scholarly journals Interleukin 8 Elicits Rapid Physiological Changes in Neutrophils That Are Altered by Inflammatory Conditions

2021 ◽  
pp. 1-17
Author(s):  
Stefan Bernhard ◽  
Stefan Hug ◽  
Alexander Elias Paul Stratmann ◽  
Maike Erber ◽  
Laura Vidoni ◽  
...  
Keyword(s):  
Systemic Inflammation ◽  
Severe Trauma ◽  
Induced Response ◽  
Neutrophil Granulocytes ◽  
Inflammatory Conditions ◽  
Flow Cytometric ◽  
Time Flow ◽  
Flow Cytometric Measurement ◽  
Detailed Kinetics

A sufficient response of neutrophil granulocytes stimulated by interleukin (IL)-8 is vital during systemic inflammation, for example, in sepsis or severe trauma. Moreover, IL-8 is clinically used as biomarker of inflammatory processes. However, the effects of IL-8 on cellular key regulators of neutrophil properties such as the intracellular pH (pH<sub>i</sub>) in dependence of ion transport proteins and during inflammation remain to be elucidated. Therefore, we investigated in detail the fundamental changes in pH<sub>i</sub>, cellular shape, and chemotactic activity elicited by IL-8. Using flow cytometric methods, we determined that the IL-8-induced cellular activity was largely dependent on specific ion channels and transporters, such as the sodium-proton exchanger 1 (NHE1) and non-NHE1-dependent sodium flux. Exposing neutrophils in vitro to a proinflammatory micromilieu with N-formyl-Met-Leu-Phe, LPS, or IL-8 resulted in a diminished response regarding the increase in cellular size and pH. The detailed kinetics of the reduced reactivity of the neutrophil granulocytes could be illustrated in a near-real-time flow cytometric measurement. Last, the LPS-mediated impairment of the IL-8-induced response in neutrophils was confirmed in a translational, animal-free human whole blood model. Overall, we provide novel mechanistic insights for the interaction of IL-8 with neutrophil granulocytes and report in detail about its alteration during systemic inflammation.

A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays

2019 ◽  
Vol 44 (6) ◽  
pp. 810-821
Author(s):  
Edibe Avci ◽  
Yeliz Z. Akkaya-Ulum ◽  
Digdem Yoyen-Ermis ◽  
Gunes Esendagli ◽  
Banu Balci-Peynircioglu
Keyword(s):  
Flow Cytometry ◽  
Cell Migration ◽  
Neutrophil Migration ◽  
Cost Effective ◽  
In Vitro Assays ◽  
Human Neutrophils ◽  
Neutrophil Granulocytes ◽  
Inflammatory Conditions ◽  
Migration Analysis

Abstract Background Neutrophil-mediated killing of pathogens is one of the most significant functions of the primary defense of the host. Neutrophil activity and migration play a key role in inflammatory conditions. To gain insights into the interactions between neutrophils and neutrophil migration-related disorders, a large number of sophisticated methods have been developed. The technical limitations of isolating highly purified neutrophil populations, minimizing both cell death and activation during the isolation process, and the short lifespan of neutrophils present challenges for studying specific functions of neutrophils in vitro. In this study, we aimed to evaluate a separation medium-based density gradient method to obtain highly purified neutrophil populations and combined this protocol with a model for studying neutrophil migration in-vitro. Materials and methods Human granulocytes were isolated using Lympholyte-poly solution. The purity and viability of isolated neutrophils were assessed by flow cytometry and morphological analysis. Neutrophil activation was confirmed by immunocytochemistry. Lastly, filter assay was performed to measure neutrophil chemotaxis. Results and discussion All validation experiments revealed that this method was capable of generating a highly purified neutrophil population for further functional in-vitro assays. Consequently, this study demonstrates a quick, cost effective, and easy-to-follow model, and may be a significant alternative to isolation methods that need extra subsequent steps such as flow cytometry-based cell sorting for reaching highly purified neutrophil population. Conclusion The suggested combination of methods for the isolation and cell migration analysis of human neutrophils is highly recommended to use for disease models involving neutrophil migration such as autoinflammatory disorders.

