Isolation Of Polymorphonuclear Neutrophils Research Articles

Blood collection technique, anticoagulants and storing temperature have minor effects on the isolation of polymorphonuclear neutrophils

In the isolation of polymorphonuclear neutrophils (PMNs) the technique and other external factors can have great influence on the quality and quantity of isolated neutrophils. To elucidate the influence of the blood collection technique, anticoagulants and storing temperature on isolated PMNs healthy volunteers provided blood samples with different needles and collection techniques, anticoagulants (EDTA, heparin, citrate) and storing temperatures (4, 22, 37 °C). From each blood sample PMNs were isolated and compared regarding number of PMNs and oxidative burst. The blood collection technique, anticoagulants and storing temperature had minor impact on isolated PMNs. All three tested cannulas and anticoagulants can be used to obtain blood samples for PMN isolation. For storing temperatures 37 °C should be preferred. Regarding time between the PMN isolation and the actual experiments, a time span of maximum 1 h should be targeted.

In Vitro Assay for Sensitive Determination of Human Blood PMN Responses.

Polymorphonuclear neutrophils (PMNs) are the most common leukocytes in the circulation and exhibit a wide range of distinct cellular phenomena as part of their microbicidal killing activities, including degranulation, phagocytosis, reactive oxygen species (ROS) production, adhesion, chemotaxis and production of PMN Extracellular Traps (NETs). As a simple in vitro test of PMN functional responses in human blood we have developed a multicolor flow cytometry-based assay of PMN cluster of differentiation (CD) surface marker expression. Short incubations of whole human blood can be performed in the presence of a wide range of agonists or inhibitors, followed by sensitive detection of changes in CD marker expression. This protocol has the advantage that small amounts of human blood are necessary and there are no PMN isolation steps, which can alter PMN activation status.

Characterization of oral polymorphonuclear neutrophils in periodontitis patients: a case-control study

BackgroundMaintaining oral health is a continuous and dynamic process that also involves the immune system. Polymorphonuclear neutrophils (PMNs) migrate from blood circulation and become apparent in the oral fluid. Controversies exist regarding the specific role of the oral PMNs (oPMNs) in the presence of chronic oral inflammation, such as periodontitis. In this study we characterized cell counts, activation status, apoptosis, and reactive oxygen species (ROS) generation by oPMNs and circulatory (cPMNs), and the salivary protease activity, in subjects with and without periodontitis.MethodsVenous blood and oral rinse samples were obtained from 19 patients with untreated periodontitis and 16 control subjects for PMN isolation. Apoptosis and expression of cell activation markers CD11b, CD63, and CD66b were analyzed using flow cytometry. Constitutive ROS generation was detected using dihydrorhodamine123. Additionally, ROS production in response to stimulation was evaluated in samples incubated with 10 μM phorbol myristate acetate (PMA) or Fusobacterium nucleatum. Total protease activity was measured using substrate PEK-054.ResultsPeriodontitis patients presented with over 4 times higher oPMN counts compared to controls (p = 0.007), which was a predictor for the total protease activity (r2 = 0.399, P = 0.007). More oPMNs were apoptotic in periodontitis patients compared to the controls (P = 0.004). All three activation markers were more expressed on the oPMNs compared to the cPMNs (p < 0.05), and a higher expression of CD11b on the oPMNs from periodontitis patients was observed compared to the control subjects (P = 0.024). Constitutive ROS production per oPMN was higher compared to the cPMN (P < 0.001). Additional analysis showed that the oPMNs retained their ability to respond to stimulation, with no apparent differences between the periodontitis and control subjects.ConclusionsHigher numbers of oral PMNs, being more apoptotic and having increased levels of degranulation markers were found in periodontitis compared to periodontal health. However, since the oPMNs in periodontitis were responsive to ex vivo stimulation, we conclude that the oPMNs are active in the oral ecosystem. It is currently unknown whether the oPMN counts, which correlated with the detected protease levels, are detrimental in the long term for the oral mucosa integrity.Trial registrationThis study was retrospectively registered at the ISRCTN registry (trial ID ISRCTN15252886). Registration date August 11, 2017.

