Incubation Of Neutrophils Research Articles

Influence of pegylated graphene oxide nanoparticles on the respiratory burst and phagocytic activity of human neutrophils

Scientific and technological progress contributes to the discovery and production of innovative materials. The emergence of graphene is a clear example of this. Graphene is considered a promising material for use in nanobiomedicine and nanobiotechnology. It is therefore important to understand how it affects human immune cells. In a study, the effects of 5 and 25 μg/mL graphene oxide nanoparticles with lateral sizes of 100-200 nm and 1-5 μm, modified with linear and branched polyethylene glycol, on human neutrophils were investigated. The formation of reactive oxygen species was evaluated with a lucigenin as a chemiluminescence activator.Inaddition, we investigated theeffect of a 60-minute incubation of neutrophils with pegylated graphene oxide nanoparticles on the viability of these cells by staining with trypan blue and a 30-minute incubation on the uptake of fluorescein isocyanate-labelled E. coli. The percentage of neutrophils which engulfed E. coli and the uptake index were determined. Samples without added nanoparticles served as controls.A decrease in lucigenin-enhanced chemiluminescence of neutrophils was observed under the influence of two types of graphene oxide nanoparticles: 1-5 μm in size coated with linear polyethylene glycol, and 100-200 nm in size coated with branched polyethylene glycol, at a concentration of 25 μg/mL in the zymosan-stimulated version of the assay. No dependence of the effect on the particle size and the type of polyethylene glycol was observed. The indicators for spontaneous chemiluminescence of neutrophils did not change with the addition of PEGylated graphene oxide nanoparticles.A thirty-minute incubation of human neutrophils at 37 °C with PEGylated graphene oxide nanoparticles with lateral dimensions of 100-200 nm and 1-5 μm had no effect on the viability of these cells and on the percentage of neutrophils that engulfed E. coli. However, 1-5 μm graphene oxide modified with linear polyethylene glycol at a concentration of 25 μg/mL increased the amount of E. coli engulfed by neutrophils per cell.Thus, in the absence of cytotoxicity, PEGylated graphene oxide particles have multidirectional immunomodulatory effects on neutrophils. In this case, their concentration is decisive and not the size of the graphene oxide particles and the type of polyethylene glycol.

Constitutive and induced forms of membrane-bound proteinase 3 interact with antineutrophil cytoplasmic antibodies and promote immune activation of neutrophils

Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFα-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigen-binding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis.

Extracellular CIRP Induces Novel Nectin-2+ (CD112+) Neutrophils to Promote Th1 Differentiation in Sepsis.

Neutrophil heterogeneity represents different subtypes, states, phenotypes, and functionality of neutrophils implicated in sepsis pathobiology. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that promotes inflammation and alters neutrophil phenotype and function through TLR4. Nectin-2 or CD112 is an Ig-like superfamily member. CD112 serves as the ligand for DNAM-1 (CD226), which induces Th1 differentiation in naive CD4+ T cells. Th1 cells produce IFN-γ to fuel inflammation. CD112 is expressed mainly on APCs, but its expression in neutrophils is unknown. We hypothesize that eCIRP induces CD112 expression in neutrophils, promoting Th1 differentiation in sepsis. Incubation of neutrophils with recombinant murine (rm)CIRP significantly increased the gene and protein expression of CD112 in neutrophils. Anti-TLR4 Ab-treated neutrophils significantly decreased CD112+ neutrophils compared with controls upon rmCIRP stimulation. After 4 h of rmCIRP injection in mice, CD112+ neutrophils were significantly increased in the blood and spleen. At 20 h after cecal ligation and puncture-induced sepsis, CD112+ neutrophils were also significantly increased. Blood and splenic CD112+ neutrophils in septic CIRP-/- mice were much lower than in septic wild-type mice. Coculture of naive CD4 T cells with rmCIRP-treated (CD112+) neutrophils significantly increased IFN-γ-producing Th1 cells compared with coculture with PBS-treated neutrophils. CD112 Ab significantly attenuated Th1 differentiation induced by rmCIRP-treated neutrophils. Thus, eCIRP increases CD112 expression in neutrophils via TLR4 to promote Th1 differentiation in sepsis. Targeting eCIRP may attenuate sepsis by reducing Th1-promoting CD112+ neutrophils.

6]-Gingerol Facilitates CXCL8 Secretion and ROS Production in Primary Human Neutrophils by Targeting the TRPV1 Channel.

