Prolactin down-regulates CD4+CD25hiCD127low/- regulatory T cell function in humans.

Among its many functions, prolactin (PRL) participates in immune responses and promotes the activation, differentiation and proliferation of T cells. However, the mechanisms by which PRL regulates regulatory T (T(reg)) cells are still unknown. Our goal was to determine whether PRL plays a role in T(reg) function. We measured the expression of PRL and its receptor in T(reg) and effector T (T(eff)) cells from 15 healthy individuals. We also evaluated the functional activity of T(reg) cells by examining proliferation and cytokine secretion in cells activated with anti-CD3/CD28 in the presence or absence of PRL. We report that T(reg) cells constitutively expressed PRL receptor, whereas T(eff) cells required stimulation with anti-CD3/CD28 to induce PRL receptor expression. Expression of PRL was constitutive in both populations. We found that the addition of PRL inhibited the suppressor effect (proliferation) mediated by T(reg) cells in vitro, reducing suppression from 37.4 to 13% when PRL was added to co-cultures of T(reg) and T(eff) cells (P<0.05). Cultures treated with PRL favoured a Th1 cytokine profile, with increased production of TNF and IFNγ. We report for the first time that PRL receptor expression was constitutive in T(reg) cells but not in T(eff) cells, which require stimulation to induce PRL receptor expression. PRL inhibited the suppressive function of T(reg) cells, apparently through the induced secretion of Th1 cytokines.

Activation of TLR2 and TLR4 by minimally modified low-density lipoprotein in human macrophages and monocytes triggers the inflammatory response.

Oxidized low-density lipoproteins and Toll-like receptors (TLR) 2 and 4 are involved in the development of atherosclerosis. The TLR are important in the pro-inflammatory response. The aim of this research was to analyze the activation of CD14, TLR4, and TLR2 in response to minimally modified low-density lipoprotein (mmLDL). Human monocytes and macrophages secreted tumor necrosis factor (TNF)-alpha in response to mmLDL, and blocking CD14 or TLR4 resulted in a approximately 60% decrease in mmLDL-induced TNF-alpha secretion. We also observed similar inhibition of TNF-alpha synthesis in human monocytes ( approximately 65%) and macrophages ( approximately 70%) when both receptors were blocked simultaneously. When TLR2 was blocked, TNF-alpha synthesis was inhibited by approximately 70% in both cell types. Moreover mmLDL induced redistribution of CD14, TLR4, and TLR2 on the cell surface. This is the first evidence that TLR2 and TLR4 are upregulated in response to mmLDL. Our results suggest that mmLDL activates CD14, TLR4, and TLR2, inducing the production of TNF-alpha and increasing the expression of TLR2 and TLR4.

Prolactin effect on CD69 and CD154 expression by CD4+ cells from systemic lupus erythematosus patients.

OBJECTIVE: The aim was to explore the role of prolactine (PRL) in the lymphocyte activation process in active and inactive systemic lupus erythematosus (SLE) patients in an in vitro model. METHODS: Peripheral blood mononuclear cells (PBMNC) were isolated from SLE patients and healthy individuals. The mRNA for prolactine and its receptor, obtained by standard techniques with an appropriate primer, were subjected to PCR and visualised. The PBMC were cultured with: a) medium alone as a negative control, b) unspecific mitogen as a positive control (PMA-ionomycin for CD154 or concanavalin A for CD69), c) PRL alone, d) mitogen plus PRL, e) mitogen plus antibody anti-PRL (1:50) and f) mitogen plus an unrelated antibody. Then CD69 and CD154 were determined by flow cytometry analysis. RESULTS: Twelve inactive and 15 active SLE patients were studied. 25% of the active patients displayed hyperprolactinemia. Under basal conditions, CD69 expression was associated with disease activity. In contrast, CD154 did not show this association. The PBMNC activated in vitro were capable of producing and secreting prolactine as measured by mRNA and Nb2 assay. In the same way the mRNA for prolactine receptor was visualized. Cells from SLE patients cultivated with PRL alone did not display increased CD69 or CD154 expression. The addition of PRL to the unspecific stimulated culture did not have an additive effect. In contrast, the addition of antibodies against PRL, in order to block the autocrine prolactine, resulted in a striking reduction in CD69 and CD154 expression. CONCLUSIONS: PRL is produced and secreted by the immune cell and acts just after the first trigger signal of activation in an autocrine way. The expression of CD69 and CD154 molecules depend partially on the prolactine.

Development of a diagnostic test for Entamoeba histolytica using idiotype expression in human.

