Losartan impairs HTR-8/SVneo trophoblast migration through inhibition of angiotensin II-induced pro-inflammatory profile in human endometrial stromal cells.

A deep interaction between the endometrium and the invading trophoblast occurs during implantation in humans, with the acquisition of uterine receptivity to the invading embryo promoted by an elevation of pro-inflammatory cytokines in the endometrium, and the invasiveness of decidualizing endometrial stromal cells, augmented by trophoblast-derived signals. Considering that usage of angiotensin II type 1 (AT1) receptor blockers, among other renin-angiotensin system (RAS) antagonists, is associated with adverse pregnancy outcomes, here we aim to analyse the involvement of AT1 receptor in the reciprocal dialogue occurring between endometrial stroma and trophoblast cells. In human endometrial stromal cells (T-HESC) pre-incubated with a decidualization cocktail, angiotensin (Ang) II increased protein expression of prolactin and FOXO1, markers of endometrial decidualization, while promoting nuclear translocation of FOXO1. In addition, Ang II treatment increased CXCL8, and matrix metalloprotease (MMP)-2 levels in T-HESC. Incubation with the AT1 receptor blocker losartan or with an NFAT signalling inhibitor, decreased Ang II-induced secretion of prolactin, CXCL8, and MMP-2 in T-HESC. In a wound healing assay, conditioned medium (CM) obtained from Ang II-treated T-HESC, but not CM from losartan-pre-incubated T-HESC, increased migration of HTR-8/SVneo trophoblasts, effect that was inhibited in the presence of a CXCL8-neutralizing antibody. An increased secretion of CXCL8 and MMP-2 was observed after treatment of T-HESC with CM obtained from HTR-8/SVneo cells, which was not observed in T-HESC pre-incubated with losartan or with the NFAT inhibitor. This study evidenced a reciprocal RAS-coded messaging between trophoblast and ESC which is affected by the AT1 receptor blocker losartan.

Differential Response of Dopamine Mediated by ß-Adrenergic Receptors in Human Keratinocytes and Macrophages: Potential Implication in Wound Healing.

OBJECTIVE: Dopamine is an immunomodulatory neurotransmitter. In the skin, keratinocytes and macrophages produce proinflammatory cytokines and metalloproteinases (MMPs) which participate in wound healing. These cells have a catecholaminergic system that modulates skin pathophysiologic processes. We have demonstrated that dopamine modulates cytokine production in keratinocytes via dopaminergic and adrenergic receptors (ARs). The aim of this study was to evaluate the effect of dopamine and its interaction with ß-ARs in human HaCaT keratinocytes and THP-1 macrophages. We evaluated the production of inflammatory mediators implicated in wound healing. METHODS: Cells were stimulated with dopamine in the absence or presence of the ß-adrenergic antagonist propranolol. Wound closure, MMP activity, and the production of IL-8, IL-1ß, and IκB/NFκB pathway activation were determined in stimulated cells. RESULTS: Dopamine did not affect the wound closure in human keratinocytes, but diminished the propranolol stimulatory effect, thus delaying cell migration. Similarly, dopamine significantly decreased MMP-9 activity and the propranolol-induced MMP activity. Dopamine significantly increased the p65-NFκB subunit levels in the nuclear extracts, which were reduced in the presence of propranolol in keratinocytes. On the other hand, dopamine significantly increased MMP-9 activity in THP-1 macrophages, but did not modify the propranolol-increased enzymatic activity. Dopamine significantly increased IL-8 production in human macrophages, an effect that was partially reduced by propranolol. Dopamine did not modify the p65-NFκB levels in the nuclear extracts in THP-1 macrophages. CONCLUSION: We suggest that the effect of dopamine via ß-ARs depends on the physiological condition and the cell type involved, thus contributing to either improve or interfere with the healing process.

Immunomodulatory effect of dopamine in human keratinocytes and macrophages under chronical bisphenol-A exposure conditions.

