Adenosine modulates LPS-induced cytokine production in porcine monocytes.

Adenosine plays an important role during inflammation, particularly through modulation of monocyte function. The objective of the present study was to evaluate the effect of synthetic adenosine analogs on cytokine production by porcine monocytes. The LPS-stimulated cytokine production was measured by flow cytometry and quantitative real-time PCR. Adenosine receptor expression was measured by quantitative real-time PCR. The present study demonstrates that adenosine analog N-ethylcarboxyamidoadenosine (NECA) down-regulates TNF-α production and up-regulates IL-8 production by LPS-stimulated porcine monocytes. The effect was more pronounced in CD163(-) subset of monocytes compared to the CD163(+) subset. Although both monocyte subsets express mRNA for A1, A2A, A2B and A3 adenosine receptors, the treatment of monocytes with various adenosine receptor agonists and antagonists proved that the effect of adenosine is mediated preferentially via A2A adenosine receptor. Moreover, the study suggests that the effect of NECA on porcine monocytes alters the levels of the cytokines which could play a role in the differentiation of naive T cells into Th17 cells. The results suggest that adenosine plays an important role in modulation of cytokine production by porcine monocytes.

Effect of fluoxetine and adenosine receptor NECA agonist on G alpha q/11 protein of C6 glioma cells.

OBJECTIVES: Trimeric G-proteins play a crucial role in the transmembrane signalling to intracellular pathways via effector phospholipase C (1,4,5 IP3) or adenylylcyclase (cAMP). G-protein modulation is considered to participate in the antidepressant mode of action by neurotransmitter G-protein coupled receptors (GPCR). Adenosine is naturally occured nucleoside and adenosine receptor belongs to GPCR family. Properties and functions of ubiquitous adenosine receptor were described with number of agonists and antagonists. METHODS: In C6 glioma cells, we studied acute administration of SSRI antidepressants - fluoxetine, sertraline and citalopram. We used immunochemical estimation (ELISA) of the main types of G-protein alpha subunits from isolated membranes of C6 glioma cells. We also estimated effect of NECA agonist on fluoxetine induced signalling via 1,4,5 IP3 and its levels. RESULTS: Results show involvement of the antidepressant drugs in the C6 glioma signal transduction cascades and their modulation in dependence on the antidepressant of SSRI type. We measured main G alpha protein profiles after fluoxetine, sertraline and citalopram administration. We found significant changes as following: decreased G alpha Gq/11 for fluoxetine, low G alpha s for sertraline and both high G alpha q/11 and high G alpha s for citalopram. Furthermore the NECA (5´-N-ethylcarboxamido- adenosine) agonist of adenosine receptor alone evoked high decrease of G alpha q/11 levels. Whereas fluoxetine influenced G alpha q/11 decline was abolished by NECA in concentration manner, especially at 10-8 and 10-9 M concentrations. These results support abolishion NECA effect on fluoxetin influenced 1,4,5 IP3 signalling via PLC. CONCLUSION: Main G alpha profiles are dependent on SSRI type antidepressant. Abolishing both fluoxetine evoked G alpha q/11 and and 1,4,5 IP3 signalling can indicate parallel interference between G-protein coupled receptors (GPCR) and the cell response. Presented data are first findings about adenosine receptor interaction with fluoxetine signalling. Thus in vitro studies contribute to the clarification of the molecular basis of antidepressant action.

The effect of adenosine on pro-inflammatory cytokine production by porcine T cells.

