Combination Of Phorbol Myristate Acetate Research Articles

Characterization of an in vitro model to study CD4+ T cells metabolism in dairy cows

Dairy cows are susceptible to several health disorders throughout their lactation. Objectives were to characterize an in vitro model to study bioenergetic measures in CD4+ T lymphocytes in dairy cows. Twenty-four healthy mid-lactating multiparous Holstein dairy cows were enrolled at a mean (±standard deviation) of 234 ± 22 d in milk (DIM). Cows were blocked according to DIM and blood was collected to isolate peripheral blood mononuclear cells followed by magnetic separation of CD4+ T lymphocytes using bovine specific monoclonal antibodies. The isolated CD4+ T lymphocytes from each cow were split into 2 tubes and randomly assigned to incubate in an assay medium as control (CON) or with a combination of phorbol myristate acetate and ionomycin (PMA+IMY) to evaluate metabolic function under resting and activated state. Mitochondrial and glycolytic functional kinetics were recorded in CD4+ T lymphocytes based on real-time measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) under basal conditions and in response to complex V inhibitor (oligomycin), a protonophore uncoupler (BAM 15), and complex I and complex III inhibitors (rotenone and antimycin A). The mean (±SD) viability and purity of CD4+ T lymphocytes was 92.5 ± 2.9% and 95.2 ± 2.9%, respectively. The basal OCR in CD4+ T lymphocytes treated with PMA+IMY was greater than CON; nevertheless, the maximal respiration rate (CON = 58.0 vs. PMA+IMY = 47.3 ± 5.7 pmol/min) and sparing respiratory capacity rate (CON = 42.0 vs. PMA+IMY = 28.7 ± 4.2 pmol/min) were decreased in activated CD4+ T lymphocytes. The ECAR in CD4+ T lymphocytes increased progressively over time in PMA+IMY compared with CON, which indicated an increase in aerobic glycolysis in PMA+IMY compared with CON (CON = 46.9 vs. PMA+IMY = 86.4 ± 7.0 pmol/min). Activated CD4+ T lymphocytes exhibit a metabolic switch from oxidative phosphorylation to aerobic glycolysis, which may support rapid cell proliferation. The results observed in this experiment demonstrate the sensitivity of the technique to detect changes in metabolic function under different cellular conditions, providing a robust framework to study immuno-metabolism in dairy cattle.

T helper 17 and regulatory T-cell profile and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in pediatric patients with beta-thalassemia

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment option for hereditary hemoglobin disorders, such as beta-thalassemia; However, this procedure is not without constraints, mainly engendering complications such as acute graft-versus-host disease (aGvHD), chronic GvHD (cGvHD), and susceptibility to infections. The clinical outcomes of allo-HSCT are highly dependant on the quality and quantity of T-cell subsets reconstitution. Following the allo-HSCT of six pediatric patients afflicted with beta-thalassemia, their mononuclear cells were isolated, and then cultured with a combination of phorbol myristate acetate (PMA)/ionomycin and Brefeldin A. The content of CD4 T-cell subsets, including T helper 17 (Th17) cells and regulatory T cells (Tregs), were determined by specific conjugated-monoclonal antibodies three and six months post-HSCT. An increased frequency of total CD4 T-cells, Tregs and Th17 cells was observed at day 90 and 180 after allo-HSCT, albeit the numbers were still lower than that of our healthy controls. In patients who developed cGvHD, a lower Th17/Treg ratio was observed, owing it to a decreased proportion of Th17 cells. In conclusion, creating balance between Th17 and Treg subsets may prevent acute and chronic GvHD in patients after allo-HSCT.

