Increased levels of IL-6 are documented in asthma, but its contribution to the pathology is unknown. Asthma is characterized by airway wall thickening due to increased extracellular matrix deposition, inflammation, angiogenesis, and airway smooth muscle (ASM) mass. IL-6 binds to a specific membrane-bound receptor, IL-6 receptor-α (mIL-6Rα), and subsequently to the signaling protein gp130. Alternatively, IL-6 can bind to soluble IL-6 recpetor-α (sIL-6Rα) to stimulate membrane receptor-deficient cells, a process called trans-signaling. We discovered that primary human ASM cells do not express mIL-6Rα and, therefore, investigated the effect of IL-6 trans-signaling on the pro-remodeling phenotype of ASM. ASM required sIL-6Rα to activate signal transducer and activator 3, with no differences observed between cells from asthmatic subjects compared with controls. Further analysis revealed that IL-6 alone or with sIL-6Rα did not induce release of matrix-stimulating factors (including connective tissue growth factor, fibronectin, or integrins) and had no effect on mast cell adhesion to ASM or ASM proliferation. However, in the presence of sIL-6Rα, IL-6 increased eotaxin and VEGF release and may thereby contribute to local inflammation and vessel expansion in airway walls of asthmatic subjects. As levels of sIL-6Rα are increased in asthma, this demonstration of IL-6 trans-signaling in ASM has relevance to the development of airway remodeling.
- soluble interleukin-6 receptor-α
- signal transducer and activator 3
- airway remodeling
il-6 is a potent pleiotropic cytokine that mediates a plethora of pathophysiological roles in inflammatory diseases. IL-6 exerts its activities via binding to two functionally different membrane glycoproteins: the ligand binding receptor IL-6 receptor-α (IL-6Rα) (also known as gp80), and the signal-transducing receptor gp130. After IL-6 specifically binds to IL-6Rα, the IL-6-IL-6Rα complex then recruits the gp130 signaling receptor, which initiates further downstream signaling via phosphorylation and nuclear translocation of signal transducer and activator 3 (STAT3) to allow resultant cellular events (32).
Although gp130 is ubiquitously expressed in the cell membrane, the expression of the membrane-bound form of IL-6Rα (mIL-6Rα) is more restricted and tightly regulated, leaving many cell types without the capacity to respond to IL-6 via the classical mIL-6Rα-gp130 complex (31). However, a soluble form of the cognate IL-6Rα (sIL-6Rα) exists and provides IL-6 with an alternative mechanism by which it can activate gp130 and thus induce signaling in cell types that cannot intrinsically respond to IL-6. This alternative mechanism, termed IL-6 trans-signaling, may prove to be critically important in the development of airway remodeling, because sIL-6Rα has been shown to be elevated in asthma (11, 57) and other inflammatory diseases (30, 31, 47).
In this study, we have investigated the effect of IL-6 trans-signaling on airway smooth muscle (ASM), a pivotal cell with important roles in airway remodeling. More than just a contractile cell responsible for bronchomotor tone alone, ASM has emerged as playing an important role in promotion of the remodeled phenotype in asthma, including ECM deposition, ASM proliferation, mast cell recruitment, and the secretion of inflammatory mediators (39). The role and importance of IL-6 in mediating these pro-remodeling effects in asthma have been an area of some considerable debate (10, 13–15, 38, 41, 43). In this study, we demonstrate that IL-6Rα is not expressed on human ASM and that, for IL-6 to signal through STAT3 via gp130, the presence of sIL-6Rα is required. As levels of sIL-6Rα are increased in asthma (11, 57), this demonstration of IL-6 trans-signaling in ASM has relevance to the development of airway remodeling.
Human ASM cells were obtained from subjects without and with asthma by methods adapted from those previously described (26, 29), in accordance with procedures approved by the Sydney South West Area Health Service Ethics Committee and the Human Ethics Committee of the University of Sydney. A minimum of three different primary cell lines was used for each experiment. Human lung fibroblasts were cultured as described previously (48, 50). Mast cell adhesion was examined using human mast cell type 1 (HMC-1), an immature-type mast cell line, kindly provided by Dr. Joseph H. Butterfield (Mayo Clinic, Rochester, MI), cultured in RPMI-1640 supplemented with 5% fetal bovine serum.
