Pharmacology, Biochemistry and Behavior 126 (2014) 73–76
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Short-term treatment with the calcineurin inhibitor cyclosporine A decreases HPA axis activity and plasma noradrenaline levels in healthy male volunteers Antje Albring a,1 , Laura Wendt a,1, Nino Harz a, Harald Engler a, Benjamin Wilde b, Oliver Witzke b,⁎, Manfred Schedlowski a a b
Institute of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany Department of Nephrology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
a r t i c l e
i n f o
Article history: Received 2 June 2014 Received in revised form 29 August 2014 Accepted 7 September 2014 Available online 16 September 2014 Keywords: Calcineurin inhibitor Cyclosporine A Neuropsychological changes Neuroendocrine parameters
a b s t r a c t Treatment with the selective calcineurin inhibitor and immunosuppressive drug cyclosporine A (CsA) is associated with neurotoxicity and neurocognitive impairments. Whether and to what extent CsA is inducing alterations of the neuroendocrine status is unknown so far. Therefore, the present study investigated the effect of short-term CsA treatment on hypothalamus–pituitary–adrenal (HPA) axis activity and catecholamine release as well as state anxiety in healthy male subjects. Treatment with CsA significantly reduced plasma concentrations of adrenocorticotropic hormone (ACTH), cortisol, and noradrenaline whereas adrenaline levels and state anxiety remained unaffected. Future studies should analyze the mechanisms of CsA-induced effects on neuroendocrine variables, neurocognitive functions and mood. © 2014 Published by Elsevier Inc.
1. Introduction The calcineurin inhibitor and immunosuppressive drug cyclosporine A (CsA) is widely employed in clinical practice, in particular in transplantation medicine for prevention and treatment of graft rejection (Olyaei et al., 2001; Merville, 2005; Boots et al., 2004). However, apart from its immunosuppressive properties, chronic treatment with CsA is associated with neurotoxicity and an increased prevalence of developing neuropsychiatric symptoms (Tombazzi et al., 2006; Lindenfeld et al., 2004; Wijdicks, 2001; de Groen et al., 1987; Palmer and Toto, 1991). In up to 20% of renal transplant patients, CsA administration is accompanied with neurological adverse effects such as tremor. Other, rare complications include depression, somnolence and burning paresthesia (Kahan et al., 1987). In addition, clinical evidence documents that 10 to 28% of patients receiving CsA experience restlessness, neuralgia and peripheral neuropathy which are possibly caused by neurotoxicity (Bechstein, 2000; Miller, 1996). The mechanisms which are responsible for the adverse neuropsychological effects after CsA treatment are largely unknown. However,
⁎ Corresponding author at: Department of Nephrology, University Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany. Tel.: +49 201 7233394; fax: +49 201 7233393. E-mail address: [email protected] (B. Wilde). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.pbb.2014.09.006 0091-3057/© 2014 Published by Elsevier Inc.
observations from animal experiments document that even acute, short-term treatment with CsA affects neural activity and behavior. Acute peripheral administration of CsA reduced activity in the open field, decreased rearing activity and reduced social interaction and motor activity while augmenting anxiety-related behavior in rodents (von Horsten et al., 1998; Sato et al., 2007). These CsA-induced behavioral effects are paralleled by observations in rats, where a single intraperitoneal injection of CsA increased neuronal activity in the insular cortex and the amygdala and enhanced amygdaloid noradrenaline release (Pacheco-López et al., 2013). Whether and to what extent these neurobehavioral changes after acute administration of CsA are associated with CsA-induced changes in HPA-axis activity and/or alterations in catecholamine release is unknown. Sympathetic activation following CsA treatment has been repeatedly documented in rodents and humans (Sander et al., 1996; Lyson et al., 1994; Morgan et al., 1991; Scherrer et al., 1990; Grobecker et al., 1995; Shimizu et al., 2001; Tavares et al., 2003). However, studies investigating the effect of CsA on the production of catecholamines remain contradictory (Reis et al., 2000; Gerkens, 1989; Duruibe et al., 1990) and only a study in rats reported a reduced activity of the hypothalamic–pituitary–adrenal (HPA) axis, reflected through reduced plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone after CsA treatment (Stephanou et al., 1992). Therefore, the present study analyzed the effect of short-term CsA treatment on cellular immune functions, neuroendocrine parameters and state anxiety in healthy male volunteers.
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A. Albring et al. / Pharmacology, Biochemistry and Behavior 126 (2014) 73–76
2. Materials and methods 2.1. Subjects Sixty healthy male volunteers (age range: 18–40 years) were recruited through public advertisement in the surrounding community. All volunteers underwent an extensive physical and psychiatric assessment (self-reported questionnaires, interview about their medical history) as well as an electrocardiogram and ultrasonography of the kidneys performed and evaluated by the physicians of the Department of Nephrology. Subjects were excluded if one of the following criteria was identified: daily intake of medication, blood donations of N200 ml within the last two months, previous participation in pharmacological studies or other medical exclusion criteria (e.g., disorders of the immune or endocrine system, previous or persistent psychiatric disorders, addiction, allergies, signs of cardiovascular, hematologic or nephrologic disorders, respiratory problems and diabetes mellitus). The baseline liver values were within the normal range (GOT: 23 ± 8 U/l, GPT: 26 ± 10 U/l, GGT: 20 ± 12 U/l, bilirubin (total): 0.7 ± 0.3 mg/dl).
