Interleukin-5-mediated Allergic Airway Inflammation Inhibits the Human Surfactant Protein C Promoter in Transgenic Mice
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m009481200
ISSN1083-351X
AutoresAnil Mishra, Timothy E. Weaver, David Beck, Marc E. Rothenberg,
Tópico(s)Pediatric health and respiratory diseases
ResumoAllergen challenge in the lung of humans and animals is associated with surfactant dysfunction, but the mechanism of this effect has not been established. By using a murine model of asthma we now report the effect of allergen-induced airway inflammation on the expression of transgenes regulated by the human surfactant protein (hSP)-C promoter. The hSP-C 3.7-kilobase pair promoter was used to direct the expression of eotaxin, an eosinophil-selective chemokine, into the lungs of several transgenic lines. As expected, the transgenic mice expressed increased amounts of eotaxin mRNA and protein compared with wild-type mice. Surprisingly, following allergen challenge, there was a marked down-regulation of transgene mRNA in three independent transgenic lines. The down-regulation was in contrast to other related proteins such as endogenous eotaxin and surfactant protein D levels, which were both increased following allergen challenge. Consistent with specific down-regulation of the eotaxin transgene, there was no increase in pulmonary eosinophil levels in the transgenic mice above that found in wild-type mice. Analysis of hSP-C transgenic mice with distinct reporter genes and 3′-untranslated regions revealed that allergen challenge was directly affecting the hSP-C promoter. We hypothesized that allergen-induced down-regulation of the hSP-C promoter was related to the eosinophilic inflammation. To test this, we blocked eosinophilic inflammation in the lungs by treating mice with neutralizing antiserum against interleukin-5. Interestingly, this treatment also blocked allergen-induced inhibition of the hSP-C promoter. These results establish that allergic airway inflammation is associated with up-regulation of the surfactant proteins primarily involved in immunity, whereas down-regulation of the surfactant protein primarily involved in maintaining airway patency. Furthermore, the marked down-regulation of the hSP-C promoter is interleukin-5-dependent, implying a critical role for eosinophilic inflammation. These results suggest that alterations in surfactant protein levels may contribute to immune and airway dysfunction in asthma. Allergen challenge in the lung of humans and animals is associated with surfactant dysfunction, but the mechanism of this effect has not been established. By using a murine model of asthma we now report the effect of allergen-induced airway inflammation on the expression of transgenes regulated by the human surfactant protein (hSP)-C promoter. The hSP-C 3.7-kilobase pair promoter was used to direct the expression of eotaxin, an eosinophil-selective chemokine, into the lungs of several transgenic lines. As expected, the transgenic mice expressed increased amounts of eotaxin mRNA and protein compared with wild-type mice. Surprisingly, following allergen challenge, there was a marked down-regulation of transgene mRNA in three independent transgenic lines. The down-regulation was in contrast to other related proteins such as endogenous eotaxin and surfactant protein D levels, which were both increased following allergen challenge. Consistent with specific down-regulation of the eotaxin transgene, there was no increase in pulmonary eosinophil levels in the transgenic mice above that found in wild-type mice. Analysis of hSP-C transgenic mice with distinct reporter genes and 3′-untranslated regions revealed that allergen challenge was directly affecting the hSP-C promoter. We hypothesized that allergen-induced down-regulation of the hSP-C promoter was related to the eosinophilic inflammation. To test this, we blocked eosinophilic inflammation in the lungs by treating mice with neutralizing antiserum against interleukin-5. Interestingly, this treatment also blocked allergen-induced inhibition of the hSP-C promoter. These results establish that allergic airway inflammation is associated with up-regulation of the surfactant proteins primarily involved in immunity, whereas down-regulation of the surfactant protein primarily involved in maintaining airway patency. Furthermore, the marked down-regulation of the hSP-C promoter is interleukin-5-dependent, implying a critical role for eosinophilic inflammation. These results suggest that alterations in surfactant protein levels may contribute to immune and airway dysfunction in asthma. surfactant protein human surfactant protein kilobase pair interleukin ovalbumin phosphate-buffered saline bronchoalveolar lavage fluid enzyme-linked immunosorbent assay N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine untranslated region The surfactant proteins compose a group of molecules with diverse biological functions. Surfactant protein (SP)1-A and SP-D are calcium-dependent lectins that have primary roles in host defense by serving as regulators for innate immune responses (1Mason R.J. Greene K. Voelker D.R. Am. J. Physiol. 