Bcl-2 Sustains Increased Mucous and Epithelial Cell Numbers in Metaplastic Airway Epithelium
J. Foster Harris,
Mark J. Fischer,
Jon R. Hotchkiss,
Brett P. Monia,
Scott H. Randell,
Jack R. Harkema and
Yohannes Tesfaigzi
Lovelace Respiratory Research Institute, Albuquerque, New Mexico; Dow Chemical Company, Midland; Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan; Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina; ISIS Pharmaceuticals, Department of Molecular Pharmacology, Carlsbad, California
Correspondence and requests for reprints should be addressed to Yohannes Tesfaigzi, Ph.D., Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive, SE, Albuquerque, NM 87108. E-mail: ytesfaig{at}lrri.org
Bcl-2, an inhibitor of apoptosis, is expressed in LPS-inducedmetaplastic goblet cells of rat airways. The present study investigatedexpression of Bcl-2 in airway mucous cells of persons with cysticfibrosis and tested in rats and mice whether its expressionis responsible for sustaining metaplastic mucous cells. A significantlyhigher percentage of mucous cells expressed Bcl-2 in humanswith cystic fibrosis compared with control subjects with nodisease or subjects with other diseases. In LPS-instilled F344/Nrats, the percentage of Bcl-2–positive mucous cells wasdecreased to background levels before the resolution of gobletcell metaplasia. Furthermore, intraperitoneal injection of ratswith antisense oligonucleotides significantly reduced Bcl-2expression and goblet cell metaplasia in nasal and pulmonaryairway epithelia in rats. In contrast, sustained expressionof Bcl-2 in transgenic mice by a metallothionein promoter causedincreased LPS-induced goblet cell metaplasia over 8 days comparedwith wild-type mice. These studies demonstrate that Bcl-2 expressionsustains goblet cell metaplasia in various species, that epithelialcell numbers are directly linked to the regulation of the numbersof goblet cells, and that downregulating Bcl-2 expression reducesgoblet cell metaplasia.
One of the main reasons for the morbidity of patients with chronicpulmonary diseases, such as cystic fibrosis (CF) and asthma,is increased mucous secretions causing airway obstruction. Reductionof mucous secretions in CF is of great interest, because interventionstrategies by gene therapy approaches to replace the CFTR geneor by reducing inflammation have largely been unsuccessful.
Inflammatory responses recruit nonciliated columnar epithelialcells into the cell cycle in large numbers (1) and thereby increasethe number of cells per millimeter of basal lamina (BL) (2).This increase is accompanied by the appearance of mucus-producingcells in areas normally devoid of these cells and is calledgoblet cell metaplasia (GCM) (3). GCM allows the epitheliumto produce larger amounts of mucus, because both preexistingand proliferating cells synthesize mucus (2). The generationof metaplastic mucous cells and the mechanisms by which inflammatorymediators induce the synthesis of mucus and the differentiationof surface epithelial cells into mucus-producing cells havebeen extensively studied. In contrast, mediators involved inreducing the numbers of metaplastic mucous cells after inflammatoryresponses subside are not well understood.
In an attempt to understand mechanisms that reduce numbers ofmetaplastic mucous cells during the resolution of inflammatoryresponses, we discovered that metaplastic mucous cells expressregulators of apoptosis from the Bcl-2 family of proteins (4–6).Exposure of rats to LPS or allergen causes airway epithelialcells to proliferate, and nonproliferating and proliferatingcells differentiate into mucous cells to establish GCM (2).Approximately 20 to 30% of the developing metaplastic mucouscells express Bcl-2 (5). Furthermore, exposure of rats to ozoneinduces GCM in nasal epithelia and Bcl-2 is expressed in epitheliawhere GCM occurs (4).
The Bcl-2 family of cytoplasmic proteins can register diverseforms of intracellular damage, gauge whether other cells haveprovided a positive or negative death stimulus, and determinethe progression or inhibition of the suicide program (7). Bcl-2,an inhibitor of apoptosis, is a member of a large group of regulatoryproteins that prevent or induce apoptosis. The activation ofproapoptotic signals leads to inactivation of Bcl-2 by Bcl-2homology region-3 domain-only family members and triggers Baxor Bak to permeabilize mitochondria to release cytochrome c,which initiates the activation of caspases and DNases responsiblefor the appearance of the apoptotic morphology (8–10).This morphology includes DNA fragmentation, chromatin condensation,membrane blebbing, cell shrinkage, and disassembly of the cell(10).
