Kawasaki disease is the most common cause of multisystem vasculitis in childhood. The resultant coronary artery lesions make Kawasaki disease the leading cause of acquired heart disease in children in the developed world. TNF-α is a pleiotropic inflammatory cytokine elevated during the acute phase of Kawasaki disease. In this study, we report rapid production of TNF-α in the peripheral immune system after disease induction in a murine model of Kawasaki disease. This immune response becomes site directed, with migration to the coronary arteries dependent on TNF-α-mediated events. Production of TNF-α in the heart is coincident with the presence of inflammatory infiltrate at the coronary arteries, which persists during development of aneurysms. More importantly, inflammation and elastin breakdown in the coronary vessels are completely eliminated in the absence of TNF-α effector functions. Mice treated with the TNF-α-blocking agent etanercept, as well as TNFRI knockout mice, are resistant to development of both coronary arteritis and coronary aneurysm formation. Taken together, TNF-α is necessary for the development of coronary artery lesions in an animal model of Kawasaki disease. These findings have important implications for potential new therapeutic interventions in children with Kawasaki disease.

Kawasaki disease (KD)3 is the most common cause of multisystem vasculitis affecting children. The acute inflammatory response is found in medium and small vessels throughout the body, but the most common site of persistent inflammation and end organ damage is the coronary arteries. Despite appropriate therapy with i.v. γ-globulin, coronary artery aneurysms continue to develop in 5% of affected children, making it the leading cause of acquired heart disease in children in the developed world (1, 2).

The exact etiology of KD is unknown, although current evidence suggests that the initial infectious trigger of KD may possess superantigenic activity leading to massive stimulation of the immune system (3). Upon superantigen activation, T lymphocytes produce proinflammatory cytokines with multiple effector functions. TNF-α is a key inflammatory cytokine, initially produced by T lymphocytes, which mediate a secondary monocyte/macrophage release of TNF-α within 24 h (4, 5). The pleiotropic effects of TNF-α work in synergy with IFN-γ to activate and recruit other immune cell populations to sites of inflammation. Adhesion molecules and chemokines are the key players involved in the process of leukocyte migration and recruitment. TNF-α mediates endothelial cell activation through the increased expression of adhesion molecules, such as ICAM-1, VCAM-1, and E-selectin (6, 7, 8, 9). TNF-α can also up-regulate the expression of chemokines such as MIP-1α and RANTES, which are important in the orchestration of leukocyte-endothelial interactions leading to activation of the vascular endothelium (6, 7, 10). TNF-α can bind to either of two receptors: TNFRp55 (TNFRI) or TNFRp75 (TNFRII). Most TNF-α inflammatory activities are mediated by TNFRI signaling (11, 12, 13).

The progression from systemic activation of the immune system to local inflammation in coronary vessels is evidenced by endothelial cell activation, up-regulation of adhesion molecules, and histopathological evidence of inflammation in affected coronary arteries (14, 15, 16, 17). The mechanisms involved in local vascular damage leading to aneurysm formation are not known. Cardiac tissue is not readily available from affected children; thus, an animal model is required. Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis in mice is a well-validated model of KD. This mouse model closely mimics the time course, response to therapy, susceptible populations, and histopathology of human KD (18, 19, 20, 21, 22). We have shown that the ability of LCWE to induce coronary arteritis in mice is directly correlated with its superantigenic activity and induction of TNF-α production (21). Although the role of TNF-α in the vascular inflammation of KD is not clearly understood, TNF-α has been shown to participate in the local inflammatory response in other autoimmune vasculitides, including Wegener’s granulomatosis and giant cell arteritis (23, 24, 25). TNF-α expression is increased in the peripheral blood of KD patients during the acute phase (15, 26, 27, 28, 29), but its role in coronary vessel damage is not known. Also, TNF-α blocking agents have been used as salvage therapy in isolated cases to treat KD unresponsive to conventional treatment (30), but the consequence of abolishing TNF-α-mediated functions and the effect on coronary outcome has not been studied.