Clarifying the function of sensory active TRP (transient receptor potential) channels in non-sensory tissue is of growing interest, especially with regard to food ingredients in nutritionally relevant concentrations. The study hypothesizes the TRPV1 agonist [6]-gingerol to facilitate cellular immune responses of primary human neutrophils, after treatment with 50nM, a concentration that can be reached in the circulation after habitual dietary intake. qRT-PCR analyses reveal a high abundancy of TRP channel RNA expression in the types of primary leukocytes investigated, namely neutrophils, monocytes, NK cells, T cells, and B cells. Incubation of neutrophils with 50nM of the known TRPV1 ligand [6]-gingerol led to increased surface expression of CD11b, CD66b, and the fMLF receptor FPR1, as shown by flow cytometry. Upon subsequent stimulation with fMLF, the neutrophils display an about 30% (p < 0.05) increase in CXCL8 secretion as well as in ROS production. Pharmacological inhibition of TRPV1 by trans-tert-butylcyclohexanol abolishes the [6]-gingerol induced effects. The TRPV1 channel is functionally expressed in human neutrophils. Activation of the channel with [6]-gingerol as a food-derived ligand in nutritionally relevant concentrations leads to an enhanced responsiveness in the cells towards activating stimuli, thereby facilitating a canonical cellular immune response in human neutrophils.

Activation of Neutrophils by Mucin–Vaterite Microparticles

Nano- and microparticles enter the body through the respiratory airways and the digestive system, or form as biominerals in the gall bladder, salivary glands, urinary bladder, kidney, or diabetic pancreas. Calcium, magnesium, and phosphate ions can precipitate from biological fluids in the presence of mucin as hybrid nanoparticles. Calcium carbonate nanocrystallites also trap mucin and are assembled into hybrid microparticles. Both mucin and calcium carbonate polymorphs (calcite, aragonite, and vaterite) are known to be components of such biominerals as gallstones which provoke inflammatory reactions. Our study was aimed at evaluation of neutrophil activation by hybrid vaterite–mucin microparticles (CCM). Vaterite microparticles (CC) and CCM were prepared under standard conditions. The diameter of CC and CCM was 3.3 ± 0.8 µm and 5.8 ± 0.7 µm, with ƺ-potentials of −1 ± 1 mV and −7 ± 1 mV, respectively. CC microparticles injured less than 2% of erythrocytes in 2 h at 1.5 mg mL−1, and no hemolysis was detected with CCM; this let us exclude direct damage of cellular membranes by microparticles. Activation of neutrophils was analyzed by luminol- and lucigenin-dependent chemiluminescence (Lum-CL and Luc-CL), by cytokine gene expression (IL-6, IL-8, IL-10) and release (IL-1β, IL-6, IL-8, IL-10, TNF-α), and by light microscopy of stained smears. There was a 10-fold and higher increase in the amplitude of Lum-CL and Luc-CL after stimulation of neutrophils with CCM relative to CC. Adsorption of mucin onto prefabricated CC microparticles also contributed to activation of neutrophil CL, unlike mucin adsorption onto yeast cell walls (zymosan); adsorbed mucin partially suppressed zymosan-stimulated production of oxidants by neutrophils. Preliminary treatment of CCM with 0.1–10 mM NaOCl decreased subsequent activation of Lum-CL and Luc-CL of neutrophils depending on the used NaOCl concentration, presumably because of the surface mucin oxidation. Based on the results of ELISA, incubation of neutrophils with CCM downregulated IL-6 production but upregulated that of IL-8. IL-6 and IL-8 gene expression in neutrophils was not affected by CC or CCM according to RT2-PCR data, which means that post-translational regulation was involved. Light microscopy revealed adhesion of CC and CCM microparticles onto the neutrophils; CCM increased neutrophil aggregation with a tendency to form neutrophil extracellular traps (NETs). We came to the conclusion that the main features of neutrophil reaction to mucin–vaterite hybrid microparticles are increased oxidant production, cell aggregation, and NET-like structure formation, but without significant cytokine release (except for IL-8). This effect of mucin is not anion-specific since particles of powdered kidney stone (mainly calcium oxalate) in the present study or calcium phosphate nanowires in our previous report also activated Lum-CL and Luc-CL response of neutrophils after mucin sorption.

The extracellular sialidase NEU3 primes neutrophils.

Some extracellular glycoconjugates have sialic acid as the terminal sugar, and sialidases are enzymes that remove this sugar. Mammals have 4 sialidases and can be elevated in inflammation and fibrosis. In this report, we show that incubation of human neutrophils with the extracellular human sialidase NEU3, but not NEU1, NEU2 or NEU4, induces human male and female neutrophils to change from a round to a more amoeboid morphology, causes the primed human neutrophil markers CD11b, CD18, and CD66a to localize to the cell cortex, and decreases the localization of the unprimed human neutrophil markers CD43 and CD62-L at the cell cortex. NEU3, but not the other 3 sialidases, also causes human male and female neutrophils to increase their F-actin content. Human neutrophils treated with NEU3 show a decrease in cortical levels of Sambucus nigra lectin staining and an increase in cortical levels of peanut agglutinin staining, indicating a NEU3-induced desialylation. The inhibition of NEU3 by the NEU3 inhibitor 2-acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these results indicate that NEU3 can prime human male and female neutrophils, and that NEU3 is a potential regulator of inflammation.

