Tayside Institute of Child Health, Maternal and Child Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, United Kingdom
Submitted 20 November 2002 ; accepted in final form 13 March 2003
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ABSTRACT |
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bronchopulmonary dysplasia; lung morphogenesis; mitogen-activated protein kinase; fibroblast growth factor; cytoprotection
Thymulin (facteur thymique sérique) is a nona-peptide neuroendocrine hormone secreted by thymic epithelial cells that regulates systemic immunity in its bioactive, Zn2+-conjugated form by augmenting the expression of factors involved in T-cell development, maturation, and migration in lymphoid tissues (15). Outwith the thymus, bioactive thymulin is consistently reported to silence proinflammatory cytokine and chemokine expression in response to a wide range of inflammatory or autoimmune disease conditions (reviewed in Ref. 40). In the lung, this effect has been observed as a suppression of C-C and C-X-C cytokines coupled with lowered leukocyte infiltration during bleomycin-induced pulmonary fibrosis (51). Additionally, thymulin+Zn2+ evokes a marked cessation in the growth rate of pulmonary metastases, suggesting that the anti-inflammatory effect has the potential to regulate cellular growth and differentiation of the lung (29, 30).
We have recently shown that the anti-inflammatory properties of bioactive thymulin are conserved in fetal distal lung epithelial cell cultures isolated from late gestation (day 19) rat fetuses (18), suggesting that pathways involved in thymulincytokine interactions are functional in the fetal lung close to term. In examining whether exogenous thymulin may modulate both form and magnitude of responses to an inflammatory agent (Escherichia coli LPS) our objectives were to 1) establish the relationship between airway surface complexity (ASC) and combinations of culture PO2 and LPS dosage in explants isolated from pseudoglandular-stage fetal rat lung, 2) determine the potential for thymulin+Zn2+ to modulate this relationship under conditions least favorable for explant airway growth, 3) examine candidate cytokine pathways through which thymulin+Zn2+ may mediate its effects, and 4) unite these observations with changes in the expression of growth factors involved in the regulation of lung morphogenesis. Our results show that bioactive thymulin simultaneously suppresses endogenous release of TNF- and potentiates IL-6 expression through a CCAAT-enhancer binding protein-
(C/EBP
)-dependent pathway, an established route for regenerative repair in other tissues. The hyperexpression of this pathway was associated with a mass proliferation of undifferentiated mesenchyme tissue involving raised expression of fibroblast growth factor 9 (FGF-9), which was matched by a loss of differentiated space encapsulating structures in the fetal lung. These results demonstrate the presence of an intact thymulin-signaling pathway in the fetal lung that may actively determine the physiological scope of regenerative repair responses to inflammatory stimuli throughout development.
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EXPERIMENTAL PROCEDURES |
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Culture of rat explants and human adenocarcinoma A549 cell line. All procedures accorded with the Animals (Scientific procedures) Act 1986, UK. Pregnant Sprague-Dawley rats were killed by cervical dislocation on day 14 of gestation (term is 22 days), and the fetuses were decapitated. Right and left lung lobes were excised and washed free of erythrocytes in ice-cold, sterile Dulbecco's modified Eagle's medium (DMEM). Single lung lobes were then mounted on 13-mm diameter Whatman Nucleopore Track Etch membranes (pore diameter of 8 µm) suspended on the surface of 2 ml of serum-free DMEM containing Ham's F-12 nutrient mix (DMEM/F-12) with 100 U/ml penicillin/streptomycin, each within a single well of a 12-well Costar cluster dish (Corning, NY). Explants were then placed into humidified 37°C incubators set to maintain either PO2s of 142 (ambient) or 23 mmHg (fetal) plus 5% CO2 and were then left to equilibrate for 12 h before the beginning of each experiment. For all experimental manipulations, care was taken to ensure that medium was pre-equilibrated to the appropriate oxygen tension and temperature. To avoid hypoxiareoxygenation effects, explants maintained at 23 mmHg were cultured in a temperature-, gas-, and humidity-controlled MACS VA500 environmental workstation (Don Whitley Scientific, Shipley, UK).
A human lung epithelial A549 cell line was routinely maintained in filter capped Cellstar 75-cm2 flasks (Greiner Bio-one, Frickhausen, Germany) in DMEM supplemented with 10% fetal calf serum and 100 U/ml penicillin/streptomycin at either fetal or ambient PO2s. Cultures were routinely passaged within 90% confluence.
LPS-, thymulin+Zn2+-, and IL-6-dependent effects. After the 12-h preincubation period, explants were washed once in phosphate-buffered saline (PBS) and then incubated in the presence of DMEM/F-12 containing 0, 0.5, 2, 10, or 50 µg/ml of E. coli LPS at fetal or ambient PO2. Digital images were captured at 0, 24, and 96 h with a DIC-HR digital camera (World Precision Instruments, Sarasota, FL) mounted on a Leica LZ7 binocular microscope under identical magnification and gain settings. At the 96-h time point, the medium was removed and frozen for determination of lactate dehydrogenase (LDH) leakage and protein and cytokine secretion.
To examine the effect of thymulin on LPS-evoked effects, we maintained explants over an identical time course without treatment or in the presence of 50 µg/ml LPS, 10 µM ZnCl2, or LPS together with ZnCl2. Thymulin was administered to LPS- and ZnCl2-exposed explants at concentrations of 0, 0.1, 10, or 1,000 ng/ml. Sulfasalazine (SSA, 1 mM), a blocker of nuclear factor (NF)-B transcriptional activation, was included with each treatment to distinguish between transcription-dependent and -independent components of the cytokine response. After 96 h, explants were digitally imaged, and the medium was retained for assessment of LDH leakage and protein and cytokine secretion. Explant tissue was divided into groups to be processed for immunohistochemistry, electrophoretic mobility shift assay (EMSA), oligonucleosome release, and immunoprecipitation.
