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. 2003 Sep;133(3):344–349. doi: 10.1046/j.1365-2249.2003.02234.x

IL-17 enhances the susceptibility of U-2 OS osteosarcoma cells to NK cell lysis

M C HONORATI *,†, S NERI *, L CATTINI *, A FACCHINI *,†
PMCID: PMC1808781  PMID: 12930359

Abstract

We investigated the effect of the proinflammatory cytokine interleukin 17 (IL-17) on the lysis of osteosarcoma cells by human NK cells. NK cells and U-2 OS, MG-63, HOS osteosarcoma cell lines express the IL-17 receptor, the highest amount being found on U-2 OS. Pre-incubation of NK cells with IL-17 did not affect the cytotoxicity against osteosarcomas, that was increased when U-2 OS were pre-incubated with IL-17. In IL-17 treated U-2 OS osteosarcoma cells FACS analysis demonstrated an increased expression of fibronectin among the panel of adhesion molecules assayed, and the treatment with anti-fibronectin antibodies decreased the NK cytotoxicity. The comparison between interferon gamma (IFN-γ) treated and IFN-γ/IL-17-treated U-2 OS showed a decreased susceptibility to NK lysis associated with a reduced expression of CD49f on U-2 OS treated with IFN-γ/IL-17. IL-17 appears to be a modulator of NK adhesion molecules on U-2 OS cells but antagonizes with IFN-γ on NK lysis.

Keywords: IL-17, NK activity, osteosarcoma, adhesion molecules

INTRODUCTION

Some cytokines can play a role as anti-tumour agents [1]. Among them, interleukin (IL)-2, IL-12, IL-18, and IFNs, can influence NK activity against tumour cells [2]. NK cells represent about 10% of peripheral blood lymphocytes (PBL) and their lytic activity constitutes an immune system's natural defence against virus-infected and neoplastic cells [3]. Adhesion molecules expressed on the cell surface can contribute to the increase of NK activity by helping the binding between NK and tumour cells [4,5], and cytokines can up-regulate the expression of some adhesion molecules [5,6]. Osteosarcomas are bone tumours derived from the malignant transformation of osteoblasts and occur mainly in young subjects. These tumours are characterized by a highly malignant and metastatic behaviour. The surgical techniques and the development of adjuvant chemotherapy cannot still avoid pulmonary metastasis, which is the major cause of death in patients with osteosarcoma [7]. Human osteosarcoma cell lines have been studied in vitro for their ability to modify expression of adhesion molecules after treatment with TNF-α, so becoming better targets for NK cells [6].

Human IL-17 is a 20–30 kD glycosylated homodimeric polypeptide of 155 amino acids produced by activated T lymphocytes [8], mainly defined as a proinflammatory cytokine [9]. The IL-17 receptor (IL-17R) is an ubiquitous transmembrane glycoprotein present in all tested tissues [10]. NK cells and some tumour cells express IL-17R [10], but the effect of the binding of IL-17 to its ligand on these cells is not well known.

A direct effect of IL-17 has been demonstrated in vitro on the secretion of IL-6, IL-8 and angiogenic factors by tumour cells [11,12], but not on cell proliferation [11–13]. The effect of IL-17 on tumour cells in vivo is more debatable. In murine models IL-17 was shown to be responsible for increased tumour size [11,12] and, in other experimental model, to act as anti-tumour factor [13,14]. Besides, it is noteworthy that IL-17 seems to increase the NK activity of spleen cells, but is not clear whether this effect of IL-17 on NK cells is indirect, due to a host-dependent mechanism [13]. IL-17 increases also the expression of adhesion molecules on fibroblasts and endothelial cells [8,15] and of intracellular adhesion molecule-1 (ICAM-1) by keratinocytes in synergism with IFN-γ[16].

The aim of this study is to clarify if IL-17 is able to influence the interaction between NK and osteosarcoma cells. Therefore, we studied in vitro the effect of IL-17 on NK cell cytolytic activity and osteosarcoma cell lines as determined by susceptibility to NK cell lysis. Moreover, we verified if IL-17 synergizes with INF-γ in modulating the expression of adhesion molecules involved in the regulation of the effector/target interaction.

