Showing papers in "Journal of Inflammation in 2005"

Mesenchymal stem cells avoid allogeneic rejection

Abstract: Adult bone marrow derived mesenchymal stem cells offer the potential to open a new frontier in medicine. Regenerative medicine aims to replace effete cells in a broad range of conditions associated with damaged cartilage, bone, muscle, tendon and ligament. However the normal process of immune rejection of mismatched allogeneic tissue would appear to prevent the realisation of such ambitions. In fact mesenchymal stem cells avoid allogeneic rejection in humans and in animal models. These finding are supported by in vitro co-culture studies. Three broad mechanisms contribute to this effect. Firstly, mesenchymal stem cells are hypoimmunogenic, often lacking MHC-II and costimulatory molecule expression. Secondly, these stem cells prevent T cell responses indirectly through modulation of dendritic cells and directly by disrupting NK as well as CD8+ and CD4+ T cell function. Thirdly, mesenchymal stem cells induce a suppressive local microenvironment through the production of prostaglandins and interleukin-10 as well as by the expression of indoleamine 2,3,-dioxygenase, which depletes the local milieu of tryptophan. Comparison is made to maternal tolerance of the fetal allograft, and contrasted with the immune evasion mechanisms of tumor cells. Mesenchymal stem cells are a highly regulated self-renewing population of cells with potent mechanisms to avoid allogeneic rejection.

Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response

Abstract: Bacterial and viral exacerbations play a crucial role in a variety of lung diseases including COPD or asthma. Since the lung epithelium is a major source of various inflammatory mediators that affect the immune response, we analyzed the inflammatory reaction of primary lung epithelial cells to different microbial molecules that are recognized by Toll-like receptors (TLR). The effects of TLR ligands on primary small airway epithelial cells were analyzed in detail with respect to cytokine, chemokine and matrix metalloproteinase secretion. In addition, the regulation of the expression of TLRs and their adaptor proteins in small airway epithelial cells was investigated. Our data demonstrate that poly(I:C), a synthetic analog of viral dsRNA, mediated the strongest proinflammatory effects among the tested ligands, including an increased secretion of IL-6, IL-8, TNF-α, GM-CSF, GRO-α, TARC, MCP-1, MIP-3α, RANTES, IFN-β, IP-10 and ITAC as well as an increased release of MMP-1, MMP-8, MMP-9, MMP-10 and MMP-13. Furthermore, our data show that poly(I:C) as well as type-1 and type-2 cytokines have a pronounced effect on the expression of TLRs and molecules involved in TLR signaling in small airway epithelial cells. Poly(I:C) induced an elevated expression of TLR1, TLR2 and TLR3 and increased the gene expression of the general TLR adaptor MyD88 and IRAK-2. Simultaneously, poly(I:C) decreased the expression of TLR5, TLR6 and TOLLIP. Poly(I:C), an analog of viral dsRNA and a TLR3 ligand, triggers a strong inflammatory response in small airway epithelial cells that is likely to contribute to viral exacerbations of pulmonary diseases like asthma or COPD. The pronounced effects of poly(I:C) on the expression of Toll-like receptors and molecules involved in TLR signaling is assumed to influence the immune response of the lung epithelium to viral and bacterial infections. Likewise, the regulation of TLR expression by type-1 and type-2 cytokines is important considering the impact of exogenous and endogenous TLR ligands on Th1 or Th2 driven pulmonary inflammations like COPD or asthma, respectively.

Suppression of neutrophil accumulation in mice by cutaneous application of geranium essential oil.

