Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation

AbstractDuring eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.

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The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness

TH Open ◽

10.1055/s-0040-1714254 ◽

2020 ◽

Vol 04 (03) ◽

pp. e163-e172

Author(s):

Juergen Koessler ◽

Philipp Klingler ◽

Marius Niklaus ◽

Katja Weber ◽

Angela Koessler ◽

...

Keyword(s):

Cold Storage ◽

Whole Blood ◽

Room Temperature ◽

Purinergic Receptor ◽

Adenosine Diphosphate ◽

Receptor Expression ◽

Activation Markers ◽

Inhibitory Pathways ◽

The Impact ◽

Induced Aggregation

Abstract Introduction Cold storage of platelets is considered to contribute to lower risk of bacterial growth and to more efficient hemostatic capacity. For the optimization of storage strategies, it is required to further elucidate the influence of refrigeration on platelet integrity. This study focused on adenosine diphosphate (ADP)-related platelet responsiveness. Materials and Methods Platelets were prepared from apheresis-derived platelet concentrates or from peripheral whole blood, stored either at room temperature or at 4°C. ADP-induced aggregation was tested with light transmission. Activation markers, purinergic receptor expression, and P2Y12 receptor function were determined by flow cytometry. P2Y1 and P2X1 function was assessed by fluorescence assays, cyclic nucleotide concentrations by immunoassays, and vasodilator-stimulated phosphoprotein (VASP)-phosphorylation levels by Western blot analysis. Results In contrast to room temperature, ADP-induced aggregation was maintained under cold storage for 6 days, associated with elevated activation markers like fibrinogen binding or CD62P expression. Purinergic receptor expression was not essentially different, whereas P2Y1 function deteriorated rapidly at cold storage, but not P2Y12 activity. Inhibitory pathways of cold-stored platelets were characterized by reduced responses to nitric oxide and prostaglandin E1. Refrigeration of citrated whole blood also led to the attenuation of induced inhibition of platelet aggregation, detectable within 24 hours. Conclusion ADP responsiveness is preserved under cold storage for 6 days due to stable P2Y12 activity and concomitant disintegration of inhibitory pathways enabling a higher reactivity of stored platelets. The ideal storage time at cold temperature for the highest hemostatic effect of platelets should be evaluated in further studies.

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Interleukin 8 Secretion from Monocytes of Subjects Heterozygous for the ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutation Is Altered

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.5.819-824.2004 ◽

2004 ◽

Vol 11 (5) ◽

pp. 819-824 ◽

Cited By ~ 52

Author(s):

Munir M. Zaman ◽

Andres Gelrud ◽

Omer Junaidi ◽

Meredith M. Regan ◽

Michel Warny ◽

...

Keyword(s):

Cystic Fibrosis ◽

Mapk Signaling ◽

Receptor Expression ◽

Interleukin 8 ◽

Healthy Controls ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Surface Receptors ◽

Cftr Mutations ◽

Cystic Fibrosis Transmembrane

ABSTRACT Patients with cystic fibrosis (CF) exhibit an excessive host inflammatory response. The aim of this study was to determine (i) whether interleukin 8 (IL-8) secretion is increased from monocytes from subjects heterozygous as well as homozygous for cystic fibrosis transmembrane conductance regulator (CFTR) mutations and (ii) whether this is due to increased cell surface lipopolysaccharide (LPS) receptors or, alternatively, increased activation of mitogen-activated protein kinases (MAPK). The basal level of IL-8 secretion was higher from monocytes from CF patients than from monocytes from healthy controls (P = 0.02) and obligate heterozygotes (parents of the CF patients). The 50% effective concentrations for LPS-induced IL-8 production for monocytes from both CF patients and obligate heterozygotes were 100-fold lower than those for monocytes from healthy controls (P < 0.05). No differences in the levels of IL-1β production were seen between these groups. Expression of the LPS surface receptors CD14 and Toll-like receptor 4 were not different between CF patients and healthy controls. In contrast, phosphorylation of the MAPKs p38 and ERK occurred at lower doses of LPS in monocytes from patients heterozygous and homozygous for CFTR mutations. These results indicate that a single allelic CFTR mutation is sufficient to augment IL-8 secretion in response to LPS. This is not a result of increased LPS receptor expression but, rather, is associated with alterations in MAPK signaling.

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Hypoxia modulates the gene expression profile of immunoregulatory receptors in human mature dendritic cells: identification of TREM-1 as a novel hypoxic marker in vitro and in vivo

Blood ◽

10.1182/blood-2010-06-292136 ◽

2011 ◽

Vol 117 (9) ◽

pp. 2625-2639 ◽

Cited By ~ 82

Author(s):

Maria Carla Bosco ◽

Daniele Pierobon ◽

Fabiola Blengio ◽

Federica Raggi ◽

Cristina Vanni ◽

...

