ARTICLE |
Correspondence to: Kazushi Fujimoto, Section of Physiological Anatomy, Fukui Prefectural U. Faculty of Nursing and Welfare, 4-1-1 Kenjojima, Matsuoka-cho, Yoshida-gun, Fukui 910-1195, Japan. E-mail: fujimoto@fpu.ac.jp
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Summary |
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The high-affinity IgE receptor (FcRI) on mast cells and basophils consists of a ligand-binding
-chain and two kinds of signaling chains, a ß-chain and disulfide-linked homodimeric
-chains. Crosslinking by multivalent antigen results in the aggregation of the bound IgE/
-chain complexes at the cell surface, triggering cell activation, and subsequent internalization through coated pits. However, the precise topographical alterations of the signaling ß- and
-chains during stimulation remain unclarified despite their importance in ligand binding/signaling coupling. Here we describe the dynamics of Fc
RI subunit distribution in rat basophilic leukemia cells during stimulation as revealed by immunofluorescence and immunogold electron microscopy. Immunolocalization of ß- and
-chains was homogeneously distributed on the cell surfaces before stimulation, while crosslinking with multivalent antigen, which elicited optimal degranulation, caused a distinct aggregation of these signaling chains on the cell membrane. Moreover, only
- but not ß-chains were aggregated during the stimulation that evoked suboptimal secretion. These findings suggest that high-affinity IgE receptor ß- and
-chains do not co-aggregate but for the most part form homogenous aggregates of ß-chains or
-chains after crosslinking.
(J Histochem Cytochem 48:17051715, 2000)
Key Words: mast cells/basophils, Fc receptors, immunogold electron microscopy
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Introduction |
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Mast cells and basophils express the high-affinity IgE receptor FcRI on their surface, which plays a pivotal role in the initiation of allergic reactions. Fc
RI is composed of two kinds of functional subunits: a ligand-binding
-chain, and signaling chains, a ß-chain and disulfide-linked homodimeric
-chains (
-chain has an extracellular domain that binds monomeric IgE with high affinity (Ka
109-10), while the ß- and
-chains contain unique cytoplasmic domains essential for downstream signaling, called immunoreceptor tyrosine-based activation motifs (ITAMs) (
RI belongs to the family of multichain immunoreceptors that includes B- or T-cell antigen receptors and immunoglobulin G receptors (Fc
R) (
RI on mast cells and basophils evokes the activation of multiple signaling pathways, culminating in degranulation, lipid mediator release, and cytokine secretion (
RI engagement also activates mitogen-activated protein kinase which, in turn, regulates cytokine gene expression (
FcRI-mediated signals have been studied extensively in the rat basophilic leukemia cell line RBL-2H3 or its variant sublines. Previous morphological studies employing scanning and transmission electron microscopy demonstrated that the engaged IgE/Fc
RI
-chain complexes aggregate at the cell surface and are subsequently internalized through coated pits (
RI ß and
at the electron microscopic level has not yet been investigated. Current biochemical evidence using chimeric receptor molecules indicates that the
-chain aggregation alone can evoke cellular responses (
-chain-mediated signal (
-chains of Fc
RI have supported such important functions of these chains (
RI at the cell surface appear to constitute a crucial event in the initiation of signal transduction. Receptor aggregation occurs within a restricted region of the cell membrane at any given time. Therefore, visualization of topographical alterations of each Fc
RI subunit at the intact cell level during stimulation will give new insight into the precise links between ligand occupancy and biochemical signal elicitation. In this study we used conventional immunocytochemical methods as well as freeze-fracture replica immunoelectron microscopy (
RI subunits, particularly signaling ß- and
-chains, on the RBL-2H3 cell membrane has been resolved at the electron microscopic level. We propose that ß- and
-chains aggregate separately at the cell surfaces immediately after crosslinking.
