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In vitro effects of everolimus and intravenous immunoglobulin on cell proliferation and apoptosis induction in the mixed lymphocyte reaction
Transplant Immunology 23 (2010) 170–173
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
In vitro effects of everolimus and intravenous immunoglobulin on cell proliferation and apoptosis induction in the mixed lymphocyte reaction Nurmamet Amet a, Mercedes Gacad a, Anna Petrosyan a, Andy Pao a, Stanley C. Jordan b, Mieko Toyoda a,⁎ a Transplant Immunology Laboratory, Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, 8700 Beverly Blvd., SSB111, Los Angeles, CA 90048, United States b Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, 8700 Beverly Blvd., suite 490W, Los Angeles, CA 90048, United States
a r t i c l e
i n f o
Article history: Received 11 June 2010 Accepted 28 June 2010 Keywords: Everolimus Intravenous immunoglobulin Synergistic effect Cell proliferation Apoptosis induction Mixed lymphocyte reaction
a b s t r a c t Targeting multiple pathways in the activation of alloimmune responses by multi-drug immunosuppressive regimens with complementary mechanisms of action enhances allograft survival and improves quality of life, owing to the reduction of adverse drug effects. In this report we investigated the effect of the combination of everolimus and intraveneous immunoglobulin (IVIG) on cell proliferation and apoptosis induction in human two-way mixed lymphocyte reaction (MLR). Everolimus alone (0.1–50 ng/ml) and IVIG (1–10 mg/ml) alone inhibited cell proliferation in a dose-dependent manner (16.4–67.2% and 12.1–66.3% inhibition, respectively). The inhibition by everolimus was not enhanced in the presence of 1 mg/ml IVIG. Addition of 10 and 50 ng/ml everolimus increased the inhibitory effect of 5 and 10 mg/ml IVIG, but only by 10–27%. Addition of 0.1 and 1 ng/ml everolimus did not increase IVIG's inhibitory effects. Apoptosis was significantly higher in IVIG (5 mg/ml)-treated CD19+ cells and less so in CD3+ cells as assessed by Annexin V and TUNEL assays. However, everolimus (0.1–50 ng/ml) did not induced apoptosis or alter apoptosis induced by IVIG. These results suggest that everolimus is a potent inhibitor of immune cell proliferation but does not act additively or synergistically with IVIG when analyzed in this in vitro system. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Transplantation has become a standard treatment option in patients with end-stage renal disease as it enhances quality of life, and dramatically reduces cost associated with dialysis. Immunosuppressive drugs that have been approved for use in clinical transplantation suffer dose-limiting toxic side-effects, including nephrotoxicity and neurotoxicity [1–3]. These adverse effects can be reduced or even avoided by using drug combinations with additive or synergistic activity, which allows each drug to be used at lower dose and thus decreases the adverse effects [4, 5]. Everolimus (Zortress®), a natural product of streptomyces hygroscopicus and analog of rapamycin, possesses favorable pharmacokinetic properties with enhanced solubility and faster steady state [6, 7] compared to rapamycin [8]. These properties allowed everolimus to be formulated as an oral agent, while maintaining rapamycin-like immunosuppressive and anti-neoplastic activities [2, 9]. After forming complexes with FK506-binding protein (FKBP-12) in the cytoplasm of target cells such as T cells and B cells, everolimus binds and prevents the kinase activity of mammalian target of rapamycin (mTOR). The ⁎ Corresponding author. Transplant Immunology Laboratory, SSB111, Dept. of Medicine, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, United States. Tel.: + 1 310 423 4975; fax: + 1 310 423 0268. E-mail address: [email protected] (M. Toyoda). 0966-3274/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2010.06.012
deactivation of mTOR activity by everolimus leads to the blockade of growth factor-dependent signal transduction, cell cycle arrest at G1 phase, and prevention of cell proliferation. Everolimus was approved in 2003 by the European Medicines Agency for use in the prophylaxis of organ transplantation in combination with cyclosporine and corticosteroids. The Food and Drug Administration (FDA) approved everolimus for use in kidney transplant patients in the United States for the prevention of organ rejection in April 2010. Everolimus (afinitor) has also been recently been approved for the treatment of advanced kidney cancer. Clinical trials evaluating the efficacy of everolimus in both heart and kidney transplant patients have shown promising results; it reduces the exposure to cyclosporine and incidence of cardiac-allograft vasculopathy [2, 4] and provides better outcome for kidney transplant patients while lowering the dose of cyclosporine [1, 10]. Intravenous immunoglobulin (IVIG) has been widely used for treatment of autoimmune and systemic inflammatory disorders [11]. We have shown that IVIG treatment reduced allo-sensitization and increased transplant rate in highly HLA-sensitized (HS) patients [12, 13]. We have also reported the successful use of IVIG for treatment of antibody-mediated allograft rejection [14]. These beneficial effects observed in IVIG-treated transplant patients are likely due to a number of recently described immunomodulatory effects of IVIG on T cells, dendritic cells, inhibition of B cell activation and antibody production, induction of anti-inflammatory cytokines, blocking of
