P19 Expression Research Articles

Transient expression of theArftumor suppressor during male germ cell and eye development inArf-Crereporter mice

The Arf tumor suppressor is expressed transiently during mouse male germ cell and eye development. Its inactivation compromises spermatogenesis as mice age and leads to aberrant postnatal proliferation of cells in the vitreous of the eye, resulting in blindness. In the testis, expression of p19(Arf) is limited to spermatogonia but is extinguished completely in spermatocytes, suggesting that Arf plays a physiologic role in setting the balance between mitotic and meiotic germ cell division. A knock-in allele encoding Cre recombinase regulated by the mouse cellular Arf promoter was used to trace Arf gene induction in vivo. Interbreeding to a reporter strain that expresses Cre-dependent YFP provided proof-of-principle that the Arf-Cre allele was appropriately expressed in the male germ cell lineage. However, Cre expression resulted in male sterility, limiting germ line transmission of the knock-in allele to females. Arf-null mice fail to resorb the hyaloid vasculature within the ocular vitreous where pericyte-like cells that express the PDGF-beta receptor (Pdgfrbeta) proliferate aberrantly and destroy the retina and lens. Interbreeding of Arf-Cre females to males containing "floxed" (FL) Arf alleles yielded Arf(Cre/FL) progeny that exhibited variably penetrant defects in visual acuity ranging to total blindness. Crossing the Arf(Cre/FL) alleles onto a Pdgfrbeta(FL/FL) background normalized all histopathology and restored vision fully.

PGE2 affects the IL-12/IL-23 balance in mature myeloid dendritic cells (38.14)

Abstract Following TLR signaling dendritic cells (DC) produce pro-inflammatory cytokines including IL-12 and IL-23. IL-12 promotes differentiation of the Th1 subset whereas IL-23 maintains the Th17 subset. PGE2, a lipid mediator, is produced by DC during inflammation. This study is focused on the effects of PGE2 on the regulation of IL-12/IL-23 production in murine DC. PGE2 inhibits LPS-induced IL-12 production through downregulation of p35, and stimulates IL-23 through upregulation of p19 expression. Changes in p35/p19 expression reduce Th1 and promote Th17 differentiation. Similar PGE2 effects on p19/p35 were observed in the presence of other TLR ligands, such as peptidoglycan (TLR2), poly I:C (TLR3) and CpG (TLR9). The effects of PGE2 on p19/p35 were mediated through EP2 and EP4 receptors, and cAMP induction. The involvement of downstream signaling pathways such as PKA and EPAC-induced activation of PI3K and GSK3, as well as the role of specific transcription factors such as CREB, cRel, and IRF5 (for p19 expression) and IRF 1, 3, 7, and RelA (for p35 expression) are currently being investigated. Research support: NIH2RO1AI052306 & NSF Pre-doctoral Fellowship (VK)

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.

Expression of phosphatidylinositol-3-kinase in the lesions of psoriasis and some skin tumors

Objective To investigate the relationship of the expression of phosphatidylinositol-3-kinase(PI3K)subunits,P85 and P110 to the pathogenesis of psoriasis.Methods Immunohistochemical staining for P85 and P110 was performed in the tissue specimens from patients with psoriasis(n=30),chronic dermatitis(n=20),seborrheic keratosis(n=20),squamous cell carcinoma(n=20),basal cell carcinoma(n=30)and normal human controls(n=10).The absorbance of immunostained tissue was quantified with image analysis system (Q550CW,Leica,Manheim,Germany).Statistical analysis was carried out by ANOVA,Results Among these groups,a significant difference was observed in the expression level of P110 in the epidermis(F=35.64,P＜0.01),as well as in that of P85(F=59.98,P＜0.01)and P110(F=323.23,P＜0.01)in the lymphocytes infiltrating the lesion.Increased expression of P110 was found in the epidermis of psoriatic lesions compared with the lesions in the other disorders,whereas no significant difference was noticed among the other disorders.In the case of P85 and P110 expression in the lesion-infiltrating lymphocytes infiltrating the lesion,psoriasis and squamous cell carcinoma significantly differed from the other disorders,while no difference was observed between psoriasis and squamous cell carcinoma (P＞0.05).Conclusions The high expression of P110 might be closely correlated to the hyperproliferation of keratinocytes;but filrther study is needed to clarify the relationship of increased expression of P85 and P110 to the activation and proliferation of lymphocytes in psoriatic lesions. Key words: Psoriasis; Carcinoma,squamous cell; Carcinoma,basal cell; Keratosis,seborrheic; 1-phosphatidylinositols 3-kinase

