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Clin Exp Immunol. Sep 2000; 121(3): 566–570.
PMCID: PMC1905728

CD14+CD16+ monocyte subpopulation in Kawasaki disease

Abstract

Kawasaki disease (KD) is an acute febrile illness caused by vasculitis, occurring in early childhood. We have demonstrated that the activation of monocytes/macrophages plays a central role during acute KD. Recently, it has been reported that the CD14+CD16+ monocyte subpopulation plays a more important role in inflammation. In this study, we investigated the peripheral blood CD14+CD16+ monocyte subpopulation by flow cytometry, and serum levels of IL-10 and IL-12 using a sandwich ELISA in 28 KD patients. We also investigated this subpopulation in patients with bacterial infections, mononucleosis and anaphylactoid purpura, since the cause of KD remains unknown. We observed an increase in the number of CD14+CD16+ monocytes with acute KD, which was a positive correlation with C-reactive protein levels, and we observed only the patients with severe bacterial infections had increased this subpopulation during the acute stage among control diseases. In addition, we found that the serum levels of IL-10, but not IL-12, were higher during acute KD. These data suggest that increased peripheral blood CD14+CD16+ monocytes are part of the regulatory system of monocyte function during acute KD.

Keywords: Kawasaki disease, CD14+CD16+ monocytes, IL-10, severe bacterial infection

INTRODUCTION

Kawasaki disease (KD) is an acute febrile illness, occurring in young children [1]. The histopathological findings in KD indicate a panvasculitis with endothelial necrosis and the infiltration of mononuclear cells into small and medium-sized blood vessels [2]. KD has been reported as one of the cytokine-induced diseases. We have demonstrated that the activation of monocytes/macrophages plays a central role during acute KD. The number of peripheral blood CD14+ monocytes increased during the acute stage of KD [3, 4]. Infiltration by macrophages is notable in the affected tissues of autopsy cases [5] and in skin biopsy specimens [6]. We have also demonstrated by electron microscopy and immunohistochemistry the activation of peripheral blood monocytes [7].

The human peripheral blood monocyte subpopulation can be detected by flow cytometry [8]. The majority of monocytes are CD16 and exhibit strong CD14 staining. CD14+ and CD16+ (CD14+CD16+) monocytes are smaller and denser cells, and are thought to play a more significant role in inflammation [9]. Recently, Hoffman et al. proposed the theory of two distinct monocyte subpopulations [10]. CD14+CD16 monocyte subpopulations generate IL-12, while the CD14+CD16+ monocyte subpopulation generates IL-10. However, it has also been reported that IL-10 induces CD16 antigen on monocytes [11, 12]. Thus, CD14+CD16+ monocytes are related closely to IL-10.

In the present study we investigated peripheral blood CD14+CD16+ monocytes by flow cytometry, and we examined serum levels of IL-10 and IL-12 in patients with acute KD using a sandwich ELISA in terms of monocyte activation. We also investigated this subpopulation in patients with bacterial infections, infectious mononucleosis (IM; it shares some clinical symptoms with KD) and anaphylactoid purpura (AP; representative of vasculitis), since the cause of KD remains unknown.

PATIENTS AND METHODS

Patients

We studied 28 patients with KD, nine with severe bacterial infections, 10 with pneumonia, 11 with IM, and 13 with AP, who were seen at our Hospital between April 1997 and March 1999. All of them are Japanese. Informed consent for participation was obtained from the subjects' parents. This study was approved by the Institutional Review Board of Yamaguchi University Hospital.

Kawasaki disease

The 28 patients who met the diagnostic criteria for KD [1] comprised 15 boys and 13 girls (0·2–9·0 years of age; mean 2·0 years). All patients received Japanese standard treatment with intravenous gamma globulin (IVGG; Venilon; Teijin Co.), 400 mg/kg per day for 5 days, oral petoxifylline (PTX; 20 mg/kg per day) and oral aspirin (30 mg/kg per day). The onset of illness was defined as the day on which fever appeared. Blood samples were obtained on days 2–7 (4·3 ± 1·2 (mean ± s.d.)) prior to treatment, on days 6–16 (9·8 ± 2·3) after IVGG treatment during the acute stage, and on days 26–355 (66·9 ± 63·3) during the convalescent stage. The mean peripheral leucocyte count was 12 861 ± 4334/mm3 and C-reactive protein (CRP) levels were 7·3 ± 5·3 mg/dl at the time of diagnosis. None of the patients had coronary artery lesions (CAL) and none was treated with a second course of IVGG.

