Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection
Marisa Freitas
a, Graça Porto
b, José L.F.C. Lima
a, Eduarda Fernandes
a,⁎
aREQUIMTE, Physical-Chemistry Department, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha 164, 4099-030 Porto, Portugal
bClinical Hematology, HGSA, Santo António General Hospital, Porto, Portugal Received 13 May 2007; received in revised form 11 November 2007; accepted 30 December 2007
Available online 11 January 2008
Abstract
Objectives:To assess the effect of different anticoagulants (EDTA, citrate and heparin) on the isolation procedure of human neutrophils and in the subsequent alterations of calcium levels and respiratory burst induced by phorbol myristate acetate (PMA).
Design and methods:Isolation of human neutrophils from whole blood was performed by the gradient density centrifugation method. PMA- induced neutrophil burst was measured by chemiluminescence. Intracellular calcium ([Ca2+]i) was measured using Fluo-3 AM, a calcium-sensitive dye.
Results: EDTA provided the highest number of isolated neutrophils/mL of blood (1.7 × 106± 1.5 × 105) when compared with citrate (0.46 × 106± 0.95 × 105) and heparin (0.66 × 106± 0.15 × 105). EDTA originated less degree of PMA-induced activation (370 ± 30%) relatively to citrate (830 ± 98%) and heparin (827 ± 77%). [Ca2+]iwas lower with EDTA (122 ± 11 nM) when compared with citrate and heparin (150 ± 13 and 230 ± 30 nM).
Conclusion:The anticoagulant used during blood collection interfered differently with the yield of isolated neutrophils as well as on their calcium levels and reactivity to PMA.
© 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Keywords:Human neutrophils; Neutrophils isolation procedure; Anticoagulants; Respiratory burst; Intracellular calcium
Introduction
Neutrophils (polymorphonuclear leukocytes; PMNs) are the most abundant leukocytes in blood and participate actively on the innate host defense response. During this response, neutrophils and other phagocytic cells are mobilized to sites of injury or infection where they ingest and kill invading microorganisms [1,2]. These are also the first type of cells to arrive at an inflammatory site, where they play a major role in the inflammation and tissue damage of non-infectious diseases
such as arthritis, inflammatory bowel disease, and ischemia- reperfusion injury [3]. During the inflammatory processes, reactive oxygen species (ROS), namely superoxide radical (O2−
U
), singlet oxygen (1O2), hydrogen peroxide (H2O2), hydroxyl radical (HOU
), and hypochlorous acid (HOCl) and nitrogen reactive species (RNS), namely nitric oxide (U
NO) andperoxynitrite anion (ONOO−) are generally formed and have been implicated in its pathophysiology[4,5]. The production of these reactive species by PMNs, usually referred as respiratory burst has been consistently studiedin vitro. After isolation of these cells, it's possible to follow the production of reactive species using several activating compounds and different probes, namely luminol[4,6], lucigenin[4,6], ferricytochrome c[4,7,8], nitroblue tetrazolium,p-hydroxyphenylacetic acid or 2,7-dichlorofluorescein[4].
During the blood collection and isolation procedure to obtain PMNs, several anticoagulants may be used. Ethylenediaminete- traacetic acid (EDTA), citrate and heparin are widely used to
Clinical Biochemistry 41 (2008) 570–575
Abbreviations: PMNs, polymorphonuclear leukocytes; ROS, reactive oxygen species; RNS, reactive nitrogen species; EDTA, ethylenediaminete- traacetic acid; [Ca2+]i, intracellular calcium; PMA, phorbol myristate acetate;
PBS, Dulbecco's phosphate buffer saline; S.E.M., mean ± ; standard error of the mean; EGTA, ethylene glycol-bis(b-aminoethyl ether)N,N,N',N'-tetraacetic acid; LPS, lipopolysaccharide.
⁎Corresponding author. Fax: +351 222004427.
E-mail address:egracas@ff.up.pt(E. Fernandes).
0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2007.12.021
inhibit blood clotting both in routine clinical analysis and inin vitro research. Since the enzymes involved in the coagulation cascade depend on free calcium as a cofactor, the calcium chelators EDTA and citrate exert their anticoagulant effect by binding free calcium ions[9,10]. Heparin is a sulphated polysac- charide with a variable molecular weight of 3000 to 30,000 Da.