MATERIALS AND METHODS

Target tumour cells

HOS, MG-63, Saos-2 and U-2 OS are human osteogenic sarcoma cell lines with an undifferentiated osteoblastic-like phenotype (ATCC, Rockville, MD, USA). They grow as adherent cells and are routinely maintained in Iscove's modified Dulbecco's medium (IMDM) (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat inactivated fetal calf serum (FCS) (Gibco BRL), 4 mm glutamine (Sigma, St.Louis, MO, USA) and 200 µg/ml gentamycin (Flow Laboratories, Biaggio, Switzerland) (complete IMDM). For the experiments, osteosarcoma cells were detached by 0·05% trypsin/0·5 mm EDTA (Sigma), washed twice and seeded in 24-well flat-bottomed plates at a concentration of 2·5 × 105 cells/ml. Cell viability, determined by the eosin dye exclusion test, was always higher than 95%. The NK-susceptible erytroleukaemia cell line K562 was used as a positive control of NK activity. K562 cells, growing in suspension in RPMI 1640 (Gibco BRL) supplemented with 10% heat inactivated FCS, 4 mm glutamine and 200 µg/ml gentamicin (complete RPMI), were used for experiments at logarithmic growth phase.

Effector cells

PBL were isolated from the venous blood of 16 healthy human volunteers (mean age ± SD, 30 ± 5 years) by density gradient centrifugation. PBL were resuspended at the concentration of 2·5 × 106/ml in complete RPMI and used for cytotoxicity assay.

Cytokine treatment

PBL (1·5 × 106/ml) and osteosarcoma cells (2·5 × 105/ml) were incubated for 24–48 h in complete RPMI and for 24-48-72-96 h in complete IMDM, respectively, either alone (control) or with the following stimuli: recombinant human IL-17 (IL-17) (25-50-100-200–400 ng/ml) (R & D Systems, Minneapolis, MN, USA), IL-2 (100 U/ml) (Boehringer, Mannheim, Germany), IFN-γ (750 U/ml) (Boehringer), as described for each experiment. Previous experiments showed that these IL-2 and IFN-γ concentrations were optimal to, respectively, stimulate NK cell cytolytic activity and U-2 OS susceptibility to NK lysis. At the end of the incubation cells were detached, if necessary, washed twice, resuspended in complete RPMI or IMDM and used for cytotoxicity assay.

Cytotoxicity assay

After stimulation, osteosarcoma or K562 cells were incubated for 30 min at 37°C in complete IMDM or RPMI with 15 µm calcein-acetoxymethyl (calcein-AM; Molecular Probes, Eugene, Oregon) at a final concentration of 1 × 106 cells/ml. A Calcein-AM cytotoxicity assay was performed in triplicate as described by Neri et al. [17], with effector/target ratios from 50 : 1 to 0·8 : 1. After 4 hours’ incubation, the plate was centrifuged, 75 µl of each supernatant were collected, and fluorescence was determined using a Spectramax Gemini dual-scanning microplate spectrofluorimeter (Molecular Device, Sunnyvale, CA, USA) (485 ± 9 nm excitation and 530 ± 9 nm emissiom). The percentage of specific lysis was calculated according to the formula:

% calcein-AM release = (experimental release − spontaneous release)/(maximum release − spontaneous release) × 100.

Spontaneous release represents calcein-AM release from target cells in medium alone and maximum release from target cells lysed in medium containing 5% Triton X-100.

Cytofluorimetric analysis

The expression of IL-17R and fibronectin were evaluated on osteosarcoma cells stained with anti-hIL-17R polyclonal antibody (R & D Systems) and hybridoma supernatants from HFN 7·1 clone (ATCC), respectively, by flow cytometry with a FACStar Plus (Becton Dickinson, St. Diego, CA, USA). U-2 OS cells were also characterized for the expression of some adhesion molecules and adhesion molecule ligands: CD29 (Endogen, Woburn, MA, USA), CD54, CD49b,c,f (Cymbus Bioscience Ltd, UK), CD49a (T Cell Diagnostic, USA), CD49e, laminin (Immunotech, France), CD56 (Becton Dickinson, Mountain View, CA, USA), CD49d, CD11a, CD11b, CD18, CD58 (hybridoma supernatants from KD4-13, TS1/22, LM2/1, TS1/18, TS2/9 clones, respectively, ATCC), αVβ3 and αVβ5 vitronectin receptor (Chemicon International, Temecula, CA, USA) monoclonal antibodies (MoAb). Ten × 104 cells were incubated for 30 min at 4°C with the primary antibody and then with the secondary antibody (goat anti-mouse FITC- or PE-conjugated immunoglobulins) (Becton Dickinson). To reduce nonspecific binding, samples were incubated with 1 : 10 normal mouse serum (Dako, Glostrup, Denmark). Negative controls were only incubated with mouse polyclonal immunoglobulins of the same isotype and with FITC-conjugated goat anti-mouse Ig.

