[CANCER RESEARCH 55. 3847-3853, September 1. 1W5]

Human Non-Small Cell Lung Cancer Cells Express a Type 2 Cytokine Pattern1

Min Huang, Jianyi Wang, Paul Lee, Sherven Stiantila, Jenny T. Mao, Horst Meissner, Koichi Uyemura,Robert Modlin, Jerome Wollman, and Steven M. Dubinett2

Pulmonary Imirntnologv Laboratory, Division of Pulmonan' and Critical Cure Medicine [M. H., J. W., P. L, S. S., J. T. M., H. M., S. M. D.\, Johnson Comprehensive CancerCenter fS. M. D.I, Division of Dermatology IK. U., R. M.], anil Department of Pathology [J. W.I. University of California-Ltis Angeles School of Medicine and the West Los

Angeles Veterans Administration Medical Center, Los Angeles, California 90073

ABSTRACT

In addition to infiltrating inflammatory cells, tumors also producecytokines and growth factors that may alter tumor growth, tumor immii-

nogenicity, and the host immune response. To characterize the expressionprofile of human non-small cell lung cancer (NSCLC)-derived cytokines,

the inRNA expression of type 1 and type 2 cytokines in five humanNSCLC lines was analyzed by reverse transcriptase-PCR. Expression ofinterleukin 5 (IL-5) and 11-10 was demonstrated in all tumor lines evaluated, whereas IL-4 was present in three of five lines and IL-13 waspresent in two of livi- lines. In contrast, none of the tumor lines expressed

IL-2 and IFN-y. Type 2 cytokine protein production by NSCLC lines wasconfirmed by immunoprecipitation and cytokine specific ELISA. Tumor-derived II.-10 secretion was significantly augmented by exogenous recombinant cytokines including IL-4 and tumor necrosis factor-a. To evaluate

whether fresh NSCLC nodules also express a type 2 cytokine pattern, thecontent of type 1 and type 2 cytokines in tissue homogenates from 13 freshNSCLC nodules and normal lung surgical specimens was assessed. Human NSCLC nodules contain significantly more type 2 cytokines thandoes normal lung tissue when corrected for total protein concentration. Toidentify the cellular source of type 2 cytokine production in tumor nodules, immunohistology was performed on sections from 5 lung squamouscell carcinomas and 5 adenocarcinomas. All of the specimens revealedpositive staining for type 2 cytokines within tumor cells. In summary, wereport that human NSCLC cells produce type 2 cytokines both in situ andin vitro, which may play an active immunoregulatory role in the lungcancer microenvironment.

INTRODUCTION

Recent studies have evaluated the cytokine network involved in thelocal immune response to tumors (1-4). A variety of lung tumor-derived factors, including cytokines such as IL-63 (5), IL-8 (6-7), and

TGF-ß(8-9), may either regulate tumor growth or alter the antitumor

immune response. Thus, human lung cancer growth may be regulatedby a variety of cytokines via both autocrine and paracrine pathways.Regulation of tumor growth by cytokines may occur directly byregulation of cell proliferation or indirectly through effects on angio-genesis or host immunity (5-9).

In both murine models and human studies, T lymphocytes havebeen found to express two distinct cytokine patterns (10-11). Type 1

Received 4/6/95; accepted 7/6/95.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1This work ¡ssupported by the American Lung Association, the Tobacco-Related

Disease research program of the University of California, Veterans Administration MeritReview Research Funds, and the Stop Cancer/Helen Neufeld Research Career Development Award. M. H. was supported by NIH Institutional Training Grant in TumorImmunology CA09120and a Jaye Haddad-Concern Foundation Immunology Fellowship.S. S. was supported by NIH Institutional Training Grant in Pulmonary Medicine HL070I4and a Jaye Haddad-Concern Foundation Immunology Fellowship. A portion of this workwas presented at the 1994 AACR annual meeting.

2 To whom requests for reprints should be addressed, at Division of Pulmonary and

Critical Care Medicine, Department of Medicine, University of California-Los AngelesSchool of Medicine, West Los Angeles VA, WillQ, 11301 Wilshire Boulevard, LosAngeles, CA 90073.

