Synergistic effect of muramyldipeptide with lipopolysaccharide or lipoteichoic acid to induce inflammatory cytokines in human monocytic cells in culture - PubMed (original) (raw)

Synergistic effect of muramyldipeptide with lipopolysaccharide or lipoteichoic acid to induce inflammatory cytokines in human monocytic cells in culture

S Yang et al. Infect Immun. 2001 Apr.

Abstract

An analog of 1alpha,25-dihydroxyvitamin D3, 22-oxyacalcitriol (OCT), differentiated human monocytic THP-1 and U937 cells to express membrane CD14 and rendered the cells responsive to bacterial cell surface components. Both THP-1 and U937 cells expressed Toll-like receptor 4 (TLR4) on the cell surface and TLR4 mRNA in the cells, irrespective of OCT treatment. In contrast, OCT-treated U937 cells scarcely expressed TLR2 mRNA, while OCT-treated THP-1 cells expressed this transcript. Muramyldipeptide (MDP) by itself exhibited only a weak ability to induce secretion of inflammatory cytokines such as interleukin-8 (IL-8) in the OCT-differentiated THP-1 cells but showed marked synergistic effects with Salmonella lipopolysaccharide (LPS) or lipoteichoic acid (LTA) from Staphylococcus aureus, both of which exhibited strong activities. Combinatory stimulation with LPS plus LTA did not show a synergistic effect on OCT-differentiated THP-1 cells. Similar results were observed in OCT-differentiated U937 cells, although combination experiments were carried out only with MDP plus LPS. Anti-CD14 monoclonal antibody (MAb) MY4, anti-TLR4 MAb HTA125, and the synthetic lipid A precursor LA-14-PP almost completely inhibited the IL-8-inducing activities of LTA as well as LPS on OCT-treated THP-1 cells, but these treatments increased MDP activity. OCT-treated THP-1 cells primed with MDP exhibited enhanced production of IL-8 upon stimulation with LPS, while the cells primed with LPS showed no change in production upon stimulation with MDP. MDP up-regulated mRNA expression of an adapter molecule to TLRs, MyD88, to an extent similar to that for LPS in OCT-treated THP-1 cells. These findings suggested that LTA as well as LPS activated human monocytic cells in a CD14- and TLR4-dependent manner, whereas MDP exhibited activity in a CD14-, TLR4-, and probably TLR2-independent manner and exhibited synergistic and priming effects on the cells for cytokine production in response to various bacterial components.

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Figures

FIG. 1

Up-regulation of mCD14 expression by OCT in THP-1 and U937 cells in culture. THP-1 and U937 cells were cultured in the presence or absence of OCT (0.1 μM/ml) for 3 days. Cells were then collected and stained with the second antibody (control) or anti-CD14 MAb (MY4) and analyzed by FACS. The results are representative of three different experiments.

FIG. 2

Influence of OCT treatment on TLR2 and TLR4 mRNA expression in THP-1 and U937 cells. THP-1 and U937 cells were cultured in the presence or absence of OCT (0.1 μM/ml) for 3 days. Total RNA was extracted from the cells and analyzed for TLR2, TLR4, and GAPDH by RT-PCR (top). Densities of the bands of TLR2 and TLR4 mRNA shown at the top were analyzed with NIH Image and expressed as mRNA expression relative to that of GADPH mRNA as an internal standard. The results are representative of three different experiments.

FIG. 3

Induction of IL-8 secretion by bacterial cell surface components in human monocytic cell cultures. OCT-differentiated THP-1 and U937 cells were stimulated with MDP, S. aureus LTA, and S. abortus-equi LPS at the indicated concentrations for 24 h in triplicate. IL-8 levels in the culture supernatants were determined by ELISA and expressed as means ± standard deviation. ∗∗, P < 0.01 versus medium alone. The results are representative of three different experiments.

FIG. 4

Synergistic effect of MDP with LPS or LTA on induction of IL-8 secretion in THP-1 cell cultures. OCT-differentiated THP-1 cells were stimulated with MDP plus S. abortus-equi LPS (A), MDP plus S. aureus LTA (B), and LPS plus LTA (C) at the indicated concentrations in triplicate. IL-8 levels in the culture supernatants were determined by ELISA, and the mean values are shown. ∗∗, ##, and ++, P < 0.01 versus control; ∗, #, and +, P < 0.05 versus control. Significant (P < 0.01) synergistic effects were detected in panel A (P < 0.01) and B (P < 0.01), but not in panel C, by ANOVA including an interaction term. The results are representative of two different experiments.