The dose-response relationship between thymulin and IL-6 or TNF- secretion in the presence and absence of LPS and ZnCl2 was assessed in a human lung A549 cell line, which shares phenotypic characteristics common to alveolar type II epithelial cells. Cells were seeded at a density of 5 x 104/ml in Costar six-well culture flasks (Corning) and were maintained at fetal or ambient PO2 in 2 ml/well DMEM with 10% fetal calf serum and antibiotics. At 80% confluence, the medium was replaced with phenol red-, serum-, and antibiotic-free DMEM, and the cells were exposed to 0.01, 1, 10, 100, or 1,000 ng/ml thymulin plus 10 µM ZnCl2 for 2 h either alone or in combination with 10 µg/ml LPS. TNF-
and IL-6 release was measured by enzyme-linked immunoabsorbent sandwich assay (ELISA) as detailed under Cytokine ELISA. Fifty-percent inhibitor constant (IC50) and effector constant (EC50) values were calculated using four-parameter sigmoidal curve regression based on the equation Y = a + (Y range)/1 + 10logEC50-X·s, where a is the background Y value and s is the Hill slope. Note that 10 µg/ml LPS was the minimum dose to produce a maximal sustained release of TNF-
and IL-6 without significant loss of cells. In an identical experiment, A549 cell proliferation was determined by supplementation of culture with 10% (vol/vol) MTT. The rate of MTT reduction by cellular oxidative phosphorylation was determined by solubilization of its formazan salt followed by spectrophotometric detection at 550 nm with background correction at 690 nm.
To establish IL-6 dependency of thymulin+Zn2+ cytoprotection, we preincubated explants for 12 h in the presence of 10 nmol/ml of control or antisense oligonucleotide targeted against rat IL-6 mRNA and then exposed them for a further 96 h to 0 or 50 µg/ml LPS together with 10 µM ZnCl2 and 0.1, 10, or 1,000 ng/ml thymulin. An additional experimental set was conducted in the presence of thymulin plus 10 µM Zn2+ in the absence of LPS. At the end of the experiment, explants were digitally imaged, and the medium plus explant tissue were processed as before.
Morphometry and immunohistochemistry. Explant surface complexity was determined from digital images as perimeter (mm)/area (mm2) using Scion Image 4.0.2 software (Scion, Frederick, MD) as described previously (16). Calibration was achieved using a 5 x 2-mm grid removed from a Fast-Read 102 disposable cell-counting chamber (ISL, Paignton, UK) that was placed onto the meniscus of the medium in each well.
Immunohistochemistry was performed on PBS-rinsed, filter-attached explants fixed for 8 h in a solution of PBS containing 10% formalin (pH 7.2). After processing, explants were embedded in paraffin, and 3-µm sections were mounted on Histogrip-coated slides (Zymed, San Francisco, CA), dried, deparaffinized, and then gradually rehydrated. We performed antigen retrieval by microwaving slides for three bursts of 7 min at 800 W in 750 ml of Antigen Unmasking Buffer (Vector Laboratories, Burlingame, CA). Sections were then blocked in 10% preimmune goat serum in Tris-buffered saline [TBS; in mM: 50 Tris (pH 8.0), 138 NaCl, and 2.7 KCl] for 1 h and then incubated overnight with anti-goat FGF-9, anti-goat FGF-10, or anti-mouse CEBP (H-7) antibodies each at 1:50 dilution. Negative controls were conducted with goat serum alone. Immunodetection was performed on TBS-washed immunoreacted sections using anti-goat IgG-FITC (FGF-9, FGF-10) or anti-mouse IgG-TRITC (C/EBP
)conjugated antibodies at 1:200 dilution followed by 5 µl of DAPI-antifade to each section before mounting. Images were obtained under a Zeiss Axioskop fluorescent microscope equipped with a Hamamatsu C474295 color digital camera using Openlab (Improvision, Coventry, UK) software. The contrast properties were optimized for each section and then referenced against the respective negative control image using identical camera and microscope settings.
The surface area of mesenchyme, airway cuboidal epithelium, and airway space was determined from immunohistochemical sections as detailed by Bolender et al. (6). Briefly, a 196-cm2 isometric grid sectioned into 1-cm2 units was superimposed onto each image, and the number of grid intersections that coincided with each of the three dominant structural features structure (mesenchyme, differentiated epithelium, and luminal airway space) was counted. We determined the fractional surface area for each by expressing these counts as a fraction of the whole. We determined the thickness of the epithelial compartment from digital images of the sections at each intersecting point using the camera-calibrated micrometer function supplied with Openlab software.
Determination of necrosis and apoptosis. The rate of necrosis was determined by the rate of appearance of LDH activity in the culture medium supporting each explant. We determined LDH activity spectrophotometrically using a Beckman Coulter (Fullerton, CA) DU 650 spectrophotometer at 340 nm by adding 20 or 100 µl of medium to 980 or 900 µl of 50 mM imidazole buffer (pH 7.0) containing 200 µM NADH at 37°C. After measurement of any endogenous rate, we started the reaction by adding 1 mM Na+-pyruvate. Units of LDH activity as an index of necrotic cell lysis were expressed as mM NADH oxidized·min-1·mg secreted protein-1.
We determined apoptosis in lysed explants using a oligonucleosome detection ELISA according to the manufacturer's instructions. Briefly, intact nuclei were removed from the lysate by centrifugation, and 20 µl of the cleared supernatant were placed into each well of a 96-well plate previously coated with an antihistone antibody. After incubation for 30 min, the plates were washed three times and a peroxidase-conjugated anti-DNA antibody was added to each well. After a further 30 min, the wells were washed as before, and the density of oligonucleosomes was captured by the anti-histone/anti-DNA complex was determined by the addition of 2'2-azino-di-[3-ethylbenzthiazoline sulfonate] chromagenic substrate. Incidence of apoptosis was expressed as the fractional difference between the background-corrected spectrophotometric absorbance at 490 nm of controls vs. experimental groups.
