Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production

Abstract

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

FIG. 1.

LPS inhibits HIV-1 infection in PBMCs but not in TZM-bl cells. LPS was tested for inhibitory activity against several different strains of HIV-1 as Env-IMC-LucR viruses expressing the indicated env genes. (A) Inhibitory activity of LPS against three different strains of PBMC-grown HIV-1 as assayed in either PBMCs (filled symbols) or TZM-bl cells (open symbols). Results are shown as the percent reduction in RLU. (B) Inhibitory activity of LPS against several additional strains of PBMC-grown HIV-1 as assayed in PBMCs. (C) Inhibitory activity of LPS in PBMCs depleted and not depleted of CD14⁺ cells, respectively. Results are the mean ± range in two independent experiments. (D) Inhibition of HIV-1 in TZM-bl cells by culture supernatants from PBMCs that were conditioned overnight with LPS. Results are the mean ± SEM of three independent experiments. In all figures, endotoxin concentrations correspond to the final volume after the addition of cells. For supernatant transfer experiments, endotoxin concentrations correspond to the concentration of LPS in the initial PBMC plate.

FIG. 2.

Identification of LPS-induced soluble factors that inhibit HIV-1 in PBMCs and TZM-bl cells. (A) LPS-induced upregulation of β-chemokine and IFN-( production in PBMCs. Results are expressed as the mean ± range in two sets of assays in PBMCs from a single donor. Positive values in the absence of LPS are due to the constitutive expression of cytokines after overnight stimulation with PHA-P. (B) Depletion of CD14⁺ cells from PBMC culture prevented the secretion of MIP-1α and MIP-1β after overnight incubation with 750 EU/ml LPS. CD14 depletion also resulted in lower levels of IFN-γ. No secretion of MIP-1α, MIP-1β, or IFN-γ was detected in isolated T cells. Graphs show concentration of cytokines above an LPS-free baseline. (C) Ability of antichemokine Abs to block the antiviral activity of LPS against PBMC-grown LucR reporter HIV-1 encoding BaL Env in PBMCs. Serial dilutions of antibodies were incubated with virus and cell culture media containing LPS at 5000 EU/ml for 1 h prior to the addition of cells. Results for the MIP-1(/MIP-1(/RANTES combination and the isotype control Ab are expressed as the mean ± range from two experiments; other points are from a single experiment. (D) Ability of anti-IFN-γ Abs to block the antiviral activity of LPS against BaL env expressing Env-IMC-LucR reporter HIV-1 in TZM-bl cells. PBMCs were incubated with LPS at 5000 EU/ml overnight. Culture supernatants were transferred into a new 96-well plate and serial dilutions of anti-IFN-γ Abs were added, followed by virus. TZM-bl cells were added after a 1-h incubation. Results are the mean ± range of two experiments. All concentrations are based on the final volume after addition of cells.

FIG. 3.

The potency of the recombinant human β-chemokine CCL3L1 against HIV-1 NL-LucR.T2A-BaL.ecto was evaluated in four different HeLa cell lines with previously established levels of CCR5: JC.10 cells (2.0 × 10³ molecules CCR5/cell), JC.37 cells (1.5 × 10⁴ molecules CCR5/cell), JC.10 cells (2.7 × 10⁴ molecules CCR5/cell), and TZM-bl/JC.53 cells (1.3 × 10⁵ molecules CCR5/cell). Viral inhibition was measured by a reduction in Renilla luciferase expression after 48 h incubation.

FIG. 4.

PBMC donors have differential sensitivity to LPS-mediated inhibition of HIV-1. Bars show the percentage of tested PBMC donors in which LPS inhibited HIV-1 NL-LucR.T2A-BaL.ecto ≥50% (white bars) or ≥80% (gray bars). The 80% bars are superimposed on the 50% bars, as any PBMCs with 80% inhibition also showed 50% inhibition. Values inset within vertical bars represent median IC₅₀ or IC₈₀ values with ranges in parentheses to show variation; all values are in EU/ml. Twenty-one different PBMCs were tested for O55:B5b, O127:B8, and S. enterica; 10 different PBMCs were tested for O55:B5a and S. enterica (R). O55:B5a and O55:B5b are LPS preparations manufactured by Lonza and Sigma, respectively.

FIG. 5.

Prolonged stimulation of PBMCs with PHA-P diminishes the antiviral effect of LPS against HIV-1 NL-LucR.T2A-BaL.ecto (A) and the amount of cytokines released (B). PBMCs from two different donors were stimulated with PHA-P either overnight (filled symbols) or for 4 days (open symbols) prior to use in a neutralization assay with LPS. Data represent mean values from two independent experiments.

FIG. 6.

High concentrations of LPS have no effect on the potency of neutralizing Abs when assayed in TZM-bl cells. Neutralizing monoclonal Abs (b12, 2G12, 2F5, 4E10) and a neutralizing polyclonal antiserum (HIVIG) were spiked with 30,000 EU/ml of E. coli O55:B5 LPS prior to assay. Neutralizing activity of the spiked samples and corresponding nonspikes samples was assayed against PBMC-grown NL-LucR.T2A-BaL.ecto in PBMCs (A) and in TZM-bl cells (B). Gray bars, LPS absent; black bars, LPS present. For comparison, the median IC80 of E. coli O55:B5 LPS in identical donor PBMCs was 1.56 EU/ml.