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1.
OBJECTIVE: To evaluate lipopolysaccharide (LPS)-induced activation of equine neutrophils in blood. SAMPLE POPULATION: Blood samples from 5 healthy adult Thoroughbreds. PROCEDURES: Neutrophil integrin (CD11/CD18) expression, size variation, degranulation, and deformability were measured with and without incubation with LPS. Time and concentration studies were done. The mechanism of endotoxin-induced neutrophil activation was investigated by inactivating complement or preincubating neutrophils with inhibitors of tumor necrosis factor-alpha (TNF-alpha) synthesis, prostaglandin-leukotriene synthesis, or platelet-activating factor. RESULTS: Incubation of equine neutrophils with LPS increased cell surface expression of CD11/CD18, decreased neutrophil deformability, increased and decreased neutrophil size, and induced neutrophil degranulation. The LPS-induced neutrophil activation was attenuated by addition of inhibitors of TNF-alpha and prostaglandin-leukotriene synthesis. CONCLUSIONS AND CLINICAL RELEVANCE: Equine neutrophils are readily activated in vitro by LPS, resulting in increased expression of integrin adhesion molecules, decreased deformability, variation in neutrophil size, and degranulation. The tests used to detect activated neutrophils in this study may be useful in detecting in vivo neutrophil activation in horses with sepsis and endotoxemia.  相似文献   

2.
OBJECTIVE: To evaluate cytokine production by equine alveolar macrophages after exposure to lipopolysaccharide (LPS), Aspergillus fumigatus, and hay dust, and determine the effect of clenbuterol on the cytokine response. ANIMALS: 6 horses. PROCEDURE: Alveolar macrophages were exposed to PBS solution (negative control), LPS, hyphae and conidia of Aspergillus fumigatus (AF), or a suspension of hay dust (HDS) and incubated for 24 hours at 37 degrees C. Concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta were measured in the supernatant. The procedure was repeated with cells that were concurrently incubated with 0.5 microM clenbuterol. RESULTS: Exposure to HDS and AF significantly increased production of TNF-alpha by equine alveolar macrophages. The increase in TNF-alpha produced in response to HDS and AF was 5 and 7 times as great, respectively, as the increase measured in response to LPS. The concentration of IL-1beta in the supernatant was significantly increased after exposure of cells to AF. Clenbuterol was effective at inhibiting TNF-alpha production by cells exposed to LPS, HDS, or AF. CONCLUSIONS AND CLINICAL RELEVANCE: Increased production of TNF-alpha and IL-1 indicated that the pro-inflammatory cytokines produced by alveolar macrophages in response to allergens may play a role in recurrent airway obstruction (RAO) in horses. Equine alveolar macrophages are not only a primary pulmonary defense mechanism but may also influence the pathogenesis of equine RAO. The beta2-adrenoceptor agonist clenbuterol, a drug that is commonly used for treatment of equine RAO, promotes immediate bronchodilation and may also contribute to downward modulation of the inflammatory response.  相似文献   

3.
A passive haemagglutination test (PHT) which has been developed for the detection of antibodies to the contagious equine metritis organism (CEMO) in serum is described. Samples from each of 30 mares with metritis were positive with titres in the range 256 to 4096. Samples from each of 239 clinically normal mares and 30 colts and fillies believed not to have been exposed to CEMO were negative with titres of less than 256, the majority of samples (97 per cent) showing a titre of 32 or less.  相似文献   