Unless otherwise specified, all chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO).
To examine expression of gp130 and mIL-6Rα protein, growth-arrested ASM cells were treated with 5% fetal bovine serum for 0, 12, and 24 h and lysed in Laemmli buffer. Proteins were separated by 6% SDS-PAGE, and immunoblotting was used to detect gp130 (mouse monoclonal IgG1, clone 29104; R&D Systems, Minneapolis, MN) and IL-6Rα (mouse monoclonal IgG1, clone 17506; R&D Systems). Human lung primary fibroblasts were used as a positive control cell line in which both mIL-6Rα and gp130 are known to be expressed (48). Ponceau staining was performed to ensure equal protein loading in each lane.
In parallel experiments, the effect of sIL-6Rα on IL-6-induced STAT3 phosphorylation in cytosolic and nuclear protein extracts of ASM cells was examined. ASM cells were growth-arrested and then treated for 0, 10, 30, 45, and 60 min with IL-6 (10 ng/ml) and sIL-6Rα (10 ng/ml), alone or in combination. Nuclear and cytoplasmic protein extracts were prepared according to previously published methodology (49). Extracts were subjected to 8% SDS-PAGE and STAT3 phosphorylation and quantified by immunoblotting using rabbit polyclonal antibodies against total and phosphorylated-STAT3 (Tyr-705) (Cell Signaling Technology, Danvers, MA). Primary antibodies were detected with goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and visualized by enhanced chemiluminescence (Perkin Elmer, Wellesley, MA).
To measure the effect of IL-6, in the presence and absence of sIL-6Rα on the production of proteins implicated in airway fibrosis, confluent growth-arrested ASM cells were treated with vehicle, IL-6 (10 ng/ml), sIL-6Rα (10 ng/ml), or IL-6 and sIL-6Rα in combination (both at 10 ng/ml). The expression of connective tissue growth factor (CTGF), collagen type I, fibronectin, or cell surface integrin subunits was examined after 24- or 48-h incubation at 37°C in 5% CO2.
CTGF, collagen type I, and fibronectin were measured by ELISA. CTGF was detected using a goat polyclonal anti-human CTGF primary antibody (L-20, Santa Cruz Biotechnology, Santa Cruz, CA) with rabbit anti-goat horseradish peroxidase-conjugated secondary antibody (DakoCytomation, Carpenteria, CA), while the antibodies for collagen type I and fibronectin were described previously (28).
The expression of integrin subunits α1, α2, α4, α5, αv, and β1 on the ASM cell surface was measured by flow cytometry. ASM cells were rapidly harvested using phenol-red free trypsin-EDTA, washed, and resuspended in phosphate-buffered saline containing 1% bovine serum albumin (30 min on ice) to prevent nonspecific antibody binding. After centrifugation, cells were incubated with the specific anti-integrin monoclonal antibodies, or appropriate isotype control (30 min on ice). Monoclonal antibodies used for flow cytometry (Chemicon International, Temecula, CA) were directly conjugated with either fluorescein-5-isothiocyanate or phycoerythrin [i.e., anti-α2-fluorescein-5-isothiocyanate (clone AK7), anti-α4-phycoerythrin (clone BU49), anti-α5-fluorescein-5-isothiocyanate (clone SAM-1), anti-αv-phycoerythrin (clone 13C2)], or unlabeled primary antibodies [i.e., anti-α1 (clone FB12), anti-β1 (clone 6S6)] used with a phycoerythrin-labeled secondary antibody. Following staining, cells were washed and fixed in 4% paraformaldehyde for 20 min. Samples were then analyzed using Cell Quest software and a FACSCalibur Sort flow cytometer (BD Biosciences, Palo Alto, CA). Typically 10,000 events were acquired in the viable region of the forward scatter/side scatter plots to examine ASM cell surface expression of integrin subunits. The geometric mean for gated events is expressed as a fold increase in fluorescence intensity for positive staining above the geometric mean determined for the relevant isotype control for each treatment.
