fMLP Stimulation Research Articles

Different innate neutrophil responses in controlled and uncontrolled asthma

Introduction: Respiratory viruses are a major cause of asthma exacerbations. Neutrophilic inflammation occurs during infections and is associated with difficult to treat asthma. The role of neutrophils in viral infections and whether neutrophil dysfunction contributes to exacerbation pathogenesis remains unclear. Aim: We aimed to investigate neutrophil immune responses in controlled (CA) and uncontrolled asthma (UnCA). We hypothesised that neutrophils from UnCA but not CA have impaired immune responses. Methods: Peripheral blood neutrophils from patients with CA (ACQ0.75) (n=7) or nonasthmatics (NA) (n=9) were stimulated with bacterial (LPS (1μg/ml), fMLP (100nM)) and viral surrogates( imiquimod (3μg/ml), R848 (1.5μg/ml), poly I:C (10μg/ml)) or rhinovirus (RV)16 (multiplicity of infection 1). Cell free supernatant was collected after 1h for measurement of neutrophil elastase (NE) by a colourimetric activity assay and matrix metalloproteinase (MMP)-9 by zymography or after 24h for CXCL8 release by ELISA. Results: fMLP stimulated neutrophils from UnCA but not CA had a 70% reduction in CXCL8 compared to NA. fMLP induced MMP9 release was also impaired by 50% in UnCA and 30% in CA compared to NA. Enhanced NE release (double) occurred constitutively and with fMLP stimulation in neutrophils from CA compared to NA. Interestingly, RV16 induced CXCL8 and R848 induced MMP-9 in CA and UnCA but not NA neutrophils. UnCA and CA responded similarly to other stimuli. Conclusion: Impaired innate responses in neutrophils from UnCA occurred only with the bacterial toxin fMLP, this might allow certain bacteria to colonise the airways and contribute to an altered lung microbiome in UnCA.

YIA2 Neutrophil Microvesicles Influence Atherogenesis and Contain Mirna

<h3>Background</h3> Many cell types are involved in the initiation and progression of atherosclerosis but neutrophils are rarely considered to be among them. However, despite their infrequent detection in plaques, neutrophils have been shown to facilitate the movement of monocytes into the vessel wall and increase plaque growth. One possible mechanism for this is through release of microvesicles that contain cargo such as microRNAs (miRNAs). <h3>Hypothesis</h3> Neutrophils influence plaque initiation and progression by releasing miRNA-containg microvesicles. <h3>Aim</h3> To determine whether neutrophil microvesicles i) influence atherosclerotic plaque development and ii) contain miRNAs relevant to atherosclerosis. <h3>Methods and results</h3> Mouse blood neutrophils were stimulated for 1h with fMLP (3 × 10⁵M) and supernatants subjected to differential centrifugation (twice at 300<i>g</i> for 6 min followed by 20,000<i>g</i> for 20 mins). Microvesicles were quantified using calibrated flow cytometry. ApoE^–/– mice were fed Western diet for 6 weeks and injected twice a week with sterile PBS or microvesicles (4 × 10⁶) isolated from wild type mice. After 6 weeks aortae were dissected and stained with Oil Red-O to identify areas of plaque formation. To detect miRNA, human neutrophils were isolated and stimulated with fMLP (10^–5M), AcLDL (20 µg/ml) or PBS for 2 h and microvesicles pelleted by differential centrifugation. MiRNA extraction and quantitative RT-PCR were performed in microvesicles, HUVEC and HUVEC incubated with microvesicles for 2 h. The number of neutrophils isolated from mice injected with microvesicles was increased (PBS = 67.3 ± 3.9/μl; microvesicles = 88.25 ± 4.0/μl, P = 0.031; n = 3 in each group). In addition neutrophil reactivity with respect to microvesicle formation was also increased (PBS = 4.5 ± 0.8 microvesicles per neutrophil; microvesicle = 8.3 ± 0.7 microvesicles per neutrophil, P = 0.0249; n = 3). Consequently plaque formation was more advanced in mice injected with microvesicles (% lesion area in aortic arch: PBS = 0.4563 ± 0.09452; microvesicles = 1.643 ± 0.3891, P = 0.0118; n = 7 in each group). MiR-223 and miR-150, found to be constitutively expressed in microvesicles and not in HUVEC, are involved in inflammation and atherosclerosis. These miRNAs were increased in after fMLP and AcLDL stimulation compared to PBS and were found to be significantly increased in HUVEC after 2h incubation with microvesicles (miR-223: 1.74 ± 0.44, P = 0.0305; 2.68 ± 0.50, P = 0.0034 and 6.79 ± 0.99, P = 0.0012 fold increase with microvesicles from PBS, fMLP and AcLDL stimulated neutrophils respectively and for miR-150: 3.79 ± 0.21, P = 0.0131 and 3.89 ± 0.78, P = 0.0109 fold increase with microvesicles from fMLP and AcLDL stimulated neutrophils respectively; n = 5 for each group). <h3>Conclusion</h3> Neutrophil microvesicles induce increased plaque formation and contain miRNA that may be transferred to endothelial cells suggesting neutrophils may be involved in atherogenesis through the release of microvesicles.

