Date of Award

2-1994

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Sciences

Supervisor

Mary H. Perdue

Abstract

Immediate hypersensitivity (allergy) is a very common disorder, which may
develop after exposure of an individual to an antigen. Intestinal hypersensitivity
to luminal antigens has been postulated as a possible cause or triggering
mechanism in the pathogenesis of ulcerative colitis, Crohn's disease, eosinophilic
gastroenteritis, celiac disease and peptic ulcer. The mechanism by which
individuals became sensitized is not known but naturally occurring adjuvants
(bacteria and their products) may be crucial for the development of
hypersensitivity. In my studies, I investigated in the role of pertussis toxin, a
product of Bordetella pertussis, in intestinal hypersensitivity, since pertussis
vaccine containing attenuated bacteria was used previously for the induction of
anaphylaxis in experimental animals. Pertussis toxin has the enzymatic activity
of ADP-ribosyltransferase. which can block the function of some G proteins,
important elements in the transduction of signals into the cell. I sensitized rats
or mast cell-deficient mice with ovalbumin (OVA) plus recombinant wild type
pertussis toxin (wPT) as adjuvant. The reaction to secondary antigen challenge
was evaluated 14 days later by determining changes in short-circuit current (Isc, indication of net ion transport) in small intestinal segments mounted in Ussing chambers. Other parameters measured included evaluation of antibody levels in the circulation and histological enumeration of mast cells in the intestinal mucosa.

Sensitization of rats with OVA and wPT resulted in enhancement of the
jejunal ion secretory response to secondary antigen challenge as compared to
rats sensitized with OVA. Injection of doses of wPT as low as 10 ng with OVA
caused significantly elevated secondary responses to OVA, while 50 ng wPT
resulted in the maximal response. The responses to OVA challenge were
enhanced 18.7 fold when OVA was added to the luminal side of the tissues
while on serosal side this effect was enhanced 2.2 fold in sensitized rats. The
induction of hypersensitivity by wPT adjuvant was dependent on the enzymatic
activity of wPT. since the enzymatically inactive mutant (mPT) did not influence
responses to secondary antigen challenge. This suggests that ADP-ribosylation
of G proteins is involved in the elevation of hypersensitivity by wPT.

Two classes of antigen-specific antibodies, IgE and IgG₂s., were measured
in the circulation of sensitized rats. The levels of both antibody isotypes were
increased at day 14 in wPT but not mPT plus OVA injected rats, which indicates
that enzymatically active wPT influences antibody production. The role of
particular classes of antibodies was examined by passive sensitization of naive
animals with serum from actively sensitized rats. Ion transport responses to
secondary antigen challenge of intestine were present in those animals.
Blockage of IgE receptors (with myeloma IgEI prior to passive sensitization
showed that the response to OVA was totally abolished. This finding shows that
in this model of hypersensitivity. IgE antibodies are crucial for the response to antigen. Experiments on mast cell-deficient mutant mice revealed that mast cells
are a necessary element for the responses to antigen. Additionally, the number
of stainable mast cells in rat intestinal mucosa was significantiy increased by
40% on day 14 in wPT but not mPT plus OVA injected rats. This indicates that
wPT-induced increases in mast cell numbers are partially responsible for elevated
hypersensitivity responses to antigen. Experiments with the neural toxin,
tetrodotoxin (TTX), showed that responses to luminal (but not serosal) OVA
were neurally regulated in that these responses to OVA were diminished by 66%
after treatment of the tissues with TTX.

Evaluation of tne responses to OVA at different days after primary
sensitization with OVA plus wPT revealed that the responsiveness to antigen
appeared between day 3 and 7 after sensitization, and it was still present on day
228. In rats sensitized with OVA, the responsiveness to OVA was highest on
day 7 and than it decreased very rapidly, showing no responses on day 56.
Evaluation of IgE levels in rats demonstrated a similar pattern: the IgE levels were
detectable for a long time in wPT plus OVA treated rats but only on day 7 in
OVA treated rats. This suggest that elevated IgE levels caused by wPT may
account for the long lasting hypersensitivity responses.

My data showed that wPT is a very potent adjuvant for hypersensitivity
reactions and that the mechanism involves elevation of antigen-specific
antibodies, increases in the number of mucosal mast cells and enhanced neurally-mediated
antigen uptake.


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