A novel in vitro sperm head decondensation protocol for rapid flow cytometric measurement of deoxyribonucleic acid content

2013 ◽  
Vol 99 (7) ◽  
pp. 1857-1861 ◽  
Author(s):  
Niki Antonucci ◽  
Sabrina Manes ◽  
Bruna Corradetti ◽  
Gian Carlo Manicardi ◽  
Andrea Borini ◽  
...  
Keyword(s):  
Acid Content ◽  
Deoxyribonucleic Acid ◽  
Sperm Head ◽  
Flow Cytometric ◽  
Rapid Flow ◽  
Flow Cytometric Measurement

scholarly journals Impaired Surface Expression of HLA-DR, TLR2, TLR4, and TLR9 in Ex Vivo-In Vitro Stimulated Monocytes from Severely Injured Trauma Patients

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
David Heftrig ◽  
Ramona Sturm ◽  
Elsie Oppermann ◽  
Kerstin Kontradowitz ◽  
Katrin Jurida ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Surface Expression ◽  
Severe Trauma ◽  
Trauma Patients ◽  
Severely Injured ◽  
Tlr4 Expression ◽  
Inflammatory Conditions ◽  
Hla Dr ◽  
Stimulation Of

Objective. Trauma patients (TP) frequently develop an imbalanced immune response that often causes infectious postinjury complications. Monocytes show a diminished capability of both producing proinflammatory cytokines and antigen presentation after trauma. TLR2, TLR4, and TLR9 recognize pathogens and subsequently activate monocytes. While there are conflictive data about TLR2 and TLR4 expression after trauma, no studies about the expression of TLR2, TLR4, TLR9, and HLA-DR on monocytes from TP after their secondary ex vivo-in vitro “hit” have been reported.Methods/Results. Ex vivo-in vitro lipopolysaccharide- (LPS-) stimulated blood from TP showed diminished interleukin- (IL-) 1β-release in TP for five postinjury days compared to healthy volunteers (HV). The recovery was observed at day 5. In parallel, monocytes from TP showed an impaired capability of TLR2, TLR4, and TLR9 expression after secondary stimulation compared to HV, while the measurement of unstimulated samples showed significant reduction of TLR4 and TLR9 at ED. Furthermore, HLA-DR decreased after trauma and was even more profound by stimulation of monocytes. Ratio of monocytes to leukocytes was significantly increased at days 6 and 7 after trauma compared to HV.Conclusion. Impaired expression of TLRs and HLA-DR in acute inflammatory conditions may be responsible for the well-described monocyte paralysis after severe trauma.

Flow cytometric measurement of cell cycle kinetics in rat Walker-256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine

Cytometry ◽  
1991 ◽  
Vol 12 (1) ◽  
pp. 33-41 ◽  
Author(s):  
Franz Fogt ◽  
Jennifer Wan ◽  
Carl O'Hara ◽  
Bruce R. Bistrian ◽  
George L. Blackburn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Pulse Labelling ◽  
Cell Cycle Kinetics ◽  
Flow Cytometric ◽  
Flow Cytometric Measurement ◽  
Walker 256

Proteomics-Based Characterization of the Effects of MMP14 on the Protein Content of Exosomes from Corneal Fibroblasts

2020 ◽  
Vol 27 (10) ◽  
pp. 979-988
Author(s):  
Kyu-Yeon Han ◽  
Jin-Hong Chang ◽  
Dimitri T. Azar
Keyword(s):  
Protein Content ◽  
Catalytic Domain ◽  
Protein Composition ◽  
Proteomics Analysis ◽  
Knock Out ◽  
Corneal Fibroblast ◽  
Flow Cytometric ◽  
Corneal Fibroblasts ◽  
Liquid Chromatography Tandem Mass

Background: Exosomes secreted by corneal fibroblasts contain matrix metalloproteinase (MMP) 14, which is known to influence pro-MMP2 accumulation on exosomes. Accordingly, we hypothesized that the enzymatic activity of MMP14 may alter the protein content of corneal fibroblast- secreted exosomes. Objective: The aim of this study was to investigate the effects of MMP14 on the composition and biological activity of corneal fibroblast-derived exosomes. Methods: Knock out of the catalytic domain (ΔExon4) of MMP14 in corneal fibroblasts was used to determine the effect of MMP14 expression on the characteristics of fibroblast-secreted exosomes. The amount of secreted proteins and their size distribution were measured using Nano Tracking Analysis. Proteins within exosomes from wild-type (WT) and ΔExon4-deficient fibroblasts were identified by liquid chromatography-tandem mass spectrometry (MS/MS) proteomics analysis. The proteolytic effects of MMP14 were evaluated in vitro via MS identification of eliminated proteins. The biological functions of MMP14-carrying exosomes were investigated via fusion to endothelial cells and flow cytometric assays. Results: Exosomes isolated from WT and ΔExon4-deficient fibroblasts exhibited similar size distributions and morphologies, although WT fibroblasts secreted a greater amount of exosomes. The protein content, however, was higher in ΔExon4-deficient fibroblast-derived exosomes than in WT fibroblast-derived exosomes. Proteomics analysis revealed that WT-derived exosomes included proteins that regulated cell migration, and ΔExon4 fibroblast-derived exosomes contained additional proteins that were cleaved by MMP14. Conclusion: Our findings suggest that MMP14 expression influences the protein composition of exosomes secreted by corneal fibroblasts, and through those biological components, MMP14 in corneal fibroblasts derived-exosomes may regulate corneal angiogenesis.

