Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists.

Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O2-) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C (PKC) activation is considered to be an important step in the signal transduction pathway for the O2- generating system, we compared O2- production by newborn and adult bovine neutrophils stimulated with 3 different PKC agonists. When the phorbol ester phorbol 12-myristate 13-acetate (PMA) was used, PKC-dependent O2- generation from newborn neutrophils was significantly reduced (P less than 0.01) for all concentrations of PMA tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly (P less than 0.01) reduced lag time for O2- generation. Similar significantly (P less than 0.01) reduced O2- generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12,13-dibutyrate); lag times were not calculated for phorbol 12,13-dibutyrate. When O2- generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O2- was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly (P less than 0.01) less O2- only at 50 microM 1,2-dioctanoyl-sn-glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay for the quantitation of Actinobacillus pleuropneumoniae cytotoxin.

Using swine neutrophils as target cells, two MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay systems, one with and one without phorbol 12-myristate 13-acetate (PMA) stimulation were established for the quantitation of Actinobacillus pleuropneumoniae cytotoxin. The MTT assays were optimized for the number of neutrophils, incubation time, and PMA concentration by a series of experiments. The optimal conditions were 25 x 10(4) cells/well incubated for four hours for the assay system without PMA stimulation, and 12.5 x 10(4) cells/well incubated for two hours for the assay system with PMA stimulation. One culture supernatant of a toxigenic Pasteurella multocida strain and five A. pleuropneumoniae cytotoxin preparations produced from three A. pleuropneumoniae strains were used to test assay reproducibility. Results showed both assays were reproducible with a coefficient of variation ranging from 7.8 to 18% for the assay system without PMA stimulation and from 10.7 to 18.2% for the assay system with PMA stimulation. The PMA-stimulated assay had 40 to 60-fold higher sensitivity than the nonstimulated MTT assay. The MTT assay also was applied to the measurement of neutralizing antibody titers against A. pleuropneumoniae cytotoxin.     

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

OBJECTIVE: 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. SAMPLE POPULATION: 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). PROCEDURES: 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. RESULTS: 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. CONCLUSIONS AND CLINICAL RELEVANCE: 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.     

Non-immune control of trypanosomosis: in vitro oxidative burst of PMA- and trypanosome-stimulated neutrophils of Boran and N'Dama cattle

An in vitro assay that measures the generation of superoxide anions (O2-) was used to assess the level of oxidative burst of phorbol myristate acetate (PMA)- and trypanosome-stimulated neutrophils isolated from healthy Boran and N'Dama cattle, and those infected with Trypanosoma congolense. PMA stimulation of healthy bovine neutrophils resulted in between 300-400 % increase in O2- generation. Neutrophils of Boran cattle exhibited slightly higher but insignificant O2- generation capacity than those of the N'Dama breed. In vitro stimulation by trypanosomes of neutrophils isolated from Trypanosoma congolense-infected cattle caused significant increases in O2- generation, especially on days 14, 28 and 42 post-infection, of both breeds of cattle. No significant differences were observed in O2- generation capacity of the neutrophils of both breeds of infected cattle throughout the period of assay. The results of this study have shown that PMA and trypanosomes do cause an enhanced in vitro oxidative burst, hence trypanosome phagocytosis and killing activity of neutrophils. Neutrophils have been shown to play very significant roles in parasite clearance, hence reduction of trypanosome parasitaemia. The rates of both in vitro generation of O2- and trypanosome phagocytosis over time did not differ significantly between Boran and N'Dama breeds of cattle, even during T congolense infection in this study. Hence, it may be inferred that sustained and higher parasitaemia, more pronounced neutropenia, inadequate bone marrow response and less effective trypanosome-specific immune response, rather than defective neutrophil trypanosome destruction, may be the problem of trypanosusceptible cattle breeds.     

