Expression Profiling of the Effect of High-Dose Intravenous Ig in Patients with Kawasaki Disease

This information is current as Jun Abe, Toshiaki Jibiki, Seiji Noma, Tosiharu Nakajima, of October 2, 2021. Hirohisa Saito and Masaru Terai J Immunol 2005; 174:5837-5845; ; doi: 10.4049/jimmunol.174.9.5837 http://www.jimmunol.org/content/174/9/5837 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Gene Expression Profiling of the Effect of High-Dose Intravenous Ig in Patients with Kawasaki Disease1

Jun Abe,2* Toshiaki Jibiki,† Seiji Noma,‡ Tosiharu Nakajima,* Hirohisa Saito,* and Masaru Terai§

Kawasaki disease (KD) is an acute vasculitis of infants and young children, preferentially affecting the coronary arteries. Intra- venous infusion of high dose Ig (IVIG) effectively reduces systemic inflammation and prevents coronary artery lesions in KD. To investigate the mechanisms underlying the therapeutic effects of IVIG, we examined profiles of PBMC and purified monocytes obtained from acute patients before and after IVIG therapy. The results suggest that IVIG suppresses acti- vated monocytes and macrophages by altering various functional aspects of the of KD patients. Among the 18 commonly decreased transcripts in both PBMC and purified monocytes, we selected six genes, FCGR1A, FCGR3A, CCR2, ADM, , and , and confirmed the microarray results by real-time RT-PCR. Moreover, the expressions of Fc␥RI and Fc␥RIII on Downloaded from monocytes were reduced after IVIG. Plasma S100A8/A9 heterocomplex, but not S100A9, levels were elevated in patients with acute KD compared with those in febrile controls. Furthermore, S100A8/A9 was rapidly down-regulated in response to IVIG therapy. Persistent elevation of S100A8/A9 after IVIG was found in patients who later developed coronary aneurysms. These results indicate that the effects of IVIG in KD may be mediated by suppression of an array of immune activation genes in monocytes, including those activating Fc␥Rs and the S100A8/A9 heterocomplex. The Journal of Immunology, 2005, 174: 5837– 5845. http://www.jimmunol.org/

awasaki disease (KD)3 is an acute systemic vasculitis ceeds 85% in Japan (9), it is imperative that we clarify the mech- that primarily affects infants and young children (1, 2). anisms of action of IVIG therapy in KD. K Although KD is, in most cases, a self-limited illness, IVIG therapy was first introduced in children with idiopathic resolving within a few weeks after fever onset, it preferentially thrombocytopenic purpura (ITP) by Imbach et al. in 1981 (10). affects coronary arteries, and without appropriate intervention, 15– Since then, it has been used for the treatment of various autoim- 25% of patients will develop coronary aneurysms or dilatation (3, mune diseases, such as vasculitis, Guillain-Barre syndrome, and 4). Intravenous infusion of high dose Ig (IVIG) was demonstrated dermatomyositis (11–13). In KD patients, IVIG was first reported to effectively reduce systemic inflammation and the incidence of by Furusho et al. in 1984 (5) to effectively reduce the incidence of by guest on October 2, 2021 coronary artery lesions (5, 6). However, the precise mechanisms coronary artery lesions. Subsequently, a single high dose Ig (2 g/kg underlying the effects of IVIG therapy in KD are unknown. More- body weight) infusion was shown to be more effective in reducing over, ϳ15% of KD patients are not responsive to this therapy and inflammation and fever than administration of the same dose di- must be given additional IVIG or immunosuppressive regimens, vided over several days (6). In responsive patients, the serum lev- such as methylprednisone pulse therapy (7, 8). Because the inci- els of a variety of inflammatory mediators, such as cytokines (IL- dence of KD in the Japanese population has increased from 88 to 1␤, IL-6, and TNF-␣) and chemokines (MCP-1, IL-8, and MIP-1) 140 per 100,000 children under 5 years of age in the 10 years since as well as acute inflammatory (C-reactive peptide (CRP) 1994, and the proportion of patients treated with IVIG now ex- and haptoglobin) are reportedly decreased after IVIG therapy (14– 16). Spontaneous Ig synthesis by PBMC was also reported to be reduced after IVIG (17). These findings indicate that the effects of *Department of Allergy and Immunology, National Research Institute for Child IVIG in KD are mediated mainly by robust suppression of acti- † Health and Development, Tokyo, Japan; Chiba Municipal Kaihin Hospital, Chiba, vated immune cells in the peripheral circulation. Ichiyama recently Japan; ‡Hachiouji Metropolitan Children’s Hospital, Tokyo, Japan; and §Graduate School of Medicine, Chiba University, Chiba, Japan reported that IVIG preparation inhibited TNF-␣-induced NF-␬B Received for publication October 1, 2004. Accepted for publication February activation in cultured monocytic cells (18). However, which cell 16, 2005. population is directly affected by IVIG remains to be clarified. The costs of publication of this article were defrayed in part by the payment of page Whether the suppressive effect is mediated though direct binding charges. This article must therefore be hereby marked advertisement in accordance of Ig to cell surface receptors or through the neutralization or with 18 U.S.C. Section 1734 solely to indicate this fact. blockade of cytokine receptor pathways is also unknown. 1 This work was supported in part by a Grant for Child Health and Development (14-2) from the Ministry of Health, Labor, and Welfare; a grant from the Japan Health With the development of DNA microarray technologies and Sciences Foundation; and a grant from the Organization for Pharmaceutical Safety functional genomics, we can now monitor the levels of Ͼ20,000 and Research of the Ministry of Health, Labor, and Welfare (Millenium Genome Project, MPJ-5). transcripts from a limited number of immune cells, allowing study of complex biological responses occurring in affected children 2 Address correspondence and reprint requests to Dr. Jun Abe, Department of Allergy and Immunology, National Research Institute for Child Health and Development, 2-10-1 Oh- (19). This method appears to be especially useful for investigating kura, Setagaya-ku, Tokyo 157-8535, Japan. E-mail address: [email protected] the unknown molecular phenomena associated with the adminis- 3 Abbreviations used in this paper: KD, Kawasaki disease; AD, average difference; tration of certain pharmaceutical agents, such as IVIG. In this ADM, adrenomedullin; CRP, C-reactive peptide; ITP, idiopathic thrombocytopenic purpura; IVIG, i.v. infusion of high dose Ig; MFI, mean fluorescent intensity; S100, study we attempted to analyze gene expression profiles in PBMC S100 calcium-binding family. and purified monocytes to reveal which cell population and which

