Although mast cells have been identified in human AAA lesions, evidence for a direct participation of mast cells in this vascular disease is lacking. The availability of mast cell–deficient mice and established mouse AAA models made it possible to determine whether these cells are essential mediators of AAA and not merely a hallmark.
This study used a BMMC reconstitution technique to determine whether mast cell–derived IL-6, IFN-γ, and TNF-α play a role in AAA formation (Figure A). TNF-α is expressed in macrophages and lymphocytes both in the atherosclerotic (34
) and AAA (35
) lesions. In ApoE–/–
mice with diet-induced atherosclerosis, a lack of TNF-α reduces atherosclerosis (36
). In this model, TNF-α plays a proatherogenic role by upregulating expression of the vascular adhesion molecules ICAM-1 and VCAM-1 and the chemokine MCP-1 in the vascular wall and by inducing expression of scavenger receptor class A and uptake of oxidized LDLs in macrophages. Although it remains to be determined whether TNF-α plays a similar role to that in atherosclerosis, both serum (37
) and aneurysmal tissues (35
) from AAA patients have increased TNF-α levels. However, our data with TNF-α–/–
mast cells did not support a direct role of mast cell–derived TNF-α in AAA. This does not exclude the possibility that TNF-α plays a role in AAA formation; rather, it is possible that TNF-α produced from other cells may compensate for its absence from mast cells.
AAA formation requires mast cell IFN-γ and IL-6 but not TNF-α. Reconstitution of mast cell–deficient KitW-sh/KitW-sh
mice with IFN-γ–/–
BMMCs did not restore the AAA phenotype (Figure A). These observations agree with the findings in IFN-γ–/–
), which are protected from CaCl2
-induced AAA. Both this study (3
) and others using a histocompatibility-mismatched aortic transplantation model in IFN-γ receptor–deficient recipient mice (6
) demonstrated that Th2-slanted immune responses promote AAA formation. Indeed, mast cells from IL-6–/–
mice produce negligible IFN-γ and IL-6, suggesting cooperative regulation of these 2 cytokines (19
). HeLa cells and monocytes exhibit IFN-γ–regulated expression of IL-6 (38
). Enhanced IL-6 and IFN-γ, which may come in part from mast cells, were detected in cultured explants of human AAA lesions and in serum of AAA patients (15
). AAA sections or tissue extracts from WT mice contained much higher levels of both IFN-γ and IL-6 than those from KitW-sh/KitW-sh
mice (Figure , C–E). In addition to their possible roles in activating macrophages and inducing MMP and cysteine protease expression (41
), which are also critical to AAA formation (4
), IL-6 and IFN-γ appeared to regulate the formation of AAA by modulating lesional SMC apoptosis (Figure , E and F). Our observations from in vitro cell-based assay and the murine AAA model not only demonstrate that mast cell–derived IL-6 and IFN-γ are essential to AAA but also suggest that other sources of IL-6 and IFN-γ, e.g., inflammatory cells such as T cells (3
) and macrophages (42
), do not compensate for their absence in mast cells. Such cell type–dependent functional specificity is not unique to IL-6 and IFN-γ, as similar activity has been reported for other cytokines. For example, mast cell TNF-α is required for recruiting neutrophils to infected tissue compartments (43
), macrophage and neutrophil TNF-α are required for resistance to intracellular Listeria
infection, and T cell TNF-α is required for protection from high bacterial loads (44
). Thus, while it may seem intuitive that, because cytokines are made by many cell types, the absence of their production by one cell type would be compensated for by their production by another cell type, when tested in experimental models, this compensation does not always prove to be true, and depending on the end point measured, a single cellular source of a cytokine can have profound influence over modulating that end point.
Our in vitro experiments demonstrate that mast cell–derived IL-6 and IFN-γ are required for apoptosis but not neovascularization. Insignificant differences in the microvessel growth from the aortic ring Matrigel assay between WT and IL-6–, IFN-γ–, and TNF-α–deficient BMMCs suggest that mast cell mediators other than IL-6, IFN-γ, and TNF-α modulate neovascularization in this assay even though IFN-γ, IL-6, and TNF-α have been implicated in angiogenesis. In fact, mast cell–derived bFGF, angiopoietin-1, chymase/tryptase, and a number of other proinflammatory cytokines and chemokines (45
) have been implicated in angiogenesis. These mast cell mediators may have obscured the effect of mast cell IL-6, IFN-γ, and TNF-α in our in vitro assays (Figure , A–C).
