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Tryptophan metabolism in allergic rhinitis: The effect of pollen allergen exposure
Human Immunology 71 (2010) 911–915
Contents lists available at ScienceDirect
Tryptophan metabolism in allergic rhinitis: The effect of pollen allergen exposure Giorgio Ciprandi a,*, Mara De Amici b, Mariangela Tosca c, Dietmar Fuchs d a
Department of Internal Medicine, Azienda Ospedaliera Universitaria San Martino, University of Genoa, Genoa, Italy Pediatric Clinic, University of Pavia, Foundation IRCCS San Matteo, Pavia, Italy c Pneumology and Allergy Unit, Istituto Giannina Gaslini, Genoa, Italy d Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria b
A R T I C L E
I N F O
Article history: Received 20 April 2010 Accepted 19 May 2010 Available online 9 June 2010
Keywords: Allergic rhinitis Serum Tryptophan Kynurenine Pollen allergen exposure
A B S T R A C T
This study evaluates serum tryptophan, kynurenine, kynurenine-to-tryptophan ratio, and neopterin levels in patients with pollen-induced allergic rhinitis (AR) during and outside of the pollen season, along with these values in healthy subjects. A total of 102 patients (56 female and 46 male, median age 28.7 years) were included in this study: 56 with seasonal AR evaluated outside of the pollen season and thus without allergic inflammation and symptoms, and 46 with seasonal AR evaluated during the pollen season with symptoms. A skin prick test and blood sampling for assessing serum concentrations of tryptophan and kynurenine and of immune activation marker neopterin were performed in all subjects. Tryptophan and kynurenine serum concentrations were higher in AR patients than in controls and were also higher out of pollen season than during this season. In conclusion, this preliminary study demonstrates that serum tryptophan metabolism could serve as a biomarker in patients with AR. 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Tryptophan catabolism-dependent mechanisms exert relevant immunoregulatory activities [1]. Tryptophan is an essential amino acid that is metabolized through two different biosynthetic pathways: the generation of the neurotransmitter serotonin and the formation of kynurenine derivatives [2,3]. This latter pathway is initiated by cleavage of the indole-ring by the enzymes tryptophan pyrrolase (tryptophan 2,3-dioxygenase, TDO) or indoleamine 2,3dioxygenase (IDO). TDO resides primarily in the liver and is regulated by tryptophan and steroid hormones [3]. IDO is the predominant extrahepatic enzyme and can be found in several cells, including macrophages, microglia, and dendritic cells, and is preferentially induced by Th1-type cytokine interferon (IFN)–␥. IFN-␥ is able to induce both the gene expression and enzymatic activity of IDO. As tryptophan is converted along the kynurenine pathway, kynurenine is the first stable intermediate produced. During an immune response, the release of IFN-␥ by activated T cells and macrophages leads to an accelerated and sustained degradation of tryptophan. Under normal conditions, kynurenine concentration is related to tryptophan level. The kynurenine (kyn) to tryptophan (trp) ratio (kyn/trp) represents an appropriate indicator of tryptophan degradation. The kyn/trp provides a better and normalized
* Corresponding author. E-mail address: [email protected] (G. Ciprandi).
measurement than absolute tryptophan or kynurenine concentration. A convenient way to substantiate that tryptophan degradation is due to activation of IDO rather than TDO is to demonstrate concomitant immune system activation. Thus, activated IDO is indicated when kyn/trp correlates with an immune activation parameter such as neopterin. Neopterin is a low-molecular-mass substance that is biosynthesized from guanosine-triphosphate and is produced preferentially by human monocytes–macrophages. Th1-type cytokine IFN-␥ was identified as the most potent cytokine that induces significant production of neopterin [4]. The extent of neopterin formation should reflect the combined effect of positively and negatively regulating influences on the population of monocytes-macrophages activated by IFN-␥. Increased neopterin concentrations in body fluids, such as serum or urine, are connected with diseases linked with cellular immune reaction, e.g., infections, autoimmune diseases, inflammatory diseases, organ transplantation rejection, and certain malignant diseases [5]. Presently, the kynurenine pathway has been implicated in a variety of diseases and disorders, including acquired immune deficiency syndrome, dementia complex, Alzheimer’s disease, schizophrenia, Huntington disease, amyotrophic lateral sclerosis, and neoplasia; and several studies have measured the levels of tryptophan and kynurenine under those conditions [2,3]. In all of these different disorders, the cellular immune system is involved in the pathogenesis and/or is affected by the underlying disease process,
