Upregulation Of IL-6 mRNA Research Articles

MyD88 or TRAM Knockdown Regulates Interleukin (IL)‐6, IL‐8, and CXCL12 mRNA Expression in Human Gingival and Periodontal Ligament Fibroblasts

In a previous report, it was shown that Toll-like receptor (TLR) 2 knockdown modulates interleukin (IL)-6 and IL-8 but not the chemokine CXCL12, an important mediator with inflammatory and proangiogenic effects, in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). This study investigates whether knocking down two important TLR adaptor molecules, such as myeloid differentiation protein 88 (MyD88) and TRIF-related adaptor molecule (TRAM), could affect mRNA expression of IL-6, IL-8, and CXCL12 in HGF and HPDLF. After small interfering (si) RNA-mediated silencing of MyD88 or TRAM, HGF and HPDLF were stimulated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) or two synthetic ligands of TLR2 (Pam2CSK4 and Pam3CSK4) for 6 hours. IL-6, IL-8, and CXCL12 mRNAs were evaluated by quantitative polymerase chain reaction. Knockdown of MyD88 or TRAM partially impaired the IL-8 mRNA upregulation in both fibroblast subpopulations. Similarly, IL-6 upregulation was partially prevented by siMyD88 or siTRAM in HGF stimulated with Pg LPS, as well as in both fibroblast subtypes challenged with Pam2CSK4. Conversely, constitutive CXCL12 mRNA levels were upregulated by MyD88 or TRAM knockdown in non-stimulated cells. These results suggest that TLR adaptor molecules knockdown, such as MyD88 or TRAM, can decrease IL-6 and IL-8 mRNA and increase CXCL12 mRNA expression in HGF and HPDLF. This can be an important step for better understanding the mechanisms that control the inflammatory cytokine and chemokine expression, which in turn contributes to periodontal pathogenesis.

Human colonic epithelial cells detect and respond to C5a via apically expressed C5aR through the ERK pathway

Intestinal epithelial cells (IECs) exhibit numerous adaptations to maintain barrier function as well as play sentinel roles by expressing receptors for microbial products and antimicrobial peptides. The complement system is another important innate sensing and defense mechanism of the host against bacteria and increasing evidence shows that complement plays a role in colitis. The split component C5a is a potent proinflammatory molecule, and the C5a receptor (C5aR) CD88 has been reported on multiple cell types. Here, we examined the question of whether human colonic cell lines can detect activated complement via C5aR and what signaling pathway is critical in the subsequent responses. T84, HT29, and Caco2 cell lines all possessed mRNA and protein for C5aR and the decoy receptor C5L2. Polarized cells expressed the proteins on the apical cell membrane. C5a binding to the C5aR on human IECs activates the ERK pathway, which proved critical for a subsequent upregulation of IL-8 mRNA, increased permeability of monolayers, and enhanced proliferation of the cells. The fact that human IECs are capable of detecting complement activation in the lumen via this anaphylatoxin receptor highlights the potential for IECs to detect pathogens indirectly through complement activation and be primed to amplify the host response through heightened inflammatory mediator expression to further recruit immune cells.

IL-23-Mediated Epidermal Hyperplasia Is Dependent on IL-6

Psoriasis is a chronic inflammatory skin disease primarily driven by Th17 cells. IL-23 facilitates the differentiation and induces complete maturation of Th17 cells. Lesional psoriatic skin has increased levels of IL-23 and recent studies show that intradermal injections of IL-23 induce a psoriasis-like skin phenotype in mice. We have now characterized the IL-23-induced skin inflammation in mice at the molecular level and found a significant correlation with the gene expression profile from lesional psoriatic skin. As observed in psoriasis, the pathogenesis of the IL-23-induced skin inflammation in mice is driven by Th17 cells. We demonstrate a dramatic upregulation of IL-6 mRNA and protein after intradermal injections of IL-23 in mice. Using IL-6(-/-) mice we show that IL-6 is essential for development of the IL-23-elicited responses. Despite producing high levels of IL-22, IL-6(-/-) mice were unable to express the high-affinity IL-22 receptor chain and produced minimal IL-17A in response to intradermal injections of IL-23. In conclusion, we provide evidence for the critical role played by IL-6 in IL-23-induced skin inflammation and show that IL-6 is required for expression of IL-22R1A.

