Expression and role of adhesion molecule CD18 on bovine neutrophils.

Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions.     

Assessment of neutrophil components as markers of lung injury in the course of bovine respiratory tract infections

To define the role of activated neutrophils in lung injury during bovine respiratory tract infections (BRTI) their in vitro function was investigated. As a means to achieve this goal the comparison of secretory action between neutrophils from the BRTI group and control was made on the basis of elastase, myeloperoxidase (MPO), alkaline phosphatase (ALK-P) release, and nitric oxide production. We noted that there is an interdependence between secretory response of neutrophils and clinical severity of BRTI. The release of elastase was greater in the BRTI group than in the control group (49.17+/-4.41 versus 46.43+/-4.95% of the total content). Neutrophils from infected heifers exhibited a significantly (p<0.05) higher value of MPO release than from healthy heifers and reached 39.23+/-10.18 versus 25.54+/-8.41% of the total content. ALK-P containing granules released significantly (p<0.001) more enzyme in the group with BRTI than in the control group (22.42+/-6.27 versus 13.74+/-2.01% of the total enzyme content). The level of nitrite accumulation rose in the culture of cells isolated from heifers with BRTI from 4+/-0.53 microM after 0.5h to 6.9+/-0.52 microM after 72 h. Our data suggest that during BRTI the increase of neutrophil secretory action results in augmentation of enzyme release including elastase, MPO and ALK-P, and the nitrite production. During an excessive secretory response of neutrophils all these factors contribute to lung injury and worsen the course of a disease and might be recognised as markers of lung injury. Moreover, such a destructive action of neutrophils must be taken into account during the introduction of new methods of BRTI treatment.     

Quantification of bovine neutrophil migration across mammary epithelium in vitro.

A microassay system was developed to quantify bovine neutrophil transepithelial migration in vitro. The bovine mammary epithelial cell line (MAC-T) formed a confluent monolayer that served as a biologically meaningful barrier for neutrophil migration. Neutrophils added into the upper compartment of an inverted monolayer were driven to migrate across the epithelium from a basal-to-apical direction by the addition of zymosan activated serum (ZAS) into the lower compartment. The numbers of migrated neutrophils were determined by assaying the neutrophil azurophilic granule marker, myeloperoxidase. Results showed that ZAS stimulated neutrophil migration across the epithelium in a time-and dose-dependent manner. In the presence of 5% ZAS and 2 x 10(6) neutrophils, approximately 2.4 x 10(5) neutrophils migrated across the epithelium in 120 min. The procedures we have developed in this study provide a simple precisely controlled system to investigate the normal dynamics of bovine neutrophil transepithelial migration and a means to detect and study impaired neutrophil migration.     

Chemotactic responses of neutrophils in cats with spontaneous feline infectious peritonitis

The culture supernatant of peritoneal exudate cells (PEC) from cats with effusive feline infectious peritonitis (FIP) was chemotactic for peripheral blood neutrophils (PBN) from healthy cats, magnitude of the chemotactic activity being approximately 10-fold lower than that in zymosan-activated fresh serum of healthy cats (ZAS). The migration profile of PBN from healthy cats was slightly different between the PEC culture supernatant and ZAS. These findings suggest that the chemotactic activity detected in the PEC culture supernatant is distinct from that in ZAS. The chemotactic responses of PBN from FIP cats to ZAS were reduced, as compared with that from healthy controls. In contrast, the neutrophil chemotactic response and sensitivity to the PEC culture supernatant in FIP cats were not remarkably different from those in healthy controls. Furthermore, the chemotactic responsiveness of PEC from FIP cats to ZAS was slightly different from that of PEC to the PEC culture supernatant. These results suggest that neutrophils from FIP cats have altered reactivities against these chemoattractants.     

The influence of TNFalpha and IL-8 on secretory action of neutrophils isolated from heifers in the course of bovine respiratory disease

The goal of this study was to give insight into the lesser known action of neutrophils in the course of bovine respiratory disease (BRD). Neutrophils are principally involved in resistance to infection but under certain conditions these cells participate in lung injury. TNFalpha and IL-8, primary cytokines which play an essential role in neutrophil activation during the inflammatory process, may lead not only to host defence reaction but also to destructive responses. Neutrophil constituents, such as elastase, myeloperoxidase (MPO), alkaline phosphatase (ALKP) and superoxide among others, contribute to tissue destruction. Also, 5-oxo-eicosatetraenoic acid (5-oxo-ETE), the potent activator of neutrophil chemotaxis and degranulation, can cause lung injury. This study revealed that neutrophils under the influence of TNFalpha exacerbated the release of elastase, MPO, ALKP and superoxide. IL-8, in turn, triggered those neutrophils which were isolated from heifers suffering from BRD, and released elastase, ALKP and 5-oxo-ETE, but not MPO or superoxide (O2(-)). The most pronounced degranulative response was observed in the acute form of BRD and a lesser response was seen in the chronic form. The secretory action of neutrophils varied not only depending on the form and phase of the disease but it also diminished successively during recovery.     