Cytokine ELISA. We determined TNF- and IL-6 secretion in duplicate samples of the medium supporting the explant using R&D Systems rat antibodies and standards by following the recommended ELISA procedure for each cytokine. We performed assays using Nunc-Immuno Maxisorp ELISA plates (Nunc) with the optical density of each well determined at 450 nm with wavelength correction at 595 nm using a Dynatech Laboratories (Guernsey, UK) MRX microplate reader. Assays were linear from 62.51,000 pg/ml (TNF-
) or 1258,000 pg/ml (IL-6 and IL-1
) and were used on condition that a R2 value >0.90 was obtained.
Nuclear extraction and EMSA. Pooled explants were mechanically disrupted in a buffer containing (in mM): 20 HEPES (pH 7.9), 1 EDTA, and 10 NaCl, plus 0.1% (vol/vol) Nonidet P-40 and 1 Complete (Roche) protease inhibitor tablet per 25 ml of lysis solution. Nuclei were collected by cold centrifugation at 5,000 g for 10 min and disrupted by a single 15-s burst of sonication in a phospho-protein lysis buffer containing (in mM): 150 NaCl, 20 Tris·HCl (pH 7.5), 2.5 Na+ pyrophosphate, 1 EDTA, 1 EGTA, 1 -glycerophosphate, and 1 Na3VO4, plus 1 tablet per 25 ml of Complete (Roche) protease inhibitor. After centrifugation at 10,000 g for 10 min at 4°C, the supernatant was retained, and the protein content was determined by the Bio-Rad protein assay.
Oligonucleotides were constructed as follows: C/EBP sense: 5'-TGCAGATTGCGCAATCTGCA-3'; C/EBP
missense: 5'-TGCAAGAGACTAGTCTGCA-3'; NF-
B sense: 5'-AGTTGAGGGGACTTTCCC-3'; NF-
B missense: 5'-AGTTGAGGCGACTTTCCC-3'. Oligonucleotides were end-labeled with [
-32P]ATP with T4 polynucleotide kinase and then purified from unincorporated radioactivity with ProbeQuant G-50 microcolumns (Amersham Pharmacia, St. Albans, UK). We set up binding reactions using 20 µg of explant nuclear protein retained as the pelleted fraction from preceding apoptosis determinations in a buffer containing (in mM): 20 Na+ HEPES (pH 7.9), 25 KCl, 5 MgCl2, 1 EDTA, and 1 dithiothreitol (DTT) plus 0.05 µg/µl poly(dI-dC). Labeled oligonucleotide probe (1 ng) was added to each reaction, and samples were then incubated on ice for 30 min. Supershift reactions were done by incorporating 1 µl of monoclonal antibody to C/EBP
or NF-
B p65 for 30 min before the addition of labeled probe. Samples were electrophoresed through a nondenaturing 6% polyacrylamide gel at 10 mA in 0.5x TBE buffer (1x TBE = 90 mM Tris-borate, 2 mM EDTA, pH 8.3). Dried gels were analyzed with a Canberra-Packard (Pangbourne, UK) Instant Imager, then subsequently exposed to film.
Mitogen-activated protein kinase-activated protein kinase 2 activity. Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) activity was determined in both explant and serum-starved A549 cultures by following the phosphorylation of its specific substrate, HSP27. Reactions were carried out with 20 µg of protein per sample at 37°C in a buffer containing 100 mM Tris-acetate (pH 7.4), 50 mM -glycerophosphate, 2.5 mM MgCl2, 1.5 mM EGTA, 1 mM DTT, 0.5 mM Na+-orthovanadate, 0.25 mM ATP, 2 µCi [
-32P] ATP, 2 µM microcystin, and 1 tablet per 25 ml of Complete proteinase inhibitor cocktail (Roche). Reactions were initiated by addition of 1 µg of recombinant human HSP27. After 30 min, we stopped the reaction by adding SDS to a final concentration of 1% and denatured the samples by heating them for 5 min to 95°C. Proteins were fractionated on a 15% SDS-polyacrylamide gel and then stained with Coomassie blue to reveal the HSP27 band. The gel was then dried, and radioactivity corresponding to phosphorylated HSP27 was observed by exposure to film.
RNA isolation and RT-PCR. Total RNA was extracted from pooled explants using TRIzol reagent (Invitrogen, Paisley, UK). Random-primed, first-strand cDNA synthesis was generated from 1 µg of total RNA per treated sample with the AMV Reverse Transcriptase System (Promega, Madison, WI) and was subsequently used for PCR. PCR primers were derived from nucleotides 3,2063,225 and 7,1877,206 of rat IL-6 cDNA to yield a 650-bp product (GenBank accession no. M26745
[GenBank]
) and were 5'-CTTCCCTACTTCACAAGTCC-3' (sense) and 5'-GACCACAGTGAGGAATGTCC-3' (antisense), respectively. A 540-bp TNF- product was generated with primers derived from nucleotides 4,5684,577 and 5,9395,958 of rat TNF-
cDNA (GenBank accession no. L00981
[GenBank]
) and were 5'-CGCTCTTCTGTCTACTGAAC-3' (sense) and 5'-TTCTCCAGCTGGAAGACTCC-3' (antisense). PCR reactions were constructed using 20 ng of each cDNA in a buffer containing 10 mM Tris·HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl2, 0.2 mM dNTP, 100 pmol of each primer, 0.1% Triton X-100, and 2.5 units Taq DNA polymerase. Polymerization was continued for 30 cycles (30 s at 94°C, 60 s at 57°C, and 120 s at 72°C) in a Techne Touchgene Gradient PCR machine, after which PCR products were fractionated on a 1% agarose gel, and the identity of each PCR product was confirmed by sequencing. Preliminary experiments demonstrated that 30 cycles were sufficient to resolve each product within the log phase of the PCR reaction.