N-acetyl cysteine reduces oxidative toxicity, apoptosis, and calcium entry through TRPV1 channels in the neutrophils of patients with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common inflammatory and oxidant disease with an uncertain pathogenesis. N-acetyl cysteine (NAC) decreases oxidative stress, intracellular free calcium ion [Ca2+]i, and apoptosis levels in human neutrophil. We aimed to investigate the effects of NAC on apoptosis, oxidative stress, and Ca2+ entry through transient receptor potential vanilloid 1 (TRPV1) and TRP melastatin 2 (TRPM2) channels in neutrophils from patients with PCOS. Neutrophils isolated from PCOS group were investigated in three settings: (1) after incubation with TRPV1 channel blocker capsazepine or TRPM2 channel blocker 2-aminoethyl diphenylborinate (2-APB), (2) after supplementation with NAC (for 6 weeks), and (3) with combination (capsazepine + 2-APB + NAC) exposure. The neutrophils in TRPM2 and TRPV1 experiments were stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 μM) and capsaicin (10 μM) as concentration agonists, respectively. Neutrophil lipid peroxidation and capsaicin-induced increase in [Ca2+]i concentrations were reduced by capsazepine and NAC treatments. However, the [Ca2+]i concentration did not change by fMLP stimulation. Neutrophil lipid peroxidation, apoptosis, caspase-3, caspase-9, cytosolic reactive oxygen species production, and mitochondrial membrane depolarization values were decreased by NAC treatment although neutrophil glutathione peroxidase and reduced glutathione levels were increased by the NAC treatment. Serum lipid peroxidation, luteinizing hormone, testosterone, insulin, interleukin-1 beta, and homocysteine levels were decreased by NAC treatment although serum vitamin A, beta-carotene, vitamin E, and total antioxidant status were increased by the NAC treatment. In conclusion, NAC reduced oxidative stress, apoptosis, cytokine levels, and Ca2+ entry through TRPV1 channel, which provide supportive evidence that oxidative stress and TRPV1 channel plays a key role in etiology of PCOS.

Pro-inflammatory cytokines and leukocyte oxidative burst in chronic kidney disease: culprits or innocent bystanders?

Pro-inflammatory cytokines are elevated in chronic kidney disease (CKD), a condition characterized by microinflammation with oxidative stress as key feature. However, their role in the inflammatory response at uraemic concentrations has not yet been defined. In this study, the contribution of cytokines on induction of leukocyte oxidative stress was investigated. Whole blood from healthy donors was incubated with 20-1400 pg/mL TNFα, 5-102.8 pg/mL IL-6, 20-400 pg/mL IL-1β and 75-1200 pg/mL IL-18 separately or in combination. Oxidative burst was measured, at baseline and after stimulation with fMLP (Phagoburst™). The effect of the TNFα blocker, adalimumab (Ada), was evaluated on TNFα-induced ROS production. Finally, the association between TNFα and the composite end point all-cause mortality or first cardiovascular event was analysed in a CKD population stage 4-5 (n = 121). While interleukin (IL)-6, IL-1β and IL-18 alone induced no ROS activation of normal leukocytes, irrespective of concentrations, TNFα induced ROS activation at baseline (P < 0.01) and after fMLP stimulation (P < 0.05), but only at uraemic concentrations in the high range (400 and 1400 pg/mL). A similar pattern was observed with all cytokines in combination, but already at intermediate uraemic concentrations (all P < 0.05, except for monocytes after fMLP stimulation: n.s.), suggesting synergism between cytokines. ROS production induced by TNFα (400 pg/mL) and the cytokine combination was blocked with Ada. Uraemia-related oxidative stress in leukocytes of haemodialysis patients was however not blocked by Ada. In patients, TNFα was not associated to adverse events (HR: 1.52, 95% CI 0.81-2.85, P = 0.13). Among several pro-inflammatory cytokines, TNFα alone was pro-oxidative but only at high-range uraemic concentrations. Adding a TNFα blocker, Ada, blocked this ROS production, but not the oxidative stress in blood samples from haemodialysis patients, suggesting that other uraemic toxins than TNFα are more crucial in this process. However, the lack of association between TNFα and mortality suggests that the role of TNFα-linked oxidative stress is limited.

