Streptococcal products and leukocyte activities.

Various streptococcal species are directly responsible for udder infections which should normally be countered by polymorphonuclear neutrophils (PMNs). In order to detect a putative inhibition of streptococcal products on the activities of bovine PMNs, we used a combination of four tests which permits an adequate evaluation of PMNs functions, e.g. PMN adherence on endothelial cells, chemotactic assay, phagocytosis of bacteria labelled with fluorescein isothiocyanate (FITC) and measurement of anion superoxide production. The conclusion is that neither of the two pathogenic streptococcal species isolated from mastitis appeared to produce in vitro factors affecting PMN activities.     

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.     

Phagocytic response of bovine polymorphonuclear leukocytes to different incubation conditions and following exposure to some effectors of phagocytosis and different anticoagulants in vitro.

The ability of bovine polymorphonuclear leukocytes (PMN) to phagocytose fluorescent beads in vitro was studied using flow cytometry. The effects of varying laboratory conditions (bead:PMN ratio, length of incubation, and temperature) were first determined, then the effects of lipopolysaccharide (LPS), phorbol myristate acetate (PMA), cytochalasin B, and formyl-met-leu-phe (fMLP) on phagocytosis were evaluated. The recommended bead:PMN ratio, incubation period, and incubation temperature are 20:1, 30 min, and 38.5 degrees C, respectively. Lipopolysaccharide increased phagocytosis at a relatively high minimum dose; PMA increased phagocytosis even at low doses; cytochalasin B increased and decreased phagocytosis at low and high doses, respectively; and fMLP had no significant effect on phagocytosis. Also, the effects of ethylene diamine tetraacetic acid (EDTA) and acid citrate dextrose (ACD) as anticoagulants were compared with heparin-treated blood PMNs. Both EDTA and ACD decreased phagocytosis. Although there are reports that demonstrated that heparin reduced PMN phagocytosis, at least among the 3 anticoagulants used, heparin remains to be the standard anticoagulant for the study of PMN phagocytosis.     

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.     

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.     

Trans-10, cis-12 conjugated linoleic acid modulates phagocytic responses of canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to Clostridium difficile toxin B

Trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) has been reported to enhance phagocyte function. Clostridium difficile toxin B (TcdB) has been known to inhibit Ras-homologous (Rho) guanosine triphosphatases (GTPases) which play essential roles in neutrophil immune functions. Here, we examined whether in vitro treatment with t10c12-CLA modulates the filamentous actin (F-actin) polymerization, phagocytic capacity, and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear neutrophilic leukocytes (PMNs) exposed to TcdB. Treatment with t10c12-CLA, but not linoleic acid, enhanced PMN F-actin polymerization, phagocytic capacity, and OBA, while TcdB suppressed these functions. t10c12-CLA reversed the suppressive effects of TcdB on these PMN functions. t10c12-CLA stimulated F-actin polymerization regardless of whether phagocytosis was stimulated by microspheres but only elevated OBA when microspheres were added. We asked whether the effects of t10c12-CLA were associated with changes in the activation of the Rho GTPase Cdc42. Treatment with t10c12-CLA augmented Cdc42 activity in both TcdB-treated and TcdB-naive PMNs during phagocytosis. Thus, t10c12-CLA up-regulates PMN phagocytic responses attenuated by TcdB. This effect is associated with an increase in actin polymerization and may involve the activation of Cdc42.     

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.     

Decreased neutrophil bactericidal activity during phagocytosis of a slime-producing Staphylococcus aureus strain

Phagocytosis and intracellular killing by bovine polymorphonuclear leukocytes (PMN) are important host defence mechanisms against mastitis caused by Staphlylococcus aureus. We compared the phagocytosis and overall killing of a non slime-producing (NSP) S. aureus and its slime-producing (SP) variant by blood PMN, using an in vitro bacteriological assay. Seven clinically healthy Holstein-Friesian dairy cows in mid-lactation stage were used for this purpose. The percentages of overall killing for the NSP and SP variant were 34+/-3% and 21+/-4% (P < 0.05) and the corresponding percentages of phagocytosis were 40+/-4% and 31+/-4%, respectively. A significant positive correlation (r = 0.79; P < 0.001) was found between phagocytosis and overall killing. These results suggest that the presence of slime was responsible for a decreased phagocytic ingestion and overall killing.     

Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro

To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.     

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.     

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.     

Evaluation In Vitro of Canine Neutrophil Function

Polymorphonuclear granulocyte (PMN) phagocytosis may be affected by many pathological changes. A panel of tests requiring relatively small volumes of blood was applied to 16 healthy dogs in order to obtain normal values and to standardize techniques. PMNs were isolated by discontinuous Percoll gradients; chemotaxis was tested in a modified Boyden chamber using the leading front method; fluorescinated yeast uptake was evaluated on a slide and superoxide (SO) production and adherence was carried out on a microtitre plate. The different aspects of phagocytosis showed no correlation with one another. Better results were obtained using a 60 min incubation period using interleukin-8 (25 ng/mL) as an activator for chemotaxis, and incubating plates for 30 min with phorbol myristate acetate (10(-6)mol/L) to assess SO production.     

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.     

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.     

