Hodgkin's lymphoma (HL) is characterised by an unbalanced cytokine secretion. Many of these cytokines have been implicated in the regulation of malignant and infiltrating cells. Interleukin-9 (IL-9) has been described to act in an autocrine fashion in HL, stimulating proliferation of the malignant cells. To investigate the potential clinical implication of this observation, a novel ELISA method was used to examine the serum levels of IL-9 in lymphoma patients. High levels of IL-9 were found in the sera from patients with HL (18/44), but not in the sera from non-Hodgkin's lymphoma patients (3/21) or healthy controls. The highest serum IL-9 levels, up to 3350 pg/ml, were observed in the nodular sclerosis subtype, and there was a correlation between IL-9 levels and the negative prognostic factors advanced stage, B-symptoms, low blood Hb and high erythrocyte sedimentation rate. Furthermore, there was no correlation between serum levels of IL-9 and IL-13, a cytokine where serum levels have been speculated to be of clinical importance. This is the first report showing that IL-9 can be measured in serum samples. A novel correlation between increased serum IL-9 levels, HL and clinical features is shown, suggesting that IL-9 is a candidate factor contributing to the development of HL.
Hodgkin's Lymphoma (HL) differs from other malignant lymphomas in that the tumours are composed mainly of benign reactive cells and just a minority of malignant cells, the so-called Hodgkin and Reed–Sternberg (HRS) cells. HRS cells produce and secrete a wide variety of cytokines in an abnormal fashion, leading to autocrine growth stimulation, recruitment and activation of the reactive infiltrate and defective host immune responses.1,2 These different cytokines may also be responsible for sclerosis, tissue eosinophilia and the B-symptoms – night sweats, fever and weight loss – seen in many HL patients.3,4
One cytokine associated with HL is interleukin-9 (IL-9).5 This is a multifunctional cytokine mainly produced by stimulated TH2-cells, first described to allow antigen-independent proliferation of murine T-helper cell clones,6 and enhance proliferation in murine mast cell lines.7 The spectrum of biological activities of IL-9 has expanded, and in humans it is believed to affect the differentiation of haematopoietic progenitors and B-cells,8 and promote the proliferation of mast cell progenitors.9 In diseases such as asthma, allergy and helminth infections, IL-9 seems to be of importance for the inflammatory responses.8 In addition to all its functions on normal cells, IL-9 has been identified as an autocrine growth factor for HRS cells.10 In HL tumours, IL-9 is also believed to work in a paracrine fashion, stimulating and activating the infiltrating TH2 type of lymphocytes. Interactions with other cells constituting the tumours such as mast cells,9,11 monocytes12 and eosinophils13 are also plausible.
Another pleiotropic TH2 cytokine implicated in autocrine stimulation of HRS cells is IL-13.14 In recent studies, the role of IL-13 in HL has been investigated by in situ hybridisation in primary lymphoma tissues,15 and by measuring protein levels in serum samples from HL patients.16 Despite the fact that IL-13 was found to be commonly expressed by HRS cells,15 only 10% of the HL patients had detectable levels of IL-13 present in the serum.16
If IL-9 plays a role in HL, it is of great interest to investigate if this cytokine can be measured in the serum, which has not been possible before, and if serum levels are of clinical relevance. In this study, we have utilised a newly developed ELISA assay to measure IL-9 in serum samples from patients with HL, non-Hodgkin's lymphoma (NHL) and healthy volunteers. We report here that increased levels of IL-9 were preferentially found in the sera from HL patients, and that increased IL-9 levels correlated to negative prognostic factors.
Materials and methods
Patients and clinical characteristics
In total, 44 cases of HL, 21 cases of NHL and 16 healthy control individuals were included in the study. Serum samples were taken at diagnosis, except for four patients with HL of which two were taken at relapse and two in complete remission. All HL cases, except the two patients in complete remission, were reclassified according to the WHO classification, and staged according to the Ann Arbor system.17 In all, 16 patients were in stage I, 15 in stage II, five in stage III and two in stage IV, and in four patients the stage was not known. Six patients had B-symptoms. The distribution in histopathological subgroups was: 30 nodular sclerosis HL (NSHL), six mixed cellularity HL (MCHL) and six nodular lymphocytic predominant HL (NLPHL). Correlation studies of clinical characteristics and serum IL-9 levels were performed in the 40 HL patients where samples were taken at diagnosis.
