Stimulation Of Human Neutrophils Research Articles

Testosterone affects type I/type II interferon response of neutrophils during hepatic amebiasis.

Differences in immune response between men and women may influence the outcome of infectious diseases. Intestinal infection with Entamoeba histolytica leads to hepatic amebiasis, which is more common in males. Previously, we reported that innate immune cells contribute to liver damage in males in the murine model for hepatic amebiasis. Here, we focused on the influences of sex and androgens on neutrophils in particular. Infection associated with neutrophil accumulation in the liver was higher in male than in female mice and further increased after testosterone treatment in both sexes. Compared with female neutrophils, male neutrophils exhibit a more immature and less activated status, as evidenced by a lower proinflammatory N1-like phenotype and deconvolution, decreased gene expression of type I and type II interferon stimulated genes (ISGs) as well as downregulation of signaling pathways related to neutrophil activation. Neutrophils from females showed higher protein expression of the type I ISG viperin/RSAD2 during infection, which decreased by testosterone substitution. Moreover, ex vivo stimulation of human neutrophils revealed lower production of RSAD2 in neutrophils from men compared with women. These findings indicate that sex-specific effects on neutrophil physiology associated with maturation and type I IFN responsiveness might be important in the outcome of hepatic amebiasis.

Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps.

Neutrophil granulocytes predominate in the lungs of patients infected with Mycobacterium tuberculosis (Mtb) in earlier stages of the disease. During infection, neutrophils release neutrophil extracellular traps (NETs), an antimicrobial mechanism by which a DNA-backbone spiked with antimicrobial components traps the mycobacteria. However, the specific mycobacterial factors driving NET formation remain unclear. Proteins from the proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family are critical to Mtb pathophysiology and virulence. Here, we investigated NET induction by PE18, PPE26, and PE31 in primary human blood-derived neutrophils. Neutrophils were stimulated with the respective proteins for 3h, and NET formation was subsequently assessed using confocal fluorescence microscopy. Intracellular ROS levels and cell necrosis were estimated by flow cytometry. Additionally, the influence of phorbol-12-myristate-13-acetate (PMA), a known NADPH oxidase enhancer, on NET formation was examined. Neutrophil integrity following incubation with the PE/PPE proteins was evaluated using transmission electron microscopy. For the first time, we report that stimulation of primary human blood-derived neutrophils with Mtb proteins PE18, PPE26, and PE31 resulted in the formation of NETs, which correlated with an increase in intracellular ROS levels. Notably, the presence of PMA further amplified this effect. Following incubation with the PE/PPE proteins, neutrophils were found to remain viable and structurally intact, as verified through transmission electron microscopy, indicating the occurrence of vital NET formation. These findings offer valuable insights that contribute to a better understanding of host-pathogen interactions during Mtb infection. Moreover, they underscore the significance of these particular Mtb antigens in triggering NET formation, representing a distinctive and previously unrecognized function of PE/PPE antigens.

Non-invasive mapping of systemic neutrophil dynamics upon cardiovascular injury

Neutrophils play a complex role during onset of tissue injury and subsequent resolution and healing. To assess neutrophil dynamics upon cardiovascular injury, here we develop a non-invasive, background-free approach for specific mapping of neutrophil dynamics by whole-body magnetic resonance imaging using targeted multimodal fluorine-loaded nanotracers engineered with binding peptides specifically directed against murine or human neutrophils. Intravenous tracer application before injury allowed non-invasive three-dimensional visualization of neutrophils within their different hematopoietic niches over the entire body and subsequent monitoring of their egress into affected tissues. Stimulated murine and human neutrophils exhibited enhanced labeling due to upregulation of their target receptors, which could be exploited as an in vivo readout for their activation state in both sterile and nonsterile cardiovascular inflammation. This non-invasive approach will allow us to identify hidden origins of bacterial or sterile inflammation in patients and also to unravel cardiovascular disease states on the verge of severe aggravation due to enhanced neutrophil infiltration or activation.