2.2. Study design Subjects received four oral doses of 2.5 mg/kg body weight CsA (Sandimmun® Optoral, Novartis) on three consecutive days (day 1 at 6 pm, day 2 at 8 am and 6 pm, day 3 at 8 am). Blood samples were drawn on day 1 (10 am) for baseline measurements and on day 3 (10 am), 2 h after the last CsA intake (CsA) to analyze the pharmacological effect of CsA on neuroendocrine and immunological variables. A last blood sample was drawn on day 8 at 10 am, five days after the last CsA intake (washout). State anxiety (STAI) (Spielberger et al., 1970) was assessed at each blood withdrawal in order to document a possible effect of short-term CsA treatment on state anxiety levels.
2.3. Endocrine parameters Plasma noradrenaline and adrenaline concentrations were determined by high-performance liquid chromatography (HPLC) with electrochemical detection (ChromSystems, Instruments and Chemicals GmbH, Munich, Germany) as previously described (Leineweber et al., 2007). Plasma levels of ACTH and cortisol were determined using enzyme-linked immunosorbent assays (ACTH and Cortisol ELISA, IBL International, Hamburg, Germany) according to the test protocol of the manufacturer and were analyzed on a Fluostar OPTIMA Microplate Reader (BMG Labtech, Offenbach, Germany). The detection limits were 0.22 pg/ml (ACTH) and 0.3 ng/ml (cortisol), respectively.
2.4. Measurement of CsA blood concentrations CsA concentrations in whole blood were assessed using Siemens Dimension Flex reagent cartridge (Cruinn Diagnostics Ltd., Dublin, Ireland) according to the manufacturer's recommendations.
2.5. Cell isolation Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Ficoll-Paque™ Plus, GE Healthcare, Munich, Germany) according to the manufacturer's protocol. Cells were washed with Hanks' Balanced Salt Solution (Life Technologies, Darmstadt, Germany), counted with an automated hematology analyzer (KX-21N, Sysmex Deutschland GmbH, Norderstedt, Germany) and adjusted to 2.5 × 106 and 1.25 × 106 cells/ml in cell culture medium (RPMI 1640 supplemented with GlutaMAX I, 25 mM Hepes, 10% fetal bovine serum, 50 μg/ml gentamicin; Life Technologies, Darmstadt, Germany).
2.5.1. IL-2 mRNA expression analysis PBMCs (2.5 × 106 cells/ml) were stimulated with 40 ng/ml of soluble mouse anti-human CD3 monoclonal antibody (clone: HIT3a; BD Pharmingen) for 4 h (37 °C, 5% CO2). Total RNA was extracted using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Single-stranded cDNA was synthesized using a high capacity cDNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). Real-time quantitative PCR was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems) using Fast qPCR MasterMix Plus Low Rox (Eurogentec, Cologne, Germany) and the following cycling conditions: 5 min at 95 °C followed by 40 cycles of 3 s at 95 °C, 20 s at 60 °C and 26 s at 72 °C. Primers (forward: 5′-CCAGGA TGCTCACATTTAAGTTTTAC-3′; reverse: 5′-GAGGTTTG AGTTCTTC TTCTAGACACTGA-3) and probe (5′–6-FAM-TGCCCAAG AAGGCCACAGAACTGAA-BHQ1–3′) were designed using Primer Express 3.0 software (Applied Biosystems) and were purchased from Eurogentec (Cologne, Germany). For quantification of IL-2 mRNA expression, serially diluted cDNA samples generated from purified specific PCR products (High Pure PCR Product Purification Kit, Roche Diagnostics, Mannheim, Germany) were used as external standards in each run. Results are expressed as fg/μg total RNA. 2.6. Determination of IL-2 in culture supernatant PBMC suspensions (2. 5 × 106 cells/ml) were transferred to 96-well flat-bottom tissue culture plates and were stimulated with 20 ng/ml of soluble mouse anti-human CD3 monoclonal antibody (clone: HIT3a; BD Pharmingen, San Diego, CA) for 24 h (37 °C, 5% CO2). Concentration of IL-2 in culture supernatants was quantified using a commercial ELISA (Human IL-2 ELISA MAX Deluxe, BioLegend, San Diego, CA) according to the manufacturer's instructions. 2.7. Proliferation analysis The percentage of proliferating CD4+ T cells was measured by flow cytometry using the Click-iT EdU cell proliferation assay (Invitrogen, Darmstadt, Germany) according to the manufacturer's instructions. Briefly, PBMCs (1.25 × 106 cells/ml) were stimulated in 96-well round bottom tissue culture plates with 2.5 μg/ml of soluble mouse antihuman CD3 monoclonal antibody (clone: HIT3a; BD Pharmingen) for 72 h (37 °C, 5% CO2). The thymidine analog 5-ethynyl-2′-deoxyuridine (EdU), which is incorporated during DNA synthesis, was added to the cells at a concentration of 10 μM for the last 48 h of culture. After incubation, cells were washed and stained with APC conjugated anti-human CD4 (clone RPA-T4, BD Pharmingen) antibody. Cells were fixed with 4% paraformaldehyde and permeabilized using a saponin-based permeabilization reagent. Afterward, cells were incubated with the Click-iT reaction cocktail. The percentage of proliferating CD4+ T cells was analyzed on a FACS Canto II flow cytometer using FACS Diva software (BD Immunocytometry Systems). 2.8. Statistical analysis Data were analyzed with analysis of variances (ANOVA) using PASW statistics (Version 18, SPSS, Chicago, IL, USA). Post hoc analyses were computed using paired t-tests. The level of significance was set at p b 0.05. All data are expressed as mean ± SEM. 3. Results Short-term CsA intake significantly increased CsA levels, however CsA was not detectable anymore after the washout phase of five days (Table 1). The immunopharmacological effectiveness of the CsA treatment was documented by a significant suppression in IL-2 mRNA expression and IL-2 concentration in culture supernatant of anti-CD3
A. Albring et al. / Pharmacology, Biochemistry and Behavior 126 (2014) 73–76 Table 1 CsA blood levels and immunological measures.
