LUNG

ELSEVIER Lung Cancer 16 (1996) 13-19

Interleukin-2 receptors in pulmonary adenocarcinoma tissue

Tokujiro Yano *, Yaw-o Fukuyama, Hideki Yokoyama, Eiji Takai, Yuichi Tanaka, Hiroshi Asoh, Yukito Ichinose

Department of’ C/jest Surgery, National Kyushu Cancer Center, 3-l-l. Notame, Minami-ku, Fukuoka 815, Japan

Received 21 May 1996; revised 5 September 1996; accepted 12 September 1996

Abstract

We previously reported that the serum soluble interleukin-2 receptor (sIL-2R) level increased with the advance of disease stage in non-small cell lung cancer. The present study was thus conducted to investigate the origin of serum sIL-2R in patients with pulmonary adenocarcinoma. Fresh tumor cell suspensions were prepared from surgically resected specimens of pulmonary adenocarcinoma. They were adjusted to a cell density of 5 x 105/ml and then cultured for 24 h at 37°C. The culture supernatants were collected and assayed to determine the sIL-2R levels using an enzyme immunoassay. The resultant cells were thereafter cytocentrifuged onto glass slides and immunochemically stained with anti-human IL-2Rc( (CD25) monoclonal antibody. In three of six cases examined, a substantial level of sIL-2R was identified in the culture supernatants. In four cases, including those three cases with the presence of sIL-2R in the culture supernatants, various proportions of tumor cells were positively stained with the anti-IL-2Ra antibody. Further examinations revealed that tumor cells expressed IL-2Ra (CD25) in seven of 16 cases with pulmonary adenocarcinoma. These results thus suggested that the tumor cells did express IL-2Ra and release sIL-2R in some cases with pulmonary adenocarcinoma. Copyright 0 1996 Elsevier Science Ireland Ltd.

Keywords: Soluble interleukin-2 receptor; Interleukin-2 receptor cr (CD25); Interleukin-2 receptor /? (~75); Pulmonary adenocarcinoma; Lung cancer; Immunochemical staining

* Corresponding author. Tel: + 81 92 5413231; fax: + 81 92 5514585

0169-5002/96/$15.00 Copyright Q 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO169-5002(96)00608-3

14 T. Yam et al. /Lung Cancer 16 (1996) 13-19

1. Introduction

A soluble form of interleukin-2 receptor (sIL-2R) is released from activated lymphoid cells [7.8]. While circulating soluble IL-2R are detected in healthy individuals at low levels, extremely high levels of sIL-2R have been detected in hemolymphopoietic neoplasms [4,11]. Recently, Lissoni et al. [2] reported that the serum levels of sIL-2R were significantly higher in cancer patients (breast, lung colon, etc.) than in controls. Furthermore, we previously reported that the serum sIL-2R level increased with the advance of disease stage in non-small cell lung cancer, especially in adenocarcinoma [9]. However, it remains to be clarified as to why the serum levels of sIL-2R increase in solid neoplasms. To address this issue, we investigated the origin of sIL-2R in patients with pulmonary adenocarcinoma using surgically resected specimens.

2. Materials and methods

2.1. Putients

Surgical specimens were obtained from patients with primary pulmonary adeno- carcinoma who had undergone surgery at the Kyushu Cancer Center. None of the patients had received any anti-cancer therapy prior to surgery.

2.2. Preparation of tumor tissue cells

Both fresh tumor tissues and normal lung tissues were excised from surgical specimens and then were transported to the laboratory immediately after removal. The specimens were minced into small pieces with scissors and filtered through a stainless mesh. The resultant cell suspension was subjected to Ficoll-Hypaque gradient (LSM, Litton Bionetics, Kensington, MD) centrifugation (1000 x g, 30 min). The interface was collected and cultured in a complete culture medium (CM) at a cell density of 5 x 105/ml. The CM consisted of RPM1 1640 supplemented with 100 U/ml penicillin, 100 pug/ml streptomycin and 10% heat-inactivated fetal bovine serum. Cell viability was usually more than 90%, as determined by Trypan blue dye exclusion. After 24 h incubation at 37°C in 5% CO?, both adherent cells (mainly macrophages) and non-adherent cells (mainly tumor cells and lymphocytes) were harvested by vigorous pipetting and a jet stream of CM through a 27 gauge needle, and cytocentrifuged onto glass slides. The cytocentrifuged specimens were stored at - 80°C until use. At the same time, the culture supernatants were also collected and stored at - 80°C until use.