In the present study, we examined the role of TNF-α in the immune response leading to vascular damage in the LCWE-induced coronary arteritis model of KD. TNF-α is rapidly produced in the peripheral immune system during the initial systemic inflammatory response and then quickly localizes to the coronary vessels. The kinetics of TNF-α production corresponds with the kinetics of the lymphocytic infiltrate in the heart. TNF-α is produced persistently during progression of coronary vessel disease to aneurysm formation in affected mice. Coronary artery inflammation and vessel wall damage are absent in wild-type mice treated with TNF-α blocking agent and in TNFRI-deficient (TNFRI−/−) mice. Local increased expression of chemokines and adhesion molecules appears to be the TNF-α-mediated effects responsible for abrogation of local disease in the heart. Thus, TNF-α-mediated functions are required for induction of inflammation and aneurysm formation in the coronary arteries. This has important clinical implications and will help direct development of new therapeutic interventions in children with KD.

Wild-type C57BL/6, TNFRI−/−, and TNFRII−/− mice were purchased from Charles River Laboratories and The Jackson Laboratory and housed under specific pathogen-free conditions at the University of Toronto under institutional guidelines. The animal care committee at the University of Toronto approved all animal protocols.

LCWE was prepared as previously described (18). The concentration of the preparation injected was based on a phenol-sulfuric acid colorimetric determination of the rhamnose content in the LCWE and expressed in micrograms per milliliter final concentration in PBS.

Mice 4–5 wk old were injected i.p. with 0.5 ml of PBS alone or containing 1 mg of LCWE. Mice were sacrificed at various time points postinjection during the course of disease, their hearts and spleens immediately homogenized in TRIzol reagent (Invitrogen Life Technologies), and total RNA was isolated per the manufacturer’s instructions. cDNA was synthesized using the GeneAmp RNA PCR kit and murine leukemia virus reverse transcriptase (Applied Biosystems). cDNA was then amplified by real time PCR in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) with appropriate controls for each run. Data were collected and analyzed using ABI Sequence Detector Software, version 1.0 (Applied Biosystems). Relative quantities of PCR products were determined off standard curves generated in each run from cDNA known to contain TNF-α and were expressed as a ratio against the housekeeping gene product GAPDH. GAPDH was amplified for each sample to control for sample-to-sample variability and differences in reverse transcriptase efficiency, thus allowing for comparisons between mRNA expression in different samples. For each experiment, the ratios of TNF-α to GAPDH from all mice at a given time point were averaged, and the SE was calculated. Real-time RT-PCR was also performed to examine the expression of ICAM-1, VCAM-1, E-selectin, MIP-1α, and RANTES, in a manner similar to that for TNF-α. Primer and probe sets for all genes except for RANTES were purchased as Assays-on-Demand from Applied Biosystems. RANTES mRNA was amplified using the forward primer 5′-GGAGTATTTCTACACCAGCAGCAA-3′, reverse primer 5′-CACTTGGCGGTTCCTTCGA-3′, and probe 5′-CTTGCAGTCGTGTTTGTC-3′ (Applied Biosystems).

Mice injected i.p. with either PBS or PBS containing 1 mg of LCWE were sacrificed at various time points during disease evolution. Serial 6-μm heart and spleen cryosections were immediately fixed in acetone for 10–15 min at −20°C. After an incubation in a permeabilizing solution of PBS containing 0.1% saponin (Sigma-Aldrich) and 2% BSA (Sigma-Aldrich), sections were then stained with appropriate dilutions of purified rat anti-mouse TNF-α (clone MP6-XT22) mAb or isotype control, followed by biotinylated goat anti-rat IgG. Ab binding was then visualized with the use of streptavidin-conjugated PE. Sections were mounted in DAKO anti-fade fluorescent mounting medium (Dako) and then viewed under a confocal microscope (Axiovert 100M; Carl Zeiss). Images were captured with LSM 510 software (Carl Zeiss). All Abs were purchased from BD Biosciences and diluted in permeabilizing solution.

Mice 4–5 wk old were injected i.p. with 0.5 ml of PBS alone or PBS containing 1 mg of LCWE and sacrificed at various time points postinjection. Cardiac tissue was removed, embedded in optimal cutting temperature compound (Tissue-Tek), snap-frozen in liquid nitrogen, and stored at −80°C. Coronary arteries from each sample were identified and 6-μm-thick serial sections of the left coronary artery, 20–50 μm away from the coronary orifice, were stained with either H&E or elastin van Gieson (EVG) stain. Blinded assessment by light microscopy was performed to determine the presence of coronary arteritis and elastin breakdown.

