Francesca L. Garcia

Systemic Arterial Expression of Matrix Metalloproteinases 2 and 9 in Acute Kawasaki Disease

Objective—Coronary artery aneurysms are the major complication of Kawasaki disease (KD). Matrix metalloproteinases (MMPs) regulate remodeling and degradation of the extracellular matrix. We hypothesized that MMP-9 expression is increased in acute KD aneurysms when compared with KD nonaneurysmal arteries and arteries from control children. Methods and Results—MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were immunolocalized in coronary arteries from children with fatal acute KD and controls. In KD coronary aneurysms, MMP-2 expression was prominent in the thickened neointima and in endothelial cells of new capillaries in areas of angiogenesis. MMP-9 was absent in control coronary arteries but was expressed in coronary artery aneurysms, nonaneurysmal coronary and noncoronary arteries, and cardiac nerves in acute KD, without an increase in TIMP-1 expression. Conclusions—MMP-2 likely participates in remodeling of the arterial wall in acute KD, particularly in the processes of neointimal proliferation and angiogenesis. MMP-9 may play a role in the development of coronary aneurysms, but its expression is not confined to aneurysmal arteries. Systemic arterial expression of MMP-9 in acute KD, even in the absence of inflammatory changes in the vessel, suggests induction by a circulating factor, or possibly by an infectious agent with tropism for arterial tissue.

Detection of antigen in bronchial epithelium and macrophages in acute Kawasaki disease by use of synthetic antibody

BACKGROUND Kawasaki disease (KD) is the most common acquired cardiac disease in children in developed nations. The etiology is unknown, but a ubiquitous infectious agent appears to be likely. Immunoglobulin A plasma cells infiltrate inflamed tissues in acute KD, producing oligoclonal, antigen-driven antibodies. METHODS To identify antigens important in the pathogenesis of KD, oligoclonal KD antibodies were prepared in vitro and tested by immunohistochemistry experiments on tissues from patients with acute KD and from control subjects and were also tested for reactivity with human inflammatory proteins. RESULTS By use of synthetic antibody A, specific binding to a cytoplasmic antigen in proximal bronchial epithelium was observed in 10 of 13 patients with acute KD but in 0 of 9 control subjects (P=.001). A subset of macrophages was positive in at least 1 inflamed tissue from all 17 patients with acute KD. Antigen was detected in 9 of 12 acute KD coronary artery aneurysms but in 0 of 10 control coronary arteries (P<.001). The antigen is not immunoglobulin or any of 40 common inflammatory proteins. CONCLUSIONS We report the first demonstration of a KD-associated antigen in the tissues targeted by the disease. Our findings are consistent with the theory that KD is caused by a previously unidentified respiratory infectious agent with tropism for vascular tissue.

Cytoplasmic Inclusion Bodies Are Detected by Synthetic Antibody in Ciliated Bronchial Epithelium during Acute Kawasaki Disease

Abstract BackgroundIn developed nations, Kawasaki disease (KD) is the most common cause of acquired heart disease in children. An infectious etiology is likely but has not yet been identified. We have previously reported that oligoclonal immunoglobulin A plasma cells infiltrate acute KD tissues and that synthetic KD antibodies detect a distinctive spheroidal antigen in acute KD ciliated bronchial epithelium MethodsTo further characterize the antigen in acute KD bronchi, we examined paraffin-embedded ciliated bronchial epithelium using light microscopy (LM) and transmission electron microscopy (TEM) ResultsThe spheroids observed by immunohistochemistry (IHC) are visualized as inclusion bodies with hematoxylin-eosin and nucleic acid stains and in methylene blue/azure II/basic fuchsin trichrome–stained plastic sections, suggesting the presence of both protein and nucleic acid. The structures visualized by LM correspond to homogeneous electron-dense perinuclear inclusion bodies (up to 1.4 microns in diameter) in ciliated bronchial epithelium from 4 patients with acute KD examined by TEM. Inclusion bodies were not present in control bronchial epithelium or in nonciliated cells ConclusionsThe antigen detected in acute KD ciliated bronchial epithelium by IHC with synthetic KD antibodies resides in cytoplasmic inclusion bodies that are consistent with aggregates of viral proteins and associated nucleic acid and may derive from the etiologic agent of KD

