Cytokine Genes In Cells Research Articles

DNA methyltransferase 3a limits the expression of interleukin-13 in T helper 2 cells and allergic airway inflammation

The inverse correlation between DNA methylation and lineage-specific gene expression during T helper cell development is well documented. However, the specific functions of the de novo methyltransferases Dnmt3a and Dnmt3b in cytokine gene regulation have not been defined. We demonstrate that the expression of Dnmt3a and Dnmt3b are induced to a greater extent in T helper 2 (Th2) cells than in T helper 1 cells during polarization. Using conditional mutant mice, we determined that Dnmt3a, but not Dnmt3b, regulated expression of T helper cell cytokine genes, with the Il13 gene most prominently affected. Dnmt3a deficiency was accompanied by decreases in DNA methylation and changes in the H3K27 acetylation/methylation status at the Il13 locus. Dnmt3a-dependent regulation of Il13 also occurred in vivo because Dnmt3a(fl/fl)Cd4cre mice exhibited increased lung inflammation in a murine asthma model, compared with littermate controls. Based on these observations, we conclude that Dnmt3a is required for controlling normal Il13 gene expression and functions as a rate-limiting factor to restrict T helper 2-mediated inflammation.

Reductive metabolism increases the proinflammatory activity of aldehyde phospholipids

The generation of oxidized phospholipids in lipoproteins has been linked to vascular inflammation in atherosclerotic lesions. Products of phospholipid oxidation increase endothelial activation; however, their effects on macrophages are poorly understood, and it is unclear whether these effects are regulated by the biochemical pathways that metabolize oxidized phospholipids. We found that incubation of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) with THP-1-derived macrophages upregulated the expression of cytokine genes, including granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-1β, IL-6, and IL-8. In these cells, reagent POVPC was either hydrolyzed to lyso-phosphatidylcholine (lyso-PC) or reduced to 1-palmitoyl-2-(5-hydroxy-valeroyl)-sn-glycero-3-phosphocholine (PHVPC). Treatment with the phospholipase A(2) (PLA(2)) inhibitor, pefabloc, decreased POVPC hydrolysis and increased PHVPC accumulation. Pefabloc also increased the induction of cytokine genes in POVPC-treated cells. In contrast, PHVPC accumulation and cytokine production were decreased upon treatment with the aldose reductase (AR) inhibitor, tolrestat. In comparison with POVPC, lyso-PC led to 2- to 3-fold greater and PHVPC 10- to 100-fold greater induction of cytokine genes. POVPC-induced cytokine gene induction was prevented in bone-marrow derived macrophages from AR-null mice. These results indicate that although hydrolysis is the major pathway of metabolism, reduction further increases the proinflammatory responses to POVPC. Thus, vascular inflammation in atherosclerotic lesions is likely to be regulated by metabolism of phospholipid aldehydes in macrophages.

The role of neuromedin U during inflammatory response in the common carp

In the current study, we cloned and characterized the neuromedin U (NMU) gene from the common carp Cyprinus carpio L., and identified its participation in immune responses in the teleost. Five isoforms of the preproNMU genes were generated by alternative splicing and isolated from carp. The longest form of the carp preproNMU1 (isoform 1) cDNA was composed of 803 bp, and contained an 18 bp 5′-UTR, a 212 bp 3′-UTR and a 573 bp open reading frame, which translates into a peptide comprising 190 amino acid (aa) residues. The remaining carp preproNMU isoforms were composed of 175 (preproNMU2), 158 (preproNMU3), 150 (preproNMU4) and 133 (preproNMU5) aa residues. Isoforms 1–3 contained four processing signals (KR or RR), while isoforms 4 and 5 contained only two processing signals. High homology was demonstrated among fish and other vertebral NMU at the biologically active C-terminal region (aa position 175–182). Carp preproNMU transcript variants were identified in various tissues, and the expression pattern has been shown to change depending on feeding status. Moreover, it was shown that the expression of preproNMU3 and preproNMU5 was increased following treatment with bacterial or viral mimics. Finally, we investigated the functional aspect of carp NMU using a synthetic NMU peptide. The peptide was found to increase the expression of inflammation-related cytokine genes in intestinal cells within 1 h of treatment. In addition, the activation of phagocytic cells was also stimulated by the NMU peptide. The discovery of NMU in carp allows for a further understanding of immune regulation by biologically active substances.