Disease was induced in 4- to 5-wk-old C57BL/6 mice as described above. Etanercept (ENBREL; Amgen) was dissolved in the supplied vehicle according to the manufacturer’s instructions and diluted in sterile PBS. Mice were given an i.p. dose of 8–10 mg/kg twice weekly, starting from the time of disease induction. Mice were sacrificed 28 and 42 days later, and cardiac tissues were harvested and prepared for cardiac histology and histological evaluation as described.

Splenocytes from TNFRI−/− and wild-type C57BL/6 mice were cultured (0.5 × 106 cells/well) in medium alone (RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS, 50 μM 2-ME, 2 mM l-glutamine, and 10 mM HEPES) or medium containing 0.4–200 μg/ml LCWE. All cultures were performed in triplicates in 96-well plates (Nunc) and incubated for 72 h in 5% CO2 at 37°C. During the last 16 h of incubation, cells were pulsed with 1 μCi/well [3H]thymidine (Amersham Pharmacia Biotech). Counts per minute from triplicate wells were averaged, and results are presented as the difference in counts per minute (Δcpm) of cells cultured with LCWE and cells cultured in medium alone.

Fisher’s exact tests were used to analyze incidence of coronary arteritis and elastin breakdown. Unpaired Student t tests were used to analyze real-time RT-PCR data. All tests were performed at a level of significance of 0.05.

To determine the kinetics of TNF-α mRNA expression in the peripheral immune system during disease development, 4- to 5-wk-old wild-type (C57BL/6) mice were injected with PBS or with PBS containing 1 mg of LCWE, and the kinetics of TNF-α mRNA expression in the peripheral immune system was determined by quantitative real time RT-PCR. In the spleen, TNF-α mRNA was rapidly produced and detected by 6 h post-LCWE injection (Fig. 1,A). Mice injected with PBS expressed very low or undetectable levels of TNF-α mRNA at all time points. This pattern of TNF-α mRNA expression was also observed in other parts of the peripheral immune system, including the mesenteric lymph nodes, post-LCWE injection (our unpublished observations). TNF-α message was translated into protein as visualized by fluorescent staining for TNF-α protein by confocal microscopy. TNF-α protein was detected at all time points from 6 h to 24 h post-LCWE injection (Fig. 1 B), consistent with the kinetics of mRNA production. TNF-α protein was not detected at any time point in either isotype controls or mice injected with PBS. These results confirm that LCWE induces rapid TNF-α mRNA expression that is quickly translated into protein in the peripheral immune system.

FIGURE 1.

TNF-α is rapidly produced in the peripheral immune system and localizes to the coronary vessel wall after LCWE stimulation. A, Real-time RT-PCR depiction of TNF-α mRNA expression in spleen (n ≥ 3, each time point). B, Confocal microscopy detection of intracellular TNF-α protein in spleen 24 h postinjection. C, Real time RT-PCR depiction of TNF-α mRNA expression in the heart (n ≥ 3, each time point). D, Confocal microscopy detection of TNF-α in hearts of mice in A. Scale bar represents 10 μm (×400) in all confocal images. Each value is representative of ≥4 independent experiments.

FIGURE 1.

TNF-α is rapidly produced in the peripheral immune system and localizes to the coronary vessel wall after LCWE stimulation. A, Real-time RT-PCR depiction of TNF-α mRNA expression in spleen (n ≥ 3, each time point). B, Confocal microscopy detection of intracellular TNF-α protein in spleen 24 h postinjection. C, Real time RT-PCR depiction of TNF-α mRNA expression in the heart (n ≥ 3, each time point). D, Confocal microscopy detection of TNF-α in hearts of mice in A. Scale bar represents 10 μm (×400) in all confocal images. Each value is representative of ≥4 independent experiments.

