Effects of human IL-8 isoforms on bovine neutrophil function in vitro

Interleukin 8 (IL-8) is a potent chemotactic and activating agent for human neutrophils and bovine IL-8 is chemotactic for bovine neutrophils; however, it is unclear whether IL-8 activates bovine neutrophils. Two isoforms of human recombinant (hr) IL-8 protein (77 and 72 amino acid) were used to stimulate bovine neutrophils in vitro. Bovine neutrophils exhibited significant migration in the presence of 0.1, 0.5, 1.0 and 5.0ngml(-1) hr IL-8 when incubated for 30min at 37 degrees C in a modified Boyden chamber assay. Both the 77 and 72 aa forms were equally effective in inducing migration in this assay. At the highest doses of IL-8 examined (1 and 5ngml(-1)), migration was similar to migration in the presence of 20% zymosan-activated serum (ZAS) or 12h lipopolysaccharide (LPS)-stimulated blood monocyte supernatants (CM). Significant (p<0. 05) release of alkaline phosphatase (ALK-P) (from specific granules) occurred but myeloperoxidase (MPO) release and superoxide anion production were not enhanced in bovine neutrophils by either form of hrIL-8 at any of the doses tested. Significant (p<0.05) alkaline phosphatase release was observed in the presence of 10 and 100ngml(-1) for the 72 aa form of IL-8 and only at the higher dose for the 77 aa form of IL-8. The ZAS and CM significantly enhanced neutrophil degranulation of ALK-P and MPO as well as inducing superoxide anion production. These results suggest that IL-8 may play a role in both neutrophil recruitment and activation during bovine inflammatory processes.     

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.     

Studies of endotoxin-induced neutrophil migration in bovine teat tissues, using indium-111-labeled neutrophils and biopsies.

Neutrophil migration through bovine teat tissues into the teat cistern, after endotoxin infusion into the teat cistern, was determined in vivo by 2 experimental procedures, indium-111 labeling of blood neutrophils, and obtaining multiple biopsy specimens from the teat cistern tissues. In both experiments, the number of leukocytes in the teat cistern flushing samples was continuously measured. A lag phase of approximately 1 hour was required between endotoxin infusion into the teat cistern and the first observed neutrophil accumulation in the teat tissues. The rate of neutrophil accumulation in the teat tissues was highest between postinfusion (PI) hours 1 and 2, and the accumulation process ceased after PI hour 3. Neutrophils migrated toward the epithelium, and intraepithelial neutrophils were observed beginning approximately 2 hours after infusion, which coincided with the first influx of cells into the teat cistern. The cell influx into the teat cistern increased continuously up to PI hour 3, peaked between PI hours 3 and 5, and was close to preinfusion value at PI hour 22. Use of indium-111-labeled neutrophils in the study of the inflammatory process provides a reliable noninvasive method to quantify cell migration in vivo. Use of biopsies allows quantification of the number of cells in different tissue areas, but has the disadvantage of being invasive. These 2 procedures complement each other, and could be of use in future studies of the local inflammatory process.     

Neutrophil migration induced by equine respiratory secretions, bronchoalveolar lavage fluids and culture supernatants of pulmonary lavage cells

Supernatants of equine respiratory secretions enhanced the migration of equine neutrophils into the lower compartments of Boyden chambers. Checkerboard analysis revealed that the neutrophil migration promoting activity (NMPA) of secretion specimens was in great part caused by chemokinesis, irrespective of the neutrophil score of the specimen. The NMPA of respiratory secretions was correlated neither with the neutrophil score of the secretion specimen nor with the severity of the chronic pulmonary disease. Respiratory secretions collected while horses were kept under low dust or under dusty housing conditions induced migration of neutrophils in the same order of magnitude. The number of migrated neutrophils and the procoagulant activity (PCA) within respiratory secretion specimens was positively correlated; however, the meaning of this finding is not yet clear. None of the nine cell-free supernatants of bronchoalveolar lavage fluid, which were assayed undiluted, induced significant neutrophil migration, although some samples contained up to 4.0 x 10(5) neutrophils/ml. In vitro culture of lung lavage cells, which mainly comprised macrophages and lymphocytes, without stimulation or with the addition of low doses of phytohemagglutinin (PHA) resulted in the secretion of NMPA which was in great part chemotactic. However, culture supernatants of lung cell preparations which were stimulated by lipopolysaccharide (LPS) or by PHA-prestimulated lymphocytes reduced the migration of neutrophils compared with the supernatants of control cells. NMPA within culture supernatants had a highly significant negative correlation with the PCA of macrophages within the lung cell preparations. Our results imply that a complicated and sophisticated regulation underlies neutrophil accumulation within the airways of horses affected with chronic pulmonary disease. Future experiments are required to assess the biological significance of the factors modulating neutrophil migration which are present in the respiratory secretions and in the culture supernatants of equine lung lavage cells.     