0198-8859/10/$32.00 - see front matter 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2010.05.017
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G. Ciprandi et al. / Human Immunology 71 (2010) 911–915
and neopterin concentrations are very closely linked with the progression of these diseases. Therefore, neopterin may be considered a diagnostic marker able to measure the degree of activation of the Th1-type immune system. Allergic rhinitis (AR) is typically an immune-mediated disease, characterized by a dysregulation of T-cell responses. The information on tryptophan metabolic changes in such patients is still very limited; it may be therefore interesting to investigate the possible role of tryptophan metabolism in AR. This preliminary study was performed to measure serum tryptophan, kynurenine, kyn/trp, and neopterin levels in seasonal AR (SAR) patients during or outside of the pollen exposure. 2. Subjects and methods A total of 102 patients (56 female and 46 male, median age 28.7 years) from Genoa and Pavia were included in this study: 56 with SAR evaluated outside of the pollen season and thus without allergic inflammation and symptoms, and 46 with SAR evaluated during the pollen season with symptoms. AR diagnosis was made according to the Allergic Rhinitis and its Impact on Asthma (ARIA) document [6]. Results were compared with the considered parameters in 96 healthy blood donors measured earlier using enzyme-linked
immunosorbent assay (ELISA) (BRAHMS, Hennigsdorf, Germany) [5]. All allergic patients were sensitized to pollens alone and had experienced moderate to severe persistent AR for at least 4 years. The Ethical Committee approved the study, and informed consent was obtained from each subject. Tryptophan and kynurenine concentrations were measured by high-performance liquid chromatography (HPLC) using 3-nitro-Ltyrosine as internal standard. Tryptophan was detected by a fluorescence detector (ProStar, Model 360, Varian, Palo Alto, CA) at an excitation wavelength of 285 nm and an emission wavelength of 365 nm. A Shimadzu SPD-6A UV-detector (Shimadzu, Kyoto, Japan) in flow stream series connection was used for detection of both kynurenine and nitrotyrosine at a wavelength of 360 nm [7]. To estimate IDO activity, kyn/trp was calculated (expressed as mol kynurenine per mmol tryptophan). Neopterin concentrations were determined by ELISA (BRAHMS, Hennigsdorf, Germany) according to the manufacturer’s instructions, with a detection limit of 2 nmol/l [2,8]. Statistical analysis was performed using the statistical software package Medcalc 9 (Frank Schoonjans, BE; MedCalc Software, Mariakerke, Belgium). Descriptive statistics were first performed, and quantitative parameters were reported as the medians (MD) with
Fig. 1. Serum concentration of tryptophan (upper left), neopterin (upper right), kynurenine (lower left), and kynurenine-to-tryptophan ratio (kyn/trp; lower right) in patients with seasonal allergic rhinitis evaluated during or outside of the pollen season and in healthy subjects. Data are represented as medians (horizontal lines), interquartile ranges (boxes), and ranges (vertical lines), excluding outliers (p values, Kruskal–Wallis test, and p values of individual group comparisons are given).
G. Ciprandi et al. / Human Immunology 71 (2010) 911–915
interquartile ranges (IQR) because of the skewed distribution. Comparisons of the serum tryptophan, kynurenine, kyn/trp, and neopterin levels in different groups of patients were performed by means of the nonparametric analysis of variance (Kruskal–Wallis test); the Wilcoxon test was used for post hoc comparisons. Correlation between quantitative variables was evaluated by means of the Spearman correlation coefficient (r). All tests were two-sided, and a p value less than 0.05 was considered statistically significant. 