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1.
Pasteurella haemolytica A1 leukotoxic culture supernatant was evaluated for its ability to cause aggregation of bovine peripheral neutrophils. Neutrophils were isolated by a hypotonic lysis method and incubated with zymosan-activated plasma (ZAP), leukotoxic culture supernatant, antileukotoxin serum, calcium and magnesium-free media, p-bromophenacyl bromide and protein kinase C inhibitors. Aggregation was evaluated by changes in infrared light transmittance. Leukotoxic culture supernatant caused neutrophils to aggregate, and this effect was significantly removed by preincubation with antileukotoxin serum. Aggregation to ZAP and leukotoxin was dependent on the presence of extra-cellular calcium. Activation of protein kinase C by phorbol myristate acetate induced aggregation which was reduced by staurosporine; however, aggregation to leukotoxin did not involve protein kinase C activation. Phospholipase A2 inhibition did not alter the aggregation response to ZAP or to leukotoxin. The in vitro measurement of neutrophil aggregation induced by the leukotoxin of P. haemolytica reflects cytoskeletal and other activation events that may contribute to the intense inflammatory process which this organism induces in the lungs of cattle.  相似文献   

2.
The bovine respiratory pathogen Pasteurella haemolytica secretes an exotoxin that is specific for ruminant leukocytes (leukotoxin). Previous studies have shown that subcytolytic concentrations of the leukotoxin stimulate bovine neutrophils to undergo a respiratory burst and degranulate. Relatively little is known about the stimulatory effects of the leukotoxin on bovine mononuclear phagocytes. In this study, we compared the relative cytolytic effects of partially purified leukotoxin on bovine peripheral blood monocytes and alveolar macrophages. We found monocytes to be approximately 8- to 10-fold more sensitive than alveolar macrophages to the cytolytic effect of leukotoxin. In addition, incubation of monocytes and alveolar macrophages with sublethal doses of leukotoxin stimulated release of IL-1 and TNF activities in a dose-dependent manner. Addition of an antileukotoxin MAb neutralized the cytolytic effects of leukotoxin, but potentiated TNF release. Heat inactivation also blocked the cytolytic activity of LKT, but only slightly reduced its ability to stimulate TNF release. Although the leukotoxin preparations were estimated to have only small amounts of lipopolysaccharide (LPS) contamination, as determined by a standard Limulus amebocyte lysate coagulation assay, a chromogenic Limulus assay indicated much greater amounts of LPS were present. Adding equivalent doses of P. haemolytica LPS largely duplicated the monokine release stimulated by leukotoxin. These results suggest that the stimulatory effects of the P. haemolytica leukotoxin on bovine mononuclear phagocytes may principally involve LPS, perhaps complexed with leukotoxin.  相似文献   

3.
Sterile, concentrated culture supernatant from Pasteurella haemolytica (biotype A, serotype 1) strain 630 was subjected to physical, chemical, and immunologic treatments to determine their influence on leukotoxin (cytotoxin) activity contained in the supernatant. Each treated sample contained approximately 8 chemiluminescence inhibitory units of leukotoxin. Treatment effects were evaluated for their ability to inactivate leukotoxin activity. Leukotoxin activity in treated samples was determined by inhibition of the luminol-dependent chemiluminescence response of bovine neutrophils. Optimal leukotoxin synthesis by P haemolytica occurred when the bacteria were at the logarithmic growth phase, whereas stationary phase cultures contained minimal amounts of leukotoxin activity in their culture supernatant. Leukotoxin activity was heat labile; activity was substantially decreased when concentrated culture supernatant samples containing leukotoxin activity were incubated at 37 C for several hours. When concentrated culture supernatant was incubated at progressively decreasing temperatures, there was a progressive increase in the length of time that the leukotoxin retained its biologic activity. Samples stored at -70 C retained activity for at least 2 months. Leukotoxin activity was nondialyzable and was able to withstand considerable extremes in hydrogen ion concentration. Leukotoxin activity could not be pelleted when subjected to forces of 100,000 X g for 1 hour. Chemical and enzymatic studies suggested that P haemolytica leukotoxin contained carbohydrate and protein moieties. Chemical treatment with 0.2% sodium lauryl sulfate, 0.5% sodium deoxycholate, 7.5 mM EDTA and 8M urea with 8 mM 2-mercaptoethanol and enzymatic treatment with lipase, ribonuclease, and deoxyribonuclease had no discernible effect on leukotoxin activity.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

