Lee, Nancy

Abstract Eosinophils comprise only 1-3% of circulating leukocytes but are often a diagnostic feature associated with a variety of inflammatory and disease states, including parasitic and fungal infections, allergic diseases (e.g., asthma, rhinitis and sensitivities to specific foods), cancer, and transplant rejection. The accurate evaluations of eosinophilia as well as the release of stored eosinophil granule proteins are important for the monitoring disease progression and/or assessing the effectiveness of a given treatment strategy(ies). Despite the availability of detection methods for each of the eosinophil granule proteins logistical difficulties limiting either the assays specificity and/or sensitivity have prevented their extensive use. We have generated unique eosinophil peroxidase specific monoclonal antibodies (EPX-mAb) and a high throughput sandwich ELISA assay as a means of overcoming these difficulties. Our EPX-mAb based ELISA is ~10 times more sensitive than traditional OPD based activity assays and allows detection of EPX in mouse BAL fluid after an acute OVA protocol as well as EPX release from mouse eosinophils stimulated ex vivo. The assay is highly specific and gives no signal in samples from EPX deficient mice even with massive eosinophilia (e.g., EPX deficient IL-5 transgenic mice). More significantly, this assay detects EPX in human tissue extracts and biological fluids and thus represents novel diagnostic assay previously unavailable in clinical settings.

Abstract Chronic asthma is characterized by pulmonary remodeling and sustained lung dysfunction. The expression of IL-13 has been demonstrated as sufficient and necessary to induce these pathologies. Despite the common assumption that T cells are the primary source of IL-13 in the lung following allergen provocation, the mechanistic events leading to IL-13 expression are unclear, including the potential importance of other cells (i.e., non-T cells). Methods: Co-expression of IL-5 and eotaxin-2 induces pulmonary histopathologies and lung dysfunction that are eosinophil-dependent (i.e., they are absent in triple transgenic I5/hE2/PHIL) animals. IL-13 knockout and CD4 knockout mice were crossed with I5/hE2 mice to assess the effects of these deficiencies on the induced pathologies occurring in this double transgenic model. Conclusions: The Th2 pulmonary pathologies associated with I5/hE2 mice included recruitment of pulmonary effector T cells and increased brochoalveolar Th2 cytokine levels. Conversely, I5/hE2/CD4-/- had only a ~50% reduction in IL-13 levels as compared to I5/hE2 mice, suggesting that a significant portion of IL-13 is non-T cell derived in these mice. I5/hE2/IL-13-/- mice are improved in lung dysfunction and mucus production as compared to I5/hE2 mice. All mice had similar levels of eosinophil degranulation. These results demonstrate a role for non-T cell derived IL-13 that contributes to mucus production and lung dysfunction in a mouse model of chronic severe asthma.

Abstract Atopic dermatitis and allergic contact dermatitis are two of the most common skin diseases. In both diseases, eosinophils have been suggested as potential contributors of the pathologies linked with these skin conditions. Our goal was to establish a causative relationship between the inflammatory response of chemically induced contact hypersensitivity and eosinophil-mediated activities. Wild type and eosinophil-less PHIL mice were sensitized and challenged with 2,4-dinitrochlorobenzene (DNFB) or alternatively trimellitic anhydride (TMA). Time lapsed videography was used to capture the number of itching events associated with treatment. Immunohistochemistry for eosinophils and nerves was performed to correlate the effect of dermatitis on innervation and eosinophil accumulation in these mice. Our studies showed that in both the DNFB and TMA models a robust eosinophil infiltrate occurred that was accompanied by copious levels of eosinophil degranulation. More importantly, these studies demonstrated that metrics of inflammation (e.g., induced ear swelling (i.e., thickness)) was reduced in PHIL mice relative to wild type. Our observations were also directly correlative with a loss of nerve growth and branching in PHIL mice. These data implicate eosinophils as contributors to the pathologies linked with chemically-induced contact hypersensitivity, including the induced itch responses associated with these models.

Abstract Eosinophilia is a hallmark of worm infection and allergy. The parasitic nematode Trichinella spiralis establishes chronic infection in skeletal muscle. We have shown previously that in eosinophil-ablated, transgenic mice, T. spiralis larvae die in large numbers in the muscle. Death is promoted by inducible nitric oxide synthase (iNOS). We report here that transcription of iNOS was upregulated in the context of an established alternative activation phenotype in local leukocytes. Infiltrating macrophages, dendritic cells, and neutrophils produced iNOS. These changes were preceded by a reduced T cell recruitment to infected muscle that was coincident with a lag in larval growth, suggesting compromise of the parasite’s habitat. The results further define the cellular interactions that occur at a site of chronic nematode infection in which the eosinophil functions as a pivotal regulator of immunity.

Abstract Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn’s disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2−/− and eotaxin-1/2−/− mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80+CD11b+ macrophages in DSS-induced epithelial injury and to CD68+ intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.