Cell lines and conditioned medium
The HL cell lines HDLM-2, DEV, KM-H2 and L540 were maintained in complete RPMI-1640 medium.18 Supernatants were obtained 24 h after passage.
ELISA of IL-9 and IL-13
The serum levels of IL-9 were measured by using an ELISA recently described.19 The sensitivity limit for quantitative determination was 6 pg/ml. In order to exclude false positives, all samples were incubated on plates coated with an anti-IL-9, or an isotype-matched irrelevant antibody, before addition of the detection anti-IL-9 antibody. Serum levels of IL-13 were measured in the serum from 40 of the HL patients, using the human IL-13 immunoassay kit (detection limit 12 pg/ml) (BioSource International, Inc. CA, USA), according to the manufacturer's instructions.
Paraffin sections from 36 HL tumours were stained with a polyclonal anti-hIL-9 antibody (R&D Systems, Abingdon, UK) and a standard ABC protocol (ABC Complex/HPRT, DAKO, Denmark) using DAB as chromogen. A case was considered IL-9 positive if unequivocal staining of the cytoplasm was present in more than 10% of HRS cells.
The Mann–Whitney U-test was used to compare IL-9 values between different groups, and the Fisher exact test was used to compare proportions between groups. The Spearman rank correlation was used to compare serum levels of cytokines.
Results and discussion
IL-9 in serum samples
IL-9 has previously been described in HL at an expressional level.5,10 In this study, we have examined if IL-9 can be detected in lymphoma serum samples, and if there is a relation to HL. IL-9 levels were measured in the serum from HL, NHL and healthy volunteers, using a newly developed ELISA. Supernatants from HL cell lines were also analysed.
The results presented in Figure 1 demonstrate that serum samples from patients with HL contained increased levels of IL-9 in a substantially higher frequency than the sera from patients with NHL and healthy controls. In fact, IL-9 could be detected in 18 out of the 44 HL patient sera. The two patients in remission were negative, indicating that IL-9 is produced within the tumours. In contrast, IL-9 was only found in three out of the 21 NHL patient sera (one B-lymphoblastic lymphoma, one low-grade malignant B-cell lymphoma and one high-grade malignant lymphoma not further possible to classify). Sera from the healthy controls contained no detectable levels of IL-9. One out of the four cell lines tested, HDLM-2, produced measurable levels of IL-9.
Correlation between serum IL-9 levels, histopathology and negative prognostic factors in HL patients
Earlier studies postulate that there is an association between different cytokines produced in the HL tissues and histopathologic subtypes.20,21,22 IL-9, among other cytokines, has been proposed to be involved in the fibrosis seen in NSHL.23 When comparing subtypes, a distinction could be made between NS and the other histopathological subtypes, since the highest concentrations (up to 3350 pg/ml) of IL-9 were found in the sera from patients diagnosed as NSHL (Figure 2). Furthermore, there was a correlation between the serum IL-9 levels and some clinical characteristics. The levels were higher in patients with B-symptoms (P=0.04) and stage III–IV disease (P=0.03), but there was no relation to sex, age or bulky disease. There was also a correlation between the serum IL-9 values and the negative prognostic factors high erythrocyte sedimentation rate (ESR) (R=0.47, P=0.006) and low B-haemoglobin (R=−0.38, P=0.03). The correlation between IL-9 and these negative prognostic factors is interesting, and warrants further investigation in a larger material to evaluate the prognostic importance of IL-9 in HL.
Serum IL-9 levels vs serum IL-13 levels in HL patients
Although IL-13 is commonly expressed by HRS cells and its role as an autocrine growth factor in HL has been emphasised,24 serum levels were not detected in the majority of cases, as reported in a recent study.16 In order to investigate this further and to compare the levels of IL-9 and IL-13, we measured IL-13 in the sera from 40 HL patients. In contrast to IL-9 that was present in 16 cases (40%), IL-13 was only measurable in five (12%). Surprisingly, no correlation between IL-9 and IL-13 could be found; rather the contrary was observed, that is, patients high in IL-9 were low in IL-13 and vice versa (Figure 3).
