tween 20 which extracts the superoxide forming activity. The observations indicate that a region of the superoxide-forming NADPH oxidase between a mercurial-sensitive site and a site sensitive to the cationic detergent is responsible for the reduction of ubiquinone.     

Effect of pH on In Vitro Phagocytosis of Streptococcus pyogenes

Phagocytosis experiments were performed with mouse peritoneal leucocytes (MPL). The natural pH of the mouse peritoneal cavity was found to be between 6.1 and 6.3. The phagocytic and intracellular killing activities of MPL by pH variations was studied. It was observed that the optimal ingestion and intracellular killing of bacteria is at the natural pH of the peritoneal cavity.     

Phagocytosis by catfish neutrophils

Channel catfish peripheral blood leucocytes were separated on a Percoll gradient to establish the phagocytic function of the neutrophils. Four fractions of leucocytes were formed on the Percoll gradient, including a fraction that contained 50–80% neutrophils at a density of 1.08–1.09 g ml⁻¹ and a fraction that contained 10% monocytes at a density of 1.071–1.074 g ml⁻¹. Phagocytic assays, using ³H-uridine, showed that the two fractions had similar phagocytic indices, although neutrophils were less phagocytic than monocytes. Neutrophils were confirmed to be phagocytic when examined with transmission electron microscopy. Staining with 3,3-diaminobenzidine-tetrahydrochloride demonstrated peroxidase-positive granules in the cytoplasm of actively phagocytic cells as well as peroxidase reaction products in a number of phagosomes containing bacteria. Phagocytosis of bacteria by channel catfish neutrophils was further confirmed by differential staining of external bacteria and cell surfaces with ruthenium red during the fixation process.     

The intracellular lifestyle of Francisella noatunensis in Atlantic cod (Gadus morhua L.) leucocytes

Francisella noatunensis causes the systemic granulomatous inflammatory disease, francisellosis in cod. Little is known about the lifestyle of this facultative intracellular bacterium within cod leucocytes. We have examined the interaction of this bacterium with phagocytic cells isolated from cod with emphasis on monocytes, macrophages, neutrophils and phagocytic B-cells. It is clear from confocal microscopy sections through adherent cell preparations that numerous bacteria were located intracellularly following in vitro infection in monocytes and macrophages. In these sections bacteria were immunostained and cell actin was stained using Alexa Fluor^® 488 phalloidin. Bacteria were observed in close association with neutrophils and intracellularly (low numbers) in B-cells. Bacteria were observed more frequently in head kidney- than in peripheral blood- and spleen- leucocytes. Following infection, bacteria were initially observed grouped together and located close to the nucleus. Later they were found spread within the cytoplasm. This indicates egression of F. noatunensis from the phagosome to the cytoplasm where replication possibly takes place. It may be hypothesised that the bacteria may alter maturation of the phagosome and thus, avoid the potent intracellular killing mechanisms of phagocytic cells. The intracellular lifestyle involving escape to cytoplasm prior to fusion with the lysosome may have consequences for vaccine development as well as antibiotic treatment of infected cod.     

Lysosomal membrane stabilization by antiinflammatory drugs

Lysosomal enzymes are important mediators of acute and chronic inflammatory diseases. The release of lysosomal enzymes into cytoplasm stimulate the inflammatory mediators like oxygen radicals, prostaglandins etc. Enfenamic acid, a fenamate, along with other antiinflammatory drugs, did not stabilize lysosomal membrane isolated from normal and activated phagocytic polymorphonuclear leucocytes of different species.     

Functional-metabolic activity of leukocytes and correction of it by lysozyme in acute cold stress]

It was shown that immersion cooling caused deminished phagocytes number and and phagocytes index of polymorphonuclear leucocytes in blood, decreased NCT test data, decreased NADPH-oxidase activity, increased activity of Ca(2+)-ATPase in the cells. Lysozyme corrected the above mentioned parameters of polymorphonuclear leucocytes. Lysozyme activity was mediated by cytokins of glass-spleen cells.     

Histochemical detection of ubiquinone in neutrophil polymorphonuclear leucocyte granules

Neutrophil polymorphonuclear leucocytes contain a special electron transport chain which is involved in the killing of bacteria in these cells. Identified components of the chain include NADPH, a flavoprotein dehydrogenase and an unusual cytochrome b, but there has been recent disagreement in the biochemical literature as to whether or not an ubiquinone is also present. This study has looked at this question by using an independent histochemical technique for ubiquinones. The results indicate that an ubiquinone is found in association with neutrophil granules, and hence may be implicated in the radical generating system.     

The determination and localization of sialic acid in guinea-pig granulocytes.