Association between Yersinia ruckeri infection, cytokine expression and survival in rainbow trout ( Oncorhynchus mykiss)

The immune response against bacterial pathogens has been widely studied in teleosts and it is evident that survival chances differ significantly within a host population. Identification of indicators for susceptibility and responsiveness will improve our understanding of this host–pathogen interaction. The present work shows that the transcripts of cytokine genes in blood cells sampled three days post-infection was significantly higher in fish which obtained a high bacteriemia and died at later time points when compared to both non-infected control fish and infected fish that survived the infection. Rainbow trout were infected by bath challenge in a bacterial suspension (LD 60 dose, 1.8 × 10 9 CFU/ml Yersinia ruckeri for 1 h) and subsequently transferred to individual aquaria for 30 days of observation. Blood samples were analyzed for presence of Y. ruckeri both by culture and quantitative RT real-time PCR (qRT-PCR) and transcript levels of 28 genes encoding molecules which are important in the immune response. The transcript levels of a number of central cytokines, chemokines and cytokine receptors (IL-1β, IL-6, IL-8, IL-10, TNF-α, IL-receptor II) were significantly increased in infected fish that died later. In addition, a significantly higher amount of Y. ruckeri was found in the blood of the fish that died when compared to survivors. The study indicates that highly susceptible trout obtain an early heavy septicemia infection, which elicits a high up-regulation of the transcript of pro-inflammatory cytokines. Thus, less susceptible fish are protected by other factors and contract merely a weak non-lethal infection eliciting no or a weak cytokine response.

Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

BackgroundArctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes.MethodsCell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction.ResultsAC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression.ConclusionAC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

Dual oxidase 2 and glutathione peroxidase gene expression are elevated in hyperimmunised sheep challenged with Haemonchus contortus

A sequential biopsy sampling method was used to investigate oxidant and antioxidant gene responses in resistant sheep challenged with Haemonchus contortus larvae or a sham saline challenge. The expression of key sheep oxidant and antioxidant producing genes were measured in sequential samples removed from the abomasums at days 0, 1, 2, 3, 5, 7 and 28 post challenge. Gene expression levels at each time point were compared to expression at day 0, and levels of the various genes were also correlated to other markers of infection including immune cell counts and cytokine gene expression. The early response to larval challenge infection in resistant animals was marked by a divergence of two groups of host oxidant producing genes: the dual oxidase group ( DUOX2/ DUOXA2) showing increases in expression to day 7, while members of the phagocytic NADPH oxidase (PHOX) group showed significant decreases in expression. The change in DUOX2 expression between days zero and seven, when host resistance to infection is mediated, was negatively correlated to final worm burden suggesting NADPH oxidase expression may play a role in parasite expulsion. Expression of the DUOX group oxidants was positively correlated to expression of the Th2 cytokine IL4. Changes in host antioxidant pathways between different members of the glutathione peroxidase family (intestinal and plasma GPX) and genes involved in glutathione metabolism were also observed. This first study of the putative roles of oxidant production by the dual oxidase group, antioxidant glutathione pathways, immune cell populations, and cytokine profiles, in the development of resistance to infection by hyperimmune sheep are discussed.

Potential Immunological Adjuvant of 'K'-Type CpG-Oligodeoxynucleotides Enhanced the Cell Proliferation and IL-6 mRNA Transcription in Canine B Cells

