Reuben P. Siraganian

Monomeric IgE Stimulates Signaling Pathways in Mast Cells that Lead to Cytokine Production and Cell Survival

Although IgE binding to mast cells is thought to be a passive presensitization step, we demonstrate herein that monomeric IgE (mIgE) in the absence of antigen (Ag) stimulates multiple phosphorylation events in normal murine bone marrow-derived mast cells (BMMCs). While mIgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE + Ag. Moreover, mIgE prevents the apoptosis of cytokine-deprived BMMCs, likely by maintaining Bcl-X(L) levels and producing autocrine-acting cytokines. The addition of Ag does not increase this IgE-induced survival. Since IgE concentrations as low as 0.1 microg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals.

Refinements in the automated fluorometric histamine analysis system.

The automated continuous flow system for the extraction and fluorometric analysis of histamine has been further improved. Samples with or without protein can be analyzed. The use of a single pump, added nitrogen, and lower concentration of o-phthalaldehyde increase the sensitivity by 5-10 fold and allow the analysis of samples at the rate of 30 per hour. Samples which are less than 0.5 ml in volume and contain 0.1 to 10 ng/ml histamine can routinely be analyzed. The sensitivity of the system is now 0.05 ng and compares favorably with isotopic techniques.

Phosphorylation of Syk Activation Loop Tyrosines Is Essential for Syk Function AN IN VIVO STUDY USING A SPECIFIC ANTI-Syk ACTIVATION LOOP PHOSPHOTYROSINE ANTIBODY

Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcεRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcεRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcεRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivoand in vitro. In FcεRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.

Protein tyrosine kinase Syk in mast cell signaling

The tyrosine kinase Syk is essential for signaling from FcrepsilonRI in mast cells. The Src homology domain mediated binding of Syk to the phosphorylated immunoreceptor tyrosine-based motif (ITAM) of the receptor subunits results in a conformational change and activation. Studies in Syk deficient mast cells have defined the pathways that are activated upstream and downstream of Syk and have demonstrated the functional importance of the linker region of Syk in signaling in mast cells.

Conformational changes induced in the protein tyrosine kinase p72syk by tyrosine phosphorylation or by binding of phosphorylated immunoreceptor tyrosine-based activation motif peptides.

A critical event in signaling in immune cells is the interaction of Syk or ZAP-70 protein tyrosine kinases with multisubunit receptors that contain an approximately 18-amino-acid domain called the immunoreceptor tyrosine-based activation motif (ITAM). Tyrosine-phosphorylated Syk from activated cells was in a conformation different from that in nonstimulated cells as demonstrated by changes in immunoreactivity. The addition of tyrosine-diphosphorylated ITAM peptides resulted in a similar conformational change in Syk from nonactivated cells. The peptides based on FcepsilonRIgamma were more active than those based on Fcepsilon RIbeta. In vitro autophosphorylation of Syk was dramatically enhanced by the addition of the diphosphorylated ITAM peptides. The conformational change and the enhanced autophosphorylation required the presence of both phosphorylated tyrosines on the same molecule. These conformational changes in Syk by tyrosine phosphorylation or binding to diphosphorylated ITAM could be critical for Syk activation and downstream propagation of intracellular signals.

Protein-tyrosine phosphorylation: an essential component of FcεRI signaling

Mast cell and basophil activation can be achieved by antigen-mediated aggregation of cell surface FceRI molecules. At least two signaling pathways are triggered by this activation. Both involve tyrosine phosphorylation. This aspect of FceRI signaling is examined here in detail and its position in a complex network of post-binding events assessed.

THE PROTEIN-TYROSINE PHOSPHATASE SHP-2 ASSOCIATES WITH TYROSINE-PHOSPHORYLATED ADHESION MOLECULE PECAM-1 (CD31)

Aggregation of many cell-surface receptors results in tyrosine phosphorylation of numerous proteins. We previously observed the tyrosine phosphorylation of the platelet/endothelial cell adhesion molecule, PECAM-1 (CD31), after FcεRI stimulation in rat basophilic leukemia RBL-2H3 cells. Here we found that PECAM-1 was also transiently tyrosine-phosphoryated after adherence of these cells to fibronectin. Similarly aggregation of the T cell receptor on Jurkat cells also induced this tyrosine phosphorylation. The protein-tyrosine phosphatase SHP-2 is a widely expressed cytosolic enzyme with two Src homology 2 (SH2) domains. SHP-2, but not the related protein-tyrosine phosphatase SHP-1, associated with PECAM-1. This association of the two proteins correlated with the extent of the tyrosine phosphorylation of PECAM-1. A fusion protein containing the two SH2 domains of SHP-2 precipitated PECAM-1 from cell lysates and also directly bound to phosphorylated PECAM-1. In immune precipitate phosphatase assays, there was tyrosine dephosphorylation of PECAM-1. Therefore, integrin and immune receptor activation results in tyrosine phosphorylation of PECAM-1 and the binding of the protein-tyrosine phosphatase SHP-2, which could regulate receptor-mediated signaling in cells.

Automated histamine analysis for in vitro allergy testing: I. A method utilizing allergen-induced histamine release from whole blood

A sensitive, automated, histamine assay system has been developed and applied for in vitro allergy testing. Nine common pollen and environmental allergens were used at three log dilutions for in vivo studies utilizing small volumes of blood (15-20 ml). The clinical evaluation was correlated with the results of the histamine release. two different procedures were utilized. The first is the commonly used histamine release from washed leukocytes. There was excellent correlation between the clinical evaluation and the results of histamine release from washed leukocytes in 17 different individuals. The second and simpler method utilized whole heparinized blood which might better reflect the immunologic reaction which occurs in vivo. Aliquots of blood and allergen were incubated for 1 hr at 37 degrees C and each supernatant was then analyzed for histamine release. There was excellent correlation between the two tests in 29 patients tested by both the whole blood and washed leukocyte methods. There was also good correlation between the clinical evaluation of the patients amd the intro tests. The precision, accuracy, and sensitivity of the automated histamine assay make feasible its routine application in the clinical study of allergic patients.

Adhesion molecules as regulators of mast-cell and basophil function

Basophils and mast cells play a role both in immediate allergic reactions and in inflammation. Both types of cells have surface adhesion receptors that can mediate binding to other cells and to extracellular matrix glycoproteins. Here Majed Hamawy and colleagues discuss the importance of these adhesion molecules in regulating basophil and mast-cell functions.

Dendritic Cell Modulation by Mast Cells Controls the Th1/Th2 Balance in Responding T Cells

The cytokines secreted by pathogen-activated human dendritic cells (DC) are strongly regulated in vitro by histamine, a major component of mast cell granules, ultimately modulating the capacity of the DC to polarize naive T cells. Because DC and mast cells are located in close proximity in peripheral compartments, we hypothesized that mast cell products would influence the maturation of DC and hence the Th balance of an immune response in vivo. In this study, we show that specific mast cell degranulation stimuli, given s.c. in mice with Ag and adjuvant, produce effector T cells that proliferate to Ag but secrete dramatically reduced levels of IFN-γ and increased amounts of IL-4 compared with control T cells primed in the absence of a mast cell stimulus. Immunization with Ag and adjuvant in the presence of a degranulation stimulus also resulted in the accumulation of DC in the draining lymph nodes that had reduced capacity to induce Ag-specific Th1 cells, in comparison with DC from mice lacking a degranulation stimulus. Therefore, by acting upon DC at sites of inflammation, mast cells play a critical role in determining the polarity of Ag-specific T cell responses in vivo.