4.
OBJECTIVE: To determine the effect of endotoxin (lipopolysaccharide [LPS]) on vasoactive mediator production by cultured equine digital vein endothelial cells (EDVECs). SAMPLE POPULATION: EDVECs obtained from forelimb digital veins of 7 healthy adult horses. PROCEDURES: EDVECs were incubated with or without LPS (1 microg/mL) for 0, 2, 4, 6, 22, and 24 hours. The EDVECs were incubated for 18 hours with LPS (10 pg/mL to 1 microg/mL) with or without ibuprofen, cycloheximide, or L-nitroarginine methyl ester. Medium concentrations of prostacyclin, cyclic guanosine monophosphate, endothelin-1, and thromboxane A(2) were determined. Changes in inducible nitric oxide synthase and cyclooxygenase-2 expression were determined. RESULTS: LPS stimulated mean 4.2- and 14.1-fold increases in EDVEC prostacyclin and cyclic guanosine monophosphate production, respectively, after 22 hours. These effects were LPS concentration-dependent (LPS concentrations that induced a response halfway between the maximum response and baseline of 1.50 and 1.22 ng/mL, respectively). The LPS-induced cyclic guanosine monophosphate production was significantly inhibited (to basal concentrations) by L-nitroarginine methyl ester, and prostacyclin production was inhibited by cycloheximide and ibuprofen. Production of thromboxane A(2) by EDVECs was not detected. Endothelin-1 accumulated in the medium, but LPS did not enhance its production. Inducible nitric oxide synthase expression in EDVECs was not detected with the available antibodies, whereas LPS stimulated cyclooxygenase-2 expression in a time- and concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: LPS stimulated vasoactive mediator production by equine endothelial cells, which may play a role in LPS-induced digital hypoperfusion.  相似文献   

5.
Lipopolysaccharide-binding protein (LBP) is an acute phase protein that binds the lipid A moiety of lipopolysaccharide (LPS) and transfers LPS monomers to soluble CD14 in plasma or membrane bound CD14 on mononuclear phagocytes. The result of these interactions is activation of the TLR4 receptor complex, and the synthesis and release of inflammatory mediators. Inclusion of LBP in cellular assays increases the sensitivity of cells expressing CD14 to LPS. Therefore, the objectives of this study were to (1) compare differentially treated sera from cattle and horses as sources of LBP activity using LPS-induced expression of procoagulant activity (PCA) by equine monocytes as a readout and (2) evaluate the use of commercial equine serum as a source of LBP activity using LPS concentration response and time course studies to validate the response. Monocytes were isolated from eight horses and incubated with five different serum preparations in the presence or absence of Escherichia coli LPS. The sera tested were heat-inactivated fetal bovine serum (HI-FBS), pooled commercial equine serum (CES), heat-inactivated pooled commercial equine serum (HI-CES), autologous equine serum (AES), and heat-inactivated autologous equine serum (HI-AES). In the absence of LPS, monocytes from half of the horses in the study had increased expression of PCA when incubated with HI-FBS alone; PCA was unaffected by incubation with the other sera. There was a four-fold increase in PCA when monocytes were incubated with LPS in the presence of CES, HI-CES or AES compared to LPS without serum. The combination of HI-FBS and LPS increased PCA 20-fold compared to LPS without serum. The HI-AES serum lacked significant LBP activity. Whereas maximal expression of PCA was induced by 1ng/ml of LPS in the absence of serum, inclusion of 1% CES reduced the LPS concentration required for maximal PCA to 30pg/ml. Monocytes incubated with LPS in the presence of CES had increased PCA at 3h and peaked at 6h. In conclusion, monocytes from many horses are directly stimulated by HI-FBS, suggesting that HI-FBS is not an optimal source of LBP for in vitro studies of LPS with equine monocytes. In contrast, CES and AES are effective sources of LBP activity for such studies, as they do not directly induce activation. Although the heat inactivation process did not affect the LBP activity in CES, it ablated LBP activity in AES. Consequently, investigators are advised to utilize either CES or AES in future studies, but not heat-inactivated AES.  相似文献   

6.
OBJECTIVE: To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses. PROCEDURE: Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively. RESULTS: Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent. Conclusion-Lipopolysaccharide induces COX-2 and iNOS in equine macrophages. CLINICAL RELEVANCE: The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses.  相似文献   

