Comparative Proteomic Analysis Reveals Varying Impact on Immune Responses in Phorbol 12-Myristate-13-Acetate-Mediated THP-1 Monocyte-to-Macrophage Differentiation.

Macrophages are sentinels of the innate immune system, and the human monocytic cell line THP-1 is one of the widely used in vitro models to study inflammatory processes and immune responses. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the most commonly used and accepted method. However, the concentrations and duration of PMA treatment vary widely in the published literature and could affect the probed phenotype, however their effect on protein expression is not fully deciphered. In this study, we employed a dimethyl labeling-based quantitative proteomics approach to determine the changes in the protein repertoire of macrophage-like cells differentiated from THP-1 monocytes by three commonly used PMA-based differentiation protocols. Employing an integrated network analysis, we show that variations in PMA concentration and duration of rest post-stimulation result in downstream differences in the protein expression and cellular signaling processes. We demonstrate that these differences result in altered inflammatory responses, including variation in the expression of cytokines upon stimulation with various Toll-like receptor (TLR) agonists. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics with one of the most common in vitro models for macrophages, which in turn has a profound impact on the immune as well as inflammatory responses being studied.

The Oxidative Response of Human Monocytes to Surface Modified Commercially Pure Titanium.

Cellular responses to implanted biomaterials are key to understanding osseointegration. The aim of this investigation was to determine the in vitro priming and activation of the respiratory burst activity of monocytes in response to surface-modified titanium. Human peripheral blood monocytes of healthy blood donors were separated, then incubated with surface-modified grade 2 commercially pure titanium (CPT) disks with a range of known surface energies and surface roughness for 30- or 60-min. Secondary stimulation by phorbol 12-myrisate 13-acetate (PMA) following the priming phase, and luminol-enhanced-chemiluminescence (LCL) was used to monitor oxygen-dependent activity. Comparison among groups was made by incubation time using one-way ANOVA. One sample from each group for each phase of the experiment was viewed under scanning electron microscopy (SEM) and qualitative comparisons made. The results indicate that titanium is capable of priming peripheral blood monocytes following 60-min incubation. In contrast, 30 min incubation time lead to reduced LCL on secondary stimulation as compared to cells alone. At both time intervals, the disk with the lowest surface energy produced significantly less LCL compared to other samples. SEM examination revealed differences in surface morphology at different time points but not between differently surface-modified disks. These results are consistent with the hypothesis that the titanium surface characteristics influenced the monocyte activity, which may be important in regulating the healing response to these materials.

Structural differences of neutrophil extracellular traps induced by biochemical and microbiologic stimuli under healthy and autoimmune milieus.

Neutrophil extracellular traps (NETs) are networks of decondensed chromatin loaded with antimicrobial peptides and enzymes produced against microorganisms or biochemical stimuli. Since their discovery, numerous studies made separately have revealed multiple triggers that induce similar NET morphologies allowing to classify them as lytic or non-lytic. However, the variability in NET composition depending on the inducer agent and the local milieu under similar conditions has been scarcely studied. In this work, a comparative study was conducted to evaluate structural and enzymatic divergences in NET composition induced by biochemical (phorbol myristate acetate [PMA] and hypochlorous acid [HOCl]) and microbiologic (Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa) stimuli, along with the presence of plasma from healthy donors or patients with systemic lupus erythematosus (SLE). The results showed a differential composition of DNA and the antimicrobial peptide cathelicidin (LL37) and a variable enzymatic activity (neutrophil elastase, cathepsin G, myeloperoxidase) induced by the different stimuli despite showing morphologically similar NETs. Additionally, SLE plasma´s presence increased DNA and LL37 release during NET induction independently of the trigger stimulus but with no enzymatic activity differences. This work provides new evidence about NET composition variability depending on the inducer stimulus and the local milieu.

Ephedra Herb, Mao, Inhibits Antigen-Induced Mast Cell Degranulation by Induction of the Affinity Receptor for IgE Internalization.

