Interleukin 4 Receptor Expression on Human Lung Tumors and Normal...

[CANCER RESEARCH 51. 261-264. January I. 199IJ

Interleukin 4 Receptor Expression on Human Lung Tumors and Normal Lung1

Muhammad F. Tungekar, Helen Turley, Michael S. Dannili, Kevin C. Gatter,2 Mary A. Ritter, and Adrian L. Harris

Nuffleld Department of Pathology Â¡M.F. T., H. T., M. S. D., K. C. C.] and ICRF Molecular Oncology Laboratory, Institute of Molecular Medicine [A. L. H.],

John Radcliffe Hospital, Headington, Oxford OX3 9DL', and Department of Immunology, Royal Postgraduate Medical School, Hammersmith Hospital,

London Â¡M.A. R.J, England

ABSTRACT

Interleukin 4 (11-4) receptors were detected by a monoclonal antibody

on tumor cells of 10 of 29 squamous cell carcinomas and 6 of 17

adenocarcinomas of the lung. None of the small cell carcinomas or

carcinoid tumors stained. Parallel sections stained for epidermal growth

factor receptors showed that all but 2 of the 11-4 receptor-positive tumors

also expressed epidermal growth factor receptors. Positive labeling for

IL-4 receptors was also obtained on nonneoplastic bronchial epithelium

and on lymphocytes and macrophages infiltrating the tumor stroma. The

role of IL-4 and its receptor in normal human lung is unknown, but the

expression of IL-4 receptors on particular subtypes of lung tumors

suggests that they may have a role in differentiation or proliferation of

squamous and adenocarcinomas.

INTRODUCTION

The discovery of peptide molecules that function as prolifer

ation signals for a range of cell types has aroused tremendous

interest in the field of tumor biology (1). Various growth factors

and lymphokines such as interleukins and interferons belong to

this family of peptide regulatory factors that exert their effects

by binding to specific receptors on the surface of target cells

(2).The interleukins were first identified as regulatory molecules

of the immune system, although the pleiotropic nature of their

effects over a wide range of other cell types is being increasingly

acknowledged (3). The importance of unraveling possible tu

mor-modulating effects of lymphokines has been highlighted in

recent years by novel therapeutic approaches utilizing interleu

kins and interferons as therapeutic agents (4). IL-4' was origi

nally described as a B-cell growth factor (5), but its receptors

are present on cells of lymphoid and myeloid lineages and have

been reported on two epithelial cell lines (6). It is known that

IL-4 may either suppress or enhance the growth of hemopoietic

progenitor cells depending on the lineage and differentiation of

target cells (7) and it is therefore a candidate for lymphokine-

based tumor therapy. IL-4 has been shown to have in vivo

antitumor effects on cell lines derived from a variety of murine

tumors on its own (8) and may potentiate similar effects of

other lymphokines such as IL-1/3 and IL-2 (9). However, to

date, there is no detailed information concerning the influence

of IL-4 on tumor progression or data on the distribution of IL-

4 receptors in any individual human organ system.

Expression of several oncogenes have been described in hu

man lung cancer and are potentially involved in its develop

ment. One such is the protooncogene c-erbB2 (also known as

neu), which shares sequence homology with the EGF receptor.

c-erbB2 is normally expressed in bronchial mucosa but may be

Received 7/25/90; accepted 10/1/90.

The cosls of publication or 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 indicale this fact.

1This work was supported in part by Kuwait University Grant MG022.

2To whom requests for reprints should be addressed, at NufTield Department

of Pathology. John RadclifTe Hospital. Headington. Oxford OX3 9DU, England.

3The abbreviations used are: IL, interleukin; IL-4-R. IL-4 receptor; EGF,

epidermal growth factor; EGFR. EGF receptor.

overexpressed in lung tumors without amplification or point

mutation (10). Its presence in breast cancer has been well

studied, although the prognostic significance of its amplifica

tion and overexpression is presently controversial (11-13). Pep

tide growth factor receptors, too, are widely distributed in

tumors and normal tissues and show different levels of expres

sion in tumors, compared with adjacent tissue. In particular

EGF receptor has been demonstrated in many tumors of lung,

breast, bladder, and brain (14-18) and, more importantly, a

correlation with pathological stage and survival for breast and

bladder carcinomas has been demonstrated (18, 19).

