Introduction

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Asthma is a complex heritable inflammatory disorder of the airway associated with clinical signs and symptoms of allergic inflammation and reversible airway obstruction. In particular, asthma is associated with enhanced airway responsiveness to various stimuli and causes airway hyper- responsiveness (AHR), chronic pulmonary eosinophilia, elevated serum immunoglobulin (Ig) E, and mucus overproduction (1). The airway epithelium is known to play an integral role in the airway defense. Evidence has shown that the epithelium is fundamentally disordered in patients with chronic asthma (4). Deleterious stimuli cause airway epithelial cells (AEC) to respond with increased secretion of mucus, altered ion transport, and changes in ciliary beating (5). Studies indicate that AEC can also produce and release biologically active mediators that recruit inflammatory cells important in the pathogenesis of airways disorders (5, 6). After injury, AEC proliferate, resulting in an expanded and continuous source of proinflammatory products as well as growth factors that drive airway remodeling (4).

Clinical and physiologic studies show a tight association between T helper (Th) 2 cytokine expression in the lung and the pathophysiology of asthma (7, 8). This is supported by the findings that Th2 cytokine expression is increased in the airways of animal models of asthma (9, 10). Functional blockade of Th2 cytokines can inhibit allergen-induced asthmalike responses in a variety of animal models (11- 14). Functional studies have revealed a pleiotropic function for each of these cytokines, including direct and indirect effects on AEC.

In particular, an important role for the Th2 cytokine interleukin (IL)-9 in asthma has been demonstrated in a number of reports (10, 15). Genetic mapping studies have linked bronchial hyperresponsiveness, atopy, and asthma to human chromosome 5q31-q33, which contains many genes involved in allergic disorders (20). The IL-9 gene was localized to this region and was identified as a candidate in allergic asthma based on linkage homology between humans and mice for bronchial hyperresponsiveness (10). A role for IL-9 as a mediator of asthma was further supported by results in two independent IL-9 transgenic models exhibiting increased mucosal mast cells, AHR, airway eosinophilia, elevated serum IgE levels, mucus overproduction, and an enhanced inflammatory response to mucosal antigen in comparison with control mice (15, 16). Additional supporting studies indicate that IL-9 has a wide range of biologic activities on many of the cell types involved in the allergic inflammatory response (17). IL-9 is distinguished as a Th2 cytokine by its ability to stimulate the production of mast cell mediators and the growth of these cells (23, 24). IL-9 also has a growth-promoting effect on T-helper cells (25) and it potentiates IgE production by B cells (17, 26). Most recently, IL-9 has been shown to induce C-C chemokine expression and mucus production in lung epithelial cells in vitro and in vivo (18, 19, 27, 28).

To better understand the mechanisms by which IL-9 influences the biologic and physiologic activities in the lung that are associated with asthma, we performed suppressive subtractive hybridization of messenger RNA (mRNA) from the whole lung of IL-9 transgenic (Tg5) mice and its parental background strain (FVB/N). Here we report the identification and characterization of mCLCA3, which was selectively induced in the airway epithelium of naive Tg5 mice, and its human homologue hCLCA1 as shared targets of Th2 cytokines.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Mice

The following studies conformed to the principles for laboratory animal research outlined by the Guide for the Care and Use of Laboratory Animals (ILAR [Institute of Laboratory Animal Resources], NRL [National Research Council]) and were approved by the Genaera Institutional Animal Care and Use Committee. IL-9 transgenic mice from two independent lines (Tg5 and Tg54) were generated in a FVB/N background as described previously (29). FVB/NJ (FVB), DBA/2J (D2), C57BL/6J (B6), and B6D2F1/J (F1) mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice 6 to 8 wk of age were used in this study. The animal housing facilities were maintained at 22°C (range, 19° to 24°C) with a cycle of 12:12 h light:dark. Food and water were provided ad libitum.