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Materials and Methods |
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Cell Culture, Activation, and Histamine Release Assays
RBL-2H3 cells (
Immunofluorescence Microscopy
RBL-2H3 cells, cultured on 5-mm glass coverslips, were stimulated with anti-DNP IgE and DNPBSA as described above. Immediately after being rinsed with ice-cold PBS, the cells were fixed with 2% formaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4C for 10 min. The cells were permeabilized with acetone at -20C for 10 min and serially rinsed in PBS containing 0.1 M glycine, 50 mM ammonium chloride, and 0.1 M glycine/5% BSA for 10 min at each step. IgE/-chain complex, ß- or
-chains of Fc
RI in the cells were detected using the sheep polyclonal anti-rat IgE antibody (Bethyl Laboratory; Montgomery, AL), the mouse monoclonal antibody JRK for the ß-chain (
-chain (
Immunoelectron Microscopy
Immunolabeling on Ultrathin Cryosections.
After stimulation with DNPBSAgold, cells were extensively washed with PBS at 4C, fixed with a mixture of 2% paraformaldehyde/0.02% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4C for 2 days, and then processed for ultrathin cryosectioning and multiple immunogold labeling (
SDS-digested Freeze-fracture Replica Labeling (SDS-FRL).
The procedure for SDS-FRL of the RBL-2H3 cells was the same as described previously (RI subunits, were captured by replicas and their cytoplasmic domains would be accessible to the antibodies. The replicas were labeled with primary antibodies and then with the gold-conjugated secondary antibodies listed above. The samples were collected on Formvar-coated grids and examined with the electron microscope described above operated at 80 kV. For quantitation, the number of gold particles for ß- and
-chains associated with well-preserved regions of the plasma membrane were counted. At least 400 gold particles for each chain were quantified in four independent experiments.
Throughout immunoelectron microscopy, nonimmune, isotype-matched control antibodies did not give any labeling beyond the background level. Specificity of labeling and absence of signaling crossover or competition were also established by examination of single-labeled samples. In addition, a competition between different sizes of immuogold particles for double labeling was examined.
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Results |
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Immunofluorescence Microscopy
To clarify the spatial fate of ß- and -chains of Fc
RI in comparison with that of the
-chain during stimulation, we first performed double immunofluorescence staining with two kinds of antibody pairs: anti-IgE antibody for the IgE/
-chain complex and anti-ß-chain antibody, and anti-ß- and -
chain antibodies. RBL-2H3 cells sensitized with anti-DNP IgE were generally spindle-shaped and showed homogeneous immunofluorescence staining on the cell membranes for anti-IgE antibody, which represented anti-DNP IgE-bound
-chains (Fig 1A), ß-chain (Fig 1D and Fig 1G), and
-chain antibodies (Fig 1J). Before and after sensitization, staining for ß- or
-chains was essentially of the same pattern (data not shown).
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In cells stimulated with 100 ng/ml DNPBSA (optimal activation) at 37C for 5 min, the staining for the IgE/-chain and ß-chain (Fig 1B and Fig 1E) and that for the ß- and
-chains (Fig 1H and Fig 1K) consisted of numerous punctate dots outlining the cell surfaces. At 20 min after stimulation, the cells became flattened and spread as reported previously (
-chain and ß-chain staining was localized mainly in large intracellular vacuoles of the cells (Fig 1C and Fig 1F). In contrast, the
-chain staining revealed a fine granular pattern in the cytoplasm (Fig 1L), and its localization was apparently distinct from that of the ß-chains (Fig 1I). However, several co-localized spots for ß- and
-chains were also observed (Fig 1I and Fig 1L, arrowheads).
Immunogold Electron Microscopy Using Ultrathin Cryosections
To determine the localization of each subunit of FcRI at the ultrastructural level, immunogold electron microscopy using ultrathin frozen sections was performed. In IgE-sensitized RBL-2H3 cells, immunoreactivity of anti-IgE (15-nm gold particles), anti-ß-chain (30-nm particles), and anti-
-chain (5-nm particles) antibodies were dispersed on the cell surface, including microvilli (Fig 2A) and were not located in any intracellular endosomal structures.
