Lori A. Wagner

Eosinophil-Derived Cationic Proteins Activate the Synthesis of Remodeling Factors by Airway Epithelial Cells

Eosinophil cationic proteins influence several biological functions of the respiratory epithelium, yet their direct contribution to airway remodeling has not been established. We show that incubation of the human bronchial epithelial cell line, BEAS-2B, or primary cultured human bronchial epithelial cells, normal human bronchial epithelial cells, with subcytotoxic concentrations (0.1, 0.3, and 1 μM) of major basic protein (MBP), or eosinophil peroxidase (EPO), augmented the transcripts of endothelin-1, TGF-α, TGF-β1, platelet-derived growth factor (PDGF)-β, epidermal growth factor receptor, metalloproteinase (MMP)-9, fibronectin, and tenascin. A down-regulation of MMP-1 gene expression was observed exclusively in BEAS-2B cells. Cationic protein-induced transcriptional effects were followed by the release of endothelin-1, PDGF-AB in the supernatants by ELISA, and by a down- and up-regulation, respectively, in the levels of MMP-1 and MMP-9 in cell lysates, by Western blot. Cell stimulation with the synthetic polycation, poly-l-arginine, reproduced some but not all effects of MBP and EPO. Finally, simultaneous cell incubation with the polyanion molecules, poly-l-glutamic acid or heparin, restored MMP-1 gene expression but incompletely inhibited MBP- and EPO-induced transcriptional effects as well as endothelin-1 and PDGF-AB release, suggesting that cationic proteins act partially through their cationic charge. We conclude that eosinophil-derived cationic proteins are able to stimulate bronchial epithelium to synthesize factors that influence the number and behavior of structural cells and modify extracellular matrix composition and turnover.

Mepolizumab as a corticosteroid-sparing agent in lymphocytic variant hypereosinophilic syndrome

BACKGROUND Mepolizumab, a monoclonal anti-IL-5 antibody, is an effective corticosteroid-sparing agent for patients with Fip1-like 1/platelet-derived growth factor receptor α fusion (F/P)-negative hypereosinophilic syndrome (HES). Lymphocytic variant hypereosinophilic syndrome (L-HES) is characterized by marked overproduction of IL-5 by dysregulated T cells. OBJECTIVE To determine whether patients with L-HES respond to mepolizumab in terms of corticosteroid tapering and eosinophil depletion to the same extent as corticosteroid-responsive F/P-negative patients with HES and a normal T-cell profile. METHODS Patients enrolled in the mepolizumab trial were evaluated for L-HES on the basis of T-cell phenotyping and T-cell receptor gene rearrangement patterns, and their serum thymus-and-activation-regulated chemokine (TARC) levels were measured. Response to treatment was compared in patient subgroups based on results of these analyses. RESULTS Lymphocytic variant HES was diagnosed in 13 of 63 patients with HES with complete T-cell assessments. The ability to taper corticosteroids on mepolizumab was similar in patients with L-HES and those with a normal T-cell profile, although a lower proportion of patients with L-HES maintained eosinophil levels below 600/μL. Increased serum TARC levels (>1000 pg/mL) had no significant impact on the ability to reduce corticosteroid doses, but a lower proportion of patients with elevated TARC achieved eosinophil control on mepolizumab. CONCLUSION Mepolizumab is an effective corticosteroid-sparing agent for patients with L-HES. In some cases however, eosinophil levels remain above 600/μL, suggesting incomplete neutralization of overproduced IL-5 or involvement of other eosinophilopoietic factors.

Identification and validation of shrimp-tropomyosin specific CD4 T cell epitopes.

