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Macrophages and T Cells during Acute Kawasaki Disease
Clinical Immunology Vol. 99, No. 3, June, pp. 373–377, 2001 doi:10.1006/clim.2001.5026, available online at http://www.idealibrary.com on
NF-B Activation in Peripheral Blood Monocytes/Macrophages and T Cells during Acute Kawasaki Disease Takashi Ichiyama, Tomomi Yoshitomi, Miki Nishikawa, Motoki Fujiwara, Tomoyo Matsubara, Takashi Hayashi, and Susumu Furukawa Department of Pediatrics, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan
Kawasaki disease (KD) is a febrile disease of childhood characterized by systemic vasculitis, and the levels of many proinflammatory cytokines are elevated in the serum at the acute stage. We investigated the activation of transcription factor NF-B for genes that encode the proinflammatory cytokines in CD14 ⴙ monocytes/macrophages and CD3 ⴙ T cells in peripheral blood by means of Western blot and flow cytometric analyses. Western blot analysis demonstrated that NF-B activation was more increased in CD14 ⴙ monocytes/macrophages than in CD3 ⴙ T cells in all children during the acute stage. Flow cytometric analysis revealed that NF-B activation in CD14 ⴙ monocytes/ macrophages was significantly higher than in CD3 ⴙ T cells at the acute stage (30.0 ⴞ 16.0% vs 11.4 ⴞ 5.0%, P < 0.01, Wilcoxon test). NF-B activation in CD14 ⴙ monocytes/macrophages was significantly decreased after high-dose intravenous immunoglobulin therapy (P < 0.05). The present findings suggest that CD14 ⴙ monocytes/macrophages play an important role in cytokine production during acute KD. © 2001 Academic Press Key Words: Kawasaki disease; NF-B activation; CD14 ⴙ cells; CD3 ⴙ cells.
INTRODUCTION
Kawasaki disease (KD) is an acute illness of early childhood characterized by prolonged fever, diffuse mucosal inflammation, indurative edema of the hands and feet, a polymorphous skin rash, and nonsuppurative lymphadenopathy (1). The histopathological findings in KD comprise panvasculitis with endothelial necrosis and the infiltration of mononuclear cells into small and medium-sized blood vessels (2). The levels of any proinflammatory cytokines, chemokines, and adhesion molecules are elevated in sera from children with KD at the acute stage (3– 8). However, the mechanism underlying cytokine production in immunocompetent cells during acute KD has been unclear. Nuclear factor kappa B (NF-B) is a pivotal transcription factor for genes that encode the proinflammatory cytokines, chemokines, and adhesion molecules
that mediate inflammation: interleukin 1 (IL-1), IL-2, IL-6, IL-8, tumor necrosis factor-␣ (TNF-␣), monocyte chemoattractant protein-1, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 (9 –15). NF-B is retained in an inactive form in the cytoplasm (16, 17); its prototypic form consists of a heterodimer of p50 and p65 that is normally bound by members of the IB family (17). Activation of NF-B requires degradation of the cytoplasmic inhibitor IB protein (18, 19). Phosphorylation of IB by drugs, cytokines, bacterial products, and viruses leads to IB degradation, translocation of NF-B to the nucleus, and transcription of proinflammatory cytokine genes (20, 21). We examined whether NF-B activation occurs in peripheral blood CD14 ⫹ monocytes/macrophages or CD3 ⫹ T cells to clarify the mechanism of cytokine production during acute KD. PATIENTS AND METHODS
Patients Twelve children (four girls and eight boys; aged from 2 months to 4 years; mean age, 1.6 years) with acute KD upon admission to our hospital between April 1999 and February 2000 were included in this study (Table 1). Coronary arteries exhibiting diameters of 4 mm or greater on two-dimensional echocardiography were regarded as having coronary artery lesions (CAL), according to the Research Committee on Kawasaki Disease (Ministry of Health and Welfare, Japan) diagnostic criteria for cardiovascular lesions with KD (22). 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 to be the first day of illness. All patients received standard Japanese treatment with high-dose intravenous immunoglobulin (IVIG; Venilon, Teijin Co., Ltd., Tokyo, Japan), 1000 mg/kg/day for 2 days, and oral aspirin (30 mg/kg/day). Samples at the acute stage were obtained on days 4 to 14 (mean ⫾ SD, 5.3 ⫾ 2.8 days) before treatment with IVIG. Sam-
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ICHIYAMA ET AL.