CpG oligodeoxynucleotides (CpG-ODNs) are ligands for toll-like receptor 9 (TLR9), signaling of which plays a role in innate immunity by inducing T helper 1 (TH1)-cell responses and pro-inflammatory cytokine production. The activation of TLR9 signaling is considered to be effective for the therapy of cancer, infectious diseases, and allergies and preclinical studies using CpG-ODNs have been performed in dogs and humans. In order to investigate the precise mechanisms responsible for the effect of CpG-ODNs in dogs, we examined their role in cell proliferation and cytokine gene expression in canine B cells. Canine B cells were collected by a magnetic cell isolation method using anti-CD21 antibody. Flow cytometric analysis for the intracellular CD79α revealed the purity of canine B cells to be as high as 90.2 ± 2.1%. Transcription of TLR2, TLR4, and TLR9 mRNA on canine CD21(+) cells was confirmed by reverse-transcript polymerase chain reaction (RT-PCR). CpG-ODNs induced dose-dependent proliferation of canine CD21(+) cells (P<0.05 compared with control-ODNs) detected by BrdU incorporation. Quantification of IL-6, IL-10, and IL-12p40 mRNA transcription on canine CD21(+) cells revealed that CpG-ODNs enhanced IL-6 mRNA transcription but not IL-10 and IL-12p40 mRNA transcription (P<0.05 compared with control-ODNs). These responses to CpG-ODNs in the canine B cells indicated that CpG-ODNs would be useful as an immunological adjuvant for vaccine in dogs.

Brevetoxin 2 alters expression of apoptotic, DNA damage, and cytokine genes in Jurkat cells

Brevetoxins are potent neurotoxins that exert their toxicity through activation of voltage-gated sodium channels. Exposure to brevetoxins cause severe respiratory inflammation in marine mammals and humans. Brevetoxin activation of voltage-gated sodium channels on immune cells can lead to several biological responses including cell proliferation, gene transcription, cytokine production and even apoptosis. Jurkat E6-1 T cells were treated with brevetoxin 2 for 4 hours at a dose previously shown to induce apoptosis and DNA damage. Changes in gene expression were then assessed via PCR arrays. Gene expression analysis revealed significant change in expression of 17 genes related to apoptosis, 21 genes related to DNA damage signaling, and 19 genes encoding common cytokines. The gene expression data supports the idea that brevetoxins trigger complex reactions involving both inflammation and cell death.

Infection-induced changes in expression of antibacterial and cytokine genes in the gill epithelial cells of Atlantic cod, Gadus morhua during incubation with bacterial pathogens

The molecular processes of immune responses in mucosal tissues, such as the gills, during infection with bacterial pathogens are poorly understood. In the present study, we analyzed the transcriptional profiles of selected antibacterial genes and cytokines in the gills of a cold-water fish, Atlantic cod, Gadus morhua following in vitro infection with bacterial pathogens, Vibrio anguillarum and atypical Aeromonas salmonicida using semi-quantitative RT-PCR. There was significant upregulation in the transcripts of the antibacterial genes: bactericidal permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme, transferrin, metallothionein, galectin and hepcidin at 3h post-incubation with the two pathogens. The expression of cathelicidin in the gills was significantly enhanced by A. salmonicida, but not by V. anguillarum. At 24h post-incubation, most of these genes were still significantly upregulated, although some genes returned to their basal expression levels. The transcription levels of cytokines such as interleukin (IL)-1beta, IL-8 and interferon (IFN)-gamma significantly increased at 3h post-incubation with the pathogens. IL-22 and CC-chemokine type 1 transcripts were enhanced by A. salmonicida, but not by V. anguillarum. There was down-regulation of expression in CC-chemokine type-2 and -3 by V. anguillarum, while the expression levels of IL-10 remained unchanged upon infection with either of the two bacterial pathogens. The early upregulation of antibacterial genes in the gills could signal the onset of the acute phase response following bacterial infection and the differential modulation of some cytokine genes could be related to host-pathogen interactions that trigger immune response cascades in mucosal tissues of the host.