Adenosine is a well described anti-inflammatory modulator of immune responses. The aim of the present study was to describe the role of common adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) in cytokine production by main porcine T cell subpopulations. TNF-α, IFN-γ, IL-2 and IL-10 were detected by multicolor flow cytometry together with cell surface markers CD3, CD4 and CD8. It was found that NECA inhibits (in a dose-dependent manner) production of pro-inflammatory TNF-α and Th1-associated cytokines IFN-γ, IL-2 in all concanavalin A-stimulated T cell subpopulations. Moreover, production of IL-10 was potentiated in all T cell subpopulations tested. These corresponded well with the fact that all T cell subsets expressed mRNA for adenosine receptor (AR) subtypes to comparable extents. Contrary to concanavalin A-stimulated cells, NECA had a moderate effect on PMA-stimulated T cells, suggesting that AR in pigs acts via signaling pathways not associated with protein-kinase C. Non-selective antagonist CGS15943 as well as allosteric modulator SCH202676 failed to reverse the effect of NECA in pigs. In conclusion, NECA has an anti-inflammatory effect on porcine T cell subpopulations.

Effect of fluoxetine or adenosine receptor NECA agonist on G-proteins of C6 glioma cells or NK immunocytes.

OBJECTIVE: Neurochemical approaches to antidepressant effects and depressive disorder are also focusing on G-protein coupled receptors (GPCR) and subsequent signalling. Trimeric G-proteins play a crucial role in transmembrane signalling, its amplification and processing. It is evident that immune system participates in antidepressant mode of action by neurotransmitter GPCR. METHODS: We studied the effect of acute administration of fluoxetine or NECA agonist of adenosine receptor (GPCR) on C6 glioma cells and natural killer (NK) cell line, innate immunity. We used immunochemical estimation (ELISA) of the main types of G-protein alpha subunits from isolated membranes of tested cells. RESULTS: Significant reduction of G alpha q/11 subunits after acute administration of fluoxetine or NECA agonist was found. In contrast, no significant influence of G alpha s or G alpha i1,2 subunit levels of C6 glioma cells were observed. Lowered Gq/11 signalling was in accordance with decreased 2nd messenger 1,4,5 IP3 formation by PLC. Acute effect of fluoxetine or NECA agonist on NK cell line resulted in significantly reduced G alpha q/11 levels without changes in G alpha s and G alpha i1,2. Furthermore, we determined that NECA agonist was able to abolish fluoxetine-evoked G alpha q/11 levels of NK cell line. CONCLUSIONS: Results show involvement of fluoxetine in the C6 glioma signal transduction and were comparable with NK cells. Similar inhibiton of G alpha q/11 by NECA agonist in both C6 glioma cells and NK cell line was determined. Furthermore NECA induced attenuation of fluoxetine evoked Galpha q/11 signalling can indicate parallel interference between GPCR and final response. Finally, we determined similarity in both interleukin 2, IL2 immunostimulator and fluoxetine evoked G q/11 levels in NK cell line and thus fluoxetine action could be related to signalling aspects of neuroimmunomodulatory activity.

Effect of mitogen lectin on lymphocyte or brain cortex cell activation.

OBJECTIVES: We studied a) mitogen lectin (PHA) evoked changes of Na+/K+-ATPase activity in functionally different lymphocytes or brain cortex cells and b) quantitative relationship between PHA- evoked early enzyme activation and late lymphocyte proliferation were analyzed. MATERIALS AND METHODS: We performed biochemical analyses of Pi released from ATP by Na+/K+-ATPase activity. Lymphocyte proliferation was assayed by 3H-thymidine incorporation. RESULTS: We demonstrated PHA stimulated Na+/K+-ATPase activity of mouse spleen lymphocytes or freshly isolated brain cortex cells. Besides this, we estimated high stimulation of Na+/K+-ATPase activity and subsequent late 3H-thymidine incorporation into pig lymphocytes as both PHA dose and K+ ion concentration dependent. CONCLUSIONS: Thus, early PHA dose-dependent stimulation of Na+/K+-ATPase activity is a more general response in different animal species and functionally different cells. We measured both cell type- and PHA-dose dependent enzyme activity stimulation. We can suggest that intensity of early PHA induced Na+/K+-ATPase activation could be in relationship to subsequent elevated level of T lymphocyte proliferation. The Na+/K+-ATPase can be a part of mitogen lectin evoked signal transduction mechanisms.

Acute and chronic effects of antidepressants on the G-protein alpha subunit profiles in vitro and in vivo.