Immunoprecipitation of FGF-9 and FGF-10. Pooled explants were sonicated by three 10-s bursts at 4°C phospho-protein lysis buffer. Explant protein (50 µg) was precleared in 10 µl of protein A agarose beads for 1 h at 4°C. Anti-goat IgG FGF-9 or anti-goat IgG FGF-10 was added at a 1:50 dilution together with 10 µl of BSA-blocked protein A-Sepharose and incubated overnight at 4°C with gentle agitation. Immunoabsorbed proteins were separated by SDS-polyacrylamide electrophoresis on a 12% gel and transferred onto nitrocellulose by Western blotting. Blots were visualized following overnight incubation with IgG FGF-9 antibody at 1:1,000 dilution using an anti-goat IgG HRP-conjugated secondary antibody and chemiluminescent detection on Kodak MXB film. Images were digitally scanned, and band intensities as pixels per cm2 were determined with Un-Scan-It software (Silk Scientific, Orem, UT).
Data handling and statistics. Data are presented as means ± SE with the number of independent repetitions provided in the legend to each figure. Statistical significance was assessed relative to control groups using one- or two-way analysis of variance with the level of significance determined using the post hoc Tukey's honestly significant difference. Where data are displayed as a fractional difference relative to control, the reported statistics were performed on the original nonreferenced data.
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RESULTS |
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Characterization of explant ASC responses to culture PO2 and LPS. As native PO2 in the midgestation lung averages 23 mmHg (midpoint between the PO2 of the amniotic/lung luminal fluid and umbilical vein PO2), experiments were conducted in explants cultured either at fetal or at hyperoxic ambient PO2 to control for oxygen-dependent effects within each experimental regime (Fig. 1, A and C). At both PO2s, explants developed fluid-filled cyst-like structures coupled with a loss of contiguous airway; however, explants cultured at fetal PO2s displayed pockets of dense airway bifurcation that maintained overall ASC statistically greater than ambient PO2 cultured explants. This regionalized hyperplasia at fetal PO2 was conserved over a range of 0.0110 µg/ml LPS, beyond which the fractional change in ASC (ASCf096h: ASC96h/ASC0h) fell significantly to 0.8 ± 0.2 (Fig. 1, B and C). Mean epithelial thickness for cultures maintained at fetal PO2 was 11.2 ± 0.6 µm, which thinned significantly (P < 0.05, n = 6) to 7.1 ± 0.3 and 4.2 ± 0.3 µm in explants maintained respectively at ambient PO2 or exposed to 50 µg/ml LPS at either PO2.
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Explants maintained at ambient PO2 showed a dynamic growth response to LPS. Despite a significant reduction in ASCf096h in control explants, those exposed to 0.510 µg/ml LPS displayed an increase in contiguous airway bifurcation and an absence of cystic structures, peaking at an ASCf096h value of 1.7 ± 0.3 at 2 µg/ml LPS. As with the fetal PO2 explants, LPS concentrations at 50 µg/ml reduced the ASCf096h to 0.7 ± 0.1; therefore, these conditions were selected to test the cytoprotective potential of thymulin+Zn2+ as being the most disruptive to airway growth.
Thymulin+Zn2+ is cytoprotective during exposure to high LPS concentrations at fetal and ambient PO2. The cytoprotective potential of thymulin-Zn2+ was tested in explants exposed to concentrations of LPS that exceeded the range shown to promote airway bifurcation at fetal or ambient PO2 (Fig. 1). ASCf096h was related to the incidence of necrosis (LDH leakage) or apoptosis (oligonucleosome release) in the presence of 50 µg/ml LPS; 10 µM ZnCl2; or 0.1, 10, or 1,000 ng/ml thymulin (Fig. 2). At fetal PO2, none of the treatments altered the incidence of apoptosis significantly from the controls; however, administration of Zn2+ or LPS+Zn2+ significantly raised LDH leakage over control values. Each dose of thymulin lowered the incidence of LPS+Zn2+-induced necrosis to a level that was not statistically significant from controls. This was not associated, however, with any recovery of ASCf096h to control values.
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At ambient PO2, tissue damage patterns evoked by moderate hyperoxia or LPS followed a different pattern. Necrotic LDH leakage remained relatively constant among all treatment groups; however, LPS+Zn2+ evoked a marked increase in apoptotic oligonucleosome release that was abolished by administration of thymulin at each concentration. ASCf096h was modestly raised by thymulin+Zn2+ treatment becoming statistically significant at the highest dosage of thymulin.
Thymulin+Zn2+ evokes IL-6, and suppresses TNF-, release at ambient, but not fetal, PO2. To determine whether thymulin+Zn2+ cytoprotection involved modulation of the LPS acute-phase response, we examined the release of two rapidly expressed cytokines, TNF-
and IL-6, which are, respectively, proapoptotic/necrotic and antiapoptotic/proliferative. Neither control nor Zn2+-treated explants showed any change in the spontaneous release of IL-6 at either PO2 (Fig. 3A); however, thymulin administered with Zn2+ increased IL-6 secretion under both conditions, becoming statistically significant at ambient PO2. Likewise, LPS evoked a significant increase in IL-6 release, which was diminished in combination with Zn2+, an effect that may stem from the increased rate of apoptosis observed under these conditions in Fig. 2. Notably, administration of thymulin at 10 and 1,000 ng/ml conserved the release of IL-6 in LPS+Zn2+-treated explants at levels that were significantly greater than controls but did not differ statistically from LPS or thymulin+Zn2+-treated explants.
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TNF- release did not change significantly in control explants maintained under either PO2 or in the presence of Zn2+ or thymulin+Zn2+ (Fig. 3B). In keeping with the key role of this cytokine in the immunogenic acute-phase response, LPS significantly raised the expression of TNF-
at ambient PO2, which was sustained on addition of Zn2+; as with the IL-6 response under these conditions, statistical significance beyond control levels remained elusive, presumably due to high rates of apoptosis. Nevertheless, thymulin administered at 0.1 and 1,000 ng/ml significantly lowered the release of TNF-
at ambient PO2 below the levels observed with LPS+Zn2+.
