- Email: [email protected]
Evaluation of interleukin-23 plasma levels in patients with polycythemia vera and essential thrombocythemia
Cellular Immunology 278 (2012) 91–94
Contents lists available at SciVerse ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Evaluation of interleukin-23 plasma levels in patients with polycythemia vera and essential thrombocythemia Sebastiano Gangemi a,b, Alessandro Allegra c, Elisabetta Pace b, Andrea Alonci c, Maria Ferraro b, Annamaria Petrungaro c, Salvatore Saitta a, Demetrio Gerace c, Sabina Russo c, Giuseppa Penna c, Caterina Musolino c,⇑ a b c
School and Division of Allergy and Clinical Immunology, Department of Human Pathology, University of Messina, Italy Institute of Biomedicine and Molecular Immunology ‘‘A. Monroy’’ (IBIM), Consiglio Nazionale delle Ricerche (CNR), Palermo, Italy Division of Haematology, University of Messina, Italy
a r t i c l e
i n f o
Article history: Received 17 April 2012 Accepted 13 July 2012 Available online 1 August 2012 Keywords: Essential thrombocythemia Polycythemia vera JAK2 mutation Interleukin-23 Interleukin-22 Interleukin-10
a b s t r a c t Essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis share the same acquired genetic lesion, JAK2V617F. It is believed that cytokines participate in the activation of JAK2V617F. In this study, we analyzed the plasma levels of interleukin (IL)-23, IL-10 and IL-22 in patients with PV and ET. In the same subjects we also performed analysis of the JAK2V617F mutation, and evaluated a possible relationship between interleukin levels and thrombotic complications or with the symptom pruritus. Plasma levels of IL-23 were significantly increased in all patients with MPN in comparison to controls. Moreover, there was a significant difference between the levels of IL-23 in patients affected by PV and those measured in controls (8.57 ± 3.69 pg/mL vs. 6.55 ± 4.125 pg/mL; p < 0.03). No difference was found between IL-23 levels in ET patients and controls. No statistically significant differences were found between the levels of IL-23, Il-22 or IL-10 in PV or ET subjects with or without thrombosis, in patients with or without pruritus, or according the JAK2V617F burden. In PV patients the JAK2 burden and Hb levels correlated with occurrence of pruritus. Our study seems to point out a possible involvement of IL-23 in the pathogenesis of PV. Ó 2012 Published by Elsevier Inc.
1. Introduction Myeloproliferative disorders are a group of clonal hematopoietic disorders resulting in aberrant production of cells of the myeloid lineage. Myeloproliferative neoplasms (MPN) that do not contain the BCR–ABL mutation include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). In 2005, multiple research groups reported the identification of a JAK2 point mutation in this kind of MPN. This mutation affects the auto-inhibitory domain of JAK2 leading to constitutive activation of JAK2 and JAK/STAT signaling. However, the detailed mechanism by which abnormal activation of JAK2 exhibits transforming activity remains to be elucidated. It is believed that cytokines participate in the activation of JAK2V617F, presumably by providing a scaffolding function where two JAK2V617F molecules could properly be juxtaposed to allow for transphosphorylation and subsequent full activation of the
⇑ Corresponding author. Address: Division of Hematology, Via Consolare Valeria, Policlinico G. Martino, University of Messina, Messina, Italy. Fax: +39 090 2935162. E-mail address: [email protected] (C. Musolino). 0008-8749/$ - see front matter Ó 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.cellimm.2012.07.003
tyrosine kinase. Pradhan et al. demonstrated that components of heterodimeric cytokine receptors can also activate JAK2V617F. Expression of IL27Ra, a heterodimeric receptor component, enhanced the activation of JAK2V617F and subsequent downstream signaling to activation of STAT5 and ERK [1]. Moreover, the role of cytokines is confirmed by recent reports that have described complete or major molecular remission in patients with PV after long-term treatment with immunomodulatory agents, while bone marrow stroma-secreted cytokines seem to protect JAK2V617F-mutated cells from the effects of a JAK2 inhibitor [2]. Finally JAK2 inhibition is able to impair the expansion of responder T helper 17 (Th17) cells, a novel population of Th cells so named because of its ability to produce IL-17A [3]. Th17 cells produce IL-22 [4]. IL-22 is also produced by Th22, Th1 cells, classical and non-classical (NK-22) NK cells, NKT cells, and lymphoid tissue inducer cells. IL-23, a member of the IL-12 family of cytokines, plays an important role in Th17 maintenance [5]. Finally IL-10 is a potent suppressor of the immune system, commonly produced by CD4(+) T cells and T regulatory cells (Th 2, Th9, Th22), which has an important role in the biology of B cells and T cells, and recently Brunsing et al. have demonstrated that IL10 is produced by Th17 cells [6].
