Effect of culture supernatants from two cancer cell lines on healthy donors'' monocytes
P. S. Novellino*, Y. G. Trejo*, M. Beviacqua*, R. H. Bordenave** and L. S. Rumi*
*Laboratorio de Inmunología. Instituto de Biología y Medicina Experimental (IByME). Buenos Aires. Argentina. **Hospital de Agudos "I. Iriarte"-Quilmes. Pcia. de Buenos Aires. Argentina.
Correspondence:
Lía S. Rumi M.D. Ph.D.
Instituto de Biología y Medicina Experimental
Vuelta de Obligado 2490 - (1428) Buenos Aires
Argentina
RESUMEN
Antecedentes: es ampliamente conocido el hecho de que las células de cultivo secretan a su medio diferentes factores que pueden alterar tanto sus propios metabolismos y funciones así como el de otras células. En el presente trabajo evaluamos el efecto de sobrenadantes de cultivo provenientes de dos líneas celulares, Calu-1 (carcinoma epidermoide de pulmón) y A-427 (adenocarcinoma de pulmón) sobre monocitos periféricos sanguíneos (MO) de 20 donantes sanos (HD).
Métodos y resultados: los MO fueron incubados con los sobrenadantes o con medio solamente (controles) durante dos o 18 horas. Fueron determinadas la expresión de antígenos HLA-DR por inmunofluorescencia directa, la formación de intermediarios reactivos del oxígeno (capacidad de reducción del nitroazul de tetrazolio) y la capacidad fagocítica (sistema Candida albicans -anti-Candida albicans). Estas determinaciones fueron también hechas en MO de 16 pacientes con cáncer de pulmón avanzado (LCP).
Resultados: los porcentajes de expresión (media ± ES) fueron: HLA-DR(+)MO: Calu-1 = 30 ± 2; A-427 = 31 ± 2; LCP = 52 ± 3 (p < 0.0001 vs HD: 77 ± 1%); NBT(+)MO: Calu-1 = 47 ± 1, A-427 = 49 ± 1, LCP = 56 ± 2 (p < 0.01 vs HD: 43 ± 2%), Ph(+)MO: Calu-1 = 38 ± 3, A=427 = 39 ± 2, LCP = 32 ± 3 (p < 0.0001 vs HD: 50 ± 1%).
Conclusiones: concluimos que este modelo in vitro reproduce características encontradas en MO de pacientes con cáncer de pulmón, ya que los sobrenadantes de cultivo indujeron en MO normales características fenotípicas y funcionales también encontradas en los MO de los pacientes analizados.
Palabras clave: Monocitos. Líneas celulares de cáncer de pulmón. Sobrenadantes de cultivo.
SUMMARY
Background: it is well known that culture cells secrete to their medium different factors that can alter their own as well as other cells'' metabolism and functions. In the present study we evaluated the effect of culture supernatants originated from two cancer cell lines, Calu-1 (lung epidermoid carcinoma) and A-427 (lung adenocarcinoma) on peripheral blood monocytes (MO) from 20 healthy donors (HD).
Methods and results: MO were incubated with supernatants or with medium only (controls) during 2 or 18 hours. There were determined the expression of HLA-DR antigen by indirect immunofluorescence technique, the formation of reactive oxygen intermediates (NBT reduction capacity) and the phagocytic capacity (Candida albicans- anti Candida albicans system). These determinations were made also in MO from 16 patients with advanced lung cancer (LCP). Percentages of expression (media ± SE) were: HLA-DR (+) MO: Calu-1 = 30 ± 2; A-427 = 31 ± 2; LCP = 52 ± 3 (p < 0.0001 vs HD: 77 ± 1%) NBT (+)MO: Calu-1 = 47 ± 1; A-427 = 49 ± 1; LCP = 56 ± 2 (p < 0.01 vs HD: 45 ± 2%), Phagocytic MO: Calu-1 = 38 ± 3; A-427 = 39 ± 2; LCP = 32 ± 3 (p < 0.0001 vs HD: 50 ± 1%).
Conclusions: we conclude that this in vitro model reproduced characteristics found in MO from lung cancer patients, since culture supernatants induced in normal MO phenotypic and functional characteristics also found in MO from patients analyzed.
Key words: Monocytes. Lung cancer cell lines. Culture supernatants.
INTRODUCTION
Interaction between tumor cells and the immune system of the host includes a complex network of cytokines and soluble factors that one and another secrete to modify each other''s metabolisms and funtions.
The host''s cellular antitumor response includes cytotoxic T cells, NK and monocytes/macrophages (1, 2) and their activation leads to the exocytosis of mediator molecules onto the surface of the tumor cells. In this regard, there can be mentioned tumor necrosis factor-alpha (TNF-*), whose action leads cells to apoptosis (3, 4) interferons, that exert antiproliferative (5) and immunoregulatory actions (6-8), and prostaglandin E2 (PGE2), who play a role in tumor associated immune suppression (9). In some cases, these factors combine to induce cells from the environment, like blood vessel cells, participate in the anti-tumor response secreting, for example, nitric oxide (NO) (10). On the other side, tumor cells respond to this situation secreting inhibitory cytokines, like TGF-ß (11, 12) or IL-10 (13), and perform a series of events to evade from the immune system.
