Short communicationStimulation of human interleukin 1 production and specific mRNA expression by microtubule-disrupting drugs
Abstract
The production of interleukin 1 (IL1), a pleiotropic monocyte-derived interleukin, can be induced in vitro by various stimuli. The present study shows that cytochalasins which inhibit actin filament polymerization in various cell types have no significant effect on IL1 production from human monocytic cells. On the contrary, microtubule disrupters such as colchicine, vinblastine, and vincristine dramatically potentiate (15- to 35-fold), in a dose-dependent fashion, cell-associated IL1 and to a lesser extent (2.5- to 7-fold) released IL1 in the myelomonocytic THP1 cell line and in adherent peripheral blood mononuclear cells. The enhancing effect of the drugs was blocked by actinomycin D and by cycloheximide and was accompanied by an increase of specific IL1 β mRNA expression as measured by Northern blot analysis, thus indicating that these drugs act at a transcriptional or post-transcriptional IL1 gene expression level.
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Microtubule disruption utilizes an NFκB-dependent pathway to stabilize HIF-1α protein
2003, Journal of Biological ChemistryHypoxia-inducible factor (HIF)-1α levels are elevated in normoxic cells undergoing physiological processes involving large scale microtubule reorganization, such as embryonic development, wound healing, and tumor cell metastasis. Although alterations in microtubules affect numerous cellular responses, no data have yet implicated microtubule dynamics in HIF-1α regulation. To investigate the effect of microtubule change upon HIF-1α regulation, we treated cells with the microtubule-depolymerizing agents (MDAs) colchicine, vinblastine or nocodazole. We demonstrate that these agents are able to induce transcriptionally active HIF-1. MDA-mediated induction of HIF-1α required microtubule depolymerization, since HIF-1α levels were unchanged in cells treated with either the microtubule-stabilizing agent paclitaxel, or an inactive form of colchicine, or in colchicine-resistant cells. HIF-1 induction was dependent upon cellular transcription, as transcription inhibitors abrogated HIF-1α protein up-regulation. The ability of transcriptional inhibitors to interfere with HIF-1α accumulation was specific to the MDA-initiated pathway, as they were ineffective in preventing hypoxia-mediated HIF-1 induction, which occurs by a distinct post-translational pathway. Moreover, we provide evidence implicating a requirement for NFκB transcription in the HIF-1 induction mediated by MDAs. The ability of MDAs to induce HIF-1α is dependent upon activation of NFκB, since inhibition of NFκB either pharmacologically or by transfection of an NFκB super-repressor plasmid abrogated this induction. Collectively, these data support a model in which NFκB is a focal point for the convergence of MDA-mediated signaling events leading to HIF-1 induction, thus revealing a novel aspect of HIF-1α regulation and function.
MEK kinase 1 (MEKK1) transduces c-Jun NH<inf>2</inf>-terminal kinase activation in response to changes in the microtubule cytoskeleton
1999, Journal of Biological ChemistryCell shape change and the restructuring of the cytoskeleton are important regulatory responses that influence the growth, differentiation, and commitment to apoptosis of different cell types. MEK kinase 1 (MEKK1) activates the c-Jun NH2-terminal kinase (JNK) pathway in response to exposure of cells to microtubule toxins, including taxol. MEKK1 expression is elevated 3-fold in mitosis and microtubule toxin-treated cells accumulated at G2/M of the cell cycle. Targeted disruption of MEKK1 expression in embryonic stem cells resulted in the loss of JNK activation and increased apoptosis in response to taxol. Targeted disruption of the MEK kinase 2 gene had no effect on activation of the JNK pathway in response to microtubule toxins demonstrating a specific role of MEKK1 in this response. Cytochalasin D-mediated disruption of actin fibers activates JNK and stimulates apoptosis similarly in MEKK1−/− and wild type cells. The results show that MEKK1 is required for JNK activation in response to microtubule but not actin fiber toxins in embryonic stem cells. MEKK1 activation can protect cells from apoptosis in response to change in the integrity of the microtubule cytoskeleton.
