Whole Blood Phagocytes Research Articles

Oxidant trade-offs in immunity: an experimental test in a lizard.

Immune system functioning and maintenance entails costs which may limit investment into other processes such as reproduction. Yet, the proximate mechanisms and ‘currencies’ mediating the costs of immune responses remain elusive. In vertebrates, up-regulation of the innate immune system is associated with rapid phagocytic production of pro-oxidant molecules (so-called ‘oxidative burst’ responses). Oxidative burst responses are intended to eliminate pathogens but may also constitute an immunopathological risk as they may induce oxidative damage to self cells. To minimize the risk of infection and, at the same time, damage to self, oxidative burst activity must be carefully balanced. The current levels of pro- and antioxidants (i.e. the individual oxidative state) is likely to be a critical factor affecting this balance, but this has not yet been evaluated. Here, we perform an experiment on wild-caught painted dragon lizards (Ctenophorus pictus) to examine how the strength of immune-stimulated oxidative burst responses of phagocytes in whole blood relates to individual oxidative status under control conditions and during an in vivo immune challenge with Escherichia coli lipopolysaccharide (LPS). Under control conditions, oxidative burst responses were not predicted by the oxidative status of the lizards. LPS-injected individuals showed a strong increase in pro-oxidant levels and a strong decrease in antioxidant levels compared to control individuals demonstrating a shift in the pro-/antioxidant balance. Oxidative burst responses in LPS-injected lizards were positively related to post-challenge extracellular pro-oxidants (reflecting the level of cell activation) and negatively related to pre-challenge levels of mitochondrial superoxide (suggesting an immunoregulatory effect of this pro-oxidant). LPS-challenged males had higher oxidative burst responses than females, and in females oxidative burst responses seemed to depend more strongly on antioxidant status than in males. Our results confirm the idea that oxidative state may constrain the activity of the innate immune system. These constraints may have important consequences for the way selection acts on pro-oxidant generating processes.

Role of histamine receptors in the effects of histamine on the production of reactive oxygen species by whole blood phagocytes

AimsThe diverse physiological functions of histamine are mediated through distinct histamine receptors. In this study we investigated the role of H2R and H4R in the effects of histamine on the production of reactive oxygen species by phagocytes in whole blood. Main methodsChanges in reactive oxygen species (ROS) production by whole blood phagocytes after treatment with histamine, H4R agonists (4-methylhistamine, VUF8430), H2R agonist (dimaprit) and their combinations with H4R antagonist (JNJ10191584) and H2R antagonist (ranitidine) were determined using the chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied compounds on the CL response, the antioxidant properties of all compounds were measured using several methods (TRAP, ORAC, and luminol–HRP–H2O2 based CL). Key findingsHistamine, 4-methylhistamine, VUF8430 and dimaprit inhibited the spontaneous and OZP-activated whole blood CL in a dose-dependent manner. On the other hand, only VUF8430 was able to inhibit PMA-activated whole blood CL. Ranitidine, but not JNJ10191584, completely reduced the effects of histamine, 4-methylhistamine and dimaprit. The direct scavenging ability of tested compounds was negligible. SignificanceOur results demonstrate that the inhibitory effects of histamine on ROS production in whole blood phagocytes were caused by H2R. Our results also suggest that H4R agonists in concentrations higher than 10−6M may also influence ROS production via binding to H2R.

Classification of infectious diseases based on chemiluminescent signatures of phagocytes in whole blood