3. Results Serum tryptophan levels significantly differed among the groups of subjects (H ⫽ 44.8, p ⬍ 0.0001, Kruskal–Wallis test; Fig. 1a). In particular, tryptophan concentrations were different between SAR patients evaluated during pollen season (MD: 61.85 mol/l, IQR: 56.5–73.8 mol/l) and evaluated outside of pollen season (MD: 81.55 mol/l, IQR: 73.55– 87.9 mol/l; U ⫽ ⫺5.86, p ⬍ 0.0001). The comparisons between healthy subjects (MD: 70.4 mol/l, IQR: 63.65–76.2 mol/l) and SAR patients evaluated during pollen season and outside of the season showed a significant difference in median serum levels of tryptophan (U ⫽ ⫺2.95, p ⬍ 0.005 and U ⫽ ⫺5.24, p ⬍ 0.0001, respectively). Serum neopterin concentrations were significantly different among the groups of subjects (H ⫽ 8.52, p ⬍ 0.02; Kruskal–Wallis Test; Fig. 1b); SAR patients evaluated during pollen season (MD: 4.7 nmol/l, IQR: 4.5–5.0 nmol/l) had lower levels than those evaluated outside of the pollen season (MD: 5.0 nmol/l, IQR: 4.5– 6.95 nmol/l; U ⫽ ⫺2.15, p ⬍ 0.05). The comparison between healthy subjects (IQR: 4.95 nmol/l, IQR: 4.7–5.7 nmol/l) and SAR patients evaluated during pollen season showed significantly different median serum neopterin levels (U ⫽ ⫺2.95, p ⬍ 0.005), but the difference between healthy subjects and SAR patients evaluated outside of the pollen season was not significant (U ⫽ ⫺0.18, p ⫽ 0.854). Also serum kynurenine concentrations differed significantly among the groups of subjects (H ⫽ 90.3, Kruskal–Wallis Test; p ⬍ 0.0001; Fig. 1c). In particular, kynurenine concentrations in SAR patients during the pollen season (MD: 2.62 mol/l, IQR: 2.24 –3.39 mol/l) were significantly lower than those outside of this season (MD: 3.32 mol/l, IQR: 2.71–3.83 mol/l; U ⫽ ⫺3.31, p ⬍ 0.001). The comparisons between healthy subjects (MD: 2.06 mol/l, IQR: 1.76 –2.3 mol/l) and SAR patients evaluated during and outside of the season showed a significant difference in median serum levels of kynurenine (U ⫽ ⫺5.96, p ⬍ 0.0001 and U ⫽ ⫺8.79, p ⬍ 0.0001, respectively). Moreover, kyn/trp significantly changed among the groups of subjects (H ⫽ 84.7, Kruskal–Wallis test; p ⬍ 0.0001; Fig. 1d). There was no difference of kyn/trp between SAR patients evaluated dur-
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ing (MD: 42.9 mol/l/mmol/l, IQR: 38.0 – 49.4 mol/l/mmol/l) versus outside of the pollen season (MD: 41.8 mol/l/mmol/l, IQR: 34.2– 46.8 mol/l/mmol/l; U ⫽ ⫺0.1; p ⫽ 0.318), but the comparisons between healthy subjects (MD: 28.4 mol/l/mmol/l, IQR: 25.0 –32.3 mol/l/mmol/l) and SAR patients evaluated during the pollen season and outside of the season revealed a significant difference in median kyn/trp (U ⫽ ⫺7.66, p ⬍ 0.0001 and U ⫽ ⫺7.40, p ⬍ 0.0001, respectively). There was a significant correlation between serum tryptophan and serum kynurenine levels in SAR patients: outside of the pollen season (r ⫽ 0.407, p ⬍ 0.005) and during the pollen season (r ⫽ 0.613, p ⬍ 0.0001; Fig. 2). There was also a significant relationship between serum neopterin and serum kynurenine levels in SAR patients outside of (r ⫽ 0.354, p ⬍ 0.01) and during the pollen season (r ⫽ 0.477, p ⫽ 0.001; Fig. 3). Moreover, there was a significant correlation between serum neopterin and kyn/trp in SAR patients outside of (r ⫽ 0.28, p ⬍ 0.05) and during the pollen season (r ⫽ 0.46, p ⫽ 0.002; Fig. 4). 4. Discussion AR is sustained by an inflammatory reaction. Various populations of effector and regulatory T cells have been shown to play a crucial role in allergic inflammation, mainly concerning Th2-type cells [8]. Th2-derived cytokines, such as IL-4 and IL-13, are the primary pathogenic factors in inducing, maintaining, and amplifying inflammatory allergic inflammation. IL-4 and IL-13 orchestrate allergic inflammation promoting IgE synthesis, up-regulating adhesion molecules selective for eosinophil recruitment, and causing increased mucus production and airway hyperreactivity. On the other hand, there is accumulating evidence that Th1-related cytokines, such as IFN-␥ and IL12, can suppress and counteract this Th2 response and vice versa, as there is functional dichotomy between Th1 and Th2 cells [9]. Therefore, allergic reaction is typically characterized by an activation of the Th2-type immune system. Tryptophan and its metabolites may be involved during activation of the immune system, and several studies have explored this issue [2,3]. However, few studies have investigated the role of tryptophan metabolism in allergic disorders. A first study evaluated 36 subjects (12 allergic SAR patients, 12 sensitized-only subjects, and 12 normal controls) during and outside of the pollen season [10]. Intergroup analysis did not evidence significant differences between groups, but the ratio kyn/trp outside-of-season intragroup analysis showed that patients only sensitized (without allergy and thus without inflammation and symptoms) had a reduction in tryptophan and an increase in both kynurenine and kyn/trp, considering the effect of
Fig. 2. Association between serum tryptophan and kynurenine concentrations in patients with seasonal allergic rhinitis evaluated outside of (left) and during the pollen season (right). Spearman’s correlation coefficient (r) and p values are given.