Mast cell adhesion.
To measure the effect of IL-6, in the absence and presence of sIL-6Rα on mast cell adhesion to confluent growth-arrested ASM cells, HMC-1 were treated with either vehicle (0.1% bovine serum albumin in DMEM), IL-6 (10 ng/ml), sIL-6Rα (10 ng/ml), or IL-6 and sIL-6Rα in combination (both 10 ng/ml; R&D Systems). After 24-h incubation at 37°C in 5% CO2, HMC-1 cells were washed, and 2 × 104 cells were added to chamber slide wells containing growth-arrested ASM (1 × 104 cells/well) for 0, 10, or 60 min at 37°C. The medium and nonadherent cells were then removed, and the wells washed gently. The remaining attached HMC-1 were fixed with Carnoy's fixative for 30 min and air dried. Attached cells were stained with Kimura Light, and only HMC-1 cells attached to ASM cells were counted in 10 consecutive fields of view across and down the slide for each well using ×200 magnification.
ASM cell proliferation was measured using [3H]thymidine incorporation, as previously described (2). We examined the effect of sIL-6Rα on IL-6-induced ASM cell proliferation in two ways: first, we measured cell proliferation using increasing concentrations of sIL-6Rα (1–100 ng/ml), in the absence and presence of 10 ng/ml IL-6; second, we measured cell proliferation in increasing concentrations of IL-6 (1–100 ng/ml), in the absence and presence of excess sIL-6Rα (i.e., 100 ng/ml). Cell proliferation to 1% FBS was measured as a positive control. Confluent, growth-arrested ASM were treated in the above manner for 16 h and then labeled with 1 μCi of [methyl-3H]thymidine (Perkin Elmer) for 24 h, and [3H]thymidine incorporation was measured as described previously (2).
Secretion of inflammatory mediators.
Confluent ASM cells were growth arrested for 48 h using DMEM with 0.1% bovine serum albumin. To examine whether sIL-6Rα is required for IL-6 to induce IL-8, VEGF, or eotaxin secretion, ASM cells were treated with IL-6 (10 ng/ml) and sIL-6Rα (10 ng/ml), alone or in combination. After 24-h incubation (37°C in 5% CO2), cell supernatants were removed and frozen at −20°C for later analysis by ELISA. IL-8, VEGF (both BD Biosciences Pharmingen), and eotaxin (R&D Systems) ELISAs were performed, according to the manufacturer's instructions.
Statistical analysis was performed using one-way analysis of variance and then Fisher's post hoc multiple-comparison test. P values < 0.05 were sufficient to reject the null hypothesis for all analyses. Data represent means ± SE.
ASM cells do not express mIL-6Rα.
For IL-6 to exert its activities, both membrane glycoproteins IL-6Rα and gp130 must be present on ASM cells. However, while gp130 is ubiquitously expressed, the expression of IL-6Rα is more restricted and tightly regulated (31). We have investigated, for the first time, the expression of the IL-6 receptor complex on ASM cells by examining the levels of mIL-6Rα and gp130 protein using cells from nonasthmatic subjects (Fig. 1A) and asthmatic subjects (Fig. 1B). The data clearly show that ASM do not express mIL-6Rα while gp130 is present. Figure 1C depicts a positive cell line (human lung fibroblasts) in which both mIL-6Rα and gp130 are expressed. Furthermore, gp130 can be regulated by serum, although there was no difference in the level of receptor expression between nonasthmatic subjects (Fig. 1A) and asthmatic subjects (Fig. 1B). These data are in contrast to those published by Lahiri et al. (38), who suggested that both receptor components were present on ASM cells. It is important to note, however, that the previous report (38) used nonquantitative PCR, and protein levels of the receptors were not shown.
sIL-6Rα is required for IL-6 to induce robust and sustained STAT3 phosphorylation.
