Neutrophil Respiratory Burst Activity Research Articles

N-acetylcysteine Protects Striated Muscle in a Model of Compartment Syndrome

To avoid ischemic necrosis, compartment syndrome is a surgical emergency treated with decompression once identified. A potentially lethal, oxidant-driven reperfusion injury occurs after decompression. N-acetylcysteine is an antioxidant with the potential to attenuate the reperfusion injury. We asked whether N-acetylcysteine could preserve striated muscle contractility and modify neutrophil infiltration and activation after simulated compartment syndrome release. Fifty-seven rats were randomized to control, simulated compartment syndrome, and simulated compartment syndrome plus N-acetylcysteine groups. We isolated the rodent cremaster muscle on its neurovascular pedicle and placed it in a pressure chamber. Chamber pressure was elevated above critical closing pressure for 3 hours to simulate compartment syndrome. Experiments were concluded at three times: 1 hour, 24 hours, and 7 days after decompression of compartment syndrome. We assessed twitch and tetanic contractile function and tissue myeloperoxidase activity. Ten additional rats were randomized to control and N-acetylcysteine administration after which neutrophil respiratory burst activity was assessed. The simulated compartment syndrome decreased muscle contractility and increased muscle tissue myeloperoxidase activity compared with controls. Treatment with N-acetylcysteine preserved twitch and tetanic contractility. N-acetylcysteine did not alter neutrophil infiltration (myeloperoxidase activity) acutely but did reduce infiltration at 24 hours, even when given after decompression. N-acetylcysteine reduced neutrophil respiratory burst activity. N-acetylcysteine administration before or after simulated compartment syndrome preserved striated muscle contractility, apparently by attenuating neutrophil activation and the resultant oxidant injury. Our data suggest a potential role for N-acetylcysteine in the attenuation of muscle injury after release of compartment syndrome and possibly in the prophylaxis of compartment syndrome.

Elevated neutrophil respiratory burst activity in essential hypertensive patients

Neutrophils play a significant role in maintaining the integrity of innate immunity via their potent respiratory burst activity. However, the uncontrolled activation of respiratory burst in neutrophils also attributes to chronic diseases such as primary hypertension and atherosclerosis. In our study, we have investigated the activation of respiratory burst function of neutrophils harvested from essential hypertensive patients. In the presence of stimuli PMA and opsonized zymosan (OZ), hypertensive patients’ neutrophils secrete significantly higher amount of superoxide anions compared to normotensive control. Although the magnitude of activation varies between both groups, yet the kinetics of activation is similar. When normotensive control’s neutrophils were pre-treated with hypertensive serum, the cells failed to migrate toward fMLP which indicates the impairment of the migration property. In conclusion, the respiratory burst activity of neutrophils is affected by hypertension and their elevated superoxide anions production could be an aggravating factor in hypertension-related complication.

Estrogenic Hormone Modulation Abrogates Changes in Red Blood Cell Deformability and Neutrophil Activation in Trauma Hemorrhagic Shock

Decreased red blood cell (RBC) deformability and activation of neutrophils (polymorphonuclear leukocytes [PMN]) after trauma-hemorrhagic shock (T/HS) have been implicated in the development of multiple organ dysfunction. Experimentally, female animals seemed to be protected from the effects of T/HS, at least in part, because of elevated estrogen levels. Thus, we examined the relative role of estrogen receptor (ER)-alpha and -beta in this protective response. To accomplish this goal, RBC deformability and neutrophil respiratory burst activity were measured in several groups of hormonally intact or ovariectomized (OVX) female rats subjected to T/HS (laparotomy plus hemorrhage to an MAP of 30 mm Hg to 35 mm Hg for 90 minutes) or trauma-sham shock (T/SS) and 3 hours of reperfusion. These groups included rats receiving vehicle, estradiol, or either an ER-alpha agonist or an ER-beta agonist administered at the end of the shock period just before volume resuscitation. RBC deformability and neutrophil activation were similar among all the T/SS groups and were not different from that observed in the non-OVX female rats subjected to T/HS. In contrast, RBC deformability was reduced and neutrophil activation was increased in the OVX, T/HS female rats as compared with the T/SS groups or the non-OVX, T/HS rats. The administration of estrogen to the T/HS, OVX rats returned RBC and neutrophil function to normal. Both the ER-alpha and -beta agonist partially, but not completely, protected the OVX rats from T/HS-induced loss of RBC deformability, whereas only the ER-beta agonist prevented the increase in neutrophil activation. The protective effects of estrogen on T/HS-induced RBC deformability are mediated, at least in part, via activation of both ER-alpha and -beta, whereas ER-beta activation is involved in limiting T/HS-induced neutrophil activation.