Abstract: Previous studies suggested that essential oils suppressed the adherence response of human neutrophils in vitro and that intraperitoneal application of geranium oil suppressed the neutrophil accumulation into peritoneal cavity in vivo. Usually, essential oils are applied through skin in aromatherapy in inflammatory symptoms. The purpose of this study is to assess the effects of cutaneous application of essential oils on the accumulation of neutrophils in inflammatory sites in skin of mice. Inflammation with accumulation of inflammatory cells was induced by injection of curdlan, a (1→3)-β-D-glucan in skin or peritoneal cavity of mice. Essential oils were applied cutaneously to the mice immediately and 3 hr after intradermal injection of curdlan. The skin with inflammatory lesion was cut off 6 hr after injection of curdlan, and the homogenates were used for myeloperoxidase (MPO: a marker enzyme of neutrophil granule) assay. The MPO activity of the skin lesion induced by curdlan was suppressed dose-dependently by cutaneous application of geranium oil. Other oils such as lavender, eucalyptus and tea tree oils also suppressed the activity, but their activities seemed weaker than geranium. Juniper oil didn't suppress the activity Cutaneous application of essential oils, especially geranium oil, can suppress the inflammatory symptoms with neutrophil accumulation and edema.

Differential signaling mechanisms regulate expression of CC chemokine receptor-2 during monocyte maturation

Abstract: Peripheral blood monocytes and monocyte-derived macrophages are key regulatory components in many chronic inflammatory pathologies of the vasculature including the formation of atherosclerotic lesions. However, the molecular and biochemical events underlying monocyte maturation are not fully understood. We have used freshly isolated human monocytes and the model human monocyte cell line, THP-1, to investigate changes in the expression of a panel of monocyte and macrophage markers during monocyte differentiation. We have examined these changes by RT-PCR and FACS analysis. Furthermore, we cloned the CCR2 promoter and analyzed specific changes in transcriptional activation of CCR2 during monocyte maturation. The CC chemokine receptor 2 (CCR2) is rapidly downregulated as monocytes move down the macrophage differentiation pathway while other related chemokine receptors are not. Using a variety of biochemical and transcriptional analyses in the human THP-1 monocyte model system, we show that both monocytes and THP-1 cells express high levels of CCR2, whereas THP-1 derived macrophages fail to express detectable CCR2 mRNA or protein. We further demonstrate that multiple signaling pathways activated by IFN-γ and M-CSF, or by protein kinase C and cytoplasmic calcium can mediate the downregulation of CCR2 but not CCR1. During monocyte-to-macrophage differentiation CCR2, but not CCR1, is downregulated and this regulation occurs at the level of transcription through upstream 5' regulatory elements.

Reversal of experimental colitis disease activity in mice following administration of an adenoviral IL-10 vector.

Abstract: Genetic deficiency in the expression of interleukin-10 (IL-10) is associated with the onset and progression of experimental inflammatory bowel disease (IBD). The clinical significance of IL-10 expression is supported by studies showing that immune-augmentation of IL-10 prevents inflammation and mucosal damage in animal models of colitis and in human colitis. Interleukin-10 (IL-10), an endogenous anti-inflammatory and immunomodulating cytokine, has been shown to prevent some inflammation and injury in animal and clinical studies, but the efficacy of IL-10 treatment remains unsatisfactory. We found that intra-peritoneal administration of adenoviral IL-10 to mice significantly reversed colitis induced by administration of 3% DSS (dextran sulfate), a common model of colitis. Adenoviral IL-10 (Ad-IL10) transfected mice developed high levels of IL-10 (394 +/- 136 pg/ml) within the peritoneal cavity where the adenovirus was expressed. Importantly, when given on day 4 (after the induction of colitis w/DSS), Ad-IL10 significantly reduced disease activity and weight loss and completely prevented histopathologic injury to the colon at day 10. Mechanistically, compared to Ad-null and DSS treated mice, Ad-IL10 and DSS-treated mice were able to suppress the expression of MAdCAM-1, an endothelial adhesion molecule associated with IBD. Our results suggest that Ad-IL10 (adenoviral IL-10) gene therapy of the intestine or peritoneum may be useful in the clinical treatment of IBD, since we demonstrated that this vector can reverse the course of an existing gut inflammation and markers of inflammation.

Proanthocyanidins, from Ribes nigrum leaves, reduce endothelial adhesion molecules ICAM-1 and VCAM-1

Abstract: Background The effects of proanthocyanidins (PACs), isolated from blackcurrant (Ribes nigrum L.) leaves, on neutrophil accumulation during inflammatory processes were investigated in vivo and in vitro.

