Clin. exp. Immunol. (1990) 80, 350-353
Detection of tumour necrosis factor/cachectin in pleural effusion of patients with lung cancer Y.
Y. UCHIYAMA*, S. HASEGAWA4, T. KINOSHITA§, K. MITSUI§, H. KOJIMAt & T. FUJITAt Departments of *Anatomy and timmunology, Institute of Basic Medical Sciences, TDepartment of Respiratory Internal Medicine, and §Thoracic Surgery, Institute of Clinical Medical Sciences, University of Tsukuba, Tsukuba, Japan
(Acceptedfor publication 4 December 1989)
SUMMARY We have found that the pleural effusion obtained from a patient with lung cancer (adenocarcinoma) 'has cytotoxic activity against the patient's lung cancer cells. This finding occurred in the course of establishing a lung cancer cell line from the patient's pleural effusion. The cytotoxic factor was partially purified from the pleural effusion and characterized. It had cytotoxicity against L-929 mouse fibroblasts in the standard 18-h killing assay of tumour necrosis factor (TNF). By molecular sieving chromatography, the activity appeared at molecular weight of 50 000. This activity was completely blocked by a monoclonal antibody to TNF. From these results, we conclude that the cytotoxic factor in the pleural effusion is TNF. The concentration of TNF in the pleural effusion was 34 5 pg/ml by radioimmunoassay. In addition, we detected TNF activity and protein in two other cases of carcinomatous pleural effusion. Therefore, it would appear that in vivo TNF displays cytotoxic activity against cancer cells. Keywords cytotoxicity tumour necrosis factor/cachectin carcinomatous pleural effusion
MATERIALS AND METHODS
Tumour necrosis factor (TNF), which induces haemorrhagic necrosis of some animal tumours, is a protein first detected by Carswell et al. (1975) in the serum of endotoxin-treated animals. It has been shown to be cytotoxic to a variety of transformed cells, including several human and murine cancer cells (Old, 1985). On the other hand, Beutler et al. (1985) isolated a polypeptide host factor, cachectin, which can induce wasting (cachexia) and a lethal state of shock. TNF and cachectin were subsequently found to be identical molecules (Beutler & Cerami, 1986). TNF/cachectin, secreted by the activated macrophage and monocyte, exerts a wide range of biological effects. Despite the clear role of TNF/cachectin in the metabolic disturbances of chronic and acute infection (Beutler & Cerami, 1987), it is unclear whether TNF functions as a cytotoxic factor under physiological conditions. It has recently been reported that TNF-like activity was detected in 50% of serum samples from cancer patients with active diseases (Balkwill et al., 1987). However, its biological significance is uncertain. We report here the detection of TNF in the pleural effusion from patients with lung cancer, and provide evidence that TNF has a cytotoxic effect on the patients' cancer cells.
Subject and sampling procedure The pleural effusion and blood were obtained from a 62-yearold man diagnosed as having adenocarcinoma of the left lung with a complication of pleuritis carcinomatosa (case 1). The patient had not been treated with any carcinostatics when the samples were obtained. The serum was separated from the blood after I h at room temperature, centrifuged for 10 min at 3000 rev/min and stored at -80'C until use. The original pleural effusion, obtained with a catheter, was centrifuged for 10 min at 3000 rev/min and its supernatant was stored as the pleural effusion at - 80'C until use. Both samples of the serum and pleural effusion were heat-treated at 56 C for 30 min before use. Two other cases of pleural effusion (cases 2 and 3) were obtained by the same methods. Antibodies A monoclonal antibody against recombinant TNF and polyclonal antibodies to recombinant lymphotoxin (LT) were kindly supplied by Dainippon Pharmaceutical Co. (Osaka, Japan) and Eisai Co. (Tsukuba, Japan), respectively.
Cells L-929, mouse fibroblast cell line was used as the target cells of TNF assay. This cell line was maintained in RPMI 1640 containing 10% heat-inactivated fetal calf serum (FCS; Flow
Correspondence: Dr Yasuo Uchiyama, Department of Anatomy, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305 Japan.