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 5838 GENE EXPRESSION PROFILING OF HIGH DOSE IVIG

transcripts are more affected by IVIG therapy in KD patients. We probes were high, gene expression was judged to be absent even if a high also investigated whether changes in gene expression are related to AD value was obtained from that particular gene. Under these conditions, the therapeutic effects of IVIG and the clinical course of KD in our we confirmed that the expression levels of genes in the same cells, analyzed twice, showed a statistically significant correlation (r ϭ 0.997). The results patients. of the GeneChip Analysis can be found on our web site at ͗www.nch.go.jp/imal/GeneChip/KAWASAKI.htm͘. Materials and Methods The data were further analyzed with GeneSpring software version 6.1 Patients (Silicon Genetics). To normalize the staining intensity variations among the chips, the AD values for all genes on a given chip were divided by the We studied 46 Japanese acute KD patients (age range, 2–76 mo; median, median AD of all measurements on that chip. To eliminate changes within 19.5 mo) between April 2002 and March 2004. Eight patients (four boys the range of background noise and to select the most differentially ex- and four girls) were treated at Hachiouji Metropolitan Children’s Hospital, pressed genes, only probes whose AD values were judged to be present by 15 patients (eight boys and seven girls) were treated at Chiba University GeneChip Analysis Suite 5.0 (Affymetrix) in at least two of four chips were Hospitals, and 23 patients (10 boys and 13 girls) were treated at Kaihin- included in the analysis. Hierarchical clustering analysis with standard cor- Chiba Municipal Hospital. All patients were diagnosed according to the relation was used to identify gene clusters. The separation ratio was set guidelines established by the Kawasaki disease research committee in Ja- at 0.5. pan. All patients were given IVIG therapy (1.0 g/kg for 1–2 days or 400 mg/kg for 4–6 days) and oral aspirin (10–30 mg/kg daily). Abnormal cardiac function and coronary artery lesions were monitored by two-di- Quantitative real-time PCR mensional echocardiography during the acute and convalescent phases of Total RNA was reverse transcribed to cDNA using SuperScript III reverse the disease. Two patients had transient mitral valve regurgitation and tri- transcriptase (Invitrogen Life Technologies) and random hexamers (Am- cuspid valve regurgitation before IVIG, and four patients (8.3%) developed ersham Biosciences). The PCR primers and probes were purchased from coronary aneurysms 1 mo after onset of the disease. Applied Biosystems (Assays-on-Demand; Gene Expression Products) for Downloaded from Venous blood was drawn from each patient before IVIG treatment (2–9 GAPDH (assay no. Hs99999905), adrenomedullin (ADM) (Hs00181605), d after the onset of fever, median, 5 d) and within 7 days after completion S100 calcium-binding protein family (S100) A8 (Hs00374264), and of IVIG therapy (7–19 d after fever onset, median, 12 d). Twenty control S100A12 (Hs00194525). The primers and probes for S100A9, CCR2, blood samples were obtained from age-matched control patients (10 boys FCGR1A, FCGR3A, IL-6, IL-8, IL-10, and TNF-␣ were designed based on and 10 girls; age range, 1–78 mo; median, 10.5 mo) who had been febrile sequences from GenBank. Primer sequences were as follows: S100A9 for- Ͼ (body temperature 38°C) for at least 3 d. Their clinical diagnoses were ward primer, 5Ј-CCGTGGGCATCATGTTGAC-3Ј; S100A9 reverse primer, acute upper respiratory infection (10), toxic shock syndrome-like exan- 5Ј-GGAAGGTGTTGATGATGGTCTCTA-3Ј; CCR2 forward primer, 5Ј-

