Interleukin 8: An Autocrine Growth Factor for Malignant Mesothelioma

MM,² unlike other pulmonary tumors, is characterized by local tumor extension and invasion of surrounding tissue without distant metastasis. It is a relatively rare tumor, but its incidence appears to be increasing. The present annual incidence of MMs is ∼11.4 per one million in men and 2.8 per one million in women in the United States (1). Despite attempts to advance early diagnosis and use combination therapies, the prognosis of the patients with MM is poor. The average survival time is about 7–9 months after diagnosis (1, 2). Unfortunately, there is no effective treatment for prolonging survival at this time.

Several tumors produce specific autocrine growth factors that improve their growth capacity and ability for metastasis (3, 4, 5). IL-8, a member of the super gene family of C-X-C chemokines, is chemotactic for and activates neutrophils (6, 7, 8, 9). By increasing expansion of adhesion molecules (10), it also facilitates leukocyte-endothelial and leukocyte-mesothelial interactions, which are involved in the inflammatory invasion of neutrophils. IL-8 has been described as an important angiogenic factor for the development of new capillaries in vivo (11, 12, 13). In empyema, IL-8 levels were reported to be severalfold higher than mesothelioma and were comparable in parapneumonic effusion; however, in tuberculosis, the IL-8 levels were very low (14). Several immune and nonimmune cells, including endothelial and mesothelial cells, have been found to produce IL-8 (15, 16). In addition, IL-8 has also been shown to be generated by a variety of tumors such as melanoma (3) and bronchogenic carcinoma (17). It serves as an autocrine growth factor, in the case of melanoma, allowing for local tumor growth and invasion (18, 19).

The aim of the present study was to determine differences in IL-8 levels from human MM effusions and CHF pleural fluids as control. It was also designed to investigate the autocrine IL-8 production by different mesothelioma and mesothelial cells and describes the potential role of IL-8 in the autocrine growth regulation of mesothelioma and mesothelial cells. We found elevated levels of IL-8 in human mesothelioma pleural fluids compared with CHF. In vitro, mesothelioma cells, but not mesothelial cells, produced high levels of IL-8. We verified that IL-8 is a direct growth-potentiating factor for mesothelioma but not for mesothelial cells.

Recombinant human IL-8, monoclonal anti-IL-8 Ab (mouse IgG1; this Ab does not cross-react with other C-X-C chemokines), mouse IgG-isotype and specific polyclonal goat anti-IL-8 Ab (polyclonal IL-8 Ab; this Ab does not cross-react with MGSA, neutrophil activating protein-2, or any other chemokine) were purchased from R & D Systems (Minneapolis, MN). Purified nonspecific goat IgG was purchased from Sigma Chemical Co. (St. Louis, MO). [³H]Thymidine was obtained from Amersham Life Science (Arlington Heights, IL).

One mesothelial cell line (CRL-9444) and three mesothelioma cell lines (CRL-2081, CRL-5915, and CRL-5820) were purchased from American Type Culture Collection (Rockville, MD). The mesothelial cells were resuspended in Ham’s 199 culture medium (Life Technologies, Inc., Grand Island, NY) containing 10% fetal bovine serum (Harlan Bioproducts, Indianapolis, IN), penicillin (100 units/ml), and streptomycin (100 g/ml). Mesothelioma cells were cultured in RPMI 1640 culture medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were plated in 75-cm² tissue culture flasks (Corning Costar Corp., Cambridge, MA) and incubated at 37°C in 5% CO₂ and 95% air. The monolayer was confluent between 8 and 10 days. Mesothelial cells had a classic cobblestone morphology (20), absence of factor VIII antigen, and presence of cytokeratin (21). All cells were used between the third and seventh passages.

Twelve patients with pleural effusions secondary to mesothelioma (n = 6) and CHF (n = 6) were studied. Pleural fluids were obtained after informed consent during diagnostic thoracocentesis, as described previously (16, 22). In all MM patients, the effusions were exudates with culture negative for infective organisms. A malignant effusion secondary to mesothelioma was defined by at least one of the following criteria: malignant mesothelioma cells in the pleural fluid on cytological examination, or mesothelioma on closed pleural biopsy. Biopsy tissue was examined by microscopy and a special panel of stains to diagnose MM. Three patients had a mixed-type mesothelioma: one was an epithelial type mesothelioma, and two were sarcomatous in nature. Transudative pleural effusions from symptomatic patients with a diagnosis of CHF served as control. The fluids were saved at −70°C for IL-8 ELISA.

