Annals of Oncology Advance Access published online on November 27, 2007
Annals of Oncology, doi:10.1093/annonc/mdm485
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© 2007 European Society for Medical Oncology
Gastric extranodal marginal zone B-cell lymphomas of MALT type exclusively express toll-like receptor 4 in contrast to other lymphomas infiltrating the stomach
1 Pathologisches Institut
2 Medizinische Poliklinik, Universität Würzburg, Würzburg, Germany
* Correspondence to: Dr P. Adam, Pathologisches Institut, Universität Würzburg, Josef-Schneider-Street 2, 97080 Würzburg, Germany. Tel: +49-931-201-47877; Fax: +49-931-201-47505; E-mail: patrick.adam{at}mail.uni-wuerzburg.de
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Abstract |
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Background: Development and growth of extranodal marginal zone B-cell lymphomas (eMZBCLs) of mucosa-associated lymphoid tissue (MALT) type are thought to be highly dependent on Helicobacter pylori and autoantigens. Receptors mediating these effects are not characterised so far. Toll-like receptors (TLRs) recognise bacterial proteins and autoantigens, which results in inflammatory reactions and influences tumour development and growth.
Materials and methods: TLR4, 5 and 9 expressions were evaluated by immunohistology and confocal microscopy in gastric eMZBCL in comparison to other lymphomas infiltrating the stomach.
Results: TLR4 was exclusively expressed on the cell surface in all eMZBCL (n = 19) and not in chronic lymphocytic leukaemia (CLL, n = 12) or mantle cell lymphoma (MCL, n = 10). TLR5 was strongly expressed in CLL and weak in some eMZBCL (15 of 19), but not in MCL. TLR4, 5 and 9 were negative in all the three lymphoma entities.
Conclusions: Exclusive TLR4 expression may enable eMZBCL to interact with H. pylori and autoantigens. Blockade of TLR4 might be a new approach for therapy of eMZBCL of MALT type.
gastric lymphoma, MALT lymphoma, TLR4, 5 and 9, toll-like receptors
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introduction |
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TopAbstractintroductiontissue samples and methodsresults and discussionfundingAcknowledgementsReferences |
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Infection of the gastric mucosa with Helicobacter pylori results in chronic active gastritis with the acquisition of a mucosa-associated lymphoid tissue (MALT), the background from which extranodal marginal zone B-cell lymphoma (eMZBCL) of MALT type arises [1, 2]. H. pylori seems to trigger the expansion of the malignant clone and eradication of the bacterium in most cases results in tumour regression [3]. But a direct recognition of H. pylori by the B-cell receptor of the lymphoma cells was not demonstrable. In addition to bacterial ligands, autoantigens, e.g. heat-shock proteins (HSPs), are discussed to drive lymphoma development and growth [4], but the receptors mediating these effects are not characterised so far.
Toll-like receptors (TLRs) as a part of the innate immune system have been shown to recognise bacterial ligands and some autoantigens, which results in the activation of proinflammatory cytokines and chemokines[5]. Recent data also indicate that TLRs influence apoptosis and cell proliferation [6]. Especially, TLR4 has been shown to recognise in addition to bacterial lipopolysaccharides (LPS) some endogenous ligands like HSP [7]. In addition, recent studies demonstrated that some malignant tumours, e.g. gastric carcinomas [8], as well as some lymphomas express TLRs [9, 10]. Therefore, TLRs expressed on tumour cells are possible candidates for interaction of the tumour with bacterial products or autoantigens.
In this study, we investigated for the first time TLR expression in gastric eMZBCL and compared these results with the TLR expression in other non-Hodgkin's lymphomas [chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL)] infiltrating the stomach, which grow independently of bacterial or endogenous ligands.
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tissue samples and methods |
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TopAbstractintroductiontissue samples and methodsresults and discussionfundingAcknowledgementsReferences |
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tissue samples
Gastric tissue specimens from 19 patients with gastric eMZBCL of MALT type (12 low-grade and seven diffuse large B-cell lymphomas with eMZBCL component) 12 patients with gastric manifestation of CLL and 10 patients with MCL infiltrating the stomach were included in this study. All tumours were reviewed and classified according to the criteria of the World Health Organisation Classification of Tumours of Haematopoietic and Lymphoid Tissues [11].
immunohistochemistry
Tissue specimens were fixed overnight in 4% buffered formaldehyde solution (pH 7.0) after paraffin embedding tissue blocks were cut into 4-µm sections. For light microscopy, immunohistochemical staining was carried out using antibodies against CD20, CD5, CD23, cyclin D1, AE1/3 and Ki67.
Cases were only included in this study if they fulfilled the morphologic and immunohistochemical diagnostic criteria. For the diagnosis of eMZBCL of MALT type, CD20 positivity of the tumour cells with negativity for CD5 and CD23 was required. Characteristic lymphoepithelial lesions had to be detectable in the staining with the pankeratin marker AE1/3.
