Epicutaneous exposure to peanut protein prevents oral tolerance and enhances allergic sensitization(1)
The key feature of the disease is a T-helper type 2 (Th2)-predominant allergen- specific immune response, with the production of IgE antibodies specific for the food allergen. Impaired function of the epidermal skin barrier is characteristic of eczema, and is seen even in areas of the skin without lesions. Considering the epidermal permeability dysfunction in eczema and other dermatoses, primary sensitization to allergens through the skin is a possibility.
The flour was de-fatted with hexane five times and then extracted with 0.1M NH4HCO3. The supernatant was removed and precipitated with 60% (NH4)2SO4 for 2 h at 4C. The precipitate was centrifuged, dissolved in a minimum volume of phosphate–buffered saline (PBS) and dialysed extensively against PBS. This method uniformly yielded 85–90% pure peanut protein.
To elicit a DTH response, mice were challenged 20 days after the last epicutaneous or subcutaneous immunization by an injection of 100 ug peanut protein in PBS into the left hind footpad. Net footpad swelling was measured using a microcalliper 24h after challenge.
Strong proliferation of T cells from both CLN and spleens was detected in response to peanut protein. The epicutaneous exposure to peanut protein induced high levels of peanut-specific IgG1 and IgE in all animals, but little or no IgG2a. This antibody pattern was evident after 20 days and is characteristic of a Th2 response. Peanut-specific DTH responses, T cell proliferation, secretion of IFN-g, IL-4, IL-10 and production of both specific IgG and IgE antibodies are abrogated or reduced, while TGF-b production is enhanced, in response to a secondary peanut challenge, compared with saline-fed controls.
Animals epicutaneously exposed to peanut protein had significantly increased DTH responses as well as in vitro T cell proliferation to peanut protein compared with tolerant animals, which were epicutaneously exposed to saline. Levels of secreted IFN-g, IL-4 and IL-10 were all significantly elevated over tolerant control animals, while TGF-b was reduced. Similarly, levels of peanut-specific IgG and IgE antibodies were all significantly higher in animals epicutaneously exposed to peanut prior to feeding than in the tolerant controls.
Three weeks later, a peanut footpad challenge demonstrated that TDTH cells were no longer hyporesponsive in animals epicutaneously exposed to peanut, but had become activated. The existing state of oral tolerance also reduced the in vitro T cell proliferation to peanut following epicutaneous peanut exposure. IgE responses, however, were no longer suppressed at all, but as high as saline fed, peanut-sensitive animals and significantly higher than toler- ant controls.
Following gastrointestinal challenge, the epicutaneously sensitized mice also developed symptoms of systemic anaphylaxis. The symptoms appeared 10–15 min after challenge and included puffiness around the eyes, decreased activity, pilar recti and cyanosis around the eyes, tail and feet. Some animals were inactive even after prodding, but no animals were lost during oral challenge and they usually recovered within 3h of challenge.