Expression of IL-9 in HL tumour tissues
If the tumour tissue produces the IL-9 present in the serum, a correlation between in situ IL-9 expression in tumour sections and serum levels can be assumed. Immunohistochemical stainings showed such a relation in this material (P=0.04) (Figure 4). In most positive cases, almost all HRS cells were positively stained (Figure 5). These results strengthen the hypothesis that IL-9 present in the serum stems from malignant cells.
The results presented in this report show that it is possible to measure IL-9 levels in serum samples. The presence of serum IL-9 correlates to HL, and to negative prognostic factors associated to this malignancy. The highest IL-9 levels are found in the sera from patients with NSHL, and within this group extremely high levels are reported. According to our data, there is an inverse relation between serum levels of IL-9 and IL-13 in HL, and IL-9 is more commonly found in HL sera compared to IL-13. Furthermore, the association between IL-9-expressing HRS cells within the tumour tissue and serum IL-9 levels indicates that the malignant cells are the source of production.
A role for IL-9 in HL would be consistent with its ability to stimulate HRS cells,5,10 and interact with infiltrating cells such as mast cells and eosinophils.8,9,13,25,26 Both mast cells and eosinophils are implicated in the stimulation of HRS cells,4,18 and the number of these cell types in affected lymph nodes correlates with poor prognosis.27,28,29 Thus, IL-9 may be of importance in HL development, regulating both HRS cells as well as infiltrating cells. Our data show the importance of IL-9 in HL, and implies a further role of IL-9 in the development of NSHL.
Maggio E, van den Berg A, Diepstra A, Kluiver J, Visser L, Poppema S . Chemokines, cytokines and their receptors in Hodgkin's lymphoma cell lines and tissues. Ann Oncol 2002; 13 (Suppl 1): 52–56.
Skinnider BF, Mak TW . The role of cytokines in classical Hodgkin lymphoma. Blood 2002; 99: 4283–4297.
Gruss HJ, Pinto A, Duyster J, Poppema S, Herrmann F . Hodgkin's disease: a tumor with disturbed immunological pathways. Immunol Today 1997; 18: 156–163.
Pinto A, Gattei V, Zagonel V, Aldinucci D, Degan M, De Iuliis A et al. Hodgkin's disease: a disorder of dysregulated cellular cross-talk. Biotherapy 1998; 10: 309–320.
Merz H, Houssiau FA, Orscheschek K, Renauld JC, Fliedner A, Herin M et al. Interleukin-9 expression in human malignant lymphomas: unique association with Hodgkin's disease and large cell anaplastic lymphoma. Blood 1991; 78: 1311–1317.
Uyttenhove C, Simpson RJ, Van Snick J . Functional and structural characterization of P40, a mouse glycoprotein with T-cell growth factor activity. Proc Natl Acad Sci USA 1988; 85: 6934–6938.
Hultner L, Druez C, Moeller J, Uyttenhove C, Schmitt E, Rude E et al. Mast cell growth-enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGFIII (interleukin 9). Eur J Immunol 1990; 20: 1413–1416.
Demoulin JB, Renauld JC . Interleukin 9 and its receptor: an overview of structure and function. Int Rev Immunol 1998; 16: 345–364.
Matsuzawa S, Sakashita K, Kinoshita T, Ito S, Yamashita T, Koike K . IL-9 enhances the growth of human mast cell progenitors under stimulation with stem cell factor. J Immunol 2003; 170: 3461–3467.
Gruss HJ, Brach MA, Drexler HG, Bross KJ, Herrmann F . Interleukin 9 is expressed by primary and cultured Hodgkin and Reed–Sternberg cells. Cancer Res 1992; 52: 1026–1031.
Renauld JC, Kermouni A, Vink A, Louahed J, Van Snick J . Interleukin-9 and its receptor: involvement in mast cell differentiation and T cell oncogenesis. J Leukoc Biol 1995; 57: 353–360.
Pilette C, Ouadrhiri Y, Van Snick J, Renauld JC, Staquet P, Vaerman JP et al. IL-9 inhibits oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human monocytes through TGF-beta. J Immunol 2002; 168: 4103–4111.
Louahed J, Zhou Y, Maloy WL, Rani PU, Weiss C, Tomer Y et al. Interleukin 9 promotes influx and local maturation of eosinophils. Blood 2001; 97: 1035–1042.
