Tumor necrosis factor induces neuroendocrine differentiation in small cell lung cancer cell lines

Kathleen J. Haley1, Kirit Patidar2, Fan Zhang2, Rodica L. Emanuel3, and Mary E. Sunday2,3

1 Pulmonary and Critical Care Division, Department of Medicine, and 3 Department of Pathology, Brigham and Women's Hospital and Harvard Medical School; and 2 Department of Pathology, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We studied tumor necrosis factor (TNF)-alpha as a candidate cytokine to promote neuroendocrine cell differentiation in a nitrosamine-hyperoxia hamster lung injury model. Differential screening identified expression of the genes modulated by TNF-alpha preceding neuroendocrine cell differentiation. Undifferentiated small cell lung carcinoma (SCLC) cell lines NCI-H82 and NCI-H526 were treated with TNF-alpha for up to 2 wk. Both cell lines demonstrated rapid induction of gastrin-releasing peptide (GRP) mRNA; H82 cells also expressed aromatic-L-amino acid decarboxylase mRNA within 5 min after TNF-alpha was added. Nuclear translocation of nuclear factor-kappa B immunostaining occurred with TNF-alpha treatment, suggesting nuclear factor-kappa B involvement in the induction of GRP and/or aromatic-L-amino acid decarboxylase gene expression. We also demonstrated dense core neurosecretory granules and immunostaining for proGRP and neural cell adhesion molecule in H82 cells after 7-14 days of TNF-alpha treatment. We conclude that TNF-alpha can induce phenotypic features of neuroendocrine cell differentiation in SCLC cell lines. Similar effects of TNF-alpha in vivo may contribute to the neuroendocrine cell differentiation/hyperplasia associated with many chronic inflammatory pulmonary diseases.

nuclear factor-kappa B; cell differentiation; gastrin-releasing peptide; aromatic-L-amino acid decarboxylase; neural cell adhesion molecule

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

TUMOR NECROSIS FACTOR (TNF)-alpha production has been reported in patients with several types of pulmonary dysfunction, including chronic obstructive lung disease (14, 16), hyperoxic lung injury (26, 31), bronchopulmonary dysplasia (5), and asthma (1, 10, 12, 60). Some of these processes have also been associated with pulmonary neuroendocrine cell hyperplasia (23, 32, 37, 44, 51, 55, 62). Because most of the clinical pulmonary conditions associated with pulmonary neuroendocrine cell hyperplasia are chronic inflammatory processes, it is possible that proinflammatory cytokines modulate the development of this hyperplasia to some degree.

We have been investigating neuroendocrine cell hyperplasia occurring during preneoplastic hamster lung injury induced by diethylnitrosamine (DEN) and hyperoxia (inspired O2 fraction 65%; DEN-O2). After 8-12 wk of exposure to DEN-O2, there was up to a 10-fold increase in the relative number of neuroendocrine cells in the hamster lung. Immunohistochemical analysis of lung sections from DEN-O2-exposed animals demonstrated only rare immunostaining (<0.5% of cells) for proliferating cell nuclear antigen in neuroendocrine cells. In contrast, there was abundant proliferating cell nuclear antigen labeling (>25% of cells) in adjacent nonneuroendocrine epithelial cells (66). Therefore, it is likely that the increased number of neuroendocrine cells in this model was predominantly due to differentiation of latent neuroendocrine cells or neuroendocrine cell precursors (66) rather than simply increased cell division.

The present study began as an investigation of potential cellular and molecular mechanisms underlying the induction of neuroendocrine cell differentiation observed in the DEN-O2 hamster lung injury model. We hypothesized that the genes involved in modulating the neuroendocrine cell response to DEN-O2 are upregulated before the appearance of the hyperplasia. Therefore, in our initial studies, we differentially screened hamster lung cDNAs for genes expressed before the appearance of neuroendocrine cell hyperplasia. Several molecules including the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) (38, 72) and the neutrophil adhesion molecule receptor CD18-beta (49), known to be upregulated by inflammatory cytokines, particularly TNF-alpha , were identified. Nitrosamines (58) and hyperoxia (26, 31) have each been associated with the production of several cytokines, including TNF-alpha , interleukin-1beta , and interleukin-6, all of which can induce differentiation in a variety of cell types (7, 34, 35, 52, 68).

The initial observation of the upregulation of genes modulated by TNF-alpha and the appearance of neuroendocrine cell differentiation led to the hypothesis that neuroendocrine cell differentiation is induced by inflammatory cytokines, specifically TNF-alpha , produced as part of the host response to DEN-O2 injury. TNF-alpha induces the expression of differentiation markers and specific enzymatic activity in a variety of cell types, including malignant myeloid cell lines and primary cell cultures (21, 28, 70) and both human and murine neuroblastoma cell lines (36, 47, 54). Several studies have also suggested that TNF-alpha triggers differentiation in normal cells such as B cells (30), thymocytes (77), macrophages (75), and dendritic cells (56). In addition, TNF-alpha can induce type II pneumocyte differentiation in murine embryonic lung bud cultures (29). Furthermore, TNF-alpha can upregulate MHC class II antigen expression (4, 38, 72) and cell surface expression of the integrin CD18 (76), thus resulting in functional modulation of immune responses.