Pharmacologic regulation of mast cell activation and degranulation directly controlled AAA formation in mice (Figure , F and G). These data not only strengthen the hypothesis that mast cells are essential mediators of AAA pathogenesis but also suggest that treatments blocking mast cell degranulation may prevent or delay AAA progression in humans. Compound 48/80 has been widely used to degranulate mast cells in live animals (46
). Although the detailed mechanisms of how C48/80 activates mast cells need to be investigated further, recent observations suggest that C48/80 acts on mast cells via activation of phospholipase D (47
) and Gαi3
G protein subunits (48
) to trigger the release of histamine as well as other granule components including cathepsins and MMP-activating chymases or cytokines, which may influence neighboring vascular cells. This hypothesis is supported by the findings of increased cysteine protease and MMP activities, elevation of AAA tissue IFN-γ and IL-6, and enhanced medial elastin degradation in aortic tissues isolated from C48/80-treated mice (Figure ). In contrast, DSCG is a drug widely used in the treatment of asthmatic patients. Observations from in vitro tests and animal models show that the effect of DSCG is related to mast cell stabilization. Importantly, this compound reduces mouse AAA by 40% (Figure ). Impaired AAA in DSCG-treated mice is likely due to reduced release of mast cell granule contents. This hypothesis is consistent with reduced AAA lesion cysteine protease and MMP activities and IFN-γ and IL-6 levels found in DSCG-treated mice (Figures and ).
While this study provides evidence that mast cells regulate AAA formation, it also leaves several important questions unanswered. First, depletion of neutrophils in mice impairs elastase perfusion–induced AAA (2
), and mice lacking T lymphocytes have protection from CaCl2
-induced AAA (3
). Determination of whether lack of mast cells in the current study affected the function of either neutrophils or T lymphocytes will require further investigation. Although we did not find significant differences of elastase activity of neutrophils from WT and KitW-sh/KitW-sh
mice, unrecognized interactions between these cell types may exist. Second, it remains unknown what mediated the increased number of mast cells in the injured aortas following elastase perfusion. Five weeks after reconstitution, we detected few if any mast cells in the aortas. In contrast, elastase perfusion enhanced mast cell numbers in the aortas. One possible explanation is that elastase perfusion induces aortic wall SMC expression of chemokines MCP-1 and RANTES (49
), which may attract mast cells (50
). Further, elastase perfusion may generate elastin degradation products that signal fibroblast, monocyte, and macrophage chemotaxis (51
). Delineation of the exact mechanism will require further investigation. Third, mast cell–mediated neovascularization and SMC apoptosis remain poorly understood. Although our data suggest that SMC apoptosis involves mast cell–derived IFN-γ and IL-6, whether these cytokines regulate SMC apoptosis directly or indirectly remains unknown. Our prior study demonstrated that mast cell IFN-γ and IL-6 augment SMC and EC expression of cysteine proteases (19
), which participate in cell apoptosis (52
). IFN-γ and IL-6 from mast cells may have induced cathepsin expression and further led to SMC apoptosis, a hypothesis that has not been tested in the current study. Fourth, it remains uncertain whether increased expression and activities of cathepsins and MMP in AAA lesions (Figure ) come from mast cells or other vascular cells. Mast cells contain large quantities of cathepsins (53
) and mast cell chymases that participate importantly in MMP activation (11
). However, mast cell cytokines (e.g., IL-6 and IFN-γ) are known stimulators of cathepsin and MMP expression, and our previous study (19
) and the current study demonstrate concurrently their importance in arterial wall remodeling. Therefore, it is possible that mast cells contribute to AAA by releasing both proteases and cytokines, a hypothesis that requires further investigation.
In summary, data from this study establish that mast cells play a role in the development of AAA by affecting matrix-degrading protease expression, medial SMC apoptosis, and microvessel growth. More importantly, our data suggest a novel and effective therapeutic strategy to control human AAA progression by preventing release of mast cell products using mast cell–stabilizing compounds.