Endogenous PGD2 acting on DP2 receptor counter regulates Schistosoma mansoni infection-driven hepatic granulomatous fibrosis.

Identifying new molecular therapies targeted at the severe hepatic fibrosis associated with the granulomatous immune response to Schistosoma mansoni infection is essential to reduce fibrosis-related morbidity/mortality in schistosomiasis. In vitro cell activation studies suggested the lipid molecule prostaglandin D2 (PGD2) as a potential pro-fibrotic candidate in schistosomal context, although corroboratory in vivo evidence is still lacking. Here, to investigate the role of PGD2 and its cognate receptor DP2 in vivo, impairment of PGD2 synthesis by HQL-79 (an inhibitor of the H-PGD synthase) or DP2 receptor inhibition by CAY10471 (a selective DP2 antagonist) were used against the fibrotic response of hepatic eosinophilic granulomas of S. mansoni infection in mice. Although studies have postulated PGD2 as a fibrogenic molecule, HQL-79 and CAY10471 amplified, rather than attenuated, the fibrotic response within schistosome hepatic granulomas. Both pharmacological strategies increased hepatic deposition of collagen fibers-an unexpected outcome accompanied by further elevation of hepatic levels of the pro-fibrotic cytokines TGF-ß and IL-13 in infected animals. In contrast, infection-induced enhanced LTC4 synthesis in the schistosomal liver was reduced after HQL-79 and CAY10471 treatments, and therefore, inversely correlated with collagen production in granulomatous livers. Like PGD2-directed maneuvers, antagonism of cysteinyl leukotriene receptors CysLT1 by MK571 also promoted enhancement of TGF-ß and IL-13, indicating a key down-regulatory role for endogenous LTC4 in schistosomiasis-induced liver fibrosis. An ample body of data supports the role of S. mansoni-driven DP2-mediated activation of eosinophils as the source of LTC4 during infection, including: (i) HQL-79 and CAY10471 impaired systemic eosinophilia, drastically decreasing eosinophils within peritoneum and hepatic granulomas of infected animals in parallel to a reduction in cysteinyl leukotrienes levels; (ii) peritoneal eosinophils were identified as the only cells producing LTC4 in PGD2-mediated S. mansoni-induced infection; (iii) the magnitude of hepatic granulomatous eosinophilia positively correlates with S. mansoni-elicited hepatic content of cysteinyl leukotrienes, and (iv) isolated eosinophils from S. mansoni-induced hepatic granuloma synthesize LTC4 in vitro in a PGD2/DP2 dependent manner. So, our findings uncover that granulomatous stellate cells-derived PGD2 by activating DP2 receptors on eosinophils does stimulate production of anti-fibrogenic cysLTs, which endogenously down-regulates the hepatic fibrogenic process of S. mansoni granulomatous reaction-an in vivo protective function which demands caution in the future therapeutic attempts in targeting PGD2/DP2 in schistosomiasis.

Eosinophils increase macrophage ability to control intracellular Leishmania amazonensis infection via PGD2 paracrine activity in vitro.

Clinical and experimental studies have described eosinophil infiltration in Leishmania amazonensis infection sites, positioning eosinophils strategically adjacent to the protozoan-infected macrophages in cutaneous leishmaniasis. Here, by co-culturing mouse eosinophils with L. amazonensis-infected macrophages, we studied the impact of eosinophils on macrophage ability to regulate intracellular L. amazonensis infection. Eosinophils prevented the increase in amastigote numbers within macrophages by a mechanism dependent on a paracrine activity mediated by eosinophil-derived prostaglandin (PG) D2 acting on DP2 receptors. Exogenous PGD2 mimicked eosinophil-mediated effect on managing L. amazonensis intracellular infection by macrophages and therefore may function as a complementary tool for therapeutic intervention in L. amazonensis-driven cutaneous leishmaniasis.

Effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema.