We tested this hypothesis using several approaches. First, we analyzed RNA from DEN-O2-treated hamster lungs for TNF-alpha gene expression. Next, we developed an in vitro model to study the possible role of TNF-alpha in directly inducing pulmonary neuroendocrine cell differentiation using undifferentiated small cell lung carcinoma (SCLC) cell lines. We evaluated changes in these cells toward a partial neuroendocrine phenotype at the level of gene expression, the presence of neuroendocrine cell-associated surface antigens and neuropeptide products, and cellular ultrastructure. We also investigated possible signal transduction pathways involved in these TNF-alpha responses, focusing on nuclear factor-kappa B (NF-kappa B), which has been implicated in molecular mechanisms promoting differentiation of other cell types (25).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Differential screening. Total RNA was prepared by the guanidinium-isocyanate-cesium chloride method (13) from hamster lung tissues harvested after 4, 8, and 12 wk of nitrosamine treatment and from age-matched control animals. The 4- to 12-wk time period was chosen for differential screening because it included both the interval preceding neuroendocrine cell hyperplasia (4-8 wk) and the point when peak numbers of neuroendocrine cells are present (12 wk), when putative genes involved in regulating neuroendocrine cell numbers might be expressed. 32P-labeled cDNAs were prepared from 1 µg of total hamster lung RNA with 50 µCi of [32P]dCTP, Moloney murine leukemia virus reverse transcriptase (2 units; Bethesda Research Laboratories, Gaithersburg, MD), RNAsin (5 units; Sigma, St. Louis, MO), and random hexamers [1 unit 5'-pd(N)6; Pharmacia LKB Biotechnology, Piscataway, NJ] in a total volume of 10 µl of 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, and 3 mM MgCl2 for 60 min at 42°C. These cDNAs were used as probes to screen duplicate nitrocellulose membrane filters bearing colony imprints of hamster cDNA libraries from animals treated for 4, 8, or 12 wk with DEN-O2.

Cell culture. NCI-H82, NCI-H526, COLO 320, NCI-H69, and NCI-H345 cells were obtained from American Type Culture Collection (Rockville, MD) and as generous gifts from Dr. Herbert Oie (National Cancer Institute, Bethesda, MD), Dr. Benjamin Neel (Beth Israel Deaconess Medical Center, Boston, MA), and Dr. Margaret Shipp (Dana Farber Cancer Institute, Boston, MA). NCI-H209, T723, NCI-H835, and OC2 cells were a generous gift from Dr. Benjamin Neel. Cell lines H82, H526, and T723 are variant SCLC cell lines; H69, H345, H209, and OC2 are classic SCLC cell lines. The classic SCLC cell lines are defined by the expression of several neuroendocrine cell markers such as aromatic-L-amino acid decarboxylase (DDC), bombesin-like immunoreactivity, gastrin-releasing peptide (GRP) gene expression, and neuron-specific enolase production. The markers characteristic of the classic SCLC cell lines are typical of well-differentiated neuroendocrine cells (11). In contrast, the variant SCLC cell lines demonstrate few or no neuroendocrine cell markers and often demonstrate high levels of c-myc RNA, consistent with undifferentiated neuroendocrine cells (11, 20). COLO 320 is a colon adenocarcinoma cell line that has some neuroendocrine features. All cell lines were maintained in RPMI 1640 medium (GIBCO BRL, Grand Island, NY) supplemented with 2.5% bovine fetal calf serum (GIBCO BRL), HITES (10-8 M hydrocortisone, 5 µg/ml of insulin, 5 µg/ml of transferrin, 10-8 M estradiol, and 5 ng/ml of sodium selenite; Sigma), 2% L-glutamine (GIBCO BRL), and 10 ml of penicillin-streptomycin (10,000 units penicillin/ml and 10 mg streptomycin/ml; Sigma) at 37°C in a humidified incubator in 5% CO2. Cells were fed by replacement of one-half of the medium every 2-3 days and were split 1:5 when a density of ~5 × 105 cells/ml was attained (about once a week). Cell viability was evaluated by trypan blue exclusion. Cultures were monitored daily for evidence of contamination; no specific testing was performed to exclude mycoplasmal contamination. Cells were counted with a hemocytometer.