Despite medical advances, infectious diseases are still a major cause of mortality and morbidity, disability and socio-economic upheaval worldwide. Early diagnosis, appropriate choice and immediate initiation of antibiotic therapy can greatly affect the outcome of any kind of infection. Phagocytes play a central role in the innate immune response of the organism to infection. They comprise the first-line of defense against infectious intruders in our body, being able to produce large quantities of reactive oxygen species, which can be detected by means of chemiluminescence (CL). The data preparation approach implemented in this work corresponds to a dynamic assessment of phagocytic respiratory burst localization in a luminol-enhanced whole blood CL system. We have previously applied this approach to the problem of identifying various intra-abdominal pathological processes afflicting peritoneal dialysis patients in the Nephrology department and demonstrated 84.6% predictive accuracy with the C4.5 decision-tree algorithm. In this study, we apply the CL-based approach to a larger sample of patients from two departments (Nephrology and Internal Medicine) with the aim of finding the most effective and interpretable feature sets and classification models for a fast and accurate identification of several infectious diseases. Whole blood samples were collected from 78 patients (comprising 115 instances) with respiratory infections, infections associated with renal replacement therapy and patients without infections. CL kinetic parameters were calculated for each case, which was assigned into a specific clinical group according to the available clinical diagnostics. Feature selection wrapper and filter methods were applied to remove the irrelevant and redundant features and to improve the predictive performance of disease classification algorithms. Three data mining algorithms, C4.5 (J48) decision tree, support vector machines and naive Bayes classifier were applied for inducing disease classification models and their performance in classifying three clinical groups was evaluated by 10 runs of a stratified 10-fold cross-validation. The results demonstrate that the predictive power of the best models obtained with the three evaluated algorithms after feature selection was found to be in the range of 63.38 ± 2.18-70.68 ± 1.43%. The highest disease classification accuracy was reached by C4.5, which also provides the most informative model in the form of a decision tree, and the lowest accuracy was obtained with naive Bayes. The feature selection method attaining the best classification performance was the wrapper method in forward direction. Moreover, the classification models exposed biological patterns specific to the clinical states and the predictive features selected were found to be characteristic of a specific disorder. Based on these encouraging results, we believe that the CL-based data pre-processing approach combined with the wrapper forward feature selection procedure and the C4.5 decision-tree algorithm has a clear potential to become a fast, informative, and sensitive tool for predictive diagnostics of infectious diseases in clinics.

The effect of increasing levels of fish oil on some immune responses of broiler chickens

There has been some interest in the enrichment of poultry meat with long-chain n-3 PUFA as a means of increasing the low consumption of these acids by human subjects consuming western diets. There is some concern, however, that at high levels of consumption, n-3 PUFA may have detrimental effects on immune function. However, research to date shows that strong controversy surrounds this immunomodulation. The aim of this experiment was to determine the effects of dietary long-chain PUFA on some aspects of immune function in broiler chickens. A total of 18 one-day-old male Ross 308 broiler chicks were reared as a single group for 21 d, and fed a common starter diet. At 21 d, the broilers were then fed for 33 d one of three wheat–soyabean meal-based diets. All diets contained 60 g/kg added oil, which was either 0, 60; 30, 30; or 60, 0 g fish oil (FO) and soya oil, respectively. Chickens between 54 and 60 d of age were sacrificed and samples of blood, bursa of Fabricus, spleen and thymus were collected. The lymphocytes were stained with four monoclonal antibodies: anti-CD3, anti-CD4, anti-CD8 and BU-1A. Immune cells were then enumerated using the FACSCalibur flow cytometer. The quantitative analysis of the phagocytic activity of peripheral mononuclear phagocytes in whole blood was performed using phagotest commercial kits. Increasing levels of FO did not affect the weights of the spleen. However, chickens fed diet with 60 g/kg FO had significantly lower bursa weights (P<0.01) than those fed diets with 0 and 30 g/kg FO. In addition, the bursae were thinner compared with bursae from chickens fed diets containing less FO (Table 1). Chickens fed diets containing 30 g/kg FO had significantly higher thymus weights compared to chickens fed 0 and 60 g/kg (P<0.05). There was no significant effect of increasing FO level on the percentage positive and mean fluorescence intensity (MFI) of the leucocyte subsets in peripheral blood, spleen and thymus. However, the proportion of B-cells in peripheral blood and the MFI of CD8 subsets in the spleen approached significance, P = 0.058 and 0.054, respectively. Results of phagocytic activity show that the different levels of FO in the experimental groups neither affected the positive percentage of cells nor MFI. However, there was a trend towards a lower proportion of monocytes being engaged in phagocytosis when broilers were fed diets containing 60 g/kg FO (P = 0.055) (Table 2).

What happens with ageing: decline or remodeling of opsonin‐independent phagocyte oxidative activity?

Changes that occur with age in the opsonin-independent oxidative activity of peripheral phagocytes in whole blood were examined by means of luminol chemiluminescence (LCL). The chemiluminescence was registered simultaneously by non-stimulated and stimulated cells and the age-related alterations of total and extracellular generation of reactive oxygen species (ROS) were studied using model systems. It was found that the rate of phagocyte activation by the glass surface of the measuring chambers, assessed by the time of the peak appearance after the start of LCL response, increased. However, the maximum oxidative activity and the integral oxidative capacity of the cells during adhesion, evaluated by the maximum LCL intensity and the area under the LCL curve, respectively, declined. No age-dependence of formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated oxidative cellular activity for total ROS generation was detected. The maximum oxidative activity and the integral oxidative capacity of peripheral phagocytes to generate extracellular superoxide in response to fMLP was decreased. The likely causes for the observed alterations in phagocyte function are discussed and an analysis of the obtained results is given on the background of the contradictory data published on phagocyte oxidative activity age-related changes.