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In this study, we analyzed the plasma levels of IL - 23, and IL-22 cytokines produced by Th17 cells, and plasma levels of IL-10 a cytokine able to regulate Th17 function, in patients with PV and ET. In the same subjects we also performed analysis of the JAK2V617F mutation, and evaluated a possible relationship between interleukin levels and thrombotic complications or with the symptom pruritus. 2. Material and methods Our population consisted of 28 patients with ET (12 M – 16 F; mean age 62 ± 8 yrs) and 20 patients with PV 13 M - 7 F; mean age 64 ± 11 yrs). Fifteen patients (7 with ET, 8 with PV) had thrombotic manifestation in the past. All patients were not receiving aspirin or any treatment for ET or PV over seven days. The study was conducted according to good clinical and laboratory practice rules and the principles of the Declaration of Helsinki. Informed written consent was obtained after the purpose, nature, and potential risks were explained to the subjects. Plasma from 28 sex-matched and age matched normal subjects (11 M -17 F; mean age 59 ± 8) were also included in this study as controls. Peripheral venous blood was collected using citrate as anticoagulant. Within 30 min after collection, samples were centrifuged for 15 min at 200 g in a 4235 A centrifuge (ALC Int S.r.L. Milan, Italy); plasma was immediately separated, aliquoted and stored in a 80 °C freezer until the assay was performed. Freeze– thaw cycles were avoided. None of the patients or control subjects had symptoms or laboratory signs of active infections, inflammatory diseases, diabetes or kidney failure.
The mean of triplicate DCT determinations (CT JAK2V617F–CT JAK2WT) was used to calculate the percentage of mutant alleles. Positive and negative controls were included in each assay; interand intra-assay variation was 3% and 5%, respectively. 2.3. Diagnosis of thrombosis Vascular thrombosis was defined as a history of occlusive vascular events. Major events included peripheral thrombosis, cardiac vascular complication, central nervous system vascular complications, and intra-abdominal vascular complications. Minor events were erythromelalgia and superficial thrombophlebitis of the extremities. No patient was studied during the acute phase of the thrombosis. 2.4. Pruritus We also evaluated the prevalence of pruritus in our patients. Pruritus was auto-assessed by the patients, using a 0–2 scale (from 0 = no itch at all, to 2 = extreme itch). The prevalence of pruritus in our study was 33.0% (grade 0: 32 patients; grade 1: 8 patients – PV 5 patients; ET 3 patients; grade 2: 8 subjects – PV 5 patients; ET 3 patients). 2.5. Routine hematologic assays White blood cell, differential count, hematocrit, hemoglobin, red blood cell, platelet counts, were determined by automated methods.
2.1. Quantification of IL-23, IL-22 and IL-10
2.6. Statistical analysis
IL-22, IL-23 and IL-10 plasma concentrations were measured by a quantitative enzyme immunoassay technique. The assays were performed by using commercially available kits (R&D Systems Europe, Abingdon, UK); a microplate reader (BioRad Laboratories, Model 550, Milan) capable of measuring absorbance at 450 nm (correction wavelength set at 540 nm) was used to measure the intensity of color developed in each well. All samples were analyzed in duplicate.
All statistical calculations were performed using the statistical package SPSS for Windows (release 11.5, 2002 software; SPSS UK, Woking, UK). The nonparametric Mann-/Whitney U-test was employed to test the differences between two groups. Spearman correlation coefficient was applied in order to evaluate the interdependence between the variables. Data were expressed as the mean ± standard deviation. p < 0.05 was considered significant. 3. Results and discussion
2.2. Analysis of the JAK2V617F mutation DNA was purified using the QIAmp DNA blood JAK2V617F allele burden and phenotype in essential thrombocythemia kit (Muta Quant Kit Ipsogen). Patients were genotyped for the JAK2V617F mutation by an allele-specific (ASO) polymerase chain reaction (PCR). To evaluate whether the mutation was carried in the homozygous or heterozygous state, PCR products were digested with BsaXI restriction enzyme (New England Biolabs, Hitchin, UK). The mutant allele burden was measured by a quantitative real time (QRT)–PCR assay, using 20 ng genomic DNA. PCR amplification and detection were performed on an ABI Prism 7300 analyzer (Applied Biosystems) using the following cycling conditions: 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Primers flanking the mutant region (forward primer 50 AAGCTTTCTCACAAGCATTTGGTTT-30 ; reverse primer 50 -AGAAAGGCATTAGAAAGCCTG TAGTT-30 ) were employed together with Taqman probes which were specific for either the wild type (VIC–50 -TCTCCACAGACACATAC–30 MGB) or the mutant JAK2 allele (FAM–50 –TCCACAGAAACATAC–30 -MGB). All samples were analyzed in triplicate and the amount of JAK2V617F allele was calculated by comparison with serial dilutions of mutant DNA, obtained from a PV patient with 100% mutant alleles, and wild-type DNA from healthy subjects.