In vitro Synthesis of a Recombinant Fungal Immunomodulatory Protein from Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (W.Curt.:Fr.) P.Karst. (Aphyllophoromycetideae) and Analysis of Its Immunomodulatory Activity

The FIP-glu (Lingzhi-8 or LZ-8) isolated from medicinal mushroom Ganoderma lucidum (Lingzhi or Reishi) was the first identified and characterized fungal immunomodulatory protein (FIP), and its biological functions have been explored extensively. On the basis of cloned LZ-8 gene sequence from the genomic DNA of G. lucidum, we expressed FIP-glu by the expression cassette vector pQE-30. The recombinant protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS). Finally, its bioactivity was examined by inducing expression of cytokine genes in mouse spleen cells. The results showed that recombinant FIP-glu protein could be expressed in E. coli successfully. The yield of recombinant FIP-glu accounted for 51.1% of the total protein of E. coli, while soluble recombinant FIP-glu accounted for 55.4% of the total soluble protein of E. coli. The purity of recombinant FIP-glu protein was more than 90% after purification. Analysis of RT-PCR demonstrated that the recombinant FIP-glu could enhance the transcription of interleukin (IL)-2, IL-3, IL-4, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-2 receptor (IL-2R) genes in mouse spleen cells.

Mapping of the lingual immune system reveals the presence of both regulatory and effector CD4+ T cells

Sublingual immunotherapy (SLIT) is safe and reduces both symptoms and medication requirements in patients with type I respiratory allergies. Nonetheless, immune mechanisms underlying SLIT need to be further documented. A detailed characterization of the lingual immune system was undertaken in mice, to investigate the presence of tolerogenic and pro-inflammatory mechanisms. Immune cells were characterized in lingual tissues from BALB/c mice using immunohistology and flow cytometry. Resident CD4(+) T cells were sorted and toll-like receptor (TLR) expression profiles as well as functional characterization were assessed by RT-PCR, T cell suppressive assays and cytokine gene expression, respectively. Eosinophils and mast cells were only detected in submucosal tissues. No NK, NK-T, gamma/delta, CD8(+) T cells, nor B-lymphocytes were detected. Potential antigen presenting cells include various subsets of dendritic cells (CD207(+) Langerhans cells, CD11b(+)CD11c(+) myeloid cells and 120G8(+) plasmacytoid DCs) together with F4/80(+) macrophages. Noteworthy, both CD103(-) and CD103(+) CD4(+) T cells expressing TLR2 and TLR4 receptors are present along the lamina propria, in vicinity of myeloid CD11b(+)CD11c(+/-) dendritic cells. Such resident lingual CD4(+) T lymphocytes comprise both suppressive T cells as well as cells with memory/effector functions (i.e. expressing IFN gamma, IL4, IL10 and IL17 genes following stimulation), irrespective of the presence of the mucosal addressing marker CD103. The sublingual route is pertinent to induce antigen-specific tolerance, due to (i) limited numbers of pro-inflammatory cells, rather located in submucosal tissues, (ii) co-localization of APCs and resident CD4(+) T cells with regulatory functions. Since the oral immune system can also elicit pro-inflammatory effector responses, the cytokine milieu in which allergens are presented by sublingual APCs needs to be controlled during immunotherapy (e.g. with adjuvants) in order to favour tolerance over inflammation.

Expression of cytokine genes in head kidney and spleen cells of Japanese flounder ( Paralichthys olivaceus) infected with Nocardia seriolae

Nocardiosis caused by Nocardia seriolae is an important disease affecting marine fish for which neither control nor preventive measures are available. In this study, we investigated cytokine gene expression in Japanese flounder ( Paralichthys olivaceus) infected with N. seriolae to understand the innate immune response. Japanese flounder were challenged with different concentrations of N. seriolae suspensions (0, 1, and 10 mg/L) by immersion for 10 min. Mortality was 75% and 95% in fish infected by 1 and 10 mg/L, respectively. The expression of cytokine genes (TNF-α, IL-1β, CC-chemokine) in head kidney and spleen cells to N. seriolae challenge was investigated 2, 24 h, 3 days, and 10 days post-challenge. TNF-α expression was significantly increased in spleen after 24 h in 1 mg/L group and in HK after 2 h in 10 mg/L group, but after 24 h and 3 days in 10 mg/L group and after 3 days in 1 mg/L group, it was significantly decreased. IL-1β expression was significantly up-regulated in spleen after 24 h in 1 mg/l group while in HK only after 2 h in 10 mg/L group before suddenly down-regulated significantly 24 h in 10 mg/L group. The expression of CC-chemokine gene in both spleen and HK was significantly up-regulated in 10 mg/L group 2 h post-challenge and down-regulated in HK after 24 h and after 10 days in 1 mg/L group in spleen, compared to the control group.