We sought to assess whether the effects of mesenchymal stromal cells (MSC) on lung inflammation and remodeling in experimental emphysema would differ according to MSC source and administration route. Emphysema was induced in C57BL/6 mice by intratracheal (IT) administration of porcine pancreatic elastase (0.1 UI) weekly for 1 month. After the last elastase instillation, saline or MSCs (1×105), isolated from either mouse bone marrow (BM), adipose tissue (AD) or lung tissue (L), were administered intravenously (IV) or IT. After 1 week, mice were euthanized. Regardless of administration route, MSCs from each source yielded: 1) decreased mean linear intercept, neutrophil infiltration, and cell apoptosis; 2) increased elastic fiber content; 3) reduced alveolar epithelial and endothelial cell damage; and 4) decreased keratinocyte-derived chemokine (KC, a mouse analog of interleukin-8) and transforming growth factor-ß levels in lung tissue. In contrast with IV, IT MSC administration further reduced alveolar hyperinflation (BM-MSC) and collagen fiber content (BM-MSC and L-MSC). Intravenous administration of BM- and AD-MSCs reduced the number of M1 macrophages and pulmonary hypertension on echocardiography, while increasing vascular endothelial growth factor. Only BM-MSCs (IV > IT) increased the number of M2 macrophages. In conclusion, different MSC sources and administration routes variably reduced elastase-induced lung damage, but IV administration of BM-MSCs resulted in better cardiovascular function and change of the macrophage phenotype from M1 to M2.

Parasitic infections: A new frontier for PGD2 functions.

Prostaglandin (PG)D2 is produced and/or triggered by different parasites to modulate the course of the infection. These findings position PGD2 as a therapeutic target and suggest potential beneficial effects of repositioned drugs that target its synthesis or receptor engagement. However, recent in vivo data may suggest a more nuanced role and warrants further investigation of the role of PGD2 during the full course and complexity of parasitic infections.

Bone marrow-derived mononuclear cells promote improvement in glomerular function in rats with early diabetic nephropathy.

BACKGROUND/AIMS: Diabetic nephropathy is one of the main causes of end-stage renal disease. The present study investigated the effect of mononuclear cell (MC) therapy in rats subjected to diabetic nephropathy. METHODS: Male Wistar rats were divided into control (CTRL), diabetic (DM), CTRL+MC and DM+MC groups. Diabetes was induced by a single injection of streptozotocin (45 mg/kg, i.p.) and, 4 weeks later, 2×10(7) MCs were injected via the jugular vein. RESULTS: The rats in the DM and DM+MC groups showed increased glycemia, glomerular filtration rate and glomerular tuff area versus control groups. The glomerular filtration rate and glomerular tuff area were normalized in the DM+MC group. No alterations were observed in the fractional excretion of electrolytes and proteinuria between the DM and DM+MC groups. TGF-ß1 protein levels in the DM group were significantly increased versus control animals and normalized in the DM+MC group. An increase in ED1(+)/arginase I(+) macrophages and IL-10 renal expression was observed in the DM+MC group versus DM group. CONCLUSIONS: Bone marrow-derived MC therapy was able to prevent glomerular alterations and TGF-ß1 protein overexpression and modulated glomerular arginase I(+) macrophage infiltration in rats subjected to early diabetic nephropathy.

Effects of mesenchymal stem cell therapy on the time course of pulmonary remodeling depend on the etiology of lung injury in mice.

