Bovine Neutrophil Function Research Articles

Biofilm and Planktonic Bacteria Differentially Express a Small Molecule (<3kD), Heat‐Stable, Protease‐Resistant Factor that Affects Bovine Neutrophil Function in a NF‐Kappa β‐Independent Mechanism

IntroductionChronic bacterial infections and the associated inflammation have a significant impact in both veterinary and human medicine. Bacterial biofilms are a major cause of the persistent inflammation observed in chronic infections and nearly 80% of all infections involve biofilms. Biofilms are typically polymicrobial in composition and many of these mixed‐infections involve anaerobic bacteria. Recent evidence demonstrates that immune cells often disregard the signals released from biofilm bacteria, and ineffectual clearance of biofilms may explain the chronicity of this sort of infection. A mixed‐species model of anaerobic biofilm growth was developed to evaluate medically relevant biofilms and their interactions with bovine neutrophils.AimsTo generate mixed‐species bacterial biofilms composed of the two opportunistic pathogens Fusobacterium necrophorum and Porphyromonas levii, and to employ the in vitro system to investigate factors associated with biofilms that may limit neutrophil functional ability.MethodsMixed‐species biofilm formation was verified with SEM, CLSM and viable cell counts. Neutrophil oxidative burst was assessed with a fluorescent microplate assay for hydrogen peroxide. Chemotaxis was measured with a Transwell transmigration assay. Extracellular factors released from planktonic cultures and biofilms were assessed by size‐exclusion filtration, lipopolysaccharide (LPS) removal, protease and heat treatment. Mechanistic experiments to study neutrophil responses to these supernatants were conducted with specific inhibitors of function, such as NF‐κβ signaling (BAY‐11) and inflammasome activation (glyburide).ResultsNeutrophils exposed to planktonic bacterial supernatants showed significantly elevated oxidative (4‐fold increase in fluorescence) and chemotactic responses compared to neutrophils exposed to biofilm products from identical bacteria. LPS plays a significant role in the stimulation of neutrophils; in this experiment biofilms produced substantially more LPS than planktonic bacteria despite inducing less neutrophil response. Removal of LPS resulted in similar neutrophil responses to planktonic and biofilm supernatants.Size‐exclusion filtration of bacterial supernatants and subsequent exposure to neutrophils revealed that the active stimulatory molecule is below 3 kD and is released strictly from planktonic cultures. Intensive heat and protease treatment of the <3kD fractions did not alter neutrophil functional responses. Treatment with a NF‐κβ inhibitor demonstrated that neutrophils respond specifically to planktonic supernatants independently of NF‐κβ signaling.ConclusionsWe describe a heat‐stable, protease‐resistant, non‐LPS, small (<3kD) factor produced by in vitro planktonic cultures of F. necrophorum and P. levii that activates bovine neutrophils. Neutrophils did not exhibit the same oxidative and chemotactic responses upon exposure to in vitro generated biofilm supernatants and these differential responses are the result of a NF‐κβ independent mechanism, perhaps through an inflammasome‐mediated process.Support or Funding InformationFunding provided by the University of Calgary Grant 10002461 to DW Morck.

The protective effect of chlorogenic acid on bovine mammary epithelial cells and neutrophil function

Chlorogenic acid (CGA) is the ester of caffeic acid and quinic acid and plays an important role in antibacterial activity and anti-inflammatory properties. The objective of this study was to examine the effects of CGA on the growth of Staphylococcus aureus and the mRNA levels of the genes encoding the inflammatory response cytokines, κ-casein, and neutrophil function in bovine mammary epithelial cells (BMEC) exposed to S. aureus. Chlorogenic acid has important antibacterial, antioxidant, and anti-inflammatory functions; however, the effect of CGA on BMEC and neutrophils exposed to S. aureus has not been investigated previously. Our results demonstrated that 10, 20, and 30 μg/mL CGA had no cytotoxic effects on BMEC in culture, and that 20 μg/mL CGA enhanced the viability of BMEC exposed to S. aureus, whereas 30 μg/mL CGA reduced S. aureus growth after 9 h compared with controls. The rate of S. aureus invasion into BMEC was also attenuated by 30 μg/mL CGA compared with controls, whereas this treatment led to reduced abundance of IL6, IL8, and TLR2 mRNA in S. aureus-exposed BMEC. Migration of bovine polymorphonuclear leukocytes was significantly decreased in S. aureus-exposed BMEC with 10 and 20 μg/mL CGA treatment when compared with S. aureus treatment alone. In addition, incubation with 20 or 30 μg/mL CGA enhanced the phagocytic ability of polymorphonuclear leukocytes compared with the control group. Importantly, levels of κ-casein were enhanced by treatment of S. aureus-exposed BMEC with CGA. Our results suggest that the use of CGA may be a potent therapeutic tool against bovine mastitis caused by S. aureus.