N. Amet et al. / Transplant Immunology 23 (2010) 170–173
anti-HLA antibodies by anti-idiotypic antibodies contained in IVIG and blocking of complement-mediated injury through inhibition of C3 activation [14, 15]. Previous studies from our lab have shown that IVIG inhibits cell proliferation in the mixed lymphocyte reaction (MLR) [16] and that IVIG induces apoptosis primarily in B cells and to a lesser degree in T cells and monocytes [17]. IVIG also reduced the expression of MHC class II and costimulatory molecules, CD40 and CD80/86 in the MLR [17]. We suggested that the reduction of intact B cell and monocyte cell numbers, modulation of surface molecule expression on B cells, and deletion of B and T cells by apoptosis may result in inhibition of optimal T cell activation. This may be one of the mechanisms responsible for suppression of the MLR by IVIG and account for many of the beneficial effects observed in IVIG-treated HS patients and those with autoimmune and inflammatory disorders. Other investigators have shown that IVIG inhibits dendritic cell activation and costimulation by inhibition of CD40 and MHC class I/II expression [18]. In a previous study, we had shown the additive effect of combination of IVIG and rapamycin on the inhibition of cell proliferation in the MLR [19] and suggested the possible utility of this combination in transplant patients, especially HS patients where additive effects for desensitization might be desired. Since everolimus is an analog of rapamycin, the combination of everolimus and IVIG might be another option in a multi-drug regimen. Here, we examined possible additive or synergistic effects of the combination of everolimus and IVIG on inhibition of immune cell proliferation and apoptosis induction in the MLR. The aim would be to determine if findings would support the use of this combination in desensitization protocols or treatment of antibody-mediated rejection (AMR). 2. Materials and methods
171
2.3. Detection of apoptosis After 3 day incubation, cells were enumerated and 1 × 106 cells were used for down-stream processing. Cells were washed with FACS buffer consisting of 1 × PBS, 1% fetal bovine serum (FBS) and 0.1% sodium azide, centrifuged for 10 min at 300 g, and then double stained with 5 μl PE-anti-CD3 antibody and 5 μl PE-Cy7-anti-CD19 antibody for 30 min on ice in the dark. After that, cells were washed, re-suspended in 900 μl FACS buffer, which was then split into 100 μl for Annexin V assay and 800 μl for TUNEL assay, as described previously [17, 19]. Briefly, Annexin V staining was accomplished by incubating cells with 5 μl FITC-Annexin V and 5 μl of 7AAD in the dark for 15 min, and TUNEL staining by incubating cells with 50 μl of FITCTUNEL reagent for 1 h in a humidified 37 °C incubator, followed by detection of apoptotic cells by a CyAn Flow Cytometer (Beckman Coulter, Fullerton, CA) and Summit software program (Beckman Coulter). The final results were expressed as the percentage of cells positive for Annexin V or TUNEL in the CD3+ and CD19+ cell populations. Data represent average ± standard deviation, with 9 different pairs of MLRs for Annexin V and 6 different pairs of MLRs for TUNEL staining. 2.4. Statistical analysis The effect of everolimus and/or IVIG on cell proliferation and apoptosis induction was assessed by a standard paired two-tailed ttest. P value b0.05 was considered as significant difference. 3. Results 3.1. The inhibitory effect of combination of Everolimus and IVIG on cell proliferation in the MLR
2.1. Reagents Everolimus (RAD001), provided by Novartis Pharmaceuticals Corp., East Hanover, NJ, was dissolved in absolute ethanol to prepare 10 mM stock solution and kept at −20 °C. The dilutions of everolimus for each experiment were made fresh in the culture media from the stock solution. IVIG was provided by Talecris Biotherapeutis Inc. (Raleigh, NC), either as a 100 mg/ml in 0.2 M glycine or 50 mg/ml in 10% maltose, and kept at 4 °C. Fresh dilutions of IVIG were prepared in cell culture media for each experiment. Glycine- or maltose-based IVIG was used in the assessment of apoptosis or cell proliferation, respectively. Monoclonal antibodies including PE-conjugated CD3 (Invitrogen Inc., Carlsbad, CA) and PE-Cy7-conjugated CD19 (eBioscience Inc., San Diego, CA) were used simultaneously to stain cell surface molecules expressed by T and B cells, respectively. Annexin V Apoptosis Detection Kit (BD Bioscience, San Jose, CA) was used to detect both early and late events of apoptosis, whereas the deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) commercial kit from Roche Diagnostics Corp. (Indianapolis, IN) was used to detect late stage apoptosis.