Suppression of LPS-induced tumor necrosis factor-α gene expression by microtubule disrupting agents
1996, ImmunobiologyMicrotubule disrupting agents such as colchicine have been shown to reduce TNF-α production in macrophages. To examine molecular mechanisms underlying the action of colchicine, TNF-α gene expression was studied in the murine macrophage cell line PU5-1.8. An LPS stimulation caused an intense up-regulation of TNF-α gene expression which was followed by a high TNF-α protein production. Simultaneous addition of colchicine (10 μM) suppressed LPS-induced TNF-α mRNA accumulation by one-third and TNF-α protein release by two-thirds. This effect was shared by vinblastine and vincristine, chemically different agents that also disrupt microtubule polymerization. For full suppressive activity on TNF-α gene expression, colchicine had to be present for 3 h in LPS-stimulated macrophage cultures. With nuclear run-on transcription experiments we could demonstrate that colchicine primarily inhibited de novo gene transcription and did not accelerate degradation of TNF-α mRNA in actinomycin D-treated macrophages. Thus, the wellknown antiinflammatory action of microtubule depolymerizing agents may be largely due to a reduced TNF-α gene expression.
Inhibition of LPS-induced tumor necrosis factor-α production by colchicine and other microtubule disrupting drugs
1996, ImmunobiologyColchicine has been shown to act as an anti-inflammatory agent. In this study, we examined whether colchicine and other microtubule-depolymerizing drugs affected the production of TNF-α. When rat peritoneal macrophages were stimulated by LPS, addition of colchicine, vincristine, vinblastine or nocodazole was found to inhibit TNF-α release in a concentration-dependent manner. Suppression of TNF-α release was not due to interference with secretion as the cytokine did not accumulate intracellularly following colchicine treatment. Colchicine markedly enhanced PGE2 release from LPS-stimulated macrophages. However, addition of the cyclooxygenase inhibitor indomethacin only partially reversed the suppressive effect of colchicine on TNF-α production. Colchicine caused a strong reduction of LPS-induced TNF-α mRNA accumulation, suggesting that a pretranslational effect may represent the primary mechanism by which colchicine reduced TNF-α production. These observations could have clinical relevance in ameliorating undesirable effects due to excessive TNF-α production, for example following LPS stimulation of monocytes/macrophages in gram-negative sepsis. Furthermore, these drugs may provide useful tools to study the apparent involvement of the microtubular system in cytokine gene expression and cytokine production.
Synthesis of interleukin-1β in primary biliary cirrhosis: Relationship to treatment with methotrexate or colchicine and disease progression
1995, HepatologyPrimary biliary cirrhosis (PBC) is a chronic, progressive, cholestatic liver disease. Interleukin-1β (IL-1β) may play a role in the pathogenesis of PBC by contributing to altered immune function and fibrosis. Colchicine or methotrexate has some beneficial effects in the treatment of PBC, and also affects interleukin-1 (IL-1β). Therefore, we prospectively studied the synthesis of IL-1β by peripheral blood mononuclear cells (PBMC) from 42 patients with PBC entered into a randomized, double-blind, double-dummy controlled trial of colchicine and methotrexate. PBMC obtained at entry, 6, 12, 18, and 24 months were stimulated to produce IL-1β with phytohemagglutinin (PHA), lipopolysaccharide (LPS), Staphylococcus epidermidis, recombinant IL-2, or mitochondrial antigen. Patients in the two treatment groups did not differ at entry in biochemical measures or liver histological stage. Over 24 months in both groups, serum bilirubin and histologic stage remained stable and alkaline phosphatase decreased significantly. For all patients, synthesis of IL-1β increased constitutively and in response to immune-mediated stimulants (PHA, IL-2, and mitochondrial antigen) but not the bacterial stimulants LPS or S epidermidis. Compared with levels of IL-1β at entry, PHA induced increases for patients treated with methotrexate (12, 18, and 24 months) or colchicine (18 and 24 months). At 24 months, IL-2-induced IL-1β synthesis was increased in patients treated with methotrexate, whereas S epidermidis-induced IL-1β was enhanced in colchicine-treated patients. Before treatment, IL-1β production did not relate to severity of disease except in response to S epidermidis. Notably, in patients with stable or improving liver histological stage, IL-1β synthesis increased dramatically at 12 months in response to IL-2 and S epidermidis, and also to LPS by 24 months. Alterations in IL-1β synthesis in patients with PBC may reflect underlying immunoregulatory changes that contribute to disease progression or stabilization.
Blood-based test for diagnosis and functional subtyping of familial Mediterranean fever
2020, Annals of the Rheumatic Diseases