help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on April 17, 2003, doi:10.1165/rcmb.2002-0309OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2002-0309OCv1
29/4/458    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nikolaidis, N. M.
Right arrow Articles by Rothenberg, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nikolaidis, N. M.
Right arrow Articles by Rothenberg, M. E.
American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 458-464, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0309OC

Trefoil Factor-2 Is an Allergen-Induced Gene Regulated by Th2 Cytokines and STAT6 in the Lung

Nikolaos M. Nikolaidis, Nives Zimmermann, Nina E. King, Anil Mishra, Samuel M. Pope, Fred D. Finkelman and Marc E. Rothenberg

Departments of Cell Biology, Molecular Genetics, and Internal Medicine, Division of Immunology, University of Cincinnati College of Medicine; and Divisions of Allergy and Immunology, and Immunobiology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

Address correspondence to: Marc Rothenberg, M.D., Ph.D., Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC 7028 Cincinnati, OH 45229. E-mail: Rothenberg{at}cchmc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Asthma, a complex chronic inflammatory pulmonary disorder, is on the rise despite intense ongoing research, underscoring the need for new scientific inquiry. In an effort to provide unbiased insight into the pathogenesis of this disease, we took an empirical approach involving transcript expression profiling of lung tissue from mice with experimental asthma. Employing asthma models induced by different allergens (ovalbumin [OVA] and Aspergillus fumigatus), we found strong induction of trefoil factor-2 (TFF2), a gene involved in epithelial restitution and mucosal secretion in the gastrointestinal tract. Using a combination of pharmacologic delivery and transgenic overexpression, TFF2 was demonstrated to be strongly induced in the lung by interleukin (IL)-4 and IL-13. Notably, TFF2 induction by both OVA and pharmacologic delivery of IL-4 and IL-13 was dependent upon signal transducer and activator of transcription (STAT)6. However, the upregulation of TFF2 by both chronic expression of IL-4 and Aspergillus fumigatus antigen was independent of STAT6. These results establish that TFF2 is an allergen-induced lung gene product differentially regulated by Th2 cytokines and STAT6. Given the important role of trefoil factors in wound healing, epithelial restitution, and maintenance of mucosal integrity in the gastrointestinal tract, these results support a potential role for TFF2, in both the acute and chronic phase of experimental asthma, via separate induction pathways.

Abbreviations: Airway hyperreactivity, AHR • interleukin, IL • ovalbumin, OVA • signal transducer and activator of transcription, STAT • T helper 2 lymphocytes, Th2 cells • trefoil factor, TFF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There is currently an epidemic of asthma and other allergic diseases in the western world, and the incidence is on the rise (1). Experimentation in the asthma field has largely focused on analysis of the cellular and molecular pathways induced by allergen exposure in sensitized animals and humans. These studies have identified elevated production of immunoglobulin E, mucus hypersecretion, airway obstruction, inflammation, and enhanced bronchial reactivity to spasmogens in the asthmatic response (2–4). Clinical and experimental investigations have demonstrated a strong correlation between the presence of CD4+ T helper 2 lymphocytes (Th2 cells) and disease severity, suggesting an integral role for these cells in the pathophysiology of asthma (5, 6). Th2 cells are thought to induce asthma through the secretion of a variety of cytokines (interleukin [IL]-4, -5, -6, -9 -10, -13, -25) that activate inflammatory and residential effector pathways both directly and indirectly (7–9). In particular, IL-4 and IL-13 are produced at elevated levels in the asthmatic lung, and are thought to be key regulators of many of the hallmark features of disease (9, 10). More recently, attention has focused on the pathogenesis of airway remodeling in the setting of chronic airway inflammation. It has also become clear that mesenchymal cell signaling, induced by Th2 cytokines, has an active role in chronic injury and repair processes in response to allergen-triggered inflammation (11). Although these collective studies have provided the rationale for the development of multiple therapeutic agents that interfere with specific inflammatory pathways (12, 13), the development of the asthma phenotype is likely to be related to the complex interplay of a large number of additional genes, and their polymorphic variants. Accordingly, we aimed to identify new genes involved in the pathogenesis of experimental asthma using an empiric approach employing DNA microarray analysis of whole lung RNA (14).

This manuscript focuses on one such gene, that encodes for trefoil factor family peptide (TFF)2. Trefoil peptides are small (7–12 kD) protease-resistant proteins, composed of a characteristic three-loop structure formed by three conserved cysteine disulfide bonds, and secreted by the gastrointestinal mucosa in a lineage-specific manner (15, 16). TFF2, also known as spasmolytic polypeptide, is expressed in the stomach (and to a lesser extent in the proximal duodenum and biliary tract), whereas TFF1 and TFF3 are expressed and secreted predominantly by gastric pit cells and intestinal goblet cells, respectively. Trefoil factors are critically involved in responses to intestinal injury, primarily by their ability to promote epithelial restitution, the rapid spreading and migration of existing epithelial cells following injury (16). Although expressed and secreted preferentially by gastric mucus neck cells, TFF2 is upregulated in diverse pathologic conditions of the gastrointestinal tract including ulceration, inflammatory bowel disease, Helicobacter pylori infection, and by injury promoted by nonsteroidal anti-inflammatory drugs (15, 16). In these conditions, TFF2 is thought to regulate acid production, stabilize the mucin gel layer (by directly interacting with mucin proteins), and promote healing, as supported by recent studies in TFF2-deficient mice (17).