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Fig. 3. Association between serum neopterin and kynurenine concentrations in patients with seasonal allergic rhinitis evaluated outside of (left) and during the pollen season (right). Spearman’s correlation coefficient (r) and p values are given.
season. No changes were observed in allergic and normal subjects. Unfortunately, the authors did not provide the mean values. Raitala et al. evaluated a cohort of 392 subjects: 149 atopic patients (not better defined) and 243 nonatopic subjects [11]. The investigators considered only the kyn/trp: atopic patients had lower ratios than normal controls. Thus it is unclear whether an increase of tryptophan or decline of kynurenine was responsible for the decline of kyn/trp. There was no difference for gender. Finally, 44 in SAR patients who were evaluated before and after subcutaneous immunotherapy (SCIT) together with 38 normal controls [12], tryptophan levels were higher in atopics, kynurenine levels were superimposable in the two groups, kyn/trp was slightly lower in atopics, and serum neopterin was higher in them. Interestingly, nonresponders to SCIT had higher tryptophan levels. Thus tryptophan levels may predict outcomes of immunotherapy. It appears that there is considerable inconsistency among these studies, and it is not easy to judge the results from these different populations. The present study shows that during spring, such as the pollen exposure season, neopterin in the allergic population is lower than in winter. A possible interpretation may be the altered balance of Th1/Th2 immunity that Th2 activation is slowed down during winter when there is no exposure to allergens. As a consequence, Th1-type immunity and thus neopterin levels increase. From that point of view, the inside-of-season/outside-of-season comparison would fit rather well with what can be expected from the current understanding of SAR pathogenesis.
Also, for the tryptophan, the present results seem to confirm what we know a little from the earlier study done outside of the pollen season [12]: patients had sometimes rather high tryptophan levels. It might mean that pollinosis patients may have a different, e.g., subnormal, baseline IDO status (with higher tryptophan levels due to smaller degradation) out of season turning to more normal values in spring when tryptophan degradation is increasing and tryptophan goes down. Thus, IDO and neopterin behave unexpectedly in the opposite direction, but this was already observed also in the previous study [12], when pollinosis patients out of season had somewhat higher neopterin concentrations despite higher tryptophan (as in out-season group). Interestingly, there was a significant relationship between tryptophan and kynurenine concentrations in our patients (Fig 3), which might result form TDO activity that produces more kynurenine when tryptophan availability is higher [3]; but still there were significant relationships between neopterin and kynurenine and also neopterin and kyn/trp (Figs. 3 and 4), either outside of or during the pollen season, and the association became even stronger during the periods with allergen exposure. Parallel development of tryptophan degradation and neopterin production in patients is reflected by the significant correlation coefficients and further substantiates the involvement of immunoregulatory pathways in the biochemical changes observed. In conclusion, this preliminary study demonstrates that serum tryptophan metabolism could serve as a biomarker in patients with AR, depending on allergen exposure, such as on inflammatory phe-
Fig. 4. Association between serum neopterin concentrations ant the kynurenine-to-tryptophan ratio (kyn/trp) in patients with seasonal allergic rhinitis evaluated outside of (left) and during the pollen season (right). Spearman’s correlation coefficient (r) and p values are given.
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nomena. However, further studies should be performed for confirming these findings, for enrolling more patients, for relating tryptophan levels to both severity and allergy markers (such as eosinophils cationic protein, tryptase, etc), and for assessing these levels also at the level of the target organs. Acknowledgments The authors thank Vania Giunta, Cristina Torre, and Giuseppe Mantegna (Clinica Pediatrica, Fondazione IRCCS Policlinico S. Matteo, Pavia) and Maria Gleinser (Innsbruck Medical University) for outstanding technical support. References [1] Fallarino F, Gizzi S, Mosci P, Grohmann U, Puccetti P. Tryptophan catabolism in IDO⫹ plasmacytoid dendritic cells. Curr Drug Metab 2007;8:209 –16. [2] Schroecksnadel K, Wirleitner B, Winkler C, Fuchs D. Monitoring tryptopham metabolism in chronic immune activation. Clin Chim Acta 2006;364:82–90. [3] Chen Y, Guillemin GJ. Kynurenine pathway metabolites in humans: Disease and healthy states. Int J Tryptophan Resour 2009;2:1–19. [4] Huber C, Batchelor JR, Fuchs D, Hausen A, Lang A, Niederwieser D, et al. Immune response-associated production of neopterin—release from macrophages primarily under control of interferon-gamma. J Exp Med 1984;160: 310 – 6.
