Becky M. Vonakis

TRPV2 has a pivotal role in macrophage particle binding and phagocytosis

Macrophage phagocytosis is critical for defense against pathogens. Whereas many steps of phagocytosis involve ionic flux, the underlying ion channels remain ill defined. Here we show that zymosan-, immunoglobulin G (IgG)- and complement-mediated particle binding and phagocytosis were impaired in macrophages lacking the cation channel TRPV2. TRPV2 was recruited to the nascent phagosome and depolarized the plasma membrane. Depolarization increased the synthesis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), which triggered the partial actin depolymerization necessary for occupancy-elicited phagocytic receptor clustering. TRPV2-deficient macrophages were also defective in chemoattractant-elicited motility. Consequently, TRPV2-deficient mice showed accelerated mortality and greater organ bacterial load when challenged with Listeria monocytogenes. Our data demonstrate the participation of TRPV2 in early phagocytosis and its fundamental importance in innate immunity.

Mapping of numerous disease-associated expression polymorphisms in primary peripheral blood CD4+ lymphocytes

Genome-wide association studies of human gene expression promise to identify functional regulatory genetic variation that contributes to phenotypic diversity. However, it is unclear how useful this approach will be for the identification of disease-susceptibility variants. We generated gene expression profiles for 22 184 mRNA transcripts using RNA derived from peripheral blood CD4+ lymphocytes, and genome-wide genotype data for 516 512 autosomal markers in 200 subjects. We screened for cis-acting variants by testing variants mapping within 50 kb of expressed transcripts for association with transcript abundance using generalized linear models. Significant associations were identified for 1585 genes at a false discovery rate of 0.05 (corresponding to P-values ranging from 1 × 10(-91) to 7 × 10(-4)). Importantly, we identified evidence of regulatory variation for 119 previously mapped disease genes, including 24 examples where the variant with the strongest evidence of disease-association demonstrates strong association with specific transcript abundance. The prevalence of cis-acting variants among disease-associated genes was 63% higher than the genome-wide rate in our data set (P = 6.41 × 10(-6)), and although many of the implicated loci were associated with immune-related diseases (including asthma, connective tissue disorders and inflammatory bowel disease), associations with genes implicated in non-immune-related diseases including lipid profiles, anthropomorphic measurements, cancer and neurologic disease were also observed. Genetic variants that confer inter-individual differences in gene expression represent an important subset of variants that contribute to disease susceptibility. Population-based integrative genetic approaches can help identify such variation and enhance our understanding of the genetic basis of complex traits.

New concepts in chronic urticaria

Chronic urticaria is a common skin disease without a clear etiology in the vast majority of cases. The similarity of symptoms and lesion pathology to allergen-induced skin reactions supports the idea that skin mast cell and blood basophil IgE receptor activation is involved; however, no exogenous allergen trigger has been identified. The presence of serum IgG autoantibodies targeting IgE or the IgE receptor in approximately 40% of CIU cases supports the theory of an autoimmune basis for the disease. However, issues remain with the assays to detect autoantibodies among other serum factors, the relationship of autoantibodies to CIU disease activity, and the occurrence of autoantibodies in healthy subjects. Other studies have identified altered IgE receptor degranulation that reverts in disease remission and is accompanied by changes in signaling molecule expression and function in mast cells and basophils in active CIU subjects. The arrival of therapies targeting IgE and the IgE receptor pathway elements has potential use in CIU.

Evidence of in vivo basophil activation in chronic idiopathic urticaria

Background Approximately 40% of chronic idiopathic urticaria (CIU) subjects have autoantibodies to either FcɛRIα or IgE. The effect of such autoantibodies on circulating basophil activation status is unknown.

Aryl hydrocarbon receptor controls murine mast cell homeostasis

We propose that the aryl hydrocarbon receptor (AhR), a unique chemical sensor, is critical in controlling mast cell differentiation, growth, and function in vitro and in vivo. In antigen-stimulated mast cells, exposure to AhR ligands resulted in a calcium- and reactive oxygen species (ROS)-dependent increase of reversible oxidation in and reduced activity of SHP-2 phosphatase, leading to enhanced mast cell signaling, degranulation, and mediator and cytokine release, as well as the in vivo anaphylactic response. Surprisingly, significant mast cell deficiency was noted in AhR-null mice due to defective calcium signaling and mitochondrial function, concomitant with reduced expression of c-kit and cytosolic STAT proteins, as well as enhanced intracellular ROS and apoptosis. Consequently, AhR-null mast cells responded poorly to stimulation, demonstrating a critical role of AhR signaling in maintaining mast cell homeostasis.

Chronic Helminth Infection Reduces Basophil Responsiveness in an IL-10–Dependent Manner

Basophils play a key role in the development and effector phases of type 2 immune responses in both allergic diseases and helminth infections. This study shows that basophils become less responsive to IgE-mediated stimulation when mice are chronically infected with Litomosoides sigmodontis, a filarial nematode, and Schistosoma mansoni, a blood fluke. Although excretory/secretory products from microfilariae of L. sigmodontis suppressed basophils in vitro, transfer of microfilariae into mice did not result in basophil suppression. Rather, reduced basophil responsiveness, which required the presence of live helminths, was found to be dependent on host IL-10 and was accompanied by decreases in key IgE signaling molecules known to be downregulated by IL-10. Given the importance of basophils in the development of type 2 immune responses, these findings help explain the mechanism by which helminths protect against allergy and may have broad implications for understanding how helminth infections alter other disease states in people.