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After crosslinking with DNPBSAgold (1:100 dilution with the PIPES buffer; optimal activation) at 37C for 5 min, these particles aggregated on the cell surface, which indicate clustered -chains. Some of them were localized in the coated pits, which was verified by immunolabeling using anti-clathrin (5-nm particles) antibody (Fig 2B). Almost all the immunogold particles for the ß- and
-chains were associated with the clusters of DNPBSAgold particles on cell surfaces. However, in many cases, labeling of either ß- or
-chains was independently co-localized with DNPBSAgold particles (Fig 2C), and the association of all three different particles was also observed (Fig 2D).
At 20 min after stimulation, the majority of the DNPBSAgold particles were concentrated in the perinuclear endosomal vacuoles. Immunoreactivity for the ß-chain was detected almost exclusively on these vacuoles (Fig 2E). Immunolabeling for the -chain, however, was present not only at the DNPBSAgold-positive endosomes (Fig 2E) but also at the DNPBSAgold-negative small vesicles (Fig 2F). Co-localization of both ß- and
-chains on the same endosomes was occasionally found. Gold labeling was essentially absent in the endoplasmic reticulum and Golgi apparatus. These results are consistent with immunofluorescence microscopic findings indicating the distinct cellular localization of ß- and
-chains during endocytosis.
SDS-digested Freeze-fracture Replica Labeling Electron Microscopy
To further investigate the two-dimensional distribution of ß- and -chains of Fc
RI on RBL-2H3 cell membranes during crosslinking, we next performed SDS-FRL electron microscopic analysis. In platinum/carbon replicas of RBL-2H3 cell membranes, smooth exoplasmic fracture faces were easily distinguished from the protoplasmic fracture faces covered by many intramembrane particles. Because the antibodies used recognize the cytoplasmic domains of these molecules, the immunogold particles for ß- and
-chains are associated with protoplasmic faces of the cell membrane. Before and after IgE sensitization, homogeneous and dispersed distribution of ß- and
-chain-associated immunogold particles on the protoplasmic faces were observed (Fig 3A), whereas the exoplasmic faces were virtually unlabeled.
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When cells were stimulated with 100 ng/ml DNPBSA (optimal activation) at 37C for 10 sec, immunolocalization for ß- and -chains showed remarkable changes. Round clusters (consisting of 420 particles about 100150 nm in diameter) of gold particles were observed (Fig 3B). Interestingly, the results indicated that ß- and
-chains did not co-aggregate but for most part formed homogeneous aggregates of ß-chains or
-chains. Freeze-fracture faces of the immunoreaction-positive regions of the cell membranes were morphologically indistinguishable from those of the surrounding membranes. After crosslinking for 5 min, patchy immunolabeled clusters became more conspicuous (Fig 3C). However, as observed by cryoimmunoelectron microscopy, co-localization of the two chains was also observed (Fig 3D). At 20 min after crosslinking, immunolabeling for either chain was observed not on protoplasmic faces of the cell membranes but in intracellular membranes of vesicles or vacuoles (Fig 3E). Aggregated immunogold particles for ß- and
-chains were found in the distinct vesicles.
Immunofluorescence Microscopy and SDS-FRL Electron Microscopy in Suboptimally Stimulated Cells
To clarify the relationships between the signaling intensity and spatial distribution of ß- and -chains, we performed immunofluorescence (Fig 4A and Fig 4B) and SDS-FRL immunogold electron microscopy (Fig 4C) in the cells stimulated with 5 ng/ml of DNPBSA that evoked a suboptimal net histamine secretion, for 5 min at 37C. In contrast to the cells stimulated with 100 ng/ml DNPBSA (optimal activation), ß-chains showed homogeneous immunofluorescence staining on the cell membranes (Fig 4A) like cells before crosslinking, while
-chains had a punctate staining pattern outlining the cell surfaces (Fig 4B). In SDS-FRL electron microscopy, consistent with the results of immunofluorescence, only
-chains were observed to be aggregated on the protoplasmic face of the cell membrane, while almost all the gold particles for ß-chains remained dispersed on the membrane (Fig 4C).