In vitro migration responses of neutrophils from cows and calves

The directional (chemotactic) and random migration activities of neutrophils from cows and newborn and 2-week-old calves were determined by use of the chemotaxis-under-agarose assay. Blood samples were stored for 2, 24, or 48 hours and at 4 or 25 C before testing. During the assay, cells were incubated at 17, 27, or 37 C. The assay was found suitable for testing the directional and random migration activities of neutrophils from cattle. Directional migration of neutrophils was diminished (P less than or equal to 0.05) when cells were incubated at 17 or 27 C, compared with data from incubation at 37 C. Random migration of neutrophils was unaffected by test incubation temperature. Significant (P less than or equal to 0.05) differences were found between cows and calves regarding the percentage number and viability and the directional and random migration activities of neutrophils. Neutrophils from cows were adversely affected to a greater extent by prolonged sample storage times or low storage temperature than were neutrophils from calves. Results indicate that a sample storage time of up to 24 hours, a sample storage temperature of 25 C, and a test incubation temperature of 37 C provided optimal conditions for testing the migratory activities of neutrophils from cattle.     

Decreased protein kinase C activity in fatty liver from cattle

Protein kinase (PK) C activity in the liver of cattle with fatty liver syndrome was evaluated and compared with that in liver of healthy cattle. The PKC activities in cytosolic and particulate fractions were reduced in fatty livers, compared with those in livers from healthy cattle. The decrease of PKC activity was more distinct in cytosolic (P = 0.0016) than particulate (P = 0.069) fractions. Protein kinase activities other than PKC were not substantially changed. Seemingly, PKC was involved in the pathogenesis of fatty liver syndrome in cattle.     

Dependence of superoxide anion production on extracellular and intracellular calcium ions and protein kinase C in PMA-stimulated bovine neutrophils.

The involvement of both intracellular and extracellular calcium, as well as the activation of protein kinase C (PKC), in phorbol myristate acetate (PMA)-stimulated respiratory burst in bovine neutrophils has been studied. PMA significantly stimulated the superoxide anion production by these cells. The increased production of superoxide anion was inhibited by BAPTA/AM, an intracellular calcium ([Ca2+]i) chelator, but not affected by EGTA, an extracellular calcium ([Ca2+]0) chelator. PMA also induced PKC activation, and a PKC inhibitor, calphostin C, blocked the stimulatory effect of PMA on superoxide anion production by the neutrophils. Therefore, we conclude that PMA-induced respiratory burst in bovine neutrophils is [Ca2+]i- but not [Ca2+]0-dependent, and also requires PKC activation.     

Cell-surface lactoferrin as a marker for degranulation of specific granules in bovine neutrophils

OBJECTIVE: To develop a rapid and accurate flow cytometric method for measuring degranulation of specific granules in bovine neutrophils. SAMPLE POPULATION: Blood samples obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: A monoclonal antibody (BL97) was generated against bovine lactoferrin and tested for applicability in ELISA, immunoprecipitation tests, immunofluorescence microscopy, and flow cytometric analyses. Using this antibody, cell-surface lactoferrin was measured concurrent with amount of secreted lactoferrin from bovine neutrophils activated with phorbol myristate acetate (PMA). Cell-surface lactoferrin also was measured on neutrophils in bovine whole blood stimulated with PMA, platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLF), and interleukin 8 (IL-8). RESULTS: Antibody BL97 recognized bovine lactoferrin in ELISA and western immunoblots and was useful for immunoprecipitation testing, immunofluorescence microscopy, and flow cytometric analyses of bovine leukocytes. Neutrophils activated with PMA had parallel increases in content of secreted lactoferrin (measured by ELISA) and cell-surface lactoferrin (measured by flow cytometry) with increasing PMA concentrations. In addition, fluorescein-conjugated BL97 antibody detected increases in cell-surface lactoferrin on neutrophils in bovine whole blood after activation with PMA, PAF, and IL-8. In contrast, increases in cell-surface lactoferrin were not detected on bovine neutrophils treated with fMLF. CONCLUSION AND CLINICAL RELEVANCE: Measurement of cell-surface lactoferrin on bovine neutrophils by flow cytometry is a valid and rapid method for assessment of release of lactoferrin from specific granules in these cells and represents a means to rapidly measure neutrophil activation. This technique allows for investigation of mechanisms of neutrophil modification in isolated cells as well as in whole blood.     

