The murine models of N. brasiliensis
infection and OVA-induced lung disease represent highly characterized models of type 2 immunity; both are critically dependent on CD4 T cells and IL-4/IL-13 as well as the shared components of their signaling, IL-4Rα and STAT6 (2
). One reflects the response to a complex array of diverse parasite antigens typically assessed at days 9 or 10, whereas the other reflects the focused response to a singular protein antigen challenge in the lung mucosa, typically generated over a period of several weeks. Despite these differences, the studies here reveal a highly stereotyped tissue response characterized by infiltrations of Th2 cells, eosinophils, and basophils. Th2 cell differentiation, as assessed by activation of IL-4 expression in lymph nodes, was independent of IL-4/IL-13 from other cell types, in support of prior studies (6
). Although CD4 T cells were necessary and sufficient to mediate the infiltration of effector cells into target tissues, IL-4/IL-13 produced by Th2 cells was neither required nor sufficient for this process. Instead, tissue infiltration by effector cells was dependent on IL-4/IL-13 produced by innate immune cells, whereas IL-4/IL-13 produced by Th2 cells was required for IgE production. Tissue infiltration by Th2 cells, eosinophils, and basophils was tightly coordinated with effective physiologic responses, including intestinal worm expulsion and induction of airway hyperreactivity, which could be mediated by IL-4/IL-13 derived solely from innate cells. These findings force a reevaluation of the mechanisms by which type 2 immunity is established and reveal previously unrecognized cellular interactions between innate and adaptive cells.
The source of IL-4, which effectively mediates Th2 differentiation in vitro and supports Th2 differentiation in vivo, has been long debated, with various studies supporting roles for autocrine IL-4 production by CD4 T cells themselves (26
) or IL-4 production by a variety of other cell types (28
). As assessed by activation of the GFP marker in the IL-4 locus, neither IL-4 nor IL-13 from other cell types is required for differentiation of Th2 cells in vivo (). The dense deficit in mediating type 2 immunity in IL-4/IL-13–, IL-4Rα–, and STAT6-deficient mice is thus explained not by an inability to produce Th2 cells, but by the inability of IL-4/IL-13–producing effector cells to enter target tissues where they mediate the diverse biologic responses required to expel worms or generate airway hyperreactivity. The accumulation of the stereotyped tissue cellular response of type 2 immunity—Th2 cells, eosinophils, and basophils—was dependent on CD4 T cells but also on IL-4/IL-13 produced by bone marrow–derived innate immune cells. In the absence of either CD4 T cells or innate IL-4/IL-13, effector biology was lost. Although prior studies using N. brasiliensis
have called attention to the requirement for IL-4Rα expression on non–bone marrow–derived cells to effect tissue responses, including worm expulsion (30
), our studies demonstrate that the source of IL-4/IL-13 required to drive these complex responses actually derives from innate immune cells rather than Th2 cells. CD4 T cell–derived IL-4/IL-13 amplified the lung inflammation that occurred after repetitive OVA stimulation, suggesting that T cells can amplify these innate responses with continued challenge and expansion, but innate cell IL-4/IL-13 was clearly capable of inducing allergic lung disease mediated by IL-4/IL-13–deficient T cells. Prior studies using Brugia malayi
demonstrated that immunity could be mediated by adoptively transferred IL-4– or IL-4Rα–deficient CD4 T cells into T cell–deficient recipients (31
), but we extend these observations by the use of doubly IL-4/IL-13–deficient T cells. Additionally, basophil accumulation in the liver after N. brasiliensis
infection was unaffected by the absence of IL-4 expression in T cells (32
These findings extend our prior report (3
) and find eosinophils and basophils to be the major infiltrating IL-4–expressing non–T cells in both allergic lung disease and in defense against migratory worms. Although mast cells accumulate under conditions of chronic mucosal stimulation (19
), such as occurs in human asthma, neither mast cells nor IgE production are required for acute immunity against N. brasiliensis
or OVA sensitization models using alum or repetitive antigen exposures (33
). Extensive mast cell infiltration of airway smooth muscles, which has been documented in human asthma (36
), does not occur in the mouse in these acute models.