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[5] Murr C, Widner B, Wirleitner B, Fuchs D. Neopterin as a marker for immune system activation. Curr Drug Metab 2002;3:175– 87. [6] Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, et al. Allergic Rhinitis and its Impact on Asthma (ARIA) 2008 update (in collaboration with the World Health Organization, GA2LEN and AllerGen). Allergy 2008;63(Suppl 86):8 –160. [7] Widner B, Werner ER, Schennach H, Wachter H, Fuchs D. Simultaneous measurement of serum tryptophan and kynurenine by HPLC. Clin Chem 1997;43: 2424 – 6. [8] Mayersbach P, Fuchs D, Schennach H. Performance of fully automated quantitative neopterin measurements in a routine voluntary blood donations setting. Clin Chem Lab Med 2010;48:373–7. [9] Akdis CA. T cells in health and diseasey. J Allergy Clin Immunol 2009;123: 1022–3. [10] Von Bubnoff D, Fimmers R, Bogdanow M, Matz H, Koch S, Bieber T. Asymptomatic atopy is associated with increased indolamine 2,3-dioxygenase activity and interleukin-10 production during seasonal allergen exposure. Clin Exp Allergy 2004;34:1056 – 63. [11] Raitala A, Karjalainen J, Oja SS, Kosunen TU, Hurme M. Indoleamine 2,3dioxygenase (IDO) activity is lower in atopic than in non-atopic individuals and is enhanced by environmental factors protecting from atopy. Mol Immunol 2006;43:1054 – 6. [12] Kositz C, Schroecksnadel K, Grander G, Schennach H, Kofler H, Fuchs D. High serum tryptophan concentration in pollinosis patients is associated with unresponsiveness to pollen extract therapy. Int Arch Allergy Immunol 2008;147: 35– 40.
Contents lists available at ScienceDirect
Tryptophan metabolism in allergic rhinitis: The effect of pollen allergen exposure Giorgio Ciprandi a,*, Mara De Amici b, Mariangela Tosca c, Dietmar Fuchs d a
Department of Internal Medicine, Azienda Ospedaliera Universitaria San Martino, University of Genoa, Genoa, Italy Pediatric Clinic, University of Pavia, Foundation IRCCS San Matteo, Pavia, Italy c Pneumology and Allergy Unit, Istituto Giannina Gaslini, Genoa, Italy d Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria b
A R T I C L E
I N F O
Article history: Received 20 April 2010 Accepted 19 May 2010 Available online 9 June 2010
Keywords: Allergic rhinitis Serum Tryptophan Kynurenine Pollen allergen exposure
A B S T R A C T
This study evaluates serum tryptophan, kynurenine, kynurenine-to-tryptophan ratio, and neopterin levels in patients with pollen-induced allergic rhinitis (AR) during and outside of the pollen season, along with these values in healthy subjects. A total of 102 patients (56 female and 46 male, median age 28.7 years) were included in this study: 56 with seasonal AR evaluated outside of the pollen season and thus without allergic inflammation and symptoms, and 46 with seasonal AR evaluated during the pollen season with symptoms. A skin prick test and blood sampling for assessing serum concentrations of tryptophan and kynurenine and of immune activation marker neopterin were performed in all subjects. Tryptophan and kynurenine serum concentrations were higher in AR patients than in controls and were also higher out of pollen season than during this season. In conclusion, this preliminary study demonstrates that serum tryptophan metabolism could serve as a biomarker in patients with AR. 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Tryptophan catabolism-dependent mechanisms exert relevant immunoregulatory activities [1]. Tryptophan is an essential amino acid that is metabolized through two different biosynthetic pathways: the generation of the neurotransmitter serotonin and the formation of kynurenine derivatives [2,3]. This latter pathway is initiated by cleavage of the indole-ring by the enzymes tryptophan pyrrolase (tryptophan 2,3-dioxygenase, TDO) or indoleamine 2,3dioxygenase (IDO). TDO resides primarily in the liver and is regulated by tryptophan and steroid hormones [3]. IDO is the predominant extrahepatic enzyme and can be found in several cells, including macrophages, microglia, and dendritic cells, and is preferentially induced by Th1-type cytokine interferon (IFN)–␥. IFN-␥ is able to induce both the gene expression and enzymatic activity of IDO. As tryptophan is converted along the kynurenine pathway, kynurenine is the first stable intermediate produced. During an immune response, the release of IFN-␥ by activated T cells and macrophages leads to an accelerated and sustained degradation of tryptophan. Under normal conditions, kynurenine concentration is related to tryptophan level. The kynurenine (kyn) to tryptophan (trp) ratio (kyn/trp) represents an appropriate indicator of tryptophan degradation. The kyn/trp provides a better and normalized
* Corresponding author. E-mail address: [email protected] (G. Ciprandi).