Cystic fibrosis neutrophils have normal intrinsic reactive oxygen species generation

Previous studies have identified abnormalities in the oxidative responses of the neutrophil in cystic fibrosis (CF), but it is unclear whether such changes relate to loss of membrane cystic fibrosis transmembrane conductance regulator (CFTR) or to the inflammatory environment present in this disease. The aim of the present study was to determine whether neutrophils from CF patients demonstrate an intrinsic abnormality of the respiratory burst. The respiratory burst activity of neutrophils isolated from stable DeltaF508 homozygote CF patients and matched healthy controls was quantified by both chemiluminscence and cytochrome C reduction. Expression of NADPH oxidase components and CFTR was determined by Western blotting and RT-PCR. The oxidative output from neutrophils from CF in response to receptor-linked and particulate stimuli did not differ from that of controls. Expression of NADPH oxidase components was identical in CF and non-CF neutrophils. While low levels of CFTR mRNA could be identified in the normal human neutrophil, we were unable to detect CFTR protein in human neutrophil lysates or immunoprecipitates. CFTR has no role in controlling neutrophil oxidative activity; previously reported differences in neutrophil function between CF and non-CF subjects most likely relate to the inflammatory milieu from which the cells were isolated.

RECOMBINANT FACTOR XIII DIMINISHES MULTIPLE ORGAN DYSFUNCTION IN RATS CAUSED BY GUT ISCHEMIA-REPERFUSION INJURY

Plasma factor XIII (FXIII) is responsible for stabilization of fibrin clot at the final stage of blood coagulation. Because FXIII has also been shown to modulate inflammation and endothelial permeability, we hypothesized that FXIII diminishes multiple organ dysfunction caused by gut I/R injury. A model of superior mesenteric artery occlusion (SMAO) was used to induce gut I/R injury. Rats were subjected to 45-min SMAO or sham SMAO and treated with recombinant human FXIII A2 subunit (rFXIII) or placebo at the beginning of the reperfusion period. Lung permeability, lung and gut myeloperoxidase activity, gut histology, neutrophil respiratory burst, and microvascular blood flow in the liver and muscles were measured after a 3-h reperfusion period. The effect of activated rFXIII on transendothelial resistance of human umbilical vein endothelial cells was tested in vitro. Superior mesenteric artery occlusion-induced lung permeability as well as lung and gut myeloperoxidase activity was significantly lower in rFXIII-treated versus untreated animals. Similarly, rFXIII-treated rats had lower neutrophil respiratory burst activity and ileal mucosal injury. Rats treated with rFXIII also had higher liver microvascular blood flow compared with the placebo group. Superior mesenteric artery occlusion did not cause FXIII consumption during the study period. In vitro, activated rFXIII caused a dose-dependent increase in human umbilical vein endothelial cell monolayer resistance to thrombin-induced injury. Thus, administration of rFXIII diminishes SMAO-induced multiple organ dysfunction in rats, presumably by preservation of endothelial barrier function and the limitation of polymorphonuclear leukocyte activation.