3 The abbreviations used are: IL-6, interleukin 6; TGF-ß,transforming growth factor

ß;Thl, type 1 T-helper lymphocytes; NSCLC, non-small cell lung cancer; TNF-a, tumornecrosis factor; RT-PCR, reverse transcriptase-PCR; ddH,O, double distilled water.

or Thl cells produce IL-2 and IFN-y, whereas type 2 or Th2 cellsproduce IL-4, IL-5, IL-10, and IL-13. Whereas type 1 cytokinespromote cell-mediated responses, type 2 cytokines promote immuno-

globulin production and inhibit the differentiation of type 1 cells andtype 1 cytokine release (12-17). Recent studies indicate that a type 2

cytokine pattern is present at the tumor site and suggest that thesecytokines may mediate immunosuppression (3, 18). The type 2 cytokine pattern at the tumor site has been ascribed to the lymphocytesinfiltrating the tumor. The purpose of this study was to determinewhether tumor cells also contribute Th2 cytokines to the lung tumormilieu. We report that NSCLC cell lines and fresh tumors produce adistinct type 2 cytokine pattern in vitro and in situ.

MATERIALS AND METHODS

Human NSCLC Lines. Lung adenocarcinoma cell lines SKLU-1, 125,

A427, lung squamous cell carcinoma line H520, and large cell lung carcinomaline H460 were obtained from Dr. J. A. Radosevich. Lung adenocarcinoma lineA549 and lung squamous cell carcinoma lines SK-MES-1 and SW 900 were

obtained from American Type Culture Collection (Rockville, MD). The humansquamous cell carcinoma line RH2 was established in our laboratory (19). Thecells were grown under an atmosphere of 5% CO2 in air as monolayers at 37°Cin 25-cm2 tissue culture flasks containing 5.0 ml of RPMI 1640 (for SKLU-1,

A549, 125, A427, RH2, H460, H520) or EMEM medium (for SK-MES-1 and

SW 900) supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin solution and 2 mM glutamine (JRH Biosciences, Lenexa, KS).

Human Cytokine PCR Primers. IL-10 and IL-13 PCR primers were

synthesized by a DNA/RNA synthesizer (Applied Biosystems, Foster City,CA). All other PCR primers were purchased from Clontech Laboratories (PaloAlto, CA). The sequences of the human cytokine PCR primers are: IL-2 senseprimer 5'-ATG-TAC-AGG-ATG-CAA-CTC-CTG-TCT-T-3' and antisenseprimer S'-GTT-AGT-GTT-GAG-ATG-ATG-CTT-TGA-C-S' (amplified DNAsize, 458 bp); IFN--y sense primer 5'-ATG-AAA-TAT-ACA-AGT-TAT-ATC-TTG-GCT-TT-3' and antisense primer 5'-GAT-GCT-CTT-CGA-CCT-CGA-AAC-AGC-AT-3' (amplified DNA size, 494 bp); TNF-a sense primer 5'-ATG-AGC-ACT-GAA-AGC-ATG-ATC-CGG-3' and antisense primer5'GCA-ATG-ATC-CCA-AAG-TAG-ACC-TGC-CC-3' (amplified DNA size,695 bp); IL-4 sense primer 5'-ATG-GGT-CTC-ACC-TCC-CAA-CTG-CT-3'and antisense primer S'-CGA-ACA-CTT-TGA-ATA-TTT-CTC-TCT-CTC-AT-3' (amplified DNA size, 456 bp); IL-5 sense primer 5'-GCT-TCT-GCA-

TTT-GAG-TTT-GCT-AGC-T-3' and antisense primer 5'-TGG-CCG-TCA-ATG-TAT-TTC-TTT-ATT-AAG-3' (amplified DNA size, 293 bp); IL-10sense primer S'-ATG-CCC-CAA-GCT-GAG-AAC-CAAGAC-CCA-S' andantisense primer S'-TCT-CAA-GGG-GCT-GGG-TCA-GCT-ATC-CCA-S'

(amplified DNA size, 352 bp); IL-13 sense primer S'-AGG-GAG-CTC-ATT-GAG-GAG-CTG-GTC-3' and antisense primer 5'-GAG-CAG-GTC-CTT-TAC-AAA-CTG-GGC-3' (amplified DNA size, 248 bp); TGF-ßsense primerS'-GCC-CTG-GAC-ACC-AAC-TAT-TGC-T-S' and antisense primer 5'-AGG-CTC-CAA-ATG-TAG-GGG-CTG-G-3' (amplified DNA size, 161 bp);ß-actinsense primer S'-ATG-GAT-GAT-GAT-ATC-GCC-GCG-S' and anti-sense primer S'-CTA-GAA-GCA-TTT-GCG-GTG-GAC-GAT-GGA-GGG-GCC-3' (amplified DNA size, 1126 bp), all used as controls.

