Low concentrations of cytokines produced by allergen-stimulated peripheral blood mononuclear cells have potent effects on nasal polyp-derived fibroblasts

Abstract

Accumulating data show that fibroblasts are important regulators in the development and maintenance of allergic airway inflammation. However, most studies so far have used individual recombinant cytokines in high concentrations, unlikely to be found in vivo. We aimed to investigate how cytokines produced by peripheral blood mononuclear cells (PBMC) affect fibroblast functions. Primary airway fibroblasts where incubated with allergen-stimulated or non-stimulated PBMC supernatants from allergic patients. The levels of cytokines in PBMC supernatants were measured and the expression of CD54, CD40 and CD106 as well as the production of eotaxin, interleukin (IL)-6 and IL-8 were assessed in fibroblasts. Although the levels of single cytokines measured in PBMC supernatants were low, a significant up-regulation of the surface molecules as well as of IL-6 and IL-8 production was found in fibroblasts cultured with allergen-stimulated PBMC supernatants as compared to non-stimulated, while the increase in eotaxin production was not significant. The evaluation of correlations between cytokines produced by PBMC and effects seen on fibroblasts did not indicate a crucial role for any single cytokine. Furthermore, the addition of comparably low concentrations of recombinant interferon (rIFN)-gamma or recombinant tumour necrosis factor (rTNF)-alpha did not induce the same effects as PBMC supernatants, the only exception being TNF-alpha as a direct inducer of CD54 expression. Our results show that synergistic mechanisms has a more important role than single mediators, highlighting important differences between in vitro experiments, where effects of individual mediators are studied, versus the actual situation in vivo.

Fig. 1

Cytokine production in PBMC supernatants (n = 6) with or without Dp stimulation for 5 days. Dp = Dp stimulation; ns = no stimulation. The following concentration ranges and mean values in pg/ml were measured: TNF-α: Dp (0–1387; 455), ns (0–762; 267); IFN-γ: Dp (0–6000; 2036), ns (0–138; 59); IL-13: Dp (146–316; 236), ns (122–275; 219); IL-6: Dp (309–10 516; 6018), ns (0–75; 13); IL-8: Dp (43 954–160 185; 88 962), ns (0–16 337; 7701).

Fig. 2

Cytokine production in fibroblast cultures, after incubation with PBMC supernatants (n = 6). Dp = Dp stimulated PBMC; ns = not stimulated PBMC. The final concentrations were corrected by subtracting the amount of the respective cytokine present in the PBMC supernatants used as stimulus of the fibroblasts. The following concentration ranges and mean values in pg/ml were measured: eotaxin: Dp (8–343; 136), ns (0–92; 37); IL-6: Dp (30 000–39 800; 33 282), ns (18 900–34 200; 26 330); IL-8: Dp (122 100–300 000; 194 522), ns (72 300–147 500; 82 470).

Fig. 3

Expression of surface molecules on fibroblasts after incubation with PBMC supernatants (n = 14). Dp = Dp stimulated PBMC; ns = not stimulated PBMC. CD54: Dp (9·6–78; 43·8), ns (5·2–20·5; 10·7); CD40: Dp (0·5–8; 4·2), ns (0·4–2·5; 1·5); CD106: Dp (0·5–14; 5·6), ns (0·5–6·1; 3·3).

Fig. 4

Eotaxin production (a) and CD54 expression (b) by fibroblasts after stimulation with two concentrations of rTNF-α (250 and 750 pg/ml), a PBMC supernatant (TNF-α: 205 pg/ml) and the same PBMC supernatant preincubated with anti-TNF-α blocking antibodies. This figure presents one representative experiment of four.

Fig. 5

Effect of four different concentrations of rTNF-α on the expression of surface molecules by primary fibroblast cultures. MFI = mean fluorescence intensity. □, CD54; ○, CD106; Δ, CD40.