To determine the constitutive production of IL-8 without stimulation, CRL-2081, CRL-5915, CRL-5820 mesothelioma, and CRL-9444 mesothelial cells were plated in 75-cm² tissue culture flasks (1 × 10⁶ cells/flask), grown to confluence, washed, and replaced with serum-free RPMI 1640 (8 ml/flask) for mesothelioma cells and Ham’s 199 for mesothelial cells. Conditioned media were collected at 24, 48, 72, and 96 h, centrifuged at 900 × g for 10 min, and saved at −70°C for IL-8 analysis by ELISA.

Extracellular immunoreactive IL-8 levels were measured by “sandwich” enzyme immunoassay (R&D System, Minneapolis, MN) as described previously (16). The samples were added to 96-well microtiter plates, which were coated with murine monoclonal Ab to IL-8. The unbound protein was washed three times, and an enzyme-linked polyclonal antibody specific to IL-8 was added. The plates were again washed three times, and substrate solution was added to the wells. After 30 min of incubation, stop solution was added to each well. The amount of IL-8 was determined by absorbance of the samples by comparing the standards at 450 nm using the ELISA reader.

Unstimulated mesothelial and mesothelioma cells were immunostained with avidin-biotin conjugate and peroxidase as described previously (7). The cells were cultured in Lab-Tek chamber slides (Nunc, Naperville, IL). The confluent cultures were fixed in absolute methanol, and endogenous peroxidase activity was quenched with 1% hydrogen peroxide. Nonspecific binding sites were blocked with 1% normal horse serum for 50 min, permeabilized in 0.1% Triton X-100 for 10 min, and overlaid with 1:1000 dilution of mouse monoclonal anti-human IL-8 Ab for 30 min. The negative controls were overlaid with mouse IgG isotype. The slides were rinsed with PBS and overlaid with avidin-biotin conjugated goat anti-mouse IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) for 60 min. The slides were washed and incubated for 5 min in peroxidase substrate solution (3,3′-diaminobenzidine) washed in PBS and counterstained by Mayer’s hematoxylin (Sigma Chemical Co.). The cells were dehydrated in graded alcohol and xylene and covered with glass.

Mesothelial and mesothelioma cell line proliferation was determined by a [³H]thymidine uptake assay as described by Lauber et al. (23) and Hott et al. (24). Cells (2 × 10⁴) were plated in 48-well plates and grown to confluence. Then the medium was replaced by RPMI 1640 or Ham’s 199 and incubated overnight. Various concentrations of IL-8 (5, 25, 50, and 100 ng/ml), polyclonal goat anti-human IL-8 specific Ab (1, 5, 10 and 20 μg/ml), or nonspecific purified goat IgG (1, 5, 10, and 20 μg/ml) as control were added. After 24-h incubation at 37°C in 5% CO₂ 95% air, 0.5 μCi of [³H]thymidine was added to each well, and the plates were reincubated for 24 h. The cells were washed three times in complete HBSS and harvested from the plates with 0.5% EDTA, precipitated in 5% trichloroacetic acid, and centrifuged at 3000 rpm for 10 min. They were resuspended in 0.1 n NaOH, placed in scintillation fluid, and counted in a β-scintillation counter. All experiments were done in triplicate, and data are expressed as percentage of stimulation (cpm produced by the test factors/cpm produced by SFMx 100). In addition, mesothelial and mesothelioma cell proliferation was measured by direct cell counts (hemocytometer). After 48 h of seeding, the cell culture medium was changed to SFM plus either 50 ng/ml IL-8 or 10 μg/ml anti-IL-8 Ab or SFM alone. Cell counts were obtained after 48 h of incubation. The selected IL-8 concentrations had no effect on the mesothelial and mesothelioma cell viability as demonstrated by trypan blue exclusion.

All values are expressed as mean ± SD. Data were compared using Student’s t test and ANOVA. Values were considered to be statistically significant when P was <0.05.

Twelve patients with pleural effusions were studied. Six had pleural effusions secondary to mesothelioma (three mixed, one epithelial, and two sarcomatous). A control group of six patients had CHF with bilateral transudative effusions. IL-8 levels (Fig. 1) were significantly (P < 0.001) elevated in patients with MM (8.78 ± 3.59 ng/ml) compared with patients with CHF (0.07 ± 0.05 ng/ml).

All three mesothelioma cell lines, tested by ELISA, produced detectable levels of human IL-8 without stimulation (Fig. 2). However, IL-8 was not detected in supernatants from mesothelial cells, even after 96 h of incubation. We observed a significant, time-dependent increase in IL-8 levels in all three mesothelioma supernatants. After 96 h of incubation, IL-8 levels ranged between 36.2 ± 6.3 pg/ml in the CRL-5820 and 2689.4 ± 60.2 pg/ml in the CRL-2081 cell line. In the CRL-5915 mesothelioma cell line, the IL-8 concentration was 516.4 ± 19.0 pg/ml at the end of the incubation period. In the CRL-5820 cell line, the IL-8 levels were insignificant throughout the experiment and are close to the minimum detectable sensitivity of the test (31.2 pg/ml).

