Stimulation Of Polymorphonuclear Neutrophils Research Articles

Neutrophils recognize and amplify IFNT signals derived from day 7 bovine embryo for stimulation of ISGs expression in vitro: A possible implication for the early maternal recognition of pregnancy

Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs’ immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs’ immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle.

TBK1/IKKε Negatively Regulate LPS-Induced Neutrophil Necroptosis and Lung Inflammation.

Cell necroptosis, a form of regulated inflammatory cell death, is one of the mechanisms that controls cell release of inflammatory mediators from innate immune cells, such as polymorphonuclear neutrophils (PMNs), and critically regulates the progress of inflammation. Cell necroptosis features receptor-interacting protein (RIPK) 1 activation and necroptosome formation. This leads to loss of plasma membrane integrity, the release of cell contents into the extracellular space, and subsequent increased inflammation. Here, we report an intra-PMN mechanism of negative regulation of necroptosis mediated through TBK1/IKKε. Using an in vivo mouse model of intratracheal injection (i.t.) of LPS and in vitro LPS stimulation of mouse PMN, we found that LPS-TLR4 signaling in PMNs activates and phosphorylates TBK1 and IKKε, which in turn suppress LPS-induced formation of the RIPK1-RIPK3-MLKL (necrosome) complex. TBK1 dysfunction by knockdown or inhibitor significantly increases the phosphorylation of RIPK1 (∼67%), RIPK3 (∼68%), and MLKL (∼50%) and promotes RIPK1-RIPK3 and RIPK3-MLKL interactions and increases PMN necroptosis (∼83%) in response to LPS, with subsequent augmented lung inflammation. These findings suggest that the LPS-TLR4-TBK1 axis serves as a negative regulator for PMN necroptosis and might be a therapeutic target for modulating PMN death and inflammation.

Direct Visualization of Circulating Neutrophil Extracellular Traps Using Cell Fluorescence during Human Septic Shock-Induced Disseminated Intravascular Coagulation

Septic shock is the most severe form of infection, defined as a subset of sepsis in which circulatory, cellular and metabolic abnormalities are profound and responsible for multiple organ failure and a high mortality-rate. Septic shock is characterized by a broad coagulation activation that can lead to uncontrolled thrombin and fibrin generation, which may evolve to disseminated intravascular coagulation (DIC). DIC increases the risk of death, thus representing a therapeutic target of interest. However, the pathophysiological mechanisms of DIC are not fully understood. Polymorphonuclear neutrophils (PMNs) have recently been identified as potential players of the response to infection by releasing their content, including DNA, histones and granules enzymes. These structures, called neutrophils extracellular traps (NETs), form a large net-like structure in which pathogens get trapped. NETs capture invading pathogens, but also represent a pro-coagulant surface at the interface between immunity and thrombosis. During septic shock-induced DIC, neutrophil activation may result in excessive NET formation. In this study, we originally report the presence of circulating NETs in human blood during septic shock-induced DIC using a simple and robust method. Blood samples were obtained from healthy human volunteers (n=3), patients with septic shock without DIC (n=3) and with DIC (n=3). PMNs were immediately purified immediately after sampling using negative immune-magnetic sorting method. 5x104 purified PMNs were subsequently spotted on microscope slides using cytocentrifugation at 35g for 5 minutes, thus preserving cell integrity and morphology. Positive controls for NET formation were obtained using PMNs isolated from healthy volunteers that were stimulated in-vitro with 4 μM ionomycin. PMNs were stained with mouse anti-human FITC anti-myeloperoxidase (MPO) antibody and the blue-fluorescent DAPI nucleic acid stain. NETs were identified as elongated extracellular DAPI stained DNA fibers associated to MPO detected by immunofluorescence microscopy. The degree of NETs formation was blindly quantified into 5 categories according to the proportion of neutrophils forming NETs per microscopic field: absence (0% of neutrophils forming NETs), scarce (1-25%), moderate (26% to 50%), high (50% to 75%) or very high degree of NETS formation (76% to 100%). PMNs from healthy donors stimulated by ionomycin released NETs, evidenced as double-stained DAPI-positive chromatin structures decorated with MPO in the extracellular space using fluorescence microscopy. NET structures similar to those observed after ionomycin stimulation were unambiguously visualized in PMNs from septic shock patients with DIC, but neither in PMNs from septic shock patients without DIC, nor in unstimulated PMNs from healthy donors. DIC patients were stratified into "high" or "very high" degree of NETs formation groups (>75 % of PMNs displaying DNA release), not different from ionomycin-stimulated PMNs (50 to 75%). Conversely, PMNs from patients devoid of DIC as well as unstimulated normal PMNs from healthy volunteers were stratified into "scarce" or "absence" of PMNs forming NET categories. Blind review of May-Giemsa-Grunwald (MGG) stained slides showed the correlation of the detection of NETs with nuclear decondensation and vacuolation of the cytoplasm of PMNs, features that are associated with neutrophil activation. Accordingly, neutrophils' side-fluorescence (NEUT-SFL), a Sysmex™ CBC analyzers parameter previously associated to NETosis (Stiel et al., 2016, Delabranche et al., 2017) was increased during septic shock induced DIC as well as following in-vitro ionomycin stimulation of normal PMNs. In this study, we showed for the first time direct evidence of circulating NETs in peripheral blood of patients with septic shock-induced DIC using immunofluorescence. These NETs are undistinguishable from NETs features observed following in-vitro ionomycin stimulation of normal PMNs. NETs images were furthermore associated with modifications of PMN morphology that could be detected using MGG staining. This observation may be of great interest in order to stratify patients eligible for future treatment protocols with molecules targeting NETs. Figure Disclosures Meziani: STAGO: Research Funding.

