Monocytic Cell Line U937 Research Articles

The role of NF-κB pathway in cancer inflammation of esophageal squamous carcinoma.

42 Background: Chronic inflammation plays an important role in tumorigenesis and tumor progression in human cancers. We aim to investigate the role of NF-kB in cancer inflammation of esophageal squamous cell carcinoma (ESCC). Methods: To generate M2-polarized macrophages, cells of human U937 monocyte cell line were treated with phorbol myristate acetate (PMA, 50 ng/ml) for 6 hours, and then cultured with PMA plus Th2 cytokines, IL-4 (20 ng/ml) and IL-13 (20 ng/ml), for another 18 hours. M2 phenotype was verified by flow cytometry and by cytokine profiling using enzyme-linked immunosorbent assay (ELISA). After co-culture with M2 macrophages, transcription nuclear factor-kB (NF-kB) activity was measured using quantitative polymerase chain reaction (Q-PCR), followed by reconfirmation with Western blot analysis for IkBα in KYSE-170 and -510 ESCC cell lines (kindly provided by Dr. Yutaka Shimada at Kyoto University, Japan). A selective inhibitor to NF-kB, Bay11-7082, was used to treat ESCC cell lines co-cultured with M2 macrophages, followed by cell proliferation, migration, invasion assays and vascular endothelial growth factor (VEGF) secretion by ELISA. The effect of Bay11-7082 (5 mg/kg) against growth of ESCC tumor was tested in xenografted tumors. Results: PMA plus Th2 cytokines treatment promoted differentiation of U937 cells into M2 macrophages. When treated with Bay11-7082, proliferation, migration, invasion and induction of VEGF expression was significantly inhibited in M2 macrophage co-cultured ESCC cells with a down-regulation of IkBα expression. Tumor growth was significantly increased in M2 macrophage co-cultured ESCC cells compared to that of the non-co-cultured controls, which was significantly retarded by treatment with Bay11-7082. Conclusions: NF-kB pathway was activated in ESCC cell lines co-cultured with M2 macrophages with an increase in cell proliferation, cell motility and angiogenic factor in vitro and tumor growth in vivo, which were significantly suppressed by a NF-kB inhibitor, Bay11-7082. These results suggest a role of M2 macrophage in promoting aggressiveness of ESCC cells, possibly through an activation of NF-kB pathway that may serve as a potential therapeutic target for ESCC.

Identification of aldo-keto reductases as NRF2-target marker genes in human cells

Transcription factor NF-E2-related factor 2 (NRF2) plays a crucial role in the cellular defense against oxidative/electrophilic stress by up-regulating multiple antioxidant genes. Numerous studies with genetically modified animals have demonstrated that Nrf2 is a sensitivity determining factor upon the exposure to environmental chemicals including carcinogens. Moreover, recent studies have demonstrated that polymorphism in the human NRF2 promoter is associated with higher risks for developing acute lung injury, gastric mucosal inflammation, and nephritis. Therefore, the identification of reliable and effective human target genes of NRF2 may allow the monitoring of NRF2 activity and to predict individual sensitivity to environmental stress-induced damage. For this purpose, we investigated genes that are tightly controlled by NRF2 to establish markers for NRF2 activity in human cells. Firstly, in the normal human renal epithelial HK-2 cells, the measurement of the expression of 30 previously reported NRF2 target genes in response to NRF2 inducers (sulforaphane, tert-butylhydroquinone, cinnamic aldehyde, and hydrogen peroxide) showed that the aldo-keto reductase (AKR) 1C1 is highly inducible by all treatments. Accordantly, the basal and inducible expressions of AKRs were significantly attenuated in NRF2-silenced HK-2 cells. Whereas, cells with stable KEAP1 knockdown, which causes a modest NRF2 activation, demonstrated substantially increased levels of AKR1A1, 1B1, 1B10, 1C1, 1C2, and 1C3. Secondly, the linkage between NRF2 and the AKRs was confirmed in human monocytic leukemia cell line U937, which can be a model of peripherally available blood cells. The treatment of U937 cells with NRF2 inducers including sulforaphane effectively elevated the expression of AKR1B1, 1B10, 1C1, 1C2, and 1C3. Whereas, the levels of both the basal and sulforaphane-inducible expression of AKR1C1 were significantly reduced in NRF2-silenced stable U937 cells compared to the control cells. Similarly, the inducible expression of AKR1C1 was observed in another human monocytic leukemia cell line THP-1 as well as in human primary blood CD14+ monocytes. In conclusion, together with the high inducibility and NRF2 dependency shown in renal epithelial cells as well as in peripherally available blood cells, current findings suggest that AKRs can be utilized as a marker of NRF2 activity in human cells.