CsA (ng/ml) IL-2 (pg/ml) IL-2 mRNA (fg/μg total RNA) Proliferating CD4+ T cells (%)
Baseline
CsA peak level
Washout
n.d. 399.6 ± 44.6 198.9 ± 16.2 27.6 ± 1.0
1297 130.7 52.2 13.4
n.d. 393.8 ± 36.2 205.4 ± 13.6 27.6 ± 27.6
± ± ± ±
32.1*** 12.3*** 5.4*** 0.7***
Results of post hoc computed paired t-test are indicated (***p b 0.001). Data are presented as mean ± SEM. n.d. = not detectable.
stimulated PBMC as well as a reduced number of proliferating CD4+ T cells (Table 1). CsA intake significantly decreased the activity of the HPA axis as reflected by decreased plasma ACTH (F = 4.9; p b 0.01) and cortisol (F = 22.7, p b 0.001) levels (Fig. 1a). In addition, CsA significantly decreased plasma noradrenaline concentrations (F = 13.4; p b 0.001) without significantly affecting adrenaline levels (F = 0.9, n.s.) (Fig. 1b). Moreover, state anxiety scores remained unaffected by CsA treatment (baseline: 34.9 ± 0.9; CsA: 35.2 ± 1.0; washout: 33.6 ± 0.9; F = 2.8, n.s.). 4. Discussion Chronic treatment with calcineurin inhibitors such as CsA is accompanied by unwanted side effects in patients such as hypertension (Scherrer et al., 1990) and neurobehavioral disturbances (Kahan et al., 1987; Bechstein, 2000; Miller, 1996). The exact mechanisms responsible for these side effects are still unclear. Data from experimental animals indicate a direct effect of acute CsA administration on neurophysiological functions (Pacheco-López et al., 2013), however, data in humans are lacking. The results presented here reveal that short-term treatment with the calcineurin inhibitor and immunosuppressant CsA reduced the production of ACTH, cortisol and noradrenaline without affecting the
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release of adrenaline and state anxiety levels in healthy male subjects. Studies in rodents demonstrated anxiety-related behavior after acute CsA administration which was paralleled by increased neural activity in the insular cortex and amygdala (von Horsten et al., 1998; Sato et al., 2007; Pacheco-López et al., 2013). Our data however, show that short-term CsA treatment does not affect state anxiety in healthy humans as previously reported (Albring et al., 2014; Ober et al., 2012). In contrast, temporary intake of CsA decreased the activity of the HPA axis and the sympathetic nervous system, reflected by significantly inhibited production of ACTH, cortisol and noradrenaline, respectively. Data on the acute effects of CsA and calcineurin inhibitors in general on neuroendocrine functions are rare. Inhibitory effects of CsA on the activity of the HPA-axis have been shown in rats (Stephanou et al., 1992) and reduced secretion of the anterior pituitary hormones, thyroid-stimulating hormone, and prolactin was detected in patients with nephrotic syndrome (Nagai et al., 1992). The decreased plasma noradrenaline content after CsA intake was surprising as we rather expected sympathetic activation following CsA treatment. However, our observation is supported by a study with rats, documenting a decrease in noradrenaline plasma levels after treatment with 5 mg/kg CsA (Gerkens, 1989). The exact mechanisms through which CsA is affecting the neuroendocrine response are poorly understood. A direct penetration of peripheral administered CsA into the brain has been negated for a long time due to the limited transport of the drug across the blood brain barrier (Cefalu and Pardridge, 1985; Sakata et al., 1994; Shiga et al., 1992). However, recent data in animals indicate a direct CsA-mediated effect on the CNS (Sato et al., 2007). A single peripheral injection of CsA increased electric activity, c-Fos expression in the insular cortex and the amygdala and amygdaloid noradrenaline release. In addition, CsA levels where detectable in the cerebellum, insular cortex and amygdala 2 to 4 h after intraperitoneal injection of CsA (Pacheco-López et al., 2013). Moreover, acute CsA injection affected behavioral responses such as rearing and motor activity and anxiety (von Horsten et al., 1998; Sato et al., 2007). The decrease in noradrenaline concentrations together with the unaffected release of adrenaline levels observed in our study is indicating a centrally mediated CsA effect on the HPA axis and sympathetic nervous system, although in contrast to data in rodents, state anxiety levels remained unaffected. Clearly, further studies are needed to analyze the mechanisms of how acute administration of calcineurin inhibitors is affecting neurobehavioral and neuroendocrine responses. These data from rodents and healthy humans will increase our understanding about the unwanted neurological side effects of immunosuppressive regimens in patients. 5. Conclusion Cyclosporine A has significant impact on the neuroendocrine system and reduces the activity of the hypothalamus–pituitary–adrenal (HPA) axis. Ethics statement The study was approved by the local ethics committee for human investigations of the University Hospital Essen and follows the rules stated in the Declaration of Helsinki. All participants gave written informed consent prior to their inclusion in the study and were reimbursed for their participation. Acknowledgments
Fig. 1. (a) Plasma ACTH and cortisol levels at baseline, after intake of four oral doses of 2.5 mg/kg CsA, and after a washout phase of five days. (b) Plasma concentrations of adrenaline and noradrenaline at baseline, after intake of four oral doses of 2.5 mg/kg CsA, and after a washout phase of five days. Results of post hoc computed paired t-test are indicated (difference from baseline) (***p b 0.001; **p b 0.01; #p b 0.01). Data are presented as mean ± SEM.