Hypoxia-induced upregulation of CD11b expression in granulocytes

The aim of this study is to evaluate leukocyte-endothelial adhesion during ischemia/reperfusion injury. Polymorphonuclear neutrophils (PMN) were isolated from healthy volunteers and incubated in the presence of 2% O2 (hypoxia) or 21% O2 (normoxia) at 37°C for two hours. In some experiments, whole blood was subjected to hypoxia or normoxia prior to PMN isolation. Flowcytometric analysis and adhesion assays were carried out using these PMN. Moreover, adhesion assay was carried out using PMN, prepared from the patients who underwent infrarenal aortic aneurysmectomy with aortic clamping at various time points as an in vivo model of ischemial reperfusion injury. Flowcytometric analysis revealed an increased expression of CD11b in PMN subjected to hypoxia compared with those subjected to normoxia prior to isolation. Interestingly, these phenomena were not observed with PMN isolated prior to being subjected with hypoxia. Enzyme-linked immunosorbent assay (ELISA) using serum prepared from whole blood subjected to hypoxia revealed elevated levels of tumor necrosis factor (TNF)-α compared with those subjected to normoxia. PMN obtained during aneurysmectomy exhibited an increased adhesion to activated human umbilical vein endothelial cells (HUVEC), compared with those taken from the same patients prior to the operation. In contrast, PMN prepared after aortic clamp, exhibited a significant reduced adhesion to activated HUVEC. Hypoxic condition, induced CD11b expression on PMN in vitro. PMN subjected to hypoxic condition in vivo exhibited an increased adhesion to activated endothelium. Elevated level of serum TNF-α may be involved in this phenomenon.

Flow‐cytometric measurement of respiratory burst in rat polymorphonuclear granulocytes: Comparison of four cell preparation procedures, and concentration–response evaluation of soluble stimulants

Polymorphonuclear neutrophils (PMNs) contribute to organ injury in sepsis, stroke, and other diseases. Evaluation of the oxidative burst by flow cytometry (FCM) is frequently applied to examine PMN status in humans, but rarely in rats. We established a method to assess granulocyte activation in rats by means of FCM analysis of oxidative burst. Two methods for PMN isolation involving Histopaque separation were investigated, and additionally two whole blood techniques. In addition, the concentration-response relation of the stimulants fMLP, PMA, TNF-alpha, and LPS has been determined, both as sole stimulants and for priming. A novel technique with diluted rat whole blood proved to be most appropriate for PMN preparation. One micromolar PMA and fMLP, respectively, are effective concentrations for PMN stimulation in rat whole blood. Priming with 0.1 mug/ml TNF-alpha and 1 mug/ml LPS, respectively, resulted in optimal additional stimulation. This study defined the appropriate conditions for evaluating the reactive oxygen derivate production in rat PMNs by flow cytometry. The rapid, simple, and reliable cell preparation procedure of whole blood dilution that preserves cell integrity and requires only small sample quantities. This is the first systematic dose-response evaluation of soluble stimulants of neutrophil respiratory burst in rats.

Down-regulation of Rac Activity during β2 Integrin-mediated Adhesion of Human Neutrophils

In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.

Hypoxia-Induced Upregulation of CD11b Expression in Granulocytes

The aim of this study is to evaluate leukocyte-endothelial adhesion during ischemia/reperfusion injury. Polymorphonuclear neutrophils (PMN) were isolated from healthy volunteers and incubated in the presence of 2% O2 (hypoxia) or 21% O2 (normoxia) at 37°C for two hours. In some experiments, whole blood was subjected to hypoxia or normoxia prior to PMN isolation. Flowcytometric analysis and adhesion assays were carried out using these PMN. Moreover, adhesion assay was carried out using PMN, prepared from the patients who underwent infrarenal aortic aneurysmectomy with aortic clamping at various time points as an in vivo model of ischemial reperfusion injury. Flowcytometric analysis revealed an increased expression of CD11b in PMN subjected to hypoxia compared with those subjected to normoxia prior to isolation. Interestingly, these phenomena were not observed with PMN isolated prior to being subjected with hypoxia. Enzyme-linked immunosorbent assay (ELISA) using serum prepared from whole blood subjected to hypoxia revealed elevated levels of tumor necrosis factor (TNF)-α compared with those subjected to normoxia. PMN obtained during aneurysmectomy exhibited an increased adhesion to activated human umbilical vein endothelial cells (HUVEC), compared with those taken from the same patients prior to the operation. In contrast, PMN prepared after aortic clamp, exhibited a significant reduced adhesion to activated HUVEC. Hypoxic condition, induced CD11b expression on PMN in vitro. PMN subjected to hypoxic condition in vivo exhibited an increased adhesion to activated endothelium. Elevated level of serum TNF-α may be involved in this phenomenon.

