Correspondence to: C.-R. Li and J. Yang, Shenzhen Children Hospital, Shenzhen 518026, China. E-mail: email@example.com
This study is designed to investigate the changes of NKG2D expression on CD8+T cells and CD3−CD56+NK cells in Kawasaki disease (KD). NKG2D/NKG2A expression on CD3−CD56+NK cells and CD8+T lymphocytes, and NKG2D ligands such as major histocompatibility complex I chain-related molecules A(MICA) and UL-16-binding proteins (ULBP-1) expression on CD14+ mononuclear cells (MC) were analysed by flow cytometry in patients with KD. Real-time polymerase chain reaction (PCR) was used to evaluate the mRNA levels of interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α in CD14+ cells. Plasma cytokine [IL-7, IL-12, IL-15, interferon (IFN)-γ and transforming growth factor (TGF)-β] concentrations were measured by ELISA. The levels of NKG2D on NK cells and CD8+T cells expression in acute phase of KD were significantly lower than those in normal controls (P < 0.05), and the levels of NKG2D expression in the patients with coronary artery lesion (KD-CAL+) were lower than those in patients with KD-CAL−. There was an upregulated tendency after treatment with IVIG. We found higher expression levels of proinflammatory cytokines from MC, such as IL-1β, IL-6 and TNF-α in patients with KD compared with the healthy controls (P < 0.05). The concentrations of IL-7 and IL-15 were significantly decreased in acute phase of KD (P < 0.05) and to some extent elevated after therapy with IVIG (P < 0.05), while antagonistic cytokines like IFN-γ were increased in acute phase of KD (P < 0.05) and reduced after therapy with IVIG (P < 0.05). These results suggest that aberrantly decreased levels of NKG2D expression on NK cells and CD8+T cells might be one of the factors led to disturbed immunological function in patients with KD. Cytokines milieu could be important factors causing reduced expression of NKG2D.
Kawasaki disease (KD) is an acute systemic vasculitis that affects infants and children. At present, the pathogenesis of KD remains to be further investigated. However, there is a large body of evidence that immunological disturbances play a key role in the pathogenesis of KD. A great many studies have found that the levels of many proinflammatory cytokines such as tumour necrosis factor (TNF)-α and interleukin (IL)-6 are elevated in acute KD, but the mechanisms resulting in aberrant immune function or overexpression of proinflammatory cytokines are not completely clear [1-3].
NKG2D is a C-type lectin-like type II transmembrane glycoprotein. It expressed on immunocompetent cells, such as natural-killer (NK) cells, CD8+ cytotoxic lymphocytes (CD8+T), NKT cells and γδT cells and participates in the regulation of innate and adaptive immune response through enhancing their killing activity. It has been demonstrated that NKG2D expression is induced on NK cells and CD8+T cells by their activation [4-6]. Accumulated evidences suggest that peripheral CD8+T cells may be functionally suppressed in acute phase of KD. Previous studies have shown a reduction in the total number of CD8+T cells in the peripheral blood of KD patients . However, the expression of NKG2D on NK cells and CD8+T cells in the acute phase of KD is still required to be investigated. In this study, flow cytometry (FCM) was used to detect the expression of NKG2A/NKG2D on CD8+T cells and CD3−CD56+ NK cells in patients with KD, both in the acute phase and after IVIG therapy. The cytokines regulating expression of NKG2D such as IL-1β, IL-6, TNF-α, IL-7, IL-12, IL-15, interferon (IFN)-γ and transforming growth factor (TGF)-β were also evaluated in this study. Aberrantly, decreased levels of NKG2D expression were found in acute phase of KD patients, suggesting that downregulation expression of NKG2D might be one of the factors led to disturbed immunological function in KD.
