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Originally published online as doi:10.1189/jlb.0904503 on February 16, 2005

Published online before print February 16, 2005
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(Journal of Leukocyte Biology. 2005;77:748-758.)
© 2005 by Society for Leukocyte Biology

Concentrations of cyclosporin A and FK506 that inhibit IL-2 induction in human T cells do not affect TGF-ß1 biosynthesis, whereas higher doses of cyclosporin A trigger apoptosis and release of preformed TGF-ß1

Jordi Minguillón*, Beatriz Morancho*, Seong-Jin Kim{dagger}, Miguel López-Botet* and José Aramburu*,1

* Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain; and
{dagger} Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

1 Correspondence: Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Carrer Dr Aiguader 80, 08003 Barcelona, Spain. E-mail: jaramburu{at}imim.es


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ABSTRACT
 
Cyclosporin A (CsA) and FK506 suppress T cell activation by inhibiting calcineurin and the calcineurin-dependent transcription factors nuclear factor of activated T cells (NFATc), which are central regulators of T cell function. It was reported that CsA up-regulated the transcription of transforming growth factor-ß1 (TGF-ß1) in lymphocytes and other cells and activated its promoter in A549 lung carcinoma cells, but the mechanisms involved are poorly understood, and it is unclear whether calcineurin plays any role. We have studied the regulation of TGF-ß1 in normal human lymphocytes and cell lines. In Jurkat T cells, the TGF-ß1 promoter was activated by calcineurin and NFATc and inhibited by CsA and FK506. However, the promoter was insensitive to both drugs in A549 cells. In human T cells preactivated with phytohemagglutinin, biosynthesis of TGF-ß1, induced by the T cell receptor (TCR) or the TGF-ß receptor, was not substantially affected by CsA and FK506 concentrations (≤1 µM) that effectively inhibited interleukin-2 production. However, pretreatment of fresh lymphocytes with CsA or FK506 during primary TCR stimulation reduced their production of TGF-ß1 during secondary TCR activation. Finally, high concentrations of CsA (10 µM), in the range attained in vivo in experiments in rodents, caused apoptosis in human T cells and the release of preformed, bioactive TGF-ß1. These effects are unlikely to owe to calcineurin inhibition, as they were not observed with FK506. Our results indicate that CsA and FK506 are not general inducers of TGF-ß1 biosynthesis but can cause different effects on TGF-ß1 depending on the cell type and concentrations used.

Key Words: calcineurin • lymphocytes • immunosuppressants


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INTRODUCTION
 
The calcium-activated Ser/Thr phosphatase calcineurin regulates gene expression in lymphocytes and other cell types by activating transcription factors such as calcineurin-dependent transcription factors nuclear factor of activated T cells (NFATc) [1 ]. The immunosuppressants cyclosporin A (CsA) and FK506 associate with endogenous immunophilins [2 ], and the resulting complexes inhibit calcineurin and prevent T cell receptor (TCR)-induced activation of lymphocytes [3 ]. CsA and FK506 are used to treat autoimmune disorders and transplant rejection. However, prolonged treatment with these compounds, particularly CsA, causes severe side effects such as nephrotoxicity and hypertension. As CsA and FK506 also inhibit the immunophilins to which they bind, it is unclear whether these side effects owe to sustained calcineurin inhibition or are mediated by calcineurin-independent mechanisms [4 ]. CsA-induced nephrotoxicity has been linked to the accumulation of transforming growth factor-ß1 (TGF-ß1) in kidney [5 , 6 ]. CsA has been shown to induce TGF-ß1 production in kidney cell lines [7 ] and to activate its promoter in A549 lung carcinoma cells [8 ]. In T cells, CsA was shown to enhance TGF-ß1 mRNA accumulation and protein production in cells stimulated with anti-CD3 antibodies or pharmacological activators of protein kinase C (PKC) and calcium mobilization [9 ]. These observations suggested that CsA might act as a general inducer of TGF-ß1 in different cell types, raising the question of whether calcineurin, the main target of CsA, is involved in TGF-ß1 expression.

TGF-ß1 is a key immunoregulatory cytokine that plays a pivotal role in immune homeostasis and prevents spontaneous autoimmunity [10 ]. TGF-ß1 is also produced by infiltrating leukocytes at sites of inflammation, as shown during allograft rejection [11 ] and experimental autoimmune encephalomyelitis [12 ]. TGF-ß1 counteracts the deleterious immune response and has been associated with remission in rodent models of multiple sclerosis and parasite-induced colitis [12 , 13 ]. Virtually all T cells, naïve or T helper (Th)0, Th1, Th2, and Th3 clones, are competent to express TGF-ß1 [14 ]. However, specialized T cell subsets, such as CD4+/CD25+, T regulatory, and Th3 cells, produce mainly TGF-ß1 and IL-10 rather than prototypical Th1/Th2 cytokines [15 16 17 ]. TGF-ß1 is synthesized and secreted as a latent precursor, which is held in an inactive conformation by its own amino-terminal propeptide, the latency-associated protein (LAP). The mature, bioactive carboxy-terminal TGF-ß1 dimer is released by conformational modifications of LAP through proteolysis or other mechanisms [18 ], occurring, for instance, during apoptosis, where bioactive TGF-ß1 is released by apoptotic cells from preformed intracellular stores [19 ].

Here, we have studied the regulation of TGF-ß1 biosynthesis by CsA and FK506 in response to TCR stimulation and autocrine TGF-ß1 induction. We show that concentrations of immunosuppressants (100 nM–1 µM), which are effective in suppressing TCR-induced production of interleukin (IL)-2, do not have a substantial effect on TGF-ß1 biosynthesis in activated, normal lymphocytes. However, pretreatment of fresh T cells with CsA and FK506 during primary TCR stimulation decreased their production of TGF-ß1 during secondary stimulation. Finally, higher concentrations of CsA (10 µM), but not of FK506, promote the release of preformed TGF-ß1, independent of de novo synthesis, by inducing apoptosis. Our results show that the activation requirements for the induction of TGF-ß1 and its sensitivity to CsA may vary depending on the cell type and CsA concentrations and highlight a greater heterogeneity in the response of TGF-ß1 to CsA than previously realized in earlier studies.


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MATERIALS AND METHODS
 
Reagents
CsA, FK506, phorbol 12-myristate 13-acetate (PMA), and the calcium ionophore ionomycin were from Calbiochem (Darmstadt, Germany). The caspase inhibitor Z-Val-Ala Asp-(Ome; z-VAD)-fluoromethylketone was from Enzyme Systems Products (Livermore, CA).

