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Original Articles: Hormones and Reproductive Health |
Laboratory of Autoimmunity, Immunology Research Unit, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México City, 06725 México
Address correspondence and requests for reprints to: Dr. Alfredo Leaños-Miranda, M.D., Ph.D., Laboratory of Autoimmunity, Immunology Research Unit, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Av. Cuauhtémoc 330, Col. Doctores, 06725 México, D.F., México. E-mail: alfredo{at}intranet.com.mx
Abstract
A woman with systemic lupus erythematosus (SLE) with marked increases in circulating 150-kDa PRL was studied from before conception, throughout pregnancy, and after pregnancy. The clinical features of the patient included idiopathic hyperprolactinemia without clinical symptoms such as amenorrhea and galactorrhea before pregnancy. No clinical lupus activity was present during follow-up. Serum PRL increase during pregnancy in this patient was considerably higher at weeks 27 and 33 than in normal pregnant women. In contrast, serum-free PRL levels were considerably lower at weeks 20, 27, and 33 than in normal pregnant women. A 150-kDa PRL (big big PRL) species persisted as the predominant circulating form of PRL throughout each measurement in this woman with SLE. In contrast, the predominant form of PRL in serum from healthy pregnant women was little PRL (or monomeric PRL). The nature of big big PRL was due to the presence of anti-PRL autoantibodies forming an IgG-23 kDa PRL complex, in accordance with the studies by affinity chromatography for IgG and Western blot analysis. The IgG-PRL complex was fully bioactive in vitro (Nb2 rat lymphoma cell assay). Injection of the serum into the rats demonstrated that the IgG-PRL complex was cleared more slowly than serum containing predominantly monomeric PRL. The data suggest that the IgG-PRL complex has biological activity; the absence of symptoms in this woman may be attributed to the fact that due to its large molecular weight, big big PRL does not easily cross the capillary walls. Delayed clearance may account for increased serum PRL levels in this SLE patient with anti-PRL autoantibodies.
IN SERUM FROM normal subjects and most
patients with hyperprolactinemia, the majority of PRL immunoreactivity
is found by gel filtration column chromatography to have an apparent
molecular weight of 
23K (little PRL). Lesser amounts are
found in the void volume (big big PRL >100 kDa) or in a position
corresponding to a molecular weight of 45–50K (big PRL). Several
authors have reported a small number of patients with asymptomatic
hyperprolactinemia, such as the absence of amenorrhea and/or
galactorrhea, because of the predominant presence of big big PRL, a
phenomenon termed macroprolactinemia (1, 2, 3).
Macroprolactinemia has been reported mainly as the cause of idiopathic
hyperprolactinemia (4, 5, 6). Although the nature of
macroprolactin is still unclear, this isoform of PRL seems to be a
postsecretory event (aggregates of monomeric PRL and PRL-bound to
binding proteins). These modifications of PRL may affect their
biological properties differently from immunoreactivity
(7). Recent evidence indicates that big big PRL is mostly
an IgG-23 kDa PRL complex (anti-PRL autoantibody); this anti-PRL
autoantibody has been reported in sera from 16% of patients with
idiopathic hyperprolactinemia (without autoimmune disorders)
(8). In contrast, we have recently shown in systemic lupus
erythematosus (SLE) patients without proven cause of hyperprolactinemia
that the frequency of macroprolactinemia due to anti-PRL autoantibodies
was higher (40.7%) (9). Interestingly, patients with
anti-PRL autoantibodies had less clinical and serological SLE activity
than those without anti-PRL autoantibodies (with only the presence of
monomeric or little PRL in their blood). PRL has effects on B and T
cells. Moreover, high serum PRL levels have been associated to lupus
activity (10, 11). A possible explanation for the lesser
lupus activity in the SLE patients with anti-PRL autoantibodies is that
PRL has attenuated biological activity when bound to its autoantibody,
interfering with PRL receptor binding on lymphocytes and/or because it
does not easily cross the capillary walls to reach lymphoid tissues due
to its high molecular weight.
