Levamisole induces interleukin-18 and shifts type 1/type 2 cytokine balance

Abstract

Immune responses can be classified, according to the predominant cytokines involved, into type 1 (featuring interferon-gamma, IFN-gamma) and type 2 (featuring interleukin-4, IL-4); imbalance between type 1 and type 2 cytokine compartments has been implicated in many human diseases. Levamisole is a drug with an unknown mode of action that has been used to boost immunity in infectious diseases including leprosy, and in some cancers. To test the hypothesis that levamisole acts by inducing a shift to a type 1 immune response, we used Brown Norway (BN) rats, which are markedly biased to type 2 responses. BN rats treated with levamisole showed a dose-dependent rise in serum IFN-gamma and fall in serum immunoglobulin E (IgE) level. Detailed analysis of cytokine gene expression showed upregulation of IFN-gamma and downregulation of IL-4 messenger RNA. This coincided with marked upregulation of IL-18, a recently characterized cytokine with potent activity in stimulating IFN-gamma production. IL-12 was not induced. Further, the type 2 response induced in BN rats by mercuric chloride was markedly attenuated when rats were pretreated with levamisole: there was a 2-log reduction in maximum serum IgE level and marked attenuation of IL-4 gene upregulation. These data indicate that levamisole acts by resetting the immune balance towards a type 1 response via induction of IL-18. Our findings provide a direction for development of more specific immunomodulating therapy.

Figure 1

Levamisole causes dose-dependent elevation of serum IFN-γ and suppression of serum IgE. Serum IFN-γ level and (b) serum IgE level by ELISA following treatment of 10 BN rats with levamisole 0·625–25 mg/kg/day intraperitoneally. There was a dose-dependent rise in serum IFN-γ and fall in serum IgE.

Figure 2

Levamisole causes elevation of serum IFN-γ and enhanced splenic IFN-γ gene expression. (a) Serum IFN-γ level by ELISA and (b) splenic IFN-γ gene expression by quantitative PCR, in 10 BN rats given levamisole 25 mg/kg/day intraperitoneally. There is a rise in serum IFN-γ by day 3, with close concordance with induction of splenic IFN-γ gene expression.

Figure 3

Levamisole causes suppression of serum IgE and of splenic IL-4 gene expression. (a) Serum IgE by ELISA and (b) IL-4 gene expression by quantitative PCR in 10 BN rats given levamisole 25 mg/kg/day intraperitoneally. Following levamisole there is a fall in serum IgE level by day 3 reaching a nadir at day 5, with close concordance with downregulation of splenic IL-4 gene expression.

Figure 4

(a) Levamisole causes upregulation of IL-18, but not IL-12, gene expression. Semiquantitative RT–PCR analysis of splenic mRNA for expression of IFN-γ, IL-12 and IL-18. cDNA was reverse transcribed from splenic mRNA on day 0 (baseline) and day 3 after levamisole 25 mg/kg/day intraperitoneally. Two animals at each time point. As seen with the fully quantitative PCR, IFN-γ is upregulated by day 3. At this time there is marked upregulation of IL-18 but IL-12 is not increased. (b) Levamisole causes early but transient upregulation of IL-18. Semiquantitative RT–PCR analysis of splenic mRNA for expression of IL-18 and IL-12. cDNA was reverse transcribed from splenic mRNA on days 0, 3, 5, 8 and 12 after levamisole 25 mg/kg/day intraperitoneally and was diluted in fivefold steps. PCR was performed on three dilutions for each sample, so that each sample is shown as a set of three lanes. n = 10, two animals at each time point. Representative gel shown: three independent experiments gave identical results. IL-18 is upregulated by day 3 then returns to baseline. IL-12 is not changed until day 8, whereafter it is slightly downregulated.

Figure 5

Levamisole blunts the Th2 response induced by HgCl₂. Serum IgE concentration by ELISA and IL-4 gene expression by quantitative PCR after HgCl₂ in BN rats with (n = 10) or without (n = 10) pretreatment with levamisole. Levamisole-treated rats had 2-log lower peak IgE and markedly attenuated upregulation of IL-4 gene expression.