In vitro effect of recombinant human granulocyte colony-stimulating factor on canine neutrophil apoptosis

Apoptosis is essential in eliminating neutrophils (polymorphonuclear leukocytes: PMNs) in animals. The suppression of PMN apoptosis is believed to be beneficial in eradicating pathogens and is implicated in the pathogenesis of human inflammatory diseases. In the present study, canine PMNs were stimulated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to investigate the in vitro effect on the apoptosis of canine PMNs. Apoptotic cell rates were assessed by flow cytometry in relation to the ability of PMNs to produce reactive oxygen species (ROS). Canine PMN apoptosis was markedly suppressed by rhG-CSF treatment, in association with the retention of the PMN ability to produce ROS. The addition of cycloheximide abolished this suppression by rhG-CSF. Moreover, canine PMNs, which were stimulated by rhG-CSF, expressed high levels of anti-apoptotic mcl-1 gene mRNA, as quantified by real-time polymerase chain reaction method. The results suggest that PMNs, stimulated by G-CSF, could work effectively over a longer period to eliminate pathogens, and that the prolongation of the PMN life-span might occasionally aggravate tissue injuries in dogs. In addition, the suppression of PMN apoptosis seems to be mediated by the induction of anti-apoptotic mcl-1 gene expression.     

Differential effects of alpha1-acid glycoprotein on bovine neutrophil respiratory burst activity and IL-8 production

During bacterial-mediated diseases, neutrophils (PMNs) play a critical role in defending the host against invading pathogens. PMN production of reactive oxygen species (ROS) contributes to the bactericidal capabilities of these cells. ROS are produced intracellularly and can be released extracellularly. The aberrant extracellular release of ROS, however, has been reported to induce injury to host tissues during mastitis and other inflammatory-mediated diseases of cattle. The acute phase response, which occurs shortly after infection or tissue injury, is characterized by the induction of a large number of plasma proteins referred to as acute phase proteins (APP). alpha1-Acid glycoprotein (AGP) is an APP that increases in response to infection or injury in cattle and humans. The precise function of AGP is unknown, but it has been reported to possess anti-inflammatory properties. The objective of this study was to evaluate the effects of bovine AGP on PMN pro-inflammatory responses, including respiratory burst activity and cytokine production. Bovine AGP dose-dependently inhibited zymosan-induced PMN extracellular release of superoxide anion and hydrogen peroxide without affecting the capacity of PMN to engulf and kill Staphylococcus aureus. Moreover, AGP exerted its effect on ROS production regardless of whether PMNs were exposed to AGP prior to or after activation. In contrast to respiratory burst activity, AGP enhanced PMN production of IL-8. The precise mechanism by which AGP regulates PMN functions remains unknown, but data presented in this study suggest that AGP may have a complex role by differentially regulating PMN pro-inflammatory activities.     

A comparative study on certain enzymes of the granulocyte from different ruminant species

In the present study the level of enzyme hydrolases (alkaline phosphatase, myeloperoxidase, elastase, arginase, lysozyme and β-galactosidase) of polymorphonuclear cell (PMN) granules in different ruminant species and their release in response to activation was studied. Buffalo PMN alkaline phosphatase activity was higher (P < 0.01) than in PMNs of cattle and goats. Interestingly, myeloperoxidase was higher in cattle PMNs and least in goat PMNs (P < 0.01), a similar pattern was observed in the distribution of enzyme arginase. As far as lysozyme is concerned, its activity was significantly higher (P < 0.01) in PMNs of buffaloes than in the case of cattle and goat PMNs. On activation, these cells released MPO and elastase, in all the species studied, while lysozyme was secreted only in buffalo PMN cells. Activity of certain enzymes related to oxidant defence systems such as glutathione peroxidase and glutathione reductase were higher in cattle and goats compared to that in buffaloes. These observations are likely to have bearing on immunodefense roles played by PMNs and reflected differences among the ruminant species studied.     

In Vitro Effects of Acellular Milk on the Bactericidal Components of Caprine Polymorphonuclear Neutrophils

The effects of acellular milk on the activity of the microbicidal cationic enzymes of the polymorphonuclear cells of goats were studied in an attempt to explain the phenomenon by which PMN functions fail in mastitis. Assays were undertaken on the myeloperoxidase, lysozyme and elastase activities in a polymorphonuclear cell (PMN) lysate, both in the presence and absence of acellular milk from homologous species. There was a significant decrease (p<0.05) in the activity of lysozyme, myeloperoxidase and elastase in the presence of acellular milk. Superoxide and H₂O₂ production following activation of caprine PMNs by lipopolysaccharide (LPS) was significantly reduced (p<0.05) in the presence of acellular milk. Thus, the microbicidal function of PMNs is significantly impaired in the presence of acellular milk and this may contribute to the development of mastitis in dairy animals.     