Intracellular reactive oxygen species production and phagocytosis of Staphylococcus aureus by milk neutrophils as tool to diagnose mastitis and identify susceptible dairy cows

ABSTRACT: The immune response capacity of the mammary gland plays a major role to determine if mastitis will or not be established. Thus, we hypothesize that a better understanding of polymorphonuclear neutrophil leukocyte (PMN) function will elucidate mechanisms that will improve our knowledge of how we could avoid an inflammatory process by increasing the immune capacity of the cow, and even further, to search for a tool to diagnose mastitis or a possible way to select and identify non-susceptible animals. The present study utilized 112 quarters from 28 Holstein dairy cows that were divided into quarters milk samples with somatic cell count (SCC) <2×105 cells mL-1 (n=72) and SCC >2×105 cells mL-1 (n=40). The percentages of milk PMNs and the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus by milk neutrophils were evaluated by flow cytometry. Our results showed a higher percentage of neutrophils in quarter milk samples with high SCC (P=0.0003), and this group also had a significantly higher percentage of neutrophils that produced ROS (P=0.008). On the other hand, the phagocytosis intensity of S. aureus by milk neutrophils was higher in quarters with low SCC (P=0.003), suggesting a better mammary gland immunity against invading pathogens. Analyzing the results of the predictive values of the measured PMN functions, they cannot be used isolated as a good diagnosis test since none of them had a satisfactory sensitivity and specificity values, which was also confirmed by the Youden index values being far from one. In conclusion, the assessment of milk bovine neutrophil functions could improve our understanding of the cellular basis of mastitis. Although, the intracellular ROS production and S. aureus phagocytosis by milk neutrophil did not have high predictive values to detect intramammary infections, our results strengthen the idea that that poor bovine mammary gland neutrophil phagocytic ability may be associated with high SCC, and might be considered to identify susceptible dairy cows to mastitis.

Interactive effects of dexamethasone and opsonized Mycoplasma bovis on bovine neutrophil function in vitro

Previously we had reported that exposure to high levels of glucocorticoids, and to unopsonized Mycoplasma bovis, has a negative interactive effect on bovine neutrophil function in vitro, and this interactive effect was a function of M. bovis strain differences. Here we hypothesized that in vitro treatment of bovine neutrophils by glucocorticoid would impair phagocytosis of opsonized M. bovis compared to non-treated neutrophils and such impairment would be a function of M. bovis strain differences. Neutrophils isolated from 20 mid-lactation cows were treated with immunosuppressive dose of 5 × 10−4 M dexamethasone or placebo and incubated with one of four opsonized M. bovis strains that had been isolated from bovine origin. After incubation neutrophil function measured included: percentage reduction in log10 of M. bovis CFU/ml, percentage of phagocytizing neutrophils, phagocytized M. bovis per neutrophil, and killed M. bovis per neutrophil. Least square means of all neutrophil groups were contrasted using linear mixed–effects models. Effects due to strain, treatment, and their interaction on neutrophil function measured by the number of phagocytized M. bovis per neutrophil and number of killed M. bovis per neutrophil were different (P < 0.05). However, no significant strain by treatment interaction effect on percentage reduction in log10 of M. bovis CFU/ml was found. Neither a strain nor a strain by treatment interaction was found to affect the percentage phagocytizing neutrophils. These findings might explain in part the association of stressful events with subsequent outbreaks of Mycoplasma bovis associated bovine diseases.