OBJECTIVE: Recent evidence suggests that mesenchymal stem cells may attenuate lung inflammation and fibrosis in acute lung injury. However, so far, no study has investigated the effects of mesenchymal stem cell therapy on the time course of the structural, mechanical, and remodeling properties in pulmonary or extrapulmonary acute lung injury. DESIGN: Prospective randomized controlled experimental study. SETTING: University research laboratory. SUBJECTS: One hundred forty-three females and 24 male C57BL/6 mice. INTERVENTIONS: Control mice received saline solution intratracheally (0.05 mL, pulmonary control) or intraperitoneally (0.5 mL, extrapulmonary control). Acute lung injury mice received Escherichia coli lipopolysaccharide intratracheally (2 mg/kg in 0.05 mL of saline/mouse, pulmonary acute lung injury) or intraperitoneally (20 mg/kg in 0.5 mL of saline/mouse, extrapulmonary acute lung injury). Mesenchymal stem cells were intravenously injected (IV, 1 × 10 cells in 0.05 mL of saline/mouse) 1 day after lipopolysaccharide administration. MEASUREMENTS AND MAIN RESULTS: At days 1, 2, and 7, static lung elastance and the amount of alveolar collapse were similar in pulmonary and extrapulmonary acute lung injury groups. Inflammation was markedly increased at day 2 in both acute lung injury groups as evidenced by neutrophil infiltration and levels of cytokines in bronchoalveolar lavage fluid and lung tissue. Conversely, collagen deposition was only documented in pulmonary acute lung injury. Mesenchymal stem cell mitigated changes in elastance, alveolar collapse, and inflammation at days 2 and 7. Compared with extrapulmonary acute lung injury, mesenchymal stem cell decreased collagen deposition only in pulmonary acute lung injury. Furthermore, mesenchymal stem cell increased metalloproteinase-8 expression and decreased expression of tissue inhibitor of metalloproteinase-1 in pulmonary acute lung injury, suggesting that mesenchymal stem cells may have an effect on the remodeling process. This change may be related to a shift in macrophage phenotype from M1 (inflammatory and antimicrobial) to M2 (wound repair and inflammation resolution) phenotype. CONCLUSIONS: Mesenchymal stem cell therapy improves lung function through modulation of the inflammatory and remodeling processes. In pulmonary acute lung injury, a reduction in collagen fiber content was observed associated with a balance between metalloproteinase-8 and tissue inhibitor of metalloproteinase-1 expressions.

Eosinophils as a novel cell source of prostaglandin D2: autocrine role in allergic inflammation.

PGD(2) is a key mediator of allergic inflammatory diseases that is mainly synthesized by mast cells, which constitutively express high levels of the terminal enzyme involved in PGD(2) synthesis, the hematopoietic PGD synthase (H-PGDS). In this study, we investigated whether eosinophils are also able to synthesize, and therefore, supply biologically active PGD(2). PGD(2) synthesis was evaluated within human blood eosinophils, in vitro differentiated mouse eosinophils, and eosinophils infiltrating inflammatory site of mouse allergic reaction. Biological function of eosinophil-derived PGD(2) was studied by employing inhibitors of synthesis and activity. Constitutive expression of H-PGDS was found within nonstimulated human circulating eosinophils. Acute stimulation of human eosinophils with A23187 (0.1-5 µM) evoked PGD(2) synthesis, which was located at the nuclear envelope and was inhibited by pretreatment with HQL-79 (10 µM), a specific H-PGDS inhibitor. Prestimulation of human eosinophils with arachidonic acid (10 µM) or human eotaxin (6 nM) also enhanced HQL-79-sensitive PGD(2) synthesis, which, by acting on membrane-expressed specific receptors (D prostanoid receptors 1 and 2), displayed an autocrine/paracrine ability to trigger leukotriene C(4) synthesis and lipid body biogenesis, hallmark events of eosinophil activation. In vitro differentiated mouse eosinophils also synthesized paracrine/autocrine active PGD(2) in response to arachidonic acid stimulation. In vivo, at late time point of the allergic reaction, infiltrating eosinophils found at the inflammatory site appeared as an auxiliary PGD(2)-synthesizing cell population. Our findings reveal that eosinophils are indeed able to synthesize and secrete PGD(2), hence representing during allergic inflammation an extra cell source of PGD(2), which functions as an autocrine signal for eosinophil activation.

Lysophosphatidic acid (LPA) as a modulator of plasma membrane Ca2+-ATPase from basolateral membranes of kidney proximal tubules.

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.

Acute catabolism of leukocyte lipid bodies: Characterization of a nordihydroguaiaretic acid (NDGA)-induced proteasomal-dependent model.

