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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2733-2741
PLENARY PAPER
From the Department of Surgery, Division of Transplantation,
University of Wisconsin, Madison, WI.
The activation of blood cells, including T cells, triggers
intracellular signals that control the expression of critical
molecules, including cytokines and cytokine receptors. We show that
T-cell receptor (TCR) ligation increases the cellular level of the
protein linker for activation of T cells (LAT), a molecule critical for T-cell development and function. T-cell activation increased LAT messenger RNA, as determined by reverse transcription-polymerase chain
reaction and by Northern blotting. The TCR-induced increase in LAT
expression involved the activation of the serine/threonine kinases PKC
and MEK, because inhibitors of these kinases blocked the increase in
LAT. Accordingly, the PKC activator phorbol myristate acetate
up-regulated LAT expression. Strikingly, the calcineurin inhibitors
cyclosporin A (CsA) and FK506 strongly potentiated TCR-induced LAT
expression, suggesting that the activation of calcineurin following TCR
ligation negatively regulates LAT expression. Accordingly,
Ca++ ionophores, which can activate calcineurin by
increasing intracellular Ca++, blocked the TCR-induced
increase in cellular LAT. CsA and FK506 blocked the Ca++
ionophores' inhibitory effect on LAT expression. Notably, CsA and FK506 preferentially up-regulated TCR-induced LAT expression; under
the same conditions, these compounds did not increase the expression of
14 other molecules that previously had been implicated in T-cell
activation. These data show that TCR-induced LAT expression involves
the activation of the PKC-Erk pathway and is negatively regulated by
the activation of calcineurin. Furthermore, the potentiation of
TCR-induced LAT expression by CsA and FK506 suggests that the action of
these agents involves up-regulating the cellular level of critical
signaling molecules. These findings may have important therapeutic implications.
(Blood. 2000;95:2733-2741)
Activated T cells are central to the immune response to
infection, tumors, and allografts. T-cell activation in vivo is
triggered by the interaction between the T-cell receptor (TCR) and its
respective antigen presented in the context of the major
histocompatibility complex. TCR engagement initiates an array of
intracellular signals that lead to cell adhesion, proliferation,
differentiation, and to the increase in the transcriptional activity of
cytokine genes. TCR-triggered signals include the stimulation of
protein tyrosine kinases (PTKs), the generation of inositol
triphosphate and diacylglycerol, the activation of protein kinase C
(PKC), the increase in intracellular Ca++, the activation
of the Ras and Rho family of GTPases, and the activation of several
mitogen-activated protein kinases (MAPKs).1,2 Stimulated
MAPKs translocate from the cytoplasm to the nucleus, where they
phosphorylate and activate several transcription
factors.3,4 The increase in intracellular Ca++
following TCR ligation stimulates key enzymes in the T-cell signal transduction cascade, such as the
Ca++/calmodulin-dependent serine/threonine
phosphatase calcineurin.5-10 Stimulated calcineurin
regulates the activity of transcription factors and, in turn, the
transcription of genes that encode cytokines, cytokine receptors,
and protooncogenes.
Proteins that become phosphorylated on tyrosine residues following
receptor ligation play important roles in the signal transduction pathways of various receptors, including TCR, and are critical for cell
growth and differentiation.1,2,11-13 Such proteins can be
divided into 2 groups: those with intrinsic enzymatic activity, such as
PTKs, and those with no known enzymatic activity (collectively known as
adapter or linker proteins). The earliest detectable biochemical event
following TCR engagement is the activation of several PTKs, resulting
in the transient tyrosine phosphorylation of numerous intracellular
substrates. Among the PTKs that become rapidly phosphorylated on
tyrosine following TCR ligation are those that belong to the Src
family, the Syk/Zap-70 family, the Tec family, the Csk family, and the
FAK family. Adapter molecules that become tyrosine-phosphorylated
following TCR ligation include SLP-76, Vav, Cbl, and LAT. Despite their
lack of enzymatic activity, adapter molecules are critical for TCR
signaling because they propagate receptor signaling by bridging the
various signaling molecules.
