Involvement of interleukin-6 in the biology and metastatic activity of B16F10 melanoma cells

A. Garcia de Galdeano; D. Boyano; I. Smith-Zubiaga; A. Alvarez; I. Canton; L. Canavate

Melanoma cells produce and/or respond to a variety of cytokines which are thought to stimulate tumor and metastatic development [1]. Some of these factors belong to the family of interleukins, soluble mediators of the immune system. Interleukin-6 (IL-6), a multi-functional cytokine [2], has been reported to induce antitumor activity against melanoma [3-5]; however, there is some evidence that IL-6 could behave as an autocrine growth factor in melanoma, at least in cell lines derived from advanced disease [6]. Interleukin-2 (IL-2), the main T cell growth factor [7], could also be involved in the biology of human melanoma. In this regard, it has been proved that certain human melanoma cell lines express the receptor for IL-2 (IL-2R) and release soluble IL-2 [8, 9]. In addition, in previous results, we demonstrated the presence of the IL-2/IL-2R system in B16F10 cells, a metastatic variant of B16 murine melanoma [10], and, even more importantly, the culture of melanoma cells in the presence of IL-2 increased the metastatic efficiency of these tumor cells [11].

Within the immune system, IL-2, IL-6 and other interleukins are members of a network in which these factors regulate each other. In this context, it has been reported that IL-2 can induce the synthesis of IL-6 [12] and that IL-1 and IL-6 can modulate the production of IL-2 and its receptor [13, 14]. Nevertheless, very little is known about the interaction of these factors outside the immune system.

In the present study, the effect of IL-6 on B16F10 cells was examined in order to clarify the role played by interleukins in melanoma. To do so, the metastatic ability of B16F10 cells and some biological properties associated with metastasis were determined, after in vitro culture in the presence of IL-6. Whether B16F10 cells express IL-6 was also analyzed. Finally, the modulation of the IL-2R expression by IL-1 and IL-6 in these melanoma cells was investigated.

Eight-week old, pathogen-free C57BL/6 mice were obtained from the Iffa Credo Laboratories (France) and used for in vivo experiments and as a source of splenocytes.

B16F10 murine melanoma cells were grown in D-MEM medium supplemented with 10% fetal calf serum (FCS). For subculture, cell monolayers were removed from culture flasks using a 2 mM solution of PBS-EDTA. Murine splenic mononuclear cells were used as controls for IL-2R expression. Single cell suspensions were prepared by teasing the spleen tissue, and mononuclear cell isolation was performed by centrifugation in a density gradient (Ficoll-paque, Pharmacia). Splenocytes were cultured in RPMI-1640 supplemented with 5 x 10^-5 M 2-mercaptoethanol, 10 mM HEPES, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml) and 10% FCS.

Metastasis colonization was evaluated in two organs: the liver and the lungs. Hepatic metastases were induced by intrasplenic injection of 5 x 10⁵ viable B16F10 cells into anesthetized C57BL/6 mice. After 7 days, the animals were killed and their livers were removed, frozen and serially cut in a cryostat. The metastatic colonization was determined in the hepatic sections by a stereological method as previously described [15]. For lung metastases, 1 x 10⁵ tumor cells were injected into a lateral tail vein. The animals were sacrificed 21 days later and the number of colonies on the surface of the lungs was determined. To study the effect of IL-6 on metastasis formation, prior to inoculation, cells were cultured for 48 hours in the presence of 200 U/ml of IL-6.

The effect of IL-6 on cell proliferation was determined in 96-well microplates. Serial dilutions of IL-6 were performed in the microplates before seeding the cells (1 x 10⁴ cl/well) in a final volume of 200 µl of D-MEM + 10% FCS. Six hours before the culture ended (24 or 48 hours), cells received a pulse of ³H-thymidine (1 µCi/well, 2 mCi/ml specific activity). Incorporated radioactivity was assesed by liquid scintillation counting. For colony formation, cells cultured for 48 hours with or without IL-6 (200 or 2000 U/ml) were obtained and then seeded in 24-well macroplates (100 cl/well) in 1 ml of D-MEM + 10% FCS. Alternatively, the assay was performed with untreated cells but in the presence of IL-6. Seven days later, the number of colonies was determined by crystal violet staining.

B16F10 cells obtained from semiconfluent cultures were washed in PBS and then, 5 x 10⁵ cells per sample were incubated with the first antibody diluted in 0.5% PBS-BSA solution for 30 min at 4° C. The following MoAb were used: anti-mouse CD44, anti-mouse VLA-4 (alpha4) (Chemicon) and AMT-13 (Boehringer Mannheim). After two washes in PBS-BSA, cells were incubated with FITC-conjugated rabbit anti-rat IgG (Dakopatts) (1:30 dilution) for 30 min at 4º C. Then, cells were washed again (three washes), resuspended in 0.5 ml PBS and analyzed using a Coulter EPICS 752. The percentage of positive cells and the fluorescence intensity were determined by Immunotest analysis (Coulter). Non-specific staining was determined from cells incubated with a rat IgG2 (Boehringer Ingelheim). Unstimulated and Con-A stimulated splenocytes were also included as controls for the IL-2Ralpha expression.