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To investigate the local inflammatory response in the coronary arteries, we examined TNF-α expression by real time RT-PCR in affected vessels. TNF-α mRNA was detected in murine hearts starting at day 3, with a second peak occurring at day 28 post-disease induction, then declining to basal levels by d 90 (Fig. 1,C). TNF-α production was maximal at day 28, coincident with maximal inflammation assessed histologically (Fig. 2,A). More importantly, expression of TNF-α protein was localized to the affected coronary vessel walls as visualized by confocal microscopy (Fig. 1 D). These data demonstrate that there is local production of TNF-α at the coronary blood vessel wall. Production begins at the transcriptional level and is followed by translation into TNF-α protein, which is found in all layers of the affected artery.

FIGURE 2.

Local immune infiltration is observed at the coronary arteries post-LCWE stimulation and is completely ablated in the absence of TNF-α activity. A, H&E staining for visualization of development of cardiac inflammatory infiltrate (×200). B, H&E staining of wild-type and TNFRI−/− mice 28 days post-disease induction (×200).

FIGURE 2.

Local immune infiltration is observed at the coronary arteries post-LCWE stimulation and is completely ablated in the absence of TNF-α activity. A, H&E staining for visualization of development of cardiac inflammatory infiltrate (×200). B, H&E staining of wild-type and TNFRI−/− mice 28 days post-disease induction (×200).

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To determine the time course of the localized inflammatory response in coronary arteries during disease evolution, coronary arteries were identified, preserved in cryosections from mice post-LCWE injection, and stained with H&E. Inflammation was first detected at day 3 post-LCWE injection, as a lymphocytic infiltrate in the adventitia of the coronary arteries (Fig. 2,A). Coronary artery inflammation continued well into disease evolution, with maximal infiltrate observed on day 28 post-LCWE injection, persisting through to day 90. No inflammatory infiltrate was detected in PBS-injected mice. Interestingly, the kinetics of TNF-α expression observed in murine hearts post-LCWE injection corresponds with the kinetics of the immune cell infiltrate during disease development (Figs. 1 and 2).

To investigate whether TNF-α activity is required for the development of coronary artery inflammation, coronary arteritis was induced in C57BL/6 mice by injection of LCWE as per protocol. Mice were given an i.p. dose of either 10 mg/kg etanercept (soluble TNF receptor fusion protein; Enbrel) or PBS, twice weekly, starting from the time of disease induction. Twenty-eight days later, mice were sacrificed, and cardiac tissues were harvested and evaluated histologically for the presence of cellular infiltrates at the coronary arteries. Consistent with earlier observations, massive cellular infiltrates were observed at the coronary arteries of LCWE-injected mice treated with PBS only. In contrast, no inflammation was detected in the hearts of LCWE-injected mice treated with the TNF blocking agent etanercept. Administration of etanercept twice weekly starting at the time of disease induction until the time of sacrifice resulted in complete protection from coronary artery inflammation (Table I).

Table I.

Coronary artery inflammation is abrogated in the absence of TNF activity

TreatmentaIncidence of Inflammationb
PBSLCWE
Without etanercept 0/6 12/13 
With etanercept 0/4 0/15 
TreatmentaIncidence of Inflammationb
PBSLCWE
Without etanercept 0/6 12/13 
With etanercept 0/4 0/15 
a

Wild-type mice were treated with either PBS or etanercept every 2–3 days after LCWE or PBS injection as described in Materials and Methods.

b

Incidence of inflammation is presented as total number of mice showing cellular infiltrate 28–42 days postinjection over total number of mice injected. Disease incidence was significantly reduced in the etanercept-treated group in comparison with that in untreated animals (p < 0.001).

Most TNF-α inflammatory activities are mediated by TNFRI signaling, whereas TNFRII plays a smaller role (11, 12, 13). To assay the role of these two signaling pathways in the development of coronary artery disease, mice with targeted gene mutations in their TNFRI or TNFRII together with their wild-type controls were injected either with PBS or with PBS containing LCWE and were examined for the presence of cellular infiltrates at the coronary arteries. Inflammatory infiltrates were observed at the coronary arteries of wild-type mice 28 days post-LCWE injection. Similarly, TNFRII-deficient mice continued to develop inflammation at the coronary artery after disease induction. In contrast, no inflammation was detected in the hearts of TNFRI-deficient mice sacrificed at day 28 or at any other time point post-LCWE injection (Fig. 2,B). Heart cryosections from PBS-injected mice did not contain infiltrating cells at any time point after injection. The incidence of coronary arteritis was 86% in wild-type mice and 93% in TNFRII-deficient mice, but in TNFRI-deficient animals it was 0%, with none showing any signs of inflammation at the coronary arteries (Table II). Thus, the TNFRI pathway is responsible for signaling TNF-α-mediated functions leading to development of coronary artery inflammation.

