Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation - PubMed (original) (raw)

Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

M L García-Hernández et al. Immunology. 2002 Feb.

Abstract

The aim of this study was to investigate the mechanisms by which interleukin-10 (IL-10) induces tumour growth in a mouse-melanoma model. A B16-melanoma cell line (B16-0) was transfected with IL-10 cDNA and three clones that secreted high (B16-10), medium and low amounts of IL-10 were selected. Cell proliferation and IL-10 production were compared in vitro, and tumour growth, percentages of necrotic areas, tumour cells positive for proliferating cell nuclear antigen (PCNA), IL-10 receptor (IL-10R) and major histocompatibility complex type I (MHC-I) and II (MHC-II), as well as infiltration of macrophages, CD4+ and CD8+ lymphocytes and blood vessels were compared in vivo among IL-10-transfected and non-transfected tumours. Proliferation and tumour growth were greater for IL-10-transfected than for non-transfected cells (P < 0.001), and correlated with IL-10 concentration (r > or =0.79, P < 0.006). Percentages of tumour cells positive for PCNA and IL-10R were 4.4- and 16.7-fold higher, respectively, in B16-10 than in B16-0 tumours (P < 0.001). Macrophage distribution changed from a diffuse pattern in non-transfected (6.4 +/- 1.7%) to a peripheral pattern in IL-10-transfected (3.8 +/- 1.7%) tumours. The percentage of CD4+ lymphocytes was 7.6 times higher in B16-10 than in B16-0 tumours (P = 0.002). The expression of MHC-I molecules was present in all B16-0 tumour cells and completely negative in B16-10 tumour cells. In B16-0 tumours, 89 +/- 4% of the whole tumour area was necrotic, whereas tumours produced by B16-10 cells showed only 4.3 +/- 6% of necrotic areas. IL-10-transfected tumours had 17-fold more blood vessels than non-transfected tumours (61.8 +/- 8% versus 3.5 +/- 1.7% blood vessels/tumour; P < 0.001). All the effects induced by IL-10 were prevented in mice treated with a neutralizing anti-IL-10 monoclonal antibody. These data indicate that IL-10 could induce tumour growth in this B16-melanoma model by stimulation of tumour-cell proliferation, angiogenesis and immunosuppression.

PubMed Disclaimer

Figures

Figure 1

Concentration and activity of IL-10 secreted by IL-10-transfected B16-melanoma cell lines. The B16-melanoma cell line was transfected with IL-10 cDNA and the production of IL-10 was quantified from cell culture supernatants by ELISA. Three clones that secreted high (B16-10), medium (B16-13) and low (B16-5) amounts of IL-10 were selected. Results are expressed in pg/ml of IL-10/2 × 106 cells/48 hr. The biological activity of the secreted IL-10 was determined by measuring the nitrite (NO2) concentrations after macrophages were incubated with cell line supernatants and then activated by LPS/IFN-γ (see Material and Methods). Labels at the left of the slash indicate the cell-line supernatant and the sign ‘−’ indicates not incubated with cell-line supernatant; ‘+’ or ‘−’ signals at the right of the slash indicate activation or not activation of macrophages. The bar representing the nitrite basal production by macrophages is indicated as ‘−/−’ (no cell line supernatant/not activated) and total nitrite production after stimulation is indicated as −/+ (no cell line supernatant/activated). The mean of triplicates is presented and error bars represent the standard errors of means.

Figure 2

Growth and IL-10 production of tumours generated with IL-10-transfected and non-transfected B16-melanoma cells. (a) Groups of 14 mice were injected subcutaneously with 3 × 105 cells from clones that secreted high (B16-10), medium (B16-13) and low (B16-5) amount of IL-10 or from non-transfected B16-melanoma cell line, and the tumour growth was measured by 18 days in six animals per group. IL-10 was determined by ELISA assay in tumour samples obtained from two mice of each group, killed at days 13, 14, 16, 17 and 18. Results are expressed in pg/ml of IL-10/0·3 g of tumour tissue (see Materials and Methods). Error bars represent the standard error of the mean in one representative experiment.

Figure 3

Tumour growth of IL-10-transfected B16-melanoma cells in mice treated with anti-IL-10 antibody. Three groups of six C57BL/6 mice were used in this experiment, two of them were inoculated with B16-10 cells and the other was inoculated with B16-0 cells. At day 11 post-inoculation, 2 days before tumour development started in B16-10 groups, one of these groups was treated with 3 µg of neutralizing antibody (anti-IL-10). The other B16-10 group and the group inoculated with B16-0 cells were treated only with PBS. All groups of mice were treated (with antibody or PBS) for 7 days, tumour growth was measured every day and mice were killed at day 18. Error bars represent the standard error of the mean of one representative experiment.