In vitro assessment of neutrophil response to proteins and antibodies of SARS-CoV-2

Abstract The SARS-CoV-2 virus caused a worldwide epidemic of respiratory disease (COVID-19). Acute respiratory distress syndrome (ARDS) has been associated with severe COVID-19. Activation of neutrophils have been linked to ARDS in COVID-19. While emerging results indicate that SARS-CoV-2 leads to neutrophil activation, it remains unknown which components of the virus are responsible for this. We assessed in vitro how the spike (S) protein and anti-spike antibodies affect neutrophils. Human neutrophils were isolated from healthy donors. Different concentrations of recombinant S protein, a monoclonal antibody (mAb) produced against the S protein and their immunocomplex were used to stimulate neutrophils. Unstimulated and PMA-treated neutrophils were used as negative and positive controls. Reactive oxygen species (ROS) production was measured by Diogenes chemiluminescence, and cell death was measured by fluorescence. While no significant increase of cell death was observed, neutrophils responded with a dose-dependent increase in the production of ROS when stimulated with either the S protein or the mAb. Neutrophils produced significantly (p<0.05) more ROS when activated by 1,000 ng of the mAb. Immune complex stimulation (1,000 ng each component) of human neutrophils also lead to significantly (p<0.005) enhanced ROS production compared to stimulations by the protein or the mAb alone. Neutrophil cell death was also significantly enhanced when stimulated by the immune complex (500 ng and 1,000 ng each component) (p<0.05, p<0.005, respectively). Our work indicates that the S protein and its immune complex formed with mAb could stimulate neutrophils in vitro and contribute to neutrophil-mediated tissue damage in COVID-19 patients. Supported by grants from: Oswaldo Cruz Foundation, International Graduate Programme (PrInt FIOCRUZ-CAPES), University of Georgia and NIH.

Identification of PDZ‐RhoGEF (PRG) as a Novel Gα i Target

Cell-migration and adhesion are fundamental biological processes regulated by chemokine G-protein-coupled receptors that couple to PTX-sensitive Gi proteins. The role of Gβγ subunits released from Gi in chemokine signaling has been well characterized. In contrast, Gαi has been thought to have no direct signaling function. Our laboratory recently uncovered a role for Gαi-GTP in regulating cell adhesion. However, a direct effector of Gαi responsible was not identified. To discover novel targets of active Gαi, we used BioID2 proximity-based labeling coupled to TMT quantitative proteomic mass spectrometry. We subsequently compared the abundance ratios of proteins in Gαi1-QL-BioID2 (constitutively active) to Gαi1-WT-BioID2 (inactive) samples. BioID2-CaaX was used as a membrane-targeted control. Experiments were done in HT1080 fibrosarcoma cells where we previously demonstrated Gαi regulation of adhesion. Known binding partners for Gαi subunits including, GPCRs, Gβ, Gγ, adenylate cyclase, and Ric8A were identified by mass spectrometry further validating the method. A candidate Gαi effector identified was PDZ-RhoGEF (PRG), a Rho guanine nucleotide exchange factor that regulates cell adhesion in neutrophils. To determine if PRG can be activated by Gαi; we co-transfected PRG with Gαi1 subunit and an SRE luciferase Rho reporter in A293 cells. Gαi1-QL but not Gαi1-WT strongly activated PRG dependent SRE luciferase, which was confirmed in a rhotekin assay for active RhoA. PRG activation was specific to the Gαi subfamily and required the PRG-RGS domain. A proximal interaction was confirmed in aproximity ligation assay in A293 cells transfected with PRG and Gαi1 cDNAs. Purified Cy5-labeled myr-His-Gαi bound specifically to GST-PRG in a TR-FRET assay. This binding was independent of the nucleotide state of Gαi1 but was inhibited by the addition of Gβγ subunits. In neutrophils, activation of the formyl peptide receptor (FPR) by fMLP, and subsequent PRG activation leads to Rho-dependent accumulation of phosphorylated-myosin light chain (P-MLC) at the trailing edge and has been proposed to result from FPR-dependent Gα13 activation. In our preliminary experiments, stimulation of primary human neutrophils with 10 nM fMLP promoted polarized P-MLC accumulation that was inhibited by PTX. This supports a model where at physiological concentrations of chemoattractant, activation of PRG/Rho is through Gαi-GTP rather than Gα13 in primary human neutrophils. Overall, we identified relatively ubiquitously expressed PRG as a novel Gαi-GTP target. Identification of a novel signaling pathway regulated by Gi-coupled GPCRs will have a significant impact on our understanding of the biology regulated by these ubiquitous and pharmacologically important receptors.