Materials and methods
Forty-six children with KD admitted to the Shenzhen Children Hospital between June 2011 and April 2012 were included in the study. The patients comprised 26 males and 20 females (mean age: 26.33 ± 23.82 months; age range: 2 months–5 years). The diagnosis was carried out according to the clinical criteria of the Kawasaki Disease Research Committee of Japan. Blood samples were obtained before treatment with 2 g/kg/day intravenous immunoglobulin (IVIG, mean duration of illness, 6.3 days; range, 3–12 days) and after IVIG treatment (mean duration of illness, 12.0 days; range, 8–20 days). All patients with KD received two-dimensional echocardiographic examination. Coronary artery lesion (CAL) was defined by internal diameter of artery >3.0 mm (<5 years); >4.0 mm (≥5 years) or coronary artery aneurysms. Patients with KD were divided into the KD-CAL+ group (n = 16) and the KD-CAL− group (n = 30) according to the echocardiographic examination results (Tables 1 and 2). Thirty age-matched healthy children (NC) (16 males and 14 females; mean age: 24.0 ± 16.4 months; age range: 1.1–4.3 years) were enrolled into this study. Informed consent was obtained from their parents, and the study was approved by the medical ethics hospital committee.
Table 1. Characteristics of patients with Kawasaki disease (KD)
SD, Standard deviation.
Forty-six children with KD admitted to the Shenzhen Children Hospital between June 2011 and April 2012 were included in the study. The diagnosis was carried out according to the clinical criteria of the Kawasaki Disease Research Committee of Japan.
26.33 ± 23.82
Duration of fever (days), mean (SD)
Oral mucosal changes
Giant peripheral aneurysm
Table 2. Clinical data of patients with KD-coronary artery lesion (KD-CAL+) and KD-CAL−
WBC, white blood cell; PLT, platelet; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; IgG, immunoglobin G.
Values are expressed as mean ± standard deviation (SD).
Venous blood (5 ml) was taken from patients with KD and normal controls using ethylene diaminetetraacetic acid (EDTA) Na2 as anti-coagulant. Blood samples were analysed immediately without stimulation of mitogens or culture in vitro unless particularly indicated. The whole blood (2 ml) was prepared for flow cytometric analysis. According to the manufacturer's instructions, CD14+T cells were immediately isolated from peripheral blood by microbead (Dynal 111.49D, US). Plasma was obtained after centrifugation and stored at −80 °C for measurement of the enzyme-linked immunosorbent assay (ELISA). Purified cells were identified as >97% with FCM, while results of cell activity were >95% by 0.05% trypan blue staining.
The antibodies CD3-FITC, CD8-PC5, CD56-PC5, CD14-PC5, NKG2A-PE and mouse IgG1-PE were obtained from Beckman Coulter, Inc. (Miami, FL, USA). NKG2D-PE, MICA-PE and ULBP-1-PE were purchased from eBioscience. (San Diego, CA, USA).Whole blood (100 μl) was incubated with relevant antibodies for 30 min at 4 °C. After incubation, red blood cells were lysed using Red Blood Cell Lysis Buffer,, and the remaining white blood cells were washed twice with phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) and 0.1% NaN3 (hereafter, PBS–0.2% BSA–0.1% NaN3). Immediately afterwards, expression of cell surface markers was analysed by flow cytometric analysis using an Epics-XL4 cytometer equipped with expo32 adc software (Beckman Coulter, San Diego, CA, USA). Data are presented as proportions of cells expressing antigen (%) and/or the relative levels of antigen levels assessed by the median fluorescence intensity (MFI).
Total RNA extraction and cDNA synthesis
Total RNA from CD14+ mononuclear cells (MC) was prepared using Versagene RNA Kit (Gentra 0050C, US) according to the manufacture's instruction. DNase I (0050D; Gentra) was used to eliminate the trace DNA during extraction. Isolated total RNA integrity was verified by an average optical density (OD) OD260/OD286 absorption to cDNA with oligodeoxythymidylic acid (oligo-dT) primer, using RevertAid™ H Minus Moloney murine leukaemia virus (MMLV) reverse transcriptase (K1632#; Fermentas, Vilnius, Lithuania). Negative control samples (no first-strand synthesis) were prepared by performing reverse transcription reaction in the absence of reverse transcriptase.
