Rapamycin antagonizes cyclosporin A- and tacrolimus (FK506)-mediated augmentation of linker for activation of T cell expression in T cells

Clifford S. Cho1, Zhen Chang1, Johny Elkahwaji1, Tara L. Scheunemann1, Eric R. Manthei1, Matthew Colburn1, Stuart J. Knechtle1 and Majed M. Hamawy1

1 Division of Transplantation, Department of Surgery, University of Wisconsin Medical School, Madison, WI 53792, USA

Correspondence to: M. M. Hamawy; E-mail: hamawy{at}surgery.wisc.edu
Transmitting editor: J. P. Allison


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The discovery of new immunosuppressive drugs such as rapamycin, cyclosporin A (CsA) and tacrolimus (FK506) has been very useful for preventing graft rejection and autoimmune disease. However, these drugs are not specific, and are associated with side-effects and toxicities. Therefore, understanding the molecular mechanisms of these drugs is important for designing specific immunosuppressants. Here, we show that in contrast to CsA and FK506, rapamycin blocks activation-induced expression of the linker for activation of T cells (LAT), a signaling molecule critical for initiating TCR signaling. Thus, whereas CsA and FK506 strongly enhanced TCR- and phorbol myristate acetate-induced LAT expression in T cells, rapamycin effectively inhibited activation-induced LAT expression. Importantly, these opposite effects were mutually antagonistic, as rapamycin acted as a potent antagonist for both CsA and FK506. Because CsA, unlike FK506 and rapamycin, does not bind to the intracellular immunophilin FK-binding protein (FKBP), the antagonism between these drugs is not simply due to competition for intracellular FKBP. Accordingly, RNA and protein stability analyses suggest inhibition by rapamycin at the translational level. Given the important role of LAT in initiating T cell activation, our data suggests that the effects of rapamycin, CsA and FK506 on T cell activation involve regulating early T cell signaling. These findings refine our understanding of the manifold effects of these immunosuppressants, thus providing insight into the drastic physiological contrasts observed between these drugs.

Keywords: immunosuppression, signaling, transplantation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The immunosuppressive drugs rapamycin and the so-called calcineurin inhibitors cyclosporin A (CsA) and tacrolimus (FK506) have been very useful for treating graft versus host, autoimmune and inflammatory diseases, and have truly revolutionized allograft transplantation (14). However, these immunosuppressive drugs have been associated with side-effects and toxicities that limit their clinical utility. This is due, at least in part, to the fact that these drugs disrupt the function of many different cell types. Thus, elucidating the molecular mechanisms of these drugs could be important for designing specific immunosuppressants with no, or low, side-effects and toxicities.

Although rapamycin, FK506 and CsA are believed to induce their immunosuppressive effects primarily by targeting T cells, the precise molecular mechanisms of their action are not fully understood. CsA and FK506 have been shown to block the activation of the calcium calmodulin-dependent serine/ threonine phosphatase calcineurin (57). Calcineurin becomes activated in response to TCR ligation, leading to the dephosphorylation of the transcription factor NF-AT (810). The dephosphorylation of NF-AT facilitates its translocation into the nucleus where it promotes the transcription of important genes including IL-2, a growth factor essential for T cell proliferation (1113). Upon entering the T cell cytoplasm, CsA and FK506 respectively bind the immunophilins cyclophilin and FK-binding protein (FKBP) (57). The drug–immunophilin complexes bind calcineurin, leading to inhibition of the phosphatase activity of calcineurin and in turn to the inhibition of IL-2 transcription (57). In contrast, rapamycin does nothing to inhibit IL-2 expression; rather, rapamycin renders the T cell unresponsive to IL-2 (14,15). Rapamycin is structurally similar to FK506 and binds the same FKBP immunophilin species to which FK506 binds. However, the resulting complex does not affect calcineurin; it inhibits the mammalian target of rapamycin (mTOR), a protein which mediates protein translation and cell cycle progression events normally induced by IL-2 receptor engagement (14).

