Maturation-related changes in the expression of Fc gamma RII and Fc gamma RIII on mouse mast cells derived in vitro and in vivo.

The expression and function of Fc gamma RII and Fc gamma RIII on three mouse mast cell populations that differ in maturity as assessed by secretory granule constituents were analyzed by cellular and immunochemical approaches. As quantified by flow cytometric analysis of the binding of the rat 2.4G2 anti-Fc gamma RII/III mAb, mouse serosal mast cells (SMC) purified from the peritoneal cavity expressed more receptors per cell than did mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC), which are progenitors of SMC. Coculture of BMMC with mouse 3T3 fibroblasts for 2 wk, which alters the secretory granule composition toward that of SMC, also increased receptor epitope expression to a level equivalent to that of SMC. As assessed by rosette assays with mouse mAb to SRBC, all three mast cell populations bound IgG1, IgG2a, and IgG2b, essentially all binding was inhibited by 2.4G2 antibody, and greater quantities of the antibody were required to block immune adherence by cocultured mast cells and SMC as compared with BMMC. Immunoprecipitation and SDS-PAGE analysis of Fc gamma RII and Fc gamma RIII from BMMC, cocultured mast cells, and SMC that were surface radiolabeled with Na125I revealed predominant native forms of 62, 57, and 56 kDa, respectively, and an additional surface form of 43 kDa in SMC. Removal of N-linked carbohydrate from immunoprecipitates demonstrated that BMMC expressed peptide cores of 38 kDa (Fc gamma RII-1 gene product) and 31 kDa (Fc gamma RII-2 gene product), and barely detectable amounts of a 28-kDa (Fc gamma RIII gene product) core. The expression of all three was increased by coculture with 3T3 fibroblasts, consistent with the increased expression of their common epitope by cytofluorographic analysis. SMC expressed primarily the Fc gamma RII-1 and some Fc gamma RIII gene product. Thus, the three populations of mast cells express different amounts and ratios of the Fc gamma RII and Fc gamma RIII gene products, and maturation of BMMC during coculture with fibroblasts in vitro and in the peritoneal cavity in vivo augments cell-surface expression of the receptors and immune adherence function.     

Mouse bone marrow-derived mast cells cocultured with fibroblasts. Morphology and stimulation-induced release of histamine, leukotriene B4, leukotriene C4, and prostaglandin D2

Mouse bone marrow-derived mast cells (BMMC), cultured for 2, 7, or 14 days in WEHI-3 conditioned medium in the absence or presence of mouse 3T3 fibroblasts, were examined morphologically and for their functional responses to IgE-Fc-mediated and calcium ionophore-mediated activation. The 7- and 14-day fibroblast-adherent and non-fibroblast-adherent populations of cocultured BMMC had more granules per cell and the granule contents were more electron dense than non-cocultured BMMC or BMMC cocultured for only 2 days. The adherent cocultured BMMC were usually located within multiple layers of fibroblasts, but did not form junctions with the fibroblasts. When activated immunologically, the adherent cocultured mast cells generally discharged their granules singly, but compound exocytosis was occasionally seen. Both the non-adherent cocultured BMMC and the BMMC that were cultured in the absence of fibroblasts were similar to one another in that they exocytosed 9 to 11% of their histamine when sensitized with anti-dinitrophenyl IgE and challenged with dinitrophenyl-bovine serum albumin and 27 to 29% of their histamine when challenged with calcium ionophore. In contrast, adherent cocultured BMMC exocytosed 27 and 61% of their histamine upon immunologic and calcium ionophore activation, respectively, representing a significant two- to three-fold increase relative to that obtained from the other populations of BMMC. When activated immunologically, BMMC cultured in WEHI-3 conditioned medium alone generated a mean of 12 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 1.6 ng of leukotriene B4 (LTB4), and 1.0 ng of prostaglandin D2 (PGD2)/10(6) cells. The immunologic response of the nonadherent 7-day cocultured BMMC was similar. Fibroblast-adherent cocultured BMMC, on the other hand, generated 56 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 6.4 ng of LTB4, and 5.6 ng of PGD2/10(6) mast cells, representing a significant increase for each product. When calcium ionophore was used as the agonist, the adherent cocultured mast cells also generated significantly more arachidonic acid metabolites than nonadherent cocultured BMMC or BMMC cultured in the absence of fibroblasts. Retention times on high performance liquid chromatography confirmed that the generated immunoreactive products were LTB4, PGD2, and LTC4. Thus, coculture of BMMC with fibroblasts induces an alteration in the composition of the secretory granules of the mast cells, as well as an augmentation of the activation-secretion response of the BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)     