On the other hand, both macroprolactinemia related or not to anti-PRL autoantibodies have the following similarities: 1) large molecular weight (>100K) and 2) lack of clinical symptoms of hyperprolactinemia. In the few cases reported, women with macroprolactinemia usually retain fertility (10, 11, 12, 13). We recently had the opportunity to prospectively follow a SLE woman with known macroprolactinemia due to the presence of anti-PRL autoantibody during pregnancy. This report describes the circulating forms of PRL and the binding of serum PRL to G protein in this woman with SLE (before, during, and after pregnancy). In addition, biological activity of PRL in fractions obtained after gel filtration and the characterization of PRL after SDS-PAGE and Western blot analysis were examined. Finally, an endocrinological evaluation and a clearance study of PRL into rats were performed.
Patients and Methods
The Human Ethical Committee and Medical Research of the Institute approved the study protocol, and informed written consent was obtained from all subjects who participated voluntarily in this study. The animals were cared for according to the Guide for the Care and Use of Laboratory Animals.
Case report
A 21-yr-old woman, who 7 yr earlier was diagnosed as having SLE,
manifested by fatigue, arthralgias, arthritis, pleuresy, leukopenia,
lymphopenia, positive antidouble-stranded DNA (ds-DNA) antibodies, and
a positive fluorescent antinuclear antibody test (1:2560), was
studied. Her menarche occurred at age 14, and she had been pregnant at
the age of 18 yr. Her menstrual cycle had always been regular. In
October 1996 (at age 19), her PRL level was measured because she was
included in another study that we recently published (9).
From October 1996 to August 1997, PRL levels ranged from 70.4–90.5
µg/L (normal, 0–20 µg/L); the patient had no evidence of pituitary
adenoma on computed tomography, nor did she have known causes for
secondary hyperprolactinemia. She again spontaneously became pregnant
in December 1997, and at 36 weeks of pregnancy she underwent cesarean
surgery due to the premature rupture of membranes. The woman did not
breast-feed her baby for medical reasons; however, she exhibited
galactorrhea for 5 days postcesarean, and menses became regular at 45
days postcesarean. Over the next 10 months, serum PRL levels fluctuated
between 66.9 and 115.5 µg/L. Blood samples were drawn at 20, 27, and
33 weeks of pregnancy. As a control, 40 pregnant but otherwise healthy
women (9 at week 20, 10 at week 27, and 21 at week 33) were also
studied. Hyperprolactinemia was evaluated at 10 months postcesarean by
the administration of metoclopramide (10 mg, iv) and
bromocriptine (BRC; 2.5 mg/day for 2 weeks). During
follow-up, she did not manifest clinical SLE activity and was treated
during this time with prednisone (5–20 mg/day). However, she continued
to exhibit the presence of antibodies to ds-DNA, Ro/SSA, La/SSB, Smith
antigen, ribonucleoprotein, and antinuclear (Table 1
).
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PRL concentrations in serum and fractions by chromatographic studies were measured by immunoradiometric assay (IRMA) (ELSA-PRL; CIS-Bio International, Gif-sur-Yvette, France). The limit of sensitivity of the assay was 0.09 µg/L. Anti-PRL autoantibody was found not to interfere with present IRMA for PRL (14). Normal levels of PRL are 5–20 µg/L. Free PRL was extracted from the sera according to the method described previously (9). Intra-assay and interassay coefficients of variation were 8% or less in all tests.
Chromatography
Gel filtration was performed using a column (60 x 1.5 cm) of Sephadex G-200 Superfine (Pharmacia, Uppsala, Sweden), as described previously (9). Serum samples of 2 mL were applied on the column, and fractions of 1.3 mL were collected for PRL assay in the same day. Recovery was 104.4 ± 14.3%, on average. Affinity chromatographic for IgG was performed using a 1-mL G protein column (HiTrap G; Pharmacia LKB, Uppsala Sweden) as previously described (9). IRMA assayed both retained and unretained fractions for PRL. When PRL standards were applied on the column, no PRL was bound to G protein (mean ± SD, 1.9 ± 3.5%).
