Phagocytic and nitroblue tetrazolium reductive properties of mature and immature neutrophils and eosinophils from blood and bone marrow from cows

Functional capabilities of morphologically mature (segmented) and immature granulocytes (neutrophils and eosinophils) from bone marrow from cows were studied and compared with similar activities of segmented granulocytes from blood. Phagocytosis of Escherichia coli and postphagocytic oxidative metabolic stimulation, measured by nitroblue tetrazolium (NBT) reduction, were evaluated simultaneously. Phagocytosis was observed readily in segmented neutrophils, neutrophilic bands, and metamyelocytes and rarely in myelocytes. Phagocytosis was not seen in promyelocytes and myeloblasts. Neutrophilic bands and metamyelocytes were phagocytically less active than were segmented neutrophils. Washed segmented bone marrow neutrophils possessed phagocytic activity similar to that of blood neutrophils, whereas the activity of unwashed segmented bone marrow neutrophils was markedly less than that of blood neutrophils. Reduction of NBT was observed only in blood segmented neutrophils and bone marrow segmented neutrophils; the magnitude of NBT reduction was significantly (P = less than 0.005) less in bone marrow neutrophils than in blood neutrophils. Eosinophils were phagocytically less competent than were neutrophils. The NBT reduction was observed only in eosinophils from blood, but not in eosinophils from bone marrow.     

Effects of glycolytic and cytoskeletal inhibitors on phagocytic and nitroblue tetrazolium reductive activities of bovine neutrophils

Phagocytic and oxidative metabolic activities of bovine blood neutrophils were determined in the presence of glycolytic (NaF) and cytoskeletal (colchicine, cytochalasin B, and prostaglandin E1) inhibitors. Phagocytosis and post-phagocytic oxidative metabolic activity, measured by nitroblue tetrazolium reduction, were determined using zymosan, Escherichia coli, Staphylococcus aureus, or Streptococcus agalactiae. Sodium fluoride (1.25 microM to 1.25 mM concentrations) did not significantly (P greater than 0.05) inhibit phagocytosis of S aureus and Str agalactiae, whereas phagocytosis of zymosan and E coli was significantly (P less than 0.05) inhibited only at 1.25 mM concentration. Colchicine at 1.25 nM to 1.25 microM concentrations significantly inhibited phagocytosis of zymosan and E coli, but not of S aureus and Str agalactiae. Cytochalasin B at 1.25 nM to 1.25 microM concentrations significantly inhibited phagocytosis of zymosan and all 3 bacteria, whereas prostaglandin E1 was noninhibitory at similar concentrations. Nitroblue tetrazolium reduction, in general, was not significantly affected by NaF and cytoskeletal inhibitors.     

Combined phagocytosis-nitroblue tetrazolium reduction test in bovine neutrophils

An assay was developed for the simultaneous evaluation of phagocytosis and oxidative metabolism of bovine blood neutrophils. Phagocytosis was evaluated by using opsonized zymosan, and oxidative metabolism was evaluated by nitroblue tetrazolium (NBT) reduction. Normal bovine neutrophils exhibited moderate variation in ability to phagocytize zymosan, but little variation in ability to reduce NBT. The subcellular location of NBT reduction to formazan was determined by electron microscopy. Electron dense formazan precipitate was observed along the inner membrane of phagosomes enclosing zymosan particles and radiating from the membrane toward the center of the phagosome.     

Differential expression analysis of proteins from neutrophils in the periparturient period and neutrophils from dexamethasone-treated dairy cows

Neutrophils play an important role in the host immune system's defense against pathogens. It has been established that neutrophil functionality is suppressed in dairy cows at parturition. The periparturient immunosuppression seen in dairy cattle is associated with an increase in the incidence of mastitis. Using amine-reactive isobaric tagging reagents we have measured relative protein expression from normal prepartum neutrophils and neutrophils isolated during immunosuppression at parturition. We found over 40 proteins that are differentially expressed at parturition compared to prepartum. In addition, we measured relative protein expression from normal neutrophils and neutrophils obtained from cows treated with an immunosuppressive dose of dexamethasone. We found over 70 proteins are differentially expressed during dexamethasone treatment. We then compared protein expression changes in dexamethasone-induced immunosuppression to periparturient immunosuppression. A number of proteins underwent similar expression changes in both dexamethasone and periparturient immunosuppressed neutrophils. Most significantly, we found a significant number of proteins whose relative expression was not the same for these two different conditions that cause neutrophil dysfunction. The data demonstrates that there are both similarities and differences in neutrophil protein expression in the naturally occurring immunosuppression observed at parturition compared to dexamethasone-induced immunosuppression in the bovine neutrophil.     

