Intravenous immunoglobulin does not increase Fc{gamma}RIIB expression on monocytes/macrophages during acute Kawasaki disease

T. Ichiyama, Y. Ueno, M. Hasegawa, Y. Ishikawa, T. Matsubara and S. Furukawa

Department of Pediatrics, Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi 755-8505, Japan.

Correspondence to: T. Ichiyama, Department of Pediatrics, Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi 755-8505, Japan. E-mail: ichiyama{at}yamaguchi-u.ac.jp


    Abstract
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 Abstract
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 Patients and methods
 Results
 Discussion
 References
 
Objectives. Intravenous immunoglobulin (IVIG) therapy has been reported to be effective for reducing the incidence of coronary artery lesions in Kawasaki disease (KD), an acute febrile vasculitis of unknown aetiology. Regarding the mechanism of IVIG in immune thrombocytopenic purpura (ITP), it has been reported that IVIG increases the expression of the inhibitory Fc receptor, Fc{gamma}RIIB (CD32B), on splenic macrophages in a murine ITP model. Regarding the mechanism of IVIG during acute KD, we investigated whether or not IVIG increases the expression of Fc{gamma}RIIB in peripheral blood CD14+ monocytes/macrophages.

Methods. The expression of Fc{gamma}RIIB in peripheral blood CD14+ monocytes/macrophages was determined before and after IVIG therapy in 13 patients with acute KD by flow cytometry.

Results. The percentage of CD14+CD32B+ monocytes/macrophages among peripheral blood mononuclear cells, the absolute number of CD14+CD32B+ monocytes/macrophages and the percentage of CD14+CD32B+ monocytes/macrophages among CD14+ monocytes/macrophages in patients with acute KD before IVIG therapy were significantly increased compared with those after IVIG therapy and in controls. CD14+CD32B+ monocytes/macrophages decreased to within the normal range soon after IVIG therapy.

Conclusions. IVIG therapy in patients with KD did not increase the expression of Fc{gamma}RIIB in peripheral blood CD14+ monocytes/macrophages during the acute stage.

KEY WORDS: Monocytes/macrophages, Kawasaki disease, Fc{gamma}RIIB, Intravenous immunoglobulin


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Kawasaki disease (KD) is an acute febrile vasculitis of unknown aetiology that may lead to cardiovascular disorders [1]. A coronary artery lesion (CAL) is the most important complication of KD and may cause significant coronary stenosis, resulting in ischaemic heart disease [2]. Intravenous immunoglobulin (IVIG) therapy has been reported to be effective for reducing the incidence of CAL in KD patients [3–5]. However, there have been few reports on the mechanism of IVIG in acute KD [4, 6, 7]. IVIG is effective for other disorders, including immune thrombocytopenic purpura (ITP) and Guillain–Barré syndrome. The mechanism of IVIG in the murine ITP model has been documented. In the murine model of ITP, IVIG increases the expression of the inhibitory Fc receptor, Fc{gamma}RIIB, on splenic macrophages [8]. The cross-linking of Fc{gamma}RIIB and the activation of the Fc receptor, Fc{gamma}RIII, by platelet–antibody immune complexes inhibits the activating signal through the recruitment of SH2-containing 5'-phosphoinositol-phosphatase and the breakdown of phosphatidylinositol 3,4,5-trisphosphate, resulting in abrogation of phagocytosis [9]. Fc{gamma}RII, which exists in three isoforms, i.e. A, B and C, is a low-affinity receptor for the Fc fragment of IgG [10]. All forms are present on monocytes, placental trophoblasts and endothelial cells [11]. In addition, the Fc{gamma}RIIB form is present on B lymphocytes and mast cells [11]. The Fc{gamma}RIIA and Fc{gamma}RIIC forms are found on neutrophils [11] and do not have the inhibitory motif that is present in Fc{gamma}RIIB [12].