Table II.

TNFRI signaling is required for induction of coronary artery inflammation

Mouse StrainIncidence of Inflammationa
PBSLCWE
Wild-type 0/22 30/35 
TNFRI−/− 0/9 0/16a 
TNFRII−/− 0/4 13/14a 
Mouse StrainIncidence of Inflammationa
PBSLCWE
Wild-type 0/22 30/35 
TNFRI−/− 0/9 0/16a 
TNFRII−/− 0/4 13/14a 
a

Incidence of inflammation is presented as total number of mice showing cellular infiltrate 28–42 days postinjection over total number of mice injected. Whereas disease incidence was significantly reduced in TNFRI-deficient mice (p < 0.001), no statistical significance was detected between the incidence of disease in the TNFRII-deficient and wild-type animals (p = 0.7).

To examine the role of TNF-α effects in the peripheral immune response to LCWE, splenocytes from either wild-type or TNFRI-deficient mice were cultured with medium alone or medium containing different concentrations of LCWE, and their proliferative responses were assayed. Consistent with our previous report (21), LCWE induced marked proliferation of naive T cells from wild-type mice in a dose-dependent manner (Fig. 3). Interestingly, splenocytes from disease-resistant TNFRI-deficient mice proliferated to LCWE at a comparable level, with intensity and dose response similar to that of the wild type. Both wild-type and TNFRI−/− splenocytes also responded similarly to the prototypic bacterial superantigen, Staphylococcus enterotoxin B (data not shown). Our findings demonstrate that the absence of TNF activity mediated by TNFRI does not affect the immune response to LCWE; thus, disease resistance in TNFRI-deficient mice was not due to an intrinsic defect in the T lymphocytes.

FIGURE 3.

Lymphocyte proliferative response to LCWE is intact in mice lacking TNF-α activity. Splenocytes from TNFRI−/− (♦) or C57BL/6 mice (▪) were cultured with varying concentrations of LCWE, and proliferation was measured 3 days later. Results are mean values of Δcpm of triplicate wells and are representative of five separate experiments.

FIGURE 3.

Lymphocyte proliferative response to LCWE is intact in mice lacking TNF-α activity. Splenocytes from TNFRI−/− (♦) or C57BL/6 mice (▪) were cultured with varying concentrations of LCWE, and proliferation was measured 3 days later. Results are mean values of Δcpm of triplicate wells and are representative of five separate experiments.

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The observation that TNFRI-deficient mice had no localized inflammation at the coronary arteries despite a normal proliferative response to LCWE suggests that TNF-α-dependent functions transduced via TNFRI may be involved in mediating local disease. One such TNF-α-dependent activity is lymphocyte trafficking. TNF-α regulates lymphocyte migration via the induction of chemokines and adhesion molecules such as MIP-1α, RANTES, ICAM-1, VCAM-1, and E-selectin (6, 7, 8, 9). We examined mRNA expression of these important migratory signals in cardiac tissue of wild-type and TNFRI-deficient mice at 0 and 24 h, as well as day 7 and 28 post-LCWE injection. LCWE induced up-regulation of RANTES, ICAM-1, VCAM-1, and E-selectin at all time points during the evolution of coronary disease (Fig. 4). More importantly, RANTES, ICAM-1, VCAM-1, and E-selectin mRNA were expressed at significantly lower levels in TNFRI-deficient mice compared with wild-type mice post-LCWE injection. This suggests that TNF-α mediated signaling via TNFRI facilitates local disease development by up-regulation of chemokines and adhesion molecules, thus allowing lymphocyte migration and infiltration into the coronary arteries.

FIGURE 4.

Absence of TNF-α activity results in the absence of local migratory signals. A--E, Real time RT-PCR depiction of MIP-1α, RANTES, ICAM-1, E-selectin, and VCAM-1 mRNA expression in wild-type and TNFRI−/− mice at 0 h (n ≥ 4), 24 h (n ≥ 4), and 7 (n ≥ 6) and 28 (n ≥ 7) days postinjection. Each value is representative of ≥3 independent experiments.