In vitro evaluation of the role of platelet-activating factor and interleukin-8 in Mannheimia haemolytica-induced bovine pulmonary endothelial cell injury

OBJECTIVE: To develop an in vitro model of the bovine alveolar-capillary interface and to evaluate the roles of interleukin-8 (IL-8) and platelet-activating factor (PAF) in neutrophil-mediated endothelial injury induced by infection with Mannheimia haemolytica. SAMPLE POPULATION: Cultured bovine pulmonary microvascular endothelial cells, freshly isolated bovine neutrophils, and monocyte-derived bovine macrophages. PROCEDURE: A coculture system was developed in which endothelial cells were grown to confluence in tissue culture inserts, neutrophils were added to the inserts, and macrophages were added to tissue culture wells. Mannheimia haemolytica-derived lipopolysaccharide (LPS) or supernatant was added to activate macrophages, and inhibitors of PAF or IL-8 were added to the insert. Endothelial cell cytotoxicity and permeability (ie, albumin leakage) and neutrophil activation (ie, adhesion, degranulation [lactoferrin expression], and superoxide production) were assessed. RESULTS: The addition of M haemolytica-derived LPS to bovine macrophages in the coculture system resulted in significant increases in endothelial cell cytotoxicity and permeability and neutrophil degranulation and adhesion. Inhibition of IL-8 reduced endothelial cell permeability and neutrophil degranulation induced by exposure to M haemolytica-derived supernatant, whereas inhibition of PAF decreased superoxide release by neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro activation of bovine macrophages by M haemolytica-derived LPS resulted in neutrophil activation and neutrophil-mediated endothelial damage. Neutrophil-mediated endothelial injury and neutrophil degranulation were, at least in part, mediated by IL8, whereas PAF promoted superoxide release by neutrophils in this in vitro system designed to mimic the in vivo events that occur during the early stages of bovine pneumonic pasteurellosis.     

The importance of interleukin-8 as a neutrophil chemoattractant in the lungs of cattle with pneumonic pasteurellosis.

Interleukin-8 (IL-8), an in vitro and in vivo neutrophil chemoattractant, is expressed at high levels in the lesions observed in bovine pneumonic pasteurellosis. Because of the role of neutrophils in the pathogenesis of pneumonic pasteurellosis, we investigated the relative importance of IL-8 as a neutrophil chemoattractant in this disease. Bronchoalveolar lavage (BAL) fluid was harvested from calves experimentally infected with bovine herpesvirus-1 and challenged with Mannheimia haemolytica. Neutrophil chemotactic activity was measured in pneumonic BAL fluid samples treated with a neutralizing monoclonal antibody to ovine IL-8, and compared to the activity in samples treated with an isotype-matched control antibody. Bronchoalveolar lavage fluid was analyzed at a dilution which induced a half-maximal response, and the concentrations of antibody were optimized in a preliminary experiment. Following incubation of replicate samples of diluted pneumonic bovine BAL fluid with 70 microg/mL of IL-8-neutralizing antibody or control antibody, the neutrophil chemotactic activities of the samples were determined using an in vitro microchemotaxis assay. Overall, pretreatment of BAL fluid samples with neutralizing anti-IL-8 antibody reduced neutrophil chemotactic activity by 15% to 60%, compared to pretreatment with control antibody. This effect was highly significant (P < 0.001), and was present in 5 of 5 samples. These data indicate that IL-8 is an important neutrophil chemoattractant in calves with pneumonic pasteurellosis, but that mediators with actions redundant to those of IL-8 must also be present in the lesions.     