The present study tested the hypothesis that disruption of mechanismsto resolve GCM could account for sustained GCM in CF where mucoushypersecretion reduces airflow. Our findings suggest that Bcl-2plays a central role in the resolution of GCM and may be a usefultarget for reducing mucus-secreting cells in chronic diseases.Some results from these studies have been previously reportedin the form of an abstract (11).
See the online supplement for additional details regarding laboratoryanimals, in situ hybridization, microdissection of airway epithelia,polymerase chain reaction, and Western blot analyses.
Tissues from Human Subjects
Paraffin-embedded lung tissues with small airways were obtainedunder the auspices of the University of North Carolina and theLovelace Respiratory Research Institute institutional reviewboards' approved protocols. Tissue sections (5-µm thick)originated from 14 subjects without lung diseases, 8 subjectswith CF, and 8 subjects with diseases such as interstitial pulmonaryfibrosis, pulmonary hypertension, and 1-antitrypsin (Table 1).All histology sections represent sections of airways and lungsexcised during transplantation for end-stage lung disease orexcess portions of donors.
Laboratory Animals
Male F344/N rats (8–10 weeks old) were purchased fromFrederick Cancer Research, Frederick, MD. Bcl-2 transgenic miceon a C57Bl/6 background with the Bcl-2 transgene under the controlof the mouse metallothionein promoter (provided by Dr. NelsonFausto, University of Washington, Seattle, WA) were bred atthe Lovelace Respiratory Research Institute. Instillation forrats and mice was as described (12, 13). All experiments wereapproved by the Institutional Animal Care and Use Committeeand were performed at the Lovelace Respiratory Research Institute,a facility approved by the Association for the Assessment andAccreditation for Laboratory Animal Care International.
Rat Nasal Explant Cultures and Treatment with Antisense Oligonucleotides
Nasal explants were treated with 100 µg/ml endotoxin inmedium (n = 4/group) for 24 hours and maintained in culturein endotoxin-free medium for 2 additional days. They were thenfixed in zinc formalin and processed for histologic analysesas described (14). They were treated with 30 µM antisenseoligonucleotides (ASODNs) in 40 µl culture medium and3 µl lipofectamine (Life Technologies, San Diego, CA).Table 2 shows the sequences of the ASODNs and control ODNs,which were 2-O-methoxy ethyl thioate derivatives (15).
TABLE 2. Sequences of the antisense oligonucleotides and their position within the bcl-2 mRNA
Injection of Rats with ASODN and LPS Instillations
Rats were injected intraperitoneally with 0.5 µl saline,control ODN, or ASODNs two times a day at 5 mg/kg in salinefor 10 consecutive days. On Day 7 or 8 of injection, rats wereintratracheally instilled with l mg LPS in 0.5 ml saline orintranasally instilled with 100 µl/naris of 1 mg/ml LPSand killed 72 or 48 hours later, respectively.
Histochemical Staining, Quantification, In Situ Hybridization, Real-Time Polymerase Chain Reaction, and Western Blot Analysis
Tissue blocks that corresponded to the same nasal anatomic siteexamined in the in vitro experiments described previously andintrapulmonary airways of the left lung lobes were obtainedas described (16). Staining and quantification of mucous cellnumbers and intraepithelial stored mucosubstances were performedas described (17–19). Bcl-2 was detected by immunohistochemistryusing two different Bcl-2 antibodies (BD-Pharmingen, San Diego,CA, or Santa Cruz Biotechnology, Santa Cruz, CA) at a dilutionof 1:1000 or 1:50, respectively, as described (12). In situhybridizations, real-time polymerase chain reaction, and Westernblot analyses were performed as described previously (20–22).See the online supplement for a detailed method descriptionand regarding RNA isolation and polymerase chain reaction.
Statistical Analysis of Data
Data were expressed as the mean group value ± SE of themean. Differences among groups were examined by analysis ofvariance and t tests using SAS software (Cary, NC) and by applicationof Bonferonni correction for multiple comparisons. Group meanswere considered significantly different when p values were 0.05or less.