Suppressive Subtractive Hybridization and Cloning of mCLCA3 and Its Human Counterpart hCLCA1

Suppressive subtractive hybridization was performed on mRNA from the lungs of naive Tg5 mice and parental FVB mice using a commercially available polymerase chain reaction (PCR)-select complementary DNA (cDNA) subtraction kit (Clontech, Palo Alto, CA). Total RNA was prepared from the lungs of Tg5 and FVB mice using Trizol as described by the manufacturer (GIBCO/BRL, Rockville, MD). mRNA was purified from total RNA with oligo(dT) cellulose columns (Pharmacia, Piscataway, NJ). Differential cDNA analysis of Tg5 and FVB lungs was carried out following the manufacturer's protocol (Clontech). This PCR-amplified subtracted cDNA library was cloned into a TA cloning vector (Stratagene, La Jolla, CA) and transformed into DH10B high efficient competent cells (GIBCO/BRL). Inserts of recombinant clones were PCR-amplified using M13 forward and reverse primers directly from bacteria colonies, and the PCR products were sequenced using a T7 primer. All the sequences were subjected to a BLAST (Basic Local Allignment Search Tool) search against GenBank.

The full-length mCLCA3 gene was cloned by screening a murine lung cDNA library (Clontech) with an EST clone (GenBank accession no. aa541829) identified from the BLAST search. The clone was ordered from IncyteGenomics (Palo Alto, CA) in 1996, with the PCR product generated from subtraction as a probe. To clone the human equivalent of mCLCA3, sequences of mCLCA3 were used to perform an EST database search, and several undescribed human EST (Expressed Sequence Tag) clones (AA508854 and AA581198) were found to share a significant homology. 5' rapid amplification of cDNA ends (RACE) (Clontech) were used to generate the full-length sequences of the human counterpart of mCLCA3, which turned out to be identical to hCLCA1.

In Situ Hybridization

Formalin-fixed, paraffin-embedded lung tissues from Tg5 and FVB mice were hybridized with biotin-uridine triphosphate (UTP)-labeled, single-strand RNA antisense and sense probes. Briefly, RNA probes were transcribed from T7 or T3 promoters of pBS-mCLCA3(1150-1377) (containing an insert corresponding to the 1150 to 1377 bp of mCLCA3 cDNA), using either T7 or T3 RNA polymerases (GIBCO/BRL) for 90 min in the presence of biotin-11-UTP. All tissue sections were pretreated with steam heat for 20 min and proteinase K for 10 min. After prehybridization (98°C for 20 min), hybridization was carried out at 42°C for 2 h with a probe concentration of 1 ng/µl. Probe diluent and poly dT (at 1.5 ng/ml) were used as negative and positive controls, respectively, to hybridize serial sections.

Cells

Human primary lung cultures were established from discarded lung sections obtained from lung resections. Lung tissues were handled under sterile conditions and first minced with scissors and passed through a wire mesh. Tissues were then digested with 175 U/ml of collagenase (Sigma, St. Louis, MO) for 1 h at 37°C. Tissue was filtered through 45- and 15-µm matrices, and then resuspended in Dulbecco Iscove's medium and plated into 10-cm tissue culture plates. Plates were then cultured for 1 h at 37°C to remove adherent macrophages. Nonadherent cells were harvested, resuspended at 2 × 10⁵ cells/ml in Dulbecco Iscove's medium supplemented with 10% fetal bovine serum and antibiotics, and cultured at 37°C in 5% CO₂ for 4 to 5 d.

Intratracheal Instillation

Recombinant mouse IL-4, IL-9, IL-13, and interferon (IFN)- (R&D Systems, Minneapolis, MN) were reconstituted in sterile phosphate-buffered saline solution (GIBCO/BRL) containing 0.1% bovine serum albumin fraction V (Sigma). B6 mice were anesthetized by inhalation of 1.5% halothane. Cytokines were administered directly into the trachea at doses of 2.5 or 5 µg (in 20 µl) per mouse under light anesthesia once daily for 10 d.

Antigen Sensitization, Antibody Treatment, and Pulmonary Function

Antigen sensitization was carried out essentially as described previously (15, 30). In brief, mice were anesthetized by methoxyflurane inhalation, and 25 µl of Aspergillus fumigatus extract antigen (1:50 wt/vol final dilution in 10% glycerol) (Bayer Pharmaceuticals, Elkhart, IN) were applied to the left nare. Mice were immunized intratracheally once per week with anti-IL-9 antibody or isotype control (PharMingen, San Diego, CA) at 200 µg/mouse and on Day 22 (i.e., Days 0, 7, 14, 21, and 22) and were phenotyped approximately 12 h after the last immunization (Day 23).

