Using mice in which the eGfp gene replaced the first exon of the Il4 gene (G4 mice), we examined production of interleukin (IL)-4 during infection by the intestinal nematode Nippostrongylus brasiliensis (Nb). Nb infection induced green fluorescent protein (GFP)pos cells that were FcɛRIpos, CD49bbright, c-kitneg, and Gr1neg. These cells had lobulated nuclei and granules characteristic of basophils. They were found mainly in the liver and lung, to a lesser degree in the spleen, but not in the lymph nodes. Although some liver basophils from naive mice express GFP, Nb infection enhanced GFP expression and increased the number of tissue basophils. Similar basophil GFP expression was found in infected Stat6−/− mice. Basophils did not increase in number in infected Rag2−/− mice; Rag2−/− mice reconstituted with CD4 T cells allowed significant basophil accumulation, indicating that CD4 T cells can direct both tissue migration of basophils and enhanced IL-4 production. IL-4 production was immunoglobulin independent and only partially dependent on IL-3. Thus, infection with a parasite that induces a “Th2-type response” resulted in accumulation of tissue basophils, and these cells, stimulated by a non-FcR cross-linking mechanism, are a principal source of in vivo IL-4 production.

The expression of interleukin-4 (IL-4) is viewed as the hallmark of a Th2 lymphocyte, whereas the subsequent action of IL-4 and IL-13, mediated through the STAT6 signaling pathway, is seen as a prerequisite for the full development of Th2 immune responses to parasites and allergens. G4 mice, whose IL-4 gene locus contains the fluorescent reporter eGFP, were used to quantify the number of Th2 cells that develop during Nippostrongylus brasiliensis-or allergen-induced immune responses under conditions where IL-4 or STAT6 was absent. Here, we show that deletion of IL-4 or STAT6 had little impact on the number or timing of appearance of IL-4-producing Th2 cells. These data indicate that in vivo differentiation of naïve CD4 T cells to Th2 status often occurs independently of IL-4 and STAT6 and that recently described pathways of Th2 cell differentiation may explain how allergens and parasites selectively induce Th2-mediated immunity.allergen ͉ cytokine ͉ asthma ͉ Nippostrongylus

Basophils are recognized as immune modulators through their ability to produce IL-4, a key cytokine required for Th2 immunity. It has also recently been reported that basophils are transiently recruited into the draining lymph node (LN) after allergen immunization and that the recruited basophils promote the differentiation of naive CD4 T cells into Th2 effector cells. Using IL-3−/− and IL-3Rβ−/− mice, we report in this study that the IL-3/IL-3R system is absolutely required to recruit circulating basophils into the draining LN following helminth infection. Unexpectedly, the absence of IL-3 or of basophil LN recruitment played little role in helminth-induced Th2 immune responses. Moreover, basophil depletion in infected mice did not diminish the development of IL-4–producing CD4 T cells. Our results reveal a previously unknown role of IL-3 in recruiting basophils to the LN and demonstrate that basophils are not necessarily associated with the development of Th2 immunity during parasite infection.

IL-4 production by leukocytes is a key regulatory event that occurs early in the type-2 immune response, which induces allergic reactions and mediates expulsion of parasites. CD4+ T-cells and basophils are thought to be the key cell types that produce IL-4 during a type-2 response. Here, we assessed the relative contribution of both CD4+ T-cell- and basophil-IL-4 production during primary and secondary responses to Nippostrongylus brasiliensis using a murine IL-4-eGFP reporter system. During infection, IL-4 producing basophils were detected systemically and tissue recruitment occurred independent of IL-4/STAT6 signaling. We observed that basophil recruitment to a tissue environment was required for their full activation. Basophil induction in response to secondary infection exhibited accelerated kinetics in comparison to primary infection. However, total basophil numbers were not enhanced, as predicted by previous models of protective immunity. Overall, the induction and migration of IL-4 producing basophils into peripheral tissues was found to be a prominent characteristic of the primary, but not memory responses to N. brasiliensis infection where CD4+ T-cells were identified as the major source of IL-4. While basophils were the major initial producers of IL-4 we determined that normal TH2 differentiation occurs independently of basophils and depletion of basophils led to an enhancement of inflammatory cell recruitment to the site of infection.

SignificanceT helper 2 (Th2) cells are defined by their ability to produce the hallmark cytokine IL-4. However, to mediate allergic inflammation in tissues, Th2 cells must secrete additional cytokines including IL-13 and IL-5. We used IL-4 and IL-13 dual-reporter mice to show that naive CD4+ T cells cultured in the presence of IL-4 and thymic stromal lymphopoietin (TSLP) generate a population of IL-4negIL-13pos Th2 cells that develop from IL-4neg precursors and express the Th2 effector cytokines IL-5 and IL-9. In vivo, high TSLP levels promote the development of a similar population of IL-4negIL-13pos T cells that also express Gata3, Il5, and Il3 transcripts. Thus, TSLP drives the early differentiation of a distinct population of effector Th2 cells with pro-inflammatory properties.