Guarea kunthiana Bark Extract Enhances the Antimicrobial Activities of Human and Bovine Neutrophils

Guarea kunthiana is used in folk remedies for the treatment of several diseases including microbial infections. The mechanism behind this phenomenon still needs to be elucidated. Here, we investigated the effect of G. kunthiana bark extract on antimicrobial functions of human and bovine neutrophils as the first line of defense against infections. For this aim, neutrophils were isolated from either human or bovine blood and treated with G. kunthiana bark extract. The antimicrobial activity of the neutrophils against Staphylococcus (S.) aureus and Escherichia (E.) coli was tested in a bacterial survival assay and a fluorescence-based phagocytosis assay. Furthermore, the formation of neutrophil extracellular traps (NETs) was visualized by immunofluorescence microscopy. We show that neutrophils treated with G. kunthiana extract distinctly increased phagocytosis of S. aureus or E. coli. Interestingly, we demonstrate that G. kunthiana bark extract induces the formation of NETs in both cell types. This effect was abolished when treating the cells with diphenyleniodonium chloride (DPI) pointing to a direct implication of the NADPH oxidase-dependent formation of reactive oxygen species in this process. In summary, our data strongly suggest that G. kunthiana bark extract boosts the antimicrobial activities of neutrophils as the first line of defense against invading pathogens.

Butyric acid stimulates bovine neutrophil functions and potentiates the effect of platelet activating factor

Increased short-chain fatty acid (SCFA) production is associated with subacute ruminal acidosis (SARA) and activation of inflammatory processes. In humans and rodents, SCFAs modulate inflammatory responses in the gut via free fatty acid receptor 2 (FFA2). In bovines, butyric acid is one of the most potent FFA2 agonists. Its expression in bovine neutrophils has recently been demonstrated, suggesting a role in innate immune response in cattle. This study aimed to evaluate if butyric acid modulates oxidative and non-oxidative functions or if it can potentiate other inflammatory mediators in bovine neutrophils. Our results showed that butyric acid can activate bovine neutrophils, inducing calcium (Ca2+) influx and mitogen-activated protein kinase (MAPK) phosphorylation, two second messengers involved in FFA2 activation. Ca2+ influx induced by butyric acid was dependent on the extracellular and intracellular Ca2+ source and phospholipase C (PLC) activation. Butyric acid alone had no significant effect on reactive oxygen species (ROS) production and chemotaxis; however, a priming effect on platelet-activating factor (PAF), a potent inflammatory mediator, was observed. Butyric acid increased CD63 expression and induced the release of neutrophil granule markers matrix metalloproteinase-9 (MMP-9) and lactoferrin. Finally, we observed that butyric acid induced neutrophil extracellular trap (NET) formation without affecting cellular viability. These findings suggest that butyric acid, a component of the ruminal fermentative process, can modulate the innate immune response of ruminants.

Effects of dexamethasone and Mycoplasma bovis on bovine neutrophil function in vitro

It is well established that exposure either to elevated levels of glucocorticoids, or to Mycoplasma bovis (M. bovis), has a negative effect on bovine neutrophil function. The objective of this research was to determine whether in vitro treatment of bovine neutrophils by M. bovis strains (n=4) and glucocorticoids would additively impair phagocyte function. Twenty, healthy, dairy cows were enrolled. Whole blood was collected from all cows for neutrophil isolation. Phagocytosis and the generation of superoxide anion (O2−) were tested in vitro by incubation of neutrophils with FITC labeled Escherichia coli (E. coli) and cytochrome c after treatment. Treatments included: NM1-4D (neutrophils treated with dexamethasone and exposed to one of the four M. bovis strains); NM1-4 (neutrophils exposed to one of the four M. bovis strains only); ND (neutrophils treated with dexamethasone only); and N (non-treated control neutrophils). The overall percentages of neutrophils phagocytizing E. coli were: 32%, 51%, 37%, and 53%±5.25% for treatments NM1-4D, NM1-4, ND, and N, respectively. The overall statistically transformed means of phagocytized E. coli per neutrophil were: 1.37, 1.72, 1.33, and 1.67±0.057 for treatments NM1-4D, NM1-4, ND, and N, respectively. The overall statistically transformed means of neutrophil O2− production were: 8.60, 11.91, 9.01, and 12.21±0.21nmol/106 for treatments NM1-4D, NM1-4, ND, and N, respectively. Exposure of neutrophils to M. bovis plus dexamethasone had an additive effect on generation of reactive oxygen species (p=0.0057), but not on the percentage of neutrophils phagocytizing E. coli (p=0.0817) or number of E. coli phagocytized per neutrophil (p=0.2946). Only one of the four M. bovis strains had a negative effect on neutrophil phagocytic function. Dexamethasone treatment consistently decreased neutrophil function as indicated by decreased percentage of neutrophils phagocytizing E. coli, decreased number of E. coli phagocytized per neutrophil, and decreased neutrophil O2− production, compared to controls (p<0.0001). Results suggested a synergistic effect of in vitro incubation of glucocorticoids and M. bovis on reduction of bovine neutrophil function as measured by generation of reactive oxygen species. These findings may explain in part the interaction between stressful events and outbreak of Mycoplasma bovis associated bovine disease.