TABLE 1 Data for 12 Children with Kawasaki Disease Patients no./age/sex
Sampling days
WBC counts (/mm 3)
Serum CRP levels (mg/dl)
Coronary artery lesions
1/4 y/M 2/2 y/F 3/4 mo/M 4/1 y/M 5/1 y/F 6/2 mo/M 7/7 mo/M 8/8 mo/F 9/1 y/M 10/4 y/M 11/11 mo/F 12/6 mo/M
4 5 5 5 6 4 4 4 14 4 4 4
8,300 11,400 9,800 11,600 11,400 14,100 19,400 22,400 18,000 24,000 20,500 10,900
12.90 4.56 2.51 8.65 26.46 6.64 4.31 10.80 21.80 5.83 2.44 9.16
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺
Note. WBC, white blood cell; CRP, C-reactive protein; y, year; mo, month; M, male; F, female.
gel and transferred to a polyvinylidene difluoride membrane. After three washes in TBST (40 mM Tris–HCl, pH 7.6, 300 mM NaCl, and 0.5% Tween 20), the membranes were incubated with rabbit polyclonal antibodies against NF-B-p65 (Santa Cruz Biotechnology, Santa Cruz, CA) in TBST containing 5% nonfat dry milk at room temperature for 1 h. After three washes in TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) for 1 h at room temperature. Immunoreactive proteins were detected by means of enhanced chemiluminescence (Amersham, Arlington Heights, IL) and analyzed by autoradiography. All experiments were repeated twice. Flow Cytometric Analysis
Mononuclear cells were isolated using Histopaque1077 (Sigma Chemical Co., St. Louis, MO) and then washed. Purification of individual cell subpopulations was achieved with a high-magnetic-gradient Mini MACS purification system (Miltenyi, Sunnyvale, CA). CD14 ⫹ monocytes/macrophages were purified using CD14 Microbeads (Miltenyi) and CD3 ⫹ T cells with CD3 Microbeads. The purity of the isolated cells was determined using the respective fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (Becton Dickinson, Mountain View, CA) by flow cytometric analysis (FACScan; Becton Dickinson).
Flow cytometric analysis was performed according to a modification of the previously published procedure (24). Whole blood was labeled with phycoerythrin-conjugated anti-CD14 monoclonal antibodies and peridinin chlorophyll protein (PerCP)-conjugated anti-CD3 monoclonal antibodies and then permeabilized in 4% paraformaldehyde in phosphate-buffered saline, pH 7.2, containing 0.1% saponin and 10 mM Hepes. The cells were then labeled with mouse anti-NF-B (nuclear localized signal) antibodies (IgG3; Boerhinger Mannheim, Mannheim, Germany) or nonspecific mouse IgG3 antibodies (Chemicon, Temecula, CA). The mouse anti-NF-B (nuclear localized signal) antibody recognizes an epitope overlapping the nuclear location signal of the NF-B-p65 and therefore selectively recognizes the activated form of NF-B. The cells were then labeled with FITC-conjugated rat anti-mouse IgG3 monoclonal antibody (Pharmingen, San Diego, CA). After washing, the cells were fixed with 1% paraformaldehyde in PBS and stored at 4°C until flow cytometric analysis. Immunofluorescence staining was analyzed with a FACScan flow cytometer equipped with CellQuest software (Becton Dickinson). All experiments were performed in duplicate.
Nuclear Extracts and Western Blot Analysis
Statistical Analysis
Nuclear extracts were harvested from CD14 ⫹ monocytes/macrophages or CD3 ⫹ T cells using a previously published procedure (23). The protein concentrations of nuclear extracts were determined using Bio-Rad (Hercules, CA) protein concentration reagent. Nuclear extracts were stored at ⫺80°C. The nuclear extracts were used as the sample for Western blotting because activated NF-B existed in the nucleus. To determine the NF-B-p65 level, samples of the nuclear extracts containing 15 g of protein were separated in a denaturing 10% polyacrylamide
The differences in the results between groups were analyzed by the means of the Wilcoxon matched-pairs signed-rank test. Correlations were analyzed using Spearman’s rank correlation coefficient test.
ples after IVIG therapy (subacute stage) were obtained on days 7 to 17 (mean ⫾ SD, 9.78 ⫾ 3.11 days). Samples at the convalescent stage were obtained on days 17 to 61 (mean ⫾ SD, 27.1 ⫾ 13.3 days). The interval between the acute and convalescent samples was 13 to 57 days (mean ⫾ SD, 21.8 ⫾ 13.5 days). Cell Purification
RESULTS
Western blot analysis demonstrated that NF-B activation occurred in CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells from children with KD at the acute stage more than at the convalescent stage (Fig. 1).
NF-B ACTIVATION DURING ACUTE KAWASAKI DISEASE
375
FIG. 2. The percentages of cells with NF-B activation in CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells of patient 6 by flow cytometric analysis. (A) CD3⫹ T cells at the acute stage; (B) CD3⫹ T cells at the convalescent stage; (C) CD14⫹ monocytes/macrophages at the acute stage; (D) CD14⫹ monocytes/macrophages at the convalescent stage.
