RESUMEN
Amplitude and kinetics of intracellular Ca2+ signals ([Ca2+]int) determine many immune cell functions. To mimic in vivo changes of [Ca2+]int in human immune cells, two approaches may be best suited: 1) Analyze primary human immune cells taken from blood under conditions resembling best physiological or pathophysiological conditions. 2.) Analyze the immune system in vivo or ex vivo in explanted tissue from small vertebrate animals, such as mice. With the help of genetically encoded Ca2+ indicators and intravital microscopy, [Ca2+]int have been investigated in murine T lymphocytes (T cells) in vivo during the last five years and in explanted lymph node (LN) during the last 10 years. There are several important reasons to compare [Ca2+]int measured in primary murine T lymphocytes in vivo and in vitro with [Ca2+]int measured in primary human T lymphocytes in vitro. First, how do human and murine data compare? Second, how do in vivo and in vitro data compare? Third, can in vitro data predict in vivo data? The last point is particularly important considering the many technical challenges that limit in vivo measurements and to reduce the number of animals sacrificed. This review summarizes and compares the results of the available publications on in vivo and in vitro [Ca2+]int measurements in T lymphocytes stimulated focally by antigen-presenting cells (APC) after forming an immunological synapse.
Asunto(s)
Señalización del Calcio/inmunología , Calcio/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , HumanosRESUMEN
AIMS: H2O2 is produced by all eukaryotic cells under physiological and pathological conditions. Due to its enormous relevance for cell signaling at low concentrations and antipathogenic function at high concentrations, precise quantification of extracellular local hydrogen peroxide concentrations ([H2O2]) originating from single cells is required. RESULTS: Using a scanning electrochemical microscope and bare platinum disk ultramicroelectrodes, we established sensitive long-term measurements of extracellular [H2O2] kinetics originating from single primary human monocytes (MCs) ex vivo. For the electrochemical techniques square wave voltammetry, cyclic and linear scan voltammetry, and chronoamperometry, detection limits for [H2O2] were determined to be 5, 50, and 500 nM, respectively. Following phorbol ester stimulation, local [H2O2] 5-8 µm above a single MC increased by 3.4 nM/s within the first 10 min before reaching a plateau. After extracellular addition of H2O2 to an unstimulated MC, the local [H2O2] decreased on average by 4.2 nM/s due to degradation processes of the cell. Using the scanning mode of the setup, we found that H2O2 is evenly distributed around the producing cell and can still be detected up to 30 µm away from the cell. The electrochemical single-cell measurements were validated in MC populations using electron spin resonance spectroscopy and the Amplex® UltraRed assay. Innovation and Conclusion: We demonstrate a highly sensitive, spatially, and temporally resolved electrochemical approach to monitor dynamics of production and degradation processes for H2O2 separately. Local extracellular [H2O2] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 29, 501-517.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Técnicas Biosensibles , Técnicas Electroquímicas , Escherichia coli/inmunología , Espacio Extracelular/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Monocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio , Análisis de la Célula IndividualRESUMEN
Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca(2+)) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca(2+) imaging, patch-clamp recordings, and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H(2)O(2)) on ORAI channel activity and human T helper lymphocyte (T(H) cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H(2)O(2). The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. T(H) cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector T(H) cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector T(H) cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector T(H) cells may tune cellular responses under oxidative stress.
Asunto(s)
Oxidación-Reducción , Canales de Calcio/metabolismo , Señalización del Calcio , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Humanos , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Interleucina-2/metabolismo , Células Jurkat , Proteína ORAI1 , Técnicas de Placa-Clamp , Isoformas de Proteínas , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno , Linfocitos T/metabolismoRESUMEN
Depletion of inositol 1,4,5 trisphosphate-sensitive Ca2+ stores generates a yet unknown signal, which leads to increase in Ca2+ influx in different cell types [J.W. Putney Jr., A model for receptor-regulated calcium entry, Cell Calcium 7 (1986) 1-12]. Here, we describe a mechanism that modulates this store-operated Ca2+ entry (SOC). Ca2+ influx leads to inhibition of protein tyrosine phosphatase 1B (PTP1B) activity in HEK 293 cells [L. Sternfeld, et al., Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells, Cell Signal 17 (2005) 951-960]. Since Ca2+ does not directly inhibit PTP1B, we assumed an intermediate signal, which links the rise in cytosolic Ca2+ concentration and PTP1B inhibition. We now show that Ca2+ influx is followed by generation of reactive oxygen species (ROS) and that it is reduced in cells preincubated with catalase. Furthermore, Ca2+-dependent inhibition of PTP1B can be abolished in the presence of catalase. H2O2 (100 microM) directly added to cells inhibits PTP1B and leads to increase in Ca2+ influx after store depletion. PP1, an inhibitor of the Src family tyrosine kinases, prevents H2O2-induced Ca2+ influx. Our results show that ROS act as fine tuning modulators of Ca2+ entry. We assume that the Ca2+ influx channel or a protein involved in its regulation remains tyrosine phosphorylated as a consequence of PTP1B inhibition by ROS. This leads to maintained Ca2+ influx in the manner of a positive feedback loop.