CYTOKININ FRACTION OF THE Hericium coralloides INCREASES OXIDATIVE METABOLISM OF MURINE PERITONEAL MACROPHAGIES

Aim. This study was aimed to examine influence of сytokinin fraction of basidiomycete Hericium coralloides on the spontaneous and induced phagocytic activity of murine peritoneal macrophages. Materials and methods. Mononuclear phagocyte (macrophage) fraction of peritoneal exudate of C57BL/6 mice was used. Macrophages were incubated under standard conditions at 37 °C, 100% humidity and 5% CO2 for 4 hours. Phorbol 12-myristate 13-acetate (PMA) was added to part of the samples to activate oxidative metabolism. Hericium coralloides was added in two concentrations that were 5 and 10 times lower than the IC50, defined as an antiproliferative effect on colon cancer cells. Incubation with samples was carried out for 2 hours. Results. When adding PMA, Hericium coralloides (0.017 and 0.035 μg/ml) and in the combination of PMA with Hericium coralloides, activation of reactive oxygen species (ROS) in peritoneal macrophages was revealed by 1.37-1.7 times, compared to the spontaneous activity of phagocytes. Conclusions. Thus, the effect of the cytokinin e.tract of the basidium fungus Hericium coralloides was manifested by an increase in the phagocytic activity of peritoneal macrophages as one of the possible mechanisms of immunomodulatory action.

Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α

The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-α in cell culture supernatants and plasma and to detect cytoplasmatic TNF-α in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-α fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines (IFN-γ, IL-10, and CCL5) and were used to develop a fluorescent bead-based assay. The range of bovine TNF-α detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-α in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-α, with subsequent recovery rates (mean ± SD) of 89% ± 9 (n = 3) in culture medium and 94% ± 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-α in bovine samples. Both bovine TNF-α mAbs also detected intracellular TNF-α in bovine CD14+ monocytes and CD4+/CD8+ lymphocytes. In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-α in a wide concentration range and to characterize intracellular TNF-α expression in bovine leukocytes.

PMA induces the differentiation of monocytes into immunosuppressive MDSCs.

The induction of immune tolerance without the use of immunosuppressive drugs is a crucial problem in organ transplantation. The use of myeloid-derived suppressor cells (MDSCs) as a cell-based adjuvant immunosuppressive therapy is a bright clinical prospect in organ transplantation. MDSCs with stable immunosuppressive activities can be used to treat immune-related diseases. In this study, macrophage colony-stimulating factor (M-CSF) was used to promote myeloid progenitor cell differentiation, and phorbol 12-myristate 13-acetate (PMA) was added to induce MDSCs at the later stage of induction in vitro. Cell phenotypes were detected by flow cytometry and mRNA was detected by real-time-polymerase chain reaction (RT-PCR). A mouse skin transplantation model was used to investigate the cell inhibitory function. The combination of PMA and M-CSF induced the differentiation of myeloid-derived monocytes into MDSCs. MDSCs were found to induce immune tolerance by inhibiting the proliferation and activation of T cells, promoting cytokine secretion and inducing T cell transformation to regulatory T cells (Treg ). PMA significantly up-regulated the expression of Arg-1 and the Arg-1 protein expression in MDSCs and arginase 1 (Arg-1) inhibitor nor-NOHA reversed the MDSC immunosuppressive activity, indicating the involvement of the Arg-1 pathway in MDSC-mediated immunosuppression. M-CSF+PMA-induced MDSCs also significantly prolonged the survival time of skin grafts in mice, showing that MDSCs exert immunosuppressive effects in vivo. We describe a novel scheme to induce immunosuppressive MDSCs in vitro. MDSCs induced by M-CSF with PMA showed stable immunosuppression. MDSCs induced by this protocol may benefit patients with organ transplantation through immune regulation.

Sialylated Cervical Mucins Inhibit the Activation of Neutrophils to Form Neutrophil Extracellular Traps in Bovine in vitro Model.

In order to combat invading pathogens neutrophils can release neutrophil extracellular traps (NETs). However, since NETs can also damage endogenous cells, several control mechanisms for the formation of NETs must work effectively. For instance, neutrophil activation is silenced within blood circulation by the binding of sialylated glycoconjugates to sialic acid binding immunoglobulin-like lectins (Siglecs) on neutrophils. As neutrophils are recruited within the female reproductive tract, after mating, a comparable mechanism may also take place within the bovine cervix to prevent an exaggerated NET formation and thus, infertility. We examined, if the highly glycosylated mucins, which are the major functional fraction of biomolecules in mucus, represent a potential regulator of NET formation. The qPCR data revealed that in polymorphonuclear neutrophils (PMNs) inhibitory Siglecs are the most frequently expressed Siglecs and might be a potential target of sialylated glycans to modulate the activation of PMNs. Remarkably, the addition of bovine cervical mucins significantly inhibited the formation of NET, which had been induced in response to lipopolysaccharides (LPS) or a combination of phorbol myristate acetate (PMA) and ionomycin. The inhibitory effects were independent of the stage of estrous cycle (estrus, luteal, and follicular mucins). PMNs retained their segmented nuclei and membrane perforation was prevented. However, the inhibitory effects were diminished, when sialic acids were released under acidic conditions. Comparable results were achieved, when sialic acids were targeted by neuraminidase digestion, indicating a sialic acid dependent inhibition of NET release. Thus, bovine cervical mucins have an anti-inflammatory capability to modulate NET formation and might be further immunomodulatory biomolecules that support fertility.