Cell culture and cytokine enzyme-linked immunosorbent assay (ELISA)
Culture medium was high-glucose Dulbecco’s modified Eagle’s medium (DMEM; 41965-039, Gibco, Pasley, UK), supplemented with L-Glut (2 mM), 2-mercaptoethanol (50 µM), and sodium pyruvate (1 mM; all supplements from Gibco). The human T cell line Jurkat [Clone E6-1, American Type Culture Collection (ATCC), Manassas, VA, #TIB 152], kindly provided by Jeremy Luban (Columbia University College of Physicians and Surgeons, New York, NY), and the human lung carcinoma cell line A549 (ATCC #CCL-185) were grown in the above culture medium plus 10% heat-inactivated fetal calf serum (FCS; Linus, Cultek, Spain). Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy volunteers by gradient sedimentation with LymphoprepTM (Axis-Shield, Oslo, Norway). PBMC (2x106 cells/ml) were stimulated for 3 days with 2.5 µg/ml phytohemagglutinin-L (PHA; Sigma-Aldrich, Steinheim, Germany) and 5.5 ng/ml IL-2 (Eurocetus, Amsterdam, the Netherlands) in culture medium plus 10% heat-inactivated FCS. After 3 days, cultures were washed, and cells were adjusted at 2 x 106 cells/ml in medium plus 10% FCS and 5.5 ng/ml IL-2. Cultures were used 24 or 48 h later and contained ≥97% T cells by flow cytometry with anti-CD3 (SpvT3b) [20 ]. Cells (2x106 cells/ml, 1 ml) were stimulated in 48-well plates with mouse anti-human CD3 (SpvT3b, 2 µg/well) bound to wells coated with rabbit anti-mouse immunoglobulin G (10 µg/well, Sigma-Aldrich), plus mouse anti-human CD28 (CD28.2 NA/LE, BD Biosciences, San Diego, CA) added in solution (1 µg/ml). Cells (2x106 cells/ml) were stimulated without IL-2 in medium with 2% FCS to reduce the background of bovine TGF-ß1 from serum, as the ELISA used (TGF-ß1 Emax Immunoassay, Promega, Madison, WI) also detects bovine TGF-ß1. Hepes buffer (50 mM, pH 7.4) was added to the medium to maintain a constant pH and prevent spurious activation of TGF-ß1 by acidification of supernatants during culture. Bioactive TGF-ß1 in supernatants was measured directly, and total TGF-ß1 (latent plus bioactive) was quantitated after acid-activation following the manufacturer’s instructions. Intracellular TGF-ß1 was measured in postnuclear supernatants of cells washed twice with phosphate-buffered saline (PBS) and lysed (3x106 cells/ml) in PBS with 1% Nonidet P-40 (NP-40) and protease inhibitors (leupeptin, antipain, chemostatin, and pepstatin, all at 2 µg/ml). NP-40 (1%) did not affect the measurement of control samples and TGF-ß1 standards. IL-2 ELISA (OptEIA human IL-2 set) was from BD Biosciences. The neutralizing anti-human TGF-ß receptor II antibody (AF-241-NA) was from R&D Systems (Minneapolis, MN).

Northern blot
Total RNA isolated using a guanidium isothiocyanate-based method (Trizol, Invitrogen, Pasley, UK) was resolved in 1% agarose formaldehyde gels, transferred to Nytran membranes (Schleicher and Schuell, Dassel, Germany), UV-cross-linked, and hybridized with a TGF-ß1 probe comprising from +348 to +653 in the coding sequence of human TGF-ß1 cDNA. The TGF-ß1 cDNA (IMAGE Clone 3510592, GenBank Accession BE312000) was from the I.M.A.G.E. consortium (UK HGMP Resource Center, Cambridge). Probe labeling, hybridization, and chemiluminescence were performed with a nonisotopic method (North2South® direct labeling and detection kit, Pierce, Rockford, IL). The signal-to-background ratio in gels and films was measured using the Bio-Rad Gel Doc 2000 imaging system and the Quantity One 4.3.1 Bio-Rad (Hercules, CA) software.

Reporter assays
The human TGF-ß1 promoter construct TG5-Luc [21 ] was made by subcloning a HincII-KpnI fragment (–453 to +11) from phTG2-CAT [21 ] into pXP2 [22 ]. The entire promoter region was sequenced and confirmed to be free of mutations. The NFATc-responsive reporter pIL-13/Luc [23 , 24 ] and expression vectors encoding the calcineurin B subunit [25 ], the NFATc-inhibitory peptide VIVIT [26 ], and the NFAT5 inhibitory dimerization domain (DD5) [27 ]—the latter two as fusions with the enhanced green fluorescent protein (EGFP)—were kindly provided by Anjana Rao (Center for Blood Research, Boston, MA) and Cristina López-Rodríguez (Center for Genomic Regulation, Barcelona, Spain). Wild-type calcineurin and the CsA-insensitive mutant V314R constructs in pBJ5 [28 ] were kindly provided by Joseph Heitman (Duke University Medical Center and Howard Hughes Medical Institute, Durham, NC). TK-Renilla (Promega) was used for normalization. The plasmid pEGFP.N1 was from BD Biosciences. Jurkat T cells (20x106 cells/400 µl in DMEM) were mixed with plasmid DNA (luciferase reporters, 1 µg/106 cells; TK-Renilla, 0.2 µg/106 cells; and expression vectors, 0.5–1 µg/106 cells) and electroporated in a Bio-Rad gene pulser (260 V, 950 µF). A549 cells (10x106 cells/400 µl in DMEM) were electroporated (260 V, 950 µF) with luciferase reporters (2 µg/106 cells) and TK-Renilla (0.5 µg/106 cells). Electroporated cells were washed in medium to remove dead cells and allowed to recover for 24 h before stimulation. Luciferase activity was measured with the dual luciferase reporter system (Promega) in a Berthold FB12 luminometer (Pforzheim, Germany).

Apoptosis and flow cytometry
Human lymphocytes (PHA blasts) or Jurkat cells (2x106 cells/ml) were treated with CsA or FK506, as indicated in the respective figure legends. Cells were stained with annexin-V-Fluos, 1/1000 dilution (Roche, Indianapolis, IN) in annexin-V buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM KCl, 1.36 mM CaCl2, 1 mM MgCl2) at 4°C for 20 min and washed to remove unbound annexin-V. Propidium iodide (PI; 2.5 µg/ml) was added 10 min before analysis by two-color flow cytometry (FACScan, BD Biosciences).

Statistical analysis
Mean and SD were calculated from at least three independently performed experiments unless otherwise indicated in figure legends. Statistical significance (P value) was determined by the ANOVA test (post-hoc analysis using the Tukey algorithm).