Extracellular signal-regulated kinase (ERK) activation in chicken heterophils stimulated with phorbol 12-myristate 13-acetate (PMA), Formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS)

The signaling pathways leading to the activation of extracellular signal-regulated kinase (ERK) by phorbol 12-myristate 13-acetate (PMA), formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) in chicken heterophils were examined. To determine the mechanism of ERK's activation and its relation with the influx of calcium ions, heterophils were stimulated by PMA, fMLP and LPS. ERK was not activated by fMLP. LPS- and PMA-stimulated activation of ERK, based on Western blotting with antibodies against the phosphorylated form of ERK, was attenuated by the pretreatment of cells with the intracellular calcium chelator BAPTA/AM (1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) but not with the extracellular calcium chelator EGTA (glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Exposure of cells to the protein kinase C (PKC) inhibitor GF109203X inhibited the LPS- and PMA-stimulated phosphorylation of ERK in a concentration-dependent manner. The LPS-stimulated phosphorylation was inhibited by pretreatment with the phospholipase C (PLC) inhibitor U73122 but not the phosphatidylinositol 3-kinase (PI₃K) inhibitor LY294002. These results indicate that the LPS-induced phosphorylation of ERK in the chicken heterophils is mediated by PLC, PKC and intracellular calcium, and the PMA-stimulated phosphorylation is dependent on intracellular calcium ion and PKC.     

Effects of maternal protein-energy malnutrition and cold stress on neutrophil function of bovine neonates

The effects of maternal protein or calorie deprivation (or both) on the bactericidal activity of neutrophils and sera from newborn calves subjected to cold stress were studied. Nutritional deficiencies in the dam had little effect on in vitro bactericidal activity of neutrophils and base-line sera taken at birth. Neutrophils obtained at birth destroyed Staphylococcus aureus but not Escherichia coli when incubated with either unheated or heated autologous base-line sera. Heat treatment of base-line sera to inactivate complement did not alter bacterial growth. When incubated in the presence of autologous base-line sera, neutrophils from 3-day-old calves were no more active in the destruction of either bacterium than were neutrophils from newborn calves. However, addition of day 3 (immunoglobulin-containing) sera enabled day 3 neutrophils to destroy E coli (P < 0.0001). The increased destruction of E coli by day 3 neutrophils and day 3 sera was not affected by heat treatment of the sera. Maternal protein deficiency significantly increased (P < 0.05) destruction of E coli by day 3 neutrophils and sera. This effect was independent of energy levels. There were no differences observed in the bactericidal activity of neutrophils and sera taken from calves exposed to 1 C or 21 C environmental chambers for 3 days. Also, cold stress-nutritional stress interactions were not detected.     

Non-specific immunity and ketone bodies. II: In vitro studies on adherence and superoxide anion production in ovine neutrophils