Both everolimus and IVIG alone inhibited cell proliferation in a dose-dependent manner (Fig. 1). The inhibition was 16.4, 44.6, 62.0, 67.2% for 0.1, 1, 10, 50 ng/ml everolimus, and 12.1, 50.9, 66.3% for 1, 5, 10 mg/ml IVIG, respectively. Addition of 1 mg/ml IVIG did not change the inhibition by everolimus at any of the tested concentrations. Addition of 0.1 and 1 ng/ml everolimus did not significantly increase the inhibitory effect of IVIG at 5 and 10 mg/ml, despite everolimus alone showing 16.4 and 44.6% inhibition at these concentrations, respectively. Addition of 10 and 50 ng/ml everolimus significantly increased the inhibitory effect of 5 mg/ml IVIG by 10.4% and 27.4%,
2.2. Cell culture The 2-way MLR was performed as previously reported [19]. Briefly, whole blood was drawn from unrelated healthy individuals to prepare peripheral blood mononuclear cells (PBMC) using the Ficoll–Hypaque (Sigma, St. Louis, MO) gradient centrifugation method. PBMCs from the two individuals were mixed at 1:1 (1× 106/ml of each PBMCs for cell proliferation and 2 × 106/ml for apoptosis experiments) with everolimus at 0, 0.1, 1, 10, 50 ng/ml, or combined with IVIG at 1, 5 or 10 mg/ ml, and then incubated for 6 days for measurement of cell proliferation by 3H-thymidine uptake and for 3 days for detection of apoptosis. The results of cell proliferation are expressed as a percentage of the cpm in the control condition without additives (100%).
Fig. 1. The effects of everolimus, IVIG or a combination of everolimus and IVIG on cell proliferation in the MLR. The two-way MLR was performed with various concentrations of everolimus and/or IVIG. The results of cell proliferation are expressed as a percentage of the cpm in the control condition without additives (100%). Mean ± standard deviation (SD) of six MLR pairs is shown, respectively. Mean values of cpm of controls without additives in Fig. 1 were 18234 ± 4955. *p b 0.02 vs. without everolimus at each IVIG concentration. #p b 0.02 vs. without IVIG at 0 ng/ml everolimus.
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respectively, compared to that by IVIG alone, and addition of 50 ng/ml everolimus to 10 mg/ml IVIG did so by 10.7%. 3.2. The effect of combination of everolimus and IVIG on apoptosis induction in the MLR Five mg/ml IVIG alone significantly increased apoptosis induction by 86.3% and 85.2% in B cells and 34.0% and 8.1% in T cells as assessed by Annexin V and TUNEL staining, respectively (Fig. 2), which is consistent with our previous study [19]. In contrast, 0.1–50 ng/ml everolimus alone did not induce apoptosis in both B cells and T cells, and had no effect on apoptosis induced by IVIG. 4. Discussion In this study, we assessed the combination of everolimus and IVIG on cell proliferation and apoptosis induction in an in vitro model of transplantation, MLR. Our study revealed that everolimus strongly inhibited cell proliferation in the MLR but this inhibition was not significantly enhanced in presence of 1 mg/ml IVIG and the inhibitory effect of 5 and 10 mg/ml IVIG was not significantly enhanced by everolimus at most concentrations. Although addition of 10 and 50 ng/ml everolimus increased the inhibitory effect of 5 and 10 mg/ml IVIG (Fig. 1), the additional inhibition was only 10–27%, much lower than 62 and 67% inhibition by everolimus alone at these concentrations. In addition, we found that everolimus at 0.1–50 ng/ml did not induce apoptosis in the MLR, or increase apoptosis induced by IVIG (Fig. 2). Inhibitory effect of everolimus on cell proliferation found in this study is consistent with the previous report showing dose-dependent inhibition of MLR by rapamycin [19]. This result is not surprising because the structure and immunosuppressive activity of everolimus is similar to that of parent drug rapamycin, except that everolimus possess hydroxyethyl group at position 40 where rapamycin has hydroxy group only [6]. However, the binding site on everolimus to its target of immunophilin remains unchanged which enables the everolimus-FKBP12 complex to bind and inhibit the kinase activity
of mTOR, which ultimately leads to the inhibition of immune cell proliferation. Our finding of no or minimal additive effect of the everolimus and IVIG combination on the inhibition of MLR was in contrast to the additive activity observed between rapamycin and IVIG as previously reported [19]. As single agents, both everolimus and rapamycin showed inhibitory effect on cell proliferation, but in combination with IVIG, they showed different results. It is possible that this observed dissimilarity could be attributable to the differences in pharmacokinetics and interaction with IVIG. The replacement of hydrogen with hydroxyethyl group at position 40 renders everolimus with favorable pharmacokinetic characteristics such as enhanced solubility thus improved bioavailability and shorter elimination half-life and thereby rapid onset of steady state, but all of these could potentially contribute to drug–drug interaction when administered together with IVIG, as compared to parent compound, rapamycin. Recent studies on the pharmacokinetics of rapamycin reported that rapamycin increases the exposure of cyclosporine by 2fold and that the concentration of rapamycin had been increased as well when administered concomitantly [8, 20]. Further, they speculate that these changes in the bioavailability of these compounds could be attributable to the fact that both rapamycin and cyclosporine are substrates for enzyme CYP3A4 and efflux pump P-glycoprotein. Although we don't have an exact answer, these studies indirectly indicate that it is possible that IVIG, especially at higher concentration (5–10 mg/ml), could limit the bioavailability of everolimus and lead to the abrogation of the inhibitory effects on cell proliferation. This may also be a singular characteristic of in vitro studies. Further analysis is required to address this issue. The finding that everolimus did not induce apoptosis in B- and Tcells was consistent with that of rapamycin [19]. We also found that everolimus did not alter apoptosis induced by IVIG, which is also consistent with our previous report for the combination of rapamycin and IVIG [19]. The effect of everolimus on apoptosis induction is controversial. Lutz et al. have shown everolimus-dependent apoptosis of tubular epithelial cells in an animal model of chronic allograft nephropathy [21]. However, several investigators reported that treatment of lung carcinoma and ovarian cancer cell lines with
Fig. 2. The effect of everolimus, IVIG and a combination of everolimus and IVIG on induction of B cell and T cell apoptosis in the MLR as detected by Annexin V and TUNEL assays. Twoway MLR was performed with various concentrations of everolimus with or without 5 mg/ml IVIG. Cells were stained for CD19 (A, C) or CD3 (B, D) and Annexin V (A, B) or TUNEL (C, D) followed by flow cytometric analysis. CD3+ or CD19+ cells were first gated, and then Annexin V+ or TUNEL+ cell number was calculated in each cell population and the results are expressed as Annexin V+ or TUNEL+ cell% in each cell population. Mean ± SD of 9 and 6 MLR pairs for Annexin V or TUNEL staining are shown, respectively. *p b 0.01 vs. without IVIG at 0 ng/ml everolimus.