The presence of IL-6 in the cultures of B16F10 was determined by immunoassay using a comercial kit for murine IL-6 (Quantikine^TM M, RD Systems). In this assay we included supernatants from B16F10 cells cultured at different concentrations of FCS (1, 5 and 10%) and also from cells cultured in the presence of IL-2 (500, 50 U/ml). Supernatants were collected 24, 48 and 72 hours after seeding the cells and then they were immediately frozen and stored at 20° C until tested.

RNA from B16F10 cells was purified by phenol/chloroform extraction and precipitation in ethanol as previously described [10]. Poly A⁺ RNA was enriched using the oligo(dT)-cellulose method.

Samples of 2 µg of total RNA were subjected to reverse transcription with oligo(dT)12-18 (Clontech) in 30 µl of a reaction mixture using MuLV-reverse transcriptase. The following oligonucleotides were used as primers for IL-6 amplification: 5'-ATG AAG TTC CTC TCT GCA AGA GAC T-3' and 5'-CAC TAC GTT TGC CGA GTA GTA CTC-3' (product size: 638 bp). The mixture was incubated at 42º C for 60 min, heated up to 95º C for 5-10 min, and then quick-chilled on ice. PCR was performed at a final concentration of 1 x PCR buffer, 200 µM dNTPs, 1µM 5' and 3' primers and 0.5 units of Taq DNA polymerase (Perkin-Elmer/Cetus). The mixture was subjected to 40 cycles of amplification and then electrophoresed on 2% agarose gels in Tris borate, EDTA buffer. Gels were stained with ethidium bromide and photographed. For Southern blot analysis, the PCR products were transferred to a nylon membrane and hybridized with specific DIG-ddUTP oligonucleotide probes for IL-6. After hybridization and blocking, DIG-labelled probes were detected by a DIG luminescent detection kit (Boehringer Mannheim Biochemica). The antisense oligonucleotide probe used (Clontech) hybridized to the 361-390 bp segment of the IL-6 gene.

Slot blot analysis was performed as follows: poly(A)⁺RNA (2 µg/ml) was blotted onto nitrocellulose filters (Amersham), using a Minifold II blotting apparatus (Schleicher & Schuell SRC 072/0). Probes specific for IL-6, IL-2Ralpha and ß-actin were ³²P-labeled and used for hybridization of blots. Filters were washed and autoradiographed after each hybridization. In order to quantify the mRNA expression levels, densitometric analysis was performed taking the data obtained from ß-actin as controls.

B16F10 cells were incubated in the presence of IL-6 (200 U/ml) for 48 hours and inoculated into syngeneic mice for metastatic evaluation. Intrasplenic inoculation of IL-6-treated B16F10 cells resulted in a remarkable increase in the hepatic occupation as was determined in histological sections. The data depicted in Table 1 show that both parameters evaluated, the number of metastatic foci/mm² which measures the clonogenic potential, and the percentage of area occupied by the metastatic foci which indicates the metastatic efficiency, were clearly higher after treatment with IL-6. A similar enhancement of metastasis was observed in the lungs, where IL-6-treated cells developed more metastatic foci (124.1 ± 41.8) than nontreated cells (35.0 ± 12.6).

The proliferative rate of the B16F10 cells cultured in the presence of different concentrations of IL-6 for 24 or 48 hours did not change significantly from the controls (Figure 1). The number of colonies produced after 7-days culture was not affected by IL-6-treatment either (Table 2).

Expression of CD44 and VLA-4 on the surface of B16F10 cells was determined by FACS analysis. Untreated and IL-6-treated cells were all positive for both antigens; however, the level of expression was different for the two adhesion molecules. The mean fluorescence intensity of B16F10 cells was very high for CD44 and much lower for VLA-4. After culturing the cells for 48 hours in IL-6 (200 U/ml), an increase (25-30%) in the cell surface expression of the two adhesion molecules was recorded (Table 3).

The expression of mRNA for IL-6 was analysed by RT-PCR and the specificity of the amplified products was tested by Southern blot. The results obtained with this analysis showed that the IL-6 gene is expressed in B16F10 cells (Figure 2). The modulation of IL-6 mRNA expression after treatment with IL-2, IL-1ß and IL-6 was also examined. Slot blot analysis revealed that the amount of IL-6 mRNA did not change when cells were incubated with IL-1ß or IL-6 while only a small increase induced by IL-2 could be observed (Figure 3). However, we could not detect soluble IL-6 in culture supernatants of B16F10 cells, even when cells had previously been stimulated with IL-2 (Figure 4).