LightCycler real-time polymerase chain reaction
The cDNA levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α were quantitated by real-time PCR, using Quantitect™ SYBR green PCR Kit (Qiagen 204143, Hilden, Germany) and a LightCycler® 480 Instrument (Roche Molecular Biochemicals, Basel, Switzerland). The primers used for real-time PCR are listed in Table 3. The second derivate maximum method was performed for CP (cross point) determination using LightCycler Software V3.5.30 (Roche Molecular Biochemicals). After normalization with Relative Quantification Software V1.0 (Roche Molecular Biochemicals), the final results were calculated as ratios of the relative transcript levels of the target genes to the relative amount of β-actin.
Table 3. Primer for real-time polymerase chain reaction
Annealing temperature (°C)
IL, interleukin; TNF-α, tumour necrosis factor-α.
Sense:5′-GAA TCT CCG ACC ACC ACT A -3′
Anti-sense:5′-ACA TAA GCC TCG TTA TCC C-3′
Sense:5′-CAA TCT GGA TTC AAT GAG GAG AC-3′
Anti-sense:5′-CTC TGG CTT GTT CCT CAC TAC TC-3′
Sense:5′-CTG GTA TGA GCC CAT CTA TC-3′
Anti-sense:5′-CGA AGT GGT GGT CTT GTT GC-3′
Sense:5′-GAG CTA CGA GCT GCC TGA CG-3′
Anti-sense:5′-GTA GTT TCG TGG ATG CCA CAG-3′
ELISA detection of plasma IL-7, IL12, IL-15, IFN-γ and TGF-β
The plasma levels of IL-7, IL12, IL-15, IFN-γ and TGF-β were measured by ELISA, using ELX-800 microplate reader (BioTek Corporation, Winooski, VT, USA) in accordance with the manufacturer's instructions (Bender MedSystems, Vienna, Austria). All samples were measured in duplicate.
All statistical analyses were performed by spss for Windows version 13.0 (SPSS, Chicago, IL, USA). Data are presented as mean ± standard deviation (SD). Differences between the values were determined using Student's t-test. A value of P < 0.05 was regarded as a significant difference.
The expression of NKG2D on CD8+T cells and CD3−CD56+NK cells in the acute phase of KD
As shown in Fig. 1, compared with healthy controls the percentage of CD8+T cells (15.63% ± 4.15% versus 21.33% ± 6.49%, t = 4.274, P < 0.05) and CD3−CD56+NK cells (5.57% ± 1.53% versus 9.07% ± 2.88%, t = 6.117, P < 0.05) were downregulated during acute phase of KD. With respect to controls, the percentage of CD8+T cells expressing NKG2D were significantly downregulated in the acute phase of KD group (50.12% ± 13.35% versus 71.15% ± 6.80%, t = 9.038, P < 0.05). Moreover, we observed the MFI of NKG2D antigen on CD8+T cells was significantly downregulated in the acute phase of KD group (5.81 ± 1.30 versus 8.82 ± 2.08, t = 7.076, P < 0.05). To further analyse the association of NKG2D expression on CD8+T cells with severity of KD children, we noticed that NKG2D proportions in the KD-CAL+ group were markedly lower than those in the KD-CAL− group (37.68% ± 6.54% versus 56.76% ± 11.11%, t = 7.327, P < 0.05; MFI: 4.90 ± 0.77 versus 6.30 ± 1.26, t = 4.667, P < 0.05). Similarly, the levels of NKG2D on CD3−CD56+NK cells expression were remarkable decreased in children with KD compared with normal controls (66.23% ± 11.16% versus 85.21% ± 7.90%, t = 8.677, P < 0.05; MFI: 10.60 ± 2.23 versus 16.24 ± 6.28, t = 4.728, P < 0.05). On CD3−CD56+NK cells, the expression levels of NKG2D was also markedly lower in the KD-CAL+ group compared with the KD-CAL− group (57.05% ± 6.21% versus 71.12% ± 10.11%, t = 5.834, P < 0.05; MFI: 8.72 ± 1.18 versus 11.60 ± 2.01, t = 6.117, P < 0.05) (shown in Fig. 2).