TLR4 interaction induced activation of human monocyte cell line U937 by sickle patient�s mmLDL. Possible experimental model for atherosclerosis initial phase.

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Antioxidant Capacity of Flaxseed Products: The Effect of In vitro Digestion

This study evaluated the effect of in vitro digestion of flaxseed products on Folin-Ciocalteu reagent reducing substances (FCRRS), its antioxidant capacity and prevention of oxidative DNA damage in human monocyte cell line U937. Flaxseed protein isolate was obtained from defatted flaxseed meal and the protein hydrolysate with high antioxidant capacity was obtained from hydrolysis of the protein isolate with Alcalase in a two factor central composite rotatable design (pH 8.5 and enzyme: substrate 1:90, w/w). The FCRRS content and antioxidant capacity measured by FRAP and ORAC in aqueous and 70% methanol extracts were the highest in protein hydrolysate, followed by protein isolate, while the defatted meal showed the lowest values. After in vitro gastrointestinal digestion, the FCRRS content of protein isolate and hydrolysate reached similar values, however the hydrolysate had the highest antioxidant capacity, measured by FRAP while the isolate had the highest ORAC values. The defatted meal showed the lowest capacity in all assays (p < 0.05). The hydrolysate did not protect against DNA damage induced by H2O2 in U937 cells under the conditions of the present study. The results suggest that flaxseed protein isolate and hydrolysate are potential functional food ingredients with antioxidant capacity.

Activated Protein C Cytoprotective Signaling in Endothelial Cells Involves apoER2 and Disabled-1

AbstractAbstract 1102Activated protein C (APC), a plasma serine protease, exerts both anticoagulant and cytoprotective signaling in vitro and in vivo. APC protects cells from apoptosis, stress, and inflammation; it also can reduce or prevent endothelial barrier leakage as well as murine death from lethal levels of radiation. APC induces selective alterations of gene expression, provides neuroprotection following ischemic stroke, and minimizes damage in chronic neurodegenerative disease animal models. When given as bolus doses, APC reduces death in animals from severe sepsis. Receptors that mediate APC-induced beneficial signaling include endothelial cell protein C receptor (EPCR), PAR1, PAR3, integrins and Tie2. We discovered that apolipoprotein E receptor 2 (ApoER2) (aka LRP8), a lipoprotein receptor, which is well known for its effects in the Reelin-signaling pathway for neurons, has remarkable APC-dependent cytoprotective actions for the monocytic U937 cell line. In neurons, ligation of ApoER2 triggers phosphorylation of disabled-1 (Dab1) by Src family kinases that further promulgate signaling via PI3K, Akt and GSK3beta. Thus, here we report studies that test and support the hypothesis that ApoER2 mediates APC-induced cytoprotective signaling in EA.hy926 endothelial cells, which involves the adaptor protein, Dab1, as well as Src family members, Akt, and GSK3beta. We used siRNA and inhibitors to probe requirements for specific receptors for phosphorylation of key signaling pathway components. APC treatment of endothelial cells induced a 2 to 3-fold increase in phosphorylation of Akt at Ser473 and GSK3beta at Ser9 over 30 to 180 min after APC treatment, as well as a substantial rapid increase in ERK1/2 phosphorylation within 5 min of APC treatment. In purified systems, APC binds with good affinity to ApoER2. APC treatment of endothelial cells caused phosphorylation of Dab1 at Tyr232, a phosphorylation known to trigger Dab1-mediated signaling in other types of cells. Knocking down of ApoER2 using siRNA reduced GSK3beta and ERK1/2 phosphorylation by circa 30–40%. Combined knockdown of EPCR and ApoER2 reduced GSK3beta and ERK1/2 phosphorylation by 50–75%. Additional studies implicated involvement of PI3K and Src kinases. When cells were pretreated with PP2, a Src kinase inhibitor, Y232-Dab1 phosphorylation was reduced by > 50%, consistent with the primary paradigm for apoER2-mediated signaling involving phosphorylations of Dab1 and Src-family kinases. Moreover, the PAR1 antagonist SCH79797 markedly reduced APC-induced phosphorylation of Ser473-Akt, Ser9-GSK3beta and ERK1/2, showing that PAR1 was also required for the full extent of these effects. Hence, both PAR1 proteolysis by APC and ligation of ApoER2 by APC appear to contribute significantly and simultaneously to signaling in this endothelial model system. In summary, these data indicate that apoER2 is an endothelial cell receptor that contributes, with other better recognized receptors, to the spectrum of APC-induced signaling that is integrated by the endothelial cell system to achieve one or more of the multiple cytoprotective effects of APC. Thus, there is a remarkable ensemble of potential endothelial receptors for APC that could variably mediate a spectrum of APC’s cytoprotective effects. Disclosures:No relevant conflicts of interest to declare.

