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. 2001 Sep;104(1):80–86. doi: 10.1046/j.0019-2805.2001.01281.x

Medroxyprogesterone acetate enhances in vivo and in vitro antibody production

Mónica Vermeulen *, Patricia Pazos †,‡, Claudia Lanari †, Alfredo Molinolo †, Romina Gamberale *, Jorge R Geffner *, Mirta Giordano *
PMCID: PMC1783281  PMID: 11576224

Abstract

In the present study we examine the effects of medroxyprogesterone acetate (MPA) on the specific antibody secretion to T-dependent antigens. Our results show that the in vivo administration of MPA to mice, 7 or 90 days before immunization with sheep red blood cells (SRBC), significantly enhanced both, primary and secondary antibody responses, without affecting delayed-type hypersensitivity (DTH). These effects could be counteracted by the anti-progestin onapristone or ZK 98299 (ZK) suggesting that MPA interacted with progesterone (PRG) receptors to increase B-cell response. To better understand the mechanisms involved in MPA activity we carried out cultures of splenocytes, bone marrow cells or lymph node cells from immunized mice in the presence of MPA, and evaluated the amount of antibody release to supernatants. We found that low doses of MPA (10−9 m and 10−10 m) significantly enhanced the in vitro production of specific immunoglobulin G (IgG) antibodies, an effect that appears to involve the interaction of the progestin with PRG receptors, as judged by the inhibition of MPA effects with ZK (10−8 m) or RU486 (10−9 m). These receptors were detected by flow cytometry analysis in a proportion of T lymphocytes. Because MPA did not increase the number of immunoglobulin-secreting cells, our findings suggest that MPA enhanced the capacity of individual cells to produce specific immunoglobulin.

Introduction

It has long been known that progesterone (PRG) and its derivates are able to modulate a variety of immune responses.1,2 In fact, the ability of progesterone to suppress cell-mediated functions has been considered of great relevance for the maintenance of pregnancy. Thus, high levels of progesterone found in the human placenta may promote survival of the fetal allograft by inhibiting maternal lymphocyte responses.3 While suppressive effects of PRG on cellular functions are well-established, its effects on antibody production are much less clear. Administration of PRG to animals has been shown to reduce4 or have no effects5 on humoral response to T-dependent antigens. However, more recent data in vitro demonstrated that PRG functions as a potent inducer of T helper 2 (Th2) type cytokines which might favour the development of antibody responses.6

Medroxyprogesterone acetate (MPA) is a synthetic progestin widely used in the treatment of mammary and endometrial adenocarcinomas,7,8 as a supportive therapy in anorexia/cachexia syndrome9 and as a long-acting contraceptive.10 Despite its wide employment in human therapy, there are few reports analysing the effects of MPA on the immune system. These studies, most of them carried out in patients receiving high dose schedules of MPA, found that it either suppresses11,12 or has no effect on lymphocyte proliferation stimulated by mitogens, such as phytohaemagglutinin (PHA) or concanavalin A (Con A).13 In animal models the only data available come from early studies which showed that MPA prolonged survival of skin allografts14 and inhibited antibody production.14,15 Because of its inhibitory activity on cell-mediated functions, MPA has been proposed as a promising substance for the treatment of autoimmune diseases.16,17

The aim of the present study was to evaluate in vivo and in vitro the effects of MPA on specific antibody secretion to T-dependent antigens. Here, we show that MPA enhances antibody production, an effect that appears to depend on the interaction of MPA with PRG receptors.

Materials and Methods

Mice

All experiments were carried out using 2-month-old virgin female BALB/c mice raised at the National Academy of Medicine, Buenos Aires, Argentina. They were housed six per cage and kept at 20 ± 2° under an automatic 12 hr light–dark schedule. Animal care was in accordance with institutional guidelines.