Effect of leukotoxin of Mannheimia haemolytica and LPS of E. coli on secretory response of bovine neutrophils in vitro

To evaluate the role of leukotoxin (LKT) of Mannheimia haemolytica and lipopolysaccharide (LPS) of E. coli 055:B5 in pathogenesis of bovine respiratory disease (BRD) we investigated their in vitro effects on cultured bovine neutrophils. Functional parameters of neutrophils including degranulation, generation of superoxide, and nitric oxide were distorted in response to both toxins. The most essential reaction of neutrophils was found in respect to release of elastase after addition of LKT as well as LPS at concentration of 300 microg/ml. Moreover, we observed an increased release of myeloperoxidase (MPO) and alkaline phosphatase (ALK-P) from polymorphonuclear cells (PMN) after addition of LKT and LPS. We also found enhanced superoxide generation by bovine neutrophils after exposure to different concentrations of LKT and LPS. In cultures of PMN treated with LKT, concentration of nitrite increased with growing concentrations of LKT. Lower values of nitrite were obtained in cultures exposed to LPS. Partial lysis of PMN, determined by LDH (lactate dehydrogenase) leakage, started at concentration of 300 microg/ml for both toxins, meanwhile LKT concentration above 300 microg/ml was lethal. Our study has revealed that neutrophils in response to both toxins exaggerate release of analysed substances, which participate in worsening the course of the disease and play a role in lung injury during BRD. Toxins introduced to the cultural medium stimulate release of studied constituents from neutrophils by combined activation and lysis of neutrophils.     

Effects of recombinant granulocyte-macrophage colony-stimulating factor on bovine peripheral blood and mammary gland neutrophil function in vitro.

Modulation of peripheral blood and mammary gland neutrophil function following in vitro exposure to recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGM-CSF) was studied. Bovine blood and mammary gland neutrophils were cultured for 9 h in media containing 0.005, 0.05 or 0.5 microgram/mL rBoGM-CSF. Neutrophils treated with rBoGM-CSF exhibited significantly more chemotactic and bactericidal activities and tended to produce more superoxide anion than control cells. The effects of rBoGM-CSF on bovine neutrophil populations appeared to be dose-dependent. The production of superoxide anion and the bactericidal activity of mammary gland neutrophils were consistently higher than blood neutrophils. Only moderate increases in lipopolysaccharide-induced mammary gland neutrophil functions were observed following incubation with rBoGM-CSF which suggests that there may be a threshold of immunomodulation for these prestimulated cells. It may be possible to augment the functional capacity of bovine neutrophil populations in vivo through the therapeutic application of rBoGM-CSF and consequently enhance resistance of dairy cattle to bacterial infections.     

Linoleic acid increases adhesion,chemotaxis, granule release,intracellular calcium mobilisation,MAPK phosphorylation and gene expression in bovine neutrophils

Neutrophils are critical to the innate immune response; therefore, the proper function of neutrophils is critical to avoid the development of certain diseases. Linoleic acid, a polyunsaturated long-chain fatty acid, is one of the most abundant long-chain fatty acids found in the plasma of cows after giving birth. In this study, we evaluated the effects of linoleic acid treatment on bovine neutrophil adhesion, chemotaxis, metalloproteinase (MMP)-9 release, CD11b expression, intracellular calcium mobilisation, mitogen-activating protein kinase (MAPK) phosphorylation and COX-2 and IL-8 expression. Bovine neutrophils isolated from healthy heifers were incubated with different concentrations of linoleic acid, and then neutrophil responses were evaluated. Our results show that the treatment of neutrophils with 100 μM linoleic acid increased their adhesion to the bovine endothelial cell line CPA47. The results of a transwell migration assay revealed that linoleic acid could also promote the chemotaxis of bovine neutrophils. Furthermore, linoleic acid treatment increased MMP-9 activity and CD11b cell surface expression in neutrophils. Fifty and 100 μM linoleic acid also increased intracellular calcium mobilisation in neutrophils loaded with Fluo-4 AM dye. Linoleic acid also rapidly (2–5 min) stimulated the phosphorylation of ERK1/2 and p38 MAPK as evaluated by immunoblot. Finally, COX-2 and IL-8 mRNA expression increased after 2 h of linoleic acid treatment. In conclusion, linoleic acid stimulates adhesion, chemotaxis, granule release and intracellular responses in bovine neutrophils.     

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils

OBJECTIVE: To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils. SAMPLE POPULATION: Blood collected from 5 healthy adult Holstein cows. PROCEDURE: Isolated neutrophils or whole blood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured. RESULTS: Incubation with TNF-alpha, IL-8, PAF, and LPS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. Incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.     