The concentration of the murine C-type viral protein p30 in mouse tissue is under host genetic control by multiple gene loci

Congenic strains on A, C57BL/6, and 129 background, which differ from the original strains for specific loci detected, are highly suitable for evaluation of the host genetic control of the expression of C-type viral protein, p30. Significant differences in the p30 concentration in the present study in some of the organs, blood, heart, kidney, lung, spleen and thymus have been demonstrated related to the loci, 77a, Thy-1, Cv-1, Gv-2, Ly-1, and Ly-3, whereas the loci, H-2, and Ly-2 evoked no effect on p30 expression. It is of particular interest that (1) the change of the genotype for two independent loci, Tla, and Thy-1 in the original A strain led to the same differences in the p30 concentration, (2) only Ly-1, and Ly-3 in the Ly-system affected the p30 expression, and (3) significant difference of the H-2 could be demonstrated for blood and thymus. Investigations of the nude locus in BALB/c, C3H, C57BL/6, and NZB congenic strains and the hairless locus in HRS strain disclosed an elevated p30 concentration compared to the coated animals, if the parental strain previously expressed a low p30 concentration. The genes investigated have the following traits: (1) presence/absence of thymus, (2) they determine cell surface antigens on thymocytes and participate in the T-cell differentiation, and (3) they all play a role in leukemogenesis.

Characterization of inbred strains and substrains of mice by determinations of C-type viral protein p30 expression in various organs

The radioimmunoassay method for detection of the major core C-type viral protein p30 is a tool for the characterization and control in inbred strains of mice. 27 mouse strains could be separated into eight groups based on examination of six organs. It is also possible to separate substrains if they are genetically different. Several genes play a role in the expression of p30, but also genotype-environmental interactions seem to affect the p30 expression.

Synergism between tumor necrosis factor alpha and interleukin-17 to induce IL-23 p19 expression in fibroblast-like synoviocytes

In order to determine the mechanisms by which a chronic inflammatory network can be maintained in the arthritic joint, we examined whether fibroblast-like synoviocytes (FLS) could provide feedback signals after their stimulation by inflammatory cytokines. FLS and dermal fibroblasts (DF) were derived from rheumatoid arthritis (RA), osteoarthritis (OA) and post-trauma patients. These two cell types were then stimulated with 10 nanogram/ml of TNFα, IL-1β and IL-17 alone or in combination treatments. Specific mRNA expression of IL-23 p19 was quantitated by real-time PCR and its protein by immunoprecipitation. A striking specific synergistic induction of IL-23 p19 versus IL-12 p35 mRNA expression was noted after stimulation with IL-17 and TNFα in FLS, and to a lesser degree in DF ( p < 0.043). This synergistic response was composed of an initial priming step by IL-17, thus making FLS hyperresponsive to TNFα-mediated stimulation. In contrast, IL-1β mediated induction of IL-23 p19 expression was cell-specific. Induction of IL-23 p19 expression by IL-1β was present in FLS but almost absent in the DF derived from the same patients. Furthermore, IL-1β did not synergize with IL-17 to induce IL-23 p19 expression. Immunoprecipitation of FLS cellular lysates after stimulation with IL-17 and TNFα detected p19 protein and this was enhanced by the addition of IL-1β. However, no co-immunoprecipitation of the p40 subunit of IL-23 was noted from the same cells. Thus, FLS are potently regulated by inflammatory cytokines to specifically express IL-23 p19. Additional byproducts of the inflammatory milieu may be required for the generation and secretion of bioactive IL-23.

P312 - Prevalence of antibodies against thyroglobulin (anti-Tg) and thyroid peroxidase (anti-TPO) in patients with inflammatory bowel disease on scheduled infliximab or conventional treatment: a two years follow up study

P312 - Prevalence of antibodies against thyroglobulin (anti-Tg) and thyroid peroxidase (anti-TPO) in patients with inflammatory bowel disease on scheduled infliximab or conventional treatment: a two years follow up study