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.     

Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide

A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion. TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected. A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period. The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells. TUNEL-positive PMN observed in normal animals showed a reduced migration capacity. The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity. Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis. The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging.     

An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E₂ (PGE₂) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE₂ at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.     

Effect of Rhodococcus equi on equine polymorphonuclear leukocyte function

A procedure was developed for isolating large numbers of purified polymorphonuclear leukocytes (PMNs) from the peripheral blood of horses. Equine PMN function was evaluated by three procedures: 1) Staphylococcus aureus ingestion, 2) nitroblue tetrazolium reduction, and 3) iodination. Four preparations of R. equi were added to polymorphonuclear leukocytes (PMNs) in each test system. Live bacteria, heat-killed bacteria, the washed pellet from heat-killed bacteria, and the supernatant fluid from heat-killed bacteria were evaluated for effects on equine PMN function. None of the R. equi preparations had an effect on S. aureus ingestion by equine PMNs. Nitroblue tetrazolium reduction by PMNs, a measure of oxidative metabolism, was suppressed by pellet and supernatant fractions. Values for the iodination reaction were depressed by all R. equi preparations, indicating decreased activity of the myeloperoxidase-H2O2-halide system of the PMN. Further evaluation of the supernatant from heat-killed R. equi showed that it retained its inhibitory effect on iodination following autoclaving and/or passage through a 10,000 MW filter. R. equi fractions did not alter the enzymatic conversion of 125I to a protein-bound form in a PMN-free assay developed to evaluate this reaction. The presence of a surface component capable of inhibiting bactericidal mechanisms of the PMN may play an important role in intracellular survival of R. equi.     

Evaluation of bovine polymorphonuclear leukocyte function

Bovine polymorphonuclear leukocytes (PMNs) were isolated from the peripheral blood of cattle. Five in vitro procedures were utilized to evaluate PMN function: 1) Random migration under agarose, 2) Ingestion of ¹²⁵I-iododeoxyuridine labeled $\text{Staphylococcus aureus}$, 3) Quantitative nitroblue tetrazolium reduction, 4) Chemiluminescence and 5) Iodination. Normal values for bovine PMNs are reported and interpretation of results is discussed. The PMN function tests were designed so that all 5 procedures may be performed in a short period of time on the same cell preparation. This allows for the detection and partial characterization of a potential PMN dysfunction.     

Evaluation of Oxygen-dependent Immunodefences of the Polymorphonuclear Cells of Some Tropical Ruminants

Activation of polymorphonuclear cells (PMNs) leads to the formation of superoxide, which is in turn dismutated to H₂O₂ by superoxide dismutase (SOD) and is partly responsible for oxygen-dependent microbicidal activity. However, no comparative information is available on the effect of SOD inhibition before PMN activation to allow simulation of the SOD defects that are known to occur in some ruminants. This paper attempts to examine the degranulative and phagocytic responses in buffalo, cattle and goat PMNs exposed to diethyldithiocarbamate, a known SOD inhibitor. The activity of glutathione peroxidase and reductase was increased in the presence of SOD inhibitor. On activation, H₂O₂ production increased significantly (p<0.01), while SOD inhibition before the activation of PMNs caused a significant decline in the production of H₂O₂ (p<0.05) in all the species studied. There was a significant increase (p<0.05) in the phagocytosis of Candida albicans spores by buffalo PMNs activated with opsonized zymosan. Activation of bovine PMNs after exposure to the SOD inhibitor resulted in a significant decline (p<0.05) in phagocytic activity; in the other species, the two values only approached significance. Among the activators, opsonized zymosan caused a significant increase in phagocytic activity as compared to lipopolysaccharide, particularly in the PMNs of buffaloes (p<0.05). Increased fungicidal activity (p<0.05) occurred with opsonized zymosan-activated PMNs of all the species studied. The fungicidal activity was found to decline in PMNs exposed to SOD inhibitor before activation (p<0.05). Interestingly, the phagocytic activity of caprine PMNs was found to be lower than that of PMNs from cattle (p<0.05).     

