Transplant Immunology 22 (2009) 72–81 Contents lists available at ScienceDirect Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m Tacrolimus causes a blockage of protein secretion which reinforces its immunosuppressive activity and also explains some of its toxic side-effects M.C. Rauch, A. San Martín, D. Ojeda, C. Quezada, M. Salas, J.G. Cárcamo, A.J. Yañez, J.C. Slebe, A. Claude ⁎ Instituto de Bioquímica, Universidad Austral de Chile, Chile a r t i c l e i n f o Article history: Received 13 March 2009 Received in revised form 6 July 2009 Accepted 13 July 2009 Keywords: Tacrolimus FK506 Brefeldin A Secretory pathway Immunosuppressive side-effects Jurkat cells MIN6 cells a b s t r a c t Background: Tacrolimus (FK506) is a macrolide immunosuppressant drug from the calcineurin inhibitor family, widely used in solid organ and islet cell transplantation, but produces significant side-effects. Objective: This study examined the effect of FK506 on interleukin-2 (IL-2) and insulin secretion, establishing a novel characteristic of this drug that could explain its diverse adverse effects, and developed an experimental model for the simultaneous analysis of mRNA expression and protein secretion affected by this drug. Methods: The IL-2 levels when tacrolimus was administered were analysed by Western blot, immunocytochemistry and RT-PCR in a T lymphocyte cellular line (Jurkat) 24 h post-stimulation. The insulin levels when tacrolimus was administered were analysed 4 h after stimulation of glucose-induced insulin secretion in a pancreatic cellular line (MIN6). Results: The previously published information describes tacrolimus as only capable of partially blocking IL-2 mRNA expression. In our hands, the cellular content of IL-2 is almost undetectable in stimulated Jurkat cells and can be detected in cellular extracts only when the secretory pathway is blocked by brefeldin A (BFA). BFA added 2 h after the beginning of stimulation was able to inhibit IL-2 secretion, without affecting IL-2 mRNA expression. Therefore BFA utilization allowed us to establish a model to analyze the effect on IL-2 secretion, separately from its expression. Tacrolimus added before stimulation inhibits only IL-2 synthesis, but blocks IL-2 protein secretion when added 2 h after stimulation. Similarly, tacrolimus is also capable of blocking the glucose-stimulated secretion of insulin by MIN6 cells. An increase of the intracellular content can be detected concomitantly with a decrease of the hormone measured in the culture medium. Conclusions: Results of this study indicate that tacrolimus possesses another important effect in addition to the inhibition of IL-2 gene transcription, namely the ability to act as a general inhibitor of the protein secretory pathway. These results strongly suggest that the diabetogenic effect of the immune suppressant FK506 could be caused by the blockade of insulin secretion. This novel effect also provides an explanation for other side-effects observed in immunosuppressive treatment. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The immunosuppressive drugs tacrolimus (FK506) and cyclosporine (CsA) belong to the class of immunosuppressant agents referred as calcineurin inhibitors, based on their proposed action mechanism and have been widely used in organ and cell transplantation and more recently in autoimmune diseases [1,2]. Several studies have demonstrated that tacrolimus exerts its immunosuppressive effects primarily by interfering with the activation of T cells [3,4]. The macrolide FK506 is a potent immunosuppressant that canonically inhibits a key step in T cell activation, blocking the interleukin-2 (IL-2) gene transcription [5,6]. This process is initiated by the binding of tacrolimus to the cytoplasmic immunophilins FKPBs, where the isoform FKBP12 is the ⁎ Corresponding author. Instituto de Bioquímica, Casilla 567, Universidad Austral de Chile, Valdivia, Chile. Tel.: +56 63 221332. E-mail address: alejandroclaude@uach.cl (A. Claude). 0966-3274/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2009.07.001 main effector in the immunosuppressive effect caused by tacrolimus [6–8]. The tacrolimus–FKBP12 complex inhibits the activity of calcineurin, a serine–threonine phosphatase that regulates the IL-2 promoter induction after T cell activation [9,10]. This inhibition of calcineurin impedes the calcium-dependent signal transduction, and inactivates the transcription factors (NF-AT's) that promote cytokine gene activation, as these are direct or indirect substrates of the calcineurin's serine– threonine phosphatase activity [11,12]. As a consequence, the transcription of cytokines IL-2, IL-3, IL-4, IL-5, interferon-γ, tumor necrosis factorα, granulocyte-macrophage colony-stimulating factor and IL-2 and IL-7 receptors is suppressed by tacrolimus [3,13–16]. Another calcineurin inhibitor, cyclosporine A (CsA), exerts similar inhibitory effects on inflammatory cytokines, although the inhibitory effect of CsA is less potent than that of tacrolimus [17]. This widely accepted mechanism, however, does not readily explain the different side-effects caused by this drug (diabetogenesis, neuropathy and nephrotoxicity). [18–23]. M.C. Rauch et al. / Transplant Immunology 22 (2009) 72–81 Post-transplant diabetes mellitus (PTDM) is a known, common side-effect of treatment with several immunosuppressive drugs [24] and often leads to the need for exogenous insulin administration to normalize glucose homeostasis in graft recipients [25]. Obviously, the diabetogenicity of immunosuppressive drugs is particularly undesiderable for pancreas and islet transplantation. Indeed, the limited