Interleukin-16 inhibits interleukin-13 production by allergen-stimulated blood mononuclear cells

Abstract

Expression of interleukin (IL)-16 is increased in bronchial mucosal biopsies of atopic asthmatics compared to normal controls. The functional significance of increased expression of IL-16 at sites of allergic inflammation is not yet clear. We have previously shown that IL-16 inhibits IL-5 secretion by allergen-stimulated peripheral blood mononuclear cells (PBMC). We investigated whether IL-16 inhibits the production of other T helper 2 cytokines, namely IL-13 and IL-4, by allergen-specific T cells. PBMC from ragweed-sensitive atopic subjects were stimulated with allergen extract for cytokine production in the presence or absence of rhIL-16. Production of cytokines was assessed by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. To evaluate whether the modulatory effect of IL-16 on cytokine synthesis was mediated by interferon-gamma (IFN-gamma), IL-10, IL-12 or IL-18, allergen-stimulated PBMC were cultured in presence of IL-16 and neutralizing concentrations of relevant antibodies. Allergen-stimulated PBMC produced significantly elevated levels of IL-13 (90-740 pg/ml) as compared to unstimulated PBMC (0-375 pg/ml, P < 0.01). Addition of rhIL-16 resulted in down-regulation of IL-13 mRNA expression as well as significantly reduced amounts of IL-13 released by allergen-stimulated PBMC (0-457 pg/ml, P < 0.001), as observed for IL-5. No effect of IL-16 was observed on IL-4 mRNA expression. Treatment with IL-16 resulted in increased levels of IL-10 and IL-18 in allergen-stimulated cell culture. Neutralization of IFN-gamma, IL-12, IL-10 or IL-18 did not alter the inhibitory effects of IL-16 on IL-13 and IL-5 secretion by allergen-stimulated PBMC. IL-16 did not modify IL-13 synthesis by anti-CD3-stimulated CD4(+) T cells, but it significantly reduced the production of IL-5. These data suggest that IL-16 may play an important immunoregulatory role in allergic states in response to allergen.

Figure 1

Kinetics of cytokine production by allergen-stimulated PBMC. PBMC were incubated with ragweed allergen extract (50 000 PNU/ml). Cytokine levels were assessed by ELISA in cell culture supernatants harvested at day 2, 3, 4, 5 and 6. Results are expressed as the mean ± SEM.

Figure 2

Effect of IL-16 on allergen-induced IL-13 production. PBMC were isolated from atopic donors (n = 11) and incubated for 6 days with culture medium, ragweed allergen extract, ragweed allergen extract plus rhIL-16 (100 ng/ml) or rhIL-16 alone. IL-13 release in cell culture supernatants was quantitated by ELISA. Results are expressed as the mean ± SEM. *P < 0·001 compared to cell cultures with culture medium only and cell cultures with allergen plus rhIL-16 (anova).

Figure 3

Effects of IL-16 on cytokine secretion by allergen-stimulated PBMC. PBMC were incubated for 6 days with ragweed allergen extract in presence of rhIL-16 (0–200 ng/ml). Results are expressed as the mean ± SEM (n = 5). *P < 0·05 and **P < 0·01 compared to allergen-stimulated cells (anova).

Figure 4

Modulation of cytokine mRNA expression by rhIL-16. (a, c) Kinetics of IL-13 and IL-5 mRNA expression in PBMC incubated in the presence or absence of allergen extract for the indicated times. (b, d) IL-16 down-regulates IL-13 and IL-5 mRNA expression in allergen-stimulated PBMC harvested on day 4. Representative results of RT–PCR products for IL-13 and IL-5 mRNA are shown for allergen-stimulated PBMC under the conditions indicated. Normalization by equivalent β-actin gene expression at subsaturating cycle number is depicted for each condition. The amplified cDNA fragments have the expected lengths of 336 bp for IL-13, 271 bp for IL-5, and 417 bp for β-actin. One representative of four independent experiments is shown.

Figure 5

Role of IFN-γ in mediating the effects of IL-16 on allergen-induced IL-13 and IL-5 production. PBMC were incubated for 6 days with ragweed allergen extract and treated with rhIL-16 in the presence or absence of anti-IFN-γ mAb (10 µg/ml) and appropriate isotype controls. Cytokine production was assayed by ELISA. Results are expressed as the mean ± SEM (n = 12).