Binding of the IL-6-IL-6Rα complex to the signal transducer gp130 induces cellular events via the phosphorylation and nuclear translocation of STAT3. IL-6 has previously been shown to be a relatively weak inducer of STAT3 phosphorylation in ASM, compared with other IL-6 family cytokines (38). As we have discovered that ASM cells are missing mIL-6Rα, a crucial piece of the IL-6 receptor complex, we investigated the effect of IL-6 on STAT3 phosphorylation in the presence of exogenous sIL-6Rα. As shown in Fig. 2, sIL-6Rα is necessary for IL-6 to induce robust and sustained STAT3 phosphorylation and nuclear translocation in ASM cells. In the absence of sIL-6Rα, IL-6-induced STAT3 phosphorylation is relatively transient, and the amount of translocation of phosphorylated STAT3 to the nucleus where it is required to mediate its effect on transcription is either small (Fig. 2A) or nonexistent (Fig. 2B). In contrast, the presence of sIL-6Rα allows IL-6 to induce robust levels of STAT3 phosphorylation, and the degree of STAT3 translocation is amplified and sustained for up to 45–60 min (Fig. 2). Although subtle differences exist between the temporal profiles of STAT3 phosphorylation and translocation using primary ASM cell lines obtained from nonasthmatic subjects (n = 3) and asthmatic subjects (n = 3), no major differences were observed. sIL-6Rα alone does not induce STAT3 phosphorylation or translocation (data not shown).
The effect of IL-6 trans-signaling on airway fibrosis.
Airway fibrosis is a hallmark of airway remodeling. ASM cells have emerged as playing a contributing role through their ability to produce ECM proteins [such as collagen type I and fibronectin (28)] and profibrotic proteins such as CTGF (7). ASM cells also express cell surface integrins that can be modulated by proinflammatory stimuli and through which the ASM cells interact with the ECM and drive the cells towards a remodeling phenotype (5, 16, 23, 27, 42, 44). To examine the hypothesis that IL-6 trans-signaling may promote airway fibrosis, we have examined the effect of IL-6 on the ASM production of CTGF, collagen type I, fibronectin, or cell surface integrin subunit expression, in the absence and presence of sIL-6Rα. As shown in Table 1, IL-6 alone did not induce changes in levels of CTGF, collagen type I, or fibronectin deposition by ASM cells from nonasthmatic subjects or asthmatic subjects. The levels of these ECM components were also unchanged by sIL-6Rα. Moreover, IL-6 did not modulate the expression of ASM cell surface integrin subunits α1, α2, α4, α5, αv, and β1, even in the presence of sIL-6Rα. There was also no difference in the degree of integrin subunit expression observed between nonasthmatic subjects and asthmatic subjects (Table 2). Taken together, these results show that IL-6 does not induce ASM production of proteins implicated in airway fibrosis, even in the presence of sIL-6Rα.
The effect of IL-6 trans-signaling on ASM mass.
Histological changes typical of remodeled airway include increased ASM mass. ASM cells are thought to undergo hyperplastic growth in response to a diverse range of mitogenic inflammatory mediators found in local proximity during the inflammatory onslaught of an acute asthma exacerbation (3, 22). Mast cells are an important source of mitogenic stimuli and in vivo are found in close proximity to the ASM layer in allergy (1) and asthma (6). In addition, IL-6 has been shown to increase mast cell integrin expression and adhesion to ECM (53). IL-6 may facilitate mast cell adhesion to ASM, leading to increased smooth muscle proliferative/synthetic activity and ASM mass. Therefore, we have taken two approaches to examine the effect of IL-6 trans-signaling: first, we examined the effect of IL-6 and sIL-6Rα, alone or in combination, on mast cell adhesion to ASM cells; second, we have measured IL-6-induced ASM cell proliferation in the absence and presence of sIL-6Rα.