We chose to focus on the expression and regulation of TFF2 in experimental asthma because this molecule has not been extensively studied in the context of allergy or lung inflammation. Because of the established role of TFF2 in gastrointestinal repair responses, TFF2 is a novel candidate gene likely to be involved in the remodeling and repair responses associated with allergic lung disorders. Furthermore, because TFFs directly interact with mucin proteins (18), molecules that are overproduced in the asthmatic lung, it was additionally relevant to examine their involvement in allergic lung responses. Our results demonstrate specific regulation of TFF2 by diverse allergens, as well as the Th2 cytokines IL-4 and IL-13. In addition, we demonstrate that signal transducer and activator of transcription (STAT)6 is required for acute TFF2 induction by IL-4, IL-13, and the allergen ovalbumin (OVA), but not by the allergen Aspergillus fumigatus or by chronic overexpression of lung IL-4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Asthma Induction
Balb/c mice were obtained from the National Cancer Institute (Frederick, MD), and STAT6-deficient mice (Balb/c) were obtained from Jackson Laboratory (Bar Harbor, ME). All mice were housed under specific pathogen-free conditions. Asthma models were induced by intraperitoneal injection with OVA and 1 mg aluminum hydroxide (alum) separated by 2 wk, followed by two doses of intranasal OVA or saline challenge 2 wk later, as previously described (19). Aspergillus fumigatus antigen-induced asthma was induced over the course of 3 wk by repeated intranasal inoculation of antigen, as described (19). IL-4 administration via intranasal delivery in lightly anesthetized (isoflourane) mice was performed as previously described (20). Briefly, recombinant murine IL-4 (2 µg in 50 µl 0.9% saline) was delivered in conjunction with monoclonal antibody (10 µg) directed against IL-4; this allows for the half-life of IL-4 to be increased from several minutes to ~ 24 h. Dosing was every other day for six doses. Recombinant murine IL-13 (4 µg in 20 µl 0.9% saline [a generous gift from Dr. Debra Donaldson, Wyeth Research]) was administered via intratracheal delivery in ketamine-anesthetized mice. Dosing was for five consecutive days.

Preparation of RNA and Microarray Hybridization
RNA was extracted using the Trizol (Invitrogen, Carlsbad, CA) reagent as per the manufacturer's instructions. After Trizol purification, RNA was repurified with phenol-chloroform extraction and ethanol precipitation. Microarray hybridization was performed by the Affymetrix Gene Chip Core facility at Cincinnati Children's Hospital Medical Center. Briefly, RNA quality was first assessed using the Agilent bioanalyzer (Agilent Technologies, Palo Alto, CA) and only those samples with 28S/18S ratios between 1.3 and 2 were subsequently used. RNA was converted to cDNA with Superscript choice for cDNA synthesis (Invitrogen), and subsequently converted to biotinylated cRNA with Enzo High Yield RNA Transcript labeling kit (Enzo Diagnostics, Farmingdale, NY). After hybridization to the murine U74Av2 GeneChip (Affymetrix, Santa Clara, CA), the gene chips were automatically washed and stained with streptavidin-phycoerythrin using a Fluidics System (Affymetrics, Santa Clara, CA). The chips were scanned with a Hewlett Packard GeneArray Scanner (Affymetrics). This analysis was performed with one mouse per chip (n >= 3 for each allergen challenge condition and n >= 2 for each saline challenge condition). For Northern blot analysis, RNA was extracted from the lungs of wild-type Balb/c mice, IL-4 Clara cell 10 lung transgenic mice (21) containing wild-type or deleted copies of the gene for STAT6 (22), and from the lungs of mice treated with saline or recombinant murine cytokines, as previously reported (23, 24). Hybridization was performed with 32P-labeled cDNA encoding the sequence-confirmed murine TFF2 (I.M.A.G.E. 438,574) or TFF3 (I.MA.G.E. 1,166,710) (obtained from American Type Tissue Culture Collection, Rockville, MD).

Data Analysis
From data image files, gene transcript levels were determined using algorithms in the Microarray Analysis Suite Version 4 software (Affymetrix). Global scaling was performed to compare genes from chip to chip; thus each chip was normalized to an arbitrary value (1,500). Each gene is typically represented by a probe set of 16–20 probe pairs. Each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide that contains a one-base mismatch at a central position. Two measures of gene expression were used: absolute call and average difference. Absolute call is a qualitative measure in which each gene is assigned a call of present, marginal, or absent based on the hybridization of the RNA to the probe set. Average difference is a quantitative measure of the level of gene expression, calculated by taking the difference between mismatch and perfect match of every probe pair and averaging the differences over the entire probe set. Differences between saline and allergen-treated mice were also determined using the GeneSpring software (Silicon Genetics, Redwood City, CA). Data were normalized to the average of the saline-treated mice. Gene lists were created that contained genes with P < 0.05 and > 2-fold change (using genes that received a present call based on the hybridization signal).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DNA Microarray Analysis Identifies TFF2 as an Allergen-Induced Gene in Experimental Asthma
To identify allergen-induced genes in the lung specifically focused on genes that were not specific to a particular experimental regime, we induced experimental asthma using two distinct experimental protocols. Using global transcript profile analysis, we aimed to identify allergen-induced genes that overlapped in the two independent models of asthma. In one model, mice were intraperitoneally sensitized with the allergen OVA in the presence of the adjuvant alum on two separate occasions separated by 14 d. Subsequently, mice were challenged with intranasal OVA or control saline on two occasions separated by 3 d. Eighteen hours after the last allergen challenge, the lung was harvested for RNA analysis. In another model, experimental asthma was induced by the Aspergillus fumigatus antigen, because this model involves a unique mucosal sensitization route (intranasal) compared with the OVA model (25, 26) and because Aspergillus fumigatus is a ubiquitous and common aeroallergen. Lung RNA was obtained 18 h after nine repetitive doses over the course of 3 wk (chronic exposure) of intranasal Aspergillus fumigatus allergen or saline. Importantly, both asthma models have similar phenotypes, including Th2-associated eosinophilic inflammation, mucus production, and airway hyperresponsiveness (AHR). We next subjected RNA from each respective saline- and allergen-challenged mouse to microarray analysis using the Affymetrix chip U74Av2 that contains oligonucleotide probe sets representing 12,422 genetic elements, one of the largest collection of characterized mouse genes commercially available. We compared allergen-challenged mice (OVA or Aspergillus) to their respective saline control mice (n = 2–3 mice in each experimental group), and genes were identified that statistically increased (P < 0.05) at least 2-fold after allergen challenge. Compared with mice challenged with saline, OVA-challenged mice had 496 genes induced and Aspergillus fumigatus–challenged mice had 527 genes induced (45). Notably, the majority (59% of OVA and 55% of Aspergillus) of the induced transcripts overlapped between the two experimental asthma models.

