Neutrophil phagocytic activity of the peripheral blood in experimental keratoconjunctivitis in a sensitized macroorganism

The study of the characteristics of the phagocytic activity of peripheral blood neutrophils (the activity and intensity of phagocytosis, the index of its completeness) in the sensitized organism in experimental keratoconjunctivitis caused by Staphylococcus aureus and Escherichia coli has revealed a decrease in the phagocytic function of neutrophils. Still more pronounced suppression of the ingestive and digestive activity of leukocytes has been observed in cases of the combined action of bacterial allergens and benzylpenicillin potassium, which probably accounts for the ineffectiveness of the penicillin treatment of bacterial keratoconjunctivitis.     

Phagocytic and bactericidal activities of leukocytes in whole blood from atomic bomb survivors

This study evaluated the phagocytic and bactericidal activities of peripheral blood leukocytes from Hiroshima and Nagasaki atomic bomb survivors for Staphylococcus aureus. The data were analyzed by multiple linear regression for age, sex, radiation exposure, city of exposure, and neutrophil counts. No significant radiation effect was observed for either blood phagocytic or bactericidal activities. The only significant variable for these functions was the neutrophil count.     

In situ lymphophagocytosis by nonlymphoreticular neoplasms

Lymphophagocytosis by nonlymphoreticular neoplasms has been observed. Twenty cancer patients having either adenocarcinoma or epidermoid carcinoma were the subjects of our study. Malignant cells were found to phagocytize autologous lymphocytes, polymorphonuclear cells (PMNs) and red blood cells (RBCs). Papanicolaou smears from cancer patients revealed that 30% (6/20) of the patients had phagocytic malignant cells in situ, the mean phagocytosis for this group was 3.7%. Different cellular elements showed differences in their susceptibility towards phagocytic activity of tumor cells: 2.6% for lymphocytes, 0.7% for PMNs and 0.4% for RBCs. The patients having phagocytic malignant cells showed complete absence of monocytes in their peripheral blood. In contrast, other patients had peripheral blood monocyte percentages within the limits of control values (7.2). However, there was no correlation between development of phagocytic activity of malignant cells and age and sex of patients or type of tumor. This study further confirms that phagocytic activity of nonlymphoreticular neoplasms is a general phenomenon which can be directed against erythroid elements. In addition, we have demonstrated that these tumor cells also phagocytize autologous leukocytes.     

Delayed hypersensitivity and nonspecific cellular immunity. The role of mono- and polynuclear phagocytes

Differences in the influence produced by sensitization with BCG vaccine and Staphylococcus aureus and by the reaction of delayed hypersensitivity (DH) induced, respectively, by the injection of old tuberculin and staphylococcal phagolysate on the phagocytic activity of peritoneal macrophages and blood leukocytes in different animals were experimentally demonstrated. A considerable activation of the bactericidal and ingesting functions of macrophages was observed in animals showing a pronounced DH reaction (rabbits, guinea pigs and mice), while in Wistar rats no such activation was noted. The latter showed no DH reaction after sensitization with BCG vaccine and the injection of the specific antigen. Among different strains of mice, the activation of macrophages occurred in the animals with the most pronounced DH reaction. Sensitization with BCG vaccine led to an insignificant sensitization of macrophages, and sensitization with S. aureus even suppressed the phagocytic activity of macrophages. The treatment of mice with antimacrophagal preparations (carrageenan, silica and trypan blue, but T-lymphocyte antiserum) before and after the injection of the specific antigen into the sensitized animals abolished the stimulation of anti-infection immunity.     

Enhanced killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes in presence of subinhibitory concentrations of carbenicillin and ticarcillin

The effect of carbenicillin and ticarcillin on the killing of Pseudomonas aeruginosa was studied with an in vitro system using peripheral blood polymorphonuclear (PMN) leukocytes collected from human donors. No corticosteroid was given to the donor prior to leukocytes collection by a continuous flow cell separator. The assay was carried out with or without serum. P. aeruginosa yield after a 4 hour-incubation was estimated by colony counting. In Hanks' balanced salt solution, P. aeruginosa strains 74 and 78 were resistant to human PMN leukocytes. The presence of subinhibitory concentrations of carbenicillin or ticarcillin (1/10th the minimal inhibitory concentration (MIC) for P. aeruginosa 74, 1/4th the MIC for P. aeruginosa 78) enhanced the bactericidal activity of human leukocytes. Difference between the numbers of bacteria recovered with PMN cells and without cells increased with concentration of carbenicillin or ticarcillin. The synergistic effect was not observed when serum (heated fetal calf serum or heated pooled human serum) was used. The mode of action of carbenicillin and ticarcillin on bactericidal activity of phagocytic cells was not elucidated, but we suggest the effect is due not to action on the phagocytic cells themselves but on the microorganisms.     