thematous disease (3), cervical lymphadenitis (2), staphylococcal scalded GCGTTTAATCACATTCGAGTGTTT-3Ј; CCR2 reverse primer, 5Ј-CC http://www.jimmunol.org/ skin syndrome (2), pneumonia (2), and enterocolitis (1). Informed consent ACTGGCAAATTAGGGAACAA-3Ј; FCGR1A forward primer, 5Ј-GGT was obtained from the patients’ parents according to the guidelines of each TCTTGACAACTCTGCTCCTTT-3Ј; FCGR1A reverse primer, 5Ј-TTG medical center. GAACACGCTGACCCAT-3Ј; FCGR3A forward primer, 5Ј-ATTGACGC Ј Ј Isolation of PBMC and enrichment of monocytes and TGCCACAGTCAAC-3 ; FCGR3A reverse primer, 5 -AGCCAGCCGA TATGGACTTCT-3Ј; IL-6 forward primer, 5Ј-CCAGTACCCCCAGGA lymphocytes GAAGAT-3Ј; IL-6 reverse primer, 5Ј-CGTTCTGAAGAGGTGAGTG Ј Ј Ј Human PBMC were isolated by centrifugation on a Ficoll-Paque Plus GC-3 ; IL-8 forward primer, 5 -CACTGCGCCAACACAGAAATTA-3 ; Ј Ј (Amersham Biosciences) density gradient. Peripheral blood monocytes or IL-8 reverse primer, 5 -ACTTCTCCACAACCCTCTGCAC-3 ; IL-10 for- Ј Ј T cells were separated from heparinized venous blood using a RosetteSep ward primer, 5 -TACGGCGCTGTCATCGATT-3 ; IL-10 reverse primer, 5Ј-GGCATTCTTCACCTGCTCCA-3Ј; TNF-␣ forward primer, 5Ј-CCCT Monocyte or T Cell Enrichment Cocktail (StemCell Technologies) accord- by guest on October 2, 2021 Ј ␣ Ј ing to the manufacturer’s instructions (20). Briefly, 2 ml of heparinized GGTATGAGCCCATCTATC-3 ; and TNF- reverse primer, 5 -AAAG Ј blood was mixed with 20 ␮l of 100 mM EDTA and 100 ␮l of RosetteSep TAGACCTGCCCAGACTCG-3 . PCR was conducted using the ABI 7700 ␮ mixture containing Abs to human CD2, CD3, CD8, CD19, CD56, and sequence detector system (Applied Biosystems) in a 25- l reaction mixture ␮ CD66b for monocytes or to human CD16, CD19, CD36, and CD56 for T containing 12.5 l of TaqMan Universal PCR Master Mix (Applied Bio- ␮ ϫ cells. After incubation for 20 min at room temperature, the sample was systems), 1.25 lof20 Assays-on-Demand Gene Expression Assay Mix- ␮ diluted with an equal volume of PBS containing 2% FBS and 1 mM EDTA ture (Applied Biosystems), or 1.25 l of a mixture of forward and reverse ␮ ␮ ␮ and was layered on top of 4 ml of Ficoll-Paque. The tubes were then primers (4.0 M each) and FAM-labeled probe (2.0 M), and 11.25 lof centrifuged at 2000 rpm at room temperature for 20 min. The interface cDNA diluted in RNase-free H2O. Samples were preincubated for 10 min between plasma and Ficoll-Paque was collected, washed, and stored in at 95°C, then subjected to 40 cycles of amplification at 95°C for 15 s for liquid nitrogen until RNA extraction. The CD14ϩ monocytes and CD3ϩ T denaturing and at 60°C for 1 min for annealing-extension. The expression cells typically represented ϳ90% and 93.5%, respectively, of the total cells of each target cDNA relative to GAPDH was calculated using a compar- on flow cytometric analysis after these enrichment procedures. ative Tc method described in the User Bulletin 2 provided by the manu- facturer (Applied Biosystems) and was determined for each sample. Extraction of RNA and GeneChip expression analysis Total RNA was isolated from the PBMC or monocyte-enriched fraction of Flow cytometry PBMC using ISOGEN (Wako Pure Chemical Industries) according to the PBMC were suspended in staining solution consisting of PBS, 5% FCS, manufacturer’s instructions. Gene expression was examined using the hu- 0.02% sodium azide, and 1 mg/ml human IgG (Mitsubishi Pharma). The man genome U133A probe array (GeneChip; Affymetrix), which contains cells were incubated with one of the mAbs, 3G8, an Ab to human CD16, the oligonucleotide probe set for 22,283 full-length genes and expressed CIKM5, an Ab to human CD32, 10.1, an Ab to human CD64 (all from sequence tags, according to the manufacturer’s protocol, and previous re- Caltag Laboratories), or 679.1Mc7, a mouse IgG1 isotype control (Beck- ports (21, 22). Five micrograms of total RNA from PBMC or 150 ng of man Coulter), followed by incubation with FITC-conjugated rat anti-mouse total RNA from monocytes was used to synthesize double-stranded cDNA. IgG1 mAb (BD Biosciences) and PE-anti-CD14 (Beckman Coulter). Flu- The cDNA was next subjected to in vitro transcription in the presence of orescence intensity was analyzed with a FACScan flow cytometer (BD biotinylated nucleoside triphosphates. In the assay of monocyte RNA, two Biosciences) and CellQuest software (BD Biosciences). cycles of cDNA synthesis and in vitro transcription reactions were con- ducted to amplify target sequences. The biotinylated cRNA was hybridized with a U133A probe array for 16 h at 45°C, and the hybridized biotinylated ELISA for S100A8/A9 heterocomplex and S100A9 homocomplex cRNA was stained with streptavidin-PE (Molecular Probes) and then in plasma scanned with a Gene Array Scanner (Hewlett-Packard). The fluorescence intensity of each probe was quantified using a computer program, Gene- ELISA was performed using MRP8/14 ELISA (Buhlmann Laboratories) Chip Analysis Suite 5.0 (Affymetrix). The expression level of a single and MRP14 ELISA (Chemicon International) kits according to the manu- mRNA was determined as the average fluorescence intensity among the facturer’s instructions. Heparinized test plasma was diluted 1/200 for the intensities obtained by 20 pairs (perfectly matched and single nucleotide- MRP8/14 kit, and 1/5 for the MRP14 kit in assay buffer, and 100 ␮l of each mismatched) of probes consisting of 25-mer oligonucleotides. The level of dilution was applied to a 96-well plate in duplicate. The absorbance was gene expression was determined as the average difference (AD) using Ge- read at 450 nm in a microplate reader, and the protein concentration was neChip Analysis Suite 5.0. In this program, if the intensities of mismatched calculated using Microplate Manager III software (Bio-Rad). The Journal of Immunology 5839

Table I. Demographic data of patients

PBMC Array Patients Monocyte Array Patients Other Patients

Age (mo after birth) 17–36 (median, 26) 3–25 (median, 22) 2–76 (median, 16) Sex (male, female) 3, 1 1, 3 18, 20 Blood drawn pre-IVIG (days after onset) 3–5 (median, 4) 5–9 (median, 6) 2–9 (median, 5) Blood drawn post-IVIG (days after onset) 8–14 (median, 12) 8–18 (median, 15) 7–18 (median, 13) Cardiac involvement (positive patients) 1 0 5 Neutrophil count/mm3 (pre, post) 9,946 Ϯ 3,792, 3,445 Ϯ 763 11,203 Ϯ 4,779, 3,322 Ϯ 2,736 9,761 Ϯ 3,353, 4,623 Ϯ 5,110 p valuea 0.04 0.02 0.002 Monocyte count/mm3 (pre, post) 862 Ϯ 540, 574 Ϯ 290 1,434 Ϯ 849, 818 Ϯ 759 856 Ϯ 700, 621 Ϯ 587 p valuea 0.42 0.16 0.06 Lymphocyte count/mm3 (pre, post) 4,580 Ϯ 735, 5,222 Ϯ 3,055 5,380 Ϯ 2,127, 5,988 Ϯ 2,556 3,469 Ϯ 1,885, 4,965 Ϯ 2,361 p valuea 0.72 0.75 0.001 C reactive protein (mg/dl; pre, post) 10.4 Ϯ 4.2, 0.8 Ϯ 0.2 7.5 Ϯ 3.4, 1.0 Ϯ 0.6 10.1 Ϯ 5.1, 1.9 Ϯ 3.0 p valuea 0.02 0.03 Ͻ0.001

a By paired t test, pre-IVIG vs post-IVIG.