Effect of Sevoflurane on Activation of Human Neutrophiles in Ex Vivo Models

The objective is to study the effect of different concentrations of sevoflurane on activation of human neutrophils in an ex vivo model.Subjects and Methods. The cell culture of venous blood neutrophils of 5 healthy men was used in this study. Neutrophil activation by lipopolysaccharide (LPS) and chemotaxis peptide N-formyl-methionine-leucine-phenylalanine (fMLP) as stimulants, was assessed by the expression level of CD11b and CD66b, IL-1β, IL-6 and IL-8, the level of phosphorylation of glycogen synthase β-kinase-3β (GSK-3β). Annexin V and propidium iodide were used to assess apoptosis. Neutrophils were exposed to 0.5, 1 and 1.5 MAC of sevoflurane to assess the effect of the drug on their activation.Results. Incubation of neutrophils with LPS and fMLP statistically significantly increased the expression of these molecules: treatment with LPS at the dose of 200 ng/ml increased CD11b and CD66b expression by 2.3 and 2.2 times (p = 0.002 and p = 0.001, respectively), while treatment with fMLP at 100 nM increased expression by 1.7 and 2.0 times (p = 0.025 and p = 0.03, respectively). When neutrophils were incubated with the same concentration of LPS after exposure to sevoflurane at a dose of 1.5 MAC, the level of CD11b and CD66b expression increased versus intact neutrophils. In this experiment, the change in CD11b expression was statistically insignificant (p = 0.055), the change in CD66b expression was statistically significant (p = 0.007). Thus, sevoflurane exposure at a dose of 1.5 MAC reduces proinflammatory activation of neutrophils induced by LPS.Conclusion. Stimulation of neutrophils by LPS was accompanied by dephosphorylation of GSK-3β, and exposure to 1.5 MAC of sevoflurane resulted in its phosphorylation. Thus, phosphorylation of GSK-3β in neutrophils by sevoflurane reduces the expression of CD11b and CD66b.

Гранулоцитарно-макрофагальный колониестимулирующий фактор: перспективы использования у детей с инфекционным мононуклеозом

Objective. To analyze the production of active oxygen species (AOS) by peripheral blood neutrophils in children with infectious mononucleosis (IM) after their exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Patients and methods. We examined 78 children aged 3 to 11 years in the acute period of IM (including 47 children aged 3–6 years and 31 children aged 7–11 years). The control group comprised 40 healthy children matched for age. Luminoldependent chemiluminescence (CL) of peripheral blood neutrophils was assessed using the method of De Sole et al. The following CL parameters were measured: time to reach the maximum (Tmax), maximum value (Imax), and the area (S) under the chemiluminescent curve. Results. Children with IM (from both age groups) demonstrated lower spontaneous lucigenin-dependent chemiluminescence of neutrophils than controls: Tmax (p &lt; 0.001 and p = 0.036, respectively), Imax (p = 0.024 and p = 0.025), and S (p = 0.01). Incubation of neutrophils with GM-CSF caused changes in luminol-dependent chemiluminescence only in children with MI from both age groups: Tmax decreased, whereas Imax and S increased. IM patients aged 7–11 years were found to have higher levels of chemiluminescence of neutrophils exposed to GM-CSF in vitro than IM patients aged 3–6 years (Tmax (p &lt; 0.001) and SGM-CSF/Ssp. (p = 0.024)). Conclusion. Our findings suggest that GM-CSF can increase functional activity of neutrophils. This might facilitate adaptive immunity at periphery. Key words: infectious mononucleosis, neutrophilic granulocytes, chemiluminescence of neutrophils, granulocyte-macrophage colony stimulating factor

Stimulation Of Neutrophil Oxidative Burst By Calcium Phosphate Particles With Adsorbed Mucin

Objective — Mucin can promote formation of gallstones via precipitation with calcium phosphate. The proinflammatory effect of mucin-coated particles is still unclear, and our aim was to study the role of mucin sorption in activation of neutrophil respiratory burst. Material and Methods — Polydisperse calcium phosphate nanowires (CP) were prepared from hot gelatin solution and according to scanning electron microscopy (SEM) had the length 1-10 μm and thickness 50-450 nm. CP were incubated in mucin or human serum albumin (HSA) giving CP-Muc and CP-HSA. Their hemolytic activity towards human erythrocytes was assayed, and neutrophil lucigenin- and luminol- chemiluminescence (Luc-CL and Lum-CL) response to CP, CP-HSA and CP-Muc was measured. Cytokine RNA was detected in neutrophils by means of reverse transcription with subsequent real-time PCR. Cytokines (IL-1β, IL-6, IL-8, IL-10) were assessed in cell medium by ELISA. Results and Conclusion — Hemolytic activity of CP was 3.0±0.5%, mucin sorption (0.019 mg/mg) reduced it to 0.24±0.04% (p&lt;0.05) as well as HSA. CP and CP-HSA stimulated neutrophil Lum-CL and Luc-Cl by 2-3 times vs. spontaneous values while for CP-Muc the effect was 10-fold and higher. No increased cytokine gene expression or cytokine secretion was detected after 1h incubation of neutrophils with samples. Obviously, sorption of mucin but not that of HSA stimulated generation of reactive oxygen and halogen species with no increase in cytokine production. Thus, the mucin-coated CP has the potential to contribute to gallstone-associated cholecystitis via oxidative damage of mucosa and epithelium.