Nurr1 dependent regulation of pro-inflammatory mediators in immortalised synovial fibroblasts.

Abstract: Background Nurr1 is an orphan member of the nuclear receptor superfamily; these orphan receptors are a group for which a ligand has yet to be identified. Nurr1 has been shown to regulate the expression of a small number of genes as a monomeric, constitutively active receptor. These Nurr1 regulated genes are primarily associated with dopamine cell maturation and survival. However, previous reports have shown an increased expression of Nurr1 in the synovium of patients with rheumatoid arthritis (RA) suggesting a pro-inflammatory role for Nurr1 in RA. In this study we investigate the potential pro-inflammatory role of Nurr1 by monitoring Nurr1 dependent gene expression in an immortalised synoviocyte cell line, K4IM.

Unaltered TNF-α production by macrophages and monocytes in diet-induced obesity in the rat

Abstract: Recent findings have established an association between obesity and immune dysfunction. However, most of the studies investigating the effects of obesity on immune function have been carried out in genetically obese rodent models. Since human obesity is mostly due to intake of a high fat diet and decreased energy expenditure, we asked whether immunological defects also occur in diet-induced obesity. Specifically, we focused on the function of monocytes and macrophages, as these cells are thought to be involved in the low-grade inflammation present in obesity. Male Sprague-Dawley rats were fed a high-fat or a standard chow diet for either 2 or 10 weeks. At the end of the intervention period animals were anaesthetised, blood collected for determination of plasma mediator concentrations and lipopolysaccharide (LPS) stimulated production of TNF-α by monocytes. LPS stimulated production of TNF-α in alveolar macrophages was also determined. High-fat feeding for either 2 or 10 weeks resulted in significant increases in fat mass and serum leptin. Although increased serum leptin has previously been linked to modulation of innate immunity, we found no significant difference in the LPS stimulated production of TNF-α by either blood monocytes or alveolar macrophages between the dietary groups. Furthermore, we failed to find a significant increase in circulating TNF-α concentrations in obese animals, as reported for genetically obese animals. Our data suggest that defects in innate immune function observed in genetically obese animals are not mimicked by dietary obesity, and may more likely reflect the gross abnormality in leptin function of these models. Further work is required delineate the effects of dietary obesity on inflammatory state and immune function.

CXCR2 is critical for dsRNA-induced lung injury: relevance to viral lung infection

Abstract: Respiratory viral infections are characterized by the infiltration of leukocytes, including activated neutrophils into the lung that can lead to sustained lung injury and potentially contribute to chronic lung disease. Specific mechanisms recruiting neutrophils to the lung during virus-induced lung inflammation and injury have not been fully elucidated. Since CXCL1 and CXCL2/3, acting through CXCR2, are potent neutrophil chemoattractants, we investigated their role in dsRNA-induced lung injury, where dsRNA (Poly IC) is a well-described synthetic agent mimicking acute viral infection. We used 6–8 week old female BALB/c mice to intratracheally inject either single-stranded (ssRNA) or double-stranded RNA (dsRNA) into the airways. The lungs were then harvested at designated timepoints to characterize the elicited chemokine response and resultant lung injury following dsRNA exposure as demonstrated qualititatively by histopathologic analysis, and quantitatively by FACS, protein, and mRNA analysis of BAL fluid and tissue samples. We then repeated the experiments by first pretreating mice with an anti-PMN or corresponding control antibody, and then subsequently pretreating a separate cohort of mice with an anti-CXCR2 or corresponding control antibody prior to dsRNA exposure. Intratracheal dsRNA led to significant increases in neutrophil infiltration and lung injury in BALB/c mice at 72 h following dsRNA, but not in response to ssRNA (Poly C; control) treatment. Expression of CXCR2 ligands and CXCR2 paralleled neutrophil recruitment to the lung. Neutrophil depletion studies significantly reduced neutrophil infiltration and lung injury in response to dsRNA when mice were pretreated with an anti-PMN monoclonal Ab. Furthermore, inhibition of CXCR2 ligands/CXCR2 interaction by pretreating dsRNA-exposed mice with an anti-CXCR2 neutralizing Ab also significantly attenuated neutrophil sequestration and lung injury. These findings demonstrate that CXC chemokine ligand/CXCR2 biological axis is critical during the pathogenesis of dsRNA-induced lung injury relevant to acute viral infections.