The effect of IVIG therapy on the expression of NKG2D on CD8+T cells and CD3−CD56+NK cells in children with KD
At present, there are no data examined whether IVIG therapy effected the NKG2D expression on CD8+T cells and CD3−CD56+NK cells in KD. In this preliminary study, the results showed that there was an upregulated tendency after treatment with IVIG,, although considerable samples was sustained low expression of NKG2D, which might be related to relative short time to be revaluated after IVIG therapy. The levels of NKG2D expression on CD3−CD56+NKG2D+ NK cells were increased in 20 children who received therapy with IVIG. The levels of NKG2D expression on CD8+T cells in 30 children with KD after IVIG treatment were detected to be higher than those before the therapy (shown in Fig. 3). The MFI of NKG2D expression on CD3−CD56+NKG2D+ NK cells was increased in 22 children who received therapy with IVIG. The MFI of NKG2D expression on CD8+T cells in 29 children with KD after IVIG treatment was detected to be higher than those before the therapy (shown in Fig. 3).
The levels of proinflammatory cytokines expression in CD14+ cells from patients with KD
Rea1-time PCR was used to evaluate the mRNA levels of cytokines such as IL-1β, IL-6 and TNF-α. As shown in Fig. 4, compared with healthy controls, the expression levels of IL-1β (5.12E-01 ± 1.78E-01 versus 8.85E-02 ± 3.13E-02, t = 14.89, P < 0.05), IL-6 (4.22E-03 ± 2.31E-03 versus 1.72E-03 ± 1.35E-03, t = 5.944, P < 0.05) and TNF-α (1.19E-01 ± 5.12E-02 versus 1.16E-02 ± 6.10E-03, t = 13.903, P < 0.05) were significantly upregulated during acute phase of KD. The levels of IL-1β (1.06E-01 ± 5.09E-02, t = 13.768, P < 0.05), IL-6 (1.48E-03 ± 8.10E-04, t = 7.590, P < 0.05) and TNF-α (3.03E-02 ± 2.48E-02, t = 10.469, P < 0.05) expression were decreased to some extents after therapy with IVIG. In addition, transcription levels of proinflammatory cytokines [IL-1β (6.12E-01 ± 2.19E-01 versus 4.59E-01 ± 1.26E-01, t = 2.576, P < 0.05), IL-6 (6.41E-03 ± 1.66E-03 versus 3.05E-03 ± 1.67E-03, t = 2.419, P < 0.05) and TNF-α (1.51E-01 ± 6.74E-02 versus 1.02E-01 ± 3.10E-02, t = 2.757, P < 0.05)] in KD-CAL+ patients with coronary artery lesion were detected to be higher than those in patients with KD-CAL−.
The concentrations of plasma cytokine in patients with KD
It has been reported that IL-7 and IL-15 induce the expression of NKG2D on immunocompetent cells, and NKG2D can be downregulated on these cells by IL-12, TGF-β and IFN-γ [8-12] (Table 4). In this study, the plasma concentration of cytokines in KD was detected by ELISA. The serum concentrations of IL-7 and IL-15 in patients with KD were significantly lower compared with the concentrations in the healthy controls and the KD patients after IVIG therapy (P < 0.05). And the IFN-γ concentration in KD was higher compared with the concentration in the healthy controls and the KD patients after IVIG therapy (P < 0.05). But there were no obvious difference to be found between the patients with KD and the healthy controls in concentrations of IL-12 and TGF-β (P > 0.05) (shown in Table 4).
Table 4. The plasma concentrations of cytokines in patients with KD ( ± s, pg/ml)
NC group (n = 30)
KD group (n = 46)
KDIVIG group (n = 46)
IL, interleukin; IFN, interferon; TGF, transforming growth factor.
Values are expressed as mean ± standard deviation (SD).
KD versus NC, aP < 0ּ05.
KD versus KDIVIG, bP < 0ּ05.