The protozoan parasite Entamoeba histolytica is the etiological agent of human amebiasis. The pathology of the disease starts with the cytolysis of the host target cells by amoebae. It is initiated by the adhesion of trophozoites to the host cells, through surface lectin via specific receptors. These adherence lectins have been demonstrated to be highly conserved, and can be recognised by serum antibodies from patients with invasive amebiasis. Some of these molecules have been used as antigens in serologic studies, which has been very helpful in the diagnosis of invasive intestinal amebiasis. However, false-positive serologic reactivity can occur using E. histolytica extracts and purified antigens. Additional problems are because the extracts display a great enzymatic activity. Several diagnostic methods, using different molecules and techniques, have been described. However, the problem still remains since these tests are not capable of differentiating between amoebic liver abscess (ALA) and intestinal amebiasis.Here, the research has been addressed to the 66-kDa antigen, which is a part of the outer membrane proteins from the E. histolytica strain HM1-IMSS trophozoites. First of all, we characterized the 66-kDa antigen in order to prove the relevance. We found that the 66-kDa antigen is a part of the plasma membranes and is distributed rather homogeneously on the cell surface of trophozoites. Apparently, the 66-kDa antigen is a glycoprotein. Using a monoclonal antibody (MAb), we found 25% of inhibition in the erythrophagocytosis by the trophozoites. Starting form one monoclonal antibody, we prepared an anti-idiotype (anti-Id) antibody reagent, with the purpose of searching for the different expressions of the idiotype between the sera from ALA and the intestinal amebiasis patients. Moreover, we produced the antibody Ab3 that is capable of recognising the 66-kDa antigen; it means that the Ab2 displays the internal image of the antigen. We found that 91.6% of the serum from ALA patients displayed the expression of the Id. In contrast, 15.7% of the E. histolytica asymtomatic cyst carriers displayed the Id expression, 6.6% of the patients with another parasite infection, and 11% of the negative controls (serum from umbilical cords of newborn babies). Our results showed that the expression of the Id could be differentiated among the AHA patients from the other groups with a 91.6% sensibility and 88.3% specificity.

Analysis of anti-prolactin autoantibodies in systemic lupus erythematosus.

Evidence has shown that prolactin is an essential component of an effective immune response. In systemic lupus erythematosus, clinical trials have produced controversial information about the role of PRL. Some results find association between serum PRL levels and disease activity. In contrast, other authors did not find this. Recently, autoantibodies against prolactin in SLE patients have been described. One hundred percent of SLE patients with anti-PRL autoantibodies had hyperprolactinemia (hPRL) and 31.7% of the SLE patients classified with idiopathic hPRL had anti-prolactin antibodies. A similar result was found in 103 pediatric SLE patients. The patients with idiopathic hyperprolactinemia and anti-PRL autoantibodies had less clinical and serological lupus activity than the SLE patients with idiopathic hyperprolactinemia, but without anti-PRL autoantibodies. This evidence suggests that anti-PRL autoantibodies or the complex with any other molecule, like macroprolactinemia (big-big PRL) could have attenuated biological activity and this could explain why some clinical studies did not find any association between serum PRL levels and disease activity in SLE patients. However, studies in vitro have shown normal or elevated biological activity in Nb2 cell lines using PRL from serum with anti-PRL autoantibodies from patients with or without autoimmune diseases. Several conclusions could be drawn. One is that while a set of hyperprolactinemic SLE patients display autoantibodies against PRL, it is not clear what role these autoantibodies play in the whole system. However, until now, we knew that the patients with antibodies to PRL lacked the clinical symptoms of hyperprolactinemia such as menstrual disturbances and/or galactorrhea and show less clinical and serological lupus activity.

Antiprolactin autoantibodies in systemic lupus erythematosus: frequency and correlation with prolactinemia and disease activity.

OBJECTIVE: To determine in patients with systemic lupus erythematosus (SLE) (1) the frequency of antiprolactin (anti-PRL) autoantibodies, and (2) the relationships among anti-PRL autoantibodies, serum prolactin (PRL) levels, and lupus activity. METHODS: In a cross sectional study 259 consecutive patients with SLE were tested for serum PRL levels and anti-PRL autoantibodies based on disease activity. RESULTS: The frequency of anti-PRL was 5% (13/259), and all SLE patients with anti-PRL had hyperprolactinemia. There was lupus activity in 110 patients (42.5%) and there was no significant difference in frequency of anti-PRL autoantibodies between patients with or without lupus activity (5.5 vs 4.7%; p = 0.99). Only a high level of serum PRL was associated with lupus activity independent from other studied variables (p = 0.024). There was a negative but nonsignificant correlation between the titers of anti-PRL autoantibody and SLEDAI (r(s) = -0.16, p = 0.59). Anti-PRL positive patients had higher levels of serum PRL than anti-PRL negative patients (33.2+/-13.8 vs 11.6+/-13.2 ng/ml; p = 0.0001) and a significantly different frequency of hyperprolactinemia (100 vs 11.4%; p = 0.00001). CONCLUSION: The presence of anti-PRL autoantibodies was associated with hyperprolactinemic status and high serum PRL levels; these data suggest that anti-PRL autoantibodies could be the cause of hyperprolactinemia in a subset of patients with SLE. An increase in serum PRL levels proved to be an important independent factor related to lupus activity, but there was no relationship between anti-PRL autoantibodies and lupus activity.

