Alpha-1-acid glycoprotein inhibits phorbol ester-induced but not Fc-receptor-induced generation of reactive oxygen species in bovine peripheral blood neutrophils

Alpha-1-acid glycoprotein (AGP) is an acute-phase protein with anti-inflammatory and immunomodulating properties. AGP is described as a potent inhibitor of the production of reactive oxygen species (ROS) in human neutrophils. However, published reports about the mechanism of inhibition are conflicting. The influence of bovine AGP on the production of ROS by bovine peripheral blood polymorphonuclear leucocytes (PMN) was studied using a highly sensitive method approaching its inhibitory mechanism. ROS production in PMN was induced with phorbol 12-myristate 13-acetate (PMA) or opsonized Staphylococcus aureus bacteria. ROS generation was quantified and evaluated by flow cytometry. AGP efficiently suppressed PMA, but did not opsonize bacteria-induced ROS generation in vitro. The suppressive effect was concentration-dependent and adversely proportional to PMA concentration. The selective inhibitory potential of AGP in comparison with ovalbumin (OVA) and bovine serum albumin (BSA) showed that ROS inhibition was not a mere protein effect. ROS production was suppressed only if AGP and PMA were simultaneously present with PMN. Pre-incubation of PMN with AGP did not alter the PMN response to PMA. Moreover, AGP could not suppress ROS production after pre-stimulation of PMN with PMA. Human and bovine AGP did not differ in their inhibitory potential to the PMA-induced ROS production in bovine, human and equine PMN. The results show that AGP does not modulate bovine neutrophil functions directly, but acts as a scavenger of PMA.     

Association among filamentous actin content, CD11b expression, and membrane deformability in stimulated and unstimulated bovine neutrophils

OBJECTIVE: To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule-independent sequestration of neutrophils in inflamed lungs of cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-alpha (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The Factin content and CD11 b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood. RESULTS: The subpopulation of the smallest-sized neutrophils (>90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11 b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells.     

Effect of isoproterenol and dexamethasone on the lipopolysaccharide induced expression of CD11b on bovine neutrophils

The present experiments investigate the changes in expression of CD11b on bovine neutrophils and its modulation by isopropylnoradrenaline (IPN, isoproterenol), dexamethasone (DX), phenylephrine (alpha-agonist) and clenbuterol (beta-agonist). Both IPN and DX caused a dose-dependent inhibition of LPS-induced CD11b expression. A combination of IPN and DX elicited a synergistical decrease of the CD11b expression. Clenbuterol mimicked the effect of IPN, whereas phenylephrine did not. The effect of IPN and DX could at least partly be mediated through a decreased TNF-alpha production by monocytes since tumor necrosis factor-alpha (TNF-alpha) is shown to mediate a dose-dependent CD11b up-regulation. Stimulation of stress hormone receptors partly immuno-suppresses neutrophil functions by inhibition of CD11b expression on the neutrophil surface upon LPS stimulation. This inhibition is probably related to a decrease in TNF-alpha production. A similar mechanism of immuno-suppression could contribute to the higher susceptibility of cattle to Gram-negative bacterial infections of the udder and lung during periods of stress.     

Differential alterations in the ability of bovine neutrophils to generate extracellular and intracellular reactive oxygen species during the periparturient period

The periparturient period of a dairy cow is associated with increased incidence and/or severity of certain infectious diseases, including mastitis. It is believed that the heightened physiological demands of calving and initiation of milk production contribute to a state of immunosuppression during this period. Previous studies have indicated that neutrophil production of reactive oxygen species (ROS), which is a critical element of the host innate immune response to bacterial infection, is impaired in the 1-2week period following calving. However, whether there is comprehensive inhibition of ROS production or selective inhibition of particular ROS remains unknown. The present study provides evidence that neutrophils isolated from cows (n=20) after calving have an increased capacity to generate intracellular ROS and an impaired ability to release extracellular superoxide anion and hydrogen peroxide.     