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RESULTS
 
Regulation of the TGF-ß1 promoter in Jurkat T cells and A549 lung carcinoma cells in response to calcineurin activation and the calcineurin inhibitors CsA and FK506
In view of an earlier work describing the activation of the TGF-ß1 promoter by CsA in the human lung carcinoma cell line A549 [8 ], we asked whether a similar response occurred in Jurkat T cells and compared the regulation of the TGF-ß1 promoter in both cell lines. In Jurkat cells, the TGF-ß1 promoter was activated by the combination of the phorbol ester PMA and the calcium ionophore ionomycin but not by each individual stimulus (Fig. 1A ). CsA and FK506 inhibited the stimulation by PMA plus ionomycin. These activation requirements were similar to that of the calcineurin and NFATc-dependent IL-13 promoter [23 ], suggesting that activation of the TGF-ß1 promoter in Jurkat T cells involved calcineurin. This was confirmed by transfecting cells with the CsA-insensitive calcineurin mutant V314R [28 ]. Activation of the TGF-ß1 promoter was specifically resistant to CsA in cells expressing the V314R mutant but not in cells transfected with wild-type calcineurin (Fig. 1B , left panel). FK506 inhibited the reporter in all cases. Activation of the promoter was repressed by 57% by the NFATc-inhibitory peptide VIVIT [26 ] but not by the NFAT5-specific inhibitor DD5 [27 ] (Fig. 1B , right panel). These results indicated that the TGF-ß1 promoter could be activated by calcineurin-dependent NFATc transcription factors in Jurkat T cells. However, in A549 cells, PMA alone activated the TGF-ß1 promoter without requiring costimulation with ionomycin (Fig. 1C , left panel). CsA and FK506 (both at 100 nM) did not repress the promoter but did not activate it either, in contrast with a previous report in which the TGF-ß1 promoter was activated by CsA in A549 cells [8 ]. We confirmed that A549 cells responded to CsA and FK506 by testing that the NFATc-dependent IL-13 promoter was activated by the combination of PMA and ionomycin and inhibited by CsA and FK506 (Fig. 1C , right panel). Differences between our results in A549 cells and those reported by Prashar et al. [8 ] 10 years ago might be attributed to the possibility that despite very similar culture conditions, this tumor cell line may display functional variations over the time in different laboratories. Nonetheless, our results indicated that calcineurin-dependent and independent pathways in different cell types can activate the TGF-ß1 promoter and that CsA and FK506 might not be general activators of TGF-ß1 transcription.



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  		Figure 1. Regulation of the TGF-ß1 promoter in Jurkat and A549 cells. (A) Jurkat T cells transfected with the human TGF-ß1 promoter (TG5-Luc reporter plasmid) and the human IL-13 promoter (IL13-Luc) were stimulated with PMA (20 nM), ionomycin (1 µM), CsA (100 nM), FK506 (100 nM), and their combinations during 24 h. Mean ± SD of three independent experiments for each promoter is shown. Activity is represented as relative light units (RLU) per minute after normalization with a cotransfected TK-Renilla control reporter. Unst, Unstimulated. (B) Jurkat T cells, transfected with the TGF-ß1 promoter and vectors encoding the CsA-insensitive calcineurin mutant (CnA) V314R, wild-type calcineurin (CnA WT), empty vector pBJ5 (left panel), or with vectors encoding GFP, the NFATc-inhibitory peptide VIVIT (VIVIT GFP), or the NFAT5-inhibitory dimerization domain (DD5 GFP; right panel), were stimulated as in A. Activity of the reporter is shown as fold-induction over the unstimulated control. Mean ± SD of three independent experiments is shown. (C) A549 cells were transfected with the TGF-ß1 promoter (left panel) or IL-13 promoter (right panel) and stimulated during 24 h as in A. Mean ± SD of four independent experiments for each promoter is shown.

Production of TGF-ß1 by normal human T cells in response to TCR stimulation and the immunosuppressants CsA and FK506
In view of the differences in the regulation of the TGF-ß1 promoter in Jurkat and A549 cells, we analyzed the regulation of TGF-ß1 in normal human T cells (PHA blasts) stimulated with anti-CD3 alone or with anti-CD3 antibody plus anti-CD28 antibody (hereafter referred to as anti-CD3/CD28) in the absence or presence of CsA or FK506. As shown in Figure 2 (upper panels), 24 h stimulation with anti-CD3/CD28 induced the production of TGF-ß1, mostly in its latent form. Stimulation with anti-CD3 alone induced almost maximal TGF-ß1 production, in contrast with IL-2, which required costimulation with anti-CD28 (Fig. 2 , lower panel). CsA or FK506, at 100 nM, did not substantially affect the production of TGF-ß1 by CD3 and CD3/CD28 but effectively suppressed IL-2 production in the same cells (Fig. 3A , lower panel).



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  		Figure 2. Production of TGF-ß1 by normal human T lymphocytes in response to TCR stimulation and immunosuppressants. (Upper panels) Total and bioactive TGF-ß1 were measured by ELISA in supernatants from human PHA blasts left unstimulated (Unst.) or activated during 24 h with anti-CD3 or anti-CD3/CD28 in the absence or presence of CsA and FK506 (both at 100 nM). Basal bovine TGF-ß1 level in 2% FCS culture medium, without cells, is shown. (Lower panel) IL-2, in the same supernatants, was measured by ELISA. Absolute values for IL-2 showed greater variation among individual experiments than TGF-ß1 values, and therefore, IL-2 production is shown as percentage of induction relative to the anti-CD3/CD28 sample (100%). Mean ± SD of three independent cultures is shown. (*) Induction of TGF-ß1 by anti-CD3/CD28 was statistically significant with respect to the unstimulated control (P<0.01, n=3). Variations between CD3 alone and CD3/CD28 were not statistically significant. CsA and FK506 did not modify (not statistically significant) the production of TGF-ß1 by anti-CD3 alone or anti-CD3/CD28, respectively.



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  		Figure 3. Autocrine production of TGF-ß1 by human T lymphocytes in response to TCR stimulation and immunosuppressants. Total TGF-ß1 (upper panel) and IL-2 (lower panel) production in PHA blasts were measured by ELISA in samples, which were left unstimulated or activated with anti-CD3/CD28 during 24 h in the absence (–) or presence of a neutralizing anti-TGF-ß receptor antibody ({alpha}TBRII; 10 µg/ml) and with or without CsA or FK506 (both at 100 nM). For the TGF-ß1 ELISA, basal TGF-ß1 levels in the culture medium containing 2% FCS and without cells are shown. IL-2 production is shown as percentage of induction relative to the anti-CD3/CD28 sample (100%). The inset shows that blockade of the TGF-ß receptor induces a modest production of IL-2 in unstimulated cells, which are not inhibited by CsA and FK506. Mean ± SD of three independent cultures are shown. (*) Inhibition of TGF-ß1 production by the anti-TGF-ß receptor antibody in unstimulated cells was statistically significant with respect to samples without antibody (P<0.01, n=3). In the same samples, CsA and FK506 had no significant effect. (**) Inhibition of TGF-ß1 production by the anti-TGF-ß receptor antibody in cells stimulated with anti-CD3/CD28 was statistically significant (P<0.01, n=3) in cells without immunosuppressants and in cells treated with CsA but not in cells treated with FK506.

In addition to TCR activation, TGF-ß1 can be induced by autocrine stimulation of its own receptor. We assessed the contribution of this pathway by incubating T lymphocytes with a neutralizing anti-TGF-ß receptor II antibody. As shown in Figure 3 , blockade of the TGF-ß receptor suppressed the basal TGF-ß1 production and partially inhibited the induction by anti-CD3/CD28 (30.5±12.7% inhibition), indicating that basal TGF-ß1 production in T cells was mainly autocrine, whereas only a small proportion of TGF-ß1 induced by TCR stimulation depended on the autocrine pathway. Neither CsA nor FK506 altered the basal autocrine production of TGF-ß1. However, FK506, but not CsA, attenuated the inhibition by anti-TGF-ß receptor antibody in CD3/CD28-stimulated cells (from 30.5±12.7% to 7.8±4%), indicating that although these drugs did not affect autocrine TGF-ß1 synthesis, FK506 could modestly enhance TGF-ß1 production induced by CD3/CD28 if the autocrine loop was blocked. Blockade of the TGF-ß receptor caused a small induction of IL-2 in unstimulated cells, which was insensitive to CsA and FK506, and it boosted the levels of IL-2 induced by CD3/CD28 costimulation. Neutralization of the TGF-ß receptor rendered IL-2 production by CD3/CD28 partially resistant to CsA and FK506. This result indicated that the low levels of bioactive TGF-ß1 produced by T cells are biologically relevant to sustain its own autocrine stimulation, down-regulate IL-2 production, and enhance the immunosuppressive effect of CsA and FK506.