Figure 3 clearly shows that the adhesion of the mast cell line HMC-1 to ASM increases over time. There was no significant difference between the amount of adhesion of vehicle-treated mast cells to ASM from nonasthmatic subjects (Fig. 3A), compared with asthmatic subjects (Fig. 3B). Furthermore, treatment of mast cells with IL-6, alone and in combination with sIL-6Rα, did not increase mast cell adhesion above that observed with vehicle-treated cells alone (Fig. 3). Thus IL-6 does not induce mast cell adhesion to ASM, even in the presence of sIL-6Rα.
Conflicting evidence exists as to whether IL-6 induces ASM proliferation. In 2000, McKay et al. (41) demonstrated that IL-6 was not a mitogen for human ASM cells. This was in contrast to earlier studies by De et al. (10), where IL-6 induced hyperplasia in ASM cells cultured from guinea pig. The lack of a promitogenic effect of IL-6 on human ASM cells, compared with guinea pig ASM cells, could simply reflect species differences, or be due to the fact that serum contains sIL-6Rα and the guinea pig cells were cultured in 1% FBS (10). Thus we examined whether the lack of a promitogenic effect of IL-6 on ASM was due to the fact that the in vitro conditions under which the study performed by McKay et al. (41) did not accurately reflect the in vivo milieu, because IL-6Rα, a crucial molecule required for IL-6 signal transduction, was missing in ASM. We examined the effect of sIL-6Rα on IL-6-induced ASM cell proliferation in two ways: first, we measured cell proliferation in response to increasing concentrations of sIL-6Rα in the absence and presence of IL-6 (Fig. 4A); second, we measured cell proliferation in increasing concentrations of IL-6, in the absence and presence of excess sIL-6Rα (Fig. 4B). As shown in Fig. 4A, IL-6 did not induce ASM cell proliferation. Furthermore, this was not augmented in the presence of increasing concentrations of sIL-6Rα. The lack of mitogenic response was not due to a limiting amount of IL-6, as increasing the concentration of IL-6 in the presence of excess sIL-6Rα had no significant effect on ASM cell proliferation (Fig. 4B). In contrast, the positive control, 1% FBS, significantly enhanced ASM cell growth by 2.7 ± 0.4-fold (data not shown) (P < 0.05). Thus, in corroboration with a previous report by McKay et al. (41), we confirm that IL-6 does not modulate ASM cell proliferation, even in the presence of sIL-6Rα.
IL-6 trans-signaling induces secretion of proinflammatory mediators.
Airway remodeling is a consequence of long-term airway inflammation. ASM cells secrete a wide range of proinflammatory mediators (24, 39); thus ASM is a pivotal airway structural cell responsible for the perpetuation and amplification of airway inflammation. Inflammatory mediators secreted by ASM cells include cytokines, such as IL-8 (25) and eotaxin (9, 18), and the angiogenic growth factor VEGF (36). Although VEGF (40) and eotaxin promoters (17, 20) do not contain GAS consequence sequences (the classical STAT3 binding element), STAT3 trans-activates VEGF transcription via a Sp1-binding element (40), while STAT3 overexpression increases eotaxin promoter activity in ASM (14). Secretion of IL-8 from ASM cells has not been shown to be STAT3 mediated.
Previous publications have shown that IL-6 is a relatively weak inducer of VEGF (15) and eotaxin (14) secretion from ASM. Importantly, the previous studies (14, 15) were performed in the absence of sIL-6Rα. As we now know that IL-6-induced STAT3 phosphorylation and translocation requires sIL-6Rα, we now wish to examine the effect of IL-6 trans-signaling on the secretion of proinflammatory mediators from ASM. As shown in Fig. 6, IL-6 alone does not induce secretion of IL-8, VEGF, or eotaxin from ASM from nonasthmatic subjects (Fig. 5A) or asthmatic subjects (Fig. 5B). sIL-6Rα alone is similarly without effect. In combination, however, IL-6 and sIL-6Rα significantly increase the secretion of eotaxin and VEGF (P < 0.05), but not IL-8. There was no significant difference between the responses of ASM cells from nonasthmatic subjects (Fig. 5A) compared with ASM cells from asthmatic subjects (Fig. 5B). Thus we can conclude that IL-6 trans-signaling induces secretion of proinflammatory and proangiogenic mediators from ASM. Further experiments are required to demonstrate whether STAT3 is directly involved.