Northern Blot Analysis and Reverse Transcription/ Polymerase Chain Reaction

Total RNA was isolated from mouse tissues, and primary lung cells and cell lines were cultured using Trizol reagent (GIBCO/ BRL) following the manufacturer's protocol. Northern blot analyses were performed by electrophoresis of 10 µg of total RNA on 1% formaldehyde gels and transferring RNA to GeneScreen Plus membranes (NEN Life Sciences, Boston, MA). Membranes were probed with [³²P]deoxycytidine triphosphate random radiolabeled mCLCA3 fragments generated by restriction digestion of the corresponding cDNA clones. Reverse transcription (RT)-PCR was performed by reverse transcribing 1 µg of total RNA and amplifying the cDNA with the appropriate primers by PCR. Products were separated by electrophoreses on 2% agarose gels and visualized by ethidium bromide staining. Primer pairs for hCLCA1 were the following: sense, 5'-GGCACAGATCTTTT CATTGCTA 3', and antisense, 5'-GTGAATGCCAGGAATGGTGCT-3', which produce a 182-bp product. hPMS2, which is a ubiquitously expressed housekeeping gene, is assayed as an internal control using primers previously described (10).

Western Blot Analysis

Antiserum to mCLCA3 protein was prepared by immunizing rabbits with multiple antigen peptides (MAPs) representing the central portion of the protein. The peptide sequences are the following: P1, N-CLVLDKSGSMLNDDRLNRMNQA-NH2 (residues 309-330); P2, QSELKQLNSGADRDLLIKHL-NH2 (residues 357-375); P3, KKKYPTDGSEIVLLTDGEDNTISSC-NH2 (residues 398-422); P4, TTHPPTIFIWDPSGVEQNGFILDC-NH2 (residues 524-546); P5, CPPITVTPVVNKNYGKFPSPVT-NH2 (residues 590-610). MAPs were synthesized by automated solid phase peptide synthesis using 9 FluorenylMethylOxycarbonyl protection chemistry and were purified by reverse phase high performance liquid chromatography. The antiserum obtained from the second and subsequent bleeds were purified with an antigen-conjugated sepharose column and then used for detection of mCLCA3 by Western blot analysis. Lung tissues from Tg5 and FVB mice were snap-frozen in liquid nitrogen. Sections (3 mm³) were lysed in 500 µl of modified RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X100, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM NaF, 10 mg/ml leupeptin, 2 mM Pefabloc). A total of 20 µl of lysates was fractionated on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels (4 to 20% Novex gel) and electrophoretically transferred to nitrocellulose (polyvinylidene difluoride; Amersham, Piscataway, NJ). Membranes were blocked in 5% nonfat milk solids and probed with purified polyclonal anti-mCLCA3 rabbit antiserum and with a secondary horseradish peroxidase-linked goat antirabbit antibody (1/10,000; Amersham). An enhanced chemiluminescence detection kit (Pierce, Rockford, IL) was used for detection.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Cloning mCLCA3 by Suppressive Subtraction Hybridization of mRNA from the Lungs of IL-9 Transgenic Mice (Tg5) and Control Mice (FVB)

To understand the molecular mechanisms associated with the pleiotropic effects of IL-9, suppressive subtraction hybridization was performed on mRNA from whole lungs of IL-9 transgenic mice (Tg5) and background mice (FVB). A subtracted cDNA library of 1,100 recombinant clones was analyzed, and several transcripts were found to be represented multiple times, each accounting for 2 to 5% of the library. One of the most prominent transcripts in the library was the IL-9 cDNA, which served as an internal control for the efficiency of subtraction (data not shown). Two other cDNA fragments that were found in multiple copies (representing 3% of the library) belonged to mCLCA3. Subsequent cloning and sequencing of the full-length clone revealed a 2,931-bp cDNA containing an open reading frame encoding a protein of 913 amino acids (Figure 1).