N. Amet et al. / Transplant Immunology 23 (2010) 170–173
everolimus alone did not induce apoptosis, although combination of everolimus with cisplatin led to apoptosis and augmented the effect of chemotherapeutic agents [22–24]. These results are supportive of our conclusions. Taken together, it is possible that everolimus might act differently in normal human PBMCs than those of non-immune cells included in other studies [21–24]. We have previously shown that IVIG inhibits cell proliferation in the MLR [16], and that IVIG induces apoptosis primarily in B cells and to a lesser degree in T cells and monocytes [17]. Incubation with IVIG in the MLR also reduced the expression of MHC Class II and costimulatory molecules. We suggested that reduction of intact B cell and monocyte cell numbers, modulation of surface molecule expression on B cells, and deletion of B and T cells by apoptosis could result in inhibition of optimal T cell activation. This may be one of the mechanisms responsible for IVIG suppression of the MLR, and account for many of the observed beneficial effects of IVIG seen in patients. In this study, everolimus did not induce apoptosis, but did not interfere with apoptosis induced by IVIG, which might be beneficial if both drugs are combined. A combination of these drugs in an in vivo model would be of interest to determine if there are synergistic effects on T cell activation and antibody production. In conclusion, we showed that everolimus is a potent inhibitor of human immune cell proliferation but it does not act additively or synergistically with IVIG to inhibit immune cell proliferation. In addition, a possible unfavorable interaction between everolimus and high concentration of IVIG was suggested. Additionally, we found that everolimus did not induce apoptosis either alone or together with IVIG in immune cells, but did not interfere with apoptosis induced by IVIG. Future investigation of combinations of everolimus and IVIG in animal models could clarify the impact of this combination of T-cell and B-cell modulation. Acknowledgement
[5]
[6]
[7] [8] [9]
[10]
[11] [12]
[13]
[14]
[15] [16]
[17]
[18]
[19]
This study was supported in part by Novartis Pharmaceuticals Corp. [20]
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cyclosporine in de novo heart transplant recipients. J Heart Lung Transplant 2007;26:700–4. Lorber MI, Ponticelli C, Whelchel J, Mayer HW, Kovarik J, Li Y, et al. Therapeutic drug monitoring for everolimus in kidney transplantation using 12-month exposure, efficacy, and safety data. Clin Transplant 2005;19:145–52. Schuler W, Sedrani R, Cottens S. H‰berlin B, Schulz M, Schuurman HJ, et al. SDZ RAD, a new rapamycin derivative: pharmacological properties in vitro and in vivo. Transplantation 1997;64:36. Augustine JJ, Hricik DE. Experience with everolimus. Elsevier; 2004. p. 500–3. Vignot S, Faivre S, Aguirre D, Raymond E. mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol 2005;16:525–37. Boulay A, Zumstein-Mecker S, Stephan C, Beuvink I, Zilbermann F, Haller R. Antitumor efficacy of intermittent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells; 2004. p. 252–61. AACR. Vitko S, Tedesco H, Eris J, Pascual J, Whelchel J, Magee JC, et al. Everolimus with optimized cyclosporine dosing in renal transplant recipients: 6-month safety and efficacy results of two randomized studies. Am J Transplant 2004;4:626. Kazatchkine MD, Kaveri SV. Immunomodulation of autoimmune and inflammatory diseases with intravenous immune globulin. N Engl J Med 2001;345:747–55. Jordan SC, Tyan D, Stablein D, McIntosh M, Rose S, Vo A. Evaluation of intravenous immunoglobulin as an agent to lower allosensitization and improve transplantation in highly sensitized adult patients with end-stage renal disease: report of the NIH IG02 trial. Am Soc Nephrol 2004:3256–62. Vo AA, Lukovsky M, Toyoda M, Wang J, Reinsmoen NL, Lai CH, et al. Rituximab and intravenous immune globulin for desensitization during renal transplantation. N Engl J Med 2008;359:242. Stanley CJ, Ashley AV, Mieko T, Dolly T, Cynthia CN. Post-transplant therapy with high-dose intravenous gammaglobulin: applications to treatment of antibodymediated rejection. Pediatr Transplant 2005;9:155–61. Jordan SC, Toyoda M, Vo AA. Intravenous immunoglobulin a natural regulator of immunity and inflammation. Transplantation 2009;88:1. Toyoda M, Zhang XM, Petrosian A, Wachs K, Moudgil A, Jordan SC. Inhibition of allospecific responses in the mixed lymphocyte reaction by pooled human gamma-globulin. Transpl Immunol 1994;2:337. Jordan SC, Vo A, Bunnapradist S, Toyoda M, Peng A, Puliyanda D, et al. Intravenous immune globulin treatment inhibits crossmatch positivity and allows for successful transplantation of incompatible organs in living-donor and cadaver recipients. Transplantation 2003;76:631–6. Bayry J, Bansal K, Kazatchkine MD, Kaveri SV. DC-SIGN and 2, 6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells. Proc Nat Acad Sci 2009;106:E24. Toyoda M, Petrosyan A, Pao A, Jordan SC. Immunomodulatory effects of combination of pooled human gammaglobulin and rapamycin on cell proliferation and apoptosis in the mixed lymphocyte reaction. Transplantation 2004;78: 1134. Mahalati K, Kahan BD. Clinical pharmacokinetics of sirolimus. Clin Pharmacokinet 2001;40:573. Lutz J, Zou H, Liu S, Antus B, Heemann U. Apoptosis and treatment of chronic allograft nephropathy with everolimus. Transplantation 2003;76:508. Beuvink I, Boulay A, Fumagalli S, Zilbermann F, Ruetz S, OíReilly T, et al. The mTOR inhibitor RAD001 sensitizes tumor cells to DNA-damaged induced apoptosis through inhibition of p21 translation. Cell 2005;120:747–59. Mabuchi S, Altomare DA, Cheung M, Zhang L, Poulikakos PI, Hensley HH, et al. RAD001 inhibits human ovarian cancer cell proliferation, enhances cisplatininduced apoptosis, and prolongs survival in an ovarian cancer model. Clin Cancer Res 2007;13:4261. Strauss WOaK-MD G. Induction of apoptosis and modulation of activation and effector function in T cells by immunosuppressive drugs. Clin Exp Immunol 2002;128:255–66.