Phagocytosis of bacteria by human leukocytes measured by flow cytometry

A new method has been developed for the evaluation of the phagocytic activity of human leukocytes using fluorescently labeled bacteria and flow cytometry. By simultaneous measurement of cellular light scatter and fluorescence, extracellular bacteria, phagocytes, and nonphagocytes could be discriminated and quantified. All leukocytes assumed to be capable of phagocytosis were phagocytosing, and about 90% of these cells were polymorphonuclear neutrophilic granulocytes. Within 15 min 85% of the bacteria were phagocytosed and each phagocyte contained an average of 15-20 bacteria. The phagocytic capacity of the leukocytes from healthy individuals showed minor interindividual and day-to-day variations. This method facilitates a rapid and accurate in vitro evaluation of the phagocytic activity of human leukocytes.     

Olfactory stress and modification of phagocytosis in peripheral blood cells of adult male mice]

Data on pheromonal influence on phagocytic activity of leukocytes in peripheral blood of adult randombred and CBA male mice have been obtained. The identified mouse pheromone 2,5-dimethylpyrazine was used, which induces some physiological effects associated with reproduction in both mouse males and females. Significant differences in spontaneous level of phagocytosis were between inbred CBA and randombred animals: the frequency of phagocytic cells was lower in CBA males by 1.4 times. The substance tested here induces phagocytosis in randombred (by 1.7 times) males. A low dose of 2,5-dimethylpyrazine (similar to the natural pheromone concentration) induces a higher increase in phagocytic activity by leukocytes. Possible mechanisms of pheromonal action on phagocytosis are discussed with the perspectives of finding highly effective immunomodulators among mammalian pheromones.     

Changes in the functional activity of neutrophils during their interaction with Yersinia pestis in vitro

The functional state and electrochemical properties of human blood neutrophil leukocytes after their in vitro interaction with Y. pestis cells, strain EV, was analyzed. A considerable decrease in the electrophoretic mobility of neutrophil leukocytes and a considerable increase in their phagocytic indices was shown. At the same time the maximum phagocytic activity in the total pool of isolated neutrophil leukocytes was registered in fractions having higher electrophoretic mobility. The dependence of electrophoretic mobility on the state of neutrophil membranes, as well as the degree of their activation, is discussed.     

Flow cytometric evaluation of leukocyte function in rat whole blood

The aim of this study was to establish a standard flow cytometric method to measure the phagocytic function of and intracellular hydrogen peroxide (H2O2) production by rat leukocytes. Thirty-six adult, male Sprague-Dawley rats were included in this study. Whole-blood specimens from the inferior vena cava were collected in a heparinized tube and ethylenediaminetetraacetic acid (EDTA) anticoagulated tube. The phagocytic function of and intracellular H2O2 generation by leukocytes were measured with FACS Vantage trade mark flow cytometer (Becton Dickinson, San Jose, CA), using fluorescent microspheres and dihydrorhodamine-123 as probes, respectively. Several conditions were optimized in this study, including anticoagulants (heparin and EDTA), fluorescent probes (0.75- and 1.72-microm-diameter microspheres), incubation time, and concentration of the chemicals used in the experiment. Neutrophils, monocytes, and lymphocytes could be clearly defined and separated in whole blood by flow cytometry and tested for phagocytosis and intracellular H2O2 generation without the need for further purification and handling of the cells. Intracellular H2O2 production by and phagocytic function of neutrophils and monocytes were inhibited in EDTA-anticoagulated blood compared with heparin- anticoagulated blood (P < 0.01). Neutrophils showed similar phagocytic function to 0.75- and 1.72-microm microspheres, but monocytes showed weak phagocytic activity to 1.72-microm beads compared with 0.75-microm beads (P < 0.01). In conclusion, a flow cytometric method to measure the phagocytic function of and intracellular H2O2 production by rat leukocytes has been developed. Quantitative flow cytometric analysis of rat leukocyte function is convenient and feasible and provides a reliable and rapid assay to assess phagocytosis and intracellular H2O2 production by rat neutrophils and monocytes.     

