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Cytometry. Part a : the Journal of the... Oct 2015The blood-brain barrier (BBB) is primarily comprised of brain microvascular endothelial cells (BMVEC) and astrocytes and serves as a physical and chemical barrier that...
The blood-brain barrier (BBB) is primarily comprised of brain microvascular endothelial cells (BMVEC) and astrocytes and serves as a physical and chemical barrier that separates the periphery from the brain. We describe a flow cytometric method using our in vitro model of the human BBB to characterize BMVEC surface junctional proteins critical for maintenance of barrier function, cell viability, and leukocyte adhesion. For this methodology, BMVEC are cocultured with astrocytes in a transwell tissue culture insert to establish the barrier, after which time the BBB are treated with specific agents, and the BMVEC collected for flow cytometric analyses. We use a standard and optimized method to recover the BMVEC from the coculture model that maintains junctional protein expression and cell viability. A novel leukocyte adhesion assay enables a quantitative analysis of peripheral blood mononuclear cell (PBMC) interactions with the BMVEC and can be used to assess the adhesion of many cell types to the BBB. Furthermore, this method enables the concomitant analysis of a large number of adhesion molecules and tight junction proteins on both the BMVEC and adherent PBMC under homeostatic and pathologic conditions. Flow cytometry is an extremely powerful tool, and this technique can also be applied to assess variables not performed in this study, including cell cycle progression, and calcium flux.
Topics: Astrocytes; Blood-Brain Barrier; Brain; Cell Adhesion; Cell Differentiation; Endothelial Cells; Flow Cytometry; Humans; Leukocytes, Mononuclear
PubMed: 25929817
DOI: 10.1002/cyto.a.22683 -
BMC Immunology Oct 2018Transcriptional profiling with ultra-low input methods can yield valuable insights into disease, particularly when applied to the study of immune cells using...
BACKGROUND
Transcriptional profiling with ultra-low input methods can yield valuable insights into disease, particularly when applied to the study of immune cells using RNA-sequencing. The advent of these methods has allowed for their use in profiling cells collected in clinical trials and other studies that involve the coordination of human-derived material. To date, few studies have sought to quantify what effects that collection and handling of this material can have on resulting data.
RESULTS
We characterized the global effects of blood handling, methods for leukocyte isolation, and preservation media on low numbers of immune cells isolated from blood. We found overall that storage/shipping temperature of blood prior to leukocyte isolation and sorting led to global changes in both CD8 T cells and monocytes, including alterations in immune-related gene sets. We found that the use of a leukocyte filtration system minimized these alterations and we applied this method to generate high-quality transcriptional data from sorted immune cells isolated from the blood of intracerebral hemorrhage patients and matched healthy controls.
CONCLUSIONS
Our data underscore the necessity of processing samples with comparably defined protocols prior to transcriptional profiling and demonstrate that a filtration method can be applied to quickly isolate immune cells of interest while minimizing transcriptional bias.
Topics: CD8-Positive T-Lymphocytes; Gene Expression Profiling; Humans; Leukocyte Reduction Procedures; Leukocytes, Mononuclear; Sequence Analysis, RNA; Transcriptome
PubMed: 30376808
DOI: 10.1186/s12865-018-0268-6 -
BMC Immunology Nov 2011Nonsteroidal anti-inflammatory drugs (NSAID) represent a one of the most widely used anti-inflammatory substances. Their anti-inflammatory effects are mainly based on...
BACKGROUND
Nonsteroidal anti-inflammatory drugs (NSAID) represent a one of the most widely used anti-inflammatory substances. Their anti-inflammatory effects are mainly based on inhibition of cyclooxygenase. The potential direct effect of NSAID on leukocyte migration was poorly investigated. Using time-lapse microscopy and 96-well fluorescence-based assay, we studied the effect of three different NSAID, ketoprofen, diclofenac and SC-560, on leukocyte haptokinesis and haptotaxis in vivo and in vitro.
RESULTS
NSAID induced an immediate inhibiting effect on leukocyte migration both in vitro and in vivo. This effect was dose-dependent and was not restricted to a specific type of leukocytes. The inhibition of leukocyte migration by NSAID was partially re-stored after removal of inhibiting agent. Only complete blockade of leukocyte migration was accompanied by a strong reduction of [Ca(2+)]i.
CONCLUSIONS
NSAID strongly supress leukocyte migration. The results of the present study may have important clinical implications since blockade of leukocyte migration can be achieved after topical application of NSAID.