A finding that has garnered considerable interest in recent years has been the observation that some states of altered T cell function thought to be related to the induction or maintenance of immunological tolerance are differentially affected by rapamycin and calcineurin inhibitors. Specific ally, it has been demonstrated that T cell anergy and T cell apoptosis, two phenomena that have been hypothesized to be involved in mechanisms of tolerance, are inhibited by calcineurin inhibitors, but facilitated by rapamycin (16,17). These in vitro observations appear to be substantiated by a growing body of small and large animal in vivo data that suggest that calcineurin inhibitors may inhibit the induction of some forms of tolerance, while rapamycin may permit or even facilitate their induction.

Considerable effort has been directed toward understanding the mechanisms for these disparate effects. Most proposed explanations have focused on late events in T cell activation, such as cell cycle progression (16), activation-induced cell death (17) and cell-surface co-stimulatory molecule expression (18). In the present report, we describe evidence that the calcineurin inhibitors and rapamycin also induce opposite influences on the early T cell signaling protein linker for activation of T cells (LAT). LAT is a 36- to 38-kDa transmembrane protein expressed almost exclusively on T cells (19). Mutation analysis and knockout mouse studies have shown that LAT is essential for T cell activation and development (1921). It has been shown to associate with the co-receptors CD4 and CD8, which may serve to bring LAT into close proximity with the TCR upon T cell–antigen-presenting cell engagement (22). Shortly after TCR engagement, the tyrosine kinases Fyn and Lck are activated, and initiate a cascade of tyrosine phosphorylation events that include the phosphorylation of the tyrosine kinase ZAP-70. LAT is a substrate for ZAP-70 in T cells and is rapidly phosphorylated on a number of tyrosine residues shortly after TCR engagement (19,20,23) or CD28 engagement (24). These phosphotyrosine residues serve as docking sites for the Src homology 2 consensus binding site regions of numerous signaling proteins, including Grb2, Cbl, phospholipase C{gamma} and Vav (19,20,25). As such, LAT functions as a non-enzymatic adapter protein that effectively links the initial tyrosine kinase cascade induced by TCR engagement with multiple downstream intracellular signaling pathways. These pathways ultimately result in protein kinase C (PKC) activation, mobilization of intracellular calcium, activation of the mitogen-activated protein kinases and transcription factor activation (2630).

We have recently reported that sustained T cell stimulation induces an up-regulation in the expression of LAT (31). We also observed that CsA and FK506 further potentiated this activation-induced expression of LAT (31). We herein report that rapamycin induces an opposite, inhibitory effect on activation-induced LAT expression, and that rapamycin, CsA and FK506 appear to exert mutually antagonistic influences on levels of LAT protein. These findings serve to enhance our understanding of these immunosuppressive agents and may have implications for their divergent effects on important aspects of T cell function.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Materials
CsA was obtained from Sandoz (East Hanover, NJ) and FK506 was purchased from Fujisawa (Deerfield, IL). Rapamycin was from Biomol (Plymouth Meeting, PA). The LumiGLO chemiluminescent substrate kit and TMB peroxidase substrate were from Kierkegaard & Perry (Gaithersburg, MD). The Oligotex mRNA mini-kit was purchased from Qiagen (Santa Clara, CA). The SV40 total RNA isolation kit was from Promega (Madison, WI). BrightStar Plus positively charged nylon membranes, the Max-Gly northern blotting kit and the BrightStar BioDetect kit was obtained from Ambion (Austin, TX). All other reagents were purchased from Sigma (St Louis, MO).

Anti-human CD3 mAb was obtained from Ancell (Bayport, MN). Anti-Csk and anti-LAT antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-mouse IgG and horseradish peroxidase (HRP)-conjugated donkey anti-mouse Ig were obtained from Jackson ImmunoResearch (Bar Harbor, ME). Goat anti-human IgG-coated Immulan beads were purchased from Biotecx (Houston, TX).

Cell preparation
Clone E6-1 Jurkat acute human leukemia T cells were obtained from ATCC (Bethesda, MD) and maintained in culture as previously described (24). Normal peripheral T cells were isolated from blood drawn from healthy volunteers, centrifuged on Ficoll-Histopaque and washed 3 times in RPMI media containing 15% FCS. T cells were enriched through negative selection by incubating the cells with goat anti-human IgG-coated Immulan beads according to the manufacturer’s recommendations. Unbound T cells were eluted and adherent cells were removed by incubating the cells in a tissue culture flask at 37°C for 30 min. T cell activation was performed using methods previously reported (24,3235).