The following amounts of TNF-alpha were used in the dose-response experiments: for H82 cells, 0, 4, 10, 40, 100, and 400 units/ml were added; for H526 cells, 0, 1, 4, 10, 40, 100, and 400 units/ml. All dose-response cultures were harvested after a 24-h incubation for RNA analyses. All of the cell lines were tested with the dose-response protocol and then screened by RT-PCR for induction of GRP mRNA after exposure to TNF-alpha . H82 and H526 cells demonstrated an induction of GRP mRNA and so were used for the remaining investigations. The other cell lines either did not demonstrate any GRP mRNA either without or with TNF-alpha treatment (COLO 320) or demonstrated baseline GRP mRNA levels without evidence of further induction after TNF-alpha treatment (H209, OC2, T723, H345, H69, and H835). For the time-course studies, we used the optimal doses of TNF-alpha : 400 units/ml for NCI-H82 cells and 4 units/ml for NCI-H526 cells. Cultures were harvested before TNF-alpha exposure, at 5, 10, 15, and 30 min, and at 1, 2, 4, 8, 12, 24, 48, and 72 h. Cultures for ultrastructural and immunohistochemical analyses were harvested after exposure to 400 (H82 cells) or 4 units/ml of TNF-alpha (H526 cells) for 7-14 days.

RNA extraction and analysis. Total RNA was prepared from cultured cells with TRIreagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. RNA integrity was evaluated by examination of 18S and 28S RNA bands after electrophoresis with ethidium bromide-stained 1.5% agarose-formaldehyde gels. Northern blotting for GRP was carried out with standard protocols with random-primed cDNA probes (13) for H82 cells. Due to the relatively low abundance of GRP and subsequent weak signal obtained on Northern blot analysis, RT-PCR was used for the routine analyses in these experiments.

RT preparation and PCR analysis. cDNA was prepared with Moloney murine leukemia virus reverse transcriptase (2 units; Bethesda Research Laboratories), RNAsin (5 units; Sigma), and random hexamers [1 unit 5'-pd(N)6; Pharmacia LKB Biotechnology] in a total volume of 10 µl of 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, and 3 mM MgCl2 for 60 min at 42°C. One microliter of this reaction was used for each PCR. Negative controls consisted of an equal volume of diethyl pyrocarbonate-treated water substituted for the volume of RNA in the above reaction.

Synthetic oligodeoxynucleotides were purchased from either Oligos Etc. (Wilsonville, OR) or The Midland Certified Reagent (Midland, TX). All primer pairs yielded products that spanned at least one intron to permit distinction between cDNA and any contaminating genomic DNA. Primer pairs are given in Table 1.

                              
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Table 1.   PCR primer sequences

PCRs were carried out with Taq polymerase (Boehringer Mannheim, Indianapolis, IN) as specified by the manufacturer. Briefly, 1 µl of the reverse-transcribed RNA mixture was added to 2.5 µl of 20× reaction buffer (20× buffer is 1.0 M Tris · HCl, pH 9.0, 400 mM ammonium sulfate, and 30 mM MgCl2; Boerhinger Mannheim), 1 µl (1 unit) Taq polymerase, 1.25 µl of 2 mM each dATP, dGTP, dCTP, and dTTP, 1 µl of each primer (final concentration 20 µM), and 38.5 µl of sterile deionized water in 500-µl Eppendorf tubes. Each reaction was overlaid with 30 µl of light mineral oil (Sigma) and subjected to the number of cycles as specified in Table 1. For each primer pair, each cycle included denaturation (0.5 min at 93°C), annealing (1.0 min), and extension (1.5 min at 72°C) with a programmable thermal cycler (MJ Research, Watertown, MA). The products of the reaction were analyzed on 1.5% agarose gels containing 0.5 µg/ml of ethidium bromide in 1× 10 mM Tris base-10 mM boric acid-40 mM EDTA buffer, pH 7.6 and blotted onto nitrocellulose membranes according to standard protocols (13). Oligonucleotide probes were end labeled with T4 kinase (13) and hybridized at 42°C for 18 h in 6× saline-sodium citrate (SSC; 1× SSC is 0.15 M NaCl and 0.015 M sodium citrate), 5× Denhardt's solution (100× Denhardt's solution is 20 mg/ml of Ficoll 400, 20 mg/ml of polyvinylpyrrolidone, and 20 mg/ml of BSA), 0.1% SDS, and 250 µg/ml of salmon sperm DNA. Blots were washed in 2× SSC with 0.1% SDS at 42-50°C before exposure to Kodak Biomax film (Kodak, Rochester, NY) at -70°C for 2-16 h. We defined our RT-PCR assay conditions to yield semiquantitative results as previously described (33). In brief, PCR cycle number and conditions were determined such that autoradiographic bands obtained by overnight exposure of RT-PCR Southern blots exposed to radiolabeled internal probes were approximately one-third of the maximal signal observed on overnight exposure. These conditions allowed for a generally linear detection of the relative amounts of specific mRNAs present in the same RT reaction mixture, which could subsequently be normalized with either actin mRNA or 18S mRNA as an internal control. Densitometry was carried out by three-dimensional integration of the area of whole bands on autoradiograms with a Hewlett-Packard Scanjet 3-P (Hewlett-Packard, Palo Alto, CA) interfaced with Biosoft Scan Analysis 2.55 software (Biosoft, Ferguson, MO). The results from the control primers were highly reproducible among experiments. The overall trends in the results from the cell lines treated with TNF-alpha were also highly reproducible, although the cell lines did demonstrate some variability in the exact timing of the responses. This variability was observed together with consistent results from the concurrent positive controls and was therefore attributed to the inherent phenotypic instability of these karyotypically unstable malignant cells.

