Title

Investigating diclofenac-specific sensitivity and tolerance following oral exposure in BALB/c mice using the potential lymph node assay

Date of Completion

January 2001

Keywords

Health Sciences, Toxicology

Degree

Ph.D.

Abstract

Drug-allergy is a significant cause of patient morbidity and mortality that is frequently associated with NSAIDs. The mechanisms involved are poorly understood and animal models cannot predict these reactions. However, it is thought that allergic reactions to drugs, like conventional antigens, are initiated and maintained by T cells. The objective of this dissertation was to use the BALB/c popliteal lymph node assay (PLNA) to examine oral sensitization and tolerance to the NSAID diclofenac (DF). DF is associated with anaphylaxis, rash, idiosyncratic hepatotoxicity and anemia, however, these occur at low frequency. Initial studies demonstrated DF induced a dose-, time-, T-cell-dependent PLN reaction. DF also induced antibody-forming-cells (AFC) in the PLN against coinjected TNP-Ficoll, and induced a dose-dependent increase in IgE and IgG 1 AFCs, with no detectable IgG2a AFCs, against co-injected TNPOVA. These observations suggested that DF induced a T-cell-dependent, type-2-like, PLN reaction in naive mice. Next, the PLNA was conducted in mice orally pretreated 21 days earlier with a single dose of diclofenac or vehicle. The PLN reaction induced by sub-optimal or optimal doses of (i) DF, (ii) DF + TNP-Ficoll or (iii) DF + TNP-OVA appeared to be, collectively, increased in magnitude, time-course, DF-specific, and resulted in augmented and accelerated AFC production against TNP-Ficoll. These observations suggested oral pretreatment rendered mice hyper responsive to DF-induced T-cell-dependent PLN reactions. In additional experiments, mice orally pretreated with a single dose of DF had reduced PLN reactions following injection of either high- or low density DF-albumin conjugates, whereas oral pretreatment had no observable effect on the PLN reaction following the injection with either high- or low-density DF-ovalbumin conjugates. These observations suggested orally pretreated mice were rendered hypo-responsive specifically against DF-albumin conjugates. To examine a potential mechanism of reduced PLN responsiveness, DF- or vehicle-primed mice were injected with free DF (hyper-responsive PLN reaction) mixed with the high-density DF-albumin conjugate (hypo-responsive PLN reaction). Reduced PLN responsiveness in DF-primed mice was noted and suggested an active suppressor mechanism of tolerance. Collectively, these results suggest mice pretreated with a single oral dose of DF may be sensitized to DF, and in addition, may be specifically protected from DF-induced anaphylaxis through the induction of tolerance. This situation may be similar to patients sensitized to DF, as evidenced by rash, hepatotoxicity or anemia, who nevertheless show no signs of DF-induced anaphylaxis. ^