IL-13 is a powerful stimulator of inflammation and tissue remodeling and is a key effector cytokine at sites of Th2 inflammation. In these responses, IL-13 is purported to use the canonical STAT6 signaling pathway to mediate its tissue inflammatory and structural effects. This widely held belief is the result of the well-documented ability of IL-13 to activate STAT6 in a variety of tissues (20
). In addition, studies from a number of laboratories have demonstrated that many of the effects of intratracheal IL-13 and Tg IL-13 are diminished in STAT6-null mice (7
). However, IL-13 can also activate MAPKs in cell lines in vitro (20
). These observations led us to hypothesize that IL-13 might also signal in vivo via MAPK-dependent pathways. To address this hypothesis, we took advantage of Tg systems established in our laboratory in which IL-13 can be selectively targeted to the murine lung (4
). The current studies demonstrate, we believe for the first time, that IL-13 is a potent and early stimulator of ERK1/2 MAPK. Our data also demonstrate that ERK1/2 activation is a proximal event in the IL-13 pathway that occurs in the absence of STAT6. Lastly, we demonstrate, using chemical and genetic approaches, that ERK1/2 MAPK activation plays a critical role in IL-13–induced inflammation and alveolar remodeling.
In accord with the importance of IL-13 in inflammation and disease, many studies have sought to define the mechanisms of IL-13–induced tissue alterations. These studies have demonstrated that the effects of IL-13 are mediated to a great extent by its ability to regulate a number of downstream genes and effector pathways (6
). Prominent in this regard are studies from our laboratory that demonstrate that IL-13 is a powerful stimulator of macrophage and epithelial chemokines and that chemokine signaling via CC chemokine receptor 2 (CCR2) and CCR1 is required for optimal IL-13–induced inflammation and remodeling (6
). Given that we found significant ERK1/2 activation in IL-13
Tg mice, studies were undertaken to define the role(s) of this activation in IL-13–induced inflammation. These studies demonstrate that chemical (PD) and genetic approaches preventing ERK1/2 activation diminish IL-13–induced tissue inflammation. They also provide insight into potential mechanisms of these inflammatory alterations by demonstrating that ERK1/2 activation plays an essential role in IL-13 stimulation of MIP-1α/CCL-3, MIP-1β/CCL-4, and MIP-2/CXCL-1. Interestingly, these effects were chemokine-specific because eotaxin/CCL-11, C10/CCL-6, and MCP-1/CCL-2 were not altered by these interventions.
As noted above, our studies demonstrate that ERK1/2-based interventions decrease tissue and BAL-fluid eosinophilia without altering IL-13 stimulation of eotaxin/CCL-11. This is surprising in light of the well-known ability of eotaxin/CCL-11 to recruit and activate eosinophils (31
). However, studies from our laboratory and others have demonstrated that IL-13–induced eosinophilia and eotaxin/CCL-11 production can be dissociated. In our experiments, the neutralization of C10/CCL6 and the genetic ablation of its putative receptor, CCR1, both diminished IL-13–induced BAL-fluid and tissue eosinophilia without altering eotaxin/CCL-11 induction (26
). Similarly, Yang et al. demonstrated that IL-13 induces eosinophilia via a mechanism that is independent of eotaxin/CCL-11 and IL-5 (13
). In evaluating these findings, it is important to appreciate that eosinophil chemotaxis is also regulated by chemokines such as MIP-1α/CCL-3, MIP-1β/CCL-4, and RANTES/CCL-5 (34
). In accord with these observations, our ERK1/2-based interventions diminished the production of all 3 of these moieties. When our studies and the literature are viewed in combination, it is clear that eotaxin/CCL-11 is induced by IL-13 via a mechanism that is largely ERK1/2, C10/CCL6, and CCR1 independent. They also demonstrate that chemokines other than eotaxin/CCL-11 are required for maximal IL-13–induced BAL-fluid and tissue eosinophil responses.
