Annals of Oncology Advance Access originally published online on November 15, 2005
Annals of Oncology 2006 17(1):110-116; doi:10.1093/annonc/mdj064
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© 2005 European Society for Medical Oncology
Gene expression profile of cytokines and chemokines in microdissected primary Hodgkin and Reed–Sternberg (HRS) cells: high expression of interleukin-11 receptor 
Departments of 1 Pathology and 3 Internal Medicine, School of Medicine, Fukuoka University, Fukuoka; 2 Department of Medicine and Biosystemic Science, Internal Medicine, Medicine and Surgery, Kyushu University Graduate School of Medical Science, Fukuoka, Japan
* Correspondence to: Dr K. Karube, Department of Pathology, School of Medicine, Fukuoka University, Nanakuma 7-45-1, Jonan-ku, Fukuoka 814-0180, Japan. Tel: +81-942-31-7547; Fax: +81-92-861-7300; E-mail: karube1975{at}yahoo.co.jp
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Abstract |
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We microdissected Hodgkin and Reed–Sternberg (HRS) cells from 14 Hodgkin's lymphoma tissue samples (nodular sclerosis = 5; mixed cellularity = 9), and after isolation and amplification of mRNA, analyzed the expression profile of 140 genes of chemokines, cytokines and their receptors by cDNA microarray methods. We also compared the profile with those of germinal center (GC) cells in reactive lymphadenitis. Unsupervised clustering revealed a relatively homogeneous expression profile in HRS cells. HRS cells tended to express mainly Th2 T cell-associated molecules rather than those of Th1, compared with GC cells. Interleukin-11 receptor
(IL-11R), a previously unknown HRS cell-specific gene, was detected in addition to known genes. Immunohistochemical staining confirmed the expression of IL-11R at the protein level. In contrast, only few cases were positive for IL-11R in B cell lymphoma, diffuse large cell lymphoma and follicular lymphoma. This is the first analysis report of tissue HRS cells with cDNA microarray technique. Key words: Hodgkin, Reed–Sternberg, chemokine, microdissection, cDNA microarray
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introduction |
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Hodgkin's lymphoma (HL), one of the most common malignant lymphomas, was first described by Thomas Hodgkin in 1832. The characteristic morphological feature of HL is the cellular composition of the tumor tissue consisting of a small number (approximately 0.1% to 1%) of neoplastic Hodgkin and Reed–Sternberg (HRS) cells, although the molecular mechanisms of HRS cells remain unknown. Recently, various cytokines and chemokines were reported to be associated with inflammatory and neoplastic state, suggesting that these molecules could play an important role in HL. Therefore, it is important to analyze the cytokine/chemokine profile using with cDNA microarray, a strong tool to analyze numerous genes at a time and to allow the discovery of new genes. A number of reports analyzed the gene expression profile of cell lines derived from HRS cells [1
–3]. However, outgrowth of such cell lines from HL patient tissues is extremely rare, and thus the few cell lines available may not represent the full spectrum of clinical and pathological features of this disease. Furthermore, the environmental difference may alter the level of gene expression in the cell lines compared with HRS cells in tissue. This is particularly important, since certain cytokines, chemokines and their receptors are often upregulated or downregulated according to the surrounding environment in HRS cell lines [4]. For these reasons, the gene expression profile of primary HRS cells in tissue should reflect the cytokine expression pattern in tissue sections, but the paucity of HRS cells in the lesion limits this approach.
The technology described by Luo et al. [5], which integrates laser-capture microscopy of individual cells with linear RNA amplification and cDNA microarray analysis, offers an approach for overcoming the aforementioned limitation. This combined technology was recently applied to the analysis of malignant and premalignant tissues [6]. We used this combination technique in the present study and analyzed the gene expression in primary HRS cells of representative subtypes of HL, mixed cellularity (MC) and nodular sclerosis (NS), compared with the profiles in the germinal center (GC) B cells, which are considered the normal counterpart of HRS cells [7].
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tissue samples
The study samples were 14 human lymphoma samples obtained from 14 patients, which comprised nine MC and five NS. Table 1 summarizes the clinical features of HL patients. We also picked up 12 lymph nodes with follicular hyperplasia. These studies were approved by the patients or their legal guardians, as well as appropriate written consent from each participating institute or hospital. A proportion of the frozen tissue was used. Details of the examination methods have been reported previously [8
]. For immunostaining, 16 cases of diffuse large cell lymphoma (DL) and 15 of follicular lymphoma (FL) were used.