Bmi1 Is Required for Hedgehog Pathway-Driven Medulloblastoma Expansion

Inappropriate Hedgehog (Hh) signaling underlies development of a subset of medulloblastomas, and tumors with elevated HH signaling activity express the stem cell self-renewal gene BMI1. To test whether Bmi1 is required for Hh-driven medulloblastoma development, we varied Bmi1 gene dosage in transgenic mice expressing an oncogenic Hh effector, SmoA1, driven by a glial fibrillary acidic protein (GFAP) promoter. Whereas 100% of SmoA1; Bmi1(+/+) or SmoA1;Bmi1(+/-) mice examined between postnatal (P) days 14 and 26 had typical medulloblastomas (N = 29), tumors were not detected in any of the SmoA1;Bmi1(-/-) animals examined (N = 6). Instead, small ectopic collections of cells were present in the region of greatest tumor load in SmoA1 animals, suggesting that medulloblastomas were initiated but failed to undergo expansion into frank tumors. Cells within these Bmi1(-/-) lesions expressed SmoA1 but were largely nonproliferative, in contrast to cells in Bmi1(+/+) tumors (6.2% vs 81.9% PCNA-positive, respectively). Ectopic cells were negative for the progenitor marker nestin, strongly GFAP-positive, and highly apoptotic, relative to Bmi1(+/+) tumor cells (29.6% vs 6.3% TUNEL-positive). The alterations in proliferation and apoptosis in SmoA1;Bmi1(-/-) ectopic cells are associated with reduced levels of Cyclin D1 and elevated expression of cyclin-dependent kinase inhibitor p19(Arf), two inversely regulated downstream targets of Bmi1. These data provide the first demonstration that Bmi1 is required for spontaneous de novo development of a solid tumor arising in the brain, suggest a crucial role for Bmi1-dependent, nestin-expressing progenitor cells in medulloblastoma expansion, and implicate Bmi1 as a key factor required for Hh pathway-driven tumorigenesis.

Decreased severity of experimental autoimmune encephalomyelitis during resveratrol administration is associated with increased IL-17 +IL-10 + T cells, CD4 − IFN-γ + cells, and decreased macrophage IL-6 expression

Experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple Sclerosis, is induced after injection of PLP 139–151 myelin peptide in complete Freund's adjuvant into SJL/J mice. During EAE, T cells and macrophages infiltrate the brain, produce cytokines IL-17, IFN-γ, TNF-α, or IL-6, and bring about autoimmune neuroinflammation. However, infiltrating T cells which simultaneously produce IL-17 and IL-10 or infiltrating CD4 − NKT cells that produce IFN-γ protect against EAE. Resveratrol, a plant polyphenol, exhibits anti-inflammatory properties. To determine if resveratrol can relieve EAE, SJL/J mice were administered diets enriched in resveratrol at EAE injection. EAE symptoms were significantly less compared with controls in mice fed resveratrol. At day 56 of EAE, splenic T cells from mice fed 0%, 0.04% or 0.08% resveratrol that were restimulated with PLP 139–151 produced similar levels while splenic T cells from mice fed 0.02% resveratrol produced significantly higher levels of IL-17, IFN-γ, and TNF-α. At peak EAE (day 14), mice fed resveratrol had higher numbers of IL-17 + T cells, IL-17 +/IL-10 + T cells, and CD4 −IFN-γ + cells in the brain and spleen compared with controls. Adoptive transfer of day 14 EAE encephalogenic T cells into mice fed resveratrol reduced the severity of EAE. In addition, resveratrol directly suppressed expression of IL-6 and IL-12/23 p40 but increased expression of IL-12 p35 and IL-23 p19 from macrophages. Therefore resveratrol protection against EAE is not associated with declines in IL-17 + T cells but is associated with rises in IL-17 +/IL-10 + T cells and CD4 -IFN-γ + and with repressed macrophage IL-6 and IL-12/23 p40 expression.

The OGF–OGFr Axis Utilizes the p16INK4aand p21WAF1/CIP1Pathways to Restrict Normal Cell Proliferation

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met(5)]enkephalin) that interacts with the OGF receptor (OGFr) and serves as a tonically active negative growth factor in cell proliferation of normal cells. To clarify the mechanism by which OGF inhibits cell replication in normal cells, we investigated the effect of the OGF-OGFr axis on cell cycle activity in human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (NHEKs). OGF markedly depressed cell proliferation of both cell lines by up to 40% of sterile water controls. Peptide treatment induced cyclin-dependent kinase inhibitor (CKI) p16(INK4a) protein expression and p21(WAF1/CIP1) protein expression in HUVECs and NHEKs, but had no effect on p15, p18, p19, or p27 protein expression in either cell type. Inhibition of either p16(INK4a) or p21(WAF1/CIP1) activation by specific siRNAs blocked OGF inhibitory action. Human dermal fibroblasts and mesenchymal stem cells also showed a similar dependence of OGF action on p16(INK4a) and p21(WAF1/CIP1). Collectively, these results indicate that both p16(INK4a) and p21(WAF1/CIP1) are required for the OGF-OGFr axis to inhibit cell proliferation in normal cells.