B-Type natriuretic peptide suppression of neutrophil superoxide generation: mechanistic studies in normal subjects.

Many acute cardiovascular disease states are associated with neutrophil infiltration of myocardium and subsequent release of superoxide (O2 (-) ) and myeloperoxidase (MPO), which contribute to inflammatory reactions. B-Type natriuretic peptide (BNP) is known to exert anti-inflammatory and antifibrotic effects, but it is not known whether these may include interactions with neutrophils. In neutrophils isolated from 20 healthy subjects, we assessed the effect of BNP on the 'neutrophil burst' (O2 (-) production and MPO release) stimulated by phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), respectively. Effects of BNP on cGMP accumulation, and the effects of the cell-permeable cGMP analogue 8-(4-chlorophenylthio) guanosine-cGMP (8-p-CPT-cGMP) and protein kinase G (PKG) inhibition with KT5823 on the neutrophil-BNP interaction were also evaluated. B-Type natriuretic peptide suppressed O2 (-) release from neutrophils by 23±6% (P<0.001) and 24±8% (P<0.05) following PMA and fMLP stimulation, respectively. Although BNP did not significantly increase cGMP formation, 8-p-CPT-cGMP suppressed both PMA- and fMLP-induced neutrophil O2 (-) release by 16% and 28%, respectively (P<0.05). The PKG inhibitor KT5823 attenuated the effects of BNP on both fMLP- and PMA-associated O2 (-) production. Neither BNP nor 8-p-CPT-cGMP significantly affected MPO release from neutrophils. Suppression of O2 (-) release from neutrophils by BNP may contribute to its anti-inflammatory and antifibrotic actions.

The autoimmune-associated genetic variant PTPN22 R620W enhances neutrophil activation and function in patients with rheumatoid arthritis and healthy individuals

ObjectivesA genetic variant of the leukocyte phosphatase PTPN22 (R620W) is strongly associated with autoimmune diseases including rheumatoid arthritis (RA). Functional studies on the variant have focussed on lymphocytes, but it...

Simvastatin down-regulates the production of Interleukin-8 by neutrophil leukocytes from dyslipidemic patients

BackgroundNeutrophil (PMN) leukocytes participate to the initial phases of atherosclerosis through the release of Interleukin 8 (CxCL8; IL-8) that contribute to amplification of inflammation. Aim of the study is to investigate the production of IL-8 by PMN leukocytes from dyslipidemic patients treated with simvastatin.MethodsIn 15 dyslipidemic subjects with moderately increased cardiovascular risk, assessed by Framingham Risk Score, blood samples were obtain to investigate PMNs IL-8 production [at baseline and after N-formyl-Met-Leu-Phe (fMLP) stimulation] before and after long-term (1-year) simvastatin treatment.ResultsThe resting release of IL-8 was higher in dyslipidemic patients at baseline when compared with control subjects (p < 0.05). One year of treatment was significantly associated with reduced IL-8 production (p < 0.01). Moreover, the fMLP-induced IL-8 production in dyslipidemic untreated patients was higher than that of controls (p < 0.05) and was reduced after simvastatin treatment (p < 0.01). IL-8 release after 1 year of treatment was reduced to levels which were lower than those observed in control subjects both for resting and stimulated cytokine production (p < 0.01).ConclusionsProlonged treatment with simvastatin is associated with a reduction of IL-8 production, suggesting the possibility of statin to modulate the pro-inflammatory response in PMNs of patients with moderately increased cardiovascular risk.