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microdissection of HRS cells and normal GC cells (Figure 1)
Four-micrometer-thick frozen sections were mounted on uncoated or silicone-coated slide glasses and stained with hematoxylin–eosin prepared using RNAase-free water. HRS and GC cells were obtained by laser capture microdissection (model PixCell II; Arcturus Engineering, Inc., Mountain View, CA, USA) from HL and follicular hyperplasia cases, respectively.
RNA isolation and amplification
Total RNA was immediately extracted and isolated from captured cells (
1000 HRS cells or three to five GCs) using the Takara RNA isolation kit (Takara, Otsu, Japan). After isolation, T7 RNA (aRNA) amplification was performed in two cycles using the Takara mRNA HS amplification kit (Takara). The isolated RNAs from normal GC cells of 12 patients were mixed and then amplified. We adopted this amplified RNA as a control.
microarray procedures
Amplified RNA (1 or 2 µg) from HRS and GC cells were directly labeled with cyanine 3-conjugated dUTP (Cy3), while Universal Human Reference RNA (Stratagene, La Jolla, CA, USA) (5 µg) was labeled with cyanine 5-conjugated dUTP (Cy5) as a reference. In all microarray studies, we used the cytokine and chemokine chip v. 2.0 (Kakengeneqs, Matsudo, Japan). Hybridizations were performed as described previously [9]. After washing, the slides were scanned using a Scan Array 4000 scanner (http://www.gsilumonics.com/; GSI Lumonics, Boston, MA, USA). Images were analyzed with QuantArray (GSI Lumonics). Two cases were subjected to repeated analysis to confirm the reproducibility of the microarray, and showed almost the same expression pattern (data not shown), indicating the reproducibility of this analysis.
data analysis
Data obtained from each hybridization analysis were entered in a database for analysis. The Cy3:Cy5 ratios were normalized to the median ratio value of all of the spots in the array. After normalization, spots with intensities for both channels of less than two times of the local background were discarded. Genes with expression of >2.5-fold upregulated or downregulated in HRS cells relative to that in GC cells in at least 50% of the samples were considered definitive. Hierarchical clustering was applied to both axes using the weighed pair-group method with centroid average as implemented in the J-Express (http://www.molmine.com/, http://www.ii.uib.no/bjarted/jexpress/;DrMolMine;contact@molmine.com) as described previously [7].
reverse transcription–PCR
Reverse transcription of amplified RNA (1 µg) was carried out using oligo(dT)18 primer, You-Prime First-Strand Beads and PuReTaq Ready-To-Go PCR Beads (Amersham Bioscience, Little Chalfont, UK). Primer pairs were the thymus and activation-regulated chemokine (TARC) (+5'-ACTGCTCCAGGGATGCCATCGTTTTT-3' and –5'-ACAAGGGGATGGGATCTCCCTCACTG-3') and macrophage-derived chemokine (MDC) (+5'-AGGACAGAGCATGGCTCGCCTACAGA-3' and –5'-TAATGGCAGGGAGGTAGGGCTCCTGA-3') and G3PDH (+5'-GCCAAGGTCATCCATGACAACTTTGG-3' and –5'-GCCTGCTTCACCACCTTCTTGATGTC-3'). Amplification conditions were denaturation at 94°C for 30 s (5 min for the first cycle), annealing at 60°C for 30 s and extension at 72°C for 30 s (5 min for the last cycle) for 32 cycles for TARC and MDC, and 27 cycles for G3PDH. Amplification products (10 µl each) were subjected to electrophoresis on 3% agarose and stained with ethidium bromide.
immunohistochemical staining
Immunohistochemical staining was performed using antibodies to IL-11R (Santa Cruz Biotechnology, Santa Cruz, CA, USA) on frozen sections.
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laser capture microdissection, RNA isolation and amplification
We succeeded in microdissecting
1000 HRS cells from HL cases and GCs from cases of reactive lymphadenitis (Figure 1). To test the quality of RNA, we isolated total RNA using the rest of each section, and the integrity of RNA was verified by electrophoresis. After RNA isolation and amplification, we were able to harvest 10–20 µg RNA in each case. Subsequently, we used 1–2 µg amplified RNA for cDNA microarray. While amplification bias is of concern, the sensitivity and reproducibility of laser-capture microdissection and T7 promoter-based RNA amplification in the detection of transcription profiles have been described previously [10]. We also compared the gene expression profile of amplified PBMC RNA with that of non-amplified PBMC RNA and confirmed a similar profile pattern (Figure 2). These results confirm the reliability of this method.