T cell activation, preparation of cell lysates and immunoblotting
T cells were washed and resuspended in RPMI media containing 5% FCS (RPMI/5% FCS). For plate-bound anti-CD3 mAb stimulation, 96-well flat-bottomed tissue culture plates were coated with the indicated concentrations of anti-CD3 mAb for 8 h at 37°C. Wells were then washed twice with RPMI/5% FCS, and 50 µl of cells (2 x 105 cells) and 50 µl of rapamycin and/or FK506 or CsA at the indicated concentrations were added to each well. For phorbol myristate acetate (PMA) stimulation, 50 µl of cells (2 x 105 cells) and 50 µl of PMA, rapamycin and/or FK506 at the indicated concentrations were mixed in 12 x 75 mm test tubes. Plates and tubes were then incubated for the indicated times at 37°C in a water-jacketed incubator. Cell lysis was performed by adding an equal volume of 2 x SDS–PAGE sample buffer. Proteins in whole-cell lysates were separated by 10% SDS–PAGE and electrotransferred onto PVDF membranes overnight. Mem branes were blocked with a 5% non-fat dry milk buffer for 4 h, then immunoblotted using the indicated primary antibodies followed by HRP-coupled donkey secondary antibodies. Signals were visualized by autoradiography using the LumiGLO detection kit.

Northern blotting
Jurkat T cells (2 x 107) were incubated in RPMI/5% FCS with the indicated concentrations of PMA and rapamycin for 16 h at 37°C. The cells were then washed in PBS and poly(A)+ RNA was extracted using the Oligotex mRNA mini-kit. The resultant mRNA was subjected to 1% agarose gel electrophoresis and then transferred to BrightStar Plus positively charged nylon membranes overnight. The RNA was cross-linked to the membranes by incubating the membranes at 80°C for 30 min. The membranes were then prehybridized for 4 h at 50°C using the Max-Gly northern blotting kit. Hybridization was performed for 16 h at 50°C with the addition of a mixture of the following 5'-biotinylated LAT antisense oligonucleotides: 5'-GAACGTTCACGTAATCATCAATGGACTCCA-3', 5'-GGCCTTTATTCTATTACACAGAGTAGGGCTGG-3' and 5'-GGCCGTTTGAACTGGATGCCCCTTGGATAC-3'. The membranes were then extensively washed and membrane-bound biotinylated probes were visualized using the BrightStar BioDetect kit. To ensure equal loading, the membranes were stripped of LAT oligonucleotides by boiling for 30 min in 0.1% SDS followed by reprobing using the 5' biotinylated ß-actin antisense oligonucleotide 5'-GGAAGGTGGACAGCGAGGCCAGGATGGAGC-3'.

RT-PCR
Jurkat T cells (5 x 107) were incubated in RPMI/5% FCS with the indicated concentrations of PMA and rapamycin for 16 h at 37°C. The cells were then washed in PBS, and poly(A)+ RNA was extracted using the Oligotex mRNA mini-kit. The resultant mRNA was reverse transcribed to generate first-strand cDNA using avian myeloblastosis virus reverse transcriptase. cDNA was then amplified using PCR with the LAT-specific oligonucleotides 5'-GTATCCAAGGGGCATCCAGTT-3' (sense) and 5'-CCTCTTCCTCCACTTCCTCTG-3' (antisense), and the GAPDH-specific oligonucleotides 5'-CCATGGAGAAGGCTGGGGG-3' (sense) and 5'-CAAAGTTGTCATGGATGACC-3' (antisense). PCR products were then subjected to 1% agarose gel electrophoresis and visualized by ethidium bromide staining.

