In vitro modelling of rat mucosal mast cell function in Trichinella spiralis infection.

Intestinal infection with the parasitic nematode, Trichinella spiralis, provides a robust context for the study of mucosal mast cell function. In rats, mucosal mast cells are exposed to parasites during the earliest stage of infection, affording an opportunity for mast cells to contribute to an innate response to infection. During secondary infection, degranulation of rat mucosal mast cells coincides with expulsion of challenge larvae from the intestine. The goal of this study was to evaluate the rat bone marrow-derived mast cells (BMMC) and the rat basophilic leukaemia cell line (RBL-2H3) as models for mucosal mast cells, using parasite glycoproteins and antibody reagents that have been tested extensively in rats in vivo. We found that BMMC displayed a more robust mucosal phenotype. Although T. spiralis glycoproteins bound to mast cell surfaces in the absence of antibodies, they did not stimulate degranulation, nor did they inhibit degranulation triggered by immune complexes. Parasite glycoproteins complexed with specific monoclonal IgGs provoked release of rat mast cell protease II (RMCPII) and ß-hexosaminidase from both cell types in a manner that replicated results observed previously in passively immunized rats. Our results document that RBL-2H3 cells and BMMC model rat mucosal mast cells in the contexts of innate and adaptive responses to T. spiralis.

Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes.

The mechanisms by which a cell uses and adapts its functional membrane organization are poorly understood and are the subject of ongoing investigation and discussion. Here, we study one proposed mechanism: the crosslinking of membrane components. In immune cell signaling (and other membrane-associated processes), a small change in the clustering of specific membrane proteins can lead to large-scale reorganizations that involve numerous other membrane components. We have investigated the large-scale physical effect of crosslinking a minor membrane component, the ganglioside GM1, in simple lipid models of the plasma membrane containing sphingomyelin, cholesterol, and phosphatidylcholine. We observe that crosslinking GM1 can cause uniform membranes to phase-separate into large, coexistent liquid ordered and liquid disordered membrane domains. We also find that this lipid separation causes a dramatic redistribution of a transmembrane peptide, consistent with a raft model of membrane organization. These experiments demonstrate a mechanism that could contribute to the effects of crosslinking observed in cellular processes: Domains induced by clustering a small number of proteins or lipids might rapidly reorganize many other membrane proteins.

Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells.

Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, and their liquid ordered (L(o)) phase appears to be important for their function. To quantify ordered lipids in biological membranes, we investigated steady-state fluorescence anisotropy of two lipid probes, 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). We show using model membranes with varying amounts of cholesterol that steady-state fluorescence anisotropy is a sensitive measure of cholesterol-dependent ordering. The results suggest that DPH-PC is a more sensitive probe than NBD-PE. In the presence of cholesterol, ordering also depends on the degree of saturation of the phospholipid acyl chains. Using DPH-PC, we find that the plasma membrane of RBL-2H3 mast cells is substantially ordered, roughly 40%, as determined by comparison with anisotropy values for model membranes entirely in a liquid ordered (L(o)) phase and in a liquid disordered (L(alpha)) phase. This result is consistent with the finding that approximately 30% of plasma membrane phospholipids are insoluble in 0.5% Triton X-100. Furthermore, detergent-resistant membranes isolated by sucrose gradient fractionation of Triton X-100 cell lysates are more ordered than plasma membrane vesicles, suggesting that they represent a more ordered subset of the plasma membrane. Treatment of plasma membrane vesicles with methyl-beta-cyclodextrin resulting in 75% cholesterol depletion leads to commensurate decreases in lipid order as measured by anisotropy of DPH-PC and NBD-PE. These results demonstrate that steady-state fluorescence anisotropy of DPH-PC is a useful way to measure the amount of lipid order in biological membranes.

Fc(epsilon)RI as a paradigm for a lipid raft-dependent receptor in hematopoietic cells.

Lipid domains or rafts are currently embraced by immunologists as critical participants in receptor-mediated signaling events occurring at the plasma membrane. This view of membrane heterogeneity and its functional importance is supported by many years of different experimental approaches. We can now refine our investigations, moving beyond the simple models to ask more detailed questions about structural properties and mechanistic interactions. As highlighted for the IgE receptor (Fc(epsilon)RI), new information about initial engagement with src family kinases, cytoskeletal regulation, and coupling with downstream signaling is beginning to emerge.

Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components.

To investigate the structural basis for membrane interactions that occur between Lyn tyrosine kinase and IgE-Fc(epsilon)RI or other components of lipid rafts, we prepared a green fluorescent protein analog of Lyn (PM-EGFP) and used cross-correlation analysis to quantify co-redistributions of aggregates that occur after IgE-Fc(epsilon)RI is cross-linked on the cell surface. PM-EGFP, which contains minimally the palmitoylation and myristoylation sites on Lyn, was compared with another inner leaflet probe, EGFP-GG, which contains a prenylation site and a polybasic sequence similar to K-ras. Confocal fluorescence microscopy was used to examine co-redistributions of these inner leaflet components with IgE-Fc(epsilon)RI and outer leaflet raft components, ganglioside GD1b and glycosylphosphotidylinositol-linked Thy-1, under conditions where the latter were cross-linked externally to form large patches at the cell surface. The cross-correlation analysis was developed and characterized with simulations representing cell surface distributions, and parameters from the cross-correlation curves, rho(o) (peak height) and A (peak area), were shown to be reliable measures of the extent of co-redistributed aggregates and their size. Cross-correlation analysis was then applied to quantify co-redistributions of the fluorescently labeled inner and outer leaflet components on RBL-2H3 cells. As visually observed and parameterized in this manner, PM-EGFP was found to co-redistribute with lipid rafts significantly more than EGFP-GG or an endogenous prenylated protein, Cdc42. These quantitative results are consistent with previous analyses of Lyn co-redistributions and support the hypothesis that the functionally important interaction of Lyn with cross-linked IgE- Fc(epsilon)RI is due to their mutual co-association with lipid rafts.

Activated Cdc42/Rac reconstitutes Fcepsilon RI-mediated Ca2+ mobilization and degranulation in mutant RBL mast cells.

Antigen stimulation of mast cells via FcepsilonRI, the high-affinity receptor for IgE, triggers a signaling cascade that requires Ca(2+) mobilization for exocytosis of secretory granules during an allergic response. This study investigates critical signaling components by using mutant RBL mast cells that are defective in antigen-stimulated phospholipase Cgamma (PLCgamma) activation, as well as other signaling activities downstream of stimulated tyrosine phosphorylation. We show that the expression of activated versions of the Cdc42 or Rac1 GTPase restores antigen-stimulated Ca(2+) mobilization necessary for degranulation in these mutant cells. Wild-type Cdc42 and Rac1, as well as activated Cdc42 containing effector domain mutations, all fail to restore antigen-stimulated signaling leading to exocytosis. Expression of oncogenic Dbl, a guanine nucleotide exchange factor for Cdc42 and Rac1, partially restores sustained Ca(2+) mobilization and degranulation, suggesting that activation of endogenous Cdc42 and/or Rac1 is impaired in the mutant cells. Overexpression of PLCgamma1 with either activated Cdc42 or Rac1 synergistically stimulates degranulation, consistent with a critical defect in PLCgamma activation in these cells. Thus, our results point to activation of Cdc42 and/or Rac1 playing an essential role in antigen stimulation of early events that culminate in mast cell degranulation.

Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases.

Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.

Heterogeneous glycosylation of immunoglobulin E constructs characterized by top-down high-resolution 2-D mass spectrometry.

Posttranslational glycosylation is critical for biological function of many proteins, but its structural characterization is complicated by natural heterogeneity, multiple glycosylation sites, and different forms. Here, a top-down mass spectrometry (MS) characterization is applied to three constructs of the Fc segment of IgE: Fcepsilon(3-4) (52 kDa) and Fcepsilon(2-3-4)(2) (76 kDa) disulfide-bonded homodimers. Fourier transform MS of a reduced sample of Fcepsilon(2-3-4) gave molecular masses of 37 527, 37 689, 37 851, and 38 014 Da, directly characterizing multiple glycoforms (hexose = 162 Da) without chromatographic separation. Limited proteolysis of the nonreduced Fcepsilon(2-3-4)(2) protein yielded a peptide mixture with molecular weight values that agreed with those expected from the DNA sequence. The single glycosylation site in these constructs was identified, and quantities were determined of five glycoforms that agreed within +/-2% of the molecular ion values. The 2-D mass spectrum of two glycosylated peptides showed these to have high-mannose structures, -GlcNAc-(hex)(n)(), demonstrating that Fcepsilon(2-3-4) has a single such structure of n = 5-9. For a mutated sample of Fcepsilon(3-4), in addition to five glycoforms, MS showed a molecular discrepancy that could be assigned with proteolysis and 2-D mass spectra to the oxidation of two methionines and an additional residue difference.