Activation and Functional Priming of Blood Neutrophils in Non-Alcoholic Fatty Liver Disease Increases in Non-Alcoholic Steatohepatitis.

IntroductionIn non-alcoholic fatty liver disease (NAFLD), neutrophils in liver infiltrates are activated, which may contribute to disease progression towards non-alcoholic steatohepatitis (NASH). However, the functional status of the blood neutrophils remains unknown and their role in the disease mechanisms is thus uncertain. We therefore characterized activation and function of blood neutrophils in patients with NAFLD in relation to clinical disease markers and the NAFLD plasma milieu.MethodsWe studied 20 patients with NAFLD, among these 6 patients with NASH, and 14 healthy persons. Neutrophil activation, interleukin (IL)-8 production and oxidative burst were measured by flow cytometry on participants´ neutrophils and on healthy neutrophils exposed in vitro to plasma from the study participants.ResultsBlood neutrophils from the NASH patients showed a doubling in their expression of the activation marker CD62L. Also, all NAFLD patients had 50–100% increased expression of CD11b. Functionally, NASH neutrophils had 30% elevated IL-8 production and more than doubled spontaneous oxidative burst. In all NAFLD patients, higher spontaneous oxidative burst was associated with worse liver function. Incubation of healthy neutrophils with NAFLD plasma paradoxically slightly reduced CD62L and CD11b expression, and NASH plasma also reduced the frequency of IL-8-producing neutrophils.ConclusionIn NAFLD, blood neutrophils are activated, and in NASH also functionally primed. This suggests a progressive neutrophil aggressiveness already present with liver fat infiltration. However, NAFLD plasma in vitro, if anything, had the opposite effect on the healthy neutrophils so the NAFLD-related neutrophil activation cannot be attributed to humoral factors and remains unexplained.

Peroxiredoxin AhpC1 protects Pseudomonas aeruginosa against the inflammatory oxidative burst and confers virulence

Pseudomonas aeruginosa is an opportunistic bacterium in patients with cystic fibrosis and hospital acquired infections. It presents a plethora of virulence factors and antioxidant enzymes that help to subvert the immune system. In this study, we identified the 2-Cys peroxiredoxin, alkyl-hydroperoxide reductase C1 (AhpC1), as a relevant scavenger of oxidants generated during inflammatory oxidative burst and a mechanism of P. aeruginosa (PA14) escaping from killing. Deletion of AhpC1 led to a higher sensitivity to hypochlorous acid (HOCl, IC50 3.2 ± 0.3 versus 19.1 ± 0.2 μM), hydrogen peroxide (IC50 91.2 ± 0.3 versus 496.5 ± 6.4 μM) and the organic peroxide urate hydroperoxide. ΔahpC1 strain was more sensitive to the killing by isolated neutrophils and less virulent in a mice model of infection. All mice intranasally instilled with ΔahpC1 survived as long as they were monitored (15 days), whereas 100% wild-type and ΔahpC1 complemented with ahpC1 gene (ΔahpC1 attB:ahpC1) died within 3 days. A significantly lower number of colonies was detected in the lung and spleen of ΔahpC1-infected mice. Total leucocytes, neutrophils, myeloperoxidase activity, pro-inflammatory cytokines, nitrite production and lipid peroxidation were much lower in lungs or bronchoalveolar liquid of mice infected with ΔahpC1. Purified AhpC neutralized the inflammatory organic peroxide, urate hydroperoxide, at a rate constant of 2.3 ± 0.1 × 106 M−1s−1, and only the ΔahpC1 strain was sensitive to this oxidant. Incubation of neutrophils with uric acid, the urate hydroperoxide precursor, impaired neutrophil killing of wild-type but improved the killing of ΔahpC1. Hyperuricemic mice presented higher levels of serum cytokines and succumbed much faster to PA14 infection when compared to normouricemic mice. In summary, ΔahpC1 PA14 presented a lower virulence, which was attributed to a poorer ability to neutralize the oxidants generated by inflammatory oxidative burst, leading to a more efficient killing by the host. The enzyme is particularly relevant in detoxifying the newly reported inflammatory organic peroxide, urate hydroperoxide.