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Introduction: Adenosine triphosphate (ATP), which is well known as energy currency of cells, is involved in both intracellular and extracellular signaling. Neutrophils (PMN) have a pivotal role in inflammation response. We have shown that ATP release and feedback via ATP receptors are essential for neutrophil activation. Meanwhile, two new fluorescent chemosensors (2-2Zn(II), 3-2Zn(II)), which detect ATP and its delivertives in specific region of living cells, were developed. Although the utility of these chemosensors has been demonstrated in cultured cells, imaging studies using human PMN have not been reported. Our objective is to establish the methods of staining human PMN with 2-2Zn(II), 3-2Zn(II) and quantify the fluorescence intensity by flow cytometry. Methods: PMN were isolated from the peripheral blood of healthy volunteers (n=12). Live PMN with these staining were observed by a confocal microscopy. Fluorescence intensity on plasma membrane (2-2Zn(II)) and in mitochondria (3-2Zn(II)) of PMN with and without fMLP stimulation were evaluated using flow cytometry. Results: These chemosensors could localize on plasma membrane surface and mitochondria in human neutrophils. Mean fluorescence intensity (MFI) of 2-2Zn(II) on plasma membrane of PMN with fMLP stimulation was significantly higher than without stimulation (274.3 ± 100.6 vs 208.4 ± 81.8 ; p< 0.01). On the other hand, MFI of 3-2Zn(II) in mitochondria with stimulation tends to be lower than without stimulation . Conclusions: Fluorescence imaging of ATP in human PMN with chemosensors (2-2Zn(II), 3-2Zn(II)) was established. The higher MFI on plasma membrane with fMLP stimulation suggests the activation of PMN. This method would contribute to understanding the dynamics of ATP in neutrophils and elucidating their diverse physiological functions in inflammation.

Uremia-Related Oxidative Stress in Leukocytes Is Not Triggered by β2-Microglobulin

Chronic kidney disease (CKD) is characterized by low-grade inflammation and increased risk for cardiovascular disease. The interest in β2-microglobulin (B2M) as a marker for cardiovascular outcome with and without CKD has grown. Clinical studies suggested that B2M could be involved in the pathogenesis of vascular disease, for which chronic leukocyte activation is a pathogenic factor. We investigated whether B2M is proinflammatory by inducing oxidative burst in leukocytes. Oxidative burst was measured at baseline and after stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP), Escherichia coli, or phorbol-12-myristate-acetate (PMA) in the whole blood of healthy volunteers in the absence (saline) and presence of human B2M (hB2M; 10 and 50mg/L) versus uremic whole blood. Because of suspicion of contamination, hB2M was dialyzed for purification and purified B2M (dB2M) and dialysates were tested in the burst test. As a comparator, reactive oxygen species (ROS) in response to lipopolysaccharide (LPS) was measured. Unpurified hB2M strongly enhanced ROS in monocytes and granulocytes after E. coli and PMA and moderately after fMLP stimulation compared with control (P < .01) and uremia (P < .01) whereas at baseline hB2M only induced ROS in granulocytes (P < .05). After purification, dB2M no longer increased burst activity, suggesting that contamination was responsible for the initial effect. An endotoxin concentration of less than 1.5 EU/mL, as observed in hB2M, could not induce oxidative stress. This study suggests that B2M, a traditional marker for middle molecule retention and a novel marker for cardiovascular outcome, may not by itself cause vascular damage by influencing inflammatory response due to induction of leukocyte free radical production. However, an effect on other cell types involved cannot be excluded. Our data further reveal that this type of research might be skewed by non-LPS contaminants, and that care should be taken to exclude this bias.

Involvement of Toll-like receptor 4 and Fc receptors gamma in human neutrophil priming by endotoxins from Escherichia coli

By using the fMLP-induced respiratory burst approach, the involvement of Toll-like receptor 4 (TLR4) in human neutrophil priming by S- or Re-glycoforms of endotoxin from Escherichia coli has been elucidated. The priming effect of Re-glycoform is more pronounced than that of the S-glycoform. Unexpectedly, fMLP-triggered generation of reactive oxygen species (ROS) by endotoxin primed neutrophils was amplified by preincubation of the cells with anti-TLR4 (HTA125) antibodies or with isotype-matched immunoglobulin IgG2a. The most significant finding of our study is that neutrophils exposed to anti-TLR4 antibodies retain their ability to distinguish between S- or Re-glycoforms being primed, respectively. Moreover, differentiated effect of HTA125 antibodies on functional responses of neutrophils during their priming and fMLP stimulation was revealed. Taking these results into consideration, it is reasonable to assume that there is a contribution of Fcγ receptors to fMLP-induced ROS generation by neutrophils preincubated with HTA125 or IgG2a and primed by endotoxins.