OBJECTIVES: Neurochemical studies on the etiopathogenesis of depression are also focusing on the transduction system beyond receptors. Trimeric G-proteins play a crucial role in the transmembrane signalling, signal amplification and intracellular processing. Abnormalities of G-protein levels are observed in subjects with depression, G-protein modulation is considered to play a role in the antidepressant mode of action. METHODS: We studied acute or chronic administration of antidepressants from different pharmacological groups. We used immunochemical estimation (ELISA) of the main types of G-protein alpha subunits from isolated membranes of C6 glioma cells and rat brain tissue. RESULTS: Significant elevation of G alpha q/11 subunits after chronic administration of sertraline and significant reduction of G alpha s subunit levels following both acute and chronic administrations of sertraline were found. In contrast, no significant effects on G alpha subunit levels following acute desipramine and moclobemide administration were observed in vitro. Chronic moclobemide effect in vivo is causing significant elevation of Galpha s and Galpha i1,2 subunit levels. CONCLUSIONS: Results show involvement of antidepressant drugs in the C6 glioma signal transduction cascades modulation in dependence on the antidepressant class. Significant influence in the cAMP system modulation is observed after administration both SSRI and MAOA inhibitors. Astrocytoma cells - C6 glioma cells also can offer a model system of the glia where modulation of cell signalization cascades can influence cell functioning and production of neurotrophic factor molecules relevant to the antidepressant treatment and depression etiopathogenesis.

Cell signalling in CNS and immune system in depression and during antidepressant treatment: focus on glial and natural killer cells.

CNS, endocrine and immune systems share the same molecules: neurotransmitters, cytokines and hormones to communicate within and among each other. Depression is associated with abnormalities in the noradrenergic, serotonergic and dopaminergic neurotransmitter systems and reductions in the level of their precursors and metabolic turnover. Most of these signalling molecules use trimeric G-proteins as a transduction system to transfer extracellular signal into cellular response. Altered levels or function of signalling proteins, especially alpha subunits of trimeric G-proteins, were found in post-mortem brain tissue and leukocytes of subject suffering from major depression. There is a considerable evidence that inflammatory response and immune system changes are the part of depression. Components of cellular immune system natural killer cells, important effectors of immune surveillance, are sensitive to stress response, and their functions are compromised in depressive subjects. Many lines of evidence also point to the loss of both neuronal and glial plasticity and neurotrophic factor support under chronic stress or in depression. There is an increasing knowledge of the role of astrocytic cells in neuroplastic processes and neurotransmitter metabolism. Alterations in the glial populations are observed in major depressive subjects. Antidepressant treatment is modulating glial signalization cascades, increasing production of neurotrophic molecules, supporting neuroplasticity processes, and also modulating functions of natural killers. At the level of membrane signalling, antidepressants show a direct influence upon G alpha subunit levels in both immune system and CNS. These findings support the view that antidepressants influence activity of natural killer and astrocytic populations, and this could be of importance in the depression etiopathogenesis and/or treatment.

Age-dependent changes of proinflammatory cytokine production by porcine peripheral blood phagocytes.

The aim of the present study was to determine postnatal ontogeny of proinflammatory cytokines IL-1beta, IL-8 and TNF-alpha production by in vitro stimulated porcine blood leukocytes. Four age categories of pigs were chosen. Cytokine production was determined using intracellular flow cytometry. It was found that IL-8 and TNF-alpha production by blood monocytes significantly increased during the postnatal period while production of IL-1beta remained unchanged. In blood neutrophils, the IL-8 production increased only during the postnatal period, while the levels of TNF-alpha and IL-1beta were undetectable during the whole postnatal period. Generally, the most intensive changes in cytokine production occurred before weaning. The production of low levels of cytokines by monocytes and neutrophils from young pigs was not caused by a delayed cytokine response because the cytokine production after 8-h stimulation was lower than that after 4-h stimulation in all age categories. The ontogenetical changes showed the same trends when two different stimulators (LPS, heat-inactivated E. coli) were used, suggesting that the ontogenetical changes are not caused by a simple defect in one signalling pathway, but it is probably a more complex process. No differences in cytokine production between the whole blood and the isolated cells supplemented with newborn or adult serum were found. Thus the ability of newborn monocytes and neutrophils to produce proinflammatory cytokines was not decreased due to the influence of composition of the microenvironment, where the cells were present. In conclusion, the ability of porcine blood leukocytes to produce cytokines develops during postnatal life.