Cytoplasmic availability of leukocyte lipid bodies is controlled by a highly regulated cycle of opposing biogenesis- and catabolism-related events. While leukocyte biogenic machinery is well-characterized, lipid body catabolic mechanisms are yet mostly unknown. Here, we demonstrated that nordihydroguaiaretic acid (NDGA) very rapidly decreases the numbers of pre-formed lipid bodies within lipid body-enriched cytoplasm of mouse leukocytes - macrophages, neutrophils and eosinophils. NDGA mechanisms driving leukocyte lipid body disappearance were not related to loss of cell viability, 5-lipoxygenase inhibition, ATP autocrine/paracrine activity, or biogenesis inhibition. Proteasomal-dependent breakdown of lipid bodies appears to control NDGA-driven leukocyte lipid body reduction, since it was Bortezomib-sensitive in macrophages, neutrophils and eosinophils. Our findings unveil an acute NDGA-triggered lipid body catabolic event - a novel experimental model for the still neglected research area on leukocyte lipid body catabolism, additionally favoring further insights on proteasomal contribution to lipid body breakdown.

Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells.

Cytoplasmic lipid bodies (also known as lipid droplets) are intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoid generation. PGE2 is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in an epithelial cell line derived from normal rat intestinal epithelium, IEC-6 cells. Lipid bodies were virtually absent in confluent IEC-6 cells. Stimulation of confluent IEC-6 cells with unsaturated fatty acids, including AA or oleic acid (OA), induced rapid lipid body assembly that was independent on its metabolism to PGE(2), but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3 K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A(2)-alpha, while facilitated AA mobilization and synthesis of PGE(2) within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA (2)-alpha-driven enhanced AA mobilization and PGE(2)production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.

Prostaglandin E2-EP1 and EP2 receptor signaling promotes apicaljunctional complex disassembly of Caco-2 human colorectal cancercells

Background: The apical junctional complex (AJC) is a dynamic structure responsible to maintainepithelial cell-cell adhesions and it plays important functions such as, polarity, mechanical integrity,and cell signaling. Alteration of this complex during pathological events leads to an impairedepithelial barrier by perturbation of the cell-cell adhesion system. Although clinical and experimental data indicate that prostaglandin E2 (PGE2) plays a critical function in promoting cellmotility and cancer progression, little is known concerning its role in AJC disassembly, an event that takes place at the beginning of colorectal tumorigenesis. Using Caco-2 cells, a cell line derivedfrom human colorectal cancer, we investigated the effects of prostaglandin E2 (PGE2) treatment on AJC assembly and function.Results: Exposition of Caco-2 cells to PGE2 promoted differential alteration of AJC protein distribution, as evidenced by immunofluorescence and immunoblotting analysis and impairs the barrier function, as seen by a decrease in the transepithelial electric resistance and an increase in the permeability to ruthenium red marker. We demonstrated the involvement of EP1 and EP2prostaglandin E2 receptor subtypes in the modulation of the AJC disassembly caused by prostanoid.Furthermore, pharmacological inhibition of protein kinase-C, but not PKA and p38MAPK significantly prevented the PGE2 effects on the AJC disassembly.Conclusion: Our findings strongly suggest a central role of Prostaglandin E2-EP1 and EP2 receptor signaling to mediate AJC disassembly through a mechanism that involves PKC and claudin-1 as important target for the TJ-related effects in human colorectal cancer cells (Caco-2).(AU)

Bone marrow-derived mononuclear cell therapy in experimental pulmonary and extrapulmonary acute lung injury.