Our results identified a set of 291 genes that were commonly involved in disease pathogenesis, rather than unique to a particular allergen or mode of disease induction. These "asthma signature" genes provide a valuable opportunity to define new pathways involved in the pathogenesis of allergic airway inflammation. In this regard, we were struck by the high level of transcripts for TFF2 in the asthmatic lung. We performed a quantitative microarray analysis for TFF2 in both the Aspergillus- and OVA-induced asthma models (Figures 1A and 1B). We also performed a kinetic analysis after the first OVA challenge, and found that TFF2 was detectable 18 h after the first allergen challenge, but not at 3 h (Figure 1C). Interestingly, microarray analysis revealed very specific dysregulation of TFF2 compared with other TFFs. For example, the hybridization signals for TFF3 was below background in the saline- and allergen-challenged lung, and although the TFF1 mRNA signal was present, it remained unchanged in response to allergen challenge (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. Expression of TFF2 by microarray analysis during induction of experimental asthma. Expression of TFF2 in Aspergillus fumigatus (Asp)- (A) and ovalbumin (OVA)-challenged mice (B and C) as measured by gene chip analysis. The average (Avg) difference for the hybridization signal after saline (black bar) and allergen (white bar) challenge is depicted. Error bars represent the standard deviation. The values represent the mean (n = 2–3 mice). Statistical significances are indicated.

 
Northern Blot Analysis Demonstrates TFF2 Induction by Allergen
We subsequently demonstrated by Northern blot analysis that Aspergillus fumigatus–challenged mice had marked expression of TFF2 compared with mice challenged with saline (Figure 2A). Additionally, we determined by Northern blot analysis that there was a time- and dose-dependent induction of TFF2 during the progression of OVA-induced experimental asthma; TFF2 was induced 18 h after the first allergen challenge and was even higher after two allergen challenges (Figure 2B). Subsequent kinetic analysis revealed that TFF2 expression was maximally expressed by 10 h after the second challenge, and this level was maintained through 120 h (Figure 2C). TFF3 mRNA was not detectable by Northern blot analysis of the same experimental asthma lung samples, although it was detected in a Northern blot prepared from gastrointestinal tissue RNA (data not shown).



View larger version (57K):
[in this window]
[in a new window]
  		Figure 2. Northern blot analysis of TFF2 expression after allergen. (A) The expression of TFF2 after repeated intranasal Aspergillus fumigatus antigen is shown. Northern blot analysis of TFF2 expression after OVA challenge is shown. Time points include 3 and 18 h after one allergen or saline challenge and 18 h after two challenges (B). Time points include 6 through 120 h after second challenge (C). The Ethidium bromide (EtBr)-stained gels are also shown. Each lane represents a separate animal.

 
Pharmacologic Delivery of IL-4 and IL-13 Induces Lung TFF2
Because asthma is a Th2-associated process, we were interested in testing the hypothesis that pharmacologic delivery of IL-4 and IL-13 was sufficient for induction of TFF2. Both cytokines have been shown to be critically involved in the development of several features of experimental asthma (27). To test this hypothesis, we administered IL-4 or IL-13 by repeated application to the respiratory tract of anesthetized mice. These protocols induce several features of experimental asthma, including eosinophilic inflammation, chemokine induction, mucus production, and AHR (9, 28, 29). Administration of both Th2 cytokines induced marked levels of TFF2 mRNA compared with saline-treated control mice (Figures 3A and 3B).



View larger version (77K):
[in this window]
[in a new window]
  		Figure 3. Regulation of TFF2 by IL-4, IL-13, and STAT6. Northern blot analyses of TFF2 mRNA expression when IL-4 or saline was delivered to wild-type (+/+) or STAT6-deficient (-/-) mice (A). (B) IL-13 or saline was delivered to wild-type (+/+) or STAT6-deficient (-/-) mice. (C) Lung RNA from IL-4 transgenic (Tg) or wild-type (WT) mice carrying wild-type (+/+) or gene deleted (-/-) copies of STAT6 were subjected to Northern blot analysis. Ethidium bromide (EtBr) staining of the RNA gels is also shown. Each lane represents a separate animal.

 
Chronic Overexpression of IL-4 Potently Induces Lung TFF2 In Vivo
We were next interested in testing the hypothesis that chronic overexpression of IL-4, particularly in the lungs, was sufficient for induction of TFF2. Mice that overexpress the IL-4 transgene in pulmonary epithelium (under the control of the Clara cell 10 promoter) have several features of asthma including eosinophil-rich inflammatory cell infiltrates, mucus production, and changes in baseline airway tone (21). We hypothesized that TFF2 mRNA would be induced by the IL-4 transgene. Indeed, IL-4 lung transgenic mice had a marked increase in the level of TFF2 mRNA compared with wild-type mice (Figure 3C).