Although in Jurkat T cells, the TGF-ß1 promoter was activated by calcineurin, the relative insensitivity of TGF-ß1 to CsA and FK506 in nontransformed lymphocytes suggested that calcineurin may not be a major regulator of this cytokine in normal T cells. The TCR activates PKC and calcineurin pathways, which can be activated with the PKC activator PMA and ionomycin, respectively. We analyzed the production of TGF-ß1 by lymphocytes activated with PMA (10 nM), ionomycin (0.3 µM), or their combination in the absence or presence of CsA (1 µM) and measured the amount of TGF-ß1 accumulated in the supernatants as well as the intracellular content of the cytokine in 1% NP-40 lysates of the same cells (Fig. 4A and 4B ). As shown in Figure 4A , PMA effectively induced the production of TGF-ß1, whereas ionomycin alone did not and even inhibited by 30% the induction by PMA. However, CsA did not cause significant variations in the production of TGF-ß1 by cells treated with PMA, ionomycin, or both, indicating that CsA did not affect pathways activated by those stimuli. As shown in Figure 4B , the levels of intracellular TGF-ß1 were not affected by PMA, ionomycin, or CsA, added individually or in combination. These results indicated that PMA enhanced the overall synthesis of the cytokine. Consistent with this, PMA increased the levels of TGF-ß1 mRNA (5.6-fold) after 5 h stimulation (Fig. 4C) . In the same experiment, secreted TGF-ß1 was detected later (24 h) in the supernatants. TGF-ß1 mRNA and protein levels in CsA- and FK506-treated cells displayed little variation with respect to untreated controls. We confirmed that 100 nM FK506 substantially prevented the activation of calcineurin, as shown by the inhibition of the calcineurin-dependent dephosphorylation of NFATc2/NFAT1 in PHA blasts (Supplementary Fig. 1).



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  		Figure 4. Stimulation of TGF-ß1 production by the phorbol ester PMA in normal human T lymphocytes. (A) Total TGF-ß1 production by PHA blasts stimulated during 24 h with 10 nM PMA (P), 0.3 µM ionomycin (I), or their combination, without or with 1 µM CsA. Mean ± SD of four independent cultures is shown. (*) Reduction of PMA-induced TGF-ß1 by ionomycin was statistically significant (P<0.01, n=4). CsA did not have a statistically significant effect on the production of TGF-ß1 by cells treated with PMA, ionomycin, or both. (B) Intracellular TGF-ß1 was measured in the same cells by lysing them in PBS plus 1% NP-40. (A and B) TGF-ß1 levels are normalized to pg/106 cells. (C) PHA blasts were stimulated during 5 and 24 h with 10 nM PMA or treated with the indicated concentrations of CsA and FK506. Total TGF-ß1 was measured in cell-free supernatants, and TGF-ß1 mRNA was detected by Northern blot using total RNA from the same samples. Error bars in the ELISA show the SD of duplicate measurements for each individual sample.

Our previous experiments were done in preactivated lymphocytes. We asked whether TGF-ß1 was regulated in a similar manner in fresh T cells during primary and secondary stimulation. Fresh PMBC were stimulated with or without CsA or FK506 as shown in the diagram in Figure 5A . Primary stimulation with anti-CD3/CD28 induced a progressive production of TGF-ß1 that was not inhibited by CsA (500 nM) or FK506 (100 nM; Fig. 5B , upper panels). Secondary stimulation with anti-CD3/CD28 elicited a stronger TGF-ß1 production than that of primary stimulation, which was also insensitive to CsA but slightly enhanced (21%) by FK506. However, induction of TGF-ß1 during secondary stimulation was decreased by 28% when cells had been continuously treated with CsA or FK506 during 5 days. In contrast with TGF-ß1, both drugs remarkably inhibited IL-2 (Fig. 2B , lower panels). IL-2 production during primary stimulation with anti-CD3/CD28 was maximal at 24 h, declining sharply at 70 h, and was inhibited by 67%, although not completely blunted by CsA and FK506. This observation agrees with earlier papers, showing that CD28 costimulation confers IL-2 production by fresh T cells a relative resistance to CsA [29 ]. With regard to secondary TCR stimulation, production of IL-2 was of greater magnitude than in the primary stimulation but was almost completely suppressed (98%) by CsA or FK506. Cells that had been pretreated with the immunosuppressants during primary stimulation produced IL-2 poorly (6±16% of nonpretreated cultures) during secondary stimulation, even when the drugs were removed, and were further inhibited by freshly added CsA or FK506. These results support the conclusion that sustained treatment of T cells with CsA or FK506 does not enhance TGF-ß1 but can moderately decrease its production during secondary TCR stimulation. Nonetheless, CsA and FK506-treated T cells still produce substantial levels of TGF-ß1.



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  		Figure 5. Production of TGF-ß1 by human T lymphocytes in response to primary and secondary TCR stimulation. (A) Diagram of the experiment. PBMC isolated from buffy coats were separated in three groups and stimulated at 3 x 106 cells/ml with anti-CD3/CD28 during 70 h in the absence (No inhibitor) or presence of 100 nM CsA or 100 nM FK506. After primary stimulation, cells were washed, adjusted to 2 x 106 cells/ml, and cultured in medium with IL-2 (5.5 ng/ml) but without anti-CD3/CD28 and cultured for another 48 h. Cells that had been pretreated with CsA or FK506 during primary stimulation were maintained with their respective immunosuppressant freshly added. Forty-eight hours later, cells from the three groups were washed, adjusted to 2 x 106 cells/ml, and restimulated during 24 h with anti-CD3/CD28 in the absence or presence of CsA or FK506 as indicated. Cells were maintained in medium with 2% FCS throughout the entire experiment. (B) Samples were collected at 24 and 70 h during primary stimulation and at 24 h during secondary stimulation. TGF-ß1 (total) and IL-2 were quantified by ELISA. Mean ± SD of three independent cultures is shown. Induction of TGF-ß1 by anti-CD3/CD8 during primary stimulation was statistically significant (*, P<0.01, n=3), whereas neither CsA nor FK506 had a significant effect at 24 or 70 h. In the secondary stimulation, FK506 enhanced the production of TGF-ß1 by anti-CD3/CD28 (*, P<0.05, n=3). Cells that had been pretreated with CsA or FK506 during the primary stimulation produced less TGF-ß1 than the untreated group during the secondary stimulation (**, P<0.01, n=3).

Altogether, our experiments indicated that CsA and FK506 concentrations, which effectively inhibited IL-2 production by fresh and preactivated T lymphocytes, did not substantially modify their ability to produce TGF-ß1 in response to CD3, CD3 plus CD28 costimulation, or an autocrine pathway. Indeed, although CsA and FK506 do not induce the synthesis of TGF-ß1 in T cells, they sharply skew the TGF-ß1/IL-2 ratio toward TGF-ß1, simply by inhibiting IL-2 production.