Real-time quantitative PCR
Jurkat T cells (5 x 107) were incubated in RPMI/5% FCS with the indicated concentrations of PMA and rapamycin for 16 h at 37°C. The cells were then washed in PBS and total RNA was extracted using the Promega SV40 total RNA isolation kit. One-step real-time RT-PCR was performed on the LightCycler using the RNA amplification kit with SYBR Green I (Roche, Indianapolis, IN) according to the manufacturer’s instructions. LAT-specific oligonucleotides 5'-GTATCCAAGGGGCATCCAGTT-3' (sense) and 5'-CCTCTTCCTCCACTTCCTCTG-3' (antisense) were used for amplification, and actin-specific oligonucleotides 5'-CCTCGCCTTTGCCGATCC-3' (sense) and 5'-GGATCTTCATGAGGTAGTCAGTC-3' (antisense) were used for internal control for relative quantitation. Using the threshold cycle numbers for LAT and actin amplification, relative expression levels of each were calculated using the LightCycler quantitation software.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Rapamycin blocks activation-induced expression of LAT in T cells
We previously reported that the inhibition of calcineurin activation using FK506 strongly potentiates activation-induced expression of LAT in T cells (31). The structurally related drug, rapamycin, binds the same intracellular receptor as FK506, but exerts different effects on T cells. To determine the effect of rapamycin on TCR-induced LAT expression, Jurkat and normal peripheral blood T cells were incubated with anti-CD3 mAb in the presence of the indicated concentrations of FK506 or rapamycin. These treatments did not affect the number or viability of T cells, as determined by Trypan blue dye exclusion assay (data not shown). After 16 h incubation, the cells were lysed with the addition of boiling SDS–PAGE sample buffer. The resulting whole-cell lysates were separated by 10% SDS–PAGE, electrotransferred to PVDF membrane and blotted with anti-LAT antibody or, for control purposes, with anti-Csk antibody. Initial studies had demonstrated that regulation of Csk expression was fairly resistant to the various treatments described in this report. In agreement with our previously published results, FK506 reproducibly potentiated activation-induced expression of LAT in a dose-dependent manner (Fig. 1A). In contrast, rapamycin reproducibly inhibited activation-induced expression of LAT in a dose-dependent manner (Fig. 1B). The inhibitory effect of rapamycin on activation-induced LAT expression was also observed in normal T cells drawn from peripheral blood (Fig. 1C). Time course experiments using Jurkat T cells demonstrated no evidence of increased LAT expression at any time with rapamycin treatment, whereas FK506 and CsA strongly potentiated LAT expression within 4 h of treatment (Fig. 2).



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Fig. 1. Rapamycin blocks activation-induced expression of LAT in T cells. (A) Jurkat T cells (2 x 105) were incubated in the absence (lane 1) or presence (lanes 2–6) of 3 µg/ml plate-immobilized anti-CD3 mAb ({alpha}CD3) with varying concentrations of FK506 (lanes 3–6) at 37°C for 16 h. Cells were then lysed and proteins were separated by 10% SDS–PAGE. Gels were electrotransferred to PVDF membranes and immunoblotted with anti-LAT or anti-Csk antibody as shown. (B) Similar experiments were conducted using Jurkat T cells with varying concentrations of rapamycin (Rapa; lanes 3–6). (C) The same reaction conditions were then repeated using 4 x 105 normal T cells collected from peripheral blood. These experiments were performed 3 times with similar results.

 


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Fig. 2. Time-course studies for rapamycin-induced inhibition of LAT expression. Jurkat T cells (2 x 105) were incubated in the absence (lane 1) or presence (lanes 2–5) of 3 µg/ml plate-immobilized anti-CD3 mAb ({alpha}CD3) in the presence of 10 nM FK506 (lane 3), 1 µM CsA (lane 4) or 10 nM rapamycin (Rapa; lane 5) at 37°C. After 2, 4 or 8 h, cells were lysed and proteins were separated by 10% SDS–PAGE. Gels were electrotransferred to PVDF membranes and immunoblotted with anti-LAT or anti-Csk antibody as shown. This experiment was performed 3 times with similar results.

