Polymorphonuclear Cell Chemotaxis and Suicidal NETosis: Simultaneous Observation Using fMLP, PMA, H7, and Live Cell Imaging

Chemotaxis and the formation of suicidal neutrophil extracellular traps (suicidal NETosis) are key functions of polymorphonuclear cells (PMNs). Neutrophil extracellular traps in particular are known to be significantly involved in the severity of inflammatory and immunological disorders such as rheumatoid arthritis and Crohn’s disease. Therefore, detailed knowledge of PMNs is essential for analyzing the mechanisms involved in, and developing new therapies for, such diseases. To date, no standard method to analyze these cell activities has been established. This study used in vitro live cell imaging to simultaneously observe and analyze PMN functions. To demonstrate this, the effects of phorbol-12-myristat-13-acetat (PMA, 0.1-10 nM), N-formylmethionine-leucyl-phenylalanine (fMLP, 10 nM), and protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) on PMN chemotaxis and suicidal NETosis were studied. PMA (1 nM-10 nM) resulted in significant concentration-dependent behavior in chemotaxis and an earlier onset of maximum oxidative burst and NET formation of up to 44%. When adding H7, PMA-triggered PMN functions were reduced, demonstrating that all three functions rely mostly on protein kinase C (PKC) activity, while PKC is not essential for fMLP-induced PMN activity. Thus, the method here described can be used to objectively quantify PMN functions and, especially through the regulation of the PKC pathway, could be useful in further clinical studies of immunological disorders.

Human neutrophils express low levels of FcγRIIIA, which plays a role in PMN activation

We have identified a rare healthy FcγRIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fcγ receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from FcγRIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of FcγRIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from FcγRIIIB WT donors and the null individual express low levels of FcγRIIIA on their surfaces. FcγRIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab′)2 antibodies alone were able to activate highly purified PMNs from the FcγRIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that FcγRIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by FcγRIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by FcγRIIIB in WT PMNs and by FcγRIIA in null PMNs. We conclude that FcγRIIIA is an important player in PMN functions, whereas FcγRIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.

In Vivo Effects of Neostigmine and Physostigmine on Neutrophil Functions and Evaluation of Acetylcholinesterase and Butyrylcholinesterase as Inflammatory Markers during Experimental Sepsis in Rats

Introduction. Recent studies have shown that acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) may serve as important diagnostic and therapeutic targets in sepsis. Since polymorphonuclear neutrophils (PMNs) play a pivotal role in the early phase of sepsis, we evaluated the potential therapeutic effects of cholinesterase inhibitors on PMN functions during cecal ligation and puncture- (CLP-) induced sepsis and investigated the roles of AChE and BChE as inflammatory markers under standardized experimental conditions.Methods. Sham surgery or CLP was performed in male Wistar rats (n=60). Animals were randomized into four groups: physostigmine, 100 μg/kg; neostigmine, 75 μg/kg; 0.9% saline (control group); and sham group, each applied four times over 24 h. The levels of reactive oxygen species (ROS) production and CD11b/CD62l expression were quantified by flow cytometry att=0, 6, 15, 20, and 24 h. Blood gas analysis as well as AChE and BChE activity levels was measured by validated point-of-care measurements. Clinical scores and survival times were determined.Results. CLP induced a significant increase in ROS production and CD11b upregulation by rat PMNs. Treatment with physostigmine or neostigmine significantly reduced ROS production and CD11b upregulation by PMNs 20 h after CLP induction. In physostigmine-treated animals, survival times were significantly improved compared to the control animals, but not in neostigmine-treated animals. While AChE activity significantly decreased in the control animals att>6 h, AChE activity did not change in the sham group. BChE activity decreased att>20 hin the control animals.Conclusion. While AChE activity may serve as an acute inflammatory marker, BChE activity shows a delayed decrease. Administration of centrally acting physostigmine in CLP-induced sepsis in rats has protective effects on PMN functions and improves survival times, which may be of interest in clinical practice.