FIG. 1. Western blot analysis demonstrated that NF-B activation was increased in CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells at the acute stage compared with activation at the convalescent stage and was higher in CD14⫹ monocytes/macrophages than in CD3⫹ T cells at the acute stage. There was no difference between the two experiments. Acute, acute stage; Convalescent, convalescent stage.
NF-B activation was obviously more increased in CD14 ⫹ monocytes/macrophages than in CD3 ⫹ T cells. Flow cytemetry analysis was performed in 9 of the 12 children. Figure 2 shows the results of activated NF-B in the CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells of patient 6. The 9 children exhibited similar results (Fig. 3). NF-B activation in CD14 ⫹ monocytes/ macrophages and CD3 ⫹ T cells at the acute stage was significantly higher than that at the convalescent stage (P ⬍ 0.01 and P ⬍ 0.01, respectively). NF-B activation in CD14 ⫹ monocytes/macrophages was significantly higher than that in CD3 ⫹ T cells at the acute stage (30.0 ⫾ 16.0% vs 11.4 ⫾ 5.0%, P ⬍ 0.01). NF-B activation in CD14 ⫹ monocytes/macrophages was significantly decreased after IVIG therapy (subacute stage) (30.0 ⫾ 16.0% vs 13.7 ⫾ 10.1%, P ⬍ 0.05) (Fig. 3). However, NF-B activation in CD14 ⫹ monocytes/macrophages at the subacute stage after IVIG therapy was still significantly higher than that at the convalescent stage (13.7 ⫾ 10.1% vs 3.3 ⫾ 1.8%, P ⬍ 0.01). The serum C-reactive protein (CRP) level at the acute stage was 9.67 ⫾ 7.53 mg/dl (range, 2.44 –26.46 mg/dl) and at the subacute stage, 1.70 ⫾ 1.47 mg/dl (range, 0.06 –3.36 mg/dl). NF-B activation in CD14 ⫹ monocytes/macrophages at the acute stage was not
correlated with serum CRP level (r ⫽ 0.142). NF-B activation in CD14 ⫹ monocytes/macrophages at the subacute stage after IVIG therapy was correlated with serum CRP level (r ⫽ 0.67, P ⬍ 0.05) (Fig. 4). There was no correlation between white blood cell counts and NF-B activation in CD14 ⫹ macrophages/monocytes. DISCUSSION
The immunological features of monocytes/macrophages observed in patients with KD can be summa-
FIG. 3. The percentages of cells with NF-B activation in CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells at the acute, subacute, and convalescent stages by flow cytometric analysis. *P ⬍ 0.01, compared with the convalescent stage. **P ⬍ 0.05, compared with the subacute stage. ***P ⬍ 0.01, compared with CD3 ⫹ cells.
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FIG. 4. The relationship between the rate of NF-B activation in CD14 ⫹ monocytes/macrophages and serum CRP level. The percentage, of cells with NF-B activation in CD14 ⫹ monocytes/macrophages at the subacute stage after IVIG therapy was correlated with serum CRP level (r ⫽ 0.67, P ⬍ 0.05).
rized as follows: (i) infiltration by the cells is notable in affected tissues in autopsy cases and in skin biopsy specimens from KD patients (25, 26); (ii) the numbers of peripheral blood CD14 ⫹ monocytes/macrophages and activated CD14 ⫹CD23 ⫹ monocytes/macrophages increase during the acute stage of KD (27); (iii) there are elevated levels of a variety of serum cytokines, such as TNF-␣, IL-1, and IL-6, considered to be produced by monocytes/macrophages during acute KD (3–5, 28); (iv) peripheral blood mononuclear cells from patients with acute KD spontaneously secrete high levels of TNF-␣ and IL-1 (29, 30); (v) increases in the number of peripheral blood CD14 ⫹ monocytes/macrophages, serum TNF-␣ level, IL-6 activity in serum, and secretion of IL-1 from mononuclear cells are more evident in KD patients with than in those without CAL (3, 5, 27, 28, 30), and (vi) KD patients with high levels of soluble TNF receptor in their serum seem to be susceptible to CAL even if they receive IVIG therapy (31); and (vii) electron microscopy showed that CD14 ⫹ monocytes/ macrophages from patients with acute KD had nuclei with complex shapes, apparent nucleoli, and abundant intracytoplasmic granules, some of which were positive for acid phosphatase (32). These findings suggest that monocytes/macrophages play an important role in the pathogenesis of KD. However, there has been no report on the mechanism underlying cytokine production in monocytes/ macrophages during acute KD. We demonstrated that NF-B activation occurred in peripheral blood CD14 ⫹ monocytes/macrophages during acute KD by means of Western blot and flow cytometric analyses. The high