Requirement for both receptor-operated and store-operated calcium entry in N-formyl-methionine-leucine-phenylalanine-induced neutrophil polarization

Tissue penetration of neutrophils is a key process in many inflammatory diseases. In response to inflammatory stimuli such as N-formyl-methionine-leucine-phenylalanine (fMLP), neutrophils polarize and migrate towards the chemotactic gradient of the stimulus. Elevated intracellular Ca2+ concentration is known to play a critical role in neutrophil polarization and migration; however, the exact mechanism remains elusive. Here, we demonstrated that fMLP stimulation caused not only store-operated calcium entry (SOCE), but also receptor-operated calcium entry (ROCE) in neutrophils by using both pharmacological and neutralizing monoclonal antibody approaches. We also investigated neither Rac2 nor Cdc42 activation could take place if either SOCE or ROCE was inhibited. This study thus provides the first evidence for coordination of Ca2+ influx by SOCE and ROCE to regulate neutrophil polarization.

P21-Activated Kinases Regulate Directional Migration and Cytoskeletal Organization in Human Neutrophils

Abstract 834Neutrophil chemotaxis is controlled by coordinated processes of directional sensing, polarization and motility. This study was designed to characterize the role of p21-activated kinases (PAKs) during the chemotaxis of human primary neutrophils. PAKs are known as effectors of the Rho GTPases Rac and Cdc42. It has been shown that PAK1 and PAK2 are strongly activated downstream of the f-Met-Leu-Phe (fMLP) receptor via Rac (Huang et al., MCB 1998). PAK1 is known to localize in lamellipodia at the leading edge of human neutrophils (Dharmawardhane et al., JLB 1999) and mediate persistent directional migration via Cdc42 in a neutrophil-like cell line (Li et al., Cell 2003). However, little is known about the specific role of PAK isoforms in spatial/temporal regulation of cytoskeletal dynamics in human neutrophils.Our data show that human neutrophils express PAK1, 2 and 4. Under an fMLP gradient, human neutrophils developed morphological polarity with a distinct leading edge and rear, and migrated up the fMLP gradient at the speed of 7.5 ± 0.56 μm/min. Inhibition of Rac or PI3K impaired directionality but did not significantly affect migration speed of chemotaxing neutrophils (6.3 ± 0.56 μm/min or 6.2 ± 0.85 μm/min, respectively). In contrast, neutrophils treated with the PAK inhibitor, PF3758309 (PF), displayed random migration, less polarization and reduced motility (3.1 ± 0.21 μm/min). These results suggest that PAK regulates neutrophil chemotaxis independently of the Rac-PI3K axis. The presence of PF did not abrogate intracellular Ca2+mobilization in fMLP-driven chemotactic condition. Instead, the decreased migratory ability by PAK inhibition was associated with multiple Ca2+ spikes. Immunofluorescence imaging shows that PAK2 but not PAK1, was phosphorylated and translocated from cytosol to actin-rich leading edge in the proximity to GTP-bound Rac within 3 min of fMLP stimulation. Notably, PF treatment resulted in partial neutrophil spreading and actin/myosin II translocation in the absence of extracellular stimuli, suggesting that basal level of PAK phosphorylation may be required for cytoskeletal integrity of resting neutrophils. Neutrophils pretreated with PF displayed less activation and translocation of PAK2 and Rac.In summary, our data demonstrate for the first time the distinct roles of PAK isoforms in human neutrophil morphological polarity and directional migration and suggest that PAK2 is activated downstream of fMLP receptor through Rho-family small GTPases. Disclosures:No relevant conflicts of interest to declare.

