Neutralization of endogenous CCL2 inhibits HIV-1 replication in MDMs by promoting the intracellular accumulation of p24 Gag Ag. (A) MDM cultures were infected with HIV-1_BaL (300 TCID₅₀/10⁶ cells) in the presence (▴) or in the absence (○) of a polyclonal anti-CCL2 Ab (2.5 μg/mL). Some cultures were maintained in the continuous presence of a control Ab (•). After 2 hours, cells were washed and maintained in complete medium either in the presence or in the absence of anti-CCL2 or control Ab. At the indicated time points, culture supernatants were collected and tested for p24 Gag Ag and CCL2 content (gray bars indicate uninfected cells; hatched bars, infected cells). Shown is 1 representative experiment of 7 independently performed. (B) Concentration-dependent effect of the Ag affinity-purified polyclonal Ab to CCL2 on HIV-1_BaL p24 Gag Ag release. MDMs were infected as described above in the presence of different concentrations of Ab to CCL2, ranging from 62.5 ng/mL to 1000 ng/mL, or control Ab. At the indicated time points, culture supernatants were collected and tested for p24 Gag Ag content. Shown is 1 representative experiment of 2 independently performed. (C) MDMs were infected with primary R5 and R5X4 isolates. At the indicated time points, culture supernatants were collected and tested for p24 Gag Ag release. (D) MDMs were infected with HIV-1_BaL. At the indicated time points, cells were harvested and lysed. The cell-associated content of p24 Gag Ag was quantified by ELISA and normalized to 10⁵ cells. The results represent an individual experiment of 3 performed. The intersample SD calculated in all experiments did not exceed 10%. Statistical analysis showed a significant decrease in terms of p24 Gag Ag released in the culture medium (A-C) and a significant increase in terms of intracellular p24 Gag Ag accumulation (D) in HIV-1-infected anti-CCL2-treated cultures with respect to control cultures (P < .05).