percentage of cells with NF-B activation in CD14 ⫹ macrophages/monocytes indicates that CD14 ⫹ macrophages/monocytes produce proinflammatory cytokines, such as TNF-␣, IL-1, and IL-6, that are elevated in the sera of children with KD at the acute stage. The patients with high serum CRP level tended to have high percentages of cells with NF-B activation in CD14 ⫹ macrophages/monocytes. It is likely that NF-B activation in CD14 ⫹ macrophages/monocytes reflects the severity of KD. IVIG therapy has been reported to be effective in reducing the incidence of CAL in patients with KD (33–35). However, the mechanism underlying the effect of IVIG therapy on KD has been unclear. We revealed that NF-B activation in CD14 ⫹ macrophages/monocytes is significantly decreased after IVIG therapy. In addition, NF-B activation in CD14 ⫹ monocytes/macrophages at the subacute stage after IVIG therapy was correlated with serum CRP level. These data suggested that the decrease of NF-B activation after IVIG therapy was related to the decrease of serum CRP level. It is not unlikely that IVIG therapy modulates inflammation through a reduction of NF-B activation in CD14 ⫹ macrophages/monocytes. In summary, NF-B activation was found to occur strongly in CD14 ⫹ monocytes/macrophages in peripheral blood from children during acute KD. REFERENCES 1. Kawasaki, T., Kosaki, F., Osawa, S., Shigemitsu, I., and Yanagawa, S., A new infantile acute febrile mucocutaneous lymph node syndrome (MLNS) prevailing in Japan. Pediatrics 54, 271– 276, 1974. 2. Fujiwara, H., and Hamashima, Y., Pathology of the heart in Kawasaki disease. Pediatrics 61, 100 –107, 1978. 3. Furukawa, S., Matsubara, T., Jujoh, K., Yone, K., Sugawara, T., Sasai, K., Kato, H., and Yabuta, K., Peripheral blood monocyte/ macrophages and serum tumor necrosis factor in Kawasaki disease. Clin. Immunol. Immunopathol. 48, 247–251, 1988 4. Maury, C. P. J., Salo, E., and Pelkonen, P., Circulating interleukin-1 in patients with Kawasaki disease. N. Engl. J. Med. 319, 1670 –1671, 1989. 5. Furukawa, S., Matsubara, T., Yone, K., Hirano, Y., Okumura, K., and Yabuta, K., Kawasaki disease differs from anaphylactoid purpura and measles with regard to tumor necrosis factor-␣ and interleukin 6 in serum. Eur. J. Pediatr. 151, 44 – 47, 1992. 6. Furukawa, S., Imai, K., Matsubara, T., Yone, K., Yachi, A., Okumura, K., and Yabuta, K., Increased levels of circulating intercellular adhesion molecule 1 in Kawasaki disease. Arthritis Rheum. 35, 672– 677, 1992. 7. Lin, C. Y., Lin, C. C., Hwang, B., and Chiang, B., Serial changes of serum interleukin-6, interleukin-8, and tumor necrosis factor ␣ among patients with Kawasaki disease. J. Pediatr. 121, 924 – 926, 1992. 8. Schiller, B., and Elinder, G., Inflammatory parameters and soluble cell adhesion molecules in Swedish children with Kawasaki disease: Relationship to cardiac lesions and intravenous immunoglobulin treatment. Acta Paediatr. 88, 844 – 848, 1999.
