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
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ABSTRACT |
We studied tumor necrosis factor (TNF)-
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-
preceding neuroendocrine cell differentiation. Undifferentiated
small cell lung carcinoma (SCLC) cell lines NCI-H82 and NCI-H526 were
treated with TNF-
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-
was added. Nuclear translocation of nuclear factor-
B immunostaining
occurred with TNF-
treatment, suggesting nuclear factor-
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-
treatment. We conclude that TNF-
can induce phenotypic features of
neuroendocrine cell differentiation in SCLC cell lines. Similar effects
of TNF-
in vivo may contribute to the neuroendocrine cell
differentiation/hyperplasia associated with many chronic inflammatory
pulmonary diseases.
nuclear factor-
B; cell differentiation; gastrin-releasing
peptide; aromatic-L-amino acid
decarboxylase; neural cell adhesion molecule
 |
INTRODUCTION |
TUMOR NECROSIS FACTOR (TNF)-
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-
(49), known to be upregulated by
inflammatory cytokines, particularly TNF-
, were identified.
Nitrosamines (58) and hyperoxia (26, 31) have each been associated with
the production of several cytokines, including TNF-
,
interleukin-1
, 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-
and the appearance of neuroendocrine cell differentiation led
to the hypothesis that neuroendocrine cell differentiation is induced
by inflammatory cytokines, specifically TNF-
, produced as part of
the host response to DEN-O2
injury. TNF-
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-
triggers differentiation in
normal cells such as B cells (30), thymocytes (77), macrophages (75), and dendritic cells (56). In addition, TNF-
can induce type II
pneumocyte differentiation in murine embryonic lung bud cultures (29).
Furthermore, TNF-
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-
gene expression. Next, we developed an in vitro model
to study the possible role of TNF-
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-
responses, focusing on nuclear factor-
B (NF-
B), which has been implicated in molecular mechanisms promoting
differentiation of other cell types (25).
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MATERIALS AND METHODS |
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-
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-
. 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-
treatment (COLO 320) or demonstrated baseline GRP mRNA
levels without evidence of further induction after TNF-
treatment
(H209, OC2, T723, H345, H69, and H835). For the time-course studies, we
used the optimal doses of TNF-
: 400 units/ml for NCI-H82 cells and 4 units/ml for NCI-H526 cells. Cultures were harvested before TNF-
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-
(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.
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-
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-
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-
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-
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 |
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-
is also expressed, with peak levels after 8 wk of
nitrosamine-O2 (Table 2). Cell
surface expression of CD18-
has been previously demonstrated to
increase after exposure to TNF-
(76). In addition, CD18 modulates
many of the effects of TNF-
, such as cell spreading and decreases in
cAMP, that precede the respiratory burst in neutrophils (49).
-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-
was selected as a candidate cytokine for testing in vitro
because it is known to modulate expression of both Ii and CD18-
.

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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.
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TNF-
production in hamster injury
model. With the use of RT-PCR analysis, TNF-
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-
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-
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-
mRNA after 6 wk of DEN-O2
treatment. Lung tissue from 4 of 16 untreated adult hamsters contained
TNF-
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)- gene expression in mRNA from DEN- and
DEN-O2-exposed hamsters but not in
healthy age-matched control animals. Maximal TNF- gene expression
occurs after 6 wk of nitrosamine exposure, ~2 wk before onset of
neuroendocrine cell hyperplasia.
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TNF-
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-
. 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-
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-
exposure. Although the induction of GRP and DDC mRNA was very
consistent, the timing and individual culture responses to TNF-
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-
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-
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-
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-
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-
effect on GRP and DDC gene expression was noted
within 5 min after TNF-
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- 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- effects on mRNA for GRP and 18S ribosomal RNA in H526 cells.
Autoradiogram is representative of at least 3 separate experiments.
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The new production of neuroendocrine cell-associated proteins was
demonstrated in TNF-
-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-
for 2 wk. H526 cells
did not manifest TNF-
-induced NCAM immunostaining but demonstrated
induction of proGRP antigen after 2 wk of culture with TNF-
(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-
-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-
in the different cell passages. No significant
immunostaining appeared in the negative controls with preimmune rabbit
serum, preabsorbed NF-
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-
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- for
2 wk. Original magnification, ×800.
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Fig. 6.
Electron micrograph of TNF- -exposed NCI-H82 cells. Arrow,
representative dense-core neurosecretory granule. Original
magnification, ×14,000.
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NF-
B. The mRNAs for
the two major components of NF-
B (p65 and p50) were present in both
H82 and H526 cells (Fig. 7). The mRNAs for
the p65 and p50 subunits of NF-
B did not change during the course of
TNF-
exposure.

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Fig. 7.
Representative Southern blot demonstrating production of mRNA for
nuclear factor- B (NF- B) components p65 and p50 throughout
duration of TNF- exposure in H82 and H526 cells. Autoradiogram is
representative of at least 2 separate experiments.
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In H82 cells, immunohistochemical analysis with
4',6-diamidino-2-phenylindole counterstaining demonstrated the
presence of the p65 component of the NF-
B protein predominantly in
the cytoplasm but not in the nuclei of untreated cells. After TNF-
exposure, the localization of the p65 immunopositivity was both
cytoplasmic and nuclear, consistent with activation and nuclear
translocation of NF-
B (Fig. 8).

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Fig. 8.
Immunohistochemistry demonstrating NF- B translocation in
TNF- -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- for 5 min.
B2: H82 cells exposed to TNF- for 5 min and DAPI stained. C1: H82 cells
exposed to 400 units/ml of TNF- for 60 min.
C2: H82 cells exposed to TNF- for
60 min and DAPI stained. D1: H82 cells
exposed to 400 units/ml of TNF- for 48 h. D2: H82 cells
exposed to 400 units/ml of TNF- for 48h and DAPI stained.
E1: H82 cells not exposed to TNF-
after 48 h. E2: H82 cells not exposed
to TNF- after 48-h culture and DAPI stained. Original magnification,
×1,000.
|
|
 |
DISCUSSION |
The present study provides evidence that TNF-
can participate in
pulmonary neuroendocrine cell differentiation. Increased TNF-
mRNA
precedes the intense neuroendocrine cell hyperplasia in
DEN-O2-treated hamsters by at
least 2 wk. TNF-
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-
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-
, we observed a rapid induction of neuroendocrine cell-related gene expression; after 5-15 min of exposure to TNF-
, 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-
, 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-
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-
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-
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-
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-
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-
. Therefore, in all of
the cultures, TNF-
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-
on SCLC cell lines have not
demonstrated any antiproliferative effects. However, the ability of
TNF-
to induce differentiation appears to be separate from its
ability to decrease cell proliferation or induce cytotoxicity (43, 53).
NF-
B probably mediates at least some of the effects of TNF-
on
human SCLC cell lines in the present study. Nuclear translocation of
p65 after TNF-
treatment of H82 cells was demonstrated by immunostaining, consistent with NF-
B activation. A recognition sequence for NF-
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-
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-
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-
, 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-
on variant SCLC cell lines suggest that similar
differentiation effects might occur with normal pulmonary neuroendocrine cells exposed to TNF-
. It appears likely that TNF-
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-
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-
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.
 |
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