Purpose: Nasal natural killer (NK)/T-cell lymphoma is associated with Epstein-Barr virus and has poor prognosis because of local invasion and/or multiple dissemination. Recently, the role of chemokines/chemokine receptors in tumor proliferation and invasion has been shown. In this study, we examined whether the specific chemokines were related to the tumor behaviors in nasal NK/T-cell lymphoma.

Experimental Design: A chemokine protein array was used to examine specific chemokines produced by SNK-6 and SNT-8 (Epstein-Barr viruspositive nasal NK/T-cell lymphoma lines). The expression of interferon inducible protein 10 (IP-10) and the IP-10 receptor CXCR3 was investigated by ELISA and flow cytometry. Cell growth and invasion were assessed by the MTT and Matrigel invasion assays, respectively. Immunohistologic staining and ELISA were used to examine IP-10 expression in biopsies and sera from patients, respectively.

Results: IP-10 was specifically produced by SNK-6 and SNT-8. Moreover, CXCR3 was expressed on the NK cell lines. Functionally, IP-10 did not affect cell proliferation but enhanced cell invasion. In biopsy samples, IP-10 and CXCR3 expressions were detected in the lymphoma cells. Serum IP-10 levels in the patients were much higher than those of healthy controls and the levels were decreased during the complete remission phase after treatments.

Conclusions: These results suggest that IP-10 may play an important role in cell invasion in nasal NK/T-cell lymphoma through an autocrine mechanism. (Clin Cancer Res 2009;15(22):67719)

Translational Relevance

In this study, we examined whether the specific chemokines were related to the tumor behaviors in nasal natural killer (NK)/T-cell lymphoma. As a result, the interferon inducible protein 10 (IP-10) was specifically produced by Epstein-Barr viruspositive nasal NK/T-cell lymphoma lines. Moreover, CXCR3, the receptor of IP-10, was expressed on the NK cell lines. Functionally, IP-10 did not affect cell proliferation but enhanced cell invasion. In biopsy samples, IP-10 and CXCR3 expressions were detected in lymphoma cells. These results suggest that IP-10 may play an important role in cell invasion in nasal NK/T-cell lymphoma through an autocrine mechanism and that IP-10 signaling pathway may be a new pharmacologic target in the treatment of the patients with nasal NK/T-cell lymphoma.

Nasal natural killer (NK)/T-cell lymphoma, previously known as lethal midline granuloma (1), has distinct epidemiologic, clinical, histologic, and etiologic features. Epidemiologically, this lymphoma is common in Asian countries but is quite rare in the United States and Europe (2,6). Clinically, this lymphoma is characterized by progressive necrotic lesions mainly in the nasal cavity and a poor prognosis resulting from rapid progression of the lesion and distinct organs (2, 7, 8). Histologically, the features of this lymphoma include angiocentric and polymorphous lymphoreticular infiltrates, which are called polymorphic reticulosis (9, 10). Original cells of the lymphoma are reported to be of the NK or T cell lineages, both of which express the NK cell marker CD56 (2, 5, 6, 11).

Regarding etiologic factors, we first indicated the presence of Epstein-Barr virus (EBV) DNA, EBV oncogenic proteins, and clonotypic EBV genome in this lymphoma, suggesting that EBV may play a role in lymphomagenesis (2, 4, 12). However, little is known about its genetic features because this lymphoma is a relatively rare disease and it is often difficult to obtain a sufficient amount of tissue from necrotic lesions. Moreover, if a sufficient amount of tissue is obtained, it is difficult to analyze the profile of gene expression in biopsy samples because the tissue specimen contains too many types of cells, including tumor cells, inflammatory cells, or normal epithelial cells, which impede the examination of gene expression of individual cells. Recently, Nagata et al. (11) established two kinds of EBV-positive cell lines of the NK or T cell lineages, SNK-6 and SNT-8, from primary lesions of nasal NK/T-cell lymphoma. They are very useful to compare the gene or protein expression of these cell lines with that of other lymphoma/leukemia cell lines to find the genes or proteins that are expressed specifically in nasal NK/T-cell lymphoma. We previously showed that interleukin (IL)9 and IL-10, which were produced specifically by nasal NK/T-cell lymphoma cell lines, enhanced the lymphoma cell proliferation directly or indirectly in an autocrine manner (13, 14). Because such cytokines were not produced by EBV-negative NK cell lymphoma lines, it is suggested that EBV may contribute to lymphoma cell proliferation through the production of these cytokines.

Chemokines are a superfamily of pro-inflammatory polypeptide cytokines that selectively attract and activate different cell types. Tissue injury, allergy, cardiovascular diseases, as well as malignant tumors are prerequisites for many pathophysiologic conditions. The role of chemokines in malignant tumors is complex: Although some chemokines may enhance innate or specific host immunity against tumor implantation, others may favor tumor growth and metastasis by promoting tumor cell proliferation, migration, or neovascularization in tumor tissue (15). Especially in EBV-related hematologic malignancies such as Hodgkin's lymphoma, chemokines are likely to have beneficial effects for tumor progression rather than unfavorable ones (16, 17). Moreover, some chemokines, including CCL20 (18), RANTES (19), interferon (IFN) inducible protein 10 (IP-10; ref. 20), and IL-8 (21) were reported to be controlled by proteins derived from EBV. Therefore, in nasal NK/T-cell lymphoma, such chemokines are possibly related to tumor progression as well. However, there is no report that describes comprehensive examination of chemokines in nasal NK/T-cell lymphoma.

To determine which chemokines are specifically produced by nasal NK/T-cell lymphoma, we compared chemokine profiles in the culture supernatants from nasal NK/T-cell lymphoma cell lines to those from the other cell lines. Chemokine protein array analysis revealed that IP-10 was specifically expressed in culture supernatants of the nasal NK/T-cell lymphoma cell lines. Moreover, functional analyses revealed that IP-10 stimulated invasive phenotype of the lines in an autocrine manner. Furthermore, in vivo studies showed IP-10 expression in biopsies and sera from patients with this lymphoma. These results suggest that IP-10 is a crucial factor in the pathogenesis of nasal NK/T-cell lymphoma.

