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Alteration of the cortisol–cortisone shuttle in leprosy type 1 reactions in leprosy patients in Hyderabad, India
Immunology Letters 109 (2007) 72–75
Alteration of the cortisol–cortisone shuttle in leprosy type 1 reactions in leprosy patients in Hyderabad, India Anna K. Andersson a , Sara E. Atkinson a , Saroj Khanolkar-Young a , MeherVani Chaduvula b , Suman Jain b , Lavanya Suneetha b , Sujai Suneetha b , Diana N.J. Lockwood a,∗ a
Department of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK b LEPRA India, Blue Peter Research Centre, Hyderabad, India Received 20 December 2006; received in revised form 14 January 2007; accepted 14 January 2007 Available online 5 February 2007
Abstract Regulation of inflammation in leprosy may be influenced by local concentrations of active cortisol and inactive cortisone, whose concentrations are regulated by enzymes in the cortisol–cortisone shuttle. We investigated the cortisol–cortisone shuttle enzymes in the skin of leprosy patients with type 1 reactions (T1R), which are characterised by skin and nerve inflammation. Gene expression of the shuttle enzymes were quantified in skin biopsies from 15 leprosy patients with new T1R before and during prednisolone treatment and compared with levels in skin biopsies from 10 borderline leprosy patients without reactions. Gene expression of 11-hydroxysteroid dehydrogenase (11-HSD) type 2, which converts cortisol to cortisone, is down-regulated in skin from T1R lesions. However expression levels of 11-HSD type 1, which converts cortisone to cortisol, were similar in skin with and without reactions and did not change during anti-leprosy drug treatment. Prednisolone treatment of patients with reactions is associated with an upregulation of 11-HSD2 expression in skin. The down regulation of 11-HSD2 at the beginning of a reaction may be caused by pro-inflammatory cytokines in the leprosy reactional lesion and may be a local attempt to down-regulate inflammation. However in leprosy reactions this local response is insufficient and exogenous steroids are required to control inflammation. © 2007 Elsevier B.V. All rights reserved. Keywords: 11-Hydroxysteroid dehydrogenase; Prednisolone; Cytokine; Leprosy
1. Introduction Leprosy type 1 reactions (T1R) complicate borderline leprosy and are characterised by delayed type hypersensitivity. Patients with T1R present with increased inflammation of skin and nerve and if not adequately treated may develop irreversible nerve damage. T1R are associated with increased cell-mediated immunity towards Mycobacterium leprae and this may occur secondarily to M. leprae antigen release during multi-drug treatment. However, reactions can occur before, during and after treatment with antibiotics and the critical mechanisms which initiate a reaction have not yet been determined. We investigated the hypothesis that the acute inflammation during a T1R is associated with a disruption of the cortisol–cortisone shuttle enzymes.
∗
Corresponding author. Tel.: +44 20 7637 4314; fax: +44 20 7637 4314. E-mail address: [email protected] (D.N.J. Lockwood).