In vitro migration responses of neutrophils from cows and calves

The directional (chemotactic) and random migration activities of neutrophils from cows and newborn and 2-week-old calves were determined by use of the chemotaxis-under-agarose assay. Blood samples were stored for 2, 24, or 48 hours and at 4 or 25 C before testing. During the assay, cells were incubated at 17, 27, or 37 C. The assay was found suitable for testing the directional and random migration activities of neutrophils from cattle. Directional migration of neutrophils was diminished (P less than or equal to 0.05) when cells were incubated at 17 or 27 C, compared with data from incubation at 37 C. Random migration of neutrophils was unaffected by test incubation temperature. Significant (P less than or equal to 0.05) differences were found between cows and calves regarding the percentage number and viability and the directional and random migration activities of neutrophils. Neutrophils from cows were adversely affected to a greater extent by prolonged sample storage times or low storage temperature than were neutrophils from calves. Results indicate that a sample storage time of up to 24 hours, a sample storage temperature of 25 C, and a test incubation temperature of 37 C provided optimal conditions for testing the migratory activities of neutrophils from cattle.     

Neutrophil, glass-adherent, nitroblue tetrazolium assay gives early indication of immunization effectiveness in rainbow trout

Neutrophil activity in rainbow trout (Oncorhynchus mykiss) is increased upon antigenic stimulation with the Yersinia ruckeri O-antigen bacterin. The characteristics of neutrophil attachment to glass and nitroblue tetrazolium (NBT) staining were used to determine the effectiveness of immunization programs with fingerling rainbow trout. Fish immunized by intraperitoneal injection with doses of 100, 10, or 1 μg of the bacterin showed the highest responses in that order in numbers of glass adherent, NBT-positive neutrophils. Studies on the kinetics of the occurrence of numbers of glass-adherent, NBT-positive staining cells from the fish injected with the 10 μg dose showed the numbers of positive cells were largest on Day 2 after injection. The specific immune response was confirmed by demonstrating the presence of plaque-forming cells by the passive hemolytic plaque assay and the rise in humoral antibody titers by passive hemagglutination 12 days after injection. The effects of immunization in trout could be detected earlier by using the neutrophil glass adherence and NBT reduction assays than by using assays based on observations of the specific immune response.     

Separation of serum glycated proteins by agarose gel electrophoresis and nitroblue tetrazolium staining in diabetic and normal cats

BACKGROUND: The total glycated protein (fructosamine) concentration in serum consists mainly of glycated albumin and lipoproteins. Measurement of fructosamine is used to diagnose and monitor diabetes mellitus in cats. OBJECTIVE: The aims of this study were to measure glycated proteins in diabetic and healthy (nondiabetic) cats using a semiquantitative technique and to determine whether measurement of any of the fractions of glycated protein could be potentially advantageous for the diagnosis and monitoring of diabetic cats. METHODS: Serum samples from 6 cats with diabetes mellitus and 10 clinically healthy adult cats were assayed for total glycated protein using a nitroblue tetrazolium (NBT) fructosamine assay. Serum proteins were separated by agarose gel electrophoresis and stained with NBT to identify individual glycated proteins within the bands. Gels were scanned by densitometry at 525 nm and the glycated protein content was calculated with reference to the total glycated protein content of the sample. RESULTS: Diabetic cats with increased total fructosamine concentrations had higher concentrations of glycated albumin and glycated alpha- and beta-lipoproteins compared with healthy cats. The concentration of glycated proteins in each of the fractions had a positive linear association with the total glycated protein content of serum, but there was large variation in the relative contributions of the 3 protein fractions to the total glycated protein concentration. CONCLUSIONS: Based on the results of this study, measurement of individual glycated fractions does not seem to offer any potential diagnostic advantage over measurement of total glycated protein (fructosamine) concentration alone. In some diabetic and healthy cats, glycated lipoproteins formed the major part of the total glycated protein, whereas in other cats albumin was the major contributor.     