measurement than absolute tryptophan or kynurenine concentration. A convenient way to substantiate that tryptophan degradation is due to activation of IDO rather than TDO is to demonstrate concomitant immune system activation. Thus, activated IDO is indicated when kyn/trp correlates with an immune activation parameter such as neopterin. Neopterin is a low-molecular-mass substance that is biosynthesized from guanosine-triphosphate and is produced preferentially by human monocytes–macrophages. Th1-type cytokine IFN-␥ was identified as the most potent cytokine that induces significant production of neopterin [4]. The extent of neopterin formation should reflect the combined effect of positively and negatively regulating influences on the population of monocytes-macrophages activated by IFN-␥. Increased neopterin concentrations in body fluids, such as serum or urine, are connected with diseases linked with cellular immune reaction, e.g., infections, autoimmune diseases, inflammatory diseases, organ transplantation rejection, and certain malignant diseases [5]. Presently, the kynurenine pathway has been implicated in a variety of diseases and disorders, including acquired immune deficiency syndrome, dementia complex, Alzheimer’s disease, schizophrenia, Huntington disease, amyotrophic lateral sclerosis, and neoplasia; and several studies have measured the levels of tryptophan and kynurenine under those conditions [2,3]. In all of these different disorders, the cellular immune system is involved in the pathogenesis and/or is affected by the underlying disease process,
0198-8859/10/$32.00 - see front matter 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2010.05.017
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G. Ciprandi et al. / Human Immunology 71 (2010) 911–915
and neopterin concentrations are very closely linked with the progression of these diseases. Therefore, neopterin may be considered a diagnostic marker able to measure the degree of activation of the Th1-type immune system. Allergic rhinitis (AR) is typically an immune-mediated disease, characterized by a dysregulation of T-cell responses. The information on tryptophan metabolic changes in such patients is still very limited; it may be therefore interesting to investigate the possible role of tryptophan metabolism in AR. This preliminary study was performed to measure serum tryptophan, kynurenine, kyn/trp, and neopterin levels in seasonal AR (SAR) patients during or outside of the pollen exposure. 2. Subjects and methods A total of 102 patients (56 female and 46 male, median age 28.7 years) from Genoa and Pavia were included in this study: 56 with SAR evaluated outside of the pollen season and thus without allergic inflammation and symptoms, and 46 with SAR evaluated during the pollen season with symptoms. AR diagnosis was made according to the Allergic Rhinitis and its Impact on Asthma (ARIA) document [6]. Results were compared with the considered parameters in 96 healthy blood donors measured earlier using enzyme-linked
immunosorbent assay (ELISA) (BRAHMS, Hennigsdorf, Germany) [5]. All allergic patients were sensitized to pollens alone and had experienced moderate to severe persistent AR for at least 4 years. The Ethical Committee approved the study, and informed consent was obtained from each subject. Tryptophan and kynurenine concentrations were measured by high-performance liquid chromatography (HPLC) using 3-nitro-Ltyrosine as internal standard. Tryptophan was detected by a fluorescence detector (ProStar, Model 360, Varian, Palo Alto, CA) at an excitation wavelength of 285 nm and an emission wavelength of 365 nm. A Shimadzu SPD-6A UV-detector (Shimadzu, Kyoto, Japan) in flow stream series connection was used for detection of both kynurenine and nitrotyrosine at a wavelength of 360 nm [7]. To estimate IDO activity, kyn/trp was calculated (expressed as mol kynurenine per mmol tryptophan). Neopterin concentrations were determined by ELISA (BRAHMS, Hennigsdorf, Germany) according to the manufacturer’s instructions, with a detection limit of 2 nmol/l [2,8]. Statistical analysis was performed using the statistical software package Medcalc 9 (Frank Schoonjans, BE; MedCalc Software, Mariakerke, Belgium). Descriptive statistics were first performed, and quantitative parameters were reported as the medians (MD) with
Fig. 1. Serum concentration of tryptophan (upper left), neopterin (upper right), kynurenine (lower left), and kynurenine-to-tryptophan ratio (kyn/trp; lower right) in patients with seasonal allergic rhinitis evaluated during or outside of the pollen season and in healthy subjects. Data are represented as medians (horizontal lines), interquartile ranges (boxes), and ranges (vertical lines), excluding outliers (p values, Kruskal–Wallis test, and p values of individual group comparisons are given).
