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Original Studies |
Departments of Reproductive Biology (F.L., V.C., G.Q., C.C.) and Immunology and Rheumatology (A.M.-C., J.A.-V., D.A.-S.), Instituto Nacional de la Nutrición Salvador Zubirán, Mexico City 14000, Mexico
Address all correspondence and requests for reprints to: Fernando Larrea, M.D., Department of Reproductive Biology, Instituto Nacional de la Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Mexico City 14000, Mexico.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immune-stimulating properties of PRL have been clearly demonstrated in both central and peripheral lymphoid organs (9), especially in the rat Nb2 T lymphoma cell line, which requires PRL for T cell proliferation (10, 11). In animal studies, PRL seems to be involved in maintaining the competence of B and T lymphocytes (2, 3) as well as enhancing macrophage cytotoxicity (4). In addition, PRL might affect thymic embryonic differentiation (12) and thereby influence neonatal T cell development (13). These observations have been further supported by the identification in a number of mammalian species, including man, of high affinity binding sites and the expression of PRL receptor genes in hematopoietic cells (14, 15, 16, 17, 18). This receptor has been shown to be a member of the superfamily of cytokine receptors involved in the growth and differentiation of lymphohematopoietic lineages (19).
A consistent finding in a number of studies has been the ability of PRL antisera to block the in vitro mitogen-stimulated murine, marmoset, and human lymphocyte proliferation (20). This effect was not seen with antibodies to GH or other trophic hormones (20). These observations suggest the presence of a locally produced PRL-like molecule that, acting in an autocrine or paracrine manner with PRL receptors in lymphocytes, could stimulate cell proliferation. In this regard, direct evidence of PRL-like message and secretion of structural and bioactive PRL-like molecules by murine (21) and human lymphocytes has been conclusively provided (17).
It is now becoming clear that conditions such as hyper-prolactinemia are accompanied by immune T cell alterations (22, 23) and reduced natural killer cell number (24) and function (5, 6, 22). These PRL-mediated abnormalities appear to predispose the susceptible host for antibody- and cell-mediated autoimmune disease. In both humans (25, 26) and the naturally occurring lupus B/W mouse model (27), systemic lupus erythematosus (SLE) is aggravated by altering normal serum PRL levels. In the present study, we have determined whether mitogen-stimulated mononuclear cells in patients with SLE differ from those in normal subjects in terms of the synthesis and secretion of PRL in vitro as an strategy to study the association of PRL with this particular autoimmune disorder.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients and controls
Eight female patients with diagnosis of SLE and with no evidence of endocrine disease were studied. Their ages ranged from 17–36 yr (mean, 26 yr). All patients fulfilled at least four of the American Rheumatism Association revised criteria (28). None of the patients were receiving corticosteroids or immunosuppressive or nonsteroidal antiinflammatory drugs at the time of the study. Clinical disease activity was scored on a 0–32 point scale according to the MEX-SLEDAI index (29). Patients with a MEX-SLEDAI score greater than or equal to 2 points were considered active. Six patients were active according to the above criteria. Eleven healthy ovulatory women (aged 21–50 yr) were studied as controls. None of the SLE or control subjects were receiving drugs known to be associated with increased PRL secretion.
Cell preparation and cultures
Human peripheral blood mononuclear cells (PBMNC) were obtained by Ficoll-Hypaque separation (30) of heparinized venous blood obtained from normal and SLE subjects. PBMNC were recovered from the interface and cultured at a concentration of 2 x 105 cells/0.15 mL in serum-free culture medium (AIM-V Medium, Life Technologies, Grand Island, NY) in the presence of various concentrations of Con-A. The viability of cells, as determined by trypan blue exclusion, was always above 95%. Cells were incubated for 72 h at 37 C in a humidified 95% air-5% CO2 atmosphere. Culture medium was kept at -20 C until analyzed for PRL bioactivity. DNA synthesis was evaluated by the addition of 1.0 µCi [3H]thymidine 8 h before harvest.
