Effects of hypoxia and/or lack of glucose on cellular energy metabolism and cytokine production in stimulated human CD4+ T lymphocytes.

Oxidative phosphorylation and/or glycolysis provide energy, mainly in the form of ATP, which ensures proper functioning of immune cells such as CD4(+) T lymphocytes. However, the main substrates, namely oxygen and glucose, are known to remain for a relatively short time in the inflamed tissue and in other clinical situations where immune cells need to function properly. Therefore, we examined the effect of hypoxia and/or lack of glucose on cellular energy metabolism and on cytokine secretion in stimulated human CD4(+) T lymphocytes. Human CD4(+) T cells were MACS-isolated using peripheral blood obtained from healthy donors. Stimulated cells were incubated in medium with or without glucose for 6h in a sealed chamber which led to cumulative hypoxia. During this incubation period, (i) oxygen saturation was measured continuously using a Clark-type electrode, and (ii) samples were taken at different time points in order to quantify for each the viability of cells, intracellular reactive oxygen species (iROS), ATP levels, glycolytic enzyme activity, mRNA expression of hexokinase-1 and superoxide dismutase-1, and concentrations of several different cytokines. Stimulated CD4(+) T cells which were incubated under normoxic conditions served as controls. Under hypoxic conditions, lack of glucose exerted a biphasic effect on cellular oxygen consumption: initially higher but later lower respiration rates were measured when compared to conditions where glucose was available. Lack of glucose strongly increased the number of dead cells and the formation of iROS under normoxia but not under hypoxia. Under both normoxic and hypoxic conditions, intracellular ATP levels remained almost unchanged during the incubation period if glucose was present, but decreased significantly in the absence of glucose, despite the enhanced glycolytic enzyme activity. Measurements of stimulated cytokine production demonstrated (i) that cumulative hypoxia stimulates especially the secretion of IL-1beta, IL-10 and IL-8, and (ii) that lack of glucose results in lower cytokine concentrations. We demonstrate that CD4(+) T cells are highly adaptive in bioenergetic terms which ensure their proper function under extreme conditions of glucose and/or oxygen availability as found under physiological and pathophysiological conditions. Hypoxia seems to facilitate inflammatory reactions and angiogenesis.

Effects of celecoxib on the expression of osteoprotegerin, energy metabolism and cell viability in cultured human osteoblastic cells.

BACKGROUND AND OBJECTIVE: The selective COX-2 inhibitor celecoxib is widely used to treat pain and inflammation in rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. The drug has well-known important effects on immune cells but its direct and/or indirect influence on osteoblasts has not yet been explored in detail. This study aimed to investigate the dose-dependent effects of celecoxib on cell viability, energy metabolism and bone remodeling processes in cultured human osteoblastic cells. METHODS: Primary human osteoblasts and MG-63 cells were incubated with celecoxib (2, 10, 50microM). Cell viability and apoptosis were determined by trypan blue, 7AAD and Annexin-V staining. Effects on cellular oxygen consumption were measured amperometrically using a Clark electrode. mRNA expression of GLUT-1 and OPG was determined by RT-PCR; OPG protein secretion by ELISA and HIF-1alpha protein expression by immunoblotting. RESULTS: While celecoxib at a concentration of 2 and 10microM showed only marginal effects, a suprapharmacological concentration of 50microM influenced viability and energy metabolism, as well as OPG expression and secretion of osteoblastic cells. Cell viability was significantly reduced by celecoxib treatment. Celecoxib at 50microM stimulated oxygen consumption significantly. Corresponding experiments with the protonophore FCCP suggest that this effect is due to mitochondrial uncoupling. After 24h, GLUT-1 mRNA expression was significantly increased. HIF-1alpha protein was not expressed under any of our experimental conditions. We also showed that celecoxib at 50microM significantly inhibits OPG protein secretion leading to a compensative increase of mRNA expression. CONCLUSION: Pronounced effects of celecoxib on cell viability (reduction), oxygen consumption (stimulation), GLUT-1 mRNA expression (stimulation) and OPG protein secretion (inhibition) in osteoblastic cells were observed only at 50microM-a concentration not reached by therapeutic doses giving plasma concentrations less than 10microM. On the contrary, celecoxib at 2 and 10microM showed only marginal effects, suggesting that celecoxib administration is probably safe with respect to bone metabolism in cases requiring potent treatment of pain and inflammation. However, higher intracellular concentrations, which might occur through accumulation, necessitate investigations with high concentrations.