Reagents

MPA depot (Medrosterone), ZK 98299 (ZK) and RU 38486 (RU486) were kindly provided by Dr Gador Laboratories (Buenos Aires), Schering AG (Berlin) and Roussel-UCLAF (Romainville, France), respectively. MPA and dexamethasone were purchased from Sigma Chemical Co (St Louis, MO). All steroid stock solutions were prepared as a 10−2 m solution in 100% ethanol. Ovalbumin (OVA), complete Freund's adjuvant (CFA) and lipopolysacharide (from E. coli O55:B5; LPS) were purchased from Sigma. Interleukin (IL)-2 and IL-4 were assessed using commercial enzyme-linked immunosorbent assay (ELISA) kits (Endogen, Cambridge, MA).

In vivo MPA treatments

Mice were injected with 40 mg of MPA depot subcutaneously (s.c.) in the back. By this procedure, MPA is slowly released and results in the inhibition of oestral cycles for at least 3 months.18 Control animals received equal volumes (0·2 ml) of vehicle alone. Seven or 90 days after MPA injection, the mice were intraperitoneally (i.p.) inoculated with 0·2 ml of sheep red blood cells (SRBC) suspended in saline at a concentration of 5 × 108 SRBC/ml. Seven days later, serum samples were obtained by retro-orbital bleeding to evaluate primary humoral response to SRBC. One month after the first inoculum, mice received a second i.p. injection of SRBC and serum samples were obtained 1 week later to evaluate secondary humoral response.

In another set of experiments, the action of an anti-progestin on MPA effects in primary antibody response was evaluated. To this aim, the mice were injected s.c. with ZK, 10 mg/kg daily, from the day of MPA (5 mg) inoculation. One week later, the mice were immunized with SRBC and primary antibody response was evaluated as described above. In the experiments in which secondary response was assessed, ZK was administered as a 1·7-mg silastic pellet implanted s.c. In a previous report we demonstrated that this dose was effective in inducing regression of progestin-dependent mammary tumours.19

In vitro MPA treatments

In order to evaluate the in vitro effects of MPA on antibody production to SRBC, mice were immunized as described above for secondary response. Two weeks after the last SRBC inoculation, cells from spleen and bone marrow were obtained by standard methods. Briefly, spleens were collected under sterile conditions and gently teased over a wire mesh to produce a single-cell suspension. Cells were washed three times and then resuspended at 1 × 106 cells/ml in RPMI medium supplemented with 10% fetal calf serum (FCS), 2-mercaptoethanol (50 mm) and antibiotics (complete medium). Bone marrow cells were obtained by flushing femurs with RPMI and processing in a similar fashion. Cell viability was always above 90% as assessed by Trypan blue exclusion. Splenocytes and bone marrow cells were placed in 24-well microplates and incubated at 37° in 5% CO2 with or without different concentrations of MPA. In some experiments, cells were also incubated with LPS as a positive control for stimulation of immunoglobulin release. After 7 days of incubation, the release of antibodies to supernatants was assessed.

To evaluate the in vitro effects of MPA on antibody production to OVA, mice were immunized with 200 µg OVA in an emulsion with CFA, given at the base of the tail. Eight days later, inguinal and paraortic lymph nodes were collected under sterile conditions, washed and resuspended in complete medium to a final concentration of 2 × 106 cells/ml. Axillary lymph nodes were used as non-immunized cells. Lymph node cells were cultured in the presence of MPA as described above and the release of antibodies to supernatants was evaluated. When the effects of anti-hormones were assessed they were added from the beginning of the cultures. Unless otherwise stated, cytokines release to supernatants and enumeration of immunoglobulin-secreting B cells were evaluated at 48 hr of culture.

Measurement of antibody production

The amount of specific antibodies to SRBC in serum was determined by direct haemagglutination using a microplate. To this aim, 1 : 2 serial dilutions of heat-inactivated sera were done in phosphate-buffered saline (PBS) supplemented with 1% FCS and equal volumes (30 µl) of a 1% SRBC suspension were added to each well. The plate was then incubated 30 min at 37° and 3 hr at 4°. In all cases, results are expressed as the highest serum dilution giving a positive haemagglutination pattern (antibody titre).