Metabolic activity of phagocytes in experimental sporotrichosis

Phagocytosis plays an important role as a protective mechanism against infections, since polymorphonuclear leukocytes (PMN) and macrophages are the first cellular lines opposed to agressive microorganisms. In patients with sporotrichosis a diminished capability of killing engulfed yeast by their PMN has been described, but the origin of this deficiency remains unknown.In this work, partial aspects of the oxidative metabolism of PMN leukocytes and peritoneal macrophages of mongolian gerbils experimentally infected with sporotrichosis were studied. For this purpose the nitroblue tetrazolium (NBT) test as described by Baehner and Nathan (1) and myeloperoxidase activity measured according to Kaplow's method were utilized.The PMN and macrophages of mongolian gerbils infected with sporotrichosis showed increased reduction of NBT when compared with the phagocytic cells of normal ones, as is usually observed in most infections. Myeloperoxidase activity was diminished in both PMN and macrophages, but this diminution was statistically significant only in PMN leukocytes. These results show that part of the oxidative mechanisms of phagocytic cells can be impaired in experimental sporotrichosis, and could be correlated with the diminished fungicidal activity of PMN leukocytes obtained from patients infected with sporotrichosis.     

Circadian variation of the phagocytic activity of polymorphonuclear leukocytes and of various other parameters in 13 healthy male adults.

Twenty-four different laboratory parameters including the phagocytic activity (phagocytic index) of polymorphonuclear leukocytes (PMNs) and various hematologic variables were investigated in 13 young healthy men during Spring 1988 in Munich, Germany. Venous blood of these volunteers was obtained under standardized conditions at 4-h intervals over a 24-h span. All parameters were analyzed by the single cosinor method and by a Kruskal-Wallis analysis of variance (ANOVA). Statistically significant circadian rhythms were found for the number of circulating lymphocytes and leukocytes (WBCs), potassium, systolic blood pressure, phagocytic index, Quick test, heart rate, and rectal body temperature (p less than 0.05; single cosinor). For all of these parameters except WBCs, rectal body temperature, and Quick test, a temporal variation was confirmed by the ANOVA (p less than 0.05; phagocytic index: p = 0.05). The circadian acrophases of WBC, number of circulating lymphocytes, and phagocytic index were all found at about 01:00 h. This temporal coincidence of the acrophases of the phagocytic index and the number of circulating lymphocytes may reflect the modulation of phagocytosis by T lymphocytes that release cytokines known to stimulate the phagocytic activity of PMNs.     

Adherent cells (macrophages?) in tumor-bearing mice suppress MLC responses

The spleens of mice bearing transplanted methylcholanthrene (MCA)-induced fibrosarcomas (MCA-1425 and MCA-1460) were shown to contain cells capable of suppressing the generation of cytolytic T lymphocytes (CTL) in mixed leukocyte cultures (MLC). The suppressive activity was first detected 21 days after tumor transplantation. No suppression was seen with lymph node cells taken at the same time as the spleen cells. The cells responsible for the suppressive activity were adherent to nylon wool and plastic dishes and they were not lysed by anti-T-cell serum plus complement. The suppressor cells were phagocytic and were resistant to irradiation (3000 rads) in vitro. Spleen cells from tumor-bearing nude mice were as suppressive as were spleen cells from tumor-bearing conventional mice. We conclude from these findings that T cells were not involved either as inducers or as effectors of the suppression observed, although the responsible adherent cells may have exerted their effect by interacting with a T-suppressor cell population in the MLC mixtures. While spleen cells of tumor-bearing mice were suppressive when added at any time during the first 4 days of a 5-day MLC, they showed no effect on the cytotoxicity of fully differentiated CTL. Indomethacin reversed suppression, suggesting that prostaglandins may have been involved.     

