Neutrophil Respiratory Burst Activity Research Articles

Comparison of neutrophil respiratory oxidative burst activity between flow cytometry using dihydrorhodamine (DHR) 123 and conventional nitroblue tetrazolium test (NBT)

Background: Neutrophil plays a defense role against bacteria and fungi specifically by the neutrophil phagoburst activity. The nitroblue tetrazolium (NBT) test is the gold standard method to measure this but the flow cytometry assays also have been developed. The aim of the study was to evaluate the performance of flow cytometry using Dihydrorhodamine (DHR) 123 and to compare with the conventional NBT test. Methods: This study was carried out to determine the phagoburst activity at Universiti Kebangsaan Malaysia Medical Centre on 26 normal healthy donors and 5 chronic myeloid leukemia (CML) patients using both methods. Results: In flow cytometry, higher fluorescence intensity was observed in Phorbol myristate acetate (PMA) stimulated neutrophils. Comparing the mean fluorescent intensity (MFI) between CML and healthy donor showed CML patients have significant lower MFI (312.15 vs 738.22; p<0.05) indicating poorer respiratory burst in CML patients. The PMA stimulated positive cells were higher in flow cytometry than the NBT (98.06% vs 87.5%). Although there was poor correlation between two methods, a good agreement between flow cytometry assay and NBT test results was observed in terms of positive and negative results in simulated and unstimulated cells. Moreover, in flow cytometry, >95% of stimulated cells suggest the higher sensitivity. All samples with negative NBT showed negative DHR, reflecting high degree of agreement between these two methods and eliminating possible false negative result in flow cytometry. Conclusion: This study showed that flow cytometry assay has superior technical advantages compared to NBT test as it is easy to perform, quick, more sensitive and requires only a small amount of blood sample. Bangladesh Journal of Medical Science Vol. 21 No. 03 July’22 Page: 626-633

Neutrophil respiratory burst activity is not exaggerated in cystic fibrosis

BackgroundExaggerated neutrophil-dominated inflammation underlies progressive cystic fibrosis (CF) lung disease. Older studies reported a defective respiratory burst in CF, but more recent studies suggest neutrophil function is normal. MethodsWe measured the amount and rate of reactive oxygen species (ROS) during PMA-stimulated respiratory burst activity in children [70 CF, 13 disease controls, 19 health controls] and adults [31 CF, 14 health controls] in neutrophils harvested from peripheral blood. Blood was collected from participants with CF when clinically stable (60 children, 9 adults) and on hospital admission (38 children, 24 adults) and discharge (18 children, 21 adults) for acute pulmonary exacerbations. ResultsWhen clinically stable, children with CF had lower ROS production [median 318,633, 25% 136,810 - 75% 569,523 RLU] than disease controls [median 599,459, 25% 425,566 - 75% 730,527 RLU] and healthy controls [median 534,073, 25% 334,057 - 75% 738,593 RLU] (p = 0.008). The rate of ROS production was also lower (p = 0.029). In neither children nor adults with CF did ROS production increase on hospital admission for acute pulmonary exacerbation, nor fall prior to discharge. There were no associations between ROS production and high-sensitivity C-reactive protein (indicating systemic inflammation) in either children or adults with CF. ConclusionsOur data do not support a role for exaggerated respiratory burst activity contributing to the exaggerated neutrophil-dominated inflammation seen with CF lung disease.

Stimulation Of Neutrophil Oxidative Burst By Calcium Phosphate Particles With Adsorbed Mucin