NF-B ACTIVATION DURING ACUTE KAWASAKI DISEASE 9. Collart, M. A., Baeuerle, P., and Vassalli, P., Regulation of tumor necrosis factor alpha transcription in macrophages: Involvement of four B-like motifs and of constitutive and inducible forms of NF-B. Mol. Cell. Biol. 10, 1498 –1506, 1990. 10. Libermann, T. A., and Baltimore, D., Activation of interleukin-6 gene expression through the NF-B transcription factor. Mol. Cell. Biol. 10, 2327–2334, 1990. 11. Hiscott, J., Marois, J., Garoufalis, J., D’Addario, M., Roulston, A., Kwan, I., Pepin, N., Lacoste, J., Nguyen, H., Bensi, G., and Fenton, M., Characterization of a functional NF-B site in the human interleukin 1 promotor: Evidence for a positive autoregulatory loop. Mol. Cell. Biol. 13, 6231– 6240, 1993. 12. Matsusaka, T., Fujikawa, K., Nishio, Y., Mukaida, N., Matsushima, K., Kishimoto, T., and Akira, S., Transcription factors NF-IL6 and NF-B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc. Natl. Acad. Sci. USA 90, 10193–10197, 1993. 13. Kunsch, C., Lang, R. K., Rosen, C. A., and Shannon, M. F., Synergistic transcriptional activation of the IL-8 gene by NF-B p65 (RelA) and NF-IL-6. J. Immunol. 153, 153–164, 1994. 14. Chen, C. C., Rosenbloom, C. L., Anderson, D. C., and Manning, A. M., Selective inhibition of E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression by inhibitors of IB-␣ phosphorylation. J. Immunol. 155, 3538 – 3545, 1995. 15. Ueda, A., Ishigatsubo, Y., Okubo, T., and Yoshimura, T., Transcriptional regulation of the human monocyte chemoattractant protein-1 gene. J. Biol. Chem. 272, 31092–31099, 1997. 16. Baeuerle, P. A., and Henkel, T., Function and activation of NF-B in the immune system. Annu. Rev. Immunol. 12, 141– 179, 1994. 17. Baldwin, A. S., Jr., The NF-B and IB proteins: New discoveries and insights. Annu. Rev. Immunol. 14, 649 – 683, 1996. 18. Grimm, S., and Baeurle, P. A., The inducible transcription factor NF-B: Structure–function relationship of its protein subunits. Biochem. J. 290, 297–308, 1993. 19. Henkel, T., Machleidt, T., Alkalay, I., Kro¨nke, M., Ben-Neriah, Y., and Baeuerle, P. A., Rapid proteolysis of IB-␣ is necessary for activation of transcriptional factor NF-B. Nature 365, 182– 185, 1993. 20. Brown, K., Gerstberger, S., Carlson, S. L., Franzoso, L. G., and Siebenlist, U., Control of IB-␣ proteolysis by site-specific, signal-induced phosphorylation. Science 267, 1485–1488, 1995. 21. Kumar, A., Haque, J., Lacoste, J., Hiscott, J., and Williams, B. R. G., Double-stranded RNA-dependent protein kinase activates transcription factor NF-B by phosphorylating IB. Proc. Natl. Acad. Sci. USA 91, 6288 – 6292, 1994. 22. Research Committee on Kawasaki Disease, “Report of Subcommittee on Standization of Diagnosic Criteria and Reporting of Coronary Artery Lesions in Kawasaki Disease,” pp. 55– 66, Ministry of Health and Welfare, Tokyo, 1984. 23. Ichiyama, T., Zhao, H., Catania, A., Furukawa, S., and Lipton, J. M., ␣-Melanocyte-stimulating hormone inhibits NF-B activaReceived December 13, 2000; accepted with revision March 2, 2001
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NF-B Activation in Peripheral Blood Monocytes/Macrophages and T Cells during Acute Kawasaki Disease Takashi Ichiyama, Tomomi Yoshitomi, Miki Nishikawa, Motoki Fujiwara, Tomoyo Matsubara, Takashi Hayashi, and Susumu Furukawa Department of Pediatrics, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan
Kawasaki disease (KD) is a febrile disease of childhood characterized by systemic vasculitis, and the levels of many proinflammatory cytokines are elevated in the serum at the acute stage. We investigated the activation of transcription factor NF-B for genes that encode the proinflammatory cytokines in CD14 ⴙ monocytes/macrophages and CD3 ⴙ T cells in peripheral blood by means of Western blot and flow cytometric analyses. Western blot analysis demonstrated that NF-B activation was more increased in CD14 ⴙ monocytes/macrophages than in CD3 ⴙ T cells in all children during the acute stage. Flow cytometric analysis revealed that NF-B activation in CD14 ⴙ monocytes/ macrophages was significantly higher than in CD3 ⴙ T cells at the acute stage (30.0 ⴞ 16.0% vs 11.4 ⴞ 5.0%, P < 0.01, Wilcoxon test). NF-B activation in CD14 ⴙ monocytes/macrophages was significantly decreased after high-dose intravenous immunoglobulin therapy (P < 0.05). The present findings suggest that CD14 ⴙ monocytes/macrophages play an important role in cytokine production during acute KD. © 2001 Academic Press Key Words: Kawasaki disease; NF-B activation; CD14 ⴙ cells; CD3 ⴙ cells.