7.
REASONS FOR PERFORMING STUDY: Lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-like peptides are Gram-negative bacterial cell wall components which, when released into the peripheral circulation in endotoxaemia, have the potential to activate leucocytes. In vitro, equine neutrophils require priming with LPS in order to generate reactive oxygen intermediates (ROI) in response to fMLP. OBJECTIVES: The aim of this study was to examine whether the release of other neutrophil products is similarly dependent on prior priming with LPS. In particular, neutrophil elastase (NE), a potent proteolytic enzyme, and its major inhibitor, alpha-1 proteinase inhibitor, were investigated. METHODS: Neutrophils were isolated from equine peripheral blood (n = 5) by discontinuous Percoll gradient preparative centrifugation and primed with LPS prior to stimulation with fMLP. ROI were measured by lucigenin dependent chemiluminescence (LDCL). Concentrations of NE and API were determined by ELISA on cell free supernatants taken at 0, 2, 10, 30, 60 and 90 mins post stimulus. Data was analysed by Kruskal-Wallis and Mann-Whitney Tests. RESULTS: Sequential exposure of Percoll purified equine blood neutrophils in vitro to LPS followed by fMLP resulted in the greatest release of NE from equine neutrophils and was required for ROI generation. However, LPS or fMLP stimulation alone resulted in an increase in NE release compared to unstimulated control cells. In contrast, significant API release was only induced by LPS stimulation or fMLP stimulation only after LPS priming, not fMLP on its own. CONCLUSIONS: These results suggest that different stimuli (fMLP or LPS) are capable of invoking similar responses from equine neutrophils with respect to NE release yet different ones with respect to API release. Potential relevance: In addition, demonstration of elastase release induced by LPS and/or fMLP suggests that monitoring serum elastase levels is a potential diagnostic tool for detecting the early onset of endotoxaemia in the horse.  相似文献   

8.
OBJECTIVE: To characterize generation of reactive oxygen species (ROS) by equine spermatozoa. SAMPLE POPULATION: Multiple semen samples collected from 9 stallions. PROCEDURE: Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluorescence microplate reader. RESULTS: Amount of H2O2 generated increased significantly with time and spermatozoa concentration for live and flash-frozen spermatozoa, and amount of H2O2 generated was significantly greater for flash-frozen than for live spermatozoa. Addition of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) significantly increased generation of H2O2 by live and flash-frozen spermatozoa. Addition of a calcium ionophore also significantly increased the amount of H2O2 generated by live spermatozoa but did not have an effect on amount of H2O2 generated by flash-frozen spermatozoa. Abnormal equine spermatozoa generated significantly greater amounts of H2O2 than did normal spermatozoa. CONCLUSIONS AND CLINICAL RELEVANCE: Equine spermatozoa generate ROS in vitro, possibly via a NADPH-oxidase reaction. Spermatozoa damaged during flash-freezing or morphologically abnormal spermatozoa generated significantly greater amounts of ROS than did live or morphologically normal spermatozoa. Damaged and abnormal spermatozoa generate greater amounts of ROS that may contribute to reduced fertility or problems related to semen preservation.  相似文献   

9.
Phosphodiesterase 4 (PDE4) inhibitors have been shown to inhibit equine neutrophil function in vitro and may be of benefit in recurrent airway obstruction (RAO), an allergy-based respiratory disease characterized by inflammatory cell recruitment and activation within the lungs following exposure of susceptible horses to allergens in mouldy hay. The aim of this study was to evaluate the inhibitory effects of the PDE4 inhibitor, rolipram, in an in vitro assay of thromboxane (Tx) production. The assay was then used to monitor the activity of this compound in vivo in normal and RAO-affected horses. Rolipram and the structurally distinct PDE4 inhibitor, denbufylline, attenuated both lipopolysaccharide (LPS)-induced and unstimulated Tx production in blood from normal horses. Thromboxane production appeared to involve a calcium-dependent interaction between leucocytes and platelets (LPS-induced Tx production = 2.3 +/- 0.4, 4.5 +/- 1.1 and 20.8 +/- 3.6 ng/mL for platelets, leucocytes and blood, respectively) and rolipram-inhibited Tx production via an effect on leucocytes. Inhibition of ex vivo LPS induced Tx production was detected after intravenous administration of rolipram (5 microg/kg) to normal ponies. This dose did not significantly affect either lung function or neutrophil accumulation when administered to three horses with clinical signs of RAO. This study suggests that inhibition of Tx production in equine blood can be used to measure PDE4 activity. However, PDE4 inhibitors with improved therapeutic profiles are required for evaluation in RAO.  相似文献   