1998; 275: L1-L13Crossref PubMed Google Scholar, 2LeVine A.M. Kurak K.E. Wright J.R. Watford W.T. Bruno M.D. Ross G.F. Whitsett J.A. Korfhagen T.R. Am. J. Respir. Cell Mol. Biol. 1999; 20: 279-286Crossref PubMed Scopus (170) Google Scholar). SP-B and SP-C are primarily involved in maintaining physiological responses specifically in the lung by reducing lung surface tension thereby promoting proper respiration. In support of the critical function of surfactant proteins, a deficiency or dysregulation of surfactant proteins can have profound effects. For example, surfactant deficiency is responsible for the respiratory distress syndrome of infancy, and surfactant replacement is an effective therapy for this disorder. Furthermore, reduced levels of surfactant constituents are found in the lungs of patients with adult respiratory distress syndrome. Experimental disruption of SP-A, SP-B, or SP-D in mice results in various lung-specific problems including impairment in pulmonary host responses to infection (SP-A and SP-D) and neonatal respiratory failure (SP-B) (3Clark J.C. Wert S.E. Bachurski C.J. Stahlman M.T. Stripp B.R. Weaver T.E. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7794Crossref PubMed Scopus (56) Google Scholar, 4Korfhagen T.R. Bruno M.D. Ross G.F. Huelsman K.M. Ikegami M. Jobe A.H. Wert S.E. Stripp B.R. Morris R.E. Glasser S.W. Bachurski C.J. Iwamoto H.S. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9594-9599Crossref PubMed Scopus (374) Google Scholar, 5Korfhagen T.R. Sheftelyevich V. Burhans M.S. Bruno M.D. Ross G.F. Wert S.E. Stahlman M.T. Jobe A.H. Ikegami M. Whitsett J.A. Fisher J.H. J. Biol. Chem. 1998; 273: 28438-28443Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 6Weaver T.E. Beck D.C. Biol. Neonate. 1999; 76 Suppl. 1: 15-18Crossref PubMed Scopus (29) Google Scholar). Hence, normal pulmonary function depends upon proper expression and function of surfactant proteins. The pleiotropic and fundamental properties of surfactant proteins underscore the importance of elucidating the role of surfactant proteins in various lung disorders. Asthma is an airway inflammatory disorder whose prevalence is on the rise worldwide despite increased availability of therapeutic options (7Holgate S.T. Nature. 1999; 402: 2-4Crossref PubMed Scopus (528) Google Scholar). An extensive amount of experimentation in humans and rodents using models of allergen-induced airway inflammation has demonstrated an essential role for Th2 lymphocytes, eosinophils, and various cytokines (e.g. IL-4, IL-5, and IL-13) (8Drazen J.M. Arm J.P. Austen K.F. J. Exp. Med. 1996; 183: 1-5Crossref PubMed Scopus (274) Google Scholar, 9Wills-Karp M. Annu. Rev. Immunol. 1999; 17: 255-281Crossref PubMed Scopus (958) Google Scholar), but the specific role of surfactant proteins has not been extensively examined. Because surfactant lipids and proteins are primary molecules involved in maintaining airway patency, an impaired process in patients with asthma, they are likely to be involved, at least in part, in the pathophysiology of asthma. Consistent with this, segmental allergen challenge in sensitized animals and humans results in loss of surfactant activity (10Hohlfeld J.M. Ahlf K. Enhorning G. Balke K. Erpenbeck V.J. Petschallies J. Hoymann H.G. Fabel H. Krug N. Am. J. Respir. Crit. Care Med. 1999; 159: 1803-1809Crossref PubMed Scopus (93) Google Scholar, 11Jarjour N.N. Enhorning G. Am. J. Respir. Crit. Care Med. 1999; 160: 336-341Crossref PubMed Scopus (60) Google Scholar, 12Liu M. Wang L. Holm B.A. Enhorning G. Clin. Exp. Allergy. 1997; 27: 802-807Crossref PubMed Scopus (24) Google Scholar). Furthermore, although no difference in bronchoalveolar lavage fluid (BALF) phospholipid levels are seen between patients with asthma and controls, following allergen challenge, only patients with asthma have a change in the ratio between phosphatidylcholine and phosphatidylglycerol, the major lipid constituents of surfactant (13Heeley E.L. Hohlfeld J.M. Krug N. Postle A.D. Am. J. Physiol. 2000; 278: L305-L311PubMed Google Scholar). In the present investigation, we were interested in testing the hypothesis that allergic airway inflammation regulates the expression of hSP-C, a major gene product of alveolar type II epithelial cells. We chose to utilize an established murine model of asthma and an established transgenic system to facilitate direct analysis of human SP-C expression. Specific transcription of the hSP-C gene in pulmonary epithelial cells is regulated by the first 3.7 kb of the SP-C promoter (14Korfhagen T.R. Glasser S.W. Wert S.E. Bruno M.D. Daugherty C.C. McNeish J.D. Stock J.L. Potter S.S. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6122-6126Crossref PubMed Scopus (93) Google Scholar). This region confers all of the necessary elements to direct specific expression of transgenes in the distal respiratory epithelium and has been instrumental for experimental analysis of lung development and disease (15Glasser S.W. Korfhagen T.R. Wert S.E. Whitsett J.A. Am. J. Physiol. 1994; 267: L489-L497PubMed Google Scholar). We therefore examined the effect of allergic airway inflammation on the expression of transgenes under the control of the hSP-C 3.7-kb promoter. Specific pathogen-free FVB mice, 8–10 weeks old were obtained from the NCI, Frederick, MD. Eotaxin or SP-B transgenic mice were generated by inserting the mouse eotaxin cDNA coding region (20–328 base pairs) or the coding region of the human SP-B cDNA (10–837 base pairs) (16Lin S. Na C.L. Akinbi H.T. Apsley K.S. Whitsett J.A. Weaver T.E. J. Biol. Chem. 1999; 274: 19168-19174Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) into the BamHI/EcoRI site of the hSP-C 3.7 transgenic cassette (construct I andII, respectively in Fig. 1). This transgenic construct utilizes the simian virus 40 (SV40) polyadenylation signal that is used as a probe for identifying transgenic mice by Southern blot analysis of tail DNA. In other experiments, transgenic mice containing a “mammalianized” SP-C 3.7 promoter followed by noncoding 5′ exons from the rabbit β-globin gene, the hSP-B cDNA, and the 3′ polyadenylation signal from bovine growth hormone were examined (construct III in Fig. 1) (17Beck D.C. Na C.L. Whitsett J.A. Weaver T.E. J. Biol. Chem. 2000; 275: 3371-3376Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). All mice (FVB/N) were housed under pathogen-free conditions and examined between 6 and 16 weeks of age. A mouse model of allergic lung disease was established using methods described previously (18Mishra A. Hogan S.P. Lee J.J. Foster P.S. Rothenberg M.E. J. Clin. Invest. 1999; 103: 1719-1727Crossref PubMed Scopus (335) Google Scholar). In brief, mice were lightly anesthetized with Metofane inhalation (methoxyfluorane; Pittman-Moore, Mundelein, IL), and 100 μg (50 μl of saline) of Aspergillus fumigatus (Bayer Pharmaceuticals, Spokane, WA) or 50 μl of normal saline alone was applied to the nares using a micropipette with the mouse held in the supine position. After instillation, mice were held upright until alert. After three treatments per week for 3 weeks, mice were sacrificed between 18 and 20 h after the last intranasal challenge. In some experiments, mice were treated with neutralizing doses (1.0 mg) of anti-IL-5 (TRFK-5) or control IgG1 (GL113 (anti-Escherichia coliβ-galactosidase); both kindly provided by Dr. Fred Finkelman, University of Cincinnati and Robert Coffman, DNAX, Palo Alto, CA) by intraperitoneal injection each week prior to the 1st weekly allergen challenge. In other experiments, a mouse model of allergic lung disease was established using intranasal ovalbumin (OVA). In brief, mice were sensitized by intraperitoneal injection with OVA (50 μg)/alum (1 mg) in 0.9% sterile saline on day 0 and day 12. Mice were then challenged intranasally with 50 μl containing 150 μg of OVA or saline on days 20, 21, 22, and 23 and then sacrificed 18–20 h after the last OVA challenge. Mice were euthanized by CO2inhalation and exsanguinated, and the trachea was cannulated and lavaged with 2 replicate volumes (0.8 ml) of normal saline containing 0.2 mm EDTA to obtain BALF. Lavage fluid was centrifuged at 250 × g for 10 min at 4 °C, and the cell pellet was resuspended in PBS and analyzed for total cell count and differential analysis. Aliquots of the BALF supernatant were taken for total protein analysis by bicinchoninic acid analysis (Pierce). A sandwich enzyme-linked immunosorbent assay (ELISA) was performed to measure eotaxin protein levels in BALF. The wells of ELISA plates (DYNEX Technologies, Chantilly, VA) were first coated with 40 ng (100 μl) of diluted mouse eotaxin-specific goat IgG (AF-420NA; R & D Systems, Minneapolis, MN) in PBS and incubated overnight at 4 °C. Following the incubation, the plate was washed with phosphate-buffered saline with 0.05% Tween 20 (PBST). Nonspecific binding sites were blocked with 300 μl of PBS containing 1% bovine serum albumin, 5% sucrose, and 0.05% NaN3 and incubated at room temperature for 1 h. Mouse eotaxin (kindly provided by PeproTech, Rocky Hill, NJ) was used as a quantitation standard. The eotaxin standard and BALF samples were diluted in the blocking solution and added to each well and incubated for 2 h at room temperature. Wells were washed three times with PBST and then further incubated with 20 ng (100 μl) of mouse eotaxin affinity-purified polyclonal secondary antibody (BAF-420, R & D Systems) for 2 h at room