Topics: Anti-Inflammatory Agents, Non-Steroidal; Calcium; Cell Movement; Cells, Cultured; Chemotaxis; Cyclooxygenase Inhibitors; Diclofenac; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Matrix; Haptens; Humans; Ketoprofen; Leukocytes, Mononuclear; Pyrazoles; Time-Lapse Imaging
PubMed: 22078067
DOI: 10.1186/1471-2172-12-64 -
American Journal of Physiology. Renal... Dec 2011The progression of IgA nephropathy (IgAN), the most frequent type of primary glomerulonephritis, is associated with high levels of mononuclear leukocyte infiltration...
The progression of IgA nephropathy (IgAN), the most frequent type of primary glomerulonephritis, is associated with high levels of mononuclear leukocyte infiltration into the kidney. These cells consist mainly of T cells and macrophages. Our previous study showed that a decoy receptor 3 (DCR3) gene therapy can prevent the development of a mouse autoimmune glomerulonephritis model by its potent immune modulating effects (Ka SM, Sytwu HK, Chang DM, Hsieh SL, Tsai PY, Chen A. J Am Soc Nephrol 18: 2473-2485, 2007). Here, we tested the hypothesis that DCR3 might prevent the progression of IgAN, an immune complex-mediated primary glomerulonephritis, by inhibiting T cell activation, renal T cell/macrophage infiltration, and protecting the kidney from apoptosis. We used a progressive IgAN (Prg-IgAN) model in B cell-deficient mice, because the mice are characterized by a dramatic proliferation of activated T cells systemically and progressive NF-κB activation in the kidney. We treated the animals with short-term gene therapy with DCR3 plasmids by hydrodynamics-based gene delivery. When the mice were euthanized on day 21, we found that, compared with empty vector-treated (disease control) Prg-IgAN mice, DCR3 gene therapy resulted in 1) systemic inhibition of T cell activation and proliferation; 2) lower serum levels of proinflammatory cytokines; 3) improved proteinuria, renal function, and renal pathology (inhibiting the development of marked glomerular proliferation, crescent formation, glomerulosclerosis, and interstitial inflammation); 5) suppression of T cell and macrophage infiltration into the periglomerular interstitium of the kidney; and 5) a reduction in apoptotic figures in the kidney. On the basis of these findings, DCR3 might be useful therapeutically in preventing the progression of IgAN.
Topics: Animals; Apoptosis; Chemotaxis, Leukocyte; Cytokines; Disease Progression; Genetic Therapy; Glomerulonephritis, IGA; Humans; Inflammation; Kidney; Leukocytes, Mononuclear; Mice; Receptors, Tumor Necrosis Factor, Member 6b
PubMed: 21900455
DOI: 10.1152/ajprenal.00050.2011 -
Blood Nov 1998A number of studies have suggested that an immune response to human leukocyte antigen (HLA) alloantigens may contribute to protection against HIV infection. In the...
A number of studies have suggested that an immune response to human leukocyte antigen (HLA) alloantigens may contribute to protection against HIV infection. In the present study, we examined the effect of alloantigen-stimulated cell lines obtained from peripheral blood mononuclear cells (PBMC) of HIV-uninfected (HIV-) individuals and the soluble factors produced by these cell lines on HIV-1 replication. Multiple in vitro restimulation with irradiated allogeneic PBMC from HIV- donors resulted in the expansion of CD8(+) T-cell lines that inhibited HIV-1 replication when cocultured with either autologous or heterologous in vitro-infected phytohemagglutinin (PHA) blasts. Supernatants from the alloantigen-stimulated cell lines also inhibited HIV replication in both PHA blasts and a chronically infected cell line. The alloantigen-stimulated cell lines and the factors they produced inhibited both T-cell-tropic (T) and macrophage-tropic (M) isolates of HIV-1. Blocking experiments using anti-chemokine antibodies suggested that this inhibition of HIV replication was not due to the beta-chemokines present in cocultures of cell lines with HIV-infected blasts. These results indicate that alloantigen-stimulation of PBMC from HIV- individuals activates CD8(+) T cells that produce soluble factor(s) that inhibit HIV replication of a wide spectrum of HIV-1 isolates through a chemokine-independent mechanism. This is a US government work. There are no restrictions on its use.
Topics: Antibodies, Monoclonal; Biological Factors; CD8-Positive T-Lymphocytes; Cell Line; Chemokines; Culture Media, Conditioned; HIV-1; Histocompatibility; Humans; Isoantigens; Leukocytes, Mononuclear; Lymphocyte Activation; Lymphokines; Macrophages; Phytohemagglutinins; Receptors, HIV; T-Lymphocytes; Virus Replication
PubMed: 9787172
DOI: No ID Found -
The Journal of Biological Chemistry Feb 2006Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that...
Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that activate leukocytic recruitment and tumor infiltration and suppress immune surveillance. Here we report that the endocrine peptide hormone, relaxin, is a regulator of leukocyte biology with properties important in recruitment to sites of inflammation. This study uses the human monocytic cell line THP-1 and normal human peripheral blood mononuclear cells to define a novel role for relaxin in regulation of leukocyte adhesion and migration. Our studies indicate that relaxin promotes adenylate cyclase activation, substrate adhesion, and migratory capacity of mononuclear leukocytes through a relaxin receptor LGR7-dependent mechanism. Relaxin-stimulated cAMP accumulation was observed to occur primarily in non-adherent cells. Relaxin stimulation results in increased substrate adhesion and increased migratory activity of leukocytes. In addition, relaxin-stimulated substrate adhesion resulted in enhanced chemotaxis to monocyte chemoattractant protein-1. These responses in THP-1 and peripheral blood mononuclear cells are relaxin dose-dependent and proportional to cAMP accumulation. We further demonstrate that LGR7 is critical for mediating these biological responses by use of RNA interference lentiviral short hairpin constructs. In summary, we provide evidence that relaxin is a novel leukocyte stimulatory agent with properties affecting adhesion and chemomigration.
Topics: Adenylyl Cyclases; Cell Adhesion; Cell Line; Cell Movement; Cell Separation; Chemokine CCL2; Cyclic AMP; Dose-Response Relationship, Drug; Female; Flow Cytometry; Humans; Lentivirus; Leukocytes; Leukocytes, Mononuclear; Male; Membrane Proteins; Models, Statistical; Protein Binding; RNA Interference; RNA, Messenger; Receptors, G-Protein-Coupled; Receptors, Peptide; Recombinant Proteins; Relaxin; Reverse Transcriptase Polymerase Chain Reaction; Time Factors
PubMed: 16303766
DOI: 10.1074/jbc.M506665200 -
Journal of Clinical Laboratory Analysis 2003In the general population, about 5% of individuals are homozygotic and 35% are heterozygotic carriers for chitotriosidase (ChT) deficiency. Activated macrophages are...
In the general population, about 5% of individuals are homozygotic and 35% are heterozygotic carriers for chitotriosidase (ChT) deficiency. Activated macrophages are considered to be the main source of plasma ChT activity, which permits the biochemical characterization of homozygote deficients. However, in the case of detecting heterozygotic carriers, the results are often inconclusive. The activities of ChT in plasma and mononuclear (MN) and polymorphonuclear (PMN) leukocytes were determined in 169 control subjects (72 males and 97 females) with a mean age (+/- SD) of 47.5+/-9.7 years (range 18-96 years). The specific enzyme activity was in PMN leukocytes >MN leukocytes >plasma, with a highly significant partial correlation being found between the activities of ChT in plasma and PMN leukocytes (r=0.578, P<0.001). A significant correlation was found between the age of the patients studied and plasma ChT activity (r=0.568, P<0.001). No significant correlation was found for enzyme activities in MN (r=0.105) or in PMN leukocytes (r=0.043). The results obtained suggest that, in normal physiological conditions, PMN leukocytes may secrete ChT to the plasma. Although the activities of ChT in MN and PMN leukocytes are not affected by demographic factors, it is not possible to use them for the biochemical detection of ChT-deficient heterozygotic carriers.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Female; Hexosaminidases; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Neutrophils; Reference Values
PubMed: 14614752
DOI: 10.1002/jcla.10108 -
The Journal of Clinical Investigation Sep 1993The CD18 mAb 60.3 and the CD49d mAb HP1/2 were given at the time of intraperitoneal instillation of either protease peptone or live Escherichia coli bacteria and at 12...
The CD18 mAb 60.3 and the CD49d mAb HP1/2 were given at the time of intraperitoneal instillation of either protease peptone or live Escherichia coli bacteria and at 12 h. Leukocyte emigration was evaluated at 4 and 24 h. PMN emigration 4 h after protease peptone instillation and injection of both mAbs was 10% of that in saline treatment. It was 15% of that in saline treatment after mAb 60.3 alone and unchanged by mAb HP1/2. At 24 h PMN emigration in response to protease peptone was not prevented by either CD18 or CD49d mAbs, however, when given together emigration was 10% of saline-treated animals. Mononuclear cell emigration to protease peptone was enhanced at 4 h by both CD18 and CD49d mAbs. The CD18 mAb did not augment mononuclear emigration in response to live bacteria. At 24 h, neither the CD18 nor the CD49d mAb alone blocked emigration of mononuclear cells, but the combination of the two did. These studies demonstrate that: (a) early (4 h) PMN emigration is CD11/CD18 dependent; (b) late (24 h) PMN emigration is CD11/CD18 independent; and (c) mononuclear cells utilize the integrins CD18 and CD49d.