The effect of citrus-derived oil on bovine blood neutrophil function and gene expression in vitro

Research on the use of natural products to treat or prevent microbial invasion as alternatives to antibiotic use is growing. Polymorphonuclear leukocytes (PMNL) play a vital role with regard to the innate immune response that affects severity or duration of mastitis. To our knowledge, effect of cold-pressed terpeneless Valencia orange oil (TCO) on bovine PMNL function has not been elucidated. Therefore, the objective of this study was to investigate the effect of TCO on bovine blood PMNL chemotaxis and phagocytosis capabilities and the expression of genes involved in inflammatory response in vitro. Polymorphonuclear leukocytes were isolated from jugular blood of 12 Holstein cows in mid-lactation and were incubated with 0.0 or 0.01% TCO for 120min at 37°C and 5% CO2, and phagocytosis (2×106 PMNL) and chemotaxis (6×106 PMNL) assays were then performed in vitro. For gene expression, RNA was extracted from incubated PMNL (6×106 PMNL), and gene expression was analyzed using quantitative PCR. The supernatant was stored at −80°C for analysis of tumor necrosis factor-α. Data were analyzed using a general linear mixed model with cow and treatment (i.e., control or TCO) in the model statement. In vitro supplementation of 0.01% of TCO increased the chemotactic ability to IL-8 by 47%; however, migration of PMNL to complement 5a was not altered. Treatment did not affect the production of tumor necrosis factor-α by PMNL. Expression of proinflammatory genes (i.e., SELL, TLR4, IRAK1, TRAF6, and LYZ) coding for proteins was not altered by incubation of PMNL with TCO. However, downregulation of TLR2 [fold change (FC=treatment/control)=−2.14], NFKBIA (FC=1.82), IL1B (FC=−2.16), TNFA (FC=−9.43), and SOD2 (FC=−1.57) was observed for PMNL incubated with TCO when compared with controls. Interestingly, expression of IL10, a well-known antiinflammatory cytokine, was also downregulated (FC=−3.78), whereas expression of IL8 (FC=1.93), a gene coding for the cytokine IL-8 known for its chemotactic function, tended to be upregulated in PMNL incubated with TCO. Incubation of PMNL with TCO enhanced PMNL chemotaxis in vitro. The expression of genes involved in the inflammatory response was primarily downregulated. Results showed that 0.01% TCO did not impair the function of PMNL in vitro. Future studies investigating the use of TCO as an alternative therapy for treatment of mastitis, including dose and duration, for cows during lactation are warranted.

D-Lactic acid interferes with the effects of platelet activating factor on bovine neutrophils

d-Lactic acidosis occurs in ruminants, such as cattle, with acute ruminal acidosis caused by ingestion of excessive amounts of highly fermentable carbohydrates. Affected animals show clinical signs similar to those of septic shock, as well as acute laminitis and liver abscesses. It has been proposed that the inflammatory response and susceptibility to infection could both be caused by the inhibition of phagocytic mechanisms. To determine the effects of d-lactic acid on bovine neutrophil functions, we pretreated cells with different concentrations of d-lactic acid and measured intracellular pH using 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and calcium flux using FLUO-3 AM-loaded neutrophils. Reactive oxygen species (ROS) production was measured using a luminol chemiluminescence assay, and MMP-9/gelatinase-B granule release was measured by zymography. CD11b and CD62L/ l-selectin expression, changes in cell shape, superoxide anion production, phagocytosis of Escherichia coli-Texas red bioparticles, and apoptosis were all measured using flow cytometry. Our results demonstrated that d-lactic acid reduced ROS production, CD11b upregulation and MMP-9 release in bovine neutrophils treated with 100 nM platelet-activating factor (PAF). d-Lactic acid induced MMP-9 release and, at higher concentrations, upregulated CD11b expression, decrease l-selectin expression, and induces late apoptosis. We concluded that d-lactic acid can interfere with neutrophil functions induced by PAF, leading to reduced innate immune responses during bacterial infections. Moreover, the increase of MMP-9 release and CD11b expression induced by 10 mM d-lactic acid could promote an nonspecific neutrophil-dependent inflammatory reaction in cattle with acute ruminal acidosis.