Abstract 402: The role of IL-6 in communication of pancreatic carcinoma cells with fibroblasts

Abstract <INTRODUCTION> Pancreatic cancer shows high mortality rates in gastroenterogical neoplasms, and there are difficulties in early diagnosis and aggressive progression. IL-6 has pleiotropic function and are produced by various immunnocompetent cells, as well as cancer cells. Some studies have been demonstrated IL-6 play an important role in evading host immune surveillance in tumor microenvironment, but interactions of fibroblasts has not been fully understood. Therefore, the aim of this study is to reveal role of fibroblasts in pancreatic tumor microenvironment. Especially, we evaluate induction and biologicial function of IL-6 in pancreatic carcinoma cells after the stimulation with various cytokines and fibroblast associated factors. <METHODS and RESULTS> Firstly, we examined IL-6 and its receptors expression by RT-PCR and Northern blotting. All 7 tested cell lines expressed gp130 and IL-6R alpha mRNA, 2 cell lines (Hs7667 and Capan1) expressed IL-6 mRNA in serum free condition. To clarify the effect of proinflammatory cytokines on IL-6 expression in Hs766T cells, cells were stimulated with or without cytokines. Northern blotting revealed TNFα and IL-1β upregulated IL-6 mRNA, but not IL-6, IL-8 and LIF. IL-6 did not affect cell proliferation by WTS assay in 7 pancreatic carcinoma cells. In contrast, IL-6 promoted cell motility significantly by gold particles phagocytosis assay (P<0.05) and cell invasion significantly by matrigel insert chamber assay (P<0.05). IL-6 induces phosphorylation of STAT3 in Hs-766T cells, but not AKT. To identify IL-6 expression by interaction between pancreatic carcinoma cells and fibroblasts, we used two established fibroblastic cell lines (MRC-9 and WI-38) isolated from human embryonal lung tissues. Serum free conditioned medium (CM) were collected after incubation for indicated periods. Hs766T originated CM (Hs766T-CM) induced IL-6 and IL-8 mRNA in MRC-9 and WI-38 cells. MRC-9 CM and WI-38-CM did not affect in Hs-766T cells. Co-culture between Hs-766T and MRC-9 cells induced IL-8 mRNA dratically. IL-6 promotes miR-146a and miR155 expression in Hs-766T cells. <CONCLUSIONS> Communication of pancreatic carcinoma cells with fibroblasts affect IL-6 expression and it could contribute to pancreatic cancer progression. Regulation of IL-6 expression in tumor microenvironment would be important for pancreatic cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 402. doi:10.1158/1538-7445.AM2011-402

Hypoxia‐Induced Pulmonary Hypertension: Possible Loss of Caveolin‐1 Function

We have previously shown an early and progressive IL-6 mRNA upregulation, IL-6 bioactivity, loss of endothelial caveolin-1 and reciprocal activation of PY-STAT3, and subsequent loss of HSP90 and eNOS in an inflammatory (monocrotaline, MCT) model of pulmonary hypertension (PH) in rats. Furthermore, rescue of caveolin-1 inhibits PY-STAT3 activation and attenuates MCT-induced PH. Since hypoxia also elicits an inflammatory response, we investigated time-dependent alterations in the expression of caveolin-1, PY-STAT3, eNOS and HSP90 during hypoxia-induced PH. Male Sprague-Dawley rats were subjected to hypobaric hypoxia and examined at 48h, 1 and 2 weeks of hypoxia. Significant PH was noted at 1 and 2 wks, and RVH at 2 wks of hypoxia. PY-STAT3 activation was observed at 48 hrs before the onset of PH, which was persistent, but there was no loss of caveolin-1 expression during hypoxia. Expression of eNOS was significantly increased during hypoxia; however the p-eNOS was decreased at 2 wks after a slight increase at 48h and 1 wk of hypoxia. HSP90 expression was unaltered. Caveolin-1 is known to inhibit PY-STAT3 activation and modulate eNOS activation. These results suggest that caveolin-1 dysfunction may play a significant role in hypoxia-induced PH.