 
A downstream result of TCR engagement is the activation of PKC, a serine/threonine kinase that has been shown to regulate protein expression in T cells (26,36,37). We have previously shown that T cell stimulation by PMA, a phorbol ester which directly binds and activates PKC, also up-regulates expression of LAT (31). To determine if rapamycin inhibited activation-induced LAT expression by blocking signals downstream of PKC, we examined the effect of rapamycin on T cells activated with PMA. As shown in Fig. 3(A), rapamycin inhibited PMA-induced expression of LAT in a dose-dependent manner, suggesting that rapamycin is exerting its inhibitory effect downstream of PKC activation. Similar results were observed in normal peripheral blood T cells (Fig. 3B). These treatments did not affect the number or viability of T cells, as determined by Trypan blue dye exclusion assay (data not shown). Together, these results strongly suggest that, although FK506 and rapamycin bind the same intracellular immunophilin receptor (FKBP), the distinct signals initiated by their interactions result in opposite regulatory effects on LAT expression.



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Fig. 3. Rapamycin inhibits PMA-induced expression of LAT. (A) Jurkat T cells (2 x 105) were incubated in the absence (lane 1) or presence (lanes 2–6) of 1 ng/ml PMA with varying concentrations of rapamycin (Rapa, lanes 3–6) at 37°C for 16 h. Western blot analysis was then performed as described above. This experiment was performed 3 times with similar results. (B) Normal T cells (4 x 105) collected from peripheral blood were incubated in the absence (lane 1) or presence (lanes 2–4) of 0.5 ng/ml PMA with varying concentrations of rapamycin (Rapa, lanes 3–4) at 37°C for 16 h. Western blot analysis was then performed as with anti-LAT and anti-actin antibody as shown. This experiment was performed 3 times with similar results.

 
Rapamycin and FK506 induce mutually antagonistic effects on activation-induced expression of LAT
Because FK506 and rapamycin share the same intracellular binding protein species (FKBP), it is conceivable that they may function as pharmacological antagonists vis-à-vis their effects on LAT expression. To explore this possibility, mixing experiments were performed by stimulating Jurkat T cells with plate-immobilized anti-CD3 mAb in the presence of suboptimal concentrations of FK506 (5 nM) and varying concentrations of rapamycin. As shown in Fig. 4(A), rapamycin inhibited the ability of FK506 to enhance activation-induced LAT expression in a dose-dependent manner. Conversely, Jurkat T cells were stimulated with plate-immobilized anti-CD3 mAb in the presence of suboptimal concentrations of rapamycin (5 nM) and varying concentrations of FK506. As shown in Fig. 4(B), FK506 overcame the ability of rapamycin to inhibit activation-induced LAT expression in a dose-dependent manner. These observations of drug antagonism suggest that FK506 and rapamycin induce their stimulatory and inhibitory effects on activation-induced LAT expression in part using a common pathway.



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Fig. 4. Rapamycin and FK506 induce mutually antagonistic effects on activation-induced expression of LAT. (A) Jurkat T cells (2 x 105) were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4–8) of 3 µg/ml plate-immobilized anti-CD3 mAb ({alpha}CD3) with 5 nM FK506 (lanes 3–8) and varying concentrations of rapamycin (Rapa, lanes 5–8) at 37°C for 16 h. Cells were then lysed and proteins were separated by 10% SDS–PAGE. Gels were transferred to PVDF membranes and immunoblotted with anti-LAT or anti-Csk antibody as shown. (B) Jurkat T cells (2 x 105) were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4–8) of 3 µg/ml plate-immobilized anti-CD3 mAb ({alpha}CD3) with 5 nM rapamycin (Rapa, lanes 3–8) and varying concentrations of FK506 (lanes 5–8) at 37°C for 16 h. Western blot analysis was then performed as described above. These experiments were performed 3 times with similar results.

