Tomoyo Matsubara

Serum levels of tumor necrosis factor, interleukin 2 receptor, and interferon-γ in Kawasaki disease involved coronary-artery lesions☆

We investigated 45 patients with Kawasaki disease (KD) and report the first simultaneous determination of tumor necrosis factor (TNF), interleukin 2 receptor (IL-2R) and interferon-gamma (IFN-gamma) in the serum during acute phase. Serum levels of TNF were measured by a sandwich enzyme-linked immunosorbent assay. Serum levels of soluble IL-2R and IFN-gamma were measured by a sandwich enzyme immunoassay and radioimmunoassay, respectively. Serum levels of TNF, IL-2R, and IFN-gamma were seen to increase during the acute phase of KD. In KD patients with coronary-artery lesions (CAL), the percentage of positive cases for TNF (greater than or equal to 10 U/ml), IL-2R (greater than or equal to 1056 U/ml), and IFN-gamma (greater than or equal to 0.3 U/ml) was higher than that in patients without CAL. Our results suggest that aggressive activation of immunocompetent cells develops in KD with CAL.

Peripheral blood monocyte/macrophages and serum tumor necrosis factor in Kawasaki disease

We analyzed the populations of peripheral blood monocyte/macrophages in 27 patients using a fluorescence-activated cell sorter, and investigated the possibility, in another 30 patients, that tumor necrosis factor (TNF) might be detectable in serum during the acute phase of Kawasaki disease (KD). Percentages of peripheral blood monocyte/macrophages among mononuclear cells and serum TNF levels were both seen to increase during the acute phase of the illness in patients with KD. The percentage of TNF positive cases in KD patients with coronary involvement was higher than that of patients without coronary involvement. These results suggest the possibility that immunological activation, accompanied by the secretion of TNF from monocyte/macrophages, is an important predisposing condition for the exacerbation of vascular damage in KD.

Kawasaki Disease: A Brief History

Tomisaku Kawasaki published the first English-language report of 50 patients with Kawasaki disease (KD) in 1974. Since that time, KD has become the leading cause of acquired heart disease among children in North America and Japan. Although an infectious agent is suspected, the cause remains unknown. However, significant progress has been made toward understanding the natural history of the disease and therapeutic interventions have been developed that halt the immune-mediated destruction of the arterial wall. We present a brief history of KD, review progress in research on the disease, and suggest avenues for future study. Kawasaki saw his first case of KD in January 1961 and published his first report in Japanese in 1967. Whether cases existed in Japan before that time is currently under study. The most significant controversy in the 1960s in Japan was whether the rash and fever sign/symptom complex described by Kawasaki was connected to subsequent cardiac complications in a number of cases. Pathologist Noboru Tanaka and pediatrician Takajiro Yamamoto disputed the early assertion of Kawasaki that KD was a self-limited illness with no sequelae. This controversy was resolved in 1970 when the first Japanese nationwide survey of KD documented 10 autopsy cases of sudden cardiac death after KD. By the time of the first English-language publication by Kawasaki in 1974, the link between KD and coronary artery vasculitis was well-established. KD was independently recognized as a new and distinct condition in the early 1970s by pediatricians Marian Melish and Raquel Hicks at the University of Hawaii. In 1973, at the same Hawaiian hospital, pathologist Eunice Larson, in consultation with Benjamin Landing at Los Angeles Childrens Hospital, retrospectively diagnosed a 1971 autopsy case as KD. The similarity between KD and infantile periarteritis nodosa (IPN) was apparent to these pathologists, as it had been to Tanaka earlier. What remains unknown is the reason for the simultaneous recognition of this disease around the world in the 1960s and 1970s. There are several possible explanations. KD may have been a new disease that emerged in Japan and emanated to the Western World through Hawaii, where the disease is prevalent among Asian children. Alternatively, KD and IPN may be part of the spectrum of the same disease and clinically mild KD masqueraded as other diseases, such as scarlet fever in the preantibiotic era. Case reports of IPN from Western Europe extend back to at least the 19th century, but, thus far, cases of IPN have not been discovered in Japan before World War II. Perhaps the factors responsible for KD were introduced into Japan after the World War II and then reemerged in a more virulent form that subsequently spread through the industrialized Western world. It is also possible that improvements in health care and, in particular, the use of antibiotics to treat infections caused by organisms including toxin-producing bacteria reduced the burden of rash/fever illness and allowed KD to be recognized as a distinct clinical entity. Itsuzo Shigematsu, Hiroshi Yanagawa, and colleagues have conducted 14 nationwide surveys in Japan. These have indicated that: 1) KD occurred initially in nationwide epidemics but now occurs in regional outbreaks; 2) there are ∼5000 to 6000 new cases each year; 3) current estimates of incidence rates are 120 to 150 cases per 100 000 children <5 years old; 4) KD is 1.5 times more common in males and 85% of cases occur in children <5 years old; and 5) the recurrence rate is low (4%). In 1978, David Morens at the Centers for Disease Control and Prevention published a case definition based on Kawasakis original criteria. The Centers for Disease Control and Prevention developed a computerized database in 1984, and a passive reporting system currently exists in 22 states. Regional investigations and national surveys suggest an annual incidence of 4 to 15 cases per 100 000 children <5 years of age in the United States. The natural history of KD reveals that coronary artery aneurysms occur as a sequela of the vasculitis in 20% to 25% of untreated children. Echocardiography can be successfully used to detect coronary artery dilatation and aneurysms in virtually all patients. Patients with no acute phase coronary artery changes detected by echocardiogram are clinically asymptomatic at least 10 years later. The Japanese Ministry of Health has established a registry of 6500 children who will be followed longitundinally to determine the natural history of the illness. No similar registry of patients exists in the United States. Studies of KD pathogenesis show a progression of arterial lesions accompanying KD vasculitis and a number of immunoregulatory changes, including a deficiency of circulating CD8+ suppressor/cytotoxic T cells; an abundance of circulating B cells spontaneously producing immunoglobulins; and circulating, activated monocytes. Biochemical and immunologic evidence suggests endothelial cell activation and injury. Although the cause of KD remains unknown, clinical trials have established effective therapies, despite the absence of a proven cause. Intravenous immunoglobulin (IVIG) plus aspirin lowers the rate of coronary artery aneurysms from 20% to between 3% and 5%. In 1988, the Committee on Infectious Diseases of the American Academy of Pediatrics endorsed IVIG treatment as recommended therapy for KD. Questions remain regarding treatment of patients who fail to respond to an initial dose of IVIG. The role of steroids or other antiinflammatory agents in the treatment of KD is controversial. Areas for further research include: 1) a more sensitive case definition that includes laboratory and echocardiographic data, as well as clinical signs and symptoms; 2) development of a diagnostic test based on the biology of inflammation and acute endothelial cell damage that, in the absence of the causative agent, could be used to identify children with KD; 3) studies of index cases and their families to identify relevant genetic factors; and 4) long-term follow-up of patients into their third and fourth decades with monitoring for late cardiovascular sequelae.