scholarly journals Anti-Inflammatory and Immunomodulatory Effects of the Grifola frondosa Natural Compound o-Orsellinaldehyde on LPS-Challenged Murine Primary Glial Cells. Roles of NF-κβ and MAPK

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 806
Author(s):  
Sarah Tomas-Hernandez ◽  
Jordi Blanco ◽  
Santiago Garcia-Vallvé ◽  
Gerard Pujadas ◽  
María José Ojeda-Montes ◽  
...  
Keyword(s):  
Natural Compound ◽  
Underlying Disease ◽  
Grifola Frondosa ◽  
Beneficial Effects ◽  
Inflammatory Conditions ◽  
Inflammatory Mechanism ◽  
Inflammatory Phenotype ◽  
Mice Brain ◽  
Anti Inflammatory

In response to foreign or endogenous stimuli, both microglia and astrocytes adopt an activated phenotype that promotes the release of pro-inflammatory mediators. This inflammatory mechanism, known as neuroinflammation, is essential in the defense against foreign invasion and in normal tissue repair; nevertheless, when constantly activated, this process can become detrimental through the release of neurotoxic factors that amplify underlying disease. In consequence, this study presents the anti-inflammatory and immunomodulatory properties of o-orsellinaldehyde, a natural compound found by an in silico approach in the Grifola frondosa mushroom, in astrocytes and microglia cells. For this purpose, primary microglia and astrocytes were isolated from mice brain and cultured in vitro. Subsequently, cells were exposed to LPS in the absence or presence of increasing concentrations of this natural compound. Specifically, the results shown that o-orsellinaldehyde strongly inhibits the LPS-induced inflammatory response in astrocytes and microglia by decreasing nitrite formation and downregulating iNOS and HO-1 expression. Furthermore, in microglia cells o-orsellinaldehyde inhibits NF-κB activation; and potently counteracts LPS-mediated p38 kinase and JNK phosphorylation (MAPK). In this regard, o-orsellinaldehyde treatment also induces a significant cell immunomodulation by repolarizing microglia toward the M2 anti-inflammatory phenotype. Altogether, these results could partially explain the reported beneficial effects of G. frondosa extracts on inflammatory conditions.

scholarly journals The Responses of Bioactive Betanin Pigment and Its Derivatives from a Red Beetroot (Beta vulgaris L.) Betalain-Rich Extract to Hypochlorous Acid

2021 ◽  
Vol 22 (3) ◽  
pp. 1155
Author(s):  
Karolina Starzak ◽  
Katarzyna Sutor ◽  
Tomasz Świergosz ◽  
Boris Nemzer ◽  
Zbigniew Pietrzkowski ◽  
...  
Keyword(s):  
Beta Vulgaris ◽  
Hypochlorous Acid ◽  
Innate Immune ◽  
Inflammatory Factors ◽  
Oxygen Species ◽  
Plant Pigments ◽  
Important Food ◽  
Inflammatory Conditions ◽  
Ph Range

Neutrophils produce hypochlorous acid (HOCl) as well as other reactive oxygen species as part of a natural innate immune response in the human body; however, excessive levels of HOCl can ultimately be detrimental to health. Recent reports suggest that betacyanin plant pigments can act as potent scavengers of inflammatory factors and are notably effective against HOCl. Comparison of the in vitro anti-hypochlorite activities of a novel betalain-rich red beetroot (Beta vulgaris L.) extract with its pure betalainic pigments revealed that the extract had the highest anti-hypochlorite activity, far exceeding the activity of all of the betalainic derivatives and selected reference antioxidants. This suggests that it may be an important food-based candidate for management of inflammatory conditions induced by excessive HOCl production. Among all pigments studied, betanidin exhibited the highest activity across the pH range.

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