Dopamine is a key neurotransmitter that links the nervous and the immune system. Bisphenol A (BPA) is an endocrine disruptor with a wide distribution in the environment that is used in the manufacturing of plastic products. Evidence shows that BPA can interfere with the central dopaminergic transmission; however, there are no previous reports of this effect outside the central nervous system. Thus, the aim of this work was to investigate the in vitro mechanisms of action involved in the response to dopamine in both human keratinocyte and macrophage cell lines chronically exposed to BPA. Dopamine modulates cytokine secretion and NF-κB expression in BPA-treated HaCaT keratinocytes, without modifying these parameters in BPA-treated THP-1 macrophages. In addition, dopamine increases MMP activity in both BPA-treated cell lines, although it decreases keratinocytes migration. We suggest the immunomodulatory effect of dopamine might be different in keratinocytes and macrophages under chronical BPA exposure conditions. These findings revealed for the first time the in vitro immunomodulatory effect of dopamine in the presence of BPA at peripheral level.

Dopamine agonists upregulate IL-6 and IL-8 production in human keratinocytes.

AIM: Catecholamines regulate functions of the nervous, neuroendocrine and immune systems. Dopamine may modulate the activity of keratinocytes, which play a role in secreting cytokines and chemokines. The aim of this study was to evaluate the effect of dopaminergic agonists on the production of IL-6 and IL-8 by a non-tumoral human keratinocyte cell line (HaCaT). METHODS: Cells were stimulated with dopamine and the D(2) dopamine receptor agonist cabergoline. Levels of IL-6 and IL-8 in culture supernatants were then determined. Cell proliferation was also assessed. Assays were carried out in the presence or absence of the dopaminergic and ß-adrenergic receptor antagonists (sulpiride and propranolol, respectively) and ascorbic acid. RESULTS: Dopamine stimulated the production of IL-6 and IL-8 in a concentration-dependent manner. The effects observed on the secretion of IL-6 were more potent than those corresponding to IL-8 and were reduced by ascorbic acid. The dopamine-induced IL-6 secretion was partially reduced by sulpiride and abrogated by propranolol. The latter drug was able to block the effect of dopamine on the secretion of IL-8. The cabergoline-induced IL-6 release was reduced by sulpiride. Cell viability was not affected by any of the drugs. CONCLUSIONS: Dopaminergic agonists can stimulate keratinocytes to produce IL-6 and IL-8 which are related to inflammatory cutaneous processes. These effects are mediated by dopaminergic and ß-adrenergic receptors and by receptor-independent oxidative mechanisms.

Brucella abortus Proliferates in Decidualized and Non-Decidualized Human Endometrial Cells Inducing a Proinflammatory Response.

Brucella spp. have been associated with abortion in humans and animals. Although the mechanisms involved are not well established, it is known that placental Brucella infection is accompanied by inflammatory phenomena. The ability of Brucella abortus to infect and survive in human endometrial stromal cells (T-HESC cell line) and the cytokine response elicited were evaluated. B. abortus was able to infect and proliferate in both non-decidualized and decidualized T-HESC cells. Intracellular proliferation depended on the expression of a functional virB operon in the pathogen. B. abortus internalization was inhibited by cytochalasin D and to a lower extent by colchicine, but was not affected by monodansylcadaverine. The infection did not induce cytotoxicity and did not alter the decidualization status of cells. B. abortus infection elicited the secretion of IL-8 and MCP-1 in either decidualized or non-decidualized T-HESC, a response also induced by heat-killed B. abortus and outer membrane vesicles derived from this bacterium. The stimulation of T-HESC with conditioned media from Brucella-infected macrophages induced the production of IL-6, MCP-1 and IL-8 in a dose-dependent manner, and this effect was shown to depend on IL-1ß and TNF-α. The proinflammatory responses of T-HESC to B. abortus and to factors produced by infected macrophages may contribute to the gestational complications of brucellosis.