Inhibition assay

Non-specific stimulation eventually due to IL-17 preparation was excluded by inhibiting the effect of the cytokine with anti-IL-17 MoAb (R & D Systems). Briefly, U-2 OS cells were cultured for 48 h with 100 ng/ml IL-17 previously incubated for 1 h at 37°C with 25–2·5–0·25 µg/ml anti-IL-17 MoAb and then the cytotoxicity test was performed. To evaluate the relevance of fibronectin on NK/U-2 OS adhesion, the calcein-AM test was performed on 48-h IL-17 stimulated U-2 OS cells after 30 min incubation at 37°C with anti-fibronectin MoAb (1·9 mg/ml).

Statistical analysis

Statistical computations were performed using CSS Statistica statistical software (Statsoft Tulsa, OK, USA). Results are expressed as means ± S.D. Differences between groups were analysed by Student's t-test and when the anova test for multiple comparison was significant, data were analysed by Student's t-test for paired data. Correlation was analysed by Pearson's test.

RESULTS

IL-17 is noneffective on NK activity

To verify the effect of IL-17 on NK cells, NK activity of PBL pre-incubated with IL-17 or IL-2 was evaluated against K562 cells. IL-2 was used as control of NK activity stimulation [18]. IL-17 had no effect on NK cells at all tested concentrations neither after 24 (Fig. 1) nor 48 h (not shown) of treatment. As expected, NK cell cytotoxicity was increased by IL-2 (Fig. 1).

Fig. 1.

Fig. 1

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Effect of IL-17 on NK cell cytotoxicity against K562 cells. PBL were incubated 24 h with medium alone, with 100 U/ml IL-2, or with various concentrations of IL-17. Results are expressed as mean ± S.D. from three independent experiments. Spontaneous release of calcein-AM was <10%. *P < 0·05 versus nonstimulated and 50-100-200 ng/ml IL-17 stimulated PBL.

Expression of IL-17R on osteosarcoma cell lines

HOS, MG-63 and U-2 OS, but not Saos-2, cell lines presented IL-17R. Cytofluorimetric analysis revealed that the U-2 OS cell line shows a higher expression of IL-17R compared with HOS and MG-63 cell lines (Fig. 2).

Fig. 2.

Fig. 2

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Expression of IL-17R on osteosarcoma cell lines. Results (mean ± S.D. of 4 independent flow cytometry assays) are expressed as mean fluorescence channel number subtracted of the negative control fluorescence. Fluorescent signals were processed by a four-decade log amplifier and displayed on a 0–1024 channel scale. *P < 0·05 versus HOS and MG63 cell lines.

Effect of IL-17and IFN-γ on osteosarcoma susceptibility to NK lysis

Since IL-17 treatment did not change the NK activity of PBL against osteosarcoma cell lines (data not shown), IL17R positive osteosarcoma cell lines were incubated 48 h with IL-17 before the cytotoxicity test, to verify if IL-17 can modulate the susceptibility of these tumour cell lines to NK cell lysis. Only U-2 OS cell line susceptibility was enhanced by the IL-17 treatment. The effect of IL-17 on U-2 OS lysis was dose and time dependent, with a peak at 200 ng/ml concentration and after 48 h of incubation, so we used these incubation conditions if not differently indicated. The specificity of this effect has been verified by incubating IL-17 with different concentrations of anti-IL-17 antibody before using it to stimulate U-2 OS culture. IL-17 biologic activity was dramatically inhibited by 25 µg/ml specific antibody (Fig. 3).

Fig. 3.

Fig. 3

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Inhibitory effect of anti-IL-17 MoAb. Results are expressed as percentage of specific lysis. U-2 OS cell line was incubated with IL-17 previously treated for 1 h at 37°C with different concentrations of anti-IL-17 MoAb. One experiment representative of three is shown.

The incubation of U-2 OS with IFN-γ for 48 h stimulated a higher susceptibility to NK lysis. It is noteworthy that when IL-17 was added to IFN-γ treated U-2 OS during the last 24 h of incubation, the susceptibility to NK lysis decreased. This decrease was not different when IL-17 was used in combination with IFN-γ during all incubation times (Fig. 4).

Fig. 4.

Fig. 4

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Susceptibility of U-2 OS cell line to NK lysis following treatment with IL-17 and IFN-γ. Results are expressed as mean percentage of specific lysis ± S.D. of PBL from 12 different subjects. Effector/target cell ratio 50/1 was used. **P < 0·01 versus non-stimulated; *P < 0·05 versus non-stimulated; §P < 0·05 versus cells treated with IL-17 or INF-γ + IL-17.