4.
Pasteurella haemolytica A1 leukotoxic culture supernatant was evaluated for its ability to induce histamine release from bovine pulmonary mast cells isolated by enzymatic dispersion of lung tissue. Histamine was measured by a radioimmunoassay technique. Leukotoxic culture supernatant of P. haemolytica significantly released histamine in a time and concentration-related manner. This effect was lost when culture supernatant was heat-inactivated or preincubated with leukotoxin neutralizing rabbit serum. Preincubation of the mast cells with propranolol or p-bromophenacyl bromide reduced the histamine-releasing effect of leukotoxin, while verapamil enhanced release. Experimental infection of calves with P. haemolytica A1 reduced the total histamine content of pulmonary mast cells recovered at postmortem. Histamine release induced by P. haemolytica leukotoxin is likely an important factor in the pathogenesis of bovine pneumonic pasteurellosis.  相似文献   

5.
Bovine neutrophil chemotactic activity was detected in the supernatant fluid of logarithmic phase cultures of P. haemolytica serotype 1. The chemoattractant was produced under culture conditions suitable for P. haemolytica leukotoxin production. An inverse correlation existed between the leukotoxin LC50 and the chemotactic activity in the culture fluid. Elimination of leukotoxin activity by heating, dilution or ultrafiltration, exposed the chemotactic activity in the culture fluid. The chemoattractant was partially resistant to heating (60 degrees C, 30 min), and had an apparent molecular weight greater than 100,000. Detection of chemotactic activity in both the concentrate and filtrate after XM300 filtration suggested that there might be more than one component with chemotactic activity or else that polymerization was occurring. Production of a potent neutrophil chemoattractant by P. haemolytica may explain the rapid infiltration of neutrophils that occurs during the early stages of bovine pneumonic pasteurellosis.  相似文献   

6.
Plasmid DNA screening experiments were conducted to determine whether a relationship existed between the presence of plasmids and antibiotic resistance in Pasteurella haemolytica or the capability to produce hemolysin or leukotoxin (cytotoxin). Regardless of plasmid content, all P haemolytica isolates produced characteristic hemolysis on blood agar plates. Similarly, standardized suspensions of living bacteria and sterile concentrated (approx 200:1) culture supernatant from strains representing each of the 15 recognized P haemolytica serotypes and 7 field strains of P haemolytica (biotype A, serotype 1) produced leukotoxin, which was detected by their capability to cause inhibition of the luminol-dependent chemiluminescence response of bovine neutrophils. However, neither living bacterial suspensions nor concentrated culture supernatant from 4 untypable P haemolytica strains or a P multocida strain caused an inhibition of the luminol-dependent chemiluminescence response. The production of neither hemolysin nor leukotoxin by P haemolytica seemed to be plasmid mediated. Leukotoxin production is apparently a stable phenotypic characteristic of pathogenic P haemolytica strains, and the gene(s) coding for this activity is probably located on the bacterial host chromosome. Antibiotic susceptibility profiles were determined for the different bacterial strains. Studies of ampicillin and penicillin resistance in 8 P haemolytica (biotype A, serotype 1) strains provided evidence that the plasmid, with size of approximately 5,200 base pairs, may code for their resistance to these compounds.  相似文献   