PRL-dependent Nb2 lymphoma cells
The Nb2 cell line was originally obtained from Dr. P. W. Gout (Vancouver, Canada). PRL bioactivity was measured using the Nb2 lymphoma cell assay as described by Tanaka et al. (11) with minor modifications. Briefly, cells were kept at 37 C in Fisher’s medium containing 10% FBS as a source of lactogen, 10% horse serum, 10-4 mol/L 2-mercaptoethanol, 50 IU/mL penicillin, and 50 µg/mL streptomycin. The cells were arrested in the early G1 phase of the cell cycle by preincubation (18–24 h) in lactogen-free medium. At this time, resumption of the cell cycle was stimulated by the addition of increasing concentrations of highly purified human pituitary PRL (NIDDK hPRL RP-2). PRL-like bioactivity was assayed in aliquots of culture medium from PBMNC at different dilutions to ascertain parallelism with the standard curve. Cultures per duplicate were further maintained in an atmosphere of 95% air-5% CO2 at 37 C for 72 h. The effects of hPRL or culture medium on cell proliferation were analyzed by the incorporation of [3H]thymidine (1.0 µCi) into Nb2 cells. The sensitivity of this assay for PRL was 1.0 pg/mL.
Complementary DNA (cDNA) synthesis and PCR amplifications
Total ribonucleic acid (RNA) was isolated from cultured PBMNC as
described by Chomzynski and Sacchi (31). One microgram of total RNA was
used as a template for cDNA synthesis, using cloned Moloney murine
leukemia virus reverse transcriptase (RT; Perkin-Elmer, Norwalk,
CT). The following are the sequences used for the sense and antisense
primers for PCR amplification of hPRL cDNA (complementary to exons 2
and 5, respectively): PRL1, 5'-TGTCAAACCTGCTCCTGTGCCAGA-3';
and PRL2, 5'-GTTGTTGTTGTGGATGATTCGGCA-3'. To monitor
efficiency for the RT reaction, we used as a control the amplification
of the ubiquitous protein cyclophilin (CF) with the following sense and
antisense primers, respectively: CF1,
5'-CCGCGTCTCCTTTGAGCTGTTT-3'; and CF2,
5'-ACCCAAAGGGAACTGTGCAGCGAGAGC-3'. These primers generate a single 633-
and 569-bp PRL and CF RT-PCR product, respectively. All
oligonucleotides were synthesized in a DNA synthesizer model 391
(Applied Biosystems, Perkin-Elmer/Cetus Co., Norwalk, CT). PCR was
carried out with the use of 20 µL of each of the RT reaction products
containing a final concentration of 0.5 µmol/L of each primer, 2.5 U
Taq polymerase, and 0.2 mmol/L deoxy (d)-NTPs in a
final incubation volume of 0.1 mL. Amplification was performed on a
Perkin-Elmer/Cetus 9600 PCR instrument beginning with a denaturation
step at 95 C for 105 s, followed by 35 cycles at 95 C for 15
s and 55 C for 30 s, with a final 7-min extension at 72 C.
Aliquots (10 µL) of each PCR were analyzed on 5% polyacrylamide gels
(Bio-Rad, Hercules, CA) to monitor the specificity and the amount of
product as described previously (32). Double distilled water or
incubations in the absence of RT were used as controls for RT-PCR
reactions. A band of the predicted size (633 bp) was confirmed as hPRL
by Southern blot analysis of previously separated DNA on 5%
polyacrylamide gels with a hPRL cDNA probe radiolabeled with
[32P]dCTP. A plasmid containing the 914-bp hPRL cDNA,
originally obtained from Dr. Nancy Cooke, University of Pennsylvania
(Philadelphia, PA), was grown in DH5
Escherichia coli,
excised and purified as previously reported (33), and radiolabeled with
[32P]dCTP with the use of a random primer kit (Life
Technologies, Gaithersburg, MD).
Western blotting and immunostaining
PBMNC extracts were homogenized in 50 mmol/L Tris (pH 8.0), 150
mmol/L NaCl, and 1% (vol/vol) Triton X-100 containing
phenylmethylsulfonylfluoride (100 µg/mL) and aprotinin (1 µg/mL)
and centrifuged at 8000 x g for 10 min, and
supernatants were saved at -20 C until assayed. Culture media from
PBMNC were immunoprecipitated using the VLS-2 anti-hPRL antiserum (Dr.
Y. N. Sinha, Whittier Institute for Diabetes and Endocrinology, La
Jolla, CA) for 72 h at 4 C. The precipitates were obtained after
the addition of goat antirabbit 
-globulin as previously described
(34), and cell extracts were resuspended and dissolved in Laemmli’s
sample buffer and heated at 95 C for 5 min. Samples were
electrophoresed on 12% polyacrylamide slab gels in the presence of SDS
(SDS-PAGE) under reducing conditions (35). After electrophoresis,
proteins were electroblotted onto nitrocellulose membranes in PAGE
buffer containing 20% methanol without SDS (36). Membranes were fixed,
blocked, and incubated overnight at room temperature in a 1:1000 final
dilution of anti-hPRL serum VLS-2 in 0.01 mol/L Tris-buffered saline,
pH 7.4, containing 0.1% Tween-20 and 3% Carnation powered non-fat dry
milk (Carnation, Los Angeles, CA). Immunocomplexes were visualized by
autoradiography after incubation in 125I-labeled protein A
(200,000 cpm/mL). After several washes in Tris-buffered saline, the
paper was dried and autoradiographed.