The effect of dexamethasone on clock gene mRNA levels in bovine neutrophils and lymphocytes

Circadian rhythms are driven by oscillating expression of a family of transcription factors called clock genes. In rodents, clock genes drive circadian rhythms in white blood cell function, and glucocorticoids are believed to regulate these rhythms. Little is known about circadian rhythms of cattle white blood cells. The objectives of this study were: (1) to quantify mRNA levels of clock genes in neutrophils and lymphocytes over 24 h in healthy steers; and (2) to quantify effects of dexamethasone on clock gene mRNA levels in bovine neutrophils and lymphocytes. We hypothesized that bovine neutrophils and lymphocytes would display 24 h variations in clock gene mRNA levels and that those patterns would be disrupted by glucocorticoid treatment. Six Holstein steers were injected with 0 or 0.10 mg/kg body weight dexamethasone according to a crossover design. Neutrophils and lymphocytes were collected from jugular blood at 0, 4, 8, 12, 16, 20, and 24 h relative to treatment administration. Neutrophil and lymphocyte mRNA levels of the clock genes Clock, Bmal1, Per1, Per2, Cry1, Cry2, Rev-erbα, and CK1ɛ were quantified. For neutrophils, an interaction between treatment and time was found for Clock, Cry1, and CK1ɛ. Time affected Clock, Per1, Cry1, Rev-erbα, and CK1ɛ. For all of those genes except Per1, neutrophils from control steers displayed 24 h changes of mRNA levels characteristic of circadian regulated cells. The dexamethasone treatment increased neutrophil mRNA levels of Per1, decreased Clock, Cry1, Cry2, and Rev-erbα, and tended to decrease Bmal1. These results suggest that circadian rhythms have the potential to impact bovine neutrophil function, and that glucocorticoid-induced disruption of neutrophil circadian rhythms may contribute to periparturient immunosuppression. For lymphocytes, an interaction between treatment and time was observed for Per1 and tended to occur for Per2 and Cry2. Although time affected Per1 and Rev-erbα, distinct 24 h patterns of lymphocyte clock gene mRNA levels were not evident as they were in neutrophils. Treatment increased Per1 and decreased Cry2, but the magnitude of the treatment effect was small. In summary, 24 h patterns in clock gene mRNA levels were observed in bovine neutrophils and to some degree in lymphocytes, and these patterns were disrupted by dexamethasone administration. Although further research is needed, individual variation in white blood cell circadian rhythms and glucocorticoid responsiveness may help to explain individual differences in periparturient disease susceptibility.

Mild Dietary Copper Insufficiency Depresses Blood Neutrophil Function in Dairy Cattle

Abstract Effects of dietary copper insufficiency on bovine blood neutrophil function were evaluated. Dairy heifers were maintained on a basal (6-7 mg Cu/kg diet) or copper-supplemented (20 mg Cu/kg diet) diet beginning 84 d prepartum. At approximately 35 d postpartum, heifers were bled for neutrophil isolation 1 d prior to and 2 d following intramammary infusion of endotoxin in 2 mammary quarters. Neutrophils were evaluated for in vitro production of superoxide anion, reduction of nitroblue tetrazolium, and bactericidal activity. Copper status did not affect neutrophil reduction of nitro-blue tetrazolium or production of superoxide anion. Neutrophil bacterial phagocytosis and number of bacteria ingested per neutrophil were similarly unaffected by diet. Prior to endotoxin infusion neutro-phils isolated from copper-supplemented heifers were significantly more effective at killing ingested bacteria than neutrophils from unsupplemented heifers. Endotoxin infusion eliminated diet-related differences in bacteri...