FIGURE 4.

Absence of TNF-α activity results in the absence of local migratory signals. A--E, Real time RT-PCR depiction of MIP-1α, RANTES, ICAM-1, E-selectin, and VCAM-1 mRNA expression in wild-type and TNFRI−/− mice at 0 h (n ≥ 4), 24 h (n ≥ 4), and 7 (n ≥ 6) and 28 (n ≥ 7) days postinjection. Each value is representative of ≥3 independent experiments.

Close modal

In some autoimmune disease models, inflammation can be disengaged from target organ damage (31). Results presented above show that the development of coronary arteritis after LCWE injection is abrogated in mice treated with TNF-α-blocking pharmacological agents or those genetically modified lacking the ability to transduce a TNF-α generated signal via the TNFRI. To address whether absence of TNF-α activity also negates vessel wall damage, cryosections of cardiac tissue were obtained from wild-type, TNFRI−/−, TNFRII−/−, and etanercept-treated animals post-LCWE injection and assayed for elastin breakdown. Elastin breakdown is the hallmark of aneurysm formation and occurs maximally at 42 days post-LCWE injection (22). Fragmentation of the elastin layers with disruption of the vessel wall is seen by EVG staining in 50–60% of the LCWE-injected mice at day 42, as determined from our previous work (22). Additionally, elastin degradation is evidenced by loss of the number of elastin layers from 5 to 3 in the coronary arterial wall together with a decrease in the elastin content evidenced by a decrease in intensity of the EVG stain. Consistent with earlier observations, immune cellular infiltrates persisted through day 42 in affected wild-type mice (Fig. 5). This was associated with elastin breakdown and vessel wall disruption in 67% of all wild-type mice and 44% TNFRII−/− mice post-disease induction (Table III). In contrast, none of the LCWE-injected animals treated with etanercept had any evidence of coronary arteritis or vessel wall breakdown as measured by elastin breakdown (Table III). Similarly, TNFRI-deficient animals did not develop coronary arteritis (Fig. 5) or elastin degradation (Table III). Therefore, abrogation of TNF-α activity mediated by TNFRI protects against elastin breakdown and aneurysm formation.

FIGURE 5.

Coronary artery aneurysms do not develop in the absence of TNF-α activity. EVG staining for visualization of elastin breakdown in wild-type and TNFRI−/− hearts 42 days post-disease induction, and corresponding H&E staining for visualization of cardiac inflammatory infiltrate (×400).

FIGURE 5.

Coronary artery aneurysms do not develop in the absence of TNF-α activity. EVG staining for visualization of elastin breakdown in wild-type and TNFRI−/− hearts 42 days post-disease induction, and corresponding H&E staining for visualization of cardiac inflammatory infiltrate (×400).

Close modal
Table III.

Coronary artery aneurysms do not develop in the absence of TNF activity or TNFRI signaling

Mouse StrainElastin Breakdowna
PBSLCWE
Wild-type 0/12 14/21 
Etanercept 0/4 0/7b 
TNFRI−/− 0/6 0/19b 
TNFRII−/− 0/4 4/9c 
Mouse StrainElastin Breakdowna
PBSLCWE
Wild-type 0/12 14/21 
Etanercept 0/4 0/7b 
TNFRI−/− 0/6 0/19b 
TNFRII−/− 0/4 4/9c 
a

Incidence of elastin breakdown is presented as total number of mice showing elastin breakage at day 42 postinjection over total number of mice injected.

b

Both the TNFRI-deficient and etanercept animals exhibited significantly reduced incidence of elastin breakdown (p < 0.001 and p < 0.05, respectively).

c

No statistical significance was detected between the incidence of elastin breakdown in the TNFRII-deficient and wild-type animals (p = 0.42).