INTRODUCTION
Kawasaki disease (KD) is an acute illness of early childhood characterized by prolonged fever, diffuse mucosal inflammation, indurative edema of the hands and feet, a polymorphous skin rash, and nonsuppurative lymphadenopathy (1). The histopathological findings in KD comprise panvasculitis with endothelial necrosis and the infiltration of mononuclear cells into small and medium-sized blood vessels (2). The levels of any proinflammatory cytokines, chemokines, and adhesion molecules are elevated in sera from children with KD at the acute stage (3– 8). However, the mechanism underlying cytokine production in immunocompetent cells during acute KD has been unclear. Nuclear factor kappa B (NF-B) is a pivotal transcription factor for genes that encode the proinflammatory cytokines, chemokines, and adhesion molecules
that mediate inflammation: interleukin 1 (IL-1), IL-2, IL-6, IL-8, tumor necrosis factor-␣ (TNF-␣), monocyte chemoattractant protein-1, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 (9 –15). NF-B is retained in an inactive form in the cytoplasm (16, 17); its prototypic form consists of a heterodimer of p50 and p65 that is normally bound by members of the IB family (17). Activation of NF-B requires degradation of the cytoplasmic inhibitor IB protein (18, 19). Phosphorylation of IB by drugs, cytokines, bacterial products, and viruses leads to IB degradation, translocation of NF-B to the nucleus, and transcription of proinflammatory cytokine genes (20, 21). We examined whether NF-B activation occurs in peripheral blood CD14 ⫹ monocytes/macrophages or CD3 ⫹ T cells to clarify the mechanism of cytokine production during acute KD. PATIENTS AND METHODS
Patients Twelve children (four girls and eight boys; aged from 2 months to 4 years; mean age, 1.6 years) with acute KD upon admission to our hospital between April 1999 and February 2000 were included in this study (Table 1). Coronary arteries exhibiting diameters of 4 mm or greater on two-dimensional echocardiography were regarded as having coronary artery lesions (CAL), according to the Research Committee on Kawasaki Disease (Ministry of Health and Welfare, Japan) diagnostic criteria for cardiovascular lesions with KD (22). 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 to be the first day of illness. All patients received standard Japanese treatment with high-dose intravenous immunoglobulin (IVIG; Venilon, Teijin Co., Ltd., Tokyo, Japan), 1000 mg/kg/day for 2 days, and oral aspirin (30 mg/kg/day). Samples at the acute stage were obtained on days 4 to 14 (mean ⫾ SD, 5.3 ⫾ 2.8 days) before treatment with IVIG. Sam-
373
1521-6616/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
374
ICHIYAMA ET AL.
TABLE 1 Data for 12 Children with Kawasaki Disease Patients no./age/sex
Sampling days
WBC counts (/mm 3)
Serum CRP levels (mg/dl)
Coronary artery lesions
1/4 y/M 2/2 y/F 3/4 mo/M 4/1 y/M 5/1 y/F 6/2 mo/M 7/7 mo/M 8/8 mo/F 9/1 y/M 10/4 y/M 11/11 mo/F 12/6 mo/M
4 5 5 5 6 4 4 4 14 4 4 4
8,300 11,400 9,800 11,600 11,400 14,100 19,400 22,400 18,000 24,000 20,500 10,900
12.90 4.56 2.51 8.65 26.46 6.64 4.31 10.80 21.80 5.83 2.44 9.16
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺
Note. WBC, white blood cell; CRP, C-reactive protein; y, year; mo, month; M, male; F, female.
gel and transferred to a polyvinylidene difluoride membrane. After three washes in TBST (40 mM Tris–HCl, pH 7.6, 300 mM NaCl, and 0.5% Tween 20), the membranes were incubated with rabbit polyclonal antibodies against NF-B-p65 (Santa Cruz Biotechnology, Santa Cruz, CA) in TBST containing 5% nonfat dry milk at room temperature for 1 h. After three washes in TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) for 1 h at room temperature. Immunoreactive proteins were detected by means of enhanced chemiluminescence (Amersham, Arlington Heights, IL) and analyzed by autoradiography. All experiments were repeated twice. Flow Cytometric Analysis
Mononuclear cells were isolated using Histopaque1077 (Sigma Chemical Co., St. Louis, MO) and then washed. Purification of individual cell subpopulations was achieved with a high-magnetic-gradient Mini MACS purification system (Miltenyi, Sunnyvale, CA). CD14 ⫹ monocytes/macrophages were purified using CD14 Microbeads (Miltenyi) and CD3 ⫹ T cells with CD3 Microbeads. The purity of the isolated cells was determined using the respective fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (Becton Dickinson, Mountain View, CA) by flow cytometric analysis (FACScan; Becton Dickinson).
Flow cytometric analysis was performed according to a modification of the previously published procedure (24). Whole blood was labeled with phycoerythrin-conjugated anti-CD14 monoclonal antibodies and peridinin chlorophyll protein (PerCP)-conjugated anti-CD3 monoclonal antibodies and then permeabilized in 4% paraformaldehyde in phosphate-buffered saline, pH 7.2, containing 0.1% saponin and 10 mM Hepes. The cells were then labeled with mouse anti-NF-B (nuclear localized signal) antibodies (IgG3; Boerhinger Mannheim, Mannheim, Germany) or nonspecific mouse IgG3 antibodies (Chemicon, Temecula, CA). The mouse anti-NF-B (nuclear localized signal) antibody recognizes an epitope overlapping the nuclear location signal of the NF-B-p65 and therefore selectively recognizes the activated form of NF-B. The cells were then labeled with FITC-conjugated rat anti-mouse IgG3 monoclonal antibody (Pharmingen, San Diego, CA). After washing, the cells were fixed with 1% paraformaldehyde in PBS and stored at 4°C until flow cytometric analysis. Immunofluorescence staining was analyzed with a FACScan flow cytometer equipped with CellQuest software (Becton Dickinson). All experiments were performed in duplicate.