Heparin acts as an anticoagulant by activating antithrombin. The antithrombin-heparin complex inactivates the complex tissue factor/VIIa, and also thrombin, factor IXa, factor Xa and factor XIa[11].
Importantly, some previous reports suggest that blood cells may behave differently depending on which anticoagulant has been used during blood collection[11–14]. Respiratory burst is one of the most important activities of neutrophils. Considering that EDTA and citrate anticoagulants interfere with calcium levels, they could influence neutrophil respiratory burst, since it may depend of intracellular calcium ([Ca2+]i), namely when neutrophils were activated by phorbol myristate acetate (PMA) [15–17]. However, the effect of previous contact with different anticoagulants in the subsequent activation of neutrophil respiratory burst is yet to be known.
The main objective of the present study was to assess the effect of three different anticoagulants (EDTA, citrate and heparin) normally used during blood collection for clinical chemistry analysis on the isolation procedure and in the subsequent neutro- phil respiratory burst induced by PMA. Considering the putative interference of the studied anticoagulants with [Ca2+]ilevels, and the fundamental contribution of [Ca2+]i for the activation of neutrophils, this parameter was also evaluated and provided a mechanistic insight for the obtained results.
Materials and methods Materials
The following reagents were obtained from Sigma Chemical Co. (St. Louis, U.S.A.): Histopaque 1077, Histopaque 1119, Dulbecco's phosphate buffer saline, without calcium chloride and magnesium chloride (PBS) [2.68 mM KCl, 0.14 M NaCl, 1.21 mM KH2PO4, 8.10 mM Na2HPO4], Dulbecco's phosphate buffer saline, with calcium chloride and magnesium chloride (PBS with Ca2+and Mg2+) [0.90 mM CaCl2·2H2O, 0.49 mM MgCl2·6H2O, 2.68 mM KCl, 0.14 M NaCl, 1.21 mM KH2PO4, 8.10 mM Na2HPO4], trypan blue solution 0.4%, luminol, phorbol myristate acetate (PMA), A23187, glycol-bis(β- aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). Acetox- ymethyl ester (AM) of Fluo-3 was purchased from Molecular Probes (Eugene, Oregon). Sodium chloride and calcium chloride were obtained from Merck (Darmstadt, Germany).
Vacuum tubes with citrate, EDTA and heparin were purchased from Vacutainer Systems (U.K.).
Isolation of human neutrophils. The gradient density centrifugation method
In accordance with Helsinki Declaration, the design and execution of the experiment were thoroughly explained to the
participants, and informed consent was obtained. Venous blood was collected by antecubital venipuncture, from each human healthy volunteer, into three vacuum tubes, each one with the respective anticoagulant, in a random order. Different antic- oagulants were used during collection of these samples, namely citrate, EDTA and heparin. The isolation of human neutrophils from whole blood was performed by the gradient density cen- trifugation method using Histopaque solutions 1077 and 1119 in polypropylene 12 mL centrifuge tubes, as reported by Costa et al.[6]. Briefly, 3 mL of Histopaque 1077 was carefully layered on top of 3 mL of Histopaque 1119 in a 12 mL polypropylene tube. Subsequently, 6 mL of the collected blood was decanted on this discontinuous density gradient. The tube was centri- fuged at 890g for 30 min at 20 °C. Once the centrifugation was complete the neutrophils were carefully removed using a Pasteur pipette. The neutrophil pellet was removed and doubled in volume using PBS without Ca2+and Mg2+(this reduces the viscosity of the Histopaque-neutrophil suspension so that the cells can be centrifuged without the need for high g forces). The neutrophils suspension was then centrifuged at 870g for 5 min at 4 °C. The supernatant was decanted and a mixture of 1.25 mL of PBS without Ca2+ and Mg2++ 5.25 mL of sterile distilled water was added to the neutrophil pellet to lyse any remaining red blood cells. The tube was gently inverted for 1.30 min, after which isotonicity was re-established by adding 2.2 mL of 3%
NaCl. This suspension was then submitted to a new centrifuga- tion at 870g for 5 min at 4 °C after which the supernatant was decanted and the neutrophil pellet resuspended in PBS with Ca2+
and Mg2+. In case of neutrophils isolated from blood treated with citrate, the lyses procedure was repeated one more time in the same conditions of the last one. The time from venipuncture to performance of the different tests was about the same in all experiments (2h). Isolated neutrophils were kept in ice until use.