Evaluation of the effects of short-chain fatty acids and extracellular pH on bovine neutrophil function in vitro

Evaluation of the effects of short-chain fatty acids and extracellular pH on bovine neutrophil function in vitro

Evaluation of the effects of short-chain fatty acids and extracellular pH on bovine neutrophil function in vitro

To investigate the effects of short-chain fatty acids (SCFAs) and pH on neutrophil oxidative burst, phagocytosis, and morphology after exposure to acetate, propionate, butyrate, or succinate at pH 5.5 and 6.7. Neutrophils isolated from bovine blood samples and Porphyromonas levii, Prevotella spp, and Bacteroides fragilis isolated from lesions of cattle with acute interdigital phlegmon (foot rot). Bacteria were cultured in strictly anaerobic conditions. Bacterial SCFA production was measured with high-performance liquid chromatography. Neutrophils were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ), and incubated with dihydroethidium or dichlorofluorescein diacetate to measure production of O(2)and H(2)O(2), respectively. Phagocytosis was assessed after exposure to serum-opsonized bacteria. Cellular morphology was assessed with differential staining. All bacteria produced at least 3 of the 4 SCFAs. Production of both O(2) and H(2)O(2) was markedly curtailed in PMA-stimulated neutrophils exposed to SCFA at pH 5.5, compared with production at pH 6.7. Succinate caused a significant dose-dependent decrease in O(2) production at pH 6.7 in OZ-stimulated neutrophils. Monoprotic SCFAs elicited a significant increase in H(2)O(2) production in OZ-stimulated neutrophils at pH 6.7 but a significant decrease at pH 5.5. Monoprotic SCFAs significantly increased phagocytosis at pH 6.7 but decreased phagocytic activity at pH 5.5. Cellular necrosis was observed in cells exposed to SCFAs at pH 5.5. Establishment and persistence of anaerobic bacteria in cattle with foot rot infection may result in part from neutrophil dysfunction secondary to the effects of bacterially secreted SCFA in acidotic microenvironments.

Expression of the estrogen receptor in blood neutrophils of dairy cows during the periparturient period

During the period around parturition, cows experience an increased susceptibility to inflammatory disorders in the mammary gland and uterus. This increased susceptibility has been correlated with a decreased functionality of neutrophils, major components in the innate immune defence. As sex steroid levels vary extensively in the period around parturition, an influence of these changes on the functionality of neutrophils has been suggested. Indeed, it has been shown that 17β-estradiol affects some functions of bovine neutrophils. In spite of these observations, receptors for 17β-estradiol have not yet been demonstrated in these cells. The investigation of the presence of estrogen receptors in bovine neutrophils was therefore the main objective of this study. The expression of estrogen receptors was evaluated at the protein level by flow cytometry, and at the mRNA level by polymerase chain reaction. A clear positive signal was obtained using flow cytometry for the estrogen receptor protein in bovine neutrophils. Further discrimination between the estrogen receptor subtypes α and β revealed the expression of the estrogen receptor β, whereas for the estrogen receptor α no reproducible positive signal could be obtained with the available antibodies. Both subtypes were found at the mRNA level. Subsequently, the estrogen receptor protein expression level in neutrophils obtained from cows in early lactation was compared with those from cows in late pregnancy. Additionally, the influence of endogenous 17β-estradiol and progesterone levels was assessed. No difference was found for the estrogen receptor protein expression in neutrophils from cows in early lactation compared with late gestation neither were the endogenous 17β-estradiol and progesterone levels correlated with the protein expression.