Impact of CCR7 on the gastrointestinal field effect

Standardized intestinal manipulation (IM) leads to local bowel wall inflammation subsequently spreading over the entire gastrointestinal tract. Previously, we demonstrated that this so-called gastrointestinal field effect (FE) is immune mediated. This study aimed to investigate the role of CCR7 in IM-induced FE. Since CCR7 is expressed on activated dendritic cells and T cells and is well known to control their migration, we hypothesized that lack of CCR7 reduces or abolishes FE. Small bowel muscularis and colonic muscularis from CCR7(-/-) and wild-type (WT) mice were obtained after IM of the jejunum or sham operation. FE was analyzed by measuring gastrointestinal transit time of orally given fluorescent dextran (geometric center), colonic transit time, infiltration of MPO-positive cells, and circular smooth muscle contractility. Furthermore, mRNA levels of the inflammatory cytokine IL-6 were determined by RT-PCR. The number of dendritic cells and CD3+CD25+ T cells separately isolated from jejunum and colon was determined in mice after IM and sham operation. There was no significant difference in IL-6 mRNA upregulation in colonic muscularis between sham-operated WT and CCR7(-/-) mice after IM. Contractility of circular muscularis strips of the colon was significantly improved in CCR7(-/-) animals following IM and did not vary significantly from sham-operated animals. Additionally, inflammation of the colon determined by the number of MPO-positive cells and colonic transit time was significantly reduced in CCR7(-/-) mice. In contrast, jejunal contractility and jejunal inflammation of transgenic mice did not differ significantly from WT mice after IM. These data are supported by a significant increase of CD3+CD25+ T cells in the colonic muscularis of WT mice after IM, which could not be observed in CCR7(-/-) mice. These data demonstrate that CCR7 is required for FE and postoperative ileus. CCR7 indirectly affects FE by inhibiting migration of activated dendritic cells and of T cells from the jejunum to the colon. These findings support the critical role of the adaptive immune system in FE.

Rosiglitazone regulates IL-6-stimulated lipolysis in porcine adipocytes

Interleukin (IL)-6, a proinflammatory cytokine, stimulates adipocyte lipolysis and induces insulin resistance in obese and diabetic subjects. However, the effects of the anti-diabetic drug rosiglitazone on IL-6-stimulated lipolysis and the underlying molecular mechanism are largely unknown. In this study, we demonstrated that rosiglitazone suppressed IL-6-stimulated lipolysis in differentiated porcine adipocytes by inactivation of extracellular signal-related kinase (ERK). Meanwhile, rosiglitazone enhanced the lipolysis response of adipocytes to isoprenaline. In addition, rosiglitazone significantly reversed IL-6-induced down-regulation of several genes such as perilipin A, peroxisome proliferators activated receptor gamma (PPARγ), and fatty acid synthetase, as well as the up-regulation of IL-6 mRNA. However, mRNA expression of PPARγ coactivator-1 alpha (PCG-1α) was enhanced by rosiglitazone in IL-6-stimulated adipocytes. These results indicate that rosiglitazone suppresses IL-6-stimulated lipolysis in porcine adipocytes through multiple molecular mechanisms.

A Novel Isoform of Microphthalmia-Associated Transcription Factor Inhibits IL-8 Gene Expression in Human Cervical Stromal Cells

Cervical ripening during pregnancy is a profound change in cervix structure and function characterized by increases in the proinflammatory cytokine IL-8 and dissolution of the cervical extracellular matrix. Relatively little is known about the molecular mechanisms that underlie these events. Here, we report identification of a novel isoform of micropthalmia-associated transcription factor in human cervical stromal cells (MiTF-CX) that is down-regulated 12-fold during cervical ripening and that represses expression of IL-8. Ectopic expression of MiTF-CX in human cervical stromal cells resulted in substantial suppression of endogenous IL-8 mRNA and protein expression, whereas expression of dominant negative MiTF-CX mutants with impaired DNA binding resulted in dramatic increases in IL-8 production. Gel shift, reporter gene, and chromatin immunoprecipitation assays revealed one strong binding site (E-box (-397) CACATG(-391)) in the human IL-8 promoter that was crucial for mediating transcriptional repression by MiTF-CX. Moreover, we show that MiTF-CX expression in the cervix was itself positively autoregulated via two E-box motifs within a 2.1-kb promoter fragment. We therefore propose that maintenance of cervical competency during pregnancy is an active process maintained through suppression of IL-8 by the transcription factor MiTF-CX. During cervical ripening, loss of MiTF-CX would result in significant up-regulation of IL-8 mRNA and protein synthesis, thereby leading to recruitment and activation of leukocytes within the cervix and dissolution of the extracellular matrix.