0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.01.004
Cortisol, a stress hormone which affects the metabolism of carbohydrates, proteins and fat, has an almost global effect in reducing inflammation [1]. It is a glucocorticoid synthesised by the adrenal cortex. Glucocorticoids, such as prednisolone, are widely used for suppression of immunity and inflammation in chronic inflammatory diseases such as leprosy reactions, asthma and autoimmune diseases. The local concentration of cortisol in any tissue is not solely dependent on the concentrations reaching the tissue from the circulation. Many tissues regulate their local concentration by inter-conversion of active cortisol (11-hydroxy) into inactive cortisone (11-keto). The enzymes involved are called the cortisol–cortisone shuttle, and they are expressed in many tissues, including skin [2]. The enzyme 11hydroxysteroid dehydrogenase (11-HSD) type 1 posses both oxidase and reductase activities and hence interconverts cortisol and cortisone requiring NADP(H) as its cofactor. In contrast, 11-HSD type 2 acts exclusively as an oxidase converting cortisol to cortisone with NAD as its cofactor, and it has a much higher affinity for its substrate than 11-HSD1. Cytokines,
A.K. Andersson et al. / Immunology Letters 109 (2007) 72–75
glucocorticoids, and progesterone, oestrogen and pregnancy affect the shuttle enzymes [3,4]. Progesterone and oestrogen are inhibitors of 11-HSD2 oxidase, resulting in higher levels of cortisol [3]. Hence, increased levels of oestrogen at puberty and during pregnancy can cause non-specific suppression of cellmediated immunity. Women with leprosy may experience T1R postpartum when the oestrogen levels drop to normal levels [5]. The pro-inflammatory cytokines TNF-␣ and IL-1 up-regulate expression levels and reductase activity of 11-HSD1 and downregulate the activity of 11-HSD2 in cell lines in vitro [6,7], so increasing cortisol levels. Increased levels of TNF-␣ and IL1 [8,9] are found in reactional skin lesions and may affect the cortisol–cortisone shuttle and the local levels of endogenous cortisol regulating inflammation. We hypothesised that skin lesions with and without T1R have different levels of 11-HSD1 and 11-HSD2 expression. A dysregulation of the cortisol shuttle could result in insufficient levels of cortisol in the skin lesion to control the inflammatory process. We tested this hypothesis by measuring 11-HSD1 and 11-HSD2 expression in skin biopsies from patients with new type 1 reactions and comparing them with newly diagnosed non-reactional patients. The study was further strengthened by taking serial biopsies from both sets of patients. In this way we were able to examine the effect of prednisolone treatment on 11-HSD1 and 11-HSD2 expression levels. 2. Materials and methods Borderline leprosy patients with T1R (n = 15) and without reaction (n = 10) were studied at the Blue Peter Research Centre (BPRC), Hyderabad, India. Patients were classified clinically and histologically according to the Ridley-Jopling classification. Of the patients in T1R, 6 had borderline tuberculoid (BT) and 9 borderline lepromatous (BL) leprosy, the 10 non-reactional patients were BT (5) and BL (5). A T1R was defined clinically as the appearance of erythema in either existing or new leprosy skin lesions and confirmed histologically. The reactions were treated with a 6-month course of oral prednisolone daily at an initial dose of 30 mg and reduced by 5 mg every month. Twelve of the T1R patients were also starting World Health Organisation (WHO) leprosy multi-drug treatment (MDT). All non-reactional patients were starting MDT at the time of recruitment. A 6 mm skin punch biopsy was taken from the reactional site at time points (months 0, 1 and 6) during prednisolone treatment and stored frozen in RNAlater (Ambion). Patients without reaction had a skin biopsy taken from a leprosy lesion at 0 and 1 month. Permission to conduct the study was obtained from the University of London, London School of Hygiene and Tropical Medicine Ethics Committee and the BPRC Research Committee. Informed consent was obtained from patients before collection of biopsies and blood samples. This study is part of a study that has been reported in detail elsewhere [8]. Total RNA was isolated from skin biopsies using RNeasy fibrous kit (Qiagen, Crawley, West Sussex, UK). Digestion of DNA with DNase I (Qiagen) was included for all RNA preparations. The RNA yield was determined with RiboGreen RNA quantitation kit (Molecular Probes), and the RNA integrity was
73