A comparison of mediators released or generated by IFN-gamma-treated human mast cells following aggregation of Fc gamma RI or Fc epsilon RI

The high affinity receptor for IgG (Fc gamma RI, CD64) is expressed on human mast cells, where it is up-regulated by IFN-gamma and, thus, may allow mast cells to be recruited through IgG-dependent mechanisms in IFN-gamma-rich tissue inflammation. However, the mediators produced by human mast cells after aggregation of Fc gamma RI are incompletely described, and it is unknown whether these mediators are distinct from those produced after activation of human mast cells via Fc epsilon RI. Thus, we investigated the release of histamine and arachidonic acid metabolites and examined the chemokine and cytokine mRNA profiles of IFN-gamma-treated cultured human mast cells after Fc gamma RI or Fc epsilon RI aggregation. Aggregation of Fc gamma RI resulted in histamine release and PGD(2) and LTC(4) generation. These responses were qualitatively indistinguishable from responses stimulated via Fc epsilon RI. Aggregation of Fc epsilon RI or Fc gamma RI led to an induction or accumulation of 22 cytokine and chemokine mRNAs. Among them, seven cytokines (TNF-alpha, IL-1beta, IL-5, IL-6, IL-13, IL-1R antagonist, and GM-CSF) were significantly up-regulated via aggregation of Fc gamma RI compared with Fc epsilon RI. TNF-alpha mRNA data were confirmed by quantitative RT-PCR and ELISA. Furthermore, we confirmed histamine and TNF-alpha data using IFN-gamma-treated purified human lung mast cells. Thus, aggregation of Fc gamma RI on mast cells led to up-regulation and/or release of three important classes of mediators: biogenic amines, lipid mediators, and cytokines. Some cytokines, such as TNF-alpha, were released and generated to a greater degree after Fc gamma RI aggregation, suggesting that selected biologic responses of mast cells may be preferentially generated through Fc gamma RI in an IFN-gamma-rich environment.     

Generation of leukotriene C4, leukotriene B4, and prostaglandin D2 by immunologically activated rat intestinal mucosa mast cells

Mucosal mast cells (MMC) were isolated from the intestine of Nippostrongylus brasiliensis-infected rats and then activated with Ag or with anti-IgE in order to assess their metabolism of arachidonic acid to leukotriene (LT) C4, LTB4, and prostaglandin D2 (PGD2). After challenge of MMC preparations of 19 +/- 1% purity with five worm equivalents of N. brasiliensis Ag, the net formation of immunoreactive equivalents of LTC4, LTB4, and PGD2 was 58 +/- 8.3, 22 +/- 4.5, and 22 +/- 3.4 ng/10(6) mast cells, respectively (mean +/- SE, n = 7). When MMC preparations of 56 +/- 9% purity were activated by Ag, the net generation of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) MMC was 107 +/- 15, 17 +/- 5.4, and 35 +/- 18 ng, respectively. These data indicate that the three eicosanoids originated from the MMC rather than from a contaminating cell. Analysis by reverse phase HPLC of the C-6 sulfidopeptide leukotrienes present in the supernatants of the activated MMC preparations of lower purity revealed LTC4, LTD4, and LTE4. In a higher purity MMC preparation only LTC4 was present, suggesting that other cell types in the mucosa are able to metabolize LTC4 to LTD4 and LTE4. The release of histamine and the generation of eicosanoids from intestinal MMC and from peritoneal cavity-derived connective tissue-type mast cells (CTMC) isolated from the same N. brasiliensis-infected rats were compared. When challenged with anti-IgE, these MMC released 165 +/- 41 ng of histamine/10(6) mast cells, and generated 29 +/- 3.6, 12 +/- 4.2, and 4.7 +/- 1.0 ng (mean +/- SE, n = 3) of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) mast cells, respectively. In contrast, CTMC isolated from the same animals and activated with the same dose of anti-IgE released approximately 35 times more histamine (5700 +/- 650 ng/10(6) CTMC), generated 7.5 +/- 2.3 ng of PGD2/10(6) mast cells, and failed to release LTC4 or LTB4. These studies establish, that upon immunologic activation, rat MMC and CTMC differ in their quantitative release of histamine and in their metabolism of arachidonic acid to LTC4 and LTB4.     