Determination of Cytokine mRNA Expression by RT-PCR. We have

shown previously that the initial copies of the cytokine templates within therange of IO2 to IO7 have a linear relationship to the amount of PCR amplifi

cation products (3). In preliminary studies, the PCR products amplified by eachcytokine PCR primer pairs matched the expected length according to thepublished cytokine cDNA sequences. The authenticity of the amplified cyto-

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kine PCR products was confirmed by hybridization to 32P-labeled cDNA or

oligonucleotide probes in Southern blot analyses (data not shown). Total RNAisolation and RT-PCR were performed as described previously (20). Briefly,

high quality total RNA from five human NSCLC cell lines was prepared byRNAzol method according to the manufacturer's instructions (Tel-Test, Inc.,

Friendswood, TX). RT-PCR was performed by using a RNA-PCR kit supplied

by Perkin Elmer Cetus (Norwalk, CT). Briefly, reverse transcription wasperformed at 42°Cfor l h in a solution containing 4 /u.1MgCl2 (25 min), 2 /u.1

10X PCR buffer (500 mm KC1, 100 mM Tris-Cl, pH 8.3), 2 pi dNTPs mix (10

mM of each), 1 /j,l RNase inhibitor (20 units/fj.1), 1 /¿Irandom hexamer (50mM), 1 /ni RT (50 units//xl), 9 /nl sterile diethyl pyrocarbonate-treated ddH20

containing 1 jxg of total RNA. For PCR amplification, the following components were added to the 20-^1 RT reaction: 3 jul 10X PCR buffer, 2 /j.1of each

PCR primer pair mix, 6 fj.1dNTPs mix (10 mM of each), 0.5 (xl Taq polymerase(5 units//il), 18.5 /¿Isterile diethyl pyrocarbonate-treated ddH20. To each PCRreaction, 50-jj.l sterile mineral oil was added on top. A PTC-100-60 thermal

cycler (MJ Research, Inc., Watertown, MA) was programmed as follows: 2min at 94°Cfor 1 cycle; 1 min at 94°Cfor 35 cycles, 1 min at 58°C,1 minat 72°C;and 7 min at 72°Cfor 1 cycle. Twenty-five ^1 of the PCR products

from each reaction was analyzed by 1.5% agarose/ethidium bromide gelelectrophoresis.

Determination of Cytokine Protein Synthesis by Immunoprecipitation.The immunoprecipitation procedure was performed as described previously(21). Briefly, 3 X IO6 cells of each line were plated in a 25-cm2 tissue culture

flask and maintained in routine culture condition. After 24-h incubation, the

medium from each culture was discarded, and the cell monolayers werewashed three times with PBS. The cells were preincubated in methionine-free

medium (JRH Biosciences, Lenexa, KS) for 2 h and labeled with 200 fiCi/ml[35S] methionine (NEN Research Products, Wilmington, DE) for 24 h. The

cultured medium from each flask was collected and anti-hIL-4, anti-hIL-5,anti-hIL-10, or anti-TGF-ß mAbs (R&D Systems, Minneapolis, MN) were

added to each sample, respectively, in a final concentration of 5 (¿g/ml.Afterincubation at 4°Covernight with gentle rotation, 50 ;u,lof protein A-Sepharose

CL-4B (Sigma Chemical Co., St. Louis, MO) in 1:1 dilution with ddH2O wasadded and incubated at 4°Cfor 4 h. The samples were pelleted by centrifu-

gation at 12,000 X g for 1 min and washed twice in buffer containing 50 mMTric-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, and 0.02%

SDS, followed by three washes in the same buffer without Triton and SDS.The cytokine/anticytokine complexes adsorbed to protein A-Sepharose from

each sample were released by boiling for 5 min in 50 /u.1buffer containing 60mM Tris-Cl (pH 6.8), 2% SDS, 2% 2-mercaptoethanol, 0.01% bromophenol,

and 10% glycerol before PAGE. The gel was then dried and exposed to KodakXAR-5 film at 25°C.