Plasma levels of IL-23 were significantly increased in all patients with MPN in comparison to controls (9.09 ± 6.03 pg/mL vs 6.55 ± 4.125 pg/mL; p < 0.02). Moreover, there was a significant difference between the levels of IL-23 in patients affected by PV and those measured in controls (8.57 ± 3.69 vs. 6.55 ± 4.125 pg/mL; p < 0.03) (Fig. 1). No difference was found between IL-23 levels in ET patients and controls (Table 1). No statistically significant differences were found between levels of IL-10 or IL-22 of PV or ET patients with respect to controls (Table 1), although we found a positive correlation between levels of IL-23 and IL-10 in all patients (r = 0.321; p < 0.031). All MPN patients were positive for JAK2V617F mutation. No statistically significant differences were found between the levels of IL-23, IL-22 or IL-10 in PV or ET subjects with or without thrombosis, in patients with or without pruritus, or according the JAK2V617F burden (Table 1). However, in PV patients the JAK2 burden and Hb levels correlated with occurrence of pruritus (r = 0.471; p < 0.001 and r = 0.335; p < 0.019 respectively), while in all patients we found a correlation between pruritus and white cell counts (r = 0.357; p > 0.012), and a correlation between JAK2 burden and white cell counts (r = 0.515; p < 0.001).
S. Gangemi et al. / Cellular Immunology 278 (2012) 91–94
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Finally in PV patients we found that JAK2 burden correlated with occurrence of pruritus. Pruritus is a common symptom in patients with Philadelphia chromosome-negative myeloproliferative disorders. The pathophysiology of MPD-associated pruritus is unclear. However in most of the studies performed in PV patients an allele burden greater than 50% was found to correlate with occurrence of aquagenic pruritus. Pieri et al. found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, and was correlated with the V617F burden. Moreover ex vivo experiments revealed that pre-treatment with a JAK2 inhibitor reduced PV basophil activation [15]. Our data seems to confirm the existence of a relationship between IAK2 mutation and pruritus. Fig. 1. IL-23 plasma levels (pg/mL) in controls and patients; ⁄p<0.02 vs. controls; # p < 0.02 vs. controls.
Table 1 IL-23, IL-22, IL-10 plasma levels (pg/mL) in controls and patients; ⁄p < 0.03 vs. controls; #p < 0.02 vs. controls.
Controls PV ET MPN
IL-23
IL-22
IL-10
6.55 ± 4.125 8.57 ± 3.68⁄ 8.35 ± 4.33 9.09 ± 6.03#
5.63 ± 5.67 6.21 ± 3.95 6.10 ± 4.62 6.05 ± 4.31
3.63 ± 2.18 3.61 ± 3.22 2.74 ± 2.28 3.18 ± 2.70
Although Th17 cells have been found in experimental animal models of cancer and in human cancers, whether these cells promote tumor growth or regulate antitumor responses remains controversial. It was demonstrated that an enhanced size of tumors developed after subcutaneous injection of stably IL-22-transfected HepG2 human hepatoma cells in nude mice. In general, favored growth of tumor cells by IL-22 may be due to induced protection against apoptosis and injury. Endogenous expression of IL-23 has been reported to promote tumor incidence and growth, and IL-23/IL-23R pathway is a potential route to facilitate the malignant progression of cancers [7]. In previous works we found that interleukin 22 is increased and correlated with CD38 expression in patients with B-chronic lymphocytic leukemia [8], while an involvement of T2677T multidrug resistance gene polymorphism in interleukin 22 plasma concentration was pointed out in the same subjects [9]. Moreover we found an increase of interleukin-23 levels in resected colorectal cancer before and after chemotherapy [10]. In the present work we found a significant difference between the plasma levels of IL-23 in patients affected by PV and those measured in controls. Several mechanisms could explain this result. Hus et al. also found increased frequencies of Th17 cells in patients with MPDs disorders such as chronic myeloid leukemia [11]. Moreover, Che Mat et al. suggested a positive feedback regulation of the IL-23 receptor via IL-23-mediated activation of the JAK/STAT pathway [12]. IL-23 is in fact a pro-carcinogenic cytokine, promoting inflammation and angiogenesis within the tumor microenvironment. The presence of these Th17 cells within the tumor microenvironment could antagonize and counter the tumor-suppressive IFN-c producing CD4+ Th1 cells [13]. In addition, a study of murine tumor formation, using the B16 melanoma cell line, found that this signaling was critical for tumor development [14]. Th17 cells antagonize the differentiation of IFN-c producing Th1 cells, and are thereby likely involved the promotion of tumor growth.