Effect of Carcinoembryonic Antigen-related Cell Adhesion Molecule-binding Recombinant Polypeptide on the Killing of Neisseria meningitidis by Human Neutrophils

Background: Neutrophils are an essential part of innate immunity and play a crucial role in controlling infection caused by Neisseria meningitidis. The Moraxella catarrhalis ubiquitous surface protein A1 (UspA1)-based recombinant polypeptide binds to the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) 1 receptor on host cells and blocks binding of the mucosal pathogens to human epithelial cells and T cells. Aim of the study: Since the CEACAM1 receptor is expressed on neutrophils, the aim of this study was to investigate the effect of CEACAM1-binding recombinant polypeptide on the ability of neutrophils to kill Neisseria meningitidis. Methods: The effect of CEACAM1-binding recombinant polypeptide on the phagocytic killing of Neisseria meningitidis by neutrophils was assessed by incubation of neutrophils with CEACAM1-binding recombinant polypeptide (UspA1 527–665) or with CEACAM1-non-binding polypeptide control (UspA1 659–863) for one hour before infection with Neisseria meningitidis. The surviving bacteria were released and counted. Results: 30 minutes after infection of neutrophils with Neisseria meningitidis, the survival of bacteria in presence of CEACAM1-binding recombinant polypeptide was 64% compared to 52% with control peptide and 43% without peptide. However, one-hour after infection, the surviving bacteria was 32% in presence of CEACAM1-binding recombinant polypeptide compared to 18% with control peptide and 22% without peptides. Conclusion: Although CEACAM1-binding polypeptide reduced the killing of Neisseria meningitidis by neutrophils, it did not entirely stop phagocytosis and killing of bacteria.

Lipid Secretion by Parasitic Cells of Coccidioides Contributes to Disseminated Disease.

Coccidioides is a soil-borne fungal pathogen and causative agent of a human respiratory disease (coccidioidomycosis) endemic to semi-desert regions of southwestern United States, Mexico, Central and South America. Aerosolized arthroconidia inhaled by the mammalian host first undergo conversion to large parasitic cells (spherules, 80–100 μm diameter) followed by endosporulation, a process by which the contents of spherules give rise to multiple endospores. The latter are released upon rupture of the maternal spherules and establish new foci of lung infection. A novel feature of spherule maturation prior to endosporulation is the secretion of a lipid-rich, membranous cell surface layer shed in vivo during growth of the parasitic cells and secretion into liquid culture medium during in vitro growth. Chemical analysis of the culture derived spherule outer wall (SOW) fraction showed that it is composed largely of phospholipids and is enriched with saturated fatty acids, including myristic, palmitic, elaidic, oleic, and stearic acid. NMR revealed the presence of monosaccharide- and disaccharide-linked acylglycerols and sphingolipids. The major sphingolipid components are sphingosine and ceramide. Primary neutrophils derived from healthy C57BL/6 and DBA/2 mice incubated with SOW lipids revealed a significant reduction in fungicidal activity against viable Coccidioides arthroconidia compared to incubation of neutrophils with arthroconidia alone. Host cell exposure to SOW lipids had no effect on neutrophil viability. Furthermore, C57BL/6 mice that were challenged subcutaneously with Coccidioides arthroconidia in the presence of the isolated SOW fraction developed disseminated disease, while control mice challenged with arthroconidia alone by the same route showed no dissemination of infection. We hypothesize that SOW lipids contribute to suppression of inflammatory response to Coccidioides infection. Studies are underway to characterize the immunosuppressive mechanism(s) of SOW lipids.

The Modulated Expression of Mo5, a Human Myelomonocytic Plasma Membrane Antigen

Mo5 is a 94-kd protein antigen expressed by human peripheral blood monocytes, neutrophils, and by all bone marrow myeloperoxidase-positive myeloid precursors (promyelocytes, myelocytes, metamyelocytes, and bands). Mo5 is borne by the malignant cells of 74% of patients (N = 27) with acute monocytic leukemia (French-American-British [FAB] group M4, M5), and 50% of patients (N = 38) with acute granulocytic leukemia (FAB M1, M2, and M3). Nonmyeloid cells in peripheral blood and bone marrow are Mo5-negative. The surface expression of Mo5 by myeloid cells is modulated by several experimental conditions: Exposure of neutrophils to calcium ionophore (1 mumol/L, 37 degrees C, ten minutes) under conditions resulting in degranulation of specific granules produces a three- to fourfold increase in the plasma membrane density of Mo5 antigen. This suggests that, in neutrophils, there is an intracellular pool of Mo5 antigen, which may be associated with specific granules, and that granule-associated Mo5 is translocated to the plasma membrane upon degranulation. Conversely, incubation of monocytes, neutrophils, U-937, and Mo5-positive leukemia cells in medium containing anti-Mo5 monoclonal antibody results in a significant decrease in surface Mo5 expression. This loss of surface Mo5 is a rapid, temperature-dependent process (occurring within 30 minutes at 37 degrees C) that is produced by divalent anti-Mo5 immunoglobulin [F(ab')2 but not F(ab)]. After down-modulation, Mo5 is reexpressed by monocytes within 48 hours. Mo5 is therefore a human myelomonocytic differentiation antigen whose expression is modulated up or down depending on the nature of extracellular stimuli.