Immunohistochemical analysis. Immunostaining was performed with a modified avidin-biotin complex technique (27). Cytospin samples were fixed in 4% paraformaldehyde for 3 min (for proGRP and p65 NF-kappa B subunit) or 1:1 acetone-methanol [for neural cell adhesion molecule (NCAM)] for 2 min and air-dried. The antibodies included rabbit polyclonal antiserum to GRP-gene-associated peptide-III (GGAP)-III (proGRP) used at a 1:1,250 dilution, the associated preimmune serum used at a 1:1,250 dilution (generous gifts from F. Cuttitta, National Cancer Institute), rabbit polyclonal antiserum to the p65 NF-kappa B subunit used at a 1:50 dilution (Santa Cruz Biotechnology, Santa Cruz, CA), a murine monoclonal IgG2a antibody to NCAM (clone UJ13A) used at a 1:10 dilution (Dako, Carpinteria, CA), and a murine irrelevant IgG2a, MOPC (clone UPC-10), used at a 1:500 dilution (Sigma). Nonspecific immunoglobulin binding was blocked with 10% normal goat serum (GIBCO BRL) for proGRP, the proGRP preimmune serum, and p65 NF-kappa B subunit and with 10% normal horse serum (GIBCO BRL) for NCAM and MOPC. The primary antiserum, diluted as above in PBS-2% BSA, was applied to tissue sections and incubated overnight at 4°C in a humidified chamber. The slides were then incubated with biotinylated IgG secondary antibody (goat anti-rabbit or horse anti-mouse depending on the primary antibody host; Vector Laboratories, Burlingame, CA) diluted 1:200 in 5% powdered milk in PBS with 5 µl/ml of normal human serum at 4°C for 2 h. Endogenous peroxidase activity was quenched with methanol containing 1% hydrogen peroxide. Avidin-biotin complex standard (Vector Laboratories) was made according to the manufacturer's instructions, applied to the sections, and incubated at room temperature for 1 h. Immunopositivity was visualized with the chromagen diaminobenzidine (0.025%) in PBS and 0.1% hydrogen peroxide. The negative controls included preabsorbed primary anti-p65 (Santa Cruz), the preimmune serum for proGRP, and the irrelevant murine IgG2a MOPC. Immunostaining with the negative controls proceeded as described for the primary antibodies. For p65 immunostaining, nuclei were localized by counterstaining the cytospins for 5 min with 4',6-diamidino-2-phenylindole (Sigma) diluted to 0.1 µg/ml in McIlvaine's buffer (17.65 ml of 0.1 M citric acid with 82.35 ml of 0.2 M Na2HPO4, pH 7.0). The other antibodies and negative controls were counterstained with 2% methyl green (Sigma).

Ultrastructural analysis. The samples for electron microscopy were harvested into 2% paraformaldehyde-0.25% glutaraldehyde (Sigma). After 2 h, the samples were transferred to Karnovsky's fixative prepared as follows: 200 ml of 0.2 M sodium cacodylate buffer, pH 7.4 (Polysciences, Warrington, PA), 20 ml of 50% glutaraldehyde solution (Ted Pella, Redding, CA), 50 ml of 16% formaldehyde solution (Ted Pella), 128.5 ml of distilled water, and 1.5 ml of CaCl2 (Fisher Scientific, Pittsburgh, PA). After overnight infiltration, the samples were routinely processed with osmium (Electron Microscopy Sciences, Fort Washington, PA), sym-collidine (Ted Pella), and propylene oxide (Polysciences) and embedded in Poly/Bed 812 embedding medium (Polysciences); 1-µm-thick sections were cut, and the samples were examined with a Jeol 100-CX electron microscope (Jeol, Tokyo, Japan).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Differential screening. Hamster lung cDNA libraries were prepared in Lambda ZAP II with poly(A)+ RNA from animals treated with nitrosamines [DEN or 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK)] plus 65% O2 for 4, 8, or 12 wk. All libraries had similar insert sizes: mean 1,065 ± 28 bp (range 300-3,000bp; n = 186) by PCR analysis with T3 and T7 primers. Six thousand primary colonies were screened with 32P-labeled cDNAs from homologous RNA versus normal adult hamster lung; 192 potential differentially expressed primary clones were selected, and the plaque lysates were used for PCR amplification. The PCR-amplified DNA inserts were used to prepare 96-well plate dot blots in duplicate for secondary screening. A total of 39 clones were confirmed as being of interest, and 32P-labeled cDNAs prepared from each isolate were used to probe multiple identical mini-Northern blots that had 10 µg/lane of pooled total lung RNA from six hamsters at four critical time points: 0 (normal, 8 wk old), 4-wk DEN-O2, 8-wk NNK-O2, and 12-wk NNK-O2. We used RNA from animals treated with either DEN-O2 or NNK-O2 because the quantities of RNA available from animals given DEN-O2 for 8 or 12 wk were not sufficient for these analyses. Hamsters given either DEN-O2 or NNK-O2 develop similar intense pulmonary neuroendocrine cell hyperplasia after 8-12 wk of treatment.