10.
Neutrophil (PMN) contributions to the acute inflammatory process and host defense include generation of bioreactive oxygen metabolites and secretion of granule enzymes. We assessed equine PMN secretion using several PMN stimuli, singly and in combination with bacterial lipopolysaccharide (LPS). LPS avidly associated with equine PMN, as shown by strong PMN labeling with FITC-conjugated LPS. LPS alone (1 or 10 micrograms ml-1) was a weak stimulus for PMN superoxide anion (O2-) generation, but preincubation with LPS followed by phorbol ester (PMA, 10 ng ml-1) significantly augmented (P less than 0.01) secretion of O2- (19.38 nmol O2- per 2 x 10(6) PMN per 5 min) over the amount generated by PMA stimulation alone (13.75 nmol O2-). A qualitatively similar, but smaller O2(-)-generation response occurred when either opsonized zymosan or recombinant human C5a was used as the PMN stimulus. Arachidonic acid (ArA; 50-200 microM) was a potent stimulus, with secreted O2- levels similar to those from PMA-stimulated PMN. Preincubation of PMN with either the formyl peptide, fMLP, or platelet-activating factor before stimulation with ArA did not significantly increase O2- generation over levels obtained using ArA alone. Release of PMN granule enzymes was also quantitated. A small amount of lysozyme secretion resulted when PMN were exposed to LPS alone (8.20% of total cell content), and PMA stimulation caused marked release of PMN lysozyme (44.45%). Non-specific proteolytic activity in PMN supernatants, assessed by cleavage of a collagen-rich substrate, was minimal with LPS as a sole stimulus (5.08%). There was significant proteolytic activity (P less than 0.01) in supernatants from PMA-stimulated PMN (27.21%), and preincubation with LPS followed by PMA stimulation slightly enhanced (P less than 0.05) the release of PMN proteases (34.62%). The activities of beta-glucuronidase, acid phosphatase, and alkaline phosphatase were minimal in PMN supernatants when using LPS and PMA as stimuli. The activity of PMN granule enzymes was found to be sensitive to the presence of normal equine serum, and proteolytic activity was markedly reduced (80.13% reduction) in the presence of 10% pooled serum.  相似文献   