We would like to thank Sonja Nissen, Zehra Yavuz, Christa Freundlieb, Alexandra Kornowski and Magdalene Vogelsang for their technical assistance and their skilled and friendly help. This work was supported by a grant of the German Research Foundation (FOR 1328; SCHE 341/17-1).
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References Albring A, Wendt L, Benson S, Nissen S, Yavuz Z, Engler H, et al. Preserving learned immunosuppressive placebo response: perspectives for clinical application. Clin Pharmacol Ther 2014;96:247–55. Bechstein WO. Neurotoxicity of calcineurin inhibitors: impact and clinical management. Transpl Int 2000;13:313–26. Boots JM, Christiaans MH, van Hooff JP. Effect of immunosuppressive agents on long-term survival of renal transplant recipients: focus on the cardiovascular risk. Drugs 2004; 64:2047–73. Cefalu WT, Pardridge WM. Restrictive transport of a lipid-soluble peptide (Cyclosporin) through the blood–brain-barrier. J Neurochem 1985;45:1954–6. de Groen PC, Aksamit AJ, Rakela J, Forbes GS, Krom RA. Central nervous system toxicity after liver transplantation: the role of cyclosporine and cholesterol. N Engl J Med 1987;317:861–6. http://dx.doi.org/10.1056/NEJM198710013171404. Duruibe VA, Okonmah A, Panton L, Blyden GT. Effect of cyclosporin A on rat kidney catecholamines. Life Sci 1990;47:255–61. Gerkens JF. Cyclosporine treatment of normal rats produces a rise in blood pressure and decreased renal vascular responses to nerve stimulation, vasoconstrictors and endothelin-dependent dilators. J Pharmacol Exp Ther 1989;250:1105–12. Grobecker HF, Riebel K, Wellenhofer T. Cyclosporine A-induced hypertension in SHR and WKY: role of the sympatho-adrenal system. Clin Exp Pharmacol Physiol Suppl 1995; 22:94–5. Kahan GD, Flechner SM, Lorber MI, Golden D, Conley S, Van Buren CT. Complication of cyclosporine–prednisone immunosuppression in 402 renal allograft recipients exclusively followed at a single center for from one to five years. Transplantation 1987;43: 197–204. Leineweber K, Bogedain P, Wolf C, Wagner S, Weber M, Jakob H-G, et al. In patients chronically treated with metoprolol, the demand of inotropic catecholamine support after coronary artery bypass grafting is determined by the Arg389Gly-beta 1-adrenoceptor polymorphism. Naunyn Schmiedebergs Arch Pharmacol 2007;375:303–9. http://dx. doi.org/10.1007/s00210-007-0166-6. Lindenfeld J, Miller GG, Shakar SF, Zolty R, Lowes BD, Wolfel EE, et al. Drug therapy in the heart transplant recipient: part II: immunosuppressive drugs. Circulation 2004;110: 3858–65. http://dx.doi.org/10.1161/01.CIR.0000150332.42276.69. Lyson T, McMullan DM, Ermel LD, Morgan BJ, Victor RG. Mechanisms of cyclosporineinduced sympathetic activation and acute hypertension in rats. Hypertension 1994; 23:667–75. Merville P. Combating chronic renal allograft dysfunction: optimal immuno-suppressive regimens. Drugs 2005;65:615–31. Miller LW. Cyclosporine-associated neurotoxicity. The need for a better guide for immunosuppressive therapy. Circulation 1996;94:1209–11. Morgan BJ, Lyson T, Scherrer U, Victor RG. Cyclosporine causes sympathetically mediated elevations in arterial pressure in rats. Hypertension 1991;18:458–66. Nagai Y, Miyakoshi H, Ohsawa K, Leki Y, Takamura T, Kobayashi K. Cyclosporine A inhibits the secretion of certain anterior pituitary hormones in patients with nephrotic syndrome. Endocrinol Jpn 1992;39:129–32.