Quantitative nitric oxide production by rat, bovine and porcine macrophages.

The aim of this work was to compare in vitro nitric oxide (NO) production by rat, bovine and porcine macrophages. NO production was induced by lipopolysaccharide (LPS) or by phorbol 12-myristate 13-acetate (PMA) with ionomycin or recombinant interferon gamma (rIFN-gamma) and was assessed by Griess reaction. NO synthase type II (NOS II) expression was quantified by immunocytochemistry, Western blot and real-time polymerase chain reaction (RT-PCR). There were differences in NO production by pulmonary alveolar macrophages (PAM) in all species tested. The largest amounts of NO were produced by rat PAM. Less NO was produced by bovine PAM. Moreover, PAM in rats and cows differed in their abilities to respond to various stimulators. Neither porcine PAM nor Kupffer cells produced NO. Stimulation of porcine PAM with alternative concentrations of LPS did not lead to inducing NO production. Stimulation of porcine PAM with rIFN-gamma together with LPS led to a significant increase in the expression of NOS II mRNA, albeit without detectable NO production or NOS II expression on the protein level.

Intracellular cytokine detection by flow cytometry in pigs: fixation, permeabilization and cell surface staining.

Intracellular flow cytometry is a method of cytokine detection that allows simultaneous detection of intracellular cytokines and cell surface markers. This important method is not extensively used in pigs, in particular due to the inaccessibility of proper methodological protocols modifying comprehensive human protocols. The aim of this study was to find the best procedure for fixation and permeabilization of porcine blood leukocytes and simultaneous cell surface staining. Permeabilization with commercial kits gave better results in most of the chosen parameters compared with combinations of different concentrations of paraformaldehyde and saponin. Among the commercial kits tested, the best results were obtained with the IntraStain kit. Cell surface markers were detected on cells stimulated for cytokine production by antibodies anti-CD14 (clone MIL-2), anti-SWC3, anti-CD4 and anti-CD8 except anti-CD14 (clone Tük4). While anti-CD8 (clone MIL-12) must be used for staining of unfixed cells, the other antibodies recognize fixed and/or permeabilized cells. Moreover, anti-SWC3 and anti-CD14 (clone MIL-2) antibodies can stain cells during the permeabilization step. These modifications of the cell surface staining protocol allow the researcher to speed up the procedure of intracellular cytokine staining or to combine cell surface staining and intracellular cytokine staining. The present study can serve as a particular protocol of intracellular cytokine detection and as a suggestion for optimization of the fixation, permeabilization and cell surface staining procedure in any laboratory.

Postnatal functional maturation of blood phagocytes in pig.

Developmental changes of functional ability of peripheral blood phagocytes from days 1 to 100 of life were investigated. Luminol enhanced chemiluminiscence was used to establish the ability of phagocytes to produce reactive oxygen species (ROS). Simple superoxide anion production was determined by spectrophotometrical measurement of cytochrome c. Activity of surface aminopeptidase N was assessed by spectrophotometrical measurement of l-alanine-p-nitroanilide. Flow cytometric measurements of CD18 and CD45 expression were performed. The ROS production per 0.5microl of blood did not show any trend; however, the values recalculated per 500 granulocytes had a decreasing course. The most noteworthy increase in production of superoxide anion occurred between days 17 and 26. Activity of aminopeptidase N decreased during the first 4 weeks. Expression of CD18 and CD45 intensively increased from days 1 to 14 with gradual decrease by day 100. Natural immunity develops during the early postnatal life and seems to be influenced by exposure of the organism to environmental antigens.