Patients and clinical evaluation

Ten Japanese patients, eight men and two women, 21 to 70 years of age with a median age of 53 years, participated in the study. Nasal NK/T-cell lymphoma was diagnosed in all of them at the Department of Otolaryngology-Head and Neck Surgery, Asahikawa Medical College (Hokkaido, Japan), between 2003 and 2008. The diagnosis was carried out according to the World Health Organization classification of hematologic malignancies (22). Pertinent clinical information and follow-up data were obtained from hospital charts for all patients (Table 1). According to the Ann Arbor classification system, they were all stage I patients. The B-symptoms were observed in 3 (30) patients. The median serum lactate dehydrogenase level was 176.5 IU/L with a range of 144 to 765 IU/L (reference range 105-210 IU/L). All patients achieved complete remission after chemoradiotherapy and are healthy without any sign of relapse. The details of chemoradiotherapy were described in our previous reports (23, 24). Briefly, the MTCOP-P regimen used in two patients consists of pirarubicin hydrochloride, cyclophosphamide, vincristine sulfate, methotrexate, peplomycin sulfate, and prednisolone; the MPVIC-P regimen used in the eight remaining patients consists of ifosfamide, carboplatin, methotrexate, peplomycin, etoposide, and prednisolone. The dose of radiotherapy was 54 to 56 Gy, and a lateral opposing field was used covering the primary site. The clinical characteristics of the 10 patients are summarized in Table 1. In vivo materials used in the studies were biopsy samples and serum before and after treatment. Serum from six healthy volunteers (all men, 29-40 years of age, median 30 years) was also used. All patients signed informed consent forms for this study, which were approved by the institutional review board.

Table 1.

Characteristics of 10 patients with nasal NK/T-cell lymphoma

Case no.Age (y)GenderClinical stageB symptomSerum LDH (IU/mL)CD56 ISHEBER ISHLMP1 IHCIP-10 IHCCXCR3 IHCSerum IP-10 levelTherapyResponsePrognosis (mo)
PretreatmentPosttreatment
48  205 464 133 MTCOPP-radiation CR Alive-55 
60  236  813 249 MTCOPP-radiation CR Alive-52 
64  162  579 117 MPVICP-radiation CR Alive-48 
48 176  761 176 MPVICP-radiation CR Alive-46 
40 144    216 220 MPVICP-radiation CR Alive-43 
70  152 150 175 MPVICP-radiation CR Alive-36 
63  219   299 278 MPVICP-radiation CR Alive-18 
21 177 634 86 MPVICP-radiation CR Alive-15 
64  151   81 133 MPVICP-radiation CR Alive-12 
10 58 765 1635 84 MPVICP-radiation CR Alive-11 
Case no.Age (y)GenderClinical stageB symptomSerum LDH (IU/mL)CD56 ISHEBER ISHLMP1 IHCIP-10 IHCCXCR3 IHCSerum IP-10 levelTherapyResponsePrognosis (mo)
PretreatmentPosttreatment
48  205 464 133 MTCOPP-radiation CR Alive-55 
60  236  813 249 MTCOPP-radiation CR Alive-52 
64  162  579 117 MPVICP-radiation CR Alive-48 
48 176  761 176 MPVICP-radiation CR Alive-46 
40 144    216 220 MPVICP-radiation CR Alive-43 
70  152 150 175 MPVICP-radiation CR Alive-36 
63  219   299 278 MPVICP-radiation CR Alive-18 
21 177 634 86 MPVICP-radiation CR Alive-15 
64  151   81 133 MPVICP-radiation CR Alive-12 
10 58 765 1635 84 MPVICP-radiation CR Alive-11 

Abbreviations: LDH, lactate dehydrogenase; ISH, in situ hybridization; IHC, immunohistochemistry; MTCOPP, pirarubicin hydrochloride, cyclophosphamide, vincristine sulfate, methotrexate, peplomycin sulfate, and prednisolone; MPVICP, ifosfamide, carboplatin, methotrexate, peplomycin, etoposide, and prednisolone; CR, complete remission.

Cell culture

The features of the cell lines used in this study are listed in Table 2. SNK-1, SNK-6, and SNT-8 were EBV-positive cell lines established from primary lesions with nasal NK/T-cell lymphoma. The cell lines were kindly provided by Dr. Shimizu (Tokyo Medical and Dental University; ref. 11). KAI-3 originated from a patient with a severe mosquito allergy (25). EBV-negative NK cell lines NK-92 and KHYG-1 were established from patients with NK cell leukemia (26, 27). Raji was an EBV-positive B-cell line that originated from Burkitt's lymphoma (28). SNK-1, SNK-6, SNT-8, KHYG-1, and KAI-3 cells were cultured in RPMI 1640 supplemented with 10 fetal bovine serum (FBS), with 50 units/mL penicillin, 50 g/mL streptomycin (Life Technologies, Inc.), and 250 units/mL recombinant human IL-2 (Takeda Pharmaceutical Company Limited). NK-92 cells were cultured in a-MEM supplemented with 12.5 horse serum, 12.5 FBS, 50 units/mL penicillin, and 50 g/mL streptomycin and 200 units/mL recombinant human IL-2. All cell lines were incubated at 37C in an atmosphere containing 5 CO2.

Table 2.