13.44 ± 5.81
7.31 ± 2.48
11.4 ± 6.11
1.9 ± 1.25
1.58 ± 1.15
1.89 ± 1.18
50.34 ± 13.27
25.82 ± 10.27
54.63 ± 15.53
25.78 ± 11.63
46.21 ± 14.97
22.24 ± 9.13
314.31 ± 141.91
253.23 ± 125.41
332.12 ± 138.37
The expression of NKG2A on CD8+T cells and CD3−CD56+NK cells
NKG2A is an inhibitory receptor expressed on NK cells and CD8+T cells. The cooperation of NKG2D and NKG2A regulated NK cells and CD8+T cells functions. When the strength of activating signals is powerful over the sum of inhibitory signals. NK cells and CD8+T cells will respond and kill the target cells . In this study, the levels of NKG2A expression on CD3−CD56+NK cells and CD8+T cells were elevated to further examine whether lower expression of NKG2D was associated with over-expression of NKG2A. The results showed that there was no difference between the KD patients and the healthy controls in the percentage of CD3−CD56+NKG2A+NK cells (56.55% ± 10.23% versus 55.89% ± 7.90%, t = 0.050, P > 0.05) and CD8+NKG2A+T cells (5.40% ± 2.10% versus 6.68% ± 2.30%, t = 0.922, P > 0.05) (Fig. 5).
The expression of MICA/ULBP-1 expression on CD14+ MC
As shown in Fig. 6, there was no obvious difference to be found between the patients with KD and the healthy controls in the percentage of CD14+MICA+MC (6.15% ± 2.44% versus 5.27% ± 1.73%, t = 1.838, P > 0.05) and CD14+ULBP-1+MC (4.58% ± 1.76% versus 3.81% ± 1.61%, t = 0.764, P > 0.05).
Kawasaki disease is currently recognized as an acute vasculitis resulted from immune dysfunction. The proinflammatory cytokines (such as TNF-α) are obviously elevated during the acute phase of KD and might be involved in the pathogenesis vasculitis in KD, but the mechanism triggering the cascade response of proinflammatory cytokine production needs further clarification. Recent work demonstrated that NKG2D is expressed on most human NK cells and CD8+T cells and is upregulated upon activation and stimulation [4, 14]. NK cells and CD8+T cells kill a variety of tumour cells, virus-infected cells and allogeneic cells in a nonmajor histocompatibility complex restricted manner and provide the first line of immune defence, thus representing a useful tool to maintain host integrity. It is becoming increasingly appreciated that NK cells or CD8+T cells may play an immunoregulatory role in limiting autoimmune responses. Elimination of activated immune cells is one mechanism by which NK cells perform this immunoregulatory role. NKG2D plays a key role in immune regulation by bridging the crosstalk between NK cells, T cells and APCs such as dendritic cells or monocytes. Moreover, a role for NKG2D-dependent NK cells and CD8+T cells killing of activated immune cells has been proposed as a mechanism to dampen immune responses. As previously mentioned, inappropriate or deregulated expression of NKG2D on NK cells or CD8+T cells can break the delicate balance between immune activation and tolerance and trigger aberrant immune response [15, 16]. It has been reported that several autoimmune diseases associated with deviant NKG2D signalling, including type I diabetes, coeliac disease, SLE and rheumatoid arthritis, which were characterized by the feature of presence and aberrantly activation of a certain population of autoreactive immune cells [13, 17, 18].