checked by agarose gel electrophoresis. cDNA was synthesised from RNA (200 ng/reaction) using Omniscript reverse transcription kit (Qiagen). Nucleotide sequences for forward and reverse primers, respectively, in this study were as follows: 11-HSD1, 5 -TAG ACA CAG AAA CAG CCA TGA AGG-3 , 5 -TCA GAA GAG TGG TCC AGA GTG A-3 ; 11HSD2, 5 -CTT GGG GGC CTA TGG AAC-3 , 5 -CTC ACT GAC TCT GTC TTG AAG C-3 ; hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1) standard, 5 -GCT GGA TTA CAT CAA AGC ACT G-3 and 5 -TGT TTC ACT CAA TAG TGC TGT GG-3 . Real-time PCR reactions were performed on the LightCycler (Roche, Idaho Technologies) using QuantiTect SYBR Green PCR Master Mix (Qiagen). The annealing temperature was set to 61 ◦ C for 11-HSD1 and 57 ◦ C for 11-HSD2. The house keeping gene HPRT1 was detected with QuantiTect Gene Expression Assay (Qiagen) and QuantiTect Probe PCR Kit (Qiagen) according to the manufacturer’s instructions. To quantify gene expression (copies/l) the unknown samples were compared against a standard curve. To prepare standards for quantification, each target sequence was amplified and gelpurified. The stocks were serially diluted from 106 to 10 copy/l in 2 mg/l Herring sperm DNA (Sigma). Results are expressed as the ratio of 11-HSD mRNA copy to HPRT1 mRNA copy in 20 ng mRNA. Differences between times and between groups of patients were determined using the Wilcoxon signed-rank test and the Mann–Whitney test, respectively. P-values of <0.05 were considered significant. 3. Results 11-HSD2 gene expression was compared in skin biopsies from patients with and without T1R before treatment. Fig. 1 shows that the levels of mRNA for 11-HSD2 were significantly (P < 0.01) lower in patients with T1R than in patients without reactions. In contrast, 11-HSD1 mRNA levels were similar in the two groups of patients (Fig. 1). The effect of prednisolone treatment on the cortisol–cortisone shuttle was assessed by quantifying gene expression of 11HSD2 in sequential biopsies from patients in T1R. Fig. 2 shows that nearly all patients had increased 11-HSD2 mRNA levels during the first month of treatment. At 6 months, by which time the prednisolone dose had been reduced, 11-HSD2 mRNA levels decreased in some patients. Levels of 11-HSD2 mRNA were significantly (P < 0.01) higher in the 1-month and the 6month biopsies than in the initial biopsies. No significant change in 11-HSD1 gene expression in skin lesions was detected during prednisolone treatment. Non-reactional patients had similar levels of 11-HSD1 and 11-HSD2 before and after 1 month of MDT. 4. Discussion This is the first study to document cortisol–cortisone shuttle enzyme gene expression levels in leprosy patients. We have demonstrated that 11-HSD2 gene expression is decreased in skin at the time a TIR is diagnosed and levels increase with
74
A.K. Andersson et al. / Immunology Letters 109 (2007) 72–75
Fig. 1. 11-HSD1 and 11-HSD2 mRNA levels in skin biopsies from patients with and without T1R before starting treatment. Levels of gene expression were quantified using real-time PCR. The result are expressed as the ratio of 11-HSD1 and 11-HSD2 mRNA to HPRT1 mRNA. Each dot represents a patient. Lines show the median levels.
prednisolone treatment. 11-HSD1 levels are unaltered during reaction and treatment. This translates into increased local levels of cortisol at the time of reaction. Previous authors have suggested, without patient data, that disruption of the cortisol–cortisone shuttle is a mechanism for the development of leprosy reactions [10]. Our data suggest that a much simpler model may pertain. We propose that leprosy patients develop a T1R with production of Th1 type cytokines (TNF-␣ and IL-1) within lesions and that these inhibit 11HSD2 mRNA expression and activity. Thus in the reactional skin lesions the effect of local cytokine production is to increase local cortisol production, but this is still inadequate to control the reactional pathology and exogenous cortisol is required. Non-reactional patients had similar levels of 11-HSD1 and 11-HSD2 before and after 1 month of MDT. This indicates that the increase in 11-HSD2 mRNA in reactional skin during treatment is due to an effect of prednisolone on 11-HSD2 or
decreased levels of TNF-␣ and IL-1 rather than MDT. However MDT itself reduces inflammation and this may influence 11-HSD2 levels during treatment. A number of studies have shown that under certain conditions TNF-␣ and IL-1 inhibit 11-HSD2 mRNA expression and activity and stimulate a rise in 11-HSD1 mRNA expression and activity [6,7,12,13]. It is also possible that during prednisolone treatment the change in 11HSD2 mRNA levels affects cytokine levels in the skin lesions. Moreover, it is possible that an increase in 11-HSD1 is required to control the inflammation of T1R and the disruption is in the failure of 11-HSD1 to increase. Our findings are consistent with a response to inflammation rather than an event that provokes inflammation. The previous authors who suggested that disruption of the cortisol–cortisone shuttle triggers a T1R suggested that this was associated with a deviation of the cytokine profile towards a Th2 type response [10,11]. However in our previously published work on leprosy
Fig. 2. Effect of treatment on 11-HSD1 and 11-HSD2 mRNA levels in skin lesions. Levels of gene expression were quantified using real-time PCR in sequential skin biopsies from patients with and without T1R. The result are expressed as the ratio of 11-HSD1 and 11-HSD2 mRNA to HPRT1 mRNA. Statistical (P < 0.01) differences compared to zero time are indicated with asterisks.