Cell lines

Cell linesDiseasePhenotypeEBV
SNK-1 Nasal NK/T-cell lymphoma NK 
SNK-6 Nasal NK/T-cell lymphoma NK 
SNT-8 Nasal NK/T-cell lymphoma 
KAI-3 Severe chronic active EBV infection NK 
Raji Burkitt's lymphoma 
KHYG-1 Aggressive NK cell leukemia NK  
NK-92 NonHodgkin's lymphoma NK  
Cell linesDiseasePhenotypeEBV
SNK-1 Nasal NK/T-cell lymphoma NK 
SNK-6 Nasal NK/T-cell lymphoma NK 
SNT-8 Nasal NK/T-cell lymphoma 
KAI-3 Severe chronic active EBV infection NK 
Raji Burkitt's lymphoma 
KHYG-1 Aggressive NK cell leukemia NK  
NK-92 NonHodgkin's lymphoma NK  

Human protein chemokine array

RayBio Human Chemokine Antibody Array I (RayBiotech) was used according to the instruction manual. Briefly, a membrane was incubated with 1 mL supernatant of SNK-6, SNT-8, or KHYG-1 cultures at 4C overnight. After washing, the membrane was incubated with 1 mL of primary biotin-conjugated antibody followed by 2 mL of horseradish peroxidaseconjugated streptavidin at 4C overnight. The membranes were developed by enhanced chemiluminescence and were exposed to X-ray film.

Flow cytometry

For flow cytometric analysis of surface molecules, cell lines were washed in cold PBS containing 1 albumin from bovine serum, centrifuged, and resuspended in PBS. Cells were incubated with Carboxyfluorescein-conjugated mouse anti-human CXCR3 monoclonal antibody (DAKO) for 30 min at 4C. Carboxyfluorescein-conjugated mouse IgG1 (DAKO) was used as isotype control. Samples were analyzed with FACScan (BD Biosciences).

ELISA

IP-10 protein in cell culture supernatants and serum was quantified using an ELISA kit, Quantikine Human CXCL10/IP-10 (R&D Systems, Inc.). Cell lines (2.5 105/mL) were cultured in 96-well round-bottomed plates, and supernatants of cell cultures were collected after 24, 48, and 72 h. Blood samples from nasal NK/T-cell lymphoma patients were obtained at pretreatment and posttreatment periods. As the healthy control, blood samples from six healthy subjects were also obtained. Serum was separated from whole blood by centrifugation and stored at 80C. The mouse anti-human IP-10 monoclonal antibody was coated onto the bottom of supplied 96-well ELISA plates. The supernatants and serum were diluted with assay diluent and added to the each well. The plates were washed with wash buffer after 2 h of incubation at room temperature. Anti-human IP-10 polyclonal antibody conjugated to horseradish peroxidase (200 L) was added to each well and incubated at room temperature for 2 h. After washing, 200 L of a substrate solution were added and the plates were incubated for 30 min in the dark. The absorbance of each well was determined at 450 nm using a microplate reader (Nalge Nunc International). For cell culture supernatants, measurements were done in triplicate, and for serum, measurements were done in duplicate. A standard curve was generated using serial dilutions of recombinant IP-10 (R&D Systems). The results correspond to mean SD.

Cell proliferation assay (MTS assay)

SNK-6 and KHYG-1 cells (2 103 or 1 104 per well) in 96-well plates were cultured in 200 L 10 FBS containing RPMI 1640 with or without 50 units/mL IL-2. Each well was treated with recombinant IP-10 (10, 100, or 1,000 ng/mL; Peprotech) or anti-human IP-10 antibody (1 or 10 g/mL; R&D Systems) for 48 h. To determine the number of viable cells, we used the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega). MTS (20 L) was added to each well, and then incubated for 4 h at 37C under 5 CO2. The absorbance at 490 nm was measured with an ELISA plate reader. Results were expressed as a percentage of untreated controls. Measurements were done in duplicate and experiments were repeated at least three times. The results correspond to mean SD.

Invasion assay

Tumor cell invasion was assayed in 24-well BioCoat Matrigel Invasion Chambers (8 m; Becton Dickinson) according to the manufacturer's protocol. Briefly, after hydration of rehydrated Matrigel inserts with DMEM for 2 h, SNK-1, SNK-6, or KHYG-1 cells (2.5 104/well) in serum-free RPMI 1640 containing recombinant human IP-10 (1 or 10 ng/mL; Peprotech), anti-humanIP-10 monoclonal antibody (1, 10 g/mL; R&D Systems), or anti-humanCXCR3 monoclonal antibody (1,10 g/mL; R&D Systems) were plated in the top chamber. RPMI 1640 with 10 FBS was placed into a bottom chamber. After incubation for 22 h, the Matrigel membranes were removed, fixed with methanol, and stained with a Giemsa-type stain (Diff-Quik, Sysmex). Migrated cells in the membranes were counted under a microscope in five arbitrary fields. As a next experiment, supernatant was obtained from culture of SNK-6 or KHYG-1 (2.5 104/mL) in RPMI 1640 with 10 FBS for 48 h, and was plated in the top chamber instead of the medium with IP-10. Assays were done in triplicates independently. Results were shown as the number of cells through the Matrigel matrix.

In situ hybridization and immunohistology

EBV-encoded small nuclear early region type 1 (EBER1) expression in tumor cells was assessed by in situ hybridization on formalin-fixed, paraffin-embedded tissue sections using a fluorescein-conjugated peptide nucleic acid probe for EBER1 (DAKO) and PNA ISH Detection Kit (DAKO) according to the manufacturer's instructions. Expression of EBV latent membrane protein 1 (LMP1) was detected by the EnVision+ system (DAKO) using anti-LMP1 monoclonal antibody CS1-4 and according to the manufacturer's instructions. The lymphoma was considered LMP1 positive if more than 50 of the cells were stained in cytoplasm by the CS1-4 antibody (29).

