Regulated Upon Activation, Normal T Cell Expressed And Secreted Secretion Research Articles

Coadministration of the cyanobacterial lipopolysaccharide antagonist CyP with antibiotic inhibits cytokine production by an in vitro meningitis model infected with Neisseria meningitidis

In this study, the objective was to determine the anti-inflammatory properties of CyP, a cyanobacterial lipopolysaccharide (LPS) antagonist, used in combination with antibiotic chemotherapy during infection of an in vitro meningitis model infected with Neisseria meningitidis (meningococcus). Monocultures of human meningioma cells and meningioma-primary human macrophage co-cultures were infected with meningococci (10(2)-10(8) cfu/monolayer) or treated with isolated outer membranes or purified LPS (0.1-100 ng/monolayer) from N. meningitidis. CyP (1-20 μg/monolayer) was added at intervals from t = 0 to 4 h, with and without benzylpenicillin (1-20 μg/monolayer). The antagonistic effect of CyP and its adjunctive properties to benzylpenicillin administration was determined by measuring cytokine levels in culture supernatants after 24 h. CyP significantly inhibited (P < 0.05) the secretion of interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 and RANTES ('regulated upon activation, normal T cell expressed and secreted') (overall reduction levels from 50% to >95%) by meningioma cell lines and meningioma-macrophage co-cultures challenged with either live meningococci or bacterial components. Inhibition was effective when CyP was added within 2 h of challenge (P < 0.05) and was still pronounced by 4 h. In the co-culture model, CyP alone partially inhibited IL-1β secretion, but did not prevent tumour necrosis factor (TNF)-α secretion, whereas penicillin alone inhibited IL-1β and TNF-α but conversely did not reduce MCP-1 and RANTES secretion. However, coadministration of CyP and penicillin in both models had an additive effect and restored the overall inhibitory profile. CyP inhibits cytokine production in an in vitro meningitis model and augments the anti-inflammatory response when combined with benzylpenicillin. Administration of an LPS antagonist with antibiotic merits consideration in the emergency treatment of patients presenting with meningococcal infection.

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.

Expression and Secretion of RANTES (CCL5) in Human Adipocytes in Response to Immunological Stimuli and Hypoxia

Obesity and related disorders represent states of systemic low-grade inflammation. Chemokine secretion by adipocytes may initiate leukocyte infiltration in obese adipose tissue and thus mediate an important step in the establishment of chronic immune activation. The chemokine RANTES (regulated upon activation normal T cell expressed and secreted)/CCL5 is a chemoattractant for various leukocyte subsets. This study was designed to examine whether RANTES is expressed and released by human adipocytes and how its expression is regulated. RANTES expression under basal conditions was studied in mature adipocytes. Cells were therefore challenged with lipopolysaccharide (LPS), interferon (IFN)-gamma, interleukin (IL)-4, monocyte chemoattractant protein (MCP)-1 or exposed to low oxygen pressure. RANTES was expressed and secreted constitutively in most samples of mature adipocytes from the omental and the subcutaneous depot. RANTES release was dependent on adipocyte size and also seemed to be higher from cells of obese donors. Hypoxia (4% O (2)) caused an approximately 36% increase of RANTES release. Human adipocytes express the chemokine RANTES and are thus identified as a novel cellular source of this immune mediator. LPS and IFNgamma do not seem to play a significant role for the expression of RANTES in contrast to moderate hypoxia, which points to a distinct role in the innate immune system.

ORIGINAL ARTICLE: Trophoblasts and Shear Stress Induce an Asymmetric Distribution of ICAM‐1 in Uterine Endothelial Cells

Problem We have used an in vitro co‐culture system consisting of early gestation macaque trophoblasts cultured on top of human uterine microvascular endothelial cells (UtMVECs) to investigate the inflammatory response of endothelial cells to trophoblasts under shear stress conditions.Method of study Uterine microvascular endothelial cells and trophoblasts were co‐cultured in a parallel plate chamber under shear stress (15 dyn/cm2) conditions. The distribution and expression of endothelial intercellular adhesion molecule‐1 (ICAM‐1) was quantified by immunofluorescence image analysis and flow cytometry. Endothelial regulated upon activation normal T‐cell expressed and secreted (RANTES) secretion was measured by enzyme‐linked immunosorbent assay and permeability was assessed using fluorescein isothiocyanate‐dextran.Results Intercellular adhesion molecule‐1, but not vascular cell adhesion molecule‐1 or platelet endothelial cell adhesion molecule‐1, was re‐distributed towards the downstream edge of endothelial cells when the cells were co‐cultured with trophoblasts under shear stress conditions. Changes in ICAM‐1 distribution were also observed when UtMVECs were co‐cultured with trophoblast‐conditioned medium under shear stress conditions. Incubation of UtMVECs with trophoblast‐conditioned medium increased endothelial permeability, RANTES secretion, and trophoblast adhesion.Conclusion These data support the idea that trophoblasts induce an inflammatory response in uterine endothelial cells that could enhance trophoblast invasion and transmigration.