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.     

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.     

Rosette nanotubes inhibit bovine neutrophil chemotaxis

Migration of activated neutrophils that have prolonged lifespan into inflamed organs is an important component of host defense but also contributes to tissue damage and mortality. In this report, we used biologically-inspired RGD-tagged rosette nanotubes (RNT) to inhibit neutrophil chemotaxis. We hypothesize that RGD-RNT will block neutrophil migration through inhibition of MAPK. In this report, RNT conjugated to lysine (K–RNT) and arginine-glycine-aspartic acid-serine-lysine (RGDSK-RNT) were co-assembled in a molar ratio of 95/5. The effect of the resulting composite RNT (RGDSK/K–RNT) on neutrophil chemotaxis, cell signaling and apoptosis was then investigated. Exposure to RGDSK/K–RNT reduced bovine neutrophil migration when compared to the non-treated group (p < 0.001). Similar effect was seen following treatment with ERK1/2 or p38 MAPK inhibitors. Phosphorylation of the ERK1/2 and p38 MAPK was inhibited at 5 min by RGDSK/K–RNT (p < 0.05). The RGDSD/K-RNT did not affect the migration of neutrophils pre-treated with αvβ3 integrin antibody suggesting that both bind to the same receptor. RGDSK/K–RNT did not induce apoptosis in bovine neutrophils, which was suppressed by pre-exposing them to LPS (p < 0.001). We conclude that RGDSK/K–RNT inhibit phosphorylation of ERK1/2 and p38 MAPK and inhibit chemotaxis of bovine neutrophils.     

Suppressive effect of deoxynivalenol, a Fusarium mycotoxin, on bovine and porcine neutrophil chemiluminescence: an in vitro study

We evaluated the immunotoxicity of deoxynivalenol (DON), a Fusarium mycotoxin, on bovine and porcine neutrophils in vitro by using two function parameters, luminol-dependent chemiluminescence and random migration under agarose. A 2-hr DON treatment suppressed the chemiluminescence of bovine and porcine cells by 42% and 35% (on average) at 10(-5)M, and by 19% and 26% at 10(-6)M. Slight suppression was observed at concentrations lower than 10(-6)M. However, after an 18-hr DON treatment, random migration of neutrophils of both species remained unaffected, even at the highest concentration (10(-5)M). Although further extensive studies are needed, to our knowledge this is the first study to have revealed in vitro that DON can affect neutrophil function.     

Sources of variation introduced into a phagocytosis assay as a result of the isolation of neutrophils from bovine blood

