ovalbumin and Filariasis

Eosinophils are now recognized as multifunctional leukocytes that provide critical homeostatic signals to maintain other immune cells and aid tissue repair. Paradoxically, eosinophils also express an armory of granule-localized toxins and hydrolases believed to contribute to pathology in inflammatory disease. How eosinophils deliver their supporting functions while avoiding self-inflicted injury is poorly understood. We have demonstrated that cystatin F (CF) is a critical survival factor for eosinophils. Eosinophils from CF null mice had reduced lifespan, reduced granularity, and disturbed granule morphology. In vitro, cysteine protease inhibitors restored granularity, demonstrating that control of cysteine protease activity by CF is critical for normal eosinophil development. CF null mice showed reduced pulmonary pathology in a model of allergic lung inflammation but also reduced ability to combat infection by the nematode Brugia malayi. These data identify CF as a "cytoprotectant" that promotes eosinophil survival and function by ensuring granule integrity. VIDEO ABSTRACT.

Topics: Animals; Brugia malayi; Cell Survival; Cells, Cultured; Cystatins; Cysteine Proteases; Cytoplasmic Granules; Eosinophils; Filariasis; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin

Helminth parasites suppress immune responses to prolong their survival within the mammalian host. Thereby not only helminth-specific but also nonhelminth-specific bystander immune responses are suppressed. Here, we use the murine model of Litomosoides sigmodontis infection to elucidate the underlying mechanisms leading to this bystander T-cell suppression. When OT-II T cells specific for the third-party antigen ovalbumin are transferred into helminth-infected mice, these cells respond to antigen-specific stimulation with reduced proliferation compared to activation within non-infected mice. Thus, the presence of parasitic worms in the thoracic cavity translates to suppression of T cells with a different specificity at a different site. By eliminating regulatory receptors, cytokines, and cell populations from this system, we provide evidence for a two-staged process. Parasite products first engage the TGF-β receptor on host-derived T cells that are central to suppression. In a second step, host-derived T cells produce IL-10 and subsequently suppress the adoptively transferred OT-II T cells. Terminal suppression was IL-10-dependant but independent of intrinsic TGF-β receptor- or PD-1-mediated signaling in the suppressed OT-II T cells. Blockade of the same key suppression mediators, i.e. TGF-β- and IL-10 receptor, also ameliorated the suppression of IgG response to bystander antigen vaccination in L. sigmodontis-infected mice.

Topics: Adoptive Transfer; Animals; Bystander Effect; CD8-Positive T-Lymphocytes; Cell Proliferation; Disease Models, Animal; Female; Filariasis; Filarioidea; Gene Expression Regulation; Host-Pathogen Interactions; Interleukin-10; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Programmed Cell Death 1 Receptor; Receptors, Transforming Growth Factor beta; Signal Transduction; T-Lymphocytes, Regulatory; Th2 Cells

In this study, we evaluated the effect chronic helminth infection has on allergic disease in mice previously sensitized to OVA. Ten weeks of infection with Litomosoides sigmodontis reduced immunological markers of type I hypersensitivity, including OVA-specific IgE, basophil activation, and mast cell degranulation. Despite these reductions, there was no protection against immediate clinical hypersensitivity following intradermal OVA challenge. However, late-phase ear swelling, due to type III hypersensitivity, was significantly reduced in chronically infected animals. Levels of total IgG2a, OVA-specific IgG2a, and OVA-specific IgG1 were reduced in the setting of infection. These reductions were most likely due to increased Ab catabolism as ELISPOT assays demonstrated that infected animals do not have suppressed Ab production. Ear histology 24 h after challenge showed infected animals have reduced cellular infiltration in the ear, with significant decreases in numbers of neutrophils and macrophages. Consistent with this, infected animals had less neutrophil-specific chemokines CXCL-1 and CXCL-2 in the ear following challenge. Additionally, in vitro stimulation with immune complexes resulted in significantly less CXCL-1 and CXCL-2 production by eosinophils from chronically infected mice. Expression of FcγRI was also significantly reduced on eosinophils from infected animals. These data indicate that chronic filarial infection suppresses eosinophilic responses to Ab-mediated activation and has the potential to be used as a therapeutic for pre-existing hypersensitivity diseases.