Ober K, Benson S, Vogelsang M, Bylica A, Gunther D, Witzke O, et al. Plasma noradrenaline and state anxiety levels predict placebo response in learned immunosuppression. Clin Pharmacol Ther 2012;91:220–6. Olyaei AJ, de Mattos AM, Bennett WM. Nephrotoxicity of immunosuppressive drugs: new insight and preventive strategies. Curr Opin Crit Care 2001;7:384–9. Pacheco-López G, Doenlen R, Krügel U, Arnold M, Wirth T, Riether C, et al. Neurobehavioural activation during peripheral immunosuppression. Int J Neuropsychopharmacol 2013; 16:137–49. Palmer BF, Toto RD. Severe neurologic toxicity induced by cyclosporine A in three renal transplant patients. Am J Kidney Dis 1991;18:116–21. Reis F, Tavares P, Teixeira F. The distribution of catecholamines between plasma and platelets in cyclosporin A-induced hypertensive rats. Pharmacol Res 2000;41:129–35. Sakata A, Tamai I, Kawazu K, Deguchi Y, Ohnishi T, Saheki A, et al. In vivo evidence for ATP-dependent and P-glycoprotein-mediated transport of cyclosporin A at the blood–brain barrier. Biochem Pharmacol 1994;48:1989–92. Sander M, Lyson T, Thomas GD, Victor RG. Sympathetic neural mechanisms of cyclosporine-induced hypertension. Am J Hypertens 1996;9:121–38. Sato Y, Takayanagi Y, Onaka T, Kobayashi E. Impact of cyclosporine upon emotional and social behavior in mice. Transplantation 2007;83:1365–70. http://dx.doi.org/10. 1097/01.tp.0000263332.65519.1f. Scherrer U, Vissing SF, Morgan BJ, Rollins JA, Tindall RS, Ring S, et al. Cyclosporine-induced sympathetic activation and hypertension after heart transplantation. N Engl J Med 1990;323:693–9. http://dx.doi.org/10.1056/NEJM199009133231101. Shiga Y, Onodera H, Matsuo Y, Kogure K. Cyclosporin A protects against ischemia– reperfusion injury in the brain. Brain Res 1992;595:145–8. Shimizu H, Kumai T, Kobayashi S. Involvement of tyrosine hydroxylase upregulation in cyclosporine-induced hypertension. Jpn J Pharmacol 2001;85:306–12. Spielberger CD, Gorusch RL, Lishene RE. Manual for the State-Trait Anxiety Inventory. Palo Alto, CA: Consulting Psychology Press; 1970. Stephanou A, Sarlis NJ, Knight RA, Lightman SL, Chowdrey HS. Effects of cyclosporine A on the hypothalamic–pituitary–adrenal axis and anterior pituitary interleukin-6 mRNA expression during chronic inflammatory stress in the rat. J Neuroimmunol 1992;41: 215–22. Tavares P, Fontes Ribeiro CA, Teixeira F. Cyclosporin effect on noradrenaline release from the sympathetic nervous endings of rat aorta. Pharmacol Res 2003;47:27–33. Tombazzi CR, Waters B, Shokouh-Amiri MH, Vera SR, Riely CA. Neuropsychiatric complications after liver transplantation: role of immunosuppression and hepatitis C. Dig Dis Sci 2006;51:1079–81. http://dx.doi.org/10.1007/s10620-006-8012-0. von Horsten S, Exton MS, Voge J, Schult M, Nagel E, Schmidt RE, et al. Cyclosporine A affects open field behavior in DA rats. Pharmacol Biochem Behav 1998;60:71–6. Wijdicks EF. Neurotoxicity of immunosuppressive drugs. Liver Transpl 2001;7:937–42. http://dx.doi.org/10.1053/jlts.2001.27475.
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Short-term treatment with the calcineurin inhibitor cyclosporine A decreases HPA axis activity and plasma noradrenaline levels in healthy male volunteers Antje Albring a,1 , Laura Wendt a,1, Nino Harz a, Harald Engler a, Benjamin Wilde b, Oliver Witzke b,⁎, Manfred Schedlowski a a b
Institute of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany Department of Nephrology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
a r t i c l e
i n f o
Article history: Received 2 June 2014 Received in revised form 29 August 2014 Accepted 7 September 2014 Available online 16 September 2014 Keywords: Calcineurin inhibitor Cyclosporine A Neuropsychological changes Neuroendocrine parameters
a b s t r a c t Treatment with the selective calcineurin inhibitor and immunosuppressive drug cyclosporine A (CsA) is associated with neurotoxicity and neurocognitive impairments. Whether and to what extent CsA is inducing alterations of the neuroendocrine status is unknown so far. Therefore, the present study investigated the effect of short-term CsA treatment on hypothalamus–pituitary–adrenal (HPA) axis activity and catecholamine release as well as state anxiety in healthy male subjects. Treatment with CsA significantly reduced plasma concentrations of adrenocorticotropic hormone (ACTH), cortisol, and noradrenaline whereas adrenaline levels and state anxiety remained unaffected. Future studies should analyze the mechanisms of CsA-induced effects on neuroendocrine variables, neurocognitive functions and mood. © 2014 Published by Elsevier Inc.