Targeting Gut T Cell Ca2+ Release-Activated Ca2+ Channels Inhibits T Cell Cytokine Production and T-Box Transcription Factor T-Bet in Inflammatory Bowel Disease

Prolonged Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels is crucial in activating the Ca(2+)-sensitive transcription factor NFAT, which is responsible for directing T cell proliferation and cytokine gene expression. To establish whether targeting CRAC might counteract intestinal inflammation, we evaluated the in vitro effect of a selective CRAC inhibitor on T cell cytokine production and T-bet expression by lamina propria mononuclear cells (LPMC) and biopsy specimens from inflammatory bowel disease (IBD) patients. The inhibitory activity of the CRAC blocker was investigated through patch-clamp experiments on rat basophilic leukemia cells and fluorometric imaging plate reader intracellular Ca(2+) assays using thapsigargin-stimulated Jurkat T cells and its detailed selectivity profile defined using a range of in vitro radioligand binding and functional assays. Anti-CD3/CD28-stimulated LPMC and biopsy specimens from 51 patients with IBD were cultured with a range of CRAC inhibitor concentrations (0.01-10 microM). IFN-gamma, IL-2, IL-8, and IL-17 were analyzed by ELISA. T-bet was determined by immunoblotting. We found that the CRAC blocker concentration-dependently inhibited CRAC current in rat basophilic leukemia cells and thapsigargin-induced Ca(2+) influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-gamma, IL-2, IL-17, but not IL-8, was observed in IBD LPMC and biopsy specimens treated with the CRAC inhibitor. In conclusion, we provide evidence that the suppression of CRAC channel function may dampen the increased T cell response in the inflamed gut, thus suggesting a promising role for CRAC inhibitor drugs in the therapeutic management of patients with IBD.

Determining the role of Ikaros in Th17 differentiation (46.18)

Abstract CD4+ Th17 T cells contribute to the pathogenesis of a number of autoimmune/inflammatory diseases. Yet the molecular events that govern their development are still poorly understood. A reciprocal relationship between Th17 and T regulatory (Tregs) cells exists such that the relative expression of cell-specific transcription factors, RORγt and Foxp3, dictates whether CD4+ T cells differentiate into Th17 or Tregs cells, respectively. We have defined a role for Ikaros in these cell fate decisions. Ikaros is a site-specific DNA-binding transcription factor involved in the epigenetic regulation of several T cell cytokine genes. We demonstrate that Ikarosnull CD4+ T cells are developmentally impaired in vitro under Th17- skewing conditions. In addition to producing less IL-17, these cells exhibit reduced RORγt and higher Foxp3 expression than their wild type counterparts. Furthermore, all Foxp3+ T cells are also CD25+, suggesting that the lack of Ikaros predisposes Th17 T cells to a regulatory T cell phenotype. The effect of Ikaros on IL-17 production is not limited to CD4+ T cells. Ikarosnull CD8+ T cells also have dramatically lower IL-17 production. However, unlike Ikaros-deficient CD4 T cells, Ikarosnull CD8+ T cells do not express markers of Treg cells but show increased IFNγ production. Thus, our data suggest Ikaros is intimately involved in the differentiation of IL-17-producing T cells. This research is supported by NIH RO1 NS047578

Effects of Salmonella enterica serovar Enteritidis on cellular recruitment and cytokine gene expression in caecum of vaccinated chickens