Nuclear Extracts and Western Blot Analysis
Statistical Analysis
Nuclear extracts were harvested from CD14 ⫹ monocytes/macrophages or CD3 ⫹ T cells using a previously published procedure (23). The protein concentrations of nuclear extracts were determined using Bio-Rad (Hercules, CA) protein concentration reagent. Nuclear extracts were stored at ⫺80°C. The nuclear extracts were used as the sample for Western blotting because activated NF-B existed in the nucleus. To determine the NF-B-p65 level, samples of the nuclear extracts containing 15 g of protein were separated in a denaturing 10% polyacrylamide
The differences in the results between groups were analyzed by the means of the Wilcoxon matched-pairs signed-rank test. Correlations were analyzed using Spearman’s rank correlation coefficient test.
ples after IVIG therapy (subacute stage) were obtained on days 7 to 17 (mean ⫾ SD, 9.78 ⫾ 3.11 days). Samples at the convalescent stage were obtained on days 17 to 61 (mean ⫾ SD, 27.1 ⫾ 13.3 days). The interval between the acute and convalescent samples was 13 to 57 days (mean ⫾ SD, 21.8 ⫾ 13.5 days). Cell Purification
RESULTS
Western blot analysis demonstrated that NF-B activation occurred in CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells from children with KD at the acute stage more than at the convalescent stage (Fig. 1).
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FIG. 2. The percentages of cells with NF-B activation in CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells of patient 6 by flow cytometric analysis. (A) CD3⫹ T cells at the acute stage; (B) CD3⫹ T cells at the convalescent stage; (C) CD14⫹ monocytes/macrophages at the acute stage; (D) CD14⫹ monocytes/macrophages at the convalescent stage.
FIG. 1. Western blot analysis demonstrated that NF-B activation was increased in CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells at the acute stage compared with activation at the convalescent stage and was higher in CD14⫹ monocytes/macrophages than in CD3⫹ T cells at the acute stage. There was no difference between the two experiments. Acute, acute stage; Convalescent, convalescent stage.
NF-B activation was obviously more increased in CD14 ⫹ monocytes/macrophages than in CD3 ⫹ T cells. Flow cytemetry analysis was performed in 9 of the 12 children. Figure 2 shows the results of activated NF-B in the CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells of patient 6. The 9 children exhibited similar results (Fig. 3). NF-B activation in CD14 ⫹ monocytes/ macrophages and CD3 ⫹ T cells at the acute stage was significantly higher than that at the convalescent stage (P ⬍ 0.01 and P ⬍ 0.01, respectively). NF-B activation in CD14 ⫹ monocytes/macrophages was significantly higher than that in CD3 ⫹ T cells at the acute stage (30.0 ⫾ 16.0% vs 11.4 ⫾ 5.0%, P ⬍ 0.01). NF-B activation in CD14 ⫹ monocytes/macrophages was significantly decreased after IVIG therapy (subacute stage) (30.0 ⫾ 16.0% vs 13.7 ⫾ 10.1%, P ⬍ 0.05) (Fig. 3). However, NF-B activation in CD14 ⫹ monocytes/macrophages at the subacute stage after IVIG therapy was still significantly higher than that at the convalescent stage (13.7 ⫾ 10.1% vs 3.3 ⫾ 1.8%, P ⬍ 0.01). The serum C-reactive protein (CRP) level at the acute stage was 9.67 ⫾ 7.53 mg/dl (range, 2.44 –26.46 mg/dl) and at the subacute stage, 1.70 ⫾ 1.47 mg/dl (range, 0.06 –3.36 mg/dl). NF-B activation in CD14 ⫹ monocytes/macrophages at the acute stage was not
correlated with serum CRP level (r ⫽ 0.142). NF-B activation in CD14 ⫹ monocytes/macrophages at the subacute stage after IVIG therapy was correlated with serum CRP level (r ⫽ 0.67, P ⬍ 0.05) (Fig. 4). There was no correlation between white blood cell counts and NF-B activation in CD14 ⫹ macrophages/monocytes. DISCUSSION
The immunological features of monocytes/macrophages observed in patients with KD can be summa-
FIG. 3. The percentages of cells with NF-B activation in CD14 ⫹ monocytes/macrophages and CD3 ⫹ T cells at the acute, subacute, and convalescent stages by flow cytometric analysis. *P ⬍ 0.01, compared with the convalescent stage. **P ⬍ 0.05, compared with the subacute stage. ***P ⬍ 0.01, compared with CD3 ⫹ cells.