Hmga2 Promotes Neural Stem Cell Self-Renewal in Young but Not Old Mice by Reducing p16 Ink4a and p19 Arf Expression

Stem cells persist throughout life in diverse tissues by undergoing self-renewing divisions. Self-renewal capacity declines with age, partly because of increasing expression of the tumor suppressor p16(Ink4a). We discovered that the Hmga2 transcriptional regulator is highly expressed in fetal neural stem cells but that expression declines with age. This decrease is partly caused by the increasing expression of let-7b microRNA, which is known to target HMGA2. Hmga2-deficient mice show reduced stem cell numbers and self-renewal throughout the central and peripheral nervous systems of fetal and young-adult mice but not old-adult mice. Furthermore, p16(Ink4a) and p19(Arf) expression were increased in Hmga2-deficient fetal and young-adult stem cells, and deletion of p16(Ink4a) and/or p19(Arf) partially restored self-renewal capacity. let-7b overexpression reduced Hmga2 and increased p16(Ink4a)/p19(Arf) expression. Hmga2 thus promotes fetal and young-adult stem cell self-renewal by decreasing p16(Ink4a)/p19(Arf) expression. Changes in let-7 and Hmga2 expression during aging contribute to the decline in neural stem cell function.

Promoter Analysis Reveals Critical Roles for SMAD-3 and ATF-2 in Expression of IL-23 p19 in Macrophages

IL-23 p19/p40, produced by macrophages and dendritic cells, is critical for development of Th17 in several autoimmune diseases. In this study, bone marrow-derived (BMM) and splenic macrophages (SPM) from SJL/J mice, susceptible to autoimmune demyelinating disease following Theiler's virus (TMEV) infection, expressed IL-23 in response to TMEV. We identified potential binding sites for IFN response factor (IRF)-3 (nt -734 to -731), Sma- and Mad-related protein (SMAD)-3 (nt -584 to -581), activating transcription factor (ATF)-2 (nt -571 to -568), IRF-7 (nt -533 to-525), and NF-kappaB (nt -215 to -209) in the murine p19 promoter. The p19(prom) in the pGL3 promoter-reporter vector responded to TMEV or poly(I:C), a TLR3 agonist in the RAW264.7 macrophage cell line. Deletions upstream from the IRF-3 site and mutations at the IRF-3, SMAD-3, ATF-2, or NF-kappaB, but not the IRF-7, sites significantly reduced promoter activity. ATF-2 or SMAD-3, but not IRF-3, short-hairpin RNA reduced p19 promoter activity and protein expression in RAW264.7 cells responding to TMEV. Chromosomal DNA immunoprecipitation assays revealed that SMAD-3 and ATF-2 bind to the endogenous p19 promoter in RAW264.7 cells and SJL/J SPM following challenge with TMEV. TGF-beta1, which activates SMAD-3, was induced in RAW264.7 cells, BMM, and SPM by TMEV. Neutralizing Ab to TGF-beta1 eliminated TMEV-induced IL-23 production and SMAD-3 activation in RAW264.7 cells, BMM, and SPM. Activation of ATF-2 was JNK, but not p38 or ERK MAPK dependent. Inhibition of the JNK, but also the ERK MAPK pathways decreased expression of p19. These results suggest that ATF-2 and SMAD-3 are transcription factors, which are, in addition to NF-kappaB, essential for IL-23 p19 expression.

Human alveolar macrophage gene responses to Mycobacterium tuberculosis strains H37Ra and H37Rv.

H37Rv and H37Ra have been widely used as models of virulent and avirulent strains, respectively, of Mycobacterium tuberculosis. Since the sequencing of H37Rv, microarrays have been used to investigate gene expression of M. tuberculosis strains under various conditions, and to compare gene expression of specific isolates of the organism. Because differences in the virulence of these organisms could also be manifest via their differential induction of host genes, we used Affymetrix Human Genome Arrays U133A and U133B to evaluate human alveolar macrophage (AM) responses to infection with H37Rv and H37Ra. H37Rv altered expression of far more genes than did H37Ra. Moreover, the genes induced by H37Rv to a greater extent than by H37Ra were predominantly associated with the development of effective immunity. H37Rv markedly increased expression of IL-23 p19, whereas neither organism significantly induced IL-12 p35 expression. Quantitative PCR confirmed that H37Rv induced significantly more AM p19 expression than did H37Ra. After low-level infection of both AM and peripheral blood monocytes (MN) with H37Rv, neither cell type produced IL-12 (by ELISA). In contrast, AM displayed significant IL-23 production in response to H37Rv, whereas MN did not. Our findings thus suggest an important role for IL-23 in human host responses to pulmonary infection with M. tuberculosis, and are consistent with epidemiologic and genetic studies that imply that H37Rv may not have unusual capacity to cause human disease.