Objective — Mucin can promote formation of gallstones via precipitation with calcium phosphate. The proinflammatory effect of mucin-coated particles is still unclear, and our aim was to study the role of mucin sorption in activation of neutrophil respiratory burst. Material and Methods — Polydisperse calcium phosphate nanowires (CP) were prepared from hot gelatin solution and according to scanning electron microscopy (SEM) had the length 1-10 μm and thickness 50-450 nm. CP were incubated in mucin or human serum albumin (HSA) giving CP-Muc and CP-HSA. Their hemolytic activity towards human erythrocytes was assayed, and neutrophil lucigenin- and luminol- chemiluminescence (Luc-CL and Lum-CL) response to CP, CP-HSA and CP-Muc was measured. Cytokine RNA was detected in neutrophils by means of reverse transcription with subsequent real-time PCR. Cytokines (IL-1β, IL-6, IL-8, IL-10) were assessed in cell medium by ELISA. Results and Conclusion — Hemolytic activity of CP was 3.0±0.5%, mucin sorption (0.019 mg/mg) reduced it to 0.24±0.04% (p<0.05) as well as HSA. CP and CP-HSA stimulated neutrophil Lum-CL and Luc-Cl by 2-3 times vs. spontaneous values while for CP-Muc the effect was 10-fold and higher. No increased cytokine gene expression or cytokine secretion was detected after 1h incubation of neutrophils with samples. Obviously, sorption of mucin but not that of HSA stimulated generation of reactive oxygen and halogen species with no increase in cytokine production. Thus, the mucin-coated CP has the potential to contribute to gallstone-associated cholecystitis via oxidative damage of mucosa and epithelium.

Silver nanoparticles as an antibacterial agent in Oreochromis niloticus and Sparus auratus fish

A trial was carried out to assess the potential use of chemically synthesized silver nanoparticles (AgNPs) with two concentrations (5 and 10 µg/L) as an antibacterial agent in Oreochromis niloticus and Sparus auratus against Streptococcus agalactiae and Vibrio alginolyticus respectively. All AgNPs were with a mean of 26 nm in diameter. The particles were negatively charged (−30.7 ± 0.45 mV). In different ways, the in vitro AgNP antibacterial activity was investigated against S. agalactiae and V. alginolyticus in different ways showing clear inhibition zones of the two AgNP concentrations dose-dependent manner. In vitro measurement of extracellular proteins of pathogenic S. agalactiae and V. algenolyticus against AgNPs and the ultrastructural interaction between AgNPs and these bacterial pathogens using transmission electron microscopy (TEM) revealed AgNPs have good antibacterial properties. O. niloticus and S. auratus were exposed to AgNPs for 28 days. Some groups were challenged with S. agalactiae and V. alginolyticus respectively. Haematological, biochemical and immunological parameters and phagocytic activity, phagocytic index, respiratory burst activity of neutrophils and serum lysozyme activity were estimated in addition to the concentration of minerals. Histopathological examinations of all fish groups were explored. From the investigation above, we concluded that AgNPs have significant deleterious effects on O. niloticus exposed to 5 and 10 µg/L of AgNPs, appearing a significant decrease in leukocytes, red blood corpuscles (RBCs) count and haemoglobin concentration the fish on day 28 of the trial, with more deleterious effect with challenging fish by S. agalactiae and V. algenolyticus. In addition, AgNPs caused a substantial increase in liver enzymes with a reduction in total protein, albumin and globulin in the fish and considerably decreased the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Additionally, there was a significant decrease in sodium (Na) and potassium (K) content, nitroblue tetrazolium (NBT) and lysozyme activity (LYZ) in all groups of O. niloticus and S. auratus. In addition, lower survival rates disturb histopathological findings; meanwhile, S. auratus exposed to 5 and 10 µg/L of AgNPs showed antibacterial potency and immunostimulatory effects, which appeared in their higher survival rates and induced immune parameters and histopathological findings. In low concentration (5 µg/ml of AgNPs), AgNPs were less cytotoxic, especially in S. auratus, representing an alternative antimicrobial compound against V. algenolyticus. Further studies are still to be explored to clarify the protective role of the AgNPs and the recovery period needed for AgNPs to be entirely safe for usage as an antibacterial for each fish species.

