Biology Reference
In-Depth Information
levels were measured by an indirect L929 fibroblast cytotoxicity assay.
Since this protocol does not provide either a quantitative or a direct
measure of TNFα concentrations, a straightforward comparison with the
above results may not be appropriate.
6.4 Ellagitannins as Lipid A Agonists
The similarity in TNFα dose-response data between the
immunostimulatory tannins and LPS (with binding via the lipid A
component) raises the real possibility that the tannin constructs may be
utilizing the previously discussed lipid A receptor system (LBP, CD14,
TLR4, etc.) to transduce the ellagitannin binding event into eventual
TNFα secretion. In order to test this premise, two types of experiments
were pursued. Initially, the role that the LPS-sensitive cell surface
receptor CD14 might play in tannin-mediated TNFα secretion from
hPBMC's was assayed using monoclonal antibodies for the CD14
receptor as a competitive blocker. Followup studies examined whether
the tannin stimulus utilized TLR4 as the signal transduction molecule
linking the cell surface binding event to gene transcription.
6.4.1 Use of CD-14 antibodies as a probe for the participation of
CD14 in tannin-stimulated TNF
α
release
Commercially available monoclonal antibodies to the human CD14
receptor have been used previously to test for the participation of this
receptor in various biological processes (Verbon
et al.
, 2001, Goyert
et al.
, 1988). This approach has proven effective when there is a
competition for the CD14 binding site between a CD14 ligand of interest
and the antibody. For the tannin series, use of a positive control (LPS)
with hPBMC's and antibody MY4 led to the expected result; nearly
complete suppression of TNFα production compared to LPS without
antibody (Fig. 6.18, columns 1 and 2). Similar experimental trials with
the naturally occurring ellagitannin coriariin A (
62
) and its non-coupled
analogue
81
provided the first glimpse that CD14 was indeed implicated
in the tannin mediated upregulation of TNFα production. The antibody