IL-4 and IL-13 Induction of Lung TFF2 is Differentially Dependent on STAT6
IL-4 and IL-13 share similar signaling requirements, such as use of the IL-4R{alpha} chain and the induction of janus kinase 1 and STAT6. A subset of their responses has been shown to be STAT6-dependent (22, 30, 31). To test the role of STAT6 in the induction of TFF2 in vivo, we delivered IL-4 to wild-type and STAT6-deficient mice. As shown in Figure 3A, IL-4–induced TFF2 expression was largely STAT6-dependent. We also delivered IL-13 to STAT6-deficient mice and found that TFF2 was largely dependent upon STAT6 (Figure 3B). We were next interested in examining IL-4 lung transgenic mice that contained wild-type or gene-targeted deletion of STAT6 (Figure 3C). These mice were generated by mating IL-4 lung transgenic mice with STAT6-deficient mice, and have been previously described (24). Notably, the IL-4 transgene induced TFF2 mRNA expression was largely STAT6-independent. This was a surprising finding, because other IL-4–induced lung genes have been reported to be STAT6-dependent (24). To verify these findings, we examined expression of eotaxin-1 in these mice, and demonstrated that IL-4–induced eotaxin mRNA expression was completely dependent upon STAT6 (Figure 3C). Collectively, these results demonstrate that TFF2 induction by pharmacologic delivery of IL-4 and IL-13 occurs by a STAT6-dependent mechanism, and that chronic overexpression of IL-4 by a transgenic approach induces TFF2 expression in the absence of STAT6.

Allergen-Induced TFF2 Expression Is Differentially Regulated by IL-13 and STAT6
We were next interested in determining if allergen-induced TFF2 expression was STAT6-dependent. This would help determine if allergen-induced TFF2 was predominantly downstream from IL-4/IL-13 signaling. Notably, mice deficient in STAT6 had reduced expression of lung TFF2 after OVA challenge; as a control, wild-type mice displayed readily detectable lung TFF2 (Figure 4A). To further examine the role of IL-13 and STAT6 signaling, we assessed OVA-induced experimental asthma in IL-13 gene-targeted mice. Notably, IL-13 gene-targeted mice had markedly reduced OVA-induced TFF2 expression. Collectively, these results indicate that OVA-induced TFF2 occurs downstream from IL-13 and STAT6 signaling (Figure 4B). In contrast, when the STAT6 requirement was examined in the Aspergillus fumigatus–induced model of experimental asthma, there was strong induction of TFF2 even in the absence of STAT6. For example, the levels of TFF2 mRNA were comparable in the wild-type and STAT6(-/-) mice (Figure 4C) after Aspergillus fumigatus antigen treatment. These results demonstrate that the mechanism of allergen-induced TFF2 induction varies with distinct experimental regimes. In particular, the OVA-induced model appears to be regulated by a Th2-associated STAT6 pathway, whereas the Aspergillus fumigatus regime induces TFF2 primarily independent of STAT6.



View larger version (69K):
[in this window]
[in a new window]
  		Figure 4. Regulation of OVA- and Aspergillus fumigatus–induced TFF2 by IL-13 and STAT6. Experimental asthma was induced by OVA (A, B) or Aspergillus fumigatus (C) in wild-type (+/+) or STAT6 gene-deleted (-/-) mice, or IL-13 gene-deleted mice. Ethidium bromide (EtBr) staining of the RNA gels is also shown. Each lane represents a separate animal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To understand the complex mechanisms involved in the pathogenesis of asthma, we employed transcript expression profile analysis to define a set of "asthma signature" genes. We focused on the discovery of TFF2 (as an asthma-associated gene) because this molecule had properties potentially important in asthmatic responses, and because TFF2 was not previously implicated in the pathogenesis of asthma. Our results have uncovered several principles concerning trefoil factors and asthma. First, allergic lung inflammation, triggered by diverse allergens and modes of disease induction, is shown to be associated with marked and specific ectopic expression of TFF2 (but not TFF1 or TFF3) in the lung. Before these findings, expression of TFFs (especially TFF2) was thought to be primarily restricted to the gastrointestinal tract (15, 16). Second, our study demonstrates that the Th2 cytokines IL-4 and IL-13 are potent inducers of TFF2 in the lung. These findings indicate that allergen-induced TFF2 is likely to be mediated, at least in part, by IL-4 and IL-13. Indeed, analysis of IL-13–deficient mice revealed that OVA-induced TFF2 expression was largely dependent on IL-13. IL-4 and IL-13 are related cytokines that share a similar signaling mechanism (e.g., use of a common receptor subunit [IL-4R{alpha} chain] and activation of STAT6) (27). Notably, although both of these cytokines are known to have an essential role in asthma, the mechanisms by which they induce various elements of the asthmatic response (e.g., AHR, mucus production, and airway remodeling) are only partially understood. Our results suggest that the pathogenesis of IL-4–/IL-13–associated allergic lung responses may be mediated by TFF2, at least in part. It is tempting to speculate that injury-associated epithelial hyperplasia and epithelial differentiation (e.g., mucus cell metaplasia), processes known to be regulated by TFF2 in the gastrointestinal tract, may also be mediated by TFF2 in the lung. Third, our results demonstrate that although TFF2 is induced by both IL-4 and IL-13, STAT6 is not a requisite for TFF2 induction. For example, the modulation of TFF2 by Aspergillus fumigatus antigen and the IL-4 transgene occurs at comparable levels in STAT6-deficient and wild-type mice. However in contrast, it is notable that TFF2 induced by pharmacologic delivery of IL-4 and IL-13 and by the OVA asthma model is attenuated in STAT6-deficient mice. These findings are consistent with prior studies that have shown distinct and overlapping mechanisms for the involvement of IL-4 and IL-13 in experimental asthma (27). Additionally, although OVA and Aspergillus fumigatus antigen both induce experimental asthma, Aspergillus is capable of inducing Th2 responses independent of adjuvant, indicating that both allergens employ distinct mechanisms for asthma induction. Collectively, these findings suggest that Th2 cytokine–mediated TFF2 induction is likely to occur by an indirect mechanism. Consistent with an indirect mechanism, the TFF2 promoter is not known to contain a STAT-binding site (32). Prior studies have shown that GATA6, a transcription factor normally expressed in the heart and gastrointestinal tract, is important for TFF2 induction (33, 34). It will certainly be important to elucidate if GATA6 is induced and required for TFF2 expression in the lung. Furthermore, our results appear to support prior studies that have suggested that certain aspects of allergic asthma are regulated via a STAT6 pathway, and that this pathway may be differentially dependent upon acute or chronic induction of experimental asthma (35–37).