A.K. Andersson et al. / Immunology Letters 109 (2007) 72–75
reactions we could find no evidence of Th2 type cytokines in skin lesions at the time of reaction. It is very difficult to tease apart the relative contributions of the cortisol–cortisone shuttle and pro-inflammatory cytokines in leprosy reactions. It is almost impossible to obtain biopsies just before the development of a reaction, even in well designed cohort studies. It is therefore difficult to establish what are precipitating factors for reactions and what are part of the pro-inflammatory response. However our data suggests that the changes we found were part of an inflammatory response rather than a trigger for reaction. We were not able to study cortisol levels in both sets of patients. Future work should investigate protein expression and activity of the cortisol–cortisone shuttle enzymes in skin biopsies during T1R. The shuttle activity in leprosy patients could be studied using methods such as vasoconstriction tests that measure skin blanching [2] and by measuring urinary steroid metabolite ratios and cortisol/cortisone ratios in skin biopsies and serum. These measurements would provide further insights into the role of the cortisol–cortisone shuttle in leprosy patients with reactions. Acknowledgements We would like to thank all the staff and patients at Blue Peter Research Centre (Hyderabad, India), in particular Syed Muzaffurullah and Mohammed Ismail for documenting clinical
75
progress and taking and maintaining skin biopsies. Blue Peter Research Centre is supported by MRC (London, UK) through Lepra-India. This work and A.K.A. are supported by a grant from the Hospitals and Homes of St. Giles (UK). References [1] Guyton AC, Hall JE. In: Guyton AC, editor. Textbook of medical physiology. 9th ed. Philadelphia: W.B. Saunders Company; 1996. p. 962– 75. [2] Teelucksingh S, Mackie AD, Burt D, McIntyre MA, Brett L, Edwards CR. Lancet 1990;335:1060–3. [3] Sun K, Yang K, Challis JRG. Biol Reprod 1998;58:1379–84. [4] Voice MW, Seckl JR, Edwards CR, Chapman KE. Biochem J 1996;317(Pt 2):621–5. [5] Duncan ME. Indian J Lepr 1996;68:23–34. [6] Escher G, Galli I, Vishwanath BS, Frey BM, Frey FJ. J Exp Med 1997;186:189–98. [7] Heiniger CD, Rochat MK, Frey FJ, Frey BM. FEBS Lett 2001;507:351–6. [8] Andersson AK, Chaduvula M, Atkinson SE, Khanolkar-Young S, Jain S, Suneetha L, et al. Infect Immun 2005;73:3725–33. [9] Khanolkar-Young S, Rayment N, Brickell PM, Katz DR, Vinayakumar S, Colston MJ, et al. Clin Exp Immunol 1995;99:196–202. [10] Rook GA, Baker R. Trop Med Int Health 1999;4:493–8. [11] Rook GA, Lightman SL, Heijnen CJ. Trends Immunol 2002;23:18–22. [12] Cai TQ, Wong B, Mundt SS, Thieringer R, Wright SD, HermanowskiVosatka A. J Steroid Biochem Mol Biol 2001;77:117–22. [13] Cooper MS, Bujalska I, Rabbitt E, Walker EA, Bland R, Sheppard MC, et al. J Bone Miner Res 2001;16:1037–44.
