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 ¡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 ¡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 lymphokinebased 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 IL4 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
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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.
3 The 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 coexpressed 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 KÌ67,
a monoclonal
antibody reactive with a nuclear proliferation-associated antigen, was
carried out to check the antigenic preservation in the tumor cells since
the KÌ67
antigen 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
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lL-4 RECEPTOR EXPRESSION IN HUMAN LUNG TUMORS
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-
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
Table 2 Details of tumor types and antibody staining results
Table 1 Summary of the antibody panel used
AntibodyMR6
45.000. IL-4 receptor
A. Ritter (I8-21)
EGFRlGISKÕ67SpecificityM,
Epidermal growth factor
ICRF(IO)Dr.
receptor
Epidermal growth factor
Gregoriou. Molecular
receptorProliferation-associated Biophysics. Oxford.
England
Dakopatts(lo)
antigenSourceM.
typeSquamous
Histológica!
EGFR-0200IL-4-R+/
EGFR-34107
EGFR+10400IL-4-R-/
EGFR+16700IL-4-R-/
carcinoma(n
cell
29)Adenocarcinoma(n=
=
17)Small
carcinoma(n
cell
10)Carcinoid
=
(n = 7)1L-4-R+/
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 antiIL-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
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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 ström.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 histocompatibility 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 IL1/3when 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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Interleukin 4 Receptor Expression on Human Lung Tumors and
Normal Lung
Muhammad F. Tungekar, Helen Turley, Michael S. Dunnill, et al.
Cancer Res 1991;51:261-264.
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