Topics: Animals; Antigens, CD; CD18 Antigens; Chemotaxis, Leukocyte; Escherichia coli Infections; Integrin alpha Chains; Leukocytes, Mononuclear; Neutrophils; Peritoneal Cavity; Rabbits; Receptors, Very Late Antigen
PubMed: 8104195
DOI: 10.1172/JCI116686 -
Frontiers in Endocrinology 2022Type 1 diabetes (T1D) is a disease of both autoimmunity and β-cells. The β-cells play an active role in their own demise by mounting defense mechanisms that are... (Review)
Review
Type 1 diabetes (T1D) is a disease of both autoimmunity and β-cells. The β-cells play an active role in their own demise by mounting defense mechanisms that are insufficient at best, and that can become even deleterious in the long term. This complex crosstalk is important to understanding the physiological defense mechanisms at play in healthy conditions, their alterations in the T1D setting, and therapeutic agents that may boost such mechanisms. Robust protocols to develop stem-cell-derived islets (SC-islets) from human pluripotent stem cells (hPSCs), and islet-reactive cytotoxic CD8 T-cells from peripheral blood mononuclear cells offer unprecedented opportunities to study this crosstalk. Challenges to develop β-cell killing models include the cluster morphology of SC-islets, the relatively weak cytotoxicity of most autoimmune T-cells and the variable behavior of expanded CD8 T-cells. These challenges may however be highly rewarding in light of the opportunities offered by such models. Herein, we discuss these opportunities including: the β-cell/immune crosstalk in an islet microenvironment; the features that make β-cells more sensitive to autoimmunity; therapeutic agents that may modulate β-cell vulnerability; and the possibility to perform analyses in an autologous setting, i.e., by generating T-cell effectors and SC-islets from the same donor.
Topics: Humans; Diabetes Mellitus, Type 1; CD8-Positive T-Lymphocytes; Leukocytes, Mononuclear; Pluripotent Stem Cells; Cell Death
PubMed: 36726462
DOI: 10.3389/fendo.2022.1076683 -
PloS One 2011This study examines the expression of IL-17A-secreting cells within the inflamed synovium and the relationship to in vivo joint hypoxia measurements.
INTRODUCTION
This study examines the expression of IL-17A-secreting cells within the inflamed synovium and the relationship to in vivo joint hypoxia measurements.
METHODS
IL-17A expression was quantified in synovial tissue (ST), serum and synovial fluid (SF) by immunohistochemistry and MSD-plex assays. IL-6 SF and serum levels were measured by MSD-plex assays. Dual immunofluorescence for IL-17A was quantified in ST CD15+ cells (neutrophils), Tryptase+ (mast cells) and CD4+ (T cells). Synovial tissue oxygen (tpO(2)) levels were measured under direct visualisation at arthroscopy. Synovial infiltration was assessed using immunohistochemistry for cell specific markers. Peripheral blood mononuclear and polymorphonuclear cells were isolated and exposed to normoxic or 3% hypoxic conditions. IL-17A and IL-6 were quantified as above in culture supernatants.
RESULTS
IL-17A expression was localised to mononuclear and polymorphonuclear (PMN) cells in inflamed ST. Dual immunoflourescent staining co-localised IL-17A expression with CD15+ neutrophils Tryptase+ mast cells and CD4+T cells. % IL-17A positivity was highest on CD15+ neutrophils, followed by mast cells and then CD4+T-cells. The number of IL-17A-secreting PMN cells significantly correlated with sublining CD68 expression (r = 0.618, p<0.01). IL-17A SF levels correlated with IL-6 SF levels (r = 0.675, p<0.01). Patients categorized according to tp0(2)< or >20 mmHg, showed those with low tp0(2)<20 mmHg had significantly higher IL-17A+ mononuclear cells with no difference observed for PMNs. Exposure of mononuclear and polymorphonuclear cells to 3% hypoxia, significantly induced IL-6 in mononuclear cells, but had no effect on IL-17A expression in mononuclear and polymorphonuclear cells.
CONCLUSION
This study demonstrates IL-17A expression is localised to several immune cell subtypes within the inflamed synovial tissue, further supporting the concept that IL-17A is a key mediator in inflammatory arthritis. The association of hypoxia with Il-17A expression appears to be indirect, probably through hypoxia-induced pro-inflammatory pathways and leukocyte influx within the joint microenvironment.
Topics: Arthritis; Cell Movement; Humans; Hypoxia; Inflammation; Interleukin-17; Joints; Leukocytes, Mononuclear; Neutrophils; Synovial Fluid; Synovial Membrane
PubMed: 21887369
DOI: 10.1371/journal.pone.0024048