Our studies demonstrate that chemical and genetic interventions blocking ERK1/2 MAPK activation ameliorate IL-13–induced alveolar remodeling. These effects could be caused by a decrease in IL-13 production or an alteration in IL-13 effector function(s). Our results suggest that effector mechanisms were altered because similar levels of IL-13 were found in BAL fluids from Tg+
mice treated with PD or its vehicle control and Tg+
mice in which dnMEK1 was or was not expressed. A variety of effector pathways might contribute to these findings, including the diminished production of important proteases or the enhanced production of antiproteases. Our studies demonstrate that the ERK1/2 MAPK pathway plays important roles in the regulation of each of these moieties. Specifically, ERK1/2 activation was required for optimal IL-13 stimulation of MMP-2, -9, -12, and -14, and cathepsin B and IL-13 inhibition of α1-AT. These are, to our knowledge, the first studies to demonstrate the importance of ERK1/2 or any MAPK in the pathogenesis of IL-13–induced tissue remodeling responses. We believe they are also the first to demonstrate that IL-13 stimulates MMPs and cathepsin B and inhibits α1-AT via ERK1/2 MAPK-dependent pathways. These findings have important implications for diseases in which IL-13 dysregulation, protease excess, and tissue destruction coexist. They have particularly intriguing implications for pulmonary emphysema since cigarette smoke exposure has been shown to induce IL-13 elaboration in the murine lung (36
), polymorphisms in IL-13 have been associated with emphysema (37
), and mice exposed to cigarette smoke as well as lung tissues from patients with emphysema exhibit increased levels of ERK1/2 MAPK activation (38
To define the role(s) of ERK1/2 MAPK in the pathogenesis of IL-13–induced tissue responses, we initially used the MEK1 inhibitor PD and its corresponding vehicle control. Because the specificity of pharmacologic inhibitors can always be questioned, we also used genetic approaches to address this issue. To accomplish this, we generated IL-13 Tg+ mice that expressed and did not express dnMEK1. As shown in Figure , mice that expressed this MEK1 inactivating construct were unable to activate downstream ERK1/2 MAPK in our modeling system. Importantly, identical results were noted with the chemical and genetic approaches. Specifically, both interventions decreased IL-13–induced inflammation, alveolar remodeling, MIP-1α/CCL-3, MIP-1β/CCL-4, RANTES/CCL-5, and MIP-2/CXCL-1 production, and MMP-2, -9, -12, and -14 and cathepsin B mRNA accumulation while increasing α1-AT expression. These mutually supportive studies demonstrate that ERK1/2 MAPK plays critical roles in these responses. Since the dnMEK1 construct is selectively targeted to the epithelial cells in our Tg mice, these studies also suggest that ERK1/2 signaling in lung epithelia is particularly crucial in these responses.
It is important to point out that in most cases, ERK1/2 inhibition decreased but did not totally reverse IL-13–induced tissue and molecular alterations. Given that we show virtually complete ERK1/2 inactivation with PD or dnMEK1 (see Figure ), we conclude that there are aspects of the IL-13 phenotype that are mediated, at least in part, by alternative signaling pathways. Previous studies from our laboratory and our associates demonstrated that STAT6 also plays an important role in the pathogenesis of selected IL-13–induced tissue events (7
). As a result, studies were undertaken to compare the relative contributions of ERK1/2 and STAT6 signaling in the tissue and regulatory effects of IL-13. These studies highlight complex relationships between ERK1/2 and STAT6 in the genesis of these responses. This can be easily seen in IL-13–induced inflammation where ERK1/2 and STAT6 play similar roles in tissue and BAL-fluid eosinophilia but only STAT6 contributes to lymphocyte and neutrophil accumulation. In accord with this finding, IL-13 stimulated pulmonary chemokines via pathways that were equally dependent on ERK1/2 and STAT6, largely dependent on STAT6 and largely dependent on ERK1/2. Similarly, both the ERK1/2 and STAT6 pathways contributed to IL-13–induced alveolar remodeling, with each pathway contributing in specific ways to the regulation of the proteases and antiproteases that mediate these responses. In contrast, STAT6 was the dominant pathway, and ERK1/2 played a minimal role in the pathogenesis of IL-13–induced mucus metaplasia. STAT6 was not, however, the only signaling pathway involved in these responses because null mutations of this gene did not completely abolish IL-13–induced goblet cell hyperplasia or Muc-5ac and Gob-5 expression. These are, to our knowledge, the first studies to appreciate the importance of ERK1/2 MAPK pathways in the pathogenesis of the in vivo effects of IL-13. We believe they are also the first to define the effector repertoires of the ERK1/2 and STAT6 pathways in this setting. Collectively, they demonstrate that the ERK1/2 and STAT6 pathways play similar and dissimilar roles in the pathogenesis of the tissue and molecular effects of IL-13. These observations provide a clear rationale for the use of ERK1/2 MAPK pathway inhibitors, alone or in combination with STAT6 inhibitors, in the treatment of IL-13–induced disorders.
In summary, our studies demonstrate that IL-13 is a potent activator of ERK1/2 MAPK and that this activation takes place even in the absence of STAT6. They also demonstrate that ERK1/2 MAPK activation plays a critical role(s) in the pathogenesis of IL-13–induced inflammation and alveolar remodeling and that the effects of this activation can be similar to and different from those mediated by STAT6. Lastly, they provide mechanistic insights by demonstrating that ERK1/2 MAPK activation is required for optimal IL-13 stimulation of chemokines (MIP-1α/CCL-3, MIP-1β/CCL-4, MIP-2/CXCL-1, and RANTES/CCL-5) and proteases (MMP-2, -9, -12, and -14 and cathepsin B) and IL-13 inhibition of α1-AT. Exaggerated IL-13 production has been implicated in the pathogenesis of a wide variety of disorders, including asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, scleroderma, hepatic fibrosis, and nodular sclerosing Hodgkin disease. The present studies suggest that the effects of IL-13 in these disorders may be beneficially controlled via interventions that control and/or prevent the activation of ERK1/2 MAPK. This establishes the ERK1/2 MAPK pathway as a worthwhile site for future investigation designed to identify therapeutic agents that can be used to treat these and other IL-13–mediated disorders.