Poly(vinyl alcohol) acetoacetate-based tissue adhesives are non-cytotoxic and non-inflammatory

Polymer-based tissue adhesives composed of poly(vinyl alcohol) acetoacetate (PVOH acac) and cross-linking amines were investigated for their effects on cell survival and inflammatory cell activation using in vitro mouse cell cultures. Cytotoxicity of tissue adhesives was evaluated by placing adhesives in direct contact with 3T3 fibroblast cells. Tissue adhesives formulated from PVOH acac and 3-aminopropyltrialkoxysilane (APS) were non-cytotoxic to fibroblasts; adhesives formulated from PVOH acac and aminated poly(vinyl alcohol) (PVOH amine) were also non-cytotoxic to fibroblasts. In contrast, a commercial adhesive composed of 2-octyl cyanoacrylate was highly cytotoxic to fibroblasts. The inflammatory potential of tissue adhesives was evaluated by exposing J774 macrophage cells to adhesives, and measuring TNF-α release from macrophages. PVOH acac-based tissue adhesives did not elicit inflammatory TNF-α release from macrophages. These results suggest that PVOH acac-based tissue adhesives are non-cytotoxic and non-inflammatory. Such tissue adhesives represent a promising technology for a variety of medical applications, including surgical wound closure and tissue engineering, and the results are also significant in the design of in vitro cell culture systems to study biomaterials.     

Biological characterization of Sorona polymer from corn-derived 1,3-propanediol

The Sorona family of polymers, based on corn-derived 1,3-propanediol, have recently been developed as novel bio-based materials for use in plastics, films, and fibers. In the present study, Sorona polymers were investigated for their effects on cell survival and inflammatory cell activation using in vitro mouse cell cultures. Cytotoxicity of Sorona polymers was evaluated by placing material samples in direct contact with 3T3 fibroblast cells. Both Sorona plastic and Sorona films were non-cytotoxic to fibroblasts. The inflammatory potential of Sorona samples was evaluated by exposing J774 macrophage cells to material samples, and measuring TNF-α release from macrophages. Sorona plastic and Sorona films did not elicit inflammatory TNF-α release from macrophages. These results indicate that Sorona polymers are non-cytotoxic and non-inflammatory. While the 1,3-propanediol component of Sorona 3GT is manufactured in a bacterial fermentation process, the absence of an inflammatory response to Sorona film and Sorona plastic is highly encouraging. The results are significant for the design of materials that utilize bio-based polymers.     

GM-CSF-induced granulation tissue formation: relationships between macrophage and myofibroblast accumulation

We have studied the formation of granulation tissue around osmotic minipumps delivering granulocyte macrophage-colony stimulating factor (GM-CSF) chronologically in the rat using electron microscopy and immunohistochemistry at the light and electron microscopic levels, with specific antibodies against α-smooth muscle (SM) actin and rat macrophages. At 2 and 3 days after pump implantation, GM-CSF application produced an extensive inflammatory reaction characterized by edema and the accumulation of polymorphonuclear cells and macrophages. Gradually, polymorphonuclear cells decreased in number and macrophages became arranged in large clusters. The expression of α-SM actin in fibroblastic cells of the granulation tissue started from the 4th day after pump implantation and progressed up to the 7th day. Double immunofluorescence staining showed macrophage clusters in relation to α-SM actinrich fibroblastic cells. Electron microscopic examination confirmed that the fibroblasts containing α-SM actinpositive stress fibers were found initially in close proximity to clustered macrophages. The delivery of plateletderived growth factor (PDGF) and tumor necrosis factor-α (TNF-α) by the osmotic minipump induced an accumulation of macrophages, but in a much smaller number compared with those seen after GM-CSF application; these macrophages were never assembled in clusters and, furthermore, TNF-α and PDGF did not stimulate α-SM actin expression in fibroblastic cells. Our results suggest that after GM-CSF administration, the cluster-like accumulation of macrophages plays an important role in stimulating α-SM actin expression in myofibroblasts. Our results may be relevant to the understanding of the processes leading to granulation tissue formation in this and other experimental models.     

Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion

Mast cell (MC) activation in the rheumatoid lesion provides numerous mediators that contribute to inflammatory and degradative processes, especially at sites of cartilage erosion. MC activation in rheumatoid synovial tissue has often been associated with tumour necrosis factor (TNF)-α and interleukin (IL)-1β production by adjacent cell types. By contrast, our in situ and in vitro studies have shown that the production of IL-15 was independent of MC activation, and was not related to TNF-α and IL-1β expression. Primary cultures of dissociated rheumatoid synovial cells produced all three proinflammatory cytokines, with production of IL-1β exceeding that of TNF-α, which in turn exceeded that of IL-15. In vitro cultures of synovial macrophages, synovial fibroblasts and articular chondrocytes all produced detectable amounts of free IL-15, macrophages being the most effective.     

Strong inhibition of TNF-α production and inhibition of IL-8 and COX-2 mRNA expression in monocyte-derived macrophages by RWJ 67657, a p38 mitogen-activated protein kinase (MAPK) inhibitor

In inflammatory processes, the p38 mitogen-activated protein kinase (MAPK) signal transduction route regulates production and expression of cytokines and other inflammatory mediators. Tumor necrosis factor α (TNF-α) is a pivotal cytokine in rheumatoid arthritis and its production in macrophages is under control of the p38 MAPK route. Inhibition of the p38 MAPK route may inhibit production not only of TNF-α, but also of other inflammatory mediators produced by macrophages, and indirectly of inflammatory mediators by other cells induced by TNF-α stimulation. Here we investigate the effects of RWJ 67657, a p38 MAPK inhibitor, on mRNA expression and protein production of TNF-α and other inflammatory mediators, in monocyte-derived macrophages. A strong inhibition of TNF-α was seen at pharmacologically relevant concentrations of RWJ 67657, but also inhibition of mRNA expression of IL-1β, IL-8, and cyclooxygenase-2 was shown. Furthermore, it was shown that monocyte-derived macrophages have a high constitutive production of matrix metalloproteinase 9, which is not affected by p38 MAPK inhibition. The results presented here may have important implications for the treatment of rheumatoid arthritis.     

Dehydroepiandrosterone and melatonin prevent Bacillus anthracis lethal toxin-induced TNF production in macrophages

The lethal toxin of Bacillus anthracis, which is composed of two separate proteinaceous exotoxins, namely protective antigen and lethal factor, is central to the pathogenesis of anthrax. Low levels of this toxin are known to induce release of cytokines such as tumor necrosis factor α (TNF-α). In the present study we investigated the effect of dehydroepiandrosterone (DHEA), melatonin (MLT), or DHEA + MLT on production of lethal toxin-induced TNF-α in mouse peritoneal macrophages. We found that treatment with DHEA significantly inhibited the TNF-α production caused by anthrax lethal toxin. Exposure of MLT to anthrax lethal toxin-treated macrophages also decreased the release of TNF-α to the extracellular medium as compared to the control. However, combined use of DHEA and MLT also inhibited TNF-α release, but not more than single therapies. These results suggest that DHEA and MLT may have a therapeutic role in reducing the increased cytokine production induced by anthrax lethal toxin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytokine expression due to Helicobacter pylori in a tissue culture model