TNF in pleural effusion
1/16 1/2 16 Sample dilution Fig. 1. Cytotoxic activity against lung cancer cells (a) and L-929 (b). (a) Results are shown for cytotoxicity of crude pleural effusion against lung cancer cells in the presence (closed circles) or absence (open triangles) of 1 jg/mi of actinomycin D. (b) Results are shown for cytotoxicity of crude pleural effusion (closed circles) and serum (open circles) samples against L-929 cells.
laboratory, McLean, VA), 100 U/ml penicillin, and 100 pg/ml streptomycin. For establishment of a lung cancer cell line, the pleural effusion was centrifuged and the precipitated cells were collected. These cells were layered on Ficoll-Hypaque (Pharmacia Japan, Tokyo, Japan) and centrifuged at 500 g for 30 min. More than 90% of mononuclear cells obtained consisted of the cancer cells; they were washed twice with RPMI 1640 medium and cultured in RPMI 1640 supplemented with 10% FCS. Some of them were also cultured in RPMI 1640 containing 10% pleural effusion. For cytotoxic assay, the lung cancer cells cultured in RPMI 1640 containing 10% FCS were used as the target cells. Assay of cytotoxic activity An assay for cytotoxic activity against L-929 cells was performed as described previously (Aggarwal et al., 1985). Briefly, 50 jyl of L-929 cells (5 x 105/ml) suspended in RPMI 1640 with 10% FCS containing 1 jug/ml of actinomycin D (Sigma Chemical Co., St Louis, MO) were layered in a 96-well, flatbottomed culture plate (Falcon Plastics, Oxnard, CA) and incubated with 50 il of a serially diluted sample in a humidified atmosphere at 370C with 5% CO2. After 18 h, these cells were stained with crystal violet, and then 100 p1 of 0-1% SDS were added to each well to solubilize the dye. The amount of stain in each well was determined by Immunoreader NJ-2000 (InterMed Co., Tokyo, Japan) at 540 nm. The amount required for 50% killing of the target cells is defined as I U/ml. The killing of lung cancer cells was tested in a dye reduction assay (Green, Reade & Ware, 1984). Briefly, I04 lung cancer cells in 50 p1 of RPMI 1640 with 10% FCS were added to 50-ju1 samples of serially diluted test materials, which were placed in a 96-well, flat-bottomed culture plate. After an incubation of 18 h at 37 C, 10 pi of a 1% MTT solution were added to each well, and the plate was further incubated for 4 h. At the end of the incubation, 100 ,l of acidified isopropanol with 0 04 N HCl were added to each well to solubilize the MTT formazan. The
amount of stain in each well reader NJ-2000 at 590 nm.
quantified by the Immuno-
Purification of cytotoxic factor Five-hundred microlitres of the pleural effusion were precipitated by ammonium sulphate precipitation by 50-75% of saturation. The precipitates were dissolved and dialysed against phosphate-buffered saline (PBS; pH 7 4) and then against 10 mm Tris-HCl, pH 8-0. The solution was loaded onto a DEAEToyopearl 650S column (Tosoh Co., Tokyo, Japan) and eluted with a linear NaCl gradient. The fractions showing cytotoxic activity against L-929 were pooled, dialysed against 10 mm TrisHCl, pH 8 0, and applied to a Mono Q FPLC column (Pharmacia Japan). The active fractions were concentrated to one-fifth volume by ultrafiltration and analysed by a TSK2000SW gel filtration column (Tosoh). Radioimmunoassay of TNF The concentration of TNF was determined by radioimmunoassay using TNF-cx IRMA kit (Medgenix, Fleurus, Belgium). The sensitivity of this assay is < 10 pg/ml. RESULTS Cytotoxic activity detected in pleural effusion To establish a cell line, the cancer cells obtained from carcinomatous pleural effusion were cultured in RPMI 1640 supplemented with 10% of either FCS or patient's pleural effusion. We noticed that the cancer cells cultured in the medium containing the pleural effusion died within a week. However, the cells cultured in the medium supplemented with FCS grew progressively. Using these lung cancer cells as the target cells, the cytotoxic activity of the pleural effusion was detected. As shown in Fig. la, cytotoxic activity was discerned in the absence of 1 jg/ml of actinomycin D, and increased when actinomycin D was added to the medium. The cytotoxicity of the pleural effusion against L-929, sensitive to TNF, was also measured. As
Y. Ishii et al. Molecular weight (kD) 67 25
Fig. 2. Molecular sieving chromatography of partially purified cytotoxic factor. Partially purified cytotoxic factor was applied to a TSK 2000SW column at a flow rate of 1 ml/min. Shaded bar graphs show cytotoxic activity against L-929 cells in each fraction. The column was calibrated with bovine serum albumin (67 kD), ovalbumin (45 kD), and chymotrypsinogen (25 kD).