Increased expression of IgG Fc receptor type I on neutrophils and monocytes from HIV-infected subjects

Interferon-gamma (IFN-gamma) induces de novo expression of IgG Fc receptor type I (FcRI) on neutrophils and significantly raises the level of these receptors on monocytes. Since increased concentrations of IFN-gamma have been observed in sera from patients with HIV infection, FcRI expression might also be increased on these subjects' phagocytes. FcRI expression was assessed by indirect immunofluorescence staining of phagocytes in whole blood from 40 healthy controls and 55 HIV+ subjects, 24 belonging to CDC class III and 31 to CDC class IV; 42 were intravenous drug abusers (IVDA) and 13 were homosexual men. Plasma levels of IFN-gamma were measured using a modified immunoradiometric assay. The mean linear fluorescence intensity, used as a relative measure of receptor expression, was significantly higher on unseparated neutrophils from HIV+ subjects in CDC classes III (P < 0.001) and IV (P < 0.0001) than from controls. Similar changes in FcRI expression were observed on monocytes from HIV+ subjects. While no differences were observed between IVDA and homosexual HIV+ patients, there was a significant association between FcRI expression and the patients' CDC stage, those in class IV having the highest FcRI levels. Plasma IFN-gamma concentrations were significantly higher in HIV+ patients than in controls and a positive correlation with the stages of HIV infection was again observed. FcRI expression was also increased on freshly purified neutrophils from five HIV+ patients in CDC class IV but did not increase further after 18 h incubation with IFN-gamma, a treatment that up-regulated FcRI expression on control neutrophils. These data suggest that: (i) FcRI evaluation may be a sensitive marker for the biological activity of IFN-gamma in vivo; (ii) phagocytes from HIV+ subjects are activated in vivo by IFN-gamma, expressing increased levels of FcRI; (iii) these IFN-gamma-activated cells may play a role in the pathogenesis of AIDS.

Local application of basic fibroblast growth factor increases the risk of local infection after trauma: An in-vitro and in-vivo study in rats

Introduction Local application of growth factors to stimulate wound and fracture healing is attracting increasing interest. We studied the effect of local application of a potent angiogenic growth factor, basic fibroblast growth factor (bFGF), on resistance to local infection after soft tissue trauma.Methods For in-vitro and in-vivo experiments, we used recombinant human bFGF. The in-vitro investigations were performed by isolation of human leukocyte fractions, cytokine analysis, phagocytosis assay, flow cytometry, and LDH assay. For the in-vivo investigation, a paired comparison of infection rates was carried out on Sprague-Dawley rats after standardized, closed soft tissue trauma and local, percutaneous bacterial inoculation of different concentrations of Staphylococcus aureus (2 × 104 to 2 × 107 colony-forming units (cfu)). The lower leg was treated with 1, 10 or 100 ng bFGF (16 animals for each concentration) and without bFGF (16 animals).Results Cytotoxic reactions due to the concentrations of bFGF used could be excluded in the in-vitro tests since incubations of isolated peripheral blood mononuclear cells (PBMCs) with increasing concentrations of bFGF for 24 h did not lead to an increase in the release of lactate dehydrogenase in the culture supernatants compared to corresponding control incubations without any bFGF added. A significant increase in cytokine release was observed after the co-incubation of PBMCs with 100 or 200 ng of the same bFGF that was used for the animal experiments. Furthermore, the capacity of phagocytes in whole blood to phagocytose bacteria was suppressed in the presence of 100 ng exogenously added bFGF. We found continuously reduced granulocytic phagocytosis in FGF-supplemented blood compared to non-supplemented blood.In the in-vivo investigation, the infection rate for the group without bFGF was 0.25. In the groups with 1, 10 and 100 ng bFGF, the infection rates were 0.5, 0.7 and 0.8, respectively. A dose-dependent increase in infection rate was observed after local application of bFGF, compared to the untreated control group. The difference in infection rates for the groups in which 10 and 100 ng bFGF was used, relative to the group without bFGF, was statistically significant.Interpretation If these initial results are confirmed for other potent angiogenic growth factors, then the local use of growth factors for stimulation of wound and bone healing—a main focus of current research in traumatology—will have to be reconsidered and preceded with a strict evaluation of the risks and benefits.