PRL-dependent Nb2 lymphoma cell
PRL bioactivity in fractions on gel filtration before pregnancy was measured using Nb2 lymphoma cell assay as described by Tanaka et al. (15), and similar to that described by Larrea et al. (16). Recombinant human PRL (rhPRL) (Genzyme, Cambridge, MA) was used as standard PRL. To inhibit the lactogenic effect of human GH (hGH), rabbit antiserum to hGH [NIDDK-anti-hGH-IC-3 A.S., CYTO (AFPC11981A)] was added to a final dilution of 1:4000. The effects of rhPRL and PRL fractions by gel filtration on cell proliferation were analyzed by incorporation of [3H]thymidine (1µCi) into Nb2 cells. When anti-hPRL was added to the Nb2 cell assay in the presence of antibodies to hGH, stimulation of the proliferation of Nb2 cell by serum fractions was completely abolished. Sensitivity of this assay for PRL was 3 pg/mL. Nb2 cell assay was carried out when the patient was not pregnant, because at the time of the study we did not have antiplacental lactogen antiserum.
Western blotting and immunostaining
The procedure used was similar to that reported by Sinha
et al. (17). Briefly, PRL samples were
immunoprecipitated with rabbit anti-hPRL antiserum
[NIDDK-anti-hPRL-IC-5, CYTO (AFP55781789SI)] for 72 h at 4 C and
subsequently with goat antirabbit 
-globulin antiserum (DAKO Corp., Carpinteria, CA) for 24 h at 4 C with continuous
shaking. Normal rabbit serum without human samples was used as control.
The precipitates were resuspended and dissolved in Laemmli’s sample
buffer and heated at 95 C for 5 min. Samples were electrophoresed on a
12% polyacrilamide gel in the presence of 0.1% SDS under reducing
conditions (18). After electrophoresis, proteins were
electroblotted onto nitrocellulose paper in PAGE buffer containing 20%
methanol without SDS (19). Paper was fixed, blocked, and
incubated for 4 h at room temperature in a 1:1000 final dilution
of same rabbit anti-hPRL serum and afterward with a 1:2000 dilution of
peroxidase-conjugated goat antirabbit serum (DAKO Corp.).
Color developing solution was added to localize the
immunocomplexes.
Clearance studies
One milliliter of serum from the woman with SLE or from a healthy pregnant woman (matched by serum PRL level) was rapidly injected via a femoral cannula into ketamine-anesthetized male Sprague Dawley rats (body weight, 300 g). Blood samples of 0.3 mL were withdrawn at 0, 1, 5, 10, 20, 40, 60, and 90 min. The rat plasmas were separated and frozen at -35 C until assayed for hPRL. Rat PRL was shown not to interfere with the present IRMA for hPRL.
Stimulation and suppression test for PRL
At 10 months postcesarean, a 10-mg dose of metoclopramide was injected as an iv bolus and blood was collected at 0, 30, 60, 90, and 180 min. Then, BRC was instituted at a dose of 2.5 mg/day for 2 weeks and blood was drawn at days 7 and 14.