RNA-Containing Cytoplasmic Inclusion Bodies in Ciliated Bronchial Epithelium Months to Years after Acute Kawasaki Disease

Background Kawasaki Disease (KD) is the most common cause of acquired heart disease in children in developed nations. The KD etiologic agent is unknown but likely to be a ubiquitous microbe that usually causes asymptomatic childhood infection, resulting in KD only in genetically susceptible individuals. KD synthetic antibodies made from prevalent IgA gene sequences in KD arterial tissue detect intracytoplasmic inclusion bodies (ICI) resembling viral ICI in acute KD but not control infant ciliated bronchial epithelium. The prevalence of ICI in late-stage KD fatalities and in older individuals with non-KD illness should be low, unless persistent infection is common. Methods and Principal Findings Lung tissue from late-stage KD fatalities and non-infant controls was examined by light microscopy for the presence of ICI. Nucleic acid stains and transmission electron microscopy (TEM) were performed on tissues that were strongly positive for ICI. ICI were present in ciliated bronchial epithelium in 6/7 (86%) late-stage KD fatalities and 7/27 (26%) controls ages 9–84 years (p = 0.01). Nucleic acid stains revealed RNA but not DNA within the ICI. ICI were also identified in lung macrophages in some KD cases. TEM of bronchial epithelium and macrophages from KD cases revealed finely granular homogeneous ICI. Significance These findings are consistent with a previously unidentified, ubiquitous RNA virus that forms ICI and can result in persistent infection in bronchial epithelium and macrophages as the etiologic agent of KD.

Cloning the Arterial IgA Antibody Response during Acute Kawasaki Disease

Kawasaki disease (KD) is the most common acquired cardiac disease in children in developed nations. The etiology of KD is unknown but likely to be a ubiquitous microbial agent. Previously, we showed that oligoclonal IgA plasma cells infiltrate coronary arteries and other inflamed tissues in acute KD. We demonstrated that a synthetic Ab made using an α H chain sequence prevalent in acute KD arterial tissue detected Ag in acute KD coronary arteries, lung, and other inflamed tissues and that Ag localized to cytoplasmic inclusion bodies in the acute KD ciliated bronchial epithelium. In this study, we synthesized a panel of mAbs from α and κ chain sequences present in the KD arterial wall and tested the Abs for binding to acute KD tissues. We report that all of the synthetic mAbs that bind to acute KD tissues detect Ag in cytoplasmic inclusion bodies in the acute KD ciliated bronchial epithelium. Abs made from α sequences that were prevalent in KD arterial tissue show stronger binding to acute KD tissues than Abs made from less prevalent sequences. These findings highlight the likely importance of the inclusion bodies in the etiopathogenesis of acute KD, confirm that the IgA Ab response in acute KD is Ag driven, and demonstrate the usefulness of cloning the Ab response in diseased tissues to identify disease-relevant Ags.

Angiogenesis in fatal acute Kawasaki disease coronary artery and myocardium

Angiogenesis has been shown to be dysregulated in coronary artery (CA) aneurysms in the chronic phase of Kawasaki disease (KD). Neovascularization may occur in inflammatory-related vascular diseases because many angiogenesis mediators are secreted by inflammatory cells. We hypothesized that inflammation of the acute KD CA aneurysm could lead to dysregulation of angiogenesis mediators and subsequent neovascularization. To investigate this hypothesis, acute fatal KD cardiac tissues were immunostained for angiogenic inducers and inhibitors. Microvessel density was determined and the degree of inflammation assessed. Marked inflammation and angiogenesis were found in acute KD CA aneurysms and myocardium, with the highest microvessel density seen in patients who died 2–3 weeks after onset of the disease. Expression of proangiogenic proteins was higher than expression of inhibitors in KD CA aneurysms and myocardium. Angiogenesis mediators were localized to inflammatory cells in the myointima, adventitia, and myocardium. We conclude that significant neovascularization occurs in acute KD CA aneurysms and myocardium much sooner after onset of the disease than has been previously reported, that multiple angiogenesis factors are involved, and that dysregulation of angiogenesis likely contributes to KD vasculopathy.

