Macrophages pre-treated with CTX demonstrated increased secretion of the IL-6 cytokine (3.19-fold at 12 h, Fig. 2A1; 80% at 24 h, Fig. 2A2) but significantly decreased secretion of the IL-1β and TNF-α cytokines at 12 h (48%, Fig. 2B1 and 57%, Fig. 2C1, Enzalutamide concentration respectively) when compared to the monoculture control. After 24 h, the levels of IL-1β were undetectable (Fig. 2B2). No differences in the levels of TNF-α secreted by the two sets of macrophages were observed (Fig. 2C2). Co-culturing macrophages in the presence of tumour cells enhanced

their IL-6 production, but pretreatment with CTX did not alter the level of this cytokine at the experimental time points, compared to the control group (Fig. 2A1 and A2). In contrast, increased secretion of IL-1β (3.7-fold at 12 h, Fig. 2B1; 3.24-fold at 24 h, Fig. 2B2) was observed. Interestingly, the level of this cytokine was decreased (76%) in co-cultures at 12 h, compared to the level detected in macrophage monocultures (Fig. 2B1), suggesting a suppressive action of the tumour cells on the secretion of this mediator. Similarly, co-cultured cells secreted

less TNF-α (20%) (Fig. 2C1) compared to monocultured cells. However, treatment with CTX did not affect TNF-α secretion by the macrophages co-cultured with tumour cells at 12 h or at 24 h (Fig. 2C1 and C2). At 48 h, secreted cytokines were not detected in selleck either the monocultures or the co-cultures (data not shown). The monocultures of LLC-WRC 256 cells secreted low levels of the cytokines analysed (data not shown). Because CTX has been demonstrated to stimulate the secretory activity of macrophages and of macrophages co-cultivated with LLC-WRC 256 cells, we evaluated the effect of macrophages treated with this toxin on tumour cell proliferation. As shown in Fig. 3, the results of the MTT assay demonstrated that tumour cell proliferation was inhibited (20%) at 48 h of co-culture with macrophage pre-treated with CTX. This effect was not due to the loss of membrane integrity because the viability of the macrophages and tumour cells in monocultures was higher than 95% after

48 h, as assessed by Trypan blue exclusion. Boc-2, a selective formyl peptide receptor Nintedanib (BIBF 1120) antagonist, abolished the stimulatory effect of pretreatment with CTX on H2O2 liberation and NO production by the macrophages in co-cultures (Fig. 4A and B, respectively), when compared to control co-cultures. Pretreatment with Boc-2 also abolished the increase in the level of secreted IL-1β observed at 12 and 24 h of co-culture (Fig. 4C1 and C2) and the increase in the level of TNF-α observed at 24 h of co-culture (Fig. 4C2). Macrophages pre-treated with Boc-2 for 15 min before CTX treatment did not inhibit the proliferative activity of tumour cells in co-cultures when compared to the control co-cultures (Fig. 5). Boc-2 per se did not have an effect on macrophage activity.