Interacting post-muscarinic receptor signaling pathways potentiate matrix metalloproteinase-1 expression and invasion of human colon cancer cells.

M3 muscarinic receptor (M3R) expression is increased in colon cancer; M3R activation stimulates colon cancer cell invasion via cross-talk with epidermal growth factor receptors (EGFR), post-EGFR activation of mitogen-activated protein kinase (MAPK) extracellular signal-related kinase 1/2 (ERK1/2), and induction of matrix metalloproteinase-1 (MMP1) expression. MMP1 expression is strongly associated with tumor metastasis and adverse outcomes. Here, we asked whether other MAPKs regulate M3R agonist-induced MMP1 expression. In addition to activating ERK1/2, we found that treating colon cancer cells with acetylcholine (ACh) stimulated robust time- and dose-dependent phosphorylation of p38 MAPK. Unlike ERK1/2 activation, ACh-induced p38 phosphorylation was EGFR-independent and blocked by inhibiting protein kinase C-α (PKC-α). Inhibiting activation of PKC-α, EGFR, ERK1/2, or p38-α/β alone attenuated, but did not abolish ACh-induced MMP1 expression, a finding that predicted potentiating interactions between these pathways. Indeed, ACh-induced MMP1 expression was abolished by incubating cells with either an EGFR or MEK/ERK1/2 inhibitor combined with a p38-α/β inhibitor. Activating PKC-α and EGFR directly with the combination of phorbol 12-myristate 13-acetate (PMA) and EGF potentiated MMP1 gene and protein expression, and cell invasion. PMA- and ACh-induced MMP1 expression were strongly diminished by inhibiting Src and abolished by concurrently inhibiting both p38-α/β and Src, indicating that Src mediates the cross-talk between PKC-α and EGFR signaling. Using siRNA knockdown, we identified p38-α as the relevant p38 isoform. Collectively, these studies uncover novel functional interactions between post-muscarinic receptor signaling pathways that augment MMP1 expression and drive colon cancer cell invasion; targeting these potentiating interactions has therapeutic potential.

PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

BackgroundCombination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells.MethodsCell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively.ResultsWe found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner.ConclusionsThese results suggest that the synergy between PMA and apicularen A is involved by PKCα activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.

Abnormal conformation and impaired degradation of propylthiouracil‐induced neutrophil extracellular traps: Implications of disordered neutrophil extracellular traps in a rat model of myeloperoxidase antineutrophil cytoplasmic antibody–associated vasculitis

Neutrophil extracellular traps (NETs) are composed of DNA and antimicrobial proteins, including myeloperoxidase (MPO). Recent studies have demonstrated that impaired regulation of NETs could trigger an autoimmune response. Propylthiouracil (PTU), an antithyroid drug, is associated with a risk of MPO antineutrophil cytoplasmic antibody (ANCA) production and MPO ANCA-associated vasculitis (MPO AAV). This study was undertaken to clarify the mechanism of MPO ANCA production, using the PTU-induced model of MPO AAV. NETs were induced by treating human neutrophils with phorbol myristate acetate (PMA) in vitro. We examined whether the addition of PTU influenced the NET formation induced by PMA and the degradation of NETs by DNase I, which is regarded as a regulator of NETs. Furthermore, we examined whether NETs generated by the combination of PMA and PTU induced MPO ANCA and MPO AAV in vivo in rats. When NETs were induced by PMA with PTU using human neutrophils in vitro, abnormal conformation of NETs was observed. Interestingly, the abnormal NETs were hardly digested by DNase I. Moreover, rats immunized with the abnormal NETs, which had been induced by PMA with PTU using rat neutrophils, produced MPO ANCA and developed pulmonary capillaritis. When rats were given oral PTU with intraperitoneal injection of PMA, pauci-immune glomerulonephritis and pulmonary capillaritis occurred with MPO ANCA production in the serum. Our findings indicate that abnormal conformation and impaired degradation of NETs induced by PTU are involved in the pathogenesis of PTU-induced MPO ANCA production and MPO AAV. These findings suggest that disordered NETs can be critically implicated in the pathogenesis of MPO AAV.

Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

The nuclear factor of activated T-cell (NFAT) proteins are a family of transcription factors (NFATc1-c4) involved in the regulation of cell differentiation. We identified REDD1, a negative regulator of mammalian target of rapamycin (mTOR) through the tuberous sclerosis complex (TSC1/2 complex), as a new molecular target of NFATc3. We show that treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus ionophore A23187 (Io), which induces NFAT activation, increased REDD1 mRNA and protein expression and inhibited mTOR signaling; pretreatment with the calcineurin inhibitor cyclosporin A (CsA), an antagonist of NFAT signaling, decreased REDD1 induction and mTOR inhibition. Knockdown of NFATc3, not NFATc1, NFATc2, or NFATc4, attenuated PMA/Io-induced REDD1 expression. Treatment with PMA/Io increased REDD1 promoter activity and increased NFATc3 binding to the REDD1 promoter. Overexpression of NFATc3 increased REDD1 mRNA and protein expression and increased PMA/Io-mediated REDD1 promoter activity. Treatment with PMA/Io increased expression of the goblet cell differentiation marker MUC2; these changes were attenuated by pretreatment with CsA or knockdown of REDD1 or NFATc3. Overexpression of NFATc3 increased, while knockdown of TSC2 decreased, MUC2 expression. We provide evidence showing NFATc3 inhibits mTOR via induction of REDD1. Our results suggest a role for the NFATc3/REDD1/TSC2 axis in the regulation of intestinal cell differentiation.

NFATc1 Regulation of TRAIL Expression in Human Intestinal Cells

TNF-related apoptosis-inducing ligand (TRAIL; Apo2) has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter.

Flavonoids targeting of IκB phosphorylation abrogates carcinogen-induced MMP-9 and COX-2 expression in human brain endothelial cells

Brain endothelial cells play an essential role as structural and functional components of the blood–brain barrier (BBB). Increased BBB breakdown and brain injury are associated with neuroinflammation and are thought to trigger mechanisms involving matrix metalloproteinase upregulation. Emerging evidence also indicates that cyclooxygenase (COX) inhibition limits BBB disruption, but the mechanisms linking metalloproteinase to COX remain unknown. In this study, we sought to investigate the nuclear factor-kappa B (NF-κB) signaling pathway, a common pathway in both the regulation of matrix metalloproteinase-9 (MMP-9) and COX-2 expression, and the inhibitory properties of several chemopreventive flavonoids. Human brain microvascular endothelial cells were treated with a combination of phorbol 12-myristate 13-acetate (PMA), a carcinogen documented to increase MMP-9 and COX-2 through NF-κB, and several naturally occurring flavonoids. Among the molecules tested, we found that fisetin, apigenin, and luteolin specifically and dose-dependently antagonized PMA-induced COX-2 and MMP-9 gene and protein expressions as assessed by qRT-PCR, immunoblotting, and zymography respectively. We further demonstrate that flavonoids impact on IκK-mediated phosphorylation activity as demonstrated by the inhibition of PMA-induced IκB phosphorylation levels. Our results suggest that BBB disruption during neuroinflammation could be pharmacologically reduced by a specific class of flavonoids acting as NF-κB signal transduction inhibitors.

