Download Variable Expression of Cytokines in Human

ICANCER RESEARCH 58. 3132-3141.
July 15. 1998]
Variable Expression of Cytokines in Human Head and Neck Squamous Cell
Carcinoma Cell Lines and Consistent Expression in Surgical Specimens1
Rendra V. Woods, Adel El-Naggar, Gary L. dayman,
and Elizabeth A. Grimm2
Departments of Tumor Biology IK. V. W., E. A. G.j, Head and Neck Surgery ¡K.V. W., G. L. C.], Pathology [A. E-N.I, and General Surgery [E. A. Gì,University of Texas M. D.
Anderson Cancer Center. Houston. Texas 77030
ABSTRACT
Head and neck squamous cell carcinomas (HNSCCs) are associated
with abnormal cell-mediated immunity at the primary tumor site. We
investigated tumor-derived cytokines as factors underlying such abnor
malities. Cytokine mRNA and protein of eight HNSCC-derived cell lines
were tested,- reverse transcription-PCR results indicated the presence of
mRNAs for interleukin la (IL-la) and transforming growth factor a (8 of
8); transforming growth factor ßand IL-lß(7 of 8); and 11-4 and IL-6 (4
of 8). IL-2, IFN-y, and tumor necrosis factor a mRNA were not detected.
Supernatants from six of these cell lines were analyzed by ELISA; IL-la,
IL-Iß,and IL-6 were found to be markedly increased compared to human
papillomavirus-16-immortalized
human oral keratinocytes. To determine
whether the cell line findings are applicable to primary tumors, we
performed immunohistochemical
analysis on tumor specimens from 12
patients with invasive HNSCC. Universal intracellular production of
IL-la, IL-l/i, and IL-6 protein was detected. We conclude that the
aberrant elaboration of biologically active IL-1 and IL-6 may contribute
lines from primary and metastatic sites; and (b) to establish whether
the IL-1 and IL-6 found in HNSCC cell lines were also present in
primary tumors.
MATERIALS
AND METHODS
Cell Lines and Culture Conditions
All cell lines used in this study are shown in Table 1. Squamous carcinoma
cell lines were grown in 1:1 DMEM-F12 culture medium containing 10% PCS
and penicillin/streptomycin.
Normal oral keratinocytes obtained by surgical
resection and HOK 16B cells were cultured in keratinocyte growth medium
(containing 0.15 mM calcium; Clonetics, San Diego, CA). Cells were incubated
in 5% CO, in air at 37°C.Stock cultures were grown to 85% confluency and
were subcultured with disruption of adherence by a 0.05% trypsin, 0.5%
EDTA solution. MDA Tu 686, MDA 686LN, MDA Tu 1186, MDA Tu 1483,
and MDA Tu 1586 (16) were kindly provided by Dr. Peter Sacks (Memorial
Sloan-Kettering Hospital, New York, NY); MDA Tu-138 and MDA Tu 177
to altered immune status in HNSCC patients.
INTRODUCTION
HNSCC3 will afflict approximately
microenviroment of primary invasive HNSCC. The specific aims
were 2-fold: (a) to determine whether IL-la, IL-Iß,IL-2, IL-4, IL-6,
IFN-y, TGF-a, TGF-ß,and/or TNF-a were expressed by HNSCC cell
67,000 people in the United
States this year (1). Despite advances in diagnosis and management,
survival rates for patients with head and neck cancer have remained
relatively unchanged for the past 30 years (2). Patients with HNSCC
manifest significant immunosuppression that is often more profound
than that in patients with other malignancies (3). Immunosuppression
in HNSCC has been postulated to occur in a hierarchical manner,
where the primary tumor region is the most affected site, followed by
draining lymph nodes (4). The level of immunosuppression in patients
who have HNSCC varies widely. Known etiological risk factors that
may contribute to this phenomenon include tobacco, alcohol
(reviewed in Ref. 5), and HPV infection (6).
Cytokines are soluble, low molecular weight, multifunctional
polypeptides that mediate cellular communication and influence many
aspects of cellular growth, differentiation, and function (7). These
fundamental biological responses are dysregulated in cancer, as immunoreactive cytokine expression has been reported in a variety of
solid tumor systems, including melanoma, ovarian carcinoma, inva
sive breast cancer, colorectal cancer, soft-tissue sarcomas, and SCC
(8-15).
The purpose of this investigation was to assess the cytokines in the
(17) cells, as well as cultured normal oral keratinocytes, were provided by Dr.
Gary dayman (University of Texas M. D. Anderson Cancer Center, Houston.
TX); UMSCC-1 (18) cells were provided by Dr. Thomas Carey (University of
Michigan, Ann Arbor, MI); and HOK 16B. a human oral keratinocyte cell line
(19), was provided by by Dr. N-H. Park (University of California at Los
Angeles, Los Angeles, CA).
Human Subjects
Twelve patients with untreated invasive HNSCC of the oral cavity and
oropharynx who presented to the Department of Head and Neck Surgery at the
University of Texas M. D. Anderson Cancer Center between 1982 and 1985
were included in this study. Patient samples were provided for research after
Institutional Review Board approval for use of incidental samples. Archival
tissue specimens were acquired from the Department of Pathology. Medical
records of each patient were reviewed for demographic and clinicopathological
information (Table 2). One patient was a former smoker, and 11 were current
smokers: 1 had no history of alcohol consumption, 2 were self-defined social
drinkers, and 8 were self-defined heavy drinkers (no data were provided
regarding alcohol consumption
for one patient).