Peptidoglycan enhances proinflammatory cytokine expression through the TLR2 receptor, MyD88, phosphatidylinositol 3-kinase/AKT and NF-kappaB pathways in BV-2 microglia

In this study, we investigated the signaling pathways involved in inflammatory production caused by peptidoglycan (PGN), a cell wall component of the gram-positive bacterium, in BV-2 microglia. PGN caused a concentration- and time-dependent increase in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein levels. In addition, PGN also induced IL-1β, TNF-α and IL-6 mRNA up-regulation in a concentration-dependent manner. Moreover, PGN also increased Toll-like receptor 2 (TLR2) expression in BV-2 microglia. Administration of TLR2 neutralizing antibody effectively inhibited PGN-induced iNOS and COX-2 expression. On the other hand, PGN-induced iNOS and COX-2 up-regulation were attenuated by PI3-kinase inhibitors (LY294002 and wortmannin), and an AKT inhibitor. Treatment of BV-2 microglia with PGN caused a time-dependent activation of PI3-kinase (p85) and AKT. PGN-induced PI3-kinase/AKT activation, iNOS and COX-2 expression were also inhibited by MyD88 inhibitory peptide. Treatment of cells with NF-κB inhibitor (pyrrolidine dithiocarbamate), IκBα phosphorylation inhibitor (Bay 117082), or IκB protease inhibitor (l-1-tosylamido-2-phenylethyl chloromethyl ketone) inhibited PGN-induced iNOS and COX-2 expression. Furthermore, stimulation of cells with PGN also activated IKKα/β, IκBα phosphorylation, IκBα degradation, p65 phosphorylation at Ser 536, and increased κB-luciferase activity. PGN-induced IKKα/β phosphorylation, IκBα phosphorylation, and IκBα degradation were further inhibited by pre-treatment with PI3-kinase inhibitors. Moreover, PGN-mediated increase of κB-luciferase activity was also inhibited by pre-transfection with dominant-negative mutants of p85, AKT, IKKα or IKKβ. Our data demonstrate that PGN-induced iNOS, COX-2 and proinflammatory cytokine expression was mediated through the TLR2/MyD88/PI3-kinase/AKT pathway, which in turn initiates IKKα/β and NF-κB activation in BV-2 microglia.

Stress activation of IL-6 neurons in the hypothalamus.

An emerging literature attests to the ability of psychological stress to alter the inflammatory cytokine environment of the body. While the ability of stress to cause cytokine release is well established, the neural pathways involved in this control have yet to be identified. This study tests the hypothesis that IL-6 neurons of the hypothalamo-neurohypophyseal system (HNS), a neural pathway proposed to secrete IL-6 into the circulation, are activated in response to psychological stress. Colocalization studies confirm robust expression of IL-6 in cell bodies and fibers of vasopressin (but not oxytocin) neurons of the paraventricular (PVN) and supraoptic nucleus (SON) of the rat hypothalamus. In response to restraint, there was a greater increase in c-Fos expression in SON IL-6-positive (IL-6+) neurons. In addition, both psychogenic (restraint) or systemic stress (hypoxia) lead to phosphorylated ERK induction only in IL-6+ magnocellular neurons, indicating selective activation of the MAPK signaling pathway in the IL-6 subset of magnocellular neurons. Finally, restraint upregulated IL-6 mRNA expression in both the PVN and SON, which was accompanied by a four-fold increase in circulating IL-6. The data indicate that noninflammatory stressors selectively activate IL-6 magnocellular neurons, upregulate IL-6 gene expression in the PVN and SON, and increase plasma IL-6. In summary, results show that IL-6 neurons of the HNS are a recruited component of the response to psychological stress.