MCL cells showed specific nuclear expression of cyclin D1 and in CLL, a co-expression of CD20, CD5 and CD23 with negativity for cyclin D1 was detectable.
For immunohistochemical detection of TLRs, polyclonal rabbit anti-TLR4 (1 : 500), polyclonal rabbit anti-TLR5 (1 : 1000, both Zymed, San Francisco, CA) and monoclonal mouse anti-TLR9 (1 : 200, Biocarta, Hamburg, Germany) were used as recently described [12]. Briefly, after microwave pretreatment and blocking of nonspecific-binding sites, primary antibodies were incubated overnight at 4°C. A biotin/streptavidin-peroxidase detection system (Super Sensitive Multilink HRP Detection System, Bio Genex, San Ramon, CA) using 3,3'- diaminobenzidine-tetra-hydrochloride solution as substrate was employed as a secondary reagent according to the manufacturer’s instructions. As negative control, polyclonal TLR4 and TLR5 antibodies were blocked using the peptide utilised for the antibody production (Zymed). For the TLR9 staining, the primary antibody was replaced by an isotype control antibody. All negative controls yielded no staining of the tissues.
TLR expression was given in percentage of positive tumour cells and semiquantitatively quantified as weak, moderate and strong.
confocal laser scanning microscopy
For confocal immunofluorescence microscopy, the pretreated sections were stained for TLR4, 5 and 9 as described above. Additional double stainings with the anti-TLR4 antibody, followed by an anti-CD79a antibody (Neomarkers, Fremont, CA) and an anti-CD3 antibody (DAKO, Hamburg, Germany) were carried out. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (Sigma, Hannover, Germany). Stainings were evaluated with a confocal laser scanning microscope (TCS SP2, Leica, Heidelberg, Germany).
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results and discussion |
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TLR4 was strongly expressed by tumour cells of all tested gastric eMZBCL (n = 19; Figure 1A). The percentage of positive tumour cells ranged from 60% to 90%. TLR5 was also expressed in 12 of 19 cases (63%; Figure 1B). TLR5 expression was weak and the range of positive tumour cells was only 10%–15%. All eMZBCL were negative for TLR9 (Figure 1C). The gastric surface epithelium as an endogenous positive control showed a specific staining with TLR4, 5 and 9 (Figure 1J–L).
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In contrast to gastric eMZBCL, all 12 cases of CLL showed no expression of TLR4 (Figure 1D). A weak, albeit specific staining for TLR5 was detectable in all cases (Figure 1E) ranging from 30% to 60% of tumour cells. Again, no expression of TLR9 was found (Figure 1F). All 10 cases of MCL turned out to be negative for the three TLRs investigated (Figure 1G–I). The gastric surface epithelium again was used as an endogenous positive control.
Confocal laser scanning microscopy paralleled the immunohistochemical results with strong TLR4 and weak TLR5 expression in gastric MALT-type lymphoma, TLR5 expression in CLL and no expression of TLRs in MCL. In detail, the TLR expression of the lymphoma cells was accentuated to the cell surface (not shown).
Additionally carried out double stainings localised TLR4 expression on the surface of CD79a co-expressing B cells (Figure 1N). Reactive CD3-positive T cells, in contrast, were negative for TLR4 (Figure 1M).
TLRs are known to recognise bacterial ligands, e.g. TLR4 LPS, TLR5 flagellin and TLR9 CpG DNA, and are able to induce an inflammatory reaction by activating nuclear factor kappa B via MyD88 [13, 14]. Moreover, TLR4 is thought to recognise endogenous ligands like HSPs and extracellular matrix breakdown products [15], which results in stimulating a signalling pathway that is indicated to enhance tumorigenesis and growth [16, 17]. Therefore, TLRs are promising candidates for a bacterial antigen or autoantigen-mediated tumour growth.
In this study, we analysed for the first time the expression of TLR4, 5 and 9 in gastric eMZBCL of MALT type and compared it to that of gastric infiltrates of CLL and MCL, which are thought to grow independently of bacterial and endogenous ligands.
In the presented study, all 19 cases of gastric eMZBCL (12 low-grade and 7 diffuse large B-cell lymphomas with eMZBCL component) expressed TLR4 specifically, whereas CLL and MCL infiltrating the stomach did not express TLR4. TLR5 was predominantly expressed by CLL, and to a lesser extent in eMZBCL. Expression of TLR9 was detectable in none of the tested lymphomas. Our data demonstrate a quite different distribution of TLRs between lymphomas infiltrating the gastric mucosa.