Adhesion molecule expression modulated by IL-17 and IFN-γ

HOS and MG-63 were constitutively positive for fibronectin and IL-17 did not enhance the fibronectin expression. The U-2 OS cell line was constitutively positive for CD56, CD58, CD29, CD49a,b,c,d,f, fibronectin, αVβ3 and αVβ5 (Table 1) and negative for CD11a, CD11b, CD18, CD54, and laminin (data not shown). Treatment with IL-17, IFN-γ or the combination of IL-17 and IFN-γ did not induce the expression of CD11a, CD11b, CD18, CD54, and laminin (data not shown). IL-17 alone only enhanced the expression of fibronectin (Table 1 and Fig. 5a). IFN-γ alone enhanced the expression of fibronectin, CD49b and CD49f (Table 1). Fibronectin up-regulation by IL-17 and IFN-γ was comparable, and the combination of IL-17 and IFN-γ had the same effect as IL-17 or IFN-γ alone, showing an antagonism between these cytokines (Table 1). To verify if the enhanced susceptibility to NK cell lysis after IL-17 stimulation depended on the up-regulation of fibronectin expression, IL-17 stimulated U-2 OS cells were incubated with anti-fibronectin antibody before calcein-AM treatment. The chosen antibody concentration of 1·9 mg/ml showed the highest fluorescence by flow cytometry in a dose-effect curve. As shown in Fig. 5b, the antibody treatment partially inhibited both the basal and IL-17 stimulated U-2 OS susceptibility to NK cell lysis.

Table 1.

Effect of treatment with IL-17 or/and IFN-γ on adhesion molecules expressed by U-2 OS

IFN-γ 48 h
Adhesion molecule non-stimulated IL-17 48 H IFN-γ 48 h + IL-17 24 h + IL-17 48 h
CD56 374 ± 18·0 362 ± 4·9 356 ± 5·7 n.d. 337 ± 30·0
CD58 225 ± 4·6 236 ± 11·0 220 ± 11·7 232 ± 24·0 221 ± 4·2
CD29 409 ± 48·7 411 ± 45·9 424 ± 43·8 421 ± 25·2 404 ± 7·5
CD49a 62 ± 7·3 55 ± 8·0 66 ± 10·0 58 ± 9·4 71 ± 8·5
CD49b 561 ± 28·3 557 ± 67·2 591 ± 17·0* 610 ± 27·0* 634 ± 12·7*
CD49c 296 ± 25·4 270 ± 17·0 272 ± 13·7 273 ± 14·2 255 ± 17·6
CD49d 329 ± 21·5 332 ± 21·7 335 ± 24·8 341 ± 23·7 360 ± 6·0
CD49e 345 ± 31·5 331 ± 41·7 302 ± 38·4 335 ± 21·7 377 ± 28·6
CD49f 315 ± 27·5 302 ± 14·6 346 ± 20·0* 327 ± 23·6 320 ± 11·5
Fibronectin 126 ± 20·0 160 ± 17·9** 157 ± 23·1** nd 156 ± 21·8*
αVβ3 322 ± 40·2 327 ± 25·4 327 ± 18·6 333 ± 14·5 nd
αVβ5 469 ± 31·6 465 ± 15·4 475 ± 21·4 466 ± 28·6 nd
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Results obtained by FACS from five independent experiments are expressed as the mean of fluorescence intensity channel number subtracted of the negative control fluorescence ± S.D.

*

P < 0·05,

**

P < 0·01 versus non-stimulated.

Fig. 5.

Fig. 5

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Role of fibronectin on U-2 OS cell line susceptibility to NK lysis. The U-2 OS cell line was treated with 200 ng/ml IL-17 for 48 h (a) The expression of fibronectin was evaluated by flow cytometry. Dashed line: isotype control; grey area: nonstimulated U-2 OS; dark line: IL-17 stimulated U-2 OS. One experiment representative of four is shown. (b) Non-stimulated (NS) and IL-17 stimulated U-2 OS were treated with 1·9 mg/ml anti-fibronectin MoAb for 30 min at 37°C before calcein-AM incubation. Results are expressed as mean percentage of specific lysis ± S.D. of PBL from 3 different subjects. Effector/target cell ratio 50/1 was used. *P < 0·05 versus NS; §P < 0·05 versus IL-17.

It is noteworthy that, in comparison with IFN-γ alone, a decreasing effect on the expression of CD49f was obtained when U-2 OS were incubated with the combination of IL-17 and IFN-γ and under this condition of incubation also the susceptibility to NK cell lysis decreased until becoming similar to that obtained with IL-17 alone (Table 1 and Fig. 4). The susceptibility of U-2 OS cells to NK cell lysis was correlated with the expression of CD49f (Pearson's correlation: r= 0·578; P= 0·010).