Biochemical and morphological modifications in dexamethasone-treated mouse bone marrow-derived mast cells

Addition of 1 microM dexamethasone (DM) to bone marrow-derived mast cells (BMMC) induced a time-dependent increase in cell histamine content. The latter reached a plateau of 2.5 micrograms/1 x 10(6) cells after 11 days in culture, compared with 100 ng/1 x 10(6) for untreated BMMC. Steroids, such as beta-estradiol, androsterone, and testosterone (1 microM), did not alter the histamine content of BMMC, whereas progesterone (1 microM) induced a moderate increase. Other glucocorticosteroids also enhanced histamine content, suggesting that the observed increase was specific for glucocorticosteroid. Treatment of BMMC with 1 microM DM for 14 days inhibited the Ag-induced, IgE-mediated release of histamine, beta-hexosaminidase, platelet-activating factor-acether, LTB4, and LTC4 by 65 +/- 3%, 66 +/- 1%, 93 +/- 3%, 66 +/- 2%, and 74 +/- 10%, respectively (mean +/- 1 SD, n = 3). In contrast with untreated cells which produce less than 2 ng/1 x 10(6) cells PGD2 after Ag challenge, DM-treated BMMC generated 16.8 +/- 0.3 ng/1 x 10(6) cells PGD2. Moreover, most of DM-treated BMMC became Alcian blue+/safranin+ and by ultrastructure, exhibited numerous cytoplasmic granules filled with abundant and uniform electron-dense matrix. The present results indicate that DM-treated BMMC exhibit biochemical and functional properties different from immature untreated cells, suggesting that a maturation-like process occurred in vitro during DM treatment.     

Prostaglandin D2 generation after activation of rat and human mast cells with anti-IgE

Anti-IgE-dependent activation of rat and human mast cells resulted in the preferential generation of the cyclooxygenase products prostaglandin D2 (PGD2) and prostaglandin I2 (PGI2) in the rat and PGD2 in the human. The average net generation of PGD2, determined by gas chromatography-mass spectrometry, was 13.1 ng/10(6) purified rat mast cells and 39.5 ng/10(6) dispersed, enriched human mast cells. After IgE-dependent activation, there was a linear relationship between the net quantities of PGD2 generated and of histamine secreted from dispersed human pulmonary cells when the number of mast cells was varied but the total number of cells was held constant, indicating that it is the number of mast cells participating in IgE-dependent activation, rather than total mast cell number, that determines PGD2 generation. A linear relationship was also shown between PGD2 generation, determined by radioimmunoassay, and the release of the granule marker beta-hexosaminidase from purified rat mast cells on the dose-response portion of the plot of their response to anti-IgE challenge. With higher concentrations of anti-IgE, PGD2 generation from rat mast cells plateaued, whereas net percent beta-hexosaminidase release increased further. In kinetic studies of rat mast cells activated with anti-IgE, the onset (1 to 2 min) and time of maximum generation (5 to 10 min) for PGD2 were delayed relative to the onset (15 to 30 sec) and completion (1 to 2 min) of beta-hexosaminidase release. Thus, the extracellular appearance of PGD2 during IgE-dependent mast cell activation represents a response additional to the secretion of granule-associated mediators.     

