Erika Baus

Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor

A method is described to facilitate flow cytometric analysis of calcium mobilization upon stimulation of murine T cell hybrids. In these transformed cell lines, the accuracy of cytometric measurement of free cytoplasmic calcium with Fluo-3 is compromised by the rapid loss of the intracellular dye. We have found that the addition of sulfinpyrazone, a known organic-anion transporter inhibitor in epithelial cells and in macrophages, severely impairs the leakage of the Fluo-3 probe from the cytoplasmic matrix. Under appropriate conditions, sulfinpyrazone has little effect on the cell physiology and permits the detection of calcium influx in a variety of murine T cell hybrids.

Dexamethasone increases intracellular cyclic AMP concentration in murine T lymphocyte cell lines

The effects of the synthetic glucocorticoid dexamethasone on the cAMP content of murine T lymphocyte cell lines has been investigated. Incubation of the 3B4.15 T cell hybrids with dexamethasone results in an average 5-fold increase in intracellular cyclic AMP levels after 6 h of treatment. This phenomenon is abolished in the presence of RU486 and of cycloheximide, indicating that it requires binding of the drug to the intracellular glucocorticoids receptor and de novo protein synthesis. Dexamethasone-induced elevation of intracellular cyclic AMP correlates with both an increase in adenylate cyclase activity and a decrease in phosphodiesterase activity in T cell hybrids. This modulation of cyclic AMP metabolism is independent of serum-derived factors, suggesting that it is not secondary to transmembrane receptor stimulation by an extracellular ligand. We propose that glucocorticoids interfere with the homeostatic control of intracellular cAMP concentration, leading to a sustained increase in the content of this important second messenger in murine T lymphocyte cell lines. This study suggests that elevation of cAMP levels may represent one way by which glucocorticoids modulate the immune response.

A novel aspect of the anti-inflammatory actions of glucocorticoids: inhibition of proximal steps of signaling cascades in lymphocytes.

Abstract. Glucocorticoid hormones are effective in inhibiting inflammatory responses, but the mechanisms that confer this action have not been completely elucidated. The prevailing view is that these compounds inhibit novel gene transcription regulated by the nuclear factor kappa B and/or activator protein-1 transcription factors. In the last few years, several reports have shown that glucocorticoids can also block signal transduction in lymphocytes at an early, postreceptor step, suggesting novel molecular targets for these hormones. These data will be briefly reviewed and the possible in vivo relevance of these findings discussed, with particular emphasis on T cell development.

Metabolic Stress Boosts Humoral Responses In Vivo Independently of Inflammasome and Inflammatory Reaction

Adjuvant formulations boost humoral responses by acting through several, yet incompletely elucidated pathways. In this study, we show that oligomycin or 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) enhances Ab production when coinjected with T cell-dependent Ags. Oligomycin and AICAR lead to intracellular ATP reduction, suggesting that metabolic stress could be sensed by immune cells and leads to increased humoral responses. AICAR promotes IL-4 and IL-21 by naive Th cells but does not affect dendritic cell activation/maturation in vitro or in vivo. Accordingly, the adjuvant effect of AICAR or oligomycin does not require MyD88 or caspase-1 expression in vivo. Because AICAR is well tolerated in humans, this compound could represent a novel and safe adjuvant promoting humoral responses in vivo with a minimal reactogenicity.

Dexamethasone inhibits invasion of murine T cells through cultured fibroblastic monolayers

Despite the wide clinical use of glucocorticoids in the chemotherapy of leukaemia and lymphoma, there have been limited efforts at understanding the effects of these hormones on metastasis formation. The purpose of this study was to investigate the effects of glucocorticoids on the tissue-infiltrating capability of lymphoid cells. Using an in vitro invasion assay, we found that dexamethasone, a synthetic glucocorticoid analogue, inhibited the invasion of a murine T-cell hybridoma through a monolayer of fibroblast-like cells. Even low doses of dexamethasone were effective at inhibiting cellular transmigration (EC50 = 0.4 nM). A maximal decrease was observed after an overnight culture in the presence of dexamethasone. The effect persisted for at least 24 h after removal of the drug and required the binding of the hormone to its intracellular glucocorticoid receptor. Our results suggest that the decreased invasiveness of dexamethasone-treated cells is not the consequence of reduced motility or deficient production of an autocrine factor required for cell migration. This in vitro study suggests that glucocorticoids may act to reduce dissemination of lymphoma cells in vivo.

Flow cytometric measurement of cytosolic calcium in lymphocytes

Calcium plays a central role in the transduction of external stimuli in many cell types. In B and T lymphocytes, stimulation of surface immunoglobulins or T cell receptors, respectively, triggers the activation of members of the src family of tyrosine kinases. These enzymes phosphorylate several intracellular substrates including phosphoinositidespecific phosphodiesterase (PLCγ), GTPase-activating protein for p2lras (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). PLCγ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate. These second messengers are responsible for the activation of protein kinase C (PKC) and an increase in the concentration of intracellular ionized calcium ([Ca2+]i). Together, these agents are capable of initiating a cascade of biochemical events that results in new gene expression leading to functional responses such as cytokine production, up-regulation of cytokine receptor expression and cellular proliferation (for a review of this subject see Weiss and Littman 1994). The role of calcium in the response of lymphocytes to extracellular stimuli is clearly demonstrated by the observation that cells cultured in medium containing low levels of extracellular calcium fail to proliferate or produce cytokines upon receptor cross-linking (Mills et al. 1985; Dennis et al. 1987).