Extraction of RNA
Tissue-cultured cells were trypsinized and washed once in PBS, and then
3-6 X IO6 cells were pelleted by centrifugation at 7000 rpm in a microcen
trifuge. The pellets were frozen at —¿
70°Cfor no longer than 72 h until RNA
Received 6/24/97; accepted 5/18/98.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by National Cancer Institute. NIH Grants ROI CA45225
could be extracted. TRIREAGENT (Molecular Research Center, Inc., Cincin
nati. OH), a commercially available reagent, was used to extract total RNA
from the cell pellets according to the manufacturer's instructions. Briefly,
and ROÕ CA64906 (to E. A. G.) and CA16672 (to the University of Texas M. D.
Anderson Cancer Center) and through the generosity of a fellowship from Drs. G. L.
dayman and Helmuth Goepfert in the Department of Head and Neck Surgery at the
University of Texas M. D. Anderson Cancer Center (to K. V. W.).
2 To whom requests for reprints should be addressed, at the Department of Tumor
lowed by precipitation of RNA with isopropanol. The RNA was then washed
with 75% ethanol and subsequently solubilized in diethyl pyrocarbonatetreated water. The RNA was finally resuspended in 24 ^1 of diethyl pyrocarbonate-treated water and frozen at —¿
70°C.
Biology, Box 79. University of Texas M. D. Anderson Cancer Center. 1515 Holcombe
Boulevard, Houston, Texas 77030.
' The abbreviations used are: HNSCC. head and neck squamous cell carcinoma; SCC.
PCR Primers
squamous cell carcinoma; HPV. human papillomavirus; IL, interleukin; TGF. transform
ing growth factor; TNF. tumor necrosis factor; DMEM-F12. DMEM-Ham's F-12;
G3PDH. glyceraldehyde-3-phosphate
dehydrogenase; RT-PCR. reverse transcriptionPCR; NF. nuclear factor.
Oligonucleotide primers for amplification of the cytokines were purchased
from Clontech Laboratories (Palo Alto, CA). Each of these Oligonucleotide
primer pairs was constructed to span an intron to distinguish cDNA transcribed
homogenization
of the cells occurred by phenol-chloroform
extraction,
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fol
VARIABLE
CYTOKINE
EXPRESSION
IN HUMAN HNSCC
RT-PCR
Table l HNSCC cell lines
After mRNA was extracted from the cell lines, the protocol for gene
amplification was accomplished in four steps: (a) reverse transcription of
mRNA; (b) establishment of proper experimental controls (optimizing the
amplification cycling to simultaneously detect each of the nine cytokines
involved varying the concentration of Mg2+, reaction buffer, and oligonucleo
nudemiceYesYes"Not
originFloor
of
lineUMSCC-1MDATu
Cell
mouthLower
of
welldifferentiatedWell
138MDATu
177M
686MDA
DA Tu
686LNMDATu
lipLarynxBase
differentiatedModerately
testedYesYesYesYesYesin differentiatedPoorly
tongueMetastasis
of
differentiatedPoorly
baseof from
differentiatedModerately
tongueSupraglottic
larynxRetromolar
differentiatedWell
1186MDA
differentiatedWell
1483MDATu
Tu
trigoneLarynxGradeModerately
differentiatedTumorigenicin
1586a
TumorigenicSite
severe combined immunodeficient mice.
Table 2 Characteristics
Age
Sex (male:female)
Race (African American:Caucasian)
Anatomical site of primary tumor
Oral tongue
Base of tongue
Floor of mouth
Palate
Histológica! grade of primary tumor
Moderately differentiated
Well differentiated
Metastatic to neck at diagnosis (yes:no)
TNM stage
T2N2bM0
tide primers); (c) amplification of the template with a portion of the G3PDH
gene to verify mRNA integrity; and (d) amplification of cell line cDNA for
cytokine expression. A reaction without any cDNA template and one in which
mRNA without reverse transcriptase were included as a negative controls.
Positive-control cDNAs corresponding to each cytokine (Clontech, Palo Alto,
CA) were amplified to confirm primer specificity. Amplification of mRNA
with primers to G3PDH and pAW109 (IL-la) RNA (supplied in the RNA PCR
kit by Perkin-Elmer Corp., Foster City, CA) also served as positive controls for
each experiment. RT-PCR was used according to a published protocol by
Brenner et al. (20). Briefly, total RNA was subjected to RT-PCR using a
Perkin-Elmer RT-PCR RNA Amplification kit. Six of p.1 total cellular RNA
were added to 34 /¿Ireverse transcription buffer containing 2.5 /mini of
random hexamers, 1 mmol/liter dNTPs (dATP, dCTP, dGTP, and dTTP), 2.5
units of MuLV reverse transcriptase (Perkin-Elmer), and 40 units of RNase
inhibitor. After initial incubation at 22°Cfor 10 min, followed by 45 min at
42°Cand 9 min at 98°Cin a Perkin-Elmer 9600 thermocycler, a 4.6-^.1 sample
of study population
40-74 yr (median. 58.4 yr)
7:5
2:10
3
5
3
1
was suspended in 24.4 jil of PCR buffer containing l /J.Meach primer, 10 HIM
Tris-HCl (pH 8.3), 50 mM KC1, 1.5 ITIMMgCU, dNTPs at a final concentration
of 200 ßM
each, and Taq polymerase at 0.8 units//xl. After an initial denaluration at 94°Cfor 5 min, 35 cycles of amplification (denaturation at 94°Cfor
15 s, annealing at 60°Cfor 12 s, and extension at 72°Cfor 90 s) were followed
by a terminal extension at 72°Cfor 7 min. Amplification was performed in the
9
3
6:6
1
4
Perkin-Elmer 9600 thermocycler.
