Polymorphonuclear leucocyte function: relationship between induced migration into the bovine mammary gland and in vitro cell activity

Low doses of 10(-7) mg Escherichia coli endotoxin applied as intramammary infusion into single bovine quarters induced a rise in milk cell count without other inflammatory signs. Significantly fewer quarters responded in early lactation than in mid lactation. Maximum cell count was also somewhat later and less pronounced in early lactation. The rise in milk cell count after infusion of E. coli endotoxin was related to in vitro chemotactic activity of blood polymorphonuclear leucocytes (PMN). PMN isolated from cows which did not respond with a rise in milk cell count upon endotoxin infusion showed a diminished chemotactic activity in vitro as compared to PMN isolated from animals which did respond to an intramammary endotoxin infusion with a rise in milk cell count. No differences in phagocytic and metabolic activity were observed in vitro between the PMN isolated from the two groups of animals.     

Effects of Pasteurella haemolytica lipopolysaccharide on selected functions of bovine leukocytes

Lipopolysaccharide (LPS) was obtained from Pasteurella haemolytica serotype 1, using phenol-water extraction, and was evaluated for its ability to modify the biological activity of bovine leukocytes. The LPS was not toxic to polymorphonuclear leukocytes (PMN). The LPS preparation had little effect on random migration of PMN or mononuclear leukocytes (MNL), but caused a 2.5- to 3- fold increase in migration of cells within a whole peripheral blood leukocyte preparation. Phagocytosis of [125I]-labeled Staphylococcus aureus by PMN decreased with low (2.5 micrograms/10(6) cells) and high (65 micrograms/10(6) cells) concentrations of LPS and increased with moderate concentrations of LPS (5 to 25 micrograms/10(6) cells). The LPS enhanced nitroblue tetrazolium reduction by PMN. Moderate LPS concentrations (25 to 50 micrograms/10(6) cells) were mitogenic for MNL, whereas high LPS concentrations (300 micrograms/10(6) inhibited [3H]thymidine incorporation by MNL.     

Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle.

Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities.     

Chemotactic factors for bovine neutrophils in relation to mastitis

The chemotactic effect of a variety of agents for bovine blood polymorphonuclear neutrophils (PMN) was evaluated in vitro in assays of migration under agarose. Activated serum and leukotriene B4 showed significant chemotactic activity whereas various bacterial products, formyl peptides, casein and platelet activating factor failed to attract bovine PMN. In vitro cultures of bovine mammary gland macrophages released chemotactic activity for homologous PMN when stimulated by addition of opsonised, killed Staphylococcus aureus, Escherichia coli or Zymosan or sterile E. coli culture filtrates or endotoxin. No significant activity was produced by unstimulated macrophages. Pharmacological levels of various inhibitors or arachidonic acid oxygenation had no significant effect on the generation of PMN chemoattractants by mammary macrophages.     

Beta-hydroxybutyrate levels in peripheral blood and ketone bodies supplemented in culture media affect the in vitro chemotaxis of bovine leukocytes.

The role of ketone bodies on chemotactic capacities of leukocytes was characterized in two experiments. Experiment I was performed to investigate the association between serum beta-hydroxybutyrate concentrations (BHB) and in vitro chemotaxis of leukocytes. Cows were divided into low-BHB, medium-BHB, and high-BHB ones and classified according to their BHB. Leukocytes from high-BHB cows had a significantly lower chemotactic differential than leukocytes from low-BHB cows (p < 0.01). The effect of adding ketone bodies into in vitro chemotaxis cultures on leukocytes chemotaxis was studied in Experiment II. Either individual or a combination of commercial ketone bodies - sodium salts of BHB (BHBA), lithium salt of acetoacetate (ACAC), and acetone (Acetone) - were diluted in culture media and divided into eight concentrations corresponding to concentrations of bovine subclinical and clinical ketosis. For leukocytes from medium- and high-BHB cow, the chemotactic indexes of leukocytes were reduced by ACAC and Acetone. Chemotactic differentials of cultures with ACAC and acetone supplementation from both sources of leukocytes were significantly lower than that of the control culture (p < 0.05). For leukocytes from high-BHB cows, chemotactic indexes were suppressed in a ketone-body environment. In conclusion, leukocytes from naturally-occurring ketotic cows have lower chemotactic differentials than those from non-ketotic cows, and a chemotactic capacity indicated by a chemotactic differential is impaired when leukocytes migrate in an environment with ketone bodies in vitro.     