As differences in size, cellular differentiation, and development presumably mask the kinetics of thymulin+Zn2+-evoked cytokine responses in explants, dose-response relationships were determined with a human pulmonary adenocarcinoma cell line (A549) that displays alveolar type II pneumocyte-like characteristics. We then correlated these results with the change in cellular proliferation by following the rate of MTT reduction in the presence of LPS+thymulin+Zn2+. As with the rat lung explants, thymulin+Zn2+ evoked a spontaneous, statistically significant rise in IL-6 release at ambient PO2 (EC50 = 0.54 ± 0.16) without detectable change in basal TNF- release (Fig. 4, A and B). In contrast to our explant observations, there was little IL-6 release in cultures incubated solely in the presence of LPS+Zn2+; however, incremental dosage with thymulin evoked a fivefold increase in the scope for IL-6 release at both PO2s [EC50 = 0.64 ± 0.34 (23 mmHg) and 1.43 ± 0.29 (142 mmHg)] over control cultures that had been similarly treated with thymulin+Zn2+ alone. In hand with the observed suppression of LPS+Zn2+-evoked TNF-
release in explants, incremental dosage with thymulin significantly inhibited the release of this cytokine from A549 cultures at either PO2 over a dose range that showed similar kinetics for IL-6 release [IC50 = 0.42 ± 0.01 (23 mmHg) and 0.53 ± 0.01 (142 mmHg)].
The amplification of IL-6 and coordinated suppression of TNF- release correlate with a significant increase in metabolic oxidative activity, interpreted as cellular proliferation, in A549 cells (Fig. 4, C and D). Cultures treated with LPS+Zn2+ in the absence of thymulin showed high spontaneous release of TNF-
with little detectable IL-6, which was coupled with a modest rate of MTT reduction. Addition of 1100 ng/ml thymulin synergistically raised IL-6 release, suppressed TNF-
expression, and significantly raised the rate of MTT reduction, which was greatest in cells maintained at fetal PO2.
Thymulin+Zn2+ augments activity of the nuclear factors of IL-6 transcription. The preceding results suggest that thymulin+Zn2+ acts as a specific agonist of IL-6 expression in lung explants and may functionally regulate the activity of nuclear factors involved in IL-6 gene expression. As IL-6 transcription is augmented by the synergistic interaction between the nuclear factor of IL-6, C/EBP, and NF-
B (1, 20, 37), we examined the effect of the transcriptional blocker SSA on IL-6 release together with C/EBP
and NF-
B DNA binding activity. SSA consistently abolished the release of IL-6 protein from explants treated with thymulin+Zn2+, LPS+Zn2+, and LPS+thymulin+Zn2+ at ambient PO2 (Fig. 5A). In the absence of SSA, fetal PO2s raised the consensus DNA binding activity of C/EBP, but not NF-
B, whereas C/EBP binding was low, and NF-
B raised, at ambient PO2 (Fig. 5, B and C). Independent administration of thymulin or Zn2+ did not result in a statistically significant effect on the activity of either transcription factor. When administered together, however, the binding of both factors displayed an increasing trend that was 4.7 ± 2.1 (C/EBP)- and 2.3 ± 0.8 (NF-
B)-fold above the ambient PO2 control. Notably, the C/EBP activation by thymulin+Zn2+ and LPS+thymulin+Zn2+ diminished to control levels on inclusion of an antisense oligonucleotide against IL-6, suggesting a positive feedback relationship between IL-6 and C/EBP expression and activation. Additionally, SSA abrogated C/EBP and NF-
B DNA binding activity in a manner that was consistent with the blockade of IL-6 release in Fig. 5A. Supershift experiments identified the C/EBP
isoform as the predominantly active species of this transcription factor in explants at either fetal PO2 or ambient PO2 in the presence of thymulin+Zn2+ (Fig. 5D).
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MAPKAP-K2 activity and mRNA abundance of IL-6 and TNF- are sustained by thymulin+Zn+. MAPKAP-K2 is the terminal kinase involved in regulating the expression of TNF-
mRNA and posttranslational stabilization of IL-6 (33, 38); therefore, we examined the potential for thymulin+Zn2+ to activate MAPKAP-K2 via the phosphorylation of a specific target substrate, HSP27 (Fig. 6). Figure 6A shows a representative experiment conducted in serum-starved A549 cultures exposed for 60 min to LPS as a positive control, LPS together with an inhibitor of the upstream kinase p38 MAPK (SB-203580) as a negative control, and two doses of thymulin+Zn2+. HSP phosphorylation was weakly evident in A549 cells at ambient PO2 and was potently induced in the presence of 100 and 1,000 ng/ml thymulin. In explants that had been exposed to each treatment regimen for 60 min, we observed a constitutive activation of HSP27 phosphorylation (Fig. 6B) that masked any specific effect thymulin+Zn2+ alone. The activation observed with thymulin+Zn2+, with or without LPS, was diminished by SB-203580.
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Figure 6C shows the overall expression of IL-6 and TNF- mRNA detected following 30 cycles of RT-PCR under a similar treatment regimen as for Fig. 5A. IL-6 mRNA was readily detected in explants cultured at fetal, but not ambient, PO2. Expression was potently induced at ambient PO2 by LPS and thymulin, administered separately or together, and was abolished in each case by SSA. TNF-
expression showed a converse oxygen sensitivity to IL-6, being weakly detected at fetal PO2, but potently expressed at ambient PO2. This expression was not altered significantly by treatment with LPS or thymulin but was abolished by SSA.
Thymulin+Zn2+ evokes mesenchyme hyperplasia in the fetal lung. To determine whether the cytoprotective effects of thymulin+Zn2+ during LPS treatment bore consequences for lung morphology, we used random sequence control and antisense oligonucleotides derived against rat IL-6 to manipulate the patterns of apoptosis and necrosis at ambient PO2 observed in Fig. 2. The differentiated epithelial fraction of each explant was calculated from histological sections as described in EXPERIMENTAL PROCEDURES (Fig. 7).