7.
Pasteurella haemolytica leukotoxin is cytotoxic to bovine leukocytes, causing increased cell membrane permeability, osmotic swelling, release of cytosolic proteins and cell lysis. These studies were designed to test if leukotoxin causes release of the cytoskeletal protein, actin, from bovine leukemia cells and if purified actin-influenced bacterial growth or leukotoxin production. Culture supernatants caused a 7-fold decrease in viability of bovine leukemia cells and increased cell permeability that was accompanied by release of beta-actin into the cell culture supernatant. Exposing P. haemolytica to purified actin solutions induced the conversion of monomeric G-actin to polymerized F-actin. This conversion was partially inhibited by bovine P. haemolytica immune, but not pre-immune, serum. Loss of streptomycin resistance following treatment of the organism with acridine orange ablated the polymerizing activity. Incubation of P. haemolytica in the presence of purified F-actin did not affect growth but resulted in culture supernatant that had 3.0-3.9-fold greater leukotoxicity compared to medium alone or medium containing G-actin, heat-denatured actin or albumin. The effect of actin on leukotoxicity was concentration-dependent and directly associated with increases in secreted leukotoxin. The interaction between P. haemolytica and actin is potentially detrimental to the host by inducing polymerization of actin into insoluble filaments and by enhancing leukotoxicity.  相似文献   

8.
The purpose of these studies was to determine mechanisms of pulmonary tissue damage mediated by Pasteurella haemolytica and interaction with bovine neutrophils. Bovine pulmonary artery endothelial cell monolayers were treated with various combinations of P. haemolytica factors including bacterial culture supernatant (CS) and purified LPS, with and without bovine neutrophils. Damage to endothelial cells was monitored by 51Cr release, cell detachment rate, and morphological changes. At 5 h post-treatment (PT) bacterial factors produced very little toxic change in cells, however, by 22 h PT both crude leukotoxin and LPS caused high levels of cytotoxicity and detachment. Neutrophils did not augment toxicity mediated by LPS, but actually protected endothelial cells from low levels of LPS. When the LPS component of CS was neutralized with polymyxin B, leukotoxin mediated neutrophil killing resulted in extensive endothelial cell damage. These results suggest that LPS may directly injure endothelial cells and this toxic effect may be reduced by neutrophils. However, neutrophil killing by leukotoxin may also contribute to endothelial cell damage in the absence of LPS.  相似文献   

9.
Dilutions of concentrated, dialyzed Pasteurella haemolytica culture supernatant were caused to react with bovine neutrophil (PMN) suspensions, and then the trypan blue dye exclusion (TBDE), 51chromium (51Cr)-release, and luminol-dependent chemiluminescence-inhibition (LDCLI) assays were done to compare their relative sensitivities in detecting biological activity of P haemolytica leukotoxin (cytotoxin). The culture supernatant was concentrated approximately 200:1, and when caused to react as an undiluted preparation with bovine PMN, it was cytotoxic for 38.6% and 80.4% of PMN as determined by TBDE and 51Cr-release assays, respectively. This undiluted leukotoxin preparation caused 100% inhibition of the luminol-dependent chemiluminescence responses of bovine PMN. The LDCLI assay was the most sensitive of the 3 in vitro assays for P haemolytica leukotoxin activity--being approximately 17 times and 2,480 times more sensitive than the 51Cr-release and TBDE assays, respectively. The relative advantages and disadvantages of the 3 assays as in vitro systems for detecting and titrating leukotoxin activity and investigating the role of leukotoxin in disease pathogenesis and immunity are discussed. Because of its sensitivity, specificity, economy, technical ease, and potential for adaptation to automation, the LDCLI assay would seem to be the in vitro assay of choice for quantitating P haemolytica leukotoxin activity. To aid standardization of studies of leukotoxin between different laboratories, it is suggested that P haemolytica leukotoxin be quantitated and expressed as chemiluminescence inhibitory units.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