Expression and Role of the Calcium-Sensing Receptor in Rat Peripheral Blood Polymorphonuclear Neutrophils

The calcium-sensing receptors (CaSRs) play an important role in many tissues and organs that are involved in inflammatory reactions. Peripheral blood polymorphonuclear neutrophils (PMNs) are important inflammatory cells. However, the expression and functions of CaSR in peripheral blood PMNs are still not reported. In this study, we collected rat peripheral blood PMNs to observe the relationship between CaSR and PMNs. From the results, we found first that the CaSR protein was expressed in PMNs, and it increased after PMNs were activated with fMLP. In addition, CaSR activator cincalcet promoted the expression of CaSR and P-p65 (NF-κB signaling pathway protein) and Bcl-xl (antiapoptosis protein), and it increased the secretion of interleukin-6 (IL-6) and myeloperoxidase (MPO); meanwhile, it decreased proapoptosis protein Bax expression and the production of IL-10 and reactive oxygen species (ROS). At the same time, cincalcet also decreased the PMN apoptosis rate analyzed by flow cytometry. However, CaSR inhibitor NPS-2143 and NF-κB signaling pathway inhibitor PDTC reverse the results cited earlier. All of these results indicated that CaSR can regulate PMN functions and status to play a role in inflammation, which is probably through the NF-κB signaling pathway.

Idelalisib Impairs TREM-1 and TLR Mediated Neutrophil Activation

Introduction: Polymorphonuclear neutrophils (PMN) play an essential role in innate inflammatory processes. Their functions are strictly regulated and many activating / inhibiting mechanisms along with their pathways are only incompletely understood. Besides toll-like receptors (TLR) and NOD-like receptors (NLR), triggering receptor expressed on myeloid cells (TREM)-1 is implicated in innate immune activation of these cells and plays a role in infectious as well as non-infectious conditions. Activation of TREM-1 results in release of pro-inflammatory chemokines and cytokines, increased surface expression of cell activation markers and degranulation. In TREM-1 downstream pathways and regulation of TREM-1 expression protein kinase A (PKA), phosphatidylinositide 3-kinase (PI3K), mitogen-activated protein kinases (p38 MAPK) and phospholipase C (PLC) are involved. During the last years, pharmacological development of small-molecule inhibitors for target therapy of hematological and oncological disease led to approval of different new drugs. Idelalisib, which is approved for B-cell malignancies functions as a selective PI3Kδ inhibitor and current data suggest that it influences innate immune defense. This may create a new high-risk group of patients vulnerable for severe infections when monitoring only for quantitative counts (as in neutropenia) but not for qualitative PMN functions. Methods: Idelalisib (Selleckchem) as a selective PI3Kδ inhibitor was used to get deeper insights in the involvement of PI3K in TREM-1 signaling in vitro. Therefore PMN from healthy humans were isolated by dextran sedimentation and Histopaque centrifugation and pretreated with idelalisib. Cells were activated with anti-TREM-1 agonistic antibody (6B1), isotope antibody (4C9) (both own production), lipopolysaccharide (LPS) or zymosan and effector functions like phagocytosis, L-selectin shedding, degranulation and oxidative burst were carried out by flow cytometry and kinetics in a fluorescence reader respectively. Interleukin 8 secretion was detected by ELISA (R&D), for apoptosis and viability Nicoletti staining and MTT assays were performed. Signal transduction pathways were evaluated by Western blot analysis of phosphorylated and non-phosphorylated molecules. For in vivo studies similarly, blood samples of patients receiving idelalisib treatment were followed up for PMN functions as described above to investigate immunomudulatory effects of this selective PI3Kδ inhibitor. Results: PMN of healthy human donors revealed functional deficiencies after pretreatment with idelalisib. We observed highly impaired oxidative burst, impaired phagocytosis, degranulation (reduced CD66b and CD11b surface expression) and L-selectin shedding (elevated CD62L surface expression) after cell activation by TREM-1 ligation as well as after activation with LPS. Cells liberated reduced amounts of IL-8, while apoptosis and viability remained nearly unchanged. Furthermore, we evaluated essential signaling molecules of the TREM-1 and TLR cascade in idelalisib-treated PMN by Western blot analysis. In vivo studies aimed to elucidate if this plays a relevant clinical role in immune responses of patients receiving idelalisib treatment by following up data and blood samples of patients receiving this therapy. Similarly, we observed impaired PMN functions, although the cells were preactivated to a much higher extend. Conclusion: So far these results provide a deeper understanding of innate immune response concerning TREM-1 signaling in PMN. By connecting the experimental data of functional impaired PMN after idelalisib pretreatment to the clinical aspects of patients receiving therapy with this small-molecule inhibitor we aim to highlight the clinical relevance of impaired PMN functions caused by new drugs, resulting in functional deficiencies but normal cell counts. In the future this may contribute to improved medical care for this patients by probably providing them with extended (prophylactic) anti-infective therapies. Disclosures Hess: Celgene: Honoraria; Pfizer: Honoraria; Roche: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Roche, CTI, Pfizer, Celgene: Research Funding.