The high sensitivity of PCR requires rigorous adherence to anticontamination precautions in the preparation of reagents and reactions. Positive displace
ment pipettes (Rainin. Wobum. MA) were used for all pipetting procedures.
Prior to the addition of template DNA, the PCR mixture was placed on a
transilluminator and irradiated with UV light for 10 min to denature any
contaminating double-stranded DNA. Reaction preparation and amplification
T4N,M0
from mRNA and DNA templates. Although these primers bind to both DNA
and RNA sites, the sizes of the bands differ. Our bands correspond to the
expected sizes of products amplified from transcribed mRNA not the larger
DNA (see Table 3). All oligonucleotide primers were tested, aliquoted. and
stored frozen at —¿20°C
according to the manufacturer's instructions. The
were performed in separate rooms. All experiments were performed in dupli
cate using both positive and negative controls as detailed above. Manipulation,
analysis, and storage of amplified products was isolated in a separate labora
tory using different equipment than that used in reagent preparation and
amplification.
primers are shown in Table 3. Amplification primers used as internal controls
to ensure the presence of intact human cellular RNA were designed to amplify
an approximately 213-bp fragment of the G3PDH gene, a gene that is present
Analysis of Amplified DNA
in every human nucleated cell.
through 4% agarose gels (3% NuSieve agarose; 1% SeaKem agarose; FMC
Fifteen ^.1 of each PCR-amplified
product was separated by electrophoresis
Table 3 PCR primers
G3PDH gene primers
Positive-strand primer
sense, 5'-ACGGATTTGGTCGTATTGGG-3'
Negative-strand primer
antisense 5'-TGATTTTGGAGGGATCTCGC-3'
Predicted size of G3PDH PCR products, 213 bp
Cytokine primers
Positive-strand primer
IL-la, 5'-ATGGCCAAAGTTCCAGACATGTTTG-3'
IL-10, 5'-ATGGCAGAAGTACCTAAGCTCGC-3'
IL-2. 5'-ATGTACAGGATGCAACTCCTGTCTT-3'
IL-4, 5'-ATGGGTCTCACCTCCCAACTGCT-3'
IL-6. 5'-ATGAACTCCTTCTCCACAAGCGC-3'
IFN--V,5'-ATGAAATATACAAGTTATATCTTGGCTTT-3'
TGF-a. 5'-ATGGTCCCCTCGGCTGGACAG-3'
TGF-ß,5'-GCCCTGGACACCAACTATTGCT- 3'
TNF-a, 5'-ATGAGCACTGAAAGCATGATCCGG- 3'
Negative-strand primer
5'-GTTTTCCAGTATCTGAAAGTCAGT-3
'
S'-ACACAAATTGCATGGTGAAGTCAGTT- 3'
5'-GTCAGTGTTGAGATGATGCTTTGAC-3
'
5'-CGAACACTTTGAATATTTCTCTCTCAT-3'
5'-GAAGAGCCCTCAGGCTGGACTG-3'
5'-GATGCTCTTCGACCTCGAAACAGCAT- 3'
5'-GGCCTGCTTCTTCTGGCTGGCA- 3'
5'-AGGCTCCAAATGTAGGGGCAGG-3 '
5'-GCAATGATCCCAAAGTAGACCTGCCC-3
'
Predicted size of cytokine PCR products (bp)
IL-la, 808
IL-Iß,802
IL-2, 458
IL-4, 456
IL-6, 628
IFN-y. 494
TGF-a, 297
TGF-ß,161
TNF-a, 695
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VARIABLE
CYTOKINE
EXPRESSION
IN HUMAN HNSCC
cell number. The supernatants were aliquoted and frozen at —¿
70°C.On the day
2 "o è "2
"e
CO.
Q
of the assay, the frozen supernatants were allowed to thaw at room tempera
ture, subjected to a pulse spin in a microcentrifuge, and then immediately
assayed in triplicate for IL-la, IL-Iß.and IL-6 according to the manufactur
er's instructions. Briefly, the supernatants were incubated in the cytokine-
O
lìs2èIÌ333Ìg|ÉÌ
specific antibody-coated
well of the supplied microtiter plate. A second distinct
anticytokine antibody conjugated to alkaline phosphatase was added to each
well. This was followed by the addition of the chromogenic substance paranitrophenyl phosphate. After 30 min, a reversible inhibitor of acetylcholines-
603
terase activity was added as a stop solution, and the absorbance at 450 nm
(reference filter 570 nm) was immediately determined by spectrophotometry
(ELISA reader, Dynatech, Chantilly, VA). The concentration of the cytokine
was determined from a standard curve. The sensitivity of the specific ELISAs
was as follows: IL-la, <2 pg/ml; IL-1/3. <1 pg/ml; and IL-6. <4 pg/ml.