The neutrophils were used from one volunteer per experiment.
Isolation of human neutrophils. Influence of anticoagulants in the yield and viability of isolated cells
Cell viability and cell yield were evaluated by the Trypan blue exclusion method, using a neubauer chamber and an optic microscope (40×). Twenty microliters of neutrophils suspension was added to an equal volume of 0.4% trypan blue in an ep- pendorf and mixed gently. After 2 min on ice, neutrophils number and viability (viable cells excluding trypan blue) were counted. Each study corresponds to ten individual experiments, performed in triplicate in each experiment.
Measurement of PMA-induced oxidative burst in human neutrophils
Essentially, this study was based on the PMA-induced acti- vation of human neutrophils, isolated from blood treated with different anticoagulants. The measurement of neutrophil burst was undertaken by chemiluminescence, by monitoring the oxi- dation of luminol by neutrophil-generated reactive species, ac- cording to a previously described procedure adapted to a microplate reader (Synergy HT, BIO-TEK)[6]. Reaction mixtures
contained the following reagents at the indicated final concentra- tions (in a final volume of 200μL): luminol (500μM), PBS with Ca2+ and Mg2+, PMA (1.6 × 10−7 M) and neutrophils (final suspension = 1 × 106 cells/mL). Each experiment was accompa- nied by a blank assay which was conducted by exactly the same method, except that the PMA was replaced by PBS with Ca2+and Mg2+. During all the assays, the reactional mixture was subjected to a soft agitation and a temperature of incubation of 37 °C.
Kinetic readings were initiated immediately after cell stimulation.
Measurements were taken at the peak of the curve. In all expe- riments, this peak was observed at around 15 min. Effects are expressed as the percentage of luminol oxidation, relatively to the blank assay. Each study corresponds to six individual experi- ments, performed in triplicate in each experiment.
Measurement of intracellular calcium ([Ca2+]i)
Measurements of intracellular calcium in human neutrophils were performed using Fluo-3 AM, a calcium-sensitive dye, as described previously [18] with modifications, adapted to a microplate reader (Synergy HT, BIO-TEK). Fluo-3 AM (excitation at 488 nm and emission at 528 nm) has a negligible fluorescence as a free acid, which is enhanced upon binding to Ca2+. Intracellular ionized calcium was calculated using the formula [Ca2+]i=Kd[F−Fmin] / [Fmax−F], whereKdrepresents the dissociation constant, reflecting the affinity of the dye to calcium, to which a value of 400 nM was attributed. The isolated neutrophils were loaded with 2.5μM of Fluo-3 AM at 37 °C, in the dark and with a soft agitation for 15 min.
Following incubation, cells were washed with PBS without Ca2+
and Mg2+. The centrifugation was performed at 870g, for 5 min at 4 °C. Thereafter, the cell suspensions (final concentration 3 × 106cells/mL) were added to 96-well plates and theFvalue was measured at the end of 10 min, representing the baseline fluorescence signal. Then, CaCl2was added in same well (final concentration = 1 mM). To obtain theFmax, a calcium ionophore A23187 (final concentration = 5 μM) was added and the fluo-
rescence was recorded after 10 min.Fminwas determined imme- diately after the addition of 10 mM EGTA to the ionophore- treated cells. The reactional mixture was subjected to a soft agitation and a temperature of incubation of 37 °C during the course of the assays. Each study corresponds to four individual experiments, performed in triplicate in each experiment.
Statistical analysis
Statistics were calculated using GraphPad Prism™(version 4.0; GraphPad Software). Results are expressed as mean ± standard error of the mean (S.E.M.) (from at least four different subjects). Statistical comparison between groups was estimated using the one-way analysis of variance (ANOVA), followed by the Bonferroni'spost-hoctest. In all cases,p-values lower than 0.05 were considered as statistically significant.