The expression of IL-2Ralpha on the surface of B16F10 melanoma cells was determined by flow cytometry. The percentage of tumor cells which were positive for IL-2Ralpha ranged from 25.31 to 32.82%. The results obtained for stimulated splenocytes included as positive controls ranged from 15 to 32% (data not shown). Several changes in the expression of IL-2Ralpha were recorded when B16 cells were cultured in the presence of IL-1ß, IL-6 or IL-2 (Figure 5). That is, the effect of IL-1ß seemed to be dose-dependent a reduction at low doses and an enhancement at 50 U/ml while IL-6 and IL-2 induced minor changes in the percentage of IL-2Ralpha expression. With regard to the modulation of IL-2Ralpha mRNA expression, IL-6 and IL-2 induced no change while IL-1ß increased the amount of IL-2Ralpha mRNA by a factor of 1.7 (Figure 6).

In this study, the effect of in vitro culture in the presence of IL-6, on the metastatic colonization of B16F10 melanoma cells was examined. The data obtained from these experiments show that IL-6 induced an increase in the metastatic activity of B16F10 cells. Up to now, most of the reports focussing on the role of IL-6 in B16 melanoma have emphasized the antitumoral effect of IL-6 against this tumor [3-5]. However, it must be taken into consideration that these results reflect the stimulation of the immune system induced by the in vivo administration of IL-6 or by the release of IL-6 from IL-6 transfected cells. In the present study, the only target of IL-6 was the B16F10 cells so, for the first time, the involvement of IL-6 in the metastatic behaviour of B16 melanoma, independently of the systemic factors, has been described. A direct prometastatic effect of
IL-6 on tumor cells has also been observed in breast carcinoma [15] and this has been also suggested to occur in human melanoma [6], so these data confirm the involvement of IL-6 in metastasis formation.

We had previously proved that the culture of B16F10 cells, in the presence of IL-2, also increased the metastatic ability of the cells, although only in the liver [11]. The increase induced by IL-6, however, was observed in the two target organs tested, the liver and the lungs, which suggests that the cellular mechanisms underlying the prometastatic effects of IL-2 and IL-6 must be different and that the modification of the metastatic phenotype induced by IL-6 seems to be non-organ-specific. In vitro, cell proliferation was also differently affected by IL-2 and IL-6: IL-2-treatment induced an increase in cell proliferation [10] whereas IL-6 did not modify this activity. Likewise, the clonogenic ability of the cells did not change after culture in the presence of IL-6, which agrees with previous results [4]. On the other hand, IL-6 induced an increase in the surface expression of CD44 and VLA-4 antigens, two adhesion molecules which have been associated with tumor progression and metastasis in melanoma [16, 17]. The same upregulating effect of IL-6 upon CD44 expression has been observed in human melanoma cell lines [18]. Moreover, VLA-4 expression has been reported to be involved in the enhancement of experimental metastasis induced by cytokines in B16 [19] as well as in human melanoma [20]; thus it could be suggested that the prometastatic effect of IL-6 on B16F10 might be due to the modulation of the adhesion properties of these tumor cells.

The analysis of IL-6 mRNA revealed that the IL-6 gene was constitutively expressed in B16F10 melanoma. The amount of IL-6 mRNA detected in B16F10 cells did not change when cells were pre-treated with IL-1 or IL-6. This absence of upregulation by IL-1 contradicts what was expected, as the induction of IL-6 by IL-1 has been described to take place in normal cells [21, 22] as well as in human melanoma [23] and other solid tumours [24]. With regard to IL-2, only a very slight increase in IL-6 mRNA expression was induced. The enhancement of IL-6 production by IL-2 has been shown in monocytes; however, in human melanoma cells, an inhibitory effect has been observed [18]. When we tested for the presence of soluble IL-6 in the cultures of B16F10 melanoma cells, no IL-6 could be detected. The production of IL-6 has been widely seen in human melanoma cell lines [23, 25, 26] and also in melanoma lesions in situ [27, 28] so a possible explanation of this lack of soluble IL-6 could be the existence of a post-transcriptional regulation and the need for appropriate stimulation. Alternatively, IL-6 could remain inside B16F10 melanoma cells and act by means of a private, endocellular loop as suggested by other authors [6].

Finally, the effect of IL-1, IL-2 and IL-6 on the expression of IL-2R was examined. IL-1 seemed to be the strongest inducer of this receptor. The increase in the IL-2R expression was recorded both at the mRNA level and on the cell surface. This result agrees with the transcriptional control of IL-1 on the IL-2 receptor seen in cells of the immune system [14]. In addition to the enhancing effect of IL-1 on IL-2R expression, in previous studies we reported that the IL-2 gene could also be upregulated by IL-1 in B16F10 cells [11]. Altogether, these data suggest that IL-1 could be important with regard to the upregulation of the IL-2/IL-2R system in B16F10 melanoma cells.

In summary, this report provides evidence for the involvement of interleukins in the biology and metastatic activity of B16 melanoma. We considered the role of IL-6 in the metastatic colonization of B16F10 cells, as well as the cytokine networks which could be regulating the activity of these tumour cells. This is of particular interest and needs to be further examined in subsequent studies.

We are grateful to Cristina Otamendi for photographic processing and Milagros Portuondo for technical assistance. This work was supported by grants UPV 075-327-EA038/94 and
UPV 075-327-EA205/96 awarded by the University of the Basque Country.

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