Helicobacter pylori, in recent years, has been recognized as the major causative agent in chronic gastritis and peptic ulcer disease in humans. H. pylori is a ubiquitous organism, with at least half of the world’s population infected. Of those individuals with peptic ulcer disease, it is estimated that 90% of cases are caused by H. pylori. Currently, the efficacy of therapies is starting to decline due to increasing resistance rates, especially towards clarithromycin. Due to this, new therapies are needed to combat this bacterium. It is hypothesized that cytokine release (especially interleukin-1β, -6, -8, and TNF-α) due to H. pylori infection and the subsequent influx of inflammatory cells causes a massive release of reactive oxygen species (ROS) during the inflammatory reaction. The ROS then cause the pathologic changes seen in the infected tissues. In this study, human gastric adenocarcinoma cell line ATCC 1739 (a cell line not previously evaluated) was examined for its production of interleukin-1β, -6, -8, and TNF-α when cocultured in a ratio of 10:1 H. pylori to adenocarcinoma cells, to determine its value as a model to demonstrate the inflammatory response. Results from this study indicated that ATCC 1739 cells only reliably produced IL-8 when cocultured with H. pylori and stimulated with TNF-α. The production of IL-1β, IL-6, and TNF-α by the ATCC 1739 cells was no different in H. pylori-exposed cells than non-exposed cells. It was concluded that the ATCC 1739 cell line is not suitable to study the effects of coculture with H. pylori on cytokine production.     

In vitro induction of tumor necrosis factor-α by ochratoxin A (OTA) from rat liver: role of Kupffer cells

Tumor necrosis factor-α (TNF-α) is released from blood-free perfused rat liver by the fungal metabolite ochratoxin A. Here we have identified Kupffer cells as the sole source of OTA-mediated cytokine release. If single cell preparation of Kupffer cells, hepatocytes, or sinusoidal endothelial cells were prepared from rat livers, only Kupffer cells released TNF-α upon incubation with 2.5 μmol/l OTA. OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blockedin vitro by 15 μmol/l gadolinium chloride. When rats were pretreatedin vivo with the Kupffer cell depleting clodronate liposomes, OTA-mediated TNF-α release was abrogated in the isolated perfused liver model.     

SSeCKS Promotes Tumor Necrosis Factor-α Autocrine via Activating p38 and JNK Pathways in Schwann Cells

Tumor necrosis factor-alpha (TNF-α) derived from activated Schwann cells (SCs) plays a critical role as an inflammatory mediator in the peripheral nervous system disease. TNF-α could act as an autocrine mediator in SC activation. In this study, we found knockdown Src-suppressed protein kinase C substrate (SSeCKS) expression suppressed TNF-α production induced by TNF-α, overexpression of SSeCKS could promoted TNF-α autocrine in SCs. Such effects might be resulted in SSeCKS promoted p38 and JNK activation in SCs treated by TNF-α. Thus present data show that while SCs activation, SSeCKS may plays an important role in the release of inflammatory mediators.     

Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements

Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.     

The effect of activation of peroxisome proliferator-activated receptor gamma (PPARγ) on human monocyte function: PPARγ ligands do not inhibit tumor necrosis factor-α release in human monocytic cell line THP-1

Peroxisome proliferator-activated receptor gamma (PPARγ) activation by its ligands reportedly inhibits monocyte function. However, because the concentrations of PPARγ ligands used in previous studies were higher than typically expected to activate PPARγ, we clarified whether PPARγ ligands influence monocyte function and cell viability of the human monocyte cell line THP-1. We determined tumor necrosis factor-alpha (TNF-α) release as a monocyte function and cell viability using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Both troglitazone and 15-deoxy-Δ^12,14-prostaglandin J₂ (15-d-PGJ2) seemed to inhibit phorbol ester-induced TNF-α release from THP-1 cells. On the other hand, neither pioglitazone nor rosiglitazone inhibited phorbol ester-induced TNF-α release. Because the cytotoxicity of troglitazone and 15-d-PGJ2 was significantly (p<0.05, Tukey–Kramer) stronger than that of pioglitazone and rosiglitazone, the inhibition of TNF-α release seemed to parallel the lack of cell viability. We concluded that PPARγ ligands did not directly inhibit TNF-α release in THP-1 cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pim protein kinase-3 is regulated by TNF-α and promotes endothelial cell sprouting