temperature followed by three washes with PBST. Fifty nanograms (100 μl) of streptavidin-horseradish peroxidase (Immunotech, Marseilles, France) was added and incubated for 1 h at room temperature. Finally, ABTS peroxidase substrate solution that contains 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulfonate) at a concentration of 0.3 g/liter in a glycine/citric acid buffer and 0.01% H2O2 (Kirkegaard & Perry, Gaithersburg, MD) was used to complete the reaction. The substrate reaction was stopped by adding 50 μl/well of 1 m H2SO4and quantitated by an ELISA plate reader at 495 nm. RNA was extracted from the lung tissue using the Trizol reagent (Life Technologies, Inc.) following the manufacturer's protocol. Twenty micrograms of total RNA from each sample were electrophoresed on 1.5% formaldehyde agarose gels and transferred to GeneScreen hybridization membrane (PerkinElmer Life Sciences) with 10× SSC. The membrane was UV cross-linked and prehybridized at 42 °C for 1 h in a 50% formamide buffer (pH 7.5), containing 10% dextran sulfate, 5× SSC, 1× Denhardt's solution, 1% SDS, 100 μg/ml of herring sperm DNA, and 20 mm Tris. The 32P-labeled probes were prepared for mouse eotaxin cDNA (19Rothenberg M.E. Luster A.D. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8960-8964Crossref PubMed Scopus (322) Google Scholar), SV40 polyadenylation signal sequence, and mouse surfactant protein cDNAs (SP-A, SP-B, SP-C, and SP-D) (kindly provided by Dr. Jeffrey Whitsett) as described previously (20D'Amore-Bruno M.A. Wikenheiser K.A. Carter J.E. Clark J.C. Whitsett J.A. Am. J. Physiol. 1992; 262: L40-L47PubMed Google Scholar,21Whitsett J.A. Dey C.R. Stripp B.R. Wikenheiser K.A. Clark J.C. Wert S.E. Gregory R.J. Smith A.E. Cohn J.A. Wilson J.M. Engelhardt J. Nat. Genet. 1992; 2: 13-20Crossref PubMed Scopus (76) Google Scholar) and hybridized overnight at 42 °C using 1–2 × 106 dpm/ml of the respective probes (22Luster A.D. Rothenberg M.E. J. Leukocyte Biol. 1997; 62: 620-633Crossref PubMed Scopus (133) Google Scholar, 23Matthews A.N. Friend D.S. Zimmermann N. Sarafi M.N. Luster A.D. Pearlman E. Wert S.E. Rothenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6273-6278Crossref PubMed Scopus (243) Google Scholar). The membranes were washed for 20 min at 42 °C, 20 min at 50 °C, 20 min at 60 °C in 2× SSC, 0.1% SDS and 20 min in 0.1× SSC, 0.1% SDS. Surfactant proteins (SP-A, SP-B, SP-C, and SP-D) in the BALF were examined by Western blot analysis. Mice were lavaged with five 1.0-ml washes of PBS (pH 7.4 containing 1% FBS and 0.2 mm EDTA). The recovered BALF (∼4.6 ml) was centrifuged at 18,000 × g for 20 min, and the pellet was resuspended in 100 μl of distilled water. Total protein in each samples was determined by bicinchoninic acid analysis (Pierce). For SP-A, SP-B, and SP-D, 10 μg of total protein was electrophoresed in 4–20% Tris glycine SDS-polyacrylamide gels (NOVEX, San Digeo, CA). In some experiments, an equal volume of BALF (100 μl) was reduced to 10 μl by centrifugation under negative pressure and subjected to electrophoresis. Protein was transferred to polyvinylidene difluoride membranes (Bio-Rad), and equal loading was verified by staining with Ponceau S (Sigma). To reduce nonspecific binding, membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBST). Immunoblotting was performed utilizing 1:5000 dilution of guinea pig anti-rat SP-A, rabbit anti-bovine SP-B, or rabbit anti-murine SP-D (kindly provided by Jeffrey Whitsett, University of Cincinnati) (24Elhalwagi B.M. Zhang M. Ikegami M. Iwamoto H.S. Morris R.E. Miller M.L. Dienger K. McCormack F.X. Am. J. Respir. Cell Mol. Biol. 1999; 21: 380-387Crossref PubMed Scopus (52) Google Scholar, 25Lin S. Phillips K.S. Wilder M.R. Weaver T.E. Biochim. Biophys. Acta. 1996; 1312: 177-185Crossref PubMed Scopus (70) Google Scholar, 26Ross G.F. Ikegami M. Steinhilber W. Jobe A.H. Am. J. Physiol. 1999; 277: L1104-L1108PubMed Google Scholar). For SP-C detection, 10–20% Tricine gels (NOVEX) were employed using an upper buffer composed of 0.1 mTris-Tricine with 0.1% SDS and a lower buffer composed of 0.2m Tris, pH 8.9. Rabbit anti-recombinant human SP-C serum (kindly provided by Jeffrey Whitsett) was diluted 1:25,000 in TBST and incubated with the membranes overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibody (Calbiochem) was diluted 1:5000 in TBST. Surfactant protein standards (5 ng) (kindly provided by Jeffrey Whitsett) were also electrophoresed as positive controls. The signal was developed using enhanced chemiluminescence (Amersham Pharmacia Biotech) according to manufacturer's instructions. Data are expressed as mean ± S.E. Statistical significance comparing different sets of mice was determined by Student's t test. The 3.7-kb 5′-flanking region of the hSP-C gene contains all of the necessary elements to promote specific expression of transgenes into pulmonary type II cells (27Glasser S.W. Korfhagen T.R. Wert S.E. Bruno M.D. McWilliams K.M. Vorbroker D.K. Whitsett J.A. Am. J. Physiol. 