A study was conducted to examine sources of variation introduced into a phagocytosis assay as a result of the isolation of neutrophils from bovine blood, including variation attributable to isolation of neutrophils from blood, variation between duplicate determinations of percentage phagocytosis, and the variation in the ability of neutrophils isolated from blood (over repeated collections from the jugular vein) to phagocytose. For the phagocytosis assay, jugular venous blood from each of 4 cows was divided into 2 equal portions. The neutrophils were isolated by lysis of red blood cells with 0.2% sodium chloride. The neutrophils (2 x 10(7)) were incubated in duplicate with 32P-labeled Staphylococcus aureus ([32P]SA; 2 x 10(8)) in skimmed milk samples (2.5% final concentration) prepared from 4 cows. This process was repeated thrice on neutrophils isolated from 4 cows at 2-week intervals. The proportions of variation in percentage of 32P-labeled S aureus phagocytosed between duplicate neutrophil isolations and between duplicate assay determinations were 0 and 1%. Differences among skimmed milk sources and among runs, using blood neutrophils taken at different times from the same donor cow, accounted for 62 and 36% of the total variation. The results indicated that variation arising from blood neutrophil isolation introduced into a phagocytosis assay within a single-day trial is of no concern. The large variation among skimmed milk sample sources indicated differences among cows in the ability of their milk to support phagocytosis. The variation in neutrophil isolations over time for any cow was considered too large to allow for evaluation of physiologic and environmental effects on phagocytosis of neutrophils isolated from blood.     

Diapedesis across mammary epithelium reduces phagocytic and oxidative burst of bovine neutrophils.

The effect of diapedesis on the phagocytic and oxidative burst activity of polymorphonuclear neutrophil (PMN) was examined, using an in vitro cell culture model consisting of a monolayer of primary mammary epithelial cells. Isolated blood PMN from 10 cows were added to the basal side of the epithelial cell monolayer. Diapedesis was induced by the addition of complement factor C5a to the apical side of the monolayer. PMN phagocytosis of Staphylococcus aureus and oxidative burst were measured before diapedesis on PMN that were non-activated and activated by incubation with C5a and on PMN after diapedesis, using flow cytometry. The percentages of PMN fluorescing due to phagocytosis of S. aureus and oxidative burst were reduced by 21.2 and 14.4%, respectively, after diapedesis. Pre-incubation in the presence of C5a had no effect on percentage PMN fluorescing due to phagocytosis or oxidative burst. The capacity for individual migrated PMN to phagocytose S. aureus and to produce an oxidative burst, as measured by the intensity of fluorescence, decreased by 34.2 and 30.3%. Activation of PMN with C5a increased intensity due to the oxidative burst, but had no effect on intensity due to phagocytosis. These data show that PMN diapedesis across mammary epithelium results in decreased phagocytosis and oxidative burst of the PMN.     

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.     

A rapid technique for the isolation of highly purified, functionally intact bovine neutrophilic granulocytes

A new method for the isolation of bovine neutrophilic granulocytes from peripheral blood based on centrifugation in a discontinuous metrizamide gradient has been developed. The procedure is rapid, taking only about 2 h, and gives highly purified (greater than 90%) neutrophils in a high yield (approximately 85%). The function of the cells, as measured by chemiluminescence and migration assays, is not significantly influenced by the isolation procedure. Eosinophils can also be isolated by a slight variation of the method. Initial applications of the separation procedure indicate its usefulness in clinical studies of bovine neutrophil function. A variation between individuals in the function of the cells was thus demonstrated. Moreover, the chemiluminescence of neutrophils from infected animals was found to be greatly increased, and neutrophil migration was shown to be stimulated by in vivo ACTH treatment.     

In vivo effect of ascorbic acid on neutrophil function in healthy and dexamethasone-treated cattle

Ascorbic acid (20 mg/kg of body weight) administered subcutaneously to otherwise nontreated cattle resulted in enhancement of neutrophil oxidative metabolism and capability of neutrophils to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Random migration, bacterial ingestion, and iodination by neutrophils was unaffected. Three dosage levels of ascorbic acid (10 mg/kg, 20 mg/kg, and 40 mg/kg) were examined for their effects on neutrophil function in cattle treated with dexamethasone (0.04 mg/kg). Dexamethasone administration caused an enhancement of neutrophil random migration and a suppression of neutrophil oxidative metabolism, iodination, and ADCC. None of the dosage levels of ascorbic acid had an effect on the alterations in the WBC count induced by dexamethasone. The ascorbic acid did tend to reverse the effects of dexamethasone on neutrophil random migration, oxidative metabolism, and ADCC in a dose-dependent manner, with the lowest dose having no discernible effect. Ascorbic acid administration also tended to enhance Staphylococcus aureus ingestion by bovine neutrophils. These results indicate that ascorbic acid should be further investigated for its potential to reduce the susceptibility of stressed or glucocorticoid-treated cattle to infective processes.     

