Modulation of monocyte-tumour cell interactions by Mycobacterium vaccae

Abstract

Immunotherapy with Mycobacterium vaccae as an adjuvant to chemotherapy has recently been applied to treatment of patients with cancer. One of the mechanisms of antitumour activity of Mycobacterium bovis bacillus Calmette-Guérin (BCG), the prototype immunomodulator, is associated with activation of monocytes/macrophages. These studies were undertaken to determine how M. vaccae affects monocyte tumour cell interactions and, in particular, whether it can prevent or reverse deactivation of monocytes that occurrs following their contact with tumour cells during coculture in vitro. Deactivation is characterised by the impaired ability of monocytes to produce tumour necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), and enhanced IL-10 secretion following their restimulation with tumour cells. To see whether deactivation of monocytes can be either prevented or reversed, three different strains of M. vaccae--B 3805, MB 3683, and SN 920--and BCG were used to stimulate monocytes before or after exposure to tumour cells. Pretreatment of monocytes with M. vaccae MB 3683, SN 920 and BCG before coculture resulted in increased TNF-alpha and decreased IL-10 production. All strains of M. vaccae and BCG used for treatment of deactivated monocytes enhanced depressed TNF-alpha secretion. Strain SN 920 and BCG increased IL-12 release but only BCG treatment inhibited an enhanced IL-10 production by deactivated monocytes. Thus, although some strains of M. vaccae may either prevent or reverse tumour-induced monocyte deactivation, none of them appears to be more effective than BCG.

Fig. 1

Production of cytokines by monocytes stimulated with different strains of M. vaccae or BCG. Monocytes were cultured with M. vaccae or BCG for 4 h (ratio 1:10), washed and then cultured in the medium for an additional 18 h. Supernatants were collected and tested for cytokine content. No IL-12 was detected. Means ± SD of four independent experiments are shown. *Significant at p<0.05

Fig. 2

Cytokine production by monocytes stimulated with HPC-4 cancer cells in the presence of different strains of M. vaccae or BCG. Monocytes were cultured either in the medium alone or were pretreated either with M. vaccae or BCG for 2 h (ratio 1:10), washed and then stimulated with HPC-4 cancer cells for an additional 18 h. Then supernatants were collected and tested for cytokine content. *Significant at p<0.05

Fig. 3

Secretion of TNF-α by monocytes treated with different strains of M. vaccae or BCG before coculture with tumour cells. Monocytes were cultured for 2 h with M. vaccae, and then HPC-4 cells were added and cocultured for an additional 2 h. CD14⁺ monocytes were isolated from the coculture by FACS sorting and restimulated with tumour cells for 18 h, when supernatants were collected and tested for cytokine content. The release of TNF-α by control monocytes stimulated with the bacteria was 2,687 ± 1,128 pg/ml; by control monocytes stimulated with tumour cells, 4,668 ± 1,388 pg/ml; and by cocultured monocytes (medium), 1,160 ± 1,241 pg/ml. Results are expressed as the percentage of response of control monocytes stimulated with tumour cells. Means ± SD of four independent experiments are shown. *Significant at p<0.05

Fig. 4

Secretion of IL-10 by monocytes treated with different strains of M. vaccae or BCG before coculture with tumour cells. Monocytes were treated as described for Fig. 3. The release of IL-10 by control monocytes stimulated with the bacteria was 1,709 ± 908 pg/ml; by control monocytes stimulated with tumour cells, 2,434 ± 916 pg/ml; and by cocultured monocytes (medium), 4,014 ± 1,045 pg/ml. Results are expressed as the percentage of the response of control monocytes stimulated with tumour cells. *Significant at p<0.05

Fig. 5

Secretion of TNF-α by monocytes treated with different strains of M. vaccae after coculture with tumour cells. Monocytes isolated from the coculture were treated either with M. vaccae strains or BCG for 3 h, washed and then restimulated with tumour cells for 18 h. Then supernatants were collected and tested for cytokine content. The TNF-α release by control monocytes stimulated with tumour cells was 3,262 ± 1,334 pg/ml, and by cocultured and restimulated monocytes (medium), 2,217 ± 1,067 pg/ml

Fig. 6

Secretion of IL-12 by monocytes treated with different strains of M. vaccae or BCG after coculture with tumour cells. Monocytes were treated as described for Fig. 5. The IL-12 release by control monocytes stimulated with tumour cells was 937 ± 536 pg/ml, and by cocultured and restimulated monocytes (medium), 200 ± 100 pg/ml

Fig. 7

Secretion of IL-10 by monocytes treated with different strains of M. vaccae after coculture with tumour cells. Monocytes were treated as described for Fig. 5. The IL-10 release by control monocytes stimulated with tumour cells was 2,500 ± 1,257 pg/ml, and by cocultured and restimulated monocytes (medium), 3,800 ± 1,706 pg/ml