survival of initially successful islets allograft, particularly in the past, may be partially explained by the diabetogenicity of the immunosuppressive regimens utilized [26]. The toxic effects observed in several tissues, such as pancreatic and central nervous system cells, remain however unexplained. Several studies have investigated the effects of these immunosuppressive agents on beta-cell function in human cell lines [27], rodent beta cells [28] and human beta cells [29–31]. These studies have shown that high doses of FK506 can cause significant exocytosis of cellular insulin [27], inhibition of insulin secretion upon stimulation by glucose [28,31], induction of apoptosis in both beta and alpha cells [29] and abolished beta-cell regeneration [32]. In vivo treatment of different animal species with oral doses of tacrolimus of at least 1 mg/ kg/day, induced glucose intolerance after 2 weeks but no hyperglycemia [33–39]. The glucose-intolerant animals showed a decreased pancreatic insulin content [34], vacuolation and degranulation of the isolated islets [33], an increased rate of islet apoptosis [39] or a decreased immunostaining of insulin, together with a diminished in situ hybridization of insulin mRNA in beta cells [38]. It is therefore clear that the FK506 immunosuppressive treatment must affect other equally important cellular pathways, different from the canonical action mechanism dependent on the transcriptional inhibition of IL-2. As an example, despite the fact that FK506 is a powerful immunosuppressive drug that inhibits the activation of several transcription factors (nuclear factors NF-AT and NF-kB) critical for T cell activation, FK506 administration induces the NF-kBregulated IL-6 transcription in vitro and in vivo in kidney, which could probably explain the nephropathy often observed during the immunosuppressive treatment [40]. Previous studies have correlated the immunosuppressive effect of these drugs with the inhibition of IL-2 expression, measuring calcineurin activity and IL-2 transcription levels. We have extended these determinations to include the effects of these drugs on protein secretion in different cell lines. We show that in the T lymphocyte cell line Jurkat J77, FK506 alters the secretion of this cytokine, similarly to the effect of brefeldin A (BFA), in addition of the expected transcriptional inhibition. Additionally, we have demonstrated that FK506 treatment also blocks the insulin secretion in the pancreatic cell line MIN6 as early as 4 h post-glucose stimulation. We therefore propose a novel effect for the immunosuppressive drug FK506, which clearly blocks IL-2 protein secretion in Jurkat cells. By extension, the diabetogenic side-effect can be explained by the blockage of insulin secretion in MIN6 pancreatic cells. 2. Methodology 2.1. Chemical compounds Tacrolimus (10 mg/ml; Tecoland Labs), CsA (1 mg/ml; MP Biomedicals LLC) and BFA (100 µM; Sigma-Aldrich) were dissolved in DMSO (Aldrich Chemicals, Milwaukee, WI, USA) and stored at 4 °C. An equivalent DMSO volume was used as a control when indicated. 2.2. Cell lines and tissue culture Jurkat J77 cells were grown in RPMI 1640, supplemented with 10% bovine calf serum, 100 U of penicillin/streptomycin and 5 mM glutamine at 37 °C in 5% CO2/O2. Mouse insulinoma (MIN6) cells were used between passages 16 and 35 at 80% confluence. MIN6 cells were grown in DMEM containing 73 25 mM glucose (DMEM full media) supplemented with 15% heatinactivated fetal calf serum, 100 µg/ml streptomycin, 100 U/ml penicillin sulfate, and 75 µM β-mercaptoethanol, equilibrated with 5% CO2, 95% air at 37 °C. Prior to treatment, the medium was removed and the cells washed twice with HEPES-balanced Krebs–Ringer bicarbonate buffer (115 mM NaCl, 5 mM KCl, 10 mM NaHCO3, 2.5 mM MgCl2, 2.5 mM CaCl2, 20 mM HEPES, pH 7.4) containing 0.5% bovine serum albumin (KRB buffer). 2.3. IL-2 and insulin secretion Secretion of IL-2 was induced by incubating 106 Jurkat cells with 10 ng/ml of phorbol 12-myristate 13-acetate (PMA) and 2% of phytohemagglutinin (PHA) in a final volume of 800 µl as described previously [41]. The total duration of stimulation was varied in different experiments (0 to 48 h) and is specified in the Results section and the appropriate figures. To determine the secreted and intracellular IL-2, cells were harvested by centrifugation (250 ×g for 10 min), the cell-free medium supernatant was collected and the Jurkat cells were washed twice with PBS to remove the secreted IL-2. Then the cells were lysed using 1.0% Triton X-100 and suspended in 100 µl of PBS. The supernatant and lysed cells were analyzed by Western blot. Secretion of insulin was measured by plating 105 MIN6 cells per well of a 6-well plate in 1 ml of complete media and grown for 3 or 4 days. Media were replaced with KRB buffer and the cells were then incubated for 2 h at 37 °C in KRB buffer prior to incubation in KRB buffer or DMEM containing 5 or 25 mM glucose for a further hour with or without drugs at various concentrations at 37 °C for 15 min, followed by buffer alone at the times indicated in each figure. Cell viability was monitored at timed intervals using Trypan blue exclusion as previously described [42]. In all cases the viability of the cells was 98–100%. The insulin released into the KRB or DMEM was then analysed by ELISA and corrected with an average cell count determined for each well as described above. 