Characterization of primate bronchoalveolar mast cells. I. IgE-dependent release of histamine, leukotrienes, and prostaglandins

Large numbers of functional mast cells were obtained by bronchoalveolar lavage (BAL) of Macaca arctoides monkeys that had been infected with the nematode Ascaris suum. These lavage cells, of which 21% were mast cells, released histamine, LTC4, and PGD2 in a concentration-dependent fashion when challenged with ascaris antigen or antibody to human IgE. However, there was no release of histamine when these cells were challenged with compound 48/80. The amount of mediator released was highly dependent on the sensitivity of the cells to immunologic challenge, but was generally in the range of 2 to 5 micrograms histamine (30 to 70% of total), 20 to 80 ng LTC4, and 100 to 300 ng PGD2 per 10(6) mast cells when maximally challenged. Other eicosanoids measured were released only in much smaller quantities. Maximal values were 4 ng LTB4, 2 ng PGE2, and approximately 10 to 20 ng PGF2 alpha per 10(6) mast cells. The amount of LTC4 and PGD2 released correlated with the release of histamine, the calculated regression line indicating that 18 ng LTC4 and 50 ng PGD2 were released per microgram of histamine released. This correlation suggests that the majority of the LTC4 and PGD2 released was probably mast cell-derived. Further support for this conclusion was given by the observation that when lavage cells were fractioned on continuous Percoll gradients, the ability to release LTC4 and PGD2 on immunologic challenge coincided with the peak of mast cells.     

Inhibition of proliferation of mouse T cell-dependent bone marrow-derived mast cells by rat serum does not change their unique phenotype

Both mouse and rat sera have been found to inhibit proliferation in vitro of interleukin 3-dependent chondroitin sulfate E proteoglycan-containing mouse bone marrow-derived mast cells (BMMC), as assessed by quantitation of 3H-labeled thymidine incorporation into DNA, cell cycle analysis, and cell number. Rat serum (9%) inhibited 3H-labeled thymidine incorporation within 60 min of exposure in a culture medium composed of 1% fetal calf serum (FCS) and 16% concanavalin A splenocyte-conditioned medium. The anti-proliferative effect of rat serum did not alter cell viability for 17 hr of subsequent culture, was dose related, with a maximal effect at 7% rat serum, and was reversible. Cytofluorographic analysis of relative DNA content per cell revealed that the proportion of cells in the S + G2 + M phases of the cell cycle was decreased in cells treated with 9% rat serum compared with cells cultured in either 1% or 10% FCS. These rat serum-treated BMMC exhibited no change in plasma membrane antigen phenotype as assessed by 15 monoclonal antibodies, and continued to synthesize chondroitin sulfate E proteoglycan. When sensitized with monoclonal IgE antibody, washed, and challenged with specific antigen, the rat serum-treated BMMC released the preformed secretory granule-associated mediators beta-hexosaminidase and histamine, and the newly generated lipid mediators leukotriene C4 (LTC4) and leukotriene B4 (LTB4) in amounts comparable to BMMC cultured in 10% FCS. Thus, the unique cell surface phenotype, the presence of chondroitin sulfate E proteoglycan rather than heparin proteoglycan, and the generation of LTC4 and LTB4 in a ratio of approximately 6:1 upon perturbation of the IgE receptor are distinctive characteristics of the interleukin 3-dependent mouse BMMC subclass, and not a functional consequence of the rapid proliferation of the cell.     