10.
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.  相似文献   

11.
Lysis of bovine platelets by Pasteurella haemolytica leukotoxin   总被引：3，自引：0，他引：3  
Pasteurella haemolytica A1 culture supernatants caused rapid cytolysis (less than 5 minutes) of isolated bovine platelets as measured by leakage of the cytoplasmic enzyme lactate dehydrogenase (LD). The platelet lytic factor had several features similar to P haemolytica leukotoxin. Like P haemolytica leukotoxin, the platelet lytic factor was produced by P haemolytica during logarithmic growth phase, was heat-labile, and was active against target cells (platelets) from ruminant species (cattle and sheep), but not from non-ruminant species (horses, pigs, and human beings). Additionally, the platelet lytic factor was neutralized with antileukotoxin rabbit serum. The amount of LD leaked by a fixed concentration of bovine platelets was proportional to the amount of toxin added at low toxic doses and became maximal at 88 +/- 11% of the total platelet LD activity for high doses of toxin. When a fixed dose of toxin was used and the platelet concentration was varied, LD leakage was initially proportional to the platelet concentration, but plateaued at higher platelet concentrations. The platelet lytic factor required Ca2+ and was inhibited by addition of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. Toxin-mediated platelet damage may be important in thrombi formation and fibrin exudation typically associated with P haemolytica pleuropneumonia of cattle.  相似文献   

12.
The leukotoxin of Mannheimia haemolytica has a very high degree of amino acid diversity because the lktA gene has a complex mosaic structure that has been derived by horizontal DNA transfer and intragenic recombination. The objective of the present study was to determine the effect of this amino acid diversity on leukotoxin cytotoxicity against bovine and ovine cell types. This was done by comparing the luminol-dependent chemiluminescence response of bovine and ovine neutrophils after pre-incubation with the various leukotoxin types. The LktA1.1-type leukotoxin associated with bovine serotype A1 and A6 strains differs from the LktA1.2-type leukotoxin produced by ovine serotype A1 and A6 strains at a single amino acid position and has enhanced activity against bovine but reduced activity against ovine neutrophils. These findings, together with the exclusive association of the LktA1.1-type leukotoxin with bovine strains, suggest that this leukotoxin type has an adaptive advantage in the bovine host. Leukotoxins LktA6-LktA10 are associated with ovine strains and have complex mosaic structures and diverse amino acid sequences but similar levels of cytotoxic activity against bovine and ovine neutrophils, respectively. However, ovine neutrophils were more sensitive to the cytotoxic activities of these leukotoxins than were bovine neutrophils. LktA8- and LktA10-type leukotoxins are associated with serotype A2 and A7 strains that are responsible for the majority of ovine disease cases, but LktA6-, LktA7- and LktA9-type leukotoxins are associated with less common serotypes. These findings contribute to the growing body of evidence suggesting that factors other than leukotoxin cytotoxicity are responsible for the full expression of virulence in M. haemolytica. Overall, the extensive recombinational exchanges within the lktA gene of M. haemolytica have had little effect on leukotoxin function which is highly conserved.  相似文献   

13.
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.  相似文献   

14.
Hybridoma-derived monoclonal antibodies (MAB) against the cell surface antigens of Pasteurella haemolytica serotype 1 were obtained by the fusion of murine myeloma cells (P3 X 63 - Ag 8.653) with splenocytes of BALB/c mice immunized with crude logarithmic growth-phase culture supernatant. Initial screening was performed, using an ELISA, with the same bacterial growth culture supernatant as coating antigens. Further selection was done, using a panel of purified antigens--either capsular polysaccharide or lipopolysaccharide--as the coating antigen in an ELISA, and then performing a leukotoxin-neutralization assay. Two MAB, designated IIB-6 and H-2, reacted specifically with the capsular polysaccharide and the other 3, designated IVG-3, IH-3, and IIC-2, reacted with the lipopolysaccharide. One MAB, designated IH-6, did not react with leukotoxin, capsular polysaccharide, or lipopolysaccharide. The MAB to the capsular polysaccharide (IIB-6 and H-2) were characterized further; both antibodies belonged to the IgM class and were agglutinating. In addition, they promoted neutrophil-mediated opsonophagocytosis and complement-mediated immune bacteriolysis of P haemolytica serotype 1. Results from 3 studies indicated that the MAB IIB-6 and H-2 were specific only to the capsular polysaccharide of serotype 1 of P haemolytica. The MAB to the lipopolysaccharide (IVG-3, IH-3, and IIC-2) were of the IgG1, IgG3, and IgM classes, respectively and were not characterized further. The availability of a MAB identifying a serotype-specific, surface-exposed determinant on the capsule of P haemolytica serotype 1 should facilitate and expand studies concerning the role of the capsular material and lipopolysaccharide in the pathogenicity of P haemolytica infection in cattle.  相似文献   

