Figure 7.

Identification of cytokines as responsible for enhancing human immunodeficiency virus type I (HIV-1) infection in unactivated CD4⁺ T-cells. (A) Unactivated CD4⁺ T-cells were stimulated with different combinations of cytokines for 72 h. Then, cells were infected and p24 antigen production was evaluated at days 4 and 7 post-infection. Each condition was compared with the corresponding RPMI condition (negative control). As a positive control, PHA stimulation was used. Percentage of living CD4⁺ T-cells (B) and percentage of infected (GFP⁺) CD4⁺ T-cells (C) after stimulation with the denoted treatments are shown. Data represent mean ± SD from four independent donors evaluated in duplicate. Concentrations of cytokines used in these experiments corresponded to the average concentrations found in monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/ml macrophage migration inhibitory factor (MIF) (peak effect) as follows: 250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β. (D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with monoclonal neutralizing antibodies. Primary CD4⁺ T-cells were incubated with supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype control antibody conditions were tested for comparison. Also, RPMI and PHA controls were included. Viral production was evaluated at day 4 post-infection. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test (all conditions versus the corresponding RMPI control) in (A) and Tukey’s post-test in (D). *p < 0.05.