Functional Expression of β-Chemokine Receptors in Osteoblasts: Role of Regulated upon Activation, Normal T Cell Expressed and Secreted (RANTES) in Osteoblasts and Regulation of Its Secretion by Osteoblasts and Osteoclasts

The expression and functions of receptors for the beta-chemokine, regulated upon activation, normal T cell expressed, and secreted (RANTES)/CCL5, were investigated in osteoblasts. Both primary osteoblasts and the MC3T3-E1 osteoblast cell line express the RANTES receptors, CCR1, 3, 4, and 5 (by RT-PCR), which encode functional receptors in osteoblasts as shown by [125I]-RANTES binding followed by Scatchard analysis. Expression of all four RANTES receptor mRNAs in osteoblast is in contrast to the reports of expression of CCR1 being the only RANTES receptor expressed by osteoclasts. Exogenous RANTES elicits chemotaxis of osteoblasts and promotes cell survival via phosphatidylinositol 3-kinase with attendant phosphorylation of Akt. Osteoclastic RANTES, obtained from the conditioned medium of receptor activator of nuclear factor-kappa B ligand-differentiated RAW264.7 cells also induces chemotaxis of MC3T3-E1 cells. Incubating the conditioned medium with an anti-RANTES neutralizing antibody attenuated this effect. RANTES secretion from osteoblast is inhibited by differentiation promoting hormones, e.g. 1,25 (OH)2D3 and dexamethasone, whereas macrophage inflammatory protein-1 alpha (but not macrophage inflammatory protein-1 beta) and elevated calcium induce it. Elevated calcium also stimulated RANTES secretion by osteoclasts. Therefore, RANTES is an osteoblast chemoattractant and a survival-promoting molecule whose regulation in osteoblast is varied. Furthermore, RANTES secreted from osteoclasts induces osteoblast chemotaxis. Therefore, expression of RANTES and its receptors in both osteoblasts and osteoclasts could enable this chemokine to act in autocrine/paracrine modes.

Peroxisome proliferator-activated receptor-γ ligand inhibition of RANTES production by human endometriotic stromal cells is mediated through an upstream promoter element

Objective To investigate the effect of peroxisome proliferator activated receptor-γ (PPAR-γ) ligands on transcription and secretion of regulated upon activation normal T-cell expressed and secreted (RANTES) in endometriotic stromal cells. Design Controlled laboratory study. Setting Academic research laboratory. Patient(s) Women in the follicular phase of the menstrual cycle undergoing laparoscopic resection for endometriosis. Intervention(s) [1] Transient transfection of endometriotic stromal cells with RANTES promoter vectors with and without a mutagenized PPAR-γ response element (PPRE), then treatment with PPAR-γ ligands; [2] co-incubation of cells with PPAR-γ ligands. Main outcome measure(s) RANTES promoter activity and RANTES secretion. Result(s) In endometriotic stromal cells, addition of PPAR-γ ligands (rosiglitazone and 15 deoxy-Δ 12,14 prostaglandin J 2) inhibited RANTES promoter activity by 51% and 50%, respectively. In cells transfected with the same promoter after site-directed mutagenesis of the 5′ PPRE, addition of PPAR-γ ligands failed to inhibit promoter activity. When endometriotic stromal cells were treated with PPAR-γ ligands, a decrease in RANTES secretion by 51% and 20%, respectively, was observed. Conclusion(s) The PPAR-γ ligands inhibit RANTES transcription and protein production in endometriotic stromal cells. Transcriptional repression appears to be mediated through a specific PPRE at −344 to −322 bp upstream from the RNA polymerase start site.