The effects of the ketone bodies beta-OH-butyrate and acetoacetate (2.4 or 4.8 mmol/l), administered singly or simultaneously in vitro, on adherence and superoxide anion (SO) production in ovine neutrophils were investigated by simultaneous assay in 96-well microplates. Because the acetoacetate used was a lithium salt, the effect of 2.4 and 4.8 mmol/l lithium chloride was also tested. Neutrophils from eight non-lactating, non-pregnant ewes were used. SO release from neutrophils was found to be very low in basal conditions and was apparently not stimulated by contact with plastic. Administration of 10(-7) mol/l phorbol myristate acetate (PMA) caused a rapid increase and release of SO production, but smaller than that induced by co-stimulation with plastic and 10(-7) mol/l PMA. LiCl (2.4 and 4.8 mmol/l) significantly increased PMA-stimulated release, but inhibited plastic and PMA co-stimulated SO release. Administration of 2.4 mmol/l ketone bodies inhibited plastic and PMA-costimulated SO release, but the effect of acetoacetate could be due to the lithium component. Administration of 4.8 mmol/l ketone bodies had no effect. Adherence was significantly increased by contact with plastic, and moreover by 10(-7) mol/l PMA. The effect was similar when PMA was acting alone or with plastic. Neither basal nor stimulated adherence were affected by 2.4 or 4.8 mmol/l ketone bodies. LiCl at a concentration of 4.8 mmol/l increased PMA and plastic co-stimulated adherence. The results suggest that, in sheep, only the ketone body beta-OH butyrate at concentrations seen in mild ketosis, could decrease bactericidal activity, while adherence is not affected. In addition to other factors that could impair the efficiency of the immune system in ketotic ruminants, the reduced bactericidal activity may contribute to the higher occurrence of infectious disease in these animals.     

A comparison of foal and adult horse neutrophil function using flow cytometric techniques

Flow cytometric assays were used to compare phagocytic and oxidative burst activity of neutrophils from healthy foals less than 7 days of age with the activity of cells from healthy adult horses. The phagocytosis of Staphylococcus aureus by foal neutrophils was less than that observed for adult neutrophils when autologous serum was used as the source of opsonins in the assay. The use of adult serum did not significantly improve the ability of foal neutrophils to attach bacteria. The oxidative burst activity of foal neutrophils was equivalent to that of adult cells. However, when serum or plasma was incorporated into the oxidative burst assay, foal neutrophils demonstrated greatly reduced autofluorescence and a suppressed response to phorbol myristate acetate (PMA), relative to that demonstrated by adult cells. These results suggest that peripheral blood neutrophils from foals have a reduced ability to phagocytose bacteria relative to that exhibited by adult horse neutrophils and that the oxidative burst activity of foal neutrophils is down-regulated in response to an unidentified serum factor(s). Such changes may contribute to the increased susceptibility of foals to septic disease.     

Pasteurella haemolytica cytotoxin: relative susceptibility of bovine leucocytes

An in vitro 51Cr-release assay was used to compare the susceptibility of various leucocytes from normal cattle to Pasteurella haemolytica cytotoxin. Neutrophils were found to be more sensitive than mammary or bronchoalveolar macrophages. Neutrophils induced with lipopolysaccharide (LPS) and mammary macrophages activated in vitro with LPS were as sensitive as homologous untreated cells. Bronchoalveolar macrophages from adult cows were significantly more resistant than those from calves. Sub-cytolytic concentrations of cytotoxin did not impair killing of para-influenza-3 virus infected MDBK cells by mammary macrophages.     

Bactericidal activity of porcine neutrophil secretions

Antimicrobial proteins in neutrophil granules exert their bactericidal activity both within the neutrophil phagolysosome and as components of neutrophil extracellular traps. This study evaluated the bactericidal activity of porcine neutrophil secretions against four bacterial pathogens of swine. Porcine neutrophils were treated with or without phorbol myristate acetate (PMA), then the resulting supernatants were incubated with Escherichia coli K-12, Streptococcus suis, Actinobacillus suis, or Pasteurella multocida, and the surviving colony forming units were enumerated. Supernatants of PMA-activated neutrophils killed an average of 95% of E. coli K-12 cells, relative to supernatants from untreated neutrophils. Inhibition of elastase activity using chloromethylketone (CMK) prior to PMA stimulation significantly reduced the bactericidal activity of the neutrophil supernatants; 57% of the PMA-induced bactericidal activity against E. coli K-12 was estimated to be elastase-dependent. The same neutrophil supernatants had lower bactericidal activity against S. suis, A. suis, and P. multocida, with 30%, 36% and 13% reduction in bacterial numbers, respectively. The cathelicidin porcine myeloid antimicrobial peptide (PMAP)-36 and lactotransferrin were among the proteins identified in the supernatants of PMA-stimulated neutrophils by mass spectrometry. These findings imply that elastase-activated proteins, such as cathelicidins, are partially responsible for the bactericidal effect of porcine neutrophil secretions, but non-elastase-dependent proteins such as lactoferrin may also contribute. Further, the secretions of activated neutrophils were effective in killing the avirulent E. coli K-12 but were less effective against the other bacteria tested, suggesting that these pathogens may have evolved mechanisms to resist neutrophil-mediated killing.     