Interleukin-8 concentration and neutrophil chemotactic activity in bronchoalveolar lavage fluid of horses with chronic obstructive pulmonary disease following exposure to hay

OBJECTIVE: To analyze effects of hay dust exposure on interleukin-8 (IL-8) concentration, percentage of neutrophils, and neutrophil chemotactic activity in bronchoalveolar lavage fluid (BALF) of horses with chronic obstructive pulmonary disease (COPD). ANIMALS: 16 healthy horses and 29 horses with COPD. PROCEDURE: IL-8 concentration, percentage of neutrophils, and neutrophil chemotactic activity in BALF were measured. Values were analyzed with respect to hay dust exposure.These variables were also measured in 5 asymptomatic horses with COPD after the induction of clinical signs by changing feed from silage to hay. RESULTS: L-8 concentrations and chemotactic activity in BALF were greater in horses with COPD, compared with healthy horses, and greater in horses with COPD exposed to hay dust, compared with nonexposed affected horses. An increase in IL-8 concentration accompanied by an increase in percentage of neutrophils in BALF and development of clinical signs of COPD were induced in asymptomatic horses with COPD by changing feed from silage to hay. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of horses with COPD to hay dust components resulted in an increase in IL-8 secretion at the bronchoalveolar surface. This chemokine may play a role in the pathogenesis of COPD, because it causes neutrophil accumulation in the bronchoalveolar space. Our results underscore the importance of eliminating dust sources for the treatment and prevention of COPD in horses.     

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.     

Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant

Interleukin-8, a member of the chemokine family of cytokines, is a potent neutrophil chemoattractant in many non-rodent species. In this study, recombinant bovine interleukin-8 (rbIL-8) was expressed in bacteria as a glutathione-S-transferase fusion protein. The fusion protein was purified by glutathione-Sepharose affinity chromatography and recombinant rbIL-8 was eluted by cleaving with thrombin. The purified rbIL-8 molecule was approximately 8 kDa and was confirmed as authentic IL-8 by Western analysis. Recombinant bovine IL-8 induced specific dose-dependent in vitro chemotaxis of neutrophils at doses as low as 1.0 ng/ml, and this activity was inhibited by pre-treatment of rbIL-8 with a monoclonal antibody to ovine IL-8. Neutrophils exposed to rbIL-8 developed pseudopodia and became elongated as determined by microscopic analysis and flow cytometry. Injection of 3.3 ng to 3.3 microg of rbIL-8 into the skin of a normal calf induced dose-dependent recruitment of neutrophils but not eosinophils. Intravascular margination of neutrophils was obvious at the injection sites from 15 to 60 min after administration of rbIL-8, and extravascular neutrophil numbers increased steadily from 1 to 18 h after injection. Neutrophils with morphologic features of apoptosis were detected in these lesions at 18 and 30 h after injection, and this correlated with reduction in the number of dermal neutrophils. These results confirm unequivocally that bovine IL-8 functions as a neutrophil, but not an eosinophil, chemoattractant in vitro and in vivo.     

Alpha(1)-acid glycoprotein is contained in bovine neutrophil granules and released after activation

The present study was designed to investigate the capability of bovine neutrophil granulocytes to produce the minor acute phase protein alpha(1)-acid glycoprotein (AGP, Orososmucoid). Bovine neutrophils contain a high MW (50-60kDa) AGP isoform (PMN-AGP), as determined by Western blotting and confirmed by fluorescence microscopy. The presence of AGP in bovine neutrophils has been confirmed by fluorescence immunocytometry. In addition, bovine neutrophils contain also a 42-45kDa isoform, which has the same MW as plasma-, liver-delivered, AGP. cDNA sequence of plasma- and PMN-AGP revealed that (i) the two proteins are products of the same gene; (ii) the differences in molecular weight are due do different post-translational modifications. This result was confirmed by deglycosylation of the two glycoforms. Exocytosis studies showed that isolated neutrophils exposed to several challengers, including Zymosan activated serum (ZAS) and phorbol 12-myristate 13-acetate (PMA), which mimic the inflammatory activation, released PMN-AGP as early as 15min. AGP's mRNA is physiologically expressed by mature resting neutrophils. Real-time PCR on LPS, ZAS and PMA challenged cells revealed that the level of expression apparently does not increase after inflammatory activation. Collectively, the findings reported in this paper proved that PMN-AGP: (i) is a hyperglycosylated glycoform of plasma AGP, (ii) is stored in granules, and (iii) is released by neutrophils in response to activation. Due to its anti-inflammatory activity, PMN-AGP may work as a fine tuning of the neutrophils functions in the inflammatory focus, i.e. it can reduce the damages caused by an excess of inflammatory response.     