C-reactive protein does not opsonize early apoptotic human neutrophils, but binds only membrane-permeable late apoptotic cells and has no effect on their phagocytosis by macrophages

Abstract: It has been reported that C-reactive protein (CRP) binds both leukocyte FcγRIIA (CD32) and the plasma membrane of apoptotic cells. Since FcγRIIA becomes functionally enabled during neutrophil apoptosis, we sought to determine whether CRP bound to apoptotic neutrophils via FcγRIIA. We prepared directly labelled CRP and demonstrated that it was essentially free of IgG. We looked for evidence of CRP binding to intact, membrane impermeable apoptotic human neutrophils and to FcγRIIA-transfected Jurkat cells. We examined the functional consequences of incubation with CRP upon phagocytosis of apoptotic cells by human monocyte-derived macrophages. We could not detect binding of purified soluble CRP to classical early apoptotic human neutrophils or to FcγRIIA-transfected Jurkat cells. In contrast, membrane-permeable late apoptotic neutrophils exhibited strong CRP binding, which comprised both Ca2+-dependent and heparin-inhibitable Ca2+-independent components. However, there was no effect of CRP binding upon phagocytosis of late apoptotic neutrophils by macrophages. Potential apoptotic cell opsonins such as CRP may bind only to intracellular structures in cells with leaky membranes that have progressed to a late stage of apoptosis.

Comparative effects of the herbal constituent parthenolide (Feverfew) on lipopolysaccharide-induced inflammatory gene expression in murine spleen and liver

Abstract: Parthenolide, a major sesquiterpene lactone present in extracts of the herb Feverfew, has been investigated for its inhibitory effects on mediators of inflammation, including the proinflammatory cytokines. Although parthenolide's anti-inflammatory effects have been investigated in vitro, little in vivo data are available. Moreover, the molecular mechanisms for these inhibitory effects are not fully understood. The objective of this study was to test the hypothesis that parthenolide suppresses lipopolysaccharide (LPS)-induced serum (interleukin) IL-6, tumor necrosis factor (TNF)-α, IL-1β and cyclooxygenase (COX)-2 expression in mice as indicated by reduced splenic and liver mRNA levels. Mice were co-treated i.p. with LPS (1 mg/kg bw) and parthenolide (5 mg/kg bw) and blood, spleen and liver collected. Serum was analyzed for IL-6, TNF-α and IL-1β by ELISA. Total RNA was extracted from spleen and liver, and real-time RT-PCR was used to determine relative mRNA expression of IL-1β, IL-6, TNF-α and COX-2. LPS induced increases in serum IL-6 and TNF-α concentrations with only IL-6 being suppressed in parthenolide-treated mice. Induction of IL-6 mRNA was reduced, TNF-α and COX-2 mRNAs unchanged, and IL-1β mRNA increased in spleens of parthenolide plus LPS co-treated animals compared to LPS-only. No significant differences were observed in inflammatory gene expression between these two groups in liver samples. Overall, mRNA expression of each proinflammatory gene was much higher in spleen when compared to liver. In summary, only one gene, IL-6, was modestly suppressed by parthenolide co-exposure which contrasts with many in vitro studies suggesting anti-inflammatory effects of this compound. Also, LPS evoked greater effects in spleen than liver on expression of proinflammatory genes. Further study of the effects of parthenolide and other herbal constituents on inflammatory gene expression using model animal systems as described here are critical to evaluating efficacy of such supplements as well as elucidating their mechanisms of action.