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FIG. 4. The relationship between the rate of NF-B activation in CD14 ⫹ monocytes/macrophages and serum CRP level. The percentage, of cells with NF-B activation in CD14 ⫹ monocytes/macrophages at the subacute stage after IVIG therapy was correlated with serum CRP level (r ⫽ 0.67, P ⬍ 0.05).
rized as follows: (i) infiltration by the cells is notable in affected tissues in autopsy cases and in skin biopsy specimens from KD patients (25, 26); (ii) the numbers of peripheral blood CD14 ⫹ monocytes/macrophages and activated CD14 ⫹CD23 ⫹ monocytes/macrophages increase during the acute stage of KD (27); (iii) there are elevated levels of a variety of serum cytokines, such as TNF-␣, IL-1, and IL-6, considered to be produced by monocytes/macrophages during acute KD (3–5, 28); (iv) peripheral blood mononuclear cells from patients with acute KD spontaneously secrete high levels of TNF-␣ and IL-1 (29, 30); (v) increases in the number of peripheral blood CD14 ⫹ monocytes/macrophages, serum TNF-␣ level, IL-6 activity in serum, and secretion of IL-1 from mononuclear cells are more evident in KD patients with than in those without CAL (3, 5, 27, 28, 30), and (vi) KD patients with high levels of soluble TNF receptor in their serum seem to be susceptible to CAL even if they receive IVIG therapy (31); and (vii) electron microscopy showed that CD14 ⫹ monocytes/ macrophages from patients with acute KD had nuclei with complex shapes, apparent nucleoli, and abundant intracytoplasmic granules, some of which were positive for acid phosphatase (32). These findings suggest that monocytes/macrophages play an important role in the pathogenesis of KD. However, there has been no report on the mechanism underlying cytokine production in monocytes/ macrophages during acute KD. We demonstrated that NF-B activation occurred in peripheral blood CD14 ⫹ monocytes/macrophages during acute KD by means of Western blot and flow cytometric analyses. The high
percentage of cells with NF-B activation in CD14 ⫹ macrophages/monocytes indicates that CD14 ⫹ macrophages/monocytes produce proinflammatory cytokines, such as TNF-␣, IL-1, and IL-6, that are elevated in the sera of children with KD at the acute stage. The patients with high serum CRP level tended to have high percentages of cells with NF-B activation in CD14 ⫹ macrophages/monocytes. It is likely that NF-B activation in CD14 ⫹ macrophages/monocytes reflects the severity of KD. IVIG therapy has been reported to be effective in reducing the incidence of CAL in patients with KD (33–35). However, the mechanism underlying the effect of IVIG therapy on KD has been unclear. We revealed that NF-B activation in CD14 ⫹ macrophages/monocytes is significantly decreased after IVIG therapy. In addition, NF-B activation in CD14 ⫹ monocytes/macrophages at the subacute stage after IVIG therapy was correlated with serum CRP level. These data suggested that the decrease of NF-B activation after IVIG therapy was related to the decrease of serum CRP level. It is not unlikely that IVIG therapy modulates inflammation through a reduction of NF-B activation in CD14 ⫹ macrophages/monocytes. In summary, NF-B activation was found to occur strongly in CD14 ⫹ monocytes/macrophages in peripheral blood from children during acute KD. REFERENCES 1. Kawasaki, T., Kosaki, F., Osawa, S., Shigemitsu, I., and Yanagawa, S., A new infantile acute febrile mucocutaneous lymph node syndrome (MLNS) prevailing in Japan. Pediatrics 54, 271– 276, 1974. 2. Fujiwara, H., and Hamashima, Y., Pathology of the heart in Kawasaki disease. Pediatrics 61, 100 –107, 1978. 3. Furukawa, S., Matsubara, T., Jujoh, K., Yone, K., Sugawara, T., Sasai, K., Kato, H., and Yabuta, K., Peripheral blood monocyte/ macrophages and serum tumor necrosis factor in Kawasaki disease. Clin. Immunol. Immunopathol. 48, 247–251, 1988 4. Maury, C. P. J., Salo, E., and Pelkonen, P., Circulating interleukin-1 in patients with Kawasaki disease. N. Engl. J. Med. 319, 1670 –1671, 1989. 5. Furukawa, S., Matsubara, T., Yone, K., Hirano, Y., Okumura, K., and Yabuta, K., Kawasaki disease differs from anaphylactoid purpura and measles with regard to tumor necrosis factor-␣ and interleukin 6 in serum. Eur. J. Pediatr. 151, 44 – 47, 1992. 6. Furukawa, S., Imai, K., Matsubara, T., Yone, K., Yachi, A., Okumura, K., and Yabuta, K., Increased levels of circulating intercellular adhesion molecule 1 in Kawasaki disease. Arthritis Rheum. 35, 672– 677, 1992. 7. Lin, C. Y., Lin, C. C., Hwang, B., and Chiang, B., Serial changes of serum interleukin-6, interleukin-8, and tumor necrosis factor ␣ among patients with Kawasaki disease. J. Pediatr. 121, 924 – 926, 1992. 8. Schiller, B., and Elinder, G., Inflammatory parameters and soluble cell adhesion molecules in Swedish children with Kawasaki disease: Relationship to cardiac lesions and intravenous immunoglobulin treatment. Acta Paediatr. 88, 844 – 848, 1999.
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