P68 RNA Helicase (DDX5) Alters Activity of Cis- and Trans-Acting Factors of the Alternative Splicing of H-Ras

BackgroundH-Ras pre-mRNA undergoes an alternative splicing process to render two proteins, namely p21 H-Ras and p19 H-Ras, due to either the exclusion or inclusion of the alternative intron D exon (IDX), respectively. p68 RNA helicase (p68) is known to reduce IDX inclusion.Principal FindingsHere we show that p68 unwinds the stem-loop IDX-rasISS1 structure and prevents binding of hnRNP H to IDX-rasISS1. We also found that p68 alters the dynamic localization of SC35, a splicing factor that promotes IDX inclusion. The knockdown of hnRNP A1, FUS/TLS and hnRNP H resulted in upregulation of the expression of the gene encoding the SC35-binding protein, SFRS2IP. Finally, FUS/TLS was observed to upregulate p19 expression and to stimulate IDX inclusion, and in vivo RNAi-mediated depletion of hnRNP H decreased p19 H-Ras abundance.SignificanceTaken together, p68 is shown to be an essential player in the regulation of H-Ras expression as well as in a vital transduction signal pathway tied to cell proliferation and many cancer processes.

P311 functions in an alternative pathway of lipid accumulation that is induced by retinoic acid

Lipid droplets are complex and dynamic intracellular organelles that have an essential role in cholesterol and lipid homeostasis, and profoundly affect cellular structure and function. Variations in lipid-droplet composition exist between different cell types, but whether there are differences in the mechanisms of lipid-droplet accumulation remains to be elucidated. Here, we report that P311, previously identified to have a function in neuronal regeneration and a potential role in distal lung generation, regulates lipid droplet accumulation. P311 upregulates several classes of genes associated with lipid synthesis, significantly increases intracellular cholesterol and triglyceride levels, and increases intracellular lipid droplets. Interestingly, P311 expression is not necessary for lipogenesis in the well-established NIH3T3-L1 cell model of adipogenic differentiation. Instead, we demonstrate a novel role for P311 in an alternative pathway of lipid-droplet accumulation that is induced by the regeneration-inducing molecule retinoic acid.

Clinical Evaluation of Proinflammatory Cytokine Inhibitors (sTNFR I, sTNFR II, IL‐1 ra), Anti‐inflammatory Cytokines (IL‐10, IL‐13) and Activation of Neutrophils after Burn‐induced Inflammation

The study was aimed at evaluating the involvement of sTNFR I, sTNFR II, IL-1 ra, IL-10, IL-13 and reactive oxygen species (ROS) in systemic inflammatory response syndrome (SIRS) development in severely burned children and at assessing the prognostic value of the immunological markers studied. The study comprised 37 patients (17 burned children and 20 controls). Serum levels of the markers determined by means of ELISA and respiratory burst of neutrophils as well as p55 and p75 tumour necrosis factor-alpha (TNF-alpha) receptor expression using flow cytometry were evaluated twice. The burned children presented significantly higher levels of IL-10 and cytokine inhibitors within the first 6-24 h after injury compared with controls (P < 0.05). The decreased oxygen metabolism of neutrophils and increased TNF-alpha receptor expression were found on admission. Moreover, a significant decrease in initially high sTNFR I, sTNFR II, IL-1 ra, IL-10, IL-13 concentrations (P < 0.05) and reduced expression of TNF-alpha receptors (P < 0.05) were observed after burn therapy, whereas ROS generation evidently augmented (P < 0.05). Four of our children who developed hypovolaemic shock revealed a significantly lower ROS generation and higher concentrations of soluble TNF-alpha receptors and IL-1 ra together with IL-10, IL-13 compared with children with good outcome (P < 0.05). Our results revealed the involvement of both ROS, soluble TNF-alpha receptors and IL-1 ra in the development of SIRS in burned children; their monitoring allows for an assessment of the systemic inflammatory reaction activity. The neutrophil BURSTTEST and IL-1 ra might have been clinically helpful markers of SIRS prognosis.