Results
During follow-up of the patient before pregnancy, she had
sustained hyperprolactinemia (70.4–90.5 µg/L), and gel filtration
showed that the majority (75.4–87.8%) of PRL immunoreactivity eluted
at position 150 kDa (big big PRL), with 6.6–16.6% eluting at the same
position of 125I-hPRL (little PRL, 23 kDa). These
proportions of big big PRL did not change significantly after pregnancy
69.2–70.7% (Table 1
). In the woman with SLE (Table 1), the serum
total PRL level at 20 weeks gestation was 214.5 µg/L, similar to mean
than that of healthy pregnant women at the same gestation time [190.4
µg/L; 95% confident interval (CI), 125.1–255.7 µg/L]. At 27 and
33 weeks, serum total PRL levels in the woman with SLE were 483.7 and
427.1 µg/L, respectively; in contrast, these concentrations were
considerably lower in healthy pregnant women at 27 weeks (254.8 µg/L;
CI 95%, 201.5–308.0 µg/L) and at 33 weeks (296.1 µg/L; CI 95%,
267.9–324.2 µg/L). On the other hand, free PRL levels in the woman
with SLE were considerably lower at weeks 20, 27, and 33 than that in
healthy pregnant women (Table 1). Figure 1A shows the gel filtration profiles of
PRL before, during, and after pregnancy in the SLE patient. As can be
seen, during pregnancy almost all (66.6–74.3%) PRL immunoreactivity
eluted as big big PRL (150 kDa). In contrast, in the healthy pregnant
women the 23-kDa PRL remained as the predominant species (
85.4% of
total PRL at the same number of weeks gestation). As pregnancy
progressed, all species of PRL increased, but the relative proportion
remained the same. However, serum-free PRL levels increased 7.4- to
10.4-fold during pregnancy in comparison with the mean before pregnancy
(8.8 µg/L). In contrast, total PRL level increases were lower (5.2-
to 5.9-fold) than the mean before pregnancy (81.7 µg/L). Figure 1B
shows affinity chromatography with G protein column profiles of serum
PRL before, during, and after pregnancy in the SLE patient. As
pregnancy progressed, there was a decrease in serum PRL retained by G
protein column at 20 weeks (45.6%), 27 weeks (35.7%), and 33 weeks
(29.5%), and serum PRL retained by the column at 4 weeks postpregnancy
returned to prepregnancy values (Table 1). In contrast, in healthy
pregnant women the proportion of PRL bound to G protein was
considerably lower (
3.6%).
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When whole serum or fractions containing big big PRL were analyzed on
SDS-PAGE displayed one band on Western blot analysis, this band was a
23-kDa protein. When whole serum from a patient with pituitary adenoma
was used, it displayed three bands (25, 23 and 22 kDa). The
pituitary hPRL [NIDKK-hPRL-SIAFP-B-3 (AFP-3855A), which was not
immunoprecipitated] also elicited 25- and 23-kDa bands as well as
others (60, 48, 20, 16.5, and 16 kDa). Finally, the same bands were
also present in rhPRL (that was not immunoprecipitated), except the
25-kDa band (glycosylated PRL) (Fig. 2).
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). More than 50% of immunoreactive PRL
was present until 90 min when the serum from the woman with SLE was
injected. In contrast, more than 50% of immunoreactive PRL was
cleared from circulation at 5 min when the serum from healthy pregnant
woman was injected.
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). Gel filtration revealed that the increase in PRL levels by
metoclopramide at 30 min consisted almost entirely of 23-kDa PRL (60.4
vs. 25.2% in the basal state). Also, PRL bound to G protein
decreased from a baseline proportion of 33.3% to 21.4% at 30 min and
slowly increased to the basal value (Table 1). Serum PRL levels were
suppressed by BRC to 17.0 µg/L at 7 days and to 15.6 µg/L at 14
days. On the other hand, gel filtration showed that when the PRL
standard was mixed with serum (1 mL) from a normal woman, the majority
of PRL immunoreactivity eluted as 23 kDa PRL (92.9%), with 4.3%
eluting as big big PRL. In contrast, these proportions changed
significantly after mixing PRL standard with serum from the woman with
SLE (at 14 days with BRC); 23.8% of PRL immunoreactivity eluted at
position 150 kDa (big big PRL) and with 67.8% as 23 kDa PRL. Also, the
proportion of PRL bound to G protein was significantly higher in PRL
standard mixed with serum from the woman with SLE (20.2 vs.