Glia Maturation Factor-Gamma Regulates Macrophage Migration Through Control Recycling of alpha5beta1 Integrin in Endosome

AbstractAbstract 3273Macrophages play an essential role in defending against invading pathogens by migrating to the sites of infection, removing apoptotic cells, and secreting inflammatory cytokines. Macrophage migration is a critical step in the regulation of immune response and is also associated with many chronic inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, atherosclerosis and cancer. Thus, understanding the mechanisms controlling macrophage migration within different environments is of paramount importance. Although it is clear that adhesion signaling via integrin receptors and the surrounding ECM play a significant role in regulating migration of macrophages to site of inflammation, the underlying cellular and molecular mechanisms responsible for these process is still not fully characterized. Defining the molecular circuits through which integrins regulate macrophage motility is therefore important for gaining a better understanding of macrophage function. Glia maturation factor gamma (GMFG), a novel ADF/cofilin superfamily protein that is predominantly expressed in inflammatory cells, has been implicated in regulating actin reorganization. We have previously demonstrated that GMFG plays a role in regulating neutrophil chemotaxis and migration. We now examine whether GMFG has similar effects on macrophage and the cellular mechanism for these effects by using small-interfering RNA technic to knockdown GMFG in human macrophage like THP-1cells. Knockdown of endogenous GMFG results in significantly reduced chemotactic migration toward fMLP and enhanced adhesion on fibronectin compared with control siRNA transfected cells. Flow cytometry analysis shows that GMFG-knockdown cells expresses significant amount of α5β1 integrin on the surface compared with control cells. Confocal microscopy analysis exhibited that GMFG was distributed throughout the cytosol and colocalized with the multiple endocytic compartment such as early endosome marker EEA1, Rab5, Rab4 and Rab11. Importantly, we found that most of the α5β1 integrins accumulated in clusters at the rear of the GMFG-knockdown cells, along with colocalization with endosome marker EEA1, rather than localization toward the leading edge in response to fMLP stimulation in control cells. These results suggest that impaired efficient α5β1 integrin recycling cause to increased adhesion, which is responsible for reduced macrophage migration on fibronectin in GMFG depletion macrophages. Accordingly, GMFG is required for proper trafficking of endosomal α5β1 integrin to the plasma membrane and is fundamental for integrin-mediated macrophage motility. Disclosures:No relevant conflicts of interest to declare.

GM-CSF induces podoplanin expression during dendritic cell differentiation (111.56)

Abstract Podoplanin is a mucin-type transmembrane glycoprotein containing extensive Oglycosylation in extracellular domain and two phosphorylation sites in a short cytoplasmic tail. It is expressed in kidney glomerular epithelial cells, lymphatic endothelium and subcapsular epithelial cells in murine thymus. Recently, podoplanin was found on murine F4/80 positive peritoneal macrophages and human follicular dendritic cells (FDCs) indicating that podoplanin is involved in immune response. We have reported that podoplanin is induced during Th9 and Th17 polarization. Here, we studied podoplanin expression on different types of murine myeloid cells. We found that podoplanin was not detectable on circulating and bone marrow derived neutrophils, monocytes, basophils and esinophils, its expression level on neutrophil and monocytes was not inducible followed by fMLP or TNF-a stimulation. Podoplanin was also not detectable on spleen and lymph node resided CD11b+macrophages, CD11c+ dendritic cells (cDCs) and CD8+ plasmacytoid dendritic cells (pDCs). However, podoplanin expression level has been dramatically increased on GM-CSF driven bone marrow-derived dendritic cells. These data suggest that podoplanin can be induced during GM-CSF driven dendritic cell differentiation, indicating that podoplanin may be a transient marker during dendritic cells differentiation and maturation. The mechanism of GM-CSF induced podoplanin and it significance need to be further investigated.

8F23 ガラス平板上におけるHL60の挙動に与えるfMLP刺激の影響に関する傾斜遠心顕微鏡を用いた実験的研究(OS16 血球運動と微小環境のバイオニクス2)

8F23 ガラス平板上におけるHL60の挙動に与えるfMLP刺激の影響に関する傾斜遠心顕微鏡を用いた実験的研究(OS16 血球運動と微小環境のバイオニクス2)

P2Y11 Purinoceptor Mediates the ATP-Enhanced Chemotactic Response of Rat Neutrophils