Hypoxia inducible factor (HIF) in rheumatology: low O2! See what HIF can do!

Maintenance of oxygen homoeostasis is the basic principle in cell proliferation, differentiation, survival, and function in all higher organisms. The transcription factor, HIF (hypoxia inducible factor) has a central role in oxygen homoeostasis, and is indispensably linked to energy metabolism. Abnormally reduced oxygen concentrations leading to dysfunctional cell metabolism are found in rheumatoid arthritis and hence, knowledge of the molecular adaptive responses to hypoxia and the involvement of HIF in the pathogenesis of RA are interesting.

Short-term effects of the 21-aminosteroid lazaroid tirilazad mesylate (PNU-74006F) and the pyrrolopyrimidine lazaroid PNU-101033E on energy metabolism of human peripheral blood mononuclear cells.

Two groups of antioxidant compounds, the 21-aminosteroids and the pyrrolopyrimidines, have been found to act as neuroprotective drugs against lipid peroxidation in the injured CNS. Like glucocorticoids at high doses they are assumed to produce their effects at least in part by direct membrane stabilizing effects. In order to prove this hypothesis, we have investigated in this study the effects of these drugs on the energy metabolism of activated human peripheral blood mononuclear cells (PBMC) since these cells have been shown to serve as a suitable test system for substances affecting processes of ATP turnover. We compared the in vitro effects of (i) the 21-aminosteroid lazaroid tirilazad, (ii) the pyrrolopyrimidine lazaroid PNU-101033E and (iii) the glucocorticoid methylprednisolone on mitogen-induced respiration rate and ATP-consumption. We show that tirilazad inhibits concanavalin A-stimulated respiration rate and sodium cycling across the plasma membrane. The effect of methylprednisolone is similar indicating corresponding cellular mechanisms. However, unlike methylprednisolone, tirilazad produced no significant effect on calcium cycling across the plasma membrane. PNU-101033E in our test system caused cytotoxic effects on PBMC that did not allow us to quantify cellular actions on energy metabolism. Our results underline the view that tirilazad, first, is mimicking the high-dose immunosuppressive pharmacology of glucocorticoids such as methylprednisolone and, second, is likely to produce its therapeutic effects by direct physicochemical interactions with cellular membranes.

Effect of glucocorticoid therapy on glucocorticoid receptors in children with autoimmune diseases.

Low-dose glucocorticoids (GC) achieve their action completely by classical genomic effects, mediated by the glucocorticoid receptor (GCR). In high doses of GC, nongenomic effects have also been found, but it is still unclear to what extent they contribute to a beneficial outcome. In this study, we present a determination of the number of lymphocyte GCR sites and the binding affinity in healthy children and children with autoimmune diseases. We further assess the effect of GC administration, especially of high-dose pulse therapy on the number of binding sites. The number of GCR sites per cell was analyzed with [(3)H]-dexamethasone radioligand binding assay and binding affinity (Kd given in nM) in peripheral blood mononuclear cells isolated from 48 healthy children and 35 patients. The patients were divided into three groups based on GC treatment: 0 mg/kg (group 1), 0.01-0.3 mg/kg orally (group 2), and 10-15 mg/kg i.v. pulse therapy (group 3) of prednisolone equivalent per day. Gender- and age-independent normal values of 4338 +/- 1687 sites/lymphocytes and Kd 6.7 +/- 2.2 nM were found. At 3463 +/- 1574, the number of receptor sites in patients without GC (group 1) was significantly lower than that of healthy volunteers (p < 0.05). In patients receiving GC treatment, this value was reduced to 2952 +/- 512 (group 2). Significant down-regulation to a minimum of 479 +/- 168 (group 3) was found after pulse therapy compared with untreated patients (p < 0.01). In pulse therapy, GC lead to a fast and dramatic receptor down-regulation. We suppose that the increase in therapeutic success of pulse-therapy may partly be mediated through additional nongenomic effects.

Norepinephrine inhibits energy metabolism of human peripheral blood mononuclear cells via adrenergic receptors.