Staining for IP-10 or CXCR3 was done as follows: formalin-fixed, paraffin-embedded sections were obtained from pretreatment biopsy samples of 10 patients with nasal NK/T-cell lymphoma. The sections were deparaffinized and treated with microwave irradiation for 8 min at 750 W for antigen retrieval. After prevention of nonspecific staining by Protein Block Serum-Free (DAKO), the sections were incubated overnight at 4C with 2.5 g/mL goat anti-human antibody IP-10 (R&D Systems) or with 1:100 rabbit anti-human antibody CXCR3 (Chemicon). After washing with PBS, the sections were incubated with Histofine Simple Stain MAX PO (G) (Nichirei Bioscience, Inc.) on IP-10 staining or EnVision+ horseradish-labeled dextran polymer (DAKO) on CXCR3 staining for 30 min at room temperature. IP-10 or CXCR3 was visualized by the DAKO liquid with the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen system (DAKO). For double staining of IP-10 or CXCR3 and CD56, the sections stained with IP-10 or CXCR3 were incubated overnight at 4C with 1:50 mouse anti-human CD56 monoclonal antibody (Novocastra) followed by incubation with anti-IgG Mouse Goat Poly Alkaline phosphatase (Chemicon) as a secondary antibody for 30 min at room temperature. CD56 was visualized by freshly prepared Fast Red substrate solution (DAKO). Finally, the sections were counterstained with Lillie-Mayer's hematoxylin. A case in which more than 30 of the CD56-positive cells were also IP-10 or CXCR3 positive was defined as IP-10 or CXCR3 positive (30).

Statistical analysis

Two group comparisons were tested using nonparametric test procedures such as the Mann-Whitney U test and Wilcoxon signed rank test. Statistical tests were based on a level of significance of P < 0.05.

Nasal NK/T-cell lymphoma cell lines express and produce IP-10

For screening of difference in profile of chemokine production among SNK-6, SNT-8, and KHYG-1 cells, we used a chemokine protein array (Fig. 1A). The measurements of the amount in culture supernatants of the cell lines revealed that SNK-6 and SNT-8 produced more IP-10 than KHYG-1. According to quantification of chemokine expression by using Image J software, IP-10 signal of SNK-6 and SNT-8 was 2.808-fold and 4.267-fold higher than the positive control signal, respectively. However, the IP-10 signal in KHYG-1 was one seventh of the positive control signal. For confirmation of the results, we examined IP-10 production using ELISA (Fig. 1B). ELISA analysis confirmed that SNK-1, SNK-6, and SNT-8 produced IP-10 in a time-dependent manner, but KHYG-1 and NK-92 did not.

Fig. 1.

IP-10 and CXCR3 expression in the nasal NK/T-cell lymphoma cell lines. A, profile of chemokine production among SNK-6, SNT-8, and KHYG-1 by chemokine protein array analysis. Arrowhead, IP-10 expression in the culture supernatant of SNK-6 or SNT-8. Spots in the ellipses show positive control signals. B, IP-10 production of the cell lines as determined by ELISA. Columns, mean expression values of three independent experiments. C, CXCR3 expression on the cell lines as determined by flow cytometric analysis. Full line, expression of CXCR3. Filled histograms, isotype control signals.

Fig. 1.

IP-10 and CXCR3 expression in the nasal NK/T-cell lymphoma cell lines. A, profile of chemokine production among SNK-6, SNT-8, and KHYG-1 by chemokine protein array analysis. Arrowhead, IP-10 expression in the culture supernatant of SNK-6 or SNT-8. Spots in the ellipses show positive control signals. B, IP-10 production of the cell lines as determined by ELISA. Columns, mean expression values of three independent experiments. C, CXCR3 expression on the cell lines as determined by flow cytometric analysis. Full line, expression of CXCR3. Filled histograms, isotype control signals.

Close modal

Nasal NK/T-cell lymphoma cell lines express CXCR3

Next, to examine whether nasal NK/T-cell lymphoma cell lines express CXCR3, the receptor of IP-10, we performed flow cytometry (Fig. 1C). The analysis revealed that CXCR3 were expressed on nasal NK/T-cell lymphoma cell lines SNK-1, SNK-6, and SNT-8, as well as in the EBV-positive NK-cell line KAI-3 and the EBV-negative NK cell lines KHYG-1 and NK-92. However, they were not detected in Raji cells as previously reported elsewhere (31).

IP-10 does not affect cell growth of nasal NK/T-cell lymphoma cell lines

According to the results above, it became clear that nasal NK/T-cell lymphoma cell lines produce IP-10 and express CXCR3. The next step was to investigate whether IP-10 plays a role as a growth factor in an autocrine manner; thus, we performed MTS assays on SNK-6 cells under culture conditions with exogenous IP-10 or antiIP-10-neutralizing antibody. KHYG-1, which has CXCR3 but does not produce IP-10, was also used for MTS assays. Administration of exogenous IP-10 affected cell growth in neither SNK-6 nor KHYG-1 (Fig. 2A). Similarly, treatment of IP-10neutralizing antibody changed cell growth in neither SNK-6 nor KHYG-1 (Fig. 2B). Even alternating the concentration of IP-10 or IP-10neutralizing antibody did not make a difference in the cell growth.

Fig. 2.

Measurement of cell growth and invasion in SNK-6 and KHYG-1 under culture conditions with exogenous IP-10 or antiIP-10-neutralizing antibody. Cell growth under culture conditions with exogenous IP-10 (A) or antiIP-10-neutralizing antibody (B) was measured by the MTS assay. A number of migrated cells under culture conditions with exogenous IP-10 (C), antiIP-10-neutralizing antibody (D), or antiCXCR3-blocking antibody (D) was measured by the Matrigel invasion assay. Columns, mean absorbance (A and B) or cell number (C and D) of three independent experiments.

Fig. 2.

Measurement of cell growth and invasion in SNK-6 and KHYG-1 under culture conditions with exogenous IP-10 or antiIP-10-neutralizing antibody. Cell growth under culture conditions with exogenous IP-10 (A) or antiIP-10-neutralizing antibody (B) was measured by the MTS assay. A number of migrated cells under culture conditions with exogenous IP-10 (C), antiIP-10-neutralizing antibody (D), or antiCXCR3-blocking antibody (D) was measured by the Matrigel invasion assay. Columns, mean absorbance (A and B) or cell number (C and D) of three independent experiments.