Carboxypeptidase A in mouse mast cells. Identification, characterization, and use as a differentiation marker

By using a conventional spectrophotometric assay with hippuryl-L-phenylalanine as the substrate, 10(6) BALB/c mouse serosal mast cells possessed 1.5 +/- 0.43 U (mean +/- SE, n = 5, range = 0.48 to 2.5) of carboxypeptidase A activity, while T cell factor-dependent, mouse bone marrow-derived mast cells (BMMC) had barely detectable levels of 0.01 +/- 0.001 U/10(6) cells (mean +/- SE, n = 3). In order to characterize the carboxypeptidase A present in the BMMC, a sensitive assay was developed that used angiotensin I as the substrate and reverse phase-high performance liquid chromatography to separate and quantify production of the cleavage product des-leu-angiotensin I. Using this assay, mouse BMMC carboxypeptidase A had a neutral to basic pH optimum and hydrolyzed angiotensin I with a Km of 0.78 mM. The antigen-induced net percent release of carboxypeptidase A from IgE-sensitized BMMC was proportional to that of the secretory granule component beta-hexosaminidase which indicates a secretory granule location for the exopeptidase. As defined by exclusion during Sepharose CL-2B chromatography, carboxypeptidase A was exocytosed as a greater than 1 X 10(7) m.w. complex bound to proteoglycans. Because BMMC cocultured with mouse skin-derived 3T3 fibroblasts are known to undergo an increase in histamine content and biosynthesis of 35S-labeled heparin proteoglycans, carboxypeptidase A activity was measured during BMMC/fibroblast coculture for 0 to 28 days. The carboxypeptidase A activity increased progressively during 28 days of co-culture from 0.004 +/- 0.002 U/10(6) starting BMMC (mean +/- SE, n = 3) to 0.36 +/- 0.10 U/10(6) co-cultured mast cells. These findings indicate that carboxypeptidase A, a neutral protease, is exocytosed from the secretory granules of mouse mast cells bound to proteoglycan and is increased during the in vitro differentiation of mouse BMMC from mucosal-like mast cells to serosal-like mast cells.     

Structural requirements of SLP-76 in signaling via the high-affinity immunoglobulin E receptor (Fc epsilon RI) in mast cells

The adapter SLP-76 plays an essential role in Fc epsilon RI signaling, since SLP-76(-/-) bone marrow-derived mast cells (BMMC) fail to degranulate and release interleukin-6 (IL-6) following Fc epsilon RI ligation. To define the role of SLP-76 domains and motifs in Fc epsilon RI signaling, SLP-76(-/-) BMMC were retrovirally transduced with SLP-76 and SLP-76 mutants. The SLP-76 N-terminal and Gads binding domains, but not the SH2 domain, were critical for Fc epsilon RI-mediated degranulation and IL-6 secretion, whereas all three domains are essential for T-cell proliferation following T-cell receptor (TCR) ligation. Unexpectedly, the three tyrosine residues in SLP-76 critical for TCR signaling, Y112, Y128, and Y145, were not essential for IL-6 secretion, but were required for degranulation and mitogen-activated protein kinase activation. Furthermore, a Y112/128F SLP-76 mutant, but not a Y145F mutant, strongly reconstituted mast cell degranulation, suggesting a critical role for Y145 in Fc epsilon RI-mediated exocytosis. These results point to important differences in the function of SLP-76 between T cells and mast cells.     

Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes

Human mast cells, dispersed from lung tissue by proteolytic treatment and enriched to a purity of 23 to 68% by density-gradient centrifugation, were maintained ex vivo for up to 13 days when co-cultured with mouse skin-derived 3T3 fibroblasts in RPMI 1640 containing 10% fetal calf serum. The human mast cells were adherent to the fibroblast cultures within 2 to 4 hr after seeding, and after 7 days of co-culture were localized between the layers of fibroblasts. The cell surfaces of the mast cells and the fibroblasts did not form tight junctions, but rather approached within 20 nm of each other. The co-cultured mast cells did not divide; they maintained their cellular content of histamine and TAMe esterase and resembled in vivo mast cells in that their secretory granules exhibited scroll patterns and their nuclei were oval. Both the freshly isolated and the co-cultured mast cells responded to activation with anti-human IgE by exocytosing histamine and generating and releasing arachidonic acid metabolites. When freshly isolated mast cells were activated immunologically, they exocytosed 38 +/- 8% of their total histamine content and released 28 +/- 1.9 ng (mean +/- range, n = 2) of immunoreactive equivalents of prostaglandin D2 (PGD2) per microgram of total cellular histamine, but did not generate significant amounts of either leukotriene C4 (LTC4) or leukotriene B4 (LTB4). The 1-wk co-cultured mast cells, on the other hand, exocytosed 43 +/- 2.4% of their total histamine content, and released 86 +/- 10, 43 +/- 20, and 5.2 +/- 5.2 ng (mean +/- SD, n = 4) of immunoreactive equivalents of PGD2, LTC4, and LTB4, respectively, per microgram of histamine. Thus, human lung-derived mast cells can be maintained ex vivo when co-cultured with fibroblasts, and, when treated with anti-IgE, they metabolize arachidonic acid via both the cyclooxygenase and the 5-lipoxygenase pathways.     

Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse PT-18 mast cell.

A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.     

Phospholipase C gamma 2 is essential for specific functions of Fc epsilon R and Fc gamma R

Phospholipase Cgamma2 (PLCgamma2) plays a critical role in the functions of the B cell receptor in B cells and of the FcRgamma chain-containing collagen receptor in platelets. Here we report that PLCgamma2 is also expressed in mast cells and monocytes/macrophages and is activated by cross-linking of Fc(epsilon)R and Fc(gamma)R. Although PLCgamma2-deficient mice have normal development and numbers of mast cells and monocytes/macrophages, we demonstrate that PLCgamma2 is essential for specific functions of Fc(epsilon)R and Fc(gamma)R. While PLCgamma2-deficient mast cells have normal mitogen-activated protein kinase activation and cytokine production at mRNA levels, the mutant cells have impaired Fc(epsilon)R-mediated Ca(2+) flux and inositol 1,4,5-trisphosphate production, degranulation, and cytokine secretion. As a physiological consequence of the effect of PLCgamma2 deficiency, the mutant mice are resistant to IgE-mediated cutaneous inflammatory skin reaction. Macrophages from PLCgamma2-deficient mice have no detectable Fc(gamma)R-mediated Ca(2+) flux; however, the mutant cells have normal Fc(gamma)R-mediated phagocytosis. Moreover, PLCgamma2 plays a nonredundant role in Fc(gamma)R-mediated inflammatory skin reaction.     

Functional comparison of Fc epsilon RI, Fc gamma RII, and Fc gamma RIII in mast cells.

The cellular responses initiated by cross-linking rodent Fc gamma RII-b1, Fc gamma RII-b2, Fc gamma RIII, and Fc epsilon RI in mast cells were compared. Individual murine Fc gamma R isoforms were transfected into rat basophilic leukemia cells and after cross-linking the FcR, changes in the phosphorylation of protein tyrosines, in the level of intracellular Ca2+, in the hydrolysis of phosphoinositides, and in the release of arachidonic acid metabolites and hexosaminidase were monitored. Cross-linking of Fc gamma RIII initiated all of these early and late biochemical functions, and although they were quantitatively somewhat smaller, the responses were qualitatively indistinguishable from those stimulated by the endogenous Fc epsilon RI. However, despite ample expression, neither Fc gamma RII-b1 nor Fc gamma RII-b2 stimulated these functions when cross-linked. The functional differences between Fc gamma RII and Fc gamma RIII were studied further by assessing the responses to cross-linking of the endogenous Fc gamma R (Fc gamma RII-b1, Fc gamma RII-b2, and Fc gamma RIII) on P815 mouse mastocytoma cells that had been transfected with normal or functionally defective Fc epsilon RI. Two types of mutant subunits had previously been observed to impair the activity of Fc epsilon RI: gamma-chains missing the cytoplasmic domain, and beta-chains missing the COOH-terminal cytoplasmic domain. In both types of transfectants the functional inhibition of the endogenous Fc gamma R paralleled that of the transfected Fc epsilon RI. These results are consistent with the gamma subunit being associated with the functions of Fc gamma RIII as well as of Fc epsilon RI. The functional results also complement the recently reported evidence that Fc gamma RIII can interact with Fc epsilon RI beta-subunits (J. Exp. Med. 175:447, 1992).     