Expression of p75 chain of IL-2 receptor in the early immunological reconstitution after allogeneic bone marrow transplantation

Expression of p55 and p75 chains of IL-2 receptor (IL-2R) on the surface of both T and natural killer (NK) circulating lymphocytes was investigated in 14 paediatric patients given allogeneic bone marrow transplantation (BMT) from HLA-identical sibling or partially matched family donors. IL-2-induced proliferative and cytotoxic responses were also studied and all analysis was performed within 45 days from transplant. We found that, early after transplant, the percentage of p55+ and of p75+ peripheral blood lymphocytes (PBL) was not significantly different in patients who had received HLA-identical BMT (p55+ 8.04 +/- 4.87%; p75+ 28.27 +/- 18.85%) compared with healthy controls (p55+ 7.26 +/- 6.17%; p75+ 19.42 +/- 10.49%), while recipients of T cell-depleted marrow included a remarkably high percentage (76-90%) of p75+ PBL, which were mostly CD3- and co-expressed CD56 molecule. Comparable values of p55+ lymphocytes were observed in all patients and controls. However, in contrast to the other two groups, in recipients of T cell-depleted BMT the majority of these cells co-expressed p75 chain and CD56 antigen. IL-2-induced proliferation and lymphokine-activated killer (LAK) activity were detectable in all patients, and their values did not correlate with expression of p55 or p75 chains. Our data suggest that expansion of NK subsets expressing IL-2R chains after T cell-depleted BMT may be related to early reconstitution of natural immunity in the presence of allogeneic stimuli.

Interleukin-4 regulates the interleukin-2 receptors on human peripheral blood B lymphocytes

The high-affinity interleukin-2 (IL-2) receptor (IL-2R) consists of the non-covalent association of at least two subunits, p55 and p70-75, capable of binding IL-2 with low and intermediate affinity, respectively. We studied the effects of cytokines on the IL-2R expressed on human peripheral blood B lymphocytes using monoclonal antibodies specific for IL-2R p55 and IL-2R p70-75, by means of two-colour flow cytometric analysis. In freshly isolated peripheral blood B lymphocytes, the p55 subunit was expressed only in a small population (7.0% of CD20+ cells), whereas the p70-75 subunit was expressed in a large population (89.0% of CD20+ cells). Of the cytokines studied, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were involved in the regulation of IL-2R on B cells. After a 2-day incubation with IL-4, expression of IL-2R p55 was markedly induced, but expression of IL2-R p70-75 was profoundly suppressed in a dose-dependent manner. These abilities of IL-4 to promote IL-2R p55 expression and suppress IL-2R p70-75 expression were inhibited by the presence of IFN-gamma. Other cytokines, including IL-1, IL-2, IL-5, and IL-6, had little effect on the expression of these two subunits. These findings suggest that IL-4 is a cytokine modulating B cell response through the regulation of IL-2R.

In Vitro Differentiation of Dendritic Cells in the Presence of Prostaglandin E2 Alters the IL-12/IL-23 Balance and Promotes Differentiation of Th17 Cells

PGE2, an endogenous lipid mediator released in inflammatory conditions, affects both dendritic cell (DC) differentiation and maturation. Whereas the effect of PGE2 on fully differentiated DC was studied extensively, little is known about its effects on DC differentiation. In this study, we show that bone marrow-derived DC generated in the presence of PGE2 (DCp) acquire a proinflammatory profile; produce higher levels of proinflammatory cytokines/chemokines; express higher levels of MHC class II, costimulatory molecules, and TLRs; and exhibit increased activation of the NF-kappaB-signaling pathway. In addition, DCp exhibit a different IL-12/IL-23 profile than DC generated in the absence of PGE2. The low IL-12 and high IL-23 production in LPS-stimulated DCp is associated with the down-regulation of p35 and the up-regulation of p19 expression, respectively. In agreement with the DCp proinflammatory phenotype and especially with the altered IL-12/IL-23 balance which strongly favors IL-23, DCp also affect T cell differentiation. In contrast to DC which favor Th1 differentiation, DCp promote Th17 and inhibit Th1/Th2 differentiation, in vitro and in vivo. Previous in vivo studies indicated that PGE2 had a proinflammatory effect, especially in models of autoimmune diseases. Our results suggest that the proinflammatory effects of PGE2 could be mediated, at least partially, through effects on differentiating DC and subsequent alterations in CD4+ T cell differentiation, resulting in the preferential development of pathogenic autoimmune Th17 cells.