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Fig 5 summarizes quantitative analysis of the distribution of ß- and -chains on the cell membrane during stimulation, as revealed by SDS-FRL labeling. The mean labeling density of immunogold particles for ß- (15-nm gold particles) and
-chains (10-nm gold particles) was 53.4 and 43.1 particles/µm2, respectively. Before crosslinking (Fig 5A), almost all the immunogold particles for both ß- and
-chains were dispersed, and no patchy clusters were observed. After suboptimal crosslinking with 5 ng/ml DNPBSA at 37C for 5 min (Fig 5B), however, 65.8% of a total of 421 gold particles for
-chains were aggregated and 31.8% were dispersed, whereas 94.0% of a total of 597 particles for ß-chains remained dispersed and only 4.2% were aggregated. Thus, 38 (80.9%) of 47 clusters counted contained
-chains alone, and only five (10.6%) and four (8.5%) clusters contained ß-chains alone and co-aggregated ß- and
-chains, respectively. After optimal stimulation with 100 ng/ml DNPBSA at 37C for 5 min (Fig 5C), 58.4% of 515 particles for ß-chain and 57.9% of 466 particles for
-chains became aggregated, without co-aggregating with each other. Under these conditions, only four (5.0%) of 80 clusters consisted of immunogold particles for both ß- and
-chains, while 34 (42.5%) consisted of ß-chains alone and 42 (52.5%) of
-chains alone. The relative density of immunolabels for ß- and
-chains did not change when the two primary antibodies were linked to a different-sized gold particle (10-nm gold particle for ß-chain and 15-nm gold particle for
-chain). These findings indicate that
-chains alone, but not ß-chains, aggregate during weak stimulation, whereas both ß- and
-chains aggregate mostly separately during optimal stimulation.
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Discussion |
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Our present findings on the dynamics of the FcRI chain distribution on crosslinking not only are consistent with the importance of the aggregation mechanism in transmembrane signaling (
RI signaling system. SDS-FRL electron microscopy has enabled us to visualize the topographical locations of ß- or
-chains and their changes occurring in a restricted cell membrane region after Fc
RI stimulation. This technique demonstrated the uniformly dispersed localization of ß- and
-chains on unstimulated RBL-2H3 cell surfaces. On antigen stimulation, the distribution of these chains changed markedly to distinctly aggregated forms. Conventional immunogold electron microscopy using ultrathin cryosections also showed that most of the ß- and
-chains are independently co-localized with clustered DNPBSAgold/IgE/
-chain complexes on the cell surface. We therefore propose that crosslinking with multivalent antigen leads to the distinct aggregation of ß- and
-chains on the cell membrane and subsequently to independent sorting of these molecules along the endocytic pathway.
Several hypothetical models could explain our observations on the predominant formation of complexes consisting of - and ß-chains or
- and
-chains: One would be that all receptors on the cell surfaces exist as an
ß
2 tetramer before stimulation and then dissociate to generate
ß and
2 partial complexes after crosslinking. Alternatively,
-chains dissociate from other subunits at some point during the internalization process. Fig 2F provides some evidence for this possiblity. The other possibility is that receptors before stimulation exist in various forms, such as partial complexes including
ß or
2 (or even as unassociated
, ß, or
2 forms) as well as
ß
2 complete tetramers, and independently aggregate after crosslinking.
Our hypothesis on the existence of partial FcRI complexes on RBL-2H3 cell surfaces presents a striking contrast to the current tetramer model of the receptor (
ß
2 tetrameric complex is also known to be easily dissociated even in mild detergent (
- and
-chains is sufficient to achieve expression of the receptor (
2 complexes have been shown to be capable of inducing cell activation (
-chain associates first with the ß-chain and
ß complex with the
-chain later (
RI complexes are considered to be assembled simply to mask the retention signal and to escape degradation in the endoplasmic reticulum before surface expression (
RI, which were obtained by analyzing the cell lysates or artificial transfectants, to the natural existence of Fc
RI on intact RBL-2H3 cells.