Prior studies have determined that Th2 responses can occur independent of STAT6 in the absence of the Bcl6 or CTLA4 molcules. Thus, the Aspergillus fumigatus antigen and the IL-4 transgene may induce TFF2 by suppressing Bcl6-associated transcription and/or by providing strong co-stimulating T cell activation. The differential regulation of STAT6 is not unique to TFF2, as indicated by our global transcript profile analysis of Aspergillus fumigatus antigen challenged STAT6-deficient mice (A. Mishra, N. Zimmermann, and M. Rothenberg, unpublished results).

Under normal physiologic conditions, TFF2 is predominantly expressed in the stomach, with lower levels in the proximal duodenum and biliary tract (15, 16). In the stomach, TFF2 is expressed by gastric mucus neck cells, and is secreted onto the mucosal surface associated with mucin proteins. TFF2 is upregulated in diverse injury-associated pathologic conditions in the gastrointestinal tract, including ulceration associated with Helicobacter pylori infection, nonsteroidal anti-inflammatory drug use, and Crohn's disease of the gastrointestinal tract (38–40). In all of these states, TFF2 expression appears to be related to the proliferative zone of the mucosa, suggesting that TFF2 may be involved in regulating epithelial proliferation in response to injury. Notably, the asthmatic lung is characterized by a large increase in epithelial proliferation (41–43). Based on homology with the lung, it is likely that TFF2 is expressed by airway epithelial and goblet cells, indeed preliminary in situ hybridization analysis has supported this (data not shown). In addition, TFF2 has been linked with inhibiting acid production in the stomach (44). Importantly, recent studies have shown that the asthmatic airway is characterized by an acidified environment that appears to be responsible for the oxidation of nitrite to nitric oxide, a process that strongly correlates with airway inflammation (45). Recent studies with TFF2-deficient mice have definitively elucidated a critical role for TFF2 in promoting mucosal healing through inhibition of acid secretion and stimulation of epithelial proliferation (17). It is thus tempting to speculate that allergen-induced TFF2 may have a critical role in regulating several critical features associated with the pathogenesis of asthma, including acidification of the airway and epithelial proliferation. These results raise the importance of subjecting TFF2-deficient mice to the induction of experimental asthma.

In conclusion, we report that (i) TFF2 is an allergen-induced gene in the asthmatic lung; (ii) the Th2 cytokines IL-4 and IL-13 induce TFF2; and (iii) TFF2 induction can occur by STAT6-dependent mechanisms in acute models (as in the case of pharmacologic delivery of Th2 cytokines and OVA-induced asthma), and independently of a STAT6-regulated pathway in chronic models (as in the case of IL-4 transgenic expression and Aspergillus fumigatus–induced asthma). Collectively, these results raise the possibility that TFF2 may have an important role in the pathogenesis of asthma, especially with processes known to be regulated by TFF2 in the gastrointestinal tract, including epithelial proliferation, mucus hypersecretion, and acid production. Finally, these results indicate that allergic lung responses may share common pathogenic mechanisms with disease processes in the gastrointestinal tract.


   Acknowledgments
 
The authors thank Andrea Lippelman for editorial assistance, and Drs. Tim Wang and Eric Brandt for helpful discussions. The authors also thank Dr. Debra Donaldson at Wyeth Research for the generous supply of IL-13. This work was supported in part by the American Heart Association Scientist Development Grant (N.Z.), NIH grants R01 AI42242–05 (M.E.R.), R01 AI45898–03 (M.E.R.), the Human Frontier Science Program (M.E.R), International Life Sciences Institute (M.E.R.), and Burroughs Wellcome Fund (M.E.R.).