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
In vitro effects of everolimus and intravenous immunoglobulin on cell proliferation and apoptosis induction in the mixed lymphocyte reaction Nurmamet Amet a, Mercedes Gacad a, Anna Petrosyan a, Andy Pao a, Stanley C. Jordan b, Mieko Toyoda a,⁎ a Transplant Immunology Laboratory, Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, 8700 Beverly Blvd., SSB111, Los Angeles, CA 90048, United States b Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA School of Medicine, 8700 Beverly Blvd., suite 490W, Los Angeles, CA 90048, United States
a r t i c l e
i n f o
Article history: Received 11 June 2010 Accepted 28 June 2010 Keywords: Everolimus Intravenous immunoglobulin Synergistic effect Cell proliferation Apoptosis induction Mixed lymphocyte reaction
a b s t r a c t Targeting multiple pathways in the activation of alloimmune responses by multi-drug immunosuppressive regimens with complementary mechanisms of action enhances allograft survival and improves quality of life, owing to the reduction of adverse drug effects. In this report we investigated the effect of the combination of everolimus and intraveneous immunoglobulin (IVIG) on cell proliferation and apoptosis induction in human two-way mixed lymphocyte reaction (MLR). Everolimus alone (0.1–50 ng/ml) and IVIG (1–10 mg/ml) alone inhibited cell proliferation in a dose-dependent manner (16.4–67.2% and 12.1–66.3% inhibition, respectively). The inhibition by everolimus was not enhanced in the presence of 1 mg/ml IVIG. Addition of 10 and 50 ng/ml everolimus increased the inhibitory effect of 5 and 10 mg/ml IVIG, but only by 10–27%. Addition of 0.1 and 1 ng/ml everolimus did not increase IVIG's inhibitory effects. Apoptosis was significantly higher in IVIG (5 mg/ml)-treated CD19+ cells and less so in CD3+ cells as assessed by Annexin V and TUNEL assays. However, everolimus (0.1–50 ng/ml) did not induced apoptosis or alter apoptosis induced by IVIG. These results suggest that everolimus is a potent inhibitor of immune cell proliferation but does not act additively or synergistically with IVIG when analyzed in this in vitro system. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Transplantation has become a standard treatment option in patients with end-stage renal disease as it enhances quality of life, and dramatically reduces cost associated with dialysis. Immunosuppressive drugs that have been approved for use in clinical transplantation suffer dose-limiting toxic side-effects, including nephrotoxicity and neurotoxicity [1–3]. These adverse effects can be reduced or even avoided by using drug combinations with additive or synergistic activity, which allows each drug to be used at lower dose and thus decreases the adverse effects [4, 5]. Everolimus (Zortress®), a natural product of streptomyces hygroscopicus and analog of rapamycin, possesses favorable pharmacokinetic properties with enhanced solubility and faster steady state [6, 7] compared to rapamycin [8]. These properties allowed everolimus to be formulated as an oral agent, while maintaining rapamycin-like immunosuppressive and anti-neoplastic activities [2, 9]. After forming complexes with FK506-binding protein (FKBP-12) in the cytoplasm of target cells such as T cells and B cells, everolimus binds and prevents the kinase activity of mammalian target of rapamycin (mTOR). The ⁎ Corresponding author. Transplant Immunology Laboratory, SSB111, Dept. of Medicine, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, United States. Tel.: + 1 310 423 4975; fax: + 1 310 423 0268. E-mail address: [email protected] (M. Toyoda). 0966-3274/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2010.06.012
deactivation of mTOR activity by everolimus leads to the blockade of growth factor-dependent signal transduction, cell cycle arrest at G1 phase, and prevention of cell proliferation. Everolimus was approved in 2003 by the European Medicines Agency for use in the prophylaxis of organ transplantation in combination with cyclosporine and corticosteroids. The Food and Drug Administration (FDA) approved everolimus for use in kidney transplant patients in the United States for the prevention of organ rejection in April 2010. Everolimus (afinitor) has also been recently been approved for the treatment of advanced kidney cancer. Clinical trials evaluating the efficacy of everolimus in both heart and kidney transplant patients have shown promising results; it reduces the exposure to cyclosporine and incidence of cardiac-allograft vasculopathy [2, 4] and provides better outcome for kidney transplant patients while lowering the dose of cyclosporine [1, 10]. Intravenous immunoglobulin (IVIG) has been widely used for treatment of autoimmune and systemic inflammatory disorders [11]. We have shown that IVIG treatment reduced allo-sensitization and increased transplant rate in highly HLA-sensitized (HS) patients [12, 13]. We have also reported the successful use of IVIG for treatment of antibody-mediated allograft rejection [14]. These beneficial effects observed in IVIG-treated transplant patients are likely due to a number of recently described immunomodulatory effects of IVIG on T cells, dendritic cells, inhibition of B cell activation and antibody production, induction of anti-inflammatory cytokines, blocking of