Transport stress increases somatic cell counts in milk, and enhances the migration capacity of peripheral blood neutrophils of dairy cows

The present study was designed to determine the effects of physiological stress on milk-somatic cell counts (SCC) and function of bovine peripheral blood leukocytes (PBL). Nine healthy lactating cows were used in the examination. Five cows were transported 100 km for 4 hr (transported group; TG), and 4 cows were penned (non-transported group; NTG). Blood and milk samples were collected at 0, 2, and 4 hr after loading, and at 2 hr, and 1, 2, 3, and 6 days after unloading. The following activities were measured: adhesion receptor (CD 18 and L-selectin) expression of neutrophils and monocytes, migration capacity and percentage of apoptotic cells of neutrophils, serum soluble L-selectin (sL-selectin), plasma cortisol, and SCC. A significant increase in plasma cortisol and milk SCC was observed in TG. Leukocytosis, derived from neutrophils was recorded in TG, and was indicated by apoptotic measurement as an increase of young cells from the marginal pool. Increased migration and decreased surface expression of both L-selectin and CD 18 in neutrophils were observed after transportation. Elevated serum sL-selectin was also noted as a result of transportation. The present study indicated that transport stress modulates peripheral blood neutrophil function, particularly enhancing migration capacity, and causes diapedesis across the mammary epithelium. Increased milk SCC in transported cattle might be due to these phenomena, and severe physiological stress may bring about an increase in SCC in milk.     

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.     

Gene expression signatures in neutrophils exposed to glucocorticoids: a new paradigm to help explain "neutrophil dysfunction" in parturient dairy cows

Neutrophils are the first line of immunity against most pathogens that infect cattle. These normally short-lived white blood cells develop from myeloid-lineage cells in bone marrow. Upon maturation, bone marrow neutrophils are released into the circulation where they marginate on inflamed blood vessel endothelial cells and migrate through them into the area of infection. Once migrated, neutrophils do not reenter the circulation, but rather, perform their bactericidal functions and die by apoptosis in the tissue. The cytokine and hormonal milieu of the blood and extracellular tissue fluid can influence neutrophil development and immunity-related activities, but the molecular basis of these phenotypic changes and physiological benefits or drawbacks of them are poorly understood. In the current paper, we review new gene expression information that resulted from two of our functional genomics studies designed to evaluate effects of glucocorticoid hormones on bovine neutrophils. This work provides one model to describe complex changes that occur in neutrophils as the cells respond to glucocorticoids, which might act to alter the cells' functional priorities and tip the delicate balance between health and disease during stress, including at parturition. A bovine immunobiology microarray and real time RT-PCR were used to study blood neutrophils collected during the natural surge of endogenous glucocorticoid (cortisol) in parturient dairy cows and bone marrow neutrophils collected from glucocorticoid (dexamethasone)-treated dairy steers. The gene expression signatures we observed led us to perform additional phenotyping of the neutrophils and correlation analyses, which together painted a picture suggesting that glucocorticoids have key roles in modulating neutrophil development, life span, and tissue defense functions during parturition and hormone therapy. Based on these observations, we postulate that glucocorticoids orchestrate adaptive changes in the entire neutrophil system that support increased cell numbers and longevity in blood and heightened remodeling activity in tissues, while at the same time decreasing some important antimicrobial defense activities of the cells. Thus, our functional genomics studies have enabled us to elucidate multiple consequences of neutrophil exposure to glucocorticoids, highlighting a probable role for this interaction in the induction of parturition and partly explaining why some parturient dairy cows may experience heightened incidence and severity of inflammatory diseases like mastitis.     