1991; 261: L349-L356PubMed Google Scholar). We initially generated transgenic mice that utilized the hSP-C promoter to direct expression of eotaxin, an eosinophil-selective chemokine (28Jose P.J. Griffiths-Johnson D.A. Collins P.D. Walsh D.T. Moqbel R. Totty N.F. Truong O. Hsuan J.J. Williams T.J. J. Exp. Med. 1994; 179: 881-887Crossref PubMed Scopus (767) Google Scholar, 29Rothenberg M.E. Am. J. Respir. Cell Mol. Biol. 1999; 21: 291-295Crossref PubMed Scopus (151) Google Scholar), in type II cells of mice to examine the consequences of eotaxin overexpression in the lungs (see transgenic construct I in Fig.1). Three different eotaxin transgenic founder lines were established that had variable but increased expression of eotaxin lung mRNA (Fig.2 A) and protein in the BALF (Fig. 2 B). For example, wild-type mice had 345 ± 205 pg/ml eotaxin in the BALF, and the transgenic lines had increased levels ranging from 687 ± 61 to 3587 ± 223 pg/ml. The specificity of the ELISA for eotaxin was determined by demonstrating undetectable eotaxin in mice genetically deficient in eotaxin (Fig.2 B). Interestingly, the base-line level of eosinophils in the BALF and lungs was not different between wild-type and eotaxin transgenic mice (data not shown). This prompted investigation of the effect of the eotaxin transgene during allergic airway inflammation, since it was hypothesized that eotaxin would cooperate with other signals generated during allergic airway inflammation (e.g.IL-5) to promote eosinophil recruitment into the lung. Allergic airway inflammation (asthma) can be triggered in mice by repeated exposure of the airways to antigens. For example, exposure of naive mice to A. fumigatus antigens 3 doses/week for 3 weeks results in marked inflammatory responses. We therefore subjected eotaxin transgenic mice to Aspergillus-induced allergic airway inflammation to determine if the eotaxin transgene would enhance eosinophil recruitment. In wild-type mice, 18 h after the last allergen exposure, there was ∼100-fold increase in the total BALF cells with the largest increase being in the eosinophil lineage (4 ± 2 × 103 to 1.9 ± 0.16 × 106) followed by the neutrophil lineage (1.3 ± 1.2 × 104 to 5.6 ± 2.4 × 105). We next examined the effect of the airway challenge on the number of eosinophils in the eotaxin transgenic mice. Surprisingly, we did not observe a significant difference in eosinophil levels between wild-type and eotaxin transgenic mice (Fig.3). Although a highly significant increase in eosinophil numbers was observed in both groups when compared with their respective saline control mice, the number of eosinophils in the allergen-challenged wild-type and eotaxin transgenic mice was comparable (1.39 ± 1.06 × 106 and 3.2 ± 1.5 × 106, respectively). Similarly, levels of eosinophils in the lung tissue did not differ between wild-type and transgenic mice (data not shown). Since we expected the eotaxin transgene to cooperate with allergen-induced signals in the lung thereby promoting increased eosinophil levels, we hypothesized that allergen challenge might be inhibiting the expression of the transgene. We therefore examined the effect of airway inflammation on the transgene expression. Interestingly, whereas eotaxin transgenic mice had readily detectable transgene expression following saline treatment, the transgene mRNA was barely detectable following allergen challenge (Fig.4 A). The decreased expression of the transgene was seen in all hSP-C eotaxin transgenic lines (data not shown). We also subjected sensitized mice to allergen challenge with OVA to determine if the allergen-induced SP-C down-regulation was applicable to other allergens. Similar to the effects of challenge withA. fumigatus, OVA exposure also down-regulated the level of the eotaxin transgene (Fig.4 B). We were next interested in determining if allergic airway inflammation was a nonspecific phenomenon associated with the down-regulation of other epithelial genes. We therefore examined the expression of the endogenous murine surfactant proteins and eotaxin. Of note, the mRNA for SP-D, and to a lesser extent SP-A, was increased following allergen challenge compared with saline challenge (Fig.5 A). Additionally, the protein level of SP-D, and to a lesser extent SP-A, was also increased. Interestingly, whereas the endogenous SP-C mRNA was not altered at this time point, the level of the SP-C protein was substantially reduced (Fig. 5 B). We also examined the expression of the endogenous eotaxin mRNA. As expected, allergen challenge markedly increased the level of eotaxin in the lung. Taken together, these results establish that the down-regulation of the hSP-C transgene by allergen was a specific event, not likely