N. Amet et al. / Transplant Immunology 23 (2010) 170–173
anti-HLA antibodies by anti-idiotypic antibodies contained in IVIG and blocking of complement-mediated injury through inhibition of C3 activation [14, 15]. Previous studies from our lab have shown that IVIG inhibits cell proliferation in the mixed lymphocyte reaction (MLR) [16] and that IVIG induces apoptosis primarily in B cells and to a lesser degree in T cells and monocytes [17]. IVIG also reduced the expression of MHC class II and costimulatory molecules, CD40 and CD80/86 in the MLR [17]. We suggested that the reduction of intact B cell and monocyte cell numbers, modulation of surface molecule expression on B cells, and deletion of B and T cells by apoptosis may result in inhibition of optimal T cell activation. This may be one of the mechanisms responsible for suppression of the MLR by IVIG and account for many of the beneficial effects observed in IVIG-treated HS patients and those with autoimmune and inflammatory disorders. Other investigators have shown that IVIG inhibits dendritic cell activation and costimulation by inhibition of CD40 and MHC class I/II expression [18]. In a previous study, we had shown the additive effect of combination of IVIG and rapamycin on the inhibition of cell proliferation in the MLR [19] and suggested the possible utility of this combination in transplant patients, especially HS patients where additive effects for desensitization might be desired. Since everolimus is an analog of rapamycin, the combination of everolimus and IVIG might be another option in a multi-drug regimen. Here, we examined possible additive or synergistic effects of the combination of everolimus and IVIG on inhibition of immune cell proliferation and apoptosis induction in the MLR. The aim would be to determine if findings would support the use of this combination in desensitization protocols or treatment of antibody-mediated rejection (AMR). 2. Materials and methods
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2.3. Detection of apoptosis After 3 day incubation, cells were enumerated and 1 × 106 cells were used for down-stream processing. Cells were washed with FACS buffer consisting of 1 × PBS, 1% fetal bovine serum (FBS) and 0.1% sodium azide, centrifuged for 10 min at 300 g, and then double stained with 5 μl PE-anti-CD3 antibody and 5 μl PE-Cy7-anti-CD19 antibody for 30 min on ice in the dark. After that, cells were washed, re-suspended in 900 μl FACS buffer, which was then split into 100 μl for Annexin V assay and 800 μl for TUNEL assay, as described previously [17, 19]. Briefly, Annexin V staining was accomplished by incubating cells with 5 μl FITC-Annexin V and 5 μl of 7AAD in the dark for 15 min, and TUNEL staining by incubating cells with 50 μl of FITCTUNEL reagent for 1 h in a humidified 37 °C incubator, followed by detection of apoptotic cells by a CyAn Flow Cytometer (Beckman Coulter, Fullerton, CA) and Summit software program (Beckman Coulter). The final results were expressed as the percentage of cells positive for Annexin V or TUNEL in the CD3+ and CD19+ cell populations. Data represent average ± standard deviation, with 9 different pairs of MLRs for Annexin V and 6 different pairs of MLRs for TUNEL staining. 2.4. Statistical analysis The effect of everolimus and/or IVIG on cell proliferation and apoptosis induction was assessed by a standard paired two-tailed ttest. P value b0.05 was considered as significant difference. 3. Results 3.1. The inhibitory effect of combination of Everolimus and IVIG on cell proliferation in the MLR
2.1. Reagents Everolimus (RAD001), provided by Novartis Pharmaceuticals Corp., East Hanover, NJ, was dissolved in absolute ethanol to prepare 10 mM stock solution and kept at −20 °C. The dilutions of everolimus for each experiment were made fresh in the culture media from the stock solution. IVIG was provided by Talecris Biotherapeutis Inc. (Raleigh, NC), either as a 100 mg/ml in 0.2 M glycine or 50 mg/ml in 10% maltose, and kept at 4 °C. Fresh dilutions of IVIG were prepared in cell culture media for each experiment. Glycine- or maltose-based IVIG was used in the assessment of apoptosis or cell proliferation, respectively. Monoclonal antibodies including PE-conjugated CD3 (Invitrogen Inc., Carlsbad, CA) and PE-Cy7-conjugated CD19 (eBioscience Inc., San Diego, CA) were used simultaneously to stain cell surface molecules expressed by T and B cells, respectively. Annexin V Apoptosis Detection Kit (BD Bioscience, San Jose, CA) was used to detect both early and late events of apoptosis, whereas the deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) commercial kit from Roche Diagnostics Corp. (Indianapolis, IN) was used to detect late stage apoptosis.