Mycophenolic acid inhibits PMA‐induced activation of the neutrophil respiratory burst

Mycophenolate mofetil (MMF) is commonly used in immunosuppressive regimens for solid organ transplantation. There is evidence that the hydrolyzed active agent mycophenolic acid (MPA) causes the endothelial depletion of intracellular guanosine 5'-triphosphate (GTP) levels. This depletion may cause inactivation of nicotinamide adenine dinucleotide phosphate oxidase. The purpose of the present study was to examine the impact of MPA on the neutrophil respiratory burst and phagocytic activity using flow cytometry. In whole blood of healthy volunteers, 2 different doses of MPA (1 and 10 mumol/L) did not alter hydrogen peroxide production of neutrophils induced by receptor-dependent activators. In contrast, MPA inhibits the protein kinase C (PKC)-mediated hydrogen peroxide production by phorbol 12-myristate 13-acetate (PMA) in a time-dependent manner (negative: 21.17 +/- 1.64 vs. 120 min: 14.46 +/- 1.28 mean fluorescence intensity, incubation with 1 mumol/L MPA). In conclusion, our results corroborated that the neutrophil respiratory burst activity of healthy volunteers, induced by either formyl-methionyl-leucylphenylalanine (fMLP), priming with tumor necrosis factor alpha followed by fMLP or Escherichia coli and neutrophil phagocytic capacity, were not significantly affected after MPA treatment. We also could demonstrate that the hydrogen peroxide production of neutrophils decreased in response to the PKC activator PMA in a time-dependent manner.

TLR Expression on Neutrophils at the Pulmonary Site of Infection: TLR1/TLR2-Mediated Up-Regulation of TLR5 Expression in Cystic Fibrosis Lung Disease

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Differential effects of α1-acid glycoprotein on bovine neutrophil respiratory burst activity and IL-8 production

During bacterial-mediated diseases, neutrophils (PMNs) play a critical role in defending the host against invading pathogens. PMN production of reactive oxygen species (ROS) contributes to the bactericidal capabilities of these cells. ROS are produced intracellularly and can be released extracellularly. The aberrant extracellular release of ROS, however, has been reported to induce injury to host tissues during mastitis and other inflammatory-mediated diseases of cattle. The acute phase response, which occurs shortly after infection or tissue injury, is characterized by the induction of a large number of plasma proteins referred to as acute phase proteins (APP). α 1-Acid glycoprotein (AGP) is an APP that increases in response to infection or injury in cattle and humans. The precise function of AGP is unknown, but it has been reported to possess anti-inflammatory properties. The objective of this study was to evaluate the effects of bovine AGP on PMN pro-inflammatory responses, including respiratory burst activity and cytokine production. Bovine AGP dose-dependently inhibited zymosan-induced PMN extracellular release of superoxide anion and hydrogen peroxide without affecting the capacity of PMN to engulf and kill Staphylococcus aureus. Moreover, AGP exerted its effect on ROS production regardless of whether PMNs were exposed to AGP prior to or after activation. In contrast to respiratory burst activity, AGP enhanced PMN production of IL-8. The precise mechanism by which AGP regulates PMN functions remains unknown, but data presented in this study suggest that AGP may have a complex role by differentially regulating PMN pro-inflammatory activities.

Effect of 2‐hydroxyethyl‐methacrylate (HEMA) on the phagocytic and respiratory burst activity of human neutrophils and monocytes

Neutrophils and monocytes/macrophages (MØ), found in oral mucosa and gingival sulcus, phagocytose and kill bacteria using products produced during a respiratory burst. 2-Hydroxyethyl-methacrylate (HEMA) is a major component released from resin glass ionomer and dental adhesives. Hence, in pulp and gingiva, phagocytes can come into contact with unpolymerized HEMA monomers. The aim of this study was to examine the effects of exposure to HEMA on neutrophil and monocyte bactericidal function. Blood collected from five female volunteers was exposed in vitro to HEMA for 2 h and then phagocytosis, respiratory burst, and cellular integrity were measured using flow cytometry. Respiratory burst was quantified by measuring fluorescent rhodamine 123 generated via oxidation of dihydrorhodamine 123. Cellular membrane integrity was evaluated by staining with propidium iodide. The respiratory burst activity of the neutrophils was significantly decreased by exposure to 7.5 and 15 mM HEMA. No significant effect of HEMA was seen on the number of granulocytes or monocytes capable of performing respiratory burst. Furthermore, there was no significant effect of HEMA on the phagocytic activity of the monocytes or the granulocytes. In conclusion, HEMA did not affect the phagocytosis activity of neutrophils; however, the ability of the cells to kill internalized prey was significantly reduced.

