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.     

Recombinant bovine interferon-gamma, but not interferon-alpha, potentiates bovine neutrophil oxidative responses in vitro

In this study we have addressed the in vitro effects of recombinant bovine interferon-gamma (rBoIFN-gamma) and interferon-alpha (rBoIFN-alpha 1) on oxidative functions of bovine neutrophils. Treatment with rBoIFN-gamma, but not rBoIFN-alpha 1, enhanced the luminol-dependent chemiluminescence (LDCL) response of bovine neutrophils to both opsonized zymosan particles and phorbol myristate acetate. Pre-incubation of neutrophils for 2 h at 39 degrees C with rBoIFN-gamma resulted in a 40% increase in both LDCL and release of hydrogen peroxide by neutrophils stimulated with opsonized zymosan. This enhancement was observed at doses ranging from 0.2 to 2000 units of rBoIFN-gamma per ml. In contrast to the results observed in the LDCL and hydrogen peroxide assays, preincubation of neutrophils with rBoIFN-gamma had no effect on the levels of superoxide anion released in response to opsonized zymosan. Pre-incubation with rBoIFN-gamma increased phorbol myristate acetate (PMA)-stimulated LDCL by 30%, although it had no effect on either superoxide anion or hydrogen peroxide release in response to PMA stimulation. Neither recombinant interferon directly elicited an oxidative burst from neutrophils in the absence of zymosan or PMA stimulation.     

Assessment of bovine mammary gland macrophage oxidative burst activity in a chemiluminescence assay

A major bactericidal mechanism of neutrophils and macrophages is the generation of toxic oxygen-free radicals upon phagocytosis of microbes. Studies were conducted to assess the oxidative metabolism of bovine mammary gland macrophages. Bovine mammary gland macrophages were challenge exposed with a variety of phagocytic stimuli in an in vitro, luminol-assisted chemiluminescence assay. A measurable oxidative burst was observed when macrophages were challenge exposed with heat-aggregated bovine immunoglobulin, opsonified zymosan, and nonosponified zymosan. Addition of superoxide dismutase decreased mammary gland macrophage chemiluminescence in a dose-dependent manner. Brucella abortus, when opsonified with antiserum, lacteal antibody, or normal serum, produced an oxidative event, whereas nonopsonified B abortus did not. When challenge exposed with phagocytic stimuli, mammary gland macrophages produced an oxidative burst similar to that produced by other phagocytes for which an oxidative event is known to be bactericidal.     

Flow cytometric study of oxidative burst activity in bovine neutrophils

A flow cytometric procedure was evaluated to measure the oxidative burst activity (hydrogen peroxide formation) of bovine neutrophils. The method involves measuring the oxidation of intracellular dichlorofluorescein to fluorescent dichlorofluorescein (DCF). Phorbol myristate acetate (PMA) was used to perturb the neutrophil plasma membrane. The sources of variation introduced into the DCF assay were also examined. The sources of variation were attributable to the isolation of neutrophils from blood, variation between duplicate assays and duplicate flow cytometric determinations of oxidative product formation, variation in neutrophil oxidative product formation among cows, and the variation (over repeated daily and weekly neutrophil isolations) in neutrophil oxidative product formation. A final objective was to determine effects of dexamethasone on oxidative product formation, and whether differences existed between blood and mammary neutrophils in oxidative product formation. There was an increasing trend in the formation of DCF with increasing time of incubation and with increasing PMA concentration. Increasing the concentration of PMA decreased lag time and increased the rate of oxidative product formation. The increase in DCF formation was statistically significant up to a PMA concentration of 10 ng/ml. This concentration was considered optimal for bovine neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Opsonin-dependent stimulation of bovine neutrophil oxidative metabolism by Brucella abortus

Nonopsonized Brucella abortus and bacteria treated with fresh antiserum, heat-inactivated antiserum, or normal bovine serum were evaluated for their ability to stimulate production of superoxide anion and myeloperoxidase-mediated iodination by neutrophils from cattle. Brucella abortus opsonized with fresh antiserum or heat-inactivated antiserum stimulated production of approximately 3 nmol of O2-/10(6) neutrophils/30 min. Similarly treated bacteria also stimulated the binding of approximately 4.3 nmol of NaI/10(7) neutrophils/h to protein. Significant (P less than 0.05) production of O2- and iodination activity by neutrophils were not stimulated by nonopsonized bacteria or organisms treated with normal bovine serum. Seemingly, B abortus stimulated oxidative metabolism in bovine neutrophils; however, the stimulation was dependent on the presence of bacterial associated opsonins.     

Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species

During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. Neutrophils are among the earliest leukocytes recruited to the site of infection and contribute to host innate immune defenses through their ability to phagocytose and kill bacteria. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). Extracellular release of ROS can induce injury to host tissue as well, and aberrant release of ROS has been implicated in the pathogenesis of certain inflammatory-mediated diseases. Due to their essential role in bacterial clearance and implicated involvement in the pathogenesis of other diseases, there is much interest in the study of neutrophil-generated ROS. Several assays have been developed to measure ROS production, however, many of these have not been evaluated with bovine neutrophils. The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS, and to compare the results of assays never previously tested with bovine neutrophils to those obtained from more well-established assays frequently used with these cells. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. The assays were evaluated in the context of their abilities to detect ROS produced in response to two agonists commonly used to induce neutrophil activation, phorbol 12-myristate, 13-acetate (PMA) and opsonized zymosan. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra- and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our knowledge, this is the first study to evaluate DHE-, MCLA-, Amplex Red-, and isoluminol-based assays for the measurement of bovine neutrophil ROS, and the most comprehensive comparative study of ROS assays under similar experimental conditions.     

Evaluation of neutrophil oxidative metabolism in canine monocytic ehrlichiosis

BACKGROUND: Canine monocytic ehrlichiosis (CME) is a tick-borne disease caused by Ehrlichia canis, a rickettsia that infects the monocytes of dogs. This infection can result in a chronic and life-threatening disease. Thrombocytopenia, mild anemia, and leukopenia are the most common hematologic findings in CME. OBJECTIVE: To investigate the role of peripheral blood neutrophils in CME, an evaluation was conducted of their functional state during the acute phase of the disease in dogs experimentally infected by E canis. METHODS: Seven dogs were inoculated with E canis, and 3 remained as uninfected controls. All dogs had physical exams and hematologic tests (CBC and nitroblue tetrazolium [NBT] reduction) during a 6-week period. RESULTS: There was no difference (P > .05) in spontaneous NBT reduction results between the 2 groups of dogs throughout the 6-week period of observation. Nevertheless, when stimulated, the neutrophils showed higher activity in the infected group (P = .01) on weeks 4 and 5 after infection. CONCLUSION: Infection by E canis has no influence on neutrophil oxidative metabolism even though during the remission period of the acute phase of the disease, the neutrophils seem to be more reactive under stimulation.     

Oxygen radical production by avian leukocytes.

Oxygen radical production by heterophils of red-tailed hawks and chickens, and by neutrophils of calves, was evaluated in a chemiluminescence microassay. Leukocytes were isolated by centrifugation of blood in capillary tubes and then challenged with opsonized zymosan in the presence of luminol. Avian heterophils produced significantly fewer oxygen radicals than did bovine neutrophils.     

Flow cytometric characterization of bovine blood neutrophil phagocytosis of fluorescent bacteria and zymosan particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered.Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.     

Adherence to bovine neutrophils and suppression of neutrophil chemiluminescence by Mycoplasma bovis

The adherence of viable and heat-treated Mycoplasma bovis to bovine peripheral blood neutrophils was studied by specific immunofluorescence staining and flow cytometry. Viable and heat-treated M. bovis cells, adhered to bovine neutrophils in dose-dependent fashion within a 30 min incubation. Fluorescence quenching using crystal violet indicated that unopsonized M. bovis cells remained on the surface of bovine neutrophils without experiencing significant ingestion. The effect of M. bovis adherence on neutrophil microbicidal function was examined by measuring luminol enhanced chemiluminescence (CL). Adherent M. bovis cells did not elicit a bovine neutrophil CL response over a 75 min incubation period. M. bovis inhibited the capacity of bovine neutrophils to mount a CL response. Inhibition occurred whether viable or heat-treated M. bovis cells were used and it occurred when neutrophils were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). Inhibition of the PMA stimulated neutrophil CL response required cytadherence by M. bovis cells. These findings suggest that activation of the bovine neutrophil respiratory burst was inhibited at or distal in the pathway to the activation of protein kinase C (PKC), the site of PMA stimulation, and that it was mediated by a direct interaction between the adhering M. bovis cells and the bovine neutrophil membrane.     