2.4. Immunoblotting Protein quantitation was performed by Western blot analysis. The samples were separated using 10% discontinuous SDS-PAGE. The resolved membrane proteins were transferred to Immobilon membranes (Millipore, Bedford, MA, USA) and then soaked in 5% non-fat dried milk in Tris–buffered saline containing Tween-20 (TBS-T; 10 mM Tris–HCl, pH 7.2, 250 mM NaCl, 0.05% Tween-20) at 4 °C overnight. The membrane was incubated with rabbit anti-IL-2 polyclonal primary antibody (1:1000 dilution in PBS-T) at room temperature for 1 h, then incubated with a biotinylated secondary antibody (1:5000 dilution in PBS-T) at room temperature for 1 h, and developed with peroxidase-conjugated streptavidin at room temperature for 1 h. Specific bands were visualized by ECL (enhanced chemiluminescence; Amersham Biosciences, Arlington Heights, IL) [43]. As controls, membranes were incubated with antibodies pre-absorbed with the respective peptide used to generate the antibodies. 2.5. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNAs were isolated by the method of Chomczynski and Sacchi [44] from Jurkat J77 cells. Only RNA samples that yielded intact 18S and 28S bands with the expected band ratio were included in subsequent experiments. The reverse transcription reaction was performed in a reaction mixture of 20 µl total volume containing 2 µg total RNA of each sample, 200 U Moloney murine leukaemia virus reverse transcriptase (BioLabs, New England), 1 mM each of the dNTPs (dATP, dCTP, dTTP, and dGTP), 20 U of ribonuclease inhibitor, 0.5 mg oligo (dT) primer, 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, and 3 mM MgCl2. The RT mix was incubated in a 74 M.C. Rauch et al. / Transplant Immunology 22 (2009) 72–81 thermal cycler at 42 °C for 50 min, followed by enzyme inactivation at 70 °C for 15 min. Subsequent PCRs were carried out in the presence of 1 mM sense and antisense primer, 3 mM MgCl2, 0.4 µl Taq DNA polymerase (Invitrogen, Los Angeles, CA), 1 µl of each dNTP (10 mM), 2.5 µl of 10× buffer and 5 µl of template cDNA, in a total volume of 25 µl. The following customized primers were used for PCR: 5′-ATG TAC AGG ATG CAA CTC CTG TCT TGC-3′ and 5′-AGT CAG TGT TGA GAT GAT GCT TTG ACA-3′ for human IL-2 (GenBank accession number X01586). The sequence for the [beta]-actin forward primer was 5′-TCA CCC ACA CTG TGC CCC ATC TAC GA-3′. The sequence for the [beta]-actin reverse primer was 5′-CAG CGG AAC CGC TCA TTG CCA ATG G-3′. Together these two primers define a 297 bp PCR product. Conditions for the PCR were denaturation at 94 °C (45 s), annealing at 55 °C (45 s), and extension at 72 °C (45 s) for 35 cycles. The PCR products were analyzed on 1.5% agarose gels, which were subsequently stained with ethidium bromide and visualized under ultraviolet light and analyzed with an analysis and documentation system (Syngene Gel Documentation, Ingenius LHR Model, Imgen Technologies, US). 2.6. Immunostaining procedures For immunoperoxidase localization, Jurkat cells were treated with 0.3% H2O2 for 5 min and incubated for 60 min at room temperature in 5% BSA–PBS pH 7.4, followed by incubation for 60 min at room temperature with anti-IL-2 polyclonal primary antibody (1:100 dilution) in 1% BSA–PBS pH 7.4 and 0.3% Triton X-100. Cells were washed and incubated with anti-rabbit IgG-horseradish peroxidase (1:500, Amersham Biosciences) for 2.5 h at room temperature. Immunostaining was developed using 0.05% diaminobenzidine and 0.03% H2O2. Stained sections were examined with a Zeiss Axioskop II microscope equipped with a digital video camera (NikonDXM1200). For immunofluorescent IL-2 and insulin localization, Jurkat and MIN6 cells respectively were washed three times with 1× PBS pH 7.4, 1 mM PMSF at 4 °C and incubated with primary antibodies, followed by the secondary antibodies anti-rabbit IgG-Alexa 488 or IgG-Alexa 594 (1:300, Invitrogen) and subsequently washed and mounted. Fig. 1. Effect of CsA, FK506 and BFA on IL-2 secretion by Jurkat cells before stimulation with PMA + PHA. Panel A: Jurkat J77 cells were stimulated with PMA (10 ng/ml) and PHA (2%) and then the cells were separated from the culture medium at the indicated times. Samples of the culture medium were taken prior to the stimulation and were labelled as SN1. The cultured medium labelled as SN2 corresponds to the culture medium separated from the cells at the end of the experiment. The collected cells were homogenized in PBS–Triton 1% and labelled as PP. Panel B: Jurkat cells were processed as indicated in panel A, with the following changes: incubated with BFA (0.1 µM), CsA (1 µg/ml) or FK-506 (100 ng/ml) and after 2 h PMA (10 ng/ml) and PHA (2%) were added for stimulation. The protein extracts and supernatants were subjected to SDS-PAGE and Western blot to detect IL-2 by chemiluminesence (ECL, Amersham). kDa: molecular weight standard. SN1: culture medium extracted before treatment; SN2: culture medium recovered after treatment; PP: cellular extract prepared from cell pellets; PMA: phorbol myristate acetate (phorbol ester); PHA: phytohemagglutinin; BFA: brefeldin A. M.C. Rauch et al. / Transplant Immunology 22 (2009) 72–81 Stained cells were examined with an Olympus Fluoview FV1000 laser scanning confocal microscope. The images obtained were processed for brightness and contrast only with Adobe Photoshop 6.0. 75 expressed as the mean ± SEM of independent measurements. Statistical significance was considered to be p b 0.05. 