Production of reactive oxygen species in neutrophils after repeated bouts of exercise in standardbred trotters

Six trained Standardbred trotters exercised on a racetrack on 2 days with a 3-day interval. On both exercise days the horses trotted three different exercise bouts with increasing intensity with 60-min intervals. Exercise-induced stress was manifested as leucocytosis, an increase in the neutrophil:lymphocyte (N:L) ratio, and increased capacity to produce reactive oxygen species in the peripheral blood as indicated by an increase in whole blood chemiluminescence. The leucocytosis was mainly due to neutrophilia, which lasted for 6 h. Production of reactive oxygen species per single neutrophil showed no significant change during a day of exercise, but was lower on the second exercise day. The cortisol concentrations and N:L ratio, used as indicators of stress, behaved differently: Cortisol did not change significantly after exercise, whereas the N:L ratio increased. These results suggest that in trained horses, the N:L ratio is a sensitive indicator of stress of short duration, and an attenuated N:L response can be taken as an indicator of adaptation to exercise stress.     

The effect of conglutinin on production of reactive oxygen species in bovine granulocytes

Conglutinin is a high molecular-weight lectin originally detected in bovine serum. It belongs to the family of collectins that bind sugar residues in a Ca²⁺-dependent manner and are effector molecules in innate immunity. Conglutinin appears to play an important role in immune defense mechanisms, showing antiviral and antibacterial activities when tested in vivo and in vitro. The present study evaluated the effect of conglutinin on the respiratory bursts in bovine peripheral phagocytes. Using nitroblue tetrazolium and hydrogen peroxide assays, we showed that sugar ligand-bound conglutinin stimulated the production of superoxide and H₂O₂ in granulocytes whereas the non-sugar-bound form of conglutinin inhibited these processes. These results indicate that both forms of conglutinin are able to interact with surface leukocyte receptors but have opposite effects on phagocytic activity. Our findings suggest that conglutinin bound to sugar residues on microbial surfaces can induce oxygen burst in phagocytes, and thereby mediates the elimination of pathogens and prevents the spread of infection.     

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.     

Modulating effects of acepromazine on the reactive oxygen species production by stimulated equine neutrophils

ObjectiveTo investigate the effect of acepromazine (ACP) on reactive oxygen species (ROS) production by stimulated equine neutrophils.Study designEx vivo biochemical experiments.AnimalsIsolated neutrophils from healthy untreated horses.MethodsNeutrophils were incubated with ACP at concentrations of 10^?4, 10^?5 or 10^?6 m and then stimulated with phorbol-myristate-acetate (PMA) before measurement of lucigenin-enhanced chemiluminescence (CL). In a second experiment neutrophils were incubated in the presence of α-keto-γ methylthiobutyric acid (KMB) and treated with ACP at concentrations of 10^?4, 10^?5 or 10^?6 m. Subsequent PMA stimulation lead to neutrophilic ROS production and decomposition of KMB to ethylene, which is measured by gas chromatography. Electron paramagnetic resonance-spin trapping (EPR) analysis was performed with PMA-stimulated neutrophils in the presence of ACP (10^?4, 10^?5 or 10^?6 m) directly added to the cell suspension. In the second experiment, the same concentrations of ACP were pre-incubated with neutrophils, then centrifuged to eliminate the excess of ACP and re-suspended in phosphate buffer before stimulation with PMA. In all experiments, the results of ACP-treated and ACP-untreated stimulated neutrophils were compared.ResultsOverall, results obtained with lucigenin-enhanced CL and KMB oxidation were in agreement with those seen in electron paramagnetic resonance spectroscopy. Acepromazine induced a dose-dependent inhibitory effect on neutrophilic ROS production. Electron paramagnetic resonance also showed, at high ACP concentration, the appearance of a cation radical derived from ACP. In contrast, electron paramagnetic resonance study performed with pre-incubated neutrophils showed an important dose-dependent inhibitory effect of ACP.ConclusionThe results indicate that ACP can neutralize O^˙?₂ or its by-products during the stimulation of neutrophils.Clinical relevanceThese findings may have a therapeutic relevance when phenothiazines are used in horses suffering from inflammatory diseases in which neutrophil activation and ROS production are implicated.     

