Elsa H. Berenstein

Phosphorylation of Tyr342 in the Linker Region of Syk Is Critical for FcεRI Signaling in Mast Cells

ABSTRACT The linker region of Syk and ZAP70 tyrosine kinases plays an important role in regulating their function. There are three conserved tyrosines in this linker region; Tyr317 of Syk and its equivalent residue in ZAP70 were previously shown to negatively regulate the function of Syk and ZAP70. Here we studied the roles of the other two tyrosines, Tyr342 and Tyr346 of Syk, in FcεRI-mediated signaling. Antigen stimulation resulted in Tyr342 phosphorylation in mast cells. Syk with Y342F mutation failed to reconstitute FcεRI-initiated histamine release. In the Syk Y342F-expressing cells there was dramatically impaired receptor-induced phosphorylation of multiple signaling molecules, including LAT, SLP-76, phospholipase C-γ2, but not Vav. Compared to wild-type Syk, Y342F Syk had decreased binding to phosphorylated immunoreceptor tyrosine-based activation motifs and reduced kinase activity. Surprisingly, mutation of Tyr346 had much less effect on FcεRI-dependent mast cell degranulation. An anti-Syk-phospho-346 tyrosine antibody indicated that antigen stimulation induced only a very minor increase in the phosphorylation of this tyrosine. Therefore, Tyr342, but not Tyr346, is critical for regulating Syk in mast cells and the function of these tyrosines in immune receptor signaling appears to be different from what has been previously reported for the equivalent residues of ZAP70.

[2] Methods of enhancing the frequency of antigen-specific hybridomas

Publisher Summary This chapter discusses the methods of enhancing the frequency of antigen-specific hybridomas. The hybridization of spleen cells from immunized mice with plasmacytoma cell lines is a powerful method for the production of monoclonal antibodies. However, the development of monoclonal antibodies has been hampered by low frequency of antigen-specific hybridomas recovered after cell fusion. In an effort to improve the yield of hybridomas, the chapter discusses two techniques to select and expand the number of spleen cells producing specific antibody prior to fusion. The first method is the adoptive transfer of spleen cells from immunized animals to X-irradiated syngeneic recipients followed by in vivo antigen boosting. In the second method, spleen cells from immunized mice have been cultured with antigen before fusion. The use of either of these methods resulted in a 10-to 50-fold increase in the percentage of antigen-specific antibody-secreting hybridomas. These methods for enhancing the yield of antigen-specific hybridomas and the techniques used for cell fusion and cloning by limiting dilution are described in this chapter.

AGGREGATION OF IGE RECEPTORS IN RAT BASOPHILIC LEUKEMIA 2H3 CELLS INDUCES TYROSINE PHOSPHORYLATION OF THE CYTOSOLIC PROTEIN-TYROSINE PHOSPHATASE HEPTP

The cDNA encoding the rat equivalent of the human hematopoietic tyrosine phosphatase, also known as leukocyte phosphatase, was isolated from a rat basophilic leukemia mast cell cDNA library. By two-dimensional electrophoresis, the protein expressed in the mast cells was of a size (40 kDa) and pI (6.9) predicted from the deduced amino acid sequence. Thus, although previously shown to be preferentially expressed in T cells and B cells, the phosphatase is also found in mast cells. By immunofluorescence microscopy, rat hematopoietic tyrosine phosphatase localized to discrete, globular compartments within the cytoplasm and was not found either in the nucleus or associated with the cell surface membrane. Aggregation of high affinity IgE receptors in the mast cells induced tyrosine phosphorylation of the phosphatase. The tyrosine phosphorylation was mimicked by stimulation with calcium ionophore A23187 but not by direct activation of protein kinase C. Since phosphorylation of the phosphatase was dramatically reduced when the cells were activated in Ca-free media, it is dependent on a rise in intracellular Ca. These data strongly suggest that hematopoietic tyrosine phosphatase may be involved in the IgE receptor-mediated signaling cascade.