shown in Fig. lb, the titre of TNF was 3 U/ml, while no cytotoxic activity was detected in the patient's serum. These results suggest that the pleural effusion contains TNF-like activity.
Characterization of the cytotoxic factor in pleural effusion The cytotoxic factor of the pleural effusion was partially purified by fractionation with ammonium sulphate precipitation and an ion-exchange chromatography by monitoring the cytotoxicity against L-929. The cytotoxic activity of partially purified cytotoxic factor showed a 40-fold higher activity (120 U/ml) than the original. The molecular weight of this cytotoxic factor was determined by a TSK 2000SW molecular sieving column under non-denaturing conditions. As shown in Fig. 2, the peak of cytotoxic activity was noted at a molecular weight of approximately 50 000, corresponding to that of TNF (Wingfield, Pain & Craig, 1987). More than 80% of the total activity were recovered from the molecular weight of 42 000 to 54 000. We then examined the effect of antibodies to TNF and to LT on the activity of partially purified cytotoxic factor. As shown in Fig. 3, a monoclonal antibody to TNF completely abolished the cytotoxic activity of the partially purified cytotoxic factor against L-929 cells. No inhibitory effect was detected by polyclonal antibodies to LT. On the basis of these findings, we conclude that the partially purified cytotoxic factor from the pleural effusion is TNF. The concentration of TNF In the light of the above results, the concentration of TNF was determined by radioimmunoassay. The concentration of TNF detected was 34-5 pg/ml in the original pleural effusion or 2050 pg/ml in partially purified cytotoxic factor (Table 1). This shows that these values correspond well to the activities as noted above. However, TNF in the serum was below the detection limit.
3 x 10-4
3 x 10-5
3 x 106
Dilution of antibody
Fig. 3. Inhibition of cytotoxic activity by antibodies to TNF and LT. Fifty microlitres of L-929 target cells (104) were mixed with 50 pi of partially purified cytotoxic factor in 96-well microtitre plate. A monoclonal antibody to TNF (closed circles) and polyclonal antibodies to LT (open circles) were added to each well at the indicated final dilution. After 18 h, cytotoxicity was measured and the percentage of inhibition was calculated. Without antibody the cytotoxicity was 49 9%.
Table 1. TNF concentrations and activities
Patient Case 1 Serum Original pleural effusion Partially purified cytotoxic factor Case 2 Original pleural effusion Case 3 Original pleural effusion
ND 34 5 2050 0
ND 3-0 140
ND, not detected.
To confirm our results, the concentration and activity of TNF were determined in two other cases of pleural effusion. As shown in Table 1, both protein and activity were recognized.