related to generalized epithelial cell toxicity. We were next interested in ruling out the possibility that the mechanism for the down-regulation of the transgene by allergen was dependent upon the presence of the eotaxin cDNA in the genetic construct. Although unlikely, it remained possible that the inhibition of the transgene was dependent upon sequences in the eotaxin cDNA or that the overexpressed eotaxin protein was inhibiting the transgene. To test these possibilities, we examined the effect of allergen challenge on an independent hSP-C transgenic mouse line. We examined transgenic mice that contained the hSP-B cDNA under the control of the same 3.7-kb hSP-C promoter used in the eotaxin transgenic mice (construct II in Fig. 1) (16Lin S. Na C.L. Akinbi H.T. Apsley K.S. Whitsett J.A. Weaver T.E. J. Biol. Chem. 1999; 274: 19168-19174Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Interestingly, down-regulation of the hSP-C-SP-B transgene in response to allergen was observed in all mice when compared with their respective saline-treated controls (Fig.6). It remained possible that the ability of allergen exposure to decrease the level of transgenes regulated by the hSP-C promoter was dependent upon the 3′-UTR of the transgenic construct, since 3′-UTR have been shown to be involved in mRNA stability (30Conne B. Stutz A. Vassalli J.D. Nat. Med. 2000; 6: 637-641Crossref PubMed Scopus (466) Google Scholar). Since the hSP-C transgenic construct contained a viral polyadenylation signal (from SV40) (14Korfhagen T.R. Glasser S.W. Wert S.E. Bruno M.D. Daugherty C.C. McNeish J.D. Stock J.L. Potter S.S. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6122-6126Crossref PubMed Scopus (93) Google Scholar), we were interested in examining the effect of allergen challenge in hSP-C transgenic mice that utilized the same 3.7-kb hSP-C promoter but a different 3′-UTR. To test this, we employed transgenic mice that contained a mammalian-derived 3′-UTR composed of the bovine growth hormone polyadenylation sequence (construct III in Fig. 1) (17Beck D.C. Na C.L. Whitsett J.A. Weaver T.E. J. Biol. Chem. 2000; 275: 3371-3376Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Interestingly, we observed down-regulation of the hSP-C-transgene in these mice in response to allergen when compared with saline-treated controls (Fig. 7). Taken together, these results demonstrate that allergen-induced inflammation down-regulates the hSP-C transgene by directly affecting the hSP-C promoter. We hypothesized that the influx of inflammatory cells during allergic airway inflammation was involved in down-regulating the SP-C transgene. Since eosinophils are the predominant inflammatory cell in A. fumigatus-induced allergic airway inflammation, we were interested in determining if these cells could directly down-regulate the SP-C transgene. To test this hypothesis, we treated mice with neutralizing doses of anti-IL-5, a well accepted approach for attenuating allergen-induced eosinophil recruitment to the lungs (31Coffman R.L. Seymour B.W. Hudak S. Jackson J. Rennick D. Science. 1989; 245: 308-310Crossref PubMed Scopus (542) Google Scholar). For example, eosinophils were 1.8 ± 0.4 × 106 and 1.0 ± 0.3 × 104 in the BALF of allergen-challenged mice following nonimmune control and anti-IL-5 serum, respectively. Interestingly, mice treated with anti-IL-5 (but not isotype matched control antiserum) were resistant to allergen-induced SP-C inhibition. Northern blot analysis of lung RNA revealed readily detectable transgene mRNA levels in mice treated with saline and in allergen-challenged mice pretreated with anti-IL-5 (Fig.8 A). In contrast, allergen-challenged mice treated with control antiserum had a markedly reduced level of transgene mRNA. Quantitative analysis of the transgene mRNA level, performed by normalization of the mRNA signal to the 28 S ribosomal protein mRNA, revealed no significant inhibition of transgene mRNA in mice treated with anti-IL-5 (Fig.8 B). These data suggest that the allergen-induced eosinophilic inflammation was responsible for the inhibition of the hSP-C promoter. Surfactant proteins have been recognized as essential molecules involved in two main critical processes in the lung, maintenance of alveolar surfactant function and host defense. As such, they have the potential to be critically involved in the pathogenesis of asthma, an allergic inflammatory disorder of the lungs characterized by airway obstruction. To examine this process, we have subjected mice to experimental models of asthma (antigen-induced allergic airway inflammation) and examined the effect on transgenes regulated by the hSP-C promoter, as well as on the level of endogenous surfactant proteins. We have found the following: 1) allergen challenge inhibits the human SP-C promoter; 2) that allergen-induced inhibition of the hSP-C promoter is