We found that 2 types of genetically mast cell–deficient mice, WBB6F1-KitW/W-v and C57BL/6-KitW-sh/W-sh mice, and C57BL/6-Mcpt4–/– mice, were more susceptible than the corresponding WT mice to the morbidity and mortality induced by the i.d. injection of the venoms of the Gila monster or of 2 species of scorpion. Our evidence indicates that mast cell–associated MCPT4 contributes to the ability of mast cells to enhance resistance to the toxicity of both helodermin, the major VIP-like peptide in Gila monster venom, and mammalian VIP, the structurally similar endogenous peptide.
Local engraftment of mast cell–deficient mice with WT mast cells only at the site of venom or peptide injection enhanced resistance to the toxicity induced by Gila monster or scorpion venoms, helodermin, or VIP to a greater extent than did engraftment with Mcpt4–/– BMCMCs. Thus, a relatively small population of mast cells containing MCPT4 appears to be sufficient, at least if these cells are present at the site of envenomation, to increase the mouse’s ability to withstand the toxic effects of such venoms. Moreover, each of the venoms or toxic peptides tested induced extensive degranulation of skin mast cells at the site of injection. Finally, we found that WT mast cells were much more effective than Mcpt4–/– mast cells in degrading helodermin or VIP in vitro. Together, these findings indicate that mast cells can enhance host resistance to the toxicity of these lizard and scorpion venoms, or to helodermin or VIP, at least partly through the local release of MCPT4, which can then degrade venom components. In contrast, our experiments in mice whose CPA3 lacked catalytic activity, or in Cpa3–/– mice, which lack both CPA3 and MCPT5 proteins, indicate that CPA3 and MCPT5 make substantially less or no contribution to resistance to these particular venoms or toxic peptides.
We think that these findings help to illuminate how mast cells can influence 2 different areas of biology: (a) innate host resistance to animal venoms and (b) regulation of the toxicity of endogenous biologically active peptides. Host resistance to the toxicity of animal venoms probably reflects the contribution of many different factors (52
); however, the mast cell has only recently been shown to deserve a place on that list (6
). Indeed, the induction of mast cell degranulation at sites of envenomation until recently has been thought to contribute to the pathology induced by the venom (6
). Notably, Higginbotham proposed in 1965 that mast cell heparin might increase resistance to Russell’s viper venom (55
) and in 1971 that mast cell heparin might also confer resistance to honeybee stings (56
). Unfortunately, mast cell–deficient mice had not yet been reported when those studies were published, and the idea that mast cells conferred more benefit than harm in snake or honeybee envenomation only recently was tested (and confirmed) using mast cell–engrafted genetically mast cell–deficient mice (6
). In the case of mice injected with A. engaddensis
venom, several lines of evidence indicate that CPA3 is the major mast cell protease that enhances host resistance (6
). Pharmacological evidence suggests that CPA3 also contributes to the mouse mast cell–dependent enhancement of resistance to the venoms of the Western diamondback rattlesnake and the Southern copperhead (6
In contrast, the evidence presented herein indicates that MCPT4 is more important than CPA3 in the mast cell–dependent enhancement of the resistance of mice to the venoms of the Gila monster or of 2 medically important scorpions, the deathstalker (yellow) scorpion (L. quinquestriatus hebraeus) and the Arizona bark scorpion (C. exilicauda). Taken together, our studies of the ability of mast cells to enhance resistance to the venoms of 3 poisonous snakes and 1 venomous lizard, as well as 3 venomous arthropods, suggest that the ability of mast cells to store and rapidly release large amounts of proteases with distinct profiles of substrate specificity may permit these cells to contribute to innate host defense against a variety of animal venoms containing diverse toxic substances. This, of course, does not rule out the possibility that the induction of mast cell degranulation by the venoms of some species of poisonous animals can increase the pathology associated with such envenomation. However, no such example has yet been reported based on studies in mice deficient in mast cells or individual mast cell–associated proteases.
