Fibroblasts inhibit the production of interleukin-12p70 by murine dendritic cells

Abstract

Interleukin-12 p70 (IL-12p70) is a key cytokine produced by dendritic cells (DC) able to drive the development of T helper type 1 (Th1) lymphocytes. We showed that thymic and other fibroblasts strongly inhibit IL-12p70 production by splenic DC stimulated by lipopolysaccharide plus either anti-CD40 or interferon-gamma (IFN-gamma) and by purified splenic DC stimulated by Pansorbin plus IFN-gamma. This IL-12p70 inhibitory activity is secreted in the conditioned medium of primary fibroblasts and fibroblast cell lines but not by haematopoietic cell lines. As IL-10 was the unique factor able to inhibit IL-12p70 produced by cultured splenic DC, we showed that a neutralizing antibody to IL-10 did not suppress the IL-12p70 inhibitory activity of thymic fibroblast-conditioned medium (FCM). This FCM potently inhibits the maturation and expression of major histocompatibility complex class II and co-stimulatory molecules induced by stimulation of spleen-derived DC. While thymic FCM suppressed the IL-12p70 expression by stimulated spleen-derived DC, tumour necrosis factor-alpha production is not affected. This inhibitory activity is able to down-regulate the IL-12p35 subunit transcription and expression, resulting in the impaired assembly of IL-12p70 heterodimer. As fibroblasts are present in the tissue microenvironment and are active players in the establishment of an immune response, the nature and role of the fibroblastic inhibitory activity remain to be established.

Figure 1

IL-12p70 production by splenic derived DC is suppressed by co-culture on thymic fibroblasts; 5 × 10⁵ day 12 splenic DC/ml were cultured for 24 hr on thymic fibroblasts monolayers and further stimulated by LPS + anti-CD40 (DS-1) or LPS + IFN-γ (DS-2) for 24 hr. Means ± SD of 11 experiments with DS-1 and seven experiments with DS-2 are depicted.

Figure 2

Thymic fibroblast-conditioned medium (FCM) contains a potent inhibitory activity of IL-12p70 production by splenic derived DC; 5 × 10⁵ day 12 splenic derived DC/ml were cultured for 24 hr in the presence of different concentrations of FCM and then stimulated for a further 24 hr with DS-1. Means ± SD are obtained from duplicate culture wells.

Figure 3

The IL-12p70 inhibitory activity contained in FCM is different from IL-10. (a) Dose–response inhibition of IL-12p70 production by splenic derived DC stimulated with DS-2 obtained in the presence of IL-10, TGF-β1, IFN-β and VEGF. (b) The inhibitory effect of IL-10 on the production of IL-12p70 by day 12 splenic derived DC is efficiently counteracted by a neutralizing anti-IL-10 antibody. (c) The inhibitory effect of thymic FCM on the production of IL-12p70 by day 12 splenic derived DC is not suppressed by a neutralizing anti-IL-10 antibody. Day 12 splenic derived DC were cultured for 24 hr in the presence of increased concentrations of the different inhibitory molecules and then stimulated with DS-2 for further 24 hr of incubation. Anti-IL-10 monoclonal antibody (5 μg/ml) or the corresponding isotype (IgG1; 5 μg/ml) were added to the cultures in (b) and (c) during the first 24 hr of incubation at the same time as IL-10 and FCM. Splenic DC cultures were then stimulated with DS-2 for a further 24 hr.

Figure 4

Dose–response curve (10⁻⁶−10⁻⁹ m) of PGE₂ inhibitory activity of IL-12p70 production by murine splenic derived DC: comparison with FCM (20%). Results are expressed as percentage inhibition of IL-12p70 produced by control DC stimulated with DS-1 or DS-2. 5 × 10⁵ day 12 splenic derived DC/ml were cultured for 24 hr in the presence of 20% of FCM or with different concentrations of PGE-2 and then stimulated for a further 24 hr with DS-1 or DS-2. Means ± SD are obtained from duplicate culture wells.

Figure 5

Thymic FCM does not inhibit TNF-α production by day 12 splenic derived DC; 5 × 10⁵ day 12 SDC/ml were cultured in the presence of increased concentrations of thymic FCM for 24 hr and then stimulated with DS-1 for a further 24 hr. ELISA measurements of TNF-α and IL-12p70 were performed on these supernatants. Means ± SD represent values from duplicate culture wells in one experiment representative of three.

Figure 6

Phenotype analysis of splenic derived DC cultured in the absence or the presence of IL-10 and thymic FCM. Day 12 splenic DC were incubated for 24 hr with or without IL-10 (10 ng/ml) or thymic FCM (20%) and then stimulated with DS-1 and PSB-2 (a) or DS-2 (b) for a further 24 hr. The cells were analysed by flow cytometry for the surface expression of the indicated antigens. Dotted line represents negative control; thick bold line represents positive control; a dashed line represents incubation with IL-10 and the bold line represents incubation with FCM.

Figure 7

Regulatory effects of MS-5 fibroblasts or thymic FCM on the transcriptional activity of IL-12p40 and p35 genes and IL-12 protein production by DC. Each figure is composed of histograms showing the IL-12p35 or p40 copies number per 1000 HPRT, and of symbols (×) showing the level of IL-12p70 (pg/ml) or IL-12p40 proteins (ng/ml). Day 12 cultured splenic DC (1 × 10⁶/ml) were cultured or not on MS-5 fibroblasts monolayers (a) or in the presence of 20% thymic FCM (b) for 20 hr and then stimulated with DS-1 for a further 9 hr. (c) Ex vivo purified splenic CD11c⁺ cells (1 × 10⁶/ml) were cultured on MS-5 fibroblast monolayers for 20 hr and then stimulated for a further 9 hr with PSB-2. Messenger RNA were extracted, reverse transcribed and amplified by real-time PCR using primers specific for IL-12p40, p35 and HPRT. IL-12p40 and p35 mRNA levels were quantified and normalized against HPRT mRNA using an internal standard curve of HPRT. Simultaneous measurements of IL-12p70 and IL-12p40 proteins were performed on culture supernatants using ELISA.