Inhibition of lipopolysaccharide-induced pro-inflammatory cytokine expression via suppression of nuclear factor-kappaB activation by Mallotus japonicus phloroglucinol derivatives

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit pro-inflammatory cytokine (tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7, or human blood monocytes. Several phloroglucinol derivatives were isolated from the pericarps as active compounds. Among these compounds, isomallotochromanol and isomallotochromene were the most potent in inhibiting cytokine production. MJE and the phloroglucinol derivatives significantly reduced these cytokine mRNA expressions. Gel shift analysis revealed that stimulation of macrophages with LPS caused an increase in the DNA binding activity of nuclear factor-kappaB (NF-kappaB), which was inhibited by isomallotochromanol and isomallotochromene. Western blot analysis showed that LPS reduced the IkappaB-alpha level in macrophages, while 10 microM isomallotochromanol and 10 microM isomallotochromene attenuated the LPS-induced decrease in IkappaB-alpha protein. We conclude that these phloroglucinol derivatives inhibit pro-inflammatory cytokine production and mRNA expression via suppression of NF-kappaB activation in activated macrophages.     

Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing NF-kappaB activation

Activation of NF-kappaB leads to the production of proinflammatory cytokines such as IL-12 and TNF-alpha that are involved in innate and adaptive immunity. We determined whether T. vaginalis-induced inflammatory response in macrophages associated with NF-kappaB. T. vaginalis adhesion led to transient NF-kappaB activation at 6 h but activation declined dramatically by 8 h. Super-shift assays showed that the gel-shifted complexes consisted of p65 (Rel A) and p50 (NF-kappaB1). NF-kappaB activation was accompanied by IkappaB-alpha degradation, and was inhibited by blocking T. vaginalis adhesion, indicating that the early NF-kappaB activation by T. vaginalis depends on IkappaB-alpha degradation. Quantitative real-time RT-PCR analyses revealed that the expression of TNF-alpha and IL-12 mRNA in T. vaginalis-adhesive cells was rapidly suppressed in comparison with LPS stimulation. We also observed that the parasite inhibited the nuclear translocation of NF-kappaB at 8 h, and diminished IL-12 and TNF-alpha production in response to LPS. In addition, inhibition of IkappaB-alpha degradation by MG-132 resulted in apoptosis. These results demonstrate that effects of T. vaginalis on NF-kappaB regulation are critical for cytokine production and the survival of macrophages. We suggest that there exist inhibitory mechanisms induced by T. vaginalis to evade host immunity.     

Interleukin-6 and interleukin-15 are selectively regulated by lipopolysaccharide and interferon-gamma in primary pig adipocytes

3T3-L1 adipocytes express the lipopolysaccharide (LPS) receptor and respond to direct stimulation with the antigen by increasing the expression of inflammatory mediators. Activation of this receptor by its ligand in the macrophage causes the activation and translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus where it regulates the expression of proinflammatory cytokines and other target genes. We investigated whether LPS could stimulate NF-kappaB translocation in primary pig adipocytes and regulate the expression and secretion of TNF-alpha and IL-6. LPS clearly induced the nuclear translocation of NF-kappaB and also upregulated (P < 0.05) the mRNA expression and secretion of IL-6 into the culture medium. An induction of TNF-alpha expression by LPS was not detected, but with extended incubation (8 h), there was a modest increase (P < 0.09) in the media concentration of this cytokine. Inhibition of either ERK1/2, PKC, or the inhibitory G protein (Gi) with U-0126, bisindolylmaleimide HCl, and pertussis toxin, respectively, blocked (P < 0.05) the increase in IL-6 expression caused by LPS. Because LPS administration in vivo increases circulating concentrations of IFN-gamma, and because this cytokine also regulates multiple immune modulators in the adipocyte, we also determined whether IFN-gamma regulates cytokine expression in primary adipocytes. Although the expression of IL-6 and TNF-alpha was unresponsive to IFN-gamma, the expression of IL-15 was markedly upregulated (P < 0.01). Furthermore, the induction of IL-15 expression by IFN-gamma was blocked by inhibition of PKC. These data indicate that NF-kappaB is responsive to LPS in the adipocyte and also identify key mediators of LPS-induced IL-6 expression. In addition, we provide novel evidence that IFN-gamma targets the adipocyte to induce IL-15 expression, thus indicating a possible role for the adipocyte in the regulation of T-cell function and muscle metabolism during the innate immune response.     