Protozoa and human macrophages infection by Legionella pneumophila environmental strains belonging to different serogroups

Three Legionella pneumophila strains isolated from municipal hot tap water during a multicentric Italian survey and belonging to serogroups 1, 6, 9 and the reference strain Philadelphia-1 were studied to determine the intracellular replication capability and the cytopathogenicity in human monocyte cell line U937 and in an Acanthamoeba polyphaga strain. Our results show that both serogroups 1 and Philadelphia-1 were able to multiply into macrophages inducing cytopathogenicity, while serogroup 6 and ever more serogroup 9 were less efficient in leading to death of the infected macrophages. Both serogroups 1 and 6 displayed a quite good capability of intracellular replication in A. polyphaga, although serogroup 1 was less cytopathogenic than serogroup 6. Serogroup 9, like Philadelphia-1 strain, showed a reduced efficiency of infection and replication and a low cytopathogenicity towards the protozoan. Our study suggests that bacterial pathogenesis is linked to the difference in the virulence expression of L. pneumophila serogroups in both hosts, as demonstrated by the fact that only L. pneumophila serogroup 1 shows the contextual expression of the two virulence traits. Serogroup 6 proves to be a good candidate as pathogen since it shows a good capacity for intracellular replication in protozoan.

Three‐dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

Podosomes, specialized actin-rich structures in macrophages (Mfs), degrade the extra-cellular matrix (ECM) and are involved in cell migration. On two-dimensional (2D) surfaces Mfs form spot-like podosomes at the ventral cell surface that develop into protrusive structures in a three-dimensional (3D) environment resembling the ECM. We have shown that the tips of these protrusive podosomes are characterized by increased accumulation of cysteine cathepsins (Cts) B, X, S, H, and L, both in human blood Mfs and in human monocytic cell line U-937. Monocyte-to-Mf differentiation induces an increase in cysteine cathepsin expression and activity, promoting their translocation to the cell surface, where they interact with ECM. This group of proteases is crucial for the extracellular as well as intracellular degradation of ECM, as demonstrated by quantitative monitoring of collagen IV degradation. Furthermore, inhibiting CtsB, X, and S significantly impairs Mf invasion through the 3D matrix. Time-lapse live-cell imaging of CtsB activity revealed that the extracellular and the intracellular ECM degradation are associated with extensive endocytosis at the tip of protrusive podosomes. The targeting of cysteine cathepsins, as the major mediators of human Mf 3D invasion, could be an approach to the treatment of inflammatory and cancerous diseases.