 
Rapamycin and CsA induce mutually antagonistic effects on activation-induced expression of LAT
Unlike FK506, CsA binds the intracellular receptor protein cyclophilin and not FKBP. Therefore, CsA and rapamycin do not share the same intracellular binding target. To examine if the antagonistic effects observed between rapamycin and FK506 were due strictly to competition for FKBP association, mixing experiments were performed by stimulating Jurkat T cells with plate-immobilized anti-CD3 mAb in the presence of suboptimal concentrations of CsA (0.5 µM) and varying concentrations of rapamycin. As seen in Fig. 5(A), rapamycin inhibited the ability of CsA to enhance activation-induced LAT expression in a dose-dependent manner. When Jurkat T cells were stimulated with plate-immobilized anti-CD3 mAb in the presence of suboptimal concentrations of rapamycin (5 nM) and varying concentrations of CsA, the inhibitory effect of rapamycin was overcome by CsA in a dose-dependent fashion (Fig. 5B). This suggests that the antagonism between the CsA, FK506 and rapamycin in their influence on activation-induced LAT expression is not simply due to competition for intracellular immunophilin binding.



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Fig. 5. Rapamycin and CsA induce mutually antagonistic effects on activation-induced expression of LAT. (A) Jurkat T cells (2 x 105) were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4–8) of 3 µg/ml plate-immobilized anti-CD3 mAb ({alpha}CD3) with 0.5 µM CsA (lanes 3–8) and varying concentrations of rapamycin (Rapa, lanes 5–8) at 37°C for 16 h. Western blot analysis was then performed as described above. (B) Jurkat T cells (2 x 105) were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4–8) of 3 µg/ml plate-immobilized anti-CD3 mAb ({alpha}CD3) with 5 nM rapamycin (Rapa, lanes 3–8) and varying concentrations of CsA (lanes 5–8) at 37°C for 16 h. Western blot analysis was then performed as described above. These experiments were performed 3 times with similar results.

 
Rapamycin-induced inhibition of LAT protein expression is not associated with a decrease in LAT mRNA
To determine if the inhibitory effect of rapamycin on activation-induced LAT expression is associated with a decrease in the level of LAT mRNA, northern blot analysis was performed on mRNA collected from Jurkat T cells stimulated for 16 h with PMA in the presence of varying concentrations of rapamycin. As shown in Fig. 6(A), levels of LAT mRNA were increased in PMA-stimulated T cells, as we have reported previously (31). However, stimulation in the presence of the indicated doses of rapamycin did not decrease LAT mRNA levels. Importantly, these doses of rapamycin were sufficient to inhibit activation-induced up-regulation of LAT protein expression. This observation was supported using RT-PCR analysis of RNA prepared from Jurkat T cells stimulated for 16 h with PMA in the presence of varying concentrations of rapamycin. Again, rapamycin did not increase the level of LAT mRNA as measured by RT-PCR of LAT-specific sequences (Fig. 6B). Similarly, amplification of LAT cDNA using quantitative real-time PCR demonstrated an increase in LAT mRNA levels in response to PMA stimulation, but these levels were not affected by the addition of various concentrations of rapamycin (Fig. 6C and D). These data suggest the inhibitory effect of rapamycin does not involve down-regulation of LAT mRNA.




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Fig. 6. Rapamycin-induced inhibition of LAT protein expression is not associated with a decrease in LAT mRNA. (A) Jurkat T cells (2 x 107) were incubated in the absence (lane 1) or presence (lanes 2–4) of 1 ng/ml PMA with 0 (lanes 1 and 2), 5 (lane 3) or 50 nM of rapamycin (Rapa; lane 4) at 37°C for 16 h. An aliquot was used for cell lysis and western blot analysis as described above. Poly(A)+ RNA was isolated from the remainder of the cells and separated by 1% agarose gel electrophoresis. The gel was transferred to a positively charged nylon membrane overnight. RNA was then cross-linked onto the membrane at 80°C for 30 min. The membrane was probed with biotinylated antisense oligonucleotides specific for LAT and actin mRNA as shown. This experiment was performed 3 times with similar results. WB (western blotting), NB (northern blotting). (B) Jurkat T cells (2 x 107) were incubated in the absence (lane 1) or presence (lanes 2–4) of 1 ng/ml PMA with 0 (lanes 1 and 2), 5 (lane 3) or 50 nM of rapamycin (Rapa; lane 4) at 37°C for 16 h. Poly(A)+ RNA was isolated and reverse transcribed to cDNA. The resultant cDNA was amplified by PCR using oligonucleotide primers specific for LAT and GAPDH cDNA. PCR products were separated by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. Densitometric analysis of LAT PCR products normalized to GAPDH PCR products from three experiments are also represented histographically. Error bars denote SD. (C) Jurkat T cells (2 x 107) were incubated in the absence of PMA (sample 1) or in the presence of 1 ng/ml PMA with 0 (sample 2), 5 (sample 3) or 50 nM of rapamycin (sample 4) at 37°C for 16 h. Total RNA was isolated and one-step real-time RT-PCR was performed using oligonucleotide primers specific for LAT and actin cDNA. Representative amplification curves representing relative fluorescence intensities after the indicated cycle numbers for both LAT and actin are shown. This experiment was performed 3 times with similar results. (D) Based on the number of amplification cycles necessary to detect the LAT and actin PCR products, the calculated LAT mRNA levels normalized by actin mRNA levels are shown as ratios to unstimulated conditions from three independent experiments are represented histographically. Error bars denote SD.

