Chloroquine interferes with lipopolysaccharide-induced TNF-alpha gene expression by a nonlysosomotropic mechanism.
Chloroquine (CQ) is a lysosomotropic weak base with over 60 years of clinical use for the treatment of malaria and rheumatologic disorders. Consistent with its anti-inflammatory properties, CQ has been shown to interfere with TNF-alpha release from mononuclear phagocytes. Because it is unclear how CQ mediates these immunomodulatory effects, we set out to elucidate its mechanism of action. CQ exhibited dose-dependent inhibition of LPS-induced TNF-alpha release from human PBMC at therapeutically attainable concentrations. Additional studies to determine the specificity of this effect showed that although CQ reduced IL-1beta and IL-6 release, secretion of RANTES was unaffected. CQ acted by reducing TNF-alpha mRNA accumulation without destabilizing its mRNA or interfering with NF-kappaB nuclear translocation or p50/p65 isoform composition of DNA-binding complexes. Intracellular cytokine staining indicated that CQ reduced TNF-alpha production pretranslationally without interfering with TNF-alpha processing or release. We utilized bafilomycin A1 pretreatment to block the pH-dependent trapping of CQ in endosomes and lysosomes. Although bafilomycin A1 alone did not interfere with TNF-alpha expression, preincubation augmented the ability of CQ to reduce TNF-alpha mRNA levels, suggesting that CQ did not act by a lysosomotropic mechanism. Using confocal microscopy, we showed that bafilomycin A1 pretreatment resulted in a dramatic redistribution of quinacrine, a fluorescent congener of CQ, from cytoplasmic vacuoles to the nucleus. These data indicate that CQ inhibits TNF-alpha gene expression without altering translocation of NF-kappaB p50/p65 heterodimers. This dose-dependent effect occurs over a pharmacologically relevant concentration range and does not require pH-dependent lysosomotropic accumulation of CQ.
Weber
S M
SM
Department of Microbiology, Evans Memorial Department of Clinical Research, and Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
Levitz
S M
SM
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D018946:Chemokine CCL5 / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007375:Interleukin-1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000276:immunology; D008247:Lysosomes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D018942:Macrolides; D016328:NF-kappa B / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D014176:Protein Biosynthesis / Q000187:drug effects / Q000276:immunology; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000276:immunology; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D051996:Transcription Factor RelA; D014158:Transcription, Genetic / Q000187:drug effects / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D000093242:Interleukin-6 Inhibitors
D000900:Anti-Bacterial Agents; D018946:Chemokine CCL5; D002738:Chloroquine; D004268:DNA-Binding Proteins; D007166:Immunosuppressive Agents; D007375:Interleukin-1; D015850:Interleukin-6; D008070:Lipopolysaccharides; D018942:Macrolides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000093242:Interleukin-6 Inhibitors; C057620:bafilomycin A