Corticosteroid insensitivity is reversed by formoterol via phosphoinositide‐3‐kinase inhibition

Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids, which has been linked to oxidative stress. Here we show that the long-acting β(2) -agonist formoterol (FM) reversed corticosteroid insensitivity under oxidative stress via inhibition of phosphoinositide-3-kinase (PI3K) signalling. Responsiveness to corticosteroids dexamethasone (Dex), budesonide (Bud) and fluticasone propionate (FP) was determined, as IC(50) values on TNF-α-induced interleukin 8 release, in U937 monocytic cell line treated with hydrogen peroxide (H(2) O(2) ) or peripheral blood mononuclear cells (PBMCs) from patients with COPD or severe asthma. PBMCs from severe asthma and COPD were less sensitive to Dex compared with those from healthy subjects. Both FM (10(-9) M) and salmeterol (SM, 10(-8) M) reversed Dex insensitivity in severe asthma, but only FM restored Dex sensitivity in COPD. Although H(2) O(2) exposure decreased steroid sensitivity in U937 cells, FM restored responsiveness to Bud and FP while the effects of SM were weaker. Additionally, FM, but not SM, partially inhibited H(2) O(2) -induced PI3Kδ-dependent (PKB) phosphorylation. H(2) O(2) decreased SM-induced cAMP production in U937 cells, but did not significantly affect the response to FM. The reduction of SM effects by H(2) O(2) was reversed by pretreatment with LY294002, a PI3K inhibitor, or IC87114, a PI3Kδ inhibitor. FM reversed oxidative stress-induced corticosteroid insensitivity and decreased β(2) adrenoceptor-dependent cAMP production via inhibition of PI3Kδ signalling. FM will be more effective than SM, when combined with corticosteroids, for the treatment of respiratory diseases under conditions of high oxidative stress, such as in COPD.

P62 The H2S-donor sodium hydrogen sulphide effectively attenuates TNF-α and IL-6 expression in the human monocyte cell line U937

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder, primarily affecting the articular structures and synovial membranes of multiple joints. Beside pharmacologically based treatments, sulphur bath therapy has long been used as a therapy for patients suffering from different rheumatic disorders. But scientific reports about the beneficial effects of H 2 S as well as about the underlying molecular mechanisms are controversial and rare. The human monocyte cell line U937 were differentiated with PMA (10 ng/ml) for 24 h and then treated for 30 min with the H 2 S-donor sodium hydrogen sulphide (NaHS) before being stimulated for 6 h with LPS (100 ng/ml). TNF-α and IL-6 release was quantified by enzyme-linked immunosorbent assay (ELISA). Modulation of the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2 were analysed by Western blotting. A significant reduction in TNF-α and IL-6 expression was already reached at 0.125 mM NaHS. The highest NaHS concentration (1.0 mM) reduced TNF-α and IL-6 production at about 40–50%. An almost completely suppression of IL-6 release was observed when the cells were treated 3 h after LPS exposure with the same dose of NaHS once again. Reduction in TNF-α and IL-6 expression levels were accompanied by deactivation of p38 and ERK1/2 MAPK. Data demonstrate that H 2 S is a gaseous molecule with potent anti-inflammatory properties. Therefore, sulphur bath therapy seems to be a valuable tool for the treatment of chronic inflammatory diseases such as RA and OA.

Inflammation‐mediated abrogation of androgen signaling: An in vitro model of prostate cell inflammation

As a link between inflammation and cancer has been reported in many studies, we established an in vitro model of prostatic inflammation to investigate the loss of androgen receptor (AR)-mediated signaling in androgen responsive prostate cell lines. First, the U937 monocyte cell line was differentiated into macrophages using phorbol acetate (PMA), and cells were induced with lipopolysaccharide (LPS) for cytokine secretion. Next, the cytokine levels (TNFα, IL-6, and IL1β) in conditioned media (CM) were analyzed. Prostate cells were then fed with CM containing varying concentrations of TNFα, and IkB degradation, nuclear factor kappa B (NFκB) translocation and transactivation, and the expression of matrix metalloproteinase-8 (MMP8) and matrix metalloproteinase-9 (MMP9) were then assessed. As a result of CM treatment, ubiquitin-mediated AR degradation, which was restored using anti-TNFα antibody neutralization, led to both a decrease in KLK4, PSA, and NKX3.1 expression levels and the upregulation of GPX2. In addition to the loss of AR, acute and chronic CM exposure resulted in p53 degradation and consequent p21 downregulation, which was also restored by either androgen administration or ectopic NKX3.1 expression via the stabilization of MDM2 levels in LNCaP cells. Additionally, CM treatment enhanced H2AX((S139)) phosphorylation (a hallmark of DNA damage) and genetic heterogeneity in the absence of androgens in prostate cells without activating mitochondrial apoptosis. Thus, the data suggest that inflammatory cytokine exposure results in the loss of AR and p53 signaling in prostate cells and facilitates genetic heterogeneity via ROS accumulation to promote prostate carcinogenesis.