Gene Expression in Human Polymorphonuclear Neutrophils (PMNs) Stimulated by Bacillus Calmette-Guérin (BCG).

Neutrophils are the most abundant leukocytes in the blood. Moreover, neutrophils form the first line of host immune defense against bacterial and fungal invasion, and also play an important part in inflammatory and immune system responses. Intravesical bacillus Calmette-Guérin (BCG) has been shown to reduce and delay tumor progression to muscle-invasive disease after transurethral resection of bladder tumors (TRUBTs). Following intravesical BCG, neutrophils gather around tissues infected by BCG in the early stage of inflammatory and immune responses. In our previous study, we reported that BCG induced the formation of neutrophil extracellular traps (NETs), which play an important role in tumor treatment. Therefore, in the present study, we analyzed the gene expression profile of neutrophils stimulated by BCG through high-throughput arrays, which helped us determine the potential roles of neutrophils in BCG immunotherapy. The results showed that the expression of neutrophil genes led to changes in the early stage of BCG stimulation. The changed genes were involved in many functions of neutrophils such as mobility, proliferation, and secretion of cytokines, chemokines, and adhesion molecules. These changes in neutrophil biological functions may play an essential role in BCG induction of inflammatory and immune responses, and in anti-tumor processes.

Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis

Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 μM, cross-linked calprotectin (5 μM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 μM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9)2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.

Neutrophil extracellular traps prevent HIV infection in the female genital tract.

Women acquire HIV mainly through sexual intercourse. However, low transmission rates per sexual act indicate that local immune mechanisms contribute to HIV prevention. Neutrophils represent 10-20% of the genital immune cells in healthy women. Neutrophils mediate mucosal protection against bacterial and fungal pathogens through different mechanisms, including the release of Neutrophil Extracellular Traps (NETs). NETs are DNA fragments associated with antimicrobial granular proteins. Despite neutrophil abundance and central contributions to innate immunity in the genital tract, their role in protection against HIV-acquisition is unknown. We found that stimulation of human genital neutrophils with HIV viral-like particles (HIV-VLP) induced NET release within minutes of viral exposure, through ROS-independent mechanisms that resulted in immediate entrapment of HIV-VLPs. Incubation of infectious HIV with pre-formed genital NETs prevented infection of susceptible cells through irreversible viral inactivation. HIV inactivation by NETs from genital neutrophils could represent a previously unrecognized form of mucosal protection against HIV-acquisition.

Entamoeba histolytica L220 induces the in vitro activation of macrophages and neutrophils and is modulated by neurotransmitters.

The neuroimmunoregulation of inflammation has been well characterized. Entamoeba histolytica provokes an inflammatory response in the host in which macrophages and neutrophils are the first line of defense. The aim of this study was to analyze the effect of the 220 kDa lectin of Entamoeba histolytica on stimulation of human macrophages and neutrophils, especially the secretion of cytokines and the relation of these to neurotransmitters. Human cells were interacted with L220, epinephrine, nicotine, esmolol and vecuronium bromide. The concentrations of IL-1β, IFN-γ, TNF-α and IL-10 were determined by ELISA at, 4 h of interaction. L220 has a cytokine stimulating function of macrophages and neutrophils for secretion of IL-1β, and IL-10 only by macrophages, which was modulated by the effect of vecuronium on cholinergic receptors in this immune cells.

