CysLTs function through their ability to interact with two homologous receptors. The CysLT type 1 receptor is prominently expressed on airway smooth muscle, eosinophils, and other immune cells, and these receptors mediate CysLT-induced bronchospasm (3
). CysLT2 receptors (4
) are prominently expressed in the heart, prostate, brain, adrenal cells, endothelium, and lung but are expressed at lower levels on eosinophils, monocytes, T and B lymphocytes, and mast cells (7
). The precise function of CysLT2 receptors in allergic disease and immunity is not known, although the CysLT2 receptor is thought to play a greater role in remodeling and fibrotic processes (14
). Both papers describing the cloning of the CysLT1 and CysLT2 receptors (3
) suggest limited expression of these receptors on circulating immune cells. In contrast, the increased expression of these receptors in asthma has been demonstrated (5
), and we have reported their increased expression in allergic rhinitis (6
). CysLT1 receptors were expressed on the majority of eosinophils and in subsets of mast cells, monocytes, macrophages, and neutrophils. CysLT2 receptors were expressed on most eosinophils, mast cells, and monocytes/macrophages, but not on neutrophils. The increased expression was observed both at the mRNA and protein levels. Similarly, in aspirin-exacerbated respiratory disease (AERD), 31% of the T cells in the nasal mucosa were positive for CysLT1 receptors (11
). Together, these studies suggest that during an allergic inflammatory insult, T cells can up-regulate their CysLT receptor expression. Alternatively, it is plausible that CysLT receptor–expressing T cells may either be selectively recruited into the nasal and sinus tissue or may selectively expand or survive under the influence of locally produced CysLTs (15
Previous studies have indicated that CysLT receptor expression can be altered depending on cell type and cytokine stimulus. IL-4 up-regulates cell surface expression of both CysLT1 and CysLT2 receptors on mast cells reportedly without altering mRNA or protein levels in the cells (16
). In contrast, IL-4 stimulated CysLT2 receptor mRNA production in endothelial cells, and both IL-4 and IL-13 stimulated mRNA and cell surface expression of the CysLT1 receptor in monocytes (13
). In the latter study, IFN-γ decreased CysLT1 receptor mRNA expression in monocytes, while IFN-γ stimulated CysLT1 and CysLT2 receptor mRNA production and CysLT1 receptor surface expression on smooth muscle cells (13
). IL-5 increased CysLT1 receptor mRNA and cell surface expression on a human eosinophil cell line (19
), and recently it has been reported that IFN-γ increases CysLT2 receptor expression on eosinophils from patients with asthma (20
). Together these studies broadly suggest the ability of cytokines associated with allergic inflammation to up-regulate expression of CysLT receptors. Our data extend these previous studies and examine a broader range of cytokines and expression on multiple immune cell types. The most profound results were observed for IL-4 stimulation of the CysLT2 receptor. Significant increases in expression of the CysLT2 receptor were seen on T and B lymphocytes and eosinophils (, ). IL-4 also up-regulated CysLT1 receptor mRNA expression on T and B cells with trends toward increased expression on monocytes and eosinophils (, ). This increase in CysLT1 receptor expression is consistent with the recent identification of a STAT6 response element in the CysLT1 receptor promoter that mediates responses to IL-4 stimulation (21
). The increase in receptor number after IL-4 stimulation resulted in increased responsiveness of T lymphocytes to LTD4
as measured by higher levels of calcium mobilization ().
Surprisingly, in addition to IL-4, IFN-γ significantly increased expression of the CysLT2 receptor on monocytes and T and B lymphocytes (, ). Asthma has traditionally been defined as a Th2 cytokine–mediated disease; however, recent evidence indicates that severe asthma is associated with increased expression of IFN-γ (22
). Our data suggest that under these conditions, the expression of CysLT2 receptors will preferentially be increased. This may have important implications for remodeling. CysLT1 receptors on smooth muscle mediate the bronchospastic effects of CysLT, whereas CysLT2 receptors may have a complementary or even a predominant role in remodeling and fibrosis pathways (14
). Thus, the proliferative effects mediated by CysLT on myofibroblasts and smooth muscle are blocked by less specific CysLT receptor antagonists but not by an antagonist specific for CysLT1 receptors (24
). CysLT2 receptors are prominently expressed in cardiac myocytes and, as such, support for a role for CysLT2 receptors in fibrosis is derived from observations that conditions associated with intravascular activation of eosinophils, such as hypereosinophil and Churg-Strauss syndromes, can lead to the development of endomyocardial fibrosis. In a bleomycin-induced model of pulmonary fibrosis, fibrosis does not develop in either 5-LO or LTC4
S knockout mice (25
). Surprisingly, when these studies were performed in the CysLT1 receptor knockout mouse, enhanced fibrosis was observed (27
). In contrast, the CysLT2 receptor knockout mouse had markedly reduced inflammation and fibrosis (28
). Conflicting results observed in a CysLT1 receptor antagonist study (29
) may reflect the different models, activity of montelukast on CysLT secretion and, at higher concentrations, directly upon CysLT2 receptors, or synergism between the two receptors. These data support the concept that severe persistent asthma with remodeling, which is characterized by high levels of IFN-γ, may be driven in part by increased expression of the CysLT2 receptor and CysLT2 receptor–mediated pro-fibrotic pathways.
Although beneficial effects of CysLT1 receptor antagonists have been anecdotally reported in chronic hyperplastic eosinophilic sinusitis/nasal polyposis (30
), the only successful controlled trial of a leukotriene modifier in this condition was reported with the 5-LO inhibitor zileuton (31
). Inhibition of 5-LO has broader implications, insofar as in addition to blockade of LTC4
, zileuton will block production of other proinflammatory lipids including LTB4
and 5(S)HETE (hydroxyeicosateraenoic acid). However, it is plausible that it is the ability of this agent to block CysLT production and therefore to inhibit activation mediated through both CysLT1 and CysLT2 receptors that is responsible for its clinical efficacy. Although combined CysLT1 and CysLT2 antagonists provide no superior benefit to selective CysLT1 antagonists in short-term studies evaluating lung function and rescue albuterol use, no studies have addressed inhibition of remodeling or fibrotic pathways.
In conclusion, we have demonstrated the ability of both Th1- and Th2-type cytokines to increase expression of both the CysLT1and -2 receptors at the mRNA and protein levels in many cell types. Combined with previous studies showing increased numbers of both CysLT1 and -2 receptor–expressing cells in inflamed tissue, our results are consistent with the concept that increased cytokine production increases expression of both receptors on mononuclear cells and granulocytes, allowing these cells to be more responsive to secreted leukotrienes.