When intact guinea-pig granulocytes (polymorphonuclear leucocytes) disrupted by sonication or with detergent were treated with neuraminidase from Vibrio cholerae, 3.1--3.2 nmol of sialic acid/10(7) cells was released. By using a chromatographic procedure for the specific determination of total cell sialic acid, this releasable portion was found to constitute 70% of the total sialate. All of the neuraminidase-releasable sialic acid of the cells could be removed by enzymic treatment of intact cells with neuraminidase. It thus seemed likely that the neuraminidase-releasable sialic acid is all on the cell surface. To make sure that the result was not due to entry of neuraminidase into the cells, the enzyme was bound covalently to Sepharose 6B, and intact polymorphonuclear leucocytes were treated with the bound enzyme. All of the neuraminidase-releasable sialic acid could still be removed, though more slowly. The cells remained intact and only 1.5--2% of the bound enzyme was released from the Sepharose during incubation. Freed enzyme could have been responsible, at the very most, for release of 18% of the sialic acid. Fractionation studies showed that the nucleus and cytoplasm contain low amounts of sialic acid and that the neuraminidase-releasable sialic acid distributes in a manner similar to the distribution of 5'-nucleotidase, an unambiguous marker for the plasma membrane in these cells. Thus neuraminidase-releasable sialate constitutes a clear marker for the membrane of polymorphonuclear leucocytes. Most of the neuraminidase-insensitive sialate was present in the granule fraction. Removal of sialic acid from intact polymorphonuclear leucocytes did not affect their ecto-AMPase, -ATPase and -p-nitrophenyl phosphatase activities.     

Interaction of Helicobacter pylori with professional phagocytes: role of the cag pathogenicity island and translocation, phosphorylation and processing of CagA

Chronic infection of the human gastric mucosa with Helicobacter pylori is a major cause of gastroduodenal pathologies, including peptic ulcerations, mucosa-associated lymphoid tissue (MALT) lymphoma and adenocarcinoma. Helicobacter pylori strains carrying the cag pathogenicity island, which encodes an active type IV protein secretion system ( cag ⁺ or type I strains), are preferentially associated with strong gastric inflammation and severe disease. We show here that cag ⁺ H. pylori strains use the type IV secretion system to inject the bacterial protein CagA into various types of professional phagocytes, including human polymorphonuclear leucocytes (PMNs) and the human and murine macrophage cell lines THP-1 and J774A.1 CagA is rapidly tyrosine phosphorylated and proteolytically processed to generate a stable 35–45 kDa C-terminally tyrosine-phosphorylated protein fragment. H. pylori was efficiently ingested by the different types of phagocytic cells. A chromosomal deletion of the complete pathogenicity island had no significant effect on the rate of ingestion. Furthermore, the survival rate of H. pylori in the phagosome was unchanged between the wild type and a deletion mutant lacking the type IV secretion system. Thus, the type IV secretion system seems to be involved neither in active phagocytosis resistance nor in prolonged survival of the bacteria in phagocytic cells.     

Studies on the NADPH oxidizing activity in polymorphonuclear leucocytes: The mode of association with the granule membrane the relationship to myeloperoxidase and the interference of hemoglobin with NADPH oxidase determination

Phospholipase C-treated polymorphonuclear leucocytes were used to study the properties of NADPH oxidase activity of stimulated polymorphonuclear leucocytes.A comparison of the effects of phospholipase C treatment of whole leucocytes on the NADPH oxidase activity with other granule enzymes showed that the activities of β-glucuronidase and acid phosphatase were un-affected, whereas the NADPH oxidase activity was stimulated 4-fold and myeloperoxidase was inhibited about 30%.The distribution of NADPH oxidase activity among subcellular fractions of polymorphonuclear leucocyte homogenates was unaffected by phospholipase C whereas the other enzymes were released into the medium in soluble form; β-glucuronidase > acid phosphatase and myeloperoxidase.A number of solubilizing agents and procedures were tested for their ability to release NADPH oxidase activity from granules of phospholipase C-stimulated polymorphonuclear leucocytes. All procedures used caused appreciable release of granule protein but no release of NADPH oxidase activity. Most of the procedures used strongly inhibited the oxidase activity. These results indicate that the enzyme is tightly bound to granule structures and that the integrity of these structures is required for activity.Some of the solubilizing agents used (KCI, guanidium chloride) were very effective in solubilizing myeloperoxidase.The differential response of myeloperoxidase and NADPH oxidase to treatment with phospholipase C or solubilizing procedures suggests that the two activities are not due to the same enzyme. However, definite conclusion cannot be drawn because of the complex nature of myeloperoxidase.It was found necessary to lyse any erythrocytes present as contaminants of polymorphonuclear leucocytes preparations, since hemoglobin was converted to methemoglobin during the NADPH oxidase assay and methemoglobin exhibits appreciable NADPH oxidase activity.     