Bcl-2 Expression in Mucous Cells from Subjects with CF
We have previously reported that Bcl-2 is expressed in metaplasticmucous cells of rats exposed to ozone, cigarette smoke (4),or LPS (5). To further study the relevance of Bcl-2 expressionin human chronic disease, we analyzed lung tissues that wereobtained from individuals with no disease and from individualswho have CF, pulmonary hypertension, or bronchiectasis. Lungsfrom patients with CF were chosen because infections with gram-negativebacteria are common in these patients and may resemble the responsewe see in the rat lung after instillation of LPS (5). Althoughan average of 1 to 2% of mucous cells were Bcl-2–positivein airways from normal subjects or from subjects with otherlung diseases, such as pulmonary hypertension and bronchiectasis,an average of 8% of mucous cells in lung epithelia of patientswith CF showed immunostaining for Bcl-2 (Figures 1A and 1B).We noticed heterogeneity in Bcl-2 positivity of mucous cellsin some tissue sections from subjects with CF. In some airways,up to 40% of goblet cells expressed Bcl-2, whereas in adjacentairways, only 1 to 2% of goblet cells were Bcl-2–positive.The identity of Bcl-2 was further verified by in situ hybridization,which showed a strong signal in epithelia where Bcl-2 was detectedby immunohistochemistry, whereas the sense control probe showedno signal (Figure 1C).
Figure 1. Increased percentages of mucous cells are Bcl-2–positive in airways of patients with cystic fibrosis (CF) compared with airways from subjects who have other lung diseases than CF or control subjects with no disease. Tissue sections were immunostained for Bcl-2 as described in METHODS. Nuclei and intraepithelial mucosubstances stain blue, and Bcl-2 immunostaining was detected with diaminobenzidine and shows brown. (A) The percentage of Bcl-2-positive mucous cells (MCs) was quantified by enumerating the number of Alcian blue (AB-)–positive MCs and the MCs that had immunostained with the Bcl-2 antibody (BD-Pharmingen). Bar represents 20 µm. N = normal; PH = pulmonary hypertension. (B) At least 250 AB-positive cells were counted to determine the percentage of positive Bcl-2 MCs. Values are group means ± SEM; n = 7 to 10 subjects/group. *Significantly different from control subjects with no disease, p < 0.05. (C) In situ hybridization of tissue sections from a subject with CF with antisense (AS) and sense (S) cRNA probes to Bcl-2. Bar represents 10 µm. (D) Increase in MCs per millimeter of basal lamina (BL) in well differentiated cultures of primary airway epithelial cells from three subjects without lung disease and three subjects with CF. *Significantly different from control subjects with no disease, p < 0.05.
To test whether CFTR deficiency is directly responsible forBcl-2 expression, primary airway epithelial cells from threesubjects with no lung disease and three subjects with CF weremaintained in culture on an air–liquid interface and allowedto differentiate into columnar cells. Cells from control subjectsand subjects with CF showed a time-dependent increase in gobletcells per millimeter of BL over 7, 14, and 21 days (Figure 1D).Significant differences in goblet mucous cells/millimeter ofbasal lamina were observed in the cultures obtained from threenormal subjects and three subjects with CF at 7 days, when cellshad just reached confluence. No differences were found at 14and 21 days, when cells had formed a differentiated columnarepithelium. This finding shows that the change in the numberof mucous cells/millimeter of BL from 7 to 14 days was greaterfor normal subjects compared with those with CF. No differenceswere observed in the percentage of Bcl-2–positive cellsin control and CF samples (data not shown).
Resolution of LPS-induced GCM in Rats
The number of cells that contain mucosubstances increased froman average of 15 to an average of 76 cells/mm BL from 2 to 4days and decreased to approximately 43 cells/mm BL at 8 daysafter instillation. The mucous cell numbers decreased to backgroundlevels at 12 days to the numbers found in rats instilled withsaline (8–20 cells/mm BL) throughout the 16 days afterinstillation (Figure 2A). The morphology of airway epithelialcells remained cuboidal in saline-instilled rats throughoutthe 16 days after instillation (Figures 2B–2D). In LPS-instilledrats, the thickness of the epithelium was already increasedat 2 days, reached maximum ( 21 ± 1.4 µm) at 3to 4 days, and returned to the original thickness (9.2 ±0.001 µm) at 12 to 16 days after instillation (Figures 2E–2G).
