Unique activation status of peripheral blood mononuclear cells at acute phase of Kawasaki disease

Abstract

Although Kawasaki disease (KD) is characterized by a marked activation of the immune system with elevations of serum proinflammatory cytokines and chemokines at acute phase, the major sources for these chemical mediators remain controversial. We analysed the activation status of peripheral blood mononuclear cells (PBMCs) by flow cytometry, DNA microarray and quantitative reverse transcription-polymerase chain reaction. The proportions of CD69+ cells in both natural killer cells and gammadeltaT cells at acute-phase KD were significantly higher than those at convalescent-phase KD. Microarray analysis revealed that five genes such as NAIP, IPAF, S100A9, FCGR1A and GCA up-regulated in acute-phase KD and the pathways involved in acute phase KD were related closely to the innate immune system. The relative expression levels of damage-associated molecular pattern molecule (DAMP) (S100A9 and S100A12) genes in PBMCs at acute-phase KD were significantly higher than those at convalescent-phase KD, while those of TNFA, IL1B and IL6 genes were not significantly different between KD patients and healthy controls. Intracellular production of tumour necrosis factor-alpha, interleukin-10 and interferon-gamma in PBMCs was not observed in KD patients. The present data have indicated that PBMCs showed a unique activation status with high expression of DAMP genes but low expression of proinflammatory cytokine genes, and that the innate immune system appears to play a role in the pathogenesis and pathophysiology of KD.

Fig. 1

Flow cytometric analysis of the activation markers on T, B and natural killer (NK) cells at acute phase of Kawasaki disease (KD). (a) The proportions of activated T, B and NK cells in the peripheral blood of seven patients with KD and 15 healthy control subjects were analysed by flow cytometry. CD69, human leucocyte antigen D-related (HLA-DR) and CD25 were used as activation markers. *P ≤ 0·05; **P ≤ 0·01; ***P ≤ 0·0001. (a) Acute phase; (c) convalescent phase; NC, healthy controls. The form of box-plot is as follows. The bottom and the top of the box correspond to 25th and 75th percentile points, respectively. The line within the box represents the median, and the whiskers indicate the values of 10th and 90th percentiles. (b,c) Representative density plot of flow cytometric analysis of CD69⁺ cells on NK, T and B cells (b) and the proportions of CD69⁺ cells in αβ and γδ T cells (c) in KD patients. The proportions of CD69⁺ cells were investigated in NK cells (n = 35), αβT cells (n = 23), γδT cells (n = 23) and B cells (n = 35). **P ≤ 0·0005; *P ≤ 0·01.

Fig. 1

Flow cytometric analysis of the activation markers on T, B and natural killer (NK) cells at acute phase of Kawasaki disease (KD). (a) The proportions of activated T, B and NK cells in the peripheral blood of seven patients with KD and 15 healthy control subjects were analysed by flow cytometry. CD69, human leucocyte antigen D-related (HLA-DR) and CD25 were used as activation markers. *P ≤ 0·05; **P ≤ 0·01; ***P ≤ 0·0001. (a) Acute phase; (c) convalescent phase; NC, healthy controls. The form of box-plot is as follows. The bottom and the top of the box correspond to 25th and 75th percentile points, respectively. The line within the box represents the median, and the whiskers indicate the values of 10th and 90th percentiles. (b,c) Representative density plot of flow cytometric analysis of CD69⁺ cells on NK, T and B cells (b) and the proportions of CD69⁺ cells in αβ and γδ T cells (c) in KD patients. The proportions of CD69⁺ cells were investigated in NK cells (n = 35), αβT cells (n = 23), γδT cells (n = 23) and B cells (n = 35). **P ≤ 0·0005; *P ≤ 0·01.

Fig. 2

Relative expression levels of S100A9, S100A12, IL8, IL6, TNF and IL1B genes in peripheral blood mononuclear cells (PBMCs) at acute phase of Kawasaki disease (KD). The gene expression levels of these cytokines were determined by the reverse transcription–polymerase chain reaction (RT–PCR) method using glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as an internal control. Gene expression levels of PBMCs from 10 KD patients, 11 healthy controls (NC), nine diseased control subjects [three patients with meningitis, three patients with acute infectious mononucleosis (IM) and three patients with measles] are shown. Only IL8 gene expression levels were analysed in 16 KD patients. The form of box-plot was the same as Fig. 1. *P < 0·05; **P < 0·01; ***P < 0·001. (a) Acute phase; (c) convalescent phase.

Fig. 3

Flow cytometric analysis of intracellular cytokine production of peripheral blood mononuclear cells (PBMCs) at acute phase of Kawasaki disease (KD). Intracellular cytokine production in PBMCs at acute and convalescent phases of KD was analysed by flow cytometry. Representative data of tumour necrosis factor (TNF)-α (a) and interleukin (IL)-10 (b) staining in monocytes, and those of interferon (IFN)-γ (c) and IL-10 (d) staining in T cells are shown. As positive and negative controls, representative data of TNF-α (a) and IL-10 (b) staining in monocytes with and without crude lipopolysaccharide (LPS) (1 µg/ml), and IFN-γ (c) and IL-10 (d) staining in T cells with and without phorbol 12-myristate 13-acetate (PMA) (25 ng/ml) plus ionomycin (1 µg/ml) are shown. The figure shows representative results of seven KD patients and three healthy controls.