Induction of apoptosis and release of bioactive TGF-ß1 by CsA and not FK506 in human T cells
Our experiments showed so far that the production of TGF-ß1 was largely insensitive to immunosuppressive doses of CsA and FK506, and we wondered whether these compounds would perform differently at higher concentrations. Whereas lower concentrations of CsA and FK506 (100 nM) did not induce the production of total or bioactive TGF-ß1 by T cells (Fig. 2) , 10 µM CsA caused the accumulation of bioactive TGF-ß1 in the supernatant after 24 h, as shown in Figure 6A . A lower CsA concentration (1 µM) had no effect and neither did FK506 at 1 and 10 µM. Visual examination of the cultures suggested that CsA was toxic to cells. Flow cytometry analysis using annexin-V and PI staining confirmed that CsA caused substantial cell death at 24 h (Fig. 6B) . CsA toxicity was not prevented by a neutralizing anti-TGF-ß receptor antibody, indicating that TGF-ß1 release was the result, rather than the cause, of cell death (Fig. 6C) . Similar results were obtained with Jurkat cells (Fig. 6D) . CsA-induced apoptosis was detected at early time-points, as shown by the presence of annexin-V+/PI– cells between 3 and 6 h of CsA treatment (Fig. 7A ). Cells died progressively (9 h onwards), as revealed by positive staining with PI. Cells treated with 10 µM FK506 did not undergo apoptosis in the same period. The results above indicated that CsA might cause the release of TGF-ß1 by inducing apoptosis. Consistent with this hypothesis, the general caspase inhibitor z-VAD prevented apoptosis and the release of TGF-ß1 in Jurkat cells exposed to 10 µM CsA during 7 h (Fig. 5B , upper and lower panels). Finally, inhibitors of transcription (actinomycin D) and protein synthesis (cycloheximide) did not affect the release of TGF-ß1 by apoptotic Jurkat T cells, indicating that CsA induced the release of TGF-ß1 from preformed stores and not by de novo synthesis (Fig. 7C) .



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  		Figure 6. CsA-induced apoptosis and release of bioactive TGF-ß1 in normal T lymphocytes and Jurkat T cells. (A) PHA blasts were left untreated or incubated with CsA (1 µM and 10 µM) or FK506 (1 µM and 10 µM) during 24 h. Bioactive TGF-ß1 was measured in cell-free supernatants. Values represent the mean ± SD of four independent donors (*, P<0.01, n=4). (B) Cell viability and apoptosis were determined by flow cytometry, assessing forward scatter (FSC) and side scatter (SSC) and PI and annexin-V staining, which correspond to the entire, ungated population, as shown in the FSC/SSC plots. One representative experiment is shown. (C) PHA blasts were incubated with neutralizing anti-TGF-ß1 receptor antibody (10 µg/ml) before addition of CsA (10 µM). Twenty-four hours later, cells were analyzed by flow cytometry. (D, upper panel) Jurkat T cells were incubated with 0.2% ethanol (EtOH; CsA solvent), 10 µM CsA, 0.1% dimethyl sulfoxide (DMSO; FK506 solvent), or 10 µM FK506 during 24 h. Bioactive TGF-ß1 in cell-free supernatants and cellular viability and apoptosis were determined as in A. Mean ± SD corresponds to duplicate wells measured for each sample. (Lower panel) Viability and apoptosis were analyzed in parallel in the same cells.



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  		Figure 7. Caspase-dependent, CsA-induced apoptosis, and TGF-ß1 release in Jurkat cells. (A) Jurkat cells left untreated ({blacksquare}) or incubated with 10 µM FK506 ({Delta}) or with 10 µM CsA ({lozenge}) were analyzed at different time-points by annexin-V-Fluos and PI staining and two-color flow cytometry. Percentages of viable cells, apoptotic cells, and dead cells are the mean ± SD of three independently performed experiments. (B) Jurkat cells were left untreated or incubated with 10 µM CsA in the absence (No inhib.) or presence of the caspase inhibitor z-VAD (50 µM) during 7 h. (Upper panel) Cell death was determined by PI and annexin-V-Fluos staining. (Lower panel) Bioactive TGF-ß1 was measured by ELISA in cell-free supernatants from the same samples. Values are the mean ± SD of duplicate wells measured for each sample. EtOH, Ethanol; Untr., Untreated. (C) Jurkat cells were left untreated or incubated with 0.2% ethanol (CsA solvent) or CsA (10 µM) for 7 h in FCS-free medium in the absence or presence of 5 µM actinomycin-D (Act-D), 20 µg/ml cycloheximide (CHX), or 50 µM z-VAD. Bioactive TGF-ß1 was measured by ELISA in cell-free supernatants. Mean ± SD is from three independently performed experiments. Actinomycin-D and cycloheximide did not cause statistically significant variations in the release of TGF-ß1 induced by 10 µM CsA.


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DISCUSSION
 
Earlier reports described that CsA activated the TGF-ß1 promoter in A549 cells and increased its mRNA levels in lymphocytes, cell lines, and kidney in mice [7 , 8 ]. These observations lent support to the notion that CsA might contribute to nephrotoxicity by inducing TGF-ß1 biosynthesis. Inhibition of calcineurin is a major mechanism underlying the immunosuppressive effect of CsA. Calcineurin is also inhibited by FK506, for which conflicting results had been reported regarding its ability to induce TGF-ß1 [30 , 31 ]. In this context, it was unclear whether TGF-ß1 can be regulated by calcineurin.

We have analyzed the production of TGF-ß1 in response to calcineurin activation and the immunosuppressants CsA and FK506 in normal human T lymphocytes and cell lines, assessing different parameters relevant to TGF-ß1 regulation. Our work has yielded three main results: The TGF-ß1 promoter can be activated by calcineurin and NFATc and inhibited by CsA and FK506 in Jurkat T cells but was insensitive to the immunosuppressants in A549 cells; the independent induction of TGF-ß1 by TCR stimulation or an autocrine pathway in normal T cells is largely unaffected, neither induced nor repressed, by CsA and FK506 at doses (≤1 µM), which effectively suppress the induction of IL-2, and this result supports the conclusion that the calcineurin inhibitors CsA and FK506 might not be general inducers of TGF-ß1 biosynthesis; and high concentrations of CsA (10 µM) cause the release of bioactive TGF-ß1 from preformed intracellular stores by inducing apoptosis in T cells. FK506 at 10 µM induced neither, indicating that down-regulation of calcineurin-dependent functions might not be sufficient to cause some of the CsA toxic effects. Our results provide an alternative angle to the long-held view that CsA is a general inducer of TGF-ß1 and suggest that the response of TGF-ß1 to CsA and FK506 in different cell types might be more heterogeneous than realized previously.