Measuring the Asian seabass (Lates calcarifer) neutrophil respiratory burst activity by the dihydrorhodamine-123 reduction flow cytometry assay in whole blood

The neutrophil oxidative respiratory burst response is a key component of the innate immune system responsible for killing microbial pathogens. Since fish rely on the innate immune system for health, monitoring the respiratory burst activity may be an effective means of gauging fish health status. Here we report that the respiratory burst of Asian seabass neutrophils can be measured in whole blood by the dihydrorhodamine (DHR)-123 reduction assay and flow cytometry. Neutrophils responded to phorbol myristate acetate (PMA) in a concentration dependent manner with significant respiratory burst activity at 100–1000 nM. Other known neutrophil agonists, such as bacterial lipopolysaccharide, tumor necrosis factor, the tripeptide f-met-leu-phe and zymosan, did not induce a significant DHR reduction. Thus, the findings enable us to propose that the DHR-123 flow cytometry whole blood assay, incorporating PMA as a stimulator, would not only facilitate future studies into fish blood neutrophil research but provides a simple, rapid and reliable assay for gauging fish natural immunity status and health.

Functional features of neutrophils in subclinical hypothyroidism compared to euthyroid status

Neutrophil or polymorphonuclear leukocytes are critical to host defense. They act by migrating (chemotaxis) to site of inflammation or infection, phagocytoze the microorganisms to form phagosome. Reactive Oxygen species and hydrolytic enzymes are released (respiratory burst activity) into this phagosome to kill ingested microorganisms (microbicidal activity). Thyroid hormones are essential for proper differentiation, growth and metabolism of an organism. Increasing evidence indicates that thyroid hormones also play an active role in immunity. Subclinical hypothyroidism is associated with normal thyroid hormone levels with increased thyroid stimulating hormone level. While studies indicate elevated TSH to have some effects on immune functions, there is very little evidence regarding its effect on functional features of neutrophils. In this cross-sectional comparative study, we evaluated the phagocytic, chemotactic, candidacidal, and respiratory burst activity of neutrophils in subclinical hypothyroid and euthyroid subjects. Phagocytosis and metabolic function of the neutrophils was assessed using Nitro-blue tetrazolium test. Chemotaxis, directional movement of the PMNs was measured using the N-Formyl methionyl-leucyl-phenylalanine as chemoattractant. Phagocytic activity was evaluated by exposing leukocytes to candida suspension and counting the number of candida ingested and killed. Microbicidal activity of neutrophils was assessed by the ability of leukocytes to kill Candida albicans in both subclinical hypothyroid and euthyroid subjects. Our study demonstrated that chemotactic and mean phagocytic activity was significantly less in those with subclinical hypothyroidism as compared to euthyroid control group (p value = 0.005 and 0.045 respectively). Respiratory burst activities of both unstimulated & stimulated neutrophils were relatively reduced in subclinical hypothyroidism (p value of 0.08 and 0.07). Candidicidal activity was similar in both groups. We conclude that subclinical hypothyroid individuals having excess of thyroid stimulating hormones are associated altered functional characteristics of neutrophils.

Neutrophil phagocytosis and respiratory burst activity of dairy cows during the transition period and early lactation.

Hormonal and metabolic changes, as well as energy imbalance, can affect health, production and reproductive performance of dairy cows. In the present study, we evaluated phagocytosis and respiratory burst neutrophil activity during the transition period and early lactation and compared it with biochemical and hematological parameters in dairy cows. Simmental cows (n = 21) were enrolled in the study. Whole blood samples were collected weekly from 3 weeks pre- calving until 6 weeks post calving. Basic metabolic and blood parameters were assessed by routine laboratory analyses, while neutrophil functions were analyzed by commercial test kits. Optimal neutrophil response was observed pre and post calving. The highest value was recorded in the 6th week after calving (89.54 ± 7.61%) and being significantly higher (p < 0.01) as compared to values recorded at two and one week before and one week after calving. The percentage of activated neutrophils was high during the entire study period: from 70.80 ± 5.22% at the beginning of the study to 89.54 ± 7.61% at the end of the study. During the study period, production of Reactive Oxidative Species by neutrophils was positively correlated with β-hydroxybutyrat and non-esterified fatty acids values (0.454** and 0.423**, respectively) and calcium levels (0.164* and 0.212**, respectively). The most prominent changes in all parameters had no influence on phagocytic and respiratory burst activity of neutrophils. Neutrophil function is preserved at the optimal level during the transition period and early lactation in Simmental cows.