11.
Neutrophil activation, whilst a key component of host defence, must be tightly regulated in order to avoid an inappropriate cellular response. Annexin-1, which is present in large amounts in neutrophils, and its N-terminal peptides, reduce neutrophil accumulation but annexin peptides have also been shown to exhibit neutrophil activating properties. We have recently shown annexin-1 to be present in equine neutrophils and demonstrated that the annexin-1-derived peptide, Ac2-26, can both reduce superoxide production by these cells in response to other stimuli and directly induce free radical production at a higher concentration. In the present study, we have further characterised the effects of Ac2-26 on equine neutrophil function. In addition, as anti-inflammatory glucocorticoids are known to up-regulate annexin-1, we have examined the effects of dexamethasone on annexin-1 expression in equine leukocytes. The effects of Ac2-26 alone and on agonist (CXCL8, leukotriene (LT)B(4) and PAF)-induced adherence and migration were examined by measuring adhesion of neutrophils to serum-coated plastic and by use of a ChemoTx migration assay. The role of formyl peptide receptors (FPRs) in mediating the effects of Ac2-26 was examined using the pan-FPR antagonist, BOC-2. Flow cytometry was used to measure the effects of dexamethasone on annexin-1 expression. Pre-incubation with Ac2-26 (10(-5)M) significantly inhibited neutrophil adhesion and migration in response to other agonists but when used alone could also induce these responses. The stimulatory and inhibitory effects of Ac2-26 were reduced by BOC-2, indicating a dependency on FPR activation. Dexamethasone increased the percentage of annexin-1 positive neutrophils and mononuclear cells by 1h post treatment (from 45±5% to 93±1% and 62±14% to 87±9% for neutrophils and monocytes, respectively) but by 4h there was no difference from control cells. No difference was seen between the percentages of annexin-1 positive cells pre- and post-treatment in animals that had undergone a dexamethasone suppression test. The attenuation of agonist-induced adherence and migration by Ac2-26 may play a part in regulating recruitment of equine neutrophils in inflammatory conditions of the horse. However, if high concentrations are produced in vivo following release of annexin-1 from activated cells, direct stimulatory effects may occur which could be either beneficial or detrimental. The therapeutic efficacy of anti-inflammatory steroids in the horse may be mediated in part by increasing annexin-1 expression although this effect appears to be short-lived.  相似文献   

12.
Objective-To examine the effect of endotoxins on metabolism and histopathologic changes of isolated perfused equine forelimbs. Sample-Forelimbs (comprising the metacarpus and digit) were collected from cadavers of 12 healthy adult horses after slaughter at an abattoir (14 limbs; 1 forelimb of 10 horses and both forelimbs of 2 horses). Procedures-Forelimbs were perfused for 10 hours with autologous blood, with and without the addition of endotoxin (80 ng of lipopolysaccharide [LPS]/L). Two limbs of the endotoxin exposure group and 2 nonperfused limbs were loaded to failure of the suspensory apparatus of the pedal bone to evaluate the effect of body weight. Metabolic and histologic variables were evaluated. Results-Blood pressure increased during the first hour and did not differ between groups. Lactate dehydrogenase activity was similar in both groups and increased significantly during the 10-hour period; glucose consumption at 5 hours and lactate concentration at 8 hours were significantly higher in limbs exposed to endotoxin. The width of secondary epidermal lamellae was greater in LPS limbs. In the primary dermal lamellae of LPS limbs, there were significantly more vessels with an open lumen and aggregates of intravascular neutrophils. Conclusions and Clinical Relevance-In the blood-perfused isolated forelimbs of equine cadavers, exposure to LPS led to significant changes in the laminar tissue as well as to metabolic changes. Therefore, endotoxin should be considered as a causative factor for laminitis and not merely as a risk factor.  相似文献   

13.
Pentoxifylline has been reported to improve peripheral vascular circulation by altering the flow properties of blood. To determine if the hemorrheological effects of pentoxifylline were mediated by alterations in neutrophil function and/or flow properties, we evaluated the drug's effects on equine neutrophils in vitro. Pentoxifylline, at a concentration of 1 x 10(-1) M, but not at concentrations of 1 x 10(-6) M to 1 x 10(-2) M, markedly suppressed neutrophil superoxide production, zymosan phagocytosis and adherence to nylon wool. Pentoxifylline failed to improve neutrophil filterability through 3 mu polycarbonate filters at any concentration tested. We conclude that equine neutrophil function and flow properties are unlikely to be affected by pentoxifylline concentrations achievable in vivo.  相似文献   