The release of anti-SRBC antibodies to the supernatants of splenocytes or bone marrow cells were evaluated by flow cytometry using anti-mouse IgG labelled with fluorescein. Briefly, 2 × 106 SRBC suspended in 50 µl of saline were incubated for 30 min at 4° with 50 µl of supernatants. Then, SRBC were washed three times with saline plus 1% FCS, resuspended at 30 µl and incubated with the fluoresceinated antibody for 30 min at 4°. Controls included SRBC cultured in 50 µl of complete medium alone, washed and incubated with or without fluoresceinated anti-mouse IgG at 4°. The cells were subjected to flow cytometry and analysed by CellQuest soltware. The results are expressed as the mean fluorescence intensity (MFI).

The release of anti-OVA antibodies to the supernatants of lymph node cells were evaluated by ELISA. Briefly, 96-well flat-bottom plates were coated with OVA (10 µg/ml in PBS) and incubated overnight at 4°. After washing three times with Tris-buffered saline Tween-20 (TTBS) and blocked with TTBS plus BSA, 100 µl per well of serial dilutions of culture supernatants were added in triplicates. The plates were incubated 90 min at room temperature, washed again with TTBS and 100 µl of biotinylated antimouse immunoglobulin κ light chain monoclonal antibody (1/500 in PBS; PharMingen, San Diego, CA) was added to each well. After 1 hr incubation at room temperature, plates were washed, streptavidin-peroxidase was applied and reactions were finally developed with the chromogenic substrate OPD (Sigma). Negative controls included wells with all reagents except supernatants and wells without antigen. Absorbancies are expressed as median specific OD at 492 nm (mean OD of sample minus mean OD of no supernatants blank).

ELISA for measure the release of different isotypes of immunoglobulin were performed essentially as described above, except that microplates were coated with IgM, IgG1, IgG2a or IgE (10 µg/ml; Caltag Laboratories, San Francisco, CA).

Flow cytometry analysis

For intracellular detection of PRG receptors, 5 × 105 lymph node cells were fixed and permeabilized by using Fix & Perm (Caltag, CA) before the addition of anti-PRG receptors (Ab-7, clone hPRa7, Neomarkers, CA) or isotype-matched monoclonal antibodies (mAb; 4 µg/ml). After 1 hr incubation at 4°, the cells were washed and treated with anti-mouse IgG–fluoroscein isothiocyanate (FITC) (F(ab′)2 fragments for 30 min at 4°. For two-colour analysis, cells were subsequently incubated with anti-CD3–phycoerythrin (PE; PharMingen).

Enumeration of immunoglobulin-secreting B cells

The number of cells secreting specific anti-OVA immunoglobulin was determined by the enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) according to Czerkinsky et al.20 Briefly, 96 flat-bottomed well, high-binding ELISA plates (Costar, Cambridge, MA) were coated with 100 µl of OVA (Sigma, 10 µg/ml) diluted in PBS. After 3 hr of incubation at 37° in a humidified 5% CO2 atmosphere, the plates were washed and blocked with complete medium. Aliquots of 100 µl of cell suspensions were placed in triplicate and serial dilutions starting at 2 × 105 cells per well up to 0·5 × 105 cells per well were performed. The plates were incubated overnight undisturbed at 37° in 5% CO2 air. Cells were washed with 0·5% Tween-20 and PBS and incubated in the presence of 100 µl of biotinylated anti-mouse immunoglobulin κ light chain mAb (PharMingen, 1/500 in PBS 1% bovine serum albumin) for 1 hr at 37°. Plates were washed and incubated with streptavidin–peroxidase (Coulter Immunotech, Marseille, France) for 1 hr. The spots were visualized by addition of the substrate 3-amino-9-ethyl carbazole–H2O2.

Immunoglobulin-secreting B cells appeared as dark spots which were counted under low magnification using a stereomicroscope (Leica AG, Switzerland). No spots were found in wells in which there were no cells but only medium plus FCS.

Delayed-type hypersensitivy (DTH)

To evaluate DTH response, the mice received an i.p. injection of SRBC as previously described and 1 week later, they were challenged with 108 SRBC suspended in 50 µl of saline inoculated into the left footpad. A comparable volume of saline was injected into the right footpad as control. The DTH reaction was recorded 24 and 48 hr later, measuring the footpad swelling with a dial caliper.

Statistical analysis

A non-parametric Wilcoxon two-samples rank test was used for statistical analysis.

