Inhibition of IL-12 synthesis of peripheral blood mononuclear cells (PBMC) stimulated with a bacterial superantigen by pooled human immunoglobulin: implications for its effect on Kawasaki disease (KD)

Abstract

The aim of this study was to further assess the role of pooled human immunoglobulin (PHIG) on cytokine production from PBMC stimulated with a bacterial superantigen. Human PBMC were cultured with Streptococcus pyrogenic exotoxin A (SPE-A) with or without PHIG and several proinflammatory cytokine levels of culture supernatants were measured. Serum cytokine levels of KD patients before and after PHIG therapy were also examined. PHIG greatly reduced the production of IL-12, interferon-gamma (IFN-gamma) and other cytokines from SPE-A-stimulated PBMC, while exogenous IL-12, but neither IL-1 nor tumour necrosis factor-alpha (TNF-alpha), restored IFN-gamma production inhibited by PHIG. Although PHIG partially adsorbed SPE-A, its inhibitory effect on cytokine production was not played by anti-SPE-A antibody. Although purified CD4+ T cells cultured with human HLA-DR-transfected mouse L cells and SPE-A could not effectively produce IFN-gamma, they produced large amounts of IFN-gamma if exogenous IL-12 was introduced. KD patients in the acute phase had higher levels of serum IFN-gamma than did controls and patients with bacterial infection. Although IL-12 levels of children with or without KD were not significantly different, IL-12 levels of children were much higher than those of adults. However, serum levels of IL-12 of KD patients were transiently but significantly decreased by PHIG therapy and IFN-gamma amounts subsequently reverted to basal levels thereafter. These findings indicate that PHIG inhibits IL-12 production of SPE-A-activated monocytes and thereby decreases IFN-gamma synthesis by T cells and suggest that inhibition of IL-12 and IFN-gamma production is an important part of the mechanisms underlying PHIG therapy on KD.

Fig. 1

Time course of IFN-γ and IL-12 production by PBMC stimulated with Streptococcus pyrogenic exotoxin A (SPE-A) and the inhibitory effect of pooled human immunoglobulin (PHIG).

Fig. 2

Inhibition by pooled human immunoglobulin (PHIG) of IL-12 p35 and p40 mRNA expression of PBMC stimulated with Streptococcus pyrogenic exotoxin A (SPE-A). PBMC were stimulated with SPE-A with or without PHIG for indicated periods and IL-12 p35 and p40 mRNA expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR).

Fig. 3

High performance liquid chromatography (HPLC) analysis of pooled human immunoglobulin (PHIG)-treated Streptococcus pyrogenic exotoxin A (SPE-A). SPE-A (100 ng/ml) was incubated with or without 5 mg/ml PHIG for 2 h. Then, buffer-diluted SPE-A (a), SPE-A + PHIG (b) and PHIG (c) were subjected to a gel permeation HPLC and areas of SPE-A and PHIG peaks were compared.

Fig. 4

The effect of exogenous IL-12 on pooled human immunoglobulin (PHIG)-reduced cytokine synthesis of PBMC stimulated with Streptococcus pyrogenic exotoxin A (SPE-A).

Fig. 5

Requirement of exogenous IL-12 for effective IFN-γ production from CD4⁺ T cells cultured with L cells and stimulated with Streptococcus pyrogenic exotoxin A (SPE-A). HLA-DR⁻ CD4⁺ T cells were purified by sorting and cultured with mitomicin C-treated L cells and SPE-A, and culture supernatants at indicated periods were subjected to ELISA.

Fig. 6

(a) Serum cytokine levels in patients with acute phase of KD, age-matched patients with bacterial infection, age-matched healthy children, and healthy adults. (b) The effect of pooled human immunoglobulin (PHIG) therapy on serum cytokine levels of KD patients. Serum cytokine levels of KD patients were compared at the acute phase, just after PHIG therapy, and at the convalescent phase. NS, Not significant.