ABSTRACT
BACKGROUND: The earliest immune events induced by allergens are poorly understood, yet are likely essential to understanding how allergic inflammation is established. OBJECTIVE: We sought to describe the earliest signaling events activated by allergen and determine their significance to allergic inflammation. METHODS: A fungal-associated allergenic proteinase (FAP) or ovalbumin was administered once intranasally to wild-type mice to determine their ability to induce allergy-associated genes and initiate allergic lung inflammation. Mice deficient in recombinase activating gene 1, C3a, the C3a anaphylatoxin receptor, and MyD88 were challenged similarly to understand the requirement of these molecules and T and B cells for allergic inflammation. Adoptive T-cell transfer experiments were further performed to determine whether signal transducer and activator of transcription 6 (STAT6) was required for cell recruitment and allergic inflammation. RESULTS: FAP, but not ovalbumin, induced eosinophilic airway inflammation and lung IL-4 production in the absence of adaptive immune cells after the transcriptional induction of allergy-specific airway chemokines. Allergen-mediated chemokine secretion and innate allergic lung inflammation occurred in the absence of STAT6, recombinase activating gene 1, C3a, C3a anaphylatoxin receptor, Toll-like receptor 4, and MyD88 but required intact proteinase activity. Furthermore, FAP induced recruitment of T(H)2 cells and eosinophils to lungs independently of STAT6, which was previously thought to be required for T(H)2 cell homing. CONCLUSION: FAP induces allergic lung inflammation through a previously unrecognized innate immune signaling mechanism. CLINICAL IMPLICATIONS: These findings reveal a new paradigm for understanding how allergic inflammation begins and suggest novel possibilities for the prevention and treatment of allergic diseases, such as asthma.
Subject(s)
Allergens/immunology , Aspergillus oryzae/enzymology , Fungal Proteins/immunology , Hypersensitivity/complications , Hypersensitivity/immunology , Peptide Hydrolases/immunology , Pneumonia/etiology , Animals , Chemokines/metabolism , Complement C3a/metabolism , Eosinophils/pathology , Gene Expression Regulation , Hypersensitivity/genetics , Interleukin-4/biosynthesis , Lung/pathology , Mice , Mice, Inbred Strains , Peptide Hydrolases/metabolism , Pneumonia/pathology , Receptors, Complement/metabolism , STAT6 Transcription Factor/metabolism , Signal Transduction , Th2 Cells/pathology , Toll-Like Receptor 4/metabolism , Transcription, GeneticABSTRACT
Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry.