Previous works showed that soluble factors from normal lung cells stimulate angiogenesis and tumor growth in vivo, and also favor the in vitro survival of tumor cells treated with cytotoxic drugs (14).
The purpose of this study was to determine the effect of two culture supernatants from lung cancer cell lines on healthy donor''s monocytes. We report that this in vitro model reproduced characteristics found in MO from lung cancer patients, since culture supernatants induced in normal MO phenotypic and functional characteristics also found in MO from patients analyzed.
MATERIALS AND METHODS
Cell lines and culture conditions
Calu-1 (lung epidermoid carcinoma) and A-427 (lung adenocarcinoma) cell lines were obtained from American Type Culture Collection (ATCC, USA). Cells were cultured in 25 cm2 tissue culture flasks (Falcon) with a nutrient mixture of RPMI-1640 medium (Gibco) supplemented with 0.1% of bovine sero-albumin (BSA), in a 5% CO2-containing 37° C atmosphere.
Conditioned media
When cell culture was semiconfluent, supernatants from Calu-1 (S/Calu-1) and from A-427 (S/A-427) cell lines were withdrawn and stored at 4° C until its use. Both supernatants were a kind gift from Dr. D. E. Bonfil.
Isolation of peripheral blood mononuclear cells (PBMCs)
PBMCs were obtained from EDTA-anticoagulated-venous blood from 20 healthy donors (HD) and 16 lung cancer patients (LCP) (clinical stages III and IV), by centrifugation over Ficoll-Hypaque (16) gradient (1,076 Hg/ml). Cells harvested from the interphase were washed 3 times with phosphate-buffered saline (PBS) for 10 minutes at 500 g and resuspended in 2 ml of RPMI-1640 medium (Gibco) containing 1% of heat-inactivated fetal calf serum (FCS), 100 IU/ml of penicillin and 100 mg/ml of streptomycin, designated as complete medium (CM). Cell concentration was adjusted to 1 x 106/ml.
Isolation of monocytes
Samples of 1 x 106 PBMCs in CM were cultured in glass 8-well-Lab-Tek chambers (Nunc) for 2 hours in the same conditions described above.
Thereafter, the non-adherent cells were removed by 3 washes with CM. At this point, the purity of the MO adhered was more than 93%, as judged by an immunofluorescent assay, using an anti-CD13 monoclonal antibody (mAb) (Sigma Diagnostic) which reacts with more than 90% of MO.
Incubation of MO with supernatants
MO from HD adhered were gently washed with PBS and then incubated for 18 hours with S/Calu-1 or S/A-427 (final concentration 20% v/v). Controls consisted in MO incubated only with RPMI-1640 medium supplemented with 0.1% of BSA. After that, the different assays described below were performed. Samples of adhered MO from HD, inmediately after 2 hours of culture were also evaluated. The same methodologies were performed in MO from LCP.
Determination of HLA-DR-positive MO (HLA-DR+MO) (Indirect immunofluorescence)
Adhered MO were washed and incubated with a 1: 20 diluted mAb against HLA-DR antigen (DAKO Corporation) for 30 minutes at 4° C. After washing 3 times with CM, FITC-conjugated rabbit anti-mouse F (ab'')2 IgG (DAKO Corporation) 1:20 diluted was added and cells were incubated for 30 minutes at 4° C. Thereafter, cells were washed and the percentage of cells expressing HLA-DR antigen was read in an epifluorescent microscope (total count: 200 cells).
Determination of MO with phagocytic activity
Preparation of the Candida-anti Candida system (Ca-anti Ca)
There were employed Candida albicans cells (Ca) from a 24-hour split in Sabouraud medium, autoclaved in 1 atm for 35 minutes. There were resuspended in PBS and conserved at -20° C until their utilization. Ca-anti Ca was prepared incubating 1 x 107 Ca in 0.1 ml of anti-Ca serum (DAKO Corp.) 1:400 diluted for 30 minutes at 37° C. Thereafter, the cell suspension was centrifugated (500 g) and the pellet obtained was resuspended in 0.2 ml of RPMI-1,640 medium, storing at 4° C until its use.
Phagocytosis assay
Adhered MO were incubated with 0.1 ml of the Ca-anti Ca at different times (between 30-240 minutes) to determine the optimum time of phagocytosis (after 30 minutes of incubation, MO phagocyted the 50% of CA). Then, cells were fixed with 100% methanol for 3 minutes and were stained with May Grünwald-Giemsa. Cells were read at optical microscope, considering as positive those MO with at least one Ca ingested.