FIG. 5

Synergistic effect of MDP with LPS on induction of IL-8 secretion in U937 cell cultures. OCT-differentiated U937 cells were stimulated with MDP plus S. abortus-equi LPS at the indicated concentrations in triplicate. The IL-8 levels in the culture supernatants were determined by ELISA, and the mean values are shown. ∗∗ and ##, P < 0.01 versus control; #, P < 0.05 versus control. A significant (P < 0.01) synergistic effect of MDP and LPS on IL-8 production was detected by ANOVA including an interaction term. The results are representative of two different experiments.

FIG. 6

Effects of anti-CD14 MAb on IL-8 secretion by THP-1 cells in response to bacterial cell surface components. OCT-differentiated THP-1 cells were preincubated with anti-CD14 MAb MY4 (10 μg/ml) or isotype-matched control antibody for 30 min and then stimulated with MDP (10 μg/ml), S. aureus LTA (1 μg/ml), or S. abortus-equi LPS (10 ng/ml) for 24 h in triplicate. IL-8 levels in the culture supernatants were determined by ELISA and expressed as means ± standard deviation. ∗∗, P < 0.01 versus control. The results are representative of two different experiments.

FIG. 7

Influence of LA-14-PP on IL-8 secretion by THP-1 cells in response to MDP, LTA, and LPS. OCT-differentiated THP-1 cells were stimulated with MDP (10 μg/ml), S. aureus LTA (1 μg/ml), or S. abortus-equi LPS (10 ng/ml) in the presence or absence of LA-14-PP (1 or 10 μg/ml) for 24 h in triplicate. IL-8 levels in the culture supernatants were determined by ELISA and expressed as means ± standard deviation. ∗∗, P < 0.01 versus control. The results are representative of two different experiments.

FIG. 8

Effects of anti-TLR4 MAb on IL-8 secretion by THP-1 cells in response to bacterial cell surface components. OCT-differentiated THP-1 cells were preincubated with anti-TLR4 MAb HTA125 (5 μg/ml) or isotype-matched antibody for 30 min and then stimulated with MDP (10 μg/ml), S. aureus LTA (1 μg/ml), or S. abortus-equi LPS (10 ng/ml) for 24 h in triplicate. IL-8 levels in the culture supernatants were determined by ELISA and expressed as means ± standard deviation. ∗∗, P < 0.01 versus control. The results are representative of two different experiments.

FIG. 9

Priming effect of MDP or LPS on IL-8 secretion by OCT-treated THP-1 cells upon stimulation with LPS or MDP, respectively. OCT-differentiated THP-1 cells were preincubated with MDP (10 μg/ml) (A) or S. abortus-equi LPS (10 ng/ml) (B) for 4 h, washed three times, and then stimulated with S. abortus-equi LPS (A) or MDP (B), respectively, at the indicated concentrations for 24 h. As reference experiments, OCT-differentiated THP-1 cells without pretreatment were stimulated with MDP (10 μg/ml) plus LPS, LPS alone (A), or MDP alone (B) at the indicated concentrations for 24 h. IL-8 levels in the culture supernatants were determined by ELISA and expressed as the mean ± standard deviation of triplicate assays. ∗∗, P < 0.01 versus control, LPS or medium alone (A) and MDP or medium alone (B). The results are representative of two different experiments.

FIG. 10

Expression of mCD14 and TLR4 in THP-1 cells treated with MDP. OCT-differentiated THP-1 cells were incubated with MDP (10 μg/ml) for 4 to 24 h. The cells were collected and stained with anti-CD14 MAb MY4 and anti-TLR4 MAb HTA125, and then mCD14 and TLR4 expression was analyzed by FACS. Representative results of mCD14 expression at 12 h and TLR4 expression at 24 h are shown. The results are representative of two different experiments.

FIG. 11

MDP and LPS up-regulated MyD88 mRNA expression in THP-1 cells. OCT-differentiated THP-1 cells were treated with MDP (10 μg/ml), S. abortus-equi LPS (10 ng/ml), and MDP plus LPS for 2 to 24 h. Total cellular RNA (20 μg/lane) extracted from the cells was electrophoresed, transferred to a nylon membrane, and then analyzed by Northern blotting using a 32P-labeled cDNA probe for MyD88. Relative induction of MyD88 mRNA expression were determined with a BAS 1500 Mac image analyzer (Fuji Photo Film) after normalization using GAPDH. The results are representative of two different experiments.

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Synergistic effect of muramyldipeptide with lipopolysaccharide or lipoteichoic acid to induce inflammatory cytokines in human monocytic cells in culture - PubMed (original) (raw)