Reduced Expression Of IL-6 Research Articles

Abstract 5055: CC Chemokine Receptor 5 Signaling Suppresses Inflammation and Attenuates Adverse Remodeling Following Myocardial Infarction by Recruiting Regulatory T Cells

Myocardial infarction triggers an inflammatory reaction that is critically involved in the pathogenesis of cardiac remodeling. Optimal cardiac repair is dependent on regulatory mechanisms that suppress inflammation preventing excessive matrix degradation. Chemokines are rapidly induced in the healing infarct mediating recruitment of leukocyte subsets with distinct properties. CCR5, the receptor for the CC chemokines MIP-1 α , MIP-1 β and RANTES plays an important role in immunoinflammatory responses. We hypothesized that CCR5 signaling may regulate the inflammatory response following myocardial infarction playing a role in the development of cardiac remodeling. CCR5 and its ligands MIP-1 α and MIP-1 β were markedly induced in reperfused mouse infarcts. Flow cytometry demonstrated that 38.3%+3.75 of the mononuclear cells infiltrating the infarcted myocardium after 24h of reperfusion expressed CCR5. CCR5 −/− mice and WT animals showed comparable macrophage and neutrophil infiltration in the infarct; however, CCR5 absence was associated with marked upregulation of pro-inflammatory cytokine (IL-1 β , TNF- α and IL-6) and chemokine (MIP-2, MIP-1 α , - β and IP-10) mRNA expression in the infarcted myocardium. CCR5+ mononuclear cells isolated from WT infarcts had anti-inflammatory properties exhibiting higher expression of the inhibitory cytokine IL-10 than CCR5- cells. In addition, mononuclear cells isolated from CCR5 −/− infarcts had reduced IL-10 expression. Enhanced inflammation in the absence of CCR5 was associated with impaired recruitment of foxp3+ regulatory T cells (Tregs), a CD4+ lymphocyte subpopulation with suppressive properties (Tregs as a percentage of T cells: WT 13.1%+ 1.1 vs. KO 7.2%+ 1.1; p<0.05). In WT mice the CCR5+ subset of Tregs exhibited enhanced expression of IL-10 reflecting potent anti-inflammatory activity. Enhanced inflammation in CCR5 −/− infarcts was associated with increased MMP and decreased TIMP expression, resulting in accentuated cardiac dilation (LVEDV: WT 52.99+2.43 mm3 vs. KO 65.13+4.53 mm3; p=0.02) without affecting the size of the infarct. CCR5-mediated recruitment of Tregs may restrain post-infarction inflammation preventing excessive matrix degradation and attenuating adverse remodeling.

Inhibition of c-Src reduces IL-6 expression in vivo and in vitro

Inhibition of c-Src reduces IL-6 expression in vivo and in vitro

Pulsatile Pulmonary Perfusion During Cardiopulmonary Bypass Reduces the Pulmonary Inflammatory Response

Pulmonary dysfunction presumably linked to an inflammatory response is frequent after cardiac operations using cardiopulmonary bypass (CPB) and pulmonary hypoperfusion. We previously demonstrated that active perfusion of the lungs during CPB reduces ischemic lung injury. We now hypothesized that avoiding ischemia of the lungs during CPB by active pulmonary perfusion would decrease pulmonary inflammatory response. Pigs were randomized to a control group with CPB for 120 minutes, followed by 120 minutes of postbypass reperfusion, or to the study groups where animals underwent active pulmonary perfusion with pulsatile or nonpulsatile perfusion during CPB (n = 7 in each group). Activation of transcription factor activity (nuclear factor [NF]-kappaB and activating protein [AP]-1) was determined by electrophoretic mobility shift assay. Levels of proinflammatory protein expression (interleukin [IL]-1, IL-6, and tumor necrosis factor [TNF]-alpha) were quantified by enzyme-linked immunoabsorbent assay. Caspase-3 activity was measured using a fluorogenic assay. The activation of transcription factor AP-1 and NF-kappaB was reduced in the pulsatile pulmonary perfusion group. The caspase-3 activity and the expression of IL-1, IL-6, and TNF-alpha revealed a significant decrease in the pulsatile and nonpulsatile pulmonary perfusion groups. Animals of the pulsatile pulmonary perfusion group showed significantly reduced IL-6 expression and caspase-3 activity compared with the nonpulsatile pulmonary perfusion group. Active pulmonary perfusion reduces the inflammatory response and apoptosis in the lungs observed during conventional CPB. This effect is greatest when pulmonary perfusion is performed with pulsatility. The reduction in cytokine expression by pulsatile pulmonary perfusion might be mediated by AP-1 and NF-kappaB.