In addition, serum levels of IL-12 and IL-10 were measured in 23 of the KD patients.

Severe bacterial infection

This study included seven boys and two girls (0·1–18·3 years of age, mean 4·8 years). Three boys were diagnosed with sepsis by clinical symptoms and laboratory findings. A 4·1-year-old boy with bronchiectasia had an Alcaligenes xylosoxidans-positive blood culture. Three patients had bacterial meningitis and sepsis. Two of them had Escherichia coli-positive infections and one had Haemophilus influenzae-positive infection. Three boys were diagnosed with severe acute bacterial lymphadenitis by clinical symptoms and laboratory findings; one boy had a Staphylococcus aureus-positive purulent discharge. The mean leucocyte count was 16 164 ± 5014/mm3 and the mean CRP level was 14·8 ± 7·7 mg/dl in all of the patients with severe bacterial infection. Samples were obtained prior to treatment. From the onset of fever to sampling, an average of 5·4 ± 3·2 days had elapsed.

Pneumonia

This study included six boys and four girls (0·1–4·8 years of age, mean 2·3 years). The diagnosis was based on clinical symptoms, chest x-ray and laboratory findings. The mean peripheral leucocyte count was 10 730 ± 3069/mm3 and the mean CRP level was 3·6 ± 2·8 mg/dl during the acute stage. Samples were obtained prior to treatment. From the onset of fever and coughing to sampling, an average of 4·7 ± 1·8 days had elapsed. All patients were hospitalized and showed improvement quickly after the administration of i.v. antibiotics.

Infectious mononucleosis

This study included seven boys and four girls (2·2–7·8 years of age, mean 4·4 years). The diagnosis was based on common clinical symptoms of acute IM and atypical lymphocytes in the peripheral blood. All patients were positive for IgM and IgG antibody to Epstein–Barr virus (EBV) capsid antigen and were negative for antibody to EBV nuclear antigen during the acute stage. Samples were obtained on admission. From the onset of fever to sampling, an average of 7·7 ± 3·6 days had elapsed.

Anaphylactoid purpura

This study included nine boys and four girls (2·8–12·3 years of age, mean 7·1 years). The diagnosis was based on common clinical symptoms, such as mild fever, purpura, arthritis and colicky abdominal pain. Samples were obtained prior to treatment. From the onset of purpura to sampling, an average of 3·8 ± 1·4 days had elapsed.

Healthy children

The control subjects were 20 healthy children, 12 boys and eight girls (0·3–6·9 years of age, mean 2·4 years). The control samples were tested in parallel with the samples from the patients with KD.

Measurement of peripheral blood CD14+CD16+ monocytes

Whole heparinized blood (100 μl) was stained with fluorescein-conjugated anti-CD14 (Becton Dickinson, Mountain View, CA) and with PE-conjugated anti-CD16 (Becton Dickinson). Erythrocytes were lysed by the addition of FACS Lysing Solution (Becton Dickinson). The cell suspensions were centrifuged and the cell pellets resuspended in PBS containing sodium azide and paraformaldehyde [3, 11]. Cells were analysed with scatter gates set specifically for mononuclear cells with a FACScan flow cytometer (Becton Dickinson).

Quantification of the serum concentrations of IL-12 and IL-10

The serum levels of IL-12 and IL-10 were determined by a sandwich ELISA, using a High Sensitivity Human IL-12 immunoassay Quantikine Kit and a Human IL-10 immunoassay Quantikine Kit (R&D Systems Co., Minneapolis, MN), based on two murine antibodies that recognize different epitopes on each of the cytokine molecules. Each assay was performed according to the manufacturers' instructions. The detection limits were 0·5 pg/ml for IL-12 and 3·9 pg/ml for IL-10.