Previous studies demonstrated that the adaptive response to stressors and inflammatory signals involves the activation of the autonomous [corrected] nervous system. Catecholamines have been shown to modulate the activity of various immune effector cells directly via membrane adrenergic receptors. Here, we investigated immediate effects of norepinephrine on energy metabolism of immune cells. Norepinephrine inhibits oxygen consumption of human peripheral blood mononuclear cells at concentrations that are relevant to its physiological range. The beta-adrenoreceptor antagonist propranolol, but not the alpha-adrenoreceptor antagonist phentolamine reversed the norepinephrine induced inhibition in quiescent cells. Conversely, phentolamine but not propranolol is capable of blocking norepinephrine mediated effects in mitogen activated human peripheral blood mononuclear cells. Our data indicate that the sensitization of alpha- and beta-adrenoreceptors on immune cells is differentially regulated, and that these processes depend on the activation state of these cells. These findings have important implications for the understanding of stress-induced suppression of immune function and may contribute to the elucidation of the pathogenesis of immunologically mediated diseases.

Bioenergetics of human peripheral blood mononuclear cell metabolism in quiescent, activated, and glucocorticoid-treated states.

The first quantitative findings on the energy metabolism of human immune cells are presented. In quiescent peripheral blood mononuclear cells (PBMC) protein biosynthesis and Na+,K+-ATPase activity each accounted for 8% of cellular oxygen consumption. Stimulation with 25, 50, and 75 microg Con A/ml (1.25, 2.5 or 3.75 microg/10(6) cells) increased total oxygen consumption within seconds by 8, 36, and 53%, respectively. After addition of 75 microg Con A/ml, the proportion of cellular oxygen consumption due to protein biosynthesis, Na+,K+-ATPase activity, and Ca2+-ATPase activity was 15% each and that due to DNA/RNA synthesis was 8%. On the basis of these findings the immediate effects of five different glucocorticoids on cellular energy metabolism were investigated. The various glucocorticoids exerted basically the same inhibitory effects on Con A-stimulated cellular respiration and individual ATP-consuming processes, but differed significantly in potency. Similar to previous studies on rat thymocytes, the relative potencies of the glucocorticoids were found to be: prednylidene (1.7) > dexamethasone (1.5) > methylprednisolone (1.0) > prednisolone (0.3) > betamethasone (< 0.2). Given their rapidity of onset, these effects must be nongenomically mediated. The differences between the relative potencies of the various glucocorticoids for these effects and those for the classical genomic effects have important clinical implications, in particular for high-dose systemic and local glucocorticoid therapy.

Glucocorticoid dose dependent downregulation of glucocorticoid receptors in patients with rheumatic diseases.

OBJECTIVE: The therapeutic success of low doses of glucocorticoids is mediated entirely by classical genomic effects, whereas that of high doses is also mediated to an as yet unknown extent by nongenomic effects. We assessed the relative therapeutic importance of these nongenomic effects in pulse therapy. METHODS: A [3H]dexamethasone radioligand binding assay was used to measure the number of glucocorticoid receptor sites (R, given as number of sites per cell) and glucocorticoid receptor binding affinity (Kd, given in nM) in peripheral blood mononuclear cells isolated from 26 healthy control blood donors and 27 patients with rheumatic diseases. Patients were divided into 4 groups on the basis of their glucocorticoid dose: 0 mg (Group A), < or = 0.25 mg (Group B), 0.25 to 1 mg (Group C), and > 1 mg (Group D) of prednisolone equivalent per kg per day. RESULTS: Sex independent normal values of 3605 +/- 1136 for R and 5.39 +/- 3.4 for Kd were found. At 5407 +/- 1968, the number of receptor sites in patients not receiving glucocorticoid therapy (Group A) was significantly higher than that of controls (p < 0.01). In patients receiving glucocorticoid therapy this value was reduced at 3855 +/- 866 (Group B), 3358 +/- 963 (Group C), and 2685 +/- 962 (Group D). The values in Groups C and D were significantly lower than those in untreated patients (p < 0.02). CONCLUSION: In pulse therapy doses of glucocorticoids that exceed receptor saturation are administered for several days, but in addition significant receptor downregulation occurs. Therefore, we assume an increase in the relative contribution of the nongenomic effects of glucocorticoids to the therapeutic success under these conditions.