Effect of phorbol myristate acetate (PMA) and ionophore A23187 on interleukin-2 levels and proliferation of activated T lymphocytes from patients with lepromatous leprosy

International Journal of Leprosy and Other Mycobacterial Diseases, Mar 1997 by Fernando Alfaro-Bustamante, Gabriela Ramirez-Flores, Amado Gonzalez-Mendoza, Alfonso Islas-Rodriguez, Mary Fafutis-Morris

Effect of Phorbol Myristate Acetate (PMA) and lonophore A23187 on Interleukin-2 Levels and Proliferation of Activated T Lymphocytes from Patients with Lepromatous Leprosy1

Fernando Alfaro-Bustamante, Gabriela Ramirez-Flores, Amado GonzalezMendoza, Alfonso Islas-Rodriguez, and Mary Fafutis-Morris2

Lepromatous leprosy is characterized, among other peculiarities, by an antispecific anergy of the cellular immune response (10). This immunodeficiency consists of the limited proliferation of T lymphocytes, and is explained in part by the impaired synthesis of interleukin-2 (IL-2) (12,19). In normal resting T cells, antigens or mitogens induce their proliferation by means of the synthesis of IL-2 and its receptor; the participation of diacylglycerol (DAG) and calcium, that produce the activation of protein kinase C (PKC) (17-23), is mandatory. Phorbol esters such as PMA can substitute for DAG and are mitogenic to human T and B cells, producing membrane alterations, modulation of cell-surface receptors (2,5,7,11,15,18) and activation of several cytokine-encoding genes (3,5,.13,15).

Ionophore A23187 increases calcium permeability across the cellular membrane into the cytosol of lymphoid cells, and is considered by several authors as a co-mitogen of T lymphocytes (1,16,24). We report here the use of PMA, an analog of DAG and ionophore A23187 (calcium increaser), in cultures of mitogen-activated T lymphocytes from lepromatous leprosy patients in order to induce the expression of the IL-2 gene, thus correcting the inadequate proliferation of T cells from these lepromatous patients.

MATERIALS AND METHODS

Study subjects. Twenty-two patients, 11 males and 11 females between the ages of 20 and 65 years, from the Instituto Dermatologico at Guadalajara, Jalisco, Mexico, were diagnosed as having polar lepromatous leprosy (LL) according to international criteria (21). All presented positive bacilloscopy and all received an irregular treatment of 100 mg of dapsone (diaminodiphenyl sulfone) per day. The length of treatment ranged from 2 to 15 years. The control group consisted of 20 normal subjects, all matched with the study group for sex and age as much as possible.

Mononuclear cells. Heparinized blood (20 IU/ml) was obtained from each subject by venipuncture. After the blood had been centrifuged on a Ficoll-Hypaque gradient (6) at 400 x g, the mononuclear cells were recovered and washed three times with Hanks' balanced salt solution (HBSS). The cells were then suspended in RPMI 1640 culture medium (GIBCO, Grand Island, New York, U.S.A.) supplemented with 5% heat inactivated fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 mM HEPES, 5 x 10^sup -5^ M 2-mercaptoethanol, and penicillin 100 U/ml and streptomycin 100 (mu)g/ml. Finally, the cells were adjusted to 1 x 10^sup 6^ cells/ml (12).

Proliferation assay. In order to activate T cells from these LL patients, 10 (mu)g/ml of phytohemagglutinin (PHA) was added to 2 x 10^sup 5^ mononuclear cells, and/or 10 ng/ml of phorbol myristate acetate (PMA), and/or 1 (mu)g of ionophore A23187 to the corresponding well, i.e., all possible combinations among the three reagents. The cultures were incubated for 48 hr at 37 deg C in a mixture of 95% air and 5% CO^sub 2^. Thereafter, they were pulsed with 1 (mu)Ci of ^sup 3^H-thymidine (specific activity 6.7 Ci/(mu)mole; New England Nuclear, Boston, Massachusetts, U.S.A.).

After a 24-hr incubation, the cells were harvested, and the incorporation of ^sup 3^Hthymidine was measured in a Packard beta counter. The results were expressed as a stimulation index (SI) according to the following (19):

SI = cpm of experimental / cpm of medium

IL-2 assay. The Quantikine Human IL-22 Immunoassay (R and D Systems, Minneapolis, Minnesota, U.S.A.) was used for the quantitative determination of human IL2 concentrations present in the culture supernatants after 48 hr (20).

RESULTS

The Table presents the results of experiments measuring the T-cell proliferation by means of ^sup 3^H-thymidine incorporation (SI) in PHA-stimulated cell cultures. It can be observed that PHA-activated T lymphocytes from LL patients can be separated into two groups: 1) 9 responders (R) with a SI of > 10 and 13 nonresponders (NR) with a SI of

Figure 1 shows the proliferative responses of cells from LL(R), LL(NR), and normal subjects stimulated with: I, PHA, PMA, PMA I, PHA PMA and PHA PMA ionophore (PPI). It can be seen that ionophore A23187 alone does not cause proliferation. The most important result occurs in LL(NR) patients since their cells that do not respond to PHA stimulation alone increase their proliferation to normal levels when they are stimulated with PHA in the presence of PMA. When lymphocytes from LL(NR) patients are stimulated with PMA I, they again are less responsive than the LL(R) patients and normal controls. On the other hand, all three groups (NR, R and normals), strongly increased their responses when they were incubated with PPI.

The significance levels of the differences in each of the following combinations used in the cell cultures from LL(R), LL(NR) and normal subjects were: for PHA, p

In Figure 2, the IL-2 concentration in the supernatants of cultures of T lymphocytes from LL(NR), LL(R) and controls stimulated with I, PHA, PMA, PMA I, PHA PMA and PPI are shown. It can be seen that cells from LL(NR) and LL(R) patients and normals do not produce detectable IL-2 levels in the presence of ionophore A23187 alone, and they produce relatively low levels of IL-2 when they are incubated with PHA or PMA. Nevertheless, the combination of ionophore and PMA causes the cells from LL(NR) patients and normals to increase their IL-2 levels. When the PHA PMA combination is used, cells from all three groups produce their highest levels of IL-2. Finally, when ionophore A23187 is added to PHA PMA there is a slight decrease in the production of IL-2 in all of these groups. The significance level of the differences of IL-2 from supernatants of normal cells versus both groups of patients was p