Statistical analysis

Statistical analyses were performed using the Mann–Whitney U-test, paired Wilcoxon signed-rank test for comparison of means and Spearman's correlation coefficient by rank.

RESULTS

Figure 1 shows flow cytometric analyses of peripheral blood mononuclear cells (PBMC) from a 1-year-old boy with KD during the acute (day 4) and convalescent (day 30) stages. This KD patient had increased CD14+ monocytes and CD14+CD16+ monocytes during the acute stage. As shown in Table 1, absolute numbers (and percentages) of CD14+ monocytes and CD14+CD16+ monocytes were higher, and the percentages of CD14+CD16+ monocytes among total CD14+ monocytes were higher in acute KD compared with those in convalescent KD and control subjects (P < 0·01). In addition, CD14+CD16+ monocytes, but not CD14+ monocytes, were decreased to within the normal range soon after IVGG treatment. As shown in Fig. 2, there was a positive correlation between the percentage of CD14+CD16+ monocytes among total CD14+ monocytes and CRP levels during the acute KD (P = 0·02). No correlation was found between CD14+CD16 monocytes and CRP levels during the acute stage (P = 0·98).

Table 1
CD14+ monocytes and CD14+CD16+ monocytes in the patients with Kawasaki disease (KD) during the acute stage and the convalescent stage, and in control subjects
Fig. 1
Flow cytometric analyses of peripheral blood mononuclear cells from a 1-year-old boy with Kawasaki disease (KD) during the acute (day 4) and convalescent (day 30) stages. Percentages of cells among the total mononuclear cells are given in the figure. ...
Fig. 2
Correlation between percentages of CD14+CD16+ monocytes among the total CD14+ monocytes and C-reactive protein (CRP) levels during acute Kawasaki disease.

Although the absolute numbers of CD14+ monocytes were increased during the acute stage in patients with severe bacterial infection, pneumonia, IM and AP (data not shown), the percentage of CD14+CD16+ monocytes among CD14+ monocytes was significantly higher in severe bacterial infection (24·8 ± 10·2% (mean ± s.d.), P < 0·01 versus controls), but not pneumonia (6·2 ± 5·9%), IM (9·3 ± 7·5%), or AP (4·8 ± 4·1%), than in control subjects (10·1 ± 4·3%) (Fig. 3).

Fig. 3
Percentages of CD14+CD16+ monocytes among the total CD14+ monocytes in patients with Kawasaki disease (KD), severe bacterial infection, pneumonia, infectious mononucleosis (IM), and anaphylactoid purpura (AP) during the acute stage. The hatched area represents ...

As shown in Fig. 4, the serum levels of IL-10 were significantly higher in patients with acute KD (44·3 ± 42·9 pg/ml) compared with the levels during convalescence (5·9 ± 2·1 pg/ml) and levels in control subjects (4·4 ± 1·5 pg/ml) (P < 0·01). There were no significant differences of levels of IL-12 in the sera among KD patients at the acute and convalescent stages, and control subjects (0·81 ± 0·34 pg/ml, 0·84 ± 0·57 pg/ml, 0·93 ± 0·63 pg/ml). Serum levels of IL-10 were not related with any clinical symptoms or immunological features (data not shown).

Fig. 4
Serum levels of (a) IL-10 and (b) IL-12 in Kawasaki disease (KD) patients during the acute and convalescent stage, and in healthy children. Serum levels of IL-10 were significantly higher in patients with acute KD compared with the levels during convalescence ...

DISCUSSION

Our previous investigations of patients with KD revealed increases in CD14+ monocyte counts [3, 4], CD14+CD23+ monocyte counts [13], serum levels of tumour necrosis factor-alpha (TNF-α) [3, 14], soluble TNF receptor [15] and soluble intercellular adhesion molecule-1 (ICAM-1) [16]. Assessment by electron microscopy showed that peripheral blood CD14+ monocytes from patients with acute KD have nuclei with complex shapes, apparent nucleoli (sometimes two or more) and abundant intracytoplasmic granules, some of which are positive for acid phosphatase [7]. Taken together, these data suggest that the activation of peripheral blood monocytes plays an important part in the development of vasculitis during acute KD.