View larger version (63K): [in this window] [in a new window]   Figure 1.   Alignment of the mCLCA3 protein with the hCLCA1 sequence. Identical amino acids are marked with asterisks, and conservative amino-acid substitutions are marked with dots. The signal sequences and the four putative transmembrane regions are underlined.

Identification of the Human Homolog of mCLCA3

Sequence comparison with the GenBank dbEST revealed several human EST clones that shared a significant homology with mCLCA3. Cloning and sequencing of these human ESTs and 5'-RACE clones yielded a cDNA containing an open reading frame encoding a protein of 914 amino acids. This human homolog of mCLCA3 was identical to the human calcium-activated chloride channel hCLAC1 described by Gruber and coworkers (31). Amino-acid sequence alignment of mCLCA3 with all the known human CLCAs (hCLCA1-3) reveals that hCLAC1 shares the highest homology with mCLCA3 (76% identity and more than 85% similarity), especially in the four putative transmembrane regions (90 to 100% identity). Similar alignment of hCLCA1 with all the known murine CLCAs (mCLCA1-3) reveals that mCLCA3 shares the highest homology with hCLCA1 (Figure 1 and data not shown). This unusually high homology between mCLCA3 and hCLCA1 strongly suggests that they are equivalent genes.

Expression of the mCLCA3 Gene in the Lungs of IL-9 Transgenic Mice

To confirm the expression of the mCLCA3 gene in the lung of IL-9 transgenic mice, Northern blot analysis of the total RNA from the lungs of Tg5 and FVB mice was performed. The RNA was hybridized with an mCLCA3-specific probe. As shown in Figure 2A, the expression of mCLCA3 mRNA was clearly elevated in the lung of Tg5 mice compared with that of FVB mice. Northern blot analysis of RNA from the lungs of an independent IL-9 transgenic line, Tg54 (23), demonstrated similar results (data not shown), indicating that induction of mCLCA3 in IL-9 transgenic mice was not owing to an artifact related to the insertion of the transgene. Western blot analysis using anti-mCLCA3-specific antiserum confirmed the significantly increased expression of the mCLCA3 protein in the lung of Tg5 mice compared with the FVB background strain (Figure 2B).

View larger version (57K): [in this window] [in a new window]   Figure 2.   Expression of mCLCA3 in IL-9 transgenic mice (Tg5) and control mice (FVB). (A) Northern blot analysis of total RNA from FVB and Tg5 lungs. Total RNA (10 µg) was hybridized with a specific mCLCA3 probe. mCLCA3 message is shown as a 3-kb band. The blot was stripped and reprobed with GAPDH as a control for equal loading. (B) Western blot analysis of tissue lysate from FVB and Tg5 lungs. Tissue lysates (50 µg) were hybridized with polyclonal anti-mCLCA3 antiserum. mCLCA3 is shown as a 90-kD band. Nonspecific hybridizing bands demonstrate equal loading. (C) Tissue distribution of mCLCA3 in FVB and Tg5 mice. Total RNA (10 µg) was hybridized with a specific mCLCA3 probe. mCLCA3 message is shown as a 3-kb band. Lanes 1 to 14 represent brain, colon, heart, kidney, liver, lung, lymph nodes, skeletal muscle, ovary, spleen, small intestine, stomach, thymus, and uterus. Blots were stripped and reprobed with GAPDH as a control (not shown).

mCLCA3 expression was not detectable in the lung of FVB mice. In contrast, mCLCA3 message was readily detected in the uterus, small intestine, and colon, and at relatively lower levels in the ovary and stomach (Figure 2C, upper panel). Similarly, hCLCA1 expression was reported in the small intestine and colon (31). The similar tissue distribution of these two genes further supports the hypothesis that mCLCA3 and hCLCA1 are equivalent genes in these two species and may serve similar biologic functions. The highly induced expression of mCLCA3 in the lung of IL-9 transgenic mice, which have demonstrated features consistent with allergic asthma, suggests the involvement of mCLCA3 in the pathogenesis of this disorder.

