- Volume 70 , Number 4
- Page: 274–7
AP-1 Is present in nuclear extracts of lymphocytes f rom lepromatous leprosy patients
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To the Editor:
Lepromatous leprosy (LL) patients present a limited T-cell in vitro proliferation to mitogens and to specific antigens, as well as a deficient interleukin-2 (IL-2) synthesis. The above mentioned is correlated with the presence of multiple M. leprae bacilli as an immunodeficiency state due, in part, to an inadequate activation of T cells (1,4,5,8).
It has been shown in different models that T cells fail to proliferate due to a defect in IL-2 gene transcription (7,11), through a deficient production of activator-protein-1 (AP-1), and other factors as well. AP-1 is an important and well studied, inducible transcription factor. AP-1 is a dimer protein containing the gene products of ihe jun (c-Jun, Jun-B and Jun-D) and fos (c-Fos, FosB, Fra-1 and Fra-2) gene families. Many signal transduction pathways involve the expression of c-fos and c-jun, formation of the dimer and union to the AP-1 consensus binding site, the oligonucleotide palindromic sequence. 5'-CGC TTG ATG ACT CAG CCG GAA-3' (6,9,10).
In the present work, we looked for the presence of the AP-1 factor in nuclear extracts of PHA activated T lymphocytes from LL patients to acknowledge if the perturbed proliferation and IL-2 response in T cells correlated with a reduced DNA binding activity of this transcription factor.
Study subjects. Ten lepromatous leprosy (LL) patients (ages 20 years to 50 years), 3 females and 7 males from La Barca, Jalisco, Mexico, were diagnosed as having LL according to international criteria (12). All were under irregular multidrug therapy treatment (dapsone, clofazimine, rifampin) according to World Health Organization (WHO) schemes, since diagnosis (13). All still presented positive bacilloscopy. The healthy control group consisted of unrelated subjects, from the same geographical rural area, sex-and-age (± 5 years) matched as the patient group.
Mononuclear cells. Heparinized blood (50 ml-100 ml) was obtained from each subject by venipuncture at the local hospital, transported to the laboratory in a 15°C ice chest and stored at 4°C overnight. The mononuclear cells were centrifuged at 400 x g, using Histopaque-1.077, washed and resuspended in RPMI 1640 culture medium (GIBCO, Grand Island, New York, U.S.A.) supplemented with 5% heat inactivated fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 mM HEPES, 5 x 10-5 M 2-mercaptoethanol, penicillin 100 U/ml, and streptomycin 100 µg/ml. Finally, the cells were adjusted to 1 x 106 cells/ml (1,8).
Proliferation and biosynthesis of IL-2 by T lymphocytes. Five µg/ml of phyto-hemagglutinin (PHA) was added to 2 x 105 mononuclear cells for culture. The cultures were incubated for 48 hr at 37°C in a mixture of 95% air and 5% CO2. Thereafter. each supernatant was assayed for IL-2 activity (ELISA) or pulsed with 1 µC of 3H-thymidine (New England Nuclear, Boston, Massachusetts, U.S.A., specific activity 6.7 Ci/µmol). After a 24-hr incubation period, the cells that had been pulsed were harvested, and the incorporation of 3H-thymidine was measured in a Beckman beta counter. The results were expressed as counts per minute (cpm) and as stimulation index (SI) according to the following: SI = cpm of experimental (with PHA) ÷ cpm of medium (1).
IL-2 assay. For the quantitative determination of human IL-2 concentrations present in culture supernatant, a specific ELIS A kit ("h-interleukin-2" ELISA kit from, Boehringer Mannheim, Germany) was used according to instruction from the manufacturer. Results were expressed as pg/ml of supernatant.
Preparation of nuclear extracts (NE). Under culture conditions, in 25 ml bottles, 5 x 106 cells were incubated (Corning, New York, New York, U.S.A.), for 6 hours only, harvested by centrifugation and then the nuclear extracts were prepared according to the method described by Digman (3). The protein content of each extract was determined by the Bradford assay and the extracts stored in aliquots at -80°C until used in the Elec-trophoretic Mobility Shift Assay (EMSA). Hela cells nuclear extracts were prepared as a positive control for AP-l presence.