Loss of Humoral and Cellular Immunity to Invasive Nontyphoidal Salmonella during Current or Convalescent Plasmodium falciparum Infection in Malawian Children.

ABSTRACTInvasive nontyphoidal Salmonella (iNTS) infections are commonly associated with Plasmodium falciparum infections, but the immunologic basis for this linkage is poorly understood. We hypothesized that P. falciparum infection compromises the humoral and cellular immunity of the host to NTS, which increases the susceptibility of the host to iNTS infection. We prospectively recruited children aged between 6 and 60 months at a Community Health Centre in Blantyre, Malawi, and allocated them to the following groups; febrile with uncomplicated malaria, febrile malaria negative, and nonfebrile malaria negative. Levels of Salmonella enterica serovar Typhimurium-specific serum bactericidal activity (SBA) and whole-blood bactericidal activity (WBBA), complement C3 deposition, and neutrophil respiratory burst activity (NRBA) were measured. Levels of SBA with respect to S. Typhimurium were reduced in febrile P. falciparum-infected children (median, −0.20 log10 [interquartile range {IQR}, −1.85, 0.32]) compared to nonfebrile malaria-negative children (median, −1.42 log10 [IQR, −2.0, −0.47], P = 0.052). In relation to SBA, C3 deposition on S. Typhimurium was significantly reduced in febrile P. falciparum-infected children (median, 7.5% [IQR, 4.1, 15.0]) compared to nonfebrile malaria-negative children (median, 29% [IQR, 11.8, 48.0], P = 0.048). WBBA with respect to S. Typhimurium was significantly reduced in febrile P. falciparum-infected children (median, 0.25 log10 [IQR, −0.73, 1.13], P = 0.0001) compared to nonfebrile malaria-negative children (median, −1.0 log10 [IQR, −1.68, −0.16]). In relation to WBBA, S. Typhimurium-specific NRBA was reduced in febrile P. falciparum-infected children (median, 8.8% [IQR, 3.7, 20], P = 0.0001) compared to nonfebrile malaria-negative children (median, 40.5% [IQR, 33, 65.8]). P. falciparum infection impairs humoral and cellular immunity to S. Typhimurium in children during malaria episodes, which may explain the increased risk of iNTS observed in children from settings of malaria endemicity. The mechanisms underlying humoral immunity impairment are incompletely understood and should be explored further.

The respiratory explosion activity of neutrophils in blood from patients with Graves' disease manifestation

&#x0D; Objective. To study respiratory burst activity in neutrophilic granulocytes in the onset of Graves’ disease (GD).&#x0D; Material and methods. Twenty-six females aged 18—55 years (mean age, 40.7±13.2 years), with the diagnosis of Graves’ disease verified for the first time, were enrolled in this study. Spontaneous and zymosan-induced chemiluminescence (CL) was assessed using a CL3606 36-channel analyzer for 90 min. The following parameters of the chemiluminescence curve were determined: time to attain the maximum (Tmax), the maximum intensity (Imax), and the area under the curve (S).&#x0D; Results. Patients with GD and lucigenin-dependent CL of neutrophils exhibited decreased zymosan-induced CL and the increased Imax of luminol-dependent spontaneous and zymosan-induced CL of neutrophils. Association between the anti-TPO level in blood serum with Tmax (r=–0,70; р=0.036) and Imax of luminol-dependent induced CL (r=–0.72; р=0.030) was found. The maximum level of synthesis of secondary reactive oxygen species (ROS) was found to be elevated in patients with GP both at relative rest and upon antigen-induced respiratory burst.&#x0D; Conclusion. In patients with GD, respiratory burst activity in neutrophils increases due to synthesis of secondary reactive oxygen species. Upon the onset of GD, already at the stage of subclinical thyrotoxicosis, the association between the indices of respiratory burst in neutrophils and thyroid hormones is lost but there emerges a relationship between the CL response and anti-TPO concentration.