A genome-wide survey of CD4+ lymphocyte regulatory genetic variants identifies novel asthma genes

BACKGROUND Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing. OBJECTIVE We evaluated 6706 cis-acting expression-associated variants (eSNPs) identified through a genome-wide eQTL survey of CD4(+) lymphocytes for association with asthma. METHODS eSNPs were tested for association with asthma in 359 asthmatic patients and 846 control subjects from the Childhood Asthma Management Program, with verification by using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by using formaldehyde-assisted isolation of regulatory elements (FAIRE) quantitative PCR and chromatin immunoprecipitation PCR in lung-derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes. RESULTS Cis-acting eSNPs demonstrated associations with asthma in both cohorts. We confirmed the previously reported association of ORMDL3/GSDMB variants with asthma (combined P = 2.9 × 10(-8)). Reproducible associations were also observed for eSNPs in 3 additional genes: fatty acid desaturase 2 (FADS2; P = .002), N-acetyl-α-D-galactosaminidase (NAGA; P = .0002), and Factor XIII, A1 (F13A1; P = .0001). Subsequently, we demonstrated that FADS2 mRNA is increased in CD4(+) lymphocytes in asthmatic patients and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity. CONCLUSIONS Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.

Histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced histamine release is enhanced with SHIP-1 knockdown in cultured human mast cell and basophil models

Previously, we demonstrated a negative correlation between histamine release to histamine‐releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of SHIP‐1 in human basophils. The present study was conducted to investigate whether suppressing SHIP‐1 using small interfering (si)RNA technology would alter the releasability of culture‐derived mast cells and basophils, as determined by HRF/TCTP histamine release. Frozen CD34+ cells were obtained from the Fred Hutchinson Cancer Research Center (Seattle, WA, USA). Cells were grown in StemPro‐34 medium containing cytokines: mast cells with IL‐6 and stem cell factor (100 ng/ml each) for 6–8 weeks and basophils with IL‐3 (6.7 ng/ml) for 2–3 weeks. siRNA transfections were performed during Week 6 for mast cells and Week 2 for basophils with siRNA for SHIP‐1 or a negative control siRNA. Changes in SHIP‐1 expression were determined by Western blot. The functional knockdown was measured by HRF/TCTP‐induced histamine release. siRNA knockdown of SHIP‐1 in mast cells ranged from 31% to 82%, mean 65 ± 12%, compared with control (n=4). Histamine release to HRF/TCTP was increased only slightly in two experiments. SHIP‐1 knockdown in basophils ranged from 34% to 69%, mean 51.8 ± 7% (n=4). Histamine release to HRF/TCTP in these basophils was dependent on the amount of SHIP knockdown. Mast cells and basophils derived from CD34+ precursor cells represent suitable models for transfection studies. Reducing SHIP‐1 protein in cultured mast cells and in cultured basophils increases releasability of the cells.

Agonists of the Mas-Related Gene (Mrgs) Orphan Receptors as Novel Mediators of Mast Cell-Sensory Nerve Interactions

IgE-dependent activation of mast cell activation is often associated with symptoms attributed to activation of sensory nerves. Depending on the tissues involved such symptoms include itching, sneezing, irritation, vasodilation, and reflex secretions. In the present study, we hypothesize that sensory neuroactive mediators released from mast cells may include agonists of recently discovered orphan receptors referred to as sensory nerve specific receptors or products of mas related genes. HEK-293 cells expressing MrgC11 receptors and wild-type HEK-293 cells were loaded with the calcium indicator Fura-2. A known stimulant of MrgC11 receptors the RF-amide, neuropeptide FF, evoked a rapid increase in cytosolic calcium in the MrgC11 expressing cells but not in the wild-type HEK-293 cells. IgE-dependent stimulation of either rat basophilic leukemia-2H3 cells (RBL-2H3 cells) or mouse bone marrow-derived mast cells, released a substance(s) that stimulated increases in cytosolic calcium in the MrgC11 expressing cells that far exceeded that seen in control cells. RT-PCR revealed that both mouse mast cells and RBL-2H3 cells express the RF-amide precursor gene proneuropeptide FF (A). Immunohistochemical analysis demonstrated RF-amide immunoreactivity in mouse skin mast cells in situ and in mast cells isolated from mouse skin. These data support the hypothesis that agonists of certain sensory nerve specific receptors or mas related genes may participate in mast cell sensory nerve interactions.

Cultured peripheral blood mast cells from chronic idiopathic urticaria patients spontaneously degranulate upon IgE sensitization: Relationship to expression of Syk and SHIP-2.

Recently, signaling changes in the FcvarepsilonRI pathway involving inositol lipid phosphatases have been identified in the basophils of chronic idiopathic urticaria (CIU) subjects. Based on the profile of basophil FcvarepsilonRI-mediated histamine degranulation, we have segregated CIU subjects into two groups, CIU Responder (CIU R) or CIU Nonresponder (CIU NR). In the present study, we compared expression of SHIP-1, SHIP-2, and Syk protein to histamine release (HR) from mast cells (MC) cultured from the peripheral blood of CIU R, CIU NR, and normal subjects. The MC of CIU R donors contained significantly increased Syk and decreased SHIP-2 as compared to CIU NR (Syk: p=0.038, SHIP-2: p=0.038) and normals (Syk: p=0.042, SHIP-2: p=0.027). Spontaneous HR from CIU donors was increased two-fold compared to normals (p=0.04). In summary, our results suggest a possible predilection for urticarial MC to spontaneously degranulate upon IgE sensitization contributing to the increased pruritus associated with CIU.