In vitro anti-inflammatory properties of Smilax campestris aqueous extract in human macrophages, and characterization of its flavonoid profile.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of Smilax campestris Griseb (Smilacaceae) have been employed in the treatment of several inflammatory diseases as a traditional herbal medicine. However, the cellular and molecular mechanisms involved in the observed effects remain elusive. Macrophages are known to play a central role in inflammatory responses. These cells are activated in response to a diversity of danger signals and produce several mediators of inflammation that eventually regulate the immune response. For all the above mentioned, scientific evidence is required to support the popular use of S. campestris. AIM OF THE STUDY: We aimed to investigate the anti-inflammatory effect of S. campestris aqueous extract (SME) in activated THP-1 human macrophages, on the production of some mediators of inflammation and oxidative stress in order to provide scientific support for its popular use. MATERIALS AND METHODS: The characterization of SME was assessed by HPLC-MS/MS. The production of the pro-inflammatory cytokines and chemokines was evaluated by ELISA. The activity of metalloproteases was evaluated by zymography. The subcellular localization of the NF-κB transcription factor was analysed by Western blot. The superoxide anion and glutathione levels were assessed by flow cytometry. The cytotoxicity induced by SME in THP-1 macrophages was also investigated by the LDH release test. RESULTS: In the present study, we have identified catechin and glycosylated derivatives of quercetin (quercetin-3-O-glucoside, quercetin-3-O-galactoside, rutin and quercetin-3-rhamnoside) as major components of the aqueous SME. We found that SME significantly decreased the production of the pro-inflammatory cytokines tumour necrosis factor (TNF)- α, interleukin (IL)-1ß, IL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 and the activity of the metalloproteinase (MMP)-9, in lipopolysaccharide-activated macrophages derived from the monocytic cell line THP-1. Furthermore, SME diminished the expression of NF-κB p65 subunit in the nuclear fraction. In addition, SME decreased the production of superoxide anion in THP-1 macrophages, without altering the levels of reduced glutathione. CONCLUSION: These results suggest that SME exerts its anti-inflammatory effects in human activated macrophages by inhibiting the production of pro-inflammatory cytokines, matrix metalloproteinases and the NF-κB transcription factor pathway along with a reduction of oxidative stress mediators. Moreover, catechin and glycosylated derivatives of were identified by HPLC-MS/MS in SME. Our findings provide scientific support for the traditional use of the S. campestris extracts.

Placental secreted factors: their role in the regulation of anti-CII antibodies and amelioration of collagen induced arthritis in rats.

Pregnancy induces collagen-induced arthritis (CIA) remission in rats. Placental hormones, cytokines and growth factors can regulate immune cell activity at the feto-maternal interface as well as at the systemic level. We assessed the effect of placental culture supernatants (PS) in CIA developed in rats after the inoculation of collagen type II (CII) in complete Freund's adjuvant. After the onset of CIA, animals were injected by ip route with seven doses of PS. On the 18th day of treatment with PS, serum anti-CII antibody (total IgG, IgG(1), IgG(2a), IgG(2b), IgG asymmetric molecules) and cytokine levels were determined by ELISA. An arthritic index was used by daily measure of joint swelling and visual signs of arthritis. Our results demonstrated that the PS treatment diminished CIA symptoms, reduced TNF-alpha, INF-gamma and anti-CII antibody serum levels, increased the proportion of asymmetric IgG anti-CII antibodies and affected IgG(1)/IgG(2a) and IgG(1)/IgG(2b) ratio. Two weeks after the last PS inoculation there was a recurrence of arthritis, a rise in IgG anti-CII and, simultaneously, the percentage of asymmetric IgG anti-CII fell. We concluded that PS have an effective CIA suppressor activity partly due to the modulation of humoral immune response and may be closely related to an inhibitory effect on TNF-alpha and INF-gamma production.

Estrogen and progesterone regulate the IL-6 signal transduction pathway in antibody secreting cells.