Immunohistochemistry
Fig. 1. Establishment
ot' optimal conditions
for cytokine mRNA amplification
by
RT-PCR. Simultaneous PCR amplification of control cDNAs corresponding to each
cylokine was performed. Fifteen fil of PCR product were loaded onto a 4% agarose gel
and resolved by electrophoresis. The DNA was visualized by staining with ethidium
bromide. Controls: second lane. Perkin-Elmer DNA kit positive control (A template)
amplified with kit primers: lliirtl lane, water instead of nucleic acid template amplified
with G3PDH primers: fourth lane. Perkin-Elmer RNA PCR kit control amplified with kit
primers (DM 151 and DM 152) after reverse transcription; fifth Itine. Perkin-Elmer RNA
PCR kit control without reverse Iranscriptase amplified with kit primers (DM 151 and DM
152).
X
O
2
e
cc
Antibodies. The following manufacturer-supplied monoclonal mouse antihuman antibodies were used in dilutions for all studies: anti-IL-la (IgGl K;
Oncogene Science, Uniondale. NY). anti-IL-lß (IgGl; Genzyme, Cambridge,
603
ee
603
bp~
Fig. 2. Analysis of cytokine mRNA detection in cultured normal oral keratinocytes for
constitutive cytokine expression. Cells were incubated in serum-free medium for 16 h
prior to RNA extraction. Reverse transcription was followed by simultaneous amplifica
tion of cytokine cDNA. Fifteen /il of PCR product were loaded onto a 4% agarose gel and
resolved by electrophoresis. The DNA was visualized by staining with ethidium bromide.
—¿
Control, cultured normal oral keratinocyte mRNA amplified with G3PDH without
reverse transcriptase.
Fig. 3. Analysis of cytokine mRNA detection in MDA Tu 177 by RT-PCR for
constitutive cylokine expression. Cell lines were incubated in serum-free medium tor 16 h
prior to RNA extraction. Reverse transcription was followed by simultaneous amplifica
tion of cytokine cDNA. Fifteen fil of PCR product were loaded onto a 4% agarose gel and
resolved by electrophoresis. The DNA was visualized by staining with ethidium bromide.
+ Control, Perkin-Elmer RNA kit positive control RNA amplified with kit primers (DM
151 and DM 152) after reverse transcription; - Control, autoclaved water in place of
nucleic acid template amplified with G3PDH primers.
500
400
Corp., Rockland, ME). The PCR products were made visible by staining with
ethidium bromide and exposure to UV light (254 nm) on a transilluminator.
The molecular weights of the amplification products were determined by
comparison with either Hindi or //udii restriction fragments derived from
bacteriophage <f>x174 DNA (United States Biochemical Corp, Cleveland, OH).
Measurement
-
300
~5t
5
200
of Secreted Cytokines (ELISAs)
100
Secreted cytokine (IL-la, IL-Iß,and IL-6) concentration was determined
by ELISA (AMAC Inc., Westbrook. ME) using the manufacturer's protocol.
Cell lines were counted and plated at equal concentrations into culture flasks
in DMEM-F12 medium with the addition of 10% PCS and penicillin/strepto
HOK16B
mycin. Cells were cultured to approximately 75% confluency and then washed
three times with HBSS. Serum-free DMEM/F12 or serum-free DMEM/F12
plus a specified concentration of recombinant cytokine (IL-la, IL-Iß,or IL-6)
was added as indicated in figure legends, and the cells were further incubated
for 72 h. Supernatants were collected, cells were trypsinized and counted, and
the total number per flask was recorded to standardize cytokine production for
i
UMSCC-1
TU 138
6861,N
1186
USI
1586
Fig. 4. Maximum cytokine secretion by HNSCC cell lines. Supernatants were collected
following a 72-h incubation in serum-free medium alone. The maximum production of
II-la, IL-1 j3, and IL-6 is shown for each of the HNSCC cell lines compared to HOK 16B.
All samples were analyzed in triplicate by ELISA, and SDs are shown. •¿.
mean was
statistically significantly different from the control value (HOK I6B) by Tukey's honestly
significant multiple comparison test (P < 0.05) after first determining that differences
existed among means by a one-way ANOVA (P < 0.05).
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VARIABLE
CYTOKINF
EXPRESSION
Table 4 Cytokine gene expression in HNSCC cell lines"
Cell line
IL- la
IL-lß
1L-4
IL-6
TGF-a
IN HUMAN HNSCC
min, rinsed once in distilled water, placed in PBS for 5 min. and then stained
immediately.