Recently, a preliminary study, in which a monoclonal anti

body, MR6, recognizing the IL-4-R complex (19, 20) was used,

showed that IL-4 receptors are present on a number of carci

nomas of various types including one case of lung cancer (21).

Because of the availability of this antibody the present study

was undertaken to investigate the detailed expression of IL-4

receptors on human lung tumors. EGF receptor expression has

been studied in parallel both as a control, since its distribution

is already well documented in lung tumors, and in order to

determine the extent of coexpression of EGF receptors with

IL-4 receptors. This study shows that IL-4 receptors are coex-

pressed with EGF receptors on approximately one-third of

cases of squamous cell carcinoma and adenocarcinoma. In

contrast, both are absent from cases of small cell carcinoma

and carcinoid tumor.

MATERIALS AND METHODS

Blocks from 63 surgically resected lung tumors were snap frozen and

stored in liquid nitrogen. The tumor types, classified according to the

World Health Organization classification (22), comprised 29 cases of

squamous cell carcinoma, 17 cases of adenocarcinoma, 10 cases of

small cell carcinoma, and 7 cases of carcinoid tumor. In 10 cases

nonneoplastic lung tissue removed from the resection specimen was

available for use as a control. The primary antibodies used are sum

marized in Table 1. In all cases, staining with KÃŒ67,a monoclonal

antibody reactive with a nuclear proliferation-associated antigen, was

carried out to check the antigenic preservation in the tumor cells since

the KÃŒ67antigen is known to be exquisitely sensitive to any form of

degradation (23). Immunohistochemical staining was performed using

the alkaline phosphatase:anti-alkaline phosphatase procedure as de

scribed previously (24).

Antibody MR6. The monoclonal antibody MR6 was raised against

an extract of thymic tissue (19) and shown to react strongly with thymic

cortical epithelial cells. By Western blotting MR6 detects a single

polypeptide of M, 200,000 which is rapidly cleaved to M, 145,000 by

proteolytic enzymes (20). This latter is known to be the approximate

molecular weight of one of the four polypeptide chains associated with

the IL-4 receptor. Flow cytometric and immunohistochemical studies

showed the antigen to be present also on T- and B-lymphocytes, cells

of myeloid lineage, and some renal epithelium (20). Recent experiments

(22) strongly suggest that MR6 binds to the IL-4 receptor complex by

demonstrating that MR6 inhibits IL-4-induced T-cell proliferation and

completely abrogates the IL-4 production of specific antigen-induced

IgE by B-cell populations. MR6 does not, however, block binding of

IL-4 to its receptor, which indicates that it recognizes a molecule closely

261

lL-4 RECEPTOR EXPRESSION IN HUMAN LUNG TUMORS

associated with the three chains of the IL-4-R but which does not form

the ligand-binding component of the receptor.

RESULTS

Staining for IL-4 receptors using monoclonal antibody MR6

was seen in tumor cells of 10 of 29 squamous cell carcinomas

and 6 of 17 adenocarcinomas (Table 2). All of the small cell

carcinomas and carcinoid tumors were unlabeled. In all samples

lymphocytes and macrophages were also positively labeled. In

the nonneoplastic lung tissue and also in many tumor samples

strong staining of bronchial epithelium was also seen. Staining

was seen both on the cell membranes and in the cytoplasm. All

of the tumors showed homogeneous staining apart from one

adenocarcinoma in which it was distributed focally.

Of the 2 monoclonal antibodies reactive with the EGF recep-

Table 1 Summary of the antibody panel used Table 2 Details of tumor types and antibody staining results

AntibodyMR6

EGFRlGISKÃ•67SpecificityM,

45.000. IL-4 receptor

Epidermal growth factor

receptor

Epidermal growth factor

receptorProliferation-associated

antigenSourceM.

A. Ritter (I8-21)

ICRF(IO)Dr.

Gregoriou. Molecular

Biophysics. Oxford.

England

Dakopatts(lo)

HistolÃ³gica!typeSquamous

cellcarcinoma(n

=29)Adenocarcinoma(n=

17)Small

cellcarcinoma(n

=10)Carcinoid

(n = 7)1L-4-R+/

EGFR-0200IL-4-R+/

EGFR+10400IL-4-R-/EGFR+16700IL-4-R-/

EGFR-34107

Fig. I. Squamous cell carcinoma express

ing IL-4 receptors (A; detail at higher power

in C). This is contrasted with the stronger

staining of the same tumor for epidermal

growth factor receptors (/?) and a different

squamous cell carcinoma unlabeled by the anti-

IL-4 receptor antibody (D). Both IL-4 recep

tors and epidermal growth factor receptors are

present on normal bronchial epithelium (ar

rows in A and B). Note that in both C and D

surrounding reactive lymphoid cells are

strongly labeled for IL-4 receptors (*).