3. Results 2.7. Statistical analysis Tests were carried out by a 1-way analysis of variance (ANOVA) followed by a Dunnett multiple comparisons post-test. Values were 3.1. IL-2 protein is detected in cellular extracts only when the secretory pathway is blocked Jurkat cells secrete IL-2 in response to appropriate stimulation; in addition IL-2 contains one disulfide bond and requires oxidative folding prior to secretion [45]. In Fig. 2. Effect of BFA on IL-2 transcription, secretion and cellular accumulation on stimulated Jurkat cells. Jurkat J77 cells were stimulated with PMA (10 ng/ml) and PHA (2%) and then BFA was added 2 or 16 h later. After a total of 24 h post-stimulation, the cells were separated from the culture media. Samples of the culture medium were taken prior to the treatment with the drugs and were labelled as SN1. The cultured medium labelled as SN2 corresponds to the culture medium separated from the cells at the end of the experiment. A fraction of the collected cells was homogenized in PBS–Triton 1% and labelled as PP. The rest of the cell pellets were processed for immunofluorescence and RNA extraction. Panel A: The extracted proteins and supernatants were analyzed by SDS-PAGE and Western blot to detect IL-2 by chemiluminescence (ECL, Amersham). Panel B: Confocal microscopy analysis of the fixed cells, where IL-2 and GBF1 were detected by immunofluorescence using specific polyclonal antibodies. The images are representative random fields from five independent determinations. Scale bars = 10 µM. Panel C: RT-PCR detection of IL-2 and β-actin mRNA and quantitation by densitometric analysis. Results were normalized to β-actin levels. Data represent the mean ± SD of four samples. The asterisk (⁎) indicates a statistically significant difference (p b 0.05) versus the control value). kDa: molecular weight standard; SN1: culture medium extracted before treatment; SN2: culture medium recovered after treatment; PP: cellular extract separated from cell pellets; PMA: phorbol myristate acetate (phorbol ester); PHA: phytohemagglutinin; BFA: brefeldin A. 76 M.C. Rauch et al. / Transplant Immunology 22 (2009) 72–81 previous studies, Manthey et al. [41] quantified the secretion of interleukin-2 by Jurkat cells in response to various inducers: phorbol-12-myristate-13-acetate (PMA), phytohemagglutinin (PHA), ionomycin, concanavalin A, and mouse anti-human CD28 cell surface marker which were used in various combinations, to choose the best conditions to stimulate proliferation. Several studies have shown that the tumor promoter phorbol myristate acetate (PMA) is a potent enhancing factor (together with PHA and IL-2) for normal T cell colony growth [46–49]. PMA apparently acts in synergy with PHA, enhancing the expression of IL-2R on normal T lymphocytes and the production of IL-2 by these cells [50,51]. Based on these antecedents, we decided to use a combination of PMA and PHA as a potent inducer of IL-2 secretion. Manthey et al. [41] established the initial time– response curves of stimulated IL-2 secretion. In these experiments, the rate of IL-2 secretion was maximal within 24 h of addition of PMA and PHA to the culture medium. Therefore, for this study we chose the following standard conditions to induce secretion of IL-2 in Jurkat cells: 1 × 106 cells were incubated with 10 ng/l of PMA and 2% of PHA in a volume of 800 µl from 0 up to 48 h. A clear IL-2 signal is detected by Western blot in the supernatant of treated cells starting at 12 h post-stimulation (SN2) (Fig. 1A). In contrast, IL-2 was not detected in the control supernatant obtained previously from the stimulation (SN1) and in the cellular extract (PP). It has been reported that some immune cells are capable of both secreting and internalizing IL-2 [52]; thus, the IL-2 detected in culture medium may be the net result of both pathways. Based on our results, we chose the 24 h post-stimulation time point for our standard secretion assay, when peak levels of IL-2 are detected in the culture supernatant. To analyze the effect of different drugs in IL-2 secretion, we compared them in Jurkat cells by Western blot (Fig. 1B). We show that IL-2 is not detected in the culture supernatant post-24 h of stimulation (SN2) when the immunosuppressive drugs FK506 or CsA were added 2 h before the PMA + PHA stimulation. These results can be readily explained by either drug inhibiting the IL-2 transcription, through the accepted calcineurin inhibition pathway. In parallel experiments, we used Brefeldin A (BFA), a heterocyclic lactone of fungal origin that blocks the protein secretion pathway [53] and causes the dramatic disappearance of the Golgi complex in most cells [54], to measure its effect in IL-2 Fig. 3. Effect of FK506 on IL-2 secretion and cellular accumulation before and after Jurkat cell stimulation. Panel A: Jurkat J77 cells were incubated with FK506 (100 ng/ml) at the indicated times, before PMA (10 ng/ml) and PHA (2%) stimulation. After 24 h of stimulation the cells (PP) were separated from culture media (SN2) and were homogenized with PBS–Triton 1%. Samples of the culture medium were taken prior to the treatment and labelled as SN1. The extracted proteins and supernatants were analyzed by SDS-PAGE and Western blot to detect IL-2 and GBF1 by chemiluminescence (ECL, Amersham). Panel B: Cells were cultured as described in panel A, with FK506 (100 ng/ml) addition at the times indicated in the figure after the stimulation with PMA and PHA. The stimulation was continued for a total of 24 h. Upper panel: Cells were separated from the culture medium and processed for immunocytochemistry to detect IL-2. The images are representative random fields from five independent determinations. Scale bars = 20 µM. Lower panel: Cells were separated from culture media and were homogenized with PBS plus Triton X-100 at 1%. The extracted proteins and supernatants were analyzed by SDS-PAGE and Western blot to detect IL-2 and GBF1 by chemiluminescence (ECL, Amersham). SN1: culture medium extracted before treatment; SN2: culture medium recovered after treatment; PP: cellular