dependent on IL-5, suggesting a critical role for eosinophilic inflammation in this process; and 3) that allergen challenge increases the surfactant proteins known to be involved in innate immune responses (SP-A and SP-D). Previous studies on the expression of surfactant proteins in patients with asthma have been limited to analysis of surfactant activity in BALF rather than surfactant protein mRNA levels, since surfactant is synthesized by small airway and alveolar cells that reside in regions that are difficult to sample experimentally in humans. Several recent clinical studies have shown that the BALF of patients with asthma at base line or following segmental allergen challenge contains dysfunctional surfactant (10Hohlfeld J.M. Ahlf K. Enhorning G. Balke K. Erpenbeck V.J. Petschallies J. Hoymann H.G. Fabel H. Krug N. Am. J. Respir. Crit. Care Med. 1999; 159: 1803-1809Crossref PubMed Scopus (93) Google Scholar, 11Jarjour N.N. Enhorning G. Am. J. Respir. Crit. Care Med. 1999; 160: 336-341Crossref PubMed Scopus (60) Google Scholar). Additionally, experimental dissection in a guinea pig model of asthma has revealed that allergen challenge decreases the surfactant activity in the BALF (32Liu M. Wang L. Li E. Enhorning G. Clin. Exp. Allergy. 1996; 26: 270-275Crossref PubMed Scopus (51) Google Scholar). Additionally, significant changes in surfactant levels have been reported in patients with interstitial pulmonary fibrosis and adult respiratory distress syndrome (1Mason R.J. Greene K. Voelker D.R. Am. J. Physiol. 1998; 275: L1-L13Crossref PubMed Google Scholar). In the present study, we have analyzed the effect of experimental allergen challenge on the expression of the hSP-C promoter. We have chosen to focus primarily on transgenic mice expressing reporter genes under the control of the hSP-C 3.7-kb promoter. Although this region may have distinct regulatory elements compared with the mouse or human SP-C promoter, it is sufficient for directing specific gene expression in type II pneumocytes, the cells that normally synthesize SP-C. This promoter construct has been used to generate over 100 different transgenic mouse lines for extensive analysis of lung development and function (15Glasser S.W. Korfhagen T.R. Wert S.E. Whitsett J.A. Am. J. Physiol. 1994; 267: L489-L497PubMed Google Scholar, 27Glasser S.W. Korfhagen T.R. Wert S.E. Bruno M.D. McWilliams K.M. Vorbroker D.K. Whitsett J.A. Am. J. Physiol. 1991; 261: L349-L356PubMed Google Scholar). Our initial results were limited to hSP-C transgenic mice overexpressing murine eotaxin cDNA and demonstrated that allergen challenge markedly inhibited the expression of this transgene. Experimental dissection of hSP-C transgenic mice that were generated with distinct reporter cDNAs (e.g. SP-B instead of eotaxin) and 3′-UTRs (e.g. bovine growth hormone instead of the SV40 polyadenylation signal) revealed that allergen challenge was affecting the hSP-C promoter, rather than other elements present in the transgenic construct. Most experimental allergen challenges were conducted utilizing A. fumigatus as an antigen. It should be noted that this antigen was selected because it represents a model antigen that has already been extensively analyzed (33Kurup V.P. Mauze S. Choi H. Seymour B.W. Coffman R.L. J. Immunol. 1992; 148: 3783-3788PubMed Google Scholar, 34Kurup V.P. Choi H. Murali P.S. Coffman R.L. J. Leukocyte Biol. 1994; 56: 593-598Crossref PubMed Scopus (51) Google Scholar, 35Kurup V.P. Seymour B.W. Choi H. Coffman R.L. J. Allergy Clin. Immunol. 1994; 93: 1013-1020Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). To prove that the results were not strictly related to this allergen, an unrelated antigen (OVA) was used to demonstrate that the effect was not allergen-specific. Additionally, airway inflammation itself, and not allergen challenge, was demonstrated to be responsible for inhibition of the hSP-C promoter. In particular, depletion of allergen-induced airway eosinophilia with antiserum against IL-5 resulted in abolishment of the inhibitory effect of allergen challenge. These data suggested that eosinophil influx into the lung was responsible for inhibition of the hSP-C promoter. Eosinophils are known to be a rich source of pleiotropic molecules including lipid mediators (e.g.leukotrienes and platelet-activating factor), cationic granule proteins (e.g. major basic protein), and cytokines (e.g.IL-1 and transforming growth factor-α/β) (36Gleich G.J. Adolphson C.R. Adv. Immunol. 1986; 39: 177-253Crossref PubMed Scopus (724) Google Scholar, 37Kita H. J. Allergy Clin. Immunol. 