Results

MPA enhances primary and secondary antibody production

To investigate the effect of in vivo administration of MPA on antibody response, mice were injected with the progestin (40 mg depot) and 7 or 90 days later they were immunized with SRBC as described in Materials and Methods. The levels of specific immunoglobulin in sera were assessed by haemagglutination. Results in Table 1 show that MPA induced a significant increase, as compared with animals injected with vehicle alone, both in primary and secondary humoral responses to SRBC. By contrast, there were no differences in DTH evaluated 1 week post-MPA injection between control and MPA-treated mice (Table 1).

Table 1.

MPA enhances primary and secondary antibody responses to SRBC

Antibody titre*
Time of immunization Treatment of mice Primary response Secondary response DTH (FPS index)†
7 days after treatment Vehicle 36·0 ± 9·8 58·7 ± 15·9 15·2 ± 4·2
MPA 946·7 ± 293·0‡ 1567·3 ± 116·8‡ 13·6 ± 3·3
90 days after treatment Vehicle 20·0 ± 4·1 64·0 ± 14·6 ND
MPA 482·0 ± 136·5§ 708·0 ± 126·5§ ND
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Mice were injected with MPA depot (40 mg, s.c.) or vehicle and 7 or 90 days later they were immunized with 108 SRBC by an i.p. inoculation. Primary antibody response was assessed one week later as described in Materials and Methods. For secondary response, mice received a second i.p. injection of SRBC 30 days after the first inoculum.

DTH was evaluated in mice immunized with 108 SRBC i.p. and challenged 1 week later with 108 SRBC into the left footpad.

Data represent the mean ± SEM of six animals.

*

Antibody titre was expressed as the highest serum dilution giving a positive haemagglutination pattern.

†

Footpad swelling index (FSI) was calculated by subtracting the thickness of the footpad on the day of challenge from that 24 hr later.

‡

P < 0·01 MPA versus vehicle;

§

P < 0·05 MPA versus vehicle.

ND, not determined.

In an attempt to determine whether the enhancement of antibody production induced by MPA could be attributed to its interaction with PRG receptors, we next evaluated the ability of the anti-progestin ZK to counteract MPA effects. It was found that the daily injection of ZK (10 mg/kg) from the administration of MPA depot to immunization with SRBC abrogated MPA-induced stimulation of primary antibody response (Fig. 1a). ZK not only impaired MPA activity, but exerted an inhibitory effect in the absence of the progestin, suggesting that physiological levels of PRG modulate immunoglobulin production. Moreover, ZK administered as a 1·7-mg silastic pellet was able to significantly, though not completely, inhibit MPA-induced increase of secondary antibody response (Fig. 1b). Taken together, these data suggest that MPA in vivo effectively interacts with PRG receptors to exert its enhancing effect on humoral response.

Figure 1.

Figure 1

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ZK98299 inhibits MPA-induced enhancement of antibody response. Mice were injected s.c. with 10 mg/kg/daily of ZK from the day of MPA depot (40 mg s.c.) inoculation until the day of immunization with SRBC. Primary antibody response (a) was evaluated as described in Materials and Methods. To determine whether the antiprogestin can counteract the enhancing effect of MPA on secondary response (b), ZK was administered as a 1·7-mg silastic pellet implanted s.c. Data represent the mean ± SEM of six animals. *P < 0·05 MPA versus control; **P < 0·05 MPA ± ZK versus MPA.

MPA enhances in vitro production of antibodies

We next analysed whether MPA was able to enhance immunoglobulin release by directly affecting immunocompetent cells. With this aim, splenocytes from mice previously immunized with SRBC were cultured in the presence of different concentrations of MPA during 7 days. Supernatants were obtained at the end of the incubation and the amount of specific IgG antibodies were evaluated as described in Materials and Methods. As shown in Fig. 2(a), MPA at low concentrations (10−9 m and 10−10 m) significantly increased the release of anti-SRBC IgG to the supernatants. The enhancing effect of MPA was similar or even greater than that induced by the well-known B-cell stimulator, LPS (Fig. 2c). Comparable results were obtained with cultures of bone marrow cells from these same animals (MFI: control = 40·1 ± 4·8; MPA 10−9 m = 64·8 ± 5·8; LPS 10 µg/ml = 57·1 ± 3·4; mean ± SEM, n = 4, P < 0·05 MPA or LPS versus control).