PURPOSE: Ephedra herb (Mao) exerts potent anti-allergic effects. This study aimed to examine the underlying mechanisms of Mao on allergic inflammation using in vitro cultured mast cells (MCs) and an in vivo model of MC-dependent anaphylaxis. METHODS: Bone marrow-derived MCs (BMMCs) were presensitized with anti-2,4-dinitrophenol (DNP) immunoglobulin E (IgE) and challenged with antigens (Ag; DNP-human serum albumin). Degranulation responses and cell surface high-affinity receptor for IgE (FcεRI) expression were assessed with/without Mao treatment. Passive systemic anaphylaxis (PSA)-treated mice were administered Mao and the pathophysiological responses were evaluated. RESULTS: Mao inhibited Ag-induced BMMC degranulation, but not polyclonal activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, indicating that Mao inhibits IgE-dependent activation of BMMCs. Mao-treated BMMCs exhibited significant reductions in expression of surface IgE and its receptor FcεRI. Analysis of subcellular localization revealed that Mao induces FcεRI internalization in BMMCs without degranulation. In the PSA mouse model, Mao administration prevented antigen-induced hypothermia. Mao administration significantly reduced cell surface expression of IgE-bound FcεRI on peritoneal MCs. CONCLUSIONS: Mao induced FcεRI internalization in MCs, thereby inhibiting Ag-induced IgE-dependent degranulation. The inhibitory effects of Mao on MC degranulation may offer a novel therapeutic approach for allergic diseases.

A semen-based stimulation method to analyze cytokine production by uterine CD56bright natural killer cells in women with recurrent pregnancy loss.

Cytokine secretion by NK cells is abnormal in some women with recurrent pregnancy loss (RPL). Cytokine production is usually evaluated after stimulation with PMA and ionomycin. However, stimulation of uterine NK cells with semen corresponds more closely to physiological conditions at the time of conception. As seminal plasma has immunomodulatory properties, we aimed to elucidate compatibility between uterine NK cells and semen. Endometrial samples were stimulated with PMA/ionomycin, semen, seminal plasma, or spermatozoa. Thereafter, cytokine production by NK (CD56bright) cells was evaluated using flow cytometry and compared between women with and without a history of RPL associated with abnormal NK cell distribution in the endometrium or unexplained RPL. The ratios (%) of NK cells producing IFN-γ and TNF-α (NK1 phenotype), IL-4 (NK1/NK2 phenotype), and IL-10 (NK1/NKr1 phenotype) were significantly lower after stimulation with semen than with PMA/ionomycin (P < 0.01). After exposure to semen, ratios (%) of NK cells producing IL-4 and IL-10 in patients with unexplained RPL were significantly lower (P < 0.05), whereas those of NK1/NK2 and NK1/NKr1 were significantly higher (P < 0.01) than those in controls. The shift of endometrial NK cells to the NK2 phenotype was more pronounced when stimulated by semen than by PMA/ionomycin. However, a semen-induced shift to NK1 in women with unexplained RPL could induce miscarriage. Couple-specific immunological compatibility tests through semen stimulation in vitro might provide important information to avoid RPL.

Mast Cells Occupy Stable Clonal Territories in Adult Steady-State Skin.

Mast cells (MCs) are tissue-resident hematopoietic cells intensely studied for their role as effectors in allergic immune responses. Yolk sac-derived embryonic MCs first populate tissues and are later replaced by definitive MCs. We show that definitive MC progenitors expand locally in skin and form clonal colonies that cover stable territories. In MC-deficient skin, colonies grow by proliferation of MCs at the border of the clonal territory. Clonal growth ceases at common borders of neighboring colonies. In steady state, colony self-renewal is independent of bone marrow contribution, and the clonal architecture remains fixed if not disturbed by skin inflammation. Inflammatory cues increase MC density setpoint, stimulating the influx of new progenitors from the bone marrow as well as proliferation of skin-resident cells. The expanding new arrivals disrespect territories of preexisting MC clones. We conclude that during a limited window early in development, definitive MC precursors efficiently enter the skin, expand, and self-maintain, occupying stable territories. In adulthood, circulating progenitors, excluded from steady-state skin, are recruited only into inflamed skin where they clonally expand alongside proliferating skin-resident MCs, disorganizing the original architecture of clonal territories.

Xanthone suppresses allergic contact dermatitis in vitro and in vivo.