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homogeneous gene expression pattern in HRS cells
We analyzed the gene expression profile in 14 HL cases and
140 genes related to cytokines, chemokines and their receptors. Epstein–Barr virus (EBV) was detected in HRS cells of seven cases of the MC type, but in none of the NS type (Table 1). Each gene expression profile of HRS cells was compared with that of GC cells. While GCs are composed of various cells such as B cells, follicular dendritic cells, blood vessels and T cells, the B cells form >90% of the nucleated cells based on counting using CD20 immunostaining (data not shown). Accordingly, we considered the expression profile of GC cells to reflect that of GC B cells. Unsupervised clustering revealed a homogenous expression profile (Figure 3A and B). However, HL subtypes, NS and MC could not be separated and EBV-infection state did not affect clustering.
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expression of various cytokines, chemokines and their receptors in HRS cells
Of 140 genes of cytokines, chemokines and their receptors, the expression of 17 genes was >2.5-fold higher and six genes was <0.4-fold lower in HR than in GC cells in more than half of HL cases (Table 2). We selected TARC and MDC and analyzed the mRNA expression level by reverse transcription–PCR to validate the results of cDNA microarray. All HL cases, except one case with MDC, showed a positive band while amplified RNA from GCs showed no bands (Figure 4). This result is compatible with the cDNA array data.
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Next, we compared EBV-positive cases and EBV-negative cases. We selected molecules that were differentially expressed as determined by Student's t-test (P <0.1) (Table 3). However, the mean score was not different between the two groups (maximum expression 2.66, minimum 0.27). On the other hand, the expression levels of IP-10 and MIG tended to be higher in EBV-positive than -negative cases (P = 0.13 and 0.12, respectively).
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high expression of IL-11R
mRNA and protein in HRS cellsWe chose IL-11R
from the genes highly expressed in HRS cells to determine the protein expression level because its expression level was definitively higher than that in GC cells and has not been reported previously. We first confirmed the expression of IL-11R protein in over half of HL cases (Figure 5A). In contrast, GC cells were negative in reactive lymphadenitis (Figure 5B and C). Comparison of the cDNA microarray and immunohistochemistry data showed a tendency for the high mRNA expression level to correlate with that of protein expression (Table 4). Furthermore, only one case was positive (though weakly) among 16 DL and none of the FL cases was positive (Table 5). These results suggest that IL-11R is a molecule specifically expressed in HRS cells.
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discussion |
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TopAbstractintroductionmaterials and methodsresultsdiscussionReferences |
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The combination of laser capture microdissection and cDNA microarray allowed the analysis of HRS cell gene expression profile and averted interference from admixed reactive lymphocytes. Linear amplification of RNA is an important step for transcript quantitation by microarray analysis. There is considerable support for the sensitivity and reproducibility of laser-capture microdissection and T7 promoter-based RNA amplification in the detection of transcription profiles even down to the single-cell level [10
]. In the present study, two evidences supported the consistency of the results. First, almost all the detected genes in this study were in agreement with previous reports, especially TARC [11], CC chemokine receptor (CCR)-7 [12] and MDC [11] (Tables 2 and 3). Secondly, the results of immunohistochemical analysis of the newly detected molecule in this study, IL-11R, matched the results of mRNA and protein expression levels (Figure 3; Table 4).
This is the first report that describes the gene expression profile of primary HRS cells. A number of previous studies analyzed the gene expression profile of HRS cell lines [1–3], and reported the expression of CCR7 [2], TARC [1, 2], IL-5 [3] and IL-13 [3] in HRS cell lines. CCR7 and TARC are representative highly expressed molecules in HRS cells in this study. Therefore, these molecules are considered essential for HRS cells and not affected by surrounding lymphocytes or cytokines. However, IL-5 and IL-13 were not definitively expressed in primary HRS cells in this study. This discrepancy may reflect the environmental difference and these cytokines may be suppressed in the primary tissue compared with the in vitro environment.
