beta-Carotene inhibits inflammatory gene expression in lipopolysaccharide-stimulated macrophages by suppressing redox-based NF-kappaB activation

beta-Carotene has shown antioxidant and anti-inflammatory activities; however, its molecular mechanism has not been clearly defined. We examined in vitro and in vivo regulatory function of beta-carotene on the production of nitric oxide (NO) and PGE(2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, TNF-alpha, and IL-1beta. beta-Carotene inhibited the expression and production of these inflammatory mediators in both LPS-stimulated RAW264.7 cells and primary macrophages in a dose-dependent fashion as well as in LPS-administrated mice. Furthermore, this compound suppressed NF-kappaB activation and iNOS promoter activity in RAW264.7 cells stimulated with LPS. beta-Carotene blocked nuclear translocation of NF-kappaB p65 subunit, which correlated with its inhibitory effect on IkappaBalpha phosphorylation and degradation. This compound directly blocked the intracellular accumulation of reactive oxygen species in RAW264.7 cells stimulated with LPS as both the NADPH oxidase inhibitor diphenylene iodonium and antioxidant pyrrolidine dithiocarbamate did. The inhibition of NADPH oxidase also inhibited NO production, iNOS expression, and iNOS promoter activity. These results suggest that beta-carotene possesses anti-inflammatory activity by functioning as a potential inhibitor for redox-based NF-kappaB activation, probably due to its antioxidant activity.     

Induction of nitric oxide synthase (NOS) by soluble glucocorticoid induced tumor necrosis factor receptor (sGITR) is modulated by IFN-gamma in murine macrophage

Earlier study showed that glucocorticoid induced tumor necrosis factor receptor (GITR), a new TNFR family, activated murine macrophages to express inducible nitric oxide synthase (iNOS) and to generate nitric oxide (NO). A possible involvement of pro-inflammatory cytokines on NO production by GITR was investigated in vitro systems and signaling molecules contributing to sGITR-induced iNOS production are determined in Raw 264.7 cells, a murine macrophage cell line. The result showed that the synergy was afforded by the combination of GITR with IFN-g in a dose-dependent manner but IFN-gamma alone was not able to induce NOS. No effects were observed with TNF-alpha, IL-1beta, or IL-6 co-treated with GITR. To determine signaling molecules contributing to sGITR-induced iNOS production, a specific inhibitor for signal pathway proteins tested showed that PDTC (NF-kappaB) and genistein (tyrosine kinase) inhibited NOS induction significantly, while sodium orthovanadate (tyrosine phosphatase) potentiated NOS expression. These results suggest that activations of NF-kappaB were involved in induction of iNOS by GITR and IFN-gamma priming caused earlier and stronger NF-kappaB activation.     

Celastrol inhibits production of nitric oxide and proinflammatory cytokines through MAPK signal transduction and NF-kappaB in LPS-stimulated BV-2 microglial cells

Excessive production of nitric oxide (NO) and proinflammatory cytokines from activated microglia play an important role in human neurodegenerative disorders. Here, we investigated whether celastrol, which has been used as a potent anti-inflammatory and anti-oxidative agent in Chinese medicine, attenuates excessive production of NO and proinflammatory cytokines such as TNF-alpha and IL-1betal in LPS-stimulated BV-2 cells, a mouse microglial cell line. We report here that the LPS-elicited excessive production of NO, TNF-alpha, and IL-1beta in BV-2 cells was largely inhibited in the presence of celastrol, and the attenuation of inducible iNOS and these cytokines resulted from the reduced expression of mRNAs of iNOS and these cytokines, respectively. The molecular mechanisms that underlie celastrol-mediated attenuation were the inhibition of LPS-induced phosphorylation of MAPK/ERK1/2 and the DNA binding activity of NF-kappaB in BV-2 cells. The results indicate that celastrol effectively attenuated NO and proinflammatory cytokine production via the inhibition of ERK1/2 phosphorylation and NF-kappaB activation in LPS-activated microglia. Thus, celastrol may be an effective therapeutic candidate for use in the treatment of neurodegenerative human brain disorders.     

