Anti-lipopolysaccharide monoclonal antibodies inhibit macrophage tnf messenger rna synthesis in vitro

Richard J. Battafarano, Randall S. Burd, Carolyn S. Cody, Todd A. Kellogg, Christopher S. Raymond, Craig A. Ratz, David L. Dunn

Research output: Contribution to journalArticle

Abstract

Gram-negative bacterial lipopolysaccharide (LPS, endotoxin) directly stimulates macrophages to produce tumor necrosis factor (TNF). TNF, in turn, produces a constellation of adverse effects that includes hypotension, systemic acidosis, arterial hypoxemia, and death. Transcription of the TNF gene occurs within minutes of LPS stimulation and appears to be a critical control point in the synthesis and secretion of TNF protein by macrophages. We hypothesized that murine monoclonal antibody (mAb) 8G9 directed against Escherichia coli 0111:B4 LPS would provide protective capacity against an E. coli 0111:B4 bacterial challenge in vivo and would concurrently inhibit LPS-induced synthesis of TNF mRNA and secretion of TNF protein in vitro. E. coli 0111:B4 LPS was used to stimulate a macrophage-derived cell line (RAW 264.7) to produce TNF in the presence or absence of mAb 8G9. Media alone and LPS without 8G9 mAb served as controls against which the effect of 8G9 mAb was compared. Total cellular RNA was purified and analyzed by a Northern blotting technique utilizing a radiolabeled cDNA probe specific for TNF mRNA. TNF mRNA levels from each sample were quantitated by autoradiograph densitometry. Pretreatment with mAb 8G9 provided protective capacity against an intraperitoneal E. coli 0111:B4 bacterial challenge in vivo when compared with saline pretreatment alone (22% versus 90% mortality respectively, P < 0.05). Preincubation of LPS with mAb 8G9 resulted in a significant inhibition of LPS-induced TNF mRNA synthesis (63 ± 20%, P < 0.01) and TNF protein secretion (88 ± 10%, P < 0.001) in vitro. We concluded that inhibition of LPS-induced TNF synthesis and secretion by mAb 8G9 is a key mechanism by which mAb 8G9 provides protection during gram-negative bacterial sepsis.

Original languageEnglish (US)
Pages (from-to)342-348
Number of pages7
JournalJournal of Surgical Research
Volume54
Issue number4
DOIs
StatePublished - Apr 1993
Externally publishedYes

ASJC Scopus subject areas

  • Surgery

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