4. Conclusions section In conclusion, although our study seems to point out a possible involvement of IL-23 in the pathogenesis of PV, a larger study is needed to confirm our results. In fact, in our study, the differences obtained between patients and controls was only around 2 pg/ml, and this could be a limitation for the interpretation of our results, as cytokines plasma levels varies depending on multifactorial aspects, namely the presence or recent resolution of an inflammatory process, the circadian cycle, time of diagnosis or treatment, and tumor burden, and a study is needed to confirm our results in enlarged MPDs and control cohorts. Further experiments should considered to investigate the role of IL-23 in PV patients, to explore the possibility that IL-23 is produced by neoplastic cells or to check for expression of the IL-23 receptor on myeloid cells. Moreover, as MPN are clonal cell disorders, which starts in a hematopoietic stem cell, the measurement of the studied cytokines in bone marrow aspirates could bring more reliable results, while an interesting approach could be to evaluate if the exogenous addition of IL-23 in cell cultures from MPN bone marrow aspirates samples, promote an increase in the expansion of JAK2 mutations positive cells. Finally a correlation between IL-23 level and progression or response to treatment or final outcome would clarify the relationship IL-23 and myeloproliferative disorders. Conflicts of interest None to declare. Acknowledgment We would like to thank Mr. Calum Stirling for reading the manuscript. References [1] A. Pradhan, Q.T. Lambert, L.N. Griner, G.W. Reuther, Activation of JAK2–V617F by components of heterodimeric cytokine receptors, J. Biol. Chem. 285 (2010) 16651–16663. [2] T. Manshouri, Z. Estrov, A. Quintás-Cardama, J. Burger, Y. Zhang, A. Livun, J. Burger, Y. Zhang, A. Livun, et al., Bone marrow stroma-secreted cytokines protect JAK2(V617F)-mutated cells from the effects of a JAK2 inhibitor, Cancer Res. 71 (2011) 3831–3840. [3] B.C. Betts, O. Abdel-Wahab, S.A. Curran, E.T. St. Angelo, P. Koppikar, G. Heller, R.L. Levine, J.W. Young, Janus kinase-2 inhibition induces durable tolerance to alloantigen by human dendritic cell-stimulated T cells yet preserves immunity to recall antigen, Blood 118 (2011) 5330–5339. [4] S. Romagnani, E. Maggi, F. Liotta, L. Cosmi, F. Annunziato, Properties and origin of human Th17 cells, Mol. Immunol. 47 (2009) 3–7. [5] N.J. Wilson, K. Boniface, J.R. Chan, Development, cytokine profile and function of human interleukin 17-producing helper T cells, Nat. Immunol. 8 (2007) 950–957. [6] R.L. Brunsing, E.R. Prossnitz, Induction of interleukin-10 in the T helper type 17 effector population by the G protein coupled estrogen receptor (GPER) agonist
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S. Gangemi et al. / Cellular Immunology 278 (2012) 91–94 G-1, Immunology (2011), http://dx.doi.org/10.1111/j.1365-2567.2011.03471.x (Epub ahead of print). M.H. Chien, C.H. Hsin, L. Shih-Shen Chou, T.T. Chung, C.H. Lin, M-S. Weng, M.Y. Chou, M.K. Chen, S.F. Yang, Interleukin-23 receptor polymorphism as a risk factor for oral cancer susceptibility, Head Neck (2011), http://dx.doi.org/ 10.1002/hed.21779. Epub ahead of print. S. Gangemi, A. Allegra, A. Alonci, E. Pace, A. Cannavò, M. Ferraro, G. Penna, S. Saitta, D. Gerace, C. Musolino, Interleukin 22 is increased and correlated with CD38 expression in patients with B-Chronic Lymphocytic Leukemia, Blood Cell Mol. Dis., in press. C. Musolino, A. Allegra, M. Ferraro, M. Aguennouz, S. Russo, A. Alonci, S. Saitta, S. Gangemi, Involvement of T2677T multidrug resistance gene polymorphism in Interleukin 22 plasma concentration in B-chronic lymphocytic leukemia patients, Acta Oncol. (2011). epub ahead of print. V. Adamo, T. Franchina, P.L. Minciullo, E. Pace, F. Colonese, G.R. Ricciardi, S. Saitta, M. Ferraro, G. Spatari, G. Gangemi, Role of interleukin-23 circulating levels increase in resected colorectal cancer before and after chemotherapy: preliminary data and future perspectives, J. Cell Physiol. 226 (2011) 3032–3034.