2.3. Immunocytochemical staining

The cytocentrifuged specimens were stained by the streptavidin-biotin method (Histofine SAB-PO (M) kit, Nichirei, Tokyo, Japan) using the following anti-IL-2R

T. Yano et al. / Lung Cancer 16 (1996) 13-19 15

monoclonal antibodies (MAbs); anti-IL-2Ra (CD25) MAb (ACT-l, IgGl, 1 pg/ml; DAKO, Denmark), anti-IL-2RP (~75) MAb (Mik-P 1, IgG2a, 1 pg/ml; Nichirei, Japan). Normal murine serum was used as a negative control for the primary antibody. To further ensure that positive staining with those anti-IL-2R MAbs was not attributed to non-specific binding through Fc receptors, two irrelevant isotype- matched MAbs (NU-T,,, against CD4, IgGl, Nichirei; NU-T3 against CD3, IgG2a, Nichirei) were also used as a negative control. The immunostained speci- mens were thereafter counterstained with Mayer’s hematoxylin. Such a nuclear staining with hematoxylin could make it easy to discriminate tumor cells from other types of cells, especially macrophages. Each slide was scored according to the percentage of positively stained cells as follows: + + , positive cells 2 50%; + , positive cells 2 10%; f , positive cells < 10%; - , totally negative.

2.4. Enzyme immunoassay to determine the sIL-2R levels of the culture supernatants

The sIL-2R levels of culture supernatants were measured with a sandwich enzyme immunoassay using commercially available kits (Immunotech, Marseille, France). The sensitivity of the EIA kit was 5 PM. The intra-assay coefficient of variation ranged from 4.9% to 6.6% while the inter-assay coefficient of variation ranged from 5.7% to 11.4%. All samples were assayed in duplicate.

3. Results

3.1. sIL-2R in the culture supernatants of cells isolated from tumor tissues

The cells prepared from six cases with lung adenocarcinoma were assayed for sIL-2R released in the culture supernatants using an enzyme immunoassay. In three of six cases (cases l-3), a substantial level (12.5-27.8 x 10W2 PM/h/lo’ cells) of sIL-2R was identified in the tumor tissue culture (Fig. 1). No correlation was observed between the presence of sIL-2R and proportions of contaminated lymphocytes in the tumor tissue culture (case 1, 43.0%; case 2, 14.8%; case 3, 36.5%; case 4, 33.1%; case 5, 32.6%; case 6, 35.8%). In contrast, no sIL-2R was detected in any normal lung tissue culture.

3.2. IL-2Ra (CD25) expression of tumor cells

To investigate the origin of sIL-2R in the culture supernatant, cells isolated from the tumor tissues were immunocytochemically stained with the anti-IL-2Ra (CD25) antibody. In four cases including the three cases with the presence of sIL-2R in the culture supernatant, various percentages of tumor cells were positively stained with the anti-IL-2Rcc antibody (Table 1, Fig. 2a). In the other two cases, no tumor cells were stained at all. In some cases (cases 2, 4) only a small proportion ( < 5%) of macrophages were stained. To ensure that positive staining with the anti-IL-2Rcc

16 T. Yano et al. 1 Lung Cancer 16 (1996) 13- 19

T: tumor tissue N: normal tissue * : not determined

TN TN TN TN TN TN Case 1 Case 2 Case 3 Case 4 Case 5 Case 6

Fig. 1. sIL-2R concentration in the tumor tissue culture supematants.