Preliminary study on overproduction of reactive oxygen species by neutrophils in diabetes mellitus.

To assess the amount and pattern of reactive oxygen species (ROS) production in diabetic patient-derived neutrophils. Blood samples from type 2 diabetes mellitus (DM) patients and volunteers (controls) were subjected to neutrophil isolation and the assessment of neutrophil oxidative burst using chemiluminescence assay. Neutrophils were activated by using phorbol myristate acetate (PMA) and neutrophils without activation were kept as a negative control. The chemiluminescence readings were obtained by transferring cell suspension into a 1.5 mL Eppendorf tube, with PMA and luminol. Reaction mixtures were gently vortexed and placed inside luminometer for a duration of 5 min. Our results showed that in the resting condition, the secretion of ROS in normal non-diabetic individuals was relatively low compared to diabetic patients. However, the time scale observation revealed that the secreted ROS declined accordingly with time in non-diabetic individuals, yet such a reduction was not detected in diabetic patients where at all the time points, the secretion of ROS was maintained at similar magnitudes. This preliminary study demonstrated that ROS production was significantly higher in patients with DM compared to non-diabetic subjects in both resting and activated conditions. The respiratory burst activity of neutrophils could be affected by DM and the elevation of ROS production might be an aggravating factor in diabetic-related complications.

Avenanthramide supplementation attenuates exercise-induced inflammation in postmenopausal women

During aging, chronic systemic inflammation increases in prevalence and antioxidant balance shifts in favor of oxidant generation. Avenanthramide (AVA) is a group of oat phenolics that have shown anti-inflammatory and antioxidant capability. The present study investigated whether dietary supplementation of avenanthramides (AVA) in oats would increase antioxidant protection and reduce inflammation after a bout of downhill walking (DW) in postmenopausal women. Women at age of 50–80 years (N = 16) were randomly divided into two groups in a double-blinded fashion, receiving two cookies made of oat flour providing 9.2 mg AVA or 0.4 mg AVA (control, C) each day for 8 weeks. Before and after the dietary regimen, each group of subjects walked downhill on a treadmill (−9% grade) for 4 bouts of 15 minutes at a speed of 4.0 km/h with 5 minutes rest between sessions. Blood samples were collected at rest, 24 h post-DW, and 48 h post-DW pre- and post-supplementation. Both DW sessions increased plasma creatine kinase activity (P < 0.05). Before supplementation, in vitro neutrophil respiratory burst (NRB) activity was increased at 24 h post-DW (P < 0.05) and C-reactive protein (CRP) was increased 48 h post-DW (P < 0.05). AVA supplementation decreased DW-induced NRB at 24 h (P < 0.05) and CRP level 48 h (P < 0.05). Plasma interleukin (IL)-1β concentration and mononuclear cell nuclear factor (NF) κB binding were suppressed at rest and during post-DW period in AVA but not C group (P < 0.05). Plasma total antioxidant capacity (P < 0.05) and erythrocyte superoxide dismutase activity were increased in AVA vs. C (P < 0.05), whereas glutathione redox status was elevated 48 h post-DW but not affected by AVA. Thus, chronic AVA supplementation decreased systemic and DW-induced inflammation and increased blood-borne antioxidant defense in postmenopausal women.

Oral pretreatment with recombinant human lactoferrin limits trauma-hemorrhagic shock–induced gut injury and the biological activity of mesenteric lymph