[11] I. Hus, J. Tabarkiewicz, M. Lewandowska, M. Wasiak, P. Wdowiak, M. Kusz, M. Legiec, A. Dmoszynska, J. Rolinski, Evaluation of monocyte-derived dendritic cells T regulatory and Th17 cells in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors, Folia Histochem. Cytobiol. 49 (2011) 153–160. [12] N.F. Che Mat, X. Zhang, C. Guzzo, K. Gee, Interleukin-23-induced interleukin23 receptor inpathway in human CD4 T cells, J. Interferon Cytokine Res. 31 (2011) 363–371. [13] W.W. Lin, M. Karin, A cytokine-mediated link between innate immunity, inflammation, and cancer, J. Clin. Invest. 117 (2007) 1175–1183. [14] L. Wang, T. Yi, M. Kortylewski, D.M. Pardoll, D. Zeng, H. Yu, IL-17 can promote tumor growth through an IL-6–Stat3 signaling pathway, JEM 206 (2009) 1457–1464. [15] L. Pieri, C. Bogani, P. Guglielmelli, M. Zingariello, R.A. Rana, N. Bartalucci, A. Bosi, A.M. Vannucchi, The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera, Haematologica 94 (2009) 1537–1545.
Contents lists available at SciVerse ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Evaluation of interleukin-23 plasma levels in patients with polycythemia vera and essential thrombocythemia Sebastiano Gangemi a,b, Alessandro Allegra c, Elisabetta Pace b, Andrea Alonci c, Maria Ferraro b, Annamaria Petrungaro c, Salvatore Saitta a, Demetrio Gerace c, Sabina Russo c, Giuseppa Penna c, Caterina Musolino c,⇑ a b c
School and Division of Allergy and Clinical Immunology, Department of Human Pathology, University of Messina, Italy Institute of Biomedicine and Molecular Immunology ‘‘A. Monroy’’ (IBIM), Consiglio Nazionale delle Ricerche (CNR), Palermo, Italy Division of Haematology, University of Messina, Italy
a r t i c l e
i n f o
Article history: Received 17 April 2012 Accepted 13 July 2012 Available online 1 August 2012 Keywords: Essential thrombocythemia Polycythemia vera JAK2 mutation Interleukin-23 Interleukin-22 Interleukin-10
a b s t r a c t Essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis share the same acquired genetic lesion, JAK2V617F. It is believed that cytokines participate in the activation of JAK2V617F. In this study, we analyzed the plasma levels of interleukin (IL)-23, IL-10 and IL-22 in patients with PV and ET. In the same subjects we also performed analysis of the JAK2V617F mutation, and evaluated a possible relationship between interleukin levels and thrombotic complications or with the symptom pruritus. Plasma levels of IL-23 were significantly increased in all patients with MPN in comparison to controls. Moreover, there was a significant difference between the levels of IL-23 in patients affected by PV and those measured in controls (8.57 ± 3.69 pg/mL vs. 6.55 ± 4.125 pg/mL; p < 0.03). No difference was found between IL-23 levels in ET patients and controls. No statistically significant differences were found between the levels of IL-23, Il-22 or IL-10 in PV or ET subjects with or without thrombosis, in patients with or without pruritus, or according the JAK2V617F burden. In PV patients the JAK2 burden and Hb levels correlated with occurrence of pruritus. Our study seems to point out a possible involvement of IL-23 in the pathogenesis of PV. Ó 2012 Published by Elsevier Inc.