Figure 2. (A) The number of MCs per millimeter of BL in saline- and LPS-instilled rats. The number of AB/periodic acid-Schiff (PAS)–positive epithelial cells were enumerated and normalized to millimeter length of BL. Values are group means ± SEM; n = 5 rats/group. *Significantly different from saline-instilled control animals, p < 0.05. (B–G) Photomicrographs of airway epithelia from rats instilled with saline (B–D) or LPS (E–G). Tissue sections were stained with hematoxylin–eosin and AB; photomicrographs are representative for 2 (B and E), 3 (C and F), and 12 (D and G) days after instillation. Bar represents 20 µm.
Accompanying this GCM, a significant increase in Bcl-2–positivemucous cells (20–30% in LPS rats compared with 3–7%in saline rats) was found at 2, 3, 4, and 6 days after instillationin LPS-instilled rats (Figure 3A). No differences between saline-and LPS-instilled rats were evident 8 to 16 days after instillation.Although the granular staining of Bcl-2 is visible in Alcianblue–positive cells at 2 to 6 days, very few cells wereimmunopositive at 12 days after instillation (Figures 3B–3D).
Figure 3. (A) Expression of Bcl-2 at various time points after LPS instillation. The percentage of Bcl-2–positive MCs in rats instilled with saline or 1000 µg LPS from 2 to 16 days after instillation. The tissue sections were immunostained for Bcl-2 and counterstained with AB to identify denoting the mucus-producing cells. At least 400 MCs were counted in each rat to determine the percentage of Bcl-2–positive MCs. Similar immunostaining was observed with two different antibodies (BD-Pharmingen and Santa Cruz) to Bcl-2. *Significantly different from saline-instilled control animals, p < 0.05. (B–D) Representative photomicrographs show that Bcl-2 is expressed in mucus-producing cells following LPS instillation. Tissue sections from the left lung at generation 5 from rats at 2 (B), 3 (C), and 12 (D) days after instillation were immunostained for Bcl-2 and AB to identify mucus-containing cells. Bcl-2 was detected with diaminobenzidine and shows brown. Bar represents 20 µm.
Bcl-2 Sustains LPS-induced GCM in Nasal Epithelia
The direct role of Bcl-2 in sustaining metaplastic mucous cellsin airway epithelia was investigated by downregulating its expressionwith ASODNs in cultured nasal explants. We screened 25 ASODNsfor inhibition of GCM and Bcl-2 mRNA expression in the organculture system where GCM can be induced by treating the distalmidseptum from the rat nose with LPS (14). This screening identifiedfour ASODNs that significantly reduced LPS-induced mucous cellsin nasal explant cultures (Figure 4A). Two of four ASODNs (64and 83) showed a significant reduction of GCM in repeat experimentsand were therefore selected for testing their efficacy by treatingnasal explant cultures at 3-, 30-, 60-, or 90-µM concentrations.ASODN 83 effectively reduced GCM, even at 3-µM concentrations,whereas ASODN 64 was most effective at the 90-µM concentration(Figure 4B). In situ hybridization for Bcl-2 mRNA showed a markedreduction of the mRNA in nasal explants treated with ASODN 64(Figure 4C). Similar results were observed for ASODN 83 (datanot shown).