Determination of Type 2 Cytokine Production from Fresh NSCLCNodules and Normal Lung Specimens. Fresh human NSCLC nodules andnormal lung samples were obtained from fresh surgical specimens at the WestLos Angeles Veterans Administration Medical Center (Los Angeles, CA). Thespecimens were rinsed in PBS with three changes. One g of tumor nodule ornormal lung tissues in 1 ml PBS was homogenized, and the tissue homogenateswere centrifuged at 12,000 X g for 1 min at 25°C.After centrifugation, the

supernatant of each sample was collected for further analysis. Cytokine production from tumor nodules and normal lung specimens were quantified bycytokine-specific ELISA. Human IL-5 (R&D Systems, Minneapolis, MN),IL-2, IFN-7, IL-4, and IL-10 (Biosource, Camarillo, CA) ELISA were per

formed as described by the manufacturers. Absorbance of each sample wasdetermined with an ELISA plate reader (Dynatech, Chantilly, VA). Thesensitivity of the assays is 15.6 pg/ml for IL-2 and IFN-7, 7.8 pg/ml for IL-4and IL-5, and 0.78 pg/ml for IL-10. The total protein content of tumor nodules

and normal lung tissues was quantified with a protein micro determination kit(Sigma). The results are expressed as pg of cytokine/mg of total protein.

Immunostaining of Paraffin-embedded NSCLC Sections. Specific bi-otinylated antibodies for immunostaining of IL-4, IL-5, and IL-10 proteins

were obtained from R&D Systems (Minneapolis, MN). The tissue sectionswere deparaffinized and hydrated through xylenes and graded alcohol series.The sections were then incubated in 0.3% H2O2 in methanol for 30 min forquenching of endogenous peroxidase activity. After washing three times withPBS, the sections were blocked with 10% FBS/PBS for 20 min. After washingwith the same buffer, biotinylated anti-hIL-4, anti-hIL-5, anti-hIL-10, or irrel

evant control antibodies were added to the sections and incubated for 30 min.

After washing, streptavidin-peroxidase was added to the sections and incu

bated for 1 h. After color development, the sections were washed in tap water,counterstained with hematoxylin, cleaned, and mounted for photography.

Induction of NSCLC-derived Type 2 Cytokines. Recombinant IL-4,IL-5, IL-8, IL-10, TGF-ß,and granulocyte-macrophage colony-stimulatingfactor were purchased from R&D systems (Minneapolis, MN). IL-6 andTNF-a were purchased from Genzyme Diagnostics, Cambridge, MA. IFN--y

was purchased from BioSource International (Camarillo, CA). IL-2 was supplied by Chiron (Emeryville, CA). IL-7 was supplied by Sterling, Inc.

(Malvern, PA). Cells from NSCLC lines SKLU1, A549, 125, A427, RH2,H460, SK-MES-1, SW900, and H520 were plated at a concentration of200,000 cells/well in 12-well tissue culture plates (Corning, Corning, NY). The

cells were maintained in RPMI 1640 supplemented with 5% human AB serum,glutamine, and antibiotics in the presence or absence of cytokines for 24 h. Thecultured supernatants from each well were collected and analyzed by cytokine-

specific ELISA. The results are expressed as pg/ml.

RESULTS

Human NSCLC Cell Lines Express Type 2 Cytokine mRNA.Total RNA from five well established human NSCLC cell lines(SKLU-1, A549, 125, A427, and RH2) were prepared, and the cyto-

kine mRNA expression profiles from these tumor cells were determined by RT-PCR using ß-actinas a positive control (Fig. 1). IL-2and IFN-7 were not detected after 35 cycle amplifications under theconditions described in "Materials and Methods." In contrast, IL-5,

IL-10, and TGF-ßwere present in all lines tested. IL-4 was present inthree of five lines (SKLU-1, 125, RH2) and IL-13 was present in twoof five lines (SKLU-1 and A549). To exclude the possibility ofcarry-over contamination, reactions containing all RT-PCR reagents

including cytokine PCR primers without sample RNA were used asnegative controls. No contamination was detected (Fig. 1, bottom). Inaddition, all of the NSCLC lines are devoid of lymphocytes and do notexpress CD3a mRNA by RT-PCR (data not shown).

Human NSCLC Cell Lines Produce Type 2 Cytokines. To determine whether the protein synthesis of tumor-derived type 2 cyto

kines correlates with the mRNA expression, isotope incorporation intothe cultured cells and immunoprecipitation with specific anticytokinemAbs were performed in these tumor lines. In addition to its highsensitivity and specificity, the advantage of immunoprecipitation isthat it only determines the newly synthesized proteins, thus eliminating the possibility of cytokine contamination from serum or othersources. The cells were maintained in culture and labeled with [35S]

methionine. New synthesis of tumor-derived type 2 cytokine proteinswas confirmed by demonstrating the specific mAb-precipitated bands

that corresponded to the appropriate molecular weight in PAGE(Fig. 2).