1996; 97: 889-892Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 38Rothenberg M.E. N. Engl. J. Med. 1998; 338: 1592-1600Crossref PubMed Scopus (929) Google Scholar). Furthermore, pulmonary epithelial cells express receptors for several of these products including tumor necrosis factor-α and transforming growth factor-α/β. Interestingly, transcription of the SP-C promoter is inhibited by certain proinflammatory triggers such as tumor necrosis factor-α (39Bachurski C.J. Pryhuber G.S. Glasser S.W. Kelly S.E. Whitsett J.A. J. Biol. Chem. 1995; 270: 19402-19407Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In the mouse SP-C gene, the tumor necrosis factor-α-sensitive elements are located 320 base pairs 5′ from the start of transcription (39Bachurski C.J. Pryhuber G.S. Glasser S.W. Kelly S.E. Whitsett J.A. J. Biol. Chem. 1995; 270: 19402-19407Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, it should be pointed out that although the 3.7-kb hSP-C promoter has been extensively characterized and appears to function similar to the endogenous promoter, it remains possible that promoter elements not included in the 3.7-kb region may be differentially affected by allergen challenge. This concern may explain the finding that the endogenous mouse SPC mRNA is not substantially reduced by allergen challenge. However, since the SPC protein level is reduced by allergen challenge, we favor the view that the mouse SPC mRNA is down-regulated at earlier points in the allergen challenge regime. Alternatively, the hSPC promoter may be distinctly inhibited by allergic airway inflammation compared with the mouse SPC promoter. Taken together, our data suggest that allergen exposure induces eosinophilic lung inflammation which inhibits the transcription of the hSP-C promoter. In contrast to the inhibition of the hSP-C promoter by allergen challenge, we have found that allergic lung inflammation increases the levels of SP-A and SP-D (but not SP-B). These proteins are known to be critically involved in innate immune responses. Interestingly, the binding of SP-A and SP-D to A. fumigatus enhances the phagocytosis of this organism by human neutrophils and alveolar macrophages (40Madan T. Eggleton P. Kishore U. Strong P. Aggrawal S.S. Sarma P.U. Reid K.B. Infect. Immun. 1997; 65: 3171-3179Crossref PubMed Google Scholar, 41Allen M.J. Harbeck R. Smith B. Voelker D.R. Mason R.J. Infect. Immun. 1999; 67: 4563-4569Crossref PubMed Google Scholar). Furthermore, these proteins can inhibit specific IgE binding to A. fumigatus allergens and block allergen-induced histamine release from human basophils (42Madan T. Kishore U. Shah A. Eggleton P. Strong P. Wang J.Y. Aggrawal S.S. Sarma P.U. Reid K.B. Clin. Exp. Immunol. 1997; 110: 241-249Crossref PubMed Scopus (133) Google Scholar). Thus, the ability of allergen challenge to increase the level of the surfactant proteins involved in immune responses indicates that these proteins are indeed likely to be involved in the host response to allergens. Finally, our results have several biological conclusions concerning eosinophil recruitment to the lung. We demonstrate that overexpression of eotaxin in the lung is not sufficient for eosinophil recruitment to this organ. This is consistent with the finding that eotaxin is constitutively produced by epithelial cells in the murine lung but not associated with substantial levels of pulmonary eosinophils (43Gonzalo J.-A. Jia G.-Q. Aquirre V. Friend D. Coyle A.J. Jenkins N.A. Lin G.-S. Katz H. Lichtman A. Copeland N. Kopf M. Gutierrez-Ramos J.-C. Immunity. 1996; 4: 1-14Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In contrast, in other organs such as the small intestine, eotaxin expression is critical for base-line eosinophil tissue homing (23Matthews A.N. Friend D.S. Zimmermann N. Sarafi M.N. Luster A.D. Pearlman E. Wert S.E. Rothenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6273-6278Crossref PubMed Scopus (243) Google Scholar). We also demonstrate that overexpression of eotaxin is not associated with eosinophil desensitization, as has been reported with other chemokine transgenic mice (44Rutledge B.J. Rayburn H. Rosenberg R. North R.J. Gladue R.P. Corless C.L. Rollins B.J. J. Immunol. 1995; 155: 4838-4843PubMed Google Scholar). In particular, we found no significant difference in the level of allergen-induced eosinophil recruitment into the lung of transgenic mice compared with wild-type mice (Fig. 3). In conclusion, these findings indicate that attention should be focused on the level of expression of transgenes regulated by the hSP-C promoter when hSP-C transgenic mice are subjected to inflammatory triggers. Additionally, these results suggest that surfactant protein dysfunction in patients with asthma is likely to be caused, at least in part, by allergen-induced and IL-5-dependent decreases in the level of SP-C. We thank Drs. Cindy J. Bachurski, Fred Finkelman, Gary Ross, and Jeffrey Whitsett for very helpful discussions and reagents. We also thank these individuals and Drs. Simon Hogan and Nives Zimmermann for critical review of the manuscript.
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