Statistical analysis Gene expression analysis in monocyte-enriched fraction Downloaded from For GeneChip microarray data, the two-tailed paired t test was performed of PBMC using normalized AD values with GeneSpring software version 6.1 (Silicon Because of the limited sample volumes obtained from patients and Genetics). For real-time RT-PCR, flow cytometry, and ELISA data, the the necessity of avoiding activation of monocytes during the pu- two-tailed paired t test was used to compare patients’ samples obtained before vs after IVIG therapy, and one-factor ANOVA and the Scheffe´F test rification procedure, we used a RosetteSep monocyte enrichment as a post-hoc test were used to compare pre-IVIG patients with KD and system to negatively select monocytes. After enrichment, we de- control patients. Correlations between S100A8/A9 and S100A9 plasma termined the percentages of T, B, and NK cells by flow cytometry. http://www.jimmunol.org/ levels and the laboratory data were assessed using Spearman’s rank test In the monocyte-enriched fraction, each cell population was re- after cell counts had been logarithmically transformed. A value of p Ͻ 0.05 Ͻ was considered statistically significant. duced to 1.0% of the total cell yield. After analyzing gene expression profiles of monocyte-enriched Results fractions obtained from four additional KD patients (Tables I and II) using the U133A probe array, 1274 transcripts Gene expression changes in PBMC after IVIG treatment were found to be differentially expressed after IVIG therapy ( p ϭ We first examined the gene expression profiles of PBMC obtained 0.05). Of these transcripts, 67 genes showed more than a doubling from four KD patients before and after IVIG therapy. The demo- of expression compared with pre-IVIG levels, and 131 genes by guest on October 2, 2021 graphic and laboratory data of patients at the time of blood draw- showed a decrease in expression to less than half the pre-IVIG ing are summarized in Tables I and II. By using the Human Ge- levels after IVIG treatment (Fig. 1A and supplemental table I4). A nome U133A probe array, which contains the oligonucleotide total of 18 genes showed consistently decreased transcripts after probe set for 22,283 transcripts, 509 transcripts were found to have IVIG therapy in both PBMC and purified monocytes, suggesting significantly changed expression levels after IVIG therapy ( p ϭ that the decreases in these gene transcripts are not attributable to a 0.05). Among them, four genes showed expression to be more than reduced number of monocytes in PBMC after IVIG. A dendrogram double the pre-IVIG levels, and 85 genes showed expression to be of the expression profiles of these 18 genes is presented in Fig. 1B less than half the pre-IVIG levels after IVIG therapy (Fig. 1A). In together with their mean AD values calculated from four pairs of total, 75 differentially expressed genes were classified according to monocyte array results. their cellular functions (Table III). Fourteen genes were excluded To confirm the GeneChip results and prove that these down- from Table II because their functions are currently unknown. Re- regulated genes were expressed mainly by monocytes in acute KD markably, most of the differentially expressed genes were down- patients, we performed a real-time RT-PCR using negatively se- regulated after IVIG. Among these down-regulated genes, cell sur- lected blood monocytes and T cells obtained from patients. We face receptors formed the largest functional group, most of which examined transcripts of six genes, FCGR1A, FCGR3A, CCR2, were mainly expressed in monocytes and macrophages. For ex- ADM, S100A9, and S100A12, as representatives of 18 genes com- ample, FCGR1A, FCGR2A, FCGR3A, and formyl peptide recep- monly decreased in both PBMC and monocytes. The protein prod- tor 1 are receptors that function in phagocytosis and transduce ucts of these genes were expected to have definite functions in the stimulatory signals to monocytes. The receptors of chemokines inflammatory process in patients, and their expression profiles in and growth factors for monocytes and macrophages, such as the monocytes from the four patients differed slightly, as illustrated in CSF-1 receptor, the CSF-2 receptor, and CCR2, were also down- Fig. 1B. In addition, the transcripts of IL-6, IL-8, IL-10, and regulated after IVIG therapy. Among the down-regulated genes of TNF-␣ were examined, because the array results did not indicate secreted proteins, there was a preponderance of monocyte-derived significant differences in their expression levels before vs after molecules, such as ADM, S100A8, S100A9, S100A12, and endo- IVIG, although many reports have indicated that these inflamma- thelial cell growth factor 1. Based on these findings, we decided to tory cytokines were overproduced in sera of acute KD patients determine whether the down-regulation of monocyte-related gene (23–27). The S100A8 transcript was also examined, because this expressions was due to a decreased number of monocytes among protein forms a heterocomplex with S100A9, which plays a role in PBMC from patients or to decreased mRNA synthesis in individ- the adhesion and chemotaxis of neutrophils and monocytes in the ual monocyte after IVIG. As an approach to this question, we examined gene expression profiles in the monocyte fraction puri- fied from patients’ PBMC. 4 The online version of this article contains supplemental material. 5840 GENE EXPRESSION PROFILING OF HIGH DOSE IVIG