Cellular uptake and reactive oxygen species modulation of cerium oxide nanoparticles in human monocyte cell line U937

Cerium oxide nanoparticles (nanoceria) are promising materials for intracellular oxygen free radical scavenging providing a potential therapy for reactive oxygen species (ROS)-mediated inflammatory processes. In this study rhombohedral-shaped nanoceria were synthesized by flame spray pyrolysis with tuneable particle diameters between 3 and 94 nm by changing the liquid precursor flow rate. Monocytes and macrophages are major players in inflammatory processes as their production of ROS species has important downstream effects on cell signalling. Therefore, this study examined the ability of the nanoceria to be internalised by the human monocytic cell line, U937, and scavenge intracellular ROS. U937 cells activated in the presence of phorbol 12-myristate 13-acetate (PMA) were found to be more responsive to the nanoceria than U937 cells, which may not be surprising given the role of monocyte/macrophages in phagocytosing foreign material. The smaller particles were found to contain more crystal lattice defects with which to scavenge ROS, however a greater proportion of both the U937 and activated U937 cell populations responded to the larger particles. Hence all nanoceria particle sizes examined in this study were equally effective in scavenging intracellular ROS.

Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway

The vitamin D receptor (VDR) mediates the pleiotropic biologic effects of 1α,25 dihydroxy-vitamin D3. Recent in vitro studies suggested that curcumin and polyunsaturated fatty acids (PUFAs) also bind to VDR with low affinity. As potential ligands for the VDR, we hypothesized that curcumin and PUFAs would induce expression of known VDR target genes in cells. In this study, we tested whether these compounds regulated two important VDR target genes — human cathelicidin antimicrobial peptide (CAMP) and 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) — in human monocytic cell line U937, colon cancer cell line HT-29 and keratinocyte cell line HaCaT. We demonstrated that PUFAs failed to induce CAMP or CYP24A1 mRNA expression in all three cell lines, but curcumin up-regulated CAMP mRNA and protein levels in U937 cells. Curcumin treatment induced CAMP promoter activity from a luciferase reporter construct lacking the VDR binding site and did not increase binding of the VDR to the CAMP promoter as determined by chromatin immunoprecipitation assays. These findings indicate that induction of CAMP by curcumin occurs through a vitamin D receptor-independent manner. We conclude that PUFAs and curcumin do not function as ligands for the VDR.

Rapamycin enhances dimethyl sulfoxide-mediated growth arrest in human myelogenous leukemia cells

Rapamycin and its derivatives have been proposed in the treatment of leukemia based on their cytostatic effects, but their possible role in differentiation therapy is less explored. The aim of the present study was to investigate the possible beneficial effects of the combination of rapamycin and dimethyl sulfoxide (DMSO) on growth arrest and differentiation of acute myelogenous leukemia (AML) cells. In myeloblastic HL-60, promyelocytic NB4, monocytic U937, immature KG-1 and erythro-megakaryocytic K562 cell lines, rapamycin alone had modest inhibitory effects, DMSO inhibited proliferation in a dose-dependent manner, and the combination of rapamycin and DMSO reduced the number of viable cells significantly more than either agent alone. In NB4 cells, rapamycin had no statistically significant effects on the DMSO-mediated increase in expression of CD11b, but increased apoptosis. These results demonstrate that rapamycin enhances DMSO-mediated growth arrest, and suggest that mTOR (mammalian target of rapamycin) inhibitors may have beneficial effects in differentiation therapy of AML.