1. Introduction The calcineurin inhibitor and immunosuppressive drug cyclosporine A (CsA) is widely employed in clinical practice, in particular in transplantation medicine for prevention and treatment of graft rejection (Olyaei et al., 2001; Merville, 2005; Boots et al., 2004). However, apart from its immunosuppressive properties, chronic treatment with CsA is associated with neurotoxicity and an increased prevalence of developing neuropsychiatric symptoms (Tombazzi et al., 2006; Lindenfeld et al., 2004; Wijdicks, 2001; de Groen et al., 1987; Palmer and Toto, 1991). In up to 20% of renal transplant patients, CsA administration is accompanied with neurological adverse effects such as tremor. Other, rare complications include depression, somnolence and burning paresthesia (Kahan et al., 1987). In addition, clinical evidence documents that 10 to 28% of patients receiving CsA experience restlessness, neuralgia and peripheral neuropathy which are possibly caused by neurotoxicity (Bechstein, 2000; Miller, 1996). The mechanisms which are responsible for the adverse neuropsychological effects after CsA treatment are largely unknown. However,
⁎ Corresponding author at: Department of Nephrology, University Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany. Tel.: +49 201 7233394; fax: +49 201 7233393. E-mail address: [email protected] (B. Wilde). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.pbb.2014.09.006 0091-3057/© 2014 Published by Elsevier Inc.
observations from animal experiments document that even acute, short-term treatment with CsA affects neural activity and behavior. Acute peripheral administration of CsA reduced activity in the open field, decreased rearing activity and reduced social interaction and motor activity while augmenting anxiety-related behavior in rodents (von Horsten et al., 1998; Sato et al., 2007). These CsA-induced behavioral effects are paralleled by observations in rats, where a single intraperitoneal injection of CsA increased neuronal activity in the insular cortex and the amygdala and enhanced amygdaloid noradrenaline release (Pacheco-López et al., 2013). Whether and to what extent these neurobehavioral changes after acute administration of CsA are associated with CsA-induced changes in HPA-axis activity and/or alterations in catecholamine release is unknown. Sympathetic activation following CsA treatment has been repeatedly documented in rodents and humans (Sander et al., 1996; Lyson et al., 1994; Morgan et al., 1991; Scherrer et al., 1990; Grobecker et al., 1995; Shimizu et al., 2001; Tavares et al., 2003). However, studies investigating the effect of CsA on the production of catecholamines remain contradictory (Reis et al., 2000; Gerkens, 1989; Duruibe et al., 1990) and only a study in rats reported a reduced activity of the hypothalamic–pituitary–adrenal (HPA) axis, reflected through reduced plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone after CsA treatment (Stephanou et al., 1992). Therefore, the present study analyzed the effect of short-term CsA treatment on cellular immune functions, neuroendocrine parameters and state anxiety in healthy male volunteers.
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A. Albring et al. / Pharmacology, Biochemistry and Behavior 126 (2014) 73–76
2. Materials and methods 2.1. Subjects Sixty healthy male volunteers (age range: 18–40 years) were recruited through public advertisement in the surrounding community. All volunteers underwent an extensive physical and psychiatric assessment (self-reported questionnaires, interview about their medical history) as well as an electrocardiogram and ultrasonography of the kidneys performed and evaluated by the physicians of the Department of Nephrology. Subjects were excluded if one of the following criteria was identified: daily intake of medication, blood donations of N200 ml within the last two months, previous participation in pharmacological studies or other medical exclusion criteria (e.g., disorders of the immune or endocrine system, previous or persistent psychiatric disorders, addiction, allergies, signs of cardiovascular, hematologic or nephrologic disorders, respiratory problems and diabetes mellitus). The baseline liver values were within the normal range (GOT: 23 ± 8 U/l, GPT: 26 ± 10 U/l, GGT: 20 ± 12 U/l, bilirubin (total): 0.7 ± 0.3 mg/dl).
2.2. Study design Subjects received four oral doses of 2.5 mg/kg body weight CsA (Sandimmun® Optoral, Novartis) on three consecutive days (day 1 at 6 pm, day 2 at 8 am and 6 pm, day 3 at 8 am). Blood samples were drawn on day 1 (10 am) for baseline measurements and on day 3 (10 am), 2 h after the last CsA intake (CsA) to analyze the pharmacological effect of CsA on neuroendocrine and immunological variables. A last blood sample was drawn on day 8 at 10 am, five days after the last CsA intake (washout). State anxiety (STAI) (Spielberger et al., 1970) was assessed at each blood withdrawal in order to document a possible effect of short-term CsA treatment on state anxiety levels.
2.3. Endocrine parameters Plasma noradrenaline and adrenaline concentrations were determined by high-performance liquid chromatography (HPLC) with electrochemical detection (ChromSystems, Instruments and Chemicals GmbH, Munich, Germany) as previously described (Leineweber et al., 2007). Plasma levels of ACTH and cortisol were determined using enzyme-linked immunosorbent assays (ACTH and Cortisol ELISA, IBL International, Hamburg, Germany) according to the test protocol of the manufacturer and were analyzed on a Fluostar OPTIMA Microplate Reader (BMG Labtech, Offenbach, Germany). The detection limits were 0.22 pg/ml (ACTH) and 0.3 ng/ml (cortisol), respectively.