Tumor necrosis factor-α (TNF-α) plays an important role in pathological angiogenesis associated with inflammatory response. Pim-3 kinase belonging to serine/threonine protein kinases is a potent suppressor of myc-induced apoptosis. We have recently demonstrated that Pim-3 plays an essential role in endothelial cell (EC) spreading and migration. In this study, we showed that TNF-α transiently increased Pim-3 mRNA expression, and this was mediated through Tumor necrosis factor-α receptor-1 (TNFR1) pathway in ECs. TNF-α could promote stabilization of Pim- 3 mRNA in ECs. Small-interfering RNA (siRNA)-mediated gene knockdown of Pim-3 significantly impaired TNF-α-induced formation of EC membrane protrusions in vitro. Furthermore, Pim-3 silencing inhibited EC sprouting in subcutaneous Matrigel in vivo. eNOS mRNA abundance was lower in Pim-3 siRNA transfected ECs compared with the control ECs. These observations suggest that Pim-3 plays a role in TNF-α-induced angiogenesis.     

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.     

Observations of Forsythia koreana methanol extract on mast cell-mediated allergic reactions in experimental models

To explore effects of Forsythia koreana methanol extract (FKME) on mast cell-mediated allergic and inflammatory properties, the effect of FKME was evaluated on compound 48/80-induced systemic anaphylaxis, ear swelling, and anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-induced passive cutaneous anaphylaxis (PCA). In addition, the effect of FKME was investigated on the histamine release from rat peritoneal mast cells (RPMCs) stimulated by compound 48/80, which promotes histamine release. The human mast cell line HMC-1 was stimulated by phorbol 12-myristate 13-acetate plus calcium ionophore A23187. Activated HMC-1 can produce several proinflammatory and chemotactic cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8. Cytokine levels in the culture supernatant were measured by an enzyme-linked immunosorbent assay. Cytotoxicity by FKME was determined by a 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. FKME inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling in mice. When 1 g/kg FKME was pretreated or posttreated with mice, compound 48/80-induced mice morality was 50 and 66.7%, respectively. One gram per kilogram of FKME pretreatment inhibited ear-swelling responses derived from compound 48/80 by 29.75%. A PCA reaction was inhibited by 17.9%. In an in vitro model, FKME (1 mg/ml) inhibited histamine release from the RPMCs by 13.8% and TNF-α, IL-6, and IL-8 production from HMC-1 cells by 71.16% (P < 0.001), 86.72% (P < 0.001), and 44.6%, respectively. However, FKME had no cytotoxic effects on cell viability. In conclusion, FKME inhibited not only systemic anaphylaxis and ear swelling induced by compound 48/80 but also inhibited a PCA reaction induced by anti-DNP IgE in vivo. Treatment with FKME showed significant inhibitory effects on histamine, TNF-α, IL-6, and IL-8 release from mast cells.     

The contact-mediated response of peripheral-blood monocytes to preactivated T cells is suppressed by serum factors in rheumatoid arthritis

Stimulation of monocytes/macrophages after cell contact with preactivated T cells has been suggested to contribute to the excessive TNF-α production in rheumatoid arthritis (RA). In this study, T cell-contact-dependent TNF-α production by peripheral-blood monocytes in vitro was investigated and found to be significantly lower in treated and untreated patients with RA than in healthy controls. This suppression was not due to a general deficiency of monocytes to respond, because responses to lipopolysaccharide were comparable in patients and controls. In agreement with the pivotal role of TNF-α in RA, T cell-dependent induction of TNF-α in synovial macrophages was fivefold to tenfold higher than in peripheral-blood monocytes from either patients or controls. The decreased response of peripheral-blood monocytes from patients with RA was found to be mediated by inhibitory serum factors, because the addition of patient sera to monocytes from healthy controls suppressed TNF-α response in the co-culture assay. Preincubation of monocytes from healthy controls with RA serum was sufficient to suppress the subsequent TNF-α response in T cell co-cultures, indicating that inhibitory factors do indeed bind to monocyte surfaces, which might represent a regulatory counter-action of the immune system to the long-standing and consuming autoimmune process in RA. There are some indications that apolipoprotein A-1 might be part of this regulatory system.     