Alteration of the cortisol–cortisone shuttle in leprosy type 1 reactions in leprosy patients in Hyderabad, India Anna K. Andersson a , Sara E. Atkinson a , Saroj Khanolkar-Young a , MeherVani Chaduvula b , Suman Jain b , Lavanya Suneetha b , Sujai Suneetha b , Diana N.J. Lockwood a,∗ a
Department of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK b LEPRA India, Blue Peter Research Centre, Hyderabad, India Received 20 December 2006; received in revised form 14 January 2007; accepted 14 January 2007 Available online 5 February 2007
Abstract Regulation of inflammation in leprosy may be influenced by local concentrations of active cortisol and inactive cortisone, whose concentrations are regulated by enzymes in the cortisol–cortisone shuttle. We investigated the cortisol–cortisone shuttle enzymes in the skin of leprosy patients with type 1 reactions (T1R), which are characterised by skin and nerve inflammation. Gene expression of the shuttle enzymes were quantified in skin biopsies from 15 leprosy patients with new T1R before and during prednisolone treatment and compared with levels in skin biopsies from 10 borderline leprosy patients without reactions. Gene expression of 11-hydroxysteroid dehydrogenase (11-HSD) type 2, which converts cortisol to cortisone, is down-regulated in skin from T1R lesions. However expression levels of 11-HSD type 1, which converts cortisone to cortisol, were similar in skin with and without reactions and did not change during anti-leprosy drug treatment. Prednisolone treatment of patients with reactions is associated with an upregulation of 11-HSD2 expression in skin. The down regulation of 11-HSD2 at the beginning of a reaction may be caused by pro-inflammatory cytokines in the leprosy reactional lesion and may be a local attempt to down-regulate inflammation. However in leprosy reactions this local response is insufficient and exogenous steroids are required to control inflammation. © 2007 Elsevier B.V. All rights reserved. Keywords: 11-Hydroxysteroid dehydrogenase; Prednisolone; Cytokine; Leprosy
1. Introduction Leprosy type 1 reactions (T1R) complicate borderline leprosy and are characterised by delayed type hypersensitivity. Patients with T1R present with increased inflammation of skin and nerve and if not adequately treated may develop irreversible nerve damage. T1R are associated with increased cell-mediated immunity towards Mycobacterium leprae and this may occur secondarily to M. leprae antigen release during multi-drug treatment. However, reactions can occur before, during and after treatment with antibiotics and the critical mechanisms which initiate a reaction have not yet been determined. We investigated the hypothesis that the acute inflammation during a T1R is associated with a disruption of the cortisol–cortisone shuttle enzymes.
∗
Corresponding author. Tel.: +44 20 7637 4314; fax: +44 20 7637 4314. E-mail address: [email protected] (D.N.J. Lockwood).