1. Introduction Myeloproliferative disorders are a group of clonal hematopoietic disorders resulting in aberrant production of cells of the myeloid lineage. Myeloproliferative neoplasms (MPN) that do not contain the BCR–ABL mutation include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). In 2005, multiple research groups reported the identification of a JAK2 point mutation in this kind of MPN. This mutation affects the auto-inhibitory domain of JAK2 leading to constitutive activation of JAK2 and JAK/STAT signaling. However, the detailed mechanism by which abnormal activation of JAK2 exhibits transforming activity remains to be elucidated. It is believed that cytokines participate in the activation of JAK2V617F, presumably by providing a scaffolding function where two JAK2V617F molecules could properly be juxtaposed to allow for transphosphorylation and subsequent full activation of the
⇑ Corresponding author. Address: Division of Hematology, Via Consolare Valeria, Policlinico G. Martino, University of Messina, Messina, Italy. Fax: +39 090 2935162. E-mail address: [email protected] (C. Musolino). 0008-8749/$ - see front matter Ó 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.cellimm.2012.07.003
tyrosine kinase. Pradhan et al. demonstrated that components of heterodimeric cytokine receptors can also activate JAK2V617F. Expression of IL27Ra, a heterodimeric receptor component, enhanced the activation of JAK2V617F and subsequent downstream signaling to activation of STAT5 and ERK [1]. Moreover, the role of cytokines is confirmed by recent reports that have described complete or major molecular remission in patients with PV after long-term treatment with immunomodulatory agents, while bone marrow stroma-secreted cytokines seem to protect JAK2V617F-mutated cells from the effects of a JAK2 inhibitor [2]. Finally JAK2 inhibition is able to impair the expansion of responder T helper 17 (Th17) cells, a novel population of Th cells so named because of its ability to produce IL-17A [3]. Th17 cells produce IL-22 [4]. IL-22 is also produced by Th22, Th1 cells, classical and non-classical (NK-22) NK cells, NKT cells, and lymphoid tissue inducer cells. IL-23, a member of the IL-12 family of cytokines, plays an important role in Th17 maintenance [5]. Finally IL-10 is a potent suppressor of the immune system, commonly produced by CD4(+) T cells and T regulatory cells (Th 2, Th9, Th22), which has an important role in the biology of B cells and T cells, and recently Brunsing et al. have demonstrated that IL10 is produced by Th17 cells [6].
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In this study, we analyzed the plasma levels of IL - 23, and IL-22 cytokines produced by Th17 cells, and plasma levels of IL-10 a cytokine able to regulate Th17 function, in patients with PV and ET. In the same subjects we also performed analysis of the JAK2V617F mutation, and evaluated a possible relationship between interleukin levels and thrombotic complications or with the symptom pruritus. 2. Material and methods Our population consisted of 28 patients with ET (12 M – 16 F; mean age 62 ± 8 yrs) and 20 patients with PV 13 M - 7 F; mean age 64 ± 11 yrs). Fifteen patients (7 with ET, 8 with PV) had thrombotic manifestation in the past. All patients were not receiving aspirin or any treatment for ET or PV over seven days. The study was conducted according to good clinical and laboratory practice rules and the principles of the Declaration of Helsinki. Informed written consent was obtained after the purpose, nature, and potential risks were explained to the subjects. Plasma from 28 sex-matched and age matched normal subjects (11 M -17 F; mean age 59 ± 8) were also included in this study as controls. Peripheral venous blood was collected using citrate as anticoagulant. Within 30 min after collection, samples were centrifuged for 15 min at 200 g in a 4235 A centrifuge (ALC Int S.r.L. Milan, Italy); plasma was immediately separated, aliquoted and stored in a 80 °C freezer until the assay was performed. Freeze– thaw cycles were avoided. None of the patients or control subjects had symptoms or laboratory signs of active infections, inflammatory diseases, diabetes or kidney failure.
The mean of triplicate DCT determinations (CT JAK2V617F–CT JAK2WT) was used to calculate the percentage of mutant alleles. Positive and negative controls were included in each assay; interand intra-assay variation was 3% and 5%, respectively. 2.3. Diagnosis of thrombosis Vascular thrombosis was defined as a history of occlusive vascular events. Major events included peripheral thrombosis, cardiac vascular complication, central nervous system vascular complications, and intra-abdominal vascular complications. Minor events were erythromelalgia and superficial thrombophlebitis of the extremities. No patient was studied during the acute phase of the thrombosis. 2.4. Pruritus We also evaluated the prevalence of pruritus in our patients. Pruritus was auto-assessed by the patients, using a 0–2 scale (from 0 = no itch at all, to 2 = extreme itch). The prevalence of pruritus in our study was 33.0% (grade 0: 32 patients; grade 1: 8 patients – PV 5 patients; ET 3 patients; grade 2: 8 subjects – PV 5 patients; ET 3 patients). 2.5. Routine hematologic assays White blood cell, differential count, hematocrit, hemoglobin, red blood cell, platelet counts, were determined by automated methods.
2.1. Quantification of IL-23, IL-22 and IL-10
2.6. Statistical analysis
IL-22, IL-23 and IL-10 plasma concentrations were measured by a quantitative enzyme immunoassay technique. The assays were performed by using commercially available kits (R&D Systems Europe, Abingdon, UK); a microplate reader (BioRad Laboratories, Model 550, Milan) capable of measuring absorbance at 450 nm (correction wavelength set at 540 nm) was used to measure the intensity of color developed in each well. All samples were analyzed in duplicate.