Sevoflurane modulates the release of reactive oxygen species, myeloperoxidase, and elastase in human whole blood: Effects of different stimuli on neutrophil response to volatile anesthetic in vitro.

Volatile anesthetics have been shown to modulate polymorphonuclear neutrophil (PMN) functions. The aim of this study was to examine the impact of clinically relevant concentrations of sevoflurane (SEVO), a volatile anesthetic, on the release of reactive oxygen species (ROS), myeloperoxidase (MPO), and elastase (EL) from human activated PMNs. For this purpose, samples of whole blood were collected from healthy volunteers and exposed in vitro to 2.3% or 4.6% SEVO in air. To assess for a stimulus-dependent effect of the volatile anesthetic, PMNs were activated using different validated protocols. Artificial stimulation of neutrophils involved either a combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). In addition, a combination of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) was also tested as a natural activation mean of PMNs. The production of ROS by PMNs was assessed by L-012 chemiluminescence. Total MPO and EL released in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, degranulation of the active fraction of MPO was also measured by specific immunological extraction followed by enzymatic detection (SIEFED). Overall, SEVO enhanced the release of ROS, MPO, and EL following artificial stimulation of PMNs but the volatile anesthetic inhibited the degranulation of active MPO and EL after neutrophil exposure to LPS and TNF-α. This study highlighted that the effect of SEVO on activated PMNs is dependent on the conditions of cell stimulation. These properties should be taken into consideration in future studies investigating immunomodulatory effects of volatile anesthetics.

IFNγ effect in the induction of the Neutrophil Extracellular Traps (NETs) in Chronic Granulomatous Disease patients

IFNγ effect in the induction of the Neutrophil Extracellular Traps (NETs) in Chronic Granulomatous Disease patients

NDS27 combines the effect of curcumin lysinate and hydroxypropyl-β-cyclodextrin to inhibit equine PKCδ and NADPH oxidase involved in the oxidative burst of neutrophils

Polymorphonuclear neutrophils (PMNs) are involved in host defence against infections by the production of reactive oxygen species (ROS), but excessive PMN stimulation is associated with the development of inflammatory diseases. After appropriate stimuli, protein kinase C (PKC) triggers the assembly of NADPH oxidase (Nox2) which produces superoxide anion (O2−), from which ROS derive. The therapeutic use of polyphenols is proposed to lower ROS production by limiting Nox2 and PKC activities. The purpose of this study was to compare the antioxidant effect of NDS27 and NDS28, two water-soluble forms of curcumin lysinate respectively complexed with hydroxypropyl-β-cyclodextrin (HPβCD) and γ-cyclodextrin (γ-CD), on the activity of Nox2 and PKCδ, involved in the Nox2 activation pathway. Our results, showed that NDS27 is the best inhibitor for Nox2 and PKCδ. This was illustrated by the combined effect of HPβCD and curcumin lysinate: HPβCD, but not γ-CD, improved the release of curcumin lysinate and its exchange against lipid or cholesterol as demonstrated by the lipid colouration with Oil Red O, the extraction of radical lipophilic probes recorded by ESR and the HPLC measurements of curcumin. HPβCD not only solubilised and transported curcumin, but also indirectly enhanced its action on both PKC and Nox2 activities. The modulatory effect of NDS27 on the Nox2 activation pathway of neutrophils may open therapeutic perspectives for the control of pathologies with excessive inflammatory reactions.

Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro

BackgroundBacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection.MethodsUsing an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot.ResultsPMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection.ConclusionsOur findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process.

An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-α in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-α, LPS and LPS/TNF-α, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-α significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity.

Human polymorphonuclear neutrophils express RANK and are activated by its ligand, RANKL

The receptor activator of NF-κB (RANK) is especially well studied in the context of bone remodeling, and RANK and its ligand, RANKL, are key molecules in the induction of bone resorbing osteoclasts. We now report that polymorphonuclear neutrophils (PMNs) contain preformed RANK, stored in secretory vesicles and in specific granules. Upon stimulation of PMNs in vitro, RANK was translocated to the cell membrane. In patients with persistent bacterial infections, RANK surface expression was enhanced compared with that of healthy individuals. The functional activity of RANK was assessed by determining migration of PMNs toward RANKL. A time- and dose-dependent migration was seen, leading to the conclusion that RANK on PMNs is functional. We presume that regulated RANK expression contributes to the fine tuning of PMN migration, for example, on and through inflamed endothelium that is known to express RANKL.

STIMULATION OF INTERLEUKIN-8 SYNTHESIS IN MONOCYTE CULTURE UNDER SOLUBLE INTERLEUKIN-8 CXCR2 RECEPTOR

Interleukin-8 (IL-8) is the main chemotactical factor for polymorphonuclear neutrophils (PMN) in inflammation. We have already shown that PMN stimulation in vitro causes a sharp decrease of IL-8 receptor expression of second type CXCR2 on the PMN membrane and an emergence of soluble glykopeptid CXCR2 (рCXCR2) in the supernatant. In this work we have studied the effect of рCXCR2 on IL-8 synthesis in the monocyte culture of the blood and bronchoepitheial human cells A549. It was shown that рCXCR2 reliably stimulated IL-8 synthesis in the monocyte culture with the evident doze-dependent effect. рCXCR2 did not cause IL-8 synthesis in the culture of the bronchoepitheial human cells A549. The influence of рCXCR2 on the monocytes was not related to the lypopolysaccharide contamination of the preparation and was caused by the protein and carbohydrade parts, included in рCXCR2 molecule.

A3 Adenosine Receptor Inhibition Improves the Efficacy of Hypertonic Saline Resuscitation

We reported previously that hypertonic saline (HS) treatment can prevent or upregulate the function of polymorphonuclear neutrophils (PMNs) via A2a-type adenosine receptors or A3-type adenosine receptors (A3R), respectively. A3R translocate to the cell surface upon PMN stimulation, and thus, HS promotes PMN responses under conditions of delayed HS treatment. Here we investigated if inhibition of A3R improves the protective effects of HS resuscitation in a mouse sepsis model. We found that HS nearly triples extracellular adenosine concentrations in whole blood and that inhibition of A3R with the selective antagonist MRS-1191 dose dependently improves the inhibitory effect of HS. MRS-1191 at a concentration of 1 nM enhanced the inhibitory effect of HS and reduced stimulatory effects of delayed HS treatment. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found that MRS-1191 reduces acute lung injury and PMN accumulation in lung tissue. Whereas delayed HS treatment (4 mL/kg of 7.5% NaCl) of mice 1 h after CLP aggravated PMN accumulation, lung tissue damage, and mortality 24 h after CLP, infusion of MRS-1191 (2 ng/kg body weight) combined with HS reduced these detrimental effects of delayed HS treatment. Our data thus show that A3 receptor antagonists can strengthen the beneficial effects of HS resuscitation by avoiding stimulatory adverse effects that result from delayed HS administration.

Neutrophils Activate Alveolar Macrophages by Producing Caspase-6–Mediated Cleavage of IL-1 Receptor-Associated Kinase-M

