The recent development of several rodent models of allergic gastrointestinal disease have started to shed important insight on immunological mechanisms leading to these poorly understood disorders (13
). To further dissect the mechanisms leading to allergic diarrhea, we developed a model of eosinophil-associated gastrointestinal hypersensitivity involving intragastric allergen challenges in mice previously sensitized to allergen in the presence of the adjuvant alum. Although somewhat similar to the protocol described by Kweon et al., which employed CFA (26
), our model displayed a stronger allergic response requiring significantly fewer allergen challenges before onset of diarrhea. The inflammation was not restricted to the large intestine, but involved marked mast cell and eosinophil infiltration in the small intestine. The location of inflammation in different regions of the intestine between these two studies may be due to the use of different adjuvants and sensitization routes (e.g., subcutaneous versus intraperitoneal) that are likely to evoke distinct DC responses. Diarrhea was accompanied by increased intestinal permeability; notably, the increased intestinal permeability persisted for at least 48 hours after antigen challenge even though the diarrhea only occurred acutely following allergen challenge. This indicates that the development of altered intestinal permeability is not by itself responsible for the allergic diarrhea. The occurrence of acute diarrhea, usually within 30 minutes of antigen exposure, the presence of marked intestinal mastocytosis, and mast cell degranulation (detected by histochemical analysis and plasma MMCP-1 levels), strongly suggested a primary type I hypersensitivity reaction.
To definitively demonstrate a critical role for mast cells in allergic diarrhea, we first attempted to induce the experimental regime in mast cell-deficient mice (W/Wv) (37
). As observed with C57Bl/6 mice, however, the strain of these mice (WWB6F1) was resistant to the experimental protocol. Therefore, we choose to deplete WT BALB/c mice of mast cells by repeated administration of ACK2 (20
). ACK2 (over a period of 2 weeks) was very effective in abrogating intestinal mastocytosis (both connective tissue and mucosal mast cells) and, importantly, completely blocked the allergic diarrhea and increased intestinal permeability. Recognizing that c-kit is not exclusively expressed by mast cells (e.g., it is also expressed by interstitial cells of Cajal), we aimed to prove the involvement of mast cells by other approaches. Indeed, anti-IgE treatment blocked allergic diarrhea. Because the mechanism of anti-IgE is not strictly dependent upon inhibition of mast cell signaling, we also analyzed FcεRI gene-targeted mice; these studies supported the involvement of mast cells, primarily through an IgE-dependent pathway. Previous studies have shown that FcεRI-deficient mice are resistant to IgE-dependent passive anaphylaxis (38
), but can undergo active anaphylaxis by an IgG/FcγRIII–dependent pathway (19
). In our model, however, allergen-induced diarrhea was not blocked by an Ab directed against FcγRII/III. The ability of FcεRI-deficient mice to eventually develop allergic diarrhea indicates that FcεRI-independent mechanisms (e.g., FcγRI, C5a, CD23 pathways) may contribute to gastrointestinal allergy under some circumstances (9
The necessity to avoid anaphylactic shock without curtailing an effective mast cell response may explain why mast cells exist as several different populations. As opposed to connective tissue mast cells, which contain numerous granules rich in histamine and serotonin, intestinal mucosal mast cells have low content of histamine and serotonin and distinct granule proteases (4
). Thus, degranulation of mucosal mast cells may only induce local mucosal and epithelial responses leading to diarrhea without provoking a massive release of histamine and serotonin in the bloodstream, where these compounds have potential to trigger life-threatening anaphylactic shock. Indeed, histamine, PAF, and serotonin have all been reported to be released during anaphylaxis, and each of these mediators can directly cause shock if injected into rodents in sufficient quantity (19
). These reports would be consistent with our observation that, despite massive mucosal (but not connective tissue) mast cell degranulation, allergic diarrhea is not accompanied by anaphylactic shock (e.g., hypothermia or death).
While the mechanisms responsible for end organ tissue damage in anaphylaxis have not been clearly elucidated (9
), our results suggest that mast cell–derived serotonin in combination with PAF has a central role in mediating allergic diarrhea. The drugs used to block serotonin receptors were unable to discriminate between the different serotonin receptors, but in combination with anti-PAF treatment, ketanserin (a 5-HT2
blocker, sometimes also described as a 5-HT1c
) was more efficient than each of the two other antiserotonin drugs. Similar inhibition, however, was observed when two antiserotonin drugs that target 5-HT3
receptors were used in combination with anti-PAF. Thus, while serotonin receptor function might display some level of redundancy, these results strongly implicate serotonin in the etiology of allergic diarrhea. Our results are consistent with studies focused on water and electrolyte movement in a rat jejunal perfusion assay that demonstrated involvement of the serotonin receptors 5-HT2
). Drugs that block 5-HT3
have also been reported to inhibit the net fluid secretion induced by cholera toxin (25
) and colonic transit in rats (45
). Interestingly, diarrhea in patients with irritable bowel syndrome is improved by treatment with alosetron, a 5-HT3
). Finally, mice deficient in a high-affinity serotonin transporter, which leads to increased serotonin accumulation in the intestinal mucosa, display watery diarrhea and increased colonic motility (47
). Collectively, these studies highlight a central role for serotonin in the elicitation of local allergic intestinal permeability. Our results do not rule out the coinvolvement of other mediators, such as MMCP-1, recently implicated in increased intestinal permeability during Trichinella spiralis
It is certainly notable that allergic diarrhea occurred by a largely histamine-independent process. Recently, histamine receptors and Th1/Th2 responses have been shown to be dynamically inversely regulated, perhaps as a protective mechanism (33
). It is interesting to speculate that the histamine-independent development of allergic diarrhea and the absence of systemic anaphylaxis (which has been shown to be histamine dependent) during our experimental regime may be a result of downregulated histamine receptors by the strong experimental Th2 protocol.
Eosinophil-associated gastrointestinal disorders are frequently associated with diarrhea (2
). In the investigation of gastrointestinal allergy, eosinophilia is often recognized before increases in mast cells are noted (due to their more ready detection by standard histological techniques). In these settings, eosinophils are often implicated in disease pathology; indeed, we have identified a critical effector role for eosinophils in the development of certain manifestations of experimental gastrointestinal allergy (13
). Furthermore, the capacity of eosinophil granule proteins (e.g., MBP) to induce mast cell degranulation has been demonstrated recently (29
). Experiments with mice containing genetically regulated levels of eosinophils revealed that eosinophils were not critical for the development of allergic diarrhea. Although we have not ruled out a role for eosinophils in other manifestations of disease in our experimental regime, these results suggest that allergic diarrhea is not dependent upon this cell population.
In summary, we have identified the mechanism of experimental oral allergen–induced diarrhea to be critically dependent upon mast cells, IgE, PAF, and serotonin. Although no murine study adequately mimics human disease, we are hopeful that these results provide paradigms for testing in humans. For example, our results highlight the critical role for mast cells in allergic diarrhea, drawing attention to the analysis of this cell type in human gastrointestinal allergic disorders, especially in patients with diarrhea. The identified mechanism also suggests that agents such as anti-IgE, recently approved for the treatment of human asthma, and the c-kit inhibitor imatinib mesylate, recently approved for various malignancies, may have use for gastrointestinal allergy. The finding that allergic diarrhea is mediated by a mechanism involving classic IgE-mediated mast cell activation (unaccompanied by signs of systemic anaphylaxis) prompts us to propose that allergic diarrhea may be better called “intestinal anaphylaxis,” at least under certain circumstances.