Acute KD is characterized by the classic signs of inflammation: redness, heat, and swelling in affected parts of the body. This is due to inflammation of small and medium-sized blood vessels. The inflammation localizes to and persists in the coronary arteries, resulting in vascular damage and aneurysm formation. T cells are important in the pathogenesis of human KD and localize to the site of aneurysm formation in coronary arteries of children with KD (32). Although still debated, current evidence suggests that the cause of KD is of an infectious origin with superantigenic activity leading to massive stimulation of the immune system, resulting in vessel wall damage and aneurysm formation in the coronary arteries (3, 33, 34). TNF-α, a well-known culprit in autoimmune disease, is produced by both T cells and macrophages after superantigenic stimulation (35, 36). Several studies have reported elevated levels of TNF-α in patient sera during the acute phase of KD (15, 26, 27, 28, 29). However, KD studies to date have focused only on TNF-α levels in the peripheral blood. No information is available regarding the relationship of peripheral blood TNF-α levels and coronary artery lesions or the role of TNF-α in the pathogenesis of coronary vessel damage. In this study, we examined the role of TNF-α in the coronary arteries using a murine model of KD. We report high levels of TNF-α in the affected vascular tissue during the development of coronary artery disease. More importantly, TNF-α-mediated actions, through TNFRI signaling, are necessary for both the local inflammatory response and vessel wall breakdown seen in a murine model of KD.

The peripheral immune response to LCWE occurred rapidly, with proinflammatory cytokines such as IFN-γ (22), and TNF-α mRNA and protein detected within 6–18 h postinjection. Migration of the immune response from the peripheral lymphoid tissue to the coronary arteries is evidenced histologically, by lymphocyte infiltration, and by determination of local production of these cytokines in the heart. Infiltrating cells were detected in the hearts of affected mice as early as day 3 and persisted until day 90 postinjection. The expression of TNF-α mRNA and protein coincided precisely with the presence of cellular infiltrate. More importantly, TNF-α protein was localized to the affected coronary artery and found in all layers of the vessel wall.

One critical role of TNF-α in promoting localized inflammation in our model is the regulation of leukocyte recruitment signals (13, 37, 38, 39, 40). This appears to be a common theme in many autoimmune diseases and their corresponding models. In particular, the vascular endothelium plays an important role in leukocyte recruitment and infiltration into affected tissue (40). TNF-α, either alone or in cooperation with other cytokines, regulates production of chemokines and expression of adhesion molecules on endothelial cells, thus participating in the multistep cascade of leukocyte migration into the target tissue (9). TNF-α is able to induce expression of MIP-1α and RANTES (7, 10, 41). It is also able to up-regulate expression of ICAM-1 (in synergy with IFN-γ and IL-1), VCAM-1 (in synergy with IL-1, IL-4, and IL-13), and E-selectin (in synergy with IL-1) in response to inflammatory stimuli (7).

Although TNF-α can signal through both TNFRI and TNFRII, most TNF responses, including regulation of leukocyte migration, are via TNFRI (42). TNFRI−/− mice demonstrated comparable lymphocyte proliferative response to LCWE in comparison to wild-type mice. Notwithstanding a robust immune response to LCWE in the peripheral immune system, inflammatory cells failed to migrate into the vascular tissue in TNFRI-deficient mice in our model of childhood KD. In wild-type mice, up-regulation of both chemokines and adhesion molecules appears to play an important role in directing local inflammation, consistent with other autoimmune disease models. These mediators of leukocyte migration and recruitment are elevated in animal models including experimental allergic encephalomyelitis, experimental autoimmune uveitis, and collagen-induced arthritis, and in human diseases including rheumatoid arthritis and KD (14, 34, 38, 39, 40, 43, 44). In addition, the important role of chemokines and adhesion molecules in development of disease has been demonstrated in various autoimmune diseases and their corresponding animal models (45, 46, 47, 48), with preliminary use of blocking agents against specific adhesion molecules and chemokines having some success in alleviating disease (49, 50, 51, 52).