Abstract 3181: Connecting EGFR activation to inflammatory signaling in epithelial ovarian cancer

Abstract Epithelial Ovarian Cancer (EOC) is the leading cause of death from gynecology cancer. EOC is initially characterized by intraperitoneal growth of tumor masses followed by an increased accumulation of ascitic fluid that contains a variable numbers of tumor cells and inflammatory leukocytes, as well as, variable levels of cytokines and chemokines. The receptor/ligand pair CXCR4/CXCL12 and the cytokines IL-1β, IL-6 and IL-8 have been found to contribute to cell proliferation of EOC. Despite our limited knowledge of initiating events of EOC, it is known that RAS oncogene mutation account for 30% of advanced EOC, activated H-RAS protein is commonly detected in EOC and activation of a receptor tyrosine kinase (RTK) could be the upstream mechanism initiating the cascade via the RAS/mitogen-activated protein kinases that lead to transcriptional activation. Indeed, in a homeostatic state EGFR, member of HER family RTK, plays a key role in normal ovarian follicle development and in the growth regulation of cells of ovarian surface epithelium (OSE) from which the EOC arises. EGFR is expressed in an estimated 35% to 70% of EOCs. Here, we wanted to assess in EOC whether the activation of the oncogene EGFR is related to an inflammation-associated signaling. Within normal and several transformed ovarian cell lines we selected IGROV-1 and OAW42 cells showing the highest EGFR expression and IL-6 release. Upon receptor activation with EGF, an up-regulation of PI3K/AKT and MEK/ERK pathways were observed. In both cell lines phosphorylation of EGFR occurred in the first 15 min of EGF treatment, followed by a concomitant phosphorylation of both AKT and MAPK. We also observed that the cited phosphorylations were completely inhibited by using the AG1478 EGFR inhibitor. In both cell lines EGF triggered NF-kB transcriptional activation as evaluated by transient transfection with a promoter-reporter gene construct. In the EOC selected cells, upon EGFR activation, an up-regulation of IL-6 mRNA was observed and immunofluorescence analysis revealed that NF-kB translocated into the nucleus. To identify the secretion of specific inflammatory molecules induced by EGFR/NFkB activation in EOC cells, a fifty-one cyto-/chemokine panel in conditioned media from EGF-treated cells have been analysed by Bioplex system. We have identified some inflammatory molecules released specifically upon EGFR activation in a time dependent way such as IL-6, PAI-1, IL-5, IL-12 and VEGF. On a limited number of ascitic fluids from EOC patients IL-6 and PAI-1 have been also analysed by ELISA assay, and the correlation with the EGFR expression on tumor cells of the same patients will be carried out. Overall the acquired information about the inflammation-related EGFR could lead to the identification of alternative approaches potentially suited for improving diagnosis and treatment of EOCs. Partially supported by Italian Health Ministry (Grant ACC1) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3181.

Human prostatic urethra expresses vitamin D receptor and responds to vitamin D receptor ligation

Chronic inflammation is now considered a determinant of benign prostatic hyperplasia (BPH), promoting, together with the hormonal milieu, prostate overgrowth and lower urinary tract symptoms (LUTS). Prostatic urethra actively participates in determining progression of LUTS associated with BPH. To investigate the expression of the vitamin D receptor (VDR) and the ability of the VDR agonist elocalcitol to reduce inflammatory responses in human prostatic urethra (hPU) cells. Human prostatic urethra, prostate and bladder neck were obtained from patients affected by BPH. Immunohistochemical studies for VDR expression were performed in tissue samples, from which primary cell cultures were also derived. In hPU cells, proliferation and chemiotaxis were studied, along with Rho kinase (ROCK) activity (MYPT-1 phosphorylation) by western blot. Quantitative RT-PCR was performed for VDR, cyclooxygenase (COX-2), and interleukin (IL)-8 expression. Urethra displays higher VDR expression compared to prostate and bladder neck tissues. The VDR agonist elocalcitol partially reverts COX-2 and IL-8 mRNA upregulation induced by a pro-inflammatory cytokine mixture (IL-17, interferon-γ, tumor necrosis factor-α) and inhibits cell migration in urethral cells. Elocalcitol prevents activation of ROCK, as previously demonstrated in bladder and prostate cell cultures. Our results suggest that prostatic urethra is, within the lower urinary tract, a novel target for VDR agonists, as shown by the capacity of elocalcitol to inhibit ROCK activity and to limit inflammatory responses in human primary urethra cells.

