Immunity is the process that protects us from infectious agents. This is accomplished by a complex interplay between innate immune cells and the T and B lymphocytes of adaptive immunity, which together mediate responses to attack by pathogens but also generate memory responses. Memory responses are initiated by long-lived T and B cells, which are able to mount rapid responses to a secondary challenge. The capacity to generate such memory responses remains the major mechanism underlying the successes of vaccines. Immunity can also drive pathologic responses when dysregulated. Host immune responses are responsible for death due to bacterial sepsis, kidney failure in systemic lupus erythematosis, and shortness of breath in asthma. Understanding how immunity activates such diverse physiologic responses will be important in pushing the balance toward protection against infections and away from these dysregulated tissue responses.
Key insights underlying the initiation of immune responses came with the discovery of the Toll-like receptors. These receptors are activated when they encounter key constituents from microbes and viruses, such as cell wall components and nucleic acids, respectively. Activation of these receptors initiates a cell response, which includes the elaboration of cytokines and chemokines important in recruiting inflammatory cells to the site of infection. Activation of Toll-like receptors on dendritic cells promotes the maturation and migration of these cells to regional lymph nodes, where they present pathogen-associated peptides to T cells and B cells, which proliferate to create cells and antibodies that eliminate the offending organisms. This combinatorial attack allows innate cells to respond early to infection while pathogen-specific adaptive T and B cells increase in numbers and mature to prompt resolution and long-term memory.
Allergic inflammation is characterized by the infiltration of tissues by eosinophils and basophils, which are normally rare myeloid cells that comprise only a few percent of circulating blood cells. The adaptive allergic immune response is characterized by increases in the numbers of Th2 cells that release interleukin-4 (IL-4) and IL-13 and the development of plasma cells that secrete immunoglobulin E (IgE). When sustained, these responses can lead to alterations at mucosal epithelial surfaces, including increases in the number of mucus-secreting cells and increased deposition of collagen in the tissues. These responses can be protective in healing the epithelium from attack by parasitic worms, such as hookworms and schistosomes. When these types of immunity become focused on common environmental exposures, however, such as inhaled dust mites or mold or consumed shellfish, the result can be allergies, including potentially life-threatening afflictions such as asthma and food allergy, which affect, respectively, more than 20 million and 2 million Americans.
Despite advances in our understanding of the immune system, relatively little is known about substances in allergens that invoke the constellation of cell responses we call allergic immunity. Our laboratory developed mice with a fluorescent reporter—green fluorescent protein—placed into the il4 gene, allowing us to track cells capable of secreting IL-4 in vivo. IL-4 and its companion, IL-13, are necessary for allergic immunity, and we could use this system to begin to understand how these cytokines are induced during immune reactions in the living mouse. Using a parasitic worm infection or a mouse model of allergic lung inflammation, we were able to identify eosinophils, basophils, and Th2 cells in tissues in response to the challenge. None of these responses occurred in mice that lacked IL-4 and IL-13, confirming that these two cytokines are essential for both defense against helminths and for the development of allergic immunity.
We used a whole-genome microarray to identify which genes are highly induced after the onset of allergic immunity. Two of the most highly induced genes belong to the chitinase-like family. This highly conserved ancient gene family is present in all vertebrates. In mice and humans, these genes are grouped at two loci, each of which contains an authentic enzymatically active chitinase, which breaks down and degrades chitin. Each of the original chitinases has been duplicated and undergone further mutations to generate various numbers of chi-lectins, which have lost the ability to degrade chitin and are of unknown function. Our finding that AMCase, one of the chitinases, is highly induced during allergic immunity suggests that chitin is part of the response to an array of common environmental allergens.
Chitin is a chain of N-acetylglucosamine sugars that can organize into a hard brittle substance. Chitin, the major component of the shells of crabs and lobsters, also forms an important structural constituent in the cell walls of molds, the pharynx and the eggs of parasitic worms, and the hard outer bodies of insects, such as cockroaches and dust mites. Chitin accumulating from these organisms is highly abundant in nature and is second only to cellulose as an environmental biopolymer. Although present in insects, crustaceans, fungi, and helminths, each of which can be associated with allergic immunity, chitin is not present in vertebrates, which use a bony skeleton for structural support. Chitinases, which degrade chitin, are expressed widely in plants, where they play a role in defense against fungi by attacking chitin in the cell wall. These findings suggested that AMCase, which is expressed by epithelial cells of the lung and the gastrointestinal tract, might represent a response to chitin polymers in fungi, worms, and insects parasitizing these surfaces.
To test this, we administered chitin to the lungs or peritoneum of mice and analyzed the cells present in the tissues. Beginning as early as 3 hours and peaking by 48 hours, eosinophils and basophils entered tissues where chitin was present. If the chitin was first digested with the recombinant chitinase, AMCase, and then given to the mice, no allergic inflammation resulted, suggesting that intact chitin is required for the response. We confirmed this by creating transgenic mice that overexpressed AMCase in the lung at levels induced after helminth infection. These mice were unaffected by the increased levels of AMCase but, when treated with chitin, they did not develop tissue eosinophils and basophils to the same extent as control mice. Finally, when the natural lung AMCase was neutralized using antibodies, the eosinophils and basophils persisted longer in tissues. These findings suggest that the IL-4 and IL-13 response induces epithelial chitinase, which in turn degrades the chitin, thus relieving the stimulus for the allergic response.
In additional studies, we identified tissue macrophages as key cells involved in recognition of the chitin particles. The macrophages attach to the chitin and release leukotriene B4, a powerful chemoattractant that mobilizes eosinophils and basophils from the blood. In addition, the macrophages express arginase-1, an enzyme that is part of an alternatively activated state known to characterize macrophages at the site of allergic inflammation. Alternatively activated macrophages sustain and prolong the tissue life of eosinophils.
Could chitin be involved in human allergy and asthma? Certain professions, such as those in crab-processing plants, have very high attack rates for asthma and are almost certainly associated with high levels of chitin exposure. Furthermore, variations in the human AMCase gene, designated CHIA, have been associated with asthma risk in various populations, suggesting that alterations in the way that chitin is degraded may be affected by different AMCase enzymes, and thus affect the infiltration of tissues by eosinophils. Indeed, we demonstrated that common human variant AMCase enzymes with diminished chitin-degrading capacity in the laboratory were significantly associated with asthma risk in several patient cohorts.
Grants from the National Institutes of Health provided partial support for this project.
As of June 09, 2010