Cellular Source of mCLCA3 in Mouse Lung Demonstrated by In Situ Hybridization

To determine the cellular source of the induced expression of mCLCA3 in the lung, sections from Tg5 mice and control FVB mice were hybridized with biotin-labeled sense and antisense RNA probes. As shown in Figure 3, antisense probe hybridization revealed minimal staining in the airway epithelium of FVB lung sections (Figure 3B), whereas strongly positive staining was observed only in the airway epithelium of Tg5 lungs (Figure 3D). In contrast, sense probe hybridization produced no staining in either FVB or Tg5 lung sections (Figures 3A and 3C).

View larger version (141K): [in this window] [in a new window]   Figure 3.   mCLCA3 expression is induced in the airway epithelium of Tg5 mice. In situ hybridization of mCLCA3 expression in the lungs of FVB (A, B) and Tg5 (C, D) mice. Hybridization with biotin-labeled antisense RNA probes showed minimal staining in the epithelium of naive FVB lung (B), but strong staining in the airway epithelium of Tg5 mice (D). Control hybridization using sense probes yielded no staining in either strain (A, C). Nuclei were counterstained with hematoxylin and eosin. Lower magnification is 16-fold and higher magnification is 80-fold of the original size.

Specific Induction of the mCLCA3 Gene by Th2 Cytokines

To examine whether the mCLCA3 expression in the lung could also be upregulated by other Th2 cytokines, we treated B6 mice with Th2 cytokines IL-4, IL-9, and IL-13, or IFN- as a control for Th1 cytokines. Recombinant murine cytokines were instilled into the tracheas of anesthetized mice daily for 10 d. Approximately 24 h after the last instillation, the mice were killed and the lungs were harvested and extracted for mRNA expression analysis. As expected (Figure 4), IL-9 induced the expression of mCLCA3, confirming the observation in IL-9 transgenic mice. In addition, IL-4 and IL-13 were also able to induce the expression of mCLCA3 in the lung, whereas no expression was observed in the lungs of mice treated with IFN- (Figure 4). These data demonstrate a role for Th2 cytokines in the induction of mCLCA3 lung expression.

View larger version (69K): [in this window] [in a new window]   Figure 4.   The effect of intratracheal administration of cytokines on mCLCA3 lung expression. Northern blot analysis of total RNA from naive B6 lungs, and B6 lungs after a daily intratracheal administration of IL-4, IL-9, IL-13, or IFN- for 10 d. Total lung RNA (10 µg) was hybridized with a specific mCLCA3 probe. mCLCA3 message was detected as a 3-kb band. Blots were stripped and reprobed with GAPDH as a control for equal loading.

Induction of mCLCA3 Expression in the Lung of Antigen-Exposed F1 Mice and Suppression of Antigen-Induced mCLCA3 Expression by Neutralizing anti-IL-9 Antibody Treatment

We have previously shown that F1 mice have intermediate lung IL-9 expression compared with the two parental strains, B6 and D2 (10). Exposure of the F1 mice to a natural antigen, A. fumigatus, induced asthmalike responses with AHR, pulmonary eosinophilia, elevated serum IgE, and mucus overproduction (11). Northern blot analysis showed minimal lung expression of mCLCA3 in naive F1 mice; however, mCLCA3 expression was significantly increased in the lung of antigen-exposed mice (Figure 5). A similar induction of mCLCA3 expression was also observed in the lungs of antigen-exposed FVB and D2 mice (data not shown), indicating a tight association of mCLCA3 expression with this inflammatory response.

View larger version (71K): [in this window] [in a new window]   Figure 5.   Induction of mCLCA3 expression in the lung of antigen-exposed F1 mice and suppression of antigen-induced mCLCA3 expression by neutralizing IL-9 antibody treatment. Northern blot analysis of total RNA from whole lung tissues of naive and antigen-exposed F1 mice treated with intratracheal anti-mIL-9 or an isotype control immunoglobulin. Total RNA (10 µg) was hybridized with a specific mCLCA3 probe. mCLCA3 message was detected as a 3-kb band. Blots were stripped and reprobed with GAPDH as a control. Aspergillus fumigatus, Asp.