LAT (linker for activation of T cells) is a recently identified 36- to
38-kd transmembrane protein that becomes tyrosine-phosphorylated after
the stimulation of various receptors, including TCR and CD28.14-25 LAT is a substrate for Src and Syk/Zap-70 PTKs
and, upon tyrosine phosphorylation, binds the SH2 domains of several signaling molecules, including Grb2, Cbl, phospholipase C (PLC)-gamma, and Vav.14,18,21,22 Interestingly, LAT has also been shown to associate with the coreceptors CD4 and CD8, a process that appears
to be important for LAT tyrosine phosphorylation by ZAP-70 upon TCR
ligation.20 LAT association with CD8 required cytosolic cysteines C227 and C229 on CD8, because replacing these residues with
alanines markedly diminished the association.20
Transfection studies and studies in knockout mice strongly suggest that
LAT is critical for T-cell development and
function.14,18,.26
We examined the effect of T-cell stimulation on the expression of LAT
in T cells and found that T-cell activation by TCR or CD28 ligation or
by phorbol myristate acetate (PMA) treatment increased the expression
of LAT. We also found that the PKC-Erk cascade is involved in
up-regulating LAT expression in T cells. Ca++ ionophores
did not increase the expression of LAT; however, they did block TCR-
and PMA-induced up-regulation of LAT expression. Remarkably, the
calcineurin inhibitors CsA and FK506, at concentrations that inhibited
interleukin (IL)-2 production, did not block LAT expression; rather,
both drugs potentiated a TCR-induced increase in LAT expression and
blocked Ca++ ionophore-induced inhibition of LAT
expression. Thus, LAT expression in T cells involves the activation of
the PKC-Erk cascade and is negatively regulated by the increase in
intracellular Ca++ and the activation of calcineurin. These
results also show that the actions of CsA and FK506 on T cells involve
the up-regulation of the expression of critical signaling molecules.
Reagents
Antibodies
Cells Resting T cells were isolated from the blood of healthy humans on Ficoll-Histopaque and washed 3 times in RPMI containing 10% fetal calf serum (FCS). The cells were then incubated with goat-antihuman IgG-coated Immulan beads according to the manufacturer's recommendations. Unbound cells (T cells) were collected, and adherent cells were removed by incubating the cells in a tissue culture flask for 30 minutes at 37°C. T-cell purity was at least 98% as determined by flow cytometry. Acute human T-cell leukemia (Jurkat) cells, clone E6-1, were obtained from American Type Culture Collection (Bethesda, MD) and maintained in suspension as described previously.19Cell activation, preparation of cell lysates, and immunoblotting T-cell activation was done as reported previously with some modification.19,27-30 Purified human normal resting T cells or Jurkat T cells were washed with RPMI containing 5% FCS (RPMI-5% FCS) and suspended in the same medium (4 × 106 cells/mL). Meanwhile 96-well tissue culture plates were coated with the indicated concentrations of the antibodies for least 2 hours at 37°C. After washing the plates, 50 µl of the cell suspension (2 × 105 cells) were added to each well, and the plates were incubated for the indicated times at 37°C in a water-jacketed incubator. After incubation, the cells were immediately lysed with boiling 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Proteins in whole cell lysates (WCL) were separated by SDS-PAGE and electrotransferred onto PVDF membranes. Membranes were immunoblotted with HRP-conjugated antiphosphotyrosine mAb PY20 or with the specific antibodies followed by HRP-coupled donkey-antimouse Ig Ab or HRP-coupled donkey-antirabbit Ig Ab (1:40 000 dilution). The signals were visualized using the LumiGLO kit.Interleukin-2 assay The IL-2 level in the supernatants of overnight cultures was determined with a sandwich enzyme-linked immunosorbent assay technique using combinations of unlabeled and biotin-labeled antibodies as described previously.38Reverse transcription-polymerase chain reaction A total of 2 × 106 Jurkat T cells in RPMI-5% FCS were stimulated with 1 ng/mL of PMA or with 3 µg/mL of anti-CD3 mAb for 8 hours at 37°C. After incubation, the cells were washed and total RNA was extracted using the RNeasy mini kit according to the manufacturer's recommendations (Qiagen). Total RNA was reverse transcribed to generate first-strand complementary DNA (cDNA), and the resultant cDNA was amplified by polymerase chain reaction (PCR). Samples in agarose gels were visualized by ethidium bromide staining. The primer sequences for LAT were 5'-GTATCCAAGGGGCATCCAGTT-3' (sense) and 5'-CCTCTTCCTCCACTTCCTCTG-3' (antisense) and for glyceraldehyde phosphate dehydrogenase (GAPDH) were 5'-CCATGGAGAAGGCTGGGG-3' (sense) and 5'-CAAAGTTGTCATGGATGACC-3' (antisense).Northern blotting A total of 2 × 107 Jurkat T cells were incubated for 8 hours at 37°C in RPMI-5% FCS or in RPMI-5% FCS containing the indicated concentrations of PMA. After incubation, the cells were washed in phosphate-buffered saline (PBS), and poly A+ RNA was extracted using the Oligotex mRNA mini kit. The resultant mRNA was subjected to 1% agarose electrophoresis and then transferred to Ambion's BrightStar-Plus positively charged nylon membranes. The RNA was cross-linked to the membranes by baking the membranes at 80°C for 30 minutes. The membranes were then prehybridized for 4 hours at 50°C using the reagents provided in the Northern blotting (Max-Gly) kit. After prehybridization, the membranes were incubated for 16 hours at 50°C with a mixture of the following 5'-biotinylated LAT antisense oligonucleotides: 5'-GAACGTTCACGTAATCATCAATGGACTCCA-3', 5'-GGCCTTTATTCTATTACACAGAGTAGGGCTGG-3', and 5'-GGCCGTTTGAACTGGATGCCCCTTGGATAC-3'. After incubation, the membranes were extensively washed, and membrane-bound biotinylated probe was visualized using the BrightStar Biodetect kit. To ensure similar loading, the membranes were stripped of LAT oligonucleotides by boiling for 30 minutes in 0.1% SDS and then were reprobed with the following 5' biotinylated -actin antisense
oligonucleotide: 5'-GGAAGGTGGACAGCGAGGCCAGGATGGAGC-3'.
Immunofluorescence confocal microscopy and flow cytometry For stimulation with anti-CD3 mAb, 2 × 107 Jurkat T cells in RPMI-5% FCS were incubated for 16 hours at 37°C in uncoated or anti-CD3 mAb-coated tissue culture dishes (100 × 20 mm Falcon polystyrene dishes; Becton Dickinson Labware, Franklin Lakes, NJ). Following incubation, nonadherent cells were aspirated and CD3-adherent cells were removed from the dishes by gentle pipetting, washed in PBS, and fixed for 60 minutes in 2% paraformaldehyde in PBS. The cells were then washed in PBS and were permeabilized for 5 minutes in 0.2% saponin in PBS. After permeabilization, the cells were washed in Tris-buffered saline containing 5% Tween (TBS/Tween). A total of 2 × 106 cells were incubated for 30 minutes at room temperature with 10 µg/mL anti-LAT Ab, washed in TBS/Tween, and then incubated for 30 minutes at room temperature with goat-antirabbit IgG conjugated to Alexa 488 diluted 1:300 in TBS/Tween. After incubation, the cells were washed in TBS/Tween and then analyzed on a Bio-Rad MRC 1024 confocal scanning laser microscope with inverted Nikon Eclipse TE300 microscope using Laser Sharp Version 3.2 software. Aliquots of the labeled cells used above were also examined for LAT expression by flow cytometry using the Becton Dickinson FACScan and the Cell Quest software.
T-cell activation by CD3 ligation up-regulates the expression of LAT To examine the effect of T-cell stimulation on the expression of LAT, we incubated purified human normal resting T cells or Jurkat T cells with the indicated concentrations of plate-immobilized anti-CD3 mAb for 16 hours at 37°C. Such incubation did not affect the number of the cells; nor did it affect the viability of the cells, as determined by trypan blue exclusion assay (data not shown). After incubation, the cells were immediately lysed with boiling SDS-PAGE sample buffer. The proteins in WCL were subjected to SDS-PAGE, electrotransferred to membranes, and blotted with anti-LAT mAb or, as a control, with anti-Csk Ab. In initial studies, we found that the expression of Csk in Jurkat T cells was resistant to the various treatments outlined in the present article. Anti-CD3 mAb reproducibly increased the level of total cellular LAT in human normal resting T cells (Figure 1A) and in the Jurkat T cell line (Figure 1B) in a dose-dependent manner. Although in some experiments Csk was slightly increased after CD3 ligation in human normal resting T cells, its level did not change in Jurkat T cells. LAT expression was also increased when T cells were incubated with anti-CD3 mAb for 16 hours at 37°C in suspension, albeit to a lesser extent than that with the plate-immobilized anti-CD3 mAb (data not shown). The results indicate that CD3 ligation initiates intracellular signals that up-regulate LAT expression in T cells.