Sodium valproate inhibits production of TNF-α and IL-6 and activation of NF-κB

Sodium valproate (VPA) is frequently used to treat epilepsy and convulsive disorders. Several reports have indicated that anti-epileptic drugs (AED) affect the immune system, but the mechanism has not been clear. We examined whether the commonly used AEDs, diazepam (DZP), carbamazepine (CBZ), phenobarbital (PB), phenytoin (PHT), and VPA, can inhibit activation of the nuclear transcription factor kappa B (NF-kappaB), in human monocytic leukemia cells (THP-1) and in human glioma cells (A-172). NF-kappaB is essential to the expression of the kappa light chain of immunoglobulin and proinflammatory cytokines. Electrophoretic mobility shift assays (EMSA) of nuclear extracts demonstrated that VPA inhibits NF-kappaB activation induced by lipopolysaccharide (LPS), but the other AEDs do not. Western blot analysis revealed that this inhibition is not linked to preservation of expression of IkappaBalpha protein. Chloramphenicol acetyltransferase (CAT) assay indicated that NF-kappaB-dependent reporter gene expression is suppressed in glioma cells pretreated with VPA. VPA significantly inhibited LPS-induced production of TNF-alpha and IL-6 by THP-1 cells, whereas other AEDs did not. The findings are consistent with the idea that VPA suppresses TNF-alpha and IL-6 production via inhibition of NF-kappaB activation. Our results suggest that VPA can modulate immune responses in vitro. These findings raise the possibility that such modulation might occur with clinical use of VPA.

Clarithromycin Inhibits NF-κB Activation in Human Peripheral Blood Mononuclear Cells and Pulmonary Epithelial Cells

ABSTRACT Macrolide antibiotics modulate the production of proinflammatory cytokines in vivo and in vitro. Transcription of the genes for these proinflammatory cytokines is regulated by nuclear factor κB (NF-κB). We examined whether or not clarithromycin inhibits the activation of NF-κB induced by tumor necrosis factor alpha (TNF-α) or staphylococcal enterotoxin A (SEA) in human monocytic U-937 cells, a T-cell line (Jurkat), a pulmonary epithelial cell line (A549), and peripheral blood mononuclear cells (PBMC). Flow cytometry revealed that clarithromycin suppresses NF-κB activation induced by TNF-α in U-937 and Jurkat cells in a concentration-related manner. Western blot analysis also demonstrated that clarithromycin inhibits NF-κB activation induced by TNF-α in U-937, Jurkat, and A549 cells and PBMC and by SEA in PBMC. Western blot analysis of cytoplasmic extracts of A549 cells revealed that this inhibition is not linked to preservation of expression of the IκBα protein. The chloramphenicol acetyltransferase assay indicated that NF-κB-dependent reporter gene expression is suppressed in U-937 cells pretreated with clarithromycin. These findings are consistent with the idea that clarithromycin suppresses the production of proinflammatory cytokines via inhibition of NF-κB activation.

