TGF-β has been suggested to be an important cytokine in the genesis of airway remodeling that underlies diverse pulmonary diseases, and which has been shown to be sufficient in mouse models to drive airway fibrosis. Numerous studies in vitro
and in vivo
have shown that TGF-β1 stimulates production of fibrotic genes, such as collagen, connective tissue growth factor (CTGF), or plasminogen activator inhibitor (PAI)-1, in diverse cell types (1
). In addition, the mouse has been reported to have TGF-β1, TGF-β2, and TGF-β3 protein expression in the lung (24
). Therefore, we investigated the production of specific TGF-β isoforms in an OVA model of allergic airway disease and showed that TGF-β1 is the predominant isoform released into the airspaces. Time-course experiments indicated that TGF-β1 release is initiated early in the disease process after OVA challenge and peaks 7 days after the first OVA exposure. We also demonstrated that the bronchial epithelium of the conducting airways is a key localization of TGF-β1 protein in control mice, its expression is decreased in the OVA model, and eosinophil recruitment is not required for TGF-β1 production.
The functional role of TGF-β signaling in the lung was then assessed using anti–TGF-β1 antibody. TGF-β1 antibody neutralization indeed blocked downstream phospho-Smad 2 accumulation in the airways, confirming that the approach used attenuates canonical TGF-β1 signaling. Anti–TGF-β1 antibody treatment failed to effect the establishment of eosinophilic or monocytic inflammation in the lung; however, it did increase Th2 cytokine profiles and it enhanced OVA-induced AHR, whereas it inhibited subepithelial collagen deposition. These data show that, although inhibition of pulmonary fibrosis with TGF-β1 neutralizing antibody may be a possible outcome, anti–TGF-β1 antibody may, in fact, exacerbate asthma pathology in terms of enhancing AHR. Furthermore, these data suggest a complex and apparently paradoxical role for TGF-β1 in disease pathophysiology: on the one hand, it may contribute to the fibrosis, whereas on the other hand, it has an ameliorating role in regard to the genesis of AHR.
The role of TGF-β signaling in lung fibrosis has been elucidated in recent years. Indeed, inhibition of TGF-β1 signaling via downstream ALK5 receptor inhibition blocks lung expression of several fibrotic genes, including type I collagen and PAI-1 (14
). Conversely, gain of function mutations in TGF-β receptors correlate to increased CTGF gene expression (25
). Treatment of OVA-induced mice with pan–TGF-β antibody reduced airway collagen deposition, epithelial mucus metaplasia, and smooth muscle cell proliferation (15
). It is therefore not surprising that, in the current study, anti–TGF-β1 antibody was effective in inhibiting pulmonary fibrosis; however, the above studies did not assess the impact of the inhibition of fibrosis on either airway function or AHR.
The observation that immunoreactive TGF-β1 protein is primarily present in airway epithelial cells is supported by recent studies using laser capture microdissection to identify the source of OVA-induced fibrotic cytokines (26
). In those studies, TGF-β1, CTGF, and PAI-1 mRNA levels were all specifically enriched in the epithelial layer and not in the underlying smooth muscle. In addition, recent studies have shown activation of signaling pathways downstream of TGF-β receptor engagement in the bronchial epithelium in both animal models and biopsies from humans with asthma (15
). Specifically, phospho-Smad 2, the activated receptor Smad, colocalizes with the epithelium in both antigen-challenged mice (including in this study) and humans with asthma. Taken together, these data implicate TGF-β1 as potentially playing an autocrine role in the airway epithelium to induce pulmonary remodeling and other pulmonary pathologies. The relative contribution of inflammatory cells to TGF-β1 production may be an important difference between human and mouse allergic airway disease, and we cannot exclude the possibility that eosinophils produced and released TGF-β1 at time points different to those studied in our model.
In the current study, anti–TGF-β1 antibody had no effect on eosinophilic inflammation but did enhance Th2 cytokine profiles induced by OVA challenge. The finding that inflammatory cell recruitment is unaltered by TGF-β1 neutralization is consistent with two recent studies using anti–pan-TGF-β antibody or ALK5 inhibition (14
). Further support for these results comes from an additional study using anti–TGF-β antibody followed by a detailed assessment of systemic immune cell function. These authors found that anti–pan-TGF-β antibody had no effect on lymphocyte proliferation, phagocytic activity, cytokine production, or immunoglobulin production (30
). However, studies conducted using the OVA model in TGF-β1 heterozygous mice, systemically lacking an allele of the TGF-β1 gene, showed increased eosinophil accumulation and increased Th2 cytokine production (31
). In these mice, TGF-β1 protein levels in the lung were reduced to 30% of normal wild-type levels after OVA challenge and it is unclear why this study produced significantly different results from our current work, although we did observe increased Th2 cytokine production similar to the results found in TGF-β1 heterozygous mice. It is important to note that disruption of the TGF-β1 gene has been shown to produce partial lethality and severe inflammatory abnormalities in mice (32
). Elevated Th2 cytokine levels have been linked with AHR and allergic airway disease severity in numerous studies, suggesting that TGF-β1 neutralization may impact AHR through modulation of Th2 cell responses (35
Interference with TGF-β1 function using neutralizing antibody had a significant effect on the pathophysiology induced by antigen challenge. Although anti–TGF-β1 antibody increased all measures of baseline (pre-Mch) lung mechanics, this effect was not significant (). However, anti–TGF-β1 antibody did significantly enhance AHR as assessed with G (tissue resistance) and H (elastance) but not RN (airway resistance), which is consistent with a peripheral (small) airway effect. These findings are both surprising and at first glance inconsistent with the commonly held view that airway remodeling is a major cause of AHR.