Both everolimus and IVIG alone inhibited cell proliferation in a dose-dependent manner (Fig. 1). The inhibition was 16.4, 44.6, 62.0, 67.2% for 0.1, 1, 10, 50 ng/ml everolimus, and 12.1, 50.9, 66.3% for 1, 5, 10 mg/ml IVIG, respectively. Addition of 1 mg/ml IVIG did not change the inhibition by everolimus at any of the tested concentrations. Addition of 0.1 and 1 ng/ml everolimus did not significantly increase the inhibitory effect of IVIG at 5 and 10 mg/ml, despite everolimus alone showing 16.4 and 44.6% inhibition at these concentrations, respectively. Addition of 10 and 50 ng/ml everolimus significantly increased the inhibitory effect of 5 mg/ml IVIG by 10.4% and 27.4%,
2.2. Cell culture The 2-way MLR was performed as previously reported [19]. Briefly, whole blood was drawn from unrelated healthy individuals to prepare peripheral blood mononuclear cells (PBMC) using the Ficoll–Hypaque (Sigma, St. Louis, MO) gradient centrifugation method. PBMCs from the two individuals were mixed at 1:1 (1× 106/ml of each PBMCs for cell proliferation and 2 × 106/ml for apoptosis experiments) with everolimus at 0, 0.1, 1, 10, 50 ng/ml, or combined with IVIG at 1, 5 or 10 mg/ ml, and then incubated for 6 days for measurement of cell proliferation by 3H-thymidine uptake and for 3 days for detection of apoptosis. The results of cell proliferation are expressed as a percentage of the cpm in the control condition without additives (100%).
Fig. 1. The effects of everolimus, IVIG or a combination of everolimus and IVIG on cell proliferation in the MLR. The two-way MLR was performed with various concentrations of everolimus and/or IVIG. The results of cell proliferation are expressed as a percentage of the cpm in the control condition without additives (100%). Mean ± standard deviation (SD) of six MLR pairs is shown, respectively. Mean values of cpm of controls without additives in Fig. 1 were 18234 ± 4955. *p b 0.02 vs. without everolimus at each IVIG concentration. #p b 0.02 vs. without IVIG at 0 ng/ml everolimus.