2.4. Measurement of CsA blood concentrations CsA concentrations in whole blood were assessed using Siemens Dimension Flex reagent cartridge (Cruinn Diagnostics Ltd., Dublin, Ireland) according to the manufacturer's recommendations.
2.5. Cell isolation Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Ficoll-Paque™ Plus, GE Healthcare, Munich, Germany) according to the manufacturer's protocol. Cells were washed with Hanks' Balanced Salt Solution (Life Technologies, Darmstadt, Germany), counted with an automated hematology analyzer (KX-21N, Sysmex Deutschland GmbH, Norderstedt, Germany) and adjusted to 2.5 × 106 and 1.25 × 106 cells/ml in cell culture medium (RPMI 1640 supplemented with GlutaMAX I, 25 mM Hepes, 10% fetal bovine serum, 50 μg/ml gentamicin; Life Technologies, Darmstadt, Germany).
2.5.1. IL-2 mRNA expression analysis PBMCs (2.5 × 106 cells/ml) were stimulated with 40 ng/ml of soluble mouse anti-human CD3 monoclonal antibody (clone: HIT3a; BD Pharmingen) for 4 h (37 °C, 5% CO2). Total RNA was extracted using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Single-stranded cDNA was synthesized using a high capacity cDNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). Real-time quantitative PCR was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems) using Fast qPCR MasterMix Plus Low Rox (Eurogentec, Cologne, Germany) and the following cycling conditions: 5 min at 95 °C followed by 40 cycles of 3 s at 95 °C, 20 s at 60 °C and 26 s at 72 °C. Primers (forward: 5′-CCAGGA TGCTCACATTTAAGTTTTAC-3′; reverse: 5′-GAGGTTTG AGTTCTTC TTCTAGACACTGA-3) and probe (5′–6-FAM-TGCCCAAG AAGGCCACAGAACTGAA-BHQ1–3′) were designed using Primer Express 3.0 software (Applied Biosystems) and were purchased from Eurogentec (Cologne, Germany). For quantification of IL-2 mRNA expression, serially diluted cDNA samples generated from purified specific PCR products (High Pure PCR Product Purification Kit, Roche Diagnostics, Mannheim, Germany) were used as external standards in each run. Results are expressed as fg/μg total RNA. 2.6. Determination of IL-2 in culture supernatant PBMC suspensions (2. 5 × 106 cells/ml) were transferred to 96-well flat-bottom tissue culture plates and were stimulated with 20 ng/ml of soluble mouse anti-human CD3 monoclonal antibody (clone: HIT3a; BD Pharmingen, San Diego, CA) for 24 h (37 °C, 5% CO2). Concentration of IL-2 in culture supernatants was quantified using a commercial ELISA (Human IL-2 ELISA MAX Deluxe, BioLegend, San Diego, CA) according to the manufacturer's instructions. 2.7. Proliferation analysis The percentage of proliferating CD4+ T cells was measured by flow cytometry using the Click-iT EdU cell proliferation assay (Invitrogen, Darmstadt, Germany) according to the manufacturer's instructions. Briefly, PBMCs (1.25 × 106 cells/ml) were stimulated in 96-well round bottom tissue culture plates with 2.5 μg/ml of soluble mouse antihuman CD3 monoclonal antibody (clone: HIT3a; BD Pharmingen) for 72 h (37 °C, 5% CO2). The thymidine analog 5-ethynyl-2′-deoxyuridine (EdU), which is incorporated during DNA synthesis, was added to the cells at a concentration of 10 μM for the last 48 h of culture. After incubation, cells were washed and stained with APC conjugated anti-human CD4 (clone RPA-T4, BD Pharmingen) antibody. Cells were fixed with 4% paraformaldehyde and permeabilized using a saponin-based permeabilization reagent. Afterward, cells were incubated with the Click-iT reaction cocktail. The percentage of proliferating CD4+ T cells was analyzed on a FACS Canto II flow cytometer using FACS Diva software (BD Immunocytometry Systems). 2.8. Statistical analysis Data were analyzed with analysis of variances (ANOVA) using PASW statistics (Version 18, SPSS, Chicago, IL, USA). Post hoc analyses were computed using paired t-tests. The level of significance was set at p b 0.05. All data are expressed as mean ± SEM. 3. Results Short-term CsA intake significantly increased CsA levels, however CsA was not detectable anymore after the washout phase of five days (Table 1). The immunopharmacological effectiveness of the CsA treatment was documented by a significant suppression in IL-2 mRNA expression and IL-2 concentration in culture supernatant of anti-CD3
A. Albring et al. / Pharmacology, Biochemistry and Behavior 126 (2014) 73–76 Table 1 CsA blood levels and immunological measures.