ARACHIDONIC ACID CORRELATION WITH SUSPENDED AND ATTACHED NEUTROPHILS

Background: Neutrophils are the blood cells for which adhesion is one of the main functions. Neutrophils adhered to biological surfaces show different characteristics of activation compared with neutrophils in suspension. The respiratory burst in suspended neutrophils in response to a chemotactic factor is lower when compared with the cells attached to the surface. Objectives: This study aimed to study the role of arachidonic acid release in suspended and attached neutrophils following stimulation of human neutrophils with LPS and PAF. Materials and methods: The suspended and attached neutrophils were primed by LPS and PAF, and inhibited by Anti-CD 14 (MY4). Results: The arachidonic acid release in attached cells is more than in suspended cells. This release is rapid with the increase of incubation time and dose-depended. The Fmet-Leu-Phe (FMLP) potentiates arachidonic acid release in attached cells pretreated with LPS and serum. Arachidonic acid release was less in suspended cells in comparison to attached cells where the addition of low concentration of LPS in presence of serum or platelet activating factor (PAF) to suspended cells produce increase in arachidonic acid release. On the other hand LPS in combination with PAF produce a large and significant increase in arachidonic acid release. The potentiative effect of LPS is mediated via CD14 receptors. The monoclonal antibodies against CD14 had no effect on arachidonic acid release in PAF treated cells where it inhibited greatly the potentiation by LPS or LPS-serum complex with PAF. Conclusion: LPS or PAF alone produced small increase in arachidonic acid release in both suspended and attached human neutrophils, where LPS in combination with PAF induced significant arachidonic acid release in suspended and attached human neutrophils.

TLR stimulation of human neutrophils lead to increased release of MCP-1, MIP-1α, IL-1β, IL-8 and TNF during tuberculosis

Neutrophils inform and shape immune responses. Toll-like receptors (TLRs) play an essential part in the perception of microbes and shape the complex host responses that occur during infection. The TLRs present on neutrophils play an indispensable role in neutrophil mediated pathogen recognition and elimination. This study was done to identify the role of significant TLRs in immune responses leading to differences in cytokine/chemokine release following stimulation. We evaluated the concentrations of various significant cytokines (IL-1β, TNF, MIP-1α, MCP-1 and IL-8) secreted by neutrophils from healthy donors and pulmonary tuberculosis patients following TLR ligand stimulation. TLR stimulation increased the release of such cytokines in both the groups. Thus it is noted that TLR stimulation of neutrophils definitely lead to increased cytokine response. Also, the release of all the studied cytokines are found to be greatly increased in patient neutrophils, affirming that neutrophils undergo secretory level modifications during tuberculosis infection.

P2Y2 receptor signaling in neutrophils is regulated from inside by a novel cytoskeleton-dependent mechanism

Functional selectivity, a process by which G-protein coupled receptors (GPCRs) can activate one signaling route while avoiding another, is regulated by ligand-mediated stabilization of specific receptor states that modulate different downstream signaling events. We propose a novel mechanism for functional selectivity, induced by the endogenous P2Y2R agonist ATP and regulated at the signaling interface by the cytoskeleton. Upon ATP stimulation of human neutrophils, a transient rise in the cytosolic concentration of free Ca2+ was not followed by activation of the superoxide anion-generating NADPH-oxidase. This was in contrast to signals generated through the formyl peptide receptor 1 (FPR1), as its activation was accompanied by both a mobilization of Ca2+ and activation of the NADPH-oxidase. The phospholipase C/Ca2+ signaling route is not modulated by the cytoskeleton-disrupting drug latrunculin A, but this drug was able to launch a new signaling route downstream of P2Y2R that led to NADPH-oxidase activation. The signaling downstream of P2Y2R was rapidly terminated and the receptors were desensitized; however, in contrast to desensitized FPR1, no P2Y2 receptor reactivation could be induced by latrunculin A. Thus, P2Y2R desensitization does not appear to involve the cytoskeleton, contrary to FPR1 desensitization. In summary, we hereby describe how ATP regulates functional selectivity via the cytoskeleton, leading to intracellular Ca2+ increase, alone or with simultaneous NADPH-oxidase activation in neutrophils.

The flavonols quercetin, myricetin, kaempferol, and galangin inhibit the net oxygen consumption by immune complex-stimulated human and rabbit neutrophils.