Close modal

IP-10 is an autocrine cell invasion factor in nasal NK/T-cell lymphoma cell lines

Because the results above indicate that IP-10 does not act as a cell growth factor for nasal NK/T-cell lymphoma cells, we subsequently performed an invasion assay using Matrigel-coated filters, which are widely used to examine invasive migration (32). Administration of exogenous IP-10 increased the number of the cells that migrated through the Matrigel membrane in SNK-1, SNK-6, and KHYG-1. The number increased in a dose-dependent manner (Fig. 2C). On the other hand, treatment with antiIP-10-neutralizing antibody or antiCXCR3-blocking antibody inhibited the number of migrated cells in SNK-1 and SNK-6 (Fig. 2D). The inhibition was enhanced in a dose-dependent manner. Because KHYG-1 did not produce IP-10, the antibodies did not affect the number of migrated cells in KHYG-1.

For the next trial, to examine whether endogenous IP-10 produced by SNK-6 has a functional role in cell invasion, we performed an invasion assay using supernatant culture fluid of SNK-6 or KHYG-1 instead of serum-free medium in cell suspension (Fig. 3). The number of migrated cells in SNK-1, SNK-6, and KHYG-1 increased in the presence of the culture supernatant of SNK-6 but did not change in the presence of culture supernatant of KHYG-1. Furthermore, the effect of the culture supernatant of SNK-6 was inhibited by antiIP-10-neutralizing antibody and antiCXCR3-blocking antibody.

Fig. 3.

Measurement of cell invasion in SNK-1, SNK-6, and KHYG-1 suspended with the supernatant culture fluid of SNK-6 or KHYG-1 with or without antiIP-10-neutralizing antibody or antiCXCR3-blocking antibody by the Matrigel invasion assay. White columns, light gray columns, charcoal gray columns, black columns, and dot columns show mean values of migrated cell number in serum free medium, KHYG-1 culture supernatant, SNK-6 culture supernatant, SNK-6 supernatant with antiIP-10-neutralizing antibody, and SNK-6 supernatant with antiCXCR3-blocking antibody, respectively. The experiments were done three times independently.

Fig. 3.

Measurement of cell invasion in SNK-1, SNK-6, and KHYG-1 suspended with the supernatant culture fluid of SNK-6 or KHYG-1 with or without antiIP-10-neutralizing antibody or antiCXCR3-blocking antibody by the Matrigel invasion assay. White columns, light gray columns, charcoal gray columns, black columns, and dot columns show mean values of migrated cell number in serum free medium, KHYG-1 culture supernatant, SNK-6 culture supernatant, SNK-6 supernatant with antiIP-10-neutralizing antibody, and SNK-6 supernatant with antiCXCR3-blocking antibody, respectively. The experiments were done three times independently.

Close modal

IP-10 is expressed on the lymphoma cells in biopsy tissues and detected at high levels in sera from patients with nasal NK/T-cell lymphoma

Finally, we examined whether IP-10 was detected in the biopsy tissues and sera from patients with nasal NK/T-cell lymphoma. The pathologic findings were shown in Fig. 4A to F and the results are summarized in Table 1. In immunohistologic single or double staining with anti-CD56 and antiIP-10 antibodies, we found that a number of CD56-positive lymphoma cells coexpressed IP-10 in the cytoplasm in 7 of 10 patients tested (Fig. 4C and D). Similarly, CXCR3 was expressed on the lymphoma cell surface in 7 (70) of 10 patients tested (Fig. 4E and F). EBER1 was detected in all 10 patients, and LMP1 was expressed in 6 (60) of 10 patients. All 6 patients with LMP1 expression showed IP-10 expression, whereas 3 (75) of 4 patients without LMP1 expression did not express IP-10 (P < 0.05; Table 1).

Fig. 4.

Expression of IP-10 and CXCR3 in in vivo materials from patients with nasal NK/T-cell lymphoma. A, H&E staining. B, negative staining for IP-10. C, single staining for IP-10. D, double staining for IP-10 (brown) and CD56 (red). E, single staining for CXCR3. F, double staining for CXCR3 (brown) and CD56 (red). E, serum IP-10 level of 10 patients with nasal NK/T-cell lymphoma and six healthy volunteers.

Fig. 4.

Expression of IP-10 and CXCR3 in in vivo materials from patients with nasal NK/T-cell lymphoma. A, H&E staining. B, negative staining for IP-10. C, single staining for IP-10. D, double staining for IP-10 (brown) and CD56 (red). E, single staining for CXCR3. F, double staining for CXCR3 (brown) and CD56 (red). E, serum IP-10 level of 10 patients with nasal NK/T-cell lymphoma and six healthy volunteers.

Close modal

ELISA results showed that IP-10 existed in serum samples from all 10 patients before treatments (Table 1). The serum IP-10 levels of the patients before treatments were significantly higher than those from healthy volunteers (P = 0.01; 81-1,635 pg/mL with median 521 pg/mL and 41-175 pg/mL with median 83 pg/mL, respectively; Fig. 4G). The serum IP-10 levels significantly decreased during the complete remission phase after treatments (P = 0.01; 84-278 pg/mL with median 154 pg/mL; Fig. 4C). Patients with LMP1 expression on the lymphoma cells showed significantly higher serum IP-10 level than the patients without LMP1 expression did (P < 0.05; 150-1,635 pg/mL with a median of 698 pg/mL and 81-579 pg/mL with a median of 258 pg/mL, respectively; Table 1).

IP-10 was first described as an -chemokine induced by IFN- in U937 monocyte-like cell line (33). IP-10 is produced by various types of cells such as human fibroblasts, endothelial cells, keratinocytes, mesangial cells, astrocytes, and neutrophils, and the production is enhanced by IFN-, IFN-, or IFN- (34). The main biological function of IP-10 is chemoattraction of human monocytes, activated T cells, and NK cells, on which CXCR3, a major receptor for IP-10, is expressed (35, 36). In hematopoietic tumors, IP-10 expression is detected in Reed-Sternberg cells of Hodgkin's lymphoma (37) and multiple myeloma cells (38). With regard to the function of IP-10 in tumor cells, there are few reports showing beneficial effects for tumor progression. Giuliani et al. (38) reported that IP-10 induced antiapoptotic effects in myeloma cells and cell lines. Additionally, Zipin-Roitman et al. (39) reported that IP-10 promoted invasive phenotype in human colorectal carcinoma cells.

