PNA is a type I hypersensitivity. During the sensitization phase, peanut allergen presented by antigen presenting cells to T cells stimulates B cells to produce specific IgE that binds to effector cells such as basophils and mast cells. During the activation phase, following antigen challenge, effector cells degranulate, and release mediators such as histamine that lead to clinical symptoms. In this study, we demonstrated that FAHF-2 protected PNA mice against oral PN challenge-induced anaphylaxis, and that this effect is persistent, as previously reported.(4
) FAHF-2 treated animals exhibited normal body temperature, no allergy symptoms, or no increased histamine release. In this study, FAHF-2 was administered at week 8, at which time PN hypersensitivity was established.(4
) This simulates a potential human treatment regimen to prevent food-induced anaphylaxis. We further demonstrated that FAHF-2 treatment reduced the number of peripheral blood basophils and peritoneal mast cells, and cutaneous mast cell degranulation. Basophil numbers began to decrease after 1 wk of treatment, reached nadir at the end of treatment (wk 14), and remained lower for at least 4 weeks post treatment (wk 18). At that time, the number of peritoneal mast cells was also significantly reduced as was their expression of FcεRI.
In our previous publications, and in this study (data not shown) we found a significant reduction in PN-specific IgE levels after four weeks of treatment.(4
) Previous publications reported that binding of monomeric IgE to FcεRI promotes mouse mast cell/basophil survival and function.(3
) Therefore, decreased peanut specific IgE levels could play a role in decreased mast cell and basophil levels after 7 weeks of treatment with FAHF-2 in this model. However, the finding that peripheral blood basophil numbers reduced as early as 1 week of treatment, prior to IgE reduction, suggests that additional mechanisms may co-exist. To test the possibility that FAHF-2 may also directly suppress basophil and mast cell activities, we employed the MC/9 mast cell line as an in vitro model and showed that MC/9 cells treated with FAHF-2 showed significantly suppressed both anti-DNP IgE induced cell proliferation, and histamine release upon DNP challenge in a non cytotoxic manner. FAHF-2 also significantly suppressed anti-DNP IgE induced FcεRI expression. These results suggested that FAHF-2 suppression of mast cell and basophil numbers and activation contributes to FAHF-2 persistent protection against peanut anaphylaxis.
Mast cell activation is triggered by binding of IgE to the high affinity receptor, FcεRI. Rodent FcεRI is a tetrameric receptor that consists of one α-chain, unique to this receptor, one β-chain and two disulfide-linked γ-chains. Our in vitro
study showed that FAHF-2 treatment suppressed γ subunit FcεRI expression by MC/9 cells. These data together with the in vivo
findings that peritoneal mast cells exhibited significantly reduced FcεRI expression suggest that FAHF-2 suppression of mast cells growth and activation may be through suppression of mast cells high affinity receptors and specific suppression of the signaling γ chain. In this study, we found FAHF-2 reduced mast cell FcεRI expression, but peripheral blood basophil FcεRI expression was unaffected although basophil numbers were significantly reduced. The mechanisms of basophil reduction were not determined. One possibility is FAHF-2 suppression of IL-3 and/or IL-33 receptors, because activation of these receptors are important for basophil maturation, survival and migration. (26
) Further investigation is required.
FAHF-2 is a water extract of 9 herbs. Previously, we began to determine how FAHF-2 acts on the allergic immune response in the murine model of peanut allergy at a single component herb level.(9
) While FAHF-2 blocked histamine release, significantly reduced peanut-specific serum IgE and IL-4, IL-5 and increased IgG2a and IFN-γ levels, no individual herb affected all these aspects. These results suggested that component herbs of FAHF-2 may work synergistically to produce the therapeutic effects produced by the whole formula. In this study, we focused on identification of active compounds in FAHF-2 that affect mast cells using rat RBL-2H3 cells, widely used in previous studies. (18
)We found that the alkaloid rich fraction 2 inhibited RBL-2H3 cell degranulation and human skin mast cell degranulation activated by FcεRIα specific mAb 22E7 as well as the compliment pathway activator C5a. We for the first time demonstrated that 3 major alkaloid compounds (berberine, palmatine and jatrorrhizine) in FAHF-2 showed dose depend responses, berberine being most effective. Interestingly, the mixture of 3 compounds showed a synergistic action (data not shown), which was associated with suppression of the phosphorylation of Syk in antigen-stimulated RBL-2H3 cells. A previous study also showed honeybee-collected pollen inhibition of mast cell degranulation associated with inhibition of IgE binding to mast cells, but no effect on FcεRI expression.(25
) However, our study showed that FAHF-2 compounds did not affect IgE binding to mast cells. This finding suggests that FAHF-2 inhibition of IgE-mediated mast cell degranulation is less likely via “a steric hindrance” or “competes off the IgE” phenomena. (27
In summary, we demonstrated for the first time that FAHF-2 reduces basophil and mast cell numbers in a murine model of PNA. FAHF-2 also suppressed FcεRI expression in vivo and directly suppressed IgE induced mast cell growth, and activation in vitro, which may be due to suppression of FcεRI γ subunit expression. The 3 major alkaloid compounds may contribute to FAHF-2 inhibition of mast cells/basophils activation.