15.
Pasteurella haemolytica A1 culture supernatant containing leukotoxin, and modifiers of cyclic nucleotide and arachidonate metabolism, were evaluated for their ability to alter oxygen radical production by pulmonary alveolar macrophages obtained from seven Holstein calves. Calves were sedated, and underwent bronchoalveolar lavage to harvest macrophages, which were then incubated with culture supernatant and/or the drugs and toxins under study, and challenged with opsonized zymosan to induce oxygen radical generation. This was measured by a chemiluminescence technique. Pasteurella haemolytica A1 culture supernatant alone delayed the time to maximum oxygen radical production, although total production was increased. The cyclooxygenase inhibitor indomethacin and the phospholipase inhibitor p-bromophenacyl bromide significantly reduced maximum oxygen radical production, but their effects were diminished in the presence of culture supernatant. Although forskolin markedly inhibited oxygen radical generation, this effect was not altered by culture supernatant. Incubation of macrophages with pertussis toxin had no effect on oxygen radical production, while incubation with cholera toxin did inhibit production. This inhibitory effect was significantly lessened by concurrent incubation with P. haemolytica A1 culture supernatant.  相似文献   

16.
Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the beta(2)-integrin CD11a/CD18 (LEA-1) on ruminant leukocytes. LKT binding induces activation, and subsequent cytolysis, of these cells. It is well known that active viral infection greatly increases the susceptibility of cattle to pasteurellosis. To better understand the mechanism by which this occurs, we investigated the effects of experimental in vivo infection of cattle with bovine herpes virus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with the M. haemolytica LKT. In this study, we demonstrated that active BHV-1 infection increased the expression of the beta(2)-integrin CD11a/CD18 (as defined by the mAb BAT75) on bovine peripheral blood neutrophils, enhanced the binding of LKT to bronchoalveolar lavage (BAL) leukocytes and peripheral blood neutrophils, and increased the killing of BAL leukocytes and peripheral blood leukocytes by LKT. In addition, BHV-1 greatly increased the number of BAL, resulting in many more LKT-responsive cells being present in the lungs. These findings might explain in part the increased susceptibility of BHV-1 infected cattle to pneumonic pasteurellosis.  相似文献   

17.
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.  相似文献   

18.
A luminol-dependent chemiluminescence (LDCL) assay was used to evaluate the response of bovine polymorphonuclear leukocytes; (neutrophils [PMN]) to living and heat-killed Escherichia coli, Pasteurella multocida (type A, serotype 3), and P haemolytica (biotype A, serotype 1), and to heat-killed P haemolytica and sterile culture supernatant from living P haemolytica. Control cultures containing PMN that had not been phagocytically stimulated with bacteria had a modest increase in LDCL during the initial 10 minutes of incubation, followed by a gradual decline throughout the 120-minute incubation period. Bovine PMN emitted LDCL more efficiently when the cells were exposed to living E coli or P multocida than when they were exposed to the same bacteria killed by heat. The mean LDCL values for reaction mixtures containing living E coli or P multocida peaked at 30 minutes of incubation and remained above values for mixtures containing the same heat-killed bacteria. Kinetics of the LDCL response of bovine PMN to heat-killed P haemolytica were similar (although reduced in amplitude) to that observed with killed E coli or P multocida. The LDCL response of bovine PMN to living P haemolytica was not like that for E coli or P multocida, and was characterized by the development of a peak response at 10 minutes followed by a precipitous decrease in responsiveness and a subsequent complete cessation of LDCL. Addition of sterile culture supernatant from living P haemolytica to test samples containing heat-killed P haemolytica induced a response similar to that obtained with the living microorganism.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
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)  相似文献   

20.
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.  相似文献   

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