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor on bovine peripheral blood neutrophil functions in vitro and in vivo

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) on bactericidal activity of bovine peripheral blood neutrophils in vitro and in vivo were studied. In in vitro experiment, bovine blood neutrophils were cultured for 9 hr in media containing 0.005, 0.05 or 0.5 microg/ml of rboGM-CSF. Neutrophils treated with rboGM-CSF showed significantly higher luminol-dependent chemiluminescence (LDCL) than control cells. In in vivo experiment, neutrophils isolated from cows injected 5.0 microg/kg of rboGM-CSF showed significantly higher Nitrobluetetrazolium (NBT) reduction value than that from control cows 24 hr post injection. Total leukocyte counts of cows injected rboGM-CSF sharply decreased 6 hr post injection and recovered to normal level 2 days post injection. Body temperature of these cows rose 6 hr post injection and back to normal level at 24 hr post injection. It was suggested that rboGM-CSF enhanced bactericidal activity of bovine neutrophils both in vitro and in vivo.     

The respiratory burst activity of bottlenose dolphin neutrophils elicited by several stimulants

To investigate the early host defense function in aquatic animals, the respiratory burst activity of bottlenose dolphin neutrophils against soluble and particulate stimulants was measured by luminol-dependent chemiluminescence assays and compared with those of bovine and human. Dolphin neutrophils generated the respiratory burst in response to phorbol 12-myristate 13-acetate (PMA), concanavalinA (ConA), heated-plasma (HP), and homologous-plasma opsonized zymosan except N-formyl-Met-Leu-Phe (fMLP). However, the respiratory burst of dolphin neutrophils stimulated by lipopolysaccharide and Staphylococcus aureus was inferior to those of bovine and human. Furthermore, DP-OZ also induced the respiratory burst of bovine and human neutrophils. In conclusion, dolphin neutrophils responded to several soluble and particulate stimulants as well as human neutrophils, but were refractory or slightly responded to bacterial agents.     

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.     

Heterogeneity in phagocytic and nitroblue tetrazolium reductive properties of neutrophils from cows.

Phagocytic and nitroblue tetrazolium (NBT) reductive activities of blood neutrophils from 19 Holstein heifers were measured by light microscopic and spectrophotometric methods, respectively. These functional properties of neutrophils correlated well (r = 0.64) and varied significantly (P less than 0.05) among animals studied. Variations in phagocytosis and NBT reductive activities attributable to the source of sera were determined in experiments in which cells from the same cows and zymogen particles opsonized with heat-inactivated autologous or homologous sera were used. Variations attributable to the source of cells were determined in experiments in which cells from different cows and particles opsonized with pooled sera from all the cows were used. Most of the variation in phagocytic properties and NBT reductive activities was attributable to the source of cells (ie, each cow). The source of sera contributed slightly to the variation in NBT reductive activities, but not to the phagocytic properties. These results support the concept of functional heterogeneity of neutrophils among cows.     

Expression and role of adhesion molecule CD18 on bovine neutrophils.

Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions.     

Costimulatory effects of complement receptor type 3 and Fc receptor for IgG (FcgammaR) on superoxide production and signal transduction in bovine neutrophils

The present study evaluated the costimulatory effects of complement receptor type 3 (CR3) and Fc receptor for IgG (FcgammaR) on superoxide production and intracellular signal transduction in bovine neutrophils. Stimulation with opsonized zymosan (OPZ) and heat-aggregated bovine IgG (Agg-IgG) resulted in much greater superoxide production and chemiluminescent (CL) responses in normal neutrophils compared with those stimulated with OPZ or Agg-IgG only. Superoxide production and CL response were closely associated with the stimulant-induced rise of the intracellular calcium ([Ca2+]i) concentration, amount of tyrosine phosphorylated 100 kDa protein, and activation of p38 mitogen-activated protein kinase (p38 MAPK). No costimulatory effect was found for these receptors on superoxide production in CR3-deficient neutrophils. Costimulation of CR3 and FcgammaR on bovine neutrophils leads to enhancement of superoxide production and their signaling pathways and appears to be associated with enhancement of neutrophil functions.     