Murine recombinant Fc gamma RIII, but not Fc gamma RII, trigger serotonin release in rat basophilic leukemia cells.

Murine Fc gamma RII and Fc gamma RIII have highly homologous extracellular domains, but unrelated transmembrane and intracytoplasmic (IC) domains. Murine Fc gamma RIIb1 and b2 are two isoforms of single-chain receptors which differ only by 47 aa in their IC domain. Murine Fc gamma RIII are composed of an IgG-binding alpha-chain, the intracellular portion of which is unrelated to that of Fc gamma RII, and of a homodimeric gamma-chain which also associates with Fc epsilon RI. Murine mast cells express Fc gamma RII, Fc gamma RIII, and Fc epsilon RI. They can be induced to degranulate by murine IgG immune complexes or by F(ab')2 fragments of the rat anti-murine Fc gamma RII/III mAb 2.4G2, complexed to mouse anti-rat (MAR) F(ab')2. In order to determine which murine Fc gamma R can activate mast cells, cDNA encoding murine Fc gamma RIIb1, Fc gamma RIIb2 or Fc gamma RIII alpha were stably transfected into RBL-2H3 cells. Murine Fc gamma RIII but not Fc gamma RIIb1 or Fc gamma RIIb2 induced serotonin release when aggregated by (2.4G2-MAR) F(ab')2 complexes. The respective roles of the IC domains of murine Fc gamma RIII subunits in signal transduction were investigated by stably transfecting cDNA encoding IC-deleted or chimeric murine Fc gamma R into RBL-2H3 cells. The substitution of the IC domain of murine Fc gamma RII for that of murine Fc gamma RIII gamma, but not that of murine Fc gamma RIII alpha, conferred the ability to trigger serotonin release. The deletion of IC sequences of the alpha subunit did not alter the ability of murine Fc gamma RIII to trigger serotonin release. It follows that 1) murine Fc gamma RIII, but not Fc gamma RII, can induce RBL cells to release serotonin, 2) the aggregation of the IC domain of the murine Fc gamma RIII gamma subunit is sufficient, but 3) the IC domain of the murine Fc gamma RIII alpha subunit is neither sufficient nor necessary for triggering serotonin release.     

IgE enhances Fc epsilon receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I expression and mediator release

We investigated the effects of IgE versus IL-4 on Fc epsilon RI surface expression in differentiated human mast cells derived in vitro from umbilical cord blood mononuclear cells. We found that IgE (at 5 micrograms/ml) much more strikingly enhanced surface expression of Fc epsilon RI than did IL-4 (at 0.1-100 ng/ml); similar results were also obtained with differentiated mouse mast cells. However, IL-4 acted synergistically with IgE to enhance Fc epsilon RI expression in these umbilical cord blood-derived human mast cells, as well as in mouse peritoneal mast cells derived from IL-4-/- or IL-4+/+ mice. We also found that: 1) IgE-dependent enhancement of Fc epsilon RI expression was associated with a significantly enhanced ability of these human mast cells to secrete histamine, PGD2, and leukotriene C4 upon subsequent passive sensitization with IgE and challenge with anti-IgE; 2) preincubation with IL-4 enhanced IgE-dependent mediator secretion in these cells even in the absence of significant effects on Fc epsilon RI surface expression; 3) when used together with IgE, IL-4 enhanced IgE-dependent mediator secretion in human mast cells to levels greater than those observed in cells that had been preincubated with IgE alone; and 4) batches of human mast cells generated in vitro from umbilical cord blood cells derived from different donors exhibited differences in the magnitude and pattern of histamine and lipid mediator release in response to anti-IgE challenge, both under baseline conditions and after preincubation with IgE and/or IL-4.     