Roles of promoter and 3′ untranslated motifs in expression of the human C5a receptor

The C5a receptor (C5aR) is a 7 transmembrane G-protein coupled receptor (GPCR) that mediates the powerful pro-inflammatory effect of the complement activation product C5a. Excess C5a generated under pathological conditions has been implicated in a variety of conditions including sepsis, asthma and rheumatoid arthritis, but very little is known about the regulation of expression of the C5aR. The 5′ promoter region and 3′ untranslated region (UTR) of the C5aR mRNA were cloned, generating enhanced green fluorescent protein (EGFP)-reporter plasmids, which were transfected into the monocytic cell line U937. Most of the cloned 2kb 5′ region was dispensable for the expression of the reporter constructs and the majority of regulatory sequences are in the first 200bp. Three motifs, a NFκB, a CCAAT and a NFAT site, were identified to be of importance by site directed mutagenesis for basal expression. Analysis of the 3′UTR of the C5aR mRNA showed that it contained two AU-rich elements (AREs), however site directed mutagenesis showed that these had no effect on basal expression. While the phorbol ester PMA and dibutyryl cAMP increased C5aR protein expression, these agents had no effect on the regulation of expression via the promoter or the 3′UTR. This is the first study to investigate the role of both the promoter and 3′UTR in regulating C5aR expression and our results show that regulation of the human C5aR is similar but not identical to that of the mouse C5aR.

Abstract 304: Proinflammatory secretory chemokine CXCL8 regulates intercellular cross-talk in the breast cancer tumor microenvironment

Abstract The cellular composition of the tumor microenvironment (TME) and soluble effector molecules mediating intercellular cross-talk are important therapeutic and preventive targets for breast cancer. Chemokines released within the TME work in a synergistic and reciprocal manner to induce malignant progression of tumor cells. CXCL8, a proinflammatory CXC chemokine shown to be released within the TME, can act as a chemoattractant, angiogenic factor, and inflammatory factor via cellular receptors CXCR1 and CXCR2. However, the specific role of CXCL8 in the signaling cascades within the tumor microenvironment has yet to be fully classified and warrants further investigation. Treatment of human breast cancer cell lines MCF-7, MDA-MB-231, and T47D with 50nM exogenous CXCL8 enhanced the expression of CXCR2. The acquisition of chemokine receptors by tumor cells, in this case CXCR2, can mediate phenotypic changes that enhance migratory and invasive potential. Treatment of these human breast cancer cell lines with 50nM exogenous CXCL8 resulted in phenotypic morphology changes characteristic of an epithelial-to-mesenchymal transition (EMT), a phenomenon commonly exhibited by metastatic tumor cells. We validated this phenomenon with 50nM exogenous CXCL8 treatment, which led to enhanced expression of both Slug (Snail2) and Twist in all three human breast cancer cell lines. Furthermore, we observed that treatment with 50nM exogenous CXCL8 promoted the migration and invasion of these breast cancer cell lines in an in vitro modified Boyden chamber assay. These results suggest that CXCL2 secreted from cells within the TME act to induce characteristic changes in human breast cancer cells, propagating the transition to more aggressive tumors. Tumor-associated macrophages (TAMs) are an important component of the heterogeneous population of cells that make up the TME. Breast cancer cells exposed to conditioned media from human monocytic cell lines THP-1 and U937, previously differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA), exhibited distinct morphology changes characteristic of EMT and enhanced migratory and invasive ability, similar to exogenous CXCL8 treatment. Together, our results suggest that CXCL8 can have profound implications in determining the fate of tumor cells by engaging in extensive cross-talk within the TME, thus providing a potential target for the treatment and prevention of breast cancer tumor progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 304. doi:1538-7445.AM2012-304