Neutrophil function is preserved in a pooled granulocyte component prepared from whole blood donations

Whole blood-derived granulocytes (buffy coats) are issued as an alternative to apheresis donations, but are heavily contaminated with red cells and platelets and there is minimal in vitro data describing their functionality. We developed a purer pooled granulocyte component (PGC) from whole blood donations by pooling 10 ABO-matched buffy coats with 400 ml of platelet additive solution (SSP+) and re-centrifuging. The PGC was irradiated (25-50 Gy) and neutrophil viability, chemotaxis, phagocytosis and respiratory burst activity were determined by flow cytometry. Results from 13 PGC at 16-20 h following donation were compared with those obtained from 20 standard individual buffy coats and with fresh whole blood. The PGC contained similar numbers of neutrophils (approximately 0.9 x 10(10)) with a reduced volume and haemoglobin content when compared with 10 individual buffy coats. Neutrophils in the PGC maintained >90% viability, oxidative burst and phagocytic activity and their ability to migrate towards a chemoattractant 16-20 h following donation, which is similar to results obtained with either fresh whole blood or standard buffy coats. Therefore, neutrophil function in the PGC was preserved 16-20 h following donation, but this product had significantly lower red cell contamination compared with 10 buffy coats, which are currently transfused.

Renal Transplantation Normalized Hydrogen Peroxide Production of Neutrophils within the First Day

Background: Hemodialysis patients are in a state of oxidant stress. In renal transplantation reactive oxygen species (ROS) are considered to be important factors of ischemia-reperfusion injury. Neutrophils produce ROS as part of the host defense against invading bacteria. This study was designed to investigate whether neutrophil function in hemodialysis patients is immediately affected by renal transplantation. Methods: We evaluated the neutrophil respiratory burst and phagocytic activity in renal transplant patients with living-related donor (LRD) and cadaveric donor (CAD) grafts using flow cytometry techniques. Twenty patients (LRD = 6, CAD = 14) and 20 healthy volunteers were included in the study. Venous blood samples were drawn before anesthesia, 5 min before reperfusion, 1 h and 1, 3 and 7 days after reperfusion. Results: Before surgery, a significant increase in hydrogen peroxide production in neutrophils was seen for both renal transplantation groups compared to healthy subjects. Within 24 h after reperfusion hydrogen peroxide production almost decreased to normal values. The phagocytic capacity of neutrophils was continuously depressed. There were no differences between the CAD and LRD groups. Conclusions: We found that the enhanced respiratory burst activity of patients with chronic renal failure decreased to normal values within 1 day following renal transplantation. Our results suggest that reduced respiratory burst activity resulting in a diminished risk of tissue damage by the uncontrolled production of ROS.

Modulation of the Glycemic Response Using Insulin Attenuates the Pulmonary Response in an Animal Trauma Model

Hyperglycemia has been shown to be an independent prognostic indicator of poor outcome in the traumatized patient. The role of insulin and the prevention of hyperglycemia in the trauma patient have as yet not been fully explored. We hypothesized that the systemic inflammatory response to trauma could be modified by modulating the glycemic response to trauma using insulin. A rodent model of end- organ (lung) injury in trauma was chosen. Two groups underwent bilateral femur fracture and 15% blood loss. The third group was anesthetized only. The treatment group immediately received subcutaneous insulin according to a sliding scale. The control groups received normal saline subcutaneously. The animals were maintained under anesthesia for 4 hours from injury. Blood samples were then taken. Bronchoalveolar lavage was performed for neutrophil content and total protein estimation. The left lower lobe was harvested for wet:dry lung weight ratios as a measure of end-organ tissue edema. Measures of end-organ injury, wet:dry lung weight ratios, and bronchoalveolar lavage neutrophil content were significantly reduced in the insulin-treated animals compared with in the controls (p < 0.05). Neutrophil respiratory burst activity was increased in insulin-treated animals compared with in controls (p < 0.05). Insulin reduces leukocyte lung sequestration and end-organ (lung) edema, indicating an endothelial protective effect in this injured-animal model without attenuating neutrophil function. This work confirms that modifying the glycemic response to trauma using insulin may have a role in reducing adult respiratory distress syndrome rates in injured patients and thereby lead to improved outcomes.