Topics: Animals; Antigen-Antibody Complex; Basophils; Cell Degranulation; Chemokine CXCL1; Chemokine CXCL2; Dermatitis, Contact; Ear; Eosinophils; Female; Filariasis; Filarioidea; Gerbillinae; Hypersensitivity, Immediate; Immune Complex Diseases; Immunoglobulin E; Immunoglobulin G; Immunosuppression Therapy; Inflammation; Leukocyte Count; Macrophages; Mast Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; Neutrophils; Ovalbumin; Receptors, IgG

The selection of allergen-specific B cells into the plasma cell (PC) pool is a critical step in the immune dysregulation that leads to the production of IgE in allergic diseases. We sought to characterize the murine IgE repertoire. In particular, we questioned whether the IgE repertoire of plasmablasts (PBs)/PCs differs from the IgE repertoire of non-PCs. Therefore, we sorted splenocytes from OVA-sensitized BALB/c mice into CD138(pos) (PBs/PCs) and CD19(pos)/CD138(neg) (non-PCs) B cell fractions. Using reverse transcription PCR, we amplified, cloned, and sequenced IgE mRNA transcripts and analyzed the Ig H chain repertoire. As a reference, we characterized the IgM repertoire of the same animals. Compared to IgM, the IgE sequences contained a significantly higher level of somatic mutations and displayed an oligoclonal expansion with clonotype restriction. Interestingly, we found two phenotypically distinct IgE-producing B cell subpopulations that differed in their repertoire of H chain transcripts; IgE transcripts from PBs/PCs showed significantly more signs of Ag-driven selection than transcripts from non-PCs, including 1) a higher number of somatic mutations, 2) increased clustering of replacement mutations in the CDRs, and 3) biased third CDR of the heavy Ig chain composition. In conclusion, PBs/PCs and non-PCs from OVA-sensitized mice express distinct IgE repertoires, suggesting that 1) the repertoire of IgE-expressing PBs/PCs represents a highly biased selection from the global B cell repertoire and 2) Ag-driven affinity maturation is a major force that selects IgE-producing B cells into the CD138(pos) PC pool.

Topics: Allergens; Animals; B-Lymphocyte Subsets; Cell Adhesion; Cell Differentiation; Clone Cells; Evolution, Molecular; Female; Filariasis; Filarioidea; Hypersensitivity, Immediate; Immunoglobulin E; Immunoglobulin Heavy Chains; Immunophenotyping; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Ovalbumin; Phylogeny; Plasma Cells; Protein Binding

Basophils are increasingly recognized as playing important roles in the immune responses of allergic diseases and helminth infections. One of the main obstacles to studying basophils has been the lack of a simple and rapid assay to measure basophil activation in mice.. The purpose of this study was to develop an assay to measure murine basophil activation.. Mouse blood cells were stained with various combinations of positive and negative markers for basophils--sorted and then assessed for basophil purity by May-Grünwald staining of cytospins. Once a flow cytometric strategy for staining basophils was determined, basophil surface expression of CD200R was assessed by multi-colour flow cytometry after stimulation of whole blood with anti-IgE, ionomycin or N-formyl MetLeuPhe (fMLP). Confirmation of basophil activation was assessed by concomitant staining of cells for intracellular IL-4. To test the ability of flow cytometric basophil CD200R measurements to assess for antigen-specific IgE-mediated activation of basophils, surface CD200R expression in response to in vitro stimulation with media alone, helminth antigen or ovalbumin was measured on basophils obtained from control mice, mice infected with helminths and mice sensitized to ovalbumin.. Using anti-IgE-FITC as a positive marker and a combination of anti-CD4-PERCP and anti-B220-PERCP as negative markers resulted in a well-separated basophil population. Additional staining with anti-CD200R-PE demonstrated that (1) basophil CD200R expression increases in response to anti-IgE, ionomycin and fMLP, (2) most CD200R-positive basophils also stain positively for IL-4 and (3) CD200R expression increases after antigen-specific activation of basophils in murine models of helminth disease and allergy.. We developed a multi-colour flow cytometry assay that measures murine basophil activation by utilizing CD200R as an activation marker. This assay is straightforward and rapid, taking approximately half a day for obtaining blood, in vitro stimulation and flow cytometric analysis.

Topics: Animals; Antibodies; Antigens; Antigens, CD; Basophils; Dose-Response Relationship, Drug; Female; Filariasis; Filarioidea; Immunization; Immunoglobulin E; Interleukin-4; Ionomycin; Kinetics; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; N-Formylmethionine Leucyl-Phenylalanine; Ovalbumin; Up-Regulation