Regulation of the immune response is necessary to allow successful pregnancy. Asymmetric IgG antibodies are involved in fetal maintenance. We have previously demonstrated that estrogen (E2) and progesterone (P4) modulate the synthesis of asymmetric antibodies but the underlying mechanisms remain unclear. Since IL-6 and a progesterone-induced blocking factor (PIBF) were shown to regulate asymmetric antibody synthesis, in this work we analyzed whether E2 and P4 were able to modulate IL-6 signal transduction pathways and the ability of P4 to induce PIBF synthesis, in hybridoma B cells was also evaluated. We found that the IL-6 treatment induced an increase in the expression of gp130 and JAK1 by the hybridoma. E2 and P4 diminished the IL-6-induced gp130 expression in a dose-dependent manner, whereas the expression of JAK1 was not significantly affected. At 10(-6)M concentration, the steroids inhibited the phosphorylation of gp130 and diminished the IL-6-induced STAT3 phosphorylation and translocation to the nucleus. Maximal PIBF expression was observed when the hybridoma was cultured with 10(-10)M P4, compared to the control (p<0.05). Results demonstrate two molecular mechanisms, the modulation of the IL-6R signal transduction pathway and PIBF induction, which could be involved in the immunoregulatory role of sexual steroids during pregnancy.

An Oral Salmonella-Based Vaccine Inhibits Liver Metastases by Promoting Tumor-Specific T-Cell-Mediated Immunity in Celiac and Portal Lymph Nodes: A Preclinical Study.

Primary tumor excision is one of the most widely used therapies of cancer. However, the risk of metastases development still exists following tumor resection. The liver is a common site of metastatic disease for numerous cancers. Breast cancer is one of the most frequent sources of metastases to the liver. The aim of this work was to evaluate the efficacy of the orally administered Salmonella Typhi vaccine strain CVD 915 on the development of liver metastases in a mouse model of breast cancer. To this end, one group of BALB/c mice was orogastrically immunized with CVD 915, while another received PBS as a control. After 24 h, mice were injected with LM3 mammary adenocarcinoma cells into the spleen and subjected to splenectomy. This oral Salmonella-based vaccine produced an antitumor effect, leading to a decrease in the number and volume of liver metastases. Immunization with Salmonella induced an early cellular immune response in mice. This innate stimulation rendered a large production of IFN-γ by intrahepatic immune cells (IHIC) detected within 24 h. An antitumor adaptive immunity was found in the liver and celiac and portal lymph nodes (LDLN) 21 days after oral bacterial inoculation. The antitumor immune response inside the liver was associated with increased CD4(+) and dendritic cell populations as well as with an inflammatory infiltrate located around liver metastatic nodules. Enlarged levels of inflammatory cytokines (IFN-γ and TNF) were also detected in IHIC. Furthermore, a tumor-specific production of IFN-γ and TNF as well as tumor-specific IFN-γ-producing CD8 T cells (CD8(+)IFN-γ(+)) were found in the celiac and portal lymph nodes of Salmonella-treated mice. This study provides first evidence for the involvement of LDLN in the development of an efficient cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases after oral Salmonella-based vaccination.

Maternal and Fetal Mechanisms of B Cell Regulation during Pregnancy: Human Chorionic Gonadotropin Stimulates B Cells to Produce IL-10 While Alpha-Fetoprotein Drives Them into Apoptosis.