A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation

Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fc epsilon receptor 1 (Fc epsilon RI), have key roles in allergic diseases. Fc epsilon RI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express Fc gamma RIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with Fc epsilon RI can block Fc epsilon RI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is gamma Hinge-CH gamma 2-CH gamma 3-15aa linker-CH epsilon 2-CH epsilon 3-CH epsilon 4. This Fc gamma Fc epsilon fusion protein was expressed as the predicted 140-kappa D dimer that reacted with anti-human epsilon- and gamma-chain specific antibodies. Fc gamma Fc epsilon bound to both human Fc epsilon RI and Fc gamma RII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human Fc epsilon RI alpha. Our results show that this chimeric protein is able to form complexes with both Fc epsilon RI and Fc gamma RII, and inhibit mast-cell and basophil function. This approach, using a Fc gamma Fc epsilon fusion protein to co-aggregate Fc epsilon RI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and Fc epsilon RI-mediated diseases.     

Leukotriene B4 potentiates the expression and release of Fc epsilon RII/CD23, and proliferation and differentiation of human B lymphocytes induced by IL-4

This study documents the influence of leukotriene (LT) B4 on human B lymphocyte responses. Incubation of freshly isolated B lymphocytes with LTB4, but not LTC4, induced a slight but significant, time- and dose-dependent increase in the surface expression of Fc epsilon RII/CD23 and class II MHC Ag and in the release of soluble CD23. These changes were maximal at 10 nM LTB4 after an incubation period of 48 h. When B lymphocytes were preactivated in vitro with Staphylococcus aureus Cowan strain I (SAC), neither LTB4 nor LTC4 was able to promote proliferation and/or IgG and IgM secretion. In contrast, when resting B lymphocytes were stimulated with a suboptimal concentration (3 U/ml) of IL-4, LTB4, but not LTC4, potentiated both the Fc epsilon RII/CD23 and the class II MHC antigen expression, and the release of soluble CD23 in a dose-dependent manner, without affecting the kinetics of these responses. Furthermore, LTB4, but not LTC4, amplified both the proliferative response and the IgG and IgM secretion induced by addition of a suboptimal dose of IL-4 (3 U/ml) to SAC-preactivated B lymphocytes. Again, LTB4 did not modify the kinetics of the proliferative response promoted by IL-4. Although LTB4 potentiated IL-4-induced IgG and IgM secretion from SAC-activated B lymphocytes, no production of IgE was observed. These data indicate that LTB4 could play a regulatory role in the modulation of IL-4-induced signaling in human B lymphocytes.     

Fc epsilon R1-mediated tyrosine phosphorylation of multiple proteins, including phospholipase C gamma 1 and the receptor beta gamma 2 complex, in RBL-2H3 rat basophilic leukemia cells.

In basophils, mast cells, and the RBL-2H3 tumor mast cell line, cross-linking the high-affinity immunoglobulin E receptor (Fc epsilon R1) stimulates a series of responses, particularly the activation of phospholipase C (PLC), that lead to allergic and other immediate hypersensitivity reactions. The mechanism of activation of PLC, however, is not clear. Here, we show that cross-linking Fc epsilon R1 on RBL-2H3 cells causes the tyrosine phosphorylation of at least 12 cellular proteins, including PLC gamma 1 (PLC gamma 1) and the receptor beta and gamma subunits. 32P-labeled PLC gamma 1 can be detected by anti-phosphotyrosine antibody as early as 10 s after the addition of antigen. The tyrosine-phosphorylated 33-kDa beta subunit and 9- to 11-kDa gamma subunit of the Fc epsilon R1 are additionally phosphorylated on serine and theonine residues, respectively, and are found as complexes with other phosphotyrosine-containing proteins in antigen-stimulated cells. Our results indicate a means by which the Fc epsilon R1 may control PLC activity in RBL-2H3 cells and raise the possibility that other receptor-mediated signalling events in mast cells may also be controlled through protein tyrosine phosphorylation.