Role of innate immune factors in the adjuvant activity of monophosphoryl lipid A

Abstract

Monophosphoryl lipid A (MPL) is a nontoxic derivative of lipopolysaccharide (LPS) that exhibits adjuvant properties similar to those of the parent LPS molecule. However, the mechanism by which MPL initiates its immunostimulatory properties remains unclear. Due to the involvement of Toll-like receptors in recognizing and transducing intracellular signals in response to LPS, the aim of the present study was to determine the ability of MPL to utilize the Toll-like receptor 2 (TLR2) and TLR4. We provide evidence that MPL differentially utilizes TLR2 and TLR4 for the induction of tumor necrosis factor alpha, interleukin 10 (IL-10), and IL-12 by purified human monocytes as well as by human peripheral blood mononuclear cells. Assessment of NF-kappa B activity demonstrated that MPL utilized TLR2 and especially TLR4 for the activation of NF-kappa B p65 by human monocytes. In addition, stimulation of human monocytes by MPL led to an up-regulation of the costimulatory molecules CD80 and CD86, an effect that could be reduced by pretreatment of cells with a monoclonal antibody to TLR2 or TLR4. Analysis of MPL-induced activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases revealed that MPL utilized both TLR2 and TLR4 for the phosphorylation of ERK1/2, while TLR4 was the predominant receptor involved in the ability of MPL to phosphorylate p38. Moreover, using selective inhibitors for MAP kinase kinase (PD98059) and p38 (SB203580), we show that ERK1/2 exhibited differential effects on production of TNF-alpha and IL-12 p40 by human monocytes, whereas MPL-induced activation of p38 appeared to be predominantly involved in production of IL-10 and IL-12 p40 by MPL-stimulated monocytes. Taken together, these findings aid in understanding the cellular mechanisms by which MPL induces host cell activation and subsequent adjuvant properties.

Figures

FIG. 1.

Involvement of TLR2 and TLR4 in MPL-induced TNF-α (A), IL-10 (B), and IL-12 p40 (C) production by human peripheral blood monocytes. Human monocytes (3 × 106/ml) were preincubated with anti-TLR2 or anti-TLR4 MAb or IC antibody for 30 min before the addition of MPL. Cell supernatants were collected 24 h after stimulation and analyzed by ELISA. Single and double asterisks indicate significant differences at P values of <0.05 and <0.01, respectively, compared to MPL-stimulated cultures. Data are expressed as means (n = 6). Standard deviations for each group were omitted for clarity.

FIG. 2.

Comparison of NF-κB activation induced by MPL in the presence or absence of anti-TLR2 or anti-TLR4 MAb. Human monocytes (106/ml) were preincubated for 30 min with the indicated anti-TLR2 or anti-TLR4 MAb or IC antibody and then incubated with MPL (10 μg/ml) for an additional 20 h. Nuclear extracts were analyzed for activation of NF-κB, and data are expressed as means optical density (OD) ± standard deviations (n = 6). Single and double asterisks indicate significant differences at P values of <0.05 and <0.01, respectively, compared to MPL-stimulated cultures.

FIG. 3.

The effects of MAbs to TLR2 or TLR4 on the ability of MPL to up-regulate the expression of the costimulatory molecules CD80 (A) and CD86 (B) on human monocytes. Human monocytes (4 × 105/ml) were preincubated for 30 min with the indicated MAb or IC antibody and then incubated with MPL for an additional 24 h. The levels of CD80 or CD86 were determined by flow cytometry. The asterisk indicates significant differences (P < 0.05) compared to the MPL-only treatment group. Data are expressed as the mean of percent positive cells ± standard deviation (n = 6).

FIG. 4.

The ability of MPL to induce TNF-α (A) and IL-10 (B) by human PBMC is differentially dependent upon both TLR2 and TLR4. PBMC (2 × 106/ml) were preincubated with the different MAbs for 30 min and then incubated for 24 h in the presence of MPL (10 μg/ml). Cell supernatants were collected and analyzed for cytokine production by ELISA. The data are expressed as means ± standard deviations (n = 5). Single and double asterisks indicate significant differences at P values of <0.05 and <0.01, respectively, compared to MPL-treated group.

FIG. 5.

MPL-induced phosphorylation of ERK1/2 (A) and p38 (B) by human monocytes. Human monocytes (3 × 106/ml) were preincubated for 45 min with PD98059 (20 μM) or SB203580 (5 μM) or for 45 min with anti-TLR2, anti-TLR4, or IC antibodies prior to the addition of MPL to cell cultures. After a 30-min incubation with MPL (10 μg/ml), total cell lysates were resolved by LDS-polyacrylamide gel electrophoresis and immunoblotted with phospho-specific ERK1/2 and p38 or anti-total ERK1/2 or p38 antibodies. Densitometer scans of pp38, total p38, pERK1/2, and total ERK1/2 were performed and recorded as the ratio of pp38 to p38 or pERK1/2 to ERK1/2. The data are expressed as means ± standard deviations (n = 5). Single and double asterisks indicate significant differences at P values of <0.05 and <0.01, respectively, compared to MPL-treated group.

FIG. 6.

The involvement of MPL-induced phosphorylation of ERK1/2 and p38 in TNF-α (A), IL-10 (B), and IL-12 p40 (C) production by human monocytes. Human monocytes (3 × 106/ml) were preincubated for 45 min with PD98059 (20 μM) or SB203580 (5 μM) or for 45 min with anti-TLR2, anti-TLR4, or IC antibodies prior to the addition of MPL to cell cultures. Cell supernatants were collected 24 h after MPL stimulation and analyzed for cytokine production by ELISA. The data are expressed as the means ± standard deviations (n = 5). Single and double asterisks indicate significant differences at P values of <0.05 and <0.01, respectively, compared to MPL-treated group.