Влияние хлорида лития на активацию нейтрофилов при развитии синдрома системного воспалительного ответа у пациентов после операций на сердце с использованием аппарата искусственного кровообращения

Цель исследования - изучение in vitro действия хлорида лития на активность нейтрофилов человека при действии сывороток пациентов с синдромом системного воспалительного ответа, развившемся после операций на сердце с искусственным кровообращением. Методика. Исследование проводили in vitro на нейтрофилах, выделенных из крови 6 здоровых доноров. Нейтрофилы активировали при помощи сыворотки пациентов с синдромом системного воспалительного ответа (ССВО), перенесших операции на сердце с искусственным кровообращением (ИК). Активность нейтрофилов оценивали с использованием флуоресцентных антител к маркерам дегрануляции CD11b и CD66b. Уровень апоптоза и некроза нейтрофилов оценивали через 22 ч после выделения из крови здоровых доноров; количественная оценка была проведена с использованием аннексина V и иодистого пропидия на проточном цитофлуориметре. Интактные и активированные нейтрофилы обрабатывали раствором хлорида лития в концентрациях 0,3; 3,0 и 9,0 мМ. Результаты. Инкубация нейтрофилов с сывороткой крови пациентов с ССВО после операций на сердце с ИК увеличивала экспрессию CD11b в 1,5 раза и экспрессию CD66b в 1,4 раза в сравнении с экспрессией на интактных нейтрофилах. Инкубация нейтрофилов с сывороткой крови пациентов с ССВО и раствором хлорида лития в концентрациях 3,0 и 9,0 мМ приводило к статистически значимому снижению уровня экспрессии CD11b CD66b на поверхности нейтрофилов в сравнении с активированными контрольными. Установлено, что хлорид лития в концентрациях 3,0 и 9,0 мМ возвращал уровни экспрессии CD11b и CD66b на активированных нейтрофилах к уровню экспрессии на интактных нейтрофилах. В концентрации 0,3 мМ хлорид лития, используемый при инкубации с активированными нейтрофилами, не вызывал значимого снижения экспрессии CD11b и CD66b относительно контрольных активированных нейтрофилов. Экспрессия CD11b и CD66b на активированных нейтрофилах при их инкубации с хлоридом лития в концентрации 0,3 мМ была значимо выше относительно экспрессии данных молекул на интактных нейтрофилах. Сыворотка пациентов с развившемся ССВО снижала спонтанный апоптоз нейтрофилов, а раствор хлорида лития в концентрации 3,0 или 9,0 мМ, добавленный в среду инкубации, увеличивал способность нейтрофилов к спонтанному апоптозу. Заключение. Хлорид лития оказывал противовоспалительный эффект снижал дегрануляцию и активацию нйтрофилов посредством уменьшения уровня экспрессии молекул CD11b и CD66b на поверхности нейтрофилов, которые предварительно были активированы сыворотками пациентов с ССВО. В концентрации 3,0 мМ и выше хлорид лития индуцировал спонтанный апоптоз нейтрофилов, активированных сыворотками пациентов с ССВО после операций на сердце с ИК. The aim of this work was to study the anti-inflammatory effect of lithium chloride on human neutrophils in vitro under the action of the serum of patients with systemic inflammatory response syndrome (SIRS), which developed after on-pump cardiac surgery. Methods. The study was performed on neutrophils isolated from the blood of five healthy donors, which was activated using serum from patients with SIRS. Neutrophil activity was assessed using fluorescent antibodies to CD11b and CD66b degranulation markers. The level of apoptosis and necrosis of human neutrophils was evaluated 22 hours after isolation. Quantification was performed using annexin V and propidium iodide on a flow cytometer. Intact and activated neutrophils were treated with 0.3, 3.0 аnd 9.0 mM lithium chlorides. Results. Incubation of neutrophils with the blood serum of patients with SIRS after on-pump cardiac surgery increased the expression of CD11b by 1.5 times and the expression of CD66b by 1.4 times compared to expression on intact neutrophils. Incubation of neutrophils with blood serum of patients with SIRS and 3.0 and 9.0 mM lithium chloride solutions led to a statistically significant decrease in the level of expression of CD11b CD66b on the surface of neutrophils in comparison with control activated neutrophils. It was found that 3.0 and 9.0 mM lithium chloride solutions returned the expression levels of CD11b and CD66b on activated neutrophils to the expression level on intact neutrophils. 0.3 mM of lithium chloride, used during incubation with activated neutrophils, did not cause a significant decrease in the expression of CD11b and CD66b relative to control activated neutrophils. The expression of CD11b and CD66b on activated neutrophils during their incubation with 0.3 mM of lithium chloride was significantly higher relative to the expression of these molecules on intact neutrophils. The serum of patients with advanced SIRS decreased the ability of neutrophils to spontaneous apoptosis. 3.0 or 9.0 mM lithium chloride solutions added to the incubation medium increased the ability of neutrophils to spontaneous apoptosis. Conclusion. Lithium chloride reduced the degranulation and activation of neutrophils by reducing the expression level of CD11b and CD66b molecules on the surface of neutrophils that were previously activated by the serum of patients with SIRS. This effect determines the anti-inflammatory influence of lithium chloride. Lithium chloride at 3.0 mM and higher induced spontaneous apoptosis of neutrophils activated by the serum of patients with SIRS after on-pump cardiac surgery.