The IL-6 receptor system consists of IL-6Rα and gp130. We have discovered that ASM cells do not express the membrane-bound ligand-binding chain IL-6Rα; hence ASM cannot intrinsically respond to IL-6. However, in the presence of exogenous sIL-6Rα, IL-6 can form an agonistic complex that can induce signaling via membrane-bound gp130. We found that sIL-6Rα is required for IL-6 to induce robust and sustained STAT3 phosphorylation and nuclear translocation. As levels of sIL-6Rα are increased in asthma, this demonstration of IL-6 trans-signaling has relevance to the development of airway remodeling. In corroboration, we have demonstrated that IL-6 trans-signaling directs ASM cells toward a pro-remodeling phenotype by increasing secretion of proinflammatory and proangiogenic mediators, possibly via a STAT3-mediated pathway, whereas increases in surrogates for ASM mass or airway fibrosis were unaffected.
There is increasing interest in understanding the role of soluble cytokine receptors in health and disease to explore their utilization as novel therapeutic targets in inflammation (30, 47). Membrane-bound and soluble receptors bind their ligands with similar affinity (47). While most soluble receptors then compete with their membrane-associated counterparts for their cytokine ligand, to thus function as antagonists, certain soluble receptors (such as sIL-6Rα) can act as agonists. With IL-6, sIL-6Rα forms an agonistic complex that binds cells that do not express the mIL-6Rα, via the ubiquitous gp130. Through this process, termed IL-6 trans-signaling, initiation of intracellular STAT3 signaling in cells occurs in cells that are usually unresponsive. Our study is the first to show that ASM cells do not express the mIL-6Rα. Hence ASM cannot intrinsically respond to IL-6, but, in the presence of sIL-6Rα, IL-6 can signal through STAT3 via gp130. In this way, ASM cells show similarity to a range of cell types, including vascular smooth muscle (35), endothelial cells (46), and T lymphocytes (4), in that they do not express mIL-6Rα and can only respond to IL-6 in the presence of sIL-6Rα.
IL-6 has been ascribed as both pro- and anti-inflammatory functions. Accumulating evidence from murine models of asthma supports a role for sIL-6Rα in directing the transition between these dual roles (30). Under noninflamed conditions, IL-6 enhances T-regulatory cell survival through mIL-6Rα (11). Perhaps this may provide an explanation for the reduction in inflammation when IL-6 is transgenically overexpressed in the airways (55). In asthma, where there is an increase in sIL-6Rα (11, 57), IL-6 may induce the expansion of proinflammatory T-helper type 2 (Th2) effector cells, as inhibition of sIL-6Rα (using a gp130Fc fusion protein) reduced CD4+ Th2 cells in the murine lung (11). Therefore, the interaction of IL-6 with different receptor components controls the balance between the levels of T-regulatory and Th2 effector cells, suggesting that modulation of IL-6 trans-signaling may be a novel molecular approach for the treatment of allergic asthma.
Airway remodeling is considered to be a consequence of long-term inflammation incurred from multiple episodes of allergic asthma. Our study is the first to examine the effect of IL-6 trans-signaling on the pro-remodeling functions of ASM, a pivotal immunomodulatory cell that has emerged as playing a key role in inflammation and airway remodeling. We have demonstrated that IL-6 trans-signaling affects the pro-remodeling functions of ASM cells in vitro by inducing the secretion of proinflammatory and proangiogenic mediators. Interestingly, the magnitude of response of ASM to IL-6 + sIL-6Rα, in comparison to other stimuli such as IL-1β, is similar. The extent of VEGF secreted by nonasthmatic or asthmatic ASM cells (∼1.3-fold), in response to IL-6 + sIL-6Rα, was similar to a 1.7-fold increase in VEGF secretion achieved with IL-1β (56). A number of publications have examined IL-1β-induced eotaxin secretion from ASM (8, 18, 19, 21), and the extent of eotaxin secretion we achieved with IL-6 in the presence of sIL-6Rα (i.e., ∼2-fold) is in line with these previous publications.
