Abstract

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.

Highlights

  • In T lymphocytes, sustained elevation in [Ca2ϩ]i following activating signals, such as engagement of T cell receptor (TCR)2

  • We found that Ca2ϩ release from both IP3R and ryanodine receptor (RyR) was activated in parallel with store-operated Ca2؉ entry (SOCE) and significantly amplified [Ca2ϩ]i signaling in T lymphocytes

  • The major finding of this study is that in T lymphocytes activation of the plasmalemmal storeoperated Ca2ϩ (SOC) channels directly stimulates Ca2ϩ release from both IP3R and RyR, which significantly amplifies [Ca2ϩ]i signaling at the expense of store refilling (Fig. 5)

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Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Chemicals—Human acute T cell leukemia line Jurkat E6 –1 (ATCC, Manassas, VA) was maintained in cell culture medium containing RPMI 1640 medium (Lonza/BioWhittaker, Basel, Switzerland), 10% FBS (Omega Scientific, Tarzana, CA), 2% L-glutamine, 2% vitamin solution, 1% RPMI amino acid solution, and 0.01% ␤-mercaptoethanol. Some cells were incubated in modified Tyrode solution containing 10 ␮M (-)-XeC, or 400 ␮M Ry, or vehicle (methanol or DMSO) alone for 30 min at 37 °C. The extracellular solution used in all Mn2ϩ quench experiments contained (in mM): 130 NaCl, 5.6 KCl, 1 MgCl2, 0.3 CaCl2, 0.5 MnCl2, 10 HEPES, 10 D-glucose, pH 7.3. Some cells were preincubated for 30 min at 37 °C with (-)-XeC (10 ␮M), or Ry (400 ␮M), or dantrolene (30 ␮M) prior to transfer into FBS-containing cell culture medium. IL-2 Production Assay—Jurkat T cells were washed three times with PBS, resuspended in FBS-free cell culture medium at a density of 0.8 ϫ 106 cells/ml, seeded into 96-well tissue culture plates (0.4 ml/well), and stimulated with 100 ␮M mitogenic lectin phytohemeagglutinin P (PHA). Statistical differences between means were accepted as statistically significant at p Ͻ 0.05, using Student’s paired or unpaired t test or Wilcoxon nonparametric test

RESULTS
SOCE Activation and Facilitates
DISCUSSION
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