Comparison of morphology, viability, and function between blood and milk neutrophils from peak lactating goats

The morphological features of blood and milk neutrophils from peak lactating goats were compared using light microscopy, scanning electron microscopy and flow cytometry in order to investigate the cytological changes of neutrophils after migration into the mammary gland. The kinetics of reactive oxygen intermediates (ROI) generation and gelatinase release of blood and milk neutrophils, with or without stimulation of phorbol 12-myristate, 13-acetate ester (PMA), were used to characterize their responses to inflammatory stimuli. Neutrophils isolated from goat milk were highly segmented and contained multi-lobed nuclei. Ultrastructurally, milk neutrophils were more ruffled on the surface compared to blood neutrophils. Approximately 30% of milk neutrophils were undergoing cell death, either necrosis or apoptosis, in contrast to 8% of blood neutrophils. The ROI production of activated milk neutrophils peaked earlier than blood neutrophils, but the duration and the intensity were much less. Neutrophils from both sources augmented the release of gelatinase in response to PMA (1 ng/mL). However, the amount of gelatinase released from milk neutrophils was lower (P < 0.05) than that of blood neutrophils. In summary, more neutrophils become apoptotic and necrotic in the mammary gland, presumably due to spontaneous aging, the process of diapedesis, and the interaction with milk components. Milk neutrophils have impaired functionalities in comparison with blood neutrophils. The information is relevant when studying mammary gland immunity and related diseases, such as mastitis.     

Adherence to bovine neutrophils and suppression of neutrophil chemiluminescence by Mycoplasma bovis

The adherence of viable and heat-treated Mycoplasma bovis to bovine peripheral blood neutrophils was studied by specific immunofluorescence staining and flow cytometry. Viable and heat-treated M. bovis cells, adhered to bovine neutrophils in dose-dependent fashion within a 30 min incubation. Fluorescence quenching using crystal violet indicated that unopsonized M. bovis cells remained on the surface of bovine neutrophils without experiencing significant ingestion. The effect of M. bovis adherence on neutrophil microbicidal function was examined by measuring luminol enhanced chemiluminescence (CL). Adherent M. bovis cells did not elicit a bovine neutrophil CL response over a 75 min incubation period. M. bovis inhibited the capacity of bovine neutrophils to mount a CL response. Inhibition occurred whether viable or heat-treated M. bovis cells were used and it occurred when neutrophils were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). Inhibition of the PMA stimulated neutrophil CL response required cytadherence by M. bovis cells. These findings suggest that activation of the bovine neutrophil respiratory burst was inhibited at or distal in the pathway to the activation of protein kinase C (PKC), the site of PMA stimulation, and that it was mediated by a direct interaction between the adhering M. bovis cells and the bovine neutrophil membrane.     

Bovine recombinant granulocyte-macrophage colony-stimulating factor enhancement of bovine neutrophil functions in vitro

Neutrophils were purified from blood of dexamethasone-treated (0.04 mg/kg of body weight) and untreated calves. Cells were untreated (controls) or cultured in media containing 5 or 10 ng of bovine recombinant granulocyte-macrophage colony-stimulating factor (rbGM-CSF)/ml for 10 to 12 hours before being tested for various functions. Dexamethasone treatment of calves decreased luminol-dependent chemiluminescence, decreased phagocytosis of Pasteurella multocida and several Staphylococcus spp by various degrees, and decreased antibody-dependent cell-mediated cytotoxicity against bovine herpesvirus-infected cells by 26 to 32%. The percentage phagocytosis of coagulase-positive S aureus and S intermedius was higher than that of coagulase-negative S epidermidis for neutrophils from all calves. Culture of neutrophils with rbGM-CSF significantly increased (P less than 0.05) all of the aforementioned functions, compared with control neutrophils; however, rbGM-CSF-induced increases in function tended to be higher in neutrophils from dexamethasone-treated calves than in neutrophils from untreated calves.     