All statistical calculations were performed using the statistical package SPSS for Windows (release 11.5, 2002 software; SPSS UK, Woking, UK). The nonparametric Mann-/Whitney U-test was employed to test the differences between two groups. Spearman correlation coefficient was applied in order to evaluate the interdependence between the variables. Data were expressed as the mean ± standard deviation. p < 0.05 was considered significant. 3. Results and discussion
2.2. Analysis of the JAK2V617F mutation DNA was purified using the QIAmp DNA blood JAK2V617F allele burden and phenotype in essential thrombocythemia kit (Muta Quant Kit Ipsogen). Patients were genotyped for the JAK2V617F mutation by an allele-specific (ASO) polymerase chain reaction (PCR). To evaluate whether the mutation was carried in the homozygous or heterozygous state, PCR products were digested with BsaXI restriction enzyme (New England Biolabs, Hitchin, UK). The mutant allele burden was measured by a quantitative real time (QRT)–PCR assay, using 20 ng genomic DNA. PCR amplification and detection were performed on an ABI Prism 7300 analyzer (Applied Biosystems) using the following cycling conditions: 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Primers flanking the mutant region (forward primer 50 AAGCTTTCTCACAAGCATTTGGTTT-30 ; reverse primer 50 -AGAAAGGCATTAGAAAGCCTG TAGTT-30 ) were employed together with Taqman probes which were specific for either the wild type (VIC–50 -TCTCCACAGACACATAC–30 MGB) or the mutant JAK2 allele (FAM–50 –TCCACAGAAACATAC–30 -MGB). All samples were analyzed in triplicate and the amount of JAK2V617F allele was calculated by comparison with serial dilutions of mutant DNA, obtained from a PV patient with 100% mutant alleles, and wild-type DNA from healthy subjects.
Plasma levels of IL-23 were significantly increased in all patients with MPN in comparison to controls (9.09 ± 6.03 pg/mL vs 6.55 ± 4.125 pg/mL; p < 0.02). Moreover, there was a significant difference between the levels of IL-23 in patients affected by PV and those measured in controls (8.57 ± 3.69 vs. 6.55 ± 4.125 pg/mL; p < 0.03) (Fig. 1). No difference was found between IL-23 levels in ET patients and controls (Table 1). No statistically significant differences were found between levels of IL-10 or IL-22 of PV or ET patients with respect to controls (Table 1), although we found a positive correlation between levels of IL-23 and IL-10 in all patients (r = 0.321; p < 0.031). All MPN patients were positive for JAK2V617F mutation. No statistically significant differences were found between the levels of IL-23, IL-22 or IL-10 in PV or ET subjects with or without thrombosis, in patients with or without pruritus, or according the JAK2V617F burden (Table 1). However, in PV patients the JAK2 burden and Hb levels correlated with occurrence of pruritus (r = 0.471; p < 0.001 and r = 0.335; p < 0.019 respectively), while in all patients we found a correlation between pruritus and white cell counts (r = 0.357; p > 0.012), and a correlation between JAK2 burden and white cell counts (r = 0.515; p < 0.001).
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Finally in PV patients we found that JAK2 burden correlated with occurrence of pruritus. Pruritus is a common symptom in patients with Philadelphia chromosome-negative myeloproliferative disorders. The pathophysiology of MPD-associated pruritus is unclear. However in most of the studies performed in PV patients an allele burden greater than 50% was found to correlate with occurrence of aquagenic pruritus. Pieri et al. found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, and was correlated with the V617F burden. Moreover ex vivo experiments revealed that pre-treatment with a JAK2 inhibitor reduced PV basophil activation [15]. Our data seems to confirm the existence of a relationship between IAK2 mutation and pruritus. Fig. 1. IL-23 plasma levels (pg/mL) in controls and patients; ⁄p<0.02 vs. controls; # p < 0.02 vs. controls.
Table 1 IL-23, IL-22, IL-10 plasma levels (pg/mL) in controls and patients; ⁄p < 0.03 vs. controls; #p < 0.02 vs. controls.