ATP and hydrolysis products of ATP like adenosine regulate the chemotaxis of neutrophils by activating purinoceptors and adenosine receptors. The present study was designed to examine exogenous ATP, activation of purinoceptors, and activation of A3 adenosine receptor as key steps in the signal cascades that control cell orientation and migration of rat neutrophils. One or more of those steps might be potential therapeutic targets for treatment of inflammatory diseases. The chemotaxis of rat neutrophils was stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and measured with an EZ-TAXIScan apparatus. The effects of apyrase, exogenous ATP, suramin (P2X and P2Y blocker), PPADS (a P2X blocker), TNP-ATP (P2X1 and P2X3 antagonist), and Reactive Blue 2 (a P2Y blocker) on the chemotactic response were also investigated. Rat neutrophil chemotaxis was significantly suppressed by apyrase. fMLP induced rat neutrophil chemotaxis was potentiated by ATP, blocked by suramin, not affected by PPADS or TNP-ATP, and significantly inhibited by RB-2. Western blotting showed that A3, P2Y2, and P2Y11 were expressed in rat neutrophils. The chemotactic response of rat neutrophils to fMLP stimulation is potentiated by ATP via P2Y11 purinoceptors but not P2X purinoceptors or A3 adenosine receptor, and that the response plays a critical role in host defense and pathogenicity.[Supplementary Movies: available only at http://dx.doi.org/10.1254/jphs.12173FP]

Coagulation Factors XIa and XIIa Modulate Neutrophil Elastase Release,

Abstract 3220Neutrophils play a vital role in innate immunity. Activated neutrophils can release proteolytic enzymes capable of neutralizing microbes and contributing importantly to host-defense. In severe sepsis, microbial components and pro-inflammatory cytokines can contribute to excess systemic neutrophil activation, resulting in tissue damage and organ failure. Thus, regulation of neutrophil activation and factor release is critical during pathologic conditions.Recent data indicate that components of the contact system modulate numerous inflammatory mediators during severe sepsis, but the exact role of the contact pathway in host-defense is not well understood. Inhibition of factor XII (FXII) in septic baboons reduces circulating neutrophil elastase (NE), a potent cytolytic enzyme that is increased during sepsis and implicated in organ failure. In vitro studies also indicate that both plasma kallikrein and FXIIa are capable of directly inducing NE release. While it is apparent that factors of the contact system interact with neutrophils, the molecular mechanisms by which these factors modulate neutrophil function have not been established. We therefore examined factor XI (FXI) neutrophil interactions and the cellular signaling pathways regulating FXIIa neutrophil stimulation.Human neutrophils were isolated from peripheral blood and resuspended in HBSS at a concentration of 0.5 ×106/ml. Cells were treated with FXI, FXIa, FXII, or FXIIa with or without fMLP (1 μM) stimulation, and the release of NE was assayed in the cell supernatants via ELISA. FXI, FXIa or FXII had no direct stimulatory effect on NE release compared to vehicle. While neither FXI nor FXII had any inhibitory effect on fMLP induced NE release, FXIa (10 μg/ml) modestly reduced fMLP-induced NE release by 20% (n=3). FXIIa (3, 10, 30 μg/ml) dose-dependently increased NE release in the presence of cytochalasin B (5 μg/ml), consistent with published data.To examine the mechanism by which FXIIa induces NE release, neutrophils were pretreated with signaling inhibitors and subsequently activated with FXIIa (30 μg/ml). Mammalian target of rapamycyin (mTOR) is a downstream serine/threonine kinase of the PI3K/AKT pathway that integrates signals from the microenvironment such as cytokines and growth factors. It is known that inhibition of mTORC2 abrogates neutrophil polarization and directed migration, thus we examined the role of rapamycin complex 1 and 2 (mTORC1/2) in mediating NE release. Pretreatment of cells with RAD001 (20 nM), an mTORC1 inhibitor had no effect on FXIIa-induced NE release, whereas the combined mTORC1/mTORC2 inhibitor, pp242 (100 nM) abrogated FXIIa-induced NE release, suggesting that components of the mTORC2 pathway contribute to NE release. Pretreatment with EHT 1864 (50 uM), a Rac inhibitor, significantly potentiated NE release induced by either fMLP or FXIIa, suggesting that Rac is also capable of modulating FXIIa signaling.Taken together, these results suggest that coagulation factors FXIa and FXIIa differentially modulate neutrophil function, and that the mTOR and Rac signaling pathways participate in FXIIa stimulated neutrophil activation. These data suggest that the contact pathway is involved in neutrophil stimulation through mTOR and Rac signaling, and thus modulating these pathways could be a potential therapeutic strategy for limiting excess neutrophil activation. Disclosures:Gruber:Aronora, LLC: Consultancy, Equity Ownership.