Degradation of IkappaBalpha in activated RAW264.7 cells is blocked by the phosphatidylinositol 3-kinase inhibitor LY294002

The mechanism by which lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) induces production of proinflammatory cytokines in murine macrophages, and the role of phosphatidylinositol 3-kinase (PI3-kinase) have not been well investigated. Activation of nuclear factor κB (NF-κB) is initiated by the phosphorylation of the inhibitory subunit, IκB, which targets IκB for degradation and leads to the release of active NF-κB. In this study we demonstrate that 2- (4-morpholinyl)-8-phenylchromone (LY294002), which inhibits PI3-kinase, specifically inhibited degradation of IκBα in RAW264.7 cells stimulated with interferon-γ (IFN-γ) plus LPS or IFN-γ plus PMA. To elucidate the importance of this activity in RAW264.7 cells, we examined tumor necrosis factor-α (TNF-α) and interleukin IL)-6 production in the activated cells. Pretreatment of the cells with LY294002 resulted in the inhibition of TNF-α and IL-6 production in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. Furthermore, LY294002 inhibited the production of nitric oxide NO) in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. LY294002 also inhibited inducible NO synthase (iNOS) mRNA expression in the activated RAW264.7 cells. In conclusion, the present results suggest that PI3-kinase is involved in the signal transduction pathway responsible for LPS- or PMA-mediated TNF-α and IL-6 production, and that LY294002 inhibits NO generation through blocking the degradation of IκBα in activated RAW264.7 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Increased c-Jun N-terminal kinase activation in human endometriotic endothelial cells

Endometriosis is a common inflammatory gynecological disease characterized by the presence of endometrial tissue outside of the uterine cavity. The c-Jun N-terminal kinase (JNK) is a subfamily of the mitogen-activated protein kinases (MAPKs) involved in cellular processes ranging from cytokine expression to apoptosis, and is activated in response to inflammation and cellular stress. We hypothesized that inflammatory cytokines in the peritoneal microenvironment increase JNK MAPK activity in endometriotic endothelial cells, and that human endometrial endothelial cells (HEECs) may be involved in inflammatory pathogenesis of endometriosis. Thus, we evaluated the expression of the total- and phosphorylated-(phospho)-JNK in endometrial and endometriotic endothelial cells in vivo, and in HEECs treated with normal peritoneal fluid (NPF), endometriotic peritoneal fluid (EPF), and the inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in vitro. Phospho-JNK immunoreactivity in HEECs in normal endometrium was significantly higher in the early proliferative and late secretory phases compared to other phases. Both eutopic and ectopic HEECs from the early secretory phase also revealed higher phospho-JNK immunoreactivity, compared to their respective cycle-matched normal HEECs. Moreover, HEECs treated with EPF showed significantly higher phospho-JNK levels compared to that in HEECs treated with NPF. In conclusion, our in vivo and in vitro findings suggest that increased phosphorylation of JNK in HEECs from women with endometriosis is likely due to high level of IL-1β and TNF-α in peritoneal fluid; this in turn may up-regulate inflammatory cytokine expression and thus play a role in the pathogenesis of endometriosis.     

TNF-α Induced Apoptosis is Accompanied with Rapid CD30 and Slower CD45 Shedding from K-562 Cells

TNF-α can induce cell death (apoptosis and necrosis), and these effects mostly depend on expression of TNF-receptor superfamily molecules. As determination of certain intracellular enzymes like LDH, released from cultured tumor cells, reflects early membrane alterations, we compared LDH release with changes in cell surface membrane molecule expression during culture of K-562 cells in the presence of TNF-α. TNF-α-mediated CD45 and CD30 shedding is shown to be to be time- and dose-dependent and associated with significant increase in LDH release, with maximal effects after 24 h of treatment. The percentage of decrease of all examined cell surface molecules on K-562 cells after TNF-α treatment was not uniform and appeared to depend on the respective constitutive level of expression and molecule type. The presence of these molecules was confirmed in supernatants using Western blot analyses. These results indicated the complexity of events on the cell membrane, including early LDH release that is associated with a difference in shedding of CD30 and CD45. Shedding of CD30 occurs before apoptosis induction, while shedding of CD45 is associated with apoptosis.