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  • Volume 69 , Number 4
  • Page: 354–7

Detection of a tumor necrosis factor-like activity in culture supernatants of armadillo leukocytes

Fernando M. Guerra-Infante; Marcela Lopez-Hurtado; Sail Flores-Medina; Angelica Zamora-Ruiz; Ma. de Jesus de Haro-Cruz

To the Editor:

Tumor necrosis factor (TNF) is one of the main cytokines involved in the immune response. It was discovered by Old in 1985 (10) as a factor with cytotoxic activity on tumor cells that is liberated in mouse serum in response to lipopolysaccharide (LPS) injection. TNF is released by several cells of the immune system, such as activated T lymphocytes and mastocytes, NK cells, monocytes, macrophages and neutrophils (3). Identification and characterization of this molecule is by means of enzymatic immunoassay and/or bioassay evaluating the cytotoxic effect on cell lines, such as L929, WeHI164, L-M, OVCAR-3 and Pkl5 (4,5,7). These assays have allowed the demonstration of the presence of TNF in various species of vertebrate and invertebrate animals (1,2,9,11). The biochemical characterization of this cytokine among the diverse animal species has revealed similarities in both structural features and biological properties whereby it has been considered as a molecule conserved evolutionarily (1,2,9,11).

The armadillo (Dasypus novemcinctus) has been considered as a feasible animal model to study the infection with Mycobacterium leprae however, this has not been completely possible due to little knowledge of their immune response mechanisms. Because M. leprae is an intracellular microorganism, the host cellular immune response plays an essential role for the elimination of this pathogen. The activation of this cellular response is mediated mainly by pro-inflammatory cytokines as TNF (2). Because TNF production has not been shown in the cells of the immune system of armadillos, we decided to investigate the presence of this factor in culture supernatants of armadillo leukocytes stimulated with phorbol myris-tate acetate (PMA). The nine-banded armadillos were captured in the municipality of Chilapa in Guerrero state in Mexico and then sent to Mexico City, where the studies were carried out.

Methods: Ten milliliters of peripheral blood was drained by heart puncture of 10 animals anesthetized with ketamine base, and was placed in tubes with 5 ml of Al-sever solution. The purification of the leukocytes was carried out by erythrocyte sedimentation for I hr in 6% dextran '70' in physiological saline solution (10 ml blood/3 ml saline). The leukocyterich plasma was recovered and centrifuged at 1500 rpm for 7 min. The resulting cell pellet was washed twice in Alsever solution. The final leukocyte pellet was resuspended at 2 x 10'' cells/ml in RPM1-1640 medium (Sigma Chemical Co., St. Louis, Missouri, U.S.A.) supplemented with 10% fetal calf serum and antibiotics (RPMl-complete). One million cells was added to each well of a 24-well fiat-bottom microplate. Then, the armadillo leukocytes were stimulated with phorbol myristatc acetate (PMA) and/or LPS (Sigma). The microplate was incubated at 37°C with 5% CO,. The culture supernatants were recovered after 24 hr and maintained at -70°C until use.

The detection of TNF was carried out by a cytotoxicity bioassay using L-929 cells as previously described (6). Briefly, 50 Ltl of the culture supernatants was added in each well in triplicate in 96-well fiat-bottom microplates that contained 20 x 101 L-929 cells/well. Then, 50 |il of RPMI-complete with 1 |Ug/ml of actinomycin D (Sigma) was added to each well. Culture supernatants of U-937 cells stimulated under the same conditions as those of the armadillo leukocytes were used as the control. The microplate was incubated at 37°C for 24 hr in 5% CO,. Supernatants were aspirated and 100 p.1 of methanol was added. After 7 min of incubation, 50 u.1 of 2% crystal violet was added. After 10 min, the microplates were washed five times with 200 u.1 per well with phosphate buffer solution (PBS). Then 100 u.1 of 33% glacial acetic acid was added. Finally, the microplates were read in a microplate reader at 600 nm for absorbance (Ceres 900Hdi, Bio-Tek Instruments, Inc.. Winooski, Vermont, U.S.A.). Results were reported as percentage of cellular cytotoxicity.

In order to confirm the results of the bioassay, an ELISA commercial kit for the determination of human TNF-α (Roche Molecular Biochemical. Mannheim, Germany) was used. The ELISA assay is based on the quantitative "sandwich enzyme immunoassay" principle, two mouse monoclonal antibodies directed against two different epitopes of TNF-α. Detection was carried out according to the manufacturer instructions.

The results obtained showed that culture supernatants of armadillo leukocytes stimulated with PMA or PMA-LPS had a 14% to 30% cytotoxic activity on L-929 cells (The Table and The Figure). A similar effect was observed in culture supernatants of U-937 cells stimulated with PMA or PMA-LPS, but with a cytotoxic activity of 37% to 70% on L-929. The quantification of TNF by means of the ELISA method showed the presence of TNF in the culture supernatants of U-937 cells, while in the culture supernatants of armadillo leukocytes this cytokine was not detected. However, it is necessary to mention that the ELISA lest was carried out using monoclonal antibodies that only recognize specific epitopes for the human TNF-α and does not react with TNF-α from a mouse, rat, dog, pig, rhesus monkey or baboon. For this reason it would be necessary to purify and to characterize this molecule to demonstrate that the cytotoxic activity observed was due to a factor similar to human TNF-α.



Fig.The Figure. Cytotoxic effect of the culture supernatants of armadillo leukocytes stimulated with PMA onthe L-929 cells. A .= culture supernatants of armadillo leukocytes not stimulated with PMA. B = Culture super-natants of armadillo leukocytes stimulated with PMA (50 ng/100 ml) for 24 hr.


- Fernando M. Guerra-Infante, Sc.D.
Marcela LOpez-Hurtado, Sc.B.
Saul Flores-Medina, Sc.B.
Angelica Zamora-Ruiz, Sc.B.
Ma. de Jesus de Haro-Cruz, Sc.B.

Department of Microbiology of Escuela Nacional de Ciencias Biologicas of Instituto Politecnico Nacional
Carpio and Plan of Ayala
Col. Santo Tonws CP 11340, Mexico



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