These findings suggest that eventually more than one

These findings suggest that eventually more than one 12/15-lipoxygenase isoform may play a role in human atherosclerosis. Humans have two 12-lipoxygenases and two 15-lipoxygenases which show different expression patterns, substrate specificities and stereo-selective metabolism [1]. However, in BCTC synthesis involved in atherosclerosis only the 12-lipoxygenase, ALOX12, and the two 15-lipoxygenases, ALOX15 and ALOX15B, seem to be expressed [15]. To identify the major 12/15-lipoxygenases in human macrophages and to better understand their role in human atherosclerosis, we investigated the basal and stimulated expression of these three 12/15-lipoxygenase isoforms ALOX12, ALOX15 and ALOX15B in human primary macrophages.
Materials and methods
Results
Discussion In addition to the differentiation of monocytes into resident macrophages, macrophages can be polarized into at least two subpopulations, identified as M1 and M2 macrophages, analogous to the well characterized Th1 and Th2 subpopulations of T-helper cells [18]. Stimulation of macrophages with INF-γ (released by Th1 cells) and LPS will lead to the classically activated pro-inflammatory M1 macrophages, which secrete IL-1β, IL-15, IL-18, TNF-α and IL-12 [19]. Such M1 cells are characterized by enhanced endocytic functions and enhanced ability to kill intracellular pathogens. In contrast, stimulation of macrophages with IL-4, IL-10 (both released by Th2 cells), IL-13, glucocorticoids and TGF-β will lead to an anti-phlogistic macrophage phenotype M2 involved in tissue regeneration and homeostasis [18], [19]. Both types of activated macrophages have been detected in atherosclerotic plaques, although the role of the differently activated macrophages in human atherosclerosis has not been fully elucidated [20], [21]. Classically activated M1 macrophages predominate in the lipid core of human carotid atherosclerotic lesions, whereas anti-phlogistic M2 macrophages prevail in the shoulder region as well as in the periphery of the plaque [22]. There is some indication from the secreted cytokine profile of polarized macrophages that classically activated M1 macrophages are pro-atherogenic, while the anti-phlogistic M2 macrophages are atheroprotective [23]. The surprising finding of our study is that human M1 macrophages stimulated by LPS express ALOX15B to high levels. So far 12/15-lipoxygenase activity was only reported for IL-4 stimulated anti-phlogistic M2 macrophages [9] but not for the classically activated M1 macrophages. Our data suggest that such M1 macrophages will specifically express ALOX15B while the M2 macrophages express both ALOX15 and ALO15B isoforms to similar levels. From an enzymatic point of view there are differences between the two 15-lipoxygenases considering substrate preference and positional specificity, which may even influence the role of the two iso-enzymes in atherosclerosis. In general, ALOX15B seems to preferentially oxygenate arachidonic acid leading to the formation of the anti-inflammatory lipid mediators while it metabolizes linoleic acid to 13(S)-hydro(per)oxy-octadecadienoic acid (13-HpODE) involved in LDL-oxidation to a lesser extend than ALOX15 [24], [25]. In addition, ALOX15B was reported to be enzymatically active for longer periods, in contrast to ALOX15 for which rapid suicide inactivation has been observed [25], [26]. These substrate preferences and the lack of suicide inactivation may suggest an even more anti-inflammatory metabolite profile for human ALOX15B than for ALOX15. ALOX15 expression was co-localized with LDL to macrophage-rich areas in early atherosclerotic lesions in humans [27], [28], [29], [30] and specific ALOX15 products were observed in these lesions [29], [31]. However, conflicting results have been observed about the temporal and regiospecific expression patterns of the peroxidizing ALOX15 enzyme in humans. ALOX15 protein and mRNA were detected in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions by in situ hybridization [31]. The expression of ALOX15 in early atherosclerotic lesions was corroborated by measuring the specific ALOX15 metabolite 13-HpODE in arterial sections [29], [32]. This study showed that the specific ALOX15 metabolite was abundant in early human atherosclerotic lesions but was not in later stages of plaque development, where non-enzymatic lipid peroxidation surpassed the ALOX15 dependent lipid peroxidation [29], [32]. Therefore, ALOX15 may play a role in the initiation of atherosclerosis but not in later stages of atherogenesis. However, the expression of ALOX15 in early atherosclerotic development was later questioned by investigations which only detected minor human ALOX15 mRNA and protein in atherosclerotic lesions and found no co-localization of ALOX15 with macrophages [15], [33]. Instead, ALOX15B expression was detected in human carotid atherosclerotic plaques and immunohistochemical analysis showed abundant ALOX15B in macrophages-rich areas of carotid lesions [13], [14], [15]. In these studies hypoxia was shown to regulate ALOX15B mRNA expression through a HIF-1 alpha mediated mechanism [14]. Our findings corroborate the upregulation of ALOX15B by hypoxia and expand our knowledge on the regulation of ALOX15B in macrophages, showing that the enzyme is not only regulated by HIF-1 alpha but also by IL-4 and LPS. Hence, a larger set of macrophages will have 15-lipoxygenase activity from ALOX15B expression including certain sets of M1 and M2 macrophages.