Short hairpin RNA-expressing oncolytic adenovirus-mediated inhibition of IL-8: effects on antiangiogenesis and tumor growth inhibition

RNA interference, due to its target specificity, may be highly effective as a novel therapeutic modality, but direct delivery of synthetic small interfering RNA still remains a major obstacle for this approach. To induce long-term expression and specific gene silencing, novel delivery vector system is also required. In this study, we have generated an efficient oncolytic adenovirus (Ad)-based short hairpin (shRNA) expression system (Ad-DeltaB7-U6shIL8) against IL-8, a potent proangiogenic factor. To demonstrate IL-8-specificity of this newly engineered Ad-based shRNA, we also manufactured replication-incompetent Ads (Ad-DeltaE1-CMVshIL8 and Ad-DeltaE1-U6shIL8) under the control of the cytomegalovirus (CMV) and U6 promoters, respectively. Ad-DeltaE1-U6shIL8 was highly effective in reducing IL-8 expression, and was much more effective in driving IL-8-specific shRNA than the CMV promoter-driven vector. The reduced IL-8 expression then translated into decreased angiogenesis in vitro as measured by migration, tube formation and rat aortic ring sprouting assays. In addition to its effect on endothelial cells, Ad-DeltaE1-U6shIL8 also effectively suppressed the migration and invasion of cancer cells. In vivo, intratumoral injection of Ad-DeltaB7-U6shIL8 significantly inhibited the growth of Hep3B and A549 human tumor xenografts. Histopathological analysis of Ad-DeltaB7-U6shIL8-treated tumors revealed an increase in apoptotic cells and a reduction in vessel density. Finally, Ad-DeltaB7-U6shIL8 was also shown to inhibit the growth of disseminated MDA-MB-231 breast cancer metastases. Taken together, these findings demonstrate the utility and antitumor effectiveness of oncolytic Ad expressing shRNA against IL-8.

455. Neutrophils Interact with Adenovirus Vectors Via the β2-Integrin CD11b

First generation adenoviruses constitute as one of the most common used vectors in gene therapy clinical trials. However, these recombinant vectors are limited by their abilities to provoke strong innate and adaptive responses in vivo. In addition, the administration of human serotypes of adenovirus to individuals previously exposed to the virus may trigger a more severe inflammatory response to the vector than their unexposed counterparts. Neutralizing antibodies are also problematic from these individuals which could blunt vector transduction entirely within hours. Previous work has demonstrated that chemical modification of adenovirus with polyethylene glycol (PEG) can protect the vectors from pre-existing and new adaptive immune responses by reducing non-specific protein-protein interaction. Here we have optimized the PEGylation process for first generation adenoviral vectors and evaluated vector transduction, vector persistence, toxicity and innate immune responses against virus. We demonstrate that increasing PEGylation of the vector ablates in vitro, but not in vivo gene delivery. In vivo biodistribution was the same despite slighty higher transduction levels for PEGylated versus non-PEGylated vectors. In addition, vector levels in the serum were up to 100-fold higher for PEGylated vectors relative to unmodified vectors 24 hour after injection. Comparison of innate immune responses using serum IL-6 levels between mice injected with PEGylated or unmodified adenovirus systemically demonstrated a reduction of up to 70% from PEG-modified viruses at 6 hour after injection. Furthermore, the integrated amount of IL-6 release from unmodified vectors was 10-fold higher than PEGylated viruses during the 48 hour period after injection. Consistent with the reduction in IL-6 levels in vivo, we found that PEGylation reduced IL-6 expression 70% in RAW264.7 macrophages when exposed to the viruses in vitro. These data suggest that macrophages may play an important role in the acute immune response to adenoviral vectors and that PEG-modified vectors may attenuate this inflammatory response by reducing macrophage activation. In summary, this work demonstrates that PEGylation of adenovirus ablates in vitro, but not in vivo transduction, increases vector persistence, and reduces innate immune responses.

