Effects of IL-6 and IL-1α on the peripheral blood polymorphonuclear neutrophil apoptosis postburn in rats

AIM:To study the effects of IL-6 and IL-1α on the blood polymorphonuclear-neutrophils(PMN) apoptosis postburn.METHODS:Wistar rats inflicted by 30% total body surface area (TBSA) Ⅲ degree scalding were employed as the model. PMN were isolated by density gradient centrifugation using Percoll-hypaque and labeled with TdT-mediated and dUTP nick end labeling (TUNEL) and analyzed by flow cytometric analysis. The intracellular caspase-3 activation and the serum levels of IL-6 and IL-1α were analyzed by fluorometric immunosorbent enzyme assay and enzyme-linked immunosorbent assay, respectively.RESULTS:The serum IL-6 levels (μg/L) in groups of 3, 6, 12, 24 and 48 h postburn (9.14±1.16, 12.49±1.14, 3.01±0.75, 1.41±0.28 and 1.56±0.43 in turn) and IL-1α (ng/L) in groups of 3, 6, 12 h postburn (90.08±8.39, 320.93±14.48 and 47.84±5.19) were much higher than IL-6 (0.24±0.07) and IL-1α (27.65±4.86) in control group (P＜0.05), respectively. The relative proportions of apoptotic PMN(%) in groups postburn were 9.89±2.00, 4.98±1.35, 1.31±0.72, 2.49±1.87 and 6.88±1.13 in turn, which were obviously less than 13.66 ± 3.88 in the control group. The results of intracellular caspase-3 activation in PMN were observed to be consistent with the results of apoptotic PMN analysis in experimental groups.CONCLUSION:IL-6 and IL-1α are the important factors to induce PMN apoptosis delay that occurred obviously postburn. And decrease in the activity of intracellular caspase-3 of PMN may be involved in their mechanisms.     

Resveratrol decreases oxidative burst capacity and alters stimulated leukocyte cytokine production in vitro

IntroductionResveratrol, a naturally-occurring phytophenol, has been shown to bolster immune surveillance and reverse immunosenescence in a dose dependent manner in rodents and humans. Although safety and pharmacokinetic studies have been completed in dogs, the immunomodulatory effects of resveratrol in dogs has not yet been investigated. The objective of this study was to determine the effect of resveratrol on canine innate immune system function in vitro. The hypothesis was that similar to other species, low concentrations of resveratrol would stimulate while high concentrations would depress innate immune system function.MethodsWhole blood was collected from six healthy, adult, client-owned dogs and was incubated with resveratrol at final concentrations of 6000 ng ml⁻¹, 3000 ng ml⁻¹, 1000 ng ml⁻¹, or control solution for 4 h. Following incubation, phagocytosis and oxidative burst were evaluated using flow cytometry, and LPS-, lipoteichoic acid (LTA) – and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay.ResultsPhagocytosis was not altered by resveratrol at any concentration compared to control. However, while the number of PMNs capable of performing oxidative burst did not change, the robustness of the reaction following stimulation with Escherichia coli and PMA was reduced in a dose dependent manner. In addition, LPS-, LTA-, PG, and PBS-stimulated TNF production was increased following incubation with all concentrations of resveratrol compared to control, and this effect was dose dependent. LTA-stimulated IL-6 was increased with resveratrol compared to control. Furthermore, LTA-stimulated IL-10 was decreased with 6000 ng ml⁻¹ and 3000 ng ml⁻¹ concentrations of resveratrol and PG-stimulated IL-10 production was decreased with all concentrations of resveratrol compared to control. The LPS-, LTA-, and PG-stimulated TNF:IL-10 ratio was increased with 6000 ng ml⁻¹ of resveratrol compared to control and lower resveratrol concentrations.ConclusionWhile resveratrol was sparing to PMN phagocytosis, it reduced the robustness of PMN oxidative burst. Resveratrol also increased pro-inflammatory and decreased anti-inflammatory leukocyte cytokine production capacity in vitro. These data suggest that resveratrol supplementation may depress oxidative burst reactions while promoting pro-inflammatory leukocyte cytokine production and decreasing anti-inflammatory cytokine production. Based on these findings, further in vivo study regarding the effects of resveratrol on PMN oxidative burst capability and leukocyte cytokine production capacity are indicated prior to routine supplementation.     

p65 Homodimer activity in distal airway cells determines lung dysfunction in equine heaves

Nuclear factor-κB (NF-κB) activity, which is a key regulator of inflammatory gene expression, is increased in bronchial epithelial cells from horses suffering from heaves (a hypersensitivity-associated inflammatory condition of the lung). To determine whether this increased activity extends to distal airways and to other pulmonary cells, cells recovered by broncho-alveolar lavage (BAL) in healthy and heaves-affected horses were assessed for NF-κB activity. NF-κB activity was much higher in BAL cells from heaves-affected horses, especially during crisis (disease exacerbation), than in cells from healthy horses. Moreover, the level of NF-κB activity found in BAL cells was positively correlated to total lung resistance and to the proportion of neutrophils present in BAL fluid. Finally, prototypical p65–p50 NF-κB heterodimers were absent from BAL cells, which mostly contained p65 homodimers. These results (1) show that increased NF-κB activity is a general feature of heaves lung; (2) demonstrate the importance of p65 homodimers in neutrophilic inflammation; and (3) suggest that the use of specific NF-κB inhibitors could improve lung function in heaves-affected horses.     

