title: Vibrio vulnificus MO6-24/O lipopolysaccharide stimulates superoxide anion, thromboxane B₂, matrix metalloproteinase-9, cytokine and chemokine release by rat brain microglia in vitro.
abstract: Although human exposure to Gram-negative Vibrio vulnificus (V. vulnificus) lipopolysaccharide (LPS) has been reported to result in septic shock, its impact on the central nervous system's innate immunity remains undetermined. The purpose of this study was to determine whether V. vulnificus MO6-24/O LPS might activate rat microglia in vitro and stimulate the release of superoxide anion (O₂⁻), a reactive oxygen species known to cause oxidative stress and neuronal injury in vivo. Brain microglia were isolated from neonatal rats, and then treated with either V. vulnificus MO6-24/O LPS or Escherichia coli O26:B6 LPS for 17 hours in vitro. O₂⁻ was determined by cytochrome C reduction, and matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatinase zymography. Generation of cytokines tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-1α), IL-6, and transforming growth factor-beta 1 (TGF-β1), chemokines macrophage inflammatory protein (MIP-1α)/chemokine (C-C motif) ligand 3 (CCL3), MIP-2/chemokine (C-X-C motif) ligand 2 (CXCL2), monocyte chemotactic protein-1 (MCP-1)/CCL2, and cytokine-induced neutrophil chemoattractant-2alpha/beta (CINC-2α/β)/CXCL3, and brain-derived neurotrophic factor (BDNF), were determined by specific immunoassays. Priming of rat microglia by V. vulnificus MO6-24/O LPS in vitro yielded a bell-shaped dose-response curve for PMA (phorbol 12-myristate 13-acetate)-stimulated O₂⁻ generation: (1) 0.1-1 ng/mL V. vulnificus LPS enhanced O₂⁻ generation significantly but with limited inflammatory mediator generation; (2) 10-100 ng/mL V. vulnificus LPS maximized O₂⁻ generation with concomitant release of thromboxane B2 (TXB2), matrix metalloproteinase-9 (MMP-9), and several cytokines and chemokines; (3) 1000-100,000 ng/mL V. vulnificus LPS, with the exception of TXB2, yielded both attenuated O₂⁻ production, and a progressive decrease in MMP-9, cytokines and chemokines investigated. Thus concentration-dependent treatment of neonatal brain microglia with V. vulnificus MO6-24/O LPS resulted in a significant rise in O₂⁻ production, followed by a progressive decrease in O₂⁻ release, with concomitant release of lactic dehydrogenase (LDH), and generation of TXB2, MMP-9, cytokines and chemokines. We hypothesize that the inflammatory mediators investigated may be cytotoxic to microglia in vitro, by an as yet undetermined autocrine mechanism. Although V. vulnificus LPS was less potent than E. coli LPS in vitro, inflammatory mediator release by the former was clearly more efficacious. Finally, we hypothesize that should V. vulnificus LPS gain entry into the CNS, it would be possible that microglia might become activated, resulting in high levels of O₂⁻ as well as neuroinflammatory TXB2, MMP-9, cytokines and chemokines.
authors.affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. amayer@midwestern.edu.
authors.forename: Alejandro M S
authors.initials: AM
authors.lastname: Mayer
authors.affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. hollandmichaela@gmail.com.
authors.forename: Mary L
authors.initials: ML
authors.lastname: Hall
authors.affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. hollandmichaela@gmail.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Holland
authors.affiliation: Department of Chemical Sciences, University of Napoli, Via Cintia 4, 80126 Napoli, Italy. decastro@unina.it.
authors.forename: Cristina
authors.initials: C
authors.lastname: De Castro
authors.affiliation: Department of Chemical Sciences, University of Napoli, Via Cintia 4, 80126 Napoli, Italy. molinaro@unina.it.
authors.forename: Antonio
authors.initials: A
authors.lastname: Molinaro
authors.affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. monica.aldulescu@mwumail.midwestern.edu.
authors.forename: Monica
authors.initials: M
authors.lastname: Aldulescu
authors.affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. jfrenk25@gmail.com.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Frenkel
authors.affiliation: Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. lottenhoff57@midwestern.edu.
authors.forename: Lauren
authors.initials: L
authors.lastname: Ottenhoff
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, 7 Greenhouse Road, Kingston, RI 02881, USA. drowley@mail.uri.edu.
authors.forename: David
authors.initials: D
authors.lastname: Rowley
authors.affiliation: Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, 10 Pine St, Baltimore, MD 21201, USA. jpowell@shire.com.
authors.forename: Jan
authors.initials: J
authors.lastname: Powell
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D018925:Chemokines / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004926:Escherichia coli / Q000472:pathogenicity*; D007113:Immunity, Innate; D008070:Lipopolysaccharides / Q000008:administration & dosage* / Q000302:isolation & purification; D020780:Matrix Metalloproteinase 9 / Q000187:drug effects / Q000378:metabolism; D017628:Microglia / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism; D013929:Thromboxane B2 / Q000378:metabolism; D041261:Vibrio vulnificus / Q000472:pathogenicity*
chemical_list: D018925:Chemokines; D016207:Cytokines; D008070:Lipopolysaccharides; D013481:Superoxides; D013929:Thromboxane B2; D020780:Matrix Metalloproteinase 9