Figure 4. (A) Four of 25 antisense oligonucleotides (ASODNs) significantly reduced the number of MCs in explant cultures. Organ cultures from rat distal nasal midsepta were placed in culture on transwell membranes in an air–liquid interface and treated with 100 µg/ml LPS for 24 hours and treated with 30 µM ASODNs to Bcl-2 mRNA throughout the 3-day culture period or were left untreated as control. Explant cultures were fixed 2 days later in zinc formalin, histochemically stained with AB/PAS to detect acidic and neutral intraepithelial mucosubstances stored in the epithelial cells, and analyzed for the number of MCs. The 25 ASODNs were tested in three separate experiments (8–9 ASODNs with an untreated group as control in each experiment). Error bars represent group mean values ± SEM (n = 4 explant cultures/experimental group). *Significantly different from untreated control explant cultures, p < 0.05. (B) A dose-dependent effect of ASODNs 64 and 83 on stored mucous substances in the nasal explant cultures. Nasal explants were placed in culture on transwell membranes, exposed to 100 µg LPS for 24 hours, and treated with the respective ASODNs at 3-, 30-, 60-, or 90-µM concentrations throughout the culture period. Tissues were processed 2 days after LPS treatment, and volume density (Vs) of stored mucosubstances in the surface epithelium was determined by morphometry. ASODN 83 shows a significant reduction at 3-µM concentration. ASODN 64 shows a significant reduction only at 90-µM concentration. Bars represent group mean values ± SEM (n = 4 explant cultures/group). *Significantly different from untreated controls, p < 0.05. (C) In situ hybridization of control ODN- or ASODN 64–treated nasal septa with antisense probe to Bcl-2 (AS) or with the sense cRNA probe as control (S). Bar represents 15 µm. (D) ASODN 64 reduces the LPS-induced goblet cell metaplasia (GCM) in the distal nasal septum in in vivo rats. Rats were injected intraperitoneally with saline control ODN or ASODN 64 mornings and evenings with 5 mg/kg for a total of 10 mg/kg/day for 9 consecutive days. On Day 7, all rats received an intranasal instillation (100 µl/naris) of a 1-mg LPS solution in saline. Two days after LPS instillation, rats were killed, and GCM in the epithelium of the distal nasal septum was quantified by morphometry after histochemical stain with AB/PAS. Bars represent group mean values ± SEM (n = 6 rats/experimental group). *Significantly different from distal septal epithelium of rats injected with saline or control ODN, p < 0.05.
We further tested whether LPS-induced GCM in distal nasal midseptacan be reduced by injection of rats with ASODNs to the Bcl-2mRNA. Rats were injected intraperitoneally with saline, controlODN, or ASODN 64 twice per day at 5 mg/kg for 7 days beforeLPS instillation and 2 days after instillation. The amount ofstored mucosubstances in respiratory epithelia of distal nasalsepta was significantly reduced in rats injected with ASODN64 compared with those injected with control ODN or saline ascontrol (Figure 4D).
Bcl-2 Sustains GCM in Pulmonary Airways
The effect of downregulating Bcl-2 expression on LPS-inducedGCM in pulmonary airways was examined by injecting rats intraperitoneallywith ASODNs 83 and 64 every day for a total of 9 days starting7 days before intratracheal instillation with 1 mg LPS. GCMwas assessed at 3 days after LPS instillation in the airwayepithelium at generation 5. The total cell number per millimeterof BL (Figure 5A), the number of mucous cells per millimeterof BL, and the amount of stored mucosubstances (Figure 5B) inthe airways of rats instilled with LPS were significantly reducedby ASODN 64. Furthermore, the percentage of Bcl-2–positivemucous cells was reduced significantly by ASODN 64 (Figure 6A).ASODN 83 caused a minor, statistically nonsignificant reductionof total epithelial cell number and mucous cells per millimeterof BL and of amount in intraepithelial stored mucosubstances.ASODN 84, which was not effective in organ culture experiments,showed similar numbers of total epithelial cells and mucouscells per millimeter of BL and similar percentages of Bcl-2–positivemucous compared with rats injected with control ODN (data notshown). No differences in inflammation were observed in lungtissues from control- or ASODN-treated rats. However, the epithelialthickness was reduced when rats were injected with ASODNs 83and 64, but not with control ODN (Figure 5D).
Figure 5. ASODN reduces LPS-induced GCM, the Vs, and the number of epithelial cells per millimeter of BL in pulmonary airways in in vivo rats. Rats were injected intraperitoneally with control (CTL) ODN, ASODN 83, or ASODN 64 twice a day for 10 consecutive days. On Day 7, all rats received an intratracheal instillation of 1 mg LPS in 500 µl. Three days after LPS instillation, rats were killed, and the numbers of MCs per millimeter of BL (A) and the amount of stored mucosubstances (B) in the epithelium of airway generation 5 were quantified by morphometry after histochemical stain with AB/PAS. Adjacent sections were stained with hamatoxylin–eosin followed by AB for quantification of the total numbers of cells per millimeter of BL (C). Bars represent group mean values ± SEM (n = 6 rats/experimental group). *Significantly different than distal septal epithelium of rats injected with control ODN, p < 0.05. (D) Photomicrographs of axial airways at generation 5 in the left lung lobe of rats injected with control ODN, ASODN 83, and ASODN 64. Tissue sections were stained with hematoxylin–eosin and counterstained with AB. Bar represents 30 µm.