A colorimetric assay for quantitating bovine neutrophil bactericidal activity

A colorimetric assay was developed for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus. The procedure used the tetrazolium compound, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay was conducted by incubating antibody-opsonized S. aureus with neutrophils in microtiter plates for 1 h at a ratio of 10 bacteria per neutrophil. Neutrophils were then lysed with saponin. The MTT was added and samples were incubated for 10 min. Live S. aureus reduced MTT to purple formazan. Dead bacteria and lysed neutrophils did not react with MTT. Bacterially-reduced formazan was solubilized by adding isopropanol and formazan production was quantitated by measuring absorption at 560 nm. Absorption of formazan was directly related to viable bacteria cell number and was used to determine the number of S. aureus not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT with known numbers of S. aureus. The colorimetric MTT assay detected suppressed bactericidal activity after in vitro treatment of bovine neutrophils with colchicine, cytochalasin B, or phorbol 12-myristate 13-acetate. In vitro treatment of neutrophils with low levels of recombinant bovine interferon gamma (rBoIFN-gamma) enhanced bactericidal activity, whereas high levels decreased activity. These results suggest the colorimetric MTT bactericidal assay is efficacious in detecting modulation of bovine neutrophil bactericidal activity. Furthermore, the MTT assay has many advantages over traditional bactericidal assays in that it is sensitive, inexpensive, requires less than 3 h to complete, and can analyze many neutrophil samples in a single day.     

Early in vivo interactions of Actinobacillus pleuropneumoniae with tonsils of pigs.

Twenty gnotobiotic piglets were inoculated with 5 x 10(8) colony forming units of an Actinobacillus pleuropneumoniae biotype 1-serotype 9 strain onto their tonsils. Five other piglets (controls) were inoculated with phosphate-buffered saline solution. Pigs were euthanized at 30 min, 90 min, 180 min, 6 h, 9 h, 12 h or 24 h after inoculation. At necropsy, samples were taken from the tonsils for bacteriological, histological, immuno-histochemical and electron microscopical examination. A. pleuropneumoniae was isolated from tonsils of all the infected pigs, but not from tonsils of the control pigs. Early after inoculation bacteria were mainly associated with the stratified squamous epithelium and detached epithelial cells. Vacuolization and desquamation of the epithelium was observed and many transmigrating neutrophils were present. At later times after inoculation, bacteria were found closely associated with the crypt-walls and with detached cells present in the crypts. A strong neutrophil migration was observed mainly in the deeper parts of the crypts. It is concluded that attachment of A. pleuropneumoniae to tonsillar epithelial cells probably constitutes a first step in establishing bacteria at this body site.     

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.     

Effect of bovine granulocyte colony-stimulating factor on the development of pneumonia caused by Mannheimia haemolytica

The role of recruited neutrophils in Mannheimia haemolytica infection is controversial. We hypothesized that the neutrophilia induced by recombinant bovine granulocyte colony-stimulating factor (GCSF) would lead to rapid bacterial clearance and less severe lesions after infection with M. haemolytica. Two experiments (A and B) were conducted in which four calves per experiment were treated daily with 5 microg/kg GCSF and four calves per experiment were treated with saline. All 16 calves were challenged with 5 x 10(9) colony-forming units (cfu)/ml (experiment A) or 4.5 x 10(8) cfu/ml (experiment B) of M. haemolytica bacteria, into the right bronchus by bronchoscope-placed catheter. The mean maximal blood neutrophil counts in non-GCSF-treated and GCSF-treated calves before bacterial challenge were 5.6 +/- 0.7 x 10(9)/liter and 25.4 +/- 2.7 x 10(9)/liter, respectively. Two untreated calves became neutropenic and were euthanatized 2 days after infection because of severe respiratory distress. GCSF-treated calves had a 37% reduction in lung lesions compared with nontreated calves, and this difference was significant (P=0.04) when the effect of previous antibody titre to leukotoxin was considered. The effect of GCSF treatment on the severity of clinical signs seemed to be influenced by the antibody titre to M. haemolytica leukotoxin, although this effect could not be conclusively addressed. In conclusion, GCSF induced neutrophilia and partially protected calves against experimental infection with M. haemolytica. These results imply that increased numbers of neutrophils may, under some circumstances, protect against severe pneumonia caused by M. haemolytica.