Table II. Background characteristics of patients in microarray study

PBMC 1 PBMC 2 PBMC 3 PBMC 4 Monocyte 1 Monocyte 2 Monocyte 3 Monocyte 4

Age (mo after birth) 36 20 32 17 3 24 19 25 Sex (male, female) M M F M M F FF IVIG (mg/kg ϫ times) 400 ϫ 4 400 ϫ 4 400 ϫ 5 400 ϫ 5 400 ϫ 5 and 1000 ϫ 2 1000 ϫ 2 1000 ϫ 2 670 ϫ 3 Aspirin (mg/kg) 30 30 0 30 30 0 30 30 Other medication None None Heparin (10 U/kg/h) None None Heparin (10 U/kg/h) None None during IVIG during IVIG Response to IVIG Well Well Well Well Well after second Well Well Well IVIG inflammatory response (28). Fig. 2 summarizes the real-time RT-PCR IL-6, IL-8, and TNF-␣ were not significantly changed by IVIG. More results of purified monocytes and T cells obtained from patients importantly, the real-time RT-PCR confirmed that all six transcripts before and after IVIG. The transcripts of six genes that were down- examined were expressed mainly in monocytes, and the contribution regulated in GeneChip analysis, in addition to S100A8 and IL-10, of T cells was much smaller than that of monocytes. Only TNF-␣ and were significantly decreased after IVIG therapy. The transcripts of IL-10 were expressed at compatible levels in monocytes and T cells. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. A, A Venn diagram of up- or down-reg- ulated genes in PBMC and/or purified monocytes. Sig- nificantly (Ͼ2.0ϫ or Ͻ0.5ϫ) changed gene expres- sions are listed from the microarray results of PBMC and monocytes and indicated as a Venn diagram. B, Clustering analysis of the down-regulated transcripts in both PBMC and monocytes. Eighteen commonly down-regulated genes in A were analyzed by Gene- Spring software, and a dendrogram was made from four paired monocyte samples obtained before and after IVIG therapy. Gene symbol, mean AD of four samples, and fold decrease in AD before vs after IVIG are pre- sented in the right columns for each gene. The Journal of Immunology 5841

Table III. Up- and down-regulated genes in PBMC

Mean AD

Gene Title Gene Symbol Before After Fold Decrease

Cell surface molecules and receptors IgG FcRIa (CD64) FCGR1A 320.9 80.7 3.98 Leukocyte Ig-like receptor B1 LILRB1 256.0 72.3 3.54 Leukocyte Ig-like receptor B2 LILRB2 644.6 195.8 3.29 IgG FcRIIa (CD32) FCGR2A 394.2 123.4 3.19 TLR2 TLR2 479.9 154.8 3.10 Adiponectin receptor 1 ADIPOR1 1356.5 450.7 3.01 Formyl peptide receptor 1 FPR1 1568.9 542.8 2.89 CSF 2 receptor b CSF2RB 646.8 225.1 2.87 IL-8Rb IL8RB 108.6 38.8 2.80 Stabilin 1 STAB1 792.1 298.0 2.66 Leukocyte Ig-like receptor B3 LILRB3 342.0 129.4 2.64 Ectonucleoside triphosphate diphosphohydrolase 1 (CD39) ENTPD1 209.5 86.2 2.43 Chemokine (CC motif) receptor 2 CCR2 467.3 194.2 2.41 IgG FcRIIIa (CD16) FCGR3A 500.6 208.7 2.40 CSF 1 receptor CSF1R 511.0 214.5 2.38 Protein tyrosine phosphatase, non-receptor type substrate 1 PTPNS1 280.8 119.1 2.36 Leukocyte-specific transcript 1 LST1 512.6 223.4 2.29 Downloaded from Apoptosis and cell growth Cold autoinflammatory syndrome 1 CIAS1 246.9 42.4 5.83 Cyclin-dependent kinase inhibitor 1C (p57, Kip2) CDKN1C 323.1 110.5 2.92 S-phase response (cyclin-related) SPHAR 162.5 59.1 2.75 IL-3-regulated NF NFIL3 441.2 168.7 2.61 Growth arrest-specific 7 GAS7 755.4 337.2 2.24

Secreted molecules http://www.jimmunol.org/ ADM ADM 255.3 54.9 4.65 S100 calcium/binding protein A9 S100A9 5820.1 1331.7 4.37 S100 calcium-binding protein A12 S100A12 1423.8 341.9 4.16 Chondroitin sulfate proteoglycan 2 (versican) CSPG2 5358.5 1714.7 3.12 S100 calcium-binding protein A8 S100A8 13143.9 4509.0 2.92 Pre-B-cell colony-enhancing factor PBEF 1593.3 558.8 2.85 Proapoptotic caspase adaptor protein PACAP 618.0 254.6 2.43 Ig ␭ L chain IGL 479.0 197.8 2.42 Endothelial cell growth factor 1 ECGF1 439.4 186.5 2.36 TNF ligand 13 (APRIL) TNFSF13 217.7 105.6 2.06 by guest on October 2, 2021 Dysferlin DYSF 544.8 150.8 3.61 2 CHN2 220.3 61.5 3.58 Hemopoietic cell kinase HCK 1084.1 322.8 3.36 Dual specificity phosphatase 1 DUSP1 5960.5 2042.8 2.92 Regulator of G-protein signalling 2 RGS2 4329.8 1565.5 2.77 RAB31 RAB31 605.0 229.3 2.64 Ribosome-binding protein 1 RRBP1 327.2 143.4 2.28 Transcription factor v-fos homolog FOS 1964.8 315.4 6.23 v-fos homolog B FOSB 643.9 111.4 5.78 Kruppel-like factor 4 KLF4 763.1 160.9 4.74 Cold shock domain protein A CSDA 1529.6 397.9 3.84 Early growth response 1 EGR1 1410.5 373.6 3.78 v-ets homolog 2 ETS2 243.7 73.2 3.33 SFFV proviral integration 1 SPI1 330.8 102.3 3.23 CALR 277.4 105.5 2.63 MHC class II transactivator MHC2TA 277.6 108.1 2.57 Transcription factor 7-like 2 TCF7L2 308.9 131.5 2.35 B cell CLL/lymphoma 6 BCL6 1242.2 507.2 2.45 Metabolism Hexokinase 3 HK3 544.5 43.3 12.57 Aminolevulinate synthase 2 ALAS2 2947.7 445.8 6.61 20 open reading frame 16 C20orf16 491.7 121.2 4.06 Guanosine monophosphate reductase GMPR 243.2 63.2 3.85 Cytochrome P450 family 1B polypeptide 1 CYP1B1 606.7 178.0 3.41 Fatty acid-coenzyme A ligase long-chain 2 FACL2 696.6 217.2 3.21 Biliverdin reductase B (NADPH) BLVRB 418.2 131.7 3.18 Histidine ammonia-lyase HAL 192.4 62.0 3.11 IFN-␥-inducible protein 30 IFI30 2168.7 773.0 2.81 Alanyl aminopeptidase (CD13) ANPEP 541.8 195.0 2.78 Flavoprotein oxidoreductase MICAL2 659.7 238.5 2.77 Neutrophil cytosolic factor 2 NCF2 941.7 351.6 2.68 Spermidine/spermine N1-acetyltransferase SAT 1215.9 480.5 2.53 Cathepsin Z CTSZ 146.6 58.0 2.53 (Table continues) 5842 GENE EXPRESSION PROFILING OF HIGH DOSE IVIG