extract separated from cell pellets. M.C. Rauch et al. / Transplant Immunology 22 (2009) 72–81 secretion. In Fig. 1B, we show that IL-2 is detected in the culture supernatant post-24 h stimulation when 0.1 µM (30 ng/ml) BFA is added to the incubation medium 2 h before the stimulation. Importantly, IL-2 is only detected in the post-24 h stimulation cellular extract (PP) when Jurkat cells are treated with BFA before the stimulation. This can be explained by an accumulation of IL-2 inside the Jurkat cells, caused by the general blockage of the secretion pathway triggered by this drug. This accumulation is not observed when the cells were treated with 1 µg/ml CsA or 100 ng/ml FK506, previously to the stimulation with PMA + PHA. This indicates that FK506 and CsA behave under these conditions in accordance with previously published results, diminishing the production and therefore the liberation of IL-2 to the culture supernatant 26 h post-treatment. Under these conditions, these immunosuppressive drugs don't cause a secretory blockage of IL-2 in Jurkat cells, in contrast with the significant block caused by BFA that can be readily detected in our experimental system. To observe the IL-2 secretion effect when BFA is added at or after the proliferation stimulation, we first treated the Jurkat cells with PMA and PHA and then we added BFA at two different concentrations (2 and 5 µM) and at different times (2 and 16 h) poststimulation (Fig. 2A). At both BFA concentrations, 2 and 16 h, IL-2 was detected in the post-24 h stimulation culture medium. However, the IL-2 detected in the culture medium when BFA was added 2 h after stimulation corresponded to only 40% of the IL-2 detected in the absence of BFA (control), and the IL-2 detected in the culture medium when BFA was added 16 h after stimulation corresponded to 70% of the control. Interestingly, IL-2 was detected in the cellular extract only when BFA (2 and 5 µM) was added 2 h after the stimulation. No IL-2 was detected in the cellular extract when BFA (2 and 5 µM) was added 16 h after the stimulation. This indicates that BFA-induced IL-2 accumulation is detected only when BFA is added shortly after the PMA/PHA stimulation because IL-2 secretion must happen rapidly after this stimulatory signal. In order to confirm the accumulation of IL-2 in the Jurkat cells, confocal microscopy analysis was used to detect IL-2 and the peripheral Golgi marker GBF1, with a BFAsensitive localization [55,56], was used as a control. In Fig. 2B we show an increase of the IL-2 signal detected at 24 h of stimulation in the BFA treated cells 2 h poststimulation compared with the DMSO-treated controls. The immunosuppressive mechanism described for FK506 and CsA is based on the inhibition of the IL-2 expression. For this reason, to obtain an experimental model that 77 discriminates between inhibition of IL-2 mRNA expression and blockage of its secretion, we analyzed the IL-2 secreted in the culture medium, and at the same time, we performed a RT-PCR analysis for IL-2 mRNA in BFA treated cells. Jurkat cells responded to PMA and PHA stimulations with increased levels of mRNA encoding IL-2. The level of IL-2 mRNA was statistically similar in PMA + PHA stimulated cells compared to BFA treated cells (Fig. 2C). This finding is consistent with the fact that BFA blocks the protein secretion pathway and not the mRNA expression. Importantly, this experimental setup also permitted us to analyze the IL-2 expression and secretion simultaneously. In Jurkat cells treated with CsA, the inhibitory effect on IL-2 mRNA expression is markedly higher than in Jurkat cells treated with FK506 (data not shown). The inhibitory effect of FK506 on IL-2 transcription has been assumed based on NF-AT's lowered activity at 3 h [57], and studies of IL-2 mRNA expression at very short times (4 h) after addition of tacrolimus [58], but we detected that this transcriptional inhibition is only partial after 22 h in our system (discussed in the next section). Therefore we cannot readily explain the dramatic effect this drug has on the release of IL-2 to the culture medium (Fig. 1B) without postulating an additional inhibitory effect of this drug on IL-2 secretion. For this reason, we decided to analyze the FK506 effect in both IL-2 and insulin secretion to test for additional effects of this drug, besides the transcriptional inhibition of IL-2 to explain its immunosuppressive effect. 3.2. The blockage of IL-2 secretion caused by tacrolimus is observed when added post-PMA + PHA stimulation We postulate that FK506 not only blocks IL-2 transcription, but also its secretion. Normally this second effect is masked by the fact that drug produces a significant diminution of this cytokine protein level when added prior to the stimulation. To demonstrate that FK506 affects the IL-2 secretion when it is added after the stimulation, we first analyzed by Western blot the effect in IL-2 protein levels when FK506 is added before the stimulation. In Fig. 3A we show that IL-2 from Jurkat cells is not detected in the culture medium, when incubated with 100 ng/ml FK506 at 6, 3, 1 and 0 h before the stimulation with PMA + PHA. IL-2 is also not detected in the cellular extracts. GBF1 and beta actin protein levels were used as controls. These results essentially confirm those already observed in Fig. 1B and indicate a clear effect in IL-2 expression. Fig. 4. Comparison between FK506 and BFA effects in IL-2 protein localization and mRNA expression in stimulated Jurkat cells. Jurkat J77 cells were cultured for 24 h in RPMI 1640 culture media supplemented with 1% serum and stimulated with PMA (10 ng/ml) and PHA (2%). After 2 h, BFA (5 µM) or FK-506 (100 ng/ml) was added. After 24 h of stimulation, the cells and media were separated. Cells were used for immunofluorescence and for RNA extraction. Panel A: Cells were fixed with HistochoiceTM and processed for IL-2 and GBF1 immunofluorescence. Scale bars = 5 µM. Panel B: The extracted RNA was used to detect IL-2 mRNA by RT-PCR. A densitometric analysis was performed and the results were normalized against β-actin content. Data represents the mean ± SD of six independent determinations. (⁎⁎) represents p b 0.01 and (⁎) represents p b 0.05 versus PMA + PHA stimulated controls. PMA: phorbol myristate acetate (phorbol ester); PHA: phytohemaggutinin; BFA: brefeldin A; FK506: tacrolimus. 