Expression of CD40 Ligand on CD4؉ T-Cells and Platelets Correlated to the Coronary Artery Lesion and Disease Progress in Kawasaki Disease Chih-Lu Wang, MD*; Yu-Tsun Wu, MD‡; Chieh-An Liu, MD*; Mei-Wei Lin, BS*; Chia-Jung Lee, BS*; Li-Tung Huang, MD*; and Kuender D. Yang, MD, PhD* ABSTRACT. Objective. Kawasaki disease (KD) is an awasaki disease (KD) is an acute multisystem acute febrile vasculitic syndrome in children. CD40 li- vasculitic syndrome of unknown cause that gand (CD40L) has been implicated in certain types of occurs in infants and children.1 Evidence in- vasculitis. We proposed that CD40L expression might be K creasingly suggests that immunoregulatory activa- correlated with coronary artery lesions in KD. tion with vascular endothelial inflammation may be Methods. Blood samples were collected from 43 pa- 2 tients with KD before intravenous immunoglobulin involved in the immunopathogenesis of KD. The (IVIG) treatment and 3 days afterward. Forty-three age- acute stage of KD is associated with overactivation of matched febrile children with various diseases were numerous immunologic parameters, such as im- studied in parallel as controls. CD40L expression on T- mune-competent cell activation,3–5 cytokines,6 nitric cells and platelets were detected by flow cytometry, and oxide production,7 autoantibody production,8 and soluble CD40L (sCD40L) levels were measured by en- adhesion molecule expression.9 Pathologic examina- zyme-linked immunosorbent assay. ؉ tion of acute coronary arteritis in the acute stage of Results. We found that CD40L expression on CD4 KD showed that KD vascular lesion formation is an T-cells was significantly higher in patients with KD than -in the febrile control (FC) group (28.69 ؎ 1.17% vs 4.37 ؎ activated T-lymphocyte–dependent process charac 0.36%). CD40L expression decreased significantly 3 days terized by transmural infiltration of activated T-lym- ϩ 10 .after IVIG administration (28.69 ؎ 1.17% vs 13.53 ؎ phocytes, with CD8 T-cell predominant 0.55%). CD40L expression on platelets from patients with CD40 ligand (CD40L, CD154, gp 39), a transmem- KD was also significantly higher than in the FC group brane protein structurally related to tumor necrosis vs 1.26 ؎ 0.12%) and decreased after IVIG factor-␣, was originally identified on activated CD4ϩ 0.41% ؎ 8.20) therapy. sCD40L levels were also significantly higher in T-cells. Both membrane-bound and soluble forms of ؎ ؎ KD patients with those of FC (9.69 0.45 ng/mL vs 2.25 this ligand may interact with CD40, which is consti- 0.19 ng/mL) but were not affected by IVIG treatment 3 tutively expressed on B-cells, macrophages, endothe- .(days afterward (9.69 ؎ 0.45 ng/mL vs 9.03 ؎ 0.32 ng/mL More interesting, we found that in KD patients, CD40L lial cells, and vascular smooth muscle cells, resulting 11 .expression on CD4؉ T-cells and platelets but not on in various immune and inflammatory responses CD8؉ T-cells or sCD40L was correlated with the occur- Interaction of CD40L and CD40 plays a central role rence of coronary artery lesions. in the activation of the immune system, such as Conclusions. CD40L might play a role in the immuno- immunoglobulin G (IgG) switching, autoimmune pathogenesis of KD. IVIG therapy might downregulate disease, antiviral effect, allograft rejection, cytokines CD40L expression, resulting in decrease of CD40L-medi- regulation, and arthrosclerosis, as well as endothelial ated vascular damage in KD. This implicates that modula- cell interaction.12 CD40L was also recently found on tion of CD40L expression may benefit to treat KD vasculitis. Pediatrics 2003;111:e140–e147. URL: http://www. activated platelets, which induce endothelial cells to pediatrics.org/cgi/content/full/111/2/e140; Kawasaki disease, secrete chemokines and to express adhesion mole- intravenous immunoglobulin, CD40 ligand, soluble CD40L. cules, for the recruitment of inflammatory cells caus- ing endothelial cell damage.13 Furthermore, Aukrust et al14 showed that levels of the soluble and the ABBREVIATIONS. KD, Kawasaki disease; CD40L, CD40 ligand; IgG, immunoglobulin G; sCD40L, soluble CD40L; FC, febrile con- membrane-bound form of CD40L were enhanced in trols; IVIG, intravenous immunoglobulin; CAL, coronary artery angina patients, suggesting that CD40L–CD40 inter- lesions; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PBS, action may play a pathogenic role in both the trig- phosphate-buffered saline; PRP, platelet-rich plasma; IL, interleu- gering and the propagation of acute coronary syn- kin; TSST-1, toxic shock syndrome toxin-1. dromes. Taken together, CD40L–CD40 interaction may not only contribute to overactivation of the im- mune system but also may be responsible for directly From the Divisions of *Allergy and Immunology and ‡Cardiology, Chang- triggering KD vasculitis syndrome and possibly even Gung Children’s Hospital at