Phagocytic and killing capacities of uterine-derived polymorphonuclear leukocytes from mares resistant and susceptible to chronic endometritis

The host defense competence of uterine-derived polymorphonuclear leukocytes (PMN) from mares considered resistant (grade I uteri) and susceptible (grade III uteri) to chronic endometritis was evaluated for phagocytic and killing (bactericidal) capacities, using a fluorochrome assay. Peripheral blood PMN from noncategorized mares and from grade I and grade III mares were used as controls. Uterine-derived PMN from mares with grade I uteri were functionally competent for phagocytosis and killing of Candida albicans, whereas uterine-derived PMN from mares with grade III uteri had significantly less phagocytic and killing capacities (P greater than or equal to 0.0001). Results of the present study, together with data obtained from chemotactic responsiveness and deformability assays of a previous study, indicated an overall deficiency in the host defense mechanism of uterine-derived PMN from mares with grade III uteri obtained 12 hours after induced Streptococcus zooepidemicus infection. This deficiency may account for the susceptibility of mares with grade III uteri to chronic endometritis.     

Comparison of peripheral blood and uterine-derived polymorphonuclear leukocytes from mares resistant and susceptible to chronic endometritis: chemotactic and cell elastimetry analysis

The functional competence of peripheral blood and uterine-derived polymorphonuclear leukocytes (PMN) from 12 mares were analyzed for chemotactic responsiveness using a chemotactic chamber (filter) assay and for deformability by cell elastimetry analysis. Peripheral blood PMN obtained from control mares and from 8 mares experimentally inoculated via the uterus with 1 x 10(9) Streptococcus zooepidemicus had similar normal chemotactic responsiveness and were highly deformable before and at 12 hours after inoculation. Uterine PMN obtained 12 hours after uterine inoculation with S zooepidemicus from resistant mares were not as deformable as peripheral blood PMN, but were within normal functional limits. The chemotactic responsiveness of uterine PMN from these mares was normal. Uterine PMN obtained from mares considered susceptible to endometritis 12 hours after uterine infection did not have chemotactic responsiveness and were nondeformable. The results indicated profound differences in the functional competence of uterine PMN between mares considered resistant and susceptible to chronic endometritis.     

Effects of four antigenic fractions of Pasteurella multocida serotype A on phagocytosis of chicken peripheral blood leukocytes.

The effects of four antigenic fractions of Pasteurella multocida serotype A isolated from a duck in the Philippines on the phagocytic activity of chicken peripheral blood leukocytes were studied by a flow cytometer. These fractions were the lipopolysaccharide-protein complex (LPS), crude capsular antigen (CCA), ribosomal fraction (RS) and outer cell layer (OCL). Among these four antigens, only CCA but not LPS RS and OCL, significantly increased the phagocytic activities of mononuclear cells (MNC) and polymorphonuclear cells (PMN). This result indicates that CCA has an immunological property enhancing the phagocytic activities of MNC and PMN.     

Examination of chemotactic properties of bovine mammary macrophages.

An in vitro model system in which polymorphonuclear leukocytes (PMN) migration under agarose was employed to examine the ability of mammary macrophages to release chemoattractants for PMN. Mammary macrophages were incubated in Hanks' balanced salt solution for up to 12 h in the presence of Staphylococcus aureus. The possibility that the chemotactic activity is mediated through release of interleukin 1 (IL-1) and prostaglandins (PGs) by mammary macrophages was investigated. The data showed that release of chemotactic activity peaked 6 h following addition of S. aureus in the culture medium of mammary macrophages. Very low levels of IL-1 were detected in the same culture medium. Addition of indomethacin, a PGs synthesis inhibitor, was ineffective in altering the chemotactic activity detected in the culture medium of macrophages. These data suggest that it is highly unlikely that the chemotactic activity is mediated through the production of IL-1 and PGs by the mammary macrophages.     