Results
Viability of human neutrophils isolated from blood treated with citrate, EDTA or heparin
The results obtained from the study of viability of human neutrophils isolated from blood treated with citrate, EDTA or heparin showed that the viability of the isolated neutrophils was always above 98%, independently of the anticoagulant used.
Yield of human neutrophils isolated from blood treated with citrate, EDTA or heparin
Fig. 1 shows the results obtained from the study of the number of human neutrophils isolated from blood treated with citrated, EDTA or heparin. Significant differences (pb0.01) were observed in the number of isolated neutrophils. The highest number of isolated neutrophils per mL of blood was obtained for EDTA (1.7 × 106± 1.5 × 105cells/mL blood), when compared with both heparin and citrate (0.66 × 106± 0.15 × 105 cells/mL blood and 0.46 × 106± 0.95 × 105cells/mL blood, re- spectively) (mean ± SEM).
Fig. 1. Number of neutrophils isolated from blood anticoagulated with citrate, EDTA and heparin, per mL of blood. Values are given as mean ± S.E.M. (n= 10).
⁎⁎pb0.01 comparatively both to the treatment with citrate and heparin.
Fig. 2. Activation, by PMA, of human neutrophils isolated from blood treated with citrate, EDTA and heparin, relatively to the blank assay (%). Values are given as mean ± S.E.M. (n= 6).⁎⁎pb0.01 comparatively both to the treatment with citrate and heparin.
Activation, by PMA, of human neutrophils isolated from blood treated with citrate, EDTA or heparin
Fig. 2shows the results obtained from the study of activation, by PMA, of human neutrophils isolated from blood treated with citrate, EDTA or heparin. The percent increase is presented relatively to the baseline for each cell treatment prior to addition of PMA. Of note, the baseline readings were similar among the three cell treatments. It was verified that the activation of neutrophils was dependent on the anticoagulant used. EDTA originated a significant (pb0.01) lower degree of activation (370 ± 30%) relatively to citrate (830 ± 98%) and heparin (827 ± 77%) (mean ± S.E.M.).
Quantification of [Ca2+]i on neutrophils isolated from blood treated with citrate, EDTA and heparin
Fig. 3shows the results obtained from the study of quan- tification of [Ca2+]i on neutrophils isolated from blood treated with citrate, EDTA or heparin. It was observed that [Ca2+]iwas significantly (pb0.05) lower for EDTA pre-treated cells (122 ± 11 nM) when compared with heparin pre-treatment (230 ± 30 nM), while [Ca2+]iin citrate pre-treated cells (150 ± 13 nM) was only tendentially lower and higher than that of heparin and EDTA pre-treatments, respectively (mean ± S.E.M.).
Discussion
The results obtained in the present study showed that the viability of the isolated neutrophils was always above 98%, independently of the anticoagulant used during blood collec- tion. On the other hand, it was demonstrated that the number of neutrophils isolated by the gradient centrifugation method depends on the anticoagulant used, EDTA being the one that provided the highest number of isolated cells when compared with citrate and heparin, which did not differ significantly between each other. It was also clearly shown that the activation
of neutrophils is dependent on the anticoagulant used, since neutrophils isolated from blood treated with EDTA had lower intracellular calcium levels and were much less activated by PMA.
Neutrophils were isolated by the gradient density centrifuga- tion method, in which two solutions of different densities Histopaque 1077 (density of 1.077) and Histopaque 1119 (density of 1.119) were used. With this methodology, we found significant differences in the number of isolated neutrophils from blood treated with different anticoagulants, citrate, EDTA and heparin.
It must be stressed that, prior to the isolation procedure, the cell count was performed, in four human healthy volunteers, using an auto-analyser Sysmex SF 3000 (Roche), which showed no significant (pN0.05) differences among anticoagulant treatments (citrate: 4.9 × 106± 0.9 × 106neutrophils/mL, EDTA:
4.6 × 106± 0.7 × 106 neutrophils/mL and heparin: 4.2 × 106± 0.9 × 106neutrophils/mL).
