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 (O
2−), 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. O
2− 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 O
2− generation: (1) 0.1–1 ng/mL
V. vulnificus LPS enhanced O
2− generation significantly but with limited inflammatory mediator generation; (2) 10–100 ng/mL
V. vulnificus LPS maximized O
2− generation with concomitant release of thromboxane B
2 (TXB
2), matrix metalloproteinase-9 (MMP-9), and several cytokines and chemokines; (3) 1000–100,000 ng/mL
V. vulnificus LPS, with the exception of TXB
2, yielded both attenuated
O
2− 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 O
2− production, followed by a progressive decrease in O
2− release, with concomitant release of lactic dehydrogenase (LDH), and generation of TXB
2, 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 O
2− as well as neuroinflammatory TXB
2, MMP-9, cytokines and chemokines.
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