Mechanisms of dimethyl sulfoxide augmentation of IL-1 beta production

Expression of the inflammatory cytokine IL-1beta occurs in various inflammatory diseases, and IL-1beta production is regulated at multiple levels. There are conflicting reports about the effects of antioxidants on IL-1beta production. In this study, we investigated the regulatory role of the antioxidant DMSO on LPS-stimulated IL-1beta gene expression in human PBMC and in vivo. This study demonstrated that 1% DMSO increased LPS-stimulated (50 ng/ml) IL-1beta secretion in a dose- and time-dependent manner without altering TNF or IL-6. DMSO also elevated IL-1beta secretion by PBMC in response to exogenous superoxide anions. Despite the increase in IL-1beta, there was no augmentation of NF-kappaB with the addition of DMSO. The steady state mRNA coding for IL-1beta following LPS stimulation was also increased. Cycloheximide studies demonstrated that the DMSO augmentation of IL-1beta mRNA did not require de novo protein synthesis, and studies with actinomycin D showed that DMSO did not alter the half-life of IL-1beta mRNA, suggesting that DMSO did not change the stability of IL-1beta mRNA. Experiments using a reporter vector containing the 5'-flanking region of the human IL-1beta gene revealed that DMSO augmented LPS-induced IL-1beta reporter activity. In vivo, treatment of mice with DMSO significantly increased plasma levels of IL-1beta after endotoxin challenge. These data indicate that DMSO directly increases LPS-stimulated IL-1beta protein production through the mechanisms of augmenting promoter activity and increasing mRNA levels.     

Differential regulation of ARE-mediated TNFalpha and IL-1beta mRNA stability by lipopolysaccharide in RAW264.7 cells

Messenger RNA degradation is a mechanism by which eukaryotic cells regulate gene expression and influence cell growth and differentiation. Many protooncogene, cytokine, and growth factor RNAs contain AU-rich element (AREs) in the 3'untranslated regions which enable them to be targeted for rapid degradation. To investigate the mechanism of ARE-mediated RNA stability, we demonstrate the expression and regulation of TNFalpha and IL-1beta mRNAs in LPS-stimulated macrophages. TNFalpha mRNA was rapidly induced by LPS and showed short half-life at 2-h induction, whereas IL-1beta mRNA was induced slowly and had longer half-life. Electrophoretic mobility shift assays showed that the LPS-induced destabilization factor tristetraprolin (TTP) could bind to TNFalpha ARE with higher affinity than to IL-1beta ARE. HuR was identified to interact with TNFalpha ARE to exert RNA stabilization activity. The expression and phosphorylation of TTP could be activated by p38 MAPK pathway during LPS stimulation. Moreover, ectopic expression with TTP and kinases in p38 pathway followed by biochemical assays showed that the activation of p38 pathway resulted in the phosphorylation of TTP and a decrease in its RNA-binding activity. The ARE-containing reporter assay presented that the p38 signal could reverse the inhibitory activity of TTP on IL-1beta ARE but not on TNFalpha ARE. The present results indicate that the heterogeneity of AREs from TNFalpha and IL-1beta could reflect distinct ARE-binding proteins to modulate their RNA expression.     

In vivo expression of IL-1 beta and IL-6 genes during viral infections in human.

Macrophage infiltration is a constant feature of human virus-infected tissues. However, the in situ functional status of these cells remains undetermined. In order to document an activation of macrophages in virus-infected tissues, the expression of IL-1 beta and IL-6 genes was analyzed using in situ hybridization. Several tissues were studied, as well as infections induced by different viruses: lymph nodes infected by HIV-1 (9 cases) or EBV (one case), lungs infected by CMV (5 cases) or adenovirus (1 case), livers infected by HBV, either chronically (2 cases) or acutely (7 cases presenting a fulminant hepatitis). With the exception of fulminant HBV hepatitis, IL-1 beta and IL-6 genes were expressed in all cases. IL-1 beta and IL-6 genes were usually coordinately regulated, as cells containing IL-1 beta or IL-6 mRNA were present in identical amounts and displayed a similar distribution. Analysis of the location and the morphology of monokine gene-expressing cells indicated that both small macrophages and endothelial cells expressed IL-1 beta and IL-6 genes. However, neither tingible body macrophages present in lymph node follicles nor Kupffer cells expressed these genes at a detectable level. Infected cells themselves were also negative for monokine gene expression. These findings indicate that expression of IL-1 beta and IL-6 genes by reactive cells may play a role in viral spreading limitation as well as virus-induced tissue damage.     