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Explant IL-6 secretion evoked by LPS+thymulin+Zn2+ was abolished in the presence of IL-6 antisense oligonucleotide but remained unaltered by addition of a random sequence control oligonucleotide (Fig. 7A). Significantly, antisense treatment did not abolish the suppressive effect of thymulin+Zn2+ on LPS-stimulated TNF- release, suggesting that the TNF-
silencing effect we observed with this hormone does not stem from a suppressive IL-6 feedback effect.
Figure 7B shows the effect of IL-6 antisense treatment on the distribution of differentiated epithelial structures as a correlate of necrosis (LDH leakage) or apoptosis (oligonucleosome release). Untreated explants maintained at 23 mmHg exhibited the highest proportional fraction of differentiated epithelium together with unperturbed fractional rates (values of 1.0 = no change) of LDH leakage and apoptosis over 96 h of culture. Exposure of ambient PO2 cultured explants to LPS + thymulin+Zn2+ in the presence of the control oligonucleotide resulted in a significant decrease in the proportion of differentiated epithelium to 8% of the total explant surface area. This was accompanied by negligible perturbation in LDH leakage, but significant inhibition of apoptosis. Similarly, exogenously applied recombinant rat (rr) IL-6 significantly reduced the rate of apoptosis but did not alter the differentiated epithelial fraction from that observed with untreated fetal or ambient PO2 explants. IL-6 antisense oligonucleotide applied under the same conditions as the control oligonucleotide raised the differentiated epithelial fraction to
30% of total explant surface area but significantly increased LDH leakage and apoptosis beyond that observed with any other treatment.
Thymulin+Zn2+ modulates FGF-9 and -10 expression to cause mesenchyme hyperplasia. As airway complexity rests on the coordination of signals among regionally expressed growth factors and repressors, we sought to determine whether the observed changes in airway complexity could be accounted for by modulation of FGF-9 and -10 expression. Figure 8A shows representative cross sections of gestation day 16 fetal lungs immunostained for FGF-9, FGF-10, and C/EBP protein. Although a focal staining of FGF-9 and -10 was observed throughout lung sections, FGF-9 protein showed a rather more diffuse pattern of staining, whereas FGF-10 was predominantly localized in the differentiated epithelial compartment of the airways. C/EBP
immunoreactivity was exclusively mesenchymal and exhibited focal pockets of intense nuclear staining around airway structures. Figure 8B demonstrates FGF-9, FGF-10, C/EBP
, and DAPI staining in explants following 96 h of culture at either 23 or 142 mmHg. Explants cultured at 142 mmHg were additionally treated with 50 µg/ml LPS, LPS and 10 µmol ZnCl2 + 1,000 ng/ml thymulin, thymulin+Zn2+ + 10 nmol/ml IL-6 antisense oligonucleotides or 10 ng/ml rrIL-6. Mesenchyme FGF-9 staining was evident in explants maintained at 23 mmHg but was largely absent from those at 142 mmHg or exposed to LPS treatment. LPS plus thymulin+Zn2+ resulted in homogeneous FGF-9 expression, mesenchyme proliferation, and a loss of differentiated epithelial structures but little fragmentation of nuclear DNA. Addition of IL-6 antisense oligonucleotides muted the mesenchyme hyperplasia but also resulted in distinct DNA fragmentation in the mesenchyme compartment. Exogenous rrIL-6 noticeably increased the proportion of FGF-9 immunoreactive tissue and significantly diminished the overall proportion of airway space without loss of epithelial structures relative to ambient PO2 controls. FGF-10 immunostaining showed regionally intense pockets of staining under all conditions but became diffuse in LPS+thymulin+Zn2+-treated explants. C/EBP
showed the same exclusively mesenchymal distribution as in gestation day 16 rat lung but became predominantly nuclear in location in explants exposed to LPS and LPS+thymulin+Zn2+. Addition of an IL-6 antisense oligonucleotide entirely abolished this effect, whereas rrIL-6 evoked strong nuclear accumulation of C/EBP
in all tissue compartments.
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Figure 9A shows a stacked histogram detailing changes in the fractional surface area of each compartment as calculated from six independent experiments. The surface area fraction of the mesenchyme compartment was significantly raised relative to fetal and ambient PO2 explants in both LPS+thymulin+Zn2+-treated explants in the presence of control or antisense oligonucleotides. Whereas mesenchyme proliferation was not observed as such with rrIL-6, the overall mesenchyme fraction was raised (P = 0.09), and total airway space was significantly (P < 0.01) diminished relative to explants maintained at ambient PO2. Notably, ambient PO2 treatment of explants with rrIL6 resulted in a distribution of airway space, mesenchyme, and epithelium that was not significantly different from explants maintained at fetal PO2. These data are complemented by mass-specific differences in FGF-9 rather than FGF-10 protein abundance (Fig. 9B).
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DISCUSSION |
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To interpret cytoprotective and immunomodulatory responses to thymulin+Zn2+, we began this study by characterizing explant morphogenic responses to culture PO2 and LPS. Our results revealed a significant increase in ASC in fetal PO2 cultured explants that was absent from those at ambient PO2. Whereas several studies have established that the low circulating PO2s characteristic of the fetal environment functionally maintain epithelial luminal fluid secretion and lung expansion (3, 4, 26, 36), evidence showing that airway branching is similarly PO2 dependent is both limited and conflicting. For example, antioxidants have been shown to promote airway proliferation under conditions similar to the mildly hyperoxic ambient PO2 treatments used here (Ref. 16 and references therein); however, culture of explants in the presence of nitric oxide donors at concentrations sufficient to invoke sustained nitrosative/oxidative stress also raises airway branching morphogenesis (52). Our results with low concentrations of LPS (0.52 µg/ml), a proinflammatory agent that promotes release of reactive oxygen and nitrogen species, similarly increased airway morphogenesis irrespective of prevailing PO2. At higher concentrations (50 µg/ml), this reverted toward a loss of mesenchyme, cystic structure formation, and epithelial flattening similar to ambient PO2 controls, suggesting permissive ranges of PO2 or LPS may facilitate airway morphogenesis. In utero, LPS promotes fetal lung maturation, resulting in improved mass-specific lung volume, alveologenesis, gas exchange surface area, surfactant protein expression, and alveolar type II cell distribution (24, 25, 31, 45, 50). Prolonged exposure to LPS in neonatal or adult lung, however, results in pulmonary fibrosis, reduced epithelial surface area, septal thickening, and enlargement of alveolar space (46). In broad terms, these studies echo the dose-dependent morphogenic effects evoked by LPS in explants and imply that developmental expression of pathways that govern the immunogenic acute-phase response and thus immune reactivity set the scope for morphogenic responses to inflammation throughout life.