Depressed neutrophil chemotactic stimuli in supernatants of ionophore-treated polymorphonuclear leukocytes from selenium-deficient goats

The effect of dietary selenium (Se) deficiency on the generation of neutrophil chemotactic factors from goat polymorphonuclear leukocytes (PMN) was studied. Polymorphonuclear leukocytes were isolated from 2 groups of crossbred goats. One group (n = 3) was fed a diet deficient in Se; the other (n = 3) was fed a diet adequate in Se. The PMN from each goat were stimulated with calcium ionophore A23187 and supernatants were collected. Each supernatant was immediately assayed for its ability to elicit neutrophil chemotaxis (NCT) and neutrophil chemokinesis by the Boyden chamber technique and neutrophil chemiluminescence (NCL) by the luminol-dependent chemiluminescence assay. Measurements from each group were compared, using the Student's t test. There was no significant difference (P greater than 0.01) in neutrophil chemokinesis elicited by supernatants derived from stimulated PMN from the 2 groups. However, supernatants derived from PMN of goats fed the Se-deficient diet had a significantly decreased capacity (P less than 0.01) to stimulate NCT and NCL. In vitro incubation of PMN with Se before ionophore treatment produced supernatants with increased NCL stimulation (P less than 0.05), but none with increased NCT stimulation (P greater than 0.05). However, both activities were restored after goats fed the Se-deficient diet (P less than 0.01) were given Se subcutaneously. Seemingly, dietary Se deficiency is associated with depressed PMN generation of factors that stimulate NCT and NCL. In contrast to NCL factors, synthesis of factors stimulating NCT probably was not associated directly with PMN glutathione peroxidase concentration.     

In vitro regulation of Mac-1 expression on bovine polymorphonuclear leukocytes by endotoxin and tumor necrosis factor-α at different stages of lactation

The purpose of this in vitro study is to clarify some of the underlying mechanisms leading to the decreased migratory capacity of polymorphonuclear leukocytes (PMN) during mastitis in dairy cows soon after calving. Surface expression of Mac-1 (CD11b, CR3) on PMN and of CD14 on monocytes was measured in early- (EL), peak- (PL), and midlactation (ML) by flow cytometric analysis. In addition, we evaluated the effect of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha on CD11b surface expression in PMN at different stages of lactation in a whole blood model. During EL, while resting monocytes expressed diminished levels of CD14, the basal expression of CD11b on PMN was not significantly altered. The relative increase of CD11b on PMN after incubation with LPS or TNF-alpha did not significantly differ among EL, PL, or ML at any of the concentrations tested. The current findings do not support an important role for basal CD11b levels nor for a defective mobilization of CD11b by LPS and TNF-alpha in the reduced migratory capacity of PMN during EL.     

Bovine leukocytes in mammary secretions during involution: surface protein changes

Changes in surface proteins of leukocytes in bovine mammary secretions were examined during mammary involution. Leukocytes were collected from mammary secretions from involution days (ID) 1 to 36 and were labeled with biotin. Labeled proteins were separated by polyacrylamide-gel electrophoresis, transferred to nitrocellulose by protein blotting, and detected with avidin-peroxidase and color reagent. The population of leukocytes in mammary secretions changed during involution from predominantly polymorphonuclear leukocytes (PMN) at ID 1 to predominantly mononuclear leukocytes by ID 36. The gel profile of surface proteins also changed during involution, and some of those changes may have been caused by shifts in leukocyte populations. Profiles of surface proteins of leukocytes from mammary secretions containing predominantly PMN (ID 1) were different from profiles of PMN from blood. A distinctive change in the pattern of surface proteins on the leukocytes occurred between ID 1 and 3, even though the proportion of PMN did not change appreciably between those days.     