Figure 6. Bcl-2 protein levels are reduced in rats injected with ASODN 64 compared with control (CTL) ODN-injected animals. Bar represents 30 µm. (A, B) The percentage of Bcl-2–positive MCs was reduced in rats injected with ASODNs 83 and 64 compared with those injected with control ODN. Bars represent group mean values ± SEM (n = 6 rats/experimental group). *Significantly different from control ODN. (C) Densitometric quantification of Bcl-xL, Bax, and Bak after normalizing to actin in proteins extracted from three rat lungs each injected with control ODN and ASODNs 83 and 64.
The effect of treatment with control ODN or ASODN 83 and 64on the levels of Bcl-2, Bcl-xL, Bax, and Bak, the major regulatorsof this protein family, was tested. The percentage of Bcl-2–positivemucous cells was more efficiently reduced by ASODN 64 comparedwith control ODN or ASODN 83 (Figures 6A and 6B). The proteinlevels of Bcl-xL, Bax, and Bak were tested in extracts preparedfrom the right lungs of three representative rats from eachtreatment group. Interestingly, not only Bcl-2 but also Bcl-xLlevels were reduced by treatment with ASODNs 83 and 64 comparedwith control ODN (Figure 6C). However, the levels of the proapoptoticproteins Bax and Bak were not affected by the ASODN treatments(Figure 6C).
Sustained GCM in Bcl-2 Transgenic Mice
Transgenic mice with Bcl-2 under the control of the MT1 promoter,which is inducible by zinc water, were used to further establishthe role of Bcl-2 expression in sustaining LPS-induced mucouscell metaplasia. We and others had previously shown that C56Bl/6mice instilled with LPS display GCM (13, 23). Bcl-2 expressionwas 10-fold greater in transgenic compared with wild-type littermates8 days after LPS instillation (Figure 7A), and goblet cellsin transgenic but not wild-type mice showed Bcl-2 positivity(Figure 7B). Furthermore, GCM in wild-type mice was decreasedat 8 days after instillation, but remained elevated in Bcl-2transgenic mice (Figure 7C).
Figure 7. Sustained Bcl-2 expression in transgenic mice sustains LPS-induced GCM. Both wild-type (WT) and human Bcl-2 transgenic (TG) mice with the Bcl-2 transgene under the control of the mouse MT1 promoter on a C57Bl/6 background were maintained on 25 mM zinc sulfate drinking water to induce transgene expression. Mice were instilled with LPS on 3 consecutive days with 180 µg in 50 µL saline, a protocol previously shown to induce maximum GCM in C57Bl/6 mice. (A) Increased expression of Bcl-2 was detected 8 days after LPS instillation in lungs of TG mice that express a zinc-inducible Bcl-2 transgene in airway epithelia compared with WT mice when they were maintained on water with zinc sulfate. Bars represent group mean values ± SEM (n = 3/group). *Significantly different from WT, p < 0.05. (B) Representative photomicrographs of Bcl-2 immunostaining cells in WT and TG (Tg) mice at 8 days after LPS instillation. Bar represents 20 µm. (C) GCM was quantified in these mice over a period of 5 and 8 days after LPS instillation. GCM was similar in both WT and TG mice at 5 days after LPS instillation. However, at 8 days, GCM was decreased in WT but sustained in TG mice.
The present study shows that Bcl-2 is present in airway mucouscells of subjects with CF and that LPS-induced epithelial proliferationand GCM is transiently sustained by the expression of this antiapoptoticprotein.