Maternal immune tolerance toward the fetus is an essential requisite for pregnancy. While T cell functions are well documented, little is known about the participation of B cells. We have previously suggested that IL-10-producing B cells are involved in pregnancy tolerance in mice and humans. By employing murine and human systems, we report now that fetal trophoblasts positively regulate the generation of IL-10-producing B cells. We next studied the participation of hormones produced by the placenta as well as the fetal protein alpha-fetoprotein (AFP) in B cell modulation. Human chorionic gonadotropin (hCG), but not progesterone, estrogen, or a combination of both, was able to promote changes in B cell phenotype and boost their IL-10 production, which was abolished after blocking hCG. The hCG-induced B cell phenotype was not associated with augmented galactosylation, sialylation, or fucosylation of IgG subclasses in their Fc. In vitro, hCG induced the synthesis of asymmetrically glycosylated antibodies in their Fab region. Interestingly, AFP had dual effects depending on the concentration. At concentrations corresponding to maternal serum levels, it did not modify the phenotype or IL-10 secretion of B cells. At fetal concentrations, however, AFP was able to drive B cells into apoptosis, which may indicate a protective mechanism to avoid maternal B cells to reach the fetus. Our data suggest that the fetus secrete factors that promote a pregnancy-friendly B cell phenotype, unraveling interesting aspects of B cell function, and modulation by pregnancy hormones and fetal proteins.

Immunostimulatory Effects Triggered by Enterococcus faecalis CECT7121 Probiotic Strain Involve Activation of Dendritic Cells and Interferon-Gamma Production.

Probiotics can modulate the immune system, conferring beneficial effects on the host. Understanding how these microorganisms contribute to improve the health status is still a challenge. Previously, we have demonstrated that Enterococcus faecalis CECT7121 implants itself and persists in the murine gastrointestinal tract, and enhances and skews the profile of cytokines towards the Th1 phenotype in several biological models. Given the importance of dendritic cells (DCs) in the orchestration of immunity, the aim of this work was to elucidate the influence of E. faecalis CECT7121 on DCs and the outcome of the immune responses. In this work we show that E. faecalis CECT7121 induces a strong dose-dependent activation of DCs and secretion of high levels of IL-12, IL-6, TNFα, and IL-10. This stimulation is dependent on TLR signaling, and skews the activation of T cells towards the production of IFNγ. The influence of this activation in the establishment of Th responses in vivo shows the accumulation of specific IFNγ-producing cells. Our findings indicate that the activation exerted by E. faecalis CECT7121 on DCs and its consequence on the cellular adaptive immune response may have broad therapeutic implications in immunomodulation.

Modified immunoglobulin G glycosylation pattern during turpentine-induced acute inflammation in rats.

Alterations in the pattern of protein glycosylation have been described during inflammation. In chronic parasitic and tumoral diseases we have reported an increase in the proportion of serum Immunoglobulin G (IgG) molecules possessing an altered Fab glycosylation pattern designated asymmetric antibodies. The alteration results in augmented concanavalin A affinity and functional univalence of the antibody. In addition, Fc agalactosylation has been described as occurring in chronically autoimmune diseases. Therefore, the aim of this paper was to evaluate by analyzing sera whether during an acute inflammatory response in rats produced by subcutaneous inoculation of turpentine oil, there was an alteration in the synthesis and glycosylation of IgG (as revealed by concanavalin A binding). We found that during acute inflammation there was a decrease in the synthesis of IgG which was not affected by prior oral administration of dexamethasone; however, the turpentine-induced increase in IgG binding to concanavalin A was found to be inhibited upon prior administration of the anti-inflammatory agent. As with turpentine, the corticoid used induced an increase in the interleukin-6 levels detected in sera by ELISA. Although we have described an improvement in asymmetric antibody synthesis by low dose of interleukin-6 previously, here we found no correlation between the observed glycosylation pattern of IgG and interleukin-6 concentration assessed in sera of treated rats, probably due to a different dexamethasone mediated pathway.

Angiotensin II activates the calcineurin/NFAT signaling pathway and induces cyclooxygenase-2 expression in rat endometrial stromal cells.