Alveolar macrophages (AMs) are exposed to respirable microbial particles. Similar to phagocytes in the gastrointestinal tract, AMs can suppress inflammation after exposure to nonpathogenic organisms. IL-1R-associated kinase-M (IRAK-M) is one inhibitor of innate immunity, normally suppressing pulmonary inflammation. During pneumonia, polymorphonuclear neutrophils (PMNs) are recruited by chemotactic factors released by AMs to produce an intense inflammation. We report that intact IRAK-M is strongly expressed in resting human AMs but is cleaved in patients with pneumonia via PMN-mediated induction of caspase-6 (CASP-6) activity. PMN contact is necessary and PMN membranes are sufficient for CASP-6 induction in macrophages. PMNs fail to induce TNF-α fully in macrophages expressing CASP-6 cleavage-resistant IRAK-M. Without CASP-6 expression, PMN stimulation fails to cleave IRAK-M, degrade IκBα, or induce TNF-α. CASP-6(-/-) mice subjected to cecal ligation and puncture have impaired TNF-α production in the lung and decreased mortality. LPS did not induce or require CASP-6 activity demonstrating that TLR2/4 signaling is independent from the CASP-6 regulated pathway. These data define a central role for CASP-6 in PMN-driven macrophage activation and identify IRAK-M as an important target for CASP-6. PMNs de-repress AMs via CASP-6-mediated IRAK-M cleavage. This regulatory system will blunt lung inflammation unless PMNs infiltrate the alveolar spaces.

Comparing effects of interleukin-2 and interleukin-4 on the expression of MHC class II, CD80 and CD86 on polymorphonuclear neutrophils

Polymorphonuclear neutrophils (PMN) have contributed only to the innate immune response, due to their phagocytosis activity. They have a short life time. Some studies of PMN cytokine production and expression of numerous cell surface proteins have suggested that PMN are likely to influence adaptive responses and may satisfy the criteria of antigen presenting cells. Flow cytometry was used in the present study for the detection of cell surface major histocompatibility complex (MHC) class II, CD80 and CD86 required for antigen presentation and subsequent T-cell activation. Human peripheral blood neutrophils were used for this purpose. In vitro stimulation of PMN with IL-2 or IL-4 for 24 h showed expression of MHC class II, CD80 and CD86. These observations, therefore, provide support to the hypothesis that human PMN have the potential to express molecules required for antigen presentation.

MT6-MMP is present in lipid rafts and faces inward in living human PMNs but translocates to the cell surface during neutrophil apoptosis

Polymorphonuclear neutrophils (PMNs) are the first line of defense against invading organisms in humans; in addition, PMNs contribute to the linking of innate and adaptive immunity. To fulfill their biological behavior, PMNs utilize an arsenal of proteolytic enzymes, including members of the matrix metalloproteinase family of zinc-dependent endopeptidases. PMNs express high levels of MT6-MMP (MMP-25), a glycosyl-phosphatidylinositol-anchored MMP, that belongs to the subfamily of membrane-anchored matrix metalloproteinases. Due to the paucity of information on MT6-MMP in primary cells, we set to investigate the localization and potential function of MT6-MMP in human PMNs. We found that MT6-MMP is present in the membrane, granules and nuclear/endoplasmic reticulum/Golgi fractions of PMNs where it is displayed as a disulfide-linked homodimer of 120 kDa. Stimulation of PMNs resulted in secretion of active MT6-MMP into the supernatants. Membrane-bound MT6-MMP, conversely, is located in the lipid rafts of resting PMNs and stimulation does not alter this location. In addition, TIMP-2, a natural inhibitor of MT6-MMP, does not co-localize with it in the lipid rafts. Interestingly, living PMNs do not display MT6-MMP on the cell surface. However, induction of apoptosis induces MT6-MMP relocation on PMNs' cell surface. Our studies suggest that metalloproteinases may play a role in respiratory burst and IL-8 secretion, but not chemotaxis or granulocyte macrophage colony-stimulating factor-induced survival. Collectively, these results provide new insights on the role of MT6-MMP in the physiology of human PMNs.

IL-4Rα, a New Member that Associates with Syk Kinase: Implication in IL-4-Induced Human Neutrophil Functions

Although Syk has been reported to be associated with IL-2R beta [corrected] and IL-15R alpha in some hematopoietic cells, its association has never been investigated in the IL-4/IL-4R system. In this study, we demonstrate for the first time that Syk is constitutively associated with IL-4R(alpha)in human polymorphonuclear neutrophils (PMNs) and that IL-4 stimulation increases the amount of Syk associated with IL-4R(alpha). Moreover, upon IL-4 treatment, a pool of Syk associated with IL-4R(alpha) is phosphorylated. We also report that such association is not unique to PMNs because Syk associates with IL-4R(alpha) in Raji and in PBMC cells. Stimulation of PMNs by IL-4 increased the amount of Syk associated with PLC-gamma2, pAkt, and alpha-tubulin. Pretreatment of cells with the Syk-selective inhibitor piceatannol or Syk inhibitor II, significantly inhibited the ability of IL-4 to enhance phagocytosis and cell adhesion and to delay apoptosis, and these results correlate with the ability of piceatannol to reduce Syk activation and its association with IL-4R(alpha). Down-regulation of Syk by antisense techniques demonstrates the importance of Syk in the antiapoptotic effect of IL-4. We conclude that association of Syk to IL-4R(alpha) is of biological significance and that IL-4R(alpha) is a new candidate to be added to the few cytokine receptor components which associate with Syk.

Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo.

Apart from the diagnostic value of anti-neutrophil cytoplasmic antibodies (ANCA), their detailed characterization and that of their corresponding antigens have opened new ways for the exploration of the pathogenesis of primary systemic vasculitis. ANCA are now thought to play an important functional role via activation of phagocytic cells (e.g. polymorphonuclear neutrophils (PMN)). In this study we examined the mechanisms by which ANCA could gain access to proteinase 3 (PR3) in intact PMN, at two levels: ex vivo by analysing the presence of PR3 on the plasma membrane of PMN from patients with ANCA-associated vasculitis, and in vitro by stimulation of PMN using different cytokines, including recombinant tumour necrosis factor-alpha (rhTNF-alpha) and two forms of IL-8 (produced by monocytic and endothelial cells). Using immunocytochemical staining techniques (FACS and immunoelectronmicroscopy) PR3 has been detected on the plasma membrane of PMN from patients with active ANCA-associated vasculitis. However, this phenomenon is also seen in patients with sepsis who do not have ANCA. In addition, TNF-alpha and both forms of IL-8 act synergistically and induce a translocation of PR3 from the intragranular loci to the cell surface of PMN. These results provide strong evidence for the hypothesis that ANCA are directly pathogenic by binding to PR3 which is expressed on the cell surface of primed/activated PMN.

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.

Effects of a Hyaluronate-Carboxymethylcellulose Membrane (Seprafilm) on Human Polymorphonuclear Neutrophil Functions

Seprafilm (Genzyme Corp., Cambridge, MA) is safe and effective for reducing adhesion formation in abdominal and pelvic surgery. However, the relationships between Seprafilm and postoperative complications, including intra-abdominal abscess formation and intra-abdominal inflammation, remain controversial. Polymorphonuclear neutrophils (PMNs) play crucial roles in the processes of intra-abdominal inflammation, infection, and abscess formation, and PMNs act as the first line in nonspecific host defense against a wide range of pathogens. The present study evaluates the effect of Seprafilm itself on human PMNs in experimental models in vitro. Human PMNs were isolated from blood samples obtained from 14 healthy volunteers. The functions of the isolated cells were assessed by examining their phagocytosis, apoptosis/necrosis rates, cytokine production, and PMN-elastase release with and without Seprafilm in an in vitro experimental model of inflammation and endotoxin-stimulation. There were no significant differences in the PMN phagocytosis, apoptosis/necrosis rates, cytokine production, or PMN-elastase release into the supernatant after coculture with bacteria between assays with and without Seprafilm. In the PMN stimulation models involving tumor necrosis factor-alpha and lipopolysaccharide, there were no significant differences in cytokine production between assays with and without Seprafilm. Furthermore, a similar experiment measuring cytokine production in the presence of well-dissolved Seprafilm produced corresponding results. Our results suggest Seprafilm itself does not affect the functions of isolated human PMNs in in vitro experimental models of inflammation and endotoxin-stimulation.

Anti-myeloperoxidase antibodies enhance phagocytosis, IL-8 production, and glucose uptake of polymorphonuclear neutrophils rather than anti-proteinase 3 antibodies leading to activation-induced cell death of the neutrophils