Staining Procedure. To quench endogenous peroxidases, the slides were
immersed in a 3% H2O2 solution in methanol for 15 min at 4°Cand air dried
TGF-0
UMSCC-1
MDA Tu 138
MDA Tu 177
MDA Tu 686
MDA 686LN
MDA 1186
MDA 1483
MDA 1586
" IL-2, INF-y. and TNF-a mRNAs were not detected in these cell lines.
for 4-5
min. The ingredients
in an avidin-biotin-peroxidase
complex
kit
(Vectastain. Vector Laboratories. Burlingame, CA) were used to complete the
staining procedure previously reported by us (9, 21). (The slides were evalu
ated independently by two of the authors (A. E-N. and K. V. W.) for confir
mation of positivity.)
Statistical Analysis. All data are presented as means ±SD. An ANOVA
was performed with Dunnett's ; test whenever two samples were compared. An
MA), and anti-IL-6 (IgGl; Genzyme. Cambridge, MA). A mouse monoclonal
antibody to cytokeratin (AE1/AE3. Boehringer Mannheim. Indianapolis, IN)
was used as a positive control for squamous epithelium. MOPC-21, a purified
mouse IgGl K immunoglobulin. and UPC-10, a clarified mouse IgG2 K
immunoglobulin (Sigma Chemical Co., St. Louis, MO), were used as nonspe
cific IgG-negative controls.
Purified Protein. Purified recombinant human IL-la. IL-Iß,and IL-6.
generously provided by Immunex Research and Development Corp. (Seattle,
WA), were used in antibody preadsorption studies for analysis of nonspecific
staining. A 10:1 molar excess of purified recombinant cytokine was incubated
with each specific monoclonal antibody for 2 h at 37°C.The antigen/antibody
ANOVA appropriate to multisample hypothesis testing was performed with
the multiple comparison ANOVA. Statistical significance was defined as
P < 0.05. When statistical significance was determined for a multiple com
parison, Tukey's honestly significant difference test was performed to ascer
tain which samples were different. Statistical analysis was performed using
SPSS Statistical Package Version 6.0 for Windows on an IBM personal
computer.
RESULTS
mixtures were centrifuged in a microcentrifuge for 3 min at 10,000 X g and
then used in immunohistochemical staining.
Tissue Preparation. All immunohistochemistry was performed on formalinfixed, paraffin-embedded specimens. The cell lines were also formalin-fixed
and paraffin-embedded as pellets prior to immunohistochemical
staining, as
reported by Tyler et al. (21 ).
Antigen Retrieval. To enhance the immunohistochemical staining, the
slides were subjected to antigen retrieval using a dilute lead thiocyanate
solution (BioGenex Laboratories. San Ramon. CA) according to the manufac
turer's specifications. Following antigen retrieval, the slides were cooled for 15
Establishment of Optimal Conditions for Cytokine mRNA
Amplification. Fig. 1 shows the result of optimizing the PCR proto
col to coamplify of all nine cytokines in a single PCR experiment. The
presence of a sharp single band, at the expected molecular weight for
each of the cytokines, supports optimal amplification while maintain
ing appropriate controls. Optimization experiments were performed
using cDNA-positive controls. Rigorous controls, including two dif
ferent sets of positive (amplification of Perkin-Elmer kit control
pAW109 and G3PDH) and negative controls (double-autoclaved wa
ter substituted for nucleic acid template and mRNA not subjected to
UMSCC-1
*
1
A
ANTI IL-1a
T,*?*
**2
:
ANTI IL-1a + IL-1a
ANTI IL-1a + IL-1B
Fig. 5. Immunohistochemical staining of paraffin-cm bedded, formalin-fixed pellet of UMSCC-l with anti-IL-la following preadsorption with purified rIL-la or rIL-lß.A, staining
of the positive cytokeratin; B. staining with the negati ve-control mouse myeloma protein UPC-IO; C. staining of tumor cells with anti-IL-la; D, at a molar ratio of 10:1 of antigen
(rIL-la) to antibody (anti-IL-la). all of the antibody had been complexed to antigen and was therefore unable to stain the cells; E, specificity of the antibody because anti-IL-la staining
is not abrogated by preadsorption with rIL-1/3.
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VARIABLE
CYTOKINE
EXPRESSION
IN HUMAN HNSCC
Fig. 6. Immunohistochemical staining of Tu 138 showing constitutive intracellular production of IL-lor, IL-10. and IL-6. A. anlicytokcralin (positive control); B. UPC-10 (negative
control); C, anti-IL-la; D, anti-IL-lß;£,anti-IL-6.
reverse transcription) were run simultaneously with each of the nine
cytokines (as seen in the control lanes of Fig. 1).
Summary of Cytokine mRNA in Cultured Normal Oral Keratinocytes. Fig. 2 shows the amplification of mRNA from cultured
normal oral keratinocytes. TGF-a is the only cytokine of the nine
expressed from these cells (positive and negative controls are shown
in the second and third lanes, respectively).
Summary of Cytokine mRNA in HNSCC Cell Lines. Fig. 3
shows the amplification of MDA Tu 177 as a SCC example. This cell
line expressed mRNA for IL-la, IL-Iß,TGF-a, and TGF-ß.Positive
and negative controls are shown in the second and third lanes, respec
tively. The cDNA from each cell line was amplified at least three
different times. A summary of all cytokine expression as detected by
RT-PCR is compiled in Table 4 from each HNSCC cell line tested. All
cell lines expressed IL-la and TGF-a; seven cell lines expressed
IL-Ißand TGF-ß;four expressed IL-4 and IL-6. IL-2, IFN--y, and
TNF-a mRNA were not detected by any cell line tested.