Oat β-Glucan Effects on Neutrophil Respiratory Burst Activity following Exercise

Fatiguing exercise has been associated with a decrease in certain functions of neutrophils, whereas moderate exercise has generally been associated with an increase. Consumption of oat beta-glucan (ObetaG), a soluble fiber and mild immune system enhancer, may offset the immunosuppression associated with intense training and perhaps further enhance the benefits of moderate exercise. To test the effects of ObetaG consumption on neutrophil function and number after both moderate and fatiguing exercise. Male mice were assigned to one of six treatment groups. Fatiguing exercise mice (Ftg-ObetaG and Ftg-H2O) ran to volitional fatigue on a treadmill for three consecutive days, and moderate exercise mice (Mod-ObetaG and Mod-H2O) ran for six consecutive days for 1 h. Control mice (Con-ObetaG and Con-H2O) were exposed to the treadmill environment but did not run. ObetaG was consumed in the drinking water (approximately 0.6 mL x d(-1)) for 10 consecutive days. After rest or exercise on the last day of training, mice were given a 1-mL i.p. injection of thioglycollate. Mice were sacrificed 3 h later; neutrophils were harvested from the peritoneal cavity and counted, and their respiratory burst activity was measured using flow cytometry. Both moderate exercise and ObetaG increased neutrophil burst activity, whereas fatiguing exercise had no effect. Neutrophil number was increased by fatiguing exercise and ObetaG, but not moderate exercise. There were no additive effects of exercise and ObetaG on either of these variables. These data suggest that although not additive in their effects, both ObetaG and exercise can alter overall neutrophil respiratory burst activity (number and/or function), but only ObetaG increased both number and function, which may have important ramifications for defense against infection.

Consumption of prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis has minor effects on selected immune parameters in polypectomised and colon cancer patients

Probiotics (PRO) modulate immunity in humans, while the effect of prebiotics (PRE) and synbiotics (SYN) on the human immune system are not well studied yet. The objective of this study was to investigate whether daily intake of a SYN modulates immune functions. In a randomised double-blind, placebo-controlled trial, thirty-four colon cancer patients who had undergone 'curative resection' and forty polypectomised patients participated. Subjects of the SYN group daily received encapsulated bacteria (1 x 10(10) colony-forming units of Lactobacillus rhamnosus GG (LGG) and 1 x 10(10) colony-forming units of Bifidobacterium lactis Bb12 (Bb12)) and 10 g of inulin enriched with oligofructose. Controls received encapsulated maltodextrin and 10 g of maltodextrin. Prior to intervention (T1), and 6 (T2) and 12 weeks after the start of the intervention (T3), phagocytic and respiratory burst activity of neutrophils and monocytes, lytic activity of natural killer cells and production of interleukin (IL)-2, IL-10 and IL-12, as well as tumour necrosis factor-alpha and interferon-gamma (IFN-gamma) by activated peripheral blood mononuclear cells (PBMC) were measured. In faeces, the concentrations of transforming growth factor-beta1 and prostaglandin E2 were measured. IL-2 secretion by activated PBMC from the polyp group increased significantly between T1 or T2 and T3 (P < 0.05). In the cancer group, SYN treatment resulted in an increased capacity of PBMC to produce IFN-gamma at T3 (P < 0.05). Other immunity-related parameters were not affected by SYN treatment, neither in the cancer nor in the polyp group. In conclusion, supplementation with this SYN has minor stimulatory effects on the systemic immune system of the two study groups. Further studies in humans should aim to focus on the gut-associated immune system.