Many animal venoms contain peptides that have structural and functional similarities to endogenous mammalian peptides (12
), and many of these venom-associated peptides can bind to the receptors for the corresponding mammalian peptides (58
). It appears likely that convergent evolution, rather than an origin from a common ancestral gene, explains the presence of some of these peptides in reptile venoms, including helodermin (12
) and sarafotoxin 6b (59
). Indeed, helodermin has been detected only in the salivary glands of the Gila monster, whereas a VIP-like peptide that may represent a true ortholog of mammalian VIP can be detected in other Gila monster tissues (12
). Thus, the ability of the Gila monster to concentrate large amounts of helodermin in its salivary secretions may have developed in large part so that the lizard’s venom can activate VIP receptors in cells of the lizard’s prey, with effects that induce disability or death in the prey animal.
Endogenous peptides such as VIP and ET-1 also can induce pathology when produced inappropriately and/or in excessively high amounts, and therefore it is important to have regulatory mechanisms to diminish the concentrations of such peptides or to counteract their effects. We previously reported evidence that mast cells can reduce the pathology and mortality associated with high levels of ET-1 (4
) or neurotensin (5
) by mechanisms that involve CPA3 (6
) or neurolysin (5
), respectively. In the present study, we found that mast cell–associated MCPT4 can degrade VIP and reduce the hypothermia and diarrhea observed in mice injected with large amounts of this peptide.
There are at least 2 implications of these findings. First, they support the general hypothesis that the ability of mast cells to produce a diverse complement of enzymes may be useful in permitting these cells to reduce the potential toxicity of a variety of endogenous peptides that can activate mast cell degranulation when present in high enough concentrations, as well as to degrade and limit the toxicity of structurally and functionally similar peptides that are present in animal venoms. More specifically, our findings suggest that mast cell–dependent degradation of VIP may either help to limit the pathology associated with excessive levels of this peptide, such as when it is produced by a tumor (44
) and perhaps in other settings, and/or may contribute to pathology associated with abnormally low levels of this peptide. For example, reduced levels of VIP have been reported in the airways of patients with severe asthma (60
), and VIP-deficient mice exhibit airway hyperresponsiveness and inflammation that can be partially rescued by administration of VIP (61
). Taken together, these findings raise the possibility that mast cell–dependent degradation of VIP might contribute to pathology in some settings, including asthma.
Mast cells have long been viewed as important contributors to disease, both in the context of anaphylaxis and allergic diseases and in many other disorders (62
). The findings presented herein and in our prior work (4
) indicate that the expression by mast cells of receptors for VIP, ET-1, neurotensin, and other endogenous peptides that can trigger mast cell degranulation, when combined with the mast cell’s ability to produce enzymes that can degrade these and related peptides, permits mast cells to contribute to health in 2 different contexts: reducing the toxicity associated with high concentrations of the endogenous peptides and limiting the pathology induced by structurally and functionally similar peptides contained in animal venoms. Indeed, we find it appealing to speculate that a mast cell–dependent mechanism that may have developed primarily to help to restore homeostasis in the face of conditions associated with elevated levels of peptides such as VIP and ET-1 (namely, the activation of mast cells by such peptides, inducing the release of proteases that can degrade the peptide) may also provide a selective advantage by enhancing host resistance to structurally and functionally similar peptides in animal venoms. It is tempting to speculate further that the occurrence of large numbers of mast cells in the skin, a frequent site of envenomation, in part reflects evolutionary pressure to position these cells where they can rapidly respond to, and thereby help to limit the toxicity of, the venoms of poisonous invertebrates and reptiles. Finally, the development of IgE antibodies to venom components has been reported for snake (66
), honeybee (69
), and scorpion (72
) venoms. It is well known that exposure of highly sensitized subjects to such venoms can result in anaphylaxis (68
). However, the possibility should be considered that the presence of anti-venom IgE may further increase the ability of mast cell degranulation to enhance resistance to such venoms, at least in those subjects whose antibody-dependent reactions to such venoms stop short of anaphylaxis.