Controls PV ET MPN
IL-23
IL-22
IL-10
6.55 ± 4.125 8.57 ± 3.68⁄ 8.35 ± 4.33 9.09 ± 6.03#
5.63 ± 5.67 6.21 ± 3.95 6.10 ± 4.62 6.05 ± 4.31
3.63 ± 2.18 3.61 ± 3.22 2.74 ± 2.28 3.18 ± 2.70
Although Th17 cells have been found in experimental animal models of cancer and in human cancers, whether these cells promote tumor growth or regulate antitumor responses remains controversial. It was demonstrated that an enhanced size of tumors developed after subcutaneous injection of stably IL-22-transfected HepG2 human hepatoma cells in nude mice. In general, favored growth of tumor cells by IL-22 may be due to induced protection against apoptosis and injury. Endogenous expression of IL-23 has been reported to promote tumor incidence and growth, and IL-23/IL-23R pathway is a potential route to facilitate the malignant progression of cancers [7]. In previous works we found that interleukin 22 is increased and correlated with CD38 expression in patients with B-chronic lymphocytic leukemia [8], while an involvement of T2677T multidrug resistance gene polymorphism in interleukin 22 plasma concentration was pointed out in the same subjects [9]. Moreover we found an increase of interleukin-23 levels in resected colorectal cancer before and after chemotherapy [10]. In the present work we found a significant difference between the plasma levels of IL-23 in patients affected by PV and those measured in controls. Several mechanisms could explain this result. Hus et al. also found increased frequencies of Th17 cells in patients with MPDs disorders such as chronic myeloid leukemia [11]. Moreover, Che Mat et al. suggested a positive feedback regulation of the IL-23 receptor via IL-23-mediated activation of the JAK/STAT pathway [12]. IL-23 is in fact a pro-carcinogenic cytokine, promoting inflammation and angiogenesis within the tumor microenvironment. The presence of these Th17 cells within the tumor microenvironment could antagonize and counter the tumor-suppressive IFN-c producing CD4+ Th1 cells [13]. In addition, a study of murine tumor formation, using the B16 melanoma cell line, found that this signaling was critical for tumor development [14]. Th17 cells antagonize the differentiation of IFN-c producing Th1 cells, and are thereby likely involved the promotion of tumor growth.
4. Conclusions section In conclusion, although our study seems to point out a possible involvement of IL-23 in the pathogenesis of PV, a larger study is needed to confirm our results. In fact, in our study, the differences obtained between patients and controls was only around 2 pg/ml, and this could be a limitation for the interpretation of our results, as cytokines plasma levels varies depending on multifactorial aspects, namely the presence or recent resolution of an inflammatory process, the circadian cycle, time of diagnosis or treatment, and tumor burden, and a study is needed to confirm our results in enlarged MPDs and control cohorts. Further experiments should considered to investigate the role of IL-23 in PV patients, to explore the possibility that IL-23 is produced by neoplastic cells or to check for expression of the IL-23 receptor on myeloid cells. Moreover, as MPN are clonal cell disorders, which starts in a hematopoietic stem cell, the measurement of the studied cytokines in bone marrow aspirates could bring more reliable results, while an interesting approach could be to evaluate if the exogenous addition of IL-23 in cell cultures from MPN bone marrow aspirates samples, promote an increase in the expansion of JAK2 mutations positive cells. Finally a correlation between IL-23 level and progression or response to treatment or final outcome would clarify the relationship IL-23 and myeloproliferative disorders. Conflicts of interest None to declare. Acknowledgment We would like to thank Mr. Calum Stirling for reading the manuscript. References [1] A. Pradhan, Q.T. Lambert, L.N. Griner, G.W. Reuther, Activation of JAK2–V617F by components of heterodimeric cytokine receptors, J. Biol. Chem. 285 (2010) 16651–16663. [2] T. Manshouri, Z. Estrov, A. Quintás-Cardama, J. Burger, Y. Zhang, A. Livun, J. Burger, Y. Zhang, A. Livun, et al., Bone marrow stroma-secreted cytokines protect JAK2(V617F)-mutated cells from the effects of a JAK2 inhibitor, Cancer Res. 71 (2011) 3831–3840. [3] B.C. Betts, O. Abdel-Wahab, S.A. Curran, E.T. St. Angelo, P. Koppikar, G. Heller, R.L. Levine, J.W. Young, Janus kinase-2 inhibition induces durable tolerance to alloantigen by human dendritic cell-stimulated T cells yet preserves immunity to recall antigen, Blood 118 (2011) 5330–5339. [4] S. Romagnani, E. Maggi, F. Liotta, L. Cosmi, F. Annunziato, Properties and origin of human Th17 cells, Mol. Immunol. 47 (2009) 3–7. [5] N.J. Wilson, K. Boniface, J.R. Chan, Development, cytokine profile and function of human interleukin 17-producing helper T cells, Nat. Immunol. 8 (2007) 950–957. [6] R.L. Brunsing, E.R. Prossnitz, Induction of interleukin-10 in the T helper type 17 effector population by the G protein coupled estrogen receptor (GPER) agonist
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