Evaluation of immunomodulatory effect of recombinant human granulocyte‐macrophage colony‐stimulating factor on polymorphonuclear cell from dogs with cancer in vitro

The objective of this in vitro study was to evaluate the immunomodulatory effects of recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF) on polymorphonuclear cell (PMN) function in dogs with cancer. PMNs were harvested from dogs with naturally developing cancer as a pre‐clinical model to evaluate the immunomodulatory effects of rhGM‐CSF on PMN phagocytic and cytotoxic functions, cytokine production and receptor expression. Some aspects of cancer‐related PMN dysfunction in dogs with cancer were restored following incubation with rhGM‐CSF including PMN phagocytosis, respiratory burst and LPS‐induced TNF‐α production. In addition, rhGM‐CSF increased surface HLA‐DR expression on the PMNs of dogs with cancer. These data suggests that dysfunction of innate immune response in dogs with cancer may be improved by rhGM‐CSF. The results of this study provided a pathophysiologic rationale for the initiation of clinical trials to continue evaluating rhGM‐CSF as an immunomodulatory therapy in dogs with cancer.     

The bitch uterine response to semen deposition and its modification by male accessory gland secretions

Little is known about the response of the bitch’s reproductive tract to semen deposition. In this study, an influx of polymorphonuclear neutrophils (PMNs) into the uterus was detected after artificial insemination, but there was normal fertility. Doppler ultrasonography showed that insemination induced an increase in uterine artery blood velocity and a decrease in the resistance index of short duration, indicating vasodilation. Semen that was extended in fluid from the sperm rich fraction of the ejaculate (seminal plasma, SP), or third fraction of the ejaculate (prostatic fluid, PF), produced a similar magnitude of effect but of longer duration. It was hypothesised that vasodilation following insemination was largely induced by SP and PF which, together with PMN influx, was part of a normal uterine response.Physiological concentrations of PMNs in vitro reduced the ability of spermatozoa to attach to uterine epithelium, most likely as a result of spermatozoa becoming attached to PMNs. However, both SP and PF increased attachment of spermatozoa to the uterine epithelium by reducing sperm attachment to PMNs, and potentially by an additional mechanism that did not involve inhibition of sperm binding to PMNs. These are the first canine studies to document an apparent physiological response by the uterus to semen, associated with uterine artery vasodilation and PMN influx. Moreover, these investigations are the first to demonstrate that canine SF and PF are part of the mechanism for increasing uterine perfusion and that both fluids have a modulatory effect on PMN-induced inhibition of spermatozoal attachment to uterine epithelium, most likely mediated by reduced sperm attachment to PMNs.     

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.     

Inflammatory cell infiltration as an indicator of Staphylococcus aureus infection and therapeutic efficacy in experimental mouse mastitis

Staphylococcus aureus intramammary colonization of the mouse mammary gland induces migration of polymorphonuclear neutrophils (PMNs) similar to that observed during bovine mastitis. In the present study, a method combining acridine orange staining, fluorescence microscopy and computer-assisted image analysis has been developed to quantitate PMN infiltration in a mouse model of mastitis. This was carried out using paraffin embedded sections, and using this method, we showed that the presence of PMNs increased with the number of bacteria present in tissues. Nearly 400 and 1100 times more PMNs were counted in the mammary gland tissue after 12 and 24 h of infection, respectively, compared to mice infected for 6 h. Treatment with the antibiotic cephapirin at 10 or 25 mg/kg reduced PMN infiltration by 71 and 85%, respectively. In conclusion, this method can be used to quantitate PMN infiltration as a marker of inflammation and bacterial burden in infected tissue sections.     

Haemophilus somnus-induced interference with bovine neutrophil functions

The effect of Haemophilus somnus on bovine polymorphonuclear leukocyte (PMN) function was examined in vitro with whole cells and fractions extracted from the surface of this bacterium. The ability of PMNs to iodinate protein and ingest Staphylococcus aureus was significantly inhibited in the presence of live cells, heat-killed whole cells or supernatant fluid from heat-killed cells, but not in the presence of washed, heat-killed cells. None of the fractions inhibited nitroblue tetrazolium (NBT) reduction by PMNs. The PMN inhibitory factors were further characterized. The material that inhibited S. aureus ingestion was found to be a heat-stable cell surface material of greater than 300 000 MW. The fraction inhibiting iodination of protein was found to be less than 10 000 MW.     

Evaluation of the polymorphonuclear cell functions of bottlenose dolphins

The functions of polymorphonuclear cells (PMN) are the important non-specific defense mechanisms in the immune system. Especially marine mammals are protected by these mechanisms from the aquatic environment with a large variety of microorganisms. Therefore, we examined the PMN functions of bottlenose dolphins in order to obtain the normal ranges and to standardize the techniques. PMNs were isolated by using lymphocyte isolate solution whose density was 1.077; superoxide production was assessed by nitroblue tetrazolium reduction test (NBT) and phagocytosis was tested by using polystyrene latex beads. We showed that the optimal incubation time was 30 min in NBT assay and 12 hr in phagocytosis assay for dolphin PMNs.     

