KD is now recognized as the leading cause of acquired heart disease in children in the United States and the developed world (1
). The underlying etiology and mechanisms leading to medium and small vessel inflammatory response and coronary artery lesions and aneurysms that are the hallmarks of KD are largely unknown. Furthermore, it is unclear how and why acute systemic immune activation leads to chronic inflammatory arterial damage that localizes to the coronary arteries. The activation of the immune system in KD seems to encompass both the innate and adaptive immunity. Both human studies and animal models indicate the presence of various activated immune cells in coronary artery lesions (15
). Intact innate immune signaling pathways are essential for such lesions to develop (15
). Neutrophils are present in the early phase of the inflammatory pattern, rapidly followed by macrophages, dendritic cells (DCs), plasma cells and T cells (33
). Over the years, considerable evidence has accumulated to suggest that T cells play a significant role in the pathogenesis of KD, and several studies have documented the presence of infiltrating T lymphocytes in the human coronary artery lesions (33
). We have also shown that human coronary lesions obtained from children with fatal KD have significant numbers of mature, activated mDCs, pDCs and macrophages in close contact with CD3+ T cells, suggesting and antigen-driven adaptive immune process (16
There are striking similarities in the histopathology and kinetics of disease between human KD and LCCWE-induced coronary arteritis mouse model (23
). Initially, we analyzed the specific innate and adaptive immune responses and infiltrating cell types between the human and mouse model of coronary artery lesions. We have shown that LCCWE-induced coronary arteritis in mice requires intact signaling via TLR2 and MyD88, both of which are key participants in innate immunity (15
). However, those studies did not address the potential participation of acquired immune system in the mouse model of coronary arteritis.
Here we report direct evidence indicating that acquired immune mechanisms participate in the development of LCCWE-induced coronary arteritis in mice. We show that T cells but not B cells are required for development of arteritis. Coronary artery lesions in LCCWE-injected mice showed positive CD3 immunostaining, but more importantly, no RAG1−/− mice develop coronary lesions after LCCWE treatment. In contrast, B cell-deficient mice developed arteritis at the same frequency and severity as wild type mice. Since RAG1−/− mice have no T cells or B cells, these results indicate that T cells are essential for coronary arteritis to develop, but B cells are not. The fact that B cells are dispensable in turn indicates that an antibody-mediated response either did not occur, or was a non-essential contributor to the pathophysiology of the disease. Our results therefore strongly implicate a major role for T cells in this murine model of coronary arteritis, and suggest that the same is likely true for patients with KD, since in both cases the lesions contain subpopulations of the same lymphocytes (16
Splenocytes from LCCWE-treated RAG1−/− mice failed to produce IFN-γ when re-stimulated ex vivo
. LCCWE causes increased local expression of IFN-γ in affected arteries in a biphasic manner, with an initial spike 3 to 7 days after LCCWE, then a late spike at 28 to 42 days (51
). However, IFN-γ expression in the arterial lesion does not appear to be essential for development of lesions, since LCCWE can still induce coronary lesions in IFN-γ-deficient mice (51
). Nevertheless, our ex vivo
results where restimulation of splenocytes with LCCWE elicited IFN-γ production clearly support the interpretation that direct activation of antigen-specific T cells occurs in response to LCCWE treatment and invariably accompanies development of arteritis. Because T cells produce a variety of cytokines and chemokines, it seems reasonable to surmise that IFN-γ is redundant, since even when T cells are genetically incapable of producing IFN-γ, they can still promote focal arteritis after LCCWE treatment (51
). Consistent with this concept, another study reported that both TNF-α receptor knockout mice and wild type mice treated with the TNF-α blocking agent etanercept were protected from development of coronary lesions (31
). These results are consistent with the interpretation that TNF-α is necessary for induction of coronary artery inflammation and aneurysm formation in the LCCWE-induced coronary arteritis mouse model. Of interest, TNF-α blocking agents have also been successfully used clinically in KD patients refractory to standard treatment with intravenous immune globulin and aspirin (52
), suggesting that TNF-α expression may similarly be required for the development and/or progression of coronary lesions in clinical KD.
In addition to T cells, we also observed that mDCs, pDCs, and macrophages accumulate in the coronary artery lesions in LCCWE-injected mice. All of these cell types are key contributors to innate and adaptive immunity. These findings are very similar to observations we described on human coronary artery tissues obtained from KD patients (16
). Collectively, these previous studies (15
) and results reported here strongly implicate the involvement of both innate and adaptive immune mechanisms in LCCWE-induced coronary arteritis, and thus suggest that both arms of the immune system also participate in the pathophysiology of clinical KD. It is interesting that we not only found pDCs in the coronary lesions in both coronary artery lesions of KD patients (16
) and LCCWE-injected mice but also that in both cases pDCs were co-localized with mDCs, suggesting that activation of mDCs could lead to exacerbation of the coronary lesions. Previous studies investigating the role of pDCs in human atherosclerosis, another example of focal arterial inflammatory disease (53
), similarly found clustering of pDCs with mDCs in atherosclerotic plaques leading to enhanced cytotoxic T cell responses. Furthermore, production of IFN-α by pDCs increased expression of TNF-α in atherosclerotic plaques, thus exacerbating the arterial inflammation (55
). Therefore, it is tempting to speculate that in both atherosclerotic coronary artery plaques and in KD lesions, pDCs may amplify focal inflammation and may contribute to the development and progression of these vascular lesions. However, this possibility requires further study.
Recently, Shaposhnik et al. showed that GM-CSF plays a key role in DC migration into atherosclerotic lesions in hypercholesterolemic mouse models of atherosclerosis and that mice deficient in GM-CSF exhibit both diminished lesion size and significantly decreased DC accumulation in the atherosclerotic plaques (42
). Indeed, we recently reported that DC accumulation in Chlamydia pneumoniae
-mediated acceleration of atherosclerotic lesions in the aortic root of ApoE-null mice were related to bacteria-induced GM-CSF production (42
). Similarly, here we observed that LCCWE induced a dose-dependent expression and release of GM-CSF in murine primary aortic ECs in a TLR2- and MyD88-dependent manner. Therefore, it is tempting to propose that the LCCWE-induced GM-CSF production may play a role in the migration and accumulation of DCs into the coronary artery lesions that we observed in this mouse model of arteritis.
In conclusion, we demonstrate that both innate and adaptive immunity are essential for development of coronary lesions in the LCCWE mouse model of coronary arteritis, and that while T cells are essential, B cells are dispensable. Additionally, we highlight the histological similarities between this mouse model of coronary arteritis and human coronary artery lesions seen in patients with KD, providing further validation for the use of this model to study the immunopathology of coronary lesions of KD. These findings expand our understanding of the cellular mechanisms regulating immune activation and localized inflammation in the coronary arteries, which may potentially lead to improved treatments and to minimize the long-term morbidity and mortality in children with KD.