Modulation of innate immune responses with synthetic lipid A derivatives

The lipid A moiety of lipopolysaccharides (LPS) initiates innate immune responses by interacting with Toll-like receptor 4 (TLR4), which results in the production of a wide range of cytokines. Derivatives of lipid A show potential for use as immuno-modulators for the treatment of a wide range of diseases and as adjuvants for vaccinations. Development to these ends requires a detailed knowledge of patterns of cytokines induced by a wide range of derivatives. This information is difficult to obtain by using isolated compounds due to structural heterogeneity and possible contaminations with other inflammatory components. To address this problem, we have developed a synthetic approach that provides easy access to a wide range of lipid A's by employing a common disaccharide building block functionalized with a versatile set of protecting groups. The strategy was employed for the preparation of lipid A's derived from E. coli and S. typhimurium. Mouse macrophages were exposed to the synthetic compounds and E. coli 055:B5 LPS, and the resulting supernatants were examined for tumor necrosis factor alpha (TNF-alpha), interferon beta (IFN-beta), interleukin 6 (IL-6), interferon-inducible protein 10 (IP-10), RANTES, and IL-1beta. It was found that for each compound, the potencies (EC50 values) for the various cytokines differed by as much as 100-fold. These differences did not follow a bias toward a MyD88- or TRIF-dependent response. Instead, it was established that the observed differences in potencies of secreted TNF-alpha and IL-1beta were due to differences in the processing of respective pro-proteins. Examination of the efficacies (maximum responses) of the various cytokines showed that each synthetic compound and E. coli 055:B5 LPS induced similar efficacies for the production of IFN-beta and IP-10. However, lipid A's 1-4 gave lower efficacies for the production of RANTES and IL-6 as compared to LPS. Collectively, the presented results demonstrate that cytokine secretion induced by LPS and lipid A is complex, which can be exploited for the development of immuno-modulating therapies.     

Theta-defensins: cyclic antimicrobial peptides produced by binary ligation of truncated alpha-defensins

The first cyclic peptide discovered in animals is an antimicrobial octadecapeptide that is expressed in leukocytes of rhesus monkeys. The peptide, termed rhesus Theta-defensin 1 (RTD-1) is the prototype of a new family of antimicrobial peptides, which like the previously characterized alpha- and beta-defensin families, possesses broad spectrum microbicidal activities against bacteria, fungi, and protects mononuclear cells from infection by HIV-1. The cyclic Theta-defensin structure is essential for a number of its antimicrobial properties, as demonstrated by the markedly reduced microbicidal activities of de-cyclized Theta-defensin analogs. Genetic and biochemical experiments disclosed that the biosynthesis of RTD-1 results from the head-to-tail joining of two nine-amino acid peptides, each of which is donated by a separate precursor polypeptide, which are in fact C-terminally truncated pro-alpha-defensins. Alternate combinations of the two nonapeptides generate two additional macaque Theta-defensins, RTD-2 and RTD-3. Humans do not express Theta-defensin peptides, but mRNAs encoding at least two Theta-defensins are expressed in human bone marrow. However, in each case the open reading frame is interrupted by a stop codon in the signal peptide-coding region. The mature Theta-defensin peptide is a two-stranded beta-sheet that, like the alpha- and beta-defensins, is stabilized by three disulfides. However, the parallel orientation of the Theta-defensin disulfide arrangement allows for substantial flexibility around its short axis. Unlike alpha- and beta-defensins, RTD-1 lacks an amphiphilic topology. This may partially explain the unusual interaction between Theta-defensins and phospholipid bilayers.     