9‐cis‐Retinoic acid induces integrin‐independent immune cell adhesion

Retinoids are essential in the proper establishment and maintenance of immunity. Although retinoids are implicated in immune related processes, their role in immune cell adhesion has not been well established. In this study, we investigated the adhesive effects of 9‐cis‐retinoic acid (9‐cis‐RA) on a variety of human hematopoietic cell lines. 9‐cis‐RA treatment specifically induced immune cell adhesion of the human B‐cell line RPMI 8866, and the human monocytic cell line U937 to select immune ligands. Due to the prominent role of integrin receptors in mediating immune cell adhesion, we sought to evaluate if 9‐cis‐RA induced RPMI 8866 and U937 cell adhesion was integrin dependent. Intriguingly, 9‐cis‐RA mediated cellular adhesion was not affected by a variety of function blocking integrin inhibitors. In addition, the adhesion of RPMI 8866 cells could be inhibited by treatment with synthetic corticosteroids, suggesting a potential role in the inflammatory response. Finally, we demonstrate that 9‐cis‐ RA treatment of primary murine B‐cells also induces ex vivo adhesion. Our study is the first to demonstrate that 9‐cis‐retinoic acid prompts the cellular adhesion of immune cells, in vitro and ex vivo, through a yet unidentified immune receptor/complex whose expression or activation is induced upon 9‐cis‐RA exposure.

FSTL1 promotes arthritis in mice by enhancing inflammatory cytokine/chemokine expression

FSTL1 is a secreted glycoprotein that exacerbates murine arthritis and is overexpressed in human arthritis. The aim of this study was to determine the mechanism by which FSTL1 promotes arthritis. Collagen-induced arthritis was induced in mice hypomorphic for FSTL1, generated with a gene trap-targeted mutant embryonic stem cell line. Arthritis was assessed by measuring paw swelling and using a qualitative arthritis index. Bone marrow-derived mesenchymal stromal cells from hypomorphic mice, as well as mouse stromal ST2 cells transduced with short hairpin RNA to suppress FSTL1 expression, were stimulated with interleukin-1β (IL-1β), tumor necrosis factor α, and IL-17. The monocyte cell line U937, which does not express FSTL1, was transfected with a plasmid encoding FSTL1 and stimulated with phorbol myristate acetate and lipopolysaccharide. Cell supernatants were assayed for IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), and FSTL1 by enzyme-linked immunosorbent assay. FSTL1 hypomorphic mice had reduced levels of FSTL1 compared to littermate controls. Following induction of arthritis, a significant correlation was observed between serum FSTL1 levels and both paw swelling and the arthritis index. Similar correlations were observed between the amount of FSTL1 produced by mesenchymal stromal cells, stromal ST2 cells, and monocytes and the secretion of IL-6, IL-8, and MCP-1. These findings demonstrate that FSTL1 up-regulates proinflammatory mediators important in the pathology of arthritis, and that serum levels of FSTL1 correlate with severity of arthritis. The latter supports the possibility that FSTL1 might be a target for treatment of certain forms of arthritis.

Increased corticosteroid sensitivity by a long acting β2 agonist formoterol via β2 adrenoceptor independent protein phosphatase 2A activation

Long-acting β2-adrenoceptor agonists (LABAs) are reported to enhance anti-inflammatory effects of corticosteroids in vitro and in vivo, although the molecular mechanisms have not yet been elucidated. We investigated the role of serine/threonine protein phosphatase 2A (PP2A) on regulation of corticosteroid sensitivity via inhibition of glucocorticoid receptor (GR) phosphorylation as the target of formoterol, an LABA. Corticosteroid sensitivity was determined as IC50 to dexamethasone (Dex) on TNFα-induced IL-8 release in a U937 monocytic cell line (Dex-IC50). Phosphorylation levels of GR-Ser226 and c-Jun N-terminal kinase (JNK) were determined by western-blotting. Phosphatase activity of immunopurified PP2A was measured by fluorescence-based assay. Exposure to IL-2/IL-4 for 48 h decreased Dex sensitivity with a concomitant increase of GR phosphorylation at Ser226 with JNK1 activation. Formoterol restored Dex sensitivity by inhibiting phosphorylation of GR-Ser226 and JNK1. PP2A inhibition by okadaic acid, a phosphatase inhibitor, abrogated formoterol-mediated effects. In addition, formoterol enhanced PP2A activity in intact or IL-2/IL-4 treated U937 cells and human peripheral blood mononuclear cells. In addition, PP2A activation by formoterol was not antagonized by ICI-118551, and formoterol could activate PP2A directly in cell free system. Taken together, formoterol increases corticosteroid sensitivity via activation of PP2A in receptor independent manner, explaining its benefits as add-on therapy for the treatment of corticosteroid-insensitive diseases, such as severe asthma.