 
Rapamycin does not induce post-translational degradation of LAT protein
Given that the level of LAT mRNA did not decrease by rapamycin treatment, we further sought to determine if the inhibitory effect of rapamycin was due to a post-translational instability of the LAT protein. Jurkat T cells were stimulated with PMA for 16 h in test tubes. After this incubation, the cells were washed in RPMI/5% FCS free of PMA. Actinomycin D was added to a final concentration of 3 µg/ml to block all new transcriptional activity along with, or without, rapamycin to a final concentration of 50 nM. Cells were then lysed at 4, 8 and 12 h after removal of PMA and inhibition of transcription. The lysates were evaluated by immunoblotting analysis. As seen in Fig. 7, at 4, 8 and 12 h after introduction of rapamycin, no decrease in LAT protein levels was seen, suggesting that protein stability was unaffected by the addition of rapamycin. This observation suggests that the inhibitory effect of rapamycin on activation-induced LAT expression does not involve protein breakdown.



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Fig. 7. Rapamycin does not induce post-translational degradation of LAT protein. Jurkat T cells (2 x 105) were incubated in the absence (lane 1) or presence (lanes 2–8) of 1 ng/ml PMA at 37°C for 16 h. Cells were then washed twice and actinomycin D was added to a final concentration of 3 µg/ml to all samples. Cells were then incubated at 37°C for the indicated time in the absence (lanes 1–3 and 5 and 7) or presence (lanes 4 and 6 and 8) of 50 nM rapamycin (Rapa). Western blot analysis was then performed as described above. This experiment was performed 3 times with similar results.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have recently reported that T cell activation using anti-CD3 mAb or the phorbol ester PMA increases levels of LAT mRNA and protein (31). We further found that treatment of T cells with CsA or FK506 further unregulated this activation-induced expression of LAT. In contrast, we now report that treatment with rapamycin, a different class of immunosuppressant widely used in clinical transplantation and in treating autoimmune diseases, results in an opposite, inhibitory effect on LAT expression. The potential relevance of these opposing regulatory effects centers around the critical significance of LAT in T cell activation and responsiveness.

When used together, rapamycin blocked the ability of FK506 to up-regulate LAT expression in activated T cells. Conversely, FK506 blocked the ability of rapamycin to down-regulate LAT expression in activated T cells. Because FK506 and rapamycin targets include a common intracellular immunophilin, it suggests that FK506 and rapamycin are antagonizing each other’s actions on LAT by competing for binding to immunophilin. However, the observation of the same opposing relationship between CsA and rapamycin suggests that this antagonism between these drugs is not simply due to competition for intracellular immunophilin binding. It has been shown that CsA and rapamycin respectively attenuate and augment T cell activation-induced increases in intracellular calcium concentration (38). Thus, it is conceivable that this functional difference may be related to their divergent effects on LAT expression. Calcium influx and mobilization induced by calcium ionophores can stimulate calcineurin function; indeed, we have previously demonstrated that calcium ionophore treatment can block activation-induced LAT expression, presumably by stimulating calcineurin (31). Thus, indirect activation of calcineurin mediated by increased calcium mobilization in rapamycin-treated cells could account at least in part for its ability to antagonize the effects of FK506 and CsA. Accordingly, the inhibition of calcineurin has been shown to induce the up-regulation of protein expression, including the up-regulation of granulocyte macrophage colony stimulating factor, IFN-{gamma}, transforming growth factor-ß, IL-5, IL-13, CD44 and CD69 expression (3942).