78 M.C. Rauch et al. / Transplant Immunology 22 (2009) 72–81 However, when FK506 was added 1 or 3 h after the stimulation with PHA + PMA, IL-2 is clearly detected inside the cells, as observed in Fig. 3B. An increased IL-2 detection by immunocytochemistry is observed after 1 h of FK506 addition, compared with 3 h and virtually none is detected 6 h post-FK506 addition. The immunolocalization control was performed with GBF-1 in Jurkat cells, stimulated and treated with BFA and FK506. The Western blot analysis shows similar results, where IL-2 can be detected in the culture medium of Jurkat cells when treated with FK506, 3 and 6 h post-stimulation. Strikingly, IL-2 is abundantly present in the cellular extracts (PP) of Jurkat cells treated with FK506 at 1 and 3 h post-stimulation. This effect was lost when the immunosuppressive drug was added 6 h post-stimulation, confirming that IL-2 synthesis and secretion occurs rapidly after stimulation with PMA + PHA. These findings are consistent with the results of Dumont et al. [59] which demonstrated that FK506 needs to be added within the first 3 h after stimulation in order to suppress the proliferative response of lymphocytes. The authors suggested that FK506 affects T cell activation at an early stage, but did not verify if IL-2 accumulated inside the cells. To demonstrate that the FK506 secretory effect is similar to that of BFA, but not the transcriptional inhibition, we analyzed the IL-2 mRNA expression by RT-PCR and protein levels by immunodetection in Jurkat cells treated with 100 ng/ml FK506 or 2 µM BFA, 2 h post-stimulation (Fig. 4A and B). A statistically significant difference in IL-2 mRNA levels was found between FK506 treated cells and stimulated controls (p b 0.05) but not between BFA treated cells and stimulated controls. These results show that, despite FK506 inhibiting the expression of IL-2 mRNA as previously published; it also blocks the secretion of this cytokine. 3.3. Tacrolimus blocks insulin secretion. This novel secretory effect provides a likely explanation for some of the side-effects observed in immunosuppressive treatment To demonstrate that the secretion blockage is a general effect, it must also occur in other cellular lines and affect other secreted proteins. We analyzed the effect of FK506 in MIN6 cells, a pancreatic cellular line that secretes insulin. The process of glucose-stimulated insulin release from β pancreatic cells has been well documented [60]. However, the effect of tacrolimus on glucose-induced insulin release has not been examined in detail. The MIN6 cells have been widely analyzed and present similar characteristics to the normal pancreatic islets in relation to the glucose metabolism and the glucose-induced insulin secretion [61,62], in contrast to other pancreatic cell lines [63,64]. Therefore, this cellular line is currently the model of choice to study the insulin secretion stimulated by glucose [65]. A timed secretion analysis (0 and 8 h) at different concentrations of glucose (5 and 25 mM) showed that insulin does not accumulate in these cells, however, the secretion of insulin to the culture medium is clearly increased compared to unstimulated controls (data not shown). MIN6 cells were stimulated with 5 or 25 mM glucose, treated with 5 µM BFA or 100 ng/ml FK506 1 h post-stimulation and harvested 4 h later. When the MIN6 cells were treated with 5 µM BFA or 100 ng/ml FK506, an increased insulin immunodetection was observed inside the cells (Fig. 5A), and a parallel diminished detection of insulin is observed in the culture medium (Fig. 5B). A statistically significant difference in secreted insulin was found in FK506 and BFA treated cells (p b 0.01), compared with control cells stimulated with 5 or 25 mM glucose. Fig. 5. Comparison between FK506 and BFA effects on insulin localization and secretion in stimulated pancreatic MIN6 cells. MIN6 cells were cultured for 4 h under low and high glucose conditions (5 and 25 mM glucose) and BFA (5 µM) or FK506 (100 ng/ml) were added as indicated. Culture media was recovered, and cells were processed for immunofluorescence studies. Panel A: Immunofluorescent detection of intracellular insulin. Scale bars = 10 µM. Panel B: ELISA determination of insulin content in the culture media collected from all samples. Data represent the mean ± SD of four samples. (⁎⁎) represents p b 0.01 and (⁎) represents p b 0.05 versus PMA + PHA stimulated controls. PMA: phorbol myristate acetate (phorbol ester); PHA: phytohemaggutinin; BFA: brefeldin A; FK506: tacrolimus. M.C. Rauch et al. / Transplant Immunology 22 (2009) 72–81 Taken together, these results clearly indicate that tacrolimus has an early inhibitory effect on the secretion pathway, producing the intracellular accumulation of IL-2 (in T lymphocytes) and insulin (in pancreatic cells). Therefore this drug has a dual effect, specifically inhibiting the expression of the interleukin-2 gene and additionally blocking the secretion of IL-2, insulin and presumably other proteins present in the secretory route. This second effect provides a simple explanation for several of the secondary effects caused by FK506 therapy. 