Some potential explanations for this outcome can be considered. One possible explanation for the results is the well-known effect that TGF-β1 has on T-cell regulation. TGF-β1 overexpression in helper T cells has been shown to inhibit OVA-induced AHR in mice (37
). Moreover, in two recent studies, one in which TGF-β1 was overexpressed in airway epithelial cells (38
) and another in which TGF-β1 was administered intratracheally (39
), both treatments were associated with increased TGF-β1 and a decrease in AHR. On the other hand, there are other reports where reduced levels of TGF-β1 (31
) or ectopically administered TGF-β1 (9
) did not alter airway resistance or AHR. Unfortunately, the determination of the mechanical response and AHR in all of the above studies was obtained noninvasively with a whole body plethysmographic technique, a measurement that has been shown to have severe shortcomings and uncertain interpretation (40
Another potential immunologic mechanism by which TGF-β1 may decrease antigen-induced AHR is through effects on regulatory T lymphocyte (Treg) activity. Treg cells were initially identified as a specific class of TGF-β1–producing T cells that make up a small percentage of the T-cell pool (41
). The Treg cells are important in the regulation of Th2 immunity and are believed to play an important role in the control of allergic responses because Treg cells have been shown to suppress OVA-induced AHR in mice, via a TGF-β1–dependent mechanism (42
). In that study, anti–TGF-β antibody blocked the inhibitory effects of Tregs on AHR, analogous to our findings. In addition, Treg cells have been shown to induce Th2 cell apoptosis (43
) and increased tolerance to inhaled antigen (44
), promoting resolution of AHR. Recent work has shown that TGF-β1 induces maturation of naive T cells to a Treg phenotype via expression of the Treg-specific transcription factor Foxp3 (45
). These data suggest that TGF-β1 activity is required for normal Treg function in the lung. By inhibiting TGF-β1 activity, it is possible that lung T-cell function is skewed toward an exacerbated allergic phenotype, which would include increased AHR. The role of Treg activity in the structure/function relationships in the lung and the link to TGF-β1 remain to be determined.
Last, it is likely that, during the inflammatory events caused by antigen challenge, elaboration of TGF-β1 inhibits AHR by a structural mechanism. Thickening of the airway walls of patients with asthma is related to asthma severity (46
) and has shown to be greatest in those who die of asthma (48
). Mathematical modeling (50
) suggests that thickening of the airway wall contributes to AHR in vivo
by a geometric mechanism, in which a thickened airway wall internal to the airway smooth muscle (ASM) will narrow the airway lumen more for any given stimuli to the ASM. MacParland and colleagues (17
) have recently reviewed the evidence that structural remodeling of the airway could hypothetically either contribute to AHR or ameliorate AHR; however, the evidence directly linking AHR to airway wall alterations is both limited and inconsistent (52
). To explain the present findings, we suggest that antigen-induced inflammation leads to the well-documented elaboration of mediators (e.g., leukotrienes and interleukins) and, in this case, TGF-β1 (), which, in turn, causes subepithelial fibrosis (). This airway wall fibrosis would then result in any one or combination of events, including the following: (1
) stiffening of the wall preventing the ASM from narrowing the lumen in response to Mch; (2
) as the fibrosis occurs within and between the cells of the ASM, this would increase the parallel elastic load to ASM which prevents shortening; or (3
) increasing the series load against which the ASM must contract (17
). It has been noted that chronic antigen challenge leads to more substantial airway wall remodeling but a decline in AHR (54
), consistent with this proposed mechanism of an ameliorating effect of airway fibrosis. Consistent with this explanation is the spatial location of the antigen-induced deposition of collagen with and outside of the ASM () and its reduction by an anti–TGF-β1 antibody. Moreover, the pattern of the temporal response to a single dose of inhaled Mch of both elastance (H) and tissue resistance (G) () shows a markedly enhanced peak response consistent with enhanced ASM narrowing. Anti–TGF-β1 antibody, by decreasing the amount of submucosal collagen, would allow ASM to contract more, leading to a marked increase in the peak response of H, a temporal pattern that we have not observed previously even with antigen challenge (55
). In this way, airway fibrosis would serve to lessen airway narrowing, but prevention of its formation by blocking the effects of TGF-β1 results in enhanced airway narrowing.
In conclusion, the data presented in this study show that, although anti–TGF-β antibody strategies may indeed provide desired antifibrotic potential, the effects of anti–TGF-β1 antibody in fact exacerbates the AHR associated with the allergic airway response. The mechanism by which TGF-β1 regulates AHR remains unclear and further study is warranted to elucidate the pathways involved. In addition, we identify TGF-β1 as the primary TGF-β isoform produced in the antigen challenge model and the airway epithelium as a potential source of TGF-β. We also demonstrate that the mechanisms of antigen-induced inflammation, remodeling, and AHR appear to be independent with regard to the TGF-β signaling pathway. We conclude that the function of TGF-β1 in allergic airway disease is to shift the lung toward a fibrotic phenotype while dampening the AHR induced by antigen challenge, perhaps by either an immunologic or mechanical mechanism. Collectively, these findings suggest that the commonly held notion that airway remodeling is directly involved in the genesis of AHR may need to be reconsidered.