Prevention of Pulmonary Injury in Isolated Perfused Rat Lungs by Activated Human Neutrophils Preincubated With Anti-Mo1 Monoclonal Antibody

Neutrophil activation results in neutrophil adherence and may subsequently cause lung injury through the generation of oxidants, release of granule proteases, and generation of a variety of mediator substances. We hypothesized that inhibition of neutrophil adherence and subsequent lung sequestration would attenuate the lung injury caused by activated neutrophils. Using isolated perfused rat lungs, we determined if anti-Mo1 monoclonal antibody (binds to the alpha subunit of a neutrophil glycoprotein [gp 155.94] that facilitates adherence) would attenuate lung neutrophil sequestration and lung injury caused by human neutrophils stimulated by phorbol myristate acetate (PMA). PMA-stimulated neutrophils but not PMA or neutrophils alone caused lung injury as assessed by accumulation of 125I-bovine serum albumin into lung parenchyma and alveolar lavage fluid. Incubation of neutrophils with anti-Mo1 antibody prior to stimulation with PMA attenuated lung injury and neutrophil sequestration. Furthermore, a histological survey revealed that anti-Mo1 antibody inhibited neutrophils present in the lung from spreading following exposure to PMA. Anti-Mo1 antibody did not inhibit PMA-stimulated neutrophil release of granule constituents or toxic O2 metabolites as evidenced by lysozyme and lactoferrin release or the reduction of ferricytochrome c in the lung perfusate. The inhibition of lung injury caused by the anti-Mo1 antibody was not likely due to a nonspecific effect of the antibody, since another murine monoclonal antibody of the same class (anti-Mo5) did not inhibit lung neutrophil sequestration or lung injury. Thus, in this experimental model, interference with the close approximation of the neutrophil to its target site inhibited the ability of the activated human neutrophil to cause injury.

The Effect of Lithium Chloride on Neutrophil Activation on Exposure to Serum of Patients with Septic Shock

The aim of the study: to examine the anti-inflammatory effect of lithium chloride by exposing the human neutrophils to serum of patients with septic shockin vitro.Material and methods. The study was carried out on neutrophils extracted from the blood of 6 healthy donors, which were activated with serum from patients with septic shock. The neutrophil activity was evaluated with fluorescent antibodies to the CD11b and CD66b markers of degranulation. The level of human neutrophil apoptosis and necrosis was assessed 22 hours after extraction; quantitative assessment was made using annexin V and propidium iodide with flow cytofluorimetry. Intact and activated neutrophils were treated with 0.3, 3.0 and 9.0 mmol lithium chloride solution.Results. The level of CD11b expression on the surface of intact neutrophils (healthy donors) was 3434.50 [3311.0-3799.0] arbitrary fluorescence units (AFU). Incubation of neutrophils with serum of patients with septic shock increased CD11b expression 2.5 times to 8589.0 [7279.0-11258.0] AFU (P=0.005) vs intact leukocytes, and increased CD66b expression 2.7 times up to 27 600.0 [22 999.0-28 989.0] AFU ((P=0.005) vs intact neutrophils. Lithium chloride in concentrations of 0.3, 3.0 and 9.0 mmol in a dose-dependent manner reduced the level of expression of CD11b and CD66b molecules on the surface of activated neutrophils. Septic serum reduced spontaneous neutrophil apoptosis, and 3.0 mmol and higher lithium chloride solution induced spontaneous neutrophil apoptosis.Conclusion. Lithium chloride reduces the activation of neutrophils preactivated by serum of patients with septic shock, reduces expression of CD11b and CD66b molecules on the neutrophil surface, inhibiting the process of their activation (degranulation). Lithium chloride in concentration of 3.0 mmol and higher is able to induce spontaneous apoptosis of neutrophils activated by serum of patients with septic shock.