In the present study, we analyzed the chemokine production of nasal NK/T-cell lymphoma cell lines SNK-6 and SNT-8 using a chemokine protein array and found that IP-10 was abundantly produced in both SNK-6 and SNT-8, compared with KHYG-1, which was established from EBV-negative NK cell leukemia. We confirmed, by using ELISA, that SNK-1, SNK-6, and SNT-8 produced the IP-10 protein in a time-dependent manner, but EBV-negative NK cell leukemia and lymphoma cell lines KHYG-1 and NK-92 did not produce the IP-10 protein. Furthermore, we clearly showed, using flow cytometry, that CXCR3, a major receptor of IP-10, was expressed on SNK-1, SNK-6, and SNT-8 cells. Because these results show that IP-10 and CXCR3 are coexpressed in nasal NK/T-cell lymphoma cell lines, we next examined, using immunohistologic double staining, whether IP-10 and CXCR3 are expressed on lymphoma cells in vivo. We succeeded to show that CD56-positive lymphoma cells coexpressed IP-10 as well as CXCR3 in a majority of biopsy samples from the patients tested.

Previously, Ohshima et al. (40) performed cDNA array analysis on biopsy tissues of nasal NK/T-cell lymphoma patients and showed a markedly increased expression of IP-10 mRNA. Teruya-Feldstein et al. (41) also showed IP-10 mRNA expression in biopsy tissues from seven nasal NK/T-cell lymphoma patients using reverse transcriptase-PCR. They found IP-10 expression in endothelial cells lining the capillary vessels, macrophages, and fibroblasts, but they failed to find IP-10 expression on the lymphoma cells in the tissue sections. We used immunohistologic double staining with anti-CD56 and antiIP-10 antibodies for tissue sections and succeeded to prove that the lymphoma cells express IP-10. On the other hand, it is an accepted fact that nasal NK/T-cell lymphoma cells express CXCR3 on the cell surface. Yagi et al. (42) showed CXCR3 expression on the cutaneous NK/T-cell lymphoma cells in all five patients tested with immunohistologic staining. Similarly, Ishida et al. (43) showed CXCR3 expression on the nasal NK/T-cell lymphoma cells in 4 (15) of 27 patients tested with immunohistologic staining. In this study, we also found CXCR3 expression on the lymphoma cells in biopsy tissues from the majority of the patients tested.

It is reported that IP-10 acts as a cell growth factor inhibiting apoptosis in myeloma cell lines (38) and as an invasion factor in human colorectal carcinoma cells (39). Coexpression of IP-10 and CXCR3 in nasal NK/T-cell lymphoma cell lines found here suggests that IP-10 may have functional roles in autocrine cell growth and/or invasion. As the results of the functional analyses, IP-10 does not act as a cell growth factor for the cells but acts as an autocrine cell invasion factor. Results of the in vivo analyses showed that IP-10 and CXCR3 were expressed on the CD56-positive lymphoma cells in biopsy tissues and that serum IP-10 levels of the patients was much higher than those from healthy volunteers. Moreover, the level dramatically decreased during the complete remission phase after treatments. These findings suggest that the role of IP-10 as a autocrine cell invasion factor possibly works not only in vitro but also in vivo.

We found here that IP-10 was expressed and produced only in EBV-positive NK-cells or T-cell lines SNK-1, SNK-6, SNT-8, and KAI-3, but never in EBV-negative NK cell lines KHYG-1 and NK-92. These findings suggest that EBV may contribute to IP-10 production in nasal NK/T-cell lymphoma. Although we cannot completely explain how EBV may contribute to IP-10 production, it can be inferred that the EBV oncogenic LMP1 may be one of the candidates involved in inducing IP-10. Recently, Vockerodt et al. (20), using LMP1-transfected cell lines derived from Burkitt's and Hodgkin's lymphomas, showed that LMP1 is sufficient to induce IP-10 expression in lymphoma cells involving transcriptional (NF-B) and posttranscriptional (p38/SAPK2) mechanisms. They further showed that LMP1-mediated IP-10 activation is independent from autocrine tumor necrosis factor-, IFN-, or IL-18, which have been described as inducers of IP-10 (33, 34). In our experiments, all of the nasal NK/T-cell lymphoma cell lines (SNK-6, SNK-1, and SNT-8) used here express LMP1 (11). After analyzing in vivo materials, we found that LMP1 expression of lymphoma cells closely correlated with high IP-10 expression in the biopsy tissues and high IP-10 levels in sera. This is evidence that LMP1 may act as an IP-10 inducer. Alternatively, it is possible that some cytokines, e.g., tumor necrosis factor-, IFN-, IL-18, or others, which has been proved to induce IP-10 (33, 34), may stimulate IP-10 production in an autocrine manner, as previously found in EBV-infected lymphoblastoid cells (44). Indeed, it has been reported that various EBV-related lymphoma cells produce such cytokines (45, 46). Furthermore, we previously confirmed that the nasal NK/T-cell lymphoma cell lines produce several cytokines such as IFN-, IL-9, and IL-10 (13, 14).

In conclusion, we present here experimental evidence that IP-10 is specifically produced by nasal NK/T-cell lymphoma cell lines, has a potential role as an autocrine invasion factor for these cell lines, and is detected in tissues and sera from patients with nasal NK/T-cell lymphoma. These results suggest that the IP-10 signaling pathway may be a new pharmacologic target in the treatment of the patients with nasal NK/T-cell lymphoma.

No potential conflicts of interest were disclosed.