Figure 2.

Figure 2

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MPA increases the release of specific antibodies in vitro. Mice were immunized with SRBC or OVA as described in Materials and Methods for primary response. Splenocytes from mice immunized with SRBC (a and c) and lymph node cells from mice immunized with OVA (b and d) were cultured in vitro with different concentrations of MPA (a and b) or MPA 10−10 m, LPS 10 µg/ml or saline (c and d) during 7 days. The production of anti-SRBC IgG was determined by flow cytometry and expressed as mean fluorescence intensity (MFI). The production of anti-OVA Immunoglobulin was determined by ELISA in samples diluted 1 : 40 and expressed as absorbance values at 492 nm. Hatched bars represent antibody released from non-immunized cells and black bars from immunized cells. Results are expressed as the mean ± SEM of seven to10 animals. *P < 0·05 MPA or LPS versus saline.

To corroborate and extend these results, we evaluated the capacity of MPA in vitro to increase the production of antibodies directed against another T-dependent antigen, OVA. In this case, OVA was injected in the base of the tail and cells from draining lymph nodes (OVA-draining lymph nodes) were compared with cells from distal lymph nodes of the same mice in their capacity to release specific immunoglobulins (IgM and IgG) to culture medium. Results in Fig. 2(b) show that, as in the case of SRBC, low doses of MPA significantly increased the secretion of immunoglobulin directed to OVA. These effects were comparable to those exerted by LPS and they were only observed in cultures from draining lymph node cells. Time-course studies indicated that there was increased levels of anti-OVA immunoglobulin induced by MPA as early as 24 hr after the initiation of culture (mean OD at 492 nm: control, 0·15 ± 0·01; MPA 24 hr, 0·35 ± 0·03; MPA 48 hr, 0·48 ± 0·12; MPA 72 hr, 0·50 ± 0·2; MPA 7 days, 0·57 ± 0·05; mean ± SEM, n = 3). Together, these results indicate that MPA enhances antibody production by directly interacting with immunocompetent leucocytes.

Next, we evaluated whether MPA differently modified the secretion of IgM, IgG1 and IgG2a, the three main isotypes involved in primary response to OVA–CFA.21 Because PRG has been shown to induce Th2-type cytokines in vitro,6 we also determined if MPA could stimulate IgE production. Results in Fig. 3 show that culture of cells from OVA-draining lymph nodes with the progestin induced the increase of IgM and IgG1 release without affecting the secretion of IgG2a or IgE.

Figure 3.

Figure 3

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MPA in vitro increases IgM and IgG1 release from OVA-immunized lymph node cells. Cells from OVA-draining lymph nodes were cultured in vitro with MPA 10−10 m (black bar) or saline (open bar). Seven days later, the release of IgM, IgG1, IgG2a and IgE was determined by ELISA in samples diluted 1 : 40 and expressed as absorbance values at 492 nm. Data represent the mean ± SEM of seven animals. *P < 0·01 MPA versus saline.

MPA activity appears to be mediated through PRG receptors on T lymphocytes

It has been previously shown that MPA at high doses is able to interact not only with PRG receptors but also with glucocorticoid receptors.22,23 The fact that only low concentrations of MPA (10−9 m and 10−10 m) could increase the release of antibodies in vitro suggests that, under our experimental conditions, MPA effects are mediated through PRG receptors. To corroborate this hypothesis, we evaluated the ability of two different progestin inhibitors, ZK (10−8 m) and RU486 (10−9 m), to counteract the enhancing effect of MPA on immunoglobulin secretion. Both inhibitors were able to completely suppress the increase in antibody secretion induced by MPA (10−9 m): (mean OD at 492 nm: control, 0·29 ± 0·03; MPA: 0·74 ± 0·09, RU: 0·30 ± 0·04, ZK: 0·24 ± 0·02, MPA + RU: 0·24 ± 0·03, MPA + ZK: 0·24 ± 0·02, mean ± SEM, n = 6, P < 0·01 MPA versus MPA + RU and MPA + ZK). These results suggest that MPA activity was mediated through the interaction with PRG receptors.