This suggests that blood treatment with the different antic- oagulants influences the separation process. The separation of gradients was more effective for EDTA comparatively to the other anticoagulants tested, which allowed an efficient recovery of the neutrophils layer and a subsequent great number of isolated neutrophils. These results corroborate previous obser- vations by Nielsen [12]who studied the influence of five dif- ferent anticoagulants on human blood monocytes isolation and functional activities. In that study, the isolation of monocytes was effectuated by a gradient density centrifugation method, with blood treated with heparin, citrate, oxalate, EDTA and ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The recovery of blood monocytes was higher with EDTA and heparin as anticoagulants, though there were no differences in viability, spontaneous migration, chemotaxis, or phagocytosis and killing of Candida albicans by monocytes isolated from blood treated with the different anticoagulants.
Considering that the methodology used for the isolation of human neutrophils is based in density differences that allow the separation in gradient of the different layers of cells, these results suggest that heparin and citrate altered cell density and the subsequent disposition of the layers, which diminished the efficiency of the recovery of the neutrophils layer. During isolation procedures, we observed that blood treated with citrate had the worst separation of gradients. With heparin, the layers were more defined than with citrate but less than with EDTA.
Our results in the isolation procedure are corroborated by previous studies performed by Mahony and Ferguson[19]who studied characteristics of blood treated with these two antic- oagulants. Ultrasonic images of heparinized blood revealed aggregation of cells. The aggregate consisted of platelet clumps resembling grapes, several neutrophils and a small number of lymphocytes. Ultrasonic images of citrated blood from the same donor revealed platelet and platelet-neutrophil aggregates, though much smaller and in fewer number than seen in hepa- rinized samples. These observations, together with our results, lead to the hypothesis that the worst visualization of the cells layers with citrate and heparin is correlated with differences of density caused by cellular aggregation. After the recovery of
Fig. 3. Quantification of [Ca2+]i in human neutrophils isolated from blood treated with citrate, EDTA and heparin. Values are given as mean ± S.E.M.
(n= 4).⁎pb0.05 comparatively to the treatment with heparin.
neutrophils layer, an erythrocyte lysing step is performed to eliminate any contamination by these cells. In this stage, it was verified that neutrophils isolated from blood anticoagulated with citrate and heparin were more contaminated by erythrocytes.
This fact results in a worse visualization of the neutrophils layer and creates difficulties in the separation of the layers. Due to this higher contamination by erythrocytes, a single lyses step was not enough to eliminate them. In the case of citrate, the lyses pro- cedure was repeated one more time to remove all erythrocytes.
This could be explained by the aggregation of cells components reported previously by Mahony and Ferguson[19]. The lowest number of isolated neutrophils verified with citrate may be mainly attributed to the worst separation of gradients, which contributes to the diminished efficiency of the recovery of the neutrophils layer and implies another lyses step.
PMA is widely used to stimulate a variety of cells, including neutrophils. According to Nixon and McPhail [20] PKC-α, PKC-β and PKC-δ are prime candidates for mediating PMA- induced responses. Indeed, this chemical compound mimics the activity of endogenous diacylglycerol (DAG) required for an active PKC that subsequently activates NADPH oxidase. Five PKC isoforms have been identified in human neutrophils. These include PKC-α,βI,βII (classified as conventional class: require Ca2+, DAG and a phospholipid such as phosphatidylserine for activation), PKC-δ(classified as novel class: requires phospha- tidylserine and DAG, but is Ca2+ independent) and PKC-ζ (named atypical class: that is Ca2+and DAG independent, but requires phosphatidylserine for activation).
The PMA-induced oxidative burst in neutrophils was severely affected by blood treatment with anticoagulants during the collection and separation process. EDTA treatment resulted in a lower degree of activation relatively to citrate and heparin.
Engstad et al.[11]have previously compared the effect of four anticoagulants (EDTA, citrate, heparin and hirudin) on mono- cytes, neutrophils and platelet function in human whole blood.
