The Influence of IgE on Cultured Human Mast Cells.

PURPOSE: The mast cell plays a pivotal role in the human immune response. Crosslinking of 2 IgE molecules bound to the high affinity IgE receptor (FcεRI) on the surface of the mast cell results in mast cell degranulation and the release of several proinflammatory mediators. Patients with type-I allergy have increased levels of IgE in the blood compared to healthy individuals. METHODS: In a 6-week culture system of stem cells to human mast cells we investigated the effect of the concentration of IgE. The mast cells were cultured with different concentrations of IgE for the last 10 days of the maturation period. It was observed how the IgE concentration affects the histamine release, FcεRI density on the mast cell surface and the concentration of other mediators. RESULTS: A clear correlation between IgE concentration in culture medium and the release of histamine upon activation was observed. It showed a bell-shaped dose response curve, with maximal response around an IgE-concentration of 250 ng/mL. Furthermore, the sensitivity of the mast cells and surface density of FcεRI on mast cell surface was also influenced by the IgE concentration in the culture medium. CONCLUSIONS: IgE in the culture medium during the last 10 days of mast cell maturation influences the release of the preformed mediator histamine after mast cell activation and the density of FcεRI on the mast cell surface. The release of the de novo synthetized mediator prostaglandin D2 and the expression of chymase and tryptase are not influenced by IgE in culture medium.

Mast cell FcϵRI density and function dissociate from dependence on soluble IgE concentration at very low and very high IgE concentrations.

OBJECTIVE: The contribution of affinity, clonality, and concentration of individual IgE species to effector cell response has recently been characterized in a model with recombinant human IgE on passively sensitized basophils. This study extends the dependence of effector cell degranulation on IgE concentration to mast cells cultured with IgE for 2 weeks. METHODS: Human mast cells cultured for 7 weeks from peripheral blood stem cells were matured for 2 weeks with interleukin-4 (IL-4) and recombinant human IgE consisting of two clones specific for Dermatophagoides pteronyssinus 2 (Derp2) (7% + 7%) and unspecific IgE at 0.8, 8, 80, and 800 kU/L. The density of the IgE receptor, FcϵRI, and mast cell function were measured after challenging with recombinant Derp2 at 14 concentrations from 10 fg/mL to 100 pg/mL. CD63 expression, histamine release, and Prostaglandin D2 (PGD(2)) synthesis were measured, and maximal expression and mast cell sensitivity were calculated. RESULTS: At 800 kU/L IgE, FcϵRI expression varied more than at 80, 8, and 0.8 kU/L IgE. There was a trend toward increased maximal expression of CD63, histamine release, and PGD(2) secretion with increasing IgE concentration. At 0.1 kU/L specific IgE, the LC50 increased up to fivefold, least so for PGD(2). CONCLUSIONS: Human mast cells cultured with rhIgE of known composition are a sensitive model for studying factors governing effector cell degranulation that is close to the in vivo situation. This model can be used to study effects of IgE concentration, clonality, and affinity and may help predict the optimal immunologic treatment for a given patient.

Cultured human mast cells are heterogeneous for expression of the high-affinity IgE receptor FcεRI.

OBJECTIVE: We determined the density of FcεRI on mast cells cultured from cord (CBMC) and peripheral blood (PBMC) and studied the kinetics of the response through FcεRI. METHODS: Mast cells were cultured from CD133+ progenitors from peripheral or cord blood. FcεRI was stabilized by culture with 2 µg/ml IgE. Cells were activated by addition of anti-FcεRI antibody (1 ng/ml-10 µg/ml). Maximal activation, sensitivity, and cooperativity were determined. RESULTS: All cultures were homogeneous for tryptase and metachromasy. All cells expressing FcεRI could be activated by cross-linking FcεRI to upregulate CD63. PBMC bind 203,000 molecules of IgE/cell. Stabilization of FcεRI with IgE doubled the number of CD63+ cells (p = 0.0001) and increased the sensitivity (from 0.083 to 0.013 µg/ml anti-FcεRI) and the slope factor (from 10.8 to 68) of PBMC but not of CBMC. Anti-IgE reversed these effects (p = 0.0002) but did not reduce activation levels below that of cell lines not stabilized with IgE. CONCLUSION: Baseline expression of FcεRI is independent of anti-IgE. The fraction of PBMC that binds high levels of IgE can be activated through FcεRI.

Molecular cloning, characterization and developmental expression of porcine beta-synuclein.

The synuclein family includes three known proteins: alpha-synuclein, beta-synuclein and gamma-synuclein. beta-Synuclein inhibits the aggregation of alpha-synuclein, a protein involved in Parkinson's disease. We have cloned and characterized the cDNA sequence for porcine beta-synuclein (SNCB) from pig cerebellum using RT-PCR. Expression analysis by quantitative RT-PCR demonstrated that SNCB transcripts were highly abundant in brain tissues. SNCB mRNA was also detected early in embryogenesis and significant increases in transcript levels were observed in several brain tissues during embryo development. Radiation hybrid mapping data indicate that the porcine SNCB maps to the q arm of chromosome 2 (2q21-22). The subcellular localization of recombinant porcine beta-synuclein was determined in three different cell types and shown to be cytoplasmic.

Porcine gamma-synuclein: molecular cloning, expression analysis, chromosomal localization and functional expression.

The gamma-synuclein protein is involved in breast carcinogenesis and has also been implicated in other forms of cancer and in ocular diseases. Furthermore, gamma-synuclein is believed to have a role in certain neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. This work reports the cloning and characterization of the porcine (Sus scrofa) gamma-synuclein cDNA (SNCG). The SNCG cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine SNCG cDNA codes for a protein of 126 amino acids which shows a high similarity to bovine (90%), human (87%) and mouse (83%) gamma-synuclein. A genomic clone containing the entire porcine SNCG gene was isolated and its genomic organization determined. The gene is composed of five exons, the general structure being observed to be very similar to that of the human SNCG gene. Expression analysis by quantitative real-time RT-PCR revealed the presence of SNCG transcripts in all examined organs and tissues. Differential expression was observed, with very high levels of SNCG mRNA in fat tissue and high expression levels in spleen, cerebellum, frontal cortex and pituitary gland. Expression analysis also showed that porcine SNCG transcripts could be detected in different brain regions during early stages of embryo development. The porcine SNCG orthologue was mapped to chromosome 14q25-q29. The distribution of recombinant porcine gamma-synuclein was studied in three different transfected cell lines and the protein was found to be predominantly localized in the cytoplasm.