Effect of environmental enrichment on methylphenidate-induced locomotion and dopamine transporter dynamics

Rats raised in an enriched condition (EC) are less sensitive to the locomotor effects of stimulant drugs than rats raised in an impoverished condition (IC). Methylphenidate (MPD), a primary pharmacotherapy for attention-deficit/hyperactivity disorder, has abuse potential. This study determined whether environmental enrichment differentially altered the effects of MPD on locomotor activity and dopamine (DA) transporter (DAT) function. Acute and repeated MPD (3 or 10mg/kg, s.c.) increased locomotion in EC, IC and social condition (SC) rats; however, EC rats showed a blunted response to repeated MPD (3mg/kg). The maximal velocity (Vmax) of [3H]DA uptake in the presence of the combination of phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator and okadaic acid, a protein phosphatase inhibitor was decreased in EC and IC rats by 68% and 40%, respectively, indicating that DAT in prefrontal cortex (PFC) is more sensitive to PKC-mediated down-regulation in EC rats. Acute MPD (10mg/kg) administration decreased the Vmax of [3H]DA uptake in PFC and striatum in EC rats, but not in IC rats. Furthermore, [3H]WIN 35,428 binding density was decreased in PFC of EC and IC rats, and in striatum of EC rats given repeated MPD (10mg/kg). These results demonstrate that environmental enrichment modulates DAT dynamics in PFC. However, since the change in DAT function was observed only following the high dose of MPH (10mg/kg), the attenuated locomotor response to repeated MPD (3mg/kg) in EC rats is not likely due to a specific DAT alteration in the brain regions examined.

Patients with acute pancreatitis complicated by organ failure show highly aberrant monocyte signaling profiles assessed by phospho-specific flow cytometry*

To outline signaling profiles and transmigration capacity of monocytes of patients with severe acute pancreatitis. Prospective study. University hospital intensive care unit. Thirteen patients with severe acute pancreatitis. All patients had organ dysfunction (acute respiratory distress syndrome in 12, renal dysfunction in eight). Healthy volunteers served as reference subjects. Blood samples were collected after admission to the intensive care unit. Phosphorylation of nuclear factor-kappaB and p38, signal transducers and activators of transcription (STATs) 1, 3, 5, and extracellular signal-regulated kinases 1/2 in appropriately stimulated and nonstimulated samples were studied using phospho-specific whole-blood flow cytometry. Monocyte chemotactic protein-1-induced transmigration of monocytes among mononuclear cells obtained by density gradient centrifugation was studied using Transwell cell culture inserts covered with confluent layer of endothelial EA-HY cells. Phosphorylation levels of nuclear factor-kappaB induced by tumor necrosis factor, bacterial lipopolysaccharide, muramyl dipeptide, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis were significantly lower in patients' monocytes than monocytes of healthy reference subjects, whereas mitogen-activated protein kinase p38 phosphorylation levels were normal. Phosphorylation levels induced by interleukin-6 in STAT1 and STAT3 and by combination of phorbol 12-myristate 13-acetate and calcium ionophore A23187 in extracellular signal-regulated kinases 1/2, members of a mitogen-activated protein kinase family, were depressed in patients' monocytes, whereas phosphorylation levels induced by granulocyte-macrophage colony-stimulating factor in STAT5 was normal. In nonstimulated samples, phosphorylation levels were normal. The transmigration percentage of patients' monocytes was significantly lower than that of reference monocytes. In severe acute pancreatitis, monocytes show impaired nuclear factor kappaB and STAT1 activation, which may increase susceptibility to secondary infections. p38 activation is normal and STAT3 activation is depressed, which may contribute to maintenance of systemic inflammation. Extracellular signal-regulated kinases 1/2 activation is impaired, which may depress monocytes' transmigration and may consequently increase risk of infection. Monitoring of monocyte signaling profiles may aid in finding new therapeutic approaches and predictors of outcome of severe acute pancreatitis.

Human Tyrosine Kinase 2 Deficiency Reveals Its Requisite Roles in Multiple Cytokine Signals Involved in Innate and Acquired Immunity

Tyrosine kinase 2 (Tyk2) is a nonreceptor tyrosine kinase that belongs to the Janus kinase (Jak) family. Here we identified a homozygous Tyk2 mutation in a patient who had been clinically diagnosed with hyper-IgE syndrome. This patient showed unusual susceptibility to various microorganisms including virus, fungi, and mycobacteria and suffered from atopic dermatitis with elevated serum IgE. The patient's cells displayed defects in multiple cytokine signaling pathways including those for type I interferon (IFN), interleukin (IL)-6, IL-10, IL-12, and IL-23. The cytokine signals were successfully restored by transducing the intact Tyk2 gene. Thus, the Tyk2 deficiency is likely to account for the patient's complex clinical manifestations, including the phenotype of impaired T helper 1 (Th1) differentiation and accelerated Th2 differentiation. This study identifies human Tyk2 deficiency and demonstrates that Tyk2 plays obligatory roles in multiple cytokine signals involved in innate and acquired immunity of humans, which differs substantially from Tyk2 function in mice.