In the present study, we used a cDNA microarray including 140 known genes associated with cytokines, chemokines and their receptors to analyze 14 HL samples. HRS cells in HL showed substantial homogeneity, confirming that HL, taken as a whole, is a distinct pathological entity. However, in the present study, the hierarchical clustering failed to separate HL subtypes, NS and MC. Furthermore, EBV-infection state did not affect clustering. We tried to detect differentially expressed molecules in NS and MC, but no such molecules were observed. A few reports have examined the differential expression of molecules in HL subtypes, except nodular lymphocyte predominance type. Newcom and Gu [13] demonstrated the preferential expression of transforming growth factor (TGF-ß) in HRS cells of NS type HL. However, in our study, TGF-ß was not significantly different between the two subtypes. The difference between the results of the two studies is probably due to the selection criteria used in the present study (positive in >50% of the cases, and ratio >2.5). This result indicates that HRS cells in NS and MC may be almost a homogeneous group with respect to cytokines and chemokines. IL-10 is a representative cytokine associated with immunosuppressive state similar to TGF-ß, and some reports described the relationship between these cytokines and HL [14]. However, in this study, IL-10 was not highly expressed in HRS cells (ratio of IL-10 in HR to GC cells 1.17). Considered collectively, these molecules may be associated with primary HL tissue, but may be expressed also by surrounding T cells (some of them are regulatory T cells [14]), rather than HRS cells themselves.
We also analyzed the relationship between HRS cells in EBV-positive and -negative cases (Table 3). While various molecules were differentially expressed in these two types of cases, the average score was not different. Only CCR7 and interleukin receptor like-1 were highly expressed in EBV-positive HRS cells (>2.5-fold), but the scores were 2.66 and 2.59, respectively. Thus, analysis of more cases is needed to confirm the expression of these molecules and EBV-infection state. IP-10 and MIG, which are reported to be more highly expressed in EBV-positive HRS cells than in EBV-negative ones [15, 16], tend to be highly expressed in EBV-positive cases in this study.
TARC, MDC and IL-6 were highly expressed in HRS cells, and they attract or activate Th2 cells [11]. On the other hand, Th1-associated chemokines and cytokines, e.g. IP-10, MIP-1, Mig, IL-2 and IL-12, were not definitively differentially expressed in HRS cells compared with diffuse large B-cell lymphoma. These molecules are expressed in some HRS cells [11]. This result indicates that Th2-associated molecules may play more important role than Th1-asoociated ones, and it is compatible with the previous report, which indicated that T lymphocytes surrounding HRS cells are predominantly Th2 cells [17]. In this study, we found a significantly higher expression of IL-11R in HRS cells than in GC cells (average ratio 15.6). Furthermore, immunohistochemistry revealed IL-11R expression at the protein level. Previous studies indicated that IL-11R mediates the actions of IL-11, which stimulates megakaryocytic maturation and platelet production [18], as well as growth stimulation of CD34+ hematopoietic progenitor cells [19]. IL-11R is a member of the cytokine receptors family, sometimes referred to as the gp130-dependent family of receptors, which also includes receptors for IL-6 [20]. Among the signaling systems activated by IL-11R and other members of this receptor family is the JAK-STAT pathway, and IL-11 indeed has been shown to activate the JAK1 and JAK2 receptor-associated kinases, triggering the activation of STAT1 and STAT3 [21]. Constitutive or aberrant activation of STAT3 has recently been associated with the malignant phenotype [22, 23]. Kube et al. [24] reported constitutive STAT-3 activation in HRS cells, but IL-6 did not affect STAT-3 activation. The signal from IL-11R may influence STAT-3 activation like other malignant neoplasms [22] and be associated with malignant phenotype.
Our results also showed downregulation of some genes in HRS cells. Among these genes, BRAK showed extremely low expression levels in HRS cells compared with GC cells. BRAK is a CXC chemokine and chemoattractant to B cells and histiocytes [25]. In the present study, no B cell chemoattractant chemokines, e.g. BLC, were upregulated in HRS cells. These findings suggest that B lymphocyte-associated cytokines/chemokines, expressed on GC cells, are downregulated in the process of malignant transformation to HRS cells and their possible involvement in T cell-rich infiltration surrounding HRS cells in contrast to GC cells, which include B cells surrounded by many B lymphocytes.
In conclusion, we analyzed the expression of 140 genes related to cytokines, chemokines and their associated receptors in primary HRS cells in HL tissue and compared their expression pattern with GC cells. Hierarchical clustering revealed HRS cells are similar to each other, regardless of their subtype and EBV-infection state. We detected IL-11R, as a new gene that was specifically expressed in HRS cells, and its expression was confirmed at the protein level by immunohistochemistry. This gene may play an important role in the pathogenesis of HL.
Received for publication April 12, 2005. Revision received September 29, 2005. Accepted for publication October 3, 2005.
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