1.0% in serum from healthy woman) (Table 1). Discussion
Hyperprolactinemic patients usually have galactorrhea, menstrual irregularity, and infertility (20, 21, 22). Conception usually occurs only after treatment with dopaminergic agonist (23, 24, 25, 26). In contrast, there is a subgroup of women with a large molecular size form of PRL who retain fertility despite sustained hyperprolactinemia (12, 13, 27). We found that the woman with SLE spontaneously became pregnant; the course of pregnancy was uneventful, and her infant was normal. These results are similar to those described in hyperprolactinemic women with macroprolactinemia related or not to anti-PRL autoantibody (12, 28).
The proportion of little PRL has been reported to be increased during pregnancy in normal women and patients with macroprolactinemia (28, 29). In the case under study, we found that as pregnancy progressed big big, big, and little PRL all increased, but the relative proportion of little PRL increased dramatically. Release of little PRL from the pituitary gland may increase as pregnancy progresses, and a part of little PRL binds to anti-PRL autoantibody, leading to an increase in big big PRL. However, an increase in little PRL may exceed the binding capacity of the autoantibody, leading to an additional increase in little PRL. PRL levels may become extremely high during pregnancy because big big PRL may not exert a physiological feedback action on the pituitary gland, as little PRL and serum PRL levels may increase until little PRL reaches its physiological level.
In normal women, serum PRL values during pregnancy increase 10- to 20-fold (29, 30). In contrast to normal women, the PRL increment in our hyperprolactinemic woman was considerably less. Lesser serum PRL increases during pregnancy also have been reported in women with other forms of hyperprolactinemia (31, 32). In fact, there is no uniform pattern of PRL increases during gestation in hyperprolactinemic women; the changes range from normal increments in PRL levels during pregnancy to decreases at the time of delivery (33). However, free PRL levels during pregnancy in the patient under study were lower than those in normal pregnant women but increased as much as 10-fold compared with before pregnancy. These results are similar to those described in normal pregnant women whose predominant circulating species was little PRL (29, 30).
Although several investigators have reported patients with macroprolactinemia, the nature of big big PRL is still controversial (12, 28, 34). In this case, it was due to the presence of a PRL-anti-PRL autoantibody complex. The diagnosis of the presence of an anti-PRL autoantibody was based on the following: 1) PRL immunoreactivity was predominantly eluted at the position of 150K molecular weight, similar to that of IgG; and 2) significantly large amounts of PRL were adsorbed by the affinity column for IgG.
A significant but lower proportion of serum PRL was retained by G protein Sepharose column than by filtration gel. This is probably because during affinity chromatography with G protein Sepharose there is a dissociation of PRL from its autoantibody, underestimating the proportion of big big PRL retained by the G protein Sepharose column. This dissociation can be explained by a low affinity of the autoantibody to PRL. However, there is an amount of big big PRL that is not IgG bound and that most likely represents PRL bound to other proteins that are not retained by G protein or other immunoglobulin isotypes binding to PRL, as reported for other autoantigens in sera SLE (IgA and IgM).
The clinical features of the present patient with macroprolactinemia include idiopathic hyperprolactinemia, without clinical symptoms such as amenorrhea and galactorrhea before pregnancy and very high levels of PRL during pregnancy. In addition, other studies have reported the presence of high PRL levels during pregnancy in women with anti-PRL autoantibody (without autoimmune disease) compared with pregnant women without macroprolactinemia (28, 35). The lack of clinical symptoms of hyperprolactinemia cannot be attributed to low biological activity of big big PRL; biological activity (Nb2 cell assay) in fractions containing big big PRL yielded the same results as in fractions containing little PRL. However, in vivo big big PRL may not exert enough action because it does not easily cross the capillary walls due to its high molecular weight. On the other hand, clearance studies in rats showed that the IgG-PRL complex is eliminated more slowly from the bloodstream than serum containing only monomeric PRL, these data according to those found by Hattori and Inagaki (36), who postulated that this could be another mechanism in which PRL is retained in circulation for a long time (prolonged half-life).