The regulation of the TGF-ß1 promoter in Jurkat cells hints at the possibility that calcineurin might activate TGF-ß1 in some cells. This is supported by a recent article describing that induction of diabetes with streptozotocin (STZ) in rats up-regulated calcineurin, activated NFATc1/2, and increased the expression of TGF-ß1 in glomeruli, tubule, and cortex [32 ]. Induction of TGF-ß1 mRNA by STZ in mesangial cells was inhibited by CsA, whereas in cortex, it was up-regulated by CsA. In view of these observations and our results, it seems plausible that calcineurin and NFATc signaling could have different effects on TGF-ß1 expression depending on the cell type. In this regard, other cytokines such as lymphotoxins (LT) require calcineurin activation in some cell types but not in others: LT{alpha} is inhibited by CsA in T cells but not in B cells [33 ], whereas LTß is CsA-sensitive in the T cell lines MOLT4 and Jurkat but not in normal T lymphocytes [26 , 33 ].

Production of TGF-ß1 induced via TCR or the TGF-ß receptor in preactivated T cells (PHA blasts) was largely insensitive to CsA and FK506 concentrations (100 nM), which suppressed TCR-induced production of IL-2 by more than 98%. Unlike IL-2, induction of TGF-ß1 did not require CD28 costimulation, and TGF-ß1 induced by anti-CD3 alone was again insensitive to CsA and FK506. However, fresh T cells that had been pretreated with CsA or FK506 during 5 days produced almost 30% less TGF-ß1 during secondary stimulation with anti-CD3/CD28. Still, this inhibition was not enhanced by the addition of fresh CsA or FK506 and was in sharp contrast with the much higher sensitivity of IL-2 to these immunosuppressants. Conversely, FK506, but not CsA, enhanced the CD3/CD28-induced production of TGF-ß1 by T cells in two types of experiments: when TGF-ß receptors were blocked (Fig. 3) and during secondary TCR stimulation of PBMC (Fig. 5) . However, these increases were of low magnitude, were not observed with CsA, and did not correlate with the relative inhibition of IL-2 by CsA or FK506. We thus propose that substantial down-regulation of calcineurin pathways by CsA and FK506, to the extent of suppressing the production of IL-2 in normal T cells, may have little effect on TGF-ß1. Independent experiments in cells stimulated with phorbol ester and calcium ionomycin showed that PMA (10 nM) alone induced the production of TGF-ß1 by T cells. Induction was 30% lower in cells costimulated with PMA and ionomycin, suggesting that calcium mobilization and perhaps calcineurin activation might reduce the stimulatory effect of other pathways. However, it is unclear whether this inhibitory effect of ionomycin represents that physiological activation of calcineurin would partially repress TGF-ß1 induction, as CsA did not enhance the production of TGF-ß1 induced by PMA or PMA plus ionomycin, and neither CsA nor FK506 enhanced TGF-ß1 induction with anti-CD3/CD28, which also mobilizes calcium and activates calcineurin.

To a certain extent, our results are in contrast with earlier papers regarding the effect of CsA on TGF-ß1 synthesis in T cells. Preceding studies had shown that CsA (100 nM–2 µM) moderately enhanced (≤ twofold) the production of TGF-ß1 in lymphocytes only when cells were stimulated with anti-CD3 antibodies or mitogens but not in cells treated with CsA alone [9 , 34 ]. More recently, other authors have found no effect of CsA on TGF-ß1 in T cells [35 ]. These studies had not included FK506. Our analysis indicates that the production of TGF-ß1 induced by the TCR or by TGF-ß1 itself in normal human T cells is quite insensitive to immunosuppressive doses of CsA and FK506 and might suggest that lymphocytes reacting to an allograft in CsA or FK506-treated transplant subjects may produce TGF-ß1 as a result of their activation by alloantigens, without requiring further stimulation from CsA or FK506. This view is supported by studies showing that TGF-ß1 was up-regulated in transplanted hearts in rats during immune-mediated rejection, acute (without CsA) or chronic (with CsA), but not in control hearts of CsA-treated animals [11 ]. An independent study also showed that the increase of TGF-ß1 induced during rejection of heart allografts in rats was neither enhanced nor repressed by CsA or FK506 [36 ]. In the same line, it has been shown that TGF-ß1 accumulation in kidney during rejection in rats correlates with the number and activation state of infiltrating lymphocytes [37 ].

We have found that CsA induced the release of bioactive TGF-ß1 in lymphocytes as result of cell death and independently of de novo synthesis. These effects required high doses of CsA (10 µM), in contrast with the protective effect of submicromolar concentrations of CsA in preventing TCR-induced cell death [38 ]. Our results suggest that CsA-induced apoptosis might contribute to regulate TGF-ß1 in experimental models of toxicity that use high doses of the drug. Nephrotoxicity protocols in rats and mice use high CsA doses (15–80 mg CsA/kg body weight) [7 ], and CsA levels in blood can reach 5.16 µg/ml (4.3 µM) after 28 days in rats injected with CsA 15 mg/kg/day [39 ]. Our titration experiments indicated that CsA could still induce detectable apoptosis in T cells and A549 cells at concentrations of 5 µM (not shown). In human patients, CsA levels in blood oscillate from 0.2 to 0.4 µM during immunosuppressive regimes and can attain peak values of 1.6–2 µM [40 ]. These concentrations are clearly lower than those measured in models of CsA nephrotoxicity in rodents, raising the question of whether the effects seen in experimentation animals accurately reproduce the situation in humans. In this regard, recent work has concluded that neither latent nor bioactive TGF-ß1 is increased in serum and plasma of CsA or FK506-treated patients, suggesting that these drugs do not trigger an overall increase in TGF-ß1 levels [41 ]. However, kidney biopsies of CsA- but not FK506-treated patients showed localized accumulation of bioactive TGF-ß1 without increasing the latent cytokine [42 ]. Processing of latent TGF-ß1 to its bioactive form occurs during apoptosis in different cell types [19 ]. Although T cells undergo apoptosis under high concentrations of CsA, other cell types such as myoblasts, endothelial cells, and kidney tubular cells die with lower doses (100 nM–1 µM) in the range measured in blood in human patients [43 44 45 ]. In this regard, it might be possible that CsA could cause the release of TGF-ß1 by inducing apoptosis of sensitive cells. This mechanism does not exclude the previous view that CsA may enhance TGF-ß1 synthesis. Indeed, both effects might combine, as TGF-ß1 locally released from apoptotic cells may induce its own production in other cells via paracrine stimulation.

Our finding that 10 µM FK506 did not cause apoptosis nor release of TGF-ß1 in T cells suggests that calcineurin inhibition may not suffice to explain CsA-induced cell death. Disruption of cyclophilins might underlie CsA toxicity, as CsA analogs (MeVal-4-CsA and MeIle-4-CsA), which do not inhibit calcineurin but block cyclophilin activity, induce apoptosis in endothelial cells comparably with CsA [43 ]. A recent article shows that transgenic mice that overexpress cyclophilin A are resistant to CsA-induced nephrotoxicity, and mice expressing a dominant-negative cyclophilin A mutant have exacerbated nephrotoxicity [46 ]. As CsA requires cyclophilins to inhibit calcineurin, one would reason that CsA should inhibit calcineurin more efficiently in the cyclophilin A transgenic. The fact that these animals present less renal damage and survived longer than nontransgenic controls indicates that mechanisms other than calcineurin inhibition are important in CsA toxicity in vivo.