Kaohsiung, Chang-Gung University, Kaohsi- ung, Taiwan. acute coronary dysfunction in KD. Thus, we hypoth- Received for publication Jul 3, 2002; accepted Oct 2, 2002. esize that CD40L expression might be involved in the Reprint requests to (K.D.Y.) Office of Vice Superintendents, 123 Ta-Pei immunopathogenesis of KD. We assessed mem- Road, Chang Gung Children’s Hospital, Niau-Sung, Kaohsiung 833, Tai- brane-bound CD40L expression on T-lymphocytes wan. E-mail: [email protected] and platelets as well as serum-soluble CD40L Drs Wang and Wu contributed equally to this study. PEDIATRICS (ISSN 0031 4005). Copyright © 2003 by the American Acad- (sCD40L) in patients with KD and correlated its ex- emy of Pediatrics. pression to disease severity and progress. e140 PEDIATRICS Vol.Downloaded 111 No. 2 from February www.aappublications.org/news 2003 http://www.pediatrics.org/cgi/content/full/111/2/ by guest on September 28, 2021 e140 TABLE 1. Demographic Data of FC and Patients With KD Before IVIG Therapy KD FC P Value (n ϭ 43) (n ϭ 43)* Age (mo) 17.5 Ϯ 1.8 21.7 Ϯ 2.7 NS Duration of fever (d) 6.7 Ϯ 0.9 5.2 Ϯ 0.7 NS Leukocyte counts (ϫ103 cells/mm3) 17.4 Ϯ 1.3 15.5 Ϯ 1.6 NS CRP (mg/dL) 76.3 Ϯ 5.9 81.3 Ϯ 6.5 NS Platelet counts (ϫ104 cells/mm3) 34.3 Ϯ 3.6 30.5 Ϯ 3.1 NS NS indicates not significant. * The 43 FC consisted of lobar pneumonia (n ϭ 8), urinary tract infection (n ϭ 6), acute gastroenteritis (n ϭ 8), hand-foot-mouth disease (n ϭ 6), anaphylactoid purpura (n ϭ 5), and simple upper respiratory tract infections (n ϭ 10). METHODS Measurement of sCD40L Levels Patients and Samples Studied Levels of sCD40L were determined by enzyme-linked immu- nosorbent assay (detection limit: 0.095 ng/mL; Chemicon Corp, Children who were admitted to Chang Gung Children’s Hos- Temecula, CA) according to the manufacturer’s instructions.14,18 pital at Kaohsiung with the diagnosis of KD were enrolled in this study after informed consent was obtained. The treatment proto- col was followed with the recommendation of the Committee on Statistical Analysis Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Results were expressed as mean Ϯ standard error of the mean. Cardiovascular Disease in the Young, American Heart Associa- When 3 groups of individuals were compared, repeated measure- tion.15 Forty-three patients with KD and 43 age-matched febrile ment by analysis of variance was used as the test for statistical controls (FCs) were studied in parallel. Blood samples from FC significance. The calculations were performed using the Statistical and from KD patients before and 3 days after intravenous immu- Package for Social Science (SPSS, version 8; SPSS Inc, Chicago, IL) noglobulin (IVIG) therapy were collected. Serum samples were software package. Statistical significance was achieved at P Ͻ .05. stored at Ϫ80°C until analysis. Blood leukocytes and platelets We used Window Multiple Document Interface for Flow cytom- were subject to flow cytometric analysis. All of the KD and FC etry (version 2.8; Scripps Research Institute, La Jolla, CA) to over- leukocytes were studied within 6 hours in parallel in each pa- lay histograms of different stages in patients with KD as well as tient’s assay. FC to better present difference of the CD40L expression on T-cells All patients with KD received 2-dimensional echocardiogram before and after treatment. examinations by a pediatric cardiologist. Coronary artery lesion Ͼ (CAL) was defined by internal diameter of coronary artery 3 RESULTS mm.3,15 Demographic Data of the Patients Studied ؉ ؉ Detection of CD40L Expression on CD4 and CD8 As shown in Table 1, 43 patients with KD and 43 T-Cells age-matched FCs were simultaneously studied. FCs Peripheral venous blood was drawn into sterile tubes contain- include lobar pneumonia (n ϭ 8), urinary tract infec- ing heparin (Becton Dickinson, Heidelberg, Germany). Within 1 ϭ ϭ hour, 200 ␮L of whole blood was mixed with 20 ␮L of appropriate tion (n 6), acute gastroenteritis (n 8), hand-foot- monoclonal antibody conjugates for 30 minutes (4°C in darkness). mouth disease (n ϭ 6), anaphylactoid purpura (He- The following antibodies were used for staining: anti-CD3 peridin noch-Schoenlein purpura, n ϭ 5), and those with chlorophyll protein, anti-CD4 and anti-CD8 fluorescein isothio- simple upper respiratory tract infections (n ϭ 10). cyanate (FITC) (all from Becton Dickinson), and CD40L phyco- There was no difference of demographic data be- erythrin (PE) (Ancell Croup, Bayport, MN). Isotype-matched FITC- and PE-conjugated mouse IgG1 (Pharmigen, San Diego, tween the 2 groups including age, duration of fever, CA) were used as negative controls. We used a protein kinase C leukocyte counts, C-reactive protein, and platelet activator phorbol myristate acetate (32 nM) and calcium iono- counts. phore (A23187; 1 ␮g/mL) to stimulate CD40L expression on CD4ϩ ϩ and CD8 T-cells.