MAb was not attributed to non-specific binding, the samples were stained with the irrelevant IgGl isotype MAb (anti-CD4). As expected, only some lymphocytes scattered around tumor cells were positive for such a MAb while tumor cells were negative (Fig. 2~). Based on the above results, extensive examinations to determine the expression of IL-2R (CD25) in pulmonary adenocarcinoma cells were con- ducted. In seven of 16 cases (43.8%) various proportions of tumor cells were positive for IL-2Ra (CD25).

3.3. IL-2Rp @75) expression of tumor cells

Since the functional IL-2R consists of an CI subunit (~55) and a p subunit (~75) the expression of IL-2RP (~75) was also immunocytochemically examined. A representative positive sample of IL-2R/3 is shown in Fig. 2b. As summarized in Table 2, IL-2RP was more frequently expressed by the tumor cells than IL-2Ra.

Table 1 The immunocytochemical staining results with anti-IL-2Ra (CD29 antibody

Case Tumor cells Macrophages Lymphocytes

1 ++ - -

2 ++ * -

3 + - -

4 - * -

5 - - -

6 + - f

+ +, positive cells250%; +, positive cells> 10%; * , positive cells< 10%; -, totally negative.

T. Yano et al. /Lung Cancer 16 (1996) 13-19 17

Fig. 2. Immunostaining of pulmonary adenocarcinoma cells with (a) anti-IL-2Ra (CD25) antibody, (b) anti-IL-2R/I (~75) antibody, and (c) anti-CD4 antibody (case 1, magnification: x 400).

4. Discussion

The present study showed that sIL-2R were present in the cellular supernatants of pulmonary adenocarcinoma tissue while, in addition, the immunoreactivity of IL-2Rcr (CD25) was localized in the tumor cells. These findings thus suggested that the cancer cells by themselves are the possible origin of high amounts of serum sIL-2R in lung cancer, while, in addition, the serum sIL-2R levels should thus depend on both the tumor burden and the proportion of tumor cells expressing IL-2R. If this is true, then our previous findings that the serum sIL-2R levels increased with the advance of disease stage, but not in all cases with advanced stages [9], can therefore be easily explained.

The presence of IL-2R has been reported on both non-hematopoietic normal [5] and malignant cells [3,6,12]. However, the biological role of IL-2R expressed by non-hematopoietic cells still remains unclear. In T lymphocytes, it is generally known that IL-2Rar (~55) alone cannot carry the signal transduction of prolifera-

Table 2 The immunocytochemical staining results with anti-IL-2Rb antibody

Case Tumor cells Macrophages Lymphocytes

1 ++ - * 2 ++ - - 3 ++ - k 4 - - - 5 - - - 6 + - -

+ + , positive cells 2 50%; + , positive cells 2 10%; + , positive cells < 10%: - . totally negative.

18 T. Yano et al. 1 Lung Cancer 16 (1996) 13-19

tion. Therefore, in the present study, we further immunocytochemically examined the expression of IL-2R/3 (~75) by pulmonary adenocarcinoma cells. Interestingly, those tumor cells also expressed IL-2Rj3 more frequently than IL-2Rc(. Recently, it has been reported that some cancer cells express both an IL-2R c( chain (~55) and a /j’ chain (p75), while, in addition, exogenously administered IL-2 can also suppress the proliferation of cancer cells expressing IL-2R [l,lO]. It is therefore suggested that IL-2R may play a role in tumor cell growth. Since the present study was conducted to investigate the origin of serum sIL-2R (a-chain, ~55) in lung cancer, the function of the IL-2R was not examined. As a result, we could not discuss the proliferative function of IL-2R on tumor cells. However, in the future, the immunosuppressive effect of sIL-2R released both locally and systemically from tumor cells should be studied further since sIL-2R can reduce the availability of IL-2 for IL-2 dependent immunity [8].

Acknowledgements

We thank Dr B.T. Quinn, Kyushu University, for his critical review and Yumiko Oshima and Yuko Ishibashi for their expert help in the preparation of this manuscript.

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