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ACKNOWLEDGEMENTS
 
This work is supported by grants from Fundació la Caixa (00/012-00), Fundació la Marató TV3 (005110), Plan Nacional (BMC2002-00380), and Distinció de la Generalitat de Catalunya to J. A. J. M. was supported by predoctoral fellowships funded by Fundació la Marató TV3 and by the Ministry of Science and Technology of Spain. B. M. was supported by a predoctoral fellowship from the Departament d’Universitats i Recerca de la Generalitat de Catalunya. We are indebted to Cristina López-Rodríguez, Anjana Rao, Patrick Hogan, Joseph Heitman, Jeremy Luban, Gabriel Gil, and Josefina Martínez-Hoyos for reagents and insightful discussion. We are grateful to Oscar Fornas for expert advice with flow cytometry and Raquel Flores for excellent technical assistance.

Received September 9, 2004; revised December 23, 2004; accepted January 14, 2005.


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REFERENCES
 
    1
  1. Hogan, P. G., Chen, L., Nardone, J., Rao, A. (2003) Transcriptional regulation by calcium, calcineurin, and NFAT Genes Dev. 17,2205-2232[Free Full Text]
    2. 2
  2. Liu, J., Farmer, J. D., Jr, Lane, W. S., Friedman, J., Weissman, I., Schreiber, S. L. (1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes Cell 66,807-815[CrossRef][Medline]
    3. 3
  3. Aramburu, J., Rao, A., Klee, C. B. (2000) Calcineurin: from structure to function Curr. Top. Cell. Regul. 36,237-295[Medline]
    4. 4
  4. Kiani, A., Rao, A., Aramburu, J. (2000) Manipulating immune responses with immunosuppressive agents that target NFAT Immunity 12,359-372[CrossRef][Medline]
    5. 5
  5. Islam, M., Burke, J. F., Jr, McGowan, T. A., Zhu, Y., Dunn, S. R., McCue, P., Kanalas, J., Sharma, K. (2001) Effect of anti-transforming growth factor-ß antibodies in cyclosporine-induced renal dysfunction Kidney Int. 59,498-506[CrossRef][Medline]
    6. 6
  6. English, R. F., Pophal, S. A., Bacanu, S. A., Fricker, J., Boyle, G. J., Ellis, D., Harker, K., Sutton, R., Miller, S. A., Law, Y. M., Pigula, F. A., Webber, S. A. (2002) Long-term comparison of tacrolimus- and cyclosporine-induced nephrotoxicity in pediatric heart-transplant recipients Am. J. Transplant. 2,769-773[CrossRef][Medline]
    7. 7
  7. Wolf, G., Thaiss, F., Stahl, R. A. (1995) Cyclosporine stimulates expression of transforming growth factor-ß in renal cells. Possible mechanism of cyclosporines antiproliferative effects Transplantation 60,237-241[Medline]
    8. 8
  8. Prashar, Y., Khanna, A., Sehajpal, P., Sharma, V. K., Suthanthiran, M. (1995) Stimulation of transforming growth factor-ß 1 transcription by cyclosporine FEBS Lett. 358,109-112[CrossRef][Medline]
    9. 9
  9. Li, B., Sehajpal, P. K., Khanna, A., Vlassara, H., Cerami, A., Stenzel, K. H., Suthanthiran, M. (1991) Differential regulation of transforming growth factor ß and interleukin 2 genes in human T cells: demonstration by usage of novel competitor DNA constructs in the quantitative polymerase chain reaction J. Exp. Med. 174,1259-1262[Abstract/Free Full Text]
    10. 10
  10. Kulkarni, A. B., Huh, C. G., Becker, D., Geiser, A., Lyght, M., Flanders, K. C., Roberts, A. B., Sporn, M. B., Ward, J. M., Karlsson, S. (1993) Transforming growth factor ß 1 null mutation in mice causes excessive inflammatory response and early death Proc. Natl. Acad. Sci. USA 90,770-774[Abstract/Free Full Text]
    11. 11
  11. Waltenberger, J., Wanders, A., Fellstrom, B., Miyazono, K., Heldin, C. H., Funa, K. (1993) Induction of transforming growth factor-ß during cardiac allograft rejection J. Immunol. 151,1147-1157[Abstract]
    12. 12
  12. Aharoni, R., Kayhan, B., Eilam, R., Sela, M., Arnon, R. (2003) Glatiramer acetate-specific T cells in the brain express T helper 2/3 cytokines and brain-derived neurotrophic factor in situ Proc. Natl. Acad. Sci. USA 100,14157-14162[Abstract/Free Full Text]
    13. 13
  13. Liu, H., Hu, B., Xu, D., Liew, F. Y. (2003) CD4+CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-ß, and CTLA4 J. Immunol. 171,5012-5017[Abstract/Free Full Text]
    14. 14
  14. Chen, W., Jin, W., Wahl, S. M. (1998) Engagement of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) induces transforming growth factor ß (TGF-ß) production by murine CD4(+) T cells J. Exp. Med. 188,1849-1857[Abstract/Free Full Text]
    15. 15
  15. Levings, M. K., Sangregorio, R., Sartirana, C., Moschin, A. L., Battaglia, M., Orban, P. C., Roncarolo, M. G. (2002) Human CD25+CD4+ T suppressor cell clones produce transforming growth factor ß, but not interleukin 10, and are distinct from type 1 T regulatory cells J. Exp. Med. 196,1335-1346[Abstract/Free Full Text]
    16. 16
  16. Groux, H., O’Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J. E., Roncarolo, M. G. (1997) A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis Nature 389,737-742[CrossRef][Medline]
    17. 17
  17. Fukaura, H., Kent, S. C., Pietrusewicz, M. J., Khoury, S. J., Weiner, H. L., Hafler, D. A. (1996) Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-ß1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients J. Clin. Invest. 98,70-77[Medline]
    18. 18
  18. Letterio, J. J., Roberts, A. B. (1998) Regulation of immune responses by TGF-ß Annu. Rev. Immunol. 16,137-161[CrossRef][Medline]
    19. 19
  19. Chen, W., Frank, M. E., Jin, W., Wahl, S. M. (2001) TGF-ß released by apoptotic T cells contributes to an immunosuppressive milieu Immunity 14,715-725[CrossRef][Medline]
    20. 20
  20. Spits, H., Keizer, G., Borst, J., Terhorst, C., Hekman, A., de Vries, J. E. (1983) Characterization of monoclonal antibodies against cell surface molecules associated with cytotoxic activity of natural and activated killer cells and cloned CTL lines Hybridoma 2,423-437[Medline]
    21. 21
  21. Kim, S. J., Glick, A., Sporn, M. B., Roberts, A. B. (1989) Characterization of the promoter region of the human transforming growth factor-ß 1 gene J. Biol. Chem. 264,402-408[Abstract/Free Full Text]
    22. 22
  22. Nordeen, S. K. (1988) Luciferase reporter gene vectors for analysis of promoters and enhancers Biotechniques 6,454-458[Medline]