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respectively, compared to that by IVIG alone, and addition of 50 ng/ml everolimus to 10 mg/ml IVIG did so by 10.7%. 3.2. The effect of combination of everolimus and IVIG on apoptosis induction in the MLR Five mg/ml IVIG alone significantly increased apoptosis induction by 86.3% and 85.2% in B cells and 34.0% and 8.1% in T cells as assessed by Annexin V and TUNEL staining, respectively (Fig. 2), which is consistent with our previous study [19]. In contrast, 0.1–50 ng/ml everolimus alone did not induce apoptosis in both B cells and T cells, and had no effect on apoptosis induced by IVIG. 4. Discussion In this study, we assessed the combination of everolimus and IVIG on cell proliferation and apoptosis induction in an in vitro model of transplantation, MLR. Our study revealed that everolimus strongly inhibited cell proliferation in the MLR but this inhibition was not significantly enhanced in presence of 1 mg/ml IVIG and the inhibitory effect of 5 and 10 mg/ml IVIG was not significantly enhanced by everolimus at most concentrations. Although addition of 10 and 50 ng/ml everolimus increased the inhibitory effect of 5 and 10 mg/ml IVIG (Fig. 1), the additional inhibition was only 10–27%, much lower than 62 and 67% inhibition by everolimus alone at these concentrations. In addition, we found that everolimus at 0.1–50 ng/ml did not induce apoptosis in the MLR, or increase apoptosis induced by IVIG (Fig. 2). Inhibitory effect of everolimus on cell proliferation found in this study is consistent with the previous report showing dose-dependent inhibition of MLR by rapamycin [19]. This result is not surprising because the structure and immunosuppressive activity of everolimus is similar to that of parent drug rapamycin, except that everolimus possess hydroxyethyl group at position 40 where rapamycin has hydroxy group only [6]. However, the binding site on everolimus to its target of immunophilin remains unchanged which enables the everolimus-FKBP12 complex to bind and inhibit the kinase activity
of mTOR, which ultimately leads to the inhibition of immune cell proliferation. Our finding of no or minimal additive effect of the everolimus and IVIG combination on the inhibition of MLR was in contrast to the additive activity observed between rapamycin and IVIG as previously reported [19]. As single agents, both everolimus and rapamycin showed inhibitory effect on cell proliferation, but in combination with IVIG, they showed different results. It is possible that this observed dissimilarity could be attributable to the differences in pharmacokinetics and interaction with IVIG. The replacement of hydrogen with hydroxyethyl group at position 40 renders everolimus with favorable pharmacokinetic characteristics such as enhanced solubility thus improved bioavailability and shorter elimination half-life and thereby rapid onset of steady state, but all of these could potentially contribute to drug–drug interaction when administered together with IVIG, as compared to parent compound, rapamycin. Recent studies on the pharmacokinetics of rapamycin reported that rapamycin increases the exposure of cyclosporine by 2fold and that the concentration of rapamycin had been increased as well when administered concomitantly [8, 20]. Further, they speculate that these changes in the bioavailability of these compounds could be attributable to the fact that both rapamycin and cyclosporine are substrates for enzyme CYP3A4 and efflux pump P-glycoprotein. Although we don't have an exact answer, these studies indirectly indicate that it is possible that IVIG, especially at higher concentration (5–10 mg/ml), could limit the bioavailability of everolimus and lead to the abrogation of the inhibitory effects on cell proliferation. This may also be a singular characteristic of in vitro studies. Further analysis is required to address this issue. The finding that everolimus did not induce apoptosis in B- and Tcells was consistent with that of rapamycin [19]. We also found that everolimus did not alter apoptosis induced by IVIG, which is also consistent with our previous report for the combination of rapamycin and IVIG [19]. The effect of everolimus on apoptosis induction is controversial. Lutz et al. have shown everolimus-dependent apoptosis of tubular epithelial cells in an animal model of chronic allograft nephropathy [21]. However, several investigators reported that treatment of lung carcinoma and ovarian cancer cell lines with
Fig. 2. The effect of everolimus, IVIG and a combination of everolimus and IVIG on induction of B cell and T cell apoptosis in the MLR as detected by Annexin V and TUNEL assays. Twoway MLR was performed with various concentrations of everolimus with or without 5 mg/ml IVIG. Cells were stained for CD19 (A, C) or CD3 (B, D) and Annexin V (A, B) or TUNEL (C, D) followed by flow cytometric analysis. CD3+ or CD19+ cells were first gated, and then Annexin V+ or TUNEL+ cell number was calculated in each cell population and the results are expressed as Annexin V+ or TUNEL+ cell% in each cell population. Mean ± SD of 9 and 6 MLR pairs for Annexin V or TUNEL staining are shown, respectively. *p b 0.01 vs. without IVIG at 0 ng/ml everolimus.
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everolimus alone did not induce apoptosis, although combination of everolimus with cisplatin led to apoptosis and augmented the effect of chemotherapeutic agents [22–24]. These results are supportive of our conclusions. Taken together, it is possible that everolimus might act differently in normal human PBMCs than those of non-immune cells included in other studies [21–24]. We have previously shown that IVIG inhibits cell proliferation in the MLR [16], and that IVIG induces apoptosis primarily in B cells and to a lesser degree in T cells and monocytes [17]. Incubation with IVIG in the MLR also reduced the expression of MHC Class II and costimulatory molecules. We suggested that reduction of intact B cell and monocyte cell numbers, modulation of surface molecule expression on B cells, and deletion of B and T cells by apoptosis could result in inhibition of optimal T cell activation. This may be one of the mechanisms responsible for IVIG suppression of the MLR, and account for many of the observed beneficial effects of IVIG seen in patients. In this study, everolimus did not induce apoptosis, but did not interfere with apoptosis induced by IVIG, which might be beneficial if both drugs are combined. A combination of these drugs in an in vivo model would be of interest to determine if there are synergistic effects on T cell activation and antibody production. In conclusion, we showed that everolimus is a potent inhibitor of human immune cell proliferation but it does not act additively or synergistically with IVIG to inhibit immune cell proliferation. In addition, a possible unfavorable interaction between everolimus and high concentration of IVIG was suggested. Additionally, we found that everolimus did not induce apoptosis either alone or together with IVIG in immune cells, but did not interfere with apoptosis induced by IVIG. Future investigation of combinations of everolimus and IVIG in animal models could clarify the impact of this combination of T-cell and B-cell modulation. Acknowledgement
[5]
[6]
[7] [8] [9]
[10]
[11] [12]
[13]
[14]
[15] [16]
[17]
[18]
[19]
This study was supported in part by Novartis Pharmaceuticals Corp. [20]
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