BackgroundLactoferrin (LF) is a pleiotropic glycoprotein that is found in bodily secretions and is postulated to enhance the gastrointestinal barrier and promote mucosal immunity. Thus, the ability of talactoferrin, an oral recombinant form of human LF, to limit gut injury and the production of biologically active gut-derived products was tested using a rat model of trauma–hemorrhagic shock (T/HS). MethodsMale rats were orally dosed with vehicle or talactoferrin (1000 mg/kg, every day) for 5 d before being subjected to T/HS or trauma–sham shock (T/SS). Subsequently, rats were subjected to a laparotomy (trauma) and hemorrhagic shock (mean arterial pressure, 30–35 mm Hg × 90 min) or to T/SS, followed by resuscitation with their shed blood. Before inducing shock, the mesenteric lymphatic duct was catheterized for collection of mesenteric lymph. Four hours after the end of the shock or sham-shock period, rats were sacrificed, a segment of the distal ileum was collected for morphologic analysis, and lymph samples were processed and frozen. Subsequently, lymph samples were tested in several pharmacodynamic assays, including endothelial cell permeability, neutrophil respiratory burst activity, and red blood cell (RBC) deformability. Total white blood cell counts in lymph samples were also quantified. ResultsPretreatment with talactoferrin reduced the incidence of T/HS-induced morphologic injury of ileum to T/SS levels. Post-T/HS lymph from vehicle-treated rats increased endothelial monolayer permeability and neutrophil priming for an augmented respiratory burst, and induced loss of RBC deformability, compared with T/SS groups. Talactoferrin pretreatment significantly reduced the biological activity of T/HS lymph on respiratory burst activity and RBC deformability, but had no effect on the lymph cell count or endothelial cell permeability. ConclusionsThese results provide a proof of principle that prophylactic dosing of oral talactoferrin can potentially protect the gut in a T/HS model and limit the production of biologically active factors in rat gastrointestinal tissue subjected to ischemia-reperfusion–type injuries.

The Effects of Plantago major on the Activation of the Neutrophil Respiratory Burst

Plantago major is a common plant that grows worldwide in temperate zones and is found in fields, lawns, and on the roadsides. Its leaves and seeds have been used in almost all parts of the world for centuries as a wound healer, analgesic, antioxidant, and antibiotic, as well as an immune system modulator, antiviral, antifungal, and anti-inflammatory agent. Baicalein and aucubin are the two most biologically active components of P. major, and both have been shown to have antioxidant, anti-inflammatory, and anticancer properties. Neutrophils have a pivotal role in wound healing and inflammation. Their principal mechanism of host defense is the killing of pathogens via the production of reactive oxygen species (ROS). The aim of the present study was to determine the in vitro effects of P. major extract, baicalein, and aucubin on human neutrophil respiratory burst activity. The cytotoxicity of the agents was assessed by lactate dehydrogenase (LDH) assays. A standard luminol-dependent chemiluminescence (CL) assay was utilized to monitor the respiratory burst of the neutrophils after exposure to P. major extract and its two active ingredients, baicalein and aucubin. Three replicates per group were included in each of the three runs of the experiments and analysis of variance (ANOVA) was used for statistical analysis. P. major and baicalein were not toxic to the cells at any of the concentrations examined. Aucubin was toxic to the cells only at the highest concentration tested (P = 0.0081). However, genistein was toxic to the cells at all of the concentrations examined except for the lowest concentration of 16.9 μg/ml (P = 0.985). P. major (−0.10 ± 0.11), aucubin (0.06 ± 0.16), baicalein (−0.10 ± 0.11), and genistein (−0.18 ± 0.07) all significantly (P < 0.0001) inhibited ROS production from the neutrophils. P. major extract inhibited neutrophil ROS production, as did aucubin and baicalein. Therefore, these components should be investigated further with relation to the regulation of destructive ROS production in conditions such as periodontal disease.

Inhibition of pre-B cell colony-enhancing factor (PBEF/NAMPT/visfatin) decreases the ability of human neutrophils to generate reactive oxidants but does not impair bacterial killing

NAMPT, also known as PBEF and visfatin, can act extracellularly as a cytokine-like molecule or intracellularly as a NAMPT, regulating NAD biosynthesis in the NAD salvage pathway. Inhibitors of NAMPT have anti-inflammatory and anticancer activity and are finding use as therapeutic agents. In view of the importance of NAD metabolism in neutrophil function, we determined the effects of NAMPT inhibition on a variety of neutrophil functions associated with their role in host protection against infections. Incubation of human neutrophils with the NAMPT inhibitor APO866 decreased neutrophil NAD(P)/H levels in a dose- and time-dependent manner but without a concomitant change in cell viability. NAMPT inhibition did not affect the expression of a number of cell-surface receptors involved in adhesion and opsono-phagocytosis, but the respiratory burst was decreased significantly. Whereas opsono-phagocytosis of Staphylococcus aureus was unaffected by NAMPT inhibition, intraphagosomal oxidant production was decreased. However, the killing efficiency of neutrophils was unaffected. These data indicate that therapeutic NAMPT inhibition is unlikely to have deleterious effects on host protection against infections, in spite of this ability to down-regulate neutrophil respiratory burst activity significantly.