Received in original form December 19, 2002

Received in final form April 14, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. ISAAC. 1998. Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee. Lancet 351:1225–1232.[CrossRef][Medline]
  2. Broide, D. H. 2001. Molecular and cellular mechanisms of allergic disease. J. Allergy Clin. Immunol. 108:S65–S71.[CrossRef][Medline]
  3. Busse, W. W., and R. F. Lemanske, Jr. 2001. Asthma. N. Engl. J. Med. 344:350–362.[Free Full Text]
  4. Lee, N. A., E. W. Gelfand, and J. J. Lee. 2001. Pulmonary T cells and eosinophils: coconspirators or independent triggers of allergic respiratory pathology? J. Allergy Clin. Immunol. 107:945–957.[CrossRef][Medline]
  5. Robinson, D. S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A. M. Bentley, C. Corrigan, S. R. Durham, and A. B. Kay. 1992. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 326:298–304.[Abstract]
  6. Hogan, S. P., K. I. Matthaei, J. M. Young, A. Koskinen, I. G. Young, and P. S. Foster. 1998. A novel T cell-regulated mechanism modulating allergen-induced airways hyperreactivity in BALB/c mice independently of IL-4 and IL-5. J. Immunol. 161:1501–1509.[Abstract/Free Full Text]
  7. Drazen, J. M., J. P. Arm, and K. F. Austen. 1996. Sorting out the cytokines of asthma. J. Exp. Med. 183:1–5.[Free Full Text]
  8. Ray, A., and L. Cohn. 1999. Th2 cells and GATA-3 in asthma: new insights into the regulation of airway inflammation. J. Clin. Invest. 104:985–993.[Medline]
  9. Wills-Karp, M. 1999. Immunologic basis of antigen-induced airway hyperresponsiveness. Annu. Rev. Immunol. 17:255–281.[CrossRef][Medline]
  10. Bochner, B. S., B. J. Undem, and L. M. Lichtenstein. 1994. Immunological aspects of allergic asthma. Annu. Rev. Immunol. 12:295–335.[CrossRef][Medline]
  11. Holgate, S. T., D. E. Davies, P. M. Lackie, S. J. Wilson, S. M. Puddicombe, and J. L. Lordan. 2000. Epithelial-mesenchymal interactions in the pathogenesis of asthma. J. Allergy Clin. Immunol. 105:193–204.[CrossRef][Medline]
  12. Boushey, H. A., Jr. 2001. Experiences with monoclonal antibody therapy for allergic asthma. J. Allergy Clin. Immunol. 108:S77–S83.[CrossRef][Medline]
  13. Barnes, P. J. 2000. New directions in allergic diseases: mechanism-based anti-inflammatory therapies. J. Allergy Clin. Immunol. 106:5–16.[CrossRef][Medline]
  14. Zimmermann, N., N. E. King, J. Laporte, M. Yang, A. Mishra, S. M. Pope, E. E. Muntel, D. P. Witte, A. E. Pegg, P. S. Foster, Q. Hamid, and M. E. Rothenberg. 2003. Dissection of experimental asthma with DNA microarray analysis: identification of arginase in asthma pathogenesis. J. Clin. Invest. 111:1863–1874.[CrossRef][Medline]
  15. Thim, L. 1997. Trefoil peptides: from structure to function. Cell. Mol. Life Sci. 53:888–903.[CrossRef][Medline]
  16. Podolsky, D. K. 2000. Mechanisms of regulatory peptide action in the gastrointestinal tract: trefoil peptides. J. Gastroenterol. 35:69–74.
  17. Farrell, J. J., D. Taupin, T. J. Koh, D. Chen, C. M. Zhao, D. K. Podolsky, and T. C. Wang. 2002. TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury. J. Clin. Invest. 109:193–204.[CrossRef][Medline]
  18. Thim, L., F. Madsen, and S. S. Poulsen. 2002. Effect of trefoil factors on the viscoelastic properties of mucus gels. Eur. J. Clin. Invest. 32:519–527.[CrossRef][Medline]
  19. Mishra, A., T. E. Weaver, D. C. Beck, and M. E. Rothenberg. 2001. Interleukin-5-mediated allergic airway inflammation inhibits the human surfactant protein C promoter in transgenic mice. J. Biol. Chem. 276:8453–8459.[Abstract/Free Full Text]
  20. Finkelman, F. D., K. B. Madden, S. C. Morris, J. M. Holmes, N. Boiani, I. M. Katona, and C. R. Maliszewski. 1993. Anti-cytokine antibodies as carrier proteins. Prolongation of in vivo effects of exogenous cytokines by injection of cytokine-anti-cytokine antibody complexes. J. Immunol. 151:1235–1244.[Abstract]
  21. Rankin, J. A., D. E. Picarella, G. P. Geba, U.-A. Temann, B. Prasad, B. DiCosmo, A. Tarallo, B. Stripp, J. Whitsett, and R. A. Flavell. 1996. Phenotypic and physiologic characterization of transgenic mice expressing interleukin 4 in the lung: lymphocytic and eosinophilic inflammation without airway hyperreactivity. Proc. Natl. Acad. Sci. USA 93:7821–7825.[Abstract/Free Full Text]
  22. Shimoda, K., J. van Deursen, M. Y. Sangster, S. R. Sarawar, R. T. Carson, R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. Vignali, P. C. Doherty, G. Grosveld, W. E. Paul, and J. N. Ihle. 1996. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 380:630–633.[CrossRef][Medline]
  23. Pope, S. M., E. B. Brandt, A. Mishra, S. P. Hogan, N. Zimmermann, K. I. Matthaei, P. S. Foster, and M. E. Rothenberg. 2001. IL-13 induces eosinophil recruitment into the lung by an IL-5- and eotaxin-dependent mechanism. J. Allergy Clin. Immunol. 108:594–601.[CrossRef][Medline]