IL-la, IL-Iß, and IL-6 Protein Secretion. ELISAs were per
formed to determine the relative levels of secreted IL-la, IL-Iß,and
IL-6 present in the supernatants obtained from six HNSCC cell lines
during 72 h of incubation in serum-free medium as compared to a
Table 5 Summary of cytokine elaboration
IL-1/3P"
I+
E
I
P
E
I+
UMSCC-1MDA
++
+
+
+
±
++
++
+
+
+
+
++
138MDA
Tu
++
+
+
+
+
++
686LNMDA
+
NT
+
±
NTIL-6P
Tu 1483IL-la
' P. PCR; E. ELISA; 1, ¡mmunohistochemistry;NT. not tested.
E
+
+
+
+
NT
HPV-16-immortalized oral keratinocyte cell line. The results of these
ELISAs are shown in Fig. 4; MDA Tu 138 is the highest secretor of
each cytokine (440.8 pg/ml of IL-la, 384.5 pg/ml of IL-6, and 27.1
pg/ml of IL-Iß).IL-1 o was secreted in the greatest quantity in all cell
lines, followed by IL-6, then followed by IL-1 ß.The concentration of
cytokines secreted and measured by ELISA were all within the range
of the cytokine standards provided in the commercial kits; therefore,
no serial dilution of supernatants was necessary.
Intracellular IL-la, II,-Iß,and IL-6. The number of cell lines
tested for intracellular cytokines was limited to the three that were
derived from tumors within the oral cavity, UMSCC-1, MDA TU 138,
and MDA 686LN, because their common location might suggest a
more homogeneous natural history and because we had available
control tissue that did not express these cytokines. Immunohistochem
ical staining was chosen to develop a cell-line model that would allow
us to test specifically whether the tumor cells themselves, rather than
tumor-infiltrating lymphocytes, were the source of the cytokines in
vivo. To control for potential nonspecific binding, antigen-adsorption
experiments were performed on all antibodies to establish the speci
ficity of each antibody. Purified recombinant antigen was incubated in
molar excess with each antibody prior to staining paraffin-embedded
formalin-fixed cell pellets of the cell lines. Purified recombinant
IL-la abolished the staining of tumor cells when preadsorbed to
anti-IL-la as demonstrated in Fig. 5 for UMSCC-1 as a representative
HNSCC cell line. Antigen preadsorption experiments were also per
formed and demonstrate the specificity of monoclonal antibodies
anti-IL-lß and anti-IL-6 (data not shown). The paraffin-embedded
tissue sections of all three paraffin-embedded cell line sections,
UMSCC-1, MDA Tu-138, and MDA 686LN, each stained positively
for IL-1 a, IL-1 ß,and IL-6, in contrast to the negative control TBS or
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VARIABLE
CYTOKINE
EXPRESSION
IN HUMAN HNSCC
Fig. 7. Immunohislochemical
staining of a primary oral tongue
tumor from a patient with negative-control MOPC-21 protein.
,
UPC-10. The data for MDA Tu 138 as a representative cell line are
shown in Fig. 6.
Summary of Cytokine Detection in Cell Lines. The combined
results of mRNA, intracellular protein, and secreted protein strongly
suggest that each HNSCC cell line tested produced IL-1 a, IL-1 ß,and
IL-6 (Table 5). Unfortunately, the cell line MDA Tu 1483 lost
viability and was no longer available for testing by immunohistochemistry. These experiments demonstrate that cytokines are pro
duced by HNSCC cell lines, and not only is cytokine mRNA ex
pressed, but also intracellular and secreted protein. IL-la and TGF-a
mRNA were expressed in 8 of 8 cell lines, IL-Ißand TGF-ßin 7 of
8, and IL-4 and IL-6 mRNA in 4 of 8. IL-la, IL-Iß,and IL-6 proteins
were secreted by 6 of 6 cell lines as detected by ELISA, and IL-1 and
IL-6 intracellular proteins were confirmed in 3 of 3 of these cell lines
by immunohistochemistry.
Intracellular Cytokine Detection in HNSCC Paraffin-embedded
Tumor Specimens. One of the concerns of working with cultured
HNSCC is the possibility that cytokine expression is an artifact of
tissue culture conditions. It was therefore imperative to demonstrate
the presence of the cytokines in resected HNSCC. We hypothesized
that the same set of cytokines as that found in the cell lines would be
detected in human tumor specimens; this hypothesis was tested by
immunostaining of paraffin-embedded tissue sections to determine
whether intracellular cytokine protein was produced by tumor cells
rather than tumor-infiltrating lymphocytes or macrophages.