Keyword(s):

Gene Expression ◽

Dendritic Cells ◽

Cell Phenotype ◽

Surface Receptors ◽

Immunoregulatory Receptors ◽

Antigen Presenting ◽

And Behavior ◽

The Impact

Abstract Dendritic cells (DCs) are a heterogeneous group of professional antigen-presenting cells functioning as sentinels of the immune system and playing a key role in the initiation and amplification of innate and adaptive immune responses. DC development and functions are acquired during a complex differentiation and maturation process influenced by several factors present in the local milieu. A common feature at pathologic sites is represented by hypoxia, a condition of low pO2, which creates a unique microenvironment affecting cell phenotype and behavior. Little is known about the impact of hypoxia on the generation of mature DCs (mDCs). In this study, we identified by gene expression profiling a significant cluster of genes coding for immune-related cell surface receptors strongly up-regulated by hypoxia in monocyte-derived mDCs and characterized one of such receptors, TREM-1, as a new hypoxia-inducible gene in mDCs. TREM-1 associated with DAP12 in hypoxic mDCs, and its engagement elicited DAP12-linked signaling, resulting in ERK-1, Akt, and IκBα phosphorylation and proinflammatory cytokine and chemokine secretion. Finally, we provided the first evidence that TREM-1 is expressed on mDCs infiltrating the inflamed hypoxic joints of children affected by juvenile idiopathic arthritis, representing a new in vivo marker of hypoxic mDCs endowed with proinflammatory properties.

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Modulation of CXC Chemokine Receptor Expression and Function in Human Neutrophils during Aging In Vitro Suggests a Role in Their Clearance from Circulation

Mediators of Inflammation ◽

10.1155/2009/790174 ◽

2009 ◽

Vol 2009 ◽

pp. 1-8 ◽

Cited By ~ 22

Author(s):

Katja C. Weisel ◽

Frank Bautz ◽

Gabriele Seitz ◽

Sedat Yildirim ◽

Lothar Kanz ◽

...

Keyword(s):

Bone Marrow ◽

Chemokine Receptor ◽

Receptor Expression ◽

Interleukin 8 ◽

Polymorphonuclear Neutrophils ◽

Human Neutrophils ◽

Chemokine Receptor Expression ◽

Chemokine Receptor Cxcr4 ◽

Cxc Chemokine

In mice, differential regulation of CXC chemokine receptor expression in circulating polymorphonuclear neutrophils (PMNs) undergoing senescence results in homing to the bone marrow. However, the role of this compartment and of the chemokine receptor CXCR4 is still under discussion, and only scarce data exist about CXCR4 function in human PMN. In our study, we provide evidence that also in human neutrophils, expression (cell surface and mRNA), chemotactic and signaling functions of the homing-related chemokine receptor CXCR4 are upregulated during aging in vitro, independent of addition of stimulatory cytokines (TNF, IL-1, IL-8, G-CSF). In contrast, interleukin-8 receptors are downmodulated (CXCR2) or remain unchanged (CXCR1), suggesting that human PMNs undergoing senescence acquire a phenotype that impairs inflammatory extravasation and favors homing to the bone marrow or other tissues involved in sequestration. Partially retained responsiveness to interleukin-8 may be important for neutrophil function when senescence occurs after extravasation in inflamed tissues.

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Regulation of interleukin-8 receptor expression in human polymorphonuclear neutrophils

Molecular Immunology ◽

10.1016/0161-5890(95)00047-i ◽

1995 ◽

Vol 32 (12) ◽

pp. 883-893 ◽

Cited By ~ 7

Author(s):

Sunil K. Manna ◽

Chitralekha Bhattacharya ◽

Sanjib K. Gupta ◽

Ajoy K. Samanta

Keyword(s):

Receptor Expression ◽

Interleukin 8 ◽

Polymorphonuclear Neutrophils

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Interleukin 8 (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own receptor expression on human neutrophils.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)40213-5 ◽

1990 ◽

Vol 265 (1) ◽

pp. 183-189

Author(s):

A K Samanta ◽

J J Oppenheim ◽

K Matsushima

Keyword(s):

Receptor Expression ◽

Chemotactic Factor ◽

Interleukin 8 ◽

Human Neutrophils ◽

Neutrophil Chemotactic Factor

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The Impact of Exercise Serum on Selected Parameters of CD4+ T Cell Metabolism

Immuno ◽

10.3390/immuno1030008 ◽

2021 ◽

Vol 1 (3) ◽

pp. 119-131

Author(s):

Jana Palmowski ◽

Kristina Gebhardt ◽

Thomas Reichel ◽

Torsten Frech ◽

Robert Ringseis ◽

...