Figure 6

Histological and immunohistochemical comparisons in tumours produced after 18 days of melanoma cell inoculation. (a) Non-transfected tumours have extensive areas of ischaemic necrosis, characterized by picnotic nuclei (arrow) surrounded by deep acidophilic cytoplasm. (b) In contrast, IL-10-transfected tumours do not have necrotic areas. (c) Necrotic areas are produced when the activity of IL-10 is suppressed using blocking antibodies in animals bearing transfected tumours. (d) There is not IL-10 immunoreactivity in tumoral non-transfected cells. (e) In contrast, the majority of neoplastic transfected cells show strong IL-10 immunostaining. (f) Many transfected neoplastic cells have cytoplasmic IL-10 immunoreactivity after administration of IL-10-blocking antibodies. (g) The immunohistochemical detection of Von Willebrand factor as a marker of endothelial cells, shows fewer blood vessels in non-transfected than in IL-10-transfected tumours (h). (i) When mice bearing IL-10-transfected tumours are treated with blocking anti-IL-10 antibodies, the number of blood vessels is similar to that in non-transfected tumours. (All micrographs ×100, except panels d–f, ×200).

Figure 4

Cell proliferation of IL-10-transfected and non-transfected B16-melanoma cells. Cell line proliferation was measured by the incorporation of BrdU with a colorimetric method. Cell lines (1440 tumour cells/well) were cultured in triplicates for 24 hr, then treated only with culture medium or 3 µg/ml of IL-10-neutralizing antibody for 48 hr. Cells were then labelled by the addition of BrdU for 5 hr and incorporation of BrdU was quantified by a colorimetric reaction measuring the absorbance at 370 or 492 nm (see Materials and methods). The mean OD of triplicates is presented and bars represent the standard error of means.

Figure 5

Representative immunohistochemical features in tumours produced after 18 days of melanoma cell inoculation. (a) Non-transfected tumours have occasional cells with nuclear immunoreactivity to PCNA. (b) In contrast, IL-10-transfected tumours have numerous PCNA-positive cells denoting a higher proliferative activity. (c) The administration of IL-10-blocking antibodies in transfected tumour-bearing mice significantly reduced the number of PCNA-positive cells. (d) Occasional neoplastic cells have immunoreactivity to IL-10R. (e) In comparison, numerous cells are IL-10R-positive in transfected tumours. (f) Animals bearing IL-10-transfected tumours treated with blocking antibodies have scarce IL-10R-positive cells. (g) Non-transfected tumours have many infiltrating macrophages (× 100). (h) Macrophages are usually settled in the periphery of the IL-10-transfected tumours (× 100). (i) A similar number and distribution of macrophages as in non-transfected tumours is observed in IL-10-transfected tumours treated with blocking antibodies (× 100). (j) The majority of neoplastic cells have MHC-I molecules in non-transfected tumours.(k) In contrast, occasional cells are MHC-I positive in IL-10-transfected tumours. (l) Many neoplastic cells express MHC-I molecules in IL-10-transfected tumours after the administration of blocking antibodies. (All micrographs ×200, except panels g–i).

References

1. Salazar-Onfray F. Interleukin-10: a cytokine used by tumors to escape immunosurveillance. Med Oncol. 1999;16:86–94. - PubMed
1. Stearns ME, García FU, Fudge K, Rhim J, Wang M. Role of interleukin 10 and transforming growth factor beta 1 in the angiogenesis and metastasis of human prostate primary tumor lines from orthotopic implants in severe combined immunodeficiency mice. Cancer Res. 1999;5:711–20. - PubMed
1. De Vita F, Orditura M, Galizia G, Romano C, Lieto E, Iodice P, Tuccillo C, Catalano G. Serum interleukin-10 is an independent prognostic factor in advanced solid tumors. Oncol Rep. 2000;7:357–61. - PubMed
1. Mosmman TR. Interleukin−10. In: Thomson A, editor. The Cytokine Handbook. London: Academic Press; 1994. pp. 223–37.
1. Fiorentino DF, Zlotnik A, Mossman TR, Howard M, O'Garra A. IL-10 inhibits cytokine production by activated macrophages. J Immunol. 1991;147:3815–22. - PubMed

Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation - PubMed (original) (raw)