14.
OBJECTIVE: To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, and interleukin- (IL)-1beta from cultured equine smooth muscle cells (SMC). SAMPLE POPULATION: Segments of palmar digital artery harvested from 6 clinically normal adult horses. PROCEDURE: Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 microg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-alpha, and IL-1beta was performed, using isolated total cellular RNA. RESULTS: Although no message was detected for IL-1beta or TNF-alpha in control or endotoxin-exposed equine vascular SMC from all horses, COX-2 underwent a distinct substantial up-regulation after endotoxin exposure. Endotoxin-exposed equine mononuclear cells had up-regulation of IL-1beta and TNF-alpha mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: Increased expression of COX-2 mRNA by equine vascular SMC may be an important early pathophysiologic event in the onset of endotoxemia in horses. Potentiated local vascular production of various prostanoids after increased expression of mRNA for COX-2 may result in vasoactive events observed with laminitis.  相似文献   

15.
Secretion of tumor necrosis factor (TNF) by equine mammary exudate macrophages (MEM phi) exposed to bacterial lipopolysaccharide (LPS) was dose-dependent and was maximal (216.5 +/- 51.9 U/ml) at 100 micrograms LPS/ml, the highest concentration tested. All concentrations of dexamethasone tested (10(-8) to 10(-4) M) significantly (P less than or equal to 0.05) inhibited TNF production by MEM phi when the agent was added 1 hour before LPS. Pretreatment with pentoxifylline at concentrations greater than 3 micrograms/ml also significantly (P less than or equal to 0.05) reduced secretion of TNF by MEM phi. The inhibitory effect of dexamethasone (10(-4) M) was observed when the agent was added to MEM phi from 30 minutes before until 4 hours after LPS. Pentoxifylline (100 micrograms/ml) significantly (P less than or equal to 0.05) suppressed TNF when added from 2 hours before until 2 hours after LPS; however, when pentoxifylline addition was delayed until 8 hours post-LPS, TNF production was enhanced. These apparent inhibitory effects of dexamethasone and pentoxifylline were not due to reduced macrophage viability or to interfering effects of the agents at the level of the TNF bioassay.  相似文献   

16.
OBJECTIVE: To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells. SAMPLE POPULATION: Mononuclear cells from 18 horses and 3 dogs. PROCEDURES: Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti-LPS-binding protein monoclonal antibody or combinations of these constituents. A 1 stage recalcification assay was used to determine procoagulant activity. RESULTS: Addition of FBS to media significantly increased procoagulant activity; equine and canine cells were stimulated by 1% and 10% FBS, respectively. Coincubation of cells with FBS and polymyxin B did not reduce this effect, suggesting that the response was not attributable to LPS contamination. Addition of HI-FBS to media did not stimulate procoagulant activity of equine or canine cells, and the sensitivity of the equine cells to LPS was significantly increased by HI-FBS. This increased LPS sensitivity was reduced 40% with monoclonal antibody directed against human recombinant LPS-binding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosan-induced procoagulant activity of canine cells. CONCLUSION AND CLINICAL RELEVANCE: Procoagulant activity production in equine and canine mononuclear cells was significantly increased by addition of FBS, whereas heat inactivation of FBS eliminated this effect. Heat inactivation did not eliminate the function of serum proteins involved in enhancement of LPS and zymosan-induced procoagulant activity. Results suggest that HI-FBS can be used as a source of serum proteins that increase the sensitivity of mononuclear cells to bacterial and yeast cell wall components.  相似文献   

17.
Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation protocol and enriched to 76% purity by adhesion to tissue culture dishes. Isolated monocytes were incubated with 0, 1, 5, 10 and 50 mM ethyl pyruvate. Cell viability, production of caspase 3/7, and caspase-3 gene expression were determined. In a separate experiment, monocytes were stimulated with LPS (0.1 ng/ml for 1h) followed by incubation with 0, 1, 5, or 10 mM ethyl pyruvate for 1 h. Proinflammatory gene expression was determined by real-time PCR. Ethyl pyruvate at 50 mM adversely affected monocyte viability. Ethyl pyruvate at 10mM or less had no significant effect on monocyte viability, and did not increase activity of caspase 3/7 nor caspase-3 gene expression. Incubation with LPS alone induced a significant upregulation in proinflammatory gene expression. Subsequent treatment of monocytes with ethyl pyruvate significantly reduced IL-8 expression in LPS stimulated monocytes at 5 mM, and IL-8, TNF-α and COX-2 at 10 mM. No beneficial effect on expression of IL-1β or IL-6 was detected. Overall, 10 mM ethyl pyruvate did not adversely affect monocyte viability and suppressed LPS-induced proinflammatory gene expression. Ethyl pyruvate may be a beneficial anti-inflammatory therapy in equine endotoxemia.  相似文献   