Accumulating evidence strongly implicates IL-6 in tissue remodeling and fibrogenesis. In vivo, IL-6 has been linked with liver fibrosis (34), fibrotic skin thickening in systemic sclerosis (51), and lung injury (54). Subepithelial fibrosis is increased in the lungs of transgenic mice with targeted overexpression of IL-6 (37). Conversely, lung fibrosis is attenuated in IL-6 knock-out mice (45). In vitro, a number of cell types (predominantly fibroblasts) have been shown to respond to IL-6 by producing proteins implicated in airway fibrosis (12, 33). Other members of the IL-6 family of cytokines, such as oncostatin M and IL-11, can also induce a profibrotic phenotype, as demonstrated both in vivo and in vitro (13, 37, 52). Although ASM are known to produce ECM proteins [such as collagen type I and fibronectin (28)] and profibrotic proteins such as CTGF (7), as well as express cell surface integrins (5, 16, 23, 27, 44), IL-6 trans-signaling did not upregulate the expression of proteins involved in airway fibrosis from ASM. Furthermore, IL-6, in either the absence or presence of sIL-6Rα, did not facilitate mast cell adhesion to ASM, or lead to increased smooth muscle proliferative activity and ASM mass. Although our laboratory's earlier studies have found mast cells in close proximity to the ASM layer in allergy (1) and asthma (6), our results demonstrate that there is no particular role for IL-6 in mediating this response.
Taken together, our data clearly show that, for IL-6 to induce STAT3 phosphorylation and nuclear translocation via gp130, the presence of sIL-6Rα is required. While VEGF and eotaxin are known to be STAT3 dependent (14, 40), our results suggest that the signaling pathways underlying increases in deposition of ECM or proteins that increase ASM proliferation are STAT3 independent.
In summary, we have demonstrated that gp130, but not IL-6Rα, is expressed on human ASM cells and clearly show that sIL-6Rα is required for IL-6 to induce STAT3 phosphorylation and nuclear translocation via gp130. IL-6 trans-signaling affects the pro-remodeling phenotype of ASM cells, possibly in a STAT3-mediated manner, to increase the secretion of the chemokine, eotaxin, and the angiogenic growth factor VEGF and, therefore, contribute to the increase of small blood vessels documented in the thickened airway wall of asthmatic subjects. The implications of our results are threefold: first, this evidence calls in question previous reports, where IL-6 has been shown to be a relatively weak stimulator of pro-remodeling functions in ASM; second, it provides an explanation for the lack of function demonstrated in vitro when the in vivo milieu contains sIL-6Rα; third, and most importantly, it demonstrates how IL-6 plays a pro-remodeling role in the presence of airway inflammation in asthma where the concentration of sIL-6Rα in the inflammatory milieu is increased. Taken together, our results suggest that targeting sIL-6Rα for pharmacological intervention has the potential to reduce and reverse the development of aspects of airway remodeling in asthma.
This work was supported by the Asthma Foundation of New South Wales-Asthma Research Funding Program and the National Health and Medical Research Council.
The authors thank Dr. Joseph H. Butterfield (Mayo Clinic, Rochester, MI) for kindly providing the HMC-1 mast cell line. The authors acknowledge the collaborative effort of the cardiopulmonary transplant team and the pathologists at St. Vincent's Hospital, Sydney, and the thoracic physicians and pathologists at Royal Prince Alfred Hospital, Concord Repatriation Hospital, Strathfield Private Hospital, and Rhodes Pathology, Sydney. We also acknowledge the contribution of Drs. Gregory King and Melissa Baraket at the Woolcock Institute of Medical Research for supplying the asthmatic biopsies, and Joanne Thompson and Pablo Britos for excellent technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society