Altered expression of cellular genes in neutrophils of periparturient dairy cows

The periparturient dairy cow undergoes a plethora of physiological changes, including changes in the immune system that lead to profound effects on animal health. Of the immune cells affected at parturition, the neutrophil has been of particular interest due to its primary role in innate immune defense against mastitis. Immune functions of bovine neutrophils are known to be depressed around parturition, but it has not been discerned at what level these alterations occur, including the possibility that parturition induces changes in expression of key genes. The hypothesis of the present study was that blood neutrophils respond to the physiology of parturition by altering the expression of genes needed for normal cellular functions. The main objectives of the study were to detect and characterize parturition induced changes in neutrophil gene expression, to determine if altered gene expression was significantly associated with the main steroid hormones of bovine parturition, and to obtain putative identities of differentially expressed neutrophil genes. Differential gene expression was detected and characterized through mRNA abundance changes in neutrophils, and steroid hormone concentrations by RIA assay of periparturient serum samples. Preliminary assessment of differential gene expression was done using differential display reverse transcription polymerase chain reaction (DDRT-PCR) followed by secondary screening using high throughput cDNA dot blot hybridization. Altered gene expression was confirmed using Northern blot hybridization and detailed expression patterns characterized using quantitative slot blot analysis. The identities of two fully characterized transcripts with clear parturition induced repression ( P≤0.02) in neutrophils had high DNA sequence homology with genes that encode bovine mitochondrial cytochrome b (cytb) and rig/ribosomal protein S15 (rig/RPS15). These proteins are critical for normal respiratory metabolism and translation in cells, respectively. The gene expression profiles for cytb were significantly related to serum progesterone concentration ( r=0.44) and for rig/RPS15 to progesterone and estradiol concentrations ( r=0.35, 0.36, respectively). Eleven additional transcripts showed evidence of parturition induced repression in neutrophils and were putatively identified as representing genes of the citric acid cycle and various DNA binding proteins. Results of this study show for the first time that genes regulating basic life functions of bovine neutrophils may be repressed by parturition, possibly due to influences of steroid hormones.

Effects of the major Pasteurella multocida porin on bovine neutrophils

Abstract Objective To evaluate in vitro effect of the major fraction of outer membrane proteins of Pasteurella multocida with porin-like activities on some biological functions of bovine neutrophils. Animals Neutrophils from 5 adult cattle. Procedure Variations in such biological processes as actin polymerization and chemotaxis and evaluation of hydrogen peroxide attributable to variable concentrations of P multocida were recorded and compared. Data were obtained, using the porin and lipopolysaccharide (LPS) isolated from a strain of P multocida cultivated in brain-heart infusion (BHI) broth. Various concentrations of porin and LPS were analyzed to evaluate changes in functional activation and microbicidal activity of bovine neutrophils. Results The 37.5-kd major polypeptide of the outer membrane of P multocida was isolated. Presence of this porin was significantly correlated with variations of some biological functions of bovine neutrophils. These immunocompetent cells had a concentration-dependent increase in actin polymerization and chemotactic activity. A concentration-dependent variation in the oxidative burst also was observed. Conclusions The porins of gram-negative bacteria affect several biological functions of cells involved in the immune response as well as in inflammation. Significant correlation of results of in vitro experiments also was identified between porin and LPS effect. Pretreatment of bovine neutrophils with various concentrations of porin always caused a concentration-dependent increase in examined biological activities. (Am J Vet Res 1998;59:1270–1274)

Functional and phenotypic characterization of monoclonal antibodies to bovine L-selectin

SUMMARY Objective To characterize 2 bovine neutrophil monoclonal antibodies (MAB) as to effects on bovine neutrophil function and their binding antigens on the cell surface of bovine neutrophils. Animals 16 healthy, lactating Holstein cattle, 1 calf with leukocyte adhesion deficiency, and 1 age-matched control calf, 2 healthy ewes, and 2 healthy human beings as neutrophil sources. Procedure Neutrophil chemotactic and respiratory burst activities and calcium influx, and binding properties of the 2 MAB were determined. Molecular mass of corresponding cell surface antigens also was determined, as was binding of human L-selectin MAB DREG56 to molecules recognized by MAB 11G10 and 2G8 on the surface of bovine neutrophils. Results MAB 11G10 and 2G8 inhibited chemotactic activity of bovine neutrophils, up-regulated amplitude of native chemiluminescence, and shortened the time to reach maximal chemiluminescence induced by serum-opsonized zymosan. Crosslinking both MAB with a second antibody induced rapid increase in intracellular free calcium concentration. Binding density of MAB 11G10 and 2G8 to bovine neutrophils treated with trypsin was increased (P &lt; 0.05), compared with that of untreated neutrophils. Neutrophils treated with phosphatidylinositol-specific phospholipase C had decreased (P &lt; 0.05) binding density of MAB 11G10 and 2G8. Binding of the various MAB to neutrophils from calves with bovine leukocyte adhesion deficiency was lower (P &lt; 0.05) than binding to neutrophils from healthy calves. Expression of antigens recognized by the aforementioned MAB on the surface of bovine neutrophils was decreased (P &lt; 0.05) within 10 minutes. Conclusion MAB 11G10 and 2G8 recognized L-selectin molecules on bovine neutrophil membrane. L-Selectin (CD62L) is involved in low-affinity adhesion reactions between leukocytes and L-selectin ligand on postcapillary venular endothelial cells. (Am J Vet Res 1997;58:1392–1401)