Detection of macroprolactinemia with the polyethylene glycol precipitation test in systemic lupus erythematosus patients with hyperprolactinemia.

The objective of this study was to determine the diagnostic performance of the percentage of serum prolactin (PRL) precipitated with polyethylene glycol (PEG) for the detection of macroprolactinemia in systemic lupus erythematosus (SLE) patients with hyperprolactinemia. Serum samples from SLE patients were examined. Serum PRL was measured by immunoradiometric assay (IRMA) and samples with hyperprolactinemia (> 20 ng/ml) were submitted to PEG precipitation, gel filtration chromatography and affinity chromatography with protein-G sepharose. A comparative survey was used. Among 259 consecutive serum samples from SLE patients, PRL was > 20.1 ng/ml in 43 samples (16.6%). Gel filtration showed a predominant pattern of macroprolactinemia (> 100 kDa) in 14 (32.6%), a predominant pattern of monomeric PRL (23 kDa) in 27 (62.7%), and a variable pattern in two (4.7%). All sera with a predominant pattern of macroprolactinemia displayed anti-PRL autoantibodies by affinity chromatography for IgG. The best cut-off point for percentage of serum PRL precipitated with PEG for detection of macroprolactinemia was > or = 58.4%. Sensitivity and specificity were 100 and 96.6%, respectively. We can conclude that PEG precipitation is a convenient and simple procedure to screen for the presence of macroprolactinemia in sera from SLE patients. Precipitations > or = 58.4% are indicative of the presence of, and those < 50% the absence of, macroprolactinemia. However, samples with precipitations between 50 and 58.3% require gel filtration chromatography to characterize the predominant molecular form of PRL. Therefore, it is important to take these findings into account in future studies that aim to establish a relationship between PRL and disease activity in SLE.

Persistence of macroprolactinemia due to antiprolactin autoantibody before, during, and after pregnancy in a woman with systemic lupus erythematosus.

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.

Biologic activity and plasma clearance of prolactin-IgG complex in patients with systemic lupus erythematosus.

OBJECTIVE: To characterize the clinical findings in hyperprolactinemic systemic lupus erythematosus (SLE) patients with or without macroprolactinemia (big, big prolactin [PRL]) due to anti-PRL autoantibodies (PRL-IgG complex), and to assess the bioactivity and structure of big, big PRL. METHODS: Twenty-seven SLE patients with hyperprolactinemia (HPRL) were studied. Patients with (n = 8) or without (n = 19) big, big PRL were identified by gel filtration chromatography and affinity chromatography for IgG. PRL concentrations in serum and fractions by gel filtration chromatography and affinity chromatography were characterized by immunoradiometric assay (IRMA), Nb2 bioassay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, and clearance studies. RESULTS: SLE patients without big, big PRL had significantly higher levels of disease activity, cause-proven HPRL, and menstrual disturbances compared with patients with big, big PRL (P < or = 0.05). The big, big PRL fractions by Western blotting revealed a single 23-kd nonglycosylated PRL. The Nb2:IRMA ratio of the samples with big, big PRL was significantly higher than that of the samples without big, big PRL (P < or = 0.02). However, bioactivity of big, big PRL in the Nb2 cells was very similar to that of 23-kd nonglycosylated PRL. Clearance studies in rats demonstrated that the PRL-IgG complex was eliminated more slowly than monomeric PRL (little PRL). CONCLUSION: We demonstrated that the PRL-IgG complex was formed by 23-kd nonglycosylated PRL that was noncovalently bound to IgG and showed that the complex was fully active in vitro. This result suggests that the absence of symptoms of HPRL or lower levels of lupus activity in these patients is not explained by lower bioactivity of the complex. Instead, because of the large molecular size of the complex, the PRL does not easily cross the capillary walls. Delayed clearance of the PRL-IgG complex may account for increased serum levels of PRL in SLE patients with anti-PRL autoantibodies.