Another mechanism that may account for the inhibition of LAT expression by rapamycin could be due to the inhibition of protein translation. We observed that rapamycin treatment did not affect LAT mRNA nor did it induce post-translational degradation of LAT protein. Therefore, whereas the up-regulation in LAT expression after T cell activation appears to be transcriptionally regulated (31), the inhibitory influence of rapamycin appears to take place at the level of protein translation. This would be in agreement with previous observations that rapamycin is capable of repressing the translation of a subset of proteins (43). Similarly, inhibition of actin translation, but not transcription, has been shown to occur during early T cell activation in rapamycin-treated T cells (44).

Others have shown that CsA, FK506 and rapamycin exert opposing influences on important aspects of T cell physiology. For example, the addition of CsA to co-stimulatory molecule blockade strategies (4558) appears to abrogate their ability to promote allograft survival, while the use of rapamycin does not (53,54,5961). It has been speculated that activation-induced cell death (AICD) of potentially alloreactive T cells may be critical for co-stimulatory molecule blockade- mediated allograft tolerance; addition of CsA to co-stimulatory molecule blockade strategies results in a significant abrogation of T cell AICD, whereas the addition of rapamycin permits AICD (5961). Also, CsA blocks the ability of activated T cells to express the cell-surface co-stimulatory molecule CD154 by NF-{kappa}B-dependent mechanisms, whereas rapamycin does not (18). These immunosuppressive agents also appear to differentially regulate the induction of T cell anergy in vitro. CsA treatment blocks the ability to induce T cell anergy using co-stimulation-deficient activation or partial activation with altered peptide ligands, suggesting that the process of anergy induction is calcineurin dependent (6265). In contrast, primary activation of T cell clones treated with rapamycin, even in the presence of co-stimulation, results in anergic hyporesponsiveness to secondary stimulation (16). It has been speculated that the specific G1 to S phase cell-cycle arrest induced by rapamycin may be responsible for this facultative effect on anergy induction (16,61).

The critical role of LAT in T cell signal transduction is underscored by the observation that T cell lines that are deficient in LAT expression demonstrate an inability to become activated and produce IL-2 in response to adequate TCR ligation. This defect is rescued by transfection of these cells with wild-type, functional LAT (2022). Furthermore, LAT knockout mice demonstrate a complete absence of mature peripheral T cells, suggesting that LAT is necessary for both T cell activation and development (20). Any potential consequences of alterations in LAT expression induced by CsA, FK506 or rapamycin on T cell physiology remain to be elucidated. Interestingly, absence of LAT function has been correlated with anergic hyporesponsiveness. Anergic T cell clones demonstrate an inability to induce LAT phosphorylation in response to TCR engagement (66), resulting in an abbreviated signal-transduction process that mimics the aborted cascade of events observed in LAT-deficient cell lines. Similarly, TCR partial agonist ligands induce incomplete early signaling manifested by absent LAT phosphorylation (67), suggesting that the ability to induce LAT phosphorylation may be an effective indicator of optimal versus suboptimal T cell stimulation (68). Given the importance of LAT in T cell responsiveness, the subnormal LAT expression induced by rapamycin might have functional implications on T cell signaling. These issues are deserving of further investigation, and their exploration may yield important insight into both the effects of these immunosuppressive agents and T cell physiology.


    Acknowledgements
 
This work was supported in part by the National Kidney Foundation–American Society of Transplant Surgeons: Folkert O. Belzer Research Grant to C. S. C., an NIH grant (PO1 AI43900) to S. J. K. and a grant from the Roche Organ Transplantation Research Foundation (ROTRF 603630057) to M. M. H.


    Abbreviations
 
AICD—activation-induced cell death

CsA—cyclosporin A

FKBP—FK-binding protein

HRP—horseradish peroxidase

LAT—linker for activation of T cells

PKC—protein kinase C

PMA–phorbol myristate acetate


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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