Inflammatory response underlies Pulmonary Hypertension

We have shown upregulation of IL‐6 mRNA, IL‐6 bioactivity and activation of PY‐STAT3, a proproliferative transcription factor in MCT‐induced pulmonary hypertension (PH), accompanied by a loss of endothelial caveolin‐1, a potent immunomodulator. To investigate the role of inflammation in hypoxia‐induced PH and compare with the MCT model, rats (¡Ö200 g) were subjected to hypobaric hypoxia or given MCT (sc, 60 mg/kg). Hemodynamic data, expression of caveolin‐1, eNOS, PY‐STAT3 were examined at 48h, 1 and 2 wks. Both models revealed progressive PH and right ventricular hypertrophy. Progressive PY‐STAT3 activation was observed at 48h, i.e. before the onset of PH (MCT, control [C], 100¡À0 vs 48h, 134¡À10*, hypoxia, C, 100¡À vs 48h, 453¡À70 *, *= P<0.05 vs C). Transient increase in eNOS expression at 1 wk was observed in both models. Unlike the MCT model, hypoxia‐induced PH did not exhibit loss of caveolin‐1. Caveolin‐1, known to inhibit PY‐STAT3 activation may be dysfunctional in the hypoxia model. In conclusion, dissimilar models of PH show activation of PY‐STAT3, indicating that inflammatory response may have a significant role in the pathogenesis of PH. (Supported in part by MF children¡ s Hosp. Res. Fund).

BS69 negatively regulates the canonical NF‐kappaB activation induced by Epstein‐Barr virus‐derived LMP1

Epstein‐Barr virus (EBV) latent membrane protein 1 (LMP1) activates NF‐kappaB signaling pathways through the two C‐terminal regions, CTAR1 and CTAR2. BS69 has previously been shown to be involved in LMP1‐induced c‐Jun N‐terminal kinase activation through CTAR2 by interacting with tumor necrosis factor (TNFR) receptor‐associated factor 6. In the present study, our experiments manipulating of BS69 expression clearly indicated that BS69 negatively regulated LMP1‐mediated NF‐kappaB activation including up‐regulated IL‐6 mRNA expression and IkappaB degradation. For the mechanisms, our immunoprecipitation experiments proposed that BS69 decreased complex formation between LMP1 and TNFR‐associated death domain protein (TRADD).

Hypothermia and Postconditioning after Cardiopulmonary Resuscitation Reduce Cardiac Dysfunction by Modulating Inflammation, Apoptosis and Remodeling

BackgroundMild therapeutic hypothermia following cardiac arrest is neuroprotective, but its effect on myocardial dysfunction that is a critical issue following resuscitation is not clear. This study sought to examine whether hypothermia and the combination of hypothermia and pharmacological postconditioning are cardioprotective in a model of cardiopulmonary resuscitation following acute myocardial ischemia.Methodology/Principal FindingsThirty pigs (28–34 kg) were subjected to cardiac arrest following left anterior descending coronary artery ischemia. After 7 minutes of ventricular fibrillation and 2 minutes of basic life support, advanced cardiac life support was started according to the current AHA guidelines. After successful return of spontaneous circulation (n = 21), coronary perfusion was reestablished after 60 minutes of occlusion, and animals were randomized to either normothermia at 38°C, hypothermia at 33°C or hypothermia at 33°C combined with sevoflurane (each group n = 7) for 24 hours. The effects on cardiac damage especially on inflammation, apoptosis, and remodeling were studied using cellular and molecular approaches. Five animals were sham operated. Animals treated with hypothermia had lower troponin T levels (p<0.01), reduced infarct size (34±7 versus 57±12%; p<0.05) and improved left ventricular function compared to normothermia (p<0.05). Hypothermia was associated with a reduction in: (i) immune cell infiltration, (ii) apoptosis, (iii) IL-1β and IL-6 mRNA up-regulation, and (iv) IL-1β protein expression (p<0.05). Moreover, decreased matrix metalloproteinase-9 activity was detected in the ischemic myocardium after treatment with mild hypothermia. Sevoflurane conferred additional protective effects although statistic significance was not reached.Conclusions/SignificanceHypothermia reduced myocardial damage and dysfunction after cardiopulmonary resuscitation possible via a reduced rate of apoptosis and pro-inflammatory cytokine expression.