Cyclic Nucleotides and Vasoactive Intestinal Peptide Production in a Rabbit Model of Escherichia Coli Septicemia

Nitric oxide and vasoactive intestinal peptide (VIP) are potent vasodilators and postulated as inducers of hypotension. These mediators activate guanylate cyclase and adenylate cyclase, respectively, with subsequent biosynthesis of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) producing vascular smooth muscle relaxation and vasodilatation. Cyclic nucleotides and VIP were evaluated during Escherichia coli septicemia in two groups of rabbits; 1) sepsis alone and 2) sepsis and a competitive inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine. Arterial blood was obtained for determination of bacteremia, lactic acidemia, nucleotides, nitrites, and VIP levels. Significant bacteremia, endotoxemia, tachycardia, lactic acidosis, and hypotension occurred in all animals (P < 0.005). Circulating blood levels of cGMP, nitrites, cAMP, and VIP (P < 0.005) increased with development of shock. The NG-monomethyl-L-arginine treated animals had less cGMP, nitrites, cAMP, and VIP produced (P < 0.01). Plasma cGMP levels remained stable, suggesting that stimulated phagocytes in whole blood were responsible for increased cGMP levels. Infusion of VIP produced profound hypotension and lactic acidemia. Results of these experiments provide definitive evidence that nitric oxide and VIP are mediators during septic shock and their messengers are cGMP and cAMP, respectively. In addition, phagocytic stimulation with increased production of cGMP may initiate shock, with these mediators acting synergistically to prolong hypotension.

The effects of glucocorticosteroids on the chemiluminescence response of bovine phagocytic cells

The effects of corticosteroids on the chemiluminescence response of bovine phagocytic cells were determined both in vitro and in vivo. The in vitro addition of hydrocortisone or dexamethasone had no significant effect on the chemiluminescence response of leukocytes in a whole blood or purified polymorphonuclear leukocyte (PMN) population. Cattle that received a single 20 mg dose of dexamethasone or three 20 mg doses of dexamethasone (given 24 hours apart) demonstrated the expected effects on the bovine leukogram (leukocytosis, neutrophilia, lymphopenia, eosinopenia, and monocytosis) and also demonstrated the expected suppressive effect on lymphocyte response to phytohemagglutinin (PHA). However, neither a single nor multiple dexamethasone treatment(s) had an effect on the chemiluminescence response of phagocytes in whole blood, but significantly enhanced the chemiluminescence response of the purified PMN leukocyte population. There was no significant difference between the two dexamethasone treatment groups in either the degree or duration of the effects observed in the chemiluminescence or lymphocyte response assays.

Phagocytosis and lysozyme activity in granulocytes from iron-deficient rat dams and pups

The effect of dietary iron deficiency during gestation and lactation on phagocyte function in rat dams and pups was investigated. Pregnant rats were fed ad libitum diets containing 7, 10 or 250 ppm iron throughout gestation and until day 17 of lactation when dams and their litters (7 pups) were sacrificed. Phagocytosis was measured by the nitroblue tetrazolium (NBT) dye reduction test. Lysozyme activity was measured in granulocytes separated from peripheral blood. A significant increase in percent phagocytes in whole blood in the 7 ppm iron pups was observed. Total white blood cell (WBC) counts were similar among groups. The 7 ppm iron pups had markedly decreased NBT reduction (per 10 8 phagocytes). On a per ml of blood basis, there were no differences in NBT reduction among groups. Plasma activities of the two granulocyte enzymes measured, lysozyme and peroxidase, were significantly increased in 7 ppm iron pups. It was shown that this increase in plasma lysozyme activity was not due to an increased concentration of lysozyme in granulocytes.

Cyanide-insensitive respiration of phagocytes in oxygen-treated whole blood

Cyanide-insensitive respiration of phagocytes has been assayed polarographically in less than 0.5 ml of oxygenated venous blood. Cell separation procedures are avoided by use of whole blood as assay sample. The method is simple and gives quantitative results. The values obtained faithfully reflect in vivo capacity of phagocytes. Blood from boys with chronic granulomatous disease showed no burst of oxygen consumption during phagocytosis. Blood from their mothers showed about one-third activity of normal blood, indicating their carrier status.