We also used immunohistological localization to demonstrate the cellular antigenic IL-8 in mesothelioma and mesothelial cells. Expression of immunoreactive cell-associated IL-8 by all three mesothelioma cell lines can be seen in Fig. 3, whereas the mesothelial cell line has no detectable IL-8 production.

To determine the effect of human IL-8 protein in regulating human mesothelioma and mesothelial cell proliferation, various concentrations of IL-8 (5, 25, 50, and 100 ng/ml), with varying concentrations of polyclonal goat anti-IL-8 Ab (1, 5, 10, and 20 μg/ml; data not shown), or nonspecific goat IgG (control) were added to the cell cultures grown in SFM. The optimal proliferation was noticed at 50 ng/ml of IL-8, and the maximum inhibition of proliferation was observed with 10 μg/ml of polyclonal IL-8 Ab; therefore, this concentration was used in neutralization studies.

IL-8 stimulated the proliferation of CRL-2081 and 5915 mesothelioma cell lines in a dose-dependent manner as determined by [³H]thymidine incorporation (Fig. 4). Maximal [³H]thymidine uptake occurred in response to 50 ng/ml of IL-8 [CRL-2081, 146.3 ± 3.6% and CRL-5915, 112.3 ± 1.6% (P < 0.001), when compared with SFM, as control]. Incubation with 100 ng/ml of IL-8 did not increase the [³H]thymidine incorporation above the level of 50 ng/ml IL-8 effect [CRL-2081, 138.2 ± 2.7% (P < 0.001) and CRL-5915, 105.9 ± 2.3% (P < 0.05), when compared with SFM]. The proliferation behavior of the CRL-5820 mesothelioma and the mesothelial (CRL-9444) cell lines, however, remained unaltered compared with untreated controls.

Furthermore, addition of specific polyclonal IL-8 Ab significantly inhibited the proliferation of CRL-2081 (61.8 ± 6.7; P < 0.001) and CRL-5915 (50.2 ± 1.5; P < 0.001) mesothelioma cell lines when compared with SFM (Fig. 5,A). However, in CRL-5820 mesothelioma and the mesothelial cells, there was no significant difference in [³H]thymidine incorporation with neutralizing IL-8 Ab. Nonspecific goat IgG was not effective (data not shown).%Direct cell count by hemocytometer also demonstrated that IL-8 induced the proliferation, and the addition of IL-8 Ab inhibited cell proliferation in CRL-2081 [152.1 ± 3.0 (P < 0.001) and 77.5 ± 2.0 (P < 0.05)] and 5915 [110.0 ± 2.4 (P < 0.05) and 68.0 ± 2.8 (P < 0.001)] mesothelioma cell lines (Fig. 5 B). There was no significant response to IL-8 as well as IL-8 Ab in the CRL-5820 and mesothelial cell lines.

Our results demonstrate that pleural fluids from patients with mesothelioma contained significantly greater IL-8 levels than did pleural fluid from CHF patients. Malignant pleural effusions, other than mesothelioma, also contain immunoreactive IL-8; however, it is present in significantly smaller quantities than we observed in mesothelioma pleural fluids (7). All three mesothelioma cell lines constitutively expressed IL-8; however, there was no detectable IL-8 in the supernatants from resting mesothelial cells. In the CRL-5820 mesothelioma cell line, the IL-8 levels were low compared with CRL-2081 and CRL-5915 mesothelioma cell lines. In CRL-2081 and CRL-5915, a significant increase in IL-8 production was observed during 96 h of incubation.

We also studied the effect of exogenous IL-8 and the requirement of IL-8 for growth and proliferation in mesothelioma and mesothelial cells as measured by the [³H]thymidine incorporation and direct cell counts. Treatment with exogenous human IL-8 showed a significant, dose-dependent increase in cell proliferation in CRL-2081 mesothelioma in which highest level of autocrine IL-8 production (2.7 ng/ml) was observed. The increase in proliferative activity was low but significant in CRL-5915 mesothelioma cells, where the IL-8 production (0.5 ng/ml) was lower. Neutralization of IL-8 in these cell lines resulted in a decrease in their proliferation. This is supported by the fact that anti-IL-8 Ab was able to decrease proliferation of these cell lines in a significant fashion. Neither [³H]thymidine incorporation nor cell counts changed after either addition of exogenous IL-8 or anti-IL-8 Ab in the CRL-5820 mesothelioma and mesothelial cell line, where the IL-8 production was nearly undetectable or absent. These findings suggest that the lack of IL-8 binding sites on these cells may be responsible for their unresponsiveness to IL-8.

