Increased interleukin-10 production and Th2 skewing in the absence of 5-lipoxygenase

Abstract

Eicosanoids (prostaglandins and leukotrienes) are important mediators of inflammatory responses. These lipid mediators may also regulate the production of peptide mediators of the immune system. In this study, we investigated the effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on interleukin (IL)-10 production. IL-10 is a key regulator of immune and inflammatory responses, and previous studies have suggested that prostaglandins effect their immunosuppressive functions in part by stimulation of IL-10 production. We therefore investigated whether leukotriene production would have a similar role in regulation of IL-10 production. We have made the striking observation that absence of 5-LO-derived leukotrienes results in increased IL-10 production with a concomitant decrease in the production of pro-inflammatory cytokines, including tumour necrosis factor (TNF)-alpha and IL-12. Moreover, T-cell cytokine production in the absence of 5-LO-derived leukotrienes results in increased IL-4 production and decreased interferon (IFN)-gamma production. This may be in part secondary to increased IL-10 production and its effects on dendritic cell function resulting in altered T-cell differentiation. These findings indicate that, in addition to the central role leukotrienes play in the acute inflammatory response, endogenous leukotrienes are also important regulators of inflammatory cytokine production, via regulation of IL-10 production and in vivo differentiation of T cells.

Figure 1

Inflammatory cytokine production from wild-type and 5-lipoxygenase (LO)-deficient spleen cells. Spleen cells from wild-type and 5LO^–/– mice (5 × 10⁶ cells/ml) were cultured in the presence or absence of lipopolysaccharide (LPS) (1 µg/ml). Supernatants were harvested after 24 hr of culture and analysed for cytokine levels by enzyme-linked immunosorbent assay (ELISA): (a) interleukin (IL)-12p70; (b) IL-6; (c) tumour necrosis factor (TNF)-α; (d) interferon (IFN)-γ. In some experiments, blocking antibody to the IL-10 receptor (1B13A; 10 µg/ml) was added at the initiation of the cultures. Data are representative of three independent experiments. *P < 0·05, for 5LO^–/– mice versus wild-type mice.

Figure 2

Interleukin (IL)-10 production from lipopolysaccharide (LPS)-stimulated wild-type and 5-lipoxygenase (LO)-deficient spleen cells. Spleen cells from wild-type and 5LO^–/– mice (5 × 10⁶ cells/ml) were cultured in the presence or absence of LPS (1 µg/ml). Supernatants were harvested after 24 hr of culture and analysed for IL-10 levels by enzyme-linked immunosorbent assay (ELISA). Data are representative of three independent experiments. *P < 0·05, for 5LO^–/– mice versus wild-type mice.

Figure 3

Effect of the leukotriene LTB₄ on interleukin (IL)-6 production from wild-type and 5-lipoxygenase deficient cells. (a) Spleen cells (5 × 10⁶ cells/ml), (b) bone-marrow-derived macrophages (2 × 10⁶ cells/ml) or (c) splenic dendritic cells (2 × 10⁶ cells/ml) from wild-type and 5LO^–/– mice were cultured in the presence or absence of lipopolysaccharide (LPS) (1 µg/ml). In some cell cultures, LTB₄ (1 µm) was added at the initiation of culture. Supernatants were harvested after 24 hr of culture and analysed for IL-6 levels by enzyme-linked immunosorbent assay (ELISA). Data are representative of three independent experiments. *P < 0·05, for 5LO^–/– mice versus wild-type mice.

Figure 4

T-cell cytokine production from wild-type and 5-lipoxygenase deficient CD4⁺ T cells. (a) Interleukin (IL)-4; (b) IL-13; (c) IL-10; (d) interferon (IFN)-γ. Purified CD4⁺ T cells (2 × 10⁶ cells/ml) were cultured in the presence or absence of plate-bound anti-CD3. Supernatants were harvested after 24 hr of culture and analysed for cytokine levels by enzyme-linked immunosorbent assay (ELISA). Data are representative of three independent experiments. *P < 0·05, for 5LO^–/– mice versus wild-type mice.

Figure 5

Comparison of antigen-specific immunoglobulin responses in wild-type and 5-lipoxygenase deficient (5LO^–/–) mice. Mice were immunized with TNP-KLH with either alum or CpG 1826 oligonucleotide as the adjuvant. Serum from 10 animals per group was collected 28 days post immunization and antigen-specific serum immunoglobulin M (IgM), IgG1, IgG2A and IgE responses were evaluated by enzyme-linked immunosorbent assay (ELISA). (a) Day 28 IgM anti-TNP response. (b) Day 28 IgG₁ anti-TNP response. (c) Day 28 IgG_2a anti-TNP response. (d) Day 28 IgE anti-TNP response. *P < 0·05, for 5LO^–/– mice versus wild-type mice.

Figure 6

Spleen cell cultures from Schistosoma egg antigen (SEA)-immunized wild-type and 5-lipoxygenase deficient (5LO^–/–) mice. Mice were immunized with SEA and non-viable Schistosoma eggs on day 0 and boosted with Schistosoma eggs on day 7. Spleens were harvested on day 12 and spleen cells (5 × 10⁶ cells/ml) placed in control medium or medium supplemented with SEA antigen (5 µg/ml). Supernatants were harvested after 5 days of culture and analysed for cytokine levels by enzyme-linked immunosorbent assay (ELISA): (a) interleukin (IL)-4; (b) interferon (IFN)-γ; (c) IL-5; (d) IL-13; (e) IL-10; (f) IL-6. Data are representative of two experiments. *P < 0·05 and ^#P < 0·001, for 5LO^–/– mice versus wild-type mice.

Figure 7

Cytokine production from wild-type and 5-lipoxygenase (LO)-deficient splenic dendritic cells. (a) Interleukin (IL)-12p70; (b) tumour necrosis factor (TNF)-α; (c) IL-10. Purified splenic dendritic cells (2 × 10⁶ cells/ml) were cultured in the presence or absence of lipopolysaccharide (LPS) (1 µg/ml). Supernatants were harvested after 24 hr of culture and analysed for cytokine levels by enzyme-linked immunosorbent assay (ELISA). Data are representative of three independent experiments. *P < 0·05 and ^#P < 0·01, for 5LO^–/– mice versus wild-type mice.

Figure 8

Phenotype of purified splenic dendritic cells. Splenic dendritic cells were purified from wild-type and 5-lipoxygenase (LO)-deficient (5LO^–/–) mice and examined by flow cytometry as described in the ‘Materials and methods’. Profiles are representative of three independent experiments. (a) CD80; (b) CD86; (c) CD40; (d) major histocompatibility complex (MHC) class II.

Figure 9

Phenotype of lipopolysaccharide (LPS)-stimulated splenic dendritic cells. Dendritic cells from wild-type and 5-lipoxygenase (LO)-deficient (5LO^–/–) mice were purified and placed in control medium or medium containing LPS (1 µg/ml). Flow cytometry was performed after 48 hr in culture. Profiles are representative of three independent experiments. (a) CD80, control; (b) CD80, LPS; (c) CD86, control; (d) CD86, LPS; (e) CD40, control; (f) CD40, LPS.