OBJECTIVE: To hypothesize that bone marrow-derived mononuclear cell (BMDMC) therapy might act differently on lung and distal organs in models of pulmonary or extrapulmonary acute lung injury with similar mechanical compromises. The pathophysiology of acute lung injury differs according to the type of primary insult. DESIGN: Prospective, randomized, controlled, experimental study. SETTING: University research laboratory. MEASUREMENTS AND MAIN RESULTS: In control animals, sterile saline solution was intratracheally (0.05 mL) or intraperitoneally (0.5 mL) injected. Acute lung injury animals received Escherichia coli lipopolysaccharide intratracheally (40 microg, ALIp) or intraperitoneally (400 microg, ALIexp). Six hours after lipopolysaccharide administration, ALIp and ALIexp animals were further randomized into subgroups receiving saline (0.05 mL) or BMDMC (2 x 10) intravenously. On day 7, BMDMC led to the following: 1) increase in survival rate; 2) reduction in static lung elastance, alveolar collapse, and bronchoalveolar lavage fluid cellularity (higher in ALIexp than ALIp); 3) decrease in collagen fiber content, cell apoptosis in lung, kidney, and liver, levels of interleukin-6, KC (murine interleukin-8 homolog), and interleukin-10 in bronchoalveolar lavage fluid, and messenger RNA expression of insulin-like growth factor, platelet-derived growth factor, and transforming growth factor-beta in both groups, as well as repair of basement membrane, epithelium and endothelium, regardless of acute lung injury etiology; 4) increase in vascular endothelial growth factor levels in bronchoalveolar lavage fluid and messenger RNA expression in lung tissue in both acute lung injury groups; and 5) increase in number of green fluorescent protein-positive cells in lung, kidney, and liver in ALIexp. CONCLUSIONS: BMDMC therapy was effective at modulating the inflammatory and fibrogenic processes in both acute lung injury models; however, survival and lung mechanics and histology improved more in ALIexp. These changes may be attributed to paracrine effects balancing pro- and anti-inflammatory cytokines and growth factors, because a small degree of pulmonary BMDMC engraftment was observed.

Leptin Elicits In Vivo Eosinophil Migration and Activation: Key Role of Mast Cell-Derived PGD2.

Eosinophils are key regulators of adipose tissue homeostasis, thus characterization of adipose tissue-related molecular factors capable of regulating eosinophil activity is of great interest. Leptin is known to directly activate eosinophils in vitro, but leptin ability of inducing in vivo eosinophilic inflammatory response remains elusive. Here, we show that leptin elicits eosinophil influx as well as its activation, characterized by increased lipid body biogenesis and LTC4 synthesis. Such leptin-triggered eosinophilic inflammatory response was shown to be dependent on activation of the mTOR signaling pathway, since it was (i) inhibited by rapamycin pre-treatment and (ii) reduced in PI3K-deficient mice. Local infiltration of activated eosinophils within leptin-driven inflammatory site was preceded by increased levels of classical mast cell-derived molecules, including TNFα, CCL5 (RANTES), and PGD2. Thus, mice were pre-treated with a mast cell degranulating agent compound 48/80 which was capable to impair leptin-induced PGD2 release, as well as eosinophil recruitment and activation. In agreement with an indirect mast cell-driven phenomenon, eosinophil accumulation induced by leptin was abolished in TNFR-1 deficient and also in HQL-79-pretreated mice, but not in mice pretreated with neutralizing antibodies against CCL5, indicating that both typical mast cell-driven signals TNFα and PGD2, but not CCL5, contribute to leptin-induced eosinophil influx. Distinctly, leptin-induced eosinophil lipid body (lipid droplet) assembly and LTC4 synthesis appears to depend on both PGD2 and CCL5, since both HQL-79 and anti-CCL5 treatments were able to inhibit these eosinophil activation markers. Altogether, our data show that leptin triggers eosinophilic inflammation in vivo via an indirect mechanism dependent on activation of resident mast cell secretory activity and mediation by TNFα, CCL5, and specially PGD2.

Eicosapentaenoic acid potentiates the therapeutic effects of adipose tissue-derived mesenchymal stromal cells on lung and distal organ injury in experimental sepsis.