It is well established that tumor cells produce a number of factors that promote tumor growth, tissue invasion, and metastatic spread. Melanoma cells secreted autocrine growth-stimulatory factors, such as MGSA (MGSA/GROα) or basic fibroblast growth factor. MGSA was found to have mitogenic activity for the melanoma cell line Hs 294T, which produces this factor (25). High levels of MGSA secretion was observed in diverse melanoma cell lines; however, it was absent in benign nevus cells (26). Recently, another member of the C-X-C supergene family, IL-8, was found to be secreted by melanoma cells in an autocrine fashion. IL-8, potentiates proliferation of melanoma cells and plays an important role in the tumorigenesis and growth of malignant melanoma (3, 18). This is supported by the fact that antisense oligonucleotides targeted against human IL-8 mRNA inhibited cell proliferation, colony formation in soft agar, and secretion of IL-8 into culture supernatants (18). In vivo experiments demonstrated that expression of IL-8 correlates with the metastatic capability of 13 human melanoma cell lines, which were injected into BALB/c mice (27). We demonstrated that mesothelioma cells similar to melanoma cells release biologically active IL-8, which causes proliferation of mesothelioma cells. The higher proliferative capacity of the tumor is directly related to its capacity to secrete more IL-8, and the differential IL-8 response in different mesothelioma cell lines noticed may be related to their aggressive growth.

Recent studies show that IL-8 receptor B (CXCR-2) is present on melanoma cells and plays a role in the cell growth (28). A specific Ab directed against the NH₂ terminus of IL-8 receptor B (28) as well as a novel inhibitor of IL-8 receptor B (25) have been shown to suppress the growth of melanoma cells. Because of high a degree of sequence homology of MGSA and IL-8, both cause receptor signal transduction when binding to the IL-8 receptor B (29). However, the presence of another, specific MGSA binding site has been theorized. Interestingly, IL-8 as well as MGSA bind with high affinity to the IL-8 receptor B, but the affinity of MGSA to the IL-8 receptor A is weak compared with the high affinity of IL-8 for this receptor. In an acute burn wound, IL-8 receptor B was restricted to the basal proliferating compartment in healing skin wounds, whereas higher concentrations of IL-8 were detected in the outer layers of the epidermis, providing a chemotactic gradient for the migration of keratinocytes (30). These results also suggest that the presence of IL-8 receptor B is critical for the proliferative effect of IL-8.

IL-8 has been classified primarily by proinflammatory properties (31), but it has multiple effects. In response to inflammatory stimuli such as lipopolysaccharide, IL-1, and tumor necrosis factor-α, IL-8 is produced by a wide variety of cell types including monocytes, neutrophils, lymphocytes, and endothelial and mesothelial cells (11, 16, 31). IL-8 activates neutrophils and increases their adhesion to endothelial cells, which leads to directional transendothelial migration (28). The effects of IL-8 is not confined only to neutrophils, but it has effects on T and B lymphocytes, basophils, and IL-2-activated natural killer cells as well. IL-8 has a strong chemotactic activity on neutrophils. The described complex effects suggest a functional role for IL-8 in the first line of host defense in vivo. In addition, IL-8 induces proliferation and chemotaxis of human umbilical vein endothelial cells (11) in vitro. In the rat sponge implant model (19) and in the corneal micropocket model (11), IL-8 has been found to be a potent angiogenic factor in a pattern similar to that seen with tumor necrosis factor-α, fibroblast growth factor, and vascular endothelial growth factor. Tumor growth beyond 1–2 mm³ was dependent on angiogenesis (32). The dysregulation of the balance between angiogenic and angiostatic factors is one of the mechanisms maintaining tumor growth by neovascularization. IL-8 is expressed in and secreted by a variety of transformed neoplastic cells (3, 33). Significantly elevated IL-8 levels were found in human non-small cell lung cancer (adenocarcinoma and squamous cell carcinoma), which were four times greater than normal lung tissue (17). These observations suggest that the more aggressive course of adenocarcinomas could be related to their capacity to generate IL-8. Inhibition of IL-8, by addition of neutralizing antisera, attenuated both corneal (17) and neoplastic (33) neovascularization. In non-small cell lung cancer, IL-8 did not act as an autocrine growth factor for proliferation; however, treatment with neutralizing antibody to IL-8 resulted in a decline of tumor-associated vascular density, a reduction in primary tumor size, and reduction of the rate of metastasis (33). This well-described angiogenic effect of IL-8 could also play an important role in the spread and growth of malignant mesotheliomas.

The findings described in this report demonstrate for the first time that IL-8, which is produced by mesothelioma cells, is a important factor in growth regulation in a subgroup of human MMs. Likewise, the progression of MM may correlate with the level of autocrine IL-8 production. However, it remains to be determined whether IL-8 mediates its proliferative effects through IL-8 receptor B or through a related molecule of the same receptor family, as in melanoma cells (25). The mechanism responsible for these findings needs to be studied further.