Several clones were identified in samples of lung from hamsters treated with DEN-O2 (Table 2). Ii was of particular interest because it is known to be upregulated in inflammatory responses (Fig. 1) (72). A representative Northern blot probed for Ii is shown in Fig. 1, demonstrating peak Ii gene expression after 8 wk of nitrosamine treatment (Fig. 1, Table 2). The integrin CD18-beta is also expressed, with peak levels after 8 wk of nitrosamine-O2 (Table 2). Cell surface expression of CD18-beta has been previously demonstrated to increase after exposure to TNF-alpha (76). In addition, CD18 modulates many of the effects of TNF-alpha , such as cell spreading and decreases in cAMP, that precede the respiratory burst in neutrophils (49). beta -Globin is probably attributable to the presence of reticulocytes in the lung vasculature of these animals; a previous description (66) of this animal model reported anemia in DEN-O2-exposed animals. Microtubule-associated protein-4 has been associated with actively dividing cells and probably reflects the nonneuroendocrine epithelial cell proliferation noted in the DEN-O2-exposed animals (66). The expression of two inflammation-associated molecules after 8 wk of nitrosamine-O2 treatment suggested to us that inflammatory cytokines might play a role in triggering increased pulmonary neuroendocrine cell differentiation-hyperplasia. TNF-alpha was selected as a candidate cytokine for testing in vitro because it is known to modulate expression of both Ii and CD18-beta .

                              
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Table 2.   Clones identified by differential screening


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Fig. 1.   Representative Northern blot demonstrating increased major histocompatibility complex chain II-associated invariant chain (Ii) in lung from hamsters exposed to diethylnitrosamine (DEN) or 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and hyperoxia (inspired O2 fraction 65%) for 4-12 wk.

TNF-alpha production in hamster injury model. With the use of RT-PCR analysis, TNF-alpha mRNA was shown to be present at high levels in lung tissue samples from hamsters exposed to DEN-O2, with peak levels occurring after 6 wk of treatment (Fig. 2). This time period immediately precedes and overlaps the period of neuroendocrine cell hyperplasia (66). TNF-alpha mRNA was identified in normal hamster lung during the neonatal period (data not shown) but not in normal adult hamster lung samples age matched to the hamsters in the DEN-O2 injury model (Fig. 2). Overall, the DEN-O2-treated animals had more pronounced levels of TNF-alpha mRNA in comparison with the animals exposed to DEN alone. Variations in the responses of individual animals, likely due to the use of outbred hamsters, did not affect the trends for the exposure groups. All hamsters exposed to DEN-O2 demonstrated high levels of TNF-alpha mRNA after 6 wk of DEN-O2 treatment. Lung tissue from 4 of 16 untreated adult hamsters contained TNF-alpha mRNA, but histological evaluation of the lung tissue from these 4 animals demonstrated acute and/or chronic aspiration pneumonia.


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Fig. 2.   Representative autoradiogram of Southern blot demonstrating tumor necrosis factor (TNF)-alpha gene expression in mRNA from DEN- and DEN-O2-exposed hamsters but not in healthy age-matched control animals. Maximal TNF-alpha gene expression occurs after 6 wk of nitrosamine exposure, ~2 wk before onset of neuroendocrine cell hyperplasia.