Transcriptional regulation of IL-6 in bile duct epithelia by extracellular ATP

The inflammatory cytokine IL-6 is essential for cell survival after liver injury. Bile duct epithelia (BDE) markedly upregulate IL-6 release after liver injury, but the mechanisms regulating this have not been defined. Purinergic signals induce multiple potent downstream effects in BDE, so the goals of this study were to determine whether extracellular ATP regulates BDE IL-6 transcription and to identify the molecular mechanisms regulating this process. Effects of extracellular nucleotides on IL-6 transcription in primary rat bile duct epithelia were assessed. The relative effects of cAMP and cytosolic calcium were determined by use of agonists and antagonists. The role of the cAMP response element (CRE) was determined by site-directed mutagenesis. We found that ATP potently upregulated IL-6 mRNA, and that the pharmacological profile for IL-6 upregulation was most consistent with the newly identified P2Y11 receptor. This occurred in a cAMP-dependent and calcium-dependent fashion. The effect of cAMP and calcium agonists on IL-6 promoter activity was synergistic, and mutation of the IL-6 CRE blocked upregulation by ATP. Taken together, these data show that extracellular ATP acts through a mechanism involving a rat P2Y receptor functionally related to the P2Y11 receptor, cAMP, and calcium signals and that the IL-6 promoter CRE to upregulate transcription of IL-6 in BDE. Since IL-6 has such critical importance in the liver, it is likely that this pathway is of great relevance to the understanding of hepatic response to injury.

Skin-Infiltrating Monocytes/Macrophages Migrate to Draining Lymph Nodes and Produce IL-10 After Contact Sensitizer Exposure to UV-Irradiated Skin

Low-dose UVB exposure induces antigen-specific unresponsiveness to antigen(s) introduced through UV-irradiated skin (tolerance). Analysis of cytokine expression in murine draining lymph nodes (DLNs) revealed that IL-12p40 mRNA and protein expression as well as IL-12p70 protein were upregulated after application of the contact sensitizer 2,4 dinitro-1-fluorobenzene (DNFB) to normal skin. The cellular source of IL-12p40 mRNA was CD11c+ cells. By contrast, following DNFB application to UV-irradiated skin (UV+DNFB), IL-12p40 mRNA was not upregulated, and DLN IL-12p40 and p70 proteins were reduced. UVB irradiation alone did not upregulate IL-10 mRNA, but UV+DNFB upregulated IL-10 mRNA as early as 3-6 hours after DNFB application, immediately preceding a decrease of IL-12p40 mRNA from the level induced by UVB. The infiltration of F4/80+ cells into UV-irradiated skin was followed by a rapid and remarkable increase of F4/80+CD11c(-) cells in DLN 3 hours following DNFB application. FITC/DNFB skin painting and subsequent enzyme-linked immunospot assay demonstrated that flow-sorted FITC+F4/80+CD11c(-) cells from the DLN produce IL-10. Thus, monocytes/macrophages that infiltrated into the skin following UVB exposure migrate to the DLN triggered by contact sensitizers. Production of IL-10 by migrating macrophages, in conjunction with IL-12 inhibition in the DLN, likely reflects a role as mobile suppressive mediators for locally induced UV tolerance.