Cell adhesion molecule expression in coronary artery aneurysms in acute Kawasaki disease

Background: The pathogenesis of coronary artery aneurysm (CAA) formation in acute Kawasaki disease (KD) remains unclear. Cell adhesion molecules mediate cell-cell and cell-matrix interactions and regulate leukocyte migration, angiogenesis and tissue remodeling. We hypothesized that cell adhesion molecules are expressed in acute KD CAA. Methods: P-selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and integrin β1 were immunolocalized in coronary arteries from 6 acute KD patients and 7 controls. Results: In endothelial cells of adventitial neovasculature in KD CAA, P-selectin and integrin β1 were expressed in all of 6 patients, and E-selectin and/or VCAM-1 were expressed in 4 of 6. Endothelial cells in controls and in nonaneurysmal KD coronary arteries expressed P-selectin and integrin β1, but not E-selectin or VCAM-1. Integrin β1 was expressed on infiltrating leukocytes in 5 of 6 KD CAA and on fibroblasts in 6 of 6; these findings were absent in controls and in nonaneurysmal KD coronary arteries. Conclusions: The lack of widespread expression of E-selectin or VCAM-1 on endothelial cells of acute KD coronary arteries was surprising and suggests that inflammatory cell infiltration into KD coronaries is not simply the result of widespread up-regulation of cell adhesion molecules on endothelial cells by circulating cytokines. Rather, inflammatory cells may be directed to specific areas of the coronary arteries targeted by a pathogen causing KD. Our results suggest that E-selectin and VCAM-1 expression on neovasculature may contribute to neoangiogenesis and prolonged CAA inflammation and that integrin β1 might be involved in fibroblastic remodeling of acute KD CAA.

CD8 T lymphocytes do not express cytotoxic proteins in coronary artery aneurysms in acute Kawasaki disease.

Coronary arterial inflammation in acute Kawasaki disease (KD) is characterized by transmural infiltration of CD8 T lymphocytes, suggesting that CD8 T lymphocyte cytotoxic activity may be important in the pathogenesis of coronary arterial damage in acute KD. We performed immunohistochemistry for the cytotoxic proteins perforin and granzyme B on paraffin-embedded, formalin-fixed coronary artery aneurysm tissue from 6 children who died in the acute stage of KD. Neither perforin nor granzyme B was detected in the KD coronary aneurysm wall. We speculate that the etiologic agent of KD interferes with expression of these cytotoxic proteins by CD8 T lymphocytes, prolonging inflammation in the arterial wall and leading to coronary artery aneurysm formation.

CD8|[plus]| T Lymphocytes and Macrophages Infiltrate Coronary Artery Aneurysms in Acute Kawasaki Disease

The pathogenesis of coronary arterial inflammation in acute Kawasaki Disease (KD) is unclear. Immunophenotypic characterization of the inflammatory cells in the KD vascular lesion has been reported previously in only a single case, by Terai and colleagues. To test the hypothesis that the vascular lesion in KD is an activated T lymphocyte-dependent process, we performed immunohistochemical studies on coronary artery aneurysms from eight fatal acute KD cases using antibodies to CD45RO (activated/memory T lymphocyte), CD8 (cytotoxic/suppressor T lymphocyte), CD4 (helper T lymphocyte), HAM56 (macrophage), and CD20 (B lymphocyte). We found that acute KD coronary arteritis was characterized by transmural infiltration of CD45RO+ T lymphocytes, with four- to five-fold more CD8+ T lymphocytes compared with CD4+ T lymphocytes. Macrophages were present primarily in the adventitial layer, and CD20+ B lymphocytes were notably absent. These data lend support to the hypotheses that KD results from infection with an intracellular pathogen such as a virus whose antigens are presented by MHC class I molecules, and that CD8+ T lymphocytes and macrophages are important in the pathogenesis of KD coronary aneurysms.

Macrophage infiltration of pancreatic acini and islets in acute Kawasaki disease.

A report of 2 patients who developed diabetes after Kawasaki disease (KD) led us to determine whether macrophages and/or T cells infiltrate acute KD pancreas. Three of 10 acute fatal KD cases had diffuse macrophage infiltration of the pancreas; T cells were not prominent. Affected islets were seen in close proximity to normal islets. These findings may explain the rarity of diabetes after KD.