Resistance of some capsular serotype D strains of Pasteurella multocida to rabbit polymorphonuclear neutrophil phagocytosis

The mechanism of resistance of Capsular Type D strains of Pasteurella multocida to killing by rabbit polymorphonuclear neutrophils (PMN) was studied using an in vitro assay that differentiates intra- from extracellular bacteria. Two Capsular Type D strains (3761 and 3766), resistant to killing by rabbit PMN, and one Type A strain (R1), susceptible to PMN destruction, were compared. After combining opsonized bacteria and PMN, the Capsular Type D Strains 3761 and 3766 remained extracellular while the Capsular Type A Strain R1 was internalized by PMN. Thus, both Type D strains were resistant to phagocytosis by rabbit PMN.     

Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes

Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.     

Strain differences in the susceptibility and resistance of Pasteurella multocida to phagocytosis and killing by rabbit polymorphonuclear neutrophils

The interactions of 2 capsular serotype A and 4 serotype D strains of Pasteurella multocida with rabbit polymorphonuclear neutrophils (PMN) were compared in vitro, using a PMN phagocytic and bactericidal assay. Bacteria and rabbit PMN were incubated for 15 minutes. The suspensions were subjected to differential centrifugation and the percentage of phagocytosis (cell association) was determined from the number of viable noncell-associated bacteria. The cell pellets and the associated bacteria were resuspended and PMN bactericidal activity was calculated from the number of remaining viable cell-associated bacteria at 45 and 75 minutes after the start of the assay. Test bacteria were not opsonized or were opsonized with immune serum containing active complement. One type A strain was ingested and killed by PMN in the presence and absence of opsonins. The 5 remaining strains were resistant to PMN killing, but only the type A strain resisted phagocytosis. Resistance of the type A strain was attributed to the hyaluronic acid capsule, since pretreatment of the bacteria with hyaluronidase rendered opsonized bacteria susceptible to ingestion and killing. The pattern of resistance of the 4 type D strains was different from that of the resistant type A strain. Both opsonized and nonopsonized type D bacteria became cell associated, but none were killed by PMN. The mechanism of resistance of these 4 strains to PMN bactericidal activity is currently unknown.     

Phagocytosis of opsonized fluorescent microspheres by equine polymorphonuclear leukocytes

Equine blood polymorphonuclear leukocytes (PMN) were isolated by buffy coat and hypotonic lysis of residual erythrocytes. A highly reproducible method is described for measuring the uptake of opsonized latex microspheres by equine PMN using flowcytometry. The use of cytochalasin D allowed for differentiation of ingested from attached particles. The kinetics of phagocytosis in vitro is shown for different experimental conditions. We developed an assay for evaluation of phagocytic capacity of PMN which allows the assessment of drugs for their influence on phagocytosis in vivo as well as in vitro.     

Association of increased estradiol and progesterone blood values with altered bovine polymorphonuclear leukocyte function

Polymorphonuclear leukocyte (PMN) function and serum concentrations of estradiol, progesterone, and cortisol (hydrocortisone) were monitored concurrently in clinically normal cows during the estrous cycle. Five parameters were used to evaluate PMN function: (i) random migration under agarose, (ii) ingestion of 125I-labeled Staphylococcus aureus, (iii) nitroblue tetrazolium (NBT) reduction, (iv) iodination, and (v) antibody-dependent cell-mediated cytotoxicity. Increased serum estradiol concentrations were associated with enhanced random migration, but had no apparent effect on NBT reduction, iodination, or ingestion of S aureus by bovine PMN. Increased serum estradiol was also associated with increased serum cortisol. Increased serum progesterone values were associated with a depression of NBT reduction and iodination by PMN, but with enhanced random migration and antibody-dependent cell-mediated cytotoxicity. These results indicate that physiologic changes in steroid hormone values during the normal estrous cycle of the cow are associated with alterations in PMN function.     

In vitro effects of very low levels of aflatoxin B₁ on free radicals production and bactericidal activity of bovine blood neutrophils

As one of the most potent and hazardous feed/food-originated mycotoxins, aflatoxin (AF) B? is regarded as a potent immunosuppressor in dairy cows. Neutrophils (PMN), as key effector cells against pathogens, have a high potential to kill engulfed microbes. To investigate the in vitro effects of very low doses of AFB? on blood PMN functions, we examined the effects of biologically relevant concentrations of AFB? on the phagocytosis and non-phagocytosis dependent luminol, representative of mainly intracellular free radicals, and isoluminol, representative of mainly extracellular free radicals, chemiluminescence (CL), necrosis and apoptosis of PMN. Isolated blood PMN from healthy dairy cows (n=12) were exposed to 0, 0.01, 0.05 and 0.5 ng/ml of AFB? for 0.5 and 18 h depending on the assay. Further, blood PMN of healthy dairy cows (n=8) were exposed to 0.5 ng/ml of AFB? for 3h and myeloperoxidase (MPO) activity, superoxide anion (O??) production, phagocytosis and killing activities against Staphylococcus (S.) aureus and Escherichia (E.) coli, were examined. Though the effect of extremely low doses of AFB? were less pronounced, at 0.5 ng/ml the production of free radicals was greatly enhanced, especially extracellularly. In contrast to isoluminol CL, the AFB?-treated PMN showed a remarkably impaired phagocytosis-depended luminol CL. PMN necrosis and apoptosis were not affected by AFB?. MPO activity, O?? production, phagocytosis rates and killing of E. coli and S. aureus by AFB?-treated PMN were significantly lower than those of non-treated ones. Our results show the extracellularly pro-oxidant and antiphagocytic properties of very low doses of AFB? for bovine PMN. The scope of the suppressive effects of the in vitro AFB? levels on cellular innate immune functions should be considered for high yielding dairy cows.