2-Aminoethoxydiphenyl borate (2-APB) reduces respiratory burst, MMP-9 release and CD11b expression, and increases l-selectin shedding in bovine neutrophils

This study describes the effect of 2-aminoethoxydiphenyl borate (2-APB), a putative store-operated calcium (Ca²⁺) entry (SOCE) inhibitor, on reactive oxygen species (ROS) production, matrix metalloproteinase 9 (MMP-9) release, CD11b and l-selectin (CD62L) expression, size changes and apoptosis in bovine neutrophils stimulated with platelet-activating factor (PAF). It was observed that doses ?1 μM 2-APB significantly reduced ROS production, whereas 50 and 100 μM 2-APB reduced MMP-9 release induced by PAF. Moreover, concentrations ?10 μM 2-APB reduced CD11b expression and increased l-selectin shedding. PAF induced size changes in neutrophils, and this effect was inhibited by 2-APB. From this work it is possible to conclude that 2-APB at concentrations that inhibit SOCE responses was able to inhibit ROS and MMP-9 release and CD11b expression, and increase l-selectin shedding, suggesting that the Ca²⁺ channel involved in SOCE is a potential target for the development of new anti-inflammatory drugs in cattle.     

Intracellular reactive oxygen species production by polymorphonuclear leukocytes in bovine leukemia virus-infected dairy cows

The present study assesses the oxidative burst activity from polymorphonuclear leukocytes (PMNLs) from bovine leukemia virus (BLV)-infected cows. Fifteen clinically healthy cows were divided into serologically positive cows without any hematological alteration, serologically positive animals with persistent lymphocytosis (PL) and healthy serologically negative cows. The oxidative burst activity from the PMNLs was evaluated by flow cytometry using 2',7'-dichlorofluorescein diacetate as a probe. PMNLs from each cow were incubated with heat-killed Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) to stimulate oxidative burst activity. The results of the present work showed no significant difference in the oxidative burst activity without any stimulus and elicited by S. aureus. Conversely, a decrease in the oxidative burst index induced by E. coli in PMNLs was observed in BLV-infected cows.     

Heat shock-derived reactive oxygen species induce embryonic mortality in in vitro early stage bovine embryos

Heat shock is known to increase the mortality of early stage embryos, but the exact mechanism is unclear. In the present study, we investigated the possibility that the increased mortality is caused by heat shock-generated reactive oxygen species (ROS). The level of ROS was controlled by using beta-mercaptoethanol (beta-ME), a scavenger of ROS. In vitro-produced 8-cell stage embryos were cultured at 38.5 C or heat-shocked by exposure to 41 C for 6 h with 0, 10 and 50 microM beta-ME. Intracellular ROS levels were measured by a fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate (DCHFDA), and intracellular reduced form of glutathione (GSH) contents were estimated by another fluorescent dye, 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin. Total glutathione content was estimated by the glutathione recycling assay. On day 8 after insemination, heat shock decreased the percentage of embryos that developed to the blastocyst stage and increased intracellular ROS levels, but there was no significant effect on the GSH and total glutathione contents. In contrast, beta-ME significantly decreased ROS levels in heat-shocked embryos and increased the GSH and total glutathione concentrations. Ten microM beta-ME significantly improved the viability of heat-shocked embryos. beta-ME caused no detrimental effects when it was added at normal culture temperature (38.5 C). These results indicate that ROS is the primary cause of increased embryonic mortality in heat-shocked early stage embryos.     

Detection of bovine herpesvirus 4 in CD11b+ leukocytes of experimentally infected rabbits

The presence and numbers of bovine herpesvirus 4 (BoHV-4) infected CD11b+ leukocytes were investigated during experimental infections of New Zealand White rabbits by Fluorescence Activated Cell Sorter (FACS) analysis. Peripheral blood leukocytes (PBL) were collected every second day, and the cells were stained with phycoerythrin-labelled CD11b-specific mouse monoclonal antibody and fluorescein-conjugated bovine herpesvirus 4-specific mouse monoclonal antibody. The numbers of double-stained cells from PBLs of the control and inoculated groups were measured and compared in FACSTREK analyser. Double-stained cells were detected in the virus-inoculated group on postinoculation days (PID) 2-5 and 9-12. The results indicated that CD11b+ PBLs were permissive for BoHV-4 infection, and are probably the main reservoir of the virus during the latent period. The data did not indicate production of infectious viral particles, but virus-specific proteins were expressed on the surface of CD11b+ cells. The two waves of double-stained cells gave similar results to the PCR assays from serum samples, which showed the presence of viral DNA in the serum on the same days when virus-infected CD11b cells were also present. Productive BoHV-4 infection of mast cells or undifferentiated leukocytes in the bone marrow and the antiviral immune response might be responsible for this periodic appearance of the virus in CD11b+ PBLs and in the serum. The paper provides evidence that CD11b+ PBLs are the main target cell populations in the blood for BoHV-4.     

