Lymphoma

Increased serum levels of interleukin-9 correlate to negative prognostic factors in Hodgkin's lymphoma

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Abstract

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.

Introduction

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.

Immunohistochemistry

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.

Statistical methods

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.

Figure 1
figure1

IL-9 levels in serum samples and conditioned medium. The IL-9 levels in serum samples from 16 healthy donors, 44 HL and 21 NHL patients, and in conditioned media from the HL cell lines DEV, HDLM-2, KM-H2 and L540, were measured by ELISA. The levels are presented as pg/ml, and the limit of detection was 6 pg/ml. The numbers of positives and totals for each group are indicated.

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.

Figure 2
figure2

Serum IL-9 levels in NSHL vs non-NSHL. IL-9 levels in the sera from 30 patients with NSHL subtype compared with the levels from 12 patients with other subtypes. The levels are presented as pg/ml, and the limit of detection was 6 pg/ml.

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).

Figure 3
figure3

Serum levels of IL-9 vs serum levels of IL-13. Levels of IL-9 and IL-13 were compared in 40 HL patients. The levels are presented as pg/ml, and the limits of detection were 6 pg/ml for IL-9 and 12 pg/ml for IL-13.

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.

Figure 4
figure4

Correlation between in situ IL-9 expression and serum levels in HL patients. In situ IL-9 expression in sections from 36 HL tumours was detected by immunohistochemistry and correlated to serum levels, measured by ELISA. A case was considered IL-9 positive if unequivocal staining of the cytoplasm was present in more than 10% of the HRS cells. The number of positive and negative cases were 19 and 17, respectively. The serum levels are presented as pg/ml, and the limit of detection was 6 pg/ml.

Figure 5
figure5

In situ expression of IL-9 in HRS cells. Section of paraffin-embedded HL tumour tissue stained with a polyclonal anti-hIL-9 antibody.

Conclusion

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.

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Acknowledgements

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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Correspondence to G Nilsson.

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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

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Keywords

  • Hodgkin's lymphoma
  • IL-9
  • IL-13
  • serum levels
  • negative prognostic factors

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