T-cell activation up-regulates the mRNA for LAT To determine whether T-cell activation up-regulates LAT mRNA, we stimulated the cells for 8 hours at 37°C with anti-CD3 mAb or PMA and then examined the cells for the level of the mRNA for LAT by the reverse transcription (RT)-PCR technique and by Northern blotting. For RT-PCR, total RNA was extracted from unstimulated and stimulated cells, reverse transcribed to generate first-strand cDNA, and the resultant cDNA was amplified by PCR. Samples in agarose gels were visualized by ethidium bromide staining. As shown in Figure 2A, both anti-CD3 mAb and PMA increased the mRNA for LAT. As a control, we measured the level of the mRNA for GAPDH. No increase in the level of mRNA for this gene was seen in cells treated with either anti-CD3 mAb or PMA (Figure 2A). To confirm these data, poly A+ RNA was isolated from unstimulated and stimulated Jurkat T cells and then used in Northern blotting as described in "Materials and methods." As shown in Figure 2B, Jurkat T-cell activation increased the level of LAT mRNA. Whether the increase in the level of LAT mRNA after T-cell activation is the result of the increase in gene transcription or due to enhanced mRNA stability is subject to further investigation.
The involvement of PKC in the stimulus-induced up-regulation of LAT expression T-cell activation through CD3 has been shown to activate the serine/threonine kinase PKC.31,32 Furthermore, the increase in LAT expression after PMA treatment suggests a role for PKC in regulating LAT expression, because PKC is the intracellular receptor for PMA.33 Therefore, we examined the involvement of PKC in regulating LAT expression by investigating the effect of the PKC inhibitors H-7 and Ro-31-8220 on a stimulus-induced increase in LAT expression. Both PKC inhibitors blocked the CD3- and PMA-induced increase in LAT expression (Figure 3, upper panels). These blockers had no effect on the viability of the cells, as determined by trypan blue exclusion assay (data not shown). Blotting the membranes with anti-Csk Ab confirmed that a similar amount of WCL was loaded in each lane, because the level of Csk was only slightly decreased in the presence of the PKC inhibitors (Figure 3, lower panels). These results implicate PKC in the signaling pathways that up-regulate LAT expression.
The MAPK MEK is involved in regulating LAT expression in T cells TCR-initiated signals in T cells have been shown to activate the MAPK mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase (MEK-Erk) cascade, leading to the phosphorylation and activation of transcription factors. Similarly, PMA-activated PKC has been shown to activate the Ras-Raf pathway, which in turn links PKC to the MEK-Erk pathway. To further define the pathways that regulate LAT expression in T cells, we examined the effect of the selective MEK blocker PD98059 on a stimulus-induced increase in LAT level. PD98059 blocked the CD3- and PMA-induced increase in LAT expression in a dose-dependent manner, suggesting that the MEK-Erk cascade is involved in regulating LAT expression (Figure 4A and 4B). The inhibition of LAT expression by PD98059 correlated with the inhibition of MEK activation by PD98059, as determined by the decrease in the tyrosine phosphorylation of Erk (Figure 4C). We do not know why PD98059 was more effective in inhibiting CD3-induced LAT expression than inhibiting PMA-induced LAT expression.