Interestingly, Bcl-2 expression was significantly reduced (asshown by the percentage of Bcl-2–positive mucous cellsand by Western blot analyses) but not completely suppressedby the ASODN treatment. This finding suggests that it is notcrucial to completely inhibit Bcl-2 expression to cause thereduction of epithelial cell numbers and GCM and that proapoptoticsignals initiate the proapoptotic cascade once Bcl-2 and Bcl-xLare reduced to a certain threshold level. It is unclear whyASODN 64 was not as effective as ASODN 83 in downregulatingGCM in organ culture but was more effective when injected inrats instilled with LPS. The presence of a large amount of inflammatorymediators in in vivo rats may result in differences in the stabilityof these ASODNs and contribute to this observed difference.Bcl-2 homology region-3–only subfamily members are knownto induce apoptosis by association with antiapoptotic Bcl-2family members or by stimulating other apoptosis-promoting familymembers (7, 9, 24, 25).
The reduction of Bcl-2 and Bcl-xL, the major antiapoptotic proteins,may have allowed the proapoptotic proteins Bax and Bak to initiatethe cell-death process. Although epithelial cells undergoingcell death are not presented in this study, terminal deoxy-nucleotidyltransferase-mediated dUTP nick end labeling (TUNEL-)–positivecells have been detected at the early times after LPS instillationin mice (26). Furthermore, we have reported that the resolutionof allergen-induced GCM is associated with the presence of TUNEL-positivecells and activated caspase 3 (27). Ongoing studies will characterizethe cell death by which epithelial cells are removed from theepithelium.
The reason why Bcl-xL levels were reduced in lungs when ratswere injected with ASODNs to Bcl-2 is not clear. The homologyof Bcl-2 and Bcl-xL mRNAs in the regions where ASODNs 83 and64 are localized is 50%. This level of homology may be sufficientto bind and downregulate Bcl-xL mRNA because of similarity inthe tertiary structures between these mRNAs. Consistent withour findings, a Bcl-2/Bcl-xL bispecific ASODN inhibits the expressionof Bcl-2 and Bcl-xL in a representative breast carcinoma cellline and inhibits the growth and induces death in cell linesfrom lung, breast, colorectal, and prostate carcinomas in vitro(28).
The reduction of Bcl-2 expression with ASODNs decreased thetotal epithelial cell number and GCM, thereby directly linkingBcl-2 expression to GCM. Because the ASODNs were administeredbefore LPS instillation, it is possible that the reduction ofBcl-2 expression affected the inflammation-induced epithelialcell proliferation. However, our previous studies demonstratethat approximately half of the Bcl-2–positive cells hadundergone a cell cycle division whereas the other half had notincorporated BrdU and were therefore derived from preexistingepithelial cells (2). These studies suggest that Bcl-2 expressionis not involved in regulating cell cycle in this system. Furthersupport for the role of Bcl-2 in sustaining GCM was establishedby time-course experiments demonstrating that the reductionof Bcl-2 expression to background levels occurs before the resolutionof GCM (5). In addition, the decrease in Bcl-2–positivemucous cells seen after injection of rats with bezafibrate wasassociated with significant reduction of LPS-induced mucouscells per millimeter of BL (12). These observations togetherwith the known antiapoptotic function of Bcl-2 (29, 30) confirmthat Bcl-2 sustains the lifespan of mucous cells after inflammatoryresponses.
The reduction of LPS-induced GCM in distal septa as well aspulmonary airways of rats injected with ASODNs to the Bcl-2mRNA suggests that the processes involved in the resolutionof GCM in epithelia lining the nasal midsepta and the pulmonaryairways are similar. Our previous studies have established thatBcl-2 is expressed in ozone- or LPS-induced GCM, both in nasaland pulmonary airways (4, 5). Because the distal region of thenasal septum and the main axial pulmonary airways are linedby a tall columnar respiratory epithelium with primarily serous-type(not mucous) secretory cells (31, 32), similar regulation ofthe resolution process of LPS-induced GCM in these epitheliais not surprising. These results suggest, therefore, that targetingBcl-2 expression with ASODN may be useful to reduce GCM andairway obstruction in humans with bronchopneumonia or rhinitis.