Keyword(s):

T Cells ◽

Oxygen Consumption ◽

T Cell ◽

Real Time ◽

Cd4 T Cells ◽

Cell Metabolism ◽

Cd4 T Cell ◽

Autologous Serum ◽

Cell Phenotype ◽

The Impact

CD4+ T cells are sensitive to peripheral changes of cytokine levels and metabolic substrates such as glucose and lactate. This study aimed to analyze whether factors released after exercise alter parameters of human T cell metabolism, specifically glycolysis and oxidative phosphorylation. We used primary human CD4+ T cells activated in the presence of autologous serum, which was collected before (CO) and after a 30-min exercise intervention (EX). In the course of activation, cells and supernatants were analyzed for cell viability and diameter, real-time oxygen consumption by using PreSens Technology, mRNA expression of glycolytic enzymes and complexes of the electron transport chain by real-time PCR, glucose, and lactate levels in supernatants, and in vitro differentiation by flow cytometry. EX did not alter T cell phenotype, viability, or on-blast formation. Similarly, no difference between CO and EX were found for CD4+ T cell activation and cellular oxygen consumption. In contrast, higher levels of glucose were found after 48 h activation in EX conditions. T cells activated in autologous exercise serum expressed lower HK1 mRNA and higher IFN-γ receptor 1. We suggest that the exercise protocol used was not sufficient to destabilize the immune metabolism of T cells. Therefore, more intense and prolonged exercise should be used in future studies.

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The impact of selenium on regulatory T cell frequency and immune checkpoint receptor expression in patients with diffuse large B cell lymphoma (DLBCL)

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-02889-5 ◽

2021 ◽

Author(s):

Mehdi Dehghani ◽

Negin Shokrgozar ◽

Mani Ramzi ◽

Mehdi Kalani ◽

Hossein Golmoghaddam ◽

...

Keyword(s):

B Cell ◽

Regulatory T Cell ◽

Immune Checkpoint ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Receptor Expression ◽

Cell Frequency ◽

Large B Cell Lymphoma ◽

The Impact ◽

Large B Cell

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COXIBs and 2,5-dimethylcelecoxib counteract the hyperactivated Wnt/β-catenin pathway and COX-2/PGE2/EP4 signaling in glioblastoma cells

BMC Cancer ◽

10.1186/s12885-021-08164-1 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Aleksandra Majchrzak-Celińska ◽

Julia O. Misiorek ◽

Nastassia Kruhlenia ◽

Lukasz Przybyl ◽

Robert Kleszcz ◽

...

Keyword(s):

Cell Cycle ◽

Cell Viability ◽

Methylation Level ◽

Molecular Mechanisms ◽

Methylation Status ◽

Receptor Expression ◽

Cell Cycle Distribution ◽

Ep4 Receptor ◽

Cox 2 ◽

The Impact

Abstract Background Glioblastoma (GBM) is the deadliest and the most common primary brain tumor in adults. The invasiveness and proliferation of GBM cells can be decreased through the inhibition of Wnt/β-catenin pathway. In this regard, celecoxib is a promising agent, but other COXIBs and 2,5-dimethylcelecoxib (2,5-DMC) await elucidation. Thus, the aim of this study was to analyze the impact of celecoxib, 2,5-DMC, etori-, rofe-, and valdecoxib on GBM cell viability and the activity of Wnt/β-catenin pathway. In addition, the combination of the compounds with temozolomide (TMZ) was also evaluated. Cell cycle distribution and apoptosis, MGMT methylation level, COX-2 and PGE2 EP4 protein levels were also determined in order to better understand the molecular mechanisms exerted by these compounds and to find out which of them can serve best in GBM therapy. Methods Celecoxib, 2,5-DMC, etori-, rofe- and valdecoxib were evaluated using three commercially available and two patient-derived GBM cell lines. Cell viability was analyzed using MTT assay, whereas alterations in MGMT methylation level were determined using MS-HRM method. The impact of COXIBs, in the presence and absence of TMZ, on Wnt pathway was measured on the basis of the expression of β-catenin target genes. Cell cycle distribution and apoptosis analysis were performed using flow cytometry. COX-2 and PGE2 EP4 receptor expression were evaluated using Western blot analysis. Results Wnt/β-catenin pathway was attenuated by COXIBs and 2,5-DMC irrespective of the COX-2 expression profile of the treated cells, their MGMT methylation status, or radio/chemoresistance. Celecoxib and 2,5-DMC were the most cytotoxic. Cell cycle distribution was altered, and apoptosis was induced after the treatment with celecoxib, 2,5-DMC, etori- and valdecoxib in T98G cell line. COXIBs and 2,5-DMC did not influence MGMT methylation status, but inhibited COX-2/PGE2/EP4 pathway. Conclusions Not only celecoxib, but also 2,5-DMC, etori-, rofe- and valdecoxib should be further investigated as potential good anti-GBM therapeutics.

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