Japanese Journal of Clinical Oncology 30:53-58 (2000)
© 2000 Foundation for Promotion of Cancer Research
Effect of IL-6 Elevation in Malignant Pleural Effusion on Hyperfibrinogenemia in Lung Cancer Patients
1Department of Internal Medicine and 2Clinical Laboratory, Toneyama National Hospital, Toyonaka, Osaka, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: The involvements of interleukin-6 (IL-6) and fibrinogen in cancer development were elucidated independently, irrespective of IL-6 activity to induce fibrinogen. This study was undertaken to clarify the clinicopathological association of these molecules in lung cancer patients with malignant pleurisy.
Methods: IL-6, fibrinogen and the related molecules in blood and pleural effusion of 38 patients were assayed at 3-day intervals.
Results: IL-6 levels were elevated in sera of 27 cases (71.1%) and in all the effusions with mean values of 20.5 and 9970.5 pg/ml, respectively. Their correlation in 22 cases who were examined on the same day was statistically strong (r = 0.902, p < 0.0001). Occasional elevations of tumor necrosis factor-
were independent of IL-6 elevation. Levels of plasma fibrinogen, fibrin(ogen) degradation products (FDP) and C-reactive protein (CRP) were more frequently elevated in the IL-6-elevated cases than those without IL-6 elevation. In all pleural effusions, fibrinogen levels were significantly decreased to <150 mg/dl with large elevations of FDP level. Immunocytologically, IL-6 was detected in cancer cells in 16 cases of adenocarcinoma in addition to host pleural cells, but its cellular positivity was not reflected in the IL-6 level in each pleural effusion.
Conclusion: Compared with lung cancer patients without malignant pleurisy, IL-6, fibrinogen, FDP and CRP levels in patients with malignant pleurisy were increased more frequently in their peripheral blood. These were basically attributed to systemic leakage of IL-6 from the affected pleural cavity, in which plasma fibrinogen induced in response to serum IL-6 was exudated and degraded predominantly to FDP.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Previous studies on the regulation of coagulation/fibrinolysis in the tumor-bearing state of lung cancer have demonstrated elevation of plasma fibrinogen levels in advanced stages of the disease (1), fibrinogen localization into the tumor tissue (2,3) and abnormal regulation in the coagulation system in malignant pleurisy by local treatment with sclerosing agents such as quinacrine (4).
Independently of those studies, the diverse effect of IL-6 on the proliferation of lung cancer cells (5), elevated levels of IL-6 in serum and malignant pleural effusion of lung cancer patients (6–8) and the elevation of IL-6 levels by local instillation of either IL-2 (9) or tetracycline (10) have been reported.
Together with the potent activity of IL-6 to produce acute-phase reactants including fibrinogen, IL-6 may affect tumor development in lung cancer patients not only directly but also indirectly through induction of hyperfibrinogenemia.
Our previous studies have demonstrated the association of serum IL-6 elevation with hyperfibrinogenemia in lung cancer patients without malignant pleural effusion (11,12). In the present study, we investigated the clinicopathological relationship between local and systemic levels of IL-6 and fibrinogen in lung cancer patients with malignant pleural effusion, in whom a high IL-6 elevation in the effusion was observed but its biological influence on the patients’ pathophysiological state had hardly been examined.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Patients
A total of 38 lung cancer patients with pleural effusion who had been admitted to our institution during the previous 3 years were studied. Their primary tumors were confirmed by cytological or histological examination and their pleural effusions were also positive for malignant cells. All cases were examined before starting the local treatment for pleural effusion. Twelve patients were recurrent cases with a mean remission interval of 283 days after prior therapy for primary lung cancer and the others had no prior therapy at all. Their peripheral venous blood and pleural effusion were obtained on the same day (22 cases) or within a 3-day period (16 cases), according to our institutional guidelines for informed consent, and the samples were kept at –70°C until assay for IL-6 and TNF-
. CRP, fibrinogen and FDP in the venous blood were examined by routine laboratory tests. Levels of CRP of >0.3 mg/dl, fibrinogen levels of >450 mg/dl and FDP levels of 10 µg/ml were regarded as elevated levels. Fibrinogen and FDP in the pleural effusions were also examined.