In the rat, the proximal midseptum is lined by a mucociliaryepithelium with endogenous mucous cells. Exposure to ozone doesnot change the numbers of mucous cells in this epithelium butinduces GCM in epithelia that line the lateral wall and thenaso- and maxilloturbinates (33). Interestingly, although thepercentage of Bcl-2–positive cells remains low in theproximal midseptum, up to 50% of mucous cells express Bcl-2where metaplastic changes occur (4). Together with our currentfinding that Bcl-2 sustains the life span of metaplastic mucouscells, we conclude that Bcl-2 is expressed only in epitheliawhere mucous cell numbers have to be regulated. In addition,the total number of epithelial cells per millimeter of BL thatwas reduced in rats injected with ASODN 64 was similar to thereduction in the number of mucous cells per millimeter of BL,suggesting that the epithelium removes primarily mucous cellsto restore the original number of epithelial cells. Therefore,targeting Bcl-2 may primarily reduce metaplastic cells withoutaffecting endogenous mucous cells, which makes this approachvaluable and relevant for therapies in chronic diseases.
The presence of Bcl-2 in mucous cells from subjects with CFindicates that this protein may be a useful target for reducingmucous secretions. Our studies with cells from subjects withCF and control subjects show that cells from subjects with CFare capable of differentiating to mucous cells, even at thetime when they reach confluence without the formation of a columnarepithelium. The fact that cells from subjects with CF do nothave more mucus-producing epithelial cells compared with controlsubjects at confluence indicates a blunted capacity for in vitrodifferentiation of CF cells to a mucus-producing phenotype.Deficiency in CF is not directly responsible for Bcl-2 expression.Therefore, it is likely that the presence of bacterial infectionsin CF (34) may be causing sustained Bcl-2 expression. It isalso possible that dysregulated expression of Bcl-2 or the presenceof inflammatory mediators sustain activation of Bcl-2 promoter,causing sustained GCM and airway obstruction. This hypothesisis supported by our finding that the sustained expression ofBcl-2 in transgenic mice, where the metallothionein promoteris activated with zinc water, prolongs the presence of GCM comparedwith wild-type mice.
Increased expression of Bcl-2 after inflammatory responses maybe crucial to sustain a transient increase of mucous cell numbers,for the epithelium to have sufficient mucus to protect injuryby further insults. However, dysregulated expression of Bcl-2may sustain GCM to cause disease. Studies have demonstratedthat, in patients with CF and in horses with reactive airwayobstruction, mucous secretions accumulate in airways and causeobstruction (35, 36). Bcl-2 is also expressed in approximately50% of mucous cells in horses with reactive airway obstruction(Y.T., personal observation). Thus, our studies showing Bcl-2expression in mice, rats, horses, and humans suggest that resolutionof GCM by this mechanism is conserved among species and supportthe hypothesis that targeting Bcl-2 for downregulation may beuseful to reduce mucous cell numbers and alleviate mucous secretions.
Acknowledgments
The authors thank Ms. Yoneko Knighton for preparing tissue samplesfor light microscopic analyses. They thank Mr. Nick Johnsonfor technical assistance and Mr. Jay Aden for support with statisticalanalyses.
FOOTNOTES
Supported, in part, by grants from the National Institutes ofEnvironmental Health Sciences (ES09237) and the National Heart,Lung, and Blood Institute (HL68111).
This article has an online supplement, which is accessible fromthis issue's table of contents at www.atsjournals.org
Conflict of Interest Statement: J.F.H. does not have a financialrelationship with a commercial entity that has an interest inthe subject of this manuscript; M.J.F. does not have a financialrelationship with a commercial entity that has an interest inthe subject of this manuscript; J.R.H. does not have a financialrelationship with a commercial entity that has an interest inthe subject of this manuscript; B.P.M. does not have a financialrelationship with a commercial entity that has an interest inthe subject of this manuscript; S.H.R. has consulted for VertexPharmaceuticals, Inc., and Novartis Institutes for BioMedicalResearch, Inc., on matters not directly related to the materialin this article and has received an honorarium for a lecturefrom Vertex Pharmaceuticals, Inc.; J.R.H. does not have a financialrelationship with a commercial entity that has an interest inthe subject of this manuscript; Y.T. does not have a financialrelationship with a commercial entity that has an interest inthe subject of this manuscript.
Received in original form August 24, 2004;accepted in final form December 9, 2004
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TOP
ABSTRACT
METHODS
1-antitrypsin (
21 ± 1.4 µm) at 3 to 4 days, and returned to the original thickness (9.2 ± 0.001 µm) at 12 to 16 days after instillation (