17β-estradiol Inhibits the Production of RANTES in Human Keratinocytes

A chemokine, regulated upon activation, normal T cell expressed and secreted (RANTES) attracts T helper-1 cells and macrophages. The production of RANTES is enhanced in keratinocytes of psoriatic skin lesions, which may contribute to the inflammatory infiltrate. It is known that estrogen regulates the natural course of psoriasis. We examined the in vitro effects of 17beta-estradiol on RANTES production by human keratinocytes. 17beta-estradiol inhibited tumor necrosis factor-alpha or interleukin-1beta-induced RANTES secretion, mRNA expression, and promoter activity in keratinocytes, and these effects of 17beta-estradiol were counteracted by estrogen receptor antagonist ICI 182 780. Two nuclear factor kappaB elements on RANTES promoter were required for tumor necrosis factor-alpha or interleukin-1beta-induced transcription and involved in the inhibition by 17beta-estradiol. 17beta-estradiol inhibited nuclear factor kappaB transcriptional activity, whereas it did not inhibit DNA binding of nuclear factor kappaB or phosphorylation or degradation of the inhibitor of nuclear factor kappaB alpha in tumor necrosis factor-alpha or interleukin-1beta-stimulated keratinocytes. 17beta-estradiol-induced inhibition of nuclear factor kappaB transcriptional activity and RANTES promoter activity was rescued by overexpression of a coactivator cyclic AMP response element-binding protein (CREB) or nuclear factor kappaB p65 but not by steroid receptor coactivator-1 or nuclear factor kappaB p50. The overexpression of CREB-binding protein rescued 17beta-estradiol-induced inhibition of transcription mediated by a chimeric protein, GAL4-p65286-551, which contained GAL4 DNA binding domain fused to C-terminal transactivating domain of p65 (amino acids 286-551). The transfection of estrogen receptor alpha or estrogen receptor beta into estrogen receptor-negative SKBR3 cells resulted in 17beta-estradiol-mediated inhibition of transcription via GAL4-p65286-551. These results suggest that 17beta-estradiol-bound estrogen receptor may inhibit nuclear factor kappaB-dependent transcription of RANTES gene by competing with p65 for limiting amounts of CREB-binding protein.

Induction of RANTES, HLA-DR, and intercellular adhesion molecule-1 on highly purified distal tubular cells from human kidney.

Expression of proinflammatory molecules by tubular epithelial cells plays an important role in renal allograft rejection and inflammatory kidney diseases. Different studies from patients with acute rejection point to the involvement of distal tubular segments. At present no in vitro system for the human distal tubule is established. Human distal tubular cells were isolated immunomagnetically. Cultured cells were stimulated with cytokines (interferon-gamma, tumor necrosis factor-alpha, interleukin-1beta, or a cytokine mix). Secretion of RANTES (regulated upon activation, normal T-cell expressed and secreted) was evaluated with an enzyme-linked immunoassay. Expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 was assessed by flow cytometric analysis and immunofluorescence studies. Our data clearly indicate that distal tubular cells express RANTES, HLA-DR, and ICAM-1 in response to a mixture of specific cytokines. Dexamethasone inhibited the induced expression of RANTES and HLA-DR significantly, but not that of ICAM-1. We demonstrate an appropriate in vitro system for the human distal tubule. The present study proves the involvement of the distal tubular segment during inflammatory kidney diseases.

Chemokines, nitric oxide and antiarthritic effects of 9-(2-phosphonomethoxyethyl)adenine (Adefovir)

Antiarthritic effects of two acyclic nucleoside phosphonates, 9-(2-phosphonomethoxyethyl)adenine (PMEA; Adefovir) and 9-(2-phosphonomethoxypropyl)adenine (PMPA), as well as their more bioavailable prodrugs, bis(pivaloyloxymethyl)ester of PMEA [bis(POM)-PMEA; Adefovir Dipivoxil] and bis(isopropyloxycarbonyloxymethyl)ester of PMPA [bis(POC)-PMPA], were investigated in a model of adjuvant-induced arthritis in Lewis rats. The drugs were injected subcutaneously at doses of 5–50 mg/kg. PMEA and its prodrug inhibited by >80% arthritic paw swelling, splenomegaly and fibroadhesive perisplenitis. Both prophylactic and therapeutic dosing regimens were effective. Neither PMPA nor bis(POC)-PMPA suppressed development of arthritic lesions. Substantially reduced nitrite+nitrate levels were detected in serum and urine of PMEA-treated animals as compared to those of untreated diseased controls. Also, complete suppression of the disease-associated, greatly enhanced systemic levels of the chemokine, RANTES (regulated upon activation, normal T cell expressed and secreted), was observed in rats injected with PMEA. Additional in vitro studies showed that PMEA does not change, PMPA enhances, and both prodrugs inhibit the immune-activated NO production. Under the same conditions PMEA inhibits, while PMPA slightly stimulates, secretion of RANTES. Collectively, these data suggest that the in vivo-inhibited production of nitric oxide (NO) is a consequence rather than a mechanism of antiarthritic action of PMEA. Possible mechanisms for the anti-RANTES activity of PMEA remains to be firmly established.