Primary tumor specimens from 12 patients were analyzed for the
intracellular production of IL-la, IL-Iß,and IL-6. Fig. 7 shows the
immunohistochemical staining of a primary tumor from one repre
sentative patient with the negative control MOPC-21. Figs. 8-10
show the results of immunohistochemical analysis of three represent
ative patients' primary tumors from the oral tongue, base of tongue,
and floor of mouth, respectively, with antibodies to cytokeratin,
IL-la, IL-Iß,and IL-6. The results are summarized in Table 6. Our
results show universal expression of IL-1 and IL-6 in invasive
.
compared to the mRNA of normal oral keratinocytes. Only TGF-a
was expressed in cultured normal oral keratinocytes. Whereas IL-la
and TGF-a mRNAs were expressed in every SCC cell line, and IL-1 ß
and TGF-ßmRNAs were expressed by all but one, IL-4 and IL-6
mRNA expression varied among the cell lines (Table 4). Even our
control reactions using IL-2 cDNA produced bands that were very
faint (Fig. 1). Thus, our inability to detect IL-2 with certainty in these
tumors may be due to suboptimal amplification conditions. Therefore,
we cannot conclude anything about IL-2 expression from these ex
periments. These cell lines were derived from all grades of SCC and
divergent anatomical sites of the head and neck region. Despite the
fact that malignancies of different sites within the HNSCC may reflect
varying carcinogenic insults and may represent unique biological
entities, the cytokine profiles among the various cell lines differed
only slightly.
Our subsequent studies focused on IL-1 and IL-6 for the following
reasons. These cytokines are immunopotentiating; they have both
been reported to synergize with IL-2. which has shown some thera
peutic efficacy in the treatment of HNSCC (22, 23). Additionally,
IL-1 and IL-6 protein and mRNA have recently been detected in the
mRNA of melanoma cells in our laboratory (9). IL-1 and IL-6 are,
therefore, candidate cytokines for investigating this phenomenon in
HNSCC. They interact through NF-IL-6 and NF-«B(24, 25).
When supernatants from six of our cell lines were analyzed by
ELISA, a marked increase in concentration of secreted IL-la and IL-6
was found in comparison to HPV 16-immortalized oral keratinocytes
(Fig. 4). HOK 16B cells are not normal but are included in this study
because they may provide a snapshot of cells early in the multistep
transformation process. Secreted cytokine products are available to
interact with cellular receptors; thus, they are able to exert paracrine
or autocrine effects. Both IL-la and IL-Ißwere produced and se
creted by each cell line and they both bind to the same receptors and
exert the same biological effects (reviewed in Ref. 26). Moreover, the
presence of secreted protein means that the mRNA transcripts have
HNSCC tumors, regardless of the histológica! grades.
been translated and processed for secretion. These proteins may be
biologically active only if they are secreted in a mature form (26). The
DISCUSSION
concentrations of cytokines secreted in this study are clearly within a
We investigated the elaboration of IL-la, IL-Iß,IL-2, IL-4, IL-6,
range expected to have biological activity; these cytokines have been
IFN-y, TGF-a, TGF-ß,and TNF-a mRNA in eight cell lines derived
shown to exert their effects in the femtomolar range (26).
The presence of IL-la, IL-Iß,and IL-6 and their localization to the
from invasive head and neck primary tumors or their métastases,
3137
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VARIABLE
CYTOKINE
EXPRESSION
IN Hl MAN HNSCV
Fig. 8. Immunohistochemica!
staining of a
primary oral tongue tumor from a patient. A. anticytokeratin (positive control): B. anti-IL-la;
C,
anti-IL-l/3; D. ami-IL-6.
^¿j&
. «CT>...*
intracellular compartment was confirmed by immunohistochemistry. The
finding that IL-1 and IL-6 are present intracellularly in HNSCC tumors
does not necessitate their secretion, specify their biological form, or
delineate their specific action. The observation of IL-IßmRNA and
intracellular IL-Ißwithout significant levels of IL-Ißsecretion is not
surprising because cleavage of the intracellular product by IL-Ißcon
verting enzyme is necessary for secretion. Whether intracellular cytokines possess autocrine activity is not known. The molecular weight that
the active forms have, as well as any potential intracellular regulatory
influences these cytokines exert, is also not known.
To determine whether our cell line findings were reflective of in
vivo tumor expression, immunohistochemistry
was performed on bi
opsy material. Intracellular production of IL-la, IL-Iß, and IL-6
protein was detected in all patients' SCCs. Previous investigators had
noted that IL-1 secretion in tumors correlated with local inflammation
and the synthesis of prostaglandins, suggesting that the cytokines were
not produced by the tumor itself, but rather by infiltrating lympho
cytes or macrophages in in lung adenocarcinomas and gliomas (27,
28). In contrast, our results support tumor cell production of cyto
kines. We have found similar production of IL-1 and IL-6 in mela
noma (9, 21). It is possible, however, that the staining reflects ILs that
have been sequestered rather than elaborated by tumor cells. How
ever, the intensity and pattern of intracellular staining support our
hypothesis that the tumor cells are more likely to be the actual
producers. The phenotypic immunohistochemical
features suggest
that (in addition to tumor infiltrating lymphocytes or tumor infiltrating
macrophages) the tumor cells are, in certain cases, producing these
cytokines. Additionally, our study of primary HNSCC tumors reveals
two patterns of cytokine staining. One is a distinct staining pattern
associated with more well-differentiated squamous cells within the
3138
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VARIABLE. CYTOKINK
F.XPRKSION
IN Hl'MAN
HNSCC
B
Fig. 9. Immunohistochcmical
staining of a
primary base of tongue tumor from a patient. A.
anticytokeratin (positive control): if. anti-IL-la; C.
anti-IL-lj3: O. anti-IL-6.
m fis
play a role in invasive bone destruction associated with HNSCC tumors
(33). To support this. Ahn et al. (14) identified IL-1 in HNSCC primary
tumors in the vicinity of osteoclasts involved in destroying osseous
structures adjacent to tumors.