Separation of mononuclear leukocytes and polymorphonuclear leukocytes from equine blood.

The present study describes a two step technique for the separation of mononuclear leukocytes or mononuclear and polymorphonuclear leukocytes from whole equine blood. First, the leukocyte rich plasma was obtained by sedimentation of erythrocytes in the undiluted blood. Subsequently, separation of the different populations of white blood cells was performed by centrifugation with different gradients overlaid with the leukocyte rich plasma. The optimal separation of the mononuclear cells was obtained by the centrifugation of the leukocyte rich plasma overlaying the gradient containing 24 parts of 9.5% ficoll and ten parts of 34% isopaque. The mononuclear leukocytes (95% lymphocytes and 5% monocytes) formed a monolayer band at the plasma-ficoll-isopaque interface and other blood cells migrated to the bottom of the tube. For the separation of mononuclear and granular leukocytes from the blood, the gradient containing 24 parts of 10% ficoll and ten parts of 34% isopaque was used. The separated monuclear leukocytes responded to stimulation with phytohemagglutin and viability of both mononuclear and polymorphonuclear leukocytes was not affected by ficoll-isopaque separation.     

Effect of cephapirin and mecillinam on the phagocytic and respiratory burst activity of neutrophil leukocytes isolated from bovine blood

Antimicrobial therapy is the most commonly used treatment of bacterial infections in dairy cows. Polymorphonuclear neutrophil leukocytes (PMN) play an important role in the first line defence against invading bacteria and it is important that the function of PMN is not compromised by antibiotics. We investigated the in vitro effect of cephapirin, a first generation cephalosporin, and mecillinam, an amidinopenicillin with activity against mainly Gram-negative bacteria, on phagocytosis and respiratory burst activity of PMN isolated from bovine blood. After in vitro incubation of PMN with different concentrations of the antibiotics, phagocytosis was evaluated by flow cytometry and respiratory burst activity was evaluated by registration of chemiluminescence (CL) with a luminometer. None of the investigated concentrations of cephapirin and mecillinam had an effect in vitro on phagocytosis of Escherichia coli by PMN. At high concentrations (100 and 1000 μg/mL), cephapirin and mecillinam reduced the respiratory burst activity of PMN. Part of these suppressive effects could be ascribed to oxidant scavenging. Inhibitory effects of cephapirin were stronger than mecillinam. In conclusion, cephapirin and mecillinam did not seem to affect antibacterial activity of PMN isolated from bovine blood in vitro at therapeutic concentrations.     

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.     

Endotoxin-induced bovine mastitis: immunoglobulins, phagocytosis, and effect of flunixin meglumine

Milk whey immunoglobulins (Ig) and phagocytosis of staphylococci by milk polymorphonuclear neutrophilic leukocytes (PMN) were measured in 12 cows (allotted to 6 pairs) during acute bovine mastitis induced by intramammary inoculation of endotoxin. Six of these cows (or 1 in each pair) were treated with flunixin meglumine and were compared with the others (given only saline solution). The endotoxin inoculation comprised 10 micrograms of Escherichia coli O26:B6 lipopolysaccharide injected into one of the rear quarters (mammae). Flunixin meglumine was administered parenterally at a dosage of 1.1 mg/kg every 8 hours (total of 7 doses) beginning at 2 hours after endotoxin was injected. Milk samples were obtained, and whey samples were prepared from each quarter of each cow 3 times before inoculation and at 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hours after endotoxin was inoculated. Significant increases (P less than 0.05) in milk whey IgG1, IgG2, IgM, and IgA concentrations were observed in whey samples from endotoxin-inoculated quarters. Greatest relative increase was seen for IgG2. Increased whey Ig concentrations were not observed in quarters which were not inoculated with endotoxin. Concentrations of whey IgG1 and IgM in endotoxin-inoculated quarters were significantly (P less than 0.05) decreased in flunixin meglumine-treated cows, compared with those in saline solution-treated cows. Significant increases in phagocytosis of staphylococci by milk PMN were observed in whey samples from endotoxin-inoculated quarters. Significant differences in PMN phagocytosis were not found in whey samples from cows given flunixin meglumine when compared with whey samples from cows given saline solution.     