BACKGROUND Shellfish allergy is an immune-mediated adverse reaction to allergenic shellfish and is responsible for significant morbidity and mortality. CD4 T cell responses play an important role in the pathophysiological mechanisms of sensitization and in production of IgE. OBJECTIVE We sought to identify and validate CD4 T cell shrimp tropomyosin-derived epitopes and characterize CD4 T cell responses in subjects with a clinical history of shellfish allergy. METHOD Using an in vitro MHC-peptide binding assay, we screened 91 overlapping peptides and identified 28 epitopes with moderate and strong binding capacities; 3 additional peptides were included based on MHC binding prediction score. These peptides were then examined in proliferation and cytokine release assays with T cells from allergic subjects. RESULT 17 epitopes restricted to DRB(∗)01:01, DRB1(∗)03:01, DRB1(∗)04:01, DRB1(∗)09:01, DQB1(∗)02:01, DQB1(∗)03:02 and DQB1(∗)05:01 alleles were identified and validated by both the MHC binding and the functional assays. Two peptides showed specificities to more than one MHC class II allele. We demonstrated that these peptides exert functional responses in an epitope specific manner, eliciting predominantly IL-6 and IL-13. CONCLUSION The identified epitopes are specific to common MHC class II alleles in the general population. Our study provides important data for the design of peptide-based immunotherapy of shrimp-allergic patients.

Penaeus monodon tropomyosin induces CD4 T-cell proliferation in shrimp-allergic patients.

Shellfish allergy affects approximately 2% of the population and can cause immediate hypersensitivity reactions such as urticaria, swelling, difficulty breathing, and, in some cases, anaphylaxis. Tropomyosin is the major shrimp allergen and binds IgE in two-thirds of patients. A total of 38 shrimp-allergic patients and 20 negative control subjects were recruited and evaluated on the basis of history, skin prick testing, specific immunoglobulin E (IgE) levels, and peripheral blood mononuclear cell proliferation in response to shrimp tropomyosin or shrimp tropomyosin-derived peptides. Of the classically allergic patients by history, 59% tested positive for serum shrimp IgE antibodies. Of patients with shrimp-specific IgE in sera, 70% also had significant IgE levels specific for shrimp tropomyosin. Peripheral blood mononuclear cells from classically shrimp-allergic patients proliferated in a dose-dependent manner in response to to tropomyosin. In addition, a T-cell line derived from a shrimp-allergic patient proliferated specifically in response to tropomyosin-derived peptides. These studies suggest a strategy for immunotherapy using a tropomyosin-derived T-cell epitope vaccination.

Developing and mature human granulocytes express ELP 6 in the cytoplasm

BACKGROUND c3orf75 is a conserved open reading frame within the human genome and has recently been identified as the Elongator subunit, ELP6 [1]. The Elongator enzyme complex has diverse roles, including translational control, neuronal development, cell migration and tumorigenicity [2]. OBJECTIVE To identify genes expressed early in human eosinophil development. METHODS Eosinophilopoiesis was investigated by gene profiling of IL-5 stimulated CD34+ cells; ELP6 mRNA is upregulated. A monoclonal antibody was raised to the recombinant protein predicted by the open reading frame. RESULTS ELP6 transcripts are upregulated in a human tissue culture model of eosinophil development during gene profiling experiments. Transcripts are expressed in most tissue types, as shown by reverse-transcriptase PCR. Western blot experiments show that human ELP6 is a 30 kDa protein expressed in the bone marrow, as well as in many other tissues. Flow cytometry experiments of human bone marrow mononuclear cells show that ELP6 is expressed intracellularly, in developing and mature human neutrophils, eosinophils and monocytes. CONCLUSIONS ELP6 is expressed intracellularly in developing and mature granulocytes and monocytes but not in lymphocytes and erythrocytes.

Cell screening assay for identifying inhibitors of eosinophil proliferation

The purpose of this study was to develop a cell‐based screening assay for identification of small molecules for the treatment of asthma. Eosinophils are leukocytes that contribute to the pathology of asthma. Lidocaine inhibits interleukin‐5 (IL‐5)‐mediated survival and activation of human eosinophils, and it is able to replace inhaled glucocorticoids for the treatment of asthma; however, lidocaine has many side effects, including anesthesia. Therefore, a collection of commercial and novel, synthesized lidocaine analogues were investigated for inhibitory activity of the IL‐5‐stimulated proliferation of TF‐1 cells, a CD34+, cytokine‐dependent, erythroleukemic cell line model for eosinophil growth. Among 74 investigated compounds, 10 were more potent inhibitors of cell proliferation than lidocaine (average IC50 = 223 µM), with IC50 values ranging within 1–119 µM. This cell‐based assay is an effective method for screening chemical compounds and has revealed promising lead compounds for the treatment of asthma. Drug Dev Res 72: 353–360, 2011. © 2011 Wiley‐Liss, Inc.