Previous studies have demonstrated that activation of monocytes/macrophages plays an important role in the pathogenesis of KD. The immunological features of monocytes/macrophages observed in patients with KD can be summarized as follows: (i) infiltration by these cells is notable in affected tissues in autopsy cases and skin biopsy specimens [13]; (ii) there are elevated levels of a variety of serum cytokines, such as TNF-{alpha}, interleukin (IL)-1 and IL-6, during acute KD [14–18]; (iii) peripheral blood mononuclear cells (PBMC) spontaneously secrete high levels of TNF-{alpha} and IL-1 [19, 20]; (iv) increases in the number of peripheral blood CD14+ monocytes/macrophages, serum TNF-{alpha} level, IL-6 activity in serum, and secretion of IL-1 from mononuclear cells are more evident in KD patients with CAL than in ones without CAL [14, 17, 20, 21]; (v) KD patients with high levels of soluble TNF receptors in their serum seem to be susceptible to CAL even if they receive IVIG therapy [22]; (vi) immunocytochemical and immunoelectron microscopic studies have shown that monocytes partly differentiate into macrophages in the peripheral circulation during the acute stage of KD [23, 24]; (vii) increased numbers of peripheral blood CD14+CD16+ (Fc{gamma}RIII) monocytes/macrophages are part of the regulatory system for monocyte/macrophage function during acute KD [25]; and (viii) nuclear factor-{kappa}B (NF-{kappa}B) activation is higher in peripheral blood CD14+ monocytes/macrophages than in CD3+ T cells in KD patients during the acute stage [26]. These findings suggest that the activation of monocytes/macrophages plays an important role during acute KD. Regarding the mechanism of IVIG during acute KD, we investigated whether or not IVIG increases the expression of Fc{gamma}RIIB on peripheral blood CD14+ monocytes/macrophages.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Thirteen children (five girls and eight boys; aged from 3 months to 4 yr; mean age 1.6 yr) with acute KD on admission to our hospital between July 2003 and May 2004 were included in this study. The children's parents gave informed consent for their participation in the study. The patients met the specific diagnostic criteria for KD [1]. The day of onset of fever was considered as the first day of illness. All patients received standard Japanese treatment with high-dose IVIG (Polyglobin N; Bayer Yakuhin, Osaka, Japan) at 1000 mg/kg/day for 2 days, and oral aspirin (30 mg/kg/day), and all responded to IVIG therapy. No patients had CAL. Samples were obtained at the acute stage, i.e. on days 3–6 (mean ± S.D., 4.6 ± 0.9 days) before treatment with IVIG. Samples were also obtained after IVIG therapy, i.e. on days 6–9 (mean ± S.D., 7.4 ± 1.4 days). The control subjects were 18 healthy children (10 males and eight females, aged from 4 months to 4 yr; median 1.8 yr). Peripheral blood labelled with phycoerythrin-conjugated anti-CD14+ antibodies was labelled with an anti-Fc{gamma}RIIB (CD32B) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibody is an affinity-purified goat polyclonal antibody raised against a peptide mapping at the carboxy terminus of CD32B of human organ. The cells were then labelled with a second fluorescein isothiocyanate-conjugated antibody (Pharmingen, San Diego, CA, USA). Immunofluorescence staining was analysed with a FACScan flow cytometer equipped with CellQuest software (Becton-Dickinson Biosciences, San Diego, CA, USA). We analysed 5000 cells for each subject in the flow cytometric studies. The values obtained from flow cytometric analyses are expressed as mean ± S.D. Statistical analysis was performed with the Wilcoxon matched pairs test and the Mann–Whitney U test, a P value of less than 0.05 being taken as significant.

The research was reviewed and approved by the Institutional Review Board of Yamaguchi University Hospital. The parents of children who were patients with KD and of healthy controls gave informed consent for their participation in the study according to the Declaration of Helsinki.