We then performed studies by flow cytometry to analyse the expression of PRG receptors on lymph node cells. Using an specific mAb, we found that almost 50% of lymphocytes from non-immunized mice expressed PRG receptors (Fig. 4a). Similar levels of expression were observed in OVA-draining lymph nodes from immunized animals (not shown). In an attempt to characterize the receptor-bearing subset, double-colour staining with anti-CD3-PE was performed. As shown in Fig. 4b, PRG receptors were expressed by a proportion of T lymphocytes, while they were absent in CD3− cells. Further analysis revealed that PRG receptors-bearing T cells belong to either CD4+ or CD8+ subsets (data not shown).

Figure 4.

Figure 4

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A subset of T lymphocytes in lymph nodes express PRG receptors. Expression of PRG receptors in lymph node cells was analysed by flow cytometry as described in Materials and Methods. (a) Solid histogram indicates control labelling with an irrelevant mAb, open histogram shows staining with specific mAb. (b) Two-colour fluorescence analysis for PRG receptors and CD3. Lymph node cells were labelled with an irrelevant mAb (left dot plot) or anti-PRG receptor mAb (right dot plot) before staining with anti-CD3-PE. Results are representative of four experiments performed.

MPA enhances immunoglobulin-secreting activity

In an attempt to further explore the cellular basis of MPA action we performed a series of additional experiments. We found that MPA did not exert a proliferating effect on lymph node cells as measured by 3H-thymidine incorporation (data not shown). Moreover, MPA was unable to induce the secretion of IL-4 or IL-2 to supernatants of cultured lymph node cells from immunized mice. In fact, these cytokines could not be detected above the lower limit of the ELISA (< 5 pg/ml). Finally, we evaluated whether MPA enhanced antibody production in vitro by increasing the number of immunoglobulin-secreting cells. To this aim, we used the ELISPOT technique by coating the plates with OVA as described in Materials and Methods. Results in Fig. 5 show that, in contrast to what was observed with the polyclonal B-cell activator LPS, treatment in vitro with MPA did not enhance the number of specific anti-OVA secreting cells. Considering that, under these experimental conditions, MPA significantly increased the release of specific antibodies from OVA-immunized lymph node cells to culture medium, our findings may indicate that MPA enhances the capacity of individual cells to produce immunoglobulin.

Figure 5.

Figure 5

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MPA does not increase the number of anti-OVA secreting lymph node cells. Cells from distal lymph nodes (hatched bars) or OVA-draining lymph nodes (black bars) were cultured in vitro with MPA (10−10 m), LPS (10 µg/ml) or saline. Five days later, the number of anti-OVA secreting cells was measured by ELISPOT. Data are expressed as the mean ± SEM of five animals. *P < 0·01 LPS versus saline.

Discussion

The results presented herein show that MPA increases both, in vivo and in vitro antibody production to T-dependent antigens. While it is difficult to determine which is the precise target(s) of MPA administered in vivo, our findings in vitro strongly suggest that the progestin exerts a direct effect on leukocytes, which results in higher antibody output per cell. In fact, culture in vitro with MPA enhanced the release of specific Immunoglobulin from lymphocytes which had been exposed in vivo to antigens, without enhancing the number of immunoglobulin-secreting cells.

Our results also show that the enhancing effect of MPA appears to be higher in vivo than in vitro. Thus, treatment of mice with MPA induced a 20-fold increase in the levels of serum anti-SRBC immunoglobulin, as measured by haemagglutination, while MPA in vitro increased two- to threefold the production of anti-SRBC IgG by splenocytes from immunized mice, as measured by flow cytometry. These differences could be explained considering that we only measured antibodies of the IgG isotype in the supernatants of splenocytes cultured in vitro, while haemagglutination assays involved not only anti-SRBC IgG but also specific IgM. On the other hand, it cannot be ruled out that the administration of MPA in vivo could trigger additional enhancing mechanisms on B-cell responses that account, at least in part, for the differences observed in vivo and in vitro.