Effects of epidural anaesthesia on surgical stress-induced immunosuppression during upper abdominal surgery

Previously, we have demonstrated that surgical stress rapidly induced transient hyporesponsiveness of blood cells to endotoxin and that monocyte mCD14 and HLA-DR expression decreased soon after the start of surgery under general anaesthesia. This study was designed to investigate the effects of epidural anaesthesia on surgical stress-induced immunosuppression in patients undergoing upper abdominal surgery. After having obtained informed consent, patients were randomly allocated to receive general anaesthesia (Group G) or general anaesthesia with epidural anaesthesia (Group E). Perioperative changes in neutrophil phagocytic activity, neutrophil respiratory burst activity, monocyte mCD14 and HLA-DR expression, plasma IL-10 concentration, and the LPS-induced TNF-alpha production in whole blood were measured. Surgical stress rapidly depressed neutrophil phagocytic activity, monocyte mCD14 and HLA-DR expression, and LPS-induced TNF-alpha production ex vivo (P < 0.05 vs preoperation) in both Group G and Group E. In contrast, the plasma IL-10 concentration increased significantly 2 h after the start of surgery (P < 0.05) in both groups. There were no significant differences between the two groups. The neutrophil respiratory burst activity did not change during the operation in either group. This study showed that the innate immune system is suppressed from the early period of upper abdominal surgery. Subgroup analysis suggested that epidural anaesthesia to T4 dermatome as well as general anaesthesia may not protect patients from this immunosuppression. These results in part explain the impairment of host-defense mechanisms seen in the perioperative period.

New Role for ATP P2X1 Receptors in the Control of Neutrophil Apoptosis and Respiratory Burst Activity.

P2X receptors are membrane non-selective cation channels that open in response to the binding of extracellular ATP. Seven genes encode P2X receptor subunits (P2X1–7) in vertebrates. Channels form homo- or hetero-multimers of several subunits that are highly permeable to Ca2+. P2X receptors are widely distributed. In recent years, increasing attention has been paid to extracellular ATP as a candidate danger signal locally released at the inception of inflammation. One of the most striking features of ATP is its ability to promote P2X7 receptor-mediated massive release of mature IL-1β from LPS-primed mononuclear phagocytes. On neutrophils, ATP rises intracellular Ca2+ concentrations, contributes to degranulation, adhesion, oxidative burst and delays apoptosis, events that may partly depend on the G-protein coupled P2Y2 receptor and on P2X7. In the present work, RT-PCR, Western blotting and immunofluorescence experiments reveal that neutrophils also express P2X1 and P2X5 receptor subtypes. A microarray analysis indicates that a 3-hour treatment of human peripheral blood neutrophils with the selective P2X1 and P2X1/5 receptor agonist, a,β-meATP, changes the expression of genes mainly involved in the control of cell fate. Accordingly, this agonist causes an increase of phosphatidylserine exposure on neutrophil membranes, maximally occurring after 3 hours and lasting until 18 hours of culture. In the presence of the protein synthesis inhibitor cycloheximide, a,β -meATP promotes caspase-3-dependent neutrophil apoptosis after 3 hours, which is correlated with highly reduced Fcγ RIII (CD16) membrane expression. In addition, a 1 min pretreatment of neutrophils with a,β -meATP potently increases tumor necrosis factor-a (TNF-α )-driven priming (30 min) of the respiratory burst induced by the bacterially derived peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Furthermore, a,β -meATP produces L-selectin (CD62L) shedding by its own and in an additive manner with TNF-α . This agonist also induces rapid and reversible phosphorylation of the survival kinases ERK (starting after 2 min) and Akt (15 min) as well as phosphorylation and degradation (after 2 min) of I-κ Bα , an inhibitor of the anti-apoptotic transcription factor NF-κ B. Hence, neutrophil P2X1 receptors might have a dual role in inflammation; they would both contribute to neutrophil activation and promote cell death of neutrophils that have reached the end of their useful life span. Activation of P2X1 receptors by extracellular ATP may thus represent novel regulatory mechanisms that govern neutrophil function.