Although vaccination of poultry is a suitable method to limit human food borne gastroenteritis caused by Salmonella (S.), the immune mechanisms responsible for a longer lasting protection against Salmonella infection in birds are not completely understood. To reveal unique protection-related immune parameters, day-old chicks were vaccinated with a commercial live S. Enteritidis vaccine and challenged with wild-type S. Enteritidis 147N at day 56 of life. The bacterial cell count was determined in gut and liver, while the immune cell composition and cytokine gene expression patterns were analysed by immunohistochemistry and quantitative real-time RT-PCR in caecum samples. The presented data suggest that the vaccine-elicited immune protection against the Salmonella wild-type infection was rather related to the bacterial count in gut mucosa and liver than to the colonisation in gut lumen. The higher number of Salmonella wild-type organisms found in caecal wall and liver of the non-immunised compared to immunised birds after challenge correlated with a more pronounced gene expression rate for IL-8, LITAF, iNOS, IL-12 and IFN-gamma. In contrast, immunised birds exhibited higher amounts of CD8(+) T cells as well as IgA than the non-immunised chickens after S. Enteritidis 147N infection in caecum. The results demonstrated a distinctive immune reaction pattern of previously vaccinated compared to non-vaccinated chickens upon S. Enteritidis wild-type challenge.

Hematopoietic Stem Cells Reduce Postischemic Inflammation and Ameliorate Ischemic Brain Injury

Systemic injection of hematopoietic stem cells after ischemic cardiac or neural lesions is one approach to promote tissue repair. However, mechanisms of possible protective or reparative effects are poorly understood. In this study we analyzed the effect of lineage-negative bone marrow-derived hematopoietic stem and precursor cells (Lin(-)-HSCs) on ischemic brain injury in mice. Lin(-)-HSCs were injected intravenously at 24 hours after onset of a 45-minute transient cerebral ischemia. Effects of Lin(-)-HSCs injection on infarct size, apoptotic cell death, postischemic inflammation and cytokine gene transcription were analyzed. Green fluorescent protein (GFP)-marked Lin(-)-HSCs were detected at 24 hours after injection in the spleen and later in ischemic brain parenchyma, expressing microglial but no neural marker proteins. Tissue injury assessment showed significantly smaller infarct volumes and less apoptotic neuronal cell death in peri-infarct areas of Lin(-)-HSC-treated animals. Analysis of immune cell infiltration in ischemic hemispheres revealed a reduction of invading T cells and macrophages in treated mice. Moreover, Lin(-)-HSC therapy counter-regulated proinflammatory cytokine and chemokine receptor gene transcription within the spleen. Our data demonstrate that systemically applied Lin(-)-HSCs reduce cerebral postischemic inflammation, attenuate peripheral immune activation and mediate neuroprotection after ischemic stroke.

Increased transcription of cytokine genes in human lung epithelial cells through activation of a TRPM8 variant by cold temperatures

Recognition of temperature is a critical element of sensory perception and allows mammals to evaluate both their external environment and internal status. The respiratory epithelium is constantly exposed to the external environment, and prolonged inhalation of cold air is detrimental to human airways. However, the mechanisms responsible for adverse effects elicited by cold air on the human airways are poorly understood. Transient receptor potential melastatin family member 8 (TRPM8) is a well-established cold- and menthol-sensing cation channel. We recently discovered a functional cold- and menthol-sensing variant of the TRPM8 ion channel in human lung epithelial cells. The present study explores the hypothesis that this TRPM8 variant mediates airway cell inflammatory responses elicited by cold air/temperatures. Here, we show that activation of the TRPM8 variant in human lung epithelial cells leads to increased expression of several cytokine and chemokine genes, including IL-1alpha, -1beta, -4, -6, -8, and -13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-alpha. Our results provide new insights into mechanisms that potentially control airway inflammation due to inhalation of cold air and suggest a possible role for the TRPM8 variant in the pathophysiology of asthma.

Hyaluronan Facilitates Transforming Growth Factor-β1-mediated Fibroblast Proliferation