The calcineurin inhibitors CsA and FK506 potentiate the TCR-induced increase in LAT expression in T cells The serine/threonine phosphatase calcineurin, also known as phosphatase 2B, is a key enzyme in TCR signal transduction pathways and has been implicated in regulating receptor-induced gene expression in T cells. The calcineurin inhibitors CsA and FK506 are widely used to block calcineurin activation. Both agents form complexes with intracellular proteins, which bind calcineurin, leading to the inhibition of its phosphatase activity.34 Thus, to examine the involvement of calcineurin in regulating receptor-induced LAT expression, T cells were stimulated with anti-CD3 mAb in the continuous presence of the indicated concentrations of CsA or FK506. Remarkably, both compounds potentiated CD3-induced LAT expression in Jurkat T cells (Figure 5A) and in human normal resting T cells (Figure 5B) in a dose-dependent manner. The effect of FK506 was dependent on the concentrations of the anti-CD3 mAb used to stimulate T cells (Figure 5C). CsA or FK506 alone increased the basal level of LAT only slightly, if at all, suggesting that inhibiting the activation of calcineurin is not by itself sufficient to modulate LAT expression, but that calcineurin inhibition appears to modulate receptor-induced LAT expression (Figure 5D). Interestingly, the effect of CsA and FK506 on LAT expression was evident at concentrations of CsA and FK506 that effectively blocked stimulus-induced IL-2 production (Figure 5E), indicating that the concentrations of FK506 and CsA used in the present studies effectively blocked calcineurin activation. These results strongly suggest that calcineurin activation negatively regulates LAT expression in T cells.
Ca++ ionophores block stimulus-induced up-regulation of LAT expression The increase in intracellular Ca++ after T-cell activation regulates the activity of various enzymes, including the protein phosphatase calcineurin. Ca++ ionophores mobilize Ca++ from intracellular stores and induce Ca++ influx by mechanisms that bypass signals activated early in TCR signaling, leading to a prolonged and sustained increase in intracellular Ca++. As described, the potentiation of TCR-induced LAT expression by CsA and FK506 strongly suggests that calcineurin negatively regulates LAT expression. To further examine this possibility, we determined whether Ca++ ionophores would down-regulate a stimulus-induced increase in LAT expression. The Ca++ ionophores A23187 and ionomycin blocked TCR- and PMA-induced expression of LAT in Jurkat T cells in a dose-dependent manner (Figure 6A and 6B). The vehicle dimethyl sulfoxide at a concentration of 1% (20 times more than that found in the 1-µM ionophore A23187 solution) did not block stimulus-induced up-regulation of LAT expression (data not shown). Notably, the inhibition of LAT expression was evident at concentrations of A23187 and ionomycin (Figure 6C) that synergized with PMA in inducing IL-2 expression, suggesting that the increase in intracellular Ca++ and the subsequent activation of calcineurin have opposing roles in IL-2 and LAT expression. The Ca++ ionophores' effect was not limited to the Jurkat T cells, because Ca++ ionophore A23187 also blocked stimulus-induced up-regulation of LAT expression in human normal resting T cells in a dose-dependent manner (Figure 6D). Notably, the LAT level in cells pretreated for 16 hours with PMA did not decrease when the cells were subsequently incubated for up to 8 hours with Ca++ ionophore A23187, suggesting that Ca++ ionophores do not down-regulate the newly expressed LAT by activating enzymes such as proteases (Figure 6E, compare lanes 2 and 5); however, this issue is subject to further investigation. These results show that Ca++ ionophores trigger signals that down-regulate receptor-induced LAT expression in T cells.
CsA and FK506 block Ca++ ionophore-mediated inhibition of activation-induced up-regulation of LAT expression The data strongly implicate calcineurin in down-regulating CD3-induced LAT expression. To further examine the involvement of calcineurin in the inhibition of LAT expression by the Ca++ ionophore A23187, T cells were treated with PMA and Ca++ ionophore A23187 in the absence or presence of CsA or FK506. Both CsA and FK506 blocked Ca++ ionophore A23187-induced inhibition of LAT expression in a dose-dependent manner (Figure 7). These results further implicate calcineurin in negatively regulating LAT expression in T cells.