TNF-alpha differentiation effects in cell lines. Screening of eight SCLC cell lines and one colon adenocarcinoma cell line with RT-PCR for GRP mRNA led to the identification of two undifferentiated SCLC cell lines, which were negative for GRP at baseline under our described assay conditions but which had mRNA for GRP induced at low cell densities after treatment with TNF-alpha . The remaining investigations were performed with these two cell lines. The other cell lines either did not demonstrate any GRP mRNA under any circumstances (COLO 320) or demonstrated baseline GRP mRNA without evidence of induction after TNF-alpha exposure (H69, H835, H345, H209, OC2, and T723). The RT-PCR results for H82 cells were confirmed by Northern blot analysis (data not shown). Cultures having a high cell density (>106 cells/ml) and/or increased attachment to the plastic tissue culture plates were noted to have trace GRP mRNA at baseline. These cultures also demonstrated upregulation of GRP mRNA after TNF-alpha exposure. Although the induction of GRP and DDC mRNA was very consistent, the timing and individual culture responses to TNF-alpha demonstrated mild variability, probably due to the inherent karyotypic instability of the cell lines because the positive controls in these experiments were very consistent.

The presence of mRNA for the p55/60 and p75/80 TNF-alpha receptors in H82 and H526 cells was confirmed by RT-PCR (data not shown). Both receptors are present in both cell lines; however, the p55 receptor is more readily detected than the p75 receptor. Treatment with TNF-alpha did not alter either the p55/60- or the p75/80-receptor mRNA levels in these cell lines under our cell culture conditions. In addition, TNF-alpha treatment neither altered either cell viability as evaluated by trypan blue exclusion nor affected the rate of cell proliferation as evaluated by average cell density (data not shown).

H82 cells demonstrated induction of mRNA for both GRP and DDC with TNF-alpha treatment, with the maximal response noted at between 100 and 400 units/ml. GRP, but not DDC, mRNA was induced in H526 cells, with the maximal response between 4 and 100 units/ml. The dose response for both cell lines varied slightly among different cell passages. The onset of the TNF-alpha effect on GRP and DDC gene expression was noted within 5 min after TNF-alpha treatment for both cell lines (Figs. 3 and 4).


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Fig. 3.   Representative autoradiogram of Southern blot demonstrating time course of TNF-alpha effects on mRNA for gastrin-releasing peptide (GRP), aromatic-L-amino acid decarboxylase (DDC), and 18S ribosomal RNA in H82 cells. Autoradiogram is representative of at least 3 separate experiments.


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Fig. 4.   Representative autoradiogram of Southern blot demonstrating time course of TNF-alpha effects on mRNA for GRP and 18S ribosomal RNA in H526 cells. Autoradiogram is representative of at least 3 separate experiments.

The new production of neuroendocrine cell-associated proteins was demonstrated in TNF-alpha -treated H82 cells. Immunohistochemical analysis demonstrated the induction of NCAM (Fig. 5, A and B) and proGRP (data not shown) immunostaining in H82 cells treated with TNF-alpha for 2 wk. H526 cells did not manifest TNF-alpha -induced NCAM immunostaining but demonstrated induction of proGRP antigen after 2 wk of culture with TNF-alpha (data not shown). The immunostaining patterns corresponded to those expected on the basis of the known antigen locations, with cell-surface immunopositivity noted for NCAM and cytoplasmic staining noted for proGRP. Although induction of proGRP and NCAM consistently occurred in 2-wk TNF-alpha -exposed cultures, the intensity of immunostaining varied among different cell passages within the same immunohistochemical experiment, thereby suggesting variable responsiveness to the effects of TNF-alpha in the different cell passages. No significant immunostaining appeared in the negative controls with preimmune rabbit serum, preabsorbed NF-kappa B, or MOPC. Finally, dense-core neurosecretory-type granules, characteristic of differentiated neuroendocrine cells, were visible in H82 cells by electron microscopy after 14 days of TNF-alpha treatment (Fig. 6). Under our culture conditions and with our ultrastructural analyses, all untreated H82 cell passages were negative for dense-core granules.


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Fig. 5.   Immunostaining demonstrating induction of neuroendocrine cell marker neural cell adhesion molecule in H82 cell line. A: untreated H82 cells. B: H82 cells treated with TNF-alpha for 2 wk. Original magnification, ×800.


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Fig. 6.   Electron micrograph of TNF-alpha -exposed NCI-H82 cells. Arrow, representative dense-core neurosecretory granule. Original magnification, ×14,000.

NF-kappa B. The mRNAs for the two major components of NF-kappa B (p65 and p50) were present in both H82 and H526 cells (Fig. 7). The mRNAs for the p65 and p50 subunits of NF-kappa B did not change during the course of TNF-alpha exposure.


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Fig. 7.   Representative Southern blot demonstrating production of mRNA for nuclear factor-kappa B (NF-kappa B) components p65 and p50 throughout duration of TNF-alpha exposure in H82 and H526 cells. Autoradiogram is representative of at least 2 separate experiments.