In vitro and in vivo 2',5'-oligoadenylate synthetase activity induced by recombinant DNA-derived bovine interferon alpha I1 in bovine alveolar macrophages and blood mononuclear cells.

Biological responses to recombinant DNA-derived bovine interferon alpha (rBoIFN-alpha I1) by bovine alveolar macrophages were examined by measuring viral yield reduction and 2',5'-oligoadenylate synthetase (2',5'-OAS) production by IFN-treated cells. In vitro IFN pretreatment of alveolar macrophages reduced viral yield in cultures challenged exposed with parainfluenza-3 virus, compared with control cultures. In vitro treatment of alveolar macrophages with IFN also resulted in increased 2',5'-OAS activity. The 2',5'-OAS activity was measured in alveolar macrophages and blood mononuclear leukocytes of calves injected IM with 3.6 x 10(6) U of rBoIFN-alpha I1/kg of body weight. The IFN action was monitored by measuring 2',5'-OAS activity of blood mononuclear leukocytes beginning 6 days before and ending 24 hours after IFN treatment. The 2',5'-OAS activity in the blood mononuclear leukocytes sharply increased 24 hours after IFN treatment, indicating response to IFN. The alveolar macrophages collected from the same calves 24 hours after IFN administration also had increased 2',5'-OAS activity, compared with alveolar macrophages from the same calves collected 6 days before treatment. Increased 2',5'-OAS activity indicates: a possible mechanism of IFN action in cattle that may be responsible for viral yield reduction; potential use of high enzyme activity as a marker for IFN induction; and potential use of 2',5'-OAS activity as a marker for determining effects of IFN on bovine macrophages and other cells of the bovine immune system.     

Local and systemic effects of endotoxin mastitis on the chemiluminescence of milk and blood neutrophils in dairy cows

The local and systemic effects of intramammary lipopolysaccharide (LPS) injection on the chemiluminescence (CL) of milk and blood polymorphonuclear leukocytes (PMN) were investigated in six healthy early lactation cows. Clinical signs of acute mastitis such as fever, increased heart rate and a decreased milk production were observed in all cows. Before LPS challenge, the CL activity of milk PMN was significantly lower than that of blood PMN (P < 0.01). A significant negative correlation was found between pre-challenge milk and blood PMN CL and, the decreased milk production in unchallenged quarters. The CL activity of milk PMN from LPS-injected quarters increased following LPS challenge, whereas it remained unchanged in control quarters. The CL activity of blood PMN showed a biphasic increase, with two peaks and a valley below pre-challenge CL activity (P < 0.01). At post-challenge hours (PCH) 6 and 12, the CL activity of milk PMN from LPS-injected quarters exceeded that of blood PMN (P < 0.05 and P < 0.001, respectively). The decreased CL activity of blood PMN and the enhanced CL activity of milk PMN during endotoxin-induced mastitis was reflected by changes in the shape of the CL curve. In blood PMN, a decrease of the second peak of the CL curve suggests that the myeloperoxidase (MPO)-H2O2 system is impaired during endotoxin-induced mastitis. In contrast, the MPO-H2O2 system was enhanced in milk PMN from challenged quarters. The highest duration and intensity of reactive oxygen intermediate (ROI) production was observed in milk PMN from LPS-injected quarters at PCH 12. The increased viability of PMN in LPS-injected quarters and to a lesser extent in control quarters suggests possible effects of both facilitated diapedesis and inflammatory mediators on milk PMN survival. In conclusion, our results suggest that a combination of local and systemic action of E. coli endotoxin is involved in the priming of milk PMN during mastitis.     

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.