IgG-aggregates rapidly upregulate FcgRI expression at the surface of human neutrophils in a FcgRII-dependent fashion: A crucial role for FcgRI in the generation of reactive oxygen species.

Autoimmune complexes are an important feature of several autoimmune diseases such as lupus, as they contribute to tissue damage through the activation of immune cells. Neutrophils, key players in lupus, interact with immune complexes through Fc gamma receptors (FcgR). Incubation of neutrophils with aggregated-IgGs caused degranulation and increased the surface expression of FcgRI within minutes in a concentration-dependent fashion. After 30minutes, IgG aggregates (1mg/mL) upregulated FcgRI by 4.95±0.45-fold. FcgRI-positive neutrophils reached 67.24%±6.88% on HA-IgGs stimulated neutrophils, from 3.12%±1.62% in non-stimulated cells, ranking IgG-aggregates among the most potent known agonists. FcgRIIa, and possibly FcgRIIIa, appeared to mediate this upregulation. Also, FcgRI-dependent signaling proved necessary for reactive oxygen species (ROS) production in response to IgG-aggregates. Finally, combinations of bacterial materials with aggregates dramatically boosted ROS production. This work suggests FcgRI as an essential component in the response of human neutrophils to immune complexes leading to the production of ROS, which may help explain how neutrophils contribute to tissue damage associated with immune complex-associated diseases, such as lupus.

CXCR4hi effector neutrophils in sickle cell anemia: potential role for elevated circulating serotonin (5-HT) in CXCR4hi neutrophil polarization

Leukocyte recruitment and heterocellular aggregate formation drive the inflammatory vaso-occlusive processes associated with sickle cell anemia (SCA). We characterized neutrophils in a population of patients with SCA and investigated whether platelet-derived molecules can induce phenotypic alterations in this cell type. Imaging flow cytometry analysis demonstrated that the frequency of circulating CXCR4hi neutrophils was significantly higher in steady-state SCA individuals than in healthy control individuals and that these cells presented increased CD11b activation and toll-like receptor-4 expression. SCA neutrophils display increased neutrophil-platelet aggregation, and CXCR4hi neutrophils demonstrated augmented neutrophil-platelet aggregate frequency with a higher mean number of platelets adhered per neutrophil. Importantly, incubation of neutrophils with platelets significantly elevated their CXCR4 expression, while SCA plasma was found to induce CXCR4hi neutrophil polarization significantly more than control plasma. SCA individuals had significantly increased plasma levels of serotonin (5-HT), and serotonin molecule and SCA plasma induced neutrophil CXCR4 expression in a serotonin-receptor-dependent manner. Thus, the augmented CXCR4hi neutrophil population may contribute to mechanisms that promote vaso-occlusion in SCA; furthermore, circulating serotonin, derived from platelet activation, may play a role in the polarization of neutrophils, suggesting that serotonin-receptor antagonists or serotonin reuptake inhibitors could represent therapeutic approaches to reduce neutrophil activation in SCA.

Role of redox imbalance and cytokines in mediating oxidative damage and disease progression of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disorder wherein the contributory role of oxidative stress has been established in the synovial fluid. As availability of synovial fluid is limited, this study aimed to evaluate in the peripheral blood of patients with RA, the relationship if any, between the extent of oxidative stress in terms of generation of reactive oxygen species (ROS) in neutrophils, plasma NADPH oxidase and myeloperoxidase activity with markers of oxidative damage, circulating cytokines and disease activity score (DAS28). In patients with RA, neutrophils in peripheral blood demonstrated an enhanced generation of ROS, coupled with depletion of free radical scavenging activity. Furthermore, the NADPH oxidase and myeloperoxidase activity was enhanced as were markers of damage. There was a positive correlation between the DAS 28 and generation of ROS, NADPH oxidase and myeloperoxidase activity as also with oxidative stress mediated protein carbonylation. Patients with RA demonstrated an increase in proinflammatory (IL-17, IL-23, and IFN-γ) and some anti-inflammatory (IL-4, IL-5, and TGF-β) cytokines. Although the levels of IL-17 correlated positively with generation of ROS, myeloperoxidase, markers of protein damage and DAS28, IL-23 correlated positively only with protein damage, and negatively with free radical scavenging activity. Importantly, incubation of neutrophils from healthy donors with plasma or SF from patients with RA translated into an enhanced generation of ROS, along with an elevation of intracellular proinflammatory cytokines. Taken together, in patients with RA, circulating neutrophils mediated a shift in the oxidant/antioxidant balance favouring the former, which translated into protein damage and contributed towards disease progression.