Early relief of osteoarthritis symptoms with a natural mineral supplement and a herbomineral combination: A randomized controlled trial (ISRCTN38432711)

Abstract: This study was designed to determine if a natural mineral supplement, sierrasil, alone and in combination with a cat's claw extract (Uncaria guianensis), vincaria, has therapeutic potential in mild to moderate osteoarthritis of the knee. Patients (n = 107) with mild to moderate osteoarthritis of the knee were randomly assigned to one of 4 groups; high dose sierrasil (3 g/day), low dose sierrasil (2 g/day), low dose sierrasil (2 g/day) + cat's claw extract (100 mg/day) or placebo, administered for 8 weeks. Treatment was double blinded. Primary efficacy variables were WOMAC scores (A, B, C and total). Visual analog score (VAS) for pain, consumption of rescue medication (paracetamol), and tolerability were secondary variables. Safety measures included vital signs and laboratory-based assays. Ninety-one of the 107 patients successfully completed the protocol. All four groups showed improvement in WOMAC and VAS scores after 8 weeks (p < 0.001), in all 3 groups receiving sierrasil the magnitude of benefits were greater vs. placebo (WOMAC Total 38–43% vs. 27%) but this was not statistically significant. In reference to baseline values sierrasil treated groups had a considerably faster onset of benefits. Placebo-treated individuals failed to show significant benefits at 4 weeks (11% reduction in total WOMAC). In contrast, after 1 or 2 weeks of therapy all the sierrasil groups displayed significant reductions in WOMAC scores (p < 0.05) and at week 4 displayed a 38–43% improvement. VAS was significantly improved at 4 weeks in all groups (p < 0.001) but was significantly greater in all sierrasil groups compared to placebo (p < 0.05). Rescue medication use was 28-23% lower in the herbomineral combination and high dose sierrasil groups although not statistically different from placebo (P = 0.101 and P = 0.193, respectively). Tolerability was good for all groups, no serious adverse events were noted and safety parameters remained unchanged. The natural mineral supplement, sierrasil alone and in combination with a cat's claw extract, improved joint health and function within 1–2 weeks of treatment but significant benefits over placebo were not sustained, possibly due to rescue medication masking. Sierrasil may offer an alternative therapy in subjects with joint pain and dysfunction.

Heat shock protein and heat shock factor 1 expression and localization in vaccinia virus infected human monocyte derived macrophages

Abstract: Viruses remain one of the inducers of the stress response in the infected cells. Heat shock response induced by vaccinia virus (VV) infection was studied in vitro in human blood monocyte derived macrophages (MDMs) as blood cells usually constitute the primary site of the infection. Human blood monocytes were cultured for 12 – 14 days. The transcripts of heat shock factor 1 (HSF1), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90) and two viral genes (E3L and F17R) were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR), and the corresponding proteins measured by Western blot. Heat shock factor 1 DNA binding activities were estimated by electrophoretic mobility shift assay (EMSA) and its subcellular localization analyzed by immunocytofluorescence. It appeared that infection with vaccinia virus leads to activation of the heat shock factor 1. Activation of HSF1 causes increased synthesis of an inducible form of the HSP70 both at the mRNA and the protein level. Although HSP90 mRNA was enhanced in vaccinia virus infected cells, the HSP90 protein content remained unchanged. At the time of maximum vaccinia virus gene expression, an inhibitory effect of the infection on the heat shock protein and the heat shock factor 1 was most pronounced. Moreover, at the early phase of the infection translocation of HSP70 and HSP90 from the cytoplasm to the nucleus of the infected cells was observed. Preferential nuclear accumulation of HSP70, the major stress-inducible chaperone protein, suggests that VV employs this particular mechanism of cytoprotection to protect the infected cell rather than to help viral replication. The results taken together with our previuos data on monocytes or MDMs infected with VV or S. aureus strongly argue that VV employs multiple cellular antiapoptotic/cytoprotective mechanisms to prolong viability and proinflammatory activity of the cells of monocytic-macrophage lineage.