The presence of IL-6 mRNA is surprising, because it usually
reflects inflammatory stimulation, which would tend to inhibit tumor
formation. IL-6 is detected in the skin and peripheral blood mononuclear cells of patients with psoriasis (34). The detection of IL-6
mRNA in HNSCC cell lines may explain the finding by Gallo et al.
(35) of high levels of IL-6 in the sera of HNSCC patients and of
elevated circulating acute-phase proteins in the serum of patients with
HNSCC and (36). IL-6 is known to stimulate acute-phase proteins.
Additionally, elevated serum levels of IL-1 and/or IL-6 have been
transcriptional level (reviewed in Ref. 32) and may provide the advantage
the tumor needs to invade the basement membrane. IL-1 is also respon
implicated in the development of cancer cachexia in animal models
sible for the prostaglandin-independent bone resorbing activity and may
(37, 38) and in human renal cell carcinoma (39), as well as HNSCC.
3139
tumor, and the other is a more diffuse staining pattern that seems to be
consistent throughout various differentiation stages within a tumor.
The physiological role of IL-1 and IL-6 in mucosa is not well estab
lished. IL-1 production by keratinocytes and interdigitating reticulum
cells of the skin has been reported (13, 29-31). IL-1 inhibitors are
secreted by keratinocytes of the gingiva and from epithelial cells of the
submandibular gland (32). IL-1 regulates some of the key enzymes of the
extracellular matrix and takes pan in the removal of tissue damaged from
infection or trauma. IL-1 also increases the proliferation of fibroblasts that
express high-affinity IL-1 receptors with the resultant secretion of neutral
proteases, such as collagenase, elastase, stromolysin, and plasminogen
activator (32). Up-regulation of these proteases by IL-1 occurs at the
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VARIABLE
CYTOKINE
EXPRESSION
IN HUMAN HNSCC
Fig. 10. Immunohistochemical staining of a pri
mary floor of mouth tumor from a patient. A, anticytokeratin (positive eontrol); H. anti-IL-la; C, anti-IL-1/3; £>.
anti-IL-6.
Although we have not tested whether the intracellular IL-6 detected in
primary tumors is actually secreted, such release into the serum and
subsequent regulation of liver function could explain the presence of
the circulating acute-phase proteins.
This investigation reports the finding of universal intracellular expres
sion of IL-la, IL-Iß,and IL-6 in invasive SCC of the oral cavity.
Although the demonstration of intracellular IL-1 and IL-6 protein in
Table 6 Cvtokine expression in invasive HNSCC lutmtrs
sile°Oral
Anatomical
la8/81/12/21/1IL-
108/81/12/21/1IL-68/81/12/21/1
tongueBase
tongueFloor
of
mouthPalateILof
" Anatomical site delineations
in this tahle refer to the actual site of the tissue
stained rather than the established site of the primary tumor.
invasive HNSCC tumors is a novel finding, the authors duly recognize
that the sample size is small. The interaction of one cytokine with another
and the regulation of one by another are not assured merely by the
presence of two distinct cytokines. Further investigations are necessary to
establish the relationship between IL-1 and IL-6 in HNSCC to determine
the putative roles these cytokines have in regulating the tumor microenvironment. Whether IL-6 in HNSCC tumors has developed resistance to
regulation by other immune mediators, including cytokines, and hence
fosters immunosuppression at that site needs to be investigated.
In conclusion, our findings support the hypothesis that the aberrant
elaboration of biologically active IL-1 and IL-6 may contribute to
altered immune status in HNSCC patients. The ectopie production of
these cytokines by tumor cells may influence the hierarchical immu
nosuppression, seen most profoundly in HNSCC patients at the level
block
of the primary tumor and decreasing with distance from the primary
3140
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VARIABLE
CYTOKINE
EXPRESSION
tumor site. However, it is important to qualify our findings because
our studies were performed on tumors and cell lines derived from
invasive oral SCCs. Future studies should address the functional
relationship between these cytokines and the differences in the ex
pression and regulation of ILs in advanced carcinomas versus premalignant and in situ carcinomas, to both elucidate the role that cytokines
play in tumor invasion and progression and to establish whether an
acquisition of altered cytokine regulation occurs.
ACKNOWLEDGMENTS
We thank Dr. Mitchell Frederick for normal oral keratinocyte mRNA. Dr.
Douglas Boyd for advice with the preadsorption assays as well as access to his
ELISA reader, Velma Harris for expert assistance with the histology speci
mens, and Pat Wolf for statistical analysis. We are grateful to Dr. Demetrios
Petropoulos for useful discussions and careful review of the manuscript.
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Variable Expression of Cytokines in Human Head and Neck
Squamous Cell Carcinoma Cell Lines and Consistent
Expression in Surgical Specimens
Kendra V. Woods, Adel El-Naggar, Gary L. Clayman, et al.
Cancer Res 1998;58:3132-3141.
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