Effects of remifentanil on neutrophil adhesion, transmigration, and intercellular adhesion molecule expression.

Anaesthetic drugs are used for pain therapy and anaesthesia. Neutrophils play a significant role during the process of inflammation. The aim of the current study was to investigate the effects of remifentanil and fentanyl on neutrophil migration through endothelial cell monolayers, and on adhesion molecule expression. After isolation of polymorphonuclear neutrophils (PMNL) we used a currently described migration assay. PMNL and/or endothelial cell monolayers (ECM) were pre-treated with remifentanil using clinically relevant, as well as higher and lower concentrations or relevant concentrations of fentanyl. Concentrations of remifentanil (50 ng/mL) similar to the relevant plasma concentration were able to inhibit PMNL migration through ECM significantly (migration compared to the control 82+/-7% SD; P<0.05), when both cell types were treated with the synthetic narcotic remifentanil. Fentanyl (30 ng/mL) showed a stronger inhibitory effect (migration compared to the control 67+/-9.2%; P<0.05). Endothelial cell adhesion molecule expression was reduced after either remifentanil or fentanyl. The results of the present investigation indicate that remifentanil influences interaction of ECM against human neutrophils. Compared to fentanyl, remifentanil seems to exhibit minor inhibitory effects on neutrophil migration.

Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a ficoll‐hypaque gradient

Polymorphonuclear neutrophils (PMN) have an important role in the host defence response to infection. These cells produce large amounts of reactive oxygen species (O2·−, H2O2 and ONOO−) with microbicidal activity. PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density (Ficoll-Hypaque gradient and dextran), yielding a highly homogeneous cellular population. However, some cellular activation due to membrane perturbation is also expected. We studied how the production of reactive oxygen species and release of myeloperoxidase (MPO) from blood PMN are affected by the use of the Ficoll-Hypaque density gradient. PMN isolated by spontaneous sedimentation and total blood were used for comparisons. Lucigenin- and luminol-enhanced chemiluminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli. The release of MPO, estimated by the chemiluminescence of the luminol/H2O2 reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B, was also higher for PMN isolated by a density gradient. In conclusion, it was shown that the PMN isolation procedure affects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results. Copyright © 2000 John Wiley & Sons, Ltd.

Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a Ficoll-Hypaque gradient.

Polymorphonuclear neutrophils (PMN) have an important role in the host defence response to infection. These cells produce large amounts of reactive oxygen species (O(2).(-), H(2)O(2) and ONOO(-)) with microbicidal activity. PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density (Ficoll-Hypaque gradient and dextran), yielding a highly homogeneous cellular population. However, some cellular activation due to membrane perturbation is also expected. We studied how the production of reactive oxygen species and release of myeloperoxidase (MPO) from blood PMN are affected by the use of the Ficoll-Hypaque density gradient. PMN isolated by spontaneous sedimentation and total blood were used for comparisons. Lucigenin- and luminol-enhanced chemiluminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli. The release of MPO, estimated by the chemiluminescence of the luminol/H(2)O(2) reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B, was also higher for PMN isolated by a density gradient. In conclusion, it was shown that the PMN isolation procedure affects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results.

Polymorphonuclear neutrophil chemiluminescence in whole blood from blunt trauma patients with multiple injuries.