Adiponectin stimulates proliferation and cytokine secretion in colonic epithelial cells

Adiponectin is a recently described mediator secreted by adipose tissue. Here we report the growth promoting and pro-inflammatory actions of adiponectin on colonic epithelial cancer cells. Full-length and globular adiponectin produced an identical stimulation of HT-29 cell growth that was blocked by inhibition of adenylate cyclase and protein kinase A and partially inhibited by a pan-specific protein kinase C inhibitor, but was unaffected by specific inhibition of extracellular signal-related kinase (ERK) or p38 MAP kinase. Globular adiponectin but not full-length adiponectin significantly increased the secretion and mRNA levels of IL-8, GM-CSF and MCP-1. Globular adiponectin doubled IL-1beta-stimulated IL-8 and GM-CSF secretion. Adiponectin-stimulated cytokine secretion was blocked by pharmacological inhibitors of NF-kappaB, ERK and p38 MAP kinase. Globular adiponectin increased phosphorylation of both ERK and p38 MAP kinase and increased the nuclear translocation of active NF-kappaB. Adiponectin has pro-proliferative and pro-inflammatory actions on colonic epithelial cells; these appear to be differentially activated by the adiponectin isoforms. Adiponectin may have a role in the regulation of gastrointestinal mucosal function, inflammation and colon carcinogenesis.     

Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid

A number of studies have investigated the effects of fish oil on the production of pro-inflammatory cytokines using peripheral blood mononuclear cell models. The majority of these studies have employed heterogeneous blends of long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which preclude examination of the individual effects of LC n-3 PUFA. This study investigated the differential effects of pure EPA and DHA on cytokine expression and nuclear factor kappaB (NF-kappaB) activation in human THP-1 monocyte-derived macrophages. Pretreatment with 100 microM EPA and DHA significantly decreased lipopolysaccharide (LPS)-stimulated THP-1 macrophage tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and IL-6 production (P<.02), compared to control cells. Both EPA and DHA reduced TNF-alpha, IL-1beta and IL-6 mRNA expression. In all cases, the effect of DHA was significantly more potent than that of EPA (P<.01). Furthermore, a low dose (25 microM) of DHA had a greater inhibitory effect than that of EPA on macrophage IL-1beta (P<.01 and P<.04, respectively) and IL-6 (P<.003 and P<.003, respectively) production following 0.01 and 0.1 microg/ml LPS stimulation. Both EPA and DHA down-regulated LPS-induced NF-kappaB/DNA binding in THP-1 macrophages by approximately 13% (P< or =.03). DHA significantly decreased macrophage nuclear p65 expression (P< or =.05) and increased cytoplasmic IkappaBalpha expression (P< or =.05). Although similar trends were observed with EPA, they were not significant. Our findings suggest that DHA may be more effective than EPA in alleviating LPS-induced pro-inflammatory cytokine production in macrophages - an effect that may be partly mediated by NF-kappaB. Further work is required to elucidate additional divergent mechanisms to account for apparent differences between EPA and DHA.     

