How the innate and adaptive immune systems cooperate in the natural history of allergic diseases has been largely unknown. Plant-derived allergen, papain, and mite allergens, Der f 1 and Der p 1, belong to the same family of cysteine proteases. We examined the role of protease allergens in the induction of Ab production and airway inflammation after repeated intranasal administration without adjuvants and that in basophil/mast cell stimulation in vitro. Papain induced papain-specific IgE/IgG1 and lung eosinophilia. Der f 1 induced Der f 1–specific IgG1 and eosinophilia. Although papain-, Der f 1–, and Der p 1–stimulated basophils expressed allergy-inducing cytokines, including IL-4 in vitro, basophil-depleting Ab and mast cell deficiency did not suppress the papain-induced in vivo responses. Protease inhibitor–treated allergens and a catalytic site mutant did not induce the responses. These results indicate that protease activity is essential to Ab production and eosinophilia in vivo and basophil activation in vitro. IL-33–deficient mice lacked eosinophilia and had reduced papain-specific IgE/IgG1. Coadministration of OVA with papain induced OVA-specific IgE/IgG1, which was reduced in IL-33–deficient mice. We demonstrated IL-33 release, subsequent IL-33–dependent IL-5/IL-13 release, and activation of T1/ST2-expressing lineage−CD25+CD44+ innate lymphoid cells in the lung after papain inhalation, suggesting the contribution of the IL-33–type 2 innate lymphoid cell–IL-5/IL-13 axis to the papain-induced airway eosinophilia. Rag2-deficient mice, which lack adaptive immune cells, showed significant, but less severe, eosinophilia. Collectively, these results suggest cooperation of adaptive immune cells and IL-33–responsive innate cells in protease-dependent allergic airway inflammation.

Cell-type-specific transcription of mouse high-affinity IgE receptor (Fc⑀RI) ␤-chain is positively regulated by the transcription factor GATA-1. Although GATA-1 is expressed in erythroid cells, megakaryocytes, and mast cells, the expression of mouse Fc⑀RI ␤-chain is restricted to mast cells. In the present study, we characterized the role of GATA-associated cofactor FOG-1 in the regulation of the Fc⑀RI ␤-chain promoter. The expression levels of FOG-1, GATA-1, and ␤-chain in each hematopoietic cell line were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. IntroductionThe high-affinity IgE receptor Fc⑀RI is composed of an ␣-chain, a ␤-chain, and a ␥-chain. Allergen-IgE antibody complex-induced cross-linking of Fc⑀RI results in activation of mast cells, which subsequently secrete various chemical mediators that induce the symptoms of an allergic response.In humans, Fc⑀RI is expressed as a tetramer (␣␤␥ 2 ) on mast cells and basophils and as a trimer (␣␥ 2 ) on Langerhans cells, monocytes, and dendritic cells. [1][2][3][4][5] Thus, while ␤-chain may facilitate cell-surface expression of Fc⑀RI, 6 it is not necessarily required for cell-surface expression of human Fc⑀RI. By contrast, in mice, Fc⑀RI is expressed as a tetramer (␣␤␥ 2 ) only on mast cells and basophils, and the ␤-chain is necessary for cell-surface expression of mouse Fc⑀RI 7 and acts as an amplifier for Fc⑀RI signaling by increasing phosphorylation of the ␥-chain 8 and by enhancing signaling protein recruitment. 9 Therefore, characterization of the mechanisms of mouse ␤-chain expression is critical for the understanding of mast-cell-and basophil-specific transcriptional regulatory systems.We previously reported that the transcription factor GATA-1 positively regulated cell-type-specific ␤-chain expression via 4 GATA motifs in the promoter. 10 GATA-1 mediates the maturation of various cell lineages, including erythroid cells, megakaryocytes, eosinophils, basophils, and mast cells. [11][12][13] However, the expression of mouse ␤-chain is limited to mast cells but not observed in other GATA-1-positive cell lineages. Thus, other factors may regulate cell-type-specific transcription of the ␤-chain.A zinc finger cofactor, FOG-1, interacts with GATA-1 and can either enhance or repress GATA-1-dependent gene expression. 14-17 FOG-1 is abundantly expressed in erythroid cells and in megakaryocytes, where it regulates growth and differentiation. Erythroid and megakaryocyte lineage development is arrested at proerythroblast stage in Gata-1 Ϫ/Ϫ or Fog-1 Ϫ/Ϫ mice, 18,19 and FOG-1 mutants that lack GATA-binding activity result in abnormal differentiation of megakaryotic cells. 19,20 Recent studies have implicated abnormalities in GATA-1 and FOG-1 in various human diseases, including thrombocytopenia and idiopathic myelofibrosis (IM). [21][22][23] Thus, the goal of the present study was to characterize the role of GATA-associated cofactor FOG-1 in the regulation of the Fc⑀RI ␤-chain promoter. Materials and methods Cell culture...

The major proteolytic allergen derived from the house dust mite Dermatophagoides pteronyssinus, Der p1, is one of the most clinically relevant allergens worldwide. In the present study, we evaluate the contribution of the proteolytic activity and structure of a highly purified rDer p 1 to immune responses. Mice were i.p. immunized with three forms of rDer p 1 adsorbed to Alum: one enzymatically active, one treated with an irreversible cysteine protease-specific inhibitor, E-64, and one heat denatured. Immunization with E-64-treated or heat-denatured rDer p 1 elicited much less production of serum total IgE and not only rDer p 1-specific IgE but also IgGs compared with immunization with active rDer p 1. Assays for Ab-binding and its inhibition and structural analyses indicated that E-64-treated rDer p 1 retained its global structure and conformational B cell epitopes. A proliferative response and production of IL-5 by spleen cells restimulated with rDer p 1 were observed on immunization with the active rDer p 1 but not E-64-treated rDer p 1. The cells from mice immunized with heat-denatured rDer p 1 exhibited the highest levels of proliferation and production of IL-5 and IFN-γ. The results indicate that the proteolytic activity of the highly purified rDer p 1 crucially commits to the sensitization process, including both IgE and IgG responses. Additionally, we demonstrated immunogenic differences by functional or structural manipulations of the rDer p 1. The findings have implications for sensitization to this relevant allergen in humans and for the design of modified allergen-vaccines for future allergen-specific immunotherapy.

The FcR β-chain, a subunit of two related multisubunit receptor complexes, the FcεRI and FcγRIII, amplifies the mast cell response and is necessary for the cell surface expression of FcεRI in mouse. The transient reporter assay indicated that −69/+4 region is required for cell type-specific transcriptional regulation of mouse β-chain gene. EMSA using Abs against transcription factors or competitive oligonucleotides demonstrated that −58/−40 region (containing overlapping three GATA-1 sites, −53/−48, −46/−51, and −42/−47) and −31/−26 region (containing one GATA-1 site) are recognized by GATA-1. The promoter activity of β-chain was decreased by nucleotide replacements of the GATA-1 sites in mouse mast cell line PT18. Furthermore, exogenously produced GATA-1 up-regulated the promoter activity in CV-1 cells, which are negative in the β-chain production and the up-regulation was apparently suppressed by GATA-1 site mutations. These results indicate that cell type-specific transcription of mouse β-chain gene is regulated by GATA-1.