Effect of cefoperazone on the activity of selected parameters of cellular immunity in mice with experimental viral-bacterial infection

We applied mixed, viral-bacterial infections of mice (with influenza virus to the respiratory tract and Staphylococcus aureus) intereperitoneally. We used Sodium Cefoperazone subcutaneously in the dose of 30 mg/kg body weight 24, 48 and 72 hours after the infection. We evaluated phagocytic activity of the granulocytes isolated from animals and bactericidal activity of these cells expressed as their chemiluminescent activity. We studied it on the 3rd, 6th, 9th, and the 14th day after the infection. Phagocytic activity of cells taken from infected mice and treated with the preparation expressed as phagocytic index was the following: 0.38, 0.19; 0.88; 0.99 respectively. In the comparative studies, concerning the effect of preincubation of cells with antibiotic (at the concentration produced in blood serum by therapeutic doses) we found the increase in chemiluminescent process by 57% on the average. Analyzing the preliminary data form the experimental studies on the influence of Cefoperazone on mixed, viral-bacterial infections in mice we found the positive effect of the antibiotic evaluated in some immunological test. Intracellular killing of bacteria is stimulated. Preincubation of granulocytes with the antibiotic gives higher chemiluminescent activity of cells. However, chemotactic and phagocytic activity of cells are not changed.     

Phagocytosis of bacteria by polymorphonuclear leukocytes suspended in liquid or adhering to a surface

Phagocytosis of Staphylococcus aureus by polymorphonuclear leukocytes (PNL) was studied in healthy men. PNL suspended in nutrient medium did not practically ingest bacteria. The intake of bacteria got considerably intensified if leukocytes and bacteria ran into each other by turning over the test tubes during incubation. A still greater rise of phagocytic activity was discovered under the conditions favouring the attachment of PNL to the surface and the possibility of chemotaxis. These conditions were created by introducing a gelatin-coated film into the test tube.     

Altered hematological and immunological parameters in silver catfish (Rhamdia quelen) following short term exposure to sublethal concentration of glyphosate

Using agrichemicals to control unwanted species has become a necessary and common worldwide practice to improve crop production. Although most currently used agrichemicals are considered relatively safe, continuous usage contributes for soil and water contamination and collateral toxic effects on aquatic species. Few studies correlated the presence of agrichemicals on fish blood cells and natural immune system. Thus, in this study, silver catfish (Rhamdia quelen) were exposed to sublethal concentrations (10% of the LC(50-96 h)) of a glyphosate based herbicide and hematological and natural immune system parameters were evaluated. Silver catfish fingerlings exposed to glyphosate for 96 h had a significant reduction on blood erythrocytes, thrombocytes, lymphocytes and total leukocytes in contrast to a significant increase in the number of immature circulating cells. The effect of glyphosate on natural immune system was evaluated after 24h or 10 days exposure by measuring the phagocytic index of coelomic cells, and lysozyme, total peroxidase, bacteria agglutination, bactericidal activity and natural complement hemolytic activity in the serum of fingerlings. A significant reduction on phagocytic index, serum bacteria agglutination and total peroxidase was observed only after 24h exposure to glyphosate. In contrast, fingerlings exposed to glyphosate for 10 days had a significant lower serum bacteria agglutination and lysozyme activity. Glyphosate had no effect on serum bactericidal and complement natural hemolytic activity after 24h or 10 days exposure. Nonetheless, the information obtained in this study indicates that glyphosate contaminated water contributes to alter blood cells parameters and to reduce the activity of natural immune components important to mediate fish resistance to infecting microorganisms.     

Determination of the bacteriostatic capacity of neutrophils and monocytes in mixed cell populations.

Cline [1, 2] described a method of determining the phagocytic and bacteriostatic activity of individual types of leukocytes within mixed cell populations. We tried to improve the applicability of this method for the investigation of clinical problems.--Bacteria in the log-phase of growth were incubated in test tubes with leukocytes separated from venous blood. After a short period of phagocytosis 3H-thymidine was added to label DNA-synthesizing organisms. Smears were prepared and processed by autoradiography. The labeling indices of extracellular bacteria and of those phagocytized by neutrophils and monocytes were determined microscopically. The intracellular inhibition of DNA-synthesis was taken as indicative of the bacteriostatic activity of the leukocytes. The proposed modification of Cline's assay is suited to investigate clinical problems of phagocyte dysfunction.     