To manipulate immune reactivity in pseudoglandular lung explants, we examined the effect of thymulin and its cofactor Zn2+ on the high-dose LPS-evoked decrease in explant ASC. Single administration of Zn2+ at concentrations representative of the umbilical circulation (21) yielded little effect on either ASC or indexes of cell death but, in combination with LPS, augmented the incidence of necrosis/apoptosis beyond other treatments at fetal and ambient PO2s. This compound effect was blocked by thymulin in the presence of a 10- to 1,000-fold molar excess of Zn2+, suggesting that the cytoprotective effect is linked to thymulin itself as opposed to incidental thymulin-Zn2+ chelation or the widely reported antiapoptotic properties of Zn2+.
Examination of the physiological mechanism behind the thymulin+Zn2+ cytoprotective effect revealed a bidirectional regulation of IL-6 and TNF- expression during an LPS inflammatory stimulus. Bioactive thymulin has previously been observed to inhibit LPS-evoked expression of both TNF-
and IL-6 in peripheral blood mononuclear cells (39, 40); however, to our knowledge, selective (ant)agonistic regulation of these cytokines in fetal lung has not previously been reported. Although TNF-
potently induces genomic expression of IL-6 along with other acute-phase response cytokines, raised IL-6 titers can suppress TNF-
expression and are thought to regulate circulating [TNF-
] during infection (28, 41, 44). IL-6 negative feedback of TNF-
synthesis therefore represents one mechanism that could account for thymulin+Zn2+ cytoprotection.
To investigate this effect, we examined the capacity for thymulin+Zn2+ to alter p38 MAPK-transduced MAPKAP-K2 activity. This terminal kinase plays a pivotal role in the phosphorylation of RNA binding proteins, particularly heterogenous ribonucleoprotein A0 (38), which govern protein interactions with the AU-rich element (ARE) in the 3'-untranslated region of both TNF- and IL-6 genes. Whereas MAPKAP-K2 phosphorylation of ARE-binding proteins is necessary for the initiation of TNF-
transcription and nucleo-cytoplasmic mRNA transport, its role in the regulation of IL-6 expression is solely linked to the stabilization of nascent mRNA (33). We reasoned that if thymulin+Zn2+ treatment abolished LPS-evoked MAPKAP-K2 activity, the potential would exist for attenuated IL-6 expression to persist under conditions that completely block TNF-
posttranscriptional processing. Rather than showing an inhibition, our results revealed a potent activation of MAPKAP-K2 in Zn2+-supplemented A549 cells in response to thymulin. Although we failed to demonstrate a similarly concise pattern of MAPKAP-K2 activation in explants, presumably due to our choice of culture conditions, we showed that the activity found in the presence of LPS and thymulin+Zn2+ is largely abolished by the p38 MAPK inhibitor SB-203580. Aside from differences in IL-6 and TNF-
mRNA abundance in control explants at either PO2, we found a sustained expression of both mRNAs that, in the case of IL-6 by thymulin+Zn2+, occurred irrespective of LPS treatment. Notably, transcriptional blockade using the NF-
B inhibitor SSA resulted in the degradation of both transcripts; therefore, this pattern of mRNA expression required continuous transcriptional activity and was not solely due to posttranscriptional mRNA stabilization. Moreover, antisense blockade of thymulin-evoked IL-6 expression did not significantly raise TNF-
mRNA abundance (data not shown) and did not relieve the suppressive effect of this hormone on LPS-evoked TNF-
protein synthesis (Fig. 7A). Our results therefore suggest that the bidirectional change in TNF-
and IL-6 protein synthesis does not stem from IL-6-inhibited TNF-
expression and cannot be accounted for by blockade of the p38 MAPK/MAPKAP-K2 pathway. The silencing of LPS-stimulated TNF-
synthesis by thymulin must occur either by an alternative posttranscriptional mechanism or by increased proteolytic targeting.
In addition to cytoprotection by the thymulin+Zn2+ blockade of TNF- synthesis, raised expression of IL-6 may directly promote mesenchyme regeneration and repair through a mitogenic response transduced by JAK/STAT, Ras/MAPK, and phosphatidylinositol 3-kinase pathways. Transgenic overexpression of IL-6 in mice markedly extends survival during exposure to 100% O2, an effect associated with conserved pulmonary expression of antiapoptotic factors such as Bcl-2 and metalloproteinase-1 (49). Moreover, IL-6 genomic knockout illustrates the key role of this cytokine in acute chemotactic signaling via VCAM-1 as well as regulating the expression levels of IL-4, -5, and 13 in bronchiolar lavage (47). Among the identified antiapoptotic properties of IL-6 is a capacity to inhibit transforming growth factor-
(TGF-
)-mediated apoptosis (10, 47), an important regulator of mesoepithelial differentiation and, possibly, alveolarization (8, 23). This suggests that when hyperexpressed, IL-6 regenerative repair may interfere with the normal clearance of tissue necessary for the formation of space encapsulating structures, as well as in promoting fibrosis, and presents one mechanism for the proliferation of mesenchyme induced by thymulin+Zn2+ treatment in this study.