Stimulated human neutrophils exhibit increased net oxygen consumption (NOC) due to the conversion of O2 into the superoxide anion by the NADPH oxidase enzymatic complex during the respiratory burst. In several inflammatory diseases, overproduction of these oxidants causes tissue damage. The present study aims to: (a) optimize the experimental conditions used to measure the NOC in serum-opsonized zymosan (OZ)- and insoluble immune complex (i-IC)-stimulated human and rabbit neutrophils; and (b) compare the effect of four flavonols (quercetin, myricetin, kaempferol, and galangin) on this activity. We used a Clark-type oxygen electrode to measure the NOC of stimulated neutrophils. Eliciting the neutrophil respiratory burst with OZ and i-IC yielded similar maximum O2 uptake levels within the same species, but the human neutrophil NOC was almost four times higher than the rabbit neutrophil NOC. The optimal experimental conditions established for both cell types were 4 x 10(6) neutrophils mL(-1), 2 mg mL(-1) OZ, and 240 microg mL(-1) i-IC. Upon stimulation with OZ or i-IC, the tested flavonols reduced the human and rabbit neutrophil NOC in the same order of potency--quercetin and galangin were the most and the least potent, respectively. These compounds were around four times more effective in inhibiting the rabbit as compared to the human neutrophil NOC, respectively. The four flavonols were not toxic to human or rabbit neutrophils. The experimental conditions used are suitable for both the determination of human and rabbit neutrophil NOC and for the assessment of the modulatory effects of natural compounds on these activities. The relationship between the level of NOC and the inhibitory potency of the flavonols suggests that rabbit neutrophils can be useful experimental models to predict the effect of drugs on immune complex-stimulated human neutrophils.

FMLP-, thapsigargin-, and H2O2-evoked changes in intracellular free calcium concentration in lymphocytes and neutrophils of type 2 diabetic patients

Type 2 diabetic (T2DM) patients are immune-compromised having a higher susceptibility to infections and long-term complications in different parts of the body contributing to increased morbidity and mortality. A derangement in the homeostasis of intracellular free calcium concentration [Ca²⁺](i) is known to be associated with several diseases in the body including T2DM. Both neutrophils and lymphocytes play active protective roles in host immune response to infection showing impairment in microbicidal functions including phagocytosis and chemotaxis which are calcium-dependent processes. This study evaluated the process of [Ca²⁺]i mobilization from both neutrophils and lymphocytes taken from blood of both T2DM patients and healthy age-matched control subjects investigating the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP), thapsigargin (TG), and hydrogen peroxide (H₂O₂) on [Ca²⁺](i) homeostasis. This study employed isolated peripheral blood neutrophils and lymphocytes from 24 T2DM patients and 24 healthy volunteers. Either neutrophils or lymphocytes were stimulated separately with fMLP, TG, or H₂O₂. Induced changes in [Ca²⁺] in both neutrophils and lymphocytes were evaluated using spectrofluorometric methods. Stimulation of human neutrophils and lymphocytes with fMLP, TG, or H₂O₂ in the presence of [Ca²⁺]o resulted in significant decreases in [Ca²⁺](i) mobilization from T2DM patients compared with healthy controls. These data indicate that neutrophils and lymphocytes from T2DM patients are less responsive to calcium mobilizing agents compared with granulocytes from healthy controls and this is possibly due to the hyperglycemia. The results suggest that agonist-evoked decrease in [Ca²⁺](i) in immune cells might be one of the possible mechanisms of impaired immunity in diabetic patients.

Synthesis and Effects of Pyrazolines and Isoxazoles on the Phagocytic Chemotaxis and Release of Reactive Oxygen Species by Zymosan Stimulated Human Neutrophils

A series of novel isoxazole and pyrazoline derivatives has been synthesized and evaluated for their effects on the chemiluminescence and chemotactic activity of human phagocytes. Their effects on the chemotactic migration of isolated polymorphonuclear leukocytes (PMNs) and on the release of reactive oxygen species (ROS) during respiratory burst of human whole blood and PMNs were carried out using the Boyden chamber technique and luminol-based chemiluminescence assay, respectively. Of the compounds tested, compounds 8, 9, 11 and 12 exhibited higher inhibitory activity on the release of ROS (with IC50 values ranging from 5.6 to 8.4 μM) than acetylsalicylic acid (IC50 = 9.5 μ M). These compounds also showed strong inhibitory activity on the migration of PMNs with compound 8 exhibiting an IC50 value lower than that of ibuprofen. The results suggest that some of these isoxazole and pyrazoline derivatives have ability to modulate the innate immune response of phagocytes at different steps, indicating their potential as a source of new immunomodulatory agents.

Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties

Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions.

Wnt5a stimulates chemotactic migration and chemokine production in human neutrophils

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (β-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.