Diapedesis across mammary epithelium reduces phagocytic and oxidative burst of bovine neutrophils.

The effect of diapedesis on the phagocytic and oxidative burst activity of polymorphonuclear neutrophil (PMN) was examined, using an in vitro cell culture model consisting of a monolayer of primary mammary epithelial cells. Isolated blood PMN from 10 cows were added to the basal side of the epithelial cell monolayer. Diapedesis was induced by the addition of complement factor C5a to the apical side of the monolayer. PMN phagocytosis of Staphylococcus aureus and oxidative burst were measured before diapedesis on PMN that were non-activated and activated by incubation with C5a and on PMN after diapedesis, using flow cytometry. The percentages of PMN fluorescing due to phagocytosis of S. aureus and oxidative burst were reduced by 21.2 and 14.4%, respectively, after diapedesis. Pre-incubation in the presence of C5a had no effect on percentage PMN fluorescing due to phagocytosis or oxidative burst. The capacity for individual migrated PMN to phagocytose S. aureus and to produce an oxidative burst, as measured by the intensity of fluorescence, decreased by 34.2 and 30.3%. Activation of PMN with C5a increased intensity due to the oxidative burst, but had no effect on intensity due to phagocytosis. These data show that PMN diapedesis across mammary epithelium results in decreased phagocytosis and oxidative burst of the PMN.     

Function of neutrophils and chemoattractant properties of fetal placental tissue during the last month of pregnancy in cows.

Neutrophils were isolated from the blood of pregnant cows on days 255, 265, and 275 of pregnancy, and on the day of parturition (n = 5/group), and in addition, simultaneously from 4 ovariectomized healthy cows (control animals). Neutrophils were subjected to neutrophil function assays (chemotaxis against zymosan-activated serum, random migration, ingestion of 125I-iododeoxyuridine [IdUR]-labeled Staphylococcus aureus, iodination of proteins, cytochrome C reduction, antibody-independent and antibody-dependent cell-mediated cytotoxicity). Results were expressed as percentage of control animals. Fetal placental tissue (cotyledon), uterine wall tissue, and skeletal muscle were obtained from the principal animals on the aforementioned days via laparotomy, and tissue suspensions were prepared. Chemotaxis of neutrophils was tested against tissue supernatants. Compared with day 255, there was an increase in ingestion of 125I-IdUR-S aureus at parturition, whereas iodination of proteins and cytochrome C reduction were reduced on the day of calving. The other neutrophil functions tested did not change over time of gestation. Fetal placental and uterine wall tissue attracted neutrophils with uterine wall tissue having a tendency to be more potent than cotyledonary tissue. Skeletal muscle tissue did not attract neutrophils. There was no change in chemotaxis response of neutrophils evoked by intrauterine and uterine tissues over time of gestation. It was concluded that at parturition, neutrophil function is impaired with respect to their bactericidal effects, which may render the animal more susceptible to bacterial infections, and that the chemoattractant properties of fetal placental and uterine wall tissues are tissue-specific, at least when compared with skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the tetrazolium salt reduction test in the detection of phagocytic activity in pigs

One of the indicators of non-specific, cell-mediated immunity can be the ability of phagocytic system to react to the presence of antigenic impulses and stimulators. The phagocytic activity induced in this way in vitro can be evaluated quantitatively by help of different methods. In this study the method of the reduction of colourless tetrazolium salt (INT) to the red formazane was used. The optimum conditions for carrying out this test in isolated peripheral pig leucocytes were determined. It was stated that 3-4 ml samples of pig blood, from which 6.10(6) leucocytes necessary for the test can be isolated, were sufficient for the examination of phagocytic activity. Further, 45 minutes were determined as the optimum time of incubation It was found that the INT test can be reproduced by 24 hours after blood sampling provided the blood is kept at the temperature of +4 degrees C. The INT test was used for examining 38 pig blood samples before and after myostress; this stress has a significant effect both on a decrease of phagocytic activity and on the ability of leucocytes to migrate in the LMI test. The INT test was further used for examining pigs before the administration of the first dose of the inactivated vaccine against the Aujeszky's disease virus and two days after; the application of the vaccine significantly increased the phagocytic activity.     