Determination of MO with nitrobluetetrazolium (NBT) reduction capacity (ROI production)
MO adhered were incubated in a 5% CO2 containing atmosphere at 37° C, with a mixture of 0.1 ml of human normal plasma (activated with Escherichia coli endotoxin), 1 ml of NBT solution (final concentration 15% v/v) and 0.1 ml of M199 culture medium. After that, it was made another incubation for 15 minutes at 4° C. Cells were fixed with methanol 100% and dyed with safranin. The lecture was made at optical microscope, considering as positive those MO that contained formazan cristals in their citoplasm, as consecuence of NBT reduction. Results were expressed as percentages.
Statistical analysis
Results were analyzed by one-way ANOVA, according to the Instat V1.0 GPIS program, followed by post-hoc comparisons using Bonferroni''s test, and p values < 0.05 were considered significant.
RESULTS
Percentage of HLA-DR(+)MO
Values (expressed as mean ± standard error, SE) for HD were: after 2 hours of culture: 77 ± 1%; after 18 hours of culture: 54 ± 5%. After treatment with supernatants, we observed an statistical significant diminution in HLA-DR expression, resulting for S/Calu-1: (30 ± 2)% and for S/A-427: 31 ± 2%. LCP value resulted: 52 ± 3% (Fig. 1A).
Figure 1A.--Percentage of HLA-DR(+)MO from HD treated with culture supernatants and from lung cancer patients (LCP).
Percentage of NBT(+)MO
Values for HD were: after 2 hours of culture: 45 ± 2%; after 18 hours: 40 ± 1%. In this case, treatment with supernatants showed higher percentages versus untreated cells, being for S/Calu-1: 47 ± 1% and for S/A-427: 49 ± 1%. Percentage for LCP was 56 ± 2, reaching this result levels over the control (Fig. 1B).
Figure 1B.--Percentage of NBT(+)MO from HD treated with culture supernatants and from LCP.
Percentage of phagocytic MO
We could observe a significant decrease in phagocytic capacity in cells treated with supernatants as in LCP MO. Values for HD were: after 2 hours of culture: 50 ± 1%; after 18 hours: 47 ± 2%; S/Calu-1: 38 ± 3%; S/A-427: 39 ± 3%. For LCP, value was: 32 ± 2 % (Fig. 1C).
Figure 1C.--Percentage of MO with phagocytic capacity from HD treated with supernatants and from LCP.
DISCUSSION
The functioning of monocytes depends on their state of maturity and on the balance of regulatory conditions of the environment, these being characterized by the presence of stimulatory or inhibitory agents (15). These agents determine characteristics like differential expression of membrane antigens and response grade to different stimuli. NBT reduction assay can be considered a measure of MO activation (16). Using this assay also as a measure of ROI production, we found that treating MO with both supernatants augmented the levels of cells with this capacity. Moreover, LCP MO showed an even higher ROI production, raising up their values over control levels. These facts are in agree with data from Hedley and Currie (17), who found that ROI production was augmented in patients with malignant melanoma.
In previous works, we have demonstrated a relationship between HLA-DR expression and ROI production in MO from untreated cancer patients, attribuiting these alterations to the enhanced production of immune complexes (18, 19).
Gruner et al (20) studied the influence of the phagocytic stimulum on the expression of HLA-DR, suggesting that the diminished expression of this antigen in MO could be a consecuence of ROI production. Our results showed a dimished percentage of phagocytic activity in MO from LCP. These data are in agree with studies done by Merendino (21), who demonstrate that in patients with metastasic carcinoma, phagocytic capacity was deteriorated. The diminished phagocytic capacity also found in supernatant-treated monocytes leads us to suppose that some soluble agent may be affecting MO function, resembling this situation that found in MO from LCP.
In our work, we verified the well-known fact that cultured monocytes diminish the expression of class II antigens (22), situation induced by phenomena of adherence. The marked diminution of HLA-DR(+)MO found in cells treated with both supernatants, also found in LCP, may be explained by the presence of soluble factors in the supernatants that cause an alteration in HLA-DR expression. This fact may be due to interleukin-10, an inhibitory cytokine, that down-regulates the expression MHC class II antigen on monocytes (23), by affecting the antigen complex arrival and recicling at the plasma membrane (24).
The fact that we cannot found any differences between both supernatants talks about a common way in which the molecule (or molecules) in the supernatant exert the inhibitory effect on monocytes, or that the same agent is present in both of them. Huang et al found that non-small cell lung cancer-derived soluble factors and exogenous prostaglandin E2 treatment, enhance the production of interleukin-10 in peripheral blood lymphocytes (25). These cell lines also constitutively produce PGE2. Also, there have been found elevated levels of prostaglandin-E (PGE) or its metabolites in plasma from cancer patients or tumor bearing-animals (26) being this factor one of the principal molecules that takes part in the inhibition of the expression of HLA-DR (27).
The data showed above let us suppose that IL-10 and PGE2 are both candidate molecules to be present in A-427 and Calu-1 supernatants, representing this a probable mechanism by which lung cancer cells may escape from host immune surveillance. In conclusion, in this in vitro model, culture supernatants induced in normal MO phenotypic and functional alterations, similar to those found in peripheral MO from lung cancer patients. Investigations in this field continue at this moment in our laboratory.
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