Oligonucleotides. Two different Ds DNA oligonucleotides were labeled by T4 kinase phosphorylation reaction in the presence of γ-P32-ATP (New England Nuclear, Boston, Massachusetts, U.S.A., specific activity 3000 µCi/mmol) according to the instructions from the manufacturer, and separated from unincorporated nucleotides by chromatography on G-50 Sephadex (BioRad Laboratories, Hercules, California, U.S.A.). The oligonucleotide was 5'-CGC TTG ATC ACT CAG CCG GAA-3'. A mutant oligonucleotide was used for heterologous competition assay, presented as "CA" → "TG" substitution in the AP-I binding motif (Santa Cruz Biotechnology, Inc., Santa Cruz, California, U.S.A.). Nonspecific competition was realized with growing amounts of poly (dldC) (2).
Binding reaction. The binding reaction (20 µl in HEPES, buffer pH 7.9) was performed with the same amount of nuclear protein in each reaction (10 µg) (equivalent to 500,000 cells) with 30 µCi (3 111) of correspondent Ds 32P-labelled oligonucleotide in the presence of 100 ng/ul of poly(dldC) as mentioned, to remove non-specific unions. The reactions were incubated on ice for 15 min.
Electrophoretic Mobility Shift Assay (EMSA). The above described samples were electrophorized on a 6% native Tris-borate-EDTA (TBE) polyacrylamide gel in 0.5 x TBE, at 200 volts, for 2 hr, at 4°C. The gels were dried under vacuum and exposed to autoradiography at -70°C using Kodak film (X-OMAT) during 24 hr (2).
Statistics. "BioStat Primer" statistical package was used to run "ANOVA" (Student "t" tests).
The Table presents experimental data of T-cell proliferation (expressed as cpm, as well as SI) and IL-2 levels. As expected, T cells from LL patients present a statistically significant (p <0.05) lower stimulation index (SI = 1.97. ± 0.33) as compared to healthy controls (SI = 3.02, ± 0.37). Lymphocytes from LL patients have a tendency to produce lower levels of IL-2 as compared to healthy controls, ( 180.07 vs 287.47 ng/ml), nevertheless, no statistically significant differences were found.
The Figure shows the results of the EMSA. One can see the presence of AP-1 in the nuclear extracts of T lymphocytes from LL patients and healthy controls, stimulated with PHA or not. It is evidenced that all of them contain the AP-1 transcription factor as they provide the specific retardation band (indicated by an arrow).
The figure. Gel Electrophoresis Mobility Shift Assays (EMSA) showing complexes formed between AP-1 consensus site γ-P32 labeled oligonucleotides (AP-1c) and nuclear extracts (NE) of lymphocytes from LL patients and healthy controls (HC) subjects: Lane 1: Control without NE; lane 2: HeLa cell control NE: lanes 3-6: AP- lc+ NE from cells of HC; lane 7-16; AP- Ic+ NE from cells of LL patients. Lanes 3, 5, 7, 9, 11, 13, 14 complexes of AP-1c + NE from cultures with medium. Lines 4, 6, 8, 10, 12, 15, 16 complexes of AP-1c + NE from cultures stimulated with PHA.
The above mentioned leads us to conclude that the T cell unresponsiveness and low IL-2 levels observed in T cells of LL patients is not due to a defect in the production of the AP-1 transcription factor and, by inference, neither of Fos and Jun proteins.
We will study other transcription factors, such as NFkB, and/or NFAT, in order to know their role in activation of T lymphocytes and IL-2 synthesis from LL patients.
- Rosario Huizar-López, M.Sc.
Ann Santerre, Ph.D.
Departamento de Biología Calillar y Molecular del Centro Universitario de
Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, .Jadisco México. Apartado Postal 5-419. CP. 45040 Colonia Chapalita, Zapopan, Jalisco, MEXICO.
- Vicente Madrid-Marina, Ph.D.
Oscar Peralta-Zaragoza, M.T.
Laboratorio de Inmunología del Instituto Nacional de Salud Publica (SSA) Cuemavaca, Morelos, México.
- Alma Villalobos-Arámbula, Ph.D.
Alfonso Islas-Rodríguez. Ph.D.
Departamento de Biología Cubilar y Molecular del Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Jalisco
México. Apartado Postal 5-419. CP 45040 Colonia Chapalita, Zapopan, Jalisco, MEXICO.
Reprint requests to Alfonso Islas-Rodríguez. Apartado Postal 5-419. CP. 45040 Colonia Chapalita, Zapopan, Jalisco, MEXICO.
Acknowledgment. M.Sc. Eduardo Gonzalez, Major of Jamay Jalisco. Dr. Genaro Paredes Romero. (SSA), La Barca, Jalisco. México. This study was supported in part by a grant from CONACYT #2578 P-M, and FOMES #98-15-12.
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