4. Discussion In this study we show that the immunosuppressive drug FK506 affects both the IL-2 expression and secretion in Jurkat cells. Tacrolimus also blocks the insulin secretion in the pancreatic cellular line, MIN6. These results could explain the cause responsible for the adverse effects in the immunosuppressive therapy and also the recently described, and unexplained inhibitory effect of FK506 in the liberation of cytokines unrelated to the cellular immune response. Recent experimental studies suggest that tacrolimus is effective in inhibiting inflammation in several experimental models [66,67]. However, these studies only determined the levels of the cytokines in the culture medium, not verifying the nature of the blockage. The present study attempts for the first time to discriminate between the tacrolimus effect on the IL-2 transcription and IL-2 secretory accumulation, analyzing the intracellular and extracellular protein levels. We standardized our assays measuring IL-2 24 h postsynthesis stimulation with PMA + PHA, although most determinations in the literature are performed at 48 h. Recently Villarino et al. [68] have reported that a classic feedback negative inhibition exists for the IL-2 transcription, with the mRNA synthesis being increasingly repressed by the accumulating protein. The authors, in accordance to our results, also observed maximum secretion of IL-2 at 24 h. The results validate our data, as measurements beyond 24 h, specifically at 48 h post-stimulation might be misleading. In this study we blocked IL-2 secretion with BFA as a model system, because this drug efficiently causes intracellular accumulation of secreted proteins [53]. Secretory inhibition mediated by BFA blocks the protein secretion pathway and causes the dramatic disappearance of the Golgi complex in many cells [54]. In addition in several studies to detect intracytoplasmic cytokines, BFA was used as secretion inhibitor in the culture medium during cell stimulation [69]. We have shown that the cellular content of the IL-2 is almost undetectable in stimulated Jurkat cells, probably because IL-2 is rapidly secreted after synthesis, but incubation with BFA, shortly before or after stimulation caused a decrease in IL-2 secretion and a readily detectable IL-2 accumulation in cellular extracts. No alteration of IL-2 mRNA expression was detected when BFA was added to the culture. Therefore this drug allowed us to establish a system to analyze the blockage on IL-2 secretion, separate from transcriptional effects. When FK506 was added before stimulation, only a partial inhibition of IL-2 mRNA synthesis and a dramatic decrease in the IL-2 secreted to the medium were detected in Jurkat cells. But when tacrolimus was added 1 or 3 h after stimulation, a clear block in IL-2 secretion was observed, with a concomitant intracellular accumulation. We postulate that FK506 has a dual action, decreasing IL-2 transcription and blocking its transit through the secretory pathway. Both effects must occur simultaneously, but the secretory effect is masked when the drug is added before the stimulation, because only small amounts of IL-2 are synthesized and likely degraded during the 24 h before the determination of intracellular levels. We postulate that the secretory blockage caused by FK506 is not limited to IL-2, but must extend to other secretory proteins. In support of our theory, it has been previously reported that the immunosuppressive drugs CsA and FK506 are diabetogenic in vivo. CsA and FK506 have direct toxic effects on pancreatic islets of several animal species [70] but only limited data is available regarding human islets [19]. Tacrolimus-associated post-transplant diabetes mellitus (PTDM) occurs more rapidly and is more frequent than the one associated 79 with CsA [71] and is associated with significant weight loss; the majority of these patients require exogenous insulin treatment [72]. These differences suggest that FK506 directly impairs human β-cell function, while CsA acts indirectly by increasing insulin resistance [73]. At the cellular level, cytoplasmic swelling, vacuolization, apoptosis, and abnormal immunostaining for insulin have been observed in biopsies from patients receiving either FK506 or CsA [19]. Apoptosis is not a general effect of FK506, since it inhibits apoptosis of astrocytes in vitro and in vivo [74]. At concentrations that inhibit the calcineurin phosphatase activity, tacrolimus has been shown to inhibit human insulin gene transcription [75]. Significant inhibition of insulin secretion by tacrolimus has also been observed in rat islets and HIT-T15 cells [28] which correlates well with several author's results concerning inhibitory effects of the drug on human insulin gene expression [76]. It is therefore clear that these immunosuppressive drugs impair human beta-cell function and survival [77], but many of these analyses were performed with a long-term pre-treatment, and the authors conclude that the main mechanism responsible for the glucose intolerance induced by FK506 is not a reduction of the beta-cell mass by increased apoptosis or a decreased proliferation, but an impairment of islet secretion due to a low rate of insulin gene transcription. Interestingly, at therapeutic concentrations, a stimulatory effect on insulin secretion was observed on human beta cells [78]. The fact that islet function was impaired, but not beta-cell