Glucocorticoid receptor down-regulation in neutrophils of periparturient cows

OBJECTIVE: To determine effects of parturition on glucocorticoid receptor (GR) expression in neutrophils, serum cortisol concentration, and total blood leukocyte and neutrophil counts in periparturient dairy cows. ANIMALS: 23 Holstein cows. PROCEDURE: Blood samples were collected from 8 multiparous and 5 primiparous periparturient cows at various times from 28 days before parturition until 14 days after parturition. Glucocorticoid receptor expression in neutrophils, serum cortisol concentration, and total blood leukocyte and neutrophil counts were determined. Results were compared with results from control samples obtained from 5 multiparous and 5 primiparous Holstein cows in midpregnancy. RESULTS: Neutrophils from periparturient cows had 49% reduction in GR expression at calving, compared with GR expression 2 to 4 weeks before calving, and 39% reduction, compared with neutrophils from cows in midpregnancy. Reduction in neutrophil GR expression began 1 week before calving and was most severe at calving and 24 hours after calving; a significant difference in GR expression was detected between primiparous and multiparous cows. Serum cortisol concentrations and total leukocyte and neutrophil counts were significantly increased at calving and returned to baseline values by 24 hours after calving. Significant negative correlations were detected between neutrophil GR expression and serum cortisol concentration, total leukocyte count, and neutrophil count. CONCLUSIONS AND CLINICAL RELEVANCE: Reduced GR expression in blood neutrophils of periparturient dairy cows was associated with increased serum cortisol concentrations, leukocytosis, and neutrophilia. Thus, GR down-regulation in neutrophils may be involved in periparturient neutrophil dysregulation and may cause increased susceptibility to mastitis.     

Partial characterization of phagocytic activity in neutrophils of the nine-banded armadillo Dasypus novemcinctus

Though the armadillo is important as a research model in leprosy studies, the activity of armadillo's neutrophils is an aspect of little research. The aim of this study was carried out to partially characterize the chemotaxis, endocytosis and bacteriocidal ability of the neutrophils found in the nine-banded armadillo (Dasypus novemcinctus). Results showed that the chemotactic activity of the neutrophils, evaluated by the movement of the neutrophils through a nitrocellulose membrane (5 microm) in response to a chemo-attractive substance, was greater towards the armadillo serum (5.16+/-1.35 migration index, p<0.05) than towards the formil methionyl leucil phenylalanine (fMLP, 1.43+/-0.18 migration index) or human serum (0.56+/-0.18 migration index). Regarding endocytic capacity of the neutrophils and the monocytes against Escherichia coli was evaluated by a flow cytometry and using opsonized and non-opsonized E. coli-FITC at the following incubation times: 5, 10, 20, 30 and 60 min. The largest percentage of endocytosis by the neutrophils was 92.32+/-0.12% with opsonized bacteria and 77.73+/-14.33% with non-opsonized bacteria at 10 min incubation time, while the largest percentage of endocytosis by monocytes was 89.94+/-1.40% with opsonized bacteria and 73.07+/-15.6% with non-opsonized bacteria at 20 min incubation time. Evaluation of the bacteriocidal capacity of neutrophils using the methyl-thiazol-tetrazolium salts (MTT) reduction color-measurement assay showed an 89.0+/-10% mortality rate of non-opsonized E. coli and 89.0+10% of opsonized E. coli. In conclusion, the armadillo neutrophils show a good phagocytosis and bacteriocidal activity; however, a deficiency in the migration towards the fMLP was observed. This deficiency could be a cause so that the armadillo neutrophils do not respond quickly to invading microorganism.     

Comparison of phagocytosis and chemiluminescence by blood and mammary gland neutrophils from multiparous and nulliparous cows