挫滅去勢あるいは副腎皮質刺激ホルモン投与ウシの血清がウシリンパ球および好中球機能に及ぼす作用

The effects of sera from calves receiving Burdizzo castration or intravenous ACTH injection (100 IU a head) and then developing an acute plasma cortisol rise on the bovine lymphocyte and neutrophil function were examined with a view to elucidating whether these treatments can induce the animals to develop a serum immunomodulatory effect on those cells. When incubated with bovine lymphocytes, the sera from ACTH-treated calves induced a significant suppression (p<0.05) of concanavalin A-induced blastogenesis. The sera from castrated animals showed similar suppression but not significantly. It can be supposed that the serum lymphocyte suppression might have been related, at least in part, to elevated cortisol concentrations due to the treatments. Those sera, however, did not affect the luminol-dependent chemiluminescence activity, a bactericidal parameter, of bovine neutrophils.

Relationship between age-dependent changes of bovine neutrophil functions and their intracellular Ca2+ concentrations.

Neutrophil functions and intracellular Ca2+ concentrations ([Ca2+]i) were evaluated in 15 Holstein cattle divided into the following 3 groups: 5 neonatal calves less than 1 week old (group 1), 5 young calves 2 to 4 weeks old (group 2) and 5 cows 2 to 3 years old (group 3). The ability of neutrophils to phagocytose Candida albicans (C. albicans) was significantly higher (p < 0.05) in neonatal and young calves than in cows, whereas the phagocytosis by neutrophils of bovine IgG-coated yeasts (IgG-yeasts) was significantly lower (p < 0.05) in neonatal and young calves than that in cows. The killing activity by neutrophils of C. albicans in neonatal and young calves was significantly lower (p < 0.05) than that in cows. Luminol dependent chemiluminescent (LDCL) responses stimulated with opsonized zymosan (OPZ), heat-aggregated IgG (H-agg.IgG) and phorbol myristate acetate (PMA) were apparently lower in neonatal and young calves than in cows. No clearly different expressions of complement receptor type 3 (CR3) on neutrophils were observed among the 3 groups of cattle, although the values due to the binding of FITC-anti-bovine IgG to neutrophils in neonatal and young calves were lower than those in group 3. The OPZ-induced [Ca2+]i of neutrophils in neonatal and young calves were significantly higher (p < 0.05) than those in cows, but they were lower in neonatal and young calves when stimulated with H-agg.IgG. These results indicate that CR3- and FcR-mediated phagocytic and killing activities of neutrophils in neonatal and young calves are different from those in cows. These phenomena may be associated with age-dependent changes in [Ca2+]i.

Effects of in vitro supplementation with α-tocopherol and selenium on bovine neutrophil functions: implications for resistance to mastitis

A low vitamin E/selenium status has been associated with increased vulnerability of dairy cattle to mastitis. Since polymorphonuclear leucocytes (PMN) provide the major cellular defence mechanism within the mammary gland, the effect of in vitro supplementation with vitamin E and selenium on the function of these cells was investigated. Both vitamin E and selenium enhanced the chemotactic and random migration of PMN and increased the production of superoxide following stimulation with phorbol myristate acetate. Vitamin E, but not sodium selenite, was also found to enhance the phagocytosis of opsonised Staphylococcus aureus by PMN. No synergistic effects of the two nutrients were observed. These results obtained in vitro may indicate the potential benefits of in vivo supplementation of dairy cows with vitamin E and selenium in terms of enhancing their natural resistance to mastitis.