DISCUSSION

We demonstrated that IL-17 in vitro does not regulate the cytolytic activity of NK cells, but can increase the susceptibility of U-2 OS osteosarcoma cells to NK cell lysis. Our findings support the hypothesis of a lack of direct action of IL-17 on cytolytic activity of NK cells, although they express IL-17R [10]. This result is apparently in contrast with the results obtained in vivo by other authors, because IL-17 transfected xenogeneic cells, intravenously injected in nude mice, stimulated a higher activity of spleen NK cells than parent cells [13]. However it is not clear whether an indirect action of IL-17 on NK and on specific immunity cells was responsible of the effect present in vivo.

In our in vitro model we verified if IL-17 induced on U-2 OS target cell line the expression of some adhesion molecules contributing to NK cell killing, or up-regulated some of those previously described on osteosarcomas [5,19]. Our findings showed an enhanced expression of fibronectin by the U-2 OS cell line treated with IL-17. Fibronectin is a well represented glycoprotein in most extracellular matrices, constitutively synthesized by bone resident cells and represents a prominent adhesive protein which mediates various aspects of cellular interactions with extracellular matrices including migration [20]. NK cells express some receptors which bind fibronectin, including very late antigens (VLA) such as CD49d and CD49e. The contribution of fibronectin to U-2 OS cell susceptibility to NK lysis was confirmed by inhibition assay with anti-fibronectin MoAb. However, the fibronectin expression on target cells is only one of the possible mechanisms responsible of the cytotoxic effect by NK cells and of the enhanced susceptibility to NK cell lysis after IL-17 treatment. In fact, anti-fibronectin MoAb could neither block the cytolytic activity against the nonstimulated osteosarcoma cell line nor fully inhibit the IL-17 enhanced susceptibility of U-2 OS.

In our experimental model, IL-17 was able to stimulate only one (U-2 OS) out of three osteosarcoma cell lines, nevertheless all were positive for IL-17R, even if at different degrees. The effect of IL-17 could be endangered by an insufficient expression of the specific receptor on target cells. The different expression of IL-17R and the effectiveness of the signal transduction for fibronectin up-regulation induced by IL-17 on osteosarcoma cell lines, might also depend on the cell differentiation level, as for the expression of other specific cellular markers [21]. U-2 OS represents the less differentiated cell line, followed by HOS, then by MG-63 and Saos-2 [21–23], being the last one the more osteoclast-like cell line and not expressing IL17R.

Costimulation of U-2 OS cell line with IL-17 and IFN-γ results in a down-regulation of both the susceptibility to NK cell lysis and the expression of CD49f in respect to IFN-γ treatment alone. Since NK cells express laminin [24], a ligand for CD49f [25], the reduced expression of CD49f on IL-17/IFN-γ treated U-2 OS seems to be one of the mechanisms responsible of the decreased susceptibility to NK cell lysis. On the contrary, we can exclude that this effect is due to IL-17/IFN-γ mediated up-regulation of MHC class I antigens that are known to inhibit NK-mediated lysis [26]. In fact, while IFN-γ enhanced the MHC-I expression, as expected [27], IL-17 had no effect neither alone nor in combination with IFN-γ (data not shown). IL-17 and IFN-γ are cytokines released by T lymphocytes [8,28] and they share some modulating effects on cell functions [8, 9, 16, 29]. Interestingly, used in combination on the same cell type in vitro they can synergize, modulate or inhibit each other's activity, and down-regulate various cell functions [16]. So, it is not surprising that the expression of fibronectin was not modulated by IL-17 in combination with IFN-γ compared with the stimulation with IL-17 or IFN-γ alone, and that these cytokines were antagonistic on CD49f expression. This study clearly shows that IL-17 may act directly on an osteosarcoma cell line up-regulating the expression of adhesion molecules, that play an important role in the interaction between an aspecific immune effector and tumour target. However, this potential therapeutic role is questionable, since the effect of well known anti-tumour cytokines such as IFN-γ can be down-regulated by IL-17.

Acknowledgments

The authors would like to thank Dr Erminia Mariani for the revision of the discussion, Dr Elettra Pignotti for statistical revision, Patrizia Rappini and Graziella Salmi for typing assistance, Luciano Pizzi for technical assistance and Keith Smith for language revision. This work was supported by grants from MIUR-Ricerca Fondamentale Orientata (ex 60%) and Ricerca Corrente I.O.R.

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