FcεRI -INDUCED ACTIVATION BY LOW ANTIGEN CONCENTRATIONS RESULTS IN NUCLEAR SIGNALS IN THE ABSENCE OF DEGRANULATION

High affinity IgE receptor (FcvarepsilonRI)-induced activation of mast cells results in degranulation and generation of leukotrienes and cytokines. FcvarepsilonRI-induced mast cell activation was analyzed at a single cell basis using a rat basophilic leukemia (RBL-2H3) cell line transfected with a reporter plasmid containing three tandem NFAT (nuclear factor of activated T cells) binding sites fused to enhanced green fluorescent protein (GFP). Surprisingly, with this sensitive detection system, there is activation of IgE sensitized cells at concentrations of antigen as low as 10pg/ml, which was 10-fold lower than was detected by degranulation. There were differences in signaling pathways leading to degranulation compared to NFAT-mediated gene activation. Both signaling to NFAT activation and degranulation required Syk and calcineurin. However inhibitors of the phosphatidylinositol 3-kinase pathway blocked degranulation but did not NFAT activation. The results also indicate that NFAT was activated at lower intracellular signals compared to degranulation. Therefore, FcvarepsilonRI activation can result in nuclear signals in the absence of the release of mediators.

Identification and Isolation of Rat Bone Marrow-derived Mast Cells Using the Mast Cell-specific Monoclonal Antibody AA4

SUMMARY Previous studies of mast cell maturation, structure, and function have been hampered by the lack of mast cell-specific markers. In this study, using a well-characterized mast cell-specific monoclonal antibody, MAb AA4, mast cells from rat bone marrow in various stages of maturation were isolated and characterized. The very immature mast cells, which have not been previously described, contained few granules and would not be recognized as mast cells by standard cytological methods. Pure populations of mast cells were isolated from the bone marrow using MAb AA4-conjugated magnetic beads. The same stages of maturation were observed in the isolated mast cells as were seen in the unfractionated bone marrow. All of these cells were immunopositive for the α-subunit of Fc∊RI, IgE, and c-kit, confirming their identity as mast cells. By direct counting of immunolabled cells and by flow cytometry, approximately 2.4% of the cells in the bone marrow are mast cells. Staining with toluidine blue and berberine sulfate, as well as RT-PCR of the cells, indicates that these cells are connective tissue-type mast cells. The use of immunological methods for identification of mast cell precursors should facilitate the study of these cells. (J Histochem Cytochem 49:219–228, 2001)

Monoclonal antibodies defining epitopes on human IgE

Twelve monoclonal antibodies (mAb) were isolated that bound to six clusters of epitopes on the constant region of the epsilon chain of human IgE. Four of the mAb bound to the C epsilon 1 or early C epsilon 2 regions; three of these bound to the IgE myeloma protein PS and to serum IgE but not to the IgE myeloma protein ND. These mAb probably recognize an allotypic marker. Another mAb reacted with heat-denatured, but not native IgE. Four of the mAb failed to release histamine; the epitopes recognized by these mAb are in the C epsilon 1, C epsilon 2 and C epsilon 3-4 regions of IgE. Three of these non-histamine releasing mAb did not bind to IgE on the basophil surface. These mAb recognize epitopes in C epsilon 2 and C epsilon 3-4 that are not accessible when IgE is bound to its receptor. Four mAb inhibited IgE binding to basophils; two of these did not release histamine, and two others that bind to epitopes in the C epsilon 2-4 domain, released histamine and therefore blocked IgE binding by steric hindrance. Inhibition of IgE binding by different mAb suggest that the Fc epsilon RI and Fc epsilon RII bind to partly overlapping regions of the IgE molecule although the sites do not appear to be identical. A number of sites on C epsilon 1 and C epsilon 3-4 were accessible when IgE is bound to its basophil receptor. The data support the concept that only part of the Fc portion of IgE is hidden in the receptor and that portions of C epsilon 1-4 are accessible on the cell surface. These mAb should be useful in determining the domains of IgE that are critical for its biological activity.

The receptor with high affinity for IgE on rat mast cells is a functional receptor for rat IgG2a