Effect of pterostilbene on development,equatorial lipid accumulation and reactive oxygen species production of in vitro-produced bovine embryos

The pterostilbene (PT) molecule is a phytoalexin with a reducing effect on reactive oxygen species (ROS) and with a capacity to block lipogenesis. However, the potential reducing effects of PT on equatorial lipid accumulation and ROS have not yet been elucidated for in vitro-derived bovine embryos. The present study evaluated the effects of concentrations of 3, 1, 0.33, 0.11 μM PT, and a vehicle group on the percentage of cleaved embryos, embryos with more than 6 cells, percentage of blastocyst on Day 7 and 8, percentage of transferable embryos on Day 7, the cell count and relative concentration of lipids. In the second experiment, the effects of 0.33 μM PT and a vehicle group within two different O₂ environments (5% and 20%) were evaluated for ROS generation and the percentage of Day 8 blastocysts. In the first experiment, no significant differences were found between the treatments with PT and the vehicle group (p > .05) concerning the percentage of cleaved embryos and embryos with more than 6 cells. Lipid reduction was observed in the groups treated with PT versus the vehicle group (p < .05). The vehicle group showed a higher rate of blastocyst production on Days 7 and 8 (p < .05) and an increase in the percentage of transferable embryos on Day 7 compared to the PT treatment groups (p < .05). Cell counts were not significantly different between treatments with PT and the vehicle group (p > .05). In the second experiment, the O₂ concentration did not significantly affect ROS generation (p > .05); however, the groups treated with PT (0.33 μM) had a reduction in ROS (p < .05). The O₂ concentration also did not significantly affect the rate of blastocyst production on Day 8 (p = .7696). Future research should be conducted to ascertain whether the reduction of lipids could enhance the cryopreservation and post-thaw viability of PT-treated embryos.     

The presence of CD25 on bovine WC1+ gammadelta T cells is positively correlated with their production of IL-10 and TGF-beta, but not IFN-gamma

WC1+ cells in cattle exhibit both regulatory and effector activities. However, it has not been elucidated whether they are so plastic that both activities co-exist in one cell or there are separate subpopulations of effector and regulatory cells. Since the production of IFN-gamma and IL-10 seems to be related to WC1+ cells' effector and regulatory function, respectively, the main aim of this study was to determine whether those cytokines are produced by separate subpopulations of WC1+, or are co-produced by the same cells. Due to increasingly frequent emphasised role of consumption of IL-2 in the mechanism of suppressor action of mouse CD25+CD4+ T regulatory cells, expression of the receptor's alpha chain for interleukin 2 (CD25) on WC1+ lymphocytes has been evaluated. An average of 5.21% of WC1+ cells obtained from PBMCs of 12-month-old heifers show constitutive expression of the CD25 molecule, with CD25(high)WC1+ and CD25(low)WC1+ cells accounting for 1.05% and 4.10% of WC1+ lymphocytes, respectively. For detection of intracellular cytokine production, PBMCs were stimulated with concanavalin A. Both IFN-gamma(-) and IL-10-producing cells within the CD25(-)WC1+ and CD25+WC1+ subpopulations were mainly separate subpopulations. The average percentage of IFN-gamma(+)IL-10(-), IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ cells among CD25(-)WC1+ lymphocytes was 4.03%, 2.67% and 0.51%, respectively. A positive correlation was observed between the presence of the CD25 molecule on WC1+ lymphocytes and production of IL-10 and TGF-beta, because the average percentage of IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ among CD25+WC1+ lymphocytes was 3 and 4.5 times higher as compared to the corresponding cells in the CD25(-)WC1+ subpopulation, whereas the percentage of IFN-gamma(+)IL-10(-) cells in both the subpopulations was not significantly different. The percentage of TGF-beta+ cells within the CD25+WC1+ subpopulation was 2.72 times as high as that of CD25(-)WC1+ lymphocytes. Therefore, with respect to the production of IFN-gamma, IL-10 and TGF-beta, CD25+WC1+ lymphocytes turn out to have a more suppressor profile than CD25(-)WC1+.     

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.     