Cyclooxygenase (COX)-2, the inducible isoform of cyclooxygenase, plays a role in the process of uterine decidualization and blastocyst attachment. On the other hand, overexpression of COX-2 is involved in the proliferation of the endometrial tissue during endometriosis. Deregulation of the renin-angiotensin-system plays a role in the pathophysiology of endometriosis and pre-eclampsia. Angiotensin II increases intracellular Ca(2+) concentration by targeting phospholypase C-gamma in endometrial stromal cells (ESC). A key element of the cellular response to Ca(2+) signals is the activity of the Ca(2+)- and calmodulin-dependent phosphatase calcineurin. Our first aim was to study whether angiotensin II stimulated Cox-2 gene expression in rat ESC and to analyze whether calcineurin activity was involved. In cells isolated from non-pregnant uteri, COX-2 expression--both mRNA and protein--was induced by co-stimulation with phorbol ester and calcium ionophore (PIo), as well as by angiotensin II. Pretreatment with the calcineurin inhibitor cyclosporin A inhibited this induction. We further analyzed the role of the calcineurin/NFAT signaling pathway in the induction of Cox-2 gene expression in non-pregnant rat ESC. Cyclosporin A abolished NFATc1 dephosphorylation and translocation to the nucleus. Cyclosporin A also inhibited the transcriptional activity driven by the Cox-2 promoter. Exogenous expression of the peptide VIVIT -specific inhibitor of calcineurin/NFAT binding- blocked the activation of Cox-2 promoter and the up-regulation of COX-2 protein in these cells. Finally we analyzed Cox-2 gene expression in ESC of early-pregnant rats. COX-2 expression--both mRNA and protein--was induced by stimulation with PIo as well as by angiotensin II. This induction appears to be calcineurin independent, since it was not abrogated by cyclosporin A. In conclusion, angiotensin II induced Cox-2 gene expression by activating the calcineurin/NFAT signaling pathway in endometrial stromal cells of non-pregnant but not of early-pregnant rats. These results might be related to differential roles that COX-2 plays in the endometrium.

Calcium/calcineurin signaling in primary cortical astrocyte cultures: Rcan1-4 and cyclooxygenase-2 as NFAT target genes.

The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway mediates important cell responses to calcium, but its activity and function in astrocytes have remained unclear. We show that primary cortical astrocyte cultures express the regulatory and catalytic subunits of the phosphatase calcineurin as well as the calcium-regulated NFAT family members (NFATc1, c2, c3, and c4). NFATs are activated by calcium-mobilizing agents in astrocytes, and this activation is blocked by the calcineurin inhibitor cyclosporine A. Microarray screening identified cyclooxygenase-2 (Cox-2), which is implicated in brain injury, and Rcan 1-4, an endogenous calcineurin inhibitor, as genes up-regulated by calcineurin-dependent calcium signals in astrocytes. Mobilization of intracellular calcium with ionophore potently augments the promoter activity and mRNA and protein expression of Rcan 1-4 and Cox-2 induced by combined treatment with phorbol esters. Moreover, Rcan 1-4 expression is efficiently induced by calcium mobilization alone. For both the genes, the calcium signal component is dependent on calcineurin and is replicated by exogenous expression of a constitutively active NFAT, strongly suggesting that the calcium-induced gene activation is mediated by NFATs. Finally, we report that calcineurin-dependent expression of Cox-2 and Rcan 1-4 is induced by physiological calcium mobilizing agents, such as thrombin, agonists of purinergic and glutamate receptors, and L-type voltage-gated calcium channels. These findings provide insights into calcium-initiated gene transcription in astrocytes, and have implications for the regulation of calcium responses in astrocytes.

Calcium-dependent expression of TNF-alpha in neural cells is mediated by the calcineurin/NFAT pathway.

We report induction of TNF-alpha via the calcium/calcineurin/NFAT pathway in PC12 neural cells. In PC12, expression of TNF-alpha mRNA, protein and TNF-alpha gene promoter activity was induced by co-stimulation with phorbol ester and either calcium ionophore A23187 or the L-type Voltage Gated Calcium Channel agonist Bay K 8644. Pre-treatment with calcineurin inhibitors CsA or FK506 inhibited the dominant calcium-dependent component of this induction, limiting it to the level achieved with phorbol ester alone. Promoter activation by Bay was abolished by nifedipine, a specific inhibitor of L-type Voltage Gated Calcium Channels. Exogenous NFAT protein transactivated the TNF-alpha promoter, and the peptide VIVIT-a specific inhibitor of calcineurin/NFAT binding-blocked calcium-inducible transactivation of the TNF-alpha promoter. Given proposed functions of TNF-alpha in spatial learning, memory and the pathogenesis of neurodegenerative diseases, the data presented suggest an important role for calcineurin/NFAT signaling in these key neurological processes.