    23. 23
  23. Macian, F., Garcia-Rodriguez, C., Rao, A. (2000) Gene expression elicited by NFAT in the presence or absence of cooperative recruitment of Fos and Jun EMBO J. 19,4783-4795[CrossRef][Medline]
    24. 24
  24. Dolganov, G., Bort, S., Lovett, M., Burr, J., Schubert, L., Short, D., McGurn, M., Gibson, C., Lewis, D. B. (1996) Coexpression of the interleukin-13 and interleukin-4 genes correlates with their physical linkage in the cytokine gene cluster on human chromosome 5q23–31 Blood 87,3316-3326[Abstract/Free Full Text]
    25. 25
  25. Luo, C., Shaw, K. T., Raghavan, A., Aramburu, J., Garcia-Cozar, F., Perrino, B. A., Hogan, P. G., Rao, A. (1996) Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import Proc. Natl. Acad. Sci. USA 93,8907-8912[Abstract/Free Full Text]
    26. 26
  26. Aramburu, J., Yaffe, M. B., Lopez-Rodriguez, C., Cantley, L. C., Hogan, P. G., Rao, A. (1999) Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A Science 285,2129-2133[Abstract/Free Full Text]
    27. 27
  27. Lopez-Rodriguez, C., Aramburu, J., Jin, L., Rakeman, A. S., Michino, M., Rao, A. (2001) Bridging the NFAT and NF-{kappa}B families: NFAT5 dimerization regulates cytokine gene transcription in response to osmotic stress Immunity 15,47-58[CrossRef][Medline]
    28. 28
  28. Zhu, D., Cardenas, M. E., Heitman, J. (1996) Calcineurin mutants render T lymphocytes resistant to cyclosporin A Mol. Pharmacol. 50,506-511[Abstract]
    29. 29
  29. June, C. H., Ledbetter, J. A., Gillespie, M. M., Lindsten, T., Thompson, C. B. (1987) T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression Mol. Cell. Biol. 7,4472-4481[Abstract/Free Full Text]
    30. 30
  30. Zawadzka, M., Kaminska, B. (2005) A novel mechanism of FK506-mediated neuroprotection: Downregulation of cytokine expression in glial cells Glia 49,36-51[CrossRef][Medline]
    31. 31
  31. Khanna, A. K. (2003) The immunosuppressive agent tacrolimus induces p21WAF/CIP1WAF1/CIP1 via TGF-ß secretion Biochem. Biophys. Res. Commun. 303,266-272[CrossRef][Medline]
    32. 32
  32. Gooch, J. L., Barnes, J. L., Garcia, S., Abboud, H. E. (2003) Calcineurin is activated in diabetes and is required for glomerular hypertrophy and ECM accumulation Am. J. Physiol. Renal Physiol. 284,F144-F154[Abstract/Free Full Text]
    33. 33
  33. Kuprash, D. V., Boitchenko, V. E., Yarovinsky, F. O., Rice, N. R., Nordheim, A., Ruhlmann, A., Nedospasov, S. A. (2002) Cyclosporin A blocks the expression of lymphotoxin {alpha}, but not lymphotoxin ß, in human peripheral blood mononuclear cells Blood 100,1721-1727[Abstract/Free Full Text]
    34. 34
  34. Ahuja, S. S., Shrivastav, S., Danielpour, D., Balow, J. E., Boumpas, D. T. (1995) Regulation of transforming growth factor-ß 1 and its receptor by cyclosporine in human T lymphocytes Transplantation 60,718-723[Medline]
    35. 35
  35. Goppelt-Struebe, M., Esslinger, B., Kunzendorf, U. (2003) Failure of cyclosporin A to induce transforming growth factor ß (TGF-ß) synthesis in activated peripheral blood lymphocytes Clin. Transplant. 17,20-25
    36. 36
  36. Wynn, C., Pan, F., Erickson, L., Ebbs, A., Kobayashi, M., Jiang, H. (2002) High level of TGF-ß in organ transplantation might be due to rejection rather than calcineurin inhibitor medication Transplant. Proc. 34,1387-1389[CrossRef][Medline]
    37. 37
  37. Hamar, P., Szabo, A., Muller, V., Heemann, U. (2002) The involvement of activated T cells and growth-factor production in the early and late phase of chronic kidney allograft nephropathy in rats Transpl. Int. 15,446-454[CrossRef][Medline]
    38. 38
  38. Takahashi, K., Reynolds, M., Ogawa, N., Longo, D. L., Burdick, J. (2004) Augmentation of T-cell apoptosis by immunosuppressive agents Clin. Transplant. 18(Suppl. 12),72-75
    39. 39
  39. Shihab, F. S., Bennett, W. M., Tanner, A. M., Andoh, T. F. (1997) Angiotensin II blockade decreases TGF-ß1 and matrix proteins in cyclosporine nephropathy Kidney Int. 52,660-673[Medline]
    40. 40
  40. Le Guellec, C., Buchler, M., Giraudeau, B., Le Meur, Y., Gakoue, J. E., Lebranchu, Y., Marquet, P., Paintaud, G. (2002) Simultaneous estimation of cyclosporin and mycophenolic acid areas under the curve in stable renal transplant patients using a limited sampling strategy Eur. J. Clin. Pharmacol. 57,805-811[CrossRef][Medline]
    41. 41
  41. Hughes, J. R., Hughes, V. F., Trull, A. K., Metcalfe, S. M. (1999) Blood levels of TGF ß1 in liver transplant recipients receiving either tacrolimus or micro-emulsified cyclosporine Transplantation 68,583-586[CrossRef][Medline]
    42. 42
  42. Mohamed, M. A., Robertson, H., Booth, T. A., Balupuri, S., Kirby, J. A., Talbot, D. (2000) TGF-ß expression in renal transplant biopsies: a comparative study between cyclosporin-A and tacrolimus Transplantation 69,1002-1005[CrossRef][Medline]
    43. 43
  43. Alvarez-Arroyo, M. V., Yague, S., Wenger, R. M., Pereira, D. S., Jimenez, S., Gonzalez-Pacheco, F. R., Castilla, M. A., Deudero, J. J., Caramelo, C. (2002) Cyclophilin-mediated pathways in the effect of cyclosporin A on endothelial cells: role of vascular endothelial growth factor Circ. Res. 91,202-209[Abstract/Free Full Text]
    44. 44
  44. Hong, F., Lee, J., Song, J. W., Lee, S. J., Ahn, H., Cho, J. J., Ha, J., Kim, S. S. (2002) Cyclosporin A blocks muscle differentiation by inducing oxidative stress and inhibiting the peptidyl-prolyl-cis-trans isomerase activity of cyclophilin A: cyclophilin A protects myoblasts from cyclosporin A-induced cytotoxicity FASEB J. 16,1633-1635[Abstract/Free Full Text]
    45. 45
  45. Healy, E., Dempsey, M., Lally, C., Ryan, M. P. (1998) Apoptosis and necrosis: mechanisms of cell death induced by cyclosporine A in a renal proximal tubular cell line Kidney Int. 54,1955-1966[CrossRef][Medline]
    46. 46
  46. Hong, F., Lee, J., Piao, Y. J., Jae, Y. K., Kim, Y. J., Oh, C., Seo, J. S., Yun, Y. S., Yang, C. W., Ha, J., Kim, S. S. (2004) Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis-trans isomerase activity Biochem. Biophys. Res. Commun. 316,1073-1080[CrossRef][Medline]



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