The effects of floor heating on body temperature, water consumption, stress response and immune competence around parturition in loose-housed sows

The aim of the present study was to study whether floor heating from 12 h after onset of nest building until 48 h after birth of the first piglet had any effect on measures related to body temperature, water consumption, stress response and immune competence in loose-housed sows (n = 23). In conclusion, the present results indicate that floor heating for a limited period around parturition did not compromise physiological and immunological parameters, water intake and body temperature in loose-housed sows. The water intake peaked the day before parturition and the body temperature peaked on the day of parturition. A cortisol peak at parturition, a transient rise in the number of leucocytes and neutrophils and a transient reduction in the number of lymphocytes, erythrocytes and in the PCV value were observed. Around and after parturition some non-specific immunological variables seemed to be stimulated while others seemed to be compromised.     

In vitro modulatory effect of ω-3 polyunsaturated fatty acid (EPA and DHA) on phagocytosis and ROS production of goat neutrophils

An in vitro study was carried out to examine the influence of two fish-oil-derived long chain ω-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on goat polymorphonuclear leukocytes (PMN). Twelve Saanen healthy goats were used as blood donors. Neutrophils were isolated from blood and incubated with increasing concentration of EPA and DHA (25, 50, 100, 200 μM). Control samples were incubated in the absence of ω-3 PUFAs. Phagocytosis was evaluated by fluorescein-labeled Escherichia coli incorporation, while extracellular Reactive Oxygen Species (ROS) production was determined by cytochrome c reduction assay, which was selected among the others due to its specificity for extracellular superoxide anion release. Phagocytic activity was significantly increased by EPA (P < 0.05) and DHA (P < 0.01). Treating PMN with EPA does not affect extracellular ROS production which is, on the contrary, down-regulated by DHA. This effect was increased in experimental conditions which mimic pro-inflammatory challenges (stimulation with PMA).This study demonstrates that EPA and DHA may have beneficial effect on neutrophil function by increasing their phagocytosis activity and, in the meanwhile, decreasing the tissue damages due to extracellular release of ROS.     

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.     

Chemotherapy and remission status do not alter pre-existing innate immune dysfunction in dogs with lymphoma

Dogs with lymphoma have altered innate immunity and little is known about the effects of chemotherapy on innate immune function in dogs. Lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PG) – induced leukocyte cytokine production capacity, and phagocytosis and respiratory burst were evaluated in dogs prior to and following 6 weeks of chemotherapy. Dogs had decreased TNF production following LPS stimulation and increased IL-10 production following PG stimulation, which did not improve following remission of lymphoma. Dogs also had reduced E. coli-induced respiratory burst function after chemotherapy induced complete or partial remission. Dogs with lymphoma have an imbalance in pro-and anti-inflammatory cytokine production which did not improve with remission, and, following treatment, a decrease in respiratory burst function. Altered immune responses following exposure to bacterial pathogen associated molecular pattern motifs and bacteria may have many implications in the management of canine lymphoma.     

Effect of EDTA on morphology of neutrophils of healthy dogs and dogs with inflammation

Increased basophilia and foamy vacuolation in the cytoplasm of neutrophils in smears made within 1 hour of blood collection suggest inflammatory disease. Due to the mild increase in foamy vacuolation which occurred with time in EDTA, the significance of mildly vacuolated neutrophils in smears prepared a few hours after blood collection is questionable. However, moderate or severe foamy vacuolation should be considered clinically significant. In vitro morphologic changes in neutrophils from healthy dogs in EDTA include clear, discrete vacuoles in the cytoplasm, uneven distribution of cytoplasmic granules, irregular cell membrane and pyknosis. These neutrophils generally lack basophilia, have minimal foamy vacuolation of the cytoplasm and are morphologically different from neutrophils associated with severe inflammation.     

The role of seasonality on the inhibitory effect of Brazilian green propolis on the oxidative metabolism of neutrophils

The reactive oxygen species (ROS) produced by neutrophils are involved in the pathogenesis of several diseases, for which the intake of antioxidants could benefit patients either as a prophylactic or therapeutic treatment. Propolis is among the known antioxidants, and its chemical composition may vary under the influence of seasonality, which may interfere in its biological properties. This work evaluates the role of seasonality on the production of some important compounds of propolis samples produced monthly from November 2001 through October 2002 as well as the effect of these samples on the oxidative metabolism of stimulated neutrophils, by using both luminol and lucigenin to produce chemiluminescence (CLlum and CLluc, respectively). The cytotoxicity of the most active extracts to neutrophils was also investigated. The inhibitory effect of the propolis samples varied significantly during the studied period for both assays (3.4 ± 1.1 to 16.0 ± 1.1 μg/mL for CLlum and 6.2 ± 2.0 to 30.0 ± 5.0 μg/mL for CLluc), which was also observed in the quantitative profile of the main analyzed compounds (aromadendrin-4′-methyl ether, artepillin C, and baccharin). This effect started to become more prominent during the fall and, among all the studied extracts, the one obtained in May displayed the highest inhibitory effect on CL production (3.4 ± 1.1 μg/mL for CLlum and 6.2 ± 2.0 μg/mL for CLluc). The HPLC qualitative profiles of the extracts of propolis samples were quite similar, but there was a huge variation in terms of quantitative profile. It seems that aromadendrin-4′-methyl ether and baccharin play an essential role in the antioxidant activity, while artepillin C is not very important for this effect. The extracts presenting the highest antioxidant activity were produced in May, June, and August, and they did not display cytotoxicity at 25 μg/mL; quercetin, used as control, was not toxic to neutrophils at 8.5 μg/mL.