Table III. (Continued)

Mean AD

Gene Title Gene Symbol Before After Fold Decrease

Exostoses 1 EXT1 109.6 49.0 2.24 Dihydropyrimidinase-like 2 DPYSL2 738.1 338.3 2.18 Ribosomal protein S11 RPS11 160.8 324.1 Ϫ2.02 Transport Aquaporin 9 AQP9 521.3 137.9 3.78 Mitochondrial solute carrier protein MSCP 558.9 190.4 2.94 Solute carrier family 11 member 1 SLC11A1 560.7 194.7 2.88 Phospholipid scramblase 1 PLSCR1 202.9 71.3 2.85 Protein disulfide isomerase-related protein P5 1123.4 444.8 2.53 Heat shock 70 kDa protein 6 (HSP70BЈ) HSPA6 592.7 238.0 2.49 Hypothetical protein BC013764 LOC115207 583.9 251.3 2.32 NK-tumor recognition sequence NKTR 40.6 93.0 Ϫ2.29

Down-regulation of Fc␥R1 and Fc␥RIII after IVIG therapy 0.50) and was not significantly elevated in KD patients compared with febrile controls (MFI, 42.3 Ϯ 3.7 vs 33.2 Ϯ 3.5; p ϭ 0.12). One proposed mechanism of the effect of IVIG therapy is a block- Downloaded from ␥ ade of Fc Rs on phagocytes. In this scenario, the bound IgG pre- Elevated plasma S100A8/A9 heterocomplex levels in pre-IVIG vents immune complexes from being phagocytosed and from de- KD patients livering an activating signal to the target cells. In a mouse model of ITP, another mechanism has been proposed that involves in- Among the 18 genes significantly decreased after IVIG therapy in duction of inhibitory Fc␥RIIb by IVIG (29). However, the effect of both PBMC and monocytes, a member of the family, ␥ S100A9, was of particular interest, because this protein as well as

IVIG on Fc R expression levels in KD has not been fully eluci- http://www.jimmunol.org/ dated. Because both GeneChip results and the real-time PCR data its partner protein, S100A8, are predominantly expressed in neu- suggested down-regulation of the activating FCGR1A and trophils and monocytes and are excreted into the circulation under FCGR3A, we examined the surface expressions of these receptors inflammatory conditions. In plasma, the S100A8/A9 heterocom- on CD14ϩ monocytes from KD patients before and after IVIG plex is the main form of the two proteins and has been shown to treatment. enhance monocyte adhesion to endothelial cells and to cause neu- As shown in Fig. 3, Fc␥RI expression in KD patients (n ϭ 12) trophil chemotaxis. Therefore, we measured plasma levels of the was elevated before IVIG therapy compared with that in febrile S100A8/A9 heterocomplex as well as the S100A9 homocomplex controls (mean fluorescent intensity (MFI), 25.8 Ϯ 3.2 vs 13.9 Ϯ in KD patients before and after IVIG therapy.

1.2; p ϭ 0.005) and decreased after IVIG treatment (MFI, 25.8 Ϯ As shown in Fig. 4, plasma S100A8/A9 heterocomplex levels by guest on October 2, 2021 ϭ 3.2 vs 17.5 Ϯ 1.6; p ϭ 0.04). Fc␥RIII expression (n ϭ 6) was also were significantly higher in pre-IVIG (n 32) than in post-IVIG Ϯ Ϯ ϭ down-regulated by IVIG treatment (% positive, 20.5 Ϯ 3.5 vs patients and febrile controls (25.3 1.5 vs 18.4 1.7 mg/ml ( p Ϯ ␮ Ͻ 10.7 Ϯ 1.9%; p ϭ 0.04), but there was no significant difference in 0.001) vs 10.7 1.0 g/ml ( p 0.0001), respectively). In con- Fc␥RIII expression levels between pre-IVIG KD patients and con- trast, plasma S100A9 homocomplex levels were significantly ϭ Ϯ trols (% positive, 20.5 Ϯ 3.5 vs 19.0 Ϯ 4.4%; p ϭ 0.79). Fc␥RII lower in pre-IVIG (n 31) than in post-IVIG patients (12.8 2.6 expression on monocytes (n ϭ 12) was not significantly changed before vs after IVIG therapy (MFI, 42.3 Ϯ 3.7 vs 38.4 Ϯ 3.7; p ϭ