0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.01.004
Cortisol, a stress hormone which affects the metabolism of carbohydrates, proteins and fat, has an almost global effect in reducing inflammation [1]. It is a glucocorticoid synthesised by the adrenal cortex. Glucocorticoids, such as prednisolone, are widely used for suppression of immunity and inflammation in chronic inflammatory diseases such as leprosy reactions, asthma and autoimmune diseases. The local concentration of cortisol in any tissue is not solely dependent on the concentrations reaching the tissue from the circulation. Many tissues regulate their local concentration by inter-conversion of active cortisol (11-hydroxy) into inactive cortisone (11-keto). The enzymes involved are called the cortisol–cortisone shuttle, and they are expressed in many tissues, including skin [2]. The enzyme 11hydroxysteroid dehydrogenase (11-HSD) type 1 posses both oxidase and reductase activities and hence interconverts cortisol and cortisone requiring NADP(H) as its cofactor. In contrast, 11-HSD type 2 acts exclusively as an oxidase converting cortisol to cortisone with NAD as its cofactor, and it has a much higher affinity for its substrate than 11-HSD1. Cytokines,
A.K. Andersson et al. / Immunology Letters 109 (2007) 72–75
glucocorticoids, and progesterone, oestrogen and pregnancy affect the shuttle enzymes [3,4]. Progesterone and oestrogen are inhibitors of 11-HSD2 oxidase, resulting in higher levels of cortisol [3]. Hence, increased levels of oestrogen at puberty and during pregnancy can cause non-specific suppression of cellmediated immunity. Women with leprosy may experience T1R postpartum when the oestrogen levels drop to normal levels [5]. The pro-inflammatory cytokines TNF-␣ and IL-1 up-regulate expression levels and reductase activity of 11-HSD1 and downregulate the activity of 11-HSD2 in cell lines in vitro [6,7], so increasing cortisol levels. Increased levels of TNF-␣ and IL1 [8,9] are found in reactional skin lesions and may affect the cortisol–cortisone shuttle and the local levels of endogenous cortisol regulating inflammation. We hypothesised that skin lesions with and without T1R have different levels of 11-HSD1 and 11-HSD2 expression. A dysregulation of the cortisol shuttle could result in insufficient levels of cortisol in the skin lesion to control the inflammatory process. We tested this hypothesis by measuring 11-HSD1 and 11-HSD2 expression in skin biopsies from patients with new type 1 reactions and comparing them with newly diagnosed non-reactional patients. The study was further strengthened by taking serial biopsies from both sets of patients. In this way we were able to examine the effect of prednisolone treatment on 11-HSD1 and 11-HSD2 expression levels. 2. Materials and methods Borderline leprosy patients with T1R (n = 15) and without reaction (n = 10) were studied at the Blue Peter Research Centre (BPRC), Hyderabad, India. Patients were classified clinically and histologically according to the Ridley-Jopling classification. Of the patients in T1R, 6 had borderline tuberculoid (BT) and 9 borderline lepromatous (BL) leprosy, the 10 non-reactional patients were BT (5) and BL (5). A T1R was defined clinically as the appearance of erythema in either existing or new leprosy skin lesions and confirmed histologically. The reactions were treated with a 6-month course of oral prednisolone daily at an initial dose of 30 mg and reduced by 5 mg every month. Twelve of the T1R patients were also starting World Health Organisation (WHO) leprosy multi-drug treatment (MDT). All non-reactional patients were starting MDT at the time of recruitment. A 6 mm skin punch biopsy was taken from the reactional site at time points (months 0, 1 and 6) during prednisolone treatment and stored frozen in RNAlater (Ambion). Patients without reaction had a skin biopsy taken from a leprosy lesion at 0 and 1 month. Permission to conduct the study was obtained from the University of London, London School of Hygiene and Tropical Medicine Ethics Committee and the BPRC Research Committee. Informed consent was obtained from patients before collection of biopsies and blood samples. This study is part of a study that has been reported in detail elsewhere [8]. Total RNA was isolated from skin biopsies using RNeasy fibrous kit (Qiagen, Crawley, West Sussex, UK). Digestion of DNA with DNase I (Qiagen) was included for all RNA preparations. The RNA yield was determined with RiboGreen RNA quantitation kit (Molecular Probes), and the RNA integrity was
73