In this study, we showed that the levels of NKG2D on NK cells and CD8+T cells expression in acute phase of KD were significantly lower than those in normal controls, and their changes were associated with the severity of the systemic vasculitis in KD. The expression of NKG2D in KD-CAL+ patients was significantly lower than that in KD-CAL− patients. Furthermore, our results showed higher expression levels of inflammatory cytokines from MC, such as IL-1β, IL-6 and TNF-α in KD patients compared with the healthy controls, and the levels of inflammatory cytokine expression in KD-CAL+ were higher than those in KD-CAL− patients. Lower the expressions of CD3−CD56+NKG2D+NK cells and CD8+NKG2D+T cells, higher the expression levels of inflammatory cytokines. The increased expression of proinflammatory cytokines seemed to be paralleling the decreased expression of NKG2D, suggesting that the lower expressions of NKG2D on NK cells and CD8+T cells in KD, which could led to the decreased elimination of MC, might be one of the factors leading to aberrant activation of MC in KD. IVIG is successfully used in the treatment of KD. The mechanisms of IVIG downregulate inflammatory response in KD are not clearly understood. In this study, we demonstrate that there was an upregulated tendency after treatment with IVIG, suggesting that IVIG might upregulate the expression of NKG2D on NK cells and CD8+T cells, but precise mechanisms of upregulated NKG2D expression about IVIG are still required to be further investigated.
It has been reported that some cytokines (such as IL-7 and IL-15) increase NKG2D transcripts, whereas others (such as IL-12, IFN-γ and TGF-β) have the opposite effect [8-12]. IL-7 synthesized by dendritic cells promotes survival and enhances cytotoxicity of NK cells through inducing NKG2D expression on the cells. IL-15 is a cytokine mainly synthesized by MC, and NKG2D signalling is coupled to IL-15 receptor signalling pathway. IL-12 is produced by APCs and act on T cells and NK cells to generate cytotoxic lymphocytes. Previous studies demonstrated that IL-12 fails to upregulate NKG2D on NK cells because the NKG2D ligand is concomitantly expressed on surrounding cells, leading to NKG2D downmodulation. Moreover, IFN-γ and TGF-β both have been found to have negative regulator properties of NKG2D. To investigate the mechanisms of reduced NKG2D expression on NK cells and CD8+ T cells in the patients with KD, we examined the serum concentration of IL-7, IL-15, IL-12, TGF-β and IFN-γ in the patients. Our data showed that the concentration of IL-7 and IL-15 was significantly decreased in acute phase of KD and to some extent elevated after therapy with IVIG, while antagonistic cytokines like IFN-γ were increased in acute phase of KD and reduced after therapy with IVIG, but IL-12 and TGF-B were not changed. Collectively, our results indicate that the changes of cytokines milieu, especially cytokines promoting expression such as IL-7, might be one of factors leading to decreased expression of NKG2D in acute KD. As we know, cell surface expression of NKG2D can be both positively and negatively regulated by cytokines. Another described mechanism for NKG2D downregulation is attributed to overexposure to NKG2D ligands. NKG2D ligands in humans are grouped into two families: the MHC class I polypeptide-related sequence A (MICA), B (MICB) and the cytomegalovirus UL16-binding protein 1-6 (ULBP1-6) . In this preliminary study, we used FCM to detect the expression of MICA/ULBP-1 on CD14+ MC in patients with KD. But there was no obvious difference to be found between the patients with KD and the healthy controls. However, the levels of other NKG2DL expression in the patients of KD are required to further investigate. NKG2A is a co-inhibiting receptor expression on NK cells and CD8+T cells. Adverse regulative signals are transmitted by NKG2A and NKG2D. Normally, the expression of NKG2A and NKG2D incline to balance [13, 18]. In this study, we found there was no difference in the percentage of CD3−CD56+NKG2A+NK cells or CD8+NKG2A+T cells between the patients and the controls. Suggesting that imbalance of NKG2A/NKG2D might not be involved in aberrant immune response of KD.
In summary, our data demonstrate aberrantly lowered expressing NKG2D on NK cells and CD8+T cells in patients with acute KD. The changes of cytokines milieu might be important factors causing low expression of NKG2D. In a physiologic condition, expression of NKG2D on NK cells or CD8+T cells could regulate immunocompetent cells function by enhancing the killing activity of NK cells and CD8+T cells. If the expression of NKG2D is exceedingly downregulated, aberrant activation of MC might result in aberrant inflammatory, indicating that aberrantly decreased levels of NKG2D expression may be one of the factors leading to disturbed immunological response in KD.
This study was supported by grants from the National Natural Science Foundation of China (no. 81102227) and Science Project of Shenzhen (no. 201002114).