Fresh Human NSCLC Nodules Produce Type 2 Cytokines.Tumor lines maintained in long-term in vitro culture may not accu

rately reflect the characteristics of tumors in situ. To evaluate whetherfresh human NSCLC nodules also produce type 2 cytokines, weobtained fresh lung tumor nodules and normal lung specimens fromsurgical resections and assessed the content of type 1 and type 2cytokines in tissue homogenates by cytokine-specific ELISA. As

shown in Table 1, fresh human NSCLC tumor nodules contain significantly more type 2 cytokines than do normal lung tissues. IL-10 is

the predominant type 2 cytokine contained in these tumor nodules.Immunohistochernical Staining Reveals Localization of Type 2

Cytokines to Lung Tumor Cells. Lung tumor nodules contain avariety of different cell types that could contribute to cytokine production. To identify the cellular source of type 2 cytokine production,we performed immunohistology on sections from 10 lung tumors (5squamous cell carcinomas and 5 adenocarcinomas). All 10 specimensrevealed positive staining for type 2 cytokines within tumor cells.

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«••»•••t»

Fig. 1. Cytokine mRNA expression pattern from five human NSCLC cell lines byRT-PCR. Top lo bottom: human NSCLC cell lines SKLU-1, A549, 125, A427, RH2, andnegative control (RT-PCR cocktails without RNA samples). Lane M, l(X)-bp molecularweight marker; Lane I, ß-actin(1,126 bp); Lane 2, IL-2 (458 bp); Lane 3, IFN--y (494 bp);Lane 4, TNF-a (695 bp); Lane 5, IL-4 (456 bp); Lane 6, IL-5 (293 bp); Lane 7, IL-10 (352bp); Lane 8, IL-13 (248 bp); Lane 9, TGF-ß(161 bp); arrows, respective PCR products.

Representative immunohistochemical staining of tumors with primarylocalization of antigenic IL-4, IL-5, and IL-10 to individual tumor

cells is shown in Fig. 3.IL-4 and TNF-a Induce NSCLC IL-10 Production. To evaluate

the regulation of tumor-derived type 2 cytokines, cell lines wereexposed to recombinant cytokines in vitro. IL-4 and IL-5 were notinduced by the following recombinant cytokines: IL-2, IL-4, IL-5,IL-6, IL-7, IL-8, IL-10, TGF-ß,TNF-a, IFN-y, and granulocyte-macrophage colony-stimulating factor (data not shown). In contrast,tumor-derived IL-10 was induced 2-14-fold in 5 of 9 NSCLC lines byrecombinant IL-4 (250 units/ml) and 1.5-15-fold in 5 of 9 NSCLClines by recombinant TNF-a (1000 units/ml) (Table 2). In addition,concanavalin A (10 /j,g/ml) induces tumor-derived IL-10 production2-13-fold in all lines (Table 2).

DISCUSSION

An important emerging concept in tumor immunology is that twodistinct cytokine patterns may be generated by T lymphocytes (10-

11). Whereas type 1 lymphocytes promote cellular immunity byproducing IL-2 and IFN-y, type 2 lymphocytes produce IL-4, IL-5,IL-10, and IL-13 and suppress cellular immune responses (12-17).Thl and Th2 cells oppose one anothers' activity, cytokine production,

and differentiation (12, 14, 22). For example, IFN-y and IL-10 haveopposing effects on macrophage antigen-presenting function (23). Inaddition, IL-10 prevents the dendritic cell costimulatory activity thatleads to IFN-y production by T lymphocytes and inhibits antigen-

specific Thl cell proliferation (24). Recent studies have also begun todefine the molecular pathways that differentiate the Thl and Th2phenotypes (14, 22, 25). Differences in costimulatory molecule expression (25), extinction of IL-12 signaling (22), modification ofpromoter binding proteins (14), and soluble factors such as prostag-landins (26-27) have been reported to modulate the Thl/Th2 axis.

The type 1 and type 2 cytokine patterns were originally thought to belimited to CD4+ lymphocytes (originally referred to as Thl and Th2lymphocytes), but it is now recognized that non-CD4* lymphocytes

can also express type 1 or type 2 cytokine patterns (28-29).