The anti-TNF-α antibody infliximab indirectly regulates PECAM-1 gene expression in two models of in vitro blood cell activation

Chronic inflammatory bowel diseases can be successfully treated with antibodies against the acute phase mediator TNF-α. The process of activation and of extravasation of inflammatory cells from the blood into the ‘stressed' tissue site is controlled by cytokines and chemokines, which attract leukocytes and by adhesion molecules, which mediate their attachment and transmigration toward the affected cell(s). The changes in the gene expression of adhesion molecules taking place in those cells before attachment have been less investigated. Changes of PECAM-1, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) gene expression were studied in phytohaemagglutinin (PHA)- and lipolysaccharide (LPS)-treated human peripheral blood leukocytes (PBLs), granulocytes and the human monocyte cell line U-937. Cells were treated either with PHA or with LPS in the presence or absence of infliximab and incubated with TNF-α, IFN-γ and/or transforming growth factor beta (TGF-β) and treated as above. Activation of PBLs by PHA or LPS treatment triggered a sharp upregulation of ICAM-1, VCAM-1 gene expression and a time-dependent downregulation of PECAM-1 gene expression reaching a minimum 4 h from start of the experiment. The anti-TNF-α antibody infliximab, by neutralizing TNF-α and IFN-γ production, completely reversed PECAM-1 mRNA downregulation and ICAM-1 and VCAM-1 upregulation. Immunostaining of PBLs cytospins with antibodies against PECAM-1 and ICAM-1 confirmed RT-PCR and western blot results. PBLs IFN-γ or TNF-α treatment downregulated PECAM-1 in parallel with the upregulation of ICAM-1 and VCAM-1 gene expression, whereas TGF-β upregulated PECAM-1- and downregulated ICAM-1 and VCAM-1 gene expression counteracting the effect of TNF-α or IFN-γ. Similar results were obtained in human U937 cells and in granulocyte cultures by TNF-α or IFN-γ treatment. Taken together, these results suggest that infliximab, blocking TNF-α and IFN-γ production, exerts its anti-inflammatory effect through inhibiting downregulation of PECAM-1 gene expression and upregulation of ICAM-1 and VCAM-1 expression in leukocytes of the peripheral blood. These results also suggest that TGF-β may thus be of therapeutic importance as an anti-inflammatory agent.

CD300a and CD300f differentially regulate the MyD88 and TRIF‐mediated TLR signalling pathways through activation of SHP‐1 and/or SHP‐2 in human monocytic cell lines

CD300a, a membrane protein expressed on myeloid lineages and specific subsets of CD4(+) T cells, has been reported to have inhibitory activities in cellular activation. However, the role of CD300a in Toll-like receptor (TLR) -mediated macrophage activation has not been investigated. The human monocytic cell lines THP-1 and U937 were stimulated with various TLR ligands after triggering of CD300a with specific monoclonal antibody. Interestingly, CD300a blocked TLR4-mediated and TLR9-mediated expression of pro-inflammatory mediators without affecting TLR3-mediated events. In contrast, CD300f, another member of the CD300 family, blocked the activation of cells induced by all TLR ligands. A transient transfection assay using luciferase reporter gene under the regulation of nuclear factor-κB binding sites indicated that co-transfection of CD300f blocked reporter expression induced by over-expression of both myeloid differentiation factor 88 (MyD88) and toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF), whereas CD300a blocked only MyD88-induced events. Synthetic peptides representing immunoreceptor tyrosine-based inhibitory motifs of CD300a or CD300f mimicked the differential inhibition patterns of their original molecules. The use of various signalling inhibitors and Western blotting analysis revealed that TLR9/MyD88-mediated signalling was regulated mainly by SH2-containing tyrosine phosphatase 1 (SHP-1), which could be activated by CD300a or CD300f. In contrast, regulation of the TLR3/TRIF-mediated pathway required the combined action of SHP-1 and SHP-2, which could be accomplished by CD300f but not CD300a. These data indicate that CD300a and CD300f regulate the MyD88 and TRIF-mediated TLR signalling pathways through differential activation of SHP-1 and SHP-2.