BACKGROUND: Even though mesenchymal stromal cells (MSCs) mitigate lung and distal organ damage in experimental polymicrobial sepsis, mortality remains high. We investigated whether preconditioning with eicosapentaenoic acid (EPA) would potentiate MSC actions in experimental sepsis by further decreasing lung and distal organ injury, thereby improving survival. METHODS: In C57BL/6 mice, sepsis was induced by cecal hligation and puncture (CLP); sham-operated animals were used as control. Twenty-four hours after surgery, CLP mice were further randomized to receive saline, adipose tissue-derived (AD)-MSCs (105, nonpreconditioned), or AD-MSCs preconditioned with EPA for 6 h (105, EPA-preconditioned MSCs) intravenously. After 24 h, survival rate, sepsis severity score, lung mechanics and histology, protein level of selected biomarkers in lung tissue, cellularity in blood, distal organ damage, and MSC distribution (by technetium-99m tagging) were analyzed. Additionally, the effects of EPA on the secretion of resolvin-D1 (RvD1), prostaglandin E2 (PGE2), interleukin (IL)-10, and transforming growth factor (TGF)-ß1 by MSCs were evaluated in vitro. RESULTS: Nonpreconditioned and EPA-preconditioned AD-MSCs exhibited similar viability and differentiation capacity, accumulated mainly in the lungs and kidneys following systemic administration. Compared to nonpreconditioned AD-MSCs, EPA-preconditioned AD-MSCs further reduced static lung elastance, alveolar collapse, interstitial edema, alveolar septal inflammation, collagen fiber content, neutrophil cell count as well as protein levels of interleukin-1ß and keratinocyte chemoattractant in lung tissue, and morphological abnormalities in the heart (cardiac myocyte architecture), liver (hepatocyte disarrangement and Kupffer cell hyperplasia), kidney (acute tubular necrosis), spleen (increased number of megakaryocytes and lymphocytes), and small bowel (villi architecture disorganization). EPA preconditioning of MSCs resulted in increased secretion of pro-resolution and anti-inflammatory mediators (RvD1, PGE2, IL-10, and TGF-ß). CONCLUSIONS: Compared to nonpreconditioned cells, EPA-preconditioned AD-MSCs yielded further reductions in the lung and distal organ injury, resulting in greater improvement in sepsis severity score and higher survival rate in CLP-induced experimental sepsis. This may be a promising therapeutic approach to improve outcome in septic patients.

Glucagon reduces airway hyperreactivity, inflammation, and remodeling induced by ovalbumin.

Glucagon has been shown to be beneficial as a treatment for bronchospasm in asthmatics. Here, we investigate if glucagon would prevent airway hyperreactivity (AHR), lung inflammation, and remodeling in a murine model of asthma. Glucagon (10 and 100 µg/Kg, i.n.) significantly prevented AHR and eosinophilia in BAL and peribronchiolar region induced by ovalbumin (OVA) challenge, while only the dose of 100 µg/Kg of glucagon inhibited subepithelial fibrosis and T lymphocytes accumulation in BAL and lung. The inhibitory action of glucagon occurred in parallel with reduction of OVA-induced generation of IL-4, IL-5, IL-13, TNF-α, eotaxin-1/CCL11, and eotaxin-2/CCL24 but not MDC/CCL22 and TARC/CCL17. The inhibitory effect of glucagon (100 µg/Kg, i.n.) on OVA-induced AHR and collagen deposition was reversed by pre-treatment with indomethacin (10 mg/Kg, i.p.). Glucagon increased intracellular cAMP levels and inhibits anti-CD3 plus anti-CD28-induced proliferation and production of IL-2, IL-4, IL-10, and TNF- α from TCD4+ cells in vitro. These findings suggest that glucagon reduces crucial features of asthma, including AHR, lung inflammation, and remodeling, in a mechanism probably associated with inhibition of eosinophils accumulation and TCD4+ cell proliferation and function. Glucagon should be further investigated as an option for asthma therapy.

Prostaglandin E2-EP1 and EP2 receptor signaling promotes apical junctional complex disassembly of Caco-2 human colorectal cancer cells.