Depolarization of neural cells induces transcription of the Down syndrome critical region 1 isoform 4 via a calcineurin/nuclear factor of activated T cells-dependent pathway.

In this study we showed that the transcriptional regulation of Down syndrome critical region isoform 4 (DSCR1.4) is mediated by the calcineurin/nuclear factor of activated T cells (NFAT) pathway in neural cells. Stimuli that elicit an increase in the intracellular concentrations of calcium, such as membrane depolarization, induced de novo transcription of DSCR1.4, with mRNA expression peaking after 4 h and then declining. Action via the physiologically relevant L-type calcium channel was confirmed by blockade with nifedipine and verapamil. This calcium-dependent transcription of DSCR1.4 was inhibited by the calcineurin inhibitors cyclosporin A and FK506. Deletional analysis showed that the calcium- and calcineurin-dependent activation is mediated by the promoter region between nucleotides -350 and -166, a region that contains putative NFAT-binding motifs. Exogenous NFATc2 potently augmented the DSCR1.4 promoter transcriptional activity, and the involvement of endogenous NFAT signaling pathway in DSCR1.4 transcription was confirmed by the suppression of depolarization-inducible promoter activity with the NFAT inhibitor peptide VIVIT. Exogenous overexpression of DSCR1 protein (calcipressin 1) resulted in the inhibition of the transcription of DSCR1.4 and NFAT-dependent signaling. These findings suggest that calcineurin-dependent induction of DSCR1.4 product may represent an important auto-regulatory mechanism for the homeostatic control of NFAT signaling in neural cells.

The immunomodulatory action of dexamethasone on monoclonal antibody-producing hybridoma cells.

As found in different studies, glucocorticoid hormones (GCs) as well as interleukin-6 (IL-6) are involved in the modulation of protein glycosylation. In this work we have investigated the immunomodulatory effect of dexamethasone by assessing in vitro IgG glycosylation by monoclonal antibody-producing hybridoma cells. As described in myeloma cell lines, cellular viability and proliferation rates of hybridoma 112D5 cells decrease when cultured with dexamethasone during 24 hours, in a dose-dependent way. Moreover, the corticosteroid triggered apoptosis of the hybridoma, which was observed as soon as 4 h after culturing cells in the presence of the drug. In line with these results, after 24 h, dexamethasone induced a drop in the anti-DNP level of antibodies synthesized by hybridoma 112D5. In previous works we described that asymmetric glycosylation of in vitro synthesized IgG correlated with induction of cell damage. Nevertheless, an increase in asymmetric IgG glycosylation was not observed here, but there was a decrease in the proportion of asymmetrically glycosylated IgG synthesized by the hybridoma after a 4-h culture with the drug. Finally, as results from assessing IL-6 production by ELISA, we conclude that the above described effects of dexamethasone on hybridoma 112D5 cells could not be due to the inhibition of IL-6 synthesis exerted by the corticoid but rather to a direct effect of the drug. Monoclonal antibody (MAb) producing hybridomas provide an excellent in vitro model for the study of the molecular mechanisms involved in immunoglobulin glycosylation.

Occurrence, properties, and function of asymmetric IgG molecules isolated from non-immune sera.