UVB Radiation-Mediated Inhibition of Contact Hypersensitivity Reactions Is Dependent on the Platelet-Activating Factor System

Through its ability to both induce immunosuppression and act as a carcinogen, UVB radiation plays a major role in cutaneous malignancies. Recent studies have indicated that UVB-mediated inhibition of delayed-type hypersensitivity reactions is mediated, in part, by the lipid mediator platelet-activating factor (PAF). The objective of this study was to further define the mechanism by which UVB inhibits contact hypersensitivity (CHS) reactions. UVB irradiation resulted in an inhibition of subsequent CHS to the chemical DNFB in wild-type, but not in PAF-R-deficient mice. UVB-mediated inhibition of CHS was also blocked by a cyclooxygenase-2 (COX-2) inhibitor or a neutralizing antibody directed against IL-10. UVB irradiation upregulated IL-10 mRNA levels in lymph nodes and spleen only to significant levels in PAF-R-expressing mice. Bone marrow transplantation studies demonstrated that UVB-mediated immunomodulatory effects were dependent on PAF-R-positive bone marrow. These studies suggest that UVB irradiation results in epidermal production of PAF agonists, which then act on PAF-R-positive bone marrow-derived cells to upregulate IL-10 through COX-2-generated prostaglandins.

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6

BackgroundMast cells play a key role in asthma and recent evidence indicates that their ongoing activation in this disease is mediated, in part, via IgE in the absence of antigen. In this study we have examined whether IgE alone enhances human lung mast cell (HLMC) survival.MethodsPurified HLMC were cultured for 4 weeks and survival assays then performed over 10 days following cytokine withdrawal in the presence or absence of human myeloma IgE. Quantitative real time RT-PCR was carried out to examine IL-6 mRNA expression and IL-6 protein was measured in HLMC supernatants by ELISA.ResultsIgE alone promoted the survival of HLMC in a dose-dependent manner following cytokine withdrawal. IgE-induced survival was eliminated with the addition of neutralising anti-IL-6 antibody but not by the addition of neutralising anti-stem cell factor. IgE sensitisation initiated profound upregulation of IL-6 mRNA in HLMC, and IL-6 concentrations were also raised in the culture supernatants of IgE-exposed cells.ConclusionThese data taken together suggest that IgE in the absence of antigen promotes HLMC survival through the autocrine production of IL-6. This provides a further mechanism through which IL-6 and IgE contribute to the pathogenesis of asthma, and through which anti-IgE therapy might achieve its therapeutic effect.

Up-Regulation of Interleukin-10 mRNA Expression in Peripheral Leukocytes Predicts Poor Outcome and Diminished Human Leukocyte Antigen-DR Expression on Monocytes in Septic Patients

None of the parameters can predict the deterioration of sepsis early enough. A new predictor allowing earlier detection of changes in septic patients' condition is indispensable. We examined whether cytokine-related mRNA expression in peripheral leukocytes, cytokine blood level, and monocyte human leukocyte antigen (HLA)-DR expression rate could be useful predictors of deterioration of sepsis. Twenty septic patients were enrolled. mRNA expression levels were quantified using real-time reverse transcriptase-polymerase chain reaction and assessed by percent changes during the first 24 h. Cytokine blood levels on admission and day 3 and monocyte HLA-DR expression rate on day 3 were also measured. Correlations between each of these parameters, and between these parameters and outcome were analyzed. Among seven kinds of cytokines studied, only IL-10 mRNA level showed significant difference between survivors and non-survivors (P < 0.05). Mortality rate of interleukin (IL)-10 mRNA level up-regulated group was significantly higher than that of IL-10 mRNA level down-regulated group (P < 0.05). HLA-DR expression rate in non-survivors were significantly lower than that in survivors (P < 0.05). Although IL-10 blood level on day 3 significantly correlated with HLA-DR expression on day 3(r = 0.54, P < 0.05), IL-10 blood level on admission did not correlate with it. Contrarily, the degree of up-regulation of IL-10 mRNA expression during the first 24 h significantly correlated with the degree of diminished HLA-DR expression on day 3 (r = 0.78, P < 0.001). Up-regulated IL-10 mRNA expression and diminished HLA-DR expression could be indicators of poor outcome. Furthermore, IL-10 mRNA expression measurement could predict the onset of immunoparalysis indicated by diminished HLA-DR expression earlier than IL-10 blood level measurement, leading to earlier commencement of the treatment.