BACKGROUND: The apical junctional complex (AJC) is a dynamic structure responsible to maintain epithelial cell-cell adhesions and it plays important functions such as, polarity, mechanical integrity, and cell signaling. Alteration of this complex during pathological events leads to an impaired epithelial barrier by perturbation of the cell-cell adhesion system. Although clinical and experimental data indicate that prostaglandin E(2) (PGE2) plays a critical function in promoting cell motility and cancer progression, little is known concerning its role in AJC disassembly, an event that takes place at the beginning of colorectal tumorigenesis. Using Caco-2 cells, a cell line derived from human colorectal cancer, we investigated the effects of prostaglandin E(2) (PGE(2)) treatment on AJC assembly and function. RESULTS: Exposition of Caco-2 cells to PGE(2) promoted differential alteration of AJC protein distribution, as evidenced by immunofluorescence and immunoblotting analysis and impairs the barrier function, as seen by a decrease in the transepithelial electric resistance and an increase in the permeability to ruthenium red marker. We demonstrated the involvement of EP1 and EP2 prostaglandin E(2) receptor subtypes in the modulation of the AJC disassembly caused by prostanoid. Furthermore, pharmacological inhibition of protein kinase-C, but not PKA and p38MAPK significantly prevented the PGE(2) effects on the AJC disassembly. CONCLUSION: Our findings strongly suggest a central role of Prostaglandin E2-EP1 and EP2 receptor signaling to mediate AJC disassembly through a mechanism that involves PKC and claudin-1 as important target for the TJ-related effects in human colorectal cancer cells (Caco-2).

The allergic response mediated by fire ant venom proteins.

Fire ants are widely studied, invasive and venomous arthropod pests. There is significant biomedical interest in immunotherapy against fire ant stings. However, mainly due to practical reasons, the physiological effects of envenomation has remained poorly characterized. The present study takes advantage of a recently-described venom protein extract to delineate the immunological pathways underlying the allergic reaction to fire ant venom toxins. Mice were injected with controlled doses of venom protein extract. Following sensitization and a second exposure, a marked footpad swelling was observed. Based on eosinophil recruitment and production of Th2 cytokines, we hereby establish that fire ant proteins per se can lead to an allergic response, which casts a new light into the mechanism of action of these toxins.

Leptin Elicits LTC4 Synthesis by Eosinophils Mediated by Sequential Two-Step Autocrine Activation of CCR3 and PGD2 Receptors.

Leptin is a cytokine, produced mainly by mature adipocytes, that regulates the central nervous system, mainly to suppress appetite and stimulate energy expenditure. Leptin also regulates the immune response by controlling activation of immunomodulatory cells, including eosinophils. While emerging as immune regulatory cells with roles in adipose tissue homeostasis, eosinophils have a well-established ability to synthesize pro-inflammatory molecules such as lipid mediators, a key event in several inflammatory pathologies. Here, we investigated the impact and mechanisms involved in leptin-driven activation of eicosanoid-synthesizing machinery within eosinophils. Direct in vitro activation of human or mouse eosinophils with leptin elicited synthesis of lipoxygenase as well as cyclooxygenase products. Displaying selectivity, leptin triggered synthesis of LTC4 and PGD2, but not PGE2, in parallel to dose-dependent induction of lipid body/lipid droplets biogenesis. While dependent on PI3K activation, leptin-driven eosinophil activation was also sensitive to pertussis toxin, indicating the involvement of G-protein coupled receptors on leptin effects. Leptin-induced lipid body-driven LTC4 synthesis appeared to be mediated through autocrine activation of G-coupled CCR3 receptors by eosinophil-derived CCL5, inasmuch as leptin was able to trigger rapid CCL5 secretion, and neutralizing anti-RANTES or anti-CCR3 antibodies blocked lipid body assembly and LTC4 synthesis induced by leptin. Remarkably, autocrine activation of PGD2 G-coupled receptors DP1 and DP2 also contributes to leptin-elicited lipid body-driven LTC4 synthesis by eosinophils in a PGD2-dependent fashion. Blockade of leptin-induced PGD2 autocrine/paracrine activity by a specific synthesis inhibitor or DP1 and DP2 receptor antagonists, inhibited both lipid body biogenesis and LTC4 synthesis induced by leptin stimulation within eosinophils. In addition, CCL5-driven CCR3 activation appears to precede PGD2 receptor activation within eosinophils, since neutralizing anti-CCL5 or anti-CCR3 antibodies inhibited leptin-induced PGD2 secretion, while it failed to alter PGD2-induced LTC4 synthesis. Altogether, sequential activation of CCR3 and then PGD2 receptors by autocrine ligands in response to leptin stimulation of eosinophils culminates with eosinophil activation, characterized here by assembly of lipidic cytoplasmic platforms synthesis and secretion of the pleiotropic lipid mediators, PGD2, and LTC4.