DISCUSSION The biological functions and molecular properties of TNF, an endogenous factor with cytotoxicity, have been clarified since its initial identification by Carswell et al. (1975). In the present study, we found the cytotoxic factor in the pleural effusion from a patient with lung cancer and identified it as TNF. Although there have been several reports of detection of TNF in serum samples from patients with cancer (Balkwill et al., 1987) and
TNF in pleural effusion infectious diseases (Waage, Halstensen & Espevik, 1987), to our knowledge there has not been any report on the production of TNF in pleural effusion or ascites in cancer patients. A key finding is that the cytotoxic factor in the pleural effusion affected our patient's cancer cells in vitro, although the titre of cytotoxicity was quite low in the absence of actinomycin D. Our patient (case 1) had not received any drugs when the pleural effusion was obtained from him. It is therefore likely that TNF inhibits the growth of cancer cells in the pleural cavity. Further, both cytotoxic activity and TNF were unable to detect cytolytic assay and radioimmunoassay in serum. It has been reported that the serum activity of TNF is labile (Balkwill et al., 1987). Herewith it is reasonable that TNF is discernible only in the pleural effusion. It is generally thought that the principal source of TNF is the macrophage; its production by isolated macrophages has been demonstrated (Beutler et al., 1985). In addition, it has been reported that blood monocytes in cancer patients show increased production of TNF when stimulated with lipopolysaccharides (Nara et al., 1987). Moreover, by means of an ELISA, TNF-like activity has been detected in 50% of freshly obtained serum samples from cancer patients with active diseases (Balkwill et al., 1987). However, it is unclear how TNF is produced in cancer patients. In the present case, it is reasonable to assume that monocytes or macrophages in the pleural effusion interact with tumour cells and produce TNF. We do not yet know whether the production of TNF in pleural effusion or ascites is a common phenomenon. This notion, however, is strongly supported by the present finding that TNF activity and protein are detected in two other cases of pleural effusion. We are currently investigating this problem using the pleural effusions and ascites from patients with various forms of cancer. Nevertheless, our findings strongly suggest that the local administration of recombinant TNF in carcinomatous pleuritis and ascites is of therapeutic benefit.
ACKNOWLEDGMENTS We thank Dr T. Hanada for measuring the concentration of TNF and Mr R. M. Haigh for reading the manuscript.
REFERENCES AGGARWAL, B.B., KOHR, W.J., HASS, P.E., MOFFAT, B., SPENCER, S.A., HENZEL, W.J., BRINGMAN, T.S., NEDWIN, G.E., GOEDDEL, D.V. & HARKINS, R.N. (1985) Human tumour necrosis factor. Production, purification, and characterization. J. biol. Chem. 260, 2345. BALKWILL, F., OSBORNE, R., BURKE, F., NAYLOR, S., TALBOT, D., DURBIN, H., TAVERNIER, J. & FiERs, W. (1987) Evidence for tumour necrosis factor/cachectin production in cancer. Lancet, ii, 1229. BEUTLER, B. & CERAmI, A. (1986) Cachectin and tumour necrosis factor as two sides of the same biological coin. Nature, 320, 584. BEUTLER, B. & CERAMI, A. (1987) Cachitin: more than a tumour necrosis factor. N. Engl. J. Med. 316, 379. BEUTLER, B., MAHONEY, J., LE TRANG, N., PEKALA, P. & CERAMI, A. (1985) Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin induced RAW 264 7 cells. J. exp. Med. 161, 984. CARSWELL, E.A., OLD, L.J., KASSEL, R.L., GREEN, S., FIORE, N. & WILLIAMSON, B. (1975) An endotoxin-induced serum factor that causes necrosis of tumours. Proc. nati Acad. Sci. USA, 72, 3666. GREEN L.M., READE, J.L. & WARE, C.F. (1984) Rapid colormetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. J. immunol. Methods, 70, 257. NARA, K., ODAGIRI, H., FuJiI, M., YAMANAKA, Y., YOKOYAMA, M.,
MORITA, T., SASAKI, M., KON, M. & ABo, T. (1987) Increased production of tumour necrosis factor and prostaglandin E2 by monocytes in cancer patients and its unique modulation by their plasma. Cancer Immunol. Immunother. 25, 126. OLD, L.J. (1985) Tumor necrosis factor (TNF). Science, 230, 630. WAAGE, A., HALSTENSEN, A. & EsPEVIK, T. (1987) Association between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet, i, 355. WINGFIELD, P., PAIN, R.H. & CRAIG, S. (1987) Tumour necrosis factor is a compact trimer. FEBS Lett. 211, 179.