Inhibition of ATP-Binding Cassette Transporter Downregulates Interleukin-1β-Mediated Autocrine Activation of Human Dermal Fibroblasts

Fibroblasts constitute an important source of cytokines during inflammatory processes in the skin. Interleukin-1 is a potent, pleiotropic cytokine that is induced in activated human dermal fibroblasts. Interleukin-1 further induces many inflammatory mediators, including the chemokine interleukin-8. As fibroblasts express both interleukin-1 and the interleukin-1 receptor complex, the cellular response may be enhanced by autocrine activation. Interleukin-1alpha and interleukin-1beta lack a signal peptide and are translocated at the plasma membrane using an alternative secretory pathway, which may involve ATP-binding cassette transporter proteins. We hypothesize that inhibition of this pathway prevents secretion of interleukin-1, thereby downregulating interleukin-1-dependent autocrine induction of interleukin-8. We used the ATP-binding cassette 1 transporter inhibitor glybenclamide, which has been previously shown to block interleukin-1beta secretion in human monocytes. Using enzyme-linked immunosorbent assay, we assessed the effect of glybenclamide on interleukin-8 production in human dermal fibroblasts. In interleukin-1beta-transfected human dermal fibroblasts, interleukin-8 was induced through an autocrine activity of interleukin-1beta. Glybenclamide disabled this activation loop and significantly reduced interleukin-8. In human dermal fibroblasts that were stimulated with tumor necrosis factor alpha to reach high interleukin-1 expression levels, glybenclamide similarly suppressed interleukin-8. In contrast, glybenclamide did not affect interleukin-8 production in cells stimulated with interleukin-1 only. Glybenclamide did not affect caspase-1 in fibroblasts, which was expressed as an inactive precursor form, irrespective of treatments with tumor necrosis factor alpha and/or glybenclamide. Using overexpressing, interleukin-1-transfected COS-1 cells, inhibition of interleukin-1alpha and interleukin-1beta secretion was directly demonstrated on Western blots. These results are consistent with glybenclamide preventing externalization of interleukin-1 and subsequent autocrine induction of interleukin-8 in human dermal fibroblasts. Acting through such a mechanism, ATP-binding cassette transporter inhibitors may downregulate inflammation locally.

Effects of pulse methylprednisolone on inflammatory mediators in peripheral blood, synovial fluid, and synovial membrane in rheumatoid arthritis.

To establish whether the clinical efficacy of pulse methylprednisolone (MP; 1,000 mg intravenously) is related to the modulation of proinflammatory cytokines within the peripheral blood, synovial membrane, or synovial fluid compartments. Eighteen patients with active rheumatoid arthritis (RA) were studied. Peripheral blood (11 patients) and knee synovial fluid (9 patients, 10 knees) were obtained before and at 4 and 24 hours after MP therapy. Interleukin-1beta (IL-1beta), IL-8, and tumor necrosis factor alpha (TNFalpha) were measured by enzyme-linked immunosorbent assay and biologic assays; prostaglandin E2 (PGE2) was measured by competitive radioimmunoassay. In 10 patients, arthroscopically directed synovial biopsies were obtained before and at 24 hours after treatment, at disease relapse (4 patients), and after retreatment (1 patient). Membranes were stained by immunohistochemical techniques with monoclonal antibodies against TNFalpha, IL-8, IL-1beta, and the IL-1 receptor antagonist protein (IL-1Ra). MP therapy was associated with a rapid (within 24 hours) and substantial decrease in the expression of TNFalpha in the lining and sublining regions of the synovial membrane, as well as substantial decreases in the levels of TNFalpha in serum and synovial fluid. There was also reduced IL-8 expression in the synovial lining, as well as reduced synovial fluid IL-8 levels. No effect on synovial membrane IL-1beta and IL-1Ra or synovial fluid IL-1beta and PGE2 was found. MP therapy rapidly reduces IL-8 and TNFalpha levels in the synovial compartment, with cytokine changes in the serum and synovial fluid reflecting the changes in the synovial membrane. Alterations in TNFalpha expression in the synovial membrane correlated with clinical response to, and subsequent relapse after, MP therapy.