In H82 cells, immunohistochemical analysis with 4',6-diamidino-2-phenylindole counterstaining demonstrated the presence of the p65 component of the NF-kappa B protein predominantly in the cytoplasm but not in the nuclei of untreated cells. After TNF-alpha exposure, the localization of the p65 immunopositivity was both cytoplasmic and nuclear, consistent with activation and nuclear translocation of NF-kappa B (Fig. 8).


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Fig. 8.   Immunohistochemistry demonstrating NF-kappa B translocation in TNF-alpha -exposed NCI-H82 cells. In all samples, 4',6-diamidino-2-phenylindole (DAPI) double staining was used to confirm nuclear location. A1: untreated H82 cells. A2: untreated H82 cells DAPI stained. B1: H82 cells exposed to 400 units/ml of TNF-alpha for 5 min. B2: H82 cells exposed to TNF-alpha for 5 min and DAPI stained. C1: H82 cells exposed to 400 units/ml of TNF-alpha for 60 min. C2: H82 cells exposed to TNF-alpha for 60 min and DAPI stained. D1: H82 cells exposed to 400 units/ml of TNF-alpha for 48 h. D2: H82 cells exposed to 400 units/ml of TNF-alpha for 48h and DAPI stained. E1: H82 cells not exposed to TNF-alpha after 48 h. E2: H82 cells not exposed to TNF-alpha after 48-h culture and DAPI stained. Original magnification, ×1,000.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study provides evidence that TNF-alpha can participate in pulmonary neuroendocrine cell differentiation. Increased TNF-alpha mRNA precedes the intense neuroendocrine cell hyperplasia in DEN-O2-treated hamsters by at least 2 wk. TNF-alpha can also trigger multiple indices of pulmonary neuroendocrine cell differentiation in human undifferentiated SCLC cell lines, including the induction of the cell-surface antigen NCAM and dense-core neurosecretory granules. These observations suggest that the neuroendocrine cell hyperplasia associated with a variety of chronic inflammatory lung conditions is due, in part, to the effects of endogenous TNF-alpha on undifferentiated neuroendocrine cell precursors.

To facilitate investigation of the molecular mechanisms of pulmonary neuroendocrine cell differentiation, we developed an in vitro system using two undifferentiated SCLC lines, H82 and H526, which grow as nonadherent loose clusters of cells in culture and are classified as "variant" SCLC cell lines (11, 20). Variant SCLC cell lines typically do not express immunoreactivity for either DDC or GRP, although other markers consistent with a neuroendocrine cell phenotype, such as neuron-specific enolase, may be present (11). The variant SCLC lines also frequently demonstrate features typical of extremely undifferentiated neuroendocrine cells, such as overexpression of c-myc mRNA (20). When we treated these cell lines with TNF-alpha , we observed a rapid induction of neuroendocrine cell-related gene expression; after 5-15 min of exposure to TNF-alpha , GRP and DDC mRNAs in H82 cells and GRP mRNA alone in H526 cells were detected.

However, the presence of mRNA for multiple cell lineages can occur in uncommitted precursor cells (24, 73). To confirm that a true phenotypic shift was being triggered by TNF-alpha , we examined the cultures after several days for evidence of changes in ultrastructural components and cell-surface antigens. We hypothesized that the induction of protein and/or structural characteristics of differentiated neuroendocrine cells would be consistent with a partial differentiation of these very undifferentiated cells. Immunohistochemical analyses demonstrated the induction of the neuroendocrine cell-related proteins NCAM and proGRP. TNF-alpha treatment of H82 cells led to a prominent induction of both cytoplasmic proGRP antigen (after 7 days) and the cell-surface antigen NCAM (after 14 days). Additionally, treatment of H82 cells with TNF-alpha for 2 wk resulted in the production of neurosecretory-type dense-core granules, which are characteristic of cells with neuronal or neuroendocrine differentiation. In contrast, TNF-alpha treatment of H526 cells resulted in induction of proGRP but not of NCAM immunostaining after 7 days. Thus H526 cells lacked DDC mRNA, NCAM immunoreactivity, and dense-core granules. These observations are consistent with previous studies (45, 57, 61, 65) indicating that GRP gene expression may be not entirely neuroendocrine cell specific.

Although the use of SCLC cell lines has the advantage of allowing the study of TNF-alpha effects on an isolated cell type, these cell lines are highly abnormal karyotypically, and phenotypic drift can occur (63, 69). This chromosomal instability and associated phenotypic drifting are characteristic of many lung cancer cells (63, 69) and can result in variations in responses to cytokines (63). In the present study, the variability in responses to TNF-alpha within the experimental treatment groups and among the cell passages was likely caused by subtle phenotypic variabilities among the cell line passages. However, the overall results of this study were not affected by this limitation because the trends in overall phenotypic features remained consistent despite some variability in individual culture responses. For example, baseline GRP and/or DDC gene expression occurred in a few cell passages, predominantly at high cell densities (>106 cells/ml) and/or were associated with spontaneous cell adhesion to the tissue culture plastic. However, even in cultures having detectable baseline production of GRP mRNA, we consistently observed increased GRP and/or DDC mRNA after exposure to TNF-alpha . Therefore, in all of the cultures, TNF-alpha rapidly induced or increased GRP and DDC mRNA.