Monocytes were activated with 5 ng/mL lipopolysaccharide (LPS), and neutrophils activation was assessed by measuring lactoferrin concentration in whole blood after 90 min of incu- bation with and without LPS. In what concerns platelets, these authors evaluated the influence of anticoagulants on the release of α-granules. Although the evaluated parameters differ from those of the present study, the relative profile of neutrophil activation was similar to the one here observed, that is, these authors reported that EDTA and citrate diminished cellular activation in neutrophils and monocytes as compared to heparin and hirudin. Calcium ions are crucial elements in the trans- duction of extracellular signals, leading to the activation of different effector responses (e.g. degranulation, O2−
U
produc-tion) of the cells. The Ca2+signal of neutrophils consists of two components: i) a rapid, transient increase of [Ca2+]idue to Ca2+
release from the internal stores induced by inositol 1,4,5- trisphosphate and ii) a sustained elevation of [Ca2+]idue to Ca2+
influx from the extracellular space[21]. The extracellular Ca2+
plays an essential role in the oxidative response, possibly through phospholipase A2activation. This enzyme hydrolyses membrane phospholipids, leading to the formation of arachi- donic acid that plays an essential role in activation of the
enzyme NADPH oxidase, which is responsible for the gener- ation of O2−
U
by neutrophils[22]. The intracellular mechanisms linking [Ca2+]ielevation to O2−U
generation are unclear. Rac-1, a monomeric G-protein, has been shown to stimulate NADPH oxidase and Valentin et al. [23] hypothesized that [Ca2+]imobilization leads to Rac-1 activation, which in turn stimulates NAPDH oxidase activity leading to O2−
U
production. Movitz et al.[24]reported that the translocation of cytosolic factors, a prerequisite for obtaining the intracellular NADPH-oxidase activity in activated neutrophils, is not induced by Ca2+per se, but rather by some additional signalling molecule(s) generated in response to an elevation of intracellular Ca2+. These molecule (s) have yet to be identified. In the present study, it was observed that treatment of blood with EDTA resulted in the lowest concentration of free calcium in neutrophils, while free calcium concentration in cells from citrate treatment showed a tendency to be higher than that of EDTA and lower than that of heparin treatments. These results could explain the lower activation of neutrophils isolated from blood anticoagulated with EDTA.The higher activation of neutrophils isolated from blood treated with heparin can be related with the stimulating effect of this anticoagulant on the expression of adhesion molecules.
According to Arnaout [25] CD11b and CD18 integrins con- stitute neutrophil activation markers and contribute to its functions, such as chemotaxis, adhesion to endothelium, phago- cytosis and activation. It has been reported by several authors that heparin, in opposition to citrate and EDTA, causes marked upregulation of adhesion molecules in neutrophils, namely the integrins CD11b and CD18 [13,14,26]. Habbal et al. [26]
reported that the selection of the optimal anticoagulant is im- portant because heparin causes an upregulation of CD11b with subsequent stimulatory effect on the neutrophils that may cause false-positive results. CD11b is easily upregulatedex vivo by handling procedures of blood samples, as is the case of blood cells isolation. In addition, alteration of the temperature from 4 °C to 37 °C upregulates CD11b. For that reason, all cell handling procedures must be on ice in order to minimize arte- factual upregulation of CD11b[13,27]. In the present study the higher sensitivity of neutrophils, isolated from blood antic- oagulated with heparin, to activators of respiratory burst, could be related by the influence of heparin on upregulation of CD11b and CD18. Temperature manipulations were rigorously main- tained for all experiments, with neutrophils being always kept on ice until use and the readings on the microplate reader ef- fectuated at 37 °C, this procedure being identical for all exper- iments performed. Repo et al.[13]reported that EDTA did not cause an upregulation of CD11b/CD18 probably because this anticoagulant exerts its inhibitory effects most probably by chelating extracellular divalent cations, which is a prerequisite for the adherence function of the integrins. Citrate, which also binds Ca2+does not depress CD11b expression, which suggests that citrate may not interfere the occupancy of the Ca2+binding sites of CD11b molecules[13].
In conclusion, the results obtained in this study demonstrate that the choice of anticoagulant influences the number of iso- lated neutrophils by gradient density method, their [Ca2+]i, and the sensibility of neutrophils to activators of respiratory burst.
These findings are considered to be of technical importance for future studies related to the isolation and activation of human neutrophilsin vitro.
Acknowledgments
The authors acknowledge Fundação para a Ciência e Tecnologia (FCT) and Fundo Europeu de Desenvolvimento Regional (FEDER) financial support for the project POCI/QUI/
59284/2004. Marisa Freitas acknowledges FCT and Fundo Social Europeu (FSE) for her PhD grant (SFRH/BD/28502/2006).
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