Rat mast cells express high-affinity receptors for IgE (Fc epsilon RI) and low-affinity receptors for IgG (Fc gamma R). In this study, the capacity of IgG to activate the rat basophilic leukemia (RBL-2H3) and rat peritoneal mast cells was investigated. Immune complexes formed with purified rat IgG and antigen as well as chemically cross-linked rat IgG induced histamine release from RBL-2H3 cells. This stimulation was inhibited by pre-incubation of the cells with saturating concentrations of monomeric IgE. With chemically cross-linked rat IgG of each subclass, only IgG2a stimulated histamine release from RBL-2H3 cells and this release was also inhibited by prior saturation of the Fc epsilon RI with monomeric IgE. Identical results were obtained with rat peritoneal mast cells. In binding experiments, IgE and cross-linked rat IgG2a bound to rat Fc epsilon RI transfected into CHO cells. Monomeric rat IgG2a, cross-linked rat IgG1, IgG2b, IgG2c and rabbit IgG did not bind to Fc epsilon RI. Stimulation of RBL-2H3 cells with aggregated IgG2a induced phosphorylation of tyrosines in the beta and gamma subunits of the Fc epsilon RI. Thus, although RBL-2H3 and rat peritoneal mast cells have Fc gamma R, the IgG-mediated stimulation of these cells for histamine release was by the Fc epsilon RI. Altogether, these data demonstrate that the rat Fc epsilon RI is a functional receptor with low affinity for rat IgG2a.

Characterization of monoclonal antibodies produced by immunization with partially purified IgE receptor complexes

A series of monoclonal antibodies (mAb) were produced following the immunization of mice with partially purified IgE receptors from the rat basophilic leukemia cell line (RBL-2H3). Twelve hybridoma cell lines were selected that secreted monoclonal antibodies capable of binding RBL-2H3 plasma membranes. These antibodies were all of the IgG1, or IgG2a subclass. All 12 antibodies bound to either intact or glutaraldehyde-fixed RBL-2H3 cells. Only one monoclonal (mAb 2AC3) inhibited 125I-labeled IgE binding (IC50 = 65 micrograms/ml compared to 1.0 microgram/ml for unlabeled IgE). This same mAb weakly precipitated the alpha component of the receptor from 125I-surface-labeled cells and directly triggered histamine secretion when incubated with RBL-2H3 cells. Therefore, this hybridoma most likely represents a low affinity anti-receptor antibody. Among the other 11 monoclonals, two caused direct histamine secretion from RBL-2H3 cells. These same two, as well as four others, released greater than 10% of total cellular histamine when rabbit anti-mouse antibody was added to cross-link mAb bound to the cell surface. One monoclonal (mA 1AD3) did not trigger histamine secretion but did inhibit IgE-mediated histamine release when incubated with pre-sensitized RBL-2H3 cells. Except for mAb 2AC3, none of the other monoclonal antibodies that caused or inhibited histamine secretion immunoprecipitated receptor or other protein components from either 125I-surface-labeled or intrinsically-labeled cells. However, two monoclonal antibodies (mAb 1CC4 and 1CD1) immunoprecipitated 45,000 and 55,000 proteins from 125I-surface-labeled cells. One hybridoma (mAb 2AA2) that failed to immunoprecipitate surface-labeled proteins did precipitate a 20,000 band from intrinsically-labeled cells. This band increased slightly in apparent mol. wt after reduction and, therefore, was not the previously described gamma component of the receptor. Because several mAb were capable of modulating histamine secretion, it appeared that some of the present monoclonal antibodies bound to undefined membrane components that are crucial to the secretory process of rat basophilic leukemia cells.

Alterations in Granule Matrix and Cell Surface of Focal Adhesion Kinase-Deficient Mast Cells

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcεRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK−/− embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing β7 integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcεRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.

The Transcription Factor Zeb2 Regulates Signaling in Mast Cells

Mast cell activation results in the release of stored and newly synthesized inflammatory mediators. We found that Zeb2 (also named Sip1, Zfhx1b), a zinc finger transcription factor, regulates both early and late mast cell responses. Transfection with small interfering RNA (siRNA) reduced Zeb2 expression and resulted in decreased FcεRI-mediated degranulation, with a parallel reduction in receptor-induced activation of NFAT and NF-κB transcription factors, but an enhanced response to the LPS-mediated activation of NF-κB. There was variable and less of a decrease in the Ag-mediated release of the cytokines TNF-α, IL-13, and CCL-4. This suggests that low Zeb2 expression differentially regulates signaling pathways in mast cells. Multiple phosphorylation events were impaired that affected molecules both at early and late events in the signaling pathway. The Zeb2 siRNA-treated mast cells had altered cell cycle progression, as well as decreased expression of several molecules including cell surface FcεRI and its β subunit, Gab2, phospholipase-Cγ1, and phospholipase-Cγ2, all of which are required for receptor-induced signal transduction. The results indicate that the transcription factor Zeb2 controls the expression of molecules thereby regulating signaling in mast cells.