18.
Lipopolysaccharide (LPS) can activate equine platelets directly or indirectly, via leukocyte-derived platelet activating factor (PAF). Thromboxane (Tx) production by LPS-stimulated equine platelets requires p38 MAPK and this kinase has been suggested as a therapeutic target in endotoxaemia. The present study has utilised selective inhibitors to investigate the role of p38 MAPK and two other kinases, phosphatidylinositol-3 kinase (PI3K) and protein kinase C (PKC), in regulating PAF-induced Tx production, aggregation and 5-HT release in equine platelets, and the modification of these responses by LPS. LPS enhanced PAF-induced 5-HT release, an effect that was reduced by the p38 MAPK inhibitor, SB203580 (60 ± 8% reduction; n  = 6). SB203580 did not affect responses to PAF alone; whereas inhibition of PKC reduced PAF-induced 5-HT release, Tx production and aggregation (maximal inhibition by the PKCδ inhibitor, rottlerin: 69 ± 13%, 63 ± 14% and 97 ± 1%, respectively; n  = 6). Wortmannin and LY249002, which inhibit PI3K, also caused significant inhibition of PAF-induced aggregation (maximal inhibition 78 ± 3% and 88 ± 2%, respectively; n  = 6). These data suggest that inhibition of platelet p38 MAPK may be of benefit in equine endotoxaemia by counteracting some of the effects of LPS. However, detrimental effects of platelet activation mediated by PAF and not enhanced by LPS are unlikely to be markedly affected.  相似文献   

19.
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.  相似文献   

20.
A study was performed to determine the effect of proadifen hydrochloride on prostacyclin (prostaglandin I2 [PGI2]) and thromboxane A2 (TxA2) synthesis by equine peritoneal macrophages and the effect of proadifen on endotoxin-induced synthesis of PGI2 and TxA2 by equine macrophages. Peritoneal macrophages (2.5 x 10(6)/ml) were incubated for 6 hours in tissue culture media containing 1) nothing (nontreated control), 2) proadifen hydrochloride (20, 100, 250, and 500 mumol/L, 3) endotoxin (5 ng/ml), or 4) the calcium ionophore A23187 (0.95 mumol/L). In a second series of experiments, peritoneal macrophages were incubated with endotoxin (5 ng/ml) and proadifen (250 umol/L), for 6 hours. Concentrations of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2, the stable metabolites of PGI2 and TxA2, were determined in the incubation media by radioimmunoassay. Proadifen caused increased synthesis of PGI2 by equine macrophages, without affecting TxA2 production. The increased PGI2 production was similar to that induced by endotoxin and calcium ionophore; however, the latter 2 agents significantly stimulated TxA2 production as well (P less than 0.05). There were no significant differences among mean concentrations of 6-keto-PGF1 alpha in media from macrophages treated with 100, 250, or 500 mumol/L proadifen, but there was a significant curvilinear regression between their concentrations. The ratio of thromboxane B2 to 6-keto-PGF1 alpha was significantly lower than baseline in incubation media from macrophages exposed to proadifen, endotoxin, and calcium ionophore. Proadifen hydrochloride did not significantly change equine peritoneal macrophage production of PGI2 or TxA2 in response to endotoxin.  相似文献   

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