CsA (ng/ml) IL-2 (pg/ml) IL-2 mRNA (fg/μg total RNA) Proliferating CD4+ T cells (%)
Baseline
CsA peak level
Washout
n.d. 399.6 ± 44.6 198.9 ± 16.2 27.6 ± 1.0
1297 130.7 52.2 13.4
n.d. 393.8 ± 36.2 205.4 ± 13.6 27.6 ± 27.6
± ± ± ±
32.1*** 12.3*** 5.4*** 0.7***
Results of post hoc computed paired t-test are indicated (***p b 0.001). Data are presented as mean ± SEM. n.d. = not detectable.
stimulated PBMC as well as a reduced number of proliferating CD4+ T cells (Table 1). CsA intake significantly decreased the activity of the HPA axis as reflected by decreased plasma ACTH (F = 4.9; p b 0.01) and cortisol (F = 22.7, p b 0.001) levels (Fig. 1a). In addition, CsA significantly decreased plasma noradrenaline concentrations (F = 13.4; p b 0.001) without significantly affecting adrenaline levels (F = 0.9, n.s.) (Fig. 1b). Moreover, state anxiety scores remained unaffected by CsA treatment (baseline: 34.9 ± 0.9; CsA: 35.2 ± 1.0; washout: 33.6 ± 0.9; F = 2.8, n.s.). 4. Discussion Chronic treatment with calcineurin inhibitors such as CsA is accompanied by unwanted side effects in patients such as hypertension (Scherrer et al., 1990) and neurobehavioral disturbances (Kahan et al., 1987; Bechstein, 2000; Miller, 1996). The exact mechanisms responsible for these side effects are still unclear. Data from experimental animals indicate a direct effect of acute CsA administration on neurophysiological functions (Pacheco-López et al., 2013), however, data in humans are lacking. The results presented here reveal that short-term treatment with the calcineurin inhibitor and immunosuppressant CsA reduced the production of ACTH, cortisol and noradrenaline without affecting the
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release of adrenaline and state anxiety levels in healthy male subjects. Studies in rodents demonstrated anxiety-related behavior after acute CsA administration which was paralleled by increased neural activity in the insular cortex and amygdala (von Horsten et al., 1998; Sato et al., 2007; Pacheco-López et al., 2013). Our data however, show that short-term CsA treatment does not affect state anxiety in healthy humans as previously reported (Albring et al., 2014; Ober et al., 2012). In contrast, temporary intake of CsA decreased the activity of the HPA axis and the sympathetic nervous system, reflected by significantly inhibited production of ACTH, cortisol and noradrenaline, respectively. Data on the acute effects of CsA and calcineurin inhibitors in general on neuroendocrine functions are rare. Inhibitory effects of CsA on the activity of the HPA-axis have been shown in rats (Stephanou et al., 1992) and reduced secretion of the anterior pituitary hormones, thyroid-stimulating hormone, and prolactin was detected in patients with nephrotic syndrome (Nagai et al., 1992). The decreased plasma noradrenaline content after CsA intake was surprising as we rather expected sympathetic activation following CsA treatment. However, our observation is supported by a study with rats, documenting a decrease in noradrenaline plasma levels after treatment with 5 mg/kg CsA (Gerkens, 1989). The exact mechanisms through which CsA is affecting the neuroendocrine response are poorly understood. A direct penetration of peripheral administered CsA into the brain has been negated for a long time due to the limited transport of the drug across the blood brain barrier (Cefalu and Pardridge, 1985; Sakata et al., 1994; Shiga et al., 1992). However, recent data in animals indicate a direct CsA-mediated effect on the CNS (Sato et al., 2007). A single peripheral injection of CsA increased electric activity, c-Fos expression in the insular cortex and the amygdala and amygdaloid noradrenaline release. In addition, CsA levels where detectable in the cerebellum, insular cortex and amygdala 2 to 4 h after intraperitoneal injection of CsA (Pacheco-López et al., 2013). Moreover, acute CsA injection affected behavioral responses such as rearing and motor activity and anxiety (von Horsten et al., 1998; Sato et al., 2007). The decrease in noradrenaline concentrations together with the unaffected release of adrenaline levels observed in our study is indicating a centrally mediated CsA effect on the HPA axis and sympathetic nervous system, although in contrast to data in rodents, state anxiety levels remained unaffected. Clearly, further studies are needed to analyze the mechanisms of how acute administration of calcineurin inhibitors is affecting neurobehavioral and neuroendocrine responses. These data from rodents and healthy humans will increase our understanding about the unwanted neurological side effects of immunosuppressive regimens in patients. 5. Conclusion Cyclosporine A has significant impact on the neuroendocrine system and reduces the activity of the hypothalamus–pituitary–adrenal (HPA) axis. Ethics statement The study was approved by the local ethics committee for human investigations of the University Hospital Essen and follows the rules stated in the Declaration of Helsinki. All participants gave written informed consent prior to their inclusion in the study and were reimbursed for their participation. Acknowledgments
Fig. 1. (a) Plasma ACTH and cortisol levels at baseline, after intake of four oral doses of 2.5 mg/kg CsA, and after a washout phase of five days. (b) Plasma concentrations of adrenaline and noradrenaline at baseline, after intake of four oral doses of 2.5 mg/kg CsA, and after a washout phase of five days. Results of post hoc computed paired t-test are indicated (difference from baseline) (***p b 0.001; **p b 0.01; #p b 0.01). Data are presented as mean ± SEM.
We would like to thank Sonja Nissen, Zehra Yavuz, Christa Freundlieb, Alexandra Kornowski and Magdalene Vogelsang for their technical assistance and their skilled and friendly help. This work was supported by a grant of the German Research Foundation (FOR 1328; SCHE 341/17-1).
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