FIGURE 3. Expressions of Fc␥Rs on CD14ϩ monocytes from KD pa- tients and febrile controls. Paired blood samples obtained before and after IVIG therapy from KD patients were double-stained with mAbs against FIGURE 2. Higher expressions of FCGR1A, FCGR3A, CCR2, ADM, CD14 and CD64 (n ϭ 12), CD32 (n ϭ 12), or CD16 (n ϭ 6), respectively, S100A9, and S100A12 genes in pre-IVIG compared with post-IVIG mono- and analyzed by flow cytometry. The results were expressed as MFI for cytes. Monocytes and T cells were negatively selected individually from CD64 and CD32 or as the percentage of cells positive for CD16 among ,ء .PBMC, and real-time RT-PCR of each transcript was performed. Results CD14ϩ monocytes. The bar indicates the mean Ϯ SEM in each group p Ͻ 0.01 (compared with ,ءء ;(are presented as relative units of each transcript compared with GAPDH. p Ͻ 0.05 (compared with post-IVIG patients .(p Ͻ 0.001 (compared with post-IVIG febrile controls ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء monocytes). The Journal of Immunology 5843

FIGURE 4. Elevated plasma S100A8/A9 levels in pre-IVIG patients. Concentrations of the S100A8/A9 heterocomplex (n ϭ 32) and the S100A9 homocomplex (n ϭ 30) in heparinized plasma were measured by ELISA in pre-IVIG and post-IVIG KD patients. F, Pa- tients with cardiac involvement; E, patients without cardiac involvement. The bar indicates the mean Ϯ] -p ϭ 0.001 (compared with post ,ء .SEM for each group -p Ͻ 0.0001 (compared with post ,ءء ;(IVIG values IVIG or febrile controls).