IL-9-blocking antibody treatment of these antigen-exposed mice has been shown to suppress the asthmalike phenotype (11). In this study, groups of mice exposed to A. fumigatus were treated with either intratracheal anti- mIL-9 antibody or an isotype control IgG. Northern blot analysis of whole lungs from antigen-exposed mice showed that anti-mIL-9 also suppressed mCLCA3 gene expression in the lungs, whereas control antibody had no effect on the mCLCA3 expression in the lungs (Figure 5). Collectively, these data indicate that IL-9 is both necessary and sufficient for the induction of mCLCA3 expression by antigen in this experimental model of asthma.

Direct Induction of the hCLCA1 Gene by Recombinant Human Th2 Cytokines

We next examined if hCLCA1 is inducible by Th2 cytokines to further test the hypothesis that hCLCA1 is the human counterpart of mCLCA3. Human primary lung epithelial cells derived from two nonasthmatic patients were analyzed for expression of the hCLCA1 gene in the presence or absence of recombinant human IL-9. RT-PCR using hCLCA1 specific primers showed that expression of hCLCA1 was induced in cells treated with IL-9, whereas no expression was observed in untreated cells (Figure 6). A similar induction was observed in lung primary epithelial cells treated with human recombinant IL-4 (data not shown). These results indicate that, like mCLCA3, hCLCA1 is a target of human Th2 cytokines.

View larger version (38K): [in this window] [in a new window]   Figure 6.   Induction of hCLCA1 expression by IL-9 in human primary lung cultures. RT-PCR analysis of hCLCA1 in 5-d-old primary lung cultures derived from unrelated nonasthmatic patients (1 and 2) grown with (+) or without () recombinant hIL-9.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this report, we demonstrated mCLCA3 as a cytokine-inducible calcium-activated chloride channel. In contrast to other known chloride channels that are expressed constitutively in the lung epithelium, expression of mCLCA3 was highly inducible by Th2 cytokines such as IL-4, IL-9, and IL-13. Importantly, we demonstrated that IL-9 is both necessary and sufficient for antigen-induced expression of mCLCA3 in the lungs of an experimental model of asthma. In contrast, mCLCA3 expression could not be upregulated by the Th1 cytokine IFN-, suggesting that mCLCA3 is selectively upregulated. Moreover, we showed that hCLCA1 is the human counterpart of mCLCA3 and is also induced by Th2 cytokines in human primary lung cultures. More important, mCLCA3 is virtually absent in the lungs of naive mice, suggesting that mCLCA3 is not involved in the normal physiologic functioning of the lung. This tight association of mCLCA3 lung expression with asthmalike disorders suggested that mCLCA3 and perhaps its human counterpart, hCLCA1, might be important in regulating antigen-stimulated epithelial cell functions in allergen-induced diseases. Our interpretation of these data is consistent with recent findings in which the introduction of mCLCA3 and hCLCA1 into NCI-H292, a mucoepidermal cell line, induced mucus production in vitro (32). Moreover, these investigators were able to demonstrate that the intratracheal administration of adenovirus-expressing mCLCA3 antisense RNA into an in vivo allergic model of asthma suppressed AHR and mucus overproduction in response to an antigen.

This chloride channel (mCLCA3 in mice and hCLCA1 in humans) was also expressed at high levels in the colon and small intestine. Expression of hCLCA1 in the intestine was restricted to basal crypts and goblet cells, suggesting the involvement of this gene in secretory or absorptive processes (31). Murine CLCA3 is identical to murine gob-5, a gene cloned from intestinal goblet cells (33). Another member of the same family, mCLCA1 is expressed constitutively in respiratory epithelium (bronchi and airways) as well as in murine submucosal glands (34). In contrast to mCLCA3, it is likely that mCLCA1 is involved in maintaining normal physiologic conditions in the respiratory tract. mCLCA1 does not appear to be inducible, and antigen exposure had no effect on the expression of this closely related channel in murine airways (Y. Zhou, unpublished results), suggesting that it may not be involved in the pathogenesis of pulmonary diseases.

Further studies will be needed to elucidate the exact role of mCLCA3 and hCLCA1 in the pathogenesis of obstructive lung diseases. Additional studies on this channel in association with other pulmonary diseases such as cystic fibrosis, chronic bronchitis, emphysema, and lung cancer may shed light on the pathogenic mechanisms of these diseases. Moreover, specific channel antagonists may offer new therapeutic strategies for these conditions.