mass, explains why all the defective islet parameters observed in rats treated with FK506 were fully reversible [79]. Therefore, the results obtained in these studies are fully compatible with a direct insulin secretion effect caused by FK506. Thus, the pathogenesis of the diabetogenic effects of CsA and FK506 in humans is still largely unknown, and we decided to analyze only the effect of tacrolimus in our experimental model. The diabetogenic effect of FK506 generally manifests itself as hyperglycemia after repeated dosing in experimental animals. Follow-up investigations, using immunocytochemistry for localization of insulin immunoreactivity and in situ hybridization histochemistry to localize insulin mRNA in the pancreas, showed that FK506 treatment resulted in a marked reduction of both insulin parameters [80]. This report indicated that the evidence of reduced mRNA levels was apparent from 1 day's exposure onwards. In contrast to mRNA levels, insulin biosynthesis was not affected after 1 day's exposure but was clearly impaired from 3 day's exposure onwards, suggesting that the initial effect of FK506 may be on a reduction of insulin mRNA levels. We propose, based on our results, that the real initial effect of FK506 may be on insulin secretion and that resulting protein accumulation will cause a reduction of the gene transcription. Our experimental setup readily detects changes in the amount of insulin secreted in the supernatant by glucose-stimulated MIN6 cells. FK506 blocks insulin secretion in these cells and causes a clear intracellular accumulation 4 h post-glucose stimulation. This brief period of time is clearly not sufficient for a significant transcriptional inhibition. We can therefore conclude that the secretory inhibition caused by FK506 also extends to insulin and is totally unrelated to the previously known transcriptional effect of this drug. Concerning kidney transplantation, chronic calcineurin inhibitor (CsA and FK506) nephrotoxicity is a major factor in chronic allograft dysfunction [81]. The long-term CsA therapy may lead to irreversible and potentially progressive nephropathy [82,83]. In studies of chronic nephropathy induced by CsA, it was suggested that the process of apoptosis in tubular cells would be responsible for the renal tubular atrophy and the observed loss of tubular mass [84–87]. This nephrotoxicity is manifested by kidney failure and by arterial hypertension [88]. The main question whether the nephrotoxicity caused by CsA and FK506 is secondary to their effect on the calcineurin-NF-AT pathway or caused by other mechanisms, remains largely unanswered. In summary, the effect of FK506 treatment of Jurkat cells poststimulation is similar to that of BFA (a well established secretion 80 M.C. Rauch et al. / Transplant Immunology 22 (2009) 72–81 inhibitor) in terms of IL-2 secretion blockage. We observed in both cases a diminution in the IL-2 detected in the culture medium and an increase in the intracellular content. Similarly, we observed an increased intracellular content of insulin in MIN6 cells incubated with both BFA and FK506 and a diminution of the insulin detected in the culture medium. By extension, the diabetogenic side-effect of tacrolimus-treated patients can be explained by the blockage of insulin secretion in pancreatic cells. Similar conclusions could be extended to comparable side-effects of this drug on organs with an important secretory function, such as the brain and kidneys [20– 23,88], pending further research in vivo and in vitro using tissuespecific cellular lines and appropriate secretion markers. Presently, we have no conclusive evidence to propose a molecular model for the secretory inhibition caused by FK506. However, circumstantial evidence can be presented to suggest a possible mechanism by which this drug can alter the secretory pathway. The immunosuppressive action of FK506 is thought to occur via binding to the immunophilin FKBP-12 and subsequent inhibition of calcineurindependent T cell activation pathways by the FK506-FKBP complex [9]. On the other hand, the large family of GTP exchange factors for ARF (ARF-GEFs) is ubiquitously represented in all known eukaryotes and is a key player in regulating the secretory pathway through the activation of ARF to the GTP-bound state and triggering the recruitment of cytosolic coat proteins, that in turn drive the formation of transport vesicles that are essential to the secretory process [89,90]. In particular, a subfamily of ARF-GEFs represented by GBF1, plays a key role in protein secretion [91–93] and also possesses a DCB domain involved in dimerization and cyclophillin/immunophilin binding [89,94]. Although the exact nature and function of this domain is unknown, its deletion produces a decrease of the GBF1 cellular levels and an increased BFA sensibility [95]. Recent studies carried out with chimeras indicate that this domain is essential to the correct function of GBF1 [96]. Therefore it is possible that the interaction between FK506 and one or more immunophilins that regulate the function of GBF1 might be sufficient to alter the secretory pathway, producing a partial or total block of protein secretion. Although far from proven, this model will suggest new research avenues and provide interesting insights on the still mysterious biological effects of tacrolimus. [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] Acknowledgments [28] We are grateful to all past and present members of the Cellular Biology and Protein Secretion Laboratory of the Instituto de Bioquímica of the Universidad Austral de Chile. 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