    Results
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 Patients and methods
 Results
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Figure 1 shows the results of flow cytometric analyses of PBMC from a 21-month-old girl with KD before and after IVIG therapy, and at the convalescent stage. The patients had an increased percentage of CD14+CD32B+ monocytes/macrophages before IVIG therapy. The results of flow cytometric analyses are presented in Table 1. The percentage of CD14+ monocytes/macrophages among PBMC, the absolute number of CD14+ monocytes/macrophages, the percentage of CD14+CD32B+ monocytes/macrophages among PBMC, the absolute number of CD14+CD32B+ monocytes/macrophages, and the percentage of CD14+CD32B+ monocytes/macrophages among CD14+ monocytes/macrophages in patients with acute KD before IVIG therapy were significantly increased compared with those after IVIG therapy (P = 0.0019, P = 0.0088, P = 0.0047, P = 0.0229 and P = 0.0309, respectively) and the controls (P < 0.001, P < 0.001, P < 0.001, P < 0.001 and P = 0.0209, respectively). CD14+CD32B+ monocytes/macrophages decreased to within the normal range soon after IVIG therapy.



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FIG. 1. Flow cytometric analyses of PBMC from a 21-month-old girl with KD before (A, day 5) and after (B, day 7) IVIG therapy, and at the convalescent stage (C, day 39). The percentages of CD14+CD32B+ monocytes/macrophages among CD14+ monocytes/macrophages are given. Anti-CD32B antibody binding is shown as a black line and the background isotype antibody (a non-specific goat IgG antibody) binding as a grey line for each sample. An area of the black line on the right of a cut-off line is the percentage of positive cells.

 

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TABLE 1. CD14+ and CD14+CD32B+ monocytes/macrophages in patients with acute KD and control subjects

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
We have already reported that CD14+ and CD14+CD16+ (Fc{gamma}RIII) monocytes/macrophages among PBMC of patients with acute KD before IVIG therapy were increased compared with at the convalescence stage, and CD14+CD16+ monocytes/macrophages decreased to within the normal range soon after IVIG therapy [25]. Activation of NF-{kappa}B through Fc{gamma}RIII has been reported [27], which agrees with our previous finding that the number of peripheral blood CD14+CD16+ monocytes/macrophages and NF-{kappa}B activation in CD14+ monocytes/macrophages decreased soon after IVIG therapy in KD [25, 26]. Recently, we revealed that Fc{gamma}RIII expression decreased transiently on the membranes of U-937 cells, a human monocytic leukaemia cell line, and peripheral blood CD14+ monocytes/macrophages after the addition of IVIG in vitro [28].

In the present study, we demonstrated that IVIG therapy in patients with KD did not increase the expression of Fc{gamma}RIIB in peripheral blood CD14+ monocytes/macrophages during the acute stage. On the contrary, CD14+CD32B+ monocytes/macrophages slightly increased during acute KD before IVIG therapy, and returned to within the normal range soon after IVIG therapy. We speculate that the slight increase in CD14+CD32B+ monocytes/macrophages during acute KD before IVIG therapy reflects negative feed-back in response to activated monocytes/macrophages associated with the increase in CD14+CD16+ monocyte/macrophage numbers. Alternatively, the slight increase in CD14+CD32B+ monocytes/macrophages during acute KD before IVIG therapy may reflect activated CD14+CD32A+ monocytes/macrophages, since the anti-CD32B antibody may react against not only CD32B but also CD32A because the extracellular and transmembrane regions of CD32A and CD32B are similar or identical [29]. Our results suggest that CD14+CD32B+ monocytes/macrophages, including CD14+CD32A+ monocytes/macrophages, do not increase in numbers after IVIG therapy in acute KD.

It is unclear whether the IVIG treatment caused the reduced numbers of CD14+CD32B+ monocytes/macrophages since we did not evaluate a control group of patients treated with only aspirin. Such a study is not possible in Japan because the Research Committee on Kawasaki Disease recommends that IVIG treatment be given to patients with KD [30]. A previous study demonstrated that there was no significant difference in the expression of CD14+CD32+ monocytes/macrophages before and after IVIG therapy [31]. We focused on CD14+CD32B+ monocytes/macrophages with an inhibitory motif, but not CD14+CD32+ ones. Regarding Fc{gamma}R expression on peripheral blood monocytes/macrophages during acute KD, the main effect of IVIG therapy may be based on a decrease in CD14+CD16+ (Fc{gamma}RIII) monocytes/macrophages, but not an increase in CD14+CD32B+ (Fc{gamma}RIIB) monocytes/macrophages.

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 15 June 2004; revised version accepted 19 October 2004.



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