Staphylococcus aureus Manganese Transport Protein C Is a Highly Conserved Cell Surface Protein That Elicits Protective Immunity Against S. aureus and Staphylococcus epidermidis

Staphylococcus aureus and other staphylococci cause severe human disease, and there are currently no vaccines available. We evaluated whether manganese transport protein C (MntC), which is conserved across the staphylococcal species group, could confer protection against S. aureus and Staphylococcus epidermidis. In vivo analysis of S. aureus MntC expression revealed that expression occurs very early during the infectious cycle. Active immunization with MntC was effective at reducing the bacterial load associated with S. aureus and S. epidermidis infection in an acute murine bacteremia model. Anti-MntC monoclonal antibodies have been identified that can bind S. aureus and S. epidermidis cells and are protective in an infant rat passive protection model and induce neutrophil respiratory burst activity. This is the first description of a protein that has the potential to provide protection across the staphylococcal species group.

Granule Exocytosis Contributes to Priming and Activation of the Human Neutrophil Respiratory Burst

The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT-SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT-SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT-SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT-SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT-SNAP-23 inhibited the increase in plasma membrane expression of gp91(phox) in TNF-α-primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase.

Assessment of Inflow of Endotoxin and Its Fragments in Patients on Maintenance Hemodialysis

Background: We estimated the flow of endotoxins (ET) from dialysates into the blood of patients on hemodialysis (HD) using limulus amebocyte lysate (LAL) assays and endotoxin activity (EA) determined by neutrophil respiratory burst activity. Methods: A cross-sectional study compared groups given ultrapure bicarbonate (n = 15; group A), acetate-free bicarbonate dialysates (n = 20; group B) and conventional bicarbonate dialysate (n = 23; group C). A prospective study of group C examined the effect of changing the purity of the dialysate. Biomarkers of inflammation and oxidative stress were measured and ET in blood was assessed by LAL assays and EA. Results: Serum ET levels did not differ among the groups, whereas EA and the biomarkers were significantly increased in group C compared with those in groups A and B. HD using conventional dialysate was independently associated with an increase in EA. Purifying the dialysate significantly decreased EA in group C. Conclusion: Measuring EA is useful to assess the influence of dialysate contamination in HD patients.

Recombinant Factor XIII Mitigates Hemorrhagic Shock-Induced Organ Dysfunction

Plasma factor XIII (FXIII) is responsible for stabilization of fibrin clot at the final stage of blood coagulation. Since FXIII has also been shown to modulate inflammation, endothelial permeability, as well as diminish multiple organ dysfunction (MOD) after gut ischemia-reperfusion injury, we hypothesized that FXIII would reduce MOD caused by trauma-hemorrhagic shock (THS). Rats were subjected to a 90 min THS or trauma sham shock (TSS) and treated with either recombinant human FXIII A(2) subunit (rFXIII) or placebo immediately after resuscitation with shed blood or at the end of the TSS period. Lung permeability, lung and gut myeloperoxidase (MPO) activity, gut histology, neutrophil respiratory burst, microvascular blood flow in the liver and muscles, and cytokine levels were measured 3 h after the THS or TSS. FXIII levels were measured before THS or TSS and after the 3-h post-shock period. THS-induced lung permeability as well as lung and gut MPO activity was significantly lower in rFXIII-treated than in placebo-treated animals. Similarly, rFXIII-treated rats had lower neutrophil respiratory burst activity and less ileal mucosal injury. rFXIII-treated rats also had a higher liver microvascular blood flow compared with the placebo group. Cytokine response was more favorable in rFXIII-treated animals. Trauma-hemorrhagic shock did not cause a drop in FXIII activity during the study period. Administration of rFXIII diminishes THS-induced MOD in rats, presumably by preservation of the gut barrier function, limitation of polymorphonuclear leukocyte (PMN) activation, and modulation of the cytokine response.