Measurements of IL-6 and TNF- in Serum and Pleural Effusion and Immunohistochemical Detection of IL-6 in Pleural Cells
Levels of IL-6 and TNF- in serum and pleural effusion were measured using commercially available ELISA kits (IL-6, Fuji Lebio, Tokyo, Japan; TNF-, JIMRO, Gumma, Japan). Each kit had a sensitivity limit of 4 pg/ml and no detectable IL-6 and TNF- were found in sera from healthy subjects (12). For detecting the source of IL-6 in the effusion, freshly obtained pleural cells were smeared on a glass slide, fixed with acetone, reacted with blocking agents and treated with polyclonal goat anti-human IL-6 antibody (Innogenetics, Zwendrecht, Belgium) for 60 min. Then the specimens were washed, treated using a Histofine SAB-PO(G) kit (Nichirei, Tokyo, Japan) and diaminobenzidine according to the manufacturer’s protocol and counterstained with hematoxylin.
Statistical Analysis
Student’s t-test was used to determine the statistical significance of differences in mean ± standard deviation (SD). The Mann–Whitney test was used for non-parametric analysis. Categorical data were compared by a 
2 test. p-Values <0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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The clinicopathological characteristics of the patients studied are shown in Table 1. Histologically, 71.1% of the total cases were adenocarcinoma. The levels of IL-6 assayed were found to be markedly elevated in all the pleural effusions and also moderately in the sera of 27 patients (71.1%). In contrast, TNF-
elevation was found in only four pleural effusions with a mean level of 13.3 pg/ml.
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No significant relationship was found between IL-6 levels in serum or pleural effusion and clinicopathological characteristics (Table 2) or between IL-6 levels in serum or pleural effusion and TNF-
elevation (data not shown).
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As shown in Fig. 1, however, a significant correlation was observed between IL-6 levels in serum and pleural effusions (r = 0.645, p < 0.001). The significance became greater (r = 0.902, p < 0.0001) when tested in the 22 patients whose paired samples of serum and pleural effusion were obtained on the same day.
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CRP, fibrinogen and FDP in the peripheral blood and pleural effusion were examined within 3 days of the IL-6 assay (Table 3). The incidences (%) of elevated levels of FDP and CRP were higher in the 27 patients who also had elevated serum IL-6 levels.
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The levels of serum IL-6 were significantly correlated with CRP but not fibrinogen in 22 patients in whom the assays were performed on the different days but within a 3-day period, as shown in Fig. 2.
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Levels of fibrinogen and FDP in pleural effusions were also examined in 31 patients. In all the effusions, the fibrinogen level was significantly lower than the level in plasma, falling to a level of <150 mg/dl (Fig. 3A). In addition, the increase in the FDP level over a wide range of values from 115 to 2436 g/ml was significantly correlated with IL-6 levels in the pleural effusions (r = 0.579, p < 0.001) (Fig. 3B). However, no close relationship was found between FDP levels in serum and pleural effusion (data not shown).
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Immunocytological detection of IL-6 in the pleural effusion was performed in 22 cases, consisting of 19 cases of adenocarcinoma and one case each of squamous cell carcinoma, large cell carcinoma and small cell carcinoma. Positive staining was observed in 16 adenocarcinomas, as shown in Fig. 4. In some cases, pleural inflammatory cells were also stained but their densities were variable even in the same specimen. No correlation was found between pleural IL-6 levels and the positivity of IL-6 staining.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Marked elevation of IL-6 was found in all of the pleural effusions examined. It ranged from 775 to 54,100 pg/ml, being far above the increased levels in the paired serum. Elevation of IL-6 in the body fluid has been documented in various types of malignant diseases other than lung cancer (13,14). It has mainly been attributed to secretion from cancer cells, based on evidence that IL-6 levels in the effusion were closely related to the amount of IL-6 released in vitro from cancer cell lines established from the effusion but not to the cytological features of the effusion (7). Of course, autonomous secretion of IL-6 from lung cancer cells has been demonstrated in different cell lines (7,15–17) and biopsied specimens (18). Although it was also immunocytologically detected in many cases of adenocarcinoma in this study (Fig. 4), no correlation with elevation of IL-6 level was observed.