Group V secretory phospholipase A2 amplifies the induction of cyclooxygenase 2 and delayed prostaglandin D2 generation in mouse bone marrow culture-derived mast cells in a strain-dependent manner.

Activation of mouse bone marrow-derived mast cells (BMMC) with stem cell factor (SCF) or IgE and antigen elicits exocytosis and an immediate phase of prostaglandin (PG) D(2) and leukotriene (LT) C(4) generation. Activation of BMMC by SCF, IL-1beta and IL-10 elicits a delayed phase of PGD(2) generation dependent on cyclooxygenase (COX) 2 induction. Cytosolic phospholipase A(2) alpha provides arachidonic acid in both phases and amplifies COX-2 induction. Pharmacological experiments implicate an amplifying role for secretory (s) PLA(2). We used mice lacking the gene encoding group V sPLA(2) (Pla2g5-/-) to definitively test its role in eicosanoid generation by BMMC. Pla2g5-/- BMMC on a C57BL/6 genetic background showed a modest reduction in exocytosis and immediate PGD(2) generation after activation with SCF or with IgE and antigen, while LTC(4) generation was not modified. Delayed-phase PGD(2) generation and COX-2 induction were reduced approximately 35% in C57BL/6 Pla2g5-/- BMMC and were restored by exogenous PGE(2). There was no deficit in either phase of eicosanoid generation by Pla2g5-/- BMMC on a BALB/c background. Thus, group V sPLA(2) amplifies COX-2 expression and delayed phase PGD(2) generation in a strain-dependent manner; it has at best a limited role in immediate eicosanoid generation by BMMC.

Antioxidant Treatment Induces Hyperactivation of the HPA Axis by Upregulating ACTH Receptor in the Adrenal and Downregulating Glucocorticoid Receptors in the Pituitary.

Glucocorticoid (GC) production is physiologically regulated through a negative feedback loop mediated by the GC, which appear disrupted in several pathological conditions. The inability to perform negative feedback of the hypothalamus-pituitary-adrenal (HPA) axis in several diseases is associated with an overproduction of reactive oxygen species (ROS); however, nothing is known about the effects of ROS on the functionality of the HPA axis during homeostasis. This study analyzed the putative impact of antioxidants on the HPA axis activity and GC-mediated negative feedback upon the HPA cascade. Male Wistar rats were orally treated with N-acetylcysteine (NAC) or vitamin E for 18 consecutive days. NAC-treated rats were then subjected to a daily treatment with dexamethasone, which covered the last 5 days of the antioxidant therapy. We found that NAC and vitamin E induced an increase in plasma corticosterone levels. NAC intensified MC2R and StAR expressions in the adrenal and reduced GR and MR expressions in the pituitary. NAC also prevented the dexamethasone-induced reduction in plasma corticosterone levels. Furthermore, NAC decreased HO-1 and Nrf2 expression in the pituitary. These findings show that antioxidants induce hyperactivity of the HPA axis via upregulation of MC2R expression in the adrenal and downregulation of GR and MR in the pituitary.