Interactions between Fc(epsilon)RI and lipid raft components are regulated by the actin cytoskeleton.

Previous studies showed that crosslinking of IgE-Fc(epsilon)RI complexes on RBL-2H3 mast cells causes their association with isolated detergent-resistant membranes, also known as lipid rafts, in a cholesterol-dependent process that precedes initiation of signaling by these receptors. To investigate these interactions on intact cells, we examined the co-redistribution of raft components with crosslinked IgE-Fc(epsilon)RI using confocal microscopy. After several hours of crosslinking at 4 degrees C, the glycosylphosphatidylinositol-linked protein Thy-1 and the Src-family tyrosine kinase Lyn co-redistribute with IgE-Fc(epsilon)RI in large patches at the plasma membrane. Under these conditions, F-actin also undergoes dramatic co-segregation with Fc(epsilon)RI and raft components but is dispersed following a brief warm-up to 37 degrees C. When crosslinking of IgE-Fc(epsilon)RI is initiated at higher temperatures, co-redistribution of raft components with patched Fc(epsilon)RI is not readily detected unless stimulated F-actin polymerization is inhibited by cytochalasin D. In parallel, cytochalasin D converts transient antigen-stimulated tyrosine phosphorylation to a more sustained response. Sucrose gradient analysis of lysed cells reveals that crosslinked IgE-Fc(epsilon)RI remains associated with lipid rafts throughout the time course of the transient phosphorylation response but undergoes a time-dependent shift to higher density that is prevented by cytochalasin D. Our results indicate that interactions between Lyn and crosslinked IgE-Fc(epsilon)RI are regulated by stimulated F-actin polymerization, and this is best explained by a segregation of anchored raft components from more mobile ones.

Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells.

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.

Quantitative analysis of phospholipids in functionally important membrane domains from RBL-2H3 mast cells using tandem high-resolution mass spectrometry.

We recently showed that ligand-mediated cross-linking of FcepsilonRI, the high-affinity receptor for immunoglobulin E, on RBL-2H3 mast cells results in its co-isolation with detergent-resistant membranes (DRM) and its consequent tyrosine phosphorylation by the co-localized tyrosine kinase Lyn that is a critical early event in signaling by this receptor [Field et al. (1997) J. Biol. Chem. 272, 4276-4280]. As part of efforts to determine the structural bases for these interactions, we examined the phospholipid composition of DRM vesicles isolated from RBL-2H3 cells under conditions that preserve FcepsilonRI association. We used positive and negative mode electrospray Fourier transform ion cyclotron resonance mass spectrometry to compare quantitatively the phospholipid composition of isolated DRM to that of total cell lipids and to a plasma membrane preparation. From these analyses, over 90 different phospholipid species were spectrally resolved and unambiguously identified; more than two-thirds of these were determined with a precision of +/-0.5% (absolute) or less. Quantitative characterization of lipid profiles shows that isolated DRM are substantially enriched in sphingomyelin and in glycerophospholipids with a higher degree of saturation as compared to total cellular lipids. Plasma membrane vesicles isolated from RBL-2H3 cells by chemically induced blebbing exhibit a degree of phospholipid saturation that is intermediate between DRM and total cellular lipids, and significant differences in the headgroup distribution between DRM and plasma membranes vesicles are observed. DRM from cells with cross-linked FcepsilonRI exhibit a larger ratio of polyunsaturated to saturated and monounsaturated phospholipids than those from unstimulated cells. Our results support and strengthen results from previous studies suggesting that DRM have a lipid composition that promotes liquid-ordered structure. Furthermore, they demonstrate the potential of mass spectrometry for examining the role of membrane structure in receptor signaling and other cellular processes.

Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of FcepsilonRI and their association with detergent-resistant membranes.

Tyrosine phosphorylation of the high affinity immunoglobulin (Ig)E receptor (FcepsilonRI) by the Src family kinase Lyn is the first known biochemical step that occurs during activation of mast cells and basophils after cross-linking of FcepsilonRI by antigen. The hypothesis that specialized regions in the plasma membrane, enriched in sphingolipids and cholesterol, facilitate the coupling of Lyn and FcepsilonRI was tested by investigating functional and structural effects of cholesterol depletion on Lyn/FcepsilonRI interactions. We find that cholesterol depletion with methyl-beta-cyclodextrin substantially reduces stimulated tyrosine phosphorylation of FcepsilonRI and other proteins while enhancing more downstream events that lead to stimulated exocytosis. In parallel to its inhibition of tyrosine phosphorylation, cholesterol depletion disrupts the interactions of aggregated FcepsilonRI and Lyn on intact cells and also disrupts those interactions with detergent-resistant membranes that are isolated by sucrose gradient ultracentrifugation of lysed cells. Importantly, cholesterol repletion restores receptor phosphorylation together with the structural interactions. These results provide strong evidence that membrane structure, maintained by cholesterol, plays a critical role in the initiation of FcepsilonRI signaling.

Membrane organization in immunoglobulin E receptor signaling.

The structure and dynamics of the plasma membrane are proposed to be critical for the initial steps of signal transduction by the high-affinity immunoglobulin E receptor. Recent experimental advances indicate that interactions between the high-affinity immunoglobulin E receptor and the tyrosine kinase Lyn with cholesterol- and sphingolipid-rich regions within the plasma membrane are important for receptor function. This accumulating evidence points to spatio-temporal control of immunoglobulin E receptor signaling by the organization of the plasma membrane; an attractive hypothesis is that ligand-dependent receptor aggregation causes the segregation of Lyn-containing ordered regions of the plasma membrane from disordered regions.

Interleukin 1 (IL-1) causes changes in lateral and rotational mobilities of IL-1 type I receptors.

To investigate IL-1-dependent interactions of IL-1 type I (IL-1 RI) receptors on intact cells, lateral and rotational mobilities and detergent insolubility were investigated. Lateral mobility was measured by fluorescence photobleaching recovery, using a Cy3-modified, noncompetitive mAb specific for IL-1RI (M5) bound to wild-type IL-1 RI or mutant IL-1 RI with a truncated cytoplasmic tail. Addition of IL-1 causes significant reduction in the mobile fraction of wild-type IL-1 RI for two different transfected cell lines. For the mutant IL-1 RI, no significant decrease in response to IL-1 is observed, indicating that the missing cytoplasmic segment is involved in IL-1-dependent interactions of IL-1 RI that lead to reduced lateral mobility on the cell surface. The rotational mobility of IL-1 RI was assessed with phosphorescence anisotropy decay measurements using erythrosin-labeled M5. IL-1 decreases the rotational mobility of cell surface IL-1 RI on the microsecond time scale and also increases the initial anisotropy, indicating loss in segmental motion. Measurements of resistance to solubilization by Triton X-100 showed that IL-1 binding increases the fraction of IL-1 RI sedimenting with cytoskeletal residues. The IL-1 receptor antagonist protein (IL-1ra) causes partial effects in reducing rotational mobility and increasing detergent insolubility of M5-lableled IL-1 RI, indicating that this ligand causes structural changes in the presence of the dimerizing M5 mAb. These ligand-dependent physical interactions of IL-1 RI on the cell surface may be related to signal initiation by this receptor.

Structural aspects of the association of FcepsilonRI with detergent-resistant membranes.