Our SDS-FRL electron microscopic study revealed only the distribution of ß- and -chains but nothing about the topographical relationship between
- and ß- or
-chains. Although immunogold electron microscopy using ultrathin cryosections showed that most of the immunogold particles for ß- and
-chains are independently associated with clustered DNPBSAgold/IgE/
-chain complexes on the cell surface, the resolution is not great enough to examine the molecular composition of each individual Fc
RI complex. In addition, particular care should be taken in how precisely our immunogold labeling procedure reflects virtual number and aggregation status of membrane protein molecules. The possibility cannot be completely ruled out that the oligomerization status of molecules concerned may affect the efficiency of antibodies used in immunocytochemical experiments, and that more than one gold-labeled antibody molecule can bind to a single primary antibody on specimens. Our result in the present study appears to reflect relative distribution and oligomeric states, but because of a certain number of technical issues and limitations, we could not discard the current established concept that the receptor exists as an
ß
2 complete tetramer after crosslinking stimuli.
If crosslinking stimuli uniformly affect each receptor complex on the cell surface, an additional mechanism should exist in either model to favor the aggregation of the same kinds of signaling chains over that to form ß
2 tetramers. A possible mechanism for differential aggregation would be that phosphorylated ß- and
-chains may undergo conformational changes leading to oligomerization of the same kind of subunits, as shown in cytokine receptor dimerization and transphosphorylation (
-chain, could participate in the receptor aggregation. Recently, Fc
RI on the cell surface was shown to be surrounded by a detergent-insoluble lipid membrane domain (
In contrast to the distinct aggregation of ß- and -chains in cells stimulated with an optimal concentration of antigen (Fig 3C and Fig 5C), only
-chains were aggregated on surfaces of cells stimulated with a suboptimal concentration of antigen, while ß-chains remained dispersed (Fig 4C and Fig 5B). These results are consistent with earlier studies on chimeric receptors (
-chains alone can evoke a cellular response. Moreover, these results are also consistent with the current model on the role of ß-chains as a signaling amplifier of the Fc
RI system (
-chains, and phosphorylated ITAM motifs of
-chains in turn recruit another protein-tyrosine kinase, Syk, which phosphorylates downstream targets. It is not clear in this model how
2 complexes generate the activation signal.
Our present data might suggest that ß-chains can elicit a signal in a -chain-independent manner. Interactions between ß-chains and Lyn might be involved in this potential ß-chain-dependent signaling. In this regard, Btk is shown to be activated by Lyn, and in turn activates downstream pathways such as Ca2+ mobilization and mitogen-activated protein kinase cascades (
-chainSyk aggregation without mobilization of the ß-chainLyn systems. The topographical segregation of Lyn and Syk is consistent with recent data (
-chains may be required for full activation of downstream pathways.
FcRI belongs to the family of multichain immunoreceptors, such as B- or T-cell receptors and Fc
R, because of homologies in structure and function (
RI, as well as these studies, suggest that multichain receptor subunits can easily dissociate from each other and function independently after ligand engagement. Fig 2E, Fig 2F, and Fig 3C provide some evidence that ß- and
-chains may segregate into distinct invaginations. However, it is not yet possible to determine whether these chains enter the cell via separate endocytotic vesicles or whether the
-chains are selectively sorted to another vesicular compartment for recycling. Further experimentation is needed to validate these possibilities.
Finally, we speculate that different partial receptor complexes have substantially different signaling characteristics and that the relative proportion of each FcRI complex existing on mast cells would determine the net signal intensity of the cells. To control the aggregation of individual receptor complexes could be the potential target of pharmacological intervention in Fc
RI-mediated allergic reactions. Further studies are necessary to provide a clearer picture of how proximal Fc
RI signaling is initiated in relation to subunit dynamics.
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Acknowledgments |
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Supported by research grants from the Ministry of Education, Science and Culture of Japan and the Fukui Prefectural Research Foundation (to K. Fujimoto).
We thank Dr Toru Noda (Kyoto University, Kyoto, Japan) for advice and encouragement throughout this study.
Received for publication July 3, 2000; accepted July 10, 2000.
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