Human gingival CD14(+) fibroblasts primed with gamma interferon increase production of interleukin-8 in response to lipopolysaccharide through up-regulation of membrane CD14 and MyD88 mRNA expression

Abstract

Gamma interferon (IFN-gamma)-primed human gingival fibroblasts (HGF) have been shown to produce higher levels of interleukin-8 (IL-8) upon stimulation with bacterial products and inflammatory cytokines than nonprimed controls. In this study, we examined whether priming of HGF with IFN-gamma up-regulates IL-8 production by the cells in response to purified lipopolysaccharide (LPS). The priming effect of IFN-gamma was clearly observed in the high-CD14-expressing (CD14(high)) HGF but not in the low-CD14-expressing (CD14(low)) HGF. The CD14(high) HGF were most effectively primed with IFN-gamma (1,000 IU/ml) for 72 h. To elucidate the mechanism of the priming effects of IFN-gamma for the LPS response by HGF, we examined whether IFN-gamma regulated expression of CD14, Toll-like receptor 2 (TLR2), TLR4, MD-2, and MyD88, all of which are molecules suggested to be associated with LPS signaling. In CD14(high) HGF, IFN-gamma markedly up-regulated CD14 and MyD88 but not TLR4 protein and MD-2 mRNA expression, while in CD14(low) HGF, IFN-gamma slightly increased MyD88 and scarcely affected CD14, TLR4 protein, and MD-2 mRNA levels. LPS-induced IL-8 production by IFN-gamma-primed CD14(high) HGF was significantly inhibited by monoclonal antibodies (MAbs) against CD14 and TLR4, but not by an anti-TLR2 MAb. These findings suggested that IFN-gamma primed CD14(high) HGF to enhance production of IL-8 in response to LPS through augmentation of the CD14-TLR system, where the presence of membrane CD14 was indispensable for the response of HGF to LPS.

FIG. 1.

Enhanced production of IL-8 by IFN-γ-primed CD14^high HGF on stimulation with LPS. CD14^high and CD14^low HGF were incubated with (closed bars) or without (open bars) IFN-γ (1,000 IU/ml) for 72 h, washed three times, and then stimulated with LPS at the indicated concentration for 48 h. After incubation, the IL-8 concentration in the culture supernatants was determined by ELISA. An additional experiment gave results similar to those shown here. Each assay was carried out in triplicate. Error bars indicate SD. ∗ and ∗∗, P < 0.05 and P < 0.01, respectively, versus respective control culture without IFN-γ-priming.

FIG. 2.

Priming effects of IFN-γ for various periods on the IL-8 production by CD14^high HGF in response to LPS. CD14^high HGF were incubated with or without IFN-γ (1,000 U/ml) for the indicated periods and then stimulated with LPS (0.1 μg/ml) for 48 h. After incubation, the IL-8 concentration in the culture supernatants was determined by ELISA. Each assay was carried out in triplicate. Error bars indicate SD. Two additional experiments gave results similar to those shown here. ∗ and ∗∗, P < 0.05 and P < 0.01, respectively, versus respective control culture stimulated with LPS without IFN-γ-priming (medium→LPS).

FIG. 3.

Priming effects of various concentrations of IFN-γ on the IL-8 production by CD14^high HGF in response to LPS. CD14^high HGF were incubated with the indicated concentrations of IFN-γ for 72 h, followed by stimulation with LPS (0.1 μg/ml) for 48 h. After incubation, the IL-8 concentration in the culture supernatants was determined by ELISA. Each assay was carried out in triplicate. Error bars indicate SD. Two additional experiments gave results similar to those shown here. ∗ and ∗∗, P < 0.05 and P < 0.01, respectively, versus the control culture stimulated with LPS without IFN-γ-priming (0 U/ml).

FIG. 4.

IFN-γ up-regulated mCD14 expression and CD14 mRNA expression in CD14^high HGF. (A) mCD14 expression determined by flow cytometry. CD14^high HGF were incubated with or without IFN-γ (1,000 IU/ml) for 3 days. Cells were then collected and stained with anti-CD14 MAb (MY4) (CD14 [solid line]) or the second Ab alone (control [dotted line]). (B) CD14 mRNA expression determined by Northern blotting. CD14^high HGF were incubated with or without IFN-γ (1,000 IU/ml) for the indicated periods. Total cellular RNA (25 μg per lane) was subjected to electrophoresis in agarose-formaldehyde gels, blotted onto nylon membranes, and then hybridized with the ³²P-labeled CD14 probe as described in Materials and Methods. Blots were further quantified using a Bio-Imaging Analyzer. The results are expressed as relative mRNA accumulation compared with GAPDH mRNA as an internal standard. Similar results were obtained in two independent experiments.

FIG. 5.

IFN-γ did not influence TLR4 expression in either CD14^high or CD14^low HGF. TLR4 expression was determined by flow cytometry. CD14^high (A) and CD14^low (B) HGF were incubated with (upper panels) or without (lower panels) IFN-γ (1,000 IU/ml) for 3 days. Cells were then collected and stained with anti-TLR4 MAb (HTA1216) (TLR4 [solid line]) or the second Ab alone (control [dotted line]). An additional experiment gave results similar to those shown here.

FIG. 6.

IFN-γ up-regulated MyD88 mRNA expression by CD14^high HGF. CD14^high (A) and CD14low (B) HGF were incubated with or without IFN-γ (1,000 IU/ml) for the indicated periods. Procedures for Northern blotting are described in the legend to Fig. 4. Three independent experiments gave results similar to those shown here.

Fig. 7.

Anti-CD14 and anti-TLR4 MAb inhibited IL-8 production by IFN-γ-primed and nonprimed CD14^high HGF in response to LPS. CD14^high HGF were preincubated with (closed bars) or without (open bars) IFN-γ (1,000 IU/ml) for 72 h. After three washes, they were incubated with or without MAb against CD14 (MY4), TLR4 (HTA125), TLR2 (TL2.1), or control Ab (IgG2b) for 30 min and then stimulated with LPS (0.1 μg/ml) for 48 h. After incubation, the IL-8 concentration in the culture supernatants was determined by ELISA. Each assay was carried out in triplicate. Error bars indicate SD. An additional experiment gave results similar to those shown here. Symbols: ∗∗, P < 0.01 versus respective control culture stimulated with LPS alone (nonpriming); ##, P < 0.01 versus respective control culture stimulated with LPS after IFN-γ-priming.