To examine this possibility further, we focused on the relationship between IL-6 and its nuclear factor, C/EBP, a basic-region leucine zipper transcription factor family member whose activity is potentiated by homo- or heterodimerization with either C/EBP
, -
, or NF-
B (1, 20, 37). Morphogenic roles have been postulated for C/EBP isoforms in the lung due to their capacity to regulate cellular turnover and differentiation. Genomic knockout of the
- and
-isoforms results in breathing difficulties from birth, cyanosis, high postnatal mortality, and, in the case of the
-isoform, hyperproliferation of alveolar type II cells and interstitial thickening (14, 17, 48). More specifically, both the
- and
-isoforms regulate the expression of Clara cell secretory protein, an important anti-inflammatory and cytoprotective agent of the bronchial airway (9). Our results highlight a specific role for the
-isoform in mediating mesenchyme proliferation in rat lung explants. We show a heterogeneous pattern of C/EBP
activation in the mesenchyme mass of gestation day 16 fetal rat lung, which was present in explants maintained at 23 mmHg but absent at ambient PO2 or in the presence of an IL-6 antisense oligonucleotide. Significantly, C/EBP
and NF-
B, but not C/EBP
or -
, were synergistically activated under treatment with thymulin+Zn2+, either with or without LPS, pointing toward a strong association between thymulin immunomodulation and C/EBP
regulation of the acute-phase response.
Precise developmental roles for C/EBP in the lung are poorly defined, partly because C/EBP family members possess conserved COOH-terminal domains and so may functionally substitute for one another in knockout experiments. In hepatic and colonic tissue, C/EBP
represents a key component of the proliferative response to oxidative injury and TGF-
-evoked ribosomal protein S-6 kinase activation and is known to mediate mesenchyme proliferation during tissue repair (7, 11). It is also involved in regulating the activation and terminal differentiation of macrophages in lymphatic tissues (43), an observation that brings it into close functional proximity with the established role of bioactive thymulin in the thymus. Our experiments showed that activation of C/EBP
by LPS and thymulin+Zn2+ initiated a hyperexpressed regenerative repair response located in the undifferentiated mesenchyme tissue fraction that, coupled with an inhibition of apoptosis, resulted in the near complete loss of differentiated epithelial structures (Figs. 7, 8, 9). Each of the core characteristics of this response (C/EBP
activation, mesenchyme proliferation, apoptosis blockade, loss of epithelial structures) was reversed by addition of IL-6 antisense oligonucleotides, which muted IL-6-evoked positive feedback of C/EBP
gene expression, an effect that sustains IL-6 acute-phase response signaling (2). Together, our observations accord with an interpretation of C/EBP
as a nuclear factor that regulates regional, proportionate, mesenchyme proliferation in the pseudoglandular stage lung and overt proliferation during lung regenerative repair.
As reciprocal signaling between mesenchyme and epithelium drives lung morphogenesis, we examined the association between IL-6- and C/EBP-coupled signaling through thymulin+Zn2+ on FGF-9 and -10 protein expression. In the pseudoglandular stage, FGF-9 mRNA is expressed in the pleura and transiently in the bronchial epithelium and stimulates proliferation and expansion of the mesenchyme compartment via the receptors FGFR1c and/or FGFR2c (13). FGF-10 is expressed in the mesenchyme adjacent to the distal termini of the developing airways, becoming diffusely expressed in mesenchyme toward the end of this developmental stage (5). Whereas FGF-10 is believed to initiate airway branching by interaction with FGFR2b, FGF-9 regulates the density of this branching through proliferation and expansion of mesenchyme (12, 13); therefore, the proportion of FGF-9 to FGF-10 expression can be used as an index of airway branching potential. Our immunohistochemical experiments showed that the distribution pattern of both factors responded to changes in culture PO2 and immunomodulation by thymulin+Zn2+. Most notable, however, was an increase in mesenchymal FGF-9 expression that mirrored the distribution of nuclear C/EBP
in response to thymulin+Zn2+ and showed a mass-specific increase in expression in response to treatments that augmented IL-6 release and mesenchyme proliferation. Given that a putative C/EBP
-binding element has been reported to reside in the 5'-flanking region of the mouse FGF-9 gene (12) and that IL-6-regulated FGF-9 expression has been shown to trigger proliferation of precursor megakaryocytes in the thrombopoietic response (27), we believe our results highlight the potential for cooperative signaling between IL-6, C/EBP
, and FGF-9 in initiating the proliferation of mesenchyme as an early phase of the tissue repair response in the lung. Notably, this proliferative effect occurred at the expense of lung mass occupied by airway and vascular structures and highlights the presence of a crucial signaling lesion at the level of mesenchyme redifferentiation. Overcoming this block will require reinstatement of the pathways that direct epithelial and endothelial redifferentiation with the temporal and spatial signaling pattern that drives lung morphogenesis.
Figure 10 summarizes our studies into the morphogenic response of fetal rat lung explants to treatment with thymulin+Zn2+ and identifies four regulatory components that account for the immunomodulatory effect of this hormone, namely, selective activation of C/EBP coupled with stress-evoked NF-
B activity, posttranscriptional regulation through p38 MAPK-dependent MAPKAP-K2, increased potential for IL-6 expression, and interference with the turnover pathway for TNF-
protein. Cessation of TNF-
synthesis in association with the antiapoptotic and proliferative properties of IL-6 results in conditions that favor the proliferation of mesenchyme, which may arise as a direct result of IL-6-C/EBP
positive feedback coupled to FGF-9 signaling. The scope for alveolar development and structural remodeling in newborn infants depends in part on the conservation of mesenchyme tissue from which new pulmonary structures develop (5, 12, 13, 19). Our study illustrates the high potential of bioactive thymulin to initiate mesenchyme proliferation in the fetal lung as a fundamental component of the tissue regenerative repair response. The therapeutic potential of this effect will require tailoring this response to operate with agents and/or conditions that facilitate the regeneration of airway and vascular structures from mesenchymal tissue.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by the Medical Research Council (UK), the Wellcome Trust, Tenovus (Scotland), and the Anonymous Trust.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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