Hormonal assays
Blood samples were allowed to clot at room temperature, then were centrifuged at 1000 x g, and supernatants were stored at -20 C until assayed. PRL was measured in duplicate by specific RIA, using reagents and protocols provided by the National Hormone Pituitary Program (Rockville, MD) as previously described (37). The within-assay coefficient of variation for PRL was 7.0 ± 0.5%, with a sensitivity of 1.5 µg/L.
Statistical analysis
Differences between groups were assessed by using the Mann-Whitney U test. P < 0.05 was considered significantly different.
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Results |
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Mean radioimmunoassayable PRL concentrations in serum from SLE and control subjects were within the normal values established in our laboratory (3–19 µg/L). However, serum PRL levels in SLE individuals (19.0 ± 5.3 µg/L) were significantly higher than those in the normal group (12.7 ± 4.9 µg/L; P < 0.01). Two SLE subjects had circulating PRL concentrations slightly above normal values (27 and 25 µg/L, respectively) without clinical manifestations of hyperprolactinemia.
Nb2 cell bioassay, parallelism and responsiveness to Con-A
As shown in Fig. 1
, Nb2 when exposed
to hPRL incorporated [3H]thymidine in a dose-response
manner (solid line). A parallel dose-response curve was
obtained when different aliquots of the supernatant from a lupic PBMNC
culture were added (dotted line). Nb2 cells were also
exposed to various concentrations of Con-A, a well known stimulator of
human lymphocytes. Over the concentration ranges tested, mitogenic
indexes were not significantly different from those obtained without
mitogen (Fig. 2). Thus, these data
indicate that Con-A at concentrations that normally stimulate human
lymphocytes had no mitogenic effects on the Nb2 cells.
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At a concentration of 2 x 105 cells in the presence of 1.5 µg/mL of Con-A, PBMNC from normal and SLE subjects produced the highest PRL-like bioactivity in the Nb2 mitogenic assay. In all cases, basal PRL-like bioactivity increased significantly in PBMNC cultured in the presence of the mitogen; however, the major increments in response to Con-A were always observed in control cells.
The production of bioactive PRL-like by PBMNC cultured in serum-free
media from SLE and normal subjects is shown in Fig. 3. As can be seen,
non- Con-A-stimulated (B) PBMNC from SLE subjects produced
significantly higher (P < 0.001) amounts of bioactive
PRL-like (195 ± 47 pg/mL) than non-stimulated PBMNC from normal
individuals (27 ± 4 pg/mL). Stimulation with Con-A (S) increased
PRL-like bioactivity in both normal (148 ± 19 pg/mL) and
SLE-PBMNC (280 ± 46 pg/mL). Although, normal cells responded
better to the mitogen, stimulated SLE cells produced significantly
higher amounts of bioactive PRL (P < 0.05
vs. normal).
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, hPRL
antiserum blocked the proliferative activity of medium from
Con-A-stimulated normal and SLE PBMNC cultures. Although, the mitogenic
activity in SLE remained higher than that in control subjects
(P < 0.05), the inhibitory effect of PRL antiserum was
statistically significant in both cases (P < 0.01;
Con-A-stimulated vs. antibody). Western blot analysis of bioactive PRL-like molecules
To further evaluate the nature of bioactive PRL-like molecules
secreted by PBMNC from normal and SLE subjects, cell extracts were
prepared and subjected to SDS-PAGE. A single 60-kDa immunoreactive PRL
product was found in Western blots from both SLE (Fig. 4, lanes 3 and 4) and control (Fig. 4, lanes 5 and 6) Con-A stimulated PBMNC. As shown, SLE lymphoid cells
contained more 60-kDa PRL than normal cells. There was no evidence for
the existence of 23-kDa PRL like that found in circulation of a patient
with SLE (Fig. 4, lane 2) or of any other PRL-like variants previously
described. A highly purified PRL preparation from human pituitaries
(NIDDK hPRL RP-2) was used as the standard for electrophoretical
mobility (Fig. 4, lane 1). Control incubations with normal rabbit serum
instead of PRL antiserum gave negative results.