Inhibition of calcineurin preferentially potentiates TCR-induced expression of LAT To examine whether inhibiting calcineurin activation enhances TCR-mediated expression of signaling molecules other than LAT, we stimulated Jurkat T cells in the absence or presence of FK506 using the same conditions described above for LAT. The cellular level of 14 signaling proteins that previously have been implicated in T-cell activation was determined by immunoblotting as described previously. These signaling proteins were arbitrarily selected, because the antibodies for these molecules were available in our laboratory at the time of the study. T-cell activation did not have a detectable effect on the cellular levels of Csk, Erk, FAK, Grb2, PLC- , or PKC
(Figure 8). T-cell activation, however,
increased the expression of some of the molecules shown in Figure
8.35-38 Thus, TCR ligation increased the expression of Cbl,
Fyn, Vav, Lck, Pyk2, SLP-76, paxillin, and ZAP-70 (Figure 8). FK506 by
itself did not induce a detectable increase in the levels of any of the molecules (Figure 8, compare lanes 1 and 8). Strikingly, under the
conditions described in the present study, FK506 strongly and
reproducibly increased the level of LAT but not of any of the other
molecules shown in Figure 8. In some experiments, however, a slight
increase was seen in the level of SLP-76 and Pyk2. Interestingly, FK506
blocked TCR-induced increase in Fyn, Lck, paxillin, and ZAP-70,
suggesting that the activation of calcineurin may be important for the
TCR-induced expression of these molecules. Similar results were
obtained with CsA (data not shown).
LAT expressed upon CD3 ligation and FK506 treatment distributes normally in the cell To examine whether the overexpressed LAT after CD3 ligation and FK506 treatment distributes normally in the cell, Jurkat T cells were incubated with the anti-CD3 mAb in the presence or absence of FK506 for 16 hours at 37°C. After washing, the cells were fixed, permeabilized, labeled with anti-LAT Ab, and then were stained with goat-antirabbit IgG conjugated to Alexa 488. The cells were then examined for LAT distribution by confocal scanning laser microscopy. As reported previously,14 LAT localized in unstimulated cells to the cell membrane and to a juxtanuclear intracellular compartment (Figure 9A2). FK506 alone induced only a slight increase in LAT expression (Figure 9A3). As reported above, anti-CD3 mAb increased LAT expression (Figure 9A4), which was markedly increased in the presence of FK506 (Figure 9A5). Notably, although LAT staining was stronger in cells stimulated with anti-CD3 mAb in the presence or absence of FK506 than in resting cells, the pattern of LAT staining in the stimulated cells was similar to that in the unstimulated cells. Thus, the overexpressed LAT localizes normally in the cells. The normal distribution of the overexpressed LAT suggests that the newly expressed LAT localizes to areas important for its involvement in TCR signaling pathways. Flow cytometry performed on aliquots of the labeled cells that were used in Figure 9A confirmed the increase in LAT expression (Figure 9B).
Proteins that become tyrosine-phosphorylated following TCR engagement are critical for the function of T cells. To better understand the involvement of the phosphoprotein LAT in T cell processes, we studied the effect of T-cell activation on the expression of LAT and examined the molecular mechanisms that regulate receptor-induced LAT expression. We showed in this report that TCR ligation initiated signals that led to the up-regulation of LAT expression in human normal resting T cells and in the Jurkat T-cell line. We also showed that the signals that regulate TCR-induced increase in LAT expression included the activation of the PKC-Erk pathway and the Ca++-calcineurin pathway. Our results strongly suggest that these pathways play opposing roles in regulating LAT expression in T cells.
We thank Drs Robert Conhaim and Donna Peters for assistance with the microscopy studies, John Fechner for assistance with flow cytometery studies, and Jackie Schultz for excellent technical assistance. We also thank Andrea Schmick for editorial assistance.
Submitted September 17, 1999; accepted December 24, 1999.
Reprints: Majed M. Hamawy, H4/749, Department of Surgery, 600 Highland Ave, Madison, WI 53792; e-mail: hamawy{at}surgery.wisc.edu.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
1. Qian D, Weiss A. T cell antigen receptor signal transduction. Curr Opin Cell Biol. 1997;9:205[Medline] [Order article via Infotrieve]. 2. Alberolaila J, Takaki S, Kerner JD, Perlmutter RM. Differential signaling by lymphocyte antigen receptors. Annu Rev Immunol. 1997;15:125[Medline] [Order article via Infotrieve].
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Abstract
Introduction