Lymph node cells from BALB/c mice with chronic visceral leishmaniasis exhibiting cellular anergy and apoptosis: Involvement of Ser/Thr phosphatase

Visceral leishmaniasis (VL) produced in BALB/c mice through intracardial administration of Leishmania donovani amastigotes was accompanied by hepatosplenomegaly with high organ parasite load and lymphadenopathy when followed up to 4-months or so. To elucidate the mechanism of immunosuppression associated with VL, we report here progressive impairment of the proliferative response of lymph node cells (lymphocytes) from infected animals (I-LNC) to in vitro stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) that could be related to the downregulation of PKC and MAP kinase (ERK 1/2) activation process. Further, pretreatment of I-LNC with the protein phosphatase inhibitor okadaic acid (OA), but not with calyculin A or sodium orthovanadate, significantly restored their proliferative response as well as PMA-induced activation of PKC. A population of LNC (primarily T-lymphocytes) from chronically infected animals was shown to undergo apoptosis, the number of which increased considerably following PMA+ Io stimulation. The apoptotic pathway, which was followed through binding of cells to Annexin V, activation of caspase-3 and fragmentation of DNA, involved destabilization of mitochondria, probably as a result of downregulation of PKC and Bcl-2. Interestingly, prior incubation of I-LNC with OA reversed the state of cell cycle arrest (anergy) and apoptosis through progression of cells from G0/G1 to S and G2/M phases with transcriptional activation of IL-2 and IL-2R genes. Our results suggest that the cellular (immune) dysfunction in VL could be attributed to dephosphorylation of key molecules in the T-lymphocyte signaling pathway by Ser/Thr phosphatase leading to their inactivation.

Raf-1 kinase activation is uncoupled from downstream MEK/ERK pathway in cells treated with Src tyrosine kinase inhibitor PP2

Recently we demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, markedly enhanced Ras-independent activation of Raf-1 by the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H 2O 2). We report here that Raf-1 knockdown cells were significantly more sensitive to treatment of PP2 than control cells. This PP2-induced growth inhibition was found to be linked to decreased ERK and p38 activity. Interestingly, the growth of Sprouty knockdown cells appeared to be inhibited at earlier time points of PP2 treatment when compared with control cells. Unexpectedly, siRNA-mediated knockdown of Spry2, which is known to modulate the Ras/Raf/MAPK signal through feedback regulation, resulted in decreased Raf-1 kinase activity. PP2 had limited effect on the ability of PMA/H 2O 2 to induce significant phosphorylation of MEK/ERK proteins in both Spry2 knockdown and control cells, indicating that PP2-mediated activation of Raf-1 did not potentiate signaling through the downstream MEK/ERK pathway. Taken together our results suggest that Raf-1 signaling may be bypassed in PP2-treated cells by uncoupling from downstream MEK/ERK pathway.

Reciprocal regulation of cyclooxygenase-2 and 15-hydroxyprostaglandin dehydrogenase expression in A549 human lung adenocarcinoma cells