  24. Zimmermann, N., S. P. Hogan, A. Mishra, E. B. Brandt, T. R. Bodette, S. M. Pope, F. D. Finkelman, and M. E. Rothenberg. 2000. Murine eotaxin-2: A constitutive eosinophil chemokine induced by allergen challenge and IL-4 overexpression. J. Immunol. 165:5839–5846.[Abstract/Free Full Text]
  25. Kurup, V. P., S. Mauze, H. Choi, B. W. Seymour, and R. L. Coffman. 1992. A murine model of allergic bronchopulmonary aspergillosis with elevated eosinophils and IgE. J. Immunol. 148:3783–3788.[Abstract]
  26. Kurup, V. P., H. Choi, P. S. Murali, and R. L. Coffman. 1994. IgE and eosinophil regulation in a murine model of allergic aspergillosis. J. Leukoc. Biol. 56:593–598.[Abstract]
  27. Wills-Karp, M. 2001. IL-12/IL-13 axis in allergic asthma. J. Allergy Clin. Immunol. 107:9–18.[CrossRef][Medline]
  28. Hamelmann, E., J. Schwarze, K. Takeda, A. Oshiba, G. L. Larsen, C. G. Irvin, and E. W. Gelfand. 1997. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am. J. Respir. Crit. Care Med. 156:766–775.[Abstract/Free Full Text]
  29. Foster, P. S., A. W. Mould, M. Yang, J. Mackenzie, J. Mattes, S. P. Hogan, S. Mahalingam, A. N. McKenzie, M. E. Rothenberg, I. G. Young, K. I. Matthaei, and D. C. Webb. 2001. Elemental signals regulating eosinophil accumulation in the lung. Immunol. Rev. 179:173–181.[CrossRef][Medline]
  30. Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, K. Nakanishi, N. Yoshida, T. Kishimoto, and S. Akira. 1996. Essential role of Stat6 in IL-4 signaling. Nature 380:627–630.[CrossRef][Medline]
  31. Ihle, J. N. 2001. The Stat family in cytokine signaling. Curr. Opin. Cell Biol. 13:211–307.[CrossRef][Medline]
  32. Ribieras, S., O. Lefebvre, C. Tomasetto, and M. C. Rio. 2001. Mouse Trefoil factor genes: genomic organization, sequences and methylation analyses. Gene 266:67–75.[CrossRef][Medline]
  33. Al-azzeh, E. D., P. Fegert, N. Blin, and P. Gott. 2000. Transcription factor GATA-6 activates expression of gastroprotective trefoil genes TFF1 and TFF2. Biochim. Biophys. Acta 1490:324–332.[Medline]
  34. Bulitta, C. J., J. V. Fleming, R. Raychowdhury, D. Taupin, I. Rosenberg, and T. C. Wang. 2002. Autoinduction of the trefoil factor 2 (TFF2) promoter requires an upstream cis-acting element. Biochem. Biophys. Res. Commun. 293:366–374.[CrossRef][Medline]
  35. Kuperman, D. A., X. Huang, L. L. Koth, G. H. Chang, G. M. Dolganov, Z. Zhu, J. A. Elias, D. Sheppard, and D. J. Erle. 2002. Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat. Med. 8:885–889.[Medline]
  36. Blease, K., J. M. Schuh, C. Jakubzick, N. W. Lukacs, S. L. Kunkel, B. H. Joshi, R. K. Puri, M. H. Kaplan, and C. M. Hogaboam. 2002. Stat6-deficient mice develop airway hyperresponsiveness and peribronchial fibrosis during chronic fungal asthma. Am. J. Pathol. 160:481–490.[Abstract/Free Full Text]
  37. Trifilieff, A., A. El-Hasim, R. Corteling, and C. E. Owen. 2000. Abrogation of lung inflammation in sensitized Stat6-deficient mice is dependent on the allergen inhalation procedure. Br. J. Pharmacol. 130:1581–1588.[CrossRef][Medline]
  38. Rio, M. C., M. P. Chenard, C. Wolf, L. Marcellin, C. Tomasetto, R. Lathe, J. P. Bellocq, and P. Chambon. 1991. Induction of pS2 and hSP genes as markers of mucosal ulceration of the digestive tract. Gastroenterology 100:375–379.[Medline]
  39. Wright, N. A., R. Poulsom, G. W. Stamp, P. A. Hall, R. E. Jeffery, J. M. Longcroft, M. C. Rio, C. Tomasetto, and P. Chambon. 1990. Epidermal growth factor (EGF/URO) induces expression of regulatory peptides in damaged human gastrointestinal tissues. J. Pathol. 162:279–284.[CrossRef][Medline]
  40. Alison, M. R., R. Chinery, R. Poulsom, P. Ashwood, J. M. Longcroft, and N. A. Wright. 1995. Experimental ulceration leads to sequential expression of spasmolytic polypeptide, intestinal trefoil factor, epidermal growth factor and transforming growth factor alpha mRNAs in rat stomach. J. Pathol. 175:405–414.[CrossRef][Medline]
  41. Holgate, S. T., P. M. Lackie, D. E. Davies, W. R. Roche, and A. F. Walls. 1999. The bronchial epithelium as a key regulator of airway inflammation and remodelling in asthma. Clin. Exp. Allergy 29:90–95.
  42. Vignola, A. M., G. Chiappara, L. Siena, A. Bruno, R. Gagliardo, A. M. Merendino, B. S. Polla, A. P. Arrigo, G. Bonsignore, J. Bousquet, and P. Chanez. 2001. Proliferation and activation of bronchial epithelial cells in corticosteroid-dependent asthma. J. Allergy Clin. Immunol. 108:738–746.[CrossRef][Medline]
  43. Hastie, A. T., W. K. Kraft, K. B. Nyce, J. G. Zangrilli, A. I. Musani, J. E. Fish, and S. P. Peters. 2002. Asthmatic epithelial cell proliferation and stimulation of collagen production: human asthmatic epithelial cells stimulate collagen type III production by human lung myofibroblasts after segmental allergen challenge. Am. J. Respir. Crit. Care Med. 165:266–272.[Abstract/Free Full Text]