Neutrophil functions in patients with inherited bone marrow failure syndromes

The inherited bone marrow failure syndromes (IBMFS) include Fanconi anemia, dyskeratosis congenita, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome (SDS). Previous studies reported decreased neutrophil chemotaxis in patients with SDS; there are no reports of neutrophil function in other IBMFS. In this study we examined neutrophil respiratory burst function in IBMFS patients. Samples from 43 IBMFS patients and 61 healthy family members were collected, shipped, and analyzed within 24 hr. We also studied samples from 12 healthy control persons immediately after collection. Neutrophils were stimulated with phorbol 12-myristate acetate (PMA) and N-formyl-methyonyl-leucyl-phenylalanine (fMLP), and respiratory burst analyzed by reduction of dihydro-rhodamine and cytochrome c. There was no significant difference in the degree of fMLP or PMA-driven respiratory burst activity between each of the IBMFS subgroups and their respective family members. There was also no difference in respiratory burst activity between any IBMFS, pooled group of all healthy family members and healthy controls. Neutrophil respiratory burst activity from IBMFS patients does not differ from that of healthy family members and controls.

Hematopoietic Pyk2 regulates migration of differentiated HL-60 cells

BackgroundPyk2 is a non-receptor cytoplasmic tyrosine kinase that belongs to the focal adhesion kinase family and has been implicated in neutrophil spreading and respiratory burst activity caused by TNF-α. However, the role of Pyk2 in neutrophil migration is incompletely defined. In this study, we tested the hypothesis that Pyk2 regulates the migration of neutrophil-like differentiated HL-60 cells subsequent to β2-integrin mediated cell adhesion.MethodsHL-60 cells were induced to differentiate into neutrophil-like cells (dHL60) by incubation in medium containing 1.25% DMSO for up to 4 days. Pyk2 expression and tyrosine phosphorylation was measured by Western blot analysis. Adhesion of dHL60 cells to plated fibrinogen was measured by residual myeloperoxidase activity. dHL60 cell migration was evaluated using a 96-well chemoTx chamber.ResultsWestern blot analysis demonstrated that hematopoietic Pyk2 was predominantly expressed after HL60 cell differentiation. Pyk2 was tyrosine phosphorylated upon adhesion of dHL60 cells to plated fibrinogen in the presence of fMLP. By contrast, tyrosine phosphorylation of Pyk2 was insignificant in dHL60 cells treated in suspension with fMLP. Antibodies against CD18 blocked both phosphorylation of Pyk2 and adhesion of dHL60 cells to fibrinogen, demonstrating that phosphorylation of Pyk2 was β2-integrin dependent. TAT-Pyk2-CT, a dominant negative fusion protein in which the TAT protein transduction domain was fused to the c-terminal Pyk2, attenuated fMLP-stimulated spreading, migration and phosphorylation of endogenous Pyk2 without blocking adhesion of dHL-60 cells to fibrinogen. Similarly, silencing of Pyk2 expression by siRNA in dHL60 cells also attenuated dHL60 cell migration caused by fMLP. Phospho-Pyk2 was evenly distributed around cell membrane circumferentially in unstimulated dHL-60 cells adherent to plated fibrinogen. In dHL60 cells treated with fMLP to cause cell spreading and polarization, Pyk2 was concentrated at the leading edge of pseudopods or at the trailing edge of uropods during migration of neutrophilic dHL-60 cells.ConclusionsWe conclude that Pyk2 is activated by β2-integrin adhesion. The activated concentration of Pyk2 and colocalization with F-actin in pseudopodia suggests that Pyk2 may regulate cell spreading and migration in dHL60 cells.