Kapp U, Yeh WC, Patterson B, Elia AJ, Kagi D, Ho A et al. Interleukin 13 is secreted by and stimulates the growth of Hodgkin and Reed–Sternberg cells. J Exp Med 1999; 189: 1939–1946.
Skinnider BF, Elia AJ, Gascoyne RD, Trumper LH, von Bonin F, Kapp U et al. Interleukin 13 and interleukin 13 receptor are frequently expressed by Hodgkin and Reed–Sternberg cells of Hodgkin lymphoma. Blood 2001; 97: 250–255.
Fiumara P, Cabanillas F, Younes A . Interleukin-13 levels in serum from patients with Hodgkin disease and healthy volunteers. Blood 2001; 98: 2877–2878.
Carbone PP, Kaplan HS, Musshoff K, Smithers DW, Tubiana M . Report of the Committee on Hodgkin's Disease Staging Classification. Cancer Res 1971; 31: 1860–1861.
Molin D, Fischer M, Xiang Z, Larsson U, Harvima I, Venge P et al. Mast cells express functional CD30 ligand and are the predominant CD30L-positive cells in Hodgkin's disease. Br J Haematol 2001; 114: 616–623.
Jenmalm MC, Van Snick J, Cormont F, Salman B . Allergen-induced Th1 and Th2 cytokine secretion in relation to specific allergen sensitization and atopic symptoms in children. Clin Exp Allergy 2001; 31: 1528–1535.
Kadin ME, Agnarsson BA, Ellingsworth LR, Newcom SR . Immunohistochemical evidence of a role for transforming growth factor beta in the pathogenesis of nodular sclerosing Hodgkin's disease. Am J Pathol 1990; 136: 1209–1214.
Ohshima K, Sugihara M, Suzumiya J, Haraoka S, Kanda M, Shimazaki K et al. Basic fibroblast growth factor and fibrosis in Hodgkin's disease. Pathol Res Pract 1999; 195: 149–155.
Newcom SR, Tagra KK . High molecular weight transforming growth factor beta is excreted in the urine in active nodular sclerosing Hodgkin's disease. Cancer Res 1992; 52: 6768–6773.
Hsu SM, Hsu PL . Autocrine and paracrine functions of cytokines in malignant lymphomas. Biomed Pharmacother 1994; 48: 433–444.
Skinnider BF, Kapp U, Mak TW . Interleukin 13: a growth factor in Hodgkin lymphoma. Int Arch Allergy Immunol 2001; 126: 267–276.
Godfraind C, Louahed J, Faulkner H, Vink A, Warnier G, Grencis R et al. Intraepithelial infiltration by mast cells with both connective tissue-type and mucosal-type characteristics in gut, trachea, and kidneys of IL-9 transgenic mice. J Immunol 1998; 160: 3989–3996.
Gounni AS, Gregory B, Nutku E, Aris F, Latifa K, Minshall E et al. Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils. Blood 2000; 96: 2163–2171.
Enblad G, Sundstrom C, Glimelius B . Infiltration of eosinophils in Hodgkin's disease involved lymph nodes predicts prognosis. Hematol Oncol 1993; 11: 187–193.
von Wasielewski R, Seth S, Franklin J, Fischer R, Hubner K, Hansmann ML et al. Tissue eosinophilia correlates strongly with poor prognosis in nodular sclerosing Hodgkin's disease, allowing for known prognostic factors. Blood 2000; 95: 1207–1213.
Molin D, Edstrom A, Glimelius I, Glimelius B, Nilsson G, Sundstrom C et al. Mast cell infiltration correlates with poor prognosis in Hodgkin's lymphoma. Br J Haematol 2002; 119: 122–124.
We wish to thank Dr Carin Backlin (Uppsala University, Sweden) for expert technical assistance, and Professor Per Venge (University Hospital, Uppsala, Sweden) for providing the serum samples of healthy volunteers. This study was supported by the Swedish Cancer Society, Lion's Cancer Research Foundation, Göran Gustafsson's Foundation and Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming.
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Cite this article
Fischer, M., Bijman, M., Molin, D. et al. Increased serum levels of interleukin-9 correlate to negative prognostic factors in Hodgkin's lymphoma. Leukemia 17, 2513–2516 (2003) doi:10.1038/sj.leu.2403123
- Hodgkin's lymphoma
- serum levels
- negative prognostic factors
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