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To determine whether the PRL gene is expressed in normal and SLE
PBMNC, PRL gene sequences were amplified by reverse transcription
followed by PCR. A 914-bp hPRL cDNA probe was used as a control for PCR
product size determination. Figure 5
shows a representative Southern blot analysis of RT-PCR products
obtained from Con-A-stimulated normal and SLE PBMNC. As depicted, a
major fragment of 633 bp was generated in both SLE and normal PBMNC
cDNAs (upper panel, lanes 2 and 3, respectively) with an
identical size as that obtained with the hPRL cDNA probe used as a
positive control (upper panel, lane 1). Control
amplifications using CF are shown in the bottom panel. In
the absence of RT, none of the mRNA samples from SLE and normal PBMNC
cDNAs for PRL and CF, respectively, gave positive results. Similar
negative results were obtained when double distilled water was used
instead of mRNA (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we have shown the presence of a PRL mRNA transcript in Con-A-stimulated PBMNC from subjects with SLE. In addition, conditioned medium from Con-A-stimulated PBMNC from SLE subjects showed a higher biological activity in the Nb2 cell bioassay than that from normal subjects. It was not possible to determine the mechanisms influencing the rate of PRL gene expression in SLE or whether it depends on clinical disease activity. Recently, Gutiérrez et al. (44) showed that both nonstimulated and stimulated PBMNC from patients with SLE have an increased production of PRL-like immunoreactive material. The immunoreactive PRL concentration in the culture medium found by these authors was nearly 3 times higher than the amount detected with the bioassay used in this study. Nothing is known concerning the immuno/bioactivity ratios of lymphocyte-derived PRL, especially under pathological conditions such as SLE. In this regard, several PRL variants with different bioactivities have been described and formed by transcriptional or translational mechanisms (45). In contrast to the 11-kDa PRL-like species reported by Gutierrez et al. (44) in peripheral lymphocytes from SLE, our results revealed the presence of a 60-kDa PRL-like variant as the major immunoreactive lymphocyte-derived PRL form. This species did express PRL-like bioactivity in the Nb2 node lymphoma bioassay, and its proliferative actions on these cells were suppressed when polyclonal anti-hPRL was added to the cultures. These data are consistent with studies that demonstrated the presence of PRL-like variants with molecular sizes ranging from 11–60 kDa in human lymphocytes (42) and may also explain in part the apparent discrepancy with our results in terms of PRL immunoreactivity and bioactivity in lymphocytes from SLE individuals. Inasmuch as the RT-PCR reaction produced a single expected 633-bp band that was further confirmed by Southern analysis with the use of hPRL cDNA as the probe, it is suggested that the size variation observed in lymphocyte-derived PRL is probably due to posttranslational modifications of a common precursor. Additional experiments will be needed to determine the molecular nature and biological properties of the various species, including the 60-kDa species found in this study. Hyperprolactinemia has been found in subsets of SLE patients; the disease is more active in those with elevated serum PRL levels (43), raising the possibility that circulating PRL might have an extrapituitary source. Inasmuch as the results from this study argued against this possibility, as the major PRL circulating species in SLE subjects had a molecular size of 23 kDa in contrast to the 60-kDa form found in both normal and SLE-PBMNC, further investigation is needed to define the source of circulating PRL in hyperprolactinemic SLE subjects.
Conventional T cell mitogens have been shown to influence the production of PRL-like activity by immune cells (46). At the concentration used (1.56 µg/mL), Con-A did not modify Nb2 responsiveness compared to that in cultures grown in the absence of mitogen, but, on the other hand, it significantly increased PRL-like secretion by both normal and SLE-PBMNC. This observation is in line with previous reports on the effects of T cell mitogens on rat Nb2 lymphoma cells (47) as well as on the effects of these agents on the secretion of bioactive PRL-like species from PBMNC (46).
Although the exact mode of action of PRL in autoimmunity is not yet completely elucidated, experimental hyperprolactinemia has been associated with characteristic Th2 cells cytokine production (48). These data suggest that PRL-driven cytokine production by Th2 cells could stimulate lymphocytes or microenvironmental cells, leading to a deterioration of the autoimmune process. Therefore, additional studies aimed at analyzing the mechanisms and pathways regulating PRL expression on lymphoid cells might provide us with new insights on the role of PRL in both normal and pathological immune conditions.
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Acknowledgments |
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Footnotes |
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Received May 11, 1997.
Revised June 25, 1997.
Accepted July 28, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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