engleski

Effect of variables in endotoxin presentation on endotoxin reactivity in the Limulus Amoebocyte Lysate Assay

The Limulus Amoebocyte Lysate (LAL) Assay is widely used in occupational and environmental health studies to assess exposures to endotoxin (lipopolysaccharide, LPS, or lipooligosaccharide, LOS), a recognized bacterial trigger of innate immunity and a causal agent for a variety of environmental lung diseases. However, it is not known if endotoxin reactivity in the LAL assay varies depending upon how endotoxin is physically presented, i.e., as aggregates of extracted and purified endotoxin vs. as integral membrane components of whole bacteria or shed “blebs” and if this parallels differences in the pro- inflammatory potency of endotoxin seen in vitro and in vivo.14C-LOS was metabolically labeled using an acetate auxotroph of Neisseria meningitidis serogroup B and used as part of intact bacteria, purified membrane blebs, and as aggregates of purified LOS (Post D et al, 2005). 14C-LOS content of each was determined by quantifying LOS-specific 14C-3-OH fatty acids. Effects of increasing doses of 14C-LOS- containing bacteria, blebs, or LOS aggregates were tested in: 1) the LAL assay ; 2) in vivo in C3HeB/FeJ mice, monitoring induced airway inflammation (e.g., accumulation of neutrophils in bronchoalveolar lavage) following intra- nasal instillation ; and 3) in vitro, using HEK293 cells ± CD14, MD-2, TLR4, monitoring LOS-triggered cell activation by the extracellular accumulation of IL-8, measured by ELISA. Doses of 14C-LOS were measured by scintillation counting. Results: Differences in endotoxin potency were observed in each of the 3 assays depending on how LOS was presented with the following rank order of reactivity: in LAL assay: membrane blebs > LOS aggregates > intact bacteria ; Airway inflammation: bacteria > blebs > LOS aggregates ; Activation of HEK/CD14/MD-2/TLR4 cells: LOS aggregates > blebs > bacteria. Maximum differences in potency ranged from 6-fold (LAL) to >10-fold (in vitro and in vivo pro-inflammatory activity). These findings demonstrate that how endotoxin is physically presented significantly affects endotoxin reactivity in the LAL assay and in endotoxin-induced pro-inflammatory responses in vitro and in vivo.

endotoxin ; LAL assay ; HEK-293 cells

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