IL-17 induces the production of IL-16 in rheumatoid arthritis

The purpose of this study was to investigate the expression of IL-16 in the rheumatoid synovium and the role of inflammatory cytokines and Toll-like receptor (TLR) ligands in IL-16 production by fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) patients. Immunohistochemical staining was performed with a monoclonal antibody to IL-16 in synovial tissues from patients with RA and likewise in patients with osteoarthritis (OA). FLS were isolated from RA synovial tissues and stimulated with IL-15, IL-1beta, IFN-gamma, and IL-17. The IL-16 mRNA level was assessed by semiquantitative RT-PCR and real time (RT) PCR and a comparison was made between IL-16 mRNA levels produced by RA-FLS and OA-FLS. Production of IL-16 was identified by a western blot assay, and IL-16 production after stimulation by specific ligands of TLR2 and TLR4 was assessed by RT-PCR. While immunohistochemical staining demonstrated strong expression of IL-16 mRNA in synovial tissues from patients with RA, similar findings were not present in the OA group. Moreover, mRNA expression of IL-16 by RA-FLS increased after treatment with IL-17 but not with IL-15, IL-1beta, and IFN-gamma. Specifically, IL-17 increased IL-16 mRNA level by RA-FLS and peripheral blood mononuclear cells in a dose-dependent manner. However, IL-17 did not stimulate IL-16 production in OA-FLS. Peptidoglycan, a selective TLR2 ligand, also increased production of IL-16 by RA-FLS dose- dependently, whereas LPS, a selective TLR4 ligand, had no such stimulatory effect. The results from our data demonstrate that IL-17 and TLR2 ligands stimulate the production of IL-16 by RA-FLS.     

Interleukin-1beta stimulates matrix metalloproteinase-2 expression via a prostaglandin E2-dependent mechanism in human chondrocytes

IL-1beta is known promote cyclooxygenase-2 (COX- 2) and matrix metalloproteinase-2 (MMP-2) expression. This study focuses on the characterization of the signaling cascade associated with IL-1beta-induced matrix metalloproteinase-2 (MMP-2) regulation in human chondrocytes. The decrease in collagen levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that IL-1beta promotes the proteolytic process leading to MMP-2 activation. IL-1beta-related MMP-2 expression was found to be dependent on prostaglandin E2 (PGE2) production. In addition, the induction of COX-2 and MMP-2 was inhibited by the pretreatment of chondrocytes with a SB203580 or Ro 31-8220, indicating the involvement of protein kinase C (PKC) or p38 mitogen-activated protein kinase (MAPK). However, there is no cross-talk between PKC and p38 MAPK in the IL-1beta-induced MMP-2 activation. Taken together, these results demonstrated that IL-1beta induces MMP-2 expression through the PGE2-dependent mechanism in human chondrocytes.     

The Effects of New Zealand Grown Ginseng Fractions on Cytokine Production from Human Monocytic THP-1 Cells

Pro-inflammatory cytokines and anti-inflammatory cytokines are important mediators that regulate the inflammatory response in inflammation-related diseases. The aim of this study is to evaluate different New Zealand (NZ)-grown ginseng fractions on the productions of pro-inflammatory and anti-inflammatory cytokines in human monocytic THP-1 cells. Four NZ-grown ginseng fractions, including total ginseng extract (TGE), non-ginsenoside fraction extract (NGE), high-polar ginsenoside fraction extract (HPG), and less-polar ginsenoside fraction extract (LPG), were prepared and the ginsenoside compositions of extracts were analyzed by HPLC using 19 ginsenoside reference standards. The THP-1 cells were pre-treated with different concentrations of TGE, NGE, HPG, and LPG, and were then stimulated with lipopolysaccharide (LPS). The levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and anti-inflammatory cytokines, such as interleukin-10 (IL-10), and transforming growth factor beta-1 (TGF-β1), were determined by enzyme-linked immunosorbent assay (ELISA). TGE at 400 µg/mL significantly inhibited LPS-induced TNF-α and IL-6 productions. NGE did not show any effects on inflammatory secretion except inhibited IL-6 production at a high dose. Furthermore, LPG displayed a stronger effect than HPG on inhibiting pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) productions. Particularly, 100 µg/mL LPG not only significantly inhibited the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, but also remarkably enhanced the production of anti-inflammatory cytokine IL-10. NZ-grown ginseng exhibited anti-inflammatory effects in vitro, which is mainly attributed to ginsenoside fractions (particularly less-polar ginsenosides) rather than non-saponin fractions.     