It is well established that, in addition to binding to PRG receptors, MPA at high concentrations (> 100 nm) is able to interact with glucocorticoid receptors.22,23 While cellular immune responses are strongly suppressed by glucocorticoid, humoral responses are poorly inhibited or even enhanced.24–27 Therefore, it was important to determine whether the enhancing effects of MPA described herein were mediated through glucocorticoid or PRG receptors. Our in vitro observations suggest that MPA action is effectively exerted through PRG receptors. First, and in agreement with previous reports,28,29 we found that a marked proportion of T lymphocytes expressed PRG receptors. Second, maximal levels of immunoglobulin release were found at 10−9 m and 10−10 m MPA, doses which have no demonstrable glucocorticoid effect.22,23 Notably, MPA did not exert any effect at doses compatible with glucocorticoid activity (10−8 m and 10−7 m), suggesting that signals delivered through glucocorticoid receptors counteracted the stimulatory effects of MPA. Finally, the fact that effective ZK doses (10−8 m) were much lower than those described to antagonize glucocorticoid receptors (10−6 m),30 strongly suggests that MPA is acting through PRG receptors.

In regard to ZK treatment in vivo, it should be noted that while daily injection of ZK completely abrogates MPA-induced stimulation of the primary antibody response, ZK administered as a silastic pellet was unable to totally counteract MPA effects. This might be explained considering the possibility that serum concentrations of ZK resulting from the depot were lower than those reached from daily administration. Alternatively, partial inhibition of MPA effects by ZK in secondary responses might be due to enhanced sensitivity of lymphocytes to MPA following the booster with antigen.

Since its development more than three decades ago, MPA is being employed in an increasing variety of clinical conditions.7–10,31 Accordingly, the doses used ranged from relative low levels (a single 150 mg intramuscular injection, every 3 months), which proved to be effective in long-term contraception32 to very high ones (2000 mg/day orally), such as those employed for endocrine therapy of hormone-related cancer.33 Likewise, the plasma steady-state concentration of MPA with these different regimens varied from 1 ng/ml to more than 0·1 µg/ml. It is important to note that the scarce data available regarding the effects of MPA on immune response has mostly been obtained from patients receiving high doses schedules.11,13 Under these conditions, MPA exerts immunosuppressive effects compatible with its interaction to glucocorticoid receptors. Taking into account the immunosuppressive properties of MPA and the fact that undesirable side-effects, such as development of Cushingoid features,34,35 is less pronounced in patients treated with high dose MPA than conventional glucocorticoids, it has been recently proposed the employment of MPA for the treatment of autoimmune diseases.16,17 The results presented herein, however, indicate that this proposal should be considered with caution as MPA, in virtue of its ability to enhance antibody production, could promote host tissue injury mediated by IgG antibodies.

Acknowledgments

We would like to thank Ms Selma Tolosa and Ms Nelly Villagra for their excellent technical assistance and Fundación de la Hemofilia for the use of the FACScan cytometer. We are grateful to Dr Gador Laboratories for the supply of Medrosterone, to Schering AG for kindly providing ZK98299 and to Roussel-UCLAF for RU38486. This work was supported by grants from: Consejo Nacional de Investigaciones Científicas y Técnicas, SECYT, Buenos Aires University School of Medicine and Fundación Alberto J. Roemmers.

Abbreviations

BSA

bovine serum albumin

Con A

concanavalin A

CFA

complete Freund's adjuvant

DTH

delayed-type hyper-sensitivity

ELISA

enzyme-linked immunosorbent assay

ELISPOT

enzyme-linked immunosorbent spot-forming assay

FCS

fetal calf serum

IgG

immunoglobulin G

LPS

lipopolysaccharide

mAb

monoclonal antibody

MPA

medroxyprogesterone acetate

SRBC

sheep red blood cells

ZK

ZK 98299

RU486

RU 38486

PHA

phytohaemagglutinin

PBS

phosphate-buffered saline

PRG

progesterone

OVA

ovalbumin

TTBS

Tris buffered saline Tween-20

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