Human lung adenocarcinoma cells, A549, possess the capacity of expressing both cyclooxygenase-2 (COX-2) and NAD+-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Resting cells express little COX-2 but significant levels of 15-PGDH. Interleukin (IL) 1beta, tumor necrosis factor-alpha (TNF-alpha) or phorbol ester [phorbol 12-myristate 13-acetate (PMA)] induced the expression of COX-2, as revealed by western blot analysis. Combination of PMA and IL-1beta or TNF-alpha induced synergistically the expression of COX-2. Interestingly, cytokines and cytokine plus PMA-induced expression of COX-2 were accompanied by a downregulation of 15-PGDH. This was evident from both the western blot analysis and activity assay of 15-PGDH. It appears that the higher the expression of COX-2 was induced, the lower the expression of 15-PGDH was found. This was further supported by the observation that overexpression of COX-2 but not COX-1 by adenovirus-mediated approach led to a decrease in 15-PGDH expression, indicating the specificity of COX-2. Furthermore, downregulation of the IL-1beta-induced expression of COX-2 by silencing RNA (siRNA) approach resulted in an increase in the expression of 15-PGDH by COX-2-siRNA but not by COX-1-siRNA, indicating that it was indeed the expression of COX-2 attenuating the expression of 15-PGDH. The IL-1beta-induced reduction of the expression of 15-PGDH was shown not to be mediated by COX-2-derived products since the presence of COX-2 inhibitors did not block the attenuation of the expression of 15-PGDH. Exogenous PGE2 also did not induce the reduction of the expression of 15-PGDH. However, overexpression of 15-PGDH by transfection with recombinant plasmid encoding 15-PGDH or adenovirus-mediated approach attenuated IL-1beta-induced expression of COX-2. On the contrary, downregulation of 15-PGDH expression by 15-PGDH-siRNA or 15-PGDH-antisense approach resulted in an increase in IL-1beta-induced expression of COX-2 but not that of COX-1. In fact, it was further observed that A549 clones expressing different degrees of 15-PGDH showed also different levels of COX-2 expression after IL-1beta induction. The levels of IL-1beta-induced COX-2 expression appeared to correlate inversely with those of 15-PGDH expression in the cells. These results support the contention that COX-2 and 15-PGDH are regulated reciprocally in A549 cells.

Possible activation of murine T lymphocyte through CD98 is independent of interleukin 2/interleukin 2 receptor system

CD98 is a widely expressed cell surface heterodimeric protein of 125 kDa. Its expression is upregulated during lymphocyte activation induced by mitogen, superantigen, conventional antigen, and a combination of phorbol myristate acetate (PMA) and ionomycin. However, the role of CD98 in the immune system is not so well understood. The role of CD98 in murine T lymphocyte proliferation was investigated, especially in correlation with the interleukin 2 (IL-2)/interleukin 2 receptor (IL-2R) system. Monoclonal antibody (mAb) directed against murine CD98 heavy chain (mCD98HC) suppressed the proliferation of lymphocytes stimulated with concanavalin A (Con A). Anti-mCD98HC mAb did not suppress the expression of IL-2Ralpha. Anti-IL-2Ralpha mAb, which suppressed DNA synthesis, did not inhibit the expression of CD98HC. Murine IL-2 (recombinant), which induced considerable DNA synthesis by lymphocytes stimulated with a sub-optimal dose of Con A, did not induce CD98HC expression in lymphocytes. In addition, anti-mCD98HC mAb did not inhibit the production of IL-2 by lymphocyte stimulated with Con A. Taken together with these findings, it was speculated that the CD98 system is independent of the IL-2/IL-2R system in murine T lymphocyte activation.

Secretion of the Human T Cell Leukemia Virus Type I Transactivator Protein Tax

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I protein Tax is well known as a transcriptional transactivator and inducer of cellular transformation. However, it is also known that extracellular Tax induces the production and release of cytokines, such as tumor necrosis factor-alpha and interleukin-6, which have adverse effects on cells of the central nervous system. The cellular process by which Tax exits the cell into the extracellular environment is currently unknown. In most cell types, Tax has been shown to localize primarily to the nucleus. However, Tax has also been found to accumulate in the cytoplasm. The results contained herein begin to characterize the process of Tax secretion from the cell. Specifically, cytoplasmic Tax was demonstrated to localize to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex. Additionally, it was demonstrated that full-length Tax was secreted from both baby hamster kidney cells and a human kidney tumor cell line, suggesting that Tax enters the secretory pathway in a leaderless manner. Tax secretion was partially inhibited by brefeldin A, suggesting that Tax migrated from the endoplasmic reticulum to the Golgi complex. In addition, combined treatment of Tax-transfected BHK-21 cells with phorbol myristate acetate and ionomycin resulted in a small increase in the amount of Tax secreted, suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies begin to provide a link between Tax localization to the cytoplasm, the detection of Tax in the extracellular environment, its possible role as an extracellular effector molecule, and a potential role in neurodegenerative disease associated with HTLV-I infection.