Interleukin-10 endogenously expressed in microglia prevents lipopolysaccharide-induced neurodegeneration in the rat cerebral cortex in vivo

A degree of brain inflammation is required for repair of damaged tissue, but excessive inflammation causes neuronal cell death. Here, we observe that IL-10 is expressed in LPS-injected rat cerebral cortex, contributing to neuronal survival. Cells immunopositive for IL-10 were detected as early as 8 h post-injection and persisted for up to 3 d, in parallel with the expression of IL-1beta, TNF-alpha, and iNOS. Double immunofluorescence staining showed that IL-10 expression was localized mainly in activated microglia. Next, we examined the neuroprotective effects of IL-10 using IL-10 neutralizing antibody (IL-10NA). Blockade of IL-10 action caused a significant loss of neurons both 3 d and 7 d after LPS injection. Further, the induction of mRNA species encoding IL-1beta, TNF-alpha, and iNOS, reactive oxygen species (ROS) production, and NADPH oxidase activation, increased after co-injection of LPS and IL-10NA, compared to the levels seen after injection of LPS alone. Taken together, these results clearly suggest that LPS-induced endogenous expression of IL-10 in microglia contributes to neuronal survival by inhibiting brain inflammation.     

Synthesis of DPIE [2-(1,2-Diphenyl-1H-indol-3-yl)ethanamine] Derivatives and Their Regulatory Effects on Pro-Inflammatory Cytokine Production in IL-1β-Stimulated Primary Human Oral Cells

Interleukin-1 beta (IL-1β) has diverse physiological functions and plays important roles in health and disease. In this report, we focus on its function in the production of pro-inflammatory cytokines, including IL-6 and IL-8, which are implicated in several autoimmune diseases and host defense against infection. IL-1β activity is markedly dependent on the binding affinity toward IL-1 receptors (IL-1Rs). Several studies have been conducted to identify suitable small molecules that can modulate the interactions between 1L-1β and 1L-1R1. Based on our previous report, where DPIE [2-(1,2-Diphenyl-1H-indol-3-yl)ethanamine] exhibited such modulatory activity, three types of DPIE derivatives were synthesized by introducing various substituents at the 1, 2, and 3 positions of the indole group in DPIE. To predict a possible binding pose in complex with IL-1R1, a docking simulation was performed. The effect of the chemicals was determined in human gingival fibroblasts (GFs) following IL-1β induction. The DPIE derivatives affected different aspects of cytokine production. Further, a group of the derivatives enabled synergistic pro-inflammatory cytokine production, while another group caused diminished cytokine production compared to DPIE stimulation. Some groups displayed no significant difference after stimulation. These findings indicate that the modification of the indole site could modulate IL-1β:IL1R1 binding affinity to reduce or enhance pro-inflammatory cytokine production.     

Coptidis rhizoma extract protects against cytokine-induced death of pancreatic beta-cells through suppression of NF-kappaB activation

We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced beta-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1beta and IFN-gamma resulted in a reduction of cell viability. CRE completely protected IL-1beta and IFN-gamma-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappaB binding activity and p65 subunit levels in nucleus, and IkappaB alpha degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated islets.     

Influence of eicosapentaenoic acid, an omega-3 fatty acid, on ultraviolet-B generation of prostaglandin-E2 and proinflammatory cytokines interleukin-1 beta, tumor necrosis factor-alpha, interleukin-6 and interleukin-8 in human skin in vivo