Effect of Cis-Urocanic Acid on Bovine Neutrophil Generation of Reactive Oxygen Species

Neutrophils play a fundamental role in the host innate immune response during mastitis and other bacterial-mediated diseases of cattle. One of the critical mechanisms by which neutrophils contribute to host innate immune defenses is through their ability to phagocytose and kill bacteria. The ability of neutrophils to kill bacteria is mediated through the generation of reactive oxygen species (ROS). However, the extracellular release of ROS can be deleterious to the host because ROS induce tissue injury. Thus, in diseases such as mastitis that are accompanied by the influx of neutrophils, the generation of large quantities of ROS may result in significant injury to the mammary epithelium. cis-Urocanic acid (cis-UCA), which is formed from the UV photoisomerization of the trans isoform found naturally in human and animal skin, is an immunosuppressive molecule with anti-inflammatory properties. Little is known about the effect of cis-UCA on neutrophils, although one report demonstrated that it inhibits human neutrophil respiratory burst activity. However, the nature of this inhibition remains unknown. Because of the potential therapeutic use that a molecule such as cis-UCA may have in blocking excessive respiratory burst activity that may be deleterious to the host, the ability of cis-UCA to inhibit bovine neutrophil production of ROS was studied. Further, because neutrophil generation of ROS is necessary for optimal neutrophil bactericidal activity, a response which is critical for the host innate immune defense against infection, the effects of cis-UCA on bovine neutrophil phagocytosis and bacterial killing were assayed. cis-Urocanic acid dose-dependently inhibited the respiratory burst activity of bovine neutrophils as measured by luminol chemiluminescence. Subsequently, the effect of cis-UCA on the production of specific oxygen radicals was investigated using more selective assays. Using 2 distinct assays, we established that cis-UCA inhibited the generation of extracellular superoxide. In contrast, cis-UCA had no effect on the generation of intracellular levels of superoxide or other ROS. At concentrations that inhibited generation of extracellular superoxide, bovine neutrophil phagocytosis and bacterial activity remained intact. Together, these data suggest that cis-UCA inhibits the tissue-damaging generation of extracellular ROS while preserving neutrophil bactericidal activity.

Bone Marrow-derived Cells Require a Functional Glucose 6-Phosphate Transporter for Normal Myeloid Functions

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the ubiquitously expressed glucose 6-phosphate transporter (Glc-6-PT). Glc-6-PT activity has been shown to be critical in the liver and kidney where a deficiency disrupts glucose homeostasis. GSD-Ib patients also have defects in the neutrophil respiratory burst, chemotaxis, and calcium flux. They also manifest neutropenia, but whether Glc-6-PT deficiency in the bone marrow underlies myeloid dysfunctions in GSD-Ib remains controversial. To address this, we transferred bone marrow from Glc-6-PT-deficient (Glc-6-PT(-/-)) mice to wild-type mice to generate chimeric mice (BM-Glc-6-PT(-/-)). As a control, we also transferred bone marrow between wild-type mice (BM-Glc-6-PT(+/+)). While BM-Glc-6-PT(+/+) mice have normal myeloid functions, BM-Glc-6-PT(-/-) mice manifest myeloid abnormalities characteristic of Glc-6-PT(-/-) mice. Both have impairments in their neutrophil respiratory burst, chemotaxis response, and calcium flux activities and exhibit neutropenia. In the bone marrow of BM-Glc-6-PT(-/-) and Glc-6-PT(-/-) mice, the numbers of myeloid progenitor cells are increased, while in the serum there is an increase in granulocyte colony-stimulating factor and chemokine KC levels. Moreover, in an experimental model of peritoneal inflammation, local production of KC and the related chemokine macrophage inflammatory protein-2 is decreased in both BM-Glc-6-PT(-/-) and Glc-6-PT(-/-) mice along with depressed peritoneal neutrophil accumulation. The neutrophil recruitment defect was less severe in BM-Glc-6-PT(-/-) mice than in Glc-6-PT(-/-) mice. These findings demonstrate that Glc-6-PT expression in bone marrow and neutrophils is required for normal myeloid functions and that non-marrow Glc-6-PT activity also influences some myeloid functions.