262

IL-4 RECEPTOR EXPRESSION IN HUMAN LUNG TUMORS

tor, EGFR1 labeled 26 of 29 squamous cell carcinomas and 11

of 17 adenocarcinomas, giving a higher positivity rate than G15

which stained 18 of 27 and 9 of 17 of these tumors. Since all

of the G15 cases were also EGFR1 positive, the results in Table

2 are given under the single heading of EGFR. All samples of

small cell carcinoma and carcinoid tumor tested were negative

for both antibodies. All of the IL-4 receptor-positive squamous

cell carcinomas also expressed EGF receptors as did 4 of the 6

IL-4 receptor-positive adenocarcinomas (Fig. 1). In all cases

such coexpression was seen in the same areas and appeared to

be involving the same cells.

In the nonneoplastic lung, more intense reactivity for EGF

receptors was seen in basal compared to luminal bronchial

epithelial cells, whereas MR6 uniformly stained the bronchial

mucosa: this was consistently seen regardless of the reactivity

of tumor itself for these markers. MR6 also stained lympho

cytes and macrophages infiltrating the strÃ¶m.i,

DISCUSSION

This study has demonstrated that IL-4 receptors are fre

quently expressed by squamous cell carcinomas and adenocar

cinomas of the lung, whereas small cell carcinomas and carci

noid tumors consistently lack them. This selective distribution

of IL-4 receptors among lung tumor types is strikingly similar

to that of EGF receptors as observed by us and others (26).

These observations may relate to mechanisms of development

of different types of lung tumors. Although many studies have

indicated that all lung tumors arise from a common stem cell

type (27) and are frequently of mixed morphological type (28),

the clinical profile of small cell carcinoma is quite distinct from

that of the other tumor types and requires different clinical

management. The absence of both EGF receptors and IL-4

receptors in small cell carcinoma and their presence in other

types of lung cancer imply a different set of regulatory mecha

nisms affecting squamous cell carcinomas and adenocarcino

mas, presumably due to differences in the routes of differentia

tion followed by the putative tumor stem cells.

The present study has shown that both EGF and IL-4 recep

tors are expressed in normal bronchial mucosa, although there

appears to be down-regulation of expression of EGF receptors

in the more differentiated luminal epithelial cells. IL-4 receptor

activation induces expression of class I and II major histocom-

patibility complex molecules on cells of monocytic lineage (29).

If this occurs in normal and malignant epithelium also, it may

be important in modulating the local immune response. It will

be of interest to assess coexpression of IL-4 receptors with these

regulatory molecules.

In the case of squamous cell carcinomas and adenocarcino

mas the present findings may be of therapeutic value. Since

both chemotherapy and radiotherapy are relatively ineffective

in their management, it may be possible in the future to influ

ence the growth of these tumors by utilizing the presence of

EGF receptors and IL-4 receptors for therapeutic manipula

tions.

In vivo antitumor effects of murine IL-4 against homografts

of a variety of murine tumor cell lines have been demonstrated

(8). One other study has shown potentiation of the tumor

inhibitory effects of a synthetic nonapeptide derived from IL-

1/3when combined with IL-4 and, to a lesser extent, with IL-2

on implants of fibrosarcoma cells in mice (9). Although these

have been assumed to be acting entirely on cells of the immune

system, the demonstration of interleukin receptors on tumor

cells suggests that a direct antineoplastic effect might be pos

sible. An obvious sequel to the present findings would be to

ascertain the direct antitumorigenic effects of toxins conjugated

to IL-4 molecules.

If this could be demonstrated for human lung tumors, then

the immunocytochemical demonstration of IL-4 receptors as

performed in this study will provide an important parameter

for selecting tumors for such treatment and for assessing their

responsiveness in vivo. It might also be possible to treat suitably

chosen tumors with IL-4 receptor antibodies or IL-4-cytotoxic

combinations if IL-4 directly affects tumor behavior. It will be

important, of course, to ascertain the effect of such therapeutic

endeavors on the normal lymphoid and epithelial cells which

also express IL-4 receptors.

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