1
Spear
GS
,
Walker
WG
 Jr
. 
Lethal midline granuloma (granuloma gangraenescens) at autopsy; report of a case and review of literature
.
Bull Johns Hopkins Hosp
1956
;
99
:
313
32
.
2
Harabuchi
Y
,
Imai
S
,
Wakashima
J
, et al
. 
Nasal T-cell lymphoma causally associated with Epstein-Barr virus: clinicopathologic, phenotypic, and genotypic studies
.
Cancer
1996
;
77
:
2137
49
.
3
Aozasa
K
,
Ohsawa
M
,
Tajima
K
, et al
. 
Nation-wide study of lethal mid-line granuloma in Japan: frequencies of Wegener's granulomatosis, polymorphic reticulosis, malignant lymphoma and other related conditions
.
Int J Cancer
1989
;
44
:
63
6
.
4
Harabuchi
Y
,
Yamanaka
N
,
Kataura
A
, et al
. 
Epstein-Barr virus in nasal T-cell lymphomas in patients with lethal midline granuloma
.
Lancet
1990
;
335
:
128
30
.
5
Kanavaros
P
,
Lescs
MC
,
Briere
J
, et al
. 
Nasal T-cell lymphoma: a clinicopathologic entity associated with peculiar phenotype and with Epstein-Barr virus
.
Blood
1993
;
81
:
2688
95
.
6
Emile
JF
,
Boulland
ML
,
Haioun
C
, et al
. 
CD5-56+ T-cell receptor silent peripheral T-cell lymphomas are natural killer cell lymphomas
.
Blood
1996
;
87
:
1466
73
.
7
Jaffe
ES
,
Chan
JK
,
Su
IJ
, et al
. 
Report of the workshop on nasal and related extranodal angiocentric T/natural killer cell lymphomas. Definitions, differential diagnosis, and epidemiology
.
Am J Surg Pathol
1996
;
20
:
103
11
.
8
Yamanaka
N
,
Harabuchi
Y
,
Sambe
S
, et al
. 
Non-Hodgkin's lymphoma of Waldeyer's ring and nasal cavity. Clinical and immunologic aspects
.
Cancer
1985
;
56
:
768
76
.
9
Harris
NL
,
Jaffe
ES
,
Stein
H
, et al
. 
A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group
.
Blood
1994
;
84
:
1361
92
.
10
Eichel
BS
,
Harrison
EG
 Jr.
,
Devine
KD
,
Scanlon
PW
,
Brown
HA
. 
Primary lymphoma of the nose including a relationship to lethal midline granuloma
.
Am J Surg
1966
;
112
:
597
605
.
11
Nagata
H
,
Konno
A
,
Kimura
N
, et al
. 
Characterization of novel natural killer (NK)-cell and T-cell lines established from primary lesions of nasal T/NK-cell lymphomas associated with the Epstein-Barr virus
.
Blood
2001
;
97
:
708
13
.
12
Minarovits
J
,
Hu
LF
,
Imai
S
, et al
. 
Clonality, expression and methylation patterns of the Epstein-Barr virus genomes in lethal midline granulomas classified as peripheral angiocentric T cell lymphomas
.
J Gen Virol
1994
;
75
:
77
84
.
13
Nagato
T
,
Kobayashi
H
,
Kishibe
K
, et al
. 
Expression of interleukin-9 in nasal natural killer/T-cell lymphoma cell lines and patients
.
Clin Cancer Res
2005
;
11
:
8250
7
.
14
Takahara
M
,
Kis
LL
,
Nagy
N
, et al
. 
Concomitant increase of LMP1 and CD25 (IL-2-receptor ) expression induced by IL-10 in the EBV-positive NK lines SNK6 and KAI3
.
Int J Cancer
2006
;
119
:
2775
83
.
15
Wang
JM
,
Deng
X
,
Gong
W
,
Su
S
. 
Chemokines and their role in tumor growth and metastasis
.
J Immunol Methods
1998
;
220
:
1
17
.
16
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
6
.
17
Laurence
AD
. 
Location, movement and survival: the role of chemokines in haematopoiesis and malignancy
.
Br J Haematol
2006
;
132
:
255
67
.
18
Baumforth
KR
,
Birgersdotter
A
,
Reynolds
GM
, et al
. 
Expression of the Epstein-Barr virus-encoded Epstein-Barr virus nuclear antigen 1 in Hodgkin's lymphoma cells mediates up-regulation of CCL20 and the migration of regulatory T cells
.
Am J Pathol
2008
;
173
:
195
204
.
19
Uchihara
JN
,
Krensky
AM
,
Matsuda
T
, et al
. 
Transactivation of the CCL5/RANTES gene by Epstein-Barr virus latent membrane protein 1
.
Int J Cancer
2005
;
114
:
747
55
.
20
Vockerodt
M
,
Pinkert
D
,
Smola-Hess
S
, et al
. 
The Epstein-Barr virus oncoprotein latent membrane protein 1 induces expression of the chemokine IP-10: importance of mRNA half-life regulation
.
Int J Cancer
2005
;
114
:
598
605
.
21
Ren
Q
,
Sato
H
,
Murono
S
,
Furukawa
M
,
Yoshizaki
T
. 
Epstein-Barr virus (EBV) latent membrane protein 1 induces interleukin-8 through the nuclear factor- B signaling pathway in EBV-infected nasopharyngeal carcinoma cell line
.
Laryngoscope
2004
;
114
:
855
9
.
22
Harris
NL
,
Jaffe
ES
,
Diebold
J
, et al
. 
The World Health Organization classification of neoplasms of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting-Airlie House, Virginia, November, 1997
.
Hematol J
2000
;
1
:
53
66
.
23
Harabuchi
Y
,
Tsubota
H
,
Ohguro
S
, et al
. 
Prognostic factors and treatment outcome in non-Hodgkin's lymphoma of Waldeyer's ring
.
Acta Oncol
1997
;
36
:
413
20
.
24
Harabuchi
Y
,
Takahara
M
,
Kishibe
K
,
Moriai
S
,
Nagato
T
,
Ishii
H
. 