Plasma concentrations of myeloperoxidase in endurance and 3‐day event horses after a competition

In man, exercise of any type has been shown to induce neutrophil degranulation and respiratory burst activity, as well as an increase in plasma myeloperoxidase (MPO), a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Until now, it is not known whether this is the same in horses. To study whether degranulation of blood neutrophils may be induced by exercise by measuring the total concentration of MPO and assess the possible influence of type of competition on this exercise-induced adjustment. Blood was sampled before, and 30 min after, the course, in 9 ponies performing the Phase D of a national 3-day event championship (CIC*), and in 7 endurance horses participating at the European endurance championship 2005. White blood cells and granulocytes, total plasma proteins, creatine phosphokinase (CK), and total MPO contents were determined from blood samples. In addition, blood was taken from all ponies and 4 of the endurance horses 5 min after completion of the course to give some idea of the intensity of exercise. The mean blood lactate was 15.8 +/- 5.8 mmol/l after the CIC* and 2.7 +/- 0.2 mmol/l after the 160 km course. Performing both competitions induced a significant increase in CK and MPO. After the endurance course, the number of granulocytes significantly increased. Whilst there was no significant correlation between the measurements in CIC* ponies, MPO was significantly correlated with granulocyte count (r2 = 0.776) and CK (r2 = 0.586) in endurance horses. Intense exercise induces an activation of blood granulocytes, with degranulation of neutrophils and release of MPO. The plasmatic MPO concentration after endurance was higher than the values reported in some inflammatory pathological conditions. This phenomenon may partly contribute to the occurrence of an exercise-induced oxidative stress and to the alteration of muscular membrane permeability. Further studies should be conducted to assess the possible relationship between MPO concentration and markers of oxidative stress in performance horses.

Lymphocyte subpopulations and neutrophil function in calves during the first 6 months of life

Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and thereafter every month until the age of 6 months. The percentages and absolute values of CD4+, CD8+ γδ TCR+ and WC1+ T cells, CD21+ B cells and NKp46+ NK cells were determined by flow cytometry, and the expression of the interleukin-2 receptor α chain (CD25) was measured to assess the level of activation of the lymphocyte subpopulations. Neutrophil phagocytosis, respiratory burst and bactericidal activity were measured in five different neutrophil function assays. Most of the parameters examined reached a stable level during the first 6 months of life. The proportions of CD4+ and CD8+ lymphocytes remained relatively stable during the study period, while there was a moderate decrease in the relative percentage of γδ T cells from birth to approximately 5 months of age. However, the absolute numbers of γδ T cells per millilitre of blood remained stable throughout the study period and did not display significant variation with age. The percentage of cells expressing the B-cell maturation marker CD21 increased significantly over the first 5 months of life. The proportion of NK cells showed substantial variation during the study. Marked differences in the relative proportions of the lymphocyte subpopulations were noted between the individual calves, and the individual ranking of the animals was largely maintained over time. CD25 expression was detected on a mean of 6.6% of the CD4+ cells, while a lower percentage of the other lymphocyte subpopulations expressed this receptor. Phagocytic activity was demonstrated in approximately 90% of the neutrophils, and this proportion remained stable during the entire study period, while respiratory burst activity showed a moderate decrease during the first 2 months of life. The present study shows that the T-cell subpopulations are present in peripheral blood of calves at levels comparable with adult values, while the B-cell population increases significantly with age. The decrease in the relative percentage of γδ T cells appears to be attributable to an increase in the absolute numbers of CD4+ and CD21+ cells, rather than a change in absolute γδ T-cell numbers. Furthermore, the results indicate that the neutrophilic granulocytes are functional and able to mount an effective response in young calves from the first week of life.