vs 20.6 Ϯ 2.8 ng/ml; p Ͻ 0.0001), but were not significantly dif- rived transcripts was also recognized in other functional catego- ferent from those in febrile controls (vs 12.5 Ϯ 5.8 ng/ml). More- ries, such as secreted peptides (ADM, S100A8, S100A9, Downloaded from over, the decrease in the S100A8/A9 heterocomplex after IVIG S100A12, endothelial cell growth factor 1, and TNF super family was absent in four of six patients who had cardiac involvement 13) and metabolic enzymes (hexokinase 3, cytochrome P450 fam- during the acute phase of the disease ( p ϭ 0.02, by ␹2 test with ily 1, subfamily B, polypeptide 1, histidine ammonia-lyase, alanyl Yates’ correction). Correlation analysis showed S100A8/A9 levels aminopeptidase, and neutrophil cytosolic factor 2). Although we in post-IVIG patients to correlate significantly with the monocyte did not examine neutrophil mRNA in this study, our findings counts, neutrophil counts, and CRP levels of post-IVIG patients clearly indicate that IVIG acts, at least in part, by suppressing http://www.jimmunol.org/ (Table IV). activated monocytes and macrophages in KD patients. The oligonucleotide array analysis of the purified monocyte Discussion fraction from KD patients confirmed the results obtained by The aim of this study was to clarify the molecular events following PBMC. By analyzing gene expression profiles of purified mono- administration of high dose Ig to KD patients. We were especially cytes, the number of transcripts differentially expressed pre-IVIG interested in which cell population in the peripheral circulation vs post-IVIG was doubled compared with the expression in was more affected by IVIG therapy with regard to the transcript PBMC. In this assay IVIG not only down-regulated the expression profile. For this purpose, we first examined gene expression in of 131 genes, but also up-regulated 67 genes in patients (Fig. 1A by guest on October 2, 2021 PBMC obtained from KD patients before IVIG therapy and just and Supplementary Table I). The increased number of down-reg- after the therapy, when acute symptoms had resolved in most of ulated genes in addition to the newly identified up-regulated genes the patients. The results suggested that IVIG acts in a broad func- might indicate increased sensitivity of the microarray assay, attrib- tional range in PBMC, favoring down-regulation. Among the 89 utable to using a more homogeneous cell population than PBMC. genes whose expressions were more than doubled or reduced to no Among the down-regulated transcripts newly identified by this more than half the pre-IVIG level, 96% (85 genes) were down- method, the cell surface receptor molecules again formed the larg- regulated. This is consistent with the suppressive effects of IVIG est functional family. These include receptors important in the ini- observed clinically in acute patients whose body temperatures as tiation of innate immune responses (TLR1 and TLR4), receptors well as other laboratory markers of inflammation dropped rapidly functioning in phagocytosis and Ag presentation (asialoglycopro- in response to therapy. More interestingly, by functional classifi- tein receptor 2, complement 3b/4b receptor 1, and stabilin 1), and cation, as many as 17 down-regulated genes (19.1%) were cell receptors involved in cell adhesion (platelet/endothelial cell adhe- surface receptor molecules, 16 of which were expressed mainly in sion molecule, selectin P ligand, and bone marrow stromal cell Ag monocytes and macrophages. The predominance of monocyte-de- 1). In addition to these surface receptors, the transcripts of some secreted proteins that work as pattern recognition molecules and enhance phagocytosis by macrophages (complement component Table IV. Correlation coefficient between S100 proteins plasma levels 1q ␣ and ␤, and ficolin 1) were newly recognized as being de- and laboratory dataa creased after IVIG therapy. These results suggest that IVIG exerts a broad range of effects, suppressing monocyte and macrophage S100A8/A9 S100A8/A9 S100A9 S100A9 Pre Post Pre Post functions. Although down-regulation of inflammatory cytokines such as TNF-␣ and IL-6 after IVIG therapy has been reported by Ϫ Ϫ Neutrophil pre 0.25 0.24 0.23 0.11 others (14–16), we found no significant differences in these cyto- Neutrophil post Ϫ0.10 0.61b Ϫ0.02 Ϫ0.08 kine transcripts between pre- and post-IVIG patients in this study. Monocyte pre 0.03 0.02 Ϫ0.13 0.26 The translation of many inflammatory cytokines has been demon- Monocyte post Ϫ0.27 0.52c 0.06 0.09 strated to be regulated by factors that control mRNA stability (30, 31). Therefore, IVIG may affect the monocyte production of these CRP pre 0.23 0.11 Ϫ0.13 Ϫ0.24 cytokines by destabilizing their mRNA. Alternatively, IVIG may CRP post Ϫ0.15 0.76d 0.15 Ϫ0.13 affect the production of these cytokines differently in PBMC and a Significance was determined by Spearman’s correlation coefficient by rank. other cells, such as vascular endothelial cells and hepatocytes. In b p Ͻ 0.001. c p Ͻ 0.01. this respect, it would be important to determine whether IVIG d p Ͻ 0.0001. affects the production of these cytokines by other cell types and 5844 GENE EXPRESSION PROFILING OF HIGH DOSE IVIG whether suppression of monocyte function by IVIG, as observed in regulated after IVIG therapy. In post-IVIG patients, S100A8/A9 this study, is due to reductions of these inflammatory signals levels correlated closely with the plasma CRP levels and neutro- outside PBMC. phil and monocyte counts. Persistent elevation of plasma Eighteen genes were down-regulated by IVIG in both PBMC S100A8/A9 levels after IVIG was related to a higher risk of de- and monocyte array experiments. Because only a limited number veloping coronary aneurysms in KD patients. These findings sug- of patients were available for the microarray analysis, we per- gest that the failure of IVIG to suppress S100A8/A9 expression in formed real-time PCR to examine six of these 18 transcripts and monocytes and other cell populations might result in the continued confirmed the microarray results. Of these genes, plasma levels of recruitment and stimulation of neutrophils and monocytes at the ADM and S100A12 have been reported to be elevated in KD pa- inflammatory sites. Alternatively, the persistent production of tients (32–34). Nomura et al. (32) demonstrated, using oligonu- S100A8/A9 might be a secondary phenomenon caused by pro- cleotide microarray analysis, that ADM and S100A12 mRNA are longed survival of activated monocytes and neutrophils. We favor highly expressed in acute KD patients, and Nishida et al. (33) the former explanation, because our findings add to the accumu- showed plasma ADM levels to be higher in patients who subse- lating evidence of direct effects of S100A8/A9 on both monocytes quently developed coronary aneurysms than in patients who did and endothelial cells (50–53). Eue et al. (50, 53) reported that not (33). Foell et al. (34) reported the plasma S100A12 level to be S100A8/A9 was secreted by activated monocytes and that protein high in pre-IVIG patients and to decrease quickly after IVIG ther- production was enhanced by the cell-cell interaction of monocytes apy. MCP-1, a ligand of CCR2, was also known to be highly with activated endothelial cells. They also reported that expressed in acute KD patients, especially in cardiac tissue (14). S100A8/A9 and S100A9 bound to resting monocytes and TNF-␣- Thus, the down-regulation of CCR2 in monocytes after IVIG ther- activated microvascular endothelial cells in a dose-dependent and apy as well as ADM and S100A12 may be beneficial for patients saturable manner and enhanced CD11b expression on monocytes. Downloaded from by reducing the accumulation of monocytes at the inflammatory It is important to clarify the role of the S100A8/A9 heterocomplex sites and preventing coronary aneurysms. Besides these genes, we in KD pathogenesis, especially in relation to the interaction be- focused on down-regulation of the activating FCGR genes and the tween activated monocytes/neutrophils and vascular endothelial S100A9 gene in response to IVIG therapy. The interaction of IVIG cells. with Fc␥Rs on monocytes/macrophages has been proposed as a In conclusion, the results of this study suggest that IVIG therapy mechanism underlying the therapeutic effect of IVIG in several in KD patients suppresses activated peripheral blood monocytes http://www.jimmunol.org/ autoimmune diseases, such as ITP and autoantibody-induced ar- and macrophages by down-regulating various functional genes. thritis in mice (29, 35). In these diseases, IVIG induces low affinity Among these genes, we focused on two pathways that IVIG may inhibitory Fc␥RIIb on a certain population of monocytes and coun- use to suppress inflammation in KD patients. One is homeostatic teracts the consumption of platelets or the inflammatory response, control of the expression of activating vs inhibitory Fc␥Rs on respectively, elicited by the interaction between immune com- monocytes. The other is suppression of S100A8/A9 heterocomplex plexes and the low affinity activating Fc␥RIIIa. In our study the production in patients. It is important to further elucidate the pre- transcripts of activating FCGR1A, FCGR2A, and FCGR3A genes cise molecular mechanisms of IVIG in these two pathways to mon- were reduced, and the expressions of Fc␥RI and Fc␥RIII on itor the effectiveness of IVIG in patients and to develop a new by guest on October 2, 2021 CD14ϩ monocytes were down-regulated after IVIG in KD pa- molecular target for treating KD patients. tients. Although we could not determine whether the expression of activating Fc␥RIIa and the inhibitory Fc␥RIIb changed indepen- Acknowledgments dently, the staining intensity for CD32 was unchanged after IVIG We are particularly grateful to the pediatricians at Hachiouji Metropolitan in our patients, and FCGR2B transcripts were not increased after Children’s Hospital and Chiba University Hospital for providing blood IVIG in the array experiment. Nevertheless, IVIG may act by mod- samples. We thank Hiromi Wakita, Naomi Wada, and Noriko Hashimoto ulating the ratio of activation to inhibitory Fc␥R expression on for their excellent technical assistance. monocytes so as to raise the threshold for monocyte excitation in KD. Reports have been accumulating that suggest dysregulation of Disclosures such a balance between activation and inhibitory Fc␥R expression The authors have no financial conflict of interest. to possibly contribute to autoimmune disease pathogenesis (36– 39). In addition, several autoantibodies have been implicated in the References 1. Kawasaki, T. 1967. Acute febrile mucocutaneous syndrome with lymphoid in- acute phase of KD (39, 40). The association of the down-regula- volvement with specific desquamation of the fingers and toes in children. Jpn. tion of these activating Fc␥Rs with the therapeutic effect of IVIG J. Allerrgol. 16:178. in KD might suggest a new venue for research into the pathogen- 2. Melish, M. E., R. M. Hicks, and E. J. Larson. 1976. 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