In the current study, we have determined that NSCLC cell lines as

1

<TGF-b

Fig. 2. Autorudiography of cytokine protein synthesis from live human NSCLC celllines by immunoprecipilation. IL-4. M, 14,000; IL-5, M, 22,000; IL-10, M, 21.(XX);TGF-ß,M, 25.IXX).Lane I, SKLU-1; Lane 2, A549; Lane 3, 125; Lane 4, A427; Lane 5,

RH2.

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Table 1 Cyiokine content of fresh lung cancer nodules vs normal lung"

IL-2Patient

code#5655321194%7837070600431318217544092805833121319777HistologicaldiagnosisAdeno*AdenoAdenoAdenoAdenoAdenoBACBACsecsecsecsecsecTumor138815541513251525Lung115492321420151114IFN-7Tumor2318000I)1(10910000Lung970017177(154(I039IL-4Tumor6314119110695668843811019263120Lung0456114510100001475129IL-5Tumor11273461121216158135903446150Lung01511147752723153002092524IL-10Tumor1,556659293146978043451892681,69535493268Lung1285771263392121192863712850149

" The results are mean values of triplicate determinations and expressed as pg of cytokine/mg total protein. The SE is within 10% of mean values.

Adeno. adenocarcinoma; BAC, bronchoalveolar carcinoma; SCC, squamous cell carcinoma.

well as fresh tumors express a distinct type 2 cytokine pattern. Otherinvestigations have shown cytokine expression at the tumor site byRT-PCR of tumor biopsy samples (3, 18). However, use of this

method did not allow distinction of the cellular source of cytokineproduction, and the cytokine pattern has been attributed only to tumorinfiltrating lymphocytes. For example, Yamamura et al. (3) reportedthat a type 2 cytokine pattern was observed in biopsy samples frombasal cell carcinoma but not in benign cutaneous lesions.

NSCLC respond only minimally to immunotherapy, perhaps because NSCLC cells produce a variety of immunosuppressive factorsand thus may escape immune recognition (8, 30-32). Some tumor-

derived products, such as epithelial growth factors, may act in anautocrine manner to enhance tumor growth (8). Others such as pros-taglandins (33-36) may regulate the cytokine release by infiltratinginflammatory cells at the tumor site. Tumor-derived TGF-ßhas been

found to be profoundly immunosuppressive, and its abrogation mayaugment antitumor immunity (19, 37-38). In addition to TGF-ß,we

found that NSCLC cells produce type 2 cytokines and speculate thatthis may also contribute to immunosuppression.

In particular, IL-10 possesses several properties that may be inhibitory to the generation of antitumor immunity (23, 39-44). IL-10inhibits a broad array of immune parameters, including proinflamma-tory cytokine production by macrophages (40-41), antigen-presentation function (24, 42-43), T-lymphocyte proliferation (44), Thl cytokine production (16, 23), and lymphokine-activated killer cellfunction (15). Pretreatment with recombinant IL-10 protects tumorcells from lysis by tumor-specific cytotoxic T cells (45-46). In amurine model, Wang et al. (47) demonstrated that tumor cells trans-fected with the IL-10 gene produce local immunosuppression andprevent the induction of tumor-specific cytotoxic T lymphocytes.

Tumor-derived IL-10 has been documented in lymphoma (48-49),

ovarian carcinoma (50), melanoma, neuroblastoma, renal cell andcolon carcinoma (51), and NSCLC (32). Colon cancer-derived IL-10

significantly inhibited the proliferative response in a mixed lymphocyte reaction (51). NSCLC-derived IL-10 inhibited TNF-a and IL-6

production by peripheral blood monocytes (32). The secretion oftumor-derived IL-10 in vivo may inhibit antigen presentation to cy

totoxic T lymphocytes (52). Recent clinical studies suggest that elevated IL-10 may serve as a marker of poor prognosis (48). The serumIL-10 level in 153 patients with non-Hodgkin's lymphoma (both

EBV-positive and EBV-negative) was found to be an independent

prognostic factor, correlating with poor prognosis in patients withintermediate or high-grade non-Hodgkin's lymphoma (48).