The pattern of TLR expression correlates well with the putative model of lymphomagenesis and progression. Tumour growth of gastric eMZBCL of MALT type seems to be highly dependent on bacterial and endogenous stimuli and eradication therapy results in most cases in tumour regression. Whereas the other two lymphoma entities probably grow independently of external stimuli and their clinical course is therapeutically barely not influenceable.
Therefore, it may be speculated that the exclusive expression of TLR4 gives eMZBCL of MALT type the possibility to interact with bacterial antigens and autoantigens.
Recently, the relevance of TLR4 in MALT-type lymphomagenesis was underlined by a study determining TLR4 polymorphisms in the peripheral blood [18]. In this study, a negative association of the TLR4 Asp299Gly allele, which is known to diminish the inflammatory response and the susceptibility to develop eMZBCL of MALT type, was found.
In summary, we show for the first time an exclusive expression of TLR4 by eMZBCL of MALT type in the stomach.
Further studies have to demonstrate whether blockade of the TLR4 may be a possible therapeutic approach in preventing development and growth of this specific lymphoma entity.
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funding |
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Deutsche Forschungsgemeinschaft, German Research Foundation (EC 203/1-3).
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Acknowledgements |
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The authors thank Eva Bachmann, Sabine Roth and Erwin Schmitt for their excellent technical assistance.
Received for publication February 6, 2007. Revision received September 16, 2007. Accepted for publication September 17, 2007.
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References |
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1. Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet (1984) 1:1311–1315.[CrossRef][Web of Science][Medline]
2. Wotherspoon AC, Ortiz-Hidalgo C, Falzon MR, Isaacson PG. Helicobacter pylori-associated gastritis and primary B-cell gastric lymphoma. Lancet (1991) 338:1175–1176.[CrossRef][Web of Science][Medline]
3. Isaacson PG, Spencer J. Gastric lymphoma and Helicobacter pylori. Important Adv Oncol (1996) 12:111–121.
4. Wotherspoon AC. Helicobacter pylori infection and gastric lymphoma. Br Med Bull (1998) 54:79–85.
5. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol (2001) 2:675–680.[CrossRef][Web of Science][Medline]
6. Sabroe I, Dower SK, Whyte MK. The role of toll-like receptors in the regulation of neutrophil migration, activation, and apoptosis. Clin Infect Dis (2005) 41(Suppl 7):S421–S426.[CrossRef][Web of Science][Medline]
7. Takenaka R, Yokota K, Ayada K, et al. Helicobacter pylori heat-shock protein 60 induces inflammatory responses through the toll-like receptor-triggered pathway in cultured human gastric epithelial cells. Microbiology (2004) 150:3913–3922.
8. Schmausser B, Andrulis M, Endrich S, et al. Toll-like receptors TLR4, TLR5 and TLR9 on gastric carcinoma cells: an implication for interaction with Helicobacter pylori. Int J Med Microbiol (2005) 295:179–185.[CrossRef][Web of Science][Medline]
9. Wysocka M, Benoit BM, Newton S, et al. Enhancement of the host immune responses in cutaneous T-cell lymphoma by CpG oligodeoxynucleotides and IL-15. Blood (2004) 104:4142–4149.
10. Henault M, Lee LN, Evans GF, Zuckerman SH. The human Burkitt lymphoma cell line Namalwa represents a homogenous cell system characterized by high levels of toll-like receptor 9 and activation by CpG oligonucleotides. J Immunol Methods (2005) 300:93–99.[CrossRef][Web of Science][Medline]
11. Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues (2001) Lyon, France: IARC Press.
12. Schmausser B, Andrulis M, Endrich S, et al. Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection. Clin Exp Immunol (2004) 136:521–526.[CrossRef][Web of Science][Medline]
13. Hasan U, Chaffois C, Gaillard C, et al. Human TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through MyD88. J Immunol (2005) 174:2942–2950.
14. Smith MF Jr, Mitchell A, Li G, et al. Toll-like receptor (TLR) 2 and TLR5, but not TLR4, are required for Helicobacter pylori-induced NF-kappa B activation and chemokine expression by epithelial cells. J Biol Chem (2003) 278:32552–32560.
15. Beg AA. Endogenous ligands of toll-like receptors: implications for regulating inflammatory and immune responses. Trends Immunol (2002) 23:509–512.[CrossRef][Web of Science][Medline]
16. Eldering E, Spek CA, Aberson HL, et al. Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res (2003) 31:e153.
17. Huang B, Zhao J, Li H, et al. Toll-like receptors on tumor cells facilitate evasion of immune surveillance. Cancer Res (2005) 65:5009–5014.
18. Hellmig S, Fischbach W, Goebeler-Kolve ME, et al. Association study of a functional toll-like receptor 4 polymorphism with susceptibility to gastric mucosa-associated lymphoid tissue lymphoma. Leuk Lymphoma (2005) 46:869–872.[CrossRef][Web of Science][Medline]
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