It is of interest that an antidopaminergic agent elicited a normal response of PRL secretion. Indeed, total PRL levels exceeded the binding capacity of anti-PRL autoantibodies, resulting in an increase in free PRL level (monomeric PRL), a finding similar to that displayed during pregnancy in this woman with SLE. BRC had a suppressive effect on PRL levels, which returned to normal values (<20 µg/L). One hundred fifty kilodaltons of PRL can be made in vitro by mixing PRL standard (23 kDa) with serum (obtained at 14 days post-BRC) from the woman with SLE, suggesting that 150 kDa PRL is the result of the binding of 23 kDa PRL to anti-PRL autoantibody, as confirmed by affinity chromatography on G protein and Western blot analysis.
In mice and humans, SLE flares increase during pregnancy; this has been associated with raising serum PRL levels (37, 38, 39, 40). We have previously shown that hyperprolactinemic SLE patients with anti-PRL autoantibodies had less SLE activity compared with those without anti-PRL autoantibodies (9), suggesting that the anti-PRL autoantibody may interact on the SLE disease spectrum. However, further research is needed to define the mechanism and importance of these findings. Nevertheless, in this woman with SLE during follow-up, the disease activity of the patient remained stable even of incurring in persistence of hyperprolactinemia.
Acknowledgments
We are indebted to the NIDDK-National Hormone and Pituitary Program (Dr. A. F. Parlow) for the hPRL reagent and specific antibodies.
Received September 13, 2000.
Revised November 17, 2000.
Accepted December 4, 2000.
References
This article has been cited by other articles:
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  A. Leanos-Miranda, J. Marquez-Acosta, G. M. Cardenas-Mondragon, Z. L. Chinolla-Arellano, R. Rivera-Leanos, S. Bermejo-Huerta, J. F. Romero-Arauz, G. Alvarez-Jimenez, J. C. Ramos-Leon, and A. Ulloa-Aguirre Urinary Prolactin as a Reliable Marker for Preeclampsia, Its Severity, and the Occurrence of Adverse Pregnancy Outcomes J. Clin. Endocrinol. Metab., July 1, 2008; 93(7): 2492 - 2499. [Abstract] [Full Text] [PDF]
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 A. Leanos-Miranda, G. Cardenas-Mondragon, A. Ulloa-Aguirre, I. Isordia-Salas, A. Parra, and J. Ramirez-Peredo Anti-prolactin autoantibodies in pregnant women with systemic lupus erythematosus: maternal and fetal outcome Lupus, May 1, 2007; 16(5): 342 - 349. [Abstract] [PDF]
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A. Glezer, C. R. J. Soares, J. G. Vieira, D. Giannella-Neto, M. T. C. P. Ribela, V. Goffin, and M. D. Bronstein Human Macroprolactin Displays Low Biological Activity via Its Homologous Receptor in a New Sensitive Bioassay J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 1048 - 1055. [Abstract] [Full Text] [PDF]
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 A. Leanos-Miranda and G. Cardenas-Mondragon Serum free prolactin concentrations in patients with systemic lupus erythematosus are associated with lupus activity Rheumatology, January 1, 2006; 45(1): 97 - 101. [Abstract] [Full Text] [PDF]
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F Blanco-Favela, K Chavez-Rueda, and A Leanos-Miranda Analysis of anti-prolactin autoantibodies in systemic lupus erythematosus Lupus, October 1, 2001; 10(10): 757 - 761. [Abstract] [PDF]
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, pregnancy at week 20; 
,
pregnancy at week 27; *, pregnancy at week 33; 
, after pregnancy).
B, Affinity chromatography profiles of immunoreactive PRL on a G
protein Sepharose column (1 mL). Samples (1 mL) were applied in the
column, and fractions of 1.5 mL were collected (•, before pregnancy;