The finding that eosinophils and basophils constitute the IL-4–expressing cells that accumulate in affected tissues forced us to consider the roles of these cells as a source of the innate IL-4/IL-13, which ultimately mediates type 2 immune effector function. Eosinophil function in parasitic disease has been much debated, despite the clear evidence that eosinophils are capable of direct toxicity to parasitic worms (37
). We document that eosinophils are completely and specifically deleted in ΔdblGATA mice, which contain a GATA-1 promoter mutation, corroborating prior studies (21
), whereas all other IL-4–producing populations were unaffected (not depicted). N. brasiliensis
infection of these eosinophil-deficient mice revealed no apparent role for eosinophils in either Th2 or basophil accumulation, IgE production, or worm expulsion during primary infection, extending prior studies using IL-5–deficient animals, although residual eosinophils are present in these mice (39
). Further, transfer of IL-4/IL-13–competent eosinophils into an IL-4/IL-13–deficient innate cell compartment, even in the presence of IL-4/IL-13–competent CD4 T cells, was not able to restore type 2 immunity (Fig. S1). Although negative experiments are difficult to interpret, we document reconstitution of transferred cells into relevant tissues. Further, if eosinophils were required to nucleate type 2 immunity in tissues, we would have anticipated greater responses in IL-5tg mice, which have numerous eosinophils in tissue. These mice, however, demonstrated attenuated type 2 immune responses after infection, as reflected by low serum IgE and a decrease in tissue-infiltrating cells, in agreement with prior findings (23
). Indeed, we were able to document a significant role for eosinophils in secondary challenges with N. brasiliensis
(), consistent with a role in attacking migratory larvae in tissues, and similar to findings reporting increased worm burdens of Trichinella spiralis
in IL-5–deficient mice (40
). Although the three- to fourfold reductions we document during secondary challenge seem modest, the contributions by eosinophils to control worm burden in endemic countries where reinfection takes place repeatedly over the human lifespan are likely considerable, and may significantly contribute to protection from helminth infections among patients with AIDS living in such endemic areas of the world.
Intriguingly, in both N. brasiliensis
and OVA-induced airway disease, basophils constitute a significant population of IL-4–producing cells that are, as we demonstrate elsewhere, capable of entering tissues in the absence of STAT6-mediated signals (3
). Thus, basophils can accumulate without IL-4/IL-13–mediated tissue-derived signals, although their recruitment is highly dependent on other signals provided by CD4 T cells (3
). Basophils have been reported to generate greater amounts of IL-4 than Th2 cells on a per-cell basis (41
), and infiltration of lungs by basophils has been linked with severe and fatal human asthma (44
). Production of IL-4 by basophils has been documented in anti-IgD–primed mice, a potent type 2 immune stimulus (46
), during secondary challenge with goat serum, although a critical role for these cells in primary type 2 immunity remains unproved. Indeed, even the precise lineage of basophils in hematopoiesis is unclear, and further work is justified in assessing the role of these cells in type 2 immune responses.
The major functional activity of Th2 cells in affected tissues was linked with the capacity to orchestrate the accumulation of eosinophils and basophils. Indeed, the accumulation of Th2 cells was dependent on innate cell sources of IL-4/IL-13 and could not be mediated by autocrine IL-4/IL-13. In CD4 T cells, these latter cytokines were critical for efficient IgE production, which may play a role in secondary infections by arming mast cells and contributing to their survival in tissues. These data suggest a model by which type 2–inciting agents, such as worms and allergens, activate dendritic cells to induce Th2 cells in draining lymph nodes, but also activate a tissue-innate noneosinophil population to produce IL-4/IL-13. These cytokines are presumably critical in recruiting Th2 cells into affected tissues, where these cells subsequently organize two myeloid populations consisting of basophils and eosinophils. Although eosinophils contribute to secondary immune responses through antigen degradation in tissues, defining a primary role for basophils in type 2 immunity will await the development of genetic tools to study these elusive cells.