Early studies of monocyte subpopulations have revealed smaller and denser cells (CD14+CD16+ monocytes), which might be more potent antigen-presenting cells and a more mature type of monocyte than regular monocytes (CD14+CD16 monocytes) [1719]. Moreover, CD14+CD16+ monocytes exhibit higher levels of very late antigen 4, the ligand for vascular cell adhesion molecule-1 and CD11a and c, which binds to ICAM-1, so these cells may have a greater tendency to adhere to endothelium and to migrate into tissues [18]. Frankenberger et al. suggested that CD14+CD16+ cells serve a function as inflammatory cells that trigger an efficient immune response [9].

In the present study we demonstrated an increase of CD14+CD16+ monocytes in KD patients during the acute stage, and our data, showing a positive correlation between CD14+CD16+ monocytes and CRP levels during the acute stage, suggest that the percentage of the CD14+CD16+ monocyte subpopulation reflects the severity of KD. The most severe course of KD leading to CAL was however, not seen in our cohort of patients. Recently, it was reported that the CD14+CD16 monocyte and CD14+CD16+ monocyte subpopulations generate IL-12 and IL-10, respectively [10]. Moreover, IL-10 has been reported to induce CD16 antigens on monocytes [11, 12]. These reports suggest that CD14+CD16+ monocytes are closely related with IL-10. Although previous reports have shown blood IL-10 levels were elevated during the acute stage of KD [20, 21], the exact role of IL-10 in KD has not yet been elucidated. No previous reports have investigated serum IL-12 levels in KD. In this study, serum levels of IL-10 and IL-12 in KD were examined in terms of monocyte activation. We detected higher levels of IL-10 but not IL-12 in the sera of KD patients during the acute stage. It is likely that increased serum levels of IL-10 reflect the activation of CD14+CD16+ monocytes rather than CD14+CD16monocytes during acute KD.

We found a rapid decrease in the CD14+CD16+ monocyte subpopulation after IVGG treatment, though no decrease was observed in the total number of CD14+ monocytes. We have already reported the efficacy of IVGG and PTX combination therapy [22]: all KD patients in this series were treated with IVGG, aspirin, and PTX. PTX blocks TNF-α production in monocytes at the messenger ribonucleic acid level, and has been used for treating diseases characterized by increases of TNF-α production [23, 24]. No previous reports have investigated the influence of CD14+CD16+ monocytes by the treatment of IVGG, aspirin, or PTX. We could not be certain that IVGG and PTX combination therapy caused the reduced number of CD14+CD16+ monocytes, since we did not evaluate a control group of patients treated only with aspirin. Although the exact mechanism of IVGG in down-regulating CD14+CD16+ monocytes remains unknown, this effect of reducing CD14+CD16+ monocytes provides new insights into the mechanisms by which IVGG reduces immune activation and prevents CAL in KD.

We also investigated the CD14+CD16+ monocyte subpopulation in patients with severe bacterial infection, pneumonia, IM and AP. IM shares some clinical symptoms with KD, and we have reported that peripheral blood monocytes are activated during the acute stage of IM [25]. AP shares some pathological findings with KD, namely vasculitis of small vessels. Therefore, we investigated the CD14+CD16+ monocyte subpopulation in these groups of patients. We found an increase in CD14+CD16+ monocytes in patients with severe bacterial infection, such as sepsis, but not in patients with other diseases, such as pneumonia, IM and AP. Although the cause of KD remains unclear, KD is similar to severe bacterial infection in terms of the peripheral blood CD14+CD16+ monocyte subpopulation.

In conclusion, our results suggest that increased peripheral blood CD14+CD16+ monocytes are part of the regulatory system of monocyte function during acute KD.

Acknowledgments

We would like to thank Dr Osamu Kondo, Dr Takashi Sugio and Dr Hiroshi Mito for help in collecting the samples.

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