Tripeptide, a fibronectin fragment, increases ingestion capacity of phagocytes

It has been found out that tripeptide Arg-Gly-Asp being under natural conditions a fibronectin fragment, responsible for adhesion, is capable in vitro to stimulate ingestion ability of the rat blood polymorphonuclear leucocytes and monocytes. Maximum increase of phagocytes index, phagocytes number and phagocyte per cent, which has ingested more than 10 latex particles, has been discovered after leuko-suspension incubation with tripeptide in initial concentrations 10(-3) mM and 10(-2) mM. Tripeptide can be regarded as a perspective phagocyte function regulator.     

The effect of bombesin-related peptides on the phagocytic function of mouse phagocytes in vitro

Bombesin (BBS) at doses of 0.1, 1.0, 10.0 and 100.0 nM stimulated chemiluminescence (CL) production by phagocytic cells (monocytes, macrophages and polymorphonuclear leucocytes) in mice in the presence of ZAP (opsonized zymosan particles containing luminol). These data suggest that BBS increased the phagocytic function of mouse phagocytes. BBS-related peptides, gastrin-releasing peptides (GRP)-27, GRP-14, GRP-10 and neuromedin B, also induced similar CL responses compared with BBS. The CL response elicited by BBS was depressed dramatically by various concentrations of EGTA (a Ca++ chelator), indicating that a Ca++ pathway may play a key role in the BBS-stimulated CL response.     

The professional phagocytes of sea bass (Dicentrarchus labrax L.): cytochemical characterisation of neutrophils and macrophages in the normal and inflamed peritoneal cavity

In order to identify the phagocytic cells of sea bass, the peritoneal leucocyte population of fish injected intraperitoneally with Photobacterium damselae subspecies piscicida was studied by light microscopy using cytocentrifuge preparations stained by the Antonow technique for peroxidase detection. Among the leucocytes present in the peritoneal exudate of the infected fish (macrophages, neutrophils, eosinophilic granular cells, lymphocytes and thrombocytes), macrophages and neutrophils were the only phagocytic cells. Neutrophils were easily distinguished from macrophages in Antonow stained preparations by the pattern of peroxidase positivity. Using ultrastructural cytochemistry, neutrophils were found to have abundant cytoplasmic granules positive for peroxidase and arylsulphatase and were negative for alpha-naphthyl butyrate (ANB) esterase. In contrast, ANB esterase activity was detected in macrophages. These leucocytes were typically negative for peroxidase, but ocasionally, some macrophages with peroxidase or arylsulphatase-positive vacuoles were observed. Both phagocytes had cytoplasmic granules positive for acid phosphatase. Glycogen particles were found in the cytoplasm of the two phagocytic cells, but they were much more abundant in neutrophils. Macrophages were much more abundant than neutrophils in the peritoneal cavity of non-injected sea bass but early after the intraperitoneal injection of bacteria, the number of neutrophils increased quickly and extensively. Higher numbers of intraperitoneally injected bacteria were found inside macrophages as compared to neutrophils because macrophages strongly predominated in the peritoneal population at the time of injection. However, when the bacteria were injected into peritoneal cavities with high numbers of neutrophils (attracted by a previous injection of 12% casein), the percentage of neutrophils with phagocytosed bacteria increased, approaching that of infected macrophages. Taken together, these results show that in sea bass, as in many other organisms, in addition to macrophages, neutrophils are important phagocytic cells, the relative participation of each of the two phagocytes in defense mechanisms against infection depending on the opportunity to encounter the invading infectious agents.     

Effect of indomethacin on selected immune parameters in different age groups of mice.

The authors studied the effect of indomethacin on the phagocytic activity of polymorphonuclear leucocytes (PMNL) and on haemolytic antibody formation (plaques) by lymphoid cells of the spleen in 3-, 6- and 12-month-old mice. In 3- and 12-month-old animals the phagocytic activity of the PMNL was significantly inhibited. Plaque formation was likewise significantly inhibited in 3-month-old mice, but it was significantly raised in 6- and 12-month-old animals.     

Phagocytic capacity of human leukocytes preincubated with Imuran and prednisone

The phagocytic and microbicidal activities of human polymorphonuclear leukocytes (PMNL) were tested after a short-term preincubation of phagocytic cells with Imuran or Prednisone, using C. albicans strains as a test organism. The tests showed that both immunosuppressive agents reduced significantly the microbicidal activity of PMNL cells by 35-40%. Detectable differences in the phagocytic activity and phagocytic index values were not statistically significant. The decreased ability of PMNLs to kill ingested C. albicans strains appears thus to sensitively reflect the altered PMNL function and can be utilized in the biologic monitoring of immunosuppression. The reduced microbicidal potential of phagocytic cells may be also one of the causes of recurrent microbial infections that are so frequent in immunosuppressant-treated patients.