checked by agarose gel electrophoresis. cDNA was synthesised from RNA (200 ng/reaction) using Omniscript reverse transcription kit (Qiagen). Nucleotide sequences for forward and reverse primers, respectively, in this study were as follows: 11-HSD1, 5 -TAG ACA CAG AAA CAG CCA TGA AGG-3 , 5 -TCA GAA GAG TGG TCC AGA GTG A-3 ; 11HSD2, 5 -CTT GGG GGC CTA TGG AAC-3 , 5 -CTC ACT GAC TCT GTC TTG AAG C-3 ; hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1) standard, 5 -GCT GGA TTA CAT CAA AGC ACT G-3 and 5 -TGT TTC ACT CAA TAG TGC TGT GG-3 . Real-time PCR reactions were performed on the LightCycler (Roche, Idaho Technologies) using QuantiTect SYBR Green PCR Master Mix (Qiagen). The annealing temperature was set to 61 ◦ C for 11-HSD1 and 57 ◦ C for 11-HSD2. The house keeping gene HPRT1 was detected with QuantiTect Gene Expression Assay (Qiagen) and QuantiTect Probe PCR Kit (Qiagen) according to the manufacturer’s instructions. To quantify gene expression (copies/l) the unknown samples were compared against a standard curve. To prepare standards for quantification, each target sequence was amplified and gelpurified. The stocks were serially diluted from 106 to 10 copy/l in 2 mg/l Herring sperm DNA (Sigma). Results are expressed as the ratio of 11-HSD mRNA copy to HPRT1 mRNA copy in 20 ng mRNA. Differences between times and between groups of patients were determined using the Wilcoxon signed-rank test and the Mann–Whitney test, respectively. P-values of <0.05 were considered significant. 3. Results 11-HSD2 gene expression was compared in skin biopsies from patients with and without T1R before treatment. Fig. 1 shows that the levels of mRNA for 11-HSD2 were significantly (P < 0.01) lower in patients with T1R than in patients without reactions. In contrast, 11-HSD1 mRNA levels were similar in the two groups of patients (Fig. 1). The effect of prednisolone treatment on the cortisol–cortisone shuttle was assessed by quantifying gene expression of 11HSD2 in sequential biopsies from patients in T1R. Fig. 2 shows that nearly all patients had increased 11-HSD2 mRNA levels during the first month of treatment. At 6 months, by which time the prednisolone dose had been reduced, 11-HSD2 mRNA levels decreased in some patients. Levels of 11-HSD2 mRNA were significantly (P < 0.01) higher in the 1-month and the 6month biopsies than in the initial biopsies. No significant change in 11-HSD1 gene expression in skin lesions was detected during prednisolone treatment. Non-reactional patients had similar levels of 11-HSD1 and 11-HSD2 before and after 1 month of MDT. 4. Discussion This is the first study to document cortisol–cortisone shuttle enzyme gene expression levels in leprosy patients. We have demonstrated that 11-HSD2 gene expression is decreased in skin at the time a TIR is diagnosed and levels increase with
74
A.K. Andersson et al. / Immunology Letters 109 (2007) 72–75
Fig. 1. 11-HSD1 and 11-HSD2 mRNA levels in skin biopsies from patients with and without T1R before starting treatment. Levels of gene expression were quantified using real-time PCR. The result are expressed as the ratio of 11-HSD1 and 11-HSD2 mRNA to HPRT1 mRNA. Each dot represents a patient. Lines show the median levels.
prednisolone treatment. 11-HSD1 levels are unaltered during reaction and treatment. This translates into increased local levels of cortisol at the time of reaction. Previous authors have suggested, without patient data, that disruption of the cortisol–cortisone shuttle is a mechanism for the development of leprosy reactions [10]. Our data suggest that a much simpler model may pertain. We propose that leprosy patients develop a T1R with production of Th1 type cytokines (TNF-␣ and IL-1) within lesions and that these inhibit 11HSD2 mRNA expression and activity. Thus in the reactional skin lesions the effect of local cytokine production is to increase local cortisol production, but this is still inadequate to control the reactional pathology and exogenous cortisol is required. Non-reactional patients had similar levels of 11-HSD1 and 11-HSD2 before and after 1 month of MDT. This indicates that the increase in 11-HSD2 mRNA in reactional skin during treatment is due to an effect of prednisolone on 11-HSD2 or
decreased levels of TNF-␣ and IL-1 rather than MDT. However MDT itself reduces inflammation and this may influence 11-HSD2 levels during treatment. A number of studies have shown that under certain conditions TNF-␣ and IL-1 inhibit 11-HSD2 mRNA expression and activity and stimulate a rise in 11-HSD1 mRNA expression and activity [6,7,12,13]. It is also possible that during prednisolone treatment the change in 11HSD2 mRNA levels affects cytokine levels in the skin lesions. Moreover, it is possible that an increase in 11-HSD1 is required to control the inflammation of T1R and the disruption is in the failure of 11-HSD1 to increase. Our findings are consistent with a response to inflammation rather than an event that provokes inflammation. The previous authors who suggested that disruption of the cortisol–cortisone shuttle triggers a T1R suggested that this was associated with a deviation of the cytokine profile towards a Th2 type response [10,11]. However in our previously published work on leprosy
Fig. 2. Effect of treatment on 11-HSD1 and 11-HSD2 mRNA levels in skin lesions. Levels of gene expression were quantified using real-time PCR in sequential skin biopsies from patients with and without T1R. The result are expressed as the ratio of 11-HSD1 and 11-HSD2 mRNA to HPRT1 mRNA. Statistical (P < 0.01) differences compared to zero time are indicated with asterisks.