Activation of bovine monocytes and neutrophils by the Bb fragment of complement factor B: demonstration by the uptake of 3H-deoxyglucose.

The Bb fragment is the enzymatically active split product of bovine complement factor B. The Bb fragment was obtained after zymosan treatment of fresh bovine serum and fractionation of the treated serum, first over diethylaminoethyl-Sephacel and then over an affinity column made up of monoclonal antibody to bovine Bb, coupled to cyanogen-bromide-activated Sepharose. Purified Bb has a molecular weight of 64,000, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The ability of purified Bb to activate phagocytes was assessed. The activation assay was based on the principle that the primary source of energy for the phagocytes is obtained from glucose. 3H-deoxyglucose, a nonmetabolizable analogue of glucose, was used to obtain the quantitative measurement of the activation process. The activation by Bb was shown by the uptake of the labelled deoxyglucose in the phagocytic cells and was comparable to the activation caused by phorbol myristate acetate and N-formyl-L-methionyl-L-leucyl-L-phenylalanine, run in parallel. These data showed that fragment Bb activates bovine monocytes and neutrophils and also suggested that, when generated after complement activation, Bb may stimulate monocytes and neutrophils for enhanced phagocytosis.     

Effects of in vitro and in vivo administration of recombinant bovine interferon-gamma on bovine neutrophil responses to Brucella abortus

The effects of in vitro and in vivo treatment of bovine polymorphonuclear leukocytes with recombinant bovine interferon-gamma on in vitro bovine polymorphonuclear leukocyte functions and the survival of Brucella abortus were determined. Activation of neutrophils in vitro with interferon-gamma resulted in enhanced production of O2- and myelopeoroxidase-H2O2-halide activity by neutrophils in the presence of B. abortus. The improved iodination responses were correlated with an enhanced ability to perform iodination in the presence of 5'-guanosine monophosphate and adenine which have previously been shown to contribute to inhibition of neutrophil myeloperoxidase-H2O2-halide activity by B. abortus. The ability of opsonized B. abortus to survive in the presence of neutrophils activated in vitro or in vivo was partially decreased by approximately 10% of control when compared to survival rates within control phagocytes. These results suggest that activation of neutrophils with recombinant interferon-gamma partially enhances their oxidative metabolic responses, resulting in a slightly enhanced ability to kill virulent B. abortus.     

In vivo effect of ascorbic acid on neutrophil function in healthy and dexamethasone-treated cattle

Ascorbic acid (20 mg/kg of body weight) administered subcutaneously to otherwise nontreated cattle resulted in enhancement of neutrophil oxidative metabolism and capability of neutrophils to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Random migration, bacterial ingestion, and iodination by neutrophils was unaffected. Three dosage levels of ascorbic acid (10 mg/kg, 20 mg/kg, and 40 mg/kg) were examined for their effects on neutrophil function in cattle treated with dexamethasone (0.04 mg/kg). Dexamethasone administration caused an enhancement of neutrophil random migration and a suppression of neutrophil oxidative metabolism, iodination, and ADCC. None of the dosage levels of ascorbic acid had an effect on the alterations in the WBC count induced by dexamethasone. The ascorbic acid did tend to reverse the effects of dexamethasone on neutrophil random migration, oxidative metabolism, and ADCC in a dose-dependent manner, with the lowest dose having no discernible effect. Ascorbic acid administration also tended to enhance Staphylococcus aureus ingestion by bovine neutrophils. These results indicate that ascorbic acid should be further investigated for its potential to reduce the susceptibility of stressed or glucocorticoid-treated cattle to infective processes.