Regulators and Effectors of Arf GTPases in Neutrophils

Polymorphonuclear neutrophils (PMNs) are key innate immune cells that represent the first line of defence against infection. They are the first leukocytes to migrate from the blood to injured or infected sites. This process involves molecular mechanisms that coordinate cell polarization, delivery of receptors, and activation of integrins at the leading edge of migrating PMNs. These phagocytes actively engulf microorganisms or form neutrophil extracellular traps (NETs) to trap and kill pathogens with bactericidal compounds. Association of the NADPH oxidase complex at the phagosomal membrane for production of reactive oxygen species (ROS) and delivery of proteolytic enzymes into the phagosome initiate pathogen killing and removal. G protein-dependent signalling pathways tightly control PMN functions. In this review, we will focus on the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) as components of signalling cascades regulating PMN responses. GEFs and GAPs are multidomain proteins that control cellular events in time and space through interaction with other proteins and lipids inside the cells. The number of Arf GAPs identified in PMNs is expanding, and dissecting their functions will provide important insights into the role of these proteins in PMN physiology.

Effect of antibiotics on polymorphonuclear leukocyte functions and myeloperoxidase activity, glutathione and malondialdehyde levels in allergic asthma

We investigated the effect of ciprofloxacin, rifampicine and doxycycline on myeloperoxidase (MPO) activity, glutathione (GSH) and malondialdehyde (MDA) levels in allergic asthma patients and healthy volunteers. Polymorphonuclear leukocytes (PMNs) were isolated with ficoll-hypaque gradient centrifugation method. MPO activity was assayed with modified o-dianisidine, GSH by Ellman's and MDA levels by Beuge's method. PMN functions and MDA levels of patients significantly decreased when compared with healthy volunteers. Ciprofloxacin significantly increased PMN functions, MPO activity and MDA levels of both groups. We have demonstrated that ciprofloxacin has beneficial effects on MPO activity and PMN functions in allergic asthma patients and healthy volunteers.

Different Modulating Effects of Adenosine on Neonatal and Adult Polymorphonuclear Leukocytes

Polymorphonuclear leukocytes (PMNs) are the major leukocytes in the circulation and play an important role in host defense. Intact PMN functions include adhesion, migration, phagocytosis, and reactive oxygen species (ROS) release. It has been known for a long time that adenosine can function as a modulator of adult PMN functions. Neonatal plasma has a higher adenosine level than that of adults; however, little is known about the modulating effects of adenosine on neonatal PMNs. The aim of this study was to investigate the effects of adenosine on neonatal PMN functions. We found that neonatal PMNs had impaired adhesion, chemotaxis, and ROS production abilities, but not phagocytosis compared to adult PMNs. As with adult PMNs, adenosine could suppress the CD11b expressions of neonatal PMNs, but had no significant suppressive effect on phagocytosis. In contrast to adult PMNs, adenosine did not significantly suppress chemotaxis and ROS production of neonatal PMNs. This may be due to impaired phagocyte reactions and a poor neonatal PMN response to adenosine. Adenosine may not be a good strategy for the treatment of neonatal sepsis because of impaired phagocyte reactions and poor response.

Neisseria gonorrhoeae-Mediated Inhibition of Apoptotic Signalling in Polymorphonuclear Leukocytes

ABSTRACTThe human pathogenNeisseria gonorrhoeaerecruits and interacts extensively with polymorphonuclear leukocytes (PMNs) during infection.N. gonorrhoeaeis able to survive the bactericidal activity of these innate immune cells and can actively modulate PMN functionsin vitro. PMNs are short-lived cells which readily undergo apoptosis, and thus the effect ofN. gonorrhoeaeinfection on PMN survival has implications for whether PMNs might serve as an important site of bacterial replication during infection. We developed and validated an HL-60 myeloid leukemia cell culture model for PMN infection and used both these cells and primary PMNs to show thatN. gonorrhoeaeinfection alone does not induce apoptosis and furthermore thatN. gonorrhoeaecan inhibit both spontaneous apoptosis and apoptosis induced by the intrinsic and extrinsic apoptosis inducers staurosporine (STS) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), respectively.N. gonorrhoeaeinfection also results in the activation of NF-κB signaling in neutrophils and induces secretion of an identical profile of proinflammatory cytokines and chemokines in both HL-60 cells and primary PMNs. Our data show that the HL-60 cell line can be used to effectively modelN. gonorrhoeae-PMN interactions and thatN. gonorrhoeaeactively inhibits apoptosis induced by multiple stimuli to prolong PMN survival and potentially facilitate bacterial survival, replication, and transmission.