The development of atherosclerotic plaques in the artery wall is driven by abnormal communication between blood cells, endothelial cells, and tissue-resident cells in a presence of inflammatory mediators and low-density lipoproteins. This process, referred to as atherogenesis, starts with adhesion of blood monocytes to activated vascular endothelium, which causes subendothelial migration of these cells and their differentiation into macrophages[1]. Monocyte extravasation to the tunica intima is regulated by endothelial cell adhesion molecules [2–4] and monocyte chemokines [5]. With the help of other tissue-resident cells, cytokines, and oxidized low-density lipoproteins (oxLDL), macrophages located in the intimal layer of the artery transform into lipid-rich foam cells, thereby causing the formation of a fatty streak, the first clinical sign of atherosclerosis [6].
The tissue-resident macrophages can be in a resting state (M0 type) or be polarized into pro-inflammatory (M1 type) or anti-inflammatory (M2 type) macrophages, depending on the stimuli present [7]. In early stages of atherosclerosis, most macrophages are M1 type [8] because IFN-γ, the major cytokine for pro-inflammatory polarization of macrophages, is more abundant than IL-4, the cytokine that induces anti-inflammatory polarization [9]. M1 macrophages initiate the immune response by releasing TNF-α and other pro-inflammatory mediators. They also produce nitric oxide or reactive oxygen intermediates (ROI) to protect against bacterial and viral pathogens. M2 macrophages assist in tissue repair [10] by inducing cell proliferation and collagen production via transforming growth factor beta 1 (TGF-β1) release [11]. Supporting the proper balance between M1 and M2 types is essential for immunity and tissue repair, but it does not occur during atherogenesis. In this pathophysiological process, when M1 macrophages switch to M2 type, they remain in this state and eventually become foam cells [12].
It is still unclear which factors interfere with macrophage polarization. However, recent experimental evidence points out that low-density lipoproteins, mast cells, and chemicals released during mast cell degranulation may be involved [13, 14]. Initially located in the adventitial layer of the artery wall, mast cells migrate to and become co-localized with macrophages in the intimal layer [15]. They contribute to the conversion of M2 macrophages to foam cells [16] and promote the atherosclerotic lesion formation through histamine release [17]. During late stages of atherosclerosis, they are located, together with macrophages, in the shoulder regions of vulnerable plaques [18]. The mechanism of how mast cells become co-localized with intimal macrophages is not yet explored.
Mast cells would migrate to the intima in response to chemotactic stimuli released by the cells present in this region. One of the chemokines involved in this process could be TGF- β1, which is known to induce the direct migration of mouse mast cells to the sites of bacterial infection at femtomolar concentrations [19, 20] through binding its serine/threonine receptors [21]. TGF- β1 can be produced by intimal M2 macrophages through SMAD2 signaling [17], but this requires re-polarization of M1 macrophages, predominantly present in early atherosclerotic sites, into M2 type. Cytokines such as interleukin-6 (IL-6) and/or lipoproteins can be involved in the process of M1 to M2 transformation. IL-6 increases the expression of CD206, a M2 macrophage marker, and the CD206/CD86 expression ratio, where CD86 is a M1 macrophage marker [22]. Macrophages are also sensitive to low-density lipoprotein (LDL), especially to its oxidized form, oxLDL. For example, LDL upregulates the expression of integrins on monocytes and accelerates their transformation into M0 macrophages [23], while oxLDL enhances cytokine (TNF-α) release from macrophages [13], and, at the same time, assists in the transformation of M1 macrophages into foam cells [24]. Clinical data support important roles of IL-6 and oxLDL in atherosclerosis and coronary artery disease. Particularly, patients with family history of premature coronary artery disease have a significantly higher level of plasma IL-6 [25]. There is also a positive correlation between the plasma level of oxLDL and coronary artery disease [26].
Based on these data, we hypothesize that IL-6 in combination with LDL induces TGF-β1 release from M1 macrophages, which in turn leads to mast cell migration toward macrophages. Adhesion and migration assays as well as ELISA are employed to test this hypothesis.