Partial differentiation of variant (undifferentiated) SCLC cell lines has been previously shown to be induced by retinoic acid (17), fibronectin (33), and v-Ha-ras (41). Doyle et al. (17) demonstrated that H82 cells incubated with retinoic acid for 8-10 days developed a classic (well-differentiated) SCLC phenotype. This effect correlated with decreased c-myc mRNA (17). Increased calcitonin mRNA and peptide production were demonstrated in the SCLC cell line DMS 53 within 7 days after transfection with a v-Ha-ras retroviral construct (41). King et al. (33) found induction of GRP mRNA after exposing H82 cells to fibronectin for 24 h. Prior studies (22, 46) evaluating the effects of TNF-alpha on SCLC cell lines have not demonstrated any antiproliferative effects. However, the ability of TNF-alpha to induce differentiation appears to be separate from its ability to decrease cell proliferation or induce cytotoxicity (43, 53).

NF-kappa B probably mediates at least some of the effects of TNF-alpha on human SCLC cell lines in the present study. Nuclear translocation of p65 after TNF-alpha treatment of H82 cells was demonstrated by immunostaining, consistent with NF-kappa B activation. A recognition sequence for NF-kappa B is present in the human DDC promoter (64). Analysis of the 5'-flanking sequence for the GRP gene promoter region demonstrates a possible consensus NF-kappa B-binding site (48). Consistent with this hypothesis, Draoui et al. (18) found that treatment with phorbol 12-myristate 13-acetate, an activator of NF-kappa B, increased the mRNA for preproGRP in the classic SCLC cell line H209.

Although the roles of the pulmonary neuroendocrine cells in disease processes are still under investigation, it is feasible that they modulate injury responses. Neuroendocrine cell hyperplasia has been documented after various lung injuries (37, 39, 44, 51, 55, 59, 66, 67) and in association with several chronic pulmonary diseases (2, 3, 23, 32). Neuropeptide products of pulmonary neuroendocrine cells, including calcitonin gene-related peptide (CGRP) (71), GRP (74), and substance P (19), can modulate inflammatory responses. GRP is a chemoattractant for macrophages and lymphocytes, with some of these effects mediated by protein kinase C (15). CGRP is chemotactic for eosinophils in rat trachea (6). In human eosinophils from allergic subjects, both CGRP and substance P augment the chemotactic response to leukotriene B4 (50). Substance P has also been found to induce the release of TNF-alpha , interleukin-6, and interleukin-1 in cultured normal human monocytes (40). In addition, the neurokinin-1-receptor knockout mouse suggests a central role for substance P in mediating immune complex lung injury (9).

Further evidence of neuroendocrine cell involvement in the pulmonary immune response is provided by animal models. A guinea pig model of allergic pulmonary inflammation identified neuroendocrine cell hyperplasia 3 wk after systemic intraperitoneal immunization with ovalbumin (8). When these animals were challenged with an aerosol of the same antigen, there was a significant decrease in the number of neuroendocrine cells, consistent with degranulation; these changes persisted for at least 6 days (8). Similarly, a guinea pig model of anaphylaxis demonstrated both pulmonary neuroendocrine cell hyperplasia 3 wk after foot pad ovalbumin immunization and evidence of bronchiolar neuroendocrine cell degranulation after intracardiac ovalbumin injection (42). These studies suggest that neuropeptides released by neuroendocrine cells might modulate pulmonary inflammatory responses.

The effects of TNF-alpha on variant SCLC cell lines suggest that similar differentiation effects might occur with normal pulmonary neuroendocrine cells exposed to TNF-alpha . It appears likely that TNF-alpha produced in hamster lungs after ~6 wk of DEN-O2 exposure might contribute to the molecular mechanisms of pulmonary neuroendocrine cell differentiation and hyperplasia after 8-12 wk of DEN-O2 exposure. Similarly, TNF-alpha or other cytokines elaborated in chronic inflammatory pulmonary conditions may induce latent neuroendocrine cells or neuroendocrine precursor cells in the human lung. Thus the effects of TNF-alpha on neuroendocrine cells may be one critical link between chronic pulmonary inflammation and pulmonary neuroendocrine cell hyperplasia.

    ACKNOWLEDGEMENTS

We thank Dr. Jeffrey M. Drazen for advice in the preparation of this manuscript.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-44984 (to M. E. Sunday).

Address for reprint requests: M. E. Sunday, Pathology Dept., Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.

Received 15 October 1997; accepted in final form 14 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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