We recently showed that aggregation of the high affinity IgE receptor on mast cells, FcepsilonRI, causes this immunoreceptor to associate rapidly with specialized regions of the plasma membrane, where it is phosphorylated by the tyrosine kinase Lyn. In this study, we further characterize the detergent sensitivity of this association on rat basophilic leukemia-2H3 mast cells, and we compare the capacity of structural variants of FcepsilonRI and other receptors to undergo this association. We show that this interaction is not mediated by the beta subunit of the receptor or the cytoplasmic tail of the gamma subunit, both of which are involved in signaling. Using chimeric receptor constructs, we found that the extracellular segment of the FcepsilonRI alpha subunit was not sufficient to mediate this association, implicating FcepsilonRI alpha and/or gamma transmembrane segments. To determine the specificity of this interaction, we compared the association of several other receptors. Interleukin-1 type I receptors on Chinese hamster ovary cells and alpha4 integrins on rat basophilic leukemia cells showed little or no association with isolated membrane domains, both before and after aggregation on the cells. In contrast, interleukin-2 receptor alpha (Tac) on Chinese hamster ovary cells exhibited aggregation-dependent membrane domain association similar to FcepsilonRI. These results provide insights into the structural basis and selectivity of lipid-mediated interactions between certain transmembrane receptors and detergent-resistant membranes.

How does the plasma membrane participate in cellular signaling by receptors for immunoglobulin E?

Accumulating evidence strongly supports the view that the plasma membrane participates in transmembrane signaling by IgE-receptors (IgE-Fc epsilon RI) through the formation of lipid-based domains, also known as rafts. Ongoing biochemical and biophysical experiments investigate the composition, structure, and dynamics of the corresponding membrane components and how these are related to functional coupling between Fc epsilon RI and Lyn tyrosine kinase to initiate signaling in mast cells.

Stimulated release of fluorescently labeled IgE fragments that efficiently accumulate in secretory granules after endocytosis in RBL-2H3 mast cells.

Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isothiocyanate (FITC)-labeled immunoglobulin E (IgE) results in slow but highly efficient accumulation of labeled IgE fragments in a pool of acidic peripheral vesicles that are visible by fluorescence microscopy after raising endosomal pH with ammonium chloride. Stimulation of cells containing these FITC-IgE fragments by aggregation of high affinity receptors for IgE (FcepsilonRI) or by Ca2+ ionophore and phorbol 12-myristate 13-acetate results in release of FITC fluorescence from the cells, which can be monitored continuously with a spectrofluorometer. The fluorescence release process corresponds to cellular degranulation: it is prevented under conditions that prevent stimulated beta-hexosaminidase release, and these two processes exhibit the same antigen dose-dependence and kinetics. Pulse-chase labeling reveals that aggregation of FITC-IgE bound to FcepsilonRI at the cell surface causes internalization and delivery to the regulated secretory vesicles with a high efficiency similar to monomeric IgE-FcepsilonRI, but more rapidly. Binding of Cy3-modified IgE to FcepsilonRI results in labeling of the same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3-labeled vesicles can be observed by fluorescence microscopy without neutralization of intracellular compartments. Simultaneous three-photon microscopy of serotonin fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these Cy3-labeled vesicles coincide with serotonin-labeled secretory granules. After stimulation of the cells via aggregation of IgE-FcepsilonRI or addition of Ca2+ ionophore and phorbol 12-myristate 13-acetate, depletion of the Cy3 label from the intracellular vesicles is observed with confocal microscopy. These results provide strong evidence for the lysosomal nature of secretory granules in these cells. In addition, they provide the basis for a direct, real-time method for monitoring single cell degranulation.

Kinetics of multivalent antigen DNP-BSA binding to IgE-Fc epsilon RI in relationship to the stimulated tyrosine phosphorylation of Fc epsilon RI.

Multivalent DNP-BSA is commonly used to cross-link anti-DNP IgE bound to Fc epsilon RI to stimulate cellular responses, although key features of the binding process are unknown. Fluorescence quenching can be used to study the kinetics of DNP-BSA binding to FITC-IgE. We observe that DNP-BSA binds more slowly to IgE than does an equimolar amount of a monovalent DNP ligand, suggesting that the average effective number of DNP groups per BSA is less than one. The binding data are well described by a transient hapten exposure model in which most of the DNP groups are unavailable for binding but have some probability of becoming exposed and available for binding during the time of the binding measurement. Additional experiments indicate that, for suboptimal to optimal concentrations of DNP-BSA, most of the FITC fluorescence quenching on the cell surface is due to cross-linking events. With these concentrations at 15 degrees C, the kinetics of FITC fluorescence quenching by DNP-BSA correlates with the kinetics of DNP-BSA-stimulated tyrosine phosphorylation of Fc epsilon RI. At 35 degrees C, the phosphorylation kinetics are biphasic during the time period in which cross-linking continues to increase. Our results establish a quantitative relationship between the time-course for cross-linking by multivalent Ag and Fc epsilon RI-mediated signaling, and they provide the means to predict the kinetics of cross-linking under a wide variety of conditions.