We have previously demonstrated that 10-20% of the IgG isolated from non-immune sera is asymmetrically glycosylated, in such a way that it fails to trigger immune effector mechanisms. As a result, a major portion of the non-immune asymmetric IgG molecules of the host could be self-specific, acting as auto-protective antibodies. In order to test this hypothesis, we investigated whether asymmetric IgG molecules are capable of recognizing self-antigens. About 40% of F(ab')2 fragment from normal rat IgG was able to react specifically with autologous rat cells. Moreover, upon being purified from normal rat sera, 78% of the asymmetric IgG sub-population showed self-reactivity. We demonstrated that about 14% of rat asymmetric IgG-F(ab')2 fragments was able to react with bacteria isolated from the intestine of uninfected rats. Lastly, in order to test whether there is a correlation between the decline of immune responses during ageing and asymmetric antibody production, we assayed IgG isolated from sera of young and old rats. There was an increase in the asymmetric:symmetric IgG ratio with ageing. We therefore suggest that asymmetric antibodies may exert a beneficial action by protecting self-antigens as well as normal intestinal flora from a deleterious immune response.

In vitro modulation of protective antibody responses by estrogen, progesterone and interleukin-6.

PROBLEM: We have previously demonstrated that the addition of placental interleukin-6 (IL-6) to murine hybridomas increased asymmetric antibody synthesis. Here we analyze whether progesterone (Pg) and estrogen (E2) affect asymmetric antibody synthesis by modulating IL-6 production in hybridoma cells. METHOD OF STUDY: Hybridoma 112D5 B cells were cultured with E2, Pg or recombinant IL-6. Cell proliferation was assessed by 3H-thymidine incorporation, and asymmetric antibodies were measured in culture supernatants by Con A fixation and enzyme-linked immunusorbant assay (ELISA). E2 and Pg-receptors (ER and PR) were evaluated in whole cell extracts by Western blot. IL-6 was measured in culture supernatants by ELISA. RESULTS: 112D5 expressed both PR and ER, which were differentially regulated. At 48 hr, Pg and E2 slightly decreased cell proliferation whereas IL-6 did not. As well as IL-6, 10(-10) M Pg but not E2 induced asymmetric antibody production. Interestingly, Pg at 10(-6) M decreased asymmetric antibody synthesis by hybridoma cells. Finally, mainly Pg but also E2 increased IL-6 synthesis, although IL-6 levels did not correlate with asymmetric antibodies synthesized in the presence of E2 or Pg. CONCLUSIONS: In cells expressing both ER and PR, we could demonstrate that steroids participate in humoral immune responses by modulating asymmetric antibody synthesis. IL-6 proved to be only partially involved. Other possible mechanisms involved in the effect of Pg on blocking antibody responses and their contribution to a successful pregnancy are discussed in the paper.

Modified immunoglobulin G glycosylation pattern during turpentine-induced acute inflammation in rats

Alterations in the pattern of protein glycosylation have been described during inflammation. In chronic parasitic and tumoral diseases we have reported an increase in the proportion of serum Immunoglobulin G (IgG) molecules possessing an altered Fab glycosylation pattern designated asymmetric antibodies. The alteration results in augmented concanavalin A affinity and functional univalence of the antibody. In addition, Fc agalactosylation has been described as occurring in chronically autoimmune diseases. Therefore, the aim of this paper was to evaluate by analyzing sera whether during an acute inflammatory response in rats produced by subcutaneous inoculation of turpentine oil, there was an alteration in the synthesis and glycosylation of IgG (as revealed by concanavalin A binding). We found that during acute inflammation there was a decrease in the synthesis of IgG which was not affected by prior oral administration of dexamethasone; however, the turpentine-induced increase in IgG binding to concanavalin A was found to be inhibited upon prior administration of the anti-inflammatory agent. As with turpentine, the corticoid used induced an increase in the interleukin-6 levels detected in sera by ELISA. Although we have described an improvement in asymmetric antibody synthesis by low dose of interleukin-6 previously, here we found no correlation between the observed glycosylation pattern of IgG and interleukin-6 concentration assessed in sera of treated rats, probably due to a different dexamethasone mediated pathway (AU)#S#a