Dietary omega-3 polyunsaturated fatty acids (omega-3 PUFA) reduce sunburn, an acute inflammatory response, in humans. We assessed whether this may be mediated by reduced ultraviolet-B (UV-B) induction of proinflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, IL-6, IL-8 and prostaglandin (PG)E(2) in healthy skin. In a double-blind, randomized study, 28 humans received 4 g daily of 95% ethyl esters of eicosapentaenoic acid (EPA) or oleic acid (OA) orally for 3 months. Skin biopsies and suction blister fluid were taken from unexposed and UV-B-exposed skin and examined for mediator expression immunohistochemically and quantitatively by immunoassay; plasma levels were also assayed. The subjects taking EPA, but not OA, showed a significant rise in their minimal erythemal dose (MED) (data reported elsewhere). Before supplementation, irradiation with 3x MED UV-B increased blister fluid TNF-alpha, IL-6, IL-8 and PGE(2) at 16 h (all P < 0.001). No significant change occurred in baseline or UV-B-induced skin levels of cytokines after either supplement, whereas UV-B induction of PGE(2) was abolished after EPA but not OA. Immunohistochemical expression of the cytokines at baseline and after UV-B was unaltered by EPA and OA; circulating cytokine and PGE(2) levels were also unchanged. Hence, in healthy skin in vivo, there was no evidence that reduction of the sunburn response by EPA is mediated by the proinflammatory cytokines examined; abrogation of UV-B-generated PGE(2) may play a role.     

B cells activated in the presence of Th1 cytokines inhibit osteoclastogenesis

Host immune response has been considered as an important disease-modifying factor of periodontitis, however, which immune cell(s) or factor(s) are involved in the destruction of periodontium remains unclear. Previously, we reported that osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8+ T cells. We present new data that B cells activated in the presence of Th1 cytokines inhibit osteoclastogenesis. Purified murine B cells were activated with anti-IgD mAb, IL-4, and anti-CD40 mAb, in the absence (BTh2) or presence of Th1 cytokines, either IL-2 (BIL-2) or IFN-gamma (BIFN-gamma). Each activated B cell population was co-cultured with RAW264.7 cells in the presence of soluble receptor activator of NF-kappaB ligand (sRANKL), and the effect on osteoclastic differentiation was evaluated. While BTh2 increased osteoclastogenesis, BIL-2 and BIFN-gamma suppressed it profoundly. To verify the mediating molecule(s), we analyzed cytokine profiles of the activated B cells. Compared to BTh2, BIL-2 expressed increased amount of IFN-gamma and BIFN-gamma expressed decreased amounts of IL-4, IL-5, and IL-10. IFN-gamma was a key negative regulator of osteoclastic differentiation, and mediated the inhibition by BIL-2. These results suggest that Th1 cytokines may have new important roles in resistance to periodontitis, acting directly on osteoclasts or indirectly through B cells.     

The stimulatory effect of IL-1beta on the insulin secretion of rat pancreatic islet is not related with iNOS pathway

Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to destroy pancreatic beta-cells, and thought to be involved in the pathogenesis of type I diabetes mellitus. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1beta may impair an islet function in rodents. Inhibition of iNOS may protect against cytokine-induced beta-cell suppression, although cytokines might also induce NO-independent impairment. To examine the role of NO in the IL-1beta treated cells, rat islets were treated with various concentrations (0, 0.5, 5, 50, 500 pmol/L) of IL-1beta with or without NG-monomethyl-L-arginine (NMMA; a competitive inhibitor of nitiric oxide synthase) for 2 or 6 h. Insulin secretion was stimulated in islets treated with 5, 50, and 500 pmol/ L of IL-1beta for 2 h and 0.5 pmol/L for 6 h, respectively. The stimulatory effect of IL-1beta on the insulin secretion of rat islets was not prevented by NMMA. Nitrate concentration was increased in a time- and concentration-dependent manner. Nitrate production was inhibited by NMMA. iNOS mRNA expression was increased at concentrations more than 5 pmol/L of IL-1beta in a dose dependent manner. iNOS mRNA was detectable after 2 h and peaked at 6 h but decreased after 24 h. These results suggested that the stimulatory effect of IL-1beta on the insulin secretion of rat islets is independent of iNOS-related NO production of IL-1beta and the enzyme activity of nitric oxide synthase.