Tumors produce IL-10 constitutively in vitro. In situ, within the

complex tumor microenvironment, the tumor is also exposed to anarray of cytokines produced by infiltrating inflammatory cells and the

tumor strema that may influence cytokine production by the tumorcells. Thus, these cytokines present within the tumor milieu mayregulate tumor IL-10 production. Of the many cytokines that could beoperative in these interactions we found IL-4 and TNF-a to beinducers of tumor-derived IL-10. This is in agreement with previousstudies demonstrating an increase in IL-10 production from coloncancer cell lines in response to IL-4 and TNF-a (51). Thus, we have

begun to define a possible cytokine network in the tumor microenvironment that may ultimately lead to diminished immune responses atthe lung tumor site. We speculate that IL-4-producing tumor-infiltrating lymphocytes may promote tumor production of IL-10. IL-10 inturn could lead to down-regulation of proinflammatory cytokine expression, decreased antigen-presenting cell MHC expression, and

maintenance of the type 2 lymphocytic infiltrate at the tumor site. Thisparacrine-inhibitory cycle could promote a greater type 2 lymphocyticinfiltration and continue induction of tumor-derived IL-10. In addition, because IL-4 is also produced by NSCLC cells, autocrine induction of tumor-derived IL-10 may occur.

IL-4 is a pleiotropic type 2 cytokine that has been found to haveboth stimulatory and inhibitory effects on antitumor immune responses (53-61). Recombinant IL-4 decreases the proliferation ofsome solid tumor cell lines including lung cancers (54-55). In contrast, IL-4 is also an autocrine growth factor produced by lymphomacells (56). Although IL-4 has been found to enhance the proliferationof tumor-infiltrating lymphocytes in vitro (57), IL-4 is also known to

inhibit macrophage production of inflammatory cytokines such asTNF-a, IL-1, and IL-8 (58-60) and inhibit lymphokine-activatedkiller cell function (61). In addition, both IL-4 and IL-10 are key

cytokines for the inhibition of the Thl cytokine response and thedevelopment of the Th2 cytokine response (12-13, 62-63). Thus,tumor-derived IL-4 and IL-10 in the NSCLC microenvironment may

favor the development of the type 2 cytokine response at the tumorsite.

Table 2 Induction of NSCLC-derived IL-ltf

CelllineSKLU-1A549125A427RH2H460SK-MES-1SW900H520Control18

±712±790±345±2019±1195

±402±0.315±919

±8IL-472

±21(4)''52

±20(4)257±73(3)46

±838±14(2)88

±2627±2(2)17

±825±14TNF-a178

±57(10)31±22(3)136±4(1.5)32

±1416±794±6330

±15(15)7±381

±8(4)Concanavalin

A196

±33(11)69±8(6)195±32(2)90±15(2)62±22(3)175±44(2)26±1(13)55±35(4)159±17(8)

*The IL-10 production is determined by ELISA and expressed as pg/ml ±SD.b Fold induction of control.

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Fig. 3. Immunostaining of paraffin-embedded NSCLC sections. Sections from the same block were stained as follows: A, H&E staining; B, nonspecific antibody staining as negativecontrol; C, specific anti-IL-10 mAb staining; D, specific anti-IL-4 mAb staining; E, specific anti-IL-5 mAb staining.

Little is known regarding the importance of IL-13 and IL-5 in the Introduction of cytokine genes into tumors is now being investi-host anti-tumor response. IL-13, the most recently described type 2 gated in animal models and in clinical trials. Before manipulation withcytokine, is similar to IL-4 in that it also decreases macrophage cytokine genes, it is becoming more clear that tumors have an endog-production of IL-1 and TNF-o (17, 64). Although the role of tumor- enous constitutive pattern of cytokine expression (5-6, 32). Thus, thederived IL-5 remains unclear, one study demonstrated that IL-5- alteration of endogenous tumor cytokine production induced by cy-transduced murine tumors escaped rejection despite eosinophil infil- tokine gene transfer may be one of the important parameters to be

tration (65). assessed in the design of cancer gene therapy (66). The development

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29.

30.

31.

of cytokine gene therapy for lung cancer may be aided by a more 27.complete understanding of the functional significance and regulationof endogenous lung tumor-derived cytokines.

We conclude that NSCLC cells contribute to the type 2 cytokine 2ii

pattern at the tumor site. Further in vivo studies will be necessary todefine the immunoregulatory activities of tumor-derived type 2 cyto

kines in the lung cancer microenvironment.

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1995;55:3847-3853. Cancer Res   Min Huang, Jianyi Wang, Paul Lee, et al.   Cytokine PatternHuman Non-Small Cell Lung Cancer Cells Express a Type 2

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