Chloroform extract of garlic (Allium sativum) stimulates production of superoxide anion and CXCL8 in human neutrophils

Garlic has garnered attention as a useful material which contains many bioactive molecules for the prevention and/or treatment of various human ailments, including infectious diseases. In this study we tested several extracts of garlic on cellular activity in human neutrophils. Stimulation of human neutrophils with chloroform extract of garlic (GCE) elicited intracellular calcium ([Ca2+]i) increases in a phospholipase C independent manner. GCE also stimulated superoxide anion production, which was completely blocked by a calcium (Ca2+) chelator. We also observed that stimulation of human neutrophils with GCE caused production of the chemokine CXCL8 in a concentration-dependent manner. GCE also stimulated activity of two important mitogen activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and p38 MAPK. Moreover, GCE-induced CXCL8 production was inhibited by a p38 MAPK inhibitor (SB203580) but not by an ERK inhibitor (PD98059). Taken together, our results demonstrate that GCE contains bioactive natural compounds that stimulate [Ca2+]i signaling, superoxide anion production, and CXCL8 production. Thus, GCE may be useful for the development of immune-modulating agents against infectious diseases.

Formyl Peptide Receptor 1 and 2 Dual Agonist Inhibits Human Neutrophil Chemotaxis by the Induction of Chemoattractant Receptor Cross-desensitization

Formyl peptide receptor 1 (FPR1) and FPR2/ALX are known to control neutrophil chemotaxis in response to various ligands. In this study, we investigated the inhibitory mechanism of compound 43 (Cpd43), an FPR1 and FPR2/ALX dual agonist, on human neutrophil chemotaxis. Precedent stimulation of human peripheral blood neutrophils with Cpd43 rendered the cells unresponsive in calcium mobilization induced by interleukin-8, C5a, or leukotriene B4. In addition, neutrophils pretreated with Cpd43 lost their chemotactic responses against these chemoattractants, wherein the expressions of chemoattractant receptors CXCR1, CXCR2, C5a receptor, and leukotriene B4 receptor 1 on the surface of neutrophils were all diminished significantly by treatment with Cpd43. By evaluating its pharmacological effect on 341 molecules, including receptors and enzymes, we also confirmed that Cpd43 has a highly specific affinity to FPR1 and FPR2/ALX and does not show binding affinity to the other chemoattractant receptors. These results indicate a previously unrecognized inhibitory mechanism of Cpd43 on neutrophil chemotaxis: the induction of cross-desensitization of multiple chemoattractant receptors in human neutrophils through its FPR1 and FPR2/ALX dual agonism.

Stimulation of human neutrophils with sera containing HLA Class I alloantibody causes preferential degranulation of azurophilic granules and secretory vesicles

The activation of neutrophils by human leukocyte antigen (HLA) Class I alloantibody is thought to be involved in transfusion-related acute lung injury. Neutrophils contain various biological substances in four groups of granules, including secretory vesicles, azurophilic granules, specific granules and gelatinase granules. To characterize the activation of neutrophils by HLA Class I alloantibody, we investigated whether HLA Class I alloantibody could cause the degranulation of these groups of granules either coordinately or selectively. Sera containing HLA-A24 alloantibody were incubated with neutrophils in a washed whole blood system. CD11b expression (secretory vesicles) on neutrophils was analysed by flow cytometry, and the secretion of markers of each granule was determined by ELISA. The treatment of cross-matching-positive neutrophils with sera containing HLA-A24 alloantibody caused the significant expression of CD11b, and the significant secretion of neutrophil elastase and myeloperoxidase, azurophilic granule markers and heparin-binding protein (HBP), which is localized in secretory vesicles and azurophilic granules when compared with cross-matching-negative neutrophils. In contrast, no significant differences were observed in the secretion of lactoferrin, a marker of specific granules, and matrix methalloproteinase-9, a marker of gelatinase granules between cross-matching-positive and cross-matching-negative cells upon stimulation with sera. CD11b expression and secretion of HBP by serum was partially inhibited by p38 mitogen-activated protein (MAP)-kinase inhibitors. Neutrophils activated with sera containing HLA Class I alloantibody caused the preferential degranulation of azurophilic granules and secretory vesicles. This process was at least in part mediated by p38 MAP kinase-involved signal transduction.