Nasal natural killer (NK)/T-cell lymphoma: clinical, histological, virological, and genetic features
.
Int J Clin Oncol
2009
;
14
:
181
90
.
25
Tsuge
I
,
Morishima
T
,
Morita
M
,
Kimura
H
,
Kuzushima
K
,
Matsuoka
H
. 
Characterization of Epstein-Barr virus (EBV)-infected natural killer (NK) cell proliferation in patients with severe mosquito allergy; establishment of an IL-2-dependent NK-like cell line
.
Clin Exp Immunol
1999
;
115
:
385
92
.
26
Yagita
M
,
Huang
CL
,
Umehara
H
, et al
. 
A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation
.
Leukemia
2000
;
14
:
922
30
.
27
Gong
JH
,
Maki
G
,
Klingemann
HG
. 
Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells
.
Leukemia
1994
;
8
:
652
8
.
28
Epstein
MA
,
Achong
BG
,
Barr
YM
,
Zajac
B
,
Henle
G
,
Henle
W
. 
Morphological and virological investigations on cultured Burkitt tumor lymphoblasts (strain Raji)
.
J Natl Cancer Inst
1966
;
37
:
547
59
.
29
Nakamura
S
,
Katoh
E
,
Koshikawa
T
, et al
. 
Clinicopathologic study of nasal T/NK-cell lymphoma among the Japanese
.
Pathol Int
1997
;
47
:
38
53
.
30
Ohshima
K
,
Karube
K
,
Kawano
R
, et al
. 
Classification of distinct subtypes of peripheral T-cell lymphoma unspecified, identified by chemokine and chemokine receptor expression: analysis of prognosis
.
Int J Oncol
2004
;
25
:
605
13
.
31
Nakayama
T
,
Fujisawa
R
,
Izawa
D
,
Hieshima
K
,
Takada
K
,
Yoshie
O
. 
Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5
.
J Virol
2002
;
76
:
3072
7
.
32
Albini
A
,
Iwamoto
Y
,
Kleinman
HK
, et al
. 
A rapid in vitro assay for quantitating the invasive potential of tumor cells
.
Cancer Res
1987
;
47
:
3239
45
.
33
Luster
AD
,
Unkeless
JC
,
Ravetch
JV
. 
-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins
.
Nature
1985
;
315
:
672
6
.
34
Luster
AD
,
Ravetch
JV
. 
Biochemical characterization of a interferon-inducible cytokine (IP-10)
.
J Exp Med
1987
;
166
:
1084
97
.
35
Taub
DD
,
Lloyd
AR
,
Conlon
K
, et al
. 
Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells
.
J Exp Med
1993
;
177
:
1809
14
.
36
Taub
DD
,
Sayers
TJ
,
Carter
CR
,
Ortaldo
JR
. 
and chemokines induce NK cell migration and enhance NK-mediated cytolysis
.
J Immunol
1995
;
155
:
3877
88
.
37
Teruya-Feldstein
J
,
Jaffe
ES
,
Burd
PR
,
Kingma
DW
,
Setsuda
JE
,
Tosato
G
. 
Differential chemokine expression in tissues involved by Hodgkin's disease: direct correlation of eotaxin expression and tissue eosinophilia
.
Blood
1999
;
93
:
2463
70
.
38
Giuliani
N
,
Bonomini
S
,
Romagnani
P
, et al
. 
CXCR3 and its binding chemokines in myeloma cells: expression of isoforms and potential relationships with myeloma cell proliferation and survival
.
Haematologica
2006
;
91
:
1489
97
.
39
Zipin-Roitman
A
,
Meshel
T
,
Sagi-Assif
O
, et al
. 
CXCL10 promotes invasion-related properties in human colorectal carcinoma cells
.
Cancer Res
2007
;
67
:
3396
405
.
40
Ohshima
K
,
Karube
K
,
Hamasaki
M
, et al
. 
Differential chemokine, chemokine receptor and cytokine expression in Epstein-Barr virus-associated lymphoproliferative diseases
.
Leuk Lymphoma
2003
;
44
:
1367
78
.
41
Teruya-Feldstein
J
,
Jaffe
ES
,
Burd
PR
, et al
. 
The role of Mig, the monokine induced by interferon-, and IP-10, the interferon--inducible protein-10, in tissue necrosis and vascular damage associated with Epstein-Barr virus-positive lymphoproliferative disease
.
Blood
1997
;
90
:
4099
105
.
42
Yagi
H
,
Seo
N
,
Ohshima
A
, et al
. 
Chemokine receptor expression in cutaneous T cell and NK/T-cell lymphomas: immunohistochemical staining and in vitro chemotactic assay
.
Am J Surg Pathol
2006
;
30
:
1111
9
.
43
Ishida
T
,
Inagaki
H
,
Utsunomiya
A
, et al
. 
CXC chemokine receptor 3 and CC chemokine receptor 4 expression in T-cell and NK-cell lymphomas with special reference to clinicopathological significance for peripheral T-cell lymphoma, unspecified
.
Clin Cancer Res
2004
;
10
:
5494
500
.
44
Yao
L
,
Setsuda
J
,
Sgadari
C
,
Cherney
B
,
Tosato
G
. 
Interleukin-18 expression induced by Epstein-Barr virus-infected cells
.
J Leukoc Biol
2001
;
69
:
779
84
.
45
Lay
JD
,
Tsao
CJ
,
Chen
JY
,
Kadin
ME
,
Su
IJ
. 
Upregulation of tumor necrosis factor- gene by Epstein-Barr virus and activation of macrophages in Epstein-Barr virus-infected T cells in the pathogenesis of hemophagocytic syndrome
.
J Clin Invest
1997
;
100
:
1969
79
.
46
Setsuda
J
,
Teruya-Feldstein
J
,
Harris
NL
, et al
. 
Interleukin-18, interferon-, IP-10, and Mig expression in Epstein-Barr virus-induced infectious mononucleosis and posttransplant lymphoproliferative disease
.
Am J Pathol
1999
;
155
:
257
65
.

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