This study aims to understand the role of the matrix polysaccharide hyaluronan (HA) in influencing fibroblast proliferation and thereby affecting wound healing outcomes. To determine mechanisms that underlie scarred versus scar-free healing, patient-matched dermal and oral mucosal fibroblasts were used as models of scarring and non-scarring fibroblast phenotypes. Specifically, differences in HA generation between these distinct fibroblast populations have been examined and related to differences in transforming growth factor-beta(1) (TGF-beta(1))-dependent proliferative responses and Smad signaling. There was a differential growth response to TGF-beta(1), with it inducing proliferation in dermal fibroblasts but an anti-proliferative response in oral fibroblasts. Both responses were Smad3-dependent. Furthermore, the two fibroblast populations also demonstrated differences in their HA regulation, with dermal fibroblasts generating increased levels of HA, compared with oral fibroblasts. Inhibition of HA synthesis in dermal fibroblasts was shown to abrogate the TGF-beta(1)-mediated induction of proliferation. Inhibition of HA synthesis also led to an attenuation of Smad3 signaling in dermal fibroblasts. Microarray analysis demonstrated no difference in the genes involved in TGF-beta(1) signaling between dermal and oral fibroblasts, whereas there was a distinct difference in the pattern of genes involved in HA regulation. In conclusion, these two distinct fibroblast populations demonstrate a differential proliferative response to TGF-beta(1), which is associated with differences in HA generation. TGF-beta(1) regulates proliferation through Smad3 signaling in both fibroblast populations; however, it is the levels of HA generated by the cells that influence the outcome of this response.

Contribution of mesothelial cells in the expression of inflammatory-related factors in omental adipose tissue of obese subjects

The objective of this study was to determine the contribution of mesothelial cells, present in human omental adipose tissue (OAT) but not in the subcutaneous depot (SAT), on the expression of inflammation-related factors. Comparison of the expression profiles of inflammation-related genes in mesothelial cells with those in the adipocyte-enriched (AEF) and stromal vascular fractions (SVF) and localization of interleukin-18 (IL-18) expression in adipose depots. Eleven obese Caucasian female subjects undergoing gastric bypass surgery (body mass index: 43.6+/-1.3 kg/m(2); age: 41.6+/-2.3 years). The expression profiles of cytokine and chemokine-related genes in mesothelial cells and in cell fractions prepared from OAT were assessed by the microarray technique. The differential expression of IL-18 was confirmed by real-time PCR and the protein was localized in adipose depots by immunohistochemistry. Microarray data analysis demonstrated that, of the 16 cytokine and chemokine-related genes that were upregulated in mesothelial cells compared with the AEF, IL-18 was the cytokine with the highest differential expression. IL-18 expression was similar in mesothelial cells and the SVF. In both SAT and OAT, IL-18 was immunolocalized in neutrophils and mast cells, but not in macrophages nor adipocytes. This cytokine was also detected in mesothelial cells in OAT. This additional source of expression may explain the higher IL-18 expression levels in OAT than SAT (+5.9-fold). By their capacity to express inflammatory-related factors, and in particular the proinflammatory cytokine IL-18 in OAT, mesothelial cells appear as a new player in the process of low-grade inflammation associated with obesity.

The Inositol 5′-Phosphatase SHIP-2 Negatively Regulates IgE-Induced Mast Cell Degranulation and Cytokine Production

Aggregation of the high-affinity IgE receptor (FcepsilonRI) on mast cells initiates signaling pathways leading to degranulation and cytokine release. It has been reported that SHIP-1 negatively regulates FcepsilonRI-triggered pathways but it is unknown whether its homologous protein SHIP-2 has the same function. We have used a lentiviral-based RNA interference technique to obtain SHIP-2 knockdown bone marrow-derived mast cells (BMMCs) and have found that elimination of SHIP-2 results in both increased mast cell degranulation and cytokine (IL-4 and IL-13) gene expression upon FcepsilonRI stimulation. Elimination of SHIP-2 from BMMCs has no effect on FcepsilonRI-triggered calcium flux, tyrosine phosphorylation of MAPKs or in actin depolymerization following activation. Rather, we observe that absence of SHIP-2 results in increased activation of the small GTPase Rac-1 and in enhanced microtubule polymerization upon FcepsilonRI engagement. Coimmunoprecipitation experiments in rat basophilic leukemia (RBL 2H3) cells show that SHIP-2 interacts with the FcepsilonRI beta-chain, Gab2 and Lyn and that unlike SHIP-1, it does not associate with SHC in mast cells. Our results report a negative regulatory role of SHIP-2 on mast cell activation that is calcium independent and distinct from the regulation by SHIP-1.