In addition to cancer cells, various host cells such as mesothelial cells and macrophages, which are predominantly involved in the affected pleural tissue and capable of producing IL-6, are also considered to be concerned. Indeed, clinical observations that IL-6 elevation in benign pleural effusion is as high as that in malignant pleural effusion (6,7,19,20) strongly suggest an important contribution from host inflammatory cells to IL-6 elevation in malignant pleural effusion. Thus, IL-6 elevation in the serum of lung cancer patients with malignant pleural effusion may be due mainly to leakage into the systemic circulation of IL-6 produced by cancer cells as well as host cells in both malignant effusion and affected pleural tissue. The correlation of IL-6 levels between serum and pleural effusion shown in Fig.1 strongly supports this idea.
IL-6 produced in the primary lung cancer site may also participate in its elevation in serum, as reported previously (11,12,18). In the present study, however, the incidence of serum IL-6 elevation was 71.1% (27/38 cases), whereas it was 44.3% in 183 lung cancer patients at the corresponding clinical stage (IIIB, IV) without pleural effusion in our previous study (12). This difference in the incidence of serum IL-6 elevations may be due to the major contribution from IL-6 produced in the affected pleural cavity. Although the maximum and mean serum IL-6 levels were low compared with those reported previously (12), the underlying mechanism still remains unknown.
Paracrine stimulation of IL-6 production by TNF-, which has been demonstrated in tumor cells (7,16,17) and host cells (21), may be involved in the IL-6 elevation in the pleural effusion. At present, however, this possibility can be excluded because elevation of TNF- level was found to be rare and not related to markedly high IL-6 levels.
The biological effect of serum IL-6 elevation was evidenced as a difference in the incidence of fibrinogen, FDP and CRP levels in venous blood (Table 3). In addition, it was even more clearly evidenced by significant correlations between levels of serum or pleural IL-6 and CRP (Fig. 2). These, taken together with other findings that no strong correlation was observed between levels of serum IL-6 and plasma fibrinogen and that fibrinogen decreased significantly and FDP increased in proportion to IL-6 levels in the pleural effusion, strongly suggested that plasma fibrinogen, increased in response to the elevated serum IL-6 level, was exudated predominantly into the affected pleural cavity. A similar process has been observed in experimental and human tumors and has been studied mainly with respect to local alterations in coagulation–fibrinolytic factors secreted from tumor tissue (3,22).
Recently, fibrin(ogen) deposition on the surfaces of tumor cells and lymphocytes (23,24) and in primary and invasive lesion of the tumor (25) have been regarded as adverse events leading to tumor development. In addition, IL-6 may also function as a growth stimulating factor (5,26,27). Thus, in patients with malignant pleural effusion in whom the serum IL-6 level is substantially increased, IL-6 may promote cancer development not only directly but also indirectly through induction of hyperfibrinogenemia. In this regard, local treatment with intrapleural instillation of agents such as bleomycin, tetracycline, quinacrine or IL-2, which have been shown to increase the levels of plasma fibrinogen and IL-6 (4,9,10), needs to be reconsidered for its therapeutic rationale, although it has generally been used to produce pleurodesis for preventing pleural effusion.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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This study was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan and a Grant from the Osaka Foundation for Promotion of Clinical Immunology.
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FOOTNOTES |
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+ For reprints and all correspondence; Takeshi Ogura, Department of Internal Medicine, Toneyama National Hospital, 5–1–1 Toneyama, Toyonaka, Osaka 560-8552, JapanAbbreviations: IL-6, interleukin-6; CRP, C-reactive protein; FDP, fibrin(ogen) degradation product; TNF-
, tumor necrosis factor- 
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Received August 27, 1999; accepted November 2, 1999.
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