Neutrophils were isolated from the blood and mammary gland of 3 multiparous lactating cows and 3 nulliparous heifers. Neutrophil function was evaluated by phagocytosis and luminol-dependent chemiluminescence. Peroxidase activity was detected by use of transmission electron microscopy. Compared with that for blood neutrophils, percentage of phagocytosis was 9.6% lower for neutrophils isolated from the mammary gland of lactating cows, but this difference was not observed between neutrophils isolated from the mammary gland and from the blood heifers. Similarly, after subtraction of chemiluminescence values in the absence of zymosan, phagocytosing neutrophils from the mammary gland of lactating cows had lower chemiluminescence than did those from the blood of such cows. For heifers, however, chemiluminescent activity by phagocytosing neutrophils obtained from the mammary gland was similar to that of blood neutrophils. Chemiluminescent activity of resting neutrophils from the mammary gland of lactating cows pretreated with cytochalasin B was not inhibited, compared with that of nontreated resting neutrophils (controls). This was attributed to xanthine oxidase activity. Transmission electron microscopy of mammary gland neutrophils from lactating cows revealed peroxidase-positive material associated with milk-fat globule membranes and with phagosomes containing zymosan. Results indicated that ingestion of fat and casein by neutrophils isolated from milk caused a decrease in phagocytic and chemiluminescent activity. Also, luminol-dependent chemiluminescence was not a reliable measure of milk neutrophil function, because of interference by xanthine oxidase.     

Use of the nitroblue tetrazolium reduction test for the evaluation of Domperidone effects on the neutrophilic function of healthy dogs

The nitroblue tetrazolium reduction test (NBT) is an assay based on the activation percentage of neutrophils in peripheral blood, that has been proposed for the follow up of canine leishmaniosis owing to the narrow relationship between the molecules involved in the oxidative burst and the leishmanicidal activity of phagocytes. Domperidone is a drug used for the treatment of canine leishmaniosis having been claimed to stimulate the dogs' cell-mediated immune response. The aim of this study was to evaluate the degree and the lasting of phagocytic activation induced by a 30-day course treatment with Domperidone (0.5 mg/kg/day) in healthy dogs, by using the NBT. A statistically significant increase in the percentages of activated phagocytes was observed in the treated group during treatment, thereafter remaining high for up to one month after the end of treatment. In contrast, untreated dogs maintained the baseline percentage of activated neutrophils all along the study. It is concluded that the NBT is a useful tool for the follow up of the stimulating effects of Domperidone on the neutrophilic response of healthy dogs and that these effects persist for up to one month after treatment with this molecule.     

PAF increases phagocytic capacity and superoxide anion production in equine alveolar macrophages and blood neutrophils

Phagocytosis exerted by alveolar macrophages and neutrophils is crucial in the clearance of exogenous particles deposited in the airways. Therefore, substances that activate these phagocytes in the airways can exert important effects on the particle clearance rate. PAF, particularly, was proved to be a potent activator of several immune cells and was shown to be present in the equine lower airways in specific conditions, such as after exercise. The present study aimed to investigate if PAF is able to increase the phagocytic capacity and the production of superoxide anion in equine alveolar macrophage and blood neutrophils. The results show that PAF increased these parameters in both phagocytes even in concentrations as low as 0.1 and 1.0 nM. On that ground, the present work suggests that PAF is involved in the process of particle clearance in equine lower airways.     

The effect of estrogens on phagocytic activity of blood leukocytes in heifers and cows]

Phagocytic activity of peripheral leucocytes (PA) was measured in heifers during the luteal phase and oestrus and after administration of various doses of oestradiol, in ovariectomized heifers and cows in the early post-partum period. PA was demonstrated in 22.63 +/- 2.49% and 50.61 +/- 3.76% of phagocytes in the luteal phase and oestrus respectively (P less than 0.01, Fig. 1). The phagocytic index (PI) rose parallelly from 2.26 +/- 0.31 to 6.55 +/- 0.64 particles per cell (Fig. 2). Intramuscular administration of a single dose 3 mg of oestradiol dipropionate resulted in an increase of PA from 28.93 +/- 3.34 to 69.60 +/- 3.32 on post-treatment day 3 (P less than 0.05, Fig. 3). A nonsignificant increase of PA was observed in heifers treated with 10 mg oestradiol. Increases of PA and PI in postparturient cows, treated with various doses of oestradiol, were nonsignificant (Fig. 5, 6) owing to a wide variance of the values obtained, which might be due to individual differences in endocrine and metabolic status or to uterine bacterial contamination. The most marked of haematological changes was the increase of eosinophil counts from 317 to 525.10(6).l-1 in a group of cows treated with 10 mg oestradiol and a decrease of the lymphocyte: neutrophil ratio (Tab. I). Both endogenous and exogenous oestrogens stimulate PA of peripheral leucocytes and 3 mg of oestradiol is a sufficient dose to obtain the effect.