Objective: To evaluate the effects of Staphylococcus aureus supernatant on the levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in co-cultures of neutrophils and retinal pigment epithelium (RPE) cells.
Methods: The co-culture system was established by co-culturing of human RPE cell line D407 and human peripheral blood neutrophils. Bacterium-free supernatant of Staphylococcus aureus ATCC29213 was added to the co-culture system for studying its effects. The volume of bacterium-free supernatant was divided into six groups: negative control, brain-heart infusion control, 50 µl, 100 µl, 250 µl and 500 µl group. The number of neutrophils was divided into four groups: negative control, 1 × 10(4), 5 × 10(4) and 5 × 10(5) group. The supernatants was collected at 6 h, 12 h, 24 h, 48 h and 72 h later and the levels of IL-1β, TNF-α and IL-6 were measured by ELISA kits.
Results: When RPE cells were cultured with different doses of bacterium-free supernatant (0, 50, 100, 250 and 500 µl), the levels of IL-1β was positively correlated with the volume of bacterium-free supernatant and the duration. The levels of IL-6 were significantly higher than that of the control group in the 500 µl group at 24 h [(23.17 ± 3.16) ng/L vs (7.61 ± 1.53) ng/L] and 48 h [(35.00 ± 4.37) ng/L vs (13.17 ± 3.27) ng/L] duration (P = 0.001 and P = 0.026, respectively). When RPE cells were co-cultured with the bacterium-free supernatant and the neutrophils (1 × 10(4), 5 × 10(4) and 5 × 10(5) cells) for 6 and 12 h, the levels of IL-1β in the 5 × 10(5) group at both 6 h [(236.62 ± 8.20) ng/L] and 12 h [(447.42 ± 35.13) ng/L] was statistically higher than that in other groups (6 h: P = 0.000, P = 0.000, P = 0.002; 12 h: P = 0.000, P = 0.000, P = 0.000, respectively). The levels of IL-6 in the 5 × 10(5) group [(46.96 ± 2.72) ng/L] was significantly higher than that in the other groups at 12 h (P = 0.000, P = 0.000, P = 0.000, respectively). TNF-α could not be detected in the conditioned media from all cultures.
Conclusions: Both IL-1β and IL-6 are expressed in the co-cultures of neutrophils and RPE cells with Staphylococcus aureus supernatant. IL-1β is upregulated at the early stage and the high level is maintained longer than that of IL-6. The virulence factor of bacteria and the neutrophil may play a role in the expression of IL-1β and IL-6 in the RPE cells.