Secretion of hydrogen peroxide by phagocytic cells from bovine non-lactating mammary glands

Phagocytic cells from non-lactating bovine mammary glands have the capacity to secrete hydrogen peroxide when exposed to the soluble membrane stimulant phorbol myristate acetate (PMA). Unfractionated cell suspensions, containing mainly neutrophils and macrophages, and cell monolayers enriched for macrophages secreted hydrogen peroxide. A correlation was observed between the amount of hydrogen peroxide secreted, the antibacterial activity of the cells and the number of neutrophils present in the cell suspensions. Pre-exposure of cells to PMA significantly impaired their antibacterial activity against Staphylococcus aureus suggesting the importance of oxygen metabolism in the bactericidal capacity of these cells.     

Tissue and intracellular distribution of rhodanese and mercaptopyruvate sulphurtransferase in ruminants and birds

Cyanide detoxification is catalysed by two enzymes: rhodanese [thiosulphate: cyanide sulphurtransferase, E.C. 2.8.1.1], and 3-mercaptopyruvate sulphurtransferase [3-MST, EC. 2.8.1.2]. In the present work, the activity of the two enzymes in the crude extracts of different tissues and in the mitochondrial and cytosolic fractions of tissues from some ruminants (camels, cattle and sheep) and birds (chickens and pigeons) have been compared. Rhodanese activity was almost exclusively present in the mitochondrial fraction. In ruminants and chickens the highest activity of rhodanese was found in the liver, followed by the kidney. In pigeons, however, the enzyme activity was the highest in the kidneys. In camels' tissues, the rhodanese activity was significantly (P < 0.05) lower than in cattle or sheep, and the enzyme activities in the two latter species were similar. The activity of 3-MST in the crude extract of tissues from camels was similar to that in sheep, but higher than that in cattle. The enzyme activity was equally distributed between the mitochondrial and cytosolic fractions in the liver and kidneys of camels, cattle and sheep.     

Modulation, in vivo and in vitro, of surface expression of CD18 by bovine neutrophils.

A series of experiments was designed to elucidate some of the factors that may influence surface expression of CD18 by bovine neutrophils. Expression of CD18 was determined by immunofluorescence flow cytometry. Neutrophils recovered from the uterus of cows (n = 9) after intrauterine administration of sterile oyster glycogen solution expressed (mean +/- SD) 123 +/- 21% more CD18 than did circulating neutrophils recovered simultaneously from the same cows (P = 0.003). In 8 cows given 20 mg of dexamethasone IM daily for 3 days, expression of CD18 on blood neutrophils was 29.6 +/- 8% less after treatment than before treatment (P = 0.0078). Neutrophils from 12 cows or bulls exposed to phorbol myristate acetate in vitro increased expression of CD18 by 137 +/- 37% (P = 0.0035). Likewise, exposure of neutrophils from 8 cattle to zymosan-activated bovine plasma increased CD18 exposure by 10.6 +/- 3.8% (P = 0.029). These findings indicate that expression of CD18 by bovine neutrophils is a dynamic system, capable of responding to inflammatory stimuli. Inadvertent activation of neutrophils may be responsible for some of the variance in expression observed when examining large groups of cattle for CD18 expression by neutrophils. The ability of bovine neutrophils to respond rapidly to various stimuli by increasing surface expression of CD18 indicates that a pool of intracellular CD18 may be available for inclusion in the plasmalemma, as has been reported for human neutrophils.