The role of TNF-α in inflammation has been demonstrated in other autoimmune diseases and their corresponding animal models, including rheumatoid arthritis (6, 53, 54), multiple sclerosis (6, 55), uveitis (56), and diabetes (57). TNF-α is also important in inflammatory heart and vessel diseases (23, 24). TNF-α is localized to the sites of vessel damage in giant cell arteritis (24, 58). Heart-specific overexpression of TNF-α has been shown to cause myocarditis in mice (25). Ablation of TNF-α activity in experimental autoimmune encephalomyelitis, experimental autoimmune uveitis, and myocarditis leads to decreased inflammation and amelioration of inflammatory disease (13, 38, 39, 40, 59). In our disease model, coronary artery inflammation is completely abrogated when TNF activity is abolished by administration of the TNF-blocking agent etanercept in mice. The TNF activity responsible for disease is mediated by the TNFRI signaling pathway. An essential role of TNF-α has also been implicated in another animal model of KD in which coronary arteritis is induced by injection of Candida albicans water-soluble fraction (CAWS). After CAWS injection, DBA/2 mice developed the most severe coronary arteritis leading to death, which corresponded to increased production of TNF-α in vitro by splenocytes from DBA/2 mice (60). Interestingly, other CAWS disease-prone mouse strains, including C57BL/6 and C3H/HeN, did not show increased TNF-α production by ex vivo splenocytes in culture. Both immune and nonimmune cell types including endothelial cells and vascular smooth muscle cells produce TNF-α (61, 62). In the LCWE model of coronary arteritis, TNF-α production is detected in vivo as de novo production of TNF-α mRNA and protein in both the peripheral lymphoid tissue and affected cardiac tissue (Fig. 1). The complex milieu of the whole animal cannot be underscored, and the contribution of TNF-α production from all tissues during disease development in vivo can explain the subtle differences detected when TNF-α production by only one tissue is studied in isolation.

In some models of autoimmune disease, end organ damage can be separated from inflammation. In rodent models of collagen-induced arthritis, ablation of TNF-α activity reduces inflammation but joint damage persists (31). Among coronary vessel diseases, aneurysm formation is almost unique to KD, with coronary artery damage most commonly detected in the subacute stage of KD. In our mouse model, vascular damage and aneurysm formation are detected from days 28 to 42 post-LCWE injection, a time period corresponding to the subacute phase in human KD. Elastin breakdown is the hallmark of aneurysm formation. Breakdown of the extracellular matrix scaffolding leads to thinning and weakening of the vessel wall and subsequent aneurysm formation (63). Evidence of elastin fragmentation and vessel wall disruption is present in ∼60% of affected mice at 42 days post-disease induction (22). Ablation of TNF-α activity, by administration of etanercept or by targeted gene disruption of TNFRI, results in complete absence of vessel wall breakdown (Table III).

Here, we show that TNF-α is critical during evolution of coronary artery damage in a murine model of Kawasaki disease. After disease induction, TNF-α is rapidly produced in the peripheral immune system and localizes to affected coronary vessel walls. TNF-α production in affected coronary arteries correlates with development of the local inflammatory response in the vessel wall. Increased expression of TNF-α-dependent leukocyte migration signals in the coronary arteries lead to lymphocyte recruitment. A sustained local immune response coupled with persistent TNF-α production leads to elastin degradation, vessel wall damage, and the characteristic coronary artery lesions seen in KD. Blocking TNF-α activity by administration of etanercept or abolishing TNF-α functions by abolishing the signal via TNFRI results in complete resistance to both inflammation and elastin breakdown in the coronary arteries. Thus, TNF-α activity is necessary for both local inflammation and tissue damage of the coronary arteries in our model of KD. TNF-α-blocking agents are already part of the therapeutic arsenal combating life-threatening primary vascular inflammation. Our results suggest that blocking TNF-α or its downstream functions, such as leukocyte recruitment or matrix degradation, may be exciting new options in the battle against coronary artery damage in children with Kawasaki disease.

We thank Lily Morikawa for the preparation of H&E- and EVG-stained cryosections, Garrick Fong for the preparation of LCWE, Andrew Lau for technical assistance, and Dr. Earl Silverman for his critical comments.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by an Operating Grant from the Canadian Institutes of Health Research. R.S.M.Y. is supported by a New Investigator Award from The Arthritis Society and the Canadian Institutes of Health Research. J.S.H.Y. is the recipient of a Graduate Scholarship from the University of Toronto, the Hospital for Sick Children Foundation, and the Ontario Student Opportunity Trust Fund.

3

Abbreviations used in this paper: KD, Kawasaki disease; EVG, elastin van Gieson; LCWE, Lactobacillus casei cell wall extract; TNFRI, TNFR p55; TNFRII, TNFR p75; CAWS, Candida albicans water-soluble fraction.

1
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