Metallothionein mRNA stability in chicken and mouse cells

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.     

Activated human Langerhans cells express mRNA for IL-1 alpha and IL-1 beta and produce these cytokines but do not secrete them.

Human Langerhans cells (LC) were isolated from epidermal cell preparations by panning with mouse anti-CD1 monoclonal antibody. RNA was prepared and probed for the presence of mRNAs for various cytokines using radiolabeled cDNAs. After stimulation with phorbol myristate acetate LC express RNA for interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta) and produce proteins but do not secrete them at detectable levels. LC-associated IL-1, particularly IL-1 alpha, may play a role in antigen presentation. PMA did not induce IL-6 expression in LC. The addition of lipopolysaccharide, a muramyl dipeptide analog, ionomycin, IL-1 alpha, tumor necrosis factor-alpha, insulin-like growth factor-1 or IL-6 did not induce IL-1 mRNA in LC. UVB augmented IL-1 beta mRNA expression. Glucocorticoids did not detectably affect IL-1 alpha or IL-1 beta mRNA levels following PMA induction, however, staurosporin inhibited IL-1 beta mRNA synthesis. Thus the inducers and regulators of IL-1 formation in human LC and monocytes are not identical.     

Expression of IL-1 beta and IL-8 by human gingival epithelial cells in response to Actinobacillus actinomycetemcomitans

The interaction between epithelial cells and microorganisms is the most important step in bacterial infections. Epithelial cells in response to exposure to pathogenic bacteria produce cytokines that initiate inflammation. However, little is known about the cytokine response of gingival epithelial cells to periodontopathogenic bacteria. Actinobacillus actinomycetemcomitans is thought to play a significant role in the initiation of periodontitis because of its bacteriological characteristics. In the present study, we investigated the cytokine induction by human gingival epithelial cells (HGEC) following exposure to A. actinomycetemcomitans in comparison with human gingival fibroblasts (HGF) in culture. Northern blot analysis showed that mRNAs of interleukin 1beta (IL-1beta) and IL-8, but not IL-6, in HGEC were induced in response to A. actinomycetemcomitans. Secretion of IL-8 by HGEC was also increased following A. actinomycetemcomitans challenge, whereas production of IL-1beta could not be detected. The levels of IL-8 and its mRNA were increased depending on the concentration of A. actinomycetemcomitans. The co-culture with HGF and A. actinomycetemcomitans resulted in an increase in the levels of IL-6 and IL-8 mRNA in HGF. However, HGF exposed to A. actinomycetemcomitans, showed no expression of IL-1beta mRNA. These findings demonstrated that HGEC and HGF stimulated with A. actinomycetemcomitans have different profiles in cytokine mRNA expression. Furthermore, A. actinomycetemcomitans may play an important role in amplifying the local immune response and in initiating inflammatory reaction through release of IL-8 from gingival epithelial cells.     

Pyrin levels in human monocytes and monocyte-derived macrophages regulate IL-1beta processing and release

Macrophages and their precursors, monocytes, are key cells involved in the innate immune response. Although both monocytes and macrophages produce caspase-1, the key enzyme responsible for pro-IL-1beta processing; macrophages are limited in their ability to activate the enzyme and release functional IL-1beta. In this context, because mutations in the pyrin gene (MEFV) cause the inflammatory disorder familial Mediterranean fever, pyrin is believed to regulate IL-1beta processing. To determine whether variations in pyrin expression explain the difference between monocytes and macrophages in IL-1beta processing and release, pyrin was studied in human monocytes and monocyte-derived macrophages. Although monocytes express pyrin mRNA and protein, which is readily inducible by endotoxin, monocyte-derived macrophages express significantly less pyrin mRNA and protein. Pyrin levels directly correlated with IL-1beta processing in monocytes and macrophages; therefore, we asked whether pyrin might promote IL-1beta processing and release. HEK293 cells were transfected with pyrin, caspase-1, apoptotic speck protein with a caspase recruitment domain, and IL-1beta. Pyrin induced IL-1beta processing and release in a dose-dependent manner. Conversely, pyrin small interference RNA suppressed pro-IL-1beta processing in both THP-1 cells and fresh human monocytes. In summary, both pyrin expression and IL-1beta processing and release are diminished upon the maturation of monocytes to macrophages. When pyrin is ectopically expressed or silenced, IL-1beta processing and release parallels the level of pyrin. In conclusion, in the context of endotoxin-induced activation of mononuclear phagocytes, pyrin augments IL-1beta processing and release.