G. Ciprandi et al. / Human Immunology 71 (2010) 911–915
interquartile ranges (IQR) because of the skewed distribution. Comparisons of the serum tryptophan, kynurenine, kyn/trp, and neopterin levels in different groups of patients were performed by means of the nonparametric analysis of variance (Kruskal–Wallis test); the Wilcoxon test was used for post hoc comparisons. Correlation between quantitative variables was evaluated by means of the Spearman correlation coefficient (r). All tests were two-sided, and a p value less than 0.05 was considered statistically significant. 3. Results Serum tryptophan levels significantly differed among the groups of subjects (H ⫽ 44.8, p ⬍ 0.0001, Kruskal–Wallis test; Fig. 1a). In particular, tryptophan concentrations were different between SAR patients evaluated during pollen season (MD: 61.85 mol/l, IQR: 56.5–73.8 mol/l) and evaluated outside of pollen season (MD: 81.55 mol/l, IQR: 73.55– 87.9 mol/l; U ⫽ ⫺5.86, p ⬍ 0.0001). The comparisons between healthy subjects (MD: 70.4 mol/l, IQR: 63.65–76.2 mol/l) and SAR patients evaluated during pollen season and outside of the season showed a significant difference in median serum levels of tryptophan (U ⫽ ⫺2.95, p ⬍ 0.005 and U ⫽ ⫺5.24, p ⬍ 0.0001, respectively). Serum neopterin concentrations were significantly different among the groups of subjects (H ⫽ 8.52, p ⬍ 0.02; Kruskal–Wallis Test; Fig. 1b); SAR patients evaluated during pollen season (MD: 4.7 nmol/l, IQR: 4.5–5.0 nmol/l) had lower levels than those evaluated outside of the pollen season (MD: 5.0 nmol/l, IQR: 4.5– 6.95 nmol/l; U ⫽ ⫺2.15, p ⬍ 0.05). The comparison between healthy subjects (IQR: 4.95 nmol/l, IQR: 4.7–5.7 nmol/l) and SAR patients evaluated during pollen season showed significantly different median serum neopterin levels (U ⫽ ⫺2.95, p ⬍ 0.005), but the difference between healthy subjects and SAR patients evaluated outside of the pollen season was not significant (U ⫽ ⫺0.18, p ⫽ 0.854). Also serum kynurenine concentrations differed significantly among the groups of subjects (H ⫽ 90.3, Kruskal–Wallis Test; p ⬍ 0.0001; Fig. 1c). In particular, kynurenine concentrations in SAR patients during the pollen season (MD: 2.62 mol/l, IQR: 2.24 –3.39 mol/l) were significantly lower than those outside of this season (MD: 3.32 mol/l, IQR: 2.71–3.83 mol/l; U ⫽ ⫺3.31, p ⬍ 0.001). The comparisons between healthy subjects (MD: 2.06 mol/l, IQR: 1.76 –2.3 mol/l) and SAR patients evaluated during and outside of the season showed a significant difference in median serum levels of kynurenine (U ⫽ ⫺5.96, p ⬍ 0.0001 and U ⫽ ⫺8.79, p ⬍ 0.0001, respectively). Moreover, kyn/trp significantly changed among the groups of subjects (H ⫽ 84.7, Kruskal–Wallis test; p ⬍ 0.0001; Fig. 1d). There was no difference of kyn/trp between SAR patients evaluated dur-
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ing (MD: 42.9 mol/l/mmol/l, IQR: 38.0 – 49.4 mol/l/mmol/l) versus outside of the pollen season (MD: 41.8 mol/l/mmol/l, IQR: 34.2– 46.8 mol/l/mmol/l; U ⫽ ⫺0.1; p ⫽ 0.318), but the comparisons between healthy subjects (MD: 28.4 mol/l/mmol/l, IQR: 25.0 –32.3 mol/l/mmol/l) and SAR patients evaluated during the pollen season and outside of the season revealed a significant difference in median kyn/trp (U ⫽ ⫺7.66, p ⬍ 0.0001 and U ⫽ ⫺7.40, p ⬍ 0.0001, respectively). There was a significant correlation between serum tryptophan and serum kynurenine levels in SAR patients: outside of the pollen season (r ⫽ 0.407, p ⬍ 0.005) and during the pollen season (r ⫽ 0.613, p ⬍ 0.0001; Fig. 2). There was also a significant relationship between serum neopterin and serum kynurenine levels in SAR patients outside of (r ⫽ 0.354, p ⬍ 0.01) and during the pollen season (r ⫽ 0.477, p ⫽ 0.001; Fig. 3). Moreover, there was a significant correlation between serum neopterin and kyn/trp in SAR patients outside of (r ⫽ 0.28, p ⬍ 0.05) and during the pollen season (r ⫽ 0.46, p ⫽ 0.002; Fig. 4). 4. Discussion AR is sustained by an inflammatory reaction. Various populations of effector and regulatory T cells have been shown to play a crucial role in allergic inflammation, mainly concerning Th2-type cells [8]. Th2-derived cytokines, such as IL-4 and IL-13, are the primary pathogenic factors in inducing, maintaining, and amplifying inflammatory allergic inflammation. IL-4 and IL-13 orchestrate allergic inflammation promoting IgE synthesis, up-regulating adhesion molecules selective for eosinophil recruitment, and causing increased mucus production and airway hyperreactivity. On the other hand, there is accumulating evidence that Th1-related cytokines, such as IFN-␥ and IL12, can suppress and counteract this Th2 response and vice versa, as there is functional dichotomy between Th1 and Th2 cells [9]. Therefore, allergic reaction is typically characterized by an activation of the Th2-type immune system. Tryptophan and its metabolites may be involved during activation of the immune system, and several studies have explored this issue [2,3]. However, few studies have investigated the role of tryptophan metabolism in allergic disorders. A first study evaluated 36 subjects (12 allergic SAR patients, 12 sensitized-only subjects, and 12 normal controls) during and outside of the pollen season [10]. Intergroup analysis did not evidence significant differences between groups, but the ratio kyn/trp outside-of-season intragroup analysis showed that patients only sensitized (without allergy and thus without inflammation and symptoms) had a reduction in tryptophan and an increase in both kynurenine and kyn/trp, considering the effect of
Fig. 2. Association between serum tryptophan and kynurenine concentrations in patients with seasonal allergic rhinitis evaluated outside of (left) and during the pollen season (right). Spearman’s correlation coefficient (r) and p values are given.