Xanthone is a phenolic compound found in a few higher plant families; it has a variety of biological activities, including antioxidant, anti-inflammatory, and anticancer properties. However, the molecular and cellular mechanisms underlying the activity of xanthone in allergic contact dermatitis (ACD) remain to be explored. Therefore, this study aimed to investigate the regulatory effects of xanthone in ACD in human keratinocytes (HaCaT cell), and human mast cell line (HMC-1 cell) in vitro and in an experimental murine model. The results demonstrated that treatment with xanthone reduced the production of pro-inflammatory cytokines and chemokines including interleukin (IL)-1ß, IL-6, IL-8, and expression of chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated HaCaT cells. Xanthone also suppressed the production of pro-inflammatory cytokines, chemokines, and allergic mediators in phorbol myristate acetate/A23187 calcium ionophore (PMACI)-stimulated HMC-1 cells. Xanthone significantly suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) and activation of caspase-1 signaling pathway in vitro model. Additionally, xanthone administration alleviated 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis like-skin lesion by reducing the serum levels of immunoglobulin E (IgE), histamine, and pro-inflammatory cytokines and suppressing MAPKs phosphorylation. Xanthone administration also inhibited mortality due to compound 48/80-induced anaphylactic shock and suppressed the passive cutaneous anaphylaxis (PCA) reaction mediated by IgE. Collectively, these results suggest that xanthone has a potential for use in the treatment of allergic inflammatory diseases.

Combining Adhesive Nanostructured Surfaces and Costimulatory Signals to Increase T Cell Activation.

Adoptive cell therapies are showing very promising results in the fight against cancer. However, these therapies are expensive and technically challenging in part due to the need of a large number of specific T cells, which must be activated and expanded in vitro. Here we describe a method to activate primary human T cells using a combination of nanostructured surfaces functionalized with the stimulating anti-CD3 antibody and the peptidic sequence arginine-glycine-aspartic acid, as well as costimulatory agents (anti-CD28 antibody and a cocktail of phorbol 12-myristate 13-acetate, ionomycin, and protein transport inhibitors). Thus, we propose a method that combines nanotechnology with cell biology procedures to efficiently produce T cells in the laboratory, challenging the current state-of-the-art expansion methodologies.

PEP-1-PEA15 suppresses inflammatory responses by regulation of MAPK in macrophages and animal models.

Phosphoprotein enriched in astrocytes 15 (PEA15) plays a multi-functional role in neuronal cell survival, however the effects of PEA15 against inflammation have not been investigated yet. To examine the effects of PEP-1-PEA15 protein against lipopolysaccharide (LPS)-induced inflammatory responses in Raw 264.7 cells and in a 12-O-tetradecanoylphobol 13-acetate (TPA)-induced mouse model, we constructed and purified PEP-1-PEA15 protein, which can transduce into cells or tissues. PEP-1-PEA15 inhibited LPS-induced damage in cells including that caused by reactive oxygen species (ROS) production and DNA fragmentation. PEP-1-PEA15 also significantly suppressed activation of mitogen activated protein kinases (MAPKs), pro-inflammatory mediator proteins and various cytokines. In a TPA-induced mouse ear edema model, PEP-1-PEA15 significantly reduced ear weight and thickness as well as MAPK activation as well as the expression levels of COX-2, iNOS, IL-6, IL-1ß, and TNF-α. These results demonstrated that PEP-1-PEA15 showed anti-inflammatory effect in cells and animal model suggesting that this fusion protein protects cells or skin tissues from inflammatory response.

Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis.

Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.

Generation of anti-porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis.

T cell-mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti-porcine CD69 mAb-producing mouse hybridomas, 01-14-22-51 (IgG2b-κ) and 01-22-44-102 (IgG2a-κ), both showing fine reactivity with phorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti-interferon-γ mAb and anti-tumor necrosis factor-α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.

Anti-inflammatory Property of ß-D-Mannuronic Acid (M2000) on Expression and Activity of Matrix Metalloproteinase-2 and -9 through CD147 Molecule in Phorbol Myristate Acetate-differentiated THP-1 Cells.

The aim of this study was to evaluate the effects of M2000, a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive property and without gastro-nephrotoxicitic effects on matrix metalloproteinases (MMP)-2 and (MMP)-9 in phorbol myristate acetate (PMA)-differentiated THP-1 cells. Gene expression and activity of MMP-2 and MMP-9 are inhibited respectively by the tissue inhibitor of matrix metalloproteinase (TIMP)-2 and (TIMP)-1 and are induced by extracellular matrix metalloproteinase inducer (CD147/EMMPRIN). In this study, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine gene expression of MMP-2, MMP-9, TIMP-1, and TIMP-2. Flow cytometry and zymography were applied to determine cellular surface expression of CD147 and activity of MMP-2 and MMP-9, respectively. Our results showed that treatment of THP-1 cells with high concentration (25 µg/mL) of M2000 significantly decreased the cellular surface expression of CD147 (p<0.05) and the gene expression of MMP-2, MMP-9 and TIMP-1 (p<0.05), and inhibited the gelatinolytic activity of MMP-2 and MMP-9 (p<0.05). According to our results, M2000 can reduce inflammation through inhibition of the cellular surface expression of CD147 and decrease the gene expression and gelatinolytic activity of MMP-2 and MMP-9 in PMA-differentiated THP-1 cells.