Effects of interleukin-1 alpha on migration of canine neutrophils in vitro and in vivo

Inflammatory cell activation by interleukin-1 (IL-1) has been the subject of studies in laboratory animals and man, and was the aim of the present study. At physiological concentrations of 0.01-100 units/ml, human recombinant IL-1 alpha was shown to be a chemokinetic and chemotactic agent for canine neutrophils in vitro and to cause dose-dependent and selective neutrophil infiltration following intradermal injection of 1 or 250 units. The findings support a putative role for IL-1, and IL-1-induced products such as IL-8, as mediator of immunoinflammatory reactions in which neutrophil infiltration is prominent.     

Alterations in bovine neutrophil function during the periparturient period

Neutrophils from 8 Holstein heifers were evaluated for function during the periparturient period. Random migration, ingestion of bacteria, superoxide anion production, native (nonenhanced) chemiluminescence, iodination, and antibody-dependent, cell-mediated cytotoxicity by neutrophils were determined. Foremilk samples were evaluated for bacteria. Significant (P less than 0.05) increases in random migration of neutrophils, iodination, and chemiluminescence were evident 2 weeks before parturition and then decreased dramatically by the first week after parturition. These impairments of neutrophil function after parturition may be manifested as a severe cumulative deficit in the native defenses afforded by the neutrophil.     

Influence of conditioned media from bovine cotyledon tissue cultures on function of bovine neutrophils.

Bovine fetal placental (cotyledon) tissue obtained from pregnant cows on days 255, 265, and 275 of gestation, as well as immediately after parturition (n = 5) was incubated in media for 48 hours, and the incubation media were collected. Neutrophils from 4 ovariectomized nonpregnant cows were incubated for 2 hours with conditioned media from placental tissue cultures or medium (control). Immediately after incubation, the neutrophils were subjected to the following leukocyte function assays: chemotaxis against zymosan-activated serum, chemotaxis against undiluted conditioned media (only neutrophils that were incubated in medium only), random migration, ingestion of 125I-iododeoxyuridine Staphylococcus aureus (125I-IdUR-S aureus), iodination of proteins, cytochrome C reduction, and antibody-independent and -dependent cell-mediated cytotoxicity. Conditioned media from cultured cotyledon tissue was chemoattractant for bovine neutrophils, and increased chemotactic response of neutrophils against zymosan-activated serum by 13%. The following neutrophil functions were decreased: random migration by 25%, iodination of proteins by 44%, cytochrome C reduction by 13%, and antibody-dependent cell-mediated cytotoxicity by 5%. Ingestion of 125I-IdUR-S aureus and antibody-independent cell-mediated cytotoxicity were not influenced by coincubation of neutrophils and conditioned media. Time of gestation did not alter the effects of conditioned media on neutrophil function. It was concluded that chemotactic properties of cotyledon tissue extracts, as has been reported earlier, may be attributable to substances released by fetal placental tissue. Those substances might also locally or systemically influence the oxygen-dependent antimicrobial system of neutrophils, thereby causing an increased susceptibility to bacterial infections in the peripartum period.     

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.     

Effects of nitric oxide on bovine polymorphonuclear functions.

The effects of nitric oxide (NO) on the functionality of polymorphonuclear neutrophils (PMNs) in bovine milk or blood were investigated. In 2 experiments, mastitis was induced by infusing both hind quarters with saline containing Escherichia coli endotoxins. In addition, the left hind quarter was infused with aminoguanidine, an inhibitor of the inducible form of NO synthase (iNOS). At various times after infusion, somatic cells were isolated from milk samples, and superoxide (O2-) production induced by phorbol myristate acetate was evaluated. In both experiments, the addition of aminoguanidine had no inhibitory effect on the number of milk somatic cells or on their O2- production. The effect of NO and iNOS inhibitors on the functionality of bovine PMNs isolated from blood was investigated in vitro. The neutrophils did not produce NO. A neutrophil:monocyte co-culture system was used to study the effect of NO derived from monocytes on O2- production by bovine neutrophils. Neither NO derived from activated monocytes nor the iNOS inhibitors aminoguanidine and L-N6-(1-iminoethyl)lysine had an effect on the ability of bovine neutrophils to release O2-. Moreover, aminoguanidine did not affect the ability of bovine neutrophils to phagocytose bacteria. These results suggest that inhibition of NO release during inflammation does not interfere with the migration of immune cells to the site of infection or the ability of these cells to destroy pathogens. Thus, NO does not appear to play a major role in the control of the functions of bovine neutrophils.