Evidence supporting a role for microfilaments in regulating the coupling between poorly dissociable IgE-Fc epsilonRI aggregates downstream signaling pathways.

Aggregation of Fc epsilonRI, the high-affinity receptor for IgE, on RBL-2H3 mast cells caused by reversible ligands such as multivalent antigen causes cellular responses that can be halted by subsequent addition of excess monovalent ligand. In contrast, Ca2+ and degranulation responses elicited by effectively irreversible streptavidin cross-linking of biotinylated IgE-Fc epsilonRI are not stopped by addition of excess biotin after stimulation is initiated. These results support previous conclusions based on studies with covalent oligomers of IgE that stable cross-links can continue to deliver stimulatory signals for extended periods of time. Dissociation measured in the presence of monovalent hapten reveals two populations of IgE-Fc epsilonRI cross-linked by multivalent antigen that differ in functional effectiveness. Aggregates with readily dissociable cross-links are normally responsible for triggering essentially all of the degranulation response, whereas aggregates with poorly dissociable cross-links apparently do not trigger this response. Treatment of RBL-2H3 cells with cytochalasin D, an inhibitor of actin polymerization, enhances downstream signaling and enables the less readily dissociable aggregates to stimulate Ca2+ and degranulation responses. Under these conditions, cytochalasin D does not affect hapten-mediated dissociation of multivalent antigen, nor does it prevent hapten from reversing tyrosine phosphorylation of Syk. Cytochalasin D alone causes tyrosine phosphorylation of a protein at approximately 75 kDa, and it reduces hapten-induced reversal of antigen-stimulated tyrosine phosphorylation of several other proteins. Taken together, these results indicate that stimulated actin polymerization normally regulates the coupling of aggregated Fc epsilonRI to downstream signaling pathways, and they provide an explanation for seeming discrepancies between responses to stable and reversible cross-links.

Altered patterns of tyrosine phosphorylation and Syk activation for sterically restricted cyclic dimers of IgE-Fc epsilonRI.

Previous studies in our laboratory established that the symmetrical bivalent ligand, N,N'-bis-[[epsilon-(2,4-dinitrophenyl)amino]caproyl]-L-tyrosyl]-L-cystin e ((DCT)2-cys), stably cross-links anti-2,4-dinitrophenyl-immunoglobulin E (IgE) bound to high affinity receptors Fc epsilonRI on the surface of RBL-2H3 cells, forming mostly cyclic dimers containing two IgE-Fc epsilonRI and two (DCT)2-cys (Posner et al. (1995) J. Immunol. 155, 3601-3609). These cyclic dimers do not trigger Ca2+ or degranulation responses under a variety of conditions. However, we find that the linearly cross-linked IgE-Fc epsilonRI formed at higher concentrations of (DCT)2-cys do trigger degranulation in the presence of cytochalasin D, an inhibitor of actin polymerization. We further investigated stimulation by (DCT)2-cys of the earliest known events in the functional response, i.e., tyrosine phosphorylation of the beta and gamma subunits of Fc epsilonRI. At the higher (DCT)2-cys concentrations corresponding to linear dimers and maximal degranulation, tyrosine phosphorylation of both beta and gamma are observed. At lower (DCT)2-cys concentrations where cross-linking is maximal and cyclic dimers are overwhelmingly dominant, only gamma tyrosine phosphorylation is observed. Cytochalasin D does not affect these phosphorylation patterns, but instead appears to enhance coupling to downstream signaling events. Phosphorylation of Syk occurs at the higher (DCT)2-cys concentrations in parallel with beta phosphorylation but does not occur in its absence at the lower (DCT)2-cys concentrations. These results suggest that cyclic dimers of IgE-Fc epsilonRI are sterically restricted such that they stimulate tyrosine phosphorylation of gamma but not beta, and this is not sufficient for Syk binding and/or activation.