Studies using isolated polymorphonuclear neutrophils (PMNs) indicate that trauma is associated with altered function of PMNs. Because isolation of PMNs can itself alter the function of these cells, we examined the relationships among measures of injury severity and several indices of PMN function using whole blood samples from trauma patients. Whole blood samples were obtained from 12 blunt trauma patients with multiple injuries in the intensive care unit of a Level I trauma center within 24 hours of admission and from 11 healthy volunteers. Samples were assayed for PMN chemiluminescence (CL) in response to a complement receptor 3 (CR3)-dependent agonist and for CD11b (CR3) expression. Common clinical parameters were correlated with CL and CR3 expression. The CL ratio (i.e., unprimed/primed CL) was significantly correlated with initial base deficit (BD), Injury Severity Score (ISS), CR3 expression, units of packed red blood cells transfused during the interval before blood sampling, and length of intensive care unit stay (survivors only). In contrast, BD did not correlate with units of red blood cells transfused or length of stay. Similarly, ISS did not correlate with length of stay. Significant correlations were observed between CL ratios and CR3 expression, ISS, initial BD, length of stay, and units of blood given. These data suggest that measuring CL produced by PMNs in whole blood is a potentially useful way to assess injury severity. Whereas the initial BD and ISS are indicators of how badly injured a patient is at the time of entry into a trauma center, the CL ratio may be a more useful indicator of both injury severity and prognosis.

Complement activates Kupffer cells and neutrophils during reperfusion after hepatic ischemia

The hypothesis that complement factors may be involved in the postischemic activation of Kupffer cells (KC) and polymorphonuclear neutrophils (PMN) was investigated in a model of hepatic ischemia (45 min) and reperfusion in male Fischer rats in vivo. Depletion of serum complement before ischemia resulted in a significant attenuation of the KC-induced oxidant stress (enhanced oxidation of plasma glutathione) and also prevented the accumulation of PMNs in the liver during the initial reperfusion period of 1 h. Complement activation through injection of cobra venom factor (CVF; 75 micrograms CVF/kg) also induced enhanced oxidation of plasma glutathione and accumulation of PMNs in the liver. Isolation of KC and PMNs from the liver 1 h after CVF treatment demonstrated a similar priming effect for stimulation with phorbol myristate acetate and opsonized zymosan as was observed in the postischemic liver. Complement-depleted animals and animals pretreated with the soluble human complement receptor type 1 (BRL 55730; 22.5 mg/kg) accumulated significantly less PMNs in the postischemic livers during longer reperfusion periods (24 h) and sustained significantly less injury. It is concluded that complement is involved in the induction of a KC-induced oxidant stress, the priming of KC and PMNs for enhanced reactive oxygen generation, and the continuous accumulation of PMNs in the liver during reperfusion.

Density changes in leukocytes following hemodialysis or exposure to chemotactic factors.

Analysis of standard Ficoll-Hypaque separation profiles of peripheral WBC from patients undergoing hemodialysis (HD) demonstrated that dialysis caused a marked alteration in the number of cells found at both the interface between the Ficoll-Hypaque and plasma which normally contains mononuclear cells and the cell pellet which normally contains granulocytes. By 30 min into dialysis, there was a 175% increase in white blood cells in the mononuclear band with a corresponding decrease in the number of cells obtained from the cell pellet. When peripheral blood samples from normal donors were pumped through various types of hemodialyzers, a shift in the cell separation profiles similar to that of patients undergoing HD was observed. Differential analysis of the cells obtained from both the interface between the Ficoll-Hypaque and plasma and the cell pellet showed that by 30 min into dialysis, the 'mononuclear' band contained 40-50% polymorphonuclear neutrophils (PMN). To ascertain whether the cell separation changes were possibly due to C5a generation resulting from complement activation by the HD membrane, whole blood was incubated with the purified chemotactic factors C5a, C5ades arg, and formyl-methionyl-leucyl-phenylalanine. This resulted in similar alterations in PMN densities. This study demonstrates that both in vivo and in vitro exposure of human peripheral blood to HD membranes as well as the chemotactic factors C5a, C5ades arg, and formyl-methionyl-leucyl-phenylalanine results in density changes in PMN.(ABSTRACT TRUNCATED AT 250 WORDS)