  44. Konturek, P. C., T. Brzozowski, S. J. Konturek, G. Elia, N. Wright, Z. Sliwowski, L. Thim, and E. G. Hahn. 1997. Role of spasmolytic polypeptide in healing of stress-induced gastric lesions in rats. Regul. Pept. 68:71–79.[CrossRef][Medline]
  45. Hunt, J. F., K. Fang, R. Malik, A. Snyder, N. Malhotra, T. A. Platts-Mills, and B. Gaston. 2000. Endogenous airway acidification. Implications for asthma pathophysiology. Am. J. Respir. Crit. Care Med. 161:694–699.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
C. C. Lewis, J. Y. H. Yang, X. Huang, S. K. Banerjee, M. R. Blackburn, P. Baluk, D. M. McDonald, T. S. Blackwell, V. Nagabhushanam, W. Peters, et al.
Disease-Specific Gene Expression Profiling in Multiple Models of Lung Disease
Am. J. Respir. Crit. Care Med., February 15, 2008; 177(4): 376 - 387.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
K. Ganguly, T. Stoeger, S. C. Wesselkamper, C. Reinhard, M. A. Sartor, M. Medvedovic, C. R. Tomlinson, I. Bolle, J. M. Mason, G. D. Leikauf, et al.
Candidate genes controlling pulmonary function in mice: transcript profiling and predicted protein structure
Physiol Genomics, November 14, 2007; 31(3): 410 - 421.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
A. Munitz, I. Bachelet, F. D. Finkelman, M. E. Rothenberg, and F. Levi-Schaffer
CD48 Is Critically Involved in Allergic Eosinophilic Airway Inflammation
Am. J. Respir. Crit. Care Med., May 1, 2007; 175(9): 911 - 918.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
I. Kouznetsova, C. E. Chwieralski, R. Balder, M. Hinz, A. Braun, N. Krug, and W. Hoffmann
Induced Trefoil Factor Family 1 Expression by Trans-Differentiating Clara Cells in a Murine Asthma Model
Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 286 - 295.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
E. A. Kurt-Jones, L. Cao, F. Sandor, A. B. Rogers, M. T. Whary, P. R. Nambiar, A. Cerny, G. Bowen, J. Yan, S. Takaishi, et al.
Trefoil Family Factor 2 Is Expressed in Murine Gastric and Immune Cells and Controls both Gastrointestinal Inflammation and Systemic Immune Responses
Infect. Immun., January 1, 2007; 75(1): 471 - 480.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
J. J. Reece, M. C. Siracusa, and A. L. Scott
Innate Immune Responses to Lung-Stage Helminth Infection Induce Alternatively Activated Alveolar Macrophages
Infect. Immun., September 1, 2006; 74(9): 4970 - 4981.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
A. L. Greene, M. S. Rutherford, R. R. Regal, G. H. Flickinger, J. A. Hendrickson, C. Giulivi, M. E. Mohrman, D. G. Fraser, and J. F. Regal
Arginase Activity Differs with Allergen in the Effector Phase of Ovalbumin- versus Trimellitic Anhydride-Induced Asthma
Toxicol. Sci., December 1, 2005; 88(2): 420 - 433.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
C K Haston, T G Tomko, N Godin, L Kerckhoff, and M T Hallett
Murine candidate bleomycin induced pulmonary fibrosis susceptibility genes identified by gene expression and sequence analysis of linkage regions
J. Med. Genet., June 1, 2005; 42(6): 464 - 473.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
N. Zimmermann, M. P. Doepker, D. P. Witte, K. F. Stringer, P. C. Fulkerson, S. M. Pope, E. B. Brandt, A. Mishra, N. E. King, N. M. Nikolaidis, et al.
Expression and Regulation of Small Proline-Rich Protein 2 in Allergic Inflammation
Am. J. Respir. Cell Mol. Biol., May 1, 2005; 32(5): 428 - 435.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
C. E. Chwieralski, I. Schnurra, L. Thim, and W. Hoffmann
Epidermal Growth Factor and Trefoil Factor Family 2 Synergistically Trigger Chemotaxis on BEAS-2B Cells via Different Signaling Cascades
Am. J. Respir. Cell Mol. Biol., November 1, 2004; 31(5): 528 - 537.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
U. Forssmann, I. Hartung, R. Balder, B. Fuchs, S. E. Escher, N. Spodsberg, Y. Dulkys, M. Walden, A. Heitland, A. Braun, et al.
n-Nonanoyl-CC Chemokine Ligand 14, a Potent CC Chemokine Ligand 14 Analogue That Prevents the Recruitment of Eosinophils in Allergic Airway Inflammation
J. Immunol., September 1, 2004; 173(5): 3456 - 3466.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Zimmermann, A. Mishra, N. E. King, P. C. Fulkerson, M. P. Doepker, N. M. Nikolaidis, L. E. Kindinger, E. A. Moulton, B. J. Aronow, and M. E. Rothenberg
Transcript Signatures in Experimental Asthma: Identification of STAT6-Dependent and -Independent Pathways
J. Immunol., February 1, 2004; 172(3): 1815 - 1824.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2002-0309OCv1
29/4/458    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nikolaidis, N. M.
Right arrow Articles by Rothenberg, M. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nikolaidis, N. M.
Right arrow Articles by Rothenberg, M. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2003 American Thoracic Society.