In vitro evaluation of the effect of trans-10, cis-12 conjugated linoleic acid on phagocytosis by canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to methylprednisolone sodium succinate

OBJECTIVE: To examine whether in vitro treatment with trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) restores the phagocytic capacity and oxidative burst activity (OBA) of canine polymorphonuclear neutrophilic leukocytes (PMNs) exposed to methylprednisolone sodium succinate (MPSS). SAMPLE POPULATION: Peripheral blood PMNs obtained from 12 healthy Beagles. PROCEDURES: The experimental design involved administration of a high dose of MPSS, which is the recommended protocol for dogs with acute spinal cord injury. To evaluate PMN function, blood samples were collected from dogs before IV injections of doses of MPSS or saline (0.9% NaCl) solution (time 0) and 2, 12, and 24 hours after injections ceased. Polymorphonuclear neutrophilic leukocytes were isolated from blood samples and incubated with t10c12-CLA alone or t10c12-CLA in combination with N-acetylcysteine (an antioxidant agent). Phagocytic capacity and OBA were measured simultaneously by use of flow cytometry. RESULTS: The phagocytic capacity and OBA of PMNs were suppressed by IV injection of MPSS and restored 12 hours after injection ceased. In vitro treatment with t10c12-CLA enhanced the phagocytic capacity and OBA of PMNs, regardless of whether dogs had been treated with MPSS. Effects of t10c12-CLA on OBA were detected only when phagocytosis was stimulated by microspheres. Use of N-acetylcysteine attenuated the stimulatory effects of t10c12-CLA. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to t10c12-CLA enhanced the phagocytic capacity and OBA of canine PMNs, and this effect may have involved t10c12-CLA-induced generation of reactive oxygen species.     

Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase

Neutrophil (PMN) contribution to the acute inflammatory processes may lead to an excessive generation of reactive oxygen metabolites species (ROS) and secretion of granule enzymes. We compared the effects of either phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) in combination with a pre-treatment by cytochalasin B (CB) on the production of ROS and the release of total and active myeloperoxidase (MPO) by isolated equine PMNs. The ROS production was assessed by lucigenin dependent chemiluminescence (CL) and ethylene release by α-keto-γ-methylthiobutyric acid (KMB) oxidation. In the supernatant of activated PMNs, total equine MPO was measured by ELISA and active MPO by the SIEFED (Specific Immunologic Extraction Followed by Enzymatic Detection) technique that allows for the study of the interaction of a compound directly with the enzyme. The stimulation of PMNs with CB-fMLP only modestly increased the release of MPO, but more than 70% of released MPO was active. PMA stimulation markedly increased the production of ROS and release of MPO, but more than 95% of released MPO was inactive. When PMNs were pre-incubated with superoxide dismutase (SOD) prior to PMA activation, the lucigenin enhanced CL, which is linked to the superoxide anion (O₂⁻) production, was much more decreased than KMB oxidation, linked to the hydroxyl-like radical production. The addition of SOD prior to the activation of PMNs by PMA also limited the loss of the activity of released MPO. These results confirm the key role of O₂⁻ generation in the ROS cascade in PMN and reveal its critical role on MPO inactivation.     

Fucoidan directly regulates the chemotaxis of canine peripheral blood polymorphonuclear cells by activating F-actin polymerization

Fucoidan was recently shown to enhance innate immune functions. The objective of this study was to examine the direct stimulatory effect of fucoidan on the chemotactic activity of canine peripheral blood polymorphonuclear cells (PMNs). The chemotactic activity of PMNs was evaluated in a modified Boyden chamber assay and total cellular filamentous (F)-actin levels were measured using a flow cytometer. The chemotactic response of PMNs was increased by exposure to recombinant canine (rc) interleukin (IL)-8. In vitro treatment with fucoidan increased the chemotactic activity of PMNs in response to rcIL-8 compared with that of untreated PMNs, and also stimulated total cellular F-actin polymerization. The increased chemotactic activity of fucoidan-treated PMNs was suppressed by cytochalasin D, an inhibitor of F-actin polymerization. These results suggest that fucoidan directly regulates PMN chemotaxis, and that this effect is associated with an increase in actin polymerization.     

Isolation of bovine polymorphonuclear leukocytes by density gradient centrifugation

A method for the isolation of an enriched population (greater than 95%) of bovine polymorphonuclear leukocytes (PMNs) was developed using density gradient centrifugation. Leukocytes were isolated from peripheral blood by centrifugation in a density gradient medium (Percoll) of specific gravity 1.092. Viability was greater than or equal to 95% and the isolated PMNs were functional in migration inhibition and chemiluminescence assays. This has proved to be a simple effective method for obtaining bovine PMNs and yields cell populations that can be utilized for a variety of measures of PMN function.