Sildenafil, a Phosphodiesterase Type 5 Inhibitor, Downregulates Osteopontin in Human Peripheral Blood Mononuclear Cells.

The aim of this study was to investigate the ability of sildenafil to regulate osteopontin (OPN) gene and protein in peripheral blood mononuclear cells (PBMCs) from healthy blood donors. OPN is expressed by a wide variety of cell types, including immune cells. OPN functions are linked to various physiological and pathological conditions. Sildenafil is a selective inhibitor of type 5 phosphodiesterase. Sildenafil has recently been found to have immunomodulatory effects in animal models and in studies performed in humans. PMA-stimulated and unstimulated PBMCs from 16 healthy blood donors (men) were cultured with sildenafil (at concentrations of 400 ng/ml and 4 µg/ml). OPN level in culture supernatants was measured by enzyme-linked immunosorbent assay. The analysis of OPN gene expression was performed by real-time PCR. Cell viability was assessed by trypan blue staining. PMA plus ionomycin stimulation of PBMCs resulted in a significant increase of OPN production and gene expression (p < 0.001). Sildenafil significantly decreased OPN secretion (p < 0.05) and gene expression (p < 0.05) in stimulated PBMCs; however, had no effect on OPN in unstimulated PBMCs. Sildenafil did not affect PBMCs viability. Sildenafil downregulates OPN in PBMCs from healthy men. Despite accumulating evidence for the immunomodulatory effects of sildenafil on human immune system cells, further studies are needed to determine if this drug affects the level of cGMP and NF-κB in PBMCs. In addition, it is needed to evaluate sildenafil's activity in PBMCs from patients with elevated OPN levels.

Enhanced T helper 1 and 2 cytokine responses at birth associate with lower risk of middle ear infections in infancy.

BACKGROUND: Respiratory tract infections and their symptoms are frequent during early childhood, but their risk factors, including the effect of early immune regulation, are less known. The aim of the study was to analyze whether stimulated cord blood cytokine production is associated with the frequency of respiratory tract infection symptoms or infections during the first year of life. METHODS: The study population consisted of children of mothers from farm or non-farm rural environment from Austria, Finland, Germany, and Switzerland who participated in a prospective birth cohort study (PASTURE: Protection against Allergy-Study in Rural Environments) (N = 550). Cord blood samples were stimulated with the combination of phorbol ester and ionomycin (P/I) for 24 h, and the production of IL-5, IL-10, TNF-α, and IFN-γ was determined using ELISA. Information about infectious morbidity was collected using weekly diaries. RESULTS: P/I-stimulated production of IL-5 (adjusted risk ratio (aRR) for ≤median production, 0.37; 95% confidence interval (CI), 0.25-0.55, aRR for >median production, 0.41; 95% CI, 0.27-0.61 vs. production median production, 0.39; 95% CI, 0.25-0.62 vs. production

Cannabidiol (CBD) induces functional Tregs in response to low-level T cell activation.

Many effects of the non-psychoactive cannabinoid, cannabidiol (CBD), have been described in immune responses induced by strong immunological stimuli. It has also been shown that CBD enhances IL-2 production in response to low-level T cell stimulation. Since IL-2, in combination with TGF-ß1, are critical for Treg induction, we hypothesized that CBD would induce CD4+CD25+FOXP3+ Tregs in response to low-level stimulation. Low-level T cell stimulation conditions were established based on minimal CD25 expression in CD4+ cells using suboptimal PMA/Io (4nM/0.05µM, S/o), ultrasuboptimal PMA/Io (1nM/0.0125µM, Us/o) or soluble anti-CD3/28 (400-800ng each, s3/28). CBD increased CD25+FOXP3+ cells from CD4+, CD4+CD25+, and CD4+CD25- T cells, as well as in CD4+ T cells derived from FOXP3-GFP mice. Most importantly, the Us/o+CBD-induced CD4+CD25+ Tregs robustly suppressed responder T cell proliferation, demonstrating that the mechanism by which CBD is immunosuppressive under low-level T cell stimulation involves induction of functional Tregs.

Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages.

This study aims to investigate the mechanisms underlying leptin-mediated crosstalk between tumor-associated macrophages (M2 macrophages) and breast cancer cells. THP1 human leukemic monocytes were induced to differentiate into M2 macrophages by PMA (100 nM) and IL-4 (20 ng/mL). Quantitative RT-PCR and Western blot revealed that leptin (100 nM) significantly increased the expression of leptin receptor (ObR) in the M2 macrophages (P < 0.01) and stimulated interleukin (IL)-8 expression in the M2 macrophages, mouse macrophage cells RAW264.7, and primary mouse peritoneal macrophages in a dose- and time-dependent manner. Leptin-induced IL-8 production was sensitive to the ERK inhibitor PD980590 (10 µmol/L), p38 MAPK inhibitor SB203580 (20 µmol/L), and anti-ObR neutralizing antibody (4 µg/mL). Leptin (100 ng/mL) substantially increased the phosphorylation of p38 and ERK1/2. Thus, leptin may induce IL-8 production in M2 macrophages by interacting with ObR to activate the p38 and ERK signaling pathways. Scratch and transwell chamber assay showed that both recombinant IL-8 and leptin-induced M2 macrophage-derived IL-8 promoted the migration and invasion of human breast cancer cells MCF7 and MDA-MB-231 (All P < 0.01). In a nude mice xenograft model of breast cancer (n = 5 per group), injection of leptin (0.1 µg/g) dramatically increased tumor volume and mass, reduced survival, exacerbated pulmonary metastasis, and elevated IL-8 and Ki67 expression in the tumor tissue (All P < 0.05) compared with PBS injection. Depletion of mouse macrophage by Clophosome®-clodronate liposome and injection of anti-mouse IL-8 neutralizing antibodies in the xenograft tumor significantly attenuated those leptin-mediated stimulations (All P < 0.05). These findings indicate that leptin may promote tumor growth and metastasis by stimulating IL-8 production in tumor-associated macrophage.

Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells.

Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase's translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation-where PKCθ plays a critical role.

Detection of Intracellular Cytokines by Flow Cytometry.

Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single-cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation dilution dyes, activation markers, and viability dyes. This capability, combined with the high throughput inherent in the instrumentation, gives ICCS an enormous advantage over other single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell surface staining are also described.

Butein suppresses ICAM-1 expression through the inhibition of IκBα and c-Jun phosphorylation in TNF-α- and PMA-treated HUVECs.

Butein (3,4,2',4'-tetrahydroxychalcone), a flavonoid derivative, has been reported to show several biological actions, including anti-inflammatory and anti-cancer. However, the possible molecular mechanisms involved are poorly understood. Treatment of human umbilical vein endothelial cells (HUVECs) with butein significantly inhibited cell surface intercellular adhesion molecule-1 (ICAM-1) expression, ICAM-1 protein synthesis, and mRNA expression induced by tumor necrotic factor-α (TNF-α) and/or phorbol 12-myristate 13-acetate (PMA). Electrophoretic mobility shift assay revealed that butein blocked activation of transcription factors, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), induced by TNF-α and PMA. Moreover, butein abolished TNF-α- and PMA-induced IκBα phosphorylation, which participates in NF-κB activation, and PMA-induced phosphorylation of c-Jun, a subunit composed of AP-1. In vitro, butein inhibited the phosphorylation of c-Jun, binding to GST beads, mediated by JNK isolated from PMA-treated cells. The inhibitory action of butein on the JNK-mediated in vitro c-Jun phosphorylation was abrogated in the presence of ATP. These results indicate that in HUVECs, butein suppresses the expression of ICAM-1 mRNA and protein through the inhibition of the activation of NF-κB and AP-1 induced by TNF-α and PMA, that the inhibitory action of butein on NF-κB activation results from the inhibition of IκBα phosphorylation by IκB kinase (IKK), and that the inactivation of PMA-activated AP-1 by butein is due to the blocking of JNK-mediated c-Jun phosphorylation through the inhibition of ATP binding.

Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus.

INTRODUCTION: Polymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE. METHODS: The following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10(-)D16(low) in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR. RESULTS: SLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10(-)CD16(low) phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation. CONCLUSIONS: Our results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.