Toxicological Sciences, Vol 51, 71-79, Copyright © 1999 by Society of Toxicology
BW Gutting, SJ Schomaker, AH Kaplan and DE Amacher
The direct popliteal lymph node assay (PLNA) is a predictive test used to
detect the immune-stimulating potential of pharmaceuticals and other low
molecular weight compounds (LMWCs) with known autoimmunogenic or
sensitizing properties. Two limitations in the PLNA are the existence of
false negatives and the inability of the assay to provide mechanistic
information. Recently the direct PLNA was modified by incorporating
reporter antigens (RA), either TNP-Ficoll or TNP-OVA. In the RA-PLNA,
immune stimulation is detected by measuring IgM or IgG TNP- specific
antibody-forming cells (AFC) using an enzyme-linked immunospot (ELISPOT)
assay. The RA-PLNA, when using potent, known autoimmunogenic compounds, may
provide greater sensitivity compared to the direct PLNA and might
distinguish LMWCs that have intrinsic adjuvant activity from those that
create neo-antigens, using TNP-OVA and TNP-Ficoll, respectively. The
purpose of this study was to rigorously compare the two assays. Our first
objective was to investigate the interlaboratory reproducibility of the
RA-PLNA using four autoimmunogenic LMWC models, plus one negative control
LMWC. Subsequently, we tested seven LMWCs with known sensitizing properties
and compared the results from the direct and modified assay. The test group
included LMWCs thought to be mechanistically distinct and similar to
compounds typically encountered in preclinical safety assessment. All
control and treatment AFC plaques were collected (76 total), pooled, coded
to conceal their source, and counted. The interlaboratory reproducibility
of the RA-PLNA was demonstrated with the model autoimmunogenic compounds
HgCl2, diphenylhydantoin, D-penicillamine, and the negative control
compound phenobarbital, by detecting TNP-specific IgM and polyclonal IgG
production to both reporter antigens. Additionally, the sensitizing effects
of streptozotocin were identified using an IgG2a ELISPOT with both TNP-OVA
and TNP-Ficoll. With the extended test group, the sensitizing effects of
aniline, a false negative LMWC in the direct PLNA, was not detected in this
study when using the direct PLNA. However, there was an increase of IgG1
AFCs using TNP-OVA, when compared to control (508 +/- 113 vs. 12 +/- 4
respectively). Glafenine, diclofenac, and ibuprofen, all associated with
drug-induced anaphylaxis in humans, produced significant increases in IgG1
production to TNP- OVA. Of these three LMWCs, only diclofenac, which has
been documented to induce neo-antigen formation, was detected with
TNP-Ficoll. Hydralazine immunomodulation could be detected only with the
direct PLNA although significant increases in IgM were identified with the
co- injection of either reporter antigen. Isoniazid and methyldopa
consistently produced negative responses in both assays. In summary, this
study has demonstrated acceptable interlaboratory reproducibility of the
RA-PLNA, using model autoimmunogenic LMWCs. Additionally, it demonstrated
that an advantage of the RA-PLNA was that it identified all
anaphylactic-associated LMWCs tested, detected the false negative compound
aniline, and revealed what is thought to be the mechanism(s) associated
with diclofenac-induced immunostimulation.
ARTICLES
A comparison of the direct and reporter antigen popliteal lymph node assay for the detection of immunomodulation by low molecular weight compounds
Drug Safety Evaluation, Central Research Division, Pfizer Inc., Groton, Connecticut 06340, USA.
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