Regulation of IκBα expression involves both NF-κB and the MAP kinase signaling pathways

Abstract: IκBα is an inhibitor of the nuclear transcription factor NF-κB Binding of IκBα to NF-κB inactivates the transcriptional activity of NF-κB Expression of IκBα itself is regulated by NF-κB, which provides auto-regulation of this signaling pathway Here we present a mouse model for monitoring in vivo IκBα expression by imaging I κB α-luc transgenic mice for IκBα promoter driven luciferase activity We demonstrated a rapid and systemic induction of IκBα expression in the transgenic mice following treatment with LPS The induction was high in liver, spleen, lung and intestine and lower in the kidney, heart and brain The luciferase induction in the liver correlated with increased IκBα mRNA level Pre-treatment with proteasome inhibitor bortezomib dramatically suppressed LPS-induced luciferase activity The p38 kinase inhibitor SB203580 also showed moderate inhibition of LPS-induced luciferase activity Analysis of IκBα mRNA in the liver tissue showed a surprising increase of the IκBα mRNA after bortezomib and SB203580 treatments, which could be due to increased IκBα mRNA stability Our data demonstrate that regulation of IκBα expression involves both the NF-κB and the p38 signaling pathways The I κB α-luc transgenic mice are useful for analyzing IκBα expression and the NF-κB transcriptional activity in vivo

Dexamethasone inhibits IL-9 production by human T cells

Abstract: Interleukin 9 (IL-9) is produced by activated CD4+ T cells. Its effects include stimulation of mucus production, enhanced mast cell proliferation, enhanced eosinophil function, and IgE production. These effects are consistent with a role in allergic diseases. Glucocorticoids have potent anti-inflammatory effects, including suppression of cytokine synthesis, and are widely used in the treatment of allergic conditions. We examined the effect of the glucocorticoid dexamethasone (Dex) on IL-9 mRNA expression and protein secretion with real-time RT-PCR and ELISA. Peripheral blood mononuclear cells (PBMC) were prepared from human volunteers and activated with OKT3. CD4+ T cells were purified from PBMC and activated with OKT3 plus PMA. IL-9 mRNA abundance and protein secretion were both markedly reduced following treatment of activated PBMC with Dex. mRNA levels were reduced to 0.7% of control values and protein secretion was reduced to 2.8% of controls. In CD4+ T cells, Dex reduced protein secretion to a similar extent. The IC50 value of Dex on mRNA expression was 4 nM. These results indicate that IL-9 production is very markedly inhibited by Dex. The findings raise the possibility that the beneficial effects of glucocorticoids in the treatment of allergic diseases are in part mediated by inhibition of IL-9 production.

Colocalization of endogenous TNF with a functional intracellular splice form of human TNF receptor type 2

Abstract: Tumor necrosis factor (TNF) is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation, differentiation, and cell death. The biological effects of TNF are mediated via two cell surface TNF receptors: p55TNFR (TNFR1; CD120a) and p75TNFR (TNFR2; CD120b). Soluble forms of these two receptors consisting of the extracellular domains are proteolytically cleaved from the membrane and act as inhibitors. A novel p75TNFR isoform generated by the use of an additional transcriptional start site has been described and was termed hicp75TNFR. We focused on the characterization of this new isoform as this protein may be involved in chronic inflammatory processes. Cell lines were retroviraly transduced with hp75TNFR isoforms. Subcellular localization and colocalization studies with TNF were performed using fluorescence microscopy including exhaustive photon reassignment software, flow cytometry, and receptosome isolation by magnetic means. Biochemical properties of the hicp75TNFR were determined by affinity chromatography, ELISA, and western blot techniques. We describe the localization and activation of a differentially spliced and mainly intracellularly expressed isoform of human p75TNFR, termed hicp75TNFR. Expression studies with hicp75TNFR cDNA in different cell types showed the resulting protein mostly retained in the trans-Golgi network and in endosomes and colocalizes with endogenous TNF. Surface expressed hicp75TNFR behaves like hp75TNFR demonstrating susceptibility for TACE-induced shedding and NFκB activation after TNF binding. Our data demonstrate that intracellular hicp75TNFR is not accessible for exogenously provided TNF but colocalizes with endogenously produced TNF. These findings suggest a possible intracellular activation mechanism of hicp75TNFR by endogenous TNF. Subsequent NFκB activation might induce anti-apoptotic mechanisms to protect TNF-producing cells from cytotoxic effects of TNF. In addition, the intracellular and not TACE-accessible splice form of the hp75TNFR could serve as a pool of preformed, functional hp75TNFR.