Patterns for RANTES secretion and intercellular adhesion molecule 1 expression mediate transepithelial T cell traffic based on analyses in vitro and in vivo.

Immune cell migration into and through mucosal barrier sites in general and airway sites in particular is a critical feature of immune and inflammatory responses, but the determinants of transepithelial (unlike transendothelial) immune cell traffic are poorly defined. Accordingly, we used primary culture airway epithelial cells and peripheral blood mononuclear cells to develop a cell monolayer system that allows for apical-to-basal and basal-to-apical T cell transmigration that can be monitored with quantitative immunofluorescence flow cytometry. In this system, T cell adhesion and subsequent transmigration were blocked in both directions by monoclonal antibodies (mAbs) against lymphocyte function-associated antigen 1 (LFA-1) or intercellular adhesion molecule 1 (ICAM-1) (induced by interferon gamma [IFN-gamma] treatment of epithelial cells). The total number of adherent plus transmigrated T cells was also similar in both directions, and this pattern fit with uniform presentation of ICAM-1 along the apical and basolateral cell surfaces. However, the relative number of transmigrated to adherent T cells (i.e., the efficiency of transmigration) was increased in the basal-to-apical relative to the apical-to-basal direction, so an additional mechanism was needed to mediate directional movement towards the apical surface. Screening for epithelial-derived beta-chemokines indicated that IFN-gamma treatment caused selective expression of RANTES (regulated upon activation, normal T cell expressed and secreted), and the functional significance of this finding was demonstrated by inhibition of epithelial-T cell adhesion and transepithelial migration by anti-RANTES mAbs. In addition, we found that epithelial (but not endothelial) cells preferentially secreted RANTES through the apical cell surface thereby establishing a chemical gradient for chemotaxis across the epithelium to a site where they may be retained by high levels of RANTES and apical ICAM-1. These patterns for epithelial presentation of ICAM-1 and secretion of RANTES appear preserved in airway epithelial tissue studied either ex vivo with expression induced by IFN-gamma treatment or in vivo with endogenous expression induced by inflammatory disease (i.e., asthma). Taken together, the results define how the patterns for uniform presentation of ICAM-1 along the cell surface and specific apical sorting of RANTES may serve to mediate the level and directionality of T cell traffic through epithelium (distinct from endothelium) and provide a basis for how this process is precisely coordinated to route immune cells to the mucosal surface and maintain them there under normal and stimulated conditions.

Immunolocalization and Regulation of the Chemokine RANTES in Human Endometrial and Endometriosis Tissues and Cells1

Retrograde menstruation is postulated as the initiating event in the histogenesis of endometriosis; however, subsequent steps in the pathogenesis of this common disorder remain poorly characterized. The ip accumulation of activated leukocytes and the infiltration of endometriosis lesions by macrophages and T cells are cytological markers of the inflammatory nature of this syndrome. The apparent recruitment of these leukocytes prompted us to search for chemokine expression by endometriosis cells. We reported previously that pelvic fluid RANTES (regulated upon activation, normal T cell expressed and secreted) concentrations correlated with the stage of endometriosis. In the current study, RANTES messenger ribonucleic acid (mRNA) was identified in normal endometrium and endometriosis lesions, and techniques were developed to localize RANTES protein within these tissues. Using isolated endometrial and endometriosis cell cultures, we demonstrated that RANTES mRNA and protein can be induced by the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma in endometrial stromal, but not in epithelial or adenocarcinoma cells. Immunocytochemical studies confirmed the biochemical findings. Metabolic labeling experiments verified that nascent RANTES secreted by cytokine-stimulated endometriosis stromal cells was the mature, 8-kDa protein predicted by the mRNA encoding this chemokine. The results indicate that RANTES is a normal constituent of the eutopic endometrium. We propose that secretion of RANTES by ectopic endometriosis implants provides a mechanism for peritoneal leukocyte recruitment.