Anti-neutrophil cytoplasmic antibodies (ANCA) not only are triggered by target protein myeloperoxidase (MPO) and proteinase 3 (PR3) of polymorphonuclear neutrophil (PMN) but also react with primed PMN to exert the inflammatory process in vasculitis syndrome. To clarify the crucial role of PMN in ANCA-associated vasculitis and the related mechanism, PMN was cultured with monoclonal antibody MPO-ANCA and PR3-ANCA to determine the function of phagocytosis, Interleukin- 8 (IL-8) production, glucose uptake, and TNF-related apoptosis induced ligand (TRAIL) production. The spontaneous membrane expression of MPO and PR3 on PMN could be significantly increased by lipopolysaccharide (LPS) and TNF-alpha, but not by IL-8 or GRO-alpha. The PMN-stimulating activity of ANCA was demonstrated by enhancing phagocytosis, IL-8 production, and glucose uptake that was more prominent by MPO-ANCA. The PMN stimulation by ANCA was not through protein kinase, H2O2, or superoxide anion radicals as their inhibitors exerted no effect on ANCA-mediated activation. On the other hand, ANCA also accelerated PMN apoptosis and increased TRAIL production. These results demonstrate that activation-induced cell death (AICD) mechanism could be initiated in PMN with existence of ANCA. In conclusion, MPO-ANCA is more potent in stimulating PMN than PR3-ANCA. ANCA-activated PMN is not only responsible for the amplified inflammatory process in blood vessel but also initiates immune circuit via triggered macrophage/monocyte by apoptotic PMN through the mechanism of AICD elicited by ANCA.

IFNα Therapy Up-Regulates Serum Soluble TRAIL Levels and Induces Marked TRAIL mRNA Expressions in Polymorphonuclear Neutrophils in Chronic Myeloid Leukemia Patients.

[Purpose] Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is an interferon α (IFN)-induced, apoptosis-inducing molecule. TRAIL is one of the promising reagents for therapeutic use in combination with imatinib in chronic myeloid leukemia (CML). A previous report showed that IFN-induced TRAIL is only weakly expressed on cell surface but is mainly secreted as a soluble form (sTRAIL) by polymorphonuclear neutrophils (PMNs) (and also by monocytes) in healthy individuals. Up to now, however, whether sTRAIL is actually released into serum in CML patients and whether its serum level is up-regulated during in vivo IFN therapy are unsolved issues. To assess the significance of sTRAIL, we examined serum sTRAIL levels by ELISA in total 40 (36 in CP, 4 in BP) CML patients either prior to or undergoing IFN therapy.[Results] Serum sTRAIL were detectable in untreated CML patients (1258±560 pg/ml, n=25), which levels were substantially comparable to those in healthy donors (1159±343, n=10). Serum sTRAIL levels significantly increased in patients during IFN therapy (1801±712, n=18) compared to those in untreated patients (P=0.009). This IFN-induced up-regulation of serum sTRAIL was further confirmed in all seven patients whom we could analyze both before and during the therapies (before 1228±396 vs. during 1956±914, P=0.037). As for initial sTRAIL levels, among the restricted patients group who later showed any cytogenetic response (n=15), there was a tendency that initially higher serum sTRAIL levels accompanied better cytogenetic responses (CCyR: 1873±719, PCyR: 1130±204, minor CyR: 830±267, P=0.03), indicating that the initial sTRAIL level may have some relation with the later response to IFN therapy. Next, to investigate the source of the sTRAIL secretion into serum, we assessed TRAIL mRNA expressions in PMNs in the peripheral blood by RQ-PCR. Again, TRAIL mRNA expressions in patients during IFN therapy (n=22) significantly increased compared to those in untreated patients (n=16) (TRAIL/GAPDH: before 1.4 vs. during 8.0, P=0.005). This IFN-induced up-regulation of TRAIL mRNA expression in PMNs was confirmed in all 16 patients whom we could analyze both before and during IFN therapies (before 3.7 vs. during 21.3, P=0.018). In vitro IFN stimulation of PMNs in five untreated patients showed marked induction (10 to 30 fold) of TRAIL mRNA at 12h, while induction of FAS mRNA stayed rather low (1 to 2 fold). Furthermore, IFN concentration-dependent accumulations of sTRAIL into the culture supernatants were observed for up to 48 h. Next, we assessed TRAIL mRNA expressions in purified CD34-positive cells in the bone marrow. TRAIL mRNA expressions were also detectable, and the mRNA levels significantly increased in patients during IFN therapy (n=37), compared to those in untreated patients (n=17) (P=0.02). This IFN-induced up-regulation of TRAIL mRNA was confirmed in nine patients whom we could analyze both before and during the therapies (P=0.01), indicating that even immature CML elements are responsive to IFN and may contribute to sTRAIL release in the microenvironment.[Conclusion] This is the first evidence showing that IFN, in vivo, provokes the release of sTRAIL when administered systematically in CML patients. The main source of the serum sTRAIL is probably PMNs in CML patients. We suspect that one of the mechanisms of anti-proliferative action of IFN on CML is sTRAIL secreted into serum. Our findings also give rationale for the use of IFN (or recombinant sTRAIL) in combination with imatinib in CML patients.