Preincubation of bovine blood neutrophils with bovine herpesvirus-1 does not impair neutrophil interaction with Pasteurella haemolytica A1 in vitro

In this study we examined the direct effects of bovine herpesvirus-1 on the interaction of bovine blood neutrophils with Pasteurella haemolytica A1. Preincubation of neutrophils for approximately 2 h in vitro with BHV-1 at a multiplicity of infection of 5:1 had no effect on neutrophil random migration and directed migration to zymosan-activated bovine serum. Neutrophils also were unimpaired in their ability to ingest and kill P. haemolytica A1. Preincubation of neutrophils with BHV-1 did not elicit an oxidative burst, as measured by luminol-enhanced chemiluminescence, nor did it alter neutrophil chemiluminescence in response to opsonized P. haemolytica A1. Prolonged preincubation with BHV-1 for 18-24 h similarly did not affect neutrophil chemiluminescence in response to opsonized P. haemolytica A1. The susceptibility of neutrophils to the lethal effects of crude P. haemolytica cytotoxin also was unaltered by preincubation with BHV-1. We observed no evidence of BHV-1 replication in bovine neutrophils as determined by indirect immunofluorescence and electron microscopy. Previous reports have indicated that active BHV-1 infection alters certain neutrophil functions and results in hypersusceptibility to pulmonary pasteurellosis. Our results suggest that these effects are unlikely to be mediated directly by BHV-1, but instead may reflect the action of endogenous mediators that are released during active BHV-1 infection.     

Molecular definition of the bovine granulocytopathy syndrome: identification of deficiency of the Mac-1 (CD11b/CD18) glycoprotein

Leukocytosis (34,600 WBC/microliter of blood) was detected in an apparently healthy 7-day-old Holstein heifer. Analysis of blood samples obtained over the next 41 days revealed chronic progressive neutrophilia, which peaked at greater than 85% neutrophils and exceeded 100,000 WBC/microliter. In vitro assessment of isolated blood neutrophils obtained from the heifer at 38 and 45 days of age revealed selected functional abnormalities. Endocytosis of immunoglobulin-opsonized Staphylococcus aureus and killing of this test organism by the calf's neutrophils were significantly diminished, as were phagocytosis-associated superoxide generation, chemiluminescence activity, and myeloperoxidase-catalyzed iodination. Diminished H2O2 elaboration by the calf's neutrophils was evident during ingestion of opsonized zymosan or on exposure to phorbol myristate acetate. Extracellular release (secretion) of elastase during ingestion of zymosan was also diminished, although total cell content of elastase was normal, compared with that of neutrophils from age-matched calves, and granular or other morphologic abnormalities of the calf's neutrophils were not evident by ultrastructural examination. Abnormalities of random migration were inconsistently detected, and normal or high degree of antibody-dependent cytotoxicity or natural killing by the calf's neutrophils was observed. Similar in vitro assessment of neutrophils obtained from the calf's dam revealed no functional abnormalities. The calf died at 48 days of age, with persistent fever and chronic diarrhea, despite administration of antibiotics. Histologic examination at necropsy revealed large numbers of intravascular neutrophils in most tissues, including massive neutrophil sequestration in spleen. However, a striking lack of extravascular neutrophils was evident in inflamed submucosa adjacent to intestinal ulcers heavily contaminated with enteric microorganisms. Bone marrow examination revealed diffuse myeloid hyperplasia, but no other abnormalities.     

Roles of reactive oxygen species in the corpus luteum

Cells living under aerobic conditions always face the oxygen paradox. Oxygen is necessary for cells to maintain their lives. However, reactive oxygen species such as superoxide radicals, hydroxyl radicals and hydrogen peroxide are generated from oxygen and damage cells. Oxidative stress occurs as a consequence of the excessive production of reactive oxygen species and impaired antioxidant defense systems. Antioxidant enzymes include superoxide dismutase (SOD), which is a specific enzyme to scavenge superoxide radicals; copper‐zinc SOD, located in the cytosol and Mn‐SOD, located in the mitochondria. Both types of SOD belong to the first enzymatic step to scavenge superoxide radicals. It has been reported that a number of local factors such as cytokines, growth factors and eicosanoids are involved in the regulation of the corpus luteum (CL) function in addition to gonadotropins. Since reactive oxygen species are generated and SOD is expressed in the CL, there is a possibility that reactive oxygen species and SOD work as local regulators of the CL function. The present review reports that reactive oxygen species and their scavenging systems play important roles in the regulation of the CL function.