Stimulation of phagocytosis and phagosome—lysosome (P-L) fusion of human polymorphonuclear leukocytes by sulfatide (galactosylceramide-3-sulfate)

Abstract Recently, extensive attention has been paid to the physiological function of glycosphingolipids (GSLs) of mammalian cell membranes. Among a variety of GSLs, sulfatide (galactosylceramide-3-sulfate) has been proposed to be a specific receptor or binding molecule to microorganisms. However, no report has appeared on the direct stimulation by sulfatide for cellular function differentiation in phagocytic cells. We found that sulfatide showed a marked stimulation for phagocytic processes of human peripheral polymorphonuclear leukocytes (PMN) using heat-killed cells of Staphylococcus aureus coated with isolated lipid. Among mammalian acidic GSLs, sulfatide showed the highest stimulative activity for adhesion, phagocytosis and phagosome—lysosome (P-L) fusion by PMN. On the other hand, neutral GSLs did not stimulate essentially. Relative phagocytic rate of sulfatide-coated staphylococci was six times higher than that of the non-coated control and P-L fusion rate was ten times at maximum, respectively. Although the promotion mechanism of sulfatide for such phagocytosis or P-L fusion is not clear, it was strongly suggested that the existence of negative charges on carbohydrate moiety may be essential for the induction of differentiation of phagocytic cell function via signal transduction systems.     

Isolation, cell culture and immunocytochemical characterization of oviduct epithelial cells of the cow

Incubation of cow oviducts flushed with 0.1 mg collagenase/ml, for 90 min helped to dislodge large numbers of ciliated and secretory cells. About 90-95% of the isolated epithelial cells were viable. The epithelial cells suspended in DMEM:F-12 + 10% serum attached to the plastic culture dish in 18-20 h after seeding. The ciliated cells which attached to the plastic dish lost their cilia after 4-5 days in culture. The attached cells, which proliferated to form a confluent monolayer 8-10 days after seeding in a 35-mm dish, could be subcultured at least 3 successive times. Some cell aggregates which did not attach to the culture dish proliferated into floating balls of cells. The ciliated cells in the unattached floating colonies maintained the ciliary movement for 9-10 days in the same culture medium. The primary cultures of the ciliated and the secretory cells maintained most of the histoarchitecture observed in intact epithelium. The secretory cells maintained their secretory activity of specific proteins in culture as indicated by immunocytology. The cultured cells contained keratin, a specific cytoskeletal component of epithelial cells.     

Influence of a muramyl dipeptide on human blood leukocyte functions and their membrane antigens.

Murabutide, which belongs to the immunomodulator family of muramyl peptides, was applied directly to fresh human blood to evaluate changes in leukocyte properties. After blood incubation with murabutide, lymphocytes presented a higher responsiveness to T-mitogens, and monocytes and polymorphonuclear cells exhibited an increase in their capacity to produce hydrogen peroxide. In addition, murabutide treatment enhanced phagocytic activity of neutrophils, whereas monocytes presented a decrease in this activity. Some surface markers were also investigated in the distinct leukocyte populations. After incubation with murabutide, a larger number of lymphocytes expressed Ta1 antigen (CD W26) and transferrin receptor (CD 71). In contrast, expression of interleukin-2 receptor (CD 25) was slightly decreased. Monocytes from treated blood displayed a larger number of receptors for C3bi (CD 11b), whereas the surface marker CD 14 and the class I receptor for the Fc portion of IgG were down-regulated. Activation of polymorphonuclear cells by murabutide was confirmed by the up-regulation of the C3bi receptor, Fc receptor, and CD 14 surface antigen. The effects of murabutide on leukocytes described in this paper may contribute to understanding mechanisms of the modulating activity of muramyl peptides on specific and nonspecific immunity.     

Phagocytic capacity of Kupffer cells during hepatic amoebiasis in guinea pigs.

Very few studies have been carried out on the role of liver macrophages (Kupffer cells) during the course of hepatic amoebiasis. The kinetics of phagocytic activity of Kupffer cells and blood monocytes was studied in guinea pigs intra-mesenterically infested with Entamoeba histolytica. The phagocytic capacity of blood monocytes of normal animals was comparatively lower than Kupffer cells for both latex and haemolysin coated sheep red blood cells. Significant decline in phagocytic response of Kupffer cells and blood monocytes of infected animals was observed right from 2nd post infection day and it kept on decreasing with the progress of infection. Depression in phagocytic response of Kupffer cells and blood monocytes was more marked in those animals who had higher grades of pathological lesions. Hence, an inverse correlation was obtained between the phagocytic capacity and severity of amoebic lesions (P less than 0.01). The significance of depression in phagocytic response of Kupffer cells and blood monocytes may be responsible for the development of hepatic lesions.