Cerebrospinal Fluid and Serum Levels of Cytokines and Soluble Tumor Necrosis Factor Receptor in Influenza Virus-associated Encephalopathy

This study determined the concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), soluble TNF receptor 1 (sTNFR1) and soluble E-selectin (sE-selectin) in cerebrospinal fluid (CSF) and serum from 15 children with influenza virus-associated encephalopathy to determine the role of cytokines in the pathogenesis. Cytokines and sTNFR1 were measured by enzyme-linked immunosorbent assay. The CSF IL-6, TNF-α and sTNFR1 concentrations were elevated in 9, 4 and 4 of 12 children, respectively. The serum concentrations of IL-6, TNF-α, sTNFR1 and sE-selectin were elevated in 10, 2, 5 and 7 of 13 children, respectively. Four children with elevated TNF-α and sTNFR1 levels in the CSF had neurological sequelae. The results suggested that cytokines not only in serum but also in CSF play a pivotal role in influenza virus-associated encephalopathy, and that the CSF TNF-α and sTNFR1 levels may be important for predicting neurological sequelae.

Kawasaki disease differs from anaphylactoid purpura and measles with regard to tumour necrosis factor-α and interleukin 6 in serum

It has been reported that tumour necrosis factor-α (TNF-α) is capable of inducing vascular injury, and interleukin 6 (IL-6) of inducing production of acute phase proteins and the maturation of megakaryocytes. Kawasaki disease (KD) is a systemic vasculitis with severe inflammation. We investigated whether TNF-α and IL-6 activities in serum from patients with KD differs from those in anaphylactoid purpura (AP) and measles. Serum TNF-α levels were measured by a sandwich enzyme immunoassay and IL-6 activities in serum were assessed by a colourimetric assay. Both KD and AP patients but not patients with measles had increased serum TNF-α levels during the acute stage. With respect to IL-6, patients with KD and measles, but not AP, had increased IL-6 activities in serum during the acute stage. IL-6 activities in serum of KD patients correlated with serum C-reactive protein levels and correlated to some extent with maximum platelet counts during the course of illness. These results suggest that KD differs from AP and measles regarding both cytokines. The combination of TNF-α, which may be responsible for severe vascular injury, and IL-6, which may be responsible for severe inflammation, may play an important role in acute KD.

CD14+CD16+ monocyte subpopulation in Kawasaki disease

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

Serum levels of p60 soluble tumor necrosis factor receptor during acute Kawasaki disease

To evaluate the role of tumor necrosis factor alpha (TNF-alpha) during acute Kawasaki disease, we measured p60 soluble tumor necrosis factor receptor (sT-NF-R) shedding into the circulation in 48 patients with acute Kawasaki disease, all of whom received intravenous infusions of gamma-globulin. Of the 48 patients, 5 had coronary artery lesions. Serum concentrations of p60 sTNF-R and TNF-alpha were measured by a sandwich enzyme immunoassay. Patients with Kawasaki disease had increased serum levels of p60 sTNF-R. We found a positive correlation between serum levels of p60 sTNF-R and levels of TNF alpha during acute Kawasaki disease. Moreover, patients with coronary artery lesions had higher levels of sTNF-R than did those without coronary artery lesions. Our findings indicate that p60 sTNF-R levels in serum may be useful for determining the severity of vascular damage during acute Kawasaki disease, and that patients with Kawasaki disease and high sTNF-R levels seem to be susceptible to coronary artery lesions even if they receive therapy with intravenous infusions of gamma-globulin.

Family-based association analysis implicates IL-4 in susceptibility to Kawasaki disease

Several compelling lines of evidence suggest an important influence of genetic variation in susceptibility to Kawasaki disease (KD), an acute vasculitis that causes coronary artery aneurysms in children. We performed a family-based genotyping study to test for association between KD and 58 genes involved in cardiovascular disease and inflammation. By analysis of a cohort of 209 KD trios using the transmission disequilibrium test, we documented the asymmetric transmission of five alleles including the interleukin-4 (IL-4) C(−589)T allele (P=0.03). Asymmetric transmission of the IL-4 C(−589)T was replicated in a second, independent cohort of 60 trios (P=0.05, combined P=0.002). Haplotypes of alleles in IL-4, colony-stimulating factor 2 (CSF2), IL-13, and transcription factor 7 (TCF7), all located in the interleukin gene cluster on 5q31, were also asymmetrically transmitted. The reported associations of KD with atopic dermatitis and allergy, elevated serum IgE levels, eosinophilia, and increased circulating numbers of monocyte/macrophages expressing the low-affinity IgE receptor (FCɛR2) may be related to effects of IL-4. Thus, the largest family-based genotyping study of KD patients to date suggests that genetic variation in the IL-4 gene, or regions linked to IL-4, plays an important role in KD pathogenesis and disease susceptibility.