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Fig. 3. Association between serum neopterin and kynurenine concentrations in patients with seasonal allergic rhinitis evaluated outside of (left) and during the pollen season (right). Spearman’s correlation coefficient (r) and p values are given.
season. No changes were observed in allergic and normal subjects. Unfortunately, the authors did not provide the mean values. Raitala et al. evaluated a cohort of 392 subjects: 149 atopic patients (not better defined) and 243 nonatopic subjects [11]. The investigators considered only the kyn/trp: atopic patients had lower ratios than normal controls. Thus it is unclear whether an increase of tryptophan or decline of kynurenine was responsible for the decline of kyn/trp. There was no difference for gender. Finally, 44 in SAR patients who were evaluated before and after subcutaneous immunotherapy (SCIT) together with 38 normal controls [12], tryptophan levels were higher in atopics, kynurenine levels were superimposable in the two groups, kyn/trp was slightly lower in atopics, and serum neopterin was higher in them. Interestingly, nonresponders to SCIT had higher tryptophan levels. Thus tryptophan levels may predict outcomes of immunotherapy. It appears that there is considerable inconsistency among these studies, and it is not easy to judge the results from these different populations. The present study shows that during spring, such as the pollen exposure season, neopterin in the allergic population is lower than in winter. A possible interpretation may be the altered balance of Th1/Th2 immunity that Th2 activation is slowed down during winter when there is no exposure to allergens. As a consequence, Th1-type immunity and thus neopterin levels increase. From that point of view, the inside-of-season/outside-of-season comparison would fit rather well with what can be expected from the current understanding of SAR pathogenesis.
Also, for the tryptophan, the present results seem to confirm what we know a little from the earlier study done outside of the pollen season [12]: patients had sometimes rather high tryptophan levels. It might mean that pollinosis patients may have a different, e.g., subnormal, baseline IDO status (with higher tryptophan levels due to smaller degradation) out of season turning to more normal values in spring when tryptophan degradation is increasing and tryptophan goes down. Thus, IDO and neopterin behave unexpectedly in the opposite direction, but this was already observed also in the previous study [12], when pollinosis patients out of season had somewhat higher neopterin concentrations despite higher tryptophan (as in out-season group). Interestingly, there was a significant relationship between tryptophan and kynurenine concentrations in our patients (Fig 3), which might result form TDO activity that produces more kynurenine when tryptophan availability is higher [3]; but still there were significant relationships between neopterin and kynurenine and also neopterin and kyn/trp (Figs. 3 and 4), either outside of or during the pollen season, and the association became even stronger during the periods with allergen exposure. Parallel development of tryptophan degradation and neopterin production in patients is reflected by the significant correlation coefficients and further substantiates the involvement of immunoregulatory pathways in the biochemical changes observed. In conclusion, this preliminary study demonstrates that serum tryptophan metabolism could serve as a biomarker in patients with AR, depending on allergen exposure, such as on inflammatory phe-
Fig. 4. Association between serum neopterin concentrations ant the kynurenine-to-tryptophan ratio (kyn/trp) in patients with seasonal allergic rhinitis evaluated outside of (left) and during the pollen season (right). Spearman’s correlation coefficient (r) and p values are given.
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nomena. However, further studies should be performed for confirming these findings, for enrolling more patients, for relating tryptophan levels to both severity and allergy markers (such as eosinophils cationic protein, tryptase, etc), and for assessing these levels also at the level of the target organs. Acknowledgments The authors thank Vania Giunta, Cristina Torre, and Giuseppe Mantegna (Clinica Pediatrica, Fondazione IRCCS Policlinico S. Matteo, Pavia) and Maria Gleinser (Innsbruck Medical University) for outstanding technical support. References [1] Fallarino F, Gizzi S, Mosci P, Grohmann U, Puccetti P. Tryptophan catabolism in IDO⫹ plasmacytoid dendritic cells. Curr Drug Metab 2007;8:209 –16. [2] Schroecksnadel K, Wirleitner B, Winkler C, Fuchs D. Monitoring tryptopham metabolism in chronic immune activation. Clin Chim Acta 2006;364:82–90. [3] Chen Y, Guillemin GJ. Kynurenine pathway metabolites in humans: Disease and healthy states. Int J Tryptophan Resour 2009;2:1–19. [4] Huber C, Batchelor JR, Fuchs D, Hausen A, Lang A, Niederwieser D, et al. Immune response-associated production of neopterin—release from macrophages primarily under control of interferon-gamma. J Exp Med 1984;160: 310 – 6.
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