Proinflammatory Effects of Bare and PEGylated ORMOSIL-, PLGA- and SUV-NPs on Monocytes and PMNs and Their Modulation by F-MLP

We wanted to test the proinflammatory effects of vinyltriethoxysilane-based organically modified silica nanoparticles (ORMOSIL-NPs) in vitro on blood leukocytes. Cell selectivity, cytokines/chemokines and O(2) (-) production were analyzed using nonpolyethylene glycol (PEG)ylated and PEGylated ORMOSIL-NPs, poly(lactic-co-glycolic acid) (PLGA)-NPs and small unilamellar vesicles (SUV)-NPs. ORMOSIL-NPs mostly bound to monocytes while other NPs to all leukocyte types similarly. Cell capture of PEGylated-NPs decreased strongly (ORMOSIL), moderately (PLGA) and weakly (SUV). Bare ORMOSIL-NPs effectively stimulated the production of IL-1β/IL-6/TNF-α/IL-8 by monocytes and of IL-8 by polymorphonuclear leukocytes (PMNs). NP PEGylation inhibited such effects only partially. Formyl-methionine-leucine phenylalanine (f-MLP) further increased the release of cytokines/chemokines by monocytes/PMNs primed with bare and PEGylated ORMOSIL-NPs. PEGylated SUV-NPs, bare and PEGylated ORMOSIL- and PLGA-NPs sensitize PMNs and monocytes to secrete O(2) (-) upon f-MLP stimulation. ORMOSIL-NPs are preferentially captured by circulating monocytes but stimulate both monocytes and PMNs per se or by sensitizing them to another agonist (f-MLP). PEG-coating confers stealth effects but does not completely eliminate leukocyte activation. Safe nanomedical applications require the evaluation of both intrinsic and cooperative proinflammatory potential of NPs.

Oxidases and oxygenases in regulation of neutrophil redox pathways in Behçet’s disease patients

This study aimed to determine plasma and neutrophil oxidase activities that may contribute to vascular inflammation in Behçet’s disease (BD) patients. Cyclooxygenase (COX), NADPH oxidase and myeloperoxidase (MPO) activity was determined in neutrophils isolated from BD patients and healthy controls. Functional assay of NADPH oxidase was significantly increased in BD patients, both at basal conditions and in response to fMLP stimulation. There was a significant increase in plasma MPO activity in the disease group as compared to controls. Total COX activity was significantly increased in BD neutrophils. The increase in total COX activity was accompanied with enhanced activity of COX-2, differentiated by using the COX-1 isoform-specific inhibitor SC-560. Neutrophil nitrate/nitrite levels showed no significant difference in BD; however, plasma nitrate/nitrite contents in BD patients were significantly greater compared to controls. In conclusion, increased plasma MPO, neutrophil NADPH and COX activities may contribute to intravascular inflammation documented in BD patients.

Effect of nifedipine on calcium status and chemiluminescence response of phagocytes during Plasmodium berghei infection in mice.

The macrophages and neutrophils from nifedipine-treated mice, both Plasmodium berghei-infected and uninfected, showed suppressed capacity to generate oxygen free radicals as compared with untreated controls. Nifedipine treatment did not affect resting state free calcium levels in these cells. But the rise in intracellular calcium levels of macrophages and neutrophils following P. berghei infection was significantly less (P < 0.05) in nifedipine-treated mice as compared with untreated groups at various parasitaemia levels. Probably this reflects a more potent effect of nifedipine on these cells in the depolarized state. Similarly, the rise in intracellular calcium levels of these cells following formyl-Met-Leu-Phe (fMLP) stimulation was also significantly less in nifedipine-treated groups than in untreated controls at different parasitaemia levels. A positive correlation between this fMLP-stimulated rise in calcium levels and the chemiluminescence response of macrophages and neutrophils was observed in nifedipine-treated and untreated groups at various parasitaemia levels. Thus the respiratory-burst responses of these cells during P. berghei infection depend on the calcium homeostasis in the cells. The disturbances of the calcium-regulating mechanisms by nifedipine treatment resulted in subnormal phagocytic cell responses which lead to more severe and rapidly fatal P. berghei infection in these animals.