NF-kappaB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa

The progression of lung disease in cystic fibrosis (CF) is characterized by an exuberant inflammatory response mounted by the respiratory epithelium that is further exacerbated by bacterial infection. Recent studies have demonstrated upregulation of nuclear factor-kappaB (NF-kappaB) in response to infection in genetically modified cell culture models, which is associated with expression of interleukin (IL)-8. Using human airway epithelial cells grown in primary culture, we examined in vitro activation of NF-kappaB in cells isolated from five CF (DeltaF508/DeltaF508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa. Immunofluorescence, gel-shift, and immunoblot assays demonstrated a rapid translocation of NF-kappaB subunits (p50 and p65) to the nucleus in both CF and NCF cell cultures. However, nuclear extracts from CF cells both before and following P. aeruginosa stimulation revealed elevated NF-kappaB activation compared with NCF cells. Additionally, elevated nuclear levels of the NF-kappaB inhibitor IkappaBalpha were detected in nuclei of CF cells after P. aeruginosa stimulation, but this increase was transient. There was no difference in IL-8 mRNA levels between CF and NCF cells early after stimulation, whereas expression was higher and sustained in CF cells at later times. Our results also demonstrated increased baseline translocation of NF-kappaB to nuclei of primary CF epithelial cell cultures, but intranuclear IkappaBalpha may initially block its effects following P. aeruginosa stimulation. Thus, IL-8 mRNA expression was prolonged after P. aeruginosa stimulation in CF epithelial cells, and this sustained IL-8 expression may contribute to the excessive inflammatory response in CF.     

Intracellular IL-1Ra type 1 inhibits IL-1-induced IL-6 and IL-8 production in Caco-2 intestinal epithelial cells through inhibition of p38 mitogen-activated protein kinase and NF-kappaB pathways

Interleukin-1 (IL-1) plays a pivotal role in the pathogenesis of inflammatory bowel disease (IBD). IL-1 action is regulated in part by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1Ra). Four splice variants of IL-1Ra gene product have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Although sIL-1Ra and icIL-1Ra1 bind to type I IL-1 receptor with equal affinity, icIL-1Ra1 may carry out unique functions inside cells. The goal of this study was to determine the role of icIL-1Ra1 in regulation of cytokine-induced IL-6 and IL-8 production in Caco-2 intestinal epithelial cells. icIL-1Ra1 inhibited IL-1-induced IL-6 and IL-8 production. IL-1 activated all three mitogen-activated protein (MAP) kinase family members: p38 MAP kinase, extracellular-regulated kinases (ERK), and c-Jun amino-terminal kinases (JNK). Specific inhibitors of each MAP kinase pathway decreased IL-1-induced IL-6 and IL-8 production. Overexpression of icIL-1Ra1 inhibited p38 MAP kinase phosphorylation, but had no effect on ERK and JNK phosphorylation. In addition, icIL-1Ra1 inhibited nuclear translocation of NF-kappaB after IL-1 stimulation. In conclusion, these data indicate that icIL-1Ra1, acting in the cytoplasm of Caco-2 cells, decreased IL-1-induced IL-6 and IL-8 production. This intracellular anti-inflammatory activity of icIL-1Ra1 was mediated through inhibition of p38 MAP kinase and NF-kappaB signal transduction pathways.     

Regulation of nuclear factor-kappaB in intestinal epithelial cells in a cell model of inflammation

BACKGROUND: Interleukin-1 (IL-1), an inflammatory cytokine whose levels are elevated in inflamed mucosa, causes part of its effect on intestinal epithelial cells (IEC) through inducing ceramide production. AIM: To study the role of nuclear factor-kappaB (NF-kappaB), a pro-inflammatory and anti-apoptotic factor, in IL-1-treated IEC. METHODS: NF-kappaB activity and levels of apoptotic proteins were assessed by electrophoretic mobility shift assay and RNA-protection assay, respectively. RESULTS: IL-1 and ceramide, which have been shown to partially mediate IL-1 effects on IEC, activated NF-kappaB levels significantly. This activation was due to a decrease in IkappaB-alpha and IkappaB-beta protein levels. Moreover, the ratio of mRNA levels of anti-apoptotic to pro-apoptotic proteins was significantly increased in IL-1-treated IEC. CONCLUSION: NF-kappaB may play a key role in the regulation of the expression of pro-inflammatory and/or apoptotic genes in inflammatory bowel disease, making this protein an attractive target for therapeutic intervention.     

Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation

GM-CSF and M-CSF (CSF-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. Of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. What the critical actions are of these CSFs on macrophages during inflammatory reactions are unknown. To address this issue, adherent macrophages (GM-BMM and BMM) were first derived from murine bone marrow precursors by GM-CSF and M-CSF, respectively, and stimulated in vitro with LPS to measure secreted cytokine production, as well as NF-kappaB and AP-1 activities. GM-BMM preferentially produced TNF-alpha, IL-6, IL-12p70, and IL-23 whereas, conversely, BMM generated more IL-10 and CCL2; strikingly the latter population could not produce detectable IL-12p70 and IL-23. Following LPS stimulation, GM-BMM displayed rapid IkappaBalpha degradation, RelA nuclear translocation, and NF-kappaB DNA binding relative to BMM, as well as a faster and enhanced AP-1 activation. Each macrophage population was also pretreated with the other CSF before LPS stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and NF-kappaB activity. Thus, GM-CSF and M-CSF demonstrate, at the level of macrophage cytokine production, different and even competing responses with implications for their respective roles in inflammation, including a possible dampening or suppressive role for M-CSF in certain circumstances.     

Effect of the alpha3beta1 integrin on the IL-1 stimulated activation of c-Jun N-terminal kinase (JNK) in CACO-2 cells

Intestinal epithelial cells (IEC) are capable of responding to IL-1 stimulation by producing a variety of pro-inflammatory cytokines. Recently, we have found that binding of the alpha3beta1 integrin may have a regulatory effect on IL-1 responses and intracellular signaling by suppressing cytokine secretion, mRNA expression and the downstream intracellular signaling events from IKK to NF-kappaB activation. In this study, we extend these findings by showing that treatment of the Caco-2 epithelial cells with a cross-linking anti-alpha3 integrin antibody resulted in a suppression in the levels of IL-1 induced AP-1 binding activity in nuclear extracts. Furthermore, suppressed levels of IL-1 induced c-Jun N-terminal kinase (JNK) phosphorylation and kinase activity were seen with the antibody treated cells. Cells cultured on purified laminin-5, the ligand for the alpha3beta1 integrin, did not show significantly elevated levels of JNK phosphorylation after IL-1 stimulation while cells cultured on fibronectin yielded significantly elevated levels of IL-1 induced JNK phosphorylation. These results indicate that binding of the alpha3beta1 integrin results in a suppression in the activation of the IL-1 induced intracellular signaling pathway from JNK to AP-1. This novel regulatory effect may be a potentially important mechanism to regulate IL-1 mediated responses by IEC.     

The effects of thalidomide on the stimulation of NF-kappaB activity and TNF-alpha production by lipopolysaccharide in a human colonic epithelial cell line

The immunomodulatory and anti-inflammatory effects of thalidomide are associated with inhibition of TNF-alpha levels. However, the mechanism by which thalidomide reduces TNF-alpha production remains elusive. NF-kappaB is known to play a central role in regulating inflammatory responses in patients with inflammatory bowel disease (IBD). We tested whether thalidomide acts through inhibiting NF-kappaB activity. HT-29 cells were stimulated with LPS (1 microg/ml) alone, or after pretreatment with thalidomide (100 microg/ml), and NF-kappaB activity was determined by gel mobility shift assays. RT-PCR was used to measure expression of the proinflammatory cytokine genes TNF-alpha, IL-1beta and IL-8. The level of TNF-alpha mRNA was also analyzed by real-time quantitative RT-PCR, and TNF-alpha protein was measured by ELISA. Thalidomide pretreatment did not affect NF-kappaB activity in HT-29 cells stimulated with LPS but production of TNF-alpha was depressed. Thalidomide was found to accelerate the degradation of TNF-alpha mRNA, but had little effect on IL-1beta or IL-8. These observations suggest that the immunomodulatory effect of thalidomide in colonic epithelial cells is associated with inhibition of TNF-alpha. However, it does not act by inhibiting NF-kappaB but rather by inducing degradation of TNF-alpha mRNA.