A.K. Andersson et al. / Immunology Letters 109 (2007) 72–75
reactions we could find no evidence of Th2 type cytokines in skin lesions at the time of reaction. It is very difficult to tease apart the relative contributions of the cortisol–cortisone shuttle and pro-inflammatory cytokines in leprosy reactions. It is almost impossible to obtain biopsies just before the development of a reaction, even in well designed cohort studies. It is therefore difficult to establish what are precipitating factors for reactions and what are part of the pro-inflammatory response. However our data suggests that the changes we found were part of an inflammatory response rather than a trigger for reaction. We were not able to study cortisol levels in both sets of patients. Future work should investigate protein expression and activity of the cortisol–cortisone shuttle enzymes in skin biopsies during T1R. The shuttle activity in leprosy patients could be studied using methods such as vasoconstriction tests that measure skin blanching [2] and by measuring urinary steroid metabolite ratios and cortisol/cortisone ratios in skin biopsies and serum. These measurements would provide further insights into the role of the cortisol–cortisone shuttle in leprosy patients with reactions. Acknowledgements We would like to thank all the staff and patients at Blue Peter Research Centre (Hyderabad, India), in particular Syed Muzaffurullah and Mohammed Ismail for documenting clinical
75
progress and taking and maintaining skin biopsies. Blue Peter Research Centre is supported by MRC (London, UK) through Lepra-India. This work and A.K.A. are supported by a grant from the Hospitals and Homes of St. Giles (UK). References [1] Guyton AC, Hall JE. In: Guyton AC, editor. Textbook of medical physiology. 9th ed. Philadelphia: W.B. Saunders Company; 1996. p. 962– 75. [2] Teelucksingh S, Mackie AD, Burt D, McIntyre MA, Brett L, Edwards CR. Lancet 1990;335:1060–3. [3] Sun K, Yang K, Challis JRG. Biol Reprod 1998;58:1379–84. [4] Voice MW, Seckl JR, Edwards CR, Chapman KE. Biochem J 1996;317(Pt 2):621–5. [5] Duncan ME. Indian J Lepr 1996;68:23–34. [6] Escher G, Galli I, Vishwanath BS, Frey BM, Frey FJ. J Exp Med 1997;186:189–98. [7] Heiniger CD, Rochat MK, Frey FJ, Frey BM. FEBS Lett 2001;507:351–6. [8] Andersson AK, Chaduvula M, Atkinson SE, Khanolkar-Young S, Jain S, Suneetha L, et al. Infect Immun 2005;73:3725–33. [9] Khanolkar-Young S, Rayment N, Brickell PM, Katz DR, Vinayakumar S, Colston MJ, et al. Clin Exp Immunol 1995;99:196–202. [10] Rook GA, Baker R. Trop Med Int Health 1999;4:493–8. [11] Rook GA, Lightman SL, Heijnen CJ. Trends Immunol 2002;23:18–22. [12] Cai TQ, Wong B, Mundt SS, Thieringer R, Wright SD, HermanowskiVosatka A. J Steroid Biochem Mol Biol 2001;77:117–22. [13] Cooper MS, Bujalska I, Rabbitt E, Walker EA, Bland R, Sheppard MC, et al. J Bone Miner Res 2001;16:1037–44.